Traditional Chinese medicine-probiotic combined preparation, preparation method thereof and application of traditional Chinese medicine-probiotic combined preparation in gastric mucosal lesion protection

Abstract

The invention discloses a traditional Chinese medicine-probiotic combined preparation, a preparation method thereof and application of the traditional Chinese medicine-probiotic combined preparation in gastric mucosal lesion protection. The preparation is composed of a traditional Chinese medicine extract and a bacillus subtilis bacterial suspension, the traditional Chinese medicine extract is prepared from radix codonopsis, rhizoma smilacis glabrae, rhizoma atractylodis macrocephalae, fructus aurantii, radix aucklandiae, bletilla striata, herba ecliptae, radix salviae miltiorrhizae, fructus arctii and fructus psoraleae according to a specific proportion through water decoction, filtration, freeze-drying and mixing with the prepared bacterial suspension. The combined preparation has remarkable protection and treatment effects on gastric mucosal lesion caused by helicobacter pylori infection and ethanol, not only can effectively inhibit the growth of helicobacter pylori, but also can relieve gastric inflammatory response and apoptosis, and the effect of the combined preparation is equivalent to or even better than that of a conventional triple therapy or omeprazole. Meanwhile, the preparation shows synergistic interaction functions of regulating body immunity and maintaining intestinal flora balance, the preparation process is simple and convenient, the safety is high, and the preparation has wide application prospects in preparation of drugs for resisting helicobacter pylori, protecting gastric mucosa and regulating intestinal flora.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a traditional Chinese medicine-probiotic combined preparation with effects of resisting Helicobacter pylori and protecting gastric mucosa, a preparation method thereof, and application of the preparation in preparation of a medicine for preventing and / or treating Helicobacter pylori infection and / or ethanol-induced gastric mucosa damage. BACKGROUND

[0002] Gastric mucosa damage is a common gastrointestinal disease, and its inducing factors are various, mainly including Helicobacter pylori (H. pylori) infection, excessive intake of stimulating substances such as ethanol, and long-term taking of non-steroidal anti-inflammatory drugs. Among them, H. pylori infection is an important cause of chronic gastritis, peptic ulcer and even gastric cancer. H. pylori penetrates the gastric mucosa mucus layer through its unique spiral structure and flagellum movement and colonizes on the surface of gastric epithelial cells. The urease secreted by the bacteria can decompose urea to produce ammonia, thereby neutralizing gastric acid and creating an alkaline microenvironment for its survival in a strong acid environment; however, this process also destroys the local acid-base balance of the gastric mucosa, and the ammonia produced has a direct toxic effect on epithelial cells. At the same time, the cytotoxin and related antigenic substances released by H. pylori can stimulate the host to produce a sustained inflammatory immune response, a large number of inflammatory cells infiltrate, and release a variety of inflammatory factors, resulting in serious damage to the function of the gastric mucosa barrier and a decrease in the repair capacity.

[0003] In the clinical treatment, the standard triple therapy of proton pump inhibitors combined with two antibiotics (such as amoxicillin and clarithromycin) or the quadruple therapy adding bismuth on this basis is the mainstream scheme for eradicating H. pylori. Although these schemes have a certain effect, their disadvantages are increasingly prominent. On the one hand, the widespread use of antibiotics leads to the continuous emergence of H. pylori drug-resistant strains, making the eradication failure rate rise. On the other hand, the treatment scheme itself can cause a series of adverse reactions, such as nausea, vomiting, diarrhea, constipation and intestinal flora disorder, which seriously affect the treatment compliance and quality of life of patients.

[0004] On the other hand, ethanol is a common exogenous irritant that causes acute gastric mucosal injury. Ethanol is lipid-soluble and can directly damage the lipid bilayer of gastric mucosal epithelial cells, increase cell membrane permeability, cause cell structure damage and chemical burns. At the same time, ethanol intake can rapidly activate immune cells in the gastric tissue, prompting the release of a large number of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), exacerbating local inflammatory response and further weakening the defense and repair capacity of the gastric mucosa. It is worth noting that there is a vicious cycle between ethanol-induced gastric mucosal injury and H. pylori infection: the damaged gastric mucosa provides more favorable conditions for the attachment and colonization of H. pylori, and the existing H. pylori infection amplifies the degree of mucosal inflammation and injury caused by ethanol.

