Application of Bacteroides fragilis in preparing medicine for preventing and treating irritable bowel syndrome

Abstract

The present invention relates to the use of Bacteroides fragilis in the preparation of a medicament for preventing and treating irritable bowel syndrome. The present invention confirms through in vivo experiments that Bacteroides fragilis ZY-312 has therapeutic and preventive effects on diarrhea-predominant irritable bowel syndrome.

Description

[0001] This application is a divisional application of Chinese patent application No. 202110977532.0, filed on August 24, 2021, with an application date of September 11, 2017, and the invention name is "Application of Bacteroides fragilis and its extracts in the preparation of drugs for the prevention and treatment of irritable bowel syndrome".

[0002] The microbial strains used in the practice of this invention were deposited on April 2, 2015, at the General Microbiology Center of the China Culture Collection Administration (CGMCC) (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing). Classification: Bacteroides fragilis ZY-312, deposit number: CGMCC No. 10685. Bacteroides fragilis ZY-312 was independently isolated and obtained by the applicant and is protected by a patent (Patent No. 201510459408.X). According to the Patent Examination Guidelines, the public can purchase it commercially or it has been authorized, so no deposit is required, i.e., no proof of deposit is required. Technical Field

[0003] The present invention relates to the technical field of application of Bacteroides fragilis, and in particular to application of Bacteroides fragilis in preparing a medicine for preventing and treating irritable bowel syndrome. Background Art

[0004] Irritable bowel syndrome (IBS) is the most common clinically recognized intestinal disorder. In recent years, it has been recognized as a psychosomatic disorder with distinct pathological and physiological underpinnings. It is a syndrome characterized by abdominal pain, bloating, constipation, diarrhea, or alternating constipation and diarrhea, without morphological or biochemical abnormalities. IBS has a high global prevalence, ranging from 10% to 15% in various regions. In Western countries, IBS accounts for 12% of family physician visits and 20% to 50% of gastroenterology outpatient visits. The prevalence of IBS symptoms in my country is similar to that in other countries, with patients primarily young and middle-aged adults aged 20 to 50. First-onset episodes over 50 years of age are rare. Women are more likely to suffer from IBS, with a male-to-female ratio of 1:5 to 1:2, and the condition tends to cluster in families. Studies have shown that IBS consumes significant medical resources and significantly impacts patients' quality of life.

[0005] The etiology and pathogenesis of IBS are still unclear. It may be related to factors such as diet, intestinal infection and mental and psychological factors, and is the result of the combined action of multiple factors. Its clinical symptoms are diverse. In addition to abdominal symptoms, it is accompanied by other symptoms, so treatment is more difficult and often requires combination medication. Some patients also have mental and psychological problems such as depression and anxiety, and often require psychotherapy. Therefore, when treating IBS, we should follow the principle of individualized treatment based on the severity of the patient's symptoms, the type of symptoms and the frequency of attacks, and adopt comprehensive treatment measures. The choice of treatment methods and drugs should vary from person to person, including: basic treatment, psychotherapy and drug therapy. At present, the main drugs for treating IBS are the following:

[0006] 1. Drugs that regulate intestinal function, including antidiarrheals, antispasmodics, prokinetics and drugs that regulate visceral sensitivity.

[0007] Antidiarrheals are commonly used to treat diarrhea in IBS patients. Commonly used drugs include loperamide, diphenoxylate, and dioctahedral montmorillonite. Loperamide (Imodium) acts on the opioid peptide receptors in the intestinal wall, preventing the release of acetylcholine and prostaglandins, inhibiting intestinal peristalsis, prolonging the retention time of intestinal contents, and enhancing the absorption of water and ions in the intestine, thereby relieving diarrhea, abdominal pain, etc. Diphenoxylate (phenoxylate) acts on intestinal smooth muscle, increasing segmental intestinal contractions, and prolonging the contact time between intestinal contents and intestinal mucosa. Dioctahedral montmorillonite (Smectite) can absorb water and pathogens, improve the protective ability of the digestive tract mucosa, and promote mucosal repair. At the same time, it can also adjust and restore colon motility and reduce colon sensitivity.

[0008] Antispasmodics are commonly used to treat abdominal pain and bloating in patients with IBS. They can be divided into three categories based on their primary mechanism of action: anticholinergics, smooth muscle relaxants, and calcium channel blockers. Many of these drugs have multiple pharmacological effects.

[0009] Anticholinergic drugs include atropine, anisodamine, and belladonna. Their clinical application is limited by atropine-like side effects. Recently developed intestinal M3-selective cholinergic receptor antagonists can inhibit postprandial intestinal motility and hold promise for the treatment of IBS.

[0010] Smooth muscle relaxants include papaverines (papaverine, dicyclomine, mebeverine) and multi-ion channel modulators (trimebutine). Papaverines act directly on smooth muscle cells and certain intestinal excitatory neurons, inhibiting the release of excitatory neurotransmitters. Trimebutine increases the excitability of smooth muscle cells by inhibiting cell membrane potassium channels, causing depolarization. On the other hand, it blocks calcium channels and inhibits calcium influx, thereby inhibiting cell contraction and relaxing gastrointestinal smooth muscle. In addition, trimebutine has a bidirectional regulatory effect on smooth muscle nerve receptors: in a low-motility state, it acts on adrenergic receptors to inhibit the release of norepinephrine and increase motor rhythm; in hypermotility, it acts on cholinergic and opioid receptors to inhibit the release of acetylcholine, thereby inhibiting smooth muscle movement.

[0011] Calcium ion antagonists selectively act on the colon calcium ion channels, blocking calcium influx, relaxing smooth muscles, inhibiting gastrocolic reflexes, regulating constipation and diarrhea, and also have a certain effect on abdominal pain, such as pinaverium bromide and otilium bromide.

[0012] Prokinetic drugs are commonly used to treat constipation in patients with IBS. 5-hydroxytryptamine (5-HT) is an important transmitter in the gastrointestinal tract and central nervous system, with a wide range of biological effects. 95% of 5-HT in the human body is distributed in the gastrointestinal tract, and multiple 5-HT receptors and 5-HT transporters are distributed in the intestinal mucosa. In recent years, the important role of the 5-HT4 receptor in regulating gastrointestinal motility and visceral sensation has gained attention. It promotes gastrointestinal motility, reduces gastrointestinal sensitivity, and stimulates the secretion of chloride ions and water molecules, making it a new target for the treatment of functional gastrointestinal disorders. The 5-HT4 receptor agonist cisapride stimulates the release of acetylcholine from postganglionic cholinergic nerves in the myenteric myenteric tract, exerting prokinetic effects throughout the gastrointestinal tract. However, due to the potential for QT prolongation, this drug should be used with caution. Tegaserod, also known as Zelmac, is a new 5-HT4 receptor agonist that partially selectively acts on the gastrointestinal 5-HT4 receptor subtype and accelerates small intestinal and colonic transit in patients with constipation-predominant IBS. Recent studies have also confirmed that tegaserod has a regulatory effect on visceral sensation and has no adverse cardiovascular reactions, making it a safe and effective new drug.

