CN114699422B

CN114699422B - Application of capsular polysaccharide extract of bacteroides fragilis in preparation of medicines for preventing and treating Alzheimer disease

Info

Publication number: CN114699422B
Application number: CN202210141343.4A
Authority: CN
Inventors: 王薇; 刘洋洋; 王晔; 智发朝
Original assignee: Guangzhou Zhiyi Biotechnology Co Ltd
Current assignee: Guangzhou Zhiyi Biotechnology Co Ltd
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2023-07-18
Anticipated expiration: 2042-02-16
Also published as: CN114699422A; WO2023155938A2; WO2023155938A3

Abstract

The invention provides an application of a capsular polysaccharide extract of bacteroides fragilis in preparing a medicament for preventing and treating Alzheimer's disease, wherein the preservation number of bacteroides fragilis is CGMCC NO.10685, and the capsular polysaccharide extract comprises capsular polysaccharide A. Compared with the prior art, the invention has the following beneficial effects: the inventor of the invention discovers that capsular polysaccharide A of bacteroides fragilis ZY-312 can effectively prevent and treat Alzheimer's disease through long-term experience accumulation and a large number of creative experimental researches, and the drug effect is verified in an animal model through Morris water maze behavior experiments. The extract of capsular polysaccharide A of bacteroides fragilis ZY-312 is used for preparing the medicine for preventing and treating the Alzheimer disease, and the medicine has the advantage of no toxic or side effect.

Description

Application of capsular polysaccharide extract of bacteroides fragilis in preparation of medicines for preventing and treating Alzheimer disease

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to application of a capsular polysaccharide extract of bacteroides fragilis in preparation of a medicament for preventing and treating Alzheimer disease.

Background

Alzheimer's Disease (AD) is also known as senile dementia, and is a neurodegenerative brain disease. In 1906 Alois Alzheimer's doctor found that the brain of the patient suffering from senile dementia is tangled with nerve fibers accompanied by degeneration of neuron cells and many plaques are present in the cerebral cortex by brain slice staining, and this nervous system disease is named "Alzheimer's disease". With the development of modern medicine, it has been found that the pathological changes characteristic of AD: cerebral amyloid beta (aβ) deposits to form senile plaques (Plaque), and Tau protein hyperphosphorylation causes neurofibrillary tangles (NFTs) and neuronal loss, accompanied by gliosis. However, the pathogenesis and treatment of AD remains a worldwide problem.

AD incidence has a rising trend year by year, becoming a high-grade disease next to cardiovascular disease and cancer. One person suffers from dementia every three seconds, and the annual cost of dementia is estimated to be $ 1 trillion, and this figure doubles by 2030, which is a significant medical burden worldwide. However, there is hardly any breakthrough progress in the development of AD drugs at present. AD is clinically manifested by reduced memory, orientation, thinking and judgment, reduced daily life, even abnormal mental behavior symptoms, and the like, which makes patient care difficult and places a heavy burden on society and families. There is currently no drug in the world that can slow down or stop neuronal damage caused by AD. Six drugs for the treatment of AD are approved by the FDA in the united states, rivastigmine, galantamine, donepezil, memantine in combination with donepezil and tacrine. In addition to memantine, which prevents the stimulation of nerve cells by overactivation of the NMDA (N-methyl-D-aspartic acid receptor) receptor in the brain, these drugs improve symptoms by temporarily increasing neurotransmitters in the brain. Mannitol sodium marketed in China in 2019 reduces accumulation of peripheral related metabolites phenylalanine/isoleucine by remodelling intestinal flora balance, reduces brain neuroinflammation, and further improves cognitive dysfunction. These drugs can alleviate the symptoms of AD but are not effective in preventing AD. Thus, there is a great clinical need to develop drugs having preventive and therapeutic effects on AD.

Disclosure of Invention

Based on the technical problems, the main purpose of the invention is to provide a new application of the capsular polysaccharide A extract of bacteroides fragilis, which mainly uses the capsular polysaccharide A extract of bacteroides fragilis ZY-312 in preparing medicines for preventing and treating Alzheimer disease.

The aim of the invention can be achieved by the following technical scheme:

the application of the capsular polysaccharide extract of the bacteroides fragilis in preparing a medicament for preventing and treating Alzheimer disease is characterized in that the preservation number of the bacteroides fragilis is CGMCC NO.10685, and the capsular polysaccharide extract is an extract of capsular polysaccharide A.

In some of these embodiments, the capsular polysaccharide extract has a content of capsular polysaccharide a of greater than 95wt%.

In some of these embodiments, the capsular polysaccharide A has a weight average molecular weight of 70KD to 90KD.

In some of these embodiments, the capsular polysaccharide A has a weight average molecular weight of 80KD to 90KD.

