Abstract:
This invention is to provide multiple specific inhibitors of cytochrome P450 isozyme CYP2C9. These inhibitors can be derived from any combinations with the following compounds including: Tamarixetin, Formononetin, isoliquritigenin, Phloretin, luteolin, Quercitrin, quercetin, myricetin, Wongonin, Puerarin, Genistein, Nordihydroguaiaretic acid, Narigenin, Capillarisin, Chrysin, Fisefin, eriodictyol, 6-Gingerol, Isorhamneti, isoquercitrin, Morin, (+)-Taxifolin, isovitexin, 3-Phenylpropyl Acetate, Oleanolic acid, ursolic acid, β-Myrcene, cinnamic acid, Luteolin-7-Glucoside, Liquiritin, (+) Limonene, Homoorientin, Swertiamarin, Embelin, Daidzein, Poncirin, (−)-Epicatechin, ergosterol. These natural products can be used to enhance the bioavailability of therapeutic agents (drugs).

Description:
RELATED APPLICATIONS 
       [0001]    This application is a continuous application of U.S. patent application Ser. No. 10/948,206, filed on Sep. 24, 2004. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    This invention is to provide inhibitors of cytochrome P450, especially inhibitors that are specific for the isoform CYP2C9. 
         [0003]    Cytochrome P450 (P450) is the most important oxidative enzymes for the metabolism of drugs and xenobiotics. P450 is classified as families and subfamilies, and is widely distributed in the liver, intestines and other tissues (Krishna D. and Klotz U., Extrahepatic metabolism of drugs in humans. Clinical Pharmacokinetics. 26:144-160, 1994). Cytochrome P450 enzymes catalyze the phase 1 reaction of drug metabolism, to generate metabolites for excretion. The classification of CYP450 is based on homology of the amino acid sequence (Slaughter R. L. and Edward D. J., Recent advances: the cytochrome P450 enzymes. The Annals of Pharmacotherapy. 29:619-624, 1995). In mammals, there is over 55% homology of the amino acid sequence of CYP450 subfamilies. The differences in amino acid sequence constitute the basis for a classification of the superfamily of cytochrome P450 enzymes into families, subfamilies and isozymes. The isozymes with similar numerical numbers (for example CYP2C9 and CYP2C11, CYP1A1 and CYP1A2) usually have high amino acid homology, and their respective genes usually locate in proximate positions on the chromosome map. For instance, CYP2C9 and CYP2C10 have only two amino acid differences; the amino acid sequence homology of CYP3A3 and CYP3A4 is 97.5%. Therefore, the nomenclature of cytochrome P450 is across all living systems and species, including animals, plants and microorganisms. Cytochrome contains an iron cation and is a membrane bound enzyme. The hemoprotein structure (heme-group, prosthetic group) and function of P450 are very similar to those of hemoglobin, it can carry out electron transfer and energy transfer. Cytochrome P450, when binds to carbon monoxide (CO), displays a maximum absorbance (peak) at 450 nm in the visible spectra, and is therefore called P450 (Omura T. and Sato R., The carbon monoxide-binding pigment of liver microsomes. The Journal of Biological Chemistry. 239:2370-2378, 1964). 
       CYP450 Tissue Distribution: 
       [0004]    Regarding tissue distribution of CYP450, there is a great similarity between rats and humans. Human CYP450 isozymes are widely distributed among tissues and organs (Zhang Q. Y., Dunbar D., Ostrowska A., Zeisloft S., Yang J., and Kaminsky L. S., Characterization of human small intestinal cytochromes P-450. Drug Metabolism and Disposition. 27:804-809, 1999). With the exception of CYP1A1, most human CYP450 isozymes are located in the liver, but are expressed at different levels (Waziers I., Cugnenc P. H., Yang C. S., Leroux J. P. and Beaune P. H., Cytochrome P450 isoenzymes, expoxide hydrolase and glutathione transferases in rat and human hepatic and extrahepatic tissues. The Journal of Pharmacology and Experimental Therapeutics. 253:387-394, 1990). For example, CYP2C family constitutes about 18.2% of the total P450 in the liver. Human intestine also has high CYP3A4 contents, approximately 50% of that in the liver. The distribution in rats is similar to humans. With the exception of CYP2B1 and CYP1A1, the majority of the known rat CYP450 isozymes are primary located in the liver. From literatures, it&#39;s also known there are species differences in the tissue distribution and expression of CYP450 enzymes between rats and humans. However, from the enzymatic and functional perspectives, the rat P450 enzymes are considered representative of the human enzymes. Consequently, Sprague-Dawley rat liver microsomes are used as an enzyme source for investigating CYP2C. 
         [0005]    Fifty-seven CYP450 isozymes have been identified from the human CYP genomics, and they have been classified into fourteen P450 subfamilies—CYP 1, 2, 3, 4, 5, 7, 8, 11, 17, 19, 21, 24, 27 and 51 (Nelson D. R., Koymans L. and Kamataki T., P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics. 6:1-42, 1996). CYP1, 2 and 3 are primary responsible for metabolism and detoxication of drugs and xenobiotics. The other 11 P450 subfamilies are responsible for the catabolism of endogenous compounds, such as hormones or steroids, etc. 
       Genetic Polymorphism 
       [0006]    Presently, four isoforms have been identified for human CYP2C subfamily. They are CYP2C8, CYP2C9, CYP2C18 and CYP2C19, and there are about 82% amino acid sequence homology among these four isoforms (Miners J. O. and Birkett D. J., Cytochrome P4502C9: an enzyme of major importance in human drug metabolism. British Journal of Clinical Pharmacology. 45:525-538, 1998). Despite the high homology, there are large differences in substrate specificity among these isoforms. It is also reported in 1980&#39;s that genetic polymorphism existed for CYP2C subfamily, as is observed for CYP2D6. Since then, many clinical studies have been performed to investigate the polymorphism of CYP2C. Results of these studies concluded that human populations can be categorized into two groups based on drug metabolism CYP450 activities: extensive metabolizers (EMs) and poor metabolizers (PMs). The ratios of this genetic polymorphism are different among different races. For example, approximately 2 to 4% of the Caucasians populations are PMs, while there are 20% in Asians. Consequently, drug-drug interactions mediated by substrate specific metabolic pathways can be a more significant issue in Asian population. 
       Drug Metabolism 
       [0007]    Following absorption and reaching systemic circulation, drug molecules undergo metabolism and elimination/excretion process. There are two major metabolic reactions—phase I reaction and phase II reactions, both leading to more hydrophilic metabolite(s). The formation of hydrophilic metabolites is to facilitate excretion from the body. Mixed function monooxygenase is the major enzyme responsible for phase I reaction. Cytochome P450 is a monooxygenase system, consisting of P450, P450 reductase, cytochrome b5. These proteins function together to catalyst the reduction/oxidation of drug molecules, the mechanism of these reactions is described in the sections follow. Phase II reactions are primary conjugation reactions, can be divided into six categories (Table 1). Glucronidation, sulfation and glutathione conjugation are the most commonly observed phase II reactions. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Drug Phase I and Phase II reactions (Shargel L., and 
               
               
                 Yu A. B. C., Hepatic elimination of drugs. Applied 
               
               
                 Biopharmaceutics and Pharmacokinetics. 4th ed., Appleton 
               
               
                 &amp; Lange, Stamford, pp. 353-398, 1999) 
               
             
          
           
               
                   
                   
                 Phase II reaction 
               
               
                   
                 Phase I reaction 
                 (High energy intermedate) 
               
               
                   
               
               
                   
                 Oxidation 
                 Glucuronide conjugation (UDPGA) 
               
               
                   
                 Aromatic hydroxylation 
                   
               
               
                   
                 Aliphatic hydroxylation 
                 Sulfate conjugation (PAPS) 
               
               
                   
                 N-, O-oxidation 
                   
               
               
                   
                 N-, O-dealkylation 
                 Glutathion conjugation (GSH) 
               
               
                   
                 Deamination 
                   
               
               
                   
                 Reduction 
                 Acetylation (Acetyl coenzyme A) 
               
               
                   
                 Azoreduction 
                   
               
               
                   
                 Nitroreduction 
                 Methylation (SAM) 
               
               
                   
                 Alcohol dehydrogenase 
                   
               
               
                   
                 Hydrolysis 
                   
               
               
                   
                 Ester hydrolysis 
                   
               
               
                   
                 Amide hydrolysis 
               
               
                   
               
               
