CN116327809A - Application of bacteroides fragilis and extract capsular polysaccharide A thereof in preparation of product for preventing and treating autoimmune arthritis - Google Patents
Application of bacteroides fragilis and extract capsular polysaccharide A thereof in preparation of product for preventing and treating autoimmune arthritis Download PDFInfo
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- CN116327809A CN116327809A CN202310182987.2A CN202310182987A CN116327809A CN 116327809 A CN116327809 A CN 116327809A CN 202310182987 A CN202310182987 A CN 202310182987A CN 116327809 A CN116327809 A CN 116327809A
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- bacteroides fragilis
- capsular polysaccharide
- arthritis
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- autoimmune arthritis
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Abstract
The invention discloses application of bacteroides fragilis and extract capsular polysaccharide A thereof in preparation of products for preventing and treating autoimmune arthritis, and the bacteroides fragilis, in particular zwitterionic capsular polysaccharide A (PSA) extracted from bacteroides fragilis ZY-312 with the preservation number of CGMCCNo.10685, can inhibit monocytes from secreting rheumatoid arthritis immune response core pro-inflammatory factors TNF-alpha and IL-1 beta in vitro; can inhibit inflammatory response by inhibiting IL-1 beta and IFN-gamma content in serum in vivo, and reduce arthritis score, ankle joint thickness and paw volume. And can reduce the content of Rheumatoid Factor (RF) and C-reactive protein (CRP) in serum. Can effectively prevent and treat autoimmune arthritis, not only can be independently used for treating autoimmune arthritis, but also can be combined with other microorganism, operation, monoclonal antibody and other treatment means for application, thereby remarkably improving comprehensive curative effect, effectively preventing occurrence and development of autoimmune arthritis and improving life quality of patients.
Description
The microbial strain used in the implementation process of the invention is preserved in China general microbiological culture Collection center (CGMCC) (No. 3 of North Chen West Lu 1 of the Korean area of Beijing city) in China general microbiological culture Collection center (CGMCC) of 4 months and 2 days of 2015. Classification naming: bacteroides fragilis ZY-312 (bacteroides fragilis ZY-312) with the preservation number of CGMCC No.10685. Bacteroides fragilis ZY-312 was isolated by the applicant's entity and has been under the authority of the patent protection (patent No. 2015175508. X), and under the provisions of the patent prosecution guidelines, the public was able to buy from commercial sources or has been granted without preservation, i.e. without providing proof of preservation.
Technical Field
The invention relates to the field of biological medicine, in particular to application of bacteroides fragilis and zwitterionic capsular polysaccharide thereof in preparation of a medicine for treating autoimmune arthritis.
Background
Autoimmune arthritis mainly refers to rheumatoid arthritis (Rheumatoid arthritis, RA), is a chronic and systemic autoimmune disease, is mainly manifested by synovial hyperplasia, synovitis, bone and cartilage erosion at joints, causes joint swelling, symmetrical pain and rigidity, finally causes joint distortion, and patients are limited in movement, is one of the main diseases causing loss of labor and disability of people, shortens the life span of patients with serious illness by 10-15 years, and seriously damages life quality.
RA occurrence and progression is a chronic, irreversible pathological process. Although research on immunology, genetics, biological cytology and biochemistry has been greatly advanced, the etiology and pathogenesis of RA are not completely defined, and it is generally considered that RA is induced by immune system disorder caused by interaction of genetic factors, viral infection, environmental factors, intestinal microbiome and other factors.
At present, the therapeutic drugs aiming at autoimmune arthritis are mainly divided into four types, namely glucocorticoid, nonsteroidal anti-inflammatory drugs, disease-improving antirheumatic drugs and biological preparations. Glucocorticoids are used primarily for controlling pain and inflammation, the former reducing the condition primarily by inhibiting inflammation, and non-steroidal anti-inflammatory drugs which inhibit cyclooxygenase. Disease-modifying antirheumatic drugs (Disease modifying anti-therapeutic drugs, DMARDs) are commonly used drugs for treating RA and are commonly used in newborns, including hydroxychloroquine, methotrexate, leflunomide, sulfasalazine, and the like. DMARDs can inhibit proliferation of immune cells, reduce deposition of deleterious factors at joints, reduce glutathione levels, reduce aerobic damage, and promote extracellular adenosine levels. Biological agents are a class of targeted inhibition drugs directed against immune responses and/or cytokines, tnfα inhibitors such as etanercept, infliximab and adalimumab, IL-1 β inhibitors such as anagold and abatacrolimus, CD20 monoclonal antibodies such as rituximab. Although these drugs are effective in alleviating the condition of RA patients, the differences in drug response rate and hysteresis of treatment are not negligible. At the same time, adverse reactions to these drugs severely affect patient compliance and thus affect the therapeutic efficacy. The long-term use of non-steroidal anti-inflammatory drugs may cause the blood system to be affected, and the leucopenia, thrombocytopenia and the like are shown. Digestive tract lesions, such as gastric ulcers and gastrointestinal bleeding, may also occur. The glucocorticoid medicine may have serious side effect after long term use, and the long term prescription is generally avoided clinically. Even short-term use can cause hormonal regulation disorder of the human body, leading to physical fatness; has effects in inhibiting immune system, and inducing infection; is easy to cause adverse reactions such as digestive tract ulcer and the like. DMARDs are slow in onset of action and usually take more than one month to achieve a relatively significant relief effect; adverse reactions include digestive tract discomfort, cardiovascular adverse reactions, infection, elevated liver transaminase, interstitial pneumonia, etc. The biological agent has higher cost, and adverse reactions include pulmonary infection, herpes zoster, tuberculosis recurrence, virus activity, malignant tumor and the like. Under the background, finding a method or means to assist in drug treatment has clinical application value.
The occurrence and progression of rheumatoid arthritis is closely related to the disturbance of local and systemic immune responses, and in combination with epidemiological studies and subclinical diagnostic features of RA, there is increasing evidence that RA immune abnormalities originate at other immune sites outside the joint, i.e. "mucosal origin hypothesis". The mucosal immune system is an important part of the immune system of the organism, and mucosa-associated lymphoid tissues and mucosa site immune cells form a relatively independent local immune system, including oral mucosa, lung mucosa, genital tract mucosa, intestinal mucosa, etc., and these mucosal sites act as barriers, which are critical to maintain a balance between local environmental damage (e.g., pollutants, toxins, pathogens) and beneficial factors (e.g., symbiotic microorganisms provide protection against colonization and infection by pathogens). Among these, the gut is considered the largest immune organ in which a large number of microorganisms are present that supply the body with the necessary nutrients and energy, and additionally shape and "acclimate" the intestinal mucosal immune system. Intestinal microorganisms interact, respond and bind to the intestinal mucosa, and the induced immune system, including the adaptive immune system and mononuclear phagocytes, is activated or inhibited, and the process of interaction between the two is achieved through the intestinal mucosa barrier. The abundance of intestinal microorganisms means that the largest number of antigenic substances are present in the intestine, and that changes in intestinal microorganisms can trigger changes in the local, systemic immune response. With the development of molecular biological sequencing technology, human beings have deeply recognized the relevance of intestinal microecology and human health, and more researches report that the occurrence and development of autoimmune diseases such as RA and the like have close relation with intestinal microecology.
