Application of salidrosin or salidrosin-containing product in preparation of urease inhibitor
By using Hongjingtianin as a drug for anti -pyloric pyloriase, the problem of susceptible degradation and severe side effects of existing pyretease inhibitors, realizing the effective suppression of Helicobacter oligraphy enzymes, and potentially the effect of treating gastric diseases Essence
Patent Information
- Application Number
- CN202411694936.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-01-24
AI Technical Summary
Existing urease inhibitors, such as acetyloxyxamic acid, have problems with prone to degradation and serious side effects, limiting their practical application in the medical field.
Rhodiola rosea was used as a candidate drug against Helicobacter pylori urease, and its effect in inhibiting Helicobacter pylori urease was verified through in vitro experiments and molecular docking technology.
Red scenery shows a good inhibitory inhibitory of the pyloric pyrhane enzyme activity, and its inhibitory effect is stronger than acetyoxic acid, which has the effects of potential treatment and prevention of gastric diseases.
Smart Images
Figure CN119345216A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the field of biomedicine technology, and specifically relates to the application of salidroside or a product containing salidroside in the preparation of urease inhibitors. Background Art
[0002] Helicobacter pylori is a Gram-negative bacillus that can cause a variety of gastric diseases, including gastritis, gastric ulcers, gastric polyps, and gastric cancer. Since 1994, the International Agency for Research on Cancer under the WHO has classified Helicobacter pylori as a Class I carcinogen for gastric cancer, which shows the severity and harm of this pathogen to humans.
[0003] Urease is an important virulence factor of Helicobacter pylori and the main cause of Helicobacter pylori pathogenicity. Urease can hydrolyze urea in the stomach to produce ammonia, neutralize gastric acid, and thus establish a suitable pH environment for the survival and colonization of Helicobacter pylori. In the catalytic process of urease, nickel ions (Ni 2+ ) and the sulfhydryl group at the active site of urease play an extremely important role. Urease activity is also considered to be an important factor in the biological utilization of nitrogen. The enzyme can decompose and reabsorb urea, leading to an abnormal increase in the environmental pH value and inducing various diseases in the human body, including inflammation, ulcers, stones, etc. Therefore, urease is considered to be a key target for the development of antibacterial drugs.
[0004] As a potential effective urease inhibitor for the treatment of Helicobacter pylori infection, active ingredients from natural sources have attracted a lot of attention and research. Salidroside is a small molecule compound extracted from the Rhodiola rosea plant. It has anti-CYP2D6 (2D6 subtype of cytochrome P450 enzyme) and AChE (acetylcholinesterase) activity, as well as antioxidant and neuroprotective activity. It can regulate the HIF-1α signaling pathway to protect the central nervous system. However, so far, there has been no report on the use of Salidroside as a Helicobacter pylori urease inhibitor. Summary of the invention
[0005] Although studies have shown that acetohydroxamic acid (AHA) can kill Helicobacter pylori by inhibiting urease activity, AHA is easily degraded in the stomach and can easily cause obvious and serious side effects in most patients (it can cause skin diseases, neurological and hematological diseases when used in large doses). These defects limit the practical application of AHA in the medical field. Therefore, the development of safer and more widely used urease inhibitors has become a new trend.
[0006] The purpose of this application is to provide the use of salidroside or a product containing salidroside in the preparation of urease inhibitors. This application has found through research that salidroside has a good inhibitory effect on Helicobacter pylori, and through in vitro experiments and molecular docking technology verification, salidroside can be used as a candidate drug for anti-Helicobacter pylori urease. In order to achieve the above invention purpose, the technical solution adopted by this application is:
[0007] In a first aspect, the present application provides the use of salidroside or a product containing salidroside in the preparation of a urease inhibitor.
[0008] In an alternative embodiment, the urease is Helicobacter pylori urease.
[0009] In a second aspect, the present application provides the use of salidroside or a product containing salidroside in the preparation of an anti-Helicobacter pylori product.
[0010] In an alternative embodiment, the anti-Helicobacter pylori activity includes inhibiting Helicobacter pylori urease activity.
[0011] In an optional embodiment, the anti-Helicobacter pylori product contains salidroside or a product containing salidroside as the sole active ingredient.
[0012] In a third aspect, the present application provides the use of salidroside or a product containing salidroside in the preparation of a digestive tract flora regulator.
