Extraction,Purification,Structure Analysis And Bioactivities Of Coptidis Radix Fibrous Polysaccharides

Objective: Coptidis Rhizoma polysaccharides have a variety of pharmacological activities.In this study,research on Coptidis Radix fibrous,a by-product during Coptidis Rhizoma process,was carried out to explore the extraction process,separation and purification,structureanalysis and biological activities of polysaccharides from it.Methods: 1.The yield of polysaccharide used as index,four factors including extraction temperature,solid-liquid ratio,extraction time and extraction times were selected to optimize the process of water bath extraction of Coptidis Radix fibrous crude polysaccharides via orthogonal experiment based on single factor experiment.And the four factors of solid-liquid ratio,ultrasonic power,ultrasonic time and ultrasonic times were selected to determine the optimal process of ultrasonic assisted extraction of Coptidis Radix fibrous crude polysaccharides adopting the above experimental design.Furtherly,the decolorization and deproteinization process of Coptidis Radix fibrous crude polysaccharides was optimized by taking decolorization rate,protein clearance rate and polysaccharide retention rate as indexes,the dedecolorized and deproteinizated polysaccharides after were named as Coptidis Radix fibrous polysaccharides（CRFP）.2.CRFP were purified via DE-52 ion exchange chromatography and Sephacry1 S-200 gel filtration chromatography,and two polysaccharides were obtained,named as CRFP-1 and CRFP-2,respectively.The purity and relative molecular weight of CRFP-1 and CRFP-2 were detected by high performance molecular exclusion chromatography（HPSEC）.The structure of the polysaccharides was analyzed by FTIR scanning,and the composition of monosaccharide was analyzed by PMP pre column derivatization.3.Vc or EDTA-2Na used as positive control,the antioxidant activity of CRFP in vitro was comprehensively evaluated by test the following items: the scavenging power of DPPH radical,ABTS radical,hydroxyl radical,superoxide anion radical and DMPD radical,total reducing power,FRAP and ferrous ion chelating power.Moreover,the scavenging power of ABTS and DPPH radical,FRAP and ferrous ion chelating force of CRFP-1 and CRFP-2 were detected respectively.OGTT,OFTT and xylene induced ear swelling test were studied to investigate the hypoglycemic,hypolipidemic and anti-inflammatory effects of CRFP on healthy mice.Intragastric administration of polysaccharides in diabetic mice,induced by intraperitoneal injection of STZ and high-fat diet,at low（100 mg/kg）,medium（200 mg/kg）and high（400 mg/kg）dose,the fasting glucose content of mice was measured every week.After 8 weeks of treatment,OGTT and OFTT were conducted,meanwhile,fasting plasma glucose,insulin level,blood lipid content,liver HK and PK,and antioxidant activity in vivo were measured to evaluate the therapeutic effect of CRFP on diabetic mice.Results: 1.The yield of CRFP was the highest,up to（1.64 ± 0.02）% under the following condition: the water bath temperature 90 ℃,the solid-liquid ratio 1:20（g/m L）,and the extraction time 1.5 h,extracting 3 times.The optimum technology of ultrasonic assisted extraction of CRFP was as follows: ultrasonic power 300 W,ultrasonic time8 min,solid-liquid ratio 1:20（g/m L）and ultrasonic times 3 times.The yield of polysaccharides reached to（3.55 ± 0.07）%;the optimal deproteinization and decolorization process was: the addition amount of resin was 10%（g/m L）,the shaking time was 1 h,the p H of polysaccharide solution was 3.0 and the polysaccharide concentration was 0.4 mg/m L.By above the process,the decolorization rate of CRFP was（81.93 ± 0.05）%,the protein removal