Composition of alginate oligosaccharin diacid

Abstract

The present invention relates to an alginate oligosaccharin diacid composition comprising mannuronic diacid of formula (IV) or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 1 to 9, m is selected from 0, 1 or 2, and m' is selected from 0 or 1, and wherein the total weight of alginate oligosaccharin diacids where n=1 to 5 is 60% or more by weight of the total composition, and the total weight of guluronic acid is 50% or less by weight of the total composition.

Description

[Technical field]

[0001] The present invention relates to an optimal alginate oligosaccharin diacid composition obtained by a bioactivity screening method using an animal model of Alzheimer's disease to evaluate the effect of different degrees of polymerization of alginate oligosaccharides and their ratios on bioactivity. Finally, the composition with the best bioactivity is obtained from the screening, and the desired target substance is prepared by a method of ultrafiltration membrane separation. [Background technology]

[0002] Alginate oligosaccharides have attracted widespread attention due to their potential medical value. Alginate oligosaccharides are usually prepared by a multi-step method using alginic acid as raw material.

[0003] The raw alginate oligosaccharide molecule contains the M segment formed from D-mannuronic acid linked by β-1,4-glucosidic bonds, the G segment formed from L-guluronic acid linked by α-1,4-glucosidic bonds, and the MG segment formed by hybridization of these two sugars (sacchorides). The structural formulae of D-mannuronic acid and L-guluronic acid are shown in the following formula (I) and formula (II). [ka]

[0004] The structural formula of alginic acid oligosaccharide is shown below by formula (III). [ka]

[0005] The M and G segments can be separated from the raw alginic acid. The general method can be briefly described as follows: Alginic acid is pre-decomposed to obtain a polysaccharide mixture of polymannuronic acid and polyguluronic acid, and then the polysaccharide mixture is subjected to acid precipitation to remove a certain amount of polyguluronic acid therein. See, for example, the methods disclosed in Chinese Patent Application Nos. 98806637.8 and CN02823707.2.

[0006] The method for preparing oligomannuronic acid is as follows: the above obtained M segment intermediate can be further subjected to acid degradation by heating under acidic conditions to obtain small fragment mannuronic acid polymers with a desired range of molecular weight. Furthermore, the degradation efficiency can be improved by oxidative degradation method, while the reducing end can be oxidized to ring-opened saccharic diacid. For details, see Chinese Patent Application No. 200580009396.5 (Patent Document 1) and U.S. Patent No. 8,835,403 B2 (Patent Document 2) filed by Meiyu Geng et al. For convenience, Patent Documents 1 and 2 are hereinafter collectively referred to as prior art documents, and are incorporated by reference in their entirety into the disclosure of this specification.

[0007] The reaction to obtain mannuronic diacid disclosed in the prior art can be represented by the following reaction formula (V): In other words, the aldehyde group at the C1 position of mannuronic acid at the reducing end of an oligomannuronic acid polysaccharide is oxidized to a carboxyl group. [ka]

[0008] In the above oxidation conversion process, the commonly used oxidizing agent is an alkaline copper sulfate solution, i.e., Fehling's reagent. Prior literature has adopted this oxidation method. Specifically, under alkaline conditions, the reaction substrate polymannuronic acid, i.e., the above M segment intermediate, is added to a copper sulfate solution and reacted in a boiling water bath for 15 minutes to 2 hours. This method uses Cu to oxidize the aldehyde group. 2+ions are used as oxidants, and a brick-red precipitate of cuprous oxide is produced in the reaction. This reaction is often used to identify reducing sugars.

[0009] Prior literature has disclosed that oligomannaric acids have effects on Alzheimer's disease (AD) and diabetes, and that oligomannaric acids with a polymerization degree of 6 have the best activity. The pathogenesis of Alzheimer's disease and type 2 diabetes is closely related to amyloid (β-amyloid and amylin). Amyloid proteins aggregate, then form protein oligomers, and further aggregate to form fibrils. These protein aggregates are cytotoxic, inducing oxidative reactions in cells to damage mitochondria, triggering cascade reactions such as inflammatory reactions, causing damage to a large number of neurons and β cells, and ultimately leading to the development of Alzheimer's disease and type 2 diabetes. Oligomannaric acids target amyloid proteins and antagonize the cascade reactions induced by amyloid proteins, and therefore have the effect of preventing and treating Alzheimer's disease and type 2 diabetes.

[0010] In order to obtain oligomannaric acid having anti-Alzheimer's disease and anti-diabetic effects as disclosed in the prior art, it is necessary to remove guluronic acid from the raw alginic acid. The content of guluronic acid in alginic acid is usually more than 30%, up to about 70%. Therefore, in order to obtain high-purity oligomannaric acid, the actual production cost is extremely high. Summary of the Invention

[0011] The first aspect of the present invention relates to a compound represented by formula (IV): [ka] (In the formula, n is an integer selected from 1 to 9, m is selected from 0, 1 or 2, and m' is selected from 0 or 1.) or a pharmaceutically acceptable salt thereof, The present invention relates to an alginate-oligosaccharin diacid composition, in which the total weight of alginate-oligosaccharin diacids, n=1-5, is 60% or more relative to the total weight of the composition, and the total weight of guluronic acid is 50% or less relative to the total weight of the composition.

[0012] Another aspect of the present invention relates to a pharmaceutical composition or health care product comprising the above alginate-oligosaccharin composition. Another aspect of the present invention also relates to the application of the alginate-oligosaccharin composition in the treatment of diseases selected from Alzheimer's disease, Parkinson's disease, inflammation, pain, diabetes or vascular dementia.

[0013] In particular, the alginate oligosaccharinic acid composition of the present invention is a mixture of mannuronic acid and guluronic acid with various degrees of polymerization, the main components of which are oligosaccharides with degrees of polymerization of 2 to 10, M segments formed from mannuronic acid linked by β-1,4-glucosidic bonds, G segments formed from guluronic acid linked by α-1,4-glucosidic bonds, and MG segments formed by the hybridization of these two sugars (sacchorides). Mannuronic diacid is known to have a specific pharmacological activity against Alzheimer's disease (AD) and diabetes. The most active sugars are pentasaccharides to decasaccharides, especially hexasaccharides. However, the present inventors have found that a mixture of mannuronic acid and guluronic acid with degrees of polymerization of 2 to 10 also has pharmacological activity against Alzheimer's disease (AD) and diabetes, but the premise is that the guluronic acid content is controlled within a specific range. In other words, the alginate oligosaccharin diacid composition of the present invention can be prepared at a significantly reduced production cost, which makes it easier to realize practical production and easier to realize industrial mass production. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the NMR spectrum of the intermediate. [Diagram 2]FIG. 2 shows the mass spectrum of the di-, tri- and tetrasaccharides in product A. [Diagram 3] FIG. 3 shows the mass spectra of the pentasaccharide, hexasaccharide, and heptasaccharide in product A. [Figure 4] FIG. 4 shows the mass spectra of the octa-, nona- and decasaccharides in product A. [Diagram 5] FIG. 5 shows the NMR spectrum of product A. [Figure 6] FIG. 6 shows the NMR spectrum of product B. [Figure 7] FIG. 7 shows the NMR spectrum of product C. [Figure 8] FIG. 8 shows the NMR spectrum of product D. [Figure 9] Figure 9 shows the effect of various oligosaccharide compositions and mannuronic diacid hexasaccharides on the number of platform crossings in AD animals. The samples corresponding to the numbers on the abscissa of the figure are as follows: i: control group; ii: model group; iii: product A; iv: product B; v: product C; vi: product D; vii: mannuronic diacid hexasaccharides. [Figure 10] FIG. 10 shows the effect of various oligosaccharide compositions and mannuronic diacid hexasaccharide on the swimming distance of AD animals, with the symbols on the abscissa of the figure being the same as in FIG. [Figure 11] FIG. 11 shows the effect of different oligosaccharide compositions and mannuronic diacid hexasaccharide on the descent time of PD animals on day 11, the symbols on the abscissa of the figure are the same as in FIG. [Figure 12] FIG. 12 shows the effect of different oligosaccharide compositions and mannuronic diacid hexasaccharides on the latency of PD animals on day 11, the symbols on the abscissa of the figure are the same as in FIG. [Figure 13a] 13a and 13b show the therapeutic effect of different oligosaccharide compositions and mannuronic diacid hexasaccharide on inflammatory bowel disease in mice, the symbols on the abscissa of the figures are the same as in FIG. [Figure 13b] 13a and 13b show the therapeutic effect of different oligosaccharide compositions and mannuronic diacid hexasaccharide on inflammatory bowel disease in mice, the symbols on the abscissa of the figures are the same as in FIG. [Figure 14] FIG. 14 shows the effect of different oligosaccharide compositions and mannuronic diacid hexasaccharide on postprandial glycemia in diabetic mice, the symbols on the abscissa of the figure are the same as in FIG. [Figure 15] Figure 15 shows the effects of various oligosaccharide compositions and mannuronic diacid hexasaccharide on the latency of writhing responses induced by acetic acid in mice, with samples corresponding to the numbers on the abscissa of the figure as follows: i: model group; ii: product A; iii: product B; iv: product C; v: product D; vi: mannuronic diacid hexasaccharide. [Figure 16] FIG. 16 shows the effect of various oligosaccharide compositions and mannuronic diacid hexasaccharides on the number of writhing responses in mice induced by acetic acid, the symbols on the abscissa of the figure being the same as in FIG. [Figure 17] FIG. 17 shows the effect of different oligosaccharide compositions and mannuronic diacid hexasaccharide on the number of head scratching bouts in nitroglycerin-induced migraine rats, the symbols on the abscissa of the figure being the same as in FIG. [Figure 18] Figure 18 shows the effects of various oligosaccharide compositions and mannuronic diacid hexasaccharide on the number of c-fos positive cells in the caudal part of the spinal trigeminal nucleus (nucleus caudalis) in rats with migraine induced by electrical stimulation of the trigeminal ganglion, with the symbols on the abscissa being the same as in Figure 9. [Figure 19] FIG. 19 shows the effect of various oligosaccharide compositions and mannuronic diacid hexasaccharides on the latency in the dark avoidance test in mice with vascular dementia induced by bilateral common carotid artery occlusion, the symbols on the abscissa of the figure are the same as in FIG. 9. [Figure 20] FIG. 20 shows the effect of various oligosaccharide compositions and mannuronic diacid hexasaccharides on the number of errors in the dark avoidance test in mice with vascular dementia induced by bilateral common carotid artery occlusion, the symbols on the abscissa of the figure are the same as in FIG. 9. [Figure 21]FIG. 21 shows the effects of various oligosaccharide compositions and mannuronic diacid hexasaccharide on the escape latency in the water maze test in mice with vascular dementia induced by bilateral common carotid artery occlusion, the symbols on the abscissa of the figure are the same as in FIG. 9. [Figure 22] FIG. 22 shows the effect of different oligosaccharide compositions and mannuronic diacid hexasaccharide on the number of platform crossings in mice with vascular dementia induced by bilateral common carotid artery occlusion, the symbols on the abscissa of the figure are the same as in FIG. 9. Description of the Invention

[0015] Various aspects of the present invention are described in detail below, but the present invention is not limited to these specific embodiments. Those skilled in the art can make some modifications and adjustments to the present invention based on the substantial disclosure below, and such modifications are also within the scope of the present invention.

