CN113730443A - Application of bacteroides fragilis and extract thereof in preparation of medicine for preventing and treating irritable bowel syndrome - Google Patents
Application of bacteroides fragilis and extract thereof in preparation of medicine for preventing and treating irritable bowel syndrome Download PDFInfo
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- CN113730443A CN113730443A CN202110977532.0A CN202110977532A CN113730443A CN 113730443 A CN113730443 A CN 113730443A CN 202110977532 A CN202110977532 A CN 202110977532A CN 113730443 A CN113730443 A CN 113730443A
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- bacteroides fragilis
- capsular polysaccharide
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- bowel syndrome
- irritable bowel
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Abstract
The invention relates to application of a bacteroides fragilis extract in preparation of a medicine or food for preventing and treating irritable bowel syndrome, wherein the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide A. The bacteroides fragilis capsular polysaccharide A with the molecular weight of 5-70KD is prepared unexpectedly, and the bacteroides fragilis capsular polysaccharide A with the molecular weight of 5-70KD is found to have a better function of preventing and treating irritable bowel syndrome, and the effect of the bacteroides fragilis capsular polysaccharide A is far better than that of the bacteroides fragilis capsular polysaccharide A with the molecular weight of 110KD extracted from NCTC 9343.
Description
The application is a divisional application of Chinese patent application 201710812676.4, which is filed on 11.9.2017 and is named as application of a bacteroides fragilis extract in preparation of medicines or foods for preventing and treating irritable bowel syndrome.
The microbial strains used in the implementation process of the invention are preserved in China general microbiological culture Collection center (CGMCC) (No. 3 Hospital No.1 Xilu Beijing, Chaoyang, respectively) 4.2 days in 2015. And (3) classification and naming: bacteroides fragilis ZY-312 (bacteriodes fragilis ZY-312), accession number CGMCC No. 10685. Bacteroides fragilis ZY-312 was isolated by the applicant and has been patented (patent No. 201510459408.X), as specified in the patent examination manual, commercially available or licensed to the public without storage, i.e., without providing proof of storage.
Technical Field
The invention relates to the technical field of application of bacteroides fragilis, in particular to application of a bacteroides fragilis extract in preparation of a medicine or food for preventing and treating irritable bowel syndrome.
Background
Irritable Bowel Syndrome (IBS) is the most common disorder of bowel function in clinic, and has recently been recognized as a type of psychosomatic disease with a special pathological and physiological basis, and is a group of syndromes manifested by abdominal pain, abdominal distension, constipation and diarrhea, or constipation and diarrhea alternating with lack of morphological or biochemical abnormalities. IBS has a high global incidence rate, and the incidence rate of IBS is between 10 and 15 percent in every region. In western countries, IBS accounts for 12% of the outpatient services of home care physicians and 20-50% of the outpatient services of gastrointestinal disorders. The rate of IBS symptoms in China is the same as that in foreign countries, patients mainly take young people and middle-aged people, the age is between 20 and 50 years, and the first-time patients above 50 years are rare. Women are more than men, the proportion of men to women is 1: 5-1: 2, and the women have a family aggregation tendency. Studies have shown that the medical resources spent on IBS are quite large, affecting the quality of life of the patient to varying degrees.
The etiology and pathogenesis of IBS are still unclear, and the IBS may be related to factors such as diet, intestinal infection and psychology, and is the result of the combined action of multiple factors. The clinical symptoms are various, and besides abdominal symptoms, other symptoms are accompanied, so that the treatment is difficult, and the combination of medicines is often needed. Some patients also have psychopsychological problems such as depression and anxiety, and usually need psychological treatment. Therefore, comprehensive treatment should be applied to treat IBS according to the severity of symptoms, type of symptoms and frequency of attacks of patients, and according to individual treatment principles. The choice of treatment and drug will vary from person to person and include: primary treatment, psychological treatment and drug treatment. At present, the following drugs are mainly used for treating IBS:
first, drugs that modulate intestinal function include antidiarrheals, spasmolytics, prokinetic drugs, and drugs that modulate visceral sensitivity.
Antidiarrheal agents are commonly used in the treatment of diarrhea in patients with IBS. Commonly used drugs are loperamide, diphenoxylate and dioctahedral smectite. Loperamide (Yimanting) acts on opioid peptide receptors on intestinal walls, prevents acetylcholine and prostaglandin from releasing, inhibits intestinal peristalsis, prolongs the retention time of intestinal contents, enhances the absorption of water and ions in intestinal tracts, and further relieves diarrhea, abdominal pain and the like. The diphenoxylate (diphenoxylate) acts on intestinal smooth muscle, increases segmental contraction of the intestine and prolongs the contact time of intestinal contents and intestinal mucosa. The dioctahedral montmorillonite (Cimpanida) can absorb water and pathogenic bacteria, improve the protective power of digestive tract mucosa, promote mucosa repair, and simultaneously can regulate and recover colon motion function and reduce colon sensitivity.
Spasmolytic is commonly used for abdominal pain and abdominal distension in patients with IBS. They can be classified into 3 groups according to their main mechanism of action, namely anticholinergic drugs, smooth muscle relaxants and calcium channel blockers, many of which have multiple pharmacological actions.
Anticholinergic agents include atropine, anisodamine, belladonna, etc. It has atropine-like adverse reaction, so it has limited clinical application. Recently developed selective cholinergic receptor antagonists of the intestinal tract M3 can inhibit the post-prandial intestinal tract motility, and are expected to be used for treating IBS.
The smooth muscle relaxant comprises papaverine drugs (papaverine, dicyclavilin, mebeverine) and a multi-ion channel regulator (trimebutine). The papaverine medicine can directly act on smooth muscle cells and certain intestinal tract excitatory neurons to inhibit the release of excitatory neurotransmitters. The trimebutine generates depolarization by inhibiting a cell membrane potassium ion channel, thereby improving the excitability of smooth muscle cells; on the other hand, the calcium ion channel is blocked to inhibit the calcium ion inflow, thereby inhibiting the cell contraction and relaxing the smooth muscle of the gastrointestinal tract. In addition, trimebutine also has a bidirectional modulating effect on smooth muscle neuroreceptors: under the condition of low movement, the compound acts on adrenergic receptors, inhibits the release of norepinephrine and increases movement rhythm; when hyperkinesia occurs, the compound acts on cholinergic receptors and opioid receptors to inhibit acetylcholine release, thereby inhibiting smooth muscle movement.
The calcium ion antagonist selectively acts on colon calcium ion channel, blocks calcium inflow, plays a role in relaxing smooth muscle, inhibiting gastric and colonic reflex, has a regulating effect on constipation and diarrhea, and also has a certain curative effect on abdominal pain, such as pinaverium bromide and oteracil bromide.
Prokinetic drugs are commonly used for the treatment of constipation in patients with IBS. 5-hydroxytryptamine (5-HT) is an important transmitter in the gastrointestinal tract and the center, has wide biological effects, 95% of 5-HT in human body is distributed in the gastrointestinal tract, and a plurality of 5-HT receptors and 5-HT transporters are distributed on intestinal mucosa. In recent years, the important role of the 5-HT4 receptor in the regulation of gastrointestinal motility and visceral sensation has been emphasized, and it has the effects of promoting gastrointestinal motility, reducing gastrointestinal sensitivity, and promoting the secretion of chloride ions and water molecules, thus becoming a new target for the treatment of functional gastrointestinal diseases. The 5-HT4 receptor agonist cisapride can promote release of acetylcholine from postganglionic cholinergic nerve of myenteric muscle, and has gastrointestinal motility promoting effect. However, it should be used with cautions because it may cause prolongation of QT interval. Tegaserod, also known as zerumab, is a novel 5-HT4 receptor agonist, partially selectively acts on the gastrointestinal 5-HT4 receptor subtype, and has the effect of accelerating small intestine and colon transmission in constipation-predominant IBS patients. Recent research also proves that tegaserod has a regulating effect on internal organ sensation, has no cardiovascular adverse reaction, and is a safe and effective novel medicine.
