WO2016188382A1 - 一种氧化型α-1，4-寡聚葡萄糖醛酸及其制备方法和用途 - Google Patents

一种氧化型α-1，4-寡聚葡萄糖醛酸及其制备方法和用途 Download PDF

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WO2016188382A1
WO2016188382A1 PCT/CN2016/082929 CN2016082929W WO2016188382A1 WO 2016188382 A1 WO2016188382 A1 WO 2016188382A1 CN 2016082929 W CN2016082929 W CN 2016082929W WO 2016188382 A1 WO2016188382 A1 WO 2016188382A1
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oxidized
acid
oligoglucuronic
oligoglucuronic acid
mixture
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PCT/CN2016/082929
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English (en)
French (fr)
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张真庆
郝杰
孙世昶
张慧灵
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上海绿谷制药有限公司
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Priority to EP16799270.0A priority Critical patent/EP3299395A4/en
Priority to US15/576,477 priority patent/US10898509B2/en
Priority to KR1020177037122A priority patent/KR20180019605A/ko
Priority to JP2017561938A priority patent/JP6908534B2/ja
Priority to CN201680029752.8A priority patent/CN107922513B/zh
Publication of WO2016188382A1 publication Critical patent/WO2016188382A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/02—Acyclic radicals
    • C07H7/033—Uronic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/18—Oxidised starch
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0033—Xanthan, i.e. D-glucose, D-mannose and D-glucuronic acid units, saubstituted with acetate and pyruvate, with a main chain of (beta-1,4)-D-glucose units; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L3/00—Compositions of starch, amylose or amylopectin or of their derivatives or degradation products
    • C08L3/04—Starch derivatives, e.g. crosslinked derivatives
    • C08L3/10—Oxidised starch
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C31/00—Saturated compounds having hydroxy or O-metal groups bound to acyclic carbon atoms
    • C07C31/02—Monohydroxylic acyclic alcohols
    • C07C31/04—Methanol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C31/00—Saturated compounds having hydroxy or O-metal groups bound to acyclic carbon atoms
    • C07C31/02—Monohydroxylic acyclic alcohols
    • C07C31/08—Ethanol

Definitions

  • the invention belongs to the technical field of pharmaceutical compounds, and particularly relates to an oxidized ⁇ -1,4-oligoglucopic acid, a preparation method thereof and use thereof.
  • Starch is mainly found in the leaves, roots and seeds of plants and is in the form of granules.
  • Starch consists of a linear sugar starch and a branched gum starch.
  • the sugar starch is ⁇ 1 ⁇ 4, which can be dissolved in hot water to obtain a clear solution.
  • the gelatin starch is also ⁇ 1 ⁇ 4 glucan, but the structure also contains ⁇ 1 ⁇ 6 branches, which are insoluble in cold water and are in the form of a gel in hot water.
  • Soluble starch is a starch derivative obtained by treating starch with an oxidizing agent, acid, glycerol, enzyme or other methods. White or light yellow powder, tasteless and odorless. Soluble starch is a denatured starch that is soluble in hot water and insoluble in cold water, alcohols and ethers. Generally, starches of rice, corn, millet and potato can be made into soluble starch, but the soluble starch prepared from sweet potato starch has the best quality. In addition to food, starch and soluble starch are used industrially to prepare dextrin, maltose, glucose, alcohol, etc., and are also used in printing, textile, pharmaceutical and other industries.
  • Glucuronic acid is a common sugar molecule that is part of the glycosaminoglycan glycosyl group in the body, such as heparan sulfate, chondroitin sulfate, etc.; it also appears in the glycosyl moiety of small molecule glycosides. However, there is no poly- or oligo-glucuronic acid in nature.
  • the present invention provides an oxidized ⁇ -1,4-oligosaccharide Uronic acid and preparation method and use thereof.
  • the method of the invention uses natural starch rich in starch, especially soluble starch, to be oxidized by sodium bromide (NaBr)-2,2,6,6-tetramethylpiperidine oxide (TEMPO)-sodium hypochlorite (NaClO).
  • NaBr sodium bromide
  • TEMPO sodium bromide
  • NaClO sodium hypochlorite
  • the system functions to oxidize all 6-position hydroxyl groups of ⁇ -1,4-polyglucose of starch to form a glucuronic acid, and at the same time, to prepare a terminal ring-opened oxidized oligogluconate by controlling the reaction conditions.
  • the compound has significant anti-ischemic activity and can be developed into a potential anti-ischemic drug.
  • One aspect of the present invention provides an oxidized ⁇ -1,4-oligoglucuronic acid having a degree of polymerization of from 1 to 20 saccharides having the structure of Formula I:
  • Another aspect of the present invention provides a mixture of the above oxidized ⁇ -1,4-oligoglucuronic acid, which comprises an oxidized ⁇ -1,4-oligogluconate having a structure of the formula I':
  • n' and m' are the average values of n and m of each of the oligomeric glucuronic acids in the mixture, n' is selected from 1.0 to 19.0; and m' is selected from 0 to 2.0. n' and m' may be an integer or a non-integer, which are arithmetical average values based on the molar amount of n and m of each oligogluconate in the mixture.
  • a further aspect of the invention relates to a process for the preparation of an oxidized alpha-1,4-oligoglucuronic acid or a mixture thereof, comprising the steps of:
  • the invention further relates to a composition
  • a composition comprising a mixture of oxidized alpha-1,4-oligoglucuronic acid of formula I of the invention, and a pharmaceutically acceptable excipient or carrier.
  • Another aspect of the present invention also provides the use of a mixture of oxidized ⁇ -1,4-oligoglucuronic acid according to the present invention for the preparation of an anti-ischemic drug or a neuroprotective drug. They can be used to treat or prevent ischemic damage to neurons caused by stroke, myocardial infarction, cerebral shock, neonatal asphyxia, and brain trauma.
  • Another aspect of the invention provides a method of treating or preventing neuronal ischemic damage in a subject or protecting a cranial nerve of a subject, comprising administering to the subject an effective amount of the oxidized form of Formula I Alpha-1,4-oligoglucuronic acid or a mixture thereof.
  • the present invention also provides an oxidized ⁇ -1,4-oligoglucuronic acid of the formula I or a mixture thereof as an agent for treating or preventing neuronal ischemic damage or as a neuroprotective agent.
  • the neuronal ischemic damage is caused by stroke, myocardial infarction, cerebral shock, neonatal asphyxia, and brain trauma.
  • the outstanding effects of the present invention include: using soluble starch as a raw material for preparing oxidized ⁇ -1,4-oligoglucosonic acid, the preparation method is simple, the conditions are mild, the cost is low, and the industrialization is easy.
  • the compounds have significant anti-ischemic activity and can be used for the treatment of ischemic damage caused by stroke, myocardial infarction, brain shock, neonatal asphyxia and brain trauma.
