Study On The Immunomodulatory Activities Mechanisms Of Wugong Mountain Purple Red Rice Bran Anthocyanin

Purple red rice is a kind of colored hybrid rice unique to Wugong Mountain area.It has high nutritional value and is known as the"seven high"nutritional rice.Purple red rice bran is a mixture of the skin layer,a small amount of rice germ and broken rice that is milled off during the processing of purple red rice.And it is often used as cheap animal feed or simply discarded.Purple red rice bran contains protein,anthocyanins,flavonoids,dietary fiber and other bioactive substances.Anthocyanins,as one of the bioactive components in purple red rice bran,have been shown to have biological functions such as antioxidant,anti-inflammatory,and anti-tumor.However,there has not yet been any study on the immunomodulatory activity of anthocyanins in purple red rice bran.In this paper,purple red rice bran anthocyanins（PRBA）were extracted from Wugong Mountain purple red rice bran.The structure were identified by Ultra-Visible Spectroscopy（UV-Vis）,Infrared Spectroscopy（FT-IR）and Ultra-High Performance Liquid Chromatography coupled with Electrospray Ionization Quadrupole Time-Of-Flight Mass Spectrometry（UPLC-ESI-QTOF-MS）,and the in vitro antioxidant activities of the anthocyanins were determined.Besides,the effects of PRBA on splenic immunity,hepatic oxidative stress and intestinal mucosal barrier immunity were investigated in a cyclophosphamide（Cy）immunosuppressed mice model,and the immunomodulatory mechanism of PRBA were elucidated in combination with gut microbiota,which may provide a theoretical basis for the in-depth study of PRBA.The main researches and results of this work are as follows:（1）PRBA were successfully prepared by the solvent extraction method,the structure were identified by combining a variety of modern analytical techniques,and the in vitro antioxidant activities were determined.The chemical composition of PRBA were measured as 6.76±0.13 mg C3G/g DW total anthocyanin,126.26±1.09mg RE/g DW total phenols and 88.46±1.44 mg GAE/g DW total flavonoids.UV-Vis demonstrated the characteristic absorption peaks of anthocyanins at 299 nm and 530nm for PRBA,while FT-IR found that PRBA had absorption peaks at 3355.59 cm^-1、2925.53 cm^-1、2856.11 cm^-1、1284.38 cm^-1、900～650 cm^-1,which suggested that PRBA might have functional groups common in anthocyanins such as pyran ring and aromatic ring.A total of seventeen anthocyanins in PRBA were identified for the first time by UPLC-Q-TOF/MS,mainly including five anthocyanin glycosides,namely cyanidin,delphinidin,malvidin,peonidin and petunidin.And twelve anthocyanins cyanidin-3-arabinoside,petunidin-3-galactoside,cyanidin-3-galactoside,delphinidin-3-arabinoside,delphinidin-3-xyloside,cyanidin-3-glucoside,peonidin-3-galactoside,peonidin-3-glucoside,delphinidin-3,5-diglucoside,malvidin-3-galactoside,malvidin-3-glucoside and delphinidin-3-sambubioside.All the twelve anthocyanins were glycoside derivatives of five aglycones such as cyanidin,indicating that PRBA mainly existed in the form of anthocyanin glycoside derivatives.In vitro antioxidant assay,the PRBA concentration was 1000μg/m L achieved a DPPH scavenging rate of90.34%,ABTS⁺scavenging rate of 43.35%,hydroxyl radical scavenging rate of36.39%and FRAP reduction rate of 44.57%,all of which showed that PRBA had strong antioxidant activity.（2）A Cy-induced immunosuppressed mice model was established to investigate the effects and mechanisms of PRBA on the growth status of mice and immune organs such as the liver and spleen.The model group mice had dull and lusterless hair,poor vitality and weight loss,which suggested that the immunosuppression model was successfully established.PRBA could alleviate the damage caused by Cy to the body of mice by increasing their body weight,reducing immune organ damage,enhancing liver antioxidant enzyme（SOD）activity and reducing oxygen radical metabolites（MDA）,thus restoring normal immune function.Moreover,PRBA could activate MAPK signaling pathway by up-regulating phosphorylated ERK,p38,and JNK proteins to induce the secretion of splenic cytokines TNF-α,IL-6,and IL-1β,and thereby restored the impaired immune function and exerted immunomodulatory effects.（3）The regulatory activity and mechanism of PRBA on intestinal immunity was investigated based on the intestinal mucosal barrier and NF-κB signaling pathway.The results showed that PRBA could alleviate intestinal mucosal barrier damage caused by Cy,including broken intestinal villi,impaired crypt structure,and decreased villi length/crypt depth ratio.And it could also increase the expression of tight junction proteins Occludin and Claudin-1 to repair the intestinal tight junction structure and improve intestinal permeability,thus reducing endotoxin（ET）and endotoxin binding protein（LBP）into the blood system,and decreasing the content of LBP and ET in serum.Meanwhile,PRBA could also repair the intestinal mucosal barrier and recover normal immune function by increasing the number of cup cells,promoting the secretion of s Ig A,and upregulating key proteins of NF-κB signaling pathway to promote the production of cytokines TNF-αand IL-1β.（4）In order to investigate the effect of PRBA on the gut microbiota of Cy-induced immunosuppressed mice,short-chain fatty acids（SCFAs）in the colonic contents of mice were measured and 16S r DNA sequencing was performed in this study to further elucidate the mechanism of PRBA on the immunomodulatory activity of Cy-induced mice.The amounts of short-chain fatty acids（SCFAs）in the contents of mice colon were determined by gas chromatography,and it was found that PRBA could promote the degradation of anthocyanins by gut microbiota to produce SCFAs.Based on the results ofα-andβ-diversity analysis in 16S r DNA sequencing,PRBA could improve the diversity of gut microbiota and modulate the structure of gut microbiota.At the species level,there were increases in the relative abundance of the beneficial bacteria Lachnospiraceae,Bacteroidaceae,and Ruminococcaceae and decreases in the relative abundance of the pathogenic bacteria Shigella after PRBA treatment.The results of the species differences and marker species analysis showed that the main marker species affected by PRBA were o＿Pseudomonadales、f＿S24＿7、f＿Streptococcaceae、f＿Pseudomonadaceae、g＿Streptococcus、g＿Sphingomonas and g＿Pseudomonas.Thus,PRBA had the effect of regulating gut microbiota and maintaining intestinal homeostasis.