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Clerodendrum petasites  S. Moore predominantly contains hispidulin (His) and nepetin (Nep) which are immunosuppressive potentials. Although the effect of individual compounds was previously confirmed, a cumulative suppression of these flavonoid mixtures is unknown. This study thus investigated their inhibitory effects and cytotoxicity on T cells by using His:Nep ratios following a naturally occurring dose (3:1) and optimized doses (1:1 and 1:3). Anti-CD3/28 stimulated peripheral blood mononuclear cells were treated with individual compounds and their mixtures. Changes in early cell activation markers in activated T cells and apoptosis were analyzed by a flow cytometer. Mixtures at 3:1 suppressed CD69 and CD25 expression in CD4 +  and CD8 +  T cells in a dose-dependent manner. At the highest concentration of 200 µM His + 66.7 µM Nep, over 90% inhibition was observed for CD25 expression in CD4 +  and CD8 +  T cells, whereas a lesser effect was observed for CD69 expression. A dose-dependent inhibition was still observed when using 1:1 and 1:3 ratios. Interestingly, 80–97% inhibition were observed in CD69 and CD25 expression without inducing cell death after treated with the highest doses of each ratio (66.7 µM His + 200 µM Nep and 200 µM His + 200 µM Nep). These mixtures were also exhibited a better suppression than individual compounds. The optimized mixture of His and Nep at 66.7:200 µM is suggested for further study due to a greater suppressive effect than a single compound or a naturally-occurring dose.

Synergistic immunosuppressive effect of hispidulin and nepetin mixtures on human T lymphocytes