Separation And Purification Of Flavonoids In Malus Toringoides (Rehd.) Hughes Leaves With Macroporous Resins And Evaluation Of Anti-inflammation Activities

There are many flavonoids in the leaves of Malus toringoides(Rehd.)Hughes,which have anti-inflammatory,anti-oxidation,anti-cancer and liver protection effects.However,the purity of flavonoids in the crude extracts of the leaves of Malus toringoides(Rehd.)Hughes is not high,and there are few reports on the separation and purification process of total flavonoids from the leaves Malus toringoides(Rehd.)Hughes(TFMT),which limits its further development and utilization.In this study,the fresh and dried leaves of Malus toringoides(Rehd.)Hughes from Qamdo,Tibet province were selected to investigate the separation and purification process of flavonoids.The structure identification and anti-inflammatory activity of the purified TMFT were studied.The purpose of this study is to provide a theoretical basis for the comprehensive utilization and development of the leaves of Malus toringoides(Rehd.)Hughes.In this study,12 macroporous resins with different polarities were used to separate and purify TFMT,and AB-8 macroporous resins with higher adsorption and desorption ratio were screened by comparing the static adsorption and desorption ability of 12 resins.Afterwards,the adsorption kinetics,desorption kinetics on AB-8 macroporous resins,and the influence of sample concentration,sample solution pH,temperature and ethanol concentration on its static adsorption and desorption were investigated.The optimum process for static adsorption and desorption of TFMT by AB-8 macroporous resin was obtained: adsorption equilibrium time of 180 min,adsorption temperature of 25 ℃,sample solution concentration of 15 mg/ml,sample solution pH of 4.0;desorption equilibrium time of 180 min,and desorption concentration of 70% ethanol.By investigating the effects of flow rate,sample volume,flow rate of eluent,volume of eluent and concentration of eluent on the dynamic adsorption and desorption of TFMT on AB-8 macroporous resin,the optimum process of dynamic adsorption and desorption was obtained: flow rate of uptake of 2.0 BV/h,volume of uptake of 9BV(144ml),concentration of uptake solution of 15 mg/ml,sample solution pH of 4.0,and eluting speed of 2.0 BV/h used with 20% ethanol solution as the favourable strippant.Under such circumstances,the content of total flavonoids in the fractions collected is the highest,and the purity of TFMT was 57.86%,which was 2.1 times higher than that of total flavonoids from crude extracts.FT-IR spectrum was used to verify the existence of purified TFMT The characteristic absorption peaks of flavonoids were found by FT-IR spectrum,which verified the hypothesis that the purified TFMT contained a large number of flavonoids.By establishing an inflammation model of macrophage RAW 264.7 induced by LPS in vitro,the effects of TFMT on the viability of RAW 264.7 cells were investigated by MTT assay,the NO content in the supernatant of induced macrophages was determined by Griess method,and the contents of IL-6,IL-1β and TNF-α in the supernatant of induced macrophages were determined by ELISA kit.The results showed that the concentration of TFMT had no effect on the cell viability,and TFMT could inhibit the excessive production of NO,IL-6,IL-1β and TNF-α in LPS-induced peritoneal macrophage inflammation model in mice at an effective dose-dependent manner.When the concentration of TFMT was more than 100 μg/ml,the total flavonoids could inhibit the above-mentioned pro-inflammatory factors and the inhibition had significant difference(P < 0.05).These results suggest that TFMT may have anti-inflammatory activity in vitro.