In some of these embodiments, the lipid content of the extract of capsular polysaccharide a is less than 0.02wt%.

In some of these embodiments, the lipid content is less than 0.01wt%.

In some embodiments, the medicament comprises the capsular polysaccharide extract and a pharmaceutically acceptable excipient.

In some embodiments, the adjunct comprises one or more of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color-flavor modulator, a solvent, a solubilizer, a co-solvent, an emulsifier, an antioxidant, a metal complexing agent, an inert gas, a preservative, a topical analgesic, a pH modulator, and an isotonic or isotonic modulator.

In some embodiments, the diluent is selected from at least one of starches, sugars, celluloses, and inorganic salts.

In some embodiments, the wetting agent is selected from at least one of water and ethanol.

In some of these embodiments, the binder is selected from at least one of starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, and polyethylene glycol.

In some embodiments, the disintegrant is selected from at least one of dry starch, sodium carboxymethyl starch, low substituted hydroxypropyl cellulose, croscarmellose sodium, crospovidone, surfactant, and effervescent disintegrant.

In some embodiments, the lubricant is selected from at least one of talc, calcium stearate, magnesium lauryl sulfate, colloidal silica, and polyethylene glycol.

In some embodiments, the color, flavor and taste modifier is selected from at least one of a color, a flavor, a sweetener, a mucilage, and a flavoring.

In some embodiments, the solvent is selected from at least one of water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, and ethyl acetate.

In some embodiments, the solubilizing agent is selected from at least one of tweens, sellers, polyoxyethylene fatty alcohol ethers, soaps, sulphates, and sulphonates.

In some embodiments, the co-solvent is selected from at least one of organic acids and salts thereof, amides and amines, inorganic salts, polyethylene glycol, povidone, and glycerin.

In some embodiments, the emulsifier is selected from at least one of span, tween, euphorbia, benzyl, glycerol fatty acid ester, higher fatty acid salt, sulfate, sulfonate, acacia, tragacanth, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxide, silica, and bentonite.

In some embodiments, the suspending agent is selected from at least one of glycerin, syrup, acacia, tragacanth, agar, sodium alginate, a cellulose derivative, povidone, carbopol, polyvinyl alcohol and a thixotrope.

In some embodiments, the antioxidant is selected from at least one of sulfite, metabisulfite, bisulfite, ascorbic acid, gallic acid, and esters thereof.

In some embodiments, the metal complexing agent is selected from the group consisting of disodium edetate and one of the polycarboxylic acid compounds

In some of these embodiments, the inert gas is selected from one of nitrogen and carbon dioxide.

In some embodiments, the preservative is selected from at least one of parabens, organic acids and salts thereof, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils.

In some embodiments, the topical analgesic is selected from at least one of benzyl alcohol, chlorobutanol, lidocaine, and procaine.

In some embodiments, the pH adjuster is selected from at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate.

In some embodiments, the isotonic or isotonic agent is selected from at least one of glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

In some embodiments, the pharmaceutical dosage form is a tablet or granule.

In some embodiments, the pharmaceutical dosage form is a pill or powder.

In some embodiments, the pharmaceutical dosage form is a capsule or an injection.

In some embodiments, the pharmaceutical is in the form of an oral liquid or a tube feeding formulation.

In some embodiments, the drug is administered by injection or orally.

In some of these embodiments, the medicament further comprises an acetylcholinesterase inhibitor and/or an n-methyl-d-aspartate receptor antagonist, and the capsular polysaccharide a is used in combination with the acetylcholinesterase inhibitor and/or the n-methyl-d-aspartate receptor antagonist.

In some embodiments, the acetylcholinesterase inhibitor comprises one or more of donepezil, galantamine, and rivastigmine.

In some of these embodiments, the n-methyl-d-aspartate receptor antagonist comprises memantine.

Compared with the prior art, the invention has the following beneficial effects:

the inventor of the invention discovers that capsular polysaccharide A of bacteroides fragilis ZY-312 can effectively prevent and treat Alzheimer's disease through long-term experience accumulation and a large number of creative experimental researches, and the drug effect is verified in an animal model through Morris water maze behavior experiments. The extract of capsular polysaccharide A of bacteroides fragilis ZY-312 is used for preparing the medicine for preventing and treating the Alzheimer disease, and the medicine has the advantage of no toxic or side effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of SEC-HPLC analysis of Bacteroides fragilis capsular polysaccharide;

FIG. 2 shows the result of SEC-MALS analysis of the capsular polysaccharide of Bacteroides fragilis.

