WO2020001611A1 - 甘露糖醛二酸的组合物在治疗炎症中的应用 - Google Patents

甘露糖醛二酸的组合物在治疗炎症中的应用 Download PDF

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WO2020001611A1
WO2020001611A1 PCT/CN2019/093656 CN2019093656W WO2020001611A1 WO 2020001611 A1 WO2020001611 A1 WO 2020001611A1 CN 2019093656 W CN2019093656 W CN 2019093656W WO 2020001611 A1 WO2020001611 A1 WO 2020001611A1
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mannuronic acid
composition
total weight
use according
acid
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French (fr)
Chinese (zh)
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耿美玉
辛现良
张真庆
丁健
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Shanghai Institute of Materia Medica of CAS
Shanghai Green Valley Pharmaceutical Co Ltd
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Shanghai Institute of Materia Medica of CAS
Shanghai Green Valley Pharmaceutical Co Ltd
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Priority to JP2020572815A priority Critical patent/JP2021529195A/ja
Priority to AU2019296419A priority patent/AU2019296419A1/en
Priority to US17/256,854 priority patent/US11406659B2/en
Priority to KR1020217001707A priority patent/KR20210040041A/ko
Priority to EP19825915.2A priority patent/EP3815693A4/en
Publication of WO2020001611A1 publication Critical patent/WO2020001611A1/zh
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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
    • A61K31/715—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
    • 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
    • A61K31/734—Alginic 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
    • A61K31/7012—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
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds 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
    • A61K31/7028—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
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the present invention relates to the application of the optimal composition obtained by the method of biological activity screening for the treatment of inflammation.
  • Inflammation refers to the physiological response that biological tissues are stimulated by trauma, bleeding or pathogen infection, foreign bodies, etc.
  • the inflammatory response involves changes in certain specific autoactive substances, such as prostaglandins and leukotrienes, and specific inflammatory cytokines, such as interleukins.
  • specific autoactive substances such as prostaglandins and leukotrienes
  • specific inflammatory cytokines such as interleukins.
  • inflammation occurs. Excessive inflammation can also damage the body's own substances.
  • commonly used anti-inflammatory drugs are mainly steroidal and non-steroidal anti-inflammatory drugs.
  • Mannuronic acid has been widely valued for its potential medicinal value.
  • Mannuronic acid is usually prepared from alginic acid through multiple steps.
  • 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 can be obtained, and after the mixed polysaccharide is precipitated by the acid method, the polyguluraldehyde can be removed therefrom Acid and further purification to obtain a homopolymannuronic acid (hereinafter also referred to as "M-stage intermediate") having a purity of 90% or more.
  • M-stage intermediate homopolymannuronic acid
  • the M-stage intermediate obtained above can be heated under acidic conditions to further acidolyze to obtain small fragments of mannuronic acid polymers in the desired molecular weight range.
  • oxidative degradation methods to improve the degradation efficiency, and at the same time, the reducing terminal can be oxidized to a ring-opened sugar diacid.
  • Chinese patent application 200580009396.5 by Geng Meiyu et al. 2 for ease of description, 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 represented by the following reaction equation (II), that is, the oxidizing group at the C1-position of the mannuronic acid 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 oligosaccharide diacid has anti-Alzheimer's disease (AD) and anti-diabetic effects.
  • the pathogenesis of Alzheimer's disease and type 2 diabetes is closely related to amyloid ( ⁇ -amyloid and amylin).
  • ⁇ -amyloid and amylin amyloid
  • protein oligomers are produced, which further aggregate to form fibers.
  • These protein aggregates are cytotoxic, induce oxidative reactions in cells to damage mitochondria, and trigger a cascade of inflammatory responses, causing a large number of neurons and ⁇ -cell damage, eventually leading to Alzheimer's disease and type II diabetes.
  • Mannan oligosaccharic acid targets amyloid and antagonizes the cascade response induced by it, thereby preventing and treating Alzheimer's disease and type 2 diabetes.
  • the previous patent CN106344594A discloses the application of mannuronic acid oligosaccharide and its derivative with a carboxyl group at the reducing end in the treatment of inflammation, and discloses the pharmacological activity of a tetrasaccharide-decasaccharide mixture in the treatment of inflammation.
