INTRODUCTION:

Anxiety is an most common psychiatric disorder, primary symptoms of that is disturbance in mood, sleep, emotional tone and phobia i.e finally results to depression. Finally, anxiety is appraised as multisystem response associated with biochemical changes in body, patient’s history, patient’s memory, and social interaction, genetic and environmental factors. The most commonly mental disorders in clinical practice are anxious behaviour. Due to physician mistake for differentiation inanxiety and physical illness symptoms are not identified so, only 23% patients receive appropriate treatment^1-2.

Anxiety disorders individuals are more prone to develop heart disease, complication in brain functioning, digestive system complications and disorders of respiratory system highly than normal individuals^3-4. The plant which shows antianxiety potential also evaluated for their antioxidant activities because oxidative stress in brain due to formation of ions and lipid peroxidation damages which promote the risk of serious diseases of brain like anxiety, depression and neuro-degenerated disorders^5-6. Various studies proof that antioxidant showed protective effect on the brain enzyme damages which were induced due to stress^7-9.In anxiety neurotransmitter level imbalance due to inactivation of GABA_1A receptor and 5-HT_1A receptor, inactivation of GABA receptor cause activation of Ca²⁺ and K⁺ channel, increase influx of ions which increase contraction of blood vessels as increase sympathetic system activity by increase in release of neurotransmitters by cell hyper polarization and inactivation of 5-HT receptor increase release of glutamate. Due to increment of glutamate release decrease acetylcholine release which decrease parasympathetic system activity¹⁰. Humans experienced anxiety when exposure to open arm and humans with high anxiety avoid open arm. Hence, humans on elevated plus maze (EPM) shows similar behaviour as observed in rodents^11-12. Anxiolytic activity in rodents are confirmed by Elevated plus maze model, light and dark apparatus and by actophotometer.Crawley developed extrapolation experiment in the light and dark apparatus, was a composer to the EPM and provides other method of analyzing anxiogenic behaviour in rodents¹³. Hence increase in extrapolation is explained as a release of exploratory prohibition^14-15. Humans follow a circadian rhythm of our biological clock a sleep-wake cycle administered by the day-night cycle. But the cycle disturbs by artificial light at night.Authors choose linalyl acetate for evaluation of its anxiolytic activity as it is major chemical constituent of lavender (Lavandula angustifolia, family-Lamiaceae) essential oil and the plant extract shows anxiolytic effect in aromatherapy and other known effect of linalyl acetate are anti-inflammatory , analgesic and antihypertensive^16-17. Antihypertensive effect of linalyl acetate by relaxing blood vessels and decrease blood pressure hence its action by controlling the over excitation of sympathetic system. In the essential oil extract of lavender plant consists two major chemicals 36.8% of linalool and 34.2% of linalyl acetate^18-19. In previous study, 400 mg/kg dose of linalyl acetate determined most potent dose (could be used for further study combination with alperazolam, buspirone and caffeine).

Anxiety Models:Elevated Plus Maze:

Anxiety behaviours are evaluated by elevated plus maze(EPM) by following Lister’s method. The %age of duration spent in open arms of maze was calculated as the total duration spent in open arm of maze/total time of exposure on maze. The %age total numbers of entrances in open area of EPM were calculated as total transitions in open part of maze/number of transitions in both open and closed arms. The increase total exposure period in open area of EPM is observed as indicator of anxiolytic potential of experimental substance and motor activity indicator is total approach to closed arm of maze in test¹¹. Other evidences of anxiety disorder observed in EPM testing are head dipping (sticking of head to the floor of maze), stretched posture, seizures, grooming (cleaning of mouth by paws)^20-21.

Light and dark model:

The light and dark (LD)model has two compartments and consists of a box (40 x 25 x 25 cm) divided into two compartment, one light compartment and another dark compartment are connected to each other and have an opening (4 x 4 cm) between them¹³.The duration of exposure in the darkened or light portions of the LD box was measured²².

Open field:

Open field apparatus is simple test apparatus for experimental animals generally performed for locomotors activity testing in rodents. The parameters recorded in the OPM are the frequency of crossing square, total numbers of squares crossed (cross of square counts by entry as well exit of four paws of rodent in that), center square entries, defecation and urination frequency²³.

