Extraction,Purification,Structure,Bioactivity Research And Analysis On Metabolic And Transcriptional Differences Of Polygonatum Kingianum Var.grandifolium Polysaccharides

Objective In this study,the extraction process,separation and purification,and their effects on antioxidant and antitumor in vitro of polysaccharides from rhizomes of Polygonatum kingianum var.grandifolium produced from farm varieties in Laifeng County,Enshi Prefecture were studied,further investigated based on metabolome and transcriptomic techniques.From the perspective of chemical composition,metabolic pathway and polysaccharide synthesis pathway,so as to point out the direction for evaluating the quality and clarifying the pharmacodynamic material basis of polysaccharides.Method Using ultrasonic-assisted cellulase method to extract crude polysaccharide from rhizome of Polygonatum kingianum var.grandifolium,and the extraction process of crude polysaccharide was optimized through orthogonal test;Papain and trichloroacetic acid were used to remove protein from polysaccharide;The polysaccharide was further separated and purified by DEAE-52 column and sephadex G-100 column,then obtained two homogeneous polysaccharide PKG-1 and PKG-2;The structure of polysaccharide was analyzed by high performance liquid chromatography,near infrared spectroscopy and scanning electron microscopy;The biological activity of polysaccharide was analyzed by antioxidant and anti-tumor tests in vitro;Finally,UPLC-MS/MS technology was used to determine the metabolites in rhizome of Polygonatum kingianum var.grandifolium and Polygonatum sibiricum Red.;Illumina high-throughput sequencing platform performs transcriptome sequencing on rhizomes.Results 1.Through single factor experiment and orthogonal design,the extraction process of Polygonatum kingianum var.grandifolium polysaccharide was optimized.Within the experimental range,the best extraction parameters were: ultrasonic time 20 min,system p H=6,ultrasonic power 220 W,cellulase concentration 2.5%.Under this condition,the crude polysaccharide PKG of Polygonatum kingianum var.grandifolium was obtained;Deproteinized by papain-trichloroacetic acid method,only a small amount of protein and nucleic acid were found under the ultraviolet spectrum scanning of 200～800nm;The polysaccharide of Polygonatum kingianum var.grandifolium was purified by DEAE-52 anion exchange column,and dextran gel G-100 column,and a single symmetric peak was obtained.2.The monosaccharides of PKG were mainly mannose,galacturonic acid,glucose and galactose,and their molar ratio was2.72:1.25:9.52:4.96.The infrared spectra of PKG,PKG-1and PKG-2 all showed typical absorption peaks of polysaccharide.By electron microscopy,the PKG showed irregular sheet structure,the surface of PKG-1 was rough and the structure of PKG-2was loose,and the surface of PKG-2 was uniform and massive.3.By measuring the scavenging ability of PKG,PKG-1 and PKG-2 on DPPH,superoxide anion and hydroxyl free radical,and comparing with the same concentration of Vc,it can be seen that the scavenging ability of three kinds of Polygonatum kingianum var.grandifolium polysaccharides on free radical is hydroxyl free radical >DPPH>superoxide anion in order;By measuring the effects of PKG,PKG-1 and PKG-2 on the viability of A549 cells,it was found that PKG-2 had the best inhibition on A549 cells.The selection of PKG-2 for ROS and apoptosis tests showed that PKG-2 could significantly promote the expression of ROS and lead to apoptosis.4.Metabolome data showed that 34 kinds of carbohydrate differential metabolites were obtained in Polygonatum kingianum var.grandifolium group and Polygonatum sibiricum Red.group.In addition,the carbohydrate metabolites with different metabolites contents were selected as characteristic biomarkers to distinguish the flavin from the two germplasms.Polygonatum kingianum var.grandifolium group contained higher amino acids,which were highly enriched in the amino acid biosynthesis pathway,phenylalanine metabolic pathway and glucosinolate biosynthesis pathway;Polygonatum sibiricum Red.group contained higher carbohydrate substances,which were highly enriched in the starch and sucrose metabolic pathway,amino sugar and nucleotide sugar metabolic pathway and galactose metabolic pathway.5.Transcriptome data showed that a total of 21553 differential genes were screened in Polygonatum kingianum var.grandifolium group and the Polygonatum sibiricum Red.group,among which 10418 genes were up-regulated and 11135 genes were down-regulated.According to the NR library annotation of differential genes,the highest matching degree was found in Asparagus officinalis related to plant stress accounted for 22.45% of all genes in GO database.KEGG database was used to analyze 14 metabolic pathways related to carbohydrate biosynthesis,and 12 differential genes were screened out.The different expression levels of different genes in rhizome were likely to indicate that these genes played a role in polysaccharide biosynthesis and stress response.Conclusions In this study,the extraction and purification process of polysaccharide from rhizome of Polygonatum kingianum var.grandifolium was established,and the structural characterization and biological activity of polysaccharides were preliminary studied.Finally,the differences and similarities in metabolism and transcription levels between Polygonatum kingianum var.grandifolium which from Enshi and Polygonatum sibiricum Red.were analyzed and compared by means of omics,which provided theoretical guidance for the quality evaluation research and subsequent development and utilization of Polygonatum kingianum var.grandifolium resources.