WO2019109348A1 - 丁酸杆菌(Butyribacter intestini)在预防和/或治疗炎症相关疾病的应用 - Google Patents
丁酸杆菌(Butyribacter intestini)在预防和/或治疗炎症相关疾病的应用 Download PDFInfo
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- WO2019109348A1 WO2019109348A1 PCT/CN2017/115278 CN2017115278W WO2019109348A1 WO 2019109348 A1 WO2019109348 A1 WO 2019109348A1 CN 2017115278 W CN2017115278 W CN 2017115278W WO 2019109348 A1 WO2019109348 A1 WO 2019109348A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Definitions
- the present invention relates to the field of microorganisms, and in particular to the use of Butyribacter intestini for the prevention and/or treatment of inflammation-related diseases.
- IBD Inflammatory bowel disease
- UC ulcerative enteritis
- CD Crohn's disease
- Ulcerative colitis is an important type of inflammatory bowel disease (IBD).
- IBD inflammatory bowel disease
- the cause of the disease is unknown.
- the main lesion is in the submucosal layer of the colonic mucosa. It is a chronic disease. Intestinal disease.
- the causes of ulcerative enteritis include host genetic susceptibility, intestinal flora and intestinal mucosal immune response.
- the clinicopathological manifestations include persistent abdominal pain, diarrhea and mucous bloody stools, and the disease is repeated. The number of patients affected showed a clear upward trend.
- the drugs for ulcerative enteritis mainly include salicylic acid, adrenal glucocorticoids, and immunological preparations.
- Salicylic acid drugs can better inhibit prostaglandin synthesis and scavenge oxygen free radicals, thereby achieving the purpose of relieving inflammation, but it can only be relieved in a short period of time and cannot be cured.
- the most common salicylate western medicine for the treatment of ulcerative enteritis (UC) is sulfasalazine (SASP), which is mainly for patients with mild, moderate and chronic UC.
- Adrenal glucocorticoids are patients with severe or explosive UC.
- Preferred drugs such as betamethasone; immunosuppressants such as cyclosporine can inhibit ulcerative enteritis (UC) by inhibiting the production of T-cell IL-2 and affecting the progression of the immune response.
- the three drugs for ulcerative enteritis can alleviate UC to a certain extent, but there are also some side effects.
- the side effects of salicylate are gastrointestinal reactions, headache, reticulocyte increase, Sperm reduction and allergic reactions caused by rash, hepatotoxicity, leukopenia, anemia, etc., these drugs also have antibacterial effects, easily lead to bacterial flora disorder and increased drug resistance.
- Adrenal glucocorticoids can cause metabolic disorders, water retention and other side effects. They can only be used as emergency medications and cannot be taken for a long time.
- Immunosuppressive therapy is highly dependent on drugs, and has a long treatment period, which is easy to cause nephrotoxicity. Sexual and secondary infections can only be used as a means of adjuvant therapy.
- a first aspect of the invention provides the use of a Butyribacter intestini for the preparation of a composition or formulation for the prevention and/or treatment of an inflammation-related disease.
- the Butyribacter intestini comprises Butyribacter intestini T01-11.
- the composition is selected from the group consisting of a food composition, a health care composition, a pharmaceutical composition, a beverage composition, a feed composition, or a combination thereof.
- the composition is an oral preparation.
- the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.
- the dosage form of the composition is selected from the group consisting of powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, and/or Or sublingual tablets.
- the food composition comprises an emulsion preparation, a solution preparation, a powder preparation, or a suspension preparation.
- the food composition comprises a dairy product, a milk powder, or an emulsion.
- the liquid formulation is selected from the group consisting of a solution preparation or a suspension product.
- the inflammation-related disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, or a combination thereof.
- the inflammation-related disease is selected from the group consisting of ulcerative enteritis, gastritis, common enteritis, or a combination thereof.
- the formulation comprises a microecological formulation.
- a second aspect of the invention provides the use of a Butyribacter intestini for the preparation of a composition or formulation for one or more uses selected from the group consisting of:
- controlling the reduction in body weight of the mammal means that the body weight of the experimental group is reduced by no more than 10%, preferably no more than 5%, more preferably than the mammal of the model group. , no more than 2%.
- the improving intestinal lesions in the mammal comprises slowing the shortening of the length of the colon, and/or reducing the inflammatory response of the colon.
- the mammal comprises a human or a non-human mammal.
- non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
- a third aspect of the invention provides a composition for preventing and/or treating an inflammation-related disease, the composition comprising:
- the Butyribacter intestini comprises Butyribacter intestini T01-11.
- the composition further comprises a growth factor (preferably, a milk growth factor).
- a growth factor preferably, a milk growth factor
- the composition is selected from the group consisting of a food composition, a health care composition, a pharmaceutical composition, a beverage composition, a feed composition, or a combination thereof.
- the composition is an oral preparation.
- the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.
- the dosage form of the composition is selected from the group consisting of powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, sublingual Tablets, or a combination thereof.
- the food composition comprises an emulsion preparation, a solution preparation, a powder preparation, or a suspension preparation.
- the food composition comprises a dairy product, a milk powder, or an emulsion.
- the liquid formulation is selected from the group consisting of a solution preparation or a suspension product.
- the composition contains 1 x 10-1 x 10 15 cfu/mL or cfu/g of Butyribacter intestini, preferably 1 x 10 4 - 1 x 10 10 cfu/ mL or cfu/g of Butyribacter intestini, based on the total volume or total weight of the composition.
- the composition contains from 0.0001 to 99% by weight, preferably from 0.1 to 90% by weight, of the butyric acid (Butyribacter intestini), based on the total weight of the composition.
- the composition is a unit dosage form (one tablet, one capsule or one vial), and the mass of the composition in each unit dosage form is from 0.05 to 5 g, preferably from 0.1 to 1 g.
- composition further contains other probiotics and/or prebiotics.
- the probiotic is selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof.
- the prebiotic is selected from the group consisting of oligofructose (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), and low soybean.
- the composition further contains a substance (e.g., a protective agent) that helps maintain the vitality of Butyribacter intestini.
- a substance e.g., a protective agent
- the substance (such as a protective agent) which contributes to maintaining the activity of Butyribacter intestini is selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, and Butylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
- the weight ratio (wt%) of the substance (such as a protective agent) which contributes to maintaining the activity of Butyribacter intestini is 0.1-2%, preferably 0.5-1.5%, More preferably, from 0.5 to 1.0%, based on the total weight of the composition.
- the content of the substance (such as a protective agent) which contributes to maintaining the activity of Butyribacter intestini is 1 mg to 20 mg, preferably 5 mg to 15 mg, more preferably 1 g of the composition. Good, 5mg-10mg.
- a fourth aspect of the invention provides a composition for preventing and/or treating an inflammatory bowel disease, the composition comprising:
- the inflammatory bowel disease is selected from the group consisting of ulcerative enteritis, gastritis, common enteritis, or a combination thereof.
- a fifth aspect of the invention provides a composition for preventing and/or treating rheumatoid arthritis, the composition comprising:
- a sixth aspect of the invention provides a group according to the third aspect of the invention to the fifth aspect of the invention
- the preparation method of the compound including the steps:
- Butyribacter intestini (i) Butyribacter intestini, (ii) a food or a pharmaceutically acceptable carrier is mixed to form the composition of the third aspect of the invention to the fifth aspect of the invention.
- the method further comprises the step of mixing with a growth factor.
- the process further comprises the step of mixing with a substance (such as a protective agent) that helps maintain the activity of the butyric acid bacteria.
- a substance such as a protective agent
- the composition further contains a substance (e.g., a protective agent) that helps maintain the vitality of Butyribacter intestini.
- a substance e.g., a protective agent
- the substance (such as a protective agent) which contributes to maintaining the activity of Butyribacter intestini is selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, and Butylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
- the method further comprises the step of mixing with a probiotic, and/or a prebiotic.
- the probiotic is selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof.
- the prebiotic is selected from the group consisting of oligofructose (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), and low soybean.
- the growth factor is a milk growth factor.
- the growth factor is selected from the group consisting of a vitamin, an anthraquinone, a pyrimidine, or a combination thereof.
- the composition is an oral preparation.
- a seventh aspect of the invention provides a method of improving intestinal lesions in a mammal, the third aspect of the invention to the composition of the fifth aspect of the invention.
- the administration comprises oral administration.
- the administration dose is 0.01 to 5 g / 50 kg body weight / day, preferably 0.1 to 2 g / 50 kg body weight / day.
- the subject comprises a human or a non-human mammal.
- non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
- the method is non-diagnostic and non-therapeutic.
- An eighth aspect of the invention provides a method of controlling body weight loss in a mammal, and/or reducing a disease activity index in a mammal, the third aspect of the invention to the composition of the fifth aspect of the invention.
- the administration comprises oral administration.
- the administration dose is 0.01 to 5 g / 50 kg body weight / day, preferably 0.1 to 2 g / 50 kg body weight / day.
- the subject comprises a human or a non-human mammal.
- non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
- the method is non-diagnostic and non-therapeutic.
- a ninth aspect of the present invention provides a method of preventing and/or treating an inflammation-related disease, comprising the steps of:
- composition of the third aspect of the invention to the fifth aspect of the invention is administered to the subject, thereby preventing and/or treating an inflammation-related disease.
- the administration comprises oral administration.
- the administration dose is 0.01 to 5 g / 50 kg body weight / day, preferably 0.1 to 2 g / 50 kg body weight / day.
