www.ijpsonline.com thymol, p-cymene, g-terpinene and other constituents Essential Oil of Thymus serpyllum L. Growing Wild in Vilnius (Lithuania). J Essent Oil Res 2004;16:236-8. of T. vulgaris due to season of harvesting is reported 10. Nickavar B, Mojab F, Dolat-Abadi R. Analysis of the essential oils of from Brazil where higher oil content and oxygenated two Thymus species from Iran. Food Chem 2005;90:609-11. compounds were found in spring[22]. 11. Mathela CS, Agarwal J, Taskinen J. Composition of essential oil of Thymus serpyllum Linn. J Ind Chem Soc 1980;57:1249-50. 12. Verma RS, Rahman L, Chanotiya CS, Verma RK, Singh A, Yadav On the basis of these results it is concluded that A, et al. Essential oil composition of Thymus serpyllum cultivated in harvesting season had considerable effect of essential the Kumaon region of western Himalaya, . Nat Prod Commun oil yield and composition of T. serpyllum growing 2009;4:987-8. 13. Kulevanova S, Ristic M, Stafilov T, Dorevski K, Ristov T. Essential oil in Kumaon region of western Himalaya. Therefore, analysis of some taxa of genera Thymus L. - environmental influences. harvesting of T. serpyllum should be done in summer Bull Chem Technol Macedonia 1996;15:33-8. season to achieve better essential oil yield. 14. Ebrahimi SN, Hadian J, Mirjalili MH, Sonboli A, Yousefzadi M. Essential oil composition and antibacterial activity of Thymus caramanicus at different phenological stages. Food Chem 2008;110:927-31. ACKNOWLEDGEMENTS 15. Loziene K, Vaiciuniene J, Venskutonis PR. Chemical composition of the essential oil of creeping thyme (Thymus serpyllum s.l.) growing wild in Lithuania. Planta Med 1998;64:772-3. Authors thank the Director, Central Institute of Medicinal 16. Raal A, Paaver U, Arak E, Orav A. Content and composition of the and Aromatic (CIMAP), Lucknow, Uttar Pradesh for essential oil of Thymus serpyllum growing wild in Estonia. Medicina providing financial support under Young Scientist Project (Kaunas) 2004;40:795-800. (YSP09.19). 17. Sefidkon F, Dabiri M, Mirmostafa SA. The composition of Thymus serpyllum L. oil. J Essent Oil Res 2004;16:184-6. 18. Paaver U, Orav A, Arak E, Maeorg U, Raal A. Phytochemical analysis REFERENCES of the essential oil of Thymus serpyllum L. growing wild in Estonia. Nat Prod Res 2008;22:108-15. 1. Stahl-Biskup E, Saez F. Thyme. The genus Thymus. London: Taylor 19. Jennings W, Shibamoto T. Qualitative analysis of flavour and fragrance and Francis; 2002. volatile by glass capillary gas chromatography. New York: Academic 2. Mukerjee SK. A revision of the Labiatae of the Indian empire. Rec Bot Press; 1980. Surv India 1940;14:93. 20. Adams RP. Identification of essential oil components by gas 3. Davidson PM, Naidu AS. Phyto-phenols. In: Naidu AS, editor. Natural chromatograph/mass spectrometry. Carol Stream, USA: Allured food antimicrobial systems. Boca Raton FL: CRC Press; 2000. p. Publishing Corporation; 1995. 265-294. 21. Poulose AJ, Croteau R. Biosynthesis of aromatic monoterpenes- 4. Bauer KD, Grabe H, Surburg H. Common fragrance and flavour conversion of gamma-terpinene to para-cymene and thymol in Thymus materials. Weinheim, Germany: Wiley-VCH Verlag; 1997. vulgaris L. Arch Biochem Biophys 1978;187:307-14. 5. Lawrence BM. Progress in essential oils. Perf Flav 1981;23:63-82. 22. Atti-Santos AC, Pansera MR, Paroul N, Atti-Serafini L, Moyna 6. Lawrence BM. Progress in essential oils. Perf Flav 1998;6:27-34. P. Seasonal variation of essential oil yield and composition of 7. Garcia-Martin D, Garcia-Vallejo MC. Chemotypes of Thymus zygis Thymus vulgaris L. (Lamiaceae) from south Brazil. J Essent Oil Res (Lofl.) L., of Guadarrama Sierra and other places in Castile (Spain). 2004;16:294-5. 9th International Congress of Essential oils, Mandarin Court B, Singapore;14-17 March 1983. Accepted 17 March 2011 8. Sajjadi SE, Khatamsaz M. Composition of the essential oil of Thymus Revised 17 February 2011 daenensis Celak. Sp lancifolium (Celak) Jalas. J Essent Oil Res 2003;15:34-5. Received 19 May 2010 9. Mockute D. 1,8-Cineole - Caryophyllene Oxide Chemotype of Indian J. Pharm. Sci., 2011, 73 (1): 233-235

