WO2019149284A1 - 一种分离的防风多糖及其用途 - Google Patents

一种分离的防风多糖及其用途 Download PDF

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WO2019149284A1
WO2019149284A1 PCT/CN2019/074601 CN2019074601W WO2019149284A1 WO 2019149284 A1 WO2019149284 A1 WO 2019149284A1 CN 2019074601 W CN2019074601 W CN 2019074601W WO 2019149284 A1 WO2019149284 A1 WO 2019149284A1
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wind
linked
polysaccharide
glucose
arabinose
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PCT/CN2019/074601
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English (en)
French (fr)
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张真庆
徐乃玉
殷翔
庞力
沈路路
薛洁
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上海绿谷制药有限公司
苏州大学
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Application filed by 上海绿谷制药有限公司, 苏州大学 filed Critical 上海绿谷制药有限公司
Priority to US16/967,345 priority Critical patent/US20210032374A1/en
Priority to KR1020207024661A priority patent/KR20200118448A/ko
Priority to EP19748215.1A priority patent/EP3750923A4/en
Priority to JP2020564308A priority patent/JP2021512997A/ja
Publication of WO2019149284A1 publication Critical patent/WO2019149284A1/zh

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    • 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
    • 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
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • 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
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L99/00—Compositions of natural macromolecular compounds or of derivatives thereof not provided for in groups C08L89/00 - C08L97/00

Definitions

  • This application relates to the field of medicine.
  • the present application relates to an isolated wind-resistant polysaccharide (Saposhnikovia Divaricata polysaccharide, SDP) and its use in the preparation of a medicament for the treatment of diabetes or hyperlipidemia.
  • carbohydrates are not only an important class of structural and energy substances, but also have important biological functions.
  • the participation of carbohydrates in the mutual recognition and information transmission between cells is considered to be another important information molecule in addition to nucleic acids in organisms.
  • carbohydrates are also a key factor in cell surface signal recognition, antigen-antibody reaction, intercellular communication and perception. Therefore, research on biologically active polysaccharides has received increasing attention. Due to the complex structure of carbohydrates, their separation and structural identification are difficult. So far, only Yunzhi polysaccharide, Polyporus polysaccharide, Lentinus edodes, Schizophyllan, and Lycium barbarum polysaccharide have been used clinically. There is a need in the art for more biologically active polysaccharides.
  • the traditional Chinese medicine is a dry root of the unsucked stem plant of the Umbelliferae plant. Wind protection is often used to treat diseases such as cold headache, rheumatism, rubella, and tetanus.
  • a wind-resistant polysaccharide is a branched polysaccharide formed by a plurality of monosaccharides.
  • the wind-resistant polysaccharide is characterized by the composition of the monosaccharide contained therein and the manner in which it is attached.
  • the monosaccharide compositions of various weather-resistant polysaccharides prepared by different extraction methods and their attachment manners are not identical to each other. Dou Hongxia et al.
  • Diabetes mellitus refers to a metabolic disease characterized by hyperglycemia due to defects in insulin secretion and/or inability of insulin to exert normal physiological effects. Diabetes can also cause a variety of complications, such as diabetic heart disease, diabetic eye disease, diabetic vascular disease. Diabetes is a chronic disease that currently poses a serious threat to public health.
  • Hyperlipoidemia refers to a metabolic disease in which one or more lipid levels in the blood are abnormal (eg, multiple lipid levels are above normal). Hyperlipidemia is manifested by excessive levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) or low levels of high-density lipoprotein cholesterol (HDL-C) in the blood. In recent years, the incidence of hyperlipidemia has continued to rise. Hyperlipidemia is also closely related to some serious cardiovascular and cerebrovascular diseases (such as atherosclerosis, coronary heart disease, etc.).
  • the present invention provides an improved method of preparing a wind-resistant polysaccharide, the method comprising the step of gradient precipitation.
  • the isolated wind-proof polysaccharide of the present invention is completely different from the known wind-proof polysaccharide structure. It has been confirmed by animal experiments that the isolated wind-proof polysaccharide of the present invention has the potential to treat diabetes and regulate blood lipids.
  • L-arabinose L-Ara
  • D-galacturonic acid D-GlaA
  • D-Man D-mannose
  • D-glucose D-Glc
  • the molar ratio of L-arabinose: D-galacturonic acid: D-mannose: D-glucose: D-galactose is 1 -15:1-10:1-10:10-40:1-15.
  • the molar ratio of L-arabinose:D-galacturonic acid:D-mannose:D-glucose:D-galactose is 1-5:5-10:1-5:20- 25:1-5.
  • the isolated wind-resistant polysaccharide comprises monosaccharide components that are linked together in a particular manner.
  • the L-arabinose comprises 1,4-linked L-arabinose and/or 1,3,4-linked L-arabinose;
  • the D-galacturonic acid comprises a terminal D-galacturonic acid
  • the D-mannose comprises 1,6-linked D-mannose;
  • the D-glucose comprises 1,4-linked D-glucose and/or 1,3,6-linked D-glucose;
  • D-galactose includes a terminal D-galactose and/or a 1,4-linked D-galactose.
  • the L-arabinose described herein comprises 1,4-linked L-arabinose and/or 1,3,4-linked L-arabinose.
  • the D-galacturonic acid described herein comprises a terminal D-galacturonic acid.
  • the D-mannose described herein comprises 1,6-linked D-mannose.
  • the D-glucose described herein comprises 1,4-linked D-glucose and/or 1,3,6-linked D-glucose.
  • the D-galactose described herein comprises a terminal D-galactose and/or a 1,4-linked D-galactose.
  • the isolated wind-resistant polysaccharide comprises 1,4-linked L-arabinose, 1,3,4-linked L-arabinose, terminal D-galacturonic acid, 1,6 - linked D-mannose, 1,4-linked D-glucose, 1,3,6-linked D-glucose, terminal D-galactose and 1,4-linked D-galactose.
  • the present application provides an isolated wind-resistant polysaccharide comprising L-arabinose, D-galacturonic acid, D-mannose, D-glucose, and D-galactose, wherein the L - arabinose comprises 1,4-linked L-arabinose and/or 1,3,4-linked L-arabinose; said D-galacturonic acid comprises terminal D-galacturonic acid; D-mannose comprises 1,6-linked D-mannose; said D-glucose comprises 1,4-linked D-glucose and/or 1,3,6-linked D-glucose; said D- Galactose includes a terminal D-galactose and/or a 1,4-linked D-galactose.
  • one or more of the monosaccharide components are pyranoses; in a preferred embodiment, the monosaccharide component is a pyranose.
  • the isolated wind-resistant polysaccharide of the present application has a molecular weight of from 5 x 10 4 to 5 x 10 5 Da, preferably from 1 x 10 5 to 3.5 x 10 5 Da.
  • the present application provides a method of preparing an isolated wind-resistant polysaccharide, the method comprising the steps of:
  • the concentration of the organic solvent in the step (2) is preferably from 17 to 28%, more preferably from 20 to 25%.
  • the step (2) is also referred to as a first gradient precipitation.
  • the concentration of the organic solvent in the step (3) is preferably from 75 to 85%, more preferably from 80 to 85%.
  • the volume to weight ratio of water to wind in the step (1) is from 8:1 to 30:1, preferably from 20:1 to 30:1.
  • the extraction temperature in the step (1) is 40-100 ° C, preferably 60-100 ° C, more preferably 80-100 ° C, most preferably 90-95 ° C.
  • the extraction time in the step (1) is from 1 to 4 hours, preferably from 1 to 2 hours.
  • the number of times of taking out windproof with water in the step (1) is one time or more.
  • the number of times of windproof extraction with water in the step (1) is 1, 2, 3 or 4 times.
  • a step (3') between the steps (3) and (4) dissolving the precipitate obtained in the step (3) with water to obtain an aqueous solution, and adding an organic solvent to the aqueous solution to obtain an organic solvent.
  • the steps (3) and/or (3') are also referred to as second gradient precipitation.
  • the organic solvent in the step (2) and/or (3) and/or (3') is selected from the group consisting of methanol, ethanol, propanol, acetone, or a mixture thereof, preferably ethanol.
  • the wind protection described in the present application includes a commercially available medicinal material that is windproof (i.e., a dried root of a plant-resistant, undrawn stem plant) and a windproof piece.
  • the wind protection described herein is a windscreen.
  • isolated weather-resistant polysaccharide refers to a wind-proof polysaccharide obtained by separating a wind-proof polysaccharide from the natural environment of its original plant material by artificial means (for example, extraction, purification, etc.).
  • the plant material may be windproof in the form of a plant in the form of a wind or a medicinal material, such as a dried root or a windproof piece of a plant-resistant, undrawn stem plant.
  • the present application provides an isolated wind-resistant polysaccharide obtained according to the method of the present application.
  • the isolated windproof polysaccharide comprises a wind-proof polysaccharide of L-arabinose, D-galacturonic acid, D-mannose, D-glucose and D-galactose, wherein L-arabinose
  • the molar ratio of D-galacturonic acid:D-mannose:D-glucose:D-galactose is from 1 to 15:1 to 10:1 to 10:10 to 40:1 to 15, preferably from 1 to 5 : 5-10:1-5:20-25:1-5.
  • the L-arabinose comprises 1,4-linked L-arabinose and/or 1,3,4-linked L-arabinose;
  • the D-galacturonic acid Including a terminal D-galacturonic acid;
  • the D-mannose comprises 1,6-linked D-mannose;
  • the D-glucose comprises 1,4-linked D-glucose and/or 1,3, 6-linked D-glucose;
  • the D-galactose comprises a terminal D-galactose and/or a 1,4-linked D-galactose.
  • the 1,4-linked L-arabinose refers to an L-arabinose linked to an adjacent group (for example, an adjacent monosaccharide residue) via a glycosidic bond at the 1 and 4 positions of the sugar ring.
  • the 1,3,4-linked L-arabinose refers to an L-arabinose linked to an adjacent group (eg, an adjacent monosaccharide residue) via a glycosidic linkage at the 1, 3, and 4 positions of the sugar ring.
  • an adjacent group eg, an adjacent monosaccharide residue
  • the terminal group D-galacturonic acid refers to D-galacturonic acid linked to an adjacent group (for example, an adjacent monosaccharide residue) via a glycosidic bond at the 1-position of the sugar ring.
  • the 1,6-linked D-mannose refers to D-mannose attached to an adjacent group (for example, an adjacent monosaccharide residue) via a glycosidic bond at the 1 and 6 positions of the sugar ring.
  • the 1,4-linked D-glucose refers to D-glucose linked to an adjacent group (eg, an adjacent monosaccharide residue) via a glycosidic bond at the 1 and 4 positions of the sugar ring.
  • an adjacent group eg, an adjacent monosaccharide residue
  • the 1,3,6-linked D-glucose refers to D-glucose linked to an adjacent group (eg, an adjacent monosaccharide residue) via a glycosidic linkage at the 1, 3, and 6 positions of the sugar ring.
  • an adjacent group eg, an adjacent monosaccharide residue
  • the terminal group D-galactose refers to D-galactose linked to an adjacent group (for example, an adjacent monosaccharide residue) via a glycosidic bond at the 1-position of the sugar ring.
  • the 1,4-linked D-galactose refers to D-galactose linked to an adjacent group (for example, an adjacent monosaccharide residue) via a glycosidic bond at the 1 and 4 positions of the sugar ring.
  • sugars described herein may be in the alpha configuration or the beta configuration.
  • the present application provides the use of the isolated wind-resistant polysaccharide obtained by the present invention for the preparation of a medicament for treating diabetes or hyperlipidemia.
  • the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the isolated windproof polysaccharide obtained by the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a tablet, a capsule, a granule, a syrup, a suspension, a solution, a dispersing agent, a sustained release preparation for oral or parenteral administration, intravenous injection.
  • the pharmaceutically acceptable carrier described herein refers to a pharmaceutically acceptable carrier well known to those skilled in the art, and the pharmaceutically acceptable carrier of the present application includes, but is not limited to, a filler, a wetting agent, a binder, a disintegrating agent, Lubricants, binders, glidants, taste masking agents, surfactants, preservatives, and the like.