Agents that modulate visceral sensitivity include 5-HT3 receptor antagonists and 5-HT4 receptor agonists. Increased visceral sensitivity is considered to be one of the pathologically and physiologically important features of IBS. Studies have shown that visceral paresthesia is present in 61% of IBS patients, and improving visceral paresthesia is an interesting approach in IBS treatment, and clinical and animal experimental studies have shown that some drugs have a modulating effect on increased visceral sensitivity.
The 5-HT3 receptor is present in enteric neurons, and promotes intestinal motility, secretion, and elicits visceral pain stimuli by releasing localized 5-HT. For patients with IBS, especially those with abdominal pain due to a decrease in visceral pain threshold, antagonists of the 5-HT3 receptor may be used. Such as alosetron, mainly inhibits the 5-HT3 receptor of a non-selective ion channel in the enteric nervous system and inhibits the visceral reflex, and recently is mainly used for female IBS patients with severe diarrhea who are ineffective by conventional treatment. Still others are ondansetron, gonissidone, and the like.
Tegaserod, a 5-HT4 receptor agonist, has dual effects of prokinetic force and reduction of visceral sensation sensitivity, and is suitable for constipation type IBS patients with marked abdominal pain symptoms. Human body research reports that tegaserod can reduce the reaction of rectal balloon dilatation injury stimulation and improve the visceral sensation of human body.
II, cathartic agent: in addition to 5-HT4 receptor agonists, laxatives may also be used in patients with constipation. It is presently believed that the use of stimulant laxatives should be cautious or avoided as much as possible, and the use of bulking laxatives is recommended. The swelling purgative such as herba plantaginis can increase the volume and water content of feces and promote defecation. Osmotic laxative polyethylene glycol binds water molecules through hydrogen bonds, increasing fecal water content and softening feces, promoting defecation. It is not absorbed, and has low toxicity, and is suitable for patients with dry stool. Lactulose is decomposed by bacteria in colon to form lactic acid and acetic acid, which can regulate intestinal cavity environment and promote intestinal peristalsis, and is more suitable for the elderly.
Thirdly, drugs for improving central emotion: patients with IBS are often associated with psychopsychological disturbances, and pharmacological treatment for IBS should include antidepressant, anxiolytic treatment. Antidepressants include tricyclic antidepressants, such as amitriptyline, and selective 5-HT reuptake inhibitors, such as fluoxetine and paroxetine. Wherein, fluoxetine has the following 4 effects on IBS, namely that psychopsychological improvement can induce the relief of gastrointestinal symptoms; 5-HT transmitter activity and its receptor action on gastrointestinal tract power for regulating visceral gastrointestinal pain; potential central analgesic effects; preventing the vicious circle between psychological disorders and physiological abnormalities. For IBS patients with severe anxiety mental symptoms, the treatment may be considered in combination with anxiolytic treatment.
Fourthly, Chinese medicine: some clinical observations in China prove that some traditional Chinese medicines can effectively relieve the symptoms of abdominal pain, diarrhea and constipation of IBS patients.
Fifthly, microecological preparation: in recent years, attention has been paid to the relationship between intestinal flora disturbance and IBS. Studies have shown that the incidence of a subset of patients is associated with a dysregulated intestinal flora, and that the supplementation of the patients with probiotics improves the symptoms of the patients to varying degrees. The mechanism of action is not well understood, and biochemical inhibition or promotion, nutritional competition, immune clearance, and adhesion receptor competition are generally considered. However, the types of probiotic preparations are not limited, and they mainly include live bifidobacterium triple, live bifidobacterium quadruple and the like.
Currently, there is no very effective treatment for IBS and there is no complete cure for IBS. There is a need to develop new effective drugs that can treat IBS.
Bacteroides fragilis (Bacteroides fragilis) is a member of the genus Bacteroides among gram-negative anaerobic bacteria, belongs to the phylum Bacteroides, and is completely different from Bifidobacterium, Lactobacillus, and the like of the phylum firmicutes. Bacteroides have 25 species, only 10 species from humans, only 10 species from animals, and 5 species from humans and animals. The bacteroides fragilis is an obligate anaerobic bacterium, the shape of the bacteroides is polymorphic according to the difference of culture media and the difference of growth stages, the bacteroides fragilis is rod-shaped, the two ends of the bacteroides fragilis are blunt and round, the coloration is dark, the middle color is light and uneven, the bacteroides fragilis, no spores and no power exist, some bacteroides have vacuoles, and the bacteroides are different in length. Bacteriodes fragilis enterotoxin (BFT) can be classified into Enterotoxigenic bacteriodes fragilis (ETBF) and non-Enterotoxigenic bacteriodes fragilis (NTBF) depending on whether they can be synthesized and secreted. Bacteroides fragilis is present mainly in the colon as part of the normal intestinal flora of humans and animals. In addition, mucous membranes of the respiratory, gastrointestinal and genitourinary tracts may colonize. Bacteroides fragilis, a opportunistic pathogen, when damaged in the host mucosa can invade the submucosa and cause infection, and can also cause purulent infection and abscess associated with it in other organs of the body, such as the intestinal tract, abdominal cavity, liver, lung, brain tissue, soft tissue, bone marrow, etc., by blood flow.
Disclosure of Invention
Based on this, the present invention provides a novel use of Bacteroides fragilis (Bacteroides fragilis) extract. The specific technical scheme is as follows:
application of a bacteroides fragilis extract in preparation of medicines or foods for preventing and treating irritable bowel syndrome, wherein the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide A.
In some embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 5-75 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 15 KD-65 KD;
in some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 25 KD-55K.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 35 KD-45 KD.
In some of these embodiments, the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide a in an amount of 60-75 wt%.
In some embodiments, the bacteroides fragilis is bacteroides fragilis ZY-312 with collection number of CGMCC No. 10685.
In some of these embodiments, the method of preparing the bacteroides fragilis extract comprises the steps of:
(1) centrifuging and precipitating the bacteroides fragilis bacterial liquid after fermentation culture, collecting a first precipitate, adding water with the temperature of 65-72 ℃ into the first precipitate, adding a phenol solution after dissolving, keeping the temperature of 65-72 ℃, stirring for 25-35min, centrifuging, and collecting a first supernatant;
(2) extracting the first supernatant collected in the step (1) by using ether to remove phenol, removing residual ether, and collecting an aqueous phase solution;
(3) adding absolute ethanol into the aqueous phase solution collected in the step (2) until the final concentration of the ethanol is 75-85 v/v%, precipitating with ethanol, centrifuging, collecting a second precipitate,
(4) and adding water into the second precipitate to prepare a suspension, adjusting the pH to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
In some embodiments, the ratio of the water, the phenol solution and the first precipitate added to the first precipitate in step (1) is 3-5 mL: 3-5 mL: 1g of a compound; the mass concentration of the phenol solution is 70-80%.
In some embodiments, the alcohol precipitation in step (3) is alcohol precipitation at a temperature of 0-8 ℃ for 8-16 hours.
