Study On The Extraction And Purification Processes Of Vitexin And Isovitexin From The Leaves Of Cajanus Cajan (L.)Millsp And Their Antibacterial Activities

In the present study, the extraction of vitexin and isovitexin from the leaves of pigeonpea [Cajanus cajan (L.)Millsp.] was carried out, the conditions for separation and purification of vitexin and isovitexin by macroporous adsorption resin and column chromatography were optimized, and the antimicrobial activities of pigeonpea leaves extracts were also studied.1. The optimum conditions of HPLC analysis were obtained as follows: chromatographic column HIQ SIL C18V (4.6mmΦ×250mm), mobile phase methanol-water-phosphoric acid (35:64.74:0.26, v/v/v), detection wavelength 330nm, flowing rate lmL/min, injection volume 10μL.2. The conditions for ultrasonic extraction of vitexin and isovitexin were optimized and the optimum extraction parameters were as follows:Solvent: ethanol-water (60:40, v/v)Ratio of liquid to solid: 20:1Extraction time: 45minExtraction power: 250wExtraction temperature: 40 ~ 50 ℃Extraction degree: 3 timesThe results showed that yield of ultrasonic extraction was much higher than that of traditional methods. The yields of vitexin and isovitexin were 0.1856% and 0.1385%, respectively.3. The separation of vitexin and isovitexin by macroporous adsorption resin was studied and the optimum adsorption and desorption parameters with the optimal resin ADS-5 were obtained. After treated by ADS-5 resin, the contents of vitexin and isovitexin were increased to 3.0146% and 3.6416%, with the recovery yields of 57.3164% and 61.8467%, respectively. The optimum parameters as follows:Optimal resin: ADS-5Temperature: 25℃pH value of sample solution: 5Concentration of sample solution: 30mg/ mLTime of adsorption equilibrium: 2hFlow rate of dynamic adsorption and desorption: 1mL/min4. Column chromatography tests with polyamide were performed for preliminary purification of vitexin and isovitexin, the crude product with the vitexin content 28.1451% and isovitexin content 6.5682% was obtained, with the recovery yields of 31.8612% and 32.1248% .Column chromatography tests with silica gel were followed for the further purification, the contents of vitexin and isovitexin were increased to 83.1216% and 68.3653%, the recovery yields were 57.4153% and 52.3514%, respectively.5. The MIC of ethanolic extract from pigeonpea leaves to staphylococcus epidermidis,staphylococcus aureus and Bacillus subtilis were all 3.00mg/mL, whereas the MIC of ethyl acetate extract to the three gram positivebacteria mentioned above were 1.50mg/mLN 1.50mg/mL> 0.75mg/mL, respectively. As shown in the dynamic curve of antimicrobial activities, when the concentrations of ethanolic and ethyl acetate extracts were 2MIC(MBC), the quantity of bacteria surviving decreased and all the bacteria were killed within 12 h.