A compound for inhibiting Helicobacter pylori and its preparation method and application

Abstract

The present invention discloses a complex for inhibiting Helicobacter pylori, and its preparation method and application. The complex is obtained by mixing a food and medicine homologous extract and Lactobacillus rhamnosus powder in a mass ratio of 5-15:1; wherein, the food and medicine homologous extract is prepared from the following raw materials in parts by weight: 40-60 parts of turmeric, 5-25 parts of saffron, 5-25 parts of bitter orange seeds, 5-25 parts of yellow mustard seeds, and 10-30 parts of torreya grandis. The complex provided by the present invention is prepared from a food and medicine homologous extract and Lactobacillus rhamnosus, can be applied to the food field, has good safety and stability, and can effectively prevent Helicobacter pylori infection when consumed in daily life; and the complex is obtained by water extraction, ultrasound, concentration, and vacuum freeze drying. This method can reduce its own bad odor, enhance taste, improve solubility, and make the extract easy to metabolize and absorb. The extract can be applied to food, broadening the application of food and medicine homologous raw materials in anti-Helicobacter pylori products and food.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine application, and particularly relates to a compound for inhibiting Helicobacter pylori, a preparation method and an application thereof. Background Art

[0002] Helicobacter pylori (H. pylori) is a common pathogen that causes gastrointestinal diseases such as gastric ulcers, gastritis, and gastric cancer. It has been classified as a Class I carcinogen by the World Health Organization, with a global infection rate exceeding 50%. H. pylori is primarily transmitted through oral-oral and fecal-oral routes. Due to my country's communal eating habits, its infection rate has been increasing annually. H. pylori is a microaerophilic, spiral-shaped, Gram-negative bacterium. Its multiple flagella on its surface enable it to penetrate the gastric mucosa, where adhesins allow it to colonize. The urease enzyme in the bacterium neutralizes gastric acid, allowing it to survive in the highly acidic environment of gastric fluid for extended periods. After colonization, Helicobacter pylori induces inflammatory response by expressing cytotoxin-associated protein A (CagA), releasing virulence factors such as vacuolar cytotoxin A (VacA), activating the NF-kB signaling pathway, and promoting cells to secrete inflammatory factors such as interleukin-8 (IL-8) and tumor necrosis factor α (TNFα), thereby causing gastric mucosal damage and inducing gastric-related diseases.

[0003] Currently, triple or quadruple therapy is often used to eradicate Helicobacter pylori and treat related diseases, with the main drugs being antibiotics such as amoxicillin, clarithromycin, and metronidazole. However, due to single or multiple gene mutations, modulation of efflux systems or membrane permeability to reduce antibiotic uptake, and other mechanisms, bacterial resistance to antibiotics is gradually increasing, resulting in a gradual decrease in Helicobacter pylori eradication rates. Furthermore, antibiotic treatment is associated with a variety of side effects such as diarrhea and vomiting. Therefore, it is of great significance to find drug alternatives and develop products related to the prevention and treatment of Helicobacter pylori.

[0004] Edible medicinal materials are edible Chinese medicinal materials, specifically referring to the edible parts of plants and animals listed in my country's "List of Items That Are Both Food and Medicine" and comply with the Pharmacopoeia of the People's Republic of China and relevant Chinese medicinal material standards. Studies have shown that various edible medicinal materials can inhibit Helicobacter pylori and offer advantages such as being less likely to induce drug resistance and having minimal side effects in preventing H. pylori infection and treating related diseases. However, due to unclear mechanisms of action, complex metabolism, insufficient research and development efforts, and an inadequate standardization system, these functional products have not been fully developed.

[0005] Probiotics are live microorganisms that have beneficial effects on host health. Numerous studies have demonstrated that various probiotics, such as Lactobacillus fermentum UCO-979C, Lactobacillus acidophilus ATCC4356, Lactobacillus reuteri ATCC 23272, Lactobacillus rhamnosus ATCC 7469, Lactobacillus plantarum ATCC 8014, and Lactobacillus casei ATCC 39392, can alleviate Helicobacter pylori infection. Probiotics can protect the stomach with mild effects and no side effects, but their therapeutic effects are not as pronounced as those of antibiotics. Therefore, in clinical practice, probiotics are often used as supplements to aid in the treatment of Helicobacter pylori infection.

[0006] In the prior art, for example, patent document CN115251381A provides a composition and beverage for combating Helicobacter pylori; patent document CN112717095A provides an anti-Helicobacter pylori preparation and a preparation method thereof. However, the use of the above patents still has the following problems:

[0007] (1) Food and medicine have rich efficacy, but they are often pungent, bitter, strong in smell or have a bad taste, so they are not easy to apply to food. There are few existing practical applications, and their edible value is ignored;

[0008] (2) Food and medicine are often taken in the form of decoctions. The active substances obtained by simple water extraction are relatively small, and their concentration is not sufficient to exert an antibacterial effect.

[0009] (3) Food and drug extracts are unstable in the digestive system, and the active substances change in a short period of time. After the effective antibacterial time, the sustained antibacterial effect cannot be guaranteed.

[0010] In view of this, the present invention is proposed. Summary of the Invention

[0011] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a complex for inhibiting Helicobacter pylori and its preparation method and application. It is prepared from food and medicine homologous extracts and Lactobacillus rhamnosus, can be applied to the food field, has good safety and stability, and can effectively prevent Helicobacter pylori infection by consumption in daily life.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] In a first aspect, the present invention provides a compound for inhibiting Helicobacter pylori, which is obtained by mixing a food and medicine homologous extract and Lactobacillus rhamnosus powder in a mass ratio of 5 to 15:1; preferably, the ratio of the two is 9:1;

[0014] The food and medicine extract is prepared from the following raw materials in parts by weight: 40 to 60 parts of turmeric, 5 to 25 parts of saffron, 5 to 25 parts of bitter orange seeds, 5 to 25 parts of yellow mustard seeds, and 10 to 30 parts of torreya nuts.

