Application of probiotic composition in preparation of medicine for preventing and / or treating colonic diverticulosis and related diseases thereof

Abstract

The invention provides an application of a probiotic composition in preparation of a medicine for preventing and / or treating colonic diverticulosis and related diseases thereof, and the probiotic composition comprises bifidobacterium infantis, lactobacillus, coccus and bacillus. The probiotic composition can improve the pain threshold value of mice with diseases and reduce the level of inflammatory factors, and has a certain treatment prospect on colonic diverticulosis and related diseases thereof.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to the use of a probiotic composition in the preparation of a drug for preventing and / or treating colonic diverticulosis and its related diseases. Background Art

[0002] Colonic diverticulosis (DC) refers to the formation of sac-like protrusions at the weak part of the intestinal wall where there are nutrient blood vessels due to increased pressure in the colon. The most common clinical manifestations of patients are abdominal pain, mostly paroxysmal dull pain, sometimes accompanied by local tenderness in the left lower abdomen or right lower abdomen, and inflammatory irritation symptoms such as fever, diarrhea, abdominal distension, nausea, vomiting, and bloody stools. The incidence of colonic diverticulosis in China has been increasing year by year. At present, for symptomatic colonic diverticulosis and its complications, medical treatment is mainly used first. Symptomatic treatment such as the use of antibiotics, a diet high in fiber content, and maintaining smooth bowel movements can be used to relieve symptoms and prevent the development of complications. The main medical treatment methods include general treatments such as dietary restriction and intestinal rest, fiber, antibiotics (including the malabsorbed antibiotic rifaximin), anti-inflammatory drugs (such as mesalazine or balsalazide), and the use of probiotics alone or in combination for treatment. Among them, the treatment method of supplementing dietary fiber belongs to lifestyle treatment and may have the disadvantage of slow effectiveness; antibiotics such as rifaximin drugs have adverse reactions, such as headache, abdominal distension, abdominal pain, nausea, vomiting, and very few patients may have urticaria-like skin reactions; anti-inflammatory drugs such as mesalazine also have certain adverse reactions, such as mild gastric discomfort and may induce chronic hepatitis. Regarding probiotic treatment, there is currently not enough evidence to show that probiotics can effectively relieve the symptoms of patients with diverticular disease.

[0003] When colonic diverticulosis is also prone to abnormal immune function, such as excessive immune response or immune tolerance, it will lead to inflammatory reactions. The inflammatory reaction initially causes macrophages to release cytokines such as IL-1β and TNF-α. These inflammatory factors can further stimulate and expand the inflammatory process. If the immunity is excessive, cytokine levels can increase suddenly, including the participation of secondary mediators and inflammatory cells, causing tissue damage. Summary of the Invention

[0004] Aiming at the above problems, the purpose of the present invention is to provide the use of a probiotic composition in the preparation of a drug for preventing and / or treating colonic diverticulosis and its related diseases. The probiotic composition can increase the pain threshold of diseased mice and at the same time reduce the level of inflammatory factors, and has a certain therapeutic prospect for colonic diverticulosis and its related diseases.

[0005] The above object of the present invention is achieved by providing the following technical solutions:

[0006] The present invention provides the use of a probiotic composition in the preparation of a medicament for preventing and / or treating colonic diverticulosis and its related diseases, wherein the probiotic composition comprises Bifidobacterium infantis, Lactobacillus, Coccus and Bacillus.

[0007] Preferably, the present invention provides the use of a probiotic composition as the sole active ingredient in the preparation of a medicament for preventing and / or treating infantile colic and its related diseases, wherein the probiotic composition comprises Bifidobacterium infantis, Lactobacillus, Coccus and Bacillus.

[0008] Preferably, the probiotic composition consists of Bifidobacterium infantis, Lactobacillus, Coccus and Bacillus.

[0009] Preferably, in the probiotic composition, the viable count ratio of Bifidobacterium infantis, Lactobacillus, Coccus and Bacillus is (1-100):(1-100):(1-100):1, preferably (1-100):(1-100):(1-10):1.

