The extraction of PHI was extracted, identified, and characterized (Fig. 1A, FigS1 to S5). In the present study, we found that PHI exhibits immunosuppressive activity in vitro . PHI was analyzed to establish appropriate levels for subsequent use that showed minimal signs of cytotoxicity on both murine splenocytes (Fig. 1B) and THP-1cells over a 24h time course growing in cultures (Fig. 1C).

Fig. 1. Phillygenin exhibited favorable immunosuppressive activity in vitro. (A) Chemical structure of PHI. (B) The cell viability of splenic lymphocytes with PHI treatment and IC₅₀ on ConA or LPS-induced lymphocyte proliferation. (C) The cell viability of THP-1 with PHI treatment. (D) Cytokine secretion levels in LPS-induced THP-1 culture. (E) The gene expression level of cytokine in LPS-induced THP-1.

Specifically, the CC50 of PHI on murine splenocytes was found to be 35.43 μg/mL, and it also exerted ideal immunosuppressive effects on ConA- and LPS-induced lymphocyte proliferation, with IC50 values of 4.357 and 1.539 μg/mL, respectively (Fig. 1B). Furthermore, the anti-inflammatory activity of PHI on PMA-induced human macrophages THP-1 was also confirmed. The ELISA assay indicated that PHI could effectively inhibit the secretion of IL-6 and TNF-α induced by LPS (Fig. 1D). On the genetic level, PHI was found to effectively inhibit expressions of IL-1β, IL-6, and TNF-α. Taken together, these results indicate that PHI is a promising candidate for immunosuppressive therapy in vitro(Fig. 1E).