WO2019149285A1 - 一种分离的白茅根多糖及其用途 - Google Patents

一种分离的白茅根多糖及其用途 Download PDF

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WO2019149285A1
WO2019149285A1 PCT/CN2019/074616 CN2019074616W WO2019149285A1 WO 2019149285 A1 WO2019149285 A1 WO 2019149285A1 CN 2019074616 W CN2019074616 W CN 2019074616W WO 2019149285 A1 WO2019149285 A1 WO 2019149285A1
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linked
polysaccharide
rhizoma imperatae
rhizoma
glucose
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PCT/CN2019/074616
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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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Priority to EP19747376.2A priority Critical patent/EP3750922A4/en
Priority to JP2020564309A priority patent/JP2021512998A/ja
Priority to US16/967,391 priority patent/US20200376021A1/en
Priority to KR1020207024485A priority patent/KR20200128668A/ko
Publication of WO2019149285A1 publication Critical patent/WO2019149285A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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
    • C08B37/0057—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Xylans, i.e. xylosaccharide, e.g. arabinoxylan, arabinofuronan, pentosans; (beta-1,3)(beta-1,4)-D-Xylans, e.g. rhodymenans; Hemicellulose; Derivatives thereof
    • 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
    • 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
    • C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
    • 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
    • C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
    • C08B37/009—Konjac gum or konjac mannan, i.e. beta-D-glucose and beta-D-mannose units linked by 1,4 bonds, e.g. from Amorphophallus species; Derivatives thereof

Definitions

  • This application relates to the field of medicine.
  • the present application relates to an isolated Imperata cylindrica polysaccharide (ICP) and its use in the manufacture of a medicament for the treatment of hyperlipidemia.
  • ICP Imperata cylindrica polysaccharide
  • 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.
  • Rhizoma Imperatae The traditional Chinese medicine Rhizoma Imperatae is the dried rhizome of Imperatora cylindrica (Linn) Beauv. var. major (Nees) C. E. Hubb.
  • the main chemical constituents of Rhizoma Imperatae are sugars, triterpenoids, lactones, and organic acids.
  • Rhizoma Imperatae polysaccharide is a branched polysaccharide formed by a variety of monosaccharides. Generally, the Rhizoma Imperatae polysaccharide is characterized by the composition of the monosaccharide contained therein and the manner in which it is attached.
  • Rhizoma Imperatae polysaccharide having a molecular weight of 8292.2, including rhamnose, xylose, fructose, mannose, glucose in a molar ratio of 1:11.45:1.26:1.02:95.23.
  • Rhizoma Imperatae polysaccharide is commonly used in the field for hemostasis, immune regulation, diuretic antihypertensive, antibacterial, anti-inflammatory and analgesic, anti-tumor, anti-oxidation, and improvement of renal function. Somatostatin has not been reported to treat hyperlipidemia.
  • 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 process for the preparation of Rhizoma Imperatae polysaccharides, the method comprising the step of gradient precipitation.
  • the isolated Rhizoma Imperata polysaccharides of the present invention are completely different from the known polysaccharide structure of Rhizoma Imperatae. It was found by animal experiments that the isolated Rhizoma Imperata polysaccharides of the present invention have the potential to regulate blood lipids.
  • L-arabinose L-Ara
  • D-xylose D-Xyl
  • D-Man D-mannose
  • D-glucose D-Glc
  • D - Isolated Rhizoma Polysaccharide a monosaccharide such as galactose (D-Gal)
  • the molar ratio of L-arabinose, D-xylose, D-mannose, D-glucose and D-galactose is 1-20: 1-15:1-15:15-40:25-60, preferably 5-10:1-5:1-5:25-30:45-55.
  • the isolated Rhizoma Imperata polysaccharide comprises monosaccharide components that are linked together by glycosidic linkages.
  • the L-arabinose comprises a terminal L-arabinose and/or a 1,2,4-linked L-arabinose;
  • the D-xylose comprises a terminal D-xylose and/or 1,3,4 - linked D-xylose;
  • the D-mannose comprises a terminal D-mannose and/or a 1,6-linked D-mannose;
  • the D-glucose comprises a 1,4-linked D-glucose And/or 1,6-linked D-glucose; or the D-galactose comprises 1,4-linked D-galactose and/or 1,4,6-linked D-galactose.
