CN109481474A - Application of the bacteroides fragilis in the drug or food of preparation prevention and treatment rhinitis - Google Patents
Application of the bacteroides fragilis in the drug or food of preparation prevention and treatment rhinitis Download PDFInfo
- Publication number
- CN109481474A CN109481474A CN201710814286.0A CN201710814286A CN109481474A CN 109481474 A CN109481474 A CN 109481474A CN 201710814286 A CN201710814286 A CN 201710814286A CN 109481474 A CN109481474 A CN 109481474A
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- CN
- China
- Prior art keywords
- bacteroides fragilis
- capsular polysaccharide
- extract
- rhinitis
- sediment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- A—HUMAN NECESSITIES
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- A23C19/00—Cheese; Cheese preparations; Making thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
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- A—HUMAN NECESSITIES
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- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
- A23G9/36—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
- A23G9/36—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
- A23G9/363—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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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
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- 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
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The present invention relates to the application of bacteroides fragilis or its extract in the drug or food of preparation prevention and treatment rhinitis, contain bacteroides fragilis capsular polysaccharide A in the bacteroides fragilis extract.The inventors found that bacteroides fragilis has the function of preventing/treating rhinitis.And the bacteroides fragilis capsular polysaccharide A that molecular weight is 5~70KD is unexpectedly prepared, has had the function of preferably preventing and treating rhinitis, effect is much better than the capsular polysaccharide A that the molecular weight extracted from bacteroides fragilis NCTC 9343 is 110KD.
Description
Technical field
The present invention relates to the applied technical fields of bacteroides fragilis, more particularly to a kind of bacteroides fragilis or its extract
Application in the drug or food of preparation prevention and treatment rhinitis.
Background technique
Rhinitis (rhinitis) is the inflammatory disease occurred in bronchia mucosal.Rhinitis can be divided into allergic rhinitis (allergy again
Property rhinitis, allergic rhinitis, AR) and anallergic rhinitis (non-allergic rhinitis, non-allergic
rhinitis,NAR)。
Allergic rhinitis (allergic rhinitis, AR) are the most common types in rhinitis.AR illness rate nearly ten years
It obviously increases, about influences the crowd in the whole world 10%~25%, it is especially higher in its national illness rate of industry prosperity.At present
U.S.'s AR illness rate is 5%~22%, is about diagnosed as AR more than 5 million peoples, annual medical expense is up to 55~7,700,000,000
Dollar.One shows that adult's AR illness rate reaches 10%~30% in survey group, youngster in the investigation that 11, Asia country carries out
Child is 10%~46%.The illness rate of China AR also dramatically increases in recent years, may add with economic development, process of industrialization
Speed, lifestyle change are related.Although AR will not threat to life, will affect quality of life, cause personal and society economy
Therefore burden, children can influence school grade, obstructive sleep respiratory disorder occurs in serious person, or even influence Maxillary region hair
It educates.
H and E factor is considered as the etiologic agent of AR.If the crowd of idiocrasy constitution is repeatedly in contact environment
Allergic disease can occur after the allergen of sensitization.In recent years " Hygiene hypothesis " is proposed, if baby is exposed to a variety of micro- lifes early stage
Object and other environment can promote the immune response of I type of body mature, reduce later period idiocrasy and asthma occurs.
Traditionally AR points are seasonal and perennial allergic rhinitis.The former is related with pollen, herding including anemophily
Grass, tree, weeds and fungal spores, the latter Ze Chang are caused by dust mite, animal scurf etc..AR is super quick anti-by I type of IgE mediation
It answers, often shows family's neurological susceptibility.The level for having proven to body generation IgE at present is associated with II class allelotype of HLA.It is a variety of thin
Intracellular cytokine, chemokines, cell adhesion molecule, inflammatory mediator etc. take part in the pathogenic process of AR.RANTES is and allergia
Disease is related, studies a widest chemotactic cytokine.Studies have found that RANTES promoter region (- 403G/A
There are 2 polymorphisms with -28C/G), AR patient RANTES-403A and the -28G allele frequency of occurrences are apparently higher than normal person
Group prompts it to can be used for genome analysis and predicts the generation of AR.Oncostatin M (Oncostatin M, OSM) is IL-6 family
One multifunctional cytokine, having proven to the reconstruction of it and asthma airways before this has important association, nearest one research shows that:
Expression up-regulation of the OSM on AR patient's schneiderian membrane, prompts it to play a role in AR inflammatory reaction injury repair.
AR treatment, which generally comprises, avoids contact with allergen, drug therapy, immunotherapy and operative treatment etc..AR occurs together chronic
Operation is contemplated that when nasal obstruction, concha nasalis inferior adenoid, hypertrophy.First generation antihistamine has central inhibitory action, and clinic does not push away
Recommend use.Antihistamine of new generation such as Desloratadine, levocetirizine, fexofenadine effect is more longlasting, and adverse reaction is few.
And anti-IgE (monoclonal antibody, leukotriene receptor antagonists (Leukotriene receptor antagonists, LTRAs),
The immunotherapy (including allergen immunostimulatory sequence) of improvement, liposome methods, DNA vaccination, anti-IL-4 antibody, anti-IL-5
Antibody, anti-VLA-4 antibody, anti-CCR antibody are the more treatment methods of current clinical application research.