[0005] In the face of the above problems, seeking an alternative therapy that can effectively inhibit H. pylori, repair gastric mucosal injury and has few side effects has become a research hotspot. Traditional Chinese medicine compounds have unique potential in regulating the spleen and stomach and protecting the gastric mucosa due to their multi-component and multi-target whole regulation advantages. For example, some traditional Chinese medicine prescriptions containing spleen-strengthening, qi-boosting, heat-clearing and detoxifying, and blood-activating and stasis-removing components can alleviate gastric discomfort symptoms, promote mucosal repair, and regulate immune function through comprehensive synergistic action. At the same time, probiotics as a kind of microecological regulator have also attracted widespread attention in inhibiting H. pylori colonization, improving antibiotic-induced intestinal flora imbalance, and enhancing mucosal immunity.

[0006] However, although there are traditional Chinese medicine treatments and probiotic therapies for H. pylori in the prior art, how to scientifically and effectively combine the two to develop a synergistic treatment regimen that can simultaneously achieve efficient antibacterial, significant anti-inflammatory, rapid mucosal repair, and simultaneous regulation of intestinal flora and systemic immunity is still a technical problem that the prior art has not solved well. In particular, providing a specific traditional Chinese medicine-probiotic combined preparation with clear components, scientific ratio, stable preparation process, and confirmed by in vitro and in vivo experiments to have a significantly better protective effect than conventional therapy on the above two gastric mucosal injury models is still a blank. Therefore, the present application aims to overcome the above-mentioned defects of the prior art and provide a completely new comprehensive solution. SUMMARY

[0007] Technical problems solved: Based on the status quo described in the background art, although the existing treatment for H. pylori infection has a certain eradication rate, it is easy to produce drug resistance and is often accompanied by gastrointestinal adverse reactions and intestinal flora disorder, leading to poor patient compliance. For acute gastric mucosal damage caused by stimulating substances such as ethanol, existing drugs often focus on single anti-acid or cell protection, and there is a lack of comprehensive anti-inflammatory, immune regulation and repair flora. At the same time, there is a lack of combined scheme in the prior art which can effectively integrate the overall regulation advantage of multiple components and multiple targets of traditional Chinese medicine and the microecological regulation effect of probiotics, and show a synergistic effect. Therefore, the present application provides a traditional Chinese medicine-probiotic combined preparation, a preparation method thereof and an application thereof in protecting gastric mucosal damage. The preparation can efficiently inhibit the growth of H. pylori, significantly protect the gastric mucosal damage caused by ethanol, and has the comprehensive effect of regulating intestinal flora balance and enhancing the body's immunity, thereby overcoming the problems of traditional therapy such as large side effects, easy drug resistance and single function, and providing a safer, more effective and comprehensive solution for the prevention and treatment of gastric mucosa-related diseases.

[0008] Technical scheme: A traditional Chinese medicine-probiotic combined preparation is prepared by mixing a traditional Chinese medicine extract and a Bacillus subtilis bacterial suspension; the traditional Chinese medicine extract is obtained by water extraction, filtration and freeze-drying of raw medicinal materials including the following components in the weight ratio: Radix Codonopsis 13-17 parts, Smilax glabra 8-12 parts, Atractylodes 8-12 parts, Aurantium 4-6 parts, Saussurea 4-6 parts, Bletilla 8-12 parts, Herba Hyperici 8-12 parts, Salvia miltiorrhiza 13-17 parts, Fructus Arctii 8-12 parts, and Psoralea 10-14 parts.

[0009] The above preparation is prepared into an oral liquid or granules.

[0010] The concentration of the Bacillus subtilis bacterial suspension in the above preparation is 1x10 9 CFU / mL to 8x10 9 CFU / mL.

[0011] The feeding ratio of the above traditional Chinese medicine extract and Bacillus subtilis is 67.6 mg: 1x10 9 CFU, based on the weight of freeze-dried powder and the number of bacterial colonies.

[0012] A method for preparing the traditional Chinese medicine-probiotic combined preparation, comprising the following steps: (1) traditional Chinese medicine extraction: weighing the raw medicinal materials according to the ratio, immersing in water and exceeding the liquid level by 2-3 cm, heating to boiling, and then three times of decoction, each time for 45 minutes, 45 minutes and 30 minutes; (2) filtration: after each decoction, filter the liquid while hot, and combine the three filtrates; (3) freeze-drying: concentrate the combined filtrate and freeze-dry to obtain a traditional Chinese medicine extract freeze-dried powder; (4) bacterial suspension preparation: culture Bacillus subtilis to the logarithmic growth phase, and prepare a bacterial suspension with a concentration of 2x109 (5) Mixing: Mix the freeze-dried powder of the Chinese herbal extract with the Bacillus subtilis suspension in proportion, stir and dissolve to obtain the final product.