  • the invention relates to the use of a mannuronic acid oligosaccharide composition in treating inflammation.
  • the invention also relates to a method for treating inflammation, which comprises administering to a patient in need of treatment a therapeutically effective amount of a mannuronic acid oligosaccharide composition according to the invention.
  • the mannuronic acid oligosaccharide composition according to the present invention comprises a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof:
  • 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 n 1-5 of the mannuronic acid accounts for more than 60% of the total weight of the composition
  • mannuronic acid compositions exhibits beneficial effects on the treatment of inflammatory reactions, and at the same time, due to its high safety derived from natural products, it is beneficial to alleviate chronic or acute pain in patients.
  • Figure 1 is the mass spectrum of disaccharide, trisaccharide and tetrasaccharide in product A.
  • FIG. 2 is a mass spectrum of pentasaccharide, hexasaccharide, and heptose in product A.
  • FIG. 2 is a mass spectrum of pentasaccharide, hexasaccharide, and heptose in product A.
  • Figure 3 is a mass spectrum of octaose, nonasaccharide, and decasaccharide in product A.
  • Fig. 4 shows the inhibitory effect of a single degree of polymerization of mannan oligosaccharides on A ⁇ -induced neuroinflammation.
  • 5a and 5b show the therapeutic effect of the oligosaccharide composition and hexasaccharide of the present invention on rheumatoid arthritis in mice; samples corresponding to the numbers on the abscissa in FIG. 5b are: i: control group; ii: model group; iii: Product A; iv: Product B; v: Product C; vi: Product D; vii: Comparative experimental sample; viii: Hexose.
  • FIG. 6a and 6b show the therapeutic effect of the oligosaccharide composition and hexasaccharide of the present invention on multiple sclerosis in mice; the abscissa in FIG. 6b is the same as that in FIG. 5b.
  • FIG. 7a and 7b show the therapeutic effect of the oligosaccharide composition and hexasaccharide of the present invention on mouse systemic lupus erythematosus; the abscissa in FIG. 7b is the same as that in FIG. 5b.
  • FIG. 8a and 8b show the therapeutic effect of the oligosaccharide composition and hexasaccharide of the present invention on inflammatory enteritis in mice; the abscissa in FIG. 8b is the same as that in FIG. 5b.
  • the invention relates to the use of a mannuronic acid oligosaccharide composition in treating inflammation.
  • the invention also relates to a method for treating inflammation, which comprises administering to a patient in need of treatment a therapeutically effective amount of a mannuronic acid oligosaccharide composition according to the invention.
  • the mannuronic acid oligosaccharide composition according to the present invention comprises a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof:
  • n is an integer selected from 1-9
  • m is selected from 0, 1 or 2
  • m ' is selected from 0 or 1
  • the weight percent content of each degree of polymerization of the mannuronic acid oligosaccharide in the mannuronic acid oligosaccharide combination of the present invention in the combination is: 5-25% of disaccharide, 15 of trisaccharide -30%, tetrasaccharide 15-28%, pentasaccharide 5-25%, hexasaccharide 2-20%, heptose 2-20%, octose 2-20%, nonaperose 2-20%, decasu 2- 20%.
  • the weight percentage content of the oligosaccharide in the combination is: 5-25% disaccharide, 15-30% trisaccharide, 15-28% tetrasaccharide, 10-20% pentasaccharide, 5 -15%, heptasaccharide 3-10%, octose 2-5%, nonaose 1-5%, and decaose 1-5%.
  • the weight percentage content of the oligosaccharide in the combination is: 10-20% disaccharide, 18-30% trisaccharide, 15-28% tetrasaccharide, 15-20% pentasaccharide, and hexasaccharide 5-10%, heptasaccharide 3-5%, octose 2-5%, nonaose 1-3%, decaose 1-3%.
  • the pharmaceutically acceptable salt is a sodium salt or a potassium salt.
  • the inventor of the present patent application found that when the above 9 oligosaccharides with new structure are compounded in a certain ratio, a highly active oligosaccharide composition can be obtained, the activity of which is higher than that of the most active hexasaccharide; especially It is a composition added with a certain proportion of disaccharides and trisaccharides, and its activity is higher than the composition without disaccharides and trisaccharides.
