CN115814093B - Application of intestinal tract bacterium APS reductase inhibitor combined with andrographolide in treating cholestatic liver injury - Google Patents
Application of intestinal tract bacterium APS reductase inhibitor combined with andrographolide in treating cholestatic liver injury Download PDFInfo
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- CN115814093B CN115814093B CN202211648587.8A CN202211648587A CN115814093B CN 115814093 B CN115814093 B CN 115814093B CN 202211648587 A CN202211648587 A CN 202211648587A CN 115814093 B CN115814093 B CN 115814093B
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- andrographolide
- bromocriptine
- reductase inhibitor
- liver injury
- cholestatic liver
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Abstract
The invention discloses an application of an intestinal tract bacterium APS reductase inhibitor combined with andrographolide in treating cholestatic liver injury. The invention discovers that the combination of the bromocriptine and andrographolide, which is an adenosine-5' -phosphosulfate (APS) reductase inhibitor, has remarkable treatment effect on cholestatic liver injury, and the APS reductase inhibitor can inhibit HSO 3 ‑ Thereby inhibiting the metabolism of andrographolide in vivo and improving the bioavailability of andrographolide. The bromocriptine is used in combination with andrographolide, so that the bioavailability of the andrographolide can be improved, the anti-cholestasis effect of the andrographolide is further remarkably improved, the level of blood biochemical indexes ALT, AST, TBA, TBIL is reduced, and pathological changes such as inflammatory cell infiltration, liver necrosis areas and the like are improved; provides experimental data support for further clinical use and popularization.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to an application of an intestinal tract bacterium APS reductase inhibitor combined with andrographolide in treating cholestatic liver injury.
Background
Cholestasis is one of the most common clinical symptoms in liver disease, a pathophysiological process due to bile secretion and excretion disorders. Accumulation of large amounts of bile in hepatocytes causes inflammation, oxidative stress, apoptosis and fibrosis of the hepatocytes, which can evolve into liver fibrosis, cirrhosis if the patient is not effectively treated, ultimately leading to liver failure.
The establishment of cholestatic animal model provides a way for basic research, treatment and screening of cholestatic liver injury prevention and cure drugs. Alpha-naphthyl-isothiocyanate (ANIT) is a cholangiotoxic agent, and single-dose oral administration can induce acute cholangitis, cause bile outflow blockage, generate intrahepatic cholestasis, and reach the highest peak 48 hours after administration, and is a model for more mature cholestasis.
Andrographolide (AP) is a main active ingredient of Andrographis paniculata Nees, and Andrographolide has effects of protecting liver injury and promoting bile flow, and can be used as an effective medicine for treating cholestatic liver disease. Andrographolide can improve ANIT-induced intrahepatic cholestatic liver injury. Andrographolide, however, undergoes rare C-sulfonated metabolism in the intestinal tract, resulting in reduced bioavailability. HSO (high speed oxygen) 3 - Is the desired prosthetic group for the formation of the sulfonated metabolite (14-deoxy-12 (R) -sulfo andrographolide, APM) at the AP C-position.
How to further provide the bioavailability of andrographolide is a problem to be solved in the art.
Disclosure of Invention
The invention aims to provide an application of an intestinal tract bacterium adenosine-5' -phosphosulfate (APS) reductase inhibitor bromocriptine combined with andrographolide in treating cholestatic liver injury.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention there is provided the use of an adenosine 5' -phosphosulfate reductase inhibitor or a pharmaceutically acceptable salt thereof in at least one of (I) to (V);
(I) Preparing a product for inhibiting the metabolism of andrographolide or derivatives thereof;
(II) preparing a product for improving the bioavailability of andrographolide or a derivative thereof;
(III) preparing a product that increases the anti-cholestasis effect of andrographolide or a derivative thereof;
(IV) preparing an andrographolide or derivative drug effect enhancer thereof;
(V) preparing a medicament for treating liver injury.
In some embodiments of the invention, the adenosine-5' -phosphosulfate reductase comprises a plant adenosine-5' -phosphosulfate reductase, a bacterial adenosine-5' -phosphosulfate reductase.
