CN109954004B - Application of bacteroides fragilis extract in preparation of composition for preventing and treating psoriasis - Google Patents
Application of bacteroides fragilis extract in preparation of composition for preventing and treating psoriasis Download PDFInfo
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- CN109954004B CN109954004B CN201711342761.5A CN201711342761A CN109954004B CN 109954004 B CN109954004 B CN 109954004B CN 201711342761 A CN201711342761 A CN 201711342761A CN 109954004 B CN109954004 B CN 109954004B
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- bacteroides fragilis
- capsular polysaccharide
- precipitate
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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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/66—Microorganisms or materials therefrom
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- 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
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- 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
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Abstract
The invention relates to application of a bacteroides fragilis extract in preparation of a medicine or food for preventing and treating psoriasis, wherein the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide A. The inventors of the present invention found that the bacteroides fragilis extract containing capsular polysaccharide a has a function of preventing/treating psoriasis.
Description
Technical Field
The invention relates to the technical field of application of bacteroides fragilis, in particular to application of a bacteroides fragilis extract in preparation of a composition for preventing and treating psoriasis.
Background
Psoriasis (Psoriasis) is a genetically regulated, T lymphocyte-mediated autoimmune skin disease. Its major histopathological changes are keratinocyte dysplasia, parakeratosis, hyperkeratosis, angiogenesis and inflammatory cell infiltration. The etiology and pathogenesis of the chronic inflammatory hyperplastic skin disease are not completely clear at present, and no effective radical treatment method exists, so that the chronic inflammatory hyperplastic skin disease is one of the most common chronic inflammatory hyperplastic skin diseases. The disease condition is stubborn and easy to recur, can cause damage to the whole system, seriously influences the life quality and even the life cycle of a patient, and is one of the diseases which are mainly concerned at home and abroad at present.
Psoriasis is a complex genetic disease of immune dysfunction, although the mechanisms of inheritance are not yet fully established. A number of environmental factors play an important role in the pathogenesis of psoriasis, including drugs, skin wounds (Koebner's phenomenons), infections and stress. Evidence that psoriasis involves immunological mechanisms includes the efficacy of immunosuppressive drugs such as methotrexate, cyclosporine (CyA), immune targeting biologics, and immunotoxins (nielloleukin). Thus, psoriasis is a hyperimmune mediated organ-specific (skin, nail and/or joint) disease in which localized lesions allow basal keratinocyte stem cells to proliferate highly and continue the disease process.
Currently, there are 2 drug treatment modalities for psoriasis, either systemically or topically. Severe skin lesions and/or extradermal distribution of diseases such as psoriatic arthritis and nail psoriasis require systemic treatment. Systemic conventional treatments are performed by using methotrexate, cyclosporine, corticosteroids, retinoids or fumarates. Of these, methotrexate, cyclosporines and corticosteroids work mostly by suppressing the immune response, while retinoids and fumarates work primarily by inhibiting the hyperproliferation of keratinocytes at the base of psoriatic lesions. However, these drugs have certain toxic side effects and limited efficacy. Recently, biologics have been introduced to treat psoriasis including alefacept, etanercept and many monoclonal antibodies, namely adalimumab, infliximab, ustekimab (usekinumab). These new drugs are more effective than traditional systemic drugs, but they require long periods of time, are very expensive to treat, and may be associated with severe toxic side effects.
In cases where the disease is localized to the skin only, topical treatment is often selected. Topical treatment of psoriasis is commonly performed using dithranol, vitamin D3 and its analogues, including calcipotriol and calcitriol, tazarotene (which is a topical retinoid), and topical glucocorticoids. The use of products for topical treatment is limited in cases where most body surfaces are affected. The patient candidates for topical treatment should present light to moderate, clinically diagnostically determined psoriasis patients with a body surface involved of < 10% or a severity index measured by the severity index PASI calculation method of < 10. The psoriasis area and PASI are the most widely used tools for measuring the severity of psoriasis. PASI combines the severity assessment and affected area of the lesion into a single score ranging from 0 (no disease) to 72 (most severe disease) (Langley RG, Ellis CN.J.Am Acad Dermatol 2004; 51: 563-9).
