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Psychopharmacological profile of Chamomile (Matricaria recutita L.) essential oil in mice.

ARTICLE INFO

Keywords: Chamomilla recutita Bisabolol Caffeine Psychostimulant Anxiogenic Tail-suspension

ABSTRACT

In this study, the effect of Matricaria recutita L. essential oil (MEO) on the central nervous system (CNS) of mice was investigated using some behavioral methods. Chemical profiling both by GC and GC MS analyses of the hydrodistilled essential oil of M. recutita revealed [alpha]-bisabolol oxide A (28%), [alpha]-bisabolol oxide B (17.1%), (Z)-[beta]-Farnesene (15.9%) and [alpha]-bisabolol (6.8%) as the main components.

Changes induced by MEO (25,50 and 100 mg/kg) and reference drug caffeine (25 mg/kg) in spontaneous locomotor activities and motor coordinations of mice were investigated by activity cage measurements and Rota-Rod tests, respectively. Open field, social interaction and elevated plus-maze tests were applied to assess the emotional state of the animals. Further, tail-suspension test was performed for evaluating the effect of MEO on depression levels of mice. As a result, at 50 and 100 mg/kg, MEO significantly increased the numbers of spontaneous locomotor activities, exhibited anxiogenic effect in the open field, elevated plus-maze and social interaction tests and decreased the immobility times of animals in tail suspension tests. The falling latencies in Rota-Rod tests did not change. This activity profile of MEO was similar to the typical psychostimulant caffeine. The exact mechanism of action underlying this stimulant-like effect should be clarified with further detailed studies.

Introduction

Chamomile (Matricaria recutita L., Chamomilla recutita [L.] Rauschert, Matricaria chamomilla L.) resides in the Asteraceae family and is one of the most popular and widely used medicinal plants in the world. The plant has a long history of application in herbal medicine, which dates back to ancient Greece and Rome where it was referenced by Hippocrates, Galen and Asclepius (Tolouee et al. 2010). Infusions and essential oils of the flower heads have been used traditionally as cholagogue, carminative and diuretic (Franke and Schilcher 2005) also for various medicinal purposes like treatmerit of indigestion and inflammation (Ganzera et al. 2006).

Extracts prepared from M. recutita have been reported for their diverse ranges of pharmacological actions including hypocholesterolernic, antispasmodic, anti-platelet, antipruritic, anti-inflammatory, antimicrobial, antiviral and antioxidant activities (McKay and Blumberg 2006). In addition, some research groups have also investigated central nervous system (CNS) effects of the extracts prepared from this plant. Della Loggia et al. (1982) reported the CNS depressant and sedative effects of aqueous Chamomile extracts. After that, Viola et al. (1995) tested a purified fraction of an aqueous Chamomile extract containing apigenin and suggested anxiolytic, sedative, myorelaxant and anticonvulsive activity potential for this flavonoid. Later studies exhibited that apigenin, major compound found in Chamomile extracts, affects benzodiazepine receptors differently than classical benzodiazepine receptor ligands (Franke and Schilcher 2005; McKay and Blumberg 2006).

Essential oil of M. recutita (MEO) prepared from fresh or dried flower heads of the plant has different phytochemical contents from the aqueous, ethanol, ethyl acetate or dichloromethane extracts prepared from the plant. The principal components of the essential oil extracted from the flowers are the terpenoids a-bisabolol and its oxides and azulenes, including chamazulene, whereas the main constituents of the extracts prepared from the flowers include several phenolic compounds primarily the flavonoids apigenin, quercetin, patuletin, luteolin and their glucosides (Franke and Schilcher 2005; McKay and Blumberg 2006).

In previous reports, MEO has been investigated for its potential pharmacological activities. Different research groups have showed antispasmodic, antipruritic, anti-inflammatory, antigenotoxic, antimicrobial, antifungal and antioxidant effects of MEO previously (McKay and Blumberg 2006). On the other hand, documents related to CNS related actions of MEO were not found in a comprehensive literature survey. Therefore, we aimed to investigate the effect of MEO on CNS of mice performing some psychopharmacological tests.

To the best of our knowledge, this is the first experimental study related to the psychopharmacological profile of MEO.