[0013] Drugs that modulate visceral sensitivity include 5-HT3 receptor antagonists and 5-HT4 receptor agonists. Increased visceral sensitivity is considered a key pathological and physiological characteristic of IBS. Studies have shown that 61% of IBS patients experience visceral abnormalities. Improving visceral sensation is an interesting approach to IBS treatment. Clinical and animal studies have shown that some drugs can modulate increased visceral sensitivity.

[0014] 5-HT3 receptors are present in enteric neurons. They promote intestinal motility and secretion, and induce visceral pain stimulation by releasing 5-HT locally. For IBS patients, especially those with a decreased visceral pain threshold and abdominal pain, 5-HT3 receptor antagonists can be tried. For example, alosetron primarily inhibits the 5-HT3 receptors of non-selective ion channels in the enteric nervous system, suppressing visceral reflexes. Recently, it has been primarily used for female patients with severe diarrhea-predominant IBS who have failed traditional treatments. Other options include ondansetron and gentamycin.

[0015] The 5-HT4 receptor agonist tegaserod has the dual effects of promoting motility and reducing visceral sensitivity, making it suitable for patients with constipation-predominant IBS accompanied by significant abdominal pain. Human studies have reported that tegaserod can reduce the response to noxious stimulation of rectal balloon distension and improve visceral sensation.

[0016] 2. Laxatives: In addition to 5-HT4 receptor agonists, laxatives can also be used for patients with constipation. It is currently believed that stimulant laxatives should be used with caution or avoided as much as possible, and bulking laxatives are recommended. Bulking laxatives, such as plantain, can increase stool volume and moisture, promoting bowel movements. The osmotic laxative polyethylene glycol binds to water molecules through hydrogen bonds, increasing stool moisture content and softening the stool, promoting bowel movements. Because it is not absorbed and has minimal toxicity, it is suitable for patients with dry stools. Lactulose is broken down by bacteria in the colon to form lactic acid and acetic acid, which regulate the intestinal environment and promote intestinal peristalsis. This drug is more suitable for the elderly.

[0017] 3. Medications that improve central affectivity: IBS patients often have psychiatric disorders, and drug treatment for IBS should include antidepressant and antianxiety treatments. Antidepressants include tricyclic antidepressants, such as amitriptyline, and selective serotonin reuptake inhibitors, such as fluoxetine and paroxetine. Fluoxetine has four effects in treating IBS: psychological improvements can induce relief of gastrointestinal symptoms; it regulates the activity of 5-HT transmitters, which are involved in visceral gastrointestinal pain sensation, and the effects of its receptors on gastrointestinal motility; it has a potential central analgesic effect; and it prevents the vicious cycle between psychological disorders and physiological abnormalities. For IBS patients with severe anxiety symptoms, anti-anxiety medications may be considered in combination with treatment.

[0018] 4. Traditional Chinese Medicine: Some clinical observations in China have confirmed that some traditional Chinese medicines can effectively relieve abdominal pain, diarrhea and constipation symptoms in IBS patients.

[0019] 5. Probiotics: In recent years, the relationship between intestinal flora imbalance and IBS has received considerable attention. Studies have shown that the onset of IBS in some patients is related to intestinal flora imbalance, and that probiotic supplementation can improve symptoms to varying degrees. The mechanism of action is not fully understood, but it is generally believed to involve biochemical inhibition or promotion, nutrient competition, immune clearance, and adhesion receptor competition. However, the number of commonly used probiotic preparations is limited, primarily including triple-acting bifidobacteria and quadruple-acting bifidobacteria.

[0020] Currently, there is still a lack of very effective treatments for IBS, and IBS cannot be completely cured. It is necessary to research new and effective drugs that can treat IBS.

[0021] Bacteroides fragilis is a member of the genus Bacteroides, a Gram-negative anaerobic bacterium belonging to the phylum Bacteroidetes, distinct from members of the phylum Firmicutes, such as Bifidobacteria and Lactobacilli. The genus Bacteroides comprises 25 species, 10 of which are exclusively human, 10 of which are exclusively animal, and 5 of which are both human and animal. Bacteroides fragilis is an obligate anaerobic bacterium with a polymorphic morphology depending on the culture medium and growth stage. Under normal conditions, the bacterium is rod-shaped, with blunt ends, dark pigmentation, and a light, unevenly colored center. It possesses a capsule, lacks spores, and is nonmotile. Some strains may be vacuolated, and the length of the bacterium varies. Based on their ability to synthesize and secrete Bacteroides fragilis enterotoxin (BFT), Bacteroides fragilis can be divided into enterotoxigenic Bacteroides fragilis (ETBF) and nontoxigenic Bacteroides fragilis (NTBF). Bacteroides fragilis, part of the normal intestinal flora of humans and animals, is primarily found in the colon. It can also colonize the mucosa of the respiratory, gastrointestinal, and genitourinary tracts. As an opportunistic pathogen, Bacteroides fragilis can invade the submucosa when the host mucosa is damaged, causing infection. It can also travel through the bloodstream and cause suppurative infections and abscesses in other organs, including the intestines, abdominal cavity, liver, lungs, brain tissue, soft tissue, and bone marrow. Summary of the Invention

[0022] Based on this, the present invention provides a new application of Bacteroides fragilis extract. The specific technical solution is as follows:

[0023] The invention discloses an application of a Bacteroides fragilis extract in preparing a medicine for preventing and treating irritable bowel syndrome. The Bacteroides fragilis extract contains Bacteroides fragilis capsular polysaccharide A.

[0024] In some embodiments, the molecular weight of the Bacteroides fragilis capsular polysaccharide A is 5 to 75 KD.

[0025] In some embodiments, the molecular weight of the Bacteroides fragilis capsular polysaccharide A is 15KD to 65KD;

[0026] In some embodiments, the molecular weight of the Bacteroides fragilis capsular polysaccharide A is 25KD to 55K.

[0027] In some embodiments, the molecular weight of the Bacteroides fragilis capsular polysaccharide A is 35KD to 45KD.

[0028] In some embodiments, the content of Bacteroides fragilis capsular polysaccharide A in the Bacteroides fragilis extract is 60-75 wt %.

[0029] In some embodiments, the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685.

[0030] In some embodiments, the method for preparing the Bacteroides fragilis extract comprises the following steps:

[0031] (1) centrifuging the fermented Bacteroides fragilis culture to collect a first precipitate, adding 65-72° C. water to the first precipitate, dissolving it, then adding phenol solution, maintaining 65-72° C. with stirring for 25-35 minutes, centrifuging, and collecting a first supernatant;

[0032] (2) extracting the first supernatant collected in step (1) with ether to remove phenol, then removing the residual ether, and collecting the aqueous phase solution;

[0033] (3) adding anhydrous ethanol to the aqueous solution collected in step (2) to a final concentration of ethanol of 75-85 v / v%, performing alcohol precipitation, centrifuging, and collecting the second precipitate.

