GC-MS ANALYSIS AND ANXIOLYTIC ACTIVITY OF ESSENTIAL OIL OF SKIMMIA ANQUETILIA OF KASHMIR REGION

GC-MS ANALYSIS AND ANXIOLYTIC ACTIVITY OF ESSENTIAL OIL OF SKIMMIA ANUETILIA OF KASHMIR REGION

Vijender Kumar ^*, Rakesh Chawla, Gazanfar Ahmad and Z. A. Bhat

School of Pharmaceutical Sciences, Delhi Pharmaceutical Sciences and Research University, New Delhi, Delhi, India.

ABSTRACT: The administration of essential oils (EOs) or some of their single constituents, either in aromatherapy, complementary medicine or folk medicine, is known for a long time or is gaining importance as a significant alternative method for the treatment of various ailments. In the last few years a lot of scientific studies have been conducted to investigate the effects and mechanisms of action of these natural compounds and their influence on the central nervous system (CNS). In the present study essential oil obtained from the leaves of Skimmia anquetilia (Rutaceae) by hydro-distillation extraction method was subjected to chemical analysis and the oil was investigated for its anxiolytic effects on rats. Chemical analysis of the leaf oil (1.2%) by gas chromatography and mass spectrometry (GC/MS) is first time study. The anxiolytic like properties was evaluated by employing the elevated plus maze, light/dark arena and hole board tests in rats. GC/MS analysis of Skimmia anquetilia oil  (SAO)  indicated the presence of  vetiverol (37.315%), pregeijerene (15.136%), linalool (10.085%), β-fenchyl-alcohol (7.715%), geranyl acetate (5.588%), terpinyl acetate (3.300%), α –pinene (3.726%), β-phallendrene (2.624%), neryl acetate (2.433%), (Z.E) - α-farnesene (2.012%), geraniol (1.463%) and limonene (1.362%) as  the major constituents of SAO. The anxiolytic studies were conducted at doses of 25 and 50 mg/kg, body weight. The SAO significantly increased number of entries and time spent in open arms on the elevated plus maze test while as in the light/dark arena test, SAO showed an increase in number of crossings and time spent in light arena. In the hole-board test, SAO significantly increased head-dipping counts and its duration. The results of in-vivo pharmacological studies are indicative that SAO has a wide range of anxiolytic potential.

Keywords: Skimmia anquetilia, Hole-board test of exploration, Elevated plus maze test, Light and dark arena

INTRODUCTION: Skimmia anquetilia (S. anquetilia) is an aromatic gregarious shrub belonging to family Rutaceae. It is mostly found in Western part of Himalayas and Kashmir in India. Traditionally, the leaf infusion of S. anquetilia is taken for treatment of headache, freshness and general fever.

The leaves are aromatic and known to contain linalool, geraniol, pinene, scopoletin, skimmianine, umbelliferone ¹. The aim of this paper was to summarize scientific literature of the last few years and thus update an already existing overview concerning the effects of essential oils of this plant on the CNS.

This time the focus is on Skimmia anquetilia oil as anxiolytic agent. Anxiolytic drugs are amongst the most frequently prescribed drugs as the disease is highly prevalent in the society. However, existing anxiolytic agents are associated with several limitations such as sedation, tachycardia, insomnia, decreased libido, cognitive impairments, alcohol interaction,  addiction with benzodiazepines,  and generally, the onset of action of 5-HT receptor ligands is slow ^{2, 3}. In this study, a possible anxiolytic property of Skimmia anquetilia essential oil was investigated using Social interaction test, hole-board test, elevated plus maze test and Light dark arena test for anxiety using rats. The elevated plus maze is one of the most extensively used models of anxiety and is based on the aversion of rodents for open spaces and anxiolytics have been found to increase the proportion of time spent on the open arms ⁴. Light and dark model based on spontaneous responses reflects a type of anxiety linked with uncontrollable stress (depressive anxiety) because animals are exposed by force to a novel and/or aversive environment from which they cannot escape ⁵. Anxiolytics have been found to increase locomotion and time spent in the light zone, whereas anxiogenics decrease them ⁶. The social interaction test is a useful animal model for evaluating anxiolytic compounds, which are prescribed for treating social phobia, social failure impairments, and emotional immaturity ⁷. In the hole-board test, it increases head-dipping counts and its duration. Because the above tests have been validated pharmacologically, behaviourally and physiologically as models of experimental anxiety in consideration to aforementioned reported psychopharmacological activities, it was thought prudent to investigate the anxiolytic potential of Skimmia anquetilia oil with the aim of finding agents that are safer and suitable for long-term treatment in anxiety like disorders.