Immunomodulatory Potential of Methanol Extract of Aegle marmelos in Animals

H. V. GOVINDA AND S. M. B. ASDAQ* Department of Phaarmacology, Krupanidhi College of Pharmacy, Varthur Hobli, Chikkabellandur Village, Carmalaram Post, Bangalore-560 035, India

Govinda and Asdaq: Immunomodulatory activity of Aegle marmelos

*Address for correspondence E-mail: [email protected]

March - April 2011 Indian Journal of Pharmaceutical Sciences 235 www.ijpsonline.com

The aim of the current research was to evaluate the immunomodulatory potential of methanol extract of Aegle marmelos in an experimental animal model of cellular and humoral immunity. Administration of methanol extract of Aegle marmelos (500 and 1000 mg/kg, p.o.) and Ocimum sanctum (100 mg/kg, p.o.), produced significant increase in adhesion of neutrophils and an increase in phagocytic index in carbon clearance assay. Both doses of Aegle marmelos prevented the mortality induced by bovine Pasteurella multocida in mice. Moreover, all treated groups demonstrated significant elevation in circulating antibody titre in the indirect haemagglunation test. From the above results, it can be concluded that methanol extract of Aegle marmelos possess immunomodulatory potential by stimulating cellular and humoral immune mechanisms. However, low dose of methanol extract of Aegle marmelos was more effective for augmenting cellular immunity, whereas, high dose was more inclined towards humoral immunity.

Key words: Aegle marmelos, carbon clearance, cellular immunity, haemagglunation test, humoral immunity, mice lethality, neutrophil adhesion

Irreversible unwanted and intolerable effects of Components such as polysaccharides, lectins, proteins conventional drugs and therapies might the underlying and peptide present in plants like Viscum album, reason for an intense research in alternative systems Panax ginseng, Tinospora cardifolia, Aspargus of medicine. Medicinal plants and its products have racemosus, have been shown to stimulate the immune been explored for variety of acute and chronic system[19]. Therefore, the chemical profile may suggest diseases across the globe. A number of ayurvedic that Aegle marmelos would be a good source of formulations containing one or more medicinal plants immunomodulatory agent. However, as of now, no have been exploited for modulation of immune biological study is performed demonstrating the system. Aegle marmelos () is commonly immunostimulatory role of the . Hence, present called Bael, found in the dry deciduous of research work was designed to study the status Himalayas[1]. Traditionally, various parts of the plant of immune system in animals subjected to Aegle are used to treat abdomen pain, palpitation of the marmelos extract using models of cellular and heart and urinary troubles. According to Bhumkas humoral immunity in animals. (local healers) of Patalkot valley in Chhindwara district of Madhya Pradesh, it acts as laxative and Laboratory bred Wistar rats (180-200 g) and febrifuge when taken fresh; it cleans and tones albino mice (20-25 g) of either sex were housed in up the intestines. Root and bark cures intermittent polypropylene cages, maintained under standardized fever. An infusion of Bael leaves is regarded as condition (12 h light/dark cycles, 28±2°) with paddy an effective remedy for peptic ulcer[2]. The plant husk bedding at the central animal house, Krupanidhi is reported to possess antiinflammatory, antipyretic College of Pharmacy, Bangalore, provided with and analgesic[3,4], antidiabetic[5-7], antidiarrhoeal[8], standard pellet food and had free access to purified antihyperlipidemic[9], antifungal[10], antimicrobial, drinking water ad libitum. The guidelines of the antiparasitic[11], anticancer[12], antimalarial[13] and committee for the purpose of control and supervision hepatoproctective activities[14]. It has been reported of experiments on animals (CPCSEA) were followed that a furanocoumarin marmesinin isolated from Aegle and prior permission was sought from the institutional marmelose exerted the protective effect against the animal ethics committee for conducting the present damage caused by experimental myocardial injury[15]. study (KCP/IAEC-25/2008-09).

