Chlorophyll a, an active anti-proliferative compound of Ludwigia octovalvis, activates the CD95 (APO-1/CD95) system and AMPK pathway in 3T3-L1 cells

Ludwigia octovalvis is an aquatic plant widely distributed in Taiwan. It is traditionally used as a diuretic and is consumed as health drink. In this study, we evaluated the anti-proliferative activity of extracts and active constituent (chlorophyll a; CHL-a) of L. octovalvis in 3T3-L1 adipocytes; its mode of action on apoptosis was also investigated. Results showed that, among the different extracts and fractions, the ethylacetate layer (EAL) possessed the most potent anti-proliferative activity. Activity guided fractionation of the EAL obtained the bioactive constituent CHL-a (IC₅₀: 24.10 ± 0.83 nM). At concentrations 5–30 nM, CHL-a exhibited a dose-dependent accumulation of the Sub-G1 peak and caused cell cycle arrest at the G0/G1 phase. At 30 nM, it significantly reduced the cell viability, induced the appearance of DNA fragments, and enhanced the activation of caspase-3. Western blot data revealed that CHL-a decreased the level of Bcl-2, and increased the expression of CD95 (APO-1/CD95) and Bax. Furthermore, CHL-a up-regulated the AMPK and p-AMPK levels, and down-regulated the expression of PPAR-γ. These results conclude that CHL-a possesses potent anti-proliferative activity, and its apoptotic effects on 3T3-L1 adipocytes are mediated through the activation of CD95 (APO-1/CD95) system and the AMPK signaling pathway.