                 UDPGA = uridine diphosphoglucuronic acid, 
               
               
                 PAPS = 3′-phosphoadenosine 5′-phosphosulfate, 
               
               
                 GSH = glutathione, 
               
               
                 SAM = S-adenoylmethionine 
               
             
          
         
       
     
         [0008]    The four CYP2C isozymes have different substrate specificity, however, metabolism of most drug molecules is carried out by CYP2C9 and CYP2C19. The relative activity of CYP2C9 and CYP2C19 in human liver is about 3:1 (Venkatakrishnan K., von Moltke L. L., Greenblatt D J., Relative quantities of catalytically active CYP 2C9 and 2C19 in human liver microsomes: application of the relative activity factor approach. Journal of Pharmaceutical Sciences. 87:845-53, 1998). One of a commonly used proton pump inhibitor, Omeprazole, is a specific substrate for CYP2C19. CYP2C9 exhibits broader substrate selectivity and metabolizes different classes of drug, including non-steroid anti-inflammatory drug (NSAID&#39;s), blood triglyceride lowering agents, anti-coagulants. Representative examples are listed in Table 2. It should be noted that phenytoin and warfarin (on the lists) are clinical agents with narrow therapeutic window. For these agents, changes in oral absorption due to individual variability or other environmental factors can lead to severe side effects and undesired treatment outcome. One of the causes in individual variability is genetic polymorphism. The pattern of genetic polymorphism is different among races. For example, CYP2D6 is an enzyme responsible for the metabolism of hydrophobic anti-depressants. About 19% of the Caucasian population is CYP2D6 poor metabolizer (PMs), in contracts, the CYP2D6 PMs among oriental populations is less than 1%. Therefore, when a standard therapeutic dose of an anti-depressant is given to a PM patient, severe side effects are often observed because of the reduced metabolism rate in a PM. These side effects compromise the quality of life and further reduce patient compliance, and even accelerate the disease progression. Similarly, when a narrow therapeutic window drug is given to a PM patient, severe adverse effects can result due to reduced metabolism rate. 
         [0009]    To address the issue of variability in drug bioavailability, one approach is to control drug absorption (for example, use of control released drug product). Another and a more direct approach is to control the rate of drug metabolism. When the rate of absorption and rate of metabolism reach a steady state, a maintenance dose can be deliver to achieve the desired drug level (systemic availability) that is required for drug efficacy. This approach will minimize the individual variability, avoid side effects. Furthermore, by searching/use of an effective P450 inhibitor, the drug metabolism rate can be regulated and drug first pass effects can be reduced. However, an effective P450 inhibitor has to process an acceptable safety profiles. For instance, natural products or Chinese herbal medicines can fulfill these safety requirements. One of most commonly observed examples for a natural product to alter (increase) the bioavailability of a drug is the effects of grape fruit juice on the pharmacokinetics of felodipine and other drug products (Edgar et al., Acute effects of drinking grapefruit juice on the pharmacokinetics and dynamics of felodipine—and its potential clinical relevance. European Journal of Clinical Pharmacology. 42:313-317, 1992; Lee et al., Grapefruit juice and its flavonoids inhibit 11 beta-hydroxysteroid dehydrogenase. Clinical Pharmacology and Therapeutics. 59:62-71, 1996; Kane et al., Drug-grapefruit juice interactions. Mayo Clinic Proceedings. 75(9):933-42, 2000). 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Substrates, Inhibitors and Inducers of CYP2C subfamilies (Rendic 
               
               
                 S., Summary of information on human CYP enzymes: human P450 
               
               
                 metabolism data. Drug Metabolism Reviews. 34: 83-449, 2002) 
               
             
          
           
               
                 Isoenzyme 
                 Substrate 
                 Inhibitor 
                 Inducer 
               
               
                   
               
               
                 CYP2C9 
                 Tolbutamide 
                 Fluconazole 
                 Rifampin 
               
               
                   
                 Diclofenac 
                 Ketoconazole 
                 Phenobarbital 
               
               
                   
                 Warfarin 
                 Metronidazole 
                 Cabamazepine 
               
               
                   
                 Phenytoin 
                 Itraconazole 
                 Ethanol 
               
               
                   
                 Torsemide 
                 Cimetidine 
                   
               
               
                   
                 Fluvastatin 
                 Sulphaphenazole 
                   
               
               
                   
                 Losartan 
                 Phenylbutazone 
                   
               
               
                   
                 Celecoxib 
                   
                   
               
               
                   
                 Meloxicam 
                   
                   
               
               
                   
                 Isoniazide 
                   
                   
               
               
                   
                 Valporic acid 
                   
                   
               
               
                   
                 Ibuprofen 
                   
                   
               
               
                   
                 Carvedilol 
                   
                   
               
               
                   
                 Naproxan 
                   
                   
               
               
                   
                 Ondansetron 
                   
                   
               
               
                 CYP2C19 
                 Omeprazole 
                 Fluoxetine 
                 Rifampin 
               
               
                   
                 Imipramine 
                 Sertraline 
                 Hexobarbital 
               
               
                   
                 Diazepam 
                 Ritonavir 
                   
               
               
                   
                 Mephenytoin 
                   
                   
               
               
                   
                 Clomipramine 
                   
                   
               
               
                   
                 Propanolol 
               
               
                   
               
             
          
         
       
     