Environmental pollutants and dietary habits are Aegle marmelos leaves were collected from the reported to influence the activity of immune system fields of Mandya, Karnataka, India. The plant were and diet containing micronutrients and antioxidants identified and authenticated by Regional Research are known to enhance the immune system[16]. From Institute (RRCBI-Mus/06, Bangalore, India). The earlier studies it is evident that, the extract of leaves were given to Phytotech Extracts Pvt. Ltd. Aegle marmelos, by its free-radical scavenging activity (Bangalore, India) to get methanol leaf extract of possess the radioprotective effect in mice[17]. Literature Aegle marmelos (LEAM). Percent yield of the study revealed the presence of many functional and extract was 17% w/w. The extract was subjected bioactive compounds such as carotenoids, phenolics, to preliminary phytochemical analysis. The ethanol alkaloids, coumarins, , terpenoids, and other extract of Ocimum sanctum (Natural remedies) was antioxidants in the leaf extract of Aegle marmelos[18]. used as standard immunomodulatory agent.

236 Indian Journal of Pharmaceutical Sciences March - April 2011 www.ijpsonline.com Leishmann’s stain and gluteraldehyde were bought blood samples. The percent neutrophil adhesion was from Merck (Mumbai, India). Indian ink was procured calculated using the following formula: Neutrophil from Hi-Media (Mumbai, India), whereas, WBC adhesion (%) = (NIu–NIt)/NIu×100, where NIu is the diluting fluid and EDTA were purchased from Nice neutrophil index of untreated blood samples and NIt Chemicals (Cochin, India). Cyclophosphamide is the neutrophil index of treated blood samples. (Endoxan Injection) was taken from German Remedies (Mumbai, India). Pasteurella multocida Swiss albino mice were treated with extract orally of bovine origin and its vaccine were obtained from for 10 days[25,26]. After 48 h of the last dose, all Institute of Animal Health and Veterinary Biologicals the animals of the different groups were given an (Bangalore, India). intravenous injection of (0.3 ml per 30 g) Indian ink via the tail vein. Blood samples were collected from Fresh sheep blood was collected from the local each animal by retro-orbital plexus at an interval of 0 slaughter house. Sheep red blood cells (SRBCs) were and 15 min after the injection. A 50-μl blood sample washed three times in large volumes of pyrogen free was added into the tube containing 4 ml of 0.1% 0.9% normal saline and adjusted to a concentration sodium carbonate solution and the absorbance of this 9 of 0.5×10 cells/ml for immunization and challenge. solution was determined at 660 nm. The phagocytic index K was calculated using the following formula: The acute toxicity study was carried out according to K= (Log OD1-Log OD2)/15 where OD1 and OD2 the up and down or stair case test described in the e e are the optical densities at 0 and 15 min, respectively. fundamentals of experimental pharmacology[20]. The animals were administered test dose of 50 mg/kg Albino mice were orally pretreated with drugs for orally and observed for a period of 24 h for mortality; 21 days in their respective groups[27]. On the 7th subsequent dose was increased by 1.5 factors. The and 17th day of the treatment, the animals were extract was found to be safe upto a dose of 10 g/ immunized with haemorrhagic septicaemic vaccine kg; p.o. According to OPPTS guidelines[21], 1/10th and (HS vaccine) through subcutaneous route. On the 1/20th of the maximum safe dose corresponding to 21st day, the animals were challenged subcutaneously 1000 mg/kg and 500 mg/kg were selected as high and with 0.2 ml of lethal dose (25×LD ) of Pasteurella low doses respectively. 50 multocida (bovine origin) containing 107 cells per ml. The drug solutions were prepared in distilled The animals were observed for a period of 72 h and water for oral administration. Evaluation of the mortality percentage was determined as follows: immunomodulatory effect was carried out by the percent mortality = 100×(number of animals dead)/ following models of cellular and humoral immunity. total number of animals. The animals were distributed into four groups consisting of six animals each. The first group served Animals of all groups were pretreated with the as control (vehicle 1 ml/100 g, p.o.), second group, drugs for 14 days and all animals of each group 9 received the ethanol extract of Ocimum sanctum were immunized with 0.5×10 sheep red blood cells [22] (OSE) at a dose of (100 mg/kg, p.o.)[22], the third (SRBCs) intraperitoneally in their respective group . and fourth groups were administered low (500 mg/ The day of immunization was considered as day kg, oral) and high dose (1000 mg/kg, oral) of LEAM, 0. The treatment was continued for 14 more days respectively. However, in mice lethality, an additional and blood samples were collected from rat at the negative control group was also present. end of the drug treatment and the titre value was estimated by titrating serum dilutions of SRBC The rats were treated orally with vehicle or extracts (0.025×109 cells) in microtitre plates. The plates were for 14 days[23,24]. On day 14, blood samples were incubated at room temperature for 2 h and examined withdrawn from the retro-orbital plexus into visually for agglutination. The minimum volume of heparinized vials and analyzed for differential serum showing heamagglutination was expressed leukocyte count (DLC). After the initial counts, as heamagglutination (HA) titre. The statistical blood samples were incubated with 80 mg/ml of significance was assessed using one-way analysis nylon fibres for 15 min at 37°. Once the incubation of variance (ANOVA) followed by Bonferroni’s was complete, blood samples were again analyzed for comparison test. The values are expressed as TLC and DLC, respectively to get neutrophil index of mean±SEM and P<0.05 was considered significant.

