The Metabolic Mechanism Of Flavonoids And Aconitine

It is well known that natural products are the main sources of chemo-preventive and chemo-therapeutic agents, since the chemical drug development is much more difficult than before. The use of flavonoids and aconite alkaloids, two kinds of efficient natural products, was limited because of low bioavailability or toxicity. Flavonoids are widely distributed in plants and Chinese herbs. Aconitine, which is used to cause side effects and clinical adverse reactions, is the typical ingredient of aconite with anti-inflammatory and analgesic effect, as well as toxicity. In order to develop and enlarge the usage of flavonoids and aconite alkaloids, it is necessary to do research on the absorption, distribution, metabolism and excretion (ADME) in vivo and in vitro for a better understand of the metabolic characterizations of flavonoids and aconitine. The aim of this thesis is to elucidate the metabolic mechanism of flavonoids and aconitine, which is helpful to the evaluations of drug therapy, drug toxicity and clinical drug safety.PartⅠThe UGT-mediated Metabolism of FlavonoidsFlavonoids are widely distributed in a variety of natural plants and edible foods including fruits and vegetables. Scientific researches have demonstrated their obvious biological activities. Despite of these claimed health benefits and demonstrated preclinical activities, there are significant challenges associated with development of isoflavones and flavones into chemo-preventive and chemo-therapeutic agents. The major challenge currently is their low bioavailabilities (<10%), as the result of extensive first-pass metabolism by phaseⅡenzymes including UGTs and SULTs. So we chose two main kinds of flavonoids:isoflavones and flavones to better understand the metabolic mechanism of flavonoids.The present studies represent a detailed and systematic study of UGT isoform-specific metabolism of 6 isoflavones and 10 flavones. We have shown for the first time that the glucuronidation of flavonoids were all UGT isoform-, substrate chemical structure-and concentration-dependent, which can be captured by metabolic "fingerprint" using expressed human UGT isoforms. We demonstrated for the first time the multifaceted utilities of UGT metabolic "fingerprint" in defining for drug interactions, genetic polymorphism, major organs for metabolism, and SMR. We believe that the approach developed here may be of general utilities in defining the metabolic "fingerprint" of other UGT substrates, which in turn could improve the safety as well as efficacy of drugs that are inactivated or eliminated by the glucuronidation pathway.1. The UGT-mediated Metabolism of Isoflavones and the Characterization of UGT Metabolic Fingerprint of IsoflavonesWe characterized the isofonm-specific glucuronidation of six isoflavones: genistein, daidzein, glycitein, formononetin, biochanin A and prunetin using 12 expressed human UGTs and human intestinal and liver microsomes. The results indicated that these isoflavones were metabolized most rapidly by one of these four UGT isoforms:UGT1A1, UGT1A8, UGT1A9 and UGT1A10. Furthermore, glycitein was usually metabolized the fastest whereas prunetin the slowest. Because prunetin was the only compound not metabolized by UGT1A1, the hydroxyl group at position 7 in the pyrane ring of isoflavones is a structural feature important for active site recognition. Using the rates of metabolism by 12 UGT isoforms as a means to establish the metabolic "fingerprint", we found that each isoflavone had distinctive concentration-dependent patterns. Determination of kinetic parameters of glucuronidation using genistein and prunetin indicated that the distinct concentration-dependent metabolic patterns were the result of differences in Km and Vmax values. We then measured how well metabolic "fingerprinting" predicted metabolism of these isoflavones by human intestinal and liver microsomes. We found that the prediction was rather successful for five isoflavones in the liver microsomes, but not successful in the intestinal microsomes. We propose that a newly discovered UGT3A1 isoform capable of metabolizing phenols and estrogens may be responsible for the metabolism of isoflavones such as formononetin in humans. In conclusion, the first systematic study of