| Objective:Our previous study found that as the polysaccharide content in the extract increased,the total amount of active ingredient in the extract increased,suggesting that the polysaccharide may have the effect of promoting the dissolution of insoluble ingredients,but whether this effect is universal,and how it works is not clear.Therefore,the feasibility of polysaccharide as the solubilizer was studied by exploring the universality of the solubilization and its affecting factors.Method:1.A method for determining the solubility of baicalin was established by the HPLC method.The effects of different polysaccharides on the solubility of baicalin were compared,and the polysaccharides with the best solubilization were selected.Using this polysaccharide as a tool polysaccharide,the solubilize ability of polysaccharides on other different compounds was screened by UV spectrophotometry.2.Influencing factors of solubilization of the polysaccharides from vinegar Bupleurum chinese:Using 30%,50%,70%,90%ethanol to separate polysaccharides from vinegar Bupleurum chinese,the average molecular weight of polysaccharides was determined by molecular gel chromatography,and the effect of different polysaccharides on the dissolution of baicalin was investigated.3.Effects of properties and structure of the polysaccharides from vinegar Bupleurum chinese on the solubility of baicalin:Using water,0.05mol/L,0.1mol/L,0.2mol/L,0.4mol/L,1mol/L Nacl and 0.5mol/L NaoH as eluent for DEAE-FF ion exchange chromatography and gel permeation chromatography to separate polysaccharides.The solubility increasing effect of different polysaccharide on baicalin was determind.The total polysaccharide content of polysaccharides was determined by phenol-sulfuric acid method;the uronic acid content was determined by the N-hydroxybiphenyl method;GC-MS was used to analysis the monosaccharides constituents of the polysaccharides after acid hydrolysis and derivatization of hydrolyzed monosaccharides by acetylation of glyconitrile.Infrared spectrometer was used to determine the type of functional groups on the polysaccharide sugar chain and NMR instrument to determine the glycosidic linkage order of polysaccharides,4.Study of the polysaccharide from vinegar Bupleurum chinese solution properties:Laser pen was used to observe the Tyndall phenomenon of the polysaccharide aqueous solution.The critical aggregation concentration of the polysaccharide aqueous solution was measured by a fluorescent probe method,the particle size,potential,and pH of the polysaccharide aqueous solution was measured by a particle size analyzer,a pH meter,and the conformation of the polysaccharide aqueous solution was observed by a transmission electron microscope.5.Study on the polysaccharides from vinegar Bupleurum chinese on the pharmacokinetics of baicalin:The pharmacokinetics experiments were conducted in SD rats to investigate the effect of polysaccharides on the bioavailability of baicalin by comparaing pharmacokentics profile after orally administration of baicalin alone or co-administration with polysaccharide from vinegar Bupleurum chinese.Result:1.A good analylaised method for baicalin was established:There are no other components in the existing method that interference the determination of baicalin,the specificity of the method is good.The linear equation of baicalin in the range of 1000-3.9?g/mL is Y=26.914X+124.79,R2=0.9997,and the method precision is RSD=0.225%.The stability in 24 h at room temperature,RSD is 1.643%.Different polysaccharides all have the effect of increasing the solubility of baicalin,and the fold increaseed is between 2-11.5 times.The polysaccharides from vinegar Bupleurum chinese has the best effect,followed by polysaccharides from Ganoderma lucidum and Glycyrrhiza.Therefore,the polysaccharides from vinegar Bupleurum chinese was selected as the model polysaccharides,and its influence on the solubility of different structural compounds was investigated.The results showed that the polysaccharides from vinegar Bupleurum chinese had solubilizing effects on all the insoluble compounds,the solubilization was between1.22-10.81 times,indicated that the solubilizing effect was a common phenomenon.2 Influencing factors of solubilization of the polysaccharides from vinegar Bupleurum chinese:the solubilization effect in the order of decreasing is:90%ethanol polysaccharide precipitated part,30%ethanol