In some embodiments, step (4) comprises: and adding water into the second precipitate to prepare a suspension with the mass concentration of 8-12%, adding a glacial acetic acid aqueous solution with the mass concentration of 8-12%, heating to boil, stirring for reaction for 1.5-2.5 hours, adjusting the pH value to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
In some of these embodiments, the method of preparing the bacteroides fragilis extract further comprises the step of degrading: degrading the bacteroides fragilis extract obtained in the step (4) by using an ultrasonic method, wherein the ultrasonic condition is as follows: 180 ℃ and 210kHz, 15-25 ℃.
In some of these embodiments, the dosage form of the medicament comprises a pill, tablet, granule, capsule, oral liquid, or tube feed formulation. The medicine comprises human medicine or animal medicine, and can be used for human or animal.
The bacteroides fragilis extract can be administered prophylactically or therapeutically alone, or with other probiotics and/or probiotic materials. In the case of combined administration, administration may be carried out in a single formulation or in separate formulations, simultaneously or at different times, using the same or different routes of administration.
The food product comprises milk powder, cheese, curd, yogurt, ice cream or fermented cereal. The food product may also be an animal food product, such as a feed or the like. The food product may also be an infant food product or a pet food product.
The invention also provides a medicine or food for preventing and treating irritable bowel syndrome. The specific technical scheme is as follows:
a medicine or food for preventing and treating irritable bowel syndrome contains a bacteroides fragilis extract, wherein the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide A.
In some embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 5-75 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 15 KD-65 KD;
in some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 25 KD-55K.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 35 KD-45 KD.
In some of these embodiments, the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide a in an amount of 60-75 wt%.
In some embodiments, the bacteroides fragilis is bacteroides fragilis ZY-312 with collection number of CGMCC No. 10685.
In some of these embodiments, the method of preparing the bacteroides fragilis extract comprises the steps of:
(1) centrifuging and precipitating the bacteroides fragilis bacterial liquid after fermentation culture, collecting a first precipitate, adding water with the temperature of 65-72 ℃ into the first precipitate, adding a phenol solution after dissolving, keeping the temperature of 65-72 ℃, stirring for 25-35min, centrifuging, and collecting a first supernatant;
(2) extracting the first supernatant collected in the step (1) by using ether to remove phenol, removing residual ether, and collecting an aqueous phase solution;
(3) adding absolute ethanol into the aqueous phase solution collected in the step (2) until the final concentration of the ethanol is 75-85 v/v%, precipitating with ethanol, centrifuging, collecting a second precipitate,
(4) and adding water into the second precipitate to prepare a suspension, adjusting the pH to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
In some embodiments, the ratio of the water, the phenol solution and the first precipitate added to the first precipitate in step (1) is 3-5 mL: 3-5 mL: 1g of a compound; the mass concentration of the phenol solution is 70-80%.
In some embodiments, the alcohol precipitation in step (3) is alcohol precipitation at a temperature of 0-8 ℃ for 8-16 hours.
In some embodiments, step (4) comprises: and adding water into the second precipitate to prepare a suspension with the mass concentration of 8-12%, adding a glacial acetic acid aqueous solution with the mass concentration of 8-12%, heating to boil, stirring for reaction for 1.5-2.5 hours, adjusting the pH value to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
In some of these embodiments, the method of preparing the bacteroides fragilis extract further comprises the step of degrading: degrading the bacteroides fragilis extract obtained in the step (4) by using an ultrasonic method, wherein the ultrasonic condition is as follows: 180 ℃ and 210kHz, 15-25 ℃.
In some of these embodiments, the dosage form of the medicament comprises a pill, tablet, granule, capsule, oral liquid, or tube feed formulation. The medicine comprises human medicine or animal medicine, and can be used for human or animal.
The medicament may comprise one or more of the following pharmaceutically acceptable excipients: diluents, excipients, binders, lubricants, suspending agents, coating agents, solubilizers, and the like. Examples of pharmaceutically acceptable excipients include: water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycols, propylene glycols, liposomes, saccharides, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, mono-and di-fatty acid glycerides, petro (petrochemical) fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and the like.
The medicament may be administered by any one or more of the following: administration by inhalation is carried out in the form of a micro-pump or nasal spray or inhalation aerosol, administration in the form of a suppository or pessary, topical administration in the form of a lotion, solution, cream, ointment or dusting powder, administration by use of a skin patch, oral administration in the form of a tablet containing an excipient such as starch or lactose or in the form of a capsule or an ovule either alone or mixed with an excipient, or administration in the form of an elixir, solution or suspension containing a flavoring or coloring agent, or parenteral injection, for example intracavernosal, intravenous, intramuscular or subcutaneous injection. For parenteral administration, the drug is preferably used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, the medicament may be administered in the form of tablets or lozenges formulated in conventional manner.
The food product comprises milk powder, cheese, curd, yogurt, ice cream or fermented cereal. The food product may also be an animal food product, such as a feed or the like. The food product may also be an infant food product or a pet food product.
The bacteroides fragilis ZY-312 of the invention has been preserved in China general microbiological culture Collection center (CGMCC) at 4.2.2015, the preservation number is CGMCC No.10685, and the preservation address is No. 3 Hospital No.1 of Xilu, Beijing, Chaoyang district.
The inventor of the invention obtains the preparation method of the bacteroides fragilis extract (the main component is capsular polysaccharide A) through long-term experience accumulation and a large number of creative experimental researches, and further experimental researches show that the bacteroides fragilis extract has the function of preventing and treating irritable bowel syndrome, has good prevention and treatment effects on diarrhea-type irritable bowel syndrome and constipation-type irritable bowel syndrome, and has a far better prevention and treatment effect on the irritable bowel syndrome than that of the bacteroides fragilis. Further, the inventors have obtained bacteroides fragilis capsular polysaccharide A with molecular weight of 5-70KD by degrading bacteroides fragilis capsular polysaccharide A with molecular weight of 70KD, and have surprisingly found that the bacteroides fragilis capsular polysaccharide A with molecular weight of 5-70KD has better function of preventing and treating irritable bowel syndrome, and the effect is far better than that of the bacteroides fragilis capsular polysaccharide A with molecular weight of 110KD extracted from NCTC 9343. The bacteroides fragilis capsular polysaccharide A provided by the invention has a good prevention and treatment effect on irritable bowel syndrome and has no side effect on organisms, and can be used for performing preventive and therapeutic administration alone or together with other probiotics and/or probiotic materials. The bacteroides fragilis capsular polysaccharide A provided by the invention has good edible and medicinal prospects, and provides a good product for health care and irritable bowel syndrome prevention and treatment suitable for human body eating for clinic.
Drawings
FIG. 1 is a characteristic diagram of the colony of Bacteroides fragilis ZY-312 of example 1;
FIG. 2 is a microscopic image of Bacteroides fragilis ZY-312 of example 1 after gram-staining.
FIG. 3 is the 1H spectrum of the capsular polysaccharide A NMR spectrometer analysis of example 1;
FIG. 4 is a 13C spectrum of capsular polysaccharide A NMR spectrometer analysis of example 1;
FIG. 5 is a COSY spectrum of capsular polysaccharide A NMR spectrometer analysis of example 1;
FIG. 6 is an HSQC spectrum of capsular polysaccharide A NMR spectrometer analysis of example 1;
FIG. 7 is a HMBC spectrum of a nuclear magnetic resonance spectrometer analysis of capsular polysaccharide A of example 1;
FIG. 8 shows the chemical structure of Bacteroides fragilis capsular polysaccharide A prepared in example 1.
