Studies On The Chemical Constituents,Processing And Structural Modification From The Polygonati Rhizoma And Phylogenetic Analysis In Plants Of Polygonatum Species

Objective:To study the differences in fingerprints of polysaccharides and alcohol extracts from three species of Polygonati Rhizoma plants in the Chinese Pharmacopoeia,and to study the transformation of various components in the traditional“Nine-steam-nine-bask”and different processing methods of P.kingianum.At the same time,using three kinds of Polygonatum rhizome as raw materials,systematic research was conducted on the extraction,purification,different chemical modification methods,structural analysis,antioxidant,hypoglycemic aspects of polysaccharides,at the same time,the phylogenetic relationship of Polygonatum and its related groups was studied using the chloroplast genome sequence and provided references for the quality evaluation and clinical application of Polygonati Rrhizoma.Method:（1）Polygonati Rhizoma polysaccharide was extracted by water extraction and alcohol precipitation,and hydrolyzed by trifluoroacetic acid（TFA）.Hydrolyzed monosaccharide product was derivative with 1-phenyl-3-methyl-5-pyrazolinone（PMP）and reused.The pre-column derivatization method was used to establish the chromatographic fingerprint of three species of Polygonati Rhizoma.At the same time,the polysaccharides were hydrolyzed by localization enzyme digestion（glycosidase）.The gel chromatography was used to analyze the differences in glycosidic bonds in the polysaccharides.（2）HPLC was used to compare the chemical constituents of alcohol extracts from three species of Polygonati Rhizoma,and the differences in chemical constituents of three species of Polygonati Rhizoma were discussed.（3）The 400 MHz nuclear magnetic resonance（NMR）fingerprint technology was used to determine the ¹H-NMR fingerprint of all species information of the three species of Polygonati Rhizoma,combined with chemometrics for chemical pattern recognition analysis.（4）Identification of the chemical composition of P.kingianum."Nine-steam-nine-bask"processed by infrared spectrum combined with PCA analysis.At the same time,HPGPC,HPLC,and UV methods were used to study the differences in polysaccharide composition,fingerprints,and polysaccharide content in different processed products.（5）Polygonati Rhizoma polysaccharide was extracted by hot water extraction method,and modified by chlorosulfonic acid-pyridine method and sodium hydroxide-acetic anhydride method.Total sugar content;used to characterize polysaccharides before and after modification by infrared spectroscopy and nuclear magnetic resonance technology;and compared the physical and chemical properties of Polygonati Rhizoma before and after modification by chemical analysis,PMP-HPLC,HPGPC Differences in modified derivatives in terms of antioxidant and hypoglycemic activity.（6）Sequencing,analysis,and comparison of chloroplast genomes of P.kingianum,P.cirrhifoliums,and P.odoratum.At the same time,based on the Genbank public database,52 chloroplast genome sequences of Polygonatum and related groups were downloaded to construct an NJ tree and infer phylogeny.Result:（1）There are differences in polysaccharide PMP-HPLC fingerprints of three species of Polygonati Rhizoma.No mannose,rhamnose,and fucose were detected in all three species,and all contained galacturonic acid,galactose,glucose,and xylose;PCA and HCA analysis showed that P.sibiricum and P.cyrtonema have similar polysaccharide chromatographic fingerprints,while P.kingianum is significantly different from the two.Polysaccharide hydrolysis results showed that all three polysaccharides hadβ-1,4-glucoside bonds,P.sibiricum polysaccharides and P.cyrtonema polysaccharides hadβ-1,3-glucoside bonds,while P.kingianum polysaccharides did not.（2）The HPLC fingerprints of three species of Polygonati Rhizoma were established for the first time.The similarity ranges of three species of Polygonati Rhizoma HPLC fingerprints were 0.869 to 0.983,0.963 to 0.998,and 0.933 to 0.988,respectively.HPLC was used to compare the alcohol-soluble components,and 3 chromatographic peaks were identified by the reference substance.Three species of Polygonati Rhizoma HPLC chromatograms all have certain differences.P.sibiricum and P.kingianum medicinal materials have higher similarity and smaller differences in fingerprint chromatograms;while P.cyrtonema differs significantly from P.sibiricum and P.kingianum;especially the alcohol-soluble components of P.cyrtonema More abundant.Fingerprints combined with chemometrics HCA and PCA analysis showed that P.cyrtonema can be clearly separated from P.sibiricum and P.kingianum,and the chemical composition of P.sibiricum and P.kingianum is more similar,and they are classified into one class;P.cyrtonema is separately classified into one class.