Lia Marliani, M.Si., Apt.

Rubi Biologi Farmasi

phenolic compounds that have one or Phenol is the structure upon
compounds ? more hydroxyl groups which the entire group is
attached directly to an based. The aromatic ring in
aromatic ring. this case is, of course, benzene

Polyphenols compounds that have more

compounds ? than one phenolic hydroxyl
group attached to one or more
benzene rings
 CLASSIFICATION
Phenolic compounds are characteristic of plants
and as a group they are usually found as esters
or glycosides rather than as free compounds

Ribéreau-Gayon (1972) :
1. Widely distributed phenols – ubiquitous to all plants,
or of importance in a specific plant
2. Phenols that are less widely distributed – limited
number of compounds known
3. Phenolic constituents present as polymers.

Harborne and Simmonds (1964)

Coumarins also have a C6-C3 skeleton, but
they possess an oxygen heterocycle as part of
the C3-unit, Isocoumarins, structure similar to
coumarins, but the position of the oxygen and
carbonyl groups within the oxygen heterocycle
are reversed

umbelliferone (1.21), 4-methylumbelliferyl β-D-

glucuronide (4-MUG); 1.22, bergenin
(Isocoumarins)(1.23)
 tonka bean
 Coumarins owe their class name to ‘Coumarou’, the vernacular name of the tonka bean (Dipteryx
odorata Willd, Fabaceae), from which coumarin, it was isolated in 1820 (Bruneton, 1999).

 Coumarins are secondary metabolites of higher plants, few microorganisms (bacteria and fungi), and
sponges

 Coumarins are widely distributed in plants, both in the free form and as glycosides, and are commonly
found in families such as the Umbelliferae/Apiaceae and Rutaceae

 There are four main coumarin sub-types:

1. Simple coumarins,

2. Furanocoumarins,

3. Pyranocoumarins,

4. Pyrone-substituted coumarins.

coumarin nucleus
 Simple coumarins
• the hydroxylated, alkoxylated and alkylated derivatives of
the parent compound, coumarin, along with their glycosides

Furanocoumarins
• consist of a five-membered furan ring attached to the
coumarin nucleus, divided into linear or angular types with
substitution at one or both of the remaining benzoid positions
 Pyranocoumarins
• analogous to the furanocoumarins, but contain a six-
membered ring
O O O

Pyrone-substituted coumarins
• Coumarins substituted in the pyrone ring include 4-
hydroxycoumarin, 3-phenylcoumarin, 3,4-benzocoumarin
 Number of coumarins presented in seven different families of plants

Maria João Matos, Lourdes Santana, Eugenio Uriarte, Orlando A.

Abreu, Enrique Molina and Estela Guardado Yordi (2015). Coumarins — An Important Class of Phytochemicals, Phytochemicals - Isolation,
Characterisation and Role in Human Health, Dr. Venketeshwer Rao (Ed.), InTech, DOI: 10.5772/59982. Available from:
http://www.intechopen.com/books/phytochemicals-isolation-characterisation-and-role-in-human-health/coumarins-an-important-class-of-
 Coumarins widely distributed in plants, both in the free form and as
glycosides, and are commonly found in families……………………..

 What are main sub-type of Coumarins ? The difference among them

are…….

 Esculetin and Umbelliferone belongs to sub-type …….