  • Figure 1 is a total ion chromatogram (TIC) of oxidized ⁇ -1,4-oligogluconoic acid in Example 5 and an ultraviolet chromatogram at 210 nm;
  • Example 2 is a first-order mass spectrum of peak 1 in Example 5, that is, an oxidized ⁇ -1,4-oligoglucuronic acid 9 and 10 sugar mass spectrum;
  • Example 3 is a first-order mass spectrum of peak 2 in Example 5, that is, an oxidized ⁇ -1,4-oligogluconoxate 8-saccharide mass spectrum;
  • Example 4 is a first-order mass spectrum of peak 3 in Example 5, that is, a mass spectrum of oxidized ⁇ -1,4-oligogluconoic acid 7-saccharide;
  • Figure 5 is a mass spectrum of the first-order mass spectrum of the peak 4 in Example 5, that is, an oxidized ⁇ -1,4-oligogluconoic acid 6-saccharide;
  • Example 6 is a first-order mass spectrum of peak 5 in Example 5, that is, an oxidized ⁇ -1,4-oligogluconoxate 5-saccharide mass spectrum;
  • Example 7 is a first-order mass spectrum of peak 6 in Example 5, that is, an oxidized ⁇ -1,4-oligogluconoxate 4-saccharide mass spectrum;
  • Figure 8 is a mass spectrum of the first-order mass spectrum of the peak 7 in Example 5, that is, an oxidized ⁇ -1,4-oligogluconoxate 3-saccharide;
  • Figure 9 is a mass spectrum of the first-order mass spectrum of the peak 8 in Example 5, that is, an oxidized ⁇ -1,4-oligogluconoxate 2-saccharide;
  • Figure 10 is a first-order mass spectrum of 10 to 20 sugars in Example 5, that is, an oxidized ⁇ -1,4-oligoglucuronic acid dp10-dp20 mass spectrum;
  • Figure 11 is a 1 H-NMR chart of the oxidized ⁇ -1,4-oligoglucuronic acid in Example 6;
  • Figure 12 is a 13 C-NMR chart of the oxidized ⁇ -1,4-oligoglucuronic acid in Example 6;
  • Figure 13 is a graph showing the effect of oxidized ⁇ -1,4-oligoglucuronic acid on the hippocampal cells of normal mice in Example 7;
  • Figure 14 is a graph showing the effect of oxidized ⁇ -1,4-oligoglucuronic acid on hippocampal cells of hypoglycemia-deficient mice in Example 7.
  • a first aspect of the invention relates to an oxidized alpha-1,4-oligoglucuronic acid having the following structural formula I:
  • the glucose molecule representing the terminal is oxidized to remove two -CH(OH)- units.
  • m 2
  • the glucose molecule representing the terminal does not remove -CH(OH)-, and only the oxidation of the hydroxyl group occurs.
  • a product of m of 0, 1, and 2 may be mixed. Exist, it can also exist alone. Oxidized alpha-1,4-oligoglucuronic acids of different m values have similar biological activities.
  • n is 1-9, that is, a disaccharide to decasaccharide corresponding to oxidized ⁇ -1,4-oligoglucuronic acid, each of which is in the present invention. Is separated and characterized.
  • n 1 corresponds to oxidized disaccharide
  • n 2 corresponds to oxidized trisaccharide
  • n 3 corresponds to oxidation Tetrasaccharide
  • n 4
  • pentoxide when n is 5, it corresponds to hexasaccharide
  • n 6, it corresponds to oxidized heptasaccharide
  • n 7, corresponds to oxidized hexose
  • n corresponds to oxidation.
  • These oxidized oligosaccharides may be used in one or more mixed forms.
  • the oxidized ⁇ -1,4-oligoglucuronic acid of the present invention is characterized by an oligosaccharide aldehyde structure having a different number of carbon atoms at the end of ring opening.
  • the oligosaccharides having different numbers of carbon atoms at the ends may be present in a mixture, and the oligosaccharides of different degrees of polymerization are also present in a mixture.
  • Another aspect of the invention relates to a mixture of oxidized alpha-1,4-oligoglucuronic acid A structure having the formula I':
  • n' and m' are the average values of n and m of each of the oligomeric glucuronic acids in the mixture, n' is selected from 1.0 to 19.0; and m' is selected from 0 to 2.0. More preferably, n' is selected from 1.0-10.0, m' is selected from 0.5 to 1.8; most preferably, n' is selected from 1.0 to 9.0, and m' is selected from 0.8 to 1.5.
  • the oxidized alpha-1,4-oligoglucuronic acid mixture of the invention comprises more than 80%, preferably more than 90%, more preferably more than 95% of the end-opening two to ten Poly alpha-1,4-glucuronic acid (corresponding to n being 1-9), wherein m' is from 0.8 to 1.5.
  • the oxidation process of the present invention comprises the steps of dissolving starch, oxidizing a starch solution, and post-treating the oxidation product to form an oxidized alpha-1,4-oligoglucuronic acid.
  • the raw material soluble starch is dissolved in water to form an aqueous solution, and the concentration of the starch solution may be about 1 to 30 mg/mL.
  • the amount of water is 50-100 ml per gram of soluble starch. Applicant found that if the concentration of the starch solution is too high, the oxidation process is not easy to complete, and the concentration of the starch solution is over-liquid, which tends to cause uneven oxidation products.
  • the present invention employs an oxidation system: sodium bromide (NaBr)-2,2,6,6-tetramethylpiperidine oxide (TEMPO)-sodium hypochlorite (NaClO) oxidation system.
  • the oxidizing properties of the oxidizing system are particularly suitable for obtaining the ⁇ -1,4-oligoglucosonic acid of the present invention, as adjusted by the reaction conditions.
  • the oxidizing system of the present invention enables the oxidation reaction to proceed more completely, obtaining a terminal ring-opened reaction product without destroying the uniformity of the reaction system.
  • the weight percentage of active sodium hypochlorite in the sodium hypochlorite solution is from 1 to 20%, preferably from 2 to 15%, more preferably from 3 to 12%.
  • the oxidation reaction was carried out under alkaline conditions. It was found that the most suitable pH range was 10-11. If the pH of the reaction system was too high, the oxidation efficiency was low and the pH value was too low, which was not favorable for oxidation.
  • the pH of the reaction is controlled by a basic compound, and the most preferred basic compound is a NaOH solution. There is no particular limitation on the solution of NaOH.
  • the oxidation temperature suitable for the oxidation reaction of the present invention is 40 to 70 ° C, preferably 45 to 60 ° C, more preferably 48 to 55 ° C. Oxidation in this temperature range is advantageous for removing residual enzymes which are not expected in the starch, and at the same time obtaining Open-ended uronic acid at the end. Without wishing to be bound by any theory, the inventors have found that a reaction temperature below 40 °C is detrimental to obtaining an end-opened oxidized alpha-1,4-oligoglucoaldehyde. Excessive reaction temperature will destroy the biological activity of the starch raw material, which is not conducive to the utilization of the reaction product.
