Chavan Preetam Nivrutti et al. / Journal of Pharmacy Research 2011,4(5),1338-1339 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Comparative study of flavonoids from Chyavanprash for anti-inflammatory activity Chavan Preetam Nivrutti* Department of Pharmacology,Appasaheb Birnale College of Pharmacy, South Shivaji Nagar,Sangli, 416 416. Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011

ABSTRACT Flavonoids being a versatile class of phytoconstituents exhibit several biological activities such as antioxidant, anti-inflammatory, antiallergic, anticarcinogenic, antiviral and antibacterial. Flavonoids from three different preparations (C1, C2, and C3) of chyavanprash were estimated by simple methods and routine quality check. Flavonoids were administered to different groups of albino rats (100 mg/kg, i.p.) and their effect on inflammation by carrageenan induced paw edema was studied. The results were demonstrated at 0 h, 1 h, 2 h, 3 h and 4 h time intervals. The C1 preparation showed more anti-inflammatory activity compared to C2 while C3 showed the least anti-inflammatory activity. Thus, flavonoids showed significant anti-inflammatory activity over a period of time in acute inflammatory models, which is attributed to the constituents of chyavanprash. Also the different preparations of chyavanprash show anti-inflammatory activity at a different extent, which may be due to the variations in their constituents, their number or their quantities.

Key words: Flavonoids; anti-inflammatory; chyavanprash. INTRODUCTION Anti-inflammatory activity of flavonoids is mediated through multiple sites and there may be Animals other cellular action mechanisms still to be elucidated.[1] The anti-inflammatory activity of Wistar rats (Rattus norvegicus) of both sexes weighing 250-300 g were used. The animals flavonoids, in vivo, to some extent, is due to their COX/LO enzyme inhibition.[1,2] Almost were kept and maintained under laboratory conditions of temperature, humidity and light; and every group of flavonoids has antioxidant property thus scavenging free radicals known to were allowed free access to food (standard pellet diet) and water ad libitum. Institutional attract various inflammatory mediators contributing to a general inflammatory response and Animal Ethics Committee (IAEC) having registration no.843/AC/04 CPCSEA has approved tissue damage. Induction of proinflammatory genes usually results from increased activity of a the experimental protocol and care of animals was taken as per guidelines of CPCSEA, subset of transcriptional factors which were inhibited by flavonoids such as inhibition of Department of Animal Welfare, Government of . transcriptional activation of COX-2 and iNOS.[1] Relevance of nitric oxide (NO) as a mediator of inflammation is more recently known.[1,3] Flavonoids demonstrated a dose dependent Carrageenan induced paw edema in rats: suppression of NO production, which acts by stimulating the synthesis of inflammatory The anti-inflammatory activity was determined by carrageenan induced paw edema in rats[10,11] prostaglandins by activating COX-2.[1,4] using plethysmometer (UGO BASILE 7140, Italy) of the standard (aceclofenac) and flavonoids separated from chyavanprash. Wistar strain of albino rats of either sex were divided into five The activity of flavonoids is weak compared with non-steroidal and steroidal anti-inflamma- groups, each consisting of five rats and were treated as follows: tory agents.[1] Chyavanprash manifests the entire human quest for immortality, freedom from disease and prevention of ageing.[5] Effect of flavonoids on lymphocyte proliferation was also Group I : vehicle-treated control group-saline (5 ml/kg, oral) studied in detail.[1,6] Since many companies manufacture chyavanprash, the recipe may differ a Group II : aceclofenac treated standard group (10 mg/kg, oral) bit. The number of used in preparation of the paste varies from 25 to 80, but the main Group III : flavonoids separated from chyavanprash preparation C1 (100 mg/kg, oral) ingredient of all chyavanprash should be amla. Other chief ingredients are dried catkins, Group IV : flavonoids separated from chyavanprash preparation C2 (100 mg/kg, oral) , asparagus, ashwagandha, turmeric, (clarified butter), dehydrated sugarcane and Group V : flavonoids separated from chyavanprash preparation C3 (100 mg/kg, oral) . Even though amla is advertised as the major component of chyavanprash, varieties sold All the doses were given 1 h prior to carrageenan injection. Percentage inhibition was in the market has beet root as the chief ingredient.[7] Chyavanprash contains the pulp of calculated by the formula[12] below and compared with control. Embilica officinalis as the prime ingredient, along with powders and extracts of several other (Vt - V0) control - (Vt - V0) treated herbs.[5] Percentage inhibition = × 100 (Vt - V0) control Chyavanprash pave the way for physical, mental and emotional well-being. It is considered as the supreme anti-stress and anti-aging formulation and is most widely revered of all the Vt = volume of carrageenan injected paw,V0 = volume of normal paw ayurvedic tonics.[8] The need for comparative study defines that more the flavonoidal content Briefly, flavonoids from samples C1, C2 and C3 at a dose of 100 mg/kg each were administered containing plants, more is the anti-inflammatory activity shown, by that preparation. Present orally to rats and the change in paw volumes were determined by four replicate measurements study aims at exploring the anti-inflammatory activity of flavonoids from chyavanprash for carried out at 60 minutes interval by plethysmometer. The anti-inflammatory potency of their potential anti-inflammatory activity. flavonoids were determined by comparing the results with those obtained from animals receiving equal volumes of aceclofenac (10 mg/kg) and control (carrageenan induced 0.2 ml/ MATERIALS AND METHODS 200g).

