TW200533364A - Inhibitor of cytochrome P450 isozyme, CYP2C9 - Google Patents

Abstract

The present invention provides various kinds of inhibitors for cytochrome P450 isozyme, CYP2C9, wherein the inhibitor is selected from the group consisted of 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. Among above, the natural product can be used to increase the bioavailability of drug.

Description

200533364 发明 Description of the invention: ~ [Technical field to which the invention belongs] The present invention provides an inhibitor of cytochrome P450 (especially as an inhibitor of cytochrome P450 isozyme) CYP2C9. [Prior art]

Cytochrome P450 (P450) is an important enzyme for redox reactions during drug metabolism in the body. P450 is divided into multiple subfamily and is widely distributed in the liver, intestine and other tissues (Krishna D. and Klotz U., Extrahepatic metabolism of drugs in humans. Clinical Pharmacokinetics · 26: 144-160, 1994), in order to carry out phase I reactin to facilitate the excretion of metabolites, various subtypes and subtypes are generally fortunate based on the similarity of amino acid sequences To distinguish (Slaughter R 丄 · and Edward DJ ·, Recent advances: the cytochrome P450 enzymes · The Annals of Pharmacotherapy · 29: 619-624, 1995), the same subfamily enzymes in mammals basically keep more than 55% from each other Amino acid sequence similarity, for example, CYP2C9 and CYP2C11 or CYP1A1 and CYP1A2. In fact, the individual enzymes with similar Arabic numbers often have a high degree of amino acid similarity, and the gene is located on the chromosome. Similar, taking CYP2C9 and CYP2C10 as examples, there are only two amino acid differences between the two, and the amino acid sequences of both CYP3A3 and CYP3A4 are: 97.5 ° /. the same. Therefore, the enzyme name of its Cytochrome P450 spans all species in the biological world, including animals, plants and microorganisms. Cytochrome P450 is translated into Chinese as "cytochrome P450". Cytochrome refers to a group of ferrous ions that are attached to the endoplasm of cells. Enzyme protein of the net, this ferritin-containing structure and function is similar to hemoglobin, which is responsible for electron transfer and energy release. It is generally called a heme-group or prosthetic group. P450 indicates that when carbon monoxide is bound to this enzyme, it is exposed to visible light. Spectral spectrometer detection (Omura T. and Sato R., The carbon monoxide-binding pigment of liver microsomes. The Journal of Biological Chemistry · 239: 2370-2378, 1964), which has a maximum absorption coefficient at a wavelength of 450 nm, is therefore defined For P450. CYP450 in vivo distribution: 200533364 For cytochrome P450, the distribution of enzymes in mice and humans is quite similar, and the distribution of isozymes in human CYP450 is quite extensive (Zhang QY, Dunbar D ". Ostrowska A.? Zeisloft S. 5 Yang J ,, and Kaminsky LS, Characterization of human small intestinal cytochromes P-450. Drug Metabolism and Disposition. ^ 27: 804-809, 1999), all types of CYP450 isoenzymes in the human body except CYP1A1 are distributed in the liver But the content ratios are different (Waziers I ·, Cugnenc PH "Yang CS ·, Leroux JP and Beaune PH, 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) »Among them, CYP2C subfamily accounts for about 1S.2%, and CY? 3A4 also has a relatively high proportion in the intestine (approximately 50% of the liver content); the situation in rats is quite similar to that in humans. Similarly, with the exception of Cyp2B1 and cypiAi, all other isoenzymes also use the liver as the main distribution area. It is known that although the ratios of various types of enzymes in mice and humans are quite different due to differences between species, the biological functions of their enzymes are still quite replaceable. Therefore, the study of CYP2C is based on Sprague Dawley. The liver of SD rats is the main source of enzyme extraction.

A total of 57 different isozymes were found in the human CYP gene and classified into 14 different CYP enzyme families (Nelson DR ·, Koymans L · and Kamataki T., P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.

