Application of red date extract in preparation of beta-glucuronidase inhibitor
By using pentacyclic triterpene acid compounds such as Lulutong acid in red date extracts, the activity of β-glucuronidase is inhibited, and the problems of drug-derived diarrhea and intestinal damage are solved, providing a safe and effective way of doing so. treatment plan.
Patent Information
- Application Number
- CN202411867823.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-02-11
AI Technical Summary
The prior art is difficult to effectively solve the problems of drug-derived diarrhea and intestinal damage caused by drugs such as irinotecan, and the excessive use of antibiotics will lead to intestinal microbial disorders.
Red date extract is used as an inhibitor of β-glucuronidase, and red date extract is prepared by ethanol extraction and heating reflux technology, including pentacyclic triterpene acid compounds such as Lulutong acid, which is used to inhibit β-glucuronidate. activity of glycase enzyme.
Red date extract can effectively inhibit the activity of β-glucuronidase, reduce the occurrence of drug-derived diarrhea and intestinal damage, and provide a safe and effective method to treat drug-derived intestinal adverse reactions.
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Figure CN119405723A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of red date extract in the preparation of beta-glucuronidase inhibitors. Background Art
[0002] Intestinal flora has been a hot topic in the field of biomedicine in recent years. Studies have shown that intestinal bacteria can participate in the metabolism of a variety of endogenous and exogenous substances, thereby affecting the occurrence and development of diseases and the absorption and metabolism of drugs. Intestinal flora can produce an important class of hydrolases, β-glucuronidase (GUS), which can catalyze the hydrolysis of glucuronic acid conjugates and produce corresponding aglycones. Intestinal bacteria GUS can catalyze the hydrolysis of glucuronide products of many drugs, releasing more toxic aglycones, and excessive accumulation of the latter in the intestine can cause severe delayed diarrhea. For example, GUS can catalyze the hydrolysis of 7-ethyl-10-hydroxycamptothecin-glucuronic acid (SN-38G) and generate a product SN-38 with strong cytotoxicity, and the accumulation of the latter in the intestine can cause intestinal mucosal shedding and cause severe delayed diarrhea and other adverse reactions. In 2010, some scholars first confirmed that inhibiting intestinal bacteria β-glucuronidase can alleviate drug-induced diarrhea caused by CPT-11.
[0003] Irinotecan (CPT-11) is a commonly used drug for the treatment of colon cancer. When it enters the human body, it is metabolized into inactive SN-38 glucuronide (SN-38G) in the liver. SN-38G is then excreted into the intestines through the bile duct and hydrolyzed into SN-38 by intestinal bacterial β-glucuronidase. When SN-38 accumulates in the intestines, it can cause severe delayed diarrhea and intestinal damage, seriously affecting the progress of chemotherapy. In addition, many non-steroidal anti-inflammatory drugs containing carboxylic acids, such as ketoprofen, diclofenac and indomethacin, can also cause similar intestinal toxicity, leading to severe drug-induced diarrhea.
[0004] A large number of studies at home and abroad have confirmed that using antibiotics to eliminate intestinal bacteria that can produce β-glucuronidase or using highly effective β-glucuronidase inhibitors can alleviate gastrointestinal adverse reactions such as diarrhea caused by chemical drugs such as irinotecan to a certain extent. However, excessive use of antibiotics can cause serious disorders in the intestinal flora of the patient's gastrointestinal system and cause fatal damage to organs such as the liver and kidneys.
[0005] Therefore, developing safe and effective drugs that can treat drug-induced diarrhea by targeting intestinal bacterial β-glucuronidase has important clinical application prospects.
[0006] At present, there is no report on whether red date extract has β-glucuronidase inhibitory activity. Summary of the invention
[0007] In order to expand the application scope of β-glucuronidase inhibitors and fully explore substances that can inhibit β-glucuronidase activity, the present invention provides the following technical solutions.