  • the product solution obtained by the oxidation was purified to obtain an oxidized ⁇ -1,4-oligogluconate.
  • the obtained oligogluconate can be purified by dialysis, and in particular, the material used for dialysis needs to retain a substance having a molecular weight of 500 Da, thereby purifying the oligosaccharide of the present invention. Purification may also be carried out in other manners known in the art as long as the purity of the oligogluconate obtained by dialysis is more than 99%, more preferably more than 99.5%.
  • the invention further relates to the use of oxidized alpha-1,4-oligoglucuronic acid or a mixture thereof as an active compound component for the preparation of a medicament for the prevention of cerebral ischemia.
  • Cerebrovascular disease is the leading cause of death among middle-aged and elderly people in China, and it is also one of the research priorities of the World Health Strategy.
  • the incidence of ischemic diseases takes the first place.
  • the compensation mechanism of the brain protects the central nervous system from damage, but when the degree of ischemia increases, Irreversible neurological damage can occur, leading to a series of clinical symptoms and even death.
  • cerebrovascular accidents such as stroke), myocardial infarction, shock, neonatal asphyxia, and brain trauma can cause ischemic damage to neurons. Therefore, it is meaningful to develop a natural source of substances that alleviate the symptoms of cerebral ischemia and improve the survival rate of brain cells. of.
  • the cell model for measuring cerebral ischemic symptoms used in the present invention is a cell produced by HT-22 cells under hypoxic and hypoglycemic conditions (OGD model).
  • HT-22 cells are a mouse hippocampal neuronal cell line and a subclone of the mouse T4 cell line, which has the characteristics of hippocampal neurons.
  • oxidized alpha-1,4-oligoglucuronic acid of the general formula I or a mixture thereof can improve the symptoms of cerebral ischemia and increase the survival rate of hypoxic ischemic brain cells. Further, the oxidized ⁇ -1,4-oligoglucuronic acid of the present invention is derived from a natural product and is easily absorbed and utilized.
  • the present invention provides a combination medicament comprising at least two oxidized alpha-1,4-oligoglucuronic acids as described above, and optionally a pharmaceutically acceptable excipient.
  • Methods of preparing various combinations of drugs containing various ratios of active ingredients are known, or will be apparent to those skilled in the art in light of this disclosure. As described by Remington's Pharmaceutical Sciences, Martin, E. W., ed., Mack Publishing Company, 19th ed. (1995). Methods of preparing the pharmaceutical compositions include the incorporation of suitable pharmaceutical excipients, carriers, diluents and the like.
  • the pharmaceutical preparations of the invention are prepared in a known manner, including conventional methods of mixing, dissolving or lyophilizing.
  • compositions of the invention are administered to a patient in a variety of ways suitable for the chosen mode of administration, such as orally or parenterally (by intravenous, intramuscular, topical or subcutaneous routes).
  • the combination of the present invention can be administered systemically, for example, orally, in combination with a pharmaceutically acceptable carrier such as an inert diluent or an edible carrier. They can be enclosed in hard or soft shell gelatin capsules and can be compressed into tablets.
  • a pharmaceutically acceptable carrier such as an inert diluent or an edible carrier.
  • the active compounds of the invention may be combined with one or more excipients and in the form of swallowable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers Used in the form of.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the ratio of such compositions and formulations may of course vary and may range from about 1% to about 99% by weight of a given unit dosage form.
  • the amount of active compound is such that an effective dosage level can be obtained.
  • Tablets, lozenges, pills, capsules and the like may also contain: a binder such as tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, Potato starch, alginic acid, etc.; a lubricant such as magnesium stearate; and a sweetener such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as mint, wintergreen or cherry.
  • a binder such as tragacanth, acacia, corn starch or gelatin
  • an excipient such as dicalcium phosphate
  • a disintegrating agent such as corn starch, Potato starch, alginic acid, etc.
  • a lubricant such as magnesium stearate
  • a sweetener such as sucrose, fructose, lactose or aspartame
  • a flavoring agent such as mint, wintergreen or cherry
  • any material used to prepare any unit dosage form should be pharmaceutically acceptable and non-toxic in the amounts employed.
  • the active compound can be incorporated into sustained release formulations and sustained release devices.
  • the active compound can also be administered intravenously or intraperitoneally by infusion or injection.
  • An aqueous solution of the active compound or a salt thereof, optionally a miscible non-toxic surfactant, can be prepared.
  • Dispersing agents 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.
  • Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions of the active ingredient (optionally encapsulated in liposomes) containing the immediate formulation of a suitable injectable or injectable solution or dispersing 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 Bulk dispersion media include, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by liposome formation, by maintaining the desired particle size in the case of a dispersing agent, or by the use of a surfactant.
  • the action of preventing microorganisms can be produced by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents such as sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use of compositions that delay the absorbent (for example, aluminum monostearate and gelatin).
  • Sterile injectable solutions are prepared by combining the required active compound in a suitable solvent with the various other ingredients enumerated above, followed by filter sterilization.
  • the preferred preparation methods are vacuum drying and lyophilization techniques which result in a powder of the active ingredient plus any additional ingredients present in the previously sterile filtration solution. .
  • Useful solid carriers include comminuted solids (e.g., talc, clay, microcrystalline cellulose, silica, alumina, etc.).
  • Useful liquid carriers include water, ethanol or ethylene glycol or a water-ethanol/ethylene glycol mixture, and the combination medicaments of the present invention may be dissolved or dispersed in an effective amount, optionally with the aid of a non-toxic surfactant.
  • Adjuvants such as fragrances
  • additional antimicrobial agents can be added to optimize the properties for a given use.
  • Thickeners can also be used with liquid carriers to form coatable pastes, gels, ointments , soap, etc., used directly on the user's skin.
  • the therapeutic or prophylactic amount of the compound or mixture thereof depends 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, ultimately depending on the decision of the attending physician or clinician.
  • unit dosage form is a unit dispersion unit containing a unit dosage unit suitable for administration to humans and other mammalian bodies.
  • the unit dosage form can be a capsule or tablet, or a plurality of capsules or tablets.
  • the amount of unit dose of the active ingredient may vary or be adjusted between about 0.1 and about 1000 mg or more, depending on the particular treatment involved.
  • Dialysis was carried out in a 500 Da dialysis bag, concentrated, and lyophilized to obtain oxidized ⁇ -1,4-oligogluconate.
  • Dialysis was carried out in a 500 Da dialysis bag, concentrated, and lyophilized to obtain oxidized ⁇ -1,4-oligogluconate.
  • Dialysis was carried out in a 500 Da dialysis bag, concentrated, and lyophilized to obtain oxidized ⁇ -1,4-oligogluconate.