Anallergic rhinitis (non-allergic rhinitis, NAR) is the general name of one group of nosal inflammation disease, by
The clinical symptoms of patient Yu it is changeable and non-specific, therefore be difficult to provide definite definition.Due to lacking specific diagnosis and row
Except standard, the classification of NAR is also multifarious.In general, all types of rhinitis in addition to AR can be included in NAR scope, main point
For idiopathic rhinitis, anallergic rhinitis is with hypereosinophilia syndrome, rhinitis medicamentosa, hormonal rhinitis, the sense of taste
Property rhinitis and coryza professionalis etc..
In treatment NAR, patient can control or change living environment, avoid contact with some irritating factors, such as fragrant
Water, cigarette, smog, cleaning supplies and certain foods etc., occupational exposure can be eliminated by putting on mask and safeguard measure.
But some environmental factors can not change, and the trigger material of such as exposure often not can avoid contact, therefore relevant drug is controlled
It is essential to treat, and drug therapy can be used for the symptom control of NAR, such as intranasal glucocorticoid (Topical Nasal
Steroids)；Antihistamine drug (Antihistamines), such as azelastine；Anticholinergic agents (Anticholinergic
Nasal Spray), such as ipratropium bromide；Nasal douche (Nasal Saline).For deviation of nasal septum, concha plumpness, nose
Rhinitis chronic caused by sinus polyp and the topography obstacle of other forms, can be used surgical operation and is treated.
Rhinitis illness rate obviously increases nearly ten years, seriously affects the quality of life of patient, and studying novel can treat
The active drug of rhinitis is of great significance.
Bacteroides fragilis (Bacteroides fragilis) is the member of Bacteroides in Gram-negative anaerobic bacteria,
Belong to Bacteroidetes, is totally different from Bifidobacterium, lactic acid bacteria of Firmicutes etc..Bacteroides has 25 strains, is only from
The mankind's has 10 strains, and be only from animal has 10 strains, has 5 strains from humans and animals.Bacteroides fragilis is
A kind of obligate anaerobes, according to the difference of culture medium and the differences of growth phase, pleomorphism is presented in thalli morphology, under general condition
Thallus be rod-shaped, both ends blunt circle, color depth, Neutral colour is shallow and uneven, has pod membrane, without brood cell, unpowered, some have vacuole,
Thallus is different in size.According to can synthesize, secrete bacteroides fragilis enterotoxin (BFT) can be classified as produce enterotoxin type fragility intend
Bacillus (Enterotoxigenic Bacteroides fragilis, ETBF) and non-production enterotoxin type bacteroides fragilis
(NontoxigenicBacteroides fragilis, NTBF).Bacteroides fragilis is as people and animal intestinal tract normal flora
A part is primarily present in colon.In addition, respiratory tract, gastrointestinal tract and urogenital mucosa can also be colonized growth.Fragility is quasi-
Bacillus is as a kind of conditioned pathogen, when host mucosal is damaged, can invade submucosa, cause to infect, can also be through blood stream
It is dynamic, cause the other organs of body, such as enteron aisle, abdominal cavity, liver, lung, brain tissue, soft tissue, marrow pyogenic infection and occur together
Abscess.
Summary of the invention
Based on this, the present invention provides a kind of bacteroides fragilis (bacteroides fragilis) and its extract is new
Using.Specific technical solution is as follows:
Application of the bacteroides fragilis in the drug or food of preparation prevention and treatment rhinitis.
Application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment rhinitis, the bacteroides fragilis are extracted
Contain bacteroides fragilis capsular polysaccharide A in object.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5~75KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 15KD~65KD；
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 25KD~55KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 35KD~45KD.
In wherein some embodiments, the content of bacteroides fragilis capsular polysaccharide A is in the bacteroides fragilis extract
60-75wt%.
In wherein some embodiments, the bacteroides fragilis is that the fragility that deposit number is CGMCC No.10685 intends bar
Bacterium ZY-312.
In wherein some embodiments, the preparation method of the bacteroides fragilis extract the following steps are included:
(1) by the bacteroides fragilis bacterium solution centrifugation after fermented and cultured, the first sediment is collected, takes first precipitating
65-72 DEG C of water is added in object, and phenol solution is added after dissolution, keeps 65-72 DEG C of stirring 25-35min, and centrifugation collects first
Supernatant；
(2) the first supernatant collected in step (1) ether is extracted into removal phenol, then removes remaining ether, received
Collect aqueous phase solution；
(3) the final concentration of 75-85v/ of dehydrated alcohol to ethyl alcohol is added in the aqueous phase solution being collected into step (2)
The second sediment is collected in v%, alcohol precipitation, centrifugation；
(4) second sediment is taken, water is added to be configured to suspension, then adjusting pH is 6.5-7.5, centrifugation collects second
Supernatant, desalination of dialysing, is freeze-dried to get the bacteroides fragilis extract.
In wherein some embodiments, the water that is added in first sediment in step (1), the phenol solution with
And the proportion of first sediment is 3-5mL:3-5mL:1g；The mass concentration of the phenol solution is 70-80%.