  • Fillers include, but are not limited to, lactose, microcrystalline cellulose, starch, powdered sugar, dextrin, mannitol, calcium sulfate, and the like.
  • wetting agents and binders include, but are not limited to, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, gelatin, sucrose, polyvinylpyrrolidone, and the like.
  • Disintegrators include, but are not limited to, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, croscarmellose sodium, low substituted hydroxypropylcellulose, 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 carboxymethylcellulose, sodium carboxymethylcellulose, glucose binders, dextrin, dextrose, ethylcellulose, gelatin, liquid glucose, guar Glue, 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.
  • 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.
  • Methods of preparing various pharmaceutical compositions 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 in 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 compositions described herein are made in a known manner, including conventional methods of mixing, dissolving or lyophilizing.
  • the proportion of active ingredient may vary from about 0.01% to about 99% by weight of a given unit dosage form.
  • the amount of active ingredient is such that an effective dosage level can be obtained.
  • the tablets, capsules and the like described herein may comprise: 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 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 such as mint, wintergreen or cherry.
  • tablets or capsules may be coated with gelatin, wax, shellac or sugar.
  • the syrup may contain the active ingredient, sucrose or fructose as a sweetener, methylparaben or propylparaben as a preservative, a dye and a flavoring such as cherry or orange flavor.
  • any material used to prepare any unit dosage form should be pharmaceutically acceptable and non-toxic in the amounts employed.
  • the active ingredient can be incorporated into sustained release formulations and sustained release devices.
  • the active ingredient can also be administered intravenously or intraperitoneally by infusion or injection.
  • An aqueous solution of the active ingredient or a salt thereof can be prepared, optionally mixed with a non-toxic surfactant.
  • 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.
  • a pharmaceutical composition suitable for injection or infusion may comprise a sterile aqueous solution of the active ingredient (optionally encapsulated in a liposome) comprising a ready-to-use preparation suitable for a sterile injectable or infusible solution or dispersion. Or a dispersing agent or a sterile powder.
  • the final dosage form must be sterile, liquid, and stable under the conditions of manufacture and storage.
  • the liquid carrier can be a solvent or liquid dispersion medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, 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 dispersing agents, or by the use of surfactants.
  • 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 ingredient in a suitable solvent with the various other ingredients listed above, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and freeze drying techniques which result in a powder of the active ingredient plus any additional ingredients present in the 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 pharmaceutical compositions of the present application 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 therapeutically effective amount of the active ingredient will depend not only on the particular salt selected, but also on the mode of administration, the nature of the condition to be treated, and the age and condition of the patient, and ultimately 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 a tablet.
  • the amount of unit dose of the active ingredient may vary or be adjusted between about 0.01 and about 1000 mg or more, depending on the particular treatment involved.
  • the present application provides the use of a pharmaceutical composition comprising a therapeutically effective amount of the isolated wind-resistant polysaccharide obtained according to the present invention for the manufacture of a medicament for the treatment of diabetes or hyperlipidemia.
  • the present application provides a method of treating diabetes or hyperlipidemia comprising administering to a subject in need thereof a therapeutically effective amount of the isolated wind-resistant polysaccharide obtained in accordance with the present invention.
  • the method of treating diabetes or hyperlipidemia comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a therapeutically effective amount of the wind-resistant polysaccharide obtained by the present invention. Things.
  • the invention also provides an isolated, weatherable polysaccharide for use in the treatment of diabetes or hyperlipidemia.
  • Treatment of diabetes as described herein includes lowering blood glucose levels (e.g., lowering fasting blood glucose levels), improving glucose tolerance, reducing islet cell damage, increasing insulin release, and the like.
  • the treatment of hyperlipidemia as described herein includes regulating blood lipid metabolism and regulating blood lipid levels (such as lowering blood lipid levels, such as lowering total cholesterol (TC), triglyceride (TG), and low density lipoprotein cholesterol in the blood ( Level of LDL-C).
  • wind-resistant polysaccharide described in the present application can also increase the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) in subjects (such as serum and liver), and reduce the activity of propylene The content of aldehyde (MDA).
  • SOD superoxide dismutase
  • GSH-px glutathione peroxidase
  • treating generally refers to obtaining the desired pharmacological and/or physiological effects.
  • the effect may be prophylactic according to the prevention of the disease or its symptoms in whole or in part; and/or may be therapeutic according to the partial or complete stabilization or cure of the disease and/or side effects due to the disease.
  • treatment encompasses any treatment for a patient's condition, including: (a) prevention of a disease or condition in a patient who is susceptible to an infectious disease or condition but has not yet diagnosed the disease; (b) inhibition of the symptoms of the disease, That is, to prevent its development; or (c) to alleviate the symptoms of the disease, that is, to cause the disease or symptoms to degenerate.
  • the percentages, ratios, ratios or parts stated herein are by volume unless otherwise indicated.
  • the volume to weight ratio described herein is a volume to weight ratio calculated in milliliters per gram (or liters per kilogram).
  • the concentration described herein is a volume concentration.
  • Figure 1 Effect of wind-resistant polysaccharide on STZ-induced islet tissue in diabetic mice.
  • Fig. 1A normal control group
  • Fig. 1B STZ 120 mg/kg group
  • Fig. 1C glibenclamide group 25 mg/kg group
  • Fig. 1D windproof polysaccharide 50 mg/kg group
  • Fig. 1E windproof polysaccharide 200 mg/kg group.
  • STZ Streptozotocin.
  • Figure 7 Effect of wind-resistant polysaccharide on liver morphology of hyperlipidemia mice.
  • Fig. 7A normal control group
  • Fig. 7B high fat model group
  • Fig. 7C force 40 mg/kg group
  • Fig. 7D windproof polysaccharide 50 mg/kg group
  • Fig. 7E windproof polysaccharide 200 mg/kg group.
  • Anti-wind drink tablets purchased from Anhui Luzhou Chinese herbal medicine market, producing Anguo; 95% ethanol, hydrochloric acid, sodium hydroxide, Coomassie brilliant blue, sulfuric acid, phenol, barium chloride, trifluoroacetic acid, sodium borohydride, dimethyl sulfoxide, etc. All purchased from Sinopharm Chemical Reagent Co., Ltd.; L-arabinose (L-Ara), D-mannose (D-Man), D-glucose (D-Glc), D-galactose (D-Gal), D - The galacturonic acid (D-GalA) control and 1-phenyl-3-methyl-5-pyrazolone (PMP) were purchased from Sigma.
  • step (3') 0.5 L of distilled water was added to the precipitate to dissolve it. Then, methanol was again added to obtain a mixture having a methanol concentration of 90%, and the mixture was centrifuged to obtain a precipitate. Repeat the operation of step (3') twice.
  • step (3') 0.5 L of distilled water was added to the precipitate to dissolve it. Then, ethanol was again added to obtain a mixture having an ethanol concentration of 80%, and the mixture was centrifuged to obtain a precipitate. The operation of the step (3') is repeated once.
  • Acetone was added to the concentrated extract to obtain a mixture having an acetone concentration of 20%, and the mixture was centrifuged to obtain a supernatant.
  • Example 1 The weather-resistant polysaccharide sulfate group content obtained in Example 8.
  • Multi-angle laser light scattering method see Ding Houqiang, Multi-angle laser light scattering instrument and size exclusion chromatography for the determination of relative molecular mass and distribution of hyaluronic acid, Food and Drug, 2009, 11(3): 24-26
  • the weight average molecular weight of the weather-resistant polysaccharide obtained in Examples 1-8 was measured.
  • the appropriate amount of the monosaccharide reference substance was weighed and formulated into a mother liquor having a concentration of 1 mg/mL. Pipette 10 ⁇ L of the mother liquor to a volume of 100 ⁇ L.
  • Derivatization treatment Take 50 ⁇ L of the reference solution, and sequentially add 100 ⁇ L of 0.3 mol/L NaOH solution, 120 ⁇ L of 0.5 mol/L 1-phenyl-3-methyl-5-pyrazolone in methanol and mix to obtain mixture. The mixed solution was reacted at 70 ° C for 60 minutes. After completion of the reaction, the solution was cooled to room temperature, and an appropriate amount of 0.3 mol/L of HCl was added to adjust the pH to neutrality, and then extracted with 1 mL of chloroform and the organic phase was discarded. A sample which was completely hydrolyzed by acidic conditions of 50 ⁇ L of the polysaccharide was taken, and derivatization treatment was also carried out in accordance with the above method.
  • the literature method (square product, methylation analysis method of polysaccharide, foreign medicine (Pharmaceutical Section), 1986, (4): 222-226) was used to methylate the windproof polysaccharide of Example 1-8, respectively.
  • the methylated product was depolymerized with 90% formic acid and hydrolyzed with 2 mol/L TFA to give a methylated monosaccharide.
  • the resulting methylated monosaccharide was then reduced with NaBH 4 and acetylated with acetic anhydride to make an aldiol acetate derivative of the methylated monosaccharide, which was then subjected to GC-MS analysis.
  • the windproof polysaccharide of Example 1-8 contained the following monosaccharides: 1,4-L-arabinose, 1,3,4-L-arabinose, terminal D-galacturonic acid, 1,6-D-mannose, 1,4-D-glucose, 1,3,6-D-glucose, terminal D-galactose, and 1,4-D-galactose.
  • the results of methylation analysis are shown in Table 4-11.
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3-Me 2 -L-Ara 1,4-L-arabinose 1 2-Me-L-Ara 1,3,4-L-arabinose 4 2,3,4-Me 3 -D-GalA Terminal D-galacturonic acid 6 2,3,4-Me 3 -D-Man 1,6-D-mannose 2 2,3,6-Me 3 -D-Glc 1,4-D-glucose 20 2,4-Me 2 -D-Glc 1,3,6-D-glucose 3 2,3,4,6-Me 4 -D-Gal Terminal group-D-galactose 1 2,3,6-Me 3 -D-Gal 1,4-D-galactose 4
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3-Me 2 -L-Ara 1,4-L-arabinose 1 2-Me-L-Ara 1,3,4-L-arabinose 1
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3-Me 2 -L-Ara 1,4-L-arabinose 1 2-Me-L-Ara 1,3,4-L-arabinose 3 2,3,4-Me 3 -D-GalA Terminal D-galacturonic acid 7 2,3,4-Me 3 -D-Man 1,6-D-mannose 4 2,3,6-Me 3 -D-Glc 1,4-D-glucose twenty four 2,4-Me 2 -D-Glc 1,3,6-D-glucose 4 2,3,4,6-Me 4 -D-Gal Terminal group-D-galactose 1 2,3,6-Me 3 -D-Gal 1,4-D-galactose 4
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3-Me 2 -L-Ara 1,4-L-arabinose 3 2-Me-L-Ara 1,3,4-L-arabinose 10 2,3,4-Me 3 -D-GalA Terminal D-galacturonic acid 9 2,3,4-Me 3 -D-Man 1,6-D-mannose 3 2,3,6-Me 3 -D-Glc 1,4-D-glucose 16 2,4-Me 2 -D-Glc 1,3,6-D-glucose 9 2,3,4,6-Me 4 -D-Gal Terminal group-D-galactose 5 2,3,6-Me 3 -D-Gal 1,4-D-galactose 9
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3-Me 2 -L-Ara 1,4-L-arabinose 4 2-Me-L-Ara 1,3,4-L-arabinose 2 2,3,4-Me 3 -D-GalA Terminal D-galacturonic acid 6 2,3,4-Me 3 -D-Man 1,6-D-mannose 7 2,3,6-Me 3 -D-Glc 1,4-D-glucose twenty two 2,4-Me 2 -D-Glc 1,3,6-D-glucose 2 2,3,4,6-Me 4 -D-Gal Terminal group-D-galactose 1 2,3,6-Me 3 -D-Gal 1,4-D-galactose 1
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3-Me 2 -L-Ara 1,4-L-arabinose 1
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3-Me 2 -L-Ara 1,4-L-arabinose 1 2-Me-L-Ara 1,3,4-L-arabinose 3 2,3,4-Me 3 -D-GalA Terminal D-galacturonic acid 7 2,3,4-Me 3 -D-Man 1,6-D-mannose 4 2,3,6-Me 3 -D-Glc 1,4-D-glucose 20 2,4-Me 2 -D-Glc 1,3,6-D-glucose 1 2,3,4,6-Me 4 -D-Gal Terminal group-D-galactose 1 2,3,6-Me 3 -D-Gal 1,4-D-galactose 2
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3-Me 2 -L-Ara 1,4-L-arabinose 3 2-Me-L-Ara 1,3,4-L-arabinose 2 2,3,4-Me 3 -D-GalA Terminal D-galacturonic acid 6 2,3,4-Me 3 -D-Man 1,6-D-mannose 3 2,3,6-Me 3 -D-Glc 1,4-D-glucose 20 2,4-Me 2 -D-Glc 1,3,6-D-glucose 5 2,3,4,6-Me 4 -D-Gal Terminal group-D-galactose 2 2,3,6-Me 3 -D-Gal 1,4-D-galactose 2
  • Example 1 Windproof polysaccharide, administered at a dose of 50 mg/kg (low dose) and 200 mg/kg (high dose), respectively.
