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Chemical Constituents and Biological Evaluation of Leaves Essential Oils of

Vitex agnus-castus L. Growing in the Southern-West Algeria

Article · September 2014

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Current Bioactive Compounds 2017, 13, 165-169
RESEARCH ARTICLE
ISSN: 1573-4072
eISSN: 1875-6646

Analgesic and Anti-Inflammatory Effects of Essential Oils of Vitex agnus-

castus L. from South-West of Algeria

Abdallah Habbab1, Khaled Sekkoum1, Nasser Belboukhari1, Abdelkrim Cheriti2 and Hassan Y. Aboul-Enein3,*

1
Bioactive Molecules & Chiral Separation Laboratory, University of Bechar, Bechar 08000, Algeria; 2Phytochemistry &
Organic Synthesis Laboratory, University of Bechar, Bechar 08000, Algeria; 3Pharmaceutical and Medicinal Chemistry
Departments, Pharmaceutical and Drug Industries Research Division, National Research Center, Dokki, Giza 12622,
Egypt

ARTICLE HISTORY
Received: May 25, 2016
Revised: September 29, 2016
Accepted: October 10, 2016
DOI:
10.2174/15734072126661610141330
31
Current Bioactive Compounds
Abstract: Background: Many species of the genus Vitex from the Verbenaceae family are used in tradi-
tional medicine for the treatment of various medical conditions such as depression, venereal diseases,
malaria, asthma, allergy, wounds, skin diseases, snake bite and body pains. One of them, Vitex agnus-
castus L. has been used to treat pain, swelling, inflammation, headaches, rheumatism, and sexual dysfunc-
tion.
Methods: The analgesic and anti-inflammaotry effects of essential oil extracts from Vitex agnus-castus L
were evaluated employing various standard experimental test models. Acetic acid was used to induce
writhes in mice and inflammation of paw edema. The analgesic activity of seeds essential oil at a dose of
150mg / kg gave a percentage of inhibition very near to diclofenac (40.13% and 40.41%, respectively).
The essential oils show an anti-inflammatory activity in the test of the edema.
Results: The evaluation of the percentage of inhibition indicates that the essential oil of Vitex agnus-
castus L. seeds possess anti-inflammatory activity (150 mg/kg) nearby and sometimes better -during the

time-than diclofenac (61,04% - 50% at 30 min; 62,04% - 83,33% at 60 min and 91,05% - 90,91% at 120
min, respectively).
Conclusion: The results of this research indicated that Vitex agnus-castus L. essential oil of seeds re-
markably inhibited inflammation and could explain the traditional usage of this plant.
Keywords: Analgesic, anti-inflammatory, essential oils, Vitex agnus-castus L, edema, traditional medicine.

1. INTRODUCTION Vitex agnus-castus L has been used to treat pain, swelling,

Based on definition of International Association for the
Study of Pain (IASP), pain is an unpleasant sensory and
emotional experience associated with actual or potential tis-
sue damage or described in terms of such damage [1]. Pain is
created by different reasons such as harmful heat, stretch,
electrical flow, necrosis, inflammation, laceration and spasm
[2]. Herbal medicines derived from plant extracts are being
increasingly utilized to treat a wide variety of clinical dis-
eases, though relatively little knowledge about their mode of
action is available [3]. Many species of the genus Vitex from
the Verbenaceae family are used in traditional medicine for
the treatment of various medical conditions [4, 5]. Previous
pharmacological studies carried out on various Vitex species
have illustrated its safety and efficacy to treat different dis-
eases, which has made Vitex a popular subject in phyto-
chemical and ethnobotanical research [5]. One of them,
inflammation, headaches, rheumatism, and sexual dysfunc-
tion [6, 7]. Essential oil of Vitex agnus-castus L contains
some important monoterpenes and sesquiterpenes [8-10].
Previous studies have indicated that some of these terpenes
have anti-inflammatory and antinociceptive effects in differ-
ent models of pain and inflammation [11]. There are many
reports proved the anti-inflammatory effect of some extracts
and isolated compounds, and the antinociceptive effect of
essential oil of leaves from Vitex agnus-castus L. [12-16].
Literature data revealed that there is no specific study on all
essential oils of Vitex agnus-castus L for its in vivo analgesic
and anti-inflammatory activities. The objective of the present
study was to evaluate the analgesic and anti-inflammatory
activities of essential oils of leaves, flowers and seeds of
Vitex agnus-castus L.
2. MATERIALS AND METHODS

