| Objective:Rehmannia glutinosa is a typical representative of different processing types with different effects.Polysaccharide of Rehmannia glutinosa(PRG)is an important part.The processing process of Rehmannia glutinosa had an effect on the content and structure of polysaccharides.Through the extraction,separation and purification of PRG,the differences in chemical composition and structure and in vitro antioxidant properties were compared,so as to further enrich the chemical changes of Rehmannia glutinosa before and after processing.These lay a foundation for the processing principle of Rehmannia glutinosa and provide scientific basis for the improvement of Rehmannia glutinosa quality standard.Base on Caenorhabditis elegans(C elegans),the anti-aging effect and the difference between the total polysaccharide and neutral polysaccharide of Rehmannia glutinosa were studied gradually,and the mechanism of action was studied deeply to establish the evaluation method of the anti-aging effect and mechanism of active polysaccharide.Methods:1.Extraction and purification of crude and refined polysaccharides from Rehmannia glutinosaDeionized water was used as the solvent for extraction,the extract was filtered and concentrated,the impurities were removed with 80%ethanol,and the partial resolubilization was precipitated.A relatively refined total polysaccharide of Rehmannia glutinosa was obtained by the method of protein removal with sevga reagent.DEAE-52 cellulose chromatographic column and Sephadex G-100 glucan gel chromatographic column was used to further refine the crude polysaccharide of Rehmannia glutinosa.Water and different concentrations of NaCl solution were used as eluents for elution,and phenol-concentrated sulfuric acid method was used for detection.Polysaccharides were combined with each other.After repeated sampling for 5 times,two kinds of processing types Rehmannia glutinosa refined polysaccharides were obtained.2.Chemical characterization of polysaccharides from two kinds of processing types Rehmannia glutinosaThe method of phenol-concentrated sulfuric acid was used to determine the content of total and refined polysaccharides in rehmannia glutinosa.Protein content in PRG samples was determined by BCA kit.The structure of polysaccharides functional groups was analyzed and identified by infrared spectroscopy.Acid hydrolysis and 1-phenyl-3-methyl-5-pyrazolone(PMP)pre-column derivatization were used to treat the total polysaccharides and refined polysaccharides.Ultrahigh performance liquid chromatography quadrupole time of flight mass spectrometer(UPLC-Q-TOF-MS)was used to analyze the monosaccharide composition and the differences between the total polysaccharides and refined polysaccharides of two kinds of processing types Rehmannia glutinosa.The total polysaccharides and refined polysaccharides were treated by acid hydrolysis and PMP pre-column derivatization,and the contents of monosaccharides in total polysaccharides and refined polysaccharides were compared by ultrahigh performance liquid chromatography tandem triple quadrupole mass spectrometer(UPLC-QQQ-MS).3.Determination and analysis of antioxidant capacity of polysaccharides from two kinds of processing types Rehmannia glutinosa in vitroThe ability of PRG to scavenge DPPH free radicals was determined by 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)method,and the antioxidant capacity of PRG in vitro was represented by Vc as a control.4.Preliminary study on the anti-aging effect of total polysaccharides of Rehmannia glutinosa on nematodesThe anti-aging effects of the total polysaccharides of Rehmannia glutinosa on nematodes were analyzed by anti-stress ability,reproduction,body length,exercise,life span and reactive oxygen species.5.Study on the anti-aging mechanism of refined polysaccharides from Rehmannia glutinosaThe antiaging activity of refined Rehmannia glutinosa polysaccharides was intuitively verified by lipofuscin experiment.And the mechanism of caloric restriction life extension was excluded by pharynx pump experiment and uracil deficient Escherichia coli OP50(E.coli OP50)growth experiment.Through the determination of antioxidant enzyme content in nematodes and the expression experiment of sod-3p::GFP fusion protein,the anti-stress mechanism of Rehmannia glutinosa polysaccharide was explained.Through the daf-16 nuclear localization experiment,the expression experiment of life-related genes and the life experiment of transgenic nematode model,the mechanism of life extension of nematodes was finally