Studay On Quality Evaluation And Effective Parts Experiment Of Achyranthes Bidentata Blume Based On The Clinical Efficacy Of Tonifying Liver, Kidney And Strong Bones

Objective:To establish multiple indexes the quality criterion for Achyranthes bidentata Blume,optimize wine-processing technology of Achyranthis bidentatae Blume by orthogonal test with muti-indexes,and observe the effects of total sterones and saponins?TSS?and total polysaccharides?TP?extracted from Achyranthes bidentata Blume on cartilage cell apoptosis.The experimental research could enrich quality control method of Achyranthes bidentata Blume,provide reasonable technical parameters for wine-processing technolog and theoretical basis for further research of Achyranthis bidentatae Radix.Methods:1.The index compounds of the content detemination were decided.An HPLC method was established for simultaneous determination of five components??-ecdysterone,25R-inokosterone,25S-inokosterone,Ginsenoside R₀,Chikusetsusaponin?a?in Achyranthes bidentata Blume.The chromatographic separations was performed on an Agilent ZORBAX SB-C₁₈ column?4.6 mm×250 mm,5?m?,with the mobile phase consisting of acetonitrile?A?and 0.5%phosphoric acid?B?in gradient elution mode?0¹8min,17%A;18²0 min,17%A?32%A;20⁶4 min,32%A;64⁶5 min,32%A?17%A?.The detection wavelength was set at 250 nm for?-ecdysterone,25R-inokosterone and25S-inokosterone,and 203 nm for Ginsenoside Ro and Chikusetsusaponin?a.2.The content of total sterones was determined by Spectrophotometry at 242nm with?-ecdysterone as reference substance.The content of total saponins was determined by Spectrophotometry at 544nm with Ginsenoside R₀ as reference substance with5%veniua-glacial acid solution and perchoric acid as developing olyes.The content of total polysaccharides was determined by Spectrophotometry at 484nm.3.Orthogonal test design was adopted to investigate influences of the wine-processing technologic factors,such as the types and amounts of wine added,moistened time.The contents of?-ecdysterone,25R-inokosterone,25S-inokosterone,Ginsenoside R₀,Chikusetsusaponin IVa,total sterones,total saponins and total polysaccharides were determined as synthesis indexes to optimize the processing technology parameters.4.According to the relevant provisions of Chinese Pharmacopoeia 2015 year edition,cubes and wine stir-fried Achyranthis Bidentatae Radix were prepared,the contents of sterones,saponins and polysaccharides in raw and processed Achyranthis bidentatae Radix were compared.5.Based on the physicochemical properties of sterones,saponins and polysaccharides in Achyranthis bidentatae Radix,TSS was extracted by ethanol,purified with macroporous resin column.TP was extracted by water,graded precipitated with ethanol.The quantity determination was carried out with multiple indexes.6.Mice blood was taken from abdominal aorta after intragastric administration for 7 days,t-hen the serum was prepared.The C28/I2 cell line was cultured,the cells in plate and SNP co ncentration for model was optimized;5%,10%,15%volume of TSSandTP were respectively added to cells with SNP for coculturing 24,48,72 hours,so the Pharmacol ogical activity,concentration and time could be made sure;The mixed TSS and TP in differ-ent proportion less than 5%were added to cells with SNP for coculturing 24 hours,so the S-ynergistic activity and the ratio of drug concentration could be made sure;The cellular prolif erative activity was tested by MTT assay.Result:1.An HPLC method for simultaneous determination of five components??-ecdyste-rone,25R-inokosterone,25S-inokosterone,Ginsenoside R₀,Chikusetsusaponin?a?in Achyranthes bidentata Blume is established.This method is simple,reproducible and accurate.The linear ranges of five components were 0.2180².7250?g?r=0.999 8?for?-ecdysterone,0.0796⁰.9950?g?r=0.999 9?for 25R-inokosterone,0.1548¹.9350?g?r=0.9999?for 25S-inokosterone,0.3520⁴.4000?g?r=0.999 8?for Ginsenoside R₀,0.3380⁴.225 0?g?r=0.9998?for Chikusetsusaponin?a,respectively.The average recoveries were 98.37%?101.66%?99.09%?99.25%and 101.53%,with RSD 0.87%?1.02%?1.74%?0.67%and 1.28%,respectively.2.The determination methods of total sterones,total saponins and total polysaccharides in Achyranthes bidentata Blume by UV are accurate and simple.The linear ranges were0.0260⁰.0940mg/mL?r=0.999 2?and 0.0090⁰.0990mg?r=0.9980?,and the average recovery rateswere98.85%and 99.32%,with RSD1.89%and1.17%,respectively.3.The optimum processing technology is as following:Achyranthis bidentatae Radix is mixed with 10%?g/g?yellow rice wine,moistened for 60 min,and stir-fried for 15 min.The process method is simple,easy to control,had good repeatability,confirmed by validation tests.4.In Achyranthis Bidentatae Radix cubes,the contents of?-ecdysterone,25R-inokosterone,25S-inokosteronetotal phytosterone and total polysaccharides were slightly lower than those in raw materials,while the contents of Ginsenoside Ro,Chikusetsusaponin IVa and total saponins were significantly reduced.In wine stir-fried Achyranthis Bidentatae Radix,the contents of sterones were slightly increased,the contents of Ginsenoside Ro and total saponins were higher than those of Achyranthis Bidentatae Radix cubes,but the contents of total polysaccharides was significantly reduced.5.The contents of 5 components and 3 chemical fractions above in three batches TSS and TP were stable.In this experiment,the methods of preparing method of TSS and TP were simple,feasible,with stable quality.6.The best condition to establish C28/I2 damage model was 15×10³ cells per hole,1 mmol-/L SNP for culturing 24h.Compared with the model group,TSS andTP could significantly i-mproved cellular proliferative activity?P<0.05?;The TP was added on the basis of TSS could significantly improved cellular proliferative activity?P<0.05?;The best TP-TSS proportion m ight be around 1.25%-5%.Conclusion:In this paper,an HPLC method is established for simultaneous determination of3 kinds of sterone and 2 kind of saponins in Achyranthes bidentata Blume,and UV methods were developed to determine the contents of total sterones,total saponins and total polysaccharides.The methods are simple,accurate,stable and reproducible,which can be used for evaluation of the quality of Achyranthis bidentatae Radix and its Praeparata.The optimized wine-processing technology is simple,easy to control and reproducible.TSS abd TP both showed effects of the inhibits cell death for C28/I2,and compatible use can enhance the curative effect.