KR100855097B1 - Composition for anti-helicobacter pylori comprising n-acetyl-n-cysteine - Google Patents

Abstract

The present invention relates to a dietary supplement, and the ammonia generation inhibitor containing H. pylori urease (urease) with the activity inhibitory ability acetylcysteine (N -acetyl- N -cysteine) and them.

Acetylcysteine (N -acetyl- N -cysteine) of the invention is Helicobacter pylori in the stomach, gastric ulcer, gastritis and various gastrointestinal-related disease caused by the Helicobacter pylori urease exhibits an antibacterial effect and inhibitory effect on (Helicobacter pylori) It can be used for treatment and prevention, and can be usefully used in the field of functional foods, such as functional drinks, health functional foods, etc., which are effective for improving gastrointestinal diseases such as gastritis. In addition, the present invention can also be applied as an environmental improving agent in that it is possible to suppress the production of ammonia caused by urease activity in livestock manure.

N-acetyl-N-cysteine, Helicobacter pylori, urease

Description

Acetylcysteine-containing anti-helicobacter pylori composition {COMPOSITION FOR ANTI-HELICOBACTER PYLORI COMPRISING N-ACETYL-N-CYSTEINE}

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 acetylcysteine ( N -acetyl- N -cysteine) of the present invention, Figure 2a is a measurement result by the indophenol method, Figure 2b is a measurement result by the phenol red method Is a diagram showing.

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 having a Helicobacter pylori urease activity inhibitory ability acetylcysteine (N -acetyl- N -cysteine) and health food using the same, and ammonia generation inhibitors and their suppression method.

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 mucosa cells, and it has been reported that urease activity is essential for colonization of Helicobacter pylori (Eaton KA, et al., Infect Immun . , 59, pp 2470-2475, 1991), ammonia produced by Helicobacter pylori urease increases the pH in gastric juice and 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 is monochloroamine ( Because it forms monochloroamines to produce reactive oxygen species, it has been reported to induce cell damage, lead to chronic inflammation, and further DNA damage to accelerate the development of cancer (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. Recent research on urease-related research involves the discovery and discovery of inhibitors that can inhibit the 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. Such synthetic urease inhibitors have relatively high toxicity and thus are only limited in use.

Cause gastroenteritis or stomach cancer and formatting in the stomach as described above, H. pylori bacteria (Helicobacter pylori) are to form a colony, and generate a strong toxicity. By the way, antibiotic therapy may be used to inhibit the activity of Helicobacter pylori, but there is a problem that the effectiveness of the side effects of antibiotics, the emergence of resistant strains, the inability to prevent re-infection, etc. is ineffective. Therefore, in recent years, various vaccines have been developed as an effective method for killing Helicobacter pylori and studies on inhibitors of urease enzyme activity, 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.

So the present invention, it was discovered in a number of natural materials, and non-toxic water-known to solve this problem to study a urease inhibitor-acetylcysteine have a urease activity inhibitory ability (N -acetyl- N -cysteine). The researchers of the present invention are many test results, the world's first-acetylcysteine (N -acetyl- N -cysteine) a Helicobacter known as a risk factor of the human body within a gastrointestinal disease pylori (Helicobacter pylori ) has been found to possess the inhibitory activity of urease activity, and this inhibition of urease activity also prevented the formation of Helicobacter pylori colonies, enabling the growth inhibition.

The present invention relates to an N-acetyl-N-cysteine-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 acetyl cysteine (N-acetyl-N-cysteine ) 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 acetylcysteine (N-acetyl-N-cysteine) having the inhibitory ability of urease activity of Helicobacter pylori .

Another invention of the present invention relates to a method for inhibiting ammonia production using a urease inhibitor containing acetylcysteine (N-acetyl-N-cysteine), which possesses the inhibitory ability of urease activity of Helicobacter pylori .

Another invention of the present invention relates to an ammonia-containing inhibitor containing acetylcysteine (N-acetyl-N-cysteine) having the ability to inhibit the urease activity of Helicobacter pylori .

Test Example  1. Helicobacter Pylori  Determination of antimicrobial activity against

The growth inhibition effect of Helicobacter pylori bacteria by the acetylcysteine (N-acetyl-N-cysteine) was confirmed. Hereinafter, the antimicrobial activity measurement illustrated in Table 1 will be described in detail.