Regardless of the treatment modality, its strong toxicity limits its long-lasting treatment, resulting in a short effective period of treatment for clinical symptoms. Thus, there is still an unsatisfactory medical need in the treatment and prevention of relapse.
Bacteroides fragilis (bacteroides fragilis) is a member of bacteroides among gram-negative anaerobic bacteria, belongs to bacteroidetes, and is completely different from bifidobacterium, lactobacillus, and the like of firmicutes. Bacteroides have 25 species, only 10 species from humans, only 10 species from animals, and 5 species from humans and animals. The bacteroides fragilis is an obligate anaerobic bacterium, the shape of the bacteroides is polymorphic according to the difference of culture media and the difference of growth stages, the bacteroides fragilis is rod-shaped, the two ends of the bacteroides fragilis are blunt and round, the coloration is dark, the middle color is light and uneven, the bacteroides fragilis, no spores and no power exist, some bacteroides have vacuoles, and the bacteroides are different in length. Bacteriodes fragilis enterotoxin (BFT) can be classified into Enterotoxigenic bacteriodes fragilis (ETBF) and non-Enterotoxigenic bacteriodes fragilis (NTBF) depending on whether they can be synthesized and secreted. Bacteroides fragilis is present mainly in the colon as part of the normal intestinal flora of humans and animals. In addition, mucous membranes of the respiratory, gastrointestinal and genitourinary tracts may colonize.
Bacteroides fragilis can express 8 different capsular polysaccharides, which are respectively designated as a-H, wherein the action of capsular polysaccharide a (PSA) is determined by its molecular weight, and the molecular weight of natural PSA is about 110 kDa.
Disclosure of Invention
Based on this, the present invention provides a novel use of Bacteroides fragilis (Bacteroides fragilis) extract.
The specific technical scheme is as follows:
application of Bacteroides fragilis extract in preparation of medicine for preventing and treating psoriasis, wherein the Bacteroides fragilis extract contains Bacteroides fragilis capsular polysaccharide A.
The dosage form of the medicine comprises pills, tablets, granules, capsules, oral liquid or tube feeding preparations. The medicine comprises human medicine or animal medicine, and can be used for human or animal.
In some of these embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 2-110 KD.
In some of these embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 5-95 KD.
In some embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 5-85 KD.
In some embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 5-75 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 40 KD-85 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 60 KD-85 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 65 KD-75 KD.
In some of these embodiments, the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide a in an amount of 60-75 wt%.
In some embodiments, the bacteroides fragilis is bacteroides fragilis ZY-312 with collection number of CGMCC No. 10685.
In some of these embodiments, the method of preparing the bacteroides fragilis extract comprises the steps of:
(1) centrifuging and precipitating the bacteroides fragilis bacterial liquid after fermentation culture, collecting a first precipitate, adding water with the temperature of 65-72 ℃ into the first precipitate, adding a phenol solution after dissolving, keeping the temperature of 65-72 ℃, stirring for 25-35min, centrifuging, and collecting a first supernatant;
(2) extracting the first supernatant collected in the step (1) by using ether to remove phenol, removing residual ether, and collecting an aqueous phase solution;
(3) adding absolute ethyl alcohol into the aqueous phase solution collected in the step (2) until the final concentration of the ethyl alcohol is 75-85v/v%, carrying out alcohol precipitation, centrifuging, and collecting a second precipitate;
(4) and adding water into the second precipitate to prepare a solution, adjusting the pH to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
In some embodiments, the ratio of the water, the phenol solution and the first precipitate added to the first precipitate in step (1) is 3-5 mL: 3-5 mL: 1g of a compound; the mass concentration of the phenol solution is 70-80%; and/or, the alcohol precipitation in the step (3) is carried out for 8 to 16 hours at the temperature of between 0 and 8 ℃.
