To Evaluate Anti-Anxiety Activity of Thymol Sanjay Singh Bhandari*1, Mahaveer Prasad Kabra2

Journal of Acute Disease (2014)136-140 136

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Document heading doi: 10.1016/S2221-6189(14)60030-5 To evaluate anti-anxiety activity of thymol Sanjay Singh Bhandari*1, Mahaveer Prasad Kabra2

1Department of Pharmacology, M.D.U. Rohtak, Haryana, India 2Department of Pharmacology, Kota College of Phramacy, Kota, Rajasthan, India

ARTICLE INFO ABSTRACT

Article history: Objective: To evaluate anti-anxiety activity of thymol (5, 10, 20 mg/kg. i.p.) in Swiss albino Received 8 August 2013 Methods: n mice. Six group ( =5) of mice were used in this study. Drug was given to each animal Received in revised form 15 September 2013 30 Accepted 24 September 2013 intraperitonealy, behavior testing was performed in animal models after min of all treatment, 5 (300 ) S Available online 20 June 2014 time spent in light area/open space was observed for min duration s . ignificant increase in percentage of time spent in open arms of EPM and significant increase in percentage of time spent Keywords: in light compartment of LDT indicate anxiolytic-like Results: effect respectively. Significant decreased in Anxiety above parameters indicates anxiogenic effect. Thymol 20 mg/kg significantly increased Thymol percentage of time spentConclusions: in open arms of EPM and light compartment of LDT as compared to their Diazepam vehicle treated group. Thymol (20 mg/kg) produced significant anti-anxiety effect Neurotransmitters as compared to vehicle (0.01% ethanol) treated mice in both EPM and LDT behavioral models. Behavioural paradigms Stress

1. Introduction inhibitory neurotransmitter in the brain. The GABAA- benzodiazepine receptor is an important target for several Anxiety disorders are common mental disorders that anxiolytic drugs and may therefore play an important share extreme or pathological anxiety as the primary role in anxiety-related disorders[5]. It is well reported disturbance in mood or emotional tone[1]. Common that Diazepam, GABAA-benzodiazepine receptor agonist denomination of all anxiety disorders is a state of increased at the dose of 2 mg/kg, i.p. significantly showed anti- fear and exaggerated version of the acute stress response[2]. anxiety activity in various behavioural paradigms of Literature revealed a link between anxiety and problems animals[6]. Several studies have shown that positive with the regulation of various neurotransmitters. The allosteric modulators (which potentiate GABA action), such large numbers of neurotransmitters, peptides, hormones, as progesterone and allopregnanolone, have anxiolytic and other neuromodulators have been implicated in fear effects in various animal models[7]. Several peptides, and anxiety[3]. 5-HT pathway originating from the dorsal such as cholecystokinin (CCK), neuro-peptide Y (NPY), raphe nucleus (DRN) and innervating the amygdala and tachykinins (substance P, neuro-kinins A and B), and frontal cortex facilitates conditioned fear. Selective 5-HT natriuretic peptides (atrial natriuretic peptide or C-type reuptake inhibitors (SSRIs) and 5-HT1A or 5-HT3 receptor- natriuretic peptide) may play important roles in fear and selective drugs can have anti-anxiety effects in certain anxiety-related behaviors[8]. There is increasing evidence anxiety disorders and animal models[4]. Several preclinical that NO may underlie anxiety in the elevated plus-maze studies have shown that stress and anxiety cause a marked test, an animal model of anxiety. NO analogues have been increase in NA release in several rat brain regions, found to reduce GABA-gated current via cGMP-dependent including the hypothalamus, the amygdala, and the LC. pathways, leading to anxiety[9]. cGMP downregulates GABA 毭-Aminobutyric acid (GABA) is the most abundant receptor function in hippocampus, an important area involved in anxiety[10]. The elevated plus-maze (EPM) test,