Bacteroides fragilis (b.fragilis) is a gram-negative, rod-shaped, blunt-ended and concentrated-stained, capsular, ungerminated, unpowered, obligate anaerobic bacterium, classified into Enterotoxigenic (ETBF) and non-enterotoxigenic (NTBF), which are part of the normal flora of the human and animal intestinal tracts, mainly present in the colon, and the mucous membranes of the respiratory, gastrointestinal and genitourinary tracts can also colonize. The Bacteroides fragilis ZY-312 used in the invention is proved to be NTBF by sequencing and experiments. Researches show that the non-enterotoxigenic bacteroides fragilis (NTBF) has important probiotics and is critical to the integrity and immune development of intestinal mucosa. The relationship of bacteroides fragilis to the host depends largely on its highly complex and dynamic capsular structure, b.fragilis capsular polysaccharide a (PSA) being the most expressed, immunocompetent one of the 8 different capsular polysaccharides expressed by bacteroides fragilis. Meanwhile, the bacteroides fragilis is used as pioneer probiotics to be part of normal flora in intestinal tracts of people and animals, and the safety of the bacteroides fragilis is guaranteed. Development of the application direction of the bacteroides fragilis extract zwitterionic capsular polysaccharide A for the medicine for treating autoimmune arthritis has prospect and is necessary.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide the application of the zwitterionic capsular polysaccharide A of the bacteroides fragilis in the treatment of autoimmune arthritis. The inventor unexpectedly discovers and proves that the bacteroides fragilis, in particular to zwitterionic capsular polysaccharide A (PSA) extracted from bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685, can inhibit monocytes from secreting rheumatoid arthritis immune response core pro-inflammatory factors TNF-alpha and IL-1 beta in vitro; the anti-inflammatory agent can inhibit inflammatory response and reduce arthritis score and ankle joint thickness of a collagen-induced mouse model of arthritis by inhibiting IL-1 beta and IFN-gamma content in serum in vivo; reducing the arthritis score and paw solvents of the adjuvant-induced model of arthritis in rats. Simultaneously, the contents of Rheumatoid Factors (RF) and C-reactive proteins (CRP) in serum are reduced, the inflammatory response of a collagen antibody-induced arthritis mouse model is relieved, and the arthritis scores and ankle thicknesses of ZY-312PSA groups are obviously reduced. Effectively relieve the symptoms of autoimmune arthritis and improve the life quality of patients.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In the first aspect, the bacteroides fragilis is the bacteroides fragilis ZY-312 with the preservation number of CGMCCNo.10685, and the application of the bacteroides fragilis in preparing a product for preventing and/or treating autoimmune arthritis.
In some embodiments, the bacteroides fragilis is a live or inactivated bacterium.
In some embodiments, the bacteroides fragilis is one or more of a live bacteroides fragilis thallus, a bacteroides fragilis lysate, a bacteroides fragilis liquid culture supernatant, a recombinant, engineered or modified, attenuated, chemically treated, physically treated or inactivated bacteroides fragilis.
In some embodiments, the inactivation means comprises one or more of heat inactivation, pasteurization, dry heat inactivation, short wave Ultraviolet (UVC) methods.
In some of these embodiments, the autoimmune arthritis is rheumatoid arthritis.
In some embodiments, the product is a food, pharmaceutical or nutraceutical.
In some embodiments, the dosage form of the pharmaceutical product comprises a pill, tablet, granule, capsule, oral liquid, or tube feeding formulation. The medicine comprises human medicine or animal medicine.
In some of these embodiments, the food product comprises milk powder, cheese, curd, yogurt, ice cream, or a fermented cereal. The food product may also be an animal food product, such as a feed or the like.
The second aspect of the invention provides an application of bacteroides fragilis capsular polysaccharide A or bacteroides fragilis extract containing polysaccharide capsular A in preparing a medicament, food or health care product for preventing and/or treating autoimmune arthritis, wherein the capsular polysaccharide A or the bacteroides fragilis extract containing polysaccharide capsular A extracts bacteroides fragilis ZY-312 with self-preservation number of CGMCC No.10685, bacteroides fragilis NCTC9343 or other non-enterotoxigenic bacteroides fragilis.
In some of these embodiments, the capsular polysaccharide a lipid is present in an amount of less than 0.02wt%.
Preferably, the capsular polysaccharide a is a lipid-free capsular polysaccharide a;
preferably, the capsular polysaccharide a has the structure shown below:
preferably, the weight average molecular weight of the bacteroides fragilis capsular polysaccharide A is 5-100kD; further preferably, the weight average molecular weight of the bacteroides fragilis capsular polysaccharide A is 40-100kD; further preferably, the capsular polysaccharide A has a weight average molecular weight of 80-90kD; it is further preferred that the fraction in which the molecular weight is distributed between 70KD and 100KD represents 70-80% of the total amount.
Preferably, the pharmaceutically effective dose of the bacteroides fragilis capsular polysaccharide A is 5-45mg/kg.
Preferably, the effective concentration of the in vitro inflammation factor inhibiting factor of the bacteroides fragilis capsular polysaccharide A is 0.1-10 mug/mL.
Preferably, the autoimmune arthritis is rheumatoid arthritis.
In some embodiments, the food product is selected from any one of milk powder, cheese, curd, yogurt, ice cream, milk-based fermented food, fermented cereal; the food may also be an animal food, such as feed, etc.; the food product may also be an infant food or a pet food.
In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier.
Preferably, the dosage form of the medicament can be pills, tablets, granules, capsules, powder, suspension, oral liquid or enema.
Preferably, the medicament may be administered orally, enema or parenterally.
Preferably, the drug may be administered intermittently, periodically, continuously or chronically.
In a third aspect of the present invention, there is provided a composition for the prevention and treatment of autoimmune arthritis, said composition comprising Bacteroides fragilis ZY-312 having a preservation number of CGMCC No.10685 and/or a pharmaceutically effective dose of Bacteroides fragilis capsular polysaccharide A.
In some embodiments, the capsular polysaccharide A extracts Bacteroides fragilis, bacteroides fragilis NCTC9343 or other non-enterotoxigenic Bacteroides fragilis with a self-preservation number of CGMCC No. 10685.
In some of these embodiments, the capsular polysaccharide a is a lipid-free capsular polysaccharide a, or the capsular polysaccharide a lipid is present in an amount of less than 0.02wt%.
In some of these embodiments, the capsular polysaccharide a has a weight average molecular weight of 5-100kD; further preferably, the weight average molecular weight of the bacteroides fragilis capsular polysaccharide A is 40-100kD; further preferably, the capsular polysaccharide A has a weight average molecular weight of 80-90kD; it is further preferred that the fraction in which the molecular weight is distributed between 70KD and 100KD represents 70-80% of the total amount.
Preferably, the pharmaceutically effective dose of the bacteroides fragilis capsular polysaccharide A is 5-45mg/kg.
Preferably, the effective concentration of the in vitro inflammation factor inhibiting factor of the bacteroides fragilis capsular polysaccharide A is 0.1-10 mug/mL.
In some embodiments, the composition is a pharmaceutical composition, a nutraceutical composition, or a probiotic composition.
In some of these embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutical composition may be in the form of a pill, tablet, granule, capsule, powder, suspension, oral liquid, or enema. The administration may be by oral, enema or parenteral forms, and the administration period may be intermittent, periodic, continuous or long-term.
