CN109954004A - Application of the bacteroides fragilis extract in the composition of preparation prevention and treatment psoriasis - Google Patents
Application of the bacteroides fragilis extract in the composition of preparation prevention and treatment psoriasis Download PDFInfo
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- CN109954004A CN109954004A CN201711342761.5A CN201711342761A CN109954004A CN 109954004 A CN109954004 A CN 109954004A CN 201711342761 A CN201711342761 A CN 201711342761A CN 109954004 A CN109954004 A CN 109954004A
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- Prior art keywords
- bacteroides fragilis
- capsular polysaccharide
- extract
- sediment
- collected
- Prior art date
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Links
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Classifications
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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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The present invention relates to application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment psoriasis, contain bacteroides fragilis capsular polysaccharide A in the bacteroides fragilis extract.The inventors found that the bacteroides fragilis extract containing capsular polysaccharide A has the function of preventing/treating psoriasis.
Description
Technical field
The present invention relates to the applied technical fields of bacteroides fragilis, are making more particularly to a kind of bacteroides fragilis extract
Application in the composition of standby prevention and treatment psoriasis.
Background technique
Psoriasis (Psoriasis) is a kind of autoimmune skin mediated by gene regulation, T lymphocyte
Disease.Its main tissue pathologies change be keratinocyte paraplasm, parakeratosis, hyperkeratinization, angiogenesis and
Inflammatory cell infiltration.Its cause of disease and pathogenesis are not yet completely clear at present, are most common slow also without effective radical cure method
One of inflammatory proliferative skin disorders of property.Its state of an illness is obstinate, easy to recur, whole body system can be caused to damage, and seriously affect patient
Quality of life in addition life cycle, be one of the disease paid close attention to both at home and abroad at present.
Psoriasis is the complicated genetic disease of immune dysfunction, although the mechanism of heredity not yet determines completely.Perhaps
Multi-environment factor plays an important role in the pathogenesis of psoriasis, including drug, skin trauma (Ke Bunei work phenomenon
(Koebner ' s phenomenon)), infection and pressure.The evidence for showing that psoriasis is related to amynologic mechanism includes immunosupress
The effect of medicine such as methotrexate, cyclosporin (CyA), immune target biology agent and immunotoxin (Buddhist nun's interleukin).Therefore, silver-colored
Bits disease is a kind of organ specificity (skin, nail and/or joint) disease that hyperimmune mediates, and wherein local patholoic change makes substrate
Cutin stem cell hyperproliferation simultaneously continues lysis.
Currently, having for psoriasis by whole body or by 2 kinds of medical treatment regimes of part.Serious cutaneous lesions
And/or be distributed outside the skin of disease, as psoriasis arthropathica and nail psoriasis need systemic therapy.Systemic tradition is controlled
Treatment is carried out by using amethopterin, cyclosporin, cortical steroid, retinoids class or fumaric acid esters.Wherein, ammonia
Methopterin, cyclosporin and cortical steroid pass through mostly inhibits immune response to work, and retinoids class and fumaric acid
Esters, which mainly pass through, inhibits the hyperproliferation of the horn cell of psoriatic lesions base portion to work.However, these drugs contain
Certain toxic side effect and offer limited effectiveness.Recently, biological products are had been incorporated into treat psoriasis, including A Laisaipu, according to
Na Xipu and many monoclonal antibodies, i.e. adalimumab, infliximab, excellent spy gram monoclonal antibody (ustekinumab).Compared to biography
The curative effect of the systemic medication of system, this kind of novel drugs is higher, but they need Time of Administration long, and medical expense is very high, and
It may be with serious toxic side effect.