polysaccharide precipitated part,70%ethanol polysaccharide precipitated part,50%ethanol polysaccharide precipitated part,and the molecular weight range of the 30%ethanol polysaccharide precipitated part was mainly distributed in the range of 1605.198 KD.;the molecular weight range of 50%ethanol polysaccharide precipitated part was mainly distributed in the range of 1394.788KD;the molecular weight range of 70%ethanol polysaccharide precipitated part was mainly distributed in the range of 2199.030KD;the molecular weight range of 90%ethanol polysaccharide precipitated part was mainly distributed in the range of26.485KD,suggesting that molecular weight was related to solubilization but not the only factor.3.Effects of properties and structure of the polysaccharides from vinegar Bupleurum chinese on the solubility of baicalin three homogeneous polysaccharides were isolated using ion exchange chromatography and gel permeation chromatography.?1?VBCP-2-A is a homogeneous acidic heteropolysaccharide with a molecular weight of 2195.757 KD,with a total polysaccharide content of89.7%,uronic acid content of 13.99%,its monosaccharide component is:rhamnose:arabinose:glucose:galactose:glucuronic acid:arabinuronic acid:galacturonicacid=1:6.97:1.29:9.27:0.78:5.65:2.25.With?1,5??-L-Araf as the main chain,there is a branch attached to Araf's C-3,and the branch is?1,5??-L-Ara or?1,2,6??D-Glcp,or?1,6??D-Glcp,or?1,3??-L-Rhap,or?1,4??-D-Galp,The?1,2,6??D-Glcp C-2 is also branched.?2?VBCP-3-A is a homogeneous acidic heteropolysaccharide with a molecular weight of 963.544 KD.Its total polysaccharide content is 93.7%,uronic acid content is 61.16%,its monosaccharide consists by:rhamnose:arabinose:galactose:galacturonic acid=1.00:2.39:0.98:5.93 and a small amount of glucose.The structure of the polysaccharide is mainly composed of?1,5??-L-Araf and 1,3,5-?-L-Araf,and is attached to a3-position on the 1,3,5-?-L-Araf,?1,5??L-Araf or?1,4??-D-Galacturonic acid.?3?VBCP-4-A is a homogeneous acidic heteropolysaccharide with a molecular weight of 1185.182 KD,with a total polysaccharide content of95.1%,uronic acid content of 56.22%,its monosaccharide component is:rhamnose:arabinose:galactose:galacturonic acid:glucose:mannose=1.00:2.33:1.04:4.64:0.43:0.14.The polysaccharide is based on?1,3??-L-Araf and is branched on the C-5 of Araf.The branched chain is?1,4??-D-Glap.?1,6??-D-GlapA,?1,2??-L-Rhap,(?1,6??-D-Glcp.4.Study of the polysaccharide from vinegar Bupleurum chinese solution properties:the aqueous of the polysaccharides from vinegar Bupleurum chinese solution has a distinct Tyndall effect.Baicalin solution changes from a suspended state to a clear and transparent state after addition of the polysaccharides,and the solubility of baicalin increases with increasing polysaccharide content.The critical aggregation concentration was determined to be 0.495 mg/mL by the limulus probe method.According to the results of transmission electron microscopy,it was found that the polysaccharides from vinegar Bupleurum chinese formed a spherical structure in the aqueous solution.The isothermal titration calorimeter was used to determine the calorimetric change of baicalin in aqueous solution of the polysaccharides from vinegar Bupleurum chinese.The Gibbs free energy,the enthalpies and entropy change values were all negative,indicating that the reaction belonged to a spontaneous endothermic reaction.5.Study on the polysaccharides from vinegar Bupleurum chinese on the pharmacokinetics of baicalin:the polysaccharides from Vinegar Bupleurum chinese increased the AUC?0-t?and Cmax of baicalin by 36%,and 33%respectively.Conclusion:The polysaccharides of traditional Chinese medicines have the universality of increasing the dissolution of poorly soluble compounds.The solubilization may be related to the uronic acid content of the polysaccharide and the structure of the main chain.Possible solubilization mechanism is that the aqueous solution of the polysaccharides from Vinegar Bupleurum chinese would form a spherical aggregate of macromolecules,and the particle size is uniform and the insoluble compound can be entrapped in it to achieve solubilizing effect,and this effect has the effect of increasing bioavailability,suggesting that traditional Chinese medicine polysaccharides can be developed as functional excipients to improve the solubility of insoluble drugs. |
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