In wherein some embodiments, step (3) alcohol precipitation be 0-8 DEG C at a temperature of alcohol precipitation 8-16 hours.
In wherein some embodiments, step (4) includes: to take second sediment, adds water to be configured to mass concentration and is
The suspension of 8-12% adds the glacial acetic acid aqueous solution that mass concentration is 8-12%, is heated to boiling, is stirred to react 1.5-2.5
Hour, adjusting pH is 6.5-7.5, and the second supernatant is collected in centrifugation, and desalination of dialysing is freeze-dried to get the bacteroides fragilis
Extract.
In wherein some embodiments, the preparation method of the bacteroides fragilis extract further includes the steps that degradation: will
Bacteroides fragilis extract obtained in step (4) is degraded by the method for ultrasound, the condition of the ultrasound are as follows: 180-
210kHz, 15-25 DEG C.
In wherein some embodiments, the dosage form of the drug includes pill, tablet, granule, capsule, oral solution or pipe
Raise preparation.The drug includes people's medication or animal-use drug, can be used for human or animal.
The bacteroides fragilis or its extract can individually carry out preventative or therapeutic, can also with it is other
Probiotics and/or prebiotic material are administered together.It, can be while or different with single formulation or separated preparation when combination medicine-feeding
When, it is administered using identical or different administration route.
The food includes milk powder, cheese, curdled milk, yoghourt, ice cream or fermented cereal food.The food can be with
It is animal foodstuff, such as feed etc..The food can also be baby food or pet food.
The present invention also provides a kind of drugs or food for preventing and treating rhinitis.Specific technical solution is as follows:
A kind of bacteroides fragilis extract that preventing and treating scorching rhinitis or drug or food contain in the drug or food
Bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide A in the bacteroides fragilis extract.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5~75KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 15KD~65KD；
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 25KD~55KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 35KD~45KD.
In wherein some embodiments, the content of bacteroides fragilis capsular polysaccharide A is in the bacteroides fragilis extract
60-75wt%.
In wherein some embodiments, the bacteroides fragilis is that the fragility that deposit number is CGMCC No.10685 intends bar
Bacterium ZY-312.
In wherein some embodiments, the preparation method of the bacteroides fragilis extract the following steps are included:
(1) by the bacteroides fragilis bacterium solution centrifugation after fermented and cultured, the first sediment is collected, takes first precipitating
65-72 DEG C of water is added in object, and phenol solution is added after dissolution, keeps 65-72 DEG C of stirring 25-35min, and centrifugation collects first
Supernatant；
(2) the first supernatant collected in step (1) ether is extracted into removal phenol, then removes remaining ether, received
Collect aqueous phase solution；
(3) the final concentration of 75-85v/ of dehydrated alcohol to ethyl alcohol is added in the aqueous phase solution being collected into step (2)
The second sediment is collected in v%, alcohol precipitation, centrifugation；
(4) second sediment is taken, water is added to be configured to suspension, then adjusting pH is 6.5-7.5, centrifugation collects second
Supernatant, desalination of dialysing, is freeze-dried to get the bacteroides fragilis extract.
In wherein some embodiments, the water that is added in first sediment in step (1), the phenol solution with
And the proportion of first sediment is 3-5mL:3-5mL:1g；The mass concentration of the phenol solution is 70-80%.
In wherein some embodiments, step (3) alcohol precipitation be 0-8 DEG C at a temperature of alcohol precipitation 8-16 hours.
In wherein some embodiments, step (4) includes: to take second sediment, adds water to be configured to mass concentration and is
The suspension of 8-12% adds the glacial acetic acid aqueous solution that mass concentration is 8-12%, is heated to boiling, is stirred to react 1.5-2.5
Hour, adjusting pH is 6.5-7.5, and the second supernatant is collected in centrifugation, and desalination of dialysing is freeze-dried to get the bacteroides fragilis
Extract.
In wherein some embodiments, the preparation method of the bacteroides fragilis extract further includes the steps that degradation: will
Bacteroides fragilis extract obtained in step (4) is degraded by the method for ultrasound, the condition of the ultrasound are as follows: 180-
210kHz, 15-25 DEG C.
In wherein some embodiments, the dosage form of the drug includes pill, tablet, granule, capsule, oral solution or pipe
Raise preparation.The drug includes people's medication or animal-use drug, can be used for human or animal.
It may include one of following pharmaceutically acceptable auxiliary material or a variety of: diluent, excipient, bonding in the drug
Agent, lubricant, suspending agent, coating agent and solubilizer etc..The example of pharmaceutically acceptable auxiliary material includes: water, salting liquid, alcohol, silicon
Ketone, wax, vaseline, vegetable oil, polyethylene glycol, propylene glycol, liposome, carbohydrate, gelatin, lactose, amylose, magnesium stearate,
Talcum powder, surfactant, silicic acid, viscous paraffin, aromatic oil, mono fatty acid glyceride and difatty acid glyceride, petrochemical industry
(petroethral) aliphatic ester, hydroxymethyl cellulose, polyvinylpyrrolidone etc..