The adenosine 5' -phosphosulfate (APS) reductase inhibitor is key enzyme produced by intestinal bacteria (such as sulfate reducing bacteria, tubercle bacillus, and Pseudomonas aeruginosa), and SO 4 2- Reduced to APS by the action of adenosine 5'-triphosphate sulfurylase, ATPS, which further produces adenosine 5' -monophosphate (AMP) and sulfite by the action of APS reductase, the intermediate of the reaction being HSO 3 - . APS reductase enzyme is prepared by introducing SO into organism 4 2- Reduction to HSO 3 - ，HSO 3 - Attack andrographolide, thereby leading the andrographolide to be metabolized in intestinal tracts. Inhibitors of APS reductase inhibit HSO 3 - Thereby inhibiting the metabolism of andrographolide in vivo and improving the bioavailability of andrographolide.
In some embodiments of the invention, the APS reductase inhibitor is bromocriptine.
The bromocriptine is an inhibitor of APS reductase and can inhibit HSO 3 - Thereby improving the bioavailability of andrographolide and further improving the anti-cholestasis effect of andrographolide. The bromocriptine combined andrographolide has remarkable treatment effect on cholestatic liver injury, and can reduce blood biochemical indexes such as glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), total Bile Acid (TBA), total Bilirubin (TBIL) level, and improve pathological changes such as inflammatory cell infiltration and liver necrosis area.
In some embodiments of the invention, the derivative comprises lianbizhi, xiyanping.
In some embodiments of the invention, the product comprises a drug, an agent.
In some embodiments of the invention, the liver injury is cholestatic liver injury.
In a second aspect of the invention there is provided a composition comprising andrographolide or a derivative thereof and an adenosine-5' -phosphosulfate reductase inhibitor or a pharmaceutically acceptable salt thereof.
In some embodiments of the invention, the adenosine 5' -phosphosulfate reductase inhibitor is bromocriptine.
In some embodiments of the invention, the mass ratio of andrographolide or a derivative thereof to the adenosine 5' -phosphosulfate reductase inhibitor or a pharmaceutically acceptable salt thereof is (0.5-50): 1.
in some preferred embodiments of the present invention, the mass ratio of andrographolide or a derivative thereof and an adenosine-5' -phosphosulfate reductase inhibitor or a pharmaceutically acceptable salt thereof is (0.5 to 20): 1.
in some more preferred embodiments of the present invention, the mass ratio of andrographolide or a derivative thereof and an adenosine-5' -phosphosulfate reductase inhibitor or a pharmaceutically acceptable salt thereof is (1.5 to 20): 1.
in some more preferred embodiments of the present invention, the mass ratio of andrographolide or a derivative thereof and an adenosine-5' -phosphosulfate reductase inhibitor or a pharmaceutically acceptable salt thereof is (2.5 to 10): 1.
in a third aspect, the invention provides the use of a composition according to the second aspect of the invention for the manufacture of a medicament for the anti-treatment of liver injury.
In some embodiments of the invention, the liver injury is cholestatic liver injury.
In some embodiments of the invention, the medicament may further comprise pharmaceutically acceptable excipients.
In some embodiments of the invention, the pharmaceutically acceptable excipients include: at least one of a diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, co-solvent, solubilizer, preservative, pH regulator, osmotic pressure regulator, surfactant, coating material, antioxidant, bacteriostat or buffer.
In some embodiments of the invention, the dosage form of the medicament comprises at least one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, an oral, a suppository, an enema, an aerosol, a patch or a drop.
In some embodiments of the invention, the route of administration of the drug comprises at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.
In a fourth aspect of the invention there is provided a medicament comprising a composition according to the second aspect of the invention.
In some embodiments of the present invention, the drug comprises an adenosine 5' -phosphosulfate reductase inhibitor or a pharmaceutically acceptable salt thereof and the andrographolide or a derivative thereof, or is in the form of a pharmaceutical composition prepared by mixing the adenosine 5' -phosphosulfate reductase inhibitor or the pharmaceutically acceptable salt thereof and the andrographolide or the derivative thereof, or is in the form of a kit consisting of independent compositions containing the adenosine 5' -phosphosulfate reductase inhibitor or the pharmaceutically acceptable salt thereof and the andrographolide or the derivative thereof.