  • Dialysis was carried out in a 500 Da dialysis bag, concentrated, and lyophilized to obtain oxidized ⁇ -1,4-oligogluconate.
  • UHPLC/Q-TOF-MS analysis was performed using an Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS (Agilent, USA) system.
  • the chromatographic conditions were as follows: ACQUITY UPLC BEH125 exclusion chromatography column (4.6 ⁇ 300 mm, Waters); detection wavelength: 210 nm; mobile phase: A was 50 mM ammonium acetate aqueous solution, B was methanol, the ratio was 80% A; flow rate: 0.1 ml /min.
  • the mass spectrometry conditions were as follows: negative ion mode; scanning range: 100 to 3000; drying gas temperature: 350 ° C; drying gas flow rate: 8 L/min; capillary voltage: 3500 V; fragmentation voltage: 80 V.
  • TIC total ion chromatogram
  • Fig. 1 each peak has a regular wavy distribution. Since a molecular exclusion chromatography column is used, it is presumed that the wave peak should be distributed from large to small according to the degree of polymerization. Further, the structure is estimated by the first-order mass spectrum (Figs. 2-9) corresponding to each chromatographic peak: Fig. 2 is the first-order mass spectrum of peak 1, in which m/z 808.1483, 793.1434 and 778.1382 both have two charges, which are calculated.
  • h (3.74ppm) is when the glucuronic acid reducing end is unopened 30 (-CH 2 O-), and the carboxyl ⁇ position H, j (3.55ppm) is when the reducing end is unopened 30 (-CH 2 O-)
  • the carboxyl group ⁇ position H or the reduction end is opened at 30° and the carboxyl group ⁇ is H.
  • the H chemical shift of the gluconic acid reducing end is not removed, the 30 is removed, and the H chemical shift associated with the hydroxyl group and the carboxyl group is close to the water peak chemical shift, so it is not visible in the hydrogen spectrum.
  • the remaining peaks are H on the unopened uronic acid ring.
  • the region 1 is a carboxyl carbon peak
  • the region 2 is a closed-loop uronic acid 1-position C under different chemical environments.
  • the culture plate was placed in an anoxic tank (flushed with 95% N 2 , 5% CO 2 ), and cultured at 37 ° C for 12 h, and the cell viability was observed and determined to determine the effect of the sample on HT-22 cells under OGD.
  • the cell viability of each group was calculated by the MTT method as shown in Tables 1, 2 and 13 and 14. As can be seen from Table 1 and Figure 13, the survival rate of cells after addition of low, medium and high doses of oxidized ⁇ -1,4-oligoglucosonic acid was not significantly different from that of the normal group, indicating oxidation. Type ⁇ -1,4-oligoglucuronic acid is not toxic to HT-22 cells.
  • the survival rate of HT-22 cells was significantly lower in the model group than in the control group (p ⁇ 0.001), indicating that OGD significantly inhibited cell survival.
  • the survival rate of cells supplemented with oxidized ⁇ -1,4-oligoglucosonic acid (10 ⁇ M) began to increase; when the drug concentration reached 100 ⁇ M, the survival rate of cells was significantly higher than that of the model group (p ⁇ 0.05), indicating Oxidized ⁇ -1,4-oligoglucuronic acid has an effect of promoting the growth and survival of HT-22 cells.
