Simultaneous determination of asiaticoside and its novel metabolites in culture of Aspergilus niger by SPE-HPLC

Abstract

Abstracts-When cultured withAspergillus niger, asiaticoside, purified from Centella Asiatica (L.) Urban, was metabolized into four metabolites including one novel alkaloid. A simple and rapid SPE-HPLC method for simultaneous determination of asiaticoside and its four metabolites in culture of A. niger was developed. The chromatographic separation was achieved on a Dikma Diamonsi C 18 ODS column (200×4.6 mm i.d.) by gradient elution with 0.1% phosphoric acid in water and 0.1% phosphoric acid in methanol as the gradient mixtures. The flow rate was 1 mL/min, the detection wavelength was 204 nm and the column temperature was kept at 28 . The retention times of asiaticoside, asiatic acid, M 1 , M 2 , and M 3 were 11.3, 9.4 and 17.1, 19.3 and 7.9 min, respectively. The mean recoveries of five analysts were all over 99%. Quantification limits were 0.02 g/mL for all tested compounds. The method was sucessively applied to the quantification of the biotransformation of asiaticoside. Asiaticoside is one of abundant triterpene glycoside in Centella Asiatica (L.) Urban. Asiaticoside is effective in wound healing [1, 2], burn wound repair [3], lung injury of mice[4, 5] and antidepressant-like effect [6]. Asiaticoside is clinically used as an agent for psoriasis, wound healing, ulceration and eczema in combination with madecassic acid and asiatic acid [7]. Wound and ulcer healing of asiaticoside are enhanced by the promotion of fibroblast proliferation and collagen synthesis [8]. Our previous research showed that, asiaticoside was biotransformed into two aglycones, asiatic acid and one novel alkaloid after 5 days of cultured with Aspgergillus niger (Figure 1). The determination of asiaticoside in Centella Asiatica by HPLC [9,10] and rat plasma by LC-MS-MS [11] and by HPLC [12] had beenreported. But the HPLC combined with solid-phase extraction (SPE) method for simultaneous determination of asiaticoside and its metabolisms, especially the novel alkaloid, has not been reported. In this paper, one simple and rapid SPE-HPLC method was developed and successively applied to quantity the production of metabolisms in the culture of Aspgergillus niger during the biotransformation.