The drug can be administered by following any one or more of mode: with micropump or nasal mist or sucking
Type aerosol etc. carries out inhalation, is administered in the form of suppository or vaginal suppository, with lotion, solution, cream, ointment
Or the form local administration dusted, it is administered by using skin patch, with the tablet of the excipient containing such as starch or lactose
Form or individually or with excipient to be blended in the oral administration of the form in capsule or ovulum, or to contain flavoring agent
Or elixir, solution or the suspended form administration of colorant, or with parenteral injection, such as cavernous body is interior, intravenous, flesh
Interior or subcutaneous administrations.When parenteral injection is administered, the drug is preferably used with sterile aqueous solutions, can be contained
Other materials, such as enough salt or monosaccharide are so that the solution and blood are isotonic.When buccal or sublingual administration, the drug can
It is administered in the form of the tablet or pastille prepared in conventional manner.
The food includes milk powder, cheese, curdled milk, yoghourt, ice cream or fermented cereal food.The food can be with
It is animal foodstuff, such as feed etc..The food can also be baby food or pet food.
Bacteroides fragilis ZY-312 of the invention is preserved in Chinese microorganism strain preservation management on April 2nd, 2015
Committee's common micro-organisms center (CGMCC), deposit number are CGMCC No.10685, and preservation address is Chaoyang District, Beijing City
The institute 3 of North Star West Road 1.
The present inventor accumulates by protracted experience and a large amount of creative experiments researchs, has excavated bacteroides fragilis
New purposes has opened up a new application field.Inventor has found that bacteroides fragilis has the function of preventing/treating rhinitis.
And the preparation method that bacteroides fragilis extract (main component is capsular polysaccharide A) is obtained by lot of experiments, leads to
It crosses further experiment research and finds that bacteroides fragilis capsular polysaccharide A of the invention has effects that significantly to prevent and treat rhinitis.Into one
Step ground, inventor by molecular weight be 70KD bacteroides fragilis capsular polysaccharide A degrade, obtain molecular weight be 5~
The bacteroides fragilis capsular polysaccharide A of 70KD, and it is surprised to find that the capsular polysaccharide A that molecular weight is 5~70KD has preferably
The function of rhinitis is prevented and treated, effect, which is much better than to extract from bacteroides fragilis NCTC 9343, obtains the pod membrane that molecular weight is 110KD
Polysaccharide A.Bacteroides fragilis provided by the invention or its extract are good to the control efficiency of rhinitis and be free from side effects to body, together
When can also be with the Drug combination of other preventing/treating rhinitis.Bacteroides fragilis extract provided by the invention has very
Good edible and prospect in medicine provides the non-defective unit of a kind of health care that suitable human body is taken and prevention and treatment rhinitis for clinic.
Detailed description of the invention
Fig. 1 is the colony characteristics figure of the bacteroides fragilis ZY-312 of embodiment 1；
Fig. 2 is that the bacteroides fragilis ZY-312 of embodiment 1 carries out the micro- sem observation figure after Gram's staining.
Fig. 3 is the 1H spectrum that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 4 is the 13C spectrum that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 5 is the COSY spectrum that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 6 is the hsqc spectrum that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 7 is the HMBC spectrogram that the capsular polysaccharide A nuclear magnetic resonance chemical analyser of embodiment 1 is analyzed；
Fig. 8 is the chemical structural formula for the bacteroides fragilis capsular polysaccharide A that embodiment 1 is prepared.
Specific embodiment
The present invention is described in further details below by specific embodiment, these embodiments are only used to illustrate this hair
It is bright, it does not limit the scope of the invention.
Bacteroides fragilis used in following embodiment is bacteroides fragilis ZY-312 (bacteroides fragilis
ZY-312), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on April 2nd, 2015
(CGMCC), deposit number is CGMCC No.10685, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The preparation of 1 bacteroides fragilis extract of embodiment
(1) fermented and cultured of bacteroides fragilis
By strain streak inoculation in blood plate, Anaerobic culturel 48h.Observe colony morphology characteristic, dyeing property, size, ball
Rod-shaped and distribution situation etc..
Colony characteristics: after bacteroides fragilis ZY-312 cultivates 48h on blood plate, round dimpling, translucent, white is presented
Color, surface be smooth, not haemolysis, colony diameter between 1-3 mm, referring to Fig. 1.
Form under microscope: bacteroides fragilis ZY-312 carries out gram stain microscopy, is gram-negative bacteria, allusion quotation is presented
Rod-shaped, both ends blunt circle and the dense dye of type, not colored part is shaped like vacuole among thallus, referring to fig. 2.
It chooses single bacterium colony to be inoculated in tryptone meat soup progress fermented and cultured 8 hours (temperature is 37 DEG C), gained bacterium
Liquid centrifugation, revolving speed 3000r/min are centrifuged 15min, remove supernatant, collect sediment.
(2) preparation of bacteroides fragilis extract
1) bacteroides fragilis bacterium mud (sediment that above-mentioned steps (1) obtain) 200g is taken, 68 DEG C of ultrapure water 750mL are added,
After dissolution, adding volume fraction is 75% phenol solution 750mL, is uniformly mixed, and keeps 68 DEG C of stirring extraction 30min,
15000g is centrifuged 20min, takes supernatant liquor.