In the aspects of the invention, the preparation method of the bacteroides fragilis capsular polysaccharide A comprises the following steps:
(1) Taking bacterial sludge of a Bacteroides fragilis ZY-312 fermentation culture, adding purified water to suspend the bacterial sludge, adjusting the pH of the bacterial sludge to 3.5 by using a hydrochloric acid solution, extracting, cooling to room temperature, centrifuging, and taking a supernatant to obtain a crude sugar solution; preferably, the pH is adjusted by using 1mol/L hydrochloric acid solution, and/or the extraction condition is 100 ℃, the extraction time is 1.5h, and/or the centrifugation condition is 12000g and the centrifugation is carried out at normal temperature for 10min;
(2) Ultrafiltering and concentrating the crude sugar solution with 10KD ultrafilter membrane to remove small molecular impurities until the conductivity is stable, and collecting the reflux;
(3) Adding 40mmol/LTris-HCl salt in equal volume into the reflux liquid; separating by DEAEDEEEeporoseFastFlow ion exchange column chromatography, tracking and monitoring by SEC-HPLC, combining components with single and symmetrical peak absorption peak at 206nm, ultrafiltering by 10KD ultrafiltration membrane, adding purified water, repeatedly ultrafiltering until conductivity is stable, collecting reflux liquid, and lyophilizing to obtain Bacteroides fragilis capsular polysaccharide A; preferably, the column used in DEAESEPHAROSeFastFlow ion exchange column chromatography is 16mm by 200mm, the flow rate is 20mL/min, the gradient elution is carried out for 25 column volumes by 20mmol/LTris-HCl, and the column volumes are collected in sections and 100 mL/bottle.
Further, the preparation method of the bacterial sludge of the bacteroides fragilis fermentation culture in the step (1) comprises the following steps:
inoculating a single colony into plant source peptone broth for fermentation culture, centrifuging and precipitating the obtained bacterial liquid, removing supernatant, and collecting precipitate to obtain Bacteroides fragilis ZY-312 bacterial mud; preferably, the conditions of the fermentation culture are 8 hours, 37 ℃, and/or the conditions of centrifugal precipitation are 3000r/min rotational speed and 15min centrifugation.
The invention has the beneficial effects that:
a large number of experiments prove that the bacteroides fragilis, in particular to bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685 and zwitterionic capsular polysaccharide A (PSA) thereof can inhibit monocytes from secreting rheumatoid arthritis immune response core pro-inflammatory factors TNF-alpha and IL-1 beta in vitro; the anti-inflammatory agent can inhibit inflammatory response and reduce arthritis score and ankle joint thickness of a collagen-induced mouse model of arthritis by inhibiting IL-1 beta and IFN-gamma content in serum in vivo; reducing the arthritis score and paw solvents of the adjuvant-induced model of arthritis in rats. Simultaneously, the contents of Rheumatoid Factors (RF) and C-reactive proteins (CRP) in serum are reduced, the inflammatory response of a collagen antibody-induced arthritis mouse model is relieved, and the arthritis scores and ankle thicknesses of ZY-312PSA groups are obviously reduced. The nonsteroidal anti-inflammatory drug has serious adverse reaction to the blood system, the digestive tract system and the like of patients, and the compliance of the patients is affected, so that the treatment effect is affected. ZY-312 and PSA thereof not only effectively relieve the symptoms of autoimmune arthritis, but also have better safety than the prior main therapeutic drugs aiming at autoimmune arthritis, and greatly help to improve the life quality of patients.
Drawings
FIG. 1 is a graph showing colony characteristics of Bacteroides fragilis ZY-312 in example 1 of the present invention;
FIG. 2 is a view of the gram-stained Bacteroides fragilis ZY-312 according to example 1 of the present invention;
FIG. 3 is a chart of the analysis of the capsular polysaccharide A nuclear magnetic resonance spectrometer of example 3 of the present invention;
A-E are respectively 1H spectrum, 13C spectrum, COSY spectrum, HSQC spectrum and HMBC spectrum analyzed by the capsular polysaccharide A nuclear magnetic resonance spectrometer in the embodiment 3;
FIG. 4 shows the chemical structural formula of the structural unit of the Bacteroides fragilis capsular polysaccharide A prepared in example 3 of the invention.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples were all commercially available. All cells were purchased from ATCC; all cell culture materials and pancreatin were purchased from Gibco; all experimental animals were purchased from Zhejiang Veitz laboratory animal technologies Co., ltd; or may be prepared by known methods. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Example 1: fermentation culture of bacteroides fragilis
The bacteroides fragilis strain is streaked and inoculated on a blood plate for anaerobic culture for 48 hours. Colony morphology, staining characteristics, size, sphere shape, distribution, etc. were observed. Colony characteristics: after the bacteroides fragilis ZY-312 is cultured on a blood plate for 48 hours, the bacteroides fragilis ZY-312 is slightly convex, semitransparent, white, smooth in surface and free from hemolysis, and the colony diameter is between 1 and 3mm, as shown in figure 1.
Morphology under microscope: the bacteroides fragilis ZY-312 was subjected to gram-stain microscopic examination to show a typical rod shape for gram-negative bacteria, and was rounded at both ends to be densely stained, and the non-colored part in the middle of the thallus was formed as a cavitation, see FIG. 2.
The bacteroides fragilis strain is streaked and inoculated on a blood plate for anaerobic culture for 48 hours. Colony morphology, staining characteristics, size, sphere shape, distribution, etc. were observed. Colony characteristics: after the bacteroides fragilis ZY-312 is cultured on a blood plate for 48 hours, the bacteroides fragilis ZY-312 is slightly convex, semitransparent, white, smooth in surface and free from hemolysis, and the colony diameter is between 1 and 3mm, as shown in figure 1.
Morphology under microscope: the bacteroides fragilis ZY-312 was subjected to gram-stain microscopic examination to show a typical rod shape for gram-negative bacteria, and was rounded at both ends to be densely stained, and the non-colored part in the middle of the thallus was formed as a cavitation, see FIG. 2.
Inoculating the single bacterial colony cultured in the culture medium to a plant source peptone liquid culture medium for fermentation culture for 8 hours (the temperature is 37 ℃), and obtaining a live bacterial liquid of the bacteroides fragilis; and (3) centrifuging the obtained bacterial liquid to precipitate at a rotating speed of 3000r/min, centrifuging for 15min, removing supernatant, and collecting precipitate to obtain bacteroides fragilis ZY-312 and NCTC9343 bacterial sludge respectively.
EXAMPLE 2 preparation of Bacteroides fragilis inactivated powder
(1) Taking the bacteroides fragilis fermentation liquor prepared by the steps, centrifuging the fermentation liquor, collecting wet thalli, and mixing the thalli according to the thalli: physiological saline=1 (10-30) (m: v) and re-suspending and washing the bacterial mud, and centrifuging again to collect the washed bacterial body. m represents mass and v represents volume.
(2) Adding an excipient mixed by 5% maltodextrin and 0.9% sodium chloride into the thalli obtained in the step (1), and mixing according to the thalli: excipient=1 (5-15) (m: m) and then stirred and dispersed, heat inactivated at (70-100) ±5 ℃ for (20-40) ±5min and centrifuged, and the bacterial sludge is collected.
(3) Adding an excipient into the inactivated bacterial sludge collected in the step (2) to enable the total weight to be consistent with the weight of the bacterial liquid before inactivation, and stirring to completely dissolve the bacterial liquid to obtain an inactivated bacterial powder stock solution.
