Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract

Betulinic acid (pentacyclic triterpenoid) is an important marker component present in Nelumbo nucifera Gaertn. rhizome. N. nucifera rhizome has several medicinal uses including hypoglycaemic, antidiarrhoeal, antimicrobial, diuretic, antipyretic, psychopharmacological activities.

To establish a simple, sensitive, reliable, rapid and validated high-performance thin-layer chromatography method for estimation of betulinic acid in hydro-alcoholic extract of N. nucifera Gaertn. rhizome.

The separation was carried out on a thin-layer chromatography aluminium plate pre-coated with silica gel 60F(254) , eluted with chloroform, methanol and formic acid (49 : 1 : 1 v/v). Post chromatographic derivatisation was done with anisaldehyde-sulphuric acid reagent and densitometric scanning was performed using a Camag TLC scanner III, at 420 nm.

The system was found to produce a compact spot for betulinic acid (R(f) = 0.30). A good linear precision relationship between the concentrations (2-10 µg) and peak areas were obtained with the correlation coefficient (r) of 0.99698. The limit of detection and limit of quantification of betulinic acid were detected to be 0.4 and 2.30 µg per spot. The percentage of recovery was found to be 98.36%. The percentage relative standard deviations of intra-day and inter-day precisions were 0.82-0.394 and 0.85-0.341, respectively.

This validated HPTLC method provides a new and powerful approach to estimate betulinic acid as phytomarker in the extract.