2) supernatant liquor extracts removal phenol with isometric ether (1.5L), collects supernatant liquor, and repetition is extracted to no benzene
Phenol residual.Heating water bath removes ether, collects water phase.
3) water phase 15000g measures volume after being centrifuged 20min, dehydrated alcohol is added, until the final concentration of 80% (volume of ethyl alcohol
Score), overnight (12 hours), 15000g is centrifuged 20min to 4 DEG C of alcohol precipitations, takes precipitating.
4) quality of the precipitating in step 3) is weighed, precipitating is configured to mass concentration by the deionized water that certain volume is added
It for 10% suspension, is uniformly mixed, the glacial acetic acid aqueous solution that mass concentration is 10% is added, is heated to boiling, persistently stir
After mixing reaction 2h, adjusts pH to 7.0,15000g and be centrifuged 20min, collect supernatant.By gained supernatant dialysis desalination, (10KD is saturating
Analyse bag), freeze-drying obtains bacteroides fragilis extract.
5) bacteroides fragilis extract described in 30mg step 4) is weighed, 0.5mLD is dissolved in2O, 1 μ l acetone of addition (1H,
2.22；13C, 30.89) calibration.1H, 13C, COSY, HSQC, HMBC are analyzed using 500MHz Bruker nuclear magnetic resonance chemical analyser
It composes (Fig. 3), the bacteroides fragilis extract that confirmation step 4) is collected is capsular polysaccharide A, and purity is about 70%.Pass through GPC (gel
Permeation chromatography) analysis, the repetitive unit molecular weight of above-mentioned capsular polysaccharide A is 781 as the result is shown, and unit repetition number n value is 89,
Molecular weight is about 70KD, and molecular formula is-[C31N3O20H47]91, chemical structure is shown in Fig. 4.
(3) preparation of different molecular weight size capsular polysaccharide A
By degrading to the capsular polysaccharide A prepared in (2), the biodegrading process includes but is not limited to the present embodiment
Chemical degradation method, physical degradation methods and biological degradation method.For the present embodiment using the method for ultrasound, the ultrasonic method is by pod membrane
In 195kHz, 20 DEG C of condition is handled 3 hours, 2 hours, 0.5 hour and 0 hour Polysaccharide A respectively, collects obtain molecular weight respectively
Size is the capsular polysaccharide A of 2KD, 5KD, 40KD, 70KD.Use the method for (2) from bacteroides fragilis NCTC 9343 (purchased from beauty
State ATCC) in extract obtain molecular weight be 110KD capsular polysaccharide A.
Influence (oral) of the 2 bacteroides fragilis capsular polysaccharide A of embodiment to rat rhinitis models
One, experimental method
In order to verify the bacteroides fragilis extract (main component is capsular polysaccharide A) of the offer of embodiment 1 for preventing/controlling
The effect of rhinitis is treated, the present embodiment selects 60 SD rats to be tested, half male and half female, and every experiment is assigned with one with rat
A unique number.Before being grouped to animal, should be marked on the label of mouse cage project number, kind/strain, gender,
Cage number and animal number.It is grouped at random using BioBook software according to the original body mass of SD rat, is divided into 6 groups, i.e., normal group
(Group1), model group (Group2), pungent a kind of reed mentioned in ancient books particle group (Group3), the bacteroides fragilis capsular polysaccharide A that embodiment 1 provides are low
(Group4), (Group5), high (Group6) dosage group in, every group SD rat 10.The present embodiment is 40KD's with molecular weight
For bacteroides fragilis capsular polysaccharide A.
SD rat rhinitis models building: basic sensitization: OVA and Al (OH)3It is dissolved in physiological saline with 1: 30 ratio, is made
OVA concentration is 0.1%, as sensitization agent solution.Above-mentioned sensitization agent solution 1ml is injected intraperitoneally in Group2~6, one time a day, continuously
10 days.Normal group is with the replacement of same amount of normal saline solution.
Local excitation: interval is after 5 days, and Group2~6 are excited with the normal saline solution collunarium containing 5%OVA, every side nostril
Be added dropwise 50 μ l, the next day 1 time, continuous 5 times.Normal group is replaced with same amount of normal saline.
Started to be administered in the 3rd same day of local excitation, Group3 is pungent a kind of reed mentioned in ancient books particle control group, Group4 is bacteroides fragilis
Capsular polysaccharide A low dose group, Group5 are bacteroides fragilis capsular polysaccharide A middle dose group, and Group6 is bacteroides fragilis pod membrane
Polysaccharide A high dose group, normal group gavage the physiological saline of same isometric(al) with model group.One time a day, continuous 7 days.Specific experiment point
Group and dosage regimen are shown in Table 1:
1 experimental group of table and dosage regimen
Animal behavioral study: after the last administration in 30min, SD rat nasal mucus secretory volume, sneeze is recorded and scratches nose number, is used
Superposition quantization point-score is scored.That is rhiocnesmus: being 0 point without nose is scratched, and slightly scratching nose is 1 point, and frequently scratching nose is 2 points, scratches nose not
It is only 3 points；Sneeze: without sneeze being 0 point in 30min, sneezing 1~3 is 1 point, and 4~10 are 2 points, and > 11 are 3 points；Nose
Tears: no nasal mucus is 0 point, and flowing to prenaris is 1 point, is 2 points beyond prenaris, it is 3 points that tears stream, which is had one's face covered with,.Concrete outcome is shown in Table 2.
