CN117562882A - Composition for selectively inhibiting activity of uridine diphosphate glucuronyltransferase 1A9 and application thereof - Google Patents

Abstract

The invention discloses a composition that selectively inhibits the activity of uridine diphosphate glucuronosyltransferase 1A9 and its application, and belongs to the technical field of biomedicine. The composition includes one or more of 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-shogaol or 10-shogaol. The composition provided by the invention has high affinity and strong inhibitory activity for UGT1A9, can selectively inhibit UGT1A9 activity, has almost no inhibition on other UGTs subtypes, and can be used for drug metabolism phenotype research, including identification of UGT1A9 Whether it participates in the metabolic reaction of specific endogenous or exogenous substances and its contribution rate. The composition is extracted from ginger and has high biological activity and good safety. It has good application prospects and can be used as a molecular tool for drug development and research.

Description

A group that selectively inhibits the activity of uridine diphosphate glucuronosyltransferase 1A9 Compounds and their applications

Technical field

The present invention relates to the field of biomedicine technology, and in particular to a composition that selectively inhibits the activity of uridine diphosphate glucuronosyltransferase 1A9 and its application.

Background technique

Uridine diphosphate glucuronosyltransferases (UGTs) are the most important phase II drug metabolizing enzymes in the body. They are involved in the metabolism and inactivation of a variety of clinical therapeutic drugs in the body and play an important role in controlling drug exposure in the body and drug therapeutic efficacy/toxicity. plays an important role. Uridine diphosphate glucuronosyltransferase 1A9 (UGT1A9) is mainly expressed in the metabolically highly active liver (∼8% of liver UGTs expressed) and kidney (the most abundant UGT isoform in the kidney, accounting for approximately 48.8% of renal UGTs expression) )middle. UGT1A9 plays a key role in the metabolism/inactivation of a large number of clinical drugs, such as propofol, mycophenolic acid mofetil, dapagliflozin, and edaravone, etc., and is also responsible for maintaining the homeostasis of endogenous signaling molecules in the body. , such as thyroid hormones, fatty acids and estrogen (Physiological Reviews. 2019.99, 1153-1222). At the same time, the expression and function of UGT1A9 are regulated by internal and external factors, such as age, gender, inhibition, induction, and genetic polymorphisms, leading to changes in the clearance of UGT1A9-metabolized drugs, therapeutic efficacy, toxic side effects, and disease sensitivity. The combination of UGT1A9 enzyme activity inhibitors and therapeutic drugs (such as mycophenolate mofetil) can reduce the glucuronidation metabolism of the drug, increase the drug's in vivo drug exposure, and achieve the expected therapeutic effect.

Since the amino acid sequences of each subtype in the UGTs family are highly homologous, their substrates usually overlap with each other, and there are few specific inhibitors for each subtype of enzymes. Currently, three selective inhibitors of UGT1A9 have been reported, namely niflufenamic acid, honokiol, and ginsenoside Rc. Niflufenamic acid is a commonly used clinical non-steroidal anti-inflammatory analgesic. Although niflufenamic acid has a high inhibitory selectivity for UGT1A9, which is 12.5 times that of other UGTs subtypes, its affinity for UGT1A9 is poor (inhibition kinetics). The constant K _i is 8.0 μM), and there are gastrointestinal adverse reactions. Honokiol is an active ingredient in the traditional Chinese medicine Magnolia officinalis bark and has a high affinity with UGT1A9 (K _i is 45nM), but its inhibitory selectivity for UGT1A9 is only about 10 times that of other UGTs subtypes. Ginsenoside Rc is a tetracyclic triterpenoid compound in the traditional Chinese medicine ginseng. Its inhibitory selectivity for UGT1A9 is high, 12.9 times that of other UGTs subtypes. However, its affinity to UGT1A9 is poor (K _i is 2.83 μM), and its chemical structure complex. Therefore, there is an urgent need to develop a specific inhibitor of UGT1A9 with high selectivity and good safety for the study of drug metabolism selectivity and catalytic molecular mechanism.

