08 Agosto

Journal of Ethnopharmacology 87 (2003) 123–142
Preliminary comparative analysis of medicinal plants used

in the traditional medicine of Bulgaria and Italy
Maria Lucia Leporatti a,∗ , Stephanie Ivancheva b
a Department of Vegetal Biology University “La Sapienza”, 00185 Roma, Italy
b Bulgarian Academy of Science, Sofia, Bulgaria
Received 12 February 2002; received in revised form 6 February 2003; accepted 6 February 2003
Abstract
Despite their geographical, historical and cultural differences, Bulgaria and Italy share a surprisingly similar patrimony as regards the
popular uses of medicinal plants. The extensive knowledge acquired over the centuries by people living in these countries and engaged in
agriculture, derives from continuous contact with natural resources. This paper compares approximately 250 medicinal plants present in both
countries and used in popular medicine. From this comparison it emerges that knowledge of medicinal plants and their uses are well founded.
In fact, more than 80% of the plants are employed in identical or similar kinds of ailments, their preparation also showing marked similarities.
The remaining 20% have very different uses, several of these being particularly noteworthy. The role played by edible plants, moreover, is
important, about 30% being employed as medicine.
© 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Ethnobotany; Bulgaria; Italy
1. Introduction order (species and/or subspecies), among the very nume-

rous examples belonging to both floras, have been exam-
Bulgaria and Italy are very different countries from sev- ined in relation to their availability and the frequency of
eral points of view, historical, phytogeographic and even their use in the two countries.
religious but, over the centuries, the people living in them The analysis was carried out firstly in accordance with the
have resorted equally to their respective patrimonies regard- respective floras: Bulgarian Flora (Stojanov et al., 1967) and
ing the knowledge of medicinal plants. Flora d’Italia (Pignatti, 1982). The Bulgarian Flora records
The landscape of both countries alternates imposing 3700 species in all, whilst 5599 species are recorded in Flora
mountains with flatlands, where people have been en- d’Italia, the respective geographical differing greatly.
gaged in agricultural activities since ancient times. Such The specific literature of the respective countries was
knowledge is, therefore, the result of experience developed then compared. As regards Italian ethnobotanical literature,
through continuous contact with natural resources down papers were mainly consulted which relate to whole re-
through the ages. gions or large geographic areas (Gastaldo, 1987; Guarrera,
1990; Tammaro, 1984) but also specific contributions about
individual, smaller areas (Bruno et al., 1959; Corsi and
2. Methodology Pagni, 1978; Lentini et al., 1987/1988, 1994, 1994/1995,
1995, 1996; Lentini and Raimondo, 1990; Leporatti et al.,
The present work consists of a preliminary compara- 1985a,b; Pagni and Corsi, 1979; Pieroni, 2000; Raimondo
tive analysis of a large number of plants claimed to have and Lentini, 1990; Uncini Manganelli and Tomei, 1999a,b;
medicinal properties in the popular traditional medicine of Reuther and Reuther, 1988).
Bulgaria and Italy. About 250 plants of different hierarchic For the Bulgarian Flora we consulted Iordanov et al.
(1977), Isaiev et al. (1977), Ivancheva and Stantcheva
(2000), Kitanov (1953), Petkov (1982), Iordanov et al.
∗ Corresponding author. (1969), Isaiev et al. (1992), and Petkov (1982).
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0378-8741(03)00047-3
124 M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142
Table 1 contrary, Crataegus laevigata is acclaimed in Bulgaria for

Most numerously represented families its anti-Herpes activity but unknown in Italy. Also the he-
Families Number of taxa Families Number of taxa patoprotective and cholagogue action of Chelidonium ma-
jus is unknown in Italy where this plant has only external
Asteraceae 24 Brassicaceae 9
uses.
Lamiaceae 19 Liliaceae 9
Apiaceae 16 Ranunculaceae 8 Most of the plants in both the countries, however, have
Rosaceae 17 Scrophulariaceae 6 the same or comparable therapeutic uses, only about 20%
Fabaceae 11 Solanaceae 6 of them being employed in a completely different way. For
example, Astragalus glycyphyllos used in Bulgaria against
menstrual pains is, instead, known in Italy as a diuretic and
antirheumatic, or again Betonica officinalis used as an anti-
3. Discussion septic in Bulgaria and for its hepatoprotective, cholagogue
and diuretic properties in Italy. In Bulgaria, Galanthus ni-
The species examined belong to 72 families and 72 valis is said to fight the effects of curare poisoning, but in
genera. Many families are present only in a small num- Italy has only an external use against foruncolosis and ab-
ber of species, e.g. Caryophyllaceae, Rhamnaceae and scesses; also Sedum acre in Italy is only used externally for
Thymeleaceae. Table 1 shows the families having a larger cicatrizing sores and infected wounds whilst in Bulgaria it
number of species. is employed as a hypotensive (in Table 2 the plants having
different therapeutic properties are marked with the symbol
䉲). Other species present several common properties and
4. Results differ in only one or two uses.
Several plants present interesting therapeutic properties
Table 2 presents a commented list of the species examined (Table 3). In particular:
and their uses.
- Viola odorata employed in cases of particular dermatitis:
only those related to the presence of arthritic illness; this
reflects notable intuition as, in fact, arthritis and dermati-
5. Discussion and conclusion
tis are often closely related as symptoms of some auto-
immune diseases.
From attentive examination of the results, some points
- Ecballium elaterium and Verbascum sinuatum considered
may be drawn: people have mainly resorted to the use of
active against psoriasis (this is also an autoimmune dis-
spontaneously occurring plants (wild or cultivated), exotic
ease). The prescription from the Italian region of Sicily is
plants accounting for only 10 cases (Aloe, Cannabis, Euca-
characteristic: “The aerial parts of the plants are crushed
lyptus, etc.).
and soaked in cold water and untreated wine. The sus-
Some general considerations may be extrapolated regard-
pension is boiled by heating with a hot flame until half
ing the different plant species or subspecies used in the two
has evaporated. It is left to stand for 12 h at room tem-
countries. For example, in Bulgaria only Origanum hera-
perature and then filtered. The filtrate must be kept in a
cleoticum is used, whilst in Italy both O. heracleoticum and
refrigerator until required for use”. (Amenta et al., 2000).
Origanum vulgare are employed, this latter being more com-
One could hypothesise that these plants, given the abun-
mon than the former; Heracleum sphondilium is used in Italy
dance of long stiff hair on the aerial parts, would have
without distinction of subspecies, whereas in Bulgaria only
a largely mechanical and abrasive action on the affected
the subspecies sibiricum is employed in popular medicine.
skin, but the complex process which the recipe demands
The same applies to Helleborus odorus used in Bulgaria,
would suggest rather more than this.
whilst in Italy several species of this genus are equally em-
- Daucus carota and Pastinaca sativa subsp. sativa consid-
ployed, as is also the case with Aristolochia sp. div. This
ered as coronary dilators and capillarotrophic agents.
may be due to the abundance or availability of these plants in
- Corylus avellana used against benign prostatic hypertro-
the respective countries, but also to well-grounded botanical
phy (BPH).
knowledge of the plants and of their therapeutic properties.
- Crataegus laevigata is considered to be effective against
This is the case of the three species of Potentilla: anserina,
Herpes simplex.
erecta and reptans considered effective in relation to three
- Chelidonium majus and Solanum melongena as hepato-
different kinds of disease.
protective agents are also interesting.
Often therapeutic properties which have been well known
since ancient times in one of the two popular medicines are Each one of these plants and their uses deserves careful
completely unknown in the other, e.g.: the anti-helmintic consideration so as to stimulate further investigation.
and antipyretic properties of Artemisia absinthium, or the In both countries the most frequently used methods for
hypotensive activity of Crataegus laevigata, common in tra- preparing traditional medicines are infusion, decoction and
ditional Italian medicine are unknown in Bulgaria. On the maceration in wine. For the small number of plants (5% ca.)
Table 2
List of examined species
Botanical name Family Local name in Local name in Therapeutic use in Therapeutic use in Italy Part used in Part used in Bulgarian Italian manipulation Notes
Bulgaria Italy Bulgaria Bulgaria Italy manipulation
Galanthus nivalis L. 䉲 Amaryllidaceae Kokitche Bucaneve Anticurare Abscesses, furunculosis, Aerial parts Bulb Source of Cataplasm, poultice, Toxic
arthritis, synovitis nivalin locally applied
ointment
Cotinus coggygria Scop. Anacardiaceae Smradlika Còtino, Scòtano Astringent, As mouth wash Leaves Leaves, Maceration in Infusion External use. Contact can
anti-inflammatory flowers, bark hot water cause skin ulcers
M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

Anethum graveolens L. Apiaceae Kopar Aneto Carminative, Appetiser, spasmolytic, Fruits, oil Fruits Crushed and Infusion Dried fruits are used to
spasmolytic diuretic, carminative boiled fruits flavour food
Angelica archangelica L. Apiaceae Letchbna Angelica Sedative, spasmolytic Stimulates digestion, tonic, Roots, fruits Roots Extract with Elixir Employed in making
Pichtjalka carminative white wine candies and liqueurs
Apium graveolens L. Apiaceae Zelina Sedano Diuretic Antiscorbutic, diuretic, Roots, fruits Leaves, fresh Fresh juice Infusion Aerial parts are eaten as
expectorant, appetiser stems, seeds, (for Vitamin salad or boiled in soups
carminative, (seeds) (fruits) C content),
infusion
Carum carvi L. Apiaceae Kim Cumino Carminative, appetiser, Antiseptic for skin and Fruits Fruits Infusion Decoction Dried fruits used to flavour
spasmolytic stimulates mucosa, to promote food
gastric secretion, peripheral circulation,
cholagogue, uterotonic externally applied to
increase milk secretion,
appetiser, uterotonic
Coriandrum sativum L. Apiaceae Koriandar Coriandolo Stimulates gastric Aromatic to flavour food Fruits Fruits Infusion Tincture, infusion Dried fruits used to flavour
secretion spasmolytic, (raw seeds), spasmolytic, food
carminative to stimulate digestion,
antiseptic
Daucus carota L. Apiaceae Morkov Carota Anti-ascaridic, source Nutritive, carminative, Roots, seeds Roots Juice of fresh Juice as drink, Roots are eaten boiled or
of carotenoids depurative, soothes the skin, root, seeds cataplasm of fresh raw as vegetable
coronary dilating (seeds) root pulp
Eryngium campestre L. Apiaceae Vetrogon Calcatreppola Prostatitis, diuretic, Anti-oedema, diuretic, Roots Roots Infusion Decoction, tincture
spasmolytic cholagogue, choleretic, to
promote perspiration
Foeniculum vulgare ssp. vulgare Apiaceae Rezee Finocchio Carminative, stimulates Carminative, diaphoretic, Fruits Fruits Infusion Infusion Base of leaves of cultivated
Miller et F.v. ssp. piperitum gastric secretion diuretic species eaten as vegetable.
(Ucria) Coutinho Seeds used to flavour food
Heracleum sphondilium L. Apiaceae -------- Spondilio, Panace -------- Sedative for CNS, against -------- Aerial parts, -------- Tincture Toxic. Subspecies used in
nervous depression fruits Italy without distinction
Heracleum sphondilium L. Apiaceae Sibirski divisil Hypotensive, Roots, fruits Maceration in The only subspecies
ssp. sibiricum (L.) Simonkai 䉲 spasmolytic, appetiser, cold water growing in Bulgaria
antidiarrhoeic,
gastrointestinal diseases
Levisticum officinale Koch Apiaceae Letcheben selim Sedano di monte Diuretic, stimulates Diuretic, carminative, Roots Roots Decoction Decoction
gastric secretion reduces intestinal gas
Pastinaca sativa L. ssp. sativa Apiaceae Pachtarnaksta Pastinaca Cardio-tonic, Dietetic, diuretic, Roots, seeds Roots, leaves Decoction Infusion
spasmolytic, cholagogue
hypotensive, coronary
dilator, capillarotrophic
agent
125
126
Table 2 (Continued )
Petroselinum crispum Mill. Apiaceae Magdanoz Prezzemolo Diuretic, spasmolytic, Diuretic, emmenagogue Fruits, roots Roots, leaves Maceration, Juice or decoction Fresh leaves are used to
abortive (abortive!). Nutritive being infusion from roots or from flavour food
rich in Vitamins A and E. fresh leaves
For insect stings (juice of
leaves rubbed locally)
Peucedanum officinale L. 䉲 Apiaceae Samodivska treva Finocchio di porco Cardio-tonic Astringent, emmenagogue Roots, fruits Roots Decoction Infusion
Pimpinella anisum L. Apiaceae Anason Anice verde Antitussive, to stimulate Aperitive, aromatic, Fruits, oil Fruits Infusion Infusion, tincture Used in making candies
gastric secretion digestive, spasmolytic and liqueurs
Pimpinella saxifraga L. Apiaceae Bedreniza Tragoselino, Antitussive Expectorant, against catarrh Fruits Roots Maceration Decoction External use for eye

Becchino diseases (Bg)
Vinca minor L. Apocynaceae Malak zimzelen Pervinca Hypotensive, sedative, Tonic, haemostatic, diuretic, Leaves Aerial parts Decoction Infusion, tincture
astringent antiscorbutic, hypotensive
Arum maculatum L. 䉲 Araceae Zmijarnik Aro, Gigaro, Pan Diseases of Rheumatic pains, against Tuber Tuber Maceration in Tincture Toxic plant
di serpe gastrointestinal tract, gout, anti-inflammatory for cold water
lung diseases, gastrointestinal and
haemorrhoids respiratory tract
Acorus calamus L. Araceae Blaten air Calamo aromatico Appetizer, Appetizer, diuretic, Rhizome Rhizome Decoction Decoction
antidiarrhoeic insecticidal, antibacterial
Hedera helix L. Araliaceae Brachljan Edera Anti-inflammatory, Expectorant, antitussive, Leaves Leaves Decoction Infusion, decoction Toxic. The fruits can cause
antitussive antineuralgic, anaesthetic in severe allergies. Internal use
rheumatic and arthritic in Italy now abandoned
pains (external use) to clean
wounds, to darken hair
Aristolochia clematitis L. Aristolochiaceae Valtcha jabalka Strallogi Inflamed wounds Vulnerary Roots Rhizome Maceration, Decoction Toxic plant; only external
decoction use in Italy; also used A.
rotunda L. and A.
pistolochia L.
Asarum europaeum L. Aristolochiaceae Kopitnik Erba Renella Antitussive, sedative, Expectorant, emetic (strong Roots Rhizome, Infusion In Bulgaria, particularly
expectorant doses) leaves against chronic alcoholism;
abandoned due to its
toxicity in Italy
Periploca graeca L. Asclepiadaceae Garbatch Topa Cardiotonic Cardiotonic, hypertensive, Bark Bark, leaves Tincture Decoction, infusion
diuretic, purgative (leaves) (leaves)
Asplenium trichomanes L. Aspleniaceae Iztravnitche Erba Rugginina Antitussive Expectorant, emollient, Aerial parts Aerial parts Infusion Decoction
externally for dandruff
Scolopendrium officinale Sm. Aspleniaceae Volski ezik Lingua di cervo, Antitussive Expectorant, diuretic Aerial parts Leaves, Infusion Infusion, decoction
(=Phyllitis s.) (L.) Newmann 䉲 Lingua di cane astringent. Externally rhizome
applied on burns and
inflamed mucosa,
anti-helmintic. Externally
used as cicatrizing agent
Achillea millefolium L. 䉲 Asteraceae Bel ravnez Millefoglio, Erba Astringent, haemostatic To improve blood Aerial parts Aerial parts Infusion Infusion
Livia circulation, haemostatic, to
aid digestion, against
menstrual pains, bitter-tonic,
haemorrhoids
Arctium lappa L. Asteraceae Repei Bardana Diuretic, ulcer Cicatrizing agent in skin Roots Leaves, roots Decoction Decoction, In Italy A. minusBernh are
and A. nemorosum Lej. 䉲 diseases, antibacterial, cataplasm of leaves used equally. Boiled leaves
antimycotic is locally applied and stalks are eaten as a
vegetable
Artemisia absinthium L. 䉲 Asteraceae Pelin Assenzio Appetizer, to stimulate Anti-helmintic, diuretic, Aerial parts Aerial parts Infusion Decoction Leaves and flowering tops
gastric secretion, emmenagogue, antipyretic, are used to give aroma to
mouth wash mouth wash, antiseptic liqueurs
Artemisia vulgaris L. Asteraceae Div pelin Assenzio selvatico, Appetizer, astringent, Tonic, aromatic, Aerial parts Aerial parts Infusion Decoction
Canapaccio sedative, haemostatic emmenagogue (not to be
used in cases
of gastric
ulcer)
Bellis perennis L. Asteraceae Paritchka Pratolina Inflamed wounds Emollient, vulnerary, against Capitula Capitula Maceration Infusion
sores and bruises (heads) (heads)
Calendula officinalis L. Asteraceae Neven Fiorrancio Spasmolytic, inflamed Soothing and emollient for Capitula Leaves and Infusion Decoction
wounds, analgesic skin, against menstrual (heads) flowering tops
pains, against warts
Carlina acanthifolia All. 䉲 Asteraceae Rechetka Carlina Diuretic, To promote sweating and Roots Roots Infusion, Decoction, alcoholic
anti-inflammatory, for secretions, diuretic, alcoholic extract

urinary tract cholagogue extract
Centaurea cyanus L. 䉲 Asteraceae Sinja metlitchina Fiordaliso Appetizer, tonic, As mouth and eye wash, Flowers Flowers Infusion Infusion
diuretic against coughs, tonic
Cichorium intybus L. Asteraceae Sinja zlatchka Cicoria Cholagogue stimulant Choleretic, hepatoprotective Roots, aerial Leaves Decoction Decoction, squashed In Italy the boiled leaves are
for gastric secretion, against jaundice mild parts fresh leaves mainly eaten as a vegetable
hypoglycaemic laxative (water after
boiling), hypoglycaemic
Cnicus benedictus L. Asteraceae Presetchka Cardo santo Appetizer, stimulates Antipyretic, to aid Aerial parts Aerial parts Infusion Infusion
gastric secretion digestion, tonic cholagogue,
expectorant, anti-acid
Helianthus annus L. Asteraceae Slantchogled Girasole Appetizer Dietetic, to promote Leaves Flowering Infusion Infusion, tincture In Italy the toasted seeds
digestion and diuresis. Mild tops, seeds are enjoyed as snacks. Oil
sedative (seeds) is used in cooking
Helianthus tuberosus L. Asteraceae Gulia Topinambur, Patata Provitamin A Source of inuline and Leaves Tuber, oil Infusion Mainly ornamental; edible
del Canada betaine, dietetic, from the tuber eaten boiled or roasted
hypoglycaemic against uric seeds
acid
Hieracium pilosella L. Asteraceae Michi uchi Pilosella Diuretic, astringent Diuretic, astringent, Aerial parts Aerial parts Infusion Infusion
antipyretic,
anti-inflammatory
Inula helenium L. Asteraceae Bjal oman Enula campana Anti-inflammatory, Against bronchitis, catarrh Roots Roots Maceration Infusion, decoction,
antitussive anti-ascaridic and coughs; bitter-tonic, tincture
choleretic, diuretic,
externally applied for
exanthemata and itching
Matricaria chamomilla L. Asteraceae Laika Camomilla, Anti-inflammatory Mild sedative, cicatrizing Flowers Capitula Infusion Infusion Used in aromatizing liqueurs
(=Ch. recutita (L.) Rauschert) Capomilla antiseptic, spasmolitic agent, emollient and (heads) (heads)
in colitis and gastritis, soothing for the skin,
choleretic, mouth wash spasmolytic for
in tooth ache gastrointestinal tract
Onopordon acanthium L. Asteraceae Magarechki bodil Cardo asinino Diuretic, stimulant of In the past claimed as Aerial parts Juice from the Infusion Abandoned
gastric secretion anticarcinoma fresh leaves
Petasites hybridus L. Asteraceae Letchebna Farfaraccio Spasmolytic Sedative, for CNS, Leaves Capitula Infusion Infusion, decoction,
ovtcharka anti-asthma and cough, (heads), tincture
diuretic, to promote leaves,
perspiration rhizome
Senecio vulgaris L. Asteraceae Sporez Calderugia, Cholinolitic, uterotonic Digestive troubles, Aerial parts Flowering Decoction Decoction Toxic
Verzellina amenorrhoea, dysmenorrhoea plant
Silybum marianum (L.) Gaertn. Asteraceae Bjal tran Cardo Mariano, Choleretic cholagogue, Choleretic, cholagogue, Seeds, fruits Fruits Decoction Decoction
Cardo di Maria hepatoprotective urinary hepatoprotective
and bladder diseases
127
128
Solidago virga-aurea L. Asteraceae Gorski entchez Verga d’oro Diuretic, antitussive, Emollient, anti-inflammatory, Aerial parts Flowering Infusion Infusion
urinary diseases astringent, diuretic, in case tops, rhizome
(nephritis) of nephritis
Tanacetum vulgare L. Asteraceae Vratiga Tanaceto Anti-ascaridic, Anti-helmintic Aerial parts Aerial parts, Infusion Infusion Toxic
antiseptic, fruits
anti-inflammatory
Taraxacum officinale Weber Asteraceae Gluchartche Taràssaco, Dente Cholagogue, choleretic Diuretic, cholagogue, Roots Rhizome Infusion Decoction Leaves are often eaten
di leone, Soffione choleretic, eupeptic, mixed in salad
digestive depurative

Tussilago farfara L. Asteraceae Podbel Fàrfara, Antitussive (whooping Expectorant, emollient for Leaves Leaves, Infusion Infusion Raw leaves are eaten as
Tassilaggine cough), anticatarral, catarrh, against cough, capitula salad
respiratory disease, soothing for the skin (heads)
(silicosis, emphysema,
tubercular cough)
antiseptic,
anti-inflammatory
Berberis vulgaris L. Berberidaceae Kisel tran Crespino Cholagogue, diuretic, Haemostatic, diuretic Roots, fruits Roots, bark, Infusion Decoction Fruits are rich in vitamins,
against diarrhoea and astringent leaves, fruits employed in preparing
dysentery home-made jams
Alnus glutinosa (L.) Gaertner Betulaceae Tchema elcha Ontàno Astringent, Mouth wash, Crushed Bark Decoction Decoction
antidiarrhoeic anti-inflammatory cones or bark
in water
Betula pendula Betulaceae Breza Betulla Diuretic, cholagogue Antipyretic, diuretic, Bark Bark Infusion, Infusion, decoction
Roth (=B. alba L.) cholagogue, diaphoretic, in decoction
skin diseases
Borago officinalis L. Boraginaceae Krasta vitchna Borrana, Borragine Diuretic, to promote Emollient, lenitive, mild Aerial parts Leaves, Maceration Infusion, decoction In Italy the aerial parts raw
treva perspiration laxative, diuretic flowering tops or boiled are eaten as a
vegetable
Pulmonaria officinalis L. Boraginaceae Meduniza Polmonaria Antitussive, Diuretic, expectorant, Aerial parts Leaves, Infusion Decoction, infusion
anti-inflammatory, vitaminic, to promote flowering tops
diuretic, expectorant perspiration
Symphitum officinale L. Boraginaceae Tcherenoman Consolida Inflamed wounds Astringent, antidiarrhoeic, Roots Roots, leaves, Decoction Infusion
maggiore vulnerary flowering tops
Armoracia rusticana Brassicaceae Hrjan Cren, Barbaforte Appetizer, to stimulate To promote digestion. As Roots Roots Decoction, Fresh roots, cataplasm Used as spice on meat and
Gaertner, Meyer and Scherb gastric secretion, spices in food, rubefacient crushed roots fish
antiscorbutic mixed with
honey
Brassica nigra (L.) Koch Brassicaceae Sinap, hardal Senape nera To stimulate flow of To stimulate blood-flow, as Seeds Crushed seeds Crushed seeds Cataplasm powdered External use
blood cataplasm locally applied with honey seeds boiled in water
on painful joints, catarrh,
bronchial diseases
Brassica oleracea L. 䉲 Brassicaceae Zele Cavolo Choleretic, anti-ulcer Antiscorbutic, cicatrizing Aerial parts Leaves Fresh Cicatrizing cataplasm, Boiled leaves, buds or the
agent for sores and burns crushed or cicatrizant, leaves whole plant are eaten as a
scalded raw or boiled as vegetable
antiscorbutic
Capsella bursa-pastoris L. Brassicaceae Ovtcharska Borsa di pastore, Astringent, menstrual Haemostatic, diuretic, Aerial parts Aerial parts Maceration Decoction, juice of
torbitchka Borsacchina diseases astringent, for menstrual fresh plant or dried +
pain powdered plant
Nasturtium officinale R. Br. Brassicaceae Poretch Nasturzio Source of vitamins Source of Vitamins A, B, Aerial parts Whole plant Fresh plant To be eaten as vegetable
and C, antiscorbutic, diuretic
Raphanus sativus L. Brassicaceae Tcherna rjapa Ravanello Cholagogue, antitussive Digestive, to stimulate Roots Roots Fresh plant Fresh roots are eaten as
Radicette blood-flow vegetable
Sinapis alba L. Brassicaceae Bjal sinap Senape bianca Laxative Laxative (whole entire Seeds Seeds Crushed Decoction of Seeds are used in preparing
seeds), aromatic stimulates crushed, powdered sauces
digestion, for painful joints seeds applied locally
(e.u.)
Cannabis sativa L. Cannabaceae Konop Canapa Anti-inflammatory, -------- Seeds -------- Maceration -------- Illegal use in Italy
spasmolytic
Humulus lupulus L. Cannabaceae Chmel Lùppolo Sedative Sedative for CNS and Cones (female Cones (female Infusion Infusion, decoction Mainly used to amortise
sexual apparatus, bitter-tonic inflorescence) inflorescence) beer
Sambucus ebulus L. Caprifoliaceae Bazak Ebbio Diuretic, antiseptic, Diuretic, antirheumatic, Roots, fruits, Roots, fruits, Infusion Decoction, infusion Toxic
antitussive laxative antineuralgic, flowers flowers, leaves
anti-inflammatory

Sambucus nigra L. Caprifoliaceae Tcheren baz Sambuco Diuretic, to promote Diuretic, antirheumatic, Flowers, fruits Flowers, fruits Infusion, Infusion, squashed Inflorescence are fried and
perspiration laxative antineuralgic, decoction leaves locally eaten, fruits are employed in
anti-inflammatory, emollient, applied preparing home-made jams
dislocated bones (e.u.)
Viburnum opulus L. Caprifoliaceae Tchervena kalinka Pallone di maggio Uterotonic, astringent, Uterotonic, anti-abortive Roots Bark Infusion Infusion (high doses
haemostatic are toxic)
Herniaria glabra L. Caryophyllaceae Golo izsipli-vtche Erniaria Diuretic, spasmolytic Diuretic, in case of Aerial parts The whole Infusion Infusion Toxic
gall-stones plant with the
root
Saponaria officinalis L. Caryophyllaceae Letchebno Saponaria Antitussive, diuretic, Expectorant, to promote Roots Roots Infusion Decoction Toxic
sapuntche stimulates sweating secretion of respiratory and
gastrointestinal tract
Stellaria media L. 䉲 Caryophyllaceae Vrabtchovitchrevza Centocchio Anti-inflammatory Astringent, diuretic, Aerial parts Aerial parts Infusion Infusion, juice of
vulnerary fresh plant
Chenopodium bonus-henricus L. Chenopodiaceae Tchuven Buon enrico, Inflamed wounds As cicatrizing agent (e.u.), Roots Flowering Infusion Decoction In Italy, the boiled aerial
Colubrina emollient, mild laxative, plant parts are also eaten as a
anti-anaemic (rich in iron) vegetable
Convolvulus arvensis L. Convovulaceae Polska povetiza, Vilucchio Strong laxative, Laxative, diuretic, against Aerial parts Aerial parts, Tincture, Tincture, infusion,
gramofontche hypertensive (flowers), furunculosis roots cataplasm cataplasm
furunculosis
Cuscuta europaea L. Convolvulaceae Kukuvitcha prezda Cuscuta Laxative, diuretic, Mild sedative, cholagogue, Aerial parts The whole Decoction Infusion, tincture
and related species 䉲 spasmolytic carminative plant
Cornus mas L. 䉲 Cornaceae Drjan Corniolo Astringent Antidiarrhoeic, Fruits Fruits fresh Decoction Decoction Fruits are used in
anti-haemorrhagic, bark, fresh home-made jams or liqueurs
antipyretic pulp of the
fruits
Corylus avellana L. 䉲 Corylaceae Obik novena leska Nocciolo Cardiac diseases, Antiseptic, intestinal Fruits, bark Fruits, bark, Infusion Decoction
prostatic hypertrophy astringent, for haemorrhoids leaves
(piles), antiseptic for minor
wounds and sores
Sedum acre L. 䉲 Crassulaceae Ljutivatlastiga Erba pignola Hypotensive, stimulant Externally applied to sores, Aerial parts Aerial parts Infusion Poultice of fresh Under medical supervision
infected wounds, warts, leaves
anti-atherosclerosis
Bryonia alba L. and Cucurbitaceae Diva tikva Vite bianca Diuretic, laxative, to Diuretic, purgative, Roots Leaves Decoction Cataplasm of fresh Toxic
Bryonia dioica Jacq. stimulate flow of blood externally applied on painful leaves (e.u.),
joints, or on furuncles infusion
Ecballium elaterium (L.) Richard Cucurbitaceae Luda krastaviza Cocomero asinino Laxative Drastic purgative, against Fruits Fruits, aerial Infusion Infusion, decoction Toxic
psoriasis part evaporated then
filtered
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130
Juniperus communis L. Cupressaceae Sinja hvoina Cipresso Diuretic, antiseptic for Balsamic, diuretic, Pseudofruits Young stem Infusion, Infusion, decoction Pseudofruits are used in
urinary tract antiseptic for urinary tract, with leaves decoction, or tincture in wine aromatizing liqueurs (I)
antipyretic, antidiarrhoeic, and pseudofruits
muscular pains (e.u.) pseudofruits maceration in
olive oil
Thuja occidentalis L. Cupressaceae Bozjo darvo Tuja Against corns Diuretic. Locally applied Leaves Leaves Decoction Decoction, tincture
against warts
Tamus communis L. Dioscoreaceae Brej Tamaro To stimulate flow of Emetic, purgative, revulsive Roots Roots Crushed and Decoction, tincture Toxic. External use only
blood mixed with

oil
Drosera rotundifolia L. Droseraceae Rosjanka Rosolida Lung diseases Against cough, bronchial Aerial parts Aerial parts, Infusion Decoction
spasmolytic roots
Equisetum telmateja Ehrh. Equisetaceae Polski hvocht Coda cavallina Diuretic, astringent, Diuretic, reconstituent to Aerial parts Aerial parts, Infusion Infusion Use must be avoided in
(=E. arvense L.) source of cartilage in lungs. To flowering tops case of urinary problems,
microelements strengthen hair and nails, can provoke haematuria
source of microelements
Vaccinum myrtillus L. Ericaceae Tchema Mirtillo nero Astringent, Astringent, antiseptic, to Leaves, fruits Leaves, fruits Decoction Juice, pulp Fresh fruits are used in
borovinka anti-inflammatory, protect retina and capillary preparing jams
hypoglicemic vessels, anti-inflammatory
for the skin, hypoglicemic
(leaves)
Vaccinium vitis-ideae L. Ericaceae Tchema borovinka Mirtillo rosso Astringent Diuretic, antiseptic, Leaves, fruits Leaves, fruits Decoction Juice from fruit Fresh fruits are used in
anti-inflammatory, anti-inflammatory pulp preparing jams
hypoglicemic
Astragalus glycyphyllos L. 䉲 Fabaceae Klinavitche Liquerizia bastarda Menstrual pains Diuretic, rheumatic pains, Aerial parts Roots, leaves Decoction Decoction
against gout
Galega officinalis L. Fabaceae Zja blek Galega, Hypoglicemic Hypoglicemic, increasing Aerial parts Flowering Infusion Infusion,
Capraggine milk secretion tops tincture
Genista tinctoria L. Fabaceae Bagrilna zjaltura Ginestrella Diuretic, laxative Diuretic, laxative, emetic Aerial parts Seeds Infusion Infusion
Glycirrhyza glabra L. Fabaceae Sladak koren Liquerizia Ulcer, antitussive, Antitussive, expectorant, Roots Roots Infusion Infusion Dried roots are chewed for
laxative, gastric emollient, sedative for sweet taste
disorders stomach-ache, antiprostatic
adenoma
Laburnum anagyroides Medicus Fabaceae Zlaten dajd Maggiociondolo To facilitate respiration Cholagogue, choleretic, Seeds Leaves Decoction Infusion Toxic
(=Cytisus l. L.) 䉲 laxative
Melilotus officinalis (L.) Lam. Fabaceae Letchebna Meliloto, Trifoglio Sedative Anti-inflammatory eye wash Aerial parts Flowering Infusion Infusion
komuniga cavallino and nostril wash, mild tops
antiseptic and antipyretic,
diuretic, sedative
Ononis arvensis L. Fabaceae Gramotran See Ononis Diuretic See Ononis spinosa Roots See Ononis Maceration See Ononis spinosa This species is rare in Italy
spinosa spinosa
Ononis spinosa L. Fabaceae Bodliv gramotan Arrestabuoi, Diuretic, kidney stones, Diuretic, anti-inflammatory Roots Roots Infusion, Decoction
Stanca bue urinary diseases for the urinary tract and for tincture
acne, mouth wash
Robinia pseudoacacia L. Fabaceae Bjal salkam Robinia Antitussive, laxative Emollient, cholagogue Flowers, bark, Flowers bark, Infusion Infusion (flowers),
(flowers), laxative or drastic leaves leaves decoction (bark)
purgative depending on
dosage
Trifolium pratense L. Fabaceae Livadna detelina Trifoglio Antitussive, diuretic Regulating mucosa secretion Flowers Flowers Infusion Infusion External use,
anti-inflammatory
Trigonella foenum-graecum L. Fabaceae Grazki sminduh Fieno greco Appetizer, anabolic, Dietetic, nutrient, Seeds Seeds Powder mixed Powdered seeds,
sedative appetizer, reconstituent with gum decoction
Quercus petraea (Mattuschka) Fagaceae Zimen dab, Gorun Quercia Astringent, for Astringent, Bark Bark, fruits Decoction Decoction External use. Roasted seeds
Liebl (=Q. sessiliflora L.) dermatitis anti-inflammatory, mild used as coffee substitute.
antiseptic, anti-haemorrhagic Acorns as food for pigs
Quercus robur L. Fagaceae Leten dab See Quercus Astringent, for See Quercus petraea Bark See Quercus Decoction See Quercus petraea External use. Roasted seeds
petraea dermatitis petraea used as coffee substitute.
Acorns as food for pigs
Centaurium erythraea Rafin. 䉲 Gentianaceae Tcherven kantarion Biondella, Appetizer Antipyretic, cicatrizing Aerial parts Aerial parts Infusion Infusion
Cacciafebbre agent for sores and minor
wounds, appetizer, digestive,

hepatoprotective,
hypoglicemic
Gentiana lutea L. Gentianaceae Zjalta tintjava Genziana Appetizer Appetizer, bitter-tonic Roots, aerial Roots, aerial Maceration Decoction, tincture Roots highly valued in
maggiore, digestive, antipyretic, part part preparing elixirs, bitter-tonic
Genziana gialla cicatrizing agent for minor
wounds (e.u. of the leaves)
Erodium cicutarium (L.) L’Her Geraniaceae Zikutovo Cicutaria Astringent Astringent, diuretic, Aerial parts Aerial parts Infusion Infusion
tchasovnitche haemostatic, antidiarrhoeic,
soothing for the skin,
anti-oedema
Geranium sanguineum L. 䉲 Geraniaceae Kraven zdravez Sanguinaria Hypotensive, antivirus, Stomatitis as mouth Roots Aerial parts Maceration Infusion
Malvaccini immuno-stimulant wash, astringent
sedative, CNS
depressant
Ribes nigrum L. Grossulariaceae Kasis Ribes Vitaminic, astringent, Vitaminic, aperitive, Fruits, leaves Fruits, leaves Fresh plant, Pulp or juice of the Fruits used in preparing
anti-inflammatory, diuretic, astringent, infusion fruits, infusion jams
diuretic anti-inflammatory protects
blood vessels and retina
Hypericum perforatum L. Guttiferae Zjalt kantarion Erba cacciadiavoli, Ulcers, Cicatrizing agent, against Aerial parts Flowering Infusion Infusion or This plant has a long
Erba di S. anti-inflammatory Herpes simplex cholagogue tops maceration, tradition in magic ritual
Giovanni decoction beliefs
Aesculus hippocastanum L. Hippocastanaceae Konski kesten Ippocastano Haemorrhoids Anti-oedema, against Bark, fruits Seeds Tincture Decoction
varicose veins and bruises
Iris florentina L. Iridaceae Bjala perunika Iris, Ireos, Antitussive, See Iris germanica Roots See Iris See Iris germanica
Giaggiolo anti-inflammatory, germanica
stomach-ache
Iris germanica L. Iridaceae Sinja perunika Iris, Ireos, Antitussive, Against coughs and catarrh, Roots Rhizome Maceration Decoction
Giaggiolo anti-inflammatory, emollient for the skin
stomach-ache
Iris pallida Lam. Iridaceae -------- Ireos, Iris, -------- Expectorant, cholagogue -------- Rhizome -------- Decoction, tincture
Giaggiolo
Iris pseudacorus L. 䉲 Iridaceae Vodna perunika Falso àcoro Neurodermatitis Astringent, emetic, Roots Rhizome Maceration Decoction, tincture
purgative applied externally
as haemostatic
Juglans regia L. 䉲 Juglandaceae Oreh Noce Astringent, Bitter-tonic, digestive, Leaves Leaves, Decoction Decoction Seeds valued “dry fruits”.
anti-inflammatory antiseptic hypoglicemic, walnut husk Husk is used to make
depurative. Externally “Nocino” liqueur (I)
applied on reddened
mucosa or skin; to darken
hair (husk)
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132
Betonica officinalis L. (=Stachys Lamiaceae Ranilist Betonica Antiseptic for inflamed Diuretic, cholagogue, Aerial parts Leaves Infusion Infusion
officinalis (L.) Trevisan 䉲 wounds hepatoprotective
Galeopis tetrahit L. Lamiaceae Budariza Erba giudaica, Antitussive Diuretic, against catarrah Aerial parts Flowering Decoction Infusion, tincture
Canapa selvitca tops
Glechoma hederaceae L. Lamiaceae Samobaika Edera terrestre Bronchitis Astringent, tonic, diuretic, Aerial parts Aerial parts Infusion Infusion
against catarrh
Lamium album L. Lamiaceae Bjala martva Ortica bianca Astringent, diuretic, Astringent, haemostatic, Flowers Infusion Infusion
kopriva tonic diuretic, uterotonic, skin

abscesses and burns (e.u.)
Lavandula angustifolia Miller Lamiaceae Lavandula Lavanda Sedative, spasmolytic Sedative, spasmolytic, Fruits, oil Flowers Infusion Infusion
externally applied antiseptic,
antihysteric
Leonurus cardiaca L. Lamiaceae Djavolska usta Cardiaca Sedative, cardiotonic Mild cardiotonic, Aerial parts Aerial parts Maceration Infusion, tincture
hypotensive and sedative
for CNS. Anti-depressive
Marrubium vulgare L. Lamiaceae Ptchelnik Marrobio Cholagogue, Choleretic, digestive, Aerial parts Flowering Maceration Infusion, tincture,
spasmolytic, antitussive, expectorant, antipyretic, tops, leaves cataplasm of boiled
menstrual disorders, anti-anaemic, antirheumatic, aerial part
hepatoprotective on painful joints (e.u.),
against tachycardia
Melissa officinalis L. Lamiaceae Matotchina Melissa, Erba Sedative, hypotensive, Sedative, spasmolytic, Leaves, aerial Leaves, aerial Infusion Infusion
limoncina spasmolytic digestive, choleretic, parts parts
externally for headache and
neuralgia
Mentha piperita L. Lamiaceae Ljutiva menta Menta Choleretic spasmolytic, Choleretic, spasmolytic, Leaves oil Leaves, Infusion Infusion, tincture Fresh or dried leaves are
antiseptic, anaesthetic antiseptic, anaesthetic, flowering tops used as herb in food
digestive refreshing,
balsamic, hepatoprotective,
against jaundice
Mentha pulegium L. Lamiaceae Blatna menta, Puleggio Choleretic spasmolytic, See Mentha piperita Leaves oil Leaves, Infusion Infusion Fresh or dried leaves are
divdjodjen antiseptic, anaesthetic, flowering tops used as herb in food (I)
jaundice,
hepatoprotective
Mentha spicata L. Lamiaceae Djodjen Menta Choleretic spasmolytic, See Mentha piperita Leaves Leaves, Infusion Infusion Fresh or dried leaves are
antiseptic, anaesthetic flowering tops used as herb in food (I)
Nepeta cataria L. 䉲 Lamiaceae Kotcha treva Erba gattaia Antiviral Mild sedative, spasmolytic, Aerial parts Flowering Infusion Infusion, tincture
aromatic, digestive tops
Origanum heracleoticum L. Lamiaceae Bjal rigan Origano Antitussive, to stimulate Aperitive, digestive. Mild Aerial parts Aerial parts Infusion Infusion, tincure Dried leaves are used as
gastric secretion antiseptic, spasmolytic, herb in food (I)
expectorant, in case of
inflamed lungs
Origanum vulgare L. Lamiaceae Tcherven rigan Origano Antitussive, stimulates Aperitive, digestive. Mild Aerial parts Aerial parts Infusion Infusion, tincture Dried leaves are used as
gastric secretion, antiseptic, spasmolytic, herb in food
choleretic, cholagogue expectorant, in case of
inflamed lungs
Rosamarinus officinalis L. Lamiaceae Rosmarin Rosmarino To stimulate gastric Aperitive, digestive, Leaves oil Leaves Infusion Infusion Fresh leaves are used as
secretion, diuretic, aromatic, diuretic, balsamic, herb in food
choleretic antiseptic, skin tonic
(promoting cutaneous blood
circulation), cholagogue,
choleretic, against
perspiration
Salvia officinalis L. 䉲 Lamiaceae Gradinski tchai Salvia Anti-inflammatory Eupeptic, digestive, Leaves Leaves and Infusion Infusion Leaves are used in cooking
cholagogue, antiseptic, flowering tops
expectorant, emmenagogue,
as mouth wash, tooth ache
Satureja hortensis L. Lamiaceae Gradinska Santoreggia Against intestinal gas, Spasmolytic, stimulating Aerial parts Aerial parts Infusion Infusion The fresh or dried leave
tchubriza spasmolytic, antiviral, digestion, eupeptic, are used to flavour food
astringent, hypotensive, depurative; by gargling
hepatoprotective, against sore throat and as
digestive mouth wash, antimycotic
Satureja montana L. 䉲 Lamiaceae Planinska tchubriza Santoreggia Dermatitis ab insectis Choleretic, stimulant, Aerial parts Aerial parts Fresh plant Infusion, fresh plant
digestive, antiseptic for
gastrointestinal tract
Teucrium chamaedrys L. Lamiaceae Tcherveno Camedrio Stomach-ache, Aperitive, digestive, Aerial parts Flowering Infusion Infusion

poddabitche antimicrobial activity bitter-tonic, antiseptic tops
Thymus sp. pl. Lamiaceae Machterka Timo Antitussive, antiviral, Antibacterial, expectorant, Aerial parts Aerial parts Infusion Infusion Leaves are used to flavour
spasmolytic balsamic, digestive, meat
depurative, aromatic, to
stimulate blood-flow,
gargling as mouth and
tooth wash
Allium cepa L. 䉲 Liliaceae Kromid Juk Cipolla Appetizer, Hypoglicemic Bulb Bulb Fresh bulb Fresh bulb Sliced raw bulb is used
stomach-ache mixed in salads, also boiled
or roasted
Allium sativum L. Liliaceae Tchesan Aglio Anti-atherosclerosis, Anti-atherosclerosis, Bulb Bulb Fresh cloves Fresh cloves Sliced cloves are used in
hypotensive, antiviral hypotensive, anti-helmintic, several recipes
antibacterial, antiviral
Allium ursinum L. 䉲 Liliaceae Levurda Aglio ursino Anti-atherosclerosis, Stimulates digestion, Aerial parts, Flowering Fresh cloves Fresh cloves Sliced cloves are mixed in
antiviral rubefacient, hypotensive bulb tops, bulb salad
Aloe arborescens Miller Liliaceae Aloe Aloe Stimulant, laxative, Aperitive, laxative Fresh leaves -------- Extract, -------- In Italy, no longer used in
choleretic infusion popular medicine. Other
(under species are rarely employed
medicinal
control)
Asparagus officinalis L. Liliaceae Asparagus Asparago Diuretic Diuretic Roots Rhizome Decoction Decoction Boiled young stems
(“turioni”) commonly eaten
as vegetable (I)
Convallaria majalis L. Liliaceae Momina salza Mughetto Cardiotonic Cardiotonic Aerial parts Flowers Tincture Infusion Toxic
Polygonatum odoratum Liliaceae Momkova salza Sigillo di Astringent diuretic, Astringent, diuretic, Roots Rhizome Decoction, Infusion External use. In ancient
(=P. officinale All.) Salomone anti-inflammatory anti-inflammatory juice or pulp times, it was believed to be
from squashed useful in healing broken
rhizome bones
Ruscus aculeatus L. Liliaceae Michi tchimchir Pungitopo Diuretic, haemorrhoids Diuretic, antigout. To Roots Rhizome Tincture Decoction, tincture Not used in cases of
(piles) promote perspiration pyelo-nephritis
astringent, anti-inflammatory,
protects capillary vessels,
against haemorrhoids (piles)
Veratrum album L. 䉲 Liliaceae Bjala tchemerika Veratro, Falso Hypotensive Applied externally as Roots Rhizome Tincture Ointment Toxic. No longer used in
elleboro analgesic on painful joints (in olive oil) Italy
Linum usitatissimum L. Linaceae Len Lino Laxative, Emollient, anti-inflammatory Seeds Seeds Maceration Decoction or
anti-inflammatory for for the skin, against cataplasm of
gastric and urinary tract itching, to regulate intestine crushed seeds; also
(whole seeds) crushed seeds in
water are drunk for
intestinal problems
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134
Viscum album L. Loranthaceae Bjal imel Vischio Hypotensive Hypotensive, diuretic, Leaves Young stems, Infusion Infusion
spasmolytic leaves
Lycopodium clavatum L. Lycopodiaceae Plavun Licopodio Mild skin emollient, Mild emollient for Aerial parts, Spores Infusion Powder of spores
uroseptic, diuretic, inflamed skin spores
antipyretic
Althaea officinalis L. Malvaceae Rouza Altea Antitussive, Emollient, soothing for the Roots Roots Decoction Decoction
anti-inflammatory for skin, as mouth wash against

respiratory system tooth ache, expectorant, mild
laxative
Malva sylvestris L. Malvaceae Gorski slez Malva Spasmolytic, Emollient, soothing, mild Flowers, Flowers Infusion Infusion Young leaves are boiled to
antitussive, sedative, laxative and expectorant leaves leaves make soups
cystitis, cholagogue
Menyanthes trifoliata L. Menyanthaceae -------- Trifoglio fibrino, Appetizer, purgative, in Bitter-tonic, antiscorbutic, -------- Leaves -------- Infusion, fresh
Trifoglio d’acqua gastric disorders diuretic, cholagogue, source leaves juice
of Vitamin A and Fe, Mn, I
Ficus carica L. Moraceae Smokinja Fico Laxative, Laxative, anti-inflammatory, Fruits Dried or Boiled with Infusion or Caustic latex seeping from
anti-inflammatory, antitussive fresh fruits milk decoction of dried the green parts is used to
antitussive are also eaten fruits, boiled with remove warts and corns
milk
Morus alba L. Moraceae Bjala tcherniza Gelso bianco Hypoglicemic, Expectorant laxative Fruits, leaves Fruits, leaves, Decoction Syrup from the Fruits are eaten fresh or
hypotensive, diuretic, (fruits), astringent, bark fruits used in preparing jams and
antitussive hypoglicemic (leaves), ices
diuretic, anti-helmintic (bark)
Morus nigra L. Moraceae Tcherniza Gelso Hypoglicemic, Expectorant laxative Fruits, leaves Fruits, leaves, Decoction Syrup from the Fresh fruits are eaten or
hypotensive, diuretic, (fruits), astringent, bark fruits employed in preparing jams
antitussive hypoglicemic (leaves), and ices
diuretic, anti-helmintic (bark)
Eucalyptus globulus Labill. Myrtaceae Evkalipt Eucalipto Spasmolytic, against Balsamic, expectorant, Leaves, oil Leaves Infusion Infusion, smoke of Used in Italy in marshy
and E. chamaldulensis Dehnh. bronchitis anticatarrhal burning leaves is areas to fight “malaria”,
inhaled given its ability to drain
water from the soil
Nymphaea alba L. 䉲 Nympheaceae Bjala vodnarosa Ninfea To attenuate freckles Narcotic, sedative, astringent Flowers Flowers, Decoction Decoction, syrup The rhizome rich in starch
(raw rhizome) rhizome from flowers was eaten in times of famine
as source of carbohydrate
Fraxinus ornus L. 䉲 Oleaceae Mazdrjan Orniello Avorniello Astringent, Laxative, antitussive Bark Juice exuding Infusion Decoction, syrup
anti-inflammatory, from the trunk
haemostatic
Fraxinus excelsior L. Oleaceae -------- Frassino -------- Antirheumatic antigout -------- Bark -------- Infusion
Olea europea L. Oleaceae Maslina Olivo Cholagogue, Cholagogue, vitaminic, Fruits, oil Fruits, oil, Tincture oil The oil used as Pickled fruits as snacks.
hypotensive, hypotensive (leaves), leaves seasoning in food, The oil is highly valued in
cardiotonic, anticholesterolemic. Locally decoction of leaves cookery for its nutritional
antiarhytmic applied to soothe and heal value
burns, mild laxative
Syringa vulgaris L. Oleaceae Ljuljak Lillà Antipyretic, Antipyretic, astringent, to Flowers, Bark, fruits, Crushed and Decoction, oil
appetizer soothe the skin (flowers) leaves leaves boiled in maceration (flowers)
water
Oxalis acetosella L. 䉲 Oxalidaceae Kiselitche Acetosella Appetizer Depurative, diuretic, Aerial parts Leaves Fresh state Juice from fresh Leaves are also eaten as
astringent, thirst quenching leaves, decoction salad
Paeonia officinalis L. Paeoniaceae -------- Peonia Sedative, spasmolytic Sedative -------- Petals, ripe -------- Infusion
seeds, root
Paeonia peregrina Mill. Paeoniaceae Tcherven bojur -------- Sedative, spasmolytic -------- Roots, flowers -------- Infusion -------- Not used for children
Chelidonium majus L. 䉲 Papaveraceae Zmijsko mljako Celidonia, Erba da Cholagogue, To remove warts and Aerial parts Latex Infusion Latex is locally
porri spasmolytic, corns (e.u.) applied
hypotensive analgesic,
hepatoprotective

Fumaria officinalis L. Papaveraceae Letcheben, Fumastrello Cholagogue, Depurative, to promote Aerial parts Aerial parts Maceration Infusion Small doses stimulate
related species also used 䉲 Rosopas spasmolytic, circulation and breathing breathing and circulation,
hypotensive high doses inhibit them
Glaucium flavum Crantz 䉲 Papaveraceae Zjaltmak Papavero cornuto Antitussive Antiseptic, cicatrizing, to Aerial parts Latex exuding Infusion External use Also used in Italy is
heal minor sores and from the Glaucium corniculatum
wounds broken stem J.H. Rudolph
(in June–
August)
Papaver rhoeas L. Papaveraceae Polski mak Papavero, Antitussive Mild sedative, against Flowers Petals Infusion Infusion Toxic
Rosolaccio cough, bronchitis
Papaver somniferum L. Papaveraceae Sanotvoren mak Papavero da Oppio Analgesic -------- Opium -------- Opium -------- Only under medical
supervision. The use and
cultivation in Italy is
forbidden
Pinus sylvestris L. Pinaceae Bjal bor Pino Antitussive, antiseptic, Balsamic expectorant, Turiones Pini Buds, young Decoction Decoction, infusion The seeds are eaten raw
diuretic, diuretic (young stems) stems, resin and also appreciated in
anti-inflammatory pastry and as snacks
Plantago sp. pl. Plantaginaceae Tsenolisten Piantaggine Emollient, to soothe Emollient for the skin, Leaves, aerial Seeds, leaves Infusion Infusion, decoction. The most highly valued
zilovljak the skin, bronchial laxative, for coughs, parts Seeds mixed in species in Italy is P. major
diseases and coughs, soothing, eye wash, mouth water drunk as L. Leaves are boiled and
mild laxative (seeds) wash laxative mixed in soups
Avena sativa L. Poaceae Oves Avena, biada Sedative for CNS Sedative emollient for the Flour, seeds Flour, seeds Maceration Maceration, Seed flour was once used
skin (bath), diuretic in decoction, decoction in making bread (I)
bladder and urinary pains, cataplasm
painful joints (e.u.)
Cynodon dactylon (L.) Pers. Poaceae Troskot Gramigna, Diuretic, laxative Diuretic, emollient for the Roots Roots Decoction Decoction, infusion
Zizzania skin, mouth wash, in renal
and urinary problems,
emollient for gastrointestinal
tract
Elytrigia repens (L.) Nevski Poaceae Pyrei Gramigna Antitussive, Diuretic Underground Rhizome Decoction Decoction, infusion
(=Agropyron repens (L.) P.S.) anti-inflammatory, stems
diuretic
Zea mays L. Poaceae Zareviza Granoturco, Mais Diuretic, cholagogue, Diuretic, depurative, Stigmata Stigmata Decoction Infusion, tincture Seed flour commonly used
astringent sudorific, hypotensive in preparing “polenta” (I).
Oil has anticholesterol
properties
135
136
Persicaria bistorta (L.) Polygonaceae Karvavitche Bistorta, Astringent Astringent, Roots Leaves, Decoction Infusion, powdered
Samp. (=Polygonum bistorta L.) Serpentaria anti-inflammatory, in anti-inflammatory rhizome rhizome
case of stomatitis antidiarrhoeic, to wash
greasy hair
Polygonum aviculare L. Polygonaceae Patcha treva Corrigiola Diuretic, astringent, Diuretic, cicatrizing agent, Aerial parts Aerial parts Infusion Infusion Being rich in Si, it was
Centinodia haemostatic astringent used in the past as a
palliative in tuberculosis
Rheum palmatum L. Polygonaceae Reven Rabarbaro Laxative, cholagogue Aperitive, depurative Roots Rhizome Tincture Decoction, powder
laxative, cholagogue