  • Experimental reagent glucose determination kit, provided by Shanghai Rongsheng Biological Pharmaceutical Co., Ltd.; insulin ELISA assay kit, provided by Shanghai Xitang Industrial Co., Ltd.; TC, TG, HDL-C, SOD, MDA and Coomassie brilliant blue protein
  • the assay kits were provided by the Nanjing Institute of Bioengineering.
  • mice were housed for 3 days at a temperature of 20 ⁇ 2 ° C and under a humidity of 50 ⁇ 5% under 12 hours of light and 12 hours of darkness, during which time the mice were free to take water.
  • Ten healthy mice were selected as the normal control group (Control).
  • the remaining mice were intraperitoneally injected with a solution of streptozotocin (STZ) at 0.2 ml/10 g body weight.
  • the fast blood glucose (FBG) of the mice was measured 72 hours later. Mice with FBG values higher than 11.1 mmol/L and less than 25 mmol/L were selected as diabetic model mice.
  • Diabetic model mice were randomly divided into a model group (STZ 120 mg/kg), a low-dose wind-resistant polysaccharide group (SDP 50 mg/kg), a high-dose wind-resistant polysaccharide (SDP 200 mg/kg) group, and a positive drug (glibenclamide, GLI 25 mg). /kg)
  • Control group 10 mice per group. For each administration group, it was administered orally at 0.2 ml/10 g body weight at 8:00 am every day. The normal control group and the model group mice were given an equal volume of distilled water. During the experiment, mice in each group were given free access to water and FBG and body weight were measured every 10 days. Mice were treated 30 days after administration, and the following indicators were measured.
  • Glucose tolerance fasting was performed at 8:00 on the day before the end of the experiment. After 4 hours, blood was taken from the eye of the mouse and FBG was measured as the blood glucose level before glucose administration (0h), and then all mice were administered at 0.2 ml/10 g body weight. A 10% glucose solution was injected intraperitoneally. Blood was taken from the eye of the mouse and the blood glucose level was measured after intraperitoneal injection of glucose for 0.5 hours (0.5 h) and 2 hours (2 h), and the area under the blood glucose curve (AUC) was calculated according to the following formula.
  • the blood glucose levels of the model group mice were significantly higher than those of the normal control group (P ⁇ 0.01).
  • the wind-resistant polysaccharide can significantly reduce the blood glucose level of diabetic mice (P ⁇ 0.05).
  • the glucose tolerance of the model group was significantly decreased, and the area under the blood glucose curve was significantly increased (P ⁇ 0.01).
  • the wind-resistant polysaccharide could significantly improve the glucose tolerance (P ⁇ 0.05 or P ⁇ 0.01) and significantly reduce the area under the blood glucose curve (P ⁇ 0.01).
  • FIG. 1B Compared with the normal control group, STZ-induced diabetic mice had nuclear pyknosis, hyaline lesions, inflammatory cell infiltration, and islet morphology in the islet tissue (Fig. 1B).
  • the number of islets in the wind-proof polysaccharide group was not significantly reduced under the low-power microscope.
  • the damage of the islets of the wind-proof polysaccharide group was significantly reduced and the morphology was relatively complete (Fig. 1D, 1E).
  • Lipid toxicity is an important factor in the pathogenesis of type 2 diabetes.
  • serum TC and TG levels were significantly increased in the STZ group (model group) compared with the normal control (P ⁇ 0.01).
  • low-dose and high-dose wind-proof polysaccharides significantly reduced serum TC and TG levels in diabetic mice in a dose-dependent manner (P ⁇ 0.05 or P ⁇ 0.01).
  • serum HDL-C levels were not significantly affected between the groups.
  • the wind-proof polysaccharide can reduce the serum MDA content of diabetic mice.
  • high-dose wind-proof polysaccharides can significantly reduce MDA content in mice serum (P ⁇ 0.01); low-dose and high-dose wind-proof polysaccharides can significantly reduce MDA content in mice (P ⁇ 0.05).
  • the wind-proof polysaccharide can increase the serum SOD activity of the mouse.
  • high-dose wind-proof polysaccharides can significantly increase serum SOD activity in mice (P ⁇ 0.05); low-dose and high-dose wind-proof polysaccharides can increase liver SOD activity in mice, and high-dose wind-proof polysaccharide can significantly increase mice. Liver SOD activity (P ⁇ 0.01).
  • the wind-resistant polysaccharide can significantly reduce the fasting blood glucose level of STZ-induced diabetic mice and significantly increase the glucose tolerance of diabetic mice. Moreover, the wind-resistant polysaccharide can also reduce the damage of mouse islets, increase the release of insulin, and significantly reduce the levels of TC and TG in the serum of hyperglycemic mice. The wind-resistant polysaccharide can also increase the activity of SOD in serum and liver and reduce the content of MDA.
  • Example 1 Windproof polysaccharide was administered at a dose of 50 mg/kg (low dose) and 200 mg/kg (high dose), respectively.
  • mice were housed for 3 days at a temperature of 20 ⁇ 2 ° C and under a humidity of 50 ⁇ 5% under 12 hours of light and 12 hours of darkness, during which time the mice were free to take water.
  • the mice were randomly divided into 5 groups: normal control group (normal group), high-fat model group, positive drug Liping (Lipanthyl, fenofibrate, 40 mg/kg) group, low-dose wind-proof polysaccharide group (SDP 50 mg/kg). ) and high-dose wind-proof polysaccharide group (SDP 200mg/kg), 10 in each group.
  • mice For each administration group, different doses of the drug were administered at a dose of 0.2 ml/10 g body weight from 8:00 to 9:00 daily. An equal volume of distilled water was administered to the normal control group and the high fat model group. Except the normal control group, each group of mice was given a high-fat diet (containing 20% lard, 10% cholesterol, 0.2% propylthiouracil, orally) at a dose of 0.2 ml/10 g body weight from 14:00 to 15:00 daily. 20% propylene glycol and 20% Tween-80) for 3 weeks to investigate the role of wind-proof polysaccharides in preventing hyperlipidemia. At the end of the experiment, the mice were fasted for 8 hours after treatment.
  • a high-fat diet containing 20% lard, 10% cholesterol, 0.2% propylthiouracil, orally
  • Blood was taken from the eye of the mouse and serum was obtained from the blood by centrifugation. Serum TC, TG, LDL-C and HDL-C were measured. Part of the liver tissue of the mice was taken and homogenized, and the contents of liver TC and TG, as well as SOD, GSH-px activity and MDA content were determined. Another part of the liver tissue of the mice was taken and fixed with 10% formaldehyde and subjected to morphological examination.
  • the liver TC and TG content (P ⁇ 0.01) and liver weight coefficient (P ⁇ 0.01) were significantly increased in the high-fat model group.
  • the wind-resistant polysaccharide can reduce liver TC, TG content and liver weight coefficient.
  • low-dose and high-dose wind-resistant polysaccharides significantly reduced liver TC content (P ⁇ 0.01).
  • high-dose wind-proof polysaccharide could significantly reduce liver TC, TG content and liver weight coefficient (P ⁇ 0.05 or P ⁇ 0.01).
  • the positive drug level could significantly reduce the liver TC content (P ⁇ 0.01), the liver weight coefficient was significantly increased. Therefore, the anti-wind polysaccharide, especially the high-dose wind-proof polysaccharide, is significantly better than the positive drug in reducing TC and TG, especially in reducing the hepatic weight coefficient.
  • FIG. 7A the liver of the normal control group was intact, the hepatic cord was clearly visible, and no significant lipid vacuoles were observed.
  • Fig. 7B A large amount of lipid vacuoles were seen in the liver of the high-fat model mice when the mice were given a high-fat diet for 3 weeks (Fig. 7B).
  • the wind-proof polysaccharide can significantly improve the lipid vacuoles of the liver; the high-dose wind-proof polysaccharide has a better effect (Fig. 7D, 7E).
  • SOD and GSH-px are antioxidant enzymes in the liver that reduce the amount of reactive oxygen species and reduce the damage of liver cells by lipid peroxidation.
  • Experimental results Figure 8 shows that compared with the normal control, the activity of SOD and GSH-px in the liver of the high-fat model group was significantly decreased (P ⁇ 0.01), and the MDA content was significantly increased (P ⁇ 0.05).
  • the wind-resistant polysaccharide increased the activity of SOD and GSH-px in the liver of mice, and significantly decreased the MDA content.
  • low-dose and high-dose wind-proof polysaccharides can significantly increase the activity of SOD (P ⁇ 0.05 or P ⁇ 0.01) in liver of mice, and significantly increase the activity of GSH-px (P ⁇ 0.05 or P ⁇ 0.01) in liver of mice. MDA content was significantly reduced (P ⁇ 0.05).
  • the wind-resistant polysaccharide can significantly reduce the levels of serum TC, TG and LDL-C in hyperlipidemic mice induced by high-fat diet, and also reduce liver weight coefficient and liver TC and TG levels, and significantly reduce lipids in liver. Empty bubble. Moreover, the wind-resistant polysaccharide can increase the activity of SOD and GSH-px in the liver, and decrease the content of MDA.

