CN117562882A - Composition for selectively inhibiting activity of uridine diphosphate glucuronyltransferase 1A9 and application thereof - Google Patents
Composition for selectively inhibiting activity of uridine diphosphate glucuronyltransferase 1A9 and application thereof Download PDFInfo
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- CN117562882A CN117562882A CN202311542628.XA CN202311542628A CN117562882A CN 117562882 A CN117562882 A CN 117562882A CN 202311542628 A CN202311542628 A CN 202311542628A CN 117562882 A CN117562882 A CN 117562882A
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- gingerol
- shogaol
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Classifications
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Abstract
The invention discloses a composition for selectively inhibiting the activity of uridine diphosphate glucuronic acid transferase 1A9 and application thereof, belonging to the technical field of biological medicines. The composition includes one or more of 6-gingerol, 8-gingerol, 10-gingerol, or 10-gingerol. The composition provided by the invention has higher affinity and stronger inhibition activity on UGT1A9, can selectively inhibit UGT1A9 activity, hardly inhibits other UGT subtypes, and can be used for drug metabolism phenotype research, including identification of whether UGT1A9 participates in metabolic reaction of specific endogenous or exogenous substances and contribution rate thereof. The composition is extracted from ginger, has high bioactivity and good safety, has good application prospect, and can be used as a molecular tool for drug development and research.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a composition for selectively inhibiting the activity of uridine diphosphate glucuronosyl transferase 1A9 and application thereof.
Background
Uridine diphosphate glucuronic acid transferase (UGTs) is the most important phase ii drug metabolizing enzyme in the body, and participates in metabolism and inactivation of various clinical therapeutic drugs in the body, playing an important role in controlling in vivo drug exposure and drug therapeutic efficacy/toxicity. Uridine diphosphate glucuronyltransferase 1A9 (UGT 1A 9) is mainly expressed in the liver (about 8% of liver UGT expression) with high metabolism and the kidney (the UGT subtype with the highest content in the kidney, which accounts for about 48.8% of kidney UGT expression). UGT1A9 plays a key role in the metabolism/inactivation of a large number of clinical drugs, such as propofol, mycophenolic acid, dapagliflozin, edaravone, etc., while being responsible for maintaining homeostasis of endogenous signal molecules of the body, such as thyroid hormones, fatty acids, and estrogens, etc. (Physiological reviews.2019.99, 1153-1222). Meanwhile, the expression and function of UGT1A9 are regulated by internal and external factors such as age, sex, inhibition, induction and genetic polymorphism, resulting in clearance of UGT1A9 metabolic drugs, changes in therapeutic efficacy, toxic side effects and disease sensitivity. The UGT1A9 enzyme activity inhibitor and a therapeutic drug (such as mycophenolate mofetil) are used together, so that the glucuronidation metabolism of the drug can be reduced, the in-vivo drug exposure of the drug can be increased, and the expected therapeutic effect can be achieved.
Because of the high homology of amino acid sequences of each subtype in the UGTs family, their substrates often overlap each other, and each subtype enzyme has very few specific inhibitors. Currently, 3 UGT1A9 selective inhibitors are reported, namely niflumic acid, magnolol and ginsenoside Rc. Niflufenamic acid is a clinically common nonsteroidal anti-inflammatory analgesic, and although it has a higher selectivity of inhibition to UGT1A9 than 12.5 times that of other UGT subtypes, it has a poor affinity to UGT1A9 (inhibition kinetic constant K i 8.0 μm) and is storedAdverse reactions in the gastrointestinal tract. Magnolol is an effective component in cortex Magnoliae officinalis, and has high affinity with UGT1A9 (K) i 45 nM), but its inhibition selectivity for UGT1A9 is only about 10-fold that of other UGTs subtypes. Ginsenoside Rc is tetracyclic triterpene compound in traditional Chinese medicine Ginseng radix, has high inhibition selectivity to UGT1A9, is 12.9 times of other UGT subtypes, but has poor affinity with UGT1A9 (K i 2.83 μm) and is complex in chemical structure. Therefore, development of a specific inhibitor of UGT1A9 with high selectivity and good safety is urgently needed for research of drug metabolism selectivity and catalytic molecular mechanism.
