Application of atractylis lactone as beta-glucuronidase inhibitor

Abstract

The invention provides an application of atractylis lactone as a beta-glucuronidase inhibitor. Experiments find that atractylenolide can act on active sites of beta-glucuronidase to generate various bonding effects and play an inhibition role, and atractylenolide shows a dose-dependent relationship on activity inhibition of beta-glucuronidase. Furthermore, the atractylenolide can be used as a beta-glucuronidase inhibitor for preventing and treating related diseases (such as drug-induced diarrhea, diabetes mellitus, calculus and the like), and has very good research and application values.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an application of atractylodes lactone as a β-glucuronidase inhibitor. Background Art

[0002] β-glucuronidase (GUS) is a key intestinal flora hydrolase that plays an important role in human physiological and pathological processes, especially in drug metabolism and detoxification. In the intestine, GUS can catalyze the hydrolysis of glucuronic acid conjugates to generate active aglycones. These aglycones are reabsorbed in the intestine, which may cause the drug's metabolic kinetics in the body to show a bimodal phenomenon, thereby increasing the risk of gastrointestinal adverse reactions, especially diarrhea.

[0003] Drug-induced diarrhea is a typical disease caused by taking certain drugs that damage the gastrointestinal mucosa or disrupt gastrointestinal function. It is a symptom of abnormally increased bowel movement frequency caused by drugs or drug interactions, often accompanied by discomfort such as abdominal pain, nausea, vomiting, and abdominal distension. In extreme cases, it may cause chills, high fever, shock, coma, and even life-threatening conditions. Diabetes is a metabolic disease that is widely present in people's daily lives, and its most significant sign is hyperglycemia. Long-term maintenance of a high blood sugar state can cause complications such as cardiovascular disease, diabetic retinopathy, diabetic nephropathy, and diabetic peripheral neuropathy, posing a major threat to human health. Studies have shown that the GUS content in diabetic patients is higher than that in normal people. By reducing the GUS content, the risk of progression of diabetic retinopathy and renal complications can be reduced. In view of this, the development of safe and effective β-glucuronidase inhibitors has important clinical application prospects.

[0004] β-glucuronidase inhibitors can be divided into natural and synthetic ones according to their sources. Synthetic β-glucuronidase inhibitors can be divided into thiotetrahydropyrimidines, thiadiazole derivatives, disulfide and sulfone hetero derivatives, cyano-substituted bisindole hetero compounds, etc.; natural β-glucuronidase inhibitors include flavonoids and terpenes, etc.

[0005] Atractylodes is the dried rhizome of Atractylodes macrocephala or Atractylodes macrocephala of the Asteraceae family. It is pungent, bitter, warm, and enters the spleen, stomach, and liver meridians. It is a sacred medicine for drying dampness and strengthening the spleen in clinical Chinese medicine. Atractylodes contains a large number of bioactive compounds, among which atractylodes lactone is a widely studied object, and it has been found to have good pharmacological activities in anti-tumor, anti-oxidation, improvement of liver damage, and the nervous system. At present, no research has reported whether atractylodes lactone has β-glucuronidase inhibitory activity. Summary of the invention

[0006] The first aspect of the present invention provides an application of atractylenolide in the preparation of a drug for treating diseases by inhibiting β-glucuronidase.

[0007] Specifically, the atractylenolide is obtained by solvent extraction from Atractylodes lancea or Atractylodes macrocephala.

[0008] The Atractylodes lancea is the rhizome of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz. of the Compositae family.

[0009] Specifically, the Atractylodes lancea is one or more of stir-fried Atractylodes lancea with bran, Atractylodes lancea processed with vinegar, stir-fried Atractylodes lancea, Atractylodes lancea processed with rice-washing water, steamed Atractylodes lancea, Atractylodes lancea processed with earth, Atractylodes lancea processed with children's urine, Atractylodes lancea processed with salt, baked Atractylodes lancea, Atractylodes lancea processed with wine, stir-fried Atractylodes lancea with gunpowder, Atractylodes lancea processed with ginger, Atractylodes lancea processed with oil, Atractylodes lancea processed with milk, Atractylodes lancea processed with bran or baked Atractylodes lancea.

[0010] The Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala Koidz. of the Compositae family.

[0011] Specifically, the Atractylodes macrocephala is one or more of stir-fried Atractylodes macrocephala, stir-fried Atractylodes macrocephala with bran, stir-fried Atractylodes macrocephala with earth, stir-fried Atractylodes macrocephala with honey and bran, Atractylodes macrocephala soaked in rice-washing water, stir-fried Atractylodes macrocephala processed with vinegar or baked Atractylodes macrocephala.

