Characterization of Superoxide Anion Scavenging Compounds in Roselle (Hibiscus sabdariffa L.) Extract by Electron Spin Resonance and LC/MS

Abstract

Roselle (Hibiscus sabdariffa L.) is known to possess strong antioxidative capacity mainly due to its high content of anthocyanins and ascorbic acid. This study aimed to elucidate the ratio of contribution of these compounds in Roselle extract towards superoxide anion scavenging capabilities through analysis using electron spin resonance (ESR) coupled with the hypoxanthine/xanthine oxidase system and DMPO spin trapping technique. The ESR analysis indicated that the IC50 of Roselle extract against superoxide anion was 0.25 mg/ml. After depleting the ascorbate in Roselle extract (0.25 mg/ml) with ascorbate oxidase, the remaining superoxide anion scavenging capacity was determined to be 30.5%, which revealed that ascorbic acid substantially facilitated the scavenging process. By both HPLC and LC/MS analyses, the major active anthocyanins in Roselle crude pigments were identified as delphinidin-3-sambubioside (71.2% of total peak area), delphinidin-3-glucoside (8.7% of total peak area) and cyanidin-3-sambubioside (16.1% of total peak area). Approximately 80% of the superoxide anion scavenging capacities of these anthocyanins was from delphinidin-derivatives (delphinidin-3-sambubioside and delphinidin-3-glucoside); the other 20% was from cyanidin-derivative (cyanidin-3-sambubioside).