| Cynanchum auriculatum Royle ex Wight is mainly grown in Binhai County,Jiangsu Province.The researches showed that there are many kinds of nutrients and more than 30 kinds of trace elements in the tuber of C.auriculatum.C.auriculatum embodies the functions of regulating immune function,anti-cancer,promoting hair growth and lowering blood lipid.At present,the research on the active components of C.auriculatum is mainly focused on C21 steroid,but the research on the extraction process,physicochemical properties and biological activity function of polysaccharide from C.auriculatum is relatively few.In this study,the extraction process,structure,chain conformation and biological activity of polysaccharide from C.auriculatum were systematically studied.The main research results are as follows:(1)The optimized method of water extraction and alcohol precipitation was used to extract polysaccharide from C.auriculatum,and CAPs was named,yield rate of CAPs is 1.19 ± 0.02%.Then the polysaccharide was extracted by ultrasonic extraction and named as UCAPs.The optimal extraction conditions were as follows: liquid/solid ratio 22:1 m L/g,ultrasonic time 44 min,ultrasonic power 350 W and ultrasonic temperature 52 ℃.Under these conditions,the yield of UCAPs was 1.35 ± 0.02% which is higher than CAPs.The total sugar content of UCAPs and CAPs was 77.24% and 60.74%,the uronic acid content was 8.05% and 28.84%,and the protein content was 5.69% and 2.16%,respectively.The results showed that the average molecular weights of UCAPs were 1.71×107,1.15×106 and 5.93×104 Da,and the average molecular weights of CAPs were 7.07×105 and 3.27×104 Da.The results of monosaccharide composition analysis showed that the neutral sugar composition of UCAPs and CAPs was similar,but CAPs species contained a large amount of galacturonic acid.(2)The polysaccharide was purified by DEAE-cellulose-52 anion exchange column,and the four components CAPW,CAP1,CAP2 and CAP3 were obtained.The main component CAP2 was further purified by Sephadex G-150 gel,and the purified component CAP2-1 was obtained.The polysaccharide contents of CAPW,CAP1,CAP2,CAP3 and CAP2-1 were94.69%,62.55%,49.89%,45.74%,70.64%,uronic acid contents were 4.53%,3.86%,37.52%,50.66%,26.24%,and protein contents were 1.18%,0.27%,0.92%,3.76%,0.29%,respectively.The molecular weight distribution of CAP2-1 was determined by molecular exclusion high performance liquid chromatography.The results showed that CAP2-1 was a homogeneous polysaccharide with a weight average molecular weight of 1.84×106 Da.The monosaccharide composition of CAP2-1 was determined by ion chromatography.The results showed that CAP2-1 was mainly composed of rhamnose,arabinose,galactose and galacturonic acid.Methylation results showed CAP2-1 is mainly composed by →5)-L-Araf-(1→,→3,5)-L-Araf-(1→,→6)-DGalp-(1→,→3,6)-D-Galp-(1→,→4)-D-Galp A-(1→,→3-L-Rhap-(1→,terminal structure for TAraf and T-Galp.An NMR results showed that the main chain of CAP2-1 is mainly composed by →6)-β-D-Galp-(1→、→3,6)-β-D-Galp-(1→、→3,6)-β-D-Galp-(1→、→3)-α-D-Rhap-(1→,side chain is mainly composed by α-L-Araf-(1→、→5)-α-L-Araf-(1→、→3,5)-α-L-Araf-1→.(3)CAP2-1 was characterized by instrumental analysis.The results of Congo red test showed that CAP2-1 did not have triple helix structure,and the results of X-ray diffractometry showed that CAP2-1 was semi-crystalline polymer.Atomic force microscopy(AFM)was used to scan the surface morphology of CAP2-1,and the results showed that CAP2-1 was irregular,with a relative height of about 15~27 nm.Scanning electron microscopy(SEM)showed that CAP2-1 was smooth sheet and filamentous with some aggregates on the surface.The results of differential scanning calorimetry showed that the endothermic process of CAP2-1 occurred in the range of 76.13~163.89 ℃,the glass transition temperature was 114.04 ℃,the exothermic process occurred in the range of 276.55~319.2 ℃,and the melting temperature was 300.30 ℃.Zeta potential analysis showed that p H value and metal cation had certain effects on the stability of CAP2-1 solution.CAP2-1 is the most stable at p H 4 and the most unstable at p H 2.The presence of metal cations can reduce the stability of CAP2-1 solution,and Al3+ ions have the greatest influence on its stability.(4)The antioxidant capacity of CAP2-1 and UCAP2-1 in vitro was measured by chemical methods(including DPPH radical,ABTS· radical and hydroxyl radical scavenging experiment and total reducing power determination experiment).The results showed that the antioxidant activity of UCAP2-1 was stronger than that of CAP2-1 in vitro.By inhibiting α-amylase and α-glycosidase enzymes experiments to evaluate the ability of fall blood sugar of CAP2-1 and UCAP2-1,the results show that CAP2-1 and UCAP2-1 can effectively inhibit α-amylase and α-glycosidase enzyme activity,CAP2-1 inhibited α-amylase more efficient than UCAP2-1,but UCAP2-1 inhibited α-glucosidase more efficient than CAP2-1.The inhibition type of UCAP2-1 on α-glucosidase is mixed inhibition.UCAP2-1 inhibits the activity of α-glucosidase by binding with α-glucosidase to form complex. |
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