- the subject comprises a human or a non-human mammal.
- non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
- the method is non-diagnostic and non-therapeutic.
- Figure 1 shows the changes in body weight of Control group, model group, VSL # 3 and T01-11 treatment groups.
- Figure 1 shows that the body weight of the mice in the control group generally maintained a slowly increasing trend. The body weight of the mice in the model group induced by DSS gradually decreased. On the 7th day, the body weight of the mice in the model group was the most obvious compared with the control group. Treatment with TF01-11 and VSL # 3 slowed the decline in body weight in UC mice. On day 7, the control of weight loss in TF01-11 and VSL # 3 mice was significant compared to the model group. This indicates that the two groups of probiotics can control the weight loss caused by UC.
- Figure 2 shows the changes in the DAI index of the Control, Model, VSL # 3, and T01-11 treatment groups.
- Figure 2 shows that the DAI of the Control group maintained a positively low level, while the DSS-induced mice gradually increased DAI due to a series of pathologies.
- the DAI of the model group began to change relative to the control group.
- the DAI of the model group mice reached the highest level on the 7th day.
- the intervention of probiotics can control the increase of DAI.
- TF01-11 reached a significant level relative to the model group, and on the 7th day, the DAI of TF01-11 and VSL # 3 groups was determined relative to the model group. Degree of control. From the DAI value on the 7th day, it was found that the DAI of the TF01-11 group was slightly lower than that of VSL # 3, indicating that TF01-11 was slightly better than VSL # 3 in controlling the increase of DAI in UC mice.
- Figure 3 shows the colon length of the Control, Model, VSL #3 and TF01-11 treated mice.
- Figure 3 shows that the colon length of the model group mice was significantly lower than that of the control group on day 7 due to a series of pathologies induced by DSS. Probiotic intervention can control the shortening of colon length, and the colon length of TF01-11 and VSL # 3 mice reached a significant level on day 7 relative to the model group. By the length of the colon on day 7, it was found that the effect of TF01-11 in controlling colon length shortening in UC mice was comparable to that of VSL # 3.
- Butyribacter intestini has prevention and/or treatment of inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), The action of rheumatoid arthritis), the active composition containing the butyric bacteria (Butyribacter intestini) of the present invention was fed to a test subject, and it was found that the composition can control weight loss, reduce disease activity index (DAI), and improve intestinal tract. The lesion can effectively alleviate inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis). On the basis of this, the inventors completed the present invention.
- inflammation-related diseases such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis)
- DAI disease activity index
- the lesion can effectively alleviate inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enter
- the term “contains” means that the various ingredients can be used together in the mixture or composition of the invention. Therefore, the terms “consisting essentially of” and “consisting of” are encompassed by the term “contains.”
- growth factor includes milk growth factors, in particular, nutrients including vitamins, terpenoids, pyrimidines, or combinations thereof.
- the vitamins include, but are not limited to: vitamin C, vitamin E, vitamin A, vitamin A precursor, vitamin B 6 , vitamin D 3 , vitamin K, folic acid, or a combination thereof;
- the terpenoid includes, but is not limited to, a purine nucleoside, wherein the purine nucleoside comprises a 5'-phosphate of a purine nucleoside; the 5'-phosphate of the purine nucleoside is selected from the group consisting of: Inosinic acid (inosine-5'-phosphate; IMP), guanylic acid (guanosine-5'-phosphate; GMP), xanthosine (xanthine-5'-phosphate; XMP), Adenylate (adenosine-5'-phosphate; AMP), or a combination thereof;
- the pyrimidine species include all substances containing a pyrimidine structure.
- controlling the decrease in body weight of a mammal As used herein, the terms “controlling the decrease in body weight of a mammal”, “reducing the decrease in body weight of a mammal”, “controlling the decrease in body weight of a mammal”, “reducing the decrease in body weight of a mammal” are used interchangeably to refer to a mammal in During the construction of the ulcerative enteritis model, the body weight of the experimental animals also decreased due to the continual inflammation, and the percentage of weight loss was the percentage of the weight loss to the original body weight. The higher the degree of weight loss, the more severe the disease, the butyric acid bacteria of the present invention can control the decrease in body weight of the experimental animals and slow down the symptoms of the disease during the treatment of ulcerative enteritis in mammals.
- DAI Disease Activity Index
- disease activity index refers to a combination of three conditions, including weight loss percentage, stool viscosity, and stool bleeding, in a patient (or diseased animal).
- butyric acid bacteria As used herein, the terms “butyric acid bacteria”, “Butyribacter intestini”, “butyric acid bacteria of the invention” are used interchangeably.
- the physiological characteristics of the butyric acid bacteria of the present invention are as follows: after the anaerobic conditions of the butyric acid bacteria on the PYG plate are cultured for 48 hours at 37 ° C, the colonies are grayish white, opaque, smooth, and the edges are irregular like pseudo roots, and the colony diameter is about 2 mm; Langer staining, microscopic observation, TF01-11 is Gram-positive bacteria, long rod-shaped, no spores, flagella, movable, diameter of the cells is about 0.5-1.0 mm, length is about 2.0-8.0 mm; and the invention
- the butyric acid bacteria are catalase-negative bacteria, the temperature growth range is 30-42 ° C, the optimum growth temperature is 37 ° C, the pH tolerance range is 6.0-8.5, and can withstand 2% NaCl. Capable of ferment
- the Butyribacter intestini comprises Butyribacter intestini T01-11.
- the present invention provides the use of Butyribacter intestini for the treatment and/or prevention of inflammation-related diseases such as inflammatory bowel diseases such as ulcerative enteritis, gastritis, common enteritis, and rheumatoid arthritis.
- inflammation-related diseases such as inflammatory bowel diseases such as ulcerative enteritis, gastritis, common enteritis, and rheumatoid arthritis.
- the subject was induced to model with DSS (sodium dextran sulfate), and the strain Butyribacter intestini T01-11 had one or more uses selected from the group consisting of: (i) controlling the decrease in body weight of the subject; (ii) Reduce disease activity index (DAI); (ii i) improve intestinal lesions.
- DAI Reduce disease activity index
- C57bl/6 mice are used as test mice, and induced modeling is performed using DSS (sodium dextran sulfate) to obtain a mouse model of ulcerative enteritis (UC), which is subjected to Butyribacter intestini T01- 11 treated UC model mice, compared with the untreated control group (model group), their weight loss decreased, and various inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common Indicators related to enteritis and rheumatoid arthritis have also been improved, such as improving the degree of intestinal lesions (including slowing the length of the colon, reducing the inflammatory response of the colon, etc.) and reducing the disease activity index (DAI). Therefore, the strain can be used for the prevention and/or treatment of inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis).
- inflammation-related diseases such as inflammatory bowel disease
- the present invention also provides a composition, preferably the composition comprises a food composition, a health care composition, a pharmaceutical composition, a beverage composition, or a feed composition, preferably a pharmaceutical composition.
- the composition comprises an effective amount of butyric acid bacteria, and in a preferred embodiment, the composition further comprises a growth factor (such as milk growth factor).
- the composition further comprises a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof; and/or a prebiotic selected from the group consisting of oligofructose ( FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligosaccharide (SOS), inulin, oligosaccharides, or combinations thereof.
- a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof
- a prebiotic selected from the group consisting of oligofructose ( FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligosacc
- the composition further comprises a substance (such as a protective agent) selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, and the like, which are selected to help maintain the activity of butyric acid bacteria.
- a substance such as a protective agent
- cysteine cysteine
- glutathione glutathione
- butylhydroxyanisole and the like, which are selected to help maintain the activity of butyric acid bacteria.
- the weight ratio of the substance (e.g., protective agent) which contributes to maintaining the activity of butyric acid bacteria is 0.1 to 2%, preferably 0.5 to 1.5%, more preferably 0.5 to 1.0, based on the total weight of the composition. %.
- the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.
- the liquid formulation is selected from the group consisting of a solution product or a suspension product.
- the dosage form of the composition is selected from the group consisting of powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, sublingual Tablet, or a combination thereof.
- composition of the present invention may be administered in the form of an oral liquid, a tablet, an injection, an orally disintegrating tablet, a lyophilized powder preparation or a capsule, preferably an enteric solvent type (e.g., a capsule), in the present invention, if there is no special
- excipients, pharmaceutically acceptable vehicles and carriers used in the present invention are mainly selected according to the characteristics of the suitable bacteria or their metabolites and the specific administration mode required, and are advantageous for the bacteria or its metabolites to be smoothly passed through the stomach.
- the drug is absorbed. These substances can be selected depending on the route of administration.
- compositions of the present invention may further comprise any additional excipients in the excipients typically used in pharmaceutical formulations, for example, to stabilize the composition itself, or to readily disperse or impart a suitable taste.
- inulin fructose, starch, xylooligosaccharide, silica, buffering agents, and flavoring agents are suitable examples.
- the pharmaceutical preparation of the present invention may further comprise an auxiliary active ingredient.
- the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, a perfume, and the like.
- the pharmaceutical composition of the present invention can be produced in a casing formulation by various known methods, so that the active ingredient of the pharmaceutical composition, that is, the microorganism, can smoothly pass through the stomach without being destroyed by gastric acid.
- the microorganism of the present invention can be used in the form of a capsule prepared by a conventional method.
- a standard excipient is mixed with the cold-dried microorganism of the present invention to prepare a pellet, and then the pellet is filled into a gelatin capsule.