*Address correspondence to this author at the Pharmaceutical and Medicinal 2.1. Animals
Chemistry Departments, Pharmaceutical and Drug Industries Research
Division, National Research Center, Dokki, Giza 12622, Egypt; Tel: Permission was obtained from the Committee of use of
+00203678948; Fax: +020233370931; E-mail: haboulenein@yahoo.com animal experiments (Approval # 1943-19).

1875-6646/17 $58.00+.00 © 2017 Bentham Science Publishers

166 Current Bioactive Compounds 2017, Vol. 13, No. 2
Swiss albino mice, male and female, weighing between
22-45 g were used in the in vivo study. These animals were
procured from the Pasteur Institute in Algiers, Algeria. They
were randomly housed in plastic cages with access to food
and water in a room with controlled temperature (25°C) and
under a 12 h light–dark cycle. Animals have benefited from
an adaptation period before use. The standard food (Bovin
fattening) was purchased. Six and three mice were used in
each group of mouse writhing assay and formaldehyde –
induced mouse paw oedema, respectively.
2.2. Drugs and Chemicals
2.2.1. Reagents
Acetic acid, Formaldehyde, Tween-20, Perfalgan
(10mg/ml) and Diclofenac sodium 75mg/3ml (SAIDAL).
2.2.2. Working Solutions
- Acetic acid 0.6%: prepared in NaCl 0.9% (sterile).
- Formaldehyde 1%: prepared in NaCl 0.9% (sterile).
- Perfalgan: solution for injection (10mg/ml) at the dose of
100 mg/kg.
- Solution of Diclofenac (SAIDAL): prepared in 0.9%
NaCl (sterile) at a dose 0.03% (10 ml/mg, 25 mg/kg).
- The solutions of essential oils used are prepared in saline
0.9% plus 4% Tween-20 (Vehicle).
2.3. Essential Oils Extraction
Plant material (leaves, flowers and seeds) of Vitex agnus-
castus L. was collected from the region between Bechar and
Beniounif (Fendi; over a distance of 90 Km), during May–
September 2012. Material was dried in the shade at room
temperature for 7 days. The air-dried of V. agnus-castus
plant material was extracted for 6 hours by hydro-
distillation. The yields of the oils were 5.5%, 6.2 and 11.26%
v/w from leaves, flowers and seeds, respectively. The ob-
tained essential oils were dried over anhydrous sodium sul-
fate and after ltration, stored at +4°C until further tests.
2.4. Toxicity Determination
Lots: we worked on five lots of 6 mice for each essential
oil.
Administred doses: 50, 100, 150 and 200 mg/kg of essen-
tial oil. The essential oils were taken over with saline (0.9%)
plus 4% Tween-20 at a rate of 10l/g body weights of mice.
- Lot I: Administration of essential oil at the dose 50
mg/kg.
- Lot II: Administration of essential oil at a dose of 100
mg/kg.
- Lot III: Administration of essential oil at the dose 150
mg/kg.
- Lot IV: Administration of essential oil at the dose 200
mg/kg.
- Lot V (Witness): Vehicle at the dose 10 l/g.
Habbab et al.
The mice were fasted for 18 hours and weighed before
moving to properly so called experiment.
The lots I, II, III and IV received respectively 50 mg/kg,
100 mg/kg, 150 mg/kg and 200 mg/kg of the essential oil by
Intraperitoneal route. Lot V was used as control, received the
vehicle at 10l/kg mice. The mice were then observed first
for 2 hours to note the immediate signs of change, of intoxi-
cation and sudden death compared to animals in the witness
group. After these 2 hours we gave them to eat and drink.
After 24 h, 48 h, we also observed again the mice to count
the dead.
2.5. Analgesic Activity
2.5.1. Mouse Writhing Assay
It consists of checking the inhibitory action of the ex-
tracts on the pain brought in mice by intraperitoneal injection
of dilute acetic acid solution.
3 lots of 6 mice; male and female of weight ranging from
22 to 29 g maintained in fasting for 18 h before the start of
the experiment.
Lot I:
- Group 1: EO of leaves: 150 mg/kg
- Group 2: EO of flowers 150 mg/kg
- Group 3: EO of seeds: 150 mg/kg
Lot II (positive control): Perfalgan (10 mg/ml) at the dose
of 100 mg/kg.
Lot III (negative control) witness: Vehicle at a dose 10
l/g.
We administered intraperitoneally our oils (150 mg/kg),
Perfalgan (100 mg/kg) or vehicle (10 l/g) 30 minutes before
intraperitoneally (IP) injection of the acetic acid solution at
the dose of 10 ml/kg according to the method of Siegmund et
al., [17] with minor changes. After 5 minutes of injection of
of acetic acid, the writhes caused by the stimulus (pain syn-
drome) was counted for each mice for 20 minutes. The pain
syndrome is characterized by stretching movements of the
hind paws and writhing of the dorsal-abdominal muscula-
ture.
2.6. Anti-Inflammatory Activity
2.6.1. Formaldehyde –Induced Mice Paw Edema
The injection of formaldehyde under the plantar aponeu-