MATERIALS AND METHODS:

Plant Material: The plant Skimmia anquetilia was collected from Gulmarg, Jammu & Kashmir, India. The plant was identified and authenticated by Dr. Zulfiqar Ali Bhat, from Department of Pharmaceutical Sciences, University of Kashmir, Srinagar-190006, India (Voucher specimen number - KUST03).

Extraction of Skimmia Oil: Dried leaves were submitted to hydro distillation in a Clevenger-type apparatus for 3 hours. After distillation the oil was collected, dried with anhydrous Na₂SO₄, and kept in a vial at a temperature of - 2°C for further analysis. Total yield of oil (1.2 % v/w) was obtained.

Gas Chromatography-mass Spectrometry Analysis of the Essential oil of Skimmia anquetilia: GC/MS analysis was performed on a Varian Gas Chromatograph series 3800 fitted with a VF-5 ms fused silica capillary column (60m x 0.25mm, film thickness 0.25μm) coupled with a 4000 series mass detector under the following conditions: injection volume 1 μL with split ratio 1:60; Helium as carrier gas at 1 mL/min constant flow mode, injector temperature 230° C, oven temperature 40° C to 250° C at 3°C/ min. Mass spectra: electron impact (EI+) mode, 70 ev and ion source temperature 2500C. Mass spectra were recorded over 50-500 amu range. Peak identification was accomplished by comparison of MS with those reported in NIST 05 and WILEY libraries.

Experimental Animals: Male Wistar rats (150-200 gm body weight) were used for anxiolytic activity study. Rats were obtained from Central Animal House of Indian Institute of Integrative Medicine, Jammu (J & K), India. They were housed in polypropylene cages in standard laboratory conditions of temperature (25±2°C) with 12h/12h light and dark cycle. They had free access to food and water ad libitum. Department of Pharmaceutical Sciences (University of Kashmir) is approved for carrying out animal studies (Approval No.801/03/ca/CPCSEA) and the protocol for the present study was approved by Institutional Animal Ethical Committee (Approval no. F-IAEC/Pharm. Sc./Approval/2011/02).

Acute Toxicity Study: Acute toxicity study was conducted as per the internationally accepted protocol drawn under the OECD guidelines 425 (OECD, 2001). Skimmia oil at a dose level of 1600 mg/kg was found safe. Doses of 25, 50 mg/kg b.w. were selected as the study dose of Skimmia oil.

Drugs: Diazepam was obtained from Ranbaxy Lab. Ltd., HPSIDC-Baddi, Solan (India). Sodium carboxy methyl cellulose was purchased from CDH-Laboratory Reagent Pvt. Ltd. Post Box. No. 7138, New Delhi-110002 (India). Diazepam and Skimmia anquetilia oil (SAO) were both suspended in a 1% sodium carboxy methyl cellulose solution. All drugs were prepared immediately before use and were given orally.

Control rats received 1% aqueous sodium carboxy methyl cellulose solution only. The effect of the drugs was estimated 60 minutes after drug administration. Drug dose, pre-treatment time and selection of 1% sodium carboxy methyl cellulose solution as vehicle were based on findings in preliminary experiments or taken from the literature. Tests were performed only after the rats had been acclimatized to the above environment for at least 7 days. All experiments were carried out between 09:00 and 16:00 h. In each experiment apparatus was cleaned using 5% ethanol before introducing the next animal to preclude the possible cueing effects of odors left by previous subjects.