March - April 2011 Indian Journal of Pharmaceutical Sciences 237 www.ijpsonline.com Incubation of neutrophils with nylon fibres (NF) was significantly increased in animals that received showed a decline in the neutrophil counts due to vaccination along with low or high dose of LEAM or adhesion of neutrophils to the fibres. Both doses of OSE compared to animals that received vaccination LEAM and OSE showed significant increase in the alone (Table 2). neutrophil adhesion when compared to control. The low dose of LEAM was found to be more effective In this paper we report that methanol extract of than high dose of LEAM. There was also rise in Aegle marmelos possessed immunomodulatory effect neutrophil count in untreated blood of all treatment in experimental models of cellular and humoral groups (Table 1). immunity in animals. The extract was found to be most effective at low dose (500 mg/kg, p.o.), Both the doses of Aegle marmelos extract and OSE whereas, high dose (1000 mg/kg, p.o.) of LEAM was showed significant increase in the phagocytic index moderately effective in modulating cellular immune when compared to control thus result suggest that system. On the contrary, high dose of (1000 mg/kg, there was increase in the clearance of colloidal carbon p.o.) Aegle marmelos (LEAM) demonstrated better from the blood after administration of these drugs. immunostimulating property than the low dose (500 However, the clearance was best with low dose of mg/kg, p.o.) in humoral mediated immunity. The LEAM and OSE (Table 2). study was carried out using four models of immunity highlighting the different stages of immunity. Mortality was found to be 100% within 72 h in control group upon administration of Pasteurella The marginations of polymorphonuclear lymphocyte multocida. There was 83.33% mortality in vaccinated in the vasculature as well as transmigration of group without any prior treatment of drug. The low neutrophils to the site of inflammation are described dose of LEAM reduced the mortality percentage by neutrophil adhesion test[24]. Both low and high to 66.66% and high dose posses 50.00% mortality doses of LEAM (500 and 1000 mg/kg, p.o.) showed with survival of three animals out of six (Table 3). a substantial rise in the neutrophil adhesion to nylon The haemmagglutinating antibody (HA) titre value fibres. This could be attributed to upregulation of β2