Each group utilizes yellow Jackets (yellow Jackets (Sigma company, lot number XS- after last dose 1h
It 20081118-00600) is anaesthetized, vena portae hepatica blood sampling, according to kit (HA ELISA kit, U.S. Rapidbio
Company, lot number 12280517；IgE ELISA kit, Tianjin Chiba Bioisystech Co., Ltd, lot number 000421) specification
Guidance, respectively to the assay of histamine (HA) and IgE in each experimental group rhinitis SD rat blood serum after administration 1h.Specific measurement knot
Fruit such as table 3.
Two, result and analysis
Oral bacteroides fragilis capsular polysaccharide A influences AR rat to the results are shown in Table 2, table 3.
Influence of the 2 bacteroides fragilis capsular polysaccharide A of table to AR rat behavior
Note: compared with model group, * * P < 0.01, * P < 0.05.
As shown in table 2, there are a large amount of clear nasal discharges, frequent sneeze, acutely scratch typical case's AR symptom such as nose in model group rats, and explanation is made
Mould success.Bacteroides fragilis capsular polysaccharide A high, middle dose group symptom integral are significantly lower than model group (P < 0.01), with pungent a kind of reed mentioned in ancient books
Grain control group is suitable；Bacteroides fragilis capsular polysaccharide A low dose group symptom integral is lower than model group (P < 0.05).Illustrate this hair
The bacteroides fragilis capsular polysaccharide A of bright offer can effectively mitigate the symptom of allergic rhinitis, have to allergic rhinitis good
Preventing/treating effect.
Influence of the 3 bacteroides fragilis capsular polysaccharide A of table to HA and IgE content in rhinitis SD rat blood serum
Note: compared with model group, * * P < 0.01, * P < 0.05.
As can be seen from Table 3, HA the and IgE level in model group ARSD rat blood serum is apparently higher than normal group of (P <
0.01)；HA and IgE level (P < lower than model group in bacteroides fragilis capsular polysaccharide A low dose group AR rat blood serum
0.05), there is statistical significance；And the middle and high dosage group of bacteroides fragilis capsular polysaccharide A (Group5, Group6) AR rat serum
HA and IgE level in clear is significantly lower than model group (P < 0.01), and significant difference, has statistical significance.Illustrate this hair
The bacteroides fragilis capsular polysaccharide A of bright offer has good preventing/treating effect to allergic rhinitis.
Influence (collunarium) of the 3 bacteroides fragilis capsular polysaccharide A of embodiment to rat rhinitis models
One, experimental method
The administration route of the present embodiment uses the form of collunarium, and give the preparation of embodiment 1 is 70KD's containing molecular weight
The extract of bacteroides fragilis capsular polysaccharide A.Except the molecular weight of administration route and the bacteroides fragilis capsular polysaccharide A given is different
Outside sample, other steps are same as Example 2.The specific experiment grouping of the present embodiment and dosage regimen are shown in Table 4:
4 experimental group of table and dosage regimen
Two, result and analysis
Bacteroides fragilis capsular polysaccharide A is administered by collunarium approach the results are shown in Table 5, table to the specific observation that AR rat influences
6:
Influence of the 5 bacteroides fragilis capsular polysaccharide A of table to AR rat behavior
Note: compared with model group, * * P < 0.01, * P < 0.05.
As shown in table 5, there are a large amount of clear nasal discharges, frequent sneeze, acutely scratch typical case's AR symptom such as nose in model group rats, and explanation is made
Mould success.After intranasal administration, bacteroides fragilis capsular polysaccharide A high, middle dose group symptom integral are significantly lower than model group (P
< 0.01), it is suitable with pungent a kind of reed mentioned in ancient books particle control group；The symptom integral of bacteroides fragilis capsular polysaccharide A low dose group is lower than model group
(P < 0.05).Illustrate that bacteroides fragilis capsular polysaccharide A provided by the invention can be used for intranasal administration, can effectively mitigate rhinitis
Symptom has good preventing/treating effect to rhinitis.
Influence of the 6 bacteroides fragilis capsular polysaccharide A of table to HA and IgE content in rhinitis SD rat blood serum
Note: compared with model group, * * P < 0.01, * P < 0.05.
As can be seen from Table 6, HA the and IgE level in model group ARSD rat blood serum is apparently higher than normal group of (P <
0.01)；HA and IgE level (P < lower than model group in bacteroides fragilis capsular polysaccharide A low dose group AR rat blood serum
0.05), there is statistical significance；And the middle and high dosage group of bacteroides fragilis capsular polysaccharide A (Group5, Group6) AR rat serum
HA and IgE level in clear is significantly lower than model group (P < 0.01), and significant difference, has statistical significance.Illustrate this hair
The bacteroides fragilis capsular polysaccharide A of bright offer has good preventing/treating effect to allergic rhinitis.