       BRIEF SUMMARY OF THE INVENTION 
       [0010]    This invention employ rat liver microsomes as an in vitro model and tolbutamide (Orinase®, a agent) as a probe (marker) substrate (tolbutamide is 90% metabolized by CYP2C9) to measure the inhibition of CYP2C9. Test compounds are purified extracts from Chinese herbal medicines and natural products. The inhibitory effects towards the in vitro microsomal metabolism of tolbutamide are measured and CYP2C9 inhibitors are identified. These inhibitors can be used as in vivo CYP2C9 inhibitors leading to improve the bioavailability of other therapeutic agents. 
         [0011]    First, this invention provides effective CYP2C9 inhibitor(s). These specific CYP2C9 inhibitors are derived from any combinations with the following compounds: Tamarixetin, Formononetin, isoliquritigenin, Phloretin, luteolin, Quercitrin, quercetin, myricetin, Wongonin, Puerarin, Genistein, Nordihydroguaiaretic acid, Narigenin, Capillarisin, Chrysin, Fisefin, eriodictyol, 6-Gingerol, Isorhamnetin, isoquercitrin, Morin, (+)-Taxifolin, isovitexin, 3-Phenylpropyl Acetate, Oleanolic acid, ursolic acid, β-Myrcene, cinnamic acid, Luteolin-7-Glucoside, Liquiritin, (+) Limonene, Homoorientin, Swertiamarin, Embelin, Daidzein, Poncirin, (−)-Epicatechin, ergosterol. 
         [0012]    Secondly, this invention is to provide a pharmaceutical combination to improve the bioavailability of drug products extensively metabolized by CYP2C9. This pharmaceutical combination(s) contain the purified ingredient(s) from the essential and adjuvant components of Chinese medicines and pharmaceutically viable drug. The purified ingredient(s) from the essential and adjuvant components of Chinese medicines act as CYP2C9 inhibitor(s), and are derived from the combination of the following: Tamarixetin, Formononetin, isoliquritigenin, Phloretin, luteolin, Quercitrin, quercetin, myricetin, Wongonin, Puerarin, Genistein, Nordihydroguaiaretic acid, Narigenin, Capillarisin, Chrysin, Fisefin, eriodictyol, 6-Gingerol, Isorhamnetin, isoquercitrin, Morin, (+)-Taxifolin, isovitexin, 3-Phenylpropyl Acetate, Oleanolic acid, ursolic acid, β-Myrcene, cinnamic acid, Luteolin-7-Glucoside, Liquiritin, (+) Limonene, Homoorientin, Swertiamarin, Embelin, Daidzein, Poncirin, (−)-Epicatechin, ergosterol). The pharmaceutically viable drug is one selected from the group consisting of tolbutamide, diclofenac, warfarin, phenytoin, torsemide, fluvastatin, losartan, celecoxib, meloxicam, isoniazide, valproic acid, ibuprofen, carvedilol, naproxen, and ondansetron. 
         [0013]    The better inhibitor from the above lists is Tamarixetin. 
         [0014]    A pharmaceutical combination contains tolbutamide and when used as a combination drug therapy, the purified ingredient(s) from the essential and adjuvant components of Chinese medicines can increase the bioavailability of tolbutamide. 
         [0015]    A pharmaceutical combination contains fluvastatin and when used as a combination drug therapy, the purified ingredient(s) from the essential and adjuvant components of Chinese medicines can increase the bioavailability of fluvastatin. 
         [0016]    These and other objectives of the present invention will become obvious to those of ordinary skill in the art after reading the following detailed description of preferred embodiments. 
         [0017]    It is to be understood that both the foregoing general description and the following detailed description are exemplary, and are intended to provide further explanation of the invention as claimed. 
         [0018]    These features and advantages of the present invention will be fully understood and appreciated from the following detailed description of the accompanying Drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0019]      FIG. 1  is the in vitro effects of Ketoconazole on 4′-hydroxylation of tolbutamide in liver microsomes. 
           [0020]      FIG. 2  is the comparison of cytochrome P450 inhibitory activities of the top ten tested essential and adjuvant components of Chinese medicines at a testing concentration of 100 μM. 
           [0021]      FIG. 3  is the comparison of cytochrome P450 inhibitory activities of the top ten tested essential and adjuvant components of Chinese medicines at a testing concentration of 10 μM. 
           [0022]      FIG. 4  is the comparison of cytochrome P450 inhibitory activities of the top ten tested essential and adjuvant components of Chinese medicines at a testing concentration of μM. 
           [0023]      FIG. 5  is the in vitro effects of tamarixetin on 4′-hydroxylation of tolbutamide in liver microsomes. 
           [0024]      FIG. 6  is the blood concentration time profiles following oral administration of fluvastatin in Sprague-Dawley rats; n=5 for dosed group and n=7 for vehicle control group. 
           [0025]      FIG. 7  is the in vitro effects of isoliquritigenin on 4′-hydroxylation of tolbutamide in liver microsomes. 
           [0026]      FIG. 8  is the in vitro effects of Genistein on 4′-hydroxylation of tolbutamide in liver microsomes. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0027]    This invention focuses on the identification of CYP2C9 inhibitors. As reported in literature, inhibition patterns of tolbutamide metabolism in rat, rabbit, dog, micropig, monkey and man liver microsomes revealed a high degree of similarity between the dog and human sytems. However, from the enzyme kinetic aspects, the kinetic parameters (Vmax/Km) values for the rat and human systems are most comparable (Bogaards et al., Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man. Xenobiotica. 30:1131-1152, 2000). When comparing the in vivo metabolism across species, it is reported that the biotransformation pathways of tolbutamide are similar in rat, rabbits and humans, and there is a species differences between dog and man (Dogterom P. &amp; Rothuizen J., A species comparison of tolbutamide metabolism in precision-cut liver slices from rats and dogs. Drug Metabolism and Disposition. 21:705-709, 1993). Furthermore, the amino acid sequence of rat and human CYP2C9 and CYP2C11 reveals 73% homology (www.drnelson.utmem.edu/CytochromeP450/html), the biological functionality of these enzymes reveals 84% similarity (www.ncbi.nlm.nih.gov/BLAST/). On the basis of these findings, it is prudent to use rat as an in vivo and in vitro model to assess the inhibition potential of testing compounds against human liver CYP2C9. 
         [0028]    This invention utilize the purified components from Chinese medicines to perform both in vitro inhibition and in vivo animal studies, the aims are to investigate their potential effects on the pharmacokinetics of drugs extensively metabolized by CYP2C9 with low bioavailability, and to identify potential CYP2C9 inhibitors from the essential and adjuvant components of Chinese medicines. 
       Materials and Methods 
       [0029]    The essential and adjuvant components Chinese medicines employed in this invention are purified chemical components from commonly used Chinese medicines (Table 3). Their chemical structures can be classified into five (5) categories: flavones, flavanones, chalcones, isoflavones and coumarins. 
         [0030]    1. Preparation of Liver Microsomes 
         [0031]    This invention use rat as the experimental animal model, therefore, in vitro enzymes used for metabolism studies are also prepared from rat liver. 
         [0032]    After sacrifice, the liver is removed from the rats and placed in 1.15% potassium chloride at 4° C. The tissue is thoroughly rinsed with cold 1.15% potassium chloride solution to remove any residual blood, blot and weighed. The rinsed tissue is then homogenized in a high speed tissue homogenizer until a complete homogenate (no residual tissue chunks) is obtained (homogenizing tubes are pre-chilled on ice). 
         [0033]    The homogenate is transferred to centrifuge tubes and centrifuged at 12,500×g for 20 minutes to remove cellular debris, nuclei, mitochondria and lysosomes. The supernatant fractions are harvested and placed into ultracentrifuges tubes (5 to 6 mL per tube). The tubes are then centrifuged in an ultracentrifuge at 100,000×g for 2 hours. The resulting supernatant (cytosol fractions) is discarded and the residual supernatant inside the centrifuge tubes are rinsed and removed cold 1.15% potassium chloride. The pellets (microsomes) are then harvested and resuspended in 0.1 M pH 7.4 phosphate buffer (one mL/g liver tissue). 
         [0034]    The final liver microsomal preparation had a protein concentration of approximately 25 mg/mL, and is stored in a −80° C. freezer. Under this storage conditions, the enzymatic activities is unchanged for at least 8 weeks, and is suitable for drug metabolism studies. To avoid any experimental artifacts, the liver microsomes preparation should be used within the recommended storage stability timeframe. The microsomes preparation is summarized in the following steps:
       (1) Animal sacrifice   (2) Removal of liver tissue   (3) Rinse liver tissue and record weigh of the tissue   (4) Cut the tissue into small pieces and mix with 1.15% KCL (1 mL/g tissue)   (5) Completely homogenize the tissue   (6) Place in high speed centrifuge tubes (12 to 15 mL per tube)   (7) Centrifuge at −4° C., 12,500×g, 20 minutes   (8) Place the supernatant into ultracentrifuge tubes   (9) Ultracentrifuge at −4° C., 100,000×g, 2 hours   (10) Discard supernatant, rinse the inside of centrifuge tubes with 1.15% KCL   (11) Remove the pellets from the centrifuge tubes   (12) Add pH 7.4 phosphate buffer, one mL per g of original tissue   (13) After respansion in phosphate buffer, dispense into micocentrifuge tubes (1 mL/tube)   (14) Store frozen at approximately −80° C. (−80° C. freezer)       
 
         [0049]    2. In Vitro CYP2C9 Activity Assay for Screening of the Essential and Adjuvant Components of Chinese Medicines 
         [0050]    After preparation and determination of microsomal protein concentrations, CYP2C9 activity assay are performed using the microsomes preparation, as a screen CYP2C9 inhibitors. Prior to screening, in vitro assay conditions are established based on enzyme kinetic principals and relevant kinetic parameters. 
         [0051]    Tolbutamide is a specific substrate for human CYP2C9. CYP2C9 catalyzes the conversion of tolbutamide to a hydrophilic metabolite, 4′-hydroxytolbutamide. This metabolic reaction has been shown to be CYP2C9 specific and does not involved other P450 isozymes. Thereby, it is considered as a reliable measurement for CYP2C9 activity. The initial substrate concentration used is 1 mM, under a enzyme saturating condition (Tang et al., Effect of albumin on phenytoin and tolbutamide metabolism in human liver microsomes: an impact more than protein binding. Drug Metabolism and Disposition. 30:648-654, 2002). 
         [0052]    The enzymatic assay conditions in microsomes are as following (total volume=1 mL):
       (1) 0.1M phosphate buffer, pH 7.4   (2) 0.5 mg microsomal protein   (3) 5 mM magnesium chloride   (4) 10 mM glucose 6-phosphate   (5) 2 IU G6P dehydrogenase   (6) 1 mM β-nicotinamide adenine dinucleotide phosphate   (7) 1 mM tolbutamide   (8) 1% methanol       
 
