Research Article ISSN: 0974-6943

Pemminati S et al. / Journal of Pharmacy Research 2010, 3(2),219-223

Available online through www.jpronline.info

Antianxiety effect of aqueous extract of fruits of Emblica officinalis(EO) on acute and chronic administration in rats .
Pemminati Sudhakar, Gopalakrishna HN, Swati B, Shreyasi C, Pai MRSM, Vinod Nair
1

Department of Pharmacology, Kasturba Medical College, Manipal University, Mangalore-575 001, karnataka,India.

Received on: 27-09-2009; Revised on: 16-11-2009; Accepted on:07-01-2010 ABSTRACT The present study was undertaken to evaluate the anti-anxiety activity of aqueous extract of fruits of Emblica officinalis (EO) on acute and chronic administration in rats. Male Wistar albino rats weighing 150-180gms were divided into five groups (n=6) for acute and chronic study separately. Diazepam (1.0mg/kg) and EO (0.8, 2.0 & 4.0 mg/kg) were suspended in 1% gum acacia and administered orally. In acute study vehicle/drugs were given 60 minutes before, while in chronic study they were given once daily for 10 days and the last dose was given 60 min prior to exposure to the experimental paradigms viz., elevated plus maze and bright and dark arena. EO significantly increased the number of entries into, time spent and rears in the open arms. Also, it increased the percentile ratio of open arm to total arm entries in elevated plus maze paradigm both on acute and chronic administration. In bright and dark arena paradigm the EO both on acute and chronic administration it significantly increases the number of entries, time spent and rears in the bright chamber, also it reduced the duration of immobility. These behavioural changes were comparable to that produced by the standard anxiolytic drug diazepam. The behavioural disinhibition produced by EO suggestive of its anxiolytic like activity. Keywords: Emblica officinalis, elevated plus maze, bright and dark arena.

INTRODUCTION Anxiety is a normal emotional behaviour. When it is severe and/or chronic, however, it becomes pathological and can precipitate or aggravate cardiovascular and psychiatric disorders. Although many drugs are available in allopathic medicine to treat anxiety disorders, they produce various systemic side effects or exhibit tolerance upon chronic use. Benzodiazepines (BZDs) are the major class of compounds used in anxiety and they remain the most commonly prescribed treatment for anxiety. However the BZDs have many unwanted side effects has prompted many researchers to evaluate new compounds in the hope of identifying other anxiolytics with fewer side effects 1. In ayurvedic medicine, many plant products have been claimed to be free from side effects and less toxic than synthetic drugs 2. memory enhancer3, antiulcer4 ,hypoglycaemic 5, hepatoprotective6 , hypolipidemic 7, antipyretic and analgesic 8, anti-inflammatory 9, antibacterial10, immunomodulatory 11, antioxidant12, gastroprotective13 , activity on central nervous system14 and anticataleptic activity15. The aqueous extract of fruits of Emblica officinalis contains 30.0% of Tannins & 13.4% of Gallic acid (estimation and purity of active principle was done by the Quality Control Laboratory, M/s. Natural Remedies, Bangalore, lab reference no.0505211, dt.31-05-2005).

From our laboratory, we have reported the anxiolytic effect of NR-ANX-C,a polyherbal product in rats 16. NR-ANX-C is composed of Withania somnifera , Ocimum sanctum, Camellia sinensis, Emblica officinallis and Shilajit. As EO is one of the components and its antianxiety activity is not reported so far. In order to evaluate Emblica officinalis Gaertn. (Phyllanthus emblica L.) the contribution or role of EO in anxiolytic activity, the present study (Family:Euphorbiaceae), popularly known as amla is a common house- was undertaken. Two pharmacologically validated experimental modhold remedy that finds use in Indian indigenous system of medicine els, elevated plus maze 17 and bright and dark box18 were employed. against several ailments. Its fruits have been reported to possess MATERIALS AND METHODS *Corresponding author.
Sudhakar Pemminati, Lecturer, Department of Pharmacology, Kasturba Medical College, Manipal University, Mangalore.India Tel.: + 91-0824-2422271, extn: 5568,9448723549(M) Telefax: +91--0824-2428183 E-mail: pemmineti@yahoo.com

Animals: Male Wistar albino rats weighing 150-180g (90-110 days old) were used for the study. They were housed in clean, clear polypropylene cages in groups of four in each cage maintained at 24 50C with 12 hours light and dark cycle with free access to food and water. The animals were acclimatized to laboratory conditions before testing. ExJournal of Pharmacy Research Vol.3.Issue 2.February 2010 219-223