In some embodiments of the invention, the medicament may further comprise pharmaceutically acceptable excipients.
In some embodiments of the invention, the pharmaceutically acceptable excipients include: at least one of a diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, co-solvent, solubilizer, preservative, pH regulator, osmotic pressure regulator, surfactant, coating material, antioxidant, bacteriostat or buffer.
In some embodiments of the invention, the dosage form of the medicament comprises at least one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, an oral, a suppository, an enema, an aerosol, a patch or a drop.
In some embodiments of the invention, the route of administration of the drug comprises at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.
The beneficial effects of the invention are as follows:
the invention discovers that the combination of the APS reductase inhibitor bromocriptine and andrographolide has remarkable treatment effect on cholestatic liver injury for the first time, and the APS reductase is a key enzyme generated by intestinal bacteria such as sulfate reducing bacteria, tubercle bacillus and the like, and plays a key role in intestinal metabolism of andrographolide. APS reductase enzyme is prepared by introducing SO into organism 4 2- Reduction to HSO 3 - ，HSO 3 - Attack andrographolide, thereby leading the andrographolide to generate intestinal metabolism and leading the bioavailability of the andrographolide to be lower. Inhibitors of APS reductase inhibit HSO 3 - Thereby inhibiting the metabolism of andrographolide in vivo and improving the bioavailability of andrographolide.
The invention discovers a novel application of an APS reductase inhibitor bromocriptine combined with andrographolide in treating cholestatic liver injury. Bromocriptine is an inhibitor of APS reductase. The invention discovers that the bromocriptine is combined with andrographolide for use, can improve the bioavailability of the andrographolide, further remarkably improve the anti-cholestasis effect of the andrographolide, reduce the level of blood biochemical indexes ALT, AST, TBA, TBIL, and improve pathological changes such as inflammatory cell infiltration, liver necrosis areas and the like. The invention clarifies and verifies the pharmacodynamic action of the APS reductase inhibitor bromocriptine combined with andrographolide in treating cholestasis, and provides experimental data support for further clinical use and popularization.
Drawings
FIG. 1 shows the AP content in the small intestine, colon, liver and kidney and HSO in the cecum of mice in the Control group (Control), inhibitor group (inhibitor) 3 - The content is as follows.
Fig. 2 is a plasma drug-time profile of AP after oral administration of AP in Control (Control), inhibitor (Inhibition) rats.
FIG. 3 shows liver and gall bladder morphology in Control mice (Control) and inhibitor mice (inhibitor).
FIG. 4 is a 200-fold magnification of H & E staining of liver from Control mice (Control) and inhibitor mice (inhibitor).
Fig. 5 shows ALT, AST, TBA, TBIL levels in plasma from normal (Control) and inhibitor (Inhibition) mice.
FIG. 6 shows the AP content in liver tissue of mice in the normal (Control) ANIT+AP200mg/kg group and mice in the inhibitor (Inhibition) ANIT+AP200mg/kg group.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Drug reagent: andrographolide standard (purity > 98%), genistein standard purchased from dupofford biotechnology limited; bromocriptine mesylate was purchased from Shanghai Secoides Corp; hydroxypropyl beta-cyclodextrin, sodium chloride, PBS, hank's basic Salts, tris, HEPES, glucose, sucrose, anhydrous sodium sulfate, sodium bicarbonate, purchased from Sigma-Aldrich; heparin sodium, basic fuchsin, sodium bisulphite, disodium ethylenediamine tetraacetate (EDTA-2 Na), formaldehyde were purchased from Dalian Mei Lun Biotechnology Co., ltd; formic acid was purchased from Shanghai Ala Biochemical technologies Co., ltd; acetonitrile, methanol were purchased from merck, usa. Alpha-naphthyl-isothiocyanate (ANIT), hydroxypropyl beta-cyclodextrin, sodium chloride were purchased from Sigma-Aldrich company; glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), total Bile Acid (TBA), and Total Bilirubin (TBIL) are purchased from Nanjing to build the institute of biological engineering.