Ginger, the fresh rhizome of Zingiber officinale Rosc., is one of the most popular dietary supplements and Chinese herbal medicines used in the treatment of different types of diseases, such as vomiting, rhinitis, neurodegenerative diseases and cancer. It has a long history of medical use (Food&Function, 2021, 12, 519-542). Ginger has a variety of biological activities, including anti-inflammatory, anti-cancer, antioxidant, anti-cough, neuropharmacological and analgesic effects. Gingerol and shogaol are the main active ingredients of ginger, including 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol, and 10-shogaol, of which 6- The content of gingerol in ginger is as high as 16.08mg/g. It is worth noting that ginger is particularly beneficial to patients with autoimmune or inflammatory diseases due to its anti-inflammatory and antioxidant effects, such as ginger’s protective effect on systemic lupus erythematosus and antiphospholipid syndrome. However, the inhibitory effect of ginger and its chemical components on UGT1A9 has not been reported, and there are no reports of using ginger and its chemical components to study drug metabolism selectivity and catalytic molecular mechanism. Therefore, there is an urgent need to better study the potential inhibitory effect of ginger and its active ingredients on UGT1A9 through in vivo and in vitro experiments, as well as the study of the drug metabolism selectivity and catalytic molecular mechanism of ginger/active ingredients.

Contents of the invention

The purpose of the present invention is to provide a composition that selectively inhibits the activity of uridine diphosphate glucuronosyltransferase 1A9 and its application, so as to solve the problems existing in the above-mentioned prior art. The composition provided by the invention has high affinity and strong inhibitory activity for UGT1A9, and can selectively inhibit the activity of UGT1A9, and has almost no inhibition on other UGTs subtypes, which is about 50 times higher than the existing inhibitors. At the same time, This type of ingredient has high biological activity and safety.

In order to achieve the above objects, the present invention provides the following solutions:

The invention provides a composition that selectively inhibits the activity of uridine diphosphate glucuronosyltransferase 1A9. The composition includes 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, Two or more of 10-gingerol or 10-shogaol.

The present invention also provides the use of the composition in inhibiting the activity of uridine diphosphate glucuronosyltransferase 1A9.

The present invention also provides the application of the composition in studying the metabolic phenotype of uridine diphosphate glucuronosyltransferase in vitro.

The present invention also provides the application of the composition in identifying metabolic reactions of specific endogenous signaling molecules or exogenous drugs mediated by uridine diphosphate glucuronosyltransferase 1A9.

Further, the specific endogenous signaling molecules include thyroid hormones, fatty acids and estrogen; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoride Prazine.

The present invention also provides the application of the composition in identifying the contribution rate in the metabolic reaction of specific endogenous substances or exogenous substances mediated by uridine diphosphate glucuronosyltransferase 1A9.

Further, the specific endogenous signaling molecules include thyroid hormones, fatty acids and estrogen; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoride Prazine.

The present invention also provides the use of the composition in inhibiting the metabolic reaction of endogenous signaling molecules or exogenous drugs mediated by uridine diphosphate glucuronosyltransferase 1A9 in vitro.

Further, the specific endogenous signaling molecules include thyroid hormones, fatty acids and estrogen; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoride Prazine.

The invention discloses the following technical effects:

The present invention has experimentally verified that 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol in the Chinese herbal ginger extract have higher affinity for UGT1A9 It has strong inhibitory activity and can selectively inhibit the activity of UGT1A9. It has almost no inhibition on other UGTs subtypes, which is about 50 times higher than the existing inhibitors. At the same time, this type of ingredient has high biological activity and safety. , can be used as a selective inhibitor of UGT1A9.

