Analysis Of Polyphenols In Osmanthus Fragrans And Study On Anti-tumor Activity Of Ergosterol Peroxide

Chapter 1: Analysis of polyphenols in three osmanthus varietiesThe polyphenols in three osmanthus cultivars(Jingui,Dangui and Yingui)were analyzed and identified by HPLC-MS/MS.The liquid chromatographic conditions were as follows: column shim-pack vp-ods(2.0×150 mm,5 μm),methanol-0.1% formic acid water as mobile phase,gradient elution,volume flow rate of 0.2 m L/min,column temperature of 30 ℃,injection volume of 5 μL.The mass spectrometry conditions were: ESI ion source,multi-reaction monitoring(MRM)with negative ion scanning mode for detection.The results showed that all three osmanthus varieties contained 7 polyphenol active ingredients(chlorogenic acid,caffeic acid,verbascoside,naringin,rutin,ferulic acid,quercetin);Chlorogenic acid,quercetin and mullein glycosides were the main components in the three osmanthus varieties,while ferulic acid,naringin,rutin and caffeic acid were the auxiliary components.Among the same varieties,the content of verbascoside was the highest;the naringin in Dangui and Jingui was the lowest,and the content of caffeic acid in Yingui was the lowest.In addition,all the 7 polyphenols had good linear relationship,precision,repeatability and stability in the concentration range.The test results meet the requirements of methodology inspection.The method established by the research institute is simple,rapid,sensitive and specific.It has certain theoretical guidance and methodological reference for the simultaneous comparison and determination of polyphenol active ingredients in various osmanthus flowers.Chapter 2:Isolation and purification of ergosterol peroxide from osmanthus fragrans and study on its anti-tumor activityThe dried osmanthus pollination was extracted by 80% ethanol,and the ethanol extracts were successively extracted by petroleum ether,ethyl acetate and n-butanol,and then separated and purified by AB-8 macroporous resin,polyamide resin and liquid phase preparation chromatography.One compound was isolated from the ethanol extract of Osmanthus fragrans,and the structure was identified by physicochemical constant,nuclear magnetic resonance,mass spectrometry and infrared spectroscopy.The compound was peroxidized ergosterol.MTT method was used to detect the effect of ergosterol peroxide on the inhibition of cell proliferation,and flow cytometry was used to detect the effect of ergosterol peroxide liver cancer on the apoptosis and cell cycle of Hep G2 cells.The results showed that the inhibition concentration of ergosterol peroxide on tumor Hep G2,He La,A549 and Mc F-7 cell lines was 56.5,70.6,112.7 and 75.2 M,respectively,which had certain anti-tumor activity,among which the inhibition effect on liver cancer Hep G2 cells was the best,but the activity was lower than vp-16.The induction rate of apoptosis of hepatocellular carcinoma Hep G2 was up to 34.7% at a concentration of 60 μM peroxidized ergosterol,which had a certain ability to induce apoptosis,but no cell cycle arrest.