[0012] In the embodiment of the present invention, the Atractylodes lancea is stir-fried Atractylodes lancea with bran.

[0013] Preferably, the solvent is selected from one or more of methanol, ethanol, pure water, ethyl acetate, chloroform, n-butanol, acetone.

[0014] Specifically, the atractylenolide is selected from one or more of atractylenolide I, atractylenolide II, atractylenolide III, preferably atractylenolide II and / or atractylenolide III.

[0015] The CAS number of atractylenolide I is 73069-13-3, and its chemical structure is shown in Formula I.

[0016]

[0017] The CAS number of atractylenolide II is 73069-14-4, and its chemical structure is shown in Formula II.

[0018]

[0019] The CAS number of atractylenolide III is 73030-71-4, and its chemical structure is shown in Formula III.

[0020]

[0021] The diseases are related to β-glucuronidase, especially the diseases that can benefit from its prevention and / or treatment by inhibiting β-glucuronidase, including drug-induced diarrhea, diabetes or calculi, etc.

[0022] The calculi are intrahepatic bile duct calculi or kidney calculi.

[0023] The drug-induced diarrhea is diarrhea caused by the side effects or adverse reactions of drugs.

[0024] Specifically, the drug that causes drug-induced diarrhea is selected from antibiotics (such as amoxicillin, cefaclor, clarithromycin), antihypertensive drugs (such as propranolol, reserpine, methyldopa), hypoglycemic drugs (such as metformin), anti-tumor drugs (such as irinotecan, fluorouracil, methotrexate, cisplatin) or non-steroidal anti-inflammatory drugs (aspirin, ibuprofen), etc.

[0025] The drugs include β-glucuronidase inhibitors.

[0026] The β-glucuronidase inhibitor is an agent that reduces the expression level of β-glucuronidase or an agent that reduces the function of β-glucuronidase.

[0027] Specifically, the β-glucuronidase inhibitor includes one or more of atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III, preferably atractylodes lactone II and / or atractylodes lactone III.

[0028] In the embodiments of the present invention, atractylodes lactone acts on the active site of β-glucuronidase to produce multiple bonding effects and thus produce an inhibitory effect.

[0029] Atractylodes lactone inhibited β-glucuronidase activity in a dose-dependent manner.

[0030] The medicine also includes pharmaceutically acceptable excipients.

[0031] Specifically, the pharmaceutically acceptable excipient is selected from: one or a combination of two or more of excipients, diluents, lubricants, wetting agents, emulsifiers, preservatives, antioxidants, buffers, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0032] Specifically, the drug can be used alone or in combination with other types of active ingredients.

[0033] The drug can be administered by any suitable route, such as enteral administration (e.g. oral) or parenteral administration (e.g. intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.).

[0034] The drug can be in any suitable dosage form, including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, effervescent tablets, pills, and gels. Preferably, the drug is an oral preparation, preferably at least one of tablets, pills, granules, capsules, and syrups.

[0035] The various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field.

[0036] The drug may contain the atractylodes lactone in a weight ratio of 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%).

[0037] In a second aspect of the present invention, a medicament is provided, which comprises atractylodes lactone and one or more pharmaceutically acceptable excipients.

[0038] In a third aspect, a method for treating a disease is provided, comprising administering an effective dose of a β-glucuronidase inhibitor or the drug to a subject in need thereof.

[0039] Specifically, the method comprises administering an effective dose of atractylodes lactone or the drug to a subject in need thereof.

[0040] In a fourth aspect, a β-glucuronidase inhibitor is provided, which includes one or more of atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III.

[0041] Beneficial effects of the present invention:

[0042] The present invention provides that atractylodes lactone can be used as a β-glucuronidase inhibitor, which can act on the active site of β-glucuronidase to produce multiple bonding effects and exert an inhibitory effect, and atractylodes lactone inhibits the activity of β-glucuronidase in a dose-dependent manner. Further, atractylodes lactone can be used as a β-glucuronidase inhibitor for the prevention and treatment of related diseases (such as drug-induced diarrhea, diabetes, stones, etc.), and has very good research and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0044] Figure 1: The results of different concentrations of Atractylodes lancea extract inhibiting β-GUS, where *** represents p<0.001.