[0013] In step (1), the decocting is done at a constant temperature.

[0014] In step (2), sterile gauze is used for filtration.

[0015] The above-mentioned combination of traditional Chinese medicine and probiotics is used in the preparation of drugs for inhibiting Helicobacter pylori.

[0016] The above-mentioned combination of traditional Chinese medicine and probiotics is used in the preparation of drugs for the prevention and / or treatment of gastric mucosal damage caused by ethanol.

[0017] Application of the above-mentioned traditional Chinese medicine-probiotic combination preparation in the preparation of drugs for regulating intestinal flora balance

[0018] Beneficial Effects: The traditional Chinese medicine-probiotic combined preparation provided by this invention exhibits multiple positive effects compared to existing technologies, with the synergistic effect between the traditional Chinese medicine components and probiotics being particularly significant. Specific experimental results show that in the *H. pylori* infection model, the high-dose group of the combined preparation showed a gastric bacterial inhibition effect similar to standard triple therapy, significantly reducing gastric inflammatory cell infiltration. Notably, the medium dose of the combined preparation also showed effects in regulating macrophages and CD4+ in gastric tissue and peripheral blood. + T cells and CD8 + In terms of T-cell levels, this combination therapy demonstrates superior immunomodulatory capabilities compared to triple therapy. Furthermore, while triple therapy leads to a significant reduction in the number of gut microbiota species, this combination effectively maintains gut microbiota diversity, bringing it close to a normal state. This characteristic overcomes the common problem of dysbiosis associated with antibiotic treatment.

[0019] In an ethanol-induced gastric mucosal injury model, all dosage groups of this combined preparation showed good protective effects. In particular, the medium and high dose groups were significantly more effective than the control group treated with omeprazole alone in reducing gastric bleeding points, decreasing the number of inflammatory cells, and inhibiting gastric mucosal cell apoptosis. Experimental data confirmed that this preparation can effectively regulate the levels of gastric inflammatory factors, with an anti-inflammatory effect superior to omeprazole, and comparable to omeprazole in improving serum oxidative stress indicators. Safety evaluation results showed that the combined preparation and its herbal components did not cause significant pathological changes in gastric epithelial cells and vital organs in in vitro cell experiments and in vivo acute toxicity experiments in mice, demonstrating good biocompatibility.

[0020] This invention organically combines a specific herbal compound with carefully selected probiotics, resulting in a significant synergistic effect. This synergistic effect is manifested in multiple aspects: it can effectively inhibit the growth of *H. pylori* while protecting the integrity of the gastric mucosa; it can regulate systemic immune function while maintaining the balance of the intestinal microecology. This multi-target and multi-level action makes this combined formulation a gastric mucosa protection strategy with significant comprehensive benefits and high safety. Attached Figure Description

[0021] Figure 1 Efficacy evaluation of H. pylori-infected mice. (A. Mouse administration timeline; B. Body weight changes during administration; C. Gastric H&E staining results; D. Number of H. pylori colonies in the stomach of each group of mice)

[0022] Figure 2 Detection of immune cells in gastric tissue of H. pylori-infected mice. (A. macrophages, CD4+) + T cells, CD8 + T cell staining image; BC. Cell quantification results)

[0023] Figure 3 Detection of peripheral blood immune cells in H. pylori-infected mice. (A. Flow cytometry detection of macrophages, CD4+) + T cells, CD8 + T cell expression level; BC. Cell quantification results)

[0024] Figure 4 Analysis of gut microbiota in H. pylori-infected mice. (A. Venn diagram from Features analysis; BC. Beta diversity analysis results; D. Alpha Diversity analysis results; E. Species taxonomy analysis at the phylum level; F. Species taxonomy analysis at the genus level; GH. LEfSe analysis results)

[0025] Figure 5 Efficacy evaluation of ethanol-induced gastric mucosal injury in mice. (A. Timeline of drug administration in mice; B. Gross observation of the stomach in each group of mice)

[0026] Figure 6 Efficacy evaluation of ethanol-induced gastric mucosal injury in mice. (A. Changes in mouse body weight during administration; B. Gastric H&E staining and TUNEL assay results; C. Quantitative results of TUNEL-positive cells)

[0027] Figure 7Detection of inflammatory factors in the stomach of mice with ethanol-induced gastric mucosal injury. (A. Fluorescent staining images of gastric inflammatory factors; B. C. Quantitative cell counts)

[0028] Figure 8 Detection of serum oxidative stress markers in mice with ethanol-induced gastric mucosal injury. (A. SOD detection results; B. MDA detection results)

[0029] Figure 9 In vitro cytotoxicity detection of traditional Chinese medicine extracts.