- the microorganisms and medicaments of the present invention allow the use of excipients such as liquid gums, celluloses, silicates or mineral oils to prepare suspensions or dispersions, which suspensions or dispersions can be filled into soft gelatin capsules. in.
- the pharmaceutical composition of the present invention can be made into an enteric tablet for oral use.
- casing as used in this application includes coatings which are permitted for use with all conventional drugs which are not degraded by gastric acid but which are sufficiently decomposed in the small intestine and rapidly release the microorganism of the present invention.
- the casing of the present invention is coated at about 16-30 mg per tablet, preferably 16-25 mg, more preferably 16-20 mg.
- the thickness of the casing is 5 to 100 ⁇ m, and the thickness is preferably 20 to 80 ⁇ m.
- the casing component is selected from conventional polymers known to the public.
- Preferred casings of the present invention are cellulose acetate phthalate polymers or trimellitate polymers and copolymers of methacrylic acid (for example, containing 40% or more of methacrylic acid and containing methylcellulose phthalic acid) Preparation of a copolymer of hydroxypropyl acrylate or its ester derivative of methacrylic acid).
- the cellulose acetate phthalate used in the casing of the present invention has a viscosity of about 45 to 90 cp, an acetyl content of 17 to 26%, and a phthalic acid content of 30 to 40%.
- the cellulose acetate trimellitate used in the casing has a viscosity of about 5-21 cp and an acetaminophen content of 17-26%.
- Cellulose acetate trimellitate is produced by Eastman Kodak Company and can be used in the casing material of the present invention.
- the hydroxypropyl methylcellulose phthalate used in the casing of the present invention has a molecular weight of generally 20,000 to 130,000 Daltons, an ideal molecular weight of 80,000 to 100,000 Daltons, and a hydroxypropyl content of 5 to 10%.
- the methoxy group content is 18-24%, and the phthaloyl group content is 21-35%.
- HP50 The hydroxypropylmethylcellulose phthalate used in the casing of the present invention is HP50, which is produced by Shin-Etsu Chemidnl Co. Ltd., Japan. HP50 contains 6-10% hydroxypropyl, 20-24% methoxy, 21-27% propyl and has a molecular weight of 84,000 Daltons.
- Another casing material is HP55, HP55 contains 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxy, 27-35% phthalic acid, and its molecular weight is 78,000 Daltons.
- the casing of the present invention is prepared by spraying a casing solution onto the core using conventional methods. All solvents in the enteric coating process are alcohols (e.g., ethanol), ketones (e.g., acetone), halogenated hydrocarbon compounds (e.g., dichloromethane), or combinations thereof. Softeners such as di-n-butyl phthalate and triacetin are added to the casing solution in a ratio of about 1 part to about 0.05 part or about 0.3 parts of softener.
- the spraying method is preferably carried out continuously, and the amount of the sprayed material can be controlled according to the conditions employed for the coating.
- the spray pressure can be adjusted at will, and in general, the desired result can be obtained at an average pressure of 1-1.5 bar.
- pharmaceutically effective amount is meant an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
- it may be prepared to contain 1 ⁇ 10 -1 ⁇ 10 15 cfu / ml or cfu / g (particularly, may contain 1 ⁇ 10 4 - 1 ⁇ 10 10 cfu / ml or cfu / g; more special A preparation of butyrobacter and/or a metabolite thereof of 1 ⁇ 10 6 - 1 ⁇ 10 10 cfu/ml or cfu/g) may be contained.
- the effective dosage of butyric acid or its metabolites used may vary depending on the mode of administration and the severity of the condition to be treated.
- a dosage form suitable for internal administration comprising about 1 x 10-1 x 10 15 cfu/ml or cfu/g intimately mixed with a solid or liquid pharmaceutically acceptable carrier (preferably, may contain 1 x 10 4 -1) ⁇ 10 10 cfu/ml or cfu/g; more preferably, 1 ⁇ 10 6 - 1 ⁇ 10 10 cfu/ml or cfu/g) of active butyric acid or a fermentatively produced active ingredient may be contained.
- This dosage regimen can be adjusted to provide an optimal therapeutic response. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
- the butyrobacterium or a metabolite thereof can be administered by oral administration or the like.
- Solid carriers include: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid carriers include: medium, polyethylene glycol, nonionic surfactants and edible oils (such as corn oil, peanut oil). And sesame oil), as long as it is suitable for the characteristics of butyric acid or its metabolites and the specific mode of administration required.
- Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavoring agents, coloring agents, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.
- compositions are solid compositions, especially tablets and/or solid filled or liquid filled capsules. Oral administration is preferred.
- composition of the invention is administered to the individual for one or more administrations per day.
- Dosage unit of administration means a dose that is formally separable and suitable for use in humans or all other mammalian individuals.
- Each unit contains a pharmaceutically acceptable carrier and a therapeutically effective amount of a microorganism of the invention.
- the amount administered depends on the patient's body weight and the severity of the inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), the supplementary active components included, and the Changes in microorganisms. Further, if possible, it can be administered separately and can be administered continuously if necessary. Therefore, the amount administered does not limit the invention.
- the "composition" in the present invention means not only a medicine but also a functional food and a health supplement.
- the composition comprises: a beverage, a food, a pharmaceutical, an animal feed, and the like.
- a food composition comprising an effective amount of butyric acid bacteria and/or a metabolite thereof, and a balance of a food acceptable carrier, said food composition
- the dosage form is selected from the group consisting of a solid, a dairy, a solution, a powder, or a suspension.
- the food composition may further comprise a growth factor (such as milk growth factor).
- the composition further comprises a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof; and/or a prebiotic selected from the group consisting of oligofructose ( FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligosaccharide (SOS), inulin, oligosaccharides, or combinations thereof.
- a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof
- a prebiotic selected from the group consisting of oligofructose ( FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligosacc
- the composition further comprises a substance selected from the group consisting of helping to maintain the activity of butyric acid bacteria (eg, Protective agent): cysteine, glutathione, butylhydroxyanisole, dibutylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipid , vitamin C (ascorbic acid), vitamin E, or a combination thereof.
- butyric acid bacteria eg, Protective agent
- composition of the composition is as follows:
- composition of the composition is as follows:
- the microecological preparation is a biological preparation containing probiotics and metabolites or a dietary supplement which can increase probiotics, and can achieve the purpose of improving human health by regulating and maintaining the micro-ecological balance in the intestinal tract. It mainly includes probiotics, prebiotics and synbiotics.
- the microecological preparation comprises (a) a safe and effective amount of butyric acid bacteria and/or a metabolite thereof; and (b) a food acceptable carrier or a pharmaceutically acceptable carrier.
- the formulation further comprises a growth factor (such as milk growth factor, preferably including a vitamin, an anthraquinone, and/or a pyrimidine).
- the formulation further comprises a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof; and/or a prebiotic selected from the group consisting of oligofructose (FOS) ), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligosaccharide (SOS), inulin, oligosaccharides, or combinations thereof.
- a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof
- prebiotic selected from the group consisting of oligofructose (FOS) ), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligo
- the composition further comprises a substance (such as a protective agent) selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, and the like, which are selected to help maintain the activity of butyric acid bacteria.
- a substance such as a protective agent
- cysteine selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, and the like, which are selected to help maintain the activity of butyric acid bacteria.
- a substance such as a protective agent
- cysteine selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, and the like, which are selected to help maintain the activity of butyric acid bacteria.
- butyric acid is cultured in PYG medium (purchased from Huanqi Microbiology Technology Co., Ltd.) at 37 ° C for 48-78 h.
- the method comprises ingesting a pharmaceutical composition, a food composition, a beverage composition, or a combination thereof of the present invention.
- the subject includes mammals such as humans.
- the method comprises: ingesting a pharmaceutical composition, a food composition, or an animal feed of the invention, or a combination thereof.
- the subject is an animal, preferably a mouse, a rabbit.
- the method comprises ingesting a pharmaceutical composition, a food composition, a beverage composition, or a combination thereof of the present invention.
- the subject includes mammals such as humans.
- the method comprises: ingesting a pharmaceutical composition, a food composition, or an animal feed of the invention, or a combination thereof.
- the subject is an animal, preferably a mouse, a rabbit.
- the method comprises ingesting a pharmaceutical composition, a food composition, a beverage composition, or a combination thereof of the present invention.
- the subject includes mammals such as humans.
- the method comprises: ingesting a pharmaceutical composition, a food composition, or an animal feed of the invention, or a combination thereof.
- the subject is an animal, preferably a mouse, a rabbit.
- the butyric acid bacteria of the present invention can significantly improve indicators associated with inflammation-related diseases such as inflammatory bowel diseases (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis (such as controlling weight loss, improvement)
- inflammation-related diseases such as inflammatory bowel diseases (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis (such as controlling weight loss, improvement)
- the degree of intestinal lesions including slowing down the length of the colon, reducing the inflammatory response of the colon, etc.
- DAI disease activity index
- the butyrobacteria of the present invention has a significant alleviating effect on ulcerative enteritis, which is specifically manifested in a significant improvement in the pathological phenotype of mice with ulcerative colitis, specifically controlling the weight loss of the mice and reducing the disease activity of the mice.
- the mouse model selected in this example is: DSS (Dextran Sulfate, Na)-induced ulcerative enteritis mouse model, continued to give mice 0.15% DSS (dextran sulfate sodium, molecular weight 36000 -50000, purchased from MPBIO, USA, 7 days, mouse strain was C57bl/6 mice (purchased from Hubei Medical Experimental Animal Center), 8 weeks old, weighing 20 g ⁇ 2 g, and reared in a SPF-class rat room environment.
