Study On The Process Of Extraction, Separation And Purification Of Astragalosides From Radix Astragli

In the present study, the effect of microwave-assisted extraction of astragalosides in Radix Astragali was investigated. Extraction yields of AstragalosidesⅠ-Ⅳ(AGsⅠ-Ⅳ) were used as index, the conditions for extraction of astragalosides were optimized. Preparative separation and purification of astragalosides from Radix Astragali extracts was carried out by macroporous resins, hydrolysis transformation of astragaloside derivatives, silica gel column chromatography, low temperature crystallization and re-crystallization. The optimum process of extraction, separation and purification of astragalosides was established and the LC-ESI/MS method for determination and quantification of astragalosides was developed, which provides the scientific basis for the rational and efficient use of Radix Astragali resources.1. LC-ESI/MS method for determination and quantification of AGs I-IV was developed as follows:Chromatographic conditions:Agilent 1100 series HPLC system; column:Agilent Eclipse XDB-C18 column (150 mm×4.6 mm i.d.,5μm); column temperature:30℃; mobile phase: 0.05% formic acid aqueous solution (A)-acetonitrile (B); gradient elution:0-6 min 66% (A),6-8 min 66-55%(A),8-13 min 55% (A),13-14 min 55-66%(A),14-17 min 66%(A); flow rate:0.7 mL/min; injection volume:10μL.MS conditions:An API3000 triple tandem quadrupole mass spectrometry; electrospray ionization in positive ion mode (ESI+); multiple reaction monitoring mode (MRM); The nebulizing gas (NEB), curtain gas (CUR) and collision gas (COL) were set at 12,10 and 6 a.u., respectively. The ion source was operated at temperature of 250℃. The ion spray voltage was 5500 V.2. Microwave-assisted extraction method was investigated on the extraction yields of AGs I-IV, the optimum parameters of the extraction process were as follows:Extraction solvent:ethanol-water (80:20, v/v) solution Ratio of solid/liquid:1:25 Extraction time:5 min Extraction temperatures:70℃Microwave power:700 W Extraction cycles:3 timesUnder the above optimum conditions, the extraction yields of AGs I-IV were 0.788 mg/g, 0.351 mg/g,0.206 mg/g and 0.278 mg/g, respectively. The recoveries of AGs I-IV were 91.415%, 89.086%,86.193% and 94.600%, respectively.3. Enrichment and separation of AGsⅠ-Ⅳby macroporous adsorption resins was studied and the optimum parameters of adsorption and desorption on the optimal SA-3 resin were obtained. Macroporous resin adsorption and desorption of the best conditions as follows: Resin type:SA-3 resin Sample concentration:AGⅠ0.483 mg/mL, AGⅡ0.460 mg/mL, AGⅢ0.168 mg/mL, AG IV 0.320 mg/mL Processing volume:30 BV (180 mL) Adsorption flow rate:1 BV/h pH value of sample solution:4 Desorption solution:ethanol-water (50:50, v/v) solution Desorption solution volume:9 BV (54 mL) Desorption flow rate:1 BV/hAfter treated by SA-3 resin under the above optimum conditions, the contents of AGs I-IV in the processing increased from 0.559%,0.532%,0.194% and 0.371% to 4.908%,6.173%,2.043% and 4.181%, which were 8.78-fold,11.60-fold,10.52-fold and 11.28-fold increased, respectively. The recoveries of AGs I-IV were 65.883%,90.920%,84.249% and 94.167%, respectively.4. By hydrolysis conversion technology, other types of acetylated astragalosides in sample solution after enrichment and separation by SA-3 resin were hydrolyzed into AGⅣ, which improved the content of AG IV, the recovery was 189.861%. Using column chromatography, low temperature crystallization and re-crystallization, the crystal of AG IV was obtained with the recovery of column chromatography 57.935% and the recovery of re-crystallization 69.854%.5. After microwave-assisted extraction, macroporous adsorption resin, hydrolysis transformation of astragaloside derivatives, column chromatography, low temperature crystallization and re-crystallization, the purity of AGⅣwas 93%, the the total recovery of AGⅣwas 68.448% and the total extraction yield of AG IV was 0.201 mg/g. Compared with traditional methods, this method is short, simple, less environmental pollution, and the product was high purity and high yield. It provides a scientific reference for the industrial production of AG IV.