  • the L-arabinose described herein comprises a terminal L-arabinose and/or a 1,2,4-linked L-arabinose.
  • the D-xylose described herein comprises a terminal D-xylose and/or a 1,3,4-linked D-xylose.
  • the D-mannose described herein comprises a terminal D-mannose and/or a 1,6-linked D-mannose.
  • the D-glucose described herein comprises 1,4-linked D-glucose and/or 1,6-linked D-glucose.
  • the D-galactose described herein comprises 1,4-linked D-galactose and/or 1,4,6-linked D-galactose.
  • the isolated Rhizoma Imperata polysaccharide comprises a terminal L-arabinose, a 1,2,4-linked L-arabinose, a terminal D-xylose, a 1,3,4-linked D-xylose, terminal D-mannose, 1,6-linked D-mannose, 1,4-linked D-glucose, 1,6-linked D-glucose, 1,4-linked D - galactose and 1,4,6-linked D-galactose.
  • the molar ratio of 4,6-linked D-galactose is 1-10:1-10:1-5:1-10:1-5:1-10:15-30:1-10:10-25 15-30, preferably 1-5:1-5:1-3:1-5:1-3:1-5:20-25:1-5:15-20:20-25.
  • one or more of the monosaccharide components are pyranoses; in a preferred embodiment, the monosaccharide component is a pyranose.
  • the isolated Rhizoma Imperata polysaccharides described herein have a molecular weight of from 1 x 10 4 to 5 x 10 5 Da, preferably from 1 x 10 5 to 3 x 10 5 Da.
  • the present application provides a method of preparing an isolated Rhizoma Imperata 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 volume to weight ratio of water to Rhizoma Imperatae 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 extracting the Rhizoma Imperatae 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.
  • Rhizoma Imperatae described in the present application includes the commercially available medicinal material Rhizoma Imperata (ie, the dried rhizome of Rhizoma Imperatae) and Rhizoma Imperatae.
  • the Rhizoma Imperatae described in the present application is a Rhizoma Imperatae.
  • isolated Rhizoma Imperatae polysaccharide refers to Rhizoma Imperatae polysaccharide obtained by artificially separating (eg, extracting, purifying, etc.) the Rhizoma Imperatae polysaccharide from the natural environment of its original plant material.
  • the plant material may be a Rhizoma Imperatae in the form of a plant in the form of Rhizoma Imperatae or a medicinal material, such as a dried rhizome of Rhizoma Imperatae or Rhizoma Imperatae.
  • the present application provides an isolated Rhizoma Imperatae polysaccharide obtained according to the preparation method described herein.
  • the isolated Rhizoma Imperata polysaccharide comprises L-arabinose, D-xylose, D-mannose, D-glucose and D-galactose of Rhizoma Imperata polysaccharide, wherein L-arabinose
  • the molar ratio of D-xylose, D-mannose, D-glucose and D-galactose is from 1-20:1 to 15:1 to 15:15 to 40:25 to 60, preferably from 5 to 10:1. -5:1-5:25-30:45-55.
  • the L-arabinose comprises a terminal L-arabinose and/or a 1,2,4-linked L-arabinose
  • the D-xylose comprises a terminal D-wood Sugar and/or 1,3,4-linked D-xylose
  • said D-mannose comprises terminal D-mannose and/or 1,6-linked D-mannose
  • said D-glucose comprises 1,4-linked D-glucose and/or 1,6-linked D-glucose
  • said D-galactose comprises 1,4-linked D-galactose and/or 1,4,6-linked D-galactose.
  • the molar ratio of 1,4,6-D-linked galactose is 1-10:1-10:1-5:1-10:1-5:1-10:15-30:1-10:10 -25:15-30, preferably 1-5:1-5:1-3:1-5:1-3:1-5:20-25:1-5:15-20:20-25.
  • the terminal group 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-position of the sugar ring.
  • the 1,2,4-linked L-arabinose refers to an L-arabinose linked to an adjacent group (e.g., an adjacent monosaccharide residue) via a glycosidic linkage at the 1, 2, and 4 positions of the sugar ring.