[0015] Helicobacter pylori's increased drug resistance may be due to single or multiple gene mutations, or to Helicobacter pylori regulating its efflux system or membrane permeability to reduce antibiotic uptake, leading to a gradual increase in the bacteria's resistance to antibiotics and a weakening of the antibiotic's effectiveness in eradicating Helicobacter pylori, resulting in a gradual decrease in the eradication rate. When the human stomach and intestines are stimulated by antibiotics, they may experience gastrointestinal discomfort and allergic symptoms such as diarrhea and vomiting, which can cause damage to the body.

[0016] To this end, the present invention uses turmeric, saffron, prunus aurantium, yellow mustard seeds, torreya grandis seeds and other edible and medicinal ingredients, as well as Lactobacillus rhamnosus, as raw materials. These edible and medicinal ingredients have both medicinal efficacy and edible value. Their extracts are rich in active substances such as polyphenols, which have a significant inhibitory effect on Helicobacter pylori, preventing the bacteria from developing drug resistance. Consumption has no side effects and does not cause harm to the human body, making them natural antibacterial ingredients.

[0017] In addition, food and medicine extracts are mostly polyphenols or flavonoids, which are unstable and easily decomposed in the digestive environment. Therefore, the effective antibacterial time is short. After the active substances are consumed, some Helicobacter pylori will regain their vitality and colonize again, weakening the antibacterial effect. In this regard, the present invention uses Lactobacillus rhamnosus powder and mixes it. The main function of Lactobacillus rhamnosus powder is to inhibit urease activity, reduce Helicobacter pylori vitality, or produce co-aggregation with Helicobacter pylori to form co-aggregates, and excrete Helicobacter pylori together. Therefore, adding probiotics to the complex can prolong the effective antibacterial time, improve the antibacterial effect, and simultaneously play a role in regulating the gastrointestinal flora.

[0018] Among them, turmeric: a perennial herb of the genus Curcuma in the Zingiberaceae family, has a pungent and bitter taste and is cold in nature. It has pharmacological effects such as anticoagulant, anti-inflammatory, anti-tumor and analgesic.

[0019] Saffron: The style and stigma are used for medicinal purposes. They are spicy and warm in nature. They have the effects of promoting blood circulation, removing blood stasis, promoting new blood, relieving pain, strengthening the stomach, and regulating menstruation.

[0020] Citrus aurantium: Its fruit or seeds with fleshy stalks can be used as medicine. It tastes sweet and sour, is neutral in nature, and has the effects of relieving thirst, detoxifying alcohol, and promoting urination.

[0021] Yellow mustard: The dried mature seeds of the cruciferous plant mustard can be used as medicine. They are pungent in taste and warm in nature. They have the effects of moistening the lungs and resolving phlegm, reducing swelling and relieving pain, warming the middle and dispersing cold, promoting diuresis and removing blood stasis, unblocking meridians, and reducing swelling and toxins. They are mainly used to treat stomach cold vomiting, heart and abdominal pain, lung cold cough, arthritis, throat paralysis, sputum, and bruises.

[0022] Torreya nuts: The seeds of Torreya grandis, a plant of the genus Torreya of the Taxaceae family, are sweet in taste and neutral in nature. They have the effects of killing insects, eliminating accumulation and moisturizing dryness. They are used to treat abdominal pain caused by insect accumulation, malnutrition in children, dry cough, constipation and other symptoms.

[0023] Lactobacillus rhamnosus can produce co-aggregation effect with Helicobacter pylori. As a probiotic, it is widely used, highly safe for consumption, has no side effects after consumption, and will not cause harm to the human body. It is a natural dietary supplement.

[0024] The combination of food and medicine with Lactobacillus rhamnosus broadens the complex's inhibitory mechanism against Helicobacter pylori, while avoiding the defects of strain resistance and antibiotic side effects.

[0025] As a preferred embodiment of the technical solution of the present invention, the food and medicine extract is prepared from the following raw materials in parts by weight: 50 parts of turmeric, 5 parts of saffron, 15 parts of bitter orange seeds, 10 parts of yellow mustard seeds, and 20 parts of torreya grandis seeds.

[0026] In a second aspect, the present invention provides a method for preparing the above-mentioned compound for inhibiting Helicobacter pylori, comprising the following steps:

[0027] (1) Water extraction: Take turmeric, saffron, bitter orange, yellow mustard seed, and torreya grandis according to weight, add distilled water, and stir and heat; after heating, the extract is obtained;

[0028] (2) Ultrasonic treatment: ultrasonically treating the extract obtained in step (1); after the treatment is completed, an ultrasonic extract is obtained;

[0029] (3) Concentration: Filtering the ultrasonic extract obtained in step (2), and then concentrating under reduced pressure to obtain a concentrated extract;

[0030] (4) vacuum freeze drying: the concentrated extract obtained in step (3) is subjected to vacuum freeze drying. After the treatment is completed, the food and medicine extract is obtained;

[0031] (5) activating the frozen Lactobacillus rhamnosus and then subculturing it; after the Lactobacillus rhamnosus is subcultured to the third generation, continuing the culture and growth to reach the logarithmic phase, then performing the initial centrifugation, discarding the supernatant, washing the resulting product, and centrifuging it again to obtain bacterial sludge; finally, vacuum freeze-drying the resulting bacterial sludge to obtain Lactobacillus rhamnosus powder;

[0032] (6) The food-drug homologous extract obtained in step (4) and the Lactobacillus rhamnosus powder obtained in step (5) are mixed to obtain a complex for inhibiting Helicobacter pylori.