Detailed Description

The present invention will be described in more detail below in order to facilitate understanding of the present invention. It should be understood, however, that the invention may be embodied in many different forms and is not limited to the implementations or embodiments described herein. Rather, these embodiments or examples are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments or examples only and is not intended to be limiting of the invention. As used herein, the optional scope of the term "and/or" includes any one of the two or more related listed items, as well as any and all combinations of related listed items, including any two or more of the related listed items, or all combinations of related listed items.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, the numerical range is referred to, and both ends of the numerical range are included unless otherwise specified.

The percentage content referred to in the present invention refers to mass percentage for both solid-liquid mixing and solid-solid mixing and volume percentage for liquid-liquid mixing unless otherwise specified.

The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system after the component is added.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.

The prevention and treatment of the invention include prevention and treatment.

Bacteroides fragilis (B.fragilis) belongs to the genus Bacteroides of the family Bacteroideaceae, the genus Bacteroides, the strict anaerobic bacillus of gram negative non-spore-forming; normally residing in intestinal tract, oral cavity and respiratory tract of human body has profound effect on host physiological function.

The invention relates to bacteroides fragilis capsular polysaccharide A which is capsular polysaccharide prepared from bacteroides fragilis ZY-312, and the invention proves that the bacteroides fragilis ZY-312 capsular polysaccharide A can improve the learning and memory capacity of AD model animals and can be applied to preparing medicines for preventing and treating AD. The application of a capsular polysaccharide extract of bacteroides fragilis in preparing a medicament for preventing and treating Alzheimer disease is provided, wherein the preservation number of bacteroides fragilis is CGMCC NO.10685, and the capsular polysaccharide extract comprises an extract of capsular polysaccharide A.

The invention discovers that the capsular polysaccharide A of the bacteroides fragilis ZY-312 can effectively prevent and treat the Alzheimer disease, and the drug effect is verified in an animal model through Morris water maze behavior experiments. The extract of capsular polysaccharide A of bacteroides fragilis ZY-312 is used for preparing the medicine for preventing and treating the Alzheimer disease, and the medicine has the advantage of no toxic or side effect.

The bacteroides fragilis used in the invention is bacteroides fragilis ZY-312 (bacteroides fragilis ZY-312), which is preserved in China general microbiological culture Collection center (CGMCC) for 4 months and 2 days in 2015, wherein the preservation number is CGMCC No.10685, and the preservation address is North Chen Xili No.1 and 3 in the Chaoyang area of Beijing city. The strain is a non-enterotoxigenic bacteroides fragilis.

In one example, the capsular polysaccharide a is present in the capsular polysaccharide extraction in an amount greater than 95wt%.

In one example, the capsular polysaccharide A has a weight average molecular weight of 70KD-90KD. The weight average molecular weight of the capsular polysaccharide A is 70KD-90KD. The weight average molecular weight of the capsular polysaccharide A of the invention is 70KD, 71KD, 72KD, 73KD, 74KD, 75KD, 76KD, 77KD, 78KD, 79KD, 80KD, 81KD, 82KD, 83KD, 84KD, 85KD, 86KD, 87KD, 88KD, 89KD and 90KD. Preferably, the capsular polysaccharide A of the present invention has a weight average molecular weight of 80KD-90KD.

In one example, the capsular polysaccharide A has a ratio of weight average molecular weight to number average molecular weight (Mw/Mn) of 1.0 to 1.3.

In one example, the fraction of the extract of capsular polysaccharide A having a weight average molecular weight distribution between 70KD and 100KD is 70% -80% of the total extract weight;

in one example, the extract of capsular polysaccharide a has a combined lipid content of < 0.02% by weight.

In the above application of the present invention, the preparation of the extract of capsular polysaccharide a may include, but is not limited to, the following steps:

(1) Centrifuging the Bacteroides fragilis fermentation liquor, and collecting bacterial sediment (bacterial sludge);

(2) Adding purified water into the bacterial sludge, and fully stirring to enable bacteria to be resuspended;

(3) Adjusting the pH of the bacterial suspension to acidic;

(4) Transferring the bacterial heavy suspension into an extraction container, heating and extracting, and centrifuging to obtain a supernatant;

(5) Ultra-filtering small molecular impurities, and collecting trapped fluid;

(6) Column exchange chromatography;

(7) The column chromatography collecting liquid is subjected to ultrafiltration for desalting, concentration, freeze drying and sealed preservation.

The preparation process is mainly summarized as follows: anaerobic fermentation of bacteroides fragilis, centrifugal collection of bacterial precipitation, bacterial sludge suspension, acid extraction, centrifugal collection of supernatant, ultrafiltration for removing small molecular impurities, ion exchange one-step chromatography and freeze-drying. The process is simpler, environment-friendly, and has high purity and high yield of capsular polysaccharide. The prepared capsular polysaccharide A can meet the following conditions: protein residue < 1%; nucleic acid residue < 0.05%; the conjugated lipid is less than 0.02%; no lipopolysaccharide or other contaminating polysaccharide component; the molecular weight distribution is uniform, the weight average molecular weight is 70KD-90KD, and Mw/Mn is less than 1.3.