[0008] In a first aspect, the present invention provides use of a red date extract in the preparation of a β-glucuronidase inhibitor.
[0009] In a second aspect, the present invention provides the use of red date extract in the preparation of a drug for treating drug-induced intestinal adverse reactions or drug-induced intestinal damage.
[0010] Preferably, the intestinal adverse reaction includes diarrhea, and the intestinal damage includes intestinal mucosal sloughing.
[0011] Furthermore, the drug-induced intestinal adverse reaction or drug-induced intestinal damage is caused by irinotecan or a carboxylic acid-containing nonsteroidal anti-inflammatory drug.
[0012] Preferably, the carboxylic acid-containing non-steroidal anti-inflammatory drug comprises one or more of ketoprofen, diclofenac or indomethacin.
[0013] Preferably, the red date extract is an ethanol extract of red dates.
[0014] Furthermore, the red date extract includes any one or more of betulinic acid, lulutong acid, oleanolic acid, ursolic acid or corosolic acid, more preferably lulutong acid.
[0015] Preferably, the preparation method of the red date extract comprises the following steps:
[0016] (1) soaking red dates in ethanol, heating under reflux, and cooling to obtain a reflux product;
[0017] (2) separating the reflux product into solid and liquid, collecting the liquid, and drying to obtain the red date extract.
[0018] Preferably, the mass ratio of red dates to ethanol in step (1) is 1:5-10, for example: 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.
[0019] Preferably, the concentration of ethanol in step (1) is ≥ 70%, for example: 70%, 75%, 80%, 85%, 90%, 95%, 100%.
[0020] Preferably, the red dates in step (1) are soaked in ethanol for 0.5 to 3 h, for example: 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h.
[0021] Preferably, the heating reflux time in step (1) is 1 to 4 h, for example: 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h.
[0022] Preferably, the solid-liquid separation in step (2) can be carried out by filtration, centrifugation or the like, more preferably by filtration.
[0023] Preferably, the drying in step (2) includes but is not limited to low-temperature drying or freeze drying, more preferably freeze drying.
[0024] Preferably, the yield of the red date extract is ≥55%, such as 55%, 58%, 60%, or 65%.
[0025] In a third aspect, the present invention provides a method for preventing and treating drug-induced intestinal adverse reactions or drug-induced intestinal damage, the method comprising administering the above-mentioned red date extract to a patient.
[0026] Preferably, the intestinal adverse reaction includes diarrhea, and the intestinal damage includes intestinal mucosal sloughing.
[0027] Furthermore, the drug-induced intestinal adverse reaction or drug-induced intestinal damage is caused by irinotecan or a carboxylic acid-containing nonsteroidal anti-inflammatory drug.
[0028] Preferably, the carboxylic acid-containing non-steroidal anti-inflammatory drug comprises one or more of ketoprofen, diclofenac or indomethacin.
[0029] Beneficial effects of the present invention:
[0030] 1. The present invention uses ethanol as a solvent and adopts a heating reflux technology to extract red dates. The obtained red date extract contains pentacyclic triterpene acid compounds (such as lulutonic acid), and the extraction process is simple and the cost is low.
[0031] 2. The red date extract of the present invention has the function of inhibiting the activity of β-glucuronidase, and can be used to treat drug-induced diarrhea and intestinal damage caused by irinotecan or non-steroidal anti-inflammatory drugs, which deeply explores the efficacy of red dates and improves the medicinal value of red dates. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 Shown is the inhibition curve of jujube extract inhibiting β-GUS;
[0033] Figure 2 Shown is the binding profile of passutonic acid based on affinity ultrafiltration technology;
[0034] Figure 3Shown are molecular docking diagrams of passutonic acid and β-GUS, where A: docking diagram of passutonic acid and β-GUS; B: docking diagram of 4-nitrobenzene-β-D-glucuronide and β-GUS;
[0035] Figure 4 Shown is the inhibition curve of β-GUS by lulutonic acid, LA represents lulutonic acid, and POS represents D-glucaroyl-1,4-lactone monohydrate. DETAILED DESCRIPTION
[0036] The technical solution of the present invention is further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are only exemplary and do not limit the scope of the present invention.