[0033] Food and medicine homologous substances belong to the category of traditional Chinese medicine and have a strong traditional Chinese medicine flavor, which makes their application range in actual products relatively narrow. Relevant research on food and medicine homologous substances tends to explain the medicinal value and ignores the edible value, so there are few existing practical applications. Targetedly, the preparation method provided by the present invention can extract a variety of active substances from food and medicine homologous substances, and the obtained extracts have reduced bad taste, improved mouthfeel, good solubility, easy metabolism and absorption, and can be applied to food. For food safety, water extraction is used to extract active substances from food and medicine homologous substances, but the active substances obtained by water extraction are less active substances and have a low concentration, and fail to reach the minimum inhibitory concentration; the present invention proposes to use ultrasound-assisted extraction method, combined with reduced pressure concentration method, to increase the concentration of active substances, and the extract is vacuum freeze-dried to obtain extract powder; the extract obtained by the above method has good solubility, no bad smell, and can be widely used in food.

[0034] Specifically, in the preparation method provided by the present invention, water extraction is first performed. The inventors have found through preliminary experiments that turmeric, saffron, bitter orange, yellow mustard seed and torreya grandis can be used as raw materials for food and medicine compatibility; through water extraction, the active ingredients of food and medicine can be extracted into water, and the obtained food and medicine extract has reduced unpleasant odor, improved taste, good solubility, and is easy to metabolize and absorb; then ultrasonic treatment is performed, and ultrasonic treatment can increase the extraction rate of active substances, and ultrasonic power can affect the content of active substances; then, concentration treatment is performed, and reduced pressure concentration can increase the concentration of active substances; then, vacuum drying treatment is performed, and the concentrated food and medicine extract is freeze-dried to obtain a solid powder of the food and medicine extract, which can be used as one of the components of the complex;

[0035] As a preferred embodiment of the technical solution of the present invention, in step (1), the material-liquid ratio is 1:10; the stirring and heating temperature is 70-100° C., and the stirring and heating time is 0.5-2 h.

[0036] As a preferred embodiment of the technical solution of the present invention, in step (2), the ultrasonic treatment power is 30 to 100 W, and the ultrasonic treatment time is 30 to 60 min.

[0037] As a preferred embodiment of the technical solution of the present invention, in step (3), the reduced pressure concentration is performed to 1 / 5 of the volume of the original solution.

[0038] As a preferred embodiment of the technical solution of the present invention, in step (5), the activation treatment of Lactobacillus rhamnosus includes: thawing the frozen Lactobacillus rhamnosus in a 37°C water bath, then inoculating into MRS broth, and culturing at 37°C for 12 hours;

[0039] The subculture process includes: re-inoculating the activated Lactobacillus rhamnosus into MRS broth until it reaches the third generation.

[0040] As a preferred embodiment of the technical solution of the present invention, in step (5), the rotation speed of the initial centrifugation is 5000-7000 rpm, and the centrifugation time is 10-20 min.

[0041] In a third aspect, the present invention further claims the use of the above-mentioned complex in the preparation of a drug for preventing or treating Helicobacter pylori.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The Helicobacter pylori-inhibiting complex provided by the present invention is obtained by mixing a food-drug extract with Lactobacillus rhamnosus. The Lactobacillus rhamnosus, used as a raw material, can co-aggregate with Helicobacter pylori, forming co-aggregates that carry the Helicobacter pylori out of the body. This property compensates for the short duration of effective antibacterial activity often associated with food-drug extracts. Furthermore, Lactobacillus rhamnosus, as a probiotic, is widely used, highly safe for consumption, and has no side effects or harm to the human body. It is a natural dietary supplement that can be used in anti-Helicobacter pylori foods.

[0044] The Helicobacter pylori inhibition complex provided by the present invention comprises a food-drug homologous extract and Lactobacillus rhamnosus in a weight ratio of 9:1. The prepared complex of the food-drug homologous extract and Lactobacillus rhamnosus is not likely to cause drug resistance, has no toxic side effects, and is highly safe. The complex can effectively reduce the urease activity and adhesion ability of Helicobacter pylori, thereby inhibiting Helicobacter pylori, and can be used in foods for preventing Helicobacter pylori infection and alleviating related symptoms.

[0045] The Helicobacter pylori-inhibiting complex provided by the present invention is obtained through water extraction, ultrasound, concentration, and vacuum freeze-drying. This method can reduce the unpleasant odor of the complex, improve the taste, increase the solubility, and make the extract easy to metabolize and absorb. The extract can be used in food, broadening the application of food and medicine homologous raw materials in anti-Helicobacter pylori products and foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is a graph showing the change in the coaggregation rate of Lactobacillus rhamnosus and Helicobacter pylori over time;

[0047] Figure 2 This is a diagram of the precipitation of the co-aggregation culture tube of Lactobacillus rhamnosus and Helicobacter pylori.

[0048] Figure 3 The urease inhibition rates of different groups of complexes against Helicobacter pylori; A: physiological saline (pH = 2); B: artificial gastric juice; C: Lactobacillus rhamnosus; D: food and drug homologous extract; E: complex of Lactobacillus rhamnosus and food and drug homologous extract;

[0049] Figure 4The in vitro adhesion inhibition rates of different groups of complexes on Helicobacter pylori; where A: physiological saline (pH=2); B: artificial gastric juice; C: Lactobacillus rhamnosus; D: food and drug homologous extract; E: complex of Lactobacillus rhamnosus and food and drug homologous extract. DETAILED DESCRIPTION

[0050] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] Unless otherwise specified, all commodities or reagents in this invention were purchased through market channels.