Through in vitro activity measurement, the present invention found that the IC ₅₀ and K _i of the above-mentioned selective inhibitor in inhibiting the UGT1A9-mediated propofol glucuronidation reaction were respectively between 0.05-30.9 and 0.07-19.31 μM. The IC ₅₀ and K _i for inhibiting the glucuronidation reaction of mycophenolate mofetil mediated by uridine diphosphate glucuronosyltransferase 1A9 are between 0.05~32.0 and 0.032~8.79μM respectively. It can be used to inhibit uridine diphosphate in vitro. Metabolic reactions of endogenous or exogenous substances mediated by phosphoglucuronosyltransferase 1A9.

The present invention investigates the recombinant uridine diphosphate glucuronosyltransferase single enzyme and the liver microsome incubation system. Through high-throughput screening experiments, spectrum-effect binding tracking experiments, inhibition kinetic reactions and inhibition selectivity, It was proved that the above-mentioned selective inhibitors can efficiently and selectively inhibit the UGT1A9-mediated glucuronidation reaction.

The selective inhibitor provided by the invention is extracted from ginger, is safe and non-toxic, has diverse pharmacological research activities, has good application prospects, and can be used as a molecular tool for drug development and research.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 shows the inhibitory effect of one hundred Chinese herbal extracts on UGT1A9 activity;

Figure 2 shows the correlation analysis of the detection activity of the fluorescent probe DDAO and the drug probe propofol;

Figure 3 is the inhibition curve of ginger extract on UGT1A9;

Figure 4 shows the chemical fingerprint of ginger (A), the spectrum of the inhibitory effect of ginger fractions on UGT1A9 (B) and the chemical structural formula after identification of the active substance;

Figure 5 shows that ginger series compounds 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-shogaol and 10-shogaol inhibit UGT1A9-mediated DDAO-O-glucosaldehyde Acidification, propofol-O-glucuronidation and mycophenolic acid-O-glucuronidation metabolism IC ₅₀ spectrum and K _i spectrum, where (a) represents 6-gingerol, 6-gingerene Phenol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol inhibit UGT1A9-mediated DDAO-O-glucuronidation metabolism IC ₅₀ spectrum, (b) represents 6-gingerol , 6-gingerol, 8-gingerol, 8-gingerol, 10-gingerol and 10-shogaol inhibiting propofol-O-glucuronidation metabolism IC ₅₀ spectrum, (c) represents 6- Gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol inhibit the metabolism of mycophenolic acid-O-glucuronidation metabolism IC ₅₀ spectrum, ( d) represents the metabolic kinetic diagram of 6-gingerol inhibiting mycophenolic acid-O-glucuronidation (e) represents the metabolic kinetic diagram of 6-gingerol inhibiting mycophenolic acid-O-glucuronidation. , (f) represents the kinetic diagram of the inhibition of mycophenolic acid-O-glucuronidation by 8-gingerol, (g) represents the inhibition of the metabolism of mycophenolic acid-O-glucuronidation by 8-gingerol. Kinetic diagram, (h) represents the metabolic kinetic diagram of 10-gingerol inhibiting mycophenolic acid-O-glucuronidation, (i) represents the inhibition of mycophenolic acid-O-glucose by 10-gingerol. Aldolation metabolic kinetics diagram

Figure 6 shows the selective preliminary screening of the inhibitory effects of 6-shogaol, 8-shogaol and 10-shogaol on the activity of 12 UGTs subtypes, as well as the inhibitory IC ₅₀ curves of UGT1A7, UGT1A8 and UGT1A9 activities; Among them, (a) is the selective preliminary screening of the inhibitory effect of 6-shogaol on the activity of 12 UGTs subtypes, (b) is the inhibitory IC ₅₀ curve of 6-shogaol on the activities of UGT1A7, UGT1A8 and UGT1A9, ( c) is the selective preliminary screening of the inhibitory effect of 8-shogaol on the activity of 12 UGTs subtypes, (d) is the IC ₅₀ curve of the inhibitory effect of 8-shogaol on the activities of UGT1A7, UGT1A8 and UGT1A9, (e) is Selective preliminary screening of the inhibitory effect of 10-shogaol on the activity of 12 UGTs subtypes, (f) is the IC ₅₀ curve of the inhibitory effect of 10-shogaol on the activities of UGT1A7, UGT1A8 and UGT1A9;