[0045] Figure 2 : Binding profiles of atractylodes lactones I, II, and III based on affinity ultrafiltration technology, wherein A is the overall binding profile of the three atractylodes lactones, B is a local magnified image of the binding profile of atractylodes lactone I, C is a local magnified image of the binding profile of atractylodes lactone III, and D is a local magnified image of the binding profile of atractylodes lactone II.

[0046] Figure 3 : Molecular docking diagrams of atractylodes lactone I, II, III and β-GUS, wherein A is the docking diagram of atractylodes lactone I and β-GUS, B is the docking diagram of atractylodes lactone II and β-GUS, and C is the docking diagram of atractylodes lactone III and β-GUS.

[0047] Figure 4 : Inhibition curves of atractylodes lactones I, II, and III against β-GUS. DETAILED DESCRIPTION

[0048] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0049] The term "pharmaceutically acceptable" as used herein refers to a substance that neither significantly irritates an organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0050] The "effective amount" of the present invention refers to the amount or dosage of the inhibitor or drug of the present invention that provides the desired treatment or prevention after single or multiple doses are administered to a subject.

[0051] The "subject" described in the present invention can be a human or non-human mammal, or a cell, tissue or organ of a human or non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0052] Atractylodes is the rhizome of Atractylodes lancea or Atractylodes lancea of ​​the Asteraceae family.

[0053] The preparation method of Atractylodes lancea slices is to pick out impurities, soak them in water until they are 70% to 80% thorough, take them out, soak them thoroughly, slice them, and dry them in the sun.

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0055] Example 1 Preparation of Atractylodes lancea Extract

[0056] Weigh a certain amount of Atractylodes lancea (fried Atractylodes lancea with bran, batch number: 220331004; manufacturer: Beijing Kangyuan Xiangrui Pharmaceutical Technology Co., Ltd.; purchased from Tongrentang Pharmaceutical Co., Ltd.), add 8 times the amount of 80% ethanol and soak for 1 hour, heat and reflux for extraction for 2 hours. Collect the filtrate, filter, freeze-dry and store. The yield is about 58%.

[0057] Example 2 Enzyme Activity Inhibition Experiment of Atractylodes lancea Extract

[0058] Using 4-nitrophenyl β-D-glucuronide (PNPG) as the specific substrate, the inhibitory activity of Atractylodes lancea extract against β-GUS was determined with the help of an in vitro incubation system of β-glucuronidase (β-GUS, batch number: GT396; manufacturer: Sigma) from Escherichia coli.

[0059] Preparation of Atractylodes lancea extract sample solutions: Based on the amount of crude drug, the extract obtained in Example 1 was prepared into Atractylodes lancea extract sample solutions with concentrations of 0.5, 1, 2, 4, and 8 mg / mL, respectively.

[0060] 1. Experimental design

[0061] The reaction system contained Atractylodes lancea extract (concentrations were 0, 0.5, 1, 2, 4, 8 mg / mL), β-GUS enzyme (20 μg / mL), PNPG (1 mM) and an appropriate amount of PBS. A total of 4 groups were set up, including: blank group, control group, drug control group, and sample group. The specific composition ratios are shown in Table 1:

[0062] Table 1 Reaction systems with different ratios

[0063]

[0064] Then, the four reaction systems were incubated at 37° C. for 30 min. After the incubation, 5 μL of 2 mol NaOH solution was added to terminate the reaction, and the absorbance was measured at 405 nm.

[0065] 2. Calculation method

[0066] According to the measured absorbance value, the residual activity value of the enzyme in each system was calculated by the formula.

[0067] Calculation formula: Enzyme residual activity = (A (样品组) -A (药物对照组) ) / (A (对照组) -A (空白对照组) )*100

[0068] 3. Experimental results

[0069] The residual enzyme activity results of the sample group are as follows Figure 1 As shown, the Atractylodes lancea extract showed significant differences in the inhibition of β-GUS activity, and the differences were statistically significant; and its inhibition of β-GUS activity showed a dose-dependent relationship, indicating that there should be specific inhibitory components in the extract.

[0070] Example 3 Affinity ultrafiltration experiment of Atractylodes lancea extract

[0071] Based on the above results, affinity ultrafiltration technology was used to screen potential components in the extract that could bind to β-GUS.

[0072] 1. Qualitative analysis of Atractylodes macrocephala extract

[0073] 1) Weigh an appropriate amount of the extract (prepared in Example 1) to prepare a solution with a concentration of 50 mg / mL (based on the amount of crude drug), centrifuge at 12000 x g for 10 min, and then inject the sample for analysis.

