ABSTRACT
We previously reported that active Astragalus polysaccharides APS-Ⅱ generate strong immune activity. Here we establish the optimal method for APS-II acid degradation. After preliminary structural studies and separation and preparation of the degradation products, the oligosaccharide active center with the strongest immune activity was identified by in vitro immune cell culture experiments. The optimum acid degradation conditions for APS-II were determined by a single factor experiment and an orthogonal experiment. Astragalus oligosaccharides prepared under the optimal conditions were subjected to structural analysis by hydrophilic interaction chromatography - electrospray ionization source - high resolution time-of-flight mass spectrometry. The products were separated and oligosaccharide fragments with different degrees of polymerization were isolated by preparative purification chromatography. Finally, fragments of the immunologically active centers were identified by in vitro immune cell cultures from multiple perspectives. The results show that the optimal acid hydrolysis conditions for APS-Ⅱ are hydrolysis temperature 80 ℃, trifluoroacetic acid concentration 1.0 mol·L-1, hydrolysis time 1 h. The degradation conditions have good repeatability. The degradation product is a six-carbon aldehyde glycan structure with the main chain 1→4 connected. The immune activity screening experiment for six oligosaccharide fragments showed that larger molecular weight oligosaccharides have stronger immune-promoting effects. It is speculated that the immunologically active center of Astragalus oligosaccharide is located in the sugar chain of DP9-DP19. The animal welfare and the experimental process in this study follow the requirements of the Animal Ethics Committee of Shanxi University. This result suggests a foundation for the structural characterization and structure-activity relationship research of Astragalus oligosaccharides, and may promote the development of Astragalus oligosaccharide drugs.
ABSTRACT
Objective: The characteristic maps and immunological activities of some acid hydrolysates of polysaccharides from Astragali Radix (APS) from different areas were compared. The quality evaluation method of Astragali Radix with oligosaccharide mixture as quality control index was established. Methods: In this study, the polysaccharides from the traditional Chinese medicine Astragali Radix were used as the research object. Firstly, the optimal partial acid hydrolysis conditions were selected by orthogonal test. The polysaccharide was hydrolyzed into oligosaccharides for analysis. The characteristic map of Astragalus oligosaccharides based on partial acid hydrolysis-hydrophilic interaction chromatography was established. Multivariate statistical analysis was performed on the data by SIMCA software to distinguish Mongolian Astragalus from different areas. The partial acid hydrolysis products of Astragalus polysaccharides were characterized by hydrophilic interaction chromatography and mass spectrometry, and the activity was evaluated by mouse peritoneal macrophage phagocytosis neutral red experiment. Results: The optimal hydrolysis conditions obtained by orthogonal experiment were temperature 90 ℃, trifluoroacetic acid concentration 1 mol/L, hydrolysis time 1 h. Under this condition, Astragalus polysaccharide was hydrolyzed into characteristic oligosaccharide fragments, and the method is reproducible. The characteristic map of Astragalus oligosaccharides based on partial acid hydrolysis-hydrophilic interaction chromatography showed that the maps of the same Astragalus oligosaccharides had good consistency, and the maps of different Astragalus oligosaccharides were quite different. PCA showed that three different kinds of Mongolian Astragalus can be distinguished. It was found by mass spectrometry that the extracted Astragalus polysaccharides were mainly 1→4 linear glucan, and gluco-oligosac-charides with the degrees of polymerization 3—8 were obtained after partial acid hydrolysis. The partial acid hydrolysate of the wild Astragalus polysaccharides from Shanxi Hunyuan had higher ability to enhance the phagocytic activity of macrophages than the transplanted Astragalus and higher than the unhydrolyzed total astragalus polysaccharide. Conclusion: This study showed that the characteristics of Astragalus polysaccharides based on partial acid hydrolysis-hydrophilic interaction chromatography and the effects on cellular immune function can be used to evaluate the quality of Astragali Radix in different habitats and different planting methods, and it is also an important supplement to the quality evaluation method of Astragali Radix. At the same time, it has a certain exemplary role in the characterization of other Chinese materia medica polysaccharides.
ABSTRACT
Water-soluble polysaccharides from traditional Chinese medicine have properties of complex structure and high molecular, resulting in hardly complete their structural characterization.However, a "bottom-up" approach could solve this problem.Glehniae Radix extract was extracted with hot water and then precipitated by 40% ethanol to obtain Glehniae Radix polysaccharides (RGP). Subsequently, a partial acid hydrolysis method was carried out and the effects of acid concentration, time and temperature on hydrolysis were investigated. Under the optimum hydrolysis condition (1.5 mol•L⁻¹ trifluoroacetic acid, 4 h, and 80 ℃), RGP were hydrolyzed to characteristic oligosaccharide fragments. Futher, a hydrophilic liquid chromatography- mass spectrometry method was used for the separation and structural characterization of the polysaccharide hydrolysates. According to MS and MS/MS analysis of several standard disaccharides, a method for determining the type of polysaccharide glycosidic linkage by mass spectrometry was established. The results showed that the polysaccharide hydrolysates were linear glucan containing 1, 4-glycosidic bonds. And gluco-oligosaccharides with the degrees of polymerization (DP) of 4-11 were obtained after partial acid hydrolysis.
ABSTRACT
Objective: To study the characteristics of partial acid hydrolysis in five different plant polysaccharides and the composition of their hydrolysic products. Methods: Polysaccharides were partially hydrolyzed with trifluoroacetic acid (TFA) at different concentration (0.05, 0.2, and 0.5 mol/L), respectively, and the hydrolytic characteristics were studied by ultra-performance liquid chromatography (UPLC) coupled with multivariate statistricts. Results: The characteristic structural segments of ephedra polysaccharide were the contents of glucuronic acid in trapped fluid hydrolyzed with 0.05 mol/L TFA (X1GlcUA), arabinose in permeation fluid hydrolyzed with 0.05 mol/L TFA (X2Ara), arabinose in trapped fluid hydrolyzed with 0.2 and 0.5 mol/L TFA (Y1Ara and Z1Ara), and galacturonic acid in the permeation fluid hydrolyzed with 0.2 mol/L TFA (Y2GalUA), which could distinguish other plant polysaccharide samples. Conclusion: The proposed method is precise and practical for the analysis on monosaccharide compositon in Chinese materia medica by UPLC coupled with multivariate statistics and partial acid hydrolytic reaction, which further provides the basis for the quality control of the plant polysaccharides.
ABSTRACT
Agaro- and carra-oligosaccharides were produced by partial acid hydrolysis of commercial agarose and kappa-carrageenan. Di- and tetrasaccharides were purified by gel filtration chromatography and characterized by NMR (1D and 2D) spectroscopy and ESIMS. The following oligosaccharides were obtained: agarobiose, agarotetraose, kappa-carrabiose and kappa-carratetraose. Agarobiose and agarotetraose were used as standards to develop a high performance size exclusion chromatography (HPSEC) method which was utilized to study the hydrolysis rate of agarose and oligosaccharide production. Six hours of hydrolysis (0.1 M TFA, 65 ºC) produced mainly di- and tetrasaccharides. The methodology for oligosaccharide production and evaluation developed in the present work shows good potential for the production of bioactive oligosaccharides.