European Journal of Integrative Medicine 4 (2012) e169–e176
Original article
Effects of Stachys tibetica essential oil in anxiety
Dinesh Kumar, Zulfiqar Ali Bhat ∗, Vijender Kumar, N.A. Khan, I.A. Chashoo, M.I. Zargar,M.Y. Shah
Department of Pharmaceutical Sciences, University of Kashmir, Srinagar 190006, India
Received 27 August 2011; received in revised form 9 January 2012; accepted 9 January 2012
bstract
thnopharmacological relevance: Stachys tibetica (Lamiaceae) is an important medicinal plant in the folk medicine of Ladakh, India and Tibetor the treatment of various mental disorders.im of the study: Present study is aimed to characterize the essential oil of S. tibetica and its anxiolytic properties.aterials and methods: The oil was extracted from the plant by hydrodistillation method. The isolated oil (0.7%) was subjected to GC–MS
or identification of its constituents and evaluated for anxiolytic effects by employing the social interaction, hole-board, elevated plus maze andight/dark arena tests in rats.esults: GC–MS of S. tibetica essential oil (SEO) indicated the presence of 33 constituents. Aciphyllene (66.415%), fenchyl alcohol (8.897%),-pinene (8.188%), caryophyllene oxide (4.648%), menthol (1.69%) and geraniol (1.315%) are the major constituents of SEO. All the anxiolytictudies were conducted at doses of 25 and 50 mg/kg body weight. In the social interaction test, SEO decreased aggressive behaviors of the animalsalbino rats) while the same significantly increased social interaction time of the high light, familiar as well as unfamiliar test conditions. In theole-board test, SEO significantly increased head-dipping counts and its duration. The SEO significantly increased number of entries and time
pent in open arms on the elevated plus maze test while as in the light/dark arena test, SEO showed an increase in number of crossings and timepent in light arena.onclusion: Results indicate that SEO has a wide range of anxiolytic properties and pave a way for new drug search for anxiety.
2012 Elsevier GmbH. All rights reserved.
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eywords: Stachys tibetica oil; Social interaction test; Hole-board test of explo
ntroduction
The use of medicinal plants for the treatment of human dis-ases has increased considerably worldwide. Evaluation of theffects of these plants on organs and systems has contributedo the development of the scientific basis for their therapeuticpplication and also has enriched considerably the therapeuticrsenal for the treatment of a number of diseases [1]. Anxiolyticrugs are among the most frequently prescribed drugs as the
isease is highly prevalent in the society. Existing anxiolyticgents are associated with several limitations such as seda-ion, addiction with benzodiazepines, tachycardia, insomnia,
∗ Corresponding author at: Department of Pharmaceutical Sciences, Univer-ity of Kashmir, Srinagar 190006, J&K, India.obile: +91 9419077701/9882120308.
ecreased libido and ineffectiveness (delayed but sustained) withuoxetine [2] an antidepressant having selective serotonin reup-
ake inhibitor (SSRI) property and being extensively used inatients of generalized anxiety disorder including social anxi-ty. These are some of the factors that led to the interest in usinglternative remedies. Several indigenous drugs are being eval-ated because of their easy availability, lack of adverse effectsnd cost-effectiveness. Traditional medicines are used by about0% of the world population in rural areas in the developingountries as well as in the developed countries where use ofodern medicine predominates [3]. Though the use of herbaledicine is steadily increasing in western world [4], the major
indrance in the amalgamation of herbal medicine into medicalractice is the lack of sufficient scientific and clinical data andetter understanding of efficacy and safety of the herbal prod-
cts [5]. The historical use of such medicine provides the sourceo study the specific plant species with potential to be used in aarticular disease.