In the case where disease is only located at skin, local treatment is generally selected.Usually using Dithranol, vitamine D3 and its
Analog, including Calcipotriol and calcitriol, tazarotene (it is local retinoids) and local glucocorticoid part
Treat psoriasis.In the case where influencing most of body surface, the use of the product for local treatment is limited.Part
The psoriatic that mild to moderate, clinical diagnosis determines, the body table being related to should be presented in the patient candidate for the treatment of
Face≤10% or Severity Index≤10 measured by Severity Index PASI calculation method.Psoriasis area and PASI are most
It is widely used for the tool of measurement severity of psoriasis.Severity assessment and affected area of the PASI by lesion
Single scoring (Langley RG, the Ellis CN.JAm being combined into 0 (no disease) to 72 (most serious disease) ranges
AcadDermatol 2004；51:563-9).
Either which kind of therapeutic modality, virulent property limit its long-lasting treatment, cause to control for clinical symptoms
The effective period for the treatment of is shorter.Therefore, medical demand unsatisfactory is still had in terms for the treatment of and preventing recurrence.
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.
Bacteroides fragilis can express 8 kinds of different capsular polysaccharides, be denoted as A-H respectively, wherein capsular polysaccharide A
The effect of (polysaccharideA, PSA) determines by its molecular size range, the molecular weight of natural PS A about 110kDa.
Summary of the invention
Based on this, the present invention provides a kind of newly answering for bacteroides fragilis (bacteroides fragilis) extract
With.
Specific technical solution is as follows:
Application of the bacteroides fragilis extract in the drug or food of preparation prevention and treatment psoriasis, the bacteroides fragilis mention
It takes and contains bacteroides fragilis capsular polysaccharide A in object.
The dosage form of the drug includes pill, tablet, granule, capsule, oral solution or tube feed preparation.The drug includes
People's medication or animal-use drug, can be used for human or animal.The food includes milk powder, cheese, curdled milk, yoghourt, ice cream or hair
Ferment cereal preparation.The food can also be animal foodstuff, such as feed etc..The food can also be baby food or pet
Food.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 2~110KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5~95KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5~85KD.
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 40KD~85KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 60KD~85KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 65KD~75KD.
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 solution, then adjusting pH is 6.5-7.5, centrifugation is collected on second
Clear liquid, 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%；And/or
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.
The present invention also provides a kind of bacteroides fragilis extract for preventing and treating psoriasis or drugs or food.
Specific technical solution is as follows:
A kind of bacteroides fragilis extract or drug or food for preventing and treating psoriasis contains in the drug or food
The bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide A in the bacteroides fragilis extract.
The dosage form of the drug includes pill, tablet, granule, capsule, oral solution or tube feed preparation.The drug includes
People's medication or animal-use drug, can be used for human or animal.The bacteroides fragilis capsular polysaccharide A can also combine with other drugs and make
With, other drugs including but not limited to salicylazosulfapyridine Salicylic Acid Formulations (such as Etiasa, mesalazine), cortex
Steroids (such as prednisone, dexamethasone) and/or immunosuppressor (such as imuran).The food includes milk powder, cheese, coagulates
Cream, yoghourt, ice cream or fermented cereal food.The food can also be animal foodstuff, such as feed etc..The food is also
It can be baby food or pet food.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 2~110KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5KD~95KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 5~85KD.
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 40KD~85KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 60KD~85KD.
In wherein some embodiments, the molecular weight of the bacteroides fragilis capsular polysaccharide A is 65KD~75KD.
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 solution, then adjusting pH is 6.5-7.5, centrifugation is collected on second
Clear liquid, 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.
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 obtains bacteroides fragilis extract by lot of experiments
(main component is capsular polysaccharide A), and it has been surprisingly discovered that the bacteroides fragilis extract containing capsular polysaccharide A has very
The effect of preventing and treating psoriasis well.
Further, inventor obtains a series of different moleculars by degrading to bacteroides fragilis capsular polysaccharide A
The bacteroides fragilis capsular polysaccharide A of amount.And it is surprised to find that molecular weight is 5~95KD (outstanding by further experimental study
It is 70KD) capsular polysaccharide A have effects that preferably to prevent and treat psoriasis.