Materials and methods

Plant material and essential oil preparation

Dried M. recutita capitula - Flores C. recutita (PhEur) was obtained from commercial sources (Wallerstein, Germany). Voucher specimen of the sample was deposited at the Department of Pharmacognosy (ESSE Archive No: 20).

The essential oil from flowers heads of the dried plant was iso-lated by hydrodistillation for 3 h using a Clevengertype apparatus. The obtained oil was dried over anhydrous sodium sulphate and stored at +4[degrees]C in the dark until analysed. Percent yield (v/w) of the hydrodistilled oil calculated on a moisture free basis was 0.55%.

Analysis and identification of components

GC-MS analysis

The GC-MS analysis was carried out using an Agilent 5975 GC-MSD system. Innowax FSC column (60 m x 0.25 mm, 0.25 [micro]m film thickness) was used with Helium as carrier gas (0.8 ml/min). GC oven temperature was kept at 60 [degrees]C for 10 min and programmed to 220 [degrees]C at a rate of4 [degrees]C/min, and kept constant at 220 [degrees]C for 10 min and then programmed to 240 [degrees]C, at a rate of 1 cC/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250 [degrees]C. Mass spectra were recorded at 70 eV where the mass range was from m/z 35 to 450.

GC analysis

The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was set to 300 [degrees]C. To obtain the same elution order with GC-MS simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions, simultaneously. Relative percentage amounts of the separated compounds were calculated from FID chromatograms.

Identification of components

Identification of the essential oil components was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Peak matching against commercial (Wiley GC/MS Library, MassFinder 3.1 Library) (Koenig et al. 2004; McLafferty and Stauffer 1989) and in-house "Baser Library of Essential Oil Constituents" built up by genuine compounds and components of known oils as well as MS literature data (Joulain and Koenig 1998) were used for the characterization of individual components.

Pharmacological studies

Animals

Adult male Balb/c mice weighing 30-35 g were used for the tests. The animals were housed in an air-conditioned room (25 [+ or -] 1 [degrees]C) with a 12-h light and 12-h dark cycle. Temperature, sound and light conditions were not altered during the course of the experiments. The experimental protocol was approved by the Local Ethical Committee on Animal Experimentation of Eskisehir Anadolu University, Turkey.

Drugs and essential oil administrations

Caffeine (Caf) (99% purity) and all other chemicals used in this study were purchased from Sigma Chemical Company, St. Louis, MO, USA.

Animals were randomly divided into three groups, namely; control group, reference group (Caf, 25 mg/kg) and MEO-treated test groups (25, 50, and 100 mg/kg). Each of the groups consists of seven animals. The control solution was sunflower oil, since MEG dissolved in it. Sunflower oil, Caf and different doses of the MEG were administrated orally 60 min before the experimental sessions (Kuribara et al. 1992; Park et al. 2010).

Behavioral tests

Activity cage test. The horizontal and vertical locomotor activities of the mice were registered by the activity cage apparatus (Ugo Basile, No. 7420, Varese, Italy), which contains two pairs of 16 photocells 3 cm and 12 cm above the floor. Interruptions of light beams to the photocells during horizontal and vertical movements of the animals were automatically recorded for 4 min (Votava et al. 2005).

Rota-Rod test. Motor performances of mice were assessed by Rota-Rod tests. Before the experimental session, three trials were given for three consecutive days on the Rota-Rod apparatus (Ugo Basile, No. 47600, Varese, Italy) set at a rate of 16 revolutions per minute. Mice remaining on the rod longer than 180 s were selected for the test. Each mouse was tested in the Rota-Rod and latency to fall from the rotating mill was recorded as previously reported (Can et al. 2010).

Open field test. The open field was made of acrylic (41 cm x 41 cm x 33 cm) with transparent walls and a black floor. To evaluate central exploratory propensity, the arena was virtually divided into the central and peripheral areas (Panlilio et al. 2007). At the beginning of the test, each mouse was placed in the centre of the arena and was allowed to explore for 5 min. The time spent in the central zone was recorded (Carnevale et al. in press; Ekong et al. 2008). Each subject was tested only once and the open field was cleaned with 70% ethyl alcohol solution at the end of the each trial.