  • the oxidized ⁇ -1,4-oligoglucuronic acid mixture of the present invention has a good anti-ischemic effect, and each of the isolated oxidized ⁇ -1,4-oligoglucuronic acids is similar. Similar results were obtained in the experiments, which can be applied in the preparation of anti-ischemic drugs.
  • the present invention has various embodiments, and all technical solutions formed by equivalent transformation or equivalent transformation are within the scope of the present invention.

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Abstract

本发明涉及一种氧化型α-1，4-寡聚葡萄糖醛酸及其制备方法和用途，属于医药化合物技术领域。本发明以自然界中丰富的淀粉，特别是可溶性淀粉为原料，通过溴化钠(NaBr)-2，2，6，6-四甲基哌啶氧化物(TEMPO)-次氯酸钠(NaClO)氧化体系作用，将淀粉这种α-1，4-多聚葡萄糖的所有6位羟基氧化成羧基形成葡萄糖醛酸，同时通过控制反应条件制备出末端开环的氧化型寡聚葡萄糖醛酸，该类化合物有显著的抗脑缺血活性，可以发展成潜在的抗脑缺血药物。

Description

一种氧化型α-1，4-寡聚葡萄糖醛酸及其制备方法和用途 技术领域
本发明属于医药化合物技术领域，具体涉及一种氧化型α-1，4-寡聚葡萄糖醛酸及其制备方法和用途。
背景技术
淀粉主要存在于植物的叶、根和种子中，呈颗粒状。淀粉由直链的糖淀粉和支链的胶淀粉组成。糖淀粉为α1→4连接，能溶于热水得到澄明溶液。胶淀粉也是α1→4葡聚糖，但是结构中还含有α1→6的支链，不溶于冷水，在热水中呈黏胶状。
可溶性淀粉是由淀粉经过氧化剂、酸、甘油、酶或其他方法处理而成的淀粉衍生物。白色或淡黄色粉末，无味无臭。可溶性淀粉是变性的淀粉，溶于热水，不溶于冷水、醇和醚。一般用大米、玉米、小米、土豆的淀粉都可制成可溶性淀粉，但以红薯淀粉制得的可溶性淀粉质量最好。淀粉、可溶性淀粉除食用外，工业上用于制备糊精、麦芽糖、葡萄糖、酒精等，也用于印刷、纺织、医药等产业。
葡萄糖醛酸是一种常见的糖类分子，在体内作为糖胺聚糖糖基组成的一部分，如硫酸乙酰肝素、硫酸软骨素等；也出现在小分子糖苷的糖基部分。但是自然界中没有多聚或寡聚葡萄糖醛酸。
近年来，随着生活水平的提高以及人口结构日益老龄化，脑血管疾病的发病率和死亡率正呈上升趋势，已成为其中一大死因。其中，缺血性脑血管病在脑血管疾病种类中占主要部分。目前具有良好治疗脑缺血疾病的药物种类有中药组合物、中药等，但是临床疗效有待确证，因此研究新的抗脑缺血疾病药物具有重要意义。
发明内容
鉴于现有技术存在的缺陷，本发明提供了一种氧化型α-1，4-寡聚葡 萄糖醛酸及其制备方法和用途。本发明的方法以自然界中丰富的淀粉，特别是可溶性淀粉为原料，通过溴化钠(NaBr)-2，2，6，6-四甲基哌啶氧化物(TEMPO)-次氯酸钠(NaClO)氧化体系作用，将淀粉这种α-1，4-多聚葡萄糖的所有6位羟基氧化成羧基形成葡萄糖醛酸，同时通过控制反应条件制备出末端开环的氧化型寡聚葡萄糖醛酸，该类化合物有显著的抗脑缺血活性，可以发展成潜在的抗脑缺血药物。
本发明的目的将通过以下技术方案得以实现：
本发明的一个方面提供一种氧化型α-1，4-寡聚葡萄糖醛酸，其聚合度为1-20糖，具有通式I的结构：
Figure PCTCN2016082929-appb-000001
其中，n＝0-19；m＝0、1或2。
本发明的另一个方面提供上述氧化型α-1，4-寡聚葡萄糖醛酸的混合物，其中由具有通式I’的结构的氧化型α-1，4-寡聚葡萄糖醛酸组成：
Figure PCTCN2016082929-appb-000002
其中，n’和m’分别为混合物中各寡聚葡萄糖醛酸的n和m的平均值，n’选自1.0-19.0；m’选自0-2.0。n’和m’可以是整数，也可以是非整数，它们是混合物中各寡聚葡萄糖醛酸的n和m的基于摩尔量计的算术平均值。
本发明的再一方面涉及氧化型α-1，4-寡聚葡萄糖醛酸或其混合物的制备方法，包括以下步骤：
(1)称取可溶性淀粉，制成水溶液；
(2)向步骤(1)中制得的水溶液中依次加入TEMPO和溴化钠，再使用碱性pH调节剂调节pH至10～11，之后加入次氯酸钠溶液于40～70℃条件下反应5～10小时，最后加入有机溶剂终止反应；
(3)置于500Da的透析袋中透析，浓缩、冻干，由此得到氧化型α-1，4-寡聚葡萄糖醛酸的混合物，或者任选地经色谱分离得到单独的所述氧化型α-1，4-寡聚葡萄糖醛酸。
本发明还涉及一种组合物，包含本发明的通式I的氧化型α-1，4-寡聚葡萄糖醛酸其混合物，以及药学上可接受的赋形剂或载体。
本发明的另一个方面还提供了根据本发明的氧化型α-1，4-寡聚葡萄糖醛酸其混合物在制备抗脑缺血药物或脑神经保护药物中的用途。它们可以用于治疗或预防中风、心肌梗塞、脑休克、新生儿窒息和脑外伤引起的神经元的缺血性损害。
本发明的另一个方面还提供了治疗或预防受试者的神经元缺血性损害或者保护受试者的脑神经的方法，包括给予受试者有效量的所述的通式I的氧化型α-1，4-寡聚葡萄糖醛酸或其混合物。
本发明的还提供了作为治疗或预防神经元缺血性损害的药剂或作为脑神经保护的药剂的通式I的氧化型α-1，4-寡聚葡萄糖醛酸或其混合物。
在上述方案中，所述神经元缺血性损害由中风、心肌梗塞、脑休克、新生儿窒息和脑外伤引起。
本发明的突出效果包括：将可溶性淀粉作为原料用于制备氧化型α-1，4-寡聚葡萄糖醛酸，制备方法简单，条件温和，成本较低，易于产业化。同时该类化合物有显著的抗脑缺血活性，可以用于治疗中风、心肌梗塞、脑休克、新生儿窒息和脑外伤引起的神经元的缺血性损害。
以下便结合实施例附图，对本发明的具体实施方式作进一步的详述，以使本发明技术方案更易于理解、掌握。
附图说明
图1是实施例5中氧化型α-1，4-寡聚葡萄糖醛酸总离子流图(TIC)和在210nm下的紫外色谱图；
图2为实施例5中峰1的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸9和10糖质谱图；
图3为实施例5中峰2的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸8糖质谱图；
图4为实施例5中峰3的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸7糖质谱图；
