In Vitro Metabolism Of Thymol And Carvacrol

Thymol and carvacrol are isomers in structure, which are predominant monoterpenic phenol widely distributed in thyme and oregano. Thymol and carvacrol are the major component of thyme and oregano essential oils, which have many diverse pharmacology activities such as antioxidant, anti inflammatory, antibacterial and anti tumor. In addition, the two compounds are also widely used as a food additive in the food industry. The study of absorption, distribution, metabolism and excretion (ADME) properties in the human body and animals tissues of the main active ingredients of Chinese medicine, which carry out in vitro technology system, not only helps to reveal the material basis of the activity of traditional Chinese medicine and its disposal of human and animal body process, then select the appropriate animal model to carry out the overall pharmacokinetics, pharmacology and toxicology; also have a significant role to explain the mechanism of traditional Chinese medicine in the human body and Chinese herbal medicine drug interactions. Thymol and carvacrol are the main active ingredients of Chinese herbal medicine homology as many different variety of food and medicine; and which the contribution of its metabolic transformation in the human body and its effects on the metabolic enyme activity is essential for the clinical safety of drugs. However, so far, metabolic pathway of the two monoterpene in vivo is not clear, the metabolic stability and metabolic behavior, and whether the cause of drug drug interactions (DDI) and other scientific issues has not been well study; and these properties for the safety and effectiveness of the Chinese medicine itself and co administer drug will have a significant impact.Phenolics compounds is widespread in Chinese herbal medicine and structurally diverse, and most have antioxidant, anti inflammatory, antibacterial activity; which not only furnish the extensive resources for the new drug development, and its metabolites further enrich the structure of various compounds. The study is to investigate the metabolic stability in vitro, enzyme pharmacokinetics, species differences, drug drug interaction and structure pharmacokinetics of thymol and carvacrol, which use human liver microsomes, animal liver microsomes and other in vitro incubation systems The different methods of high performance liquid detection for thymol and carvacrol were established at first. The method has good specificity, which the precision & accuracy and stability of it are in line with methodology requirements; and the method can meet the quantitative requirements for the follow up study. On this basis, the paper focuses on in vitro phase I andâ…¡metabolism of thymol and carvacrol, and the metabolic pathways of the two compounds have been elucidation.In vitro phase I of thymol and carvacrol were investigated in this study, and the phase I pathway of the two compounds in HLMs were illustrated. There were one hydroxylated metabolite (M 1) found in the metabolic reaction of thymol and two hydroxylated metabolites (M 1, M 2) generated in the metabolic reaction of carvacrol. Throughout the investigation of recombine CYP and inhibitory effects, we found CYP1A2, CYP2A6 and CYP2B6 were involved in the metabolism of thymol in the hydroxylation reaction; CYP1A2, CYP2A6 involved in the formation of M 1, and CYP1A2, CYP2A6 and CYP2B6 were involved in the formation of M 2. The diversity of the ability of metabolism was shown in different species, so we can select animal model according as the object of the study. For the toxicity study of former drug, the animal which have the comparatively slow clear rate was the better choice; but for the metabolite, the quicker the better. The diversity of the CYP in the different species prompt us the study of pharmacodynamics and toxicity of metabolite were very essentialMeanwhile, the investigation of the in vitro phaseâ…¡of thymol and carvarol were carried out, the phaseâ…¡pathyway of it in HLMs and HIMs were clarified. There were one glucuronidation metabolite found in the HLMs and HIMs. The study also validated there were more enzymes involoved the reaction. Chmical inhibition studies and related screening of recombinant human enzyme were experimented, and found UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT2B7 involoved in the thymol glucuronidation metabolic response, UGT1A3, UGT1A6, UGT1A7, UGT1A9 and UGT2B7 in carvacrol. Furthermore, we indentified UGT1A9 as the major isozyme responsible for carvacrol glucuronidation in HLMs; and the UGT1A7 as the major isozyme responsible in HIMs. In dispite of there is not much different in zymogram between the different spices, the ability of metabolism have shown the huge diversity. So, the selection of the animal model should be in line with the orders.The kinetic paraments showed that carvacrol have own the relatively high metabolic activities and clearance rate than thymol in terms of phase I or II metabolic reactions, especially in the glucuronidation of carvacrol. Meanwhile, the higher clearance rate of thymol hydroxylation in the phase I and II shown that the CYP might be the main metabolic pathway of thymol in human; and the result of the carvacrol in the phase I and II indicate the CYP and UGT metabolism might have the identical contribution in human body. The results shown that the interspace between the hydrophobic group and hydroxyl group have the significant effects in determining the pathyway and speed of metabolism.The study also extremely investigated the inhibitory effects of carvacrol and thymol on major CYP and UGT isoformes, and the carvacrol have the ability of inhibition on UGT1A9 isoformes. The probe substrate for all tested UGT isoforms is 4 MU which is a nonselective substrate of UGTs, and the Propofol, mainly metabolized by UGT1A9, could be used as a probe substrate for UGT1A9. The results showed that carvacrol could inhibit the activity of UGT1A9 with negligible effects on other UGT isoforms. The carvacrol competitively inhibit UGT1A9 mediated 4 MU glucuronidation reaction (Ki=5.7 M), but the noncompetitively inhibiton found in carvacrol towards UGT1A9 mediated propofol glucuronidation(Ki=25.0 M). To date, mechanism of substrate dependent inhibition of drug metabolizing enzymes was still unclear. Therefore, in vitro data on the inhibition of UGT1A9 by carvacrol should be interpreted with caution.We found the compounds that the hydrophobic group far from the phenolic hydroxyl group have the tendency of occurrence of phase II, through compare the structure between the two compounds and analysis the literature at home and abroad. Meanwhile, there were some different on the metabolic enzyme and metabolic capability, which may be due to the stereospecific blockade effects of hydrophobic group. These findings suggest that small changes in the structure of small molecule compounds, may lead to a huge difference in the metabolic pathways. It should be selected with the people with similar metabolic behavior and metabolic capability to carry out the whole animal experiments, if the inappropriate animal models were be use, the pre clinical study may generate vary significantly, thus affecting the design of clinical trials and data reliability.In this study, the in vitro metabolism of thymol and carvacrol were carried out, and the metabolism pathway of the two compounds were first clarified, and the consequence is very useful to asses the'medicine'for the candidate compounds in the early development. However, the result enriches the pharmacokinetic study of monoterpenic phenol, and provide a theoretical basis and experimental support for such compounds in the follow up study.