CN117643611A - Helicobacter pylori resistant composition and application thereof - Google Patents

Abstract

The invention provides a helicobacter pylori resistant composition and application thereof, wherein the helicobacter pylori resistant composition comprises grape seed extract and fucoidin; and the grape seed extract is calculated according to mass: fucoidan=8 to 10:1. the composition for resisting helicobacter pylori can reduce the possibility of infection of helicobacter pylori by inhibiting the expression of virulence genes of helicobacter pylori, and is effective in preventing or treating helicobacter pylori.

Description

An anti-Helicobacter pylori composition and its application

Technical field

This patent belongs to the technical field of functional compositions, and specifically relates to an anti-Helicobacter pylori composition and its application.

Background technique

Helicobacter pylori (HP) is a spiral-shaped, slightly anaerobic Gram-negative bacillus that requires very demanding growth conditions. It is the only microbial species currently known to be able to survive in the human stomach. Helicobacter pylori mainly enters the human stomach through the oral route and colonizes on the surface of epithelial cells. After colonization, it is difficult for the body to clear it spontaneously, and almost always causes active inflammation of the gastric mucosa. On the basis of active inflammation, chronic gastritis, A series of common diseases such as chronic atrophic gastritis, peptic ulcer and gastric cancer seriously endanger people's health. Typically, HP colonizes the human stomach during childhood and survives in the human stomach throughout the lifetime of the carrier.

There are many types of Helicobacter pylori strains, different strains have different tolerances to drugs, and the pathogenesis of Helicobacter pylori is complex. Generally speaking, after Helicobacter pylori enters the stomach, it mainly exerts its pathogenic effects through the following mechanisms: 1. The bacteria use the movement of flagella to move toward the gastric epithelial cells. 2. The bacteria use adhesins to adhere firmly to gastric epithelial cells. 3. The bacteria will secrete urease, thereby adjusting the pH value of gastric acid so that gastric acid cannot completely kill the bacteria. 4. Bacteria secrete vacuolating toxins, which can induce gastric epithelial cells to produce endosomal vacuoles and inhibit T cell proliferation in vitro, leading to gastric mucosal damage and immune disorders.

The various pathogenic mechanisms mentioned above all require Helicobacter pylori to be realized through specific gene expression. Therefore, in view of the above pathogenic mechanism, it is necessary to seek a composition that can inhibit the gene expression of Helicobacter pylori, thereby reducing the possibility of Helicobacter pylori infection and effectively preventing or treating Helicobacter pylori.

Contents of the invention

The object of the present invention is to provide an anti-Helicobacter pylori composition and its application. The anti-Helicobacter pylori composition inhibits the gene expression of Helicobacter pylori, thereby reducing the possibility of Helicobacter pylori infection and effectively preventing or treating it. Helicobacter pylori.

According to a first aspect of the present invention, an anti-Helicobacter pylori composition is provided, including grape seed extract and fucoidan; and based on mass calculation, grape seed extract: fucoidan = 8 to 10:1.

Grapes (Vitis vinifera) belong to the grape family and are a traditional edible fruit. Grape skins and grape seeds are usually rich in polyphenols, especially proanthocyanidins, which have a variety of effects. Proanthocyanidins can inhibit the urease activity of Helicobacter pylori. The grape seed extract used in the present invention can inhibit the gene expression of Helicobacter pylori related to adhesin, thereby affecting the adhesion performance of Helicobacter pylori by inhibiting the expression of adhesin-related genes.

Fucoidan is one of the main polysaccharides in brown algae (Phaeophyta). It contains high contents of fucose and sulfate and has strong anti-cancer, anti-viral, anti-pathogen adhesion and anti-infective activities. Fucoidan can prevent Helicobacter pylori from attaching to gastric epithelial cells and effectively inhibit Helicobacter pylori infection. In addition, fucoidan has been shown to inhibit the production of pro-inflammatory cytokines by downregulating MAPK and NF-κB-mediated signaling pathways. Fucoidan contains sulfate groups that can bind to the donor protein on Helicobacter pylori, thereby inhibiting the adhesion of Helicobacter pylori to gastric epithelial cells. When fucoidan enters the stomach, Helicobacter pylori, whether it has adhered to the stomach wall or is free in the stomach, will be coated by fucoidan. Fucoidan is a macromolecular polysaccharide that will not be broken down and digested by the stomach. Eventually, Helicobacter pylori will be excreted from the body as it is wrapped in fucoidan as the body metabolizes it.