(4) Vacuum freeze drying the inactivated bacteria powder stock solution obtained in the step (3), pre-freezing for 1-3 h at minus 40+/-2 ℃, pre-freezing for 0.5-1 h at minus 20+/-2 ℃, pre-freezing for 0.5-2 h at minus 40+/-2 ℃ and finally, performing primary drying (-5+/-2 ℃ and 0+/-2 ℃) and analytical drying (35+/-2 ℃) at the vacuum degree of 0.25mbar to prepare the inactivated bacteria powder, wherein the bacterial number of the bacteria powder reaches 1 multiplied by 10 11 Cells/g or more.
NCTC9343 inactivated bacterial powder is prepared by the same method.
Samples were prepared for the following examples.
Example 3: preparation of Bacteroides fragilis capsular polysaccharide A
(1) Taking 50g of the bacterial sludge prepared in the example 1, adding 300g of purified water to suspend the bacterial sludge, adjusting the pH of the bacterial sludge to 3.5 by using 1mol/L hydrochloric acid solution, extracting for 1.5 hours at 100 ℃, cooling to room temperature, centrifuging for 10 minutes at 12000g of room temperature, and taking the supernatant to obtain a crude sugar solution;
(2) Ultrafiltering and concentrating the crude sugar solution with 10KD ultrafilter membrane to remove small molecular impurities until the conductivity is stable, and collecting the reflux;
(3) Adding an equal volume of 40mmol/LTris-HCl (pH 8.5) salt; DEAESepharoseFast Flow ion exchange column chromatography (16 mm. Times.200 mm), flow rate 20mL/min, gradient elution of 20mmol/LTris-HCl (pH 8.5, containing 0.2 mol/LNaCl) for 25 column volumes, sectional collection, 100 mL/bottle (component), SEC-HPLC tracking and monitoring, combining components with 206nm absorption peak as single and symmetrical peak, ultrafiltering with 10KD ultrafiltration membrane, adding purified water for repeated ultrafiltration until conductivity is stable, collecting reflux liquid, and lyophilizing to obtain Bacteroides fragilis ZY-312 extract;
(4) Weighing 30mg of the Bacteroides fragilis extract described in the step (3), and dissolving in 0.5. 0.5mLD 2 O, 1. Mu.l of acetone (1H, 2.22;13C, 30.89) was added for calibration. Analysis of the 1H and 13C, COSY, HSQC, HMBC spectra (see FIGS. 3A-E) using a 500MHz Bruker NMR spectrometer confirmed that the Bacteroides fragilis extract collected in step (3) was capsular polysaccharide A, the bound lipid content was less than 0.02%, the protein residue was less than 1%, and the nucleic acid residue was less than 0.05%. The molecular weight of the obtained capsular polysaccharide A is 80-90kDa, mw/Mn is 1.0-1.2 by GPC (gel permeation chromatography), and the chemical structure is shown in figure 4. The capsular polysaccharide of NCTC9343 was also prepared using this method.
(5) Preparation of capsular polysaccharide A with different molecular weights: the capsular polysaccharide produced is degraded by methods including, but not limited to, chemical degradation, physical degradation, and biological degradation. In the embodiment, the capsular polysaccharide A with different molecular weights is obtained by adopting an ultrasonic method, wherein the capsular polysaccharide A is treated for 3 hours, 2 hours and 0 hour at 195kHz and 20 ℃ respectively, and the capsular polysaccharide A with the molecular weights of 2kDa, 5kDa, 40kDa and the original molecular weight is respectively collected. Are denoted as ZY-312PSA_1, ZY-312PSA_2, ZY-312PSA_3 and ZY-312PSA, respectively.
Example 4: bacteroides fragilis capsular polysaccharide A inhibits LPS-induced inflammatory factor up-regulation in vitro
1. Materials and Experimental design
This example uses the human monocyte THP-1 cell line for endotoxin tolerance testing. Pretreatment was performed with ZY-312 extract PSA of different molecular weights for 24 hours. Subsequently, the cells were washed with fresh medium, and 10ng/mL of LPS (Sigma, L2886) was formulated to stimulate the cells for 24 hours. Cell culture supernatants were collected and levels of the cytokines TNF- α (R & DSsystems, DTA 00D) and IL-1β (R & DSsystems, QK 201) were measured in the supernatants.
2. Culture method
(1) Resuscitation and passaging of THP-1 cells
THP-1 cells were grown in DMEM medium containing 10% FBS, 1% penicillin/streptomycin was addedAt 37℃ 5% CO 2 Culturing in an incubator with concentration and saturated humidity. The medium was changed every two days.
a) The cell lines were removed from the liquid nitrogen and rapidly thawed in a 37℃thermostat water bath. The freezing tube was opened under sterile conditions, the liquid was transferred to a 15mL centrifuge tube, the cells were resuspended in 2-3mL of DMEM complete medium, and centrifuged at 1000rpm for 5min. After discarding the supernatant, 5mL of DMEM complete medium was added, and the cells were inoculated into a flask at 37℃in 5% CO 2 The culture was performed in a concentration incubator for 48 hours.
b) The stock culture was discarded, 1mL of 0.25% trypsin was added, digested for 5min, 1mL of DMEM medium containing 10% fetal bovine serum was added to stop the reaction, and the mixture was transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 5 min; removing supernatant, adding appropriate DMEM complete medium containing 10% foetal calf serum, tapping cells into culture flask, and extracting with 5% CO at 37deg.C 2 The culture was performed in a concentration incubator for 24 hours.
(2) Preparation of bacteroides fragilis extract capsular polysaccharide A
ZY-312 extract PSA and products of different molecular weights were prepared as in examples 1 and 3.
3. ZY-312PSA upregulates inflammatory factors by inhibiting LPS-induced THP-1 cells
(1) 1mL of THP-1 cells (10 6 Respectively inoculating into 6-well plates at 37deg.C, 5% CO 2 The culture is carried out for 24 hours in a concentration incubator until the cells adhere to the wall. 2mL of the crude molecular weight ZY-312 extract PSA (ZY-312 PSA) was added, respectively, and 2mL of 10. Mu.g/mL of ZY-312PSA_1, ZY-312PSA_2, ZY-312PSA_3 were added to 3 groups, respectively, to perform pretreatment for 24 hours, followed by washing the cells three times with fresh medium, and stimulating the cells with 10ng/mL of LPS for 24 hours. Cell culture supernatants were collected.
(2) Cytokine content in cell supernatants was measured using the Elisa kit:
a) After 24h of LPS incubation, the cell supernatant culture was collected in a 1.5ml centrifuge tube, centrifuged at 4000rpm for 20min and the supernatant was taken to a new centrifuge tube. The samples were stored in a-80 ℃ refrigerator.
b) The harvested cell supernatant was detected with reference to the Elisa kit instructions.
4. Experimental results:
TABLE 1 cytokine effects induced by LPS after administration (mean+ -SD)
Note that: compared with the model group, the P value between the two groups is calculated according to an unpaired-test (two-charged) method. * Indicating significant difference P <0.05; * Represents a very significant difference P <0.01.
