KR20080035912A - Natural urease inhibitor including glutathione and healthy foods thereof, the inhibiting method of ammonia formation thereby - Google Patents

Abstract

A natural urease inhibitor is provided to inhibit activity of urease of Helicobacter pylori without side effects, thereby inhibiting colony formation of Helicobacter pylori. A method for inhibiting ammonia formation is also provided to inhibit ammonia formation from the excretions of domestic animals by using the urease inhibitor. A natural inhibitor of Helicobacter pylori urease contains 0.1-5.0 mM of glutathione. A health food for inhibiting Helicobacter pylori urease contains glutathione. The ammonia formation from the excretions of domestic animals is inhibited by spraying the urease inhibitor containing glutathione to the domestic animal excretions.

Description

Natural Urease Inhibitor including Glutathione and Healthy foods etc, the inhibiting method of Ammonia formation according to Helicobacter pylori Glutathione (을 ｌｕｔａｔｈｉｏｎｅ) and the health functional food using the same

1 is a scheme showing the urease-mediated hydrolysis action of urea.

Figure 2 is a graph showing the Helicobacter pylori urease inhibitory activity of glutathione (Glutathione) of the present invention, Figure 2a is a measurement result by the indophenol method, Figure 2b is a view showing the measurement result by the phenol red method.

Figure 3 is a graph showing the results of measuring the inhibitory effect of the production of ammonia caused by the urease activity in pig manure.

The present invention relates to glutathione (Glutathione) having an inhibitory activity of Helicobacter pylori urease, a health functional food using the same, and an ammonia production inhibitor.

Helicobacter pylori was discovered in 1983 by Marshall and Warren of Australia and was observed as a Gram-negative curved bacilli from gastric mucosal biopsy of gastritis and gastric ulcer patients. And is known as the primary determinant of gastric cancer.

Helicobacter pylori secretes urease, a urease, to hydrolyze one molecule of urea (H ₂ NCONH ₂ , urea) in gastric juice to form two molecules of ammonia (NH ₃ ) (FIG. 1). Close associations have been reported with urease infecting Helicobacter pylori in human gastrointestinal epidermal cells and helping with colonization. Specifically, Helicobacter pylori strains that inactivate urease do not colonize in gastric mucosal cells, and it has been reported that urease activity is essential for colonization of Helicobacter pylori (Eaton KA, et al., Infect Immun . , 59, pp2470-). 2475, 1991), ammonia produced by Helicobacter pylori urease increases the pH in gastric juice, damages the gastric mucus layer (Sidebotham RL, et al., J. Clin . Pathol . , 44, pp52-57, 1991), Ammonia itself inhibits oxygen consumption of gastric mucosa cells and ATP production in mitochondria (Tsujii M., et al., Gastroenterology , 102, pp1881-1888, 1992), and ultimately ammonia forms monochloroamine Because of the production of reactive oxygen species, it has been reported to induce cell damage, lead to chronic inflammation, and further to DNA damage, thereby promoting the cancer development process. (Hahm KB, et al., Am. J. Gastroenterol . , 92, pp 1853-1857, 1997). Therefore, in order to prevent such damage, the bacterium Helicobacter pylori should be disinfected or alternatively, the urease function should be weakened or destroyed.

Antibiotic therapy may be used to inhibit the activity of Helicobacter pylori as described above, but there is a problem that the effectiveness of antibiotics such as side effects, the emergence of resistant strains, and the inability to prevent reinfection may be prevented. Therefore, there have been recent efforts to develop various vaccines and studies on inhibitors of urease enzyme activity as an effective way to eradicate Helicobacter pylori, most of which are known as urease, which is known as a specific target for the pathogenicity of Helicobacter pylori. Proteins such as, VacA, CagA, and Neutrophil-Activating Protein (NAP) have been applied to animal experiments as targets, but more research is needed on their effectiveness. Recently, research related to urease is related to the discovery and discovery of inhibitors capable of inhibiting urease activity of Helicobacter pylori. Synthetic urease inhibitors include acetohydoxamic acid (AHA), cyclohexylphosphoric triamide, phenyl phosphorodiamidate and n- (n-butyl). thiophosphoric triamide and its structural analogs / isomers are known (Odake S et al., Biol) Pharm Bull 1994; 17: 1329-1332). Among them, AHAs and hydroxamic acids (HXAs, R-NHOH: R-acyl groups) are known to be effective and potent urease inhibitors and have been used to treat urolithiasis or urolithiasis (Griffith DP et al, J Urol 1978). 119: 9-15).

