Validated HPTLC Fingerprint Analysis for Simultaneous Estimation of Quercetin, Kaempferol and Asiatic Acid in Leaves of Two Different Chemotypes of Centella asiatica C Joshi, J Savai * , A Varghese, N Pandita. Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s NMIMS, Vile Parle (West), Mumbai-400056. India. E-mail ID: jaysavai08@gmail.com ABSTRACT Aim: A High-performance thin layer chromatography (HPTLC) method for the simultaneous estimation of Quercetin, Kaempferol and Asiatic acid in the methanolic extract of leaves of Two Chemotypes of Centella asiatica was developed and validated. Methods: The HPTLC method was developed using a suitable mobile phase Toluene: Ethyl acetate: Chloroform: Formic acid (6:6:4:1). The plates were derivatized with anisaldehyde reagent and densitometric determination was performed at wavelength 530nm. Results: The R f values for Asiatic acid, Quercetin, and Kaempferol was 0.33, 0.44 and 0.55 respectively. The linear regression analysis for the calibration showed a linear relationship with r 2 = 0.9764 and 0.9823 for Quercetin and Kaempferol respectively in the concentration range of 100-1000ng/band. In case of Asiatic acid, the linear regression analysis data for the calibration plots showed a linear relationship with r 2 =0.9996 in the concentration range of 100-500ng/band. Precision was confirmed by performing replicate analysis on a single day and different days. The accuracy of the method was validated by conducting recovery studies using the standard addition method. The average recovery for Quercetin was 98.60% for Kaempferol was 90.08% and for Asiatic acid was 97.66%. System suitability parameter was evaluated by spotting five replicates of all three standards. The %RSD was found to be below 2% with respect to peak area as well as R f value. Robustness of method was evaluated by changing mobile phase concentration from Toluene: Ethyl Acetate: Chloroform: Formic Acid (6:6:4:1) to (5:5:5:1), for which %RSD of peak areas was found to be below 10%. Conclusion: The HPTLC method developed was found to be simple, precise, accurate, suitable and robust for routine quality control of the powder of leaves of Centella asiatica. KEYWORDS: Centella asiatica, Chemotypes, HPTLC, Linear, Precision, Robust. INTRODUCTION Centella asiatica (L.) Urban is dried fragmented aerial parts of herb, belonging to family Apiaceae [1] is a small perennial trailing and slightly aromatic herb with orbicular- crenate leaf on long petiole, found in damp and shady banks of water bodies and other moist places of tropical and subtropical India and Sri Lanka up to 1800 m in hills. It is indigenous to South Africa, southern United States and tropical Africa [2]. Study supported by clinical data has proven that plant can be used in treatment of wounds, burns, and ulcerous skin ailments, and prevention of keloid and hypertrophic scars [3-9]. Extracts of the plant have been employed to treat second and third degree burns [5]. Extracts have been used topically to accelerate healing, particularly in cases of chronic postsurgical and post-trauma wounds [5]. Extracts have been administered orally to treat stress induced stomach and duodenal ulcers [3]. Pharmacopoeias and traditional system of medicines Centella asiatica has reported that Centella asiatica can be used for treatment of leprous ulcers and venous disorders [10, 11, 12, 13, 14]. Studies indicate that Centella asiatica causes regression of inflammatory infiltration of the liver in cirrhosis patients [3, 9]. Centella asiatica has prominent mention in Sushrut samhita- an ancient Ayurvedic medicinal text. However one hurdle in its acceptance globally is the lack of standard quality control profile. Unlike synthetic medicines, it has number of phytoconstituents and it is therefore difficult to maintain their quality. Asiatic acid, Quercetin and Kaempferol are phytoconstituents present in Centella asiatica can be used to assure the authenticity of the plant. C Joshi et al. / International Journal of Pharmaceutical and Biological Research (IJPBR) ISSN : 0976- 285X Vol 3 Issue 5 Oct-Nov 2012 202