         [0061]    The activity assay mixture is placed on ice to maintain a 4° C. After the addition of the cofactor cocktails, it is pre-incubated in a 37° C. water bath for 1 minute. Reaction is initiated by the addition of the substrate and is terminated by 1N hydrochloric acid (0.1 mL). The metabolic reaction product is extracted using 2 mL methylene chloride. After separation by centrifugation, the organic fraction is concentrated to dryness, constituted in appropriate solvent and then analyzed for the metabolite (product) concentration. 
         [0062]    The assay conditions are established as such product formation is linear with respect to incubation time and protein concentrations. In additions, initial substrate concentrations are selected based on the values of kinetic parameters, Km and Vmax. 
         [0063]    The reaction product (metabolite) is analyzed using high performance liquid chromatotograhy (Shimadzu Model LC-10AD), UV detector (Shimadsu SPD-10A) at wavelength 230 nm (Miners et. al 1988). The LC conditions are, C-18 column (150×4.6 mm), mobile phase (10 mM acetate, pH 4.4/acetonitrile 25:75 v/v), flow rate 1.3 mL/min, ambient temperature. The retention time for the metabolite, internal standard and the substrate is 4.6, 14.2 and 26.5 minutes, respectively. 
         [0064]    Ketoconazole is used as the positive control and are tested under different concentrations to demonstrate concentration dependency (Results shown in  FIG. 1 ). At 100 μM concentrations, ketoconazole completely abolished the activity of the microsomes, exhibiting 100% inhibition. A 80.1 and 45.9% inhibition is observed at 10 and 1 μM, respectively. 
         [0065]    On the basis of inhibitory activity observed for the positive control, screening of inhibitors from essential and adjuvant components of Chinese medicines is carried out at high, mid and low concentrations. However, the aqueous solubility of the essential and adjuvant components of Chinese medicines is relatively poor, and organic co-solvents (such as methanol, ethanol, acetonitrile) are usually used under the assay conditions. Consequently, solvents effects (vehicle control) on the enzymatic activities are assessed to eliminate experimental artifacts due to organic co-solvents. 
         [0066]    3. In Vivo Study in Rodents 
         [0067]    Potential inhibitors identified from the in vitro screen (using rat liver microsomes as an enzyme source and triglyceride lowering drug tolbutamide as a probe substrate) are subject to further in vivo evaluation in small animals. The test system used is the Sprague-Dawley rat. However, since the oral bioavailability of tolbutamide in rats is 90%, therefore, it is not an appropriate model compound for in vivo assessment. Blood cholesterol lowering agent, fluvastatin is used as a model compound. Fluvastatin is a synthetic HMG-CoA reductase inhibitor, its oral bioavailability is about 25 to 30% and it is predominantly metabolized by CYP2C9. Absorption of fluvastatin sodium following oral administration is about 90%, therefore, the low bioavailability of 25 to 30% is due to high first pass effects. Fluvastatin is metabolized in liver, forming four major metabolites (Scripture et. al 2001). Liver CYP2C9 is responsible for approximately 80% of fluvastatin metabolism, and other isozymes are responsible for 20%. 
         [0068]    After overnight fast, rats are anesthetized and prepared with a jugular catheter. Dosing group received 9.32 mg/kg tamarixetin (dissolved in DMSO at 10 mg/mL), control group received only DMSO. After 30 minutes, both groups are administered fluvastatin at a dose of 1.5 mg/kg (dissolved in water at 2 mg/mL). Twelve blood samples (including pre dose blank) are collected over 24 hours—0, 10, 20, 40, 60, 120, 240, 360, 480, 720, 1080 and 1440 minutes. Each sample (0.5 to 0.6 mL blood) is collected into microfuge tubes containing 20 uL of 10 IU heparin (anti-coagulant). After separation, plasma samples are protected from light and stored at −80° C. freezer. 
         [0069]    Fluvastatin plasma concentration is determined using high performance liquid chromatography with fluorescence detector (excitation 309 nm, emission 390 nm). The LC conditions are, C-18 reverse phase column (5μ, 150×4.6 mm), mobile phase (0.1 M TBAF:0.1M phosphate, pH 6.0:Methanol (15:25:60 v/v/), flow rate 1.0 mL/min, column temperature (50° C.). Analytical procedure is as reported by Toreson et al., (Determination of fluvastatin enantiomers and the racemate in human blood plasma by liquid chromatography and fluorometric detection. Journal of Chromatography A. 729:13-18, 1996).
       (1) thaw samples on ice   (2) pippet 250 μL plasma sample into screw cap test tube   (3) add 50 μL of internal standard (celecoxib, 20 μg/mL in MeOH)   (4) add 250 μL of acetonitrile and vortex mixing for 5 seconds   (5) add 250 μl of 0.5 M phosphate buffer, pH 5.0   (6) add 2.5 mL MTBE (methyl tert-butyl ether), shake for 30 minutes   (7) transfer the organic levels into another test tube, evaporate under reduced pressure   (8) dissolve extracted residue in mobile phase   (9) transfer the extract and centrifuge at 13000 rpm for 5 minutes   (10) remove the clear supernatant (150 μL) and inject onto HPLC       
 
         [0080]    Experimental Results 
         [0081]    In vitro screening is conducted the essential and adjuvant components of Chinese medicines HUCHE001 to HUCHE070 depicted as Table 3. Inhibition of tolbutamide metabolism in liver microsomes are evaluated at three different concentration range, 1, 10 and 100 μM. For compounds with limited solubility, the highest testing concentration is the highest soluble concentration. The inhibition potential of test compounds is ranked within each testing concentration. The best inhibitors found are: isoliquritigenin 95.5% inhibition at 100 μM, Tamarixetin 88.2% at 10 μM, Genistein 49.6% at 1 μM. (Tables 4 to 6). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 introduction of the essential and adjuvant 
               
               
                 components of Chinese medicines 
               
             
          
           
               
                 Code 
                 Test article 
                 Source 
               
               
                   
               
               
                 HUCHE001 
                 Genkwanin 
                   Astemisiae  Capillaris 
               
               
                 HUCHE002 
                 apigenin 
                   Chamomiliae  Flos 
               
               
                 HUCHE003 
                 luteolin 
                 Digitals Folium 
               
               
                 HUCHE004 
                 Luteolin-7-Glucoside 
                 Digitals Folium 
               
               
                 HUCHE005 
                 Homoorientin 
                   Swertiae  Herba 
               
               
                 HUCHE006 
                 sovitexin 
                   Swertiae  Herba 
               
               
                 HUCHE007 
                 Neohesperidin 
                   Aurantii Fructus  Immaturus 
               
               
                 HUCHE008 
                 Formononetin 
                   Astragali  Radix 
               
               
                 HUCHE009 
                 isoliquritigenin 
                   Astragali  Radix 
               
               
                 HUCIIE010 
                 kaempferol 
                 Sennae Folium 
               
               
                 HUCHE011 
                 Isorhamnetin 
                 Sennae Folium 
               
               
                 HUCHE012 
                 isoquercitrin 
                 Hydrangeae Dulcis Folium 
               
               
                 HUCHE013 
                 (+)-epicatechin 
                 Gambir 
               
               
                 HUCHE014 
                 ergosterol 
                 Ergota 
               
               
                 HUCHE015 
                 (+)Catechin 
                   Paeoniae  Radix 
               
               
                 HUCHE016 
                 6- Gingerol 
                   Zingiberis  Rhizoma 
               
               
                 HUCHE017 
                 Liquiritin 
                   Glycyrrhizae  Radix 
               
               
                 HUCHE018 
                 3-Phenylpropyl Acetate 
                 Cinnamami Cortex 
               
               
                 HUCHE019 
                 (−)-Epicatechin 
                 Gambir 
               
               
                 HUCHE020 
                 Narigenin 
                   Aurantii Fructus  Immaturus 
               
               
                 HUCHE021 
                 Umbelliferone 
                   Aurantii Fructus  Immaturus 
               
               
                 HUCHE022 
                 Rutin 
                   Sophorae  Flos 
               
               
                 HUCHE023 
                 Hesperidin 
                   Aurantii Fructus  Immaturus 
               
               
                 HUCHE024 
                 Diosmin 
                 — 
               
               
                 HUCHE025 
                 Hesperetin 
                 Citri Reticulatae 
               
               
                 HUCHE026 
                 Wongonin 
                   Scutellariae  Radix 
               
               
                 HUCHE027 
                 baicalin 
                   Scutellariae  Radix 
               
               
                 HUCHE028 
                 Baicalein 
                   Scutellariae  Radix 
               
               
                 HUCHE029 
                 Puerarin 
                   Pueraria  Radix 
               
               
                 HUCHE030 
                 Daidzein 
                   Pueraria  Radix 
               
               
                 HUCHE031 
                 Daidzin 
                   Pueraria  Radix 
               
               
                 HUCHE032 
                 Quercitrin 
                 
                   Viscum Coloratum 
                 
               
               
                 HUCHE033 
                 quercetin 
                 
                   Viscum Coloratum 
                 
               
               