Pharmacogenetics 6: 1-42, 1996), which are CYP 1, 2, 3, 4, 5, 7, 8, 11, 17, 19, 21, 24, 27, and 51, among which the three families of CYPI ~ 3 Enzymes are responsible for most of the metabolism and detoxification of drugs and foreign substances, while the remaining ten-different types of enzyme groups are responsible for the metabolism of endogenous substances in the organism, such as the hormones or steroids mentioned above Matter, etc ... Genotypes · The human CYP2C family is currently known to have four defined subtypes, namely cyp2C8, CYP2C9, CYP2C18, and CYP2C19. The amino acid sequences of the four have 82% similarity (Miners JO and Birkett DJ, 5 Cytochrome P4502C9: an enzyme of major mportance in human drug metabolism, British Journal of Clinical Pharmacology. 200533364 45: 525 Ma 1998), although the amino acid sequence has such a high degree of similarity, however, all types of enzymes are However, there are considerable differences in drugs. It has been reported that cYp2c subfamity has genetic polymorphism characteristics like CYP2D6. It has also been clinically engaged in many cyp2c gene polymorphism studies. Among them, the ability of human cytochromes to drug metabolism can be summarized as There are two types of Extensive metabolizers (EMs) and Poor metabolizers (PMs), and the proportion of different genotypes also varies from race to race. Take (^ monk) as an example, the white population is about 2 to 4%, and Asians are about 20%, so more attention needs to be paid to drug interactions with enzyme-specific metabolism. Drug Metabolism: Drug Via After the drug is absorbed into the body through this route of administration, it follows the kinetic model to continue the process of deluchet and elimination. The metabolism of drugs in the body is mainly divided into one-phase reaction and two-phase reaction (phase Π reaction), both mechanisms are It can form hydrophilic metabolites to facilitate drug excretion. Mixed monooXygenase is the main enzyme responsible for phaselreaction in the body, and p45〇 belongs to this type of enzyme, that is, via P450, P450 reductase, cytochrome b5 Other groups of proteins are responsible for the redox and hydrolysis reactions of the drug. The sound of its metabolic mechanism will be described in the next section, and the phase. The Π reaction is mainly conjugation. It can be roughly divided into six categories (Table 1) Among them, three kinds, sulfation and glutathione conjugation are the most common. @ 表 一 、 One-phase and two-phase metabolic reactions in vivo (Shargel L ·, and Yu ABC ·, Hepatic elimination of drugs. Applied Biopharmaceutics and Pharmacokinetics. 4th ed ”Appleton & Lange, Stamford, pp. 353-398, 1999) 200533364 phase I reaction phase Π reaction (High energy intemiedate) Oxidation Aromatic hydroxylation Glucuronide conjugation (UDPGA) Aliphatic hydroxylation N-, O-oxidation Sulfate conjugation (PAPS) N-? O-dealkylation Deamination Glutathion conjugation (GSH) Reduction Azoreduction Acetylation (Acetyl coenzyduction Aethylation) ) Alcohol dehydrogenase Hydrolysis Ester hydrolysis Amide hydrolysis Table 1. UDPGA = uridine diphosphoglucuronic acid, PAPS = = 3'-phosphoadenosine S ^ phosphosulfate, GSH = glutathione, SAM = S-adenoylmethionine "CYP2C subfamily four subtypes Although enzymes have different metabolic drugs, but in terms of clinical drug metabolism, CYP2C9 and CYP2C19 are the mainstays. In terms of liver CYP2C content ratio, the proportion of the two households is about 3: 1 (Venkatakrishnan K ·, von Moltke LL , Greenblatt DJ5 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) Among them, Omeprazole, a commonly used drug that inhibits gastric acid secretion, is the specific metabolic substrate of CYP2C19. As for the types of drugs that CYP2C9 is responsible for metabolism, it covers a wide range. Generally, non-steroidal anti-inflammatory drugs (NSAIDs), hypoglycemic drugs, anticoagulants The drugs are all in the category of CYP2C9 responsible for metabolism. Only the commonly used drugs are listed in (Table 2). Among them, phenytoin and warfarin 200533364 are both commonly used in clinical treatment with low therapeutic index (na_ 细 叩 _d_ ) Drugs, once the differences between individuals or external Wei cause oral or variability of drugs, it is very likely to cause serious drugs: 1 effects and lead to adverse treatment consequences, among which the main reason for the difference in absorption between individuals is the gene It ’s difficult. The genetic polymorphism is between different races. Taking CYP2D6 as an example, CYP2D6 is mainly responsible for the lipophilic antipsychotic drug metabolism. About η% of the Western population has the CYP2D6PM genotype. Less than 1% of patients, so if the general dose of antipsychotic drugs is used in patients with PM, drug metabolism often slows down, causing severe application The quality of life is so much more advanced-it reduces the patient's medication compliance and accelerates the deterioration of the condition, the same result; if a drug with a low therapeutic index is used in pM patients, it may get more serious drug side effects and cause medical tragedy. First, to resolve the differences in drug absorption between individuals, in addition to controlling the amount of drug absorption (such as long-acting controlled release dosage forms), the most direct method is to control the rate of drug metabolism. When the amount of drug absorption and the rate of metabolism between individuals are controlled -致 性 ， 则 温馨 # In the ideal situation, similar oral bioavailability of drugs should be obtained to achieve the same therapeutic effect, which can reduce the difference in absorption between individuals, _ avoid the occurrence of vices, except Looking for effective cytochrome regulators outside the state, you can still slow down the metabolism of high flrst_pass effect drugs by temporary enzyme inhibition. However, effective enzyme regulators must take into account the prerequisites for safety and consumption in daily life. Or medicinal natural substances and Chinese herbal medicines can meet this condition. The most common natural substance that affects the oral bioavailability of a drug is the significant increase in blood concentration of the drug when combined with grapefruit juice (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. 5 Grapefruit juice and its flavonoids inhibit 11 beta-hydroxysteroid dehydrogenase. Clinical Pharmacology and Therapeutics. 59: 62 ^ 71, 1996; Kane et al .5 Drug-grapefruit juice interactions. Mayo Clinic Proceedings. 75 (9): 933-42, 2000). Table 2. Clinical drugs for inhibiting, promoting and responsible for metabolism of CYP2C subfamily (Rendic s., Summary of information on human CYP enzymes: human P450 metabolism 200533364 data. Drug Metabolism Reviews. 34: 83-449, 2002)

Isoenzyme Substrate Inhibitor inducer CYP2C9 Tolbutamide Diclofenac Warfarin Phenytoin Torsemide Fluvastatin Losartan Celecoxib Meloxicam Isoniazide

Fluconazole Ketoconazole Metronidazole Itraconazole Cimetidine Sulphaphenazole Phenylbutazone

Rifampin Phenobarbital Cabamazepine Ethanol

Valporic acid Ibuprofen

Carvedilol Naproxan

Ondansetron CYP2C19 Omeprazole Fluoxetine Imipramine Sertraline Diazepam Ritonavir Mephenytoin Clomipramine

Rifampin Hexobarbital

This "Ming selects rat liver microsomal enzymes as the experimental model, extracts herbal medicine and natural products, and conducts in vitro cytochrome inhibition tests to make blood sugar lowering drugs. She said that this is the standard drug (such as coffee) (9G% is exclusively metabolized by CYP2C9) 'Take microsomal enzyme metabolism and suppress the king as a teacher's selection indicator, from which to find effective CYP2C9 R engineering enzyme regulators, and by this 200533364 effect shoot regulators to improve the body of the drug The purpose of availability. SUMMARY OF THE INVENTION The present invention. The first aspect is to provide effective inhibitors of cytochrome P450 in order to increase the bioavailability of the drug. The inhibitor is selected from the group consisting of the following compounds: Tamarixetm, flavonoid isoflavones ―), Isoglycosides such as 〇1 coffee harpy, Phloretm, luteolin (iuteolin quercetin (Querdtrin), quercetinized _ coffee), myricetin, myricetin, Wongonin ), Puerarin (taken as Ru), Genistein, Nordihydroguaiaretic acid, Narigenin, Capillarisin, Chrysin , Fisefm, eriodictyol, 6-Gmgerol, isorhamnetin, isocercitrin, Morin, (+)-Taxifolin, Isobaric (Isovitexin), phenylpropyl acetate (3_phenylprOpyi Acetate), oleanolic acid, ursolic acid, β-myrcene, cinnamic acid , Luteolin; 0 · glucoside (Luteolin-7_Ghic〇side), licorice extract (Liquiritin), lime Oil ((+) Limonene), Homoorientin, Swertiamarin, Embelin, Lignin Isoflavones (Daid2ein), Poncirin, Epicatechin ((-)- Epicatechin), erg0sterol. This inhibitor is specifically targeted at the cytochrome P450 isoenzyme CYP2C9 with inhibitory effect. The inhibitor of cytochrome P450 isoenzyme CYP2C9 is preferably selected from the group consisting of the following compounds: Tamarixetin, Formononetin, Isoliquritigenin, Phloretin, Osmanthus Luteolin, Quercitrin 'quercetin, myricetin, wongonin, puerarin < (Puerarin), genistein , Nordihydroguaiaretic acid, naringin. The inhibitor of cytochrome P450 isoenzyme CYP2C9. The inhibitor is more preferably Tamarixetin. A second aspect of the present invention is to provide a pharmaceutical composition for increasing the bioavailability of a drug, the composition comprising a pure ingredient of a traditional Chinese medicine and a pharmaceutically acceptable carrier. The traditional Chinese medicine drug pure ingredients 11 200533364 can be used as an inhibitor of cytochrome P450. The pure ingredients of the traditional Chinese medicine are selected from the group consisting of the following compounds: Tamarixetin, Formononetin, Isoliquritigenin, Phloretin, luteolin, and luteolin. Quercitrin, quercetin, bayberry bark (niyricetin), wongonin, puerarin, genistein, nordihydroguaiaretic acid , Narigenin, Capillarisin, Chrysin, Fisefin, eriodictyol, 6-Gingerol, Isorhamnetin, Isoquercetin Isoquercitrin, Morin, (+)-Taxifolin, isovitexin, 3-Phenylpropyl Acetate, Oleanolic acid, ursolic acid, β- Laurel dilute (β-Myrcene), cinnamic acid (cinnamic acid),. Luteolin-7.0-glucose (Luteolin-T * Glucoside), licorice extract (Liquiritin), (+) cup lemon essential oil ((+) Limonene), isoxetolin. (Homoorientin), Swertiamarin, Embelin, Daidzein 'Poncirin, ㈠-Epicatechin, ergosterol. The pure component of this traditional Chinese medicine extract inhibits cytochrome P450 isoenzyme CYP2C9. The pure ingredients of the traditional Chinese medicine are preferably selected from the group consisting of the following compounds: Tamarixetin, flavonoid isoflavones (FormOnonetin), isoglycone,