Detailed Description
The present invention is further illustrated by the following specific examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
The Bacteroides fragilis used in the following examples is Bacteroides fragilis ZY-312 (bacteriodes fragilis ZY-312), which was deposited in China general microbiological culture Collection center (CGMCC) at 4.2.2015, with the collection number of CGMCC No.10685, and the collection address of No. 3, Xilu 1, North Cheng Yang district, Beijing.
Example 1 preparation of Bacteroides fragilis extract
(1) Fermentation culture of bacteroides fragilis
The strain is streaked and inoculated on a blood plate, and anaerobic culture is carried out for 48 h. Observing the morphological characteristics, staining characteristics, size, club shape, distribution and the like of colonies.
Colony characteristics: after culturing the bacteroides fragilis ZY-312 on a blood plate for 48h, the bacteroides fragilis ZY-312 presents a round and slightly convex shape, is semitransparent, white, has a smooth surface and is not hemolyzed, and the diameter of a colony is between 1 and 3mm, as shown in figure 1.
Microscopic morphology: gram-stained bacteroides fragilis ZY-312 was used as gram-negative bacteria, and was typically rod-shaped, with blunt and densely stained ends, and non-staining areas in the middle of the cells, such as vacuoles, as shown in FIG. 2.
Selecting single colony, inoculating into tryptone broth, fermenting and culturing for 8 hours (at 37 ℃), centrifuging the obtained bacterial liquid for precipitation, rotating at 3000r/min, centrifuging for 15min, removing supernatant, and collecting precipitate.
(2) Preparation of Bacteroides fragilis extract
1) Taking 200g of bacteroides fragilis bacterial mud (the precipitate obtained in the step (1)) and adding 750mL of ultrapure water at 68 ℃, after dissolving, adding 750mL of phenol solution with the volume fraction of 75%, uniformly mixing, keeping at 68 ℃, stirring and extracting for 30min, centrifuging for 20min at 15000g, and taking supernatant.
2) The supernatant was extracted with an equal volume of ether (1.5L) to remove phenol, the supernatant collected and extracted repeatedly until no phenol remained. The ether was removed by heating in a water bath and the aqueous phase was collected.
3) Centrifuging the water phase at 15000g for 20min, measuring volume, adding anhydrous ethanol until the final concentration of ethanol is 80% (volume fraction), precipitating with ethanol at 4 deg.C overnight (12 hr), centrifuging at 15000g for 20min, and collecting precipitate.
4) Weighing the mass of the precipitate in the step 3), adding a certain volume of deionized water to prepare the precipitate into a suspension with the mass concentration of 10%, stirring and mixing uniformly, adding a glacial acetic acid aqueous solution with the mass concentration of 10%, heating to boil, continuously stirring and reacting for 2h, adjusting the pH value to 7.0, centrifuging for 20min at 15000g, and collecting the supernatant. The resulting supernatant was dialyzed to remove salts (10KD dialysis bag), and freeze-dried to obtain Bacteroides fragilis extract.
5) Weighing 30mg of Bacteroides fragilis extract of step 4) in 0.5mL D2O, 1. mu.l of acetone (1H, 2.22; 13C, 30.89) was added for calibration. Analyzing the spectra of 1H, 13C, COSY, HSQC and HMBC (figure 3) by using a 500MHz Bruker nuclear magnetic resonance spectrometer, and confirming that the bacteroides fragilis extract collected in the step 4) is capsular polysaccharide A and the purity is about 70%. GPC analysis showed that the above capsular polysaccharide A had a molecular weight of 781 in repeating units, a number of repeating units n of 89, a molecular weight of about 70KD and a molecular formula of- [ C ]31N3O20H47]91The chemical structure is shown in FIG. 4.
(3) Preparation of capsular polysaccharide A of different molecular weights
This example is carried out by degrading capsular polysaccharide A prepared in (2) by methods including, but not limited to, chemical degradation, physical degradation and biodegradation. In the embodiment, an ultrasonic method is adopted, wherein the capsular polysaccharide A is treated for 3 hours, 2 hours, 0.5 hour and 0 hour at 195kHz and 20 ℃, and capsular polysaccharide A with molecular weights of 2KD, 5KD, 40KD and 70KD is collected respectively. Capsular polysaccharide A with molecular weight of 110KD is extracted from Bacteroides fragilis NCTC 9343 (purchased from ATCC in USA) by the method of (2).
Example 2 Effect of Bacteroides fragilis capsular polysaccharide A on diarrhea-causing IBS in folium sennae
First, experimental design
In order to verify the effect of the bacteroides fragilis extract (capsular polysaccharide a as a main ingredient) provided in example 1 on the prevention/treatment of irritable bowel syndrome, 60C 57BL/6 mice were selected for the experiment. The 60C 57BL/6 mice were hermaphroditic and each experimental mouse was assigned a unique number. Before animals are grouped, the squirrel cage labels should be labeled with item number, species/strain, gender, cage number, and animal number. The mice were randomly assigned using BioBook software based on initial body weight of C57BL/6 mice into 6 groups, i.e. normal Group (Group1), model Group (Group2), loperamide capsule Group (2.4mg/kg) (Group3), bacteroides fragilis capsular polysaccharide a low (Group4), medium (Group5), high (Group6) dose groups, 10 mice per Group C57 BL/6. This example illustrates capsular polysaccharide A, which has a molecular weight of 5 KD.
Mice in each group were gavaged with the corresponding drug, and normal and model groups were given an equal amount of physiological saline 1 time a day for 5 consecutive days. All C57BL/6 mice were fasted for 24h, and 1h after the last administration, 1g/mL senna leaf decoction was administered to each Group of C57BL/6 mice except for the normal Group (Group1) which was administered with the same amount of physiological saline. The C57BL/6 mice were housed in cages with 1 mouse per cage, the wet feces count was determined by placing filter paper under the cages, the degree of diarrhea was indicated by the amount of wet feces, and the filter paper was changed 1 time every 1 hour. And observing and counting the total number of shit, the total number of shit and the number of shit stages of the mice within 6 h.
Second, judging standard
The number of free feces was determined according to the Zhou's method (Wednung, mussaka, Wanyixian, et al. preparation of diarrhea model and application of diarrhea index in mice [ J ]. Chinese herbal medicine, 1994, 250(4):195-196 ]. The diameter of the stain is 1 grade when the diameter is less than l cm, 2 grades when the diameter is 1-1.9 cm, 3 grades when the diameter is 2-3 cm, and 4 grades when the diameter is more than 3 cm. Each animal had a stool dilution rate (%) (total stool count/total stool count x 100%) and a diarrhea Index (ID) (stool dilution rate x stool dilution series).
Third, results and analysis
All data are expressed in x +/-s, statistical analysis is carried out by using SPSS 17.0 software, single-factor analysis of variance is adopted for comparison among groups, and P is less than 0.05, so that statistical significance is achieved. Specific results are shown in table 1.
TABLE 1 Effect of Bacteroides fragilis capsular polysaccharide A on diarrhea-predominant IBS in mice caused by folium sennae: (
n＝10)
Note P < 0.01 compared to normal group; compared with the model group, P is less than 0.01.
