Feed-derived Natural Plant Ruminant Database

Construction method[25] and representative chromatogram of the fingerprint of Astragalus membranaceus

（1）Instruments and Reagents

Jasco High Performance Liquid Chromatography (HPLC) system (Jasco PU1580 pump,MD-1510 controller,Absorbance Detector). Reference substances of Astragalus membranaceus:calycosin,calycosin-7-O-β-D-glucoside,formononetin,ononin,(6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan,9,10-dimethoxypterocarpan-3-O-β-D-glucopyranoside,(3R)-8,2'-dihydroxy-7,4'-dimethoxyisoflavane.Their structures weredetermined by spectroscopic methods.

（2）Chromatographic conditions

Chromatographic column:YMC-Pack ODS-A (250mm x 4.60mm,5μm).Mobile phase:Acetonitrile and 0.1% phosphoric acid aqueous solution with linear gradient elution.Flow rate:1 mL/min.Detection wavelength:210 nm.Column temperature:Room temperature.Injection volume:10 μL.

（3）Preparation of sample solution and reference substance solution

Preparation of sample solution:Precisely weigh 2.0 g of the dried powder of Astragalus membranaceus root and fruit.Precisely add 50 mL of 50% methanol,then conduct ultrasonic extraction for 45 min.Filter the solution.Recover the solvent from the filtrate under reduced pressure until nearly dry.Dissolve the residue in 2 mL of water.Then,pass the solution through a column chromatography packed with 10 mL of HP-20 macroporous adsorption resin.Elute the column with 50 mL of H₂O and 100 mL of 95% ethanol successively.Recover the solvent from the 95% ethanol eluate under reduced pressure until nearly dry.Dissolve the residue with 50% methanol and transfer it to a 10-mL volumetric flask.Make up to the mark and shake well to obtain the test sample solution.Filter the solution through a 0.45-μm microporous membrane for later use. Preparation of reference substance solution:Weigh appropriate amounts of the reference substances,namelycalycosin-7-O-β-D-glucoside,calycosin,ononin,formononetin,9,10-dimethoxypterocarpan-3-O-β-D-glucopyranoside,(6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan,and (3R)-8,2'-dihydroxy-7,4'-dimethoxyisoflavane respectively.Prepare a reference substance solution with a mass concentration of 40 μg/mL using 50% methanol.

（4）Investigation of the stability of the fingerprint chromatogram of Astragalus membranaceus

The HPLC fingerprint chromatograms of the Astragalus membranaceus sample solution were collected at 0,2,4,8,12,24,and 48 hours respectively.The retention times and peak areas of the fingerprint peaks were investigated.The results showed that the relative standard deviation (RSD) was less than 5%,which met the detection requirements for fingerprint chromatograms.