Enzymatic Preparation And Immuno-enhancing Activities Of Symphytum Officinale L Polypeptides

The present study aimed to prepare immunomodulatory peptides of Symphytum officinale L to improve its utilization values.Symphytum officinale L nutrients and amino acid composition of Symphytum officinale L leaf protein were determined.The results showed that dry grass contained 24.3%of crude protein,29.52%of total dietary fiber,1.2%of fat,19.3%of carbohydrate,19.7%of ash and 6.02%of water.Leaf protein contained 18 amino acids and its amino acid score was 40.37%,its essential amino acid score was 1.2487.Therefore,Symphytum officinale L is of high quality protein source.Based on the basic principles of Box-Behnken design,a mathematical model was established by response surface method and the optimum conditions for preparing Symphytum officinale L polypeptides were determined.The results showed that the polypeptide content in the hydrolysate reached to 0.664mg·mL^-1 and the optimized condition for Symphytum officinale L polypeptides preparation was as following:smashing the leaves at a ratio of 8%（material to water）,adding 3%of cellulose,adjusting initial pH to 6.0,water-bathing at 45℃ for 2h,then adding 4.57%of alcalase to the system and adjusting initial pH to 8.91,maintaining at 43.4℃ for 56min.The ConA-induced spleen lymphocyte proliferation system（MTT method）and the LPS-promoted celiac macrophages phagocytosis system（neutral red method）in mice were established to explore the immuno-enhancing activity of the resulted Symphytum officinale L polypeptides.Results suggested that 66,0.66 and 0.066μg·mL^-1 Symphytum officinale L polypeptides significantly enhanced the proliferation of mice spleen lymphocytes（P<0.05）.66,0.66 and 0.066μg·mL^-1 polypeptides dramatically increased the phagocytic capacity of mice celiac macrophages（P<0.05）.Hence,the optimal concentration of Symphytum officinale L polypeptides was 66mg·mL^-1.Symphytum officinale L polypeptides were separated with ultrafiltration centrifugation to obtain three fractions containing P1（>10kDa）,P2（3～10kDa）和 P3（<3kDa）,which were explored the immuno-enhancing activity.The results showed that P3(0.597 mg·mL^-1)significantly enhanced the proliferation of mice spleen lymphocytes（P<0.05）and increased the phagocytic capacity of mice celiac macrophages（P<0.05）.