Study On Isolation And Identification Of Active Ingredients From Rhodiola Rosea And Their Bioactivities

Rhodiola rosea,belonging to the family crassulaceae,is a perennial herb mainly distributed in xinjiang,with the title of"highland ginseng".The main ingredients are saponins,polysaccharides,flavonoids and multivitamins.Recent pharmacological studies have indicated that R has various bioactivities such as anti-hypoxia,anti-fatigue,anti-aging,anti-depression and immune regulation,and has been recognized as a health medicine and food resource.At present,researches of the Rhodiola rosea are focused on the extraction process of active components and separation of salidroside,there are few researches on other ingredients and pharmacological activies.Based on anti-aging activity as the research key,Rhodiola rosea had the best effect from four natural products.Starting from the chemical composition and biological activity of Rhodiola rosea,its active ingredients were separated and purified,and the anti-aging activity was focused on.The total crude ethanol extracts of Polygala tenuifolia,Suanzaoren,Radix ophiopogonis and Rhodiola rosea were obtained by the extraction of heating reflux with 75%ethanol.After fractional extraction,four polar fractions?petroleum ether phase,ethyl acetate phase,n-butanol phase,and water phase?were obtained.UVB irradiation damage models were established for HSF and HaCaT cells to determine cell irradiation energy.The skin cells were screened for anti-photo aging for 5 samples of 4 varieties,and the best one is Rhodiola rosea.Different biological models?including anti-oxidation,anti-tumor,anti-aging of cells and celegans experiments?were used to evaluated the activities of crude ethanol extract and four parts of Rhodiola rosea.Radical-scavenging and antioxidant activities were evaluated by DPPH,ABTS and FRAP assay,the results showed that samples of Rhodiola rosea had strong antioxidant activity,especially ethyl acetate phase and n-butanol phase;The anti-tumor results that at low concentrations,there is almost no effect;Anti-aging,the effect of the ethyl acetate fraction and the n-butanol fraction of R are the most obvious.Considering the results comprehensively,it was confirmed that the main active ingredients were concentrated in the ethyl acetate fraction and n-butanol fraction after solvent extractions of Rhodiola rosea.Therefore,the ethyl acetate fraction and the n-butanol fraction were the focus of further research.In order to analyze the active ingredients of Rhodiola rosea comprehensively,four chemical constituents?saponins,flavonoids,polysaccharides,and volatile oils?were obtained from R by different extraction methods.Their contents were determined by standard curves of four reference substance,be 56.82%,44.14%,55.58%,and the extraction rate of volatile oils is0.12%.In addition,the antioxidant,antitumor and anti-aging activities of the four chemical constituents were investigated.Results showed that the antioxidant activity of flavonoids and saponins is better than polysaccharides and volatile oils;the anti-tumor effect of the four is not obvious;In the anti-aging activity,the effect of saponin is the best,and is better than the positive control.The effect of polysaccharide and volatile oil is general.The chemical compositions of ethyl acetate and n-butanol fraction were analyzed by silica gel column chromatography,Sephadex LH-20 gel column chromatography,polyamide column chromatography and ODS column chromatography.Four compounds were obtained and their structures were characterized via physicochemical properties and modern spectroscopic techniques(including MS,¹H-NMR,¹³C-NMR),namely Kaempferol,arbutin,salidroside and rhodiola.Antioxidant,antitumor and anti-aging activities of the four compounds were investigated.Results showed that the antioxidant activity of monomeres was strong,and kaempferol was better than the positive control,arbutin effect was comparable to the positive control.The order was:kaempferol>arbutin>salidroside>salidroside;In the antitumor activity,there are no killing effect on cancer cells at low concentrations,and at high concentrations,salidroside exhibits strong anti-hepatocarcinoma and anti-lung cell capacity,which is stronger than R;In the photoaging antiaging activity,monomers showed strong photoaging repairing ability.Among them,the ability of kaempferol and arbutin were better than the positive control,and salidroside and rhodiolacin were slightly weaker than positive control,the effect are very strong and worth studying in-depth.Finally,the effect of monomeres on the injured HSF cells were studied,to measure the inflammatory cytokines in the cell supernatants to further determine the mechanisms of photoaging and aging of cells.The results showed that:compared with the blank group,a certain amount of UVB irradiation had cause an increase in inflammatory cytokines.The levels of IL-6 and TNF-?in the supernatant of the model group were significantly higher than those in the blank group;Compared with the model group,IL-6 and TNF-?in the cell supernatant of the samples group gradually decreased with increasing concentrations,that indicating monomeres can indeed reduce the release of inflammatory cytokines IL-6 and TNF-?.This may be the first step for the monomeres to act as a photoaging antiaging agent,and it acts as an anti-aging agent by reducing the release of inflammatory cytokines to hinder the expression of its genes and proteins,and the further anti-aging mechanism of related genes and proteins remains to be followed study.