In some embodiments, step (4) comprises: and adding water into the second precipitate to prepare a suspension with the mass concentration of 8-12%, adding a glacial acetic acid aqueous solution with the mass concentration of 8-12%, heating to boil, stirring for reaction for 1.5-2.5 hours, adjusting the pH value to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
In some of these embodiments, the method of preparing the bacteroides fragilis extract further comprises the step of degrading: degrading the bacteroides fragilis extract obtained in the step (4) by using an ultrasonic method, wherein the ultrasonic condition is as follows: 180 ℃ and 210kHz, 15-25 ℃.
The invention also provides a bacteroides fragilis extract or a medicament for preventing and treating psoriasis.
The specific technical scheme is as follows:
a Bacteroides fragilis extract or a medicament for preventing and treating psoriasis, wherein the medicament contains the Bacteroides fragilis extract, and the Bacteroides fragilis extract contains Bacteroides fragilis capsular polysaccharide A.
The dosage form of the medicine comprises pills, tablets, granules, capsules, oral liquid or tube feeding preparations. The medicine comprises human medicine or animal medicine, and can be used for human or animal. The bacteroides fragilis capsular polysaccharide a may also be used in combination with other drugs including, but not limited to: sulfasalazine salicylic acid preparations (e.g., adisha, mesalamine), corticosteroids (e.g., prednisone, dexamethasone), and/or immunosuppressants (e.g., azathioprine). In some of these embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 2-110 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 5 KD-95 KD.
In some embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 5-85 KD.
In some embodiments, the Bacteroides fragilis capsular polysaccharide A has a molecular weight of 5-75 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 40 KD-85 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 60 KD-85 KD.
In some embodiments, the bacteroides fragilis capsular polysaccharide A has a molecular weight of 65 KD-75 KD.
In some of these embodiments, the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide a in an amount of 60-75 wt%.
In some embodiments, the bacteroides fragilis is bacteroides fragilis ZY-312 with collection number of CGMCC No. 10685.
In some of these embodiments, the method of preparing the bacteroides fragilis extract comprises the steps of:
(1) centrifuging and precipitating the bacteroides fragilis bacterial liquid after fermentation culture, collecting a first precipitate, adding water with the temperature of 65-72 ℃ into the first precipitate, adding a phenol solution after dissolving, keeping the temperature of 65-72 ℃, stirring for 25-35min, centrifuging, and collecting a first supernatant;
(2) extracting the first supernatant collected in the step (1) by using ether to remove phenol, removing residual ether, and collecting an aqueous phase solution;
(3) adding absolute ethyl alcohol into the aqueous phase solution collected in the step (2) until the final concentration of the ethyl alcohol is 75-85v/v%, carrying out alcohol precipitation, centrifuging, and collecting a second precipitate;
(4) and adding water into the second precipitate to prepare a solution, adjusting the pH to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
In some embodiments, the ratio of the water, the phenol solution and the first precipitate added to the first precipitate in step (1) is 3-5 mL: 3-5 mL: 1g of a compound; the mass concentration of the phenol solution is 70-80%.
In some embodiments, the alcohol precipitation in step (3) is alcohol precipitation at a temperature of 0-8 ℃ for 8-16 hours.
In some embodiments, step (4) comprises: and adding water into the second precipitate to prepare a suspension with the mass concentration of 8-12%, adding a glacial acetic acid aqueous solution with the mass concentration of 8-12%, heating to boil, stirring for reaction for 1.5-2.5 hours, adjusting the pH value to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
In some of these embodiments, the method of preparing the bacteroides fragilis extract further comprises the step of degrading: degrading the bacteroides fragilis extract obtained in the step (4) by using an ultrasonic method, wherein the ultrasonic condition is as follows: 180 ℃ and 210kHz, 15-25 ℃.
The bacteroides fragilis ZY-312 of the invention has been preserved in China general microbiological culture Collection center (CGMCC) at 4.2.2015, the preservation number is CGMCC No.10685, and the preservation address is No. 3 Hospital No.1 of Xilu, Beijing, Chaoyang district.