Elevated plus-maze test. Anxiety levels of animals were measured using the elevated plus-maze test. The apparatus was consisted of two open arms (30 cm x 5 cm) and two closed arms of the same dimensions with walls 15 cm high. The arms were connected in the middle by a central platform (5 cm x 5 cm). The maze was elevated 60 cm off the ground. At the start of the session, mice were individually placed on the central platform facing an open arm. The number of entries and the time spent in both closed and open arms were recorded during a 5-min observation period. Arm entries were defined as entry of all four paws into an arm (Peng et al. 2000).

The percentage of open arm entries (POAE) and the percentage of time spent on the open arms (PTOA) were calculated for each animal by the following formulas:

POAE = (open arm entries)/(total arm entries) x 100

PTOA = (open arm time)/(total arm time) x 100

Social interaction test. The social interaction arena was an open topped box (60 cm x 60cm x 35cm) (Min et al. 2005). Mice were isolated for I Ii before the test. After introduction to the test arena, mice were observed for cumulative time spent in genital investigation, sniffing a partner, climbing over and under and neck licking in 5 mm. Percentage of time spent in social interaction was recorded (Gilhotra and Dhingra 2010).

Tail-suspension test. Tail-suspension test was carried out by a method described earlier by Steru and co-workers (1985). Mice were suspended 30 cm above the floor by an adhesive tape placed approximately 1 cm from the tip of the tail and considered immobile oniy when they failure to make any struggling movements and hung passively. Immobility times of animals were scored during the last 4 mm of 6 mm test duration (Can et al. 2009).

Statistical analyses

GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA, USA) was used for statistical analyses of the experimental data. Correlation coefficient was calculated by Correlation analysis and significant differences between groups (n = 7 for each) were determined by One-way analysis of variance (ANOVA) followed by Tukey's test. The results were expressed as mean [+ or -] standard error of mean (SEM). Differences between data sets were considered as significant when p value WS less than 0.05.

Results

Essential oil analysis

The characteristic MEO was obtained by hydrodistillation, with a moderate yield. Thereafter, thirty-nine compounds of the essential oil were identified using simultaneous GC and GC-MS, which represented 96.6% of the total oil of M. recutita flowers. [alpha]-Bisabolol oxide A (28%), [alpha]-bisabolol oxide B(17.1%), (Z)-[beta]-Farnesene (15.9%) and [alpha]-bisabolol (6.8%) were found as main components. Results of the constituents (> 1%) of MEN are given in Table 1.
Table 1
Chemical composition of the MEO.

            Compound           RKI     %   Identification method

1   (Z)-[beta]-Farnesene       1668  15.9  a
2   Spathulenol                2144   2.5  a
3   [alpha]-Bisabolol oxide B  2156  17.1  a
4   [alpha]-Bisabolon oxide A  2200   5.0  a
5   Methyl hexadecanoaie       2226   2.6  a
6   [alpha]-Bisabolol          2232   6.8  a, b
7   Decanoic acid              2298   3.0  a, b
8   Chamazulene                2430   4.2  a
9   [alpha]-Bisabolol oxide A  2438  28.0  a
10  Methyl linoleate           2509   1.0  a, b
11  Methyl linolenate          2583   1.1  a, b
12  Hexadecanoic acid          2931   1.5  a, b
13  Spiro ether                2937   1.1  a

RRI, relative retention indices calculated against n-alkanes;
%, calculated from FID data: a, comparison of mass spectra
with the Wiley and Mass Finder libraries and retention times;
b, comparison with genuine compounds on the HP Innowax column


Pharmacological tests

Comparative results of the activity cage measurements are given in Table 2. MEO when applied at 50 and 100 mg/kg doses caused significant increase in the recorded numbers of both horizontal and vertical locomotor activities compared to the control group. Whereas, MEO applied at 25 mg/kg was ineffective. Besides, MEO administration did not change the falling latencies of the mice from the rotating mill in Rota-Rod tests at none of the applied doses (data not shown).
Table 2
Effects of the MEO on spontaneous locomotor activity parameters of
mice in activity cage tests.

