Journal of Chemical, Biological and Physical Sciences

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1 August-October, 2012, Vol. 2, No. 4, e- ISSN: Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online at Section A: Chemical Sciences CODEN (USA): JCBPAT Research Article Quantitative Analysis of Quercetin in Pueraria Tuberosa by Using High Performance Liquid Chromatography Bharti Jain* and Rashmi Raghuvanshi *Department of Chemistry, Sarojini Naidu Govt. Girls Post Graduate (Autonomous) College, Shivaji Nagar, Bhopal , India Received: 5 July 2012; Revised: 20 August 2012 Accepted: 23 August 2012 ABSTRACT A simple, reproducible and efficient reverse phase high performance liquid chromatographic method has been developed and described for the quantitative determination of quercetin the root tuber powder of Pueraria tuberosa. The analysis was done at room temperature with a Promosil C18 column (250mm 4.60mm i.d.,5μm particle size) as stationery phase at a wavelength of 267nm for detection and determination. The mobile phase consisted of water: acetonitrile : acetic acid (90:10:0.2 v/v/v) at a flow rate of 1ml/min.The quercetin content of 66.7% was found in the ethanolic extract of crude Pueraria tuberosa. The proposed method is precise, sensitive, reproducible and easy to perform which can be effectively used for detection, monitoring and quantification of the flavonoid quercetin in Pueraria tuberosa. Keywords: HPLC, Flavonoids, Quercetin, Pueraria tuberose. INTRODUCTION Pueraria tuberosa (Wild.)D.C.(Fabaceae) commonly known as Vidarikand or Indian Kudzu is mainly found in Nepal, Pakistan and India. It is a perennial woody climber which grows upto 6cm tall. The leaves are compound, opposite, trifoliate, orate and coriaceous. Pods are flat, constricted between seeds. The plant perinnates by producing large tubers (up to 10kg) at a depth of 1-2meters. Pueraria.tuberosa is an important and potential medicinal plant in traditional and folklore system 1. In folk medicine the root tuber is applied for blood purification and to improve sperm production. The shade dried root powder controls overgrowth in stomach. The consumption of raw root for one month leads to sterilization in women (Venkata Ratnam, 2006) J. Chem. Bio. Phy. Sci. Sec. A, 2012, Vol.2, No.4,

2 The tribles of Kesla block of Hoshangabad district, Madhya Pradesh have been using tubers of this plant for the treatment of diabetes 2. Quercetin is a plant derived flavonoid used as a nutritional supplement found in fruits and vegetables.. Flavonoids, also collectively known as Vitamin-P and citrin are a class of secondary metabolites. Flavonoids are almost universal pigments of plants 3. They are important parts of Human diet and considered as active principles of many medicinal plants. Quercetin has many medicinal properties like antioxidant, anti tumor, antiviral and anti inflammatory 4. Quercetin is mainly found in many often consumed fruits include green apple, onion, green tea, lemon as well as many seeds, flowers, barks, and leaves. It is also found in medicinal botanicals those are Solanum Trilobatum, Ginkgobiloba, Hypercerium perforatum and in many others. There has been increasing interest in Quercetin in the sports, science and athletic communities due to scientific and clinical research results that show Quercetin has antioxidant, anti-inflamatory, anti cancerous and other properties as likely to improve mental and physical performance 5, 6. Quercetin reduces blood glucose level and improves plasma insulin levels in Alloxan induced diabetic rats 7.Quercetin may have a pharmalogical application in treating cardiovascular disease in diabetes mellitus patients. In the present study HPLC has been used for the quantitative analysis of flavonoid quercetin from the medicinal plant Pueraria tuberosa tubers. MATERIALS AND METHOD Pueraria. tuberosa root tubers were collected from the forest of Kesla block of Hoshangabad district of Madhya Pradesh.The voucher specimens were identified and deposited with the help of local flora and deposited at the department of Botany, Sarojini Naidu Govt.Girls Post Graduate College, Bhopal. Preparation of extract: The collected root tubers were cut in to small pieces, shade dried, powdered and extracted with 250 ml of petroleum ether, ethyl acetate and ethanol using Soxhlet apparatus. The extracts were filtered and concentrated under reduced pressure below 40 o C to dryness. Crude extracts were screened for their photochemical and anti diabetic properties. Photochemical Analysis: Photochemical analysis of Pueraria tuberosa root tuber in different solvent extracts show the presence of different groups of secondary metabolites viz. alkaloid cumarines, flavonoids, terpenoids, anthocyanidins, volatile oils and glycosides, which are of medicinal importance. Of the test extracts, the flavonoids were rich in ethanolic extract. Estimation of quercetin in Alcoholic Fraction by HPLC Chromatographic system: For High Performance Liquid Chromatography system Young Linn. (YL-Clarity 9100) and PDA (UV-Vis) detector equipped with universal injector have been used. The injection volume is 20 µl, The stationary phase is formed by a Promosil C18 column ( mm) with particle size 5µm. (Table.1) Preparation of standard: About 100 mg Standard quercetin was accurately weighted and taken into 100ml volumetric flask and dissolved in mobile phase and make up to the mark. The flask was shaken for 10 min and the volume was made up to the mark with diluent to obtain standard stock solution of Quercetin (1000 µg/ml). Stock solution was filtered through a 0.2 µm membrane filter. The 10ppm working standard solution of quercetin was prepared. Suitable aliquots of stock solution were pipette out and volumes were made up to the mark with mobile phase as diluent. Preparation of sample: To prepare stock solution of sample, 10 gm of accurately weighed alcoholic extract of the tubers of plant Pueraria tuberosa were taken in a 100ml volumetric flask and dissolved in the mobile phase and made upto the mark. From this, the working sample solution (10 ppm) was prepared J. Chem. Bio. Phy. Sci. Sec. A, 2012, Vol.2, No.4,