Influence (collunarium) of the bacteroides fragilis capsular polysaccharide A of 4 different molecular weight of embodiment to rat rhinitis models
One, experimental method
The administration route of the present embodiment uses the form of collunarium, gives containing different molecular weight (2KD, 5KD, 40KD, 70KD
And 110KD) bacteroides fragilis capsular polysaccharide A bacteroides fragilis extract, concentration is 0.5mg/mL.2KD,5KD,
The capsular polysaccharide A of 40KD, 70KD and 110KD prepare gained by embodiment 1.Other steps are same as Example 2.The present embodiment
Specific experiment grouping and dosage regimen are shown in Table 7:
7 experimental group of table and dosage regimen
Two, result and analysis
Bacteroides fragilis capsular polysaccharide A is administered by collunarium approach the results are shown in Table 8, table to the specific observation that AR rat influences
9:
Influence of the 8 bacteroides fragilis capsular polysaccharide A of table to AR rat behavior
Note: compared with model group, * * P < 0.01, * P < 0.05.
As shown in table 8, there are a large amount of clear nasal discharges, frequent sneeze, acutely scratch typical case's AR symptom such as nose in model group rats, and explanation is made
Mould success.After intranasal administration, Group4~6 group rat AR symptom integral is significantly lower than model group (Group2) (P <
0.01)；Group4~6 group rat AR symptom integral is lower than Group3, Group7 group (P < 0.05).
Influence of the 9 bacteroides fragilis capsular polysaccharide A of table to HA and IgE content in rhinitis SD rat blood serum
Note: compared with model group, * * P < 0.01, * P < 0.05.
As can be seen from Table 9, HA the and IgE level in model group ARSD rat blood serum is apparently higher than normal group of (P <
0.01)；HA and IgE level in the AR rat blood serum of Group3~6 group is lower than model group (P < 0.05), has statistics meaning
Justice；HA and IgE level in the AR rat blood serum of Group4~6 group is significantly lower than Group3, Group7 group, and significant difference (P
< 0.05), there is statistical significance.
The testing result of the present embodiment illustrates the bacteroides fragilis capsular polysaccharide A of different molecular weight provided by the invention to change
Answering property rhinitis has good preventing/treating effect.The present invention is expected by degrading to bacteroides fragilis capsular polysaccharide A
Less than ground discovery degradation after bacteroides fragilis capsular polysaccharide A (molecular weight 5KD-70KD) to rhinitis have preferably prevention/
Therapeutic effect (see Table 8 for details, the testing result of table 9Group3~7).
Curative effect of the 5 difference bacteroides fragilis bacterial strain capsular polysaccharide A of embodiment to rhinitis
The ultrasonic method that the present embodiment is recorded using embodiment 1 degrades to the capsular polysaccharide A that molecular weight is 110KD
(ultrasound condition: 195kHz, 25 DEG C, 0.5 hour), and the capsular polysaccharide A that molecular weight is 70KD is collected, it is denoted as NCTC 9343-
70KD group, and the capsular polysaccharide A (being denoted as ZY-312-70KD group) for being 70KD with the molecular weight extracted from ZY-312 is carried out
Comparison, evaluates its curative effect to ARSD rat model.The method that the present embodiment reference implementation example 4 is recorded, detects high concentration respectively
(0.5mg/mL) bacteroides fragilis capsular polysaccharide A is to the situation of change of HA and IgE content in AR rat behavior, serum, specifically
As a result as follows:
Curative effect of the 10 difference bacteroides fragilis bacterial strain capsular polysaccharide A of table to rhinitis
It can be seen from the results above that the bacteroides fragilis capsular polysaccharide of NCTC9343-70KD group and ZY-312-70KD group
A will be extracted from NCTC9343 bacterial strain and be divided to the influence indifference of HA and IgE content in AR rat behavior, serum
The capsular polysaccharide A that son amount is 110KD degrades, and may be implemented with the capsular polysaccharide A that extracts from ZY-312 bacterial strain to rhinitis phase
Close therapeutic effect.
By the above experimental result as it can be seen that bacteroides fragilis capsular polysaccharide A provided by the invention can pass through oral, collunarium etc.
Number of ways administration, can effectively mitigate the symptom of rhinitis, while can effectively inhibit the release of HA and IgE in serum.Pass through
It degrades to bacteroides fragilis capsular polysaccharide A, it has been surprisingly discovered that A pairs of the bacteroides fragilis capsular polysaccharide of 5KD~70KD
Rhinitis has better preventing/treating effect.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. application of the bacteroides fragilis in the drug or food of preparation prevention and treatment rhinitis.
2. application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment rhinitis, which is characterized in that the fragility
Contain bacteroides fragilis capsular polysaccharide A in bacteroid extract.
3. application according to claim 2, which is characterized in that the molecular weight of the bacteroides fragilis capsular polysaccharide A be 5~
75KD。
4. application according to claim 3, which is characterized in that the molecular weight of the bacteroides fragilis capsular polysaccharide A is
35KD~45KD.
