阅读说明：本技术 一种含壳寡糖的抗幽门螺旋杆菌制剂 (A preparation containing chitosan oligosaccharide for resisting helicobacter pylori ) 是由 韩旺全 韩睿乾 于 2020-05-19 设计创作，主要内容包括：本发明公开了一种包含壳寡糖的抗幽门螺旋杆菌制剂,所述的抗幽门螺旋杆菌制剂包括壳寡糖、ε-聚赖氨酸,所述的抗幽门螺旋杆菌制剂至少还包括溶菌酶、乳铁蛋白中的一种；添加薄荷醇、纯化水,或添加天然菊粉、麦芽糊精、淀粉,可制成抗幽门螺旋杆菌制剂的含潄剂或喷剂或颗粒剂。该发明制剂可以克服现有抗幽门螺旋杆菌制剂存在严重副作用、效果差等缺陷,以具有杀菌功能的纯天然成分壳寡糖等为主要原料制成的制剂,生产成本低,杀菌功能强、无毒副作用、不产生耐药性。可以在临床上用于口腔和胃部预防和治疗幽门螺旋杆菌感染以及与之相关疾病。(The invention discloses a helicobacter pylori resistant preparation containing chitosan oligosaccharide, which comprises chitosan oligosaccharide and polylysine, wherein the helicobacter pylori resistant preparation at least comprises lysozyme and lactoferrin; adding menthol and purified water, or adding natural inulin, maltodextrin and starch, and making into liquid soap, spray or granule for resisting helicobacter pylori. The preparation can overcome the defects of serious side effect, poor effect and the like of the existing helicobacter pylori resistant preparation, is prepared by taking chitosan oligosaccharide and the like which are pure natural components with sterilization function as main raw materials, and has the advantages of low production cost, strong sterilization function, no toxic or side effect and no drug resistance. Can be clinically used for preventing and treating helicobacter pylori infection and related diseases in oral cavity and stomach.)

1. The helicobacter pylori resistant preparation containing chitosan oligosaccharide is characterized by comprising chitosan oligosaccharide and polylysine, and the helicobacter pylori resistant preparation at least comprises lysozyme and lactoferrin.

2. The helicobacter pylori resistant preparation according to claim 1, wherein the helicobacter pylori resistant preparation comprises chitosan oligosaccharide, -polylysine, lysozyme, menthol and purified water, wherein the chitosan oligosaccharide is used in an amount of 35 to 40 g, the polylysine is used in an amount of 3 to 5g, the lysozyme is used in an amount of 8 to 15 g, and the menthol is used in an amount of 0.001 g in 1000ml of the helicobacter pylori resistant preparation, and the above component materials are sequentially added into the purified water to form the helicobacter pylori resistant preparation.

3. The helicobacter pylori resistant preparation containing chitosan oligosaccharide according to claim 1, wherein the helicobacter pylori resistant preparation comprises chitosan oligosaccharide, -polylysine, lactoferrin, native inulin, maltodextrin and starch, wherein in 1000g of the helicobacter pylori resistant preparation, the content of the chitosan oligosaccharide is 40-45 g, the content of the-polylysine is 3-15 g, the content of the lactoferrin is 0.1-0.5 g, the content of the native inulin is 9-11 g and the content of the maltodextrin is 29-31 g, and the above component materials are mixed with the starch to form the helicobacter pylori resistant preparation.

4. The anti-helicobacter pylori agent comprising chitosan oligosaccharide according to claim 2, wherein the following production method is employed: heating purified water to 40 ℃, sequentially adding chitosan oligosaccharide and polylysine according to the usage amount of the components, fully stirring until the components are dissolved, cooling to room temperature, adding lysozyme and menthol, stirring again until all the contents are completely dissolved, filtering with a 0.22 mu m filter membrane, adjusting the required pH value with lactic acid, subpackaging, and making into a liquid cleaning agent or a spray according to the requirements.

5. The preparation of claim 3, which comprises chitosan oligosaccharide, and is prepared by the following steps: adding the materials into a stainless steel tank in sequence according to the content, then uniformly mixing, using 70% ethanol as a wetting agent, preparing into granules by a wet method, sieving by a sieve, and then drying to obtain the helicobacter pylori resistant preparation granules.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a helicobacter pylori resistant preparation containing chitosan oligosaccharide.

