Bioactivity-guided Isolation of Immunomodulatory Compounds from the Fruits of Ligustrum Lucidum.

Ethnopharmacological relevance: The fruits of Ligustrum lucidum (FLL) W.T. Aiton (Oleaceae) is included in the 2020 "Chinese Pharmacopoeia" and is widely used in traditional Chinese medicine as a tonic. In recent years, FLL has been reported to improve immune function, but the bioactive compounds and mechanisms of FLL remain poorly characterized. Aim of the study: To identify FFL compounds with strong immune activity and explore their molecular mechanisms. Materials and methods: The phagocytic activity of RAW264.7 macrophages and proliferation activity of spleen lymphocytes were used to guide the isolation of bioactive compounds from FLL extracts. Lymphocyte subpopulations, Ca2+ concentrations, and surface molecule expression were analyzed using flow cytometry. Cytokine secretion was examined using ELISA. FITC-OVA uptake was observed using fluorescence microscopy. NF-kappa B activation was analyzed using western blotting. Results: The extraction and isolation produced ten compounds, namely oleuropeinic acid, nuezhenide, isonuezhenide, salidroside, isoligustrosidic acid, ligulucidumosides A, 8(E)-nuezhenide, hydroxytyrosol, oleuropein, and p-hydroxyphenethyl 7-beta-D-glucosideelenolic acid ester were isolated and identified from FLL-Bu-30%. Immunoactivity experiments showed that hydroxytyrosol had the strongest macrophage phagocytotic and lymphocyte proliferation-promoting activities. Further studies showed that hydroxytyrosol could significantly enhance lymphocyte subsets CD3+, CD4(+)/CD8(+), and CD3(+)CD4(-)CD8(-), promote IL-4, IFN-gamma, and TNF-alpha secretion, and increase intracellular Ca2+ concentrations. In addition, the results from RAW264.7 macrophages showed that hydroxytyrosol increased FITC-OVA uptake, induced TNF-alpha and IL-1 beta production, upregulated MHC-II, CD80, and CD86 expression, promoted cytoplasmic I kappa B-alpha degradation, and increased nuclear NF-kappa B p65 levels. Conclusion: Our study provides substantial evidence regarding the mechanism of the immunomodulatory effects of compounds from FLL.