[0037] Example 1 Preparation of red date extract
[0038] The specific process includes the following steps:
[0039] (1) 50 g of red dates were added to 400 g of 80% ethanol and soaked for 1 hour, then heated under reflux for extraction for 2 hours, and cooled to obtain a reflux product.
[0040] (2) filtering the reflux product obtained in step (1), collecting the filtrate, and freeze-drying to obtain a red date extract with a yield of 58%.
[0041] Example 2 Enzyme Activity Inhibition Experiment of Red Date Extract
[0042] The inhibitory activity of red jujube extract against β-glucuronidase (β-GUS) from Escherichia coli was determined by in vitro incubation system of 4-nitrobenzene-β-D-glucuronide (PNPG) as the specific substrate.
[0043] The specific process is as follows:
[0044] (1) Set a series of red date extract concentrations: based on the amount of raw medicine, equivalent to 0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.5mg / mL, 1mg / mL, 2mg / mL, 4mg / mL, 8mg / mL, 16mg / mL, 32mg / mL. The mixed reaction system contains drugs, β-GUS enzyme (20μg / mL), PNPG (1mM) and an appropriate amount of PBS. A total of 4 groups were set up, namely blank group, control group, drug control group and sample group.
[0045] Blank group system: drug (0μL) + β-GUS (0μL) + PBS (100μL) + PNPG (20μL) Control group system: drug (0μL) + β-GUS (20μL) + PBS (80μL) + PNPG (20μL)
[0046] Drug control system: drug (20 μL) + β-GUS (0 μL) + PBS (80 μL) + PNPG (20 μL)
[0047] Sample group system: drug (20μL) + β-GUS (20μL) + PBS (60μL) + PNPG (20μL)
[0048] (2) According to the above reaction system, the drug, β-GUS and an appropriate amount of PBS were incubated at 37°C for 30 min, and then 20 μL PNPG was added and incubated for 30 min. After the incubation, 5 μL of 2 mol NaOH solution was added to terminate the incubation, and the absorbance was measured at 405 nm to calculate the enzyme activity of β-GUS.
[0049] Calculation formula: Enzyme activity = (A (样品组) -A (药物对照组) ) / (A (对照组) -A (空白对照组) )×100
[0050] like Figure 1 As shown, the red date extract inhibited β-GUS activity in a dose-dependent manner, indicating that there are specific inhibitory components for β-GUS in the red date extract.
[0051] Example 3 Affinity ultrafiltration experiment of red date extract
[0052] According to the results of Example 1, potential components in the red date extract that can bind to β-GUS were screened using affinity ultrafiltration technology.
[0053] The specific process is as follows:
[0054] (1) Qualitative analysis: Weigh an appropriate amount of red jujube extract to prepare a solution with a concentration of 50 mg / mL (based on the amount of crude drug), centrifuge at 12,000 × g for 10 min, and then inject the sample for analysis.
[0055] The specific instrument parameters are as follows:
[0056] Chromatographic analysis was performed using an ACQUITY UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm); the mobile phase was 0.1% formic acid water (A)-acetonitrile (B), gradient elution (0-1 min, 99% A; 1-35 min, 99%-1% A; 35-36 min, 1%-1% A, 36-36.5 min, 1%-99% A; 36.5-40 min, 99%-99% A); column temperature was 40°C; flow rate was 0.3 mL / min; injection volume was 3 μL.