                 HUCHE034 
                 Nordihydroguaiaretic acid 
                 — 
               
               
                 HUCHE035 
                 Capillarisin 
                   Artemisia  Capillaris 
               
               
                 HUCHE036 
                 Swertiamarin 
                   Swertiae  Herba 
               
               
                 HUCHE037 
                 Genistein 
                   Puerariae  Radix 
               
               
                 HUCHE038 
                 trans-Cinnamaldehyde 
                 Cinnamami Cortex 
               
               
                 HUCHE039 
                 protocatechuic acid 
                 Cinnamami Cortex 
               
               
                 HUCHE040 
                 gallic acid 
                 — 
               
               
                 HUCHE041 
                 paeoniflorin 
                   Paeoniae  Radix 
               
               
                 HUCHE042 
                 eriodictyol 
                 
                   Pyracantha Fortuneana 
                 
               
               
                 HUCHE043 
                 Poncirin 
                   Aurantii Fructus  Immaturus 
               
               
                 HUCHE044 
                 α-Naphthoflavone 
                 Synthesis 
               
               
                 HUCHE045 
                 β-Myrcene 
                 
                   Amomum cardamomum 
                 
               
               
                 HUCHE046 
                 α-terpineol 
                 Cinae Flos 
               
               
                 HUCHE047 
                 +) -Limonene 
                 Cardamomi Fructus 
               
               
                 HUCHE048 
                 Lauryl Alcohol 
                 Synthesis 
               
               
                 HUCHE049 
                 Ethyl Myristate 
                 Cardamomi Fructus 
               
               
                 HUCHE050 
                 Cineole 
                 Cinae Flos 
               
               
                 HUCHE051 
                 glycyrrhizin 
                   Glycyrrhizae  Radix 
               
               
                 HUCHE052 
                 Oleanolic acid 
                   Zizyphi  Fructus 
               
               
                 HUCHE053 
                 ursolic acid 
                   Zizyphi  Fructus 
               
               
                 HUCHE054 
                 Narigin 
                   Aurantii Fructus  Immaturus 
               
               
                 HUCHE055 
                 β-Naphthoflavone 
                 Synthesis 
               
               
                 HUCHE056 
                 trans-cinnamic acid 
                 Cinnamoni Cortex 
               
               
                 HUCHE061 
                 Morin 
                 Mori Radix Cortex 
               
               
                 HUCHE062 
                 (+)-Taxifolin 
                   Paeoniae  Radix 
               
               
                 HUCHE063 
                 Chrysin 
                 Propolis 
               
               
                 HUCHE064 
                 Galangin 
                   Zingiberis  Rhizoma 
               
               
                 HUCHE065 
                 Fisefin 
                   Paeoniae  Radix 
               
               
                 HUCHE066 
                 myricetin 
                 Hibiscus  Abelmoschus   
               
               
                 HUCHE067 
                 chrysoeriol 
                 
                   Vernonia Cinerea 
                 
               
               
                 HUCHE068 
                 Phloretin 
                 Apple 
               
               
                 HUCHE069 
                 Embelin 
                   Ardisia  Squamulosa 
               
               
                 HUCHE070 
                 Tamarixetin 
                 
                   Tamarix Ramosissima 
                 
               
               
                 HUCHE071 
                 sciadopitysin 
                 
                   Ginko Biloba 
                 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Inhibition of CYP2C9 activity at 100 μM concentration. 
               
             
          
           
               
                   
                   
                 Test article 
                 % inhi- 
                   
               
               
                 Rank 
                 Test article 
                 conc 
                 bition 
                 SD 
               
               
                   
               
             
          
           
               
                 — 
                 Ketoconazole 
                 100 
                 μM 
                 100.00 
                 0.00 
               
               
                 1 
                 isoliquritigenin 
                 100 
                 μM 
                 95.47 
                 0.15 
               
               
                 2 
                 Phloretin 
                 100 
                 μM 
                 95.13 
                 0.62 
               
               
                 3 
                 luteolin 
                 100 
                 μM 
                 93.20 
                 0.94 
               
               
                 4 
                 quercetin 
                 100 
                 μM 
                 91.92 
                 0.52 
               
               
                 5 
                 Tamarixetin 
                 100 
                 μM 
                 90.18 
                 0.43 
               
               
                 6 
                 myricetin 
                 100 
                 μM 
                 88.84 
                 3.37 
               
               
                 7 
                 Wongonin 
                 100 
                 μM 
                 84.03 
                 2.22 
               
               
                 8 
                 Genistein 
                 100 
                 μM 
                 82.71 
                 2.82 
               
               
                 9 
                 Nordihydroguaiaretic acid 
                 100 
                 μM 
                 81.18 
                 0.50 
               
               
                 10 
                 Narigenin 
                 100 
                 μM 
                 79.70 
                 — 
               
               
                 11 
                 Capillarisin 
                 100 
                 μM 
                 79.49 
                 3.22 
               
               
                 12 
                 Chrysin 
                 50 
                 μM 
                 75.11 
                 6.05 
               
               
                 13 
                 Fisefin 
                 100 
                 μM 
                 72.89 
                 3.37 
               
               
                 14 
                 eriodictyol 
                 100 
                 μM 
                 69.62 
                 5.68 
               
               
                 15 
                 6- Gingerol 
                 100 
                 μM 
                 66.21 
                 1.94 
               
               
                 16 
                 Isorhamnetin 
                 75 
                 μM 
                 65.74 
                 4.99 
               
               
                 17 
                 isoquercitrin 
                 100 
                 μM 
                 61.80 
                 15.60 
               
               
                 18 
                 Formononetin 
                 50 
                 μM 
                 57.94 
                 0.84 
               
               
                 19 
                 Morin 
                 100 
                 μM 
                 51.00 
                 4.55 
               
               
                 20 
                 (+)-Taxifolin 
                 100 
                 μM 
                 50.47 
                 10.38 
               
               
                 21 
                 isovitexin 
                 100 
                 μM 
                 45.36 
                 0.97 
               
               
                 22 
                 3-Phenylpropyl Acetat 
                 100 
                 μM 
                 42.62 
                 2.00 
               
               
                 23 
                 Oleanolic acid 
                 100 
                 μM 
                 41.13 
                 11.52 
               
               
                 24 
                 ursolic acid 
                 100 
                 μM 
                 38.47 
                 3.37 
               
               
                 25 
                 Puerarin 
                 100 
                 μM 
                 33.30 
                 17.52 
               
               
                 26 
                 β-Myrcene 
                 100 
                 μM 
                 29.85 
                 4.31 
               
               
                 27 
                 trans-cinnamic acid 
                 100 
                 μM 
                 26.10 
                 3.57 
               
               
                 28 
                 Luteolin-7-Glucoside 
                 100 
                 μM 
                 25.08 
                 1.57 
               
               
                 29 
                 Liquiritin 
                 100 
                 μM 
                 24.77 
                 8.72 
               
               
                 30 
                 (+)-Limonene 
                 100 
                 μM 
                 22.29 
                 4.04 
               
               
                 31 
                 Homoorientin 
                 100 
                 μM 
                 20.19 
                 11.59 
               
               
                 32 
                 Swertiamarin 
                 100 
                 μM 
                 18.44 
                 2.11 
               
               
                 33 
                 Embelin 
                 50 
                 μM 
                 17.98 
                 4.20 
               
               
                 34 
                 Daidzein 
                 25 
                 μM 
                 15.74 
                 3.24 
               
               
                 35 
                 Poncirin 
                 100 
                 μM 
                 14.99 
                 12.51 
               
               
                 36 
                 Quercitrin 
                 100 
                 μM 
                 13.48 
                 15.69 
               
               
                 37 
                 (−)Epicatechin 
                 100 
                 μM 
                 5.44 
                 4.90 
               
               
                 38 
                 glycyrrhizin 
                 100 
                 μM 
                 4.87 
                 2.73 
               
               
                 39 
                 ergosterol 
                 30 
                 μM 
                 3.57 
                 2.64 
               
               
                 40 
                 Diosmin 
                 50 
                 μM 
                 3.51 
                 1.99 
               
               
                 41 
                 (+)Catechin 
                 100 
                 μM 
                 −0.22 
                 6.94 
               
               
                 42 
                 gallic acid 
                 100 
                 μM 
                 −0.97 
                 16.40 
               
               
                 43 
                 Daidzin 
                 25 
                 μM 
                 −1.16 
                 6.54 
               
               
                 44 
                 Daidzin 
                 100 
                 μM 
                 −1.33 
                 7.67 
               
               
                 45 
                 paeoniflorin 
                 100 
                 μM 
                 −1.77 
                 3.49 
               
               
                 46 
                 Umbelliferone 
                 100 
                 μM 
                 −2.02 
                 5.27 
               
               
                 47 
                 Rutin 
                 100 
                 μM 
                 −6.46 
                 13.80 
               
               
                 48 
                 (+)-epicatechin 
                 100 
                 μM 
                 −11.54 
                 0.77 
               
               
                 49 
                 Narigin 
                 100 
                 μM 
                 −24.21 
                 10.50 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Inhibition of CYP2C9 activity at 10 μM concentration. 
               
