CN117643611A - Helicobacter pylori resistant composition and application thereof - Google Patents
Helicobacter pylori resistant composition and application thereof Download PDFInfo
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- CN117643611A CN117643611A CN202311411981.4A CN202311411981A CN117643611A CN 117643611 A CN117643611 A CN 117643611A CN 202311411981 A CN202311411981 A CN 202311411981A CN 117643611 A CN117643611 A CN 117643611A
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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
Technical Field
The patent belongs to the technical field of functional compositions, and particularly relates to a helicobacter pylori resistant composition and application thereof.
Background
Helicobacter pylori (Helicobacter pylori, HP) is a spiral, micro-anaerobic, gram-negative bacillus that is very demanding in terms of growth conditions, the only species of microorganism currently known to survive in the human stomach. Helicobacter pylori enters the stomach of a human body through an oral cavity and is fixedly planted on the surface of epithelial cells, the organism is difficult to spontaneously remove after the fixation, the active inflammation of gastric mucosa can be almost caused, a series of common diseases such as chronic gastritis, chronic atrophic gastritis, peptic ulcer, gastric cancer and the like can also occur on the basis of the active inflammation, and the health of people is seriously endangered. Typically, HP colonizes the human stomach during childhood and survives the life of the carrier.
The helicobacter pylori has a plurality of strains, different strains have different drug tolerance, and the pathogenic mechanism of the helicobacter pylori is complex. In general, helicobacter pylori enters the stomach and plays a pathogenic role mainly by the following mechanisms: 1. the cells move to the gastric epithelial cells by the movement of flagella. 2. The thallus is firmly adhered to the gastric epithelial cells by using the adhesin. 3. The bacteria secrete urease, thereby regulating the pH value of gastric acid, so that the gastric acid cannot completely kill the bacteria. 4. The bacterial body can secrete vacuolated toxin, and the vacuolated toxin can induce gastric epithelial cells to generate endosomal vacuoles and inhibit T cell proliferation in vitro, so that gastric mucosa injury and immune disorder are caused.
The above-mentioned various pathogenic mechanisms are all realized by specific gene expression of helicobacter pylori. Thus, in view of the above-described pathogenic mechanism, there is a need for a composition capable of achieving a reduced possibility of infection with helicobacter pylori by inhibiting helicobacter pylori gene expression, and effectively preventing or treating helicobacter pylori.
Disclosure of Invention
The present invention is directed to a helicobacter pylori resistant composition and its use, which is effective in preventing or treating helicobacter pylori by reducing the possibility of infection with helicobacter pylori through inhibiting gene expression of helicobacter pylori.
According to a first aspect of the present invention, there is provided an anti-helicobacter pylori composition comprising grape seed extract and fucoidan; and the grape seed extract is calculated according to mass: fucoidan=8 to 10:1.
grape (grape vinifera) belongs to the family of Vitaceae and is a traditional edible fruit, grape skin and grape seeds are usually rich in polyphenols, and particularly procyanidins in grape skin and grape seeds have multiple effects and can inhibit helicobacter pylori urease activity. The grape seed extract adopted by the invention can inhibit the gene expression of helicobacter pylori about the adhesin, and the adhesion performance of the helicobacter pylori is influenced by inhibiting the gene expression related to the adhesin.
Fucoidan is one of the main polysaccharides in brown algae (Phaeophyta), contains high content of fucose and sulfate, and has strong anticancer, antiviral, antipathogen adhesion and anti-infectious activities. The fucoidin can prevent helicobacter pylori from adhering to gastric epithelial cells, and is effective in inhibiting helicobacter pylori infection. Furthermore, fucoidan has also been shown to inhibit the production of pro-inflammatory cytokines by down-regulating MAPK and NF- κB mediated signaling pathways. Fucoidan contains sulfate groups and is capable of binding to donor proteins on helicobacter pylori, thereby inhibiting adhesion of helicobacter pylori to gastric epithelial cells. After the fucoidan enters the stomach, helicobacter pylori, whether already adhered to the stomach wall or dissociated in the stomach, is encapsulated by the fucoidan. The fucoidin is a macromolecular polysaccharide, and the fucoidin is not decomposed and digested by stomach, and finally helicobacter pylori can be discharged out of the body along with metabolism of the body in a state of being wrapped by the fucoidin.