After long-term exploration and experiments by the inventor, it was discovered that there is a synergistic effect between grape seed extract and fucoidan, which can further inhibit the gene expression of Helicobacter pylori. When grape seed extract and fucoidan are combined according to the above mass ratio, the composition prepared has obvious inhibitory activity against the standard strain of Helicobacter pylori and inhibits the colonization of Helicobacter pylori. Its mechanism of action is to inhibit the expression of various genes related to flagella, adhesion properties, urease, and secreted toxins of Helicobacter pylori, thereby inhibiting the movement ability of the flagella and inhibiting the secretion of adhesins, urease, vacuolating toxins, etc. by the bacteria. It significantly increases the difficulty for Helicobacter pylori to colonize and invade the human body, and effectively prevents or treats Helicobacter pylori.

Preferably, based on mass calculation, the grape seed extract:fucoidan=9-10:1.

Preferably, based on mass calculation, grape seed extract: fucoidan = 9.3:1.

When the mass ratio of grape seed extract to fucoidan falls within the above range, it can further inhibit the expression of various genes of Helicobacter pylori related to flagella, adhesion properties, urease, and secreted toxins, thereby potentially effectively inhibiting Helicobacter pylori. It also reduces the activity of urease, reduces flagellar motility, and reduces the adhesion ability of Helicobacter pylori, thereby inhibiting the ability of Helicobacter pylori to survive and grow in the human body.

Preferably, the proanthocyanidin content in the grape seed extract is ≥68%; the total sugar content in the fucoidan is ≥50%, the fucose content is ≥15%, and the sulfate group content is ≥15%. Among the industry standards commonly used in this field, the content of sulfate group SO ₄ ^2- can be used as one of the general physical and chemical indicators of fucoidan.

According to a second aspect of the present invention, there is provided the use of the above-mentioned anti-Helicobacter pylori composition in preparing products for preventing or treating Helicobacter pylori.

Preferably, Helicobacter pylori is selected from at least one strain SS1, ATCC 43504 strain, and ATCC 700392 strain. Helicobacter pylori includes a variety of different strains, and different strains have differences in gene expression and pathogenic proteins. In actual tests, the inventor found that the above-mentioned anti-Helicobacter pylori composition has a good inhibitory effect on SS1 strain, ATCC 43504 strain, and ATCC700392 strain. This can be seen from the strain's urease activity, flagellar shaping and flagellar motility, and adhesion ability. , toxicity and other aspects are inhibited at the same time, thereby providing users with a good anti-Helicobacter pylori effect.

Preferably, Helicobacter pylori is strain ATCC 700392. After many explorations and tests by the inventor, it was found that the above-mentioned anti-Helicobacter pylori composition has the most obvious inhibitory effect on the ATCC 700392 strain, and has a better inhibitory effect on the urease activity, flagellar movement activity, adhesion ability, and toxicity of the bacteria. , can achieve a better effect of inhibiting the colonization of Helicobacter pylori at a smaller concentration.

According to a third aspect of the present invention, there is provided the use of the above-mentioned anti-Helicobacter pylori composition in inhibiting the expression of flagellar genes of Helicobacter pylori ATCC 700392 strain. The flagellar genes include at least one of the flaB gene and the flgK gene. Among them, the flaB gene has been considered by industry researchers as an essential gene for the complete movement of Helicobacter pylori. The flgK gene is not only involved in the structural assembly of flagellar hook protein, but also affects the antigenicity of the flagellum. After many experiments, the inventor found that the above-mentioned anti-Helicobacter pylori composition can significantly inhibit the expression of flaB gene and flgK gene, thereby inhibiting the flagellar activity of Helicobacter pylori ATCC700392 strain.

According to a fourth aspect of the present invention, there is provided the application of the above-mentioned anti-Helicobacter pylori composition in inhibiting the expression of adhesion property genes of Helicobacter pylori ATCC 700392 strain. The adhesion property genes include at least one of alpA gene and babA gene. The adhesion of Helicobacter pylori to the gastric epithelial surface is a key step for the successful colonization of Helicobacter pylori. In the absence of the alpA gene, the amount of Helicobacter pylori colonization in animals will be reduced. The blood group antigen-binding adhesin obtained through babA gene expression is one of the adhesins necessary for Helicobacter pylori colonization and infection. After many experiments, the inventor found that the above-mentioned anti-Helicobacter pylori composition can inhibit the expression of alpA gene and babA gene, thereby reducing the adhesion performance of Helicobacter pylori ATCC 700392 strain.