Bacteroides fragilis capsular polysaccharide A provided by the invention is good to the control efficiency of psoriasis and makees to body without secondary
With, while can also be with the Drug combination of other preventing/treating psoriasis.Bacteroides fragilis pod membrane provided by the invention is more
Sugared A has edible well and prospect in medicine, provides a kind of the good of the preventing/treating psoriasis that suitable human body is taken to be clinical
Product.
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；
Fig. 9 is that the HE section staining of embodiment 2 detects skin histology's result of variations figure.
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
The preparation of bacteroides fragilis extract, comprising the following steps:
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, obtains supernatant liquid.
2) supernatant liquid extracts removal phenol with isometric ether (1.5L), collects supernatant liquid, 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 obtained in step 3) is weighed, precipitating is configured to quality by the deionized water that certain volume is added
The solution that concentration is 10%, is uniformly mixed, and the glacial acetic acid aqueous solution that mass concentration is 10% is added, is heated to boiling, continues
After being stirred to react 2h, adjusts pH to 7.0,15000g and be centrifuged 20min, collect precipitating.By gained supernatant dialysis desalination, (10KD is saturating
Analyse bag), freeze-drying obtains bacteroides fragilis extract.
5) weigh bacteroides fragilis extract described in 30mg step 4), be dissolved in 0.5mL D2O, be added 1 μ l acetone (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-Fig. 7), 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, unit repetition number n value
It is 89, molecular weight is about 70KD, and molecular formula is-[C31N3O20H47]91, chemical structure is shown in Fig. 8.
(3) preparation of different molecular weight size capsular polysaccharide A
By degrading to the capsular polysaccharide 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.The present embodiment obtains different molecular weight size using the method for ultrasound
Capsular polysaccharide A, the ultrasonic method are by capsular polysaccharide A in 195kHz, and 20 DEG C of condition handles 3 hours, 2 hours, 0.5 respectively
Hour and 0 hour collect obtain the capsular polysaccharide A that molecular size range is 2KD, 5KD, 40KD, 70KD respectively.
(4) molecular weight is the preparation of 110KD capsular polysaccharide A
It uses the method for (2) to extract from bacteroides fragilis NCTC 9343 (being purchased from U.S. ATCC) to obtain.
The curative effect for the mouse psoriasis that 2 various dose bacteroides fragilis capsular polysaccharide A of embodiment induces imiquimod
One, experimental design
Then the present embodiment is made by 5% imiquimod induction Female BALB/c mouse psoriasis model using embodiment 1
Standby obtained bacteroides fragilis extract (main component is capsular polysaccharide A) is to 5% imiquimod induction Female BALB/c mouse
Psoriasis is treated, its therapeutic effect is detected.The present embodiment by taking molecular weight is the capsular polysaccharide A of 70KD as an example, detect it is low,
Therapeutic effect of the capsular polysaccharide A of middle and high dosage to psoriasis.
Mouse psoriasis abductive approach: 60 female BAl BIcs of the present embodiment/c mouse, every laboratory mice are assigned with
One unique number.Before being grouped to animal, project number, kind/strain, property should be marked on the label of mouse cage
Not, cage number and animal number.6 groups, i.e. Normal group the (the 1st are randomly divided into according to the original body mass of mouse using BioBook software
Group), model group (the 2nd group), positive controls (the 3rd group), bacteroides fragilis capsular polysaccharide A low dose group (the 4th group), middle dosage
Group (the 5th group), high dose group (the 6th group), every group 10.
6 groups of back of mices are lost hair or feathers, area about 2cm × 1.5cm, the 0th day starts, and removes the 1st group of (Normal group) mouse
Distilled water about 0.5ml is smeared at back without hair-fields daily, and the 2nd group (model group) to the 6th group back of mice is coated with 5% without hair-fields daily
Imiquimod, 60mg/ days (active drug 3mg/d), continuous 6 days.6th day, the 1st group of (Normal group) back of mice was hairless
Area's skin without the obvious erythema scales of skin that peel off, and the 2nd group (model group) to the 6th group back of mice without hair-fields after the processing of 5% imiquimod,
Skin of back gradually appears erythema, and grows tackness silvery white scale, illustrates the present embodiment modeling success.
