Study On High-effective Induction And Extraction Of Luteolin And Apigenin From The Leaves Of Cajanus Cajan (L.) Millsp.

In the present study, the effects of acid induction, enzyme induction and UV radiationinduction on the contents of luteolin and apigenin in pigeonpea [Cajanus cajan(L.) Millsp.]leaves were investigated, the ultrasonic extraction of luteolin and apigenin from enzyme-treated pigeonpea leaves was carried out, the conditions for separation and purification ofluteolin and apigenin by macroporous adsorption resins and polyamides columnchromatography were optimized.1. An RP-HPLC-PAD method for simultaneous determination of luteolin and apigeninwas developed as follows:Chromatographic column HIQ SIL C18V (250 mm×4.6 mm I.D.), mobile phaseacetonitrile-water-acetic acid (30:69.3:0.7, v/v/v), flow rate 1 mL/min, PAD at 345 nm,injection volume 10μL, and column temperature 30℃.2. Effects of acid induction, enzyme induction on the contents of luteolin and apigeninwere studied, the main factors affecting the contents were optimized.Acid induction hydrolysis:Hydrolysis mode: bidirectional acid hydrolysisConcentration of hydrochloric acid: 0.5 mol/LHydrolysis time: 8 hHydrolysis temperature: 90℃After acid induction hydrolysis treatment, the contents of luteolin and apigenin were0.124 mg/g and 0.056 mg/g, which increased 61.1％and 43.6％, respectively, comparing to theuntreated ones.Enzyme induction:Concentration of pectinase: 0.4 mg/mLInduction time: 36 hpH value of pectinase solution: 3.5-4Induction temperature: 30-35℃After enzyme induction treatment, the contents of luteolin and apigenin were 0.268 mg/gand 0.132 mg/g, which increased 248.1％and 238.5％, respectively, comparing to the untreatedones.3. Ultrasonic extraction conditions of luteolin and apigenin from the enzyme-treatedleaves were optimized, and the optimum extraction parameters were as follows:Extraction solvent: ethanol-water(80:20, v/v) solutionRatio of liquid/solid: 20:1Extraction time: 3 times, each 15 min, total 45 min Extraction power: 250 WUnder the above optimum conditions, the extraction yields of luteolin, apigenin were0.257 mg/g and 0.126 mg/g, the contents in extracts were 0.169％and 0.083％, respectively.4. Separation of luteolin and apigenin by macroporous adsorption resins was studied andthe optimum adsorption and desorption parameters on the optimal AL-2 resin were obtained.Sample concentration: luteolin 52.4μg/mL, apigenin 25.6μg/mLProcessing volume: 3 BVAdsorption flow rate: 1.5 BV/hpH value of sample solution: 5Desorption solution and volume: ethanol-water (50:50, v/v) solution 2 BV and followedby ethanol-water (60:40, v/v) solution 2 BVDesorption flow rate: 1 BV/hOperating temperature: 25℃After treated by AL-2 resin under the above conditions, the contents of luteolin andapigenin were increased 15.1-fold and 9.2-fold from 0.169％, 0.083％to 2.55％and 0.76％, therecovery yields were 78.54％and 72.31％, respectively.5. Pigeonpea leaves extracts after separated by AL-2 resin were purified by polyamidescolumn chromatography. After eluted by ethanol-water (60:40, v/v) solution, the content ofluteolin was increased to 42.39％, after eluted by ethanol-water (80:20, v/v) solution, thecontent of apigenin was increased to 14.52％, the recovery yields were 69.42％and 72.86％,respectively.