However, only AHA had an inhibitory effect on the urease activity of Helicobacter pylori, while the rest of the synthetic urease inhibitors had no inhibitory effect on the activity of Helicobacter pylori derived urease. In addition, such synthetic urease inhibitors have relatively high toxicity and are only used in a very limited manner.

Cause gastroenteritis or stomach cancer and formatting in the stomach as described above, H. pylori (Helicobacter pylori ) form colonies and produce strong toxicity. By the way, antibiotic therapy may be used to inhibit the activity of Helicobacter pylori, but there are problems in that the effectiveness of antibiotics such as side effects, the emergence of resistant strains, the inability to prevent reinfection, and the like are poor. Therefore, there have been recent efforts to develop various vaccines and studies on inhibitors of urease enzyme activity as an effective way to eradicate Helicobacter pylori, most of which are known as urease, which is known as a specific target for the pathogenicity of Helicobacter pylori. Proteins such as, VacA, CagA and Neutrophil-activating protein (NAP) have been applied to animal experiments as targets, but their effectiveness is questionable.

Thus, in the present invention, in order to solve this problem, many natural materials and natural products known to be non-toxic have been studied by urease inhibitors and have found glutathione (Glutathione) having the ability to inhibit urease activity. Researchers of the present invention have long found that glutathione has the world's first inhibitory effect of urease activity of Helicobacter pylori , which is known as a risk factor for gastrointestinal diseases in humans. Helicobacter pylori ( Hylicobacter pylori ) was not allowed to form a colony to inhibit the growth.

The present invention relates to a glutathione-containing urease inhibitor having the ability to inhibit the urease activity of Helicobacter pylori .

Another invention of the present invention relates to a growth inhibitor for Helicobacter pylori comprising glutathione (Glutathione) containing urease inhibitors have a urease activity inhibitory ability of Helicobacter pylori (Helicobacter pylori).

Another invention of the present invention relates to a health functional food containing glutathione (Glutathione) having the inhibitory ability of urease activity of Helicobacter pylori .

Another invention of the present invention relates to a method for inhibiting ammonia production by using a glutathione-containing urease inhibitor having the ability to inhibit urease activity of Helicobacter pylori .

Another invention of the present invention relates to a glutathione-containing ammonia production inhibitor having the ability to inhibit the urease activity of Helicobacter pylori .

Test Example  1. Helicobacter Pylori  Determination of antimicrobial activity against

Growth inhibition of Helicobacter pylori by glutathione was confirmed.

Specifically, Helicobacter pylori KCCM 41351 was suspended in 0.9% physiological saline to 10 ⁹ cfu / ㎖, diluted 10-fold with sterile distilled water to prepare a strain dilution. Glutathione was then added at 9 mM, 0.5 mM, 1.0 mM and 5.0 mM concentrations to 9 ml of sterilized 100 mM phosphate buffer solution (pH 7), followed by incubating for 30 minutes by adding 1 ml of the strain dilution solution. Afterwards, the culture was serially diluted with sterile saline. 0.1 ml of the diluted solution was inoculated into Columbia blood agar medium, and cultured in an aerobic state at 37 ° C. for 72 hours, and then viable bacterial count was calculated from colony water. As a control, a phosphate buffer solution containing no glutathione was used.

As a result, as shown in Table 1, when the concentration of glutathione (Glutathione) was 1.0 mM, the growth inhibitory effect was about 210 times compared to the control group, and at the concentration of 5.0 mM, almost all Helicobacter pylori strains were killed. Indicated.