图5为实施例5中峰4的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸6糖质谱图；
图6为实施例5中峰5的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸5糖质谱图；
图7为实施例5中峰6的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸4糖质谱图；
图8为实施例5中峰7的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸3糖质谱图；
图9为实施例5中峰8的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸2糖质谱图；
图10为实施例5中10到20糖的一级质谱图即氧化型α-1，4-寡聚葡萄糖醛酸dp10-dp20质谱图；
图11为实施例6中氧化型α-1，4-寡聚葡萄糖醛酸的1H-NMR图谱；
图12为实施例6中氧化型α-1，4-寡聚葡萄糖醛酸的13C-NMR图谱；
图13为实施例7中氧化型α-1，4-寡聚葡萄糖醛酸对正常小鼠海马细胞的作用；
图14为实施例7中氧化型α-1，4-寡聚葡萄糖醛酸对缺糖缺氧小鼠海马细胞的作用。
具体实施方式
下面通过具体实施例对本发明的方法进行说明，但本发明并不局限 于此。
本发明的第一方面涉及一种氧化型α-1，4-寡聚葡萄糖醛酸，其具有如下结构通式I：
Figure PCTCN2016082929-appb-000003
其中n＝0-19；m＝0、1或2。
其中m＝0时，代表末端的葡萄糖分子经氧化脱除两个-CH(OH)-单元，当m＝1时，代表末端的葡萄糖分子经氧化脱除一个-CH(OH)-单元，当m＝2时，代表末端的葡萄糖分子未脱除-CH(OH)-，仅发生羟基的氧化。在一种氧化型α-1，4-寡聚葡萄糖醛酸，即n为某一数值的氧化型α-1，4-寡聚葡萄糖醛酸中，m为0、1和2的产物可以混合存在，也可以单独存在。不同m值的氧化型α-1，4-寡聚葡萄糖醛酸具有相似的生物活性。
在本发明的一个优选实施方案中，n为1-9，也即对应于氧化型α-1，4-寡聚葡萄糖醛酸的二糖至十糖，它们的每一种均在本发明中被分离并表征。更具体地，在式I的氧化型α-1，4-寡聚葡萄糖醛酸中，n为1时对应于氧化二糖，n为2时对应于氧化三糖，n为3时对应于氧化四糖，n为4时对应于氧化五糖，n为5时对应于氧化六糖，n为6时对应于氧化七糖，n为7时对应于氧化八糖，n为8时对应于氧化九糖，n为9时对应于氧化十糖。这些氧化的寡聚糖可以以一种或多种混合的形式使用。
本发明的氧化型α-1，4-寡聚葡萄糖醛酸的特征在于具有末端开环的不同碳原子数的寡聚糖醛酸结构。在一个实施方案中，末端不同碳原子数的寡聚糖醛酸可以以混合物的形式存在，不同聚合度的寡聚糖醛酸也以混合物形式存在。
本发明的另一个方面涉及氧化型α-1，4-寡聚葡萄糖醛酸的混合 物，其具有通式I’的结构：
Figure PCTCN2016082929-appb-000004
其中，n’和m’分别为混合物中各寡聚葡萄糖醛酸的n和m的平均值，n’选自1.0-19.0；m’选自0-2.0。更优选地，n’选自1.0-10.0，m’选自0.5-1.8；最优选地，n’选自1.0-9.0，m’选自0.8-1.5。
在另一个优选的实施方案中，本发明的氧化型α-1，4-寡聚葡萄糖醛酸混合物中包含80％以上、优选90％以上，更优选95％以上的末端开环的二至十聚α-1，4-葡萄糖醛酸(对应于n为1-9)，其中m’为0.8-1.5。
本发明的氧化方法包括如下步骤：溶解淀粉、氧化淀粉溶液、以及后处理氧化产物形成氧化型α-1，4-寡聚葡萄糖醛酸。
1.淀粉的溶解
为制备本发明的氧化型α-1，4-寡聚葡萄糖醛酸，将原料可溶性淀粉溶于水中，形成水溶液，淀粉溶液的浓度可以是约1-30mg/mL。具体操作时，水的量为50-100ml每克可溶性淀粉。申请人发现，淀粉溶液的浓度过高，则氧化过程不易进行完全，淀粉溶液的浓度过液，易造成氧化产物不均匀。
2.淀粉溶液的氧化
本发明采用了如下氧化体系：溴化钠(NaBr)-2，2，6，6-四甲基哌啶氧化物(TEMPO)-次氯酸钠(NaClO)氧化体系。经反应条件的调整，该氧化体系的氧化性尤其适用于获得本发明的α-1，4-寡聚葡萄糖醛酸。相比于常规氧化剂，本发明的氧化体系能够使得氧化反应更完全地进行，获得末端开环的反应产物，但不会破坏反应体系的均匀性。另外，以摩尔量计的氧化体系中各物质的比例可以是，例如TEMPO∶NaBr＝1∶5到1∶50； 淀粉中葡萄糖单元∶活性NaClO＝1∶0.5到1∶5；TEMPO∶淀粉中葡萄糖单元＝1∶1到1∶5。在本发明的一个实施方案中，次氯酸钠溶液中活性次氯酸钠的重量百分比为1至20％，优选2至15％，更优选3-12％。
氧化反应在碱性条件下进行，研究发现，最适合的pH值范围为10-11，反应体系的pH值过高，则氧化效率低下，pH值过低，则不利于氧化发生。反应的pH值通过碱性化合物控制，最优选的碱性化合物为NaOH溶液。对NaOH的溶液没有特别的限制。
适用于本发明的氧化反应的氧化温度为40至70℃，优选45-60℃，更优选48-55℃，此温度范围内进行氧化有利于除去淀粉中不期望存在的残留的酶，同时获得末端开环的糖醛酸。不囿于任何理论，发明人发现，反应温度低于40℃不利于获得末端开环的氧化型α-1，4-寡聚葡萄糖醛。反应温度过高会破坏淀粉原料的生物学活性，不利于反应产物的利用。
加入有机溶液终止反应以后，对氧化所获得的产物溶液进行纯化，获得氧化型α-1，4-寡聚葡萄糖醛酸。作为一种优选纯化的方式，可以通过透析法纯化所获得的寡聚葡萄糖醛酸，特别地，透析所使用的材料需截留分子量为500Da的物质，由此纯化本发明的低聚寡糖。纯化也可以采用其他本领域中已知的方式，只要确保透析所获得的寡聚葡萄糖醛酸的纯度高于99％、更优选高于99.5％以上即可。
本发明还涉及将氧化型α-1，4-寡聚葡萄糖醛酸或其混合物作为活性化合物成分用于制备抗脑缺血药物的用途。
脑血管疾病是导致我国中老年人死亡的第一大病因，也是世界卫生战略研究重点之一。在脑血管疾病中以缺血性疾病的发病率占据首位，通常在轻度缺血/缺氧的情况，脑的补偿机制保护着中枢神经系统免受损伤，但当缺血程度加重时，便会发生不可逆的神经损害，导致一系列的临床症状，甚至死亡。临床上，脑血管意外(例如中风)、心肌梗塞、休克、新生儿窒息和脑外伤都可引起神经元的缺血性损害。因此，开发出一种缓解脑缺血症状，提高脑细胞存活率的天然来源物质是很有意义 的。
本发明中使用的测定脑缺血症状的细胞模型是HT-22细胞在缺氧缺糖条件下产生的细胞(OGD模型)。HT-22细胞是一种小鼠海马神经元细胞系，是小鼠T4细胞系的一种亚克隆，具有海马神经元的特性。相关的内容可参见，例如，Jeney Ramírez-Sánchez et al.，Neurochemistry International 90(2015)215-223，“Neuroprotection by JM-20against oxygen-glucose deprivation in rat hippocampal slices：Involvement of the Akt/GSK-3βpathway”；Xiao-Jing Li et al.，Journal of Ethnopharmacology141(2012)927-933，Neuroprotective effects of TongLuoJiuNao in neurons exposed to oxygen and glucose deprivation；TIAN-ZHI ZHAO et al.，Neuroscience 328(2016)117-126，“GPER1Mediates Estrogen-Induced Neuroprotection Against Oxygen-Glucose Deprivation In The Primary Hippocampal Neurons”；等等。