Drugs and chemicals Statistical Analysis Methanol (75 %) was used as a reflux solvent. Solvent ether and Ethyl acetate were used for The statistical analysis of all the results was carried out using one way ANOVA followed by successive fractionations using a separating funnel. Aceclofenac (Alembic Pharmaceuticals Dunnet’s multiple comparisons test and all the results obtained in the study were compared Ltd.) was used as a standard drug; carrageenan (Research-Lab, Fine chemical Industries, with the vehicle control group. P values < 0.01 were considered statistically significant. Mumbai) was used to induce paw edema. RESULTS Separation of flavonoids The rats treated with flavonoids separated from different samples of chyavanprash showed anti- The chyavanprash (40 g) from three different preparations are refluxed with methanol 75 % (200 inflammatory activity in terms of significant decrease in paw volume in comparison to the ml) for 30 minutes. It was cooled and filtered. The methanolic extracts were reduced to small control group. Decrease in paw volume with flavonoids separated from C1 was more than those volume by evaporation of solvent at low temperature, under reduced pressure. The concen- treated with C2 and C3 (p<0.01) in rats (Table 1). trated extracts were mixed with 100 ml water, extracted thrice with each 50 ml of solvent ether and ether layers were discarded. The remaining aqueous layer was again extracted thrice with each 50 ml ethyl acetate. The combined ethyl acetate extracts were dried by evaporation of Table 1: Effect of flavonoids separated from different samples of chyavanprash on carrag- solvent under reduced pressure. The separated flavonoids were analyzed qualitatively. [9] eenan induced paw edema in rats

Treatment Dose Paw volume (ml) *Corresponding author. 0 h 1 h 2 h 3 h 4 h Chavan Preetam Nivrutti, Control Saline 1.528 ± 0.03421 1.636 ± 0.03847 1.998 ± 0.03194 2.20 ± 0.03162 2.398 ± 0.03899 “Sai Shraddha”, Row House no. 1, (5 ml/kg) Aceclofenac10 mg/kg 1.488 ± 0.05119 1.338 ± 0.03834 0.934 ± 0.03130 0.868 ± 0.01241 0.824 ± 0.01817 Wasan Nagar, Pathardi Phata, C1 100 mg/kg 1.478 ± 0.05215 1.396 ± 0.01949 1.078 ± 0.06099 0.928 ± 0.01068 0.898 ± 0.01304 C2 100 mg/kg 1.458 ± 0.06301 1.434 ± 0.02966 1.214 ± 0.03050 1.038 ± 0.01655 0.94 ± 0.02550 Nashik-422009, Maharashtra. India. C3 100 mg/kg 1.47 ± 0.4416 1.454 ± 0.03050 1.244 ± 0.04037 1.12 ± 0.01140 1.02 ± 0.02702 Journal of Pharmacy Research Vol.4.Issue 5. May 2011 1338-1339 Chavan Preetam Nivrutti et al. / Journal of Pharmacy Research 2011,4(5),1338-1339 Table 2: Percentage inhibition for anti-inflammatory activity of flavonoids separated from ACKNOWLEDGEMENTS different samples of chyavanprash in carrageenan induced rat paw edema I would like to thank the entire department of pharmacology of Appasaheb Birnale college of Treatment Dose Percentage inhibition Pharmacy, Sangli, especially Dr. Smt. N. S. Naikwade and all my batchmates and friends 0 h 1 h 2 h 3 h 4 h whose promising help during the period of study was gracious and overwhelming.