Through long-term exploration and experiments by the inventor, the synergistic effect exists between the grape seed extract and the fucoidin, and the gene expression of helicobacter pylori can be further inhibited. When grape seed extract and fucoidin are matched according to the mass ratio, the prepared composition has obvious inhibition activity on helicobacter pylori standard strains, and plays a role in inhibiting helicobacter pylori colonization. The action mechanism is that the movement capability of the flagella is inhibited by inhibiting the expression of various genes of helicobacter pylori about the flagella, the adhesion performance, the urease and the secretion toxin, the secretion of adhesin, the urease, the vacuolated toxin and the like by thalli is inhibited, the difficulty of the helicobacter pylori in colonisation and invasion of human bodies is obviously increased, and the helicobacter pylori is effectively prevented or treated.
Preferably, the grape seed extract is calculated by mass: fucoidan=9 to 10:1.
Preferably, the grape seed extract is calculated by mass: fucoidan = 9.3:1.
when the mass ratio of the grape seed extract to the fucoidan falls within the above range, it is possible to further inhibit the expression of various genes of helicobacter pylori with respect to flagella, adhesion property, urease, and secretory toxin, thereby making it possible to effectively inhibit the activity of helicobacter pylori urease, and to reduce the ability of the flagella to move and the adhesion ability of helicobacter pylori, thereby playing a role in inhibiting the survival and growth of helicobacter pylori in the human body.
Preferably, 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 percent, the fucose content is more than or equal to 15 percent, and the sulfate group content is more than or equal to 15 percent. In industry standards commonly used in the art, sulfate groups SO 4 2- The content of (2) can be used as one of the general physicochemical indexes of fucoidan.
According to a second aspect of the present invention there is provided the use of a composition as described above against helicobacter pylori for the preparation of a product for the prevention or treatment of helicobacter pylori.
Preferably, helicobacter pylori is selected from at least one of SS1 strain, ATCC 43504 strain, ATCC 700392 strain. Helicobacter pylori includes a number of different strains, the gene expression of which differs from that of pathogenic proteins. In practical tests, the inventor finds that the helicobacter pylori resistant composition has good inhibition effect on SS1 strain, ATCC 43504 strain and ATCC 700392 strain, and can inhibit the urease activity, flagella forming and flagella movement capacity, adhesion capacity, toxicity and other aspects of the strain at the same time, thereby providing good helicobacter pylori resistant effect for users.
Preferably, helicobacter pylori is the ATCC 700392 strain. Through repeated researches and tests by the inventor, the composition for resisting helicobacter pylori has the most obvious inhibiting effect on ATCC 700392 strain, has better inhibiting effect on urease activity, flagella exercise activity, adhesion capability and toxicity of thalli, and can achieve better inhibiting effect on helicobacter pylori colonization at a smaller concentration.
According to a third aspect of the present invention, there is provided the use of the above composition for inhibiting the expression of a flagella gene of helicobacter pylori ATCC 700392 strain, the flagella gene comprising at least one of a flaB gene and a flgK gene. Among them, the flaB gene has been recognized by the researchers in the industry as an essential gene for complete movement of helicobacter pylori, and the flgK gene is involved not only in structural assembly of flagellin but also in influencing antigenicity of flagella. The inventors have found through a plurality of experiments that the above composition for anti-helicobacter pylori can significantly inhibit the expression levels of the flaB gene and the flgK gene, thereby inhibiting the flagellum activity of the helicobacter pylori ATCC 700392 strain.