According to a fifth aspect of the present invention, an anti-Helicobacter pylori product is provided, comprising the above-mentioned anti-Helicobacter pylori composition. The product provided by the invention uses the above-mentioned anti-Helicobacter pylori composition as an active ingredient, providing a new way to prevent or treat Helicobacter pylori infection.

Preferably, the anti-H. pylori product is an oral product.

Preferably, oral products include drinks, tablets, capsules, powders, jelly, and candies. The product provided by the present invention has various dosage forms, a wide range of use, and multiple application scenarios, making it convenient for consumers to take the product in different application scenarios.

Description of drawings

Figure 1 shows the effects of Example 1 and the blank control group on the expression of virulence genes;

Figure 2 is the scanning electron microscope image corresponding to the blank control group;

Figure 3 is a scanning electron microscope image corresponding to Example 1.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the drawings in the embodiments. Obviously, the described implementation The examples are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present invention.

Example 1

This example uses commercially available grape seed extract and fucoidan to prepare an anti-Helicobacter pylori composition.

The anti-Helicobacter pylori composition is mixed with 46.5 parts of grape seed extract and 5 parts of fucoidan. The grape seed extract used contains 95wt% procyanidins; the fucoidan used contains 50wt% total sugar, 15wt% fucose, and 20wt% sulfate group (calculated as SO ₄ ^2- ).

Test example 1

Participant: the anti-Helicobacter pylori composition provided in Example 1.

Test method: Use the broth microdilution method to determine the minimum inhibitory concentration (MIC) of different Helicobacter pylori strains in the test subjects. The Helicobacter pylori strains selected in this test example are: SS1 strain, ATCC 43504 strain, and ATCC 700392 strain. The above strains were purchased from the American type culture collection (ATCC).

The specific test methods are as follows:

1. Medium preparation

Blood plate preparation: Weigh 3.9g of Columbia agar basic culture medium into 100 mL of distilled water, shake and mix, and autoclave at 121°C for 20 minutes, then cool to 55°C, and add 5% sterile defibrinated sheep blood to the system. Mix well, invert while hot, and let it dry overnight in a biological safety cabinet. The prepared Columbia blood plate should be used within 2 weeks.

Preparation of brain heart infusion (BHI): Weigh 3.7g of brain heart infusion powder into 100 mL of distilled water, shake and mix, and autoclave at 121°C for 20 minutes, then store in a 4°C refrigerator. The prepared brain heart infusion needs to be Use within 2 weeks.

2. Recovery and passage of bacteria

Take out the frozen strains (SS1 strain, ATCC 43504 strain, ATCC 700392 strain) from the ultra-low temperature refrigerator. After the strains naturally thaw, take 100 μL of each strain and spread it evenly on the Columbia blood plate, place it in a three-gas incubator, and incubate at 37°C. Invert culture under microaerophilic (5% O ₂ , 10% CO ₂ , 85% N ₂ ) conditions for 48 to 72 h. When the strain reaches the surface of the blood plate, it can be passaged. Use a moistened sterile cotton swab to scrape the bacterial lawn from the blood plate, and suspend the bacterial lawn in sterile PBS to make a bacterial liquid. Take 100 μL of bacterial liquid and spread it evenly on a new Columbia blood plate, place it in a three-gas incubator, and incubate it upside down at 37°C under microaerophilic conditions for 48 to 72 hours.

3. Determination of minimum inhibitory concentration MIC by broth microdilution method:

Dissolve the test object in BHI ultrasonically, vortex and mix, centrifuge at 12000 rpm for 3 minutes, filter with a 0.22 μm filter, and prepare compositions with multiple concentration gradients such as 9, 6, 3, 1.5, 1, and 0.75 mg/mL. solution.

Add 50 μL of the above prepared composition solution to a 96-well plate, set at least 3 duplicate wells for each drug concentration, and set up a negative control group (containing only drugs, without inoculation of strains) and a growth control group (containing no drugs, only inoculated strains).

Take out the strains from the incubator, scrape the bacterial lawn with a moistened sterilized cotton swab into PBS, adjust the turbidity of the bacterial suspension to 1 McF with BHI containing 20% FBS, dilute it 10 times and inoculate it into the above-mentioned 96-well plate containing medicine ( The final turbidity of the bacterial suspension in the 96-well plate is about 1×10 ⁶ CFU/mL). Immediately place the 96-well plate in the incubator and incubate it for 72 hours under microaerobic conditions at 37°C with shaking at 150 rpm and then take it out for observation. result.

