Extraction Process Of Supercritical CO₂ Of Pueraria Crude Extract And Its Antioxidant Properties And Study On Intestinal Epithelial Protection

Aluminum（Al）is the most abundant and ubiquitous metal element in the environment.The main way humans are exposed to aluminum is through food and water intake.Aluminum compounds are widely used in food additives,antacids,medicines and food packaging,cooking utensils,etc.,which may also cause excessive accumulation of aluminum in the human body,thereby threatening human health.Pueraria,the dried root of the leguminous plant Pueraria,is a traditional Chinese medicinal material in my country.It has high nutritional,edible,medicinal and health-care value.Modern research shows that Pueraria has rich biological activities,such as isoflavones and Flavonoids are the main constituents of the antioxidant and anti-inflammatory activities of Pueraria root,and saponins have a protective effect on liver damage.Therefore,explore the extraction process of the effective components of Pueraria and explore the antioxidant capacity of the extract,as well as the intestinal epithelium caused by aluminum.The protective effect of barrier integrity is of great significance.As a new and green extraction technology,supercritical fluid extraction technology is gradually applied to natural products.This research explored the supercritical CO₂ extraction（PCE）and ethanol-assisted supercritical CO₂ extraction（PAE）process,it was found that the extraction rate of the former was 2.58±0.04%,and the extraction rate of the latter was increased to 5.24±0.12%.The effects of traditional ultrasonic-assisted ethanol extraction（PUE）and supercritical CO₂ extraction process on the extraction of total flavonoids were compared.The results showed that the total flavonoid content of PCE was 1.52 mg/g,the total flavonoid content of PAE was 25.24 mg/g,and the total flavonoid content of PUE was 4.56 mg/g.The crude extracts of the three extraction were further analyzed by GC-MS and HPLC.GC-MS results show that ethyl linoleate is the main compound of PCE and PAE,and the content of 4-isopropylbenzaldehyde,2,3-dihydrothiophene and ligustrazine in PAE is significantly higher than that of PCE.HPLC results showed that the content of Puerarin in PUE was 31.63 mg/g,daidzein 7.75 mg/g,daidzein 1.43 mg/g,and genistein0.57 mg/g.In order to explore the antioxidant activity of the crude extract of Pueraria,we first investigated its ability to scavenge DPPH,ABTS,and hydroxyl free radicals in vitro.The results showed that the ability of supercritical extraction Pueraria extract to scavenge free radicals was stronger than that of traditional extraction methods.Based on this,human colon cancer HT-29 cells were used to establish an intestinal epithelial model in vitro to further explore the protection of Pueraria on the integrity of the intestinal epithelium induced by aluminum in vivo.TEER and monolayer cell permeability were used to detect the integrity of the intestinal epithelial barrier.The experimental results showed that Pueraria extract can effectively protect the intestinal epithelial barrier damage induced by aluminum.The results of quantitative evaluation of ROS reactive oxygen species showed that Pueraria extract can reduce the accumulation of ROS reactive oxygen species caused by Al stimulation.In order to explore the protective mechanism of Pueraria extract on the intestinal epithelial barrier,RT-PCR was used to detect related molecules.The results showed that Pueraria extract can reverse the down-regulation of Occludin and Claudin-4 molecules caused by the breakdown of the intestinal epithelial barrier induced by aluminum.The expression of inflammatory factors TNF-αand IL-1βdecreased:WB results were consistent with RT-PCR results.After Pueraria extract pretreatment,the expressions of Occludin and Claudin-4 proteins were up-regulated.In summary,Pueraria extract can inhibit aluminum-induced intact intestinal epithelium Sexual destruction.