Preliminary Study On Isolation And Purification Of Total Flavonoids From Lycium Ruthenicum Murr. And Its Lipid-lowering And Antihepatocarcinogenic Activity

Objection:The response surface method was used to isolate and purify the total flavonoids of Lycium ruthenicum Murr.,to establish HLP model to investigate its lipid-lowering activity and to preliminarily study the inhibitory effect of total flavonoids of Lycium ruthenicum Murr.on liver cancer in vitro.Method:1)Through static and dynamic adsorption experiments,the sample concentration,sample volume,sample pH,eluent concentration,eluent liquid volume and eluent flow rate were investigated as factors,and the content of total flavonoids of Lycium ruthenicum Murr.was investigated as an indicator for single factor investigation.On this basis,a three-factor,three-level Box-Behnken experimental design was selected to screen and validate the parameters of the total flavonoid purification process of Lycium ruthenicum Murr..2)A model of HLP in SD rats was established by gavage of 90 SD rats on high-fat diet.And 60hyperlipidemic SD rats were randomly divided into 6 groups?n=10?:normal control group,HLP model group,Simvastatin group?3.3 mg/kg?and the high,medial and low doses of total flavonoids treated groups.The normal rats were fed with normal diet for 4weeks and the other groups were fed with high fat diet.After administration,the concentrations of TG,TC,HDL-C and LDL-C in serum and liver tissue fluid and the serum concentrations of MDA,SOD and GSH-Px were detected by enzyme labeling instrument.Meanwhile the change rate of body weight and liver index were measured and the liver lesions of SD rats were observed after the trial.3)The MTT method was used to determine the survival rate of LO-2 and the inhibition rate of HepG-2 at different concentrations of total flavonoid purified from Lycium ruthenicum Murr.to determine the optimal administration time and concentration,and the three identified concentrations of total flavonoid purified?25,50 and 100?g/mL?and 20?g/mL cisplatin were administered for 48 h,followed by flow cytometry to detect apoptosis and cycle changes,and the activity of apoptosis-related enzymes was detected by ELISA method.Result:1)NKA-9 macroporous resin was the best resin for the purification of total flavonoids from Lycium ruthenicum Murr..The optimum purification conditions were as follows:the concentration of total flavonoids was 4 mg/mL,pH was 4.0,the sample was added at 5 BV,and eluted with 80%ethanol of 3 VB at the flow rate of 2 mL/min,the content of total flavonoids of Lycium ruthenicum Murr.could be increased from 4.54%to43.19%.2)Compared with the HLP model group,the levels of TG,TC and LDL-C in the serum and liver tissue of the high and middle dose groups were significantly lower than those of the model group while the values of HDL-C were elevated?P<0.01?.Compared with the HLP model group,the weight gain rate and liver index were significantly reduced in SD rats with high and medium dose group of total flavonoids from Lycium ruthenicum Murr.,the difference was statistically significant?P<0.05?.The serum concentrations of SOD and GSH-Px increased and MDA decreased significantly compared with the HLP model group?P<0.01?.3)MTT results showed a gradual decrease in LO-2 survival with time to administration and increasing concentration.At low concentrations?Con.?100?g/mL?the drug was less cytotoxic to LO-2 and cell survival was more complete;The inhibition rate of HepG-2 increased with time and concentration of administration.At high concentrations,HepG-2 cells showed massive deformation and apoptosis.With increasing drug concentration,the ratio of G₀/G₁ cells in the HepG-2 cell cycle gradually decreased and increased in the S phase,and the apoptosis rate of HepG-2 cells also increased,with the highest apoptosis rate reaching 78.8%,and the Bax and Caspase-3 enzyme activities associated with apoptosis increased,while the Bcl-2 enzyme activities decreased.Conclusion:1)The purification process parameters of total flavonoid of Lycium ruthenicum Murr.were accurate and reliable,and the content of the three batches of samples prepared by this process was basically stable,with an average value of 43.19%and RSD value of 1.12%,suitable for industrial production.2)Total flavonoid extract of Lycium ruthenicum Murr.can effectively regulate the body lipid metabolism and improve the body's antioxidant capacity,which has certain application value in the prevention and treatment of hyperlipidemia.3)Total flavonoid purified from Lycium ruthenicum Murr.inhibits the proliferation of HepG-2 cells,the inhibition rate could be as high as 98.27%.Meanwhile,it may induce apoptosis of HepG-2 cells by up-regulating Bax and Caspase-3 enzyme activities and down-regulating Bcl-2 enzyme activities.Lycium ruthenicum Murr.total flavonoid purification induces HepG-2 cell cycle blockade in S phase,which in turn stops cell division.