The inventor of the invention discovers new application of bacteroides fragilis and develops a new application field through long-term experience accumulation and a large amount of creative experimental research. The inventor obtains the bacteroides fragilis extract (the main component is capsular polysaccharide A) through a large number of experimental researches, and unexpectedly finds that the bacteroides fragilis extract containing capsular polysaccharide A has good effect of preventing and treating psoriasis.
Further, the inventors obtained a series of bacteroides fragilis capsular polysaccharide a of different molecular weights by degrading bacteroides fragilis capsular polysaccharide a. And further experimental researches show that the capsular polysaccharide A with the molecular weight of 5-95 KD (especially 70KD) has better effect of preventing and treating psoriasis.
The bacteroides fragilis capsular polysaccharide A provided by the invention has a good effect of preventing and treating psoriasis, has no side effect on organisms, and can be used in combination with other medicines for preventing/treating psoriasis. The bacteroides fragilis capsular polysaccharide A provided by the invention has good edible and medicinal prospects, and provides a good product for preventing/treating psoriasis, which is suitable for human body eating clinically.
Drawings
FIG. 1 is a characteristic diagram of the colony of Bacteroides fragilis ZY-312 of example 1;
FIG. 2 is a microscopic view of Bacteroides fragilis ZY-312 of example 1 after gram-staining;
FIG. 3 is the 1H spectrum of the capsular polysaccharide A NMR spectrometer analysis of example 1;
FIG. 4 is a 13C spectrum of capsular polysaccharide A NMR spectrometer analysis of example 1;
FIG. 5 is a COSY spectrum of capsular polysaccharide A NMR spectrometer analysis of example 1;
FIG. 6 is an HSQC spectrum of capsular polysaccharide A NMR spectrometer analysis of example 1;
FIG. 7 is a HMBC spectrum of a nuclear magnetic resonance spectrometer analysis of capsular polysaccharide A of example 1;
FIG. 8 is the chemical structural formula of Bacteroides fragilis capsular polysaccharide A prepared in example 1;
FIG. 9 is a graph showing the results of detecting histological changes in skin by HE section staining in example 2.
Detailed Description
The present invention is further illustrated by the following specific examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
The Bacteroides fragilis used in the following examples is Bacteroides fragilis ZY-312 (bacteriodes fragilis ZY-312), which was deposited in China general microbiological culture Collection center (CGMCC) at 4.2.2015, with the collection number of CGMCC No.10685, and the collection address of No. 3, Xilu 1, North Cheng Yang district, Beijing.
Example 1 preparation of Bacteroides fragilis extract
(1) Fermentation culture of bacteroides fragilis
The strain is streaked and inoculated on a blood plate, and anaerobic culture is carried out for 48 h. Observing the morphological characteristics, staining characteristics, size, club shape, distribution and the like of colonies.
Colony characteristics: after culturing the bacteroides fragilis ZY-312 on a blood plate for 48h, the bacteroides fragilis ZY-312 presents a round and slightly convex shape, is semitransparent, white, has a smooth surface and is not hemolyzed, and the diameter of a colony is between 1 and 3mm, as shown in figure 1.
Microscopic morphology: gram-stained bacteroides fragilis ZY-312 was used as gram-negative bacteria, and was typically rod-shaped, with blunt and densely stained ends, and non-staining areas in the middle of the cells, such as vacuoles, as shown in FIG. 2.
Selecting single colony, inoculating into tryptone broth, fermenting and culturing for 8 hours (at 37 ℃), centrifuging the obtained bacterial liquid for precipitation, rotating at 3000r/min, centrifuging for 15min, removing supernatant, and collecting precipitate.
(2) Preparation of Bacteroides fragilis extract
Preparation of a bacteroides fragilis extract comprising the steps of:
1) taking 200g of bacteroides fragilis bacterial mud (precipitate obtained in the step (1)) and adding 750mL of ultrapure water at 68 ℃, after dissolving, adding 750mL of phenol solution with the volume fraction of 75%, uniformly mixing, keeping at 68 ℃, stirring and extracting for 30min, and centrifuging for 20min at 15000g to obtain upper-layer liquid.
