Constituents And Quality Control Of The Traditional Mongolian Medicine RUBI RAMULUS

Objective: To study quality control index components,quality control methods,and its impact on immune proliferation of Mongolian Medicine RUBI RAMULUS.Select the quality control index components through the chemical constituents system study and the fingerprints with HPLC-MS / MS analysis.Establishing identification and content determination methods and the quality of different origin,processing methods,cultivated materials and commercial materials of RUBI RAMULUS.The effects of aqueous extracts and different polar extracts of RUBI RAMULUS on the immune proliferation of splenic lymphocytes in mice were examined.This paper provides the basis for clarifying the pharmacological basis,establishing quality control method and ensuring the safe and reliable clinical application of RUBI RAMULUS.Methods: 1)Chemical composition isolation: The compounds in the Ethyl acetate fraction were isolated and purified by reversed phase chromatography,sephadex LH-20 column and reverse-phase preparative HPLC.The structures were identified by various spectroscopic data such as 1D、2D-NMR,HRMS.2)HPLC fingerprints were established by HPLC and the chemical constituents in the fingerprints of RUBI RAMULUS were identified by HPLC-MS / MS,the structure of the compounds was deduced by comparison with the mass spectrometric information of the first and second mass spectra and literatures.3)TLC identification and HPLC determination of catechins and its main components of caffeic acid and ellagic acid.4)The aqueous extract and different polar extract petroleum ether layer,chloroform layer,ethyl acetate layer,n-butanol layer and water layerof RUBI RAMULUS on the proliferation of mouse splenic lymphocytes was detected by CCK-8 method.Results: 19 compounds were isolated from theRubus sachalinensis Leveille,three new compounds were 1α,2β,3β,19α-tetrahydroxyurs-l2-en-28-oic acid β-D-glucopyranospylester(5),1,6-di-caffeoylglucopyranoside(11),Rubuside lignan A(14),Respectively.16 known compounds were Geranyl-1-O-α-L-Arabinofuranosyl-(1→6)-β-D-glucopyranoside(1),2α,3α,19α,24-tetrahydroxy-urs-12-en-28-oic acid β-D-glucopyranospyl ester(2),2α,3α,19α-trihydroxy-23-formyl-urs-12-en-28-oic acid β-D-glucopyranospyl ester(3),3α,19α,24-trihydroxy-urs-12-en-28-oic acid β-D-glucopyranospyl ester(4),2α,3β,19α-trihydroxy-23-formyl-urs-12-en-28-oic acid β-D-glucopyranospyl ester(6),2α,3β,19α,23-tetrahydroxy-urs-12-en-28-oic acid β-D-glucopyranospylester(7),2α,3β,19α-trihydroxy-23,28-dioic acid-urs-12-en-28-oic acid β-D-glucopyranospyl ester(8),Ellagic acid(9),Quercetin-3-O-β-D-butyl glucopyranoside(10),7S,8S-threo-3’,4,7,9-tetrahydroxy-5’,3-dimethoxy-8-O-4’-lignan-9’-O-α-L – rhamnopyranoside(12),7S,8S-threo-3’,4,9-trihydroxy-3-methoxy-7,8-dihydrobenzofuran-1’-propanol-based lignan-9’-O-L-rhamnopyranoside(13),P-Hydroxy-cinnamic acid(15),Esculetin(16),Caffeic acid(17),Epicatechin(18),Catechin(19).2)Established HPLC fingerprint method for the determination of RUBI RAMULUS and the preliminary fingerprints of RUBI RAMULUS from 9 different batches were studied.HPLC-MS / MS analysis identified 17 chemical constituents of RUBI RAMULUS fingerprint including 5 flavonoids,6 triterpenoid saponins,5 phenolic compounds and 1 esculetin.The dissociation law of triterpenoids was summarized.3)Accurate TLC identification and HPLC determination of caffeic acid,epicatechin and ellagic acid in 7 different batches of medicine.4)The activity results showed that the extract of RUBI RAMULUS has the effect of promoting the proliferation of splenic lymphocytes in mice,but petroleum ether layer,ethyl acetate layer,chloroform layer,n-butanol layer and water layer have no obvious lymphocyte proliferation.Conclusion: Compounds 1,3,4,5-12,18,19 wrer isolated from RUBI RAMULUS for the first time,compounds 5,11,14 were new compounds.The method of HPLC fingerprint and HPLC-MS / MS were established for the first time,which laid the foundation for the follow-up study on serum drug chemistry.The quality control method of RUBI RAMULUS was established for the first time.The first method of CCK-8 method was used to detect the proliferation of splenic lymphocytes in mice,and the polysaccharides were suggested to be the active substances.