  • the ratio of each oligosaccharide in the high-activity oligosaccharide composition needs to be combined according to the following proportional relationship:
  • the medicament for treating inflammation comprises a mannuronic acid oligosaccharide composition comprising a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof, and one or more medicaments Acceptable carrier.
  • the medicament according to the invention may be tablets, hard capsules, soft capsules, enteric capsules, microcapsules, granules, syrups, injections, granules, emulsions, suspensions, solutions and for oral or parenteral administration In the form of a slow-release preparation.
  • the pharmaceutically acceptable carrier in the present invention refers to a pharmaceutically acceptable carrier well known to those skilled in the art.
  • the pharmaceutically acceptable carrier in the present invention includes, but is not limited to, fillers, wetting agents, adhesives, and disintegrants. , Lubricants, adhesives, glidants, taste-masking agents, surfactants, preservatives, etc.
  • Fillers include, but are not limited to, lactose, microcrystalline cellulose, starch, powdered sugar, dextrin, mannitol, calcium sulfate, and the like.
  • Wetting agents and binders include, but are not limited to, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, gelatin, sucrose, polyvinylpyrrolidone, and the like.
  • Disintegrating agents include, but are not limited to, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, croscarmellose sodium, low-substituted hydroxypropyl cellulose, and the like.
  • Lubricants include, but are not limited to, magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, magnesium lauryl sulfate, and the like.
  • Binders include, but are not limited to, gum arabic, alginic acid, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, glucose binding agents, dextrin, dextrose, ethyl cellulose, gelatin, liquid glucose, guar Gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, magnesium aluminum silicate, maltodextrin, methyl cellulose, polymethacrylate, polyvinylpyrrolidone, pregelatinized starch , Sodium alginate, sorbitol, starch, syrup and tragacanth.
  • Glidants include, but are not limited to, colloidal silica, powdered cellulose, magnesium trisilicate, silica, and talc.
  • Taste-masking agents include, but are not limited to, aspartame, stevioside, fructose, glucose, syrup, honey, xylitol, mannitol, lactose, sorbitol, maltitol, glycyrrhizin.
  • Surfactants include, but are not limited to, Tween-80, Poloxamer.
  • Preservatives include, but are not limited to, paraben, sodium benzoate, potassium sorbate, and the like.
  • the present invention also relates to a mannuronic acid oligosaccharide composition for treating inflammation, comprising a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof:
  • n is an integer selected from 1-9
  • m is selected from 0, 1 or 2
  • m ' is selected from 0 or 1
  • the ratio of the total weight of the acid oligosaccharides is between 1.0 and 3.5, preferably between 1.0 and 3.0.
  • the weight percent content of each degree of polymerization of the mannuronic acid oligosaccharide in the mannuronic acid oligosaccharide composition for treating inflammation according to the present invention in the combination is: disaccharide 5 -25%, trisaccharide 15-30%, tetrasaccharide 15-28%, pentasaccharide 5-25%, hexasaccharide 2-20%, heptasaccharide 2-20%, octose 2-20%, nonaose 2- 20%, deca sugar 2-20%.
  • the weight percentage content of the oligosaccharide in the combination is: 5-25% disaccharide, 15-30% trisaccharide, 15-28% tetrasaccharide, 10-20% pentasaccharide, 5 -15%, heptasaccharide 3-10%, octose 2-5%, nonaose 1-5%, and decaose 1-5%.
  • the weight percentage content of the oligosaccharide in the combination is: 10-20% disaccharide, 18-30% trisaccharide, 15-28% tetrasaccharide, 15-20% pentasaccharide, and hexasaccharide 5-10%, heptasaccharide 3-5%, octose 2-5%, nonaose 1-3%, decaose 1-3%.
  • 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.
  • treatment generally refers to obtaining the desired pharmacological and / or physiological effect.
  • the effect may be preventive based on the complete or partial prevention of the disease or its symptoms; and / or based on the partial or complete stabilization or cure of the disease and / or side effects due to the disease, which may be therapeutic.
