Extraction And Purification Of Flavonoids From Euonymus Fortunei And Its Effect On Aging Model

Objective:Using modern Chinese medicine research methods,such as mouse model experiment,in vitro cell culture technology and other molecular biology experimental technology,explore the extraction process of Euonymus fortune,a common drug of zhuangyao nationality and study its effects on aging models to explore its possible Anti-aging mechanism,so as to improve the theoretical basis for further develop of new anti-aging and anti-aging related diseases drugs with clear mechanism.Methods(1)Ultrasound-assisted extraction-ethyl acetate extraction-D101 macroporous resin adsorption as extraction method.Through the single factor experiment,the optimal reaction temperature,the best material ratio and the optimal reaction time of the total flavonoids extracted from Euonymus fortunei were obtained.On the basis of this,Total flavonoids in Euonymus fortunei was used as the evaluation,the reaction temperature,the ratio of material to liquid and extraction time were the influencing factors.The orthogonal extraction experiment L9(3~3)method was used to optimize the extraction process to determine the optimal conditions for the extraction of total flavonoids from Euonymus fortunei.Under these conditions,the total flavonoids of Euonymus fortunei were extracted and purified.(2)A D-galactose-induced aging mouse model was constructed and 60clean Kunming mice were randomly divided into 6 groups.The normal group,the model group,the Astragalus polysaccharide-positive control group(40mg/kg),and the total flavonoid extract of Euonymus fortunei were high(0.8g/ml).,medium(0.4 g/ml),low(0.2 g/ml)dose group.After 6 weeks of feeding,examine the activities of catalase(CAT),total antioxidant capacity(T-AOC),and glutathione peroxidase(GSH-PX)in serum,myocardial tissue,and liver tissue of each group of mice.(3)Extraction and purification of flavonoids from Euonymus chinensis by high performance liquid chromatography.(4)Angiotensin ?(Ang-?)-induced senescence model of human umbilical vein endothelial cells(HUVEC)was used as a pharmacodynamic research object,and cell proliferation activity was detected by MTT assay,cellular senescence was detected by?-galactosidase assay,to study the effects of different concentrations of flavonoids solution on the aging model cells.Result(1)The optimum conditions for the extraction of total flavonoids from Euonymus fortunei were determined by orthogonal test:extraction temperature(60°C),ratio of material to liquid(1:50),extraction time(30 min).Total flavonoids in Euonymus fortunei finally extracted according to this condition reached 3.57%.Under the conditions of large-scale extraction,the purity of total flavonoids of Euonymus fortunei was 68.7%after final purification.(2)The D-galactose-induced aging mouse model was successfully constructed.In this experiment,the mice were given subcutaneous injection of D-galactose into the neck.After 4 weeks,the aging signs such as arch back,slow weight gain,rough hair and decreased activity were gradually appeared.,and over time,the more obvious the sign,the successful modeling.Through the drug test,compared with the negative group,the high,medium and low dose groups of the total flavonoids of Euonymus fortunei and the positive group could improve the color and reactivity of the mice,and the total flavonoid extract of Euonymus fortunei could improve the aging model mice.The levels of T-AOC,GSH-PX and CAT in serum,heart tissue and liver tissue indicate that the total flavonoids of Euonymus fortunei can effectively improve the body of mice and have a certain anti-aging effect.(3)The final separation and purification by HPLC gave three flavonoid monomers:FFT006,FFT007,and FFT008,and the purity was over 95%.(4)The angiotensin ?-induced senescence model of human umbilical vein endothelial cells was successfully constructed,and the optimal concentration of angiotensin ? stimulation was determined to be 10~-66 g/ml,and the optimal time was 12 h.The senescence cell model reacted with three flavonoids.After MTT drug toxicity test,FFT006,FFT007 and FFT008 all promoted cell proliferation,and the concentration of 10?g/ml was the highest to promote cell proliferation.effect.Compared with the model group,the survival rate of the positive group and the FFT006,FFT007,and FFT008 groups were significantly improved by?-galactosidase assay.Conclusion:The extracted and purified total flavonoids of Euonymus fortunei showed anti-oxidation effect on D-galactose-induced aging mice and had anti-aging effects.Purified and purified flavonoids can promote the proliferation of HUVEC cells induced by Ang-? and have anti-aging effects.