The observation results are only available when the growth control group appears button-shaped or obviously turbid. When observed with the naked eye from bottom to top, the drug concentration at which the bacterial turbidity is significantly reduced is the MIC.

Test results: As shown in Table 2.

Table 2. Minimum inhibitory concentrations measured on test subjects

Group Minimum inhibitory concentration MIC (mg/mL) SS1 strain 6 ATCC 43504 strain 3 ATCC 700392 strain 1.5

Result analysis:

It can be seen from the minimum inhibitory concentration shown in Table 2 that the anti-Helicobacter pylori composition provided in Example 1 has an inhibitory effect on various strains mentioned above. Among them, the anti-Helicobacter pylori composition provided in Example 1 has the most obvious inhibitory effect on ATCC700392 strain, and only 1.5 mg/mL is needed to achieve a better effect of inhibiting Helicobacter pylori. This shows that the anti-Helicobacter pylori composition provided by the present invention has a limited inhibitory effect on the ATCC 700392 strain and can effectively reduce the infection and colonization possibility of this strain.

Test example 2

Participant test subject: According to the preparation method in Test Example 1, the anti-Helicobacter pylori composition provided in Example 1 was prepared into a composition solution with a concentration of 1/2 MIC, and the composition solution was used as the test subject.

Test method: Targeting the four pathogenic mechanisms of Helicobacter pylori, RT-qPCR experiments were conducted to detect specific gene expression levels. The primer information related to this test example is shown in Table 3. The specific test methods are as follows:

Take the ATCC 700392 strain with a turbidity of 1 McF and culture it to the logarithmic phase of growth. Add 1 mL of bacterial liquid to the culture medium containing the test object and incubate for 24 hours. And set up a blank control group: take the ATCC 700392 strain with a turbidity of 1 McF, culture it to the logarithmic phase of growth, add 1 mL of bacterial liquid to the culture medium without the test object, and incubate for 24 hours.

The culture medium after the above incubation was centrifuged to collect the bacterial cells, and RNA was extracted using an RNA extraction kit. The concentration of RNA was determined using NanoDrop 2000. Reverse transcription was performed using the PrimeScript ^TM RT kit. The PremixEx Taq ^TM Ⅱ kit was used for real-time fluorescence quantification, the results were normalized to the 16S gene, and the relative expression was calculated using the 2 ^-ΔΔCT method.

Table 3. Primer information

Test results: The gene expression corresponding to Example 1 and the blank control group is shown in Figure 1. Note: "*" in Figure 1 indicates that the statistical analysis is the difference between the sample after treatment and the blank control group, P≤0.05.

Result analysis:

Regarding the flagellum formation and movement of Helicobacter pylori, the inventors selected the flaB gene and flgK gene as key detection targets. Among them, flaB has been considered by industry researchers as an essential gene for the complete movement of Helicobacter pylori. flgK not only participates in the structural assembly of flagellar hook protein, but also affects the antigenicity of the flagellum. In view of the adhesion ability of Helicobacter pylori, the inventors selected alpA gene and babA gene as key detection targets. Among them, the adhesion of Helicobacter pylori to the gastric epithelial surface is a key step for the successful colonization of Helicobacter pylori. In the absence of alpA, the amount of Helicobacter pylori colonization in animals will be reduced. The blood group antigen-binding adhesin obtained through babA expression is one of the adhesins necessary for Helicobacter pylori colonization and infection. For the urease of Helicobacter pylori, urease is mainly composed of structural proteins and auxiliary proteins. The inventors selected the ureB gene and ureI gene as the key detection targets. Among them, ureB regulates structural proteins, and ureI regulates auxiliary proteins. ureI is a gene unique to Helicobacter pylori urease, encoding a urease-specific channel protein, and plays an important role in the colonization of Helicobacter pylori on the acidic stomach surface. Urea in the stomach can be ingested, allowing the urea to enter the interior of Helicobacter pylori and react with urease to produce ammonia and carbon dioxide, so that Helicobacter pylori can colonize on the acidic stomach surface. In view of the virulence factors of Helicobacter pylori, the inventors selected the VacA gene as a key detection target. Among them, VacA is an important virulence gene of Helicobacter pylori, and vacuolating toxin is expressed through VacA.