Actophotometer:

The measure of locomotors activity in rodents done by actophotometer, it gives an accurate measure of locomotors activity. The principle of actophotometer based on the total number of photocell beam crossed by rodent during testing session²².

Amputation and Homogenization:

There upon, after the last behavioral experiment, mice were placed according to groups; cervical dislocation method was used for scarification of miceand whole brain of each mouse was collected by dissection and used for estimation of antioxidants levels and neurotransmitters levels (n = 5/group). Whole brain from each mouse was collected and weighed. From whole brain, homogenate was prepared by using 10% (w/v) tissue in freshly prepared in phosphate saline buﬀer (0.1 M) having pH 7.6 and homogenization of all mice brain samples were done using pestle- mortar and centrifuged themin a centrifuge at 5,000 rpm for 20 min and supernatants were separated for oxidative stress indicators and neurotransmitters estimations. After amputation and homegenization estimation of antioxidants levels and neurotransmitters level was completed.

MATERIAL AND METHODS:

Oxidative stress parameters:Estimation of protein:

The development of blue colour in reaction mixture is the result reducing action of amino acids present in protein on FCR and this process is termed as biuret reaction (reaction of alkaline copper solution with protein content of sample). And the absorbance is read at λ value 660 nm²⁴.

Reagents/Solutions:

A) 2 % Na₂CO₃ was made by 2 gm of Na₂CO₃ was added in 0.2 N NaOH solution (0.8 gm of NaOH dissolved in 100 ml distilled H₂O). B) 0.5 % Copper Sulphate was made by mixing 0.5 gm of CuSO₄ in 1 % Potassium sodium tartarate solution (1 gm of potassium Sodium tartarate dissolved in 100 ml H₂O). C) Preparation of alkaline copper reagent: By taking 2 ml of reagent B and made up to 100 ml with reagent A.D) Folin-Ciocalteau reagent: Mix 2 ml of folin-Ciocalteau and 4 ml of distilled water. Stock standard solution: BSA (100 mg) was mixed in 100 ml of D.W.

Standard working solution:

By adding 20 ml of stock solution in D.W. and made up to 100 ml.From working solution pipette out 0.2 ml to 1 ml volume and transferred that in 5 different tubes and made up to 1 ml with D.W. Then 0.1 ml from each tube were pipette out and transferred in another 5 tubes and different tubes of samples were also prepared by adding 0.1 ml of sample in each tube. After that volume in each tube made 1 ml by using distilled water(D.W). Then 5 ml of copper solution was acquired in donominator tube and shake properly and left them for incubation about 10 min. After incubation 0.5 ml of folin reagent was added to each tube, mixed well them and left for 30 min incubation at normal conditions. And after that, the colored mixture absorbance was decoded at 660 nm besides using blank.Using standard plot methodprotein concentration was measured. Standard plot was developed by using different concentration of Bovine serum albumin solution and result was expressed in mg/ml.

Estimation of Reduced Glutathione (GSH) level in mice:

Estimation of GSH levels in mice were done by Ellman’s method. In this method a yellow colour compound is formed by reaction of DTNB with GSH present in sample and asorbance of that compound is read at λ value 412 nm.

Reagents/Solutions: DTNB (Ellman's reagent) (5,5′-dithiobis-(2-nitrobenzoic acid) dye:

1 mM DTNB was made in 1% citrate solution: TCA solution: 4 gm of tri-chloro acetic acid was added into 100 ml distilled H₂O.

Buffer:

0.3M phosphate buffer having pH 8.4 was prepared.Tissue homogenates (2 ml of 10% w/v) has been transferred into 2 ml of TCA solution and left for 10 min incubation at 4°C, the precipitates has been separated by centrifugation at 5000 rpm for 23 min duration, 1 ml of supernatants were mixed into 4 ml of phosphate buffer and 0.8 ml of H₂O, 0.5 ml of DTNB dye was mixed to above solution and left for incubation about 10 min at normal conditions. The yellow colour was developed and change in absorbance recorded at 412 nm using UV-spectrophotometer against appropriate blank (represented in n moles/mg of protein).