As can be seen from the results in Table 1, the total number of loose stools, the rate of loose stools and the diarrhea index comparison between the model Group (Group2) and the normal Group (Group1) are all significantly different (P < 0.01), which indicates that the IBS model of the mice diarrhea caused by senna leaves is successfully modeled. Compared with the model group, the loperamide group and the bacteroides fragilis capsular polysaccharide A low, medium and high dose groups provided in example 1 can obviously inhibit the degree of diarrhea (P is less than 0.01) of mice, and the bacteroides fragilis capsular polysaccharide A provided by the invention has obvious inhibition effect on diarrhea-type IBS caused by folium sennae.
Example 3 Effect of Bacteroides fragilis capsular polysaccharide A on Castor oil induced diarrheal IBS
First, experimental design
In order to verify the effect of the bacteroides fragilis extract (capsular polysaccharide a as a main ingredient) provided in example 1 on the prevention/treatment of irritable bowel syndrome, 60C 57BL/6 mice were selected for the experiment. The 60C 57BL/6 mice were hermaphroditic and each experimental mouse was assigned a unique number. Before animals are grouped, the squirrel cage labels should be labeled with item number, species/strain, gender, cage number, and animal number. The mice were randomly assigned using BioBook software based on initial body weight of C57BL/6 mice into 6 groups, i.e. normal Group (Group1), model Group (Group2), loperamide capsule Group (2.4mg/kg) (Group3), bacteroides fragilis capsular polysaccharide a low (Group4), medium (Group5), high (Group6) dose groups, 10 mice per Group C57 BL/6. This example illustrates capsular polysaccharide A, which has a molecular weight of 40 kD.
Mice in each group were gavaged with the corresponding drug, and normal and model groups were given an equal amount of physiological saline 1 time a day for 5 consecutive days. All C57BL/6 mice were fasted for 24h and 1h after the last administration, except for the normal Group (Group1) which was administered with an equal amount of physiological saline, the other groups of C57BL/6 mice were administered castor oil 1 time (20 mL/kg). The C57BL/6 mice were housed in cages with 1 mouse per cage, the wet feces count was determined by placing filter paper under the cages, the degree of diarrhea was indicated by the amount of wet feces, and the filter paper was changed 1 time every 1 hour. And observing and counting the total number of shit, the total number of shit and the number of shit stages of the mice within 6 h.
Second, judging standard
The same criteria as in example 2.
Third, results and analysis
All data are as follows
It shows that SPSS 17.0 software is used for statistical analysis, single-factor analysis of variance is adopted for comparison among groups, and P is less than 0.05, which is of statistical significance. Specific results are shown in table 2.
TABLE 2 Effect of Bacteroides fragilis capsular polysaccharide A on Castor oil-induced diarrhea-type IBS in mice: (
n＝10)
Note P < 0.01 compared to normal group; compared with the model group, P is less than 0.01.
As can be seen from the results in Table 2, the total number of loose stools, the rate of loose stools and the diarrhea index comparison between the model Group (Group2) and the normal Group (Group1) are all significantly different (P < 0.01), which indicates that the IBS model of the mice diarrhea caused by castor oil is successfully modeled. Compared with the model group, the loperamide group can obviously inhibit the total number of loose stools of 6h, the loose stool rate and the diarrhea index (P < 0.05) of mice with castor oil diarrhea. The low dose (0.125g/kg) of Bacteroides fragilis capsular polysaccharide A has certain inhibition effect on total number of loose stools, loose stool rate and diarrhea index after 6 hours compared with the model group, but has no significant difference (P is more than 0.05); but in medium and high dose, the bacteroides fragilis capsular polysaccharide A can remarkably inhibit the total number of loose stools of 6h, the loose stool rate and the diarrhea index (P < 0.05) of mice with castor oil diarrhea.
Example 4 Effect of Bacteroides fragilis capsular polysaccharide A on neostigmine-induced hyperkinetic movement of mouse Small intestine
First, experimental design
In order to verify the effect of the bacteroides fragilis extract (mainly comprising capsular polysaccharide a) provided in example 1 on preventing/treating irritable bowel syndrome, 60C 57BL/6 mice were selected for experiments in this example. The 60C 57BL/6 mice were hermaphroditic and each experimental mouse was assigned a unique number. Before animals are grouped, the squirrel cage labels should be labeled with item number, species/strain, gender, cage number, and animal number. The mice were randomized using BioBook software based on initial body weight of C57BL/6 mice and divided into 6 groups, i.e. normal (Group1), model (Group2), pinaverium bromide (0.1g/kg) Group (Group3), bacteroides fragilis capsular polysaccharide a low (Group4), medium (Group5), high (Group6) dose groups, 10 mice per Group, C57 BL/6. This example illustrates capsular polysaccharide A, which has a molecular weight of 70 kD.
Mice in each group were gavaged with the corresponding drug, and normal and model groups were given an equal amount of physiological saline 1 time a day for 5 consecutive days. 1h after the last administration, neostigmine was subcutaneously injected in the other 5 groups except the normal group at 0.15mg/kg, causing the small intestine to be excited, and the normal group was subcutaneously injected with the same amount of physiological saline. After 15min, the stomach was perfused with a suspension containing 5% activated carbon powder, after 20min, cervical vertebrae were taken off and sacrificed, small intestines were separated by laparotomy, the total length of the small intestines and the length of the small intestines into which carbon powder was pushed were measured, and the percent pushing of carbon powder was calculated. Percent carbon-pushed (length of small intestine pushed by carbon/total length of small intestine) × 100%.
Second, results and analysis
All data are as follows
It shows that SPSS 17.0 software is used for statistical analysis, single-factor analysis of variance is adopted for comparison among groups, and P is less than 0.05, which is of statistical significance. Specific results are shown in table 3.
TABLE 3 Effect of Bacteroides fragilis capsular polysaccharide A on neostigmine-induced hypermotility of mouse small intestine: (
n＝10)
Note P < 0.01 compared to normal group; compared with the model group, P is less than 0.01.
As can be seen from Table 3, neostigmine can cause the mouse small intestine hyperkinesia, compared with the model group, the pinaverium bromide group can obviously reduce the carbon-end propulsion rate and inhibit the mouse small intestine hyperkinesia caused by neostigmine (P is less than 0.01); the Bacteroides fragilis capsular polysaccharide A with medium and high dose can also remarkably reduce carbon dust propulsion rate (P < 0.05), and inhibit mouse intestinal hypermotility caused by neostigmine.
Example 5 Effect of Bacteroides fragilis capsular polysaccharide A on Constipation-type IBS
First, experimental design
In order to verify the effect of the bacteroides fragilis extract (capsular polysaccharide a as a main component) provided in example 1 on prevention/treatment of constipation-predominant irritable bowel syndrome, 60 SD rats were selected for experiments in this example. The 60 SD rats are half male and half female, and each experimental rat is assigned a unique number. Before animals are grouped, the squirrel cage labels should be labeled with item number, species/strain, gender, cage number, and animal number. Groups were randomized using BioBook software based on initial body weight of SD rats into 6 groups, namely a normal Group (Group1), a model Group (Group2), tegaserod maleate (solution formulation: 1.2mg tegaserod maleate in 10ml sterile saline) Group (Group3), a bacteroides fragilis capsular polysaccharide a low (Group4), medium (Group5), high (Group6) dose Group, 10 SD rats per Group. This example illustrates capsular polysaccharide A, which has a molecular weight of 70 kD.
The SD rat constipation type IBS model was constructed according to the method (Pen L H, Yang Y S, Sun G, et al. A new model of coherence-preventive bilateral synthesis in rats [ J ]. World Chinese Journal of Digestology,2004,12(1): 112-116.). Except for the normal Group (Group1), the rest groups are perfused with ice physiological saline (0-4 ℃) once a day, 2ml of ice physiological saline is added each time, and a constipation type rat IBS model is established for 14 consecutive days. During the molding period, rats in each group can drink water freely.