In one example, the medicament comprises the capsular polysaccharide extract and a pharmaceutically acceptable excipient.

It is understood that the auxiliary materials are, for example, diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusters, isotonic or isotonic adjusting agents, etc., and may be contained in one kind or two or more kinds. The adjuvants may be selected from, but are not limited to, the following: the diluent is starch, saccharide, cellulose and inorganic salt; the wetting agent is at least one selected from water and ethanol; the binder is at least one selected from starch slurry, dextrin, sugar, cellulose derivative, gelatin, povidone and polyethylene glycol; the disintegrating agent is at least one selected from dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crosslinked sodium carboxymethyl cellulose, crosslinked povidone, surfactant and effervescent disintegrating agent; the lubricant is at least one selected from talcum powder, calcium stearate, magnesium dodecyl sulfate, micro powder silica gel and polyethylene glycol; the color, smell and taste regulator is at least one selected from pigment, perfume, sweetener, mucilage and corrigent; the solvent is at least one selected from water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil and ethyl acetate; the solubilizer is at least one selected from tween, herba Euphorbiae Helioscopiae, polyoxyethylene fatty alcohol ether, soap, sulfate, and sulfonate; the cosolvent is at least one selected from organic acid and salts thereof, amide and amine compounds, inorganic salts, polyethylene glycol, povidone and glycerin; the emulsifier is at least one selected from span, tween, herba Euphorbiae Helioscopiae, benzyl, glycerin fatty acid ester, higher fatty acid salt, sulfate, sulfonate, acacia, tragacanth, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silicon dioxide and bentonite; the suspending agent is at least one selected from glycerol, syrup, acacia, tragacanth, agar, sodium alginate, cellulose derivative, povidone, carbopol, polyvinyl alcohol and thixotrope; the antioxidant is at least one selected from sulfite, metabisulfite, bisulfite, ascorbic acid, gallic acid and esters thereof; the metal complexing agent is selected from one of disodium ethylenediamine tetraacetate and polycarboxylic acid compounds; the inert gas is selected from one of nitrogen and carbon dioxide; the preservative is at least one selected from nipagin, organic acid and salts thereof, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols and volatile oil; the local analgesic is at least one selected from benzyl alcohol, chlorobutanol, lidocaine and procaine; the pH regulator is at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate and citrate; the isotonic or isotonic regulator is at least one selected from glucose, sodium chloride, sodium citrate, sorbitol and xylitol.

It will be appreciated that the medicament may be formulated into suitable dosage forms such as tablets, granules, pills, powders, capsules, injections, oral liquids, tube feeding preparations and the like according to clinical requirements.

It will be appreciated that the medicament may be administered by any suitable route of administration, such as injection, oral administration, etc., according to clinical requirements.

In one example, the medicament further comprises an acetylcholinesterase inhibitor and/or an n-methyl-d-aspartate receptor antagonist, and the capsular polysaccharide a is used in combination with the acetylcholinesterase inhibitor and/or the n-methyl-d-aspartate receptor antagonist.

In one example, the acetylcholinesterase inhibitor comprises one or more of donepezil, galantamine, and rivastigmine.

In one example, the n-methyl-d-aspartate receptor antagonist comprises memantine.

EXAMPLE 1 preparation of Bacteroides fragilis capsular polysaccharide A

And (5) centrifuging the bacteroides fragilis fermentation liquor, and collecting bacterial sludge.

(1) Method 1: acid extraction method for preparing bacteroides fragilis capsular polysaccharide A

1) Taking 50g of bacterial mud, adding 300g of purified water to re-suspend the bacterial mud, adjusting the pH of the bacterial mud to 4.5 by using 1mol/L hydrochloric acid solution, extracting for 1.5h at 90 ℃, cooling to room temperature, centrifuging for 10min at 12000g of room temperature, and taking the supernatant to obtain a crude sugar solution.

2) The crude sugar solution is ultrafiltered and concentrated by a 10KD ultrafiltration membrane, and the cut-off liquid is collected.

3) Adding an equal volume of 40mmol/L Tris-HCl (pH 8.5) buffer solution into the cut-off solution for salt conversion; DEAE Sepharose Fast Flow ion exchange column chromatography (16 mm. Times.200 mm), gradient eluting with 20mL/min flow rate, 20mmol/L Tris-HCl (pH 8.5, containing 0.2mol/L NaCl) buffer solution for 25 column volumes, collecting in sections, 100 mL/bottle (component), tracking and monitoring by SEC-HPLC, combining components with 206nm absorption peak as single and symmetrical peak, ultrafiltering with 10KD ultrafiltration membrane, repeatedly ultrafiltering with purified water until conductivity is stable, collecting reflux liquid, and lyophilizing; 2 experiments are performed in parallel, and the obtained bacteroides fragilis capsular polysaccharide is named as S1 and S2 respectively, and the detection results are shown in figures 1 and 2.

