Study On Efficient Extraction Of Calycosin And Formononetin From Radix Astragali

In the present study, Incubation pretreatment with the function of endogenous enzyme combined with negative-pressure cavitation extraction (IP-NPCE) was established to extract calycosin and formononetin from Radix Astragali for the first time, and the parameters of the extraction process for IP-NPCE was optimized. Furthermore, the extraction yield, energy consumption of two aglycones by different extraction methods and antioxidant activity of crude extracts was studied respectively. The results as follows:1. A HPLC-UV analysis method for simultaneous determination of calycosin and formononetin was established as follows:Chromatographic column A Phenomenex Gemini C18110A reverse column (250mm×4.6mm ID,5μm), flow rate1mL/min, column temperature30℃, detection wavelength248nm, injection volume10μL. A mobile phase was acetonitrile (A)-0.05%formic acid (B). The following gradient elution program was used for separation:0-50min,10-40%(A);50-55min.40-50%(A);55-80min,50-100%(A).2. The enzyme biotransformation process parameters of calycosin and formononetin were optimized. The optimal endogenous enzyme incubation pretreatment parameters as follows:Incubation solvent:sterile distilled waterIncubation temperature:35℃Incubation time:60minIncubation pH value:4Incubation negative pressure:-0.07MPaSolid-to-liquid ratio:1:25Incubation cyeles:1timeUnder the above optimized incubation pretreatment conditions, the contents of calycosin and formononetin were0.626,0.297mg/g (n=3), the R.S.D. were2.13%and1.78%, respectively, which were88.55%and72.67%higher than those without incubation pretreatment, respectively.3. Significant factors involved in the NPCE process of calycosin and formononetin were selected using Plackett-Burman factorial design (PBD) and then were optimized by central composite design (CCD). The optimum experimental conditions as follows:Negative pressure (NP):-0.08MPaEthanol concentration (EC):60%ethanol solutionParticle size (PS):60meshExtraction cycle (ECY):2times pH value (pH):natural pHExtraction time (ET):30minSolid-to-liquid ratio (SLR):1:25Validation experiments were conducted under conditions optimized above. The observed values of the extraction yields of calycosin and formononetin from experiments were0.650and0.307mg/g (n=3), the R.S.D. were2.13%and1.78%, respectively, the relative yield of IP-NPCE increased even two-fold to that of other extraction methods. Furthermore, the antioxidant activities of extracts of IP-NPCE were1.845mg/mL (IC50) and0.703mmol Fe(II)/g according to the DPPH and FRAP assays, respectively. The above results demonstrated that the novel extraction technique not only gave higher yields of target compounds but also enhanced antioxidant activities of crude extracts against other extraction methods.The results of this study fully proved the IP-NPCE was very applicable for efficient extraction of calycosin and formononetin from Radix Astragali due to high the extraction efficiency, high the throughput, low the equipment expenditure and energy consumption. It may provide valuable data for process design and pilot-scale as well as industrial scale-up applications, and as a potential eco-friendly technology. it is promising for extensive industry applications in the nature product extraction fields.