Anxiolytic Activity:

Procedures:

Social Interaction test: This model is based on the observation that animals tend to reduce or even suppress their interaction with other animals under conditions of new environments or excessive light. The test procedure for social interaction test was similar to that described by File and Pellow (1985) ⁴. The test arena was a black Plexiglas box, 60 × 60 × 35 cm, with the base divided into 9 cm squares by lines of white tape. Two test conditions were performed: high light (380 lx), unfamiliar arena (HLUF) and high light, familiar arena (HLF).

On day 1 of testing, each rat was randomly assigned according to body weight (<10 g difference) to an unfamiliar partner in groups of 12 animals (six pairs) which were subsequently administered the appropriate drug. These rats were then replaced into their home cage until testing. Following appropriate pre-treatment time, members of each pair of unfamiliar rats were placed in opposite corners of the arena and observed for social interaction behaviors for 10 min.

Social interaction time (s) per pair of rats was measured as time of sniffing, mutual grooming, adjacent lying, climbing over and crawling under the partner, and approximation ⁸, Aggressive-type behaviors (e.g. kicking, aggressive grooming, biting, boxing and jumping ⁹ were also scored. These behaviours were treated as separate entities and same were modulated by different pharmacological agents than social behaviors ¹⁰. After day 1, test rats were returned to their home cages. On days 2 and 3, the rats were placed individually, without drug, in the same box for 10 min per day to familiarize them with the apparatus. On the fourth day, the same pairs of rats were once again placed in the test arena for 10 min and the same test procedure was carried out ¹¹. Pairs of rats were allocated randomly to the following test groups: vehicle control, diazepam (1 mg/kg po), SAO (25 and 50 mg/kg p.o.).

Elevated Plus-maze Test: The wooden maze consists of two open arms (50 x 10 cm) and two closed arms of the same size enclosed with 40 cm high walls and a central square of 10 x 10 cm. The maze was 60 cm above the floor. A slight raised edge on the open arms (0.25 cm) provided additional grip for the animals, whereas open arm activity was further encouraged by testing under dim red light (4 x 25 W).

The experiment was conducted as described ¹² in sound proof room during the dark phase of the light cycle (9 –16:00 h). To facilitate adaptation to new surroundings, rats were transported to the laboratory at least 1 h prior to testing. The trial was started by placing an animal on the central platform of the maze facing an open arm.  Standard 5-min test duration was used and between subjects, the maze was thoroughly cleaned by 5% ethanol with damp and dry towels.

Rats were randomly allocated to the following groups: vehicle control, diazepam (1 mg/kg po, SAO (25 and 50 mg/kg po). Similarly, study was carried out on 1^st, 3^rd and 7^th day for acute, subacute and chronic model. In acute study 60 min. after the first dose, in subacute 60 min. after the 3^rd dose and in chronic study 60 min. after the last dose on the 7^th day of drug or vehicle administration. The animal was placed at the central platform of the maze facing an open arm.

Standard 5-min test duration was used and between subjects, the maze was thoroughly cleaned with damp and dry towels. The experiments were performed with an observer unaware of the treatment of the rats inside the room. The following parameters are classically measured in this test: frequency and duration (s) of arm visits, separately for open and closed arms. A mouse was considered to have entered an arm when all four paws were on the arm. The percentage of entries into open arms (open arm entries/total arm entries × 100; % open arm entries) and the percentage of time spent in open arms (open arm time/total arm time×100; % open arm time) are used as traditional indices of the anxiety. In addition, head dips (exploratory head/shoulder movement over sides of maze) and closed arm returns (exiting from an arm with only two paws, and then returning/doubling back into the same arm) were also recorded. Head dips were further differentiated as protected (occurring on or from the relative security of the closed arms or central platform) or unprotected (occurring on or from the open arms). Datas were given as percentage protected scores (protected/total×100; % Head-dips) ¹³.

Light / Dark Box Test: Light/dark test The test apparatus consisted of a wooden box (50 cm long x 30 cm high x 35 cm wide) divided in to two equal- size compartments by a barrier possessing a door way (10 cm high x10 cm wide) on the floor level at the centre of the partition. One of the compartments was painted black and covered with a lid, where as the other was painted white and illuminated with a 60-W light bulb set 40 cm above the box ¹⁴. On the day of the test, rats were transported to the darkened testing room and left in their home cages for 2 h. Animals were then placed in the middle of the lit compartment, facing away the dark chamber.