             
          
           
               
                   
                   
                 Test article 
                 % inhi- 
                   
               
               
                 Rank 
                 Test article 
                 conc 
                 bition 
                 SD 
               
               
                   
               
             
          
           
               
                 — 
                 Ketoconazole 
                 10 μM 
                 80.11 
                 0.71 
               
               
                 1 
                 Tamarixetin 
                 10 μM 
                 88.12 
                 0.69 
               
               
                 2 
                 apigenin 
                 25 μM 
                 76.88 
                 1.37 
               
               
                 3 
                 Genistein 
                 10 μM 
                 67.70 
                 2.28 
               
               
                 4 
                 Isorhamnetin 
                 10 μM 
                 61.53 
                 3.57 
               
               
                 5 
                 Chrysin 
                 10 μM 
                 60.62 
                 2.07 
               
               
                 6 
                 Wongonin 
                 10 μM 
                 51.31 
                 1.43 
               
               
                 7 
                 Narigenin 
                 10 μM 
                 49.98 
                 — 
               
               
                 8 
                 quercetin 
                 10 μM 
                 44.80 
                 2.37 
               
               
                 9 
                 Oleanolic acid 
                 10 μM 
                 42.35 
                 9.56 
               
               
                 10 
                 Puerarin 
                 10 μM 
                 39.02 
                 10.00 
               
               
                 11 
                 kaempferol 
                 10 μM 
                 38.29 
                 15.43 
               
               
                 12 
                 luteolin 
                 10 μM 
                 37.89 
                 14.42 
               
               
                 13 
                 ursolic acid 
                 10 μM 
                 37.46 
                 3.31 
               
               
                 14 
                 isovitexin 
                 10 μM 
                 37.38 
                 5.79 
               
               
                 15 
                 Genkwanin 
                 10 μM 
                 37.37 
                 3.64 
               
               
                 16 
                 α-Naphthoflavone 
                 10 μM 
                 37.27 
                 7.06 
               
               
                 17 
                 Capillarisin 
                 10 μM 
                 34.79 
                 3.04 
               
               
                 18 
                 Phloretin 
                 10 μM 
                 34.41 
                 7.95 
               
               
                 19 
                 (−)Epicatechin 
                 10 μM 
                 33.75 
                 13.74 
               
               
                 20 
                 (+)-Taxifolin 
                 10 μM 
                 31.16 
                 8.11 
               
               
                 21 
                 Formononetin 
                 10 μM 
                 30.57 
                 3.69 
               
               
                 22 
                 isoliquritigenin 
                 10 μM 
                 29.66 
                 14.74 
               
               
                 23 
                 Hesperetin 
                 10 μM 
                 29.09 
                 2.10 
               
               
                 24 
                 eriodictyol 
                 10 μM 
                 28.65 
                 15.29 
               
               
                 25 
                 6- Gingerol 
                 10 μM 
                 27.72 
                 10.54 
               
               
                 26 
                 isoquercitrin 
                 10 μM 
                 27.02 
                 17.78 
               
               
                 27 
                 Fisefin 
                 10 μM 
                 26.52 
                 7.25 
               
               
                 28 
                 Quercitrin 
                 10 μM 
                 21.10 
                 15.81 
               
               
                 29 
                 Liquiritin 
                 10 μM 
                 18.35 
                 1.97 
               
               
                 30 
                 β-Myrcene 
                 10 μM 
                 16.60 
                 6.31 
               
               
                 31 
                 Swertiamarin 
                 10 μM 
                 16.56 
                 3.84 
               
               
                 32 
                 Poncirin 
                 10 μM 
                 16.34 
                 10.77 
               
               
                 33 
                 protocatechuic acid 
                 10 μM 
                 16.22 
                 1.72 
               
               
                 34 
                 trans-cinnamic acid 
                 10 μM 
                 15.82 
                 9.04 
               
               
                 35 
                 Daidzein 
                 10 μM 
                 13.45 
                 4.49 
               
               
                 36 
                 Morin 
                 10 μM 
                 11.63 
                 17.51 
               
               
                 37 
                 Embelin 
                 10 μM 
                 11.23 
                 9.18 
               
               
                 38 
                 myricetin 
                 10 μM 
                 10.57 
                 13.21 
               
               
                 39 
                 (+)-Limonene 
                 10 μM 
                 10.55 
                 4.18 
               
               
                 40 
                 Nordihydroguaiaretic acid 
                 10 μM 
                 9.76 
                 5.26 
               
               
                 41 
                 ergosterol 
                 10 μM 
                 8.12 
                 2.19 
               
               
                 42 
                 baicalin 
                 25 μM 
                 7.77 
                 3.08 
               
               
                 43 
                 Hesperidin 
                 10 μM 
                 6.68 
                 3.32 
               
               
                 44 
                 (+)-epicatechin 
                 10 μM 
                 6.30 
                 3.72 
               
               
                 45 
                 Baicalein 
                 25 μM 
                 5.06 
                 8.64 
               
               
                 46 
                 Diosmin 
                 10 μM 
                 4.70 
                 0.75 
               
               
                 47 
                 β-Naphthoflavone 
                 10 μM 
                 4.64 
                 3.02 
               
               
                 48 
                 Homoorientin 
                 10 μM 
                 2.45 
                 13.94 
               
               
                 49 
                 glycyrrhizin 
                 10 μM 
                 2.23 
                 4.65 
               
               
                 50 
                 paeoniflorin 
                 10 μM 
                 0.70 
                 3.50 
               
               
                 51 
                 Luteolin-7-Glucoside 
                 10 μM 
                 −0.32 
                 5.20 
               
               
                 52 
                 Daidzin 
                 10 μM 
                 −2.46 
                 4.10 
               
               
                 53 
                 gallic acid 
                 10 μM 
                 −2.47 
                 10.16 
               
               
                 54 
                 Umbelliferone 
                 10 μM 
                 −6.64 
                 4.94 
               
               
                 55 
                 (+)Catechin 
                 10 μM 
                 −8.46 
                 3.53 
               
               
                 56 
                 (−)-Epicatechin 
                 10 μM 
                 −8.61 
                 5.95 
               
               
                 57 
                 Narigin 
                 10 μM 
                 −13.25 
                 4.33 
               
               
                 58 
                 Rutin 
                 10 μM 
                 −13.97 
                 14.31 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Inhibition of CYP2C9 activity at 1 μM 
               
             
          
           
               
                   
                   
                 Test article 
                 % inhi- 
                   
               
               
                 Rank 
                 Test article 
                 conc 
                 bition 
                 SD 
               
               
                   
               
             
          
           
               
                 — 
                 Ketoconazole 
                 1 μM 
                 45.88 
                 3.13 
               
               
                 1 
                 Genistein 
                 1 μM 
                 49.60 
                 1.37 
               
               
                 2 
                 Tamarixetin 
                 1 μM 
                 41.96 
                 6.63 
               
               
                 3 
                 Puerarin 
                 1 μM 
                 38.15 
                 0.57 
               
               
                 4 
                 3-Phenylpropyl Acetate 
                 1 μM 
                 36.57 
                 7.30 
               
               
                 5 
                 isovitexin 
                 1 μM 
                 35.56 
                 7.96 
               
               
                 6 
                 ursolic acid 
                 1 μM 
                 33.62 
                 0.99 
               
               
                 7 
                 eriodictyo 
                 1 μM 
                 32.78 
                 4.41 
               
               
                 8 
                 Genkwanin 
                 1 μM 
                 30.85 
                 1.68 
               
               
                 9 
                 6- Gingerol 
                 1 μM 
                 30.17 
                 2.36 
               
               
                 10 
                 Wongonin 
                 1 μM 
                 28.82 
                 1.41 
               
               
                 11 
                 trans-cinnamic acid 
                 1 μM 
                 26.92 
                 4.26 
               