Instrument apparatus: the Acquity ultra-high performance liquid chromatograph and the Micromass Quattro Premier XE mass spectrum are manufactured by Volter company in the United states; the electronic analytical balance is Style BT21S/BS124S; the test tube vortex instrument is German IKAMS3basic/MS3digital; the ultra-pure water system is Millpore company in U.S.; the surgical instrument and the injector are made by Ningbo Xinzhi biotechnology company; the pipettor is Eppendorf, germany; the ultralow temperature refrigerator with the temperature of 80 ℃ below zero is China sea company.
Statistical analysis: statistical analysis in the invention adopts Graph Pad Prism 7, experimental data are represented by Mean ± standard error (Mean ± SEM. Variance alignment is performed by using Tukey method, one-way ANOVA is used when variance is aligned, dunnett's T3 method is used when variance is uneven. Significance standard is alpha=0.05, and difference between p <0.05 is statistically significant.
EXAMPLE 1 study of the influence of APS reductase inhibitors on the tissue distribution of andrographolide
1.1 laboratory animals
SPF-class healthy male C57 mice, 6-8 weeks old, weight 20-22g.
1.2 pharmaceutical formulation
1.2.1 preparation of bromocriptine (bromocriptine mesylate) solution: precisely weighing 100.00mg of bromocriptine, adding 50mL of physiological saline, mixing uniformly by vortex, preparing 2mg/mL of bromocriptine solution by ultrasonic dissolution, and preserving at 4 ℃ for later use;
1.2.2 preparation of Andrographolide (AP) solution: precisely weighing a proper amount of AP, adding 40% hydroxypropyl-beta cyclodextrin, mixing uniformly by vortex, respectively preparing solutions with concentration of 20mg/mL by ultrasonic dissolution, and preserving at 4 ℃ for later use;
1.2.3 preparation of hydroxypropyl-beta Cyclodextrin (beta-CD) solution: precisely weighing 20.00g of beta-CD, adding 50mL of distilled water, vortex suspending to prepare 40% beta-CD solution, and preserving at 4 ℃ for later use.
1.2.4 Preparation of 0.9% sodium chloride solution (physiological saline): 9.00g of NaCl is precisely weighed, 1L of distilled water is added, and the mixture is dissolved in ultrasonic waves at the temperature of 4 ℃ for standby.
1.3 animal modeling and Experimental grouping
Healthy male C57 mice were divided into two groups, a Control group (Control) and an inhibitor group (inhibitor), 6 each. The bromocriptine 20mg/kg is orally and intragastrically administered to the mice in the inhibitor group, once a day, for 7 days continuously, and an equivalent dose of physiological saline is administered to the control group; after the last administration, 100mg/kg of AP solution is orally and gastrolavaged according to the body weight, timing is started after administration, eyeball blood collection is carried out after 90 minutes of administration, the mice are killed by cervical dislocation, and plasma samples, liver tissue, kidney tissue, small intestine, cecum and colon samples are collected.
1.4UPLC-MS/MS method for determining the AP content in plasma and tissue samples
1.4.1 liquid phase conditions and Mass Spectrometry conditions
1.4.1.1 chromatographic conditions: chromatography column, acquity UPLC BEH C column (50×2.1mm i.d.,1.7 μm, waters, milford, MA, USA); flow rate, 0.3mL/min; mobile phase a:100% acetonitrile; mobile phase B: the water gradient elution is shown in table 1.
TABLE 1 gradient elution procedure
1.4.1.2 mass spectrometry conditions: ionization mode, ESI (-); capillary voltage, 3KV; drying gas (nitrogen) temperature, 350 ℃; ion source temperature, 110 ℃; the flow rate of the desolventizing gas is 600L/Hr; the flow rate of the taper hole airflow is 50L/Hr. The MRM detection mode is shown in table 2.