Croups (cone.)    Number of horizontal          Number of vertical
                        movements                   movements

Control              421.0 [+ or -] 36.7         103.0 [+ or -] 8.6
Caf(25mg/kg)     621.3 [+ or -] 36.0 ***     161.8 [+ or -] 11.2 **
MEO (25 mg/kg)       461.0 [+ or -] 33.1          97.6 [+ or -] 8.6
MEO (50 mg/kg)     558.3 [+ or -] 29.4 *  142.4 [+ or -] 9.9 *, (a)
MF.O(100mg/kg)  689.4 [+ or -] 29.7 ***,    183.3 [+ or -] 8.4 ***,
                                 (c), &                      (c), &

Values are given as mean [+ or -] SEM. One-way ANOVA, post hoc Tukey
test. Control-doses correlation: for horizontal movements r = +0.9936,
p = 0.0064; for vertical movements r = +0.9531, p = 0.0469.

(d) p < 0.05 significance against 25 mg/kg group.
(c) p< 0.001 significance against 25 mg/kg group.
* p < 0.05 significance against control values.
** p<0.01 significance against control values.
*** p < 0.001 significance against control values.
& p <0.05 significance against 50 mg/kg group.


Tables 3 and 4 show the effects of essential oil in the open field and elevated plus-maze tests, respectively. In open field test, percentage time spent in the central area was decreased by 50 and 100 mg/kg doses. Furthermore, both POAE and PTOA were significantly and dose dependently decreased with the same doses of MEO.

Effect of the essential oil in the social interaction test is given in Table 5. MEO reduced the durations of social interaction behaviors in the social interaction tests when applied at 50 and 100 mg/kg doses.
Table 3
Effect of the MEO on percentage of time spent in the
central zone in open field tests.

Groups (cone.)   Time spent in the central area (%)
Control                            9.4 [+ or -] 0.7
Caf(25mg/kg)                   2.9 [+ or -] 0.5 ***
MEO(25mg/kg)                       7.0 [+ or -] 0.9
MEO(50mg/kg)                      6.1 [+ or -]0.6 *
MEO (100 mg/kg)             2.9[+ or -]0.5 ***, b &

Values are given as mean [+ or -] SEIVl. One-way ANOVA,
post hoc Tukey test. Control-doses correlation:
r = -0.9908, p = 0.0092.
(b) p<0.01 significance against 25 mg/kg group,
* p < 0.05 significance against control values.
*** p< 0.001 significance against control values.
& p < 0.05 significance against 50 mg/kg group.
Table 4
Effects of the MEO on exploratory parameters of mice in elevated
plus-maze tests.

Groups (cone.)           POAE                  PTOA

Control              39.4 [+ or -] 2.9      38.4 + or -] 3.7
Caf (25 mg/kg)   14.1 [+ or -] 1.6 ***  5.6 [+ or -] 2.4 ***
MEO (25 mg/kg)       32.8 [+ or -] 2.9     35.3 [+ or -] 3.4
MEO (50 mg/kg)    27.0 [+ or -] 1.1 **   25.9 [+ or -] 2.5 *
MEO (100 mg/kg)      17.9 [+ or -] 1.7     i3.i [+ or -] 2.1
                             ***, c, &             ***, c, &

Values are given as meaniSEM. One-way ANOVA, post hoc Tukey test.
Control-doses correlation: for POAE r= -0.9957, p = 0.0043; for
PTOA r = -0.9910. p = 0.009.
(c) p< 0.001 significance against 25 mg/kg group,
* p < 0.05 significance against control values.
** p<0.01 significance against control values.
*** p<0.001 significance against control values.
& p < 0.05 significance against 50 mg/kg group.


Table 6 shows the change in immobility times of animals, a parameter of behavioral despair, following MEO administrations in tail-suspension test. Immobility times of mice treating with MEO (50 and 100 mg/kg) were significantly and dose dependently shorter than the control values.
Table 5
Effect of the MEO on social interaction times of mice in
social interaction tests.