[0016] Alginate Oligosaccharin Diacid Composition The first aspect of the present invention relates to a compound represented by formula (IV): [ka] (In the formula, n is an integer selected from 1 to 9, m is selected from 0, 1 or 2, and m' is selected from 0 or 1.) or a pharmaceutically acceptable salt thereof, The total weight of the alginic acid oligosaccharin diacids, n=1 to 5, is 60% or more based on the total weight of the composition; The present invention relates to an alginate oligosaccharin diacid composition, wherein the total weight of guluronic acid is less than or equal to 50% by weight relative to the total weight of said composition.

[0017] The alginate oligosaccharinic acid composition of the present invention is a mixture of mannuronic acid and guluronic acid with various degrees of polymerization, and its main components are oligosaccharides with degrees of polymerization of 2 to 10, M segment formed from mannuronic acid linked by β-1,4-glucosidic bonds, G segment formed from guluronic acid linked by α-1,4-glucosidic bonds, and MG segment formed by hybridization of these two sugars (sacchorides). According to the previous application, mannuronic diacid is known to have pharmacological activity against Alzheimer's disease (AD) and diabetes, and the most active sugars in mannuronic diacid are pentasaccharides to decasaccharides, especially hexasaccharides. However, unlike the known prior art, the present inventors have found that a mixture of mannuronic acid and guluronic acid with degrees of polymerization of 2 to 10 also has pharmacological activity against Alzheimer's disease (AD) and diabetes, but the guluronic acid content needs to be controlled within a certain range.

[0018] In the actual preparation process, the guluronic acid content of the product after the initial pre-decomposition of alginic acid as described above is usually more than 30%, up to about 70%. According to the prior application, in order to obtain oligomannaric acid with high activity, guluronic acid must be removed as much as possible by separation. However, based on the above findings of the present inventors, there is no need to separate and remove guluronic acid from the decomposition product. Furthermore, the present inventors have found that by controlling the proportion of guluronic acid within a certain range by controlling the conditions of the acid precipitation reaction, the activity of the obtained composition can be equal to or even better than that of the oligomannaric acid hexasaccharide disclosed in the prior application. Also, since there is no need to remove guluronic acid as an impurity, the product yield is significantly higher than that disclosed in the prior application. Thus, it greatly reduces the production cost and reduces the discharge of waste, which makes it easier to realize practical production and industrial large-scale production.

[0019] According to a preferred embodiment, in the alginate oligosaccharin diacid composition of the present invention, the total weight of the alginate oligosaccharin diacids with n=1-5 is 80-95% relative to the total weight of the composition, and the total weight of guluronic acid is 50% relative to the total weight of the composition. below It is.

[0020] According to a preferred embodiment, in the alginate oligosaccharin diacid composition of the present invention, the ratio of the total weight of alginate oligosaccharin diacids with a low degree of polymerization, n=1-3, to the total weight of alginate oligosaccharin diacids with a low degree of polymerization, n=4-7, is between 1.0 and 3.5.

[0021] According to a preferred embodiment, in the alginate oligosaccharin diacid oligosaccharide composition of the present invention, the total weight of the alginate oligosaccharin diacids where m+m'=1 or 2 is 50% or more, preferably 60% to 90%, more preferably 70% to 90% based on the total weight of the composition. In particular, in the alginate oligosaccharin diacid composition, the total weight of the alginate oligosaccharin diacids where m+m'=1 is 10% or more, preferably 30 to 40% based on the total weight of the composition. In another preferred embodiment, in the alginate oligosaccharin diacid composition, the total weight of the alginate oligosaccharin diacids where m+m'=2 is 10% or more, preferably 30 to 50% based on the total weight of the composition.

[0022] According to a preferred embodiment, in the alginate oligosaccharin diacid composition of the present invention, the total weight of the alginate oligosaccharin diacids, n=1-5, is 80-95% based on the total weight of the composition.

[0023] According to a preferred embodiment, in the alginate oligosaccharin diacid composition of the present invention, the total weight of the alginate oligosaccharin diacids with n=1-3 is 20-70% based on the total weight of the composition.

[0024] According to a preferred embodiment, in the alginate oligosaccharin diacid composition of the present invention, the ratio of the total weight of alginate oligosaccharin diacids with n=1-3 to the total weight of alginate oligosaccharin diacids with n=4-7 is between 1.0 and 3.5, preferably between 1.0 and 3.0.

[0025] According to a preferred embodiment, in the alginate oligosaccharin diacid composition of the present invention, the weight percentage content of alginate oligosaccharin diacid of each degree of polymerization in said composition is 5-25% disaccharides, 15-30% trisaccharides, 15-28% tetrasaccharides, 10-25% pentasaccharides, 5-15% hexasaccharides, 3-10% heptasaccharides, 2-5% octasaccharides, 1-5% nonasaccharides, and 1-5% decasaccharides. In particular, in the composition, the weight percentage content of oligosaccharides in said composition is 10-20% disaccharides, 18-30% trisaccharides, 15-28% tetrasaccharides, 15-20% pentasaccharides, 5-15% hexasaccharides, and 1-5% decasaccharides. 10 %, heptasaccharides 3-5%, octasaccharides 2-3%, nonasaccharides 1-3%, and decasaccharides 1-3%.

[0026] According to a preferred embodiment, the alginate oligosaccharin diacid composition of the present invention comprises , G The total weight of the uronic acid is 0.1 to 50%, preferably 1 to 30%, based on the total weight of the composition.

[0027] In the alginate oligosaccharin diacid composition of the present invention, the pharma- ceutically acceptable salt is a sodium salt or a potassium salt.

[0028] Methods for preparing alginate oligosaccharin diacid compositions The method for preparing the alginate oligosaccharin diacid of the present invention is as follows.

[0029] After preliminary degradation of alginic acid, a polysaccharide mixture of polymannuronic acid and polyguluronic acid can be obtained. The polysaccharide mixture is then precipitated by an acidic method to remove a certain amount of polyguluronic acid. In the process of this acidic precipitation, the higher the pH, the higher the polyguluronic acid content in the resulting polysaccharide mixture. See, for example, the methods disclosed in Chinese Patent Applications Nos. 98806637.8 and CN02823707.2. In the presence of an oxidizing agent, the sugar chains of the aforementioned polysaccharide mixture undergo oxidative degradation to obtain oxidized oligosaccharides with different degrees of polymerization. These oxidized oligosaccharides are characterized by the oxidation of mannuronic acid or guluronic acid at the reducing end of the oligosaccharide to saccharin diacids with 3 to 6 carbons.

[0030] An oxidizing agent particularly advantageous for the reaction of the present invention is ozone. When ozone is introduced into a solution containing a polysaccharide mixture during the reaction process, an oxidative decomposition reaction of the sugar chains can occur. The temperature at which the oxidative decomposition step is carried out is preferably 0 to 70°C, more preferably 10 to 45°C. The pH value of the oxidative decomposition step is 3 to 13, preferably 4 to 10, more preferably 6 to 8.

[0031] The oxidative decomposition reaction using ozone in the present invention, the acidic hydrolysis in the presence of alkaline copper sulfate (prior literature) or hydrogen peroxide and sodium hypochlorite (Chinese Patent Application No. 01107952.5) ​​used in the prior art are common in that these three methods can decompose glycans. The difference is that the reducing end structures of the glycans of the decomposition products are different. The reducing end of the oxidative decomposition product mannuronic acid or guluronic acid obtained in the present invention comprises a diacid structure having 3 to 6 carbons. In addition, the method used in the oxidative decomposition step of the present invention also has other advantages: 1. The reaction conditions are mild and no special reaction conditions are required; 2. The ozone used is prepared on the reaction site, which can reduce the transport pressure in industrial production; 3. After the reaction, the ozone is automatically decomposed into oxygen without the risk of residual reagents and does not cause environmental pollution. The reaction process is shown in the following formula (VI). [ka]

[0032] In the above schematic diagram of reaction formula (VI) and compound of general formula (IV), An oligosaccharide with m=2 and m'=1 is a saccharinic acid with six carbons at the end; An oligosaccharide with m=1 and m'=1 or m=2 and m'=0 is a saccharinic acid with five carbons at the end; An oligosaccharide where m=1 and m'=0 or m=0 and m'=1 is a saccharinic acid having four carbons at the end; Oligosaccharides with m=0 and m'=0 are saccharinic acids with three carbons at the ends.

[0033] In the composition, the total weight of alginic acid oligosaccharin diacids with n=1-5 is 80-95% of the total weight of the composition, the total weight of alginic acid oligosaccharin diacids with n=1-3 is 20-70% of the total weight of the composition, and the ratio of the total weight of alginic acid oligosaccharin diacids with n=1-3 to the total weight of alginic acid oligosaccharin diacids with n=4-7 is between 1.0-3.5, preferably between 1.0-3.0. The total weight of guluronic acid is less than 50% of the total weight of the composition, preferably between 0.1%-50%, most preferably between 1%-30%.

[0034] In an exemplary embodiment, the preparation method of the present invention comprises the steps of:

[0035] (1) Preparation of alginate oligosaccharin diacid product: Preparation of polysaccharide mixture of polymannuronic acid and polyguluronic acid. As mentioned above, the polysaccharide mixture of polymannuronic acid and polyguluronic acid, which is the raw material used in the present invention, can be prepared by the methods known in the prior art, such as the methods disclosed in Chinese Patent Application Nos. 98806637.8 and CN02823707.2. The general method can be briefly described as follows: alginic acid is pre-decomposed to obtain a polysaccharide mixture of polymannuronic acid and polyguluronic acid. After the polysaccharide mixture is subjected to acid precipitation again, the content of the polyguluronic acid portion can be adjusted to obtain a polysaccharide mixture of polymannuronic acid and polyguluronic acid.

[0036] Ozone oxidation decomposition. The polysaccharide mixture is dissolved in an appropriate amount of water and stirred at room temperature or under heating conditions. Ozone is continuously introduced to start the reaction. The pH value of the reaction can be adjusted to 3 to 13, preferably 4 to 10, more preferably 6 to 8, by dropping dilute hydrochloric acid or dilute NaOH solution. The temperature is preferably 0 to 70°C, more preferably 10 to 45°C. After the reaction is completed, the introduction of ozone is stopped and the pH is adjusted to neutral.

