CN108938653B

CN108938653B - Application of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide in preparation of medicine for treating Alzheimer disease

Info

Publication number: CN108938653B
Application number: CN201811136880.XA
Authority: CN
Inventors: 张真庆; 唐蓉; 郝杰; 吴芳霞; 李笃信
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2018-09-28
Filing date: 2018-09-28
Publication date: 2021-03-02
Anticipated expiration: 2038-09-28
Also published as: CN108938653A

Abstract

The invention discloses application of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide in preparation of a medicine for treating Alzheimer disease, and application of a derivative or pharmaceutically acceptable salt thereof in preparation of a medicine for treating Alzheimer disease. The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the mixture thereof remarkably improve the survival rate of neuron cells damaged by beta amyloid, remarkably improve the learning and memory abilities of AD model mice, and have obvious dose dependence; the structure is simple, no obvious side effect group exists, the safety and the controllability are higher, and in the aspect of exerting the anti-AD curative effect, the combination mode of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide is more diversified; the raw material can be obtained by specific oxidation modification of cellulose rich in natural resources, and compared with other polysaccharides, the polysaccharide is safer and controllable, and has great drug potential.

Description

Application of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide in preparation of medicine for treating Alzheimer disease

Technical Field

The invention relates to application of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and derivatives thereof or pharmaceutically acceptable salts thereof in treating Alzheimer's disease.

Background

Alzheimer's Disease (AD), also known as senile dementia, is a neurodegenerative disease occurring in the brain, and is clinically manifested as memory disorder, aphasia, disuse, cognitive dysfunction, and character behavioral changes. With the increase of age, the incidence rate of AD gradually increases, and according to statistics, the incidence rate of 65-80 years old people is 5% -8%, and the incidence rate of people over 80 years old is more than 20%. With the increasing aging of the population, the global AD patients are expected to exceed 1 billion, 3 thousand, and 7 million by 2050, at which time the global investment for this disease will amount to billions of dollars, placing a heavy burden on society and many families. At present, the drugs clinically used for preventing and treating AD comprise central stimulant, cholinergic improving substance, Chinese herbal medicine, pyrrolidone compound and the like, such as aricept, memantine, tacrine and the like. However, these drugs can only improve the symptoms of mild and moderate AD to some extent, and cannot prevent, reverse or even delay the disease process, thereby limiting their wide application. As such, AD is a disease that is currently not cured clinically. In the face of such severe conditions of AD onset and treatment, the development of drugs that can prevent, or even reverse the progression of AD is becoming urgent. Recently, with the intensive research on the pathogenesis of the disease, not only a variety of therapeutic targets, such as beta amyloid, tau protein, intestinal flora, neuroinflammation, etc., have been proposed, but also a large number of active compounds directed against these targets have been reported.

Cellulose is a polysaccharide which is widely distributed and has the largest content in the nature, and accounts for more than 50 percent of the carbon content in the plant. The polymerization degree of the glucan is 3000-5000, the linear glucan combined through beta 1 → 4 glycosidic bonds has a linear molecular structure, has certain strength and rigidity, is not easy to hydrolyze by dilute acid or alkali, and is an important component of plant cell walls. Since human beings and meat animals rarely contain beta-glucosidase, cellulose cannot be digested and utilized. Microcrystalline cellulose (MCC) is a purified and partially depolymerized cellulose, and is a straight-chain polysaccharide substance with the main component combined by 1, 4-beta-D-glucoside bonds, is white, odorless and tasteless, is crystalline powder consisting of porous particles, improves the use efficiency of the cellulose, and is widely applied to industries such as pharmacy, cosmetics, foods, light chemical engineering and the like due to the special properties of low polymerization degree, large specific surface area and the like.

Disclosure of Invention

The invention aims to provide oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and derivatives or pharmaceutically acceptable salts thereof, and solves the problems.

The invention has a technical scheme that: the application of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and derivatives or pharmaceutically acceptable salts thereof in preparing medicaments for treating Alzheimer's disease.

Further, the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the derivatives or pharmaceutically acceptable salts thereof comprise the following general formula (I):

wherein,

n in the formula (I) is selected from any one or more integers from 0 to 19;

m is selected from 0,1 or 2;

one or more oxidized 1, 4-beta-D-glucuronic acid oligosaccharides with a structure of a general formula (I) and derivatives thereof or pharmaceutically acceptable salts thereof are obtained by selecting one or more integers from n.

Further, n is selected from any one or more integers from 1 to 9.

Further, n is 1,2, 3, 4, 5, 6, 7, 8 and 9, corresponding to the oxidized 1,4- β -D-

glucuronic acid oligosaccharide

2, 3, 4, 5, 6, 7, 8, 9 and 10 sugars, respectively.

Further, the component with n of 1-9 accounts for more than 80% of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the derivative or pharmaceutically acceptable salt thereof.

Further, the component with n of 1-9 accounts for more than 90% of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the derivative or pharmaceutically acceptable salt thereof.

Further, the component with n of 1-9 accounts for more than 95% of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the derivative or pharmaceutically acceptable salt thereof.

Further, the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the derivative or pharmaceutically acceptable salt thereof are applied to improving the survival rate of beta amyloid protein-damaged neuronal cells.