Groups (cone)   Social interaction times (s)

Control                    52.9 [+ or -] 3.7
Caf (25mg/kg)           33.8 [+ or -] 3.4 **
MEO (25mg/kg)              44.5 [+ or -] 2.7
MEO (50mg/kg)            38.9 [+ or -] 3.2 *
MEO (100mg/kg)  25.4[+ or -] 2.7 ***, (b), &


Values are given as mean [+ or -] SEM. One-way ANOVA, post hoc
Tukey test. Control-doses correlation: r = -0.9982, p = 0.0018.

(b) p<0.01 significance against 25 mg/kg group.

* p<0.05 significance against control values.

** p<0.01 significance against control values.

*** p< 0.001 significance against control values.

& p<0.05 significance against 50 mg/kg group.

Table 6
Effect of the MEO on immobility times of mice in tail-suspension test.

Groups (cone.)             Immobility times (s)

Control                       129.1 [+ or -] 93
Caf (25 mg/kg)            79.7 [+ or -] 10.3 **
MEO (25 mg/kg)               124.9 [+ or -] 9.8
MEO (50 mg/kg)              89.9 [+ or -] 7.2 *
MEO (100 mg/kg)   47.9 [+ or -] 6.2 ***, (c), &

Values are given as mean [+ or -] SEM. One-way ANOVA, post
hoc Tukey test. Control-doses correlation: r = -0.9800, p = 0.02.
(c) p< 0.001 significance against 25 mg/kg group.
* p<0.05 significance against control values.
** p<0.01 significance against control values.
*** p< 0.001 significance against control values.
& p < 0.05 significance against 50 mg/kg group.


Reference drug Caf, when applied at 25 mg/kg dose, increased the spontaneous locomotor activities in activity cage tests and decreased all of the measured behavioral parameters in open-field, elevated plus-maze and social interaction tests (Tables 1-6). On the other hand, the falling latencies in Rota-Rod tests did not change with Caf administrations.

Discussion

The effects of MEO on CNS of mice were investigated by various psychopharmacological methods, in this present study. Changes induced by MEO in the spontaneous locomotor activities and motor coordinations of mice were investigated by activity cage measurements and Rota-Rod test, respectively. Open field, elevated plus-maze and social interaction tests were applied for assessing the effect of MEO on emotional state of the animals. Further, tail-suspension test was performed for evaluating the effect of the essential oil on the depression levels of mice.

MEO dosed at 50 and 100 mg/kg induced a significant increase in the total number of horizontal and vertical spontaneous loco-motor activities of animals in the activity cage tests. It is generally stated that locomotor activities result from brain activation, which is manifested as an excitation of central neurons involving different neurochemical mechanisms and an increase in cerebral metabolism (Zapata-Sudo et al. 2010). Psychostimulants like caffeine are one of the well-known drug groups increasing the spontaneous locomotor activities of animals after acute administrations (Lynch et al. 2011).

Data obtained from the MEO-treated animals in Rota-Rod tests were not significantly different from the controls indicating that it did not change the motor coordination of animals or induce a non-specific effect at neuromuscular junction level.

In open field test, natural tendency of a rodent, named as "thigmotaxis", is to prefer the peripheral zone and walk close to the walls (Treit and Fundytus 1988). The increased activities of animals in the central zone are usually considered as an increase in exploratory behavior and reduction in anxiety level. Conversely, decrease in the central zone activity reflected on the high anxiety level of animals (Prut and Belzung 2003). In this study, MEO dosed at 50 and 100 mg/kg induced a tigmotactic effect, as indicated by a significant decrease in the percentage of time spent in the central zone. These findings pointed out the anxiogenic effects of MEO at applied doses.

The elevated plus-maze is an etiologically valid animal model of anxiety because it uses natural stimuli (fear of a novel open space and fear of balancing on a relatively narrow, raised platform) that can induce anxiety in humans. Anxiolytic agents increase POAE and PTOA parameters in plus-maze tests whereas anxiogenics decreases both of them (Sampath et al. 2011). In this study, results of the plus-maze test were supported the open field findings indicating the presence of anxiogenic effect, since MEO caused significant decreases in both POAE and PTOA values.

In the social interaction tests using MEO, percentage of the time spent in social interaction decreased, significantly. This data confirmed the anxiogenic effect of the tested essential oil at 50 and 100 mg/kg doses, additionally supporting the data of our previous tests. Psychostimulant drugs like caffeine are also known to show anxiogenic effects in behavioral tests (Bhattacharya et al. 1997; Park et al. 2010).

