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@#To investigate the effect of nucleoside diphosphate kinase (NDK) on the synthesis of hyaluronic acid, nucleoside diphosphate kinase gene (ndk) was overexpressed along with the hyaluronic acid-producing genes in recombinant B.subtilis. Two engineered strains named Hp8tg and Pn8tg were constructed.Uniform hyaluronic acid (HA) could be obtained from both engineered strains.HA produced by both recombinant strains was confirmed by monosaccharide composition analysis, Fourier transform infrared spectometry and nuclear magnetic resonance spectroscopy.Inducing conditions of HA fermentation were optimized by response surface methodology.Overexpression of ndk could increase the production and molecular weight of HA by 1.3-fold and 1.1-fold, respectively. This study revealed for the first time that overexpression of ndk could relieve the inhibition effect of uridine diphosphate (UDP) on Class II HA synthase and increase the production and molecular weight of HA, which proves to be an efficient strategy for the production of HA, and the preparation of other polysaccharides.
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Objective:To study the relative molecular weight and distribution of polysaccharides in Polygonati Rhizoma （PR） before and after processing， as well as the effects of different polysaccharide fractions on immune function and inflammatory response of mouse peritoneal macrophages. Method:High-performance gel permeation chromatography （HPGPC） was used to determine the relative molecular weight and distribution of polysaccharides in PR （named SC） and polysaccharides in PR processed with wine （named JC）， and polysaccharide fractions with different relative molecular weights were obtained by dialysis. Different polysaccharide fractions were applied to mouse peritoneal macrophages， which was normal or induced by lipopolysaccharide （LPS）. Cell counting kit-8 （CCK-8） method was used to select the optimal administration concentration. Enzyme linked immunosorbent assay （ELISA） was used to determine the concentrations of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） and interleukin-1<italic>β</italic> （IL-1<italic>β</italic>） in the cell supernatant. The Griess method was used to detect the concentration of nitric oxide （NO）. Result:SC and JC could be divided into four parts according to relative molecular weight and its distribution range， including part Ⅰ（14 800-2 273 kDa）， part Ⅱ（2 148-296 kDa）， part Ⅲ（12-1 kDa） and part Ⅳ（818-362 Da）. Based on the differences of part Ⅰ and part Ⅲ after processing， the SC and JC were divided into two-part according to the weight-average relative molecular weight （<italic>M</italic><sub>W</sub>）. For normal mouse peritoneal macrophages， JC could significantly promote the secretion of TNF-<italic>α</italic> （<italic>P</italic><0.01）， while SC had no significant effect. Four polysaccharide fractions， named SD （SC fraction with <italic>M</italic><sub>W</sub>>50 kDa）， JD （JC fraction with <italic>M</italic><sub>W</sub>>50 kDa）， SX （SC fraction with <italic>M</italic><sub>W</sub><50 kDa） and JX （JC fraction with <italic>M</italic><sub>W</sub><50 kDa）， also could<italic> </italic>significantly promote the secretion of TNF-<italic>α</italic> （<italic>P</italic><0.01）， but only JX could significantly promote the secretion of NO （<italic>P</italic><0.05）. In addition， the effect of JX group stimulated secretion of TNF-<italic>α</italic> was better than the JD group （<italic>P</italic><0.01）. For the LPS-induced macrophage model， JC and SC group could<italic> </italic>significantly inhibit the secretion of TNF-<italic>α</italic> and IL-1<italic>β</italic> （<italic>P</italic><0.01）， and the effect of JC was stronger. To compare different polysaccharide fractions， the impact of JX on inhibiting the secretion of TNF-<italic>α</italic> and IL-1<italic>β</italic> was<italic> </italic>significantly stronger than JD （<italic>P</italic><0.01）， and SX inhibited the secretion of TNF-<italic>α</italic> was significantly stronger than SD （<italic>P</italic><0.01）. Conclusion:The relative molecular weight and distribution of polysaccharides in PR before and after processing have changed. JC and SC improve the immune regulation mainly by inhibiting the inflammatory response， the fraction of <italic>M</italic><sub>W</sub><50 kDa is the main effective part， and the effect of PR polysaccharides in inhibiting the inflammatory is enhanced after processing with wine.