[0034] (4) taking the second precipitate, adding water to prepare a suspension, adjusting the pH to 6.5-7.5, centrifuging, collecting the second supernatant, dialyzing to remove salt, and freeze-drying to obtain the Bacteroides fragilis extract.

[0035] In some embodiments, the ratio of water, the phenol solution and the first precipitate added to the first precipitate in step (1) is 3-5 mL: 3-5 mL: 1 g; and the mass concentration of the phenol solution is 70-80%.

[0036] In some embodiments, the alcohol precipitation in step (3) is carried out at a temperature of 0-8° C. for 8-16 hours.

[0037] In some embodiments, step (4) includes: taking the second precipitate, adding water to prepare a suspension with a mass concentration of 8-12%, then adding a glacial acetic acid aqueous solution with a mass concentration of 8-12%, heating to boiling, stirring and reacting for 1.5-2.5 hours, adjusting the pH to 6.5-7.5, centrifuging, collecting the second supernatant, dialyzing for desalination, and freeze-drying to obtain the Bacteroides fragilis extract.

[0038] In some embodiments, the method for preparing the Bacteroides fragilis extract further comprises a degradation step: degrading the Bacteroides fragilis extract obtained in step (4) by ultrasonication, wherein the ultrasonication conditions are: 180-210 kHz, 15-25°C.

[0039] In some embodiments, the dosage form of the drug includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations. The drug includes human drugs or animal drugs and can be used for humans or animals.

[0040] The Bacteroides fragilis extract can be administered alone for preventive or therapeutic purposes, or can be administered together with other probiotics and / or prebiotic materials. When administered in combination, the extract can be administered in a single formulation or in separate formulations, simultaneously or at different times, using the same or different routes of administration.

[0041] The present invention also provides a drug for preventing and treating irritable bowel syndrome. The specific technical solution is as follows:

[0042] A medicine for preventing and treating irritable bowel syndrome contains a Bacteroides fragilis extract, and the Bacteroides fragilis extract contains Bacteroides fragilis capsular polysaccharide A.

[0043] In some embodiments, the molecular weight of the Bacteroides fragilis capsular polysaccharide A is 5 to 75 KD.

[0044] In some embodiments, the molecular weight of the Bacteroides fragilis capsular polysaccharide A is 15KD to 65KD;

[0045] In some embodiments, the molecular weight of the Bacteroides fragilis capsular polysaccharide A is 25KD to 55K.

[0046] In some embodiments, the molecular weight of the Bacteroides fragilis capsular polysaccharide A is 35KD to 45KD.

[0047] In some embodiments, the content of Bacteroides fragilis capsular polysaccharide A in the Bacteroides fragilis extract is 60-75 wt %.

[0048] In some embodiments, the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685.

[0049] In some embodiments, the method for preparing the Bacteroides fragilis extract comprises the following steps:

[0050] (1) centrifuging the fermented Bacteroides fragilis culture to collect a first precipitate, adding 65-72° C. water to the first precipitate, dissolving it, then adding phenol solution, maintaining 65-72° C. with stirring for 25-35 minutes, centrifuging, and collecting a first supernatant;

[0051] (2) extracting the first supernatant collected in step (1) with ether to remove phenol, then removing the residual ether, and collecting the aqueous phase solution;

[0052] (3) adding anhydrous ethanol to the aqueous solution collected in step (2) to a final concentration of ethanol of 75-85 v / v%, performing alcohol precipitation, centrifuging, and collecting the second precipitate.

[0053] (4) taking the second precipitate, adding water to prepare a suspension, adjusting the pH to 6.5-7.5, centrifuging, collecting the second supernatant, dialyzing to remove salt, and freeze-drying to obtain the Bacteroides fragilis extract.

[0054] In some embodiments, the ratio of water, the phenol solution and the first precipitate added to the first precipitate in step (1) is 3-5 mL: 3-5 mL: 1 g; and the mass concentration of the phenol solution is 70-80%.

[0055] In some embodiments, the alcohol precipitation in step (3) is carried out at a temperature of 0-8° C. for 8-16 hours.

[0056] In some embodiments, step (4) includes: taking the second precipitate, adding water to prepare a suspension with a mass concentration of 8-12%, then adding a glacial acetic acid aqueous solution with a mass concentration of 8-12%, heating to boiling, stirring and reacting for 1.5-2.5 hours, adjusting the pH to 6.5-7.5, centrifuging, collecting the second supernatant, dialyzing for desalination, and freeze-drying to obtain the Bacteroides fragilis extract.

[0057] In some embodiments, the method for preparing the Bacteroides fragilis extract further comprises a degradation step: degrading the Bacteroides fragilis extract obtained in step (4) by ultrasonication, wherein the ultrasonication conditions are: 180-210 kHz, 15-25°C.

[0058] In some embodiments, the dosage form of the drug includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations. The drug includes human drugs or animal drugs and can be used for humans or animals.

[0059] The drug may contain one or more of the following pharmaceutically acceptable excipients: diluents, excipients, binders, lubricants, suspending agents, coating agents, solubilizers, etc. Examples of pharmaceutically acceptable excipients include: water, saline solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, aromatic oils, mono- and di-glycerides of fatty acids, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0060] The drug may be administered by any one or more of the following: by inhalation via a micropump or nasal spray or inhalation aerosol, orally in the form of a suppository or vaginal suppository, topically in the form of a lotion, solution, cream, ointment, or dust, by use of a skin patch, or orally in the form of a tablet containing an excipient such as starch or lactose, or in a capsule or ovule, alone or mixed with an excipient, or in the form of an elixir, solution, or suspension containing flavorings or coloring agents, or by parenteral injection, e.g., intracavernous, intravenous, intramuscular, or subcutaneous. For parenteral administration, the drug is preferably administered in the form of a sterile aqueous solution, which may contain other substances, e.g., sufficient salts or simple sugars to render the solution isotonic with the blood. For buccal or sublingual administration, the drug may be administered in the form of tablets or lozenges formulated in a conventional manner.

[0061] The Bacteroides fragilis ZY-312 of the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on April 2, 2015, with a deposit number of CGMCC No. 10685 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0062] After long-term experience accumulation and a large number of creative experimental studies, the inventors of the present invention have obtained a preparation method for a Bacteroides fragilis extract (the main component of which is capsular polysaccharide A). Through further experimental studies, it was found that the Bacteroides fragilis extract of the present invention has the function of preventing and treating irritable bowel syndrome, and has a good preventive and therapeutic effect on both diarrhea-type irritable bowel syndrome and constipation-type irritable bowel syndrome, and its preventive and therapeutic effect on irritable bowel syndrome is much better than that of Bacteroides fragilis itself. Furthermore, the inventors obtained a Bacteroides fragilis capsular polysaccharide A with a molecular weight of 5 to 70 KD by degrading the Bacteroides fragilis capsular polysaccharide A with a molecular weight of 70 KD, and unexpectedly found that the capsular polysaccharide A with a molecular weight of 5 to 70 KD has a better function of preventing and treating irritable bowel syndrome, and its effect is much better than the capsular polysaccharide A with a molecular weight of 110 KD extracted from Bacteroides fragilis NCTC 9343. The Bacteroides fragilis capsular polysaccharide A provided by the present invention has a good preventive and therapeutic effect on irritable bowel syndrome and has no side effects on the body. It can be administered alone for preventive or therapeutic purposes, or in combination with other probiotics and / or prebiotic materials. The Bacteroides fragilis capsular polysaccharide A provided by the present invention has great prospects for edible and medicinal uses, providing a clinically effective product suitable for human consumption for health care and the prevention and treatment of irritable bowel syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a colony characteristic diagram of Bacteroides fragilis ZY-312 in Example 1;

[0064] Figure 2 This is a microscopic observation picture of Bacteroides fragilis ZY-312 in Example 1 after Gram staining.