Since the 7th day, the 1st, the 2nd group of mouse smeared distilled water daily, and positive controls (the 3rd group) are coated with daily
0.5% Dithranol, the bacteroides fragilis pod membrane that the 4th~6 group of molecular weight for being coated with basic, normal, high dosage respectively daily is 70KD are more
Sugared A.
Specific experiment grouping and dosage regimen are as shown in table 1:
1 experimental group of table and dosage regimen
Two, sample collection
Mouse is put to death with cervical dislocation after experiment.Skin histology biopsy acquires back skin lesion tissue, average mark
It is 3 parts, wherein two parts are immediately placed on -80 DEG C of refrigerators and save backup, the 10% formalin fixer that portion investment prepares in advance
Middle fixation.
Three, observation index and judgement
1, appearance
Bearing Mice Life sign is examined in therapeutic process, the daily same time photographs to record the erythema scales of skin that peel off at skin lesion and changes
Situation.
2, HE section staining detection skin histology variation
HE stained slice is organized at production mouse skin lesion, observes the variation of inflammatory cell infiltration number and epidermal thickness.Every
Slice is observed under microscope × 200 times, and 3 visuals field is taken to take pictures, and inflammatory cell leaching is calculated after amplifying using image processing software
Moisten number, be averaged record, unit is a/HP；Image processing software, using basement membrane zone as boundary, cropping vertical table are used simultaneously
Surface product is simultaneously converted into numerical value, takes the average value in 3 visuals field to record, unit 4/9in2/HP
3, statistics and analysis
Calculate separately the infiltrating cells number (a/HP) and longitudinal epidermis side product (4/9in of each group mouse2/ HP), with mean value ±
Standard false statistic.Using Graphpad Prism, SPSS or Sigmaplot software is for statistical analysis.Specific data are to scheme
The form of table is presented.P < 0.05 is considered to have statistical difference.
Three, result and analysis
Infiltrating cells number (a/HP) and longitudinal epidermis side product using means standard deviation (Mean ± SD) statistics mouse
(4/9in2/HP).And multiple analysis is carried out with ANOVA combination Dunnett ' s.As P < 0.05, then it is assumed that statistically have
Difference.The result that specific testing result is shown in Table 2, HE slice dyeing is shown in Fig. 9.
2 each group infiltrating cells number of table and longitudinal epidermis side product are relatively (Mean ± SD)
In the present embodiment, positive drug (0.5% Dithranol) reduces the mouse psoriasis clinic of 5% imiquimod induction
Symptom, including infiltrating cells number and longitudinal epidermis side product, compared with model group, all have statistical difference.
The molecular weight that embodiment 1 provides is that the bacteroides fragilis capsular polysaccharide A high dose group of 70KD is shown significantly
The drug effect for the mouse psoriasis for inhibiting 5% imiquimod to induce, showing its significantly reduces infiltrating cells number and vertical table
Surface product, compared with model group, the above index all has statistical difference (P < 0.01), and see Table 2 for details.
In the present embodiment, by comparing 6 groups of laboratory test results it can be found that basic, normal, high dosage provided by the invention
Bacteroides fragilis capsular polysaccharide A can be effectively reduced 5% imiquimod induction mouse psoriasis clinical symptoms (see Table 2 for details,
Fig. 9).
As it can be seen that bacteroides fragilis capsular polysaccharide A provided by the invention has the mouse psoriasis that 5% imiquimod induces
Good therapeutic effect, and any toxic side effect is not shown.
