Determination of antioxidant constituents in the herb Cissus quadrangularis Linn.

The herb Cissus quadrangularis Linn. has been widely used as antihemorrhoid preparations in Thailand. In order to establish the quality control procedures, both qualitatively and quantitatively determination of the antioxidant constituents of the herb were investigated by using the HPLC method. The antioxidant constituent of the plant was identified by the standard DPPH and ABTS methods, together with the conventional fractionation and isolation process, consequently, quercetin was proposed as a marker for analysis. A quantitative analysis by a RP-HPLC was developed and validated, and the quercetin content in the plant samples collected from various locations and different months in a year were also determined, including the finish tablets product. The main antioxidant, quercetin was found in the ethyl acetate fraction which exhibit the strongest antioxidant properties with the IC50 of 94.54 + 0.62 ug/ml (for the DDPH method) and 34.7 + 0.013 ug/ml (for the ABTS method). The developed HPLC method comprises of the Aquasil C18 column (5 µm, 4.6 x 250 mm id.), the mixture of 0.05% ortho-phosphoric acid: acetonitrile (65: 35) as mobile phase (flow rate 1.0 ml/min), the detector wavelengh at 260 nm and ethyl paraben as internal standard. Validation of the methods were investigated and proved to conform to the ICH guideline criteria on accuracy, precision, specificity, linearity, range, system suitability test (tailing factor, resolution factor). The content of quercetin varies in the range 1.5-13.6 mg% when the plants were collected from six different months and range 0.8-1.5 mg% in three different locations at the same period. The content in finish preparations was found to corresponse to the content in raw material used The developed HPLC method is applicable to the quercetin analysis in plant extract or plant preparation with accurate and precise results.