- DSS Extran Sulfate, Na
- mice A total of 48 experimental mice were randomly divided into 4 groups, 12 in each group, including:
- control group feeding with normal feed
- TF01-11 treatment group - DSS model each mouse was given 0.2ml TF01-11 (acquired from Shenzhen Huada Life Science Research Institute, the deposit number is CGMCC 10984).
- VSL # 3 treatment group - DSS modeling each mouse was given 0.2ml VSL # 3 per day (purchased from Alfasigma, USA, clinically used for the treatment of probiotics for UC);
- TF01-11 bacterial solution was cultured for 24 hours, and the cells were collected by centrifugation, suspended in PBS, adjusted to a concentration of 10 9 cfu/ml, and each mouse was intragastrically administered with 200 ul of TF01-11 per day.
- VSL # 3 was also suspended in PBS, and the concentration was adjusted to 10 9 cfu/ml, and each mouse was given 200 ul per day.
- TF01-11 and VSL # 3 were administered in the first 3 days of DSS modeling.
- DSS was added to the drinking water of mice, and UC was modeled by free drinking for 7 days.
- the body weight, diet and water intake of the mice were recorded every day.
- the fecal traits and fecal occult blood of the mice were observed, and the disease activity index (DAI) of the mice was calculated on the first day, the third day, the fifth day, and the seventh day, respectively.
- the DAI scoring standards are shown in Table 1.
- the mice were sacrificed. All mice were bled, necked, colonic, photographed, weighed, and the length of the colon was measured. Colon tissue was stored in a -80 ° C refrigerator and paraformaldehyde.
- Stool characteristics in the table normal stool - forming stool; loose stool - paste-like, semi-formed stool that does not adhere to the anus; loose stool - a thin watery stool that can adhere to the anus.
- blood in the stool normal mice have positive blood in the stool; the blood in the naked eye is red or brown; the occult blood is positive in the blood of the naked eye, and the test is performed using tetramethylbenzidine.
- the DAI index is equal to the sum of the three points of weight, stool traits and fecal occult blood.
- mice The body weight changes of mice before and after treatment are shown in Table 2 and Figure 1:
- the colon tissue of the UC model mice changes, as the ulcer tissue and inflammation cause the colon tissue to shorten. After the end of the treatment, the colon length of the mouse measured by anatomy is shown in Table 4 and Figure 3.
- the raw materials are shown in Table 5.
- the raw material ratio is shown in Table 6.
- lactose, yeast powder and peptone are mixed uniformly with purified water, preheated to 60-65 ° C, homogenized at 20 Mpa, sterilized at 90 ° C for 20-30 minutes, cooled to 36-38 ° C, mixed with vitamin C, and connected.
- Butyribacter intestini TF01-11 freeze-dried product and mix it with maltodextrin, protective agent (such as vitamin C, cysteine) and put it into capsules to make a drug containing Butyribacter intestini TF01-11 combination.
- maltodextrin such as vitamin C, cysteine
- Example 4 A method for preparing a medicament for treating an inflammation-related disease such as ulcerative enteritis (UC)
- UC ulcerative enteritis
- pharmaceutical dosage form production 5 volumes (ml) of growth factors and 1 volume (ml) of a protective agent (such as vitamin C, cysteine) added to 100 volumes (ml) of Butyribacter intestini TF01-11 fermentation of bacterial liquid
- a protective agent such as vitamin C, cysteine
- a starch adjuvant such as maltodextrin
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Abstract
本发明提供了一种丁酸杆菌(Butyribacter intestini)在预防和/或治疗炎症相关疾病的应用,具体地,本发明首次发现丁酸杆菌(Butyribacter intestini)具有预防和/或治疗炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)的作用。
Description
本发明涉及微生物领域,具体地,涉及丁酸杆菌(Butyribacter intestini)在预防和/或治疗炎症相关疾病的应用。
炎症性肠道疾病(inflammatory bowel disease,IBD)是一种病因不明的慢性炎症性肠道疾病,该疾病易反复,严重影响患者的生活质量。现代医学认为,引发炎症性肠道疾病(IBD)的因素有遗传、饮食、感染、自身免疫、心理因素以及环境等。炎症性肠道疾病包括溃疡性肠炎(UC)和克罗恩病(CD),均属于炎症相关疾病。
溃疡性肠炎(ulcerative colitis,UC)是炎症性肠道疾病(inflammatory bowel disease,IBD)的一种重要疾病类型,其发病原因不明,主要病变部位是在结肠粘膜的粘膜下层,属于一种慢性的肠道疾病。基于目前的研究,主要认为引发溃疡性肠炎的病因有宿主遗传易感性、肠道菌群以及肠粘膜的免疫反应,临床病理表现为持续腹痛、腹泻和黏液血便,且病情反复,近年来我国UC的患病人数呈明显上升趋势。
目前,临床上针对溃疡性肠炎(UC)的用药主要有水杨酸类、肾上腺糖皮质激素类、免疫制剂。水杨酸类药物可以比较好的抑制***素合成,清除氧自由基,从而达到缓解炎症反应的目的,但也只能短期缓解,无法实现根治。临床上治疗溃疡性肠炎(UC)常见的水杨酸类西药主要是柳氮磺胺吡啶(SASP),主要针对轻度、中度以及慢性UC患者;肾上腺糖皮质激素是重症或者爆发性UC患者的首选用药,比如倍他米松;免疫抑制剂如环孢素可以通过抑制T细胞IL-2的产生,影响免疫反应的进展,从而对溃疡性肠炎(UC)进行抑制。
现有针对溃疡性肠炎(UC)的三类药物均可以一定程度上对UC进行缓解,但是也都存在一定的副作用,水杨酸类的副作用是引发消化道反应、头痛、网织红细胞增多、***减少及过敏反应引起的皮疹、肝毒性、白细胞减少、贫血等,这类药物同时也具有抗菌作用,容易引起菌群紊乱以及耐药性增强。肾上腺糖皮质激素会导致机体代谢紊乱,水潴留等副作用,仅可作为应急用药,不能长期服用。免疫抑制剂治疗对药物依赖性较大,治疗周期长,容易引起肾毒
性及二次感染,只能作为一种辅助治疗的手段。