The application of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the derivative or the pharmaceutically acceptable salt thereof in the aspect of treating the Alzheimer disease has the main advantages that:

1. the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the mixture thereof remarkably improve the survival rate of neuron cells damaged by beta amyloid, remarkably improve the learning and memory abilities of AD model mice, and have obvious dose dependence;

2. the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide related to the invention has simple structure, no obvious side effect group, and is safer and more controllable, and the combination mode of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide is more diversified in the aspect of exerting the anti-AD curative effect;

3. compared with other polysaccharides, the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide is safer and controllable, and has larger medicament potential.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein,

FIG. 1 is a SEC-LC chromatogram of an oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture at 210 nm;

FIG. 2 is a mass spectrum of oligosaccharide with different polymerization degrees in an oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid;

FIG. 3 is an extracted ion current chromatogram (EIC) of oxidized oligosaccharide of 1,4- β -D-glucuronic acid with single degree of polymerization; wherein dotted dashed lines indicate the OX1 structure type, solid lines indicate the OX2 structure type, and dashed short lines indicate the OX3 structure type;

FIG. 4 is a graph showing the THT fluorescence value variation of the oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid for inhibiting the aggregation of Abeta 1-40;

FIG. 5 is a schematic diagram showing the protective effect of oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid on SH-SY5Y cell injury caused by A beta 25-35;

FIG. 6 is a schematic diagram showing the regulation effect of oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid on the mRNA level of Bcl2, p53, Drp1 and Parkin gene;

FIG. 7 is a graph showing the effect of oxidized oligosaccharide mixture of 1,4- β -D-glucuronic acid on the level of Bcl2, p53, Drp1, and Parkin genes;

FIG. 8 is a graph showing the improvement effect of oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid on A beta 1-40 induced cognitive impairment of mice and the experimental results of a mouse movement track tracking system.

FIG. 9 is a graph showing the experimental results of the escape latency of Morris water maze, the improvement effect of oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid on the cognitive impairment of mice caused by Abeta 1-40.

FIG. 10 is a graph showing the results of experiments on the number of times that the oxidized oligosaccharide mixture of 1,4- β -D-glucuronic acid causes cognitive impairment of mice due to Abeta 1-40, and passes through the Morris water maze.

FIG. 11 is a schematic diagram of the protective effect of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide with a single degree of polymerization on SH-SY5Y cell injury caused by A beta 25-35.

Detailed Description

The invention provides an application of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and derivatives thereof in treating Alzheimer's disease. The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide and the derivatives thereof or the pharmaceutically acceptable salts of the oligosaccharide and the derivatives thereof have the structure shown in the structural formula (I):

n in formula (I) is selected from one or more integers from 0 to 19; for example n is one or more integers selected from 0,1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

In the formula (I), n is preferably 1 to 9, more preferably 2 to 8, and most preferably 4. The reason why 1,4- β -D-glucuronic acid oligosaccharides with n ═ 1 to 9 (preferably n ═ 2 to 8, most preferably n ═ 4) have a better biological effect is not clear, and it is likely that they are easily recognized and accepted by body cells. In some embodiments, n may also be one or more integers selected from the range of 1 to 9. N in the formula (I) is selected from 1,2, 3, 4, 5, 6, 7, 8 and 9 and respectively corresponds to oxidized 1, 4-beta-D-glucuronic acid oligosaccharide disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide and decasaccharide.

In the formula (I), m is selected from 0,1 or 2. Respectively correspond to 1, 4-beta-D-glucuronic acid oligosaccharide-

OX

3, 1, 4-beta-D-glucuronic acid oligosaccharide-

OX

2 and 1, 4-beta-D-glucuronic acid oligosaccharide-OX 1, and the structures of the oligosaccharides are respectively shown as structural formulas (II), (III) and (IV):

the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide is obtained by specifically oxidizing cellulose by a sodium bromide (NaBr) -2,2,6, 6-tetramethylpiperidine oxide (TEMPO) -sodium hypochlorite (NaClO) system, and the specific preparation process flow and the structure analysis method can refer to Chinese patent 201510267009.3.

In a preferred embodiment of the present invention, the oxidized 1,4- β -D-glucuronic acid oligosaccharide represented by the structural formula (I) is prepared by:

(1) weighing microcrystalline cellulose (MCC) powder, and carrying out mercerization treatment to obtain a mercerized MCC solution with the concentration of 10-20 mg/ml;

(2) sequentially adding 2,2,6, 6-tetramethylpiperidine oxide (TEMPO) and sodium bromide into the mercerized MCC solution prepared in the step (1), adjusting the pH to 10-11 by using an alkaline pH regulator, then adding a sodium hypochlorite solution to react for 5-10 hours at the temperature of 40-80 ℃, and finally adding an organic solvent to terminate the reaction;

(3) dialyzing in 500Da dialysis bag, concentrating, ultrafiltering, and lyophilizing to obtain mixture of oxidized 1, 4-beta-D-glucuronic acid, or optionally performing chromatographic separation to obtain oxidized 1, 4-beta-D-glucuronic acid oligosaccharide with single polymerization degree.

The derivative of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide of the formula (I) or the pharmaceutically acceptable salt thereof comprises ester of which one or more hydroxyl groups are esterified by inorganic acid or organic acid. The organic acids capable of forming esters with one or more hydroxyl groups of the oxidized 1,4- β -D-glucuronic acid oligosaccharide of formula (I) of the present invention or a pharmaceutically acceptable salt thereof include, but are not limited to: formic acid, acetic acid, oxalic acid, glycolic acid, propionic acid, malonic acid, butyric acid, succinic acid, valeric acid, acrylic acid, oxalic acid, maleic acid, fumaric acid, malic acid, succinic acid, citric acid, tartaric acid, lactic acid, methanesulfonic acid, lactic acid, salicylic acid, acetylsalicylic acid, benzenesulfonic acid, p-toluenesulfonic acid, pyruvic acid, hydroxybutyric acid, adipic acid, phthalic acid, mandelic acid, benzoic acid, boric acid, and the like. The inorganic acids capable of forming an ester with one or more hydroxyl groups of the oxidized 1,4- β -D-glucuronic acid oligosaccharide of formula (I) of the present invention or a pharmaceutically acceptable salt thereof include, but are not limited to: sulfuric acid, sulfurous acid, phosphoric acid, metaphosphoric acid, phosphorous acid, hypophosphorous acid, pyrophosphoric acid, polyphosphoric acid, and the like.