Cytokines play an important role in the regulation of the immune response of rheumatoid arthritis, and TNF- α and IL-1β, both of which are important pro-inflammatory cytokines, are positively correlated to the condition of rheumatoid arthritis to reflect the degree of activity of inflammation, playing a key role in synovitis and bone destruction. Inhibiting the amounts of TNF- α and IL-1β in vivo can inhibit the symptoms of autoimmune arthritis.
As shown in Table 1, the amounts of inflammatory cytokines TNF-. Alpha.and IL-1β secreted by the THP-1 cells of the model group (induced inflammation by LPS addition) were significantly up-regulated. The pretreatment of the Bacteroides fragilis ZY-312 original molecular weight PSA can inhibit THP-1 cells under inflammatory conditions from secreting TNF-alpha and IL-1 beta, and has statistical significance compared with a model group. LPS tolerance experiments were performed using different molecular weight PSAs of ZY-312PSA_1, ZY-312PSA_2, ZY-312PSA_3. The results show that PSA with molecular weight of more than or equal to 5kDa has LPS tolerance capability, remarkably inhibits TNF-alpha and IL-1 beta secretion compared with a model group, but compared with a PSA treatment group with original molecular weight, ZY-312PSA_2 and ZY-312PSA_3 with small molecular weight have weaker functions of inhibiting LPS-induced TNF-alpha and IL-1 beta secretion than ZY-312PSA with original molecular weight.
Therefore, bacteroides fragilis ZY-312PSA can treat autoimmune arthritis by inhibiting the secretion of TNF-alpha and IL-1β by monocytes, and the effect of the primary molecular weight is optimal.
Example 5: pharmacodynamic test of Bacteroides fragilis capsular polysaccharide A for treating collagen-induced arthritis mice
1. Collagen-induced arthritis model construction
Type II collagen was dissolved in 0.1mol/L acetic acid (mass concentration: 2 g/L) at 4℃and stirred to be sufficiently dissolved, followed by standing in a refrigerator at 4℃overnight. Then mixing Complete Freund's Adjuvant (CFA) with type II collagen in equal volume, emulsifying to obtain CII emulsion, injecting 0.1mL of the emulsion into tail root of each mouse for inflammatory, and injecting 0.1mL of the emulsion into 20d abdominal cavity as excitation injection.
2. Grouping and administration
Except for 10 normal animals, the remaining model mice were randomized into 5 groups at 32d according to the clinical score for arthritis, 10 per group: model group, ZY-312PSA low (10 mg/kg), medium (20 mg/kg), high (30 mg/kg) dose group, celecoxib group (80 mg/kg).
Model group: normal saline 10mL/kg, 1 time a day, and 33-46d continuous gastric lavage.
ZY-312PSA low, medium, high dose group: the capsular polysaccharide A (PSA) of Bacteroides fragilis extract is prepared into 1, 2 and 3mg/mL respectively by physiological saline, and is administrated by intragastric administration of 10mL/kg for 1 time/day, and 33-46d continuous intragastric administration is carried out.
Celecoxib group: the positive control drug was celecoxib. The dosage of the mice calculated by the body surface area is 80mg/kg according to 200mg and 2 times a day, namely 400 mg/day. Celecoxib is prepared into 8mg/mL suspension by using physiological saline, and is administrated by lavage for 1 time/day according to 10 mL/kg.
3. Detection index and method
(1) Clinical score for arthritis
4 paws were observed at 32, 36, 40, 44, 46d, clinical scores were made based on severity of arthritis, each paw scored 0-3 points, and the total sum score of 4 paws per mouse was the clinical score. The scoring criteria were:
1 minute, slight inflammation appears locally;
2 minutes, the paw is moderately swollen;
3 minutes, severe inflammation and swelling occurred in all parts of the paw.
(2) Ankle joint thickness measurement
Ankle thicknesses (mm) of left and right hind legs of the mice were measured with vernier calipers having precision of 0.01mm at 32, 36, 40, 44, and 46d, respectively, and the average was counted. When measuring the thickness of the ankle, the thickest part of the ankle and the most serious part of inflammatory edema are measured.
(3) Cytokine detection
Levels of IFN-gamma (R & DSsystems, DY 485), IL-1β (R & DSsystems, MLB 00C) cytokines were measured in mouse serum using Elisa.
4. Test results
(1) Clinical score for arthritis
TABLE 2 influence on arthritis score after dosing (mean+ -SD)
Note that: compared with the model group, the P value between the two groups is calculated according to an unpaired-test (two-charged) method. * Indicating significant difference P <0.05; * Represents a very significant difference P <0.01.
As can be seen from table 2, the arthritis score was reduced for each of the ZY-312PSA groups compared to the model group, with statistical differences at 40, 44, 46 d; PSA low dose groups were statistically different at 44, 46 d. Celecoxib, a positive drug, can also treat collagen-induced arthritis in mice, but has no significant difference from ZY-312 PSA. Demonstrating that ZY-312PSA is effective in alleviating inflammatory swelling of joints in mice in a collagen-induced model of arthritis in mice.
(2) Ankle joint thickness measurement
TABLE 3 influence on ankle thickness after administration (mean+ -SD)
Note that: compared with the model group, the P value between the two groups is calculated according to an unpaired-test (two-charged) method. * Indicating significant difference P <0.05; * Represents a very significant difference P <0.01.
As can be seen from Table 3, the model group gradually increased in ankle thickness from 32-46d mice, with 44d peaking. The bacteroides fragilis ZY-312PSA low dose group had statistical differences at 44, 46d compared to the model group; the medium and high dose groups of PSA were statistically different at 40, 44, 46d and had no significant difference in efficacy from the positive drug celecoxib.
(3) Cytokines and methods of use
TABLE 4 effects on mouse serum cytokines after dosing (mean+ -SD)
Note that: compared with the model group, the P value between the two groups is calculated according to an unpaired-test (two-charged) method. * Indicating significant difference P <0.05; * Represents a very significant difference P <0.01.
The cytokine IL-1 beta plays a key role in synovitis and bone destruction, and is to some extent positively correlated with rheumatoid arthritis conditions to reflect the degree of activity of inflammation. IFN-gamma is also closely related to the onset of rheumatoid arthritis, is an interferon with general immunoregulatory action, and is also a marked Thl-type cytokine. As can be seen from Table 4, the serum IL-1. Beta. And IFN-gamma. Content of the model group was significantly increased. Compared with the model group, the bacteroides fragilis ZY-312PSA has statistical differences in serum IL-1 beta and IFN-gamma contents in low, medium and high dose groups, and has no obvious difference with the positive drug celecoxib.
In conclusion, the bacteroides fragilis ZY-312 extract capsular polysaccharide A can effectively treat collagen-induced arthritis mice.
Example 6: efficacy test of Bacteroides fragilis and capsular polysaccharide A treatment adjuvant-induced arthritis rats
1. Test design and flow
Wistar rats were purchased and after quarantine, 100 rats were randomly divided into 10 groups of 10 rats: normal group (normal saline), model group (normal saline), ZY-312PSA low (5 mg/kg), medium (15 mg/kg), high (45 mg/kg) dose group, ZY-312 group (10) 9 CFU/only), inactivated ZY-312 group (10) 9 Cells/only), NCTC-9343 group (10) 9 CFU /) NCTC-9343PSA (45 mg/kg), meloxicam group (0.5 mg/kg of Wilson pharmaceutical industry, suzhou). Except for 10 animals in the normal group, the other animals were modeled. Firstly, preparing a molding reagent CFA, wherein the weight ratio of liquid paraffin to anhydrous lanolin is 2:1 into 1mL of mixed solution, autoclaving, and adding attenuated BCG vaccine inactivated at 80deg.C for 1 hr. The CFA with the final mass concentration of 10g/L is injected into the bilateral hind feet of the rat at 0.1 mL/side/time by subcutaneous injection of the plantar region. The 8 th to 27 th doses were orally administered 1 time per day at the designed dose.