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Abstract

本申请涉及医药领域。本申请涉及一种分离的防风多糖及其在制备用于治疗糖尿病或高脂血症的药物中的用途。特别地，本申请涉及一种包含L-阿拉伯糖、D-半乳糖醛酸、D-甘露糖、D-葡萄糖和D-半乳糖的分离的防风多糖，其中L-阿拉伯糖∶D-半乳糖醛酸∶D-甘露糖∶D-葡萄糖∶D-半乳糖的摩尔比为1-15∶1-10∶1-10∶10-40∶1-15，优选为1-5∶5-10∶1-5∶20-25∶1-5。

Description

一种分离的防风多糖及其用途 技术领域
本申请涉及医药领域。具体而言，本申请涉及一种分离的防风多糖(Saposhnikovia Divaricata polysaccharide，SDP)及其在制备用于治疗糖尿病或高脂血症的药物中的用途。
背景技术
近年的研究发现，糖类物质不仅是一类重要的结构物质和能量物质，而且还具有重要的生物学功能。糖类物质参与细胞间的相互识别及信息传递过程，被认为是生物体内除核酸以外的又一类重要的信息分子。而且，糖类物质还是细胞表面信号识别、抗原抗体反应、细胞间信息传递和感受的关键因子。因此，对具有生物活性的多糖的研究日益受到重视。由于糖类物质结构复杂，其分离及结构鉴定均很困难。到目前为止，只有云芝多糖、猪苓多糖、香菇多糖、裂褶多糖、茯苓多糖等用于临床。本领域中需要更多具有生物活性的多糖。
中药防风为伞形科植物防风的未抽花茎植株的干燥根。防风常用于治疗感冒头痛、风湿麻痹、风疹瘙痒、和破伤风等疾病。
防风主要的化学成分有挥发油、色原酮、香豆素、有机酸、防风多糖等。防风多糖是一种由多种单糖连接成的支链多糖。通常，通过其中所含的单糖组成及其连接方式来表征防风多糖。通过不同提取方法制得的各种防风多糖的单糖组成及其连接方式彼此并不相同。窦红霞等(防风的化学成分与药理作用研究进展，中医药信息，2009，26(2)，15)从防风中提取得到多种防风多糖：XC-1(平均分子量为13100)、XC-2(平均分子量为73500)、Saponikovan A、B、C(分子量分别为54000、280000、132000)。防风多糖在本领域中通常用于抗肿瘤、抗氧化、提高机体免疫力等。迄今尚未报道防风 多糖用于治疗糖尿病或高脂血症。
糖尿病(diabetes mellitus)是指由于胰岛素分泌缺陷和/或胰岛素不能发挥正常生理作用所致的以高血糖为特征的代谢疾病。糖尿病还可以引发多种并发症，例如糖尿病性心脏病、糖尿病性眼病、糖尿病性血管病等。糖尿病是当前严重威胁公共健康的慢性疾病。
高脂血症(Hyperlipoidemia)是指血液中一种或多种脂质水平异常(例如，多种脂质水平高于正常水平)的代谢疾病。高脂血症表现为血液中总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白胆固醇(LDL-C)水平过高或高密度脂蛋白胆固醇(HDL-C)水平过低。近年来，高脂血症的发病率不断上升。高脂血症还与一些严重的心脑血管疾病(比如动脉粥样硬化、冠心病等)密切相关。
发明内容
由于糖类物质结构复杂，不同的提取方式会直接影响到多糖的结构组成，从而影响药效。本发明提供一种改进的制备防风多糖的方法，所述方法包括梯度沉淀的步骤。经结构分析，发现本发明所述的分离的防风多糖与已知的防风多糖结构完全不相同。经动物试验验证，发现本发明所述分离的防风多糖具有潜在的治疗糖尿病和调节血脂的作用。
本申请的一个方面提供了一种包含L-阿拉伯糖(L-Ara)、D-半乳糖醛酸(D-GlaA)、D-甘露糖(D-Man)、D-葡萄糖(D-Glc)和D-半乳糖(D-Gal)等单糖的分离的防风多糖，其中L-阿拉伯糖∶D-半乳糖醛酸∶D-甘露糖∶D-葡萄糖∶D-半乳糖的摩尔比为1-15∶1-10∶1-10∶10-40∶1-15。在一种实施方式中，L-阿拉伯糖∶D-半乳糖醛酸∶D-甘露糖∶D-葡萄糖∶D-半乳糖的摩尔比为1-5∶5-10∶1-5∶20-25∶1-5。
在一种实施方式中，所述分离的防风多糖包含的单糖组分以特定的方式相互连接在一起。所述L-阿拉伯糖包括1，4-连接的L-阿拉伯糖和/或1，3，4-连接的L-阿拉伯糖；所述D-半乳糖醛酸包括端基D- 半乳糖醛酸；所述D-甘露糖包括1，6-连接的D-甘露糖；所述D-葡萄糖包括1，4-连接的D-葡萄糖和/或1，3，6-连接的D-葡萄糖；所述D-半乳糖包括端基D-半乳糖和/或1，4-连接的D-半乳糖。
在一种优选的实施方式中，本申请所述L-阿拉伯糖包括1，4-连接的L-阿拉伯糖和/或1，3，4-连接的L-阿拉伯糖。
在一种优选的实施方式中，本申请所述D-半乳糖醛酸包括端基D-半乳糖醛酸。
在一种优选的实施方式中，本申请所述D-甘露糖包括1，6-连接的D-甘露糖。
在一种优选的实施方式中，本申请所述D-葡萄糖包括1，4-连接的D-葡萄糖和/或1，3，6-连接的D-葡萄糖。
在一种优选的实施方式中，本申请所述D-半乳糖包括端基D-半乳糖和/或1，4-连接的D-半乳糖。
在一种实施方式中，所述分离的防风多糖包含1，4-连接的L-阿拉伯糖、1，3，4-连接的L-阿拉伯糖、端基D-半乳糖醛酸、1，6-连接的D-甘露糖、1，4-连接的D-葡萄糖、1，3，6-连接的D-葡萄糖、端基D-半乳糖和1，4-连接的D-半乳糖。在另一优选实施方式中，所述1，4-连接的L-阿拉伯糖∶1，3，4-连接的L-阿拉伯糖∶端基D-半乳糖醛酸∶1，6-连接的D-甘露糖∶1，4-连接的D-葡萄糖∶1，3，6-连接的D-葡萄糖∶端基D-半乳糖∶1，4-连接的D-半乳糖的摩尔比为1-5∶1-10∶1-10∶1-10∶10-30∶1-10∶1-5∶1-10，优选为1-3∶1-5∶5-10∶1-5∶15-25∶1-5∶1-3∶1-5。
在一种优选的实施方式中，本申请提供一种包含L-阿拉伯糖、D-半乳糖醛酸、D-甘露糖、D-葡萄糖和D-半乳糖的分离的防风多糖，其中所述L-阿拉伯糖包括1，4-连接的L-阿拉伯糖和/或1，3，4-连接的L-阿拉伯糖；所述D-半乳糖醛酸包括端基D-半乳糖醛酸；所述D-甘露糖包括1，6-连接的D-甘露糖；所述D-葡萄糖包括1，4-连接的D-葡萄糖和/或1，3，6-连接的D-葡萄糖；所述D-半乳糖包括端基D-半乳糖和/或1，4-连接的D-半乳糖。在进一步优选的实施方式中，所述1，4- 连接的L-阿拉伯糖∶1，3，4-连接的L-阿拉伯糖∶端基D-半乳糖醛酸∶1，6-连接的D-甘露糖∶1，4-连接的D-葡萄糖∶1，3，6-连接的D-葡萄糖∶端基D-半乳糖∶1，4-连接的D-半乳糖的摩尔比为1-5∶1-10∶1-10∶1-10∶10-30∶1-10∶1-5∶1-10，优选为1-3∶1-5∶5-10∶1-5∶15-25∶1-5∶1-3∶1-5。
在另一种实施方式中，所述单糖组分中的一种或多种是吡喃糖；在一种优选的实施方式中，所述单糖组分都是吡喃糖。
在一种优选的实施方式中，本申请所述分离的防风多糖分子量为5×10 4至5×10 5Da，优选为1×10 5至3.5×10 5Da。
本申请在另一个方面提供一种制备分离的防风多糖的方法，所述方法包括以下步骤：
(1)用水一次或多次提取防风，得到防风水提取液，任选浓缩所述防风水提取液；
(2)向所述任选浓缩的防风水提取液加入有机溶剂以得到有机溶剂浓度为15-30％的混合物，离心处理所述混合物以得到上清液；
(3)向所述上清液中加入有机溶剂以得到有机溶剂浓度为70-90％的混合物，离心处理所述混合物以得到沉淀；
(4)干燥所述沉淀，以得到所述的分离的防风多糖。
在一种实施方式中，所述步骤(2)中的有机溶剂浓度优选为17-28％，更优选20-25％。在一些实施方式中，所述步骤(2)也被称为第一梯度沉淀。
在一种实施方式中，所述步骤(3)中的有机溶剂浓度优选为75-85％，更优选80-85％。
在一种实施方式中，所述步骤(1)中水与防风的体积重量比为8∶1至30∶1，优选20∶1至30∶1。
在一种实施方式中，所述步骤(1)中所述提取温度为40-100℃，优选60-100℃，更优选80-100℃、最优选90-95℃。
在一种实施方式中，所述步骤(1)中所述提取时间为1-4小时，优选1-2小时。
在一种实施方式中，所述步骤(1)中用水提取防风的次数为一次以上。
在另一种优选实施方式中，所述步骤(1)中用水提取防风的次数为1、2、3或4次。
在一种实施方式中，所述步骤(3)和(4)之间还存在步骤(3’)：用水溶解步骤(3)所得沉淀以得到水溶液，向所述水溶液加入有机溶剂以得到有机溶剂浓度为70-90％、优选为75-85％、更优选80-85％的混合物，离心处理所述混合物以得到沉淀；步骤(3’)可以重复一次或多次，优选1、2或3次。
在一些实施方式中，所述步骤(3)和/或(3’)也被称为第二梯度沉淀。
在一种实施方式中，所述步骤(2)和/或(3)和/或(3’)中所述有机溶剂选自甲醇、乙醇、丙醇、丙酮、或其混合物，优选乙醇。
本申请所述防风包括商业上可获得的药材防风(即植物防风的未抽花茎植株的干燥根)和防风饮片。在一种实施方式中，本申请所述防风是防风饮片。
术语“分离的防风多糖”是指通过人工手段(例如提取、纯化等)将防风多糖从其原始植物原料的自然环境中分离出来而得到的防风多糖。所述植物原料可以是植物形式的防风或药材形式的防风，例如植物防风的未抽花茎植株的干燥根或防风饮片。
本申请在另一个方面提供一种分离的防风多糖，其是根据本申请所述方法获得的。在一种优选的实施方式中，所述分离的防风多糖包含L-阿拉伯糖、D-半乳糖醛酸、D-甘露糖、D-葡萄糖和D-半乳糖的防风多糖，其中L-阿拉伯糖∶D-半乳糖醛酸∶D-甘露糖∶D-葡萄糖∶D-半乳糖的摩尔比为1-15∶1-10∶1-10∶10-40∶1-15，优选为1-5∶5-10∶1-5∶20-25∶1-5。在一种优选的实施方式中，所述L-阿拉伯糖包括1，4-连接的L-阿拉伯糖和/或1，3，4-连接的L-阿拉伯糖；所述D-半乳糖醛酸包括端基D-半乳糖醛酸；所述D-甘露糖包括1，6- 连接的D-甘露糖；所述D-葡萄糖包括1，4-连接的D-葡萄糖和/或1，3，6-连接的D-葡萄糖；所述D-半乳糖包括端基D-半乳糖和/或1，4-连接的D-半乳糖。在一种进一步的优选实施方式中，所述1，4-连接的L-阿拉伯糖∶1，3，4-连接的L-阿拉伯糖∶端基D-半乳糖醛酸∶1，6-连接的D-甘露糖∶1，4-连接的D-葡萄糖∶1，3，6-连接的D-葡萄糖∶端基D-半乳糖∶1，4-连接的D-半乳糖的摩尔比为1-5∶1-10∶1-10∶1-10∶10-30∶1-10∶1-5∶1-10，优选为1-3∶1-5∶5-10∶1-5∶15-25∶1-5∶1-3∶1-5。
所述1，4-连接的L-阿拉伯糖是指通过糖环1和4位上的糖苷键与相邻基团(例如相邻单糖残基)连接的L-阿拉伯糖。
所述1，3，4-连接的L-阿拉伯糖是指通过糖环1、3和4位上的糖苷键与相邻基团(例如相邻单糖残基)连接的L-阿拉伯糖。
所述端基D-半乳糖醛酸是指通过糖环1位上的糖苷键与相邻基团(例如相邻单糖残基)连接的D-半乳糖醛酸。
所述1，6-连接的D-甘露糖是指通过糖环1和6位上的糖苷键与相邻基团(例如相邻单糖残基)连接的D-甘露糖。
所述1，4-连接的D-葡萄糖是指通过糖环1和4位上的糖苷键与相邻基团(例如相邻单糖残基)连接的D-葡萄糖。
所述1，3，6-连接的D-葡萄糖是指通过糖环1、3和6位上的糖苷键与相邻基团(例如相邻单糖残基)连接的D-葡萄糖。
所述端基D-半乳糖是指通过糖环1位上的糖苷键与相邻基团(例如相邻单糖残基)连接的D-半乳糖。
所述1，4-连接的D-半乳糖是指通过糖环1和4位上的糖苷键与相邻基团(例如相邻单糖残基)连接的D-半乳糖。
本申请所述糖可以是α构型或β构型。
本申请在另一个方面提供了本发明所获得的分离的防风多糖在制备治疗糖尿病或高脂血症药物中的用途。
本申请在另一个方面提供了一种药物组合物，包含治疗有效量的本发明所获得的分离的防风多糖，以及药学上可接受的载体。
在一种优选的实施方式中，所述药物组合物是片剂、胶囊剂、颗粒剂、糖浆剂、悬浮液、溶液、分散剂、用于口服或非口服给药的缓释制剂、静脉注射制剂、皮下注射制剂、吸入制剂、透皮制剂、直肠或阴道栓剂。
