Structural Characterization,in Vitro Digestion And Fermentation Properties Of Polysaccharide From Semen Astragali Complanati

Semen astragali complanati,a kind of leguminous plants,is the dried ripe seed of Astragalus complanatus R.Br.It is traditional Chinese Medicine distributed in Shaanxi and also a kind of raw materials of health foods in our country.Current studies showed that Semen Astragali complanati had antihypertensive,hypolipidemic,anti-tumor,anti-oxidation,anti-fatigue and other biological effects.Polysaccharide is a kind of complex biological macromolecule formed by more than 10 monosaccharides connected through glycosidic bond.Due to its various bioactive functions and physicochemical properties,it has been widely used in food industry,medical and health care,material field and other areas.In this study,we used Semen Astragali complanati from Weinan,Shaanxi Province,as the raw materials,separated and purified the polysaccharide fractions.Then these fractions were conducted to investigate the physicochemical properties,molecular weight distribution and monosaccharide composition,etc.,and the main purified fraction was further characterized the structural properties and solution conformation.Finally,the characteristics of in vitro digestion and fermentation of the polysaccharides from Semen Astragali complanati（SACP）were also studied.The results of this study will help to provide theoretical basis for the development and utilization of Semen Astragali complanati resources,and the main research contents and conclusions were summarized as follows:（1）Water extract and ethanol precipitation,as well as deproteinization were applied to obtain the polysaccharides from Semen Astragali complanati（SACP）,with the yield of 5.96%,and total sugar content of 80.81%.SACP-20,SACP-25 and supernatant（SACP-25-S）were obtained using gradient ethanol precipitations to fractionate SACP.Their total sugar contents were 56.20%,87.40%and 39.51%,respectively.Their respective uronic acid contents were 1.67%,1.06%and 23.62%and protein contents were 6.15%,0%,and 3.99%.Monosaccharide composition analysis showed that the major monosaccharides in SACP were mannose（47.08%）,galactose（45.57%）,arabinose（3.03%）,xylose（1.76%）,glucose（0.29%）and glucuronic acid（1.99%）.For SACP-20,it consisted of mannose（54.02%）,galactose（45.98%）with trace amounts of glucose and arabinose.SACP-25 was only composed of mannose（57.08%）and galactose（42.92%）.SACP-25-S was composed of mannose（36.62%）,galactose（25.45%）,xylose（15.69%）,arabinose（6.29%）,glucuronic acid（19.55%）and trace amounts of glucose.Molecular weight distribution of SACP and its purified fractions was analyzed by high performance gel permeation chromatography,and all of them showed similar molecular weight distribution,but only SACP-25 had a single symmetrical peak in chromatograms that means good homogeneity.The molecular weight of SACP-25 was 1.17×10⁶ Da.The infrared spectra showed that acetyl groups existed in SACP and SACP-25-S,indicating that both of them contain ED uronic acid.（2）The detailed structure properties of SACP-25,including the types of glycosidic bond,anomeric configuration and sequences of sugar units.Methylation analysis,combined with gas chromatography-mass spectroscopy（GC-MS）indicated that the main glycosidic bonds in SACP-25 were 1,4-linked-Manp,1,4,6-linked-Manp and T-Galp,with the contents of 27.67%,32.02%and 32.76%,respectively.From the result of NMR spectrum,SACP-25 was demonstrated as a kind of galactomanans,with the main chain is composed ofβ-1,4-linked-Manp and T-Galp as side chains that attached to C-6 of the Manp residues.Size exclusion chromatography coupled with muti-angle laser light scattering,differential pressure viscometer and refractive index detectors technology was used to study the solution conformation of SACP-25.The number-average molecular weight M_n of SACP-25 was 4.873×10⁵ Da,the weight-average molecular weight M_w was1.040×10⁶ Da,the Z-average molecular weight M_z was 1.895×10⁶ Da,and the exponentαof Mrak-Houwink equation was 0.515.By fitting the log function of root mean square radius of rotation<R_g>_z and the hydrodynamic radius R_h to M_w,the conformational parametional parameters v_g and v_h of the two were 0.48 and 0.51,respectively.The results indicated that the high-level structure of SACP-25 in 0.1 M Na NO₃ is a random coil comformation.（3）The in vitro gastrointestinal tract digestion and fermentation characteristics of SACP and SACP-25 were studied through the static in vitro digestion and in vitro fermentation models.The results of in vitro digestion showed that the molecular weight distribution of SACP and SACP-25 was basically unchanged after gastrointestinal digestion,and there was no free monosaccharide produced.This indicated that the polysaccharides could not be directly digested and absorbed by humen digestive system.Simulating the metabolism of polysaccharides in large intestine by researching in vitro fermention.Compared with the blank control group,SACP-25 and SACP could reduce the p H value of the fermentation broth of intestinal flora,and the total sugar content decreased significantly during the process of fermentation indicating that intestinal microflora could use polysaccharides as carbon source which had potential of prebiotics in regulating human intestinal health,and it had increased the amount of short-chain fatty acids significantly such as acetic acid,propionic acid and butyric acid to promote the growth of human intestinal flora.