Ginger is a fresh rhizome of ginger (Zingiber officinale rosc) belonging to the genus Zingiber of the family zingiberaceae, is one of the most popular dietary supplements and herbal medicines, and has a long history of medical use in the treatment of different types of diseases, such as emesis, rhinitis, neurodegenerative diseases, cancer, etc. (Food & Function,2021,12,519-542). Ginger has various biological activities including anti-inflammatory, anticancer, 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, 10-shogaol, wherein the content of 6-gingerol in ginger is up to 16.08mg/g. Notably, ginger is particularly beneficial to patients with autoimmune or inflammatory diseases due to its anti-inflammatory and antioxidant effects, such as the protective effect of ginger against systemic lupus erythematosus and antiphospholipid syndrome. However, the inhibition of UGT1A9 by ginger and its chemical components has not been reported, nor has it been reported that ginger and its chemical components are utilized for research of drug metabolic selectivity and catalytic molecular mechanism. Therefore, there is an urgent need to better study the potential inhibition of UGT1A9 by ginger and its active ingredients through in vitro and in vivo experiments, and the study of the drug metabolism selectivity and catalytic molecular mechanism of ginger/active ingredients.
Disclosure of Invention
The invention aims to provide a composition for selectively inhibiting the activity of uridine diphosphate glucuronosyl transferase 1A9 and application thereof, so as to solve the problems of the prior art. The composition provided by the invention has higher affinity and stronger inhibition activity on UGT1A9, can selectively inhibit UGT1A9 activity, hardly inhibits other UGT subtypes, improves the activity by about 50 times compared with the existing inhibitors, and has higher biological activity and safety.
In order to achieve the above object, the present invention provides the following solutions:
the present invention provides 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.
The invention also provides application of the composition in inhibiting the activity of uridine diphosphate glucuronosyl transferase 1A 9.
The invention also provides application of the composition in researching the metabolic phenotype of the uridine diphosphate glucuronosyl transferase in vitro.
The invention also provides application of the composition in identifying a specific endogenous signal molecule mediated by uridine diphosphate glucuronyltransferase 1A9 or a metabolic reaction of an exogenous drug.
Further, the specific endogenous signaling molecules include thyroid hormones, fatty acids, and estrogens; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoperazine.
The invention also provides application of the composition in identifying the contribution rate of uridine diphosphate glucuronyltransferase 1A9 mediated metabolic reaction of specific endogenous substances or exogenous substances.
Further, the specific endogenous signaling molecules include thyroid hormones, fatty acids, and estrogens; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoperazine.
The invention also provides application of the composition in inhibiting the metabolic reaction of endogenous signal molecules or exogenous drugs mediated by uridine diphosphate glucuronosyltransferase 1A9 in vitro.
Further, the specific endogenous signaling molecules include thyroid hormones, fatty acids, and estrogens; the exogenous drugs include mycophenolic acid, propofol, DDAO, 4-methylumbelliferone and trifluoperazine.
The invention discloses the following technical effects:
according to the invention, experiments prove that the 6-gingerol, the 8-gingerol, the 10-gingerol and the 10-gingerol in the Chinese herbal medicine ginger extract have higher affinity and stronger inhibition activity on UGT1A9, can selectively inhibit UGT1A9 activity, hardly inhibit other UGT subtypes, and can improve the activity by about 50 times compared with the existing inhibitors at present, and meanwhile, the components have higher biological activity and safety and can be used as selective inhibitors of UGT1A 9.
The invention discovers that the selective inhibitor inhibits the IC of the UGT1A9 mediated glucuronidation reaction of the propofol through in vitro activity assay 50 And K i Between 0.05 and 30.9 and 0.07 and 19.31 μm respectively. IC for inhibiting glucuronide diphosphate glucuronidation reaction mediated by mycophenolic acid glucuronidation 1A9 50 And K i Can be used for inhibiting the metabolism reaction of endogenous or exogenous substances mediated by uridine diphosphate glucuronosyltransferase 1A9 in vitro between 0.05 and 32.0 and 0.032 and 8.79 mu M respectively.
The invention is examined from a recombinant uridine diphosphate glucuronyl transferase single enzyme and liver microsome incubation system, and the evidence of high throughput screening experiments, spectrum effect combination tracking experiments, inhibition of dynamic reaction and inhibition of selectivity proves that the selective inhibitor can efficiently and selectively inhibit the glucuronidation reaction mediated by UGT1A 9.