               
                 12 
                 Embelin 
                 1 μM 
                 24.71 
                 6.18 
               
               
                 13 
                 Quercitrin 
                 1 μM 
                 24.19 
                 1.71 
               
               
                 14 
                 β-Myrcene 
                 1 μM 
                 24.06 
                 3.08 
               
               
                 15 
                 Phloretin 
                 1 μM 
                 23.76 
                 6.21 
               
               
                 16 
                 Formononetin 
                 1 μM 
                 23.33 
                 0.43 
               
               
                 17 
                 apigenin 
                 2.5 μM     
                 21.69 
                 1.37 
               
               
                 18 
                 isoquercitrin 
                 1 μM 
                 20.94 
                 1.96 
               
               
                 19 
                 protocatechuic acid 
                 1 μM 
                 20.26 
                 9.00 
               
               
                 20 
                 luteolin 
                 1 μM 
                 20.09 
                 21.27 
               
               
                 21 
                 Isorhamnetin 
                 1 μM 
                 19.63 
                 6.32 
               
               
                 22 
                 Capillarisin 
                 1 μM 
                 19.33 
                 7.81 
               
               
                 23 
                 Liquiritin 
                 1 μM 
                 18.10 
                 9.70 
               
               
                 24 
                 (+)-epicatechin 
                 1 μM 
                 16.99 
                 2.53 
               
               
                 25 
                 Oleanolic acid 
                 1 μM 
                 16.79 
                 1.67 
               
               
                 26 
                 Swertiamarin 
                 1 μM 
                 16.33 
                 0.92 
               
               
                 27 
                 quercetin 
                 1 μM 
                 15.11 
                 1.03 
               
               
                 28 
                 Morin 
                 1 μM 
                 14.26 
                 2.86 
               
               
                 29 
                 (+)-Limonene 
                 1 μM 
                 14.12 
                 3.63 
               
               
                 30 
                 paeoniflorin 
                 1 μM 
                 10.11 
                 4.34 
               
               
                 31 
                 Luteolin-7-Glucoside 
                 1 μM 
                 9.37 
                 3.17 
               
               
                 32 
                 Poncirin 
                 1 μM 
                 7.76 
                 6.36 
               
               
                 33 
                 Chrysin 
                 1 μM 
                 6.86 
                 2.17 
               
               
                 34 
                 Fisefin 
                 1 μM 
                 5.49 
                 7.50 
               
               
                 35 
                 Narigenin 
                 1 μM 
                 5.20 
                 — 
               
               
                 36 
                 glycyrrhizin 
                 1 μM 
                 5.14 
                 6.63 
               
               
                 37 
                 Homoorientin 
                 1 μM 
                 3.37 
                 8.22 
               
               
                 38 
                 Hesperidin 
                 1 μM 
                 2.57 
                 2.07 
               
               
                 39 
                 β-Naphthoflavone 
                 1 μM 
                 2.35 
                 4.87 
               
               
                 40 
                 Baicalcin 
                 2.5 μM     
                 1.76 
                 2.53 
               
               
                 41 
                 Diosmin 
                 1 μM 
                 1.51 
                 0.82 
               
               
                 42 
                 Daidzein 
                 1 μM 
                 1.35 
                 1.54 
               
               
                 43 
                 (−)-Epicatechin 
                 1 μM 
                 1.11 
                 4.15 
               
               
                 44 
                 ergosterol 
                 1 μM 
                 1.00 
                 0.59 
               
               
                 45 
                 Daidzin 
                 1 μM 
                 0.95 
                 3.51 
               
               
                 46 
                 isoliquritigenin 
                 1 μM 
                 0.87 
                 5.00 
               
               
                 47 
                 α-Naphthoflavone 
                 1 μM 
                 −0.05 
                 6.26 
               
               
                 48 
                 (+)-Taxifolin 
                 1 μM 
                 −1.29 
                 8.16 
               
               
                 49 
                 Rutin 
                 1 μM 
                 −2.59 
                 12.71 
               
               
                 50 
                 gallic acid 
                 1 μM 
                 −3.05 
                 5.18 
               
               
                 51 
                 (+)Catechin 
                 1 μM 
                 −3.05 
                 0.78 
               
               
                 52 
                 myricetin 
                 1 μM 
                 −3.19 
                 16.64 
               
               
                 53 
                 Hesperetin 
                 1 μM 
                 −3.58 
                 11.11 
               
               
                 54 
                 baicalin 
                 2.5 μM     
                 −5.36 
                 6.97 
               
               
                 55 
                 Umbelliferone 
                 1 μM 
                 −7.17 
                 3.59 
               
               
                 56 
                 Narigin 
                 1 μM 
                 −11.48 
                 2.10 
               
               
                 57 
                 Nordihydroguaiaretic acid 
                 I μM 
                 −16.06 
                 2.77 
               
               
                 58 
                 kaempferol 
                 1 μM 
                 −22.27 
                 18.96 
               
               
                   
               
             
          
         
       
     
         [0082]    Student T-test is performed on the inhibition data to assess the statistical significance of observed effects relative to the control group. Results from the best 10 test compounds at 100, 10 or 1 μM concentration are depicted in  FIGS. 2 to 4 . 
       Specific Example 1 
       [0083]    Using the procedure described in previous section, the inhibitory effect of Tamarixetin against the microsomal metabolism of tolbutamide is evaluated at different concentrations. The reaction conditions are: tolbutamide 1 mM, microsomal protein 0.5 mg, reaction time 7.5 minute. Test results indicated Tamarixetin is an inhibitor. The % inhibition is 90.2, 88.1 and 42.0% at the high, mid and low concentration, respectively ( FIG. 5  and Table 7). It is concluded that Tamarixetin is an effective CYP2C9 inhibitor. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 In vitro effects of Tamarixetin on the metabolism 
               
               
                 of tolbutamide in microsomes (n = 3) 
               
             
          
           
               
                   
                 Concentration 
                 4′-hydroxytolbutamide (ng) 
                 % inhibition 
               
               
                   
               
             
          
           
               
                   
                 Control 
                 368.5409 ± 35.3091 
                 0.0000 
               
             
          
           
               
                 1 
                 μM 
                 213.5696 ± 24.4309 
                 41.9620 
               
               
                 10 
                 Mm 
                 43.10052 ± 2.5372  
                 88.1204 
               
               
                 100 
                 μM 
                 35.49297 ± 1.5825  
                 90.1803 
               
               
                   
               
             
          
         
       
     
         [0084]    Effects of Tamarixetin on oral bioavailability of fluvastatin in Sprague Dawley rats are summarized in Tables 8 and 9. Pharmacokinetic parameters obtained for both treatment groups are presented in Table 10. Plasma fluvastatin concentration verus time curves are depicted in  FIG. 6 . Pharmacokinetic analysis indicated that there are differences in the Cmax and AUC (area under the plasma concentration time curve) values. The Cmax for the treatment group is 141.4±15.8 ng/mL, about two-fold higher than the value (63.1±10.4 ng/mL) for the control group. Estimates of plasma clearance (CL) and volume of distribution (Vd) are also different between the treatment and the control groups, suggesting inhibition of hepatic metabolism. There is no apparent changes of terminal elimination rate constant (k), and therefore the half-life (T 1/2 ) of both groups are not different. These results indicated that Tamarixetin did not exhibit a persisted inhibition of the metabolic activity, and fluvastatin is eliminated and excreted from the animal body by the regular pathways. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Blood concentration of fluvastatin in Sprague-Dawley rats following 
               
             
          
           
               
                 Time 
                 Concentration (ng/ml) 
                   
               
             
          
           
               
                 (min) 
                 C-1 
                 C-2 
                 C-3 
                 C-4 
                 C-5 
                 C-6 
                 C-7 
                 mean 
                 SE 
                 CV % 
               
               
                   
               
             
          
           
               
                 10 
                 13.56 
                 16.39 
                 19.78 
                 24.70 
                 0.27 
                 8.94 
                 16.41 
                 14.3 
                 3.0 
                 55.2 
               
               
                 20 
                 28.95 
                 18.03 
                 18.10 
                 24.48 
                 1.15 
                 19.79 
                 31.50 
                 20.3 
                 3.8 
                 49.1 
               
               
                 40 
                 47.38 
                 22.74 
                 25.67 
                 29.33 
                 7.35 
                 63.98 
                 42.36 
                 34.1 
                 7.0 
                 54.5 
               