After 14 days, each test group was administered the corresponding drug by gavage, wherein the normal group and the model group were administered 10ml of normal temperature sterile physiological saline, respectively; positive control Group (Group3) gavage tegaserod maleate; group 4-6 administered low, medium and high doses of Bacteroides fragilis capsular polysaccharide A, respectively, specific experiments and dosing regimens are shown in Table 4:
table 4 experimental groups and dosing regimens
Second, results and analysis
The number of fecal particles in each group of rats was collected at 24 hours on day 1, 14 and 28, respectively, and one contamination blot was used as one if any diarrhea was observed (see Table 5 for details). The collected feces were weighed, dried, and the moisture content of the feces was calculated (see table 6 for details).
TABLE 5 variation of stool particle count for 24h in SD rats (particles) (C)
n＝10)
| | Day | 1 | Day 14 | |
| Group1 | 49.13±6.34 | 48.53±6.48 | 48.10±5.43◆ | |
| Group2 | 47.37±5.27 | 33.07±5.32*▲ | 32.44±6.23 | |
| Group3 | 48.22±7.34 | 32.46±5.72*▲ | 47.43±6.35★◆ | |
| Group4 | 47.78±5.39 | 32.73±4.39*▲ | 40.04±7.94★◆ | |
| Group5 | 48.05±7.91 | 34.07±5.83*▲ | 43.90±8.06★◆ | |
| Group6 | 47.88±6.54 | 33.77±5.60*▲ | 49.03±5.71★◆ |
Note that P < 0.01 for each group of experiments compared to the first day; on the 14 th day, comparing Group 2-6 with normal control Group, wherein P is less than 0.01; the ratio of: (i) on day 28 to day 14 in each experiment,: (P) 0.01; on day 28, each experimental group was compared to the model group, P < 0.01.
TABLE 6 fecal moisture content Change in SD rats (
n＝10)
| | Day | 1 | Day 14 | |
| Group1 | 0.51±0.04 | 0.49±0.08 | 0.48±0.07◆ | |
| Group2 | 0.47±0.07 | 0.39±0.04*▲ | 0.35±0.06 | |
| Group3 | 0.48±0.08 | 0.37±0.07*▲ | 0.56±0.06★◆ | |
| Group4 | 0.49±0.10 | 0.38±0.09*▲ | 0.44±0.07★◆ | |
| Group5 | 0.46±0.05 | 0.40±0.05*▲ | 0.47±0.05★◆ | |
| Group6 | 0.47±0.06 | 0.39±0.06*▲ | 0.53±0.09★◆ |
Note that P < 0.05 for each group of experiments compared to the first day; on the 14 th day, comparing Group 2-6 with normal control Group, wherein P is less than 0.05; the ratio of the first day 28 to the second day 14 in each experiment is: < 0.05; on day 28, each experimental group was compared to the model group, P < 0.05.
As can be seen from Table 5, in Group 1-6, the number of fecal particles was not very different in each Group of rats 24 hours the first day; the number of fecal particles in the rats of the other groups on day 14 is obviously reduced compared with that of the rats on the first day except the normal group, and the difference has statistical significance (P is less than 0.05); on day 14, the numbers of fecal particles in Group 2-6 were significantly reduced compared to the number in the normal Group (Group1), and the differences were statistically significant (P < 0.05), indicating that this example successfully constructed a constipation-predominant IBS rat model. On day 28, the number of fecal particles in Group 2-6 was significantly increased compared to that on day 14, with the difference being statistically significant (P < 0.05). The low, medium and high doses of bacteroides fragilis capsular polysaccharide A provided by the invention can effectively increase the number of fecal particles of constipation-type IBS rats.
As can be seen from Table 6, in Group 1-6, the water content of feces of rats in each Group is not greatly different in 24 hours in the first day; except for the normal Group, the water content of the feces of the rats in the other groups on the 14 th day is obviously reduced compared with that of the rats on the first day, the difference has statistical significance (P is less than 0.05), and on the 14 th day, the water content of the feces of the groups 2-6 is obviously reduced compared with that of the rats in the normal Group (Group1), and the difference has statistical significance (P is less than 0.05). On day 28, the water content of the feces of Group 2-6 is obviously increased compared with that on day 14, and the difference has statistical significance (P is less than 0.05). The low, medium and high doses of bacteroides fragilis capsular polysaccharide A provided by the invention can effectively increase the water content of feces of constipation IBS rats.
From the results, the bacteroides fragilis capsular polysaccharide A provided by the invention has a good treatment effect on constipation type IBS.
Example 6 therapeutic Effect of Bacteroides fragilis and Bacteroides fragilis capsular polysaccharide A
First, experimental design
In order to compare the effects of the bacteroides fragilis extract (mainly comprising capsular polysaccharide a) and bacteroides fragilis itself provided in example 1 on the prevention/treatment of irritable bowel syndrome, 60C 57BL/6 mice were selected for the experiment in this example. The 60C 57BL/6 mice were hermaphroditic and each experimental mouse was assigned a unique number. Before animals are grouped, the squirrel cage labels should be labeled with item number, species/strain, gender, cage number, and animal number. The mice were randomly assigned using BioBook software based on initial body weights of C57BL/6 mice into 6 groups, namely, senna diarrhea-causing IBS model Group (Group1), castor oil diarrhea-causing IBS model Group (Group2), high-dose bacteroides fragilis capsular polysaccharide a versus treatment Group for senna diarrhea-causing IBS (Group3), high-dose bacteroides fragilis itself (10)10CFU/ml) treatment Group for diarrhea-causing IBS with senna (Group4), treatment Group for diarrhea-causing IBS with high-dose Bacteroides fragilis capsular polysaccharide A with castor oil (Group5), and high-dose Bacteroides fragilis itself (10)10CFU/ml) treatment Group for castor oil diarrhea IBS (Group6), 10 mice per Group C57 BL/6. The molecular weight of capsular polysaccharide A in the embodiment is 40KD, and the concentration is 0.5 mg/mL; the concentration of Bacteroides fragilis is 1010CFU/ml。
The mice in each Group were gavaged with the corresponding drug, and the model Group (Group1, Group2) was given an equal amount of physiological saline 1 time a day for 5 consecutive days. All C57BL/6 mice were fasted for 24h, 1h after the last dose, and 1g/mL senna leaf water decoction was administered to Group1, Group3 and Group 4C 57BL/6 mice, and castor oil was administered to Group2, Group5 and Group 6C 57BL/6 mice 1 time (20 mL/kg). The C57BL/6 mice were housed in cages with 1 mouse per cage, the wet feces count was determined by placing filter paper under the cages, the degree of diarrhea was indicated by the amount of wet feces, and the filter paper was changed 1 time every 1 hour. And observing and counting the total number of shit, the total number of shit and the number of shit stages of the mice within 6 h.
Second, judging standard
The same criteria as in example 2.
Third, results and analysis
All data are as follows
It shows that SPSS 17.0 software is used for statistical analysis, single-factor analysis of variance is adopted for comparison among groups, and P is less than 0.05, which is of statistical significance. Specific results are shown in table 6.