Plants belonging to the family Lamiaceae are rich in essen-ial oils. Previous studies have shown that a number of plantsrom this family like Salvia officinalis, Salvia elegans, Salviaeuterana, and Scutellaria baicalensis have shown anti-anxietyctivity [6]. Salvia sclarea and Lavender also have shown anti-nxiety activity and the activity has been attributed to theirssential oils [7]. Moreover another species of stachys namelytachys lavandulifolia has shown anti-anxiety activity becausef its essential oil [8].
Stachys L. (Lamiaceae) is a large genus of herbs and shrubsomprising 300 species distributed in temperate and tropicalegions of the world, with the exception of Australasia [9]. Plantsf this genus have been used in folk medicine for centurieso treat genital tumors, sclerosis of the spleen, inflammatoryiseases, cough and ulcers [10]. Stachys tibetica is an herb dis-ributed in tropical and subtropical regions of the world. Theerb is distributed in Tibet, China, India, etc. In India, it is foundn cold desert region of Ladakh valley and in the mountainsf Himachal Pradesh. The traditional practitioners in Ladakhegion of India use the drug for the treatment of various mentalisorders and phobias. In traditional practice, the whole plant isoiled and made into a decoction. A tea cup of the decoction isiven twice a day to treat fever [11].
Different drugs, known for their sedative properties at highoses, such as minor tranquillizers, also exhibit an anticonflictffect. Angelica and Ducrosia anethifolia essential oil were pre-iously evaluated for anxiolytic activity by oral route in murineests [12,13]. In this study, a possible anxiolytic property of S.ibetica essential oil was investigated using murine tests for anx-ety: social interaction test, hole-board test, elevated plus mazeest and light–dark arena test in rats. The elevated plus mazes one of the most extensively used models for the investiga-ion of drug effects on anxiety-related behavior in laboratoryodents. It is based on the aversion of rodents for open spacesnd anxiolytics have been found to increase the proportion ofime spent on the open arms [14]. The light/dark test is based onhe innate aversion of rodents to brightly illuminated areas andn the spontaneous exploratory behavior of rodents in responseo mild stressors, that is, novel environment and light [15]. Anx-olytics have been found to increase locomotion and time spentn the light zone, whereas anxiogenics decrease them [16]. In theocial interaction test, anxiolytic decreases aggressive behaviorsnd increases social interaction time of the high light, unfamiliarest condition and also prolong social interaction time of the highight, familiar test condition. In the hole-board test, it increasesead-dipping counts and its duration. Because the above testsave been validated pharmacologically, behaviorally and phys-ologically as models of experimental anxiety, we used them toalidate the putative anxiolytic effect of stachys essential oil andiazepam treatment was included as a positive control.
aterials and methods
lant material
The plant specimen of S. tibetica Vatke was collected fromargil Valley of Ladakh, Jammu & Kashmir, India. The plant
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as identified and authenticated by Dr. Zulfiqar Ali Bhat, fromepartment of Pharmaceutical Sciences, University of Kashmir,rinagar 190006, India (Voucher specimen number – KUST01).
xtraction of stachys oil
100 g of S. tibetica drug was subjected to hydrodistillation in Clevenger apparatus according to the method recommended inritish Pharmacopoeia (1988) [17] and 0.7 ml of the oil (0.7%,/w) was collected.
as chromatography–mass spectrometry analysis of thessential oil of S. tibetica
About 1 �L of aliquot of oil sample, appropriately diluted inexane, was subjected to gas chromatography–mass spectrom-try (GC–MS) analysis. The GC–MS analysis was performedsing a Varian GC–MS series 3800 with a VF-5MS column60 cm × 0.25 mm; film thickness, 0.25 �m). The column tem-erature was kept at 60 ◦C for 3 min programmed to 280 ◦C at
rate of 3 ◦C/min and kept constant at 250 ◦C for 1 min. Flowate of helium as a carrier gas was 1 mL/min. The sample wasnalyzed twice.