The invention relates to a pharmaceutically acceptable salt of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide shown in formula (I) and a derivative thereof, which comprises the following components: inorganic salts such as lithium salt, sodium salt, potassium salt, beryllium salt, magnesium salt, calcium salt, iron salt, zinc salt, selenium salt, vanadium salt, tin salt, silicon salt, strontium salt, or basic addition salts with a basic amino acid such as lysine, arginine, ornithine, etc., among which sodium salt is preferred. The pharmaceutically acceptable salts can be prepared by conventional methods.

The medicine for treating the Alzheimer disease comprises the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide shown in the formula (I) and a derivative thereof, or pharmaceutically acceptable salts of the oligosaccharide and the derivative thereof, and one or more pharmaceutically acceptable carriers. The drug of the present invention may be in the form of tablets, hard capsules, soft capsules, enteric capsules, microcapsules, granules, syrups, injections, granules, emulsions, suspensions, solutions, and sustained-release preparations for oral or non-oral administration.

The pharmaceutically acceptable carrier of the present invention refers to pharmaceutically acceptable carriers well known to those skilled in the art, and the pharmaceutically acceptable carriers of the present invention include, but are not limited to: fillers, wetting agents, binders, disintegrants, lubricants, binders, glidants, taste masking agents, surfactants, preservatives, and the like. Fillers include, but are not limited to, lactose, microcrystalline cellulose, starch, powdered sugar, dextrin, mannitol, calcium sulfate, and the like. Wetting agents and binders include, but are not limited to, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gelatin, sucrose, polyvinylpyrrolidone, and the like. Disintegrants include, but are not limited to, sodium carboxymethyl starch, crospovidone, croscarmellose sodium, low substituted hydroxypropyl cellulose, and the like. Lubricants include, but are not limited to, magnesium stearate, aerosil, talc, hydrogenated vegetable oils, polyethylene glycols, magnesium lauryl sulfate, and the like. Binders include, but are not limited to, acacia, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, dextrates, dextrin, dextrose, ethylcellulose, gelatin, liquid glucose, guar gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, magnesium aluminum silicate, maltodextrin, methylcellulose, polymethacrylates, polyvinylpyrrolidone, pregelatinized starch, sodium alginate, sorbitol, starch, syrup, and tragacanth. Glidants include, but are not limited to, colloidal silicon dioxide, powdered cellulose, magnesium trisilicate, silicon dioxide, and talc. Taste-masking agents include, but are not limited to, aspartame, stevioside, fructose, glucose, syrup, honey, xylitol, mannitol, lactose, sorbitol, maltitol, glycyrrhizin. Surfactants include, but are not limited to, tween-80, poloxamers. Preservatives include, but are not limited to, parabens, sodium benzoate, potassium sorbate, and the like.

Methods of preparing various pharmaceutical compositions containing various proportions of active ingredients are known or will be apparent to those skilled in the art in light of the present disclosure. As described in Remington's Pharmaceutical Sciences, Martin, e.w., ed., Mack Publishing Company,19th ed. (1995). The process for preparing the pharmaceutical composition comprises incorporating suitable pharmaceutical excipients, carriers, diluents and the like.

The pharmaceutical formulations of the present invention are manufactured in a known manner, including conventional mixing, dissolving or lyophilizing processes.

The medicaments of the invention are administered in various routes suitable for the selected mode of administration, for example orally or parenterally (by intravenous, intramuscular, topical or subcutaneous routes).

Thus, the medicaments of the invention may be administered systemically, e.g. orally, in a suitable pharmaceutically acceptable carrier, such as an inert diluent or an edible carrier. They may be enclosed in hard or soft shell gelatin capsules and may be compressed into tablets. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of swallowable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such formulations should contain at least 0.1% active compound. The proportion of active compound in such formulations may, of course, vary and may range from about 0.01% to about 99% by weight of a given unit dosage form. In such therapeutically useful pharmaceutical formulations, the amount of active compound is such that an effective dosage level is obtained.

Tablets, troches, pills, capsules and the like may also contain: binders, such as gum tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrating agents, such as corn starch, potato starch, alginic acid, and the like; lubricants, such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavor. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a vegetable oil or polyethylene glycol. Various other materials may be present, as coatings, or to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl or propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained release formulations and sustained release devices.

The active compounds may also be administered intravenously or intraperitoneally by infusion or injection. Aqueous solutions of the active compounds or their salts, optionally in admixture with non-toxic surfactants, may be prepared. Dispersants in glycerol, liquid polyethylene glycols, triacetin and mixtures thereof, and oils may also be prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders of the active ingredient, optionally encapsulated in liposomes, containing ready-to-use preparations of injectable or infusible solutions or dispersions suitable for sterility. In all cases, the final dosage form must be sterile, liquid and stable under the conditions of manufacture and storage. The liquid carrier can be a solvent or liquid dispersion medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersants, or by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of compositions which delay absorption of the agent (e.g., aluminum monostearate and gelatin).

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional required ingredients present in the sterile-filtered solution.

Useful solid carriers include finely divided solids (e.g., talc, clay, microcrystalline cellulose, silicon dioxide, alumina, and the like). Useful liquid carriers include water, ethanol or ethylene glycol or water-ethanol/ethylene glycol mixtures in which the combination of the invention may be dissolved or dispersed in effective amounts, optionally with the aid of non-toxic surfactants. Adjuvants (such as fragrances) and additional antimicrobial agents may be added to optimize the properties for a given use.

Thickeners (e.g., synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified inorganic materials) can also be used with liquid carriers to form coatable pastes, gels, ointments, soaps, etc., for direct application to the skin of the user.

The therapeutically required amount of the compound or its active salt or derivative will depend not only on the particular salt selected, but also on the mode of administration, the nature of the disease to be treated and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or clinician.

The formulations may be presented in unit dosage form comprising physically discrete units of a unit dose suitable for administration to the human or other mammalian body. The unit dosage form may be a capsule or tablet, or a plurality of capsules or tablets. The amount of unit dose of the active ingredient may be varied or adjusted from about 0.01 to about 1000 mg or more depending upon the particular treatment involved.