本申请所述药学上可接受载体是指本领域技术人员熟知的药学上可接受载体，本申请的药学上可接受载体包括但不限于：填充剂、润湿剂、黏合剂、崩解剂、润滑剂、粘合剂、助流剂、掩味剂、表面活性剂、防腐剂等。填充剂包括但不限于乳糖、微晶纤维素、淀粉、糖粉、糊精、甘露醇、硫酸钙等。润湿剂与黏合剂包括但不限于羧甲基纤维素钠、羟丙基纤维素、羟丙基甲基纤维素、明胶、蔗糖、聚乙烯吡咯烷酮等。崩解剂包括但不限于羧甲基淀粉钠、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、低取代羟丙基纤维素等。润滑剂包括但不限于硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇、月桂醇硫酸镁等。粘合剂包括但不限于阿拉伯胶、藻酸、羧甲基纤维素钙、羧甲基纤维素钠、葡萄糖结合剂、糊精、右旋糖、乙基纤维素、明胶、液体葡萄糖、瓜尔胶、羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素、硅酸铝镁、麦芽糖糊精、甲基纤维素、聚甲基丙烯酸酯、聚乙烯吡咯烷酮、预明胶化淀粉、藻酸钠、山梨醇、淀粉、糖浆和黄蓍胶。助流剂包括但不限于胶体二氧化硅、粉状纤维素、三硅酸镁、二氧化硅和滑石粉。掩味剂包括但不限于阿斯巴坦、甜菊苷、果糖、葡萄糖、糖浆、蜂蜜、木糖醇、甘露醇、乳糖、山梨醇、麦芽糖醇、甘草甜素。表面活性剂包括但不限于吐温-80、泊洛沙姆。防腐剂包括但不限于尼泊金酯、苯甲酸钠、山梨酸钾等。
制备各种含有各种比例活性成分的药物组合物的方法是已知的，或根据本申请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 的方法制造本申请所述药物组合物，包括常规的混合、溶解或冻干方法。
在本申请所述药物组合物中，活性成分的比例可以变化，可占给定的单位剂型重量的大约0.01％至大约99％。在这种治疗有用的药物组合物制剂中，活性成分的量使得能够获得有效剂量水平。
本申请所述的片剂、胶囊剂等可以包含：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，片剂或胶囊剂可以用明胶、蜡、虫胶或糖等包衣。糖浆可以包含活性成分，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，活性成分可以掺入缓释制剂和缓释装置中。
活性成分也可以通过输注或注射到静脉内或腹膜内施用。可以制备活性成分或其盐的水溶液，任选可混和无毒的表面活性剂。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组合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成分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，本申请的药物组合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。
活性成分的治疗有效量，不仅取决于选择的特定的盐，而且取决于施药方式、待治疗的疾病的性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。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0.01到大约1000毫克或更多之间进行变化或调整。
本申请在另一个方面提供了含有治疗有效量的本发明所获得的分离的防风多糖的药物组合物在制备用于治疗糖尿病或高脂血症的药物中的用途。
在还有另一个方面，本申请提供一种治疗糖尿病或高脂血症的方法，包括向有需要的受试者施用治疗有效量的本发明所获得的分离的防风多糖。
在一种优选的实施方式中，所述的治疗糖尿病或高脂血症的方法，包括向有需要的受试者施用治疗有效量的含有治疗有效量的本发明所获得的防风多糖的药物组合物。
在一个方面，本发明还提供分离的防风多糖，用于治疗糖尿病或高脂血症。
本申请所述治疗糖尿病包括降低血糖水平(例如降低空腹血糖水平)、改善糖耐受量、减轻胰岛细胞损伤、增加胰岛素释放等。本申请所述治疗高脂血症包括调节血脂代谢、调节血脂水平(比如降低血液中脂质的水平，例如降低血液中总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白胆固醇(LDL-C)的水平)。另外，本申请所述防风多糖还能增加受试者(例如血清和肝中)的超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-px)的活性，降低丙二醛(MDA)的含量。
本申请使用的术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病的症状，即阻止其发展；或(c)缓解疾病的症状，即，导致疾病或症状退化。
除非特别说明，本申请所述百分数、比例、比率或份数是按体积计。本申请所述体积重量比是以毫升/克(或升/千克)计算的体积重量比。本申请所述浓度是体积浓度。
附图说明
图1：防风多糖对STZ诱导的糖尿病小鼠胰岛组织的影响。图1A：正常对照组；图1B：STZ 120mg/kg组；图1C格列本脲组25mg/kg组；图1D防风多糖50mg/kg组；图1E防风多糖200mg/kg组。STZ：链脲佐菌素。
图2：防风多糖对STZ诱导的糖尿病小鼠血清胰岛素水平的影响(
Figure PCTCN2019074601-appb-000001
n＝10)。与正常对照组(正常组)比较， ##P＜0.01(LSD法检验)；与模型组(STZ 120mg/kg)比较， *P＜0.05， **P＜0.01(LSD法检验)；GLI：格列苯脲；SDP：防风多糖；STZ：链脲佐菌素。
图3：防风多糖对STZ诱导的糖尿病小鼠血清脂质的影响(
Figure PCTCN2019074601-appb-000002
n＝10)。与正常对照组(正常组)比较， ##P＜0.01(LSD法检验)；与模型组(STZ 120mg/kg)比较， *P＜0.05， **P＜0.01(LSD法检验)；GLI：格列苯脲；SDP：防风多糖；STZ：链脲佐菌素。
图4：防风多糖对STZ诱导的糖尿病小鼠MDA含量和SOD活性的影响(
Figure PCTCN2019074601-appb-000003
n＝10)。与正常对照组(正常组)比较， ##P＜0.01(LSD法检验)；与模型组(STZ 120mg/kg)比较， *P＜0.05， **P＜0.01(LSD法检验)；GLI：格列苯脲；SDP：防风多糖；STZ：链脲佐菌素。
图：5：防风多糖对高脂血症小鼠血脂水平的影响(
Figure PCTCN2019074601-appb-000004
n＝10)。与正常对照组(正常组)比较， #P＜0.05， ##P＜0.01(LSD法检验)；与高脂模型组比较， *P＜0.05， **P＜0.01(LSD法检验)。
图6：防风多糖对高脂血症小鼠肝TC、TG以及肝重系数的影响(
Figure PCTCN2019074601-appb-000005
n＝10)。与正常对照组(正常组)比较， ##P＜0.01(LSD法检验)；与高脂模型组比较， *P＜0.05， **P＜0.01(LSD法检验)。
图7：防风多糖对高脂血症小鼠肝形态学的影响。图7A：正常对照组；图7B：高脂模型组；图7C：力平之40mg/kg组；图7D：防风多糖50mg/kg组；图7E：防风多糖200mg/kg组。
图8：防风多糖对高脂血症小鼠肝脏SOD、GSH-px活性和MDA含量的影响(
Figure PCTCN2019074601-appb-000006
n＝10)。与正常对照组(正常组)比较， #P＜0.05， ##P＜0.01(LSD法检验)；与高脂模型组比较， *P＜0.05， **P＜0.01(LSD 法检验)。
具体实施方式
下面，本申请将通过实施例展示本申请的有益效果。本领域技术人员会知道，这些实施例是示例性的，而不是限制性的。这些实施例不会以任何方式限制本申请的范围。下述实施例中所述实验操作，如无特殊说明，均为常规操作；所述试剂和材料，如无特殊说明，均可从商业途径获得。
主要试剂与材料
防风饮片，购自安徽亳州中药材市场，产地安国；95％乙醇、盐酸、氢氧化钠、考马斯亮蓝、硫酸、苯酚、氯化钡、三氟乙酸、硼氢化钠、二甲基亚砜等均购自国药集团化学试剂有限公司；L-阿拉伯糖(L-Ara)，D-甘露糖(D-Man)，D-葡萄糖(D-Glc)，D-半乳糖(D-Gal)，D-半乳糖醛酸(D-GalA)对照品和1-苯基-3-甲基-5-吡唑啉酮(PMP)均购自Sigma公司。
主要仪器
1260型高效液相色谱仪(DAD和RID检测器，美国Agilent公司)；DAWN HELEOS-II型18角度激光光散射仪(美国Wayyat公司)；7890B型气相色谱质谱联用仪(美国Agilent公司)；Infinite M200型酶标微孔板读数仪(美国Tecan公司)。
实施例1：防风多糖的制备
(1)向300g防风饮片中加入6L蒸馏水。在90℃下用所述蒸馏水提取防风饮片2小时，以得到提取液。分离提取液后，重复提取2次，每次使用6L蒸馏水，每次2小时。将所得提取液合并并浓缩至2L，得到浓缩提取液。
(2)向所述浓缩提取液中加入乙醇得到乙醇浓度为20％的混合物，离心处理所述混合物以得到上清液。
(3)向所述上清液中加入乙醇以得到乙醇浓度为80％的混合物，离心处理所述混合物以得到沉淀。
(3’)向所述沉淀加入0.5L蒸馏水，以使之溶解。然后，再次加入乙醇以得到乙醇浓度为80％的混合物，离心处理所述混合物以得到沉淀。
(4)干燥所得沉淀，以得到防风多糖6g，收率2％。
实施例2：防风多糖的制备
(1)向300g防风饮片中加入9L蒸馏水。在40℃下用所述蒸馏水提取防风饮片4小时，以得到提取液。分离提取液后，重复提取3次，每次使用9L蒸馏水，每次4小时。将所得提取液合并并浓缩至2L，得到浓缩提取液。
(2)向所述浓缩提取液中加入甲醇得到甲醇浓度为30％的混合物，离心处理所述混合物以得到上清液。
(3)向所述上清液中加入甲醇以得到甲醇浓度为90％的混合物，离心处理所述混合物以得到沉淀。
(3’)向所述沉淀加入0.5L蒸馏水，以使之溶解。然后，再次加入甲醇以得到甲醇浓度为90％的混合物，离心处理所述混合物以得到沉淀。重复步骤(3’)的操作2次。
(4)干燥所得沉淀，以得到防风多糖4.8g，收率1.6％。
实施例3：防风多糖的制备
(1)向300g防风饮片中加入3L蒸馏水。在90℃下用所述蒸馏水提取防风饮片2小时，以得到提取液。分离提取液后，重复提取3次，每次使用3L蒸馏水，每次2小时。将所得提取液合并并浓缩至2L，得到浓缩提取液。
(2)向所述浓缩提取液中加入乙醇得到乙醇浓度为15％的混合物，离心处理所述混合物以得到上清液。
(3)向所述上清液中加入乙醇以得到乙醇浓度为80％的混合 物，离心处理所述混合物以得到沉淀。
(3’)向所述沉淀加入0.5L蒸馏水，以使之溶解。然后，再次加入乙醇以得到乙醇浓度为80％的混合物，离心处理所述混合物以得到沉淀。重复步骤(3’)的操作1次。
(4)干燥所得沉淀，以得到防风多糖6.2g，收率2.1％。
实施例4：防风多糖的制备
(1)向300g防风饮片中加入2.4L蒸馏水。在100℃下用所述蒸馏水提取防风饮片1小时，以得到提取液。分离提取液后，重复提取4次，每次使用2.4L蒸馏水，每次1小时。将所得提取液合并并浓缩至2L，得到浓缩提取液。
(2)向所述浓缩提取液中加入丙醇得到丙醇浓度为20％的混合物，离心处理所述混合物以得到上清液。
(3)向所述上清液中加入丙醇以得到丙醇浓度为70％的混合物，离心处理所述混合物以得到沉淀。
(3’)向所述沉淀加入0.5L蒸馏水，以使之溶解。然后，再次加入丙醇以得到丙醇浓度为70％的混合物，离心处理所述混合物以得到沉淀。
(4)干燥所得沉淀，以得到防风多糖4.9g，收率1.6％。
实施例5：防风多糖的制备
(1)向300g防风饮片中加入6L蒸馏水。在60℃下用所述蒸馏水提取防风饮片3小时，以得到提取液。分离提取液后，重复提取2次，每次使用6L蒸馏水，每次3小时。将所得提取液合并并浓缩至2L，得到浓缩提取液。
(2)向所述浓缩提取液中加入丙酮得到丙酮浓度为20％的混合物，离心处理所述混合物以得到上清液。
