Isolation,Purification,Structural Characterization Of Polysaccharide APS-1a From Astragalus Membranaceus And Its Activity Of Stimulating EPO Secretion In HepG2 Cells

Astragalus propinquus Schischkinis a commonly used traditional Chinese medicine.It was first published in the"Shen Nong’s Herbal Classic"of the Eastern Han Dynasty.It was originally named"Astragalus membranaceus"and has been recorded on the herbal classic.The dried roots of Astragalus are mainly produced in Inner Mongolia,Shanxi,Heilongjiang,Gansu,and Jilin province in China.The main components of Astragalus are astragalus polysaccharides(APS),astragaloside,trace elements and Amino acids,etc.Among them,astragalus polysaccharide is the main active ingredient in Astragalus,and it is a kind of substance which possesses the highest content and strong pharmacological effect in Astragalus.Therefore,studying the structure and pharmacological effects of astragalus polysaccharide is important for developing this kind of medicinal materials.In this study,an environmentally friendly and simple process of water extraction and alcohol precipitation method was used to extract crude astragalus polysaccharides.After repeated freeze-thaw protein removal,dialysis,and column chromatography were used to separate and purify,a refined astragalus polysaccharide was obtained and named as APS-1a.Its structure was analyzed by means of chemical analysis and spectral analysis,and its blood-reinforcing effect is preliminary explored.Part Ⅰ Preparation of Astragalus Polysaccharide APS-1aIn this part of the experiment,Astragalus produced in Inner Mongolia were used as raw materials.The crude extract of astragalus polysaccharide was obtained by water extraction and alcohol precipitation method.Through repeated freeze-thaw,dialysis,and column chromatography,a homogeneous refined astragalus polysaccharide APS-1a was obtained.It was a kind of white-cotton-floc substance and had soft texture and was easily soluble in water.The sugar content of APS-1a was 95.10%tested by phenol-sulfuric acid method.The uronic acid content of APS-1a was 84.22%calculated by the m-hydroxybiphenyl method,which meant that the polysaccharide was an acidic polysaccharide with a high uronic acid content.By high-performance gel permeation chromatography,a narrow and symmetrical single peak could be observed in liquid chromatography,which meant that the astragalus polysaccharide was refined and its molecular weight was 2.2×10~5 Da.Part Ⅱ Structural identification of APS-1aIn this part of the experiment,two chemical methods,complete acid hydrolysis and methylation analysis,along with infrared and nuclear magnetic spectroscopy were used to analyze the structure of APS-1a.The characteristics of the polysaccharide could be observed in the Fourier transform infrared spectrum scanning spectrum.By complete acid hydrolysis,pre-column PMP derivatization combined with high-performance liquid chromatography,the monosaccharide composition of APS-1a was detected.It was observed that it was mainly composed of galacturonic acid,arabinose,galactose and rhamnose,and the ratio of these four kinds of monosaccharides was calculated:galacturonic acid:arabinose:galactose:rhamnose=8.61:6.38:2.29:1.00.After the reduction of polysaccharides,through methylation analysis,6 different sugar residue connection methods and their content ratios were determined:(1)Araf-(1→(2)→2)-Araf-(1→(3)Galp-(1→(4)→3,4)-Galp-(1→(5)→2,4)-Rhap-(1→and(6)→4)-Gal(A)p-(1→.Combining one-dimensional(1H spectrum,13C spectrum)and two-dimensional(1H-1H COSY,HSQC,HMBC)nuclear magnetic resonance spectroscopy,the type and order of sugar chain attachment of APS-1a was further determined.The possible main repeated unit of APS-1a was speculated to be:Part Ⅲ Study on APS-1a Promoting Erythropoietin(EPO)Secretion in vitro:In the practice of traditional Chinese medicine,Astragalus is commonly used in many herbal formulas to treat various body disorders and diseases.As the main component of Danggui Buxue Decoction,it is clinically recommended as a formula for nourishing Qi and nourishing blood for the treatment of anemia.This article explores the pharmacological mechanism of blood initiation,and this section investigates the effect of astragalus polysaccharides on the secretion of erythropoietin(EPO)in vitro.Erythropoietin(EPO)is a kind of hormone which could maintain the survival,proliferation and differentiation of red blood cell progenitor cells in bone marrow.It is the main driving factors for red blood cell production,which is mainly produced by kidney and a small amount is produced by liver.This study found that APS-1a could significantly promote human liver cancer cell Hep G2 in the secreting EPO at the protein and gene levels in vitro,which provided new ideas for the mechanism research of its blood replenishing effect.