[0010] Preferably, the total viable count of the medicament is not less than 1×10 5 cfu / g, preferably 2×10 5 to 4×10 9 cfu / g, more preferably 1×10 6 to 6×10 6 cfu / g.

[0011] Preferably, the viable count of Bifidobacterium infantis in the medicament is not less than 1×10 5 cfu / g, preferably 1×10 6 to 1×10 9 cfu / g.

[0012] Preferably, the viable count of Lactobacillus in the medicament is not less than 1×10 5 cfu / g, preferably 1×10 6 to 1×10 9 cfu / g.

[0013] Preferably, the viable count of Coccus in the medicament is not less than 1×10 4 cfu / g, preferably 1×10 5 to 1×10 8 cfu / g.

[0014] Preferably, the viable count of Bacillus in the medicament is not less than 1×10 3 cfu / g, preferably 1×10 4 to 1×10 7 cfu / g.

[0015] Preferably, the lactobacillus is selected from one or more of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. Bulgaricus (Lactobacillus bulgaricus), Lactobacillus delbrueckii subsp. Lactis, Lactobacillus fermentium, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei, and Lactobacillus curvatus, and preferably Lactobacillus acidophilus.

[0016] Preferably, the Enterococcus is selected from Enterococcus faecalis; Streptococcus, such as Streptococcus thermophilus; Galactococcus, such as Lactococcus Lactis subsp. lactis, Lactococcus Lactis subsp. cremoris, Lactococcus Lactis subsp. diacetylactis; Leuconostoc, such as Leuconostoc mesenteroides subsp. mesenteroides; Pediococcus, such as Pediococcus acidilactici, Pediococcus pentosaceus; and Staphylococcus, such as Staphylococcus vitulinus, Staphylococcus xylosus, Staphylococcus carnosus, and one or more of them, preferably Enterococcus faecalis.

[0017] Preferably, the Bacillus is Bacillus cereus and / or Bacillus coagulans, preferably Bacillus cereus.

[0018] More preferably, the Bifidobacterium infantis is Bifidobacterium infantis with the preservation number of CGMCC No. 0460.1.

[0019] More preferably, the Lactobacillus acidophilus is Lactobacillus acidophilus with the preservation number of CGMCC No. 0460.2.

[0020] More preferably, the Enterococcus faecalis is Enterococcus faecalis with the preservation number of CGMCC No. 0460.3.

[0021] More preferably, the Bacillus cereus is Bacillus cereus with the preservation number of CGMCC No. 0460.4.

[0022] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0023] Preferably, the pharmaceutically acceptable excipients are selected from one or more of fillers, moisture regulators, sour taste regulators, glidants, and flavors.

[0024] Preferably, the filler is selected from one or more of trehalose, lactose, and mannitol.

[0025] The inventors of the present invention found that if mannitol is included in the pharmaceutically acceptable excipients, mannitol has a high water activity and slightly poor compatibility with Bifidobacterium infantis and Lactobacillus acidophilus. Therefore, a moisture regulator needs to be added to the pharmaceutically acceptable excipients to reduce the moisture and improve the compatibility of mannitol with Bifidobacterium infantis and Lactobacillus acidophilus.

[0026] Preferably, the moisture regulator is soluble starch.

[0027] Preferably, the soluble starch is corn soluble starch and / or potato soluble starch.

[0028] The inventors of the present invention found that the hygroscopicity of potato soluble starch is lower than that of corn soluble starch or soluble starch from other sources in different environments, and thus the stability of the powder preparation prepared with potato soluble starch is higher.

[0029] Preferably, the sour taste regulator is citric acid and / or sodium citrate.

[0030] Preferably, the glidant is silicon dioxide.

[0031] Preferably, the target group of the drug is patients of all ages.

[0032] Preferably, the drug is a tablet, powder, or capsule.

[0033] The present invention has at least the following beneficial effects:

[0034] The present invention uses a probiotic composition at a lower dose alone to intervene in diseased mice, which can effectively increase their pain threshold, reduce the expression of inflammatory factors IL-1β and TNF-α, effectively relieve abdominal pain caused by colonic diverticulosis, and has a certain therapeutic prospect for colonic diverticulosis and its related diseases.

