Food and Chemical Toxicology 49 (2011) 533536

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Food and Chemical Toxicology

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Brief communication

Chemical composition and antioxidant activity of essential oils and solvent

extracts of Ptychotis verticillata from Morocco
El Mokhtar El Ouariachi a, Pierre Tomi b, Abdelhamid Bouyanzer a, Belkheir Hammouti a,
Jean-Marie Desjobert b, Jean Costa b, Julien Paolini b,
a
b

LCAE-URAC18, Facult des Sciences, Universit Mohammed Premier B.P. 717, 60 000 Oujda, Morocco
Universit de Corse, Laboratoire de Chimie des Produits Naturels, UMR-CNRS 6134, BP 52, 20250 Corte, France

a r t i c l e

i n f o

Article history:
Received 21 July 2010
Accepted 12 November 2010

Keywords:
Ptychotis verticillata
Essential oil
Solvent extract
Antioxidant activity
Chemical composition

a b s t r a c t
The objective of this study was to characterize the chemical composition of the essential oil and extracts
of Ptychotis verticillata. The antioxidative activities of this species were also evaluated to suggest it as a
new potential source of natural antioxidants. Analysis of the chemical composition of P. verticillata essential oil from Morocco was carried out using GC and GCMS. The oil was dominated by phenolic compounds (48.0%) with carvacrol (44.6%) and thymol (3.4%) as the main compounds. Plant phenolics
constitute one of the major groups of components that act as primary antioxidant free radical terminators. The amounts of total phenolics and avonoids in the solvent extracts (diethyl ether and ethyl acetate) were determined spectrometrically. Furthermore, the antioxidant activities of the essential oil and
extracts were determined using a DPPH test system. The DPPH scavenging activity of extracts increased
in the order ethyl acetate > ascorbic acid > diethyl ether > essential oil. Finally, a relationship was
observed between the antioxidant activity potential and total phenolic and avonoid levels of the extract.
Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.

1. Introduction
Phenolic compounds are the main agents that can donate
hydrogen to free radicals and thus break the chain reaction of lipid
oxidation at the rst initiation step (Agraval, 1989). This high potential of phenolic compounds to scavenge radicals may be explained by their phenolic hydroxyl groups (Havsteen, 2002).
Polyphenolic compounds are also known for their ability to prevent fatty acids from oxidative decay (Fecka et al., 2007). The oxidation is caused by the rancidity of unpreserved aliments rich in
unsaturated fatty acids (Li et al., 2008). Furthermore, many synthetic antioxidant components (BHA and BHT) have shown toxic
and/or mutagenic effects; therefore, plant antioxidants are suggested as an interesting alternative. Numerous studies exhibited
a strong relationship between total phenolic content and antioxidant activity in fruits, vegetables, and medicinal plants (Dorman
et al., 2003; Velioglu et al., 1998). Flavonoid constituents possess
a wide spectrum of chemical and biological activities, including
radical scavenging properties (Shimoi et al., 1996). Indeed, Shimoi
et al. (1996) reported that plant avonoids that show antioxidant
activity in vitro also function as antioxidants in vivo. Malkowski
(2006) showed the role of these compounds in the defense mech-

Corresponding author. Tel.: +33 04 95 45 01 93; fax: +33 04 95 45 01 62.

E-mail address: paolini@univ-corse.fr (J. Paolini).

