Determination of antioxidant constituents in the medicinal plant Cissus quadrangularis Linn

Abstract

Cissus quadrangularis Linn. (C.quadrangularis), or “Phet-SangKhat”, a climbing herb of the Vitaceae family that has been widely used in Thai traditional medicine as remedy for haemorrhoid. The identification and quality control of active principle in raw materials and finished product using as a marker to ensure consistent therapeutic efficacy of the product has never been reported. In this study, the activities (DPPH radical scavenging activity and antioxidant capacity) guided fractionation technique was used to identify the potential active principle in the plant, which will be used as the marker for quality control purpose. A flavonoid was isolated from the most active ethyl acetate fraction from the ethanolic extract of the plant and the compound was identified as quercetin by compare to the authentic quercetin. A quantitative HPLC method for determining quercetin in Cissus quadrangularis Linn. has been developed and validated. The optimum HPLC system was comprised of C-18 reverse phase (Aquasil column 5 um, 250 x 4.6 mm i.d.), isocratic elution with 0.05 % ortho-phosphoric acid and acetonitrile (65 : 35) as the mobile phase and UV detection at 260 nm. The retention time of quercetin is at 10.68 min while ethyl paraben which was used as internal standard is at 13.68 min. Validation of the method was assessed from the specificity, linearity, precision, accuracy and system suitability test. The linearity with R[superscript 2] = 0.9996 was found in the range of 0.3 - 1.5 micorgram / ml for quercetin. The precision of quercetin calculated from the relative standard deviation was less than 2. The mean recovery of quercetin was found to be 97.53 % (% RSD = 1.31) in plant samples. The validated HPLC method has been used to determine the quercetin contents in plant raw materials which were collected in different period of time through out a year (February, April, June, August, October and December) from Phitsanuloke and the contents of quercetin was found as following 10.15, 1.54, 2.54, 3.25, 7.88 and 13.59 mg %. Determination of quercetin in the plant collected from Phitsanuloke, Prachinburi and Prachuapkhirikhan province were found 1.54, 1.88 and 0.83 mg %. In addition, determination of quercetin in 2 lots of GPO finished product was also found 13.45 and 9.95 mg %, respectively