ABSTRACT
OBJECTIVE： To establish pre- column derivatization-HPLC fingerprint of polysaccharide from Achyranthes bidentata，and to determine the contents of monosaccharide components ，so as to provide reference for quality evaluation of A. bidentata decoction pieces. METHODS ：Taking 10 batches of A. bidentata decoction pieces from different manufacturers as samples，the polysaccharides were extracted by water extraction and alcohol precipitation ，hydrolyzed by trifluoroacetic acid ，and then derivatized by 1-phenyl-3-methyl-5-pyrazolone for HPLC analysis. The determination was performed on Hanbon Hedera C 18 column with column temperature of 30 ℃ at the flow rate of 1.2 mL/min. The mobile phase consisted of acetonitrile- 0.02 mol/L ammonium acetate solution （gradient elution ）. The detection wavelength was set at 250 nm，and sample size was 20 μL. HPLC fingerprint was established and similarity evaluation was performed for 10 batches of A. bidentata polysaccharide by using TCM Chromatogramic Fingerprint Similarity Evaluation System （2012A edition ）. The common peak was identified by comparing with the reference substance ，and cluster analysis was performed by using SPSS 25.0 software. The contents of identified monosaccharides were determined by HPLC. RESULTS ：The similarity of 10 batches of A. bidentata polysaccharide were all higher than 0.95. Nine common peaks were fixed and a total of 5 common peaks were identified ，namely anhydrous glucose （peak 1）， mannose（peak 2），rhamnose（peak 4），galacturonic acid （peak 5）and arabinose （peak 7）. Results of cluster analysis showed that S1 sample was classified into one category ；S2，S5，S8 and S 9 samples were clustered into one category ；S3，S4，S6，S7 and S10 samples were clustered into one category. Results of content determination showed that the contents of anhydrous glucose in 10 batches of samples ranged from 6.17 to 17.55 mg/g；those of mannose ranged from 3.31 to 7.66 mg/g；those of rhamnose ranged from 38.80 to 73.97 mg/g；those of galacturonic acid ranged from 2.49 to 8.95 mg/g；those of arabinose ranged from 11.30 to 28.58 mg/g. CONCLUSIONS ：Established pre-column derivatization HPLC fingerprints and content determination method can provide reference for quality evaluation of A. bidentata decoction pieces.
ABSTRACT
OBJECTIVE：The establ ish the qu ality standard of Achyranthes bidentata decoction pieces and establish the fingerprint of decoction pieces of different origins. METHODS ：TLC method was used to identify A. bidentata decoction pieces. The contents of water ，total ash and ethanol-soluble extract in A. bidentata decoction pieces were determined according to the method in 2015 edition of Chinese Pharmacopoeia （part Ⅳ）. UV spectrophotometry was used to determine the content of total steroid. The content of β-ecdysterone in A. bidentata decoction pieces was determined by HPLC. HPLC method was used to establish the fingerprint of 10 batches of A. bidentata decoction pieces. Using β-ecdysterone（No. 10 peak）as reference ，similarity evaluation was conducted by using Similarity Evaluation System for TCM Chromatographic Fingerprints （2012 edition），and the common peaks were determined. SPSS 21.0 software was used for cluster analysis and principal component analysis so as to evaluate the comprehensive quality of A. bidentata decoction pieces. RESULTS ：Results of TLC identification showed that the spots with the same color on the corresponding positions of β-ecdysterone and ginsenoside Ro control in chromatogram of test sample. The average water content of 10 batches of A. bidentata decoction pieces was 4.07％ -6.33％ . The total ash content was 5.04％ -6.43％ . The ethanol-soluble extract was 6.57％ -11.12％ . The linear range of total sterone （by β-ecdysterone） and β-ecdysterone were 0.01-0.08 mg/mL and 68.5-479.5 µg/mL （R2＞0.999）， respectively. RSDs of precision ，stability and repeatability tests were all less than 3％. The average recovery rates were 98.85％（RSD＝1.89％，n＝6）and 100.34％（RSD＝ 2.12％，n＝9），respectively. The average contents were 0.34％-0.56％ and 0.07％-0.09％，respectively. There were 24 common peaks in 10 batches of A. bidentata decoction pieces ，and the similarity was all over 0.900. Cluster analysis results showed that 10 batches of A. bidentata decoction pieces could be grouped into 4 categories，among which S 1，S3，S6 and S 10 were one category ， S2，S7 and S 8 were one category ，S4 and S 9 were one category ，and S 5 was one category. The results of principal component analysis showed that the cumulative variance contribution rate of the first four principal components was 86.774％. The order of comprehensive quality is S 8＞S5＞S9＞S4＞S3＞S7＞S6＞S10＞S2＞S1. CONCLUSIONS ：The established quality standard ， content deter mination method and HPLC fingerprint are stable and accurate ，and can be used for the quality evaluation of A. bidentata decoction pieces. Δ 基金项目：国家科技部重点研发计划（No.2018YFC1707105） ＊硕士研究生。研究方向：中药质量控制。E-mail：995354085@