TABLE 1: EFFECT OF LEAF EXTRACT OF AEGLE MARMELOS ON NEUTROPHIL ADHESION TEST Treatment TLC (103/mm3) [A] Neutrophil % [B] Neutrophil index [A×B] Neutrophil UB NFTB UB NFTB UB NFTB adhesion (%) Control 6.6±0.16 6.5±0.16 24.3±0.80 23.5±0.8 160.58±5.4 153.5±4.5 4.4±0.6 OSE 100 mg/kg 7.6±0.18 6.8±0.15 27.6±1.08 19.6±0.4 209.23±6.4 135.0±4.2 35.4±0.6* LEAM 500 mg/kg 7.29±0.97 6.70±0.70 28.16±1.13 19.8±0.54 205.60±10.6 132.2±4.46 35.3±1.43 LEAM 1000 mg/kg 7.71±0.16 6.98±0.11 27.50±1.78 21.6±1.28 211.95±16.4 151.3±10.4 28.3±1.13* All values are expressed as mean±SEM of six observations. *P<0.001 when compared to control. UB: Untreated blood, NFTB: Nylon fiber treated blood; OSE: Ocimum sanctum extract; LEAM: Leaf extract of Aegle marmelos.

TABLE 2: EFFECT OF LEAF EXTRACT OF AEGLE MARMELOS ON PHAGOCYTIC INDEX AND HAEMAGGLUTINATION TITRE Treatment Phagocytic index in carbon clearance assay Haemagglutination (HA) titre Control 0.0096±0.0035 0.0875±0.2562 OSE (100 mg/kg, p.o.) 0.0168±0.0033* 0.0019±0.0003* LEAM (500 mg/kg, p.o.) 0.0143±0.0037* 0.0038±0.0006* LEAM (1000 mg/kg, p.o.) 0.0126±0.0043* 0.0077±0.0015* All values are expressed as mean±SEM of six observations. *P<0.001 when compared to control. OSE: Ocimum sanctum extract; LEAM: Leaf extract of Aegle marmelos.

TABLE 3: EFFECT OF LEAF EXTRACT OF AEGLE MARMELOS ON MICE LETHALITY TEST Treatment Observation 24 h 48 h 72 h Mortality percentage No Drug, No vaccination 2 4 100 No Drug, Vaccination 1 3 1 83.33 OSE (100 mg/kg, p.o.) + Vaccination - 1 3 66.66 LEAM (500 mg/kg, p.o.) + Vaccination - 2 2 66.66 LEAM(1000 mg/kg, p.o.) + Vaccination - 1 2 50.00 OSE: Ocimum sanctum extract; LEAM: Leaf extract of Aegle marmelos.

238 Indian Journal of Pharmaceutical Sciences March - April 2011 www.ijpsonline.com integrins, situated on the membrane of the neutrophils In conclusion, both low dose (500 mg/kg, p.o) as well by that they adhere firmly to the nylon fibres[28]. as high dose (1000 mg/kg, p.o) of Aegle marmelos Hence, it was inferred that LEAM causes stimulation stimulates immune system by acting through cellular of neutrophils towards the site of inflammation. and humoral immunity in experimental models of immunity in animals. However, low dose was The influence of drugs on reticuloendothelial system found to be most effective in cell mediated immune (RES) was assessed by carbon clearance test. The response, whereas, in humoral immunity, high dose reticuloendothelial system (RES) is a diffuse system was best effective. consisting of phagocytic cells. Cells of the RES have a direct influence on the clearance of particles from ACKNOWLEDGEMENTS the bloodstream. When colloidal carbon particles in the form of ink are injected directly into the systemic The authors are thankful to Prof. Suresh Nagpal, Chairman, circulation, the rate of clearance of carbon from the Krupanidhi Educational Trust, Prof. Sunil Dhaminigi, blood by macrophage is governed by an exponential Secretary, Krupanidhi Educational Trust and Dr. Amit equation[26]. Both doses of LEAM as well as OSE Kumar Das, Principal, Krupanidhi College of Pharmacy for showed remarkable augmentation in the phagocytic providing facilities to carry out the work. index due to their ability to increase the activity of the reticuloendothelial system. REFERENCES