本发明的发明人发现，通式I的氧化型α-1，4-寡聚葡萄糖醛酸或其混合物可以改善脑缺血症状，增加缺血缺氧脑细胞的存活率。另外，本发明的氧化型α-1，4-寡聚葡萄糖醛酸来自于天然产物，易于吸收和利用。
本发明提供了这样的组合药物，其中包含至少两种如上所述的氧化型α-1，4-寡聚葡萄糖醛酸，和任选的药学上可以接受的赋形剂。
制备各种含有各种比例活性成分的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或预防需要量，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。
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
实施例1一种氧化型α-1，4-寡聚葡萄糖醛酸的制备方法
(1)称取1g可溶性淀粉，溶解于50ml水中溶解，制成水溶液；
(2)向步骤(1)中制得的水溶液中依次加入5mg TEMPO和50mg溴化钠，用5％NaOH溶液调节pH至10，加入5ml次氯酸钠溶液，其以活性次氯酸计的浓度为5％重量，于50℃条件下反应5小时，加入无水乙醇终止反应；
(3)置于500Da的透析袋中透析，浓缩、冻干得到氧化型α-1，4-寡聚葡萄糖醛酸。
实施例2一种氧化型α-1，4-寡聚葡萄糖醛酸的制备方法
(1)称取1g可溶性淀粉，溶解于75ml水中溶解，制成水溶液；
(2)向步骤(1)中制得的水溶液中依次加入30mg TEMPO和350mg溴化钠，用20％NaOH溶液调节pH至10，加入10ml次氯酸钠溶液，其以活性次氯酸计的浓度为5％重量，于45℃条件下反应8小时，加入无水乙醇终止反应；
(3)置于500Da的透析袋中透析，浓缩、冻干得到氧化型α-1，4-寡聚葡萄糖醛酸。
实施例3一种氧化型α-1，4-寡聚葡萄糖醛酸的制备方法
(1)称取1g可溶性淀粉，溶解于100ml水中溶解，制成水溶液；
(2)向步骤(1)中制得的水溶液中依次加入50mg TEMPO和500mg溴化钠，用30％NaOH溶液调节pH至11，加入15ml次氯酸钠溶液，其以活性次氯酸计的浓度为5％重量，于55℃条件下反应10小时，加入甲醇终止反应；
(3)置于500Da的透析袋中透析，浓缩、冻干得到氧化型α-1，4-寡聚葡萄糖醛酸。
实施例4一种氧化型α-1，4-寡聚葡萄糖醛酸的制备方法
(1)称取1g可溶性淀粉，溶解于100ml水中溶解，制成水溶液；
(2)向步骤(1)中制得的水溶液中依次加入50mg TEMPO和600mg溴化钠，用50％NaOH溶液调节pH至11，加入15ml次氯酸钠溶液，于50℃条件下反应10小时，加入甲醇终止反应；
(3)置于500Da的透析袋中透析，浓缩、冻干得到氧化型α-1，4-寡聚葡萄糖醛酸。
实施例5一种氧化型α-1，4-寡聚葡萄糖醛酸质谱检测
5.1方法
称取实施例1中获得的2mg的氧化型α-1，4-寡聚葡萄糖醛酸用1ml的纯水溶解后，用0.22μm的微孔滤膜过滤后，进行超高效液相串联四级杆飞行时间质谱(UHPLC/Q-TOF-MS)分析。
UHPLC/Q-TOF-MS分析使用Agilent 6540UHD Accurate-Mass Q-TOF LC/MS(安捷伦公司，美国)系统。其色谱条件如下：ACQUITY UPLC BEH125分子排阻色谱柱(4.6×300mm，Waters)；检测波长：210nm；流动相：A为50mM乙酸铵水溶液，B为甲醇，比例为80％A；流速：0.1ml/min。质谱条件如下：负离子模式；扫描范围：100～3000；干燥气温度：350℃；干燥气流速：8L/min；毛细管电压：3500V；碎裂电压：80V。
5.2结果
氧化型α-1，4-寡聚葡萄糖醛酸通过UHPLC/Q-TOF-MS分析后，得到如下的总离子流图(TIC)和紫外色谱图(见图1)。从图1中可以看出，各峰呈规则的波浪状分布，因为使用的是分子排阻色谱柱，推测波浪峰应是按照聚合度由大到小分布。进一步通过各色谱峰对应的一级质谱图(图2～9)推测其结构：图2为峰1的一级质谱图，其中m/z808.1483、793.1434和778.1382都带2个电荷，经计算后得到这三个信号代表的化合物分子量分别为1618、1588和1558Da，对应的结构为氧化型α-1，4-寡聚葡萄糖醛酸9糖，如式I(n＝8，m＝0、1或2)。此外，该质谱图中还有少许带2个电荷的质谱信号m/z 896.1640、881.1638和866.1564，经计算后确认它们分别为三种分子量为1794、1764和 1734Da，对应的结构为氧化型α-1，4-寡聚葡萄糖醛酸10糖，如式I(n＝9，m＝0、1或2)。同理，图3～9为峰2～8的质谱图，经解析结构分别为氧化型α-1，4-寡聚葡萄糖醛酸8糖到2糖，如式I(n＝7→1，m＝0、1或2)。在色谱图中10到20糖没有很好的分离，但是在质谱中它们有较清晰的信号，如图10所示。
实施例6氧化型α-1，4-寡聚葡萄糖醛酸核磁结构确认
6.1方法
准确称取实施例1的产物样品25mg用0.5ml重水(D≥99.96％)溶解，内标三甲基硅烷基丙酸钠(TSP)浓度为0.2μg/ml，用600MHz核磁共振仪(Agilent，美国)分析。氢谱扫描时间1h，试验温度为室温。碳谱扫描时间12h以上，试验温度为室温。
6.2结果
核磁结果如图11(1H-NMR)和图12(13C-NMR)所示。从氢谱中可以看出，a(5.45～5.49ppm)表示与开环糖醛酸直接相连或邻近的α构型环状葡萄糖醛酸的1位H，b(5.23ppm)为与开环糖醛酸相隔较远的α构型环状葡萄糖醛酸1位H。h(3.74ppm)为葡萄糖醛酸还原端开环未脱30(-CH2O-)时羧基β位H，j(3.55ppm)为还原端开环未脱30(-CH2O-)时羧基γ位H或还原端开环脱30时羧基β位H。此外，葡萄糖醛酸还原端开环未脱、脱30、脱60结构中同时和羟基、羧基相连的H化学位移由于与水峰化学位移接近，所以在氢谱中看不到。其余峰为未开环糖醛酸环上的H。从碳谱中可以进一步看出区域1为羧基碳峰，而区域2为不同化学环境下的闭环糖醛酸1位C。结合氢谱、碳谱分析可以得出，α葡萄糖结构单元中的6位被成功氧化成羧基，且葡萄糖醛酸寡糖的还原端发生开环并伴随着一定程度的降解，进一步验证了质谱分析结果。
实施例7一种氧化型α-1，4-寡聚葡萄糖醛酸在缺氧缺糖(OGD)条件下对HT-22细胞的影响
7.1OGD模型的建立及实验分组