[0074] 2) Instrument parameter setting: The chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column (2.1 mm×100 mm, 1.8 μm). The mobile phase system is: 0.1% formic acid water (A)-acetonitrile (B). The elution gradient is shown in Table 2.

[0075] Table 2 Elution gradient

[0076]

[0077] The mass spectrometry analysis used an electrospray ionization source with simultaneous positive and negative ion detection mode. The column temperature was 40°C; the flow rate was 0.3 mL / min, and the injection volume was 3 μL. The sheath gas flow rate was 35 arb, the auxiliary gas flow rate was 10 arb, the capillary temperature was 320°C, and the spray voltage was 3.5 kV (+) and 2.5 kV (-). The m / z acquisition range was 100-1500, the full scan resolution was (full MS) 60 000, the secondary resolution was (MS / MS) 15 000, and the collision energy was (multi-level mode) 20 V / 40 V / 60 V.

[0078] 3) Atractylodes lancea extracts with different components were obtained through sample analysis, and potential components in the extracts that can bind to β-GUS were screened.

[0079] 2. Screening of components of Atractylodes lancea extract that bind to β-GUS

[0080] 1) Preparation of inactivated enzyme: Weigh an appropriate amount of β-GUS and dissolve it in PBS solution to make the final concentration 0.5 mg / mL. Then heat it in a metal bath at 100°C for 1 hour to inactivate the enzyme for use.

[0081] 2) Operation method: Referring to the above enzyme activity inhibition experimental steps, the reaction system was set as 20 μL Atractylodes extract (50 mg / mL) + 20 μL enzyme + 60 μL PBS. The Atractylodes extract was incubated with β-GUS enzyme and β-GUS inactivating enzyme at 37°C for 1 hour, and centrifuged at 12000xg for 10 minutes.

[0082] The mixed solution was transferred to a 100KDa ultrafiltration tube, allowed to stand at low temperature for 30 minutes, and centrifuged at 12000xg for 10 minutes. The ultrafiltration tube was rinsed with 240μL of PBS, and the standing-centrifugation process was repeated twice. The ultrafiltration tube was transferred to a new EP tube, and 240μL of 80% methanol was added to the ultrafiltration tube, allowed to stand at low temperature for 1 hour, and centrifuged at 12000xg for 10 minutes. Repeat 3 times.

[0083] 3) Sample analysis: The centrifuged solutions were combined and dried with N2. They were reconstituted with 50% methanol before use and the samples were analyzed by LC-MS.

[0084] 4) Binding force calculation: Calculate the binding rate (BD) based on the peak area results.

[0085] The calculation formula is: BD = (Aa-Ab) / Aa*100

[0086] Wherein, Aa is the chromatographic peak area of ​​the corresponding compound after the drug reacts with the inactivated enzyme; Ab is the chromatographic peak area of ​​the corresponding compound after the drug reacts with the active enzyme.

[0087] 5) Experimental results: After comparison with the database and the reference substance, the Atractylodes lancea extract prepared in Example 1 contains atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III. The binding strength of atractylodes lactone to β-GUS is shown in Figure 2 As shown in Table 3, all three components can strongly bind to β-GUS, indicating that atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III may be specific inhibitors of β-GUS.

[0088] Table 3. Binding rate of atractylodes lactones I, II, and III from Atractylodes lancea extracts to β-GUS

[0089]

[0090] Example 4 Molecular docking experiment of atractylodes lactones I, II, III and β-GUS

[0091] With the help of molecular docking technology, 4-nitrophenyl β-D-glucuronide (PNPG) was used as a positive inhibitor to clarify the binding site of lulutonic acid with β-GUS, and compare the differences in their binding abilities with the positive inhibitors.

[0092] 1. Experimental design

[0093] 1) Ligand processing: The mol2 format of atractylodes lactone I, II, III, and 4-nitrophenyl β-D-glucuronide (10344-94-2) was obtained from the PubChem database, and the energy minimization was performed using Chem 3D software and saved in pdb format. AutodockTools 1.5.6 further detected the root of the ligand, searched and defined the rotatable bond, and saved it as a pdbqt file.

[0094] 2) Receptor processing: The sdf format of β-GUS (3LPG) was obtained from the PDB database, and the PyMOL 2.1.1 software was used to remove the solvent and save it in pdb format. The AutodockTools software was used to remove water, add hydrogen, define atomic types, calculate charges, and save it in pdbqt file.

