Extraction,separation,structural Analysis And Anti-tumor,anti-biofilm Activity Of Hemicellulose From The Residue Of Astragalus Root

RATIONALES: Astragalus,one of the most important medicinal materials in China.Annual consumption of astragalus in China is over one million tons,so there is a large amount of Astragalus residue every year.At present,the treatment of traditional Chinese medicine dregs has been concentrated on making base materials for edible fungi,making fertilizers,and animal feed,but these products have low added value and may even cause secondary pollution of the environment during the production process.The residue of traditional Chinese medicine is mostly plant cell wall component.Modern research has proved that many active polysaccharides are contained in plant cell wall component(pectin,hemicellulose).Hemicellulose contains the most active polysaccharides,so it is called the potential "drug repository".Zhou Sumei et al.found that the antitumor activity of arabinoxylan in hemicellulose of wheat bran cell wall was relatively high.Previously,our group found that the hemicellulose contained in the slag of Astragalus membranaceus contains mainly monosaccharides such as arabinose and xylose,and it is speculated that the polysaccharide component may have anti-tumor and other biological activities.Therefore,the development of polysaccharide products with anti-tumor and other biological activities from the Astragalus residue is of great significance for the full utilization of the Astragalus resources.OBJECTIVE: To extract and purify hemicellulose from residue of Astragalus Root,its structure was analyzed,and its antioxidant activity,anti-tumor activity and anti-biofilm activity were evaluated.It provids a theoretical basis for the comprehensive development and utilization of polysaccharides in residue of Astragalus Root.METHOD&CONTENT : The hemicellulose was extracted fromresidue of Astragalus Root by fractional extraction method,and the extracted hemicellulose was purified by ion exchange chromatography and gel filtration chromatography.The relative molecular mass was determined by GPC.The monosaccharide composition was determined by GC-MS.The site of the monosaccharide was determined by methylation and the structure was characterized by IR and NMR.In vitro antioxidant activity of hemicellulose were evaluated.The inhibitory effect of hemicellulose on the proliferation of human lung adenocarcinoma cell A549 and human normal lung epithelial cell BEAS-2B were evaluated.An in vitro biofilm model was established and used to screen anti-biofilm actives.The damage effect of hemicellulose and microbial metabolites on biofilm of S.aureus and C.albicans was evaluated.RESULTS: Residue of Astragalus Root were separated and purified to obtain four components(AX-I-1,AX-I-2,AX-I-3,AX-I-4).The structure of polysaccharide AX-I-1,AX-I-2,AX-I-3 and AX-I-4were studied.The results showed that AX-I-1 contained rhamnose,arabinose,xylose,mannose,glucose and galactose in a molar ratio of 0.006: 14.113:8.284: 0.116: 0.468: 1.The main chain consisted of arabinose and xylose through β-(1→2),(1→3)and(1→4)glycosidic bonds,and the branch consists of(1→ 4)βArap(1→ 3)βGalp and(1→2)βMan composition,non-reducing end consisting of αRhap,βGclp and βGalp.AX-I-2 contained arabinose and xylose in a molar ratio of 11.2: 8.9.The connection method of monosaccharide residues in AX-I-2 was: →4)-D-Xylp-(1→,→4)-D-Arap-(1→.AX-I-3 contains arabinose,xylose and glucose in a molar ratio of 10.4: 79.3: 1.1.The methylation analysis confirmed that the linkage of monosaccharide residues in AX-I-3 was: →4)β-D-Arap,→2,3,4)β-D-Xylp(1→,→5)β-D-Glcp(1→.AX-I-4 contains arabinose,Xylose and glucose in a molar ratio of 12.6: 76.1: 2.4.Confirmed that the monosaccharide residues in AX-I-4 were connected by: →4)β-D-Arap(1→,→2,3,4)β-D-Xylp(1→,→5)β-D-Glcp(1→.When concentration is 0.1 ~ 0.5 mg/mL,the scavenging ability of AX-I-1,AX-I-2,AX-I-3 and AX-I-4 does not change with the change of mass concentration.The scavenging rate of hydroxyl radicals of AX-I-1,AX-I-2,AX-I-3 and AX-I-4 were all about 5%.When concentration is 0.1 ~ 0.5mg/mL,the scavenging effect of AX-I-1,AX-I-2,AX-I-3 and AX-I-4 on superoxide anion free radical had no positive correlation between mass concentration.The clearance rate of AX-I-1 on superoxide anion free radical was about 10%.The clearance rate of AX-I-2 and AX-I-3 on superoxide anion free radical was about 30%.The clearance rate of AX-I-4 on superoxide anion free radical was about 40%.When concentration is 0.1~0.5 mg/mL,the clearance rate of AX-I-2,AX-I-3 and AX-I-4 on DPPH was about 30%.The clearance rate of AX-I-1 on DPPH was about 5%.When concentration of the AX-I-1 was 1 mg/mL,AX-I-1 had certain killing effect on A549 cells,and the cell vitality of A549 cells after administration was about 70%.At this concentration,AX-I-1 also had a strong killing effect on BEAS-2B cells.When the concentration of AX-I-3is between 100 and 1000 μg/mL,AX-I-3 had strong killing effect on A549 cells.When the concentration of AX-I-3 was 1 mg/mL,the cell vitality of A549 cells after administration was about 50%.When the concentration of AX-I-4 was between 200 and 2000 μg/mL,AX-I-4 had certain killing effect on A549 cells,and the cell vitality of A549 cells after administration was about 50%.AX-I-1,AX-I-2,AX-I-3 and AX-I-4 had no anti-biofilm activity on biofilm of C.albicans and S.aureus.Secondary metabolites of E.coli,P.aeruginosa and lactobacillus L1 / L5 / L7 had significant anti-biofilm activity on biofilm of C.albicans and S.aureus.And found that anti-biofilmactive ingredients are protein in metabolites of E.coli,the protein play a role in anti biofilm by cutting S.aureus biofilm formation related gene transcription.Secondary metabolites of E.coli,P.aeruginosa and lactobacillus L1/L5/L7 had no significant influence on the growth of C.albicans.Secondary metabolites of E.coli and P.aeruginosa have certain effects on the growth of S.aureus.Secondary metabolites of lactobacillus L1/L5/L7 completely inhibit the growth of S.aureus.CONCLUSION:Residue of Astragalus Root were isolated and purified to obtain four components.The structure of AX-I-1,AX-I-2,AX-I-3 and AX-I-4 were analyzed by HPLC gel permeation chromatography,GC-MS monosaccharide composition analysis,GC-MS methylation analysis.The antioxidant,anti-tumor and anti-biofilm activities of the four components were evaluated.The results showed that AX-I-1,AX-I-2,AX-I-3 and AX-I-4all had certain antioxidant activity in vitro.AX-I-3 had stronger killing effect on human lung adenocarcinoma A549 cells.The cytotoxicity of AX-I-1,AX-I-2,AX-I-3 and AX-I-4 was also showed by killing the normal human lung epithelial cells BEAS-2B cells.AX-I-1,AX-I-2,AX-I-3 and AX-I-4 had no anti-biofilm activity.