Phloretin, luteolin, Querc trin, quercetin (myncetm), wonongin, puerarin, genistein Teacher), dihydroguaiaretic acid (run bump _〇 酔 丨 batch coffee heart), sleeve skin Su Qing kiss gold). 4 The better component of traditional Chinese medicine is Tamarixetin. "This group of oral products contains a combination of tolbutamide, wherein the pure ingredients of the traditional Chinese medicine can increase the bioavailability of 7tolbutamide. The combination of oral and oral products containing xanbuta, wherein the traditional medicine is pure Ingredients can increase the bioavailability of fluvastatin. [Embodiment] This month, we focus on finding CYP2C9 lyostat, according to literature reports: the blood glucose lowering drug 12 200533364 tolbutamide is used as a model drug in rats' rabbits, dogs, pigs and monkeys In vitro experiments of liver microsomal enzymes obtained by animals and other animals were found to be evaluated in inhibition pattern. Dogs and humans have the highest similarity to CYP2C9. However, enzyme activity and kinetic parameters were observed from the perspective of enzyme kinetics. € (Vmax / Km) The atmosphere is closest to humans (Bogaards et al., Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat5 rabbit, dog, micropig, monkey and man · Xenobiotica · 30: 1131-1152, 2000), if we observe the metabolic pathway of tolbutamide in various animals (Dogterom P. & Rothuizen J., A species comparison of t olbutamide metabolism in precision-cut liver slices from rats and dogs · Drug Metabolism and Disposition · 21: 705-709, 1993) found that rats, rabbits, and humans follow the same metabolic pathway for this drug, and the metabolites they produce are also the same, However, dogs belong to different metabolic pathways. In addition, the human and rat CYP2C9 and CYP2C11 polypeptide sequences are compared (http://ww.dmelson.utmem.edu/CytochromeP450/html). Has 73% amino acid sequence identity, and functional evaluation has 84% biological functional similarity (http://www.ncbLnlm.nih.gov/BLAST/), so the present invention chooses to use rats as the ideal animal model It replaces human liver microparticles in vitro and in vitro tests, and evaluates the enzyme inhibitory effect of drugs on CYP2C9. This test is to use the pure ingredients of traditional Chinese medicine for in vitro metabolism inhibition experiments and small animal live negative tests to further study Chinese medicine. Lead to low organism availability. The pharmacokinetic properties of the drug affect the screening of Chinese medicines with potential for cytochrome inhibition. . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be further described with reference to the following examples, but these examples are only for illustrative purposes 3 and should not be construed as limitations to the practice of the present invention. Materials and methods: The classification of M Yinai, a common chemical compound in tirr, can be classified as Da'an, fiavones, flavanones, chalcones, isnf! T # (microsomes preparation) coumarins 〇 Experimental animals of the present invention It is a rat, so the enzyme for drug metabolism is also taken from rat liver homogenate 13 200533364 II First, the liver is taken out immediately after the animal is sacrificed and placed in the generation]. I5 化 钟 '_Face sound fine solution to remove The excess blood in the tissue is reduced and freshly spread with human nails. There is no longer a piece to be homogenized and then packed in a centrifuge tube. However, frost on the tube wall should be avoided. g and 1,250 g of preliminary centrifugation for 20 minutes to remove excess extracellular tissue. After centrifugation, carefully remove the supernatant, which contains soluble liver microsomes, hepatocytes, and potassmm) solution to clean the microsome gel particles (peiiet) at the bottom of the tube. After removing the remaining centrifugal π liquid 'washing' strips in the tube, remove the microsome gelatinous particles (pellet), add the same weight as the liver's pH 7 4 0 · 1M phosphate buffer, and perform a second homogenization to A microsome suspension was obtained at a concentration of 100% (w / w). Organelles such as cell debris, nuclei, and glandular glands, and the part of the cell microsomes required for the experiment can be prepared by preliminary separation: the contact with the upper layer fluid and ultra-high-speed centrifugation: the upper layer fluid obtained from the appropriate talent is divided into super-speed Isolate ~ officer '5 ~ 6 m each, and tighten the cap, set ^ Lin g ultra-high speed centrifugation for 2 hours, remove the supernatant solution, and use U5% potassium chloride (contact The prepared microsome suspension has a protein content of about 25 g / perml liver homogenate. The suspension can be stored in a -80 ° C freezer to maintain the enzyme activity for about eight weeks without affecting the changes. The suspension prepared by the process of microsomal enzymes can be applied to the study of drug metabolism, and the microsomal enzymes used in the experiments should be used within the time limit as soon as possible to avoid serious errors in the birth and misjudge the experimental results. Preparation Brief description of the process is as follows: (1) Animal sacrifice (spinal cord dislocation); (2) take the liver; (3) weigh after washing with KC1 (U5%); ⑷ add KC1 (U5%) after cutting, weight ratio (Liver: KC1 = 1: 2); ,, ⑸ homogeneous completely; High-speed centrifuge tubes, about 12 ~ 15ml per tube; ⑺ High-speed centrifugation 4t: 520min512500g; 14 200533364 (8) Take the supernatant and place it in an ultra-high-speed centrifuge tube; „(9) Ultra-high-speed centrifugation at 4 ° C, 2hr, l〇〇 (00); (10) Discard the supernatant and wash the remaining liquid in the tube with KC1 (U5%); (11) Scrape off the pellets in the tube; (12) Add a phosphate buffer solution equal to the liver pH 7.4 "(13) After homogenization, aliquot each tube in a microcentrifuge tube; (14) Place in -80 C refrigerator for refrigerating. 2. Perform traditional Chinese medicine bow | screening | screening | microparticles with CYP2C9 in vitro study After the body enzyme preparation and protein concentration measurement are completed, the activity measurement and inhibitor screening of the enzyme cYp2c can be performed. Before this, the experimental mode of this enzyme must be established to find the relevant kinetic parameters of the brother 'to determine the in vitro test (in in vitro study) conditions and reaction environment. tolbutamide is a highly specific metabolizing drug of human CYP-2C9, which can be transformed into a metabolite with a hydrophilic functional group 4-hydroxytolbutamide after activation and metabolism, and the reaction has been confirmed to be single Approach it This enzyme does not involve its metabolic process, so it can be regarded as an ideal experimental drug and can accurately detect the activity of CYP2C9. The concentration of the substrate response is set to ig, which can reach the enzyme saturation state (Tang et al ”Effect of albumin on phenytoin and tolbutamide metabolism in human liver microsonies: an impact more than protein binding.