[0037] Membrane separation and purification. The reaction product obtained above is prepared into a solution with a concentration of about 10%, and separated by a molecular cut-off membrane to remove the decomposition products below monosaccharides. The retentate is collected. The molecular cut-off membrane used has a MWCO of 1000 Da to 3000 Da, preferably 2000 Da. The collected liquid is concentrated by a rotary evaporator and dried under vacuum to obtain an oligomeric alginic acid oligosaccharide mixture. After analysis, all of these products are found to be oligosaccharide compositions with a content of disaccharides to decasaccharides within a certain percentage range. Examples 1 to 3 illustrate the method.

[0038] (2) Comparison of activity of oligosaccharide compositions The pharmacological activity of the oligosaccharide composition of the present invention is compared with that of the oligomannaric acid hexasaccharide of the prior application. The results show that the pharmacological activity of the oligosaccharide composition of the present invention is significantly higher than that of the oligomannaric acid hexasaccharide of the prior application. Without being bound by theory, it is believed that the pharmacological activity of the oligosaccharide composition of the present invention is significantly higher than that of the oligomannaric acid hexasaccharide of the prior application. below It is believed that when the percentage of guluronic acid is greater than 60%, the composition is most active, but when the percentage of guluronic acid is greater than 60%, the activity of the composition also decreases.

[0039] The present invention also provides a medicament or health care product comprising an alginate oligosaccharide composition as described above and optionally a pharma- ceutically acceptable carrier or excipient.

[0040] Methods for preparing oligosaccharide compositions comprising various ratios of active ingredients are known or will be apparent to those skilled in the art based on the present disclosure, as described in Remington's Pharmaceutical Sciences, Martin, EW, ed., Mack Publishing Company, 19th Edition (1995). Methods for preparing pharmaceutical compositions include incorporating suitable pharmaceutical excipients, carriers, diluents, and the like.

[0041] The pharmaceutical preparations of the present invention are prepared by known methods, including conventional mixing, dissolving or lyophilizing processes.

[0042] The pharmaceutical compositions of the present invention may be administered to a patient by a variety of routes appropriate to the selected mode of administration, such as orally or parenterally (by intravenous, intramuscular, topical or subcutaneous routes).

[0043] Thus, the compositions of the present invention formulated with a pharma- ceutical acceptable carrier (e.g., an inert diluent or an edible carrier) can be administered systemically, e.g., orally. They can be enclosed in hard or soft gelatin capsules and compressed into tablets. For oral therapeutic administration, the active compounds of the present invention can be formulated with one or more excipients and used as swallowable tablets, lozenges, troches, capsules, elixirs, suspensions, syrups, round tablets, and the like. Such compositions and preparations should contain at least 0.1% of the active compound. The percentage of such compositions and formulations can, of course, vary and may comprise from about 1% to about 99% by weight of a given unit dosage form. In such therapeutically useful compositions, the amount of active compound is such that an effective dosage level is obtained.

[0044] Tablets, troches, pills, capsules, etc. may also contain binders such as gum tragacanth, gum arabic, corn starch, or gelatin; excipients such as dicalcium phosphate; and disintegrating agents such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or aspartame; or flavoring agents such as peppermint, wintergreen oil, or cherry flavor. When the unit dosage form is a capsule, in addition to the above types of materials, it may also contain a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may contain gelatin, wax, shellac, or sugar. etc.The syrup or elixir may comprise the active compound, sucrose or fructose as a sweetener, methylparaben or propylparaben as a preservative, a dye and a flavor (e.g., cherry flavor or orange flavor). Any material used to prepare unit dosage form must be pharma-ceutically acceptable and non-toxic in the amount used. In addition, the active compound can be incorporated into sustained release preparations and devices.

[0045] The active compound can also be administered intravenously or intraperitoneally by infusion or injection.The aqueous solution of the active compound or its salt can be prepared, if necessary, using a miscible non-toxic surfactant.Dispersions in glycerin, liquid polyethylene glycol, triacetin and their mixtures and oils can also be prepared.Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0046] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders (optionally encapsulated in liposomes) of the active ingredient, comprising the extemporaneous formulation suitable for sterile injectable or infusible solutions or dispersions. In all cases, the final dosage form must be sterile, liquid and stable under the conditions of manufacture and storage. Liquid carriers can be, for example, solvents or liquid dispersion media, including water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size, or by the use of surfactants. To prevent microorganisms, various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.) can be used. In many cases, it is preferable to include isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in compositions which delay absorption, for example, aluminum monostearate and gelatin.

[0047] Sterile injectable solutions are prepared by mixing the required amount of active compound in a suitable solvent with various other ingredients as listed above, as needed, and then filtering and sterilizing. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of the active ingredient and other required ingredients that are previously present in the solution that has been filtered and sterilized.

[0048] Useful solid carriers include ground solids (e.g., talc, clay, microcrystalline cellulose, silica, alumina, etc.). Useful liquid carriers include water, ethanol or ethylene glycol or water-ethanol / ethylene glycol mixtures, in which the combination of the present invention can be dissolved or dispersed at effective content, optionally with the aid of non-toxic surfactants. Adjuvants (e.g., fragrances) and additional antimicrobial agents can be added to optimize the properties for a given use.

[0049] Thickening agents (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, and the like, which are applied directly to the skin of the user.

[0050] The therapeutically required amount of the compounds or mixtures thereof will vary depending not only on the compounds themselves but also on the method of administration, the nature of the disease being treated, and the age and condition of the patient, and ultimately on the judgment of the physician or clinician at the time.

[0051] The above-mentioned preparations can be provided in unit dosage forms, which are physically discrete units comprising a unit dose suitable for administration to humans and other mammals. The unit dosage form can be one capsule or tablet, or a number of capsules or tablets. Depending on the specific treatment involved, the amount of the unit dose of the active ingredient can vary or be adjusted from about 0.1 to about 1000 mg or more.

[0052] Another aspect of the present invention provides a pharmaceutical composition or health care product comprising the alginate-oligosaccharide composition of the present invention and optionally a suitable carrier.

[0053] Another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating Alzheimer's disease.

[0054] Yet another aspect of the present invention provides a method for treating a patient having Alzheimer's disease comprising administering to a patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

[0055] Another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating Parkinson's disease.

[0056] Yet another aspect of the present invention provides a method for treating a patient having Parkinson's disease, comprising administering to a patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

[0057] Yet another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating inflammation.

[0058] Another aspect of the present invention provides a method for treating a patient having inflammation comprising administering to a patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

[0059] Another aspect of the invention provides the use of an alginate oligosaccharide composition for treating pain responses.

[0060] Another aspect of the present invention provides a method for treating a patient having pain, comprising administering to a patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

[0061] Another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating diabetes.

[0062] Another aspect of the present invention provides a method for treating a patient with diabetes comprising administering to a patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

[0063] Yet another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating vascular dementia.

[0064] Another aspect of the present invention provides a method for treating a patient with vascular dementia, comprising administering to a patient in need thereof an effective amount of an alginic acid oligosaccharide composition of the present invention.

[0065] Pain referred to herein includes a variety of pain including acute pain, chronic pain, neuropathic pain, post-operative pain, chronic back pain, cluster headache, herpes neuralgia, phantom limb pain, central pain, dental pain, opioid resistant pain, visceral pain, post-operative pain, bone injury pain, fatigue and pain during labor, pain caused by burns including sunburn, post-partum pain, migraine, angina, and genitourinary tract related pain (including cystitis), vascular pain, trigeminal neuralgia, intercostal neuralgia, surgical incision pain, chronic fasciitis pain, heel pain, muscle pain, bone pain, joint pain, cancer pain, non-cancer pain, and the like.

[0066] Inflammation as referred to in this specification includes various inflammations including acute inflammation, chronic inflammation, vascular inflammation, neuroinflammation, central nervous system inflammation (e.g., multiple sclerosis including encephalomyelitis), peripheral nerve inflammation, arthritis (e.g., osteoarthritis, sacroiliitis, psoriatic arthritis, rheumatoid arthritis, rheumatoid arthritis, etc.), ankylosing spondylitis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), inflammatory diabetic ulcers, systemic lupus erythematosus, inflammatory skin diseases (e.g., psoriasis, atopic dermatitis, eczema), and the like.

[0067] The alginic acid oligosaccharide composition of the present invention is prepared by a method different from the prior art, and does not need to separate the M segment and the G segment. It greatly reduces the complexity of the production process, greatly reduces the production cost, the preparation method involves simple reactions, the active ingredient has a high content, and there is no residual reaction reagent. From the test, it is proved that the alginic acid oligosaccharide composition of the present invention has the potential to prevent and treat Alzheimer's disease, diabetes, Parkinson's disease, various inflammatory reactions, pain and vascular dementia.

[0068] Animal models and procedures for evaluation of pharmacodynamic activity 1. Animal model for anti-AD pharmacodynamic evaluation: The AD model was induced using unilateral intracerebroventricular injection of Aβ, and the learning and memory behaviors of the AD model rats were evaluated using the Morris water maze.

[0069] Male Wistar rats (weight 180-220g each) were obtained and randomly divided into a sham-operated control group, a model group, and a drug-administered group, with 14 rats in each group. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40mg / kg) and fixed in a stereotaxic apparatus. The skin was prepared and disinfected as usual, and the bregma was exposed. The hippocampal CA1 region was located according to the “Rat Brain Stereotactic Atlas” (BAO Xinming, SHU Siyun, Beijing, People's Medical Press, 1991, 28) at a location 3.0mm posterior to the bregma, 2.2mm lateral to the midstitch, and 2.8mm below the dura. In each of the model and drug-administered groups, a needle was inserted vertically into the right hippocampal CA1 region of the skull using a microinjector. 5 μl of condensed Aβ (Aβ1-40 was prepared as a 1.4 mg / mL solution in PBS and incubated in a 37°C incubator for 5 days to form aggregates) was slowly injected at a flow rate of 1 μL / min. After the injection was completed, the needle was kept in place for 5 min to allow complete dispersion of Aβ, and then the needle was slowly removed. The surgical wound was sutured, and the rats were warmed and awakened. The control group was injected with the same volume of sterile PBS, and other steps were the same as above. The corresponding drugs were administered 7 days before surgery and continued until the end of the study.

[0070] The Morris water maze test was performed on the 11th day after surgery.

