| Buckwheat, which belongs to the family Polygonaceae, genus Fagopyrum, is an edible and medicinal crop acknowledged by the Food and Agriculture Organization of the United Nations. As the original country and major producer of buckwheat, China owns quite abundant germplasm resources, long cultivation history and extensive distribution of buckwheat. It is suggested that buckwheat has both nutritional and medicinal benefits at very high levels, which had been recorded in many Chinese ancient books. Modern medicine also demonstrates buckwheat and its extracts possess a variety of pharmacological activities including antioxidation, anti-inflammation, anticancer, antihyperglycemia, antihyperlipemia, antihypertension and so on, thereby buckwheat consumption is capable of decreasing morbidities or ameliorating symptoms of: breast cancer, colorectal cancer, diabetes mellitus and cardiovascular diseases effectively. Nowadays, because of the prevalence of dietary therapy, buckwheat has attracted increasing attention from people as an important raw material for formulating various flavor food and a series of functional food.Xenobiotics can be absorbed into human body via multiple pathways and then react directly with nucleophilic centers of macromolecules such as proteins and DNA to initiate a host of diseases(even cause cancer to start) that threaten human health severely. The human body has developed a complex metabolic system in the long process of evolution to eliminate these toxic substances, which mainly involves two families of enzymes that are Phase I enzymes and Phase II enzymes. While Phase I enzymes are reported to be able to activate certain unreactive procarcinogens and promutagens, Phase II enzymes, such as QR, GSTs and UGTs, are typically responsible for facile excretion of numerous electrophiles(e.g., carcinogens and mutagens) and reactive oxygen species by Phase II conjugation reactions exerting highly detoxifying and antioxidative functions to maintain the stability of the human body’s internal environment. Therefore, it is suggested that the elevation of Phase II enzymes is a primary protective mechanism for human body against toxic xenobiotics and a significant strategy for achieving cancer chemoprotection as well.However, surprisingly little is known about the chemopreventive activity of buckwheat and its related chemical determinants, particularly, there are no literatures reporting the chemopreventive effects of buckwheat and buckwheat compounds by means of inducing the Phase II enzyme activity. Thus, in the present study we used the murine hepatoma Hepa 1c1c7 cell model and the typical Phase II enzyme- QR as a biomarker to guide the isolation and identification of potentially QR-inducing agents from different polar extracts of buckwheat flour, and those isolated bioactive compounds were tested alone and in binary combination in cultured cells to assess features of their action. Consequently, the objective of this study was to isolate, identify and evaluate potential QR-inducing agents from buckwheat systematically.The principal contents and findings of the present study are summarized as follows:1. Isolation and identification of QR-inducing agents from nonpolar extracts of buckwheat. Buckwheat flour was successively extracted with n-hexane and ethyl acetate to obtain the hexane extract and the ethyl acetate extract that made up the nonpolar buckwheat extracts. These crude extracts were then partitioned between n-hexane and 80% aqueous methanol, by using the QR induction bioassay-guided strategy, the active 80% methanol-soluble fractions were subjected to subsequent fractionation with normal phase silica gel column chromatography, TLC and preparative thin-layer chromatography. As a result, three pure compounds were isolated. After the spectroscopic analyses(e.g., NMR and HR-ESI-MS) and comparison with previous literature reports, these isolated compounds were identified as ferulic acid ethyl ester(FAEE, 1), furaneol(2) and protocatechuic acid(3).2. Isolation and identification of QR-inducing agents from methanolic extract of buckwheat. Buckwheat flour methanolic extract was sequentially partitioned with ethyl acetate, n-butanol and water. Guided by the QR induction bioassay in vitro, the active ethyl acetate-soluble fraction was then subjected to fractionation with normal and C18-reversed phase silica gel column chromatography, preparative thin-layer chromatography and Sephadex LH-20 chromatography to yield four pure compounds consequently. After the spectroscopic analyses(e.g., NMR and HR-ESI-MS) and comparison with previous literature reports, these isolated compounds were identified as N-trans-feruloyltyramine(4), syringic acid(5), quercetin(6) and myricetin(7).3. Investigation of the QR induction potency of each isolated buckwheat compound by the murine Hepa 1c1c7 cells. It was indicated that all seven compounds exhibited QR-inducing capabilities and could induce QR activity in dose-dependent manners. Of these active buckwheat compounds, the QR induction potency decreased in the following order: FAEE(CD = 2.5 μM, IC50 = 80 μM) > quercetin(CD = 3.0 μM, IC50 = 32 μM) > N-trans-feruloyltyramine(CD = 23.8 μM, IC50 > 200 μM) > myricetin(CD = 57.5 μM, IC50 = 74 μM) > furaneol(CD = 206 μM, IC50 = 283 μM) > protocatechuic acid(CD = 2.0 m M, IC50 = 2.1 m M) > syringic acid(CD = 5.4 m M, IC50 = 9.3 m M), and moreover, in the tested dose range, their maximum induction ratios were 6.5, 4.2, 4.2, 3.5, 5.3, 2.9 and 3.9, respectively.4. Based upon evaluation of the QR-inducing activities of binary mixtures involving all seven compounds isolated from buckwheat, the combined interactions were investigated using the method of Chou-Talalay via calculating the interaction factor(IF) values. Twelve combinations were tested totally, and results showed that there were antagonism or additivity presenting between the combined components regarding QR induction. Three kinds of dose responses were summarized below:① The combined compounds showed an antagonistic interaction in the tested dose range with IF > 1. There were four of mixtures falling into this category, including FAEE : furaneol(1, at 1.00 : 5.85 mole ratio), FAEE : quercetin(4, at 1 : 1 mole ratio), FAEE : myricetin(5, at 1 : 2 mole ratio) and quercetin : myricetin(12, at 1 : 2 mole ratio).② The combined compounds showed a mixed effect in the tested dose range, i.e., antagonism was indicated at the lower doses with IF > 1 and additivity was indicated at the higher doses with IF = 1. There was only one mixture falling into this category, which was FAEE : N-trans-feruloyltyramine(3, at 1 : 2 mole ratio).③ The combined compounds showed a mixed effect in the tested dose range, i.e., additivity was indicated at the lower doses with IF = 1 and antagonism was indicated at the higher doses with IF > 1. There were seven of mixtures falling into this category, including FAEE : protocatechuic acid(2, at 1.00 : 48.66 mole ratio), furaneol : protocatechuic acid(6, at 1.00 : 8.31 mole ratio), N-trans-feruloyltyramine : syringic acid(7, at 1.00 : 25.23 mole ratio), N-trans-feruloyltyramine : quercetin(8, at 2 : 1 mole ratio), N-trans-feruloyltyramine : myricetin(9, at 1 : 1 mole ratio), quercetin : syringic acid(10, at 1.00 : 50.46 mole ratio) and myricetin : syringic acid(11, at 1.00 : 25.23 mole ratio).In the present study, by employing cultured murine hepatoma Hepa 1c1c7 cells, efforts were made for the first time to isolate and identify potential QR inducers from different polar extracts of buckwheat using a QR induction bioassay-directed strategy as well as evaluate the QR-inducing activities of isolated compounds alone or in combination. Of the seven active compounds isolated from buckwheat, ferulic acid ethyl ester is reported for the first time, and no previous report was found on QR-inducing activity of furaneol, in addition, the interactive relationships of these combined compounds in twelve combinations regarding QR induction are also reported for the first time. The findings here can broaden the cognition scope of bioactive agents in buckwheat, provide the evidence for its chemopreventive effect and theoretical basis for further comprehensive utilization and exploitation of this medicinal and edible plant resource. |
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