Control Saline(5 ml/kg) - - - - - Aceclofenac 10 mg/kg 14.76 43.43 93.44 99.13 99.48 REFERENCES C1 100 mg/kg 12.67 38.37 81.96 95.07 96.08 1. Jadhav RB, Kharya MD, Plant Flavonoids: A versatile class of phytoconstituents with potential anti- C2 100 mg/kg 16.90 33.72 71.31 87.32 91.30 inflammatory activity, Indian drugs, 42, 2005, 485-495. C3 100 mg/kg 14.08 31.39 68.85 81.69 85.03 2. Harborne JB, The flavonoids: Advances in research since 1980. Chapman and Hall. London, 1988. 3. Lidia S, Fitoterapia, 71, 2000, 48-57. 4. Pant N, Jain DC, Inducible nitric oxide synthase (iNOs) inhibitors from plant, Indian J. Pharma- DISCUSSION ceutical Sciences, 64, 2002, 183-190. Flavonoids being polyphenolic in nature and the derivatives of benzo-?-pyrone show anti- 5. Kasar RP, Laddha KS, Chaudhary J, Shukla A, ‘Chyawanprash-Truth or Mythy’, Pharmacognosy reviews, 1, 2007, 185-190. inflammatory activity and have great hopes for these natural compounds to emerge out as a new 6. Kim HP, Son KH, Chang HC, Kang SS, J. Natural Products, 6, 2000, 170-178. class of anti-inflammatory agents.[1] The anti-inflammatory activity varies according to the 7. http://en.wikipedia.org/wiki/chyawanprash content of flavonoids present in it. The more content of flavonoids the more degree of anti- 8. http://www.ayurvedacollege.com/ThepathtoLongevity.htm 9. Jain UK, Dixit VK, Indian Drugs, 43, 2006, 714-717. inflammatory activity was shown by that sample of chyavanprash. From results it can be 10. Moncada S, Ferreira SH, Vane JR, Prostaglandins, Aspirin-like Drugs and the Oedema of Inflam- viewed clearly that the significant anti-inflammatory activity was C1>C2>C3. mation, Nature, 246, 1973, 217-219. 11. Suleyman H, Odabasoglu F, Aslan A, Cakir A, Karagoz Y, Gocer F, Halici M, Atir Y, Effects of calcium channel blockers on hyaluronidase-induced capillary vascular Permeability, Chyavanprash is a polyherbal formulation made up of extracts of more than 40 crude drugs Phytomedicine, 10, 2003, 552-557. containing a variety of phytoconstituents. Keeping this in view, according to European 12. Asongalem EA, Foyet HS, Ngogang J, Folefoc GN, Dimo T, Kamtchouing P, Analgesic and anti- guidelines, chyavanprash may be considered as ‘Herbal Medicinal Product’ made up of inflammatory activities of Erigeron floribundus, J. Ethnopharm., 91, 2004, 301- 308. multiple numbers of constituents in minute quantities.[9] Thus, from the overall data available 13. Pietta PG, Antioxidative constituents from lycopus lucidus, J. Natural Products, 63, 2000, 1035- 1042. on the biochemical aspects of flavonoids, it appears that they have a great potential to emerge 14. Sadik CD, Sies H, Schewe T, Inhibition of 15-lipoxygenases by flavonoids: structure- activity as a new class of anti-inflammatory agents. A detailed study of comparative antioxidant relations and mode of action, Biochem. Pharmacol., 65, 2003, 773-781. potential of flavonoids has been worked out by many investigators.[13-18] Also from the study 15. Paul C, Mario C, Jean-Bosco S, Jess BB, Kanyanga, C, Luc P, Arnold JV, Dirk VB,Planta Med., 67, 2001, 515-519. it is evident that there is a wide variation in the anti-inflammatory activity of flavonoids 16. Shetgirl P, D’Mello PM, Antioxidant activity of flavonoids: A comparative study, Indian Drugs, 40, separated form different preparations which may be due to the variations in their constituents, 2003, 567-569. their number or their quantities. Thus, there is a need to explore the percentage edema 17. Dugas AJ, Jose Castaneda-Acosta, Gloria CB, Kimberly LP, Nikolaus HF, Wisnston GW, J. Natural Products, 63, 2000, 327-331. inhibition by the flavonoids separated from different preparations of chyavanprash. 18. Kessler M, Ubeaud G, Jung L, Anti and pro-oxidant activity of rutin and quercetin derivatives, J. Pharm. Pharmacol., 55, 2003, 131-142. Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 5. May 2011 1338-1339