| [Objective] Gingkgo flavones possess anticarcinogenic activity, antioxidation potential and prevention on chronic diseases, such as hypertension, diabetes, adiposity, cardiopathy and malignancy. They have been widely used in fields of dietary supplements, drugs and consumer products. Dietary supplements are being increasingly consumed world-wide as part of complementary and alternative medicine or self-medication in western world. This has substantially increased human (especially the old people and patients) exposure to phytochemicals, because they often take other clinical medicines at the same time. As a consequence, variability in the pharmacokinetics of drugs may appear, as well as drug-drug interactions, or even therapeutic failure.P450 (CYPs) form a superfamily of mixed-function monooxygenases involved in the biotransformation of a variety of chemically diverse substances including endogenous compounds and xenobiotics like steroid, food compounds, pollutants and drugs. The CYP3A family of enzymes constitutes the most predominant phase-I drug metabolizing enzymes and accounts for approximately 30% of hepatic CYPs and more than 70% of intestinal CYPS activity, present primarily in the hepatocytes. Moreover, CYP3A is estimated to metabolize between 50% and 70% of currently administered drugs. Inhibition of CYP-mediated metabolism often causes drug-druginteractions and limits the use of a drug because of adverse clinical effects.The present study was undertaken to investigate the effects of kaempferol and quercetin on the activity of cytochrome 450. Both ENRD and ADM were selected in this study because they are highly affected by xenobiotics, and together they are involved in the metabolism of approximately 70% of all marketed drugs. The alteration of CYP3A4 activity can influence the metabolism of substrates. Some compounds have been shown to induce or inhibit CYP3A4 and interactions occurred, such as drug-drug and food-drug interactions.The study will help us to illustrate the possible mechanisms of ginkgo flavones in body, predict interactions between the food and other compounds (drug), prevent adverse reactions and offer the instruction for all the people, especially the physician, patients and nutritionists.[Method] Hepatocytes were isolated using collagenase digestion procedure. After isolation, viability of the hepatocyte suspensions was measured using Trypan blue exclusion test;only hepatocytes with >80% viability were used and purified by a Percoll gradient. Isolated hepatocytes were transferred to collagen precoated 24-well plate. Primary cultured hepatocytes were exposed to kaempferol or quercetin in 0.1 to 10 umol/L for 12h, 24h, and 48h.The protein concentration was measured by Lowery method. Hepatocytes CYP isoemzymes-erythromycin N-demethylase (ERND) and aminopyrine N-demethylase (ADM) activities were determined by Nash methods.The OD values were read from multi-count machine. Set vehicle controls (hepatocytes dosed with 0.1%DMSO without any test article) and positive controls (hepatocytes dosed with incubation medium containing 10 nmol/L erythromycin). Rat hepatocytes CYP450 isoform was reported as specific activity (umol/mg /minxprotein) .Human hepatocytes CYP3A4 activty results were expressed as fold compare to the 0.1%DMSO. All the datas were expressed as meandbS.D.Statisticals evaluation of thedata was analyzed by Dunnet-t test and one-way analysis of varians. When P<0.05,there was statistical markedness difference .[Result] In the primary cultured rat hepatocytes assay, kaempferol and quercetin inhibited ENRD activity in dose- and time-dependent manner. In dose-response study, the ENRD activities in kaempferol (0.1, 1 and 10 umol/L) treated groups were (0.088±0.008), (0.074±0.006), and (0.041±0.006) umol/mg/min, respectively. ENRD activity in quercetin treated groups at the same concentrations were (0.082±0.007), (0.063±0.007), and (0.034±0.005) umol/mg/min, respectively. In time-courses study, the ENRD activity exposed to 10 umol/1 kaempferol or quercetin for 12h and 48h were (0.053±0.006) and (0.037±0.007) umol/mg/min, or (0.067±0.005) and (0.032±0.004) umol/mg/min. ADM activity was inhibited only by kaempferol in lOumol/L at 24h, but was not significantly altered by quercetin at any concentration tested.Primary cultured human hepatocytes were treated with 0.1~10umol/L kaempferol and exposed for 12 n 24 n 48h,no evident cell toxicity was observed. Comparing with rat, kaempferol inhibitional rate was larger in human hepatocytes and kaempferol inhibited ENRD activity also in dose- and time-dependent manner. After lOumol/L kaempferol treated for 12h ,the ENRD activity inhibition effects show (PO.05) , and the inhibition rate was 64.2%.After 1 -. lOumol/L kaempferol treated 24h,the inhibition rate was 39.7% and 68.4% respectively. After 0.K 1 and lOumol/L kaempferol treated 48h,the ENRD activity was lower than solvent control(P<0.01),the inhibition rate was 40.4%,66.1% and 90.8% respectively.The kaempferol inhibitional rate was larger than positive control erythromycin.[Discussion] Kaempferol and quercetin is the major effective components of ginkgo flavones. The old people often take some dietary supplement of ginkgo blob leaf extract (GBE) with other drugs, therefore the food-drug interactions may happen. Sowe need to know the effects of kaempferol and quercetin on major drag- metabolizing enzymes.The measure of ENRD and ADM reflected the activity of CYP3 A4 and CYP2A^ 2B> 2C subfamily(such as 2A2> 2BK 2B2s 2C11) respectively. In our laboratory, we found kaempferol and quercetin can induce CYP3A4 transcription in HepG2 cells through activing nuclear receptor PXR, which illuminate they have induction effects on CYP3A4. We also found the CW, AUC0-8 and the mean retention time (MRTo-g)of NFP were significantly increased by simultaneous oral treatment with kaempferol, which suggests that kaempferol might reduce the first-pass metabolism of NFP.Our investigation in primary cultured rat hepatocytes shows that the two gingkgo flavones could inhibite CYP3A4 activity. To accurate predication of human from animal data, human hepatocytes were used .We found the inhibitional effect of kaempferol in human was accordant with in rat .We can presume that the action of kaempferol and quercetin on CYP3A4 may occur after protein expression by non-competitive inhibition or certain allosteric effect. Whereas, appropriate endpoints for assessing induction or inhibition potential of a agent include catalytic activity, gene expression at the level of transcriptional activity and the protein espression, so further study should be done on the two latter, we can guess that other similar structured gingkgo flavones compounds have the same actions on CYPs.For example, quercetin may has inhibition effects on ENRD in human hepatocytes.Repeated and abundant taking gingko flavones may change some important drug-metabolism enzymes,such as CYP3A4> CYP2A2> CYP2B1 ^ CYP2C and so on,and induce food-drug or drug-drug interactions,even adverse effects.The CYP3A4 activity in human hepatocytes (nmol/mg/min) was much lower than rat (umol/mg/min) ,compare with the rat, human may be more sensitive to enzymes inhibitors.In the test of inhibiting ENRD,the kaempferol inhibitional ability was better than erythromycin.In human hepatocytes,kaempferol showed more obviousinhibitioal rate and time-dose effect ,the highest inhibitional rate was 90.8% in human after 48h treatment and only 51.8% in rat.The results suggest kaempferol has better inhibition ability in human than rat. When people took ginkgo flavones CAM and clinical drugs at the same time,the blood-drug concentration may get virtual value ,which could change metabolize enzymes activity, the side-effect may happen. [Conclusion] In the present condition, kaempferol and quercetin acts as a potential CYP3A4 inhibitor as they can significantly inhibit ENRD in primary cultured rat hepatocytes but not for ADM. Kaempeferol has obvious inhibiting effect on CYP3A4 in human hepatocytes. Our investigation shows that the interactions between ginkgo flavones dietary supplements and drugs may happen. |
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