[0035] When the probiotic composition provided by the present invention is used alone, it can achieve a therapeutic effect under the condition of a lower dose, which is beneficial to reducing the treatment cost and can avoid the adverse reactions caused by current drug treatments. Description of the Drawings

[0036] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0037] Figure 1 The AWR scores of mice in the non-intervention group and the drug-intervention group after modeling with trinitrobenzenesulfonic acid (TNBS).

[0038] Figure 2 The intestinal inflammatory factor levels of mice in the non-intervention group and the drug-intervention group after TNBS modeling. (a) is TNF-α; (b) is IL-1β. Specific implementation manners

[0039] The present invention will be further described in detail below in conjunction with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention.

[0040] Example 1 Preparation of probiotic composition

[0041] 1.1 Freeze-drying of probiotics

[0042] After secondary activation of Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis, and Bacillus cereus, the probiotic powder is made into freeze-dried bacteria powder.

[0043] Among them, the strain preservation number of Bifidobacterium infantis is CGMCC No.0460.1 (preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: June 20, 2000);

[0044] The strain preservation number of Lactobacillus acidophilus is CGMCC No.0460.2 (preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: June 20, 2000);

[0045] The strain preservation number of Enterococcus faecalis (also known as Streptococcus faecalis) is CGMCC No.0460.3 (preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: June 20, 2000);

[0046] The strain preservation number of Bacillus cereus is CGMCC No.0460.4 (preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: June 20, 2000).

[0047] 1.2 Preparation of the Bacterial Solution of the Probiotic Composition

[0048] Mix the bacterial powders of Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis, and Bacillus cereus in ddH2O according to the calculated amounts to obtain the bacterial solution of the probiotic composition.

[0049] Among them, the total viable count of the bacterial solution of the probiotic composition is 4.22×10 7 cfu / ml, the viable count of Bifidobacterium infantis is 2.0×10 7 cfu / ml, the viable count of Lactobacillus acidophilus is 2.0×10 7 cfu / ml, the viable count of Enterococcus faecalis is 2.0×10 6 cfu / ml, and the viable count of Bacillus cereus is 2.0×10 5 cfu / g; the ratio of the viable counts of Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis, and Bacillus cereus is 100∶100∶10∶1.

[0050] Example 2 Animal Experiment

[0051] 2.1 Experimental Animals

[0052] The experimental animals are 6-week-old ICR mice, purchased from Beijing Speyford Biotechnology Co., Ltd., and raised in an animal house. The mice are raised in an SPF environment, and the temperature and humidity are controlled (22±2°C, 45%) under a strict 12∶12-hour dark-light cycle. Food and water are available ad libitum. Male and female mice are housed together. All animal experiments have been approved by the Animal Experiment Ethics Committee of Qilu Hospital of Shandong University.

[0053] 2.2 Experimental Methods

[0054] 2.2.1 Grouping

[0055] Grouping of mice: The mice are randomly grouped by body weight into an untreated group and a drug intervention group; the drug intervention group is further divided into: a probiotic group (administered the bacterial solution of the probiotic composition prepared in Example 1) and a control probiotic combination group.

[0056] Probiotic group: The mice are gavaged with the bacterial solution of the probiotic composition prepared in Example 1 (Bifidobacterium infantis and Lactobacillus acidophilus are both 2.0×10 7 cfu / kg, Enterococcus faecalis is 2.0×10 6 cfu / kg, Bacillus cereus is 2.0×10 5 cfu / kg), for a total of 4.22×10 7cfu / kg, continuously intragastrically administered for 4 weeks, and then TNBS was used to establish the model. Mice were fasted but allowed to drink water 24 hours before enema. 0.1 mL of 1.75 mg TNBS was injected into the descending colon through the anus, and the end of the catheter was 4 cm away from the anus. After the injection, the mice were kept in an inverted position for 30 s. The control group was given the same volume of normal saline.