rosis of the hind paw of the mice or the rat causes an in-
flammatory reaction which can be reduced by anti-
inflammatory substances.
The pleurisy induced by the formaldehyde (1%) in mice
was evaluated according to the method of Winter et al., 1962
[18] with minor changes.
In this study, 3 groups; each group contains 3 lots of 3
male and female mice weighting between 35 and 46 g are
formed and maintained fasting for 18 h before the start of the
experiment. The mice constituting the three test groups are
treated by intra-planta (IP) with 10 .l/g (150 mg/kg, 150
mg/kg and 150 mg/kg for EO of leaves, flowers and seeds,
Analgesic Effects of Essential Oils of Vitex agnus-castus L
respectively) of fruits EO, leaves EO and seeds EO of Vitex
agnus-castus L. thirty minutes before the injection of 0.025
ml of formaldehyde 1%. The mice of positive control lots are
treated with 10 l/g of Diclofenac by intra-planta thirty min-
utes before the intra-pleural injection of formaldehyde.
The mice of negative control Lots receive by intra-planta
injection 10 l/g of the vehicle thirty minutes before the in-
tra-pleural injection of formaldehyde.
2.6.1.1. Substances and Administered Doses
Lot I:
- Group 1: Leaves EO: 150 mg/kg
- Group 2: Flowers EO 150 mg/kg
- Group 3: Seeds: EO 150 mg/kg
Lot II (positive control): Diclofenac solution (standard
drug) at the dose 0.03% (10 l/mg, 25 mg/kg).
Lot III (negative control) witness: Vehicle at a dose 10 l/g.
Administer different products by the intraperitoneal
route.
30 minutes after the intraperitoneal (IP) injection, we
injected 0.025 ml of formaldehyde 1% in the plantar aponeu-
rosis of the right hind paw of the mice.
Measuring foot volume over a given period at regular
intervals: we measured the volume of the hind paw of each
mice at 30, 60, 120, and 180 min after formaldehyde.
2.6.2. Evaluation of the Anti-inflammatory Activity
We noted the variation in volume of the paw in the time
(Vt).
The percent of increase (% Increase) in mouse paw is
given by the formula,
With Vo = initial volume of the mouse paw.
Vt = volume after injection of formaldehyde 1% and
treatment.
The percent of inhibition (% Inh) of the edema is calcu-
lated for each treated group of mice compared with the nega-
tive control. It is given by the following formula:
Table 1. Results of toxicity test by the intraperitoneal route of EO of different parts of Vitex agnus-castus L.
Product Doses
mg/kg Hypoactivity
Eos 50
100
150
200
- : No signs; +: Low signs.
Current Bioactive Compounds 2017, Vol. 13, No. 2 167
With Po = % increase in the paw of the witness.
Pt = % increase in the treated paw of the treated Lot.
3. RESULTS AND DISCUSSION
3.1. Toxicity Determination
The immediate signs of change, intoxication and sudden
death compared to the control group animals were followed
continuously for 2 hours after intraperitoneal administration
of Essential Oil. After 24 h, 48 h, we also observed again the
mice to count the dead, to detect the delayed effects of EO.
The taking of different doses of essential oil from different
parts of Vitex agnus-castus L., by intraperitoneal route, in-
duced grave changes in physical activity and behavior of
mice that can go to the death. Table 1 summarizes the imme-
diate signs registered during this experiment.
In our work, it is very important to note the animals re-
ceived a dose at 200 mg/kg showed weak signs of change:
hypoactivity, drowsiness and tachycardia, and did not show
any mortality during (2 h, 24 h and 48 h). Due to these re-
sults and to avoid all risk of undesirable symptoms we have
chosen a dose of 150 mg/kg.
3.2. Mouse Writhing Assay
The analgesic activity of seeds essential oil at a dose of
150 mg/kg gave a percentage of inhibition very near to di-
clofenac (33.17% and 33.33%, respectively) as shown in Fig.
(1). The others essential oils give a weak analgesic activity
than EO of seeds (22.45% for EO of leaves and 25.15% for
flowers) as shown in Fig. (2).
3.3. Formaldehyde –Induced Mice Paw Oedema
The injection of the vehicle without treatment (witness),
formaldehyde leads to a significant increase in the volume of
the mice paw of 0.11 ± 0.005; 0.13 ± 0.01; 0.136 ± 0.01 and
0.14 ± 0.02 at 30 min, 60 min and 120 min and 180 min,
respectively, as shown in Fig. (3).
The injection of the treatment (essential oils and Di-
clofenac 10 l/mg) by intraperitoneally route prevents sig-
nificantly the increase in volume of the mice paw after the
injection of formaldehyde 1%.
Symptoms Mortality
Drowsiness Tachycardia
- - - 0
- - - 0
- - - 0
+ + + 0
168 Current Bioactive Compounds 2017, Vol. 13, No. 2 Habbab et al.