  • Treatment encompasses any treatment of a patient's disease, including: (a) preventing a disease or symptom that occurs in a patient who is susceptible to a disease or condition that has not yet been diagnosed; (b) suppressing the symptoms of the disease, That is, preventing its development; or (c) alleviating the symptoms of the disease, that is, causing the disease or the symptoms to deteriorate.
  • the mannuronic acid oligosaccharide composition for treating inflammation comprises a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof:
  • n is an integer selected from 1-9
  • m is selected from 0, 1 or 2
  • m ' is selected from 0 or 1
  • the mannuronic acid oligosaccharide combination according to the present invention is a mixture of mannuronic acid with different polymerization degrees, and its main component is a mannuronic acid oligosaccharide with a polymerization degree of 2 to 10. It is known that among mannuronic acids, the most active sugars are 4-10 sugars, especially 6 sugars. However, the inventor's research found that adding a certain proportion of the less active 2-3 sugars to the 4-10 sugars with the highest activity, the biological activity did not decrease or even increased at the same quality of the dosage, which may be due to Although 2-3 sugars with smaller molecular weight cannot work alone, they can play a synergistic effect when mixed with other oligosaccharides. However, when the ratio of 2-3 sugars is too high, the overall activity of the composition decreases. Therefore, the ratio of 2-3 sugars in the composition must be controlled within a certain range.
  • composition activity can reach or even be better than the composition disclosed in the earlier application, and because the 2-3 sugars are not removed as impurities, the yield of the product is theoretically significantly higher than that of the product disclosed in the earlier application, which greatly reduces Production costs and reduction of waste emissions are easier to achieve in actual production and easier to achieve large-scale industrial production.
  • the method for preparing the mannuronic acid oligosaccharide composition for treating inflammation includes the following steps:
  • the M-stage intermediate of the raw materials used in the present invention can be prepared by methods known in the art. For example, the methods disclosed in Chinese Patent Application No.98806637.8 and CN02823707.2.
  • the general method can be simply described as: after the initial degradation of alginic acid, a mixed polysaccharide of polymannuronic acid and polyguluronic acid can be obtained, and after the mixed polysaccharide is precipitated by the acid method, the polyguluraldehyde can be removed therefrom Acid, further purification can obtain a homopolymannuronic acid with a purity of more than 90%, that is, an M-stage intermediate.
  • the M-stage intermediate is dissolved in an appropriate amount of water at room temperature or under heating conditions, stirred, and ozone is continuously introduced, and the reaction starts.
  • 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 vacuum dried to obtain an oligomannuronic acid mixture. After analysis, it was found that these products are all disaccharide-decasaccharide oligosaccharides whose composition is in a certain ratio range. The proportion and structure of oligosaccharides in some compositions are confirmed in Examples 1-3.
  • the oligosaccharide mixture obtained in step (1) is dissolved, formulated to a concentration of about 10%, separated by a P6 gel chromatography column, and detected by UV, and each effluent component is collected, and components with the same degree of polymerization are combined.
  • Nine components of 2-10 sugars were collected, desalted by G10 gel column chromatography, concentrated on a rotary evaporator, and dried under vacuum.
  • a specific purification preparation process is shown in Example 4. These column chromatography, desalting and drying operations are known to those skilled in the art.
  • the nine oligosaccharides with a single degree of polymerization were evaluated for their pharmacological activity using anti-inflammatory cell models, and it was found that the activity of hexasaccharide was the best.
  • the pharmacological activity of the composition of the present invention and the composition and the purified hexasaccharide were compared at the same time.
  • the results show that the oligosaccharide composition of the present invention is better than the most active hexasaccharide in a single degree of polymerization oligosaccharide, and contains a higher proportion.
  • the combination of disaccharides and trisaccharides is slightly less active than hexasaccharides. Without being bound by any theory, it is speculated that the percentage content of di- and tri-saccharides in the oligosaccharide composition is within a specific range so that the combination of oligosaccharides can play a synergistic effect.
  • 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
  • a clinical score of 0-4 is used to reflect the degree of disease progression.