Rumex acetosa L. Polygonaceae Kiselez Acetosa Antiscorbutic, diuretic, Diuretic, antipyretic, as Aerial parts Aerial parts Decoction Decoction Fresh leaves are eaten as
tonic mouth wash for gingivitis salad
and stomatitis
Dryopteris filix-mas L. Polypodiaceae Mazka paprat Felce maschio Anti-ascaridic Anti-helmintic (mainly Roots Rhizome Decoction Decoction Given its toxicity it is no
(=Aspidium f.m. L.) against Taenia solium) longer used in Italy
Polypodium vulgare L. Polypodiaceae Sladka paprat Felce dolce Antitussive Expectorant, diuretic, Roots Rhizome Maceration Decoction
choleretic, laxative,
anti-inflammatory,
anti-helmintic
Anagallis arvensis L. 䉲 Primulaceae Ognivtche Mordigallina, Diuretic, antitussive, Diuretic, astringent, Aerial parts Flowering Decoction Infusion, foot bath Under medical supervision
Centocchio inflamed wounds antitussive, to stimulate tops (poisonous (not in case of
flow of bile, expectorant, plant) ulcerated skin)
stimulates glandular
secretion, soothes swollen
legs and feet
Cyclamen hederifolium Aiton Primulaceae Ciklama Ciclamino Sedative Drastic purgative, Tuber Tuber Infusion Powdered tuber Toxic, tuber contains
and C. repandum Sm. 䉲 emmenagogue, substances not resistant to
anti-helmintic heat. No longer used in Italy
Primula veris L. Primulaceae Zjaltaiglika Primula, Primavera Antitussive, Diuretic, spasmolytic, Roots, flowers Roots, Infusion Decoction, infusion A mixture with Althaea
(=P. officinalis L.) 䉲 expectorant, expectorant, sedative for flowers, leaves officinalis and Verbascum
anti-inflammatory for CNS, externally applied thapsiforme calms
lungs (decoction) is useful in tubercular coughs
case of painful joints
Punica granatum L. Punicaceae Nar Melograno Anti-helmintic (mainly Anti-helmintic, astringent, Bark Bark of the Infusion Decoction of seeds Flowers can be used in
against tapeworm) thirst quenching (seeds), root, stem, with sugar drunk medicines to improve
anticatarrhal, in gingivitis peel, seeds for catarrh flavour. Fresh seeds are
and pyorrhoea refreshing
Adonis vernalis L. Ranunculaceae Gorizvet Adonide giallo, Cardiac diseases, Cardiac diseases, diuretic Aerial parts Aerial parts Tincture -------- No longer present in Italy
Occhio del diavolo diuretic, sedative
Clematis vitalba L. 䉲 Ranunculaceae Obiknoven povet Vitalba Revulsive Against rheumatic pains Leaves, roots, Leaves Infusion Maceration of Toxic; external use. Several
and gout, revulsive ( the flowers crushed leaves in different species, such as
fresh plant locally applied alcohol mixed with C. recta L. or C. flammula
can cause skin ulcers), as fat, tincture L. are used in Italy. Young
wash for vein ulcers and leaves and buds are smoked
furuncolosis as tobacco substitute.
Young stems can be boiled
or fried with omelettes
Consolida regalis S.F. Gray Ranunculaceae Obiknovena raliza Speronella, Erba Curare like activity Diuretic Aerial parts Aerial parts, Infusion Infusion Toxic
(=Delphinium c. L.) 䉲 cornetta seeds
Helleborus odorus Waldst et Kit Ranunculaceae Kuturiak Elleboro -------- Drastic cardiotonic, -------- Roots Decoction -------- Not frequent in Italy, no
anaesthetic, active on CNS longer used in popular
medicine
Helleborus sp. pl. Ranunculaceae -------- Elleboro -------- -------- Cardiotonic -------- -------- Decoction Toxic
anti-helmintic,
to promote
blood-flow
(leaves)
Nigella sativa L. Ranunculaceae Polskatchelebitka Damigella, Carminative, laxative, Diuretic, carminative, Seeds Seeds Decoction Decoction
Fanciullaccia anti-ascaridic emmenagogue, increasing
milk secretion
Pulsatilla vulgaris Miller 䉲 Ranunculaceae Sassanka Pulsatilla Sedative, anaphrodisiac Antineuralgic, promotes Aerial parts Flowering Tincture Infusion No longer used in Italy
blood-flow tops given its toxicity
Frangula alnus Miller Rhamnaceae Elchoviden Frangola, Alno Laxative Laxative Bark Bark Decoction or
zarnastez nero powdered
bark

Agrimonia eupatoria L. Rosaceae Kamchik Eupatoria Astringent, Anti-helmintic, astringent, as Aerial part, Leaves and Infusion Fresh leaves on
anti-inflammatory in mouth wash, anticatarrhal, roots flowering tops sores as cataplasm
urinary and liver hepatoprotective, mild
diseases hypotensive and antistaminic
Alchemilla vulgaris L. Complex 䉲 Rosaceae Chapitche Erba stella, Erba Astringent, inflamed Against catarrh, against Aerial part Leaves Infusion Infusion
ventaglina wounds menstrual pains
Crataegus laevigata (Poiret) DC. Rosaceae Obiknoven glog Biancospino Antivirus effect against Cardiac diseases, coronary Flowers, Flowers, Decoction Infusion (flowers),
(C. oxyacantha All.) Herpes simplex protecting, against leaves, fruits fruits, bark decoction (fruits,
tachycardia hypotensive, bark)
sedative for CNS, emollient
for the skin, antipyretic
(bark), atherosclerosis
Crataegus monogyna Jacq. Rosaceae Glog Biancospino Cardiac diseases, See Crataegus laevigata Flowers, See Crataegus Infusion See Crataegus
coronary diseases, leaves, fruits laevigata laevigata
myocardial ischemia
Cydonia oblonga Miller 䉲 Rosaceae Djulja Cotogno Antitussive, astringent Anti-inflammatory for the Seeds Fresh fruit Decoction Decoction Fresh pulp is used in
skin (seeds), astringent, pulp, seeds, making home-made jams
dietetic, mild sedative leaves
(leaves)
Filipendula ulmaria (L.) Maxim Rosaceae Blaten taznik Filipendula, Erba Diuretic, against Rheumatic pains, nephritis, Aerial parts Flowering Maceration Infusion
peperina rheumatism gout, oedema tops
Fragaria moschata Duchesne Rosaceae Gradinska jagoda Fragola Diuretic, astringent, Emollient for the skin Fruits, leaves Fruits, leaves, Infusion Infusion (leaves, Mainly valued as edible
anti-inflammatory, (fruits) astringent rhizome rhizome) squeezed fruits, may cause skin
anti-sclerosis pulp of fresh fruits allergy. Used in making
locally applied jams and jellies
Fragaria vesca L. Rosaceae Gorska jagoda Fragola Diuretic, astringent, Emollient for the skin Pulp of the Fruits, leaves Infusion Decoction Mainly valued as edible
anti-inflammatory, (fruits) astringent fruits, leaves, (rhizome), squeezed fruit, but it can cause skin
anti-atherosclerosis rhizome fresh fruit is locally allergy. Used in making
applied jams and jellies
Fragaria viridis Duchesne Rosaceae Planiza Fragola Diuretic, astringent, See Fragaria vesca Fruits, leaves Fruits, leaves Infusion Infusion (leaves, Used less than the
anti-inflammatory, rhizome rhizome) preceding two species
anti-sclerosis
Geum urbanum L. Rosaceae Omajnitche Cariofillata, Anti-inflammatory, Astringent, Roots, aerial Rhizome, Infusion Infusion, tincture
Ambretta astringent, antimicrobial anti-inflammatory, parts aerial parts
antipyretic
Potentilla anserina L. Rosaceae Patchiotchibolez Argentina Intestinal gas, Astringent, antipyretic, Aerial parts Aerial parts, Infusion Decoction
dismenorrhea, antidiarrhoeic rhizome
stomach-ache
Potentilla erecta (L.) Rauschel Rosaceae Buturak Tormentilla Astringent, See Potentilla anserina Roots See Potentilla Infusion See Potentilla
anti-inflammatory for anserina anserina
mouth and throat (by
gargling), in gastric
and digestive disorders
137
138
Potentilla reptans L. Rosaceae Galitcheva treva Cinquefoglio Diarrhoea See Potentilla anserina Roots See Potentilla Infusion See Potentilla
anserina anserina
Prunus spinosa L. 䉲 Rosaceae Tranka Prugnolo Laxative, diuretic, Astringent, Flowers, fruits Bark, fruits, Infusion, Decoction Fruits are also used in
astringent anti-inflammatory, flowers decoction preparing home-made
depurative, diuretic, liqueurs
anti-asthma, hypoglycemic
Rosa canina L. Rosaceae Chipka Rosa di macchia Antiscorbutic, Antiscorbutic, diuretic, Cynorrhods Leaves, Decoction Decoction In the past hips were

cholagogue, diuretic, astringent, anti-inflammatory (hips) cynorrhods, collected as source of
astringent for the skin, nutritive (hips), petals vitamins. They are still
used in making syrups,
jams and jellies
Rubus sp. pl. Rosaceae Kapina Rovo Astringent, antiseptic Astringent, Roots, leaves, Leaves, fruits Infusion Decoction, syrup by Fruits are eaten raw, or
anti-inflammatory, fruits fruits, fresh leaves used in preparing
antiscorbutic, diuretic, locally applied home-made jams
thirst quenching, laxative
Rubus idaeus L. Rosaceae Malina Lampone Astringent Astringent, Roots, leaves, Leaves, fruits Infusion Decoction Fruits are eaten or used in
anti-inflammatory anti-inflammatory for fruits making home-made jams
intestinal tract
Sanguisorba officinalis L. Rosaceae Letchebna dinka Salvastrella Astringent, Astringent, Roots Whole plant Decoction Decoction
anti-inflammatory anti-haemorrhagic, against
haemorrhoids
Sorbus aucuparia L. 䉲 Rosaceae Ofika, kalina Sorbo degli Antirheumatic, Dietetic, soothing for Fruits Fruits Decoction Juice, decoction
uccellatori, Sorbo astringent, diuretic throat and skin
rosso
Sorbus domestica L. 䉲 Rosaceae -------- Sorbo -------- See Sorbus aucuparia -------- See Sorbus -------- See Sorbus Fruits are eaten
aucuparia aucuparia
Galium aparine L. Rubiaceae Lepka Attacca veste Diuretic, laxative, Diuretic, spasmolytic, Aerial parts Flowering Infusion Infusion
analgesic sedative tops
Galium odoratum (L.) Scop. Rubiaceae Lazarkinja Stellina odorosa Diuretic, antitussive Spasmolytic, sedative, mild Aerial parts Flowering Infusion Infusion
(=Asperula odoratum L.) 䉲 hypnotic, astringent for tops
chapped skin (e.u.)
Galium verum L. Rubiaceae Enjovtche Caglio Diuretic, laxative, Diuretic, sedative for CNS; Aerial parts Flowery tops Infusion Infusion It is also used in dyeing
analgesic externally used in case of clothes
inflamed skin
Rubia tinctorum L. Rubiaceae Broch Robbia Spasmolytic, kidney Kidney stones, gall stones Roots Roots, Tincture Infusion, decoction Used after meals
stones choleretic, cholagogue, rhizome
locally applied on inflamed
skin
Dictamnus albus L. Rutaceae Rocen, Dı̀ttamo Diuretic To stimulate digestion Roots Flowering Infusion Infusion Toxic
samodivsko bile tops
Ruta graveolens L. Rutaceae Sedeftche Ruta Sedative, Protecting blood vessels, Aerial parts Tops before Maceration Rarely used given Fresh stems are put into
anti-inflammatory emmenagogue, abortive, flowering its toxicity and rooms infested by mice and
abortive, anti-ascaridic anti-helmintic. To aromatize ability to inflame fleas to repel them
liqueurs skin
Populus alba L. Salicaceae Bjala topola Pioppo, Gattice Astringent diuretic, Antirheumatic, astringent Gemmae Bark, gemmae Tincture Decoction
antiseptic bitter-tonic, diuretic, (buds) (buds)
diaphoretic, absorbs
intestinal gas (char wood)
Populus nigra L. Salicaceae Tcherna topola Pioppo nero, Astringent diuretic, Anti-arthritis, antigout, Gemmae Bark Tincture Decoction
Pioppo cipressino antiseptic bronchial sedative (buds)
Populus tremula L. Salicaceae Trepetlika Pioppo tremulo Astringent diuretic, See Populus alba Gemmae See Populus Tincture See Populus alba
antiseptic (buds) alba
Salix alba L. Salicaceae Bjala varba Salice Antirheumatic, Antirheumatic, antipyretic, Roots Bark Decoction Decoction Used externally in
antipyretic antineuralgic gynaecology (Bg)
Euphrasia officinalis L. s.l. Scrophulariaceae Otchanka Eufrasia Astringent, Emollient, eye wash Aerial parts Whole plant Decoction Gauze soaked in
anti-inflammatory infusion, or
antiviral, eye diseases decoction, are
locally applied
Gratiola officinalis L. Scrophulariaceae Letchebna sirotiza Graziella Laxative, diuretic Cardiac stimulant, diuretic Aerial parts Leaves Infusion Infusion, decoction Toxic

Gratia-Dei
Linaria vulgaris Mill. Scrophulariaceae Lulitchka Linaiola Diuretic, laxative, Astringent, Aerial parts Flowering Infusion Infusion, cataplasm
haemorrhoids anti-inflammatory, laxative, tops of the crushed drug
anti-haemorrhoids
Verbascum densiflorum Bertol. Scrophulariaceae Visok lopen Verbasco, Tasso Antitussive, See Verbascum thapsus L. Leaves See Infusion See Verbascum
barbasso anti-inflammatory, Verbascum thapsus L.
spasmolytic thapsus L.
Verbascum phlomoides L. 䉲 Scrophulariaceae Lopen Verbasco, Tasso Hypotensive, See Verbascum thapsus L. Leaves See Component of See Verbascum
barbasso spasmolytic Verbascum the remedy thapsus L.
thapsus L. “Verbaskan”
Verbascum sinuatum L. Scrophulariaceae -------- Verbasco, Tasso -------- Against psoriasis, -------- Aerial parts, Decoction
barbasso haemorrhoids (piles), to leaves evaporated
wash infected wounds then filtered.
Cataplasm of
crushed
leaves used
on wounds
Verbascum thapsus L. 䉲 Scrophulariaceae -------- Verbasco, Tasso Antitussive Emollient against catarrh -------- Flowers Cataplasm,
barbasso and coughs. Cataplasm ointment (in
applied externally to soothe olive oil),
gangrenous sores decoction
Veronica officinalis L. Scrophulariaceae Letchebno Veronica Appetizer, antitussive, Appetizer, Aerial parts Aerial parts Infusion Infusion
velikdentche anti-inflammatory anti-inflammatory, soothing
expectorant, asthma, agent, diuretic, antitussive
pharingitis
Atropa belladonna L. Solanaceae Ludobile Belladonna Spasmolytic, in Spasmolytic, sedative in Leaves, Leaves, Tincture, Infusion, decoction
Parkinson’s disease gastric and intestinal flowers, roots flowers, roots alcoholic
“Cura Bulgarica” troubles, externally for extract
rheumatism (alcohol rub)
Capsicum annum L. Solanaceae Piper Peperoncino Stimulates blood-flow, To stimulate digestion, Fruits Raw fruits Tincture Cataplasm of fresh Mixed with foods to give
appetizer antiscorbutic, against or dried and flavour
haemorrhoids (piles), crushed fruits
painful rheumatism in joints mixed with vinegar
Datura stramonium L. Solanaceae Tatul Stramonio Spasmolytic, Sedative for CNS, Leaves Leaves, Tincture Tincture, infusion Toxic
anti-asthmatic antispasmodic, chilblains flowers, seeds
(foot or hand bath)
Hyosciamus albus L. Solanaceae Bel bojour Giusquiamo Spasmolytic, against Spasmolytic, against asthma, Leaves Leaves, seeds Infusion Infusion, maceration Toxic. Seeds used to make
asthma anaesthetic, analgesic, in olive oil locally decayed teeth to fall out
hypnotic, rheumatic pains applied
Hyosciamus niger L. Solanaceae Tcheren bljan Giusquiamo Spasmolytic, against Spasmolytic, against Leaves Leaves, seeds Infusion Infusion Toxic
asthma asthma, anaesthetic,
analgesic, hypnotic
139
140
Physalis alkekengi L. Solanaceae Mechunka Alkekengi Diuretic, Diuretic, in case of kidney Fruits Fruits Decoction Infusion, decoction Toxic
anti-inflammatory and gall stones antipyretic, powdered fruits
rheumatism and gout
Solanum dulcamara L. 䉲 Solanaceae Razvodnik Dulcamara To promote perspiration Hypnotic, anaphrodisiac, Aerial parts Young stems, Decoction Decoction Toxic
anti-asthma aerial parts
Solanum melongena L. 䉲 Solanaceae Sin domat Melanzana Antibiotic Hepatoprotective, diuretic, Fruits Fruit peel Juice Decotion The frruit is mainly eaten
depurative with outer as vegetable

part of pulp
Solanum nigrum L. Solanaceae Tchemo Erba morella Spasmolytic, sedative Spasmolytic, sedative, Aerial parts Aerial parts Tincture Infusion Toxic
kutchechko grodze antalgic, sliced fresh pulp
externally applied in skin
diseases, itching and painful
joints
Solanum tuberosum L. Solanaceae Kartof Patata Anti-acid, Anti-acid, anti-inflammatory. Tuber Tuber Juice Pulp It is used mainly as food
anti-inflammatory Sliced fresh pulp is
externally applied to minor
burns and reddened skin
Daphne mezereum L. Thymeleaceae Bjasnodarvo Fior di stecco Revulsive Revulsive Barks Barks Powder mixed Decoction Toxic. External use only
with fat
Tilia cordata Miller Tiliaceae Drebnolistna lipa Tiglio To promote Mild sedative, diaphoretic, Flowers Flowers and Infusion Infusion
perspiration, soothes the mucosa, bracts
anti-inflammatory antitussive
Tilia platiphyllos Scop. Tiliaceae Edrolistna lipa Tiglio To promote See Tilia cordata Flowers See Tilia Infusion See Tilia cordata
perspiration, cordata
anti-inflammatory,
sedative
Ulmus minor Miller Ulmaceae Kolski brjast Olmo Astringent, against Diuretic, depurative, Barks Bark Decoction Decoction
diarrhoea, anti-inflammatory
anti-inflammatory
Urtica dioica sp. pl. Urticaceae Kopriva Urtica Stimulant, astringent, Diuretic, depurative, Leaves Leaves Fresh plant, Infusion, decoction Boiled leaves are eaten in
diuretic soothing for the intestinal infusion soups or in omelettes
tract, antigout to strengthen
hair and fight dandruff,
haemostatic
Valeriana officinalis L. Valerianaceae Diljanka Valeriana Sedative for CNS Sedative for CNS Roots Roots Decoction Decoction
Verbena officinalis L. Verbenaceae Varbinka Verbena To promote Diuretic, bitter-tonic Aerial parts Flowering Infusion Infusion, fresh
perspiration, depurative, anti-pyretic, tops squeezed pulp,
antipyretic, sedative antirheumatic, antineuralgic, decoction evaporated
to protect liver and spleen, and then filtered
trigeminal neuralgia, against
psoriasis
Viola odorata L. Violaceae Gorska temenuzka Viola mammola Antitussive, diuretic, Emollient, expectorant for Aerial parts, Flowers Infusion Infusion Flowers are used in making
against atherosclerosis coughs, sudoriferous, roots rhizome candies
diuretic, mild laxative
Viola tricolor L. Violaceae Trizvetna Viola tricolore Antitussive, diuretic, Used against dermatitis Aerial parts Flowering Infusion Cataplasm with Flowers are used in making
temenuzka against dermatitis, when arthritic pain is also plant infusion, decoction candies
against atherosclerosis present. Against psoriasis evaporated and then
filtered
M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142 141
Table 3
Noteworthy therapeutic uses
Antibiotic Solanum melongena Cardiotonic Geranium macrorrhizum
Corylus avellana
Antipyretic Cornus mas
Antiviral Crataegus laevigata Coronary dilating and Daucus carota
Euphrasia officinalis capillarotrophic Pastinaca sativa subsp. sativa
Geranium sanguineum
Geum urbanum
Hypericum perforatum Jaundice Mentha pulegium
(only against Herpes simplex)
Nepeta cataria Mentha spicata
Satureja hortensis
Thymus sp. pl.
Antiviral and immuno-stimulant Geranium sanguineum Hypotensive Centaurium erytrhaea
Antineuralgic Sambucus ebulus Hepatoprotective Chelidonium majus
Sambucus nigra Solanum melongena
Verbena officinalis (only Mentha spicata
trigeminal neuralgia)
Anti-atherosclerosis Sedum acre Prostatic hypertrophy Corylus avellana
Viola tricolor
Against dermatitis when arthritic Viola odorata Psoriasis Ecballium elaterium
pains are also present
Verbascum sinuatum
used externally, the direct employment of fresh plant juice References

is common. This use is probably a form of first aid in cases
of burns or small cuts, or as an anti-inflammatory and soo- Amenta, R., Camarda, I., Di Stefano, V., Lentini, F., Venza, F., 2000.
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Anti-inflammatory activity of (E)-1-(3,4-dimethoxyphenyl)

butadiene from Zingiber cassumunar Roxb.
Rattima Jeenapongsa a,∗ , Krongtong Yoovathaworn b ,
Kittima M. Sriwatanakul b , Ubonwan Pongprayoon c ,
Kampon Sriwatanakul b
aDepartment of Pharmacy Practice, Faculty of Pharmaceutical Sciences,
Naresuan University, Muang, Phitsanulok 65000, Thailand
b Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand
c Thailand Institute of Scientific and Technological Research, Bangkok, Thailand
Received 3 September 2002; received in revised form 22 February 2003; accepted 13 March 2003
Abstract
This study aimed to investigate the anti-inflammatory activity of (E)-1-(3,4-dimethoxyphenyl) butadiene (DMPBD), isolated from Zingiber
cassumunar Roxb., using in vivo and in vitro models. The results show that DMPBD dose-dependently inhibited the rat ear edema induced
by ethyl phenylpropiolate (EPP), arachidonic acid (AA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) and it was more potent than any
other standard drugs being used. In EPP-induced edema IC50 of DMPBD and oxyphenbutazone were 21 and 136 nmol per ear, respectively.
The IC50 of DMPBD and phenidone were 60 and 2520 nmol per ear, respectively, in AA-induced edema whereas DMPBD was 11 times more
potent than diclofenac in TPA-induced edema (IC50 = 660 and 7200 pmol per ear, respectively). DMPBD and diclofenac inhibited the rat
paw edema induced by carrageenan but not by platelet activating factor (PAF). In in vitro study DMPBD, aspirin and phenidone inhibited
collagen-induced platelet aggregation with IC50 of 0.35, 0.43 and 0.03 mM, respectively. Whereas IC50 of these agents in ADP, AA and
PAF inductions were 4.85, 3.98 and 1.30 mM; 0.94, 0.13 and 0.04 mM; and 1.14, 6.96 and 2.40 mM, respectively. These results indicate that
DMPBD possesses a potent anti-inflammatory activity through the inhibition of CO and LO pathways and seems to have more prominent
effects on the LO pathway.
© 2003 Published by Elsevier Science Ireland Ltd.
Keywords: Zingiber cassumunar; DMPBD; Anti-inflammatory activity; Platelet aggregation; Ear edema; Paw edema
1. Introduction prostaglandin-like activity of the exudates (Panthong et al.,

1990).
In many Asian countries, Zingiber cassumunar Roxb. (E)-1-(3,4-Dimethoxyphenyl) butadiene (DMPBD) was
(Zingiberaceae) is widely used in folklore remedies as a isolated from the hexane extract of Zingiber cassu-
single plant or as a component of herbal recipes. Com- munar Roxb. by Thailand Institute of Scientific and
pounds with known chemical structures have been isolated Technological Research (TISTR). Its chemical structure
from Zingiber cassumunar Roxb. Among these the hex- is shown in Fig. 1. Preliminary studies suggested that
ane extract seemed to possess a potent anti-inflammatory DMPBD is an active ingredient of the essential oil derived
activity. Compound D, isolated from the hexane extract, from Zingiber cassumunar Roxb. Our in vivo preliminary
exhibited a strong inhibitory activity on the edema forma- study revealed the effect of DMPBD on both cyclooxy-
tion in carrageenan-induced rat paw edema. In rat pleurisy genase (CO) and lipoxygenase (LO) pathways. Therefore,
model, it exerted markedly inhibitory activity on the ex- this study aimed to characterize the anti-inflammatory
udates formation, the accumulation of leukocytes and the activity of DMPBD using in vivo and in vitro mod-
els. Several inducers and standard drugs were employed
∗ Corresponding author. Tel.: +66-55-261000-4 Ext 3620; so that possible mechanisms of action of DMPBD on
fax: +66-55-261057. arachidonic acid (AA) metabolic pathway could be postu-
E-mail address: rattimaj@yahoo.com (R. Jeenapongsa). lated.
0378-8741/03/$ – see front matter © 2003 Published by Elsevier Science Ireland Ltd.
doi:10.1016/S0378-8741(03)00098-9
144 R. Jeenapongsa et al. / Journal of Ethnopharmacology 87 (2003) 143–148
the experiment. Prior to any treatment, ear thickness was

determined as a basal value. In the EPP- and AA-induced
ear edema, ear thickness was determined at 0.5, 1 and 2 h
and 0.5, 1, 1.5, 2, 3 and 4 h after the treatment, respectively.
In the TPA treated group, the thickness was determined at
2, 4, 6, 8, 10 and 12 h after the TPA application.
Drug was applied simultaneously with EPP or AA.
Fig. 1. Chemical structure of (E)-1-(3,4-dimethoxyphenyl) butadiene Oxyphenbutazone or DMPBD was dissolved in the 5% EPP
(DMPBD). solution to obtain the final drug concentrations of 0.005,
0.05, 0.5 and 5%. In the AA treatment group, phenidone or
DMPBD was dissolved in 10% AA solution to obtain the
The in vivo studies were carried using rat ear edema and
drug concentration of 5%.
rat paw edema models whereas platelet aggregation model
In the TPA-induced edema, diclofenac was prepared in
was employed as an in vitro study.
a mixture of ethanol and acetone (4:6) and DMPBD was
prepared in acetone. The solution of diclofenac or DMPBD
was applied on the ear at 1 h after the TPA application.
2. Materials and methods
2.3.2. Rat paw edema
2.1. Animals Paw edema was induced in male Sprague–Dawley rats
weighing 120–130 g by subplantar injection of 0.1 ml car-
Male Sprague–Dawley rats, 50–70 and 120–130 g, were rageenan or PAF by a modified method described by Winter
obtained from the National Laboratory Animal Center of et al. (1962) with some modifications.
Mahidol University. Before conducting any studies animals Carrageenan (1%, w/v) in 0.9% normal saline solution
were acclimatized for 1 week at 24–26 ◦ C with food and was injected into the left hind paw. Paw volume was mea-
water ad libitum. sured before and at 1, 2, 3, 4, 5 and 6 h after the injection
Adult male and female albino rabbits weighing 3–5 kg of carrageenan. Diclofenac or DMPBD was dissolved in
were supplied by the Department of Animal Science, Faculty acetone and 0.06 ml of the drug was applied over the upper
of Agriculture, Kasetsart University, Bangkok, Thailand. and lower surfaces of the injected paw at 1 h after the car-
rageenan injection. Edema volume was determined plethys-
2.2. Materials mographically at 1, 2, 3, 4, 5 and 6 h after the treatment.
PAF was dissolved in 0.9% normal saline to obtain a
DMPBD was supplied by TISTR in the form of white concentration of 8 ␮g/ml. Diclofenac and DMPBD were
crystal. Ethyl phenylpropiolate (EPP), AA, 12-O-tetrade- prepared at the same concentration as those used in the study
canoylphorbol 13-acetate (TPA), carrageenan, platelet induced by carrageenan. A solution of salbutamol (5%) was
activating factor (PAF), oxyphenbutazone, diclofenac, obtained by dissolving salbutamol in a mixture of methanol
phenidone, salbutamol, collagen and adenosine diphosphate and acetone (1:1). Then 5% salbutamol was diluted with
(ADP) were obtained from Sigma (St. Louis, MO). All acetone to give the concentrations of 1, 0.5 and 0.05%.
other solvents and chemicals employed were of analytical Drug solutions were applied over the upper and lower sur-
grade. Compounds were prepared as solutions in acetone faces of the paw 30 min before the injection of PAF. Edema
(reagent grade) just prior to application. volume was determined at 0.5, 1, 1.5, 2 and 3 h after the
PAF injection.
2.3. In vivo study
2.3.3. Platelet aggregation
2.3.1. Rat ear edema
Ear edema was induced in male Sprague–Dawley rats 2.3.3.1. Preparation of platelet suspension. Blood was
weighing 50–70 g, according to the modified method of taken from the central ear artery of the rabbits. An aliquot
Brattsand et al. (1982) and Pongprayoon et al. (1991) with (9 ml) of blood sample was placed into a polystyrene
some modifications. The edema was induced by an applica- tube containing 1 ml of 3.2% (w/v) sodium citrate as an
tion of a solution of EPP, AA or TPA in acetone at concen- anti-coagulant. Citrated blood was centrifuged at 160 × g
trations of 5, 10 and 0.02%, respectively. Each solution in for 10 min and the supernatant was removed as platelet
volume of 0.01 ml was applied to each of inner and outer rich plasma (PRP). The remaining blood was further cen-
surfaces of both ears by means of an automatic micropipette. trifuged at 4000 × g for 10 min to obtain the supernatant
For ear thickness determination, a dial caliper (no. 7039, as platelet-poor plasma (PPP). Platelet concentration of the
Mitutoyo) was applied to the tip of the ear. In order to PRP was adjusted to 3×105 to 5×105 /ml by diluting with the
minimize technical variations among measurements, the PPP as required. Platelet aggregation tests were performed
same investigator performed the measurements throughout during 30 min and 3 h after the blood had been drawn.
R. Jeenapongsa et al. / Journal of Ethnopharmacology 87 (2003) 143–148 145
2.3.3.2. Preparation of chemical reagents. Collagen and gradually. DMPBD and oxyphenbutazone when applied con-
ADP were dissolved in distilled water at the concentration comitantly with 5% EPP concentration-dependently inhib-
of 10 g/ml and 0.2 mM, respectively. AA was dissolved in ited the EPP-induced edema formation with IC50 of 21 and
a small volume of absolute ethanol (not higher than 5% 136 nmol per ear, respectively (Table 1). DMPBD was about
in final concentration) and then adjusted with 0.9% normal six times more potent than oxyphenbutazone.
saline to obtain the final concentration of 12.38 mM. PAF AA produced a rapid edema formation with a maximum
was prepared by evaporating the solution of PAF in chloro- effect at 30–60 min and the edema then gradually decreased.
form under nitrogen gas and reconstitute with normal saline DMPBD and phenidone exhibited concentration-related in-
to achieve a final concentration of 10 mg/ml. hibitory profile with the maximal activity at 30 min after the
Aspirin was dissolved in distilled water at the concentra- topical administration. DMPBD (IC50 of 60 nmol per ear)
tions of 1 and 5 mg/ml. Phenidone (1%, w/v) was prepared was much more potent than phenidone (IC50 2520 nmol per
by dissolving the drug in propylene glycol (60% in final ear) in inhibiting AA-induced ear edema in rats.
concentration) and then the final volume was adjusted with TPA-induced edema formation was clearly observed at 2 h
normal saline. DMPBD was dissolved in propylene glycol after the topical application and reached its maximum effect
(40% in final concentration) and adjusted the volume with at 8 h after the application. DMPBD and diclofenac applied
normal saline. 1 h after TPA application inhibited ear edema with the maxi-
mum activity at 2 h. DMPBD (IC50 of 660 pmol per ear) was
2.3.3.3. Platelet aggregation study. PRP (450 ␮l) was approximately 11 times more potent than diclofenac (IC50
placed into siliconized glass cuvettes. The aggregating agent of 7200 pmol per ear) in inhibiting TPA-induced ear edema.
was added into the stirred platelet suspension to induce
platelet aggregation. When studying the inhibitory effects 3.2. DMPBD inhibits paw edema induced by carrageenan
of aspirin, phenidone and DMPBD on platelet aggregation but not by PAF
induced by these four agents, the studied compound was
added into the stirred PRP 2 min before the addition of The injection of carrageenan resulted in paw edema within
the aggregating agent. The extent of platelet aggregation 1 h and the maximum effect was reached at 3 h. This re-
was shown as a percentage of change in light transmission action was inhibited by topical application of diclofenac in
through the cuvette comparing with that of PPP suspension. a concentration-dependent fashion with an IC50 at 3 h of
The inhibitory effect was observed as the decrease in light 14 ␮mol per paw. The topical application of DMPBD also
transmission comparing with that observed in the aggrega- showed a concentration-dependent effect with an IC50 at 3 h
tion without an inhibitor. Absence of platelet aggregation of 22 ␮mol per paw.
was defined by having no changes in light transmission The subplantar injection of PAF into a rat hind paw rapidly
after the period of 5 min. induced edema formation. The topical application of di-
clofenac or DMPBD showed no significant inhibition on
PAF-induced paw edema. However, salbutamol significantly
3. Results inhibited the PAF-induced paw edema in a dose-related man-
ner with IC50 of about 1.9%.
3.1. DMPBD inhibits ear edema induced by EPP,
AA and TPA 3.3. DMPBD inhibits platelet aggregation induced by
collagen, ADP, AA and PAF
Topical application of 5% EPP produced ear swelling
within 30 min. Erythema was also observed. The maximum Collagen-induced platelet aggregation in a concentra-
effect of EPP was at 1 h, thereafter the ear swelling decreased tion-dependent manner with a lag period of 2–3 min.
Table 1
Inhibitory effects of DMPBD and standard drugs on rat ear edema and rat paw edema. Data are expressed as IC50 values
IC50
DMPBD Standard drugs
Oxyphenbutazone Phenidone Diclofenac Salbutamol
Ear edema
EPP 21 nmol/ear 136 nmol/ear – – –
AA 60 nmol/ear – 2520 nmol/ear – –
TPA 660 pmol/ear – – 7200 pmol/ear –
Paw edema
Carrageenan 22 ␮mol/paw – – 14 ␮mol/paw –
PAF No effect – – No effect 1.9%
– is non-applicable.
Table 2 provided a new source of mediators that contributed to the

Inhibitory effects of DMPBD, aspirin and phenidone on platelet aggre- modulation of the blood flow. The response to EPP dur-
gation induced by collagen, ADP, AA and PAF
ing the first hour was partially suppressed by pretreatment
Aggregating agents DMPBD Standard drugs with indomethacin, aprotinin and mechlorethamine (Patrick
Aspirin Phenidone et al., 1987).
The topical application of TPA onto a rat ear induced
Collagen 0.35 0.43 0.03
ADP 4.85 3.98 1.30
a long-lasting edema formation. The majority of TPA ac-
AA 0.94 0.13 0.04 tions appear to involve or be dependent on AA release and
PAF 1.14 6.96 2.40 metabolism (Patrick et al., 1987). At the biochemical level,
Data are shown as IC50 (mM) values. cAMP, PGI2, PGE2 and PGF2 are elevated within minutes to
hours after TPA application (Ashendel and Boutwell, 1979;
Furstenberger and Marks, 1980). Phospholipase inhibitor,
Preincubation of platelets with aspirin, phenidone and CO inhibitors and LO inhibitors as well as corticosteroids
DMPBD inhibited collagen-induced platelet aggregation are effective at edema suppression after topical application
with IC50 of 0.43, 0.03 and 0.35 mM, respectively (Table 2). of high dose of TPA (Young et al., 1983; Carlson et al.,
The lag phases were prolonged by these agents. The inhibi- 1985), confirming a role of AA release and metabolism.
tion was concentration-related and among these phenidone DMPBD, if possessing the same pharmacological effect as
was the most potent inhibitor. diclofenac, should possess inhibitory activity on the CO
ADP-induced platelet aggregation differently from that pathway. However, when comparing the IC50 of both com-
of collagen, in that the lag period was not observed with pounds in inhibiting TPA-induced ear edema, DMPBD with
ADP. The aggregation was observed immediately after the IC50 of 660 pmol per ear is more potent than diclofenac.
addition of ADP solution into the platelet suspension. The The topical application of AA provokes a rapid and
response to ADP increased as ADP concentration was in- intense inflammatory response of the ear. This response
creased. Higher concentrations of aspirin, phenidone and is not affected by CO inhibitors (Young et al., 1983) but
DMPBD were needed to inhibit platelet aggregation induced is inhibited by agents that inhibit both CO and LO path-
by ADP than those required in the collagen induction. The ways of AA metabolism (Young et al., 1984). It was found
IC50 of aspirin, phenidone and DMPBD were 3.98, 1.30 and that AA-induced ear edema is predominantly mediated by
4.85 mM, respectively. leukotriene C4 and other LO products while PGE2 (in the
Platelet aggregation profile induced by AA was similar presence of LTs) acts to facilitate ear swelling (Inoue et al.,
to that induced by ADP. The platelets aggregated immedi- 1988). In this study DMPBD and phenidone, a dual CO and
ately after the addition of the aggregating agents. Aggrega- LO inhibitor, inhibited AA-induced ear edema with IC50
tion profiles in the presence of the inhibitors were similar of 60 and 2520 nmol per ear, respectively, suggesting that
but it was slightly delayed when DMPBD was used. Aspirin, DMPBD may also acts on the LO pathway.
phenidone and DMPBD concentration-dependently inhib- In the rat paw edema model, carrageenan induces edema
ited AA-induced platelet aggregation with IC50 of 0.13, 0.04 formation in three distinct phases according to the mediators
and 0.94 mM, respectively. involved (Di Rosa et al., 1971). The initial phase occurring
Platelet response occurred immediately after the addition during the first hour after subplantar injection of carrageenan
of PAF solution into the stirred PRP suspension. Aspirin, into rat hind paw is mediated by histamine and 5-HT. Af-
phenidone and DMPBD inhibited platelet aggregation with ter that the increased vascular permeability in the second
IC50 of 6.96, 2.40 and 1.14 mM, respectively. phase is maintained by kinin release up to 2.5 h. Then the
third phase, from 2.5 to 6 h, the mediators involved are PGs
(Di Rosa et al., 1971; Hwang et al., 1986). The biosynthe-
4. Discussion sis of LTs is suggested to be involved in the fourth phase of
carrageenan-induced edema formation (Holsapple and Yim,
Murine models of inflammation provide a relative system 1984). All non-steroidal anti-inflammatory drugs (NSAIDs)
for studying the physiological and biochemical mechanisms are effective in inhibiting edema formation especially in the
of anti-inflammatory agents (Jacobson and Jacobs, 1992). In late phase in which PGs involves (Niemegeers et al., 1964).
this study, DMPBD was clearly shown to exert its effects on In this study, diclofenac showed the maximum inhibitory
all models tested, except in PAF-induced rat paw edema. effect on edema formation at 5 h. This corresponds with
DMPBD showed a marked inhibitory activity on EPP- the period of PG phase (Di Rosa and Willoughby, 1971).
induced ear edema. This action is more potent than oxyphen- DMPBD also inhibited edema formation with maximum ac-
butazone. Immediately after the application of EPP, the tivity at the same period as diclofenac. These results support
vascular permeability was increased (Patrick et al., 1987). the suggestion from the rat ear edema model that DMPBD
The combined actions of kinins and PGs on vascular per- may interfere with the CO pathway of AA metabolism.
meability seemed to be involved in the early response to It has been observed that the injection of PAF into rat hind
EPP while increased in inflammatory cells in the tissues paw provokes an inflammatory response characterized by an
R. Jeenapongsa et al. / Journal of Ethnopharmacology 87 (2003) 143–148 147
acute edema (Cordeiro et al., 1986). The subplantar injec- pholipase C and phosphorylation of several proteins occurs
tion of PAF to rat hind paw-induced dose-related edema for- (Dhar et al., 1990). TXA2 , an AA metabolic product, is not
mation with the maximum response at 1 h (data not shown). found to be important in the mechanism of PAF-induced
The results are in agreement with the study reported by aggregation (McCulloch and Vandongen, 1990). Aspirin
Castro-Faria-Neto et al. (1990). In this study, PAF-induced and other NSAIDs do not affect this response (Kuster and
paw edema was suppressed by ␤2 -adrenergic agonist, salbu- Frolich, 1986). In this study, DMPBD strongly inhibited
tamol. It strongly inhibited paw edema while diclofenac and platelet response to PAF with IC50 of 1.14 mM. Aspirin
DMPBD did not, suggesting that DMPBD and diclofenac and phenidone also inhibited aggregation with lower po-
do not affect the pathways involved in PAF-induced paw tency than DMPBD (Table 1). Aspirin was effective only
edema. at much higher concentration than those of DMPBD and
The platelet aggregation study represents an attempt phenidone. This may suggest that the effects of aspirin on
to explore the effects of DMPBD on AA metabolism in PAF action may occur via pathways other than CO inhibitor
platelet, since AA metabolites, PGs and thromboxane A2 and DMPBD may also affect pathways other than the CO
(TBA2 ) are involved in the platelet aggregation. The ef- and LO pathways in PAF-induced aggregation.
fects of the anti-platelet aggregating agents, aspirin and The results obtained from the in vivo models of inflamma-
phenidone, on collagen-induced platelet aggregation pro- tion suggest that DMPBD exerts a clear anti-inflammatory
duced characteristics similarly to that reported in the pre- effect. The differences in the potency of DMPBD in in-
vious study (Karniguian et al., 1990). Collagen-induced hibiting platelet aggregation induced by each inducer point
platelet aggregation in a concentration-related fashion and out the specificity of DMPBD on the pathways involved in
a lag phase was observed before the aggregation occurred. platelet aggregation. The in vivo data suggest that DMPBD
The earlier study suggested that the lag phase is the time may inhibit CO and LO activities and seems to have more
that is needed for collagen to activate phosphatidylinosi- prominent effects on the LO pathway. However, further stud-
tol 4,5-diphosphate-specific phospholipase C (Karniguian ies should be performed to explore the activities of DMPBD
et al., 1990). on the enzymatic levels of the inflammation before any final
The results are in agreement with the previous stud- conclusion can be made.
ies that aspirin could inhibit collagen-induced aggregation This study is the first to demonstrate that DMPBD is a
(O’Brien, 1968; Kuster and Frolich, 1986). Phenidone, a unique topically active anti-inflammatory agent. Possibly it
CO and LO inhibitor, was slightly more potent than as- is a modulator of AA metabolism having activities on both
pirin. In this study, phenidone was the most potent inhibitor CO and LO enzymes. It has a potential for local therapeutic
of collagen-induced platelet aggregation. Regarding the applications in inflammatory diseases. A drug development
greater potent anti-aggregating effects of phenidone com- program should be systematically initiated to determine the
pared to aspirin, it may be postulated that LTs, which are possibility of making this compound a new pharmacological
synthesized via the LO pathway, may also be involved in agent for the treatment of inflammatory disorders.
the platelet aggregation. DMPBD may affect both CO and
LO pathways but it is less potent than phenidone in the
collagen-induced aggregation. Acknowledgements
NSAIDs including aspirin are reported to be effective in-
hibitors of ADP-induced platelet aggregation (Zucker and This work is supported by the University Developing
Peterson, 1968; Chars et al., 1977). In this study, phenidone Commissions Grant, Ministry of University Affairs, and the
seemed to have a higher potency than aspirin and DMPBD. Faculty of Graduate Studies, Mahidol University, Thailand.
This observation suggests that both CO and LO pathways The authors would like to thank all the staff at TISTR for
are involved in the activity of ADP and DMPBD may exert their technical assistance and valuable suggestion.
its inhibitory effect on these pathways.
In the AA-induced platelet aggregation, AA is converted
to endoperoxides and thromboxanes by platelets and these
compounds are further responsible for platelet aggregation References
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Comparative effect of Prunus persica L. BATSCH-water extract

and tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride)
on concentration of extracellular acetylcholine
in the rat hippocampus
Yeon-Kye Kim a,b , Byung-Soo Koo b , Dae-Jong Gong b , Young-Choon Lee c ,
Jeong-Heon Ko d , Cheorl-Ho Kim a,b,∗
a National Research Laboratory for Glycobiology (NRLG), Korean Ministry of Science and Technology, Kyungju City, Kyungbuk 780-714, South Korea
b Department of Biochemistry, Molecular Biology and Neurobiology, DongGuk University COM, Kyungju City, Kyungbuk 780-714, South Korea
c Faculty of Life Science and Bio-Resources, Dong-A University, Saha-Ku, Pusan 608-714, South Korea
d Proteomic Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-600, South Korea
Received 8 November 2002; received in revised form 13 March 2003; accepted 13 March 2003
Abstract
Prunus persica L. BATSCH seed-water extract (PPE) has been used in the treatment of the degenerative disorders, such as hypermenorrhea
and dysmenorrhea, in Taiwan, China, Japan and Korea. In this study, the effects of oral administration of PPE on the extracellular acetylcholine
concentration in the hippocampus of rats were evaluated, and compared to that of tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride),
a well-known and centrally acting acetylcholinesterase (AChE) inhibitor, which had been developed for the treatment of Alzheimer’s disease.
We measured the inhibition of brain AChE. PPE at 2.5 g/kg and tacrine at 5 mg/kg showed significant effects for more than 6 h. At these doses,
the maximum increases were observed at about 1.5 h after administration of PPE, and at about 2 h with tacrine, and were 454 and 412% of the
pre-level, respectively. The results suggest that oral administration of PPE and tacrine increases acetylcholine concentration in the synaptic
cleft of the hippocampus mostly through AChE inhibition, and that PPE has a potent and long-lasting effect on the central cholinergic system.
Keywords: Semen Persicae (Prunus persica L. BATSCH); Acetylcholinesterase inhibitor; Acetylcholine; Microdialysis; Tacrine
1. Introduction 1995; Kosuge et al., 1985). The chemical constituents of

the herb include the cyanogenic glycosides, amygdalin and
Semen Persicae (Prunus persica L. BATSCH) water ex- prunasin as major components, along with the glycerides
tract (PPE) has long been used as an agent in China (Tounin and sterols (Kosuge et al., 1985; Ministry of Health and
and Taoren in Chinese), Japan (Donin in Japanese) and Ko- Welfare, 2001; Arichi et al., 1985; Isoza et al., 2001; He and
rea (Doin in Korean) in the treatment of the degenerative Li, 1988).
disorders, such as hypermenorrhea, dysmenorrhea, leiomy- Preliminary pharmacological studies in vitro have revea-
oma and infertility (Sakamoto et al., 1988, 1992; Wang et al., led that PPE is comprised of constituents having reversible
1998; Ge et al., 1983). It is frequently used as an ingredient and non-competitive cholinesterase inhibitory activity. PPE
in a variety of traditional prescriptions, particularly those produces marked and long-lasting inhibition of brain acetyl-
used to treat women’s diseases (Fukuda et al., 2003). It cholinesterase (AChE) and increases the brain content of
was also reported that Semen Persicae showed a relatively acetylcholine in vivo. Tacrine is the first drug approved
strong anti-tumour promoter and anti-Oketsu syndrome by the US FDA for the treatment of Alzheimer’s dis-
(stagnation of blood circulations) effects (Okuyama et al., ease, although it has adverse effects related to its actions
on the peripheral nervous system and to hepatic toxicity
(Beermann, 1993; Kosasa et al., 1999). Alzheimer’s disease
∗ Corresponding author. Tel.: +82-54-770-2663; fax: +82-54-770-2281. is a progressive neurodegenerative disease characterised by
E-mail address: chkimbio@dongguk.ac.kr (C.-H. Kim). deficits in memory and cognitive function. One of the most
doi:10.1016/S0378-8741(03)00106-5
150 Y.-K. Kim et al. / Journal of Ethnopharmacology 87 (2003) 149–154
pronounced changes in the brain of Alzheimer’s disease extracted three times with 1 l of distilled water in the hot
patients occurs in the cholinergic systems. Cholinesterase water bath for 5 h, and after filtration the filtrate was con-
inhibitors are the only class of drugs currently approved for centrated at reduced pressure for convenience. The extract
the treatment of Alzheimer’s disease. solution was stored at 4 ◦ C.
Since the extracellular acetylcholine concentration mea-
sured by the intracerebral microdialysis technique reflects 2.3. Implantation of microdialysis probe
the acetylcholine concentration in the synaptic cleft, it is
a useful parameter with which to compare the potential Rats were anaesthetised with pentobarbital Na (50 mg/kg,
efficacy of various cholinesterase inhibitors in the central i.p.) and placed in a stereotaxic frame (Narishige, Tokyo,
cholinergic system. The effects of PPE and tacrine on ex- Japan). The skull was exposed and a hole was drilled for
tracellular acetylcholine concentration have been studied by implantation of a microdialysis probe. In order to increase
microdialysis. the recovery of acetylcholine, a probe with a long mem-
In the present study, we have examined and compared the brane (3.0 mm membrane length, 0.5 mm diameter; I-4-03
effects of orally administered PPE and tacrine on the basal Eicom, Kyoto, Japan) was used. The probes were implanted
concentration of extracellular acetylcholine in the hippocam- according to the methods of Maysinger et al. (1988) into
pus of rats under the same experimental conditions and they the right lateral hippocampus at 4.5 mm lateral and 5.1 mm
were compared. Moreover, in order to validate the micro- posterior to the bregma and to a depth of 8.0 mm from
dialysis data, we measured the inhibition of brain AChE and the brain surface according to the atlas of Paxinos and
the brain concentrations of these drugs. Watson (1986). The probe was then fixed with dental cement.
After surgery, the rats were housed in their home cages.
Placement of the probe within the hippocampus was con-
2. Materials and methods firmed by visual inspection of the probe track at the end of
the experiment.
2.1. Animals and reagents
2.4. Microdialysis
Male Wistar rats (210–290 g) at 6 weeks of age were
purchased from Experimental Animal Station, Genetic Re- One day after surgery, the microdialysis probe was per-
sources Center, Korea Research Institute of Bioscience and fused with Ringer’s solution (145 mM NaCl, 4.5 mM KCl,
Biotechnology (Daejon, South Korea). They were allowed 0.5 mM MgSO4 , 2.5 mM CaCl2 , 5.0 mM HEPES) at a flow
at least 1 week to adapt to the environment (25 ± 3 ◦ C, rate of 1.0 ␮l/min. The perfusate was discarded during the
55 ± 5% humidity and a 12 h light/dark cycle, and start at first 1 h of perfusion and then collected at 20-min intervals
07:00 h) for at least 1 week before experiments. The animals into microtubes containing 5 ␮l of 0.1 M phosphate buffer
were given free access to food and water. All animal experi- (pH 3.5). Tacrine or PPE was orally administered after three
ments were approved by the Animal Care and Use Commit- fractions had been collected. The perfusate was collected
tee of DongGuk University Medical and Oriental Medical until 6 h after administration.
Hospital.
Tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochlo- 2.5. Acetylcholine assay
ride) was obtained from Sigma Co. (St. Louis, MO, USA).
All other chemicals were commercial products of reagent Acetylcholine concentration in dialysis samples was mea-
grade. Tacrine was dissolved in distilled water and admin- sured by high-performance liquid chromatography (HPLC)
istered by gavage. with electrochemical detection (Kosasa et al., 1999). As an
internal standard, 500 fmol of isopropylhomocholine was
2.2. Water-extract preparation from Prunus added to the sampling tubes, and the mixture was subjected
persica L. BATSCH to HPLC. The HPLC system (Shimazu LC-930A) consisted
of a degasser, an HPLC pump system, an autosampler,
Prunus persica L. BATSCH-water extract (300 g) was a column temperature controller, and an electrochemical
obtained from the Oriental Medical Hospital (Specimen No. detector with a platinum electrode (5 mm in diameter). A
P-75), DongGuk University College of Oriental Medicine, guard column and an enzyme reactor (3.0 mm × 4.0 mm;
Kyungju City, Kyungbuk, South Korea, as an oral adminis- AC-Enzympak, Eicom) were placed before and after the
tration grade for human. Also, for a large scale production analytical column (2.0 mm × 160 mm; Shimpak AC-Gel,
in our laboratory, Prunus persica L. BATSCH samples Shimadzu CO., Tokyo, Japan), respectively. Following its
from South Korea were purchased from Kyungju market, separation from the analytical column, acetylcholine was
Kyungju, South Korea. A voucher specimen has been de- converted to hydrogen peroxide inside the enzyme reac-
posited at the Kyungju Oriental Medical Hospital, DongGuk tor. The analytical and enzyme columns and the electrode
University, Kyungju City, Kyungbuk, South Korea, under were kept at 30 ◦ C in a column temperature controller.
acquisition number P-W-17. Sliced seeds (200 g) finely was The mobile phase was 0.1 M phosphate buffer (pH 8.3)
Y.-K. Kim et al. / Journal of Ethnopharmacology 87 (2003) 149–154 151
Fig. 1. Effect of oral administration of PPE on the basal concentration of extracellular acetylcholine in the hippocampus of rats. Data are expressed as
percentages of the pre-levels (average of three samples prior to administration = 100%). Values are means ± S.E.M. (n = 6). Pre-levels were: control:
102.6 ± 19.1 fmol/tube; 0.625 g/kg: 75.4 ± 6.3 fmol/tube; 1.25 g/kg: 88.2 ± 17.1 fmol/tube; 2.5 g/kg: 73.2 ± 6.2 fmol/tube. ∗ P < 0.05 vs. control (Dunnett’s
multiple comparison test).
containing 150 mg/l sodium 1-decanesulphonate, 50 mg/l 2.6. Measurement of AChE activity
tetramethylammonium chloride and 40 mg/l EDTA·2Na.
The flow rate of the mobile phase was 0.1 ml/min. Peaks Rats were decapitated at 0.5, 1, 2, 4, 8 and 12 h after re-
were recorded on a Powerchrom integrator (Shimazu). ceiving PPE (2.5 g/kg) and tacrine (10 mg/kg) by oral ad-
Acetylcholine in the dialysate sample was quantified by the ministration. Control animals received no treatment. The
internal standard method. The data are expressed as per- whole brain, excluding cerebellum and olfactory bulb, was
centages of the pre-level (average of three samples prior to removed and the two hemispheres were split for assays of
administration). AChE inhibition and brain concentrations of the drugs.
Fig. 2. Effect of oral administration of tacrine on the basal concentration of extracellular acetylcholine in the hippocampus of rats. Data are expressed as
percentages of the pre-levels (average of three samples prior to administration = 100%). Values are means ± S.E.M. (n = 6). Pre-levels were: control:
101.5 ± 15.4 fmol/tube; 1.25 mg/kg: 90.2 ± 9.5 fmol/tube; 2.5 mg/kg: 81.3 ± 7.5 fmol/tube; 5 mg/kg: 74.2 ± 12.1 fmol/tube. ∗ P < 0.05 vs. control (Dunnett’s
multiple comparison test).
AChE activity was measured using the radiometric ing Dunnett’s multiple comparison test was applied between
method, as described by Thomsen et al. (1989) and Sherman doses for individual fractions. Cholinesterase inhibition data
(1991). [3 H]Acetylcholine iodide (Amersham Korea, Seoul, were analysed with Dunnett’s multiple comparison test. A P
South Korea) was used as a substrate. In order to minimise value of less than 0.05 was considered significant. Statisti-
the dilution effect which is seen in ex vivo assays of the cal analysis was conducted using the software package SAS
inhibition by reversible cholinesterase inhibitors, the brain ver. 6.12 (SAS Institute Japan, Tokyo, Japan), available on
hemisphere was homogenised in 4 vol. of buffer, and finally the statistical analysis support system.
10 mg of brain tissue was diluted in 100 ␮l of assay solution.
Data are presented as percentages of the intact level.
4. Results
3. Statistical analysis 4.1. Comparative effects of PPE and tacrine on

hippocampal extracellular acetylcholine concentration
The data are expressed as means ± S.E.M. Microdialy-
sis data were analysed using a repeated measures analysis We examined the effects of PPE and tacrine on the
of variance with dose and fraction as factors. If a signifi- extracellular acetylcholine concentration in the hippocam-
cant dose×fraction interaction existed, post hoc analysis us- pus of rats. These cholinesterase inhibitors produced
Fig. 3. Comparison of the hippocampal extracellular brain AChE inhibition induced by PPE (A) and tacrine (B). Extracellular acetylcholine data are
expressed as percentages of the pre-level (average of three samples prior to administration = 100%). Values are means ± S.E.M. (n = 6). AChE activity
is expressed as a percentage of the intact level shown at 0 h (100%). Values are means ± S.E.M. (n = 5). ∗ P < 0.05 vs. intact (Dunnett’s multiple
comparison test).
Y.-K. Kim et al. / Journal of Ethnopharmacology 87 (2003) 149–154 153
dose-dependent increases in the extracellular acetylcholine less potent than those of tacrine at 10 mg/kg. Moreover, PPE
concentration (Figs. 1 and 2). PPE had a significant effect and tacrine showed long-lasting effects on both the extracel-
at a dose of 2.5 g/kg. The maximum increase produced lular acetylcholine concentration and the brain AChE activ-
by this dose was seen at about 1.5 h, when acetylcholine ity. These results are consistent with the idea that the effect
reached 465% of the pre-treatment level. The duration of of these compounds on the extracellular acetylcholine con-
the acetylcholine increase produced by the drug at this dose centration is based on the inhibition of AChE in the brain.
was more than 6 h (Fig. 1). The increase in the extracel-
lular acetylcholine concentration produced by tacrine at
5 mg/kg was maximum at 2 h when the level was 418% of 6. Conclusion
the pre-treatment level. The acetylcholine-increasing action
at this dose continued for more than 6 h (Fig. 2). The findings of this study demonstrated that centrally act-
ing cholinesterase inhibitors, PPE and tacrine, potently in-
4.2. Effects of PPE and tacrine on rat brain AChE activity crease the extracellular acetylcholine concentration in the
synaptic cleft of the hippocampus of rats mostly through
Fig. 3 shows the effects of PPE (2.5 g/kg) and tacrine AChE inhibition, and that PPE has a potent activity and a
(10 mg/kg) on rat brain AChE activity ex vivo. PPE pro- long-lasting effect on the central cholinergic system. PPE
duced maximal inhibition at 1 h after administration, when may be one of the more useful cholinesterase inhibitors for
AChE activity was 44% of the intact level. AChE activ- the treatment of Alzheimer’s disease.
ity gradually recovered thereafter, and reached 78% of the
intact level at 12 h after administration. The maximal inhibi-
tion of AChE by tacrine was observed at 1–2 h after admin- Acknowledgements
istration, although AChE inhibition remained at a plateau
for 0.5–8 h after administration (0.5 h, 41%; 1 h, 40%; 2 h, This work was in part supported by National Research
41%; 4 h, 42%). The AChE activity was 73% of the intact Laboratory Program, KISTEP, MOST, South Korea (No.
level at 12 h after administration. Acetylcholine elevation in M10203000024-02J0000-01300 to C.-H. Kim).
the hippocampus was a mirror image of AChE inhibition.
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5. Discussion
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Ethnopharmacology 23, 151–158.
The use of medicinal plants in self-care

in rural central Ethiopia
Teferi Gedif∗ , Heinz-Jürgen Hahn
Institute of Pharmacoepidemiology and Pharmaco Economics, School of Pharmacy, Martin-Luther University,
Wolfgang-Langenbeck Street 4, Room # 107, 06120 Halle/Saale, Germany
Received 17 July 2002; received in revised form 20 January 2003; accepted 14 March 2003
Abstract
Medicinal plants are an important element of Ethiopian traditional medicine. This questionnaire survey examined the extent and type of
medicinal plants used in self-care by rural Ethiopian community. Six hundred mothers were interviewed using a semi-structured questionnaire.
The prevalence of the use of herbal drugs in self-care was found to be 12.5%. Twenty-five plant species belonging to 21 families were reported,
each with local names, methods of preparation and parts used. This study showed that self-care using medicinal plants is a major part of health
care options in Butajira community.
Keywords: Medicinal plants; Self-care; Prevalence; Rural Ethiopia
1. Introduction communities of the developing countries including Ethiopia

are the de facto healers of the family treating accidents
The use of plants as medicines predates written human and ailments with medicinal plants (Lambert et al., 1997).
history and almost all cultures in the world have a body of The present research, therefore, attempted to document
expertise concerned with the therapeutic properties of the the medicinal plants used in self-care in a rural Ethiopian
local flora (Houghton, 1995). community using mothers as informants.
Ethiopian traditional medicine is composed of a number of
specific skills, namely, the use of plants, animal products and
minerals as well as magic and superstition (Pankrust, 1976). 2. Subjects and methods
The main body, however, is based on the use of ethnobotany
(Vicchiato, 1993). 2.1. Description of study area
Though most practices and treatments in herbal medicine
require specialists or professionals which are referred gen- Butajira is a district located 130 km south of Addis
erally to as herbalists, self-care using plants is common in Ababa, capital city of Ethiopia (Fig. 1). The population size
Ethiopia (Kitaw, 1987; Gedif, 1995). Although few studies extrapolated from 1994 census is estimated to be 257,000.
on the medicinal plant resources of Ethiopia have been con- Generally the demographic pattern is typical of developing
ducted (Abebe and Ayehu, 1993; Tadesse and Demissew, countries where children below the age of 15 constitute the
1992; Abebe, 1986; Jansen, 1981), the extent and types of majority (Berhane, 2000). The dominant ethnic group is
herbs used in self-care by the vast majority of the popula- Gurage of meskan dialects. Farming is the main economic
tion particularly in rural areas has not been documented. activity and the main cash crops being pepper, coffee and
Knowing factors involved in the selection of different treat- khat. The estimated size of the district is 797 km2 and lies
ment options at household level is also important for health at an average of 2100 m above sea level, ranging from 1750
service planning and to incorporate herbal medicine in a in the lowlands to 3400 m in mountainous areas (Berhane
country’s health care delivery system. Mothers in most rural et al., 1999). During the study time, the district had 1 health
centre, 2 health stations, 11 private clinics, 11 health posts
∗ Corresponding author. Fax: +49-345-55-2-72-92. and 8 drug retail outlets. Although the health centre is the
E-mail address: Gedif66@yahoo.com (T. Gedif). highest level of health institutions in the district, it has
doi:10.1016/S0378-8741(03)00109-0
156 T. Gedif, H.-J. Hahn / Journal of Ethnopharmacology 87 (2003) 155–161
for health related researches (Berhane, 2000; Shamebo,

1993).
2.2. Data collection and analysis
Information on demographic characteristics, prevalence

of perceived illness, the extent and type of herbs used in
self-care; and factors associated with the choice of treatment
options were collected by using a semi-structured question-
naires from mothers (or woman who assumed the role of
a mother) in 600 households. When mother in a house-
hold was absent in the first visit, repeated visits were made
up to three times. The 600 households were selected using
systematic random sampling technique. Enumerators who
are workers of Butajira Rural Health Project were given
additional training for two days on the data collection in-
strument. Before the initiation of the interview, oral con-
sent was obtained from each respondent who participated
Fig. 1. Map of Ethiopia showing the location of the Butajira district. in the study.
EPI-INFO Version 6.0 statistical software was used for
data entry and analyses.
no capacity to manage surgical and obstetric emergencies
(Berhane, 2000). Communicable diseases including malaria,
ARI, diarrhoeal diseases and tuberculosis are the major pub- 3. Results
lic health problems of the district. This study was carried out
in villages that are under a continuous demographic surveil- 3.1. Perceived illness
lance by the Butajira Rural Health Programme (BRHP).
There are 10 study communities under BRHP which were The distribution of illness and the corresponding action
selected based on probability proportional to size out of 84 taken against the illness by background factors is presented
rural and 4 urban kebeles (the lowest administrative units) of in Table 1. One hundred thirty-six persons were reported
Butajira sub-district (Fig. 2). The demographic surveillance to have an illness episode during a four weeks recall pe-
has been going on since 1987 to provide sampling frame riod preceding the interview date. Females were reported to
Fig. 2. Location of the 10 study villages in the district.