(3)向所述上清液中加入丙酮以得到丙酮浓度为75％的混合物，离心处理所述混合物以得到沉淀。
(3’)向所述沉淀加入0.5L蒸馏水，以使之溶解。然后，再次加入丙酮以得到丙酮浓度为75％的混合物，离心处理所述混合物以得到沉淀。
(4)干燥所得沉淀，以得到防风多糖5.2g，收率1.7％。
实施例6：防风多糖的制备
(1)向300g防风饮片中加入6L蒸馏水。在80℃下用所述蒸馏水提取防风饮片2小时，以得到提取液。分离提取液后，重复提取2次，每次使用6L蒸馏水，每次2小时。将所得提取液合并并浓缩至2L，得到浓缩提取液。
(2)向所述浓缩提取液中加入乙醇得到乙醇浓度为25％的混合物，离心处理所述混合物以得到上清液。
(3)向所述上清液中加入乙醇以得到乙醇浓度为80％的混合物，离心处理所述混合物以得到沉淀。
(4)干燥所得沉淀，以得到防风多糖5.8g，收率1.9％。
实施例7：防风多糖的制备
(1)向300g防风饮片中加入9L蒸馏水。在70℃下用所述蒸馏水提取防风饮片2小时，以得到提取液。分离提取液后，重复提取3次，每次使用9L蒸馏水，每次2小时。将所得提取液合并并浓缩至2L，得到浓缩提取液。
(2)向所述浓缩提取液中加入乙醇得到乙醇浓度为20％的混合物，离心处理所述混合物以得到上清液。
(3)向所述上清液中加入乙醇以得到乙醇浓度为85％的混合物，离心处理所述混合物以得到沉淀。
(3’)向所述沉淀加入0.5L蒸馏水，以使之溶解。然后，再次加入乙醇以得到乙醇浓度为85％的混合物，离心处理所述混合物以得到沉淀。
(4)干燥所得沉淀，以得到防风多糖5.7g，收率1.9％。
实施例8：防风多糖的制备
(1)向300g防风饮片中加入6L蒸馏水。在95℃下用所述蒸馏水提取防风饮片1小时，以得到提取液。分离提取液后，重复提取1次。将所得提取液合并并浓缩至2L，得到浓缩提取液。
(2)向所述浓缩提取液中加入乙醇得到乙醇浓度为20％的混合物，离心处理所述混合物以得到上清液。
(3)向所述上清液中加入乙醇以得到乙醇浓度为80％的混合物，离心处理所述混合物以得到沉淀。
(3’)向所述沉淀加入0.5L蒸馏水，以使之溶解。然后，再次加入乙醇以得到乙醇浓度为80％的混合物，离心处理所述混合物以得到沉淀。
(4)干燥所得沉淀，以得到防风多糖6.1g，收率2.0％。
实施例9：防风多糖的结构鉴定
(1)总糖、糖醛酸、蛋白及硫酸基含量测定
根据硫酸-苯酚法(参见张泽庆，防风多糖的提纯、结构分析及生物活性研究，陕西师范大学硕士学位论文，2008，77页)测定实施例1-8所得防风多糖总糖含量。
(2)根据间羟联苯法(参见高林，MCP中糖醛酸的含量测定，化学工业与工程，2005，22(6)：487-489)测定实施例1-8所得防风多糖糖醛酸含量。
(3)根据考马斯亮蓝法(参见张婕，盐炙前后关黄柏多糖基本含量测定及对免疫功能的影响，辽宁中医杂志，2017，44(6)：1263-1267)测定实施例1至实施例8所得防风多糖蛋白含量。
(4)根据硫酸钡-比浊法(参见陈乾，硫酸钡-比浊法测定褐藻糖胶中硫酸根的含量，药学实践杂志，2012，30(2)：118-120)测定实施例1至实施例8所得防风多糖硫酸基含量。
测定结果见下表1：
表1
实施例编号 总糖含量％ 糖醛酸含量 蛋白含量％ 硫酸基含量
1 76.33 16.41 1.17 未检测到
2 71.48 18.22 2.46 未检测到
3 77.89 14.14 1.33 未检测到
4 72.35 16.72 2.07 未检测到
5 75.53 13.66 1.24 未检测到
6 76.47 17.36 2.21 未检测到
7 74.92 18.41 1.53 未检测到
8 79.62 13.74 1.83 未检测到
(5)重均分子量测定
采用多角度激光光散射法(参见丁厚强，多角度激光光散射仪与尺寸排阻色谱法联用测定透明质酸相对分子质量及其分布，食品与药品，2009，11(3)：24-26)测定实施例1-8所得防风多糖的重均分子量。
测定方法
将10mg待测样品置于1.5mL离心管中。然后加入1mL去离子水，以使所述样品使溶解。在14000rpm转速下将所述离心管离心10min，以得到上清液。使用Agilent 1260 HPLC色谱仪对所述上清液进行测定，以确定重均分子量。
色谱条件：
色谱柱：XBridge Protein BEH SEC 
Figure PCTCN2019074601-appb-000007
Column(3.5μm，7.8×300mm)；柱温：25℃；RID温度：35℃；流动相：0.1mol/L NaOAc溶液；流速：0.5mL/min；进样量：30μL。
测定结果见表2：
表2
实施例编号 重均分子量/Da
1 3.5×10 5
2 1.8×10 5
3 4.4×10 5
4 2.7×10 5
5 8.5×10 4
6 2.4×10 5
7 1.3×10 5
8 3.4×10 5
(6)单糖组成分析
将2mg实施例1-8所得防风多糖分别溶解于安瓿瓶中的1mL的3mol/L的三氟乙酸(TFA)水溶液中，然后将所述安瓿瓶封口。将所述安瓿瓶中的防风多糖在105℃水解4小时。将所述安瓿瓶中的水在减压条件下蒸干后，向所述安瓿瓶中加入2mL甲醇，然后蒸干。重复添加乙醇并蒸干的操作2次以除去TFA。然后，向所述安瓿瓶中加入100μL水，得到所述多糖的酸性条件完全水解的样品。
再称取适量单糖对照品配制成浓度为1mg/mL的母液。吸取母液10μL，定容至100μL。
衍生化处理：取50μL对照品溶液，依次加入100μL 0.3mol/L NaOH溶液、120μL 0.5mol/L的1-苯基-3-甲基-5-吡唑啉酮的甲醇溶液并混匀以得到混合溶液。将所述混合溶液在70℃反应60分钟。反应结束后，将所述溶液冷却至室温，加入适量0.3mol/L的HCl以调节pH值至中性，然后用1mL氯仿萃取并弃去有机相。取50μL所述多糖的酸性条件完全水解的样品，同样按照以上方法进行衍生化处理。
色谱条件：
Agilent Eclipse XDB-C18色谱柱；流动相：0.1mol/L磷酸盐缓冲液(pH＝6.7)∶乙腈(v/v＝83∶17)；柱温25℃；检测波长245nm；流速1.0mL/min；进样体积10μL。
测定结果见下表3：
表3
实施例编号 摩尔比(L-Ara∶D-GalA∶D-Man∶D-Glc∶D-Gal)
1 5∶6∶2∶23∶5
2 6∶6∶7∶24∶2
3 6∶2∶5∶20∶3
4 4∶6∶3∶25∶4
5 5∶8∶3∶25∶4
6 3∶6∶5∶23∶2
7 4∶7∶4∶21∶3
8 3∶7∶5∶22∶4
(7)甲基化分析
参照文献方法(方积年，多糖的甲基化分析方法，国外医学(药学分册)，1986，(4)：222-226)分别对实施例1-8防风多糖进行甲基化。甲基化后的产物用90％甲酸解聚，并用2mol/L TFA全水解，得到甲基化单糖。然后所得甲基化单糖用NaBH 4还原、并用醋酸酐乙酰化以制成所述甲基化单糖的阿尔迪醇乙酸酯衍生物，然后对所述衍生物进行GC-MS分析。
根据甲基化分析结果，可确定实施例1-8防风多糖包含以下单糖：1，4-L-阿拉伯糖、1，3，4-L-阿拉伯糖、端基D-半乳糖醛酸、1，6-D-甘露糖、1，4-D-葡萄糖、1，3，6-D-葡萄糖、端基D-半乳糖、和1，4-D-半乳糖。甲基化分析结果见表4-11。
表4 实施例1所得防风多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3-Me 2-L-Ara 1，4-L-阿拉伯糖 1
2-Me-L-Ara 1，3，4-L-阿拉伯糖 4
2，3，4-Me 3-D-GalA 端基D-半乳糖醛酸 6
2，3，4-Me 3-D-Man 1，6-D-甘露糖 2
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 20
2，4-Me 2-D-Glc 1，3，6-D-葡萄糖 3
2，3，4，6-Me 4-D-Gal 端基-D-半乳糖 1
2，3，6-Me 3-D-Gal 1，4-D-半乳糖 4
表5 实施例2所得防风多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3-Me 2-L-Ara 1，4-L-阿拉伯糖 1
2-Me-L-Ara 1，3，4-L-阿拉伯糖 1
2，3，4-Me 3-D-GalA 端基D-半乳糖醛酸 6
2，3，4-Me 3-D-Man 1，6-D-甘露糖 9
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 10
2，4-Me 2-D-Glc 1，3，6-D-葡萄糖 2
2，3，4，6-Me 4-D-Gal 端基-D-半乳糖 1
2，3，6-Me 3-D-Gal 1，4-D-半乳糖 1
表6 实施例3所得防风多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3-Me 2-L-Ara 1，4-L-阿拉伯糖 1
2-Me-L-Ara 1，3，4-L-阿拉伯糖 3
2，3，4-Me 3-D-GalA 端基D-半乳糖醛酸 7
2，3，4-Me 3-D-Man 1，6-D-甘露糖 4
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 24
2，4-Me 2-D-Glc 1，3，6-D-葡萄糖 4
2，3，4，6-Me 4-D-Gal 端基-D-半乳糖 1
2，3，6-Me 3-D-Gal 1，4-D-半乳糖 4
表7 实施例4所得防风多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3-Me 2-L-Ara 1，4-L-阿拉伯糖 3
2-Me-L-Ara 1，3，4-L-阿拉伯糖 10
2，3，4-Me 3-D-GalA 端基D-半乳糖醛酸 9
2，3，4-Me 3-D-Man 1，6-D-甘露糖 3
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 16
2，4-Me 2-D-Glc 1，3，6-D-葡萄糖 9
2，3，4，6-Me 4-D-Gal 端基-D-半乳糖 5
2，3，6-Me 3-D-Gal 1，4-D-半乳糖 9
表8 实施例5获得的防风多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3-Me 2-L-Ara 1，4-L-阿拉伯糖 4
2-Me-L-Ara 1，3，4-L-阿拉伯糖 2
2，3，4-Me 3-D-GalA 端基D-半乳糖醛酸 6
2，3，4-Me 3-D-Man 1，6-D-甘露糖 7
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 22
2，4-Me 2-D-Glc 1，3，6-D-葡萄糖 2
2，3，4，6-Me 4-D-Gal 端基-D-半乳糖 1
2，3，6-Me 3-D-Gal 1，4-D-半乳糖 1
表9 实施例6所得防风多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3-Me 2-L-Ara 1，4-L-阿拉伯糖 1
2-Me-L-Ara 1，3，4-L-阿拉伯糖 2
2，3，4-Me 3-D-GalA 端基D-半乳糖醛酸 6
2，3，4-Me 3-D-Man 1，6-D-甘露糖 5
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 18
2，4-Me 2-D-Glc 1，3，6-D-葡萄糖 5
2，3，4，6-Me 4-D-Gal 端基-D-半乳糖 1
2，3，6-Me 3-D-Gal 1，4-D-半乳糖 1
表10 实施例7所得防风多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3-Me 2-L-Ara 1，4-L-阿拉伯糖 1
2-Me-L-Ara 1，3，4-L-阿拉伯糖 3
2，3，4-Me 3-D-GalA 端基D-半乳糖醛酸 7
2，3，4-Me 3-D-Man 1，6-D-甘露糖 4