Drug Metabolism and Disposition · 30: 648-654, 2002) o Microsomal enzyme activity response environment is set as follows (total volume == 1 ml): (1) 0 · 1 Μ phosphate buffer ρΗ 7 · 4; · (2) 0.5 mg microsomal enzyme protein; (3) 5 mM magnesium chloride solution; (4) 10 mM gluconic acid; (5) 2 IU glucose phosphate dehydrogenation gland; (6) 1 mM nucleoside diphosphonic acid adenine ; (7) 1 mM Tolbutamide ί '(8) 1% MeOH〇15 200533364 The activity measurement reactions were all performed on ice to maintain the environment. After the coenzyme was added, it was placed in a 37 ° C water bath for 1 minute, and then added to the mode. The drug reaction time was calculated, and the reaction was terminated with Hushan m hydrochloric acid, and extracted with 2 mi of digas methane. The lower solvent was removed by centrifugation, and the mobile phase solution was dissolved after blow drying, and then the drug analysis was performed. Reaction time_set, must depend on the degree of acceptance, to determine the correlation between metabolite production and reaction time gate: take a linear segment with positive correlation and convert metabolite generation at each time point ϊ: j decides the most suitable for The reaction time of the analysis, and the initial concentration of the model drug need to be compared with the concentration of metabolites produced by the microsomal enzyme metabolism at different concentrations in the case of solid reaction. And Vmax to determine the initial reaction concentration that is most suitable for analysis. The analysis of 7-mode drugs and metabolites was performed using a high-level liquid phase analyzer (sw leg ^ LC_1〇Ad). Echocardiography uses an ultraviolet light detector (madzuSpD_1〇A), with a wavelength set to 230 (Qing et al, 1988), and a chromatography tube using 15 × 4 6 dirty, cμ Γ Qingqing, Qing-Example Wenka 4.6 , mI 2T5 ^ 144 B " J ^ ^ Enzyme benefit l == conazole is the normal phase control group (posit— ^ concentrated production is 10 'system, and the result of determining the anti-light system's positive impurity' is shown in Figure 1. 4% inhibitory effect μ 4 ^ e = az ° le can complete _w_mes generation of warship power, and the screening mode of up to 101 〒 乐 # primer inhibitor is the same concentration inhibition _ for enzyme activity ^ ;! Normal phase control group · ' The lower, middle, and high ear shapes are often used in the experiment. Methanol, B '=,' But when the drug is not good in water solubility, different organic solvents with different price are used for enzyme activity. By comparing the test names, you can correctly judge the transit = 11 when the deduction of the solvent is turned off and the control is turned 3. The effect of the small animal in vivo experiment on enzyme activity. Hypoglycemic tolbutamide is an in vitro experiment from rat liver microparticles Noodle 16 200533364-type medicine for towel index screening, the screening results will be based on the degree of inhibition, choose the effective ingredients of pure towel medicine for small In vivo experiments in animals, experimental animals still use money as the animal model. Since tdbutamide can be taken up to 9G% in oral organisms, it is not suitable for threatening experiments. The degree of absorption and metabolism of Echa drugs in animals is called. Fluvastatin (Lescol ⑨), a lipid-lowering drug, was selected as the model drug for intracranial tests. Fluvastatin is the first completely chemically synthesized HMG-CoA reductase inhibitor. The oral bioavailability is about 25-30%, and it is specifically affected by CYP2C9. Metabolism, fluvastatin sodium can be absorbed up to 90% orally. Therefore, the severe primary effect is the main reason for the low rate of biotic stricture. This drug can be metabolized by the liver to produce four different metabolites (ScriptureetaL, 2001), of which CYP2C9 is responsible for metabolism. 80% fluvastatin, while the remaining isoenzymes are responsible for the other 20%. Rats were fasted before the test for another day, and then carotid arterial intubation was performed after anesthesia. The rats in the experimental group were orally administered with 9 32 mg / kg Tamarixetin (soluble Organic solvent DMSO, 10 mg / ml), the control group was given DMSO only, and after 30 minutes, both groups were given fluvastatin 1.5 mg / kg (soluble in water, 2 mg / ml), and the blood samples were taken with blank control (Bl ank) A total of twelve o'clock lasts for twenty-four hours, which are 〇, 10, 20, 40, 60, 120, 240, 360, 480, 720, 1080, 1440 mm, and each point draws about 0.5 ~ 0.6 ml (Plasma can be separated from about 250 to 300 μΐ) 'The micro tube for taking blood is added with 20 μΐ 10HJ heparin (heparin) in advance to prevent blood clotting. After plasma separation, it should be refrigerated at _80 ° C in the refrigerator after being separated from light.