Background

Helicobacter pylori, HP for short, is a spiral, micro-anaerobic bacterium with severe requirements on growth conditions, and after foreign researchers separate the helicobacter pylori from the gastric mucosa of a patient with chronic active gastritis, years of research show that the helicobacter pylori infection is considered as a main pathogenic factor of chronic gastritis, peptic ulcer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma and is closely related to the occurrence of gastric cancer, and the helicobacter pylori is determined as a carcinogen by the world health organization/International cancer institute (WH0/1ARC) in 1994, and is also a cause of some non-digestive tract diseases;

since then, researchers abroad separate and culture helicobacter pylori (Hp) from dental plaque and saliva successively, the Hp in the oral cavity is homologous with the Hp in the stomach, and the oral environment is also suitable for Hp aggregation and colonization;

the repeated infection of helicobacter pylori in stomach is closely related to the infection of helicobacter pylori in oral cavity, and has become the mainstream view of the international medical community at present, therefore, comprehensive intervention must be carried out synchronously aiming at Hp infection of the stomach and the oral cavity, so as to thoroughly and radically cure the infection caused by Hp, according to the relevance of the oral cavity and the Hp of the stomach, the invention provides a novel clinical solution with systematicness and creativity, and the chitosan oligosaccharide without side effect and drug resistance is adopted as a main raw material, firstly kills helicobacter pylori in the oral cavity, cuts off an input source, and then radically cures the helicobacter pylori in the stomach;

at present, the oral cavity helicobacter pylori resistant bacteria mainly comprise two types:

(1) the toothpaste containing the traditional Chinese medicines has the defects of complex traditional Chinese medicine components, weak sterilization capability, side effect, long administration time, poor effect and great functional defects of the dosage form of the toothpaste, and because the tooth brushing time is generally short and only takes 1-2 minutes, and the toothpaste has a local effect and can not fully contact all parts of the oral cavity in an all-round way, the sterilization is not thorough, and the sterilization effect is poor, the traditional Chinese medicines are not selected for the oral cavity HP killing component, and the gargle is selected for the dosage form.

(2) The bactericide containing chemical components has too large side effect and can destroy the oral micro-ecological balance, so the clinical proposal of the type has a plurality of technical defects;

currently, an antibiotic therapy is mainly adopted for diagnosis and treatment of helicobacter pylori in the stomach clinically, but long-term administration of antibiotics can generate a large amount of side effects and drug resistance, the curative effect is poor, the antibiotics are widely used, the antibiotics are one of the reasons for inducing the drug resistance of the helicobacter pylori, the eradication failure rate is high, Hp infection combined with the oral cavity is one of important reasons which cannot be ignored, the other one is treatment by using traditional Chinese medicines, although the side effects of the traditional Chinese medicines are relatively small, the medicine taking period is long, the sterilization effect is poor, the radical treatment rate is low, and the two clinical treatment modes have the condition that the effect of the preparation is damaged by the gastric acid environment;

therefore, overcoming the serious drawbacks and side effects of the existing anti-Hp clinical protocols, and developing a reliable, low-cost formulation for anti-Hp infection and treatment of diseases associated therewith is a very important and necessary new topic, which can serve more Hp-infected patients.

Disclosure of Invention

The invention aims to provide an application of chitosan oligosaccharide in a helicobacter pylori resistant preparation and a preparation method thereof, solves the problems of drug resistance, low cure rate and the like in the existing clinical treatment scheme, provides a biological preparation taking natural raw material chitosan oligosaccharide and the like as components for killing helicobacter pylori, and in order to enable a compound formula to generate antibacterial synergistic interaction, the preparation mainly comprises the chitosan oligosaccharide, -polylysine, lactoferrin, lysozyme and the like, and the components are all purely natural and have no side effect on a human body.

The invention provides the following technical scheme that the helicobacter pylori resistant preparation containing chitosan oligosaccharide comprises chitosan oligosaccharide and polylysine, and the main component of the helicobacter pylori resistant preparation at least comprises lysozyme and lactoferrin.

Preferably, the helicobacter pylori resistant preparation comprises chitosan oligosaccharide, polylysine, lysozyme, menthol and purified water, wherein in 1000ml of the helicobacter pylori resistant preparation, the usage amount of the chitosan oligosaccharide is 35-40 g, the usage amount of the polylysine is 3-5 g, the usage amount of the lysozyme is 8-15 g, and the usage amount of the menthol is 0.001 g, and the above component materials are sequentially added into the purified water to form the helicobacter pylori resistant preparation.