Figure 7 shows the metabolic spectrum of ginger extract series compounds inhibiting mycophenolic acid at the HepRG cell level (a); the combined cytotoxicity of ginger monomer components and mycophenolic acid at the HepRG cell level (b); ginger extract Effects of a series of compounds on the concentration of mycophenolic acid and mycophenolic acid glucuronide in the supernatant of HepRG cells (c); during the combined use of mycophenolic acid and 6-gingerol The metabolism of mycophenolic acid (d); the formation of mycophenolic acid glucuronide during the combined use of mycophenolic acid and 6-gingerol (e).

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range, and any other stated value or value intermediate within a stated range, is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples of the present invention are exemplary only.

The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to.

The inventors have carried out research on the inhibition of UGT1A9 by more than a hundred kinds of Chinese herbal medicine extracts and their main chemical components (Figure 1), in order to find safe and efficient UGT1A9 inhibitors from Chinese herbal medicines with the same origin as medicine and food, for drug metabolism selectivity and catalysis. Study of molecular mechanisms. Studies have found that Chinese herbal ginger extract and its monomer components can strongly inhibit UGT1A9 activity. Among them, the Chinese herbal ginger extract contains gingerol and shogaol phenolic compounds containing aliphatic side chain structures, including 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, and 10-gingerol. and 10-shogaol, their structural formulas are as follows:

In the following examples, the above-mentioned Chinese herbal ginger extract is collectively referred to as ginger extract H7. The extraction method is: add 50g of ginger to 500 mL of 95% ethanol, seal with plastic wrap, and soak at room temperature for 4 hours. After the medicinal materials are completely soaked, ultrasonic for 2 hours, filter to remove the medicinal residue, spin-evaporate in a large bottle until thick or dry, directly scrape off and transfer to a white plastic bottle. After cooling at room temperature, tighten the bottle cap and mark it at -20°C for storage.

Example 1 Determination of inhibitory activity of ginger extract on human UGT1A9

Using DDAO glucuronidation metabolism as a probe reaction, using an in vitro UGTs enzyme incubation system and a multi-functional enzyme plate assay method, high-throughput screening of medicinal materials with inhibitory activity against human UGT1A9 was carried out. The specific experimental process is as follows:

(1) In vitro UGT metabolism reaction system, including Tris-Hcl buffer (50mM) at pH 7.4, MgCl ₂ solution (5mM), mixed human liver microsomes (0.1mg/mL), DDAO final concentration is 3.0μM, ginger extract Compound H7 (10 μg/mL), pre-incubated with shaking at 37°C for 5 minutes;

(2) Add the initiating factor UDPGA (final concentration 2.5mM) to the reaction system to start the reaction;

(3) After 30 minutes, add 200 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

(4) Under the conditions of 4°C and 20,000×g, after high-speed centrifugation for 10 minutes, take the supernatant and conduct fluorescence detection (Ex=465nm, Em=608nm);

The results are shown in Figure 2-3. Ginger extract H7 showed strong inhibitory activity against UGT1A9. The residual activity of UGT1A9 in this reaction system was 21.4%, and the IC ₅₀ value was 0.17 μg/mL.