3 Procedure: After setting the instrument 10 µl of standard solution was injected and chromatogram recorded. From this, the area of chromatogram and the percentage of quercetin content were calculated from the peak areas. Table-1: Chromatographic conditions: Selection of Separation variable Variable Condition Column Dimension. 250mm 4.60mm Particle Size 5µm Bonded Phase Octadecylsilane (C18) Mobile Phase Water (ml) 90 Acetonitril (ml) 10 Acetic Acid (ml) 0.2 Flow rate 1 ml/min Temperature Room temp. Sample Size 20 µl Detection wavelength 267 nm Structure of Flavonoid Quercetin IUPAC Name: 2-(3, 4- dihydroxyphenyl)- 3,5,7- trihydroxy- 4H-chromen- 4-one Molecular Formula: C 15 H 10 O 7 Results: The obtained chromatogram shows a sharp peak at the retention time min. From the peak area µV.s. The percentage of quercetin in the sample, calculated with the help of the chromatogram of the standard quercetin is found to be 66.7% J. Chem. Bio. Phy. Sci. Sec. A, 2012, Vol.2, No.4,

4 REFERENCES. 1. A.Gupta, Quality standards of Indian Medicinal plant,icmr,new Delhi, 2003, pp K. Jain, Ph.D thesis, Ethnobotanical studies in the tribal region of Hoshangabad District (M.P.) with special Reference to Phytochemical Analysis of some Predominate Plant, F. Galeotti, E. Barile, P. Curir, M. Dolci, and V.Lanzotti, Flavonoids from carnation (Dianthus caryophyllus) and their anti fungal. Phytochemistry Letters. 2008;1: Spencer et al., Flavonoids: Modulators of brain function., British Journal of Nutrition.2008; 99: T.P.T. Cushnie,. and A.J.Lamb,., Antimicrobial activity of flavonoids,. International Journal of Antimicrobial Agents,. 2005,26 (5): Sousa et al., Phosphoprotein levels,mapk activities and NF kappa B,expression are affected by fisetin. J.Enzyme Inhib., Med. Chem. 2007,.22 (4): Rashmi Raghuwanshi and Bharti Jain Hypoglycemic effect of Pueraria tuberosa tubers in healthy and Alloxan diabetic Rats, Journal of Chemical, Biological and Physical Sciences Jan.2012 volume (2)Issue J. Chem. Bio. Phy. Sci. Sec. A, 2012, Vol.2, No.4,

5 8. Y.S. Tanwar, S. Goyal,. and K.G. Ramawat, Hypolipidemic effects of tubers of Indian Kudzu, Journal of Herbal Medicine and Toxicology, 2008, (1) B.S. Ashok Kumar, K. Lakshman, K.N.Jayaveera, N. Vamshi Krishna1, M.Manjunath and M.V. Suresh, Estimation of Rutin and Quercetin in Amaranthus viridis Linn by HPLC. 10. K. Venkata Ratnam, Medico-botanical, phytochemical and antimicrobial properties of certain rare and endemic plants from Gundlabrahmeswaram wild life sanctuary, Andhra Pradesh, India Ph.D Thesis S.K.University, Anantapur,2006. *Correspondence Author: Bharti Jain; Department of Chemistry, Sarojini Naidu Govt. Girls Post Graduate (Autonomous) college, Shivaji Nagar, Bhopal , India 1692 J. Chem. Bio. Phy. Sci. Sec. A, 2012, Vol.2, No.4,