  • 0 points are without erythema and redness; 1 points are near the metatarsal bone or near the ankle joint or sacrum, with redness or swelling, and 1 toe is red and swollen; 2 points are ankle and sacrum with redness and swelling, or more than two toes 3 points for moderate erythema and swelling of the ankle, wrist, and sacrum; 4 points for severe redness and swelling of the ankle, wrist, and sacrum and toes; 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 About 12 days after the immunization, the immunized mice will develop symptoms and begin to observe closely and record the body weight and clinical score every day. 0 to 4 points indicate different degrees, 0 to normal performance, no obvious signs of disease; 1 to weak tail drooping, unilateral weakness of hind limbs; 2 to weak tail sagging, weakness of both hind limbs gait; 3 points It is paralysis of unilateral hind limb weakness paralysis; 4 is divided into paralysis of both hind limb weakness 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.
  • 0-6 points indicate different degrees, 0 points are normal; 1 points are less than 1cm in diameter at two points; 2 points are less than 1cm in diameter at two points; 3 are points at three sides Location diameter is less than 1cm; 4 points are larger than 1cm in diameter on one side and the other two points are smaller than 1cm; 5 points are larger than 1cm in diameter on both sides and the other two Less than 1cm; 6 points with a diameter greater than 1cm at three points on both sides.
  • IBD Inflammatory bowel disease
  • DSS model-dextran 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.
  • Step 1) Preparation of mannuronic acid oligosaccharide mixture
  • the method of preparing the M-stage intermediate as disclosed in the previous patent is briefly described as follows: 5Kg of sodium alginate is prepared into a solution of about 10%, and diluted hydrochloric acid is added to adjust the pH to about 3.0, and the temperature is raised to 80 ° C, stirred, and reacted. 10hr, stop heating, cool to room temperature, add NaOH to adjust pH to 9.0, add dilute hydrochloric acid to adjust pH to 2.85, centrifuge at 5000rpm for 10min, collect supernatant, add HCl to adjust pH to 1.0, centrifuge, collect precipitate, rotate The evaporator was concentrated and dried under vacuum to obtain 1500 g of M-stage intermediate.
  • Step 2) Proportion and structure analysis of oligosaccharides with various polymerization degrees in mannuronic acid product A
  • disaccharides-decasaccharides are represented by dp2-dp10, respectively, dp2 is 19%, dp3 is 25%, dp4 is 22%, dp5 is 13%, dp6 is 9%, dp7 is 6%, and dp8 is 3 %, Dp9 is 2%, and dp10 is 1%.
  • Step 3) LC-MS analysis of the structure of oligosaccharides with various degrees of polymerization in Mannuronic acid 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.
  • Example 1 100 g of the M-stage intermediate in Example 1 was weighed, dissolved in distilled water, and prepared into a volume of 0.8 L. The solution was adjusted to pH 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. After 10 hours of reaction, stop introducing ozone, add appropriate amount of water to adjust the solution concentration to about 15%, and filter with an ultrafiltration membrane with a molecular weight cut off of 1000 Da. Collect the impervious liquid, concentrate on a rotary evaporator, and dry in vacuo to obtain 80 g of mannaldehyde Acid Product B.
  • GE Superdex peptide
  • MALS multi-angle laser scattering
  • dp2-dp10 Disaccharides-decasaccharides are represented by dp2-dp10, respectively, dp2 is 20%, dp3 is 25%, dp4 is 19%, dp5 is 12%, dp6 is 9%, dp7 is 5%, and dp8 is 5 %, Dp9 is 3%, and dp10 is 2%.
  • Example 1 100 g of the M-stage intermediate in Example 1 was weighed, dissolved in distilled water, and then a 1.5 L volume solution was prepared. 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. After reacting for 2 hours, stop introducing ozone, add an appropriate amount of water to adjust the solution concentration to about 5%, and filter through an ultrafiltration membrane with a molecular weight cut-off of 3000 Da. Collect the impervious liquid, concentrate on a rotary evaporator, and dry in vacuo to obtain 60 g of mannaldehyde. Acid Product C.
  • GE Superdexpeptide
  • MALS multi-angle laser scattering
  • dp2-dp10 disaccharides-decasaccharides are represented by dp2-dp10, respectively, dp2 is 8%, dp3 is 20%, dp4 is 28%, dp5 is 19%, dp6 is 13%, dp7 is 6%, and dp8 is 3 %, Dp9 is 2%, and dp10 is 1%.
