Linking propolis chemical composition and geographical origin to immunomodulatory effects in human immune cells

Ethnopharmacological relevance Propolis is a resinous bee product traditionally used for its anti-inflammatory activity; however, its chemical complexity limits standardization and ethnopharmacological validation. This study integrated chemical profiling with functional immune assays to identify compounds underlying its anti-inflammatory effects. Methods 52 ethanolic propolis extracts from Australia (N = 7), Denmark (N = 38), and Norway (N = 7) were analysed by high-performance liquid chromatography (HPLC). 8 polyphenols with reported anti-inflammatory activity were used as reference standards. Human peripheral blood mononuclear cells (PBMCs) were stimulated with lipopolysaccharide (LPS) or phytohemagglutinin-L (PHA), and effects of propolis extracts and individual polyphenols on interleukin-1β (IL-1β) secretion were quantified by ELISA. Log 10 -transformed HPLC fingerprints and IL-1β data were analysed using multivariate methods, including partial least squares (PLS) regression, selectivity ratios (SRs), forward stepwise selection (FSS), and corrected Akaike's Information Criterion (AICc). Additional cytokines were measured using a mesoscale multiplex immunoassay. Results HPLC-analysis showed that propolis from Denmark and Norway contained higher levels of aromatic compounds than Australian samples. Propolis extracts reduced IL-1β secretion by 13%, 41%, and 37% for samples from Australia, Denmark, and Norway, respectively. Chemometric modelling identified the total extract load as key for anti-inflammatory effect, however, following log 10 -transformation, the chrysin-associated peak emerged as the most consistently supported candidate for independent, compound-specific IL-1β suppression. Multiplex cytokine profiling further demonstrated that propolis decreased IL-1β, IL-10, and IFN-γ while increasing IL-4, IL-6, and TNF-α, indicating a multifaceted immunomodulatory effect, including a Th1-to-Th2 shift. Conclusion Propolis demonstrated anti-inflammatory and immunomodulatory properties supporting its traditional use. Geographic variation in chemistry was associated with differences in IL-1β suppression. Chemometric analysis identified overall composition and the chrysin-associated signal as the most consistently supported putative candidate for compound-specific anti-inflammatory activity, while cytokine profiling further indicated that propolis acts as a complex immunomodulator rather than a broad immunosuppressant.