60 55,67 0,16

0,14
50
43,17 0,12
41,67
40 0,1 EO-Leaf
33,17 33,33
0,08 EO-Seed
30 EO-Flower
Number of writhes 0,06
0,04 Drug
20
vehicle
0,02
10 0
0 30 60 120 180
0 Times
vehicle Drug EO-Leaf EO-Seed EO-Flower

Fig. (1). Number of writhes; the values of the number of writhes Fig. (3). Curve of the edema evolution, before and after formalde-
presented by the mean of 6 mice. hyde1% injection (0.025 ml).

EO-Leaf 22,45 180

120 %Inc-vehicle
EO-Flower 25,15

Times
%Inc-Drug
% Inhibition 60 %Inc-Flower
EO-Seed 40,13 %Inc-Seed
%Inc-Leaf
30

Drug 40,41
0 20 40 60 80 100
%
0 10 20 30 40 50

Fig. (2). Average percentage of analgesic activity of treatments. Fig. (4). Percentage of increase of average volumes of the paw.
The injection of Diclofenac at a dose of 10 l/mg pre-
vents following way: 0.09±0.02; 0.08±0.01%; 0.076±0.005 100
and 0.072±0.005 at 30, 60, 120 and 180 min after the injec- 90
tion of formaldehyde 1%. 80
70
The essential oils did prevent the increase in the volume
60 %Inh-Leaf
of the mouse paw, as follows: 0.096±0.02; 0.090±0.01; 50
%

0.090±0.01 and 0.086±0.01 for the EO of leaves, and %Inh-Seed

40
0.103±0.011; 0.11±0.01; 0.090±0.017 and 0.083±0.0057 for %Inh-Flower
30
the EO of seeds, and 0.103±0.0057; 0.116±0.015; 20 %Inh-Drug
0.110±0.01 and 0.090±0.01 for the EO of flowers at 30, 60, 10
120 et 180 min after the injection of formaldehyde 1% as 0
shown in Fig. (4). 30 60 120 180
Times
The treated groups present significant anti-inflammatory
activity compared to the negative control (witness) group as
shown in Fig. (5). Fig. (5). Percentage of inhibition of volumes in the time.
Essential oils show an anti-inflammatory activity in the
test of the edema. The evaluation of the percentage of inhibi-
tion indicates that the essential oil of Vitex agnus-castus L. The seeds EO displays the best inhibition compared to
seeds possess anti-inflammatory activity nearby and some- the others essential oils of Vitex agnus-castus L., this anti-
times better -during the time- than diflonac (61.04% - 50% inflammatory activity could be to the presence of some bio-
at30 min ; 62.04% - 83.33% at 60 min and 91.05% - 90,91% active compounds are absent in others EOs (according to GC
at 120 min, respectively). / MS [9, 10, 19-21]).