[0013] In an optional embodiment, the efficacy of the digestive tract flora regulator includes downregulating the abundance of Helicobacter pylori in the digestive tract.
[0014] In a fourth aspect, the present application provides the use of salidroside or a product containing salidroside in the preparation of a method for treating and / or preventing gastric diseases caused by Helicobacter pylori infection.
[0015] In an optional embodiment, the gastric disease caused by Helicobacter pylori infection includes at least one of gastritis, gastric ulcer, gastric polyp or gastric cancer caused by Helicobacter pylori infection.
[0016] In an optional embodiment, the product containing salidroside contains salidroside as the only active ingredient.
[0017] Among them, the chemical structural formula of Rhodiola rosea involved in this application is:
[0018]
[0019] Based on the above technical solution, this application has at least the following beneficial effects:
[0020] The present application discloses for the first time that salidroside has a good inhibitory effect on Helicobacter pylori urease, and verifies the above findings through in vitro experiments and molecular docking technology. Therefore, salidroside can be used as a candidate drug against Helicobacter pylori urease, and further can be used to prepare drugs for the treatment and / or prevention of gastritis, gastric ulcer and other stomach-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0022] Figure 1A -B is a graph showing the results of a test of salidroside inhibiting the urease activity of Helicobacter pylori in the examples of the present application;
[0023] Figure 2A -B is a result diagram of the study on the inhibition type of Helicobacter pylori urease by Rhodiola rosea in the examples of the present application;
[0024] Figure 3 is a result diagram of the effect of thiol-containing compounds on the urease activity of Helicobacter pylori in the examples of the present application;
[0025] Figure 4 is a result diagram of the effect of inorganic compounds on the urease activity of Helicobacter pylori in the examples of the present application;
[0026] Figure 5 This is a result diagram of a test on the reactivation of Helicobacter pylori urease inhibited by salidroside in the examples of the present application;
[0027] Figure 6 This is a result diagram of the molecular docking study of salidroside and the active site of Helicobacter pylori urease in the examples of this application. DETAILED DESCRIPTION
[0028] In order to further explain the technical means and results taken by this application to achieve the predetermined invention purpose, the specific implementation methods, technical solutions and features of this application are described in detail below with preferred embodiments. The specific features, structures, or characteristics of the multiple embodiments in the following description can be combined in any suitable form.
[0029] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.
[0030] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.
[0031] 1. Experimental Materials
[0032] 1. Test drug
[0033] Rhodiola rosea was purchased from MedChemExpress with a purity of 99.36%; urea, dithiothreitol (DTT), cysteine (L-cys), glutathione (GSH), sodium fluoride (NaF), and boric acid (BA) were all purchased from Beijing Solebow Technology Co., Ltd.; sodium salicylate, sodium nitroprusside, sodium hydroxide, and sodium hypochlorite were all purchased from Maclean and were of analytical grade.
[0034] 2. Cultivation of Helicobacter pylori
[0035] Helicobacter pylori (ATCC 43504) was inoculated on Columbia agar containing bovine serum albumin and resuscitated for 72 hours at 37°C, 5% O2, 10% CO2 and 85% N2 under microaerobic conditions. The biological characteristics and biochemical reactions of Helicobacter pylori were identified on the plates after 3 days of resuscitation. The identified and uncontaminated Helicobacter pylori were subcultured. The subculture method is: scrape the bacterial mass from the mother plate with an inoculation loop, inoculate it in Brucella broth with 10% inactivated horse serum, and then culture it in a can for 24 hours.
[0036] 3. Extraction of Helicobacter pylori urease
[0037] Take 50mL of Helicobacter pylori bacterial liquid, centrifuge at 5000g and 4℃ to separate and collect Helicobacter pylori precipitate, wash the precipitate twice with pH 7.4 phosphate buffer, store it at -80℃ for 24h, then take it out and let it cool to room temperature, add 3mL of distilled water and protease inhibitors, ultrasonicate for 1min, centrifuge at 15000g and 4℃ for 10min, take out the upper clear layer and desalt it through Sephadex G-25 column to obtain urease solution, add an equal volume of glycerol and store it at 4℃.