According to a fourth aspect of the present invention, there is provided the use of the above composition for inhibiting the expression of adhesion property genes of helicobacter pylori ATCC 700392 strain, the adhesion property genes comprising at least one of alpA gene and babA gene. Adhesion of H.pylori to the surface of the gastric epithelium is a key step in successful colonization by H.pylori, which in the absence of the alpA gene would be reduced in animals. Blood group antigen binding adhesins obtained by expression of the babA gene are among the adhesins necessary for H.pylori colonization and infection. The inventors have found through a plurality of experiments that the above composition for anti-helicobacter pylori can inhibit the expression levels of alpA gene and babA gene, thereby reducing the adhesion property of helicobacter pylori ATCC 700392 strain.
According to a fifth aspect of the present invention, there is provided an anti-helicobacter pylori product comprising the above anti-helicobacter pylori composition. The product provided by the invention takes the helicobacter pylori resistant composition as an active ingredient, and provides a new path for preventing or treating helicobacter pylori infection.
Preferably, the anti-helicobacter pylori product is an oral product.
Preferably, the oral products include drinks, tablets, capsules, powders, jellies, candies. The product provided by the invention has various dosage forms, wide application range and multiple application scenes, and is convenient for eaters to take the product in different application scenes.
Drawings
FIG. 1 is the effect of example 1 and blank on virulence gene expression;
FIG. 2 is a diagram of a scanning electron microscope corresponding to a blank group;
fig. 3 is a scanning electron microscope image corresponding to embodiment 1.
Detailed Description
In order that the manner in which the above-recited embodiments of the invention are attained and can be readily understood by those skilled in the art, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1
In this example, a composition against helicobacter pylori was prepared using commercially available grape seed extract, fucoidan.
The helicobacter pylori resistant composition is prepared by mixing 46.5 parts of grape seed extract and 5 parts of fucoidin. The grape seed extract used comprises 95wt% procyanidins; the fucoidan used contained 50wt% total sugar, 15wt% fucose, 20wt% sulfuric acid group (in SO 4 2- Meter).
Test example 1
A reference subject: the helicobacter pylori resistant composition provided in example 1.
The testing method comprises the following steps: the Minimum Inhibitory Concentration (MIC) of the individual H.pylori strains was determined by means of a mini-broth dilution method. The helicobacter pylori strains selected in this test example were: SS1 strain, ATCC 43504 strain, ATCC 700392 strain. The above strains were all purchased from American type culture Collection (American type culture collection, ATCC).
The specific test method is as follows:
1. preparation of culture medium
Preparing a blood plate: weighing 3.9g of Columbia agar basal medium into 100mL of distilled water, shaking and mixing uniformly, sterilizing at 121 ℃ for 20min under high pressure, cooling to 55 ℃, adding 5% of sterile defibrinated sheep blood into the system and mixing uniformly, inverting while hot, standing and drying overnight in a biosafety cabinet, wherein the prepared Columbia blood plate is used up within 2 weeks.
Brain heart leachate formulation (BHI): weighing 3.7g of brain-heart leaching solution powder into 100mL of distilled water, shaking and mixing, sterilizing at 121deg.C for 20min under high pressure, and storing in a refrigerator at 4deg.C until the obtained brain-heart leaching solution is used up within 2 weeks.
2. Resuscitating and passaging of bacteria
Taking out frozen strains (SS 1 strain, ATCC 43504 strain, ATCC 700392 strain) from ultra-low temperature refrigerator, after the strains are naturally melted, respectively sucking 100 μL of the strains, uniformly coating on Columbia blood plates, placing in a three-gas incubator, and microaerophilizing (5% O) at 37deg.C 2 、10％ CO 2 、85％ N 2 ) And culturing for 48-72 h in an inversion mode under the condition. When the strain grows to the surface of the blood plate, passage can be performed. Scraping the lawn from the blood plate with a wet sterile cotton swab, and suspending the lawn in sterile PBS to obtain a bacterial solution. 100 mu L of bacterial liquid is absorbed and evenly coated on a new Columbia blood plate, and the new Columbia blood plate is placed in a three-gas incubator to be cultivated for 48 to 72 hours in an inverted way under the microaerophilic condition at 37 ℃.