The blood concentration of Fluvastatin was analyzed by High Performance Liquid Chromatography (HPLC). The detector was a fluorescence spectrophotometer. The excitation and scattered light were set to 309 nm and 390 nm, respectively. 5 μ, 150 X 4.6 mm, C18 reverse phase column, mobile phase was 〇 · Μ TBAF: 0 · 1 Μ phosphate buffer (pH 6.0 ·): MeOH (15: 25: 60 v / v), temperature control 50 The flow rate in ° C is set to 1 ml / min. For the blood test procedure, refer to Kalafsky et aL, (High-pbrformance liquid chromatographic method for the determination of fluvastatin in human plasma. Journal of Chromatography. 614: 307-313, 1993) And Toreson et al "(Determination of fluvastatin enantiomers and the racemate in human blood plasma by liquid chromatography and fluorometric detection. Journal of Chromatography A. 17 200533364 729: 13-18, 1996), as follows: (1) After the ice / dong test sample is taken out, put it on crushed ice to dissolve the beads; (2) Take 250 μ1 plasma test sample and make it in a spiral test tube; (3) Add 50 μ1 internal standard (c elec〇xib 2〇_ml ries; (4) Add Acetonitrile 250 μΐ and vibrate i 5, s Γ (5) add 0.5 M phosphate buffer ρΗ ^^. Ο 250 μΐ; ⑹ add 2.5 ml ΜΤΒΕ (methyl -tert seven utyl ether), shaking for 30 min; (7) Take the organic layer solution in a flat-mouth test tube, and concentrate it under reduced pressure to volatilize; (8) Re-dissolve with 200 μΐ mobile phase solution; * ⑼ Take the solution to the south speed Centrifuge at 13,000 rpm for 5 min; (10) Take the supernatant 150 μΐ and inject it into HPLC. Experimental results:. In vitro screening of traditional Chinese medicines cited HUCHE001 ~ HUCHE070, the degree to which each component inhibits the metabolism of microsomes tolbutamide is divided into 1,1. , 100μM, some traditional Chinese medicines have poor solubility, only the soluble concentration is the highest inhibitory concentration, and then diluted to the middle and low settings in order. The experimental results are set according to the inhibitor settings of high, medium and low. Different concentrations are divided into three categories. The best inhibitors are: HUCHE009 isdiquritigenin 100μM inhibits microsomes up to 95.5%, HUCHE070 Tamarixetin 10 μM inhibits 88.2%, HUCHE037 genistein isoflavone Genistein 1 μM inhibits 49 6% According to traditional Chinese medicine-numbered finishing shown in Table 4 to Table VI. Table 3. Introduction to Chinese Medicine Sources 200533364 No. Pure Ingredient Name Raw Medicine Source Raw Medicine Name HUCHE001 Genkwanin Astemisiae Capillaris HUCHE002 Apigenin Chamomillae Flos HUCHE003 Digitalis Folium HUCHE004 Luteolin-7-Glucoside Digitals Folium HUCHE005 Homoorientin Swerticie Herba HUCHE006 Isovitexin Swerttae Herba HUCHE007 New Orange Skin (Neeohesperidin) Anrantii Fructus Immatums HUCHE008 Formononetin Astragali Radix HUCHE009 Isoliquritigenin Astragali Radix HUCHE010 Kaempferol Sennae Folium Plum (Isorhamnetin) Senna Sennae Folium HUCHE012 isoquercitrin (Isoquercitrin) Sweet tea Hydrangeae Dulcis Folium HUCHE013 (+)-epicatechin (+)-epicatechin 児 茶 Gambir HUCHE014 Ergot ergot. Ergota HUCHE015 (+) Cate chin Paeoniae Radix HUCHE016 6-Gingerol Ginger Zingiberis Rhizoma HUCHE017 Liquiritin Glycyrrhizae Radix HUCHE018 'Phenylpropyl Acetate Cinnamami Cortex HUCHE019 ㈠ epicatechin (-) -Epicatechin Cosmos Gambir HUCHE020 Narigenin Aurantii Fructus Immatums HUCHE021 Umbelliferone Aurantii Fructus Immaturns HUCHE022 Rutin Sophorae Flos HUCHE023 Lemon Yellow Aurantii Fructus Immaturus HUCHE024 Diosmin —— _ HUCHE025 Hesperetin Citri Reticulatae HUCHE026 Wongonin Scutellariae Radix HUCHE027 Baicalin Scutellariae Radix HUCHE028 HUCHE029 Puerariain Pueraria Radix HUCHE030 Lignin Isoflavone Daidzein Pueraria Radix HUCHE031 Daidzin Pueraria Radix HUCHE032 Quercitrin) 獬 皮 Viscum Coloratum HUCHE033 Quercetin or quercetin Viscnm Coloratum HUCHE034 ~: (Nordihydroguaiaretic acid) Table III 19 200533364 Table III. Introduction to Chinese Medicine Sources (continued) No. Pure Ingredients Name of crude drug, source of raw drug name HUCHE035 fe] Chen Se original surface I (Capillarisin) HUCHE036 Swertiamarin HUCHE037 Genistein HUCHE038 trans-Cinnamaldehyde HUCHE039 protocatechuic acid HUCHE040 not Gallic acid HUCHE041 paeoniflorin HUCHE042 eriodictyol HUCHE043 Poncirin HUCHE044 flavones (a -Naphthoflavone) HUCHE045 laurel roast (0 -Myrcene) HUCHE046 α-terpineol (α-terpineol) terpineol) HUCHE047 (+)-Lemon essential oil (+) -Limonene HUCHE048 Lauryl Alcohol HUCHE049 Ethyl Myristate HUCHE050 Cineole HUCHE051 Glycyrrhizin HUCHE052 Oleanolic acid) HUCHE053 ursolic acid HUCHE054 Pomelo bitter Narigin HUCHE055 yS-Naphthoflavone HUCHE056 trans-cinnamic acid HUCHE061 Morin HUCHE062 (+)-Taxifolin HUCHE063 Chrysin HUCHE064 Galangin HUCHE065 Fisefin HUCHE066 Myrictin St. HUCHE0 Chrysoeriol HUCHE068 Phloretin HUCHE069 Embelin HUCHE070 Tamarixetin HUCHE071 sciadopitysin Cassia gallic acid paeony fire thorn wood sago flower. Myristyl alcohol 1 Artemisia sylvestris var. Artemis sylvestris Artemisia radix bark Yindang Gegui Berberis sylvestris scabra licorice Jujube Jujube Jujube Cinnamon mulberry peony Peony Propolis Ginger Paeonia oleifera Mallow typhus apple purple golden cow holy willow ginkgo

Artemisia Capillaris Swertiae Herba Puerariae Radix

Cinnamami Cortex Cinnamami Cortex

Paeoniae Radix Pyracantha Fortnneana Aurantii Fructus lmmaturus

Amomum cardamomum Cinae Flos Cardamomi Fructus

Cardamomi Fructus

Cinae Flos Glycyrrhizae Radix Zizyphi Fructus Zizyphi Fructus Aurantii Fructus lmmaturus

Cinnamoni Cortex

Mori Radix Cortex Paeoniae Radix Propolis

Zingiberis Rhizoma Paeoniae Radix Hibiscus Abelmoschas Vernonia Cinerea Apple

Ardisia Squamulosa Tamarix Ramosissimci Ginko Biloba Table III (continued) 20 200533364 Table IV. Inhibition of Enzyme Activity by Pure Ingredients of Chinese Medicine 100 μΜ Ranking of Pure Ingredient Name Inhibitor Concentration% inhibition SD-Ketoconazole 100 μΜ 100.00 0.00 1 Isoglycone (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 Η) 0 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 Naringin 100 μΜ 79.70-11 Capillarisin 100 μΜ 79.49 3.22 12 Chrysin 50 μΜ 75.11 6.05 13 Fisefin 100 μΜ »72.89 3.37 f4 eriodictyol 100 μΜ 69.62 5.68 15 6-Gingerol 100 μM 66.21 1.94 16 Isorhamnetin 75 μM 65.74 4.99 17 isoxperidin ( i soquerdtri η) 100 μΜ 61.80 15.60 18 Formononetin 50 μΜ 57.94 0.84 19 Morin 100 μΜ 51.00 4.55 20 (+)-Taxifolin 100 μΜ 50.47 10.38 21 isovitexin 100 μΜ 45.36 0.97 22 3-Phenylpropyl Acetate 100 μΜ 42.62 2.00 23 Oleanic acid 100 μΜ 41.13 11.52 24 Ursolic acid 100 μΜ 38.47 3.37 25 Puerarin 100 μM 33.30 17.52 26 Myrcene 100 μM " 29.85 4.31 27 trans-cinnamic acid 100 μM 26.10 3.57 28 Luteolin-7-Glucoside 100 μΜ 25.08 1.57 29 Liquiritin 100 μΜ 24.77 8.72