Rats were allowed to freely explore the box for 5 min. Study was carried out on 1^st, 3^rd, 7^th day for acute, subacute and chronic model. In acute study 60 min. after the first dose, in sub acute 60 min after the 3^rd dose and in chronic study 60 min. after the last dose on the 7^th day of drug or vehicle administration, the animal was placed at the centre of the brightly lit arena in the light and dark box. The number of entries into and the time spent in the bright arena, the number of rears in the bright and dark arenas were noted. Following each trial, the apparatus were cleaned by 5% ethanol to mask the odour left by the animal in the previous experiment. Hand operated counters and stop watches were used to score the behaviour of animals and experiments were performed with an observer inside the room ^{13, 15}.

RESULTS:

Identification of Constituents of SAO: GC-MS of Skimmia anquetilia oil (SAO) indicated the presence of 19 constituents. The constituents were identified by comparing with the NIST library of mass spectrometry at Indian Institute of Integrative Medicine, Jammu (J & K), India. Important phytoconstituents like vetiverol (37.315%), linalool (10.085%), β-fenchyl-alcohol (7.715%), geranyl acetate (5.588%), terpinyl acetate (3.300%), α –pinene (3.726%), β-phallendrene (2.624%), neryl acetate (2.433%), (Z.E)- α-farnesene (2.012%), geraniol (1.463%) and limonene (1.362%) areidentified as major constituents  in S. anquetilia oil.

Anxiolytic Activity

Social Interaction Test: The time of active social interaction of the animals treated with both 25 and 50 mg/kg doses of SAO and diazepam (1 mg/kg po) significantly (p<0.005) increased social interaction time compared with the control group of animals.

In the HLUF test condition there was a significant drug-induced increase in social interaction (**P<0.01). Further analysis confirmed that both 25 and 50 mg/kg doses of SAO and diazepam (1 mg/kg po) significantly increased social interaction time compared with the control group and also markedly enhanced active social interaction (**P <0.01) Table 1.

In the HLF test condition there was again a significant drug-induced increase in social interaction (**P<0.01) at doses of 25 & 50 mg/kg of SAO. Although the diazepam (1 mg/kg) had effect on time spent in social interaction but the same was less than the effect shown by 50 mg/kg of SAO Table 1. The data of varied behavioural categories are shown in Table 2.

The results showed that the increase of social interaction time was based on the enhancing duration of sniffing and mutual grooming and/or adjacent lying for rats (**P <0.01 or *P <0.05).

On the other hand there was a significant decrease in the duration of aggressive behaviourswith SAO at 25 and 50 mg/kg (**P <0.01 or *P<0.05) in both HLUF and HLF test conditions. However, diazepam also decreases the duration of aggressive behaviours, results are depicted in Table 1.

TABLE 1: SOCIAL INTERACTION TEST - ANXIOLYTIC AND ANTIDEPRESSANT EFFECTS OF SAO

*SAO- Skimmia anquetilia oil as test drug; Diazepam used as standard drug. Drug administered (n =6 pair/group) 40 min before (oral). Test conditions were high light unfamiliar (HLUF) and high light familiar (HLF).  *P <0.05, **P <0.01, significantly different from control. Data’s statistically analysed by ANOVA followed by Dunnet’s test.

Hole-board Test: The rats were grouped and evaluated as vehicle control, diazepam (1 mg/kg po), as standard and SAO (25 and 50 mg/kg p.o.) were evaluated as test drug for various parameters of hole board activity. SAO (25 mg/kg/p.o.) treated rats showed a reduction in head-dip latency time, increase head-dip count, head-dip duration and rearing duration similar to standard diazepam (*P <0.01). However, at dose of 50mg/kg/p.o. of SAO and diazepam increased the locomotion time as compare to control untreated rats. Results show increased head dip latency as compared to lesser dose of test and standard drug Table 2. All the groups were compared with the control group. Data statistically analyzed by ANOVA followed by Dunnet test.

