Helicobacter pylori urease inhibitor and application thereof

Abstract

The invention relates to application of trimethylglycine and salts thereof as helicobacter pylori urease inhibitors. The compound has an efficient inhibition effect on helicobacter pylori urease, and can be used as a novel medicine for treating gastric ulcer, duodenal ulcer and gastritis caused by helicobacter pylori infection.

Description

Technical Field

[0001] The present invention relates to the inhibitory activity of trimethylglycine on Helicobacter pylori urease and the application of trimethylglycine in treating gastric ulcer, duodenal ulcer and gastritis caused by Helicobacter pylori infection. Background Art

[0002] At present, with the development of society and changes in people's lifestyles, the incidence of peptic ulcers caused by factors such as smoking, drinking, emotional stress, drug stimulation, Helicobacter pylori infection, etc. is gradually increasing, becoming a common and frequently occurring disease, bringing great pain to patients.

[0003] Proton pump inhibitors (PPIs) are currently the most advanced class of drugs for the treatment of peptic ulcers (Li H, Meng L, Liu F, et al. H + / K + -ATPase inhibitors: a patentreview. Expert Opin Ther Patents, 2013, 23: 99–111). However, after acid suppression treatment with proton pump inhibitors, peptic ulcers are prone to recurrence. Helicobacter pylori is a Gram-negative bacterium that lives in the human stomach. It is the main pathogenic factor of gastritis, gastric ulcers, and duodenal ulcers, and is listed as the main cause of gastric cancer by the World Health Organization. Currently, triple therapy (1 proton pump inhibitor or colloidal bismuth + 2 antibiotics) or quadruple therapy (1 proton pump inhibitor + colloidal bismuth + 2 antibiotics) is usually used to treat Helicobacter pylori infection. However, this therapy takes a long time to take medicine, is prone to side effects such as upper abdominal distension and pain, antibiotic resistance, and has a high cost of medication.

[0004] Urease in Helicobacter pylori is a hydrolase that catalyzes the decomposition of urea into ammonia and carbon dioxide, increasing the ammonia concentration in gastric acid, thereby neutralizing part of the gastric acid and forming a nearly neutral liquid film on the surface of the bacteria, providing a suitable microenvironment for Helicobacter pylori, thereby enabling Helicobacter pylori to survive in the highly acidic environment of the stomach (Krajewska B. Ureases I. Functional, catalytic and kinetic properties:A review. J Mol Catal B Enzym, 2009, 59 (1): 9-21). Therefore, urease has become an important target for the design of anti-Helicobacter pylori drugs in recent years.

[0005] Trimethylglycine is a quaternary ammonium alkaloid. Its pure form is prismatic or leaf-shaped white crystals, soluble in water, methanol and ethanol. As a natural plant endogenous component and food nutrient, trimethylglycine has the advantages of safety and non-toxicity, and has been synthesized in large quantities and is inexpensive.

[0006] This study discovered for the first time that trimethylglycine has significant Helicobacter pylori urease inhibitory activity, and has the advantages of high cost-effectiveness and good biosafety. It can be used as a new type of therapeutic drug for peptic ulcer and gastritis caused by Helicobacter pylori infection. Summary of the invention

[0007] The object of the present invention is to provide a novel highly active Helicobacter pylori urease inhibitor trimethylglycine, the structural formula of which is: .

[0008] The Helicobacter pylori urease inhibitor is used in preparing medicine for treating gastric ulcer, duodenal ulcer and gastritis caused by Helicobacter pylori infection.

[0009] The inhibitors also include acceptable salts including hydrochloride, citrate, malate, salicylate, fumarate, sulfonate or methanesulfonate.

[0010] Another technical solution of the present invention is to provide a Helicobacter pylori urease inhibitor, which is trimethylglycine or an acceptable salt thereof, including hydrochloride, citrate, malate, salicylate, fumarate, sulfonate or methanesulfonate.

[0011] Helicobacter pylori urease inhibition activity assay: 1. Helicobacter pylori strain culture Helicobacter pylori was inoculated in Brucella broth containing horse serum (10%) and cultured at 37 °C under microaerobic conditions (85% N2, 10% CO2 and 5% O2) for 24 h.

[0012] 2. Preparation of Helicobacter pylori urease The cultured Helicobacter pylori culture was centrifuged at 5000g for 15 min at 4℃, and the Helicobacter pylori was collected and washed three times with pH 7.4 phosphate buffer. The Helicobacter pylori was stored at -80℃ for 24 h, then taken out and restored to room temperature, 3 mL of distilled water and protease inhibitors were added, ultrasonicated for 1 min, and centrifuged at 12000g for 10 min at 4℃. The supernatant was desalted through a SephadexG-25 column, and an equal volume of glycerol was added to the crude enzyme, and stored at 4℃ for activity determination.

[0013] 3. Determination of Helicobacter pylori urease inhibition activity The activity test of urease was carried out according to the method reported by Weatherburn. The specific steps are as follows: add 25 μL of Helicobacter pylori urease solution (10 U) and 25 μL of the test compound (the concentration is set according to the experiment and prepared with DMSO-phosphate buffer solution) to a 96-well plate, co-culture at 37°C for 30 minutes, add 50 μL of phosphate buffer solution containing 25 mmol of urea, co-culture at 37°C for 30 minutes, then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl), co-culture at 37°C for 30 minutes, and measure the absorbance value at 620 nm. A blank group, a normal control group, and a positive control group (acetohydroxamic acid) were set up to calculate the inhibition rate and half inhibition concentration IC of the inhibitor. 50 .