Preferably, the helicobacter pylori resistant preparation comprises chitosan oligosaccharide, polylysine, lactoferrin, natural inulin, maltodextrin and starch, wherein in 1000g of the helicobacter pylori resistant preparation, the content of the chitosan oligosaccharide is 40-45 g, the content of the polylysine is 3-15 g, the content of the lactoferrin is 0.1-0.5 g, the content of the natural inulin is 9-11 g, the content of the maltodextrin is 29-31 g, and the starch is added into the above components to form the helicobacter pylori resistant preparation.

Preferably, the following production method is adopted: heating purified water to 40 ℃, sequentially adding chitosan oligosaccharide and polylysine according to the usage amount of the components, fully stirring until the components are dissolved, cooling to room temperature, adding lysozyme and menthol, stirring again until all the contents are completely dissolved, filtering with a 0.22 mu m filter membrane, adjusting the required pH value with lactic acid, subpackaging, and making into a liquid cleaning agent or a spray according to the requirements.

Preferably, the following production method is adopted: adding the materials into a stainless steel tank in sequence according to the content, then uniformly mixing, using 70% ethanol as a wetting agent, preparing into granules by a wet method, sieving by a sieve, and then drying to obtain the helicobacter pylori resistant preparation granules.

Chitosan oligosaccharide is selected because the bacteriostatic function is enhanced in the strong acid environment of the stomach. To date, no other component has been found that can sufficiently exert antibacterial effect in a strong acid environment in the stomach, other than chitosan oligosaccharide. Most of the components have serious damage to the efficacy in a strong acid environment of gastric acid, but the chitosan oligosaccharide has better antibacterial effect in the strong acid environment, which is the unique point of the invention. Chitosan oligosaccharide (beta-1, 4-oligosaccharide-glucosamine) is called chito-oligosaccharide, is a natural aminopolysaccharide high molecular compound, and is dietary fiber with little positive charge found in nature. Is known as the 'sixth vital element' after protein, fat, sugar, vitamins and minerals by the international academia. Low molecular weight, good water solubility, easy absorption by human body, high bioactivity, etc. Not only has good biocompatibility and degradability, but also has broad-spectrum antibacterial, anti-infection and strong blood coagulation effects, and has various physiological activities such as wound healing promotion, immunity enhancement, tumor resistance and the like. Under the acidic condition, free amino groups in chitosan oligosaccharide molecules are protonated, and the protonated amino groups act with cell membranes with positive charges of bacteria to interfere the functions of the cell membranes of the bacteria and cause cytoplasm loss in the bacteria, so that the chitosan oligosaccharide has a strong killing effect on pathogenic microorganisms.

The application of the chitosan oligosaccharide in the helicobacter pylori resistant preparation comprises the following steps:

the factors influencing the bacteriostatic effect of the chitosan oligosaccharide are determined through a bacteriostatic test.

And (3) bacteriostatic test:

A. test materials:

helicobacter pylori standard strain Hp-ss1, chitosan oligosaccharide (molecular weight 1500), anticoagulated sheep blood, Columbia agar, Brookfield broth, autoclave, and distilled water.

B. The test method comprises the following steps:

(1) preparing a culture medium:

preparing a selective Columbia blood culture medium, 20g of Columbia agar, 2.5g of beta-cyclodextrin, 2ml of selective bacteriostatic mother liquor, 40ml of anticoagulated sheep blood, adjusting the pH value to 7.0, and adding purified water to 500 ml. Placing in an autoclave, autoclaving at 121 deg.C for 20min, cooling to 55 deg.C, adding anticoagulated sanguis caprae seu ovis and bacteriostatic mother solution, mixing, pouring into 8cm sterilized culture dish, making into selective Columbia blood culture medium plate with thickness of about 6mm, and cooling and solidifying.

Brookfield broth containing 30% glycerol 500 ml: contains glucose 0.5g, tryptone 5g, yeast extract 1g, peptone 10g, sodium chloride 2.5g, glucose 0.5g, and glycerol 150 ml.

