Research Article ISOLATION OF STIGMASTEROL AND βSITOSTEROL FROM PETROLEUM ETHER EXTRACT OF AERIAL PARTS OF AGERATUM CONYZOIDES (ASTERACEAE) ANJOO KAMBOJ * , AJAY KUMAR SALUJA 1 Guru Gobind Singh College of Pharmacy, Yamuna Nagar135001 (Haryana), India*, 1 A.R. College of Pharmacy, Vallabh Vidhyanagar, 388120 (Gujarat), India Email: anjookamboj@gmail.com, anjoo73_kamboj@indiatimes.com Received: 24 Sep 2010, Revised and Accepted: 27 Oct 2010 ABSTRACT General phytochemical screening of the aerial parts of Ageratum conyzoides (Asteraceae) revealed the presence of steroids, terpenes, phenolic compounds, saponins, fatty acids, alkaloids. The aim of this study is to identify and characterize the bioactive principle from the aerial parts of the plant. It has wide folk medicinal use. For isolation of the compound, the dried aerial parts powder of Ageratum conyzoides was subjected to hot extraction with petroleum ether; this extract was saponified with alcoholic KOH and subjected to chromatography. Isolated compound were purified by chloroform. The isolation and purification afforded white crystalline powder which was subjected to physical, chemical and spectral identification by IR, 1 H‐NMR, 13 C‐NMR and GC‐MS. The compound was concluded as stigmasterol and β‐sitosterol. Keywords: Ageratum conyzoides, Phytochemical, Stigmasterol, β‐sitosterol, Fatty acids. INTRODUCTION The use of plants and their preparations to treat infectious diseases is an age‐old practice and in the past possibly the only method available. However, the systemic study of plants for detecting antimicrobial activity is of comparatively recent origin 15 . These investigations have been triggered by the emergence and spread of antibiotic resistant microorganisms causing the effective life‐span of existing antibiotics limited. Hence the plant kingdom is being screened for newer and effective chemotherapeutic agents. Higher plants can serve both as potential antimicrobial crude drugs as well as a source of new anti‐infective agents. Ageratum conyzoides is a small herbaceous plant belongs to the family Asteraceae 1,2 . It is softly hairy, erect, branched, annual weed up to 80‐90 cm in height. It is a tropical plant used in various parts of Africa, Asia and South America for curing various diseases. The plant has been reputedly being used as purgative, febrifuge, for opthalmia, colic, treatment of ulcers and wound dressing 3‐5 . The antienteralgic and antipyretic properties of the plant were also indicated in a review on ‘Medicinal plants from Senegal’ 6 . In some African countries, the plant has been popularly use for skin diseases, wound healing, mental and infectious diseases, headaches and dyspnea, used in traditional medicine for its antiasthmatic, antispasmodic and haemostatic effects, uterine troubles, pneumonia by rubbing them on the chest of the patient 7‐10 . Its roots, leaves, flowers and whole plants are used as medicine. A wide range of chemical compounds including alkaloids, coumarins, flavonoids, chromenes, benzofurans, sterols and terpenoids are present in this species. The purpose of this study is to identify and characterize the bioactive principles from the aerial part of Ageratum conyzoides. In this paper, we report the isolation and characterization of known compounds from Ageratum conyzoides namely stigmasterol and beta‐sitosterol. EXPERIMENTAL Collection, Identification and preparation of plant materials The aerial parts of the plant were collected from Herbal Nature Park, Chuharpur, Yamuna Nagar, Haryana in the month September 2007. The plant was taxonomically identified, authenticated by Professor Dr. J. S. Sodhi, HOD, Botany Department, Guru Nanak Khalsa College, Yamuna Nagar, Kurukshetra University, Haryana and deposited with A. R. College of Pharmacy, Vallabh Vidhya Nagar, Gujarat. The aerial parts of the plant were manually separated was air dried, powdered, sieved, weighed and stored in air tight container and subsequently referred to as powdered drug. Extraction and Isolation Powdered (400g) aerial parts of Ageratum conyzoides was defatted exhaustively with petroleum ether (60‐80 o c) in a soxhlet extractor. The solvent was recovered under pressure to obtained dark greenish brown oily mass (5.6g), which was labeled as petroleum ether extracts (PEE) and kept in the refrigerator. The resulting marc was air dried at room temperature and then exhaustively extracted successively with solvents with increased polarity and concentrated under reduced pressure and labeled accordingly. The petroleum ether extract of aerial parts of the plant was saponified using 1M alcoholic KOH, to remove fatty material and then subsequently picked up in petroleum ether and the solvent was evaporated to yield 3g of unsaponified matter. This fraction contains lesser number of components than the unsaponified extract 16 . Chromatographic separation A small quantity of unsaponifiable matter was dissolved in chloroform and this solution is spotted on TLC plates using pre‐ coated aluminium with silica gel 60 F254. Then the TLC plates were run by specific solvent system and viewed individually under UV light and also (5%) sulphuric acid in methanol reagent. Through several pilot experiments it was found that the compounds of unsaponifiable fraction were separated by the solvent system of chloroform and ethanol in the proportion of 9.8:0.2. The chromatograms when developed in iodine chamber yielded six to seven spots respectively and three spots at Rf (0.43, 0.64, 0.95) becomes reddish brown soon turns to purple or violet indicate zones for steroidal nucleus. Column chromatography of PEE was conducted using silica gel (Mesh 60‐120) that was packed using wet packing method in hexane. The column was run using hexane, chloroform and methanol by gradient elution technique. TLC was used to monitor the eluates. A total of 158 eluates were collected. Similar fractions were pooled together. Further purification is carried out using preparative TLC. Spots were identified, scraped and eluates using petroleum ether and chloroform as solvents 15, 16 . Finally eluate ST yielded a single spot when subjected to TLC using several solvent systems including chloroform: ethanol (9.8:0.2), ethyl acetate: ethanol (9.8: 0.2), chloroform: ethyl acetate (4:1) and it showed to be homogenous compound. ST a white crystalline powder (100mg) with melting point (144‐146 o C) was further subjected to IR, Proton NMR (400MHz), Carbon‐13 NMR (100 MHz) and GC‐MS to ascertain the chemical structure. Tests for alcohol 4g of cerric ammonium nitrate was dissolved in 10ml of 2N HNO3, on mild heating. A few crystals of isolated compound were dissolved in International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 3, Issue 1, 2011