5. application according to claim 1-4, which is characterized in that the bacteroides fragilis is that deposit number is
The bacteroides fragilis ZY-312 of CGMCCNo.10685.
6. application according to claim 1-4, which is characterized in that the preparation side of the bacteroides fragilis extract
Method the following steps are included:
(1) by the bacteroides fragilis bacterium solution centrifugation after fermented and cultured, the first sediment is collected, first sediment is taken to add
Enter 65-72 DEG C of water, phenol solution is added after dissolution, keep 65-72 DEG C of stirring 25-35min, the first supernatant is collected in centrifugation
Liquid；
(2) the first supernatant collected in step (1) ether is extracted into removal phenol, then removes remaining ether, collect water
Phase solution；
(3) final concentration of 75-85v/v% of the dehydrated alcohol to ethyl alcohol, alcohol are added in the aqueous phase solution being collected into step (2)
Heavy, the second sediment is collected in centrifugation；
(4) second sediment is taken, water is added to be configured to suspension, then adjusting pH is 6.5-7.5, the second supernatant is collected in centrifugation
Liquid, desalination of dialysing, is freeze-dried to get the bacteroides fragilis extract.
7. application according to claim 6, which is characterized in that the water that is added in first sediment in step (1),
The phenol solution and the proportion of first sediment are 3-5mL:3-5mL:1g；The mass concentration of the phenol solution is
70-80%；And/or
Step (3) alcohol precipitation be 0-8 DEG C at a temperature of alcohol precipitation 8-16 hours.
8. application according to claim 6, which is characterized in that step (4) includes: to take second sediment, and water is added to match
The suspension that mass concentration is 8-12% is made, adds the glacial acetic acid aqueous solution that mass concentration is 8-12%, is heated to boiling, stir
Mix reaction 1.5-2.5 hour, adjustings pH be 6.5-7.5, centrifugation, collect the second supernatant, dialysis desalination, be freeze-dried to get
The bacteroides fragilis extract.
9. application according to claim 6, which is characterized in that the preparation method of the bacteroides fragilis extract further includes
The step of degradation: bacteroides fragilis extract obtained in step (4) is degraded by the method for ultrasound, the ultrasound
Condition are as follows: 180-210kHz, 15-25 DEG C.
10. a kind of bacteroides fragilis extract for preventing and treating rhinitis or drug or food, which is characterized in that the drug or food
Contain bacteroides fragilis extract in product, contains bacteroides fragilis capsular polysaccharide A in the bacteroides fragilis extract.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710814286.0A CN109481474A (en) | 2017-09-11 | 2017-09-11 | Application of the bacteroides fragilis in the drug or food of preparation prevention and treatment rhinitis |
| PCT/CN2017/120111 WO2019047438A1 (en) | 2017-09-11 | 2017-12-29 | Application of bacteroides fragilis in preparation of drug or food for preventing rhinitis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710814286.0A CN109481474A (en) | 2017-09-11 | 2017-09-11 | Application of the bacteroides fragilis in the drug or food of preparation prevention and treatment rhinitis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023134212A1 (en) * | 2022-01-12 | 2023-07-20 | 广州知易生物科技有限公司 | New use of bacteroides fragilis and/or zwitterionic capsular polysaccharide of bacteroides fragilis |
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|---|---|---|---|---|
| WO2004089407A2 (en) * | 2003-03-31 | 2004-10-21 | The Brigham And Women's Hospital, Inc. | Zwitterionic immunomodulators for the treatment of asthma and allergy |
| US20140243285A1 (en) * | 2011-07-12 | 2014-08-28 | The Brigham and Women's Hospital, Inc, | Lipid-containing psa compositions, methods of isolation and methods of use thereof |
| CN105434476A (en) * | 2015-10-29 | 2016-03-30 | 广州知易生物科技有限公司 | Application of bacteroides fragilis to prevention and/or treatment of inflammatory bowel diseases (IBDs) |
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2017
- 2017-09-11 CN CN201710814286.0A patent/CN109481474A/en active Pending
- 2017-12-29 WO PCT/CN2017/120111 patent/WO2019047438A1/en active Application Filing
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|---|---|---|---|---|
| WO2004089407A2 (en) * | 2003-03-31 | 2004-10-21 | The Brigham And Women's Hospital, Inc. | Zwitterionic immunomodulators for the treatment of asthma and allergy |
| US20140243285A1 (en) * | 2011-07-12 | 2014-08-28 | The Brigham and Women's Hospital, Inc, | Lipid-containing psa compositions, methods of isolation and methods of use thereof |
| CN105434476A (en) * | 2015-10-29 | 2016-03-30 | 广州知易生物科技有限公司 | Application of bacteroides fragilis to prevention and/or treatment of inflammatory bowel diseases (IBDs) |
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| DENG H: "A novel strain of Bacteroides fragilis enhances phagocytosis and polarises M1 macrophages", 《SCIENTIFIC REPORTS》 * |
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| WO2023134212A1 (en) * | 2022-01-12 | 2023-07-20 | 广州知易生物科技有限公司 | New use of bacteroides fragilis and/or zwitterionic capsular polysaccharide of bacteroides fragilis |
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