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 20
2，4-Me 2-D-Glc 1，3，6-D-葡萄糖 1
2，3，4，6-Me 4-D-Gal 端基-D-半乳糖 1
2，3，6-Me 3-D-Gal 1，4-D-半乳糖 2
表11 实施例8所得防风多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3-Me 2-L-Ara 1，4-L-阿拉伯糖 3
2-Me-L-Ara 1，3，4-L-阿拉伯糖 2
2，3，4-Me 3-D-GalA 端基D-半乳糖醛酸 6
2，3，4-Me 3-D-Man 1，6-D-甘露糖 3
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 20
2，4-Me 2-D-Glc 1，3，6-D-葡萄糖 5
2，3，4，6-Me 4-D-Gal 端基-D-半乳糖 2
2，3，6-Me 3-D-Gal 1，4-D-半乳糖 2
实施例10：降血糖作用实验
实验药物：实施例1防风多糖，分别以50mg/kg(低剂量)和200mg/kg(高剂量)的剂量施用。
实验试剂：葡萄糖测定试剂盒，由上海荣盛生物药业有限公司提供；胰岛素ELISA测定试剂盒，由上海西唐实业有限公司提供；TC、TG、HDL-C、SOD、MDA和考马斯亮兰蛋白测定试剂盒，均由南京建成生物工程研究所提供。
实验动物：健康清洁级的雄性昆明小鼠(体重18-22g，上海斯莱克实验动物有限责任公司提供)。
实验仪器：高速冷冻离心机，由德国Eppendorf公司生产；电子天平，由Mettler-Toledo公司生产；多功能酶标仪，由美国伯腾仪器有限公司生产。
实验方法：将小鼠在20±2℃的温度下和在50±5％的湿度下在12小时光照和12小时黑暗的条件下饲养3天，其间小鼠可自由进食摄水。选取10只健康小鼠作为正常对照组(Control)。以0.2ml/10g体重向剩余小鼠腹腔注射链脲佐菌素(STZ)溶液。72小时后测定小鼠的空腹血糖(fast blood glucose，FBG)。选取FBG值高于11.1mmol/L并且小于25mmol/L的小鼠为作为糖尿病模型小鼠。将糖尿病模型小鼠随机分成模型组(STZ 120mg/kg)、低剂量防风多糖组(SDP 50mg/kg)、高剂量防风多糖(SDP 200mg/kg)组以及阳性药物(格列本脲，GLI 25mg/kg)对照组，每组10只小鼠。对于各给药组，在每天上午8：00，按0.2ml/10g体重灌胃给药。正常对照组和模型组小鼠给予等体积蒸馏水。在实验期间，各组小鼠自由饮水，每10天测定FBG和称体重一次。给药30天后处理小鼠，测定下列指标。
(1)糖耐受量：实验结束前一天8：00禁食，4h后从小鼠眼眶取血并测定FBG作为给予葡萄糖前(0h)的血糖值，然后以0.2ml/10g体重向所有小鼠腹腔注射10％葡萄糖溶液。在腹腔注射葡萄糖0.5小时(0.5h)和2小时(2h)后从小鼠眼眶取血并测定血糖值，按下列公式计算血糖曲线下面积(AUC)。
Figure PCTCN2019074601-appb-000008
(2)于实验结束前一天22：00禁食不禁水，实验于当日8：00开始。从小鼠眼眶取血，并通过离心从血中获得血清。测定血清中的胰岛素、总胆固醇(TC)、甘油三酯(TG)和高密度脂蛋白胆固醇(HDL-C)，以及SOD活性和MDA含量。取部分小鼠肝脏组织，匀浆，测定肝SOD(超氧化物歧化酶)活性和MDA(丙二醛)含量。 取部分小鼠的胰腺组织，用10％甲醛固定并进行形态学检查。
实验结果：
(1)防风多糖对STZ诱导的糖尿病模型小鼠空腹血糖和糖耐受量的影响
如表12所示：与正常对照组比较，模型组小鼠的血糖水平显著升高(P＜0.01)。与模型组相比，防风多糖能够显著降低糖尿病小鼠血糖水平(P＜0.05)。
表12防风多糖降低链脲佐菌素(STZ)诱导糖尿病小鼠空腹血糖(FBG)的作用(
Figure PCTCN2019074601-appb-000009
n＝10)
Figure PCTCN2019074601-appb-000010
注：与正常对照组比较， ##P＜0.01(LSD法检验)；与模型组组比较， *P＜0.05(LSD法检验)。GLI：格列苯脲；SDP：防风多糖；STZ：链脲佐菌素。
如表13所示：与正常对照组比较，模型组小鼠的糖耐受量明显下降，血糖曲线下面积显著增加(P＜0.01)。与模型组相比，防风多糖能够显著改善糖耐受量(P＜0.05或P＜0.01)，显著减少血糖曲线下面积(P＜0.01)。
表13.防风多糖降低链脲佐菌素(STZ)诱导糖尿病小鼠空腹 血糖(FBG)的作用(
Figure PCTCN2019074601-appb-000011
n＝10)
Figure PCTCN2019074601-appb-000012
注：与正常对照组比较， ##P＜0.01(LSD法检验)；与STZ(120mg/kg)组比较， *P＜0.05(LSD法检验)。GLI：格列苯脲；SDP：防风多糖；STZ：链脲佐菌素。
(2)防风多糖对链脲佐菌素(STZ)诱导的糖尿病小鼠胰岛组织和血清胰岛素水平的影响
如图1所示：与正常对照组比较，STZ诱导的糖尿病小鼠胰岛组织中细胞发生核固缩、玻璃样病变、炎性细胞浸润，胰岛形态不完整(图1B)。给予防风多糖后，与STZ组比较，低倍镜下，防风多糖组胰岛数量没有明显减少；高倍镜下，防风多糖组小鼠胰岛的损伤明显减轻，形态比较完整(图1D，1E)。
同时测定小鼠血清胰岛素水平。实验结果如图2所示。与正常对照组比较，STZ组(模型组)小鼠的血清胰岛素水平显著降低(P＜0.01)。而给予防风多糖后，与STZ组相比，防风多糖(200mg/kg)高剂量组的血清胰岛素水平显著升高(P＜0.01)。
(3)防风多糖对STZ诱导的糖尿病小鼠血清脂质的影响
脂毒性是2型糖尿病发病机制的一个重要因素。如图3所示：与正常对照比较，STZ组(模型组)小鼠血清TC和TG水平均显著 升高(P＜0.01)。而给予防风多糖后，与STZ组相比，低剂量和高剂量防风多糖均能够显著降低糖尿病小鼠血清TC和TG水平，并呈剂量依赖性(P＜0.05或P＜0.01)。另外，血清HDL-C水平在各组之间未见有明显影响。
(4)防风多糖对链脲佐菌素(STZ)诱导的糖尿病小鼠血清和肝MDA含量和SOD活性的影响
在STZ诱导的糖尿病大鼠模型中，会产生大量的氧自由基。脂质在氧自由基的作用下发生过氧化作用，从而产生大量的醛类、醇类等物质，其中丙二醛(MDA)是一种代表性性质。丙二醇含量反映过氧化作用的程度。而SOD的活性则反映机体清除氧自由基的能力。实验结果图4显示：与正常对照比较，STZ组(模型组)小鼠血清和肝中MDA的含量均显著升高(P＜0.01)，同时肝中SOD的活性显著降低(P＜0.01)。而给予防风多糖后，与STZ组相比，防风多糖都能够降低糖尿病小鼠血清MDA含量。特别地，高剂量防风多糖能够显著降低小鼠血清MDA含量(P＜0.01)；低剂量和高剂量防风多糖都能够显著降低小鼠肝MDA含量(P＜0.05)。与STZ组相比，防风多糖都能够升高小鼠血清SOD活性。特别地，高剂量防风多糖能够显著升高小鼠血清SOD活性(P＜0.05)；低剂量和高剂量防风多糖都能够升高小鼠肝SOD活性，其中高剂量防风多糖能够显著升高小鼠肝SOD活性(P＜0.01)。
实验结论：防风多糖可明显降低STZ诱导的糖尿病小鼠空腹血糖水平并显著增加糖尿病小鼠糖耐受量。而且，防风多糖还可以减轻小鼠胰岛的损伤，增加胰岛素的释放，并且明显降低高血糖小鼠血清中TC和TG水平。防风多糖还能增加血清和肝中SOD活性而降低MDA的含量。
实施例11 防风多糖降血脂作用实验
实验药物：实施例1防风多糖，分别以50mg/kg(低剂量)和 200mg/kg(高剂量)的剂量施用。
实验试剂：TC、TG、LDL-C、HDL-C、SOD、GSH-px、MDA和考马斯亮兰蛋白测定试剂盒，均由南京建成生物工程研究所提供。
实验动物：健康清洁级的雄性昆明小鼠(体重18-22g，上海斯莱克实验动物有限责任公司提供)。
实验仪器：高速冷冻离心机，由德国Eppendorf公司生产；电子天平，由Mettler-Toledo公司生产；多功能酶标仪，由美国伯腾仪器有限公司生产
参照文献方法(孙立彦，刘振亮，孙金霞，等.白茅根多糖对小鼠耐缺氧作用的影响，中国医院药学杂志，2008，28(2)：96-99；冷斌，白茅根多糖对IgA肾病大鼠免疫调节及肾纤维化的干预，桂林医学院学位论文，2013；吕世静，龙启才，何德袁等，白茅根多糖对乙肝患者淋巴细胞增殖及T细胞亚群的调节作用，[会议论文]2001-第二届全国中医药免疫学术研讨)，建立高脂血症模型。实验方法：将小鼠在20±2℃的温度下和在50±5％的湿度下在12小时光照和12小时黑暗的条件下饲养3天，其间小鼠可自由进食摄水。将小鼠随机分为5组：正常对照组(正常组)、高脂模型组、阳性药力平之(Lipanthyl，非诺贝特，40mg/kg)组、低剂量防风多糖组(SDP 50mg/kg)和高剂量防风多糖组(SDP 200mg/kg)，每组10只。对于各给药组，在每天8：00-9：00按0.2ml/10g体重给予不同剂量的药物。向正常对照组与高脂模型组给予等体积的蒸馏水。除正常对照组外，各组小鼠于每天14：00-15：00按0.2ml/10g体重灌胃给予高脂饮食(含有20％猪油、10％胆固醇、0.2％丙基硫氧嘧啶、20％丙二醇和20％吐温-80)，连续3周以考察防风多糖预防高血脂症的作用。实验结束时，小鼠禁食不禁水8h后处理。从小鼠眼眶取血，并通过离心从血中获得血清。测定血清TC、TG、LDL-C和HDL-C。取小鼠部分肝脏组织，匀浆，测定肝TC和TG含量，以及SOD、GSH-px活性和MDA含量。另取小鼠部分肝脏组织，用10％甲醛固定并进行形态学检查。
实验结果：
(1)防风多糖对高脂血症小鼠血脂水平的影响
如图5所示：与正常对照组比较，高脂模型组中小鼠的血清TC和LDL-C水平均显著升高(P＜0.01)。与高脂饮食组相比，防风多糖能够降低血清TC、TG和LDL-C水平。特别地，低剂量和高剂量防风多糖能够显著降低血清TC水平(P＜0.05)，高剂量防风多糖能够显著降低血清TC、TG和LDL-C水平(P＜0.05)。防风多糖对血清HDL-C水平未见有明显影响。
(2)防风多糖对高脂血症小鼠肝TC、TG以及肝重系数的影响
如图6所示：与正常对照组比较，高脂模型组中小鼠肝TC和TG含量(P＜0.01)、以及肝重系数(P＜0.01)均显著升高。与高脂模型组相比，防风多糖能够降低肝TC、TG含量和肝重系数。特别地，低剂量和高剂量防风多糖能够显著降低肝TC含量(P＜0.01)。与高脂模型组相比，高剂量防风多糖能够显著降低肝TC、TG含量和肝重系数(P＜0.05或P＜0.01)。阳性药力平之虽然也能显著降低肝TC含量(P＜0.01)，但对肝重系数却明显升高。因此，防风多糖尤其是高剂量防风多糖在降低TC、TG，尤其在降低肝重系数方面的作用明显优于阳性药力平之。
(3)防风多糖对高脂血症小鼠肝形态学的影响
如图7所示，正常对照组小鼠肝结构完整，肝索清晰可见，未见有明显的脂质空泡(图7A)。当给予小鼠高脂饮食3周后，高脂模型组小鼠肝中可见有大量的脂质空泡(图7B)。防风多糖能明显改善肝脏的脂质空泡；高剂量防风多糖具有更好的效果(图7D、7E)。
(4)防风多糖对高脂血症小鼠肝SOD、GSH-px活性和MDA含量的影响
SOD和GSH-px是肝中的抗氧化酶，可以减少活性氧的量，减 轻脂质过氧化作用对肝细胞的损伤。实验结果图8显示：与正常对照比较，高脂模型组小鼠肝中SOD和GSH-px活性均显著降低(P＜0.01)，同时MDA含量显著升高(P＜0.05)。而给予防风多糖后，与STZ组相比，防风多糖升高小鼠肝SOD和GSH-px活性，显著降低MDA含量。特别地，低剂量和高剂量防风多糖都能够显著升高小鼠肝SOD(P＜0.05或P＜0.01)活性，显著升高小鼠肝GSH-px(P＜0.05或P＜0.01)活性，显著降低MDA含量(P＜0.05)。
实验结论：防风多糖可明显降低高脂饮食诱导的高脂血症小鼠血清TC、TG和LDL-C水平，同时亦可降低肝重系数以及肝TC和TG含量，明显减少肝中的脂质空泡。而且，防风多糖可以增加肝中SOD和GSH-px活性，而降低MDA的含量。

