Ultrasound-assisted Extraction Of Flavonoids From Ziziphi Spinosae Semen And Its Mechanisms Of Anti-A?_1-42 Damage

Alzheimer's disease?AD?is an age-related neurodegenerative disease.The amyloid hypothesis holds that amyloid beta protein(A?_1-42)is abnormally secreted in brain tissue,which is the main cause of AD symptoms such as oxidative damage and dementia.Ziziphi Spinosae Semen?ZSS?,as a traditional Chinese medicine,has many pharmacological effects.The latest research shows that the flavonoids of ZSS have strong antioxidant capacity,and are a natural antioxidant with great potential.Therefore,it has good practical development and application prospects.At present,there is no report on the relationship between the antioxidant activity and extraction methods of Flavonoids from ZSS and the mechanism of antioxidant damage protection.Therefore,Plackett-Burman design and response surface methodology were used to optimize the ultrasonic-assisted extraction process of Flavonoids from ZSS.The relationship between the antioxidant activity of Flavonoids from ZSS and the methods of heat reflux extraction and ultrasound-assisted extraction was investigated by using a variety of in vitro antioxidant activity detection methods.The effects of 6'''-Feruloylspinosin?6-FS?on nematode behaviour?paralysis test,life test,heat stress test and chemotaxis test?,antioxidant capacity and lysosome content of autophagic markers were studied by using AD transgenic Caenorhabditis elegans GMC101.At the same time,AD cell model was established by PC12 cells,and the effects of 6-FS on cell viability,reactive oxygen species,mitochondrial membrane potential,amyloid protein aggregation level and expression of autophagy-related proteins of A?_1-42 oxidative damaged cells were investigated.The research results are as follows:?1?Response Surface Methodology?RSM?was used to optimize the ultrasonic-assisted extraction of total flavonoids?TF?from ZSS:Single factor experiments and Packett-Burman design experiments were used to screen the factors and levels that had significant effects on the extraction yield of TF from ZSS.Taking these factors as independent variables,TF extraction yield was the response value.Box-Behnken response test method was established to obtain the best ultrasonic extraction conditions.The optimum extraction conditions were as follows:ultrasonic power 404 W,liquid-solid ratio15.02:1 mL/g,extraction time 60.03 min,ethanol concentration 70.6 v/v,the extraction yield of total flavonoids was 1.590%.It is close to 1.588%of the validation test results,which proves that the theoretical predicted value of the model fits well with the actual value.Therefore,this study can provide a reference for ultrasound-assisted extraction of flavonoids.The extraction yield of total flavonoids from ZSS was 1.590%,which was17.11%higher than that of heat reflux extraction.?2?In this study,we compared the antioxidant activities of ultrasound-assisted extraction total flavonoids?UTF?and heat reflux extraction total flavonoids?HTF?in vitro antioxidant activity assays.The results showed that the total flavonoid extracts of two kinds of ZSS showed strong total reduction ability and significant scavenging ability of free radicals such as OH?ABTS⁺?O^2-and DPPH.At the same time,flavonoids from ultrasound-assisted extraction showed stronger antioxidant activity than heat-reflux extraction of flavonoids.In conclusion,ultrasound-assisted extraction is a more efficient and green extraction method for flavonoids of ZSS.?3?In this study,the effects of 6-FS on paralysis time,longevity time,heat stress resistance and chemotaxis of GMC101 were investigated,and the mechanism of 6-FS against AD symptoms of Caenorhabditis elegans was discussed by using the results of reactive oxygen species?ROS?and autophagic lysosome microfluorescence staining.The results showed that 6-FS could significantly delay the paralysis time,prolong the lifespan,and enhance resistance to adverse environmental conditions such as high temperature and the chemotaxis ability of chemical perception of sodium chloride.The learning index of nematodes increased gradually and the ability of chemical perception was recovering slowly with the increase of concentration of 6-FS.6-FS significantly reduced ROS accumulation induced by A?_1-42 in GMC101.6-FS reduced the degree of amyloid protein aggregation and the degree of A?_1-42 injury.Restoring the autophagy level of GMC101,weakening oxidative damage and alleviating the AD symptoms of GMC101.?4?In order to investigate the effects of 6-FS on cell viability,ROS,mitochondrial membrane potential?MMP?,amyloid aggregation and autophagy-related protein expression of A?_1-42 oxidative damaged cells and its protective mechanism,AD cell model was established by PC12 cells.The results showed that A?1-42 could reduce cell viability,increase ROS content and aggregation,decrease MMP and the ratio of autophagic marker protein LC3II/I and increase the expression of autophagic receptor protein p62,and increase the expression of mitochondrial transporters Tim23 and Tom20,thereby reducing the level of autophagy in injured cells.6-FS can improve oxidative stress,mitochondrial oxidative damage induced by A?1-42,restore the normal expression of Tim23 and Tom20,and improve the protective effect of damaged mitochondria by reducing the accumulation of ROS in cells and restoring MMP.Increased LC3II/I ratio and decreased expression of autophagic protein p62,suggesting that 6-FS can significantly restore the level of autophagy of mitochondria.At the same time,the results of western blotting showed that the expression of autophagy regulatory protein Pink1 and Parkin in PC12 cells pretreated with A?1-42 decreased significantly compared with the blank group,suggesting that the autophagy regulatory level of PC12 cells was inhibited by A?_1-42.The expression levels of Pink1 and Parkin in PC12 cells pretreated with A?_1-42 increased significantly under the action of 6-FS,indicating that 6-FS could alleviate the damage of A?_1-42 to autophagy through Pink1/Parkin protein.It can be concluded that 6-FS protect damaged mitochondria by regulating Pink1/Parkin-mediated mitochondrial autophagy pathway and reduce the degree of oxidative damage caused by amyloid protein,alleviating AD symptoms.