[0071] Spatial orientation test: Each group of rats was trained (i.e., spatial orientation test) once a day for 5 consecutive days. The time it took the animals to find the platform (i.e., escape latency) was recorded. If the platform was not found after about 90 seconds, the rats were guided to swim in a straight line to the platform and stand on it for 30 seconds to promote learning and memory.

[0072] Spatial exploration test: One day after completing the spatial orientation test, the platform was removed and the rats were placed in the water through the entry point, and the number of times the rats crossed the platform and the percentage of the total distance swum within the quadrant where the platform was located were recorded. The learning and memory functions of the animals were evaluated.

[0073] 2. Animal models for pharmacodynamic evaluation of anti-Parkinson's disease (PD) drugs Mice were randomly divided into blank control group, MPTP model group and drug-treated group, with 14 mice in each group. Animals were divided into groups and given drugs on the same day. Blank control group and MPTP model group were given saline by intragastric administration, while other groups were given the corresponding drugs once a day for 17 consecutive days. Drugs to establish the model were given from the 6th day. Animals in blank control group were given 10ml / kg saline subcutaneously, while other animals were given 25mg / kg MPTP subcutaneously once a day for 5 days.

[0074] Behavioral tests were performed on days 11, 14, and 17, respectively. Mice were gently placed head-up on the rough surface of the rod (8 mm diameter, 55 cm high). The time it took for the mouse to adjust its head from up to down was recorded as the latency (T-turn), and the time it took for all paws to reach the bottom of the rod after moving down was recorded as the descent time (T-LA). If it exceeded 30 seconds, it was recorded as 30 seconds. Each mouse was tested five times and the results were averaged.

[0075] MPTP has selective destructive effects on dopamine neurons in the substantia nigra. The MPTP-induced PD animal model is the most classical animal model that resembles the pathological changes and clinical characteristics of human Parkinson's disease. The main symptoms of PD are resting tremor, increased muscle tone, decreased movement, etc. The turning and descending times of the rod climbing test can represent the overall activity and coordination ability of the mouse.

[0076] 3. Animal models for pharmacodynamic evaluation of anti-inflammatory responses (1) Rheumatoid arthritis model - Collagen-induced arthritis mouse model Male DBA / 1 mice weighing 19-22g were obtained and randomly divided into blank control group, model group, and drug-treated group, with 8 mice in each group. Except for the blank control group, the remaining mice were sensitized by subcutaneous injection of 10mg / kg bovine collagen type II-complete Freund's adjuvant (CII-CFA) emulsion into the tail base on day 0, and on day 23, 1.5mg / kg lipopolysaccharide (LPS) was intraperitoneally injected. The administration started on day 28, and the blank control group and model group were orally administered saline, while the other groups were administered the corresponding drugs (once a day for 14 consecutive days). After LPS injection, the mice were observed daily for their pathology. When the mice started to develop the disease (appearance of clinical symptoms of arthritis), clinical scoring was performed according to the various degrees of the disease (redness, joint deformation) and based on a 0-4 point scale to indicate the degree of disease progression. 0 means no erythema and swelling; 1 means erythema or mild swelling near the tarsus or near the ankle or metatarsus, and redness and swelling of one toe; 2 means mild erythema and swelling of the ankle and metatarsus, or redness and swelling of two or more toes; 3 means moderate erythema and swelling of the ankle, carpal joints and metatarsals; 4 means severe redness and swelling of all of the ankles, carpal joints, metatarsals and toes; the highest score for each limb is 4 points, and the highest score for each animal is 16 points.

[0077] (2) Multiple sclerosis model: MOG-induced multiple sclerosis mouse model Female C57BL / 6 mice weighing 17-20 g were obtained, and five of them were randomly selected to serve as the blank control group. The remaining animals were sensitized by subcutaneous injection of myelin oligodendrocyte glycoprotein-complete Freund's adjuvant (MOG-CFA) emulsion on the back on day 0 (10 mg / kg MOG, 20 mg / kg CFA), and intraperitoneally injected with 10 μg / kg pertussis toxin on days 0 and 2. Administration began on day 1. The blank control group and model group were orally administered saline, while the other groups were administered the corresponding drugs (once a day for 24 consecutive days). Approximately 12 days after immune induction, the immunized mice developed symptoms. Detailed daily observation and recording of body weight and clinical scores were started to indicate the degree of disease progression. A score of 0 to 4 was used to indicate different degrees: 0 means normal appearance with no obvious signs of disease; 1 means drooping tail, weakness of one hind leg; 2 means drooping tail, weakness of both hind legs and a staggering gait; 3 points means weakness and paralysis of one hind leg; 4 means weakness and paralysis of both hind legs.

[0078] (3) Systemic lupus erythematosus model - MRL / lpr lupus erythematosus mouse model MRL / lpr transgenic mice, which have a homozygous mutation in the Faslpr gene, can spontaneously develop lymphoid tissue hyperplasia. The mice began to develop symptoms of systemic lupus erythematosus at around 10-14 weeks of age. Female MRL / lpr transgenic mice (9 weeks old) were randomly divided into blank control and drug-treated groups, with 8 mice in each group. The blank control group was orally administered saline, and the other groups were administered the corresponding drugs (once a day for 4 consecutive weeks). Lymph nodes were scored weekly. 0 to 6 points indicate different degrees: 0 means normal; 1 means one place on each side less than 1 cm in diameter; 2 means two places on each side less than 1 cm in diameter; 3 means three places on each side less than 1 cm in diameter; 4 means one place on each side more than 1 cm in diameter and two other places on each side less than 1 cm in diameter; 5 means two places on each side more than 1 cm in diameter and the other places on each side less than 1 cm in diameter; 6 means three places on each side more than 1 cm in diameter.

[0079] (4) Inflammatory bowel disease (IBD) model - dextran sulfate sodium (DSS)-induced colitis mouse model Female C57 mice (7-8 weeks old) weighing 18-20g were obtained and randomly divided into blank control group, model group, and drug-treated group, with 8 mice in each group. The mice in the model group and drug-treated group were administered 2.5% high molecular weight polymer dextran sulfate sodium (DSS) as drinking water on days 1-7, and administration began on day 1. The control group and model group were orally administered saline, and the other groups were administered the corresponding drug (once a day for 30 consecutive days). On day 31, the mice were killed by cervical dislocation, the abdominal cavity was opened, and the mesentery was isolated. The ileocecal origin to anal terminal portion of each mouse was removed. Samples were removed sequentially from each group. The length of the colon was measured.

[0080] 4. Animal models for evaluating antidiabetic pharmacodynamics Male NIH mice were randomly divided into normal control group, model group, and drug administration group, with 10 mice in each group. On the test day, all mice except the standard group were Movement of The animals were intraperitoneally injected with 150 mg / kg streptozotocin. The corresponding drugs were administered for 10 days. On the 11th day, the eyes were removed and blood was taken to measure blood glucose concentration.

[0081] 5. Animal Models for Anti-pain Pharmacodynamic Evaluation (1) Acetic acid-induced pain model in mice Kunming mice, half male and half female, weighing 18-22g, were randomly divided into blank control group, model group, and drug-treated group, with 10 mice in each group. From the day of grouping, the blank control group was intragastrically administered 20ml / kg distilled water once a day for 7 consecutive days, while the other groups were intragastrically administered the corresponding drug once a day for 7 consecutive days. One hour after the last administration, the mice in each group were intraperitoneally administered 0.2ml of 0.6% acetic acid solution, and the writhing latency (the time from acetic acid injection to the occurrence of a writhing response) and the number of writhings of the mice within 20 minutes after acetic acid injection were recorded.

[0082] Injection of chemicals such as acetic acid solution into the abdominal cavity of mice can irritate the peritoneum of the mice and produce intermittent persistent pain, which is manifested by abdominal concavity, anterior abdominal wall touching the bottom of the cage, flexion of the hips and extension of the hind legs, a peculiar posture called writhing reaction. The writhing latency (the time from acetic acid injection to occurrence of writhing reaction) and the number of writhings within a certain time can represent the severity of pain. The shorter the writhing latency and the more the number of writhings, the more severe the pain.

[0083] (2) Nitroglycerin-induced migraine in rats SD male rats, weighing 180-220g, were randomly divided into blank control group, model group, and drug-treated group, with 8 rats in each group. The administration started on the day of grouping. The blank control group and model group were intragastrically administered with distilled water once a day for 28 consecutive days, and the other groups were intragastrically administered with the corresponding drug once a day for 28 consecutive days. Thirty minutes after the last administration, the animals except the blank control group were given saline, and the other groups were subcutaneously injected with 10mg / kg nitroglycerin in the right shoulder to establish the model. After the model establishment, the appearance and duration of redness of the rats' ears, as well as the number of head scratches within 30-45 minutes after the model establishment, were observed. The 5-HT content in brain tissue was determined by fluorescence spectrophotometry and measured at Ex356nm / Em483nm wavelength. The results are shown in ng / g brain weight.

[0084] Migraine is a vascular and neural dysfunction caused by the interaction of vascular and neural mechanisms. Nitroglycerin can cause migraine by inducing a hypersensitive reaction of trigeminal nerve fibers, dilating meningeal blood vessels, forming neurogenic inflammation, and enhancing the function of hypothalamic, brainstem, and spinal cord neurons. The nitroglycerin model is an animal model established in 1995 and is now a classic animal migraine model. According to the pathogenic mechanism of nitroglycerin, the detection of the ear redness time induced by vasodilation, the number of head scratchings caused by pain, and the content of serotonin (5-HT) (a pain-sensing factor in brain tissue) was used to evaluate the severity of migraine. The longer the ear redness, the more the number of head scratchings, and the higher the 5-HT content, the more severe the migraine.

[0085] (3) A rat model of migraine induced by electrical stimulation of the trigeminal ganglion SD rats (5 months old, male, weighing 200-240 g) were randomly divided into a blank control group, a sham operation group, a model group, and a drug administration group, with 10 rats in each group.

[0086] The corresponding drugs were orally administered to each group, while the blank control group, sham-operated group, and model group were orally administered distilled water. After 10 days of continuous administration, all rats except the blank control group were anesthetized by intraperitoneal injection of 350 mg / kg chloral hydrate. The rats were fixed in a stereotaxic apparatus, and a midline incision was made on the parietal region. The skin and muscle were cut in layers to expose the radius at the center of the sagittal suture. A hole was made 3 mm posterior and 3 mm lateral from the bregma using a dental drill, and then an electrode was inserted into the trigeminal ganglion (9.5 mm deep from the dura). Anesthesia was continued after surgery. All surgeries were performed under aseptic conditions. The stimulation electrode was debugged. The electrical stimulation parameters were 200 ms period, 10v amplitude, and 5 ms wave width for 10 min of stimulation. In the sham-operated group, the electrode was inserted but no stimulation was given. 50 mg / kg Evans blue was injected into the right femoral vein 7 min before stimulation, followed by perfusion and fixation within 20 min after stimulation.