[0065] Figure 3 This is the 1H spectrum of the capsular polysaccharide A of Example 1 analyzed by nuclear magnetic resonance spectroscopy;

[0066] Figure 4 This is the 13C spectrum of the capsular polysaccharide A of Example 1 analyzed by nuclear magnetic resonance spectroscopy;

[0067] Figure 5 COSY spectrum of the capsular polysaccharide A of Example 1 obtained by nuclear magnetic resonance spectroscopy analysis;

[0068] Figure 6 HSQC spectrum of the capsular polysaccharide A of Example 1 obtained by nuclear magnetic resonance spectroscopy analysis;

[0069] Figure 7 This is the HMBC spectrum of the capsular polysaccharide A of Example 1 analyzed by nuclear magnetic resonance spectroscopy;

[0070] Figure 8 This is the chemical structural formula of Bacteroides fragilis capsular polysaccharide A prepared in Example 1. DETAILED DESCRIPTION

[0071] The present invention is further described in detail below through specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0072] The Bacteroides fragilis used in the following examples is Bacteroides fragilis ZY-312 (bacteroides fragilis ZY-312), which was deposited in the China General Microbiology Center (CGMCC) on April 2, 2015, with a deposit number of CGMCC No. 10685 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0073] Example 1 Preparation of Bacteroides fragilis extract

[0074] (1) Fermentation culture of Bacteroides fragilis

[0075] Streak the bacteria onto a blood plate and incubate anaerobically for 48 hours. Observe the colony morphology, staining properties, size, club shape, and distribution.

[0076] Colony characteristics: After 48 hours of culture on a blood plate, Bacteroides fragilis ZY-312 appears as a round, slightly convex, translucent, white, smooth surface, and non-hemolytic colony with a diameter of 1-3 mm. Figure 1 .

[0077] Microscopic morphology: Gram staining of Bacteroides fragilis ZY-312 revealed that it is a Gram-negative bacterium with a typical rod shape, blunt and intensely stained ends, and a non-stained middle portion shaped like a vacuole. Figure 2 .

[0078] A single colony was selected and inoculated into tryptone broth for fermentation culture for 8 hours (temperature was 37°C). The obtained bacterial solution was centrifuged at a speed of 3000 r / min for 15 minutes, the supernatant was removed, and the precipitate was collected.

[0079] (2) Preparation of Bacteroides fragilis extract

[0080] 1) Take 200 g of Bacteroides fragilis sludge (the precipitate obtained in step (1) above), add 750 mL of 68°C ultrapure water, dissolve it, then add 750 mL of 75% phenol solution by volume, mix well, maintain stirring and extract at 68°C for 30 min, centrifuge at 15,000 g for 20 min, and collect the supernatant.

[0081] 2) Extract the supernatant with an equal volume of ether (1.5 L) to remove phenol. Collect the supernatant and repeat the extraction until no phenol remains. Heat in a water bath to remove the ether and collect the aqueous phase.

[0082] 3) After centrifugation at 15,000 g for 20 min, the volume of the aqueous phase was measured, and anhydrous ethanol was added to a final ethanol concentration of 80% (volume fraction). The mixture was precipitated at 4°C overnight (12 hours), and centrifuged at 15,000 g for 20 min to collect the precipitate.

[0083] 4) Weigh the precipitate from step 3) and add a certain volume of deionized water to a 10% suspension. Stir and mix thoroughly. Add 10% glacial acetic acid solution, heat to boiling, and continue stirring for 2 hours. Adjust the pH to 7.0, centrifuge at 15,000 g for 20 minutes, and collect the supernatant. The resulting supernatant is dialyzed for desalination (10KD dialysis bag) and freeze-dried to obtain a Bacteroides fragilis extract.

[0084] 5) Weigh 30 mg of the Bacteroides fragilis extract described in step 4) and dissolve it in 0.5 mL of D2O. Add 1 μl of acetone (1H, 2.22; 13C, 30.89) for calibration. Analyze the 1H, 13C, COSY, HSQC, and HMBC spectra using a 500 MHz Bruker NMR spectrometer. Figure 3 ), confirming that the Bacteroides fragilis extract collected in step 4) is capsular polysaccharide A with a purity of approximately 70%. GPC (gel permeation chromatography) analysis showed that the capsular polysaccharide A had a repeating unit molecular weight of 781, a unit repeat number n value of 89, a molecular weight of approximately 70 kD, and a molecular formula of -[C 31 N3O 20 H 47 ] 91 -, chemical structure see Figure 4 .

[0085] (3) Preparation of capsular polysaccharide A with different molecular weights

[0086] In this example, the capsular polysaccharide A prepared in (2) was degraded, and the degradation methods include but are not limited to chemical degradation, physical degradation, and biodegradation. This example uses an ultrasonic method, wherein the capsular polysaccharide A is treated at 195 kHz and 20°C for 3 hours, 2 hours, 0.5 hours, and 0 hours, respectively, to collect capsular polysaccharides A with molecular weights of 2KD, 5KD, 40KD, and 70KD, respectively. Capsular polysaccharide A with a molecular weight of 110KD was extracted from Bacteroides fragilis NCTC 9343 (purchased from ATCC, USA) using the method in (2).

[0087] Example 2 Effect of Bacteroides fragilis capsular polysaccharide A on diarrhea-type IBS induced by senna leaves

[0088] 1. Experimental Design

[0089] In order to verify the effect of the Bacteroides fragilis extract (main component is capsular polysaccharide A) provided in Example 1 for preventing / treating irritable bowel syndrome, the present embodiment selects 60 C57BL / 6 mice to be experimented. 60 C57BL / 6 mice are male and female, and each experimental mouse is all assigned a unique number. Before the animals are grouped, the project number, species / strain, sex, cage number and animal number should be marked on the label of the mouse cage. BioBook software is used to carry out random grouping according to the initial body weight of C57BL / 6 mice, and is divided into 6 groups, i.e. normal group (Group 1), model group (Group 2), loperamide capsule group (2.4mg / kg) (Group 3), Bacteroides fragilis capsular polysaccharide A low (Group 4), medium (Group 5), high (Group 6) dosage group, 10 C57BL / 6 mice per group. The present embodiment is an example of a capsular polysaccharide A with a molecular weight of 5KD.

