Taweesak Dhammaraj
Phoobet Kotseekieo
Tunnathon Chotikarn
Narumol Phosrithong
Wattanakarn Praison
Tossapol Prasomsub
Panupong Lumthong
Warangrat Supaporn
Bunleu Sungthong
Chawannuch Mudjupa
Pawitra Pulbutr

Introduction: Xanthine oxidase inhibitors with strong antioxidant activity are promising candidates for the treatment of gout and reactive oxygen species (ROS)-related disorders. ۳,۴,۵-Trihydroxycinnamic acid (THCA), a natural hydroxycinnamic acid, exhibits strong antioxidant activities. This study investigated its xanthine oxidase inhibitory and antioxidant activities in comparison with sinapic acid, caffeic acid, and allopurinol. Methods: In vitro xanthine oxidase inhibitory assay and a Lineweaver-Burk plot were used to measure enzyme inhibition activity and pattern. A docking study was used to explore hydroxycinnamic acid-xanthine oxidase interactions. Antioxidant activity was determined by ۲,۲-Diphenyl-۱-picrylhydrazyl (DPPH) radical scavenging assay. Results: THCA (IC۵۰ = ۶۱.۶۰±۸.۰۰ µM) inhibited xanthine oxidase more potently than sinapic acid and caffeic acid (IC۵۰s = ۱۱۷.۰۰±۴.۰۰ and ۲۱۴.۰۰±۵.۰۰ µM), but less than allopurinol (IC۵۰ = ۲.۸۴±۰.۴۱ µM) (P<۰.۰۵). THCA and allopurinol were competitive xanthine oxidase inhibitors with inhibition constants (Ki ) of ۱۷۰ and ۲.۱۲ µM, respectively. The docking investigation revealed that hydroxycinnamic acids occupied the enzyme active site near the molybdopterin core. THCA formed one more hydrogen bond with the enzyme active site than the other hydroxycinnamic acids, which could account for its higher inhibitory potency. THCA had significantly the strongest DPPH-radical scavenging activity (IC۵۰ = ۱۶.۴۵±۳.۳۵ μM) and higher than ascorbic acid (IC۵۰ = ۳۳.۱۶±۷.۳۸ μM) (P<۰.۰۵). Conclusion: THCA inhibits xanthine oxidase and has good antioxidant properties even more than sinapic acid and caffeic acid. Thus, it is a promising natural active compound that should be further investigated for the treatment of gout and other ROS-related disorders.