nimals
Albino rats (Wistar Strain) of either sex weighing 150–200 gespectively were used for studies. The albino rats were obtainedrom animal house of Indian Institute of Integrative Medicine
Jammu, Jammu and Kashmir, India. They were housed inolypropylene cages with standard pellet chow and water adibitum. In social interaction test, six pairs of rats were usedn HU and HF behavioral study and in all other experimen-al sets, 10 rats were used for each dosage. This Institutions approved for carrying out animal studies (Approval No.01/03/ca/CPCSEA) and the protocol for the present study waspproved by Institutional Animal Ethical Committee [Approvalo. F-IAEC (Pharm. Sc.) APPROVAL/2011/01].
cute toxicity study: (OECD guidelines-425, 2001)Acute toxicity study was conducted as per the interna-
ionally accepted protocol drawn under the OECD guidelines25 (OECD, 2001). Overnight fasted, healthy rats (n = 6) weredministered orally the stachys essential oil in the doses of 100,00, 400, 800, 1600 mg/kg body weight and observed continu-usly for 4 h and 24 h for any abnormality and mortality. Stachysil at a dose level of 1600 mg/kg was found safe. 25 and 50 mg/kgere selected as the study dose of stachys oil.
rugsDiazepam was obtained from Ranbaxy Lab. Ltd., HPSIDC-
addi, Solan (India). Sodium carboxy methyl cellulose wasurchased from CDH-Laboratory Reagent Pvt. Ltd., Post Box
o. 7138, New Delhi 110002 (India). Diazepam and stachys
ssential oil (SEO) were both suspended in a 1% sodium carboxyethyl cellulose solution. All drugs were prepared immediately
efore use and were given orally. Control mice received 1%
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queous sodium carboxy methyl cellulose solution only. Theffects of the drugs were estimated 60 min after drug administra-ion. Drug dose, pretreatment time and selection of 1% sodiumarboxy methyl cellulose solution as vehicle were based on find-ngs in preliminary experiments or taken from the literature.ests were performed only after the rats had been acclimatized
o the above environment for at least 7 days. All experimentsere carried out between 09:00 and 16:00 h. In each experiment
pparatus was cleaned using 5% ethanol before introducing theext animal to preclude the possible cueing effects of odors lefty previous subjects.
nxiolytic activity
roceduresocial interaction test. The test procedure for social interactionest was similar to that described by File and Pellow [18]. Theest arena was a black Plexiglas box, 60 cm × 60 cm × 35 cm,ith the base divided into 9 cm squares by lines of white tape.he light intensity of the arena floor was 380 lx. Two test con-itions were performed: high light, unfamiliar arena (HU) andigh light, familiar arena (HF). On day 1 of testing, each rat wasandomly assigned according to body weight (<15 g difference)o an unfamiliar partner in groups of 12 animals (six pairs) whichere subsequently administered the appropriate drug. These ratsere then replaced into their home cage until testing. Following
ppropriate pretreatment time, members of each pair of unfamil-ar rats were placed in opposite corners of the arena and observedor social interaction behaviors for 10 min. At the end of thiseriod the rats and any fecal boluses were removed and the arenaas wiped with a damp cloth. Social interaction time (in s) perair of rats was measured as time of sniffing and mutual groom-ng, adjacent lying, climbing over and crawling under the partner,pproximation and following [19]. Aggressive-type behaviors,.g. kicking, aggressive grooming, biting, boxing and jumping20] were also scored. These were treated as separate entitiesince such behaviors are modulated by different pharmacologi-al agents than social behaviors [21]. Following completion ofhe first test, rats were returned to their home cages. On days 2nd 3, the rats were placed individually, undrugged, in the sameox for 10 min per day to familiarize them with the apparatus.n the fourth day, the same pairs of rats were once again placed
n the test arena for 10 min and the same test procedure wasarried out. Pairs of rats were allocated randomly to the follow-ng test groups: vehicle control, diazepam (1 mg/kg po), SEO25 and 50 mg/kg po) [22].