The inhibition is remarkable at thirty minute after treat- The essential oil of the leaves and the essential oil of
ment for a dose of 150 mg / kg of seeds EO, and maximal flowers exhibited an anti-inflammatory activity less efficient
after 180 min (100%). Also the essential oil of flowers gives than those obtained with the essential oil of seeds and Di-
a very significant inhibition after 180 min (81.58%). clofenac during the same period.
 Analgesic Effects of Essential Oils of Vitex agnus-castus L
4. CONCLUSION
The analgesic activity of seeds essential oil at a dose of
150mg / kg caused a percentage of inhibition very near to
diflonac (40.13% and 40.41%, respectively). The essential
oils show an anti-inflammatory activity in the test of the
edema. The evaluation of the percentage of inhibition shows
that the essential oil of Vitex agnus-castus L. seeds possess
anti-inflammatory activity (150 mg/kg) nearby and some-
times better -during the time- than diclfenac (61.04% - 50%
at 30 min ; 62.04% - 83.33% at 60 min and 91.05% - 90.91%
at 120 min, respectively). In conclusion, the results of the
present study provide evidences for the anti-inflammatory
and analgesic activity of Vitex agnus-castus L. growing in
Algeria. This finding could explain the traditional usage of
this plant.
ABBREVIATIONS
EO = Essential oil
EOs = Essential oils
IP = Intraperitoneal
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
Declared none.
REFERENCES
[1] Benzon, H.T.; Rathmell, J.P.; Wu, C.L.; Turk, D.C.; Argoff, C.E.;
Hurley, R.W. Practical Management of Pain, 5th ed.; Elsevier:
Philadelphia, 2014; pp. 13.
[2] Bahmani, M.; Shirzad, H.; Majlesi, M.; Shahinfard, N.; Rafieian-
Kopaei, M. A review study on analgesic applications of Iranian
medicinal plants. Asian Pac. J. Trop. Med., 2014, 7(Suppl. 1), S43-
S53.
[3] Ratheesh, M.; Helen, A. Anti-inflammatory activity of Ruta
graveolens Linn on carrageenan induced paw edema in wistar male
rats. Afr. J. Biotechnol., 2007, 6(10), 1209-1211.
[4] Sheng-Hong, L.; Hong-Jie, Z.; Sheng-Xiang, Q.; Xue-Mei, N.;
Bernard, D.S.; Andrew, D.M.; Harry, H.S.F.; Norman, R.F.; Han-
Dong, S. Vitexlactam A, a novel labdane diterpene lactam from the
fruits of Vitex agnus-castus L. Tetrahedron Lett., 2002, 43, 5131-
5134.
[5] Nyiligira, E.; Viljoen, A.M.; Van Heerden, F.R.; Van Zyl, R.L.;
Van Vuuren, S.F.; Steenkamp, P.A. Phytochemistry and in vitro
pharmacological activities of South African Vitex (Verbenaceae)
species. J. Ethnopharmacol., 2008, 119, 680-685.
[6] Latoui, M.; Aliakbarianb, B.; Casazza, A.A.; Seffen, M.; Convert,
A.; Perego, P. Extraction of phenolic compounds from Vitex agnus-
castus L. Food Bioprod. Process., 2012, 90, 748-754.
View publication stats
Current Bioactive Compounds 2017, Vol. 13, No. 2 169
[7] Webster, D.E.; Lu, J.; Chen, S.N.; Farnsworth, N.R.; Jim, W.Z.
Activation of the -opiate receptor by Vitex agnus-castus L.
methanol extracts: Implication for its use in PMS. J. Ethnopharma-
col., 2006, 106, 216-221.