anism against oxidative stress from oxidizing agents and free

radicals.
The scavenging of reactive oxygen species (ROS) is a possible
mechanism of action for antioxidant compounds (Havsteen,
2002; Matkowski, 2008; Peterson and Dwyer, 1998). ROS may be
the causative factor involved in many human degenerative diseases, and antioxidants are known to have some degree of preventive and therapeutic effects on these disorders. The ROS hydrogen
peroxide caused lipid peroxidation and DNA oxidative damage in
cells (Nordberg and Arner, 2001; Pervaiz and Clement, 2007).
Moreover, high levels of ROS such as superoxide and hydrogen peroxide are observed in various cancer cells (Ushio-Fukai and
Nakamura, 2008). Small molecular weight antioxidants are considered possible protection agents that reduce oxidative damage in
the human body when the internal enzymatic mechanisms fail or
are inadequately efcient (Halliwell, 1995).
Ptychotis verticillata Nnkha (Apiaceae family), otherwise
known as Ammoides pusilla (Brot.) Breistr., Psychotis ammoides
Koch (Bellakhdar, 1997), Ammoides verticillata Briq. and Petroselium
ammoides Rchb. l. (Wehmer, 1931; Quezel and Santa, 1963), is an
aromatic herbaceous species (1035 cm tall) widespread in northern Africa (Quezel and Santa, 1963).
In Morocco, this species is currently used in folk medicine as a
febrifuge and for its antispasmodic, antiseptic and antidiabetic
properties (Bnouham et al., 2007, 2010). Bekhchi and Abdelouahid
(2004) showed the antimicrobial and antifungal properties of

0278-6915/$ - see front matter Crown Copyright 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.11.019

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E.M. El Ouariachi et al. / Food and Chemical Toxicology 49 (2011) 533536

essential oils from P. verticillata. Moreover, the antimicrobial activity of essential oil was studied using the agar diffusion test on eight
strains of bacteria and against fungus and yeast. A twofold oil solution showed important antimicrobial activity (Laouer et al., 2003).
These authors was also described the effect of P. verticillata oil on
the growth of two pathogenic bacteria (Pseudomonas syringae pv.
syringae and Pseudomonas syringae pv. mosprunorum) using agar
diffusion test (Laouer et al., 2004). While the essential oil composition has only been the subject of a few investigations, the oils were
found to be rich in thymol (Bellakhdar, 1997). Indeed, the essential
oil of the aerial parts of plant was analyzed using both GC and GC/
MS. The results revealed no less than 46 constituents, among
which thymol (44.5%), c-terpinene (32.9%), and p-cymene (13.5%)
were the most abundant. More recently, Bnouham et al. (2007,
2010) have reported the antidiabetic and antihyperglycemic activities of the water extract from this species. The toxicology tests
used in this paper suggested no adverse effect of the use of this
plant.
In the present work, the antioxidant activity of essential oil and
extracts of P. verticillata were determined using the 2,2-diphenyl1-picrylhydrazyl (DPPH) test system. Antioxidants react with the
stable free radical 1,1-diphenyl-2-picrylhydrazyl (deep violet color) and convert it to 1,1-diphenyl-2-picrylhydrazine with discoloration. The degree of discoloration indicates the free radical
scavenging potential of the sample/antioxidant, and it has been
found that antioxidants such as cysteine, glutathione, ascorbic acid,
tocopherol, and polyhydroxy aromatic compounds (hydroquinone,
pyrogallol, etc.) reduce and decolorize 1,1-diphenyl-2-picrylhydrazyl by their donating ability (Blois, 1958). The aim of this
work is to evaluate the antioxidative properties of the essential oil
and extracts of P. verticillata. Additionally, the total phenolic and
avonoid contents of diethyl ether and ethyl acetate extracts and
the chemical composition of the essential oil have been
determined.
2. Materials and methods
2.1. Plant material

280 C. Samples were injected in the split mode (1/50) using helium as a carrier gas
(1 mL/min) and a 0.2 lL injection volume of pure oil. Retention indices (RI) of compounds were determined relative to the retention times of a series of n-alkanes
(C5C30) (Restek, Lisses, France) with linear interpolation using the Van den Dool
and Kratz (1963) equation and software from PerkinElmer.

2.5. GCMS analysis

Samples were analyzed with a Perkin-Elmer turbo mass detector (quadrupole)
coupled to a PerkinElmer Autosystem XL equipped with the fused-silica capillary
columns Rtx-1 and Rtx-wax. Carrier gas: helium (1 mL/min), ion source temperature: 150 C, oven temperature programmed from 60 to 230 C at 2 C/min and then
held isothermally at 230 C (35 min), injector temperature: 280 C, energy ionization: 70 eV, electron ionization mass spectra were acquired over the mass range
35350 Da, split: 1/80, injection volume: 0.22 lL of pure oil.