2) The upper layer was extracted with an equal volume of ether (1.5L) to remove phenol, the upper layer was collected and the extraction was repeated until no phenol remained. The ether was removed by heating in a water bath and the aqueous phase was collected.
3) Centrifuging the water phase at 15000g for 20min, measuring volume, adding anhydrous ethanol until the final concentration of ethanol is 80% (volume fraction), precipitating with ethanol at 4 deg.C overnight (12 hr), centrifuging at 15000g for 20min, and collecting precipitate.
4) Weighing the mass of the precipitate obtained in the step 3), adding a certain volume of deionized water to prepare the precipitate into a solution with the mass concentration of 10%, stirring and mixing uniformly, adding a glacial acetic acid aqueous solution with the mass concentration of 10%, heating to boil, continuously stirring and reacting for 2h, adjusting the pH value to 7.0, centrifuging for 20min at 15000g, and collecting the precipitate. The resulting supernatant was dialyzed to remove salts (10KD dialysis bag), and freeze-dried to obtain Bacteroides fragilis extract.
5) 30mg of the Bacteroides fragilis extract of step 4) was weighed, dissolved in 0.5mL of D2O, and 1. mu.l of acetone (1H, 2.22; 13C, 30.89) scaling. Analyzing the spectrum of 1H, 13C, COSY, HSQC and HMBC (figures 3-7) by using a 500MHz Bruker nuclear magnetic resonance spectrometer, and confirming that the bacteroides fragilis extract collected in the step 4) is capsular polysaccharide A with the purity of about 70%. GPC analysis showed that the above capsular polysaccharide A had a molecular weight of 781 in repeating units, a number of repeating units n of 89, a molecular weight of about 70KD and a molecular formula of- [ C ]31N3O20H47]91The chemical structure is shown in FIG. 8.
(3) Preparation of capsular polysaccharide A of different molecular weights
This example is carried out by degradation of the capsular polysaccharide prepared in (2) by methods including, but not limited to, chemical, physical and biological degradation. In the embodiment, capsular polysaccharide A with different molecular weights is obtained by an ultrasonic method, wherein the capsular polysaccharide A is treated for 3 hours, 2 hours, 0.5 hour and 0 hour at 195kHz and 20 ℃, and capsular polysaccharide A with molecular weights of 2KD, 5KD, 40KD and 70KD are obtained by collection respectively.
(4) Preparation of capsular polysaccharide A with molecular weight of 110KD
Extracted from Bacteroides fragilis NCTC9343 (purchased from ATCC in USA) by the method of (2).
Example 2 therapeutic efficacy of different doses of Bacteroides fragilis capsular polysaccharide A on Imquimod-induced psoriasis in mice
First, experimental design
In the present example, a female BALB/c mouse psoriasis model was induced by 5% imiquimod, and then the treatment effect was examined by treating the psoriasis model in the 5% imiquimod-induced female BALB/c mouse with the bacteroides fragilis extract (capsular polysaccharide a as a main ingredient) prepared in example 1. In the present example, capsular polysaccharide A with molecular weight of 70KD is taken as an example, and the therapeutic effect of low, medium and high doses of capsular polysaccharide A on psoriasis is tested.
The psoriasis induction method of the mice comprises the following steps: this example gives 60 female BALB/c mice, each of which is assigned a unique number. Before animals are grouped, the squirrel cage labels should be labeled with item number, species/strain, gender, cage number, and animal number. The mice were randomly divided into 6 groups, i.e., a normal control group (group 1), a model group (group 2), a positive control group (group 3), a bacteroides fragilis capsular polysaccharide a low dose group (group 4), a medium dose group (group 5), a high dose group (group 6), 10 per group, based on the initial body weight of the mice using BioBook software.
The backs of 6 groups of mice were dehaired to an area of about 2cm × 1.5cm starting on day 0, except for the hairless area on the back of group 1 (normal control group) mice which were coated with about 0.5ml of distilled water daily, and the hairless area on the back of group 2 (model group) to group 6 mice which were coated with 5% imiquimod daily at 60 mg/day (3 mg/d of effective drug) for 6 consecutive days. On day 6, the skin of the hairless area on the back of the group 1 (normal control group) mice had no obvious erythema scale, while the skin on the back of the hairless area from the group 2 (model group) to the group 6 mice gradually appeared with erythema and adhesive silvery white scales after being treated with 5% imiquimod, indicating that the molding of the embodiment was successful.