Pemminati S et al. / Journal of Pharmacy Research 2010, 3(2),219-223 periments were conducted between 9 00 to14 00 hours. Each rat was Following the elevated plus maze test, the animal was placed at the used only once. The study was approved by the Institutional Animal centre of the brightly lit arena in the light and dark box. The number of Ethical Committee (IAEC) and the study was conducted according to entries into and the time spent in the bright arena, the number of rears the Indian National Science Academy guidelines for the use and care in the bright and dark arenas and the duration of immobility were noted. Following each trial, the apparatus were cleaned to mask the of experimental animals. odour left by the animal in the previous experiment. Hand operated counters and stop watches were used to score the behaviour of aniDrugs and Chemicals The standard anxiolytic, diazepam (Ranbaxy Pvt ltd., Mumbai) mals. and the test drug, a dry powder of aqueous extract of fruits of Emblica officinalis (Natural Remedies, Bangalore) were suspended in 1% gum acacia solution19. Each drug solution was prepared freshly just before the administration. Drugs and vehicles were administered orally. The doses of each drug were selected on the basis of earlier finding with NR-ANX-C16. Control group of animals were received appropriate volume of vehicle, 1% gum acacia solution. Drugs, dosage and number of animals used per treatment were shown in (Table 1). Statistical Analysis: All results were expressed as mean standard error (SEM) and analysed by one-way ANOVA with drug treatment as the independent factor. Post-hoc comparisons were performed by applying Dunnets test. P <0.05 was considered statistically significant.

Acute Toxicity Test: Acute toxicity study was carried out for EO on healthy swiss albino rats of both sexes of body weight 150-180 gm by using up and Table 1. Groups, treatment and dose down or stair case method. All the animals were housed in polypropyGroups Treatment ( n = 6) lene cages at room temperature, fed on standard pellet diet and water I Control- 1%Gum acacia - 10.0ml/kg ad libitum. Graded doses (0.25,0.5,1.0,2.0 and 4.0 gm/kg) of EO was II Diazepam - 1.0mg/kg III Emblica officinalis 0.8 mg/kg given by orally or i.p. the animals were observed for gross effects IV Emblica officinalis - 2.0 mg/kg continuously for first 6 h and then at 6 hourly interval upto 72 h. V Emblica officinalis 4.0 mg/kg Emblica officinalis was well tolerated by rats in doses upto 4g/kg. At In acute study, drugs/vehicle was administered 60 min prior doses above 4g/kg animals appeared drowsy and dull soon after the to experiment. In chronic study they were administered once daily for EO administration and were active again after 1-3 hours. No mortality 10 days and the last dose was given on the 10th day, 60 min prior to was observed and EO found to be safe at the given doses. experiment. Apparatus Elevated plus maze The wooden maze consisted of two open arms (length 50 cm X breadth 10 cm) and two closed arms of the same size (height 40 cm). The arms of the same type were opposite to each other, with a central square of 10 cm. The maze was elevated to a height of 50 cm above the floor17. RESULTS: a. Elevated plus maze Acute Study:

Diazepam(1.0mg/kg) treated rats showed a significant increase in the number of open arm entries , percentile ratio of open arm to total arm entries and time spent in the open arms. They showed a reduction in time spent in the closed arms.EO at all the doses tested(0.8,2.0 and 4.0mg/kg) siginificantly increased and decreased the time spent in open arms and total arms respectively. In addition, Bright and dark box The apparatus consisted of an open top wooden box. Two the extract significantly increased number of rears in open arms ( at 2 distinct chambers, a black chamber (20 X 30 X 35 cm) painted black and 4mg/kg doses), the number of entries to open arms and the percentile ratio of open arm to total arm entries (at 4mg/kg dose) and illuminated with dimmed red light and a bright chamber (30 X 30 X [Table 2] 35 cm) painted white and brightly illuminated with 100 W white light sources, were located 17 cm above the box. The two chambers were Chronic Study: connected through a small open doorway (7.5 X 5 cm) situated on the Diazepam (1.0 mg/kg) significantly increased the number of 18 floor level at the centre of the partition . entries into and the time spent in open arms and total arm entries. Repeated administration of EO at all the doses tested (0.8,2.0 &4.0mg/ Behavioral assessment. kg) significantly increased and decreased the time spent in open and Each animal was tested initially in plus maze and, then, in closed arms respectively. The extract significantly increased the numbright and dark arena paradigm in a single setting. In acute study 60 ber of total arm entries and the percentile ratio of open arm to total min after and in chronic study 60 min after the last dose on the 10th arm entries ( at 2.0 and 4.0mg/kg doses) and number of entries and day of drug or vehicle administration, each animal was placed in the rears in open arms (at 4.0mg/kg). [Table - 3]. centre square of the plus maze, facing one of the open arms. The number of entries into and the time spent in open and closed arms and the number of rears in each arm in a five-minute period was noted. Journal of Pharmacy Research Vol.3.Issue 2.February 2010 219-223