[0052] Among them, the strain preservation number of Lactobacillus rhamnosus is: CGMCC NO.26088, and it was deposited in the China General Microbiological Culture Collection Center on November 8, 2022.

[0053] Example 1

[0054] A compound for inhibiting Helicobacter pylori, obtained by mixing a food and medicine homologous extract and Lactobacillus rhamnosus powder in a mass ratio of 9:1;

[0055] Among them, the food and medicine extract is prepared from the following raw materials by weight: 50g of turmeric, 5g of saffron, 15g of bitter orange, 10g of yellow mustard seeds, and 20g of torreya grandis.

[0056] In this embodiment, a method for preparing the above-mentioned compound for inhibiting Helicobacter pylori is also provided, comprising the following steps:

[0057] (1) Water extraction: Take turmeric, saffron, bitter orange, yellow mustard seed, and torreya grandis by weight, then add distilled water at a material-liquid ratio of 1:10, and then stir and heat at 90°C for 1 hour; after heating, the extract is obtained;

[0058] (2) Ultrasonic treatment: The extract obtained in step (1) was ultrasonically treated at a power of 40 W for 45 minutes; after the treatment, an ultrasonic extract was obtained;

[0059] (3) Concentration: The ultrasonic extract obtained in step (2) was filtered through gauze and allowed to stand for 2 h. The supernatant was centrifuged at 5000 rpm / min for 10 min, the precipitate was discarded, and the obtained extract was concentrated under reduced pressure to 1 / 5 of the original volume to obtain a concentrated extract;

[0060] (4) Vacuum freeze drying: The concentrated extract obtained in step (3) is vacuum freeze dried for 36 hours using a freeze dryer. After the treatment is completed, the food and medicine extract is obtained;

[0061] (5) The frozen Lactobacillus rhamnosus was placed in a 37°C water bath for rapid thawing, inoculated into fresh MRS broth, and cultured at 37°C for 12 hours. This is the first generation of bacteria, and activation is completed. The activated Lactobacillus rhamnosus was then suspended by oscillation and inoculated into fresh MRS broth at a 3% (v / v) inoculation rate. This is the second generation of bacteria. When the Lactobacillus rhamnosus is transferred to the third generation, the culture was continued for 12 hours to grow to the logarithmic phase. The product was then centrifuged at 6000 rpm / min for 15 minutes using a desktop centrifuge. After discarding the supernatant, the product was washed and centrifuged again to obtain bacterial sludge. Finally, the obtained bacterial sludge was resuspended in a 14% skim milk powder solution, suspended by oscillation, mixed evenly, and vacuum-freeze-dried using a freeze dryer until the sample was dry. The resulting freeze-dried powder was divided into sterile ziplock bags and stored at 4°C for later use. The Lactobacillus rhamnosus powder was obtained.

[0062] (6) The food-drug homologous extract obtained in step (4) and the Lactobacillus rhamnosus powder obtained in step (5) are mixed in proportion to obtain a complex for inhibiting Helicobacter pylori.

[0063] Example 2

[0064] A compound for inhibiting Helicobacter pylori, obtained by mixing a food and medicine homologous extract and Lactobacillus rhamnosus powder in a mass ratio of 9:1;

[0065] Among them, the food and medicine extract is prepared from the following raw materials by weight: 55g of turmeric, 6g of saffron, 12g of bitter orange, 8g of yellow mustard seeds, and 22g of torreya grandis.

[0066] In this embodiment, a method for preparing the above-mentioned compound for inhibiting Helicobacter pylori is also provided, comprising the following steps:

[0067] (1) Water extraction: Take turmeric, saffron, bitter orange, yellow mustard seed, and torreya grandis by weight, then add distilled water at a material-liquid ratio of 1:10, and then stir and heat at 90°C for 1 hour; after heating, the extract is obtained;

[0068] (2) Ultrasonic treatment: The extract obtained in step (1) was ultrasonically treated at a power of 40 W for 45 minutes; after the treatment, an ultrasonic extract was obtained;

[0069] (3) Concentration: The ultrasonic extract obtained in step (2) was filtered through gauze and allowed to stand for 2 h. The supernatant was centrifuged at 5000 rpm / min for 10 min, the precipitate was discarded, and the obtained extract was concentrated under reduced pressure to 1 / 5 of the original volume to obtain a concentrated extract;

[0070] (4) Vacuum freeze drying: The concentrated extract obtained in step (3) is vacuum freeze dried for 36 hours using a freeze dryer. After the treatment is completed, the food and medicine extract is obtained;

[0071] (5) The frozen Lactobacillus rhamnosus was placed in a 37°C water bath for rapid thawing, inoculated into fresh MRS broth, and cultured at 37°C for 12 hours. This is the first generation of bacteria, and activation is completed. The activated Lactobacillus rhamnosus was then suspended by oscillation and inoculated into fresh MRS broth at a 3% (v / v) inoculation rate. This is the second generation of bacteria. When the Lactobacillus rhamnosus is transferred to the third generation, the culture was continued for 12 hours to grow to the logarithmic phase. The product was then centrifuged at 6000 rpm / min for 15 minutes using a desktop centrifuge. After discarding the supernatant, the product was washed and centrifuged again to obtain bacterial sludge. Finally, the obtained bacterial sludge was resuspended in a 14% skim milk powder solution, suspended by oscillation, mixed evenly, and vacuum-freeze-dried using a freeze dryer until the sample was dry. The resulting freeze-dried powder was divided into sterile ziplock bags and stored at 4°C for later use. The Lactobacillus rhamnosus powder was obtained.

[0072] (6) The food-drug homologous extract obtained in step (4) and the Lactobacillus rhamnosus powder obtained in step (5) are mixed in proportion to obtain a complex for inhibiting Helicobacter pylori.