取正常培养的HT-22细胞，调整细胞数目至2×104个/ml，按100μl/ 孔接种在96孔培养板上，每组设4个平行(n＝4)。预培养12h后，给药组分别加入10μl的实施例1的氧化产物用培养基配制的不同浓度的样品(1、10、100μM)，正常组加入相同体积的培养基。将培养板置于5％CO2、37℃恒温培养箱中培养12h，观察并测定细胞存活率以确定样品对正常培养的HT-22细胞的影响。
取正常培养的HT-22细胞，吸去原培养液，细胞用无糖DMEM培养液洗涤2次，加入DMEM无糖培养液调整细胞数目至2×104个/ml。按100μl/孔接种在96孔培养板上。给药组分别加入10μl的实施例1的氧化产物用培养基配置的不同浓度的样品(1、10、100μM)，模型组加入相同体积的培养液。将培养板置于缺氧罐中(冲入95％N2、5％CO2)、37℃恒温培养12h，观察并测定细胞存活率以确定样品在OGD下对HT-22细胞的影响。
7.2MTT法测定细胞存活率
待上述各组细胞培养完毕后，每孔加入10μL MTT(5mg/ml)溶液，继续培养4h，加入100μl 10％SDS，待紫色结晶完全溶解后，测定其在570nm的OD值，并计算出细胞存活率。
7.3结果
通过MTT法计算出各组细胞存活率如表1、2和图13、14所示。从表1和图13中可以看出，加入低、中、高剂量的氧化型α-1，4-寡聚葡萄糖醛酸后细胞的存活率与正常组相比，无显著性差异，表明氧化型α-1，4-寡聚葡萄糖醛酸对HT-22细胞无毒性。
从表2中可以看出，模型组与对照组相比HT-22细胞的存活率显著降低(p＜0.001)，表明OGD对细胞的存活有明显的抑制作用。而加入氧化型α-1，4-寡聚葡萄糖醛酸(10μM)细胞存活率开始升高；当药物浓度达到100μM后细胞的存活率与模型组相比显著升高(p＜0.05)，表明氧化型α-1，4-寡聚葡萄糖醛酸有促进HT-22细胞生长存活的作用。
表1氧化型α-1，4-寡聚葡萄糖醛酸对正常HT-22细胞存活率的影响
Figure PCTCN2016082929-appb-000005
数据以mean±SEM表示
与对照组比较：p＜0.05有显著性差异(LSD法检验)
表2氧化型α-1，4-寡聚葡萄糖醛酸对OGD下HT-22细胞存活率的影响
Figure PCTCN2016082929-appb-000006
数据以mean±SEM表示
###表示与对照组比较p＜0.001(LSD法检验)
*表示与模型组比较p＜0.05(LSD法检验)
由上可见，本发明中的氧化型α-1，4-寡聚葡萄糖醛酸混合物具有良好的抗脑缺血作用，各分离的氧化型α-1，4-寡聚葡萄糖醛酸在类似的实验中也具有类似的结果，它们在制备抗脑缺血药物中的可以得到应用。
本发明尚有多种实施方式，凡采用等同变换或者等效变换而形成的所有技术方案，均落在本发明的保护范围之内。

Claims (23)

  1. 一种氧化型α-1，4-寡聚葡萄糖醛酸，其特征在于，聚合度为1-20糖，其具有通式I的结构：
    Figure PCTCN2016082929-appb-100001
    其中，n选自0-19；m选自0、1或2。
  2. 根据权利要求1的氧化型α-1，4-寡聚葡萄糖醛酸，其中m为0。
  3. 根据权利要求1的氧化型α-1，4-寡聚葡萄糖醛酸，其中m为1。
  4. 根据权利要求1的氧化型α-1，4-寡聚葡萄糖醛酸，其中m为2。
  5. 根据权利要求1的氧化型α-1，4-寡聚葡萄糖醛酸，其中n为1-19。
  6. 根据权利要求1的氧化型α-1，4-寡聚葡萄糖醛酸，其中n为1-9。
  7. 根据权利要求1的氧化型α-1，4-寡聚葡萄糖醛酸，其中n为1、2、3、4、5、6、7、8或9，各自对应于氧化型α-1，4-寡聚葡萄糖醛酸的二糖、三糖、四糖、五糖、六糖、七糖、八糖、九糖和十糖。
  8. 一种氧化型α-1，4-寡聚葡萄糖醛酸混合物，其由具有通式I’的结构的氧化型α-1，4-寡聚葡萄糖醛酸组成：
    Figure PCTCN2016082929-appb-100002
    其中，n’和m’分别为混合物中各寡聚葡萄糖醛酸的n和m的平均值，n’选自1.0-19.0；m’选自0-2.0。
  9. 根据权利要求8的混合物，其中n’选自1.0-10.0，m’选自0.5-1.8；优选n’选自1.0-9.0，m’选自0.8-1.5。
  10. 根据权利要求8或9所述的混合物，其中，n为1-9的组分占混合物的重量计为80％以上，优选90％以上，更优选95％以上。
  11. 一种根据权利要求1所述的氧化型α-1，4-寡聚葡萄糖醛酸的制备方法，包括以下步骤：
    (1)称取可溶性淀粉，制成水溶液；
    (2)向步骤(1)中制得的水溶液中依次加入2，2，6，6-四甲基哌啶氧化物和溴化钠，再使用碱性pH调节剂调节pH至10～11，之后加入次氯酸钠溶液于40～70℃条件下反应5～10小时，最后加入有机溶剂终止反应；
    (3)置于500Da的透析袋中透析，浓缩、冻干得到所述氧化型α-1，4-寡聚葡萄糖醛酸。
  12. 根据权利要求11所述的制备方法，其特征在于，所述步骤(1)中可溶性淀粉与水的质量体积比为1g∶(50～100)ml。
  13. 根据权利要求11所述的制备方法，其特征在于，所述步骤(2)中所述可溶性淀粉与2，2，6，6-四甲基哌啶氧化物的质量比为1000∶(5～50)。
  14. 根据权利要求11所述的制备方法，其特征在于，所述步骤(2)中溴化钠与2，2，6，6-四甲基哌啶氧化物的质量比大于等于10∶1。
  15. 根据权利要求11所述的制备方法，其特征在于，所述步骤(2)中可溶性淀粉与次氯酸钠溶液的质量体积比为1g∶(5～15) ml。
  16. 根据权利要求11所述的制备方法，其特征在于，所述步骤(2)中的有机溶剂为无水乙醇或甲醇。
  17. 根据权利要求11所述的制备方法，其特征在于，所述步骤(2)中碱性pH调节剂为5～50％NaOH溶液。
  18. 根据权利要求1-7中任一项所述的氧化型α-1，4-寡聚葡萄糖醛酸或者根据权利要求8-10任一项所述的氧化型α-1，4-寡聚葡萄糖醛酸混合物在制备抗脑缺血药物中的用途。
  19. 根据权利要求18所述的用途，所述抗脑缺血药物用于治疗或预防由中风、心肌梗塞、脑休克、新生儿窒息和脑外伤引起的神经元的缺血性损害。
  20. 根据权利要求1-7中任一项所述的氧化型α-1，4-寡聚葡萄糖醛酸或者根据权利要求8-10任一项所述的氧化型α-1，4-寡聚葡萄糖醛酸混合物在制备脑神经保护药物中的用途。
  21. 一种药物组合物，包含如权利要求1-7任一项所述的通式I的氧化型α-1，4-寡聚葡萄糖醛酸或者如权利要求8-10任一项所述的氧化型α-1，4-寡聚葡萄糖醛酸混合物，以及药学上可接受的赋形剂或载体。
  22. 一种治疗或预防受试者的由中风、心肌梗塞、脑休克、新生儿窒息和脑外伤引起的神经元缺血性损害的方法，包括给予需要的受试者有效量的如权利要求1-7任一项所述的通式I的氧化型α-1，4-寡聚葡萄糖醛酸或者如权利要求8-10任一项所述的氧化型α-1，4-寡聚葡萄糖醛酸混合物。
  23. 作为治疗或预防由中风、心肌梗塞、脑休克、新生儿窒息和脑外伤引起的神经元缺血性损害的药剂的权利要求1-7任一项所述的通式I的氧化型α-1，4-寡聚葡萄糖醛酸或者如权利要求8-10任一项所述的氧化型α-1，4-寡聚葡萄糖醛酸混合物。
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