[0038] 4. Preparation of drugs
[0039] Weigh an appropriate amount of rhodiola rosea, prepare it into corresponding concentrations with dimethyl sulfoxide solution (DMSO), dilute it in gradients, store it at -80°C in the dark for later use.
[0040] 5. Preparation of urea
[0041] Weigh an appropriate amount of urea, dissolve it in phosphate buffer (pH 7.4) to prepare a 20 mM urea solution, and store it in a refrigerator at 4°C for later use.
[0042] 6. Preparation of Berthelot colorimetric solution
[0043] Solution A: Weigh appropriate amounts of sodium nitroprusside (sodium nitrosoferricyanide) and sodium salicylate powder respectively, dissolve them in phosphate buffer (pH 7.4) to prepare a colorimetric solution A containing 9.73 mM sodium nitroprusside and 700 mM sodium salicylate, and store at 4°C away from light for later use;
[0044] Solution B: Weigh 18 g of sodium hydroxide and dissolve it in phosphate buffer (pH 7.4). After cooling, add 24 mL of sodium hypochlorite, mix well, make up to 100 mL, and store at 4°C away from light for later use.
[0045] 2. Experimental Methods
[0046] 1. Test on the inhibition of Helicobacter pylori urease activity by Rhodiola rosea
[0047] A series of concentrations of rhodiola rosea solution (5-320 μM) and 100 μL of a certain concentration of Helicobacter pylori urease solution were mixed and incubated at 37°C for 20 min, then 100 μL of urea solution was added at room temperature, and the reaction was protected from light for 20 min. Then, berthelot color development solution (first add solution A and then solution B, 100 μL each) was added, and the color was protected from light for 10 min. Then 200 μL of the incubation solution was aspirated onto a 96-well plate, and the OD at 630 nm was measured by an enzyme reader. 绝对 The corresponding OD is obtained according to formula 1. 相对 The residual enzyme activity RA was calculated according to formula 2, and the corresponding half inhibition concentration IC50 was calculated through the concentration-residual enzyme activity curve. The solvent with added substances was used as blank control and the measurement was repeated 3 times.
[0048] OD 相对 =OD 绝对 –OD 空白 (Formula 1);
[0049] RA(%)=OD 相对 sample / OD 相对 control ×100% (Formula 2).
[0050] 2. Study on the inhibitory type of salidroside on Helicobacter pylori urease
[0051] Take 100 μL of each series of rhodiola rosea solution (0, 40, 80, 120 μM), mix with a certain concentration of Helicobacter pylori urease solution, and incubate at 37°C for 20 min. Then add 100 μL of a series of urea solutions (1, 1.5, 2, 6, 10, 15 mM) at room temperature, react in the dark for 20 min, and then perform berthelot color development and measure OD according to the method in "Test 1". 绝对 The corresponding OD 相对 The solvent with added substances was used as blank control and the test was repeated three times. The experiment was carried out by measuring the inverse of the reaction rate (1 / V, i.e. 1 / OD 相对 ) and the inverse of the substrate concentration (1 / [urea]) were plotted as a Lineweaver-Burk plot, and the kinetic parameter K was finally obtained by combining the LB curve with formula 3 M 、V max The inhibition constant K was obtained by secondary plotting of the LB curve. i .
[0052] 1 / OD 相对 =1 / V max +K M / V max *1 / [urea](Formula 3).
[0053] 3. Effects of sulfhydryl compounds on urease activity of Helicobacter pylori
[0054] Take the Helicobacter pylori urease solution, the thiol compound solution (DTT solution, L-Cys solution and GSH solution, all with a concentration of 1.25 mM) and the 200 μM shodrogenin solution, first mix two of the solutions, place them at 37 ° C for 20 minutes, then add the third solution, mix them, and incubate them at 37 ° C for another 20 minutes, then add urea to the co-incubation solution at room temperature and react for 20 minutes, then perform berthelot color development according to the method in "Test 1" and measure the OD 绝对 The corresponding OD 相对 The value was calculated and the corresponding residual enzyme activity RA was calculated. The solvent with added substances was used as blank control and the measurement was repeated 3 times.
