WO2020001632A1 - 褐藻胶寡糖二酸的组合物 - Google Patents

褐藻胶寡糖二酸的组合物 Download PDF

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WO2020001632A1
WO2020001632A1 PCT/CN2019/093778 CN2019093778W WO2020001632A1 WO 2020001632 A1 WO2020001632 A1 WO 2020001632A1 CN 2019093778 W CN2019093778 W CN 2019093778W WO 2020001632 A1 WO2020001632 A1 WO 2020001632A1
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alginate oligosaccharide
acid
diacid
total weight
composition according
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PCT/CN2019/093778
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English (en)
French (fr)
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耿美玉
张真庆
晋迎申
肖中平
丁健
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上海绿谷制药有限公司
中国科学院上海药物研究所
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Priority to KR1020217001852A priority Critical patent/KR20210041556A/ko
Priority to JP2020572823A priority patent/JP2021529197A/ja
Priority to CA3104959A priority patent/CA3104959A1/en
Priority to EP19825647.1A priority patent/EP3815692A4/en
Priority to EA202190080A priority patent/EA202190080A1/ru
Priority to BR112020026849-6A priority patent/BR112020026849A2/pt
Priority to US17/256,853 priority patent/US11464794B2/en
Priority to AU2019296843A priority patent/AU2019296843A1/en
Publication of WO2020001632A1 publication Critical patent/WO2020001632A1/zh
Priority to PH12020552287A priority patent/PH12020552287A1/en
Priority to ZA2021/00350A priority patent/ZA202100350B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7012—Compounds having a free or esterified carboxyl group attached, directly or through a carbon chain, to a carbon atom of the saccharide radical, e.g. glucuronic acid, neuraminic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/328—Foods, ingredients or supplements having a functional effect on health having effect on glycaemic control and diabetes

Definitions

  • the invention relates to an optimal composition of alginate oligosaccharide diacid obtained by a biological activity screening method.
  • the method uses an animal model of senile dementia to evaluate the effect of different degrees of polymerization of alginate oligosaccharide and its ratio on biological activity.
  • the composition with the best biological activity is screened and the desired target substance is prepared by means of ultrafiltration membrane separation.
  • Fucoidan oligosaccharides have received widespread attention due to their potential medicinal value. Fucoidan oligosaccharides are usually prepared by alginic acid through several steps.
  • alginate oligosaccharide molecules there are M segments formed by the connection of mannuronic acid (D-mannuronic acid) through ⁇ -1,4-glycosidic bonds, and the guluronic acid (L-guluronic acid) through ⁇ -1 , G segment formed by 4-glycosidic linkage, and MG segment formed by hybridization of these two sugars.
  • the structural formulas of mannuronic acid and guluronic acid are as shown in formulas (I) and (II):
  • M and G sections can be separated from the raw alginic acid.
  • the general method can be simply described as: after the initial degradation of alginic acid, a mixed polysaccharide of polymannuronic acid and polyguluronic acid is obtained. After the mixed polysaccharide is precipitated by the acid method, a certain amount of polygulose can be removed. Alkyd. For example, see the methods disclosed in Chinese Patent Application No. 99866637.8 and CN02823707.2.
  • the method for preparing oligomannuronic acid is as follows: the M-stage intermediate obtained above is heated under acidic conditions and further acidolyzed to obtain small fragments of mannuronic acid polymers in a desired molecular weight range.
  • the reducing terminal can be oxidized to a ring-opened sugar diacid.
  • Patent Documents 1 and 2 are hereinafter collectively referred to as prior patents, which are all incorporated herein by reference.
  • reaction process of the mannuronic acid disclosed in the prior patent can be expressed by the following reaction equation (V), that is, the mannuronic acid C1-position aldehyde group at the reducing end of the oligomannuronic polysaccharide is oxidized to a carboxyl group.
  • a common oxidant is a basic copper sulfate solution, that is, a film reagent.
  • This oxidation method was adopted in a prior patent. Specifically, under alkaline conditions, the reaction substrate is polymannuronic acid, namely The above M-stage intermediate is added to the copper sulfate solution and reacted in a boiling water bath for 15 minutes to 2 hours. This method uses Cu 2+ ions as oxidant to oxidize aldehyde groups. Brick red cuprous oxide precipitates during the reaction. This reaction is often used to identify reducing sugars.
  • mannan-oligosaccharic acid has anti-Alzheimer's disease (AD) and anti-diabetic effects, and the mannan-oligosaccharic acid having a polymerization degree of 6 has the best activity.
  • the pathogenesis of Alzheimer's disease and type 2 diabetes is closely related to amyloid ( ⁇ -amyloid and amylin). After the amyloid aggregates, protein oligomers are produced, which further aggregate to form fibers. These protein aggregates are cytotoxic, induce oxidative damage to mitochondria in the cells, and trigger a cascade of inflammatory responses, causing a large number of neurons and ⁇ -cell damage, eventually leading to Alzheimer's disease and type 2 diabetes. Mannan oligosaccharic acid targets amyloid and antagonizes its cascade response, thereby preventing and treating Alzheimer's disease and type 2 diabetes.
  • the guluronic acid in the alginic acid needs to be removed, and the content of the guluronic acid in the alginic acid is usually Above 30%, up to about 70%, so in order to obtain high-purity mannan oligosaccharide diacid, the actual production cost is very high.
  • a first aspect of the invention relates to a composition of alginate oligosaccharic acid comprising a mannuronic acid and / or a guluronic acid or a pharmaceutically acceptable salt thereof having the formula (IV):
  • n is an integer selected from 1-9
  • m is selected from 0, 1 or 2
  • m ' is selected from 0 or 1
  • Another aspect of the present invention relates to a pharmaceutical composition or a health supplement, which comprises a composition of alginate oligosaccharide diacid as described above.
  • Another aspect of the present invention also relates to the use of a composition of alginate oligosaccharic acid in the treatment of a disease selected from the group consisting of senile dementia, Parkinson's disease, inflammation, pain, diabetes, or vascular dementia.
  • the alginate oligosaccharide diacid composition of the present invention is a mixture of mannuronic acid and guluronic acid with different degrees of polymerization, and its main component is that mannuronic acid passes through a ⁇ -1,4-glycosidic bond
  • the most active sugars are 5-8 sugars, especially 6 sugars.
  • a mixture of mannuronic acid and guluronic acid oligosaccharic acid having a degree of polymerization of 2 to 10 also has pharmacological activity against Alzheimer's disease and anti-diabetes, provided that The content of guluronic acid is controlled within a certain range. That is, the alginate oligosaccharide diacid composition of the present invention can be prepared at a greatly reduced production cost, which is easier to achieve in actual production, and easier to realize industrialized large-scale production.
  • Figure 2 is a mass spectrum of a disaccharide, a trisaccharide, and a tetrasaccharide in Product A.
  • FIG. 3 is a mass spectrum of pentasaccharide, hexasaccharide and heptose in product A.
  • Figure 4 is a mass spectrum of octaose, nonasaccharide, and decasaccharide in product A.
  • Figure 5 shows the NMR spectrum of Product A.
  • Figure 6 shows the NMR spectrum of Product B.
  • Figure 7 shows the NMR spectrum of product C.
  • Figure 8 shows the NMR spectrum of product D.
  • Figure 9 shows the effect of different oligosaccharide compositions and mannuronic acid hexasaccharides on the number of times AD animals cross the platform; the numbers corresponding to the numbers on the horizontal axis in the figure are: i: control group; ii: model group; iii: product A; iv: product B; v: product C; vi: product D; vii: mannuronic acid hexaose.
  • FIG. 10 shows the effects of different oligosaccharide compositions and mannuronic acid hexasaccharides on the swimming distance of AD animals; the abscissa reference numerals are the same as those in FIG. 9.
  • FIG. 11 shows the effect of different oligosaccharide compositions and mannuronic acid hexaose on the climbing time of PD animals on the 11th day; the abscissa reference numerals are the same as those in FIG. 9.
  • FIG. 12 shows the effects of different oligosaccharide compositions and mannuronic acid hexaose on the incubation period of the 11th day of PD animals; wherein the abscissa reference numerals are the same as those in FIG. 9.
  • Figures 13a and 13b show the therapeutic effect of different oligosaccharide compositions and mannuronic acid hexaose on inflammatory bowel disease in mice; the abscissa in the figure is the same as that in Figure 9.
  • FIG. 14 shows the effect of different oligosaccharide compositions and mannuronic acid hexaose on postprandial blood glucose in diabetic mice; the abscissa in the figure is the same as that in FIG. 9.
  • Figure 15 shows the effect of oligosaccharide composition and mannuronic acid hexaose on the latency of mouse writhing response induced by acetic acid; the numbers on the abscissa in the figure correspond to the samples: i: model group; ii: product A ; Iii: product B; iv: product C; v: product D; vi: mannuronic acid hexaose.
  • FIG. 16 shows the effects of different oligosaccharide compositions and mannuronic acid hexaose on the number of writhing reactions in mice induced by acetic acid; wherein the abscissa reference numerals are the same as those in FIG. 15.
  • FIG. 17 shows the effect of different oligosaccharide compositions and mannuronic acid hexaose on the number of scratches in migraine rats induced by nitroglycerin; the abscissa reference numerals are the same as those in FIG. 9.
  • FIG. 18 shows the effect of different oligosaccharide compositions and mannuronic acid hexaose on the number of c-fos positive cells in the caudal side of the nucleus of the trigeminal spinal tract induced by trigeminal ganglion induced by electrical stimulation;
  • Figure 9 shows the effect of different oligosaccharide compositions and mannuronic acid hexaose on the number of c-fos positive cells in the caudal side of the nucleus of the trigeminal spinal tract induced by trigeminal ganglion induced by electrical stimulation
  • FIG. 19 shows the effects of different oligosaccharide compositions and mannuronic acid hexaose on the latency of the dark avoidance experiment in vascular dementia mice induced by bilateral common carotid artery ligation; the abscissas are the same as those in FIG. 9.
  • FIG. 20 shows the effect of different oligosaccharide compositions and mannuronic acid hexaose on the number of errors in dark avoidance experiments in vascular dementia mice induced by bilateral common carotid artery ligation; the abscissas are the same as those in FIG. 9.
  • FIG. 21 shows the effect of different oligosaccharide compositions and mannuronic acid hexaose on the avoidance of the latency period of the water maze experiment of vascular dementia mice induced by bilateral common carotid artery ligation; the abscissas are the same as those in FIG.
  • Fig. 22 shows the effect of different oligosaccharide compositions and mannuronic acid hexaose on the number of times of crossing the platform of mice with vascular dementia induced by bilateral common carotid artery ligation; wherein the abscissa reference numerals are the same as Fig. 9.
  • a first aspect of the invention relates to a composition of alginate oligosaccharic acid comprising a mannuronic acid and / or a guluronic acid or a pharmaceutically acceptable salt thereof having the formula (IV):
  • n is an integer selected from 1-9
  • m is selected from 0, 1 or 2
  • m ' is selected from 0 or 1
  • the total weight of guluronic acid is less than 50% of the weight of the composition.
  • the alginate oligosaccharide diacid composition of the present invention is a mixture of mannuronic acid and guluronic acid with different degrees of polymerization, and its main component is formed by the connection of mannuronic acid through ⁇ -1,4-glycosidic bonds
  • mannanedioic acid has pharmacological activity against Alzheimer's disease and anti-diabetes, among which the most active sugar is 5-8 sugars, especially 6 sugars.
  • a mixture of mannuronic acid and guluronic acid oligosaccharic acid having a degree of polymerization of 2 to 10 is also resistant to Alzheimer's disease and diabetes Pharmacological activity, but the content of guluronic acid needs to be controlled within a certain range.
  • the content of guluronic acid in the product after the initial degradation of the alginic acid is usually above 30% and up to about 70%. If you apply according to the previous application in order to obtain highly active manna Glyuronic acid should be separated and removed as far as possible. However, based on the above findings of the inventors, it may not be necessary to separate and remove the guluronic acid from the degradation products. Further, the inventors have found that by controlling the conditions of the acid precipitation reaction, and controlling the ratio of guluronic acid to a certain range, the activity of the obtained composition can reach or even exceed that of the mannan oligosaccharides disclosed in the earlier application. Diacid 6 sugars.
  • the yield of the product is theoretically significantly higher than the yield of the product disclosed in the previous application, which greatly reduces production costs and reduces waste emissions, which is easier to achieve in actual production. It is easier to realize large-scale industrial production.
  • the proportion of the total weight of the diacid is between 1.0 and 3.5.
  • the weight percent content of the alginic oligosaccharide diacid in each of the degrees of polymerization of the algin oligosaccharide diacid composition in the present invention is 5-25% of the disaccharide and 15 ⁇ 15 of the trisaccharide. 30%, tetrasaccharides 15-28%, pentasaccharides 10-25%, hexasaccharides 5-15%, heptasaccharides 3-10%, octose 2-5%, nonasaccharides 1-5%, decasaccharides 1-5 %.
  • the weight percentage content of the oligosaccharides in the combination is: 10-20% disaccharides, 18-30% trisaccharides, 15-28% tetrasaccharides, 15-20% pentasaccharides, and 5 hexasaccharides. ⁇ 10%, heptasaccharide 3-5%, octose 2-3%, nonaose 1-3%, decasuose 1-3%.
  • the total weight of guluronic acid in the alginate oligosaccharide diacid composition of the present invention accounts for 0.1-50%, preferably 1-30%, of the weight of the composition.
  • the pharmaceutically acceptable salt is a sodium salt or a potassium salt.
  • a mixed polysaccharide of polymannuronic acid and polyguluronic acid can be obtained, and the mixed polysaccharide can be precipitated by the acid method, and a certain amount of polyguluronic acid can be removed; during the acid method precipitation process
  • the higher the pH control the higher the polyguluronic acid content in the obtained mixed polysaccharide.
  • the mixed polysaccharides described above undergo oxidative degradation of sugar chains to obtain oxidized oligosaccharides with different degrees of polymerization.
  • the oxidized oligosaccharides are characterized by mannuronic acid or guluronic acid at the reducing end of the oligosaccharide. It is oxidized to 3-6 carbon sugars.
  • An oxidant particularly advantageous for the reaction of the present invention is ozone.
  • the oxidative degradation reaction of sugar chains can occur by passing ozone into a solution containing mixed polysaccharides.
  • the temperature at which the oxidative degradation step is performed is preferably 0-70 ° C, and more preferably 10-45 ° C.
  • the oxidative degradation step is performed at a pH of 3-13, preferably 4-10, and more preferably 6-8.
  • the oxidative degradation reaction using ozone in the present invention is similar to the oxidative degradation of basic copper sulfate (prior patent) or acid hydrolysis (Chinese patent application 01107952.5) in the presence of hydrogen peroxide and sodium hypochlorite used in the prior art in three ways. Both can degrade sugar chains, the difference is that the reducing end structure of the sugar chain of the degradation product is different, and the reducing end of the oxidative degradation product mannuronic acid or guluronic acid obtained in the present invention contains 3-6 carbon Acid structure.
  • the process used in the oxidative degradation step of the present invention also has other advantages: 1. Mild reaction conditions without special reaction conditions; 2. The ozone used can be prepared at the reaction site, reducing transportation pressure in industrial production; 3. Ozone is automatically decomposed into oxygen after the reaction. There is no danger of reaction reagent residue, and it will not cause pollution to the environment.