(2) Method 2: phenol/water method for preparing bacteroides fragilis capsular polysaccharide A

(1) 50g of bacterial sludge is taken, 200mL of 0.15mol/L sodium chloride solution is added, 12000g of the bacterial sludge is centrifuged at normal temperature for 20min after washing, and the bacterial sludge is collected.

(2) 750mL of purified water was added to allow the bacteria to re-suspend, then an equal volume of 75% phenol aqueous solution (m/m) was added, stirred at 68℃for 30min, and 16000g was centrifuged at room temperature for 30min to remove the precipitate.

(3) Extracting the supernatant with an equal volume of diethyl ether for 3 times, and collecting a water phase; the aqueous phase was concentrated by rotary evaporator, dialyzed and lyophilized.

(4) The sample was prepared using a 0.1mol/L sodium acetate solution (containing 10mmol/L CaCl) ₂ And 10mmol/LMgCl ₂ ) Dissolving, adding 2mg of DNase and 10mg of RNase, stirring at 37deg.C for 2h, adding 2mg of DNase and 10mg of RNase again, and stirring at 37deg.C overnight; adjusting pH to 7.0, adding 20mg pronase, stirring at 37deg.C for 2 hr, adding 20mg pronase again, and stirring at 37deg.C overnight; absolute ethanol was added to a final concentration of 80%, and the mixture was centrifuged at 12000g for 10min at 4℃overnight to obtain a precipitate.

(5) Adding 5% acetic acid solution into the precipitate for redissolution, hydrolyzing at 100deg.C for 1 hr, dialyzing for desalting, separating with DEAE Sepharose Fast Flow ion exchange column chromatography, collecting in sections, tracking and monitoring by SEC-HPLC, mixing components with single and symmetrical absorption peak at 206nm, ultrafiltering for desalting, and lyophilizing; in parallel 2 experiments, the obtained bacteroides fragilis capsular polysaccharide A is named as 'S3' and 'S4', respectively.

(3) Experimental results

The comparison results of the capsular polysaccharides of Bacteroides fragilis obtained by the two preparation methods are shown in Table 1.

Table 1 comparison of quality Properties of Bacteroides fragilis capsular polysaccharide A obtained by two preparation methods

The above samples were respectively noted as: S1-PSA, S2-PSA, S3-PSA, S4-PSA.

Example 2 demonstration of the Effect of Bacteroides fragilis capsular polysaccharide A on controlling AD

1. Experimental method

Animals: the experiment selects 90 SPF SD rats, 8 weeks old, male and female half, and the mass is 200g-240g.

Grouping animals: 90 SD rats, each half of the male and female, were divided into 9 groups according to the random number table method, each group of 5 male and female animals was a sham operation group, a model group, a S1-PSA low, medium and high dose group, a S3-PSA low, medium and high dose group, and a donepezil hydrochloride group (positive drug group), as shown in Table 2.

AD model preparation: the 9 groups of rats are all anesthetized by 10% chloral hydrate intraperitoneal injection, partial hair on the top of the brain is shaved off, the head of the rat is fixed on a brain stereotactic instrument, the iodophor is used for sterilizing the conventional skin, a longitudinal incision is cut at the midline position of the scalp by a sterile scalpel, the length of the incision is about 1.5cm, the pre-halogen is fully exposed, 2.0mm is left and right by the midline of the brain according to the three-dimensional positioning map of the rat brain, and two sides 3.0mm behind the pre-halogen are respectively drilled by sterile dental drills, wherein the drilling depth is 3mm, and the intracranial bleeding is avoided. Except for sham operated groups, rats of each group were slowly injected with 5 μl of aggregated aβ _1-40 (beta-amyloid protein, 1. Mu.L/min), the depth of penetration was 4.0mm. The sham operation group is injected with an equal volume of physiological saline solution, needles are left for 5min after the injection is finished, then wounds are sutured, and amikacin spraying is used at the wounds to prevent wound infection. The animals were routinely kept for 1 week after the operation. After 1 week, all rats were subjected to the water maze test, and the model formation was judged to be successful if the escape latency of the model formation rats was long and significantly different from that of the sham operation group, and finally all model formation was successful.