T. Gedif, H.-J. Hahn / Journal of Ethnopharmacology 87 (2003) 155–161 157
Table 1
Types of action taken by those with perceived illness in four weeks recall period
Background factors Modern health facilities (%) Herbal medicine (%) No action (%) Total ill
Sex
Male 77.6 14.3 8.2 49
Female 71.3 16.1 12.6 87
Age in years
0–4 82.5 14.3 3.6 28
5–14 82.1 – 17.9 28
15–54 72.3 15.4 12.3 65
55+ 46.7 46.7 6.7 15
Marital statusa
Married 65.4 21.2 13.5 52
Others 71.4 21.4 7.1 28
Religion
Muslim 73.3 16.3 10.5 86
Christian, others 74.0 14.0 12.0 50
Educationb
Illiterate 64.8 22.5 12.7 71
Literate 81.8 3.0 15.2 33
Household size
1–4 69.6 21.4 8.9 56
5–9 89.2 10.8 13.5 74
10+ 83.3 6.7 – 6
a Excludes too young to get married.
b Excludes too young to be literate.
have more morbidity than males. Being illiterate was also 3.2. Health care options
associated with high morbidity. Febrile illnesses including
malaria, cough/cold, diarrhoea, and skin disorders were the The overall action taken for those with reported perceived
most frequently reported illnesses in the area. illness was 89%; out of which 17.4% used herbal medicine
Table 2
Self-care with herbal drugs and factors associated with it in Butajira community, 2000
Category Number ill Self-care with herbs (%) Chi-square d.f. P-value
Sex
Male 49 10.2
Female 87 13.8 0.43 1 NS
Age in years
0–14 56 3.6
15–54 65 13.8
55+ 15 40.0 44.3 2 0.0000
Religion
Muslim 86 14.0
Christian, others 50 10.0 0.43 1 NS
Marital statusa
Married 52 19.2
Others 28 17.9 0.03 1 NS
Educationb
Illiterate 71 29.4
Literate 33 3.0 23.25 1 0.000014
Household size
1–4 56 17.9
5+ 80 8.8 2.74 1 NS
a Excludes too young to get married.
b Excludes too young to be literate; NS = P > 0.05.
Table 3
Medicinal plants used in self-care by Butajira community, south central Ethiopia
Family/species Local name Part used Citation Uses, preparation
Acanthaceae
Adhatoda schimperiana Sensel Leaf 6 For scabies, fresh leaves are crushed and macerated in
Hochst. ex Nees water, and then the affected area is washed with the
macerate
Alliaceae
Allium sativum Nech-shinkurt Bulb 97 To treat diarrhoea and malaria, the bulb are chewed and
swallowed. For cough/cold treatment, the bulb is
crushed and boiled with tea and drunk
Compositae
Vernonia amygdalina Girawa Leaf 7 For skin rashes, fresh leaves are crushed and macerated
Delile in water, then wash the affected area with the macerate
Cucurbitaceae
Zehneria scabra Areg-ressa Leaf 4 To treat diarrhoea, headache and fever, the juice of
Sond. fresh squeezed leaves is drunk
Crucifereae
Lepidum sativum Feto Seed 43 For diarrhoea and dysentery, most said grind the seed and
make pellet with honey and swallowed; few said solution
in water is drunk. To treat skin disorders, the powdered
seed is mixed with fresh butter and applied topically
Euphorbiaceae
Croton macrostachyus Bissana Leaf; areal part 3 To treat skin problems, the juice from the apical parts
Hoechst of the plant is applied on the affected area
Fabaceae
Taverniera abyssinica Dingetegna Root 90 To treat stomachache, vomiting and fever, the root is
A. Rich pounded in water and the juice is drunk
Labiatae
Ajuga integrifolia Anamaro/(armagussa) Leaf 26 For diarrhoea and malaria, decoction of the fresh leaves
Buch-Ham is drunk; for treating wound, the leaves are pounded
into a paste and is applied on the affected part
Thymus serrulatus Tosegne Leaf 6 Decoction or infusion of pounded leaves is drunk to
Hoechst ex. Benth treat respiratory problems
Ocimum lamiifolium Damkesse Leaf 195 To treat diarrhoea, amoeba (diarrhoea with blood) and
Hochest ex. Benth cough/cold, fresh leaves are pounded in water and the
juice is drunk
Linaceae
Linum usitatissimum L. Telba Seed 118 To treat diarrhoea and amoeba, the water solution of
toasted and powdered seed is drunk in an empty stomach
Malvaceae
Sida ovata Forsk Chifrig Leaf 4 For eczema, the leaves are pounded and the paste
applied on the affected area
Myrsinaceae
Embelia schimperi Enkoko Fruit 2 To expel tape worm, the ground fruit is macerated in tela
Vatke (local alcoholic beverage) or water and left over night,
and then the macerate is drunk in an empty stomach
Myrtaceae
Eucalyptus globulus Nech-bahirzaf Leaf 27 To treat cough/cold, the fresh leaves are decocted and
Labill the steam is inhaled
Poaceae
Oryza sativa L. Ruz Seed 3 To treat diarrhoea, rice water is drunk
Phytolaccaceae
Phytolacca dodecandra Endod Leaf 5 To treat scabies, chopped leaves are immersed in water
L’Herit and then the affected part is washed with the solution
Polygonaceae
Rumex helpalensis Tult Leaf 3 To treat amoeba, the solution of the dried and powdered
leaves is drunk
Family/species Local name Part used Citation Uses, preparation
Rumex nervosus Vahl. Embatcho/dengago Leaf 9 To treat skin disorders, leaves are crushed and mixed with
butter, and then it is applied on the affected area
Rosaceae
Hagenia abyssinica Kosso Flower 70 To expel tape worm, water/local alcoholic extract of the
(Bruce) Gmel. flower is drunk in the morning in an empty stomach or the
flower is pasted with honey and taken
Rubiaceae
Coffea arabica Linn. Buna Seed 18 Roasted and powdered coffee is pasted with honey and
taken orally for the treatment of diarrhoea and amoebiasis
Rutaceae
Ruta chalepensis L. Tenadam Leaf 97 A decoction of the leaves mixed with tea is drunk against
headache, fever, and cough/cold
Solanaceae
Datura stramonium L. Itsefaris/astenagir Leaf 6 For treating eczema, powdered leaves is mixed with butter
or fat; and the ointment is applied on the affected area
Solanum campylacanthum Emboye Fruit 7 To treat eczema and scabies, the juice inside the fruit is
Hochst applied
Verbenaceae
Verbena officinalis Atuch Root 4 The crushed roots are left in water for some time and then
subsp. africana the extract is drunk to stop diarrhoea
Zingiberaceae
Zingiber officinale Rosc. Zingibel Rhizome 68 To treat stomachache, the rhizome is chewed; for
cough/cold the decoction is mixed with tea and drunk
and 82.4% modern medicine. There was a relatively higher were the most frequently used plants (Table 3). Leaves were
rate of action taken among males than females. Belief on ef- the part of the plant commonly used.
ficacy (57.2%), and economic (28.5%) and geographical in-
accessibility of modern health care (14.3%) were mentioned
as reasons for choosing herbal medicine as the first line of 4. Discussion and conclusions
health care option in Butajira area.
Proportions of those with perceived illness and chose to Perceived morbidity is the main initiator for seeking
use herbal medicine in self-care were compared between health care. The perception of an illness by a person in
subgroups (e.g. among different ages, males versus females, a household was, therefore, used as a starting point of
illiterate versus literate, etc.) using chi-square tests (Table 2). our inquiry to reveal the extent and types of herbs taken
Females were more likely to use herbal medicine than against illnesses in Butajira community. The morbidity pat-
males but the association was not statistically significant tern reported in this study concurs with what was reported
(P > 0.05). Age was found to have a significant association by Shamebo et al. (1994) and other similar study done in
with the use of herbal medicine (P < 0.0001). Chi-square the rift valley (Kitaw, 1987). Like other studies, this study
for linear trend for age also indicated that the tendency to showed that the burden of illness is less pronounced in males
use herbal medicine increases with age. Illiterates (who are than females (Kitaw, 1987; Gedif, 1995). This might have
not able to read and write) were significantly more likely a strong association with the status of women in Butajira
to use herbal medicine than literates (P < 0.0001). community. As documented by Berhane (2000), women in
rural Butajira are in a very disadvantaged position because
3.3. Self-care with herbal drugs of very low literacy levels, high workloads and low access
to modern health facilities. This study also showed that
The prevalence of self-care with herbal drugs in Buta- perceived efficacy, economic and geographic accessibility
jira community in four weeks recall period was 12.5%. are main reasons for popularity of herbal medicine and its
Twenty-five species belonging to 21 families were claimed practitioners in Butajira community.
to be used in self-care. Taverniera abyssinica A. Rich. Although the association was not statistically significant,
(Fabaceae), Ocimum lamiifolium Hochst. ex Benth (Labi- females were more likely found to use herbal medicine in
atae), Allium sativum (Alliaceae), Ruta chalepensis (Ru- self-care than males. This is consistent to the result of pre-
taceae), Linum usitatissimum L. (Linaceae), Hagenia vious study done in Hong Kong about the general use of
abyssinica (Bruce) Gmel (Rosaceae), Zingiber officinale herbal medicine (Wong et al., 1997). It was also interesting
Rosc. (Zingiberaceae) and Lepidum sativum (Crucifereae) to note that education and age had a significant association
with the use of herbal medicine. In this regard, illiterates and

older residents are significantly more likely to use herbal
medicine than literate and younger people, respectively. A
study conducted in rural Tanzania also showed that age and
education were the main factors that seemed to influence the
choice of health care (Satimia et al., 1998).
Almost 1/3rd of the medicinal plants reported to be used
in self-care in this study have also long been used as food
and/or spices in food and widely sold in Ethiopian markets
(Kloos et al., 1978). Recognising their frequent use there
is a need to promote phytochemical and pharmacological
investigations on these plants.
Acknowledgements
The financial assistance from Katholischer Akademischer

Ausländer-Dienst (KAAD) is highly acknowledged. We
extended our deepest appreciation to Professor Dr. R.
Neubert for his all rounded support to make this study a
reality. Ethiopian Science and Technology Commission and
Butajira Rural Health Project are acknowledged for facili-
tating the field work. Special thanks go to enumerators who
skilfully collected the data. Finally, we wish to express
our gratitude to mothers in Butajira who shared with us
information about their personal lives and experiences.
Appendix A
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modern or traditional. Archives of Dermatology 134, 1363–1366. Wong, T.W., Yu, T.S., Liu, J.L.Y., Lee, N.L., Lloyd, O.L., 1997. Factors
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Biological activity of extracts from Catalpa bignonioides

Walt. (Bignoniaceae)
D. Muñoz-Mingarro a,∗ , N. Acero b , F. Llinares c , J.M. Pozuelo d , A. Galán de Mera b ,
J.A. Vicenten b , L. Morales e , L.F. Alguacil e , C. Pérez e
a Sección de Quı́mica Analı́tica, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU,
Apartado 67, 28660 Boadilla del Monte, Madrid, Spain
b Sección de Biologı́a Vegetal, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU,

c Sección de Microbiologı́a, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU,

d Sección de Biologı́a Celular, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU,

e Sección de Farmacologı́a y Toxicologı́a, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU,
Received 19 February 2002; received in revised form 17 March 2003; accepted 21 March 2003
Abstract
Catalpa bignonioides Walt. (Bignoniaceae) is a species that belongs to a tropical family but has been introduced in many countries as
ornamental. Although this plant is consumed by indigenous cultures of South America for medical uses, experimental studies of the biological
properties of Catalpa bignonioides are lacking. The aim of this work was to study the biological activity of crude extracts from either pods,
seeds or leaves of Catalpa bignonioides which were collected in Spain. Ethyl ether, butanolic and aqueous fractions of the pod extract were also
prepared and studied. We have examined the antimicrobial activity against five bacteria and one yeast, the cytotoxic activity against HepG2
cells and the anti-inflammatory and antinociceptive effects in rodents. A preliminary phytochemical analysis of the extracts and fractions was
also conducted. Results showed no antimicrobial or antitumoral effects, but prominent anti-inflammatory and antinociceptive actions of the
extracts. These last activities may be a result of the presence of either of saponins, sterols or phenols, mainly found in the leaves and pods of
the plants.
Keywords: Catalpa bignonioides; Extracts; Biological activities
1. Introduction species are found in cultivation as greenhouse ornamentals.

Some extracts from the Catalpa species have shown inter-
Species belonging to the Bignoniaceae are widely used for esting biological properties: extracts from Catalpa ovata
many different purposes in medicinal practice by the indige- G. Don fructus have mutagenic activity towards Salmonella
nous cultures of South America (Rasadah and Houghton, typhimurium (Nozaka et al., 1989), while its stem-bark
1998). Some of these species, like Jacaranda micranta extracts have antitumoral activity (Fujiwara et al., 1998).
Cham., Tabebuia caraiba (Mart.) Bureau, Tecoma sambuci- Regarding properties and uses of Catalpa bignonioides,
folia Kunth and Tecoma stans (L.) Juss. ex Kunth are known commonly known as Bean-tree, which were already men-
for the anti-inflammatory, antirheumatic, antinociceptive, tioned in the physiomedical dispensatory of Cook (1869),
narcotic or antisyphilitic activity of their extracts. The the bark of Catalpa bignonioides is cited as a stimulat-
Catalpa Scop. genus, which belongs to this family, com- ing tonic in syrup form, the decoction of the pods as a
prises 11 species of trees and shrubs native to East Africa demulcent with relaxant and stimulant properties and the
and North and South America. Outside the tropics, several leaves are described as useful for irritable ulcers. Catalpa
bignonioides has also been described for the treatment of
respiratory diseases (decoction of pods and seeds), irritable
∗ Corresponding author. scrofulous ulcers (cataplasms of bruised leaves), strumous
doi:10.1016/S0378-8741(03)00111-9
164 D. Muñoz-Mingarro et al. / Journal of Ethnopharmacology 87 (2003) 163–167
ophthalmia, cutaneous affections (juice of leaves and roots), and sterols by using the methods previously described by
scrofulous maladies and helmintic infections (decoction or Tona et al. (1998).
powder of barks). Despite all these potential beneficial ef-
fects, experimental studies of the biological properties of 2.4. In vitro cytotoxicity assay
Catalpa bignonioides extracts are lacking.
Phytochemical studies (phytochemDB) show the pres- The cell line used was human hepatoma cell line (HEpG2)
ence of several classes of compounds in extracts of Catalpa obtained from the American Type Culture Collection
bignonioides, which could be linked to some of the tra- (ATCC, HB 8065). The cell line was grown on Dulbeco’s
ditional uses of Bignoniaceae. These include fats, sugars, Mem medium supplemented with 10% fetal bovine serum
terpenes, alkaloids, tannins and, particularly, flavonoid and (FBS), and incubated at 37 ◦ C in an atmosphere of 5%
phenolic compounds, which were detected in high amounts. CO2 in air (95% humidity). At a log phase of their growth
Previously Rau (1870; King’s American Dispensatory) made cycle, the cells were treated in triplicate with various con-
an examination of the inner bark and found it to contain tan- centrations of the extracts (between 25 and 0.1 mg/ml)
nin and a nauseating matter soluble in ether. Sugar, tannin, with a final dimethylsulfoxide (DMSO) concentration of
resin and fixed oil are constituents of the seeds. 0.1%. This concentration of DMSO did not affect cell
In this paper we report a preliminary phytochemical viability.
analysis and biological screening on bactericidal, cytotoxic, The assay was performed using a 96-well plate which con-
anti-inflammatory and antinociceptive activities of aqueous tained 2500 cells per well incubated with the corresponding
extracts from leaves, pods and seeds of Catalpa bignon- extract, during 72 h, as previously described. Test wells con-
ioides. These activities were chosen in part on the basis of taining the cells alone, free of plant extracts were used as
previous findings of our team regarding the biological ac- controls. The MTT assay, according to Borenfreund et al.
tivity of Tecoma sambucifolia, another Bignoniaceae plant (1988), was used to obtain the effective dose that inhibits
(Alguacil et al., 2000). The pod extract was further frac- 50% growth after the incubation period (IC50 ).
tionated in order to approach the isolation of the possible
active principles. 2.5. Antimicrobial assays
The bacterial growth inhibition assays were performed

2. Methodology using cultures of Escherichia coli (ATCC 35219), Pseu-
domonas aeruginosa (ATCC 9027), Staphylococcus aureus
2.1. Plant material (ATCC 24213), Enterococcus faecalis (ATCC 29212),
Salmonella tiphymurium (ATCC 13311), and the yeast
Catalpa bignonioides specimens were collected in Boad- Candida albicans (ATCC 10231). Bacteria strains were
illa del Monte (Madrid, Spain) and identified by Dr. Galán maintained on Mueller-Hinton broth and the yeast on
de Mera, USP (San Pablo University), Madrid. A voucher Sabourand’s dextrose agar.
specimen was deposited in the USP herbarium under the The diluted extract suspension was homogenized and
number 250399. Collected material was dried under dark- the screening was then performed according to the liq-
room conditions. uid dilution method (Vanden Berghe and Vlietinck, 1991).
Minimum inhibitory concentration (MIC) was determined
2.2. Plant extracts by incorporating various amounts (1–200 mg/ml) of recon-
stituted extract solution into the medium.
Crude extracts of leaves, pods, and seeds were prepared The MIC was interpreted as the lowest concentration of
by decoction of 10 g of each pulverized material in 200 ml the extracts that did not permit any visible growth when
of water for 30 min. The resultant extracts were then filtrated compared with that of the control.
and concentrated to dryness under reduced pressure. The
yield was 22.9% (w/w) for pods, 14.1% (w/w) for leaves 2.6. In vivo pharmacological assays
and 5.3% (w/w) for seeds.
The concentrated aqueous extract of pods was fractionated 2.6.1. Animals and sample preparation
by successive extraction with ethyl ether (3 × 100 ml) and Male Sprague–Dawley rats (200–250 g; San Pablo-CEU
butanol (3 × 100 ml). An ethyl ether (P-E, 3.94% w/w), University breeding) and male OF1 mice (25–30 g,
a butanol (P-B, 28% w/w) and an aqueous fraction (P-A, Iffa-Credo, France) were used. The animals were housed in
55.2% w/w) were obtained. cages with water and food available ad libitum, kept in a
controlled environment (temperature, 20–22 ◦ C; dark/light,
2.3. Preliminary phytochemical analysis 12 h/12 h, humidity, 45–55%) and randomly assigned to the
different experimental groups. The aqueous extracts were
Plant materials were screened for the presence of dissolved in physiological saline for i.p. administration
saponins, tannins, total phenols, anthraquinones, flavonoids (10 ml/kg) at a dose of 1 g/kg. The doses of the fractioned
D. Muñoz-Mingarro et al. / Journal of Ethnopharmacology 87 (2003) 163–167 165
pod extracts were calculated to provide an amount of the Table 1

different substances equivalent to that of the crude pod Phytochemical screening and cytotoxic activity, against HEpG2 cells, of
crude aqueous extracts and fractions from Catalpa bignonioides
extract, i.e. 40 mg/kg for P-E, 300 mg/kg for P-B and
555 mg/kg for P-A. Anth. Sap. Este. Tan. T. Ph. Flav. IC50
(mg/ml)
2.6.2. Acetic acid writhing in the mouse Crude extract
Seeds − + + − + − 28
The test was performed as described by Koster et al.
Leaves − + + − + − 23.6
(1959). Nociception induced by acetic acid was character- Pods − + + + + + 10.9
ized by the presence of abdominal constrictions, consisting
Pod fractions
of the contraction of the flank muscles associated with P-A − + + − + − 3.5
inward movements of the hind limbs or with whole body P-B − − + + + + 0.68
stretching. Animals were injected with the extracts from P-E − − − − − + 3.58
Catalpa bignonioides, saline or indomethacin (3 mg/kg; Anth.: anthraquinones, Sap.: saponins, Este.: sterols, Tan.: tannins, T.
Sigma, Spain), which was used as a reference compound. Ph.: total phenols, Flav.: flavonoids. P-A: aqueous fraction, P-B: butanol
Thirty minutes after treatment, all the animals received fraction, P-E: ethyl ether fraction.
2% acetic acid i.p., and 10 min afterwards, the number of
abdominal constrictions was recorded for 15 min by visual 4.2. Cytotoxicity assay
observation of the animals.
The IC50 (mg/ml) values obtained in the cytotoxicity assay
2.6.3. Carrageenan-induced hind paw edema in the rat against the HepG2 cell line are shown in Table 1. A very low
Carrageenan-induced inflammation was initially de- toxicity was found in all extracts and fractions. The highest
scribed by Winter et al. (1962) and has become a widely toxicity was found in the crude extract of pods and fraction
used model for screening anti-inflammatory agents. At the P-B.
beginning of the study, the baseline paw volume was de-
termined by submerging the right hind paw up to the tibio-
4.3. Antimicrobial activity
tarsal joint into a water cell of a plethysmometer (Digital
Pletysmomet, L37500; Letica). The volume of displace-
The inhibitory bacterial growth by extracts, indicated by
ment, which is equal to the paw volume, was then read on
their MIC values, is summarized in Table 2. None of the
a digital display. Each determination was the average of
extracts examined showed a significant bactericidal activity,
three repeated measures. After baseline determination, the
since the lower MIC (showed by pods against Salmonella
rats were injected with the extracts from Catalpa bignon-
tiphymurium) was 3.12 mg/ml. No effects were observed
ioides, saline or indomethacin (5 mg/kg; Sigma), which
in yeast analysis, as the MIC obtained was higher than
served as a positive control. One hour afterwards, the rats
200 mg/ml in all cases.
were subcutaneously injected with 0.1 ml of 1% lambda
carrageenan (Sigma) into the surface of the right hind paw,
and the effect on paw volume was studied 1, 2 and 3 h post 4.4. Anti-inflammatory activity
injection.
The results of this study are shown in Table 3. The extracts
of leaves and pods exhibited a significant anti-inflammatory
3. Statistical analysis activity of similar magnitude 2 and 3 h after carrageenan in-
jection, while the extract of seeds was devoid of significant
Statistical analysis was performed with ANOVA followed effects during all the periods considered. Although the three
by multiple range tests (least-squares difference test, LSD). pod fractions exhibited some activity in the test, only P-A
Differences were considered significant at P < 0.05. provided a marked and sustained effect throughout the eval-
uation period considered. P-E only produced a transient de-
crease of paw edema, and P-B also provided a slight effect
4. Results that achieved statistical significance 3 h post injection.
4.1. Phytochemical study 4.5. Antinociceptive activity
The results of our assay on the crude extracts and frac- The effects obtained in the antinociception experiments
tions are shown in Table 1. All crude extracts showed the were qualitatively similar to those of the anti-inflammatory
presence of saponins (triterpenic and steroidic), sterols and study (Fig. 1). Although the three crude extracts exhibited
phenols and were negative for anthraquinones. Regarding some pharmacological activity, the extract of seeds was the
pod fractions, saponins were detected in P-A, sterols in P-A least potent and the extracts of pods and leaves behaved
and P-B and flavonoids in P-B and P-E. similarly. Concerning the pod fractions, P-A was again the
166 D. Muñoz-Mingarro et al. / Journal of Ethnopharmacology 87 (2003) 163–167
Table 2
Antibacterial activity of crude aqueous extracts and fractions from Catalpa bignonioides (MIC, mg/ml)
Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Enterococcus faecalis Salmonella tiphymurium
Crude extract
Seeds 100 100 50 50 50
Leaves 6.25 100 12.5 12.5 6.25
Pods 6.25 50 12.5 12.5 3.12
Pod fractions
P-A 25 50 25 25 6.25
P-B >200 >200 100 >200 >200
P-E >200 >200 100 100 >200
P-A: aqueous fraction, P-B: butanol fraction, P-E: ethyl ether fraction.
Table 3
Anti-inflammatory effect of crude aqueous extracts and fractions from Catalpa bignonioides on carrageenan-induced hind paw inflammation in the rat
Treatment n Paw volume (ml) after carrageenan injection
Baseline 1h 2h 3h
Saline 15 1.53 ± 0.02 2.00 ± 0.03 2.64 ± 0.10 2.91 ± 0.10

Indomethacin 14 1.55 ± 0.02 1.79 ± 0.03∗ 2.18 ± 0.04∗ 2.43 ± 0.04∗
Crude extract
Seeds 6 1.56 ± 0.01 1.92 ± 0.07 2.62 ± 0.20 2.86 ± 0.25
Leaves 6 1.59 ± 0.04 1.93 ± 0.03 2.15 ± 0.10∗ 2.31 ± 0.22∗
Pods 16 1.61 ± 0.02 1.92 ± 0.04 2.10 ± 0.11∗ 2.25 ± 0.11∗
Pod fractions
P-A 7 1.51 ± 0.04 1.77 ± 0.06∗ 2.15 ± 0.13∗ 2.37 ± 0.15∗
P-B 9 1.53 ± 0.03 1.88 ± 0.04 2.35 ± 0.10 2.56 ± 0.12∗
P-E 7 1.48 ± 0.02 1.81 ± 0.04∗ 2.33 ± 0.07 2.68 ± 0.12
P-A: aqueous fraction, P-B: butanol fraction, P-E: ethyl ether fraction.
∗ P < 0.05 vs. saline.
Fig. 1. Effect of aqueous extracts of Catalpa bignonioides on abdominal constrictions produced by acetic acid. Bars are means ± S.E.M. ∗ P < 0.05 vs.
saline. (P-A: aqueous fraction, P-B: butanol fraction, P-E: ethyl ether fraction).
most active; on the other hand, the effect of P-E injection phytochemical analysis of aqueous extracts from Catalpa
did not reach statistical significance. bignonioides show the presence of phenolic compounds, the
cytotoxic effect observed is considerably low and has little
relevance against the human hepatoma cell line (HEpG2),
5. Discussion and conclusions it has been suggested that the reputed antimicrobial activ-
ity of some species of Bignoniaceae like Kigelia pinnata
The antitumoral activity of species such as Catalpa ovata (Jacq.) DC (Akunyili et al., 1991), Jacaranda mimosifolia
(Kingston and Rao, 1982; Fujiwara et al., 1998) has been re- D. Don, Tecoma stans, Crescentia cujete L. (Binutu and
lated to phenolic compounds like naphtoquinones. Since the Lajubutu, 1994) Tabebuia spectabilis (Planch. and Linden)
D. Muñoz-Mingarro et al. / Journal of Ethnopharmacology 87 (2003) 163–167 167
G. Nicholson, and Oroxylum indicum (L.) Vent. (Rasahad References

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Acknowledgements Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan-induced edema
in hind paw of the rat as an assay for anti-inflammatory drugs. In:
We wish to express our gratitude to Linda Hamalainen Proceedings of the Society for Experimental Biology and Medicine
for her linguistic help. III, pp. 554–547.
Comparison of the gastroprotective effect of a diterpene lactone

isolated from Croton cajucara with its synthetic derivatives
Patricia Silva Melo a , Nelson Durán b,c , Clélia Akiko Hiruma-Lima d ,
Alba Regina Monteiro Souza-Brito e , Marcela Haun a,∗
a Departamento de Bioquı́mica, Instituto de Biologia, Universidade Estadual de Campinas, cp. 6110, CEP 13081-970, Campinas, SP, Brazil
b Laboratório de Quı́mica Biologica, Instituto de Quı́mica, Universidade Estadual de Campinas, cp. 6154, CEP 13081-970, Campinas, SP, Brazil
c Núcleo de Ciências Ambientais, Universidade Mogi das Cruzes, CEP 08790-911, Mogi das Cruzes, SP, Brazil
d Departamento de Fisiologia, Instituto de Biociencias, Universidade Estadual Paulista, cp. 510, Rubião Junior s/n, CEP 18618-000, Botucatu, SP, Brazil
e Departamento de Fisiologia e Biofı́sica, Instituto de Biologia, Universidade Estadual de Campinas, cp. 6109, CEP 13084-970, Campinas, SP, Brazil
Received 30 November 2002; received in revised form 27 March 2003; accepted 27 March 2003
Abstract
The effect of three new derivatives from dehydrocrotonin (DHC—compound I) on gastric damage in different animal models including gastric
ulceration induced by a necrotic agent and hypothermic restrained-stress was studied: compound II (produced by reducing the cyclohexenone
moiety of DHC with NaBH4 ); compound III (produced by reducing the carbonyls with LiAlH4 ); and compound IV (produced by transforming
the lactone moiety into an amide). Their structures were confirmed on the basis of chemical and physicochemical evidence. When previously
administered (p.o.) at a dose of 100 mg/kg, compound II significantly (P < 0.01) reduced gastric injury induced by HCl/ethanol (78%) and
indomethacin (88%) better than did reference compound I (48 and 43%, respectively). But the anti-ulcerogenic activity of compound II was
completely abolished by the stress-induced ulcer. Reduction of carbonyls with LiAlH4 (compound III) caused decreased activity, markedly
when no protective effect in any of the models was applied (P > 0.05). However, compound IV, in which the lactone moiety was changed into
an amide, when administered at the same dose (100 mg/kg, p.o.), was more effective. The presence of a lactone moiety or Michael acceptor
is probably essential for the anti-ulcerogenic effect of these compounds.
Keywords: Gastroprotective activity; DHC; Croton cajucara
1. Introduction through protection of the gastric mucosa by an increase of

prostaglandins-E2 (Hiruma-Lima et al., 1999). Clerodane
In Brazil Croton cajucara Benth. (Euphorbiaceae), pop- diterpenes with anti-ulcerogenic activity have also been
ularly known as “Sacaca” is used in popular medicine as a isolated from Croton sublyratus (Kitasawa et al., 1980) and
cytoprotective agent for gastric ulcers and to treat several Aparisthmium cordatum (Hiruma-Lima et al., 2000, 2001).
illnesses, such as diarrhea, diabetes, and liver inflamma- The etiology of gastroduodenal ulcers is influenced by
tion (Di Stasi et al., 1989). As a start for our studies on various aggressive and defensive factors, such as acid-pepsin
anti-ulcer substances, we reported the anti-ulcerogenic secretion, parietal cell, mucosal barrier, mucus secretion,
activity of trans-dehydrocrotonin (DHC—compound I, blood flow, cellular regeneration, and endogenous protec-
Fig. 1), the primary compound (nor-clerodane diterpene tive agents, such as prostaglandins and epidermic growth
lactone) isolated from Croton cajucara bark, in different factors (Repetto and Llesuy, 2002). It is well known that
ulcerogenic models, using mice and rats (Souza-Brito et al., peptic ulcers are the outcome of an imbalance between de-
1998). The protective effect of DHC on induced gastric fensive factors (forces of mucosal resistance) and aggres-
lesions occur through the suppression of acid secretion and sive factors (gastric acid and pepsin secretion) (Yamamoto
et al., 1998). There are many products used for the treatment
of gastric ulcers, such as antacid, proton pump inhibitor or
∗ Corresponding author. Fax: +55-19-3788-6129. H2 -blockers, but most of these drugs produce some adverse
E-mail address: marcelah@unicamp.br (M. Haun). reactions (Brunton, 1996). Therefore, numerous efforts have
doi:10.1016/S0378-8741(03)00139-9
170 P.S. Melo et al. / Journal of Ethnopharmacology 87 (2003) 169–174
Fig. 1. DHC and derivatives.
been pursued to find a novel class of anti-ulcer agent which cers in mice with the aim to compare the structure–activity
strengthens defensive mechanisms with less toxic effect. relationship.
Studies of the acute toxicity of DHC in vivo (Souza-Brito
et al., 1998) have shown that this compound has a rela-
tively low oral toxicity when administered for a short pe- 2. Material and methods
riod of time. Moreover, in subchronic toxicity experiments,
a significant dose-dependent increase in liver weight was 2.1. Animals
observed, whereas a significant reduction in both plasma
alkaline phosphatase and cholesterol levels, as well as an All experiments were performed using male albino Swiss
increase in ␥-glutamyl transpeptidase were observed in the mice (30 ± 3.0 g) from the Central Animal House of the
DHC-treated animals. Despite the good anti-ulcerogenic ac- State University of Campinas (CEMIB/UNICAMP). The
tivity of DHC, the long-term use of this compound can in- mice were fed a standard chow (Nuvilab CR-a, Nuvital)
duce liver damage based on in vitro and in vivo evaluation and had free access to water under fixed conditions of illu-
(Rodriguez and Haun, 1997, 1999). mination (12/12 h light/dark cycle), humidity (55 ± 1.0%),
According to Melo et al. (2001, 2002), derivatives of and temperature (22 ± 1.0%). The mice were fasted before
DHC differ from DHC in their toxicity on V79 cells and all assays, because standard drugs were administered orally
hepatocytes. Therefore, the present research was undertaken (by gavage) using a 12% solution of Tween 80 (10 ml/kg) as
to determine the anti-ulcerogenic activity of three DHC vehicle. Experimental Protocols were approved by the Ethic
derivatives in different experimental models of gastric ul- Committee for Animal Research (UNICAMP), and the
P.S. Melo et al. / Journal of Ethnopharmacology 87 (2003) 169–174 171
experiments were conducted according to the recommen- of HCl/ethanol, and the extent of the gastric lesions was
dations of the Canadian Council on Animal Care (Olfert determined, expressed as a lesion index, corresponding to
et al., 1993). the sum of the lesions. The results were expressed as an
ulcerative index (UI) as described by Szeleyi and Thiemr
2.2. DHC and derivatives (1978).
DHC was obtained from Croton cajucara bark as de- 2.4.2. Hypothermic restraint-stress ulcers
scribed by Souza-Brito et al. (1998). The lactone DHC The anti-ulcerogenic activity of DHC and DHC deriva-
molecule was opened through a dimethylamine reaction to tives in this model was assessed in mice using the method of
yield compound IV, as described by Cromwell and Cook Levine (1971) with some modifications. After 24 h of star-
(1958): 0.5 g of DHC was dissolved in 2.0 ml of MeOH and vation, the animals were given an oral dose of compounds
3.0 ml of aqueous 25% solution of dimethylamine heated I, II, III or IV (100 mg/kg). One hour after treatment, ulcer-
in a sealed tube at 75 ◦ C for 72 h. The reaction mixture ation was induced by immobilizing the animals in a closed,
was evaporated to dryness, and the product purified by cylindrical cage at 4 ◦ C. After 3 h, the mice were killed by
thin-layer chromatography to yield compound IV (Fig. 1) cervical dislocation, and the stomachs removed and exam-
as a yellow oil. The reduction of the cetone group in DHC ined for ulcers as described earlier.
was done as described by Itokawa et al. (1989), using a
methanol solution of DHC (100 mg) treated with an excess 2.4.3. Indomethacin-induced ulcers
of sodium borohydride (NaBH4 , 30 mg). After work-up Mice (8/group) were randomly divided into four groups
in the usual way, the product was purified by thin-layer and fasted for 24 h. Thirty minutes after the oral adminis-
chromatography (n-hexane–EtOAc–MeOH, 7:2:1 v/v), af- tration of compounds I, II, III or IV (100 mg/kg), cimeti-
fording compound II (Fig. 1) as a white powder (Itokawa dine (100 mg/kg) or 12% Tween 80 (10 ml/kg), 30 mg/kg
et al., 1989). For compound III, DHC (300 mg) in 50 ml of of indomethacin was administered subcutaneously to the
tetrahydrofuran (THF) was added to a solution of LiAlH4 mice from each group as described by Hayden et al. (1978).
(130 mg) in THF. The mixture was refluxed by stirring for The animals were killed 4 h later, the stomachs removed
2 h to give a crude crystalline material which was sepa- and opened and the gastric lesions determined as described
rated by thin layer chromatography (EtOAc–hexane, 1:1 earlier.
v/v), yielding a white powder (Shimoma et al., 1998). The
purity and structure of compounds II–IV was character- 2.5. Statistical analysis
ized by NMR, UV, IR, and MS techniques (Melo et al.,
2002). The results were expressed as mean ± S.D. Statistical
significance was determined by one-way ANOVA followed
2.3. Drugs by Dunnet’s pairwise test, with the level of significance set
at P < 0.05.
The drugs and reagents were prepared immediately be-
fore use. The following drugs were used: cimetidine and
indomethacin from Sigma Chemical Co. (St. Louis, MO); 3. Results
Tween 80® (Synth) obtained commercially in Brazil. All
other reagents used were of analytical grade. 3.1. DHC and derivatives
2.4. Anti-ulcerogenic activity Bark of Croton cajucara were collected in our experi-
mental plantation in Benfica, near Belém, Pará, Brazil. A
The gastroprotective activity of DHC derivatives was as- voucher specimen number 247 has been deposited in the
sessed on three experimentally induced gastric ulcer models. Herbarium of Museu Paraense Emı́lio Goeldi. Powdered
plant material (20 kg dry bark) was extracted with hex-
2.4.1. HCl/ethanol-induced ulcers ane in a Soxhlet apparatus, and the crude crystals formed
The anti-ulcerogenic activity of DHC and DHC deriva- in a concentrate hexane solution were recovered after a
tives on HCl/ethanol-induced gastric ulcers was assessed in few days. A pure compound (111 g) was obtained after re-
mice as described by Mizui and Doteuchi (1983). Mice were peated crystallization from isopropanol, showing physic-
divided into groups of eight animals and fasted for 24 h prior ochemical properties in perfect accordance with reported
to receiving an oral dose of the vehicle solution (12% Tween data for the structure of trans-DHC, as depicted in Fig. 1.
80, 10 ml/kg), cimetidine (100 mg/kg), and compounds I, II, Compounds II, III, and IV (Fig. 1) were obtained at good
III, and IV (100 mg/kg). Fifty minutes later, all groups were yields (80, 70, and 30%, respectively) and were character-
dosed orally with 0.2 ml of a 0.3 M HCl/60% ethanol so- ized by standard spectroscopic methods, such as IR, UV-Vis,
lution (HCl/ethanol) to induce gastric ulcers. Animals were MS, and 1 H NMR according to the literature (Melo et al.,
killed by cervical dislocation 1 h after the administration 2002).
Table 1
Comparative effects of compounds I (DHC), II, III, and IV on the different
methods of induced gastric lesions in mice
Treatment (p.o.) Ulcer inhibition (%)
HCl/ethanol Indomethacin Stress
Control – – –
Cimetidine 49∗ 70∗ 75∗
Compound I 48∗ 43∗ 53∗
Compound II 78∗ 88∗ −14
Compound III −22 21 18
Compound IV 89∗ 90∗ 74∗
All compounds were administered at 100 mg/kg. Data are expressed as
mean ± S.D. ANOVA followed by Dunnett’s test.
∗ P < 0.01.
domethacin. Cimetidine, as well as compound I, signifi-

Fig. 2. Effects of compounds I (DHC), II, III, and IV on gastric ulcer in- cantly protected the gastric mucosa against gastric lesion in-
duced in mice by HCl/ethanol (positive control: cimetidine), hypothermic- duced by indomethacin (12 ± 5 and 26 ± 6, P < 0.05) when
stress (positive control: cimetidine), and indomethacin (positive control: compared with the control group. In this model, the activity
cimetidine). Results are expressed as mean ± S.D. Asterisks indicate
significant difference from corresponding control (ANOVA followed by
of compound IV was more potent than that of the reference
Dunnett’s test, where ∗ P < 0.01). compound, cimetidine, at the same dose. The same results
were also obtained with compound II. However, the deriva-
3.2. HCl/ethanol-induced ulcers tive III also abolished the anti-ulcerogenic activity markedly.
Compounds II and IV significantly decreased the lesions in-
The effects of the DHC and DHC derivatives in the three duced by indomethacin.
assays of gastric lesions are shown in Fig. 2. All the tested
compounds were administered orally at a dose of 100 mg/kg. 3.5. Anti-ulcerogenic activity
This dose was chosen because it is the same as our standard
drug, cimetidine, thus allowing us to compare the potencies The relative potency of DHC (compound I) and its
of the compounds. derivatives were observed in Table 1 at a fixed oral dose of
Compounds II, III, and IV obtained in this study were 100 mg/kg. This allowed direct comparison of the potency
evaluated for anti-ulcer activity in the HCl/ethanol model. of these compounds. In this table, we can observe that the
In this method, cimetidine, as well as compounds I, II, and oral administration of compound III presented less activity
IV significantly protected the gastric mucosa against HCl/ than did other derivatives (P > 0.05) on the ulceration
ethanol (15±5 mm, 13±6 mm, 9±4 mm, and 5±3 mm, P < caused by stress, ethanol or indomethacin (Fig. 2) when
0.05) when compared with the control group. Meanwhile, compared with control animals. However, in this series, the
the treatment with compound III resulted in the total disap- most active compound was IV which yielded inhibition of
pearance of the anti-ulcer activity (54 ± 5 mm, P > 0.05). ulcers induced by stress (74%), HCl/ethanol (89%), and
indomethacin (90%) at the test dose (Table 1).
3.3. Hypothermic restraint-stress ulcers
The oral administration of compound IV inhibited ul- 4. Discussion

cer formation in the hypothermic restraint-stress model
(Fig. 2). The derivative IV presented good activity which In this study, we changed synthetically the DHC moi-
was equipotent with that of cimetidine and two-folds more ety with the aim to improve the anti-ulcerogenic activity, as
active than DHC at the same dose. But at this model, well as to reduce the toxic effects of DHC. The three DHC
the anti-ulcerogenic activities of compounds II and III derivatives obtained were tested against various acute ulcer
were markedly abolished. These results indicate that re- models in animals. Research on new gastroprotective agents
ducing the cyclohexenone moiety of DHC with NaBH4 , commonly include the use of different models of experimen-
or reducing the carbonyls with LiAlH4 , resulted in loss of tally induced gastric lesions that act by different mechanism
gastroprotective activity. of ulcerogenesis. These models allow the determination of
the potential of anti-ulcer substances, and they represent the
3.4. Indomethacin-induced ulcers first approach to the possible mechanism involved in their
gastroprotective activity.
The anti-ulcerogenic effects of DHC and DHC deriva- The 60% ethanol/0.3 M HCl induces an acute ulcer model
tives were assayed against gastric lesions induced by in- that allows the evaluation of the capacity of the drug to
P.S. Melo et al. / Journal of Ethnopharmacology 87 (2003) 169–174 173
protect the gastric mucosa. Ethanol induces the formation in the lactone, the molecule retained its anti-ulcerogenic
of gastric ulcers by a direct action on the mucosa, with little activity because of the presence of a Michael acceptor sub-
involvement of gastric acid in the formation of this lesion; stitute [(C(O)N(CH3 )2 )]. In contrast, compound III lost its
the presence of HCl only accelerates the process. More- anti-ulcerogenic activity due to the absence of a Michael
over, ethanol induces solubilization of mucus constituents acceptor.
in the stomach with a concomitant decrease in the trans- In recent report about the cytotoxic effects of these com-
mucosal potential difference, and increases the Na+ and pounds, it was demonstrated that compounds II, III, and IV
K+ flux into the lumen, while enhancing pepsin secretion, differed in toxicity from DHC (Melo et al., 2001, 2002).
the loss of H+ ions, and the histamine content in the lu- The data obtained with rat cultured hepatocytes further indi-
men (Szabo, 1987). The results obtained in this work in the cated that compound II, a derivative with a lactone moiety,
HCl/ethanol model suggest that compounds II and IV may produced similar toxicological manifestations to those seen
enhance gastric-mucosal defensive factors, such as mucus with DHC. However, compound IV showed lower hepato-
and prostaglandin production. toxic effect than DHC and compound II, when evaluated
The cytoprotective activities of the compounds were also in rat’s cultured hepatocytes (Melo et al., 2002). A balance
examined on the hypothermic restraint-stress model. This between the therapeutic versus toxicological effects of a
assay induces the formation of gastric ulcer through distur- compound is an important parameter, when verifying its
bances in the gastric-mucosal microcirculation, alteration in applicability as a pharmacological drug. Cell culture can be
gastric secretion, abnormal motility, and gastric-mucus de- used to evaluate basal cytotoxicity and in some cases, may
pletion (Koo et al., 1986). However, the most important fac- provide information on the lethal dose in vivo. According
tor in the genesis of stress ulcers is an increase in gastric to recent results, compound IV was less toxic in the cell
acid secretion, often termed the aggressive factor (Goa and models used (Melo et al., 2001, 2002) than the other com-
Monk, 1987). Compound IV was the most effective to pro- pounds; so it is a potential compound to be investigated in
tect the gastric mucosa in this induced-ulcer model. relation to its pharmacological potential, since it was also
Anti-inflammatory agents, such as indomethacin, reduce the better anti-ulcer derivative.
gastric cyclooxygenase activity and decrease endogenous In conclusion, compounds II and IV had significant
prostaglandin levels. There is increasing evidence that an anti-ulcer activity following oral administration. This effect
elevation of certain endogenous prostaglandins can en- could be related to an increase of gastric-mucosal defensive
hance gastric-mucosal resistance to ulcerogenic agents, mechanisms. The presence of the lactone ring or a Michael
such as NSAID (Vane and Botting, 1995), and that a rise acceptor substitute is probably essential for the anti-ulcer
in prostaglandin levels significantly increases blood flow in activity of these compounds. Further experiments are cur-
the stomach (Droy-Lefaix, 1988). rently underway to determine the anti-ulcer mechanisms of
Compounds II and IV significantly decreased the lesions these DHC derivatives and to assess their structure–activity
induced by indomethacin (88 and 90%, respectively). These relationships.
results also suggest a probable involvement of compound
II and, primarily, compound IV in prostaglandin synthesis
and/or release. Hiruma-Lima et al. (1999) also suggested that Acknowledgements
the excellent preventive effect of DHC on gastric ulcers oc-
curred mainly by the suppression of acid secretion through This work was supported by the Brazilian foundations
non-competitive antagonism with receptors involved in gas- CAPES, FAPESP and PADCT-FINEP. The authors thank
tric acid secretion, and by protecting the gastric mucosa Nelson A. Rosa by identification of the voucher specimen
through an increase in PGE2 levels. used in the study.
According to Giordano et al. (1992), structure–activity
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Effect of Holotrichia diomphalia larvae on liver fibrosis

and hepatotoxicity in rats
Woo-Yong Oh a , Suhkneung Pyo a , Kang-Ro Lee a , Bum-Koo Lee a ,
Dae-Hee Shin b , Sung Ig Cho c , Sun-Mee Lee a,∗
a College of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Changan-gu, Suwon 440-746, Kyunggi-do, South Korea
b Institute of Complementary Medicine, Conmaul Oriental Hospital & Medical Clinic, Seoul 137-881, South Korea
c Department of Pharmacology, Ajou University School of Medicine, Suwon 442-749, South Korea
Received 30 April 2002; received in revised form 28 March 2003; accepted 28 March 2003
Abstract
Holotrichia diomphalia larvae, one of the most widely used Korean folk medicinal preparations, have long been used for the treatment of
chronic liver cirrhosis. The present study was undertaken to clarify whether extract of Holotrichia diomphalia larvae could prevent acute liver
damage and liver fibrosis in rats. A single administration of Holotrichia diomphalia protected rats from acute liver damage induced by carbon
tetrachloride (200 ␮l/kg, i.p.) and ␤-d-galactosamine (600 mg/kg, i.p.). This was evidenced by the lowered serum aminotransferase (ALT,
AST) activities in rats treated with Holotrichia diomphalia. The hepatic cirrhosis was induced by 28 days of bile duct ligation/scission in
rats. The four-week treatment with Holotrichia diomphalia reduced the serum ALT, AST, alkaline phosphatase activities, and hydroxyproline
content in the liver and improved the histological appearance of the liver sections. The present results led us to conclude that Holotrichia
diomphalia larvae can reduce the degree of hepatocellular damage and may become a promising antifibrotic agent for liver fibrosis/cirrhosis.
Keywords: Holotrichia diomphalia larvae; Acute hepatotoxicity; Liver cirrhosis
1. Introduction et al., 1994). Prophenoloxidase from the hemolymph of

Holotrichia diomphalia larvae has also been purified and
Recently, the number of cases of liver disease has tended characterized (Kwon et al., 1997). Furthermore, our own re-
to increase year by year, and more than 15,000 deaths from cent studies have shown that Holotrichia diomphalia larvae
this disease occur annually in Korea. Hepatic fibrosis is an augment macrophage function and stimulate the synthesis
important feature of, and a common sequel to, most forms and release of cytotoxic mediators (Kang et al., 2002). How-
of chronic liver disease and is an essential component in the ever, the hepatoprotective effects of Holotrichia diomphalia
development of cirrhosis (Friedman, 1993). Therefore, the larvae have not been investigated.
prevention or suppression of fibrotic changes in the liver or The present study was undertaken to investigate the hep-
protection from and treatment of cirrhosis is important. At atoprotective effect of Holotrichia diomphalia larvae against
present, there is no effective therapy to cure cirrhosis or to acute liver injury and liver fibrosis.
prevent its complications. Furthermore, there are no drugs
to suppress fibrosis, therefore, it is important to prevent the
development of hepatic fibrosis. 2. Materials and methods
Holotrichia diomphalia (Coleoptera, Scarabaeidae) lar-
vae (Scarab beetle) have been traditionally used in Korea 2.1. Animals and treatments
for the treatment of liver cirrhosis, contusion, edema, fu-
runcle and apoplexy. Recently, potent antibacterial proteins Male, Sprague–Dawley rats weighing 240 ± 20 g were
have been isolated from Holotrichia diomphalia larvae (Lee obtained from Jeil Animal Breeding Company of Ko-
rea and were acclimated to the laboratory conditions at
Sungkyunkwan University for at least one week. During
∗ Corresponding author. Tel.: +82-31-290-7712; fax: +82-31-292-8800. this period, food (Samyang Co., Korea) and tap water were
E-mail address: sunmee@yurim.skku.ac.kr (S.-M. Lee). supplied ad libitum. The experimental animals were kept in
doi:10.1016/S0378-8741(03)00140-5
176 W.-Y. Oh et al. / Journal of Ethnopharmacology 87 (2003) 175–180
a temperature- and humidity-controlled room (25 ± 1 ◦ C, p.o.) and Holotrichia diomphalia (100, 300 mg/10 ml/kg,
55 ± 5%, respectively) with a light–dark cycle of 12 h, and p.o.). After 48 h ␤-d-galactosamine administration, blood
were fasted 18 h before the experiment. was taken from the abdominal aorta for the assay of serum
aminotransferase activity.
2.2. Chemicals
2.5. Liver cirrhosis
Carbon tetrachloride, ␤-d-galactosamine hydrochloride,
silymarin, p-dimethylaminobenzaldehyde, and p-toluene- Secondary biliary cirrhosis was induced by double lig-
sulfon-chloramide (chloramine T) were supplied by the ation and division of the common bile duct (Kountouras
Sigma Chemical Co., USA. All the other chemicals used in et al., 1984; Gross et al., 1987). Under ether anesthesia,
this study were reagent grades and are locally and commer- a midline incision was made to the abdomen, the com-
cially available. mon bile duct was isolated, and the proximal bile duct
was held with two silk sutures and then dissected between
2.3. Preparation of crude extracts the double ligations. In sham-operated group, the bowel
and mesentery were manipulated and replaced. After op-
Holotrichia diomphalia larvae (1 kg) were purchased eration, saline (10 ml/kg), silymarin (12.5 mg/10 ml/kg) or
at the herbal drug market in Cheju-Do, Korea, in August Holotrichia diomphalia (6.25, 12.5 mg/10 ml/kg) were fed
1999 and identified by Dr. B.G. Lee of the Institute for orally to each group of rats once a day for four weeks.
Traditional Medicine, Sungkyunkwan University, Suwon, At four weeks, blood was taken from the abdominal aorta
Korea. A voucher specimen (SKK-H001) is deposited in for the assay of serum aminotransferases and alkaline phos-
the College of Pharmacy at Sungkyunkwan University. phatase activities. The medium and left lobes of the liver
Air-dried and chopped Holotrichia diomphalia larvae (1 kg) were removed and used for histology and hydroxyproline
were refluxed with 70% ethanol (2 l) two times for 8 h. measurements.
The materials were filtered, and the clear supernatant was
then concentrated under reduced pressure at 40 ◦ C with a 2.6. Assay of serum ALT, AST and ALT activities and
vacuum rotary evaporator. The concentrated ethanol extract hydroxyproline content
(100 g) was partitioned between water (1 l) and n-hexane
(0.5 l × 2, 20 g). After removing the n-hexane fraction, the The serum alanine aminotransferase (ALT), aspartate
aqueous layer was partitioned again with methylene chlo- aminotransferase (AST), alkaline phosphatase (ALP) activ-
ride (0.5 l × 2, 10 g), followed by n-butanol (0.5 l × 2, 40 g). ities were determined by spectrophotometric procedures,
The extract was evaporated and the residue was used for the using Sigma Kits (Sigma Chemical Co., St. Louis, MO,
experiment. USA). The hydroxyproline (HOPro) contents in the liver
were measured according to the method of Jamall et al.
2.4. Acute hepatotoxicity (1981). Briefly, the liver tissue was homogenized in 6N hy-
drochloride (HCl) and then hydrolyzed at 110 ◦ C for 18 h.
Five groups of animals were studied. All animals were After cooling, chloramine T was added to the hydrolysate.
treated humanely under Sungkyunkwan University Ani- After 5 min, p-dimethylaminobenzaldehyde was added and
mal Care Committee guidelines. The rats in group I (ve- the mixture was incubated for 30 min at 60 ◦ C. The sam-
hicle) received only olive oil (2 ml/kg, i.p.). In groups ples were read at 560 nm against a reagent blank, which
II–V, carbon tetrachloride (CCl4 ) was dissolved in olive contained the complete system without added tissue, using
oil (1:9) (v/v), then intraperitoneally administered (final a spectrophotometer (Milton Roy Spectonic 1201, USA).
concentration: 200 ␮l/kg). Four hours after the CCl4 treat-
ment, groups I (vehicle) and II (control) were treated with 2.7. Histology
saline (10 ml/kg, p.o.), and groups III–V were treated with
silymarin (positive control, 300 mg/10 ml/kg, p.o.) and A small piece of liver tissue from the anterior portion
Holotrichia diomphalia (100, 300 mg/10 ml/kg, p.o.). Fol- of the left lateral lobe was taken for light microscopy.
lowing 24 h CCl4 administration, blood was taken from the Paraffin blocks were prepared after fixation in 10% neu-
abdominal aorta for the assay of serum aminotransferase tral formalin and stained with hematoxylin and eosin. The
activity. degree of necrosis and fibrosis was assessed according
In order to induce hepatitis, five other groups of rats to Frei et al. (1984). The severity of liver alteration was
were given an intraperitoneal injection of 600 mg/kg of semi-quantitatively graduated on a scale of 0 to IV (0:
␤-d-galactosamine dissolved in saline. Group I (vehicle) absent; I: minimal; II: mild; III: modest; IV: severe), and
was given an intraperitoneal injection of saline (1 ml/kg). separate scores were obtained for each of the following:
Four hours after the ␤-d-galactosamine treatment, group cell necrosis, inflammatory cell infiltration, fibrosis and
II (control) was treated with saline (10 ml/kg, p.o.), and steatosis. The bile duct proliferation was rated as present or
groups III–V were treated with silymarin (300 mg/10 ml/kg, absent.
W.-Y. Oh et al. / Journal of Ethnopharmacology 87 (2003) 175–180 177
2.8. Statistical analysis Table 1