determined.Results:1.Total polysaccharides and neutral polysaccharides were preparedAfter extracting the total polysaccharides from rehmannia glutinosa,the polysaccharides from Rehmannia glutinosa were further purified by column chromatography.Because the content of acid polysaccharide is very low,compared with neutrophils can be ignored,so the refined Rehmannia glutinosa polysaccharide is mainly neutrophils.2.Chemical characterization of polysaccharides from Rehmannia glutinosa was carried outThe yield of crude polysaccharide was 22.92%and 18.75%,respectively.After purification,the yield of the polysaccharides of two kinds of processing types Rehmannia glutinosa was 12.09%and 8.33%,respectively.Through the determination of the total polysaccharides,the total polysaccharides from Rehmanniae radix(TPRR)was 71.26%and the total polysaccharides from Rehmanniae radix praeparata(TPRRP)was 66.71%,respectively.The neutral polysaccharides from Rehmanniae radix(NPRR)was 83.35%,the neutral polysaccharides from Rehmanniae radix praeparata(NPRRP)was 81.22%respectively,but they still contained 13.32%and 19.6%of protein,respectively.Through the infrared spectrum analysis,the infrared spectrum absorption peaks of polysaccharides of two kinds of processing types Rehmannia glutinosa were almost the same,and they all had similar characteristics of the absorption peaks of neutral polysaccharides.There were mannose,rhamnose,galacturonic acid,glucose,galactogalactose and arabinose in the monosaccharide composition of the total polysaccharides of Rehmannia glutinosa,indicating that there was no difference in the monosaccharide composition of the total polysaccharide of Rehmannia glutinosa.The neutral polysaccharides contained mannose,rhamnose,glucose,galactose and arabinose but not galactonic acid.The glucose and galactose in the polysaccharide of Rehmannia glutinosa accounted for the majority,while the content of other monosaccharides did not exceed 5%.The proportion of glucose and galactose in PRRP is higher than PRR.3.polysaccharides of two kinds of processing types Rehmannia glutinosa had certain antioxidant activity in vitroVc,TPRR and TPRRP half maximal inhibitory concentration(IC50)of polysaccharides were 0.0021,0.1291,0.1845 mg/mL,namely TPRR and TPRRP removal ability of DPPH free radical is 1.60%and 1.10%compared with Vc,which suggests that RPG has certain in vitro antioxidant capacity.The antioxidant capacity of TPRRP was stronger than that of TPRR in vitro,but weak in vitro antioxidant capacity compared with Vc.4.The total polysaccharide of Rehmannia glutinosa has a certain anti-aging effectPRRP can prolong the life of nematodes without affecting their reproduction and motility.PRG can improve the resistance of nematodes to heat stress and oxidative stress.In addition,PRRP can promote the growth and development of nematodes.The ROS formation experiment further verified that the mechanism of PRRP to prolong the life of nematodes was to reduce reactive oxygen species(ROS)content,improve the oxidative stress capacity of nematodes and reduce oxidative damage.5.The neutral polysaccharides of Rehmannia glutinosa prolonged the lifespan of nematodes through insulin/IGF-1 signaling pathway(IIS)The mechanism of action of the neutral polysaccharides of Rehmannia glutinosa for inhibiting the transcription activity of daf-2,thereby inhibiting insulin receptor signal of C.elegans DAF-2 negative regulation function of the signal path,relative to the normal state,the transcription factor DAF-16 will more into the nuclear expression,thus regulate gene expression of resistance to stress,improve the nematodes anti-stress ability,and extend the life of the worm.Conclusion:In this study,a method was established to extract and purify PRG,and the content of PRG before and after processing was studied by chemical characterization.Through the analysis of the monosaccharide composition and content of PRG,the similarities and differences between TPRR,TPRRP,NPRR,NPRRP were compared.Using C.elegans model,the antiaging effects of PRG were compared.The preliminary study on the anti-aging effect of the total polysaccharide of Rehmannia glutinosa had laid a foundation for the study on the anti-aging mechanism of the refined neutral polysaccharide.Through the life-related experiment and biomolecular technology of C.elegans,the signaling pathway for the life-extending of C.elegans was excluded in essence,and the IIS was determined as the anti-aging signaling pathway of PRG. |
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