Table 2MRM detection procedure
| Compounds of formula (I) | Parent(m/z) | Daugther(m/z) | Dwell(s) | Cone(v) | Collision(v) |
| AP | 349.1 | 287.2 | 0.2 | 18 | 15 |
1.5 preparation of standard curve
1.5.1 preparation of solutions
(1) Preparing Andrographolide (AP) mother solution: precisely weighing 3.50mg of AP standard substance, dissolving in 1mL of DMSO, vortexing and ultrasonically dissolving to prepare 10mM AP solution, and preserving at-20deg.C for later use;
(2) Preparing genistein internal standard mother solution: precisely weighing 5.40mg of genistein, adding 1mL of DMSO, mixing by vortex, dissolving by ultrasonic, preparing into an internal standard mother solution of 20mM, preserving at-20deg.C for standby, and diluting the mother solution with acetonitrile to 0.1 μm concentration for standby when in use;
(3) Preparation of Hank's Balanced salt buffer (HBSS): precisely weighing 1.9604g of Hank's balanced salt, 1.1928g of HEPES powder, 0.7004g of glucose, 0.0744g of sodium bicarbonate and 0.2328g of sodium chloride, adding 200mL of ultrapure water, uniformly mixing by vortex, ultrasonically dissolving, adjusting the pH value of the solution to be=6.5, and preserving at 4 ℃ for later use;
(4) Preparation of 0.9% sodium chloride solution: 9.00g of NaCl is precisely weighed, 1L of distilled water is added, and the mixture is dissolved in ultrasonic waves at the temperature of 4 ℃ for standby.
1.5.2 preparation of standard curve
10mM AP was diluted with acetonitrile to a 20, 10,5,2.5,1.25,0.625,0.3125,0.15625,0.078125,0. Mu.M series of concentration solutions, 10 1.5mL centrifuge tubes were taken for each standard curve, 90. Mu.L HBSS was added, 10. Mu.L of the above series of gradient solutions and 100. Mu.L of internal standard solution were then added, vortexed for 3min, and centrifuged at 13000rpm for 30min, and 10. Mu.L of supernatant was taken for UPLC-MS/MS analysis. Integrating the peak areas of the AP and the internal standard, processing the data by using a weighted least square method, and carrying out linear regression on the peak areas/drug concentration to obtain the standard curve of the AP.
1.5.3 preparation of biological sample Standard Curve
Weighing 1.0g of small intestine, colon, liver and kidney of a blank mouse, adding 3mL of physiological saline to homogenate for 2min, centrifuging the homogenate at 13000rpm at 4 ℃ for 15min, and collecting supernatant to obtain blank homogenate of each tissue. AP and APM are diluted into 20, 10,5,2.5,1.25,0.625,0.3125,0.15625,0.078125,0 mug/mL series concentration solutions by acetonitrile, 10 centrifuge tubes with 1.5mL are taken for each standard curve, 100 mug of blank plasma and 100 mug of blank homogenate of each tissue are respectively added, 10 mug of the series gradient solutions and 100 mug of internal standard solutions are respectively added, 500 mug of acetonitrile is added, vortex is carried out for 3min, and then the mixture is placed at the temperature of minus 20 ℃ for 20min. The sample was centrifuged at 13000rpm for 30min, the supernatant was aspirated and the organic solvent was evaporated in a vacuum oven. The volatilized sample was reconstituted by adding 100. Mu.L of 70% acetonitrile water, vortexing for 3min, centrifuging at 13000rpm for 30min, and collecting 10. Mu.L of supernatant for UPLC-MS/MS analysis. Integrating the peak areas of the AP and the internal standard, processing the data by using a weighted least square method, and carrying out linear regression on the peak areas/drug concentration to obtain the plasma or tissue standard curve of the AP.
Determination of AP content in 1.5.4 plasma and tissue
The AP content of the plasma sample and the tissue sample is measured according to standard curve operation.
1.5.5 HSO in cecum 3 - Determination of the content
(1) Preparation of the solution
1)1mg/mL NaHSO 3 Preparing a solution: accurately weighing 1.00mg NaHSO 3 Dissolving the standard substance in 1mL of 0.01% EDTA-disodium, mixing by vortex, and dissolving by ultrasonic to prepare 1mg/mL NaHSO 3 The solution is prepared for use.