Effect of Holotrichia diomphalia on serum aminotransferase activities in
carbon tetrachloride-induced acute hepatic injury
All results were expressed as means ± S.E.M. The un-
paired Student’s t-test was used to analyze the difference Group Dose (mg/kg) ALT (U/l) AST (U/l)
between groups. Values of P < 0.05 were considered to be Vehicle 46 ± 4 74 ± 6
significant.
CCl4
Control 871 ± 71∗∗ 1310 ± 146∗∗
Holotrichia 100 402 ± 156+ 577 ± 45∗∗,++
3. Results diomphalia
300 187 ± 31∗,++ 410 ± 51∗∗,++
Silymarin 300 286 ± 64∗,++ 778 ± 144∗∗,+
3.1. Acute hepatotoxicity
Each value is the mean ± S.E.M. for six to ten rats per group.
The serum levels of ALT and AST in the vehicle-treated (∗, ∗∗) significantly different (P < 0.05, P < 0.01) from vehicle-treated
group.
rats were 46 ± 4 U/l and 74 ± 6 U/l, respectively, which (+, ++) significantly different (P < 0.05, P < 0.01) from control group.
were similar to those of the normal rats. In the CCl4 -treated
control group, the ALT and AST increased to 871 ± 71 U/l
and 1310 ± 146 U/l, respectively. These higher levels
were markedly suppressed in a dose-dependent manner
Table 2
by Holotrichia diomphalia. The increase in ALT and Effect of Holotrichia diomphalia on serum aminotransferase activities in
AST was also attenuated by silymarin (Table 1). In the ␤-d-galactosamine-induced hepatitis
galactosamine-treated control group, the ALT and AST in- Group Dose (mg/kg) ALT (U/l) AST (U/l)
creased to 1638 ± 282 U/l and 2342 ± 408 U/l, respectively.
Holotrichia diomphalia and silymarin treatments prevented Vehicle 46 ± 4 74 ± 6
the elevations of serum aminotransferases. The hepatopro- ␤-d-Galactosamine
tective effect of Holotrichia diomphalia was stronger than Control 1638 ± 282∗∗ 2342 ± 408∗∗
Holotrichia 100 704 ± 82∗∗,+ 694 ± 204∗,+
that of silymarin (Table 2).
diomphalia
300 368 ± 99∗,++ 552 ± 132∗,++
3.2. Liver cirrhosis Silymarin 300 615 ± 75∗∗,+ 1266 ± 124∗∗
Each value is the mean ± S.E.M. for six to ten rats per group.
The liver of the sham-operated rats was normal. His- (∗, ∗∗) significantly different (P < 0.05, P < 0.01) from vehicle-treated
tology of the bile duct ligation/scission (BDL/S) rat liver group.
showed excessive bile duct proliferation, inflammation and (+, ++) significantly different (P < 0.05, P < 0.01) from control group.
Table 3
Quantitative summary of histological observation on Holotrichia diomphalia protection of BDL/S-induced liver cirrhosis
Histological changes (%) Sham BDL/S
Control Holotrichia diomphalia Silymarin
6.25 mg/kg 12.5 mg/kg
Necrosis
No change 100 0 0 0 0
Grades I–II 0 20 60 70 40
Grades III–IV 0 80 40 30 60
Inflammatory infiltration
No change 100 0 0 0 0
Grades I–II 0 30 60 70 50
Fibrosis
No change 100 0 0 0 0
Grades I–II 0 20 50 60 40
Bile duct proliferation Absent Present Present Present Present
Scores are the numerical values of individual 10 rats per group. Necrosis and fibrosis were assessed according to Frei et al. (1984). Inflammatory
infiltration grading was made according to five severity grades (0: absent, I: minimal, II: mild, III: modest and IV: severe). Bile duct proliferation was
rated as present or absent.
Table 4
Serum biochemical values in rats with cirrhosis induced by BDL/S treated with Holotrichia diomphalia
Group Dose (mg/kg) ALT (U/l) AST (U/l) ALP (U/l)
Sham 24 ± 4 177 ± 39 157 ± 10

BDL
Control 308 ± 11∗∗ 665 ± 52∗∗ 929 ± 31∗∗
Holotrichia diomphalia 6.25 141 ± 26∗∗,++ 318 ± 55++ 576 ± 87∗∗,++
12.5 126 ± 11∗∗,++ 356 ± 65∗,++ 670 ± 20∗∗,++
Silymarin 12.5 241 ± 33∗∗ 592 ± 35∗∗ 750 ± 39∗∗,++
Each value is the mean ± S.E.M. for seven to ten rats per group.
(∗, ∗∗) significantly different (P < 0.05, P < 0.01) from sham-operated group.
(++) significantly different (P < 0.01) from control group.
Fig. 1. Hydroxyproline content of liver from cirrhotic rats with BDL/S treated with Holotrichia diomphalia. (∗∗) significantly different (P < 0.01) from
sham-operated group. (++) significantly different (P < 0.01) from control group. Values are means ± S.E.M. for seven to ten rats per group.
connective tissue deposition resulting in destruction of the 4. Discussion and conclusions

lobular architecture. In the Holotrichia diomphalia-treated
rats, there was a tendency towards less pronounced de- In the present study, the hepatoprotective effect of
struction of the liver architecture. In the silymarin-treated Holotrichia diomphalia larvae in three experimental liver
BDL/S rats, the liver histology was similar to that of the injury models was investigated. The sequence of events
BDL/S rats (Table 3). The serum biochemical parameters after CCl4 administration has been well characterized by
of the BDL/S rats are shown in Table 4. The levels of many investigators (Slater, 1966; Recknagel, 1967). After
serum ALT, AST and ALP were significantly elevated in the administering CCl4 , the histological changes of the liver in-
control BDL/S rats. In the Holotrichia diomphalia-treated clude ballooning degeneration, fatty metamorphosis in the
rats, the serum ALT, AST and ALP levels were signif- adjacent hepatocyte, cell necrosis, cell inflammation and
icantly lower relative to the control BDL/S rats. In the the infiltration of lymphocytes and Kupffer cells around the
BDL/S rats treated with silymarin, the serum level of central vein.
ALP was significantly reduced, and there were no signifi- The mechanism of CCl4 -induced liver damage is con-
cant changes in the serum ALT and AST levels compared sidered to be due to the enzymatic activation (cytochrome
with those of the control BDL/S rats. BDL/S increased P450) of CCl4 into the trichloromethyl free radical (• CCl3 )
the hydroxyproline content about three-fold. Compared within the membrane of the endoplasmic reticulum. This is
with the control BDL/S group, treatments with Holotrichia followed by chloromethylation, saturation, peroxidation and
diomphalia and silymarin reduced the hydroxyproline progressive destruction of the unsaturated fatty acid of the
content in the liver by up to 64 and 68%, respectively endoplasmic reticulum membrane phospholipids (Reynolds
(Fig. 1). and Moslen, 1979). These processes are known as lipid
W.-Y. Oh et al. / Journal of Ethnopharmacology 87 (2003) 175–180 179
peroxidation, leading to its functional and structural dis- ALT, AST, ALP and also lowered the hydroxyproline in the
ruption (Recknagel, 1983). In the CCl4 -treated control liver, and improved its morphology.
of the present study, the serum aminotransferases levels In conclusion, treatment using Holotrichia diomphalia
were elevated significantly. The hepatoprotective effects prevents hepatocellular damage and improves liver fibrosis.
against CCl4 -induced hepatic injury were clearly demon- Our results suggest that Holotrichia diomphalia could be a
strated in the rats treated with Holotrichia diomphalia. promising antifibrotic agent. Further study is needed to fully
This effect may be attributable to the inhibition of cy- understand the mechanism by which Holotrichia diomphalia
tochrome P450 activity as well as the prevention of lipid larvae inhibit collagen deposition and to establish its clinical
peroxidation. applicability.
␤-d-Galactosamine-induced acute liver injury was con-
sidered in an experimental model of hepatitis. Decker and
Keppler (1974) have shown that the metabolite of ␤-d- Acknowledgements
galactosamine, uridindiphosphogalactosamine (Rasenack
et al., 1980), may deplete several uracil nucleotides in- This work was supported by research grant (99-B-
cluding UDP-galactose, UDP-glucose and UTP, impairing 5-2) from the Kyunggi Pharmaceutical Research Center,
mRNA and glycoprotein synthesis and altering the compo- Korea.
sition of the cellular membranes. These phenomena may
lead to cellular damage and cellular inflammation resulting
in a histological and biochemical picture closely resembling
viral hepatitis (Lin et al., 1996). References
The results presented here show a significant increase in
ALT and AST activities after administration of ␤-d-galac- Decker, K., Keppler, D., 1974. Galactosamine hepatitis: key role of the
nucleotide deficiency period in the pathogenesis of cell injury and cell
tosamine. In contrast to the control rats, the elevated serum death. Reviews of Physiology, Biochemistry and Pharmacology 71,
aminotransferase levels were reduced by treatment with 77–106.
Holotrichia diomphalia. Furthermore, the hepatoprotec- Frei, A., Zimmermann, A., Weigand, K., 1984. The N-terminal propeptide
tive effect of Holotrichia diomphalia seemed to be higher of collagen type III in serum reflects activity and degree of fibrosis in
than that of silymarin, which has been used as a potent patients with chronic liver disease. Hepatology 4, 830–834.
Friedman, S.L., 1993. Seminars in medicine of the Beth Israel Hospital
hepatoprotective agent. The present results of the CCl4 - Boston. The cellular basis of hepatic fibrosis. Mechanisms and treat-
and ␤-d-galactosamine-induced liver injuries suggest that ment strategies. The New England Journal of Medicine 328, 1828–
Holotrichia diomphalia may have potential clinical appli- 1835.
cation for treating liver diseases. Gross Jr., J.B., Reichen, J., Zeltner, T.B., Zimmermann, A., 1987. The
Therapy for hepatic fibrosis should affect the production evolution of changes in quantitative liver function tests in a rat model
of biliary cirrhosis: correlation with morphometric measurement of
of hepatic connective tissue proteins in particular (Schuppan, hepatocyte mass. Hepatology 7, 457–463.
1991). The best therapeutic strategies can be designed only Jamall, I.S., Finelli, V.N., Que, S.S., 1981. A simple method to determine
with a full understanding of the mechanism involved in fi- nanogram levels of 4-hydroxyproline in biological tissues. Analytical
brogenesis, which has yet to be completed. Nonetheless, any Biochemistry 112, 70–75.
intervention that blocks collagen deposition will probably Kang, N.S., Park, S.Y., Lee, K.-R., Lee, S.-M., Lee, B.G., Shin, D.-H.,
Pyo, S., 2002. Modulation of macrophage function activity by ethano-
be effective in reducing hepatic fibrosis, regardless of the
lic extract of larvae of Holotrichia diomphalia. Journal of Ethnophar-
mechanisms involved. macology 79, 89–94.
In this study, we used BDL/S cirrhotic rats. Cirrhosis in- Kountouras, J., Billing, B.H., Scheuer, P.J., 1984. Prolonged bile duct
duced by BDL/S showed a slight decrease in body weight obstruction: a new experimental model for cirrhosis in the rat. British
due to the operation during the first week, and then re- Journal of Experimental Pathology 65, 305–311.
Kwon, T.H., Lee, S.Y., Lee, J.H., Choi, J.S., Kawabata, S., Iwanaga, S.,
turned to normal weight afterwards. Between the control
Lee, B.L., 1997. Purification and characterization of prophenoloxidase
BDL/S rats and Holotrichia diomphalia-treated BDL/S rats, from the hemolymph of coleopteran insect, Holotrichia diomphalia
there was no significant difference in body weight. In the larvae. Molecules and Cells 7, 90–97.
BDL/S rats, the liver weight increased markedly 28 days Lee, S.Y., Moon, H.J., Kurata, S., Kurama, T., Natori, S., Lee, B.L.,
after biliary obstruction. The liver-to-body weight ratio of 1994. Purification and molecular cloning of cDNA for an inducible
antibacterial protein of larvae of a coleopteran insect, Holotrichia
the Holotrichia diomphalia-treated BDL/S rats was slightly
diomphalia. Journal of Biochemistry (Tokyo) 115, 82–86.
lower than that of the control BDL/S rats, but the difference Lin, S.C., Lin, C.C., Lu, F.J., Lin, Y.H., Chen, C.H., 1996. Protective
was not significant (data not shown). and therapeutic effects of huanglian-jie-du-tang on hepatotoxin-induced
We were able to show that this procedure resulted in liver injuries. American Journal of Chinese Medicine 24, 219–
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Rasenack, J., Koch, H.K., Nowack, J., Lesch, R., Decker, K., 1980.
erately increased serum aminotransferases and alkaline
Hepatotoxicity of d-galactosamine in the isolated perfused rat liver.
phosphatase. Furthermore, a significant increase in the hy- Experimental and Molecular Pathology 32, 264–275.
droxyproline content in the liver was observed. Treatment Recknagel, R.O., 1967. Carbon tetrachloride hepatotoxicity. Pharmaco-
with Holotrichia diomphalia reduced the serum levels of logical Reviews 19, 145–208.
Recknagel, R.O., 1983. A new direction in the study of carbon tetrachlo- Schuppan, D., 1991. Connective tissue polypeptides in serum as param-
ride hepatotoxicity. Life Sciences 33, 401–408. eters to monitor antifibrotic treatment in hepatic fibrogenesis. Journal
Reynolds, E.S., Moslen, M.T., 1979. Environmental liver injury: halo- of Hepatology 13, 17–25.
genated hydrocarbons. In: Farber, E., Fisher, M. (Eds.), Toxic Injury Slater, T.F., 1966. Necrogenic action of carbon tetrachloride in the rat: a
of the Liver. Marcel Dekker Inc., New York and Basel. speculative mechanism based on activation. Nature 209, 36–40.
Evaluation of immunomodulatory activity of Ipomoea carnea

on peritoneal cells of rats
I.M. Hueza a , E.S.M. Fonseca a , C.A. Paulino b , M. Haraguchi c , S.L. Górniak a,∗
a Research Centre for Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine,
University of São Paulo, SP, CEP 05508-900, Brazil
b University Bandeirante of São Paulo, SP, CEP 02071-013, Brazil
c Biological Institute of São Paulo, SP, CEP 04014-002, Brazil
Received 31 July 2002; received in revised form 28 March 2003; accepted 28 March 2003
Abstract
In the present study, animals of the experimental groups were treated with an aqueous fraction (AF) of Ipomoea carnea diluted in drinking wa-
ter in order to obtain daily doses of 3 g dry leaves/kg/body weight (bw) and 15 g/kg/bw for 14 and 21 days, or by gavage 15 g/kg/bw administered
for 14 days, respectively. Peritoneal macrophages were collected and submitted to the spreading, phagocytosis, and hydrogen peroxide release
tests. AF administration in drinking water for 14 and 21 days promoted increased macrophage phagocytosis activity and hydrogen peroxide
release. However, the administration of 15 g/kg/bw of AF by gavage for 14 days resulted in no alteration in macrophage activity. These results
suggest that low dosages of Ipomoea carnea induced enhanced phagocytosis activity and hydrogen peroxide production by macrophages.
Keywords: Ipomoea carnea; Swainsonine; Macrophages; Immunomodulation
1. Introduction species known as locoweed, belonging to the genera As-

tragalus and Oxytropis (North America; van Kampen and
Ipomoea carnea Jacq. spp. fistulosa (Choisy) D.F. Austin James, 1969). The SW mechanism of intoxication has been
(Convolvulaceae) is a toxic plant found throughout Brazil extensively investigated and has been found to be due to
and other tropical countries (Austin and Huaman, 1996). the potent inhibition of the lysosomal ␣-mannosidase which
Natural intoxication occurs when different animal species, causes a similar genetic disease, ␣-mannosidosis, described
such as cattle, sheep, and goats, chronically ingest the plant principally in Angus cattle and felines, but rarely in humans
(Hoehne, 1939). Such intoxication is clinically characterized (Jolly and Walkley, 1997). The effect of the acquired lyso-
by inappetence, soft feces, and weight loss, and by central somal ␣-mannosidase inhibition, a characteristic lysosomal
nervous system (CNS) signs, such as head shaking, hyper- storage disease, showing lysosomal accumulation of incom-
esthesia, and death (De Balogh et al., 1999). pletely processed oligosaccharides, loss of cellular function,
The toxic principles of the plant have been recently iden- and ultimately cell death (Tulsiani et al., 1988) has been ob-
tified as two nortropane alkaloids (De Balogh et al., 1999), served in different organs, including thyroid, liver, kidneys,
calystegines B2 and C1, and indolizidine alkaloid, swainso- and mainly CNS (Stegelmeier et al., 1998; De Balogh et al.,
nine (SW). The first two are powerful glycosidase inhibitors 1999).
affecting ␣-glucosidase and ␣- and ␤-galactosidases, lead- SW also inhibits Golgi mannosidase II, which is involved
ing to the production of phenocopies of the human genetic in the N-linked glycoprotein metabolism (Elbein, 1989). The
lysosomal storage defects, Gaucher’s and Fabry’s disease, resulting alteration in the synthesis, processing, and trans-
respectively (Dorling, 1984). The latter alkaloid has also port of glycoproteins causes impairment and dysfunction of
been isolated from other Leguminosae plants, such as Swain- cell adhesion molecules, circulating hormones, and various
sona canescens (Australia; Dorling et al., 1978) and certain membrane receptors (Stegelmeier et al., 1998). This effect
is seen clinically as abnormal embryogenesis (Nelson et al.,
∗ Corresponding author. Tel.: +55-11-30917693; 1980), abnormal endocrine (Stegelmeier et al., 1995) and
fax: +55-11-30917829. gastrointestinal functions (Pan et al., 1993), and alteration
E-mail address: gorniak@usp.br (S.L. Górniak). of the immune system (Karasuno et al., 1992).
doi:10.1016/S0378-8741(03)00138-7
182 I.M. Hueza et al. / Journal of Ethnopharmacology 87 (2003) 181–186
The modification resulting from glycoprotein metabolism 2.3. Animals and experimental design
has received much attention because of its potential im-
munomodulatory properties. Studies involving field ob- Fifty-one female Wistar rats aged about 60 days (150–
servations reported that SW causes immunodeficiency 200 g), inbred in the Departament of Pathology, School of
in animals exposed to plants containing this compound Veterinary Medicine, were used. The animals were housed
(Stegelmeier et al., 1998). Animals also became susceptible in temperature-controlled (24–26 ◦ C) and artificially lighted
to the occurrence of pneumonia, foot rot, or “pink eye” rooms on a 12-h light/12-h dark cycle (lights on at 07:00 h)
(Sharma et al., 1984). Experimental studies conducted in with free access to food and water, and used in accordance
vitro, however, showed that SW has potential immunomod- with the guidelines of the National Research Council, USA.
ulatory properties, such as increasing murine natural-killer The animals were divided at random into eight groups:
(NK) activity (Humphries et al., 1988), enhancing the gen- five experimental groups (DW14A, DW14B, DW21A,
eration of lymphokine-activated killer (LAK) cell activity DW21B, and G) and three control groups (CDW14, CDW21,
(Bowlin et al., 1989), increasing human large granular lym- and CG). Animals from the DW groups were treated with
phocytes citotoxicity against NK-resident colon carcinoma AF diluted in drinking water in order to obtain the target
cells (Yagita and Saksela, 1990), and activation of resident doses of 3 or 15 g/kg/day of Ipomoea carnea dry leaves.
tissue macrophages (Das et al., 1995). Rats from the DW14A and DW21A groups were to receive
The purpose of the present study was to evaluate in vivo 3 g/kg/day of the plant leaves for 14 or 21 days, respec-
the effect of Ipomoea carnea on macrophage activity in rats, tively, and those from the DW14B and DW21B groups
specifically, macrophage spreading, phagocytosis, and hy- were to ingest 15 g/kg/day of the plant leaves for 14 or 21
drogen peroxide (H2 O2 ) production by peritoneal cells after days, respectively. Animals from the G group were dosed
lipopolysaccharide (LPS) activation. by gavage for 14 consecutive days with 15 g/kg of Ipomoea
carnea leaves. Control groups received only tap water for
14 days (CDW14) or 21 days (CDW21). CG rats received
2. Material and methods tap water by gavage for 14 days.
Every day the amount of AF administered in the drink-
2.1. Plant material ing water was adjusted to the body weight and to the wa-
ter consumption, and a fresh solution of AF was provided.
Leaf sample of Ipomoea carnea was collected from the Twenty-four hours before euthanasia, the rats were given
cultivated plants at the Research Centre for Veterinary Toxi- an intraperitoneal (i.p.) injection of LPS (1 mg/kg) in or-
cology (CEPTOX), University of São Paulo (USP), Pirassu- der to induce peritoneal inflammatory cell activation. The
nunga, Brazil, in May 2000. A voucher herbarium specimen macrophage activity was evaluated using the protocols pre-
was deposited in the Botanical Institute of São Paulo (SP), viously described by Rabinovitch and DeStefano (1973a,b).
Brazil, under number SP-360911. Taxonomic identification
was performed by Dr. Rosangela Simão Bianchini, Botani- 2.4. Evaluation of spreading and phagocytosis of
cal Institute of São Paulo. Dry leaf sample (800 g) was mac- peritoneal macrophages
erated in 96% ethanol (3 l). After total solvent evaporation
under reduced pressure at 50 ◦ C, a dark green extract was Peritoneal exudate cells were obtained by lavage of the
obtained (140 g, 17.5% w/w), which was suspended in wa- peritoneal cavity 24 h after injection of LPS. The cells
ter to remove the waxy residue (60.2 g, 7.5% w/w) and con- were centrifuged and resuspended in PBS and adjusted to
secutively fractionated with n-butanol saturated with water 2 × 106 cells/ml. To study macrophage spreading, 200 ml
(25.2 g, 3.2% w/w). The remaining aqueous solution was of each cell suspension was prepared in duplicate on glass
lyophilized to give an aqueous fraction (AF), which by pre- slide monolayer that was kept in multiwell (6 wells) tis-
vious assay, revealed the presence of the active principles. sue culture plate (Corning Costar). Within 20 min, the
wells were washed several times with cold PBS (4 ◦ C);
2.2. Reagents RPMI-1640-supplemented medium was added to each well.
The culture plates were incubated for 60 min at 37 ◦ C in a
Trypan Blue Stain, RPMI-1640 culture medium supple- humidified atmosphere of 5% CO2 . After incubation, the
mented with 10 mM HEPES, 11 mM sodium bicarbonate, wells were rinsed with cold (4 ◦ C) PBS and the adherent
2 mM l-glutamine, and 10% fetal bovine serum (FBS) were cells fixed with 0.5% glutaraldehyde for 10 min. These
used to maintain the cell cultures; these reagents were pur- cells were then counted with a phase contrast microscope
chased from Gibco. Zymosan A, LPS 0127:B8 100 mg, and (Nikon, Inc.) at 40× magnification. Using an ocular grid,
phenol red (0.5%) were purchased from Sigma Chemical 200 macrophages were scored as either round or spread.
Co. Glutaraldehyde-25% was purchased from Nuclear, São An index of macrophage spreading (SI) was then calculated
Paulo. Phorbol myristate acetate (PMA) was purchased from for each monolayer of each well: SI = number of spread-
ICN and hydrogen peroxide was purchased from Merck, São ing macrophages × 100/200 adherent cells counted; hence,
Paulo. SI = percent of spreading macrophages.
I.M. Hueza et al. / Journal of Ethnopharmacology 87 (2003) 181–186 183
Macrophage phagocytosis was performed using the same 2.6. Statistical analysis
method as described above. One milligram of zymosan A
solution (5.0 mg/ml) was added to each well 1 h before the Student’s t-test was used to compare two groups and
60-min incubation period (37 ◦ C). Using the same micro- ANOVA followed by Dunnet’s post hoc test was used for
scope, and the same objective and methods described above, more than two groups, with the level of significance set at
an index of phagocytosis (PI) was then obtained: PI = P < 0.05. Data are expressed as mean ± S.D.
number of macrophages with phagocytic activity × 100/200
adherent cells counted; hence, PI = percent of macrophages
with zymosan particles phagocytized. 3. Results
The mean of four counts obtained from the two slides of
each rat was used to express the SI or PI index. 3.1. Daily consumption of AF and weight gain
2.5. Hydrogen peroxide release The actual dose of AF received from DW14A and DW21A
was near the target dose (3 g/kg/day); however, animals from
Spontaneous and PMA-induced H2 O2 release from the DW14B and DW21B groups showed a dramatic depres-
macrophages was measured by a method described pre- sion of water consumption resulting in a much lower inges-
viously (Russo et al., 1989). Briefly, the peritoneal cells tion of the plant extract than expected (P < 0.05). Exposure
adjusted to 2 × 106 cell/ml were centrifuged for 10 min and to the higher concentration of Ipomoea carnea in drinking
resuspended in 1 ml of phenol red solution (PRS, contain- water or Ipomoea carnea administered by gavage reduced
ing 140 mM NaCl, 10 mM potassium-phosphate buffer, pH the food consumption of the experimental rats as compared
7.0, 0.5 mM dextrose, 0.28 mM phenol red, and 8.5 U/ml to their respective controls. In addition, body weight gain
HRPO) for H2 O2 detection. The solutions were prepared was lower in the animals from groups DW14B, DW21A,
as described elsewhere (Pick and Mizel, 1981). The cell and DW21B compared to their control groups (P < 0.05),
suspensions were added to 96-well flat-bottom microplates whereas animals from the G group did not show a signif-
and incubated in a humidified 5% CO2 atmosphere at icant difference in body weight gain compared to the CG
37 ◦ C for 1 h. Subsequently, the wells containing PRS re- group (Table 1).
ceived 10 ␮l of 1N NaOH to stop the reaction. Hydrogen
peroxide-dependent phenol red oxidation was measured 3.2. Number of peritoneal cells harvested
spectrophotometrically at 620 nm with a Titertek Multiscan
apparatus (Flow Laboratories). The same procedure was The number of peritoneal cells collected from ani-
employed to determine H2 O2 release after stimulation with mals from the DW groups ranged from 2.4 × 106 to
PMA, with 10 ␮l of 10 ng/ml PMA added to each well be- 12.4 × 106 cells/ml peritoneal exudate. This variability
fore incubation. The concentration of H2 O2 was calculated among animals in the number of cells harvested from the
from absorbance measurements as described previously peritoneum under the same experimental conditions did not
(Pick and Mizel, 1981). show significant differences between these groups (data
Spontaneous and PMA-induced H2 O2 production exper- not shown). However, G group rats showed a significant
iments were repeated four times for each rat in each group, decrease (P < 0.05) in the number of peritoneal cells
and the mean value of the four counts was used for H2 O2 harvested (1.90 ± 1.03) when compared to CG animals
determination. (5.30 ± 3.20).
Table 1
Target and actual doses, food consumption, water consumption, and total weight gain of rats treated with aqueous fraction of Ipomoea carnea dry leaves
Group n Treatment (days) Dose (g/kg/day) Food consumption Water consumption Total weight
(g/day) (ml/day) gain (g)
Target Actual
CDW14 7 14 0.0 0.0 16.60 ± 1.32 31.66 ± 3.14 18.85 ± 6.31

DW14A 7 14 3.0 2.34 ± 0.14 16.37 ± 0.98 23.48 ± 1.40a 17.28 ± 5.56
DW14B 6b 14 15.0 7.63 ± 0.16 13.77 ± 1.10a 15.26 ± 1.67a 2.83 ± 5.87a
CDW21 6 21 0.0 0.0 17.79 ± 1.37 32.28 ± 4.82 20.2 ± 6.67
DW21A 6 21 3.0 1.90 ± 0.47 16.14 ± 1.02 19.0 ± 4.78a 4.48 ± 6.74a
DW21B 6 21 15.0 10.60 ± 0.64 14.74 ± 1.11a 21.20 ± 6.45a 0.40 ± 7.89a
CG 6 14 0.0 0.0 16.37 ± 0.44 29.74 ± 2.02 12.56 ± 6.83
G 6 14 15.0 15.0 14.72 ± 0.46a 27.58 ± 2.69 8.83 ± 3.73
a Student’s t-test was used when comparing two groups and for more than two groups data were analyzed statistically by ANOVA followed by
Dunnet’s post hoc test, with the level of significance set at P < 0.05. Data are expressed as mean ± S.D.
b One animal of the DW14B group died on the last day of treatment.
Fig. 1. Effect of administration of aqueous fraction of Ipomoea carnea on macrophage spreading index (SI) in rats treated with daily doses of 3 g/kg/body
weight (bw) (DW14A) and 15 g/kg/bw (DW14B) for 14 days (A), with the daily doses of 3 g/kg/bw (DW21A) and 15 g/kg/bw (DW21B) for 21 days
(B), and with a dose of 15 g/kg/bw (G) for 14 days (C).
Fig. 2. Effect of administration of Ipomoea carnea aqueous fraction on macrophage phagocytosis index (PI) in rats treated with daily doses of 3 g/kg/body
weight (bw) (DW14A) and 15 g/kg/bw (DW14B) for 14 days (A), with daily doses of 3 g/kg/bw (DW21A) and 15 g/kg/bw (DW21B) for 21 days (B),
and with a dose of 15 g/kg/bw (G) for 14 days (C). Data are mean ± S.D. of n ≥ 6 animals. ∗ P < 0.05, compared with the respective control groups.
3.3. Effect of AF administration on macrophage spreading macrophages of rats treated with AF by gavage for 14 days
and phagocytosis activity (G group) did not show any significant increase in H2 O2
release, with PMA stimulation or not, when compared to
The spreading activity of peritoneal macrophages from the CG group (Fig. 3).
animals of all experimental groups was not affected by Ipo-
moea carnea administration (Fig. 1). On the other hand,
rats from groups DW14A, DW14B, DW21A, and DW21B
showed increased macrophage phagocytosis (P < 0.05). No 4. Discussion and conclusion
difference in this parameter was found between animals from
the G and CG groups (Fig. 2). Our first attempt was to deliver the plant sample in drink-
ing water since this route permits administration without any
stress. However, apparently the AF of Ipomoea carnea has
3.4. Effect of AF administration on spontaneous and a very poor palatability and experimental rats drastically re-
induced H2 O2 release by peritoneal macrophages fused to ingest water containing AF, especially those from
group DW14B (target dose of 15 g/kg/day) that consumed
Administration of AF for 14 and 21 days (DW14A, about half the expected amount of Ipomoea carnea AF. This
DW14B, DW21A, and DW21B groups) increased the spon- would have probably led the animals from the DW14B and
taneous H2 O2 release by peritoneal macrophages in all of DW21B groups to ingest small quantities of food, with a con-
these experimental groups (P < 0.05) compared to their sequent reduction in body weight gain. For this reason, we
respective untreated controls (CDW14 and CDW21); the administered the plant material by gavage (G group) which
same was observed when peritoneal macrophages were in- assured the ingestion of 15 g/kg/day of the AF of Ipomoea
duced to release H2 O2 by PMA (P < 0.05). Nevertheless, carnea. However, while no significant difference in water
I.M. Hueza et al. / Journal of Ethnopharmacology 87 (2003) 181–186 185
Fig. 3. Effect of administration of Ipomoea carnea aqueous fraction on spontaneous and induced H2 O2 release by peritoneal macrophages in rats treated
with daily doses of 3 g/kg/body weight (bw) (DW14A) and 15 g/kg/bw (DW14B) for 14 days (A), with daily doses of 3 g/kg/bw (DW21A) and 15 g/kg/bw
(DW21B) for 21 days (B), and with a dose of 15 g/kg/bw (G) for 14 days (C). Data are mean ± S.D. of n ≥ 6 animals. ∗ P < 0.05, compared with the
respective control groups.
intake or body weight gain was detected in these rats from we propose that receptor alterations promoted by SW could
the G group, a decrease in food consumption was observed. be responsible for the enhanced activation of these effector
These results signify that a decrease of food intake is re- cells.
lated to a direct toxic effect of the plant. In fact, it was well An important finding of the present study was the ab-
demonstrated that SW, the main toxic principle of Ipomoea sence of immune stimulation verified in rats treated with the
carnea, produces anorexic effects (Pritchard et al., 1990). highest dose of Ipomoea carnea AF (CG group). A possi-
While several in vitro investigations have shown unequ- ble explanation for this lack of activation of peritoneal cells
ivocally that SW produces immune stimulation in different is given by the dual effects produced by SW. Thus, since
effector cells, such as lymphocytes, NK cells (Humphries SW inhibits not only Golgi mannosidase II but also lyso-
et al., 1988; Bowlin et al., 1989), and peritoneal macrophages somal ␣-mannosidase, the impairment of the latter enzyme
(Das et al., 1995), there is a lack of information in the lit- could lead to vacuolization of different tissue cells, includ-
erature showing this effect in an in vivo trial. The present ing those of the myeloid system, the monocytes, as observed
study reveals that the administration of AF at moderate in ␣-mannosidosis disease (Alroy et al., 1989). Hence, this
doses (about 2–10 g/kg/day) produces stimulatory effects monocyte vacuolization in animals that received the high-
on macrophage phagocytosis and hydrogen peroxide pro- est dose of the plant extract could be the responsible factor
duction by peritoneal cells, even without PMA stimulation, for the unsuccessful recruitment elicitation of macrophages
during both periods of 14 and 21 days of administration. after LPS activation. This hypothesis is supported by the
It is known that the immune responses are largely regu- decrease in the number of harvested cells found in the peri-
lated by cell surface and secreted glycoproteins. In addition, toneum of rats from the G group.
interaction between sugars and lectins might be functionally The disparity of the results obtained for rats that received
involved in the immune recognition and activation, includ- lower doses and rats in the gavaged group could also help
ing the receptors involved in phagocytosis (Linehan et al., explain the discrepancy between field observations, which
2000). Thus, one explanation for the enhanced phagocyto- shows that animals intoxicated with plants containing SW
sis activity of macrophages observed here could be the al- are more susceptible to infections (Sharma et al., 1984;
teration of the synthesis and processing of a hybrid type of Stegelmeier et al., 1998), and in vitro experiments, which
oligosaccharide caused by SW. Further supporting this hy- show the immune stimulation produced by SW. Thus, im-
pothesis was the observation that peritoneal macrophages of mune stimulation or suppression is probably directly related
rats treated with Ipomoea carnea showed enhanced perox- to the dose of SW ingested. However, to better investigate
ide production of the same intensity as that observed when this theory, future experiments will be conducted in our
PMA was added. It is known that PMA increases hydro- laboratory, administering higher doses than that used here
gen peroxide production acting directly on protein kinase (15 g/kg/kg) to verify the possible immunosuppressive ef-
C (PKC). PKC participation could be involved in the trans- fects of Ipomoea carnea.
duction of phagocytic signals generated by various recep- In conclusion, this study shows that the administration
tors (Kwiatkowska and Sobota, 1999). Since Breton et al. of AF of Ipomoea carnea at moderate doses, for a pe-
(1990) suggested that SW indirectly mediates the same event riod of up to 3 weeks, clearly up-regulates phagocytosis in
as that induced by PMA in the modulation of PKC activity, macrophages, as well as metabolic pathways yielding H2 O2 .
On the other hand, at higher doses the plant extract appar- Jolly, R.D., Walkley, S.U., 1997. Lysosomal storage diseases of animals:
ently causes non-specific immunosuppression. an essay in comparative pathology. Veterinary Pathology 34, 527–548.
Karasuno, T., Kanayama, Y., Nishiura, T., Nakao, H., Yonezawa, T., Tarui,
S., 1992. Glycosidase inhibitors (castanospermine and swainsonine)
and neuraminidase inhibit pokeweed mitogen-induced b-cell matura-
Acknowledgements tion. European Journal of Immunology 22, 2003–2008.
Kwiatkowska, K., Sobota, A., 1999. Signaling pathways in phagocytosis.
We thank Professors Drs. H.S. Spinosa and M.L.Z. Dagli BioEssays 21, 422–431.
Linehan, S.A., Martı́nez-Pomares, L., Gordon, S., 2000. Macrophage
for their comments on the manuscript and for reviewing this lectins in host defense. Microbes and Infection 2, 279–288.
paper. This research was supported by grants from FAPESP, Nelson, B.K., James, L.F., Sharma, R.P., Cheney, C.D., 1980. Locoweed
Brazil. embryotoxicity in rats. Clinical Toxicology 16, 149–166.
Pan, Y.T., Ghidoni, J., Elbein, A.D., 1993. The effects of castanospermine
and swainsonine on the activity and synthesis of intestinal sucrase.
Archives of Biochemistry and Biophysics 303, 134–144.
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In vitro antioxidant and antithrombotic activity of

Hemidesmus indicus (L) R.Br.
N.K. Mary, C.R. Achuthan, B.H. Babu, J. Padikkala∗
Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala 680 553, India
Received 4 July 2002; received in revised form 31 March 2003; accepted 2 April 2003
Abstract
The methanolic extract of Hemidesmus indicus (L) R.Br. (Asclepiadaceae) roots was found to inhibit lipid peroxidation and scavenge
hydroxyl and superoxide radicals in vitro. The amount required for 50% inhibition of lipid peroxide formation was 217.5 ␮g/ml. The
concentrations needed to scavenge hydroxyl and superoxide radicals were 73.5 and 287.5 ␮g/ml, respectively. The intravenous administration
of this extract (5 mg/kg body weight) in rabbits delayed the plasma recalcification time and enhanced the release of lipoprotein lipase enzyme
significantly. The extract also inhibited ADP-induced platelet aggregation in vitro (50–250 ␮g), which was comparable to commercial heparin.
Keywords: Hemidesmus indicus; Antioxidant activity; Antithrombotic activity; Antiplatelet aggregation; Anticoagulant activity; Lipoprotein lipase
1. Introduction eases, the indigenous drugs with long descended heritage

of traditional use, are of most significance, in this period of
The traditional Indian medicine and the use of plant high rate of the disease.
drugs against various diseases receive considerable attention Present study is a scientific approach to reestablish the
nowadays. Hemidesmus indicus (L) R.Br. is a twining shrub traditional uses of the plant Hemidesmus indicus and evalu-
which has been used as folk medicine and as ingredient in ates its antioxidant and antithrombotic properties.
Ayurvedic and Unani preparations against diseases of blood,
inflammation, etc. (Vaidya and Kulkarni, 1991). It has also
been used in combination with other drugs for snake bite 2. Materials and methods
(Kirthikar and Basu, 1935; Mors, 1991). Recently, this plant
was used to treat viper venom (haemotoxic)-induced lethal- 2.1. Plant material
ity (Alam et al., 1996) and against hypercholesterolaemia
in hyperlipidaemic rats (Bopanna et al., 1997). The plant Hemidesmus indicus was collected locally
Coronary heart diseases are primarily lipid disorders; and identified by Dr. Sasidharan, Taxonomist, Kerala For-
yet being initiated and associated with the increased intra- est Research Institute (KFRI), Thrissur, Kerala, India. A
cellular generation of reactive oxygen species leading to voucher specimen was kept in the herbarium of our Institute
tissue injury with a variety of pathological processes like (ACRH-24). The dried root of the plant was used for the
ischaemia, inflammation, atherosclerosis, and thrombosis. experiment.
Hence compounds, which can scavenge the excess of free
radicals formed or inhibit their production or protect mem-
branes from peroxidation are of wide therapeutic value 2.2. Preparation of the drug
(Diaz et al., 1997). The hypolipidaemic and anticoagulant
agents are also playing major roles in preventing cardiovas- Dried and powdered roots of Hemidesmus indicus (10 g)
cular diseases. Although several chemicals and drugs are were extracted twice with 70% MeOH by continuous stir-
generally used against atherosclerosis and related heart dis- ring. The extract was pooled and evaporated to dryness
under reduced pressure. The yield of the extract was 14%
(w/w). The extract was re-suspended in water and subjected
∗ Corresponding author. Fax: +91-487-211020. to various studies after preliminary phytochemical screen-
E-mail address: jpadikkala@rediffmail.com (J. Padikkala). ing (Wagner et al., 1984) which showed positive reaction
doi:10.1016/S0378-8741(03)00119-3
188 N.K. Mary et al. / Journal of Ethnopharmacology 87 (2003) 187–191
for flavonoids, terpenoids, polyphenols, and coumarins 2.6. Hydroxyl radical scavenging activity
(Harbone, 1976; Stahl, 1969). Alkaloids are not found in
the extract. Hydroxyl radical scavenging was measured by studying
the competition between deoxyribose and the extract for hy-
2.3. Animals droxyl radicals generated from the Fe3+ /ascorbate/EDTA/
H2 O2 system. The hydroxyl radicals attack deoxyribose,
Male New Zealand White rabbits and Male Wistar rats which eventually results in TBARS formation (Elizabeth
were purchased from Veterinary College, Mannuthy, Thris- and Rao, 1990). The reaction mixture contained deoxyribose
sur. They were housed in ventilated cages and fed with pellet (2.8 mM), FeCl3 (0.1 mM), EDTA (0.1 mM), H2 O2 (1 mM),
diet (Lipton India Ltd.) and water ad libitum. ascorbate (0.1 mM), K H2 PO4 –KOH buffer (20 mM, pH
7.4), and various concentrations of the drug (10–500 ␮g/ml)
in a final volume of 1 ml. The reaction mixture was incu-
2.4. Superoxide radical scavenging activity bated for 1 h at 37 ◦ C. Deoxyribose degradation was mea-
sured as TBARS by the method of Ohkawa et al. (1979) and
Superoxide radical scavenging activity was determined percentage inhibition was calculated. Curcumin (1–100 ␮g)
by the Nitroblue tetrazolium (NBT) reduction method of was used as reference.
Mc Cord and Fridovich (1969). The reaction mixture con-
tained EDTA (0.1 M) containing 0.0015% NaCN, riboflavin 2.7. Anticoagulant activity by plasma recalcification
(0.12 mM), NBT (1.5 mM), various concentrations of the ex- method
tract (10–500 ␮g/ml), and phosphate buffer (M/15, pH 7.5)
in a final volume of 3 ml. The tubes were uniformly illumi- Blood was collected from normal rabbits through the ear
nated under an incandescent lamp for 15 min and the optical vein in EDTA (0.1 M) added tubes. The plasma was sepa-
density was measured at 530 nm before and after illumina- rated by centrifugation (1000 rpm × 5 min). 200 microlitre
tion. The percentage inhibition of superoxide generation was of M/100 CaCl2 was added to 100 ␮l of the plasma pre-
evaluated by comparing the absorbance values of the con- warmed at 37 ◦ C. The time taken for the formation of a
trol and experimental tubes. A known antioxidant, curcumin firm clot was noted immediately by the help of a stopwatch
(1–100 ␮g) was used as reference. (Achuthan et al., 1997). Similarly the plasma recalcification
time was noted 10 min after the intravenous administration
2.5. Inhibition of lipid peroxide formation of the drug (5 mg/kg body weight) and compared with the
anticoagulant heparin (1 mg/kg) as reference.
2.5.1. Induction by Fe2+ /ascorbate system
The peroxide formation was measured by the method 2.8. Antiplatelet aggregation activity—ADP induced
of Ohkawa et al. (1979) by measuring the colour of thio-
barbituric acid reactive substances (TBARS) formed at Platelet rich plasma (PRP) was prepared by centrifugation
the end of the reaction. Malonaldehyde (MDA), which is (1000 rpm × 5 min) of blood collected from normal aspirin
formed, as the end product in lipid peroxidation will re- free blood bank donors. 1.5 millilitre of acid citrate dextrose
act with thiobarbituric acid (TBA) to give TBARS, which (ACD) was used as anticoagulant for every 8.5 ml of blood.
is pink in colour, measured at 530 nm. The reaction mix- PRP was taken into siliconized glass cuvettes. Platelet poor
ture contained rat liver homogenate (0.1 ml, 25% (w/v)) in plasma (PPP) collected by centrifugation (3000 rpm×5 min)
Tris–HCl buffer (20 mM, pH 7.0), KCl (150 mM), ferrous was kept as reference. The cuvettes were incubated at 37 ◦ C
ammonium sulphate (0.8 mM), ascorbic acid (0.3 mM) and for 5 min. The aggregation was initiated by adding 20 ␮l of
various concentrations of the drug (10–500 ␮g) in a final ADP (10 ␮M) to 1 ml of PRP. The aggregation was recorded
volume of 0.5 ml, was incubated for 1 h at 37 ◦ C (Bishayee for 5 min using spectrophotometer at 600 nm. The effect of
and Balasubramanian, 1971). different concentrations (50–250 ␮g) of extract was studied
The incubated reaction mixture (0.4 ml) was treated with by incubation of PRP and the drug at 37 ◦ C for 5 min before
0.2 ml of 8% sodium dodecyl sulphate (SDS), thiobarbituric the addition of ADP. Commercial heparin (20 ␮g/ml) was
acid (1.5 ml, 8%) and acetic acid (1.5 ml, 20%, pH 3.5). used as reference (Subramaniam and Satyanarayana, 1989).
The total volume was then made up to 4 ml by adding dis-
tilled water and kept in a water bath at 100 ◦ C for 1 h. After 2.9. Lipoprotein lipase releasing activity
cooling, 1 ml of distilled water and 5 ml of a mixture of
n-butanol-pyridine (15:1 (v/v)) were added and shaken vig- The lipoprotein lipase releasing activity of the drug was
orously. The absorbance of the organic layer was measured determined by the method of Korn (1962). Blood was col-
at 560 nm after centrifugation. The percentage inhibition of lected from normal rabbits through the ear vein in EDTA
lipid peroxide formation was determined by comparing the (0.1 M) added tubes. The plasma was collected by centrifu-
results of the drug-treated and untreated samples. Curcumin gation (3000 rpm × 5 min) and used as the enzyme source.
(1–100 ␮g) was used as reference. The substrate was lipimic serum obtained from rabbits 3 h
N.K. Mary et al. / Journal of Ethnopharmacology 87 (2003) 187–191 189
after feeding 35 g of saltless butter. The substrate (0.1 ml), Table 2

enzyme (0.1–0.4 ml), albumin (20%, 0.4 ml, pH 8.5), and Anticoagulant activity of Hemidesmus indicus
(NH4 )2 SO4 (0.1 ml) were mixed at a low temperature and Sample Plasma recalcification time (s)
made up to a final volume of 1 ml. The mixture was incu- Normal rabbit blood 58 ± 2
bated at 37 ◦ C for 1 h by transferring 0.05 ml of aliquots at Heparin 142 ± 4
0 h and at the intervals of 30 min into centrifuge tubes con- Hemidesmus indicus (5 mg/kg) 130 ± 5
taining 0.1 ml of 1N H2 SO4 for glycerol determination. The Values are mean ± S.D. of triplicates.
samples treated with 0.1 ml of sodium periodate (0.05 M)
and 0.1 ml of sodium arsenate (0.05 M) were kept in boil-
of the extract needed for 50% inhibition was 217.5 ␮g/ml
ing water bath for 30 min after adding 9 ml of chromotropic
where as curcumin needed was only 8.9 ␮g/ml (Table 1).
acid. The volume was adjusted to 10 ml and after cooling the
optical density was measured at 570 nm. Heparin (1 mg/kg)
was used as reference. 3.4. Hydroxyl radical scavenging activity
The assay was standardized with glycerol solution of
known molarity and the glycerol liberated was calculated. Degradation of deoxyribose mediated by hydroxyl rad-
The same experiment was repeated after the administration icals generated by the Fe3+ /ascorbate/EDTA/H2 O2 system
of the drug (5 mg/kg) for a period of 10 min. The glycerol lib- was also found inhibited by Hemidesmus indicus root ex-
erated was calculated and compared with normal untreated tract. The concentration of root extract needed for 50% inhi-
groups. bition was 287.5 ␮g/ml and that of curcumin was 2.7 ␮g/ml
(Table 1).
3. Results 3.5. Effect on plasma recalcification time
3.1. Phytochemical screening Normal plasma recalcification time noticed was 58 ± 2 s.