[0057] The mass spectrometry analysis used an electrospray ionization source, with positive and negative ion detection modes. The sheath gas flow rate was 35arb, the auxiliary gas flow rate was 10arb, the capillary temperature was 320°C, and the spray voltage was 3.5kV (+), 2.5kV (-). The acquisition range was m / z 100-1500, the full scan resolution (full MS) was 60000, the secondary resolution (MS / MS) was 15000, and the collision energy (multi-level mode) was 20V / 40V / 60V.
[0058] (2) 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 h to inactivate the enzyme for later use.
[0059] (3) Incubation: Referring to the enzyme activity inhibition experimental steps in Example 2, the reaction system was set to 20 μL of drug + 20 μL of enzyme + 60 μL of PBS. The red date extract was incubated with β-GUS enzyme and β-GUS inactivating enzyme at 37°C for 1 hour, and centrifuged at 12,000×g for 10 minutes.
[0060] (4) Separation: The mixed solution was transferred to a 100 KDa ultrafiltration tube, allowed to stand at low temperature for 30 min, and centrifuged at 12,000 × g for 10 min. The ultrafiltration tube was rinsed with 240 μL of PBS, and the standing-centrifugation process was repeated twice.
[0061] (5) Dissociation: Transfer the ultrafiltration tube to a new EP tube, add 240 μL of 80% methanol to the ultrafiltration tube, let stand at low temperature for 1 hour, centrifuge at 12,000 × g for 10 minutes, and repeat 3 times.
[0062] (6) Sample analysis: The centrifuged solutions were combined, dried with N2, reconstituted with 50% methanol, and analyzed by LC-MS.
[0063] (7) Binding force calculation: Calculate the binding rate (BD) based on the peak area results.
[0064] BD=(Aa-Ab) / Aa×100%
[0065] Aa: chromatographic peak area of the corresponding compound after the drug reacts with the inactivated enzyme;
[0066] Ab: The chromatographic peak area of the corresponding compound after the drug reacts with the active enzyme.
[0067] (8) Experimental results: After comparison with the database and reference substances, the red date extract in this experiment contained chlorellanic acid.
[0068] As shown in Table 1, the binding rate of the lulutonic acid to β-GUS was 90.11%.
[0069] like Figure 2As shown, the affinity ultrafiltration results showed that the component could strongly bind to β-GUS, suggesting that lulutonic acid is a specific inhibitor of β-GUS.
[0070] Table 1 Binding rate of β-GUS to lulutong acid in red date extract
[0071]
[0072] Example 4 Molecular docking experiment of thiamethoxam acid and β-GUS
[0073] With the help of molecular docking technology, 4-nitrobenzene-β-D-glucuronide (PNPG) was used as a positive control to clarify the binding site of lulutonic acid and β-GUS, and compare the differences in their binding abilities with PNPG.
[0074] The specific process is as follows:
[0075] (1) Ligand processing: The mol2 format of passutonic acid (4481-62-3) and 4-nitrobenzene-β-D-glucuronide (10344-94-2) was obtained from the PubChem database, and the energy minimization was performed using Chem3D software and saved in pdb format. AutodockTools1.5.6 further detected the root of the ligand, searched and defined the rotatable bonds, and saved them as pdbqt files.
[0076] (2) Receptor processing: The sdf format of β-GUS (3LPG) was obtained from the PDB database, and the PyMOL2.1.1 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.
[0077] (3) Molecular docking: Autodockvina 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 image was visualized using PyMOL.
[0078] (4) Experimental results: Figure 3 The molecular docking results show that lulutonic acid can produce multiple bonding interactions with β-GUS, and has the same binding site LEU-18 as PNPG, and exhibits strong binding efficiency overall, with a binding force of -8.8Kcal / mol, close to the positive control 4-nitrobenzene-β-D-glucuronide (-7.2Kcal / mol). This suggests that lulutonic acid produces a competitive binding effect similar to 4-nitrobenzene-β-D-glucuronide by acting on the active site of β-GUS.