（3）The¹H-NMR fingerprints of three species of Polygonati Rhizoma have obvious differences,which can truly and comprehensively reflect the characteristic components and intrinsic qualities in Polygonati Rhizoma.The main difference components are organic acids and fatty acids;PCA,PLS-DA,and HCA analysis can distinguish three species of Polygonati Rhizoma.（4）With the increase of the number of processing,the color and sexual taste of P.kingianum gradually changed,from the yellow and white of the raw product to the black gradually;the difference between the average IR spectrum and the second derivative spectrum of different processed products is obvious,and the PCA mode can P.kingianum processed into different types is divided into 3 categories;HPGPC shows that the polysaccharide composition distribution of P.kingianum processed products is more different from raw products;the differences between processed products are also significantly different,with the increase of processing times,The proportion of small-molecular polysaccharide components decreased significantly,while the macromolecular polysaccharide component gradually dissolved out as the number of processing times increased;HPLC results showed that after processing,the fingerprints of different processed products of P.kingianum were different,which was mainly reflected in a certain These peak positions and areas are different;P.kingianum processed by PMP-HPLC does not have Man,Rib,Gal UA,Glc UA,and Arab;the content measurement results show that the polysaccharide content of P.kingianum raw materials is the highest.With increasing,polysaccharide content gradually decreases.（5）Sulfated P.kingianum polysaccharides with different degrees of substitution have stronger scavenging effects on hydroxyl radicals and DPPH radicals than P.kingianum polysaccharides,but their reducing ability is lower than that of P.kingianum polysaccharides.The difference is not much.S-PKP significantly enhancedα-amylase andα-glucosidase activity inhibitory activity.Compared with PKP,the three types of S-PKP have lower total sugar content and different types and molar ratios of monosaccharide composition.（6）The degree of substitution of A-PKP_1-1,A-PKP_1-2and A-PKP_1-3is 0.2113,0.4287,and 0.5328,respectively.The acetylated P.kingianum polysaccharides with different degrees of substitution have stronger scavenging effects on hydroxyl radicals and DPPH radicals than P.kingianum polysaccharides,but the reducing ability is lower than that of P.kingianum polysaccharides,and the scavenging effect on superoxide anions is not different Large;A-PKP significantly enhancedα-amylase andα-glucosidase activity inhibitory activity.Compared with PKP,the three types of A-PKP have lower total sugar content and different types and molar ratios of monosaccharide composition.（7）The results support the treatment of related families by the APG system.Polygonatum formed a single line and belonged to Asparagaceae.Conclusion:（1）There are differences in the polysaccharide composition,glycoside bonds and alcohol-soluble components of the three legal-based protoxanthin species.The established method is simple,accurate and reproducible.It can be used for the analysis of the sugar composition of Polygonati Rhizoma polysaccharides and the quality of materials evaluation.（2）The color,taste,chemical composition,polysaccharide molecular weight,content and monosaccharide composition of P.kingianum before and after processing has changed,which provides a certain basis for the mechanism research and standardized processing of P.kingianum.（3）The polysaccharides of P.kingianum was chemically modified to increase their antioxidant activity by changing the polarity of the polysaccharides.The antioxidant and hypoglycemic activity of P.kingianum polysaccharides is different from different modification methods.（4）This study analyzes the chloroplast genome structure of some of the plants of the genus polygonatum,and discusses the phylogenetic relationship between the species of polygonatum and related species,It can provide a basis for the classification of polygonatum.