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Objective:To characterize the structure of polysaccharide isolated from Linggui Zhugan Tang（LGZGT）,including monosaccharide composition and functional group detection, investigate the difference of the antioxidant activities of crude polysaccharide（CP） and pure polysaccharide（PP）, and provide the basis for the quality evaluation of LGZGT by in vitro bioassay. Method:The average molecular weight of CP was analyzed by high performance gel chromatography（HPGPC）. Gas chromatography-mass spectrometry（GC-MS） and fourier transform infrared（FT-IR） were employed to determine the structure of the polysaccharide. The antioxidant activities of CP and PP samples were evaluated on the basis of 1-diphenyl-2-picrylhydrazyl（DPPH） radical scavenging activity and OH radical scavenging activity. Result:The total polysaccharide was composed of single peaks, with a molecular weight of 3 689 Da. It was mainly composed of arabinose, mannose, glucose, galactose and fructose with a molar ratio of 6.85∶1.00∶109.21∶1.04∶21.82. Among them，glucose and fructose were the predominant components. In addition， IR study indicated the presence of pyranose and anomeric configurations in glycan structure, with two stereoisomers of glycosidic bond (α-glycosidic bond and β-glycosidic bond). It was found that the total polysaccharide had the ability of scavenging DPPH and hydroxyl radicals, and the activity of crude polysaccharide was better than that of refined polysaccharide. It was found in antioxidant research that the total polysaccharide had the ability of scavenging DPPH and hydroxyl radicals, and the activity of CP was better than that of PP. Furthermore, LC-Q-TOF-MS was used to qualitatively analyze the other components in CP, which indicated that it was related to the adsorption of pentacyclic triterpenoids in Glycyrrhiza uralensis. Conclusion:The polysaccharides and pentacyclic triterpenoids in LGZGT are the material basis for the antioxidative effect of LGZGT. The antioxidative activity determined by in vitro bioassay can be used as an evaluation index for the overall quality control of LGZGT.
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Objective:To establish a method for the determination of polysaccharide and monosaccharide composition of Tremella fuciformis, and to analyze the difference of polysaccharide content in T. fuciformis from different sources and cultivation methods, so as to provide reference for the quality determination.Method:High performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detection (HPSEC-MALLS-RID) was employed to determine the content and relative molecular weight distribution of T. fuciformis polysaccharides. The monosaccharide types and proportions of T. fuciformis polysaccharides were analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) precolumn derivative high performance liquid chromatography (HPLC).Result:The weight-average relative molecular weight (Mw) and the content of polysaccharides in T. fuciformis cultivated by cut-log from different sources were distributed in 2.618×106-3.503×106 Da and 307.12-609.06 g·kg-1, respectively. These two parameters of polysaccharides in T. fuciformis with substitute cultivation from different sources were 2.723×106-3.886×106 Da and 366.38-647.37 g·kg-1, respectively. The T. fuciformis polysaccharides mainly consisted of mannose, glucuronic acid, glucose, galactose, xylose and fucose, their ratios in samples with cut-log and substitute cultivation were 4.4∶0.7∶1.0∶0.2∶1.4∶1.6 and 4.4∶0.8∶1.0∶0.1∶1.5∶1.5, respectively. The contents of the above six monosaccharides in 39 batches of T. fuciformis from different sources were mannose of 36.71-191.31 g·kg-1, glucose of 10.46-76.10 g·kg-1, galactose of 1.00-6.72 g·kg-1, xylose of 16.73-70.54 g·kg-1, glucuronic acid of 9.74-32.12 g·kg-1, fucose of 17.16-68.20 g·kg-1.Conclusion:The content of polysaccharides in T. fuciformis from different sources has a certain difference, the developed method can be used as a routine method for the quality evaluation of polysaccharides in T. fuciformis.
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Objective: To establish a method for determining the content of total polysaccharides in decoction pieces of Polyporus,analyze the content of total polysaccharides in samples with different sources and grades. Method: The relative molecular weight and the polydispersity index of polysaccharides in decoction pieces of Polyporus were measured by a high performance gel chromatography coupled with a multi-angle laser light scattering and refractive index system.Dextran with similar molecular weight as polysaccharides was selected as the reference substance.Orthogonal experiment and single factor tests were used to optimize the pretreatment conditions for the determination of total polysaccharides in Polyporus.Polysaccharides in Polyporus with different areas and grades were determined by anthrone-sulfuric acid colorimetric method at 630 nm. Result: The linearity,stability,precision,repeatability and recovery rate of the established method all reached the standards,respectively.The content of total polysaccharides in samples from different areas ranged from 0.87% to 1.39%.The content of total polysaccharides in samples with different grades was 1.40% for first-grade pieces,1.21% for second-grade pieces, and 1.03% for third-grade pieces. Conclusion: The established method is simple,accurate and reproducible,and it can be used for the determination of polysaccharides in decoction pieces of Polyporus.The content of polysaccharides in samples from different origins varies greatly.The content of polysaccharides in samples with different grades shows a certain regularity.The content of polysaccharides is the highest in the first-grade pieces,followed by the content in the second-grade,and the lowest in the third-grade.The results can provide a reference for formulating limits for the content of total polysaccharides and the grade standard of decoction pieces of Polyporus.