Claims (17)

  1. 一种分离的防风多糖，包含L-阿拉伯糖、D-半乳糖醛酸、D-甘露糖、D-葡萄糖和D-半乳糖，其特征在于，所述L-阿拉伯糖∶D-半乳糖醛酸∶D-甘露糖∶D-葡萄糖∶D-半乳糖的摩尔比为1-15∶1-10∶1-10∶10-40∶1-15，优选为1-5∶5-10∶1-5∶20-25∶1-5。
  2. 如权利要求1所述的分离的防风多糖，其特征在于，
    所述L-阿拉伯糖包括1，4-连接的L-阿拉伯糖和/或1，3，4-连接的L-阿拉伯糖；
    所述D-半乳糖醛酸包括端基D-半乳糖醛酸；
    所述D-甘露糖包括1，6-连接的D-甘露糖；
    所述D-葡萄糖包括1，4-连接的D-葡萄糖和/或1，3，6-连接的D-葡萄糖；
    所述D-半乳糖包括端基D-半乳糖和/或1，4-连接的D-半乳糖。
  3. 如权利要求2所述的分离的防风多糖，其特征在于，所述1，4-连接的L-阿拉伯糖∶1，3，4-连接的L-阿拉伯糖∶端基D-半乳糖醛酸∶1，6-连接的D-甘露糖∶1，4-连接的D-葡萄糖∶1，3，6-连接的D-葡萄糖∶端基D-半乳糖∶1，4-连接的D-半乳糖的摩尔比为1-5∶1-10∶1-10∶1-10∶10-30∶1-10∶1-5∶1-10，优选为1-3∶1-5∶5-10∶1-5∶15-25∶1-5∶1-3∶1-5。
  4. 如权利要求1-3中任一项所述的分离的防风多糖，其特征在于，所述分离的防风多糖分子量为5×10 4至5×10 5Da，优选为1×10 5至3.5×10 5Da。
  5. 一种制备如权利要求1-4任一项所述的分离的防风多糖的方法，其特征在于，所述方法包括以下步骤：
    (1)用水一次或多次提取防风，得到防风水提取液，任选浓缩所述防风水提取液；
    (2)向所述任选浓缩的防风水提取液加入有机溶剂以得到有机溶剂浓度为15-30％、优选17-28％、更优选20-25％的混合物，离心 处理所述混合物以得到上清液；
    (3)向所述上清液中加入有机溶剂以得到有机溶剂浓度为70-90％、优选为75-85％、更优选80-85％的混合物，离心处理所述混合物以得到沉淀；
    (4)干燥所述沉淀，以得到所述的分离的防风多糖。
  6. 如权利要求5所述的方法，其特征在于，所述步骤(1)中水与防风的体积重量比为8∶1至30∶1，优选20∶1至30∶1。
  7. 如权利要求5-6中任一项所述的方法，其特征在于，所述步骤(1)中提取温度为40-100℃，优选60-100℃，最优选90-95℃。
  8. 如权利要求5-6中任一项所述的方法，其特征在于，所述步骤(1)中提取时间为1-4小时，优选1-2小时。
  9. 如权利要求5-6中任一项所述的方法，其特征在于，所述步骤(1)中用水提取防风一次以上，优选1-4次，最优选2-3次。
  10. 如权利要求5所述的方法，其特征在于，所述步骤(3)和(4)之间还存在步骤(3’)：用水溶解步骤(3)所得沉淀以得到水溶液，向所述水溶液加入有机溶剂以得到有机溶剂浓度为70-90％、优选为75-85％、更优选80-85％的混合物，离心处理所述混合物以得到沉淀；步骤(3’)可以重复一次或多次，优选1、2、或3次。
  11. 如权利要求5或10所述的方法，其特征在于，所述步骤(2)和/或(3)和/或(3’)中所述有机溶剂选自甲醇、乙醇、丙醇、丙酮、或其混合物，优选乙醇。
  12. 如权利要求5-6中任一项所述的方法，其特征在于，所述步骤(1)中所述防风是防风饮片。
  13. 如权利要求1-4任一所述的分离的防风多糖在制备治疗糖尿病或高脂血症药物中的用途。
  14. 一种药物组合物，包含如权利要求1-4中任一项所述的分离的防风多糖，以及药学上可接受的载体。
  15. 如权利要求14所述的药物组合物在制备用于治疗糖尿病或高脂血症的药物中的用途。
  16. 如权利要求1-4中任一项所述的分离的防风多糖，用于治疗糖尿病或高脂血症。
  17. 一种治疗糖尿病或高脂血症的方法，包括向有需要的受试者施用治疗有效量的如权利要求1-4中任一项所述的分离的防风多糖。
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EP19748215.1A EP3750923A4 (en) 2018-02-05 2019-02-02 SEPARATED SAPOSHNIKOVIA DIVARICATA POLYSACCHARIDE AND USE OF IT
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1135900A (zh) * 1995-05-10 1996-11-20 北京中医药大学 防风多糖及其制剂的制备方法
CN107632096A (zh) * 2017-10-19 2018-01-26 佛山科学技术学院 一种测定防风多糖的单糖组成的方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101601705A (zh) * 2008-06-11 2009-12-16 上海玉森新药开发有限公司 一种防风多糖的制备方法
CN101935359B (zh) * 2010-09-10 2012-05-30 中国食品药品检定研究院 一种短葶山麦冬多糖及其制备方法
CN102775509A (zh) * 2012-07-20 2012-11-14 崔振华 一种用中药材分级提取粗多糖的方法
CN103073650B (zh) * 2012-11-26 2015-01-07 兰州大学 红芪多糖4及其有效成分的制备和应用
CN104387489A (zh) * 2014-11-21 2015-03-04 中国人民解放军南京军区福州总医院 一种具有降血糖作用的白子草酸性均一多糖的制备方法
CN105884922A (zh) * 2014-12-09 2016-08-24 张政 一种热浸提取植物性粗多糖方法
CN104558231A (zh) * 2015-01-08 2015-04-29 广西大学 一种浸提取植物性多糖方法
CN106474435A (zh) * 2016-09-30 2017-03-08 安徽华澳生物技术有限公司 一种促进鸡毛生长的营养剂
CN107163162A (zh) * 2017-07-06 2017-09-15 西北大学 一种枸杞多糖精制的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1135900A (zh) * 1995-05-10 1996-11-20 北京中医药大学 防风多糖及其制剂的制备方法
CN107632096A (zh) * 2017-10-19 2018-01-26 佛山科学技术学院 一种测定防风多糖的单糖组成的方法

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
"REMINGTON'S PHARMACEUTICAL SCIENCES", 1995, MACK PUBLISHING COMPANY
BIN LENG: "Intervention of immunoregulation and renal fibrosis by Imperata cylindrica polysaccharide in rats with IgA nephropathy", DISSERTATIONS OF GUILIN MEDICAL UNIVERSITY, 2013
HONGXIA DOU ET AL.: "Research progress on the chemical components and pharmacological effects of Saposhnikovia divaricata", INFORMATION ON TRADITIONAL CHINESE MEDICINE, vol. 26, no. 2, 2009, pages 15
HOUQIANG DING: "Determination of molecular weight and distribution of hyaluronic acid by combination of multi-angle laser light scattering spectrometer and size exclusion chromatography", FOOD AND DRUG, vol. 11, no. 3, 2009, pages 24 - 26
JIE ZHANG: "Determination of the basic content of Phellodendron amurense polysaccharides before and after stir-heating with a salt solution and its effect on immune function", LIAONING JOURNAL OF TRADITIONAL CHINESE MEDICINE, vol. 44, no. 6, 2017, pages 1263 - 1267
LIN GAO: "Quantitative Determination of Uronic Acid in MCP", CHEMICAL INDUSTRY AND ENGINEERING, vol. 22, no. 6, 2005, pages 487 - 489
LIYAN SUNZHENLIANG LIUJINXIA SUN ET AL.: "Effect of Imperata cylindrica polysaccharide on hypoxia tolerance in mice", CHINA JOURNAL OF HOSPITAL PHARMACY, vol. 28, no. 2, 2008, pages 96 - 99
QIAN CHEN: "Determination of the sulfate content in fucoidan by barium sulfate-turbidimetry", JOURNAL OF PHARMACEUTICAL PRACTICE, vol. 30, no. 2, 2012, pages 118 - 120
See also references of EP3750923A4
SHIJING LVQICAI LONGDEYUAN HE ET AL.: "Regulation of lymphocyte proliferation and T cell subpopulation by Imperata cylindrica polysaccharide in patients with hepatitis B", CONFERENCE PAPER] 2001 - THE SECOND NATIONAL ACADEMIC CONFERENCE ON IMMUNOLOGY OF TRADITIONAL CHINESE MEDICINE, 2001
WANG SONGBAI: "Isolation , purification and chemical structure analysis of saposhnikovia divaricata polysaccharide", MASTER THESIS, no. 11, 30 June 2006 (2006-06-30), CN, pages 1 - 55, XP009522776 *
ZEQING ZHANG: "Purification, structural analysis and biological activity study of Saposhnikovia divaricata polysaccharides", MASTER'S THESIS OF SHAANXI NORMAL UNIVERSITY, 2008, pages 77

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