[0087] Five minutes after the end of stimulation, the left ventricle was perfused for 2 minutes. The craniotomy was performed, and the whole brain was removed and fixed for identifying c-fos by immunohistochemistry in pathological sections. The location of the electrode was also identified, and the dura at the electrode insertion site and the corresponding location in the other cerebral hemisphere were separated, then washed with deionized water, spread flat on a slide glass, dried at 37°C for 15 minutes, and fixed with 70% glycerol. The fluorescence intensity of the indicated areas on the stimulated and control sides was detected under a confocal microscope with an excitation wavelength of 647 nm and an emission wavelength of 680 nm. The ratio of the fluorescence intensity on the stimulated side / control side was calculated to indicate plasma protein extravasation (PPE). Serial frozen coronal sections of the whole brain with a section thickness of 10 μm were prepared, and c-fos positive cells were immunohistochemically fluorescently labeled. Five fields were randomly selected under a confocal microscope to determine the number of positive cells on the experimental and control sides of the caudal part of the spinal trigeminal nucleus, and the average of the five fields was taken as the average number of positive cells.

[0088] Activation of the trigeminovascular system is an important part of the pain generation in migraine patients, and meningeal neuroinflammation plays an important role in the generation and maintenance of migraine pain. When the trigeminal nerve innervating the dura is stimulated, this nerve releases vasoactive substances, which cause meningeal vasodilation, extravasation of plasma components, degranulation of mast cells, and activation of platelets, resulting in migraine. In addition, neurotransmitters released after pain stimulation bind to corresponding receptors on cell membranes. Under the action of this second messenger, the c-fos mRNA gene is expressed, translated, and synthesized into c-fos protein in the nucleus, which exerts long-term physiological effects on the body. Thus, during the development of migraine, the number of c-fos mRNA and c-fos protein expressing cells in the spinal tract nucleus of the trigeminal nerve and the raphe magnus increases. Thus, the severity of migraine can be expressed by measuring the amount of serum protein exuded from the dura of migraine animals and the number of c-fos positive cells in the caudal part of the spinal trigeminal nucleus (nucleus caudalis). The fewer the number of cells, the less severe the migraine.

[0089] 6. Animal models for pharmacodynamic evaluation of anti-vascular dementia drugs (1) Animal model for pharmacodynamic evaluation of anti-vascular dementia drugs: A mouse model of vascular dementia induced by bilateral common carotid artery occlusion (BCCAo) The bilateral common carotid artery occlusion (BCCAo) model is a vascular dementia model commonly used in the art, established by global cerebral ischemia and reperfusion.

[0090] 1.1 Animal Grouping and Dosing Male C57BL / 6 mice, weighing 22±2 g, were selected and randomly divided into a sham operation group, a 30-minute bilateral common carotid artery occlusion (BCCAo) model group (abbreviated as the 30-minute BCCAo group), and a drug administration group, with 10 animals in each group. After the animals were divided into groups, the mice in the sham operation group and the 30-minute BCCAo group were intragastrically administered with distilled water once a day for 5 consecutive days, and then underwent BCCAo surgery. The mice in the drug administration group were intragastrically administered with the corresponding drug once a day for 5 consecutive days, and then underwent BCCAo surgery. The BCCAo surgery consisted of anesthetizing the mice in each group with sodium pentobarbital, isolating and occluding the bilateral common carotid arteries of the mice in the model group and the drug administration group for 30 minutes, removing the occlusion, and suturing the neck wound. For the sham operation group, the bilateral common carotid arteries were isolated and not occluded, and the neck incision was sutured as it was. 24 hours after BCCAo, each group of mice continued to intragastrically administer corresponding drugs or distilled water according to preoperative administration schedule for another 23 consecutive days of administration.To evaluate the improving effect of mannuronic diacid composition on the learning and memory of mice, dark avoidance test was performed on the 7th day after BCCAo, and Morris water maze test was started on the 13th day.After this behavioral test, mice were sacrificed and their brain tissues were fixed.The neuronal damage of the hippocampus of mice after BCCAo and the protective effect of mannuronic diacid composition on damaged neurons were evaluated by methods such as HE staining.

[0091] 1.2 Dark avoidance test The dark avoidance test is used to test the learning and memory of mice in spatial discrimination. Memory impairment of spatial location can only appear when the hippocampus or the area around the hippocampus is damaged. The dark avoidance test box is an apparatus designed to exploit the mouse's tendency to track in the dark and avoid the light. Half of the box is a dark chamber, the other half is a light chamber, with a small hole in the middle to connect them. The floor of the dark chamber is covered with a copper grid. When the animal enters the dark chamber, it receives a shock and escapes back to the light chamber. The animal is trained for 24 hours and then tested again. The latency of the dark avoidance test refers to the time from when the animal is placed in the light chamber to when it first enters the dark chamber. The longer the latency of the dark avoidance test and the fewer the number of avoidance errors, the better the animal's memory.

[0092] 1.3 Morris water maze behavioral test The Morris water maze (MWM) test is a test in which experimental animals are made to swim and learn to find a platform hidden in the water. This test is mainly used to test the learning and memory of experimental animals regarding spatial location and orientation (spatial location recognition). The mouse Morris water maze mainly consists of a cylindrical pool with a diameter of 80 cm and a height of 70 cm and a movable platform with a diameter of 8 cm. A digital camera suspended above the pool is connected to a computer. Before the test, transparent water is poured into the pool in advance. The water depth is 15 cm, and the water surface is 0.5 cm above the surface of the platform. Milk is added to make the pool water opaque. The position of the platform remains unchanged during the test. Morris water maze behavior includes the following two indices:

[0093] The spatial orientation test is used to measure the ability of mice to learn and acquire memory in the water maze. This test was performed in the BCCA oThe training started on the 13th day after the experiment and continued for 4 days. The mice were trained once in both the morning and the afternoon, a total of 8 times. During the training, the mice entered the pool in the 1 / 2 arc of the west quadrant and entered the water with their heads facing the wall of the pool. If the platform was not found within 120 seconds, the experimenter guided the mice to the platform and left them for 30 seconds to induce learning and memory. The route map and the time it took the mice to find and climb the platform were observed and recorded, i.e., the escape latency and swimming speed of the Morris water maze test were recorded. The escape latency of the Morris water maze test refers to the time from when the mice entered the water to when they found the platform. The shorter the escape latency of the Morris water maze test, the better the memory of the animals.

[0094] The spatial exploration test is used to measure the ability of mice to retain the memory of the spatial location of the platform after learning to find it. After the spatial orientation test was completed, the platform was removed after a one-day delay. Mice were placed in the water from the same entry point and the number of times they crossed the original platform was measured. Data acquisition and processing were completed by an image automatic monitoring and processing system.

[0095] (2) A rat model of vascular dementia induced by middle cerebral artery occlusion (MCAO) The middle cerebral artery occlusion (MCAO) model is a vascular dementia model established by focal cerebral ischemia and commonly used in the art.

[0096] 2.1 Animal Grouping and Dosing Male Wistar rats were selected and randomly divided into blank control group, sham operation group, model group (MCAO group), and drug administration group, with 10 rats in each group. The animals in the blank group, sham operation group, and MCAO group were orally administered distilled water, and the animals in the alginate oligosaccharide group were orally administered distilled water. AnimalsAll rats were orally administered the corresponding dose of alginate oligosaccharides. After 7 consecutive days of treatment in each group, the rats in the other groups, except for the rats in the blank group, were anesthetized by intraperitoneal injection of 350 mg / kg chloral hydrate and fixed in the left lateral decubitus position on a rat board. Under an operating microscope, the skin was incised along the midpoint connecting the external auditory meatus and the canthus of the eye to expose the zygomatic arch. The distance between the phosphate bone and the mandible was widened with a small distractor. A 2 mm × 2 mm bone window was opened at the base of the skull. The dura was opened to expose the middle cerebral artery, and one side of the middle cerebral artery was coagulated by high-frequency electrocautery to produce focal ischemia (in the animals in the sham-operated group, the middle cerebral artery was only exposed, but not coagulated). The incision was sutured in layers. The room temperature during and after the operation was strictly controlled at 24-25°C. After surgery, each group continued to receive the drug or distilled water according to the preoperative administration schedule. On the 11th day after surgery, each group was subjected to the Morris water maze test.

[0097] In this test, each group of rats was trained once a day for 5 consecutive days (i.e., spatial orientation test). The time it took the animals to find the platform (i.e., escape latency in Morris water maze test) was recorded. If an individual failed to find the platform for about 120 seconds, they were guided to swim in a straight line toward the platform and stand on it for 30 seconds to promote learning and memory. After the spatial orientation test was completed, the platform was removed after a day. The rats were placed in the water from the entry point, and the time it took for the rat to reach the original platform and the number of times the rat crossed the original platform were recorded (i.e., spatial exploration test). The learning and memory functions of the animals were evaluated. The escape latency in Morris water maze test refers to the time it took for the rat to enter the water and find the platform. The shorter the escape latency in Morris water maze test, the better the animal's memory. EXAMPLES

[0098] The advantages of the present invention are further illustrated in the following non-limiting examples. However, the specific materials and amounts thereof used in the examples, as well as other experimental conditions, should not be construed as limiting the present invention. Unless otherwise specified, all parts, ratios, percentages, and the like in the present invention are calculated by weight.

[0099] Example 1: Step 1): Preparation of alginate oligosaccharide mixture 5 kg of sodium alginate was prepared into a solution of about 10% and the pH was adjusted to about 3.0 by adding dilute hydrochloric acid. The solution was heated to 80°C and stirred. After reacting for 10 hours, the heating was stopped. After cooling to room temperature, the pH was adjusted to 9.0 by adding NaOH, and further adjusted to 3.2 by adding dilute hydrochloric acid. The solution was centrifuged at 5000 rpm for 10 minutes. The supernatant was collected. ,B The mixture was concentrated using a Tarley evaporator and dried in vacuum to obtain 1500 g of an intermediate.

[0100] For the NMR spectrum of the intermediate, see Figure 1. The NMR measurement method is as follows: Sample preparation: 30 mg of test sample was weighed, dissolved in 0.5 ml of DO, and freeze-dried; 0.5 ml of deuterated heavy water was further added for dissolution; freeze-dried again; finally, the freeze-dried sample powder was dissolved in an appropriate amount of heavy water and transferred to an NMR tube to prepare a 100 mg / ml test solution; 0.01% (w / v) deuterated TSP (trimethylsilylpropionate) sodium salt was added as an internal standard.