  • the reaction process is shown in the following equation (VI):
  • the total weight of guluronic acid accounts for less than 50% of the weight of the composition, preferably 0.1-50%, and most preferably 1-30%.
  • the method of the present invention includes the following steps:
  • the mixed polysaccharides of the raw materials polymannuronic acid and polyguluronic acid used in the present invention can be prepared by methods known in the prior art. For example, the methods disclosed in Chinese Patent Application No.98806637.8 and CN02823707.2. The general method can be briefly described as: after the initial degradation of alginic acid, a mixed polysaccharide of polymannuronic acid and polyguluronic acid can be obtained. After the mixed polysaccharide is precipitated by the acid method, some of the polyguluronic acid can be adjusted. Acid content to obtain a mixed polysaccharide of polymannuronic acid and polyguluronic acid.
  • the mixed polysaccharide was dissolved in an appropriate amount of water at room temperature or under heating conditions, stirred, and ozone was continuously introduced, and the reaction started.
  • the reaction pH can be adjusted to between 3-13, preferably 4-10, more preferably 6-8, by adding dilute hydrochloric acid or dilute NaOH solution.
  • the temperature is preferably 0-70 ° C, and more preferably 10-45 ° C.
  • the reaction product obtained above was prepared into a solution having a concentration of about 10%, and was separated by a molecular cut-off membrane to remove degradation products below monosaccharides, and the impermeable liquid was collected.
  • the molecular retention membrane MWCO used has a specification of 1000 Da to 3000 Da, preferably 2000 Da.
  • the collected solution was concentrated on a rotary evaporator and dried under vacuum to obtain a mixture of oligofucoidan oligosaccharides. After analysis, it was found that these products are all disaccharide-decasaccharide oligosaccharides whose composition is in a certain ratio range. Examples 1-3 illustrate this method by way of example.
  • the pharmacological activity of the oligosaccharide composition of the present invention was compared with that of the mannooligosaccharic acid hexaose in the earlier application at the same time. Sour hexaose. Without being bound by any theory, it is believed that when the proportion of di-hexasaccharide in the composition is higher than 60%, and the total weight of guluronic acid accounts for less than 50% of the weight of the composition, the activity of the composition is the highest; but When the ratio of guluronic acid exceeds 60%, the activity of the composition is also reduced.
  • the invention also provides a medicament or health supplement comprising the alginate oligosaccharide combination as described above and optionally a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical preparation of the present invention is manufactured by a known method, including a conventional mixing, dissolving or lyophilizing method.
  • the pharmaceutical composition of the present invention is administered to a patient by various routes suitable for the selected mode of administration, such as oral or parenteral (by intravenous, intramuscular, topical or subcutaneous route).
  • the combination drug of the present invention in combination with a pharmaceutically acceptable carrier can be administered systemically, for example, orally. They can be enclosed in hard or soft shell gelatin capsules and compressed into tablets.
  • a pharmaceutically acceptable carrier such as an inert diluent or an edible carrier
  • the active compounds of the present invention may be combined with one or more excipients and in swallowable tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, discs And other forms.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the ratio of such compositions and preparations may, of course, be varied and may comprise from about 1% to about 99% of the weight of a given unit dosage form.
  • the amount of active compound enables an effective dose level to be obtained.
  • Tablets, lozenges, pills, capsules, etc. may also contain: binders, such as tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, Potato starch, alginic acid, etc .; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, fructose, lactose, or aspartame; or flavoring agents, such as mint, wintergreen, or cherry flavor.
  • a liquid carrier such as a vegetable oil or polyethylene glycol.
  • any material used to prepare any unit dosage form should be pharmaceutically acceptable and non-toxic in the amount used.
  • the active compounds can be incorporated into sustained-release preparations and devices.
  • the active compounds can also be administered intravenously or intraperitoneally by infusion or injection.
  • Aqueous solutions of the active compounds or their salts, optionally miscible non-toxic surfactants, can be prepared.
  • Dispersants in glycerol, liquid polyethylene glycols, triacetin and mixtures thereof, and oils can also be prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injection or infusion can include sterile aqueous solutions or dispersions containing the active ingredients (optionally encapsulated in liposomes) of an instant preparation suitable for sterile injectable or infusible solutions or dispersants.
  • Agent or sterile powder In all cases, the final dosage form must be sterile, liquid and stable under the conditions of manufacture and storage.
  • the liquid carrier can be a solvent or a liquid dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof.
  • Appropriate fluidity can be maintained, for example, by the formation of liposomes, by maintaining the desired particle size in the case of dispersants, or by the use of surfactants.
  • a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. can be used to prevent microorganisms.
  • isotonic agents for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of injectable compositions can be produced by using compositions that delay the absorption (eg, aluminum monostearate and gelatin).
  • Sterile injectable solutions are prepared by combining the required amount of active compound in a suitable solvent with the various other ingredients listed above as needed, and then sterilizing by filtration.
  • the preferred methods of preparation are vacuum drying and freeze-drying techniques, which results in a powder of the active ingredient plus any additional previously required ingredients present in the sterile filtered solution .
  • Useful solid carriers include comminuted solids (such as talc, clay, microcrystalline cellulose, silica, alumina, etc.).
  • Useful liquid carriers include water, ethanol or ethylene glycol or water-ethanol / ethylene glycol mixtures, and the combination drugs of the present invention can optionally be dissolved or dispersed in effective amounts with the help of non-toxic surfactants.
  • Adjuvants such as fragrances
  • additional antimicrobial agents can be added to optimize properties for a given use.
  • Thickeners (such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified inorganic materials) can also be used with liquid carriers to form coatable pastes, gels, ointments , Soap, etc., directly on the user's skin.
  • the therapeutic requirements of a compound or a mixture thereof depend not only on the compound itself, but also on the mode of administration, the nature of the disease to be treated, and the age and condition of the patient, and ultimately on the decision of the attending physician or clinician.
  • the above-mentioned preparations may exist in a unit dosage form, which is a physically dispersed unit containing a unit dose and is suitable for administration to human bodies and other mammalian bodies.
  • the unit dosage form can be a capsule or tablet, or many capsules or tablets.
  • the amount of unit dosage of active ingredient may be varied or adjusted between about 0.1 to about 1000 milligrams or more, depending on the particular treatment involved.
  • Another aspect of the present invention provides a pharmaceutical composition or a health product comprising the alginate oligosaccharide composition of the present invention and an appropriate carrier if necessary.
  • Another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating senile dementia.
  • Yet another aspect of the present invention provides a method of treating a patient with senile dementia, which comprises administering to a patient in need thereof an effective amount of the alginate oligosaccharide composition of the present invention.
  • Yet another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating Parkinson's disease.
  • Yet another aspect of the present invention provides a method of treating a patient with Parkinson's disease, which comprises administering to a patient in need thereof an effective amount of the alginate oligosaccharide composition of the present invention.
  • Another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating an inflammatory response.
  • Yet another aspect of the present invention provides a method of treating a patient suffering from inflammation, comprising administering to a patient in need thereof an effective amount of the alginate oligosaccharide composition of the present invention.
  • Yet another aspect of the present invention provides the use of an alginate oligosaccharide composition to treat a pain response.
  • Yet another aspect of the present invention provides a method for treating a patient suffering from pain, comprising administering to a patient in need thereof an effective amount of the alginate oligosaccharide composition of the present invention.
  • Another aspect of the present invention provides the use of alginate oligosaccharide composition for treating diabetes.
  • Yet another aspect of the present invention provides a method of treating a patient suffering from diabetes, which comprises administering to a patient in need thereof an effective amount of the alginate oligosaccharide composition of the present invention.
  • Another aspect of the present invention provides the use of an alginate oligosaccharide composition for treating vascular dementia.
  • Yet another aspect of the present invention provides a method of treating a patient suffering from vascular dementia, which comprises administering to a patient in need thereof an effective amount of the alginate oligosaccharide composition of the present invention.
  • the pain described in the present invention includes various pains, including but acute pain, chronic pain, neuropathic pain, postoperative pain, chronic lower back pain, cluster headache, herpes neuralgia, phantom limb pain, central pain, toothache, opioid Substance-resistant pain, visceral pain, surgical pain, bone injury pain, pain during labor and childbirth, pain due to burns including sunburn, postpartum pain, migraine, angina, and pain associated with the urogenital tract (including Cystitis), vascular pain, trigeminal neuralgia, intercostal neuralgia, surgical incision pain, chronic fasciitis pain, heel pain, muscle pain, bone pain, joint pain, cancerous pain, non-cancerous pain, etc.
  • various pains including but acute pain, chronic pain, neuropathic pain, postoperative pain, chronic lower back pain, cluster headache, herpes neuralgia, phantom limb pain, central pain, toothache, opioid Substance-resistant pain, visceral pain, surgical pain, bone injury pain, pain during labor
  • the inflammation in the present invention includes various inflammations, including but not limited to acute inflammation, chronic inflammation, vascular inflammation, neuroinflammation, central nervous inflammation (such as multiple sclerosis, including encephalomyelitis, etc.), peripheral neuroinflammation, arthritis ( (Such as osteoarthritis, sacroiliitis, etc., psoriasis arthritis, rheumatoid arthritis, rheumatoid arthritis, etc.), ankylosing spondylitis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), Inflammatory diabetic ulcer, systemic lupus erythematosus, inflammatory skin diseases (such as psoriasis, atopic dermatitis, eczema), and the like.
  • arthritis such as osteoarthritis, sacroiliitis, etc., psoriasis arthritis, rheumatoid arthritis, rheumatoid arthritis, etc.
  • the alginate oligosaccharide composition of the present invention is prepared by using a method different from the prior art, and does not need to separate the M stage and the G stage, which greatly reduces the complexity of the production process, and also greatly reduces the production cost. Simple, high content of active ingredients, no residual reagents. It has been experimentally proven that the alginate 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.
  • AD model was induced by unilateral intraventricular injection of A ⁇ , and learning and memory behaviors of AD model rats were evaluated by Morris water maze.
  • Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40mg / kg) and fixed on a brain stereotactic device, routine skin preparation and disinfection, incision of the skin, extremity exposure, hippocampal CA1 area refer to "Rat brain stereotactic atlas” (Bao Xinming, Shu Siyun, Beijing, People's Medical Publishing House, 1991, 28) "Anterior and posterior 3.0mm, 2.2mm next to the middle slit, 2.8mm subdural" for positioning.
  • the model group and the administration group were injected into the right hippocampal CA1 area with a micro-injector, and then slowly injected with condensed A ⁇ (A ⁇ 1-40 was formulated in PBS solution to 1.4 mg / mL, and incubated in a 37 ° C incubator for 5 minutes. It was brought into an aggregated state) 5 ⁇ l at a flow rate of 1 ⁇ L / min. After the injection was completed, the needle was left for 5 minutes to fully disperse A ⁇ , and then the needle was slowly withdrawn. The surgical incision is sutured and the body is warmed up. The control group was injected with an equal amount of sterilized PBS, and the remaining steps were the same as before. The corresponding drugs were given 7 days before the operation and continued to the end of the experiment.
  • the Morris water maze experiment was performed on the 11th day after the operation.
  • Positioning and sailing experiment Each group of rats is trained once a day for 5 consecutive days, which is the positioning and sailing experiment, and the time it takes for the animal to find the platform (ie, the escape latency) is recorded. Those who did not find a platform in about 90s, guided them to swim in a straight direction toward the platform and stood on the platform for 30s to induce their learning and memory.
  • mice were randomly divided into 8 groups: a blank control group, an MPTP model group, and an administration group, with 14 animals in each group. Animals were dosed on the same day. The blank control group and the MPTP model group were perfused with saline. The other groups were given the corresponding drugs, once a day for 17 consecutive days. From the 6th day, modeling drugs were given. The animals in the blank control group were subcutaneously given 10ml / kg of saline, and the remaining animals were given subcutaneously MPTP 25mg / kg once a day for five days.
  • MPTP selectively destroys dopaminergic neurons in the brain substantia nigra.
  • MPTP-induced PD animal model is the most classic animal model similar to the pathological changes and clinical characteristics of human Parkinson's disease.
  • the main symptoms of PD are resting tremor, increased muscle tone, and decreased exercise.
  • the head-turning time and climbing-down time of the pole climbing experiment can represent the overall activity coordination ability of mice.
  • mice Male DBA / 1 mice, weighing 19-22 g, were randomly divided into blank control group, model group and administration group, with 8 mice in each group. Except for the blank control group, the remaining animals were subcutaneously injected with bovine type II collagen-complete Freund's adjuvant (CII-CFA) emulsion 10 mg / kg at the root of the tail on day 0. On day 23, lipopolysaccharide (LPS) was injected intraperitoneally. 1.5mg / kg. Administration was started on the 28th day, the blank control group and the model group were orally administered with normal saline, and the other groups were given the corresponding drugs, administered once a day for 14 consecutive days. After the LPS injection, the mice were observed daily for morbidity.
  • CII-CFA bovine type II collagen-complete Freund's adjuvant
  • LPS lipopolysaccharide
  • the clinical score is based on a scale of 0-4 according to the degree of the disease (redness, swelling, joint deformation) to reflect the degree of disease progression.
  • the degree of the disease redness, swelling, joint deformation
  • 1 point is near the metatarsal bone or near the ankle joint or sacrum, with redness or mild swelling, 1 toe is red and swollen
  • 2 points is ankle and sacrum with slight erythema and swelling, or more than two toes Redness and swelling
  • the highest score for each limb is 4 points, and the highest score for each animal is 16 points ).
  • mice Female C57BL / 6 mice, weighing 17-20 g, were randomly selected as blank control groups. The remaining animals were subcutaneously injected with myelin oligodendrocyte glycoprotein-complete Freund's adjuvant (MOG-CFA) emulsion on the 0th day to sensitize, MOG 10mg / kg, CFA 20mg / kg, and on the 0th day and On the second day, pertussis toxin was injected intraperitoneally at 10ug / kg. The administration was started on the first day. The blank control group and the model group were orally administered with normal saline. The other groups were given the corresponding drugs, which were administered once a day for 24 consecutive days.
  • MOG-CFA myelin oligodendrocyte glycoprotein-complete Freund's adjuvant
  • mice On the 12th day after immunization, the immunized mice will develop symptoms, and begin to observe and record the body weight and clinical scores every day (0-4 points indicate different degrees, 0 points are normal performance, and there are no obvious signs of disease; 1 points are drooping tails Weakness, unilateral weakness in the hind limbs; 2 points for sagging tail and weakness in both hind legs; 3 points for unilateral hind limb weakness and paralysis; 4 points for both hind limb weakness and paralysis) to reflect the degree of disease progression.
  • MRL / lpr transgenic mice have homozygous mutations in the Faslpr gene, which can spontaneously form hyperplasia of lymphoid tissues. The mice begin to develop disease at about 10-14 weeks of age and develop systemic lupus erythematosus symptoms.
  • Lymph node scores are performed once a week (0-6 points indicate different degrees, 0 points are normal; 1 point is less than 1cm in diameter on one side of the point; 2 points are less than 1cm in diameter on both sides of the point; 3 points In order to be less than 1cm in diameter on three sides, 4 points are larger than 1cm in diameter on one side, and the diameter is less than 1cm in two points; The diameter of the other two points is less than 1cm; 6 points are divided into three sides and the diameter is greater than 1cm).