[0090] Each group of mice was gavage-administered the corresponding drug. The normal and model groups were given an equal volume of saline once daily for 5 consecutive days. All C57BL / 6 mice were fasted for 24 hours. One hour after the last dose, all C57BL / 6 mice, except the normal group (Group 1), were gavage-administered with an equal volume of saline. All other C57BL / 6 mice, except for the normal group (Group 1), were gavage-administered with a 1g / mL decoction of senna leaves. C57BL / 6 mice were housed in separate cages, one per cage. Filter paper was placed under the cage to count wet feces, with the amount of wet feces indicating the severity of diarrhea. The filter paper was changed every hour. The total number of stools, the total number of loose stools, and the degree of loose stools were observed and counted over a 6-hour period.

[0091] II. Judging Criteria

[0092] The loose stool grade was determined according to Zhou's method (Zhou Gannan, Hu Zhihua, Wang Yaxian, et al. Preparation of a mouse diarrhea model and application of the diarrhea index [J]. Chinese Herbal Medicine, 1994, 250(4):195-196). Stain diameters <1 cm were assigned to grade 1, 1-1.9 cm to grade 2, 2-3 cm to grade 3, and >3 cm to grade 4. The loose stool rate (%) for each animal was calculated as: total loose stools / total stools × 100%. The diarrhea index (ID) was calculated as: loose stool rate × loose stool grade.

[0093] 3. Results and Analysis

[0094] All data are expressed as x ± s. Statistical analysis was performed using SPSS 17.0 software. Intergroup comparisons were performed using one-way analysis of variance, with P < 0.05 considered statistically significant. The specific results are shown in Table 1.

[0095] Table 1 Effects of Bacteroides fragilis capsular polysaccharide A on diarrhea-type IBS in mice induced by senna leaves ( n=10)

[0096]

[0097] Note: Compared with the normal group, *P < 0.01; compared with the model group, ▲P < 0.01.

[0098] As shown in Table 1, there were significant differences (P < 0.01) in the total number of loose stools, loose stool rate, and diarrhea index between the model group (Group 2) and the normal group (Group 1), indicating that the senna-induced IBS model in mice with diarrhea was successfully established. Compared with the model group, the loperamide group and the low-, medium-, and high-dose groups of Bacteroides fragilis capsular polysaccharide A provided in Example 1 significantly inhibited the degree of diarrhea in mice (P < 0.01), demonstrating that the Bacteroides fragilis capsular polysaccharide A provided by the present invention has a significant inhibitory effect on senna-induced diarrhea-predominant IBS.

[0099] Example 3 Effect of Bacteroides fragilis capsular polysaccharide A on castor oil-induced diarrhea-type IBS

[0100] 1. Experimental Design

[0101] In order to verify the effect of the Bacteroides fragilis extract (main component is capsular polysaccharide A) provided in Example 1 for preventing / treating irritable bowel syndrome, the present embodiment selects 60 C57BL / 6 mice to be experimented. 60 C57BL / 6 mice are male and female, and each experimental mouse is all assigned a unique number. Before the animals are grouped, the project number, species / strain, sex, cage number and animal number should be marked on the label of the mouse cage. BioBook software is used to carry out random grouping according to the initial body weight of C57BL / 6 mice, and is divided into 6 groups, i.e. normal group (Group 1), model group (Group 2), loperamide capsule group (2.4mg / kg) (Group 3), Bacteroides fragilis capsular polysaccharide A low (Group 4), medium (Group 5), high (Group 6) dosage group, 10 C57BL / 6 mice per group. The present embodiment is an example of a capsular polysaccharide A with a molecular weight of 40KD.

[0102] Each group of mice was gavage-administered the corresponding drug. The normal and model groups were given an equal volume of normal saline once daily for 5 consecutive days. All C57BL / 6 mice were fasted for 24 hours. One hour after the last dose, all C57BL / 6 mice, except the normal group (Group 1), were gavage-administered with an equal volume of normal saline. Castor oil (20 mL / kg) was administered once per gavage to all other C57BL / 6 mice, with one C57BL / 6 mouse per cage. Filter paper was placed under the cage to count wet feces, with the amount of wet feces indicating the degree of diarrhea. The filter paper was changed every hour. The total number of stools, the total number of loose stools, and the degree of loose stools in the mice were observed and counted over a 6-hour period.

[0103] II. Judging Criteria

[0104] The same evaluation criteria as in Example 2.

[0105] 3. Results and Analysis

[0106] All data are SPSS 17.0 software was used for statistical analysis, and one-way analysis of variance was used for comparison between groups. P < 0.05 was considered statistically significant. The specific results are shown in Table 2.

[0107] Table 2 Effects of Bacteroides fragilis capsular polysaccharide A on castor oil-induced diarrhea-type IBS in mice ( n=10)

[0108]

[0109]

[0110] Note: Compared with the normal group, *P < 0.01; compared with the model group, ▲P < 0.01.

[0111] As shown in Table 2, there were significant differences in the total number of loose stools, loose stool rate, and diarrhea index between the model group (Group 2) and the normal group (Group 1) (P < 0.01), indicating that the castor oil-induced IBS model in mice with diarrhea was successfully established. Compared with the model group, loperamide significantly inhibited the total number of loose stools, loose stool rate, and diarrhea index in the 6-hour period in mice with castor oil-induced diarrhea (P < 0.05). A low dose (0.125 g / kg) of B. fragilis capsular polysaccharide A had a modest inhibitory effect on the total number of loose stools, loose stool rate, and diarrhea index in the 6-hour period compared with the model group, but no significant differences were found (P > 0.05). However, at medium and high doses, B. fragilis capsular polysaccharide A significantly inhibited the total number of loose stools, loose stool rate, and diarrhea index in the 6-hour period in mice with castor oil-induced diarrhea (P < 0.05).

[0112] Example 4 Effect of Bacteroides fragilis capsular polysaccharide A on neostigmine-induced small intestinal hypermotility in mice

[0113] 1. Experimental Design

[0114] In order to verify the effect of the Bacteroides fragilis extract (main component is capsular polysaccharide A) provided in Example 1 in preventing / treating irritable bowel syndrome, this embodiment selects 60 C57BL / 6 mice to carry out the experiment. 60 C57BL / 6 mice are half male and half female, and each experimental mouse is assigned a unique number. Before the animals are grouped, the project number, species / strain, gender, cage number and animal number should be marked on the label of the mouse cage. BioBook software is used to randomly group the C57BL / 6 mice according to their initial body weight and divide them into 6 groups, i.e., a normal group (Group 1), a model group (Group 2), a pinaverium bromide (0.1 g / kg) group (Group 3), a low (Group 4), a medium (Group 5) and a high (Group 6) dose group of Bacteroides fragilis capsular polysaccharide A, with 10 C57BL / 6 mice in each group. This embodiment is an example of a capsular polysaccharide A with a molecular weight of 70KD.