因此本领域迫切需要开发一种新的,无毒副作用的,用于治疗和/或预防炎症相关疾病的药物。
发明内容
本发明的目的在于提供一种新的,无毒副作用的,用于治疗和/或预防炎症相关疾病的药物。
本发明第一方面提供了一种丁酸杆菌(Butyribacter intestini)的用途,用于制备组合物或制剂,所述组合物或制剂用于预防和/或治疗炎症相关疾病。
在另一优选例中,所述丁酸杆菌(Butyribacter intestini)包括Butyribacter intestini T01-11。
在另一优选例中,所述组合物选自下组:食品组合物、保健组合物、药物组合物、饮料组合物、饲料组合物、或其组合。
在另一优选例中,所述的组合物为口服制剂。
在另一优选例中,所述的组合物为液态制剂、固态制剂、半固态制剂。
在另一优选例中,所述的组合物的剂型选自下组:粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、和/或舌下含片。
在另一优选例中,所述的食品组合物包括乳液制品、溶液制品、粉末制品、或悬浮液制品。
在另一优选例中,所述的食品组合物包括乳品、乳粉、或乳液。
在另一优选例中,所述的液态制剂选自下组:溶液制品或悬浮液制品。
在另一优选例中,所述炎症相关疾病选自下组:炎症性肠道疾病、类风湿性关节炎、或其组合。
在另一优选例中,所述炎症相关疾病选自下组:溃疡性肠炎、胃炎、普通肠炎、或其组合。
在另一优选例中,所述制剂包括微生态制剂。
本发明第二方面提供了一种丁酸杆菌(Butyribacter intestini)的用途,用于制备组合物或制剂,所述组合物或制剂用于选自下组的一种或多种用途:
(i)控制哺乳动物体重的降低;
(ii)降低哺乳动物的疾病活动指数(DAI);
(iii)改善哺乳动物的肠道病变。
在另一优选例中,所述控制哺乳动物体重的降低指与模型组哺乳动物相比,实验组哺乳动物体重的降低幅度为不超过10%,较佳地,不超过5%,更佳地,不超过2%。
在另一优选例中,所述改善哺乳动物的肠道病变包括减缓结肠长度的缩短、和/或减轻结肠炎症反应。
在另一优选例中,所述哺乳动物包括人或非人哺乳动物。
在另一优选例中,所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
本发明第三方面提供了一种用于预防和/或治疗炎症相关疾病的组合物,所述组合物包括:
(a)安全有效量的丁酸杆菌(Butyribacter intestini);和
(b)食品上或药学上可接受的载体。
在另一优选例中,所述丁酸杆菌(Butyribacter intestini)包括Butyribacter intestini T01-11。
在另一优选例中,所述组合物还包括生长因子(较佳地,牛奶生长因子)。
在另一优选例中,所述组合物选自下组:食品组合物、保健组合物、药物组合物、饮料组合物、饲料组合物、或其组合。
在另一优选例中,所述的组合物为口服制剂。
在另一优选例中,所述的组合物为液态制剂、固态制剂、半固态制剂。
在另一优选例中,所述的组合物的剂型选自下组:粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、舌下含片、或其组合。
在另一优选例中,所述的食品组合物包括乳液制品、溶液制品、粉末制品、或悬浮液制品。
在另一优选例中,所述的食品组合物包括乳品、乳粉、或乳液。
在另一优选例中,所述的液态制剂选自下组:溶液制品或悬浮液制品。
在另一优选例中,所述组合物含有1×10-1×1015cfu/mL或cfu/g的丁酸杆菌(Butyribacter intestini),较佳地1×104-1×1010cfu/mL或cfu/g的丁酸杆菌(Butyribacter intestini),按所述组合物的总体积或总重量计。
在另一优选例中,所述的组合物中,含有0.0001-99wt%,较佳地0.1-90wt%所述的丁酸杆菌(Butyribacter intestini),以所述组合物的总重量计。
在另一优选例中,所述的组合物为单元剂型(一片、一粒胶囊或一小瓶),每个单元剂型中所述组合物的质量为0.05-5g,较佳地为0.1-1g。
在另一优选例中,所述的组合物还含有其它益生菌和/或益生元。
在另一优选例中,所述的益生菌选自下组:乳酸菌、双歧杆菌、嗜酸乳杆菌、或其组合。
在另一优选例中,所述的益生元选自下组:低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)、寡聚糖、或其组合。
在另一优选例中,所述组合物还含有有助于保持丁酸杆菌(Butyribacter intestini)活力的物质(如保护剂)。
在另一优选例中,所述有助于保持丁酸杆菌(Butyribacter intestini)活力的物质(如保护剂)选自下组:半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。
在另一优选例中,所述有助于保持丁酸杆菌(Butyribacter intestini)活力的物质(如保护剂)的重量比(wt%)为0.1-2%,较佳地,0.5-1.5%,更佳地,0.5-1.0%,以所述组合物的总重计。
在另一优选例中,以组合物1g计,所述有助于保持丁酸杆菌(Butyribacter intestini)活力的物质(如保护剂)的含量为1mg-20mg,较佳地,5mg-15mg,更佳地,5mg-10mg。
本发明第四方面提供了一种用于预防和/或治疗炎症性肠道疾病的组合物,所述组合物包括:
(a)安全有效量的丁酸杆菌(Butyribacter intestini);和
(b)食品上或药学上可接受的载体。
在另一优选例中,所述炎症性肠道疾病选自下组:溃疡性肠炎、胃炎、普通肠炎、或其组合。
本发明第五方面提供了一种用于预防和/或治疗类风湿性关节炎的组合物,所述组合物包括:
(a)安全有效量的丁酸杆菌(Butyribacter intestini);和
(b)食品上或药学上可接受的载体。
本发明第六方面提供了一种本发明第三方面至本发明第五方面所述的组
合物的制法,包括步骤:
将(i)丁酸杆菌(Butyribacter intestini)、与(ii)食品上或药学上可接受的载体混合,从而形成本发明第三方面至本发明第五方面所述的组合物。
在另一优选例中,所述制法还包括与生长因子混合的步骤。
在另一优选例中,所述制法还包括与有助于保持丁酸杆菌活力的物质(如保护剂)混合的步骤。
在另一优选例中,所述组合物还含有有助于保持丁酸杆菌(Butyribacter intestini)活力的物质(如保护剂)。
在另一优选例中,所述有助于保持丁酸杆菌(Butyribacter intestini)活力的物质(如保护剂)选自下组:半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。
在另一优选例中,所述制法还包括与益生菌、和/或益生元混合的步骤。
在另一优选例中,所述的益生菌选自下组:乳酸菌、双歧杆菌、嗜酸乳杆菌、或其组合。
在另一优选例中,所述的益生元选自下组:低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)、寡聚糖、或其组合。
在另一优选例中,所述生长因子为牛奶生长因子。
在另一优选例中,所述生长因子选自下组:维生素类物质、嘌呤类物质、嘧啶类物质、或其组合。
在另一优选例中,所述的组合物为口服制剂。
本发明第七方面提供了一种改善哺乳动物肠道病变的方法,给所述对象施用本发明第三方面至本发明第五方面所述的组合物。
在另一优选例中,所述的施用包括口服。
在另一优选例中,所述的施用剂量为0.01-5g/50kg体重/天,较佳地,0.1-2g/50kg体重/天。
在另一优选例中,所述的对象包括人或非人哺乳动物。
在另一优选例中,所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
在另一优选例中,所述方法为非诊断性和非治疗性的。
本发明第八方面提供了一种控制哺乳动物体重降低、和/或降低哺乳动物疾病活动指数的方法,给所述对象施用本发明第三方面至本发明第五方面所述的组合物。
在另一优选例中,所述的施用包括口服。
在另一优选例中,所述的施用剂量为0.01-5g/50kg体重/天,较佳地,0.1-2g/50kg体重/天。
在另一优选例中,所述的对象包括人或非人哺乳动物。
在另一优选例中,所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
在另一优选例中,所述方法为非诊断性和非治疗性的。
本发明第九方面提供了一种预防和/或治疗炎症相关疾病的方法,包括步骤:
给所述对象施用本发明第三方面至本发明第五方面所述的组合物,从而预防和/或治疗炎症相关疾病。
在另一优选例中,所述的施用包括口服。
在另一优选例中,所述的施用剂量为0.01-5g/50kg体重/天,较佳地,0.1-2g/50kg体重/天。
在另一优选例中,所述的对象包括人或非人哺乳动物。
在另一优选例中,所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
在另一优选例中,所述方法为非诊断性和非治疗性的。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
图1显示了Control组、模型组、VSL#3和T01-11治疗组小鼠的体重的变化。图1可见control组小鼠的体重基本维持缓慢升高的趋势,DSS诱导的模型组小鼠的体重逐渐下降,第7天,模型组小鼠体重相对于control组最明显。而TF01-11和VSL#3的治疗可以减缓UC小鼠体重的下降,在第7天,TF01-11
和VSL#3小鼠的体重下降的控制相对于模型组比较显著。说明这两组益生菌可以控制UC引起的体重下降情况。通过比较第7天各组的体重数值可以发现TF01-11组小鼠的体重略高于VSL#3,说明TF01-11在控制UC小鼠体重降低的能力略好于VSL#3。
图2显示了Control组、模型组、VSL#3和T01-11治疗组小鼠的DAI指数的变化。图2表明,Control组小鼠的DAI维持一个正恒定的低水平,而DSS诱导的小鼠由于出现一系列病理,导致DAI逐渐升高,第三天模型组小鼠DAI相对于对照组开始变得显著,第7天模型组小鼠DAI达到最高水平。益生菌的干预可以控制DAI的升高,TF01-11在第5天DAI相对于模型组达到了显著水平,并在第7天TF01-11和VSL#3组的DAI相对于模型组得到了一定程度的控制。通过第7天的DAI数值可以发现TF01-11组小鼠的DAI略低于VSL#3,可以说明TF01-11在控制UC小鼠DAI升高的效果略优于VSL#3。
