Study On The Structure And Biological Activity Of K4 Polysaccharide Andits Sulfated Derivatives

Chondroitin sulfate(CS)is an acidic chain polysaccharide consisting of glucuronic acid and N-acetylgalactosamine repeat disaccharide units.It is widely distributed on the extracellular matrix and cell membrane surface of animal tissues.The form of glycans(PG)exists.Studies have found that chondroitin sulfate has many important biological activities such as anti-inflammatory,immunoregulatory,cardio-cerebral vascular protection,anti-oxidation,cell adhesion,anti-tumor,and long-term use of small side effects,can be widely used as health care products and drugs.At present,the source of chondroitin sulfate is mainly extracted from animal cartilage tissue.The chondroitin sulfate extracted from different animal tissues has differences in molecular size and degree of sulfation,and the animal extraction has the disadvantages of high cost,low yield,and environmental pollution.It is hoped that the biochemical method can be adopted.Synthesis of chondroitin sulfate analogs with controlled sulfation and molecular weight.The sugar chain structure of Escherichia coli K4 polysaccharide(K4)is a repeating disaccharide unit composed of glucuronic acid and N-acetylgalactosamine,and has a fructose side chain on glucuronic acid C-3.The same carbon chain skeleton as chondroitin sulfate can be used as a precursor for synthesizing chondroitin sulfate.In this study,K4 polysaccharide fermentation liquid was separated and purified through high-density fermentation to obtain pure K4.The mild acid hydrolysis of K4 removes the side chain of fructose to obtain the defructosylated K4(K4de),and the structure of the K4 is characterized by NMR,IR,SEC-MALLS and HPLC.The obtained K4 and K4 de were studied for their immunomodulatory activity in vitro using the macrophage cell line to develop their potential as natural immune enhancers.At the same time,the obtained K4 de was sulfated and sulfated to varying degrees,and the structure of the sulfated product was characterized to study the physiological activity of the sulfated product.The results of the study are as follows:(1)K4de was obtained through high-density biological fermentation,and the uronic acid production in the fermentation broth reached 790 mg/L.Ethanol precipitation and savage deproteinization,anion exchange chromatography and other methods of purification,the resulting K4 purity.The resulting K4 was characterized by NMR and IR.Its Mw was-MALLS.(2)K4 and K4 de were treated with different concentrations of K4 and RAW264.7(macrophages).The results showed that K4 and K4 de have immunoenhancing activity on macrophages,can activate macrophages,extend pseudopods,and stimulate giants.Phagocyte proliferation increases the secretion of NO and related inflammatory cytokines in a concentration-dependent manner.(3)K4de with a molecular weight of 12 kDa was chemically degraded to produce L-K4de(low molecular weight K4de)with a molecular weight of 3 kDa,which was sulphated separately to produce a non-site-sulfated derivative K4deOS(H).),K4de-NS,K4de-ONS,and high and low sulphated derivatives of two molecular weights,K4de-4OS,K4de-6OS,K4de-4.6OS,L-K4de-4OS,L-K4de-6OS,L-K4de-4.6OS,both determined by SEC-MALLS.The agarose gel electrophoresis and elemental analysis showed that the degree of sulfation was examined.The sulfated product was digested with the chondroitinase ABC and analyzed by HPLC for disaccharide analysis.(4)Preparation of L-K4deOS(L)with low molecular weight and low degree of sulfation,acting on CIA model of arthritis mice,to investigate the effect of arthritis on mice.After 8 weeks of oral administration of L-K4deOS(L),the mouse had a late onset of disease,a low polyarthritis index,and no significant adverse reactions in mice.