Xanthine Oxidase inhibitors from selected Malaysian medicinal plants as potential remedies for gout

Abstract

Xanthine oxidase (XO) is an enzyme that catalyses the metabolism of hypoxanthine and xanthine to uric acid. It is accountable for the medical condition known as gout due to deposition of uric acid in the joints, causing painful inflammation. Inhibition of XO leads to remission in gout. Malaysia houses abundant of medicinal plants, which can be introduced as new natural sources of gout medication and as alternative to synthetic xanthine oxidase inhibitors (XOI), like allopurinol which brings many side effects. The preliminary screening study of different parts of plant from several plant species revealed that aqueous extract of Carica papaya L. leaves possess promising XO inhibitory activity at a concentration of 100 μg/ml. Thus, response surface methodology (RSM) from Design Expert® v.6.0.8 was used to study the effects of temperature (ºC), time (hour), agitation speed (rpm), and ratio of sample to the solvent (g/ml) on enhancement of XOI production. The analysis of variance (ANOVA) demonstrated that the F-value was 15.07, which implies that the model is significant with low probability value (p<0.0001); having the residuals distributed along a well randomized straight line. Coefficient of determination (R2) was 93.36%. The p-value of 0.2349 for the lack of fit indicated the model has no lack of fit that further validates the model. Statistical optimization helped in developing the process conditions and the percentage of XO inhibition was enhanced to 88.68 ± 1.82%, obtained at 30ºC, 15 hours, 125 rpm and 1g/20ml. The inhibition was less by 5% as compared to allopurinol (93.69 ± 0.2%). Qualitative analysis on the optimized distilled water extract of Carica papaya L. leaves (ODEC) showed the presence of secondary metabolites such as flavonoids, alkaloids, xanthine alkaloids, saponins, anthranol glycosides and terpenoids. Quantitative analysis of ODEC showed the presence of 27.51-33.15% flavonoids, 14.43-16.72% alkaloids and 1.12-1.97% saponins. The ODEC was subjected to reversed-phase flash column chromatography and high performance thin layer chromatography for rapid separation and purification. Fraction EEA1 with Rf value of 0.767 was isolated and demonstrated 95.70 ± 2.57% of XO inhibition, much higher than allopurinol. Partial identification via HPLC on the ODEC and EEA1 against phenolic acids and flavonoids standards revealed the presence of three phenolic acids and four flavonoids. They were caffeic acid, p-coumaric acid, ferulic acid, myricetin, quercetin, kaempferol and apigenin whereas EEA1 has only quercetin (now denoted as purified compound, PC), which could explain the pharmacological properties of this plant and demonstrate its importance in daily intake especially for gout patient. Quercetin demonstrated competitive mode of inhibition against XO. Antioxidant activities were investigated with BHT, a renowned antioxidant used primarily as food additive as positive control. ODEC and PC scavenged 2, 2-diphenyl-1-picrylhydrazyl (DPPH) activity at an IC50 of 2.86 μg/ml and 3.14 μg/ml, respectively. ODEC inhibited β-carotene bleaching at an IC50 of 2.13 μg/ml whereas PC at 2.16 μg/ml. Ferric reducing antioxidant power (FRAP) of ODEC was 1146.89 ± 44.24 μmol FeSO4.7H2O/g dry, 1.8-fold higher than unoptimized extract (UODEC), 1.3-fold higher than PC and 1.5-fold higher than BHT. However, ODEC and PC exhibited minimal anti-diabetic activities. The data grants imperative breakthrough to discover natural XOI from plants and the findings are encouraging to plan clinical studies in hyperuricemic patients, and in the formulation of nutraceuticals or pharmaceuticals, or cosmeceuticals products incorporating natural-based XOI.