[0115] Each group of mice was gavaged with the corresponding drug, while the normal and model groups were given an equal amount of normal saline once daily for five consecutive days. One hour after the last dose, all five groups except the normal group were subcutaneously injected with 0.15 mg / kg of neostigmine to induce small intestinal hyperactivity, while the normal group was subcutaneously injected with an equal amount of normal saline. Fifteen minutes later, all groups were gavaged with a suspension containing 5% activated charcoal powder. Twenty minutes later, the mice were sacrificed by cervical dislocation. The small intestine was then opened and isolated, and the total length of the small intestine and the length of the small intestine propelled by charcoal powder were measured. The percentage of charcoal powder propelled was calculated. The percentage of charcoal powder propelled = (length of the small intestine propelled by charcoal powder / total length of the small intestine) × 100%.

[0116] 2. Results and Analysis

[0117] All data are SPSS 17.0 software was used for statistical analysis, and one-way analysis of variance was used for comparison between groups. P < 0.05 was considered statistically significant. The specific results are shown in Table 3.

[0118] Table 3 Effects of Bacteroides fragilis capsular polysaccharide A on neostigmine-induced small intestinal hypermotility in mice ( n=10)

[0119]

[0120] Note: Compared with the normal group, *P < 0.01; compared with the model group, ▲P < 0.01.

[0121] As can be seen from Table 3, neostigmine can cause intestinal hypermotility in mice. Compared with the model group, the pinaverium bromide group can significantly reduce the carbon propulsion rate and inhibit the intestinal hypermotility of mice caused by neostigmine (P < 0.01). Medium and high doses of Bacteroides fragilis capsular polysaccharide A can also significantly reduce the carbon propulsion rate (P < 0.05) and inhibit the intestinal hypermotility of mice caused by neostigmine.

[0122] Example 5 Effect of Bacteroides fragilis capsular polysaccharide A on constipation-type IBS

[0123] 1. Experimental Design

[0124] In order to verify the effect of the Bacteroides fragilis extract (main component is capsular polysaccharide A) provided in Example 1 on the prevention / treatment of constipation-type irritable bowel syndrome, this example selected 60 SD rats for experiment. The 60 SD rats were half male and half female, and each experimental rat was assigned a unique number. Before grouping the animals, the project number, species / strain, gender, cage number and animal number should be marked on the label of the mouse cage. BioBook software was used to randomly divide the SD rats into 6 groups according to their initial weight, namely a normal group (Group 1), a model group (Group 2), a tegaserod maleate (solution preparation: 1.2 mg tegaserod maleate was added to 10 ml sterile saline) group (Group 3), a low (Group 4), medium (Group 5) and high (Group 6) dose group of Bacteroides fragilis capsular polysaccharide A, with 10 SD rats in each group. This example takes capsular polysaccharide A with a molecular weight of 70KD as an example.

[0125] A constipation-predominant IBS model was established in SD rats according to the method of (Peng LH, Yang YS, Sun G, et al. A new model of constipation-predominant irritable bowel syndrome in rats [J]. World Chinese Journal of Digestology, 2004, 12(1): 112-116.) Except for the normal group (Group 1), all other groups were gavaged with 2 ml of ice-cold saline (0-4°C) once daily for 14 consecutive days to establish the constipation-predominant IBS model in rats. During the modeling period, rats in each group had free access to food and water.

[0126] After 14 days, each experimental group was gavaged with the corresponding drug. The normal group and the model group were each given 10 ml of normal temperature sterile saline; the positive control group (Group 3) was gavaged with tegaserod maleate; Groups 4 to 6 were given low, medium, and high doses of Bacteroides fragilis capsular polysaccharide A, respectively. The specific experiments and dosing schedules are shown in Table 4:

[0127] Table 4 Experimental groups and dosing regimens

[0128]

[0129]

[0130] 2. Results and Analysis

[0131] Fecal pellets were collected from each group of rats for 24 hours on days 1, 14, and 28. If diarrhea occurred, one contaminated pellet was counted as one pellet (see Table 5). The collected feces were weighed, dried, and the fecal water content was calculated (see Table 6).

[0132] Table 5 Changes in the number of fecal particles in SD rats over 24 hours (particles) n=10)

[0133] experimental group Day 1 Day 14 Day 28 Group 1 49.13±6.34 48.53±6.48 <![CDATA[48.10±5.43 ◆ ]]> Group2 47.37±5.27 <![CDATA[33.07±5.32 *▲ ]]> 32.44±6.23 Group3 48.22±7.34 <![CDATA[32.46±5.72 *▲ ]]> <![CDATA[47.43±6.35 ★◆ ]]> Group4 47.78±5.39 <![CDATA[32.73±4.39 *▲ ]]> <![CDATA[40.04±7.94 ★◆ ]]> Group5 48.05±7.91 <![CDATA[34.07±5.83 *▲ ]]> <![CDATA[43.90±8.06 ★◆ ]]> Group6 47.88±6.54 <![CDATA[33.77±5.60 *▲ ]]> <![CDATA[49.03±5.71 ★◆ ]]>

[0134] Note: *P<0.01 for the comparison between the experimental group and the first day; ▲P<0.01 for the comparison between Group 2-6 and the normal control group on the 14th day; ★P<0.01 for the comparison between the experimental group and the 14th day on the 28th day; ◆P<0.01 for the comparison between the experimental group and the model group on the 28th day.

[0135] Table 6 Changes in fecal water content of SD rats ( n=10)

[0136]

[0137]

[0138] Note: *P<0.05 for the comparison between the experimental group and the first day; ▲P<0.05 for the comparison between Group 2-6 and the normal control group on the 14th day; ★P<0.05 for the comparison between the experimental group and the 14th day on the 28th day; ◆P<0.05 for the comparison between the experimental group and the model group on the 28th day.

[0139] As can be seen from Table 5, in Groups 1 to 6, the difference in the number of fecal pellets in the first 24 hours of each group of rats was not significant; except for the normal group, the number of fecal pellets in the remaining groups of rats on the 14th day was significantly reduced compared with that on the first day, and the difference was statistically significant (P < 0.05); on the 14th day, the number of fecal pellets in Groups 2 to 6 was significantly reduced compared with that in the normal group (Group 1), and the difference was statistically significant (P < 0.05), indicating that this embodiment successfully constructed a constipation-type IBS rat model. On the 28th day, the number of fecal pellets in Groups 2 to 6 was significantly increased compared with that on the 14th day, and the difference was statistically significant (P < 0.05). This shows that the low, medium, and high doses of Bacteroides fragilis capsular polysaccharide A provided by the present invention can effectively increase the number of fecal pellets in constipation-type IBS rats.

[0140] As can be seen from Table 6, in Groups 1 to 6, the fecal water content of the rats in each group on the first 24 hours was not much different; except for the normal group, the fecal water content of the rats in the other groups on the 14th day was significantly reduced compared with that on the first day, and the difference was statistically significant (P < 0.05). On the 14th day, the fecal water content of Groups 2 to 6 was significantly reduced compared with that of the normal group (Group 1), and the difference was statistically significant (P < 0.05). On the 28th day, the fecal water content of Groups 2 to 6 was significantly increased compared with that on the 14th day, and the difference was statistically significant (P < 0.05). This shows that the low, medium, and high doses of Bacteroides fragilis capsular polysaccharide A provided by the present invention can effectively increase the fecal water content of rats with constipation-type IBS.