rate was（28.74 ± 0.87）%,the polysaccharide retention rate was（79.18 ± 0.22）%,and the weighted score was（65.15 ± 0.26）.2.CRFP-1 and CRFP-2 were obtained by ion exchange chromatography followed by gel filtration chromatography,which were homogeneous polysaccharides with relative molecular weights of 35 341 Da and 34 366 Da via HPSEC detection,respectively,Structural analysis showed that CRFP-1 wasɑ-typepolysaccharide,composing of eight monosaccharides: mannose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,arabinose and fucose,with the molar ratio 0.245﹕0.071﹕0.003﹕0.001﹕0.122﹕0.425﹕0.1235﹕0.01;CRFP-2was β-Type one,composing of ten monosaccharides: mannose,ribose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,xylose,arabinose and fucose.The molar ratio is 0.061﹕0.01﹕0.083﹕0.128﹕0.127﹕0.038﹕0.301﹕0.048﹕0.112﹕0.093.3.The scavenging power of DPPH radical,ABTS radical,hydroxyl radical,superoxide anion radical and DMPD radical of CRFP were（24.46 ± 2.12）μmol Vc equivalent/mg 、（101.63 ± 10.62）μmol Vc equivalent/mg,（0.47 ± 0.17）mmol Vc equivalent/mg,（2.16 ± 0.6）μmol Vc equivalent/μg,（16.61 ± 4.25）μmol Vc equivalent/ng,respectively.Its FRAP was（45.80 ± 4.05）μmol Vc equivalent/mg,ferrous ion chelating capacity was（77.55 ± 9.53）μmol EDTA-2Na equivalent/mg,total reducing force（53.56 ± 2.99）μmol Vc equivalent/mg.The ABTS radical scavenging capacities of CRFP-1 and CRFP-2 were（11.28 ± 1.27）and（65.83 ± 5.24）μmol Vc equivalent/mg,respectively.The DPPH free radical scavenging capacitieswere（4.63 ± 1.18）and（6.74 ± 1.08）μmol Vc equivalent/mg,respectively.And the FRAP were（2.11 ± 0.46）and（22.57 ± 3.34）μmol Vc equivalent/mg,respectively.Ferrous ion chelating capacity were（2.47 ± 1.73）and（17.47 ± 1.51）μmol EDTA-2Na equivalent/mg.The results in vivo showed that CRFP could accelerate the metabolism of glucose and fat in healthy mice,and the AUC of blood glucose and TG decreased in CRFP-L、CRFP-M、CRFP-H dose groups significantly compared with the model group（p < 0.01）.CRFP-L、CRFP-M、CRFP-H dose could significantly inhibit the ear swelling rate of mice caused by xylene（p < 0.01）in a dose-dependent manner.After treatment for 8 w,the blood glucose level of the diabetic group declined significantly compared with that of the model group（p <0.05）.The AUC of OGTT and OFTT in CRFP-L、CRFP-M、CRFP-H groups were significantly lower than those in model group（p < 0.01）.Compared to the（7.87 ±0.54）m IU/L of model mice,the fasting insulin content of CRFP-L 、 CRFP-M 、CRFP-H groups were increased to（13.06 ± 0.77）,（13.92 ± 0.53）,（15.88 ± 0.30）m IU/L,respectively,showing a very significant difference（p < 0.01）.The level of TC in the administration group was lower than that in the model group,with a significant difference in the CRFP-M and CRFP-H dose groups（p < 0.05）.The contents of LDL-C,TG and FFA in diabetic mice after treatment of CRFP declined significantly（p < 0.01）and the HDL-C level increased（p < 0.05）.After treatment of CRFP,the three doses could significantly increase the activities of HK and PK（p <0.01）in the liver of diabetic mice.In addition,CRFP at low,medium and high dose could significantly increase the activities of SOD、CAT and GSH-Px,and value of T-AOC（p < 0.01）,and significantly reduce the content of MDA in serum（p < 0.01）.CRFP significantly increased the activities of SOD,CAT,GSH-Px and T-AOC（p <0.05）,and decreased the MDA content in the liver of diabetic mice（p < 0.01）.Conclusion: Coptidis Radix fibrous polysaccharidesare potential anti-diabetic and anti-inflammatory drugs with hypoglycemic,hypolipidemic,anti-oxidation and anti-inflammatory effects.This study could provide an experimental basis for the in-depth development and utilization of Coptidis Rhizoma.