  • IBD Inflammatory bowel disease
  • DSS distaln sodium sulfate
  • mice Female C57 mice, 7-8 weeks old, weighing 18-20 g, were randomly divided into: blank control group, model group, administration group, 8 mice in each group.
  • the mice in the model group and the administration group were given 2.5% high-molecular-weight polymer dextran sodium sulfate (DSS) for modeling on the 1st to 7th days by drinking water, and the administration was started on the 1st day, blank
  • the control group and the model group were orally administered with normal saline, and the other groups were given the corresponding drugs, administered once a day for 30 consecutive days.
  • the mice On day 31, the mice were sacrificed by cervical dislocation, the abdominal cavity was opened, and the mesentery was separated. Each mouse was taken from the beginning to the end of the anesthesia, and each group was sampled sequentially to measure the colon length.
  • mice Male NIH mice were randomly selected into a normal control group, a model group, and an administration group, with 10 mice in each group. On the day of the test, all animals except the normal group were injected with streptozotocin 150 mg / kg intraperitoneally. The corresponding drugs were continuously administered for 10 days. On the 11th day, the eyeballs were removed to take blood, and the blood glucose concentration was measured.
  • Kunming mice half male and half male, weighing 18-22 g, were randomly divided into blank control group, model group, and administration group, with 10 mice in each group. From the day of grouping, the blank control group was orally administered with 20 ml / kg of distilled water daily, and the remaining groups were orally administered with the corresponding drugs, once a day for 7 consecutive days. One hour after the last dose, mice in each group were intraperitoneally injected with 0.2 ml of a 0.6% acetic acid solution, and the writhing latency of the mice (i.e., the time from the injection of acetic acid to the occurrence of writhing response) and the writhing were recorded within 20 minutes after the acetic acid injection Body times.
  • Injecting chemicals such as acetic acid solution into the abdominal cavity of mice can stimulate the peritoneum of the mouse and cause intermittent pain, which is manifested by the abdomen recession, the front wall of the abdomen close to the bottom of the cage, the hips twisted, and the hind legs extended.
  • a special posture is called a writhing body response.
  • the writhing incubation period (the time from the injection of acetic acid to the writhing response) and the number of writhing in a certain period of time can represent the severity of pain. The shorter the incubation period of writhing body, the more the number of writhing body, the more severe the pain.
  • SD male rats weighing 180-220g, were randomly divided into: blank control group, model group, administration group, 8 rats in each group. Administration was started on the day of grouping. The blank control group and the model group were administered with distilled water by gavage. The remaining groups were given the corresponding drugs once a day for 28 consecutive days. 30 minutes after the last administration, the animals in the blank control group were given physiology. In addition to saline, the other groups were subcutaneously injected with nitroglycerin 10 mg / kg under the right shoulder for modeling. Observe the time and duration of ear redness after modeling, and the number of scratches in the period of 30-45 minutes after modeling. The content of 5-HT in brain tissue was measured by fluorescence spectrophotometry. Measured at the wavelength of Ex356nm / Em and 483nm, the results are expressed in ng / g brain weight.
  • Migraine is a vascular and nerve dysfunction disease under the interaction of blood vessels and neural mechanisms.
  • Nitroglycerin can cause the hypersensitivity of trigeminal nerve fibers and cause migraines by dilating meningeal blood vessels, forming neurogenic inflammation, and activating neuronal functions in the hypothalamus, brainstem, and spinal cord segments.
  • the nitroglycerin model is an animal model established in 1995 and has now become a classic animal migraine model. According to the pathogenic mechanism of nitroglycerin, the ear redness time due to vasodilation, the number of scratching heads due to pain, and the serotonin (5-HT) content in the brain tissue can be evaluated to evaluate the severity of migraine. The longer the ear redness lasts, the more times the head is scratched, and the higher the 5-HT content, the more severe the migraine.
  • the corresponding drugs were given orally in each group, while the blank control group, sham operation group, and model group received oral distilled water. After 10 days of continuous administration, except for the blank control group, all rats were anesthetized with intraperitoneal injection of chloral hydrate 350mg / kg, and then the rats were fixed on a stereotactic device, a midline incision was made on top of the head, and the skin and muscle were cut in layers. An opening in the middle of the sagittal suture of the brain exposes the skull.
  • the parameters of the electrical stimulation are 200ms period, amplitude 10v, wave width 5ms, and stimulation for 10 minutes.
  • the animals in the sham operation group were only inserted with electrodes and not stimulated.
  • the right femoral vein was injected with 50 mg / kg of Evans blue 7 minutes before the stimulation, and the perfusion was fixed within 20 minutes after the stimulation.
  • Activation of the trigeminal vascular system is a key link in the pain of migraine patients, and the occurrence of neurological inflammation of the meninges plays an important role in the generation and maintenance of migraine pain.
  • the trigeminal nerve distributed in the dura mater When the trigeminal nerve distributed in the dura mater is stimulated, it releases vasoactive substances, which causes meningeal blood vessels to dilate, extravasation of plasma components, mast cell degranulation, and activation of platelets, resulting in migraine headaches.
  • the neurotransmitter released after pain stimulation binds to the corresponding receptor on the cell membrane. Under the action of the second messenger, the C-fos mRNA gene is expressed, and the c-fos protein is translated and synthesized in the nucleus. effect.
  • the degree of migraine can be reflected by measuring the dural serum protein exudation of migraine animals and the number of c-fos positive cells on the caudal side of the nucleus of the trigeminal spine. A lower number of cells indicates a less severe migraine.
  • the bilateral common carotid artery ligation (BCCAo) model is a vascular dementia model commonly used in the art and established by global cerebral ischemia-reperfusion.
  • mice Male C57BL / 6 mice, weighing 22 ⁇ 2g, were randomly divided into: sham operation group, 30 minutes bilateral common carotid artery occlusion (BCCAo) model group (referred to as 30min BCCAo group), administration group, There are 10 animals in each group. After the animals were divided into groups, mice in the sham operation group and the 30-minute BCCAo group were perfused with distilled water once a day. After 5 consecutive days of intragastric administration, BCCAo surgery was performed. The mice in the administration group were given the corresponding drugs by gavage. They were administered by gavage once a day. After 5 days of continuous administration, BCACo surgery was performed.
  • BCCAo bilateral common carotid artery occlusion
  • mice of each group are anesthetized with sodium pentobarbital; the common carotid arteries of the model group and the administration group are separated and ligated for 30 minutes, and then the ligature is removed and the neck wound is sutured; In the group, bilateral common carotid arteries were not ligated and the neck incisions were directly sutured. Twenty-four hours after BCCAo, the mice in each group continued to be orally administered with the corresponding drugs or distilled water according to the pre-operative dosing regimen, and each was continuously administered for 23 days.
  • the dark avoidance test was performed on the 7th day after BCCAo, and the Morris water maze test was started on the 13th day to evaluate the improvement effect of mannuronic acid composition on the learning and memory ability of mice.
  • the mice were sacrificed and the brain tissues were fixed.
  • the staining of the hippocampal nerve cells after BCCAo and the protective effect of the mannuronic acid composition on the damaged neurons were evaluated by HE staining and other methods.
  • the dark avoidance experiment is used to detect the spatial discrimination of learning and memory ability of mice.
  • the memory impairment of spatial localization can only occur if the hippocampus or the area around the hippocampus is damaged.
  • the dark-proof experiment box is a device designed by using the mouse's habit of darkening and avoiding light. Half of it is a dark room and half is a bright room. There is a small hole in the middle. The bottom of the dark room is covered with a copper grid that is energized. Animals are shocked when they enter the dark room. Escape to the bright room. After the animals are trained for 24 hours, the test is performed again. The time from when the animals are placed in the bright room to when they enter the dark room for the first time is the latency period of the dark avoidance experiment. The longer the incubation period of the dark avoidance experiment, the fewer the number of avoidance errors, indicating that the animal's memory is better.
  • the Morris water maze (MWM) experiment is an experiment that forces experimental animals to swim and learn to find hidden platforms in the water. It is mainly used to test the experimental animals' learning and memory of spatial position and orientation (spatial positioning). ability.
  • the mouse Morris water maze is mainly composed of a cylindrical pool with a diameter of 80cm and a height of 70cm and a movable platform with a diameter of 8cm. A digital camera is connected to the computer over the pool. Before the experiment, inject fresh water into the pool, the water depth is 15cm, and the water surface is 0.5cm higher than the platform surface. Add milk to make the pool water opaque, and keep the platform position unchanged during the experiment.
  • Morris water maze behavior includes the following two test indicators.
  • Place navigation experiments are used to measure the ability of mice to learn and memorize water mazes.
  • the experiment started on the 13th day after BCCAo and lasted 4 days.
  • the mice were trained once in the morning and afternoon in total, 8 times in total.
  • the mouse enters the pool at 1/2 radian of the west quadrant, and heads into the pool wall. If the platform is not found within 120 seconds, the experimenter will guide them to the platform and leave it for 30 seconds to guide their learning and memory.
  • the experimental observation and recording of the route map and the time required for the mice to find and climb onto the platform, that is, the escape latency and swimming speed of the Morris water maze experiment were recorded.
  • the Morris water maze experiment avoidance latency refers to the time from when a mouse enters the water to when it finds a platform. The shorter the escape latency of the Morris water maze experiment, the better the animals' memory.
  • a spatial search experiment (spatial probe test) is used to measure the ability of a mouse to retain the platform's spatial position memory after it learns to find a platform. After the positioning and navigation experiment was completed, the platform was removed at an interval of one day, and the mice were put into the water from the same water entry point, and the number of times they crossed the original platform was measured. Data acquisition and processing are performed by an automatic image monitoring and processing system.
  • the middle cerebral artery ligation (MCAO) model is a vascular dementia model commonly used in the art established by focal cerebral ischemia.
  • the rats in the other groups were anesthetized with intraperitoneal injection of chloral hydrate 350mg / kg, and the left side was fixed on the rat plate, and the outer ear canal and eyes were under the operating microscope.
  • the pulse sequence is a 45 ° pulse, each acquisition is 4 seconds, the relaxation time is 1 second, and the accumulation is 20 times, and the spectral width is from -2 ppm to 10 ppm.
  • One-dimensional hydrogen spectrum was obtained by Fourier transform after data collection, and the TSP methyl hydrogen signal was set to 0.00ppm.
  • the intermediate contains a mannuronic acid fragment (M-block, chemical shift of 5.1 ppm) and a guluronic acid fragment (G-block, chemical shift of 5.5 ppm), and a mannuronic acid and gulose Uric acid mosaic fragment (MG-block, chemical shift 5.3 ppm).
  • M-block mannuronic acid fragment
  • G-block guluronic acid fragment
  • MG-block mannuronic acid and gulose Uric acid mosaic fragment
  • Step 2) Proportion and structure analysis of oligosaccharides of various polymerization degrees in alginate oligosaccharide diacid product A
  • test results disaccharide-decaose are expressed as dp2-dp10, respectively, dp2 is 18%, dp3 is 24%, dp4 is 23%, dp5 is 14%, dp6 is 8%, dp7 is 7%, and dp8 is 2 %, Dp9 is 2%, and dp10 is 2%.
  • Step 3) LC-MS analysis of the structure of oligosaccharides of various polymerization degrees in the alginate oligosaccharide diacid product A
  • Mass spectrometry conditions Agilent 6540 QTOF; ion source: ESI collision voltage 120V; negative ion mode.
  • the acquisition signal (m / z) width is 100-1000.
  • Sample preparation Weigh 50mg sample to be dissolved in 0.5ml D2O, freeze-dry, add 0.5ml deuterated heavy water to dissolve, re-lyophilize, and finally dissolve the lyophilized sample powder with an appropriate amount of heavy water, and transfer it to the nuclear magnetic tube. A 100 mg / ml test solution was prepared, and 0.01% (w / v) deuterated TSP (trimethylsilylpropionic) sodium salt was added as an internal standard.
  • TSP trimethylsilylpropionic
  • a 400M Fourier transform nuclear magnetic resonance instrument collects one-dimensional hydrogen spectra at room temperature.
  • the pulse sequence is a 45 ° pulse, each acquisition is 4 seconds, the relaxation time is 1 second, and the accumulation is 20 times, and the spectral width is from -2 ppm to 10 ppm.
  • One-dimensional hydrogen spectrum was obtained by Fourier transform after data collection, and the TSP methyl hydrogen signal was set to 0.00ppm.
  • the NMR spectrum of product A is shown in Figure 5.
  • the multiple peak with a chemical shift of 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 guronic acid (G)
  • 4.9 ppm It is the C-1 hydrogen signal of mannuronic acid and guluronic acid chimeric fragment (MG).
  • the formula for the content of guluronic acid is:
  • I4.6, I5.0, and I4.9 are mannuronic acid (M), guluronic acid (G), mannuronic acid, and guluronic acid chimeric fragment (MG), respectively.
  • M mannuronic acid
  • G guluronic acid
  • MG guluronic acid chimeric fragment
  • a 400M Fourier transform nuclear magnetic resonance apparatus was used to determine the guluronic acid content in product B at 60 ° C at 50%.
  • the determination method is the same as that in the relevant part of Example 1.
  • the nuclear magnetic resonance hydrogen spectrum is shown in FIG. 6. It can be seen from the figure that the integrated area of mannuronic acid (M, chemical shift value of 4.6 ppm) and guluronic acid (G, chemical shift value of 5.0 ppm) is relatively close, while that of mannuronic acid and guluronic acid is The integrated area of the mosaic fragment (MG, chemical shift is 4.9 ppm) is small.
  • Formula for calculating content of guluronic acid product (G) According to Example 1, the content of G is 50%.
  • Example 1 Weigh 100g of the intermediate in Example 1. After adding water to suspension, add NaOH to adjust the pH value to alkaline, so that the powder is completely dissolved, and finally prepare a 1L solution, and then add HCl to adjust the pH value to 2.95. Partial white precipitation appears. The precipitate was removed by centrifugation, and the supernatant was collected and further diluted with distilled water to prepare a 1.5L volume solution. The pH was adjusted to 9.0 with NaOH, and the reaction was performed at 45 ° C in a water bath. The gas flow at the outlet of the oxygen cylinder and the power of the ozone generator were adjusted so that the ozone mass concentration flow reached 3 g / hr and passed into the reaction solution.
  • a 400M Fourier transform nuclear magnetic resonance apparatus was used to determine the guluronic acid content in product C at 10 ° C at 10%. The determination method was the same as that in the relevant part of Example 1. The test results are shown in Figure 7.
  • the mannuronic acid M, chemical shift value of 4.6 ppm
  • G chemical shift value of 5.0 ppm
  • Mannuronic acid and guluraldehyde The integrated area of the acid mosaic fragment (MG, chemical shift 4.9 ppm) is close to the integrated area of guluronic acid.
  • Formula for calculating content of guluronic acid product (G) According to Example 1, the content of G is 10%.
  • the product with high G content is prepared by referring to the preparation method of the foregoing Example 2.
  • the raw material of sodium alginate is a high G content sample provided by Qingdao Haizhilin Biotechnology Development Co., Ltd.
  • the preparation method is the same as that in Example 2. Specifically: 500 g of sodium alginate powder with high G content, mixed with distilled water, swelled, and prepared into a 5 L volume solution, adjusted the pH to 4.0 with NaOH, and reacted at room temperature at 25 ° C.
