| Rationales:Traditional Chinese medicine injection is a combination of modern pharmaceutical preparation technology and traditional Chinese medicine theory,which has unique curative effect.The treatment is used by approximately 400 million people every year in China,and the rate is increasing by 30%per year.However,with the rapid growth of the use of traditional Chinese medicine injections in recent years,the adverse reactions caused by this dosage form have also increased rapidly.Astragalus polysaccharides for injection(APS)is a traditional Chinese medicine intravenous powder injection developed by the Shanxi Research Institute of Traditional Chinese Medicine in the early 1990s on the basis of the advantages of astragalus resources in Shanxi Province.APS aims to improve the leukocytes level of patients undergoing chemotherapy and enhance their immune function.Its role in increasing leukocytes is stable and sustainable,and comparable to that of the clinically preferred imported drug Human Granulocyte Colony Stimulating Factor.APS is the first Chinese patent medicine to improve the hemogram of patients undergoing chemotherapy comprehensively.Since the drug was developed for injection at that time,a part of the astragalus polysaccharide with good water solubility(molecular weight range of about 10 kDa to 1000 kDa)was obtained by multiple alcohol precipitation.However,although the fingerprints are used for APS quality control,as a macromolecular polar substance group,the drug's specific molecular conformation,medicinal effect components and pharmacological mechanism are not clear,and even adverse reactions will occur,so it is difficult to enter the international market.Therefore,how to find a compound with a single structure,a clear chemical structure,and a clear pharmacological mechanism has become the key to the secondary development of APS.Objective:According to the fingerprint,the drug was separate into two parts and characteristic structure was analyzed.And activity screening was performed based on the innate and adaptive immunities function of APS at the cell level and the immune mechanism was further analyzed using cell metabolomics technology.Activity verification was performed at the animal level.Thereby,the active polysaccharide was screened out,and its structure-activity relationship was clarified.The research results not only provide the material basis for the screening of new drugs with guaranteed efficacy and risk reduction,but also provide molecular models for in-depth research on glycobiology.Method:APS was separated into two polysaccharides with different molecular weights by gel filtration chromatography.The relative molecular mass of the polysaccharide was determined by high-performance gel chromatography,and the monosaccharide composition was determined by high performance liquid chromatography(HPLC-UV).The connection sites between the monosaccharides were determined by the methylation method,the characteristic functional groups were analyzed by infrared spectroscopy(IR),and the glycosidic bond configuration was determined by nuclear magnetic resonance spectroscopy(NMR).Screening of the immune activity of polysaccharides of different molecular weight in APS at the cellular level includes determination of phagocytic activity of macrophages,killing activity of NK cells,proliferation of T and B lymphocytes,and IgG secreted by B lymphocytes.LC-MS metabolomics was used to study the mechanism of active polysaccharides regulating macrophage metabolism.Animal level activity determination includes determination of leukocyte levels,immune organ index,immune cytokine levels,and T lymphocyte subsets in cyclophosphamide immunosuppressed mice.Results:1.Dextran gel(Sephadex G-100)was used to separate APS into two parts with different molecular weights,named APS-?(>500 kDa)and APS-?(10 kDa).2.Chemical structure analysis of two polysaccharides with differen t molecular weights showed that APS-I consisted of glucose,galactose,arabinose,rhamnose,and galacturonic acid,with a ratio of approximat ely 1.5:1:5.4:0.08:0.1.And monosaccharide residue was linked as follow s:D-Glcp-(1?,?4)-D-Glcp-(1?,?2)-L-Rhap-(1?,D-Araf(1?,?5)-D-Araf-(1?,?2,5)-D-Araf-(1?,?4)-D-Galp-(1?.Meanwhile,APS-I I consisted of glucose,galactose,arabinose,rhamnose,and galacturonic acid,with a molar ratio of 9:1:1.4:0.04:0.001.And monosaccharide res idue was connected as follows:?-D-Glcp-(1?,?4)-?-D-Glcp-(1?,?6)-?-D-Glcp-(1?,?4,6)-?-D-Glcp-(1?,?3,4,6)-?-D-Glcp-(1?,?2)-?-L-Rhap-(1?,?-D-Araf-(1?,?5)-?-D-Araf-(1?,?4)-?-D-Galp-(1?.3.Screening experiments of the activity of polysaccharides at the cell level showed that APS-? was better than APS and APS-? group in promoting the phagocytic activity of macrophages and enhancing the killing activity of NK cells.APS-? is superior to APS and APS-? group in promoting the proliferation of T and B lymphocytes and the level of IgG secreted by B lymphocytes.4.After the active polysaccharide APS-? was applied to Raw 264.7,41 different metabolites were found inside and outside the cell,mainly related to amino acid metabolism,energy metabolism and antioxidant effects,suggesting that the active constituents of APS may be involved in the regulation of the above physiological processes.5.Verification experiments of the activity of polysaccharides at the animal level showed that APS-? can increase the levels of leukocytes and immune organs,increase the levels of cytokines IL-2,IL-6,and GM-CSF in cyclophosphamide immunosuppressed mice,and adjust the levels of CD4+,CD8+,CD4+/CD8+.Conclusion:APS was separated and purified to obtain two polysaccharides with molecular weights>500 kDa and 10 kDa.The structures of these two polysaccharides were analyzed by monosaccharide composition analysis,methylation analysis,IR and NMR analysis.Cell experimental analysis showed that APS-? can improve the body's innate immunity and adaptive immunity.The in vivo experiments showed that APS-? can increase the leukocytes level and immune effect of mice after chemotherapy.And it mainly regulated the metabolism of macrophages through amino acid metabolism,energy metabolism and antioxidant effect.Therefore,APS-? is the main active ingredient of APS,which is expected to become a new generation of APS. |
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