TABLE 6 Effect of Bacteroides fragilis capsular polysaccharide A on diarrheal IBS in mice: (
n＝10)
Note: group3, Group4 compare P < 0.05 with Group1, respectively; group5, Group6 and Group2,▲p is less than 0.05; group3 compared to Group4, Group5 compared to Group6,★P＜0.05。
as can be seen from the results in Table 6, the total number of loose stools, the rate of loose stools and the diarrhea index comparison of the Group3 and the Group4 with the model Group (Group1) are all significantly different (P < 0.05), which indicates that the Bacteroides fragilis per se and the Bacteroides fragilis capsular polysaccharide A have treatment and prevention effects on IBS caused by senna leaf in mice. Compared with the total number of loose stools, the loose stool rate and the diarrhea index of the Group5 and the Group6 of the model Group (Group2), the total number, the loose stool rate and the diarrhea index are all significantly different (P is less than 0.01), which indicates that the bacteroides fragilis and the bacteroides fragilis capsular polysaccharide A have treatment and prevention effects on IBS caused by castor oil. Compared with Group4, the total number of loose stools, the loose stool rate and the diarrhea index of Group3 are small and have significant differences (P is less than 0.05), which indicates that the Bacteroides fragilis capsular polysaccharide A has better treatment and prevention effects on IBS (IBS) caused by diarrhea of mice due to senna leaves than the Bacteroides fragilis per se; compared with Group6, the total number of loose stools, the loose stool rate and the diarrhea index of Group5 are small and have significant differences (P is less than 0.05), which indicates that the Bacteroides fragilis capsular polysaccharide A has better IBS treatment and prevention effects on castor oil-induced mouse diarrhea than Bacteroides fragilis per se.
Experiments also prove that the bacteroides fragilis capsular polysaccharide A provided by the invention has better treatment and prevention effects on mouse small intestine hyperkinesia and constipation type IBS caused by neostigmine than the bacteroides fragilis per se.
Example 7 therapeutic Effect of Bacteroides fragilis capsular polysaccharide A of different molecular weights on IBS
In this example, the bacteroides fragilis extracts containing bacteroides fragilis capsular polysaccharide A with 2KD, 5KD, 40KD and 70KD prepared in the invention example 1 were used for preventing/treating constipation type IBS rat model, and the therapeutic effect of bacteroides fragilis capsular polysaccharide A with different molecular weights on constipation type IBS was examined. This example illustrates high doses of 2kD, 5kD, 40kD and 70kD Bacteroides fragilis capsular polysaccharide A.
First, experimental design
Referring to the experimental grouping method of example 5, 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.
The SD rat constipation type IBS model was constructed according to the method (Pen L H, Yang Y S, Sun G, et al. A new model of coherence-preventive bilateral synthesis in rats [ J ]. World Chinese Journal of Digestology,2004,12(1): 112-116.). Except for the normal Group (Group1), the rest groups are perfused with ice physiological saline (0-4 ℃) once a day, 2ml of ice physiological saline is added each time, and a constipation type rat IBS model is established for 14 consecutive days. During the molding period, rats in each group can drink water freely.
After 14 days, each test group was administered the corresponding drug by gavage, wherein the normal group and the model group were administered 10ml of normal temperature sterile physiological saline, respectively; groups 3-7 were administered capsular polysaccharide A with molecular weight of 2KD (Group3), 5KD (Group4), 40KD (Group5), 70KD (Group6) and 110KD (Group7), respectively, and the specific experiments and administration schedule are shown in Table 7:
table 7 experimental groups and dosing regimens
Second, results and analysis
The number of fecal particles in each group of rats was collected at 24 hours on day 1, 14 and 28, respectively, and one contamination blot was used as one if any diarrhea was observed (see Table 8 for details). The collected feces were weighed, dried, and the moisture content of the feces was calculated (see table 9 for details).
TABLE 8 variation of stool particle count for 24h in SD rat: (particle) (C)
n＝10)
Note that P < 0.01 for each group of experiments compared to the first day; on the 14 th day, comparing Group 2-7 with normal control Group, wherein P is less than 0.01; the ratio of: (i) on day 28 to day 14 in each experiment,: (P) 0.01; on day 28, each experimental group was compared to the model group, P < 0.01.
TABLE 9 fecal moisture content Change in SD rats (
n＝10)
| | Day | 1 | Day 14 | |
| Group1 | 0.50±0.03 | 0.48±0.05 | 0.49±0.08◆ | |
| Group2 | 0.49±0.06 | 0.37±0.04*▲ | 0.34±0.05 | |
| Group3 | 0.48±0.08 | 0.38±0.07*▲ | 0.41±0.07★◆ | |
| Group4 | 0.48±0.07 | 0.36±0.06*▲ | 0.54±0.08★◆ | |
| Group5 | 0.49±0.09 | 0.37±0.10*▲ | 0.53±0.06★◆ | |
| Group6 | 0.48±0.08 | 0.39±0.08*▲ | 0.55±0.09★◆ | |
| Group7 | 0.47±0.08 | 0.38±0.05*▲ | 0.45±0.08★◆ |
Note that P < 0.05 for each group of experiments compared to the first day; on the 14 th day, comparing Group 2-6 with normal control Group, wherein P is less than 0.05; the ratio of the first day 28 to the second day 14 in each experiment is: < 0.05; on day 28, each experimental group was compared to the model group, P < 0.05.
As can be seen from Table 8, in Group 1-7, the number of fecal particles was not very different in each Group of rats 24 hours the first day; except for the normal Group (Group1), the number of the fecal particles of the rats in the other groups on the 14 th day is obviously reduced compared with that on the first day, the difference is extremely obvious (P is less than 0.01), and the statistical significance is achieved; on the 14 th day, the numbers of the Group 2-7 and the number of the normal Group (Group1) feces particles are all obviously reduced, the difference is extremely obvious (P is less than 0.01), and the statistical significance is achieved. This example illustrates the successful construction of a constipation-predominant IBS rat model. On the 28 th day, except for Group3, the number of the fecal particles in Group 4-7 is obviously increased compared with that on the 14 th day, the difference is very obvious (P is less than 0.01), and the statistical significance is achieved. The bacteroides fragilis capsular polysaccharide A with different molecular weights provided by the invention can effectively increase the number of fecal particles of constipation IBS rats. Meanwhile, the fecal particle number of the Group 3-7 in 28 days is found by comparing, and the fecal particle number of the Group4, the Group5 and the Group6 is obviously more than that of the Group2 and the Group7, the difference is extremely obvious (P is less than 0.05), and the statistical significance is achieved.
As can be seen from Table 9, in Group 1-7, the water content of feces of rats in each Group is not greatly different in 24 hours the first day; except for the normal Group, the water content of the feces of the rats in the other groups on the 14 th day is obviously reduced compared with that of the rats on the first day, the difference has statistical significance (P is less than 0.05), and on the 14 th day, the water content of the feces of the groups 2-7 is obviously reduced compared with that of the rats in the normal Group (Group1), and the difference has statistical significance (P is less than 0.05). On day 28, except for Group3, the moisture content of feces from groups 4-7 was significantly increased compared to that on day 14, with statistical significance of the difference (P < 0.05). Meanwhile, the water content of the feces among the 28-day groups 3-7 is compared, and the feces containing water of Group4, Group5 and Group6 is obviously more than that of Group3 and Group7, so that the difference is extremely obvious (P is less than 0.05), and the statistical significance is achieved.
The results show that the bacteroides fragilis capsular polysaccharide A with the molecular weight of 5 KD-70 KD provided by the invention can effectively increase the stool particle number and the stool water content of constipation type IBS rats, and has a good treatment effect on constipation type IBS. Meanwhile, the degradation of the Bacteroides fragilis capsular polysaccharide A is also demonstrated, so that the molecular weight and viscosity of the capsular polysaccharide A are reduced, and the treatment effect on constipation-predominant irritable bowel syndrome can be enhanced.