The mouse lethality test is one of the commonly 1. Available from: http://www.divineremedies.com/angle_marmelos. htm [Last accessed on 2008 Nov 23]. employed tests to assess serological responses in 2. Acharya D, Shrivastava A. Indigenous Herbal Medicines: Tribal animals immunized with vaccines. Pasteurella Formulations and Traditional Herbal Practices. Jaipur: Aavishkar multocida is pathogenic to mice. The mouse lethality Publishers Distributors; 2008. p. 252-7. 3. Arul V, Miyazaki S, Dhananjayan R. Studies on the anti-inflammatory, test involves injecting mice with the vaccine before antipyretic and analgesic properties of the leaves of Aegle marmelos. J administration of the bacterial culture and determining Ethnopharmacol 2005;96:159-63. the mortality percentage[29]. The vaccination induces 4. Shankarananth V, Balakrishnan N, Suresh D, Sureshpandian G, Edwin E, Sheej E. Analgesic activity of methanol extract of Aegle marmelos humoral immunization. The survival of animals leaves. Fitoterapia 2007;78:258-9. is dependent on the ability of drug to produce 5. Kamalakkannan N, Stanely P, Prince M. Hypoglycaemic effect of adequate number of antibodies, which can counter water extracts of Aegle marmelos fruits in streptozotocin diabetic rats. J Ethnopharmacol 2003;87:207-10. the pathogen. The low dose of LEAM prevented 6. Arumugam S, Kavimani S, Kadalmani B, Ahmed AB, Akbarsha MA, the death of 33.33% of animals at the end of 72 h, Rao MV. Antidiabetic activity of leaf and callus extracts of Aegle and OSE showed only 33.33% mortality, whereas marmelos in rabbit. Sci Asia 2008;34:317-21. high dose of LEAM resulted in survival of 50% of 7. Kesari AN, Gupta RK, Singh SK, Diwakar S, Watal G. Hypoglycemic and antihyperglycemic activity of Aegle marmelos seed extract in animals. Hence it is speculated that LEAM in low normal and diabetic rats. J Ethnopharmacol 2006;107:374-9. dose and OSE carries moderate immunopotential 8. Shoba FG, Thomas M. Study of antidiarrhoeal activity of four in humoral immunity in increasing the number of medicinal plants in castor-oil induced diarrhea. J Ethnopharmacol 2001;76:73-6. survival, while the LEAM in high dose is remarkably 9. Vijaya C, Ramanathan M, Suresh B. Lipid lowering activity ethanolic effective in preventing the mortality. extractof leaves Aegle marmelos in hyperlipidaemic models Wistar albino rats. Indian J Exp Biol 2009;47:182-5. 10. Rana BK, Singh UP, Taneja V. Antifungal activity and kinetics of The confirmation of potency of LEAM on inhibition by essential oil isolated from leaves Aegle marmelos. J antibody-mediated immune response was done Ethnopharmacol 1997;57:29-34. by indirect haemagglutination test. It is mainly 11. Ulahannan RK, Thomas T, Sadasivan C. Antibacterial Action of Leaves of Aegle marmelos. Int J Sci 2008;2:134-8. composed of interacting B cell with antigens and 12. Gangadevi V, Muthumary J. Taxol, an anticancer drug produced by subsequently proliferating and differentiating into an endophytic fungus Bartalinia robillardoides Tassi, isolated from a antibody producing cells. Antibody works by binding medicinal plant, Aegle marmelos Correa ex Roxb. World J Microbiol with antigens and neutralizing it or facilitating its Biotechnol 2008;24:717-24. 13. Elango G, Abdul Rahuman A, Bagavan A, Kamaraj C, Abduz Zahir elimination by cross linking to form latex that is A, Venkatesan C. Laboratory study on larvicidal activity of indigenous more readily ingested by phagocytic cells[25]. The plant extracts against Anopheles subpictus and Culex tritaeniorhynchus. results showed that levels of circulating antibodies are Parasitol Res 2009;104:1381-8. 14. Singh R, Rao HS. Hepatoprotective effect of the pulp/seed of Aegle increased if the test animals are pretreated with Aegle marmelos Correa ex Roxb against carbon tetrachloride induced liver marmelos extract or OSE. damage in rats. Int J Green Pharmacy 2008;2:232-4.