[0101] Acquisition and processing of nuclear magnetic data: One-dimensional hydrogen spectra were collected at 60 °C by a 400M Fourier transform nuclear magnetic resonance instrument. The pulse sequence was 45° pulse, each acquisition was 4 seconds, the relaxation time was 1 second, the accumulation was 20 times, and the spectral width was -2 ppm to 10 ppm. After data collection, one-dimensional hydrogen spectra were obtained using Fourier transform, and the TSP methyl hydrogen signal was set to 0.00 ppm.

[0102] It can be seen from FIG. 1 that the intermediate contained a mannuronic acid segment (M-block, chemical shift 5.1 ppm) and a guluronic acid segment (G-block, chemical shift 5.5 ppm), as well as a chimeric segment of mannuronic acid and guluronic acid (MG-block, chemical shift 5.3 ppm). 500 g was weighed and dissolved in distilled water to prepare a solution with a volume of 5 L. The solution was adjusted to pH 6.5 with NaOH and heated in a water bath to control the reaction temperature at 75° C. The gas flow rate at the outlet of the oxygen cylinder and the output of the ozone generator were adjusted so that ozone was introduced into the reaction solution at a mass concentration flow rate of 8 g / h. After 4 hours of reaction, the introduction of ozone was stopped and a suitable amount of water was added to adjust the concentration of the solution to about 10%. The solution was filtered through an ultrafiltration membrane with a molecular weight cutoff of 2,000 Da, and the retentate was collected. The collected liquid was concentrated on a rotary evaporator and dried under vacuum to give 350 g of product A.

[0103] Step 2): Analysis of the proportion and structure of oligosaccharides with different degrees of polymerization in alginate oligosaccharin diacid product A Accurately weigh 100mg of the above dried alginic acid oligosaccharin diacid product A, dissolve it in water to a concentration of 10mg / mL, and pass through a 0.22μm filter membrane to obtain test sample solution.The percentage of oligosaccharides with different polymerization degrees in the composition is measured by Superdex peptide molecular exclusion chromatography (GE Co.) combined with multi-angle light scattering (MALS, Wyatt Co.).The experimental conditions are as follows: Chromatography column: Superdex Peptide 10 / 300Gl Mobile phase: 0.1mol / L NaCl Injection volume: 10μL Flow rate: 0.3mL / min Test results: Disaccharides to decasaccharides are represented by dp2 to dp10, respectively, with dp2 being 18%, dp3 being 24%, dp4 being 23%, dp5 being 14%, dp6 being 8%, dp7 being 7%, dp8 being 2%, dp9 being 2% and dp10 being 2%.

[0104] Step 3): LC-MS analysis of the structures of oligosaccharides with different degrees of polymerization in alginate oligosaccharin diacid product A Test conditions: Chromatography column: Superdex Peptide 10 / 300Gl Mobile phase: 20% methanol + 80% 80mmol / L NH4Ac Flow rate: 0.1mL / min Column temperature: 25℃±0.8℃ Mass spectrometry conditions: Agilent 6540 QTOF; ion source: ESI collision voltage 120 V; negative ion mode. The width (m / z) of the acquired signal was 100-1000.

[0105] Mass spectra of oligosaccharides having various degrees of polymerization are shown in Figures 1 to 3. Various signal peaks in the mass spectra were assigned to confirm the molecular structures of all oligosaccharides in product A, i.e., the structures shown in general formula (III). For signal assignments and structures corresponding to the signals, see Table 1 below.

[0106] [Table 1]

[0107] From the above mass spectrometry structural analysis, it was found that the mannuronic acid or guluronic acid at the reducing end of the glycan in product A was oxidized to a saccharin diacid structure (see general formula IV for the structure), which may be a mannaric diacid or guluronic diacid structure (m+m'=3) containing 6 carbon atoms with a content of about 10-30%, or a decarboxylation product of the mannaric diacid or guluronic diacid, i.e., saccharin diacid (m+m'=2) containing 5 carbon atoms (30-50%) and saccharin diacid (m+m'=1) containing 4 carbon atoms (30-40%).

[0108] Step 4) NMR analysis of the guluronic acid content of alginate oligosaccharin diacid product A Sample preparation: 50 mg of test sample was weighed, dissolved in 0.5 ml of DO, and freeze-dried; 0.5 ml of deuterated heavy water was further added for dissolution; freeze-dried again; finally, the freeze-dried sample powder was dissolved in an appropriate amount of heavy water, and all of them were transferred to an NMR tube to prepare a 100 mg / ml test solution; 0.01% (w / v) deuterated TSP (trimethylsilylpropionate) sodium salt was added as an internal standard.

[0109] Acquisition and processing of nuclear magnetic data: One-dimensional hydrogen spectra were collected at room temperature by a 400M Fourier transform nuclear magnetic resonance instrument. The pulse sequence was 45° pulse, each acquisition was 4 seconds, the relaxation time was 1 second, the accumulation was 20 times, and the spectral width was -2 ppm to 10 ppm. After data collection, one-dimensional hydrogen spectra were obtained using Fourier transform, and the TSP methyl hydrogen signal was set to 0.00 ppm. The proton nuclear magnetic resonance spectrum of product A is shown in Figure 5. In Figure 5, the multiplet at chemical shift 4.6 ppm is the hydrogen signal at the C-1 position of mannuronic acid (M), 5.0 ppm is the hydrogen signal at the C-1 position of guluronic acid (G), and 4.9 ppm is the C-1 hydrogen signal of the chimeric segment of mannuronic acid and guluronic acid (MG). The formula for calculating the guluronic acid content is as follows:

number

[0110] In the above formula, I4.6, I5.0 and I4.9 are the integrals of the hydrogen signals at the C-1 position of mannuronic acid (M), guluronic acid (G) and the chimeric segment of mannuronic acid and guluronic acid chimera (MG), respectively. The calculated guluronic acid content of A is 30%.

[0111] Example 2: Weigh out 100g of commercially available sodium alginate (purchased from the website of Sinopharm Reagent Co., CAS No. 9005-38-3, CP Planning, Shanghai Testing), add distilled water and mix uniformly. After swelling, the volume1 L solution. The solution was adjusted to pH 4.0 with NaOH, and the reaction was carried out at room temperature (25°C). The gas flow rate at the outlet of the oxygen cylinder and the output of the ozone generator were adjusted so that ozone was introduced into the reaction solution at a mass concentration flow rate of 1 g / h. After 10 hours of reaction, the introduction of ozone was stopped, and a suitable amount of water was added to adjust the concentration of the solution to about 15%. The solution was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1,000 Da, and the retentate was collected. The collected liquid was concentrated in a rotary evaporator and dried under vacuum to obtain 80 g of product B.

[0112] The proportions of oligosaccharide components with different polymerization degrees in B were determined by Superdex peptide molecular exclusion chromatography (GE Co.) combined with multi-angle light scattering (MALS, Wyatt Co.). The measurement method was the same as the relevant part of Example 1. Test results: disaccharides to decasaccharides are represented by dp2 to dp10, respectively, dp2 is 25%, dp3 is 24%, dp4 is 18%, dp5 is 13%, dp6 is 10%, dp7 is 5%, dp8 is 2%, dp9 is 2% and dp10 is 1%.

[0113] The guluronic acid content of product B was determined to be 50% at 60°C using a 400M Fourier transform nuclear magnetic resonance instrument. The measurement method was the same as that in the relevant part of Example 1. The proton nuclear magnetic resonance spectrum is shown in Figure 6. It can be seen from the figure that the integral areas of mannuronic acid (M, chemical shift value 4.6 ppm) and guluronic acid (G, chemical shift value 5.0 ppm) are relatively close, while the integral area of ​​the chimeric segment of mannuronic acid and guluronic acid (MG, chemical shift 4.9 ppm) is small. According to the formula for calculating the content of guluronic acid product (G), the content of G is 50%.

[0114] Example 3: 100 g of the intermediate of Example 1 was weighed out. After adding water for suspension, NaOH was added to adjust the pH value to basic to allow complete dissolution of the powder. The solution was finally prepared as 1 L of solution, and HCl was further added to adjust the pH value to 2.95. A portion of white precipitate appeared, which was removed by centrifugation. The supernatant was collected. Distilled water was added to the solution to a volume of 1.5 L for further dilution. The solution was adjusted to pH 9.0 with NaOH, and the reaction was carried out in a water bath at 45°C. The gas flow rate at the outlet of the oxygen cylinder and the output of the ozone generator were adjusted so that ozone was introduced into the reaction solution at a mass concentration flow rate of 3 g / h. After 2 hours of reaction, the introduction of ozone was stopped, and a suitable amount of water was added to adjust the concentration of the solution to about 5%. The solution was filtered through an ultrafiltration membrane with a molecular weight cutoff of 3,000 Da, and the retentate was collected. The collected liquid was concentrated in a rotary evaporator and dried under vacuum to obtain 60 g of product C.

[0115] The percentages of oligosaccharides with different polymerization degrees in C were determined by Superdex peptide molecular exclusion chromatography (GE Co.) combined with multi-angle light scattering (MALS, Wyatt Co.). The measurement method was the same as the relevant part of Example 1. Test results: disaccharides to decasaccharides are represented by dp2 to dp10, respectively, dp2 is 9%, dp3 is 21%, dp4 is 27%, dp5 is 18%, dp6 is 13%, dp7 is 6%, dp8 is 3%, dp9 is 2%, and dp10 is 1%.

[0116] The content of guluronic acid in product C was determined to be 10% at 60°C using a 400M Fourier transform nuclear magnetic resonance apparatus, and the determination method was the same as that in the relevant part of Example 1. The test result is shown in Figure 7. By integrating the corresponding signals respectively, the integral area of ​​mannuronic acid (M, chemical shift value 4.6 ppm) is 13 times that of guluronic acid (G, chemical shift value 5.0 ppm), and the integral area of ​​the chimeric segment of mannuronic acid and guluronic acid (MG, chemical shift 4.9 ppm) is close to that of guluronic acid. According to the formula for calculating the content of guluronic acid product (G) shown in Example 1, the content of G is 10%.

[0117] Example 4: Evaluation of pharmacological activity between alginate oligosaccharin diacid compositions and mannuronic diacid hexasaccharide Sample preparation: 1. Preparation of Mannuronic Diacid Hexasaccharide 20 g of mannuronic diacid hexasaccharide was prepared by referring to the method disclosed in Examples 1 and 2 of Prior Patent No. 200580009396.5.

[0118] The oligosaccharide percentages and guluronic acid content of Products A, B, and C prepared in Examples 1, 2, and 3 above of this application are shown in Table 2 below.