2) Preparation of 0.5% basic fuchsin: precisely weighing 5.00mg of basic fuchsin standard substance, dissolving in 0.5mL of concentrated hydrochloric acid, mixing by vortex, adding 10mL of distilled water, and dissolving by ultrasonic to prepare 0.5% basic fuchsin solution.
3) Preparation of 0.2% formaldehyde solution: precisely sucking 50 mu L of 40% formaldehyde solution, adding 10mL of distilled water, mixing uniformly by vortex, and dissolving by ultrasonic to prepare 0.2% formaldehyde solution.
4) Preparation of 0.01% EDTA-disodium solution: precisely weighing 4.00mg of EDTA-disodium, adding 40mL of distilled water, mixing uniformly by vortex, and dissolving by ultrasonic to prepare 0.01% EDTA-disodium solution.
(2) Preparation of a Standard Curve
Sodium bisulphite solution with concentration of 1mg/mL is prepared into a series of gradient solutions by using 0.01% EDTA-disodium solution, wherein the concentration is 0, 0.0078125, 0.015625, 0.03125, 0.0625, 0.125, 0.25 and 0.5mg/mL respectively. Then, 20. Mu.L of a series of gradient solutions were taken, 180. Mu.L of a 0.01% EDTA-disodium solution was added, and then 20. Mu.L of a 0.2% formaldehyde solution and 20. Mu.L of a 0.05% basic fuchsin solution were added and mixed by blowing. The sample was left at room temperature for 40min, and the absorbance was measured at 560nm using a microplate reader. And drawing a standard curve by taking the difference between the OD value of the sample and the OD value of the blank HBSS solution as an abscissa and taking the concentration as an abscissa.
(3) HSO in cecum samples 3 - Is measured according to the content:
precisely weighing 100.00mg of cecum sample, homogenizing with 500 μl of physiological saline for 2min, centrifuging at 4deg.C and 13000rpm for 15min, collecting 20 μl of supernatant, and measuring HSO under standard curve operation 3 - Is contained in the composition.
AP and HSO 3 - The content detection results of (1) are shown in figure 1, and the results show that the content of the AP in each tissue of the inhibitor group (Inhibition) mice is obviously improved; HSO in cecum of inhibitor group (Inhibition) mice 3 - The content of the bromocriptine is obviously reduced, which indicates that the bromocriptine can inhibit the metabolism of AP and inhibit HSO 3 - Is generated.
Example 2 improvement of bioavailability of andrographolide by the APS reductase inhibitor bromocriptine
This example further investigated the effect of the APS reductase inhibitor bromocriptine on andrographolide pharmacokinetics.
2.1 laboratory animals
SPF-grade male SD rats, 6-8 weeks old, weight 180-220g.
2.2 pharmaceutical formulation
Formulation of bromocriptine (Inhibition) solution, AP solution, 40% hydroxypropyl-beta cyclodextrin (beta-CD) solution reference example 1.
2.3 grouping and administration
Healthy male SD rats were divided into two groups, a Control group (Control) and an inhibitor group (inhibitor), each group being 6, at random, according to body weight. The bromocriptine 10mg/kg was orally administered to the rats in the inhibitor group by lavage once a day for 7 days, and the rats in the control group were administered an equivalent dose of physiological saline. All rats were dosed orally by intragastric administration of 120mg/kg of AP per body weight after the last day of dosing.
2.4 detection of plasma samples
After AP administration, 500 mu L of blood is collected from the eye sockets of each group of rats after 0,5, 10, 20, 30, 40, 60, 90, 120, 180, 240, 360 and 480min, the rats are placed in a heparin tube, and centrifugation is carried out at 8000r/min for 5min at 4 ℃, and blood plasma is taken and stored at-80 ℃ to be tested. Measurement of AP content in plasma reference is made to example 1.
Pharmacokinetic parameters of AP in rat plasma are shown in Table 3, and the AP content measurements are shown in FIG. 2.