2. Grouping and administration
100 rats were randomly divided into 10 groups of 10: normal, model, ZY-312PSA low (5 mg/kg), medium (15 mg/kg), high (45 mg/kg) dose groups, ZY-312 (10) 9 CFU/only), inactivated ZY-312 group (10) 9 Cells/only), NCTC-9343 group (10) 9 CFU/alone), NCTC-9343PSA (45 mg/kg), meloxicam group (0.5 mg/kg).
Model group: normal saline 10mL/kg, 1 time a day, 8-27d continuous gastric lavage.
ZY-312PSA low, medium, high dose group: the bacteroides fragilis capsular polysaccharide A (PSA) is respectively prepared into 0.5 mg/mL, 1.5 mg/mL and 4.5mg/mL by physiological saline, and is administrated by intragastric administration of 10mL/kg for 1 time/day, and continuous intragastric administration is carried out for 8-27 days.
ZY-312, inactivated ZY-312 and NCTC-9343 groups: three groups of fragile quasi-rods The bacteria are respectively prepared into 5×10 with physiological saline 8 CFU/mL was administered at 2 mL/lavage, 1 time/day, 8-27d continuous lavage.
NCTC-9343PSA group: the capsular polysaccharide A (PSA) of the Bacteroides fragilis standard strain is prepared into 4.5mg/mL with physiological saline, and is administrated by lavage of 10mL/kg for 1 time/day, and 8-27d continuous lavage is carried out.
Meloxicam group: the positive control drug was meloxicam. The dosage of the rat is 0.5mg/kg calculated by body surface area. Meloxicam is prepared into liquid medicine of 0.05mg/mL with physiological saline, and the liquid medicine is administrated by irrigating the stomach for 1 time per day according to 10 mL/kg.
3. Detection index and method
(1) Clinical score for arthritis
4 paws were observed at 8, 14, 20, 26d, clinical scores were made based on the severity of arthritis, each paw was scored 0-4 points, and the total score of 4 paws per rat summed to the clinical score. The scoring criteria were:
1 minute, erythema and slight swelling of the ankle joint;
2 minutes, erythema and slight swelling of ankle to toe joint
3 minutes, erythema and moderate swelling of ankle to toe joints;
4 minutes, erythema and severe swelling occurred from ankle to toe.
(2) Foot paw volume measurement
The paw volumes were measured by drainage at 8, 14, 20, 26d, respectively. Firstly, marking a circle at a position about 1cm above an ankle joint of a rat, filling water into a 10mL glass graduated tube, keeping the foot of the rat vertically inserted into the water until the marked position, taking out, adding water to the original water surface height by using a syringe filled with water, recording the residual water quantity in the syringe, wherein the difference value is the water quantity discharged by foot paws, each measurement is carried out by a special person, and each foot paw is measured for 2 times and the average value is taken to minimize the error.
(3) Cytokine detection
Levels of cytokines such as IFN-. Gamma.s (R & DSsystems, RIF 00) and IL-1β (R & DSsystems, RLB 00) in rat serum were measured using Elisa.
4. Test results
The model group gradually increased from 8-27d rat arthritis and paw volumes, with 20d peaking. As can be seen from table 5, the arthritis score and paw volume were reduced in the ZY-312PSA, the high dose group, compared to the model group, with statistical differences at 14, 20, 26 d; the PSA low dose group, ZY-312 group, inactivated ZY-312 group, NCTC-9343 group and NCTC-9343PSA had statistical differences at 20, 26 d. The positive drug meloxicam can also treat adjuvant-induced arthritis in rats, but has no obvious difference with ZY-312PSA in curative effect. From the statistics of 20 and 26d, the results of arthritis scores and paw volumes of the ZY-312 live bacteria and inactivated bacteria administration groups are statistically different from those of the model group, and are better than the treatment effect of the NCTC-9343 group, and the results of the arthritis scores and paw volumes of the ZY-312 live bacteria group 26d are obviously lower than those of the NCTC-9343 group (P < 0.05); the treatment effect of the ZY-312 PSA-dosed group was better than that of the standard strain PSA at the same dose, and there was a statistical difference at 20 d. It is demonstrated that ZY-312PSA and live bacteria and inactivated bacteria can effectively relieve joint inflammation and swelling of rats and reduce paw volume in a collagen-induced rat arthritis model.
(1) Clinical score for arthritis
TABLE 5 influence on arthritis score after dosing (mean+ -SD)
Note that: the P-value between the two groups was calculated according to the unpaired-test (two-charged) method, compared to the model group. * Indicating significant difference P <0.05; * Represents a very significant difference P <0.01.ZY-312PSA high was compared to NCTC-9343PSA group, and the P values between the two groups were calculated according to the unpaired-test (two-charged) method. # represents significant difference P <0.05; ZY-312 is compared to NCTC-9343, and the P value between the two groups is calculated according to the unpaired-test (two-measured) method, where @ indicates that the difference is significant P <0.05.
(2) Foot paw volume measurement
TABLE 6 influence of paw volumes after administration (mean+ -SD)
Note that: the P-value between the two groups was calculated according to the unpaired-test (two-charged) method, compared to the model group. * Indicating significant difference P <0.05; * Represents a very significant difference P <0.01.ZY-312PSA high was compared to NCTC-9343PSA group, and the P values between the two groups were calculated according to the unpaired-test (two-charged) method. # represents significant difference P <0.05; ZY-312 is compared to NCTC-9343, and the P value between the two groups is calculated according to the unpaired-test (two-measured) method, where @ indicates that the difference is significant P <0.05.
(2) Cytokines and methods of use
TABLE 7 cytokine levels (mean+ -SD) for groups of mice
Note that: compared to model group, x represents significant difference P <0.05; * Represents very significant difference P <0.01; ZY-312PSA high was compared to NCTC-9343PSA group, the P values between the two groups were calculated according to the unpaired-test (two-measured) method, # indicating significant difference P <0.05; # denotes very significant difference P <0.01; ZY-312 is compared with NCTC-9343, and the P value between the two groups is calculated according to the unpaired-test (two-measured) method, where @ represents that the difference is significant P <0.05, and @ represents that the difference is extremely significant P <0.01.
The cytokine IL-1 beta plays a key role in synovitis and bone destruction, and is to some extent positively correlated with rheumatoid arthritis conditions to reflect the degree of activity of inflammation. IFN-gamma is also closely related to the onset of rheumatoid arthritis, is an interferon with general immunoregulatory action, and is also a marked Thl-type cytokine. As can be seen from Table 7, the serum IL-1. Beta. And IFN-gamma. Content of the model group was significantly increased. The serum IL-1. Beta. And IFN-. Gamma.contents of Bacteroides fragilis ZY-312PSA were statistically different from those of the model group, the medium-high dose group, the ZY-312 group, the inactivated ZY-312 group, the NCTC-9343 group and the NCTC-9343PSA group. The effect of the ZY-312PSA administration group on down-regulating IL-1 beta and IFN-gamma inflammatory factors is superior to that of the standard strain PSA at the same dosage, and the effect is statistically different. ZY-312 has better IL-1 beta and IFN-gamma inhibition than NCTC-9343 group and has statistical difference.