Administration: the dosage of the experimental medicine is converted according to the body surface area ratio, and 5mg/kg, 3mg/kg and 1mg/kg of gastric lavage are respectively configured for the high, medium and low dosage groups of the S1-PSA and the high, medium and low dosage groups of the S3-PSA. Donepezil hydrochloride group donepezil hydrochloride Ji Pian was ground into powder to prepare a suspension of 1.02 mg/kg ^-1 The stomach is irrigated, which is equivalent to 6 times of daily dose for adults. Rats in sham surgery and model groups were perfused with normal saline daily. Each group had a lavage volume of 10mL/Kg and was continued for 10 weeks, as detailed in Table 2.

Table 2, animal grouping and administration

Morris water maze introduction: morris water maze is an experimental means which is originally designed and applied to study of brain learning and memory mechanism by Morris of the UK psychologist in the generation 80 (1981) of the 20 th century, and is very popular in AD study. The more classical Morris water maze, the test procedure mainly comprises two parts of a positioning navigation test and a space exploration test. The positioning sailing test is carried out for several days, the rat is put into water from 4 water inlet points for several times each day, and the time (escape latency) for finding the platform hidden under the water surface is recorded. Generally, the more severe the AD, the longer the escape latency of the rat. The space exploration test is to remove the platform after the positioning navigation test, then to put the rat into the pool by selecting a water inlet point, to record the swimming track of the rat in a certain time, to examine the memory of the rat to the original platform. Generally, the more severe the AD, the less the number of times the rat traverses the platform, the shorter the effective area residence time and the shorter the effective area movement path.

Morris water maze behavioral experiments: after the continuous gastric lavage is completed for 10 weeks, morris water maze behavior experiments are performed. The experiment was continued for 5 days, and divided into positioning navigation and space exploration experiments. The positioning sailing experiment was carried out for 4 days, and the time required for the animals to find and climb up the platform from the time of putting in water, i.e. the escape latency, was recorded. If no stations are found within 120s, latency is recorded as 120s. Space searching experiments are carried out on the 5 th day, the original platform is removed, and the number of times of crossing the platform, the residence time of an effective area (the range of 2 times of the diameter of the platform) and the swimming distance of the effective area in 120s of the rat are recorded.

Statistical analysis: data toThis indicates that SPSS 23.0 was used for statistical analysis. The comparison between groups adopts single factor analysis of variance (ANOVA), and when the variances are uniform, the average value between groups is compared pairwise by adopting an SNK method; if the variance is irregular, dunnett T3 (Dunnett T3) is used instead to test for P<0.05 indicates that the difference is statistically significant.

2. Results and analysis

The effect of oral bacteroides fragilis capsular polysaccharide a on aβ -induced SD rats is shown in tables 3 and 4.

TABLE 3 influence of capsular polysaccharide A on Abeta-induced SD rats on escape latency in the water maze test

Note that in comparison to the sham surgery group, ^* P＜0.05， ^** p is less than 0.01; comparison with model group ^# P＜0.05， ^## P＜0.01。

As shown in table 3, the escape latency of the male and female rats in each administration group in the water maze was shortened with the increase of training time and frequency. On day 4, compared with a sham operation group, the escape latency of the male and female rats of the model group is obviously prolonged (P is less than 0.01), which indicates that the model building is successful; compared with the model group, the escape latency of the male and female rats of the bacteroides fragilis capsular polysaccharide A administration group is obviously shortened (P is less than 0.05 and P is less than 0.01), which proves that the capsular polysaccharide A of the bacteroides fragilis can improve AD symptoms of SD rats induced by Abeta.

TABLE 4 influence of capsular polysaccharide A on Abeta-induced SD rats spatial exploration in water maze test

Group of	Number of passes/times of traversing the platform	Effective area residence time/s	Effective area movement distance/mm
				False operation	5.38±0.74	5.98±0.68	837.31±75.03
Model	3.25±0.74 ^**	3.71±0.54 ^**	624.74±82.42 ^**
				Donepezil	4.13±0.43 ^##	4.34±0.60 ^##	691.60±43.00 ^##
S1-PSA low dose group	4.19±0.63 ^#	4.43±0.45 ^#	703.58±73.55 ^##
				Dose group in S1-PSA	4.62±0.60 ^#	5.18±0.66 ^##	757.38±93.64 ^##
S1-PSA high dose group	4.35±0.77 ^#	4.75±0.76 ^#	733.21±64.29 ^##
				S3-PSA low dose group	4.33±0.63 ^#	4.56±0.35 ^#	722.53±80.25 ^##
Dose group in S3-PSA	4.89±0.45 ^#	5.43±0.62 ^##	789.64±87.20 ^##
				S3-PSA high dose group	4.45±0.77 ^#	4.82±0.79 ^#	754.29±68.39 ^##

Note that: in comparison with the sham-operated group, ^* P＜0.05， ^** p is less than 0.01; comparison with model group ^# P＜0.05， ^## P＜0.01。

As can be seen from the results in Table 4, compared with the sham operation group, the number of times of crossing the platform, the effective area residence time and the movement path of the male and female rats in the model group are obviously reduced (P < 0.01); compared with a model group, the administration of donepezil and the bacteroides fragilis capsular polysaccharide A can improve the number of times of crossing the platform, the residence time of the effective area and the movement path of rats, wherein the increase of the number of times of crossing the platform, the residence time of the effective area and the movement path of the male and female rats of the bacteroides fragilis capsular polysaccharide A is more remarkable (P is less than 0.05 and P is less than 0.01).