  • an adjacent group e.g., an adjacent monosaccharide residue
  • the terminal group D-xylose refers to D-xylose linked to an adjacent group (e.g., an adjacent monosaccharide residue) via a glycosidic bond at the 1-position of the sugar ring.
  • an adjacent group e.g., an adjacent monosaccharide residue
  • the terminal group D-mannose refers to D-mannose 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 1,6-linked D-glucose refers to D-glucose linked to an adjacent group (eg, an adjacent monosaccharide residue) via a glycosidic linkage at the 1 and 6 positions of the sugar ring.
  • an adjacent group eg, an adjacent monosaccharide residue
  • 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, in another aspect, the use of the isolated Rhizoma Imperata polysaccharide obtained by the present invention for the preparation of a medicament for the treatment of hyperlipidemia.
  • the therapeutic hyperlipidemia drug is a lipid metabolism regulator.
  • 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 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 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 may be present, as a coating, or otherwise alter the physical form of the solid unit dosage form.
  • 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 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 ingredient in a suitable solvent with the various other ingredients listed 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 sterile filtration solution.
  • 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 therapeutic hyperlipidemia drug is a lipid metabolism regulator.
  • the present application provides an isolated Rhizoma Imperatae polysaccharide obtained according to the method described herein.
  • the present application provides a method of treating hyperlipidemia comprising administering to a subject in need thereof a therapeutically effective amount of the isolated Rhizoma Imperatae polysaccharide obtained in accordance with the present invention.
  • the treating hyperlipidemia refers to modulating lipid metabolism.
  • the method of treating hyperlipidemia comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a therapeutically effective amount of the Rhizoma Imperata polysaccharide obtained according to the present invention. .
  • the invention also provides an isolated Rhizoma Imperatae polysaccharide for use in the treatment of hyperlipidemia.
  • the treating hyperlipidemia refers to modulating lipid metabolism.
  • 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).
  • 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).
  • the Rhizoma 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 C activity.
  • SOD superoxide dismutase
  • GSH-px glutathione peroxidase
  • MDA dialdehyde
  • 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.
  • # P ⁇ 0.05 (LSD test method)
  • ## P ⁇ 0.01 (LSD test method)
  • * P ⁇ 0.05 (LSD test method)
  • ** P ⁇ 0.01 (LSD method test).
  • Figure 3 Effect of Rhizoma Imperatae polysaccharide on liver morphology in hyperlipidemia mice.
  • Figure 3A Normal control group
  • Figure 3B High fat model group
  • Figure 3C Lipping 40 mg/kg group
  • Figure 3D Rhizoma Imperatae polysaccharide 100 mg/kg group
  • Figure 3E Rhizoma Imperatae polysaccharide 200 mg/kg group
  • Figure 3F Rhizoma Imperatae polysaccharide 400mg/kg group.
  • # P ⁇ 0.05 (LSD test method)
  • ## P ⁇ 0.01 (LSD test method)
  • * P ⁇ 0.05 (LSD test method)
  • ** P ⁇ 0.01 (LSD method test).
  • Baimaogen Decoction Tablets purchased from Anhui Chinese Medicine Market, originating in Anguo; 95% ethanol, hydrochloric acid, sodium hydroxide, Coomassie Brilliant Blue, sulfuric acid, phenol, barium chloride, trifluoroacetic acid, sodium borohydride, dimethyl Sulfone and the like are all purchased from Sinopharm Chemical Reagent Co., Ltd.; L-arabose (L-Ara), D-xylose (D-Xyl), D-mannose (D-Man), D-glucose (D-Glc) A control substance such as D-galactose (D-Gal) 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 (4) Determination of Example 1 according to the BaCl 2 turbidimetric method (see Chen Gan, barium sulfate-turbidimetric determination of sulfate in fucoidan, Journal of Pharmaceutical Practice, 2012, 30(2): 118-120) The content of the sulfate base of the Rhizoma Imperatae polysaccharide 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 Rhizoma Imperatae polysaccharides obtained in Examples 1 to 8 was measured.
  • Rhizoma Imperatae polysaccharide obtained in Example 1 was dissolved in 1 mL of a 3 mol/L aqueous solution of trifluoroacetic acid (TFA) in an ampoule, and then the ampoule was sealed.