[0030] Figure 10 The mixed formulation was subjected to acute toxicity tests in mice. (AB. Changes in mouse body weight; C. H&E staining results of vital organs in mice) Detailed Implementation

[0031] Example 1

[0032] Step 1, Extraction of Traditional Chinese Medicine: Weigh the following traditional Chinese medicine materials according to weight: Codonopsis pilosula (Franch.) Nannf. 15g, Smilax glabra Roxb. 10g, Atractylodes macrocephala Koidz. 10g, Citrus aurantium L. 5g, Radix Aucklandiae 5g, Bletilla striata 10g, Herba Ecliptae 10g, Salvia miltiorrhiza Bunge 15g, Arctium lappa L. 10g, Psoralea corylifolia Linn. 12g. Place them in a 500mL beaker, add purified water until the liquid completely submerges the medicinal materials and exceeds the surface by 2~ 3cm, heat to boiling and then simmer at a constant temperature three times, each simmering for 45 minutes, 45 minutes and 30 minutes respectively.

[0033] The second step is filtration: while the medicine residue is still warm, remove it and filter it with three layers of sterile gauze to remove any residue fragments, and collect the filtrate; then, add an equal amount of purified water back to the medicine residue and continue the decoction process according to the above conditions.

[0034] The third step is freeze-drying: the three filtrates are combined and filtered again, the filtrate is concentrated, and then freeze-dried using a freeze dryer to obtain freeze-dried powder of the prescription, which is kept for later use.

[0035] Step 4, Preparation of bacterial suspension: Bacillus subtilis was cultured to the logarithmic growth phase and prepared into a suspension with a concentration of 2×10⁻⁶. 9The bacterial suspension of CFU / mL was stored in a refrigerator at 4°C for later use.

[0036] Step 5, Formulation Mixing: Calculate the dosage based on each 20g body weight mouse, i.e., "67.6mg of lyophilized formula powder + 1×10 probiotics". 9 "CFU" is prepared by mixing freeze-dried powder of traditional Chinese medicine extract with Bacillus subtilis suspension and stirring thoroughly until completely dissolved.

[0037] Example 2

[0038] Step 1, Chinese herbal medicine extraction: Weigh the Chinese herbal medicines according to their weight (Codonopsis pilosula 15g, Smilax glabra 10g, Atractylodes macrocephala 10g, Citrus aurantium 5g, Aucklandia lappa 5g, Bletilla striata 10g, Eclipta prostrata 10g, Salvia miltiorrhiza 10g, Arctium lappa 10g, Psoralea corylifolia 10g), place them in a 500mL beaker, add purified water until the liquid level completely submerges the Chinese herbal medicines and exceeds them by 2-3cm, heat to boiling, and then decoct three times at a constant temperature, with each decoction lasting 45 minutes, 45 minutes, and 30 minutes respectively.

[0039] The second step is filtration: while the medicine residue is still warm, remove it and filter it with three layers of sterile gauze to remove any residue fragments, and collect the filtrate; then, add an equal amount of purified water back to the medicine residue and continue the decoction process according to the above conditions.

[0040] The third step is freeze-drying: the three filtrates are combined and filtered again, the filtrate is concentrated, and then freeze-dried using a freeze dryer to obtain freeze-dried powder of the prescription, which is kept for later use.

[0041] Step 4, Preparation of bacterial suspension: Bacillus subtilis was cultured to the logarithmic growth phase and prepared into a suspension with a concentration of 2×10⁻⁶. 9 The bacterial suspension of CFU / mL was stored in a refrigerator at 4°C for later use.

[0042] Step 5, Formulation Mixing: Calculate the dosage based on each 20g body weight mouse, i.e., "67.6mg of lyophilized formula powder + 1×10 probiotics". 9 "CFU" is prepared by mixing freeze-dried powder of traditional Chinese medicine extract with Bacillus subtilis suspension and stirring thoroughly until completely dissolved.

[0043] Example 3

[0044] Step 1, Chinese herbal medicine extraction: Weigh the Chinese herbal medicines according to their weight (Codonopsis pilosula 15g, Smilax glabra 10g, Atractylodes macrocephala 10g, Citrus aurantium 5g, Aucklandia lappa 5g, Bletilla striata 10g, Eclipta prostrata 10g, Salvia miltiorrhiza 15g, Arctium lappa 10g, Psoralea corylifolia 12g), grind them into powder, coarsely sieve them and place them in a 500mL beaker, add purified water until the liquid level completely submerges the Chinese herbal medicines and exceeds them by 2-3cm, heat to boiling and then decoct three times at a constant temperature, each time for 45 minutes, 45 minutes and 30 minutes respectively. After each decoction, filter through a 100-mesh sieve and then let stand overnight.

