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Estimation of Quercetin in Acacia Catechu Ethanolic Bark Extract by HPLC Method

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Estimation of Quercetin in Acacia Catechu Ethanolic Bark Extract by HPLC Method International Journal of PharmTech Research CODEN (USA): IJPRIF ISSN : 0974-4304 Vol.4, No.1, pp 501-505, Jan-Mar 2012 Estimation of Quercetin in Acacia catechu Ethanolic Bark Extract by HPLC method Anitha Roy1, Lakshmi. T.*1, Geetha R.V.2 1Faculty of Pharmacology, Saveetha Dental College, Chennai,India. 2Faculty of Microbiology, Saveetha Dental College, Chennai,India. *Corres. author: [email protected] Abstract: High-performance liquid chromatography (HPLC) is a powerful analytical technique because of its reliability, simplicity, reproducibility, and speed. The aim of this work was to develop an accurate, specific, repeatable and robust method for the estimation of Quercetin content in Acacia catechu ethanolic bark extract. The method was validated in compliance with International Conference on Harmonization guidelines. The data obtained from our study conclude that the amount of Quercetin present in Acacia catechu ethanolic bark extract is 0.070 % w/w. Keywords: Acacia catechu Bark, Quercetin, HPLC, ICH guidelines. INTRODUCTION Quercetin is a plant pigment found in many foods such white and heart wood is small and red in colour. The as onions, apples, berries, tea, grapes and red wine. It's leaves, bark and heartwood have many nutritional and not a nutrient, but is classified as a flavonoid. Like many medicinal uses. [3,4] other plant chemicals, it is sold as a supplement. Oral The chief constituents of the plant are catechin and quercetin is relatively well absorbed, and it is [5] [5] catechutannic acid. The wood contains epicatechin , metabolized mainly to isorhamnetin, tamarixetin and [1] Atzelchin, catechin tetramer, dicatechin, gallochin, kaempferol. It often occurs in plants as glycosides, [ 6,7,8] gossypetin, phlobatannin, kaempferol, quercetin . such as rutin (quercetin rutinoside) in tea. Quercetin and Catechin is biologically highly active. It is used as a rutin are used in many countries for blood vessel health haemostatic. Taxifolin an other important constituent and are ingredients of numerous multivitamin has antibacterial8, anti-fungal , antiviral, anti- preparations and herbal remedies .Quercetin posses [6] inflammatory, and antioxidant activity . antioxidant, anti inflammatory, anti cancer, anti microbial property. The dry skin of wild onion is used The extract of Acacia catechu extract have been as a yellow dye, it contains Quercetin, which is an anti reported to have various pharmacological effects like allergic agent and is helpful in treating IBD.[2] immuno modulatory13, anti pyretic [ 7], hypoglycaemic, hepatoprotective activity. [7, 11] Cutch is astringent, Acacia catechu (AC) (Family: Fabaceae, subfamily: [9, cooling, and digestive. It is useful in cold and cough Mimosoideae) known as Black cutch.AC is medium 10] ulcers, boils and eruptions of the skin, bleeding piles, sized thorny deciduous tree mainly found in India and uterine haemorrhages, atonic dyspepsia, chronic also found in deciduous forests around the world. It bronchitis etc. In stomatitis, halitosis, dental caries and grows up to 13 meters in height. The foliage is softly [16] cavities. The bark is anthelmintic, antipyretic, textured; light green and oval-shaped. The branches are antiinflammatory and antileprotic. Bark and cutch are thin and spike like due to tiny thorns grown around the antidiarrhoeal, astringent and stomachic. Catechu or exterior. The sap wood of AC is large and yellowish Lakshmi.T et al /Int.J.PharmTech Res.2012,4(1) 502 Cutch tree bark is useful in melancholia, conjunctivitis stated CTO-10AVP column oven compartment, an and haemoptysis.[17] auto sampler, and a SPD-M10AVP diode array detector and Rheodyne 7725i injector with 20 μl Our literature survey revealed that there are no loop volume and a flow rate of 1.0 ml/min. The scientific studies carried out regarding the output signal was monitored and processed using phytochemical activity of Acacia catechu ethanolic SPI Software LC Solutions, Version 1.21 on a Bark extract compared to standard Quercetin by Pentium computer (Hewlett Packard). All analyses HPLC method. Hence in our present study the were performed at a ambient temperature of 37ºC, Ethanolic Bark extract of Acacia catechu were with a mobile phase of orthophosporic acid examined for its presence of Quercetin content in it /Acetonitrile, which is responsible for its pharmacological actions. The analysis was performed using a high- performance liquid chromatographic system an MATERIALS AND METHODS injection volume of 20 μl, and a flow rate of 1.0 Extract and Chemicals ml/min. The UV absorbance of the eluent was The Ethanolic Bark extract of Acacia catechu willd and measured at 350 nm. Quercetin standard was obtained from Green Chem, The developed method was validated according to Herbal Extracts & Formulations, Bangalore. Acetonitrile International conference on Harmonization and methanol HPLC grade solvents; all analytical grade guidelines (ICH).