[0055] 4. Effects of inorganic compounds on urease activity of Helicobacter pylori
[0056] Take 100 μL of Helicobacter pylori urease solution and inorganic compound solution (BA solution, NaF solution, both with a concentration of 1.25 mM) and mix them evenly, place them in a 37°C incubator for pre-incubation for 20 min, then add 100 μL of rhodiola rosea solution (200 μM) and mix them evenly, place them in a 37°C incubator for another 20 min, then take them out, then add 100 μL of urea at room temperature and react for 20 min, then perform berthelot color development according to the method in "Test 1" and measure the OD 绝对 The corresponding OD 相对 The value was calculated and the corresponding residual enzyme activity RA was calculated. The solvent with added substances was used as blank control and the measurement was repeated 3 times.
[0057] 5. Experiment on the inhibition of reactivation of Helicobacter pylori urease by shodiola rosea
[0058] Equal volumes of Helicobacter pylori urease solution and rhodiola rosea solution (200 μM) were mixed and incubated in a 37°C incubator for 20 min. Half of the incubation solution was taken out and added with an appropriate amount of GSH solution (1.25 mM). The other half of the incubation solution was not treated and incubated in a 37°C incubator for 0-90 min. At each designated time point, two incubation solutions, 40 μL each, were taken out and 40 μL of urea solution was added to each of them. The mixture was reacted in the dark at room temperature for 20 min, and then berthelot color development was performed according to the method in "Test 1" to measure the OD 绝对 The corresponding OD 相对 The value was calculated and the corresponding residual enzyme activity RA was calculated. The solvent with added substances was used as blank control and the measurement was repeated 3 times.
[0059] 6. Molecular docking study of salidroside and the active site of Helicobacter pylori urease
[0060] In order to study the binding mode of salidroside and Helicobacter pylori urease, the present application uses AutoDock Vina1.2.3 software 1 to perform molecular docking work, wherein the Helicobacter pylori ureB protein crystal structure used for docking is downloaded from the PDB database, and the PDB ID is 6zja, and the 3D structure of salidroside is downloaded from the PUBCHEM database, and energy is minimized under the MMFF94 force field. Before docking begins, PyMol 2.5.52 is used to process the receptor protein, including removing water molecules, salt ions and small molecules. The docking box is then set to wrap the entire protein structure. In addition, ADFRsuite 1.03 is used to convert all processed small molecules and receptor proteins into the PDBQT format required for AutoDock Vina1.2.3 docking. During docking, the exhaustiveness of the global search is set to 32, and the remaining parameters remain the default settings. The docking conformation with the highest score output is considered by us to be the binding conformation, and finally the PyMol 2.5.5 docking results are used for visual analysis.
[0061] 3. Experimental Results
[0062] 1. Test on the inhibition of Helicobacter pylori urease activity by Rhodiola rosea
[0063] The results are as follows Figure 1A and 1B As shown, the half inhibitory concentration (IC50) of salidroside for inhibiting Helicobacter pylori urease activity is 82.38±5.46μM, while the half inhibitory concentration (IC50) of acetohydroxamic acid, a recognized standard urease inhibitor, for inhibiting Helicobacter pylori urease activity is 88.10±7.50μM, indicating that the inhibitory activity of salidroside for Helicobacter pylori urease is stronger than that of acetohydroxamic acid.
[0064] 2. Study on the inhibitory type of salidroside on Helicobacter pylori urease
[0065] The results are as follows Figure 2A and 2B As shown in the figure, in the absence of salidroside, the Michaelis constant K of the enzymatic reaction of Helicobacter pylori urease M The maximum reaction rate was 6.41±2.43μM, and the maximum reaction rate was V max It can be seen from the Lineweaver-Burk double reciprocal plot that these nearly parallel lines pass through different points on the y-axis intercept. After adding different concentrations of salidroside, the kinetic parameters of its inhibition of Helicobacter pylori urease lead to K M 、V maxAll of them were reduced, so it can be preliminarily inferred that the type of action of salidroside in inhibiting Helicobacter pylori urease is the anti-competitive inhibition type. In addition, combined with the type of inhibition of Helicobacter pylori urease by salidroside, the intercept of the Lineweaver-Burk double reciprocal plot was plotted twice to obtain the inhibition constant Ki of salidroside in inhibiting Helicobacter pylori urease, which is 146.40±2.19μM.