[0045] The second step is freeze-drying: the three filtrates are combined and filtered again, the filtrate is concentrated, and then freeze-dried using a freeze dryer to obtain freeze-dried powder of the prescription, which is kept for later use.

[0046] The third step is the preparation of the bacterial suspension: Bacillus subtilis is cultured to the logarithmic growth phase and prepared into a suspension with a concentration of 2×10⁻⁶. 9 The bacterial suspension of CFU / mL was stored in a refrigerator at 4°C for later use.

[0047] Step 4, Formulation Mixing: Calculate the dosage based on each 20g body weight mouse, i.e., "67.6mg of lyophilized formula powder + 1×10 probiotics". 9 "CFU" is prepared by mixing freeze-dried powder of traditional Chinese medicine extract with Bacillus subtilis suspension and stirring thoroughly until completely dissolved.

[0048] 1. Materials

[0049] 1.1 Sample

[0050] Purchase Chinese herbal medicines and prepare them using Example 1.

[0051] 1.2 Strains

[0052] (1) H. pylori strains: including standard strains 26695 and G27; and Bacillus subtilis were provided by the Research Center for Prevention and Control of Drug-Resistant Microorganisms of Youjiang Medical College for Nationalities.

[0053] 1.3 Main culture media and reagents: Columbia medium, brain heart infusion medium, nutrient agar medium, nutrient broth medium, MH medium, Sabouraud dextrose medium, standard fetal bovine serum, LB medium.

[0054] 1.4 Main instruments: 30℃ and 37℃ incubators, 30℃ and 37℃ shakers, centrifuge, microplate reader, electronic balance.

[0055] 1.5 Consumables: EP tubes, tip heads, centrifuge tubes.

[0056] 2. Methods and Results

[0057] 2.1 H. pylori gastric mucosal injury model

[0058] 2.1.1 The minimum inhibitory concentration (MIC, 100 μL system) of the main active ingredients of traditional Chinese medicine prescriptions against H. pylori was determined by microdilution method.

[0059] (1) Prepare 101 mg / mL of Chinese herbal extract.

[0060] (2) Preparation of MIC plates: Add 173.6 μL of culture medium to the first well, then add 6.4 μL of antibacterial drug, and serially dilute to the 10th well; add only culture medium to the 11th well, and do not add drug to the 12th well. Retain 90 μL of culture medium as a control with added bacteria but no drug.

[0061] (3) Preparation of bacterial suspension: Take the strains growing in the logarithmic phase on solid plates and prepare bacterial suspensions with appropriate culture media: H. pylori and bacteria are adjusted to OD 600 It is 0.3 (1×10 8 CFU / mL), H. pylori diluted 10-fold, yielding 1×10⁻⁶. 7 CFU / mL; bacteria diluted 100-fold, yielding 1×10⁻⁶. 6 CFU / mL; Fungal adjustment concentration OD 600 0.5 (5×10 6 CFU / mL), diluted 5000 times, yields 1×10⁻⁶. 3 CFU / mL, for later use.

[0062] (4) Inoculation solution: Add 10 μL to wells 1-8 (concentration of fungi per well: 1 × 10⁻⁶) 2 CFU / mL, H. pylori is 1×10 6 CFU / mL, other bacteria 1×10⁻⁶ 5 (CFU / mL). Results were determined after culturing *H. pylori* for 72 h, fungi for 48 h, and other strains for 24 h. Drug concentrations in wells 1 through 6 were 6.2, 3.25, 1.625, 0.813, 0.406, and 0.203 mg / mL, respectively.

[0063] (5) Result Interpretation: The lowest drug concentration that completely inhibits bacterial growth within the well is defined as the MIC. The test is only meaningful when bacteria show significant growth in well 12 (i.e., without antibiotic) and no growth in well 11 (sterile). When a single skipped well occurs in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple skipped wells occur, the results should not be reported, and the test should be repeated. Each drug should be tested three times.

[0064] (6) Results: The minimum inhibitory concentration of the extract of the traditional Chinese medicine formula against H. pylori 26695 and G27 was 1.625 mg / mL, and against Bacillus subtilis 25 mg / mL.