metabolic "fingerprinting" of six common isoflavones showed that each isoflavone has UGT isoform-specific metabolic patterns that are concentration-dependent and predictive of metabolism of the isoflavones in liver microsomes.2. The UGT-mediated Metabolism of Flavones and the Characterization of UGT Metabolic Fingerprint of FlavonesThe present study aims to predict the region-specific glucuronidation of three dihydroxyflavones and seven mono-hydroxyflavones in human liver and intestinal microsomes using recombinant UGT isoforms. Seven mono-hydroxyflavones (HFs), 2'-,3'-,4'-,3-,5-,6-, and 7-hydroxyflavone, and three di-hydroxyflavones (diHFs), 3,7-,3,5-and 3,4'-dihydroxyflavone were chosen. The results indicated that the position of glucuronidation of three diHFs could be determined by using the UV spectra of relevant HFs. The results also indicated that UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 were the most important six UGT isoforms for metabolizing the chosen flavones. Regardless of isoforms used,3-HF was always metabolized the fastest whereas 5-HF was usually metabolized the slowest, probably due to the formation of an intra-molecular hydrogen bond between 4-carbonyl and 5-OH group. Relevant UGT isoform-specific metabolism rates were found to correlate well with the rates of glucuronidation in human intestinal and liver microsomes, except for 3'-HF. In conclusion, the glucuronidation "fingerprint" of seven selected mono-hydroxyflavones was affected by UGT isoforms used, positions of the-OH group, and the substrate concentrations, and the rates of glucuronidation by important recombinant UGTs correlated well with those obtained using human liver and intestinal microsomes.PartⅡAconitine Toxicokinetics and Its CYP-mediated Metabolism,and the Effect of Flavonoids on the Metabolism of AconitineAconitine, a famous aconite alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. Our previous research has found that the security window of aconitine was 0.06879-0.08708 mg/kg (ED95-LD50). And it was suggested that the high toxicity and narrow security window were correlated with the metabolism by enzyme in vivo. In order to understand the molecular mechanism on the toxicity, we paid attention to the toxicokinetics of aconitine and how the phase I reaction affect the toxicokinetics, which could give more evidence to the evaluation of clinical safety of aconitine. In the end, we chose two isoflavones, genistein and daidzein, to investigate if flavonoids could affect the metabolism of aconitine.1. Toxicokinetics of Aconitine in RatIn order to investigate the pharmacokinetic behaviors of aconitine in rats, a sensitive and fast UPLC-MS/MS method was successfully developed. Aconitine was absorbed fleetly after oral administration at 0.504 mg/kg. The concentration reached the highest level in rat plasma (Cmax 7.43±3.84 ng/ml) around 0.5 hour. We presumed it related to the effect of metabolic enzyme in vivo.2. The Characterization of Metabolites and CYP-mediated Metabolic Mechanism of AconitineAn UPLC/MS/MS and a high resolution mass spectrogram (HRMS) method were developed to determine the characteristics of metabolism of aconitine. We found 11 metabolites of aconitine in phase I reaction system in human, rat, mice and dog liver microsomes. In order to distinguish the specific-CYP isoforms responsible for metabolising aconitine, six selective CYP-inhibitors were used in phase I reaction system. The results indicated that CYP2D6 could not metabolize aconitine. CYP3A4 produced 16-O-demethyl-aconitine. CYP1A2 and CYP2C could metabolize aconitine moderately. 3. The Effect of Flavonoids on the Metabolism of AconitineWe determined the effect of two famous flavonoids, genistein and daidzein, on the metabolism of aconitine in CYP reaction system. The results indicated that genistein could affect the metabolism of aconitine distinctly in vitro while daidzein did not.In summary, we established the UGT fingerprints of sixteen flavonoids and elucidated the metabolic mechanism by UGT for the first time. Also we demonstrated the metabolic pathways of aconitine in human, rat, mice and dog liver microsomes are similar and elucidated the CYP-mediated metabolic mechanism. In the end, we demonstrated the obvious effect of flavonoids on the metabolism of aconitine.