Functional Evaluations on Immunomodulatory Activities of Different Protease Hydrolysates and Isolated Peptides from Yam Storage Proteins, Dioscorin

The main purpose of this two-year proposal will be focused on the immunomodulatory effects of active peptides from different protease hydrolysates of yam tuber storage protein, dioscorin. In our reported research, it was found that the dioscorin could stimulate phagocytic activity and cytokine productions in vitro (Food Chem. Toxicology, 2007, 45, 2312-2318) and oral administration to BALB/c mice in vivo (J. Agric. Food Chem., 2009, 57, 9274-9279). In our preliminary results (please see the proposal section), it was found that protease hydrolysates (such as peptic and tryptic hydrolysates) exhibited the activities to stimulate NO (nitric oxide) productions in treated-RAW264.7 cells. Base on these results, it will be important to investigate and find out the bioactive peptides from dioscorin hydrolysates and to evaluate the immumomodulatory effects in a series of experiments. For this propose, we propose this two-year project as followed. In the first year, the purified dioscorin will be hydrolyzed by different enzymes, and the immunomodulatory activities will be evaluated by RAW 264.7 cell lines (with or without reporter gene transfection, the RAW264.7/Luc-P1 cells consisted NF-κB-responsive region from endothelial leukocyte adhesion molecule I (ELAM1), followed by the reporter gene firefly luciferase which the overall produced luminescence was positively correlated with the active peptide fraction binding to TLR4 which might be the immunomodulatory peptides, see method section). Moreover, the bioactive peptides will be purified based on those immunomodulatory activity-guided screenings. The protease hydrolysates will also be assayed for the antioxidant activities. Those bioactive peptides will be sequenced for further synthesis. In the second year, the bioactive peptides will be synthesized by solid-phase peptide synthesis method based on our self-synthesized experiences. The RAW 264.7 cell line and the primary cell culture, especially the peripheral blood mononuclear cell (PBMC), will be used to make the platform much more closely to the immune system of human beings. Furthermore, the bioactive peptides will be orally administrated to BALB/c mice for 21-days experiments. The sub-population of peripheral blood, spleen cell proliferation, phagocytosis activity, nature killer cell activity and the changes of cytokine production will be analyzed. And the Peyer’s patches will also be counted for much more complete evaluation.