[0079] Example 5 Experiment on the inhibition of β-GUS enzyme activity by lulutong acid
[0080] Using 4-nitrobenzene-β-D-glucuronide (PNPG) as the specific substrate, the β-glucuronidase (β-GUS) from Escherichia coli was incubated in vitro and a positive inhibitor group (D-glucaroyl-1,4-lactone monohydrate, POS) was set up to determine and compare the inhibitory activity of lulutonic acid on β-GUS.
[0081] The specific process is as follows:
[0082] (1) The concentrations of D-glucaric acid-1,4-lactone monohydrate and lulutonic acid were set to 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM, and 640 μM, respectively.
[0083] (2) The mixed reaction system includes drugs, β-GUS enzyme (20 μg / mL), PNPG (1 mM) and appropriate amount of PBS. A total of 4 groups were set up, namely blank group, control group, drug control group and sample group.
[0084] Blank group system: drug (0μL) + β-GUS (0μL) + PBS (100μL) + PNPG (20μL) Control group system: drug (0μL) + β-GUS (20μL) + PBS (80μL) + PNPG (20μL)
[0085] Drug control system: drug (20 μL) + β-GUS (0 μL) + PBS (80 μL) + PNPG (20 μL)
[0086] Sample group system: drug (20μL) + β-GUS (20μL) + PBS (60μL) + PNPG (20μL)
[0087] (3) According to the above reaction system, the following experiments were carried out using lulutonic acid (LA) and D-glucaric acid-1,4-lactone monohydrate (POS) as drug samples:
[0088] The drug, β-GUS and an appropriate amount of PBS were incubated at 37°C for 30 min, followed by the addition of 20 μL PNPG 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.
[0089] Calculation formula: Enzyme activity = (A (样品组)- A (药物对照组) ) / (A (对照组)- A (空白对照组) )×100
[0090] (4) Experimental results: Figure 4As shown, the inhibition of β-GUS activity by lulutonic acid showed a dose-dependent relationship, and the IC50 value was close to that of a positive inhibitor. As the concentration of the drug was greater than 160 μM, the inhibitory ability of lulutonic acid on β-GUS activity was higher than that of D-glucaric acid-1,4-lactone monohydrate, indicating that lulutonic acid can be used as a specific inhibitor of β-GUS.
[0091] 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.
Claims
1. The use of red date extract in the preparation of β-glucuronidase inhibitors, characterized in that: The application is an in vitro application.
2. Application of red date extract in the preparation of drugs for treating drug-induced intestinal adverse reactions or drug-induced intestinal damage.
3. The use according to claim 2, characterized in that: The intestinal adverse reactions include diarrhea, and the intestinal damage includes intestinal mucosal sloughing.
4. The use according to claim 2, characterized in that: The drug-induced intestinal adverse reaction or drug-induced intestinal damage is caused by irinotecan or a carboxylic acid-containing nonsteroidal anti-inflammatory drug; preferably, the carboxylic acid-containing nonsteroidal anti-inflammatory drug includes one or more of ketoprofen, diclofenac or indomethacin.
5. The use according to any one of claims 1 to 4, characterized in that: The red date extract contains one or more of betulinic acid, lulutonic acid, oleanolic acid, ursolic acid or corosolic acid.
6. The use according to any one of claims 1 to 4, characterized in that: The preparation method of the red date extract comprises the following steps: (1) soaking red dates in ethanol, heating under reflux, and cooling to obtain a reflux product; (2) separating the reflux product into solid and liquid, collecting the liquid, and drying to obtain the red date extract.
7. The use according to claim 6, characterized in that: The mass ratio of red dates to ethanol in step (1) is 1:5-10.
8. The use according to claim 6, characterized in that: The concentration of ethanol in step (1) is ≥70%.
9. The use according to claim 6, characterized in that: The time for soaking the red dates in ethanol in step (1) is 0.5 to 3 hours.
10. The use according to claim 6, characterized in that: The heating reflux time in step (1) is 1 to 4 hours.