Separation And Purification Of Coptis Chinesis Polysaccharides And Its Impact On Advanced Glycation End-products Formation In Vitro And In Vivo

Coptis chinensis Franch is a common medicine and widely used in clinic in China,which has the very high nutritional value and medicinal value. The initial research of Coptis chinensis Franch mainly focus on alkaloid compounds, while the polysaccharide was rarely reported. Coptis chinensis Polysaccharide is an important active ingredients with value for research and development. It has become a hotspot in the research of natural medicine. In the experiment, the antioxidant activity, hypoglycemic ability and inhibition to advanced glycosylation end products(AGEs) of Coptis chinensis Polysaccharide(CCP) were inverstigated to provide a basis for CCP’s clinical application.PARTⅠExtraction, separation and purification and antioxidant activity of Coptis chinensis polysaccharideObjective: To obtain the CCP from the root of Coptis chinensis Franch, extraction,separation and purification technology will be established and optimized. And in vitro antioxidant activity of CCP will be studied.Method:(1) Extraction of raw Coptis chinensis polysaccharide. On the basis of single factor tests, four elements including solid-liquid ratio, extraction temperature, extraction time and extraction times taking effect on the yield of the Coptis chinensis polysaccharide were researched by orthogonal experimental design. Considering the quality of polysaccharides, the final optimized the best concentration of ethanol.(2) Separation and purification of raw Coptis chinensis polysaccharide. The protein of raw Coptis chinensis polysaccharide was removed by sevage method. Then the rawCoptis chinensis polysaccharide was separated by DEAE-Cellulose, and further purified by Sephadex G-100 gel column.(3) Antioxidant activity of Coptis chinensis polysaccharide in vitro. The raw Coptis chinensis polysaccharide and the CCP compounds with different charge could effectly eliminate the hydroxyl free radical, superoxide anion and DPPH. It can be measured that CCP and the CCP compounds with different charge inhibit hydroxyl free radical on the basis of safranine fading by hydroxyl free radical in Fenton system. In addition, we estimated their inhibition for superoxide anion by UV-757 depending on the fact that there is ultraviolet absorption for the intermediate which is produced by pyrogallol self oxidation releasing superoxide anion. CCP and the CCP compounds with different charge can eliminate the purple colour of ethanol solution caused by DPPH in different degrees. Therefore, their eliminating ability can be compared according to it.Result:(1) Extraction of raw Coptis chinensis polysaccharide. Considering the extraction efficieney and quality of polysaccharides, the final optimization extracting condition was that the ratio of material to liquid ratio of l:70, 1.5 h, 80℃,and the ethylalcohol concentration of 80%.(2) Separation and purification of raw Coptis chinensis polysaccharide. CCP-Ⅰ 、CCP-Ⅱ、CCP-Ⅲ and CCP-Ⅳ were obtained from raw Coptis chinensis polysaccharide after removing protein and purifying by DEAE-Cellulose. CCP-Ⅱtreated with Sephadex G-100 gel column, we got a purification polysaccharide: CCP-Ⅱa.(3) Antioxidant activity in vitro. The CCP could eliminate hydroxy radical, superoxide anion and DPPH. The inhibition ratio of hydroxy radical, superoxide anion and DPPH of the raw Coptis chinensis polysaccharide were 11.29%, 38.38% and 14.51%. The inhibition ratio of superoxide anion of CCP-Ⅰ、CCP-Ⅱ、CCP-Ⅲ and CCP-Ⅳ were56.85%, 33.51%, 44.43% and 56.30% respectively.Conclusion: Optimized extraction condition applied to extraction of CCP reasonably.The CCP can be a natural antioxidant and good free-radical scavenger.PART ⅡEffect of polysaccharides from Coptis chinensis on advanced glycation end-products formation and aldose reductase activityObjective: This study investigated inhibitory effect of polysaccharides from Coptis chinensis on advanced glycation end-products formation and aldose reductase activity.Method:(1) The reaction mixture contained bovine serum albumin(BSA, 50 mg/m L),glucose(500 m M), 0.02% sodium azide and phosphate buffer(200 m M, p H 7.4). Test samples with different concentrations such as 0.01, 0.1, 0.5, 1.0 mg/m L dissolved in phosphate buffer were added to the reaction