The selective inhibitor provided by the invention is extracted from ginger, is safe and nontoxic, has various pharmacological research activities, has good application prospect, and can be used as a molecular tool for drug development and research.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the inhibition of UGT1A9 activity by one hundred Chinese herbal extracts;
FIG. 2 is a correlation analysis of fluorescence probe DDAO and drug probe propofol detection activity;
FIG. 3 is a graph of ginger extract versus UGT1A9 inhibition;
FIG. 4 is a chemical structural formula of the ginger chemical fingerprint (A), the ginger fraction after UGT1A9 inhibition effect spectrum (B) and active substance identification;
FIG. 5 inhibition of UGT1A 9-mediated DDAO-O-glucuronidation, isopropoxy-O-glucuronidation and mycophenolic acid-morpholino acid metabolism IC for ginger series compounds 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol 50 Spectrogram and K i Spectrogram, wherein (a) represents 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol inhibiting UGT1A 9-mediated DDAO-O-glucuronidation metabolism IC 50 The spectrum (b) represents 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol for inhibiting the isopropylphenol-O-glucuronidation metabolism IC 50 Spectrogram, (c) represents 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol inhibiting mycophenolic acid-O-glucuronidation metabolism IC of mycophenolate mofetil 50 Spectrogram, (d) represents 6-gingerol-inhibited mycophenolic acid-O-glucuronidation metabolic dynamics map (e) represents 6-gingerol-inhibited mycophenolic acid-O-glucuronidation metabolic dynamics map, (f) represents 8-gingerol-inhibited mycophenolic acid-O-glucuronidation metabolic dynamics map, (g) represents 8-gingerol-inhibited mycophenolic acid-O-glucuronidation metabolic dynamics map, (h) represents 10-gingerol-inhibited mycophenolic acid-O-glucuronidation metabolic dynamics map, (i) represents 10-gingerol-inhibited mycophenolic acid-O-glucuronidation metabolic dynamics map
FIG. 6 shows 6-shogaol, 8-shogaol and 10-shogaol vs. 12 UGTs subtype activitiesSelective prescreening of inhibitory effect on UGT1A7, UGT1A8 and UGT1A9 Activity and inhibition IC 50 A curve; wherein (a) is a selective primary screen for the inhibitory effect of 6-shogaol on the activity of 12 UGT subtypes and (b) is an IC for the inhibition of the activity of 6-shogaol on UGT1A7, UGT1A8 and UGT1A9 50 Curve, (c) is a selective initial screen for inhibition of 12 UGT subtype activities by 8-shogaol, (d) is an inhibition IC of UGT1A7, UGT1A8 and UGT1A9 activities by 8-shogaol 50 Curve, (e) is a selective initial screen for inhibition of 12 UGT subtype activities by 10-shogaol, (f) is an inhibition IC of UGT1A7, UGT1A8 and UGT1A9 activities by 10-shogaol 50 A curve;
FIG. 7 is a graph of mycophenolic acid metabolism of a ginger extract series of compounds inhibiting mycophenolic acid at the HepRG cell level (a); hepRG cell level ginger monomer component combined cytotoxicity with mycophenolic acid of mycophenolate mofetil (b); effect of ginger extract series compounds on concentrations of mycophenolic acid and mycophenolic acid glucuronide in HepRG cell supernatants (c); a metabolic rule (d) of mycophenolic acid in the course of mycophenolic acid and 6-shogaol combination; rules of formation of mycophenolic acid glucuronide in mycophenolic acid and 6-shogaol are (e) in the course of combination.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 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 present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The inventor develops inhibition researches (figure 1) of more than hundred Chinese herbal medicine extracts and main chemical components thereof on UGT1A9, so as to search safe and efficient UGT1A9 inhibitors from Chinese herbal medicines with homology of medicine and food, and the inhibitors are used for researching medicine metabolism selectivity and catalytic molecular mechanism. Researches show that the Chinese herbal medicine ginger extract and the monomer components thereof can effectively inhibit UGT1A9 activity. The Chinese herbal medicine ginger extract is gingerol and shogaol compounds containing aliphatic side chain structures, and comprises 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol, wherein the structural formulas of the gingerol and shogaol compounds are shown as follows:
in the following examples, the above Chinese herbal ginger extract is collectively called ginger extract H7, and the extraction method is as follows: 50g of ginger is added with 500mL of 95% ethanol, sealed by a preservative film and soaked for 4 hours at room temperature. After the medicinal materials are completely soaked, carrying out ultrasonic treatment for 2 hours, filtering to remove residues, carrying out rotary steaming on a large bottle until the residues are sticky or dry, directly scraping off the residues, transferring the residues to a white plastic bottle, cooling the white plastic bottle at room temperature, and then screwing the bottle cap to a temperature of minus 20 ℃ for marking and preserving.