Pemminati S et al. / Journal of Pharmacy Research 2010, 3(2),219-223 Table 2. Effect of EO on rat behaviour in elevated plus maze (acute study)
Drug groups (n=6) 1%Gumacacia (10.0ml/kg) Diazepam 1.0mg/kg E.O-0.8mg/kg E.O-2.0mg/kg E.O-4.0mg/kg F DF 4,25 Number of open arm entries 1.50.22 3 0.25** 1.3 0.33 2.5 0.34 5.0 0.36** 22.82 Number of total arm entries 5.83 0.30 6.66 0.76 5.1 0.79 6.0 0.57 10.6 0.7** 17.01 Percentage of open /total arm entries 25.31 3.0 46.50 3.68* 43.0 3.40 44.5 8.5 48.3 6.4* 2.90 Time spent in open arm (seconds) 27.66 0.55 106.33 5.80** 63.3 4.41* 84.0 4.50** 101.3 10.90** 23.04 Time spent in closed arm(seconds) 221.66 0.55 150.66 3.28* 200.6 4.41* 195.0 4.50** 172.0 10.90** 23.04 Number of rears in open arms 1.33 0.21 1.5 1.02 1.6 0.41 6.3 0.55** 5.1 0.47** 16.69

(All values are mean SEM; statistical analysis by one-way ANOVA followed by Dunnetts test; *P < 0.05 **P < 0.01 compared with control)

Table 3. Effect of EO on rat behaviour in elevated plus maze (chronic study)

Drug groups (n=6) 1%Gumacacia (10.0ml/kg) Diazepam 1.0mg/kg E.O-0.8mg/kg E.O-2.0mg/kg E.O-4.0mg/kg F DF Number of open arm entries 2.330.21 4.5 0.34* 3.00.57 3.10.75 5.80.75** 9.01 4,25 Number of total arm entries arms entries 6.33 0.49 9.33 0.55* 5.60.84 9.01.15* 9.30.3* 5.71 Percentage of open/total arms(seconds) 32.33 1.15 48.14 1.68 52.27.02 63.07.6* 63.59.08* 3.24 Time spent in open arm(seconds) 40 3.87 125 3.41** 100.35.71* 111.34.5** 119.04.25** 56.98 Time spent in closed 200.0 5.16 145.4 2.88** 180.65.7** 170.74.5** 152.04.25** 56.98 No of rears in open arms 2.83 0.40 5 0.63 4.00.68 5.10.40 6.30.55** 3.49

(All values are mean SEM; statistical analysis by one-way ANOVA followed by Dunnets test ; *P < 0.05 **P < 0.01 compared with control)

Table 4. Effect of EO on rat behavior in bright and dark arena (acute study)
Drug groups (n=6) 1%Gumacacia (10.0ml/kg) Diazepam 1.0mg/kg E.O-0.8mg/kg E.O-2.0mg/kg E.O-4.0mg/kg F DF Number of bright chamber entries 1.00 3.3 0.21** 0.80.30 1.60.3 2.50.42** 12.72 4,25 Time spent in bright chamber (seconds) 14 0.5164 30.33 1.054** 22.83.85* 24.01.36* 27.52.53** 37.60 Number of rears Number of rears in brigh in dark chamber chamber 2.66 0.4216 4.66 0.2108* 1.80.30 2.60.42 6.60.61** 18.72 9 1.506 14.83 1.869* 7.60.84 11.50.80 13.01.36* 5.06 Duration of immobility (seconds) 75 5.774 67.5 7.013 65.03.41 66.13.37 45.82.86** 22.11

(All values are mean SEM; statistical analysis by one-way ANOVA followed by Dunnets test; *P < 0.05 **P < 0.01 compared with control)