The experimental results show that the bacteroides fragilis capsular polysaccharide A provided by the invention can prevent and treat AD.

Example 3 demonstration of the Effect of Bacteroides fragilis capsular polysaccharide A in combination with donepezil in controlling AD

1. Experimental method

Grouping animals: 110 SD rats, each half of which was divided into 11 groups according to the random number table method, each group of 5 females and males was respectively a sham operation group, a model group, a S2-PSA medium dose group, a S2-PSA low, medium and high dose combination donepezil group, a S4-PSA medium dose group, a S4-PSA low, medium and high dose donepezil group, and a donepezil hydrochloride group (positive drug group), as detailed in Table 5.

AD model preparation: all 11 groups of rats are anesthetized by 10% chloral hydrate intraperitoneal injection, partial hair on the top of the brain is shaved off, the head of the rat is fixed on a brain stereotactic instrument, the conventional skin of iodophor is disinfected, a longitudinal incision is cut at the midline position of the scalp by a sterile scalpel, the length of the incision is about 1.5cm, the front halogen is fully exposed, 2.0mm is left and right by the midline of the brain according to the three-dimensional positioning map of the rat brain, and two sides 3.0mm behind the front halogen are drilled by sterile dental drills respectively, wherein the drilling depth is 3mm, and intracranial bleeding is avoided. Except for sham operated groups, rats of each group were slowly injected with 5 μl of aggregated aβ _1-40 (beta-amyloid protein, 1. Mu.L/min), the depth of penetration was 4.0mm. The sham operation group is injected with an equal volume of physiological saline solution, needles are left for 5min after the injection is finished, then wounds are sutured, and amikacin spraying is used at the wounds to prevent wound infection. The animals were routinely kept for 1 week after the operation. After 1 week, all rats were subjected to the water maze test, and the model formation was judged to be successful if the escape latency of the model formation rats was long and significantly different from that of the sham operation group, and finally all model formation was successful.

Administration: the dosage of the experimental medicine is converted according to the body surface area ratio, the dosage of the S2-PSA is in a group of high, medium and low dosages of the S2-PSA, the dosage of the S4-PSA is in a group of the S4-PSA, the dosage of the S4-PSA is in a group of the high, medium and low dosages of the S4-PSA, and the dosage of the high, medium and low dosages of the PSA is respectively configured with 5mg/kg, 3mg/kg and 1mg/kg of gastric lavage. Donepezil hydrochloride group donepezil hydrochloride Ji Pian was ground into powder to prepare a suspension of 1.02 mg/kg ^-1 The stomach is irrigated, which is equivalent to 6 times of daily dose for adults. Rats in sham surgery and model groups were perfused with normal saline daily. Each group had a lavage volume of 10mL/Kg and was continued for 10 weeks, as detailed in Table 5.

Table 5, animal grouping and administration

The more classical Morris water maze, the test procedure mainly comprises two parts of a positioning navigation test and a space exploration test. The positioning sailing test is carried out for several days, the rat is put into water from 4 water inlet points for several times each day, and the time (escape latency) for finding the platform hidden under the water surface is recorded. Generally, the more severe the AD, the longer the escape latency of the rat. The space exploration test is to remove the platform after the positioning navigation test, then to put the rat into the pool by selecting a water inlet point, to record the swimming track of the rat in a certain time, to examine the memory of the rat to the original platform. Generally, the more severe the AD, the less the number of times the rat traverses the platform, the shorter the effective area residence time and the shorter the effective area movement path.

2. Results and analysis

TABLE 6 influence of capsular polysaccharide A on Abeta-induced SD rats with donepezil on escape latency in the water maze test

Note that in comparison to the sham surgery group, ^* P＜0.05， ^** p is less than 0.01; comparison with model group ^# P＜0.05， ^## P＜0.01， ^### P＜0.001。

As shown in table 6, the escape latency of the male and female rats in each administration group in the water maze was shortened with the increase of training time and frequency. Compared with a sham operation group, the escape latency of the male and female rats in the model group is obviously prolonged (P is less than 0.05 and P is less than 0.01), which indicates that the model is successful; the escape latency of the male and female rats of Bacteroides fragilis capsular polysaccharide A group and donepezil was significantly reduced (P < 0.01) compared to the model group, and the combined group had a shorter escape latency (P < 0.001) than the single group.