*Corresponding author: Sanjay Singh Bhandari, Department of Pharmacology, M.D.U. one of the most popular animal models for research on Rohtak, Haryana, India. Tel: +919672625112 anxiety, is based on the natural aversion of rodents for open E-mail: [email protected] spaces and uses an elevated plus-maze with two open and Sanjay Singh Bhandari et al./ Journal of Acute Disease (2014)136-140 137 two closed arms[11,12]. This test is rapid and was found to (5, 10, 20 mg/kg, i.p.), Diazepam (2 mg/kg, i.p.)[29]. be sensitive to the effects of both anxiolytic and anxiogenic 2.4. Behavioral paradigms agents. The Light/Dark exploration test (LDT) is another commonly used model for anxiety[13], devised by Crawley[14], 2.4.1. Elevated plus maze this test is based on the innate aversion of rodents to brightly illuminated areas and on the spontaneous exploratory The elevated plus maze (EPM) was first proposed as behaviour of rodents in response to mild stressors, i.e. an animal model of anxiety by Handley and Mithani[30]. novel environment and light. The LDT has been widely Elevated plus maze consisted of two close arms, 16 cm伊5 adopted as an anxiolytic screening test in mice[15]. Thymol cm伊12 cm, and two open arm, 16 cm伊5 cm, connected to a (2-isopropyl-5-methylphenol) is naturally occurring central platform (5 cm伊5 cm). The maze was elevated to a phenolic monoterpene derivative of cymene, which is height of 25 cm above the floor. During the experiment each Thymus found in the oils of thyme and plants such as mouse was placed in the central compartment face towards vulgaris Thymus glandulosus Thymus hyemalis [16], [17], [18], either of open arm, observed for 5 min to record the time Thymus zygis [19]. It exhibits multiple biological activities: spent in open arms, the total observation of experiment was antibacterial[20], antifungal[21], anti-oxidant[22], free radical 5 min (300 s). An entry was counted when all four paws of scavenging[23] and anti-lipid peroxidative[24] properties. the mouse entered an open or closed arm. Arm entry was Thymol was also shown to have strong anti-inflammatory defined as all four paws having crossed the dividing line action by decreasing the release of inflammaroty metabolites between and arm and the central area[28]. Maze was washed like prostanoids, interleukins and leukotrienes[25,26]. Thymol thoroughly with 5% ethanol after each observation to remove acts as a GABAA receptor agonist/modulator[27]. odor[31]. Percent of time spent by mice in open arms was calculated as follows: 2. Materials and methods Number of seconds spent in open arms % = 伊100 300 total sec (5 min observation time) 2.1. Animals 2.4.2. Light/ dark test