[0141] It can be seen from the above results that the Bacteroides fragilis capsular polysaccharide A provided by the present invention has a good therapeutic effect on constipation-type IBS.

[0142] Example 6 Therapeutic Effects of Bacteroides fragilis and Bacteroides fragilis Capsular Polysaccharide A

[0143] 1. Experimental Design

[0144] In order to compare the effects of the Bacteroides fragilis extract (main component is capsular polysaccharide A) provided in Example 1 and Bacteroides fragilis itself on the prevention / treatment of irritable bowel syndrome, this example selected 60 C57BL / 6 mice for experiment. The 60 C57BL / 6 mice were half male and half female, and each experimental mouse was assigned a unique number. Before grouping the animals, the project number, species / strain, gender, cage number and animal number should be marked on the label of the mouse cage. The C57BL / 6 mice were randomly divided into 6 groups according to their initial weight using BioBook software, namely, senna-induced diarrhea-type IBS model group (Group 1), castor oil-induced diarrhea-type IBS model group (Group 2), high-dose Bacteroides fragilis capsular polysaccharide A treatment group for senna-induced diarrhea-type IBS (Group 3), high-dose Bacteroides fragilis itself (10 10 CFU / ml) for the treatment group of diarrhea-type IBS induced by senna leaves (Group 4), high-dose Bacteroides fragilis capsular polysaccharide A for diarrhea-type IBS induced by castor oil (Group 5), and high-dose Bacteroides fragilis itself (10 10 CFU / ml) for the treatment group of castor oil-induced diarrhea-type IBS (Group 6), with 10 C57BL / 6 mice in each group. The molecular weight of the capsular polysaccharide A described in this example is 40KD and the concentration is 0.5mg / mL; the concentration of Bacteroides fragilis is 10 10 CFU / ml.

[0145] Each group of mice was gavage-administered the corresponding drug. The model groups (Group 1 and Group 2) received an equal volume of normal saline once daily for 5 consecutive days. All C57BL / 6 mice were fasted for 24 hours. One hour after the last dose, Groups 1, 3, and 4 received a 1g / mL senna leaf decoction, while Groups 2, 5, and 6 received a single gavage of castor oil (20mL / kg). C57BL / 6 mice were housed in separate cages, one per cage. Filter paper was placed under the cage to count wet feces, with the amount of wet feces indicating diarrhea severity. The filter paper was changed every hour. The total number of stools, the total number of loose stools, and the degree of loose stools were observed and counted over a 6-hour period.

[0146] II. Judging Criteria

[0147] The same evaluation criteria as in Example 2.

[0148] 3. Results and Analysis

[0149] All data are SPSS 17.0 software was used for statistical analysis, and one-way analysis of variance was used for comparison between groups. P < 0.05 was considered statistically significant. The specific results are shown in Table 6.

[0150] Table 6 Effects of Bacteroides fragilis capsular polysaccharide A on diarrhea-type IBS in mice ( n=10)

[0151]

[0152]

[0153] Note: Group 3 and Group 4 were compared with Group 1, *P < 0.05; Group 5 and Group 6 were compared with Group 2, ▲ P<0.05; Group3 compared with Group4, Group5 compared with Group6, ★ P<0.05.

[0154] As shown in Table 6, there were significant differences in the total number of loose stools, loose stool rate, and diarrhea index between Groups 3 and 4 and the model group (Group 1) (P < 0.05), indicating that Bacteroides fragilis itself and Bacteroides fragilis capsular polysaccharide A have therapeutic and preventive effects on senna-induced diarrhea-induced IBS in mice. There were also significant differences in the total number of loose stools, loose stool rate, and diarrhea index between Groups 5 and 6 and the model group (Group 2) (P < 0.01), indicating that Bacteroides fragilis itself and Bacteroides fragilis capsular polysaccharide A have therapeutic and preventive effects on castor oil-induced diarrhea-induced IBS in mice. Compared with Group 4, the total number of loose stools, loose stool rate, and diarrhea index of Group 3 were all smaller, with significant differences (P < 0.05), indicating that the therapeutic and preventive effects of Bacteroides fragilis capsular polysaccharide A on IBS caused by diarrhea in mice induced by senna leaves were better than those of Bacteroides fragilis itself; compared with Group 6, the total number of loose stools, loose stool rate, and diarrhea index of Group 5 were all smaller, with significant differences (P < 0.05), indicating that the therapeutic and preventive effects of Bacteroides fragilis capsular polysaccharide A on IBS caused by diarrhea in mice induced by castor oil were better than those of Bacteroides fragilis itself.

[0155] Experiments have also shown that for neostigmine-induced small intestinal hypermotility and constipation-type IBS in mice, the therapeutic and preventive effects of the Bacteroides fragilis capsular polysaccharide A provided by the present invention are also better than those of Bacteroides fragilis itself.

[0156] Example 7 Therapeutic Effects of Bacteroides fragilis Capsular Polysaccharide A with Different Molecular Weights on IBS

[0157] In this example, Bacteroides fragilis extracts containing 2KD, 5KD, 40KD, and 70KD Bacteroides fragilis capsular polysaccharide A prepared in Example 1 of the present invention were used to prevent and treat constipation-predominant IBS rat models, and the therapeutic effects of Bacteroides fragilis capsular polysaccharide A of different molecular weights on constipation-predominant IBS were tested. This example uses high doses of 2KD, 5KD, 40KD, and 70KD Bacteroides fragilis capsular polysaccharide A as examples.

[0158] 1. Experimental Design

[0159] Referring to the experimental grouping method of Example 5, the mice were divided into a normal control group, a model group, a 2KD group, a 5KD group, a 40KD group, a 70KD group, and a 110KD group.

[0160] A constipation-predominant IBS model was established in SD rats according to the method of (Peng LH, Yang YS, Sun G, et al. A new model of constipation-predominant irritable bowel syndrome in rats [J]. World Chinese Journal of Digestology, 2004, 12(1): 112-116.) Except for the normal group (Group 1), all other groups were gavaged with 2 ml of ice-cold saline (0-4°C) once daily for 14 consecutive days to establish the constipation-predominant IBS model in rats. During the modeling period, rats in each group had free access to food and water.

[0161] After 14 days, the corresponding drugs were administered to each experimental group by gavage. The normal group and the model group were given 10 ml of normal temperature sterile saline respectively; Groups 3 to 7 were given capsular polysaccharide A with molecular weights of 2KD (Group 3), 5KD (Group 4), 40KD (Group 5), 70KD (Group 6), and 110KD (Group 7), respectively. The specific experiments and dosing schedules are shown in Table 7:

[0162] Table 7 Experimental groups and dosing regimens

[0163]

[0164]

[0165] 2. Results and Analysis

[0166] Fecal pellets were collected from each group of rats for 24 hours on days 1, 14, and 28. If diarrhea occurred, one contaminated pellet was counted as one pellet (see Table 8). The collected feces were weighed, dried, and the fecal water content was calculated (see Table 9).