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. 1 ml of the strain dilution solution was added to 9 ml of 100 mM phosphate buffer solution (pH 7) sterilized by adding acetylcysteine (N-acetyl-N-cysteine) at concentrations of 0.1 mM, 0.5 mM, 1.0 mM and 5.0 mM, respectively. After addition and incubation for 30 minutes, the culture was serially diluted with sterile saline. 0.1 ml of the dilution solution was inoculated into Columbia blood agar medium, and cultured in an aerobic state at 37 ° C. for 72 hours, and then viable bacterial counts were calculated from colony water. As a control, a phosphate buffer solution containing no acetylcysteine (N-acetyl-N-cysteine) was used.

As a result, as shown in Table 1, when the concentration of acetylcysteine (N-acetyl-N-cysteine) is 1.0mM showed more than 210 times the growth inhibitory effect compared to the control, acetylcysteine (N-acetyl- N-cysteine) showed the effect of killing almost all Helicobacter pylori strains at 5.0 mM.

Concentration of N-acetyl-N-cysteine Acetylcysteine 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 measure Helicobacter pylori urease inhibitory activity of the acetylcysteine (N-acetyl-N-cysteine) (Fawcett JK and Scott JE, J. Clin . Pathol . , 13, pp156-159, 1960; Chaney AL and Marbach EP, Clin . Clem . , 8, pp 130-132, 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, it was observed that acetylcysteine (N-acetyl-N-cysteine) shows 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 the acetylcysteine (N-acetyl-N-cysteine) (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. After diluting the acetylcysteine (N-acetyl-N-cysteine) by concentration, 100 μl was prepared, and 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 the experiment was performed with acetohydroxamic acid (AHA), which is commonly used as a urease inhibitor, in a positive control group. It was used as compared to the results. In order to verify the statistical significance, the results obtained by experimenting more than 3 at each concentration were used as the average and standard error.

As a result, as shown in Figure 2b is shown a maximum of urease inhibitory activity up to 96.65 ± 0.47 in the above-acetylcysteine (N -acetyl- N -cysteine) 5 mM concentration.

Test Example  4. From pig manure

Urease  Inhibitor of production of ammonia caused by activity and method of removing ammonia using the inhibitor

It was confirmed that the acetylcysteine (N-acetyl-N-cysteine) exhibits an effect of inhibiting ammonia production caused by the urease activity generated by 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 acetylcysteine (N-acetyl-N-cysteine) 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, this test example 4 can be applied as a livestock environment improving agent by suppressing ammonia generation by reducing urease activity in manure when used alone or mixed with acetylcysteine (N-acetyl-N-cysteine) for livestock manure and livestock. Could confirm.

Example  Acetylcysteine (N- acetyl -N- cysteine Manufacture of Functional Foods Containing

N-acetyl-N-cysteine 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 functional food containing acetylcysteine (N-acetyl-N-cysteine) in the preferred embodiment 1, but may be modified arbitrarily After mixing the above components according to a conventional health food production method, and then to prepare a granule, it can be used for preparing a health food composition according to a conventional method.

If the above effects of the present invention are applied industrially in consideration of the cost, it can be said that it is likely to be used as an economical acetylase (N-acetyl-N-cysteine) -containing urease inhibitor. The discovery of N-acetyl-N-cysteine-containing urease inhibitors, which are known to be non-toxic, suggests that H. pylori , a known risk factor for human gastrointestinal disease, can be used as a growth inhibitor, and therefore, several strains can be used. It is possible to overcome the disadvantages and disadvantages of eggplants, to induce a decrease in the inflammatory response due to fungal infection, and to be expected to have significant anti-cancer efficacy as a result of long-term inhibition. In addition, ammonia production inhibitors containing acetylcysteine (N-acetyl-N-cysteine) have a high possibility of being used as various environmental improvers since they can suppress the production of ammonia caused by urease activity in livestock manure. .

Claims (5)

delete N-acetyl-N-cysteine-containing anti-helicobacter pylori composition. The composition of claim 2, wherein the composition is used as a dietary supplement. delete delete

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