[8] Habbab, A.; Sekkoum, K.; Belboukhari, N.; Cheriti, N.; Aboul-
Enein, H.Y. Chemical composition and biological evaluation of
Vitex agnus-castus L. essential oils from Southern-West Algeria.
Curr. Bioact. Compd., 2016, 12, 51-60.
[9] Stojkovic, D.; Sokovic, M.; Glamoclija, J.; Dzamic, A.; Ciric, A.;
Ristic, M. Chemical composition and antimicrobial activity of Vitex
agnus-castus L. fruits and leaves essential oils. Food Chem., 2011,
128, 1017-1022.
[10] Ghannadi, A.; Bagherinejad, M.R.; Abedi, D.; Jalali, M.; Absalan,
B.; Sadeghi, N. Antibacterial activity and composition of essential
oils from Pelargonium graveolens L’Her and Vitex agnus-castus L.
Iran. J. Microbiol., 2012, 4(4), 171-176.
[11] Guimarães, A.G.; Quintans, J.S.S.; Quintans-Júnior, L.J. Monoter-
penes with analgesic activity - A systematic review. Phytother.
Res., 2013, 27, 1-15.
[12] Choudhary, M.I.; Saima Jalil, A.; Nawaz, S.A.; Khan, K.M.;
Tareen, R.B.; Rahman, A. Antiinflammatory and lipoxygenase in-
hibitory compounds from Vitex agnus-castus L. Phytother. Res.,
2009, 23, 1336-1339.
[13] Bashir, A.; Sadiq, A.; Shumaila, B.; Khan, I.; Adhikari, A.; Choud-
hary, M.I. Anti-inflammatory and enzyme inhibitory activities of a
crude extract and a pterocarpan isolated from the aerial parts of
Vitex agnus-castus L. Biotechnol. J., 2010, 5, 1207-1215.
[14] Khalilzadeh, E.; Saiah, G.V.; Hasannejad, H.; Ghaderi, A.;
Ghaderi, S.; Hamidian, G.; Mahmoudi, R.; Eshgi, D.; Zangisheh,
M. Antinociceptive effects, acute toxicity and chemical composi-
tion of Vitex agnus-castus essential oil. Avicenna J. Phytomed.,
2015, 5(3), 218-230.
[15] Ramezani, M.; Amin, G.; Jalili, J. Antinociceptive and anti-
inflammatory effects of hydroalcohol extract of Vitex agnus-castus
L. Fruit. Int. J. Biolog. Biomol. Agric. Food Biotechnol. Eng.,
2010, 4(4), 214-216.
[16] Nasri, S.; Sourmaghi, M.H.S.; Amin, G.; Mohebali, S.; Sharifi, A.
Major essential oil components, antinociceptive and anti-
inflammatory effects of hexane extract of Vitex agnus-castus L.
fruits and possible mechanism in male mice. J. Paramed. Sci.,
2013, 4(3), 10-16.
[17] Siegmund, E.; Cadmus, R.; Lu, G. A method for evaluating both
non-narcotic and narcotic analgesics. Proc. Soc. Exp. Biol. Med.,
1957, 95, 729-731.
[18] Winter, C.A.; Risley, E.A.; Nuss, G.W. Carrageenan-induced
edema in hind paw of the rat as an assay for anti-inflammatory
drugs. Proc. Soc. Exp. Biol. Med, 1962, 111, 544-547.
[19] Zoghbi, M.G.B.; Andrade, E.H.A.; Maia, A.G.S. The essential oil
of Vitex agnus-castus L. growing in the Amazon region. Flavour
Frag. J., 1999, 14, 211-213.
[20] Galletti, G.C.; Russo, M.T.; Bocchin, P. Essential oil composition
of leaves and berries of Vitex agnus-castus L. from Calabria,
Southern Italy. Rapid Commun. Mass Spectrom., 1996, 10, 1345-
1350.
[21] Senatore, F.; Napolitano, F.; Ozcan, M. Chemical composition and
antibacterial activity of essential oil from fruits of Vitex agnus-
castus L. (Verbenaceae) growing in Turkey. J. Essent. Oil Bear.
Pl., 2003, 6(3), 185-190.