[0073] Example 3

[0074] A compound for inhibiting Helicobacter pylori, obtained by mixing a food and medicine homologous extract and Lactobacillus rhamnosus powder in a mass ratio of 9:1;

[0075] Among them, the food and medicine extract is prepared from the following raw materials by weight: 45g of turmeric, 7g of saffron, 9g of bitter orange, 6g of yellow mustard seeds, and 18g of torreya grandis.

[0076] In this embodiment, a method for preparing the above-mentioned compound for inhibiting Helicobacter pylori is also provided, comprising the following steps:

[0077] (1) Water extraction: Take turmeric, saffron, bitter orange, yellow mustard seed, and torreya grandis by weight, then add distilled water at a material-liquid ratio of 1:10, and then stir and heat at 90°C for 1 hour; after heating, the extract is obtained;

[0078] (2) Ultrasonic treatment: The extract obtained in step (1) was ultrasonically treated at a power of 40 W for 45 minutes; after the treatment, an ultrasonic extract was obtained;

[0079] (3) Concentration: The ultrasonic extract obtained in step (2) was filtered through gauze and allowed to stand for 2 h. The supernatant was centrifuged at 5000 rpm / min for 10 min, the precipitate was discarded, and the obtained extract was concentrated under reduced pressure to 1 / 5 of the original volume to obtain a concentrated extract;

[0080] (4) Vacuum freeze drying: The concentrated extract obtained in step (3) is vacuum freeze dried for 36 hours using a freeze dryer. After the treatment is completed, the food and medicine extract is obtained;

[0081] (5) The frozen Lactobacillus rhamnosus was placed in a 37°C water bath for rapid thawing, inoculated into fresh MRS broth, and cultured at 37°C for 12 hours. This is the first generation of bacteria, and activation is completed. The activated Lactobacillus rhamnosus was then suspended by oscillation and inoculated into fresh MRS broth at a 3% (v / v) inoculation rate. This is the second generation of bacteria. When the Lactobacillus rhamnosus is transferred to the third generation, the culture was continued for 12 hours to grow to the logarithmic phase. The product was then centrifuged at 6000 rpm / min for 15 minutes using a desktop centrifuge. After discarding the supernatant, the product was washed and centrifuged again to obtain bacterial sludge. Finally, the obtained bacterial sludge was resuspended in a 14% skim milk powder solution, suspended by oscillation, mixed evenly, and vacuum-freeze-dried using a freeze dryer until the sample was dry. The resulting freeze-dried powder was divided into sterile ziplock bags and stored at 4°C for later use. The Lactobacillus rhamnosus powder was obtained.

[0082] (6) The food-drug homologous extract obtained in step (4) and the Lactobacillus rhamnosus powder obtained in step (5) are mixed in proportion to obtain a complex for inhibiting Helicobacter pylori.

[0083] The complex prepared in the example was subjected to relevant anti-Helicobacter pylori tests. The test method is as follows:

[0084] Test 1: Antibacterial Effect of Food and Drug Extracts

[0085] (1) Principle: The inhibitory effects of food and medicine extracts prepared under different conditions on Helicobacter pylori are different. By measuring the in vitro antibacterial effect of the extracts on Helicobacter pylori, the optimal preparation conditions of the food and medicine extracts (based on the preparation method and raw material dosage ratio provided in Example 1) are determined.

[0086] (2) Method: The in vitro antibacterial effect of food and drug extracts on Helicobacter pylori was determined using the drug-sensitive paper antibacterial zone method. Different combinations of food and drug extracts were appropriately dissolved, and 20 μL of the solution was pipetted onto a sterile blank drug-sensitive paper. After the paper completely absorbed the liquid and dried, the paper was placed on a Columbia blood agar plate evenly smeared with Helicobacter pylori. The plate was cultured at 37°C under microaerobic conditions for 2-4 days, and the diameter of the antibacterial zone was measured. In the experiment, distilled water under the corresponding extraction conditions was set as the negative control, and the metronidazole drug-sensitive paper was set as the positive control.

[0087] (3) Results

[0088] The effects of different extraction temperatures on the Helicobacter pylori inhibition of food and medicine extracts are shown in Table 1. It was found that the antibacterial effect of the food and medicine combination raw materials was significantly better than the blank group and the single raw material (P<0.01). The antibacterial effect of the same group at different extraction temperatures was affected by temperature, with the antibacterial effect at 90°C being better than at other temperatures. The antibacterial effect of food and medicine extracts against Helicobacter pylori showed an overall trend of first increasing and then decreasing with increasing temperature, and the effect was optimal at 90°C. The test results are shown in Table 1 below.

[0089] Table 1 Results of inhibition zone test of food and medicine extracts at different extraction temperatures

[0090]

[0091] Note: Letters A, B, and C represent significant differences in the antibacterial effects of different groups at the same temperature (P<0.05); letters a, b, and c represent significant differences in the antibacterial effects of the same group at different temperatures (P<0.05) (the same below)

[0092] The effects of different extraction times on the antibacterial effects of food and medicine extracts on Helicobacter pylori are shown in Table 2. It was found that the antibacterial effect of the food and medicine combination raw materials was significantly better than the blank group and the single raw material (P<0.01). The antibacterial effect of the same group at different extraction times was affected by the heating time. The antibacterial effect of the 1-hour extraction was better than that of the other groups. The antibacterial effect of the food and medicine extracts on Helicobacter pylori showed an overall trend of first increasing and then decreasing with increasing heating time, and the effect was optimal under the 1-hour condition. The test results are shown in Table 2 below.

