Inhibition Kinetics of Sida rhombifolia Extract toward Xanthine Oxidase by Electrochemical Method

Abstract

Uric acid is final product of purine metabolism. Abnormal conditions of uric acid metabolism will cause precipitation of sodium urate crystals in joints, a condition termed gout. Xanthine oxidase (XO) is an enzyme that plays an important role in purine metabolism and functions to catalyze hypoxanthine oxidation into xanthine and, xanthine into uric acid. The well known xanthine oxidase inhibitors (XOIs) is allopurinol, which is one option out of the many synthetic drugs used in modern medicine for the treatment of gout. Nevertheless, the use of allopurinol can cause side effects such as allergies, fever, and gastrointestinal disorders. The side effects of synthetic drug use such as allopurinol have prompted people to turn to traditional medicine that utilizes herbs (medicinal herbs). Sida rhombifolia is one of the traditional medicinal plant with potential as anti-gout. The previous research showed that flavonoids crude extract from this plant could in vitro inhibit the activity of xanthine oxidase up to 71% and the kinetic study by spectrophotometric resulted that the type of flavonoids crude extract inhibition was a competitive inhibition. The purpose of research was to investigate the type of inhibition kinetics of S. Rhombifolia’s ethanol extract by electrochemical method and to compare the study of linearity and sensitivity between electrochemical and spectrophotometric methods. The determination of inhibition kinetics type which is formed can subsequently explain the inhibitory mechanism formed and describe the affinity formed between XO enzyme as a target with drug candidate compounds, whether it is temporary (competitive inhibition and uncompetitive inhibition) or permanent (non-competitive inhibition). The results showed that the yield of S. Rhombifolia’s ethanol extract was 9.82% with the inhibition activity of 13.64% to 82.69% (5.00-200 ppm) and the value of IC50 was 91.15±5.74 ppm. Allopurinol as a control showed the inhibition activity of 15.26-70.95% (0.10-4.00 ppm) and the value of IC50 was 2.45±2.21 ppm. Inibition kinetic of the ethanol extract caused a change of KM (0.187 mM or increase 68.73%) and unchange of Vmax. Based on the data, the type of inhibition kinetic was a competitive inhibition, that had the value of inhibitor affinity (α) of 3.18. The inhibitor affinity value (α) of the enzyme can be determined by calculating the ratio between the value of KM with inhibitors and KM values without inhibitor. Linearity of xanthine oxidase activity assay by electrochemical and spectrophotometric methods showed the range of 0.01-1.00 mM and 0.05-0.70 mM respectively. The sensitivity of electrochemical method was reported higher (0.95 μA mM-1) than the spectrophotometric method (0.007 min-1).