Estimation of MDA (Malondialdehyde):

MDA has been defined as the index of lipid peroxidation. The estimation of MDA level in sample is based on the reaction of MDA present in sample with TBA(thiobarbituric acid) and this reaction form a compound which gives red colour to the reaction mixture and absorbance is recorded at λ value 535 nm²⁵.

Reagents /Solutions:Stock solution:

0.25 N hydrochloric acid15% w/v C₂HCl₃O₂ 0.375 w/v and C₄H₄N₂O₂S in 100 ml water. Solution was prepared by mixing 15 gm of tricholroacetic acid (TCA) and 0.375 gm of thiobarbituric acid in 100 ml H₂O and boiled on water bath for complete disintegration and after 0.91 ml of HCl was transferred to above mixture.2 ml supernatant were mixed into 4 ml of TCA-TBA-HCL reagent and shake properly and heated on double boiler for 25 min and subsequently cooling the precipitates were removed by centrifugation carried at 2,000 rpm for 15 min. Optical density has been recorded at 535 nm using appropriate blank sample that contain all the solutions except the sample by using UV-spectrophotometer.The results were represented in n moles of MDA formed/mg protein.

Estimation of Catalase:

Catalase activities in samples were estimated by Luck’s method. In this method the presence of catalase is determined by the amount of H₂O₂breaks by catalase enzyme present in sample and absorbance is recorded at 240 nm²⁶.

Reagents/Solutions: 0.05 M Phosphate buffer pH 7:

(a) 6.81 gram of KH₂PO₄ in 100 ml water, (b) 8.9 gram of Na₂HPO₄ in 100 ml water.a:b= 1:1.5 , and pH adjusted to 7.

H₂O₂ solution:

0.019 M Hydrogen peroxide solution was prepared in distilled water. The 0.05 ml of supernatant has been transferred in 1.95 ml phosphate buffer and 1ml of H₂O₂ solution was to above mixture and allowed to stand about 10 min under normal conditions. After 10 min optical density was recorded at 240 nm using UV-spectrophotometer against an appropriate blank (represented in µ mole H₂O₂ decomposed/mg of protein).

Estimation of Nitrite levels:

Estimation of NO^2- level in samples has been done using Greiss reagent by colorimetric method. The measurement of nitrite in sample is used as an index of NO production²⁷.

Reagents/Solutions:

Greiss reagent: 0.2% N- (1-napthyl) ethylene di-amine di-hydrochloride and 2% sulfanilamide was mixed in 5% m- phosphoric acid solution in distilled water and after proper mixing incubate at 0°C for 60 min.Greiss reagent was prepared using Green’s method. 2 ml of supernatant transferred in 2 ml of Greiss reagent and allowed to stand for 10 min under dark at room temperature and optical density was recorded at 540 nm using UV-spectrophotometer against appropriate blank.The level of nitrite in sample was manifested in µM of NO/mg of protein.

Estimation of Superoxide Dismutase (SOD):

SOD was measured by using the method of Kono²⁸.

Reagents/Solutions:

Solution A: 100 mM Na₂Co₃, 0.2 M Ethylene diamine tetra acetic acid, pH 10.2; Solution B: 192 µM Nitrobluetetrazolium; Solution C: 1.2% of Triton X- 100; Solution D: 40 mM Hydroxylamine hydrochloride, pH 6.2. Solution A (8 ml) was mixed with solution B (2ml) and solution C (4ml) and mixture was left for 10 min at 37°C for incubation. After incubation addition of 100 µl of solution D reaction was started and to that reaction added 10 µl of test sample and the rate of % inhibition of NBT by SOD present in sample was recorded by UV-Visible Spectrophotometer at λ value 560 nm against blank.SOD activity was manifested in µg/mg protein.

Neurotransmitters Estimation:

Estimation of GABA:

Estimation of GABA was done by Maynert’s method²⁹.

