Characterization Of Mulberry Leaf Albumin And Its Alleviative Effect On Immunosuppressive Mice

Mulberry leaf protein is a potentially functional food component and health care agent with antioxidant and anti-inflammatory properties.However,its composition,immunoregulatory and gut microbial regulatory effects are unclear.Herein,the secondary structure of a gel-fractionated and ultra-filtrated mulberry leaf protein(GUMP)was analyzed.Its effects on cyclophosphamide(CP)-induced immunosuppressive mice were further investigated.Results are as the follows:1.Only one retained component of ultra-filtrated mulberry leaf albumin(UMP)appeared in column chromatography,which was collected as GUMP.The subunits of MP,UMP and GUMP were compared by SDS-PAGE,and it was found that the subunits of MP,UMP and GUMP were composed of 14 k Da small subunit and 52 k Da large subunits.These results suggested that GUMP may be Rubisco commonly found in plant leaves.After separation and purification,the protein content of GUMP increased significantly(p < 0.05),while the total phenol content(TPC)decreased significantly(p < 0.05),but the sugar content in the protein did not change significantly.The purified GUMP was further confirmed as glycoprotein by PAS staining.The binding mode between glycogroup and protein is O-glycopeptide bond.Ion Chromatography(IC)was used to analyze the monosaccharide composition of GUMP glycogroup.The results showed that glucose and arabinose were the main components of GUMP glycogroup,accounting for 2.48 and 2.22 ppm,respectively.The secondary structure of GUMP was analyzed by FTIR and CD,and the results showed that the primary secondary structure of GUMP was β-sheet(> 40%).The protein fragments of GUMP were identified by LC-MS/MS to several proteins in the NCBI Morus database,including the large subunit(52.7k Da)of Rubsico with a confidence of 155.87.These results suggest that mulberry leaf albumin is a glycoprotein that contains Rubisco.After separation and purification,the purity of mulberry leaf albumin can be improved but the sugar group can not be removed.2.CP induced immunosuppressive BALB/C mouse model was used to verify the immunoregulatory effect of GUMP.Intraperitoneal injection CP for 3 days,followed by intervention with gradient GUMP(30,90,270 mg/kg)and 40 mg/kg levomisole(LH).The results showed that the intervention of GUMP could significantly alleviate the decrease of thymus index caused by CP in mice.Serum levels of cytokines(IL-2,IL-6,and TNF-α)were increased in mice given GUMP,and serous IL-6 exhibited a dose-dependent manner.Meanwhile,the contents of immunoglobulin Ig A and Ig G in serum of mice were significantly increased by GUMP(p < 0.05),thus restoring the immunity of mice.Observation and analysis of mouse spleen showed that the red and white pulp boundary of mice in GUMP intervention groups was gradually clear,the number of megakaryocyte precursors cells in the visual field decreased,and the occurrence of cell apoptosis in the visual field decreased.This was especially true in the medium-dose group.These results suggest that GUMP,as a nutritional supplement,could alleviate the decrease of cytokines and immunoglobulin caused by CP and reduce the depletion of immune cells in important immune organs.3.The intestinal microflora and its metabolites of the mice were analyzed.Short-chain fatty acids(SCFAs)were analyzed by Gas Chromatography(GC).The results showed that the medium dose of GUMP could increase the content of total fatty acids,especially n-butyric acid(p < 0.05).The sequencing results of 16 S r RNA gene of intestinal microflora in mice showed that the αdiversity of intestinal microflora decreased after CP treatment,which could be recovered by gavage of GUMP.According to the results of β diversity,the composition of microflora in mice was significantly affected by medium and high doses of GUMP,which was manifested as the deviation of grouping ellipse.These results indicate that compared with the positive control group(LH),GUMP as dietary supplement can better restore the intestinal microecology and improve theα diversity of intestinal flora in mice.At phylum level,the abundance of Firmicutes in the medium dose GUMP group was significantly increased.GUMP intervention can also reduce the abundance of desulphurization bacteria.At the genus level,medium dose of GUMP could significantly increase the abundance of butyric-producing bacteria Odoribacter(p < 0.05),and the abundance of Desulfvibrio at low and high concentrations of GUMP could be significantly reduced(p < 0.05).These results suggest that GUMP could improve dysbiosis in immunosuppressed mice,increased the diversity of microflora,enhanced the abundance of potentially beneficial bacteria and reduce the abundance of harmful bacteria.The findings and results above prove that GUMP is a complex glycoprotein containing Rubisco large subunit and its glycopeptide bond is linked by O-glycopeptide bond.GUMP has an alleviating effect on immunosuppressed mice,which is embodied in the promotion of cytokines and immunoglobulin in serum and slowing down the damage of CP to spleen.At the same time,GUMP can also alleviate the intestinal microflora imbalance caused by CP in mice and restore the diversity of microflora.In conclusion,GUMP,as a renewable plant protein with abundant resources,has the potential to become a new nutritional supplement for people with low immune system or impaired immune system.