Identification of Phorbol Ester and its Degradation by Traditional Purification Method in the Root of Baliospermum montanum (Willd.) Muell. Arg

Abstract

Background Currently five types of phorbol esters are reported in the root of Balliospermum montanum Muell. Here we observed the presence of 12-myristate 13-acetate (C36H56O8) in the root, an additional phorbol ester which restricted its use in food or medicine due to its carcinogenic property. No specific method is reported that can remove or disintegrate the phorbol esters in Balliospermum montanum Muell. root. Aim Identification of 12 myristate13 acetate phorbol ester (C36H56O8) in B. montanum Muell. root and its purification and degradation by traditional method recommended by Acharya Charak.Materials and Methods: The traditional ‘putpak’ (similar to traditional oven method) method was employed to detoxify the raw sample of B.montanum root. Ethanolic extract of root of B. montanum. unprocessed and processed samples were analysed on the Camag HPTLC system. Results The HPTLC analysis gave a band at R f 0.49 corresponding to the standard 12 myristate 13acetate (C36H56O8). Further the band corresponding to the identified phorbol ester (biomarker standard, 12 myristate 13acetate, Sigma Aldrich, USA) was scraped out, the phorbol ester was separated by vortexing with methanol. The resulting sample was then subjected to LC-MS analysis using Impact HD system employing Electrospary Ionization (ESI) in positive ion mode to identify the isolated phorbol ester. Conclusion The LC-MS analysis indicated the presence of this phorbol ester. The present ‘putpak (similar to traditional oven method)’ method of detoxification was able to reduce the identified new phorbol ester by 75% when compared to the raw/unprocessed sample.

Keywords: Phorbol esters; 12 myristate13 acetate; Baliospermum montanum; putpak; Traditional; Detoxification; Purification