[0119] 2. Preparation of Product D A product with a high G content was prepared with reference to the preparation method of Example 2 above. The sodium alginate raw material was a sample with a high G content prepared by Qingdao Haizhilin Biotechnology Development Co., Ltd., and the preparation method was the same as the corresponding part of the example. Specifically, 500 g of sodium alginate powder with a high G content was mixed uniformly with distilled water and prepared as a solution with a volume of 5 L after swelling, and then adjusted to pH 4.0 with NaOH and reacted at room temperature of 25 ° C. The gas flow rate at the outlet of the oxygen cylinder and the output of the ozone generator were adjusted so that ozone was introduced into the reaction solution at a mass concentration flow rate of 1 g / h. After 12 hours of reaction, the introduction of ozone was stopped and a suitable amount of water was added to adjust the concentration of the solution to about 15%. The solution was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1,000 Da, and the retentate was collected. The collected liquid was concentrated by a rotary evaporator and dried under vacuum to obtain 350 g of product D.

[0120] The proportions of oligosaccharide components with different polymerization degrees in D were determined by Superdex peptide molecular exclusion chromatography (GE Co.) combined with multi-angle light scattering (MALS, Wyatt Co.). The measurement method was the same as the relevant part of Example 1. Test results: disaccharides to decasaccharides are represented by dp2 to dp10, respectively, dp2 is 18%, dp3 is 26%, dp4 is 20%, dp5 is 15%, dp6 is 8%, dp7 is 7%, dp8 is 3%, dp9 is 2%, and dp10 is 1%.

[0121] The content of guluronic acid in product D was determined to be 60% at 60°C using a 400M Fourier transform nuclear magnetic resonance apparatus, and the measurement method was the same as that in the relevant part of Example 1. The hydrogen nuclear magnetic resonance spectrum is shown in Figure 8. It can be seen from the figure that the integral area of ​​guluronic acid (G, chemical shift value 5.0 ppm) is larger than that of mannuronic acid (M, chemical shift value 4.6 ppm), and the integral area of ​​the chimeric segment of mannuronic acid and guluronic acid (MG, chemical shift 4.9 ppm) is smaller. According to the formula for calculating the content of guluronic acid product (G) in Example 1, the content of G is 60%.

[0122] [Table 2]

[0123] 10 g of each of the above four kinds of samples A, B, C, and D and the mannuronic diacid hexasaccharide sample were collected. According to the methods described in "Animal Model for Pharmacodynamic Evaluation of Anti-AD", "Animal Model for Pharmacodynamic Evaluation of Anti-PD", "Animal Model for Pharmacodynamic Evaluation of Anti-inflammatory Response", "Animal Model for Pharmacodynamic Evaluation of Anti-diabetic", "Animal Model for Pharmacodynamic Evaluation of Anti-pain", and "Animal Model for Pharmacodynamic Evaluation of Anti-vascular Dementia", the pharmacological activities of these alginate oligosaccharin diacid compositions were compared with that of mannuronic diacid hexasaccharide.

[0124] 1. Pharmacodynamic evaluation of anti-AD drugs In this study, the latency to find the platform was significantly longer in the model group compared with the sham-operated control group, indicating that the evaluation model was successfully established. Compared with the model group, the latency to find the platform in each drug-administered group was significantly shorter.

[0125] After the spatial orientation test was completed, the platform was removed one day later, and the spatial exploration test was started. The number of times the animals crossed the platform and the percentage of the swimming distance in the quadrant where the platform was located relative to the total distance were observed and recorded. The learning and memory functions of the animals were evaluated. The results showed that, as shown in FIG. 9, compared with the sham-operated control group, the number of platform crossings in the model group was significantly decreased, and the number of platform crossings in the drug-administered group was significantly increased. The percentage of the swimming distance in the quadrant where the platform was located relative to the total distance also showed a similar trend to the number of platform crossings. As shown in FIG. 10, compared with the sham-operated control group, the percentage of the swimming distance in the quadrant where the original platform was located relative to the total distance was significantly decreased in the model group, and the percentage of the swimming distance in the quadrant where the original platform was located relative to the total distance was significantly increased in the drug-administered group.

[0126] The test results showed that the pharmacodynamic activity of products A, B and C was stronger than that of mannuronic diacid hexasaccharide, indicating that the oligosaccharide compositions containing a certain amount of guluronic acid and having a ratio of disaccharide to hexasaccharide higher than 60% had a synergistic effect. However, the activity of oligosaccharide composition D with a higher guluronic acid content was reduced.

[0127] 2. Pharmacodynamic evaluation of anti-PD drugs In this study, the latency and fall time of the model group were significantly longer than that of the blank control group. Compared with the model group, the latency and fall time of each drug administration group were shortened to various degrees. Among them, the pharmacodynamic activity of products A, B, and C was better than that of mannuronic diacid hexasaccharide with a single degree of polymerization, which was previously expected to be the most active. However, the activity of product D was weaker than that of mannuronic diacid hexasaccharide. Without being bound by theory, it is speculated that the content of guluronic acid and the ratio of disaccharide to hexasaccharide in the composition have a significant effect on the activity of the product, but if the ratio of guluronic acid is too high, the activity of the composition will decrease. See Figures 11 and 12.

[0128] 3. Pharmacodynamic evaluation of anti-inflammatory responses (1) Collagen-induced arthritis mouse model In this study, compared with the normal control group, the model group showed obvious arthritis symptoms as well as moderate erythema and swelling of the ankle joints, wrist joints and metatarsals. The clinical score reached 6 points, indicating that the arthritis model had been successfully established. Compared with the model group, the morbidity of each drug administration group was reduced to various degrees. Products A, B, and C significantly delayed the onset time of the mice compared with mannuronic diacid hexasaccharide with a single degree of polymerization, and the clinical score was also lower compared with mannuronic diacid hexasaccharide, indicating that the pharmacodynamic activity of Products A, B, and C was better than that of mannuronic diacid hexasaccharide. However, the onset of Product D was earlier and the clinical score was higher, indicating that the activity of Product D was weaker than that of mannuronic diacid hexasaccharide. This demonstrates that the content of guluronic acid and the ratio of disaccharide to hexasaccharide in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition will decrease.

[0129] (2) MOG-induced multiple sclerosis mouse model test In comparison with the normal control group, most of the mice in the model group showed weakness and paralysis in both hind limbs. The average clinical score of the model group reached 3 points, indicating that the multiple sclerosis model had been successfully established. Compared with the model group, the inflammatory progression of each drug administration group was alleviated to various degrees. The clinical scores of products A, B, and C during and at the end of the whole experiment were lower than mannuronic diacid hexasaccharide, indicating that the pharmacodynamic activity of products A, B, and C was better than that of mannuronic diacid hexasaccharide, but the clinical score of product D during and at the end of the whole experiment was somewhat higher, indicating that the anti-inflammatory activity of product D was the weakest. This demonstrates that the content of guluronic acid and the ratio of disaccharide to hexasaccharide in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition will decrease.

[0130] (3) MRL / lpr lupus erythematosus mouse model Starting from the 10th week, the transgenic mice began to develop disease, lymph node assertion was observed, and the lymph node score continued to increase over time, indicating that the disease development in the model group was successful and the disease progressed rapidly. Compared with the model group, the disease progression in each drug administration group was alleviated to various degrees. Products A, B, and C significantly delayed the onset time of the mice compared with mannuronic diacid hexasaccharide, and the lymph node score was also lower than that of mannuronic diacid hexasaccharide, indicating that the pharmacodynamic activity of Products A, B, and C was better than that of mannuronic diacid hexasaccharide. However, the onset time of Product D was earlier and its lymph node score was also higher, representing that the activity of Product D was weaker than that of mannuronic diacid hexasaccharide. This demonstrates that the content of guluronic acid and the ratio of disaccharide to hexasaccharide in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition will decrease.

[0131] (4) Dextran Sodium Sulfate (DSS)-Induced Colitis Mouse Model After the completion of the test, compared with the normal control group, the colon of the model group was significantly shorter due to inflammation, and most of the mice lost weight. Nearly half of the animals in the model group died, indicating that the intestinal inflammation was extremely severe. Compared with the model group, the intestinal inflammation of each drug administration group was alleviated to various degrees, which was reflected in the recovery of colon length and the improvement of survival rate. From Figures 13a and 13b, it can be seen that Products A, B, and C improved the colon length and animal survival rate of mice better than mannuronic diacid hexasaccharide, indicating that the pharmacological activities of Products A, B, and C were all better than that of mannuronic diacid hexasaccharide. However, Product D showed a shorter colon length and a slightly lower survival rate compared with mannuronic diacid hexasaccharide, indicating that the activity of Product D was weaker than that of mannuronic diacid hexasaccharide. Similarly, these results are consistent with previous studies and show that the content of guluronic acid and the ratio of disaccharides to hexasaccharides in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition decreases.

[0132] 4. Pharmacodynamic evaluation of antidiabetic effects In this study, the model group was compared with the normal control group, and the postprandial blood glucose of the model group was significantly higher, indicating that the evaluation model was successfully established. Compared with the model group, the postprandial blood glucose of each drug administration group was significantly lower. Among them, the pharmacodynamic activity of products A, B, and C was all The pharmacodynamic activity of product D was better than that of mannuronic diacid hexasaccharide, but the activity of product D was weaker than that of mannuronic diacid hexasaccharide. These test results are consistent with previous tests, and show that the content of guluronic acid and the ratio of disaccharides to hexasaccharides in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition decreases. See Figure 14.

[0133] 5. Anti-pain pharmacodynamic evaluation (1) Acetic acid-induced pain mouse model In the experiment, compared with the blank control group, the writhing latency of the model group was significantly shorter and the number of writhings was significantly higher, indicating that the evaluation model was successfully established. Compared with the model group, the writhing latency of each drug administration group was significantly extended and the number of writhings was significantly reduced. Among them, products A, B, and C can extend the mouse writhing latency more than mannuronic diacid hexasaccharide, and can reduce the number of writhings more than mannuronic diacid hexasaccharide, and products A, B, and C can extend the mouse writhing latency more than mannuronic diacid hexasaccharide, and can reduce the number of writhings more than mannuronic diacid hexasaccharide, indicating that .... of Pharmacodynamic activity all The results show that the activity of Product D was better than that of mannuronic diacid hexasaccharide. However, Product D showed a shorter writhing latency and a slightly higher number of writhings compared to mannuronic diacid hexasaccharide, reflecting the fact that the activity of Product D was weaker than that of mannuronic diacid hexasaccharide. Similarly, these test results are consistent with previous tests, showing that the content of guluronic acid and the ratio of disaccharides to hexasaccharides in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition decreases. See Figures 15 and 16.