  • the gas flow at the outlet of the oxygen cylinder and the power of the ozone generator were adjusted so that the ozone mass concentration flow reached 1 g / hr and passed into the reaction solution.
  • a 400M Fourier transform nuclear magnetic resonance apparatus was used to determine the guluronic acid content in product D at 60 ° C, and the determination method was the same as in the relevant part of Example 1.
  • the nuclear magnetic resonance spectrum is shown in FIG. 8. It can be seen from the figure that the integrated area of guluronic acid (G, chemical shift value of 5.0 ppm) is greater than the integrated area of mannuronic acid (M, chemical shift value of 4.6 ppm), while mannuronic acid and guluraldehyde The integrated area of the acid mosaic fragment (MG, chemical shift is 4.9 ppm) is small.
  • the formula for calculating the content of guluronic acid (G) According to Example 1, the content of G is 60%.
  • the swimming distance in the quadrant of the original platform in the model group decreased significantly as a percentage of the total distance, and the percentage of the swimming distance of the administration group in the quadrant in the original platform increased significantly in the total distance, see FIG. 10.
  • the latency and climbing time of the former were significantly prolonged.
  • the incubation period and climbing time of each administration group were shortened to different degrees.
  • the product A, B, and C had better pharmacological activity than the previously expected single-polymerized mannanuronic acid hexaose with the highest activity, but the activity of product D was weaker than that of mannuronic acid hexaose.
  • mice in the model group developed paralysis of paralysis in both hind limbs.
  • the average clinical score of the model group reached 3 points, indicating that the multiple sclerosis model was successfully modeled.
  • the inflammation progress of each administration group was alleviated to varying degrees.
  • the clinical scores of products A, B, and C were lower than that of mannuronic acid hexaose throughout the experiment and at the end point, indicating that the pharmacological activity of products A, B, and C was better than that of mannuronic acid hexaose;
  • the clinical score of Product D was slightly higher throughout the experiment and at the end point, reflecting that the anti-inflammatory activity of Product D was the weakest, indicating that the content of guluronic acid and the proportion of di-hexaose in the composition had an effect on the activity of the product. Significant effect, but when the content of guluronic acid is too high, the activity of the composition will be reduced.
  • transgenic mice began to develop disease, and lymphadenopathy appeared, and the lymph node score continued to increase with time, indicating that the model group had successfully developed the disease and the disease progressed rapidly.
  • the disease progression of each administration group was alleviated to varying degrees.
  • the model group showed a significant shortening of the colon due to inflammation, and most of the mice lost significantly weight. Nearly half of the model group died later, indicating that the intestinal inflammation was very serious. Compared with the model group, the intestinal inflammation in each administration group was alleviated to varying degrees, which was reflected in the recovery of colon length and improved survival rate.
  • products A, B, and C make the incubation latency of mice longer than mannuronic acid hexaose, and the number of twists is less than that of mannuronic acid hexaose, indicating that products A, B, and C have better pharmacological activity than Mannuronic acid hexaose;
  • the twisting latency of product D is short, and the number of twists is slightly higher than that of mannuronic acid hexaose, reflecting that the activity of product D is weaker than that of mannuronic acid.
  • the rats developed ear redness, which lasted for about 2.5 hours.
  • the number of scratches in the model group was significantly higher than that of the blank control group in the 30-45 minute period.
  • the ear redness of the rats in the administration group was significantly delayed, the duration of ear redness was shortened, and the number of scratches in the 30-45 minute period was reduced.
  • products A, B, and C made the number of scratches of rats less than that of mannuronic acid hexaose, indicating that the pharmacological activity of products A, B, and C was better than that of mannuronic acid hexaose; but product D group of rats The number of scratches is slightly higher than that of mannuronic acid hexaose, reflecting that the activity of product D is weaker than that of mannuronic acid hexaose.
  • the number of c-fos positive cells in products A, B, and C was less than that of mannuronic acid hexaose, indicating that the pharmacological activity of products A, B, and C was better than that of mannuronic acid hexaose; but product D
  • the number of c-fos positive cells in the group was slightly higher than that of mannuronic acid hexaose, reflecting that the activity of product D was weaker than that of mannuronic acid hexaose.
  • the latency period of the former dark avoidance experiment was significantly shortened, and the number of errors increased significantly, indicating that the memory ability of the model group mice was significantly reduced, and the evaluation model was successfully established.
  • the incubation period of the avoidance experiment in each administration group was significantly increased, and the number of errors was significantly reduced.
  • the incubation period of the mice in products A, B, and C was longer than that of mannuronic acid hexaose, and the number of errors was less than that of mannuronic acid hexaose, indicating that the pharmacological activity of products A, B, and C was better than that of mannaldehyde.
  • Diacid hexaose Diacid hexaose; however, the incubation period of product D is slightly shorter than that of mannuronic acid hexaose, and the number of errors is slightly higher, reflecting that the activity of product D is weaker than that of mannuronic acid hexaose.
  • the experimental results are consistent with the previous experiments, indicating that the content of guluronic acid and the proportion of di-hexaose in the composition have a significant effect on the activity of the product, but the combination will be reduced when the content of guluronic acid is too high ⁇ ⁇ ⁇ Activity. See Figures 19 and 20.
  • the products A, B, and C group mice crossed the platform more times than the mannuronic acid hexaose, indicating that the product A, B, and C had better pharmacological activity than the mannuronic acid hexaose; but the product D group The number of times of crossing the platform is slightly lower than that of mannuronic acid hexaose, reflecting that the activity of product D is weaker than that of mannuronic acid hexaose. See Figure 22.