[0093] Table 2 Results of inhibition zone test of food and medicine extracts at different extraction times

[0094]

[0095] The effects of different ultrasonic powers on the Helicobacter pylori inhibition of food-drug extracts are shown in the table. The antibacterial effect of the food-drug combination was significantly better than that of the blank group and the single ingredient (P<0.01). The antibacterial effect of the same group under different ultrasonic powers was affected by the power level, with the best inhibition effect at 40W. The test results are shown in Table 3 below.

[0096] Table 3 Inhibition zone test results of food and medicine extracts at different ultrasonic powers

[0097]

[0098] The food and medicine were combined in different weight proportions to prepare extracts, and the inhibitory effect of the extracts on Helicobacter pylori was determined. It was found that when 50 parts of turmeric, 5 parts of saffron, 15 parts of bitter orange, 10 parts of yellow mustard seeds and 20 parts of torreya grandis (ie, Example 1) were added, the antibacterial effect was the best.

[0099] The above test results show that the food and medicine extracts can inhibit the growth of Helicobacter pylori, and the antibacterial effect of the combined extract is better than that of a single raw material. The weight ratio of the food and medicine is selected as 50 parts of turmeric, 5 parts of saffron, 15 parts of bitter orange, 10 parts of yellow mustard seeds and 20 parts of torreya grandis. The extraction conditions are selected as extraction temperature 90℃, extraction time 1h and ultrasonic power 40W.

[0100] Test 2: Coaggregation of Lactobacillus rhamnosus and Helicobacter pylori

[0101] (1) Principle: Lactobacillus rhamnosus can co-aggregate with Helicobacter pylori to form co-aggregates, which can be excreted from the body together with Helicobacter pylori. The co-aggregation ability of Lactobacillus rhamnosus and Helicobacter pylori can be determined by measuring the change of the co-aggregation rate of Lactobacillus rhamnosus and Helicobacter pylori over time.

[0102] (2) Methods: Lactobacillus rhamnosus and Helicobacter pylori cultured to the third logarithmic phase were centrifuged, washed twice with sterile PBS buffer, and resuspended in simulated gastric fluid (0.3% pepsin, 0.5% NaCl, pH = 3.0). The absorbance at 600 nm was measured and the bacterial solution concentration was adjusted to 1 × 10 9 CFU / mL. Mix equal volumes of Lactobacillus rhamnosus and Helicobacter pylori culture solutions and shake thoroughly. Incubate at 37°C for 4 h. Measure the absorbance of the supernatant at 600 nm after 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h of incubation. Calculate the coaggregation rate of probiotics and Helicobacter pylori according to (1).

[0103]

[0104] Where:

[0105] A1——Absorbance of Lactobacillus rhamnosus culture;

[0106] A2——absorbance of Helicobacter pylori culture;

[0107] A mix ——Absorbance of the supernatant after co-aggregation of mixed bacterial liquid.

[0108] (3) Results

[0109] The results of the study on the co-aggregation of Lactobacillus rhamnosus and Helicobacter pylori are as follows.

[0110] Table 4 Changes in the coaggregation rate of Lactobacillus rhamnosus and Helicobacter pylori over time

[0111]

[0112] Note: a, b, c represent significant differences in copolymerization rates at different times (P<0.05)

[0113] The change pattern of the coaggregation rate of Lactobacillus rhamnosus and Helicobacter pylori over time is shown in Table 4. The overall coaggregation rate shows a trend of gradually increasing over time. Within 0-2 hours, the coaggregation rate increased significantly (P<0.05). Within 2-4 hours, the coaggregation rate increased slightly but not significantly (P<0.05). This shows that Lactobacillus rhamnosus can coaggregate with Helicobacter pylori to produce coaggregates, and the coaggregation effect is significantly enhanced within 2 hours. The coaggregation phenomenon can also be judged by observing the formation of white flocs with the naked eye. Figure 2 As shown, the coaggregates appeared as white flocs that precipitated at the bottom of the culture tube. The experimental results show that Lactobacillus rhamnosus can effectively inhibit Helicobacter pylori by coaggregating with Helicobacter pylori.

[0114] Test 3: Effect of the complex on Helicobacter pylori urease activity

[0115] (1) Principle: Urease in Helicobacter pylori can neutralize gastric acid, allowing it to survive in the high-acid environment of gastric juice for a long time. Therefore, urease is an important marker of Helicobacter pylori. By measuring the urease activity of Helicobacter pylori under the intervention of different groups of complexes, the inhibition rate of the complex on urease is calculated, and the effect of the complex on the urease activity of Helicobacter pylori is determined.

[0116] (2) Method: The activated Helicobacter pylori was centrifuged and washed twice with sterile PBS buffer, then resuspended in the complex and incubated for 2 h. The urease activity of Helicobacter pylori was measured. The sample preparation method for each group of complexes was as follows:

[0117] Sample group A: physiological saline (0.9% NaCl, pH=2).

[0118] Sample group B: artificial gastric fluid [1 mL CH3COONa, 23.6 mg pepsin, 25 mg lipase added to 100 mL gastric electrolyte (1.10 g / L KCl, 0.25 g / L CaCl2, 0.60 g / L NaHCO3, 3.10 g / L NaCl), adjusted to pH 2 with 0.1 M HCl solution].

[0119] Sample group C: Lactobacillus rhamnosus powder was dissolved in artificial gastric fluid and shaken evenly.

[0120] Sample group D: an appropriate amount of the food and medicine extract obtained in Example 1 was dissolved in artificial gastric juice.

[0121] Sample Group E: The complex prepared in Example 1 was dissolved in artificial gastric fluid and shaken to mix evenly.

[0122] Blank group: physiological saline (0.9% NaCl).