Administration of heparin could delay the time to 142 ± 4 s
The phytochemical screening of the root extract showed (Table 2). The methanolic extract of Hemidesmus indicus
positive reaction for flavonoids, terpenoids, polyphenols and delayed the time to 130±5 s, which is much greater than the
coumarins. Alkaloids are not found in the extract. recalcification time of normal plasma. Compared to heparin
this drug is more or less equally effective.
3.2. Superoxide scavenging activity
3.6. Effect on platelet aggregation
The root extract of Hemidesmus indicus was found to
scavenge the superoxide generated by photoreduction of Addition of ADP to a suspension of washed human
riboflavin. The concentration needed for 50% scavenging platelets caused a marked decrease in optical density at
of superoxide was found to be 73.5 and 6.3 ␮g/ml for 600 nm indicating the aggregation of platelets. The aggre-
Hemidesmus indicus and curcumin, respectively (Table 1). gation effect was greater at 37 ◦ C compared to that at room
temperature. The methanol extract of Hemidesmus indicus
(50–250 ␮g/ml) interestingly inhibited platelet aggregation
3.3. Inhibition of lipid peroxidation
(Fig. 1). Greater inhibition of aggregation was noticed with
greater concentrations.
The generation of lipid peroxides by Fe2+ /ascorbate in
rat liver homogenate was found inhibited by the addition
of root extract of Hemidesmus indicus. The concentration 3.7. Lipoprotien lipase releasing activity
The drug-treated animals for a period of 10 min showed

Table 1 increased production of glycerol as an index of the greater
Effect of Hemidesmus indicus on free radical generation release and activity of enzyme from the arterial intima. The
Percentage Hydroxyl Superoxide Lipid peroxide glycerol liberated in the drug-treated animals was found to
inhibition radical (␮g/ml) radical (␮g/ml) (␮g/ml)
10 17.5 ± 0.7 9.5 ± 0.05 15.0 ± 0.2
20 35.5 ± 1.2 20.9 ± 0.6 33.7 ± 0.4 Table 3
30 50.0 ± 1.3 43.0 ± 0.9 47.5 ± 0.8 Effect of Hemidesmus indicus on lipoprotein lipase releasing activity
40 170.2 ± 2.1 56.9 ± 0.5 128.7 ± 0.7
Sample Glycerol liberated (mg/dl)
50 287.5 ± 1.7 73.5 ± 1.3 217.5 ± 1.5
50 (curcumin) 2.7 ± 0.07 6.3 ± 0.06 8.9 ± 0.05 Normal rabbits 3.6 ± 0.2
60 375.3 ± 2.5 90.2 ± 1.2 220.5 ± 1.7 Heparin (1 mg/kg) 12.7 ± 1.7
70 427.7 ± 2.7 106.7 ± 2.1 302.2 ± 1.9 Hemidesmus indicus (5 mg/kg) 9.0 ± 0.7
Values are mean ± S.D. of triplicates. Values are mean ± S.D. of triplicates.
190 N.K. Mary et al. / Journal of Ethnopharmacology 87 (2003) 187–191
Fig. 1. Effect of methanolic extract of Hemidemus indicus on the aggregation of human platelets (washed).
be 9 mg/dl where in the normal animals it was only 3.6 mg/dl for ameliorating the mechanisms related to atherogen-
(Table 3). esis (Cimmniello and Toschi, 1999) and this drug has
interestingly inhibited platelet aggregation. Hemidesmus
indicus root extract incubated with viper venom antago-
4. Discussion nized coagulant and haemorrhagic activity (Alam et al.,
1996). The plasma recalcification time was also delayed
India recently increased research in Traditional Ayurvedic significantly by the intravenous administration of the root
Herbal Medicines after observations that they are effective extract.
for conditions to which they have traditionally been ap- Lipoprotein lipase has been reported in the post-heparin
plied. The present investigation has explored the use of one plasma of rabbits and has a major role in the transport
such plant Hemidesmus indicus, abundantly found in the and metabolism of triglycerides of exogenous origin (Korn,
Indian continent, for preventing coronary artery diseases. 1962). It is the key enzyme, the metabolic gatekeeper regu-
The vascular endothelium is the principal site of action of lating the disposal of lipid fuels in the body (Fielding and
cardiovascular risk factors and early atherogenesis (Ross, Frayn, 1998). The glycerol liberated by the action of lipopro-
1993). The imbalance between prooxidants and antioxidants tein lipase enzyme in the drug-treated animals was found
in the development of atherosclerosis has prompted the in- to be three times greater than the normal untreated animals.
vestigation of antioxidants as a possible therapy (Khan and This drug thus has an enhancing role of releasing and acti-
Butler, 1998). The process of atherogenesis is initiated by vating the enzyme, resulting in the metabolic degradation
the oxidation of lipids in low-density lipoproteins (LDL), of lipids. Bopanna et al. (1997) have also reported the hy-
termed lipid peroxidation (Diaz et al., 1997; Camejo et al., polipidaemic effect of the cell culture derived Hemidesmus
1976). The screening of the antioxidant activity of this plant indicus. The various in vitro studies have shown earlier that
has revealed its capacity to scavenge the superoxide and certain flavonoids are the potent inhibitors of the oxidative
hydroxyl radicals at low concentrations. The lipid peroxida- modification of LDL by macrophages (Havsteen, 1983). The
tion was also found inhibited by low concentrations of the flavonoids (Mors, 1991), terpenoids (Gupta et al., 1992),
root extract. polyphenols and coumarins (Nandkarni, 1976) present in
Platelets play an important role in the process of athero- the plant extract may account for the above-mentioned
thrombosis by adhering to the damaged regions (caused properties.
by reactive oxygen species) of the endothelial surface. With all these wide spectrum of the antioxidant, anti-
The activated platelets form platelets to platelets bonds, coagulant, hypolipidaemic, antiplatelet aggregation, anti-
binds also to leucocytes bringing them into a complex haemorrhagic and lipoprotein lipase releasing properties,
process of plaque formation and growth (Prentice, 1999). Hemidesmus indicus can be considered an effective an-
The antiplatelet therapy constitutes the best available tool tiatherogenic agent preventing coronary artery diseases.
N.K. Mary et al. / Journal of Ethnopharmacology 87 (2003) 187–191 191
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119.
Inhibition of experimental gastric lesion and inflammation

by Phyllanthus amarus extract
K. Regi Raphael, Ramadasan Kuttan∗
Amala Cancer Research Centre, Thrissur, Kerala 680 553, India
Received 16 July 2002; received in revised form 28 March 2003; accepted 2 April 2003
Abstract
Methanolic extract of Phyllanthus amarus Shum & Thonn (Euphorbiaceae) 50, 200, and 1000 mg/kg body weight significantly inhibited
gastric lesions, induced by intragastric administration of absolute ethanol (8 ml/kg). Mortality, increased stomach weight, ulcer index, and
intraluminal bleeding were reduced significantly by Phyllanthus amarus. Biochemical analysis indicated that reduced glutathione (GSH) of
gastric mucosa produced by ethanol administration was significantly elevated by treatment with Phyllanthus amarus extract. Aqueous and
methanol extracts of Phyllanthus amarus produced an inhibition of rat paw edema up to 42% compared to control in 3 h and continued up to
8 h. Anti-oxidant activity of the extract as well as presence of tannins in the extract may be responsible for these observed activities.
© 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Phyllanthus amarus; Ethanol toxicity; Gastric lesion; Anti-inflammatory
1. Introduction activity of Phyllanthus amarus using experimental paw

edema produced by carrageenan administration. We have
Phyllanthus amarus is traditionally used to treat flu, also looked the protection of gastric lesions by Phyllanthus
dropsy, diabetes, and jaundice (Foo, 1993). It is also be- amarus extract.
ing used to treat hepatic and urolitic diseases and have
diuretic activity. Phyllanthus amarus inhibited hepatitis B
virus polymerase activity and decreased episomal hepatitis 2. Materials and methods
B virus DNA content and suppressed viral release into the
culture medium (Lee et al., 1996). Simultaneous adminis- 2.1. Extraction of Phyllanthus amarus
tration of Phyllanthus amarus extract along with carcinogen
has been reported to inhibit the hepatocellular carcinoma Leaves and stems of Phyllanthus amarus were collected
development induced by NDEA and increased the life span from Thrissur district of Kerala and were dried at 50 ◦ C.
of hepatocellular carcinoma harbouring animals (Joy and A voucher specimen of the plant was identified by Regi
Kuttan, 1998; Rajesh and Kuttan, 2000). In chemically Raphael K, Botanist, Amala Cancer Research Centre, Thris-
induced liver toxicity models Phyllanthus amarus signifi- sur, Kerala (Voucher No: EUP. 9) and has been kept at Amala
cantly protected the liver tissue (Prakas et al., 1995). Phyl- Ayurvedic Hospital and Research Centre.
lanthus amarus has potent free radical scavenging activity
and could scavenge superoxides and hydroxyl radicals and 2.1.1. Preparation of alcoholic extract
can inhibit lipid peroxides (Joy and Kuttan, 1995). As the Dried parts of Phyllanthus amarus were powdered and
inflammation is mainly produced by the oxidative burst this powder was extracted twice in five volumes of 75%
of the macrophages, many anti-oxidants may be effective methanol by stirring overnight and centrifuged at room
to reduce the inflammation. Infusion of the young shoots temperature. This supernatant was evaporated to dryness
of Phyllanthus amarus has been recommended to lessen at 50 ◦ C under reduced pressure using a rotary evaporator.
the edematous swelling and ulcers (Mhaskar et al., 2000). The yield of the extract was 8%.
In the present study, we have checked anti-inflammatory
2.1.2. Preparation of aqueous extract
∗ Corresponding author. Fax: +91-487-307020. Powdered Phyllanthus amarus (50 g) was extracted twice
E-mail address: amalaresearch@rediffmail.com (R. Kuttan). overnight with 250 ml of distilled water at room temperature.
0378-8741/03/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0378-8741(03)00120-X
194 K.R. Raphael, R. Kuttan / Journal of Ethnopharmacology 87 (2003) 193–197
The supernatant was collected and evaporated to dryness at method of Lowry et al. (1951) with bovine serum albumin
50 ◦ C under reduced pressure. The yield of the extract was as the standard.
10%.
Both methanolic and aqueous extracts had almost similar 2.4. Histopathology
TLC pattern. Major extracted products in both cases were
tannins as seen by ferric chloride test. However, yield of the A portion of the stomach from each group was fixed in
aqueous extract was higher than methanol extract. Extracts 10% formalin. The formalin fixed specimens were embedded
were prepared freshly before each experiment. in paraffin and sectioned (3–5 ␮m) and stained with hema-
toxylin and eosin and histochemical sections were evaluated
2.2. Induction of ethanol-induced gastric lesion by light microscopy.
Adult male Wistar rats weighing 120 g were used for the 2.5. Statistical evaluation
experiment. They were grouped into four groups of five ani-
mals each. All the animals were fasted for 16 h and deprived The values are expressed as mean ± standard deviations.
of water for 12 h prior to the experiments. Group I acted The results were analyzed statistically by analysis of vari-
as animals treated with alcohol alone. Group II–IV were ance. Values of P less than 1% (P < 0.01) were considered
treated with 1000, 200, and 50 mg of Phyllanthus amarus to be statistically significant.
extract/kg body weight as a single dose 30 min prior to
the experiment. Acute gastric lesion was induced by abso- 2.6. Determination of anti-inflammatory activity
lute ethanol (Robert et al., 1979). Briefly, absolute ethanol
(8 ml/kg body weight) was administered intragastrically to Anti-inflammatory activity was determined by carra-
control and drug-treated animals. Each animal was sacri- geenan-induced mice paw edema method of Langrange
ficed by ether overdose 1 h after administration of ethanol et al. (1974). Female inbred strains of Balb/c mice weigh-
and stomach was excised, opened along the greater curva- ing 25–28 g (6–7 weeks old) were used for the experiment.
ture and washed gently with ice cold solution. The stomach They were divided into four groups of three animals each.
weight and intraluminal bleeding were recorded. Group 1 acted as control. Groups 2–4 received 500, 250,
The extent of erosion of stomach mucosa was assessed and 100 mg/kg body weight of Phyllanthus amarus extract
from a scoring system designed by Merazzi–Uberti Turba as orally as a single dose 1 h prior to the experiment. Paw
follows: 0, no erosions; 1, one to three small erosions (4 mm edema was induced by injecting carageenan (200 ␮g/20␮l)
or smaller); 2, more than three small erosions or one large into the sub-plantar region of the left paw. The thickness of
erosion; 3, two large erosions; 4, three to four large erosions; paw edema was measured by venire calipers before treat-
and 5: more than four large erosions or lesion proliferation ment and after injection with carageenan. Measurement
(Giordano et al., 1990). The results were expressed in terms was continued at 60 min intervals up to 8 h and further at
of an ulcer index, which is the average severity of erosions the 24th hour. The inhibition of paw edema was calculated
per rat each group on the scale from 0 to 5. by comparing the difference in paw thickness of the con-
trol and treated group. Experiment was repeated twice and
2.3. Biochemical analysis average values were taken.
The mucosa of glandular stomach was removed by scrap-

ing with a blunt knife and 10% homogenate was prepared. 3. Results
Reduced glutathione (GSH) in the gastric mucosa was de-
termined by Ellman’s reaction using 5 5 -dithio-bis-2-nitro 3.1. Effect of Phyllanthus amarus extract in gastric lesion
benzoic acid (DTNB) was described by Moron et al. (1979).
Briefly 125 ␮l of 25% trichloro acetic acid (TCA) was added The present investigation indicate that rat mucosal gastric
to 0.5 ml of homogenate to precipitate proteins. The tubes injury induced by ethanol was significantly and dose depen-
were cooled in ice for 5 min and the mixture was further di- dently reduced by methanolic extract of Phyllanthus amarus
luted with 0.6 ml of 5% TCA and centrifuged at 9000×g for (Table 1). Administration of absolute ethanol to fasted rats
10 min. 0.3 ml of the supernatant was taken for estimation. resulted in severe gastric damage visible from the outside of
For this purpose the volume of the aliquot was made up to the stomach as thick reddish-black lines. After opening, the
1 ml with 0.2 M sodium phosphate buffer (pH 8.0) and 2 ml gastric lesions were found in the mucosa and consisted of
of freshly prepared DTNB solution (0.6 mM in 0.2 M phos- elongated bands, 1–10 mm long, usually parallel to the long
phate buffer pH 8.0) was added to the tubes. After 10 min the axis of the stomach. They were located mostly in the cor-
intensity of the yellow color formed was read at 412 nm in pus (the portion of the stomach secreting acid and pepsin).
a spectrophotometer. Reduced glutathione (Sisco Research No gross lesions developed in the fore stomach (the nonse-
Laboratories, Mumbai, India) was used as a standard. The cretory part of the stomach). Intragastric administration of
protein content of the gastric mucosa was quantified by the the absolute ethanol to rats resulted in 50% mortality due to
K.R. Raphael, R. Kuttan / Journal of Ethnopharmacology 87 (2003) 193–197 195
Table 1
Effect of Phyllanthus amarus administration on mortality, stomach weight, and intraluminal bleeding of rats treated with absolute ethanol
Treatment Dose (mg/kg) Mortality Stomach weight Intraluminal bleeding
(g/100 g body weight ± S.D.)
Number % Number %
Normal rats 0 0/5 0 0.68a ± 0.05 0/5 0

Ethanol 0 5/10 50 1.02d ± 0.13 5/5 100
Ethanol +
Phyllanthus amarus 50 1/5 20 0.85c ± 0.10 2/4 50
Ethanol +
Phyllanthus amarus 200 0/5 0 0.79b,c ± 0.05 0/5 0
Ethanol +
Phyllanthus amarus 1000 0/5 0 0.70a,b ± 0.08 0/5 0
a,b,c,d Result of the significance test done by ANOVA method.
acute reaction of the alcohol and its metabolites. Increased of the mucosa which are infiltered with polymorphonuclear
mortality in the controls were found to be aggrevated due to leucocytes. The depth of the lesion extended up to the mus-
the fasting (16 h) and deprivation of water (12 h). The rats, cularis mucosae with red blood corpuscles extravasation.
which died, had perforated lesions and severe intraluminal The submucosa of the corpus was markedly thickened by
bleeding. Stomach weight in alcohol-treated rats was in- edema but devoid of polymorphonuclear leucocytes. Histo-
creased to 1.02 ± 0.013 g/100 g body weight as compared to logically, the stomach of the Phyllanthus amarus pretreated
normal rat stomach weight 0.68 ± 0.05 g/100 g body weight groups (250 and 1000 mg/kg) showed superficial erosion in
possibly due to inflammation. In the treated animals because the mucosa and moderate degree of sub-mucosal edema with
of scavenging of the oxygen radicals generated by ethanol, neutrophilic infiltration.
the mortality rate and increase in stomach weight induced
by ethanol was found to be significantly less (Table 1). All 3.2. Anti-inflammatory activity of Phyllanthus amarus
animals treated with absolute ethanol caused intraluminal
bleeding in the glandular portion of the stomach, while all Development of paw edema was observed in both control
animals in the Phyllanthus amarus (200 and 1000 mg/kg) and treated group after carrageenan injection. Thickness of
pretreated group were found to be significantly protected the paw was found to be increased initially upon injection of
from intraluminal bleeding. carrageenan due to volume effect. Difference in the thickness
Ethanol administration to rats produced gastric damage of mice paw edema was further increased during the time
with an ulcer index of 4.75 ± 0.5 and 48.8% reduction in interval of 60–180 min in control group. Water extract of
gastric mucosal GSH. Phyllanthus amarus pretreatment (50, Phyllanthus amarus (100, 250, and 500 mg/kg) produced an
200, and 1000 mg/kg) significantly reduced the ulcer index inhibition of 26, 33, and 39%, respectively at 3 h (Fig. 1).
to 3.5±0.6, 2.0±0.5, and 0.6±0.5, respectively and reduced While the methanol extract of Phyllanthus amarus (100,
the depletion of GSH to 36, 16.5, and 5.5%, respectively 250, and 500 mg/kg) produced an inhibition of 29, 37, and
(Table 2). 42%, respectively at 3 h (Fig. 2) and significant inhibition
Histological analysis of ethanol-treated rat stomach re- of paw oedema was observed throughout the course of the
vealed the presence of necrotic debrii in the lamina propria experiment up to 8 h.
Table 2
Effect of Phyllanthus amarus administration on the ulcer index and glutathion (GSH) content of the mucosa of rats treated with absolute alcohol
Treatment Dose (mg/kg) Ulcer index ± S.D. Inhibition (%) GSH (nmol/mg (%) reduction protein) in GSH
Normal rats 0 0 100 12.7a ± 0.8 –

Ethanol 0 4.75a ± 0.5 – 6.5b ± 0.6 48.8
Ethanol +
Phyllanthus amarus 50 3.5b ± 0.6 26.3 8.1c ± 1.1 36
Ethanol +
Phyllanthus amarus 200 2.0c ± 0.5 57.9 10.6d ± 1.1 16.5
Ethanol +
Phyllanthus amarus 1000 0.6d ± 0.5 87.4 12.0a ± 0.5 5.5
a,b,c,d Result of the significance test done by ANOVA method.
196 K.R. Raphael, R. Kuttan / Journal of Ethnopharmacology 87 (2003) 193–197
Fig. 1. Anti-inflammatory activity of water extract of Phyllanthus amarus. (䊉) Control treated with the extract; (䊏) 100 mg/kg; (䉱) 250 mg/kg; (×)
500 mg/kg.
Fig. 2. Anti-inflammatory activity of methanolic extract of Phyllanthus amarus. (䊉) Control treated with the extract; (䊏) 100 mg/kg; (䉱) 250 mg/kg;
(×) 500 mg/kg.
4. Discussion eration, (b) suppression of activation of carcinogen, and (c)

anti-oxidant activity of the extract.
In the present study we have checked the anti-lesion and The present study indicating that the extract has signif-
anti-inflammatory activity of Phyllanthus amarus extract icant effect in reducing inflammation and lesion is again
which is a very important plant in the herbal medicine prac- reflective of its activity as scavenging oxygen radicals. Re-
tice. Phyllanthus amarus has been shown to be an effective active oxygen species has been shown to have significant
medicine against viral hepatitis as it has been shown to effect on the cellular system, damages its structure, and
suppress the mRNA transcription of hepatitis B virus (Lee induces alteration especially in its high molecular weight
et al., 1996). It has been shown to be useful to reduce the components. Large doses of ethanol has been shown to
Hbs/Ag antigen found in human hepatitis carriers (Unander specifically effect the inner lining of the stomach producing
and Blumberg, 1992). Our recent findings indicate that the erosion. In liver ethanol converted to ethanal (aldehyde)
extract had a significant activity to suppress the chemi- which is more toxic. Ethanal has been shown to reduce
cal carcinogenesis induced by chemicals such as 3-methyl GSH levels in liver tissues. Fall in GSH increases lipid
cholanthrene (Rajesh and Kuttan, 2001) and N-nitroso di- peroxidation. Pretreatment with the Phyllanthus amarus ex-
ethylamine (Joy and Kuttan, 1998). The mechanism of tract may either produce a protective lining on the stomach
action of the extract seems to be (a) suppression of prolif- and reduces oxygen radical production and thereby reduces
K.R. Raphael, R. Kuttan / Journal of Ethnopharmacology 87 (2003) 193–197 197
its effect on the liver. Similar mechanism could also be Gowrishankar, B., Vivekanandan, O.H., 1994. In vivo studies of a crude
postulated for its anti-inflammatory activity of Phyllanthus extract of Phyllanthus amarus L by tannery effluents. Mutation Re-
search 322, 185–192.
amarus. Inflammation produced by carrageenan is mainly Joy, K.L., Kuttan, R., 1995. Antioxidant activity of selected plant extracts.
due to macrophage activation and there by the producing of Amala Research Bulletin 15, 68–71.
oxygen radicals. Phyllanthus amarus extract could inhibit Joy, K.L., Kuttan, R., 1998. Inhibition by Phyllanthus amarus of hepato-
the oxygen radicals and there by reduces the inhibition. carcinogenesis induced by N-nitrosodiethylamine. Journal of Clinical
Another possible mechanism for the activity of the extract Biochemistry and Nutrition 24, 133–139.
Langrange, P.H., Mackaness, G.B., Miller, T.E., 1974. Influence of dose
to inhibit gastric lesion produced by alcohol may be due to and route of antigen injection on the immunological induction of
the formation of a protective layer of the polyphenolic com- T-cells. Journal of Experimental Medicine 139, 528–530.
pounds present in the extract with the protein of the stom- Lee, C.D., Ott, M., Thyagarajan, S.P., Shfritz, D.A., Burk, R.D., Gupta,
ach lining by hydrophobic interaction. Moreover the extract S., 1996. Phyllanthus amarus down regulates hepatitis B virus m RNA
may inhibit the prostaglandin synthesis like nonsteroidal transcription and translation. European Journal of Clinical Investiga-
tions 26, 1069–1076.
anti-inflammatory drugs. It has been shown that Phyllanthus Lowry, H.D., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein
amarus extract could inhibit the onset of diarrhea induced measurement with folin phenol reagent. Journal of Biological Chem-
by castor oil and reduced frequency of defecation and also istry 193, 265–275.
reduced gut meal travel distance significantly (Odetola and Mhaskar, K.S., Blatter, E., Caius, J.F., 2000. Kirtikar and Basu’s Illustrated
Akojenu, 2000). This effect has been attributed to the inhibi- Indian Medicinal Plants, vol. 9. Sri Satguru Publications, Delhi, India,
p. 3074.
tion of prostaglandin synthesis. Extract may also inhibit the
Moron, M.A., Depierre, J.W., Mannervick, B., 1979. Levels of glutathion,
macrophage migration which can reduce the inflammatory glutathion S-transferase activities in rat liver. Acta Biochemistry and
response produced by carrageenan. Biophysics 582, 67–68.
Several active compounds have been identified in Phyl- Nara, T.K., Glyeye, J., Cerval, E.L., Stanislan, E., 1977. Flavonoids
lanthus amarus extracts. Most common among them are lig- of Phyllanthus niruri, Phyllanthus urinaria, Phyllanthus orbiculatus.
Plantes Medicinales et Phytotherapie 11, 82–86.
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Odetola, A.A., Akojenu, S.M., 2000. Anti-diarrhoeal and gastrointesti-
et al., 1993), flavonoids like quercetin, astragalin (Nara et al., nal potentials of the aqueous extract of Phyllanthus amarus (Euphor-
1977), ellagitannins like amarinic acid (Foo, 1995) as well biaceae). African Journal of Medicine and Medical Sciences 29, 119–
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Kaji, A., Kaji, H., 1992. HIV-1 reverse transcriptase inhibitor from
amarus were found to be potent inhibitors of rat liver cyclic
Phyllanthus niruri. AIDS Research in Human retroviruses 11, 1937–
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nin inhibitors are the most specific and potent plant-derived Polya, G.M., Wang, B.H., Fooly, L.Y., 1995. Inhibition of signal regulated
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Prakas, A., Satyan, K.S., Washi, S.P., Singh, R.P., 1995. P. urinaria, P.
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niruri and P. simplex, on carbon tetrachloride induced liver injury in
Vivekanandan, 1994). Phyllanthin, a diaryl butane lignan, the rat. Phytotherapy Research 9, 594–596.
isolated from Phyllanthus amarus showed a significant pro- Qian-Cutrone, J., Huang, S., Trimble, J., Li, H., Lin, P.F., Alam, M.,
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and significantly increased protein level (Syamasundar binding inhibitor from Phyllanthus niruri. Journal of Natuaral Products
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Rajesh Kumar, N.V., Kuttan, R., 2000. Phyllanthus amarus ex-
et al., 1992) and niruriside (Qian-Cutrone et al., 1996) iso- tract administration increases the life span of rats with hep-
lated from Phyllanthus amarus were shown to inhibit HIV atoprotective carcinoma. Journal of Ethnopharmacology 73, 215–
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Anti-inflammatory and analgesic activities of mature

fresh leaves of Vitex negundo
M.G. Dharmasiri, J.R.A.C. Jayakody, G. Galhena, S.S.P. Liyanage, W.D. Ratnasooriya∗
Department of Zoology, University of Colombo, Colombo 3, Sri Lanka
Received 8 November 2002; received in revised form 6 February 2003; accepted 3 April 2003
Abstract
This study confirmed the oral anti-inflammatory, analgesic and antihistamine properties of mature fresh leaves (MFL) of Vitex ne-
gundo L. (Verbenaceae) claimed in the Ayurveda medicine by orally treating a water extract of the leaves to rats. The early phase (2 h)
of carrageenan-induced rat paw oedema was significantly (P < 0.01) suppressed in an inversely does-dependent (r2 = 1, P < 0.01) manner
by MFL. The EC50 was 2 g/kg of MFL. In the formaldehyde-induced rat paw oedema test, the 2.5 and 5 g/kg leaves significantly (P < 0.05)
suppressed the inflammation on days 4–6 of the test. In the hot plate test, 2.5 and 5 g/kg of MFL showed a significant (P < 0.05) and directly
dose-dependent analgesic activity at 1 h of treatment while the activity was absent in the tail flick test in rats. The EC50 for the analgesic
activity was 4.1 g/kg. In the formalin test, 1.25, 2.5 and 5 g/kg of MFL significantly (P < 0.05) suppressed the pain in both the phases of the
test like aspirin. The leaves showed an inversely dose-dependent in vivo antihistamine and in vitro prostaglandin (PG) synthesis inhibition,
membrane stabilising and antioxidant activities. Naloxone did not abolish the analgesic activity in the hot plate test. A 5 g/kg of MFL did not
impair muscle strength and co-ordination and did not induce sedation. The treatment of 5 g/kg of MFL did not show signs of acute toxicity
or stress. Fourteen-day oral treatment of 5 g/kg of MFL significantly increased the serum activity of AST. Flowering of the tree did not
abolish the analgesic and anti-inflammatory activities of the leaves. These observations revealed that the fresh leaves of Vitex negundo have
anti-inflammatory and pain suppressing activities possibly mediated via PG synthesis inhibition, antihistamine, membrane stabilising and
antioxidant activities. The antihistamine activity can produce the anti-itching effect claimed in Ayurveda medicine.
© 2003 Published by Elsevier Science Ireland Ltd.
Keywords: Vitex negundo fresh leaves; Anti-inflammatory activity; Analgesic activity; Antihyperalgesic activity; Antihistamine activity; Prostaglandin
synthesis inhibition activity; Antioxidant activity; Membrane stabilisation
1. Introduction gesic and anti-itching agents internally and externally

(Gunatillake, 1994). The tree is distributed in Sri Lanka, In-
As a result of adverse side effects, like gastric lesions, dia, Malaya, The Philippine Islands and East Africa. Flowers
caused by NSAIDs and tolerance and dependence induced occur throughout the year (Jayaweera, 1981). However, the
by opiates, the use of these drugs as anti-inflammatory and claimed activities of the leaves have not been investigated
analgesic agents have not been successful in all the cases. using controlled experiments in detail. The purpose of this
Therefore, new anti-inflammatory and analgesic drugs lack- study was to investigate in rats (a) the effectiveness of the
ing those effects are being searched all over the world as leaves as anti-inflammatory, analgesic and antihistaminic
alternatives to NSAIDs and opiates. During this process, the agents, (b) whether flowering of the tree abolishes the
investigation of the efficacy of plant-based drugs used in anti-inflammatory and analgesic activities and (c) any pos-
the traditional medicine have been paid great attention be- sible toxic effect caused after short-term use of the leaves.
cause they are cheap, have little side effects and according
to WHO still about 80% of the world population rely mainly
on plant-based drugs (Kumara, 2001). 2. Materials and methods
Vitex negundo L. (Verbenaceae), Nika in Sinhala, is a
small tree of which water extract of fresh mature leaves 2.1. Chemicals
are used in Ayurveda medicine as anti-inflammatory, anal-
Carrageenan (Sigma Chemical Co., St. Louis, MO,
∗ Corresponding author. Tel.: +94-1-503-399. USA), aspirin, indomethacin, chlorpheniramine (State Phar-
E-mail address: sd@chem.cmb.ac.lk (W.D. Ratnasooriya). maceutical Corporation, Colombo, Sri Lanka), naloxone
0378-8741/03/$ – see front matter © 2003 Published by Elsevier Science Ireland Ltd.
doi:10.1016/S0378-8741(03)00159-4
200 M.G. Dharmasiri et al. / Journal of Ethnopharmacology 87 (2003) 199–206
hydrochloride, formaldehyde (Fluka, Buchs, Switzerland), 2.5. Analgesic activity

assay kits (Randox Laboratories Ltd., Co., Antrim, UK).
2.5.1. Hot plate and tail flick tests
2.2. Collection of plants and preparation of extracts Female rats (n = 6/group) were treated with 1.25, 2.5
and 5 g/kg of MFL at preflowering stage, 5 g/kg of MFL
Mature fresh leaves (MFL) of Vitex negundo at 1–3 at flowering stage, 5 ml/kg of water and 100 mg/kg aspirin.
weeks before flowering and 1–2 weeks after flowering were The reaction times of these rats were measured 1 h prior to
collected from a tree in the campus garden of the Uni- the treatment, 1 and 3 h after the treatment using hot plate
versity of Colombo, Colombo, Sri Lanka, between March and tail flick techniques as described by Langerman et al.
and July 2002 and authenticated by Prof. A.S. Seneviratne, (1995). In the hot plate test, the rat was placed in a hot
Department of Botany, University of Colombo. Voucher plate analgesia meter (Model MK 35 A, Muromachi Kikai
specimen (23-VN) has been deposited at the museum of Co. Ltd., Tokyo, Japan) at 50 ◦ C and the time taken to lick
the Department of Zoology, University of Colombo. Fifty the hind paw or to jump was recorded. In the tail flick test,
grams of MFL was macerated with 100 ml of distilled wa- the tail of the rat 4–5 cm from its tip was immersed in a
ter (Jayasinghe, 1975) using an electric blender for 3 min, water bath at 55 ◦ C and the time taken to flick the tail was
squeezed through muslin cloth and 50% (w/v) extract was recorded. Rats showing a pre-treatment reaction time greater
obtained. Doses were determined according to the informa- than 15 s in the hot plate test and 5 s in the tail flick test
tion provided by an Ayurvedic physician, Dr. S. Ediriweera, were not used in the experiment. A cut off time of 25 s was
and animals were orally treated with the extract so that they set to avoid tissue damage. The hot plate test was repeated
received MFL 1.25, 2.5 and 5 g/kg of body weight (5 g/kg with naloxone (5 mg/kg s.c.) and 5 g/kg dose.
dose per rat is approximately equal to the human dose when
extrapolated). The extract was prepared every day for the 2.5.2. Formalin test
experiments. Rats of either sex (n = 10/group) were treated respec-
tively with 1.25, 2.5 and 5 g/kg of MFL, 5 ml/kg of water
and 100 mg/kg of aspirin. One hour later, these rats were in-
2.3. Animals jected with 0.05 ml of 2.5% formaldehyde in normal saline
into the foot pad of one of the hind paw (Dubuisson and
Wistar male and female rats (150–200 g) housed under Dennis, 1977), immediately placed in a transparent plastic
standardised animal house conditions were used in all the ex- cage separately and the licking time and frequency of the
periments. They had access to pelleted food (Vet House Ltd., injected paw were recorded from the first 0 to 5 and 20 to
Colombo, Sri Lanka) and water ad libitum. The animals were 25 min (Hunskaar et al., 1985).
assigned to different groups to be treated in experiments.
2.6. Mechanisms of anti-inflammatory and analgesic
2.4. Anti-inflammatory activity activities
2.4.1. Carrageenan-induced paw oedema 2.6.1. Prostaglandin (PG) synthesis inhibition

Male rats (n = 9/group) were treated with 1.25, 2.5 The experiment was carried out according to Lindsey et al.
and 5 g/kg of MFL at preflowering stage of the tree, 5 g/kg (1999) and Dharmasiri et al. (2003). One centimetre por-
of MFL at flowering stage, 5 ml/kg water and 5 mg/kg in- tions of isolated dioestrous rat uteri were suspended in a
domethacin, respectively (Forestieri et al., 1996). After 1 h, 50 ml organ bath containing Krebs–Henseleit solution (pH
these rats were injected with 0.05 ml of 1% carrageenan sus- 7.4), maintained at 37 ◦ C and aerated with 5% CO2 and
pension into the foot pad of left hind paw (Winter et al., 95% O2 gas mixture. The spontaneous contractions of the
1962). The paw volumes of these rats were measured using uteri were recorded using an isometric sensor (Star Med-
a Plethysmometer (Letica Scientific Instruments, Barcelona, icals, Tokyo, Japan) for 10 min and then, the organ bath
Spain) at 1 h before, and 2 and 4 h after the carrageenan in- was treated in triplicate with the extracts so that the final
jection and the paw oedema was calculated. concentrations of MFL in the organ bath became 100, 200,
300 and 400 ␮g/ml and the contractions were recorded for
2.4.2. Formaldehyde-induced paw oedema further 10–15 min. Aspirin was used as the positive refer-
Male rats (n = 9/group) were treated with 1.25, 2.5 and ence drug. The latent period for the initiation of contractions
5 g/kg/day of MFL and 5 ml/kg/day of water for 7 consecu- and the percent reduction of the amplitude and frequency
tive days. After 1 h on days 1 and 3 of treatment, these rats of contractions with respect to the normal contractions was
were injected with 0.1 ml of 2% formaldehyde into the foot calculated.
pad of left hind paw (Selye, 1949). Paw oedema was mea-
sured 1 h before formaldehyde injection and at 4 h after the 2.6.2. Membrane stabilising activity
injection on day 1 and everyday at 1 h after the treatment The experiment was done using heat-induced haemolysis
for 7 consecutive days. of rat erythrocytes in vitro as described by Perez et al. (1995)
M.G. Dharmasiri et al. / Journal of Ethnopharmacology 87 (2003) 199–206 201
and Dharmasiri et al. (2003). Vials containing 20 ␮l fresh strength) followed by the bridge test (to evaluate muscle
rat blood in 1 ml of phosphate-buffered saline were treated co-ordination) and the latency to fall and slide off was
in triplicate with the extract so that the final concentration determined, respectively (Plaznic et al., 1993).
of the MFL in the vials became 2.5, 5, 7.5 and 10 mg/ml.
Fifteen microlitres of saline was used as the control while 2.7. Toxicity
aspirin was used as the positive reference drug. The vials
were then incubated for 15 min at 37 ◦ C followed by 54 ◦ C Male rats (n = 6/group) were treated either with
for 25 min, centrifuged and the absorbance of the super- 5 g/kg/day of MFL or 5 ml/kg/day of water for 14 consecu-
natant was measured at 540 nm spectrophotometrically. The tive days. After the treatment, these rats were continuously
percent inhibition of haemolysis with respect to the control observed for 1 h for overt clinical signs of acute toxic-
was calculated. ity or stress. They were daily observed for overt signs of
toxicity or stress during the period of treatment. The rats
2.6.3. Antioxidant activity were weighed using an animal balance (MP 6000, Chyo
The experiment was carried out using thiobarbitiuric acid Balance Corporation, Tokyo, Japan), prior to the start of
reactive substances assay as described by Dorman et al. the experiment and on day 1 of the post-treatment. On day
(1995). The vials containing the reagents were treated in trip- 1 of the post-treatment, blood was taken out from the tail
licate with the extract so that the final concentrations of the under mild ether anaesthesia, serum separated out. The
MFL in the vials became 1.25, 1.875, 2.5 and 3.125 mg/ml. serum activities of aspartate and alanine transaminases,
A 100 ␮g/ml of butylated hydroxytoluene (BHT) was used serum concentrations of glucose, urea and creatinine were
as the positive reference and distilled water was used in the determined using Randox assay kits. Then, the rats were
control. The vials were mixed well and incubated at 95 ◦ C for killed, stomachs were taken out, opened and examined for
60 min, allowed to cool, 5 ml of butanol was added, mixed macroscopic haemorrhagic gastric lesions.
well and centrifuged at 1500 × g. The absorbance of the
butanol layer was measured at 532 nm and the antioxidant 2.8. Analysis of data
index was calculated as follows:
Data are given as means±S.E.M. Statistical analyses were
T
Antioxidant index = 1 − × 100 done by using Student’s t-test, linear regression analysis and
C
Pearson’s correlation analysis, one-way ANOVA followed
where T is the absorbance of test and C absorbance of con- by Tukey’s Family Error Rate test. P = 0.05 was considered
trol. as significant.
2.6.4. Antihistamine activity

Rats of either sex (n = 9/group) of which fur on poste- 3. Results
rior lateral side have been shaved 24 h earlier were treated
with 1.25, 2.5 and 5 g/kg of MFL, 0.67 mg/kg of chlorpheni- 3.1. Anti-inflammatory activity
ramine and 5 ml/kg of water, respectively. After 1 h, these
rats were subcutaneously injected with 0.05 ml of 200 ␮g/ml 3.1.1. Carrageenan-induced paw oedema test
histamine dihydrochloride into the fur removed area of the When compared with the control, treatment with MFL
skin (Spector, 1956) under mild ether anaesthesia and the significantly (P < 0.05) and dose-dependently reduced the
area of the wheal formed after 1.5 min was calculated. paw oedema only at the 2 h after carrageenan injection
while there was no significant suppression at 4 h. There was
2.6.5. Sedative activity no significant difference between the paw oedema of rats
Rats of either sex (n = 9/group) were treated either with treated with MFL at preflowering and flowering stages of
5 g/kg of MFL or 5 ml/kg of water. After 1 h, all these rats the tree (data not shown). However, 5 mg/kg indomethacin
were tested on rat hole-board apparatus to determine seda- significantly suppressed paw oedema at both 2 and 4 h
tion as described by File and Wardill (1975). The rats were (Table 1). The suppression of paw oedema by MFL was
individually placed in the centre of rat hole-board apparatus inversely dose related (r 2 = 1, P < 0.01). The EC50 for the
and the number of rears, number of head dips, locomotory suppression of paw oedema by MFL was 2 g/kg.
activity and the number of faecal boluses produced were
recorded for 7.5 min. The time spent per head dip was then 3.1.2. Formaldehyde-induced paw oedema test
calculated. The treatment with 2.5 and 5 g/kg/day of MFL for 7
days significantly (P < 0.05) reduced the paw oedema
2.6.6. Muscle strength and co-ordination from days 4 to 6 when compared with the control. By
Rats of either sex (n = 9/group) were treated either day 7 of the treatment, the paw oedema in control and
with 5 g/kg of MFL or water. One hour later, these rats treated groups had almost disappeared. However, the treat-
were subjected to bar holding test (to evaluate muscle ment with 1.25 g/kg/day did not significantly suppressed the
Table 1 trol. The frequency of licking was significantly (P < 0.05)

Effect of oral treatment of Vitex negundo mature fresh leaves (MFL) on reduced only at the late phase. However, aspirin significantly
the carrageenan-induced paw oedema of rats
(P < 0.05) reduced the licking time and frequency at both
Dose Paw oedema (ml) the phases of the test. The licking time was significantly
2h 4h (P < 0.05) higher at the early phase than the late phase in
both MFL- and aspirin-treated rats (Table 4).
5 ml/kg water 0.52 ± 0.05 0.49 ± 0.02
1.25 g/kg MFL 0.23 ± 0.03∗∗ 0.45 ± 0.04
2.5 g/kg MFL 0.28 ± 0.04∗∗ 0.49 ± 0.06 3.3. Mechanisms of the anti-inflammatory and analgesic
5 g/kg MFL 0.38 ± 0.03∗ 0.43 ± 0.04 activities
5 mg/kg indomethacin 0.11 ± 0.02∗∗ 0.28 ± 0.03∗∗
Values are means ± S.E.M. (n = 9). 3.3.1. PG synthesis inhibition activity
∗ P < 0.05 as compared with the control (Student’s t-test).
The treatment with MFL does-dependently reduced the
∗∗ P < 0.01 as compared with the control (Student’s t-test).
amplitude and frequency of spontaneous contractions of
isolated dioestrous rat uterus indicating a PG synthesis in-
paw oedema (Table 2). The paw oedema suppression showed hibition (Table 5). The percent inhibition was inversely pro-
a bell-shaped dose–response relationship. portional to the concentration (for amplitude, r = −0.98,
P < 0.05; for frequency, r2 = 0.88, P < 0.05). For aspirin,
3.2. Analgesic activity percent inhibition of amplitude (r = 0.96, P < 0.05) and
frequency (r = 0.98, P < 0.05) was directly proportional
3.2.1. Hot plate and tail flick tests to the concentration. The EC50 for the inhibition of am-
As compared with the controls, the treatment with MFL plitude: MFL versus aspirin: 228.1 ␮g/ml versus 3.0 ␮g/ml
produced a significant and dose-dependent (r = 0.92, P < and the frequency: MFL versus aspirin: 257.7 ␮g/ml ver-
0.05) increase in the reaction time of rats at 1 h after treat- sus 3.6 ␮g/ml. The latent period for the initiation of the
ment in the hot plate test showing an analgesic effect. A inhibition: MFL versus aspirin: 228.0 ± 30.6 s versus
significant difference could not be observed between the re- 202.8 ± 33.0 s.
action times of rats treated with MFL at preflowering and
flowering stages of the tree (data not shown). The increase 3.3.2. Membrane stabilising activity
of the reaction time was significant only with the 2 and MFL dose-dependently inhibited the heat-induced
5 g/kg doses. The EC50 for the increase in reaction time was haemolysis of rat erythrocytes in vitro indicating a mem-
4.1 g/kg. However, aspirin produced a significant increase brane stabilising activity as seen with aspirin (Table 6).
in the reaction time at 1 and 3 h after treatment. There was However, the activity was inversely related to the concen-
no significant increase in the reaction time with 1.25 g/kg tration (r 2 = 0.9, P < 0.05) with MFL while with aspirin it
in the hot plate test and any of the doses in the tail flick was directly related to the concentration. EC50 for the mem-
test (Table 3). The treatment with 5 mg/kg of naloxone did brane stabilising activity: MFL versus aspirin: 2.6 mg/ml
not significantly reduce the reaction time of MFL-treated versus 44.4 ␮g/ml.
rats excluding an opioid receptor-mediated action (reaction
time: MFL + naloxone versus MFL + saline: 12.8 ± 1.4 s 3.3.3. Antioxidant activity
versus 13.6 ± 0.9 s). MFL showed a dose-dependent (r2 = 0.68) antioxidant
activity as indicated by the antioxidant index. However, BHT
3.2.2. Formalin test showed a higher antioxidant index than MFL (Table 7).
The treatment with MFL significantly (P < 0.05) reduced The dose–response relationship was curvilinear (r 2 = 0.68)
the licking time of the formalin-injected paw both in the and the EC50 for the antioxidant activity was 2.3 mg/ml
early and late phases of the test as compared with the con- MFL.
Table 2
Effect of oral treatment of mature fresh leaves (MFL) of Vitex negundo on the formaldehyde-induced paw oedema of rats
Dose Paw oedema (ml)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
5 ml/kg/day water 0.26 ± 0.03 0.24 ± 0.05 0.22 ± 0.05 0.46 ± 0.05 0.39 ± 0.02 0.31 ± 0.05 0.16 ± 0.03
1.25 g/kg/day MFL 0.31 ± 0.03 0.35 ± 0.04 0.15 ± 0.05 0.47 ± 0.08 0.36 ± 0.03 0.29 ± 0.04 0.09 ± 0.04
2.5 g/kg/day MFL 0.41 ± 0.03 0.21 ± 0.04 0.25 ± 0.04 0.26 ± 0.03∗∗ 0.18 ± 0.02∗∗ 0.13 ± 0.02∗∗ 0.11 ± 0.03
5.0 g/kg/day MFL 0.46 ± 0.04 0.22 ± 0.04 0.32 ± 0.04 0.31 ± 0.05∗ 0.24 ± 0.05∗∗ 0.18 ± 0.04∗ 0.11 ± 0.03
Values are means ± S.E.M. (n = 9).
Table 3
Effect of oral treatment of mature fresh leaves (MFL) of Vitex negundo on the reaction time of rats
Reaction time(s)
Hot plate Tail flick
Pre-treatment 1h 3h Pre-treatment 1h 3h
5 ml/kg water 11.8 ± 0.8 11.1 ± 0.7 12.3 ± 0.9 1.5 ± 0.2 1.8 ± 0.2 1.6 ± 0.1
1.25 g/kg MFL 12.2 ± 1.0 9.1 ± 0.8 11.1 ± 0.8 1.7 ± 0.2 1.4 ± 0.8 1.8 ± 0.2
2.5 g/k MFL 10.9 ± 0.5 15.2 ± 1.5∗ 11.3 ± 2.4 1.8 ± 0.1 1.9 ± 0.2 1.7 ± 0.1
5 g/kg MFL 12.2 ± 0.6 18.8 ± 1.6∗∗ 13.3 ± 1.6 1.5 ± 0.2 1.2 ± 0.1 1.3 ± 0.1
100 mg/kg aspirin 9.5 ± 1.2 15.3 ± 1.7∗ 16.4 ± 1.4∗ 1.5 ± 0.1 1.3 ± 0.1 1.1 ± 0.1
Table 4
Effect of oral treatment of mature fresh leaves (MFL) of Vitex negundo on the licking time and frequency of rats in the formalin test
Dose Early (first) phase (0–5 min) Late (second) phase (20–25 min)
Licking time (s) Licking frequency (min−1 ) Licking time (s) Licking frequency (min−1 )
5 ml/kg water 59.3 ± 5.3 12.4 ± 1.4 48.8 ± 7.4 10.1 ± 1.2
1.25 g/kg MFL 37.9 ± 4.5∗ 8.4 ± 0.8 21.9 ± 5.1∗∗ 6.0 ± 1.9∗
2.5 g/kg MFL 43.0 ± 6.1∗ 12.0 ± 1.4 14.8 ± 5.2∗∗ 9.0 ± 4.0
5 g/kg MFL 39.4 ± 6.0∗ 8.8 ± 1.0 14.2 ± 4.1∗∗ 4.1 ± 0.9∗∗
100 mg/kg aspirin 20.8 ± 3.0∗∗ 7.0 ± 1.5∗∗ 8.3 ± 2.2∗∗ 4.9 ± 1.5∗∗
3.3.4. Antihistamine activity 3.3.5. Sedative activity

As compared with the control treatment with MFL signif- The treatment with MFL failed to significantly alter the
icantly (P < 0.01) and dose-dependently reduced the area parameters of rat hole-board test compared to control (data
of the wheal formed on the rat skin by the injection of his- not shown), showing that there is no sedative action in MFL.
tamine indicating an antihistamine activity. A 0.67 mg/kg of
chlorpheniramine also significantly (P < 0.01) reduced the 3.3.6. Muscle relaxation and co-ordination
area of the wheal (Fig. 1). The antihistamine activity of MFL When compared with the control, the treatment with MFL
is inversely dose related (r = −0.98, P < 0.05). EC50 for failed to significantly alter the latency to fall in the bar
the antihistamine activity was 0.99 g/kg of MFL. holding test (control versus treatment: 60.0 ± 0.1 versus
Table 5
Prostaglandin synthesis inhibition activity of different concentrations of Table 6
mature fresh leaves (MFL) of Vitex negundo and aspirin as indicated by Membrane stabilising effect of different concentrations of Vitex negundo
the reduction of spontaneous contractions of isolated rat uterus at dioestrus mature fresh leaves (MFL) and aspirin as indicated by the inhibition of
with respect to normal contraction heat-induced haemolysis of rat erythrocytes in vitro
Concentration % reduction % reduction Concentration % inhibition

(␮g/ml) of amplitude of frequency 2.5 mg/ml MFL 31.2 ± 0.9
100 MFL 71.3 ± 3.1 70.1 ± 9.0 5 mg/ml MFL 26.8 ± 2.8
200 MFL 57.5 ± 6.8 50.9 ± 6.3 7.5 mg/ml MFL 16.5 ± 2.5
300 MFL 51.1 ± 15.1 48.9 ± 7.6 10 mg/ml MFL 6.6 ± 2.6
400 MFL 37.2 ± 6.5 33.8 ± 10.0 5 ␮g/ml aspirin 17.6 ± 1.6
2 aspirin 36.9 ± 3.5 19.4 ± 4.0 10 ␮g/ml aspirin 20.9 ± 1.8
Values are means ± S.E.M. (n = 3/concentration). Values are means ± S.E.M.
There was a significant relationship (P < 0.05) between the concentration There was a significant (P < 0.05) relationship between the concentration
and percent reduction (linear regression, Pearson’s correlation). and percent inhibition (linear regression analysis).
Fig. 1. Antihistamine activity of Vitex negundo mature fresh leaves as indicated by the area of the wheal. Data are given as means ± S.E.M. ∗ P < 0.01
as compared with the control (one-way ANOVA, Tukey’s Family Error Rate test).
56.9 ± 2.1) or latency slide off in the bridge test (control 4. Discussion
versus treatment: 58.3 ± 1.7 versus 55.6 ± 2.2).
Experimental investigations revealed that the MFL of
3.4. Toxicity Vitex negundo have dose-dependent activity against inflam-
mation as revealed in the carrageenan and formaldehyde
The treatment with 5 g/kg/day of MFL for 14 days failed models. Further, hot plate test and the formalin test re-
to produce any overt clinical signs of toxicity or stress. The vealed that MFL can also suppress acute pain. However,
treatment also did not significantly alter the body weights the anti-inflammatory activity is 1.7 times lower than in-
(control versus treatment: 232.0 ± 8 g versus 251.8 ± 5.6 g), domethacin in the carrageenan model while the analgesic
serum creatinine (control versus treatment: 1.7 ± 0.3 mg/dl activity is 1.2 times lower than aspirin in the formalin test.
versus 1.1 ± 0.2 mg/dl), urea (control versus treatment: MFL demonstrated a dose-dependent PG synthesis inhibi-
33.6 ± 3.0 mg/dl versus 38.6 ± 1.2 mg/dl), random glu- tion, membrane stabilising, antihistamine and antioxidant
cose (control versus treatment: 136.2 ± 8.0 mg/dl versus activities. The inverse dose–response relationship shown
141.5 ± 6.6 mg/dl) and the activity of ALT (control versus by acute anti-inflammatory, antihistamine, PG synthesis
treatment: 12.0±2.1 U/l versus 15.0±2.0 U/l). However, the inhibition and membrane stabilising activities may be due
treatment caused a significant (P < 0.05) increase in serum to reduction of the effectiveness of the active principle at
activity of AST (control versus treatment: 24.3 ± 5.1 U/l its high concentrations. However, further investigations are
versus 74.7 ± 5.5 U/l). The treatment also did not cause needed to confirm this suggestion. This type of relation-
haemorrhagic lesions in the gastric mucosa after 14 days of ship indicates that the concentrations used in these tests
treatment. are within the therapeutic window of MFL in which certain
drugs exert their maximum curative effect (Tripathi, 1994).
According to Vinegar et al. (1987) and Antonio and Brito
Table 7 (1998) in the carrageenan model, the early phase (1–2 h) is
Antioxidant activity of different concentrations of mature fresh leaves mainly mediated by histamine, serotonin and the increase
(MFL) of Vitex negundo of PG synthesis in the surroundings of the damaged tissues
Concentration (mg/ml) Antioxidant index while the late phase is mainly mediated by bradykinin,
leukotrienes, polymorphonuclear cells and PGs produced
1.25 MFL 40.2 ± 0.1
1.875 MFL 38.8 ± 1.2 in tissue macrophages. In this experiment, the suppression
2.5 MFL 33.7 ± 0.5 of inflammation at the early phase of inflammation can be
3.125 MFL 45.6 ± 0.1 contributed by PG synthesis inhibition and antihistamine
0.1 BHT 57.4 ± 1.2 activities shown by MFL. The lack of anti-inflammatory
Values are means ± S.E.M. activity at the second phase may indicate the short duration
of action of MFL and the increase of the leukotriene at the analgesic activity in the hot plate test, analgesic action op-
second phase caused by the inhibition of PG synthesis in erating through opioid receptors can be ruled out. Muscle
the first phase because inhibition of PG synthesis diverts the relaxants can produce false positive results in the hot plate
reaction towards increase in leukotrienes synthesis (Mayes, test indicating analgesic activity (Gracioso et al., 1998).
1996). Although opioid receptor-mediated activities can However, there was no muscle relaxant activity induced by
suppress the inflammation in the carrageenan-induced paw MFL in the bar holding test. Stress can produce analgesia
oedema (Planas et al., 2000), such activity can be excluded (Ganong, 1995). There was no sign of stress observed in
here because the naloxone test revealed that the leaves do the rats treated with the MFL.
not act via opioid receptors. The anti-inflammatory and analgesic activities of the
In the formaldehyde-induced paw oedema model, the leaves did not disappear after the flowering of the tree in
anti-inflammatory activity was evident from days 4 to 6 contrast to Anisomeles indica which lost these activities
of the treatment, indicating that MFL is effective against after flowering of the plant (Dharmasiri et al., 2002, 2003).
the establishment of chronic inflammation which happens The treatment of MFL for 14 days did not produce de-
at the later stage of acute inflammation (Hanna and Poste, tectable toxic effect in terms of body weight, serum con-
1991). This action can be contributed by the PG synthesis centrations of urea, creatinine, glucose and serum activity
inhibition, membrane stabilising and antioxidant activities of ALT. The treatment did not cause gastric lesions which
of MFL as reduced PG synthesis and oxidation and the is a beneficial effect compared to the modern NSAIDs. The
membrane stabilisation play key roles in countering the reason for the increase of serum activity of AST has to be
establishment of chronic inflammation (Perez et al., 1995; further investigated. Therefore, people should be cautious
Rang et al., 1995). As seen in this experiment, the ability when the leaves are used in oral preparations.
of a drug to suppress inflammation when it is applied after In conclusion, these observations provide evidence for
the onset of inflammation is likely to be due to the genuine the anti-inflammatory and analgesic properties of mature
anti-inflammatory activity of the drug (Duwiejua et al., fresh leaves of Vitex negundo claimed in Ayurveda medicine.
1994). These observations provide the support for the use Also, it uncovered some of the possible mechanisms of these
of MFL of Vitex negundo as anti-inflammatory agent in the actions. Further studies will be undertaken to correlate the
Ayurveda medicine while the strong antihistamine activity pharmacological activities with the chemical constituents.
confirms the use of MFL to counter skin itching.
The analgesic activity shown only in the hot plate test
reveals that the activity is supraspinally mediated (Hough Acknowledgements
et al., 1999) and can be brought about by the PG synthesis
inhibition activity of MFL. Dr. G.A.S. Premakumara of Industrial Technology Insti-
In the formalin test, the pain in the early phase is caused tute, Sri Lanka, is acknowledged for testing the antioxidant
due to the direct stimulation of the sensory nerve fibres activity of leaves.
by formalin while the pain in the late phase is due to in-
flammatory mediators, like histamine, PGs, serotonin and
bradykinin (Murray et al., 1988; Tjolsen et al., 1992). The
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Hypoglycaemic effect of water extracts of Aegle marmelos

fruits in streptozotocin diabetic rats
N. Kamalakkannan, P. Stanely Mainzen Prince∗
Department of Biochemistry, Annamalai University, Annamalai Nagar, Tamil Nadu 608002, India
Received 8 August 2002; received in revised form 12 February 2003; accepted 16 April 2003
Abstract
Aegle marmelos Corr. (Rutaceae) is widely used in Indian Ayurvedic medicine for the treatment of diabetes mellitus. The hypoglycaemic
effect of the water extract of the fruits of Aegle marmelos was examined in streptozotocin-induced diabetic Wistar rats. Oral administration of
the water extract (125 and 250 mg kg−1 ) twice a day for 4 weeks resulted in significant reductions in blood glucose, plasma thiobarbituric acid
reactive substances, hydroperoxides, ceruloplasmin and ␣-tocopherol and a significant elevation in plasma reduced glutathione and Vitamin
C in diabetic rats. The effect of the extract at a dose of 250 mg kg−1 was more effective than glibenclamide in restoring the values of these
parameters. The results of this study clearly shows the hypoglycaemic activity of the fruit extract.
© 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Aegle marmelos; Streptozotocin diabetes; Fruit extract; Plasma lipid peroxides; Antioxidants
1. Introduction of diarrhoeas, dysenteries and diabetes mellitus (Chopra

et al., 1958; Saheb Dass, 1969). Preliminary reports indi-
Diabetes mellitus is a metabolic disease as old as mankind cate Aegle marmelos leaf extract exhibits antidiabetic action
and its incidence is considered to be high (4–5%) all over in glucose-induced hyperglycaemic rats (Sachdewa et al.,
the world (Pickup and Williams, 1997). Active oxygen 2001) and in alloxanized diabetic rats (Ponnachan et al.,
metabolism plays a key role in the normal functioning of 1993a,b). Karunanayake et al. (1984) have reported that
the ␤-cells of pancreas. Free oxygen radicals and oxidative aqueous decoction of root bark exhibits hypoglycaemic
stress appears to be an important element of the produc- action in rats. There are no available reports on the pharma-
tion of secondary complications in diabetes (Thomson and cological actions of Aegle marmelos fruit extract. This in-
McNeil, 1993; Thornalley et al., 1996). Hyperglycaemia vestigation has been carried out to see, whether the aqueous
generates reactive oxygen species which in turn cause lipid extract of Aegle marmelos fruits (AMFEt) exert any effect
peroxidation and membrane damage (Hunt et al., 1988). on blood glucose, plasma lipid peroxides and non-enzymatic
Elevated levels of circulating lipid peroxides have been antioxidants in streptozotocin (STZ)-induced diabetic rats.
registered in diabetic patients and experimental diabetic The effect of the extract was compared with glibenclamide,
animals (Srinivasan et al., 1997; Stanely Mainzen Prince a well-known hypoglycaemic drug.
and Menon, 1998). Circulating lipid peroxides were shown
to be capable of initiating atherosclerosis which is a well
known late feature of diabetes (Hessler et al., 1983). 2. Materials and methods
Many indigenous drugs have been used by practitioners
of Ayurvedic system for the treatment of diabetes mellitus 2.1. Plant extract
in India. Different parts of Aegle marmelos Corr. (Rutaceae)
(leaves and ripe fruits) have been used in the treatment An aqueous Aegle marmelos fruit extract (brown dry pow-
der) was received as a gift from Chemiloids (manufactur-
ers and exporters of herbal extracts), Vijayawada, Andhra
∗ Corresponding author. Tel.: +91-4144-238343. Pradesh (India). Herb-to-product ratio is 8:1. The extract
E-mail address: psmprince@rediffmail.com (P.S.M. Prince). was suspended in distilled water prior to use.
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0378-8741(03)00148-X
208 N. Kamalakkannan, P.S.M. Prince / Journal of Ethnopharmacology 87 (2003) 207–210
2.2. Drugs and chemicals et al. (1973), respectively. Plasma ceruloplasmin and
␣-tocopherol were determined according to the procedures
All the drugs and biochemicals used in this experiment of Ravin (1961) and Desai (1984), respectively.
were purchased from Sigma, St. Louis, MO, USA. All other
chemicals were of analytical grade. 2.7. Statistical analysis
2.3. Induction of diabetes in rats Statistical analysis was performed using one way anal-
ysis of variance (ANOVA) followed by Duncan’s Multiple
Female albino Wistar rats of body weight 160–190 g proc- Range Test (DMRT). Results were expressed as mean±S.D.
ured from the Central Animal House, Department of Ex- from six rats in each group. P-values <0.05 were considered
perimental Medicine, Rajah Muthiah Medical College and significant.
Hospital, Annamalai University were used in the present
study. The rats were fed on a standard pellet diet (Kamad-
henu Agencies, Bangalore, India) and water was freely avai- 3. Results
lable. Diabetes was induced in rats by a single intraperitoneal
injection of freshly prepared streptozotocin (45 mg kg−1 Glucose levels measured in blood of normal and ex-
body weight) in 0.1 M citrate buffer (pH = 4.5) in a volume perimental rats are given in Table 1. STZ-treated diabetic
of 1 ml kg−1 (Siddique et al., 1987). Forty-eight hours after rats showed significantly increased levels of blood glu-
STZ administration, blood glucose level of each rat was de- cose. Aqueous Aegle marmelos extract treatment showed a
termined. Rats with a blood glucose range of 250–300 mg/ significant decrease in blood glucose in diabetic rats.
100 ml were considered diabetic and included in the study. The levels of lipid peroxides, thiobarbituric acid reactive
substances and hydroperoxides in plasma in normal and ex-
2.4. Experimental design perimental animals are depicted in Table 2. Diabetic animals
showed significantly increased levels of lipid peroxides and
In the experiment, a total of 30 rats (6 normal; 24 STZ hydroperoxides. Aqueous Aegle marmelos extract treatment
diabetic surviving rats) were used. The rats were divided resulted in significantly lower levels of lipid peroxides and
into five groups of six rats each. hydroperoxides in diabetic rats.
Vitamin C and reduced glutathione levels measured in
Group 1: Normal untreated rats.
plasma of normal and experimental animals are shown
Group 2: STZ-treated diabetic rats.
in Table 3. Diabetic rats had significantly lower levels of
Groups 3 STZ-treated diabetic rats administered
plasma Vitamin C and reduced glutathione. Aqueous Aegle
and 4: AMFEt orally (125 and 250 mg kg−1
body weight) in distilled water using an
Table 1
intragastric tube twice a day for 4 weeks.
Effect of Aegle marmelos extract on blood glucose in diabetic rats
Group 5: STZ-treated diabetic rats given
glibenclamide orally (300 ␮g kg−1 body Group Blood glucose (mg/dl)
weight) in distilled water using an Initial Final

intragastric tube twice a day for 4 weeks. Normal 83.7 ± 5.4 87.6 ± 6.2a
Streptozotocin-treated 268.1 ± 15.2 309.8 ± 31.5b
After 4 weeks of treatment, all the rats were decapitated Streptozotocin + extract (125 mg) 260.6 ± 17.9 198.2 ± 19.9c
after fasting overnight. Blood was collected in potassium Streptozotocin + extract (250 mg) 262.3 ± 20.1 98.7 ± 8.3a
oxalate and sodium fluoride containing tubes for the es- Streptozotocin + glibenclamide 270.6 ± 19.3 129.0 ± 8.2d
timation of fasting blood glucose. Plasma was separated (300 ␮g)
for the estimation of thiobarbituric acid reactive substances Each value is the mean ± S.D. of six rats in each group. Values not
(TBARS), hydroperoxides (HP), Vitamin C, reduced glu- sharing a common superscript differ significantly at P < 0.05 (DMRT).
tathione (GSH), ceruloplasmin and ␣-tocopherol.
Table 2
Effect of Aegle marmelos extract on plasma TBARS and HP in diabetic
2.5. Estimation of blood glucose and lipid peroxides rats
Group TBARS HP (values ×
Fasting blood glucose was estimated by the method of (nM/ml) 10−5 mM/dl)
Sasaki et al. (1972). Plasma thiobarbituric acid reactive sub-
Normal 2.2 ± 0.2a 7.2 ± 0.3a
stances and hydroperoxides were determined by the meth- Streptozotocin-treated 3.9 ± 0.3b 11.3 ± 0.7b
ods of Yagi (1976) and Jiang et al. (1992), respectively. Streptozotocin + extract (125 mg) 3.2 ± 0.3c 9.3 ± 0.9c
Streptozotocin + extract (250 mg) 2.7 ± 0.3d 7.5 ± 0.5a,d
2.6. Determination of non-enzymatic antioxidants Streptozotocin + glibenclamide 3.0 ± 0.2c,d 8.1 ± 0.5d
(300 ␮g)
Plasma Vitamin C and reduced glutathione were esti- Each value is the mean ± S.D. of six rats in each group. Values not
mated by the methods of Omaye et al. (1979) and Rotruck sharing a common superscript differ significantly at P < 0.05 (DMRT).
N. Kamalakkannan, P.S.M. Prince / Journal of Ethnopharmacology 87 (2003) 207–210 209
Table 3 ministration of Aegle marmelos fruit extract decreases blood

