Study On The Separation Of Chemical Constituents And Content Determination Of Active Ingredients Of Tinospora Sinensis (Lour.) Merr.

Tinospora sinensis?Lour.?Merr.the Tibetan name Lezhe,is a dry cane of the Chinese green stenan in the genus Biliary,and is a commonly used medicinal material in Tibetan medicine.It mainly produced in southeastern Tibet,Hainan,Yunnan Guangdong,Guangxi and other places.It has anti-inflammatory,liver-protecting,anti-oxidation,immune regulation and other pharmacological effects.The main chemical components are sesquiterpenes,diterpenoids,phenylpropanoids,lignans,alkaloids and other compounds.The results of the research literature showed that most of the studies were focused on the identification of chemical constituents,few were quantitative detection of chemical constituents,and there was no report on the basis of pharmacological effect and mechanism of action on AD prevention and control of Tinospora sinensis?Lour.?Merr.It is a commonly used medicine for preventing and curing senility in Tibetan areas,but its basic research is insufficient and its material basis is not clear.With the support of the National Natural Science Foundation of China,this research group will study the anti-aging mechanism and active ingredients of Tinospora sinensis?Lour.?Merr.Previous experiments have confirmed that the extract of Tinospora sinensis?Lour.?Merr.extract,chloroform extract and ethyl acetate extract have obvious anti-aging effects,and some active components of chloroform are separated.The main contents and results of this paper are as follows: 1.The study on the chemical constituents of Tinospora sinensis?Lour.?Merr.In this paper,the extract of ethyl acetate extract from Tinospora sinensis?Lour.?Merr.was systematically separated and purified by silica gel column chr-omatography,ODS column chromatography,Sephadex LH-20 gel column chromatography,and preparative HPLC.By using MS,UV,1H-NMR,¹³C-NMR and other spectroscopic techniques combined with literature analysis,and 10 compounds were obtained,vanillic acid?1?,vanillin?2?,4-Hydroxybenzoic acid?3?,3-hydroxy-1-?4-hydroxy-3,5-dimethoxyphenyl?propan-1-one?4?,syringic acid?5?,daucosterol?6?,?-sitosterol?7?,5-hydroxymethyl furfural?8?,l-?2-formyl-l-pyrro lyl?butanoic acid?9?,5-Ethoxymethyl-1-[3'-?N-methyl-hydrazino?-propyl]-1H-pyrr ole-2-carbaldehyde?10?.Among them,compound 10 is a new compound,and compounds 4,5,8,and 9 are isolated from the plant for the first time.2.Analysis and identification of the ethyl acetate fraction of Tinospora sinensis?Lour.?Merr.based on UPLC-ESI-Q-TOF-MS/MSIn this paper,the extract of ethyl acetate extract of Tinospora sinensis?Lour.?Merr.was roughly chopped into 23 fractions by silica gel column chromatography.The chemical composition of ethyl acetate extract of Tenospora sinensis?Lour.?Merr.was analyzed by UPLC-ESI-Q-TOF-MS/MS technology.According to its polarity and separation under positive and negative ion mode,12 fractions were selected for analysis,and 492 compounds were identified.The identified chemical constituents are heterocyclic compounds:86;alkaloid: 146;terpenoids:2;glycosides: 8;flavonoids: 2;saccharides: 8;steroids: 6;terpenoids:1;organicacid: 111;other compounds: 122.Where in the alkaloid comprises anamide alkaloid: 79;a pyrrolidine alkaloid: 7;an organic amine alkaloid: 18;animidazole alkaloid: 14;a thiol alkaloid: 10;a piperidine alkaloid: 4;Indolealkaloids:9;indole alkaloids: 4;Pyrimidine alkaloids:1.3.Determination of active ingredients in Tinospora sinensis?Lour.?Merr.In the early stage of this research,MTT method was used to detect the AD injury model of PC12 cells induced by A?_25³5.The results showed that syringin could significantly improve the survival rate of PC12 cell AD injury model induced by A?_25³5.Therefore,a method for the determination of syringenin Tinospora sinensis?Lour.?Merr.was established,which laid an experimentalfoundation for objective evaluation of the quality of Tinospora sinensis?Lour.?Merr..A Waters Symmetry C18?4.6 mm × 250 mm,5 ?m?column was usedwith acetonitrile?A?-water?B?as the mobile phase,gradient elution?0-8 min,5% A-10% A;8-9 min,10% A-11% A;9-20 min,11% A-11% A?,flow rate was 1.0m L/min,injection volume was 10 ?L,detection wavelength was 265 nm,and column temperature was 25 ?.The above established chromatographic conditions were used to determine the content of syringin in 8 batches of Tinospora sinensis?Lour.?Merr.collected in different areas and at different times.The results show that the method is simple,stable and reliable,which providesan important reference value for the quality control of Tinospora sinensis?Lour.?Merr.