Extraction Of Polyphenols From Red Sorghum Bran And Its Regulation On Intestinal Microenvironment In Aging Mice

In this paper,sorghum bran was used as raw material to compare the effects of four different extraction methods on the content,composition and activity of bound polyphenols in red sorghum bran,and the SAE extraction process was optimized.On this basis,with the oxidative aging mouse model,the preventive effect of sorghum bran bound polyphenol（RSBBP）on aging mice and its regulation on intestinal microenvironment were evaluated.Through the research of this paper,it is expected to establish a green and efficient extraction method of combined polyphenols,which provided a theoretical foundation for the efficient preparation of bound polyphenols by subcritical water extraction technology.At the same time,it not only enriched the resources of bound polyphenols,but also provided new ideas for the development and utilization of sorghum bran.The main research contents and results are as follows:1.Effects of different extraction methods on the composition and activity of bound polyphenols from sorghum branAlkaline hydrolysis（AH）,subcritical water assisted extraction（SAE）,ultrasonic assisted extraction（UAE）,microwave assisted extraction（MAE）RSBBP were used and purified by macroporous adsorption resin.To screen out the best extraction method,the content,composition,antioxidant and anti-tumor proliferation activities of the bound polyphenols obtained by the four methods were compared.The results showed that the contents of bound polyphenols obtained by the four methods were0.61 mg GAE/g dw（AH）,1.99 mg GAE/g dw（SAE）,1.98 mg GAE/g dw（UAE）and1.90 mg GAE/g dw（MAE）;Ahter analysis by LC-MSⁿ,16 phenols were detected in four groups of RSBBP samples,in which 10 phenolic substances were identified by SAE;Through the determination of ABTS⁺·scavenging ability,DPPH·scavenging ability,NO₂^-scavenging ability and reducing power,the order of evaluating the antioxidant ability of RSBBP is:SAE>UAE>MAE>AH;The anti-proliferative activity of Hep G2 cells of RSBBP was detected by MTT assay,and the order of evaluating the inhibitory rates of RSBBP on Hep G2 cells when the final concentration was 400μg/m L is:SAE>UAE>MAE>AH.In conclusion,the content,antioxidant and anti-proliferation activities of bound polyphenols extracted by SAE were significantly higher than AH（p<0.05）and MAE（p<0.05）,slightly higher than UAE,and the polyphenol components were the most abundant.Therefore,SAE was selected for the next process optimization.2.Study on subcritical water extraction of polyphenols from sorghum branBased on Plackett Burman experiment and Steepest Ascent experiment,the significant factors affecting the bound polyphenols content were screened.The SAE process of RSBBP was optimized by Response Surface Methodology and corrected in combination with the actual operation.Finally,the optimal extraction process of SAE was obtained as follows:extraction time 30 min,extraction temperature 190℃and2M Na OH addition 83%.Under these conditions,the content of bound polyphenols was 2.70 mg GAE/g dw,which was 3.45 times higher than AH（p<0.05）,The extraction time was reduced by 50%,and the addition of 2M Na OH was reduced by17%;Under this condition,the ability to scavenge ABTS⁺·,DPPH·,NO₂^-and reducing power of RSBBP were significantly higher than AH（p<0.05）.The anti-proliferation activity of Hep G2 was 49.32%when the RSBBP were 200μg/m L.The above results shown that SAE was a better preparation method of RSBBP.3.Preventive effect of sorghum bran bound polyphenols on aging induced by oxidative stressD-galactose（D-gal）was used to induce mice to establish a classic oxidative stress-induced aging model.The effects of RSBBP（low dose 50 mg/kg,high dose100 mg/kg）on body weight,organ index,biochemical indexes related to oxidation in liver and serum were studied.The results showed that RSBBP significantly prevented the inhibition of aging on body weight growth（p<0.05）,reduced liver index,alleviated liver swelling,increased spleen and thymus index,and improved the atrophy of spleen and thymus.RSBBP improves the oxidation level of serum and liver tissue in a dose-dependent manner reduces the level of inflammatory factors in mouse serum,alleviates dyslipidemia and reduces liver function injury.RSBBP could improve the swelling and deformation,disordered arrangement,blurred boundary and uneven cytoplasm of liver cells caused by D-gal.4.Regulation of sorghum bran bound polyphenols on intestinal microenvironment in aging miceThe effects of RSBBP on intestinal microenvironment of aging mice were evaluated by the effects of oxidative damage markers,water content of colonic contents,p H,short chain fatty acids（SCFAs）content and intestinal flora structure.The results showed that compared with the control group,the water content of colonic contents in RSBBP-L and RSBBP-H groups increased and the p H of colonic contents decreased（all p<0.05）.The contents of four SCFAs of acetic acid,propionic acid,n-butyric acid and isovaleric acid increased to varying degrees（p<0.05）,and the level of oxidation in colonic tissue increased significantly（p<0.05）.Through the alpha diversity analysis of intestinal microorganisms,the results showed that compared with the control group,low dose RSBBP could increase the relative abundance of beneficial bacteria（p<0.05）,reduce the relative abundance of harmful bacteria（p<0.05）,increase the F/B value,inhibit the proliferation of inflammatory factors and pathogenic bacteria,maintain the stability of mouse intestinal environment and protect the function of intestinal barrier.