From day 7, mice in groups 1 and 2 were coated with distilled water daily, positive control group (group 3) was coated with 0.5% dithranol daily, and groups 4-6 were coated with low, medium and high doses of bacteroides fragilis capsular polysaccharide A with molecular weight of 70KD daily.
Specific experimental groups and dosing regimens are shown in table 1:
table 1 experimental groups and dosing regimens
Second, sample collection
After the experiment, the mice were sacrificed by cervical dislocation. Skin tissue biopsy was performed to collect the skin lesion tissue on the back, and the tissue was divided into 3 parts on average, two of the parts were immediately stored in a refrigerator at-80 ℃ for later use, and one part was fixed in a previously prepared 10% formalin fixing solution.
Third, observe the index and judge
1. Appearance of the product
Carefully observing the vital signs of the mice in the treatment process, and taking pictures at the same time every day to record the change condition of erythema and scale at the skin lesion.
2. HE section staining method for detecting histological changes of skin
And (3) preparing an HE stained section of the tissue at the skin lesion of the mouse, and observing the infiltration number of inflammatory cells and the change of the thickness of the epidermis. Observing each section under a microscope multiplied by 200 times, taking 3 visual fields for photographing, calculating the infiltration number of inflammatory cells after amplifying by adopting image processing software, and taking an average value for recording, wherein the unit is HP; simultaneously, image processing software is adopted, a substrate membrane band is used as a boundary, the longitudinal epidermis area is delineated and converted into a numerical value, and the average value of 3 visual fields is recorded, wherein the unit is 4/9in2/HP
3. Statistics and analysis
The number of infiltrating cells (number/HP) and the area of the longitudinal epidermis (4/9 in) were calculated for each group of mice2/HP), as mean. + -. standard error statistics. Statistical analysis was performed using Graphpad Prism, SPSS or Sigmaplot software. The specific data is presented in the form of a chart. P<0.05 was considered to have a statistical difference.
Third, results and analysis
The Mean ± standard deviation (Mean ± SD) was used to count the number of infiltrating cells (number/HP) and the longitudinal epidermal area (4/9in2/HP) of the mice. And multiple analyses were performed using ANOVA in combination with Dunnett's. When P <0.05, a statistical difference was considered. The specific detection results are shown in Table 2, and the staining results of HE sections are shown in FIG. 9.
TABLE 2 comparison of the number of infiltrating cells and the area of the epidermis in the longitudinal direction (Mean. + -. SD)
In this example, the positive drug (0.5% dithranol) reduced the clinical symptoms of psoriasis in mice induced by imiquimod by 5%, including the number of infiltrating cells and the area of the longitudinal epidermis, all statistically different from the model group.
The bacteroides fragilis capsular polysaccharide a high dose group with a molecular weight of 70KD provided in example 1 showed significant efficacy in inhibiting 5% imiquimod-induced psoriasis in mice as it significantly reduced the number of infiltrating cells and the longitudinal epidermal area, all of which were statistically different (P <0.01) compared to the model group, as detailed in table 2.
In this example, it can be found by comparing the test results of 6 groups of experiments that the low, medium and high doses of bacteroides fragilis capsular polysaccharide a provided by the present invention can effectively reduce the clinical psoriasis symptoms induced by 5% imiquimod in mice (see table 2, fig. 9 for details).
Therefore, the bacteroides fragilis capsular polysaccharide A provided by the invention has a good treatment effect on psoriasis of mice induced by 5% imiquimod, and does not show any toxic or side effect.