Example 2 Tracking of UGT1A9 inhibitors in ginger under activity guidance

With the help of high-performance liquid chromatography, a fingerprint spectrum of ginger extract was established, the eluates of the spectrum were collected, and DDAO glucuronidation metabolism was used as the probe reaction to determine the inhibitory activity of all eluents against UGT1A9, and the fingerprints and inhibition spectra were compared. Figure, through comparison with the retention time, ultraviolet spectrum, and high-resolution mass spectrum of the standard product, the main monomer components of ginger that have an inhibitory effect on UGT1A9 are identified. The specific experimental process is as follows:

(1) Establishment of fingerprint spectrum: With the help of Waters high performance liquid chromatography and Acquity UPLC HSS T3 C18 (2.1×100mm, 1.8μm) chromatographic column, the fingerprint spectrum of ginger extract was established. The mobile phase composition is acetonitrile (A) and water (B), the flow rate is 0.8mL/min, and the detection wavelength is 285nm. The effluent was collected directly into a black 96-well plate every 60 seconds, dried under vacuum, and used for UGT1A9 inhibition experiments.

(2) Establish an inhibition map: Use the UGT1A9-specific fluorescent probe substrate DDAO to evaluate the inhibitory effect of monomeric compounds in ginger on UGT1A9 activity. The incubation system contained HLM (0.1mg/mL), 50mM Tris-HCl (pH 7.4), 5mMgCl ₂ , 3.0μM DDAO, ginger chromatography elution fraction, and was preincubated at 37°C for 5 minutes. Add 10 μLUDPGA to the reaction system to start the reaction. After 30 minutes, add 200 μL of ice-cold acetonitrile and shake vigorously to terminate the reaction. After high-speed centrifugation for 10 minutes at 4°C and 20,000×g, take the supernatant and perform fluorescence detection (Ex=465nm, Em=608nm).

The results are shown in Figure 4. There are 6 components in ginger extract H7 that show strong inhibitory effects on UGT1A9. By comparison with the retention time, ultraviolet spectrum, and high-resolution mass spectrum of the standard, they were identified as 6-gingerol. , 6-shogaol, 8-shogaol, 8-shogaol, 10-shogaol and 10-shogaol.

Example 3 In vitro inhibition test to quantitatively evaluate the inhibitory ability of ginger series compounds on UGT1A9

Using mycophenolic acid, propofol and DDAO glucuronidation metabolism as probe reactions, using an in vitro UGTs enzyme incubation system and a multi-functional enzyme plate assay method, high-throughput evaluation of different concentrations of ginger extract monomers was carried out IC ₅₀ and K _i values of ingredients (6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol, 10-shogaol) inhibiting UGT1A9, the specific experimental process is as follows :

(1) In vitro UGT metabolism reaction system, including pH 7.4 Tris-Hcl buffer (50mM), MgCl ₂ solution (5mM), mixed human liver microsomes (0.1mg/mL), mycophenolic acid mofetil/isopropyl The final concentrations of phenol/DDAO were 30/100/1.0μM respectively. Different concentrations of ginger extract monomer components (6-gingerol 2-80μM, 8-gingerol 0.5-15μM, 10-gingerol 0.5-20μM, 6-gingerol) Shogaol 10-500nM, 8-shogaol 5-200nM, 10-shogaol 10-800nM) were pre-incubated with shaking at 37°C for 3 minutes;

(2) Add the initiating factor UDPGA (final concentration 2.5mM) to the reaction system to start the reaction;

(3) After 30 minutes, add 200 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

(4) Under the conditions of 4°C and 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant and perform fluorescence detection and mass spectrometry detection. The maximum excitation and emission wavelengths of DDAO-glucuronide are 465nm and 608nm respectively, and Moticumab Phenolic acid glucuronide was detected in the negative ion mode, and the precursor ion was 495.0, and the product ion was 319.2. Propofol glucuronide was detected in the negative ion mode, and the precursor ion was 353.0, and the product ion was 177.0.