In some embodiments, the present invention also relates to oxidized 1,4- β -D-glucuronic acid oligosaccharide and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof for treating Alzheimer's disease, which are oxidized 1,4- β -D-glucuronic acid oligosaccharide and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof represented by the following structural formula (I),

wherein n in formula (I) is one or more integers selected from 0 to 19. In the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide represented by the formula (I) and derivatives thereof, or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof for treating Alzheimer's disease, n is one or more integers selected from 1 to 9. In the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide represented by the formula (I) and derivatives thereof, or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof for treating Alzheimer's disease, n is one or more integers selected from 2 to 8. In the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide represented by the formula (I) and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof for treating Alzheimer's disease, n is 4.

In some embodiments, the present invention also relates to a method of treating alzheimer's disease comprising administering to a patient in need thereof a therapeutically effective amount of oxidized 1,4- β -D-glucuronic acid oligosaccharide and derivatives thereof according to the invention or pharmaceutically acceptable salts of said oligosaccharide and derivatives thereof. In some embodiments, the method for treating alzheimer's disease comprises administering to a patient in need of treatment a therapeutically effective amount of oxidized 1,4- β -D-glucuronic acid oligosaccharide and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof according to the present invention, wherein the oxidized 1,4- β -D-glucuronic acid oligosaccharide and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof are oxidized 1,4- β -D-glucuronic acid oligosaccharide and derivatives thereof represented by the following structural formula (I) or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof,

wherein n in formula (I) is one or more integers selected from 0 to 19. In some embodiments, the method for treating alzheimer's disease comprises administering to a patient in need of treatment a therapeutically effective amount of oxidized 1,4- β -D-glucuronic acid oligosaccharide represented by formula (I) and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof according to the present invention, wherein n in the oxidized 1,4- β -D-glucuronic acid oligosaccharide represented by formula (I) and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof is one or more integers selected from 1 to 9. In some embodiments, the method for treating alzheimer's disease comprises administering to a patient in need of treatment a therapeutically effective amount of oxidized 1,4- β -D-glucuronic acid oligosaccharide represented by formula (I) and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof according to the present invention, wherein n in the oxidized 1,4- β -D-glucuronic acid oligosaccharide represented by formula (I) and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof is one or more integers selected from 2 to 8. In some embodiments, the method for treating alzheimer's disease comprises administering to a patient in need of treatment a therapeutically effective amount of oxidized 1,4- β -D-glucuronic acid oligosaccharide represented by formula (I) and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof according to the present invention, wherein n in the oxidized 1,4- β -D-glucuronic acid oligosaccharide represented by formula (I) and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof is 4.

The term "treating" as used herein generally refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic, in terms of preventing the disease or its symptoms, in whole or in part; and/or may be therapeutic in terms of partially or completely stabilizing or curing the disease and/or side effects due to the disease. As used herein, "treatment" encompasses any treatment of a disease in a patient, including: (a) preventing a disease or condition in a patient susceptible to the disease or condition but not yet diagnosed as having the disease; (b) inhibiting the symptoms of the disease, i.e., arresting its development; or (c) alleviating the symptoms of the disease, i.e., causing regression of the disease or symptoms.

In all embodiments of the present invention, the oxidized 1,4- β -D-glucuronic acid oligosaccharide and derivatives thereof described in the present invention further include a mixture comprising the one or more oxidized 1,4- β -D-glucuronic acid oligosaccharides and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharides and derivatives thereof. For example, n and m may also be a plurality of integers selected from within the numerical ranges recited for each; 1, 4-beta-D-glucuronic acid oligosaccharide-OX 3, di-to-decasaccharide, 1, 4-beta-D-glucuronic acid oligosaccharide-OX 2, di-to-decasaccharide, 1, 4-beta-D-glucuronic acid oligosaccharide-OX 1, and di-to-decasaccharide can be mixed with each other in any form to play a role.

On the other hand, in all embodiments of the present invention, the numerical ranges of the present invention are intended to cover any integer within the ranges. For example, in all embodiments of the present invention, n in the oxidized 1,4- β -D-glucuronic acid oligosaccharide represented by formula (I) and derivatives thereof or pharmaceutically acceptable salts of the oligosaccharide and derivatives thereof may be any integer in the range of 0 to 19, for example, n may be one or more integers selected from 0,1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19. Unless otherwise specified, percentages recited herein refer to weight percentages.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.

First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The present invention is described in detail by using the schematic structural diagrams, etc., and for convenience of illustration, the schematic diagrams are not enlarged partially according to the general scale when describing the embodiments of the present invention, and the schematic diagrams are only examples, which should not limit the scope of the present invention. In addition, the actual fabrication process should include three-dimensional space of length, width and depth.

Example 1

Preparation of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide:

(1) weighing 10g of MCC powder, and carrying out mercerization treatment for 24h by using 500mL of 10% NaOH solution to prepare a mercerized MCC solution;

(2) adding the dialyzed MCC solution into about 100mL of concentrated hydrochloric acid solution, adjusting the pH to 7.0, cooling, adding into a dialysis bag, and dialyzing for 24h to remove salt.

(3) Adding 320mg of TEMPO and 3.2g of sodium bromide into the MCC solution prepared in the step (2) in sequence, adjusting the pH to 10.0 by using a 10% NaOH solution, adding 85ml of sodium hypochlorite solution with the concentration of 5% by weight in terms of active hypochlorous acid for 5 times, reacting for 4 hours at the temperature of 50 ℃, and adding absolute ethyl alcohol to stop the reaction;

(4) dialyzing in a dialysis bag of 500Da, concentrating, and lyophilizing to obtain oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture a.