[0123] 2 mL of Helicobacter pylori culture incubated with the complex was placed in a sterile centrifuge tube. After centrifugation, the supernatant was discarded, an appropriate amount of extract was added, and the bacteria were broken by ultrasound (ice bath, power 200 W, ultrasound 3s, interval 10s, repeated 30 times). The supernatant was taken and placed on ice for testing. According to the instructions of the urease (UE) kit, the ammonia produced by urease hydrolysis of urea was determined by indigo blue colorimetry. The corresponding reagent was added to the sample to be tested, and an equal amount of distilled water was added to the control group. After mixing, the sample was placed in a 37℃ water bath for 1h, centrifuged at 10000g at 25℃ for 10min, and the supernatant was taken and diluted 10 times. The corresponding reagent was added to a 96-well plate, and the absorbance at 578nm was measured. A control tube was set for each test tube. The urease activity was calculated according to (2).

[0124] Urease activity (μg / min / 10 4 CFU)=0.1092×(A1-A0)(2)

[0125] Where:

[0126] 0.1092 – Urease activity coefficient calculated based on bacterial density (one unit of enzyme activity is defined as 1 μg of NH3-N produced per 10,000 bacteria per minute);

[0127] A0——absorbance of control tube;

[0128] A1——Measurement tube absorbance.

[0129] The urease inhibition rate was calculated according to (3).

[0130]

[0131] (3) Results

[0132] The urease inhibition rate of the complex against Helicobacter pylori is as follows Figure 3 As shown in the figure, assuming the inhibition rate of the blank group on urease is 0, compared with the blank group, groups A and B had no inhibitory effect on urease activity, but instead increased its activity; compared with the blank group, groups C, D, and E all significantly inhibited the urease activity of Helicobacter pylori (P<0.01), with the inhibition rates of groups D and E being higher than that of group C. This experiment shows that Lactobacillus rhamnosus and edible medicinal extracts and their complexes can inhibit urease activity, thereby inhibiting the activity of Helicobacter pylori, and the inhibitory effect of the extracts and complexes is more pronounced.

[0133] Test 4: Effect of the complex on the adhesion of Helicobacter pylori in vitro

[0134] (1) Principle: This study determines the adhesion inhibition rate of Helicobacter pylori in vitro under the intervention of different combination complexes to determine the adhesion inhibition effect of the complex on Helicobacter pylori.

[0135] (2) Method: The H. pylori to be labeled was cultured to the third generation, centrifuged at 4°C for 10 min to obtain bacterial slurry, washed twice with sterile PBS, resuspended in carbonate buffer (0.5 mol / L pH = 9.5), and added with isothiocyanate solution to make the final concentration of isothiocyanate in the bacterial suspension 100 μg / mL. The suspension was stirred at room temperature for 1-2 h, centrifuged and the supernatant was discarded. The suspension was washed twice with PBS, and the obtained bacterial slurry was prepared with sterile saline (pH = 3.0) to a concentration of 1×10 9 CFU / mL labeled bacterial suspension. Weigh an appropriate amount of mucin and dissolve it in Tris buffer solution. Take 200 μL of the mucin solution in a 96-well plate and place it at 37°C for 24 hours. Rinse the plate twice with sterile PBS to remove unfixed mucin. On the 96-well plate with fixed mucin, add the labeled Helicobacter pylori liquid to each well, incubate at 37°C for 2 hours, discard the excess liquid, and wash twice with buffer. Prepare different groups of complexes according to Example 1, add different groups of complexes and commercially available anti-Helicobacter pylori drug solutions to the wells respectively, and incubate for 2 hours. Rinse the plate twice with sterile PBS, and use a multifunctional microplate reader to measure the relative fluorescence intensity at an excitation wavelength of 480 nm. Set up 3 parallels for each sample, and calculate the adhesion inhibition rate of each group of samples to Helicobacter pylori according to (4).

[0136]

[0137] (3) Results

[0138] In vitro adhesion inhibition rates of different combination complexes on Helicobacter pylori Figure 4 As shown, compared with groups A and B, the in vitro adhesion inhibition rates of groups C, D, and E were significantly increased (P < 0.01), indicating that Lactobacillus rhamnosus, the edible and medicinal extracts, and the complex of the two all inhibited the in vitro adhesion of H. pylori. Compared with groups C and D, the in vitro adhesion inhibition rate of group E was significantly increased (P < 0.01), indicating that the complex had a higher inhibition rate on H. pylori adhesion than either extract alone. This analysis suggests that Lactobacillus rhamnosus and the edible and medicinal extracts interact in inhibiting the in vitro adhesion of H. pylori. These results suggest that Lactobacillus rhamnosus, the edible and medicinal extracts, and the complex can inhibit H. pylori by reducing its adhesion rate, with the complex having a more significant effect than either extract alone, indicating that the complex can effectively inhibit the in vitro adhesion of H. pylori.

[0139] SPSS was used to analyze the interaction between the two factors. The results showed that the number of Lactobacillus rhamnosus had a significant effect on the adhesion inhibition rate of Helicobacter pylori (P<0.01), the concentration of the food and medicine extract had a significant effect on the adhesion inhibition rate of Helicobacter pylori (P<0.01), and the interaction effect between the number of Lactobacillus rhamnosus and the concentration of the food and medicine extract had a significant effect on the adhesion inhibition rate (P<0.05).

[0140] Test 5: Effect of the complex on the expression of genes related to Helicobacter pylori adhesion characteristics

[0141] (1) Principle: Among the genes related to the adhesion characteristics of Helicobacter pylori, sialic acid binding adhesion (SabA) and blood group antigen binding adhesion (BabA) are adhesin genes, and their expression affects the adhesion ability of Helicobacter pylori. The urease structural gene (ureA), urease auxiliary genes (ureE, ureG), and urease specific gene (ureI) are urease-related genes, and their expression affects the urease activity of Helicobacter pylori. This experiment determines the effect of the complex on the expression of genes related to the adhesion characteristics of Helicobacter pylori by measuring the expression levels of the above genes.