In conclusion, the bacteroides fragilis ZY-312 and the extract capsular polysaccharide A thereof can effectively treat the rats with arthritis induced by the adjuvant.
Example 7: drug effect test I, test design and flow of bacteroides fragilis capsular polysaccharide A for treating collagen antibody induced arthritis mice
And purchasing 7-week-old male DBA/1 mice, and performing model construction on the rest animals except 10 normal animals after quarantine is qualified. Mice at 0d were intraperitoneally injected with 5mg of anti-collagen antibody mixture (Chondrex) and mice at 3d were intraperitoneally injected with 50 μg of lps. Randomly 60 mice were randomly divided into 6 groups of 10: normal (saline), model (saline), ZY-312PSA low (10 mg/kg), medium (20 mg/kg), high (30 mg/kg) dose, meloxicam (0.5 mg/kg). 1-15d are orally administered 1 time per day at the designed dose.
2. Grouping and administration
60 mice were randomly divided into 6 groups of 10: normal, model, ZY-312PSA low (10 mg/kg), medium (20 mg/kg), high (30 mg/kg) dose groups, meloxicam group (0.5 mg/kg).
Model group: normal saline 10mL/kg, 1 time a day, and 1-15 days for continuous gastric lavage;
ZY-312PSA low, medium, high dose group: the capsular polysaccharide A (PSA) of Bacteroides fragilis extract is prepared into 1, 2 and 3mg/mL respectively by physiological saline, and is administrated by intragastric administration of 10mL/kg for 1 time/day and 1-15d continuously.
Meloxicam group: the positive control drug was meloxicam. The dosage of the mice calculated by the body surface area is 0.5mg/kg. Celecoxib is prepared into a suspension of 0.05mg/mL by using normal saline, and is administrated by intragastric administration of 10mL/kg for 1 time/day and for 1-15 days.
3. Detection index and method
(1) Clinical score for arthritis
4 paws were observed at 7, 11, 15d, clinical scores were made according to the severity of arthritis, each paw was scored 0-4 points, and the total sum score of 4 paws per mouse was the clinical score. The scoring criteria were:
1 minute, erythema and slight swelling of the ankle joint;
2 minutes, erythema and slight swelling of ankle to toe joint
3 minutes, erythema and moderate swelling of ankle to toe joints;
4 minutes, erythema and severe swelling occurred from ankle to toe.
(2) Ankle joint thickness measurement
Ankle thicknesses (mm) of left and right hind legs of the mice were measured with vernier calipers having a precision of 0.01mm at 7, 11, and 15d, respectively, and the average was counted. When measuring the thickness of the ankle, the thickest part of the ankle and the most serious part of inflammatory edema are measured.
(3) Cytokine detection
A biochemical index detection program of the mouse serum is selected, and a biochemical analyzer is adopted to measure the content of rheumatoid factors (Rheumatoid factor, RF) and C-reactive protein (CRP) in the serum.
4. Experimental results
The foot paw of the mice in experiment 3d starts to be swollen, the front foot paw and the rear foot paw are sequentially red and swollen and are gradually aggravated, meanwhile, the arthritis score and the ankle joint thickness are continuously increased along with the disease development, the 11d swelling reaches the peak, and the 15d foot paw swelling is gradually resolved. As can be seen from Table 8, the ZY-312PSA low, medium and high dose groups had reduced arthritis scores and ankle thickness compared to the model group, and had statistical differences at 7, 11, and 15 d. The positive drug meloxicam can also treat collagen antibody induced arthritis in rats, but has no obvious difference with ZY-312PSA in curative effect. It is demonstrated that ZY-312PSA was effective in reducing the mouse arthritis score and ankle thickness and paw swelling in a collagen antibody-induced mouse arthritis model.
(1) Clinical score for arthritis
TABLE 8 influence on arthritis score after dosing (mean+ -SD)
Note that: the P-value between the two groups was calculated according to the unpaired-test (two-charged) method, compared to the model group. * Indicating significant difference P <0.05; * Represents a very significant difference P <0.01.
(2) Ankle joint thickness measurement
TABLE 9 influence on ankle thickness after administration (mean+ -SD)
Note that: compared with the model group, the P value between the two groups is calculated according to an unpaired-test (two-charged) method. * Indicating significant difference P <0.05; * Represents a very significant difference P <0.01.
(2) Cytokines and methods of use
Table 10 levels of mouse cytokines (mean+ -SD) for each group
Note that: compared to the model group, x represents a significant difference p <0.05; * Represents a very significant difference p <0.01.
CRP and RF are the most commonly used biochemical indicators for clinical diagnosis and evaluation of rheumatoid arthritis, and the elevation of RF and CRP in the serum of rheumatoid arthritis patients is positively correlated with disease severity and activity. As can be seen from table 10, the serum CRP and RF content of the model group were significantly increased compared to the normal control group. The serum RF and CRP levels were reduced in the high dose group, as compared to the model group, with statistical differences in the Bacteroides fragilis ZY-312 PSA. Serum RF and CRP levels were reduced in PSA low dose groups, with RF alone being statistically significant compared to the model group.
In conclusion, the bacteroides fragilis ZY-312 extract capsular polysaccharide A can effectively treat collagen antibody induced arthritis mice.
Of course, the present invention is capable of other various embodiments and its several details are capable of modification and variation in light of the present invention, as will be apparent to those skilled in the art, without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. An application of bacteroides fragilis in preparing a medicine, food or health care product for preventing and/or treating autoimmune arthritis, wherein the bacteroides fragilis is bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685;
preferably, the bacteroides fragilis is a live bacterium or an inactivated bacterium;
preferably, the bacteroides fragilis is one or more of live bacteroides fragilis thallus, bacteroides fragilis subjected to gene recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation, bacteroides fragilis lysate and bacteroides fragilis liquid culture supernatant;
preferably, the inactivation means comprises one or more of heat inactivation, pasteurization, dry heat inactivation, short wave Ultraviolet (UVC) methods;
Preferably, the autoimmune arthritis is rheumatoid arthritis.
2. The use according to claim 1, wherein,
the food is selected from any one of milk powder, cheese, curd, yogurt, ice cream, milk-based fermented food, and fermented cereal; the food may also be an animal food, such as feed, etc.; the food product may also be an infant food or a pet food.
3. The use according to claim 1, wherein,
the medicine comprises one or more of the following pharmaceutically acceptable auxiliary materials: diluents, excipients, binders, lubricants, suspending agents, coating agents and solubilizing agents; preferably, the pharmaceutically acceptable excipients are: one or more of water, saline solution, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposomes, saccharides, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, aromatic oil, mono-and di-fatty acid glycerides, petrochemical (fatty acid esters), hydroxymethyl cellulose, polyvinylpyrrolidone;
preferably, the dosage form of the medicine comprises pills, tablets, granules, capsules, oral liquid or tube feeding preparation;
Preferably, the drug may be administered intermittently, periodically, continuously or chronically;
preferably, the medicament comprises a human medicament or an animal medicament, which is useful for humans or animals;
preferably, the bacteroides fragilis can be administered prophylactically or therapeutically alone or in combination with other probiotics and/or probiotic materials; when administered in combination, the administration may be performed in a single formulation or in separate formulations, simultaneously or at different times, using the same or different routes of administration.
