Original Article IDENTIFICATION, QUANTIFICATION AND VALIDATION OF β-SITOSTEROL FROM HOLOPTELEA INTEGRIFOLIA (Roxb.) PLANCH USING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY METHOD RAVINDRA C. SUTAR 1* , SANJAY B. KASTURE 1 , V. K. KALAICHELVAN 2 1 Department of Pharmacology, Sanjivani College of Pharmaceutical Education and Research, Kopargaon.At-Sahajanandnagar,Post- Shinganapur (Pin code-423603), Tal- Kopargaon, Dist-Ahmednagar, Maharashtra, India. 2 Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram (Pin code- 608002), Tamilnadu, India. Email: ravi_sutar1980@yahoo.com Received: 12 Mar 2014 Revised and Accepted: 02 Apr 2014 ABSTRACT Objective: The Present study was designed to develop a new simple, precise, rapid and selective high‐performance thin‐layer chromatographic (HP TLC) method for the determination of beta‐sitosterol in methanolic leaf extract of holoptelea integrifolia (Roxb.) Planch. Methods: As per ICH guidelines we have applied different concentrations of beta‐sitosterol as standard on HPTLC plates for the quantification of beta sitosterol from the plant extract. Concentration of standard beta‐sitosterol was 0.1µg/µl Results: The retention factor of beta‐sitosterol was 0.38. Linearity was obtained in the range of 100 ng‐500 ng for beta sitosterol. The developed and validated HPTLC method was employed for beta‐sitosterol in methanolic leaf extract of holoptelea integrifolia (Roxb.) Planch for Standardization of the content of the marker. Satisfactory recoveries of 85.0 ‐86.0 % were obtained for beta‐sitosterol. Conclusion: The results obtained in validation assays indicate the accuracy and reliability of the developed HPTLC method for the quantification of beta‐sitosterol in methanolic leaf extract of holoptelea integrifolia (Roxb.) Planch. Keywords: holoptelea integrifolia (Roxb.) Planch, Beta‐sitosterol, HPTLC INTRODUCTION Nature has blessed mankind with a treasure of medicinal plants. Natural products have always remained a profile source for the discovery of new drugs and are used since Vedic period [1]. Ayurveda has been a vibrant system of health care in India and has been practiced since 6000 years but growth as an industry has commenced only a few years back. India’s share in the global exports of herbal medicines is at around 10 per cent only which is low. Therefore, there is a need to transform Ayurveda into a dynamic, scientifically validated and proof based industry which takes its roots from rich knowledge base of old tradition and solemn [2‐4]. It is necessary to develop methods for rapid, precise and accurate identification and estimation of active constituents or marker compound/s as the qualitative and quantitative target to assess the authenticity and inherent quality [5,6].Through various analytical techniques like TLC, HPLC and HPTLC we can ascertain the presence of these compounds in plants and also quantify them. HPTLC offers many advantages over other chromatographic techniques such as unsurpassed flexibility (esp. stationary and mobile phase), choice of detection, user friendly, rapid and cost effective [7]. Thus, HPTLC is most widely used at industrial level for routine analysis of herbal medicines. Holoptelea Integrifoila belongs to the family ulmaceae commonly called as Indian Elm and commonly used in India by the tribal people for it’s medicinal properties. The mucilaginous bark is boiled and the juice squeezed out and applied to rheumatic swellings[8]. In traditional system of medicine, bark and leaves of Holoptelea Integrifoila used as bitter, astringent, acrid, thermogenic, anti‐inflammatory, digestive, carminative, laxative, anthelmintic, depurative, repulsive, urinary astringent and in rheumatism [9,10]. The plant Holoptelea integrifolia is used traditionally for the treatment of inflammation, gastritis, dyspepsia, colic, intestinal worms, vomiting, wound healing, leprosy, diabetes, haemorrhoids, dysmenorrhoea and rheumatism[11,12]. By considering the demand of this plant, there is a need of simple and rapid analytical method. for the manufacturer of plant based medicines. Thus, the objective of the present work was to Identify, Quantify and validate a High Performance Thin Layer Chromatography method For estimation of the biomarker beta‐sitosterol present in Holoptelea Integrifolia (Roxb.) planch. MATERIALS AND METHODS Materials Standard and reagents β‐sitosterol (purity 98%), was purchased from Sigma‐Aldrich Chemie GmbH (Aldrich, Division, Steinbeim, Germany). Ethyl acetate, toluene, glacial acetic acid, methanol and sulphuric acid used in the present research work were of HPLC grade and were procured from E. Merck Mumbai, India. Plant material The leaves of Holoptelea Integrifolia (Roxb.) planch were collected in the Month of August from the agricultural fields of Tirunelveli distri ct, Tamil Nadu, India. The plant was identified and leaves of Holoptel ea Integrifolia were authenticated and confirmed from Dr.V.Chelladu rai, Research Officer, Botany, C.C.R.A.S. (Retired), Govt of India by co mpairing morphological features (leaf and stem arrangement, flower /inflorescence arrangement, fruit and seed morphology etc.). The leaves of Holoptelea Integrifolia (Roxb.) planch ., were dried in a preset oven at 45 o C, and powdered using motor and pestle and then sieved through BSS mesh size 85 and stored at 25 o C, in an airtight container. HPTLC instrumentation Chromatographic conditions The sample solutions were spotted in the form of bands of width 8.0 mm with a Camag microlitre syringe on precoated silica gel aluminium plate 60F254 (20 cm × 10 cm with 250 μm thickness; E. Merck, Darmstadt, Germany, supplied by Anchrom Technologists, Mumbai) using a Camag Linomat V (Switzerland). The plates were prewashed by methanol and activated at 120°C for 20 min International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Issue 5, 2014 Innovare Academic Sciences