Effect of Aegle marmelos extract on plasma Vitamin C and GSH in glucose in diabetic rats. This shows the hypoglycaemic ef-
diabetic rats fect of the fruit extract. Phytochemical studies have shown
Group Vitamin C GSH (mg/dl) the presence of coumarins such as marmelosin, alloimpera-
(mg/dl) torin, ␤-sitosterol and d-␣-phellandrene in Aegle marmelos
Normal 1.5 ± 0.1a 23.2 ± 1.8a fruits (Chopra et al., 1958). Shani et al. (1974) have reported
Streptozotocin-treated 0.9 ± 0.1b 14.7 ± 1.1b that coumarins exhibit hypoglycaemic action in diabetic rats.
Streptozotocin + extract (125 mg) 1.0 ± 0.0b,c 19.4 ± 1.8c
The hypoglycaemic effect may be due to the presence of
Streptozotocin + extract (250 mg) 1.2 ± 0.1d 22.8 ± 1.6a
Streptozotocin + glibenclamide 1.1 ± 0.1c 21.4 ± 1.5a,c coumarins in the fruit extract.
(300 ␮g) We also observed elevated levels of thiobarbituric acid
Each value is the mean ± S.D. of six rats in each group. Values not reactive substances and hydroperoxides in plasma of
sharing a common superscript differ significantly at P < 0.05 (DMRT). STZ-induced diabetes in rats. Elevated levels of perox-
ides observed in plasma are a consequence of increased
Table 4 production and liberation of lipid peroxides into the cir-
Effect of Aegle marmelos extract on plasma ceruloplasmin and culation due to pathological changes (Stanely Mainzen
␣-tocopherol in diabetic rats
Prince and Menon, 1998). Similar findings were reported
Group Ceruloplasmin ␣-Tocopherol for STZ-induced diabetes in rats (Kinalski et al., 2000).
(mg/dl) (mg/dl)
Alterations in the non-enzymatic antioxidants were ob-
Normal 20.7 ± 2.9a 1.6 ± 0.1a served in diabetic animals. The plasma protein, ceruloplas-
Streptozotocin-treated 36.3 ± 2.8b 3.6 ± 0.3b
Streptozotocin + extract (125 mg) 27.8 ± 1.9c 2.4 ± 0.2c
min is a powerful non-enzymatic antioxidant that inhibits
Streptozotocin + extract (250 mg) 22.0 ± 1.7a,d 1.9 ± 0.0d lipid peroxidation by binding to copper (Halliwell and
Streptozotocin + glibenclamide 24.9 ± 1.9c,d 2.1 ± 0.1e Gutteridge, 1990). Dormandy (1980) has shown that ceru-
(300 ␮g) loplasmin levels increase under conditions leading to the
Each value is the mean ± S.D. of six rats in each group. Values not generation of superoxide radicals and hydrogen peroxide.
sharing a common superscript differ significantly at P < 0.05 (DMRT). The observed increase in plasma ceruloplasmin in diabetic
animals may be due to elevated lipid peroxides. Analogous
marmelos extract treatment resulted in significant increase findings were observed in experimental diabetes (Stanely
in plasma Vitamin C and reduced glutathione in diabetic Mainzen Prince and Menon, 1998).
rats. ␣-Tocopherol is the most important lipid soluble, radical
The levels of plasma ceruloplasmin and ␣-tocopherol in scavenging antioxidant. It reduces lipid hydroperoxides gen-
normal and experimental animals are illustrated in Table 4. erated during the process of peroxidation and protects the
Diabetic rats had significantly increased levels of cerulo- cell structures against damage (Kinalski et al., 2000). The
plasmin and ␣-tocopherol. Aqueous Aegle marmelos extract elevated levels of ␣-tocopherol noted in diabetic rats play
treatment resulted in significant reduction in plasma cerulo- a protective role against increased peroxidation in diabetes
plasmin and ␣-tocopherol in diabetic rats. (Stanely Mainzen Prince and Menon, 1998). Similar results
For all the parameters studied, Aegle marmelos at a dose have been observed by other workers for STZ-induced dia-
of 125 mg kg−1 showed a significant effect and 250 mg kg−1 betes in rats (Asayama et al., 1994).
showed a highly significant effect and brought back all the Glutathione protects the tissues from free radical damage.
parameters to near normal levels. The effect at a dose of It inhibits free radical-mediated lipid peroxidation (Meistor
250 mg kg−1 was better than that of glibenclamide. and Anderson, 1983). We observed a decrease in the levels
of GSH in plasma in diabetic rats. It appears that generation
of oxygen radicals by increased levels of glucose causes
4. Discussion utilization of GSH and thus diminishes GSH levels in the
plasma in diabetes (Stanely Mainzen Prince and Menon,
Oral administration of an aqueous extract of Aegle 1998). In this context, Sajithlal et al. (1998) also reported di-
marmelos fruit has shown hypoglycaemic effect against minished levels of serum GSH in experimental diabetic rats.
streptozotocin-induced diabetes in rats. The extract lowered Vitamin C is an excellent hydrophilic antioxidant in
significantly the levels of blood glucose, plasma thiobarbi- plasma, because it disappears faster than other antioxidants
turic acid reactive substances, hydroperoxides, ceruloplas- when plasma is exposed to reactive oxygen species (Frei
min and ␣-tocopherol and significantly increased the levels et al., 1986). The observed decrease in plasma Vitamin
of plasma GSH and Vitamin C. C may be due to increased utilization as an antioxidant
To our knowledge, this is the first study in which a follow defence against increased reactive oxygen species or due
up was made on the effect of Aegle marmelos extract in the to a decrease in GSH level, since GSH is required for the
levels of glucose, lipid peroxides and non-enzymatic antiox- recycling of Vitamin C (Inefers and Sies, 1988). Similar ob-
idants in diabetic animals. In our study, we observed elevated servations were reported in STZ-induced diabetes (Sajithlal
levels of blood glucose in streptozotocin diabetic rats. Ad- et al., 1998).
210 N. Kamalakkannan, P.S.M. Prince / Journal of Ethnopharmacology 87 (2003) 207–210
In conclusion, aqueous extract of Aegle marmelos fruit ex- Pickup, J.C., Williams, G., 1997. Epidemiology of diabetes mellitus.
hibited hypoglycaemic effect in streptozotocin-induced dia- In: Text Book of Diabetes, 2nd ed., Vols. 1–2. Blackwell, Oxford,
pp. 3.1–3.28.
betes in rats. The effect of the extract on plasma antioxidants Ponnachan, P.T.C., Paulose, C.S., Panikkar, K.R., 1993a. Hypoglycaemic
is due to reduction in blood glucose concentration in dia- effect of alkaloid preparation from leaves of Aegle marmelos. Amala
betic rats. The extract at a dose of 250 mg kg−1 was more Research Bulletin 13, 37–41.
effective than glibenclamide. Further studies are necessary Ponnachan, P.T.C., Paulose, C.S., Panikkar, K.R., 1993b. Effect of leaf
to determine the exact nature of the active principle and the extract of Aegle marmelos in diabetic rats. Indian Journal of Experi-
mental Biology 31, 345–347.
mechanism of action of the fruit extract. Ravin, H.A., 1961. An improved colorimetric enzymatic assay of ceru-
loplasmin. Journal of Laboratory and Clinical Medicine 589, 161–
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Potential risk of acute hepatotoxicity of kodo poisoning

due to exposure to cyclopiazonic acid
Mary Antony a , Yogeshwar Shukla b , K.K. Janardhanan c,∗
a National Environmental Engineering Research Institute (NEERI), CSIR Complex South Kalamassery, Cochin 693 109, India
b Industrial Toxicology Research Centre, M.G. Marg, Lucknow 226 001, India
c Amala Cancer Research Centre, Amala Nagar, Trichur 680 553, India
Received 19 August 2002; received in revised form 9 April 2003; accepted 16 April 2003
Abstract
Kodo millet (Paspalum scrobiculatum L.) is a staple food of some sections of people of North India. Consumption of Kodo millet is
often found to cause intoxication and poisoning. The grains are frequently infested with Aspergillus tamarii Kita, which produced substantial
amount of a mycotoxin, cyclopiazonic acid (CPA). Investigations were carried out to evaluate the hepatotoxic/preneoplastic changes in rat liver
following single and multiple dose administration of CPA. Results showed a marked increase in the activity of glutamate pyruvate transaminase
(GPT) and glutamate oxaloacetate transaminase (GOT) following CPA exposures, suggesting acute hepatotoxicity. Significant increase was
also observed in gamma glutamyl transpeptidase (GGT) activity following CPA exposures, indicating preneoplastic changes in the liver. The
results reveal that Kodo poisoning might cause acute hepatotoxicity in men and animals. The findings thus suggest that the consumption of
contaminated Kodo millet is a serious health hazard due to exposure to CPA produced by Aspergillus tamarii associated with the millet.
Keywords: Kodo millet; Aspergillus tamarii; Cyclopiazonic acid; Hepatotoxic; Hepatocarcinogenic
1. Introduction Aspergillus oryzae, Aspergillus flavus (Holzapfel, 1968;

Ohmomo et al., 1973; Orth, 1977; Luk et al., 1977; Still
Kodo millet (Paspalum scrobiculatum L.) is a staple food et al., 1978). Morrissey et al. (1985) reported that CPA was
of some sections of people in parts of North India. However, acutely toxic to rats and produced focal necrosis in many
Kodo poisoning is a toxic syndrome encountered in men and organs. In chickens, CPA exposure caused reduced weight
animals in areas where this millet is grown and consumed. gain and produced proventricular lesions (Dorner et al.,
The consumption of Kodo millet is often reported to cause 1983). Neuhring et al. (1985) found that main target organs
intoxication and poisoning. The millet is reported to be toxic of CPA-induced toxicity in dogs were gastrointestinal tract
to animals when consumed (Bazlur, 1960). The symptoms and kidneys. In this communication, we report the acute
of Kodo poisoning in the affected people are characterized hepatotoxicity and preneoplastic changes in rat liver follow-
by nausea, vomiting, delirium, depression, intoxication, ing oral administration of CPA, isolated from Aspergillus
and unconsciousness. Kodo millet is often found heavily tamarii associated with Kodo millet collected from Uttar
infested with Aspergillus tamarii Kita. Janardhanan et al. Pradesh, India, in the month of September. The voucher
(1984) isolated fumigaclavin A from Aspergillus tamarii as- specimen of Aspergillus tamarii was deposited in CAB
sociated with Kodo millet and suggested that the compound International Mycological Institute, UK (IMI-260311).
might be responsible for part of Kodo poisoning symptoms.
The fungus was also reported to produce significant amount
of a mycotoxin, cyclopiazonic acid (CPA; Fig. 1) (Dorner, 2. Materials and methods
1983; Rao and Husain, 1985). CPA has been reported to
be isolated from a number of fungi namely Penicillium cy- Gamma glutamyl-p-nitroanilide hydrochloride was pur-
clopium, Penicillium camembertii, Aspergillus versicolor, chased from Sigma Chemical Co. (St. Louis, MO). Glycyl-
glycine and 2,4-dinitrophenyl hydrazine were obtained from
∗ Corresponding author. Fax: +91-487-2307020. BDH, UK. The rest of the chemicals used were of analytical
E-mail address: kkjanardhanan@yahoo.com (K.K. Janardhanan). grade.
doi:10.1016/S0378-8741(03)00146-6
212 M. Antony et al. / Journal of Ethnopharmacology 87 (2003) 211–214
(5 mg in 200 ␮l vegetable oil) once; Group III: vegetable oil

(200 ␮l/animal) three times alternate days; Group IV: CPA
(5 mg in 200 ␮l vegetable oil/animal) three times alternate
days.
2.3. Assay for biochemical parameters
All animals were sacrificed 48 h after the last dose by

Fig. 1. Structure of CPA.
stunning and their blood was collected by cardiac puncture
into clean dry centrifuge tubes. The livers were immediately
2.1. Isolation of CPA from Aspergillus tamarii taken out, cleaned, and weighed. The blood was allowed to
clot at 8–10 ◦ C and clear serum was collected after centrifu-
CPA was isolated by the method described by Rao and gation. One-fourth of the liver was homogenized (10% w/v)
Husain (1985) with some modifications. The fungus was in 0.05 M Tris buffer (pH 7.0) for the estimation of enzyme,
grown in several 1 l Erlenmeyer flasks containing 200 ml gamma glutamyl transpeptidase (GGT), and another part
modified Richards medium (g/l: KNO3 10.0, KH2 PO4 5.0, was homogenized in 0.25 M sucrose for the assay of gluta-
MgSO4 ·7H2 O 2.5, FeCl3 0.01, sucrose 50.0, and yeast ex- mate oxaloacetate transaminase (GOT), glutamate pyruvate
tract 1.0) for 10 days at 25–27 ◦ C. After the incubation pe- transaminase (GPT), and lipid peroxidation. Third portion of
riod, the cultures were harvested, centrifuged at 4000 rpm the liver was homogenized in 0.15% KCl containing 1 nmol
for 15 min, and mycelial biomass and culture filtrate were nicotinamide and used for the assay for aryl hydrocarbon
separated. The mycelium was washed, dried at 45–50 ◦ C hydroxylase (AHH) and aminopyrene-N-demethylase. The
for 48 h, and powdered. The culture filtrate (10 l) and dried fraction used for non-protein thiols (NPSH) was homoge-
mycelial powder were extracted separately with chloroform. nized in mannitol–EDTA.
The chloroform extract of culture filtrate and mycelium were GGT activity was assayed according to the method of
pooled and dried over anhydrous Na2 SO4 . The solvent evap- Roomi and Goldenburg (1981) in liver and in sera according
orated to dryness under vacuum. The residue, crude toxin to the method of Naftalin et al. (1969). GOT and GPT were
(3.5 g), was obtained. The crude toxin was analyzed for the assayed by the method of Wootton (1964) in liver and serum.
presence of CPA by TLC on silica gel G plates impregnated AHH was estimated by the method of Dehnean et al. (1973)
with 0.4N oxalic acid using chloroform:methylethylketone and aminopyrene-N-demethylase was estimated according to
(80:20) as solvent system. The plates were sprayed with Schenkman et al. (1967). Non-protein thiols were estimated
Ehrlich reagent (1 g p-dimethyl benzaldehyde in 30 ml of according to the method of Sedlak and Lindsay (1968). Lipid
50% HCl). CPA was detected as violet-colored spot (Rao peroxidation was measured by the method of Utley et al.
and Husain, 1985). The crude toxin was further purified (1967) and protein content of the samples was estimated by
by column chromatography using cellulose powder impreg- the method of Lowry et al. (1951).
nated with formamide:oxalic acid and then eluted with hex-
ane followed by hexane:benzene mixture (Holzapfel, 1968). 2.4. Statistical analysis
The hexane:benzene mixture fractions were combined and
the solvent evaporated completely at low temperature. Fur- Experimental data were expressed as mean ± S.D. The
ther purification of the toxin was done by preparatory TLC Student’s t-test was applied for detecting the significance.
on silica gel G impregnated with 0.4N oxalic acid using P < 0.05 was regarded as significant.
chloroform:methylethylketone (80:20) solvent system (Rao
and Husain, 1985). CPA band was removed carefully eluted
with chloroform–acetone. The fraction was washed several 3. Results and discussion
times with water to remove oxalic acid. The solvent was
completely evaporated and the residue contained chromato- Both single and multiple dose administration of CPA
graphically pure (99%) CPA (280 mg). had shown some behavioral changes like breathlessness
and lethargy, reluctance to move even on provocation. But
2.2. Treatment schedule a few hours after treatment, they became apparently nor-
mal. An oral dose of 50 mg/kg body weight CPA has been
Male Wistar rats (100 ± 5 g) maintained under environ- reported to cause severe liver lesions and cell necrosis in
mentally controlled conditions and standard solid pellet diet rats (Purchase, 1974). Hence, this dose was selected for the
were used for the experiments. The CPA (99% pure) was studies. Activity of aminopyrene-N-demethylase was found
dissolved in vegetable oil and administered at a dose of to be decreased in animals exposed to CPA (Table 1). The
50 mg/kg body weight (5 mg in 200 ␮l vegetable oil/animal). induction of aminopyrene-N-demethylase is considered to
The animals were divided in four groups. Group I: untreated be related with synthesis of cytochrome P-450 (Pelissier
control (vegetable oil 200 ␮l/animal) once; Group II: CPA and Albrecht, 1976). A significant increase in the AHH
M. Antony et al. / Journal of Ethnopharmacology 87 (2003) 211–214 213
Table 1
Effect of CPA on hepatic aryl hydrocarbon hydroxylase (AHH) and aminopyrene-N-demethylase
Parameter Group
I II III IV
AHH(nmol 3-OH B (a) P formed/h/mg protein) 2.90 ± 0.11 4.80 ± 0.31∗∗ 2.50 ± 0.26 5.60 ± 0.22∗∗∗
N-demethylase (nmol formaldehyde formed/min/mg protein) 3.90 ± 0.57 3.45 ± 0.48 4.40 ± 0.45 2.00 ± 0.25∗∗
Each value represents mean ± S.D. of six rats.
∗∗ P < 0.01.
∗∗∗ P < 0.001.
activity was observed in both single (Group II) and mul- normally high levels of GGT were also observed in tumors
tiple dose (Group IV)-treated animals (Table 1) which of a variety of tissues including hepatocellular carcinomas
indicated that there might be some involvement of poly- (Brelsterili, 1979).
cyclic aromatic hydrocarbon (PAH)-specific modulation The parameters examined to evaluate hepatic damage in
of aminopyrene-N-demethylase. The two-fold increase in this study were tissue and serum glutamate oxaloacetate
AHH activity indicated the possibility of CPA to induce transaminase (GOT and SGOT) and tissue and serum glu-
only CYP1A1- and CYP1A2-dependent induction, which tamate pyruvate transaminase (GPT and SGPT). Significant
might convert CPA to its toxic metabolite forms that could increase in the activity of both the enzymes in tissue and
bind to the macromolecules inside the cell (Ioannides et al., serum of CPA-exposed animals were observed. Induction in
1984). Therefore, it may be suggested that the AHH thus the activity of these two enzymes are reported to be associ-
induced may activate CPA to its intermediate forms for the ated with generalized hepatotoxicity.
expression of their mutagenic/preneoplastic/carcinogenic The levels of lipid peroxidation in the liver of CPA-exposed
potential. animals did not show any appreciable variation. This in-
A significant increase in the activity of GGT, which is a dicates that free radicals are not involved in CPA-induced
marker enzyme for the assessment of preneoplastic changes hepatotoxicity (Table 3). However, significant increase in
(Hanigan and Pitot, 1985; Periano et al., 1983), was ob- the non-protein thiols was found in animals exposed to
served in both liver and serum in the animals of the Groups CPA (Table 3). The increase in the levels of non-protein
II and IV (Table 2). GGT is known to catalyze the transfer of thiols may be due to the formation of deoxyribonucleo-
gamma glutamyl group of compounds containing this group side diphosphate (dNdp). The formation of dNdp in rel-
to a wide variety of amino acceptors (Meister, 1973). Ab- atively more efficient manner appears to be of metabolic
Table 2
Effect of CPA on gamma glutamyl transpeptidase (GGT), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) in
liver and serum of rats
Parameter Group
I II III IV
GGT (nmol p-nitroaniline liberated/min/mg protein) 3.89 ± 0.35 6.44 ± 0.45∗∗ 4.5 ± 1.1 18.0 ± 2.7∗∗∗
GOT (␮mol/min/g tissue) 7.84 ± 0.75 9.24 ± 0.82∗ 6.22 ± 0.43 16.33 ± 1.77∗∗
GPT (␮mol/min/g tissue) 39.87 ± 5.1 48.4 ± 2.7∗ 38.91 ± 3.20 98.53 ± 6.30∗∗∗
SGGT (nmol p-nitroaniline liberated/min/mg protein) 29.42 ± 1.42 35.49 ± 1.30∗ 27.33 ± 1.7 61.24 ± 2.98∗∗∗
SGOT (␮mol/min/g protein) 0.306 ± 0.029 0.459 ±0.017∗∗ 0.497 ± 0.013 0.752 ± 0.025∗∗∗
SGPT (␮mol/min/g protein) 3.269 ± 0.250 4.68 ± 0.23∗∗ 2.89 ± 0.17 4.949 ± 0.15∗∗∗
Each value represents mean ± S.D. of six rats.
∗ P < 0.05.
∗∗ P < 0.01.
∗∗∗ P < 0.001.
Table 3
Non-protein thiols and lipid peroxidation in liver of rats following exposure to CPA
Parameter Group
I II III IV
Non-protein thiols (nmol/mg protein) 17.80 ± 0.97 21.40±1.02∗ 20.2 ± 1.18 34.3 ± 1.62∗
Lipid peroxidation (nmol malonaldehyde formed/h/mg protein) 0.478 ± 0.024 0.56 ± 0.370 0.475 ± 0.04 0.60 ± 0.04
Each value represents mean ± S.D. of six animals.
∗ P < 0.05.
214 M. Antony et al. / Journal of Ethnopharmacology 87 (2003) 211–214
importance in cell proliferation subsequent to cellular dam- Ioannides, C., Lum, P.Y., Parke, D.V., 1984. Cytochrome P 448 and the
age (Holmgren, 1976). activation of toxic chemicals and carcinogens. Xenobiotica 14, 119–
127.
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peated oral administration of CPA may exert hepatotoxic/ clavin A by Aspergillus tamarii Kita. Canadian Journal of Microbiol-
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Antioxidant and antimicrobial activity of the essential oil and methanol

extracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae)
Ferda Candan a,∗ , Mehmet Unlu b , Bektaş Tepe c , Dimitra Daferera d ,
Moschos Polissiou d , Atalay Sökmen c , H. Aşkın Akpulat c
a Department of Chemistry, Faculty of Science and Literature, Cumhuriyet University, Sivas 58140, Turkey
b Department of Microbiology, Faculty of Medicine, Cumhuriyet University, Sivas 58140, Turkey
c Department of Biology, Faculty of Science and Literature, Cumhuriyet University, Sivas 58140, Turkey
d Laboratory of General Chemistry, Agricultural University of Athens, Iera Odos 75, Athens 118 55, Greece
Received 6 October 2002; received in revised form 14 April 2003; accepted 16 April 2003
Abstract
The in vitro antimicrobial and antioxidant activities of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium Afan.
(Asteraceae) were investigated. GC-MS analysis of the essential oil resulted in the identification of 36 compounds constituting 90.8% of the
total oil. Eucalyptol, camphor, ␣-terpineol, ␤-pinene, and borneol were the principal components comprising 60.7% of the oil. The oil strongly
reduced the diphenylpicrylhydrazyl radical (IC50 = 1.56 ␮g/ml) and exhibited hydroxyl radical scavenging effect in the Fe3+ –EDTA–H2 O2
deoxyribose system (IC50 = 2.7 ␮g/ml). It also inhibited the nonenzymatic lipid peroxidation of rat liver homogenate (IC50 = 13.5 ␮g/ml). The
polar phase of the extract showed antioxidant activity. The oil showed antimicrobial activity against Streptococcus pneumoniae, Clostridium
perfringens, Candida albicans, Mycobacterium smegmatis, Acinetobacter lwoffii and Candida krusei while water-insoluble parts of the
methanolic extracts exhibited slight or no activity. This study confirms that the essential oil of Achillea millefolium possesses antioxidant and
antimicrobial properties in vitro.
Keywords: Achillea millefolium; Antioxidant activity; Antimicrobial activity; Methanol extracts; Essential oil; GC-MS
1. Introduction essential oils of several Achillea species have been cited in

the literature. (Rustaiyan et al., 1998; Chalchat et al., 1999;
It has long been recognized that naturally occurring Simic et al., 2000).
substances in higher plants have antioxidant activity. Re- The genus Achillea (Family: Asteraceae, Section: San-
cently, there is a growing interest in oxygen-containing free tolinoidea) is represented by about 85 species mostly
radicals in biological systems and their implied roles as found in Europe and Asia and a handful in North America
causative agents in the aetiology of a variety of chronic dis- (Könemann, 1999). Forty species of Achillea are widely
orders. Accordingly, attention is focused on the protective distributed in Turkey (Davis, 1982). As far as ethnophar-
biochemical functions of naturally occurring antioxidants in macologic background is concerned, Achillea millefolium
the cells of the organisms containing them (Larson, 1988; is a well-known species amongst the members of Achil-
Halliwell, 1997). Antioxidants in the oils are important lea (Asteraceae) (Chandler et al., 1982). It is known as
in the stabilization of free fatty acids (Six, 1994; Baldioli “Civanperçemi” and used in folk remedies as an appetizer,
et al., 1996). The antioxidant activity of phenols and other wound healer, diuretic, carminative or menstrual regulator
compounds present in oils has been well and widely studied (Baytop, 1999). To the best of our knowledge, information
by several authors (Litridou et al., 1997; Visioli et al., 1998; concerning the in vitro antioxidant features of Achillea mille-
Yoshida and Takagi, 1999). Besides, reports concerning the folium subsp. millefolium has not been found in the literature.
local uses, pharmacological features of the extracts and the As a part of our continuing research project on the in vitro
antimicrobial and antioxidant activities of several Achillea
∗ Corresponding author. Tel.: +90-346-219-1010x1417; species, we previously reported antimicrobial activities of
fax: +90-346-219-1606. the essential oils isolated from Achillea setacea and Achil-
E-mail address: candan@cumhuriyet.edu.tr (F. Candan). lea teretifolia (Ünlü et al., 2002). The aim of this study was
doi:10.1016/S0378-8741(03)00149-1
216 F. Candan et al. / Journal of Ethnopharmacology 87 (2003) 215–220
to evaluate the in vitro antioxidant and antimicrobial prop- 2.5. Antioxidant activity
erties of the essential oil and methanol extracts of Achillea
millefolium subsp. millefolium (Asteraceae). 2.5.1. Hydroxyl radical scavenging activity
Hydroxyl radical scavenging was carried out by mea-
suring the competition between deoxyribose and the ex-
2. Materials and methods tract or crude oil for hydroxyl radicals, which attack
to deoxyribose leading to the formation of thiobarbi-
2.1. Collection of plant material turic acid reaction system (TBARS), generated from the
Fe3+ –ascorbate–EDTA–H2 O2 system (Kunchandy and
The herbal parts of Achillea millefolium subsp. mille- Rao, 1990). The formed TBARS were measured by using
folium were collected in Kızıldaǧ Pass, 130 km east of Sivas, the method described elsewhere (Ohkawa et al., 1979). Ex-
when flowering late-July 2001. The voucher specimens have periments were carried out in triplicate. All reagents were
been deposited at the Herbarium of the Department of Biol- prepared freshly.
ogy, Cumhuriyet University, Sivas, Turkey (CUFH-Voucher Inhibition (I) of deoxyribose degradation in percent was
No.: ED 6358). calculated in following way:

A0 − A1
2.2. Preparation of the methanolic extracts I= × 100
A0
The air-dried and finely ground sample was extracted by
where A0 is the absorbance of the control reaction (con-
using the method as described elsewhere (Sökmen et al.,
taining all reagents except the test compound), and A1 is
1999). The resulting extract (19.78%, w/w) was suspended in
the absorbance of the test compound. The IC50 value rep-
water and partitioned with chloroform (CHCI3 ) to separate
resented the concentration of the compounds, that caused
less polar, water-insoluble compounds. The water-soluble
50% inhibition.
(16.36%, w/w) and non-soluble parts (3.42%, w/w) were
then lyophilised and kept in the dark at +4 ◦ C until tested.
2.5.2. Inhibition of superoxide radicals
Superoxide radical generated by the xanthine–xanthine
2.3. Extraction of the essential oil oxidase system was determined spectrophotometrically by
monitoring its ability to reduce nitroblue tetrazolium (NBT)
The air-dried and ground aerial parts of plants col- (Robak and Gryglewski, 1988). Percent scavenging of super-
lected were submitted for 3 h to water-distillation using a oxide was calculated from the optical density of the treated
Clevenger-type apparatus to produce an oil in 0.6% (v/w) and control samples.
yield. Oil was dried over anhydrous sodium sulphate and,
after filtration, stored at +4 ◦ C until tested and analyzed. 2.5.3. DPPH assay
Radical scavenging activity was determined by a spec-
2.4. GC-MS analysis trophotometric method based on the reduction of a methanol
solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) as de-
The analysis of the essential oil was performed using a scribed elsewhere (Cuendet et al., 1997). Tests were carried
Hewlett Packard 5890 II GC, equipped with a HP-5 MS out in triplicate.
capillary column (30 m × 0.25 mm i.d., 0.25 ␮m) and a HP
5972 mass selective detector. For GC-MS detection, an elec- 2.5.4. Inhibition of lipid peroxide formation
tron ionisation system was used with ionisation energy of The reaction mixture contained 0.1 ml of 25% (w/v) rat
70 eV. Helium was the carrier gas, at a flow rate of 1 ml/min. liver homogenate in 40 mM, pH;7.0 Tris–HCl buffer, 30 mM
Injector and MS transfer line temperatures were set at 220 KCl, 0.16 mM ferrous iron, various concentrations of the
and 290 ◦ C, respectively. Column temperature was initially extract, and positive controls; BHT, curcumin, 0.06 mM
at 50 ◦ C, then gradually increased to 150 ◦ C at a 3 ◦ C/min ascorbic acid in a final volume of 0.5 ml. As positive con-
rate, held for 10 min and finally increased to 250 ◦ C at trols, BHT and curcumin had their own control reactions
10 ◦ C/min. Diluted samples (1/100 in acetone) of 1.0 ␮l were containing all related reagents except the test compounds.
injected manually and splitless. The components were iden- The mixture was then incubated at 37 ◦ C for 1 h (Bishayee
tified based on the comparison of their relative retention time and Balasubramanian, 1971). The lipid peroxide formation
and mass spectra with those of NBS75K library data of the was measured by using the method described elsewhere
GC-MS system, literature data (Adams, 2001) and standards (Ohkawa et al., 1979). The percentage inhibition of lipid
of the main components. The results were also confirmed by peroxidation was determined by comparing the results of the
the comparison of the compounds elution order with their test compounds with those of controls not treated with the
relative retention indices on non-polar phases reported in the extracts. Calculations were done as mentioned in hydroxyl
literature (Adams, 2001). radical scavenging method.
F. Candan et al. / Journal of Ethnopharmacology 87 (2003) 215–220 217
2.6. Antimicrobial activity were incubated at 37 ◦ C for 24 h for bacteria and at 30 ◦ C

for 48 h for the yeasts. The diameters of the inhibition zones
2.6.1. Microbial strains were measured in millimetres. Amikacin, clindamycin and
The methanolic extracts (both water-soluble and ciprofloxacin were individually used as positive controls for
water-insoluble parts) and the essential oil of Achillea bacteria.
millefolium were individually tested against a panel of
microorganisms including Staphylococcus aureus ATCC 2.6.5. Determination of minimum inhibitory concentration
#25923 and for minimum inhibitory concentration (MIC) (MIC)
test, ATCC #29213, Streptococcus pneumoniae ATCC A broth microdilution broth susceptibility assay was used,
#49619, Moraxella catarrhalis ATCC #49143, Bacillus as recommended by NCCLS, for the determination of MIC
cereus ATCC #11778, Acinetobacter lwoffii ATCC #19002, (NCCLS, 1999). All tests were performed in Mueller Hin-
Enterobacter aerogenes ATCC #13043, Escherichia coli ton broth (MHB; BBL) supplemented with Tween 80 deter-
ATCC #25922, Klebsiella pneumoniae ATCC #13883, gent (final concentration of 0.5% (v/v), with the exception
Proteus mirabilis ATCC #7002, Pseudomonas aeruginosa of the yeasts (Sabouraud dextrose broth-SDB + Tween 80).
ATCC #27853, Clostridium perfringens KUKENS-Turkey, Bacterial strains were cultured overnight at 37 ◦ C in MHA
Mycobacterium smegmatis CMM 2067, Candida albicans and the yeasts were cultured overnight at 30 ◦ C in SDB. Test
ATCC #10239 and Candida krusei ATCC #6258. Bacte- strains were suspended in MHB to give a final density of 5×
rial strains were cultured overnight at 37 ◦ C in Mueller 105 cfu/ml and these were confirmed by viable counts. Ge-
Hinton agar (MHA), with the exception of Streptococcus ometric dilutions ranging from 0.036 mg/ml to 72.00 mg/ml
pneumoniae (MHA containing 50 ml citrate blood/l) and of the essential oil were prepared in a 96-well microtiter
Clostridium perfringens (in anaerobic conditions). Yeasts plate, including one growth control (MHB + Tween 80) and
were cultured overnight at 30 ◦ C in Sabouraud dextrose one sterility control (MHB+Tween 80+test oil). Plates were
agar. incubated under normal atmospheric conditions at 37 ◦ C for
24 h for bacteria and at 30 ◦ C for 48 h for the yeasts. The
2.6.2. Antimicrobial screening MIC of amikacin, clindamycine and ciprofloxacine was in-
Two different methods were employed for the determina- dividually determined in parallel experiments in order to
tion of antimicrobial activities; agar well-diffusion method control the sensitivity of the test organisms. The bacterial
for the methanol extracts (water-soluble and water-insoluble growth was indicated by the presence of a white “pellet” on
parts) and agar disc diffusion method for the essential oil. the well bottom.
MICs of the essential oil against the test organisms were
determined by broth microdilution method. All the tests
were performed in duplicate and repeated twice. Modal 3. Results and discussion
values were selected.
3.1. Chemical composition of the essential oil
2.6.3. Agar well-diffusion method
The water-soluble extracts were weighed and dissolved Thirty-six compounds were identified and constituted
in phosphate buffer saline (PBS; pH 7.0–7.2), 10 mg/ml, 90.8% of the total oil. The essential oil of Achillea mille-
water-insoluble parts were dissolved in dimethylsulphoxide folium was characterized by a high number of monoter-
(DMSO), 10 mg/ml. Both extracts were filter-sterilised using penes. Eucalyptol (24.6%), camphor (16.7%), ␣-terpineol
a 0.45 ␮m membrane filter. Each microorganism was sus- (10.2%), ␤-pinene (4.2%), and borneol (4.0%) were the
pended in sterile saline and diluted at ca. 106 colony forming principal components comprising the 59.7% of the essential
unit (cfu)/ml. They were “flood-inoculated” onto the surface oil (Table 1).
of MHA. The wells (8 mm in diameter) were cut from the Changes in the composition of Achillea millefolium es-
agar and 0.06 ml of extract solution was delivered into them. sential oil were reported as being related to maturation of
After incubation for 24 h at 37 ◦ C, all plates were examined the plant, with increasing amounts of monoterpenes in rela-
for any zones of growth inhibition, and the diameter of these tion of the sesquiterpenes (Rohloff et al., 2000).
zones were measured in millimetres. Eucalyptol, camphor, and/or ␣-terpineol have been
found as major compounds in many other Achillea species
2.6.4. Disc diffusion method (Rustaiyan et al., 1998; Chalchat et al., 1999; Simic et al.,
Disc diffusion method was employed for the determina- 2000).
tion of antimicrobial activities of the essential oil (NCCLS, The studied essential oil displayed different chemical
1997). Briefly, a suspension of the tested microorganism profile from those observed from Achillea millefolium
(0.1 ml of 108 cells per ml) was spread on the solid media plants of other geographical origin. Achillea millefolium
plates. Filter paper discs (6 mm in diameter) were impreg- essential oil from Cuba (Pino et al., 1998) was found to
nated with 15 ␮l of the oil or the fraction and placed on the include caryophyllene oxide at a percentage of 20%, while
inoculated plates. These plates, after staying at 4 ◦ C for 2 h, Achillea millefolium oil from five clones from Russia (Orth
Table 1 lipid peroxides, superoxides, and hydroxyl radicals. Since

Chemical composition of the essential oil from Achillea millefolium the water-insoluble part of the extract is partly soluble in
subsp. millefolium
aqueous test media and its colour interfered the spectro-
Compounda Rt b Composition (%) scopic measurements, only water-soluble part and essential
Thujene 9.412 0.1 oil could be tested for their antioxidative capacity. All re-
␣-Pinene 9.669 2.4 sults are reported in Table 2.
Camphene 10.323 2.4 As can be seen from Table 2, the extract improved 50%
Benzaldehyde 10.948 0.2 inhibition at higher concentrations, indicating lesser antiox-
Sabinene 11.523 2.8
␤-Pinene 11.592 4.2
idant capacity than positive controls, but in the superoxide
2,3-Dehydro-1,8-cineole 12.355 0.6 radical inhibition case, it was found to be more effective
␣-Terpinene 13.545 0.5 than ascorbic acid. Hydroxyl radical scavenging and lipid
Eucalyptol 14.496 24.6 peroxidation were not tested with ascorbic acid since this
␥-Terpinene 15.666 1.0 chemical was already present in the test medium. On the
Terpinolene 17.123 0.2
Linalool 17.807 0.6
other hand, results in Table 2 demonstrated the strong abil-
Camphor 20.166 16.7 ity of the essential oil to act as a donor for hydrogen atoms
Borneol 21.117 4.0 or electrons. The reduction of the stable radical DPPH to
Terpinen-4-ol 21.692 2.8 yellow coloured diphenylpicrylhydrazine was obtained with
␣-Terpineol 22.515 10.2 an IC50 = 1.56 ␮g/ml instead of 3.90, 7.92, 19.30 ␮g/ml
Myrtenol 22.663 0.3
Fragranol 23.486 0.5
for ascorbic acid, curcumine, and BHT, respectively. Both
7-Methyl-3-methylene-6-octen-1-ol 23.694 0.2 hydroxyl radical scavenging and the lipid peroxidation inhi-
3,7-Dimethyl-3,6-octadien-1-ol 24.408 0.6 bition of the oil were also better than curcumine and BHT.
Chrysanthenyl acetate 25.716 0.8 This could be assigned to the presence of some phenolic
Bornyl acetate 26.866 0.1 compounds (Table 1). The antioxidative effectiveness in nat-
Myrtenyl acetate 28.888 0.1
Eugenol 30.196 0.2
ural sources was reported to be mostly due to phenolic com-
␣-Copaene 31.019 0.1 pounds (Hayase and Kato, 1984). Phenolic compounds were
Caryophyllene 32.991 0.4 reported to play an important role in inhibiting autoxidation
␤-Farnesene 34.002 0.3 of the oils (Ramarathnam et al., 1986). In order to deter-
␥-Curcumene 35.588 0.2 mine the antioxidant nature of the oil, its main components,
Zingiberene 36.262 0.3
Nerolidol 39.464 0.1
e.g. eucalyptol, camphor, ␤-pinene, borneol, terpinen-4-ol,
Caryophyllene oxide 40.604 0.7 ␣-pinene were all tested individually and none exhibited
␥-Eudesmol 43.647 1.8 antioxidative activity in all methods employed. Antioxidant
␤-Eudesmol 44.945 1.6 activity of eucalyptol and terpinen-4-ol were previously
Bisabolol oxide II 45.381 3.8 reported using two different methods; aldehyde/carboxylic
Bisabolone oxide 47.354 3.3
␣-Bisabolol 47.443 2.1
acid assay and lipid peroxidation (Lee and Shibamoto,
Others not identified (33) 9.2 2001) with prolonged incubation periods (30 days and
18 h, respectively) in contrast to our experimental proce-
Total 100
dure employing 30–60 min incubation period. These results
a Compounds listed in order of elution from a HP-5 MS column.
b
implicate that the main components in the total oil might
Retention time (as minutes).
synergize each other or other components involve in these
activities.
et al., 1999) were characterized by sesquiterpenes with
high chamazulene contents (46–74%) in the four clones 3.3. Antimicrobial activity
and high ␤-caryophyllene content (38–45%) in the one
clone. Water-insoluble parts of the methanolic extracts were
The fraction of sesquiterpenes in the essential oil of Achil- found to have moderate activity against Clostridium per-
lea millefolium (section Santolinoidea) of Turkey origin has fringens and the yeasts. No activity was exhibited by the
been found in remarkable amounts, but was qualitatively water-soluble parts. The water-insoluble parts exhibits, in
different from the mentioned above (Table 1). many cases, greater activity than the water-soluble (aquatic)
ones (Sökmen et al., 1999).
3.2. Antioxidant activity The essential oil possessed stronger antimicrobial activity
than the extracts tested. The oil exhibited moderate activity
The antioxidant activity of Achillea millefolium extract against Streptococcus pneumoniae, Clostridium perfringens
(water-soluble part) was examined by comparing it to the and Candida albicans, and weak activity against Mycobac-
activity of known antioxidants such as curcumin, ascorbic terium smegmatis, Acinetobacter lwoffii and Candida krusei
acid and BHT by the following four in vitro assays; in- (Table 3). The growth inhibitions of test microorganisms
hibition of DPPH radical and the oxygen radicals such as ranged from 4.5 mg/ml (w/v) to 72.00 mg/ml (w/v) with
F. Candan et al. / Journal of Ethnopharmacology 87 (2003) 215–220 219
Table 2
Effects of methanolic extracts (water-soluble part) and essential oil of Achillea millefolium and positive controls on the in vitro free radical (DPPH,
superoxide and hydroxyl) and lipid peroxidation generation
Sample IC50 (␮g/ml)
DPPH Superoxide Hydroxyl Lipid peroxidation
Extract 45.60 ± 1.30 304.00 ± 5.10 407.30 ± 4.05 892.67 ± 13.0

Oil 1.56 ± 0.03 nt 2.70 ± 0.03 13.50 ± 0.07
Curcumin 7.92 ± 0.30 11.04 ± 0.17 14.28 ± 0.08 40.83 ± 0.15
Ascorbic acid 3.90 ± 0.15 1390.00 ± 2.90 nt nt
BHT 19.30 ± 0.05 nt 32.00 ± 1.20 17.80 ± 0.04
nt: not tested.
Table 3
Antimicrobial activity of the essential oil and the methanolic extracts of Achillea millefolium subsp. millefolium
Microorganism Essential oil MeOHc The MIC of Antibioticsd
DDa MICb H2 O CHCl3 AK CF CM FC
Staphylococcus aureus 8 72.00 nae na 2.00 0.25 ntf nt

Streptococcus pneumoniae 14 4.50 na na nt nt 0.125 nt
Moraxella catarrhalis na na na na nt nt nt nt
Bacillus cereus 10 72.00 na 10 nt nt nt nt
Acinetobacter lwoffii 15 18.00 na na nt nt nt nt
Enterobacter aerogenes 7 72.00 na na nt nt nt nt
Escherichia coli na na na na 2.00 0.015 nt nt
Klebsiella pneumoniae 9 72.00 na na nt nt nt nt
Proteus mirabilis na na na na nt nt nt nt
Pseudomonas aeruginosa na na na na 1.00 0.25 nt nt
Clostridium perfringens 12 4.50 na 12 nt nt 0.25 nt
Mycobacterium smegmatis 12 9.00 na na nt nt nt nt
Candida albicans 21 4.50 na 12 nt nt nt 128.00
Candida krusei 16 18.00 na 12 nt nt nt 64.00
a DD, disc diffusion method as recommended by NCCLS. Diameter of zone of inhibition (mm) including disk diameter of 6 mm.
b MIC, minimum inhibitory concentration. Values given as mg/ml (for the essential oil) and as ␮g/ml (for antibiotics).
c MeOH, methanolic extracts. Diameter of zone of inhibition (mm) including well diameter of 8 mm.
d AK, amikacin; CF, ciprofloxacin; CF, clindamycin, FC, fluconazole.
e na, not active.
f nt, not tested.
the lowest MIC value against Streptococcus pneumoniae, References

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In vitro antiplasmodial activity and cytotoxicity of

ethnobotanically selected Ivorian plants
Catherine Vonthron-Sénécheau a , Bernard Weniger b,∗ , Modibo Ouattara c ,
Fezan Tra Bi d , Alphonse Kamenan a , Annelise Lobstein b ,
Reto Brun e , Robert Anton b
a UFR des Sciences et Technologie des Aliments, Université d’Abobo-Adjamé, 02 BP 801 Abidjan 02, Ivory Coast
b Unité de Pharmacognosie, UMR ULP/CNRS 7081, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg, France
c UFR des Sciences Pharmaceutiques et Biologiques, Université de Cocody, 01 BP V34 Abidjan, Ivory Coast
d UFR des Sciences de la Nature, Université d’Abobo-Adjamé, 02 BP 801 Abidjan 02, Ivory Coast
e Swiss Tropical Institute, Medical Parasitology and Infection Biology, Socinstrasse 57, CH-4002 Basel, Switzerland
Received 3 January 2003; received in revised form 9 April 2003; accepted 16 April 2003
Abstract
Eight extracts from four Ivorian medicinal plants, traditionally used to treat malaria, were tested for their antiplasmodial activity in vitro
by assessing their ability to inhibit the uptake of [3 H]hypoxanthine into the Plasmodium falciparum K1 chloroquine-resistant strain. The
most active extract was the methylene chloride extract of Anogeissus leiocarpus which exhibited an IC50 value of 3.8 ␮g/ml. Inhibition of the
growth of Plasmodium falciparum was also observed with the methylene chloride extract of Cochlospermum planchonii and Microdesmis
keayana as well as with both methylene chloride and methanolic extracts of Hymenocardia acida.
Keywords: Malaria; Traditional medicine; Ivory Coast
1. Introduction In this framework, data on plants traditionally used in the

treatment of malaria in the Savannah and the forest regions
Malaria remains the first world endemic parasitic disease of Ivory Coast were collected, by personal contact with local
and is responsible for the death of over 1 million people an- traditional healers during an ethnobotanical survey of medic-
nually in the belt of Africa, within which lies Ivory Coast inal plants, conducted from April to August 2001. A biblio-
(Snow et al., 1999). Most malaria mortality in this coun- graphical survey permitted to retain four native plant species
try is caused by Plasmodium falciparum, the most com- among the most used by traditional healers and for which
mon species in the highly malaria endemic areas of Africa. antiplasmodial activity had never been evaluated (Anogeis-
Present drugs have become ineffective because Plasmodium sus leiocarpus (DC.) Guill. and Perr., Hymenocardia acida
falciparum is developing resistance to most of them, particu- Tul. and Microdesmis keayana Leon.) or at least only
larly to chloroquine, the cheapest and previously very effec- partially studied (Cochlospermum planchonii Hook. f. ex
tive antimalarial drug, and to pyrimethamine (Peters, 1998; Planch.).
Wellems and Plowe, 2001). Thus, there is an urgent need Apolar (methylene chloride) and polar (methanol) extracts
to discover new antiplasmodial agents. History shows that were prepared from the part of each selected plant indicated
biodiversity and traditional medicine knowledge are useful as traditionally used in decoction by the traditional healers,
to open new ways in the field of antimalaria therapy, as i.e. leaves for Anogeissus leiocarpus, Hymenocardia acida
it was the case for artemisinin (Quinghaosu Antimalarial and Microdesmis keayana and roots for Cochlospermum
Coordinating Research Group, 1979). planchonii. Each extract was then evaluated for antiplas-
modial activity in vitro, by assessing its ability to inhibit
the uptake of [3 H]hypoxanthine by the parasite. The cy-
∗ Corresponding author. Tel.: +33-390-244238; fax: +33-390-244311. totoxicity of the most active extracts was evaluated using
E-mail address: weniger@pharma.u-strasbg.fr (B. Weniger). L6 cells.
doi:10.1016/S0378-8741(03)00144-2
222 C. Vonthron-Sénécheau et al. / Journal of Ethnopharmacology 87 (2003) 221–225
Table 1
Plant species analyzed for antiplasmodial activity
Botanical name/family Traditional names Collection area Part used Voucher no.
Anogeissus leiocarpus (DC.) Guill. and Perr./Combretaceae Kèrèkètè (M) S Leaf CNF 14798
Cochlospermum planchonii Hook. f. ex Planch./Cochlospermaceae Tourougbebourou (M) S Root CNF 14643
Hymenocardia acida Tul./Euphorbiaceae Djoun (M) S Leaf CNF 8705
Microdesmis keayana J. Leon./Pandaceae Ibrélégni (B) A Leaf CNF 17789
M: Malinke; B: Bowle; S: Savannah region near Seguela (Northern Ivory Coast); A: forest region near Abidjan (Southern Ivory Coast).
2. Methodology ing the methods of Trager and Jensen (1976). Plant ex-
tracts were tested on K1 strain (multidrug pyrimethamine/
2.1. Plant material chloroquine-resistant strain; Thaithong and Beale, 1981).
Initial concentration of the plant extracts was 30 ␮g/ml
The selected plants (Table 1) were collected between diluted with two-fold dilutions to make seven concentra-
June and August 2001 in their natural habitats in Ivory tions, the lowest being 0.47 ␮g/ml. After 48 h incubation of
Coast, i.e. in the Savannah region in Bouandougou near the parasites with the extracts at 37 ◦ C, [3 H]hypoxanthine
Seguela (Northern Ivory Coast) for Anogeissus leiocarpus, (Amersham, UK) was added to each well and the incubation
Cochlospermum planchonii and Hymenocardia acida and was continued for another 24 h at the same temperature.
in the forest region near Abidjan (Southern Ivory Coast) The extract concentrations at which the parasite growth
for Microdesmis keayana. Botanical determination was per- (=[3 H]hypoxanthine uptake) was inhibited by 50% (IC50 )
formed by Dr. L. Aké Assi (Centre National de Floristique, was calculated by linear interpolation between the two drug
Université de Cocody, Abidjan). Voucher specimens are de- concentrations above and below 50% (Huber and Koella,
posited at the Herbarium of the Centre National de Floris- 1993). Chloroquine and artemisinin were used as positive
tique (CNF). references. The values given in Table 2 are means of two
independent assays; each assay was run in duplicate.
2.2. Preparation of crude extracts
2.4. Cytotoxicity assay
Air dried plant material of each species (10 g) was ground
(0.2 mm sieve) and defatted with hexane (200 ml) overnight Cytotoxicity assay of the plant extracts was done follow-
at room temperature. The plant material was extracted ing the method of Pagé and Noel (1993) with the modifica-
twice for 30 min with methylene chloride (60 ml) at 40 ◦ C, tion of Ahmed et al. (1994).
then twice for 30 min with methanol (60 ml) at 64 ◦ C, in a Cell line L6 (rat skeletal muscle myoblasts) were seeded
semi-automated Soxhlet extractor (Soxtec Avanti 2055 ap- in 96-well Costar microtiter plates at 2.2×105 cells/ml, 50 ␮l
paratus, Foss Tecator AB, Höganäs, Sweden). The filtrates per well in MEM supplemented with 10% heat inactivated
were taken to dryness under vacuum and the residues were fetal bovine serum (FBS). A three-fold serial dilution rang-
stored at room temperature until testing. Tannins were re- ing from 500 to 0.07 ␮g/ml of crude extracts in test medium
moved from the crude methanolic extracts using Sephadex was added. Plates with a final volume of 100 ␮l per well
LH-20 exclusion chromatography, according to the method were incubated at 37 ◦ C for 72 h in a humidified incubator
described by Houghton and Raman (1998). Methylene containing 5% CO2 . Alamar Blue was added as viability
chloride and methanol extract (after tannin removal) yields indicator according to Ahmed et al. (1994). After an addi-
were 5 and 0.7% for Anogeissus leiocarpus; 2.3 and 0.4% tional 2 h of incubation, the plate was measured with a fluo-
for Cochlospermum planchonii; 7.7 and 0.8% for Hymeno- rescence scanner using an excitation wavelength of 536 nm
cardia acida; 14.3 and 6.7% for Microdesmis keayana, and an emission wavelength of 588 nm (SpectraMax Gemi-
respectively. niXS, Molecular Devices). IC50 values were calculated from
the sigmoidal inhibition curve.
2.3. Antimalarial assay
Quantitative assessment of antimalarial activity in vitro 3. Results

was determined by means of the microculture radioisotope
technique based upon the method previously described by Traditional healers in the area of Seguela and Abidjan
Desjardins et al. (1979) and modified by Ridley et al. (1996). were consulted directly in collecting the basic ethnobotani-
The assay uses the uptake of [3 H]hypoxanthine by parasites cal information about the plants studied. Questions concen-
as an indicator of viability. trated mainly on plants which are traditionally used against
Continuous in vitro cultures of asexual erythrocytic malaria. Based on this information, different databases
stages of Plasmodium falciparum were maintained follow- (NAPRALERT Database, MEDLINE/PubMed, ISI Web of
C. Vonthron-Sénécheau et al. / Journal of Ethnopharmacology 87 (2003) 221–225 223
Table 2
In vitro antiplasmodial activity of the selected plant extracts against K1-resistant strain of Plasmodium falciparum and in vitro cytotoxicity towards L6
cells for the extracts exhibiting IC50 below 20 ␮g/ml
Plant species Plant part Extract Antiplasmodial activity Cytotoxic activity (L6 cells)
(Plasmodium falciparum K1 strain)
IC50 (␮g/ml) 95% Confidence intervala IC50 (␮g/ml) Selectivity index (SI)
Anogeissus leiocarpus Leaf Db 3.8 2.5–5.1 71.9 19