 Family-specie Use Referenc
Coumarin
(vernacular name) * e
Apiaceae/ Umbelliferae
Imperatorin, bergapten, oxypeucedanin, pabulenol, marmesin, xanthotoxin,
Ammi majus (Bishop's flower) M [72]
isopimpinellin and heraclenin.
A. visnaga (Pick-tooth,
PyranoCoumarins M [58, 73]
Toothpickweed)
Anethum graveolens M,
Aesculetin, bergapten, scopoletin [60]
(Dill/Adas sowa) F
Angelicin, osthol (major constituent in rhizome/root at 0.2%), bergapten, imperatorin,
Angelica archangelica M,
isoimperatorin (major constituent in fruit), oreoselone, oxypeucedanin, umbelliferone, [57, 58]
(Angelica) F
xantonin, xanthotoxin, xanthotoxol
Apigravin, apiumetin, apiumoside, bergapten, celerin, celereoside, isoimperatorin,
M,
Apium graveolens (Celery) isopimpinellin, osthenol, rutaretin, seselin, umbelliferone, 8-hidroxy-5- [57, 58]
F
methoxypsoralen.
Coriandrum sativum M,
Umbelliferone, [58]
(Coriander) F
M,
Cuminum cyminum (Cumin) Escopoloetina, bergapten [58]
F
Daucus carota subsp. carota 8-methoxypsoralen, 5-methoxypsoralen (0.01–0.02 ug/g) in fresh plant, concentration
M [57]
(Wild Carrot) increased in the disease plant.
M,
Foeniculum vulgare (Fennel) Umbelliferone, esculetin, bergapten, seselin, psoralen [58]
F
Ferula assafoetida Umbelliferone, coumarin-sesquiterpene complexes e.g. asacoumarin A and asacoumarin
M [57]
(Asafoetida) B.
Bergapten and oxypeucedanin as major constituent (up to 0.02% and 0.01%,
Petroselinum crispum M,
respectively); also 8-metoxypsoralen, imperatorin, isoimperatorin, isopimpinellin, [57, 58]
(Parsley) F
psoralen, xanthotoxin (up to 0.003%).
 Rutaceae
Sesquiterpenic coumarin ethers, diterpenic coumarin ethers, triterpenic coumarin
Aegle marmelos (Bael fruit) M, F [75-77]
ethers, sesterterpenic coumarin ethers, auraptene, epoxyauraptene, marmin.
Citrus aurantium (Bitter Volatile Coumarins (0.09%): aurapteno, auraptenol, bergapteno, bergaptol,
M, F [58]
Orange tree) escoparona, citropteno.
C. limonum (Lemon tree) Escopoletin, umbelliferone, bergamotin, bergapten, bergaptol, citropten M, F [58]
C. sinensis (Orange tree) Herniarin, scopoletin M, F [60]
Melicope spp. Coumarins, chromones, dichromones M [56]
Murraya paniculata (M.
exotica) (Orange Jessamine, Coumarins M [74]
Chinese box)
Paramygnya monophylla Poncitrin, nordentatin M [56]
Stauracanthus perforates Coumarins M [78]
Tetradium daniellii (Euodia
Coumarins M [79, 80]
daniellii)
Toddalia aculeata (T. asiatica)
Ulopterol M [74, 81]
(Orange climber)
Zanthoxilum americanum Xanthyletin, xanthoxyletin, alloxanthoxyletin, 8-(3,3-dimethylallyl)-
M [57]
(Northern Prickly Ash) alloxanthoxyletin.
Z. syncarpum Coumarins M [82]
 Asteraceae/Compositae
Achillea millefolium
Coumarins (0.35% ) M [58]
(Yarrow)
Ageratum conyzoides
1-2 benzopirone M [83]
(Mexican ageratum)
Arnica montana (Arnica) Scopoletin, umbelliferone M, F [57, 58]
Chamaemelum nobile
Scopoletin-7-glucoside M, F [57, 58]
(Roman Chamomile)
Cichorium intybus
Coumarins M, F [73]
(Chicory)
Conyza sumatrensis
Osthol M [56]
(Fleabane)
Eupatorium triplinerve
Coumarins M [84]
(White Snakeroot)
Hieracium pilosela
Coumarins (0.2–0.6%): 7-glucosil- umbeliferone M [58]
(Mouse Ear)
Lactuca virosa (Wild
Aesculin, cichoriin M [58]
Lactuce)
Matricaria recutita
Umbelliferone and its methyl ether, heniarin. M, F [57, 58]
(Chamomille)
Mikania glomerata
Coumarins M [85]
(Guaco)
Mikania hirsutissima Coumarins M [86]
 Hypericaceae
Hypericum perforatum (Saint
Umbelliferone, escopoletin, M [58]
John Wort)
Lamiaceae/Labiadae
Lavandula angustifolia [58,
Coumarins: 1,500 ppm, 0.25%: hernairin, santonin M
(Lavender) 84]
L. latifolia (Aspic) Coumarins: 22 ppm M [84]
Lycopus europeus (European
Coumarins: 1,200 ppm M [84]
Bugle)
Ocimum basilicum (Basil) Aesculetin, aesculin M, F [60]
Salvia officinalis (Garden
Esculetin M [58]
Sage)
Lauraceae
Cinnamomum cassia (C.
aromaticum) (Chinese Coumarins M, F [57]
cinnamon)
C. verum (C. zeylanicum) [57,
Coumarins (0.65%) M, F
(Cinnamon) 58]
Laurus nobilis (laurel, sweet
Coumarins M, F [57]
bay)
Persea americana (Avocado) Scopoletin M [60]
Lytraceae
Lawsonia inermis (Henna) Coumarins M [58]

Maria João Matos, Lourdes Santana, Eugenio Uriarte, Orlando A.