[0065] 2.1.2 Detection of the inhibitory effect of customized H. pylori in the mouse stomach

[0066] Forty-eight SPF-grade, 6-8 week old female C57BL / 6 mice were randomly divided into a negative control group (n=6) and an infection group (n=42). Mice in each group were weighed, and the average body weight was calculated. Mice in the infection group were gavage: The 42 infected mice were fasted for 12 hours before gavage and then administered a suspension of *H. pylori* (HPBS001) prepared with BHI at a concentration of 1×10⁻⁶. 9 The concentration of CFU / mL was administered to each mouse at a dose of 0.5 mL. The mice were then kept on a 4-hour fast (no food or water). This was repeated every other day for a total of 5 times.

[0067] Model verification: 14 days after the last gavage, mice in the infected group and control group were weighed and their average body weight was calculated. Three mice in the infected group were randomly selected, fasted for 12 hours, and then euthanized by cervical dissection. The stomachs of the mice were dissected, and a portion of the stomach tissue was preserved in formalin for pathological examination. The other portion of stomach tissue was weighed, homogenized, and diluted 10, 100, and 1000 times. 100 μL of each portion was evenly spread on Columbia agar plates containing 10% serum and incubated in a tri-gas incubator (85% N2, 10% CO2, 5% O2) for 72–96 h. The characteristics of bacterial colonies on the culture medium were observed, and the number of pinhead-sized transparent colonies was counted.

[0068] Strain identification: The culture was pinhead-sized and translucent. Single colonies were Gram-stained and examined under a microscope.

[0069] Model construction assessment: 10% of mouse stomach tissue was used to detect H. pylori colonization, with a colonization amount ranging from 1×10⁻⁶. 5 ~1×10 6 When the concentration of CFU / g was reached, colonization was considered successful, and the test results showed that H. pylori colonization was present in all samples.

[0070] 2.1.3 Detection of the therapeutic effect of the combined formulation on H. pylori-infected mice in vivo

[0071] (1) Grouping: The successfully modeled infection group was divided into 8 groups: the stomach negative control group (Normal) of 6 mice that were not infected with H. pylori; PBS group; triple therapy (OPZ+AC); traditional Chinese medicine group (TCM); probiotic group (BS); traditional Chinese medicine + probiotic low group (TCM+BS-Low); traditional Chinese medicine + probiotic medium group (TCM+BS-Medium); traditional Chinese medicine + probiotic high group (TCM+BS-High).

[0072] (2) Administration: The medication was administered by gavage. In the triple therapy group, omeprazole (138.2 mg / kg) was administered 30 minutes before the other drugs, followed by amoxicillin (28.5 mg / kg) and clarithromycin (14.3 mg / kg); in the traditional Chinese medicine group (3380 mg / kg); and in the probiotic group (0.5 mL, 2×10⁻⁶ mg / kg). 9 CFU / mL); Low-dose group of traditional Chinese medicine + probiotics: Traditional Chinese medicine (482.85 mg / kg) + probiotics (0.5 mL, 5 × 10⁻⁶ CFU / mL); 8 CFU / mL); Traditional Chinese Medicine + Probiotics Medium-Dose Group: Traditional Chinese Medicine (3380mg / kg) + Probiotics (0.5mL, 2×10) 9 CFU / mL); High-dose group of traditional Chinese medicine + probiotics: Traditional Chinese medicine (13520mg / kg) + probiotics (0.5mL, 8×10) 9 The dose was administered at CFU / mL. Patients were not allowed to eat or drink for 4 hours after administration. The medication was administered once daily for 14 consecutive days. The negative control group was given PBS solution, with the same dosage and frequency as the other groups.

[0073] (3) On day 14 after drug withdrawal, the mice in the infected group were weighed to calculate the average body weight and then euthanized by cervical dislocation. Subsequently, stomach tissue was extracted from these mice, and H. pylori was isolated, cultured, and identified according to standardized operating procedures. In addition, pathological sections were prepared and H&E staining was performed to detect macrophages and CD4+ in the tissue samples. + CD8 + Expression of inflammatory factors. Simultaneously, the gut microbiota was analyzed to assess the impact of H. pylori infection on the gut microbiota.

