Study On The Enrichment, Purification And Antioxidant Activity Of Calycosin And Formononetin From Radix Astragali

Radix Astragali, known as as a traditional Chinese herbal medicines, has been reported to have a wide range of pharmacological activity. In the present study, HPLC method was established to determine the contents of calycosin and formononetin from extracts of Radix Astragali residues. Preliminary enrichment and purification of calycosin and formononetin from extracts of Radix Astragali residues by macroporous resin, silica gel column chromatography, low temperature crystallization and re-crystallization were performed, and the antioxidant activity was evaluated by DPPH. These studies provide a scientific basis for rational and efficient use of Radix Astragali resources and the modernization of traditional Chinese medicine industry.1. HPLC method for determination of calycosin and formononetin was established as follows:Column:Phenomenex Gemini C18110A (250mm×4.6mm ID,5μm); mobile phase: methanol-water-formic acid (60:39.95:0.05, v/v/v); flow rate:1mL/min; injection amount:10μL; column temperature:25℃; detection wavelength:219nm for calycosin,248nm for formononetin. In methodology study, all indexes of detection method meet the analysis requirements.2. Calycosin and formononetin were enrichment and separation through macroporous resins from extracts of Radix Astragali residues, the optimum parameters of adsorption and desorption on the HPD500resin were as follows:Resin type:HPD500resinSample concentration:Calycosin0.69mg/mL, Formononetin0.31mg/mLpH value of sample solution:4Sample solution volume:12BV (108mL)Adsorption flow rate:0.1mL/minDesoiption solution:60%ethanolDesorption solution volume:15BV (135mL)After one run treatment with HPD500resin, the most of impurities were separated out, and the contents of calycosin and formononetin in elutes increased4.41-fold and4.60-fold from1.75%and0.73%to6.49%and2.78%, respectively. The recoveries of calycosin and formononetin were75.16%and77.06%, respectively.3. Sample treated by macroporous adsorption resin processing was further separated by column chromatography, low temperature crystallization and re-crystallization. After a series of separation and purification, the crystal of calycosin and formononetin were obtained with the total recovery were37.80%,32.37%and the purity were95%,94%, respectively.4. The antioxidant capacities of extracts of Radix Astragali residues and its components were assessed with1,2-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay. The results showed that the isoflavones of Radix Astragali has a good DPPH· radical scavonging ability. The concentration of sample required to scavenge50%of DPPH radicals (IC50) were0.02,0.03and 0.05mg/mL for calycosin,60%ethanol eluate and extracts of Radix Astragali residues, respectively. The antioxidant capacities of calycosin were higher than formononetin.In the present study, HPLC method was established to determine the contents of calycosin and formononetin from extracts of Radix Astragali residues. Enrichment, purification and antioxidant activity were carried out and the crystal of calycosin and formononetin with good antioxidant activity were obtained. These studies provide a solid theoretical basis and technical support for the rational development and utilization of Radix Astragali resources, large-scale industrial production and as a natural antioxidant.