图3显示了Control组、模型组、VSL#3和TF01-11治疗组小鼠的结肠长度。图3表明,由于DSS诱导的小鼠由于出现一系列病理,第7天模型组小鼠结肠长度相对于对照组显著降低。益生菌的干预可以控制结肠长度的缩短,TF01-11和VSL#3组小鼠的结肠长度在第7天相对于模型组达到了显著水平。通过第7天的结肠长度可以发现TF01-11在控制UC小鼠结肠长度缩短的效果与VSL#3相当。
本发明人经过广泛而深入的研究和实验,意外地发现,丁酸杆菌(Butyribacter intestini)具有预防和/或治疗炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)的作用,将含有本发明的丁酸杆菌(Butyribacter intestini)的活性组合物饲喂食实验对象,发现该组合物能够控制体重降低,降低疾病活动指数(DAI),改善肠道病变,可有效减轻炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)等病症。在此基础上本发明人完成了本发明。
如本文所用,术语“含有”表示各种成分可一起应用于本发明的混合物或组合物中。因此,术语“主要由...组成”和“由...组成”包含在术语“含有”中。
如本文所用,术语“生长因子”包括牛奶生长因子,具体地,包括维生素类物质、嘌呤类物质、嘧啶类物质、或其组合的营养物质。
其中,所述维生素类物质包括(但并不限于):维生素C、维生素E、维生素A、维生素A前体、维生素B6、维生素D3、维生素K、叶酸、或其组合;
所述嘌呤类物质包括(但并不限于):嘌呤核苷,其中所述嘌呤核苷包括嘌呤核苷的5’-磷酸酯;所述嘌呤核苷的5’-磷酸酯选自下组:肌苷酸(肌苷-5’-磷酸酯;IMP)、鸟苷酸(鸟苷-5’-磷酸酯;GMP)、黄苷酸(黄嘌呤核苷-5’-磷酸酯;XMP)、腺苷酸(腺苷-5’-磷酸酯;AMP)、或其组合;
所述嘧啶类物质包括所有含嘧啶结构的物质。
如本文所用,术语“控制哺乳动物体重的降低”、“减缓哺乳动物体重的降低”、“控制哺乳动物体重的下降”、“减缓哺乳动物体重的下降”可互换使用,是指哺乳动物在进行溃疡性肠炎模型构建过程中由于炎症的不断严重,实验动物的体重也随之下降,体重下降的百分比为下降体重占原始体重的百分比。体重下降的程度越高,疾病越严重,本发明的丁酸杆菌在对哺乳动物溃疡性肠炎治疗过程中可以控制实验动物体重的降低,减缓疾病的症状。
疾病活动指数(DAI)
如本文所用,术语“疾病活动指数”是指结合患者(或患病动物)的体重下降百分率、大便粘稠度和大便出血等3种情况进行综合评分。
丁酸杆菌及其应用
如本文所用,术语“丁酸杆菌”,“Butyribacter intestini”、“本发明的丁酸杆菌”可互换使用。本发明的丁酸杆菌的生理特性如下:丁酸杆菌在PYG平板上厌氧条件37℃培养48h之后,菌落呈现灰白色,不透明,平滑,边缘不规则似假根状,菌落直径约2mm;通过革兰氏染色、显微观察,TF01-11为革兰氏阳性菌,长杆状,无芽孢,具有鞭毛,可运动,菌体直径约0.5-1.0mm,长度约2.0-8.0mm;并且本发明所述的丁酸杆菌为过氧化氢酶阴性菌,温度生长范围是30-42℃,最适生长温度是37℃,pH值耐受范围是6.0-8.5,能耐受2%的NaCl。能够发酵几种碳水化合物,包括木糖、半乳糖、棉子糖、葡萄糖、
麦芽糖、纤维二糖、蔗糖、淀粉和糖原,主要产生乙酸和丁酸。
在一优选实施方式中,所述丁酸杆菌(Butyribacter intestini)包括Butyribacter intestini T01-11。
本发明提供了丁酸杆菌(Butyribacter intestini)在治疗和/或预防炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)的应用。受试者用DSS(葡聚糖硫酸钠)进行诱导造模,菌株Butyribacter intestini T01-11具有选自下组的一个或多个用途:(i)控制该受试者体重的降低;(ii)降低疾病活动指数(DAI);(ii i)改善肠道病变程度。根据本发明的一个优选例,采用C57bl/6小鼠作为试验小鼠,用DSS(葡聚糖硫酸钠)进行诱导造模,从而获得溃疡性肠炎(UC)模型小鼠,经Butyribacter intestini T01-11治疗的UC模型小鼠,与未接受治疗的对照组(模型组)相比,其体重降低幅度减缓,并且各种与炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)相关的指标也得到改善,如改善肠道病变程度(包括减缓结肠长度缩短、减轻结肠炎症反应等)、降低疾病活动指数(DAI)等。因此,所述菌株能够用以预防和/或治疗炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)。
组合物及其应用
本发明还提供了一种组合物,较佳地,所述组合物包括食品组合物、保健组合物、药物组合物、饮料组合物、或饲料组合物,优选地,为药物组合物。所述组合物包括有效量的丁酸杆菌,在一个优选例中,所述组合物还包括生长因子(如牛奶生长因子)。在一个优选例中,所述组合物还包括选自下组的益生菌:乳酸菌、双歧杆菌、嗜酸乳杆菌、或其组合;和/或选自下组的益生元:低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)、寡聚糖、或其组合。在一个优选例中,所述组合物还包括选自下组的有助于保持丁酸杆菌活力的物质(如保护剂):半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。以组合物的总重计,所述有助于保持丁酸杆菌活力的物质(如保护剂)的重量比为0.1-2%,较佳地,0.5-1.5%,更佳地,0.5-1.0%。
在一优选例中,所述的组合物为液态制剂、固态制剂、半固态制剂。
在一优选例中,所述的液态制剂选自下组:溶液制品或悬浮液制品。
在一优选例中,所述的组合物的剂型选自下组:粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、舌下含片、或其组合。
本发明的组合物可以以口服液、片剂、针剂、口崩片、冻干粉制剂或胶囊的任一种形式给药,优选肠溶剂型(如胶囊),在本发明中,如无特别说明,本发明所用的赋形剂、药物允许的媒介和载体主要是根据适合菌或其代谢产物特性和所需的特定给药方式进行选择,有利于菌或其代谢产物顺利通过胃而被给药者吸收。这些物质可根据给药途径进行选择。
本发明的组合物可进一步包含那些通常用于药物制剂的赋形剂中任何附加的赋形剂,为的是例如稳定组合物自身,或使其容易地分散或赋予其适宜的味道。
在所述赋形剂当中,菊粉、果糖、淀粉、低聚木糖、二氧化硅、缓冲试剂以及风味剂是合适的实例。
本发明中药物制剂可进一步包含辅助的活性组份。
乳糖、麦芽糊精、葡萄糖、蔗糖、山梨糖醇、甘露糖、淀粉、***胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮(PVP)、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁或矿物油等都可用作本发明中药物组合物的载体、赋形剂或稀释剂等。
此外,本发明的药物组合物可进一步包括润滑剂、润湿剂、乳化剂、悬浮液稳定剂、防腐剂、甜味剂和香料等。本发明的药物组合物可通过多种公知的方法以肠衣制剂生产,以便于药物组合物的活性成分即微生物能顺利通过胃而不被胃酸所破坏。
另外,本发明的微生物可以常规方法制备的胶囊形式使用。例如,标准赋形剂和本发明的冷干微生物混合制成小球药丸,然后将药丸装填入明胶胶囊中。此外,本发明的微生物和药物允许使用的赋形剂如液体胶、纤维素、硅酸盐或矿物油等混合制作悬浮液或分散液,这种悬浮液或分散液可装入软的明胶胶囊中。
本发明的药物组合物可制成肠衣片供口服使用。本申请中的术语-“肠衣”,包括所有常规药物允许使用的包衣,这些包衣不被胃酸降解,但在小肠中能充分分解并快速释放出本发明的微生物。本发明的肠衣能在合成胃酸如
pH=1的HCl溶液中在36-38℃维持2小时以上,并优选在合成肠液如pH=7.0的缓冲液中在1.0小时内分解。
本发明的肠衣为以每片约16-30mg进行包衣,较佳地16-25mg,更佳地16-20mg进行包衣。本发明中肠衣厚度为5-100μm,理想的厚度为20-80μm。肠衣成分选自己公开知晓的常规聚合物。
本发明优选的肠衣由纤维素乙酸邻苯二甲酸酯聚合物或偏苯三酸酯聚合物以及异丁烯酸的共聚物(例如,含有40%以上异丁烯酸和含有甲基纤维素邻苯二甲酸羟丙酯或其酯类衍生物的异丁烯酸的共聚物)制备。
本发明中肠衣所使用的纤维素乙酸邻苯二甲酸酯的粘度为约45-90cp,乙酰含量17-26%,邻苯二甲酸含量30-40%。用于肠衣中的纤维素乙酸偏苯三酸酯粘度为约5-21cp,乙酞含量17-26%。纤维素乙酸偏苯三酸酯由Eastman科达公司生产,可用于本发明中的肠衣材料。
用于本发明肠衣中的羟丙基甲基纤维素邻苯二甲酸酯,分子量一般为20,000-130,000道尔顿,理想分子量为80,000-100,000道尔顿,羟丙基含量为5-10%,甲氧基含量为18-24%,邻苯二甲酰基含量为21-35%。
用于本发明肠衣中的羟丙基甲基纤维素邻苯二甲酸酯为HP50,由日本Shin-Etsu Chemidnl Co.Ltd.生产。HP50含有6-10%羟丙基,20-24%甲氧基,21-27%的丙基,其分子量为84,000道尔顿。另一种肠衣物质为HP55,HP55含有5-9%的羟丙基甲基纤维素邻苯二甲酸酯,18-22%甲氧基,27-35%的邻苯二甲酸,其分子量为78,000道尔顿。
本发明肠衣如下制备:使用常规方法将肠衣溶液喷雾到核心上。该肠包衣方法中所有溶剂为醇类(如乙醇)、酮类(如丙酮)、卤代烃化合物(如二氯甲烷)、或其组合物。将软化剂如二-正丁基邻苯二甲酸酯和三乙酸甘油酯加入到肠衣溶液中,其比例为1份包衣物对约0.05份或约0.3份软化剂。喷雾方法优选连续执行,所喷雾的料量可根据包衣所采用的条件进行控制。喷雾压力可随意调节,一般而言,能在平均1-1.5巴压力下获得理想的结果。
说明书中“药物有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。比如,在本发明中,可制备含有1×10-1×1015cfu/ml或cfu/g(特别的,可含有1×104-1×1010cfu/ml或cfu/g;更特别地,可含有1×106-1×1010cfu/ml或cfu/g)的丁酸杆菌和/或其代谢产物的制剂。
当用于制备药物组合物时,所用的丁酸杆菌或其代谢产物的有效剂量可
随施用的模式和待治疗的疾病的严重程度而变化。适用于内服的剂量形式,包含与固态或液态药学上可接受的载体密切混合的约1×10-1×1015cfu/ml或cfu/g(较佳地,可含有1×104-1×1010cfu/ml或cfu/g;更佳地,可含有1×106-1×1010cfu/ml或cfu/g)的活性丁酸杆菌或发酵产生的活性成分。可调节此剂量方案以提供最佳治疗应答。例如,由治疗状况的迫切要求,可每天给予若干次分开的剂量,或将剂量按比例地减少。