Table IV 21 200533364 Table IV. Inhibition of enzyme activity by 100 μM pure ingredients of traditional Chinese medicines (continued) Ranking pure ingredient name inhibitor concentration% inhibition SD 30 (+)-lemon essential oil (+) -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 Lignin Isoflavone Daidzein 25 μM 15.74 3.24 35 Poncirin β 100 μM 14.99 12.51 36 Quercitrin 100 μM 13.48 15.69 37 (-) Epicatechin-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 μΜ 3.51 1.99 41 (+) Catechin 100 μM -0.22 6.94 42 gallic acid 100 μM -0.97 16.40 43 Daidzin 25 μΜ ΛΛ6 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 Rutin) 100 μM -6.46 13.80 48 (+)-epicatechin (+)-epicatechin 100 μM -11.54 0.77 49 Naringin Narigin 100 μM -24.21 10.50 Table 4 (continued) 22 200533364 Table 5. Purified ingredients of traditional Chinese medicine Degree of inhibition of enzyme activity by 10 μΜ Ranking pure ingredient name Inhibitor concentration% inhibition SD-Ketoconazole 10 μΜ 80.11 0.71 1 Tamarixetin 10 μΜ 88.12 0.69 2 apigenin 25 μΜ 76.88 1.37 3 Genistein 10 μM 67.70 2.28 4 Isorhamnetin 10 μM. 61.53 3.57 5 Chrysin 10 μΜ 60.62 2.07 6 Wongonin 10 μΜ 51.31 1.43 7 Narigenin 10 μΜ 49.98-8 Quercetin 10 μΜ 44.80 2.37 9 Oleanic acid 10 μM 42.35 9.56 10 Puerarin 10 μM. 39.02 10.00 11 kaempferol 10 μΜ 38.29 15.43 12 luteolin 10 μΜ 37.89 14.42 13 ursolic acid 10 μΜ 37.46 3.31 14 isovitexin 10 μΜ 37.38 5.79 15 Genkwanin flavones 10 μM 37.37 3.64 16 (a-Naphthoflavone) 10 μM 37.27 7.06 17 Capillarisin 10 μM. 34.79 3.04 18 'Phloretin 10 μΜ 34.41 7.95 19 Epi-catechin (-)-Epicatechin 10 μΜ 33.75 13.74 20 (+)-Taxifolin 10 μΜ 31.16 8.11 21 Formononetin 10 μΜ 30.57 3.69 22 Isoglycoside Isoliquritigenin 10 μM 29.66 14.74 23 Hesperetin 10 μM 29.09. 2.10 24 eriodictyol 10 μΜ 28.65 15.29 25 6-Gingerol 10 μΜ 27.72 10.54 26 isoquercitrin 10 μΜ 27.02 17.78 27 Fisefm 10 μΜ 26.52 7.25 28 Quercitrin) 10 μΜ 21.10 15.81 29 Liquiritin 10 μΜ 18.35 1.97 Table 5 23 200533364 Table 5: Inhibition of Enzyme Activity by Pure Ingredients of Traditional Chinese Medicine 10 μΜ (Continued) Ranking Pure Ingredient Name Inhibitor Concentration% inhibition SD 30 Myrcene (/ 3 -Myrcene) 10 μΜ 16.60 6.31 31 Swertiamarin 10 μΜ 16.56 3.84 32 Poncirin 10 μΜ 16.34 10.77 33 protocatechuic acid 10 μΜ 16.22 L72 34 trans- Trans-cinnamic acid 10 μΜ 15.82 9.04 35 lignin isoflavone (Daidzein) 10 μΜ 13.45 4.49 36 Morin 10 μΜ 11.63 17.51 37 Embelin 10 μΜ 11.23 9.18 38 myricetin 10 μΜ 10.57 13.21 39 ( +)-Lemon essential oil (+)-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 6.68 332 44 (+)-epicatechin (+)- epicatechin 10 μΜ 6.30 3.72 45 Huang Dajie Baicalein 25 μΜ 5.06 8.64 46 Diosmin 10 μΜ 4.70 0.75 47 yS-Naphth flavone 10 μΜ 4.64 3.02 48 Homoorientin 10 μΜ 2.45 13.94 49 & quot Glycyrrhizin 10 μM 2.23 4.65 50 paeoniflorin-Luo-glucoside 10 μM 0.70 3.50 51 (Luteolin-7-Glucoside) 10 μM -0.32 5.2〇52 Daidzin 10 μΜ '-2.46 4.10 53 gallic acid 10 μΜ -2.47 10.16 54 Umbelliferone 10 μΜ -6.64 4.94 55 (+) Catechin (+) Catechin 10 μΜ -8.46 3.53 56 Epiatechin-Epicatechin 10 μM -8.61 5.95 57 Citrus ^ (Narigin) 10 μM -13.25 4.33 58 Rutin 10 μM -13.97 14.31

Table 5 (continued) 24 200533364 Table 6. Degree of inhibition of enzyme activity by 1 μΜ of pure ingredients of traditional Chinese medicines Ranking Pure ingredient name Inhibitor concentration% inhibition SD-Ketoconazole 1 μΜ 45.88. 3.13 1 genistein 8¾ Genistein 1 μM 49.60 1.37 2 Tamarixetin 1 μM 41.96 6.63 3 Puerarin 3-Phenylpropyl ϊ μΜ 38.15 0.57 4 Acetate) 1 μM 36.57 7.30 5 isovitretin (isovitexin) 1 μΜ 35.56 7.96 6 ursolic acid 1 μΜ 33.62 0.99 7 eriodictyol 1 μΜ 32.78 4.41 8 Genkwanin 1 μΜ 30.85 1.68 9 6-Gingerin (6- Gingerol) 1 μΜ 30.17 2.36 10 Wongonin 1 μΜ 28.82 1.41 11 trans-cinnamic acid 1 μΜ 26.92 4.26 12 Embelin 1 μΜ 24.71 6.18 13 Quercitrin 1 μΜ 24.19 .1.71 14 Laurel (/ 3 -Myrcene) 1 μΜ »24.06 3.08 15 Phloretin 1 μΜ 23.76 6.21 16 Formononetin 1 μΜ 23.33 0.43 Π apigenin 2.5 μΜ 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 CKKpicatechin ΙμΜ. 16.99 2.53 25 Oleanic acid 1 μM 16.79 L67 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 (+)-Lemon essential oil (+) -Limonene 1 μM 14.12 3.63

