| Sagittaria sagittifolia L.is a perennial aquatic herb of family alisma(Alismataceae),its underground bulbs are edible,also know as Cigu Polysaccharides are one of the main active components of Sagittaria sagittifolia L.,but there are few studies on the S.sagittifolia L.polysaccharides,which seriously hinder the development and application of S.sagittifolia L.resources.Based on this,in the present study,the crude polysaccharides of S.sagittifolia L.was extracted by ultrasound assisted method,then separated by graded alcohol precipitation and column chromatography,and its chemical structure,chain conformation and biological activity were also studied.This research may not only lay a theoretical foundation for high efficiency preparation technology of S.sagittifolia L polysaccharides by ultrasonic-alcohol precipitation,but also develop healthy products with high activity to improve the national health,which has important theoretical value and practical significance.The general contents are(1)Ultrasonic assisted extraction process optimization of S.sagittifolia L polysaccharidesFour major affecting factors(Extraction temperature,Ultrasonic time,Ultrasonic power and Liquid-material ratio)were investigated,and three significant factors were selected on the basis of single factor for response surface test.The optimum extraction conditions of S.sagittifolia L.polysaccharides were as follows extraction temperature 85?;extraction time 15 min;liquid-material ratio 43 mL/g The actual extraction rate of polysaccharide was 34.78%,which increased by about 168%compared with conventional hot water extraction.These results showed that ultrasonic assisted extraction can significantly improve the extraction efficiency and is an effective method for extracting S.sagittifolia L.polysaccharides(2)Alcohol precipitation and antioxidant activity classification of S.sagittifolia L.polysaccharidesThe crude extract of S.sagittifolia L.polysaccharide was preliminarily separated and purified.Four fractions were obtained by the method of graded alcohol precipitation,which were respectively SPU60,SPU70,SPU80 and SPU90 with purity over 70%,their average molecular weight(Mw)were respectively 52.0 kDa?394.9 kDa?230.6 kDa and 229.4 kDa,in which SPU60 had the highest sugar content(90.64%).In addition,SPU90 has the highest yield(3.47%),while SPU70 protein,uronic acid,sulfate group and molecular weight were the highest.All four polysaccharides were a-pyranose,which were composed of rhamnose,arabinose,xylose,mannose,glucose and galactose.However,the molar ratio was significantly different.Antioxidant activity results showed that SPU70 had the strongest scavenging ability against DPPH,ABTS and hydroxyl radicals(50.23%,55.65%,90.15%,respectively)compared with the other three fractions,which may be related to its higher molecular weight,sulfate group,rhamnose and uronic acid contents.Therefore,SPU70 was screened as the sample for further exploration(3)Preparation of homogeneous component of SPU70 and its structure identificationAfter separation and purification by cellulose anion exchange column and glucan gel column,a uniform polysaccharide component,SPU70-W1 was obtained,with the polysaccharide content of 99.28%and yield of 56.87%.The structure of SPU70-W1 was identified by chemical methods combined with chromatographic,spectrum and spectral analysis.The Mw and Mn were 7.70 kDa and 7.24 kDa,respectively,by HPSEC-MALLS-RI;The monosaccharide composition was mannose,glucose and galactose,and the molar ratio was 2.06:93.58:4.36 by glyconitrile acetate derivatization combined with gas chromatography;Periodate oxidation,Smith degradation and partial acid hydrolysis were explored,and it was found that the main chain glucose and branch chain glucose and galactose might be 1?,1?6,1?2 and 1?2.The structure of SPU70-W1 was mainly connected by?2,6)-a-Glcp-(1?,?3,6)-a-Glcp-(1?,a-Glcp-(1?and?6)-a-Glcp-(1?configuration.There was no triple helix structure in SPU70-W1.The surface of SPU70-W1 was flaky or clastic,with a few pores and uneven size.In addition,some curl shape can be observed(4)Preliminary study on the immunoactivity and antitumor activity of SPU70-W1The immunomodulatory activity of SPU70-W1 was investigated by measuring the proliferation,phagocytic index and the ability to secrete NO and cytokines of macrophages.Human hepatoma cells(HepG2)were used as a model to detect the effect of SPU70-W1 on the proliferation of tumor cells by MTT analysis.The results showed that in the range of experimental concentration(25-200 ?g/mL),to a certain extent,SPU70-W1 could significantly stimulate the proliferation of macrophages and enhance their phagocytosis in a dose-dependent manner(p<0.05).SPU70-W1 could stimulate macrophages to release NO,TNF-? and IL-6 cytokines,indicating that SPU70-W1 can stimulate immune cell activity at a lower concentration(25?mL)to induce immune response,showing significant immunomodulatory activity;in addition,SPU70-W1 can significantly inhibit the proliferation of HepG2 cells(p<0.05),indicating that SPU70-W1 possess the ability to kill liver cancer cells directly. |
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