  • Step 1) The preparation of mannuronic acid oligosaccharide with a single degree of polymerization is as follows:
  • Sample preparation Take 300g from the mannuronic acid product A prepared in Example 1, dissolve it with water, configure it into a 1000mL concentrated solution, and place it in a 4 ° C refrigerator for later use. After each use, 50 mL was taken out and diluted with water, and then filtered with 0.22um ultrafiltration membrane.
  • Chromatographic separation conditions The chromatograph is AKTA pure 150 (purchased from GE), equipped with UV detector and automatic collector. Separation chromatographic column: 1.2kg BioGel P6 (purchased from Bole Company) mixed with deionized water, vacuum degassed, manually packed into a glass column (10cm inner diameter), washed with pure water 10 times the column volume, the column bed is stable , The height is 1.0m. Then use 0.02M NaCl solution as the mobile phase. After equilibrating 10 times the column volume, start loading.
  • the flow rate of the pump is set to 1 mL / min. After 100 mL of the sample solution is pumped to the top of the column by the pump that comes with the chromatograph, switch to the mobile phase and elute at a flow rate of 5 mL / min. After the volume of the stagnant water flowed out, automatic collection was started, and 50 mL was collected per tube.
  • IL-1 ⁇ a major functional inflammatory factor in microglia
  • a ⁇ stimulation was measured after A ⁇ stimulation to reflect the inhibitory effect of the drug on neuroinflammation, and the efficacy of each oligosaccharide was evaluated. It was found that compared with the blank control group, the neuron inflammation in the A ⁇ model group was significantly enhanced.
  • Each single degree of polymerization oligosaccharide has a tendency to reduce neuroinflammation. Among them, a single degree of polymerization with a degree of polymerization of 4-10 can significantly reduce the expression of IL-1 ⁇ .
  • Sugar works especially well. Hexose has the best activity, and the effect of 2-3 sugars is weak. See Figure 4.
  • Composition product D Mannuronic acid oligosaccharide having a single degree of polymerization prepared in Example 4 was accurately weighed according to the degree of polymerization from disaccharide to decasaccharide, and the weight of each sugar was as follows: 3.0 g of disaccharide , 3.0g of trisaccharide, 1.5g of tetrasaccharide, 1.5g of pentasaccharide, 0.4g of hexasaccharide, 0.2g of heptasaccharide, 0.2g of octose, 0.1g of nonaperose, 0.1g of decasuose, and mix to obtain 10g of composition product D.
  • the neutral liquid was slowly added to 4 times the volume of ethanol in the liquid, alcohol-precipitated, and left to stand overnight.
  • the solid matter obtained by alcohol precipitation was separated by filtration, and the solid matter obtained by filtration and washing was separated by filtration during the filtration and separation to obtain a white filter cake.
  • the filter cake was dried in an oven at 60 ° C to obtain a crude alginate oligosaccharide.
  • a fresh oxidant copper hydroxide was prepared by adding 25 ml of a 5% (weight percent) copper sulfate solution to 50 ml of a 10% (weight percent) sodium hydroxide solution and immediately mixing.
  • the fresh oxidizing agent copper hydroxide was immediately added to 40 ml of the above-mentioned 5% (wt%) algin gum oligosaccharide solution, and simultaneously heated by a boiling water bath until no red brick precipitate was generated.
  • the reaction system was centrifuged to remove a precipitate to obtain a supernatant.
  • the eluent as a mobile phase was 0.2 mol ⁇ L-1NH 4 HCO. 3 .
  • the eluate was collected from the column chromatography using a plurality of 5 ml test tubes in order, and then the sugar content of the eluate in each of the headers was detected by the sulfuric acid-carbazole method. According to the detection results, eluates containing alginate oligosaccharide components with different molecular weights were collected. The eluates containing alginate oligosaccharide components with different molecular weights were respectively concentrated under reduced pressure and freeze-dried, and component 1 was discarded to obtain alginate oligosaccharide components 2-12 having different molecular weights respectively.
  • 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.
  • Dextran sodium sulfate (DSS) -induced colitis model in mice
  • 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.

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