ole-board test. The hole-board apparatus was an open-fieldrena with four equally spaced holes of 3.8 cm in diameter inhe floor, similar to the box used in social interaction test. Theenter of each hole was 10 cm from the nearest wall [23]. Theoor of the box was positioned 25 cm above the ground andivided into squares of 10 cm × 10 cm with a water-resistant
arker. Rats were randomly allocated to the following groups:
ehicle control, diazepam (1 mg/kg po), SEO (25 and 50 mg/kgo). The rats were injected with drugs or vehicle. Thirty minutesater, each animal was placed in the center of the hole-board,
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nd allowed to freely explore the apparatus for 5 min. Totalocomotor activity (numbers of squares crossed), number anduration of head dipping and latency to the first head-dip wereecorded. A head dip was scored if both eyes disappeared intohe hole [24–28]. In the above two tests, the behavior of the ani-
als was recorded with an observer inside the room, comprisedoth conventional and ethological parameters. The apparatusas wiped with a damp cloth after each trial and any feces
emoved [19,29,30].
levated plus-maze test. The test procedure and scoringethodology for elevated plus-maze test have been described in
etail elsewhere [14]. In brief, the apparatus composed of twopen arms (50 cm × 10 cm) and two enclosed arms of the sameize with 40 cm high wall arranged so that the arms of the sameype were opposite to each other with a central square of 10 cmo form a plus sign. The apparatus was wooden and was elevatedo a height of 50 cm above floor level by a single central sup-ort. A slight raised edge on the open arms (0.25 cm) provideddditional grip for the animals, whereas open arm activity wasurther encouraged by testing under dim red light (4× 25 W).he experiment was conducted during the dark phase of the lightycle (9–16:00 h). To facilitate adaptation to new surroundings,ats were transported to the laboratory at least 1 h prior to testing.he trial was started by placing an animal on the central platformf the maze facing an open arm. Standard 5-min test duration wassed and between subjects, the maze was thoroughly cleanedith damp and dry towels. Rats were randomly allocated to the
ollowing groups: vehicle control, diazepam (1 mg/kg po), SEO25 and 50 mg/kg po). Similarly, study was carried out on 1st,rd and 7th day for acute, subacute and chronic model. In acutetudy 60 min after the first dose, in subacute 60 min after the 3rdose and in chronic study 60 min after the last dose on the 7thay of drug or vehicle administration, the animal was placed athe central platform of the maze facing an open arm. Standard 5-
in test duration was used and between subjects, the maze washoroughly cleaned with damp and dry towels. The experimentsere performed with an observer unaware of the treatment of the
ats inside the room. The following parameters are classicallyeasured in this test: frequency and duration (s) of arm visits,
eparately for open and closed arms. A mouse was consideredo have entered an arm when all four paws were on the arm.he percentage of entries into open arms (open arm entries/totalrm entries × 100; % open arm entries) and the percentage ofime spent in open arms (open arm time/total arm time × 100; %pen arm time) are used as traditional indices of the anxiety. Inddition, head dips (exploratory head/shoulder movement overides of maze) and closed arm returns (exiting from an arm withnly two paws, and then returning/doubling back into the samerm) were also recorded. Head dips were further differentiateds protected (occurring on or from the relative security of thelosed arms or central platform) or unprotected (occurring on orrom the open arms). Data were given as percentage protected
cores (protected/total × 100; % head-dips) [14].