The results are shown in Figure 5 and Table 1. The six monomeric components of ginger showed dose-dependent inhibition of UGT1A9-mediated mycophenolic acid mofetil, propofol and DDAO, 6-gingerol and 6-gingerene. The IC ₅₀ values of mycophenolic acid-O-glucuronidation of UGT1A9 inhibition by phenol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol are 32.0, 0.13, 3.94, 0.05, 4.18 respectively. and 0.38 μM, with K _i of 8.79, 0.032, 2.00, 0.034, 1.46, and 0.085 μM, respectively.

Table 1 Ginger monomer components inhibit UGT1A9-mediated DDAO-O-glucuronidation, propofol-O-glucuronidation and mycophenolic acid-O-glucuronidation metabolism IC ₅₀ and K _i values

Example 4 In vitro determination of UGT1A9 enzyme activity inhibition selectivity

Using 4-methylumbelliferone (4-MU) and trifluoperazine glucuronidation metabolism as probe reactions, with the help of in vitro UGTs enzyme incubation system, high performance liquid chromatography tandem mass spectrometry was used to evaluate 6-gingerol. The selectivity of 6-shogaol, 8-shogaol, 8-shogaol, 10-shogaol and 10-shogaol in inhibiting UGT1A9. The specific experimental process is as follows:

(1) In vitro UGT metabolism reaction system, including pH 7.4 Tris-Hcl buffer (50mM), MgCl ₂ solution (5mM), recombinant UGTs single enzyme, 4-MU/trifluoperazine, and different concentrations of raw extracts Monomer components, pre-incubated with shaking at 37°C for 3 minutes; UGT1A1(1A1), UGT1A3(1A3), UGT1A6(1A6), UGT1A7(1A7), UGT1A9(1A8), UGT1A9(1A9), UGT1A10(1A10), UGT2B4 (2B4), UGT2B7(2B7), UGT2B15(2B15) and UGT2B17(2B17) protein concentrations were 37.5, 15, 7.5, 15, 7.5, 15, 15, 7.5, 15, 60 and 15 μg/mL, respectively, 4-MU The substrate concentrations are 110, 1200, 110, 30, 750, 30, 30, 1000, 350, 250 and 2000 μM respectively; the UGT1A4 protein concentration is 30 μg/mL, and the final concentration in the trifluoperazine substrate system is 50 μM;

(2) Add the initiating factor UDPGA (final concentration 2.5mM) to the reaction system to start the reaction;

(3) After 150min (UGT1A1, 1A3, 1A10, 2B4, 2B7, 2B15 and 2B17) or 60min (UGT1A4, 1A6, 1A7, 1A8 and 1A9), add 200 μL of ice-cold acetonitrile and shake vigorously to terminate the reaction;

(4) After high-speed centrifugation for 20 minutes at 4°C and 20,000×g, take the supernatant and perform mass spectrometry measurement;

The results are shown in Figure 6 and Table 2. 6-shogaol, 8-shogaol, and 10-shogaol showed the strongest inhibitory effect on the enzyme activity of recombinant human UGT1A9, and hardly inhibited the activity of other UGTs subtypes. Or slight inhibition, the inhibition selectivity is between 43.8-656.5 times.

Table 26 - IC ₅₀ values of shogaol, 8-shogaol and 10-shogaol for inhibiting UGT1A7, 1A8 and 1A9

Example 5 HepRG cell level determination of the effect of ginger series compounds on the metabolism of mycophenolic acid

UGT1A9 overexpressing HepRG cells were established, and the content of mycophenolic acid metabolite mycophenolic acid glucuronide in the supernatant of HepRG cells at different time points was determined with the help of high-performance liquid chromatography tandem mass spectrometry, and the effect of ginger monomer components on The impact of mycophenolic acid metabolism, the specific operation process is as follows:

(1) Establishment of mycophenolic acid metabolism system at the HepRG cell level: HepRG cells were cultured in a cell culture incubator (37°C, 5% CO ₂ , 95% humidity), and the culture medium contained 10% fetal bovine serum and 1 1640 medium with % antibody (100 units/mL penicillin, 100 μg/mL streptomycin), the cells were seeded in a 24-well cell culture plate, and after adherent culture for 1-2 days, mycophenolic acid mofetil (5 μM) and different After incubating the ginger extract monomer components at a concentration (0-20 μM) for 12 hours, take the cell supernatant, centrifuge it at high speed for 20 minutes, take the supernatant, and perform mass spectrometry measurement;

(2) HepRG cells were seeded in a 96-well cell culture plate, and mycophenolic acid mofetil (5 μM) and different concentrations of ginger extract monomer components (0-20 μM) were added. After incubation for 12 hours, the culture medium was aspirated. Add 100 μL CCK-8 solution to each well, place it in the incubator away from light for 50 minutes, and detect the OD value at 450 nm with a microplate reader;

(3) HepRG cells were seeded in a 24-well cell culture plate, and mycophenolic acid mofetil (5 μM) and different ginger extract monomer components (20 μM) were added. After incubation for 12 hours, the cell supernatant was taken and centrifuged at high speed for 20 minutes. Afterwards, take the supernatant and perform mass spectrometry to determine the changes in the content of mycophenolic acid and mycophenolic acid glucuronide;

(4) HepRG cells were seeded in a 24-well cell culture plate, mycophenolic acid mofetil (5 μM) and 6-shogaol (20 μM) were added, and incubated for 0, 1, 2, 4, 6, 8, 10 and After 12 hours, take the cell supernatant, centrifuge it at high speed for 20 minutes, take the supernatant, and conduct mass spectrometry to determine the metabolism of mycophenolic acid and the production of mycophenolic acid glucuronide.

The results are shown in Figure 7 and Table 3. 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol can inhibit the growth of HepRG living cells. Metabolism of mycophenolic acid, Table 3 shows that the IC ₅₀ are 13.14, 5.75, 9.69, 4.79, 13.6 and 19.6μM respectively. In UGT1A9 overexpressing HepRG cells, all six monomeric components of ginger can significantly inhibit the metabolism of mycophenolic acid in HepRG cells and increase the concentration of mycophenolic acid in the cell supernatant by about 10 times. . At the same time, 6-shogaol can extend the metabolic half-life of mycophenolic acid from 2.93h to 22.9h in UGT1A9-overexpressing HepRG cells.

Table 3 IC ₅₀ values of ginger extract series compounds inhibiting mycophenolic acid metabolism at the HepRG cell level

The above-described embodiments only describe the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.

Claims (9)

1. A composition for selectively inhibiting the activity of uridine diphosphate glucuronyltransferase 1A9, said composition comprising two or more of 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol, or 10-shogaol.

2. Use of a composition according to claim 1 for inhibiting the activity of uridine diphosphate glucuronosyl transferase 1 A9.

3. Use of the composition of claim 1 for studying the metabolic phenotype of uridine diphosphate glucuronosyl transferase in vitro.

4. Use of the composition of claim 1 for identifying a specific endogenous signaling molecule or an exogenous drug metabolic response mediated by uridine diphosphate glucuronyltransferase 1 A9.

5. The use according to claim 4, wherein the specific endogenous signal molecules comprise thyroid hormones, fatty acids and estrogens; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoperazine.

6. Use of a composition according to claim 1 for identifying the contribution rate of uridine diphosphate glucuronyltransferase 1A9 mediated by a specific endogenous signal molecule or an exogenous drug metabolic response.

7. The use according to claim 6, wherein the specific endogenous signal molecules comprise thyroid hormones, fatty acids and estrogens; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoperazine.

8. Use of a composition according to claim 1 for inhibiting in vitro a uridine diphosphate glucuronosyl transferase 1A9 mediated metabolic reaction of endogenous signaling molecules or exogenous drugs.

9. The use according to claim 8, wherein the specific endogenous signal molecules comprise thyroid hormones, fatty acids and estrogens; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoperazine.