| As a traditional medicinal and edible resource,Colla corii asini has attracted attention due to its immune-enhancing activity.The production and processing of Colla corii asini include complex chemical reactions which are protein degradation,aggregation and Maillard reaction,as well as the degradation of donkey skin glycosaminoglycans.Therefore,there are some differences in the structure and functional quality of Colla corii asini products produced by different processing conditions.However,it is still not clear that the immune-enhancing activity of the different molecular weight(Mw)components of Colla corii asini is different or not,which restricts the standardization and processing control of product quality.In this study,Colla corii asini was preliminarily divided into three components according to Mw by ultrafiltration.The immune activity in vitro and in vivo were explored through cell and animal models,and then the fraction was separated by Superdex 75 pg column and studied its immune activity in vitro.Finally,the reasons for the differences in immune activity between different Colla corii asini components were explored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),sulfuric acid phenol method,liquid chromatograph/mass spectrometer(LC/MS)to preliminarily.This study could provide reference for the processing and manufacture of Colla corii asini with high immune activity.The main research contents and results are as follows:Firstly,F0,F1,F2 and F3 with weight average Mw of 25730 Da,40831 Da,6075 Da and2989 Da were obtained from Colla corii asini by using ultrafiltration membranes with molecular weight cut off(MWCO)of 100 k Da and 10 k Da,respectively.Their immuneenhancing activity on RAW264.7 macrophages were researched.The results showed that the ultrafiltration fractions of 62.5 μg/m L～1000 μg/m L did not show obvious cytotoxicity(p > 0.05)after treatment for 24 h.The 1000 μg/m L F1 had the highest pinocytosis rate and phagocytosis rate which were 33.91% and 95.53% higher than those of the blank control group,respectively,and the content of reactive oxygen species(ROS)was 1.5 times than that of the blank control group.The nitric oxide(NO)concentration of F1 was 20.40 ± 0.78 μmol/L,and the secretion of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were 4395.95 ± 260.90 pg/m L and6959.44 ± 379.49 pg/m L which were significantly higher than other fractions(p < 0.05),indicating that F1 had the strongest activity in promoting the immune function of macrophages.Then the four ultrafiltration fractions were subjected to in vitro digestion,and the weightaverage Mw of the digested products F0 D,F1 D,F2 D,and F3 D were 6196 Da,6901 Da,5085 Da,and 2546 Da,respectively.Experimental results showed that after the digestion of different Colla corii asini components,the immune activity was enhanced.1000 μg/m L F0 D promoted the release of NO by 51.13% before digestion,which was significantly different(p< 0.01).F1 D treated cells had the strongest endocytosis ability and promoted the cells to secrete the NO,ROS,and IL-6,indicating that the F1 fraction still had the strongest immune-enhancing activity after digestion.To explore the in vivo immune activity of different Colla corii asini components,the immunosuppressive mice model induced by cyclophosphamide(CTX)was used to evaluate the immune-enhancing ability of Colla corii asini components in vivo.The results showed that the F3’ group mice had the highest spleen index and Ig M.The F2 group had the highest spleen ACP and LDH enzyme activities,the F1 group had the highest number of blood leukocytes,which were 7.52% and 51.06% higher than the F2 and F3’ groups,respectively.The serum levels of Ig G,Ig A,and granulocyte-macrophage colony-stimulating factor(GM-CSF)of the mice in the F1 group were the highest,which were 112.71%,64.49%,and 74.39% higher than those in the model group,respectively.In addition,the F1 group had the strongest phagocytic ability of peritoneal macrophages and the best spleen lymphocyte activity.The four experimental groups all increased the expression of MAPK signaling protein,and the expression of MAPK protein in the F1 group was the highest,indicating that the F1 component had the best immuneenhancing activity,which was consistent with the results of cell experiments.The F1 fraction was then separated by Sephadex column to obtain F11(Mw: < 10 k Da),F12(Mw: 10 k Da ～30 k Da)and F13(Mw: > 30 k Da).It was found that the contents of NO and TNF-α in the culture medium of the cells treated with F12 were 38.87 ± 1.26 μM and 6577.29± 51.02 pg/m L which were higher than those of the cells treated with F11,respectively.And the contents of NO and TNF-α in the culture medium of the cells treated with F12 were 38.87%and 15.25% higher than those of the cells treated with F13.Different concentrations of F12 could promote cell endocytosis and stimulate cells to secrete ROS and IL-6 in a dose-dependent manner,indicating that F12 with Mw ranging from 10 k Da to 30 k Da in Colla corii asini has the strongest immune activity.The results of real-time quantitative PCR(q-PCR)and Westernblot experiments(Western-blot)showed that F12 could bind to Toll-like receptor 4(TLR4)on the surface of macrophages and promote the phosphorylation of MAPK signaling pathway proteins.Finally,the amino acid composition,protein-bound sugar content and protein composition of different Colla corii asini components were analyzed by high performance liquid chromatography(HPLC),sulfated phenol method,SDS-PAGE and LC-MS.The results showed that there was no significant difference in the amino acid composition ratio of different components.There was a difference in the protein-bound sugar content.The protein-bound sugar content of the F1 component was 0.65%,which was 4 times that of the F3’ component.The results of the proteomic analysis showed that the collagen content in the F12 fraction was the highest,and the keratin content in the F13 fraction was the highest.These results indicated that the fraction of Colla corii asini with Mw between 10 k Da and30 k Da has the strongest immune activity,and the differences in the immune activity of different fractions of Colla corii asini may be related to Maillard reaction and glycosaminoglycan degradation.The content of glycosylated protein produced by polymerization is related to the content of collagen in the Colla corii asini fraction.The results have a positive guiding role for the processing and quality control of Colla corii asini. |
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