TABLE 3 pharmacokinetic parameters of AP in rat plasma from Control (Control) and inhibitor (inhibitor) groups
The results show that the concentration of AP in rat plasma is significantly increased following administration of the APS reductase inhibitor bromocriptine. C of AP in Control group (Control) and inhibitor group (inhibitor) max Is 1.52+/-1.01 mug/mL; AUC (0- ≡) is 227.07 + -88.08 mg/L min. The relative bioavailability of inhibitor group AP was increased by 115.3% compared to the control group. The bromocriptine can inhibit the metabolism of the AP and improve the bioavailability of the AP.
Example 3 Effect of APS reductase inhibitors on andrographolide anti-cholestasis
3.1 laboratory animals
SPF-class male C57 mice, 6-8 weeks old, weight 20-22g.
3.2 pharmaceutical formulation
3.2.1 preparation of bromocriptine solution, AP solution, 40% hydroxypropyl-beta cyclodextrin (beta-CD) solution the same as in example 1.
3.2.2S preparation of ademetionine (S-AMe) solution (positive drug): 100.00mg of S-AMe powder is precisely weighed, 10mL of physiological saline is added, vortex ultrasonic dissolution is carried out, 10mg/mL of S-AMe solution is prepared, and the solution is preserved at 4 ℃ for standby.
3.2.3 preparation of alpha-naphthyl-isothiocyanate (ANIT) solution: accurately weighing 120.00mg of ANIT, adding 12mL of olive oil, performing vortex ultrasonic dissolution, preparing 10mg/mL of ANIT solution, and preserving at 4 ℃ for later use.
3.3 grouping and administration
The 60 male C57 mice were randomly divided into two groups according to body weight, namely a Control group (Control) and an inhibitor group (inhibitor), and the specific grouping situation and intervention scheme are as follows:
wherein the Control component is Vehicle, ANIT, AP200, ANIT+AP200, ANIT+S-AMe; the Inhibition components are bromocriptine 20, ANIT+bromocriptine 20, AP200+bromocriptine 20, ANIT+bromocriptine 20+AP200, ANIT+bromocriptine 20+S-AMe. Each group had 6. The duration of the experimental period was 28 days in total. The Inhibition group mice were orally administered with bromocriptine 20mg/kg by gastric lavage, 1 time a day, for 28 days, and the Control group mice were given an equivalent dose of physiological saline. On day 15, all Vehicle mice and ANIT model mice (ANIT) were orally gavaged with an equal dose of 40% β -CD solution, AP-dosed mice were orally gavaged with AP200mg/kg, S-AMe positive control mice were given S-AMe mg/kg by intraperitoneal injection, and all mice were given a continuous dose of 14 days 1 time per day. On day 26, all mice containing the ANIT administration group were orally gavaged with ANIT 50mg/kg and the remaining mice were given an equal dose of olive oil.
3.4 detection of the collection of plasma and tissue samples
3.4.1 sample collection: after 48h of ANIT intragastric administration, all mice were subjected to blood collection of 500. Mu.L by eyeball, centrifugation at 8000rpm at 4℃for 5min, and the supernatant was aspirated into an EP tube and stored at-80℃for further use. The mice were then sacrificed by cervical dislocation, liver tissue and gall bladder were immediately isolated and photographed, while kidney, small intestine, cecum and colon tissue were isolated and stored in a-80 ℃ freezer, with a portion of liver tissue fixed in 4% paraformaldehyde at room temperature.
3.4.2 histopathological examination
Liver tissue partially fixed in 4% paraformaldehyde was taken, embedded in paraffin according to conventional methods of histopathology, cut into 4 μm slices, and sectioned. Subsequently, staining (H & E staining) was performed with hematoxylin and eosin, and each sample section was subjected to 200-fold mirror observation of liver tissue change for histopathological analysis.
3.4.3 Biochemical index detection
The mice were tested for plasma ALT, AST, TBA, TBIL levels, strictly following the kit protocol.
3.4.4 determination of AP content in liver tissue: reference is made to example 1.
The level of plasma ALT, AST, TBA, TBIL is used as a biochemical index, the level of liver injury and cholestasis are estimated by assisting in H & E staining and liver gall bladder morphology, and the influence of the APS reductase inhibitor bromocriptine on the anti-cholestasis effect of the AP is examined by detecting the content of the AP in liver tissues.