Mc >20 – – –
Cochlospermum planchonii Root D 4.4 3.1–5.7 67.3 15
M >20 – – –
Hymenocardia acida Leaf D 6.9 5.9–7.9 65.4 10
M 10.7 7.1–14.3 71.8 6
Microdesmis keayana Leaf D 12.2 10.4–14 nd nd
M >20 – – –
Chloroquine 0.064
Artemisinin 0.0015
Selectivity index, ratio of cytotoxic activity on L6 cells to antiplasmodial activity against K1 strain of Plasmodium falciparum; nd: not determined; IC50
values are means of two experiments.
a 95% confidence intervals were determined with Student’s t-test (Snedecor and Cochran, 1980), P < 0.05.
b Methylene chloride extract.
c Methanolic extract after tannins removal.
Science, Chemical abstracts and PRELUDE Database) were 4. Discussion

consulted to survey the literature concerning these plants.
Consequently, four plant species, belonging to four genera Anogeissus leiocarpus (DC.) Guill. and Perr. (Combre-
and four families, were chosen and collected in the field taceae): Leaves are used in Nigeria and in Guinea as anti-
(Table 1). Traditional uses of these species are listed in malarial (Adjanohoun et al., 1991; Bhat et al., 1990; Gbile
Section 4. The part of the selected plant species tradition- et al., 1990; Pobeguin, 1911). Other medicinal uses in Africa
ally used was extracted to yield eight extracts, which were are reported, like emmenagogue (Berhault, 1974), antitus-
tested for antiplasmodial activity. In vitro antiplasmodial sive (Baoua et al., 1976), disinfection of syphilitic ulcers,
activity of these eight extracts is summarized in Table 2. against diarrhea after parturition, against leprosy (Vasileva,
Finally, active plant extracts (IC50 < 20 ␮g/ml) were inves- 1969) and for cleaning teeth (Bhat et al., 1990). This species
tigated for their cytotoxicity towards L6 cells (rat skeletal showed antimicrobial activity against Haemophilus influen-
muscle myoblasts). zae, Staphylococcus aureus, Streptococcus pneumoniae,
The strongest antiplasmodial activities were found in the Streptococcus pyogenes, and Moraxella catarrhalis (Sanogo
dichloromethane extract of Anogeissus leiocarpus (IC50 = et al., 1998). The presence of 3,4,3 -tri-O-methylflavellagic
3.8 ␮g/ml) and of Cochlospermum planchonii (IC50 = acid and its glucoside were recently described in this
4.4 ␮g/ml). Methanolic and methylene chloride extracts species. This glucoside showed antimicrobial activity on
of Hymenocardia acida as well as the methylene chloride Staphylococcus aureus, Escherichia coli, Pseudomonas
extract of Microdesmis keayana showed greater IC50 val- aeruginosa and Candida albicans (Adigun et al., 2000).
ues, ranging from 6.9 to 12.2 ␮g/ml. Methanolic extracts Cochlospermum planchonii Hook. f. ex Planch. (Cochlos-
of Anogeissus leiocarpus, Cochlospermum planchonii and permaceae): The rhizome of this species is used as anti-
Microdesmis keayana exhibited IC50 values greater than malarial in Niger (Adam et al., 1972; Dakuyo, 1989). The
20 ␮g/ml. related species Cochlospermum trinctorium and Cochlosper-
Table 2 also summarizes the cytotoxicity towards L6 cells mum angolense are used for the same purpose in Ivory Coast
and the selectivity index (SI) of extracts which exhibited (Benoit-Vical et al., 1995) and in Angola (Presber et al.,
IC50 < 20 ␮g/ml in the antiplasmodial assay. SI is defined 1992). Essential oil, long-chain triacylbenzenes and unusu-
as the ratio of cytotoxicity to biological activity. Cytotoxic ally high levels of manganese and zinc have been found
activities of the four extracts tested are all in the same range, in this species (Addae-Mensah et al., 1985; Aliyu et al.,
the highest cytotoxic activity was found for the methano- 1995). A crude leaf extract and essential oil prepared from
lic extract of Hymenocardia acida (IC50 = 65.4 ␮g/ml) and the leaves of this species showed both antiplasmodial ac-
the lowest for the methylene chloride extracts of Hymeno- tivity and the leaf oil yielding the best antimalarial effect
cardia acida and Anogeissus leiocarpus (IC50 = 71.9 and (IC50 = 22–35 ␮g/ml) (Benoit-Vical et al., 1999). The rhi-
71.8 ␮g/ml, respectively). The best SI was found for the zome is also used in decocotion by native medical prac-
methylene chloride extract of Anogeissus leiocarpus (SI = titioners in the treatment of jaundice in Northern Nigeria
19) and the lowest one for the methylene chloride extract of (Aké Assi and Guinko, 1991). Aliyu et al. (1995) isolated a
Hymenocardia acida (SI = 6). zinc salt from an aqueaous extract of the rhizome which acts
224 C. Vonthron-Sénécheau et al. / Journal of Ethnopharmacology 87 (2003) 221–225
as an hepatoprotective agent by inhibiting the cytochrome were determined. It is generally considered that biological
P450 enzymes. efficacy is not due to in vitro cytotoxicity when SI ≥ 10. In
Hymenocardia acida Tul. (Euphorbiaceae): The roots are this study, three extracts out of four showed SI ≥ 10; only
used as antimalarial in Nigeria (Ainslie, 1937). Other reports the methanolic extract of Hymenocardia acida showed poor
mention the traditional use of this species against sleeping selectivity (SI = 6).
sickness (Kerharo, 1974), in case of skin diseases, diabetes, The three plant extracts that showed both significant an-
anemia, cough, infected wounds, urinary tract infections tiplasmodial activity and good selectivity in in vitro tests
and dental caries (Muanza et al., 1994) and as an aphro- have been selected for bioguided fractionation. Phytochem-
disiac (Muanza et al., 1994; Bouquet and Debray, 1974; ical studies of these plants are being performed in order to
Alvaro Viera, 1959). A peptide alkaloid, hymenocardine, identify the most effective fraction or compound, bearing in
was isolated from the root bark of this species (Pais et al., mind that the optimal product may not always correspond
1968). The methanol extract from the same part of the plant to single compounds.
exhibited cytotoxic activity against a panel of human tu-
mor cell lines (Muanza et al., 1995), antimicrobial activity
against Streptococcus mutans and Salmonella typhimurium Acknowledgements
(Muanza et al., 1994) and spasmolytic activity (Kambu et al.,
1990). The ethanolic extract exhibited antitrypanosomal ac- The authors wish to thank the traditional healers from
tivity (Freiburghaus et al., 1997) and antiinflamatory effect Ivory Coast for their willingness to share with us their knowl-
(Sackeyfio, 1998). edge about plants, especially A. Kamgate, L. Ouattara and
Microdesmis keayana J. Leon. (Pandaceae): A related N. Samake in Bouandougou, near Seguela. Special thanks
species, Microdesmis puberula was used in Ivory Coast to retired Professor L. Aké Assi who kindly performed the
as an emmenagogue and as an aphrodisiac (Bouquet and botanical determinations.
Debray, 1974). Neither biological nor chemical data about
this species could be found in the literature.
All these four plant species, selected for their traditional
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Antioxidant activity of Thespesia populnea bark extracts against

carbon tetrachloride-induced liver injury in rats
Raju Ilavarasan a,∗ , Mani Vasudevan a , Sockalingam Anbazhagan a ,
Subramanian Venkataraman b
a Department of Pharmacology, C. L. Baid Metha College of Pharmacy, Jyothi Nagar,
Old Mahabalipuram Road, Thorapakkam, Chennai 600096, India
b Department of Pharmacology and Environmental Toxicology, Dr. A. L. Mudaliar
P.G. Institute of Basic Medical Sciences, University of Madras,

Taramani, Chennai 600113, India
Received 7 January 2003; received in revised form 14 April 2003; accepted 16 April 2003
Abstract
Antioxidant activity of the aqueous (AET) and methanolic extracts (MET) of the Thespesia populnea bark was investigated in rats by
inducing liver injury with carbon tetrachloride:olive oil (1:1). The extracts exhibited significant antioxidant activity showing increased levels
of glutathione peroxidase (GPX), glutathione S-transferase (GST), glutathione reductase (GRD), superoxide dismutase (SOD) and catalase
(CAT) and decreased level of lipid peroxidation (LPO). Thespesia populnea bark extracts, AET and MET, at a dose level of 500 mg/kg showed
significant antioxidant activity against carbon tetrachloride-induced liver injury in rats.
Keywords: Thespesia populnea; Carbon tetrachloride; Marker enzymes in liver; Antioxidant activity
1. Introduction 2. Materials and methods
Thespesia populnea Soland ex Correa (Family: Mal- 2.1. Plant material

vaceae) is a large avenue tree found in the tropical regions
and coastal forests in India. The bark, leaves, flowers and The fresh bark of Thespesia populnea was collected
fruits are useful in cutaneous infections, such as scabies, from Chennai, India in the month of August and the plant
psoriasis, eczema, ringworm and guinea worm. A decoction was identified and authenticated by the Research Officer
of the bark is commonly used for the treatment of skin and (Botany), Central Research Institute (Siddha), Ministry of
liver diseases. Oil of bark mixed with vegetable oil is useful Health and Family Welfare, Govt. of India. The voucher
in urethritis and gonorrhea. The astringent bark, roots and specimen has been kept in the Department of Pharmacology,
fruits were used in dysentery, cholera and hemorrhoids; the C. L. Baid Metha College of Pharmacy, Chennai, India.
mashed bark is employed as a poultice or hot fomentation
for wounds. The leaves were reported to be employed lo- 2.2. Preparation of aqueous and methanolic extracts
cally as anti-inflammatory in swollen joints (Anonymous,
1995). Based on the above reports, the present study has The freshly collected bark of this plant was chopped,
been undertaken to investigate the antioxidant activity of shade dried and coarsely powdered. The powder was then
aqueous (AET) and methanolic extracts (MET) of the bark successively extracted with methanol and distilled water
of Thespesia populnea in CCl4 -intoxicated liver injury using a Soxhlet extractor. The aqueous and methanolic ex-
in rats. tracts were dried under reduced pressure using a rotary flash
evaporator and they were kept under refrigeration. Both the
extracts were administered to the animals as a suspension
∗ Corresponding author. in propyleneglycol.
doi:10.1016/S0378-8741(03)00147-8
228 R. Ilavarasan et al. / Journal of Ethnopharmacology 87 (2003) 227–230
2.3. Chemicals determined by reported methods (Misra and Fridovich,

1972; Bergmeyer et al., 1994). LPO was estimated by
Reduced glutathione (GSH), 1-chloro-2,4-dinitrobenzene the methods of Ohkawa et al. (1979). The total protein
(SD Fine Chem. Ltd., Mumbai), 5,5 -dithio-bis-2-nitroben- content was estimated by biuret methods (Doumas et al.,
zoic acid, nicotine adenine dinucleotide phosphate, epine- 1971).
phrine (Sigma, MO, USA), ethylenediamine-tetra-acetic
acid, hydrogen peroxide (Qualigens Fine Chemicals, Mum- 2.6. Statistical analysis
bai), thiobarbituric acid (Rolex Laboratory, Mumbai) were
used for the study. All other reagents used were of analytical The statistical analysis was carried out using one way
grade. analysis of variance (ANOVA) followed by Dunnet’s t-test.
P-values <0.05 were considered as significant.
2.4. Animals
Adult Wistar rats (120–200 g) of either sex (King Insti- 3. Results

tute of Preventive Medicine, Chennai) were maintained in
well-ventilated room temperature with natural day–night The results of antioxidant activity of AET and MET on
cycle in large polypropylene cages. They were fed balanced CCl4 -intoxicated rats are shown in Table 1.
rodent pellet diet (Poultry Research Station, Tamilnadu
Veterinary and Animal Sciences University, Chennai) and 3.1. Glutathione peroxidase (GPX)
water ad libitum through out the experimental period. The
animals were quarantined for one week, prior to the exper- GPX activity in liver homogenates was significantly
iments to acclimatize to laboratory conditions. The study (P < 0.001) reduced in CCl4 -treated animals when com-
protocol was approved by the IAEC (Institutional animal pared to control. The AET and MET treatment (500 mg/kg
ethics committee of CPCSEA, Govt. of India). dose level) significantly increased (P < 0.001) the GPX
levels when compared to CCl4 -treated animals. However,
2.5. Antioxidant activity AET and MET (250 mg/kg) showed less significant increase
in GPX levels (P < 0.02 and P < 0.01, respectively) in
The adult Wistar rats of either sex were divided into liver homogenate when compared to CCl4 -treated animals.
seven groups of six animals each. Group I received only Silymarin (25 mg/kg p.o.)-treated animals also showed sig-
propyleneglycol (5 ml/kg per day p.o.) for seven days and nificant (P < 0.001) increases of GPX level in the liver
served as control. Group II animals received single dose of homogenate compared with CCl4 -treated animals.
equal mixture of carbon tetrachloride and olive oil (50% v/v,
5 ml/kg i.p.) on the seventh day. Group III and IV animals 3.2. Glutathione S-transferase (GST)
were treated with AET at a dose level 250 and 500 mg/kg
per day p.o., respectively, for seven days. On the seventh GST level in liver was significantly reduced (P < 0.001)
day, a single dose of equal mixture of carbon tetrachloride in CCl4 -treated animals when compared with normal
and olive oil was given (50% v/v, 5 ml/kg i.p.). Group V animals. Treatment with AET and MET at 500 mg/kg
and VI animals were treated with MET at doses of 250 and dose showed significant increase (P < 0.001) in GST
500 mg/kg per day p.o., respectively, for seven days and on when compared to CCl4 -treated group. AET and MET
the seventh day, a single dose of equal mixture of carbon (250 mg/kg) also showed less significant (P < 0.05 and
tetrachloride and olive oil (50% v/v, 5 ml/kg i.p.) was ad- P < 0.01) increase of GST in liver homogenate. A signifi-
ministered. Group VII animals were treated with silymarin cant (P < 0.001) increase of GST in the liver homogenate
(25 mg/kg per day p.o.) for seven days and on the seventh of Silymarin-treated animals was observed.
day, a single dose of equal mixture of carbon tetrachloride
and olive oil (50% v/v, 5 ml/kg i.p.) was administered. 3.3. Glutathione reductase (GRD)
All animals were sacrificed by cervical decapitation un-
der light ether anesthesia on the eighth day. Immediately In the present study, GRD activity was significantly
after sacrifice, the liver were dissected out, washed in the decreased (P < 0.001) in CCl4 -treated animals when com-
ice-cold saline, and the homogenate was prepared in 0.1 M pared to control. A significant increase (P < 0.001) in the
Tris–HCl buffer (pH 7.4). The homogenate was centrifuged level of GRD was observed in AET and MET (500 mg/kg)-
and the supernatant was used for the assay of marker en- treated rats when compared with CCl4 -treated animals.
zymes namely glutathione peroxidase (GPX), glutathione AET and MET (250 mg/kg) also showed less significant
S-transferase (GST) and glutathione reductase (GRD) by (P < 0.02 and P < 0.01) increase in the GRD when com-
reported methods (Necheles et al., 1968; Habig et al., 1974; pared with CCl4 -treated animals. Silymarin-treated group
Dubler and Anderson, 1981, respectively). The activi- also showed significant (P < 0.001) increase in the level of
ties of superoxide dismutase (SOD), catalase (CAT) were GRD when compared to CCl4 -treated animals.
R. Ilavarasan et al. / Journal of Ethnopharmacology 87 (2003) 227–230 229
3.4. Superoxide dismutase (SOD)

(25 mg/kg) + CCl4
9.6b,c
0.8b,c
3.5b,c
4.7b,c
0.7b,c
SOD level was significantly reduced (P < 0.001) in

12b,c
CCl4 -treated animals when compared with normal ani-

Silymarin
±
±
±
±
±
±
mal. The AET (500 mg/kg) and MET (250 and 500 mg/kg)
302.4
279.1
21.8
77.2
165
5.8
showed significant increase (P < 0.001) in SOD when com-

pared to CCl4 -treated animals; AET at 250 mg/kg showed
only less significant increase (P < 0.01) in SOD in liver ho-
17.2b,c
5.1b,c
0.9b,c
4.3b,c
3.9b,c
0.8b,c
mogenate compared with CCl4 -treated animals. Silymarin-

treated animals also showed significant (P < 0.001)
±
±
±
±
±
±
289.84
252.45
18.42
73
150.5
6.5
increase in SOD when compared to CCl4 -treated animals.

500
3.5. Catalase (CAT)

8.9b,e
4.9b,c
4.2c,e
1.3c,e
3.7c,e
0.9c,e
MET (mg/kg)
CAT activity was significantly (P < 0.001) reduced in

±
±
±
±
±
±
CCl4 treatment when compared to control. AET and MET

256.2
229.8
15.6
59.8
74.8
8.7
at 500 mg/kg dose significantly increased (P < 0.001) CAT

250
Thespesia populnea bark extracts + CCl4
in liver homogenate when compared to CCl4 -treated ani-

mals. Treatment of AET and MET at 250 mg/kg dose also
13.1b,c
10.9b,c
0.9b,c
3.5b,c
9.9b,c
1.5b,c
showed less significant (P < 0.02 and P < 0.01) increase

of CAT when compared with CCl4 -challenged animals.
±
±
±
±
±
±
Silymarin-treated group also showed significant (P < 0.001)

271.4
240.4
17.2
65.8
140
7.4
500
increase of CAT when compared to CCl4 -treated animals.

12.7c,d
3.6. Lipid peroxidation (LPO)

1.2c,d
3.1c,e
0.6c,e
5.2c,f
2.1c,f
AET (mg/kg)
±
±
±
±
±
±
LPO level of liver homogenates significantly increased

249.9
207.7
14.6
56.9
68.7
9.1
(P < 0.001) in CCl4 -challenged rats when compared to

250
control rats. Treatment with AET and MET of 500 mg/kg

dose showed significant (P < 0.001) decrease in LPO when
21.5a,b
16.9a,b
0.9a,b
2.3a,b
4.8a,b
1.3a,b
compared with CCl4 -treated animals. AET and MET at

Antioxidant activity of Thespesia populnea bark extracts against CCl4 -induced liver damage
CCl4 -treated
250 mg/kg dose also showed less significant (P < 0.01) de-
±
±
±
±
±
±
crease in LPO in liver homogenate when compared with

group
189.1
162.9
10.8
40.2
48
13.5
CCl4 -treated animals. The silymarin-treated group showed a

significant (P < 0.001) decline in the LPO when compared
to CCl4 -treated animals.
22.7
26.8
1.5
4.4
6.7
0.8
±
±
±
±
±
±
Control
314.6
292.4
22.3
83.6
198
4.2
4. Discussion
c CCl -treated animals compared with AET and MET.
GPX plays a pivotal role in H2 O2 catabolism (Eaton,

GST (nmol of CDNB conjugate formed/min/mg protein)
1991) and the detoxification of endogenous metabolic per-

Control compared with CCl4 -treated animals.
CAT (nmol of H2 O2 decomposed/min/mg protein)
oxides and hydroperoxides which catalyses GSH (Floka,

GRD (nmol of GSSG utilized/min/mg protein)
GPX (nmol of GSH oxidized/min/mg protein)
1971). GPX activity was significantly reduced after CCl4

treatment when compared to control. The reversal of
the GPX activity to normal after pretreatment with AET
and MET is due to antioxidant activity by scavenging/
LPO (nmol of MDA/mg protein)
detoxifying the endogenous metabolic peroxides generated

after CCl4 injury in the liver tissues.
Many investigators have suggested that GST offers pro-
tection against LPO by promoting the conjugation of toxic
SOD (Kat/g protein)
electrophiles with GSH (Jakoby, 1988). GST plays a phys-

P < 0.001.
d P < 0.02.
e P < 0.01.
f P < 0.05.
iological role in initiating the detoxification of potential

Parameters
alkylating agents. Chemicals like chloroform and CCl4 alter

Table 1
the hepatic GST activity (Aniya and Anders, 1985). GST

b
a
level was significantly reduced in CCl4 -treated animals and

230 R. Ilavarasan et al. / Journal of Ethnopharmacology 87 (2003) 227–230
upward reversal was observed after the treatment with AET Although the precise mechanism of action of AET and
and MET. This may be attributed to a direct action of the ex- MET has not been elucidated, it can be safely assumed that
tract on the hepatic GST activation, the mechanism of which the lowering of enzyme levels is responsible for cell injury
is not known. An increase in GRD activity implies that AET and enhancing the enzymes responsible for antioxidant ac-
and MET protect the liver tissue from oxidative damage by tivity. It can be concluded that the AET and MET possess
GSH regenerated from its oxidized form (GSSG). definite antioxidant activities either through stabilization of
In the present study, the SOD activity is significantly cellular membrane or antiperoxidase activity.
reduced in CCl4 -intoxicated rats. The SOD activity was
brought to near normal after treatment with the extracts
in CCl4 -intoxicated rats. Decreased activity of CAT was References
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and inactivation of the enzyme protein in the lipid perox- transferase and release into serum after treatment with bromobenzene
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The CAT activity was restored to normal after treatment Bergmeyer, H.V., Gawehn, K., Grassik, M., 1994. Methods of enzymatic
with extracts evidently shows the antioxidant property of analysis. In: Bergmeyer, H.V., Chemie, V., Weinhein, S. (Eds.). Aca-
the extracts against oxygen free radicals (OFRs). demic Press, New York, p. 348.
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branes. The level of thiobarbituric acid relative substance catalytic activities of yeast glutathione reductase by N-alkyl meleim-
(TBARS) is an indirect measurement of lipid peroxidation imdes. Biochemica Biophysica Acta 659, 70.
(Halliwell et al., 1995). Lipid peroxide levels in tissue were Eaton, J.W., 1991. Catalase, glutathione peroxidase and hydrogen perox-
found to be significantly elevated in CCl4 -challenged rats. idase. Journal of Laboratory and Clinical Medicine 118, 3–4.
Floka, L., 1971. Glutathione peroxidase enzymologic and biological as-
This toxic effect is the consequence of CCl4 activation by pects. Klin Nochenschr 32, 49.
cytochrome P450 to trichloromethyl radical (CCl3 • ) which Habig, W.H., Pabst, M.J., Jocoby, W.B., 1974. The first enzyme step
readily reacts with oxygen to form trichloromethyl peroxyl in mercaptouric acid formation. Journal of Biological Chemistry 249,
(CCl3 O2 • ) radical (Tappel, 1973). These free radicals trig- 7130–7139.
Halliwell, B., Aeschbach, R., Loligger, J., Aruoma, O.I., 1995. The char-
ger cell damage through two mechanisms namely covalent
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binding to cellular macromolecules and lipid peroxidation Jakoby, W.B., 1988. Detoxification, conjugation and hydrolysis in liver
which affect the ionic permeability of the membrane pre- biology and pathology. In: Arias, I.M., Jakoby, W.B. (Eds.), Raven
venting the disintegration and solubilization of membrane Press, New York, pp. 375–385.
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tooxidation of epinephrine and a simple assay for superoxide dismu-
the extracts may be attributed to the antioxidant activity of
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the metabolic transformation of CCl4 in the liver. assay method. Journal of Pediatrics 72, 319.
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progress of hepatocellular diseases (Wilkinson, 1962). Arnold, E. (Ed.). Academic press, London, p. 84.
Screening and comparison of antioxidant activity of solvent

extracts of herbal medicines used in Korea
Dae Gill Kang a , Chi keun Yun b , Ho Sub Lee a,∗
a Department of Herbal Resources, Professional Graduate School of Oriental Medicine and Medicinal Resources Research Center (MRRC),
Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea
b Department of Health Administration, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea
Received 20 March 2002; received in revised form 7 April 2003; accepted 17 April 2003
Abstract
The hexane, ethylacetate, n-butanol, and water extracts of 10 Korean herbal medicines were screened and compared for their antioxidant
activities in a range of lipid peroxidation system using rat brain homogenates, antihemolysis assay of red blood cells, and other in vitro assays to
determine their ability to scavenge superoxide and hydroxyl radicals. All of the 10 Korean herbal medicines have potent antioxidant activities.
Among the four solvent extracts, the antioxidant activities of more-polar solvent extracts (BuOH and water extracts) were relatively higher
than that of non-polar solvent extracts (hexane and EtOAC extracts). These results will be useful to further analyze those herbal medicines
that contain the most antioxidant activity in order to identify the active principles.
Keywords: Antioxidant; Korean herbal medicines; Solvent extracts; Screening
1. Introduction and Lee, 2001). Solvent extraction is frequently used for

isolation of the antioxidants and both extraction yield and
Reactive oxygen species (ROS), such as hydroxyl radical, antioxidant activity of the extracts are strongly dependent
hydrogen peroxide, and superoxide anions, are produced as on the solvent, due to the different antioxidant potentials
byproducts in aerobic organisms, and have been implicated of compounds with different polarity. For these reasons,
in the pathology of a vast variety of human diseases includ- comparative studies for selecting the optimal solvent pro-
ing cancer, atherosclerosis, diabetic mellitus, hypertension, viding maximum antioxidant activity are required for each
AIDS, and aging (Halliwell and Gutteridge, 1984; Wallace, substrate. Although the use of different polarity substances
1999; Lee et al., 2000). Therefore, antioxidant activity is an can provide more exhaustive information on the properties
important in-view of the free radical theory of aging and as- of the extracts, the literature contains few reports of the
sociated diseases. It has long been recognized that naturally polarity-based solvent extraction of medicinal plants.
occurring substances in higher plants have antioxidant ac- The present study was undertaken to perform the screen-
tivity. A great number of plant origin substances have been ing of antioxidant properties of 10 traditional medicines
suggested to appear as antioxidants. Flavonoids and other grown in Korea, and we selected hexane, ethylacetate,
phenolic compounds of plant origin have been reported as n-BuOH, and water as extract solvents which permits com-
scavenger ROS, thus they are viewed as promising therapeu- parison of the antioxidant properties among the polarity-
tic drugs for free radical pathologies (Parshad et al., 1998; based solvent extracts of medicinal plants.
Lee et al., 2000).
The antioxidant activity of plant origin is dependent on
the type and polarity of the extracting solvent as well as on 2. Materials and methods
the test system and the substrate to be protected by the an-
tioxidant (Heinonen et al., 1998; Moure et al., 2000; Kang 2.1. Chemicals
Hexane, ethylacetate (EtOAC), and n-butanol (n-BuOH)

∗ Corresponding author. Tel.: +82-63-850-6841; fax: +82-63-850-7324. were purchased from Merck (Darmstate, Germany).
E-mail address: host@wonkwang.ac.kr (H.S. Lee). Phenazine methosulfate (PMS)-␤-nicotinamide adenine
doi:10.1016/S0378-8741(03)00142-9
232 D.G. Kang et al. / Journal of Ethnopharmacology 87 (2003) 231–236
Table 1
Latin names, herbarium voucher specimen numbers, plant parts, and uses in Korea
Herbal medicines Voucher specimen numbers Plant parts Uses in Korea
Inula helenium L. DH-43 Root Antibacterial, disinsection

Astragalus membranaceus BUNGE DH-14 Root Cardiotonic, diuretic
Atratylodes koreana NAKAI DH-07 Root Sedative, hypoglycemic
Gardenia jasminoides for. Grandiflora MAKINO DH-08 Fruit Sedative, cholagogue
Magnolia liliflora DESR. DH-34 Flower Antihypertensive
Scutellaria baicalensis GEORGI DH-05 Root Antipyretic, antiallergy
Siegesbeckia orientalis L. DH-57 Whole Antihypertensive
Sinomenium acutum REHDER et WILS. DH-15 Root Analgesia, antiinflammation
Sorbus amurensis KOEHNE DH-41 Bark Expectorant
Xanthium strumarium L. DH-58 Fruit Analgesia
dinucleotide (reduced form, NADH), nitroblue tetrazolium USA). l-Ascorbic acid was used as a positive control. The
(NBT), l-ascorbic acid, cytochrome c, dl-dithiothreitol, superoxide radical scavenging ratio (%) was calculated
thiobarbituric acid (TBA), Butylated hydroxyanisole (BHA), using the following formula:
and 2,2 -azo-bis-(2-amidinopropane)dihydrochloride (AAPH)
A − A1
were purchased from Sigma (St. Louise, MO, USA). All Superoxide radical scavenging ratio (%) = × 100
other unstated chemicals and reagents were of analytical A
grade. where A is the absorbance of positive control, and A1 is the
absorbance of the test samples.
2.2. Plant material
2.5. Scavenging activities of hydroxyl radicals
All herbal medicines were purchased from a herbal mar-
ket in Iksan, Jeonbuk Province, South Korea. Dr. Kyu Kwan Scavenging activity of hydroxyl radicals was determined
Jang at the Botanical Garden of Wokwang University iden- by the Liu and Ng method (2000), which was slightly mod-
tified plant materials. Herbarium voucher specimens were ified by Kang and Lee (2001). The hydroxyl radicals were
prepared and deposited in the herbarium of the Professional generated in a l-ascorbic acid–CuSO4 system by reduction
Graduate School of Oriental Medicine, Wonkwang Univer- of Cu2+ and were assayed by the oxidation of cytochrome c
sity, Iksan, Jeonbuk, South Korea (Table 1). in the 96-well microplate. The hydroxyl radicals were gener-
ated in 200 ␮l of 10 mM sodium phosphate buffer (pH 7.4),
2.3. Extraction containing 100 ␮M l-ascorbic acid, 100 ␮M CuSO4 , 12 ␮M
cytochrome c and the samples to be tested at different con-
For the partitioning by solvent, the Korean herbal centrations. The reduced cytochrome c was produced by ad-
medicines (100 g) were air-dried at room temperature and dition of excess dl-dithiothreitol and followed by Sephadex
reduced to fine powder by milling. The resulting powder G-15 chromatography (bed volume, 10 ml) to remove excess
was subjected to extraction with 200 ml of methanol, three dl-dithiothreitiol. The change in absorbance caused by the
times, 24 h each. The methanol extract was evaporated oxidation of cytochrome c was measured at 550 nm using a
and resuspended in H2 O, and sequentially partitioned with microplate reader (Molecular Devices). Thiourea was used
n-hexane, EtOAC, and BuOH. as a positive control. The scavenging activity of hydroxyl
radical by 500 ␮g/ml of thiourea was taken as 100%. The
2.4. Scavenging activities of superoxide radicals scavenging activity of hydroxyl radical was calculated using
the following formula:
Scavenging activity of superoxide radicals was deter- A − A0
mined by the Liu and Ng method (2000), which was slightly Hydroxyl radical scavenging activity (%) = ×100
AT − A 0
modified by Kang and Lee (2001). Superoxide radicals were
generated in a PMS-␤-nicotinamide adenine dinucleotide where A is the absorbance of samples, and AT and A0 are
(reduced form, NADH) system by oxidation of NADH and the absorbance of the thiourea and the control, respectively.
were assayed by the reduction of NBT in the microplate.
The superoxide radicals were generated in 200 ␮l of 16 mM 2.6. Inhibitory effects on erythrocyte hemolysis
Tris–HCl buffer (pH 8.0), which contained 78 ␮M NADH,
50 ␮M NBT, 10 ␮M PMS and samples (10 ␮l) to be tested Whole blood was obtained carefully by cannulation of
at different concentrations. The color reaction between su- femoral artery in Sprague–Dawley rats and collected in hep-
peroxide radicals and NBT was detected at A560 nm using arinized tubes. Erythrocytes were isolated from plasma by
a microplate reader (Molecular Devices, Synnyvate, CA, centrifugation (1000 × g for 20 min) and washed three times
D.G. Kang et al. / Journal of Ethnopharmacology 87 (2003) 231–236 233
with 10 volumes of saline solution. Erythrocyte hemolysis 3.1. Scavenging effects of solvent extract on O2 •− radical
was mediated by peroxyl radicals in this assay system (Niki
et al., 1988). A 10% suspension of erythrocytes in 10 mM The four solvent extracts of 10 herbal medicines were
of phosphate-buffered saline (PBS, pH 7.4) was added to screened for their superoxide-scavenging activity in the
the same volume of 200 mM AAPH in PBS solution con- PMS/NADH–NBT system, and the results are shown in
taining samples to be tested at different concentrations. The Table 2. In the PMS/NADH–NBT system, superoxide an-
reaction mixture was shaken gently while being incubated ion derived from dissolved oxygen by PMS/NADH cou-
at 37 ◦ C for 2 h. The reaction mixture was then removed pling reaction reduces NBT. The decrease of absorbance at
by centrifugation (1000 × g for 20 min), diluted with eight 560 nm with antioxidants thus indicates the consumption of
volumes of PBS and centrifuged at 1000 × g for 20 min. superoxide anion in the reaction mixture. There was a dif-
The absorbance (A) of supernatant was read at 540 nm using ference in the overall scavenging ability among the extract
spectrophotometer (Milton Roy, Rochester, NY, USA). Sim- solvents from the 40 extracts and even among the same
ilarly, the reaction mixture was treated with eight volumes species. Seven of the extracts, at 200 ␮g/ml assay, displayed
of distilled water to achieve complete hemolysis, and the ab- scavenging activities that were greater than 50%, while
sorbance (B) of the supernatant obtained after centrifugation seven extracts exhibited a nearly zero-scavenging activity
was measured at 540 nm. The inhibition of hemolysis (%) of the superoxide radical. Among them, the water extract of
was calculated by the equation (1 − A/B) × 100. l-Ascorbic Sinomenium acutum showed the highest scavenging activity
acid was used as a positive control. of superoxide radical in this system.
2.7. Inhibitory effects on lipid peroxide (LPO) 3.2. Scavenging effects of solvent extract on OH• radical
production in brain homogenates
Table 3 shows the scavenging effect of solvent extracts
For the in vitro studies, the brains of normal Sprague– of 10 Korean herbal drugs on hydroxyl radicals generated
Dawley rats were isolated and homogenized with Polytron by l-ascorbic acid/CuSO4 –cytochrome c system. The hy-
homogenizer (Switzerland) in ice-cold Tris–HCl buffer droxyl radicals were generated in a l-ascorbic acid/CuSO4
(20 mM, pH 7.4) to produce a 10% (w/v) homogenate. system by reduction of Cu2+ and were assayed by the oxi-
The homogenate was centrifuged at 10,000 × g for 10 min. dation of cytochrome c. High-scavenging activity (≥50% at
The supernatant (0.5 ml) was incubated with the test sam- 200 ␮g/ml assay) was found in the BuOH extracts of Astra-
ples in the presence of 10 ␮M FeSO4 and 0.1 mM ascor- galus membranaceus, Siegesbeckia orientalis, and Sorbus
bic acid at 37 ◦ C for 1 h. The reaction was stopped by amurensis, and EtOAC extracts of Scutellaria baicalensis
addition of 0.5 ml trichloroacetic acid (TCA, 28%, w/v) and Sinomenium acutum, and hexane extracts of Sorbus
and 0.75 ml thiobarbituric acid (TBA, 1%, w/v) in suc- amurensis.
cession, and the solution was then heated at 100 ◦ C for
15 min. After centrifugation to remove precipitated pro- 3.3. Inhibitory effects on erythrocyte hemolysis
tein, the color of the malondialdehyde (MDA)-TBA com-
plex was detected at OD 532 nm using spectrophotometer The azo compound generates few radicals by its uni-
(Milton Roy). BHA was used as a positive control. The molecular thermal decomposition. The rate of generation of
inhibition ratio (%) was calculated using the following peroxyl radicals can be easily controlled and measured by
formula: adjusting the concentration of AAPH (Miki et al., 1987).
A − A1 Therefore, hemolysis induced by AAPH must provide suit-
Inhibition ratio (%) = × 100 able means for studying the oxidative erythrocyte membrane
A
damage by peroxyl radical attack from the outside of the
where A is the absorbance of control, and A1 is the ab- membrane (Ng et al., 2000). Of the extracts studied, BuOH
sorbance of the test samples. extracts of Astragalus membranaceus, Atratylodes koreana,
Magnolia liliflora, Xanthium strumarium, and Scutellaria
baicalensis, and water extracts of Gardenia jasminoides
3. Results and discussion and Sinomenium acutum, and EtOAC extracts of Scutellaria
baicalensis, tested at 100 and 200 ug/ml, markedly inhibited
The methanol extracts of 10 Korean herbal drugs were erythrocyte hemolysis in this system (Table 4).
divided into four fractions with different polarities by par-
titioning it in various solvents such as hexane, EtOAC, 3.4. Inhibitory effects on LPO production in brain
n-BuOH, and H2 O. Then these solvent extracts were tested homogenates
for their antioxidant activity in a range of lipid peroxidation
systems using rat brain homogenates, red blood cells and Quantification of MDA, one of the products of lipid
other in vitro assay to determine their ability to scavenge peroxidation, with TBA is the most common assay used
ROS. for determination of the rate extent of lipid peroxidation.
Table 2
Effect of solvent extracts of Korean herbal medicines on superoxide radicals generated by PMS/NADH system
Samples Concentration (␮g/ml) Solvents
Hexane EtOAC n-BuOH H2 O
Inula helenium 100 7.5 ± 4.2 5.76 ± 2.3 21.7 ± 1.8 22.3 ± 0.9
200 18.2 ± 3.7 12.4 ± 2.8 31.6 ± 9.1 23.2 ± 8.3
Astragalus membranaceus 100 nz nz 20.2 ± 5.8 29.0 ± 8.0
200 nz nz 40.1 ± 6.5 33.2 ± 4.3
Atratylodes koreana 100 nz nz 38.2 ± 1.3 6.5 ± 7.0
200 nz nz 56.5 ± 3.6 13.5 ± 3.2
Gardenia jasminoides 100 17.1 ± 7.0 18.8 ± 3.5 29.8 ± 3.5 54.6 ± 0.4
200 45.1 ± 1.4 47.9 ± 3.2 44.4 ± 5.6 68.8 ± 1.2
Magnolia liliflora 100 nz 5.8 ± 1.1 27.9 ± 1.2 19.8 ± 3.6
200 nz 12.4 ± 2.2 37.9 ± 3.4 52.5 ± 1.5
Scutellaria baicalensis 100 nz 65.9 ± 1.4 56.8 ± 1.7 26.3 ± 3.8
200 16.7 ± 1.0 83.8 ± 1.3 75.6 ± 1.7 30.1 ± 1.1
Siegesbeckia orientalis 100 9.4 ± 2.7 nz 30.7 ± 5.6 53.5 ± 5.5
200 16.7 ± 2.4 20.0 ± 6.3 49.6 ± 2.7 67.6 ± 0.6
Sinomenium acutum 100 6.1 ± 1.2 13.9 ± 2.9 nz 90.2 ± 0.5
200 8.2 ± 2.1 16.0 ± 0.1 14.3 ± 3.1 91.7 ± 0.1
Sorbus amurensis 100 nz nz 60.1 ± 1.6 34.6 ± 1.1
200 nz 5.5 ± 7.8 78.1 ± 3.5 41.1 ± 4.1
Xanthium strumarium 100 nz 14.7 ± 3.5 31.9 ± 6.5 14.6 ± 4.0
200 nz 31.7 ± 1.3 61.7 ± 2.6 49.1 ± 4.1
Results show mean ± S.E. (n = 3) of the inhibition of superoxide radical (%); nz: nearly zero.
Table 3
Effect of solvent extracts of Korean herbal medicines on hydroxyl radicals generated by l-ascorbic acid/Cu2+ system
Inula helenium 100 12.4 ± 1.9 31.5 ± 2.6 6.7 ± 0.4 5.7 ± 1.9
200 16.7 ± 3.7 45.8 ± 2.5 17.1 ± 0.3 10.3 ± 0.5
Astragalus membranaceus 100 11.4 ± 2.7 23.5 ± 2.2 40.6 ± 7.7 20.7 ± 3.3
200 15.7 ± 3.7 28.8 ± 2.6 49.8 ± 4.6 31.5 ± 7.3
Atratylodes koreana 100 15.8 ± 5.0 13.7 ± 7.0 19.1 ± 2.1 6.2 ± 1.0
200 27.6 ± 6.1 24.3 ± 5.3 31.4 ± 5.9 21.1 ± 2.2
Gardenia jasminoides 100 nz 32.1 ± 4.6 16.7 ± 7.0 24.8 ± 1.4
200 nz 46.7 ± 0.9 40.2 ± 4.1 39.1 ± 1.0
Magnolia liliflora 100 12.9 ± 1.5 10.0 ± 0.8 12.8 ± 1.9 5.23 ± 1.2
200 29.3 ± 2.9 28.8 ± 1.0 30.9 ± 0.9 9.96 ± 1.6
Scutellaria baicalensis 100 25.8 ± 2.5 32.5 ± 4.8 34.0 ± 2.8 11.0 ± 1.2
200 38.7 ± 1.9 63.4 ± 3.7 44.8 ± 1.0 10.5 ± 2.8
Siegesbeckia orientalis 100 9.2 ± 0.9 25.3 ± 3.6 67.1 ± 0.3 26.5 ± 9.7
200 19.1 ± 1.6 28.1 ± 2.2 84.5 ± 4.4 41.4 ± 2.0
Sinomenium acutum 100 5.5 ± 2.3 34.7 ± 3.8 27.0 ± 4.4 34.6 ± 1.7
200 13.6 ± 1.6 85.5 ± 3.7 33.6 ± 0.5 41.8 ± 3.7
Sorbus amurensis 100 32.8 ± 0.8 10.7 ± 1.1 28.1 ± 2.0 7.1 ± 3.4
200 51.9 ± 8.8 15.0 ± 1.5 48.9 ± 3.1 16.3 ± 1.7
Xanthium strumarium 100 13.4 ± 2.9 24.5 ± 6.0 11.1 ± 2.8 5.5 ± 1.4
200 14.9 ± 2.4 28.2 ± 1.5 30.2 ± 0.5 10.2 ± 1.4
Results show mean ± S.E. (n = 3) of the inhibition of hydroxyl radical (%); nz: nearly zero.
D.G. Kang et al. / Journal of Ethnopharmacology 87 (2003) 231–236 235
Table 4
Effect of solvent extracts of Korean herbal medicines on the inhibition of hemolysis by AAPH system
Inula helenium 100 18.1 ± 3.5 20.9 ± 2.3 58.8 ± 2.0 12.3 ± 3.7
200 28.2 ± 0.7 40.8 ± 2.2 73.6 ± 0.2 21.6 ± 2.9
Astragalus membranaceus 100 6.5 ± 4.8 8.4 ± 1.9 94.9 ± 1.0 38.8 ± 2.5
200 24.4 ± 2.8 14.6 ± 0.3 94.5 ± 0.4 48.6 ± 2.3
Atratylodes koreana 100 8.7 ± 6.8 12.2 ± 2.5 96.4 ± 1.1 7.2 ± 5.9
200 12.0 ± 0.1 13.2 ± 0.2 97.8 ± 0.1 14.3 ± 4.9
Gardenia jasminoides 100 23.8 ± 3.4 37.6 ± 3.3 56.3 ± 0.8 92.8 ± 0.2
200 24.5 ± 2.0 65.9 ± 7.4 79.3 ± 1.6 96.7 ± 0.4
Magnolia liliflora 100 12.2 ± 2.0 13.4 ± 1.7 97.2 ± 1.0 33.3 ± 2.5
200 16.1 ± 0.6 25.2 ± 0.4 98.7 ± 0.2 55.2 ± 1.3
Scutellaria baicalensis 100 9.8 ± 0.2 96.9 ± 0.9 87.9 ± 1.4 26.9 ± 2.2
200 12.2 ± 0.8 97.6 ± 1.6 97.1 ± 0.3 54.5 ± 0.7
Siegesbeckia orientalis 100 21.7 ± 1.1 16.1 ± 2.8 56.3 ± 3.9 41.1 ± 4.6
200 21.6 ± 0.9 22.1 ± 1.0 76.3 ± 0.2 75.2 ± 2.1
Sinomenium acutum 100 17.0 ± 2.9 32.2 ± 3.8 11.4 ± 3.6 93.2 ± 1.3
200 25.2 ± 3.5 43.9 ± 2.7 30.2 ± 5.2 98.5 ± 0.2
Sorbus amurensis 100 11.4 ± 1.5 13.5 ± 3.3 64.6 ± 2.0 23.0 ± 0.2
200 16.4 ± 2.7 20.9 ± 1.5 78.9 ± 2.1 28.9 ± 1.3
Xanthium strumarium 100 8.6 ± 5.6 43.2 ± 0.5 94.8 ± 0.4 43.0 ± 1.4
200 12.7 ± 2.3 58.3 ± 1.1 95.3 ± 0.1 69.4 ± 1.2
Results show mean ± S.E. (n = 3) of the inhibition of erythrocyte hemolysis (%).
Our experiment proved that incubation of the rat brain and food chemistry (Bravo, 1998). It has been revealed that
homogenate with Fe2+ /ascorbate at pH 7.4 causes rapid various phenolic antioxidants, such as flavonoids, tannins,
peroxidation, detectable by the TBA method. Table 5 shows coumarins, xanthones and more recently procyanidins scav-
the activities of the representative 10 extracts, which showed enge radicals dose-dependently, thus they are viewed as
high-antihemolysis activity, against lipid peroxidation of the promising therapeutic drugs for free radical pathologies (Lee
rat brain homogenate. All the extracts markedly inhibited et al., 2000). Flavonoids are 15-carbon aromatic pigments
LPO production in the brain homogenates (Table 5). found in green plants and include chalcones, flavonones,
Medicinal herbs are known to contain a variety of an- flavones, biflavonoids, dihydroflavonols, anthrocyanidins,
tioxidants. Numerous substances have been suggested to and flavonols (VanderJagt et al., 2002). More than 4000 nat-
appear as antioxidants. The most detailed investigations so urally occurring flavonoids have been described (Hollman
far were concerned with reactions involving phenolic com- and Katan, 1998).
pounds, ranging from polymer chemistry to biochemistry The present study suggests that more-polar components
present in herbal medicinal extracts contributed towards
Table 5 the increased ROS-scavenging activity. Although there are
Inhibitory effect of solvent extracts of Korean herbal medicines on the no direct evidences in this study, the antioxidant activities
lipid peroxide production in brain homogenates shown by BuOH extracts and/or water extracts of herbal
Plants Concentration Solvents Inhibition (%) medicines could be related to the presence of phenolic
(␮g/ml) compounds such as tannins and flavonoids because they
Inula helenium 200 BuOH 88.8 ± 0.2 contain an aromatic hydroxyl moiety (Yesilada et al., 2000;
Astragalus membranaceus 200 BuOH 90.8 ± 0.1 Ramezani et al., 2001). As well known, polyphenols are
Atratylodes koreana 200 BuOH 90.9 ± 1.5 very important constituents because of their scavenging
Gardenia jasminoides 200 H2 O 83.8 ± 0.8 ability with ROS and chelating ability with divalent cations
Magnolia liliflora 200 BuOH 93.7 ± 0.1
Scutellaria baicalensis 200 EtOAC 96.2 ± 0.0
due to their hydroxyl groups (de las Heras et al., 1998;
Siegesbeckia orientalis 200 BuOH 90.5 ± 1.1 Hatano et al., 1989; Lopes et al., 1999).
Sinomenium acutum 200 H2 O 91.2 ± 0.2 Although the active principles responsible for the antiox-
Sorbus amurensis 200 BuOH 90.7 ± 0.7 idant activity of the tested extracts have not yet been iso-
Xanthium strumarium 200 BuOH 88.8 ± 1.4 lated in this study, it will be useful to further analyze those
Results show mean ± S.E. (n = 3) of the inhibition of production of LPO. herbal medicines that contain the most antioxidant activity
in order to identify the active principles. Then it would lead damage after transient global ischemia in gerbils. Neuroscience Letter
to a better understanding of the kinds of antioxidants used 287, 191–194.
Lee, Y.M., Kim, H., Hong, E.K., Kang, B.H., Kim, S.J., 2000. Water
in Korea as herbal medicines. extract of 1:1 mixture of Phellodendron cortex and Aralia cortex has
inhibitory effects on oxidative stress in kidney of diabetic rats. Journal
of Ethnopharmacology 73, 429–436.
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This study was supported by the Brain Korea 21 Project Lopes, G.K., Schulman, H.M., Hermes-Lima, M., 1999. Polyphenol tan-
nic acid inhibits hydroxyl radical formation from Fenton reaction by
and grant of the Oriental Medicine R&D Project, Ministry complexing ferrous ions. Biochimica Biophysica Acta 1472, 142–
of Health & Welfare, Republic of Korea (HMP-00-CO- 152.
03-0003). Miki, M., Tamai, H., Mino, M., Yamamoto, Y., Niki, E., 1987. Free-radical
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Antihepatotoxic activity of seeds of Cichorium intybus

Bahar Ahmed a,b,∗ , Tawfeq A. Al-Howiriny b , Abu B. Siddiqui a
a Antihepatotoxic Research Laboratory, Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
b Department of Pharmacognosy, Medicinal, Aromatic and Poisonous Plants Research Center,
College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

Received 5 June 2002; received in revised form 2 April 2003; accepted 17 April 2003
Abstract
The different fractions of alcoholic extract and one phenolic compound AB-IV of seeds of Cichorium intybus Linn were screened for
antihepatotoxic activity on carbon tetrachloride (CCl4 )-induced liver damage in albino rats. The degree of protection was measured using
biochemical parameters like aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALKP), and total protein
(TP). The methanol fraction and compound AB-IV were found to possess a potent antihepatotoxic activity comparable to the standard drug
Silymarin (Silybon-70). The histopathological study of the liver was also carried out, wherein the methanolic fraction and compound AB-IV
showed almost complete normalization of the tissues as neither fatty accumulation nor necrosis was observed.
Keywords: Cichorium intybus; Antihepatotoxic activity; Histopathology; Silymarin
1. Introduction ethyl acetate fraction of the seeds, namely cichosterol, char-

acterized as 13,14-seco-stigma 5(6),14(15)-diene-3-␤-ol and
The crude extracts of about 100 Indian medicinal plants stigma 5(6)-ene-3-␣-O-(␤-d-glucopyranoside), respectively
belonging to 40 families are used in the herbal formula- (Ahmed et al., 2002).
tions for the treatment of various diseases of the liver. In We now report a thorough pharmacological screening
addition, about 600 commercial preparations, mainly plant for the antihepatotoxic activity of different fractions of the
crude extracts with claimed liver-protecting activity, are seeds of the plant on carbon tetrachloride (CCl4 )-induced
available all over the world (Handa et al., 1986). The plant liver damage in rats. The study showed different degrees of
Cichorium intybus Linn (Family: Compositae, Asteraceae) activity on measuring the different biochemical parameters
commonly known as Chicory or Kasni is also used as liver like aspartate transaminase (AST), alanine transaminase
tonic, cardiotonic, diuretic, stomachic, cholagogue, depu- (ALT), alkaline phosphatase (ALKP), and total protein (TP),
rative, emmenagogue, hepatomegaly, cephalalgia, inflam- wherein the methanol fraction and compound AB-IV were
mations, anorexia, dyspepsia, flatulence, colic, jaundice, found to be most active. The histopathological study of the
splenomegaly, amenorrhea dysmenorrhea, and asthma, etc. liver of the methanolic fraction and compound AB-IV (iso-
(The Wealth of India, 1992; Sala, 1994). The seed of the lated from methanol fraction) also showed almost complete
plant is also one of the main ingredients of Jigrine, a normalization of the liver tissues as neither fatty accumula-
commercial product of Hamdard (Waqf) Dawakahna, New tion nor necrosis was observed. The central vein appeared
Delhi, which is used for the treatment of various diseases clearly indicating a potent antihepatotoxic activity.
of liver.
The literature survey revealed that neither the systematic
assessment of antihepatotoxic activity nor phytochemical in- 2. Materials and methods
vestigation of the seeds of the plant have been done so far.
We have recently reported one new and one rare sterol from 2.1. Plant material
∗ Correspondingauthor. Tel.: +966-1-467-7210; fax: +966-1-467-7245.