Swiss albino mice of either sex (20-30 g) were employed The light/dark test (LDT) model was first described by [32] in the present study. Animals were procured from Disease the Crawely and Goodwin . The apparatus consisted of Free Small Animal House, LLRUVAS, Hisar, Haryana, rectangular shaped box (45 cm伊27 cm伊27 cm) partition into India. Animals were provided normal diet and water ad two compartments connected by a 7.5 cm伊7.5 cm opening in libitum and were exposed to natural light and dark cycle at the wall between the compartments. An animal was placed controlled room temperature of 20-25 曟. The animals were in the centre of the light compartment face toward opening acclimatized to the laboratory condition before experiments. of the wall and observed for 5 min, for time spent in open The animals were kept fasted 2 h before drug administration, (white/light) compartment. Apparatus was thoroughly washed [31] all the behavioral paradigm ware performed during day with 5% ethanol after each observation to remove odor . time between 9 a.m. and 2 p.m.[28]. Experimental protocol Percent of time spent by mice in light area was calculated as follows: was approved by Institutional Animal Ethics Committee Number of seconds spent in light compartment ( ) IAEC . Care of the animals was taken as per guidelines of % = 伊100 the Committee for the Purpose of Control and Supervision of 300 total sec (5 min observation time) Experiments on Animals (CPCSEA), Ministry of Environment 2.5. Experimental protocol and Forests, Government of India. 2.2. Drugs and chemicals There were six group of mice used in this study. Each group consisted of five mice. Behavior testing was performed i.e Thymol, Diazepam, Ethanol and Sodium chloride were carefully in stepwise manner . mice in each group were used in this study. Thymol, was procured from C®entral Drug subjected to maximum two behavioral tests of anxiety, one House (CDH) Ltd. India. Diazepam (Calmpose ); Ranbaxy followed by another to minimize the stress, that may arise Laboratories, Ltd., Gurgaon, India. Normal saline (0.9% NaCl) from continuous exposure to these paradigms. Doses and was used as vehicle for Diazepam while absolute ethanol routes of administration of drugs were selected according to solution (0.01%) was used as vehicle for Thymol. Volume of previous studies as reported in the literature. All treatments injection for mouse was 10 mL/kg. (vehicle, 10 mL/kg; Thymol 5, 10 and 20 mg/kg; Daizepam 2.3. Selection of doses 2 mg/kg) were administered intraperitoneally (i.p.) in a fixed volume of 1 mL/100 g body weight in separate groups of i.e. i.e. mice. Behavior testing was performed drug was given to Doses were selected on the basis of literature, , Thymol Sanjay Singh Bhandari et al./ Journal of Acute Disease (2014)136-140 138 each animal, after 30 min behavior testing was performed in in light compartment of light/dark test. Thymol (20 mg/kg) EPM and LDT. significantly increased percentage of time spent in light compartment of light/dark test as compared to vehicle (0.01% 2.6. Statistical analysis ethanol) treated mice. Therefore thymol (20 mg/kg) produced significant anti-anxiety effect as compared to vehicle (0.01% All the results were expressed as mean依SEM. All statistical ethanol) treated mice (Table 2; Figure 2). analysis was done using one way analysis of variance ’ P Table 1 (ANOVA) followed by the Tukey s post hoc test. <0.05 was considered as significant when compared to their respective Effect of different treatments on percentage of time spent by mice in control group. open arms of elevated plus maze. % of time spent in Treatments Dose (mg/kg, i.p.) open arms 3. Results Vehicle 1 (0.01% ethanol) 10 mL 12.46依0.75 Vehicle 2 (0.9% NaCl) 10 mL 13.01依2.07 b In elevated plus maze and light/dark test, significant Diazepam 2 33.80依2.29 increase in percentage of time spent in open arms and Thymol 5 12.13依2.56 Thymol 10 13.46依3.01 significant increase in percentage of time spent in light a Thymol 20 17.13依2.50 compartment indicate anxiolytic-like effect respectively. On n Values are expressed as Mean 依 SEM, =5 in each group. Data the other hand, significant decreased in above parameters ’ was analyzed by one-way ANOVA followed by Tukey s Post-hoc indicates anxiogenic effect. aP test. <0.05 significant difference from vehicle 1 treated group, P 3.1. Effect of different drug treatments on mice behavior in b <0.001 significant difference from vehicle 2 treated group. elevated plus maze Table 2 Effect of different treatments on percentage of time spent by mice in Diazepam significantly increased percentage of time light compartment of light/dark test. T 20 spent in open arms of elevated plus maze. hymol mg/kg % of time spent in Treatments Dose (mg/kg, i.p.) significantly increased percentage of time spent in open light compartment ( % ) arms as compared to vehicle 0.01 ethanol treated mice. Vehicle 1 (0.01% ethanol) 10 mL 15.73依2.65 Therefore thymol (20 mg/kg) produced significant anti- Vehicle 2 (0.9% NaCl) 10 mL 16.46依2.82 b anxiety effect as compared to vehicle (0.01% ethanol) treated Diazepam 2 37.93依2.41 mice (Table 1; Figure 1). Thymol 5 16.86依2.87 Thymol 10 16.86依2.50 a Thymol 20 21.00依2.30 n Values are expressed as Mean 依 SEM, =5 in each group. Data was ’ analyzed by one-way ANOVA followed by Tukey s Post-hoc test. aP bP <0.05 significant difference from vehicle 1 treated group, <0.001 significant difference from its vehicle 2 treated group.

Figure 1. n Values are expressed as Mean 依 SEM, =5 in each group. ’ Data was analyzed by one-way ANOVA followed by Tukey s Post- aP hoc test. <0.05 significant difference from vehicle 1 treated group, bP <0.001 significant difference from vehicle 2 treated group. Figure 2. n 3.2. Effect of different drug treatments on mice behavior in Values are expressed as Mean 依 SEM, =5 in each group. ’ light/dark test Data was analyzed by one-way ANOVA followed by Tukey s Post- aP hoc test. <0.05 significant difference from vehicle 1 treated group, bP <0.001 significant difference from its vehicle 2 treated group. Diazepam significantly increased percentage of time spent Sanjay Singh Bhandari et al./ Journal of Acute Disease (2014)136-140 139 4. Conclusion Acknowledgement

E (EPM) L (LDT) levated plus maze and ight/dark test box I would like to thanks to Department of Pharmaceutical are two best models of anxiety. These both come under Sciences, M.D.U. Rohtak, Haryana for their financial ethologically based animal models of fear and anxiety and ’ support. involves the animal s spontaneous or natural reactions e.g. ( flight, avoidance and freezing) to stress stimuli that do not explicitly involve pain or discomfort[33]. In the present References study, only thymol 20 mg/kg dose produced significant D anti-anxiety like activity among its three doses. iazepam [1] Ninan PT. Recent perspectives on the diagnosis and treatment of GABAA Am J Managed Care 7 binds to a specific subunit on the receptor at a site generalized anxiety disorder. 2001; : S367- GABA distinct from the binding site of the endogenous S376. T GABAA molecule, known as an allosteric site. he receptor [2] Barbee JG. 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