Time-space Variations, Efficient Extration And Antioxidant Activity Of Effective Constituents In Pigeonpea [Cajanus Cajan (L.) Millsp.]

In the present study,the time-space variations of active constituents in pigeonpea [Cajanus cajan(L.) Millsp.]i.e.vitexin,isovitexin and orientin were investigated,the negative-pressure vavitation extraction conditions for vitexin,isovitexin and orientin were optimized.Moreover,the antioxidant activity of ethanol extracts of leaves,stems and stems of pigeonpea as well as vitexin,isovitexin and orientin was studied.1.An RP-HPLC method for simultaneous determination of vitexin,isovitexin and orientin 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 340 nm,injection volume 10μL.A gradient programme was used with the mobile phase combining solvent A(water containing 0.1%formic acid,v/v) and solvent B(methanol) as follows:33%B(0-10 min),33-52%B(10-28 min),52-33%B(28-30 min).2.Content variations of vitexin,isovitexin and orientin in pigeonpea during the seed germination and seedling growth stages were studied.The best materials for obtaining vitexin, isovitexin and orientin were pigeonpea leaves,the best harvesting time was pre and post 80 days after pigeonpea seeds was planted.The contents of vitexin,isovitexin and orientin in pigeonpea leaves at this time were 0.994,6.634 and 30.889 mg/g,respectively.In both seed germination and seedling growth stages,the contents of orientin were the highest,followed by isovitexin,the contents of vitexin were low;the contents of vitexin,isovitexin and orientin in stems were lower than those in leaves;no vitexin,isovitexin or orientin was detected in roots.3.Negative-pressure cavitation extraction conditions for vitexin,isovitexin and orientin in pigeonpea leaves were optimized,the main parameters influencing the recovery were studied. The optimum extraction process was as follows:Extraction solvent:ethanol-water(60:40,v/v) solutionRatio of liquid/solid:10:1(each time)Number of extraction cycles and extraction time:3 cycles,each 20 min,total 60 minNitrogen flow:30 mL/minExtraction temperature:room temperatureUnder the above optimum conditions,the extraction yields of vitexin,isovitexin and orientin were 0.718,0.707 and 3.512 mg/g,the contents in extracts were 0.182,0.180 and 0.893%,respectively.4.The antioxidant activity of ethanol extracts of leaves,stems and stems of pigeonpea was studied.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.The antioxidant activity of three active constituents in pigeonpea i.e.vitexin,isovitexin and orientin was also investigated,orientin exhibited much better antioxidant activity than vitexin and isovitexin.In both DPPH radical scavenging assay andβ-carotene bleaching test, orientin demonstrated moderate antioxidant activity,the IC₅₀ were 0.329 and 0.407 mg/mL, respectively,which were obviously lower than those of vitexin and isovitexin.