The seeds of Cichorium intybus L. were procured from
E-mail addresses: drbahmed@rediffmail.com, bahmed@ksu.edu.sa Khari Bawli market, Delhi, India and were identified by
(B. Ahmed). a taxonomist, Department of Botany, Hamdard University,
doi:10.1016/S0378-8741(03)00145-4
238 B. Ahmed et al. / Journal of Ethnopharmacology 87 (2003) 237–240
New Delhi, where a voucher specimen no. 765 has been of 1.5 ml/kg by oral route. After 24 h of CCl4 administra-
deposited for future reference. tion, blood of rats was obtained by puncturing retro-orbital
plexus. The livers of animals from each group were taken for
2.2. Preparation of the plant extracts histopathological studies. The blood samples of each animal
were taken and allowed to clot for 45 min at room temper-
The dried seeds were crushed to a coarse powder (9.0 kg) ature. Serum was separated by centrifugation at 2500 rpm
and were exhaustively extracted with ethanol by cold perco- at 30 ◦ C for 15 min and analyzed for various biochemical
lation. The crude alcoholic extract was concentrated under parameters.
reduced pressure to get a viscous mass (950 g). It was dis-
solved in boiling methanol and kept at room temperature, 2.5. Assessment of the liver function
solidified fats (250 g) were removed by suction, and the
filtrate so obtained was concentrated to get a viscous solid, The biochemical parameters such as AST, ALT, and
and then subsequently fractionated into petroleum ether AKLP were estimated by reported methods (Reitman and
(60–80 ◦ C) (200 g), ethyl acetate (250 g), and methanol sol- Frankel, 1957; Kind and King, 1954). The TP was also mea-
uble (150 g) fractions. The petroleum ether fraction could sured according to the reported methods (Wooton, 1964;
not be analyzed for its chemical components, since it was Dumas et al., 1971).
an oily material. The ethyl acetate fraction afforded a new
steroid named as cichosterol and a rare steroid namely 2.6. Statistical analysis
stigma (Ahmed et al., 2002). The methanol fraction (100 g)
on column chromatography (CHCl3 –MeOH = 3:2) afforded The results of the biochemical estimations are reported as
a phenolic compound AB-IV (5.0 g) as viscous solid, which mean ± S.E. Total variation present in a set of data was es-
gave a positive test for phenols. All fractions and isolated timated by one-way ANOVA, student’s t-test and Dennett’s
compounds were filtered using a 0.2 ␮m nylon filter. The test were used for determining the significance (Woolson,
purity of all compounds was checked by TLC and HPLC. 1987; Dennett, 1964).
After evaporating the solvents, the above fractions, com-
pound AB-IV and the total alcoholic extract (100 g) were 2.7. Histopathological studies of the liver
prepared with gum acacia (1:1) for oral administration to
albino rats. The histopathological studies were carried out by reported
method (Luna, 1968). The rats were sacrificed under light
2.3. Experimental animals ether anesthesia after 24 h of the last dosage, and the liv-
ers removed and washed with normal saline. Small pieces
The study was carried out on Wistar albino rats (150– of liver tissues were processed and embedded in paraffin
200 g) of either sex as reported in the literature (Handa wax. Sections of 5–6 ␮m in thickness were cut, stained with
and Singh, 1995). The rats were bred in a colony in the hematoxylin and eosin, and then studied under an electron
Central Animal House of Jamia Hamdard. They were fed microscope.
with a standard pellet diet (Gold Mohar, Lipton India Ltd.,
Kolkata) and water ad libitum. Before their use in the ex-
periment, the rats were kept in standard environmental con- 3. Results and discussions
ditions (25–28 ◦ C, 60–70% relative humidity and 12/12 h
light/dark cycle). Eight animals in each group were used in As shown in Table 1, activities of liver enzymes, ALT,
all sets of experiments. AST, and ALKP, were markedly elevated, while the TP level
was decreased in CCl4 -treated animals in comparison to nor-
2.4. Testing of antihepatotoxic activity mal values. Administration of different extracts of the seeds
of Cichorium intybus markedly prevented CCl4 -induced el-
Animals were divided into eight groups of six rats in each evation of AST, ALT and ALKP, and diminution of TP.
for all the experiment. The first group served as vehicle The petroleum ether, ethyl acetate, methanol, total alco-
control and received normal saline only. The second group holic extracts, and compound AB-IV decreased the levels
served as CCl4 -intoxicated control and received by gavage of ALT, AST, and ALKP, while the level of TP increased
vehicle (normal saline) and CCl4 diluted with liquid paraf- against CCl4 -intoxicated control. The methanol fraction
fin (1:1, single dose). The third group was given standard and compound AB-IV were found to be most active at the
drug Silymarin at the dose of 70 mg/kg body weight and the dose levels of 500 and 250 mg/kg, respectively, exhibiting a
remaining groups were given different extracts at the dose decrease in ALT, AST, and ALKP as compared to standard
of 500 mg/kg body weight (group 8 received 250 mg/kg) drug Silymarin against intoxicated control in comparison to
and CCl4 . The vehicle (1% gum acacia in distilled water) normal values. The level of TP was increased by all frac-
or test drugs were administered orally for 5 days. CCl4 ditions in different proportions, wherein methanol and com-
luted with liquid paraffin (1:1) was administered in a dose pound AB-IV were found to be most active as compared to
B. Ahmed et al. / Journal of Ethnopharmacology 87 (2003) 237–240 239
Table 1
Effect of different fractions and the isolated compound of the total alcoholic extract of the seeds of Cichorium intybus on biochemical parameters in
albino rats intoxicated with CCl4
Groups Treatment Dosea ALT (units/l) AST (units/l) ALKP (units/l) TP (g/dl)
1 Normal (control) Normal saline only 65.50 ± 173 76.66 ± 5.77 31.75 ± 2.05 9.41 ± 0.36
2 CCl4 (toxicity control) 1.5 ml/kg (p.o.) 248.16 ± 16.17 180.83 ± 17.61 59.33 ± 4.04 5.68 ± 0.141
3 Silymarin (standard drug) 70 mg/kg (p.o.) 38.88 ± 21.17∗∗∗ 39.66 ± 4.04∗ 31.25 ± 2.16∗ 7.48 ± 0.29∗
4 Petroleum ether fraction 500 mg/kg (p.o.) 105.55 ± 8.89∗∗∗ 39.66 ± 4.04∗ 30.77 ± 3.35∗ 6.55 ± 0.32∗∗
5 Ethyl acetate fraction 500 mg/kg (p.o.) 99.99 ± 12.61∗∗∗ 26.83 ± 2.02∗ 32.30 ± 3.84∗ 6.84 ± 0.26∗
6 Methanol fraction 500 mg/kg (p.o.) 24.0 ± 3∗∗∗ 35.0 ± 7∗ 31.25 ± 2.17∗ 8.89 ± 0.66
7 Total alcoholic extract 500 mg/kg (p.o.) 108.88 ± 21.17∗∗∗ 32.66 ± 4.04∗ 35.12 ± 3.63∗∗ 6.72 ± 0.40∗∗
8 Compound AB-IV 250 mg/kg (p.o.) 29.99 ± 5.77∗∗∗ 35.0 ± 0∗ 27.53 ± 5.60∗ 8.59 ± 1.35∗
Values are mean ± S.E. of six rats. ALT, alanine transaminase; AST, aspartate transaminase; ALKP, alkaline phosphatase; TP, total protein; p.o., per oral.
a Single dose of CCl on first day and daily dose of drug/extract were given for 5 days.
4
∗ P < 0.001.
∗∗ P < 0.01.
∗∗∗ P < 0.1 vs. intoxicated control using Student’s t-test.
Table 2
Histopathological studies of liver tissues
Groups Treatment Observations
1 Normal (control) On first day: The section of its liver showed normal hepatocytes without any necrosis and fatty depositions.
On fifth day: Neither the change nor any sign of degeneration was observed in the liver sections.
2 CCl4 (toxicity control) On second day of CCl4 administration: The liver showed centrilobular necrosis with prominent and enlarged
central vein. Fatty depositions were also seen. The section showed a classic view of degenerating liver.
After 5 days: The liver did not show any significant recovery. The section showed focal and centrilobular fatty
change with necrosis.
3 Silymarin (standard drug) The section showed good recovery with absence of necrosis and fatty depositions. The central vein was clear.
4 Petroleum ether fraction The liver section showed partial disappearance of fatty deposits and necrosis. Hepatic cells in focal areas
showed various degrees of degenerative changes like cloudy swelling and hydropic degeneration. However, other
areas showed hepatic cell with prominent nucleus and nucleolus indicating mild antihepatotoxic activity.
5 Ethyl acetate fraction The liver section did not show any significant activity.
6 Methanol fraction It showed significant recovery with disappearance of fatty deposition and necrosis, the central vein appeared
clearly indicating a potent antihepatotoxic activity.
7 Total alcoholic extract There was a significant recovery except mild fatty change.
8 Compound AB-IV The section revealed a tremendous progress with disappearance of fatty deposits and necrosis. It showed
superiority as compared with other groups.
standard drug Silymarin against CCl4 -intoxicated control in any gross behavioral changes or mortality. It can, therefore,
comparison to normal values. In addition, decrease in liver be said to be non-toxic and may be used as a safe drug.
enzymes and increase in TP was also observed by other
fractions (Table 1).
The histopathological studies of the liver showed swelling 4. Conclusion
and necrosis in hepatocytes in CCl4 -treated rats in com-
parison to normal control rats. Administration of different The above observations lead to the conclusion that the
extracts of the plant exhibited a significant recovery of methanolic fraction possessed most active chemical com-
hepatocytes in different sections of the liver, wherein the ponent, which has also been isolated and named as AB-IV.
methanolic fraction and compound AB-IV showed almost The preliminary identification has shown it to be a pheno-
complete normalization of the tissues as neither fatty ac- lic compound. Further elucidation of its structure is under
cumulation nor necrosis was observed. The central vein progress in the laboratory.
appeared clearly indicating a potent antihepatotoxic activ-
ity. The compound AB-IV showed superiority as compared
with other groups. Other fractions also showed a consider- Acknowledgements
able recovery of the liver tissues (Table 2).
Thus, the different extracts/fractions of the seeds of Cicho- The authors are thankful to the Head, Department of
rium intybus have various degrees of antihepatotoxic activ- Pharmaceutical Chemistry for providing necessary research
ity. The methanol fraction and compound AB-IV possessed facilities, and to the in-charge, Central Animal Facility,
better antihepatotoxic activity. Further, they did not produce Jamia Hamdard, New Delhi for providing rats and related
240 B. Ahmed et al. / Journal of Ethnopharmacology 87 (2003) 237–240
facilities. One of the authors (A.B.S.) is also thankful to Handa, S.S., Sharma, A., Chakarborti, K.K., 1986. Natural products and
UGC, New Delhi for awarding GATE Scholarship. plants as liver protecting drugs. Fitoterapia 57, 307–351.
Kind, P.R.N., King, E.J.J., 1954. Estimation of plasma phosphatase by
determination of hydrolyzed phenol with aminopyrines. Journal of
Clinical Pathology 7, 332.
References Luna, L.G., 1968. Manual of Histology: Staining Methods of Armed
Force Institute of Pathology, 3rd ed. McGraw-Hill, New York.
Ahmed, B., Bawa, S., Siddiqui, A.B., Alam, T., Alam, S.A., 2002. Com- Reitman, S., Frankel, S.A., 1957. Colorimetric method for the deter-
ponents from seeds of Cichorium intybus Linn. Indian Journal of mination of serum glutamic oxalo acetic acid and glutamic pyruvic
Chemistry 41B, 2701–2705. transaminases. American Journal of Clinical Pathology 28, 56–63.
Dennett, C.W., 1964. New tables for multiple comparisons with a control. Sala, A.V. (Ed.), 1994. Indian Medicinal Plants: A Compendium of 500
Biometrics 20, 482–491. Species, 1st ed. Origent Longmen Ltd., Chennai, p. 74.
Dumas, B.T., Watson, W.A., Biggs, H.G., 1971. Albumin standards and The Wealth of India, vol. III, 1992. Publication & Information Directorate,
the measurement of serum albumin with bromocresol green. Clinica Council of Scientific and Industrial Research, New Delhi, p. 555.
Chimica Acta 31, 87–96. Woolson, R.F., 1987. Statistical Methods for the Analysis of Biomedical
Handa, S.S., Singh, A., 1995. Hepatoprotective activity of Apium grave- Data. Wiley, New York.
olens and Hygrophila auriculata against paracetamol and thioacetamide Wooton, I.D.P., 1964. Microanalysis in Medical Biochemistry, 4th ed.
intoxication in rats. Journal of Ethnopharmacology 49, 119–126. Churchill, London, pp. 138–140.
Screening of antimutagenicity via antioxidant

activity in Cuban medicinal plants
A. Ramos∗ , A. Visozo, J. Piloto, A. Garcı́a, C. A. Rodrı́guez, R. Rivero
Centro de Investigación y Desarrollo de Medicamentos, Avenue 26, No. 1605, Nuevo Vedado, Ciudad de La Habana, CP 10600, Cuba
Received 11 September 2002; received in revised form 23 April 2003; accepted 25 April 2003
Abstract
The reducing activity on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, • OH radical scavenging potential, in vitro inhibition of lipid
peroxidation and modulation of mutagenicity induced by ter-butyl hydroperoxide (TBH) in Escherichia coli were sequentially screened in
45 species of plants used with medicinal purposes in Cuba, in a search for antioxidant agents which protect DNA against oxidative stress.
Five species, e.g. Tamarindus indica L., Lippia alba L., Pimenta dioica (L.) Merr, Rheedia aristata Griseb. and Curcuma longa L.
displayed IC50 < 30 ␮g/ml in the DPPH radical reduction assay and IC50 < 32 ␮g/ml in lipid peroxidation inhibition testing. Pimenta dioica
and Curcuma longa L. showed also a 20% inhibition of the in vitro induced • OH attack to deoxyglucose. Further antimutagenesis assay in
Escherichia coli IC 188 evidenced that only Pimenta dioica prevents DNA damage by TBH to the test bacteria. A role of antioxidant enzymes
is presumed in this case, as judged by a different response in the isogenic Escherichia coli IC 203 deficient in catalase and alkyl hydroperoxide
reductase and the discrete inhibition of oxidative mutagenesis also observed when pre-treatment of the extract was assayed. Eugenol, the main
constituent of the essential oil of Pimenta dioica, also inhibited oxidative mutagenesis by TBH in Escherichia coli, at concentrations ranging
from 150 to 400 ␮g/plate.
Keywords: Antioxidant; Lipid peroxidation; Escherichia coli; Antimutagenesis; Pimenta dioica; Eugenol
1. Introduction in vitro assays followed by testing of the best candidates

for bacterial reversion in Escherichia coli.
The inhibition of permanent (transmissible) damage
of DNA in living organisms, known as antimutagenesis,
encompasses several enzymatic and non-enzymatic mecha- 2. Materials and methods
nisms (De Flora and Ramel, 1988) to counteract the mod-
ification of DNA components such as nitrogen bases and 2.1. Plant material
sugars by electrophiles. Free radicals released during ox-
idative stress are among the most widespread intracellular Medicinal plants screened in this study were collected in
DNA modifiers (Hochstein and Atallah, 1988; Møller and 2001 at the Medicinal Plants Experimental Station “Dr. Juan
Wallin, 1998). Their involvement in carcinogenesis, inflam- Tomás Roig”, in Güira de Melena, La Habana. The species
mation, diabetes, atherosclerosis, brain and heart ischaemia, are listed alphabetically in Table 1, indicating the parts of
ageing, etc. has been intensely addressed during the last the plant studied. A voucher specimen (number indicated in
years. the table) was deposited at the herbarium of this institution.
Search for novel antimutagens acting in chemoprevention
of relevant biomolecules oxidation is a promising field in 2.2. Extracts preparation
phytotherapy. This study presents the screening for antiox-
idant activity leading to inhibition of DNA damage of 45 The species collected were dried and 1 kg was subjected to
botanical species traditionally used in Cuba as herbal reme- hydroalcoholic percolation (Martin and Cook, 1961), yield-
dies, in a sequential strategy including three non-enzymatic ing 1 l of extract. It was concentrated under reduced pressure
to obtain a crude, kept in sealed containers at 4 ◦ C. Before
∗ Corresponding author. testing, the crude was dissolved in 70% methanol (v/v) up
E-mail address: arruiz2001@yahoo.com (A. Ramos). to 5 mg/ml and appropriately diluted.
doi:10.1016/S0378-8741(03)00156-9
242 A. Ramos et al. / Journal of Ethnopharmacology 87 (2003) 241–246
Table 1
Medicinal plants screened for antioxidant antimutagenicity IC50 values in the DPPH reduction assay of the hydroalcoholic extracts screened
No. Species Family Voucher ID Parts used IC50 (␮g/ml)
1 Anethum graveolens L. Apiaceae ROIG 4644 Seed 87

2 Annona reticulata L. Annonaceae ROIG 5663 Aerial parts 54
3 Artemisia absinthium L. Asteraceae ROIG 4640 Aerial parts 121
4 Bidens pilosa L. Asteraceae ROIG 4598 Aerial parts 127
5 Boerhavia erecta L. Nyctaginaceae ROIG 4642 Aerial parts 51
6 Bromelia pinguin L. Bromeliaceae ROIG 4667 Leaves, fruits 645
7 Brugmansia candida Pers. Solanaceae ROIG 4691 Leaves, flowers 314
8 Calendula officinalis L. Asteraceae ROIG 4625 Flowers 76
9 Capsicum frutescens L. var. frutescens Solanaceae ROIG 4650 Fruit 418
10 Cassia grandis L. Caesalpinaceae ROIG 4692 Seed 1108
11 Chrysantellum americanum (L.) Vatke Asteraceae ROIG 4699 Aerial parts 109
12 Curcuma longa L. Zingiberaceae ROIG 4693 Rhizome 47
13 Cyperus rotundus L. Cyperaceae ROIG 4688 Whole plant 72
14 Hamelia patens Jacq. Rubiaceae ROIG 4684 Leaves 116
16 Indigofera suffructicosa Mill. Fabaceae ROIG 4594 Aerial parts 341
15 Justicia pectoralis Jacq. var. Pectoralis Acanthaceae ROIG 4636 Aerial parts 368
17 Lepidium virginicum L. Brassicaceae ROIG 4626 Aerial parts 752
18 Lipia alba (Mill) NE Brown Verbenaceae ROIG 4611 Aerial parts 23
19 Matricaria recutita L. Asteraceae ROIG 4692 Flowers 351
20 Melia azederach L. Meliaceae ROIG 4687 Aerial parts 732
21 Melissa officinalis L. Lamiaceae ROIG 4586 Aerial parts 33
22 Mentha spicata L. Lamiaceae ROIG 4621 Aerial parts 4571
23 Mentha x piperita L. Lamiaceae ROIG 4590 Aerial parts 90
24 Momordica charantia L. var. abreviata Ser. Cucurbitaceae ROIG 4694 Aerial parts 169
25 Musa x paradisiaca L. Musaceae ROIG 4695 Juice 918
26 Nerium oleander L. Apocynaceae ROIG 4665 Aerial parts 104
27 Ocimum basilicum L. Lamiaceae ROIG 4638 Aerial parts 109
28 Ocimum tenuiflorum L. Lamiaceae ROIG 4675 Aerial parts 76
29 Parthenium hysterophorus L. Asteraceae ROIG 4626 Aerial parts 254
30 Pedilanthus tithymaloides (L.) Poit Euphorbiaceae ROIG 4697 Stem 251
31 Petiveria alliacea L. Phytolaccaceae ROIG 4678 Whole plant 255
32 Pimenta dioica (L.) Merr. Myrtaceae ROIG 4609 Aerial parts 21
33 Piper aduncum L. s.l. Piperaceae ROIG 4679 Whole plant 346
35 Plantago lanceolata L. Plantaginaceae ROIG 4588 Aerial parts 534
34 Plantago major L. Plantaginaceae ROIG 4589 Aerial parts 290
36 Plectranthus amboinicus (Lour) Spreng. Lamiaceae ROIG 4579 Leaves 181
37 Portulaca oleraceae L. Portulacaceae ROIG 4685 Whole plant 253
38 Pouteria mammosa (L.) Cronquist Sapotaceae ROIG 4683 Fruit, seed 1253
39 Rheedia aristata Griseb. Clusiaceae ROIG 4698 Bark 41
40 Ruta graveolens L. Rutaceae ROIG 4630 Aerial parts 459
41 Stachitarpheta jamaicensis (L.) Vahl Verbenaceae ROIG 4641 Aerial parts 79
42 Tamarindus indica L. Caesalpinaceae ROIG 4670 Bark 16
43 Teloxys ambrosioides (L.) W.A. Weber Chenopodiaceae ROIG 4639 Whole plant 153
44 Thuja occidentalis L. Cupressaceae ROIG 4632 Aerial parts 112
45 Zebrina pendula Schnizl Commelinaceae ROIG 4674 Aerial parts 48
2.3. DPPH reduction assay 2.5. Scavening of hydroxyl radical activity
Reduction of the 1,1-diphenyl-2-picrylhydrazyl radical Inhibition of hydroxyl radical damage to deoxyglucose

(DPPH) by the crude was performed in 96-well microplates. was assayed according to Jodynis-Liebert et al. (1999). OD
The absorbance at 515 nm of a methanolic solution of DPPH at 535 was recorded and the inhibition of • OH attack at
was recorded at different concentrations of the extract, ac- 100 ␮g/ml of extract expressed as a percentage of control.
cording to Navarro et al. (1992). IC50 values were derived Mannitol (50 mM) was used as reference • OH radical scav-
from the inhibition curves obtained. enger.
2.4. Lipid peroxidation assay 2.6. Antimutagenesis assay in Escherichia coli
Inhibition of lipid peroxidation was assessed as described Inhibition of bacterial mutagenesis reversion was tested
by Ramos et al. (2001). in Escherichia coli WP2 trp65 uvrA rfa/pKM 101 (IC 188)
A. Ramos et al. / Journal of Ethnopharmacology 87 (2003) 241–246 243
Table 2 to test suppression of the mutagenic response. Results are

In vitro hydroxyl radical scavenging and lipid peroxidation inhibition shown in Fig. 1, where a plot was shown for each species.
values of some species screened for antioxidant activity
The Tamarindus indica extract did not exert any effect
No. Species % Inhibition upon Escherichia coli mutagenicity by TBH. However, a
• OH Scavenging Lipid peroxidationa lowering to 25–50% in the revertant count was observed
when Escherichia coli was treated with the extracts of Lippia
5 Boerhavia erecta 0.00 2.23
12 Curcuma longa 22.52 100.00 (6.13)
alba L. and Rheedia aristata in the concentration ranging
18 Lippia alba 9.06 100.00 (31.30) from 2.5 to 10 mg/plate, even in the absence of TBH, which
21 Melissa officinalis 3.84 0.00 indicates some toxicity by the species assayed. This effect
32 Pimenta dioica 23.00 100.00 (29.30) was even more drastic in the case of Curcuma longa, where
39 Rheedia aristata 1.37 100.00 (14.83) a decrease of 25% was recorded at 4 mg/plate.
42 Tamarindus indica 6.23 100.00 (18.70)
45 Zebrina pendula 0.00 25.57
A different response was observed with the extract of
Pimenta dioica. A clear reduction up to 25% in the rever-
a Assayed for 100 ␮g/ml of extract; values in parentheses indicate IC
50 tant frequency was observed at no appreciable cytotoxicity.
in ␮g/ml.
The effect was confirmed when bacteria were pre-treated
(5 mg/ml) with the Pimenta dioica extract before TBH chal-
and its isogenic oxyR30 derivative (IC 203), kindly sent by lenge, a moderate 35% inhibition of oxidative mutagenesis
Dr. Manuel Blanco (Instituto de Investigaciones Citológicas, being recorded. On the other hand, post-treatment did not
Valencia, España). The basic plate incorporation protocol result in a significant decrease in mutagenicity. Escherichia
described by Blanco et al. (1998) was followed. Briefly, a coli strain IC 203, unable to express key antioxidant en-
mixture of fresh overnight culture of the strain, plant extract zymes such as catalase and alkylhydroperoxide reductase,
(typically 0–10 mg/ml) and tert-butylhydroperoxide (TBH) proved sensitive to the Pimenta dioica extract alone, with
at 100 ␮g/plate was poured in minimal agar plates and less than a 25% revertant survival at 10 mg/plate.
incubated at 37 ◦ C. Revertants were scored 48 h later and Eugenol, the main component of the Pimenta dioica es-
expressed as percentage to the untreated control (without sential oil, was also tested in the Escherichia coli IC 188
extract), an indicator of oxidative mutagenesis inhibition. system and the results were similar than those observed for
Concurrent testing to discriminate cytotoxicity from true the extract (Fig. 2). At low levels of cytotoxicity in a range
antimutagenesis was performed by treating bacteria with of 300–400 ␮g/plate, a significant decrease of 50% in the
the extract only. Pre- and post-treatment assays were peroxidative mutagenesis by TBH was recorded.
formed with the inclusion of a pre-incubation step (Yahagi
et al., 1977). For pre-treatment, Escherichia coli was treated
with 0–5 mg/ml of extract during 4 h at 37 ◦ C with shak- 4. Discussion
ing and plated at 100 ␮g/plate of TBH. On the other hand,
challenging of the cells with 100 ␮g/ml TBH was followed Antimutagenic properties elicited by plant species have a
by post-treatment with 0–5 mg/plate of the extract. full range of prospective applications in human healthcare.
Herbal remedies and phytotherapy drugs containing active
principles are currently developed to protect against elec-
3. Results trophile (e.g. free radical) attack to DNA and its widespread
outcomes such as ageing and cancer. Even for populations
DPPH reduction by the extracts is shown in Table 1. which use herbs traditionally, encouraging the use of species
Eight extracts (Boerhavia erecta L., Melissa officinalis L., with chemopreventive actions could be helpful as part of
Zebrina pendula L., Tamarindus indica L., Lippia alba L., life expectancy improvement strategies: costs are signifi-
Pimenta dioica (L.) Merr., Rheedia aristata Griseb. and cantly low, herbs have usually little or no toxicity during
Curcuma longa L.) exhibit IC50 values below 30 ␮g/ml, long-term oral administration and are relatively available at
indicating good potential as free radical scavengers. When large scale. Such is the case of curcumin, a polyphenol iso-
the screening proceeded to in vitro lipid peroxidation only lated from Curcuma sp. widely known in Asiatic medicinal
five extracts out of the above mentioned were really ac- practice, though a dietary component also (Commandeur and
tive as inhibitors of oxidative injury (Table 2). Besides, Vermeulen, 1996).
Curcuma longa and Pimenta dioica displayed some inhibi- Evidence of antioxidative properties has been gathered
tion (approximately 20%) of the hydroxyl radical attack to previously for some medicinal species scored as positive
deoxyglucose at 100 ␮g/ml, a relevant endpoint for DNA in our screening. Three active components (2-hydroxy-3 ,
protection against damage induced by free radicals. 4 -dihydroxyacetophenone, methyl 3,4-dihydroxybenzoate,
Antimutagenicity was then assayed in the five extracts 3,4-dihydroxyphenyl acetate and (−)-epicatechin) were iso-
displaying a positive response in at least two of the former lated by Tsuda et al. (1994) from the coat seed of Tamarindus
biochemical testing systems, using Escherichia coli rever- indica. There is a wide amount of literature supporting the
sion upon oxidative damage induced by TBH as a model use of Curcuma longa in the chemoprevention of pathologies
Fig. 1. Influence of hydroalcoholic extracts of Pimenta dioica, Curcuma longa, Tamarindus indica, Lippia alba and Rheedia aristata upon the oxidative
mutagenesis induced by TBH in Escherichia coli strains IC 188 and IC 203.
associated to free radical damage (Aráujo and León, 2001). Results of the screening tests indicate that antioxidant
Those effects have been attributed to curcumin and re- potential estimated in biochemical assays, though demon-
lated compounds (Commandeur and Vermeulen, 1996), strated in 18% of the species tested, does not confer protec-
well-known hydroxyl radical scavengers and inhibitors of tion to the cellular genome per se against oxidative stress
lipid peroxidation in vitro (Joe and Lokesh, 1994; Ruby in the experimental conditions described. Out of the five
et al., 1995). Finally, methanolic extracts of Pimenta dioica species which positively reduced the DPPH radical and in-
were also reported as antioxidant by Oya et al. (1997), hibited lipid peroxidation, only two were moderate hydroxyl
activity being attributed to the presence of pimentol and scavengers and just one of them, Pimenta dioica, resulted
biflorin. Apart from eugenol, Kikuzaki et al. (1999) isolated in an effective antimutagen in Escherichia coli.
five phenilpropanoids with antioxidant activity from the The case with Curcuma longa is particularly surprising,
berries of Pimenta dioica. due to the huge amount of experimental data accumulated
A. Ramos et al. / Journal of Ethnopharmacology 87 (2003) 241–246 245
Fig. 2. Effect of eugenol on the oxidative mutagenesis induced by TBH in Escherichia coli IC 188.
which supports its antioxidant potential (Aráujo and León, References

2001). However, as in other phenolic phytochemicals
(e.g. flavonoids), the question of reactive intermediaries Aráujo, C.A.C., León, L.L., 2001. Biological activities of Curcuma longa
and auto-oxidation through redox cycling, leading to a L. Memorias do Instituto Oswaldo Cruz 96, 723–728.
pro-oxidant state, should be addressed (De Groot and Rauen, Armstrong, M.J., Bean, C.L., Galloway, S.M., 1992. A quantitative as-
sessment of the cytotoxicity associated with chromosomal aberration
1988). In fact, Kelly et al. (2001) have reported that 25 ␮M detection in Chinese hamster ovary cells. Mutation Research 265, 45–
curcumin, the main biologically active principle of Cur- 50.
cuma longa, damages the DNA of Jurkat-T-lymphocytes as Blanco, M., Urios, A., Martı́nez, A., 1998. New Escherichia coli WP2
measured by single cell gel electrophoresis (comet assay). strains highly sensitive to reversion by oxidative mutagens. Mutation
As for Pimenta dioica, eugenol seems to be involved Research 413, 95–101.
Commandeur, J.N.M., Vermeulen, N.P.E., 1996. Cytotoxicity and cytopro-
in the protective response against the genotoxic injury by tective activities of natural compounds. The case of curcumin. Xeno-
TBH, though the contribution from closely related phyto- biotica 26, 667–680.
chemicals (e.g. phenylpropanoids) should not be excluded. De Flora, S., Ramel, C., 1988. Mechanisms of inhibitors of mutagenesis
As TBH releases reactive oxygen species (ROS) intracellu- and carcinogenesis. Classification and overview. Mutation Research
larly (Kappus, 1987), inactivating enzyme induction could 202, 285–306.
De Groot, H., Rauen, U., 1988. Tissue injury by reactive oxygen species
be involved in the lower levels of bacterial reversion ob-
and the protective effect of flavonoids. Fundamental and Clinical Phar-
served, apart from chemical interaction between ROS and macology 12, 249–255.
the extract components. In this sense, results with strain IC Ellahueñe, M.F., Pérez-Alzola, L.P., Orellana-Valdebenito, M., Muñoz, C.,
203 differed markedly, indicating a cytotoxic effect of the Lafuente-Indo, N., 1994. Genotoxic evaluation of eugenol using the
extract alone that might be due to its inability to cope with bone marrow micronucleus assay. Mutation Research 320, 175–180.
Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., Zeiger, E., 1983.
reactive species released after extract uptake. Additionally,
Salmonella mutagenicity test results for 250 chemicals. Environmental
it should be pointed out that pre-treatment with the extract Mutagenesis 5, 3–142.
also lead to a moderate inhibitory response in the strain IC Hochstein, P., Atallah, A.S., 1988. The nature of oxidants and antioxidant
188. In fact, it has been reported that eugenol induces phase systems in the inhibition of mutation and cancer. Mutation Research
II enzymes in mammals (Yokota et al., 1986), though data 202, 363–375.
on metabolism in prokaryotes is not available. On the other Jodynis-Liebert, J., Murias, M., Bloszyk, E., 1999. Effect of several
sesquiterpene lactones on lipid peroxidation and glutathione level.
hand, eugenol was not mutagenic in the bacterial reversion Planta Medica 65, 320–324.
(Ames) assay at concentrations of 3.3–333.3 ␮g/plate, as- Joe, B., Lokesh, B.R., 1994. Role of capsaicin, curcumin and dietary n-3
sayed in the standard battery of Salmonella strains (Haworth fatty acids in lowering the generation of reactive oxygen species in rat
et al., 1983) and its clastogenic potential in vitro and in vivo peritoneal macrophages. Biochimica Biophysica Acta 1224, 255–263.
is confined to doses near toxicity (Armstrong et al., 1992; Kappus, H., 1987. Oxidative stress in chemical toxicity. Archives of
Toxicology 60, 144–149.
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is not clear, in vivo assays with transgenic mice being neg- similar phenolic phytochemicals as inhibitors of oxidative damage to
ative (Rompelberg et al., 1996). So the response described cellular DNA. Mutation Research 485, 309–318.
herein deserves further attention. At present, experiments Kikuzaki, H., Hara, S., Kawai, Y., Nakatani, N., 1999. Antioxidative
are carried out in peripheral blood lymphocytes to get more phenylpropanoids from berries of pimenta dioica. Phytochemistry 52,
1307–1312.
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Martin, E.W., Cook, E.F., 1961. Remington’s Practice of Pharmacy, 12th
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Planta Medica 59, 312–314. Osawa, T., 1994. Antioxidative components isolated from the seed of
Oya, T., Osawa, T., Kawakishi, S., 1997. Spice constituents scavenging tamarind (Tamarindus indica L.). Journal of Agricultural and Food
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Evaluation of antioxidant activity of leaf extract of Seabuckthorn

(Hippophae rhamnoides L.) on chromium(VI)
induced oxidative stress in albino rats
S. Geetha a , M. Sai Ram a , S.S. Mongia a , Virendra Singh b , G. Ilavazhagan a , R.C. Sawhney a,∗
a Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi 110 054, India
b H.P. Agriculture University, Palampur, Himachal Pradesh, India
Received 12 December 2002; received in revised form 17 April 2003; accepted 25 April 2003
Abstract
The present study reports the antioxidant activity of Seabuckthorn (Hippophae rhamnoides), family Elaegnaceae, on chromium induced
oxidative stress in male albino rats. Oxidative stress was induced in the rats by force-feeding of potassium dichromate equivalent to a dose
of 30 mg/kg body weight (BW) of chromium(VI) for 30 days. Administration of chromium decreased the body weight and increased organ
to body weight ratio significantly. Chromium treatment significantly decreased reduced glutathione (GSH), and increased malondialdehyde
(MDA) and creatine phosphokinase (CPK) levels; further it also enhanced glutamate oxaloacetate transferase (GOT) and glutamate pyruvate
transferase (GPT) levels in the serum. Different doses of the alcoholic leaf extract of Seabuckthorn were evaluated for the protection against
the chromium induced oxidative stress. The results show that the leaf extract at a concentration of 100 and 250 mg/kg BW protected the
animals from the chromium induced oxidative injury significantly.
Keywords: Seabuckthorn; Chromium; Oxidative stress; Antioxidants
1. Introduction thetic antioxidants have been shown to have one or the other
side effects (Musk et al., 1994; Nocentini et al., 2001), there
Cellular exposure to exogenously or endogenously gen- has been an upsurge of interest in the therapeutic potential
erated oxidants causes macromolecular damage, including of medicinal plants as antioxidants in reducing free radical
protein oxidation, lipid peroxidation, and nucleic acid insta- induced tissue injury (Siddique et al., 2000; Engelhart et al.,
bility, and mutation (Ames and Shigenaga, 1992; Halliwell, 2002; Koleva et al., 2002). Numerous plant products have
1998). Chromium is a naturally occurring heavy metal found been shown to have the antioxidant activity (Aruoma and
commonly in the environment in the trivalent Cr(III) and Cuppelt, 1997; De-Groot and Rauen, 1998; Koleva et al.,
hexavalent Cr(VI) forms. Cr(VI) compounds have been de- 2002; Scartezzini and Speroni, 2000), and the antioxidant
clared as potent occupational carcinogens among workers in vitamins, flavonoids, and polyphenolic compounds of the
chrome plating, stainless steel, and pigment industries. The plant origin have been extensively reported as scavengers of
reduction of Cr(VI) to Cr(III) results in the formation of re- free radicals and inhibitors of lipid peroxidation (Hanasaki
active intermediates together with the oxidative tissue dam- et al., 1994; Formica and Regelson, 1995; Tapiero et al.,
age and a cascade of cellular events, including modulation of 2002).
apoptosis regulatory gene p53, and contribute to the cytotox- Seabuckthorn (Hippophae rhamnoides L., Elaegnaceae)
icity, genotoxicity, and carcinogenicity of Cr(VI)-containing is a thorny nitrogen fixing deciduous shrub, native to Eu-
compounds (Flores and Perez, 1999; Bagchi et al., 2001). rope and Asia (Rousi, 1971). All parts of the plant are con-
In recent years, the clinical importance of the herbal sidered to be a good source of a large number of bioactive
drugs has received considerable attention. As many syn- substances. The ripe fruit has been reported to be a rich
source of Vitamins A, C, E, and K, carotenoids, and organic
acids (Chen et al., 1990; Yao and Tigerstedt, 1992; Pintea
∗ Corresponding author. Tel.: +91-11-394-6546; fax: +91-11-393-2869. et al., 2001; Kallio et al., 2002). Many medicinal effects of
E-mail address: em dipas@yahoo.com (R.C. Sawhney). Seabuckthorn against flu, cardiovascular diseases, mucosal
doi:10.1016/S0378-8741(03)00154-5
248 S. Geetha et al. / Journal of Ethnopharmacology 87 (2003) 247–251
injuries, and skin disorders (Xiao, 1980; Beveridge et al., dislocation and weight of various organs of the body was
1999; Eccleston et al., 2002,) have been suggested to be due determined.
to the high contents of antioxidant substances present in this
plant. 2.4. Determination of biochemical parameters
In an earlier study (Geetha et al., 2002), we reported that
the alcoholic leaf extract has a significant antioxidant and Reduced glutathione (GSH) was estimated in the blood
immunomodulatory activity against the chromium induced by the method of Kum-Talt and Tan (1974). Malondialde-
oxidative stress, in vitro, in rat lymphocytes. Alcoholic leaf hyde (MDA) was determined by the method of Dousset
extract at a concentration of 500 ␮g/ml provided significant et al. (1983). Superoxide dismutase (SOD), glutathione
cytoprotection against the chromium induced free radical peroxidase (GPx), serum glutamate oxaloacetate transami-
production, apoptosis, and DNA fragmentation. To confirm nase (SGOT), and serum glutamate pyruvate transaminase
whether the antioxidant activity obtained from in vitro ap- (SGPT) activity in erythrocytes was determined as per the
plies to in vivo also, a study has been carried out to eval- manufacturer’s instructions using kits obtained from M/s
uate the antioxidant activity of the leaf extract against the Randox.
chromium induced oxidative injury using Sprague–Dawley All the experiments were conducted on two different oc-
albino rats as a model system. casions and data were analyzed using Student ‘t’-test. Since
there was no significant difference in any of the parameters
in the animals fed with the leaf extract alone, the data have
2. Methodology not been incorporated.
2.1. Plant material

3. Results
Seabuckthorn (Hippophae rhamnoides) leaves were col-
lected from the hilly regions of Western Himalayas, in the 3.1. Body weight and food and water consumption
month of September where the plant grows wildly under
natural conditions. The fresh leaves were cleaned and dried The effect of alcoholic leaf extract of Seabuckthorn on
under shade. The extraction was carried out using powdered body weight changes during the chromium induced oxidative
leaf (10 g) by refluxing with 500 ml of 70% ethanol at 80 ◦ C stress is shown in Fig. 1. Chromium feeding resulted in a
in a Soxhlet for 10 h. The extract was filtered and the fil- significant decrease in the body weight with the duration of
trate was dried at 50 ◦ C under reduced pressure in a rotary treatment; however, in animals fed with both the leaf extract
evaporator and dissolved in 70% alcohol to provide a con- and chromium, there was no significant change as compared
centration of 100 mg/ml. The crude extract was then diluted to the control group. Administration of chromium did not
in sterile saline containing 0.1% Tween-80. cause any significant change in the food and water intake
(data not shown).
2.2. Animals
3.2. Organ to body weight ratio
The experiments were conducted on male Sprague–
Dawley albino rats weighing 180–200 g maintained at
Table 1 shows organ to body weight ratio in the
25 ± 2 ◦ C with food and water ad libitum. The animals were
chromium- and Seabuckthorn-treated animals. Adminis-
housed three rats per cage, and maintained on 12 h day and
tration of chromium caused a significant increase in the
night cycle. Three different concentrations of the leaf ex-
tract were given orally, 50, 100, and 250 mg/kg body weight
(BW) with the help of gastric canula and the control group
was maintained on saline containing 0.1% Tween-80. The
leaf extract was administered an hour prior to the chromium
feeding. The study was approved by the Institute’s Animal
Ethical Committee and confirms to National guidelines on
the care and use of laboratory animals.
2.3. Oxidative stress
Oxidative stress was induced in rats by force-feeding of

1 ml potassium dichromate equivalent to a dose of 30 mg/kg
BW of chromium(VI) for 30 days. Food and water intake
by the animals was monitored daily and body weight was Fig. 1. Body weight changes in the chromium- and Seabuckthorn leaf
measured weekly. The animals were sacrificed by cervical extract (LE)-treated animals.
S. Geetha et al. / Journal of Ethnopharmacology 87 (2003) 247–251 249
Table 1
Organ to body weight ratio in the chromium- and Seabuckthorn leaf extract (LE)-treated animals
Control Chromium LE (50 mg) + chromium LE (100 mg) + chromium LE (250 mg) + chromium
Heart 3.52 ± 0.03 4.37 ± 0.21a 3.41 ± 0.12b 3.15 ± 0.13b 2.89 ± 0.16b
Liver 0.03 ± 0.06 0.05 ± 0.01a 0.030 ± 0.01b 0.031 ± 0.03b 0.029 ± 0.01b
Lung 5.25 ± 0.75 5.56 ± 0.49 5.41 ± 0.19 5.30 ± 0.41 5.03 ± 0.32
Spleen 1.7 ± 0.3 2.51 ± 0.31a 1.67 ± 0.26b 1.83 ± 0.15b 1.62 ± 0.18b
Kidney 7.26 ± 0.78 9.45 ± 0.48a 7.20 ± 0.54b 7.36 ± 0.39b 6.94 ± 0.62b
a P < 0.05 vs. control.
b P < 0.05 vs. chromium.
heart, liver, spleen, and kidney to body weight ratio in all

the animals. However, the lung to body weight ratio was
not altered in the chromium-treated animals. Pretreatment
with Seabuckthorn leaf extract in all the doses (50,100,
and 250 mg) maintained the organ to body weight ratio
comparable to that of control values.
3.3. GSH and MDA levels
Erythrocyte GSH levels were significantly decreased

following the chromium treatment, whereas a significant
increase in plasma MDA levels was observed (Fig. 2). Ad-
ministration of leaf extract in 100 and 250 mg/kg BW dose
reverted the GSH and MDA levels to that of control values. Fig. 3. Glutathione peroxidase (GPx) and superoxide dismutase (SOD)
activity in the chromium- and Seabuckthorn leaf extract-treated animals.
3.4. GPx and SOD activity
leaf extract significantly inhibited the chromium induced in-
Administration of chromium caused a significant increase crease in enzyme levels and restored to that of control values
(P < 0.05) in the erythrocyte GPx levels but did not affect
SOD levels (Fig. 3). The leaf extract in a dose of 100 and
250 mg/kg BW was able to restore the GPx levels to that of 4. Discussion
control values.
Three different concentrations of the leaf extract, 50, 100,
3.5. SGOT, SGPT, and creatine phosphokinase (CPK) and 250 mg/kg BW, were evaluated for the antioxidant ac-
activity tivity against the chromium induced oxidative stress in male
albino rats. The results of the present study demonstrate that
CPK, SGOT, and SGPT levels were increased (P < 0.05) the ethanolic leaf extract of Seabuckthorn at a concentra-
in all the animals treated with chromium (Fig. 4). Adminis- tion of 100 and 250 mg/kg BW protected the animals sig-
tration of 100 and 250 mg/kg BW dose of the Seabuckthorn nificantly from the chromium induced oxidative damage.
Oral feeding of chromium resulted in a significant de-
crease in body weight and increase in organ to body weight
ratio. Chromium(VI) compounds are well-known oxidiz-
ing agents capable of directly inducing tissue damage and
possess carcinogenic, mutagenic, and teratogenic potency
(Sugiyama, 1992). Chromium(VI) compounds are easily
taken up by the cells and are subsequently reduced to Cr(III)
species. This reduction generates free radicals, which play
a major role in the adverse biological effects of these com-
pounds (Shi et al., 1999). Administration of chromium
significantly increased the lipid peroxidation as evident by
the increase in MDA levels. To cope with the oxidative
stress, there was a significant increase in GPx activity in the
erythrocytes, which in turn caused a marked reduction in
Fig. 2. Effect of various doses of the leaf extract (LE) of Seabuckthorn the GSH levels. No significant change in the SOD activity
on reduced glutathione (GSH) and malondialdehyde (MDA) levels. was observed in the chromium-treated animals and our re-
250 S. Geetha et al. / Journal of Ethnopharmacology 87 (2003) 247–251
Fig. 4. Serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), and creatinine phosphokinase (CPK) activity
in the chromium- and Seabuckthorn leaf extract-treated animals.
sults fall in confirmation with earlier studies (Bagchi et al., and phytochemical analysis are also in progress in this
1995). Besides activating the oxidative stress, chromium laboratory.
also caused a marked increase in CPK, SGOT, and SGPT
activity suggesting that the chromium treatment also causes
hepatic damage. Many workers have also demonstrated the Acknowledgements
hepatotoxic effects of chromium (Ueno et al., 1995; Dartsch
et al., 1998), which is mainly due to the lipid peroxidation. The authors are grateful to Director, DIPAS, for providing
These adverse effects of chromium could be significantly all the facilities to carry out the experimental work. Secretar-
curtailed by pretreating the animals with the leaf extract of ial assistance of Mr. Parveen Kumar is also acknowledged.
Seabuckthorn.
In animals fed with 50 mg/kg BW of the leaf extract, no
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Short communication
Antimalarial activity of Cinchona-like plants used to
treat fever and malaria in Brazil
V.F. Andrade-Neto a , M.G.L. Brandão b , J.R. Stehmann c , L.A. Oliveira a , A.U. Krettli a,∗
a Laboratory of Malaria, Centro de Pesquisas René Rachou/FIOCRUZ and Department of Parasitology,
Universidade Federal de Minas Gerais (UFMG), Av. Augusto de Lima 1715, Belo Horizonte, CEP 30190-002 MG, Brazil
b Laboratory of Pharmacognosy, Faculty of Pharmacy, UFMG, Belo Horizonte, Brazil
c Department of Botany, UFMG, Belo Horizonte, Brazil
Received 30 September 2002; received in revised form 27 March 2003; accepted 27 March 2003
Abstract
For centuries, malaria was treated with the bark of Cinchona calisaya and Cinchona succirubra plants named “quinas” in Brazil, from which
the quinine molecule was isolated. Other plant species known also as “quinas” are used to treat fever and malaria, like Deianira erubescens
(roots and leaves), Strychnos pseudoquina (bark), and Remijia ferruginea (bark). Based on this popular knowledge, we evaluated the in vivo
antimalarial activity of the ethanol crude extracts of these plant species in mice infected with Plasmodium berghei. Only Remijia ferruginea
showed antimalarial activity, reducing parasitaemia and mortality at the highest dose tested. Its phytochemical analysis showed the presence
of alkaloids but not quinine. The other two plant species were inactive.
© 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Antimalarial tests; Ethnopharmacology; Cinchona; Remijia ferruginea; Plasmodium berghei
1. Introduction over 350 years (Meshnick, 1997; Meshnick and Dobson,

2001), especially in cases of chloroquine-resistant Plasmod-
Malaria is a human protozoan disease widespread in the ium falciparum (Ridley, 2002). The antimalarial activity of
Amazon region. In Brazil, most malaria cases at present about 40 medicinal plant species used in Brazil have been
are caused primarily by Plasmodium vivax, followed by tested in vivo and in vitro (Carvalho et al., 1991; reviewed
Plasmodium falciparum (Ministry of Health/Funasa, 2002). in Krettli et al., 2001). In this paper, we studied species
Several derivatives of the quinine molecule are used to treat known as “quinas” used to treat fever and malaria, namely,
acute symptomatic malaria including chloroquine, amodi- Deianira erubescens Cham. and Schltdl. (Gentianaceae;
aquine and mefloquine or, to prevent Plasmodium vivax roots and leaves), Remijia ferruginea (A.St.-Hil.) DC.
late relapses, as primaquine and other 8-aminoquinolines (Rubiaceae; bark) and Strychnos pseudoquina A.St.-Hil.
(Greenwood, 1995; Ridley, 2002). At present, malaria (Loganiaceae; bark) (Wasick, 1944; Correa, 1975; Balbach,
chemotherapy is complicated by drug-resistant strains of 1980) for their antimalarial activity.
Plasmodium falciparum, including in the Amazon region
(Segurado et al., 1997), thus, new antimalarials are needed.
Quinine is an alkaloid first identified and isolated from 2. Material and methods
the barks of the Peruvian plants Cinchona calisaya Wedd.
and Cinchona succirubra Pav. ex Klotzsch (Rubiaceae) 2.1. Plant samples
(Bruce-Chwatt, 1988), popularly known as “quinas” in
Brazil. Quinine has been used to treat human malaria for The roots and leaves of Deianira erubescens and the bark
of Remijia ferruginea and Strychnos pseudoquina, popu-
larly used as remedies, were collected and dried at room
∗ Corresponding author. Tel.: +55-31-3295-3566; temperature. Plant samples were collected in the “cerrado”
fax: +55-31-3295-3115. (savanna-like) vegetation of the Serra do Cabral, northern
E-mail address: akrettli@cpqrr.fiocruz.br (A.U. Krettli). Minas Gerais, in July 2000. Voucher herbarium specimens
0378-8741/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0378-8741(03)00141-7
254 V.F. Andrade-Neto et al. / Journal of Ethnopharmacology 87 (2003) 253–256
were prepared and deposited in the herbarium BHCB, Fed- ined (1000× magnification). Parasitaemia was determined
eral University of Minas Gerais. Plants were identified in coded blood smears by randomly counting 2000–6000
by one of us (J.R.S.) as Deianira erubescens Cham. and erythrocytes in the case of low parasitaemia (≤10%); or
Schltdl. (BHCB 47133), Remijia ferruginea (A.St.-Hil.) up to 1000 erythrocytes in the case of higher parasitaemia.
DC. (BHCB 47136), and Strychnos pseudoquina A.St.-Hil. Overall mortality was monitored daily in all groups during
(BHCB 4720). a period of four weeks following inoculation. The inhibition
of parasite growth in the drug-treated groups was calculated
2.2. Ethanol extract preparation as follows: parasitaemia in the control (non-treated) group
minus parasitaemia in the drug-treated group, divided by
Plant samples (100 g each) were pulverized, then extracted parasitaemia in the control (non-treated) group, expressed as
by percolation with 80% ethanol, as described earlier by percentages. The extracts were considered partially active if
Brandão et al. (1997). The dried extracts (4.82 g from root parasitaemia was reduced by 30% or more (Carvalho et al.,
and 23.1 g from leaves of Deianira erubescens, 6.75 g from 1991). All extracts were tested in three independent exper-
Remijia ferruginea bark, and 11.2 g from Strychnos pseudo- iments at daily doses of 500 and 1000 mg/kg body weight.
quina bark) were stored at 4 ◦ C and tested as a fresh water
suspension in Tween 20 at 1% final concentration as anti- 2.6. Statistical analysis
malarial drug.
The ANOVA and Student’s t-test were used for compari-
2.3. Phytochemical screening son of average parasitaemia and the Kruskal–Wallis test by
the Biostat 1.0 Software for Windows (MCT-CNPq) for sur-
For identification of alkaloids, the dried extract was vival analysis.
solubilized in ethanol and water (1:2), alkalinized to pH
9.0 with NH4 OH and extracted with CH2 Cl2 . The ex- 2.7. Ethical approval
tracts were chromatographed (TLC) using concentrated
dichloromethane phase and applied on silica gel. The solvent The present work was approved by the Ethical Commit-
system used was toluene–ethyl acetate–diethylamine (7:2:1) tee for Using Animals at Fundação Instituto Oswaldo Cruz,
or chloroform–diethylamine and Draggendorff/NaNO2 or FIOCRUZ (CEUA P0094-01).
10% ethanol/H2 SO4 (and UV-365 nm) as a spray reagent.
Quinine (Sigma Ref. 1125, St. Louis, MO, USA) was
used as the reference compound. For the identification of 3. Results and discussion
flavonoids, saponins and tannins, solvent systems and de-
tection methods as described by Wagner and Bladt (1996) The phytochemical screening of “quina” plants showed
were used. the presence of flavonoids and tannins in Deianira
erubescens leaves, and tannins in its root; alkaloids were de-
2.4. Malaria parasites tected in the Strychnos pseudoquina and Remijia ferruginea
barks. The antimalarial activity of these plants in Plasmod-
The Plasmodium berghei, strain NK-65 was originally ium berghei-infected mice is shown in Table 1 for one repre-
received from the New York University Medical School. sentative experiment in a total of three performed. Extracts
It was maintained through weekly blood passages in mice, from the bark of Strychnos pseudoquina and the root and
by intraperitoneal route (i.p.), using 3.8% sodium citrate as leaves of Deianira erubescens were inactive and caused no
anticoagulant and also in liquid nitrogen with glycerolyte. significant reduction in parasitaemia or mortality. Extracts
from Remijia ferruginea bark caused up to 48% inhibition
2.5. Antimalarial tests of parasite growth at the dose of 1000 mg/kg, but only a bor-
derline activity at the dose of 500 mg/kg in one experiment.
We used the traditional suppressive test of Peters (1965) Malaria mortality was slightly but not significantly re-
as modified by Carvalho et al. (1991). Briefly, adult Swiss duced by Remijia ferruginea in relation to the mortality in
albino mice weighing 18–20 g were inoculated by i.p. route non-treated mice (P > 0.05). However, the mice survival
with 1 × 105 Plasmodium berghei-infected red blood cells. time was reduced in groups treated with Deianira erubescens
The mice were randomly divided in groups of five per cage, extracts from leaves and root (Table 1). Indeed, one dose of
and treated during 4 consecutive days with daily doses of 4000 mg/kg of Deianira erubescens ethanol extract caused
the extracts, by oral route. Two control groups were used 20% mortality to non-malaria mice, whereas, similar doses
in each experiment, one treated with chloroquine at low of Strychnos pseudoquina and Remijia ferruginea bark ex-
non-curative doses (≤25 mg/kg, orally), the other group tracts caused no mortality (not shown). Chloroquine, a stan-
was kept untreated. On the 5th day after parasite inocula- dard antimalarial used in non-curative doses to mice, caused
tion, blood smears were prepared from all mice, fixed with a significant reduction of parasitaemia (51–95%) and mor-
methanol, stained with Giemsa, then microscopically exam- tality (Table 1) in all experiments.
V.F. Andrade-Neto et al. / Journal of Ethnopharmacology 87 (2003) 253–256 255
Table 1
Antimalarial activity of ethanolic extracts of Cinchona-like plants and chloroquine in mice infected with Plasmodium berghei
Plant species/part used or Dose (mg/kg) Reduction of Half-survival time Antimalarial
reference drug orally 4× parasitaemia (%)a in days ± S.D. activityb
Strychnos pseudoquina
Bark 1000 10 10 ± 2.0 None
500 0 10 ± 1.5
0c 10 ± 2.0
Remijia ferruginea
Bark 1000 48∗ 12 ± 2.6 Partial
500 34 11 ± 2.4
0c 10 ± 2.1
Deianira erubescens
Leaves 1000 0 9 ± 1.2 None
500 0 9 ± 2.3
0c 10 ± 0.6
Root 1000 0 8 ± 1.7+ None
500 18 9 ± 2.6
0c 11 ± 0.6
Chloroquine (control) 25 95∗∗ 28 ± 3.0++ Active
12.5 51∗∗∗ 18 ± 1.5+++ Partial
0c 11 ± 1.0
S.D.: standard deviation. The significance of parasitaemia inhibition evaluated by the Student’s t-test was ∗ P = 0.045; ∗∗ P = 0.0001; ∗∗∗ P = 0.001;
and, of mortality by the Kruskal–Wallis test was + P = 0.015; ++ P = 0.007; +++ P = 0.04.
a Reduction of parasitaemia in relation to control non-treated mice.
b Extracts which reduce ≥30% parasitaemia are considered partially active.
c Control non-treated group.
Cinchona species, a source of quinine, do not occur in Brandão, M.G.L., Krettli, A.U., Soares, L.S.R., Nery, C.G.C., Marinuzzi,
Brazil. Remijia ferruginea known as “quina” has been used H.C., 1997. Antimalarial activity of extracts and fractions from Bidens
pilosa and other Bidens species (Asteraceae) correlated with the pres-
as a substitute of quinine to treat malaria (Wasick, 1944; ence of acetylene and flavonoid compounds. Journal of Ethnopharma-
Souza, 1951). Other plants named “quinas” are used in tra- cology 57, 131–138.
ditional medicine to treat fever and malaria. In the present Bruce-Chwatt, L.J., 1988. Cinchona and its alkaloids: 350 years later.
study, the antimalarial effect of three species of “quinas” New York State Journal of Medicine 88, 318–322.
tested in rodent malaria proved to be disappointing. Only Carvalho, L.H., Brandão, M.G.L., Santos-Filho, D., Lopes, J.L.C.,
one, Remijia ferruginea, displayed some activity; Strychnos Krettli, A.U., 1991. Antimalarial activity of crude extracts from
Brazilian plants studied in vivo in Plasmodium berghei-infected
pseudoquina and Deianira erubescens were totally inactive. mice and in vitro against Plasmodium falciparum in culture.
It has been previously shown that Strychnos pseudoquina ex- Brazilian Journal of Medical and Biological Research 24, 1113–
tracts tested in Plasmodium cathemerium, an avian malaria, 1123.
were inactive (Wasick, 1944). In an attempt to characterize Correa, M.P., 1975. Dicionário de Plantas uteis do Brasil e das Exóticas
the active compounds in Remijia ferruginea, we performed e Cultivadas, 3 ed., vol. 1–6. Instituto Brasileiro de Desenvolvimento
Florestal, Rio de Janeiro.
TLC tests of the alkaloid extract. We found no quinine. Other
Greenwood, D., 1995. Conflicts of interest: the genesis of synthetic anti-
alkaloids are described in Remijia plants (McCalley, 2002), malarial agents in peace and war. Journal of Antimicrobial Chemother-
but further work has to be undertaken to elucidate whether apy 36, 857–872.
they are responsible for the antimalarial activity of Remijia Krettli, A.U., Andrade-Neto, V.F., Brandão, M.G.L., Ferrari, W.M.S.,
ferruginea. 2001. The search for new antimalarial drugs from plants used to treat
fever and malaria or plants randomly selected: a review. Memórias do
Instituto Oswaldo Cruz 96, 1033–1042.
McCalley, D., 2002. Analysis of the Cinchona alkaloids by
Acknowledgements high-performance liquid chromatography and other separation tech-
niques. Journal of Chromatography A 967, 1–19.
We express thanks to the Brazilian Agencies (CNPq Meshnick, S.R., 1997. Why does quinine still work after 350 years of
and CAPES) for fellowships, to PRONEX, FAPEMIG and use? Parasitology Today 13, 89–90.
FIOCRUZ (PAPES-II) for financial support. Meshnick, S.R., Dobson, M., 2001. The history of antimalarial drugs.
In: Rosenthal, P. (Ed.), Antimalarial Chemotherapy. Mechanisms of
Action, Resistance, and New Directions in Drug Discovery. Humana,
Totowa, New Jersey, pp. 15–16.
References Ministry of Health, Brazil/Funasa, 2002. Casos de Malária no Brasil.
Available at www.funasa.gov.br/index.
Balbach, A., 1980. A Flora nacional na Medicina Doméstica, 17th ed., Peters, W., 1965. Drug resistance in Plasmodium berghei I. Chloroquine
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antimalarial drugs. Nature 415, 686–693. 130.
Segurado, A.A., Di Santi, S.M., Shiroma, M., 1997. In vivo and in vitro Wagner, H., Bladt, S., 1996. Plant Drug Analysis. A Thin Layer Chro-
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Tropical de São Paulo 39, 85–90. Anais da Associação Quı́mica do Brasil 3, 44–59.

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Journal of Ethnopharmacology 87 (2003) 123–142

Preliminary comparative analysis of medicinal plants used

Keywords: Ethnobotany; Bulgaria; Italy

1. Introduction order (species and/or subspecies), among the very nume-

Table 1 contrary, Crataegus laevigata is acclaimed in Bulgaria for

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123–142

used externally, the direct employment of fresh plant juice References

Anti-inflammatory activity of (E)-1-(3,4-dimethoxyphenyl)

1. Introduction prostaglandin-like activity of the exudates (Panthong et al.,

the experiment. Prior to any treatment, ear thickness was

DMPBD Standard drugs

Oxyphenbutazone Phenidone Diclofenac Salbutamol

Table 2 provided a new source of mediators that contributed to the

Comparative effect of Prunus persica L. BATSCH-water extract

1. Introduction 1995; Kosuge et al., 1985). The chemical constituents of

3. Statistical analysis 4.1. Comparative effects of PPE and tacrine on

The use of medicinal plants in self-care

Keywords: Medicinal plants; Self-care; Prevalence; Rural Ethiopia

1. Introduction communities of the developing countries including Ethiopia

for health related researches (Berhane, 2000; Shamebo,

2.2. Data collection and analysis

Information on demographic characteristics, prevalence

Fig. 2. Location of the 10 study villages in the district.

with the use of herbal medicine. In this regard, illiterates and

The financial assistance from Katholischer Akademischer

Biological activity of extracts from Catalpa bignonioides

Apartado 67, 28660 Boadilla del Monte, Madrid, Spain

Apartado 67, 28660 Boadilla del Monte, Madrid, Spain

Apartado 67, 28660 Boadilla del Monte, Madrid, Spain

Keywords: Catalpa bignonioides; Extracts; Biological activities

1. Introduction species are found in cultivation as greenhouse ornamentals.

The bacterial growth inhibition assays were performed

pod extracts were calculated to provide an amount of the Table 1

4.1. Phytochemical study 4.5. Antinociceptive activity

Saline 15 1.53 ± 0.02 2.00 ± 0.03 2.64 ± 0.10 2.91 ± 0.10

G. Nicholson, and Oroxylum indicum (L.) Vent. (Rasahad References

Comparison of the gastroprotective effect of a diterpene lactone

Keywords: Gastroprotective activity; DHC; Croton cajucara

1. Introduction through protection of the gastric mucosa by an increase of

Fig. 1. DHC and derivatives.

HCl/ethanol Indomethacin Stress

domethacin. Cimetidine, as well as compound I, signifi-

The oral administration of compound IV inhibited ul- 4. Discussion

Effect of Holotrichia diomphalia larvae on liver fibrosis

Keywords: Holotrichia diomphalia larvae; Acute hepatotoxicity; Liver cirrhosis

1. Introduction et al., 1994). Prophenoloxidase from the hemolymph of

2.8. Statistical analysis Table 1