(5) Weighing 1g of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture sample (a) obtained in the previous step, dissolving the sample (a) in 10mL of purified water, and preparing a sample solution of 100mg/mL for later use. 0.6mL of each sample was loaded, and 500mL of each sample was isocratically eluted with 0.1M NaCl (column: Superdex TM 30pg-Superdex TM 75 pg). The automated collection was performed using an apparatus (AKTA pure M150) from a 96-well preparation plate, and 2mL of mobile phase was collected per well. After the elution is finished, the compositions of the same chromatographic peak are combined together according to the elution volume of the chromatographic peak.

(6) Desalting the collected fractions with 500Da dialysis bag in pure water, dialyzing for 24 hr, and freeze drying to obtain white powder of oligosaccharide mixture (OX1, OX2 and OX3) with single degree of polymerization (dp2, 3, 4, 5, 6, 7, 8, 9 and 10) respectively

Example 2

Structure confirmation of oxidized 1,4- β -D-glucuronic acid oligosaccharide mixture samples (a) and oligosaccharide mixtures (OX1, OX2 and OX3) at single degrees of polymerization (dp2, 3, 4, 5, 6, 7, 8, 9 and 10):

weighing 4mg of a sample to be detected, fully dissolving the sample by using 200 mu L of ultrapure water in a vortex mode, and carrying out UPLC-Q/TOF-MS analysis after passing through a filter membrane of 0.22 mu m. The samples were separated using an Agilent 1290 ultra high pressure liquid chromatography system under the following chromatographic conditions: waters ACQUITY UPLC @ BEH 125 size exclusion chromatography column (1.7 μm, 4.6X 300 mm); mobile phase, 80% 50mM NH4OAc in water and 20% methanol; the flow rate is 0.1 ml/min; the sample amount is 5 mul; the column temperature is 25 ℃; the detection wavelength is 210 nm. The mass spectrum data acquisition uses Agilent 6540Q/TOF-MS high-resolution mass spectrum equipped with an electrospray ionization source (ESI), and the acquisition is carried out in a negative ion mode, and the scanning mass number range is 100-3000 m/z. The remaining mass spectral parameters were as follows: capillary voltage 3500V; fragmentation voltage 120V; the drying airflow rate is 8 l/min; the temperature of the drying gas is 325 ℃; the atomization gas pressure was 40 psig. Data analysis used massvhunter software. The chromatogram and mass spectrum of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture are shown in figures 1 and 2, and the chromatogram of oligosaccharide mixture with single polymerization degree is shown in figure 3.

Example 3

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) inhibits A beta 1-40 aggregation:

1. experimental Material

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide (n is 1-9, namely the components dp2-dp10 in the figure, m is 0,1,2) mixture of the formula I is prepared according to the method of the first example, and the mixture is white powder and is easy to dissolve in water; SensoLyte Thioflavin T beta-Amyloid (1-40) Aggregation Kit (containing A beta 1-40 and THT), ANASPEC corporation, USA.

2. Experimental methods

(1) Preparing working solution

Preparing a THT working solution: mu.L of 20mM ThT was added to 900. mu.L buffer to give a 2mM working solution.

Preparing an Abeta 1-40 solution: 1mL of ice-cold buffer was added to the A β 1-40 vial and the solution was inverted and sonicated for 5min to facilitate dissolution, but no vortexing was possible. Centrifuging at 10000rpm in a four-degree centrifuge for 5min, sucking the supernatant, and placing on ice for later use.

(2) Preparation of aggregation assay

In a black 96-well plate, 10. mu.L of 2mM ThT was added.

Add 85. mu.L of freshly prepared Abeta.1-40 and 5. mu.L of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide solution, including the control group. So that the molar ratio of A.beta.to sample (calculated as the average molecular mass of the mixture) was 1:4 in a 100. mu.L system.

Another set of 10. mu.L of 2mM ThT + 85. mu.L buffer + 5. mu.L samples was set up. So as to eliminate the interference of the sample on the THT fluorescence value.

(3) Detection of

The mixed 96-well plate was immediately transferred to the Biotek rotation 5 cell imaging microplate detection system, at which time the system had been preheated to 37 ℃. The fluorescence at Ex/Em 440nm/484nm was read immediately and recorded as 0 dots. Fluorescence was read every 10min thereafter, with 15s shaking between readings. The data is processed and analyzed.

3. Results of the experiment

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) can inhibit the aggregation beta amyloid cascade hypothesis of A beta 1-40, which is one of the main hypotheses for the pathogenesis of Alzheimer's disease, and the A beta 1-40 plays an important role in the pathogenesis of Alzheimer's disease, and the aggregation of the A beta 1-40 can cause the cytotoxicity of neurons to a certain degree. THT is a fluorescent dye and can be specifically combined with A beta protein, and the higher the aggregation degree of the A beta protein, the higher the fluorescence value of THT, thereby being capable of representing the aggregation degree of the A beta.

The results of the experiments on the inhibition of A beta 1-40 aggregation by the oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid (a) are shown in Table 1. The A beta 1-40 gradually generates aggregation in the incubation process at 37 ℃, the aggregation is more obvious along with the prolonging of time, the fluorescence value of the Control group is steadily increased, and the fluorescence value is increased from 12053 at 0min to 56936 at 180min and almost reaches the equilibrium within the third hour. The addition of the oxidized oligosaccharide mixture (a) of 1, 4-beta-D-glucuronic acid can inhibit the aggregation of Abeta 1-40 to a certain extent, and the fluorescence value of the group is 53257 at 180 min.

TABLE 1 oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid (a) inhibits Ass 1-40 aggregation

Example four

Protection of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) against SH-SY5Y cell injury caused by A beta 25-35:

1. experimental Material

Cell: human neuroblastoma cell line SH-SY5Y, ATCC origin.