[0095] 3) Molecular docking: Autodock vina 1.1.2 was used for semi-flexible docking, and the best conformation with affinity (minimum binding energy) was selected as the final docking conformation. The docking images were visualized using PyMOL.

[0096] 2. Experimental results

[0097] The molecular docking results are as follows Figure 3 As shown, atractylodes lactones I, II, and III can produce multiple bonding interactions with β-GUS, and overall exhibit strong binding efficiency, with a binding force of -8.8 Kcal / mol, close to the positive inhibitor 4-nitrobenzene-β-D-glucuronide (-7.2 Kcal / mol). This indicates that atractylodes lactones I, II, and III may produce an inhibitory effect similar to 4-nitrobenzene-β-D-glucuronide by acting on the active site of β-GUS.

[0098] Example 5 Experiment on the inhibition of β-GUS enzyme activity by atractylodes lactones I, II, and III

[0099] The inhibitory activity against β-glucuronidase (β-GUS) from Escherichia coli was determined using 4-nitrophenyl β-D-glucuronide (PNPG) as the specific substrate in an in vitro incubation system.

[0100] Atractylodes lactone preparation: Atractylodes lactone I, II, and III were prepared into samples with concentrations of 0, 10, 20, 40, 80, 160, 320, and 640 μM, respectively.

[0101] 1. Experimental design

[0102] The reaction system contained atractylodes lactone (I, II, III) at concentrations of 0, 10, 20, 40, 80, 160, 320, 640 μM, β-GUS enzyme (20 μg / mL), PNPG (1 mM) and an appropriate amount of PBS. A total of 4 groups were set up, including: blank group, control group, drug control group, and sample group. The specific composition ratios are shown in the following table:

[0103] Table 4 Reaction systems with different ratios

[0104]

[0105] The reaction system obtained according to the above ratio was incubated at 37° C. for 30 min. After the incubation, 5 μL of 2 mol NaOH solution was added to terminate the reaction, and the absorbance was measured at 405 nm.

[0106] 2. Calculation method

[0107] According to the measured absorbance value, the residual activity value of the enzyme in each system was calculated by the formula.

[0108] Calculation formula: Enzyme residual activity = (A (样品组) -A (药物对照组) ) / (A (对照组) -A (空白对照组) )*100

[0109] 2. Experimental results

[0110] like Figure 4 As shown, atractylodes lactones I, II, and III inhibited β-GUS activity in a dose-dependent manner, indicating that atractylodes lactones I, II, and III can be used as specific inhibitors of β-GUS.

[0111] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0112] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. The application of atractylodes lactone in the preparation of drugs for treating diseases by inhibiting β-glucuronidase.

2. The use according to claim 1, characterized in that: The atractylodes lactone is selected from one or more of atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III, preferably atractylodes lactone II and / or atractylodes lactone III.

3. The use according to claim 1, characterized in that: The disease is selected from drug-induced diarrhea, diabetes or stones.

4. The application according to claim 3, characterized in that: The drug-induced diarrhea is diarrhea caused by the side effects or adverse reactions of drugs; The drug is selected from antibiotics, antihypertensives, hypoglycemics, antitumor drugs or non-steroidal anti-inflammatory drugs.

5. The application according to claim 4, characterized in that: The drug is selected from the group consisting of amoxicillin, cefaclor, clarithromycin, propranolol, reserpine, methyldopa, metformin, irinotecan, fluorouracil, methotrexate, cisplatin, aspirin, and ibuprofen.

6. The use according to claim 1, characterized in that: The drugs include β-glucuronidase inhibitors; The β-glucuronidase inhibitor is an agent that reduces the expression level of β-glucuronidase or an agent that reduces the function of β-glucuronidase.

7. The use according to claim 6, characterized in that: The β-glucuronidase inhibitor is selected from one or more of atractylodes lactone I, atractylodes lactone II and atractylodes lactone III, preferably atractylodes lactone II and / or atractylodes lactone III.

8. The use according to any one of claims 1 to 7, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

9. The use according to claim 8, characterized in that: The pharmaceutically acceptable excipients are selected from: excipients, diluents, lubricants, wetting agents, emulsifiers, preservatives, antioxidants, buffers, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors, or a combination of two or more thereof.

10. The use according to claim 1, characterized in that: The medicine is an oral preparation, preferably at least one of tablets, pills, granules, capsules and syrups.

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