25 200533364 Table 6. Inhibition of Enzyme Activity by Pure Ingredients of Chinese Medicine 1 μM (continued) Ranking Pure Ingredient Name Inhibitor Concentration% Inhibition SD 30 Paeoniflorin Luteolin-7-0-glucose 1 μM 10.11 4.34 31 (Luteol iri-7 ~ Glucoside) 1 μM 937. 3.17 32 Pondrin 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 Isorhodin (Homoorientin) 1 μΜ 3.37 8.22 38 Hesperidin 1 μΜ ^ 2.57 2.07 39 Flavones (/ 3-Naphthoflavone) 1 μΜ 2.35 4.87 40 Yellow A * Baicalein 2.5 μΜ 1.76 2.53 41 Diosmin 1 μΜ 1, 51 0.82 42 Lignin Isoxanthin Daidzein 1 μM 1.35 1.54 43 Epiatechin-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 yellow (a-Naphthoflavone) 1 μM -0.05 6.26 48 (+)-Taxifolin 1 μM -1.29 8.16 49 Rutin 1 μM -2.59 12.71 50 Gallia Acid (gallic acid) 1 μM -3.05 5.18 51 (+) Catechin (+) Catechin 1 μM. : 3.05 0.78 52 · Myricetin 1 μΜ -3.19 16.64 53 Hesperetin ΙμΜ -3.58 11.11 54 baicalin 2.5 μΜ -5.36 6.97 55 (Umbelliferone) ΙμΜ- 7.17 3.59 56 Naringin Narigin Ordihydroguaiaretic 1 μM -11.48 2.10 57 acid 1 μM -16.06. 2.77 58 kaempferol ΙμΜ -2227 18.96

Table 6 (continued) 26 200533364... (Stat T-test). · The degree is relative to the value of ⑽ plus 丨 g calendar p has a significant difference, medium _ cited Gang, called 丨 _ three kinds of inhibitory concentration of the inhibitory effect obtained by screening, the name of the towel is shown in Figures 2, 3 and 4 Show. Detailed description of the preferred embodiment 1 + The above-mentioned experimental method depends on the effects of different concentrations of tamarinxetin on the liver microsomal enzyme rhyme tdbutamide. The prostitutes are ... tGlbutamide concentration is mM, Mi__ protein content is 0 5 mg with a reaction time of 7.5 minutes. The test wire shows the number HUCHE070Tamarixetin inhibitor, which has 90.2%, 88.1%, and 42.0% metabolic inhibition levels at three different concentrations: high, medium, and low (as shown in Table 7 and Figure 5).

Effective CYP450 isoenzyme modulator. Table VII. In vitro screening of Chinese herbal medicines HUCHE〇7〇The effect of different concentrations of Tamarixetin on liver microsomal enzyme metabolism tolbutamide (n = 3) 〇Concentration 4-〇Η (ng)% inhibition Control 368.5409 ± 35.3091 0.0000 1 μΜ 213.5696 ± 24.4309 41.9620 10Mm 43.10052 ± 2.5372 88.1204. 100 μM 35.49297 ± 1.5825 90.1803 Table 7 ---- j

Experimental study on the effect of Tamarixetin on oral absorption of the lipid-lowering drug fluvastatin in SD rats. Experimental group (Table 9) SD rat oral fluvastatin 1.5 mg / kg in combination with jjuche〇7〇 〇 Pitysin) 9.32 mg / kg (n = 5). The blood concentration data of the drug are shown in Table 9. The control group (Table 8) SD rat (rv = 7) was combined with a dissolution inhibitor solvent. The calculated kinetic parameters are listed in Table 10. The curve of blood concentration over time is shown in Figure 6. The kinetic parameters show that the area under the curve (areauncierthecurve (AUC)) has significant differences. The most ancient blood concentration (Cmax) experiment The control group was 141.4 ± β · 8 ng / m. The control group was 63 ″ ± 104 ng / ml. The concentration was approximately doubled. The drug clearance rate (c1t) and distribution volume (Vd) had significant differences. It indicates that the ability of liver enzymes to metabolize drugs has been significantly inhibited. Because of the exclusion phase 27 200533364 in the latter stage, there is no significant difference in the division constant (k), so it does not change the half-life of drug exclusion in the body (Ti /). Agent HUCHEOTO Tamarixetin does not blame for a long time _ No, but fluvastatin can still excrete P enzyme metabolism in the body according to the normal elimination process. Table VIII. SD rat blood concentration data of oral fluvastatin control group — °

Time ^ C_2 C " 3 C " 4 c-5 C-6 C-7 mean SB Concentration (ng / ml) CV〇 / 0 l〇2〇4〇6〇! 2〇24〇36〇48〇720 13 · 56 16.39 19.78 24.70 0.27 8.94 16.41 14.3 3 〇28.95 18.03 18.10 24.48 1.15 19.79 31.50 20.3 3.8 47.38 22.74 25.67 29.33 7.35 63.98 42.36 34.1 7q 69.48 24.74 34.71 41.24 7.57 99.41 44.17 45.9 \ χ 4 61.69 41.13 11.91 43.91 41.19 11.13 43.91 54.64 38.66 66.97 73.42 14.48 68.72 38.28 50.7 8 1 40.68 45.74 60.75 83.84 20.41 50.80 27.07 47 · 〇§ 〇37.37 57.30 45.05 54.92 21.57 37.68 24.92 39.8 5 2 22.45 37.37 25.63 39.30 21.75 18.47 15.40 25.8 35 10.5 δ. 10.67 11.68 21.1 〇11 · 47 22.33 21.04-10.74 6.33 8.58 13.4 2/7 Table 8 55.2 49.1 54.5 65.9 56.6 42.1 45.2 34.4 35.6 52.6 51.

Table 9.Experimental data of oral fluvastatin and HUCHE070 Tamarixetin (mt blood) Agricultural 28 200533364

Concentration (ng / ml)

Time (min) Sl 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 〇4 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 9735 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 2761 10.1 9.40 1961. Fluvastatin pharmacokinetic parameters in SD, SD rat control group and experimental group

Fluvastatin (A) PK parameter (unit) Fluvastatin (5) control, n = 7 with Tamarixetin? N = 5 / β

