| Objectives:Using the rhizome of Megacarpaea delavyi Franch from Cangshan,Dali as the raw material,the Megacarpaea delavyi rhizome polysaccharide(MDRP)was extracted,separated,and purified.The homogeneous fraction MDRPa with a high rate of acquisition was selected as the experimental object.The structure of MDRPa was analyzed.The antitumor activity in vitro and the mechanism at the molecular level were researched.Meanwhile,the effect of MDRPa on acute liver injury in mice induced by CCl4 was also investigated.This study may provide basic research data for screening new and promising Megacarpaea delavyirhizome polysaccharide drugs Methods:1.MDRP extraction,separation and purificationPolysaccharides were extracted from the roots of Megacarpaea delavyi rhizome by water extraction and alcohol precipitation method,and deproteinized by Sevage method.The dialysis,alcohol precipitation,and freeze-drying were used to obtain the total polysaccharides(MDRP)of Megacarpaea delavyi rhizomes.The mass fraction of polysaccharides in MDRP samples and the extraction rate of polysaccharides in raw materials were determined respectively.MDRP was fractionated into different fractions by using DEAE-52 cellulose column chromatography and eluted by distilled water,then The different fractions were lyophilized.The higher yield MDRPa fraction was selected and further purified on Saphadex-G75 column chromatography for subsequent experiments.2.Observation of MDRPa physical and chemical properties,determination homogeneity and molecular weight of MDRPaThe physical and chemical properties of MDRPa were measured by experiments such as solubility determination,iodine-potassium iodide reaction,ferric chloride reaction,and sulfuric acid-carbazole reaction.High performance gel permeation chromatography(HPGPC)was used to measure the weight average molecular weight(Mw),number average molecular weight(Mn),and molecular weight distribution coefficient(D)of MDRPa,respectively,and determine its purity based on the peak shape of HPGPC.3.MDRPa structural analysisPre-column derivatization with PMP(1-phenyl-3-methyl-5-pyrazolinone)and high-performance liquid chromatography(HPLC)analysis were used to determine the MRPPa monosaccharide composition.The potassium bromide tablet method was used for infrared spectrum analysis.After MDRPa samples were subjected to methylation,hydrolysis,reduction,and acetylation reactions,partially methylated alditol acetates(PMAA)derivatives were obtained,and GC-MS analysis was performed on an Agilent 7890A/5975C gas chromatography-mass spectrometer.4.Study on the antitumor activity and antitumor mechanism of MDRPa in vitro The CCK-8 method was used to investigate the inhibitory effect of different dose groups of MDRPa(100μg/mL、150μg/mL、300μg/mL、600μg/mL)on human breast cancer cell MCF-7.RT-qPCR experiments were used to detect MCF-7 cell’s changes in mRNA expression levels of akt1,bax,bcl-2,and pten related genes.5.Effect of MDRPa on acute liver injury in mice induced by CCl4A mouse model of CCl4 acute liver injury was established by conventional methods,and was administered orally with MDRPa 80 ug/g,160 ug/g,and 320 ug/g for 7 consecutive days.Twenty-four hours after the last administration,blood was collected from the eyeballs,and the mice were sacrificed.The serum alanine aminotransferase(AST)and aspartate aminotransferase(ALT)activities of the mice were measured,and the liver was dissected,and the liver index,liver malondialdehyde(MDA)content,reduced glutathione(GSH)content,and superoxide Dismutase(SOD)activity were tested and the histological changes of liver cells was observed.Results:1.Extraction,isolation and purification of MDRPThe extraction rate of total polysaccharide(MDRP)from rhizome of Megacarpaea delavyi was 1.86%after water extraction and alcohol precipitation,deproteinization,dialysis and freeze drying.After deproteinization,the MDRP samples were separated by DEAE-52cellulose column chromatography,and four components were collected,named as MDRPa,MDRPb,MDRPc and MDRPd.In this study,MDRPa component with high yield was purified by Saphadex-G75 column chromatography,and a single symmetrical peak appeared,indicating that MDRPa was a single component,which could be used for subsequent experimental studies.2.Preliminary structural analysis of MDRPaThere was no blue in the reaction between MDRPa and iodine-potassium iodide,indicating that MDRPa did not contain starch or cellulose.The reaction with ferric chloride showed no purple color,indicating that MDRPa did not contain polyphenols.The sulfate-carbazole reaction showed no purplish red,indicating that MDRPa did not contain uronic acid.HPGPC showed a single symmetrical peak,indicating that MDRPa was homogeneous polysaccharide.The results showed that MDRP was composed of four monosaccharides,D-Man,D-Glc,D-Gal and D-Xyl.The molar ratio was 9.98:8.33:7.76:7.58.The results of infrared spectrum analysis showed that MDRPa had an absorption peak near 896cm-1 and 854cm-1,indicating that both α-and β-configurations existed in the molecule.There were absorption peaks near 765cm-1 and 719cm-1,suggesting that MDRPa contained furans.In addition,there were absorbents in 854cm-1 and 896cm-1,suggesting that polysaccharides might contain mannose.3.Anti-tumor activity and mechanism of MDRPaThe results of CCK8 experiment showed that each dose of MDRPa group(100μg/mL,150μg/mL,300μg/mL,600μ/mL)could inhibit the growth of tumor cell of MCF-7 to different degrees,with inhibition rates of 16.87%,25.59%,47.27%and 73.46%,respectively.RT-qPCR results showed that compared with the tumor model group,MDRPa significantly promoted the expression of bax and pten genes,and decreased the expression of akt1 and bcl-2 genes.4.Biological activity and mechanism of MDRPa in vitroThe results showed that compared with the model group,the medium and high dose MDRPa group could significantly improve the liver injury effect of mice in the CCl4 group.MDRPa could significantly reduce the serum AST and ALT activity and significantly increased the content of GSH and SOD activity in hepatocytes of the mice.On the other hand,HE staining results showed that,compared with the tumor model group,the hepatic lobular reticular structure of the MDRPa groups became more complete with the increase of MDRPa dose under light microscope.Conclusion:1.MDRPa is a neutral polysaccharide with weight-average molecular weight of 1.4791×105 Da,number-average molecular weight of 1.0582×105 Da,and the distribution coefficient of D=1.40.There are D-Man,D-Glc,D-Gal and D-Xyl monosaccharides in MDRPa and the molar ratio is 9.98:8.33:7.76:7.58.The glycosidic linkage types of MDRPa are 1,4-Xylf,1,4,6-Manp,1,4-Manp,1,6-Glcp,1,3,6-Glcp and 1,6-Galp.The molar percentage is 7.08:1.75:31.2:28.41:1.52:20.25.MDRPa is a branched structure,1,4,6-Manp,and 1,3,6-Glcp are the sugar chain branch points.2.MDRPa has anti-tumor activity in vitro and can significantly inhibit the increase of MCF-7 tumor cells.The anti-tumor mechanism is closely related to its promotion of bax and pten gene expression and reduction of akt1 and bcl-2 gene expression.3.MDRPa can antagonize CCl4 induced acute liver injury in mice... |
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