Example 1 determination of inhibitory Activity of ginger extract on human UGT1A9
DDAO glucuronidation is used as a probe reaction, and a multifunctional ELISA plate determination method is adopted by an in-vitro UGT enzyme incubation system to screen medicinal materials with inhibitory activity to human UGT1A9 in a high throughput manner. The specific experimental procedure is as follows:
(1) An in vitro UGT metabolic reaction system comprises Tris-HCl buffer solution (50 mM) with pH of 7.4 and MgCl 2 Mixing human liver microsomes (0.1 mg/mL) with a final concentration of DDAO of 3.0 μM, ginger extract H7 (10 μg/mL), and pre-incubating with shaking at 37deg.C for 5min;
(2) Adding an initiation factor UDPGA (final concentration of 2.5 mM) into the reaction system to initiate reaction;
(3) After 30min, 200 μl of glacial acetonitrile is added, and after vigorous shaking, the reaction is terminated;
(4) After centrifugation at 20,000Xg at high speed for 10min at 4℃the supernatant was taken and subjected to fluorescence detection (ex=460 nm, em=608 nm);
the results are shown in FIGS. 2-3, ginger extract H7 shows strong inhibitory activity against UGT1A9, and the residual UGT1A9 activity under the reaction system is 21.4%, IC 50 The value was 0.17. Mu.g/mL.
Example 2 Activity directed tracking of UGT1A9 inhibitors in ginger
Establishing a fingerprint of the ginger extract by means of a high performance liquid chromatograph, collecting eluent of the fingerprint, performing a probe reaction by using DDAO glucuronidation metabolism, determining the inhibition activity of all eluents on UGT1A9, comparing the fingerprint with the inhibition spectrum, and identifying the main monomer component of the ginger with the inhibition effect on UGT1A9 by comparing the retention time, the ultraviolet spectrum and the high resolution mass spectrum of a standard substance, wherein the specific experimental flow is as follows:
(1) Fingerprint establishment: fingerprint of rhizoma Zingiberis recens extract was established by Waters high performance liquid chromatography, acquity UPLC HSS T C18 (2.1X100 mm,1.8 μm) column. The mobile phase consists of acetonitrile (A) and water (B), the flow rate is 0.8mL/min, and the detection wavelength is 285nm. The effluent was directly collected into a black 96-well plate every 60s and vacuum dried for UGT1A9 inhibition experimental study.
(2) Establishing an inhibition map: the inhibition of UGT1A9 activity by monomeric compounds in ginger was evaluated with the aid of the UGT1A 9-specific fluorogenic probe substrate DDAO. The incubation system contained HLM (0.1 mg/mL), 50mM Tris-HCl (pH 7.4), 5mM MgCl 2 3.0. Mu.M DDAO, rhizoma Zingiberis recens chromatographic eluate, pre-incubated at 37deg.C for 5min. The reaction system was charged with 10. Mu. LUDPGA to initiate the reaction. After 30min, 200 μl of glacial acetonitrile was added and the reaction was terminated after vigorous shaking. After centrifugation at 20,000Xg at 4℃for 10min, the supernatant was taken and subjected to fluorescence detection (Ex=460 nm, em=608 nm).
As shown in FIG. 4, the ginger extract H7 has 6 inhibition components which are strong against UGT1A9, and is identified as 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol in sequence by comparison with the retention time, ultraviolet spectrum and high resolution mass spectrum of the standard.