ight and dark box. The apparatus consisted of an openop wooden box. Two distinct chambers, a black chamber
e172 D. Kumar et al. / European Journal of Integrative Medicine 4 (2012) e169–e176
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20 cm × 30 cm × 35 cm) painted black and illuminated withimmed red light and a bright chamber (30 cm × 30 cm × 35 cm)ainted white and brightly illuminated with 100 W white lightource, were located 17 cm above the box. The two chambersere connected through a small open doorway (7.5 cm × 5 cm)
ituated on the floor level at the center of the partition. Each ani-al was in bright and dark arena paradigm. Study was carried
ut on 1st, 3rd, 7th day for acute, subacute and chronic model. Incute study 60 min after the first dose, in subacute 60 min afterhe 3rd dose and in chronic study 60 min after the last dose on theth day of drug or vehicle administration, the animal was placedt the center of the brightly lit arena in the light and dark box.he number of entries into and the time spent in the bright arena,
he number of rears in the bright and dark arenas were noted.ollowing each trial, the apparatus were cleaned to mask thedor left by the animal in the previous experiment. Hand oper-ted counters and stop watches were used to score the behaviorf animals and experiments were performed with an observernside the room, comprised both conventional and ethologicalarameters [31].
tatistical analysis
All observations were presented as mean ± S.E.M. All results
ere analyzed using one-way analysis of variance (ANOVA) fol-
owed by Dunnet’s test [*P < 0.05, **P < 0.01]. P values lowerhan 0.05 were considered statistically significant.
a[(t
chys tibetica oil.
esults
. tibetica oil composition
GC–MS of S. tibetica oil indicated the presence of 33onstituents. The constituents were identified by comparingith the NIST library of mass spectrometry (Indian Institutef Integrative Medicine, Sanat-Nagar, J&K, India). Chemicaldentification of S. tibetica oil is the first time report with aciphyl-ene (66.415%), fenchyl alcohol (8.897%), �-pinene (8.188%),aryophyllene oxide (4.648%), menthol (1.690%), and geraniol1.315%) as major constituents. All the constituents, major asell as minor are depicted in Fig. 1.
nxiolytic activity
ffect of SEO on social interaction of ratsIn the high light, unfamiliar test condition there was a signif-
cant drug-induced increase in social interaction [**P < 0.01].urther analysis confirmed that both 25 and 50 mg/kg po dosesf SEO and diazepam (1 mg/kg po) significantly increasedocial interaction time in comparison to the control group andlso markedly enhanced active social interaction (**P < 0.01)Fig. 2). In the high light, familiar test condition there was
gain a significant drug-induced increase in social interaction**P < 0.01] at the dose of 25 mg/kg of SEO, although diazepam1 mg/kg) also increased social interaction time but it was foundo be less than the test drug at a dose of 50 mg/kg of SEO
D. Kumar et al. / European Journal of Integrative Medicine 4 (2012) e169–e176 e173
Table 1Mean or median time (s) spent in varied behavioral categories for pairs of rats in a 10-min social interaction trial.
Time spent in varied behavioral categories (s) Control Diazepam SEO 25 SEO 50
HU test conditionSniffing and mutual grooming 25.50 ± 1.34 53.67 ± 2.17** 52.17 ± 3.69** 96.83 ± 5.24**
Diazepam (DZ, 1 mg/kg) and Stachys tibetica Vatke oil (SEO 25–50 mg/kg) administered po 40 min before testing. n = 6 pairs per group. Test conditions were highlight, unfamiliar (HU) and high light, familiar (HF).
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* P < 0.05, significantly different from vehicle. Statistically analyzed by ANO** P < 0.01, significantly different from vehicle. Statistically analyzed by ANO
Fig. 2). The data of varied behavioral categories are shownn Table 1. The results displayed that the increase of social inter-ction time was due to the enhancing duration of “sniffing andutual grooming” and/or “adjacent lying” for rats (**P < 0.01
r *P < 0.05). On the other hand, ANOVA followed by Dunnet’sest revealed a significant decrease in the duration of aggressiveehaviors with SEO at 25 and 50 mg/kg (**P < 0.01 or *P < 0.05)n both HU and HF test conditions. However, diazepam alsoecreased the duration of aggressive behaviors (Table 1).
ffect of SEO on hole-board test of ratsHole-board measures are summarized in Table 2. ANOVA
emonstrated significant treatment effects on head-dip counts*P < 0.05], head-dip duration [*P < 0.05] and head-dip latency**P < 0.01]. All the test groups significantly shortened head-ip latency compared with the control group (**P < 0.01 orP < 0.05).