Curative effect of the bacteroides fragilis capsular polysaccharide A of 3 different molecular weight of embodiment to psoriasis
The method of the present embodiment reference implementation example 2 prepares mouse Animal Models of Psoriasis, then real using the present invention respectively
The bacteroides fragilis capsular polysaccharide A for applying 2KD, 5KD, 40KD, 70KD, 110KD that example 1 is prepared induces 5% imiquimod
Mouse psoriasis carry out preventing/treating, detect the bacteroides fragilis capsular polysaccharide A of different molecular weight and the treatment of psoriasis imitated
Fruit.The present embodiment is by taking the bacteroides fragilis capsular polysaccharide A of 2KD, 5KD, 40KD, 70KD and 110KD of high dose as an example.
Referring to embodiment 2 experimental group group technology, by mouse be divided into Normal group, model group, 2KD, 5KD group,
40KD group, 70KD group and 110KD group.
Dosage regimen is referring to embodiment 2.
Specific experiment grouping and dosage regimen are as follows:
3 experimental group of table and dosage regimen
The method detection each group infiltrating cells number of reference implementation example 2 and longitudinal epidermis side product compare, concrete outcome such as 4 institute of table
Show:
4 each group infiltrating cells number of table and longitudinal epidermis side product are relatively (Mean ± SD)
Molecular weight provided by the invention is the bacteroides fragilis capsular polysaccharide A of 5KD, 40KD, 70KD as can be seen from the above table
The drug effect for showing the significant mouse psoriasis for inhibiting the induction of 5% imiquimod, is in particular in that its is reduced significantly
Infiltrating cells number caused by psoriasis and longitudinal epidermis side product, see Table 4 for details.Compared with 2KD group and 110KD group, 5KD, 40KD,
The capsular polysaccharide A of 70KD causes infiltrating cells number and longitudinal epidermis side product to show better activity, P < in reduction psoriasis
0.05, statistical difference is significant.
Curative effect of the 4 different strains bacteroides fragilis capsular polysaccharide A of embodiment to psoriasis
The biodegrading process that the present embodiment is recorded using embodiment 1 extracts the pod that resulting molecular weight is 110KD to embodiment 1
Film Polysaccharide A is degraded, and collects the capsular polysaccharide A that molecular weight is 70KD, is denoted as NCTC9343-70KD group, and with from ZY-
The molecular weight extracted in 312 is that the capsular polysaccharide A (being denoted as ZY-312-70KD group) of 70KD is compared, and evaluates high dose
The curative effect of NCTC 9343-70KD, ZY-312-70KD to psoriasis.The method that the present embodiment reference implementation example 3 is recorded, is examined respectively
It surveys infiltrating cells number and longitudinal epidermis side product, concrete outcome is as shown in table 5:
5 infiltrating cells number of table and longitudinal epidermis side product comparison
It can be seen from the results above that obtaining the capsular polysaccharide that molecular weight is 110KD for extracting from 9343 bacterial strain of NCTC
A degrades, and therapeutic effect of the capsular polysaccharide A to psoriasis of ZY-312 bacterial strain extraction may be implemented.
The clinical trial result of 5 bacteroides fragilis capsular polysaccharide A of embodiment
The present embodiment is evaluated to assess the effect of bacteroides fragilis capsular polysaccharide A is to psoriatic by PASI, together
When also evaluate bacteroides fragilis capsular polysaccharide A safety.The present embodiment selects 3 clinical diagnosises to suffer from psoriasis at least 3 years
Patient studies.Patient does not take and controls with topical corticosteroid class or whole body before and during Capsular polysaccharides A is treated
The combination therapy for the treatment of.The body surface area being related at baseline is value≤10≤10% and PASI.
Situation is as follows before patient medication:
Patient 1, male, 39 years old, more than 10 years of illness, both feet it is the most serious, upper body and on hand relatively slightly.It is inclined to have used Chinese medicine
Square oral administration and externally (specific medicine name is unknown) energy Iterim Change symptom (as normal skin) is still easy recurrence, it is believed that
No effect.Nearly 5-6 years, drug therapy is rarely employed, loses confidence.According to reflection, the every winter exacerbation of symptoms of patient, weather becomes
Exacerbation of symptoms is obvious when change, and summer can fully recover as normal skin.Currently, both legs and both feet are serious, redness continues, when and
Itch very much, pain.