mixture. Then, the mixture was incubated at 37℃ for 4 weeks to obtain glycated materials. Aminoguanidine(50mmol/L) was used as the positive control and normal control without any drug. During the incubation time, after 0, 7th, 14 th, and 30 th days, some glycated materials were taken from the system for the following experiments. Their ultraviolet absorption were measured by UV at 530 nm after 14 day at 37℃. Four weeks later, AGEs’ fluorescence value was measured(excitation wavelength 370 nm, emission wavelength 371 nm, slot 1.5 nm)and the concentration of Girard-T was estimated by its ultraviolet absorption according to the standard curve.1m L of glycated material(30th day) was precipitated by adding an equal volume of10% TCA and centrifuged at 6000 rpm for 15 min. The precipitate was washed with 5%TCA, redissolved in SDS-PAGE sample buffer, denatured in boiling water for 2-10 min,and applied to SDS-PAGE. The samples were separated on 30% polyacrylamide gelcontaining 1% SDS. The inhibition ratio of CCP to glycation products was estimated depending on the bands.(2) The blood(1 m L) was obtained from Kunming rats, and was centrifugated at 3000r/min for 15 min To got the erythrocyte. Then it was washed for 3 times by adding to 10 volumes of saline at 4℃. The erythrocyte was destroyed by adding 4 volumes of 10mmol/L PBS buffer(p H 6.0) and shocking for 10 min, and then centrifugated. NAPPH,DL-glyceraldehyde, ammonium sulphate, phosphate buffer and appropriate supernatant was reacted at 37℃ for 5 min. The reaction was stopped by adding 2 m L 0.3 mol/L Na OH solution. And adding 3 m L 11.8 mol/L Na OH solution containing 0.1% H2O2 reacted for 5 min at 37℃ to produce fluorescence. Aldose reductase activity was measured by the fluorescence spectrophotometer.Result:(1) In glycosylation reaction, inhibitory effect of polysaccharides from Coptis chinensis on initial fructosamine formation depend on its concentration in vitro. The inhibition ratio of 1.0 mg/m L CCP was higher than 50 mmol/L aminoguanidine significantly. It could also inhibit further dicarbonyl compounds. The fluorescence intensity of AGEs showed different concentrations of the polysaccharide decreased significantly 30 days later(P<0.01). The inhibition of 1.0 mg/m L CCP solution could reach 90%. In addition, SDS-PAGE electrophoresis had proved it.(2) The fluorescence intensity of AGEs showed that CCP with different concentration and aminoguanidine could inhibit aldose reductase activity. The inhibition ratio of 30μg/m L CCP could reach 93.01%.Conclusion: Inhibitory effect of polysaccharides from Coptis chinensis on advanced glycation end-products formation and aldose reductase activity.PART ⅢInhibitory effect of Coptis chinensis polysaccharide on advanced glycation end-products of diabetic miceObjective: To study the effect of coptis chinensis polysaccharide on blood-glucose and advanced glycation end-products of diabetic mice, and discuss the pathological change of heart, liver and kidney of diabetic mice.Method: After feeding for 7 days, The rat model of diabetic were established model by intraperitoneal injection of streptozocin(STZ, 125 mg/kg). The diabetic mice were randomly assigned to the model group, administration group with CCP by intragastric administration and positive control group with AG, which was a reference hypoglycemic drug, by intragastric administration, with the normal rats as the blank control group. The weight and the fasting blood glucose(FBG) were measured for each mice at certain time. After 15 days, the mice were sacrificed and the content of AGEs in heart, liver and kidney was measured. Finally, Hematoxylin-eosin(HE) stain were used to observe the structural changes in heart, kidney and liver of mice.Result: After diabetic model preparation, FBG, GSP, AGEs in serum and AGEs levels in liver tissue homogenate of STZ-induced diabetic rats were significantly higher than those of normal rats(P<0.01). The administration of coptis chinensis polysaccharide for15 days in STZ-induced diabetic rats could significantly reduce FBG, serum levels of GSP(P<0.01) and AGEs(P<0.01), and AGEs in liver tissue(P<0.05). Tissue dyeing assays also showed significantly improvement on liver structure.Conclusion: The administration of CCP could decrease FBG, serum levels of GSP and AGEs, and AGEs in liver tissue. CCP has a role in protecting the liver.