Extraction And Purification Of Total Flavonoids From Acanthopanax Senticosus And Their Effects On Oxidative Stress-induced Apoptotic

Acanthopanax senticosus flavonoids are secondary metabolites in Acanthopanax senticosus,which have important biopharmacological activities,especially their antioxidant capacity,which can reduce the damage caused by oxidative stress in the body.As an important antioxidant additive,flavonoids have been used in livestock and poultry production,such as curcumin has been used as a feed additive.A large number of previous studies have also confirmed that Acanthopanax senticosus flavonoids are strong antioxidants,which can reduce the oxidative stress of livestock and poultry.In this study,modern extraction technology was used to separate and purify flavonoids from Acanthopanax senticosus,and to study the antioxidant mechanism of flavonoids from Acanthopanax senticosus in vitro and in vivo.1.Optimization of the extraction process of total flavonoids from Acanthopanax senticosus:Combined enzyme-assisted ultrasonic extraction of total flavonoids from Acanthopanax senticosus was used to analyze the influence of different single factors on the yield of total flavonoids,and the extraction conditions were optimized by response surface methodology.The results showed that the ratio of cellulase:pectinase was 3:2,the amount of compound enzyme was 6960 U·g^-1,the enzymatic hydrolysis time was 59.80min,the enzymatic hydrolysis temperature was 53.70℃,the enzymatic hydrolysis p H value was 6.05,the total flavonoids The yield reached 36.95±0.05 mg·g^-1.2.Different polar solvent extraction process and antioxidant capacity evaluation of total flavonoids from Acanthopanax senticosus The content of bioactive substances in the phase was analyzed,and the distribution range of Acanthopanax senticosus flavonoids in different polar solvents was analyzed.The scavenging abilities of different relative DPPH radicals,ABTS radicals,hydroxyl radicals and superoxide anions were measured,and the relationship between the content of biologically active substances and antioxidants was analyzed by pearson correlation.The results showed that the total flavonoids of Acanthopanax senticosus had the highest content in the n-butanol phase,and was positively correlated with the antioxidant properties.3.The protective effect of Acanthopanax senticosus total flavonoids n-butanol against H₂O₂-induced apoptosis of RAW264.7 cells.In the experiment,RAW264.7 cells were stimulated by H₂O₂to establish an oxidative stress apoptosis model,and the effect of total flavonoids of Acanthopanax senticosus on ROS level and MDA content was investigated.Giemsa staining,Hoechst staining and flow cytometry were used to detect the apoptosis rate of each group.Real-time fluorescence quantitative PCR was used to detect the gene expression levels of cellular oxidative stress response and apoptosis mechanism-related molecules（Nrf2,HO-1,keap-1,NQO1,Bcl-2,Bax）.The results showed that the total flavonoids of Acanthopanax senticosus could promote the proliferation of RAW264.7 cells,reduce the level of intracellular ROS and MDA,and increase the activity of SOD.HO-1,Keap-1,NQO1,Bax gene expression was significantly down-regulated（P<0.05）.4.The protection of the total flavonoids of Acanthopanax senticosus n-butanol relative to CCl₄-induced liver injury in mice.The mouse liver injury model was established by CCl₄,and the treatment mode of Acanthopanax senticosus flavonoids and CCl₄ was administered at the same time.CCl₄ was administered by intraperitoneal injection of 20%CCl₄ soybean oil solution 1 m L·kg^-1 to mice,3 times·week^-1.The flavonoids of Acanthopanax senticosus were administered by intragastric gavage,and the doses were 20mg·m L^-1,40 mg·m L^-1,and 80 mg·m L^-1,respectively,for 2 consecutive weeks.To explore the protective mechanism of Acanthopanax senticosus total flavonoids n-butanol against liver injury in mice.The contents of ALT,AST,ALP and LDH in serum were detected;the pathological changes of liver tissue HE staining and Masson staining were observed;the contents of SOD and MDA in liver tissue were detected;the apoptosis of hepatocytes in each group was observed by TUNEL staining;fluorescence quantitative PCR was detected The relative expression trends of related oxidative stress genes,apoptosis genes,and liver fibrosis genes;immunohistochemical detection of apoptosis proteins Bax and Bcl-2 in liver tissue;detection of liver fibrosis-specific proteinsα-SMA and TGF-βexpression.The results showed that the total flavonoids n-butanol phase of Acanthopanax senticosus could effectively prevent hepatic inflammatory infiltration and tissue damage caused by oxidative stress,reduce the histopathological score of mice,and significantly reduce the contents of ALT,AST,ALP and LDH in serum.The content of SOD activity in liver tissue increased,and the content of MDA decreased;the expression of apoptosis factor Bax gene in liver tissue decreased,and the gene expression of Bcl-2 increased;liver tissue fibrosis was alleviated to different degrees.The experiment optimized the extraction and purification process of the total flavonoids from Acanthopanax senticosus and obtained the optimal extraction conditions,which improved the yield of total flavonoids from Acanthopanax senticosus,which is of great significance for the full clinical application of Acanthopanax senticosus.The total flavonoid free radical scavenging test study shows that the n-butanol phase of the total flavonoids of Acanthopanax senticosus has strong antioxidant activity in vitro;the research on the antioxidant mechanism shows that the total flavonoids of Acanthopanax senticosus can activate the Keap1-Nrf2-ARE signaling pathway,and reduce the oxidative stress.The induced macrophage apoptosis has obvious protective effect;the total flavonoids of Acanthopanax senticosus can achieve the protective effect on CCl₄-induced liver injury by balancing free radicals in animals.The research results have high reference significance for the development and application of total flavonoids from Acanthopanax senticosus,and lay a theoretical foundation for the application of traditional Chinese medicine to prevent and treat livestock and poultry diseases caused by oxidative stress leading to cell damage or apoptosis.