TABLE 2: EFFECTS OF LEAVES OF SKIMMIA ANQUETILIA OIL (SAO) IN HOLE-BOARD TEST IN RATS

Effects of SAO on anxiety in hole board test in rats; Results are expressed as means ± S.E.M., n = 6 per group. SAO25, and 50 means S. anquetilia oil at dose level 25, and 50mg/kg/p.o. respectively. The values ^aP<0.01 vs. control group. Statistically analyzed by ANOVA followed by Dunnetts test.

DISCUSSION AND CONCLUSION: Essential oils, especially those of Rutaceae family, are popularly used in therapies for their effects on mood states (anxiety, depression) and are believed to induce an effect of mental relaxation. The results of present study suggest that the essential oil of Skimmia anquetilia, at the doses of 25 and 50 mg/kg/b.w./p.o., induced a decrease in the level of emotionality, evaluated in social interaction test models of anxiety. Such response might be correlated with the activation of the olfactory system by the volatile components present in the oil, which suggests a possible central action. We would highlight the fact that anxiolytic drugs lessen fear and increase the exploratory activity of animals ^15-16.

The anxiolytic and antidepressant effects on the central nervous system which are likely to be attributed by a specific component of the essential oil such as vetiverol, linalool, limonene and citronellal are scientifically recognized for such activity ¹⁷.

The social interaction test of anxiety was developed to provide an ethologically based test that was sensitive to both anxiolytic and anxiogenic effects. An increase in social interaction is indication of an anxiolytic effect, whereas a specific decrease in social interaction indicates an anxiogenic effect. This test provided a new approach to the neurobiological mechanisms underlying anxiety disorders.

The aversive test condition of the social interaction test (HLUF) increases 5-HT and DA turnover throughout the rat brain. In brief, the social interaction test is an extremely useful animal model for evaluating anxiolytic compounds, which are prescribed for treating social phobia, social failure/impairments and emotional immaturity ^{13, 18}. In Social interaction test, SAO and DZ decreased aggressive behaviors at the doses of 25, 50 mg/kg and 1 mg/kg respectively while the same doses significantly increased and prolonged social interaction time of the HLUF test condition. Whereas, SAO (25 mg/kg/p.o.) treated rats in hole board test showed a reduction in head-dip latency time, increase head-dip count, head-dip duration and rearing duration similar to standard diazepam (*P <0.01). However, at dose of 50mg/kg/p.o. of SAO and diazepam increased the locomotion time as compare to control untreated rats.

The results indicate that SAO has a significant anxiolytic-like effect in rats. The activity is more in SAO 50mg/kg as compared to SAO 25 mg/kg and the same was exhibited on all the days of our recordings which clearly indicate a dose dependant activity with maximum activity in SAO 50mg/kg. The pharmacological mechanism that might account for the anxiolytic effect of SAO has not been clearly identified yet. In this study we have reported that SAO contain vetiverol, linalool, limonene and linalyl acetate which are well recognized potential anxiolytic agentsand these can interact and modulate the GABA_A receptors, N-methyl-D- aspartate (NMDA) receptor in cerebral cortex and nicotinic receptor at the neuromuscular junction. It can be suggested that the GABAergic system is at least partly involved in the pharmacological activity of SAO. Skimmia anquetilia oil was evaluated for its constituents and was found to contain 19 constituents; some of these were in major quantity especially vetiverol (37.315%), pregeijerene (15.136%), linalool (10.085%) and limonene (1.362%). This oil could be a rich sources for isolation of vetiverol. Further investigations are going on to isolate bioactive components from Skimmia anquetilia oil to study the molecular pharmacological mechanism for its anxiolytic activity in animal models.

ACKNOWLEDGMENTS: Dr. Vijender Kumar and Dr. Z. A. Bhat would like to thank University of Kashmir, Srinagar and CSIR, New Delhi, India for provided research fellowship for the same work.

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Kumar V, Chawla R, Chauhan S and Bhat ZA: GC-MS analysis and anxiolytic activity of essential oil of Skimmia anuetilia of Kashmir region. Int J Pharm Sci & Res 2024; 15(2): 506-12. doi: 10.13040/IJPSR.0975-8232.15(2).506-12.

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