ffect of SEO on elevated plus-maze test of ratsIn the EPM, the behavior observed confirmed the anxiolytic
ctivity of diazepam as reported previously. In order to deter-ine the anxiolytic effect of SEO on the EPM, SEO at doses 25
nd 50 mg/kg were tested. SEO at a dose of 50 mg/kg (1st, 3rd,
ig. 2. Mean time ± S.E.M. (s) spent in active social interaction for pairs ofale rats given a 10-min trial, 40 min after drug administration. n = 6 pairs per
roup. Test conditions were high light, familiar (HF) and high light, unfamiliarHU). DZ = diazepam 1 mg/kg body weight. SEO 25 = Stachys tibetica Vatke oil25 mg/kg). SEO 50 = Stachys tibetica Vatke oil (50 mg/kg). Significance of dif-erence: **P < 0.01 compared with control condition and statistically analyzedy ANOVA followed by Dunnet’s test.
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llowed by Dunnet’s test.llowed by Dunnet’s test.
th day) significantly increased the percentage of time spentnd percentage of arm entries in the open arms (*P < 0.05,*P < 0.01; Table 3) and decreased the closed arms entries, %ead-dips, closed arm returns (*P < 0.05, **P < 0.01; Table 3).n a similar fashion, diazepam increased the percentage of timepent and percentage of arm entries in the open arms (*P < 0.05,*P < 0.01; Table 3).
ffect of SEO on behavior of rat in light and dark arena testTreatment with diazepam significantly increased the time
pent (*P < 0.05, **P < 0.01) in the light arena as well as numberf crossing (*P < 0.05, **P < 0.01) between the light and darkrena, whereas the time spent in dark arena (**P < 0.01) anduration of immobility (**P < 0.01) were significantly reduced.he SEO at dose 25 and 50 mg/kg treated rat also showed sig-ificant increase (*P < 0.05, *P < 0.01) in the time spent in theight arena and the number of crossing (*P < 0.05, **P < 0.01)etween the light and dark arena. However, the time spent in darkrena (**P < 0.01) and duration of immobility (**P < 0.01) wereignificantly reduced as compared to control (Table 4).
iscussion and conclusion
There has been a considerable popular interest in the usef the so-called natural remedies, or herbal products, to treatnxiety and depression. St. John’s wort is the most well-knownerbal product available over the counter for the treatment ofnxiety and other ailments. Recently, several plants have beeneported to possess anxiolytic effects through animal models ofnxiety [32]. The social interaction test of anxiety was devel-ped to provide an ethologically based test that was sensitiveo both anxiolytic and anxiogenic effects. Generally speaking,n increase in social interaction is indicative of an anxiolyticffect, whereas a specific decrease in social interaction indi-
ates an anxiogenic effect. This test provided a new approacho the neurobiological mechanisms underlying anxiety disor-ers. Ge et al. have found that the aversive test condition ofhe social interaction test (HU) increases 5-HT and DA turnover
e174 D. Kumar et al. / European Journal of Integrative Medicine 4 (2012) e169–e176
Table 2Measures recorded from rats given a 5-min hole-board test, 40 min after vehicle or drug administration po.
Drug Head-dip latency (S) Head-dip counts Head-dip duration Locomotion (SC) Rearing counts Rearing duration
Values are median ± range, n = 10 per group. SC = squares crossed.* P < 0.05, significantly different from (control) vehicle condition. Statistically analyzed by ANOVA followed by Dunnet’s test.
** P < 0.01, significantly different from (control) vehicle condition. Statistically analyzed by ANOVA followed by Dunnet’s test.
Table 3Effects of SEO and diazepam in the elevated plus-maze test in rats.