Example 3 in vitro inhibition assay quantitative assessment of UGT1A9 inhibition ability of ginger series Compounds
The method comprises the steps of taking mycophenolic acid, isopropyl phenol and DDAO glucuronidation metabolism of mycophenolic acid to be probe reaction, and evaluating IC of different concentrations of ginger extract monomer components (6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol) for inhibiting UGT1A9 in a high throughput manner by using an in vitro UGT enzyme incubation system and adopting a multifunctional ELISA plate measuring method 50 And K i The numerical values, the specific experimental procedure are as follows:
(1) An in vitro UGT metabolic reaction system comprises Tris-HCl buffer solution (50 mM) with pH of 7.4 and MgCl 2 Mixing human liver microsomes (0.1 mg/mL) with final concentrations of mycophenolic acid, isopropyl phenol and DDAO of 30/100/1.0 mu M respectively, and shake pre-incubating for 3min at 37deg.C with different concentrations of rhizoma Zingiberis recens extract monomer components (6-gingerol 2-80 mu M, 8-gingerol 0.5-15 mu M, 10-gingerol 0.5-20 mu M, 6-shogaol 10-500nM, 8-shogaol 5-200nM, 10-shogaol 10-800 nM);
(2) Adding an initiation factor UDPGA (final concentration of 2.5 mM) into the reaction system to initiate reaction;
(3) After 30min, 200 μl of glacial acetonitrile is added, and after vigorous shaking, the reaction is terminated;
(4) And (3) carrying out high-speed centrifugation for 20min at the temperature of 4 ℃ under the condition of 20,000Xg, taking a supernatant, carrying out fluorescence detection and mass spectrum detection, wherein the maximum excitation and emission wavelengths of DDAO-glucuronide are 465nm and 608nm respectively, and carrying out mycophenolic acid glucuronide negative ion mode detection, parent ion 495.0, child ion 319.2, propofol glucuronide negative ion mode detection, parent ion 353.0 and child ion 177.0.
The results are shown in FIG. 5 and Table 1, that 6 monomer components of ginger showed dose-dependent inhibition of UGT1A9 mediated mycophenolic acid, propofol and DDAO, 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol inhibited UGT1A9 by mycophenolic acid-O-glucuronidation IC 50 Values of 32.0, 0.13, 3.94, 0.05, 4.18 and 0.38. Mu.M, K, respectively i 8.79, 0.032, 2.00, 0.034, 1.46 and 0.085 μm, respectively.
TABLE 1 inhibition of UGT1A 9-mediated DDAO-O-glucuronidation, propofol-O-glucuronidation and mycophenolic acid-O-glucuronidation metabolism IC by ginger monomeric components 50 And K i Numerical value
Example 4 in vitro determination of UGT1A9 Activity inhibition selectivity
The selectivity of 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol on UGT1A9 inhibition is evaluated by using 4-methylumbelliferone (4-MU) and trifluoperazine glucuronidation as probe reaction and by using an in vitro UGT enzyme incubation system and adopting a high performance liquid chromatography tandem mass spectrometry method, wherein the specific experimental procedures are as follows:
(1) An in vitro UGT metabolic reaction system comprises Tris-HCl buffer solution (50 mM) with pH of 7.4 and MgCl 2 Solutions (5 mM), recombinant UGT single enzyme, 4-MU/trifluoperazine, different concentrations of radix rehmanniae extract monomer components, shake pre-incubation for 3min at 37deg.C; UGT1A1 (1A 1), UGProtein concentrations of T1A3 (1 A3), UGT1A6 (1 A6), UGT1A7 (1 A7), UGT1A9 (1 A8), UGT1A9 (1 A9), UGT1a10 (1 a 10), UGT2B4 (2B 4), UGT2B7 (2B 7), UGT2B15 (2B 15) and UGT2B17 (2B 17) were 37.5, 15,7.5, 15, 60 and 15 μg/mL, respectively, and 4-MU substrate concentrations were 110, 1200, 110, 30, 750, 30, 1000, 350, 250 and 2000 μΜ, respectively; UGT1A4 protein concentration is 30 mug/mL, and final concentration in a trifluoperazine substrate system is 50 mu M;
(2) Adding an initiation factor UDPGA (final concentration of 2.5 mM) into the reaction system to initiate reaction;
(3) After 150min (UGT 1A1,1A3,1A10,2B4,2B7,2B15 and 2B 17) or 60min (UGT 1A4,1A6,1A7,1A8 and 1 A9) 200 μl of glacial acetonitrile was added and after vigorous shaking the reaction was terminated;
(4) Centrifuging at high speed for 20min at 4deg.C under 20,000Xg, collecting supernatant, and performing mass spectrometry;
as shown in FIG. 6 and Table 2, 6-shogaol, 8-shogaol and 10-shogaol showed the strongest inhibition of recombinant human UGT1A9 enzyme activity, little or slight inhibition of other UGT subtype activities, and inhibition selectivity was between 43.8-656.5 times.