[0066] 3. Effects of sulfhydryl compounds on urease activity of Helicobacter pylori
[0067] Studies have shown that sulfhydryl compounds can interact with the sulfhydryl group (-SH) of urease and are recognized as urease activators. Therefore, this experiment used three thiol compounds (DTT, GSH, L-Cys) to study the potential inactivation sites of salidroside on urease. Figure 3 It was shown that in the salidroside-sulfhydryl compound-urease interaction test, even in the presence of salidroside, Helicobacter pylori urease can still maintain a high enzyme activity, and the residual urease activity is related to the order of adding salidroside, urease and sulfhydryl compounds. From this, we can infer that one of the action sites of salidroside in inhibiting the activity of Helicobacter pylori urease is the sulfhydryl group in the urease amino acid series.
[0068] 4. Effects of inorganic compounds on urease activity of Helicobacter pylori
[0069] Inorganic compounds (NaF and BA) interact with nickel ions (Ni 2+ ) reaction, and is usually used as a competitive inhibitor to study whether the inhibitory target of the inactivator is the enzyme active site Ni 2+ The results are as follows Figure 4 As shown in the figure, the inhibition degree of urease by salidroside, NaF and BA was inconsistent. When H. pylori urease was simultaneously present with salidroside and BA or NaF in the system, the residual urease activity was reduced, slightly higher than the residual enzyme activity when salidroside acted alone on H. pylori urease, indicating that BA and NaF had a weak protective effect on the activity of H. pylori urease.
[0070] 5. Experiment on the inhibition of reactivation of Helicobacter pylori urease by shodiola rosea
[0071] In this experiment, GSH was used to determine the stability of the urease-salidroside complex. Figure 5As shown in the results, after incubation with salidroside for 20 minutes, the activity of urease decreased from 100% to 30%. After adding 1.25mM GSH, the urease activity gradually recovered within 90 minutes, and finally the urease activity of Helicobacter pylori could be restored to 47.80% of the initial activity. The results showed that the reaction between urease and salidroside was reversible. The experimental results further supported that the inactivation of urease was related to the binding of salidroside to the sulfhydryl group of the urease active site.
[0072] 6. Molecular docking study of salidroside and the active site of Helicobacter pylori urease
[0073] After running and calculating the molecular docking program, the docking score of salidroside and Helicobacter pylori urease was -8.678 kcal / mol, indicating that it binds well to the ureB protein. Figure 6 As shown in the diagram of the interaction between salidroside and urease ureB protein, it can be seen that it does not interact with the active site Ni of urease 2+ Instead of interacting, it forms hydrogen bonds with ALA-365, ALA-169, HIS-221, GLY-279, ARG-338, and HIS-322 on the ureB protein. The formation of hydrogen bonds makes the protein and small molecules bind more tightly. Among them, ALA-365, ALA-169, HIS-221, and HIS-322 are essential groups in the flap region of urease. The strong hydrogen bonds formed by salidroside and these essential groups keep the flap conformation open, thereby changing the enzyme activity.
[0074] The above is only a preferred specific implementation of the present application; however, the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved ideas of the present application within the technical scope disclosed in the present application, which should be included in the protection scope of the present application.
Claims
1. Application of salidroside or products containing salidroside in the preparation of urease inhibitors.
2. The use according to claim 1, characterized in that: The urease is Helicobacter pylori urease.
3. Application of salidroside or products containing salidroside in the preparation of anti-Helicobacter pylori products.
4. The use according to claim 3, characterized in that: The anti-Helicobacter pylori activity includes inhibiting the urease activity of Helicobacter pylori.
5. The use according to claim 3, characterized in that: The anti-Helicobacter pylori product uses salidroside or a product containing salidroside as the only active ingredient.
6. Use of salidroside or products containing salidroside in the preparation of digestive tract flora regulators.
7. The use according to claim 6, characterized in that: The efficacy of the digestive tract flora regulator includes downregulating the abundance of Helicobacter pylori in the digestive tract.
8. Use of salidroside or a product containing salidroside in the preparation of a drug for treating and / or preventing gastric diseases caused by Helicobacter pylori infection.
9. The use according to claim 8, characterized in that: The gastric disease caused by Helicobacter pylori infection includes at least one of gastritis, gastric ulcer, gastric polyp or gastric cancer caused by Helicobacter pylori infection.
10. The use according to any one of claims 1 to 9, characterized in that The product containing rhodiola rosea contains rhodiola rosea as the only active ingredient.