所述的丁酸杆菌或其代谢产物可通过口服等途径给予。固态载体包括:淀粉、乳糖、磷酸二钙、微晶纤维素、蔗糖和白陶土,而液态载体包括:培养基、聚乙二醇、非离子型表面活性剂和食用油(如玉米油、花生油和芝麻油),只要适合丁酸杆菌或其代谢产物特性和所需的特定给药方式。在制备药物组合物中通常使用的佐剂也可有利地被包括,例如调味剂、色素、防腐剂和抗氧化剂如维生素E、维生素C、BHT和BHA。
从易于制备和给药的立场看,优选的药物组合物是固态组合物,尤其是片剂和/或固体填充或液体填充的胶囊。口服给药是优选的。
将本发明组合物施用给所述个体,每天给药1次或多次。给药剂量单位表示其形式上能分开且适用于人类或其他所有哺乳动物个体的剂量。每一单位含有药物允许的载体和有效治疗量的本发明微生物。给药量随病人的体重和炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)的严重程度、所包括的补充活性组份和所使用的微生物而变化。此外如可能,可分开给药,并且如需要可连续给药。因此,所述给药量不会对本发明造成限制。此外,本发明中的“组合物”不仅意味着药品而且表示可作为功能性食品和健康补充食品。在一个优选例中,所述组合物包括:饮料、食品、药品、动物饲料等。
在本发明的一个优选例中,还提供了一种食品组合物,它含有有效量的丁酸杆菌和/或其代谢产物,以及余量的食品上可接受的载体,所述的食品组合物的剂型选自固体、乳品、溶液制品、粉末制品、或悬浮液制品。在一优选例中,所述食品组合物还可含有生长因子(如牛奶生长因子)。在一优选例中,所述组合物还包括选自下组的益生菌:乳酸菌、双歧杆菌、嗜酸乳杆菌、或其组合;和/或选自下组的益生元:低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)、寡聚糖、或其组合。在一个优选例中,所述组合物还包括选自下组的有助于保持丁酸杆菌活力的物质(如
保护剂):半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。
在一优选例中,所述组合物的配方如下:
1×10-1×1015cfu/mL的丁酸杆菌和/或其代谢产物;以及食品上或药学上可接受的载体,和/或赋形剂。
在另一优选例中,所述组合物的配方如下:
1×104-1×1010cfu/mL的丁酸杆菌和/或其代谢产物;以及食品上或药学上可接受的载体,和/或赋形剂。
微生态制剂
微生态制剂是一种包含益生菌及代谢产物的生物制剂或者是可以增加益生菌的膳食补充剂,可以通过调节、维持肠道内微生态平衡,达到提高人体健康水平的目的。主要包括益生菌、益生元和合生元。
在本发明中,所述微生态制剂包括(a)安全有效量的丁酸杆菌和/或其代谢产物;以及(b)食品上可接受的载体或药学上可接受的载体。在一优选例中,所述制剂还包括生长因子(如牛奶生长因子,较佳地,包括维生素类物质、嘌呤类物质、和/或嘧啶类物质)。在一个优选例中,所述制剂还包括选自下组的益生菌:乳酸菌、双歧杆菌、嗜酸乳杆菌、或其组合;和/或选自下组的益生元:低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)、寡聚糖、或其组合。在一个优选例中,所述组合物还包括选自下组的有助于保持丁酸杆菌活力的物质(如保护剂):半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。
丁酸杆菌的培养方法
在本发明中,将丁酸杆菌于PYG培养基(购自环凯微生物科技公司)中,37℃厌氧培养48-78h。
改善哺乳动物肠道病变的方法
在另一优选例中,所述方法包括:摄取本发明的药物组合物、食品组合物、饮料组合物、或其组合。所述实验对象包括哺乳动物,如人。
在另一优选例中,所述方法包括:摄取本发明的药物组合物、食品组合物、或动物饲料,或其组合。所述实验对象为动物,较佳地为鼠类,兔类。
控制哺乳动物体重的下降、和/或降低哺乳动物的疾病活动指数(DAI)的方法
在另一优选例中,所述方法包括:摄取本发明的药物组合物、食品组合物、饮料组合物、或其组合。所述实验对象包括哺乳动物,如人。
在另一优选例中,所述方法包括:摄取本发明的药物组合物、食品组合物、或动物饲料,或其组合。所述实验对象为动物,较佳地为鼠类,兔类。
预防和/或治疗炎症相关疾病的方法
在另一优选例中,所述方法包括:摄取本发明的药物组合物、食品组合物、饮料组合物、或其组合。所述实验对象包括哺乳动物,如人。
在另一优选例中,所述方法包括:摄取本发明的药物组合物、食品组合物、或动物饲料,或其组合。所述实验对象为动物,较佳地为鼠类,兔类。
本发明的主要优点包括:
(a)本发明的丁酸杆菌能够显著改善与炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)相关的指标(如控制体重下降、改善肠道病变程度(包括减缓结肠长度缩短、减轻结肠炎症反应等)、降低疾病活动指数(DAI)等)。
(b)本发明的丁酸杆菌对UC小鼠的治疗具体表现为可有效控制DSS诱导的UC小鼠体重的下降,抑制小鼠疾病活动指数(DAI)的升高,改善小鼠结肠的变化。
(c)本发明的丁酸杆菌对溃疡性肠炎有明显的缓解作用,具体表现在能够显著改善溃疡性结肠炎小鼠的病理表型状态,具体为控制小鼠体重下降,降低小鼠疾病活动指数(DAI),并控制UC小鼠结肠缩短。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说
明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照《微生物:实验手册》(James Cappuccino和Natalie Sherman编,Pearson Education出版社)中所述的条件,或按照制造厂商所建议的条件。
如无特别说明,实施例所用的材料和试剂均为市售产品。
实施例1:丁酸杆菌(Butyribacter intestini T01-11)对UC小鼠的治疗
本实施例所选取的小鼠模型为:DSS(硫酸葡聚糖钠Dextran Sulfate,Na)诱导的溃疡性肠炎小鼠模型,续给小鼠饮用0.15%的DSS(葡聚糖硫酸钠,分子量36000-50000,购自美国MPBIO)7天,小鼠品系为C57bl/6小鼠(购自湖北医学实验动物中心),8周龄,体重20g±2g,在SPF级别的鼠房环境中饲养。
试验小鼠总计48只,随机分为4组,每组12只,包括:
正常组(control组)——采用普通饲料进行饲喂;
模型组——DSS造模,每只鼠每天灌胃0.2ml PBS;
TF01-11治疗组——DSS造模,每只鼠每天灌胃0.2ml TF01-11(获自深圳华大生命科学研究院,保藏号为CGMCC 10984)的菌液;
VSL#3治疗组——DSS造模,每只鼠每天灌胃0.2ml VSL#3(购自Alfasigma,USA,临床用于UC的治疗益生菌药物);
TF01-11处理过程为:培养24h的TF01-11菌液,离心收集菌体,用PBS进行悬浮,调整菌浓度至109cfu/ml,每只小鼠每天灌胃200ul的TF01-11。VSL#3也采用PBS进行悬浮,同样调节浓度至109cfu/ml,每只小鼠每天灌胃200ul。
在DSS造模的前3天开始灌胃TF01-11和VSL#3,DSS添加在小鼠饮水中,通过自由饮用进行UC的造模,持续7天,每天记录小鼠体重、饮食和饮水情况,同时观察小鼠的粪便性状及粪便隐血情况,分别在第1天、第3天、第5天和第7天计算小鼠的疾病活动指数(DAI),DAI评分标准详见表1。实验结束后处死小鼠,所有小鼠取血、脱颈、取结肠、拍照、称重、量取结肠长度。结肠组织保存于-80℃冰箱和多聚甲醛中。
表1.DAI指数评分表
表中的大便性状:正常大便-成形大便;松散大便-不粘附于***的糊状、半成型大便;稀便-可粘附于***的稀样水便。其中便血情况:正常小鼠便血为阳性;肉眼血便为红色或褐色;隐血阳性为不明显的肉眼血便,使用四甲基联苯胺进行检测。DAI指数等于体重、大便性状以及大便隐血三个积分之和。
以下,分别通过比较体重下降、DAI和结肠长度来考察TF01-11对DSS诱导的UC模型小鼠的治疗效果。
1、体重变化
治疗前后小鼠的体重变化如下表2和图1所示:
表2
表2的结果显示,Control组小鼠的体重呈缓慢升高的趋势,DSS诱导的3组小鼠的体重均持续下降,在第3天,模型组相对于对照组体重下降开始显著(*P<0.05),第7天,模型组和对照组之间的差异显著程度更加明显(**P<0.01)。而TF01-11和VSL#3的干预可以减缓UC小鼠体重的下降,在第7天,这两组小鼠的体重下降的控制相对于模型组比较显著(▲P<0.05)。说明TF01-11和VSL#3均可以控制UC引起的体重下降情况。在第7天TF01-11组小鼠的体重略高于VSL#3,说明TF01-11在控制UC小鼠体重降低的效果与VSL#3效果相当,甚至略优于VSL#3。
2、DAI的变化
DSS诱导的溃疡性肠炎的小鼠由于体重下降、大便性状和便血情况的变化引起DAI指数的变化,小鼠DAI指数在治疗前后的变化如表3和图2:
表3
表3和图2的数据表明,Control组小鼠的DAI基本持平,而随着DSS诱导,模型组、TF01-11和VSL#3组小鼠的DAI逐渐升高,第三天模型组小鼠DAI相对于对照组变得开始显著(*P<0.05),第7天模型组小鼠DAI达到最高水平(相对于control组**P<0.01)。益生菌的干预可以控制DAI的升高,TF01-11组小鼠在第5天的DAI相对于模型组得到了显著的控制(▲P<0.05),在第7天TF01-11和VSL#3组小鼠的DAI同样显著低于模型组(▲P<0.05),且TF01-11组小鼠的DAI略低于VSL#3,说明TF01-11在控制UC小鼠DAI升高的效果与VSL#3的效果相当,甚至略优于VSL#3。
3、结肠长度的变化
UC模型小鼠的结肠组织会发生改变,因为溃疡和炎症的发生导致结肠组织缩短,治疗结束后,通过解剖测量的小鼠结肠长度见表4和图3。
表4
表4和图3结果显示,在进行DSS诱导7天后的小鼠(模型组)的结肠组织缩
短情况比较严重,与control组相比较非常显著(**P<0.01)。而益生菌TF01-11和VSL#3的干预可以显著控制小鼠结肠的缩短(相对于模型组显著,*P<0.05)。通过表4中数据可以发现,TF01-11在控制UC小鼠结肠长度缩短的效果与VSL#3相当。
实施例2含丁酸杆菌Butyribacter intestini TF01-11的食品组合物
原料配比如表5。
表5
原料 | 质量百分比(%) |
Butyribacter intestini TF01-11 | 0.5 |
牛奶 | 90.0 |
白糖 | 9.0 |
维生素C | 0.5 |