Group % open arm time % open arm entries Total arm entries
Results are expressed as means ± S.E.M. (n = 10). follo follo
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* P < 0.05, compared with (control) vehicle. Statistically analyzed by ANOVA** P < 0.01, compared with (control) vehicle. Statistically analyzed by ANOVA
hroughout the rat brain [33]. In brief, the social interaction test isn extremely useful animal model for evaluating anxiolytic com-ounds, which are prescribed for treating social phobia, socialailure/impairments and emotional immaturity [34].
The acute toxicity of EO of S. tibetica was initially checked
nd it was found that there are neither behavioral changes norortality and the oil is devoid of any kind of irritations. In
he social interaction test, SEO and DZ decreased aggressive
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able 4ffect of SEO on behavior of rat in light and dark arena test.
esults are expressed as means ± S.E.M. (n = 10).* P < 0.05, compared with (control) vehicle. Statistically analyzed by ANOVA follo
** P < 0.01, compared with (control) vehicle. Statistically analyzed by ANOVA follo
wed by Dunnet’s test.wed by Dunnet’s test.
ehaviors at the doses of 25, 50 mg/kg and 1 mg/kg while theame doses significantly increased social interaction time ofhe high light, unfamiliar test condition and also prolongedocial interaction time of the high light, familiar test condi-ion. We may postulate that increased social interaction in rats
eceiving SEO and DZ treatment is merely an artifact of theyperactivity induced by the drug. However, the data of variedehaviors of rats in this study indicated that the increased social
nteraction elicited by DZ (1 mg/kg), SEO (25 and 50 mg/kg)riginated from the increase in sniffing, grooming and adjacentying rather than any other behavior.
The hole-board test has been widely used to assess emo-ionality, anxiety and/or responses to stress in animals [26].everal behaviors can be readily observed and quantified in the
est, which makes a comprehensive description of the animals’ehavior possible. It has been established that head dippingehavior in mice and rats reflects exploration distinct from gen-ral locomotor activity [35]. Based on previous reports, Takedat al. indicated that head-dipping behavior was sensitive tohanges in the emotional state of the animal, and suggestedhat the expression of an anxiolytic state in animals might beeflected by an increase in head-dipping behavior [27].
Our results showed an increase in the frequency and durationf exploratory head-dips in rats which received oral administra-ion of non-sedative dose (1 mg/kg in this study) of diazepam on
hole-board. This model, as noted above, also yielded a con-istent anxiolytic action of SEO. It seems that future behavioraltudies of SEO in rats could usefully explore even lower doses ofhe drug. It is obvious that there are some differences between theutcomes of the two tests, such as the locomotor activity of theested animals and the anxiolytic doses of SEO and diazepam.lthough we do not know the reasons for the differences, the
wo tests are quite different. File and Pope (1974) found thatrug effects that are present when one rat is tested alone in theole-board may not be present when two rats are tested together36]. This may be due to the fact that the behavior of one ratnfluences the behavior of the other. Both File and Belzung ande Pape have reported that different measures of anxiety (e.g.s recorded in the plus-maze, social interaction, Vogel conflict,ight/dark exploration, hole-board, free-exploration, and neo-hobia tests) correlated very poorly with one another [37,38].ndeed, they can actually yield separate anxiety factors (e.g.lus-maze anxiety, social interaction anxiety, and Vogel anx-ety), thereby conforming the growing view that the inherentnconsistency exists in the tapping into different facets of anx-ety. In summary, this research expanded findings with SEO tohow consistent anxiolytic effects in both the social interactionnd hole-board tests which would further substantiate predic-ion of clinical efficacy of the agent. For example, since there is
significant decrease in the duration of aggressive behaviors ofats with SEO in social interaction test, the volatile oil mighte a novel therapeutic approach to reduce or inhibit height-ned aggressiveness and possibly to treat aggressive behaviorssociated with psychiatric disorders.