Reagents/Solutions: Mobile phase: 50 ml of n-butanol and 12 ml CH₃COOH mixed with 60 ml distilled water;Ninhydrin reagent: 0.25 gm ninhydrin was added to 50 ml ethanol;Homogenation of brain sample was prepared 5.0 ml and transferred into 10 ml ice cold alcohol and allowed to stand for 1 hr at 0°C. The sample was centrifuged at 3000 rpm for 30 min. The upper part centrifuged samples were put in china dish and samples were placed for evaporation till dried at 80°C on water bath. 2 ml of water transferred to the dried masses and 4 ml chloroform and centrifugation was done at 4000 rpm for 10 min. The upper part of centrifuged samples were separated and spotted on Whatman’s filter paper 2 µl. The mobile phase was put in chamber for activation. The chromatogram was developed by ascending capillary method on papers. The papers were taken out from chamber after ¾ chromatogram developed and drained in air and then sprayed ninhydrin reagent on papers. Then papers were dried for 4 min at 100°C in oven. Blue colour spots were observed on papers and spots area were incise and boiled with 5 ml of ninhydrin solⁿ on boiling water bath about 10 minutes and then 5 ml of water was transferred to that and keep on water bath for 1 hr. After that 2 ml of samples were taken and absorbance of each sample was recorded at λ value 570 nm by assessing UV-Spectrophotometer against blank.The concentration of GABA was manifested in µ moles/gram weight.

Estimation of Glutamate: Glutamate level in samples was estimated by paper chromatography.

Reagents/Solutions:Mobile phase: N- butanol: acetic acid: water (24:6:10); Ninhydrin reagent: 1.5 gm ninhydrin mixed in 150 ml ethanol; Copper sulphate solution (0.010%): 5 mg copper sulphate added to 50 ml distilled H₂O.Standard glutamate solution: 5mg glutamate was added to 20ml distilled water.Supernatant 0.5 ml of brain sample was evaporated in hot air oven at 100°C till dryness. After dryness residues were transferred to 50 ml H₂O. Standard solution of glutamate was prepared. The samples were spotted on what man filter paper 2µl by micropipette. The mobile was put into developing chamber for activation, after chamber activation papers were placed in chamber. After chromatogram developed paper were dried in air. After air dried sprayed with ninhydrin solution and then dried in hot air oven at 100°C for 4 min. The portion which contained glutamate compatible near standard sample and that separated and transferred in CuSo₄ solution for dissolution. Read the absorbance at 515 nm by UV-spectrophotometer against blank.The concentration of glutamate was expressed in µmoles/gram weight.

Statistical analysis:

Values have been manifested in form of mean ± SEM. One-way ANOVA pursued by the Tukey’s multiple comparison test has been used for data exploration. The statistical analysis was carried out using Graph pad Instat. The level of significant in results were determined by p value, results were considered to be significant at p<0.001, p<0.01 and p<0.05.

Procurements of animals:

Swiss albino female mice (weight 25-35 g) have been purchased from LUVAS’ animal house, Hisar (Haryana, India). Rodents were procured to the Central Animal House of MDU, Rohtak after aproval of Institutional Animal Ethics Committee (IAEC) in a meeting held at Maharshi Dayanand University, Rohtak, Haryana, India. They were corroborated under standard conditions for two weeks. After that mice were divided randomly into 5 groups (n=5). The handling of animal has been taken as per guidelines of CPCSEA, Dept. of Animal Husbandry and Dairying Ministry of Agriculture and Farmers Welfare, Government of India.

Selection of doses:

On the basis of literature portfolio the doses of following drugs are selected: Caffeine (100 mg/kg i.p.), Alprazolam (1 mg/kg orally) and buspirone (10 mg/kg orally)^30-31.

Experimental groups:

Groups/sets (n=4) that will be used for the assessment of following activity:

For most potent dose estimation of linalyl acetate:

Set 1: Control.

For GABA (1A) receptor agonistic activity:

Set 2: Alprazolam (1 mg/kg p.o.) + Linalyl acetate (most potent).

For 5-HT (1A) receptor agonistic activity:

Set 3: Buspirone (10 mg/kg p.o.) + Linalyl acetate (most potent).

For anxiolytic potential of linalyl acetate in drug induced anxiety:

Set 4: Caffeine (100 mg/kg i.p.) + Linalyl acetate (most potent).Linalyl acetate is active chemical constituent of various spices and flowering plants has been suspended in 1% (v/v) polysorbate tween 80 solution in water solution and delivered per oral according to body weight, 400 mg/kg dose of Linalyl acetate selected. GABA modulators (alprazolam) were dissolved in distilled water and administered to standard control. And another procedure for determination of mechanism of action of linalyl acetate was carried in which pre-treatment with Linalyl acetate most potent dose 15 min before administration of buspirone 5-HT1A agonist (per oral); in other group administration of alprazolam after 15 min pretreatment with linalyl acetate most potent dose and administration of Caffeine (i.p.) after 15 min pretreatment with linalyl acetate most potent dose. All sets of mice were treated with selected doses once daily for 15 days.