按照上述配方比例混合牛奶、白糖,搅拌至完全混合,预热,20Mpa压力均质,90℃左右杀菌5-10分钟,冷却至40-43℃,混入保护剂维生素C,接种1-100×106cfu/g的Butyribacter intestini TF01-11菌,即制成含Butyribacter intestini TF01-11菌的食品组合物。
实施例3含丁酸杆菌Butyribacter intestini TF01-11的药物组合物
原料配比见表6。
表6
原料 | 质量百分比(%) |
Butyribacter intestini TF01-11 | 1.0% |
乳糖 | 2.0% |
酵母粉 | 2.0% |
蛋白胨 | 1.0% |
纯净水 | 93.5% |
维生素C | 0.5% |
按照比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀,预热到60-65℃,20Mpa压力均质,90℃左右杀菌20-30分钟,冷却至36-38℃,混入维生素C,接入Butyribacter intestini TF01-11活菌(1-50×106cfu/mL),36-38℃发酵至pH值为
6.0,离心,冷冻干燥至水份含量小于3%,即制备Butyribacter intestini TF01-11菌冷冻干燥物。称取0.5克Butyribacter intestini TF01-11冷冻干燥物与麦芽糊精、保护剂(如维生素C、半胱氨酸)等量混合后装入胶囊中,即制成含Butyribacter intestini TF01-11菌的药物组合物。
实施例4一种用于治疗炎症相关疾病(如溃疡性肠炎(UC))的药物的制备方法
1、菌液准备:将Butyribacter intestini TF01-11(1×109cfu/ml)进行厌氧培养,厌氧培养基采用PYG培养基,经过37℃厌氧发酵2-3天。
2、生长因子制备:将脱脂牛奶、酪蛋白进行混合、离心、超滤获得牛奶生长因子粗提物(含有维生素类物质、嘌呤类物质、和/或嘧啶类物质的营养成分)。
3、药物剂型制作:将5体积(ml)生长因子和1体积(ml)的保护剂(如维生素C、半胱氨酸)加入到100体积(ml)的Butyribacter intestini TF01-11发酵的菌液中,充分搅拌混匀,然后加入淀粉辅料(如麦芽糊精),从而制备含Butyribacter intestini TF01-11菌的药物或药物剂型。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (15)
- 一种丁酸杆菌(Butyribacter intestini)的用途,其特征在于,用于制备组合物或制剂,所述组合物或制剂用于预防和/或治疗炎症相关疾病。
- 如权利要求1所述的用途,其特征在于,所述丁酸杆菌(Butyribacter intestini)包括Butyribacter intestini T01-11。
- 如权利要求1所述的用途,其特征在于,所述炎症相关疾病选自下组:炎症性肠道疾病、类风湿性关节炎、或其组合。
- 一种丁酸杆菌(Butyribacter intestini)的用途,其特征在于,用于制备组合物或制剂,所述组合物或制剂用于选自下组的一种或多种用途:(i)控制哺乳动物体重的降低;(ii)降低哺乳动物的疾病活动指数(DAI);(iii)改善哺乳动物的肠道病变。
- 一种用于预防和/或治疗炎症相关疾病的组合物,其特征在于,所述组合物包括:(a)安全有效量的丁酸杆菌(Butyribacter intestini);和(b)食品上或药学上可接受的载体。
- 如权利要求5所述的组合物,其特征在于,所述丁酸杆菌(Butyribacter intestini)包括Butyribacter intestini T01-11。
- 如权利要求5所述的组合物,其特征在于,所述的组合物还含有其它益生菌和/或益生元。
- 如权利要求7所述的组合物,其特征在于,所述的益生菌选自下组:乳酸菌、双歧杆菌、嗜酸乳杆菌、或其组合。
- 如权利要求7所述的组合物,其特征在于,所述的益生元选自下组:低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)、寡聚糖、或其组合。
- 如权利要求5所述的组合物,其特征在于,所述组合物还含有有助于保持丁酸杆菌(Butyribacter intestini)活力的物质。
- 一种权利要求5-10任一项所述组合物的制法,其特征在于,包括步骤:将(i)丁酸杆菌(Butyribacter intestini)、与(ii)食品上或药学上可接 受的载体混合,从而形成权利要求5-10任一项所述的组合物。
- 如权利要求11所述的制法,其特征在于,所述制法还包括与有助于保持丁酸杆菌(Butyribacter intestini)活力的物质混合的步骤。
- 一种改善哺乳动物肠道病变的方法,其特征在于,给所述对象施用权利要求5-10任一项所述的组合物。
- 一种控制哺乳动物体重降低、和/或降低哺乳动物疾病活动指数的方法,其特征在于,给所述对象施用权利要求5-10任一项所述的组合物。
- 一种预防和/或治疗炎症相关疾病的方法,其特征在于,包括步骤:给所述对象施用权利要求5-10任一项所述的组合物,从而预防和/或治疗炎症相关疾病。
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US16/769,011 US11338000B2 (en) | 2017-12-08 | 2017-12-08 | Use of Butyribacter intestini in preventing and/or treating inflammation-related diseases |
CN201780094565.2A CN111093682B (zh) | 2017-12-08 | 2017-12-08 | 丁酸杆菌(Butyribacter intestini)在预防和/或治疗炎症相关疾病的应用 |
DK17934313.2T DK3721889T3 (da) | 2017-12-08 | 2017-12-08 | Butyribacter intestini til anvendelse til forebyggelse og/eller behandling af inflammatorisk tarmsygdom |
EP17934313.2A EP3721889B1 (en) | 2017-12-08 | 2017-12-08 | Butyribacter intestini for use in preventing and/or treating inflammatory bowel disease |
PCT/CN2017/115278 WO2019109348A1 (zh) | 2017-12-08 | 2017-12-08 | 丁酸杆菌(Butyribacter intestini)在预防和/或治疗炎症相关疾病的应用 |
JP2020531065A JP7189954B2 (ja) | 2017-12-08 | 2017-12-08 | 炎症関連疾患の予防及び/又は治療におけるBUTYRlBACTER INTESTINIの使用 |
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CN113304175B (zh) * | 2021-07-08 | 2022-07-19 | 温州医科大学 | 丁酸杆菌在制备预防和/或治疗川崎病药物中的应用 |
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CN105687252A (zh) * | 2016-03-22 | 2016-06-22 | 温州医科大学附属第二医院 | 丁酸杆菌在制备治疗和/或预防急性肝损伤药物中的应用 |
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US20040047868A1 (en) * | 2000-07-03 | 2004-03-11 | Gerald Pang | Treating endotoxemia and related disorders with probiotics |
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US9125935B1 (en) * | 2011-05-31 | 2015-09-08 | Nutech Ventures | Probiotics and methods of obtaining same |
EP2995314A1 (en) * | 2014-09-12 | 2016-03-16 | Swecure AB | Use of collinsella for treatment of inflammatory bowel disease |
CN106479906B (zh) | 2015-08-27 | 2020-08-25 | 深圳华大生命科学研究院 | 肠道细菌丁酸杆菌(Butyribacter intestini)及其应用 |
CN106974262B (zh) * | 2016-01-15 | 2021-05-25 | 深圳华大生命科学研究院 | 肠道益生杆菌在治疗和预防肥胖及其相关疾病中的应用 |
CN106994134B (zh) * | 2016-01-25 | 2020-08-25 | 深圳华大生命科学研究院 | 肠道益生菌在预防和/或治疗糖尿病及其相关疾病中的应用 |
US11376289B2 (en) * | 2018-05-31 | 2022-07-05 | Bgi Shenzhen | Composition and uses thereof |
WO2019227417A1 (zh) | 2018-05-31 | 2019-12-05 | 深圳华大生命科学研究院 | 一种组合物及其应用 |
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CN111093682A (zh) | 2020-05-01 |
DK3721889T3 (da) | 2023-07-03 |
EP3721889A1 (en) | 2020-10-14 |
JP2021505609A (ja) | 2021-02-18 |
US20210085730A1 (en) | 2021-03-25 |
EP3721889B1 (en) | 2023-05-17 |
US11338000B2 (en) | 2022-05-24 |
JP7189954B2 (ja) | 2022-12-14 |
CN111093682B (zh) | 2024-01-16 |
EP3721889A4 (en) | 2021-05-19 |
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