3. Minimum inhibitory concentration MIC was determined by micro broth dilution:
the test subjects were sonicated in BHI, vortexed, centrifuged at 12000rpm for 3min, and filtered through a 0.22 μm filter to prepare composition solutions having a plurality of concentration gradients of 9, 6, 3, 1.5, 1, 0.75mg/mL, etc.
50. Mu.L of the above-prepared composition solution was added to a 96-well plate, and at least 3 duplicate wells were set for each drug concentration, and negative control groups (containing only drug, not inoculated with strain) and growth control groups (containing no drug, only inoculated with strain) were set.
The strains were removed from the incubator, the wet sterilized cotton swab was used to scrape the lawn into PBS, the turbidity of the bacterial suspension was adjusted to 1McF with BHI containing 20% FBS, and the bacterial suspension was diluted 10 times and inoculated into the above-mentioned drug-containing 96-well plate (the bacterial suspension turbidity in the 96-well plate was finally about 1X 10) 6 CFU/mL), the 96-well plate was immediately placed in an incubator, and after shaking culture at 150rpm for 72 hours at 37 ℃, the observation was taken out.
When the growth control group shows button shape or obvious turbidity, the observation result is available, and when the naked eye is observed from bottom to top, the concentration of the drug with obviously reduced bacterial turbidity is MIC.
Test results: as shown in table 2.
TABLE 2 minimum inhibitory concentration measured in reference subjects
| Group of | Minimum inhibitory concentration MIC (mg/mL) |
| SS1 Strain | 6 |
| ATCC 43504 Strain | 3 |
| ATCC 700392 Strain | 1.5 |
Analysis of results:
as can be seen from the minimum inhibitory concentrations shown in Table 2, the helicobacter pylori resistant composition provided in example 1 has inhibitory effects on all of the above-mentioned various strains. Among them, the composition for anti-helicobacter pylori provided in example 1 had the most remarkable inhibitory effect on ATCC 700392 strain, and only 1.5mg/mL was required to achieve the excellent helicobacter pylori inhibitory effect. Therefore, the helicobacter pylori resistant composition provided by the invention has a limited inhibition effect on ATCC 700392 strain, and can effectively reduce the infection and colonization possibility of the strain.
Test example 2
A reference subject: the helicobacter pylori resistant composition provided in example 1 was prepared as a composition solution having a concentration of 1/2MIC in the same manner as that of test example 1, and the composition solution was used as a subject.
The testing method comprises the following steps: specific gene expression levels were detected by RT-qPCR experiments against 4 pathogenic mechanisms of H.pylori, respectively. Primer information related to this test example is shown in Table 3. The specific test method is as follows:
the ATCC 700392 strain with 1McF turbidity is taken and cultured until the growth log phase, and 1mL of bacterial liquid is added to the culture medium containing the test subjects for 24 hours for incubation. And a blank group is set: the ATCC 700392 strain with 1McF turbidity is taken, the strain is cultivated to a growth log phase, and 1mL of bacterial liquid is added to a culture medium without a test object for incubation for 24 hours.
Then, the culture medium after the incubation is centrifuged to collect the bacterial cells, RNA is extracted by using an RNA extraction kit, the concentration of RNA is measured by using Nanodrop 2000, and PrimeScript is used TM Reverse transcription of RT kit,Premix Ex Taq TM II kit for real-time fluorescence quantification, normalization of the results with 16S gene, use of 2 -ΔΔCT The relative expression level was calculated by the method.
TABLE 3 primer information
Test results: example 1 Gene expression corresponding to the blank is shown in FIG. 1, annotated: in FIG. 1, ", indicates that the statistical analysis is the difference between the treated samples and the blank control group, and P.ltoreq.0.05.