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) of the formula I' is prepared according to the method of the first example (n is 1-9, namely the components dp2-dp10 in the figure, m is 0,1,2), and the mixture is white powder and is easy to dissolve in water; amyloid beta 25-35(a β 25-35), manufactured by sigma aldrich trade ltd; fetal bovine serum, DMEM medium, penicillin/streptomycin solution, Gibco, usa; CCK-8 detection kit, Shanghai Biyuntian biotechnology Limited.

2. Experimental methods

(1) The A beta 25-35 was dissolved in a 1mg/mL solution according to the instructions of the reagents, incubated at 37 ℃ for seven days and aged into an oligomeric form with toxicity.

(2) SH-SY5Y cells cultured normally were collected, the number of cells was adjusted to 5X 104 cells/ml, and the cells were seeded in a 96-well culture plate at 100. mu.l/well, each group was provided with 3 parallel wells (n: 3), and a control group, a model group, and an administration group were provided.

(3) After 24 hours of culture, the old medium was discarded, a serum-free medium was added, 10. mu.L of each sample solution of different concentrations prepared from the medium for the product obtained in example one was added to the administration group so that the final concentration of the system was 25, 50, and 100. mu.g/mL, and the same volume of the medium was added to the normal group.

(4) After further culturing for 2h, the aged A beta solutions were added to the model group and the administration group, respectively, so that the final A beta concentration was 30. mu. mol/L.

(5) After the culture is continued for 48h, 10 mu L of CCK-8 solution is added into each well, the absorbance at 450nm is detected after 4h, and the cell survival rate is calculated.

3. Statistical treatment

The results were statistically analyzed and expressed as "mean ± standard error" (mean ± SEM) and compared using analysis of variance (ANOVA). The difference is significant when p is less than 0.05, and the difference is very significant when p is less than 0.0 l.

4. Results of the experiment

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) has the protective effect on SH-SY5Y cell injury caused by A beta 25-35.

The Abeta 25-35 can induce SH-SY5Y cell damage, and the cell viability is reduced, which is shown in that the absorbance of the model group is reduced compared with that of the control group. The lower the absorbance, the poorer the cell viability.

The experimental results of the protective effect of the oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid (a) on SH-SY5Y cell damage caused by A beta 25-35 are shown in Table 2. The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) has a protective effect on the existence of SH-SY5Y cell injury caused by A beta 25-35, and shows a dose-dependent relation among dose groups, and the effect of a high dose group is best. Compared with the control group, the administration group and the model group have different cell viability among the groups, and the high-dose group has statistical significance. The protective effect of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) on SH-SY5Y cell injury caused by A beta 25-35 is shown.

TABLE 2 protective effect of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) on SH-SY5Y cell damage caused by A beta 25-35 (n ═ 3, mean + -SEM)

Model group to control group comparison: #, p < 0.01;

the dosing group was compared to the model group: p < 0.05.

EXAMPLE five

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) can protect neuronal cells by regulating the mRNA and protein levels of related genes.

1. Experimental Material

Cell: human neuroblastoma cell line SH-SY5Y, ATCC origin.

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) of the formula I' is prepared according to the method of the first example (n is 1-9, namely the components dp2-dp10 in the figure, m is 0,1,2), and the mixture is white powder and is easy to dissolve in water; amyloid beta 25-35(a β 25-35), manufactured by sigma aldrich trade ltd; fetal bovine serum, DMEM medium, penicillin/streptomycin solution, Gibco, usa; primer synthesis, Invitrogen, usa; RNA extraction reagent, reverse transcription kit and SYBR Green Mix kit, TAKARA, japan); primary and secondary antibodies to Bcl2, p53, Drp1, Parkin, and GAPDH, CST inc.

2. Experimental methods

(2) SH-SY5Y cells cultured normally are taken, the number of the cells is adjusted to 5 × 104/mL, and the cells are inoculated in a 24-well culture plate at 500 μ L/well or in a 6-well plate at 2 mL/well, each group is provided with 3 parallel wells (n is 3), and a control group, a model group and an administration group are provided.

(3) After 24 hours of culture, the old medium was discarded, a serum-free medium was added, samples of different concentrations prepared from the medium for the product obtained in example one were added to the administration groups so that the final concentration of the system was 25, 50, 100. mu.g/mL, and the same volume of medium was added to the normal group.

(4) After further culturing for 2h, the aged A beta solution was added to the model group and the administration group, respectively, so that the final concentration of A beta 25-35 was 30. mu. mol/L.

(5) After continuing culturing for 48h, extracting total RNA from the cells in the 24-well plate; after the concentration is quantified, carrying out reverse transcription to obtain cDNA according to the kit specification; then, the mRNA level of the target gene is quantitatively detected in real time according to the specification of a SYBR Green Mix kit and the primer of the target gene.

(6) Continuously culturing the 6-hole plate for 48 hours after the step (4), using the cells for extracting protein, quantifying the total protein concentration of each hole and normalizing; changes in the protein level of the gene of interest among the groups were examined according to the Western Blot protocol.

3. Statistical treatment

4. Results of the experiment

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) can protect nerve cells and reduce the damage of oligomeric beta amyloid to the nerve cells by regulating the mRNA and protein levels of a B lymphoma-2 gene Bcl2, a human cancer suppressor gene p53, a mitochondrial dynamics protein Drp1 and a Parkin gene.

The oligomeric state of a β 25-35 can change the mRNA levels of some genes in the neural SH-SY5Y cells as shown in tables 3, 4, 5, 6 (n ═ 3, mean ± SEM), the mRNA levels of B lymphoma-2 gene Bcl2 are reduced, the mRNA levels of human tumor suppressor gene p53 are increased, the mRNA levels of mitochondrial dynamics protein Drp1 are reduced, and the mRNA levels of Parkin gene are reduced, whereas different doses of oxidized 1,4- β -D-glucuronic acid oligosaccharides can reverse the change, with a dose-dependent relationship, and the high dose group 100 μ g/mL has statistical significance.