RESULTS AND DISCUSSION:

For determination of mechanism of action of Linalyl acetate(LA) for its anxiolytic activity:

Procedure N= 5 in each set, Values are manifested as mean ± S.E.M. One way ANOVA pursued by Tukey’s test has been used for data exploration. C: Control group; LA 400: Linalyl acetate 400 mg/kg;

Elevated plus maze (EPM):

· Number of accesses: Increase in number of accesses in C (Control) vs LA 400, C vs LA+A, C vs LA+B, C vs LA+C show significant at p<0.001, LA 400 vs LA+A (Alperazolam), LA 400 vs LA+B(Buspirone), LA 400 vs LA+C(caffeine) do not show any significant as represented in figure (1).

· Total period spent: Increase in total time spent in open arm of maze in C vs LA 400, C vs LA+A, C vs LA+B, C vs LA+C, LA 400 vs LA+A show significant at P<0.001, LA 400 vs LA+A, LA 400 vs LA+B, LA 400 vs LA+C do not show significant as represented in figure (2).

Figure. 1. Influence of different treatment on numbers of accesses in open arm of EPM in Mice.

Figure 2. Infulence of different treatment on time spent in open arm of EPM in Mice

LA+A: Linalyl acetate (400 mg/kg) in addition of Alprazolam (1 mg/kg); LA+B: Linalyl acetate (400 mg/kg) in addition with Buspirone (1 mg/kg); LA+C: Linalyl acetate (400 mg/kg) within combination of caffeine {(100 mg/kg) in number of accesses}: caffeine {(1 mg/kg) in total period spent}.

Light and dark box:

· Number of accesses in light chamber: Increase in number of accesses/entrances/entries in light chamber in C vs LA 400, C vs LA+A, C vs LA+C, LA 400 vs LA+B show significant at P<0.001; C vs LA+B, LA 400 vs LA+A; LA 400 vs LA+C do not show any significant as represented in figure (3).

· Time spent in light compartment: Increase in time spent in light compartment in C vs LA 400, C vs LA +A, C vs LA+B, C vs LA+C, LA 400 vs LA+C show significant at p<0.001, LA 400 vs LA+A, ; LA 400 vs LA+B do not show any significant do not show any significant as represented in figure (4).

Figure 3. Impact of multiple treatments on number of accesses in light and Dark compartment in Mice

Figure 4. Multiple treatments impact on time spent in high part of LD apparantus in Mice

*LA+A: Linalyl acetate (400 mg/kg) in combination of Alprazolam (1 mg/kg); LA+B: Linalyl acetate (400 mg/kg) in addition with Buspirone (10 mg/kg); LA+C: Linalyl acetate (400 mg/kg) in addition of caffeine {(100 mg/kg)innumber of accesses}: caffeine{(1 mg/kg) in total period spent}.

Actophotometer:

Increase in number of beams crossed in C vs LA 400, C vs LA+B, C vs LA+C show significant at p<0.001; C vs LA+A, LA 400 vs LA+A, LA 400 vs LA+B, LA 400 vs LA+C do not show any significant as represented in figure (5).

Open field:

· Number of squares crossed at other than centre: Increase in numbers of squares covered at periphery in C vs LA 400, C vs LA+A, C vs LA+B, C vs LA+C show significant at p<0.001, LA 400 vs LA+A, LA 400 vs LA+B; LA 400 vs LA+C do not show any significant as represented in figure (6).

· Number of crossing of squares at center: Increase in number of squares covered at center in C vs LA, C vs LA+A, C vs LA+B 400 show significant at p<0.001; C vs LA+C show significant at p<0.05; LA 400 vs LA+A, LA 400 vs LA+B; LA 400 vs LA+C do not show any significant as represented in figure (7).

Protein level:

Increase in protein concentration in C vs LA 400, C vs LA+A, C vs LA+B, C vs LA+C show significant at p<0.001;LA 400 vs LA+A; LA 400 vs LA+B do not show any significant as represented in figure (8).