  • the Rhizoma Imperata polysaccharide in the ampoule was hydrolyzed at 105 ° C for 4 hours.
  • 2 mL of methanol was added to the ampoule, followed by evaporation to dryness. The operation of adding ethanol and evaporating to dryness was repeated twice to remove TFA. Then, 100 ⁇ L of water was added to the ampoule to obtain a sample in which the acidic condition of the polysaccharide was completely hydrolyzed.
  • 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 the end 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 methylation of the polysaccharide of Example 1 was carried out by reference to the literature method (Fang Ji Nian, methylation analysis method of polysaccharide, Foreign Medicine (Pharmaceutical Section), 1986, (4): 222-226).
  • 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 Rhizoma Imperatae polysaccharides obtained in Examples 1-8 can be determined, mainly containing L-arabinose, D-xylose, D-mannose, D-glucose and D-galactose. And linked by the following monosaccharide forms: terminal group L-arabinose, 1,2,4-L-arabinose, terminal D-xylose, 1,3,4-D-xylose, terminal D-mannose Sugar, 1,6-D-mannose, 1,4-D-glucose, 1,6-D-glucose, 1,4-D-galactose, 1,4,6-D-galactose.
  • the results of methylation analysis are shown in Table 5-12.
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3,4-Me 3 -L-Ara End group L-arabinose 4 3-Me-L-Ara 1,2,4-L-arabinose 4 2,3,4-Me 3 -D-Xyl End group D-xylose 1 2-Me-D-Xyl 1,3,4-D-xylose 2 2,3,4,6-Me 4 -D-Man End group D-mannose 1 2,3,4-Me 3 -D-Man 1,6-D-mannose 3 2,3,6-Me 3 -D-Glc 1,4-D-glucose twenty four 2,3,4-Me3-D-Glc 1,6-D-glucose 5 2,3,6-Me 3 -Gal 1,4-D-galactose 20 2,3-Me 2 -D-Gal 1,4,6-D-galactose 25
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3,4-Me 3 -L-Ara End group L-arabinose 10 3-Me-L-Ara 1,2,4-L-arabinose 1 2,3,4-Me 3 -D-Xyl End group D-xylose 4 2-Me-D-Xyl 1,3,4-D-xylose 10 2,3,4,6-Me 4 -D-Man End group D-mannose 2 2,3,4-Me 3 -D-Man 1,6-D-mannose 3 2,3,6-Me 3 -D-Glc 1,4-D-glucose 15 2,3,4-Me 3 -D-Glc 1,6-D-glucose 1 2,3,6-Me 3 -Gal 1,4-D-galactose 13 2,3-Me 2 -D-Gal 1,4,6-D-galactose 30
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3,4-Me 3 -L-Ara End group L-arabinose 2 3-Me-L-Ara 1,2,4-L-arabinose 5 2,3,4-Me 3 -D-Xyl End group D-xylose 2 2-Me-D-Xyl 1,3,4-D-xylose 1 2,3,4,6-Me 4 -D-Man End group D-mannose 1 2,3,4-Me 3 -D-Man 1,6-D-mannose 1 2,3,6-Me 3 -D-Glc 1,4-D-glucose twenty two 2,3,4-Me 3 -D-Glc 1,6-D-glucose 5 2,3,6-Me 3 -Gal 1,4-D-galactose 15 2,3-Me 2 -D-Gal 1,4,6-D-galactose 25
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3,4-Me 3 -L-Ara End group L-arabinose 8 3-Me-L-Ara 1,2,4-L-arabinose 10 2,3,4-Me 3 -D-Xyl End group D-xylose 2 2-Me-D-Xyl 1,3,4-D-xylose 1 2,3,4,6-Me 4 -D-Man End group D-mannose 1 2,3,4-Me 3 -D-Man 1,6-D-mannose 8 2,3,6-Me 3 -D-Glc 1,4-D-glucose 30 2,3,4-Me 3 -D-Glc 1,6-D-glucose 10 2,3,6-Me 3 -Gal 1,4-D-galactose 25 2,3-Me 2 -D-Gal 1,4,6-D-galactose 30
