Antioxidant Activities of Plant Extracts (Ammannia multiflora, Ammannia coccinea, and Salix gracilistyla) Activate the Nrf2/HO-1 Signaling Pathway

To identify potent plant extracts with strong antioxidant activity, we evaluated the free radical scavenging activity of 184 plant extracts obtained from the Freshwater Bioresources Culture Collection (FBCC) of Nakdonggang National Institute of Biological Resources (Republic of Korea), as various plant extracts have been used therapeutically to prevent chronic diseases associated with oxidative stress. From them, three plant extracts (FBCC-EP858 from Ammannia multiflora, FBCC-EP920 from Ammannia coccinea, and FBCC-EP1014 from Salix gracilistyla) were selected based on their abilities to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with more than 80% efficiency. We found that these extracts had in vitro half maximal inhibitory concentration (IC50) values ranging from 11.89 to 14.26 μg/mL and strong total antioxidant activity (corresponding to approximately 0.18, 0.22, and 0.23 mM Trolox, respectively). We also studied the effect of these extracts on RAW 264.7 macrophages and found that FBCC-EP920 significantly downregulated relative cell viability at a concentration of 100 μg/mL. However, the other two extracts, FBCC-EP858 and FBCC-EP1014, did not affect cell viability at the same concentration. Additionally, all three extracts inhibited hydrogen peroxide (H2O2)-induced reactive oxygen species (ROS) production and depolarization of mitochondrial membrane potential in RAW 264.7 macrophages. An additional experiment in zebrafish larvae showed that the three extracts reduced 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent intensity induced by H2O2. The extracts also upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression, and an HO-1 inhibitor, zinc protoporphyrin (ZnPP), attenuated the extract-induced antioxidant activity both in vivo and in vitro. Taken together, these findings suggest that the extracts from A. multiflora, A. coccinea, and S. gracilistyla have potential free radical scavenging and antioxidant capacities both in vivo and in vitro by activating the Nrf2/HO-1 signaling pathway. These results could be useful for the prevention and treatment of various oxidative stress-mediated human diseases.