(2) The operation method of the bacteriostasis test comprises the following steps:

0.3ml of the strain-containing 4 × 10⁷ml^-1Uniformly coating the broth liquid on culture medium, drying, sticking filter paper sheets with different samples on the surface of the culture medium by using filter paper sheet method, collecting filter paper sheets with sterilized purified water, horizontally placing the filter paper sheets in a sealed box, and placing a micro-oxygen bag (gas component 85% N)₂、10％CO₂、5％O₂) After incubation in an incubator at 37 ℃ for 48h, the diameter of the zone of inhibition was measured with a vernier caliper, and the average value was taken for at least 5 replicates for each sample.

C. Test results

(1) The effect of concentration on the inhibition test of helicobacter pylori.

The method comprises the steps of respectively detecting the bacteriostatic effect of solutions with the concentration of 1%, 2%, 3%, 4% and 5% by adopting a punching method, measuring the diameter of a bacteriostatic ring by using a vernier caliper, taking the average value of the diameters, and showing the result of the inhibition effect of chitosan oligosaccharide solutions with different concentrations on helicobacter pylori according to the test result of the following graph as shown in figure 1.

According to the curve in figure 2, it can be seen that chitosan oligosaccharide with different concentrations has inhibition effect on helicobacter pylori, and the inhibition effect is also obviously enhanced along with the increase of the concentration.

(2) The pH value influences the bacteriostasis test of the helicobacter pylori.

Preparing 5% chitosan oligosaccharide solution, adjusting pH to 2, 3, 4, 5, 6, and 7, respectively, and soaking the filter paper sheet therein for use. Pouring the sterilized culture medium into a culture dish, uniformly spreading and solidifying, sucking 0.3mL of bacterial liquid on the culture medium by using an aseptic injector, coating by using an aseptic coater, then placing filter paper sheets in the culture dish by using tweezers, horizontally placing four filter paper sheets in an incubator at 37 ℃ for 24 hours, measuring the diameter (mm) of a bacteriostatic ring, taking an average value, as can be seen from figure 3, the bacteriostatic effect of the chitosan oligosaccharide on helicobacter pylori is in the best state when the pH value is 3-4, the bacteriostatic effect is gradually enhanced along with the reduction of the pH value, the bacteriostatic effect is more obvious when the pH value is 3-4, because the chitosan oligosaccharide has a certain dissociation constant (pKa), when the pH value is lower than the pKa, the amino group of the chitosan oligosaccharide is protonated, so that the whole molecule has positive charge, and the crosslinking effect formed with the outer membrane of Hp bacteria is strongest, therefore, the anti-Hp effect is also strongest, and the interaction of the chitosan oligosaccharide and the substance with negative charge on the surface of the bacteria causes the membrane function to be disordered and generates the sinking, deformation and leakage. Meanwhile, the DNA enters the interior of the bacteria through the damaged cell outer membrane, further reacts with cytoplasm, and disturbs the transcription of the bacterial DNA, so that the bacterial reproduction is inhibited.

(3) Selection of Minimal Inhibitory Concentration (MIC), Minimal Bactericidal Concentration (MBC):

(a) adding chitosan oligosaccharide dilution solution with different concentration gradients into corresponding culture medium, simultaneously using culture medium containing equal amount of purified water as blank control, sterilizing at 121 deg.C for 20min, pouring the mixture into a plate, after the mixture is condensed, sucking 10 mu L of the bacterial liquid on a culture medium, dripping 3 drops (each drop is 10 mu L) on each plate, simultaneously inoculating a culture medium without chitosan oligosaccharide as a blank control test, repeating the test for 3 times, after the bacterial liquid is absorbed, the plate is horizontally placed in an incubator at 37 ℃ for culture, the growth condition of bacterial colonies is continuously observed for 4 days, taking the concentration of the non-growing bacteria after 24h culture as the Minimum Inhibitory Concentration (MIC) of the chitosan oligosaccharide and the derivatives thereof to the yeast, the test details are shown in Table 1 (note: + indicates the presence of bacteria, -indicates the absence of bacteria) with the concentration of 4d of non-growing bacteria in continuous culture as the minimum antimicrobial concentration (MBC).

TABLE 1 Minimum Inhibitory Concentration (MIC), minimum inhibitory concentration (MBC) determination Table

Concentration of

0.5％

1.0％

1.5％

2.0％

2.5％

3.0％

3.5％

4.0％

4.5％

24h

+

+

-

-

-

-

-

-

-

72h

+

+

+

+

-

-

-

-

-

96h

+

+

+

+

+

+

-

-

-

(b) As can be seen from Table 1, the minimum inhibitory concentration of chitosan oligosaccharide to helicobacter pylori is 1.5%, the minimum antimicrobial concentration is 3.5%, and the results of the whole experiment conclude that the chitosan oligosaccharide has the strongest inhibitory effect between ph values of 3-4, and the minimum antimicrobial concentration is 3.5%.