[0057] Control probiotic combination group: Commercially available products Bifid Triple Viable Capsules and Yuanshou (Bacillus cereus var. mycoides viable capsules) were selected for isolation and culture, and a combination of four viable bacteria containing Bifidobacterium longum, Lactobacillus acidophilus, Enterococcus faecalis, and Bacillus cereus (the ratio of the four bacteria was the same as that of the probiotic group) was obtained. The intragastric administration dose was controlled at 4.22×10 7 cfu / kg, and then the TNBS model was established according to the same method as the probiotic group.

[0058] Non-intervention group: Mice were directly subjected to TNBS model establishment according to the same method as the probiotic group without drug intervention.

[0059] After model establishment, the abdominal wall withdrawal reflex was scored, and ELISA was used to detect the contents of inflammatory factors such as IL-1β and TNF-α in intestinal tissues.

[0060] 2.2.2 Index detection

[0061] 2.2.2.1 Abdominal wall withdrawal reflex (AWR) score

[0062] Before the experiment, mice were fasted but allowed to drink water for 24 h. A No. 6 pediatric urinary catheter with a balloon was lubricated with liquid paraffin and slowly inserted into the colorectum of the mice anesthetized with ether inhalation (about 2 cm from the anus) and fixed to the tail of the mice. After the mice woke up and adapted to the balloon for 30 min, air was quickly injected into the balloon to reach the specified pressure (60 mmHg, 80 mmHg) and maintained for 10 s, with an interval of 60 s. Each pressure was repeated 3 times. The abdominal wall withdrawal reflex was observed and scored, and the average score of the 3 times was taken.

[0063] AWR scoring criteria: 0 point, no response to dilation; 1 point, only slight head movement; 2 points, abdominal muscle contraction; 3 points, abdominal lifting; 4 points, body arching and pelvic lifting.

[0064] 2.2.2.2 Content of inflammatory factors

[0065] Total RNA was extracted from the intestine using TRIzol reagent, and then cDNA was prepared using the Toyobo qPCR kit. On the Applied Biosystems StepOne real-time PCR system, all reactions were performed in triplicate. GAPDH was used as an endogenous control.

[0066] 2.2.3 Statistical methods

[0067] The gene expression was analyzed by t-test. The data were expressed as Mean±SEM. Among them, p<0.05 (*) indicated significant difference, and p<0.01 (**) indicated extremely significant difference.

[0068] 2.3 Experimental results

[0069] 2.3.1 Abdominal withdrawal reflex score

[0070] As Figure 1 shown, when the pressure was 60 mm Hg, the AWR score of the mice intervened with probiotics was extremely significantly lower than that of the non-intervened group, and compared with the control probiotic combination group, the decrease amplitude of the probiotic group was greater; when the pressure increased to 80 mm Hg, there was still a significant difference in the AWR score between the probiotic group and the non-intervened group, while there was no significant difference between the control probiotic combination group and the non-intervened group, and even slightly higher than the non-intervened group. This indicated that the pain threshold of the mice in the probiotic group was higher than that of the mice in the non-intervened group, and the probiotic composition of the present invention had a better effect on improving the pain threshold than the control probiotic combination group.

[0071] 2.3.2 Content of inflammatory factors

[0072] As Figure 2 (a) shown, the level of TNF-α in the probiotic group was extremely significantly lower than that in the non-intervened group, while there was no significant difference between the control probiotic combination group and the non-intervened group.

[0073] As Figure 2 (b) shown, the level of IL-1β in the probiotic group was extremely significantly lower than that in the non-intervened group, while the control probiotic combination group was only significantly lower than the non-intervened group.

[0074] This indicated that the probiotic composition of the present invention had a better effect on alleviating the intestinal inflammation caused after modeling than the control probiotic combination group.

[0075] 3 Experimental summary

[0076] In the present invention, mice were intervened with a probiotic composition and TNBS enema was used. Three weeks after TNBS modeling, the rectal pain threshold of the mice was reflected by AWR score. When the rectal pressure was relatively high, the score of the mice in the probiotic group was significantly lower than that of the mice in the non-intervened group, indicating that the intervention with the probiotic composition of the present invention could effectively improve the pain threshold of the diseased mice; in addition, the intervention with the probiotic composition of the present invention also significantly reduced the levels of IL-1β and TNF-α, and alleviated the inflammatory response to a certain extent.

