Study On Extraction And Purification Of Genistein And Apigenin From Pigeonpea [Cajanus Cajan (L.) Millps.] Roots And Their Antioxidant Activities

In the present study, the negative-pressure cavitation extraction (NPCE) of genistein and apigenin from the roots of pigeonpea [Cajanus cajan (L.) Millsp.] was studied for the first time, and the process for separation and purification of genistein and apigenin by macroporous absorption resin, silica gel column chromatography, recrystallization,etc. was optimized. Moreover, the antioxidant activity of ethanol extracts of leaves, stems and roots of pigeonpea as well as genistein and apigenin was studied. The results as follow:1.An RP-HPLC method for simultaneous determination of genistein and apigenin was developed as follows:Chromatographic column Diamonsil C18V (5μm,250 mm×4.6 mm I.D.),flow rate 1 mL/min, column temperature 30℃, detection wavelength 260 nm, injection volume 10μL.A mobile phase programme was methanol-water-formic acid (65:34.935:0.065,v/v/v).2.Negative-pressure cavitation extraction conditions for genistein and apigenin in pigeonpea roots were optimized, and the main parameters influencing the recovery were studied. The optimum extraction process was as follows:Extraction solvent:ethanol-water(70:30, v/v) solutionParticle size:50 meshRatio of liquid/solid:44:1Extraction time:45 minNegative-pressure:-0.05 MPaNumber of extraction cycles:3 timesExtraction temperature:room temperatureUnder the above optimum conditions, the extraction yields of genistein and apigenin were 0.418 mg/g and 0.118 mg/g,the RSD of them were 1.06% and 1.53%,respectively.3.The optimum process for the purification of genistein and apigenin from pigeonpea roots was obtained firstly, and the results were as follows:(1)Separation of genistein and apigenin by macroporous adsorption resins was studied and the optimum adsorption and desorption parameters on the optimal ADS-5 resin were obtained.Sample concentration:genistein 0.3307 mg/mL, apigenin 0.0475 mg/mLProcessing volume:20 BVAdsorption flow rate:1 mL/minpH value of sample solution:4.5Desorption solution and volume:ethanol-water(20:80, v/v) solution 4 BV and followed by ethanol-water (70:30, v/v) solution 10 BVDesorption flow rate:1 mL/minOperating temperature:25℃After treated by ADS-5 resin under the above conditions, the contents of genistein and apigenin were increased 9.36-fold and 11.09-fold from 1.62%,0.22% to 15.16% and 2.44%, the recovery yields were 89.78% and 93.41%,respectively.(2) The optimum conditions of normal phase medium pressure silica gel column chromatography:an gradient elution was performed with Ethylacetate-Petroleum Ether(12:1, v/v) on the column, in which filled silica gel of 300 to 400 meshes, the quantity ratio of sample/silca gel was 1:50, the flow rate was 30 mL/min, the ratio of diameter/height was 1:3.5. And after this process the content of genistein and apigenin were 73.26% and 20.13%.(3) The purification of reverse-phase medium pressure ODS column chromatography:the quantity ratio of sample/ODS silica gel was 1:100;the flow phase was methanol-water-formic acid (60:39.935:0.065,v/v/v).(4) Crystallization was done in methanol, recrystallization was finished with solution of CHCl3-Petroleum Ether(1:3,v/v).The yellow crystal for genistein and light yellow crystal for apigenin with more than 95% in purity were obtained.(5) The products were identified as the target compounds by the reaction of physical and chemical property combined with FT-IR, ESI-MS-MS,1H-NMR and 13C-NMR.4. The antioxidant activity of ethanol extracts of leaves, stems and roots of pigeonpea and two active constituents in pigeonpea i.e.genistein and apigenin were investigated. In the DPPH radical scavenging assay, the stems extracts exhibited better activity; when the concentrations were lower, the DPPH radical scavenging activity of leaves and roots extracts were weak, when the concentration was higher than 2 mg/mL,their activity was much better; the IC50 for leaves, stems and roots extracts were 1.407,0.844 and 1.184 mg/mL, respectively. In theβ-carotene bleaching test, the roots extracts showed notable antioxidant activity (IC50 0.206 mg/mL), which is nearly comparable to the synthetic reference BHT (IC50 0.196 mg/mL);the IC50 for leaves and stems and roots extracts were 0.378 and 0.311 mg/mL.In both DPPH radical scavenging assay andβ-carotene bleaching test, both of genistein and apigenin demonstrated moderate antioxidant activity, the IC50 were 0.341 and 0.312 mg/mL in DPPH radical scavenging assay,0.185 and 0.176 mg/mL inβ-carotene bleaching test, which were nearly comparable to the synthetic reference BHT (IC50 0.196 mg/mL).