Analysis of results:
aiming at the formation and movement of flagella of helicobacter pylori, the inventor selects a flaB gene and a flgK gene as key detection objects. Among them, flaB has been recognized by the researchers in the industry as an essential gene for complete movement of helicobacter pylori, and flgK is involved not only in structural assembly of flagellin but also in the antigenicity of flagella. Aiming at the adhesion capacity of helicobacter pylori, the inventors selected alpA gene and babA gene as important detection objects. Wherein, the adhesion of helicobacter pylori to the surface of gastric epithelium is a key step in successful colonization of helicobacter pylori, and the colonization amount of helicobacter pylori in animals is reduced in the absence of alpA. The blood group antigen binding adhesins obtained by expression of babA are among the adhesins necessary for H.pylori colonization and infection. Urease mainly consists of structural proteins and auxiliary proteins aiming at helicobacter pylori, and the inventor selects ureB genes and ureI genes as key detection objects. Wherein, the ureB regulatory structural protein and the ureI regulatory auxiliary protein. ureI is a gene unique to helicobacter pylori urease, codes urease specific channel protein, and plays an important role in helicobacter pylori colonization on the surface of acid stomach. Can absorb urea in the stomach, so that the urea enters the helicobacter pylori to react with urease to generate ammonia and carbon dioxide, and thus the helicobacter pylori can colonise on the surface of the acid stomach. The inventors selected the VacA gene as an important test object for the virulence factor of helicobacter pylori. Among them, vacA is an important virulence gene of helicobacter pylori, and vacuolar toxins are obtained by VacA expression.
By comparing the virulence gene expression of example 1 with that of the control group in FIG. 1, it was found that the expression levels of the flaB and flgK genes, alpA and babA genes corresponding to example 1 were significantly lower relative to the gene expression levels of the control group, and that the flaB and flgK genes were related to the flagellum formation and motility of helicobacter pylori; the alpA and babA genes are associated with the adhesion capacity of H.pylori. Thus, it was demonstrated that the composition of helicobacter pylori provided in example 1 can significantly inhibit gene expression of helicobacter pylori with respect to flagella and adhesion property, thereby inhibiting the motility of flagella and the adhesion property of thallus. Meanwhile, the expression levels of the ureI and ureB genes and VacA genes corresponding to example 1 were low relative to the gene expression levels of the blank group, and the ureI and ureB genes were related to urease of helicobacter pylori; the VacA gene is an important virulence gene for helicobacter pylori. As described above, the helicobacter pylori composition provided in example 1 also has the effect of inhibiting the expression of various genes of helicobacter pylori with respect to urease and toxins, and further inhibiting the colonization of helicobacter pylori.
Test example 3
A reference subject: the helicobacter pylori resistant composition provided in example 1 was prepared as a composition solution having a concentration of 1/2MIC in the same manner as that of test example 1, and the composition solution was used as a subject.
The testing method comprises the following steps: the ATCC 700392 strain with 1McF turbidity is taken and cultured until the growth log phase, and 1mL of bacterial liquid is added to the culture medium containing the test subjects for 24 hours for incubation. And a blank group is set: the ATCC 700392 strain with 1McF turbidity is taken, the strain is cultivated to a growth log phase, and 1mL of bacterial liquid is added to a culture medium without a test object for incubation for 24 hours.
And centrifuging the incubated culture medium to collect thalli, washing the thalli for 2 times by using PBS, and adding 2.5% glutaraldehyde electron microscope fixing solution for overnight fixation at 4 ℃. Sequentially dehydrating 30%, 50%, 70%, 80% and 95% ethanol, soaking in tert-butanol for replacement, drying, attaching to a table, spraying gold, and observing with a scanning electron microscope.
Test results: the scanning electron microscope image corresponding to the blank group is shown in fig. 2. The scanning electron microscope diagram corresponding to the embodiment 1 is shown in fig. 3.
Analysis of results: the morphology of the whole helicobacter pylori cells was observed by a Scanning Electron Microscope (SEM), and the cell morphology shown in fig. 2 and 3 was compared, so that it was found that the cell corresponding to the blank group exhibited a regular shape and the surface was uniform. The cell surface corresponding to example 1 was damaged, the cell was dented and shrunken, and the cell shape was remarkably destroyed.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit 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.
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