[0142] (2) Method: The expression level of Helicobacter pylori genes was determined by Real-Time PCR. Different groups of complexes were prepared according to Example 1. The activated Helicobacter pylori was centrifuged and washed twice with sterile PBS, and then resuspended in each group of complex samples and incubated at 37°C for 2h. The total RNA of Helicobacter pylori was extracted using a total RNA extraction kit, and cDNA was obtained using a reverse transcription kit. After the operation according to the fluorescence quantitative PCR kit, the expression levels of sialic acid binding adhesion (SabA), blood group antigen binding adhesion (BabA), urease structural gene (ureA), urease auxiliary gene (ureE, ureG), and urease specific gene (ureI) were determined using a fluorescence quantitative PCR instrument. The 16S gene was used as the internal reference gene, and the expression level of the blank group was used as the reference to calculate the relative expression level of the target gene in each group. Three parallels were set for each sample, and the results of Real-Time PCR were used 2 -△△Ct Method for data analysis.

[0143] (3) Results

[0144] The effects of different groups of complexes on the expression of some genes related to the adhesion ability of Helicobacter pylori are shown in Table 5. Compared with the blank group, Lactobacillus rhamnosus, edible and medicinal extracts and the complex can significantly reduce the expression of SabA, uraE, uraI and uraG (P<0.05), while the expression of BabA and uraA did not change significantly (P>0.05), indicating that Lactobacillus rhamnosus, edible and medicinal extracts and the complex can inhibit Helicobacter pylori by reducing the expression of some genes related to the adhesion ability of Helicobacter pylori. The effect of the complex is more significant than that of the two alone, that is, the complex can effectively inhibit Helicobacter pylori by reducing gene expression.

[0145] Table 5 Helicobacter pylori gene expression levels under the intervention of different groups of complexes

[0146]

[0147]

[0148] Note: a, b, c, d, e represent significant differences in Helicobacter pylori gene expression under intervention in different groups (P<0.05)

[0149] Among them, group A: normal saline (pH=2); B: artificial gastric juice; C: Lactobacillus rhamnosus; D: food and medicine homologous extract; E: complex of Lactobacillus rhamnosus and food and medicine homologous extract.

[0150] While the present invention is illustrated by the aforementioned embodiments, the present invention is not limited to these embodiments, nor does it necessarily rely on these embodiments for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent replacements for individual raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A compound for inhibiting Helicobacter pylori, characterized in that: The food and medicine homologous extract and Lactobacillus rhamnosus powder are mixed in a mass ratio of 5 to 15:1; The food and medicine extract is prepared from the following raw materials in parts by weight: 40-60 parts of turmeric, 5-25 parts of saffron, 5-25 parts of Hovenia dulcis seeds, 5-25 parts of yellow mustard seeds, and 10-30 parts of Torreya grandis seeds.

2. The compound for inhibiting Helicobacter pylori according to claim 1, characterized in that: The food and medicine extract is prepared from the following raw materials in parts by weight: 50 parts of turmeric, 5 parts of saffron, 15 parts of Hovenia dulcis seeds, 10 parts of yellow mustard seeds, and 20 parts of Torreya grandis seeds.

3. A method for preparing the Helicobacter pylori inhibiting compound according to claim 1 or 2, characterized in that: The steps include: (1) Water extraction: Take turmeric, saffron, Hovenia dulcis, yellow mustard seeds, and Torreya grandis according to weight, add distilled water, and stir and heat; after heating, the extract is obtained; (2) Ultrasonic treatment: ultrasonically treat the extract obtained in step (1); after the treatment is completed, an ultrasonic extract is obtained; (3) Concentration: Filter the ultrasonic extract obtained in step (2), and then concentrate under reduced pressure to obtain a concentrated extract; (4) Vacuum freeze drying: The concentrated extract obtained in step (3) is subjected to vacuum freeze drying. After the treatment is completed, the food and medicine extract is obtained; (5) Activating the frozen Lactobacillus rhamnosus and then subculture it; after the Lactobacillus rhamnosus is subcultured to the third generation, continuing to grow and reach the logarithmic phase, then performing the initial centrifugation, discarding the supernatant, washing the resulting product, and centrifuging it again to obtain bacterial sludge; finally, vacuum freeze-drying the resulting bacterial sludge to obtain Lactobacillus rhamnosus powder; (6) The food-drug homologous extract obtained in step (4) and the Lactobacillus rhamnosus powder obtained in step (5) are mixed to obtain a complex for inhibiting Helicobacter pylori.

4. The method according to claim 3, characterized in that In step (1), the material-liquid ratio is 1:10; the stirring and heating temperature is 70-100°C, and the stirring and heating time is 0.5-2h.

5. The method according to claim 3, characterized in that In step (2), the ultrasonic treatment power is 30~100W, and the ultrasonic treatment time is 30~60min.

6. The method according to claim 3, characterized in that In step (3), when concentrating under reduced pressure, the volume of the solution is concentrated to 1 / 5 of the original volume.

7. The method according to claim 3, characterized in that In step (5), the activation treatment of Lactobacillus rhamnosus includes: thawing the frozen Lactobacillus rhamnosus in a 37°C water bath, then inoculating it into MRS broth, and culturing it at 37°C for 12 hours; the subculture treatment includes: inoculating the activated Lactobacillus rhamnosus into MRS broth again until it is subcultured to the third generation.

8. The method according to claim 3, characterized in that In step (5), the speed of the initial centrifugation is 5000-7000 rpm, and the centrifugation time is 10-20 min.

9. Use of the complex according to claim 1 or 2 in the preparation of a drug for inhibiting Helicobacter pylori.

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