Figure 5. Influence of various treatments on number of beams crossed in actophotometer in Mice.

Figure 6. Influence of different treatments on number of squares coverd at periphery in open field in Mice

Figure 7. Influence of multiple treatments on number of squares covered at center in OPM in Mice.

Figure 8. Influence of different treatments on protein level in Mice

*LA+A: Linalyl acetate (400 mg/kg) in conjunction with Alprazolam (1 mg/kg); LA+B: Linalyl acetate (400 mg/kg) in conjunction with Buspirone (1 mg/kg); LA+C: Linalyl acetate (400 mg/kg) in addition with caffeine (100 mg/kg).

MDA: Decrease in MDA level in C vs LA 400, C vs LA+A, C vs LA+B, C vs LA+C, LA 400 vs LA+C show significant at p<0.001; LA 400 vs LA+A do not show any significant; LA 400 vs LA+B show significant at p<0.05 as represented in figure (9).

GSH: Increase in GSH level in C vs LA 400, C vs LA +A, C vs LA+B, C vs LA+C show significant at p<0.001; LA 400 vs LA +A, LA 400 vs LA+B, LA 400 vs LA+C do not show any significant as represented in figure (10).

Catalase: Increase in catalase activity in C vs LA 400, C vs LA+A, C vs LA+B, C vs LA+C show significant at p<0.001; LA 400 vs LA+A; LA 400 vs LA+B do not show significant as represented in figure (11).

Nitrite Level: Decrease in nitrite level in C vs LA 400, C vs LA+A, C vs LA+B, C vs LA+C, LA 400 vs LA+A show significant at p<0.001; LA 400 vs LA+B not show any significant as represented in figure (12).

SOD: Increase in SOD level in C vs LA 400, C vs LA+A, C vs LA+B, C vs LA+C show significant at p<0.001; LA 400 vs LA+A, LA 400 vs LA+B do not show any significant as represented in figure (13).

GABA: Increase in GABA level in C vs LA 400 show significant at p<0.001;C vs LA+A show significant at p<0.001; C vs LA+B show significant at p<0.001; C vs LA+C show significant at p<0.001. LA 400 vs LA+A show significant at p<0.001 as represented in figure (14).

Figure 9. Impact of diverse treatments on MDA in Mice

Figure 10. Different treatments effects on GSH Level in Mice.

Figure 11. Different treatments influence on catalase level in Mice.

Figure 12. Multiple treatments impact on nitrite level in Mice

Figure 13. Influence of different treatments on SOD level in Mice

*LA+A: Linalyl acetate (400 mg/kg) in addition with Alprazolam (1 mg/kg); LA+B: Linalyl acetate (400 mg/kg) in conjunction with Buspirone (1 mg/kg); LA+C: Linalyl acetate (400 mg/kg) in conjunction with caffeine (1 mg/kg).

