Isolation of Apigenin from Sungkai (Peronema canescens) Leaves and Its Immunomodulatory Effects: An In Vivo Study on Granzyme B, Interferon-γ, and Perforin Expression with Supporting In Silico Analysis

Background Paronema canescens Jack., commonly known as sungkai, is a medicinal plant native to Southeast Asia, particularly abundant in the forests of Sumatra and Borneo, Indonesia. Traditionally, sungkai has been used to treat various ailments, likely due to its rich content of bioactive secondary metabolites. This study aimed to isolate, characterize, and evaluate the immunostimulatory and anti-inflammatory potential of compounds from sungkai leaves based on in silico and in vivo analyses. Methods Apigenin was isolated from Paronema canescens leaves via ethanol extraction, liquid-liquid partitioning, and chromatographic purification, then characterized by UV-Vis, FT-IR, and NMR spectroscopy. Molecular docking was conducted using MOE software to assess apigenin’s binding to granzyme B, perforin, and IFN-γ, with levamisole as a reference. In vivo, 25 male mice were randomized into five groups and administered apigenin (1, 25, or 50 mg/kg BW) intramuscularly for seven days, alongside COVID-19 vaccination. Granzyme B and IFN-γ serum levels were quantified using ELISA. Statistical analysis employed one-way ANOVA with Duncan’s test (p < 0.05). Results The in silico analysis demonstrated that apigenin exhibited favorable binding affinities and multiple stabilizing interactions with granzyme B, perforin, and interferon-γ, supporting its potential role in enhancing cellular immune responses through direct molecular modulation of key cytotoxic effector proteins. To assess its immunostimulatory activity in vivo, apigenin was orally administered to mice (Mus musculus) at doses of 1, 25, and 50 mg/kg body weight. Mice were pre-induced with a COVID-19 vaccine to simulate immune system activation. Immunological responses were evaluated through the measurement of granzyme B, perforin, and interferon-γ expression levels. The results demonstrated that apigenin significantly increased the expression of all three markers in a dose-dependent manner. Conclusion Collectively, the chemical, computational, and biological data confirm that apigenin from sungkai leaves holds strong immunostimulant and selective anti-inflammatory potential, supporting its development as a natural immune booster or vaccine adjuvant.