               
                 60 
                 69.48 
                 24.74 
                 34.71 
                 41.24 
                 7.57 
                 99.41 
                 44.17 
                 45.9 
                 11.4 
                 65.9 
               
               
                 120 
                 61.69 
                 29.13 
                 43.91 
                 54.63 
                 11.19 
                 98.41 
                 41.14 
                 48.6 
                 10.4 
                 56.6 
               
               
                 240 
                 54.64 
                 38.66 
                 66.97 
                 73.42 
                 14.48 
                 68.72 
                 38.28 
                 50.7 
                 8.1 
                 42.1 
               
               
                 360 
                 40.68 
                 45.74 
                 60.75 
                 83.84 
                 20.41 
                 50.80 
                 27.07 
                 47.0 
                 8.0 
                 45.2 
               
               
                 480 
                 37.37 
                 57.30 
                 45.05 
                 54.92 
                 21.57 
                 37.68 
                 24.92 
                 39.8 
                 5.2 
                 34.4 
               
               
                 720 
                 22.45 
                 37.37 
                 25.63 
                 39.30 
                 21.75 
                 18.47 
                 15.40 
                 25.8 
                 3.5 
                 35.6 
               
               
                 1080 
                 16.11 
                 35.39 
                 22.58 
                 36.80 
                 14.10 
                 10.67 
                 11.68 
                 21.1 
                 4.2 
                 52.6 
               
               
                 1440 
                 11.47 
                 22.33 
                 21.04 
                 — 
                 10.74 
                 6.33 
                 8.58 
                 13.4 
                 2.7 
                 51.5 
               
               
                   
               
               
                 oral administration of fluvastatin only in the control group. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Blood concentration of fluvastatin in Sprague-Dawley rats following 
               
               
                 oral administration of fluvastatin and tamarixetin in the test group. 
               
             
          
           
               
                 Time 
                 Concentration (ng/ml) 
                   
               
             
          
           
               
                 (min) 
                 S-1 
                 S-2 
                 S-3 
                 S-4 
                 S-5 
                 mean 
                 SE 
                 CV % 
               
               
                   
               
             
          
           
               
                 10 
                 59.54 
                 18.79 
                 29.52 
                 100.09 
                 25.45 
                 46.68 
                 15.07 
                 72.2 
               
               
                 20 
                 137.55 
                 16.78 
                 35.58 
                 127.52 
                 32.38 
                 69.96 
                 25.79 
                 82.4 
               
               
                 40 
                 186.33 
                 38.82 
                 45.28 
                 153.00 
                 49.34 
                 94.55 
                 31.16 
                 73.7 
               
               
                 60 
                 190.51 
                 45.28 
                 — 
                 150.29 
                 74.50 
                 115.15 
                 33.48 
                 58.1 
               
               
                 120 
                 155.29 
                 107.75 
                 83.64 
                 150.05 
                 102.20 
                 119.78 
                 14.03 
                 26.2 
               
               
                 240 
                 110.95 
                 110.19 
                 160.57 
                 185.52 
                 97.33 
                 132.91 
                 17.02 
                 28.6 
               
               
                 360 
                 88.95 
                 97.35 
                 146.50 
                 157.95 
                 106.03 
                 119.36 
                 13.81 
                 25.9 
               
               
                 480 
                 71.17 
                 86.62 
                 116.44 
                 153.96 
                 136.59 
                 112.95 
                 15.32 
                 30.3 
               
               
                 720 
                 55.50 
                 60.50 
                 97.19 
                 103.45 
                 38.63 
                 71.05 
                 12.53 
                 39.4 
               
               
                 1080 
                 28.45 
                 — 
                 — 
                 50.99 
                 — 
                 39.72 
                 11.27 
                 40.1 
               
               
                 1440 
                 27.61 
                 — 
                 — 
                 41.51 
                 18.00 
                 29.04 
                 6.82 
                 40.7 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Pharmacokinetics of fluvastatin in Sprague-Dawley rats following 
               
               
                 oral administration of fluvastatin with or without tamarixetin. 
               
             
          
           
               
                   
                   
                   
                 Fluvastatin with 
                   
               
               
                 PK parameter 
                 (unit) 
                 Fluvastatin only (B) 
                 tamarixetin (A) 
                 A/B 
               
               
                   
               
             
          
           
               
                 C max   
                 (ng/mL) 
                  63.14 ± 10.36 
                 141.40 ± 15.76* 
                 2.4 
               
               
                 T max   
                 (hr) 
                  4.7 ± 1.7 
                 4.2 ± 1.1 
                 0.9 
               
               
                 AUC t   
                 (hr*ng/mL) 
                 710.57 ± 81.55 
                 1389.20 ± 166.14* 
                 2.0 
               
               
                 AUC INF   
                 (hr*ng/mL) 
                  949.86 ± 133.48 
                 2281.00 ± 386.56* 
                 2.4 
               
               
                 K 
                 (l/hr) 
                  0.074 ± 0.005 
                 0.065 ± 0.009 
                 0.9 
               
               
                 T 1/2   
                 (hr) 
                  9.7 ± 0.65 
                 11.3 ± 1.33 
                 1.2 
               
               
                 Cl/F 
                 (mL/min/kg) 
                 29.12 ± 4.05 
                  12.33 ± 2.10** 
                 0.4 
               
               
                 Vz/F 
                 (mL/kg) 
                 24846.64 ± 4721.23 
                 11163.54 ± 861.69*  
                 0.4 
               
               
                 AUMC INF   
                 (hr*hr*ng/mL) 
                 15156.0 ± 2864.6 
                 42896.4 ± 12379.8 
                 2.8 
               
               
                 MRT INF   
                 (hr) 
                 15.82 ± 1.56 
                 17.31 ± 2.27  
                 1.1 
               
               
                   
               
               
                 PK = pharmacokinetic, 
               
               
                 Data = mean ± SE, 
               
               
                 *p &lt; 0.05, 
               
               
                 **P &lt; 0.01 
               
             
          
         
       
     
       Specific Example 2 
       [0085]    Using the procedure described in previous section, the inhibitory effect of isoliquritigenin against the microsomal metabolism of tolbutamide is evaluated at different concentrations. The reaction conditions are: tolbutamide 1 mM, microsomal protein 0.5 mg, reaction time 7.5 minute. Test results (Table 11 and  FIG. 7 ) indicated isoliquritigenin inhibited 95.46% of the activity at the high concentration. It is considered that isoliquritigenin is an effective CYP2C9 inhibitor. 
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                 TABLE 11 
               
             
             
               
                   
               
               
                 In vitro effects of isoliquritigenin on the metabolism 
               
               
                 of tolbutamide in microsomes (n = 3) 
               
             
          
           
               
                   
                 Concentration 
                 4′-hydroxytolbutamide (ng) 
                 % inhibition 
               
               
                   
               
             
          
           
               
                   
                 Control 
                 374.8785 ± 54.8521 
                 0.0000 
               
             
          
           
               
                 1 
                 μM 
                 371.5965 ± 18.7272 
                 0.8737 
               
               
                 10 
                 Mm 
                 263.4592 ± 55.2455 
                 29.6603 
               
               
                 100 
                 μM 
                 16.25213 ± 0.5544  
                 95.4680 
               
               
                   
               
             
          
         
       
     
       Specific Example 3 
       [0086]    Using the procedure described in previous section, the inhibitory effect of Genistein against the microsomal metabolism of tolbutamide is evaluated at different concentrations. The reaction conditions are: tolbutamide 1 mM, microsomal protein 0.5 mg, reaction time 7.5 minute. Test results indicated Genistein is an inhibitor. The % inhibition is 82.7, 67.7 and 49.6% at the high, mid and low concentration, respectively (Table 12 and  FIG. 8 ). It is concluded that Genistein is an effective CYP2C9 inhibitor. 
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                 TABLE 12 
               
             
             
               
                   
               
               
                 In vitro effects of Genistein on the metabolism 
               
               
                 of tolbutamide in microsomes (n = 3) 
               
             
          
           
               
                   
                 Concentration 
                 4′-hydroxytolbutamide (ng) 
                 % inhibition 
               
               
                   
               
             
          
           
               
                   
                 Control 
                 479.3314 ± 56.4829 
                 0.0000 
               
             
          
           
               
                 1 
                 μM 
                 241.2098 ± 6.5885  
                 49.5979 
               
               
                 10 
                 Mm 
                  154.311 ± 10.9480 
                 67.6979 
               
               
                 100 
                 μM 
                 82.24342 ± 13.3679 
                 82.7088