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3,4-Me 3 -L-Ara End group L-arabinose 3 3-Me-L-Ara 1,2,4-L-arabinose 2 2,3,4-Me 3 -D-Xyl End group D-xylose 3 2-Me-D-Xyl 1,3,4-D-xylose 1 2,3,4,6-Me 4 -D-Man End group D-mannose 1 2,3,4-Me 3 -D-Man 1,6-D-mannose 3 2,3,6-Me 3 -D-Glc 1,4-D-glucose 25 2,3,4-Me 3 -D-Glc 1,6-D-glucose 4 2,3,6-Me 3 -Gal 1,4-D-galactose 16 2,3-Me 2 -D-Gal 1,4,6-D-galactose twenty four
  • Methylated sugar residue Monosaccharide The molar ratio of 2,3,4-Me 3 -L-Ara End group L-arabinose 2 3-Me-L-Ara 1,2,4-L-arabinose 2 2,3,4-Me 3 -D-Xyl End group D-xylose 1 2-Me-D-Xyl 1,3,4-D-xylose 1 2,3,4,6-Me 4 -D-Man End group D-mannose 2 2,3,4-Me 3 -D-Man 1,6-D-mannose 1 2,3,6-Me 3 -D-Glc 1,4-D-glucose 16 2,3,4-Me 3 -D-Glc 1,6-D-glucose 1 2,3,6-Me 3 -Gal 1,4-D-galactose 15 2,3-Me 2 -D-Gal 1,4,6-D-galactose 20
  • Example 1 Rhizoma Imperatae polysaccharides were administered at doses of 100 mg/kg (low dose), 200 mg/kg (medium dose) and 400 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.
  • mice were randomly divided into 6 groups: normal control group (normal group), high-fat model group, positive drug Liping (Lipanthyl, fenofibrate, 40 mg/kg) group, low-dose Rhizoma Imperata polysaccharide group (ICP 100 mg/ Kg), medium dose of Rhizoma Imperatae polysaccharide group (ICP 200mg/kg) and high dose of Rhizoma Imperatae polysaccharide group (ICP 400mg/kg), 10 in each group.
  • different doses of the drug were administered by intragastric administration at 0.2 ml/10 g body weight from 8:00 to 9:00 daily.
  • the normal control group and the high fat diet group were given an equal volume of distilled water.
  • mice On the 4th day of administration, except for the normal control group, each group of mice was orally administered with a high-fat diet (containing 20% lard, 10% cholesterol, 0.2%) at a dose of 0.2 ml/10 g body weight from 14:00 to 15:00 every day. Propylthiouracil, 20% propylene glycol and 20% Tween-80) for 3 weeks. At the end of the experiment, the mice were fasted for 8 hours after treatment. Blood was taken from the eye of the mouse and serum was obtained from the blood by centrifugation. Serum TC, LDL-C and HDL-C were measured and the LDL-C/HDL-C ratio was calculated.
  • 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.
  • Rhizoma Imperatae polysaccharides significantly reduced serum TC levels (P ⁇ 0.05) (LSD assay); high doses of Rhizoma Imperatae polysaccharides (400 mg/kg) significantly reduced serum TC, LDL-C and LDL- C/HDL-C ratio (P ⁇ 0.05 or P ⁇ 0.01) (LSD test).
  • Rhizoma Imperatae polysaccharide can significantly improve lipid vacuoles in the liver; high doses of Rhizoma Imperatae polysaccharides have better effects (Fig. 3D, 3E, Fig. 3F).
  • SOD and GSH-px are antioxidant enzymes in the liver that reduce the amount of reactive oxygen species and reduce the damage of lipid peroxides to liver cells.
  • the activity of SOD and GSH-px in the liver of the high-fat model group was significantly decreased (P ⁇ 0.01) (LSD test), and the MDA content was significantly increased (P ⁇ 0.05). (LSD method test).
  • Rhizoma Imperatae polysaccharide can increase the activity of SOD and GSH-px in liver of mice (P ⁇ 0.05 or P ⁇ 0.01) (LSD test) and reduce MDA content.