Cmax (ng / mL) 63.14 ± 10.36 141.40 ± 15.76 2.4 Tmax (hr) 4.7 ± 1.7. 4.2 ± 1.1 0.9 AUCt (hr * ng / mL) 710.57 ± 8L55 1389.20 ^ 166.14 * 2.0 AUCinf (hr * ng / mL) 949.86 ± 133.48 2281.00 ± 386.56 * 2.4 K (1 / hr) 0.074 soil 0.005 0.065 ± 0.009 0.9 Ti / 2 (hr) 9.7 ± 0.65 11.3i 1.33 1.2 Cl / F (mL / min / kg) 29.12 ± 4.05 12.33 Soil 2.10 ** 0.4 Vz / F (mL / kg) 24846.64 ± 4721.23 11163.54 ± 861.69 * 0A AUMCinf (hr * hr * ng / mL) 15156.0 ± 2864.6 42896.4 ± 12379.8 2.8 MRTinf (hr) 15.82 i 1.56 17.31 ± 221 1.1 PK = pharmacokinetic, Data = mean soil SE, *: p < 0.05, **: P < 0.01 Table 10 29 200533364 Detailed description of the preferred embodiment 2 The above experimental method is used to test the effects of different concentrations of HUCHE009 isoliquritigenin on liver microsomal enzyme metabolism tolbutamide. The reaction conditions are: tolbutamide concentration is 1 mM, Microsomes protein content It was 0.5 mg, and the reaction time was 7.5 minutes. The test results are shown in Table 11 and Figure 7. The number of HUCHE009 selenium grass 〇00 is 111 * 丨% 6! 1 丨 11) The inhibitor has a degree of metabolic inhibition of 95.46% at high concentrations, which can be regarded as _ effective CYP450 isoenzyme Regulators. Table 11: Effect of different concentrations of HUCHE009 isoliqUritigenin on the metabolism of tolbutamide in liver microsomal enzymes (n = 3) Concentration 4-〇Η (ng)% inhibition Control 1 μΜ 10 μΜ 100 μM 374.8785 ± 54.8521 0.0000 371.5965 ± 18.7272 0.8737 263.4592 ± 55.2455 29.6603 16.25213 ± 0.5544 95.4680 Table 11 Detailed description of Example 3 Use the above experimental method to test different concentrations of HUCHE037 Gin Isoflavone Genistein for liver microsomal enzyme metabolism The reaction conditions are: the tolbutamide concentration is 1 mM, the Microsomes protein content is 0.5 mg, and the reaction time is 7.5 minutes. The test results are shown in Table 12 and Figure 8. The numbered genistein isoflavone Gemstein inhibitors have 82.7%, 67.7%, and 6 ° / 〇 metabolic inhibition at three different concentrations: high, medium, and low, which can be regarded as effective. CYP450 isoenzyme modulator. Table 12. In vitro screening of traditional Chinese medicine cited HUCHE〇3 7 different effects of genistein ag Genistein at different concentrations on liver microsomal enzyme metabolism tolbutamide (n = 3) 30 200533364

Category 4-ΟΗ (ng)% inhibition Control 479.3314 ± 56.4829-0.0000 1 μΜ 241.2098 ± 6.5885 49.5979 10 μΜ 154.311 ± 10.9480 67.6979 100 μΜ 82.24342 ± 13.679 82.7088 Table 12 Although the present invention has been described by the above detailed description and better implementation Explained by way of example, the present invention = should be interpreted as being affected by; minus the ground 'the invention is covered by the present invention, #know when the skilled person from the technical disclosure of the invention specification disclosed in this case _ capacity and real silk consideration, There are many other different equivalent changes and decorations that can be made. Under the meaning of the invention of the rat and Lin Pianxian, all the relatively small changes and modifications made in the application for the special fiber circumference and the content of the county book according to the present invention should still fall within the scope of the invention patent. [Brief description of the figure] Figure 1 is a comparison chart of in vitro screening of Keto_zole affecting liver microsomal enzyme metabolism tdbutamide formation 44iydroxyt〇lbutamide. Figure-is a comparison chart of the top ten inhibitory enzyme metabolism activities of pure ingredients of lGGpM Chinese medicine. Figure 3 is a comparison chart of the top ten inhibitory enzyme metabolic activities of pure ingredients of 10 μM traditional Chinese medicine. Figure 4 is a comparison chart of the top ten of 1—Chinese herbal medicine pure ingredients inhibiting enzyme metabolism. Figure 5 is a comparison chart of the in vitro screening of the traditional Chinese medicine drug inhibitor Tamarixetin on liver microsomal enzyme metabolism tolbutamide to form 4-hydroxytolbutamide. Figure 6 shows the changes of blood concentration of fluvastatin in SD rat control group (n = 7) and experimental group (n = 2) over time. Figure 7 is a comparison chart of the in vitro screening of the traditional Chinese medicine isoliquriti horse enin on the effect of liver microsomal enzymes on tolbutamide to form 4-hydroxytolbutamide. 31 200533364 Figure 8 shows the comparison of in vitro screening of the traditional Chinese medicine drug inhibitor genistein (Genistein) on liver microsomes, enzyme metabolism, tolbutamide formation and 4-hydroxytolbiitamide. 32

Claims (1)

200533364 The scope of patent application: 1. An inhibitor of cytochrome P450 isoenzyme CYP2C9, wherein the inhibitor is selected from the group consisting of the following compounds: Tamarixetin, xanthophyllin Yellow (Formononetin), isoliquritigenin, Phloretin, luteolin, Quercitrin, quercetin, myricetin, Wongonin, Puerarin, Genistein, Nordihydroguaiaretic acid, Narigenin, Capillarisin, Chrysin, Fisefin, eriodictyol , 6-Gingerol 6-Gingerol), Isorhamnetin, isoquercitrin, Morin, (+)-Taxifolin, isovitexin, phenylpropionate acetate (3- Phenylpropyl Acetate), oleanolic acid, ursolic acid, β-Myrcene, cinnamic acid, lignan-7-0 • glucose bitter ( Luteolin-7-Glucoside >, Liquiritin, (+ ) Lemon Essential Oil ((+) Limonene), Homoorientin, Swertiamarin, Embelin, Lignin Isoflavone Daidzein, Pondrin, Epiatechin (-)-Epicatechin Ergosterol 〇2. If the cytochrome (Cytochrome) P450 isoenzyme CYP2C9 inhibitor of item 1 of the scope of patent application 'inhibitor is selected from the group consisting of the following compounds: Tamarixetin, Formononetin, isoliquritigenin, Phloretin, luteolin, Quercitrin, quercetin, myricin myricetin), Wongonin, Puemrin, Genistein, Nordihydroguaiaretic acid, Narigenin. 3. The inhibitor of cytocbrome P450 isoenzyme CYP2C9 'according to item 1 of the scope of patent application, wherein the inhibitor is Tamarixetin. 4. A pharmaceutical composition for increasing the bioavailability of a drug, comprising: (a) a pharmaceutically effective amount of a CYP2C9 inhibitor such as the one in the scope of patent application; and 33 200533364 (b) —pharmaceutically acceptable Accepted vehicle. 5. If the pharmaceutical composition according to item 4 of the patent application is applied, wherein the pharmaceutical composition is administered in combination with another pharmaceutically effective amount of a hypoglycemic drug tolbutamide: 6. If the pharmaceutical composition according to item 4 of the patent application, ° The pharmaceutical composition is administered in combination with another pharmacologically effective amount of a lipid-lowering drug, fluvastatin. 34