4. An application of bacteroides fragilis capsular polysaccharide A or bacteroides fragilis extract containing polysaccharide capsular A in preparing a medicine, food or health care product for preventing and/or treating autoimmune arthritis, wherein the capsular polysaccharide A or the bacteroides fragilis extract containing polysaccharide capsular A extracts bacteroides fragilis ZY-312 with self-preservation number of CGMCCNo.10685, bacteroides fragilis NCTC9343 or other non-enterotoxigenic bacteroides fragilis;
preferably, the capsular polysaccharide a lipid is present in an amount of less than 0.02wt%;
preferably, the capsular polysaccharide a is a lipid-free capsular polysaccharide a;
preferably, the capsular polysaccharide a has the structure shown below:
Preferably, the weight average molecular weight of the bacteroides fragilis capsular polysaccharide A is 5-100kD; further preferably, the weight average molecular weight of the bacteroides fragilis capsular polysaccharide A is 40-100kD; further preferably, the capsular polysaccharide A has a weight average molecular weight of 80-90kD; further preferably, the fraction in which the molecular weight is distributed between 70KD and 100KD represents between 70 and 80% of the total amount;
preferably, the autoimmune arthritis is rheumatoid arthritis.
5. The use according to claim 4, wherein,
the food is selected from any one of milk powder, cheese, curd, yogurt, ice cream, milk-based fermented food, and fermented cereal; the food may also be an animal food, such as feed, etc.; the food product may also be an infant food or a pet food.
6. The use according to claim 4, wherein,
preferably, the medicament further comprises a pharmaceutically acceptable carrier;
preferably, the dosage form of the medicament can be pills, tablets, granules, capsules, powder, suspension, oral liquid or enema;
preferably, the medicament may be administered orally, enema or parenterally;
preferably, the drug may be administered intermittently, periodically, continuously or chronically.
7. The use according to any one of claims 4-6, wherein,
the preparation method of the bacteroides fragilis capsular polysaccharide A comprises the following steps:
(1) Taking bacterial sludge of a Bacteroides fragilis ZY-312 fermentation culture, adding purified water to suspend the bacterial sludge, adjusting the pH of the bacterial sludge to 3.5 by using a hydrochloric acid solution, extracting, cooling to room temperature, centrifuging, and taking a supernatant to obtain a crude sugar solution; preferably, the pH is adjusted by using 1mol/L hydrochloric acid solution, and/or the extraction condition is 100 ℃, the extraction time is 1.5h, and/or the centrifugation condition is 12000g and the centrifugation is carried out at normal temperature for 10min;
(2) Ultrafiltering and concentrating the crude sugar solution with 10KD ultrafilter membrane to remove small molecular impurities until the conductivity is stable, and collecting the reflux;
(3) Adding 40mmol/LTris-HCl salt in equal volume into the reflux liquid; separating by DEAEDEEEeporoseFastFlow ion exchange column chromatography, tracking and monitoring by SEC-HPLC, combining components with single and symmetrical peak absorption peak at 206nm, ultrafiltering by 10KD ultrafiltration membrane, adding purified water, repeatedly ultrafiltering until conductivity is stable, collecting reflux liquid, and lyophilizing to obtain Bacteroides fragilis capsular polysaccharide A; preferably, the column used in DEAESEPHAROSeFastFlow ion exchange column chromatography is 16mm by 200mm, the flow rate is 20mL/min, the gradient elution is carried out for 25 column volumes by 20mmol/LTris-HCl, and the column volumes are collected in sections and 100 mL/bottle.
8. The use according to claim 7, wherein,
the preparation method of the bacterial sludge of the bacteroides fragilis fermentation culture in the step (1) comprises the following steps:
inoculating a single colony into plant source peptone broth for fermentation culture, centrifuging and precipitating the obtained bacterial liquid, removing supernatant, and collecting precipitate to obtain Bacteroides fragilis ZY-312 bacterial mud; preferably, the conditions of the fermentation culture are 8 hours, 37 ℃, and/or the conditions of centrifugal precipitation are 3000r/min rotational speed and 15min centrifugation.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310182987.2A CN116327809A (en) | 2023-02-27 | 2023-02-27 | Application of bacteroides fragilis and extract capsular polysaccharide A thereof in preparation of product for preventing and treating autoimmune arthritis |
| PCT/CN2024/078796 WO2024179462A1 (en) | 2023-02-27 | 2024-02-27 | Use of bacteroides fragilis and extract capsular polysaccharide a thereof in preparation of product for preventing and treating autoimmune arthritis |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310182987.2A CN116327809A (en) | 2023-02-27 | 2023-02-27 | Application of bacteroides fragilis and extract capsular polysaccharide A thereof in preparation of product for preventing and treating autoimmune arthritis |
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| CN202310182987.2A Pending CN116327809A (en) | 2023-02-27 | 2023-02-27 | Application of bacteroides fragilis and extract capsular polysaccharide A thereof in preparation of product for preventing and treating autoimmune arthritis |
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| CN (1) | CN116327809A (en) |
| WO (1) | WO2024179462A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024179462A1 (en) * | 2023-02-27 | 2024-09-06 | 广州知易生物科技有限公司 | Use of bacteroides fragilis and extract capsular polysaccharide a thereof in preparation of product for preventing and treating autoimmune arthritis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110002965A1 (en) * | 2007-11-09 | 2011-01-06 | Round June L | Immunomodulating compounds and related compositions and methods |
| WO2013052099A2 (en) * | 2011-10-03 | 2013-04-11 | Mazmanian Sarkis K | Generation of psa-only producing mutant strain |
| CN109954004B (en) * | 2017-12-14 | 2021-07-09 | 广州知易生物科技有限公司 | Application of bacteroides fragilis extract in preparation of composition for preventing and treating psoriasis |
| CN110151794A (en) * | 2019-06-03 | 2019-08-23 | 山东大学齐鲁医院 | Application of Bacteroides fragilis YCH46 in the treatment or adjuvant treatment of autoimmune diseases |
| CN112587552B (en) * | 2020-09-17 | 2023-09-12 | 大连图腾生命科学发展有限公司 | Application of bacteroides fragilis839 in preparing medicament for treating or assisting in treating immune related diseases |
| CN118345133A (en) * | 2021-06-15 | 2024-07-16 | 广州知易生物科技有限公司 | Preparation method of bacteroides fragilis capsular polysaccharide A |
| CN114470003B (en) * | 2022-01-12 | 2023-07-25 | 广州知易生物科技有限公司 | Application of bacteroides fragilis or zwitterionic capsular polysaccharide thereof in preparing medicines for preventing and treating digestive system tumors |
| CN116327809A (en) * | 2023-02-27 | 2023-06-27 | 广州知易生物科技有限公司 | Application of bacteroides fragilis and extract capsular polysaccharide A thereof in preparation of product for preventing and treating autoimmune arthritis |
-
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- 2023-02-27 CN CN202310182987.2A patent/CN116327809A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024179462A1 (en) * | 2023-02-27 | 2024-09-06 | 广州知易生物科技有限公司 | Use of bacteroides fragilis and extract capsular polysaccharide a thereof in preparation of product for preventing and treating autoimmune arthritis |
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