Concentration of Glutathione Viable cell count (cfu / ml) Control 7.8 × 10 ⁶ 0.1 mM 37.5 × 10 ⁴ 0.5 mM 21 × 10 ³ 1.0 mM 2.9 × 10 ² 5.0 mM Less than 10

Test Example  2. Helicobacter Pylori Urease  Measurement of inhibitory activity

Indophenol method was used to determine the inhibitory activity of Helicobacter pylori urease of glutathione (Fawcett JK and Scott JE, J. Clin . Pathol. , 13, pp156-159, 1960; Chaney AL and Marbach EP, Clin . Clem., 8, pp130-132, 1962).

Briefly explaining the principle of the method, one molecule of urea (H ₂ NC = ONH ₂ ) and one molecule of water (H ₂ O) are converted into two ammonia (NH ₃ ) molecules and one molecule of carbon dioxide by urease. . The generated ammonia (NH ₃ ) is reacted with 2 molecules of phenol and alkaline hypochlorite by sodium nitroprusside (Na ₂ Fe [CN] ₅ NO) catalyst to indophenol 1 Form a molecule. Since the indophenol produced by such a reaction is directly proportional to the amount of ammonia, it becomes a quantitative method of converting the amount of ammonia generated when absorbance is measured at 570 nm for blue indophenol.

Specifically, 2 μl of the sample prepared in Example 1 was added to 50 μl of urease solution (100 unit / 15 mL) such that the final concentration was 1, 10, 100, 1000 μM. After mixing well with slow stirring, the reaction tube was placed in a 37 ° C. water bath and allowed to stand for 10 minutes so that the urea was hydrolyzed to ammonia.

Thereafter, 200 µl of phenol nitroprusside (Sigma, Cat. No. 535-30) was added to the tube, followed by 200 µl of alkaline hypochlorous acid solution (Sigma, Cat. No. 640-3). 1.0 ml of distilled water was added thereto, mixed well, and left at room temperature for 30 minutes. Then, 100 μl of each sample was taken and placed in 96 wells, and the absorbance was measured at 570 nm with an ELISA reader. The results were compared with the case where no urease inhibitor was added to calculate the inhibition rate (%).

As a result, glutathione (Glutathione) was observed to exhibit an inhibitory effect of 100 or more at all 0.1, 0.5, 1.0, 5.0 mM doses (Fig. 2a). Here, the numerical value of 100 or more means that the absorbance was measured by selecting the vehicle as a blank and measuring the absorbance.

Test Example  3. Helicobacter Pylori Urease  Measurement of inhibitory activity

The phenol red method was used to measure the Helicobacter pylori urease inhibitory activity of glutathione (Uesato S. et al., Chem) . Pharm Bull. , 50, pp 1280-1282, 2002; Kobashi K. et al., Biochem Biophys Acta , 65, pp 380-383, 1962).

Indophenol method of Test Example 2 has the disadvantage that the substance having the ammonia adsorption effect can also come out as a false positive (false positive). The phenol red method is a method for checking the change in pH due to ammonia production. In other words, by reacting urease and urease inhibitor candidate with 1M Tris-HCl buffer (pH 6.8) containing indicator phenol red, the urease converts urea to ammonium ions and increases the pH value at 560 nm. How to measure.

Specifically, 20 μl (2 units) was used after diluting urease to 0.1 unit / μl. The glutathione (Glutathione) was diluted by concentration to prepare 100 μl, and then 380 μl of distilled deionized water (DDW) was added to adjust the volume to 0.5 mL. 0.5 ml of urease solution (2% urea in 0.1M Tris-HCl pH 6.8) was added to the solution, followed by incubation at 37 ° C. for 30 minutes. Thereafter, absorbance at 560 nm was measured for each sample. The results were compared with the case where no urease inhibitor was added to calculate the inhibition rate (%). Since screening methods for urease inhibitory activity of Test Examples 2 and 3 have advantages and disadvantages, two methods were used together, and experiments were performed using acetohydroxamic acid (acetohydroxamic acid; AHA) commonly used as a urease inhibitor. The results were compared using the positive control. And, in order to verify the statistical significance, the results obtained by experimenting at least 3 for each concentration were used as the average and standard error.