  • the escape latency of the rats in group A, B, and C was shorter than that of mannuronic acid hexaose, indicating that the pharmacological activity of products A, B, and C was better than that of mannuronic acid hexaose; but group D of product escaped
  • the incubation period is slightly longer than that of mannuronic acid hexaose, reflecting that Product D is less active than mannuronic acid hexaose.

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Abstract

本发明涉及一种褐藻胶寡糖二酸组合，其包含具有式(IV)的甘露糖醛二酸或其药学上可接受的盐，其中n为选自1-9的整数，m选自0，1或2，m'选自0或1，并且其中n＝1-5的褐藻胶寡糖二酸的重量总和占所述组合总重量的60％以上，古罗糖醛酸的重量总和占所述组合物重量的50％以下。

Description

褐藻胶寡糖二酸的组合物 技术领域
本发明涉及通过生物活性筛选的方法得到褐藻胶寡糖二酸的最佳组合物，该方法采用老年痴呆动物模型评价褐藻胶寡糖的不同聚合度及其配比对生物活性的影响。最终筛选到具有最佳生物活性的组合物并通过超滤膜分离的办法制备得到所需的目标物质。
背景技术
褐藻胶寡糖由于其潜在的药用价值已经受到广泛的重视。褐藻胶寡糖通常以海藻酸为原料经过多步骤制得。
在褐藻胶寡糖分子中，有由甘露糖醛酸(D-mannuronic acid)通过β-1,4-糖苷键连接形成的M段、古罗糖醛酸(L-guluronic acid)通过α-1,4-糖苷键连接形成的G段，以及由这两种糖杂合形成的MG段。甘露糖醛酸和古罗糖醛酸的结构式如下(Ⅰ)式和(II)式所示：
Figure PCTCN2019093778-appb-000001
褐藻胶寡糖的结构式如下(III)式所示:
Figure PCTCN2019093778-appb-000002
M段和G段可以从原料海藻酸中分离。通常的方法可以简单描述为：将海藻酸初步降解后得到聚甘露糖醛酸和聚古罗糖醛酸的混合多糖，混合多糖再经酸法沉淀后，可以除去其中一定量的聚古罗糖醛酸。例如，可参见中国专利申请No.98806637.8以及 CN02823707.2所披露的方法。
制备寡聚甘露糖醛酸的做法如下：将上述得到的M段中间体在酸性条件下加热进一步酸解得到所需分子量范围的小片段甘露糖醛酸聚合物。另外，也有通过氧化降解的办法提升降解效率，同时可以将还原末端氧化为开环的糖二酸，详见耿美玉等人的中国专利申请200580009396.5(专利文献1)及美国专利US 8835403B2(专利文献2)。为了方便叙述，专利文献1和2在下文中统称为在先专利，它们以引证的方式全部并入本文。
在先专利披露的甘露糖醛二酸的反应过程可通过如下反应方程式(V)表示，即寡聚甘露糖醛酸多糖还原端的甘露糖醛酸C1-位醛基氧化成羧基。
Figure PCTCN2019093778-appb-000003
在上述氧化转化过程中，常用的氧化剂有碱性硫酸铜溶液，即菲林试剂，在先专利即采用了该氧化方法，具体为：在碱性条件下，将反应底物聚甘露糖醛酸即上文的M段中间体加入硫酸铜溶液中，在沸水浴中反应15分钟至2小时。该法是以Cu 2+离子为氧化剂氧化醛基，反应中产生砖红色的氧化亚铜沉淀，这个反应常用于鉴定还原性糖。
在先专利公开了甘露寡糖二酸具有抗阿尔茨海默病(Alzheimer’s disease,AD)和抗糖尿病的作用，且聚合度为6的甘露寡糖二酸活性最佳。阿尔茨海默病与Ⅱ型糖尿病的发病过程与淀粉样蛋白(β-amyloid及amylin)密切相关。淀粉样蛋白聚集以后产生蛋白寡聚体，进一步聚集形成纤维。这些蛋白聚集物有细胞毒性，在细胞内诱导氧化反应损伤线粒体以及引发炎症反应等级联反应，造成大量的神经元和β细胞损伤，最终导致阿尔茨海默病与Ⅱ型糖尿病的 发生。甘露寡糖二酸靶向淀粉样蛋白并拮抗其诱导的级联反应，由此具有预防和治疗阿尔茨海默病与Ⅱ型糖尿病的作用。
为了获得在先专利公开的具有抗阿尔茨海默病和抗糖尿病的作用甘露寡糖二酸，需将原料海藻酸中的古罗糖醛酸除去，而海藻酸中古罗糖醛酸的含量通常在30％以上，最高可达70％左右，因此为了获得高纯度的甘露寡糖二酸，实际生产成本非常之高。
发明内容
本发明的第一个方面涉及一种褐藻胶寡糖二酸的组合物，其包含具有式(IV)的甘露糖醛酸和/或古罗糖醛酸或其药学上可接受的盐：
Figure PCTCN2019093778-appb-000004
其中n为选自1-9的整数，m选自0，1或2，m’选自0或1，
并且其中，
n＝1-5的褐藻胶寡糖二酸的重量总和占所述组合总重量的60％以上；古罗糖醛酸二酸的重量总和占所述组合物重量的50％以下。
本发明的另一个方面涉及一种药物组合物或保健品，其包含上文所述的褐藻胶寡糖二酸的组合物。本发明的其他方面还涉及褐藻胶寡糖二酸的组合物在治疗选自老年性痴呆、帕金森病、炎症、疼痛、糖尿病或血管性痴呆等疾病中的应用。
特别地，本发明的褐藻胶寡糖二酸组合物是不同聚合度的甘露糖醛酸和古洛糖醛酸的混合物，其主要成分是由甘露糖醛酸通过β-1,4-糖苷键连接形成的M段、古罗糖醛酸通过α-1,4-糖苷键连接形成的G段，以及由这两种糖杂合形成的MG段，聚合度为2至10的寡糖。已知甘露糖醛二酸具有一定的抗阿尔茨海默病和抗糖尿病的药理活性。活性最高的糖为5-8糖，特别是6糖。但是，发明人发现，聚合度为2至10的甘露糖醛酸和古洛糖醛酸的寡糖二酸混合物， 同样具有抗阿尔茨海默病和抗糖尿病的药理活性，但前提是需要将古洛糖醛酸的含量控制在一定范围内。也即，本发明的褐藻胶寡糖二酸组合物可以以大大降低生产成本来制备，在实际生产中更容易实现，更易于实现工业化大生产。
附图说明
图1中间体核磁图谱。
图2是产品A中二糖、三糖和四糖的质谱图。
图3是产品A中五糖、六糖和七糖的质谱图。
图4是产品A中八糖、九糖和十糖的质谱图。
图5表示产品A的核磁图谱。
图6表示产品B的核磁图谱。
图7表示产品C的核磁图谱。
图8表示产品D的核磁图谱。
图9表示不同寡糖组合物及甘露糖醛二酸六糖对AD动物穿越平台次数的影响；图中横坐标的编号分别对应的样品为：i:对照组；ii:模型组；iii:产品A；iv:产品B；v:产品C；vi:产品D；vii:甘露糖醛二酸六糖。
图10表示不同寡糖组合物及甘露糖醛二酸六糖对AD动物游泳路程的影响；其中横坐标附图标记同图9。
图11表示不同寡糖组合物及甘露糖醛二酸六糖对PD动物第11天爬下时间的影响；其中横坐标附图标记同图9。
图12表示不同寡糖组合物及甘露糖醛二酸六糖对PD动物第11天潜伏期的影响；其中横坐标附图标记同图9。
图13a和13b表示不同寡糖组合物及甘露糖醛二酸六糖对小鼠炎性肠炎的治疗作用；图中横坐标附图标记同图9。
图14表示不同寡糖组合物、甘露糖醛二酸六糖对糖尿病小鼠餐后血糖的影响；图中横坐标附图标记同图9。
图15表示寡不同糖组合物、甘露糖醛二酸六糖对醋酸所致小鼠 扭体反应潜伏期的影响；图中横坐标的编号分别对应的样品为：i:模型组；ii:产品A；iii:产品B；iv:产品C；v:产品D；vi:甘露糖醛二酸六糖。
图16表示不同寡糖组合物、甘露糖醛二酸六糖对醋酸所致小鼠扭体反应次数的影响；其中横坐标附图标记同图15。
图17表示不同寡糖组合物、甘露糖醛二酸六糖对硝酸甘油致偏头痛大鼠挠头次数的影响；其中横坐标附图标记同图9。
图18表示不同寡糖组合物、甘露糖醛二酸六糖对电刺激三叉神经节致偏头痛大鼠三叉神经脊束核尾侧c-fos阳性细胞数的影响；其中横坐标附图标记同图9。
图19表示不同寡糖组合物、甘露糖醛二酸六糖对双侧颈总动脉结扎致血管性痴呆小鼠避暗实验潜伏期的影响；其中横坐标附图标记同图9。
图20表示不同寡糖组合物、甘露糖醛二酸六糖对双侧颈总动脉结扎致血管性痴呆小鼠避暗实验错误次数的影响；其中横坐标附图标记同图9。
图21表示不同寡糖组合物、甘露糖醛二酸六糖对双侧颈总动脉结扎致血管性痴呆小鼠水迷宫实验逃避潜伏期的影响；其中横坐标附图标记同图9。
图22表示不同寡糖组合物、甘露糖醛二酸六糖对双侧颈总动脉结扎致血管性痴呆小鼠穿越平台次数的影响；其中横坐标附图标记同图9。
具体实施方式
下文将对本发明的各个方面进行具体说明，但本发明并不限于这些具体的实施方式。本领域技术人员可以根据下文公开内容的实质对本发明进行一些修改和调整，这些调整也属于本发明的范围。
褐藻胶寡糖二酸组合物
本发明的第一个方面涉及一种褐藻胶寡糖二酸的组合物，其包含具有式(IV)的甘露糖醛酸和/或古罗糖醛酸或其药学上可接受的盐：
Figure PCTCN2019093778-appb-000005
其中n为选自1-9的整数，m选自0，1或2，m’选自0或1，
并且其中，
n＝1-5的褐藻胶寡糖二酸的重量总和占所述组合总重量的60％以上；
其中，古罗糖醛酸二酸的重量总和占所述组合物重量的50％以下。
本发明的褐藻胶寡糖二酸组合物是不同聚合度的甘露糖醛酸和古洛糖醛酸的混合物，其主要成分是由甘露糖醛酸通过β-1,4-糖苷键连接形成的M段、古罗糖醛酸通过α-1,4-糖苷键连接形成的G段，以及由这两种糖杂合形成的MG段，聚合度为2至10的寡糖。根据在先申请已知，甘露糖醛二酸具有抗阿尔茨海默病和抗糖尿病的药理活性，其中活性最高的糖为5-8糖，特别是6糖。但是，与已知的现有技术不同，发明人发现，聚合度为2至10的甘露糖醛酸和古洛糖醛酸的寡糖二酸混合物，同样具有抗阿尔茨海默病和抗糖尿病的药理活性，但是需要将古洛糖醛酸的含量控制在一定范围内。
在实际制备过程中，如前文所述将原来海藻酸初步降解后的产物中古罗糖醛酸的含量通常在30％以上，最高可达70％左右，若按照在先申请为了获得高活性的甘露寡糖二酸应将古罗糖醛酸尽可能分离去除。而基于发明人的上述发现，可以不需要将降解产物中的古罗糖醛酸分离除去。进一步地，发明人发现通过控制酸法沉淀反应的条件，将古罗糖醛酸的比例控制在一定的范围之内，获得的组合物活性能达到甚至优于在先申请所公开的甘露寡糖二酸6糖。且 因不用将古罗糖醛酸作为杂质去除，产品得率理论上也显著高于在先申请所公开的产品得率，大大降低生产成本，减少废弃物的排放，在实际生产中更容易实现，更易于实现工业化大生产。
根据一个优选的实施方案，本发明的褐藻胶寡糖二酸组合物中，n＝1-5的褐藻胶寡糖二酸的重量总和占所述组合总重量的80-95％，且古罗糖醛酸的重量总和占所述组合物重量的50％以下。
根据一个优选的实施方案，本发明的褐藻胶寡糖二酸组合物中，n＝1-3的低聚褐藻胶寡糖二酸的重量总和与n＝4-7的低聚褐藻胶寡糖二酸重量总和的比例在1.0-3.5之间。
根据一个优选的实施方案，本发明的褐藻胶寡糖二酸组合中m+m’＝1或2的褐藻胶寡糖二酸的重量总和不低于所述组合总重量的50％以上，优选60％-90％，更优选70％-90％。特别地，本发明的褐藻胶寡糖二酸组合物中m+m’＝1的褐藻胶寡糖二酸的重量总和不低于所述组合总重量的10％，优选30-40％。在另一个优选实施方案中，本发明的褐藻胶寡糖二酸组合物中m+m’＝2的褐藻胶寡糖二酸的重量总和不低于所述组合总重量的10％，优选30-50％。
根据一个优选实施方案，本发明的褐藻胶寡糖二酸组合物中n＝1-5的褐藻胶寡糖二酸的重量总和占所述组合总重量的80-95％。
根据一个优选实施方案，本发明的褐藻胶寡糖二酸组合物中n＝1-3的褐藻胶寡糖二酸的重量总和所述组合总重量的20-70％。
根据一个优选实施方案，本发明的褐藻胶寡糖二酸组合物中n＝1-3的褐藻胶寡糖二酸的重量总和与n＝4-7的褐藻胶寡糖二酸重量总和的比例在1.0-3.5之间，优选在1.0-3.0之间。
根据一个优选实施方案，本发明的褐藻胶寡糖二酸组合物中各聚合度褐藻胶寡糖二酸在所述组合中的重量百分含量为：二糖5～25％，三糖15～30％，四糖15～28％，五糖10～25％，六糖5～15％，七糖3～10％，八糖2～5％，九糖1～5％，十糖1～5％。特别地，组合中寡糖在所述组合中的重量百分含量为：二糖10～20％，三糖 18～30％，四糖15～28％，五糖15～20％，六糖5～10％，七糖3～5％，八糖2～3％，九糖1～3％，十糖1～3％。
根据一个优选实施方案，本发明的褐藻胶寡糖二酸组合物中古罗糖醛酸的重量总和占所述组合物重量的0.1-50％，优选1-30％。
本发明的褐藻胶寡糖二酸组合物中，其中所述药学上可接受的盐是钠盐或钾盐。
褐藻胶寡糖二酸组合物的制备方法
本发明的褐藻胶寡糖二酸的制备过程概括如下：
将海藻酸初步降解后可得到聚甘露糖醛酸和聚古罗糖醛酸的混合多糖，混合多糖再经酸法沉淀，可以除去其中一定量的聚古罗糖醛酸；酸法沉淀过程中pH值控制越高，获得的混合多糖中聚古罗糖醛酸的含量也越高。例如，可参见中国专利申请No.98806637.8以及CN02823707.2所披露的方法。上文所述的混合多糖在氧化剂存在下，糖链发生氧化降解得到不同聚合度的氧化型寡糖，该氧化型寡糖的特征为寡糖还原端的甘露糖醛酸或古罗糖醛酸被氧化为3-6个碳的糖二酸。
特别有利于本发明反应的氧化剂为臭氧。反应过程中，将臭氧通入含混合多糖的溶液中即可发生糖链的氧化降解反应。氧化降解步骤进行的温度优选为0-70℃，更优选10-45℃。上述氧化降解步骤进行的pH值为3-13，优选4-10，更优选6-8。
本发明中使用臭氧的氧化降解反应与现有技术中使用的碱性硫酸铜(在先专利)或者双氧水和次氯酸钠存在下的酸水解(中国专利申请01107952.5)氧化降解的相同之处为三种方法均可使得糖链降解，不同之处在于降解产物的糖链还原末端结构不同，本发明中得到的氧化降解产物甘露糖醛酸或古罗糖醛酸的还原末端包含3-6个碳的二酸结构。另外，本发明的氧化降解步骤所采用的工艺还具有其他方面的优势：1、反应条件温和，无需特殊反应条件；2、所用臭氧可以在反应现场制备，在工业生产中减少了运输的压力；3、 反应后臭氧自动分解成氧气，无反应试剂残留的危害，也不会给环境造成污染。反应过程如下方程式(VI)所示：
Figure PCTCN2019093778-appb-000006
上述反应方程式(VI)和化合物通式(IV)的示意图中，
m＝2且m’＝1的寡糖为末端6个碳的糖二酸；
m＝1且m’＝1或者m＝2且m’＝0的寡糖为末端5个碳的糖二酸；
m＝1且m’＝0或者m＝0且m’＝1的寡糖为末端4个碳的糖二酸；
m＝0且m’＝0的寡糖为末端3个碳的糖二酸。
组合物中n＝1-5的褐藻胶寡糖二酸的重量总和占所述组合总重量的80-95％，n＝1-3的褐藻胶寡糖二酸的重量总和所述组合总重量的20-70％。其中n＝1-3的褐藻胶寡糖二酸的重量总和与n＝4-7的褐藻胶寡糖二酸重量总和的比例在1.0-3.5之间，优选在1.0-3.0之间。 古罗糖醛酸的重量总和占所述组合物重量的50％以下，优选0.1-50％，最优选1-30％。
在一个示例性的实施方案中，本发明的方法包括如下几个步骤：
(1)褐藻胶寡糖二酸产品的制备：
聚甘露糖醛酸和聚古罗糖醛酸的混合多糖的制备。如前文所述，本发明中采用的原料聚甘露糖醛酸和聚古罗糖醛酸的混合多糖可以通过现有技术中已知的方法制备。例如中国专利申请No.98806637.8以及CN02823707.2所披露的方法。通常的方法可以简单描述为：将海藻酸初步降解后可得到聚甘露糖醛酸和聚古罗糖醛酸的混合多糖，混合多糖再经酸法沉淀后，可以调节其中部分聚古罗糖醛酸的含量，得到聚甘露糖醛酸和聚古罗糖醛酸的混合多糖。
