Immunomodulatory Potential of Kaempferol Isolated from Peronema canescens Jack. Leaves Through Inhibition of IL-6 Expression

Sungkai leaves were selected due to their herbal medicine prevalence and documented biological activities. This study explores the immunomodulatory potential of kaempferol isolated from Sungkai (<i>Peronema canescens</i> Jack.) through a combination of in silico and in vitro methods. <i>P. canescens</i> leaves were extracted with ethanol using maceration, followed by fractionation with n-hexane, ethyl acetate, and water using a separatory funnel. Among all the fractions, the ethyl acetate fraction demonstrated the strongest inhibitory effect on IL-6 (Interleukin 6) expression, leading to further separation for the enhanced analysis of its activity. The resulting sub-fractions were purified by vacuum liquid chromatography with n-hexane and ethyl acetate gradient. Sub-fraction E was isolated through preparative thin-layer chromatography to obtain a pure compound identified as kaempferol using UV, FTIR, MS, and NMR analyses. The isolated kaempferol was then evaluated by molecular docking and molecular dynamics simulations, employing MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) for binding affinity calculations. Kaempferol showed a binding affinity (ΔG) of -5.98 kcal/mol, slightly stronger than TLA (tartaric acid) (-5.90 kcal/mol). Key interactions with amino acid residues, such as Gln175, Arg182, and Arg179, were observed. Additionally, molecular dynamics simulation demonstrated that kaempferol exhibited better stability than TLA between 15 ns and 100 ns. The MM-PBSA analysis showed that kaempferol has strong van der Waals (-17.02 kcal/mol) and electrostatic interactions (-293.16 kcal/mol), with binding free energy (-17.85 kcal/mol) significantly stronger than TLA (-1.00 kcal/mol). This stability, combined with its ability to reduce IL-6 expression in vitro, highlights kaempferol's immunomodulatory potential.