Example 3 therapeutic effects of Bacteroides fragilis capsular polysaccharide A of varying molecular weights on psoriasis
In this example, a mouse psoriasis animal model is prepared by referring to the method in example 2, then 5% imiquimod-induced mouse psoriasis is prevented/treated by using 2KD, 5KD, 40KD, 70KD and 110KD bacteroides fragilis capsular polysaccharide a prepared in example 1 of the invention, and the treatment effect of bacteroides fragilis capsular polysaccharide a with different molecular weights on psoriasis is tested. This example illustrates high doses of 2kD, 5kD, 40kD, 70kD and 110kD Bacteroides fragilis capsular polysaccharide A.
Referring to the experimental group grouping method of example 2, mice were divided into normal control group, model group, 2KD, 5KD group, 40KD group, 70KD group, and 110KD group.
The dosing schedule was according to example 2.
The specific experimental groups and dosing regimens are as follows:
table 3 experimental groups and dosing regimens
The number of infiltrating cells and the area of the longitudinal epidermis in each group were compared by the method of reference example 2, and the specific results are shown in table 4:
TABLE 4 comparison of the number of infiltrating cells and the area of the epidermis in the longitudinal direction (Mean. + -. SD)
From the table above, the bacteroides fragilis capsular polysaccharide A with the molecular weight of 5KD, 40KD and 70KD provided by the invention shows the significant drug effect of inhibiting psoriasis in mice induced by 5% imiquimod, and particularly shows that the bacteroides fragilis capsular polysaccharide A remarkably reduces the number of infiltration cells and the longitudinal epidermal area caused by psoriasis, which is detailed in table 4. Compared with the 2KD group and the 110KD group, the 5KD, 40KD and 70KD capsular polysaccharide A has better activity in reducing the number of infiltrating cells and longitudinal epidermal area caused by psoriasis, P is less than 0.05, and the statistical difference is obvious.
Example 4 therapeutic effects of different strains of Bacteroides fragilis capsular polysaccharide A on psoriasis
In this example, the capsular polysaccharide A with a molecular weight of 110KD extracted in example 1 was degraded by the degradation method described in example 1, and the capsular polysaccharide A with a molecular weight of 70KD was collected and recorded as NCTC9343-70KD group, and compared with capsular polysaccharide A with a molecular weight of 70KD extracted from ZY-312 (recorded as ZY-312-70KD group), the therapeutic effects of high-dose NCTC9343-70KD and ZY-312-70KD on psoriasis were evaluated. In this example, the number of infiltrating cells and the area of the epidermis in the longitudinal direction were measured by the method described in example 3, and the results are shown in table 5:
TABLE 5 number of infiltrating cells and longitudinal epidermal area contrast
From the results, the capsular polysaccharide A with the molecular weight of 110KD extracted from the NCTC9343 strain is degraded, so that the treatment effect of the capsular polysaccharide A extracted from the ZY-312 strain on psoriasis can be realized.
Example 5 clinical trial Effect of Bacteroides fragilis capsular polysaccharide A
This example evaluates the efficacy of bacteroides fragilis capsular polysaccharide a on psoriasis patients by PASI evaluation, while also evaluating the safety of bacteroides fragilis capsular polysaccharide a. This example selects 3 patients with clinical diagnoses of psoriasis for at least 3 years for the study. Patients did not take combination therapy with topical corticosteroids or systemic therapy before and during treatment with mucopolysaccharide a. The body surface area involved at baseline is 10% or less and the PASI value is 10 or less.
The patient had the following pre-medication conditions:
The disease is affected for more than 10 years in the age of 2 male and 66 years, the focus is mainly distributed on hands, feet and heads, and the disease is mainly manifested as itching, white dandruff and coexistence of old and new focus in 10 years, and is repeated for more than 10 years.
The patients use the high dose of bacteroides fragilis capsular polysaccharide A with molecular weight of 70KD prepared in example 1 every day, and the bacteroides fragilis capsular polysaccharide A is uniformly smeared on affected parts. PASI scores were recorded before trial (baseline), 6 weeks after drug administration, and 12 weeks after drug administration, respectively. The specific detection results are shown in table 6:
TABLE 6 PASI evaluation
The average PASI at baseline was 5.43 (standard deviation 0.74); the average PASI value was 3.87 (standard deviation 0.29) after 6 weeks of administration, and 2.30 (standard deviation 0.43) after 12 weeks of administration.