The detection results are shown in fig. 3-6, and the results show that:
(1) On the control group model, compared with the Vehicle group, the mouse gall bladder of the ANIT model group is obviously enlarged, the bile is dark green, and more white spots of the liver are deposited and are matt; compared with the ANIT model group, the normal control group mice have no obvious improvement effect on the gallbladder and liver morphology by orally administering 200mg/kg of AP; administration of positive drug S-AMe100mg/kg significantly improved the gallbladder and liver morphology caused by ANIT; oral administration of AP200mg/kg on the APS reductase inhibitor model had a significant improvement in both gallbladder and liver morphology (fig. 3).
(2) H & E staining results show that, on the control group model, compared with the Vehicle group, the liver tissue of the mice in the ANIT model group has a plurality of hepatic cell focal necrosis, the nucleus is dissolved, the cytoeosinophilia is enhanced, and a small amount of inflammatory cells infiltrate; compared with the ANIT model group, the normal control group mice have no obvious improvement effect on the liver necrosis area by orally administering 200mg/kg of AP; administration of positive drug S-AMe at 100mg/kg significantly improved liver necrosis caused by ANIT; oral administration of AP200mg/kg on the APS reductase inhibitor model had a significant improvement effect on liver necrosis area (fig. 4).
(3) On the control group model, compared with the ANIT model group, the normal control group mice orally administrate 200mg/kg of AP without obvious improvement effect on the ALT, AST, TBA, TBIL level in the blood plasma; administration of the positive drug S-AMe at 100mg/kg significantly improved the levels of ALT, AST, TBA, TBIL in plasma; on the APS reductase inhibitor model, oral administration of AP200mg/kg significantly improved the level of ALT, AST, TBA, TBIL in plasma (fig. 5).
(4) Compared to the normal control group, the content of AP in liver tissue of mice administered with the enterobacteria APS reductase inhibitor was significantly increased (fig. 6).
It is shown that bromocriptine can significantly improve its anti-cholestasis effect by inhibiting AP metabolism in the gut.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (6)
1. Use of bromocriptine or a pharmaceutically acceptable salt thereof in the preparation of a product which is at least one of (I) - (IV);
(I) A product that inhibits andrographolide metabolism;
(II) a product that increases the bioavailability of andrographolide;
(III) a product that enhances the anti-cholestatic liver injury effect of andrographolide;
(IV) an agent for enhancing the efficacy of andrographolide against cholestatic liver injury.
2. The use according to claim 1, wherein the product comprises a medicament or an agent.
3. A composition for preventing cholestatic liver injury, comprising andrographolide and bromocriptine or a pharmaceutically acceptable salt thereof.
4. A composition according to claim 3, wherein the mass ratio of andrographolide to bromocriptine or a pharmaceutically acceptable salt thereof is from 0.5 to 50:1.
5. use of a composition according to any one of claims 3 to 4 in the manufacture of a medicament for the treatment of cholestatic liver injury.
6. An anti-cholestatic liver injury agent comprising the composition of any one of claims 3 to 4.
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Non-Patent Citations (4)
| Title |
|---|
| Poor Oral Bioavailability of a Promising Anticancer Agent Andrographolide is Due to Extensive Metabolism and Efflux by P-Glycoprotein;LING YE等;《JOURNAL OF PHARMACEUTICAL SCIENCES》;第100卷(第11期);第5007-5017页 * |
| Preparation of Andrographolide-Loaded Solid Lipid Nanoparticles and Their In Vitro and In Vivo Evaluations: Characteristics, Release, Absorption, Transports, Pharmacokinetics, and Antihyperlipidemic Activity;TAO YANG等;《JOURNAL OF PHARMACEUTICAL SCIENCES》;第4414-4425页 * |
| Strategies for formulation development of andrographolide;Atmaram Pawar等;《RSC Advances》;第69282-69300页 * |
| 大鼠在体肠吸收穿心莲内酯的特征研究;叶玲等;《中草药》;第41卷(第2期);第219-222页 * |
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