The elevated plus-maze is a well-established animal modelnd is currently the first choice test for anxiolytic drugs andas been validated for both rats and mice [38,39]. It is basedn the natural conflict between the drive to explore a new envi-onment and the tendency to avoid potentially dangerous area.
ore recently, it has been argued that the incorporation of aange of ethological parameters may enhance the utility of this
aradigm [14]. In the present study, it was found that in rats,EO (25 and 50 mg/kg) and diazepam (1 mg/kg) increased both
he percentage of time spent and entries into the open armsf the maze and decreased the percentage of head-dips and
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losed-arm returns, indicating a significant anxiolytic-likeffect. Our results indicate that SEO has a significant anxiolytic-ike effect in rats. The activity is more in SEO 50 as comparedo SEO 25 and the same was exhibited on all the days of ourecordings which clearly indicate a dose dependant activity withaximum activity in SEO 50. The results also indicated a slight
ncrease in anxiolytic activity with the passage of time and dos-ng on each day. This is quite evident from the results with thectivity reaching its maximum on the seventh day as comparedo the activity on previous days.
The light/dark test is another widely used rodent anxietyodel for screening anxiolytic or anxiogenic-like drugs. In the
resent study, rat spent more time in the light box and exhibitedery insignificant change in the number of transitions betweenhe two boxes, which indicated an anxiolytic activity. In the ratsreated with SEO 25, the time spent by the rats in the light boxas insignificant as compared to either diazepam or SEO 50 on
ll the days of the experiment. The activity of SEO 50 was quiteignificant and comparable with diazepam on all the days ofhe treatment and experimentation with the seventh day readinghowing a slight increase in activity as compared to the standardiazepam. It will be interesting if the studies are carried beyondhe seventh day to see if the slight advantage of SEO 50 can be
aintained over diazepam.Synthetic drugs including diazepam are currently the first
ine of treatment in anxiety however some side effects like psy-hological dependency, physiological dependency, withdrawalymptoms, and depression are major hurdles for their use in longerm treatment of diseases which is always associated with anyind of CNS disorder including anxiety. On the contrary herbalrugs including essential oils are easily acceptable to patients forong term treatment. Thus any progress in this field will be ofremendous significance to people in the whole world and thirdorld countries in particular where majority of the people relyn alternative system of medicine for their healthcare needs.
Animal models help to understand the information aboutolecular mechanisms involved in anxiety and for screening
nd developing new medications for their treatment that woulde impossible in humans. The human studies have establishedhe genetic basis of anxiety and animal studies have been usedo attempt to further clarify its genetic determinants. The firstnvolves the animal’s conditioned responses to stressful andften painful events (e.g. exposure to electric foot shock) andhe second includes ethologically based paradigms and involveshe animal’s spontaneous or natural reactions (e.g. flight, avoid-nce and freezing) to stress stimuli that do not explicitly involveain or discomfort (e.g. exposure to a novel highly illuminatedest chamber or to a predator). The models used in this study areell established and scientifically validated models of anxiety
40].Numerous psychological and brain disorder research stud-
es have been conducted using traditional medicinal plants inhe form of specific oils, herbal extracts, and combinations to
reat specific diseases including anxiety, depression, insomnia,lzheimer, Convulsions, and Parkinson in an effort to discoverew therapeutic agents that lack the toxic side effects associatedith current agents. Stachys oil was evaluated for its constituents
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176 D. Kumar et al. / European Journal o
nd was found to contain 33 constituents; some of these weren major quantity especially aciphyllene (66.415%). This coulde one of the rich sources for aciphyllene. Further investigationsre needed to isolate and identify active components of the stud-ed oil to confirm the pharmacological activity in more detail innimal models.
onflict of interest
None.
cknowledgments
Dinesh Kumar, Z.A. Bhat, and M.Y. Shah would like to thankhe Department of Pharmaceutical Sciences, University of Kash-
ir, Jammu and Kashmir, India, IIIM-Srinagar for providingC–MS facility and UGC, New Delhi, for financial assistance
or the same work.
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