臭氧氧化降解。在室温或者加热条件下使混合多糖溶解于适量的水中，搅拌，持续通入臭氧，反应开始进行。反应pH值可以通过滴加稀盐酸或者稀NaOH溶液调节至3-13之间，优选4-10，更优选6-8。温度优选为0-70℃，更优选10-45℃。反应完成以后，停止通入臭氧，调节pH至中性。
膜分离纯化。将上述所得的反应产物配成约10％浓度的溶液，通过分子截留膜分离，去除单糖以下的降解产物，收集未透过液。所采用的分子截留膜MWCO规格为1000Da-3000Da，优选2000Da。收集液经旋转蒸发仪浓缩、真空干燥即得寡聚褐藻胶寡糖的混合物。经分析发现这些产品均是以二糖-十糖的寡糖且其含量是在一定比例范围的组合物。实施例1-3示例性地示出了该方法。
(2)寡糖组合物的活性比较
将本发明的寡糖组合物与在先申请中的甘露寡糖二酸六糖同时比较药理活性，结果表明本发明的寡糖组合物的药理活性明显高于在先申请提供的甘露寡糖二酸六糖。不囿于任何理论，认为当组合物中二-六糖的比例高于60％，且古罗糖醛酸的重量总和占所述组合 物重量的50％以下时，组合物的活性最高；但古罗糖醛酸的比例超过60％时，组合物的活性也会降低。
本发明还提供了包含如上所述的褐藻胶寡糖组合以及任选的药学上可以接受的载体或赋形剂的药物或保健品。
制备各种含有各种比例活性成分的寡糖组合药物的方法是已知的，或根据本发明的公开内容对于本领域技术人员是显而易见的。如Remington’s Pharmaceutical Sciences,Martin,E.W.,ed.,Mack Publishing Company,19th ed.(1995)所述。制备所述药物组合物的方法包括掺入适当的药学赋形剂、载体、稀释剂等。
以已知的方法制造本发明的药物制剂，包括常规的混合、溶解或冻干方法。
本发明的药物组合物，并向患者以适于选定的施用方式的各种途径施用，例如口服或肠胃外(通过静脉内、肌内、局部或皮下途径)。
因此，本发明的组合药物结合药学上可以接受的载体(如惰性稀释剂或可食用的载体)可以全身施用，例如，口服。它们可以封闭在硬或软壳的明胶胶囊中，可以压为片剂。对于口服治疗施用，本发明的活性化合物可以结合一种或多种赋形剂，并以可吞咽的片剂、颊含片剂、含片、胶囊剂、酏剂、悬浮剂、糖浆、圆片等的形式使用。这种组合物和制剂应该包含至少0.1％的活性化合物。这种组合物和制剂的比例当然可以变化，可以占给定的单位剂型重量的大约1％至大约99％。在这种治疗有用的组合物中，活性化合物的量使得能够获得有效剂量水平。
片剂、含片、丸剂、胶囊剂等也可以包含：粘合剂，如黄蓍胶、阿拉伯胶、玉米淀粉或明胶；赋形剂，如磷酸氢二钙；崩解剂，如玉米淀粉、马铃薯淀粉、藻酸等；润滑剂，如硬脂酸镁；和甜味剂，如蔗糖、果糖、乳糖或阿司帕坦；或调味剂，如薄荷、冬青油或樱桃香味。当单位剂型是胶囊时，除了上面类型的材料，它还可以包含液体载体，如植物油或聚乙二醇。各种其他材料可以存在，作为 包衣，或以其他方式改变固体单位剂型的物理形式。例如，片剂、丸剂或胶囊剂可以用明胶、蜡、虫胶或糖等包衣。糖浆或酏剂可以包含活性化合物，蔗糖或果糖作为甜味剂，对羟苯甲酸甲酯或对羟苯甲酸丙酯作为防腐剂，染料和调味剂(如樱桃香料或桔子香料)。当然，用于制备任何单位剂型的任何材料应该是药学上可以接受的且以应用的量为无毒。此外，活性化合物可以掺入缓释制剂和缓释装置中。
活性化合物也可以通过输注或注射到静脉内或腹膜内施用。可以制备活性化合物或其盐的水溶液，任选的可混和的无毒的表面活性剂。也可以制备在甘油、液体聚乙二醇、甘油三乙酸酯及其混合物以及油中的分散剂。在普通的储存和使用条件下，这些制剂包含防腐剂以防止微生物生长。
适于注射或输注的药物剂型可以包括包含适于无菌的可注射或可输注的溶液或分散剂的即时制剂的活性成分(任选封装在脂质体中)的无菌水溶液或分散剂或无菌粉末。在所有情况下，最终的剂型在生产和储存条件下必须是无菌的、液体的和稳定的。液体载体可以是溶剂或液体分散介质，包括，例如水、乙醇、多元醇(例如，甘油、丙二醇、液体聚乙二醇等)、植物油、无毒的甘油酯及其合适的混合物。可以维持合适的流动性，例如，通过脂质体的形成，通过在分散剂的情况下维持所需的粒子大小，或通过表面活性剂的使用。可以通过各种抗细菌剂和抗真菌剂(如对羟苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等)产生预防微生物的作用。在许多情况下，优选包括等渗剂，如糖、缓冲剂或氯化钠。通过使用延缓吸收剂的组合物(例如，单硬脂酸铝和明胶)可以产生可注射的组合物的延长吸收。
通过将合适的溶剂中的需要量的活性化合物与需要的上面列举的各种其他成分结合，然后进行过滤灭菌，制备无菌可注射溶液。在用于制备无菌注射溶液的无菌粉末的情况下，优选的制备方法是真空干燥和冷冻干燥技术，这会产生活性成分加上任何另外需要的 以前无菌过滤溶液中存在的成分的粉末。
有用的固体载体包括粉碎的固体(如滑石、粘土、微晶纤维素、二氧化硅、氧化铝等)。有用的液体载体包括水、乙醇或乙二醇或水-乙醇/乙二醇混合物，本发明的组合药物可以任选在无毒的表面活性剂的帮助下以有效含量溶解或分散在其中。可以加入佐剂(如香味)和另外的抗微生物剂来优化对于给定用途的性质。
增稠剂(如合成的聚合物、脂肪酸、脂肪酸盐和酯、脂肪醇、改性纤维素或改性无机材料)也可和液体载体用于形成可涂覆的糊剂、凝胶、软膏、肥皂等，直接用于使用者的皮肤上。
化合物或其混合物的治疗需要量，不仅取决于化合物本身，而且取决于施药方式、待治疗的疾病的本质和患者的年龄和状态，最终取决于在场医师或临床医生的决定。
上述制剂可以以单位剂型存在，该单位剂型是含有单位剂量的物理分散单元，适于向人体和其它哺乳动物体给药。单位剂型可以是胶囊或片剂，或是很多胶囊或片剂。根据所涉及的具体治疗，活性成分的单位剂量的量可以在大约0.1到大约1000毫克或更多之间进行变化或调整。
本发明的另一个方面提供一种药物组合物或保健品，其包含本发明的褐藻胶寡糖组合物和必要时适当的载体。
本发明的再一个方面提供一种褐藻胶寡糖组合物治疗老年性痴呆的用途。
本发明的再一个方面提供一种治疗患有老年性痴呆的患者的方法，其包括给予有需要的患者有效量的本发明的褐藻胶寡糖组合物。
本发明的再一个方面提供一种褐藻胶寡糖组合物治疗帕金森病的用途。
本发明的再一个方面提供一种治疗患有帕金森病的患者的方法，其包括给予有需要的患者有效量的本发明的褐藻胶寡糖组合物。
本发明的再一个方面提供一种褐藻胶寡糖组合物治疗炎症反应的用途。
本发明的再一个方面提供一种治疗患有炎症的患者的方法，其包括给予有需要的患者有效量的本发明的褐藻胶寡糖组合物。
本发明的再一个方面提供一种褐藻胶寡糖组合物治疗疼痛反应的用途。
本发明的再一个方面提供一种治疗患有疼痛的患者的方法，其包括给予有需要的患者有效量的本发明的褐藻胶寡糖组合物。
本发明的再一个方面提供一种褐藻胶寡糖组合物治疗糖尿病的用途。
本发明的再一个方面提供一种治疗患有糖尿病的患者的方法，其包括给予有需要的患者有效量的本发明的褐藻胶寡糖组合物。
本发明的再一个方面提供一种褐藻胶寡糖组合物治疗血管性痴呆的用途。
本发明的再一个方面提供一种治疗患有血管性痴呆的患者的方法，其包括给予有需要的患者有效量的本发明的褐藻胶寡糖组合物。
本发明所述疼痛包括各种疼痛，包括但是急性疼痛、慢性疼痛、神经性疼痛、术后痛、慢性下背痛、丛集性头痛、疱疹神经痛、幻肢痛、中枢性痛、牙痛、阿片样物质抗性疼痛、内脏痛、手术痛、骨损伤痛、劳累和分娩过程中的疼痛、因灼伤包括晒伤导致的疼痛、产后痛、偏头痛、心绞痛和与泌尿生殖道相关的疼痛(包括膀胱炎)、血管性疼痛、三叉神经痛、肋间神经痛、手术切口痛、慢性筋膜炎痛、足跟痛、肌肉疼痛、骨骼疼痛、关节疼痛、癌性疼痛、非癌性疼痛等。
本发明所述炎症包括各种炎症，包括但不限于急性炎症、慢性炎症、血管炎症、神经炎症、中枢神经炎症(例如多发性硬化症，包括脑脊髓炎等)、外周神经炎症、关节炎(例如骨关节炎、骶髂关节炎等、牛皮癣关节炎、类风湿性关节炎、风湿性关节炎等)、强直性脊柱炎、炎症性肠病(例如克罗恩病和溃疡性结肠炎)、炎症性糖尿病性溃疡、系统性红斑狼疮、炎症性皮肤病(例如银屑病、特应性皮炎、湿疹)等。
本发明的褐藻胶寡糖组合物使用不同于现有技术的方法制备，不需要将M段和G段分离，大大降低了生产工艺的复杂程度，也大幅度降低了生产成本，且制备方法反应简单，有效成分含量高，无反应试剂残留。经实验证明，本发明的褐藻胶寡糖组合物具有潜在的预防和治疗阿尔茨海默病、糖尿病、帕金森病、各类炎症反应、疼痛以及血管性痴呆的作用。
动物模型及药效活性评价步骤
1、抗AD的药效评价动物模型：采用Aβ单侧脑室注射诱导AD模型，采用Morris水迷宫对AD模型大鼠的学习记忆行为进行评价。
取雄性Wistar大鼠，每只体重在180-220g之间。随机分组：假手术对照组、模型组、给药组，每组14只动物。大鼠经戊巴比妥钠(40mg/kg)腹腔注射麻醉后固定于脑立体定位仪上，常规备皮消毒，切开皮肤，暴露前囟，海马CA1区参照《大鼠脑立体定向图谱》(包新明，舒斯云，北京，人民卫生出版社，1991，28)“前囟后3.0mm，中缝旁开2.2mm，硬膜下2.8mm”进行定位。模型组及给药组分别于右侧海马CA1区用微量进样器垂直颅骨进针，缓慢注入凝聚态Aβ(Aβ1-40以PBS溶液配制成1.4mg/mL，于37℃培养箱中孵育5天使其形成聚集态)5μl，流速1μL/min，注射完成后留针5min，以使Aβ充分弥散，然后缓慢撤针。缝合手术切口，保温苏醒。对照组注入等量灭菌PBS，余步骤同前。术前7天开始给予相应药物，连续给药至实验结束。
术后第11天进行Morris水迷宫实验。
定位航行实验：各组大鼠每天训练1次，连续训练5天，即定位航行实验，记录动物找到平台所用时间(即逃避潜伏期)。约90s未找到站台者，引导其按直线方向游向平台并在平台上站立30s，诱导其学习记忆。
空间探索实验：定位航行实验结束后，间隔1天，撤去平台， 将大鼠从入水点放入水中，记录其穿越平台次数、在平台所在象限的游泳距离占总路程的百分比。评价动物的学习记忆功能。
2、抗帕金森病(PD)的药效评价动物模型
取小鼠随机分为8组：空白对照组、MPTP模型组、给药组，每组14只动物。动物分组当天开始给药，空白对照组和MPTP模型组灌胃生理盐水，其余各组均给予相应药物，每天给药1次，连续给药17天。从第6天起给予造模药物，空白对照组动物皮下给予生理盐水10ml/kg，其余动物皮下给予MPTP 25mg/kg，每天一次，共五天。
分别于实验第11、14、17天进行行为学检测。将小鼠头向上轻柔的放在粗糙的杆顶(直径8毫米，高55厘米)。小鼠从头向上调整至头完全向下的时间记录为潜伏期(T-turn)，小鼠从向下运动至四肢全部到达杆底的时间记录为爬下时间(T-LA)，超过30秒按照30秒记录。每只小鼠重复检测5次取平均值。
MPTP对脑黑质多巴胺能神经元有选择性的破坏作用，MPTP导致的PD动物模型是最为经典的类似人类帕金森病病理变化及临床特征的动物模型。PD的主要症状表现为为静止性震颤、肌张力增高、运动减少等，爬杆实验的调头时间和爬下时间可以代表小鼠的整体活动协调能力。
3、抗炎症反应的药效评价动物模型
(1)类风湿性关节炎模型—胶原诱导的小鼠关节炎模型
取雄性DBA/1小鼠，体重19-22g，随机分组：空白对照组、模型组、给药组，每组8只。除空白对照组外，其余动物于第0天尾根部皮下注射牛二型胶原-完全弗氏佐剂(CII-CFA)乳剂10mg/kg免疫致敏，第23天，腹腔注射脂多糖(LPS)1.5mg/kg。第28天开始给药，空白对照组和模型组口服生理盐水，其余各组均给予相应药物，每天给药1次，连续给药14天。LPS注射后，每天观察小鼠发病情况。当小鼠开始发病之后(出现关节炎的临床症状)，根据病 变的不同程度(红肿，关节变形)按照0-4分的标准进行临床评分来反应疾病进展程度。(0分为无红斑和红肿；1分为近跗骨附近或踝关节或跖骨出现红斑或轻度红肿，1个脚趾红肿；2分为踝关节和跖骨轻微红斑和肿胀，或超过两个脚趾红肿；3分为踝、腕关节和跖骨中度红斑和肿胀；4分为踝、腕关节，跖骨和脚趾全部严重红肿；每个肢体的最高评分为4分，每只动物最高评分为16分)。
(2)多发性硬化症模型—MOG诱导的小鼠多发性硬化症模型
取雌性C57BL/6小鼠，体重17-20g，随机选出5只作为空白对照组。其余动物于第0天背部皮下注射髓鞘少突胶质细胞糖蛋白-完全弗氏佐剂(MOG-CFA)乳剂免疫致敏，MOG 10mg/kg，CFA 20mg/kg，并于第0天和第2天腹腔注射百日咳毒素，10ug/kg。并于第1天开始给药，空白对照组和模型组口服生理盐水，其余各组均给予相应药物，每天给药1次，连续给药24天。免疫后第12天左右，免疫的老鼠会出现病症，开始每天密切观察记录体重和临床评分(以0-4分表示不同程度，0分为正常表现，没有明显的疾病征兆；1分为尾巴下垂无力，后肢单侧无力；2分为尾巴下垂无力，双后肢均无力步态蹒跚；3分为单侧后肢无力麻痹瘫痪；4分为双后肢均无力麻痹瘫痪)，来反应疾病进展程度。
(3)系统性红斑狼疮模型—MRL/lpr红斑狼疮小鼠模型
MRL/lpr转基因小鼠，具有Faslpr基因的纯合子突变，可以自发形成淋巴组织增生，小鼠在第10-14周龄左右开始发病，出现系统性红斑狼疮症状。雌性MRL/lpr转基因小鼠，9周龄，随机分组：空白对照组、给药组，每组8只。空白对照组口服生理盐水，其余各组均给予相应药物，每天给药1次，连续给药4周。每周进行一次淋巴结评分(以0-6分表示不同程度，0分为正常；1分为在一个两侧点位置直径小于1cm；2分为在两个两侧点位置直径小于1cm；3分为在三个两侧点位置直径小于1cm；4分为在一个两侧点位置直径大于1cm，另外两个两侧点位置直径小于1cm；5分为在两个两侧点位置 直径大于1cm，另外一个两侧点位置直径小于1cm；6分为在三个两侧点位置直径大于1cm)。
(4)炎症性肠病(IBD)模型-葡聚糖硫酸钠(dextran sulfate sodium，DSS)诱导小鼠结肠炎模型
取雌性C57小鼠，7-8周龄，体重18-20g，随机分组：空白对照组、模型组、给药组，每组8只。模型组、给药组小鼠于第1-7天以饮水方式给予2.5％的高分子量聚合物葡聚糖硫酸钠(dextran sulfate sodium，DSS)造模，并于第1天开始给药，空白对照组和模型组口服生理盐水，其余各组均给予相应药物，每天给药1次，连续给药30天。第31天，小鼠颈椎脱臼处死，打开腹腔，分离肠系膜。取每只小鼠回盲部起始端到肛门末端，每组顺次取样，计量结肠长度。
4、抗糖尿病的药效评价动物模型
取雄性NIH小鼠，随机为正常对照组、模型组、给药组，每组10只。试验当天，除正常组外，其余动物均腹腔注射链脲霉素150mg/kg。连续给予相应药物10天，第11天摘眼球取血，测血糖浓度。
5、抗疼痛的药效评价动物模型
(1)醋酸致小鼠疼痛模型
昆明种小鼠，雌雄各半，体重18-22g，随机分组：空白对照组、模型组、给药组，每组10只。从分组当天起，空白对照组每天灌胃20ml/kg蒸馏水,其余各组灌胃给予相应药物，每天给药1次，连续给药7天。于末次给药后1小时，各组小鼠均腹腔注射0.6％醋酸溶液0.2ml，记录注射醋酸后20分钟内小鼠的扭体潜伏期(即从注射醋酸至扭体反应发生的时间)和扭体次数。
醋酸溶液等化学物质注射到小鼠腹腔，可刺激小鼠腹膜，引起间歇发作的持久性疼痛，表现为腹部收内凹、腹前壁紧贴笼底、臀部歪扭和后肢伸张，呈一种特殊姿势，称为扭体反应。扭体潜伏期(即 从注射醋酸至扭体反应发生的时间)和和一定时间内的扭体次数，可以代表疼痛的严重程度。扭体潜伏期越短，扭体次数越多，代表疼痛程度越严重。
(2)硝酸甘油致大鼠偏头痛模型
SD雄性大鼠，体重180-220g，随机分组：空白对照组、模型组、给药组，每组8只。于分组当天开始给药，空白对照组和模型组灌胃给予蒸馏水，其余各组给予相应药物，每天给药1次，连续给药28天，末次药后30分钟，除空白对照组动物予生理盐水外，其余各组均在右肩皮下注射硝酸甘油10mg/kg造模。观察大鼠造模后耳红出现时间和持续时间，以及造模后30-45分钟时间段中的挠头次数；采用荧光分光光度法测定脑组织中5-HT的含量。于Ex356nm/Em 483nm波长处测定,结果以ng/g脑重表示。