Effect of MEO on depression parameters of mice was screened by tail-suspension test, which is the most widely preferred model for assessing antidepressant-like activity in rodents (Cryan et al. 2005). In the case of the tail-suspension test, the stressful situation involves the haemodynamic stress of being hung in an uncontrollable fashion by their tail. This short-term, inescapable stress causes the occurrence of an immobile posture following the initial escape-oriented movements of the animal. Various antidepressant medications are known to reverse the immobility and promote the occurrence of escape-related behavior in tail-suspension test (Cryan et al. 2005). In this study, MEO caused significant and dose dependent decrease in the immobility times of animals. However, this shortening in the immobility time may have not been caused by an actual antidepressant like activity of the MEO, since drugs, which are increasing the locomotor activity such as psychostimulants, are known to cause false positive responses in tail-suspension test (Cryan et al. 2005).

In summary, when MEO was administrated at 50 and 100 mg/kg doses increased the numbers of total horizontal and vertical locomotor activities, exhibited anxiogenic effect in the open field, elevated plus-maze and social interaction tests and decreased the immobility times of animals in tail-suspension test. The falling latencies in Rota-Rod tests did not change with MEO administrations. The activity profile of MEO exhibited in all these performed tests was very similar to the activities of typical psychostimulant caffeine (Cryan et al. 2005; Listos et al. 2010; Lynch et al. 2011; Park et al. 2010). However, the exact mechanism of action underlying this stimulant effect should be clarified with further detailed studies.

The major constituents of the tested essential oil were identified as [alpha]-bisabolol oxide A (28%), [alpha]-bisabolol oxide B (7.4-17.1%), (Z)-[beta]-Farnesene (15.9%) and [alpha]-bisabolol (6.8%). Also in previously reported studies [alpha]-bisabolol oxide A (3.1-56%), [alpha]-bisabolol oxide B (3.9-30.6%) and [alpha]-bisabolol (0.1-44.2%) have been found as the main compounds, which correlates with our results (Sashidhara et al. 2006; Kowalski and Wawrzykowski 2009; Orav et al. 2010; Raal et al. 2011). The observed stimulant effect in this study was probably associated with these major compounds detected in the tested essential oil. Furthermore, these components may act synergistically or other minor constituents may also contribute to the observed activity. Investigations on the CNS-related pharmacological activities of the major constituents are still under progress in our laboratory.

Conclusion

Results obtained from this study interestingly exhibited the stimulant effect of MEO comparable with caffeine for the first time to the best of our knowledge. Compounds having psychostimulantlike activities may be evaluated as new drug candidates for the treatment of attention deficit hyperactivity disorder (Caballero et al. 2011; Gibson et al. 2006). Therefore, more detailed and comprehensive experiments with this essential oil and its volatile components are worthwhile to investigate.

Acknowledgements

The authors would like to thank Mr. E. Schmidt for providing the plant material from a local Pharmacy in Wallerstein, Germany. Also Prof. Fatih Demirci is acknowledged for his valuable phytochemical approaches.

Abbreviations: ANOVA, analysis of variance; Caf, caffeine; CNS, central nervous system; FED, flame ionization detector; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry; GC-MSD, gas chromatography-mass spectrometry detector; MEO, essential oil of Matricaria recutita; POAE, percentage of open arm entries; PTOA, percentage of time spent on the open arms; RRI, relative retention index; SEMI standard error of mean.

* Corresponding author. Tel.: +90 222 3350580x3749; fax: +90 222 3350750. E-mail address: ozgurdt@anadolu.edu.tr (O.D. Can).

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Ozgur Devrim Cans (a), *, Umide Demir Ozkaya, Hulya Tuba Kiyan (b), Betul Demirci (b)

(a.) Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 Eskisehir, Turkey

(b.) Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, 26470 Eskisehir, Turkey

0944-7113/$ - see front matter [C] 2011 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2011.10.001
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Author:Can, Ozgur Devrim; Ozkay, Umide Demir; Kiyan, Hulya Tuba; Demirci, Betui
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:7TURK
Date:Mar 1, 2012
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