Intestinal Metabolic Transformation Of Pomegranate Peel Polyphenols And Their Interaction With Microflora

Pomegranate polyphenols have anti-oxidation,anti-atherosclerosis,anti-obesity effect,and so on.Studies have shown that the applications of pomegranate in the field of medicine are closely related to its high content of polyphenols.However,few studies have been conducted on the absorption and transformation of pomegranate polyphenols in the gut and the biologically active forms that ultimately work in the body.Therefore,in this study,polyphenols was extracted from pomegranate peel,and neomycin sulfate was used to inhibit intestinal flora of rats,so as to evaluate the effect of inttestinal flora on metabolic transformation of pomegranate peel polyphenols in rats.This experiment was designed to study three groups of rats.Group A was fed with normal diet(n=10),Group B was given pomegranate peel polyphenol extracts(n=10),Group C was given pomegranate peel polyphenol extracts and neomycin sulfate(n=10).Samples of plasma and feces were collected within 35 days and urine was collected weekly.Indicators such as serum creatinine,urea nitrogen,and glutamic oxalacetic transaminase were measured by automatic biochemical analyzer to evaluate the renal and hepatic toxicity of pomegranate peel polyphenols in rats.The oxidative damage kit was used to measure the antioxidant capacity of pomegranate peel polyphenols.High-throughput sequencing technology was used to analyze part of the variable region sequence of 16S rRNA gene,conduct in-depth quantitative analysis on the flora composition,and study the changing effects of pomegranate peel polyphenols and antibiotics on the flora.The contents of short-chain fatty acids in rat feces were determined by GC-MS,so as to the analyze the effects of pomegranate peel polyphenols on short-chain fatty acids in the gut.The abundance of pomegranate polyphenol metabolites in urine and feces of rats and the changes in the content of Urolithin A were analyzed by LC-MS,to further elaborate the interaction between intestinal flora and metabolism of pomegranate polyphenols.Results are as follows:(1)The results of serum biochemical indexes showed that the antibiotics and pomegranate peel polyphenols applied in this experiment did not cause obvious side-effects on SD rats,and pomegranate peel polyphenols had a certain anti-obesity and anti-oxidation effect.Compared with Group A,the weight,total cholesterol and total triglyceride of the rats in Group B all decreased.Serum ALT,AST,urea and creatinine levels were the highest in Group C,but there was no significant difference between Group C and the other two groups.Under the intervention of pomegranate peel polyphenols,MDA levels in the liver tissues and serum of rats in Group B and Group C were lower than those in Group A,and the MDA level in the liver tissue of Group B was significantly lower than that in the normal group(P<0.05).Compared with the normal group,pomegranate peel polyphenol intervention can significantly increase the activity of SOD in the serum of rats in Group B(P<0.05),the content of GSH-Px in Group B increased by 15.7%,while that in Group C did not increase.The liver GSH-Px activity of Group B was significantly higher than that of the normal group(P<0.05).These results showed that the pomegranate peel polyphenols extract and the dose of antibiotics had no harmful effect on rats,which met the requirements of subsequent experimental animals.(2)Pomegranate peel polyphenols did not significantly change the diversity of intestinal flora,while neomycin sulfate had a significant inhibitory effect on intestinal flora in rats.The number of OTU in feces of rats in Group C was significantly lower than that in normal group and pomegranate peel polyphenols group.Shannon diversity index analysis results showed that Group B>Group A>Group C,in which the diversity of Group C was significantly lower than that of Group B(P<0.05).Pomegranate peel polyphenols could significantly increase the relative abundance of firmicutes in faeces of rats,and this increase was related to Ruminococcaceae and Lachnospiraceae of the Class Clostrida.At the genus level,pomegranate peel polyphenols could significantly increase the contents of Ruminococcaceae_UCG-014 and Ruminococcaceae_NK4A214_group(P<0.05).Short-chain fatty acids detection results showed that the highest concentrations of short-chain fatty acids in feces samples of each groups were acetic acid and propionic acid.The content of short-chain fatty acids in faeces of rats in Group C was lower than that in Group A,and the concentration of total short-chain fatty acids in faeces of rats in Group B was significantly higher than that in Group A(P<0.05).Pomegranate peel polyphenols could increase the concentrations of acetic acid,propionic acid,and butyric acid.Short-chain fatty acids test results showed that the diversity and structure of intestinal flora were positively correlated with the concentration of short-chain fatty acids,and the increase in short-chain fatty acids content was related to the increase in relative abundance of Ruminococcaceae NK4A214_group and Lachnospiraceae NK4A136_group.(3)The results of LC-MS showed that the abundance of pomegranate peel polyphenol metabolites in the urine and feces of rats in Group B was higher than that in Group C.In this study,the daily intake of punicalagin in Group B and Group C rats was between 33.5-35.4mg.The content of Urolithin A in Group B increased significantly compared with the normal group,and the average content of Urolithin A increased by 1.06 mg during the experiment period.The content of Urolithin A excreted in the feces and urine of rats in Group B accounted for 4%of the punicalagin intake.After antibiotic intervention,the content of Urolithin A in the urine of rats in Group C decreased sharply,but the content of Urolithin A in the feces increased significantly and was proportional to the time,and the increase of Urolithin A in the feces was consistent with the decrease of the content of Urolithin A in the urine.In summary,Ruminococcaceae_NK4A 214_group,Ruminococcaceae_UCG-014,Ruminococcaceae_UCG-005 in the intestinal flora could promote the metabolic transformation of pomegranate peel polyphenols,and improve the utilization of Urolithin A.The decrease of intestinal flora diversity could significantly reduce the abundance of metabolites of pomegranate peel polyphenols in urine and feces,and lower the conversion and absorption efficiency in vivo of pomegranate peel polyphenols and their metabolites.A small portion of the ingested punicalagin was metabolized and/or absorbed,while the vast majority was converted into undetected metabolites or accumulated in the tissue.In this research,the effects of pomegranate peel polyphenols on intestinal flora and short-chain fatty acids were systematically studied,the toxicological effects of antibiotics on liver and kidney of rats and the antioxidant effects of pomegranate peel polyphenols in vivo were evaluated,and the effect of bacterial flora alternation on the metabolism of pomegranate peel polyphenols was further studied.