[0077] In summary, the research of the present invention shows that the intervention with the probiotic composition of the present invention can increase the pain threshold of diseased mice and reduce the levels of inflammatory factors, effectively relieve the abdominal pain caused by colonic diverticulosis, and has a certain therapeutic prospect for colonic diverticulosis and its related diseases.

[0078] The above are only several exemplary embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention is disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications using the technical content disclosed above to obtain equivalent or equivalent embodiments, which all belong to the scope of the present invention.

Claims

1. Use of a probiotic composition in the preparation of a medicament for preventing and / or treating colonic diverticulosis and its related diseases, wherein, The probiotic composition contains Bifidobacterium infantis, Lactobacillus, coccus, and Bacillus.

2. Use of a probiotic composition as the sole active ingredient in the preparation of a medicament for preventing and / or treating colonic diverticulosis and related diseases, wherein, The probiotic composition contains Bifidobacterium infantis, Lactobacillus, coccus, and Bacillus.

3. Use according to claim 1 or 2, wherein The probiotic composition consists of Bifidobacterium infantis, Lactobacillus, coccus, and Bacillus.

4. Use according to any one of claims 1 to 3, wherein In the probiotic composition, the viable count ratio of Bifidobacterium infantis, Lactobacillus, coccus, and Bacillus is (1-100):(1-100):(1-100):1, preferably (1-100):(1-100):(1-10):

1.

5. Use according to any one of claims 1 to 4, wherein The total number of viable bacteria contained in the drug is not less than 1×10 5 cfu / g, preferably 2×10 5 to 4×10 9 cfu / g.

6. Use according to any one of claims 1 to 5, wherein The live bacteria count of Bifidobacterium infantis contained in the drug is not less than 1×10 5 cfu / g, preferably 1×10 6 to 1×10 9 cfu / g; Preferably, the viable count of Lactobacillus contained in the drug is not less than 1×10 5 cfu / g, preferably 1×10 6 to 1×10 9 cfu / g; Preferably, the viable count of cocci contained in the drug is not less than 1×10 4 cfu / g, preferably 1×10 5 to 1×10 8 cfu / g; Preferably, the viable count of Bacillus contained in the drug is not less than 1×10 3 cfu / g, preferably 1×10 4 to 1×10 7 cfu / g.

7. Use according to any one of claims 1 to 6, wherein, The Lactobacillus is selected from one or more of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei, and Lactobacillus curvatus, preferably Lactobacillus acidophilus; Preferably, the coccus is selected from one or more of Enterococcus faecalis, Streptococcus, Lactococcus, Leuconostoc, Pediococcus, and Staphylococcus, preferably Enterococcus faecalis; Preferably, the Bacillus is Bacillus cereus and / or Bacillus coagulans, preferably Bacillus cereus.

8. The use according to claim 7, wherein, The Bifidobacterium infantis is Bifidobacterium infantis with the deposit number of CGMCC No. 0460.1; Preferably, the Lactobacillus acidophilus is Lactobacillus acidophilus with the deposit number of CGMCC No. 0460.2; Preferably, the Enterococcus faecalis is Enterococcus faecalis with the deposit number of CGMCC No. 0460.3; Preferably, the Bacillus cereus is Bacillus cereus with the deposit number of CGMCC No. 0460.

4.

9. Use according to any one of claims 1 to 8, wherein The drug further contains pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients are selected from one or more of fillers, moisture regulators, sour taste regulators, glidants, and flavors; Preferably, the filler is selected from one or more of trehalose, lactose, and mannitol; Preferably, the moisture regulator is soluble starch; more preferably, the soluble starch is corn soluble starch and / or potato soluble starch; Preferably, the sour taste regulator is citric acid and / or sodium citrate; Preferably, the glidant is silicon dioxide.

10. Use according to any one of claims 1 to 9, wherein, The target group of the drug is patients of all ages; Preferably, the drug is in the form of tablets, powders, or capsules.

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