TABLE 3 Regulation of Bcl2 gene mRNA by oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid (a) (mean + -SEM, n-3)

Model group to control group comparison: #, p < 0.01;

the dosing group was compared to the model group: p < 0.01.

TABLE 4 Regulation of p53 gene mRNA by oxidized oligosaccharide mixture of 1, 4-beta-D-glucuronic acid (a) (n ═ 3, mean. + -. SEM)

Model group to control group comparison: #, p < 0.05;

the dosing group was compared to the model group: p < 0.05; p < 0.01.

TABLE 5 Regulation of Drp1 gene mRNA by oxidized oligosaccharide mixture of 1,4- β -D-glucuronic acid (a) (n ═ 3, mean. + -. SEM)

Model group to control group comparison: #, p < 0.05;

the dosing group was compared to the model group: p < 0.05.

TABLE 6 Regulation of mRNA of Parkin gene by oxidized oligosaccharide mixture of 1,4- β -D-glucuronic acid (a) (mean. + -. SEM, n ═ 3)

Model group to control group comparison: #, p < 0.05;

the dosing group was compared to the model group: p < 0.01.

The western blot experiment result is shown in fig. 6, oligomeric state of a β 25-35 can reduce the protein level of Bcl2, increase the protein level of human cancer suppressor gene p53, reduce the level of mitochondrial dynamics protein Drp1, and reduce the protein level of Parkin gene in the SH-SY5Y cells of nerve cells, and different doses of oxidized 1,4- β -D-glucuronic acid oligosaccharide can reverse the change, thereby reducing the damage of a β 25-35 to nerve cells and improving the cell viability.

EXAMPLE six

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) has the effect of improving the cognitive disorder of mice caused by A beta 1-40.

1. Experimental Material

1.1 drugs and reagents

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (a) of the formula I' is prepared according to the method of the first example (n is 1-9, namely the components dp2-dp10 in the figure, m is 0,1,2), and the mixture is white powder and is easy to dissolve in water; amyloid beta 1-40(A beta 1-40).

1.2 Experimental animals

C57 mouse, 8 weeks old, male, weight-22 g, supplied by Shanghai Ling Chang Biotech limited.

1.3 Experimental instruments

Brain stereotaxic apparatus, micro-syringe pump, STOELTING; model 204 dental bur, STRONG; mouse motion trajectory tracking system EthoVision XT, Noldus; morris Water maze, Shanghai Xin Soft information technology, Inc.

2. Test method

2.1 animal grouping and administration

Male C57 mice were randomly divided into a blank control group, a sham operation group, a model group, and administration groups of 100, 200, and 400mg/kg, each group consisting of 15 animals. The corresponding drugs are orally taken to each group, and distilled water is orally taken to a blank control group, a sham operation group and a model group.

2.2 animal Molding operation

After 7 days of continuous administration, except for the blank control group, all mice were anesthetized by 400mg/kg of chloral hydrate via intraperitoneal injection, the mice were fixed on a stereotaxic apparatus, a central incision was made at the vertex of the head, the skin was incised layer by layer, and the skull cap bone was exposed at a central opening of the sagittal suture of the brain. After moving 2mm backward on the chimney, 1.5 mm to the right, inserting a hole by a dental drill, then downwards moving a microsyringe by 1.5 mm at the injection position of the hippocampus CA1 area, and injecting 3 microliter of aged Abeta 1-40 (with the concentration of 1.4mg/mL, aged for 5 days) or PBS at a constant speed for 5 minutes. All manipulations were performed under sterile conditions. Injecting PBS into the sham operation group, injecting Abeta 1-40 into the model group and the administration group, staying for 5 minutes after injection, pulling out the needle, suturing the scalp, smearing a proper amount of gentamicin for disinfection, and putting back into the cage for reviving.

2.3 behavioural testing

The administration is continued on the day of surgery, and is carried out for 10 days after surgery, and then an ethological experiment is carried out to test the learning and memory abilities of the mice.

Firstly, the learning ability of each group of mice is detected by using a mouse motion trail tracking system, the mouse motion trail tracking system is used for placing the mouse in an observation box, and the spontaneous motion of the mouse is monitored by an infrared camera at the top of the observation box. The lower left corner of the observation box is provided with a triangular plate, the triangular plate is provided with three holes, the mouse has the nature of drilling holes, when the mouse continuously enters the left hole five times, the system rewards one food, and when the success rate of the mouse entering the left hole reaches 80%, the event is defined as learning. The learning ability of the mouse is judged by comparing the total hole entering times of the total attempts of the mouse to learn the event, and the smaller the total times, the better the learning ability of the mouse is.

And then detecting the learning and space memory abilities of the mouse by using the Morris water maze, training the mouse to search for a platform in the first 5 days generally, taking a rest in the sixth day, removing the platform in the seventh day, and detecting the escape latency and the times of passing through the platform of the mouse. The shorter the escape latency of the mouse and the more times of passing through the platform, the better the learning and spatial memory ability of the mouse is.

3. Statistical treatment

The results were statistically analyzed and expressed as "mean ± standard error" (mean ± SEM) and compared using analysis of variance (ANOVA). Differences with p <0.05 were statistically significant.

4. Test results

The hippocampal structure belongs to an important structure in the limbic system of the brain, is bilaterally symmetric, and is involved in learning, memory, and cognitive functions, especially short-term memory and spatial memory. The position of injecting Abeta is CA1 area of right hippocampus, which is vulnerable area and can simulate Alzheimer disease to some extent. The research results are shown in tables 7 and 8, and the injection of Abeta 1-40 in the area of the mouse CA1 obviously causes the reduction of the learning and memory functions of the mouse, which shows that the total times of the events of the student in the experiment of the mouse motion trail tracking system, which need to try to enter the hole, is obviously higher than that of the control group in the model group compared with the control group; in the water maze experiment, the escape latency of the model group is higher than that of the control group, and the platform crossing times of the model group are lower than that of the control group. The continuous administration of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide can reverse the conditions, has an improvement effect on mouse cognitive impairment caused by Abeta 1-40, and is in a dose-dependent relationship. The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide has the potential of being developed into an anti-Alzheimer disease medicament.