[0167] Table 8 Changes in the number of fecal particles in SD rats over 24 hours (particles) n=10)

[0168]

[0169]

[0170] Note: *P<0.01 for the comparison between the experimental group and the first day; ▲P<0.01 for the comparison between Group 2-7 and the normal control group on the 14th day; ★P<0.01 for the comparison between the experimental group and the 14th day on the 28th day; ◆P<0.01 for the comparison between the experimental group and the model group on the 28th day.

[0171] Table 9 Changes in fecal water content of SD rats ( n=10)

[0172] experimental group Day 1 Day 14 Day 28 Group 1 0.50±0.03 0.48±0.05 <![CDATA[0.49±0.08 ◆ ]]> Group2 0.49±0.06 <![CDATA[0.37±0.04 *▲ ]]> 0.34±0.05 Group3 0.48±0.08 <![CDATA[0.38±0.07 *▲ ]]> <![CDATA[0.41±0.07 ★◆ ]]> Group4 0.48±0.07 <![CDATA[0.36±0.06 *▲ ]]> <![CDATA[0.54±0.08 ★◆ ]]> Group5 0.49±0.09 <![CDATA[0.37±0.10 *▲ ]]> <![CDATA[0.53±0.06 ★◆ ]]> Group6 0.48±0.08 <![CDATA[0.39±0.08 *▲ ]]> <![CDATA[0.55±0.09 ★◆ ]]> Group7 0.47±0.08 <![CDATA[0.38±0.05 *▲ ]]> <![CDATA[0.45±0.08 ★◆ ]]>

[0173] Note: *P<0.05 for the comparison between the experimental group and the first day; ▲P<0.05 for the comparison between Group 2-6 and the normal control group on the 14th day; ★P<0.05 for the comparison between the experimental group and the 14th day on the 28th day; ◆P<0.05 for the comparison between the experimental group and the model group on the 28th day.

[0174] As can be seen from Table 8, in Groups 1 to 7, the difference in the number of fecal particles in the first 24 hours of each group of rats was not significant; except for the normal group (Group 1), the number of fecal particles in the remaining groups of rats on the 14th day was significantly reduced compared with that on the first day, and the difference was extremely significant (P < 0.01), which was statistically significant; on the 14th day, the number of fecal particles in Groups 2 to 7 was significantly reduced compared with that in the normal group (Group 1), and the difference was extremely significant (P < 0.01), which was statistically significant. This shows that this example successfully constructed a constipation-type IBS rat model. On the 28th day, except for Group 3, the number of fecal particles in Groups 4 to 7 was significantly increased compared with that on the 14th day, and the difference was extremely significant (P < 0.01), which was statistically significant. This shows that the Bacteroides fragilis capsular polysaccharide A of different molecular weights provided by the present invention can effectively increase the number of fecal particles in constipation-type IBS rats. At the same time, by comparing the number of fecal particles between Group 3 to 7 on day 28, it was found that the number of fecal particles in Group 4, Group 5 and Group 6 was significantly more than that in Group 2 and Group 7. The difference was extremely significant (P < 0.05) and statistically significant.

[0175] As shown in Table 9, there was little difference in fecal water content over the first 24 hours for Groups 1 to 7. With the exception of the normal group, the fecal water content of all groups on day 14 was significantly lower than that of the first day, with statistically significant differences (P < 0.05). On day 14, the fecal water content of Groups 2 to 7 was significantly lower than that of the normal group (Group 1), with statistically significant differences (P < 0.05). On day 28, with the exception of Group 3, the fecal water content of Groups 4 to 7 increased significantly compared to that of Group 14, with statistically significant differences (P < 0.05). Furthermore, a comparison of fecal water content between Groups 3 to 7 on day 28 revealed that the fecal water content of Groups 4, 5, and 6 was significantly higher than that of Groups 3 and 7, with extremely significant differences (P < 0.05), demonstrating statistical significance.

[0176] These results demonstrate that the Bacteroides fragilis capsular polysaccharide A with a molecular weight of 5-70 kD provided by the present invention can effectively increase the number of fecal particles and fecal water content in rats with constipation-predominant IBS, demonstrating a significant therapeutic effect for constipation-predominant IBS. They also demonstrate that degradation of Bacteroides fragilis capsular polysaccharide A, reducing its molecular weight and viscosity, can enhance its therapeutic efficacy for constipation-predominant IBS.

[0177] At the same time, the present invention also proves through experiments that the effect of Bacteroides fragilis capsular polysaccharide A with a molecular weight of 5KD to 70KD on reducing the diarrhea index of diarrhea-type IBS is much better than that of Bacteroides fragilis capsular polysaccharide A with a molecular weight of 2KD or 110KD.

[0178] Example 8 The efficacy of capsular polysaccharide A of different Bacteroides fragilis strains on constipation-type IBS

[0179] In this example, the ultrasonic method described in Example 1 was used to degrade capsular polysaccharide A with a molecular weight of 110 kD (ultrasound conditions: 195 kHz, 25°C, 0.5 hour). Capsular polysaccharide A with a molecular weight of 70 kD was collected and designated as the NCTC 9343-70 kD group. This was compared with the capsular polysaccharide A with a molecular weight of 70 kD extracted from ZY-312 (designated as the ZY-312-70 kD group) to evaluate their efficacy in treating constipation-type IBS. This example, referring to the method described in Example 7, measured changes in fecal particle count and fecal water content in rats. The specific results are as follows:

[0180] Table 10 Changes in the number of fecal particles in SD rats over 24 hours (particles) n=10)

[0181]

[0182]

[0183] Note: *P<0.01 for each group compared with the first day; ★P<0.01 for each group compared with the 14th day.

[0184] Table 11 Changes in fecal water content of SD rats ( n=10)

[0185]

[0186] Note: *P<0.01 for each group compared with the first day; ★P<0.01 for each group compared with the 14th day.

[0187] From the above results, it can be seen that degrading the capsular polysaccharide A with a molecular weight of 110KD extracted from the NCTC 9343 strain can achieve a therapeutic effect on constipation-type IBS similar to that of the capsular polysaccharide A extracted from the ZY-312 strain.

[0188] From the results of the above examples, it can be seen that the Bacteroides fragilis capsular polysaccharide A provided by the present invention has a good preventive and therapeutic effect on both diarrhea-type and constipation-type IBS, and has a bidirectional regulatory effect.

[0189] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0190] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of Bacteroides fragilis ZY-312 in the preparation of a medicament for preventing and / or treating diarrhea-predominant irritable bowel syndrome, characterized in that: The deposit number of the Bacteroides fragilis is CGMCC No.10685.

2. The use according to claim 1, characterized in that The diarrhea-type irritable bowel syndrome is caused by senna leaves or castor oil.

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