Abreu, Enrique Molina and Estela Guardado Yordi (2015). Coumarins — An Important Class of Phytochemicals, Phytochemicals - Isolation,
Characterisation and Role in Human Health, Dr. Venketeshwer Rao (Ed.), InTech, DOI: 10.5772/59982. Available from:
http://www.intechopen.com/books/phytochemicals-isolation-characterisation-and-role-in-human-health/coumarins-an-important-class-of-
 Biosynthesis

The shikimate pathway provides an

alternative route to aromatic
compounds, particularly the aromatic
amino acids l-phenylalanine, l-
tyrosine, and l-tryptophan
Bourgaud et.al, Phytochem Rev (2006) 5:293–308
Bourgaud et.al, Phytochem Rev (2006) 5:293–308
 Coumarins Biosynthesis are via ……………..pathway

 Shikimic acid  ……………………  cinnamate acid

Dicoumarol and Warfarin
• oral blood anticoagulant in the treatment of thrombosis, where the risk of blood clots
becomes life threatening
• Warfarin was initially developed as a rodenticide and has been widely employed for many
years as the first-choice agent, particularly for destruction of rats
Psoralens
• Plants containing psoralens have been used internally and externally to promote skin
pigmentation and suntanning. Bergamot oil obtained from the peel of Citrus aurantium
ssp. bergamia (Rutaceae) can contain up to 5% bergapten and is frequently used in
external suntan preparations.
• The psoralen, because of its extended chromophore, absorbs in the near UV and allows
this radiation to stimulate formation of melanin pigments
Musa et al., 2013
  There have been a variety of methods described for extraction of coumarins. Generally,
coumarins extraction can be performed either on dry or fresh material, with solvents of
different polarities, depending on the type of structure. Some coumarins are sparingly
soluble in apolar solvents and often they can be crystallized directly by cooling or
concentrating the solvent

 Soxhlet extracts the dry powdered material with petroleum ether continuously for 3
days. The ether extract is concentrated until 1/5 of the original volume and it is cooled
to obtain the crystallization of the extract. Coumarins are presented in the obtained
solid.

Maria João Matos, Lourdes Santana, Eugenio Uriarte, Orlando A.

Abreu, Enrique Molina and Estela Guardado Yordi (2015). Coumarins — An Important Class of Phytochemicals, Phytochemicals - Isolation,
Characterisation and Role in Human Health, Dr. Venketeshwer Rao (Ed.), InTech, DOI: 10.5772/59982. Available from:
http://www.intechopen.com/books/phytochemicals-isolation-characterisation-and-role-in-human-health/coumarins-an-important-class-of-
  The dry material is removed and sprayed with ethanol, continuously using the Soxhlet for 2–3
days, at least. The extract is concentrated under vacuum to an oily residue. This residue is
repeatedly washed with portions of hot water. The aqueous washings are combined and
concentrated to the minimum volume, acidifying with hydrochloric acid solution 10%. The
mixture is refluxed for 30 minutes. If some precipitate appears, it is filtered (hot filtration) and
the solution is allowed to cool. The crystals are collected by filtration and found therein the
coumarins.

 Available materials are extracted with ethyl ether or by successive macerations with the Soxhlet
apparatus. The extract is concentrated to dryness and the residue contains the coumarins.

 The dried and ground plant material is extracted with acetone continuously in a Soxhlet
apparatus, for at least 3 days, and the extract is concentrated to dryness giving a residue that
contains the coumarins.

Maria João Matos, Lourdes Santana, Eugenio Uriarte, Orlando A.

