| In the present study, an efficient, fast and accurate method was developed for the analysis of flavonoids in pigeon pea leaves. The variations in contents of flavonoids during growth and post-harvest storage of pigeon pea seedlings were studied. The parameters for negative-pressure cavitation extraction of flavonoids were optimized. The pER8-GFP transgenic Arabidopsis was used to study the estrogenic activity of extract of pigeon pea leaves and its different fractions, ethyl acetate fraction was found as the active fraction. Reversed-phase ODS medium-pressure column chromatography in combanition of recrystallization technique was used to separate and purify the flavonoids in ethyl acetate fraction. The estrogenic activity of the purified flavonoids was further screened by using the pER8-GFP transgenic Arabidopsis, cajanol was confirmed as the active constituent. Furthermore, the preliminary antitumor activity of cajanol was investigated.1. A LC-MS/MS method was developed for the simultaneous separation and determination of flavonoids in pigeon pea leaves.Chromatographic conditions:HiQ Sil C18V reversed-phase column (250 mm×4.6 mm I.D.), mobile phase consisted of methanol and 0.1% formic acid aqueous solution, flow rate 0.7 mL/min, injection volume 10μL and column temperature 30℃.MRM transitions:apigenin m/z 269.0→117.0, luteolin m/z 284.8→133.0, isorhamnetin m/z 315.1→151.0, vitexin m/z 431.0→283.0, isovitexin m/z 431.0→283.0, orientin mlz 447.3→327.0, cajanol m/z 315.1→178.9 and genistin m/z 431.3→268.0.The flavonoids in pigeon pea leaves were detected with CID-MS/MS analysis at negative-ion mode, eight flavonoids were successfully separated, identified and quantified for the first time. The developed method was selective, sensitive and accurate, which was suitble for the determination of flavonoids in pigeon pea leaves as well as in other plants.2. The variations in contents of flavonoids during growth and post-harvest storage of pigeon pea seedlings were investigated, the results were as follows:(1) Vitexin, isovitexin and orientin were mainly accumulated in pigeon leaves, while the contents in stems and roots were significantly lower. Genistin was mainly accumulated in roots. Apigenin, luteolin, isorhamnetin and cajanol could be obtained from all three parts of pigeon pea seedlings, their contents in different parts were similar. In a comprehensive consideration of the contents of eight flavonoids, pigeon pea leaves are the main accumulative part, the 40th day after the emergence of leaves is the optimal harvesting time. Pigeon pea stems and roots were also the sources of flavonoids.(2) In post-harvest storage, slight and steady increases in contents of eight flavonoids were found in room temperature (25℃) stored pigeon pea leaves up to 120 days. The highest accumulation of eight flavonoids in chilling temperature (4℃) stored leaves was observed at the 45th day, after which the contents decreased sharply.The obtained results underline the important role of the pre-and post-harvest factors on the phytochemical characteristics of pigeon pea.3. A negative-pressure cavitation extraction (NPCE) device was designed for the efficient extraction of flavonoids in pigeon pea leaves. The effects of liquid/solid ratio, extraction time, air flow, pressure and number of extration cycles on the recovery of flavonoids were optimized. The optimum parameters were as follows:liquid/solid ratio 10:1 (mL/g), extraction time 20 min, air flow 30 mL/min, pressure-0.04--0.05 Mpa,3 extration cycles and extraction solvent 70% ethanol.Under the above optimum conditions, the relative recoveries of apigenin, luteolin, isorhamnetin, vitexin, isovitexin, orientin, cajanol and genistin were all higher than 95%.In the NPCE process, air was continuously introduced in the device under the action of negative pressure. The resulting tiny bubbles entered in the gas-liquid-solid system comprised of bubbles, liquid drops and solid particles. Under the energy created by the collapse of these bubbles, the equilibrium of three phases was broken, the interactions among target constituents, solid particles and extraction solvents were enhanced, hence, favored the rapid transfer of target constituents from the matrix to the solvent, increased the mass transfer and extraction efficiency.4. The estrogenic activity of extract of pigeon pea leaves as well as its four fractions was studied by using the pER8-GFP transgenic Abrbidopsis, the results were as follows:(1) Extract of pigeon pea leaves possessed estrogenic activity at 200μg/mL and higher concentrations;(2) Among the four fractions of extract of pigeon pea leaves, the activity of ethyl acetate fraction was the best, strong green fluorescence could be observed at the concentration of 100μg/mL; followed by petroleum ether fraction, which exhibited weak estrogenic activity at 400μg/mL; the n-butanol and water fractions were inactive at 400μg/mL. The ethyl acetate fraction was the main active fraction.The pER8-GFP transgenic Abrbidopsis screening method is sensitive and efficient, which is applicable in the confirmation of active fractions with estrogenic activity as well as in estrogenic activity-guided separation tests.5. The separation and purification of flavonoids in ethyl acetate fraction of extract of pigeon pea leaves were studied.The conditions for reversed-phase ODS medium-pressure column chromatography of apigenin, luteolin, isorhamnetin and cajanol were gradient elution by 25% acetontrile-water system with flow rate of 1.5 mL/min. After purification, the purities of apigenin, luteolin, isorhamnetin and cajanol were 92.98%,93.51%,92.42% and 91.56%, with the recoveries being 78.93%,83.12%,75.62% and 77.96%, respectively.The conditions for reversed-phase ODS medium-pressure column chromatography of vitexin, isovitexin, orientin and genistin were gradient elution by 20-30% methanol-water system with flow rate of 1.5 mL/min. After purification, the purities of vitexin, isovitexin, orientin and genistin were 93.12%,92.39%,91.67% and 90.37%, with the recoveries being 81.33%,84.00%,80.14% and 78.24%, respectively.6. The estrogenic activity of eight flavonoids obtained from ethyl acetate fraction of extract of pigeon pea leaves was studied by using the pER8-GFP transgenic Abrbidopsis, the results were as follows:cajanol had the strongest estrogenic activity, it induced high level of GFP protein expression at the concentration of 50μM. Cajanol is the main active constituent. Genistin demonstrated relatively stronger estrogenic activity at 200μM and higher concentrations; apigenin showed weak estrogenic activity at 400μM, while luteolin, isorhamnetin, vitexin, isovitexin and orientin were inactive at 400μM.The pER8-GFP transgenic Abrbidopsis is suitable for screening constituents with estrogenic activity from pigeon pea and other plants.7. The effect of cajanol on PC-3 prostate cancer cells was studied, the results were as follows:(1) Cajanol inhibited the growth of PC-3 prostate cancer cells at concentration and time-dependent manners. The survival rate of cells decreased and the inhibition effect was enhanced with the increases of treatement time and cajanol concentration. When treating PC-3 cells for 24,48 and 72 h, the IC50 were 23.9,16.2 and 12.2μg/mL, respectively;(2) Cajanol induced cell cycle arrest at G2/M phase after treatment of PC-3 prostate cancer cells at the concentrations of 5-15μg/mL. The proliferation of cancer cells was inhibited;(3) Cajanol inhibited the expression of ERa and ERβ, after the treatment of PC-3 prostate cancer cells at 15μg/mL for 48 h, the relative expression levels of ERa and ERβwere 39.73±2.36% and 73.12±2.33%, respectively. The down-regulation of ER expression decreased the proliferation of cancer cells enhanced by estrogens.
|
PDF Full Text Request