Formulation and characterization of hydrogel containing asiaticoside fraction for minimizing hyperpigmentation

Abstract

Centella asiatica (L) Urban bioactive component, asiaticoside is known to exhibit many therapeutic applications, especially wound healing. Few studies found that asiaticoside affected melanogenesis pathway by inhibiting the activity of microphtalmia-associated transcription factor (MITF) which regulates the enzymes responsible for melanogenesis. This study aimed to develop new fractionation method using activated charcoal for asiaticoside fraction from C. asiatica, evaluate the bioactivity on melanogenesis of the fraction, as well as the optimisation and characterisation of 3:2 (methanol:dichoromethane) fraction from C. asiatica (CAEMD) hydrogel. Thin layer chromatography (TLC) was used to detect the presence of asiaticoside in CAEMD fraction. The quantification of asiaticoside was done by high performance liquid chromatography (HPLC) using water-acetonitrile (75:25, v/v) as mobile phase. LC-DAD-ESI-ITMS in positive ion mode was also adopted to further confirm the presence of asiaticoside in CAEMD fraction. TLC, HPLC, and LC-DAD-ESI-ITMS analysis were successfully identified and quantified the asiaticoside in CAEMD fraction. The presence of asiaticoside was detected in CAEMD fraction with Rf value of 0.6 and HPLC showed that the concentration of asiaticoside found in CAEMD was 1126.86 µg/mL which was 11.27 % of the CAEMD fraction. The amount of asiaticoside in CAEMD fraction was increased by more than two-fold compared to the crude extract of C. asiatica. In LC-DAD-ESI-ITMS, asiaticoside was found to be the second most abundant compound in CAEMD fraction and several fragments were produced at m/z 453.33, 471.08, 489.33, 635.17, 797.17, 813.17, 959.17, and 976.25. The main fragment ions formed were at m/z 976.25 and m/z 453.33 which might derived from [M+NH4]+ and [M+H-Glu-Glu-Rha-2H2O] respectively which indicate ammonium adduct and loss of glycoside moiety in asiaticoside molecule. The anti-tyrosinase activity of CAEMD fraction had increased by two-fold compared to the crude extract (p < 0.05) while asiaticoside alone did not show any activity. Carbopol 940 gel containing CAEMD was successfully optimised using three-factor, three-level Box-Behnken design, and the formulation with Carbopol 940 (C940) 0.6 % w/v, triethanolamine (TEA) 0.27 % w/v, and mixing speed at 650 rpm were selected as the optimised formulation. The optimised CAEMD hydrogel was subjected to further in vitro and in vivo studies. The CAEMD hydrogel was characterised by its efficacy of anti-microbial preservation for in vitro study and skin irritation study was conducted on New Zealand white rabbits. The optimised hydrogel did not show any microbial growth after being challenged by the microorganisms as required by European Pharmacopoeia for efficacy of anti-microbial preservation test and no irritation was found upon the application of hydrogel on rabbits’ skin. The fractionation using activated charcoal had successfully produced CAEMD fraction which contained higher amount of asiaticoside and anti-tyrosinase activity compared to the crude extract before fractionation is adopted. CAEMD fraction showed two-fold effect on inhibiting melanogenesis pathway compared to the crude extract using anti tyrosinase assay and the CAEMD formulation has successfully developed in this study.