[0134] (2) Nitroglycerin-induced migraine in rats The rats showed ear redness about 3 minutes after subcutaneous injection of nitroglycerin, which lasted for about 2.5 hours. The number of head scratching within 30-45 minutes after model establishment in the model group was significantly higher than that in the blank control group. Compared with the model group, the drug-administered group showed a significant delay in the appearance of ear redness, a shortened duration of ear redness, and a reduced number of head scratching within 30-45 minutes. Among them, products A, B, and C could reduce the number of head scratching in rats more than mannuronic diacid hexasaccharide, and products A, B, and C. The pharmacological activities of all the products were better than those of mannuronic acid hexasaccharide. However, product D had slightly more pharmacological activity than mannuronic acid hexasaccharide. RatThe head scratching times shown in Figure 17 indicate that the activity of product D was weaker than that of mannuronic diacid hexasaccharide. Similarly, these test results are consistent with previous tests, and indicate that the content of guluronic acid and the ratio of disaccharides to hexasaccharides in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition decreases. See Figure 17.

[0135] (3) Migraine model induced by electrical stimulation of the trigeminal ganglion Electrical stimulation of the rat trigeminal ganglion obviously induced the exudation of dural serum proteins. Compared with the blank control group and the sham-operated group, the PPE rate significantly increased and the number of c-fos expression positive cells significantly increased in the model group. Compared with the model group, the PPE rate significantly decreased and the number of c-fos expression positive cells significantly decreased in the drug-administered group. Among them, products A, B, and C Group of The number of c-fos-positive cells in the mannuronic acid hexasaccharide was smaller than that in the A, B, and C, and C. All of the pharmacological activities were better than those of mannuronic diacid hexasaccharide. However, product D Group of showed a slightly higher number of c-fos expression positive cells compared to mannuronic diacid hexasaccharide, indicating that the activity of product D was weaker than that of mannuronic diacid hexasaccharide. Similarly, these test results are consistent with previous tests, indicating that the content of guluronic acid and the ratio of disaccharides to hexasaccharides in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition decreases. See FIG. 18.

[0136] 6. Pharmacodynamic evaluation of anti-vascular dementia (1) A mouse model of vascular dementia induced by bilateral common carotid artery occlusion (BCCAo) 1.1 Dark avoidance test results In this study, the model group was compared with the sham-operated control group. In the model group, the latency in the dark avoidance test was significantly shorter and the number of errors significantly increased, indicating that the memory of the mice in the model group was significantly reduced, and the evaluation model was successfully established. Compared with the model group, the latency in the dark avoidance test of each drug administration group was significantly increased and the number of errors significantly decreased. Among them, the latency of the mice in the product A, B, and C groups was longer than that of mannuronic diacid hexasaccharide, The number of errors in this group is also less than that of mannuronic acid hexasaccharide. Products A, B, and C. All of the pharmacological activities were better than those of mannuronic diacid hexasaccharide. However, product D Group of showed slightly shorter latencies in mice compared with mannuronic diacid hexasaccharide, There are a few more errors, It shows that the activity of product D was weaker than that of mannuronic diacid hexasaccharide. Similarly, these test results are consistent with previous tests, and show that the content of guluronic acid and the ratio of disaccharides to hexasaccharides in the composition have a significant effect on the activity of the product. However, if the content of guluronic acid is too high, the activity of the composition decreases. See Figures 19 and 20.

[0137] 1.2 Results of the Morris water maze test In this study, the escape latency of the model group mice in the Morris water maze test was significantly longer than that of the sham-operated group, indicating that the establishment of a BCCAo-induced vascular dementia mouse model was successful. Compared with the model group, the escape latency of each drug administration group was significantly shorter. Among them, the escape latency of the mice in the product A, B, and C groups was shorter than that of mannuronic acid hexasaccharide, and the escape latency of the mice in the product A, B, and C groups was shorter than that of mannuronic acid hexasaccharide, and the escape latency of the mice in the product A, B, and C groups was shorter than that of mannuronic acid hexasaccharide. and C. All of the pharmacological activities were better than those of mannuronic diacid hexasaccharide. However, product D Group of showed a slightly longer escape latency compared to mannuronic diacid hexasaccharide, indicating that the activity of product D was weaker than that of mannuronic diacid hexasaccharide. See FIG. 21.

[0138] Four days after the water maze spatial orientation test, the platform was removed. A spatial exploration test was performed to observe the number of times the animals crossed the platform. Compared with the sham-operated group, the number of times the mice crossed the original platform in the model group was significantly decreased, indicating that the memory of the BCCAo mice was significantly decreased, while the number of times the mice crossed the original platform in each drug administration group was increased. Among them, the number of times the mice crossed the platform in the product A, B, and C groups was higher than that of mannuronic diacid hexasaccharide, and the number of times the mice crossed the original platform in the product A, B, and C groups was higher than that of mannuronic diacid hexasaccharide, and the number of times the mice crossed the platform in the product A, B, and C groups was higher than that of mannuronic diacid hexasaccharide. and C. All of the pharmacological activities were better than those of mannuronic diacid hexasaccharide. However, product D Group of In the case of the mannuronic diacid hexasaccharide, the number of times the mice crossed the platform was slightly less than that of the mannuronic diacid hexasaccharide, indicating that the activity of the product D was weaker than that of the mannuronic diacid hexasaccharide. See FIG. 22.

[0139] (2) Effects in rats with vascular dementia induced by middle cerebral artery occlusion (MCAO) In this study, the escape latency of the rats in the model group in the Morris water maze test was significantly longer than that of the sham-operated group, indicating that the establishment of the MCAO-induced mouse vascular dementia model was successful. Compared with the model group, the escape latency of each drug administration group was significantly shorter. Among them, the escape latency of products A, B, and C was significantly longer than that of the control group. Group The escape latency of rats was shorter than that of mannuronic acid hexasaccharide, and products A, B, and C. All of the pharmacological activities were better than those of mannuronic diacid hexasaccharide. Group of showed a slightly longer escape latency compared to mannuronic diacid hexasaccharide, indicating that the activity of product D was weaker than that of mannuronic diacid hexasaccharide.

[0140] One day after the spatial orientation test was completed, a spatial exploration test was conducted to observe and determine the number of times the animals crossed the platform within 2 minutes. Compared with the sham-operated group, the number of times the rats crossed the original platform in the model group was significantly decreased, indicating that the memory ability of the rats in the MCAO group was significantly decreased, while the number of times the rats in each drug-administered group crossed the original platform increased. Among them, products A, B, and C Group of The number of times that rats crossed the platform in the mannuronic acid hexasaccharide was greater than that in the mannuronic acid hexasaccharide. and C. All of the pharmacological activities were better than those of mannuronic diacid hexasaccharide. However, product D Group of In the control group, the number of times the rats crossed the platform was slightly less than that of the control group containing mannuronic diacid hexasaccharide, indicating that the activity of product D was weaker than that of mannuronic diacid hexasaccharide.

Claims

1. Formula (IV): 【Chemistry 1】 wherein n is an integer selected from 1 to 9, m is selected from 0, 1 or 2, and m' is selected from 0 or 1. or a pharmaceutically acceptable salt thereof, The total weight of the alginic acid oligosaccharin diacids, n=1 to 5, is 60% or more based on the total weight of the composition; the total weight of guluronic acid is 50% or less based on the total weight of the composition; An alginate-oligosaccharin diacid composition, wherein the total weight of alginate-oligosaccharin diacids where m+m'=1 and 2 is 50% or more based on the total weight of the composition.

2. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the total weight of alginate oligosaccharin diacids with m+m'=1 and 2 is from 60% to 90% by weight relative to the total weight of the composition.

3. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the total weight of alginate oligosaccharin diacids with m+m'=1 and 2 is from 70% to 90% by weight relative to the total weight of the composition.

4. 4. The alginate oligosaccharin diacid composition according to any one of claims 1 to 3, wherein the total weight of alginate oligosaccharin diacids, where m+m'=1, is 10% or more relative to the total weight of the composition.

5. 4. The alginate oligosaccharin diacid composition according to any one of claims 1 to 3, wherein the total weight of alginate oligosaccharin diacids, where m+m'=1, is between 30% and 40% relative to the total weight of the composition.

6. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the total weight of alginate oligosaccharin diacids, m+m'=2, is 10% or more by weight relative to the total weight of the composition.

7. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the total weight of the alginate oligosaccharin diacid, m+m'=2, is between 30% and 50% by weight relative to the total weight of the composition.

8. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the total weight of the alginate oligosaccharin diacids, n=1-5, is 80%-95% by weight based on the total weight of the composition.

9. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the total weight of alginate oligosaccharin diacids with n=1-3 is 20%-70% by weight relative to the total weight of the composition.

10. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the ratio of the total weight of alginate oligosaccharin diacids, n=1-3, to the total weight of alginate oligosaccharin diacids, n=4-7, is between 1.0 and 3.

5.

11. 11. The alginate oligosaccharin diacid composition of claim 10, wherein the ratio of the total weight of the alginate oligosaccharin diacids, n=1-3, to the total weight of the alginate oligosaccharin diacids, n=4-7, is between 1.0 and 3.

0.

12. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the total weight of guluronic acid is from 0.1% to 50% by weight relative to the total weight of the composition.

13. 2. The alginate oligosaccharin diacid composition of claim 1, wherein the total weight of guluronic acid is from 1 to 30% relative to the total weight of the composition.

14. 14. The alginate oligosaccharin diacid composition according to any one of claims 1 to 13, wherein the weight percentage content of the alginate oligosaccharin diacids of each degree of polymerization in said composition is: disaccharides 5-25%, trisaccharides 15-30%, tetrasaccharides 15-28%, pentasaccharides 10-25%, hexasaccharides 5-15%, heptasaccharides 3-10%, octasaccharides 2-5%, nonasaccharides 1-5%, decasaccharides 1-5%.

15. 15. The alginate oligosaccharin diacid composition of claim 14, wherein the weight percentage content of alginate oligosaccharin diacids of each degree of polymerization in said composition is: disaccharides 10-20%, trisaccharides 18-30%, tetrasaccharides 15-28%, pentasaccharides 15-20%, hexasaccharides 5-10%, heptasaccharides 3-5%, octasaccharides 2-3%, nonasaccharides 1-3%, decasaccharides 1-3%.

16. 16. The alginate oligosaccharin diacid composition according to any one of claims 1 to 15, wherein the pharma- ceutically acceptable salt is a sodium salt or a potassium salt.

17. A pharmaceutical composition or health care product comprising an effective amount of the alginic acid oligosaccharinic diacid composition according to any one of claims 1 to 16 and optionally a suitable carrier.

18. 18. A pharmaceutical composition or health care product according to claim 17 for the treatment of Alzheimer's disease, Parkinson's disease, inflammation, pain, diabetes or vascular dementia.

Images