Table 5. Effect of EO on rat behavior in bright and dark arena (chronic study)
Drug groups (n=6) 1%Gumacacia (10.0ml/kg) Diazepam 1.0mg/kg E.O-0.8mg/kg E.O-2.0mg/kg E.O-4.0mg/kg F DF Number of bright Chamber entries 1.830.1667 2.66 0.210 2.80.30 2.80.30 5.80.40** 21.34 4,25 Time spent in bright chamber(seconds) 20 0.7303 30 1.155** 26.82.56* 42.30.91** 50.31.83** 59.24 Number of rears in bright Chamber 2.33 0.333 4.83 0.7032** 3.80.40 6.00.44** 7.60.55** 22.48 Number of rears in dark Chamber 6.5 0.4282 9.33 0.667* 11.10.83** 12.11.01** 14.50.61** 22.26 Duration of immobility (seconds) 147.33 3.383 61.33 1.520** 30.30.61** 31.32.60** 22.53.12** 436.84

(All values are mean SEM; statistical analysis by one-way ANOVA followed by Dunnets test; *P < 0.05 **P < 0.01 compared with control)

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Pemminati S et al. / Journal of Pharmacy Research 2010, 3(2),219-223 b. Bright and dark arena Acute Study: Diazepam (1.0mg/kg) treated rats significantly increased the number of entries into the bright arena the time spent and the rears in bright arena. EO at the highest dose tested(4.0mg/kg) significantly increased the number of entries into, time spent and rears in bright arena and decreased the duration of immobility. However increase time spent was significant at all the doses tested (0.8,2.0 &4.0mg/ kg)[Table - 4] Chronic Study: Repeated administration of diazepam (1.0 mg/kg) significantly increased the time spent and the rears in light arena and decreased the duration of immobility. However, increase in the number of entries into bright arena was not significant. EO at all the doses tested(0.8,2.0 & 4.0mg/kg) increased the time spent in bright chamber, number of rears in dark chamber and decreased duration of immobility, The extract also increased the the number of rears at higher doses( 2.0 and 4mg/ kg)and the number of entries in bright chamber at the highest dose, 4mg/kg. [Table - 5] DISCUSSION: The two experimental models of anxiety, elevated plus maze and bright and dark arena, are based on the assumption that unfamiliar, non-protective and brightly lit environmental stress provokes inhibition of normal behaviour. This normal behavioural inhibition is further augmented in the presence of fear or anxiety like state. In the elevated plus maze, the open arms are more fear provoking than the closed arms. The ratio of entries, time spent and rearing behaviour in open arms to closed arms reflects the safety of closed arms with relative fearfulness of open arms 17. The reduction in entry, time spent, rearing in open arms, ratio of open arm to total arm entries and increased defecation are the indications of high level of fear or anxiety. Anxiolytic drugs increase the proportion of entries, time spent and rearing in open arms. They also increase the ratio of open arm to total arm entries. In the bright and dark box paradigm, the brightly lit environment is a noxious environment stressor that inhibits the exploratory behaviour of rodents. Reduction in the number of entries, time spent and rearing behaviour in the light chamber is regarded as markers of anxiety18. Rearing reflects an exploratory tendency20 of the animal that can be reduced due to a high level of fear. An increase in the number of entries to open arm , time spent and rears in open arms, and percentile ratio of open to total arm entries in elevated plus maze paradigm and increase in the number of transition to , time spent and rears in bright arena in bright and dark arena paradigm by the rats treated with standard anxiolytic diazepam suggest the decreased fear, decreased aversion to light and increased exploratory behaviour21. The test compound, Emblica officinalis,also produced the behavioural changes in both paradigms similar to that produced by diazepam indicating that the test drug has behavioural disinhibitory or anxiolytic like activity.The anxiolytic effect of EO was consistent with our earlier report of anxiolytic effect of NR-ANX-C16, in which EO is one of the components. Despite the wide spread traditional use of EO for treating various disorders, there are no reports of any scientific evaluation of its antianxiety effect. Earlier reports on the chemical constituents of plants and their pharmacology suggest that plants containing flavonoids, saponins and tannins possess activity against many CNS disorders 22. Further studies are required to elucidate the possible mechanism of anxiolytic activity and its usefulness in human beings. ACKNOWLEDGMENTS. The authors are grateful to Natural Remedies Pvt. Ltd, Bangalore for providing the aqueous extract of fruits of Emblica officinalis powder. REFERENCES
1. Grundman O., Nakajima J., Seo S., Butterweck V. Antianxiety effects of Apocymum venetum L. in the elevated plus maze test. J Ethnopharmacol 2007;110:406-411. Pari L, Maheshwari JU. Hypoglycemic effects of Musa sapientum L in alloxan induced diabetic rats. J Ethnopharmacol 1999;38:1-5. Vasudevan M, Parle M. Effect of Anwala churna (Emblica officinalis Gaertn.): an ayurvedic preparation on memory deficit rats. Yakugaku Zasshi. 2007; 127(10):1701-1707. Battacharya S, Chaudhuri SR, Chattopadhyay S, Bandyopadhyay SK. Healing Properties of Some Indian Medicinal Plants against Indomethacin-Induced Gastric Ulceration of Rats. J Clin Biochem Nutr 2007;41(2):106114.5. Sabu MC, Kuttan R. Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property. J Ethnopharmacol 2002; 81:155-160. Achilya GS. Evaluation of hepatoprotective effect of Emblica officinalis against carbon tetrachloride induced hepatic damage in rats. J.Ethnopharmacol 2004; 90(2-3):229-232. Yokozawa T, Kim HY, Kim HJ, Okubo T, Chu DC, Juneja LR. Amla ( Emblica officinalis Gaertn.) prevents dyslipidaemia and oxidative stress in the ageing process. Br J Nutr. 2007;97(6):1187-1195. James B Perianayagam., S.K. Sharma., Aney Joseph., A.J.M. Christina. Evaluation of anti-pyretic and analgesic activity of Emblica officinalis Gaertn. Journal of ethnopharmacology 2004; 95:83-85. Sharma S.K., Perianayagam J.B., Joseph A., Christina A.J.M. Anti-inflammatory activity of ethanol and aqueous extracts of Emblica officinalis Gaertn fruits. Hamdard Medicus 2003; XLVI:71-73. Jayashri S et al Phytochemical antibacterial and pharmacological investigations on Mamordica chirantia and Emblica officinalis Ind J Pharm Sci 1993;1:6-13. Sai Ram M., Neetu D., Yogesh B., Anju B., Dipti P., Pauline T., Sharma S.K., Sarada S.K., Ilavazhagan G., Kumar D., Selvamurthy W. Cytoprotective and Immunomodulating properties of amla (Emblica officinalis) on lymphocytes: an in-vitro study. J Ethnopharmacol 2002;81:5-10. Battacharya A., Ghoshal S., Bhattacharya S.K. Antioxidant activity of tannoid principles of Emblica officinalis (amla) in chronic stress induced changes in rat brain. Indian J Exp Biol 2000;38:877-880. Al-Rehaily, A.J., Al-Howiriny, T.A., Al-Sohaibani,M.O., Raftullah S. Gastroprotective effects of AmlaEmblica officinalis on in vivo test models in rats. Phytomedicine 2002;9:515-522. Tripathi B. Charaka Samhita 3rd ed., Chaudhamba Surbharati Prakashan, Varanasi.1994;p.417. Sudhakar Pemminati, V Nair, Dorababu.P, Gopalakrishna HN, Pai MRSM. Effect of aqueous fruit extract of Emblica officinalis on haloperidol induced catalepsy in albino mice. Journal of Clinical and Diagnostic Research 2009;3(4):1657-1662. Herur N.Gopala Krishna, R.B.Sangha, N.Misra, MRSM.Pai. Antianxiety activity of NR-ANX-C, a polyherbal preparation in rats.Indian J Pharmacol