Table 7. improving effect of oxidized 1,4- β -D-glucuronic acid oligosaccharide mixture (a) on a β 1-40 induced cognitive impairment of mice, and results of experiments of a mouse movement trajectory tracking system. (n 15 mean ± SEM)

Model group to control group comparison: #, p < 0.01;

the dosing group was compared to the model group: p < 0.01; p < 0.001.

Table 8. improving effect of oxidized 1,4- β -D-glucuronic acid oligosaccharide mixture (a) on a β 1-40 induced cognitive impairment in mice, Morris water maze experimental results. (n 15 mean ± SEM)

Model group to control group comparison: #, p < 0.01; # #, p < 0.001;

the dosing group was compared to the model group: p < 0.05; p < 0.001.

EXAMPLE seven

The protective effect of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (OX1, OX2 and OX3) at a single polymerization degree (dp2, 3, 4, 5, 6, 7, 8, 9 and 10) on SH-SY5Y cell damage caused by A beta 25-35.

1. Experimental Material

Cell: human neuroblastoma cell line SH-SY5Y, ATCC origin.

The oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture (OX1, OX2 and OX3) with single polymerization degree (n is 1-9, namely, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide and decasaccharide) shown in the formula I' is prepared according to the method of the first embodiment, is white powder and is easy to dissolve in water; amyloid beta 25-35(a β 25-35), manufactured by sigma aldrich trade ltd; fetal bovine serum, DMEM medium, penicillin/streptomycin solution, Gibco, usa; CCK-8 detection kit, Shanghai Biyuntian biotechnology Limited.

2. Experimental methods

(3) After 24 hours of culture, the old medium was discarded, a serum-free medium was added, and 10. mu.L of each of the samples of different concentrations prepared in the medium for the product obtained in example one was added to the administration group so that A.beta.: oligosaccharide molar ratios were identical (as converted to average molecular weight at a final concentration of the mixture of 100. mu.g/mL) and the same volume of medium was added to the normal group.

(4) After further culturing for 2h, the model group and the administration group were added with aged A β to dissolve, respectively, so that the final concentration of A β was 30. mu. mol/L.

3. Statistical treatment

4. Results of the experiment

Protection effect of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide mixture with single polymerization degree on SH-SY5Y cell injury caused by Abeta 25-35

The Abeta 25-35 can induce SH-SY5Y cell damage, and the cell viability is reduced, which is shown in that the absorbance of the model group is reduced compared with that of the control group. The mixture of oxidized 1, 4-beta-D-glucuronic acid oligosaccharides with different polymerization degrees has a protective effect on SH-SY5Y cell damage caused by A beta 25-35, the experimental results are shown in Table 9, and the higher the polymerization degree is, the better the activity is.

TABLE 9 protective Effect of the mixture of oxidized 1, 4-ss-D-glucuronic acid oligosaccharides with a single degree of polymerization on SH-SY5Y cell damage caused by A ss 25-35 (n ═ 3, mean. + -. SEM)

Model group to control group comparison: # #, p < 0.001;

the dosing group was compared to the model group: p < 0.05; p < 0.01; p <0.001

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. The application of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide or pharmaceutically acceptable salt thereof in preparing a medicament for treating Alzheimer's disease is characterized in that: the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide or the pharmaceutically acceptable salt thereof comprises the following general formula (I):

wherein,

n in the formula (I) is selected from any one or more integers from 0 to 19;

m is selected from 0,1 or 2;

one or more oxidized 1, 4-beta-D-glucuronic acid oligosaccharides with the structure of the general formula (I) or pharmaceutically acceptable salts thereof are obtained by selecting one or more integers from n.

2. Use of oxidized 1,4- β -D-glucuronic acid oligosaccharide or a pharmaceutically acceptable salt thereof according to claim 1, for the preparation of a medicament for treating alzheimer's disease, characterized in that: and n is selected from any one or more integers from 1 to 9.

3. Use of oxidized 1,4- β -D-glucuronic acid oligosaccharide or a pharmaceutically acceptable salt thereof according to claim 2, in the preparation of a medicament for treating alzheimer's disease, characterized in that: n is 1,2, 3, 4, 5, 6, 7, 8 and 9, respectively corresponding to the sugars of oxidized 1, 4-beta-D-glucuronic acid oligosaccharide 2, 3, 4, 5, 6, 7, 8, 9 and 10.

4. Use of oxidized 1,4- β -D-glucuronic acid oligosaccharide or a pharmaceutically acceptable salt thereof according to claim 1, for the preparation of a medicament for treating alzheimer's disease, characterized in that: the component with n of 1-9 accounts for more than 80% of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide or the pharmaceutically acceptable salt thereof.

5. Use of oxidized 1,4- β -D-glucuronic acid oligosaccharide or a pharmaceutically acceptable salt thereof according to claim 4, for the preparation of a medicament for treating alzheimer's disease, characterized in that: the component with n of 1-9 accounts for more than 90% of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide or the pharmaceutically acceptable salt thereof.

6. Use of oxidized 1,4- β -D-glucuronic acid oligosaccharide or a pharmaceutically acceptable salt thereof according to claim 5, for the preparation of a medicament for treating alzheimer's disease, characterized in that: the component with n of 1-9 accounts for more than 95% of the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide or the pharmaceutically acceptable salt thereof.

7. Use of oxidized 1,4- β -D-glucuronic acid oligosaccharide or a pharmaceutically acceptable salt thereof according to claim 1, for the preparation of a medicament for treating alzheimer's disease, characterized in that: the oxidized 1, 4-beta-D-glucuronic acid oligosaccharide or the pharmaceutically acceptable salt thereof is applied to improving the survival rate of beta amyloid protein-damaged neuron cells.