Optimization of Extraction Conditions using Response Surface Methodology and HPTLC Fingerprinting Analysis of Indian Propolis

Abstract

The potential of Indian propolis as a suitable therapeutic agent is still to be explored to the fullest. As variation in the chemical composition across different geographical regions has been a major concern, it is, therefore, worthwhile to systematically investigate, and assess the quality of Indian propolis and set nationalized standards. The propolis collected from three different regions were authenticated for the major plant source, followed by the evaluation of physicochemical properties. Different extraction methods were compared, with their influence on balsam content, polyphenol, and flavonoid content. The sample coded HAR using ultrasonication gave the highest extraction yield of 71.87%. Further, the process variables in the ultrasonication method (i.e. Amplitude, extraction time, the concentration of ethanol) at three levels were optimized using the Box-Behnken response design. The model was found significant with the concentration of ethanol having a maximum influence on the responses. The model was successfully verified by setting the amplitude at 48%, extraction time to 5.6 mins, and ethanol concentration to 87% v/v. The extraction efficiency of 83.86±1.17% was achieved with a substantial diminution of extraction time. The total phenolic, total flavonol, and total flavanone content of the optimized batch were 28.74±0.13 mgGAE/g, 4.66±0.78 mgQE/g, and 3.16±0.14 mgPE/g of propolis respectively. Further HPTLC fingerprint image of the optimized extract revealed the presence of quercetin, p-coumaric acid, kaempferol, chrysin, pinocembrin, and galangin.