TABLE 7 influence of capsular polysaccharide A in combination with donepezil on Abeta-induced SD rats on spatial exploration in water maze test

As can be seen from the results in Table 7, compared with the sham operation group, the number of times of crossing the platform, the effective area residence time and the movement path of the male and female rats in the model group are obviously reduced (P < 0.01); compared with the model group, the number of times of crossing the platform, the effective area residence time and the movement path of the male and female rats of the single group of the bacteroides fragilis capsular polysaccharide A and the group of the donepezil Ji Shan are increased (P is less than 0.01), and the number of times of crossing the platform, the effective area residence time and the movement path of the rats of the combined group of the bacteroides fragilis capsular polysaccharide A and the donepezil are increased more obviously (P is less than 0.01 and P is less than 0.001).

The experimental results show that the combination of the bacteroides fragilis capsular polysaccharide A and donepezil provided by the invention has more remarkable AD prevention and treatment effect.

The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. The application of a capsular polysaccharide extract of bacteroides fragilis in preparing a medicament for preventing and treating Alzheimer disease is characterized in that the preservation number of the bacteroides fragilis is CGMCC NO.10685, and the capsular polysaccharide extract comprises capsular polysaccharide A;

the weight average molecular weight of the capsular polysaccharide A is 80KD-90KD;

the lipid content of the capsular polysaccharide A is less than 0.02wt%.

2. Use according to claim 1, characterized in that the capsular polysaccharide a content of the capsular polysaccharide extract is greater than 95wt%.

3. Use according to claim 1, characterized in that the lipid content of capsular polysaccharide a is less than 0.01wt%.

4. Use according to any one of claims 1 to 3, wherein the medicament comprises the capsular polysaccharide extract and a pharmaceutically acceptable adjuvant.

5. The use according to claim 4, wherein the adjuvant comprises one or more of diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH modifiers and isotonic or isotonic agents.

6. The use according to claim 5, wherein the diluent is selected from at least one of starches, sugars, celluloses and inorganic salts; or/and the wetting agent is selected from at least one of water and ethanol; or/and the binder is at least one selected from starch slurry, dextrin, sugar, cellulose derivative, gelatin, povidone and polyethylene glycol; or/and, the disintegrating agent is at least one selected from dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, crospovidone, surfactant and effervescent disintegrating agent; or/and the lubricant is at least one selected from talcum powder, calcium stearate, magnesium dodecyl sulfate, micro powder silica gel and polyethylene glycol; or/and the color, smell and taste regulator is at least one selected from pigment, perfume, sweetener, mucilage and corrigent; or/and the solvent is at least one selected from water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin and ethyl acetate; or/and the solubilizer is at least one selected from tween, herba Euphorbiae Helioscopiae, polyoxyethylene fatty alcohol ether, soap, sulfate and sulfonate; or/and the cosolvent is selected from at least one of organic acid and salts thereof, amide and amine compounds, inorganic salts, polyethylene glycol, povidone and glycerin; or/and the emulsifier is at least one selected from span, tween, herba Euphorbiae Helioscopiae, benzyl Euphorbiae, glycerin fatty acid ester, higher fatty acid salt, sulfate, sulfonate, acacia, tragacanth, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silica and bentonite; or/and the antioxidant is selected from at least one of sulfite, metabisulfite, bisulfite, ascorbic acid, gallic acid and esters thereof; or/and the metal complexing agent is selected from one of disodium ethylenediamine tetraacetate and a polycarboxylic acid compound; or/and the inert gas is selected from one of nitrogen and carbon dioxide; or/and the preservative is at least one selected from nipagin, organic acid and salts thereof, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols and volatile oil; or/and, the local analgesic is at least one selected from benzyl alcohol, chlorobutanol, lidocaine and procaine; or/and the pH regulator is at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate and citrate; or/and the isotonic or isotonic regulator is at least one selected from glucose, sodium chloride, sodium citrate, sorbitol and xylitol.

7. The use according to any one of claims 1 to 3, 5 and 6, wherein the medicament is in the form of a tablet, granule, pill, powder, capsule, injection, oral liquid or tube feeding; or/and the administration route of the medicine is injection administration or oral administration.

8. The use according to any one of claims 1 to 3, 5 and 6, wherein the medicament further comprises an acetylcholinesterase inhibitor and/or an n-methyl-d-aspartate receptor antagonist, and wherein the capsular polysaccharide a is used in combination with the acetylcholinesterase inhibitor and/or the n-methyl-d-aspartate receptor antagonist.

9. The use according to claim 8, wherein the acetylcholinesterase inhibitor comprises one or more of donepezil, galantamine and rivastigmine.

10. The use according to claim 8, wherein the n-methyl-d-aspartate receptor antagonist comprises memantine.