2.6. Identication of essential oil constituents

Identication of individual components was based (i) on comparison of calculated RI, on polar and apolar columns, with those of authentic compounds or literature data (Knig et al., 2001; National Institute of Standards and Technology,
2008); and (ii) on computer matching with commercial mass spectral libraries
(Adams, 2001; Knig et al., 2001; National Institute of Standards and Technology,
1999) and comparison of mass spectra with those of our own library of authentic
compounds or literature data (Adams, 2001; Knig et al., 2001).

2.7. Determination of total phenolic contents

Total phenolic contents of the extracts were determined using FolinCiocalteu
reagent according to the method previously reported by Slinkard and Singleton
(1977), using caffeic acid as a standard, and as modied by Li et al. (2008).
200 lL of the dilute extract solution containing 40 lg of the extract was added to
1 mL of Folin-Ciocalteu reagent (diluted in distillated water). After 4 min, 800 ll
of Na2CO3 (75 mg/mL) solution was added and the mixture was allowed to stand
for 45 min at room temperature. At the end of the incubation, the absorbance
was measured at 760 nm. The same procedure was also applied to the standard
solutions of caffeic acid, and a standard curve was obtained. The concentrations
of phenolic compounds expressed as lg caffeic acid equivalent per mg of extract

Table 1
Essential oil composition of Ptychotis verticillata.

The aerial parts of P. verticillata were harvested in May 2009 (full bloom) from
Ahr, Morocco. Voucher specimens were deposited in the herbarium of Mohamed
1st University, Oujda, Morocco.
2.2. Essential oil isolation
Fresh vegetal material was water distillated (3 h) using a Clevenger-type apparatus according to the method recommended in the European Pharmacopoeia
(Council of Europe, 1996). The essential oil yields were 2% (w/w). The oils were
dried over anhydrous sodium sulfate and then stored in sealed glass vials at
45 C prior to analysis.
2.3. Preparation of the extracts
Boiling water extracts (100 mL) of plant samples obtained under reux conditions (hydrodistillation process) were extracted three times (3  20 mL) with organic solvents (diethyl ether and ethyl acetate). Water extract residues were then
extracted by boiling acidied water (2 N HCl) prior to liquidliquid extraction.
The diethyl ether and ethyl acetate extracts were ltered and concentrated under
vacuum to obtain two extracts in yields of 0.15% and 0.33% (w/w), respectively.
The organic solvent extracts were dried over anhydrous sodium sulfate and then
stored in sealed glass vials at 45 C prior to analysis. Each extraction was performed in triplicate.
2.4. GC analysis
a

GC analysis was carried out using a Perkin-Elmer Autosystem XL GC apparatus

(Waltham, MA, USA) equipped with a dual ame ionization detection (FID) system
and the fused-silica capillary columns (60 m  0.22 mm I.D., lm thickness
0.25 lm) Rtx-1 (polydimethylsiloxane) and Rtx-wax (polyethyleneglycol). The oven
temperature was programmed from 60 to 230 C at 2 C/min and then held isothermally at 230 C for 35 min. Injector and detector temperatures were maintained at

Na

Components

RI lb

RI ac

RI pd

%e

Identication

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

a-Thujene
a-Pinene

924
936
973
978
987
1015
1025
1024
1051
1086
1150
1164
1176
1226
1267
1278
1335
1362
1578

923
931
966
972
982
1015
1025
1025
1052
1084
1151
1163
1175
1227
1266
1279
1334
1364
1569

1031
1028
1123
1113
1160
1268
1203
1210
1244
1513
1691
1592
1686
1540
2153
2169
1668
1751
1967

0.2
1.0
2.2
0.7
1.3
9.4
18.4
1.4
9.5
0.2
0.2
0.3
0.2
0.2
3.4
44.6
0.7
4.7
0.3

GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,
GC,

Sabinene
b-Pinene
Myrcene
p-Cymene
Limonene
Cineole-1,8
c-Terpinene
Linalol
Borneol
Terpinen-4-ol
a-Terpineol
Carvacryl methyl ether
Thymol
Carvacrol
a-Terpinyl acetate
Geranyl acetate
Caryophyllene oxyde
Total identied

98.9

Monoterpene hydrocarbons
Phenolic components
Oxygenated Monoterpenes
Oxygenated Sesquiterpenes

33.3
48.2
6.9
0.3

CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS
CGMS

The numbering refers to elution order on apolar column (Rtx-1).