By comparing the expression of virulence genes in Example 1 and the blank control group in Figure 1, it can be found that compared to the gene expression levels in the blank control group, the expression of the flaB and flgK genes, alpA and babA genes corresponding to Example 1 The amount is significantly lower, while the flaB and flgK genes are related to the flagellum formation and movement of Helicobacter pylori; the alpA and babA genes are related to the adhesion ability of Helicobacter pylori. This shows that the Helicobacter pylori composition provided in Example 1 can significantly inhibit the expression of genes related to flagella and adhesion properties of Helicobacter pylori, thereby inhibiting the motility of the flagella and the adhesion ability of the bacteria. At the same time, compared with the gene expression levels in the blank control group, the expression levels of the ureI and ureB genes and the VacA gene corresponding to Example 1 are low, and the ureI and ureB genes are related to the urease of Helicobacter pylori; the VacA gene is important for Helicobacter pylori. virulence genes. This shows that the Helicobacter pylori composition provided in Example 1 can also inhibit the expression of various genes of Helicobacter pylori related to urease and toxins, and further inhibit the colonization of Helicobacter pylori.

Test example 3

Participant test subject: According to the preparation method in Test Example 1, the anti-Helicobacter pylori composition provided in Example 1 was prepared into a composition solution with a concentration of 1/2 MIC, and the composition solution was used as the test subject.

Test method: Take the ATCC 700392 strain with a turbidity of 1McF, culture it to the logarithmic phase of growth, add 1mL of bacterial liquid to the culture medium containing the test object, and incubate for 24 hours. And set up a blank control group: take the ATCC 700392 strain with a turbidity of 1 McF, culture it to the logarithmic phase of growth, add 1 mL of bacterial liquid to the culture medium without the test object, and incubate for 24 hours.

The culture medium after the above incubation was centrifuged to collect the bacterial cells, washed twice with PBS, and then added with 2.5% glutaraldehyde electron microscope fixative and fixed overnight at 4°C. Dehydrate in 30%, 50%, 70%, 80% and 95% ethanol series, immerse and replace in tert-butyl alcohol, dry, mount and spray gold and observe with scanning electron microscope.

Test results: The corresponding scanning electron microscope images of the blank control group are shown in Figure 2. The scanning electron microscope image corresponding to Example 1 is shown in Figure 3.

Result analysis: The morphology of the entire Helicobacter pylori cell was observed with a scanning electron microscope (SEM). Comparing the morphology of the cells shown in Figure 2 and Figure 3, it can be found that the cells corresponding to the blank control group showed a regular shape and a uniform surface. . The surface of the bacterial cells corresponding to Example 1 was damaged, the bacterial cells were sunken and shrunk, and the morphology of the bacterial cells was obviously damaged.

The above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out. Modifications or equivalent substitutions may be made without departing from the essence and scope of the technical solution of the present invention.

Claims (10)

1. A composition for combating helicobacter pylori, comprising a grape seed extract and fucoidan; and the grape seed extract is calculated according to mass: fucoidan=8 to 10:1.

2. the helicobacter pylori resistant composition according to claim 1, characterized in that the grape seed extract is, by mass: fucoidin=9-10:1; preferably, the grape seed extract: fucoidan = 9.3:1.

3. the helicobacter pylori resistant composition according to any one of claims 1 to 2, characterized in that,

the procyanidine content in the grape seed extract is more than or equal to 68%;

the total sugar content in the fucoidin is more than or equal to 50%, the fucose content is more than or equal to 15%, and the sulfate group content is more than or equal to 15%.

4. Use of a helicobacter pylori resistant composition as defined in any one of claims 1 to 3 for the preparation of a helicobacter pylori product.

5. The use as claimed in claim 4, wherein the helicobacter pylori is at least one selected from the group consisting of SS1 strain, ATCC 43504 strain, ATCC 700392 strain; preferably, the helicobacter pylori is ATCC 700392 strain.

6. Use of the helicobacter pylori resistant composition according to any one of claims 1 to 3 for inhibiting expression of flagella genes of helicobacter pylori ATCC 700392 strain, including at least one of a flaB gene and a flgK gene.

7. Use of the helicobacter pylori resistant composition according to any one of claims 1 to 3 for inhibiting the expression of adhesion property gene of helicobacter pylori ATCC 700392 strain, the adhesion property gene comprising at least one of alpA gene, babA gene.

8. An anti-helicobacter pylori product comprising the anti-helicobacter pylori composition as defined in any one of claims 1 to 3.

9. The anti-helicobacter pylori product according to claim 8, characterized in that the anti-helicobacter pylori product is an oral product.

10. The anti-helicobacter pylori product according to claim 9, characterized in that the oral product comprises a drink, a tablet, a capsule, a powder, a jelly, a candy.