偏头痛是血管与神经机制相互作用下的一种血管、神经的功能紊乱疾病。硝酸甘油可以通过扩张脑膜血管、形成神经源性炎症及激活下丘脑、脑干及脊髓节段神经元功能等作用,引起三叉神经纤维的超敏性，造成偏头痛。硝酸甘油模型是1995年建立的一种动物模型，目前已成为经典的动物偏头痛模型。根据硝酸甘油致病机理，检测模型动物由于血管扩张引起的耳红时间、由于疼痛引起的挠头次数及脑组织致疼敏感因子5-羟色胺(5-HT)含量，可以评估偏头痛的严重程度。耳红时间持续越长、挠头次数越多、5-HT含量越高，代表偏头痛程度越严重。
(3)电刺激三叉神经节致偏头痛大鼠模型
SD大鼠，5月龄，雄性，体重200～240g，随机分组：空白对照组、假手术组、模型组、给药组，每组10只。
各组均口服给予相应药物，空白对照组、假手术组、模型组口服蒸馏水。连续给药10天后，除空白对照组外，所有大鼠经水合氯醛350mg/kg腹腔注射麻醉后，将大鼠固定在立体定位仪上，头顶正中切口，逐层切开皮肤、肌肉，于脑矢状缝中部开口暴露颅盖骨。在前囱后移3毫米，旁开3毫米处，用牙科钻插孔，然后将电极插 入三叉神经节(以硬脑膜算起深度为9.5mm)，术后持续麻醉。所有操作均在无菌条件下进行。调试好刺激电极，电刺激参数为周期200ms，幅度10v，波宽5ms，刺激10分钟。假手术组动物只插入电极，不给刺激。刺激前7分钟右股静脉注射50mg/kg伊文氏蓝，刺激结束20分钟内灌注固定。
刺激结束后5分钟，左心室灌流2分钟，开颅，取全脑，固定，待做病理切片免疫组化测定c-fos；另确定电极位置，分离电极插入处及另一脑半球对应位置脑硬膜，去离子水洗涤后平铺在载玻片上，37度干燥15分钟，70％甘油固定。共聚焦显微镜上以647nm激发波长，680nm发射波长检测刺激侧及对照侧指定区域的荧光强度，计算刺激侧/对照侧荧光强度的比值，用以指示血浆蛋白渗出率(Plasma Protein Extravasation，PPE)。全脑连续冰冻冠状切片，片厚10μm，免疫组化荧光标记c-fos阳性细胞。共聚焦显微镜下，随机选择5个视野，测定三叉神经脊束核尾侧实验侧及对照侧阳性细胞数目，再取5个视野的平均值，则为平均阳性细胞数。
三叉神经血管系统的激活是偏头痛患者疼痛产生的关键环节，其中脑膜的神经性炎症的发生在偏头痛的疼痛产生和维持中起着重要的作用。当分布于硬脑膜的三叉神经受到刺激时，释放血管活性物质，使脑膜血管扩张、血浆成分外渗、肥大细胞脱颗粒和血小板激活，产生偏头痛。另外，疼痛刺激后释放的神经递质与细胞膜上的相应受体结合，在第二信使作用下，C-fos mRNA基因表达，在细胞核翻译合成c-fos蛋白，产生对机体长时程的生理效应。故偏头痛时，三叉神经脊束核和中缝大核的C-fos mRNA和c-fos蛋白表达的细胞数增多。因此，通过测定偏头痛动物硬脑膜血清蛋白渗出量及三叉神经脊束核尾侧c-fos阳性细胞数量可以反映偏头痛的程度。细胞数量越低代表偏头痛的程度越轻。
6、抗血管性痴呆的药效评价动物模型
(1)双侧颈总动脉结扎(BCCAo)致血管性痴呆小鼠模型
双侧颈总动脉结扎(BCCAo)模型是经全脑缺血再灌注建立的 本领域常用的血管性痴呆模型。
1.1动物分组与给药
取雄性C57BL/6小鼠，体重22±2g，随机分组：假手术组、30分钟双侧颈总动脉结扎(Bilateral Common Carotid Artery occlusion，BCCAo)模型组(简称30min BCCAo组)、给药组，每组10只。动物分组后，假手术组和30min BCCAo组小鼠灌胃蒸馏水，每天灌胃1次，连续灌胃5天后，进行BCCAo手术。给药组小鼠分别灌胃给予相应药物，每天灌胃给药1次，连续给药5天后，进行BCCAo手术。所述BCCAo手术是先以戊巴比妥钠麻醉各组小鼠；将模型组、给药组中小鼠双侧颈总动脉分离并结扎30分钟，然后去除结扎并缝合颈部伤口；将假手术组中小鼠双侧颈总动脉分离后不结扎，直接缝合颈部切口。BCCAo后24小时，各组小鼠按所述术前给药方案分别继续灌胃给予相应药物或蒸馏水，再各自连续给药23天。BCCAo后第7天进行避暗实验的测试，第13天开始Morris水迷宫测试，评价甘露糖醛二酸组合物对于小鼠学习记忆能力的改善作用。行为学测试结束后，处死小鼠，脑组织固定，通过HE染色等方法，评价小鼠经BCCAo后海马区神经细胞损伤情况及甘露糖醛二酸组合物对于受损神经元的保护作用。
1.2避暗实验测试
避暗实验用于检测小鼠空间辨别的学习记忆能力，空间定位的记忆障碍只有在海马或海马周围区域受到损伤的情况下才会出现。避暗实验箱是利用小鼠具有趋暗避明的习性设计的装置，一半是暗室，一半是明室，中间有一小洞相连，暗室底部铺有通电的铜栅，动物进入暗室即受到电击而逃回明室。将动物训练24小时后，再进行测试，从动物放入明室至首次进入暗室的时间即为避暗实验潜伏期。避暗实验潜伏期越长，逃避错误次数越少，表明动物记忆力越好。
1.3 Morris水迷宫行为测定
Morris水迷宫(Morris water maze,MWM)实验是一种强迫实验 动物游泳，学习寻找隐藏在水中平台的一种实验，主要用于测试实验动物对空间位置感和方向感(空间定位)的学习记忆能力。小鼠Morris水迷宫主要由一直径80cm、高70cm的圆柱型水池和一直径8cm的可移动位置的平台组成，水池上空通过一个数字摄相机与计算机相连接。实验前预先在水池中注入清水，水深15cm，水面高出平台表面0.5cm，加入牛奶使池水变为不透明，实验过程保持平台位置不变。Morris水迷宫行为包括如下两项测试指标。
定位航行实验(place navigation)，用于测量小鼠对水迷宫学习和记忆的获取能力。实验自BCCAo后第13天开始，历时4天，上、下午各训练小鼠1次，共计8次。训练时小鼠入池位置为西象限1/2弧度处，头朝池壁入水。120秒内未找到站台者，实验人员将其引至站台，放置30秒，引导其学习和记忆。实验观察和记录小鼠寻找并爬上平台的路线图及所需时间，即记录其Morris水迷宫实验逃避潜伏期和游泳速度。所述Morris水迷宫实验逃避潜伏期是指从小鼠入水至找到站台的时间。Morris水迷宫实验逃避潜伏期越短，表明动物记忆力越好。
空间搜索实验(spatial probe test)，用于测量小鼠学会寻找平台后，对平台空间位置记忆的保持能力。定位航行实验结束后，间隔1天，撤去平台，将小鼠从同一个入水点放入水中，测其穿越原平台的次数。数据采集和处理由图像自动监视和处理系统完成。
(2)大脑中动脉结扎(MCAO)致血管性痴呆大鼠模型
大脑中动脉结扎(MCAO)模型是经局灶性脑缺血建立的本领域常用的血管性痴呆模型。
2.1动物分组与给药
取雄性Wistar大鼠，随机分组：空白对照组、假手术组、模型组(简称MCAO组)、给药组，每组10只。空白组、假手术组、MCAO组动物口服给予蒸馏水，褐藻胶寡糖组均口服给予所述相应剂量的褐藻胶寡糖。各组连续给药7天后，除空白组大鼠外，其余组中大鼠经水合氯醛350mg/kg腹腔注射麻醉后，左侧卧位固定于 鼠板上，于手术显微镜下沿外耳道与眼眦连线中点切开皮肤，暴露出颧弓，用小牵张器将磷状骨和下颌骨间距撑开，于颅骨底开一2mm×2mm的骨窗，撕开硬脑膜，暴露出大脑中动脉，用高频电刀电凝阻断一侧大脑中动脉造成局部脑缺血(假手术组动物仅暴露大脑中动脉，不进行电凝阻断)，逐层缝合切口。术中术后室温严格控制在24～25℃。术后各组继续依照各自术前给药方案给药或给予蒸馏水，术后第11天各组均进行Morris水迷宫实验。
在该实验中，各组大鼠每天训练1次，连续训练5天，即定位航行实验，记录动物找到平台所用时间(即Morris水迷宫实验逃避潜伏期)。约120秒未找到站台者，引导其按直线方向游向平台并在平台上站立30秒，诱导其学习记忆。定位航行实验结束后，间隔1天，撤去平台，将大鼠从入水点放入水中，记录第一次到达原平台时间以及穿越原平台的次数，即空间搜索实验。评价动物的学习记忆功能。所述Morris水迷宫实验逃避潜伏期是指从大鼠入水至找到平台的时间。Morris水迷宫实验逃避潜伏期越短，表明动物记忆力越好。
本发明的优点在以下非限制性的实施例中进一步进行说明。但实施例中采用的具体材料及其用量，以及其他实验条件并不应理解为对本发明的限制。除非特别指明，本发明中份数、比例、百分比等均以质量计。
实施例1：
步骤1)：褐藻胶寡糖混合物的制备
将5Kg海藻酸钠配成约10％的溶液，加稀盐酸调pH至3.0左右，升温至80℃，搅拌，反应10hr，停止加热，冷却至室温后，加NaOH调pH值至9.0，再加稀盐酸回调pH至3.2，离心机5000rpm离心10min，收集上清，旋转蒸发仪浓缩，真空干燥得中间体1500g。
中间体的核磁图谱见附图1。核磁共振测定方法如下：样品准备： 称取30mg待测样品溶于0.5ml D2O，冷冻干燥，再加0.5ml氘代重水溶解，重新冻干，最后把冻干样品粉末用适量重水溶解后，全部转移至核磁管中，配成100mg/ml的待测溶液，加入0.01％(w/v)的氘代TSP(trimethylsilylpropionic)钠盐做内标。核磁数据采集和处理：400M傅里叶变换核磁共振仪在60℃采集一维氢谱。脉冲序列为45°脉冲，每次采集4秒，弛豫时间1秒，累积20次，谱宽从-2ppm到10ppm。数据收集后采用傅里叶变换得到一维氢谱，将TSP甲基氢信号设定为0.00ppm。
由图1可知，该中间体含有甘露糖醛酸片段(M-block，化学位移5.1ppm)和古罗糖醛酸片段(G-block，化学位移5.5ppm)以及甘露糖醛酸和古罗糖醛酸镶嵌片段(MG-block，化学位移5.3ppm)。称取500g该中间体，加蒸馏水溶解后，配成5L体积的溶液，NaOH调pH至6.5，水浴加热，控制反应温度到75℃。调节氧气钢瓶出口的气流量和臭氧发生器的功率，使得臭氧质量浓度流量达到8g/hr，通入反应液中。反应4hr后停止通入臭氧，加适量水调整溶液浓度至10％左右，以截留分子量为2000Da的超滤膜过滤，收集未透过液，旋转蒸发仪浓缩，真空干燥，得350g产品A。
步骤2)：褐藻胶寡糖二酸产品A中各聚合度寡糖的比例和结构分析
精密称取100mg上述干燥的褐藻胶寡糖二酸产品A，加水溶解配制成10mg/mL的浓度，过0.22um滤膜，做为供试样品溶液。采用Superdex peptide(GE公司)分子排阻色谱联用多角度激光散射(MALS，怀雅特公司)测定组合物中不同聚合度寡糖的比例。实验方法如下：
色谱柱：Superdex peptide 10/300Gl
流动相：0.1mol/L NaCl
进样量：10uL
流速：0.3mL/min
测试结果：二糖-十糖分别以dp2-dp10表示，分别为dp2为18％， dp3为24％，dp4为23％，dp5为14％，dp6为8％，dp7为7％，dp8为2％，dp9为2％，dp10为2％。
步骤3)：LC-MS分析褐藻胶寡糖二酸产品A中各聚合度寡糖的结构
实验条件：
色谱柱：Superdex peptide 10/300Gl
流动相：20％甲醇+80％80mmol/L NH4Ac
流速：0.1mL/min
柱温：25℃±0.8℃。
质谱条件：Agilent 6540 QTOF；离子源：ESI碰撞电压120V；负离子模式。采集信号(m/z)宽度为100-1000。
各聚合度寡糖的质谱图见附图1-3所示。对质谱图中各信号峰进行归属，验证了产品A中所有寡糖的分子结构，即通式(Ⅲ)所示的结构。信号归属及该信号所对应的结构见下表1。
Figure PCTCN2019093778-appb-000007
由上述质谱结构解析发现，产品A中糖链还原末端的甘露糖醛酸或古罗糖醛酸氧化为糖二酸结构(结构见通式IV)，该糖二酸可以是含6个碳(m+m’＝3)的甘露糖二酸或古罗糖二酸结构，含量约为10％～30％，也可以是甘露糖二酸或古罗糖二酸的脱羧产物，即5个碳(m+m’＝2)的糖二酸(30～50％)和4个碳(m+m’＝1)的糖二酸(30％～40％)。
步骤4)核磁分析褐藻胶寡糖二酸产品A中古罗糖醛酸含量
样品准备：称取50mg待测样品溶于0.5ml D2O，冷冻干燥，再加0.5ml氘代重水溶解，重新冻干，最后把冻干样品粉末用适量重水溶解后，全部转移至核磁管中，配成100mg/ml的待测溶液，加入0.01％(w/v)的氘代TSP(trimethylsilylpropionic)钠盐做内标。
核磁数据采集和处理：400M傅里叶变换核磁共振仪在室温采集一维氢谱。脉冲序列为45°脉冲，每次采集4秒，弛豫时间1秒，累积20次，谱宽从-2ppm到10ppm。数据收集后采用傅里叶变换得到一维氢谱，将TSP甲基氢信号设定为0.00ppm。产品A的核磁共振氢谱如图5所示。附图5中，化学位移为4.6ppm的多重峰为甘露糖醛酸(M)C-1位的氢信号，5.0ppm为古罗糖醛酸(G)C-1位的氢信号，4.9ppm为甘露糖醛酸和古罗糖醛酸嵌合片段(MG)的C-1位氢信号。古罗糖醛酸的含量计算公式为：
Figure PCTCN2019093778-appb-000008
上式中，I4.6，I5.0和I4.9分别为甘露糖醛酸(M)，古罗糖醛酸(G)，甘露糖醛酸和古罗糖醛酸嵌合片段(MG)的C-1位氢信号积分值。通过计算，A的古罗糖醛酸含量为30％。
实施例2：
称取100g的市售海藻酸钠(国药试剂网采购，CAS号9005-38-3，规格CP，沪试)，加蒸馏水混匀，溶胀后，配成1L体积的溶液，NaOH调pH至4.0，室温25℃反应。调节氧气钢瓶出口的气流量和臭氧发生器的功率，使得臭氧质量浓度流量达到1g/hr，通入反应液 中。反应10hr后停止通入臭氧，加适量水调整溶液浓度至15％左右，以截留分子量为1000Da的超滤膜过滤，收集未透过液，旋转蒸发仪浓缩，真空干燥，得80g产品B。
采用Superdex peptide(GE公司)分子排阻色谱联用多角度激光散射(MALS，怀雅特公司)测定B中各聚合度寡糖组分的比例。测定方法同实施例1中相关部分。测试结果：二糖-十糖分别以dp2-dp10表示，分别为dp2为25％，dp3为24％，dp4为18％，dp5为13％，dp6为10％，dp7为5％，dp8为2％，dp9为2％，dp10为1％。
采用400M傅里叶变换核磁共振仪在60℃测定产品B中古罗糖醛酸含量为50％，测定方法同实施例1中相关部分，核磁共振氢谱图见附图6。由图可知，甘露糖醛酸(M，化学位移值4.6ppm)和古罗糖醛酸(G，化学位移值5.0ppm)的积分面积比较接近，而甘露糖醛酸和古罗糖醛酸的镶嵌片段(MG，化学位移为4.9ppm)的积分面积较小。古罗糖醛酸产物(G)的含量计算公式根据实施例1中所示，G的含量为50％。
实施例3：
称取100g实施例1中的中间体，加水混悬后加NaOH调pH值至碱性，使粉末完全溶解，最终配成1L的溶液，再加HCl调pH值至2.95，出现部分白色沉淀，离心去除沉淀，收集上清，再加蒸馏水进一步稀释，配成1.5L体积的溶液，NaOH调pH至9.0，水浴45℃反应。调节氧气钢瓶出口的气流量和臭氧发生器的功率，使得臭氧质量浓度流量达到3g/hr，通入反应液中。反应2hr后停止通入臭氧，加适量水调整溶液浓度至5％左右，以截留分子量为3000Da的超滤膜过滤，收集未透过液，旋转蒸发仪浓缩，真空干燥，得60g产品C。
采用Superdex peptide(GE公司)分子排阻色谱联用多角度激光 散射(MALS，怀雅特公司)测定C中各聚合度寡糖组分的比例。测定方法同实施例1中相关部分。测试结果：二糖-十糖分别以dp2-dp10表示，分别为dp2为9％，dp3为21％，dp4为27％，dp5为18％，dp6为13％，dp7为6％，dp8为3％，dp9为2％，dp10为1％。
采用400M傅里叶变换核磁共振仪在60℃测定产品C中古罗糖醛酸含量为10％，测定方法同实施例1中相关部分。测试结果见附图7。通过对相应信号分别积分，甘露糖醛酸(M，化学位移值4.6ppm)是古罗糖醛酸(G，化学位移值5.0ppm)的积分面积13倍，甘露糖醛酸和古罗糖醛酸的镶嵌片段(MG，化学位移4.9ppm)的积分面积和古罗糖醛酸的积分面积接近。古罗糖醛酸产物(G)的含量计算公式根据实施例1中所示，G的含量为10％。
