Development of a new analytical method for determination of Asiaticoside content in Centella Asiatica

Abstract

Asiaticoside, an active triterpene glycoside in Centella asiatica L., has weak UV absorption due to the lack of chromophore in its structure, and this causes an ineffective asiaticoside analysis by standard UV-Vis HPLC. In this study, a simple TLC-densitometic method was developed for rapid and effective analysis of the triterpenoid glycosides in C. asiatica crude extracts and its commercial products. This new analytical method is based on the derivatization of asiaticoside structure with 2-naphthol acid reagent on a TLC silica gel plate to form a chromophore-containing compound that can be detected by UV-visible based TLC-densitometric analysis (λmax at 530nm). In practice, crude C. asiatica extracts were prepared from the plant materials under sonication with 80% (v/v) methanol. The analysis was then performed on a silica gel 60 F254 TLC plate (20x10 cm.) with chloroform/methanol/water (30:15:1.2 volume ratio) system as the mobile phase. Densitometric analysis is performed at 530 nm after post-chromatographic derivatization with 2-naphthol sulfuric acid reagent (brownish band for glycoside). This new method showed good accuracy comparable to the UV-HPLC method but is more sensitive and effective. The linear range for the analysis of asiaticoside was 100-1000 ng/band (r2≥0.99) with good precision and accuracy (1.15- 1.9 %RSD, 98-104% recovery) and the values of asiaticoside contents determined by the developed TLC method and HPLC method were well correlated in range 99-101%. The developed densitometric TLC appeared to be simple, accurate, precise and fast as eighteen chromatographic runs could be performed simultaneously per plate within 15 minutes.