Study On Extraction And Enrichment And Separation Of Four Main Flavonoids From Dalbergia Odorifera T.Chen Leaves

Dalbergia odorifera T. Chen has been used as a rare traditional Chinese medicine since ancient times, and modern researches show that dalbergia have various bioactive, such as anti-allergic, anti-inflammatory, anti-tumor and cardiovascular protection. But the plant resource of Dalbergia odorifera T. Chen is scarce, and most research is focused on its heartwood, while Dalbergia odorifera T. Chen leaves has little research. Preliminary studies indicate that isoflavones in Dalbergia odorifera T. Chen leaves show excellent bioactivities (i.e. antioxidant and antitumor activity). And four high levels of isoflavones from Dalbergia odorifera T. Chen leaves has been identified as prunetin, tectorigenin, genistein and biochanin A. Therefore, our study focused on extraction, and enrichment and separation of these four important isoflavones from Dalbergia odorifera T. Chen leaves, developed a fast and easy analysis methods of HPLC, and established an efficient extraction process. Our study provided theoretical guidance for the full development of Dalbergia odorifera T. Chen leaves, also produced a positive effect on protection of the plant resource of Dalbergia odorifera T. Chen.The main findings are as follows:1. A rapid analysis method of HPLC was established for determination of prunetin, tectorigenin, genistein and biochanin A:Column:Phenomenex CuroSil-PFP (250mm X4.6mm ID,5?m); mobile phase: acetonitrile (A)-0.1%formic acid (B); flow rate:1mL/min; column temperature:30?; detection wavelength:260nm; injection volume:5uL. Gradient elution conditions:0-15min,28-40%(A);15-25min,40-100%(A).2. The optimal parameters of negative pressure cavitation assisted deep eutectic solvent extraction process:Deep eutectic solvents:Choline chloride/ethylene glycol (1:2, mol/mol)The ratio of deep eutectic solvent to water:74/26Extraction pressure:-0.07MPaExtraction temperature:45?Extraction time:20minLiquid/solid ratio:20:1mL/gExtraction cycle:3cyclesUnder these optimum extraction conditions, the average extraction yields of prunetin, tectorigenin, genistein and biochanin A was1.204mg/g,1.057mg/g,0.911mg/g and2.448mg/g. And negative pressure cavitation assisted deep eutectic solvent extraction process is better than ultrasonic extraction and heat reflux extraction. 3. Macroporous resins for enrichment and separation of prunetin, tectorigenin, genistein and biochanin A in deep eutectic solvent:Type of resin:AB-8, ME-2and SA-3AB-8macroporous resin made the recovery yields of prunetin, tectorigenin, genistein and biochanin A reached79.3%,83.9%,78.8%and86.3%; SA-3macroporous resin made the recovery yields of prunetin, tectorigenin, genistein and biochanin A reached83.2%,84.7%,79.4%and80.1%; ME-2macroporous resin made the recovery yields of prunetin, tectorigenin, genistein and biochanin A reached78.2%,88.7%,75.1%and88.6%. The enrichment and separation of these three macroporous resins made the content of prunetin increased6.5-6.9fold, the content of tectorigenin increased6.9-7.3fold, the content of genistein increased6.2-6.6fold and the content of biochanin A increased6.7-7.3fold, compared with their contents in crude ethanol extract.