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15. Vimal V, Devaki T. Linear furanocoumarin protects rat myocardium Pharmacol 2003;35:51-4. against lipidperoxidation and membrane damage during experimental 24. Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf myocardial injury. Biomed Pharmacother 2004;58:393-400. MN. Preliminary studies on the immunomodulatory activity of Cedrus 16. Available from: http://www.clevelandclinic.org/health/healthinfo/ deodara wood oil. Fitoterapia 1999;70:333-9. docs/0900/0955.asp?index=5429 [Last accessed on 2007 Sep 21]. 25. Jayathirtha MG, Mishra SH. Preliminary immunomodulatory 17. Jagetia GC, Venkatesh P, Baliga MS. Evaluation of radioprotective activities of methanol extracts of Eclipta alba and Centella asiatica. effect of bael leaf (Aegle marmelos) extract in mice. Int J Radiat Biol Phytomedicine 2004;11:361-5. 2004;80:281-90. 26. Gokhale AB, Damre AS, Saraf MN. Investigations into the 18. Bioactive compounds and volatile compounds of Thai bael fruit immunomodulatory activity of Argyreia speciosa. J Ethnopharmacol (Aegle marmelos (L.) Correa) as a valuable source for functional food 2003;84:109-14. ingredients. Available from: http://www.ifrj.upm.edu.my/galley%20proof/ 27. Ramanatha KR, Lakshminaryana R, Gopal T. Potency test of Duck Suvimol.pdf [Last accessed on 2008 Nov 22]. Pasteurella vaccine in mice. Mysore J Agric Sci 1995;29:155-7. 19. Oladunmoye MK. Immunomodulatory Effects of Ethanolic Extract of 28. Srikumar R, Narayanaperumal JP, Rathisamy SD. Immunomodulatory Tridax procumbens on Swiss Albino Rats Orogastrically Dosed with activity of Triphala over neutrophil functions. Biol Pharm Bull Pseudomonas aeruginosa (NCIB 950) Trends Med Res 2006;1:122-6. 2005;28:139-403. 29. Finco Kent DL, Galvin JE, Suiter BT, Huether MJ. Pasteurella 20. Ghosh MN. Fundamentals of experimental pharmacology. 2nd ed. multocida toxin type D serological assay as an alternative to the toxin Kolkata: J. Sinha Publishers; 1984. neutralisation lethality test in mice. Biologicals 2001;29:7-10. 21. Available from: http://www.epa.gov/oppts/ [Last accessed on 2009 Nov 11]. 22. Sharma M, Kishore K, Gupta SK, Joshi S, Arya DS. Cardioprotective Accepted 18 March 2011 potential of Ocimum sanctum in rats. Mol Cell Biochem Revised 18 February 2011 2001;225:75-83. Received 25 February 2010 23. Fulzele SV, Satturwar PM, Joshi SB, Dorle AK. Study of the immunomodulatory activity of Haridradi ghrita in rats. Indian J Indian J. Pharm. Sci., 2011, 73 (1): 235-240

Simultaneous Determination of Withanolide A and Bacoside A in Spansules by High-Performance Thin-Layer Chromatography

P. B. SHINDE*, P. D. ARAGADE, M. R. AGRAWAL, U. A. DEOKATE1 AND S. S. KHADABADI1 Sitabai Thite College of Pharmacy, Shirur, Pune-412 210, 1Government College of Pharmacy, Amravati-444 604, India

Shinde, et al.: Estimation of Bacoside A and Withanolide A in Spansules

The objective of this work was to develop and validate a simple, rapid, precise, and accurate high performance thin layer chromatography method for simultaneous determination of withanolide A and bacoside A in combined

dosage form. The stationary phase used was silica gel G60F254. The mobile phase used was mixture of ethyl acetate: methanol: toluene: water (4:1:1:0.5 v/v/v/v). The detection of spots was carried out at 320 nm using absorbance reflectance mode. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 200 to 800 ng/spot for withanolide A and 50 to 350 ng/spot for bacoside A. The limit of detection and limit of quantification for the withanolide A were found to be 3.05 and 10.06 ng/spot, respectively and for bacoside A 8.3 and 27.39 ng/spot, respectively. The proposed method can be successfully used to determine the drug content of marketed formulation.

Key words: Bacoside A, HPTLC, simultaneous estimation, withanolide A

Withanolide A is a steroidal lactone present in A is a glycoside present in leaves and stem of dried roots of Withania somnifera (linn.)(Physalis Bacopa monnieri (linn.) or Herpestis monnieri (linn.) somnifera) belonging to family Solanaceae. Bacoside belonging to family Scrophulariaceae[1]. Both the is important plant in the treatment of stress[2,3]. *Address for correspondence Effective formulations are present in market for both E-mail: [email protected] plant extracts individually and also in combination.

240 Indian Journal of Pharmaceutical Sciences March - April 2011