2. 3.

4.

6.

7.

8.

9.

10.

11.

12.

13.

14. 15.

16.

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2006; 38(5):330-335. catalepsy in albino mice. Indian J Pharmacol 2007; 39(2):87-89. 17. Pellow S, Chopin P, File SE, Briley M. Validation of open-closed arm 20. Van der poel AM. A note on stretched attention, a behavioural element entries in elevated plus maze as a measure of anxiety in the rat.J Neurosci indicative of an approach-avoidance conflict in rats. Animal Behav 1979; Methods 1985; 14:149-167. 27:446-50. 18. Costall B, Domeney AM, Gerrard PA, Kelly ME, Naylor RJ. 21. Rang HP., Dale MM., Ritter JN. Pharmacology: Churchill Livingstone; Zacopride:Anxiolytic profile in rodent and primate models of anxiety. J 2003.p.483-494. Pharm Pharmacol 1988; 40: 302-305. 22. Battacharya SK., Satyan KS. Experimental methods for evaluation of psy19. Sudhakar Pemminati, V Nair, P Dorababu, HN Gopalakrishna. Effect of chotropic agents in rodents: Anti-anxiety agents. Indian J Exp Biol 1997; ethanolic extract of leaves of Ocimum sanctum on haloperidol-induced 35:565-575. Source of support: Nil, Conflict of interest: None Declared

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