实施例4
褐藻胶寡糖二酸组合物与甘露糖醛二酸六糖之间的药理活性评价。
样品准备：
1、甘露糖醛二酸六糖制备
参照在先专利200580009396.5实施例一和二公开的方法制备获得甘露糖醛二酸六糖20g。
本申请前述实施例1、2、3中分别制备得到的产品A、B、C的寡糖比例及古罗糖醛酸含量如下表2所示。
2、产品D制备
参照前述实施例2的制备方法制备高G含量的产品。海藻酸钠原料由青岛海之林生物科技开发有限公司提供的高G含量的样品，制备方法同实施例2相应部分。具体为：500g高G含量的海藻酸钠粉末，加馏水混匀，溶胀后，配成5L体积的溶液，NaOH调pH至4.0，室温25℃反应。调节氧气钢瓶出口的气流量和臭氧发生器的功率，使得臭氧质量浓度流量达到1g/hr，通入反应液中。反应12hr 后停止通入臭氧，加适量水调整溶液浓度至15％左右，以截留分子量为1000Da的超滤膜过滤，收集未透过液，旋转蒸发仪浓缩，真空干燥，得350g产品D。
采用Superdex peptide(GE公司)分子排阻色谱联用多角度激光散射(MALS，怀雅特公司)测定D中各聚合度寡糖组分的比例。测定方法同实施例1中相关部分。测试结果：二糖-十糖分别以dp2-dp10表示，分别为dp2为18％，dp3为26％，dp4为20％，dp5为15％，dp6为8％，dp7为7％，dp8为3％，dp9为2％，dp10为1％。
采用400M傅里叶变换核磁共振仪在60℃测定产品D中古罗糖醛酸含量为60％，测定方法同实施例1中相关部分，核磁共振氢谱图见附图8。由图可知，古罗糖醛酸(G，化学位移值5.0ppm)的积分面积要大于甘露糖醛酸(M，化学位移值4.6ppm)的积分面积，而甘露糖醛酸和古罗糖醛酸的镶嵌片段(MG，化学位移为4.9ppm)的积分面积较小。古罗糖醛酸(G)的含量计算公式根据实施例1中所示，G的含量为60％。
表2褐藻胶二酸寡糖组合物产品中的寡糖百分比及古罗糖醛酸含量
Figure PCTCN2019093778-appb-000009
以上A、B、C三个样品及甘露糖醛二酸六糖样品各取10g，按照“抗AD的药效评价动物模型”、“抗PD的药效评价动物模型”、“抗炎症反应的药效评价动物模型”、“抗糖尿病的药效评价动物模型”、“抗疼痛的药效评价动物模型”以及“抗血管性痴呆的药效评价动物模 型”所描述的方法，比较这些褐藻胶寡糖二酸组合物与甘露糖醛二酸六糖的药理活性。
1、抗AD的药效评价
实验中，模型组与假手术对照组相比，前者寻找平台潜伏期明显延长，说明该评价模型造模成功。与模型组相比，各个给药组寻找平台潜伏期明显缩短。
在结束定位航行训练后，休息一天。然后撤去平台，开展空间探索试验，观测动物穿越平台次数、在原平台所在象限的游泳距离占总路程的百分比，评价动物的记忆功能。结果发现模型组与假手术对照组相比，穿越平台次数明显减少，且给药组穿越平台次数明显增加，见附图9。在原平台所在象限的游泳距离占总路程的百分比这一指标与穿越平台次数具有相似的趋势。模型组与假手术对照组相比，在原平台所在象限的游泳距离占总路程的百分比明显减少，且给药组在原平台所在象限的游泳距离占总路程的百分比明显增加，见附图10。
实验结果表明，产品A、B、C各自的药理活性比甘露糖醛二酸六糖活性更强，说明含有一定量古罗糖醛酸的寡糖组合物，且组合物中二-六糖的比例高于60％时有增效作用。但古罗糖醛酸的含量较高的寡糖组合物D的活性降低。
2、抗PD的药效评价
实验中，模型组与空白对照组相比，前者潜伏期和爬下时间明显延长。与模型组相比，各给药组潜伏期和爬下时间有不同程度缩短。其中产品A、B、C的药效活性均好于之前预期的活性最高的单一聚合度的甘露糖醛二酸六糖，但产品D的活性弱于甘露糖醛二酸六糖。不囿于任何理论，推测组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的比例过高时则会降低组合物的活性。见附图11和12。
3、抗炎症反应的药效评价
(1)胶原诱导的小鼠关节炎模型
实验中，模型组与正常对照组相比，出现明显的关节炎症状，踝、腕关节和跖骨中度红斑和肿胀。临床评分达到6分，说明该关节炎模型造模成功。与模型组相比，各个给药组发病程度均有不同程度的减轻。产品A、B、C使得小鼠发病时间较单一聚合度的甘露糖醛二酸六糖明显延迟，临床评分也低于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D发病时间较早，临床评分较高，反映出产品D的活性弱于甘露糖醛二酸六糖。说明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。
(2)MOG诱导的小鼠多发性硬化症模型
实验中，模型组与正常对照组相比，大部分小鼠出现双后肢均无力麻痹瘫痪症状，模型组平均临床评分达到3分，说明该多发性硬化症模型造模成功。与模型组相比，各给药组的炎症进展均有不同程度的减轻。产品A、B、C在整个实验过程中以及终点时的临床评分均低于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；而产品D在整个实验过程中以及终点时的临床评分略高,反映出产品D的抗炎活性最弱,说明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。
(3)MRL/lpr红斑狼疮小鼠模型
从第10周开始，转基因小鼠开始发病，出现淋巴结肿大情况，且淋巴结评分随着时间进展不断增大，说明模型组已经成功发病，且疾病进展迅速。与模型组相比，各给药组的发病进展均有不同程度的减轻。产品A、B、C使得小鼠发病时间较甘露糖醛二酸六糖明显延迟，淋巴结评分也低于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D发病时间较早，淋巴结评分较高，反映出产品D的活性弱于甘露糖醛二酸六糖。说明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著 影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。
(4)葡聚糖硫酸钠(dextran sulfate sodium，DSS)诱导小鼠结肠炎模型
实验结束后，模型组与正常对照组相比，结肠出现由于炎症造成的明显缩短，大部分小鼠体重明显下降，近半数模型组动物后期出现死亡，说明肠部炎症非常严重。与模型组相比，各给药组的肠部炎症均有不同程度的减轻，反映在结肠长度恢复，存活率提高上。从附图13a和13b可以看出产品A、B、C使得小鼠结肠长度大于甘露糖醛二酸六糖，动物存活率也高于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D结肠长度较小，存活率也略低于甘露糖醛二酸六糖，反映出产品D的活性弱于甘露糖醛二酸六糖。同样，实验结果与前述实验一致，表明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。
4、抗糖尿病药效评价
实验中，模型组与正常对照组相比较，前者餐后血糖明显升高，说明该评价模型造模成功。与模型组相比，各个给药组餐后血糖明显降低，其中产品A、B、C的药效活性均好于甘露糖醛二酸六糖，但产品D的活性弱于甘露糖醛二酸六糖。实验结果与前述实验一致，表明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。见附图14。
5、抗疼痛药效评价
(1)醋酸致小鼠疼痛模型
实验中，模型组与空白对照组相比，前者扭体潜伏期明显缩短，扭体次数明显增加，说明该评价模型造模成功。与模型组相比，各给药组扭体潜伏期延长、扭体次数减少。其中，产品A、B、C使得小鼠扭体潜伏期大于甘露糖醛二酸六糖，扭体次数少于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖； 但产品D扭体潜伏期较短，扭体次数略高于甘露糖醛二酸六糖，反映出产品D的活性弱于甘露糖醛二酸六糖。同样，实验结果与前述实验一致，表明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。见附图15和16。
(2)硝酸甘油致大鼠偏头痛模型
硝酸甘油皮下注射后3分钟左右，大鼠出现耳红，耳红持续2.5小时左右；模型组与空白组相比，造模后30-45分钟时间段中的挠头次数显著多于空白对照组；而给药组与模型组相比，大鼠耳红出现时显著延迟，耳红持续时间缩短，在30-45分钟时间段中的挠头次数减少。其中产品A、B、C使得大鼠挠头次数少于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D组大鼠挠头次数略高于甘露糖醛二酸六糖，反映出产品D的活性弱于甘露糖醛二酸六糖。同样，实验结果与前述实验一致，表明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。见附图17。
(3)电刺激三叉神经节致偏头痛模型
电刺激大鼠三叉神经节明显导致硬脑膜血清蛋白渗出，模型组与空白对照组和假手术组相比，模型组PPE率明显升高，c-fos表达阳性细胞数明显增加；而给药组与模型组相比，PPE率明显降低，c-fos表达阳性细胞数明显减少。其中产品A、B、C组c-fos表达阳性细胞数少于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D组c-fos表达阳性细胞数略高于甘露糖醛二酸六糖，反映出产品D的活性弱于甘露糖醛二酸六糖。同样，实验结果与前述实验一致，表明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。见附图18。
6、抗血管性痴呆药效评价
(1)双侧颈总动脉结扎(BCCAo)致血管性痴呆小鼠模型
1.1避暗实验测试结果
实验中，模型组与假手术对照组相比较，前者避暗实验潜伏期明显缩短，错误次数明显增加，说明模型组小鼠记忆能力明显下降，该评价模型造模成功。与模型组相比，各个给药组避暗实验潜伏期明显增加，错误次数明显减少。其中产品A、B、C组小鼠潜伏期长于甘露糖醛二酸六糖，错误次数也少于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D组潜伏期略短于甘露糖醛二酸六糖，错误次数也略高，反映出产品D的活性弱于甘露糖醛二酸六糖。同样，实验结果与前述实验一致，表明组合物中古罗糖醛酸的含量及二-六糖的比例对于产品的活性有显著影响，但当古罗糖醛酸的含量过高时则会降低组合物的活性。见附图19和20。
1.2 Morris水迷宫测试结果
实验中，模型组与假手术组相比，小鼠的Morris水迷宫实验逃避潜伏期明显延长，说明BCCAo致小鼠血管性痴呆模型建立成功。与模型组相比，各个给药组逃避潜伏期明显缩短。其中产品A、B、C组小鼠逃避潜伏期短于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D组潜伏期略高于甘露糖醛二酸六糖，反映出产品D的活性弱于甘露糖醛二酸六糖。见附图21。
水迷宫定位航行实验4天后，撤去平台进行空间探索实验，观测动物穿越平台次数。模型组与假手术组相比，小鼠穿越原平台的次数明显减少，说明BCCAo小鼠记忆能力明显下降，而各给药组小鼠穿越原平台的次数增加。其中产品A、B、C组小鼠穿越平台的次数高于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D组穿越平台的次数略低于甘露糖醛二酸六糖，反映出产品D的活性弱于甘露糖醛二酸六糖。见附图22。
(2)大脑中动脉结扎(MCAO)致血管性痴呆大鼠的作用
实验中，模型组与假手术组相比，大鼠的Morris水迷宫实验逃避潜伏期明显延长，说明MCAO致大鼠血管性痴呆模型建立成功。与模型组相比，各个给药组逃避潜伏期明显缩短。其中，产品A、B、C组大鼠逃避潜伏期短于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D组逃避潜伏期略长于甘露糖醛二酸六糖，反映出产品D的活性弱于甘露糖醛二酸六糖。
定位航行实验结束后，间隔1天，进行空间探索实验，观测动物2分钟内穿越平台次数。模型组与假手术组相比，大鼠穿越原平台的次数明显减少，说明MCAO组大鼠记忆能力明显下降，而各给药组小鼠穿越原平台的次数增加。其中产品A、B、C组大鼠穿越平台的次数高于甘露糖醛二酸六糖，说明产品A、B、C的药效活性均好于甘露糖醛二酸六糖；但产品D组穿越平台的次数略低于甘露糖醛二酸六糖，反映出产品D的活性弱于甘露糖醛二酸六糖。

Claims (16)

  1. 一种褐藻胶寡糖二酸组合物，其包含具有式(IV)的甘露糖醛酸和/或古罗糖醛酸或其药学上可接受的盐：
    Figure PCTCN2019093778-appb-100001
    其中n为选自1-9的整数，m选自0，1或2，m’选自0或1，
    并且其中，
    n＝1-5的褐藻胶寡糖二酸的重量总和占所述组合总重量的60％以上；
    其中，古罗糖醛酸的重量总和占所述组合物重量的50％以下。
  2. 根据权利要求1的褐藻胶寡糖二酸组合物，其中m+m’＝1或2的褐藻胶寡糖二酸的重量总和不低于所述组合总重量的50％以上，优选60％-90％，更优选70％-90％。
  3. 根据权利要求2的褐藻胶寡糖二酸组合物，其中m+m’＝1的褐藻胶寡糖二酸的重量总和不低于所述组合总重量的10％，优选30-40％。
  4. 根据权利要求1的褐藻胶寡糖二酸组合物，其中m+m’＝2的褐藻胶寡糖二酸的重量总和不低于所述组合总重量的10％，优选30-50％。
  5. 根据权利要求1的褐藻胶寡糖二酸组合物，其中n＝1-5的褐藻胶寡糖二酸的重量总和占所述组合总重量的80-95％。
  6. 根据权利要求1的褐藻胶寡糖二酸组合物，其中n＝1-3的褐藻胶寡糖二酸的重量总和占所述组合总重量的20-70％。
  7. 根据权利要求1的褐藻胶寡糖二酸组合物，其中n＝1-3的褐藻胶寡糖二酸的重量总和与n＝4-7的褐藻胶寡糖二酸重量总和的比例在1.0-3.5之间。
  8. 根据权利要求7的褐藻胶寡糖二酸组合物，其中n＝1-3的褐藻胶寡糖二酸的重量总和与n＝4-7的褐藻胶寡糖二酸重量总和的比例在1.0-3.0之间。
  9. 根据权利要求1的褐藻胶寡糖二酸组合物，其中古罗糖醛酸的重量总和占所述组合物重量的0.1-50％，优选1-30％。
  10. 根据权利要求1-9任一项所述的褐藻胶寡糖二酸组合物，其中各聚合度褐藻胶寡糖二酸在所述组合中的重量百分含量为：二糖5～25％，三糖15～30％，四糖15～28％，五糖10～25％，六糖5～15％，七糖3～10％，八糖2～5％，九糖1～5％，十糖1～5％。
  11. 根据权利要求10所述的褐藻胶寡糖二酸组合物，其中各聚合度褐藻胶寡糖二酸在所述组合中的重量百分含量为：二糖10～20％，三糖18～30％，四糖15～28％，五糖15～20％，六糖5～10％，七糖3～5％，八糖2～3％，九糖1～3％，十糖1～3％。
  12. 根据权利要求1-11任一项所述的褐藻胶寡糖二酸组合物，其中所述药学上可接受的盐是钠盐或钾盐。
  13. 一种药物组合物或保健品，其包含有效量的权利要求1-12 任一项所述的褐藻胶寡糖二酸组合物和必要时适当的载体。
  14. 如权利要求1-12任一项所述的褐藻胶寡糖二酸组合物在制备用于治疗选自老年性痴呆、帕金森病、炎症、疼痛、糖尿病或血管性痴呆的药物或者保健品中的用途。
  15. 作为抗老年性痴呆、帕金森病、炎症、疼痛、糖尿病或血管性痴呆的药物或保健品的根据权利要求1-12任一项所述的褐藻胶寡糖二酸组合物。
  16. 一种治疗患有选自如下疾病的患者的方法：老年性痴呆、帕金森病、炎症、疼痛、糖尿病或血管性痴呆，该方法包括给予需要的患者有效量的根据权利要求1-12任一项所述的褐藻胶寡糖二酸组合物。
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