[0074] (4) Results: such as Figure 1 As shown: The combined formulation did not have a significant effect on body weight. Figure 1 (BC) The high-dose combination therapy significantly inhibited H. pylori and significantly reduced the production of inflammatory cells in the stomach, with effects comparable to triple therapy. Figure 1 :D), the high-dose combination therapy also showed a similar bactericidal effect in the stomach as triple therapy. Figure 1 :E). For example Figure 2As shown: In the combined formulation, the dosage of gastric tissue macrophages and CD4... + CD8 + Cell counts were significantly reduced, and immune regulation was superior to the triple therapy group. For example... Figure 3 As shown: Macrophages and CD4 in the blood + CD8 + Cell count results and Figure 2 Consistent. For example... Figure 4 As shown: The Normal group, PBS group, OPZ+AC group (triple therapy), and TCM+BS-Medium group (medium-dose combination therapy) had 608, 441, 57, and 532 OTU species, respectively. Figure 4 (A) The number of gut microbiota in the triplet group was significantly decreased, with an increase in harmful bacteria such as Klebsiella spp., while the number of beneficial bacteria decreased. Figure 4 (EH). This demonstrates that the combined preparation has good effects in reducing gastric inflammation, regulating immunity, and restoring intestinal flora.

[0075] 2.2 Ethanol-induced gastric mucosal injury model

[0076] 2.2.1 In vivo efficacy detection of the combined formulation for treating ethanol-induced gastric mucosal injury

[0077] (1) Grouping: Forty-eight SPF-grade 6-8 week old female C57BL / 6 mice were randomly divided into groups of six, with six mice serving as the negative control group and the remaining 42 mice serving as the model group. The groups were: ddH2O group; omeprazole group (OPZ); traditional Chinese medicine group (TCM); probiotic group (BS); traditional Chinese medicine + probiotic low group (TCM+BS-Low); traditional Chinese medicine + probiotic medium group (TCM+BS-Medium); and traditional Chinese medicine + probiotic high group (TCM+BS-High).

[0078] (2) Administration: Omeprazole (138.2 mg / kg) was administered by gavage; traditional Chinese medicine group (3380 mg / kg); probiotics 0.5 mL (2 × 10) 9 Group CFU / mL; Low dose: Traditional Chinese medicine (482.85 mg / kg) + probiotics 0.5 mL (5 × 10) 8 CFU / mL); Medium dose: Traditional Chinese medicine (3380mg / kg) + probiotics 0.5mL (2×10) 9 CFU / mL); High dose: Traditional Chinese medicine (13520mg / kg) + probiotics 0.5mL (8×10) 9The dose was CFU / mL. Patients were kept NPO (no food or water) for 4 hours after administration. The medication was administered once daily for 7 consecutive days. The negative control group received the same dose and frequency as the ddH2O group. On day 7, patients were kept NPO for 24 hours but allowed free water. Two hours after administration, 75% ethanol (0.5 mL / 100 g) was administered, and the patients were sacrificed 4 hours later.

[0079] (3) After blood was collected from the eyes of the mice, they were euthanized by cervical dislocation. The stomach was photographed and recorded, and then the stomach tissue was taken for subsequent testing. Pathological sections of the stomach tissue were prepared and subjected to H&E staining and immunohistochemical analysis. At the same time, the expression levels of inflammatory factors such as IL-6, TNF-α and IL-1 in the tissue samples and serum SOD and MDA were detected.

[0080] (4) Results: such as Figure 5 As shown: the low-dose group of the combined formulation had fewer gastric bleeding points than the omeprazole group, and the medium and high doses had fewer bleeding points than the low-dose group. Figure 6 As shown: the combined treatment reduced the number of inflammatory cells in the stomach and significantly decreased the number of apoptotic cells, while apoptosis persisted throughout the entire course of the disease in the omeprazole group, indicating that the combined treatment was significantly more effective than the omeprazole group. Figure 7 As shown: the combined treatment significantly modulates gastric inflammatory factors, and is superior to the conventional treatment group using omeprazole alone. Figure 8 As shown: the serum levels of oxidative stress SOD and MDA in the combined treatment group were comparable to those in the omeprazole group.

[0081] 2.3 Safety Evaluation

[0082] 2.3.1 Cytotoxicity detection of traditional Chinese medicine extracts

[0083] (1) Prepare GES-1 cell suspension and adjust the concentration to 2×10⁻⁶. 5 .

[0084] (2) Inoculate into 96-well plates: 100 μL per well, and make 3 replicates of the same sample.

[0085] (3) Incubate at 37℃ for 24 hours.

[0086] (4) Set up experimental and control groups, and add the same volume of Chinese herbal extract and ddH2O solution respectively, with working concentrations of 16.25 mg / mL, 8.125 mg / mL and 1.625 mg / mL respectively. Set up pure culture medium group.

[0087] (5) Incubate at 37℃ for 24 hours.

[0088] (6) Add 10 μL of MTT to each well, gently tap to mix, and incubate for 4 hours.

