| A large number of studies have indicated that the pharmacological effects of plantpolysaccharides include enhancing host immunity, anti-tumor, anti-radiation, and so on.Therefore, relevant studies have been the focal point of current research fields. Angelicapolysaccharide, as one of the main active ingredients of Angelica sinensis(Oliv.) Diels(Umblliferae), which is used as traditional Chinese medicine for thousands years, hasattracted more and more attentions. However, limited by a variety of factors, the extraction,separation, purification process of Angelica polysaccharide are undetermined, the successorresearches including structural analysis and pharmacological activity don’t be conducted indetail. In this study, we obtained a refined of Angelica polysaccharide component ASP1from water-soluble Angelica crude polysaccharide, which was extracted from Angelicasinensis(Oliv.) Diels (Minxian county, Gansu province, China), by a series of purificationand separation process; Then we got the refined of Angelica polysaccharide componentsASP2, ASP3from the alkali-soluble Angelica crude polysaccharide, with purification andseparation, which was obtained from the dregs aforementioned, by soaked it with a certainconcentration of NaOH solution, alcohol precipitation. We identified the primary and thesenior chemical structure of the three refined Angelica polysaccharide components, ASP1,ASP2, ASP3, by a combination of chemical analysis, spectral analysis and moderninstrumental analysis techniques. And on this basis, further study of the three componentson human hepatoma HepG2cells, human lung carcinoma A549cells, and human breastcancer MCF-7cells to determine their anti-tumor activity in vitro had been done, whichlaid solid material and theoretical basis for subsequent anti-tumor activity in vivo andmechanism research.Part ⅠPreparation of refined Angelica polysaccharidesWith the root of Angelica sinensis(Oliv.) Diels (Minxian county, Gansu province, China), a famous genuine Chinese medicine materials as the raw materials, we obtained thewater-soluble Angelica crude polysaccharide by water extraction, acid-base purification,alcohol precipitation, then after eight-repeated freezing and thawing method to deproteinize,the resulting polysaccharide solution was purified by dialysis(3500Da) to remove the metalsalt and a large number of small molecules of monosaccharides, oligosaccharides, pigmentsand other substances, finally, through the gel filtration chromatgraphy on SephadexG-50,we got the refined Angelica polysaccharide ASP1; Through the extraction of a certainconcentration of NaOH, concentration after the addition of acetate, ethanol precipitation,the alkali-soluble Angelica crude polysaccharide was extracted from the remaining dregsaforementioned. After H2O2decolorization and ultrafiltration, we got the other two refiningthe Angelica polysaccharides, ASP2and ASP3. The research of the physical and chemicalproperties indicated the three refined Angelica polysaccharides did not belong to thereducing sugar; the sugar content, the relative molecular weight and UV scanningwere used to identify their purity. The sugar content of the three refined Angelicapolysaccharides were approximately95%,92%,88%, respectively, which wasmeasured by phenol-sulfuric acid method. The result of UV scanning demonstratedthat they didn’t contain nucleic acid and protein, the relative molecular weight of thethree components were determined by HPGPC to be122kDa,172kDa,80.9kDa,respectively, and the chromatographic peak was sharp and symmetrical. Inconclusion, the three refined Angelica polysaccharides not only possess good traits andhigh purity, but also meet the requirements of the successor experiments.Part Ⅱ Study on the structure of the refined AngelicapolysaccharidesTo determine the structure by chemical analysis firstly, including: complete acidhydrolysis for monosaccharide composition, periodate oxidation and Smithdegradation for the type and proportion of their sugar residues, Methylation analysis for the sugar residues’ ratio.Then spectral analysis further confirms the structure,infrared spectroscopy scanning showed characteristic absorption peaks and the sugarresidues’ configuration of each purified component;1D,2D NMR analysises the typeof sugar residue, the order of connection, and substitution sites. Modern instrumentalanalysis technique, that is atomic force microscope scanning investigates themolecular morphology of each purified component. Finally, comprehensive analysisto identify the structure of polysaccharides. Experimental results show that thefractions’ composition are all of rhamnosus, arabinose, glucose and galactose, andtheir composition ratio were as follows: ASP1,1:4:10:30; ASP2,1:2.5:1:5; ASP3,1:2:2:3.5. Comprehensive of the chemical analysis indicates that each of the fractionsinclude1-6and end:1-4and1-6type sugar residue, and the proportions of them areas follows, respectively: ASP1,2:3:5; ASP2,1.3:3.6:5.1; ASP3,3:3:4. FT-IR scanningalso shows that they all have characteristic absorption peaks of polysaccharide, andthe structures are all β-D-pyranose. The NMR data vested the hydrocarbon atoms andestimated the correlation, which could further verify the results of the chemicalanalysis; AFM results confirmed intuitively the results of chemical analysis andspectroscopic analysis. The Angelica polysaccharide fractions’ composition isextremely complex, their molecular main chains were not long and multi-branch,while the branched chains were more complex, resulting in its spatial structure in theaqueous state, due to hydrogen bond association and the van der Waals force andcross-linked with each other, eventually gathered into one body.Part Ш Study on the antitumor activity in vitro of the refinedAngelica polysaccharidesInvestigate the refined Angelica polysaccharide fractions’(ASP1, ASP2, ASP3)inhibitory effect of proliferation of HepG2cells, A549cells and MCF-7cells in vitro by MTT assay, respectively. The experimental results showed that each refined Angelicapolysaccharide fraction had a significant inhibitory effect on the proliferation of threetumor cells in vitro, and with the increase of drug concentration, the inhibition rateincreased, namely dose-dependent manner, and showed good linear relationship. Whereas,the fractions’ inhibitory effect on different tumor cells was selective. Against HepG2cell,the inhibition rate up to about34%, about33%inhibition rate to A549cells, and MCF-7cells were inhibited by approximately29%. The different fractions’ inhibition also variedfor the same tumor cell line, at the same time. The water-soluble component ASP1hadsignificantly higher inhibition against HepG2cells than the alkali-soluble component ASP2and ASP3, while for the A549cells, quite the contrary. Meanwhile, the molecular weight ofthe polysaccharide component had a certain influence on its anti-tumor activity. For HepG2cells, low molecular weight components ASP1and ASP3possessed a higher inhibition ratethan the high molecular weight component ASP2; for the A549cells, the higher molecularweight component ASP2of the two alkali-soluble components owned higher inhibition rate;however, the inhibition of the three fractions had no significant difference for MCF-7cells. |
PDF Full Text Request