[19] solvents obtained from E-Merck Ltd, Mumbai, India. Potassium dihydrogen orthophosphate Analytical Chromatography Reagent grade was procured from Qualigens Fine HPLC Column C18 ODS thermo Chemical, Mumbai, India. (250 x 4.6 mm) 5μm Wavelength 350 nm Instrumentation Injection volume 20 μl The output signal was monitored and processed Flow rate 1.0ml/min. using LC Solutions Version 1.21 SP1 software on a Mobile phase Solvent A-0.1 % Pentium computer (Hewlett Packard). orthophosphoric acid Preparation of Standard Solution Mobile phase ratio Solvent B-Acetonitrile The standard was prepared by accurately weighing 80:20v/v about 10 mg Quercetin in 10 ml of methanol in a volumetric flask. It was then sonicated for 10-15 minutes .The contents of flask were filtered through HPLC ANALYSIS Whatman no.41 paper (Merck, Mumbai, India) to attain the stock solution of 1 mg/ml. Equation 1 Calculation of Assay of Quercetin Preparation of Sample Solution (Std Wt / Sample Wt) x (Sample Area / STD Area) 500 mg Sample (Acacia catechu Bark extract) is x Assay of Standard = Assay of Sample. accurately weighed and dissolved in 50 ml of methanol in a volumetric flask. It was then Calculation of assay of Quercetin in Acacia sonicated for 10-15 minutes then the contents of the catechu Bark (Ethanolic) Extract flask were filtered through Whatman No. 41 paper Sample Weight taken : 10.02mg (Merck, Mumbai, India) to attain the stock solution Area of sample : 410112 of 10 mg/ml. Standard Weight taken : 1 mg Area of Standard : 5754965 Methodology Assay of Quercetin : 100% High-performance liquid chromatography [18] method development Assay of Quercetin The separation was performed by using column C18 (1.0 /10.02) x (410112 /5754965) x 100 =0.070% ODS thermo 250 × 4.6 mm, 5 μm on a Shimadzu w/w. class LC(California ,USA), which consisted of a FCV-10 ACVP pump, DGU-14A degasser, a thermo Lakshmi.T et al /Int.J.PharmTech Res.2012,4(1) 503 CHROMATOGRAM ANALYSIS GRAPH 1 ESTIMATION OF STANDARD (QUERCETIN) Sample Name : QUERCETIN/STD/100% Vial : 1 Injection Volume : 20ul TABLE 1 –CHROMATOGRAM ANALYSIS OF QUERCETIN Lakshmi.T et al /Int.J.PharmTech Res.2012,4(1) 504 GRAPH 2 ESTIMATION OF QUERCETIN CONTENT IN ACACIA CATECHU ETHANOLIC BARK EXTRACT Sample Name : Acacia catechu ethanolic Bark extract Vial : 2 Injection Volume : 20ul TABLE 2 –CHROMATOGRAM ANALYSIS OF ACACIA CATECHU ETHANOLIC BARK EXTRACT RESULT AND DISCUSSION Equation 1 .The retention times observed for the Flavonoids like Quercetin are a group of polyphenolic authentic sample of Quercetin and those of Acacia compounds, which possess many biochemical effects like catechu ethanolic Bark extract are given in Table 1 and inhibition of enzymes, regulatory role on different 2. hormones and pharmacological activities like The present study reveals that the antioxidant, antimicrobial, antioxidant, and anticancer, antihepatotoxic, antimicrobial, antiinflammatory, anticancer activity is protection of cardio vascular system. not only due to presence of phytochemical constituent Acacia catechu willd contains many active like Catechin and Epicatechin, it is also due to the constituents in it like Catechin, Epicatechin, Quercetin, presence of Quercetin which possess lots of medicinal Taxifolin etc., the HPLC studies revealed that the plant value, since antiquity . extract were also rich in Quercetin (0.070%w/w), the assay of Quercetin was calculated according to the Lakshmi.T et al /Int.J.PharmTech Res.2012,4(1) 505 CONCLUSION present is 0.070%w/w in Acacia catechu ethanolic Bark extract determined by HPLC method. Herbal medicines have been used for many years. Phytomedicine with medicinal properties are a useful ACKNOWLEDGEMENT and effective source of treatment for various diseases. The authors are thankful to Mr.Rajendran.R, Green Many of the common drugs used by us today are Chem PVT LTD. Bangalore for providing us the derived from herbs. Screening of medicinal plants for Ethanolic extract of Bark of Acacia Catechu & phytochemical analysis are important for finding Quercetin as a gift sample for our research Study. potential new compounds for therapeutic use .Hence our present study concludes that the Quercetin content REFERENCES 1. Quercetin supplements benefit and side effects by Indian Journal of Pharmacology, 29(6), 1997, 426- Ray Sahelian, M.D. A flavonoid used for prostate, 428 allergy and blood vessel health and as an 12. Nagaraja T.G , S.V. Sarang and D. C. Jambhale antioxidant Evaluation of anti-mycotic activity of Acacia http://www.raysahelian.com/quercetin.html catechu Willd.(Mimosaceae) Anti-mycotic activity of Acacia catechu Journal of Biopesticides, 2. Ashok Kumar et al Estimation of rutin and 1(2):197 - 198 (2008) Quercetin in Amaranthus viridis by HPLC 13. Syed Ismail and Mohammed Asad., Ethnobotanical leaflets 13:437-42, 2009. Immunomodulatory activity of Acacia catechu. 3. Anonymous, the Wealth of India, Raw Material, Indian journal of Pharmacology 2009:53 (1), 25 - Vol 1, CSIR, New Delhi, 2004, 11 33 4. Lakshmi.T Black cutch (Acacia catechu willd) -A 14. Ammara Hassan, Salma Rahman, Farah Deeba and potent medicinal plant INTERNATIONALE Shahid Mahmud. Antimicrobial activity of some PHARMACEUTICA SCIENCIA vol 1 issue 2 plant extracts having hepatoprotective effects April-june 2011 .Journal of Medicinal Plants Research Vol.
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