After 15 days administration of linalyl acetate (400 mg/kg) with alprazolam (1 mg/kg) and Buspirone (10 mg/kg), caffeine (100 mg/kg) expressed anti-anxiety effect as compare to control. Antianxiety effect also demonstrated by a meaningful rise in number of beams cut in actophotometer and significant elevation in protein concentration, glutathione level, SOD activities and catalase activities and reduction in lipid peroxidation level in relation with normal mice. This is supported by literature studies as alprazolam induces significant decreases in the activity of acetylcholine esterase in the brain, hence enhance cholinergic system neurotransmitters level in the treated mice³⁰. It means in combination alprazolam sedative effect is comparatively less than its alone treatment, that can be as the number of receptors are limited and all receptors can be occupied by linalyl acetate alone and less number of receptors are available for alprazolam binding. The combination antianxiety effect indicated by an insignificant increase in protein level, an insignificant increase in GSH level, an insignificant increase in catalase activity and an insignificant increase in SOD activities as compared to linalyl acetate, an insignificant decrease in MDA level by mice produced in oxidative stress parameter test. An insignificant reduction in glutamate level in mice as compared to linalyl acetate was recorded^32-33. Linalyl acetate is basically bioactive monoterpene presents in various plants essential oils and monoterpenes have potential to modulate GABA_A receptor by decrease in noradrenaline release^34-35. The inhibitory effect of Buspirone on sympathetic neurotransmission in brain and activate effects on peripheral sympathetic system already reported by previous scholars³⁶. In present study, combination of linalyl acetate with Buspirone produced anxiolytic effect comparatively equal to linalyl acetate. The antianxiety effect of combination indicated by an insignificant rise in total accesses and extent of exposure in open arm of EPM as compared to linalyl acetate, a meaningful elevation in total number of transitions in bright compartment and an insignificant elevation in duration of exposure in bright chamber by mice in LD box test. An insignificant increase in protein level, an insignificant increase in GSH level, a meaningful increase in catalase activity and an insignificant reduction in nitrite level in oxidative stress parameter test as compared to linalyl acetate. A meaningful reduction in glutamate level was recorded as compared to linalyl acetate. This supported by literature as buspirone is a 5-HT receptor (serotonin) agonist and used as standard anxiolytic agent. In present study use of buspirone in combination with linalyl acetate was used for evaluation of 5-HT synergistic potential of linalyl acetate. In previous study, buspirone alone as well as in combination with sesamol shows anxiolytic activity at 5-HT_1A receptor site. On exposure to elevated plus maze mice experienced stress conditions and serotonergic system activation impaired and dysregulation of serotonergic system cause anxiety due to less availability of serotonin inhibitory neurotransmitters at receptor site³¹.The binding of linalyl acetate on NMDA receptor has been mentioned in previous study. The NMDA receptor antagonistic potential of linalyl acetate itself due to direct binding on NMDA receptor in amygdala of brain was explained in previous study³⁷. Linalyl acetate in addition to caffeine produced antianxiety effect that indicated by a meaningful rise in open arm exposure, a meaningful rise in total exposure period in open arm of EZM (elevated-Z-maze). A reduction in MDA level and nitrite level but a meaningful elevation in SOD, catalase and GSH level expressed in oxidative stress parameters test as compared to untreated mice. A valuable rise in GABA level and a significant reduction in glutamate level were recorded as compared to normal. This is supported by literature studies asCaffeine is a psychoactive agent, high dose of caffeine induce anxiety as panic to generalised anxiety disorder on long term use of caffeine. Caffeine induces anxiety by its inhibitory action on adenosine receptor³⁸. High dose of caffeine (>400 mg) induce anxiety in humans^39-40. Antianxiety effect of linalyl acetate in combination with caffeine indicated by non-valuable rise in number of entrances in open arm and total exposure period to open arm of EPM during experiment. An insignificant rise in GSH level as compared to linalyl acetate was recorded. Hence, linalyl acetate in combination with caffeine produced comparatively equal or less anxiolytic effect than linalyl acetate treatment.

CONCLUSION:

Linalyl acetate 400 mg/kg dose did not expressed decrease in locomotors activities alone as well as in combination with alprazolam, that was due to all receptors are occupied by linalyl acetate alone and no free receptors available for binding of further administered alprazolam hence alprazolam not expressed its sedative effect. Another combination of linalyl acetate with caffeine did not express any decrease in locomotors activities as caffeine have no effect on locomotors activities, it means linalyl acetate not expressed sedative effect at low dose. The results proposed that anxiolytic effect of linalyl acetate can be due to action on GABA receptor, 5-HT receptor, A₁ and A₂ and NMDA receptor. Potential of linalyl acetate can be due to its 5-HT_1A agonistic action, GABA_1A agonistic action, direct action on NMDA receptor and can be due to activation of A₁A₂ receptors. Linalyl acetate 400 mg/kg was recorded as most potent dose for its anxiolytic action and linalyl acetate 400 can be comparatively equal or less effective than Linalyl acetate in combination with alprazolam and linalyl acetate in combination with buspirone but, linalyl acetate 400 can be comparatively equal or better effective than linalyl acetate in combination with caffeine.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are grateful to MDU, Rohtak which runs Department of Pharmaceutical Sciences, for providing funds to carry out the present investigation. The authors alone are responsible for the content and writing of the paper.

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Received on 29.11.2023 Revised on 27.02.2024

Accepted on 02.04.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):117-126.

DOI: 10.52711/0974-360X.2025.00018

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