As a result, as shown in Figure 2b at the concentration of glutathione (Glutathione) 5 mM showed a maximum urease inhibitory effect of up to 99.58 ± 0.75.

Test Example  4. From pig manure Urease  Inhibitor of production of ammonia caused by activity and method of removing ammonia using the inhibitor

The glutathione (Glutathione) was confirmed to exhibit an effect of inhibiting the ammonia produced by the urease activity produced by the pig manure microorganisms.

The following experiment was carried out in a pig farm in Icheon, Gyeonggi-do. The experimental pigs raised 700 pigs (100 days old) and the area was 200 pyeong. Ammonia was measured using a gas meter (GasTec Model GV-100, Japan). The formulation used in this test was prepared by dissolving 100 g of glutathione in 1 L of propylene glycol. The prepared 1 L of inhibitor was diluted in 100 L of water and then sprayed inside the pig house using a high pressure sprayer, and the spraying of the preparation was performed at intervals of 3 to 4 days. Ammonia was measured three times a week before the test and at three places in the experimental pig house.

As a result, as shown in Figure 3, when spraying the formulation inside pig pigs, ammonia generated in pigs was reduced by 50-70% for three days from the day after the spraying, three times the formulation every 3-4 days In case of repeated spraying, it was confirmed that the ammonia was maintained at 50% or more.

Therefore, in Test Example 4, when glutathione alone or mixed was used for livestock manure and livestock farming, it could be confirmed that it can be applied as a livestock environment improving agent by reducing urease activity in manure and suppressing ammonia generation.

Example  1. Preparation of dietary supplements

Glutathione 100 mg Vitamin mixtures Quantity Vitamin A Acetate 70 μg Vitamin E 1.0 mg Vitamin B ₁ 0.13 mg Vitamin B ₂ 0.15 mg Vitamin B ₆ 0.5 mg Vitamin B ₁₂ 0.2 μg Vitamin c 10 mg Biotin 10 μg Nicotinic acid amide 1.7 mg Folic acid 50 μg Calcium Pantothenate 0.5 mg Mineral mixture Quantity Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Potassium phosphate monobasic 15 mg Dicalcium Phosphate 55 mg Potassium citrate 90 mg Calcium carbonate 100 mg Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixtures of Table 2 is a relatively suitable composition for the health food in a preferred embodiment, but the composition ratio may be arbitrarily modified, mixing the above components according to a conventional health food production method The granules can then be prepared and used to prepare the health food composition according to conventional methods.

If the above effects of the present invention are applied industrially in consideration of cost, it can be said that it is highly likely to be used as an economical urease inhibitor. The discovery of urease inhibitors in natural products known to be non-toxic suggests the potential use of H. pylori as a growth inhibitor, known as a risk factor for human gastrointestinal diseases, thus overcoming various disadvantages and shortcomings of bacterial strains. In addition, it is possible to induce a decrease in the inflammatory response due to bacterial infection, and significant anti-cancer efficacy is expected as a result of long-term inhibition. In addition, since the production of ammonia caused by urease activity can be suppressed in livestock manure, it can be said that the use as an environmental improvement agent is also great.

Claims (5)

A glutathione-containing urease inhibitor having the ability to inhibit the urease activity of Helicobacter pylori . A growth inhibitor of glutathione-containing Helicobacter pylori, comprising a glutathione-containing urease inhibitor of claim 1. A glutathione-containing health functional food having an inhibitory activity of urease activity of Helicobacter pylori of claim 1. Glutathione-containing ammonia production inhibitor having the ability to inhibit the urease activity of Helicobacter pylori of claim 1. A method of inhibiting ammonia production using a glutathione-containing ammonia production inhibitor having a urease activity inhibitory effect of Helicobacter pylori of claim 4.

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