TABLE 26 IC inhibition of UGT1A7,1A8 and 1A9 by shogaol, 8 shogaol and 10 shogaol 50 Value of
Example 5HepRG cell level determination of the Effect of ginger series Compounds on mycophenolic acid metabolism
Establishing UGT1A9 over-expression HepRG cells, measuring the content of mycophenolic acid metabolite mycophenolic acid glucuronide of mycophenolic acid in supernatants of the HepRG cells at different time points by means of high performance liquid chromatography tandem mass spectrometry, and examining the influence of ginger monomer components on mycophenolic acid metabolism of mycophenolic acid, wherein the specific operation flow is as follows:
(1) Establishment of a mycophenolic acid metabolic system at the level of HepRG cells: hepRG cells were cultured in a cell incubator (37 ℃,5% CO) 2 95% humidity), the culture medium is1640 medium containing 10% of fetal bovine serum and 1% of antibody (100 units/mL penicillin, 100 mu g/mL streptomycin), inoculating cells into a 24-well cell culture plate, culturing for 1-2 days by adherence, adding mycophenolic acid (5 mu M) and ginger extract monomer components with different concentrations (0-20 mu M), incubating for 12 hours, taking cell supernatant, centrifuging at a high speed for 20min, taking the supernatant, and carrying out mass spectrometry;
(2) HepRG cells are inoculated into a 96-well cell culture plate, mycophenolic acid (5 mu M) and ginger extract monomer components (0-20 mu M) with different concentrations are added, after the culture is incubated for 12 hours, 100 mu L of CCK-8 liquid is added into each well, the mixture is placed in a incubator for 50min in a dark place, and an enzyme-labeled instrument detects OD value at 450 nm;
(3) HepRG cells are inoculated into a 24-hole cell culture plate, mycophenolic acid (5 mu M) and different ginger extract monomer components (20 mu M) are added, after the cells are incubated for 12 hours, cell supernatant is taken, after high-speed centrifugation is carried out for 20min, the supernatant is taken, and mass spectrometry is carried out to determine the content change of mycophenolic acid and mycophenolic acid glucuronide;
(4) HepRG cells are inoculated in a 24-hole cell culture plate, mycophenolic acid (5 mu M) and 6-shogaol (20 mu M) are added, after incubation for 0, 1, 2, 4, 6, 8, 10 and 12 hours respectively, cell supernatants are taken, after high-speed centrifugation for 20min, the supernatants are taken, and mass spectrometry is carried out to determine the mycophenolic acid metabolism rule and mycophenolic acid glucuronide generation rule.
The results are shown in FIG. 7 and Table 3, and the results show that 6-gingerol, 6-shogaol, 8-gingerol, 8-shogaol, 10-gingerol and 10-shogaol can inhibit the metabolism of mycophenolic acid mycophenolate mofetil at the HepRG viable cell level, table 3 shows that IC 50 13.14, 5.75, 9.69, 4.79, 13.6 and 19.6 μm, respectively. In the UGT1A9 over-expressed HepRG cells, the metabolism of mycophenolic acid in the HepRG cells can be obviously inhibited by all 6 ginger monomer components, and the concentration of mycophenolic acid in cell supernatant is increased by about 10 times. Meanwhile, the 6-shogaol can prolong the metabolic half-life of mycophenolic acid in UGT1A9 over-expressed HepRG cells from 2.93h to 22.9h.
TABLE 3 inhibition of mycophenolic acid metabolism by ginger extract series compounds at HepRG cellular levelIC 50 Numerical value
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311542628.XA CN117562882A (en) | 2023-11-20 | 2023-11-20 | Composition for selectively inhibiting activity of uridine diphosphate glucuronyltransferase 1A9 and application thereof |
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