[0014] This study found that trimethylglycine and its salts have significant Helicobacter pylori urease inhibitory activity, with a half inhibitory concentration IC 50 The concentration of trimethylglycine is 2.2-3.6 µM, and it has the advantages of high cost performance and good biosafety. Trimethylglycine and its salts can be used as a new type of drug for the treatment of peptic ulcer and gastritis caused by Helicobacter pylori infection. DETAILED DESCRIPTION

[0015] Example 1 Add 25 μL of Helicobacter pylori urease solution (10 U) and 25 μL of trimethylglycine solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with phosphate buffer solution) to a 96-well plate, co-culture at 37°C for 30 min, add 50 μL of phosphate buffer solution containing 25 mmol of urea, co-culture at 37°C for 30 min, then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl), co-culture at 37°C for 30 min, and measure the absorbance at 620 nm. Set up a blank group, a normal control group, and a positive control group (acetohydroxamic acid) to calculate the half-maximal inhibitory concentration IC of trimethylglycine on Helicobacter pylori urease. 50 The positive control group acetohydroxamic acid IC 50 is 18.1 µM.

[0016] Example 2 Add 25 μL of Helicobacter pylori urease solution (10 U) and 25 μL of trimethylglycine hydrochloride solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with phosphate buffer solution) to a 96-well plate, co-culture at 37°C for 30 min, add 50 μL of phosphate buffer solution containing 25 mmol of urea, co-culture at 37°C for 30 min, then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl), co-culture at 37°C for 30 min, and measure the absorbance at 620 nm. Set up a blank group, a normal control group, and a positive control group (acetohydroxamic acid) to calculate the half-maximal inhibitory concentration IC of trimethylglycine hydrochloride on Helicobacter pylori urease. 50 The positive control group acetohydroxamic acid IC was 2.5µM. 50 is 18.1 µM.

[0017] Example 3 25 μL of Helicobacter pylori urease solution (10 U) and 25 μL of trimethylglycine citrate solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with phosphate buffer solution) were added to a 96-well plate and co-cultured at 37°C for 30 min. 50 μL of phosphate buffer solution containing 25 mmol of urea was added and co-cultured at 37°C for 30 min. Then 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) were added and co-cultured at 37°C for 30 min. The absorbance value was measured at 620 nm. A blank group, a normal control group, and a positive control group (acetohydroxamic acid) were set up to calculate the half-maximal inhibitory concentration IC of trimethylglycine citrate on Helicobacter pylori urease. 50 The positive control group acetohydroxamic acid IC 50 is 18.1 µM.

[0018] Example 4 25 μL of Helicobacter pylori urease solution (10 U) and 25 μL of trimethylglycine malate solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with phosphate buffer solution) were added to a 96-well plate, and co-cultured at 37°C for 30 min. 50 μL of phosphate buffer solution containing 25 mmol of urea was added, and co-cultured at 37°C for 30 min. Then 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) were added, and co-cultured at 37°C for 30 min. The absorbance value was measured at 620 nm. A blank group, a normal control group, and a positive control group (acetohydroxamic acid) were set up to calculate the half-maximal inhibitory concentration IC of trimethylglycine malate on Helicobacter pylori urease. 50 The positive control group acetohydroxamic acid IC 50 is 18.1 µM.

[0019] Example 5 25 μL of Helicobacter pylori urease solution (10 U) and 25 μL of trimethylglycine salicylate solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with phosphate buffer solution) were added to a 96-well plate and co-cultured at 37°C for 30 min. 50 μL of phosphate buffer solution containing 25 mmol of urea was added and co-cultured at 37°C for 30 min. Then 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) were added and co-cultured at 37°C for 30 min. The absorbance value was measured at 620 nm. A blank group, a normal control group, and a positive control group (acetohydroxamic acid) were set up to calculate the half-maximal inhibitory concentration IC of trimethylglycine salicylate on Helicobacter pylori urease. 50 The positive control group acetohydroxamic acid IC 50 is 18.1 µM.

[0020] Example 6 25 μL of Helicobacter pylori urease solution (10 U) and 25 μL of trimethylglycine fumarate solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with phosphate buffer solution) were added to a 96-well plate, and co-cultured at 37°C for 30 min. 50 μL of phosphate buffer solution containing 25 mmol of urea was added, and co-cultured at 37°C for 30 min. Then 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl) were added, and co-cultured at 37°C for 30 min. The absorbance value was measured at 620 nm. A blank group, a normal control group, and a positive control group (acetohydroxamic acid) were set up to calculate the half-maximal inhibitory concentration IC of trimethylglycine fumarate on Helicobacter pylori urease. 50 The positive control group acetohydroxamic acid IC 50 is 18.1 µM.

Claims

1. A use of trimethylglycine as a Helicobacter pylori urease inhibitor, characterized in that: The structural formula of the trimethylglycine is: 。 2. Use of the Helicobacter pylori urease inhibitor according to claim 1 in the preparation of a medicament for treating gastric ulcer, duodenal ulcer and gastritis caused by Helicobacter pylori infection.

3. The use according to any one of claims 1 to 2, characterized in that: The inhibitor includes acceptable salts including hydrochloride, citrate, malate, salicylate, fumarate, sulfonate or methanesulfonate.

4. A Helicobacter pylori urease inhibitor, characterized in that: The inhibitor is trimethylglycine or an acceptable salt thereof, including hydrochloride, citrate, malate, salicylate, fumarate, sulfonate or methanesulfonate.

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