[0089] (7) Measure the absorbance at 450 nm and calculate the cell viability using the formula: Cell viability = [(As-Ab)] / [(Ac-Ab)]×100%, where As represents wells containing cell culture medium, drug, and MTT, Ac represents wells containing cell culture medium and MTT but no drug, and Ab represents wells containing neither cells nor drug but only culture medium and MTT. Establish a survival curve based on the viability.

[0090] (8) Results: such as Figure 9 As shown: Extracts from traditional Chinese medicine formulas with a MIC of 1 to 10 times have little damage to GES-1 cells, exhibiting low toxicity and high safety.

[0091] 2.3.2 Safety experiment in mice

[0092] SPF-grade C57BL / 6 mice aged 6-8 weeks were housed in an SPF environment and randomly divided into a treatment group and a negative control group (n=8 per group). The mice were administered a 10-fold moderate therapeutic dose once daily for 7 days. The negative control group received PBS solution with the same frequency and dosage as the treatment group. Mice were weighed daily for 7 days, starting one day before administration. On day 8 after drug withdrawal, the mice in the treatment group were weighed, and the average weight was calculated. Blood was collected from the eyeballs, and the mice were euthanized by cervical dislocation. Heart, lung, stomach, kidney, liver, and spleen tissues were collected for pathological sectioning and H&E staining.

[0093] The results are as follows Figure 10 As shown: The body weight of mice in the 10-fold combined formulation administered by gavage was not significantly different from that in the normal group; no obvious pathological damage was observed in the heart, stomach, liver, spleen, kidneys, and lungs.

Claims

1. A traditional Chinese medicine-probiotic combined preparation, characterized in that, The preparation is prepared by mixing Chinese medicine extract and Bacillus subtilis suspension; the Chinese medicine extract is obtained by water extraction, filtration and freeze-drying of raw medicinal materials in the following proportions: Radix Codonopsis 13-17 parts, Smilax glabra 8-12 parts, Atractylodes 8-12 parts, Aurantii Fructus 4-6 parts, Saussurea 4-6 parts, Bletilla 8-12 parts, Eclipta 8-12 parts, Salvia miltiorrhiza 13-17 parts, Arctium lappa 8-12 parts, Psoralea 10-14 parts. 2.The traditional Chinese medicine-probiotics combined preparation of claim 1, characterized in that, The preparation is prepared into oral liquid or granules. 3.The traditional Chinese medicine-probiotics combined preparation of claim 1, characterized in that, The concentration of the Bacillus subtilis bacterial suspension in the preparation is from 1 x 10 9 CFU / mL to 8 x 10 9 CFU / mL. 4.The traditional Chinese medicine-probiotics combined preparation of claim 1, characterized in that, The feeding ratio of the traditional Chinese medicine extract and Bacillus subtilis is 67.6 mg: 1x10 9 CFU according to the weight of the freeze-dried powder and the number of colonies.

5. A method for preparing the traditional Chinese medicine-probiotic combined preparation according to any one of claims 1-4, characterized in that, The method comprises the following steps: (1) Chinese medicine extraction: according to the proportion, each raw medicinal material is weighed, and then immersed in water and exceeds the liquid level by 2-3 cm; after being heated to boiling, three times of decoction are carried out, and the decoction time of each time is 45 minutes, 45 minutes and 30 minutes respectively; (2) filtration: after each time of decoction, the medicinal liquid is filtered while hot, and the three times of filtrates are combined; (3) freeze-drying: the combined filtrate is concentrated and then freeze-dried to obtain a freeze-dried powder of Chinese medicine extract; (4) preparation of bacterial suspension: Bacillus subtilis is cultured to logarithmic growth phase, and a bacterial suspension with a concentration of 2x10 9 CFU / mL is prepared; (5) mixing: the freeze-dried powder of Chinese medicine extract and the bacterial suspension of Bacillus subtilis are mixed in proportion, stirred and dissolved, and then the product is obtained.

6. The method of claim 5, wherein, In step (1), the decoction is constant temperature decoction.

7. The method of claim 5, wherein, In step (2), sterile gauze is used for filtration.

8. Use of the Chinese medicine-probiotic combined preparation according to any one of claims 1-4 in the preparation of a medicament for inhibiting Helicobacter pylori.

9. Use of the Chinese medicine-probiotic combined preparation according to any one of claims 1-4 in the preparation of a medicament for preventing and / or treating ethanol-induced gastric mucosal injury.

10. Use of the Chinese medicine-probiotic combined preparation according to any one of claims 1-4 in the preparation of a medicament for regulating intestinal flora balance.

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