Meanwhile, experiments prove that the bacteroides fragilis capsular polysaccharide A with the molecular weight of 5 KD-70 KD has better effect on reducing diarrhea index of diarrhea-type IBS than the bacteroides fragilis capsular polysaccharide A with the molecular weight of 2KD or 110 KD.
Example 8 therapeutic Effect of different Bacteroides fragilis strains capsular polysaccharide A on Constipation-type IBS
In this example, capsular polysaccharide A with a molecular weight of 110KD was degraded using the sonication method described in example 1 (sonication conditions: 195kHz, 25 ℃, 0.5 hour), capsular polysaccharide A with a molecular weight of 70KD was collected and assigned as NCTC 9343-70KD, and compared with capsular polysaccharide A with a molecular weight of 70KD extracted from ZY-312 (assigned as ZY-312-70 KD), and the therapeutic effect on constipation-type IBS was evaluated. In this example, referring to the method described in example 7, the change of the number of fecal particles and the change of the moisture content of rat feces were measured, and the specific results are as follows:
TABLE 10 variation of stool particle count for 24h in SD rats: (1)
n＝10)
Note that P < 0.01 for each group of experiments compared to the first day; the ratio of::.0.01 on day 28 and day 14 of each experiment.
TABLE 11 changes in fecal water content in SD rats (
n＝10)
Note that P < 0.01 for each group of experiments compared to the first day; the ratio of::.0.01 on day 28 and day 14 of each experiment.
From the above results, it can be seen that the capsular polysaccharide A with molecular weight of 110KD extracted from the NCTC 9343 strain is degraded, and the therapeutic effect similar to that of the capsular polysaccharide A extracted from ZY-312 strain on constipation IBS can be realized.
The results of the above examples show that the bacteroides fragilis capsular polysaccharide A provided by the invention has good prevention and treatment effects on diarrhea type and constipation type IBS, and has a bidirectional regulation effect.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The application of the bacteroides fragilis in preparing the medicine for preventing and/or treating irritable bowel syndrome is characterized in that the bacteroides fragilis ZY-312 with the preservation number of CGMCC No. 10685.
2. The use according to claim 1, wherein the medicament comprises one or more of the following pharmaceutically acceptable excipients: diluents, excipients, binders, lubricants, suspending agents, coating agents and solubilizers; preferably, the pharmaceutically acceptable excipients are: water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycols, propylene glycols, liposomes, saccharides, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, mono and di fatty acid glycerides, petro (petrochemical) fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone.
3. The use according to claim 1, wherein the bacteroides fragilis can be administered prophylactically or therapeutically alone or in combination with other probiotics and/or probiotic materials; in the case of combined administration, administration may be carried out in a single formulation or in separate formulations, simultaneously or at different times, using the same or different routes of administration.
4. The use according to any one of claims 1 to 3, wherein the irritable bowel syndrome is a diarrhea type, a constipation type or a irritable bowel syndrome caused by hypermotility of the small intestine.
5. The application of the bacteroides fragilis capsular polysaccharide A in preparation of the medicine for preventing and/or treating irritable bowel syndrome is characterized in that the bacteroides fragilis is bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685, and the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5-70 KD.
6. The use according to claim 5, wherein the medicament comprises one or more of the following pharmaceutically acceptable excipients: diluents, excipients, binders, lubricants, suspending agents, coating agents and solubilizers; preferably, the pharmaceutically acceptable excipients are: water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycols, propylene glycols, liposomes, saccharides, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, mono and di fatty acid glycerides, petro (petrochemical) fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone.
7. The use according to claim 5, wherein the medicament is in the form of a pill, tablet, granule, capsule, oral liquid or tube feed.
8. The use according to claim 5, wherein the Bacteroides fragilis capsular polysaccharide A can be administered prophylactically or therapeutically alone, or in combination with other probiotics and/or probiotic materials; in the case of combined administration, administration may be carried out in a single formulation or in separate formulations, simultaneously or at different times, using the same or different routes of administration.
9. The use according to claim 5, wherein the preparation process of Bacteroides fragilis capsular polysaccharide A comprises the following steps:
(1) centrifugally precipitating the bacteroides fragilis bacterial liquid after fermentation culture, collecting a first precipitate, adding water with the temperature of 65-72 ℃ into the first precipitate, adding a phenol solution after dissolving, keeping the temperature of 65-72 ℃, stirring for 25-35min, centrifuging, collecting a first supernatant, wherein the proportion of the water added into the first precipitate, the phenol solution and the first precipitate is 3-5 mL: 3-5 mL: 1g of a compound; the mass concentration of the phenol solution is 70-80%;
(2) extracting the first supernatant collected in the step (1) by using ether to remove phenol, removing residual ether, and collecting an aqueous phase solution;
(3) adding absolute ethanol into the aqueous phase solution collected in the step (2) until the final concentration of the ethanol is 75-85 v/v%, carrying out alcohol precipitation, wherein the alcohol precipitation is carried out for 8-16 hours at the temperature of 0-8 ℃, centrifuging, collecting a second precipitate,
(4) adding water into the second precipitate to prepare a suspension with the mass concentration of 8-12%, adding a glacial acetic acid aqueous solution with the mass concentration of 8-12%, heating to boil, stirring for reaction for 1.5-2.5 hours, adjusting the pH to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract;
(5) degrading the bacteroides fragilis extract obtained in the step (4) by using an ultrasonic method, wherein the ultrasonic condition is as follows: 180 ℃ and 210kHz, 15-25 ℃.
10. The use according to any one of claims 5 to 9, wherein the irritable bowel syndrome is a diarrhea type, a constipation type or a irritable bowel syndrome caused by hypermotility of the small intestine.
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| CN114470003A (en) * | 2022-01-12 | 2022-05-13 | 广州知易生物科技有限公司 | Application of bacteroides fragilis or zwitter-ion capsular polysaccharide thereof in preparation of medicine for preventing and treating digestive system tumor |
| WO2023134204A1 (en) * | 2022-01-12 | 2023-07-20 | 广州知易生物科技有限公司 | Application of capsular polysaccharide a of bacteroides fragilis combined with pd-1 and pd-l1 antibodies in treatment of respiratory system tumors |
| WO2023134194A1 (en) * | 2022-01-12 | 2023-07-20 | 广州知易生物科技有限公司 | Application of bacteroides fragilis capsular polysaccharide a in combination with pd-1 inhibitor in preparation of pharmaceutical for treating skin tumors |
| CN114558036A (en) * | 2022-01-25 | 2022-05-31 | 广州知易生物科技有限公司 | Application of bacteroides fragilis in improvement and treatment of diarrhea |
| WO2023142877A1 (en) * | 2022-01-25 | 2023-08-03 | 广州知易生物科技有限公司 | Use of bacteroides fragilis in improvement and treatment of diarrhea |
| CN116004486A (en) * | 2023-03-24 | 2023-04-25 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Bacteroides fragilis BFS17 for relieving irritable bowel syndrome and intestinal tract hypersensitivity and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113730443B (en) | 2022-10-04 |
| CN115252652A (en) | 2022-11-01 |
| CN109481473A (en) | 2019-03-19 |
| WO2019047439A1 (en) | 2019-03-14 |
| CN109481473B (en) | 2023-03-21 |
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