Simultaneous Method Development and Validation of Berberine, Rubiadin and 3-O-acetyl-11-keto-β-boswellic acid By Reverse Phase High Performance Liquid Chromatography In Polyherbal Formulation

Abstract

Berberine, Rubiadin and 3-O-acetyl-11-keto-β-boswellic acid (AKBA) are reported mainly for their anti-inflammatory activity among other uses like antidiarrheal, anti-fungal, antitumor, anti-oxidant, anti-diabetic and immunomodulation. The polyherbal gel formulation contains hydrolalcoholic extracts of Berberis aristata DC., Rubia cordifolia L., Boswellia serrata Roxb. And it is claimed for anti-inflammatory and anti-bacterial activity. Hence, the current work was planned to estimate Berberine, Rubiadin and AKBA in the polyherbal formulation. A Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method was planned for the simultaneous estimation of Berberine, Rubiadin and AKBA in accordance with International Council for Harmonisation (ICH) guidelines. The chromatographic separations of the active ingredients were achieved in the C18 column (250×4.6 mm;5 μm), the mobile phase consisted of acetonitrile: phosphate buffer pH 3.5 (20:80 %v/v), the flow rate was adjusted to 1 ml/min and Ultra Violet (UV) detection was carried out at 261 nm. The retention time for Berberine, Rubiadin and AKBA was 6.76, 8.37 and 5.76 min, respectively. The detector showed linear responses for all three compounds in the concentration range 1-5 µg/ml, which had a good correlation coefficient (0.996). The developed method was also validated as per ICH guidelines. This RP-HPLC method is simple, precise, sensitive and accurate. It can be used for quality control of raw materials and evaluation of herbal formulations containing Berberine, Rubiadin and AKBA.