RI l = retention indices on the apolar column of literature (Knig et al., 2001;
National Institute of Standards and Technology, 2008).
c
RI a = retention indices on the apolar column (Rtx-1).
d
RI p = retention indices on the polar column (Rtx-Wax).
e
Relative percentages of components (%) are calculated on GC peak areas on the
apolar column (Rtx-1); Values expressed are means of three parallel measurements.
b

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E.M. El Ouariachi et al. / Food and Chemical Toxicology 49 (2011) 533536

Table 2
Total phenol and avonoid contents of Ptychotis verticillata solvent extracts.
Solvent extract

Total polyphenol
content (lg CA/mg extract)

Total avonoid content

(lg quercetin/mg extract)

Diethyl ether
Ethyle acetate

251 6
343 18

27 2
73 4

published on biological activities (antitermitic, antibacterial, nematicidal and insecticidal) of phenol rich oils (with thymol, carvacrol
and eugenol derivatives) from various species as Origanum sp.,
Thymus sp., or Trachystermum ammi (Kaur and Arora, 2009; Mayaud
et al., 2008; Pandey et al., 2009; Park et al., 2007; Seo et al., 2009).
3.2. Assays of total phenolics and avonoids from solvent extracts

Values expressed are means SD of three parallel measurements.

were calculated according to the standard caffeic acid graph. All experiments were
carried out in triplicate, and caffeic acid equivalent values were reported as X SD
of triplicates.
2.8. Determination of total avonoids contents
Total avonoid contents were determined using the Dowd method as adapted
by Arvouet-Grand et al. (1994). One milliliter of 2% aluminium trichloride (AlCl3)
in methanol was mixed with the same volume of extracts (200 lg). The absorption
at 430 nm was measured after 10 min against a blank sample consisting of 1 mL
methanol without AlCl3. The concentrations of avonoid compounds expressed as
lg quercetin equivalent per mg of extract were calculated according to the standard
quercetin graph. All experiments were carried out in triplicate, and quercetin equivalent values were reported as X SD of triplicates.
2.9. Antioxidant activity
The free radical-scavenging activities of essential oil and solvent extracts were
measured using DPPH as described by Hatano et al. (1988). Various concentrations
(0.1 mL) of the oil (0.252.00 mg/mL), the diethyl ether extract (28224 mg/L), and
ethyl acetate (1767 mg/L) in ethanol and water were added to 4 mL of a DPPH radical solution in ethanol (the nal concentration of DPPH was 0.05 mM). The mixture
was strongly shaken and left to stand at room temperature for 30 min in the dark.
The absorbance was measured at 517 nm against a blank. Inhibition of the free radical, DPPH, in percent (I%) was calculated according to the formula:

I% 100  Acontrol  Asample =Acontrol ;

where Acontrol is the absorbance of the control reaction and Asample is the absorbance
of the test compound. The sample concentration providing 50% inhibition (IC50) was
calculated from the graph of inhibition percentage against sample concentration.
Tests were carried out in triplicate. Ascorbic acid was used as a positive control.

3. Results and discussion

3.1. Essential oil composition
The chemical composition of essential oil of P. verticillata from
Morocco was characterized by 19 constituents, which accounted
for 98.9% of the total oil. The identied compounds and the essential oil compositions are reported in Table 1. The oil was dominated
by phenolic compounds (48.0%), with high amounts of carvacrol
(44.6%) and thymol (3.4%). The other major components were limonene (18.4%), g-terpinene (9.5%), p-cymene (9.4%), and geranyl
acetate (4.7%). It should be noted that several studies have been