Abreu, Enrique Molina and Estela Guardado Yordi (2015). Coumarins — An Important Class of Phytochemicals, Phytochemicals - Isolation,
Characterisation and Role in Human Health, Dr. Venketeshwer Rao (Ed.), InTech, DOI: 10.5772/59982. Available from:
http://www.intechopen.com/books/phytochemicals-isolation-characterisation-and-role-in-human-health/coumarins-an-important-class-of-
  Purification and separation of coumarins contained in various extracts could be
performed by using chromatographic columns, using as a carrier aluminum oxide
and as solvent the eluotropic series: benzene-hexane (1:2.5); benzene; chloroform;
chloroform-acetone, in proportions of a linear gradient to pure acetone. For
recognition of the described structures some trials were described, within which there
are:
 Those that recognize coumarin’s phenolic substitutions where Emerson's Reagent is used,
developing color.

 The presence of lactone groups can be observed leading to changes of pH in the medium.
When coumarins are dissolved in ethanol, solutions change the color when acidified (yellow
color disappears).

 The furan ring can be recognized by using the Erlich test. The extract is treated with a
solution of dimethylamino-benzaldehyde (5% ethanol), and then acidified by bubbling
gaseous hydrochloric acid. The orange color indicates a positive test.

Maria João Matos, Lourdes Santana, Eugenio Uriarte, Orlando A.

Abreu, Enrique Molina and Estela Guardado Yordi (2015). Coumarins — An Important Class of Phytochemicals, Phytochemicals - Isolation,
Characterisation and Role in Human Health, Dr. Venketeshwer Rao (Ed.), InTech, DOI: 10.5772/59982. Available from:
http://www.intechopen.com/books/phytochemicals-isolation-characterisation-and-role-in-human-health/coumarins-an-important-class-of-
  the isolation and analysis of coumarins diverse methods have been used:
chromatography (paper chromatography, thin layer chromatography, gas
chromatography, and high-performance liquid chromatography), titrimetric,
and spectrophotometric (colorimetric and polarographic) methods.

 Due to coumarin-characteristic chromophore groups and its strong UV

absorption at around 300 nm, it is routinely possible to be detected by
feasible methods such as ultraviolet–visible spectroscopy (UV–vis). UV–vis
detector is used in high performance liquid chromatography (HPLC) and also
other hyphenated techniques are employed to characterize and quantify
natural products such as Liquid Chromatography (LC)–Photo Diode Array
Detector (PDA), coupling of Mass Spectrometry to LC-MS or Ultra
Performance Liquid Chromatography coupled with Mass Spectrometry
(UPLC-MS)

Maria João Matos, Lourdes Santana, Eugenio Uriarte, Orlando A.

Abreu, Enrique Molina and Estela Guardado Yordi (2015). Coumarins — An Important Class of Phytochemicals, Phytochemicals - Isolation,
Characterisation and Role in Human Health, Dr. Venketeshwer Rao (Ed.), InTech, DOI: 10.5772/59982. Available from:
http://www.intechopen.com/books/phytochemicals-isolation-characterisation-and-role-in-human-health/coumarins-an-important-class-of-
 They display the fluuorescence in UV light (365 nm)  spot on TLC can
be easily located and recovered without the use of a chromogenic reagent

 In general, furanocoumarin posses a dull yellow or ochre fluorescence;

except psoralen and 6-methoxyangelicin (the blue fluorescence); and
angelicin and 8-hydroxyl-5-methoxypsoralen (the purple fluorescence)

 Ammonia vapour  indicative of phenolic groups in the coumarin.

Spektrum UV
Gojiberi
• The extract was a column chromatographed on silica gel with a
gradient elution of n-hexane/EtOAc (1:0→0:1) and afford to nine
fractions. F6 was subjected to column chromatography with n-
hexane/ EtOAc (7:3→0:1) and finally purified by preparative thin
layer chromatography with n-hexane/ EtOAc (2:8) to afford impure
compound 1. Further purification of this impure crude by preparative
1. Scopoletin
thin layer chromatography with chloroform/methanol (9:1) obtained
pure compound 1 (310 mg).
• F8 was subjected to column chromatography with n-hexane/EtOAc
(6:4→0:1) and then loaded on preparative thin layer chromatography
with n-hexane/EtOAc (1.5:8.5) to afford impure further purification
using preparative TLC with chloroform/methanol (9:1) afford to pure
compound 2 (220 mg)
2. Esculetin