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To determine relative molecular weight of astragalus polysaccharides (APs), we used Shodex GS620 gel permeation chromatographic column and differential refraction detector (GPC-RI) with dextran as a reference. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and GPC combined with multi-angle laser light scattering detection (GPC-MALLS) were also used．GPC-RI measure showed four peaks of APs, with the Mw of 1 380 000, 231 000, 18 000, and 480, respectively. Three main peaks were found by GPC-RI-MALLS with the Mw as 1 148 000, 180 000, and 43 000, respectively. Strong signals in 155 000 and 18 000 were detected by MALDI-TOF-MS, which also indicated the sugar moieties of the APs as hexoses. From our study, we found that the GPC-RI method with universal correction is most suitable for Mw determination of the APs. Nevertheless, the three methods should be combined and contrasted with each other to obtain accurate information in research of polysaccharides from Chinese medicine.
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Chondroitin sulfate (CS) is an anionic linear polysaccharide having natural bioactivity, which is widely utilized as a drug and nutraceutical. Due to the structure complexity and source variety of chondroitin sulfate, the specifications of chondroitin sulfate in different pharmacopoeias were different. The related references published in recent years and pharmacopeias were analyzed, sorted, and summarized. This review summarized the methods and limits that were used to for the assay and inspection of impurities, disaccharide and relative molecular weight of chondroitin sulfate. This article would be beneficial to improve the quality control system of chondroitin sulfate.
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Objective: To establish a high performance size exclusion chromatography for the determination of high molecular weight substance in Reduning Injection. Methods: The chromatography on TSK G2000 SWxl column (300 mm × 7.8 mm, 5 μm) was used with 0.1% trifluoroacetic acid aqueous solution-acetonitrile (70:30) as the mobile phase at a flow rate of 0.7 mL/min, and the detection wavelength was at 214 nm at column temperature of 30°C. Results: In the range of 3108-66446, the molecular weight was linear with retention time (r=0.9920). The high molecular weight substances were not detected in 11 batches of Reduning Injection. Conclusion: This method is simple, quick, and accurate, and suitable for the quality control of Reduning Injection.
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Objective To investigate the relative molecular weight of polysaccharides in Bletilla striata by matrix-assisted laser desorption ionizatior/time-of-flight mass (MALDI-TOF-MS). Methods 2, 5-Dihydroxybenzoic acid (DHB) as a matrix was used. By drop drying at room temperature the proportion of the matrix and polysaccharide sample was 1.5∶1. Baseline mode and laser intensity at 1 500-2 500 units were carried out. Results The relative molecular weights of polysaccharides in B. striata were 6?104-3?105, whose distribution rang was wide. Conclusion The relative molecular weights of polysaccharides in B. striata are obtained by MALDI-TOF-MS technique in the proportion of DHB-polysaccharides in B. striata 1.5∶1, which is valuable for the preparation and good quality by the direct drop drying method.
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Objective To determine the relative molecular weight(M_r) of polysaccharide-2b of moutan(PSM_(2b)) to establish the standard of reference substance.Methods PSM_(2b) was separated by a Sepharose 4B column and then PSM_(2b-A) and PSM_(2b-B) were obtained after cryodesiccation.Then HPLC was used to determine their M_r.Ultrapure water was used as mobile phase to compare with 0.7% Na_2SO_4 solution.Results PSM_(2b-A) was pure.The M_r and polydispersity(D) of PSM_(2b-A) obtained by use of dextran as standard were 3.596 2?10~(5) and 4.15 in 0.7% Na_2SO_4 solution phase,respectively,and in ultrapure water phase the M_r and D were 4.758 5?10~(5) and 1.03,respectively.Conclusion Different results are detected by different mobile phases in determination of M_r and D of PSM_(2b-A) by HPLC.And the ultrapure water as mobile phase is more suitable than 0.7% Na_2SO_4 solution.