| Objective Through compaired pharmacological experimental study with the laboratory of Radix Tetrastigmae polysaccharide and the alcohol extraction,Radix Tetrastigmae polysaccharide have a good antipyretic on animal.While polysaccharide is one of the main soluble components,Radix Tetrastigmae polysaccharide can get a maximum efficiency of extraction and purification by processing optimization and charactering of its structure.As well as for the clinical application of the polysaccharide,it lays a good foundation for the benefit of mankind to provide a scientific basis for clinical application.Methods(1)The antipyretic experiment was carried out in a dry yeast induced mouse model.The method of water extract and alcohol precipitation was used to extract Radix Tetrastigmae polysaccharide,and the alcohol extraction was extracted by the aerial part of Radix Tetrastigmae.60 ICR mice were randomly divided into 9 groups,blank group,model group,control group(aspirin),Radix Tetrastigmae polysaccharide low-dose(150 mg/kg),medium-dose(250 mg/kg)and high-dose(350 mg/kg)groups,Radix Tetrastigmae alcohol extraction low-dose(150 mg/kg),medium-dose(250 mg/kg)and high-dose(350 mg/kg)groups.Except the blank group,the other groups were subcutaneously injected with 20%20mL/kg dry yeast suspension to establish the fever mouse model induced by yeast.When the temperature rises,the model group was given physiological saline 10 mL/kg,aspirin positive group 200 mg/kg,the treatment groups were given corresponding 150 mg/kg,250 mg/kg,the drug concentration of 350 mg/kg.After the temperature rises,it is administered once each 1h,6h.Subsequent groups will measure rectal temperature and observe the cooling effect after administration by 1h,2h,4h,6h.(2)The hot water extraction of Radix Tetrastigmae polysaccharide the absorbance wavelength of 490nm using phenol sulfuric acid method.In a certain range of concentration,the value of A has a linear relation with the content of polysaccharide,thus the colorimetric determines its content;and it will have a methodological study,including the establishment of standard curve,precision,stability,repeatability,recovery rate.And the effects of the method under the optimal extraction process include single factor experiment,orthogonal test.(3)Uniform component of Radix Tetrastigmae polysaccharide was got by separation of DEAE Sepharose Fast Flow anion exchange resin and purification of Sephacryl S-300 High Resolution gel resin.UV scanning was measured its purity.High performance liquid(HPLC)united evaporative light scattering detector was to determine the molecular weight.After trifluoroacetic acid(TFA)hydrolysis,reduction by sodium borohydride(NaBH4)and acetylation with acetic anhydride,the composition of monosaccharides was analyzed on gas phase-mass spectrometry(GC-MS).The monosaccharide composition of PMP was determined and its molar ratio and content ratio were analyzed quantitatively;Energy groups were analyzed by Infrared spectrum.After methylation reaction,TFA hydrolysis,reduction by NaBH4 and acetylation with acetic anhydride,the connection was analyzed on GC-MS.Results(1)Each group of experimental animal with 20%dry yeast suspension were fever,indicating that the model was successful;Radix Tetrastigmae polysaccharide group after administration compared with the model control group,there was significant difference between the aspirin group and the Radix Tetrastigmae polysaccharide of high dose group in one hour(P<0.001),Radix Tetrastigmae polysaccharide of middle dose group is highly significant(P<0.01);there was significant difference between the aspirin group and the Radix Tetrastigmae polysaccharide of high dose group in two hours(P<0.001),the difference in middle dose group provided Radix Tetrastigmae polysaccharide was statistically significant(P<0.01):the difference was statistically significant about the aspirin group in four hours(P<0.001),Radix Tetrastigmae polysaccharide of high dose group and Radix Tetrastigmae polysaccharide of middle dose group were statistically significant differences(P<0.01),the differences were provided in Radix Tetrastigmae polysaccharide of low dose group was statistically significant(P<0.05);In six hours,Radix Tetrastigmae polysaccharide in each dose group and aspirin group were no Statistical significance(P>0.05);there were no significant compared with Radix Tetrastigmae polysaccharide group and the model group(P>0.05),Radix Tetrastigmae polysaccharide of dried yeast suspension in mice induced by fever has a good cooling effect,and it is compared to the aspirin is equivalent to its antipyretic effect.(2)The establishment and the method of chromogenic reaction standard curves of phenol-sulfuric acid method in the study,the results show that the linear correlation study,precision,repeatability and recovery rate are consistent with the methodology validation requirements.The glucose standard curve y=5.0095x-0.0024,r=0.9992,which was lined in the concentration range of 0.0063-0.2000 mg/mL,the average recovery was 98.2%,and the RSD was 1.7%.The result was measured Radix Tetrastigmae polysaccharide content is about 12%,and the best extraction process of Radix Tetrastigmae polysaccharide was optimized by single factor experiment and orthogonal experiment,finally through the optimum theory of orthogonal test for optimization of material liquid ratio 1:15,extraction temperature 80,extraction time 30 min,extraction for 3 times.(3)RP1-1 was got after separation of DEAE Sepharose Fast Flow anion exchange resin and purification of Sephacryl S-300 High Resolution gel resin.UV scanning showed that the component was uniform component.The molecular weights of RP1-1 18205,determined by HPLC united evaporative light scattering detector.PMP analysis of monosaccharide composition,known RP1-1 is composed of five known mannose,monosaccharide rhamnose,galacturonic acid,galactose,arabinose.The mole ratio of monosaccharide was 1:3:48:3:3 and the ratio of the corresponding content was 4:49:503:29:22.GC-MS analysis of monosaccharide composition,known RP1-1 is composed of five known monosaccharide rhamnose,arabinose,glucose,mannose,galactose and the speculated composition of methyl-β-D-glucopyranoside;Infrared spectrum scanning results showed that the RP1-1 which was belonged to pyranoside and contained β-glycosidic bond was a polymerization sugar made up of monosaccharide containing acid residues;RP1-1 had four connection modes,respectively:Glcp(1→,→4)Glcp(1→,→6)Glap(1→,→3)Manp(1→.Conclusion(1)Radix polysaccharide of each group can be reduced in different degrees of mouse fever caused by dried yeast,validated the effect of Radix Tetrastigmae polysaccharide to dry yeast induced mouse model of heat antipyretic,provide a pharmacological basis for its clinical application.(2)The extraction process of Radix Tetrastigmae polysaccharide was optimized,which could provide the main raw material for the development of health care products.(3)Further study on the Radix Tetrastigmae polysaccharide structure was provided some experimental basis by utilizing the method of separation and purification of polysaccharides and using the modern analysis method for the structure identification of uniform components RP1-1. |
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