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 IC 50 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 identifi ed 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).

Antioxidant properties and color of Hibiscus sabdariffa extracts

“…Figure 1 illustrates the anthocyanin separation by HPLC, which occurred in the following order: delphinidin-3-O-sambubioside, delphinidin-3-O-glucoside, and cyanidin-3-O-sambubioside. Hong and Wrolstad (1990) and Hsieh et al (2008) reported chromatograms similar to that depicted in Figure 1, suggesting that delphinidin-3-O-sambubioside, delphinidin-3-O-glucoside, and cyanidin-3-O-sambubioside are the three mayor anthocyanins in Roselle calyces. An area of 22,962,409 units was obtained for the standard cyanidin-3-O-glucoside chloride suspended in ethanol:water (50:50) after the HPLC analysis performed at 520 nm.…”