Patient 2, male, and 66 years old, in more than 10 years of illness, lesion is mainly distributed to be mainly shown as over 10 years with hand, foot, head
Itch, white bits, new and old lesion is simultaneously deposited, 10 Yu Nianlai, again and again.
Patient 3, and male, 68 years old, illness 4 years, lesion was distributed mainly on four limbs, cardinal symptom: it is red and swollen, itch, white bits；Four limbs are red
Swollen area is big, and the new old tinea of tinea is distributed.
The molecular weight for the high dose that the above patient uses embodiment 1 to prepare daily is the bacteroides fragilis capsular polysaccharide of 70KD
A is uniformly applied to affected part.(baseline), the PASI scoring after medication 6 weeks, after medication 12 weeks before the record trial period respectively.Specific inspection
The results are shown in Table 6 for survey:
6 PASI of table evaluation
Average value of the PASI at baseline is 5.43 (standard deviation=0.74)；After medication 6 weeks, the average value of PASI is
3.87 (standard deviation=0.29), and the average value of PASI is 2.30 (standard deviation=0.43) after medication 12 weeks.
Above-mentioned testing result show molecular weight provided by the invention be 70KD bacteroides fragilis capsular polysaccharide A in medication
In contrast to the statistically significant reduction of the PASI value of (baseline) before medication after 12 weeks (Student t examines P < 0.05).
In addition to the reduction of PASI value, all patients indicate that the clinical symptoms of disease are greatly improved, and treat resistance to
It is fine by property, without any toxic side effect.
In short, can effectively improve PASI value to the treatment of psoriasis using bacteroides fragilis capsular polysaccharide A, this prompt is in silver
Considering to be worth doing in the treatment of disease can be used bacteroides fragilis capsular polysaccharide A, the bacteroides fragilis for being 5~70KD especially with molecular weight
Capsular polysaccharide A.
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 extract in the drug or food of preparation prevention and treatment psoriasis, which is characterized in that described crisp
Contain bacteroides fragilis capsular polysaccharide A in weak bacteroid extract.
2. application according to claim 1, which is characterized in that the molecular weight of the bacteroides fragilis capsular polysaccharide A be 2~
110KD。
3. application according to claim 2, which is characterized in that the molecular weight of the bacteroides fragilis capsular polysaccharide A be 5~
95KD。
4. application according to claim 3, which is characterized in that the molecular weight of the bacteroides fragilis capsular polysaccharide A is
60KD~85KD.
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 CGMCC No.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 solution, then adjusting pH is 6.5-7.5, the second supernatant is collected in centrifugation,
Dialysis desalination, 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 or 7, which is characterized in that step (4) includes: to take second sediment, adds water
It is configured to the suspension that mass concentration is 8-12%, the glacial acetic acid aqueous solution that mass concentration is 8-12% is added, is heated to boiling,
It is stirred to react 1.5-2.5 hours, adjusting pH is 6.5-7.5, and the second supernatant is collected in centrifugation, desalination of dialysing, freeze-drying, i.e.,
Obtain the bacteroides fragilis extract.
9. application according to claim 6 or 7, which is characterized in that the preparation method of the bacteroides fragilis extract is also
Include the steps that degrading: bacteroides fragilis extract obtained in step (4) is degraded by the method for ultrasound, it is described super
The condition of sound are as follows: 180-210kHz, 15-25 DEG C.
10. a kind of bacteroides fragilis extract or drug or food for preventing and treating psoriasis, which is characterized in that the drug or
Contain the bacteroides fragilis extract in food, contains bacteroides fragilis capsular polysaccharide A in the bacteroides fragilis extract.
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