The amounts of total phenolics in the extracts were determined

spectrometrically according to the FolinCiocalteu procedure and
calculated as caffeic acid equivalent. The amounts of total phenols
found in the plant extracts are shown in Table 2. The total phenolics and contents of the diethyl ether and ethyl acetate extracts of
P. verticillata were 251 6 and 343 18 lg/mg, respectively. The
results showed that the ethyl acetate extract has higher total phenol and avonoid components than the diethyl ether extract. Similarly, the ethyl acetate extract was found to be richer in avonoids
(73 4 lg/mg) than the diethyl ether extract (27 2 lg/mg).
3.3. Antioxidant properties
The antioxidant activity of the essential oil and extracts was
determined by the DPPH test system. Table 3 demonstrates DPPH
scavenging activity, expressed in percentage, caused by different
concentrations of essential oil and solvent extracts from P. verticillata. The weakest radical scavenging activity (23%) was exhibited
by the essential oil at a concentration of 6 lg/mL, whereas the
strongest activity (67%) was exhibited by the ethyl acetate extract
at a concentration of 1 lg/mL. The next highest activity (47%) was
for the diethyl ether extract at a concentration of 1 lg/mL. In this
system, the ethyl acetate extracts showed activity as strong as
the synthetic antioxidant (54% at 1 lg/mL concentration), and
diethyl ether extracts exhibited similar activity to ascorbic acid.
At shown in Table 3, the antioxidant activity of extracts and essential oil also increased with an increase in their concentrations. At
higher concentrations, the antioxidant activity of extracts was closer to the scavenging effect of ascorbic acid. For instance, at 2.0 lg/
mL, the scavenging activity of ascorbic acid was around 82%, and a
diethyl ether extract solution of 2.8 lg/mL had a scavenging activity of 87%. The same value was obtained for the ethyl acetate extract at a concentration of 1.5 lg/mL.
Therefore, DPPH scavenging activity is usually presented by the
IC50 value. Concentrations of the antioxidant providing 50% inhibition of DPPH in the test solution (IC50) were calculated and presented in Table 3. The ethyl acetate extracts of P. verticillata had
the highest radical scavenging activity with the lowest IC50 value
of 0.64 lg/mL. This was higher than the diethyl ether extracts with
an IC50 value of 0.92 lg/mL and essential oil with an IC50 value
of 18.75 lg/mL. In addition, DPPH scavenging abilities of ethyl

Table 3
DPPH radical-scavenging of essential oil and solvent extracts (diethyl ether and ethyl acetate) from Ptychotis verticillata measurated at different concentrations.
Sample

Antioxidant activities

Essential oil

Essential oil concentration (lg/mL)

Scavenging effect on DPPH (%)
DPPH IC50 (lg/mL)
Extract concentration (lg/mL)
Scavenging effect on DPPH (%)
DPPH IC50 (lg/mL)
Extract concentration (lg/mL)
Scavenging effect on DPPH (%)
DPPH IC50 (lg/mL)
Extract concentration (lg/mL)
Scavenging effect on DPPH (%)
DPPH IC50 (lg/mL)

Diethyl ether

Ethyl acetate

Ascorbic acid

Values expressed are means of three parallel measurements.

6.2
23

12.5
38

18.7
50

25.0
54

37.5
60

0.7
43

1.0
57

1.4
62

2.8
87

5.6
93

0.4
35

0.6
48

1.0
67

1.2
75

1.5
87

0.2
21

0.35
26

0.5
34

1.0
54

2.0
82

18.75

0.92

0.64

0.97

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E.M. El Ouariachi et al. / Food and Chemical Toxicology 49 (2011) 533536

acetate were higher than that of the standard (ascorbic acid with
an IC50 value of 0.97 lg/mL).
4. Conclusions
The solvent extracts and essential oil of P. verticillata were found
to be effective antioxidants by in vitro assays, and can therefore be
proposed as new potential sources of natural additives for the food
and/or pharmaceutical industries. According to these results, there
is a relationship between the total phenol content and antioxidant
activity. Indeed, it is extremely important to point out that there is
a positive correlation between the antioxidant activity potential
and the amount of phenolic compounds in the extracts. Moreover,
as reported in literature data (Bellakhdar, 1997), the antioxidant
activity of essential oil could be attributed to its relatively high
content (48.2 lg/mg) of the phenolic compounds carvacrol and
thymol. However, the components responsible for the antioxidant
activities of the extracts were not identied and further work
should be conducted to isolate and identify these bioactive
compounds.
Conict of Interest
The authors declare that there are no conicts of interest.
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