Study On The Extraction And Purification Of Biochanin A And Genistein From Dalbergia Odorifera T. Chen Leaves

In this study, the RP-HPLC method for determination and quantification of biochanin A and genistein was developed. An efficient microwave-assisted enzymatic extraction (E-MAE) technique was developed to simultaneously extract biochanin A and genistein from Dalbergia odorifera T. Chen leaves. Box-Behnken Design combined with response surface methodology was applied to optimize the operating parameters of MA-ATPE. After, simple rand efficient protocols were developed for preparation of biochanin A and genistein from Dalbergia odorifera T. Chen leaves using macroporous resin followed by flash chromatography. The optimum process of extraction, separation and purification of biochanin A and genistein were established, which provides the scientific basis for the rational and efficient use of Dalbergia odorifera T. Chen resources.1. RP-HPLC method for determination and quantification of biochanin A and genistein was developed as follows:Chromatographic conditions:HIQ Sil C18V reversed-phase column (250mm×4.6mm I.D.,5μm); column temperature:30℃; mobile phase:acetonitrile (A)-0.1%formic acid aqueous solution (B); gradient elution:0-5min,5-10%A;5-10min,10-28%A;10-30min,28-60%A;30-35min,60%A;35-37min,60-80%A;37-42min,80-100%A;42-55min,100%A; flow rate:1.0mL/min; injection volume:5μL.2. Microwave-assisted enzymatic extraction method was investigated on the extraction yields of biochanin A and genistein, the optimum parameters of the extraction process were as follows:Amount of enzyme:3.5mg/gpH:5.2Extraction time:15minExtraction temperatures:34℃Microwave power:300WUnder the above optimum conditions, the extraction yields of biochanin A and genistein were2.012and1.023mg/g, respectively.The extraction yields of biochanin A and genistein were about17.45%and63.94%higher than those of heat reflux extraction (HRE); the extraction yields of biochanin A and genistein were about5.07%and13.79%higher than those of ultrasound extraction (USE), respectively.3. Enrichment of biochanin A and genistein by macroporous adsorption resins was studied. Macroporous resin adsorption and desorption of the best conditions as follows:Macroporous resin:AL-2Macroporous resin/sample ratio:5:1 After treatment with AL-2resin, the contents of biochanin A and genistein in the enriched product were6.60-fold and6.41-fold increased with recovery yields of87.13%and84.60%, respectively.4. Separation and purification of biochanin A and genistein by flash chromatography was studied. The operating parameters of flash chromatography were optimized. The optimal conditions were as follows:stationary phase:silica gel, elution system:n-hexane/ethyl acetate, sample/silica gel ratio:1.3:40and flow rate:50mL/min, gradient program:0-8min, n-hexane-ethyl acetate (10:1, v/v);8-35min, n-hexane-ethyl acetate (6:1, v/v).After one flash chromatography run, purities of biochanin A and genistein effectively reached over95%, their recovery yields were80.13%and73.11%, respectively. The developed protocol was simple, efficient, scalable and economical, which represented an excellent alternative for the separation and purification of bioactive compounds from plants.5. After microwave-assisted enzyme extraction, macroporous adsorption resin, flash chromatography, the purity of biochanin A and genistein were more than95%, the total recovery of biochanin A and genistein were69.82%and61.85%.