In conclusion, the chitosan oligosaccharide has the characteristic of stronger bacteriostatic effect in an acid environment, (1) when the chitosan oligosaccharide is applied to an oral clinical diagnosis and treatment use scheme, microorganisms in the oral cavity coexist in the oral cavity in symbiotic, competitive, antagonistic and other modes, so that a compound formula is needed to kill helicobacter pylori and other pathogenic bacteria in the oral cavity under the condition of protecting the oral micro-ecological environment; (2) the chitosan oligosaccharide is applied to a scheme for killing helicobacter pylori in the stomach, and can stimulate stronger bactericidal capacity and neutralize gastric acid by just utilizing the acidic environment of gastric acid.

The invention selects the natural antibacterial components existing in human body such as polylysine, lysozyme, lactoferrin and the like to be compatible with the chitosan oligosaccharide components, so as to ensure that the formula is more optimized and play a role in antibacterial synergy, and the composite preparation not only has enhanced antibacterial capability, but also can broaden antibacterial spectrum.

Polylysine, a polypeptide with bacteriostatic action, can be decomposed into lysine in human body, and the lysine is one of 8 amino acids necessary for human body, so that polylysine is a safe, non-toxic and nutrient type bacteriostatic agent, and has broad bacteriostatic spectrum, and can obviously inhibit and kill many pathogenic bacteria such as helicobacter pylori in gram-negative bacteria. Polylysine is rich in cations, has strong electrostatic interaction with harmful bacteria bearing anions and has good penetration into biological membranes, and on the basis of this property, polylysine can be used as a carrier for certain drugs and is therefore widely used in medical and pharmaceutical fields. In the invention, polylysine is used, and the chitosan oligosaccharide is matched to kill helicobacter pylori under different clinical application scenes, thereby playing a synergistic effect.

Lysozyme is a biologically active protein with no toxicity or side effects, and is widely present in various tissues of the human body. Hundreds of millions of active enzyme molecules can be activated instantaneously, mainly by breaking the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, so that the insoluble mucopolysaccharide of the cell wall is decomposed into soluble glycopeptide, and the content of the broken cell wall overflows to dissolve the bacteria. Can directly dissolve the cell wall of the oral bacteria to force the oral bacteria to die, promote the repair of damaged tissues and help balance the oral flora. Thus, various oral problems can be effectively improved.

Lactoferrin (LF) is an important non-heme iron-binding glycoprotein in milk, a monomeric glycoprotein with bactericidal activity in neutrophil granules. The sterilization mechanism is as follows: first capturing iron ions through the ion binding zone, thereby depriving the bacteria of iron necessary for their growth, causing a delay in bacterial growth; and interacts with lipid a in Lipopolysaccharide (LPS) on gram-negative bacteria, causing release of LPS from the cell wall, resulting in bacterial death.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention mainly selects the components of chitosan oligosaccharide and also selects natural antibacterial components such as polylysine, lysozyme, lactoferrin and the like existing in human bodies. The formula of the compound preparation not only enhances the antibacterial ability, but also can broaden the antibacterial spectrum. More importantly, the formula is safe, has no side effect, does not generate drug resistance and has high radical cure rate. Is beneficial to large-scale production and can benefit more patients.

2. At present, a plurality of diagnosis and treatment means and inventions only consider that the helicobacter pylori is killed theoretically and the special strong acid environment of gastric acid is not considered, but the chitosan oligosaccharide component selected by the invention happens to meet the harsh condition of enhancing the antibacterial ability in the acid environment. The technical defect that the efficacy of a plurality of clinical schemes in a gastric acid environment is damaged is overcome.

Drawings

FIG. 1 is a graph showing the results of the inhibition of H.pylori by the solutions of the present invention at different concentrations.

FIG. 2 is a graph showing the effect of different concentrations of the present invention on the inhibition of H.pylori.

FIG. 3 is a graph showing the effect of different pH values on the inhibition of helicobacter pylori.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides the following technical scheme: a helicobacter pylori resisting preparation containing chitosan oligosaccharide comprises chitosan oligosaccharide, -polylysine, and at least one of lysozyme and lactoferrin;