The detection results show that the bacteroides fragilis capsular polysaccharide A with the molecular weight of 70KD provided by the invention has a statistically significant reduction in PASI value after administration for 12 weeks compared with that before administration (baseline) (Student t test P < 0.05).
All patients showed a great improvement in the clinical symptoms of the disease, with the exception of a reduction in the PASI values, and the treatment was well tolerated without any toxic side effects.
In conclusion, the treatment of psoriasis using bacteroides fragilis capsular polysaccharide a is effective in improving PASI values, which suggests that bacteroides fragilis capsular polysaccharide a may be used in the treatment of psoriasis, especially with bacteroides fragilis capsular polysaccharide a having a molecular weight of 5-70 KD.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (9)
1. The application of the bacteroides fragilis extract in the preparation of the medicine for preventing and treating psoriasis is characterized in that the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide A, the bacteroides fragilis is bacteroides fragilis ZY-312, and the bacteroides fragilis ZY-312 is stored in the China general microbiological culture Collection center (CGMCC) on 4-2 days 2015 with the preservation number of CGMCC No. 10685;
the molecular weight of the bacteroides fragilis capsular polysaccharide A is 65-75 KD.
2. The use according to claim 1, wherein the Bacteroides fragilis capsular polysaccharide A has the formula- [ C31N3O20H47]91-。
3. The use of claim 2, wherein the bacteroides fragilis extract contains bacteroides fragilis capsular polysaccharide a in an amount of 60-75 wt%.
4. The use according to claim 3, wherein the medicament is in the form of a pill, tablet, granule, capsule, oral liquid or tube feed.
5. The use according to any one of claims 1 to 4, wherein the Bacteroides fragilis ZY-312, after 48h of culture on a blood plate, appears to be round and slightly convex, translucent, white, smooth in surface, non-hemolytic, with a colony diameter between 1 and 3 mm.
6. The use according to any one of claims 1 to 4, wherein the Bacteroides fragilis extract is prepared by a process comprising the steps of:
(1) centrifuging and precipitating the bacteroides fragilis bacterial liquid after fermentation culture, collecting a first precipitate, adding water with the temperature of 65-72 ℃ into the first precipitate, adding a phenol solution after dissolving, keeping the temperature of 65-72 ℃, stirring for 25-35min, centrifuging, and collecting a first supernatant;
(2) extracting the first supernatant collected in the step (1) by using ether to remove phenol, removing residual ether, and collecting an aqueous phase solution;
(3) adding absolute ethyl alcohol into the aqueous phase solution collected in the step (2) until the final concentration of the ethyl alcohol is 75-85v/v%, carrying out alcohol precipitation, centrifuging, and collecting a second precipitate;
(4) and adding water into the second precipitate to prepare a solution, adjusting the pH to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
7. The use of claim 6, wherein the ratio of the water added to the first precipitate in step (1), the phenol solution and the first precipitate is 3-5 mL: 3-5 mL: 1g of a compound; the mass concentration of the phenol solution is 70-80%; and/or the presence of a catalyst in the reaction mixture,
and (3) carrying out alcohol precipitation at the temperature of 0-8 ℃ for 8-16 hours.
8. Use according to claim 6 or 7, characterized in that step (4) comprises: and adding water into the second precipitate to prepare a suspension with the mass concentration of 8-12%, adding a glacial acetic acid aqueous solution with the mass concentration of 8-12%, heating to boil, stirring for reaction for 1.5-2.5 hours, adjusting the pH value to 6.5-7.5, centrifuging, collecting a second supernatant, dialyzing to remove salt, and freeze-drying to obtain the bacteroides fragilis extract.
9. The use according to claim 6 or 7, wherein the preparation method of the Bacteroides fragilis extract further comprises the step of degrading: degrading the bacteroides fragilis extract obtained in the step (4) by using an ultrasonic method, wherein the ultrasonic condition is as follows: 180 ℃ and 210kHz, 15-25 ℃.
Priority Applications (1)
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