  • Rhizoma Imperatae polysaccharide can significantly reduce serum TC and LDL-C levels and LDL-C/HDL-C ratio in hyperlipidemic mice induced by high-fat diet, and also reduce liver TC and TG levels, significantly reduce liver Lipid vacuoles in the medium.

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Abstract

一种分离的白茅根多糖及其在制备用于治疗高脂血症的药物中的用途，该白茅根多糖包含L-阿拉伯糖、D-木糖、D-甘露糖、D-葡萄糖和D-半乳糖，其中L-阿拉伯糖、D-木糖、D-甘露糖、D-葡萄糖和D-半乳糖的摩尔比为1-20:1-15:1-15:15-40:25-60。

Description

一种分离的白茅根多糖及其用途 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白茅根多糖(Imperata cylindrica polysaccharide，ICP)及其在制备用于治疗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。
中药白茅根为伞形科植物白茅根(Imperata cylindrica(Linn)Beauv.var.major(Nees)C.E.Hubb.)的干燥根茎。白茅根主要的化学成分有糖类、三萜类、内酯类、有机酸类等。白茅根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白茅根多糖。通过不同提取方法制得的各种白茅根多糖的单糖组成及其连接方式彼此并不相同。邹一可等(邹一可、张明月、王彩云等，白茅根多糖IC1的分离及其相对分子质量和单糖组成的测定，中国实验方剂学杂志，2012，18(2)：80-82)报道了一种白茅根多糖，其分子量为8292.2，包括摩尔比为1∶11.45∶1.26∶1.02∶95.23的鼠李糖、木糖、果糖、甘露糖、葡萄糖。白茅根多糖 在本领域中通常用于止血、调节免疫、利尿降压、抑菌、抗炎镇痛、抗肿瘤、抗氧化、改善肾功能等作用等。迄今尚未报道白茅根多糖用于治疗高脂血症。
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白茅根多糖的方法，所述方法包括梯度沉淀的步骤。经结构鉴定，发现本发明所述分离的白茅根多糖与已知的白茅根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白茅根多糖具有潜在的调节血脂的作用。
本申请的一个方面提供了一种包含L-阿拉伯糖(L-Ara)、D-木糖(D-Xyl)、D-甘露糖(D-Man)、D-葡萄糖(D-Glc)和D-半乳糖(D-Gal)等单糖的分离的白茅根多糖，其中L-阿拉伯糖、D-木糖、D-甘露糖、D-葡萄糖和D-半乳糖的摩尔比为1-20∶1-15∶1-15∶15-40∶25-60，优选为5-10∶1-5∶1-5∶25-30∶45-55。
在一种实施方式中，所述分离的白茅根多糖包含的单糖组分通过糖苷键相互连接在一起。所述L-阿拉伯糖包括端基L-阿拉伯糖和/或1，2，4-连接的L-阿拉伯糖；所述D-木糖包括端基D-木糖和/或1，3，4-连接的D-木糖；所述D-甘露糖包括端基D-甘露糖和/或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1，6-连接的D-葡萄糖；或者所述D-半乳糖包括1，4-连接的D-半乳糖和/或1，4，6-连接的D-半乳糖。
在一种优选实施方式中，本申请所述L-阿拉伯糖包括端基L-阿拉伯糖和/或1，2，4-连接的L-阿拉伯糖。
在一种优选实施方式中，本申请所述D-木糖包括端基D-木糖和/或1，3，4-连接的D-木糖。
在一种优选实施方式中，本申请所述D-甘露糖包括端基D-甘露糖和/或1，6-连接的D-甘露糖。
在一种优选实施方式中，本申请所述D-葡萄糖包括1，4-连接的D-葡萄糖和/或1，6-连接的D-葡萄糖。
在一种优选实施方式中，本申请所述D-半乳糖包括1，4-连接的D-半乳糖和/或1，4，6-连接的D-半乳糖。
在一种实施方式中，所述分离的白茅根多糖包含端基L-阿拉伯糖、1，2，4-连接的L-阿拉伯糖、端基D-木糖、1，3，4-连接的D-木糖、端基D-甘露糖、1，6-连接的D-甘露糖、1，4-连接的D-葡萄糖、1，6-连接的D-葡萄糖、1，4-连接的D-半乳糖和1，4，6-连接的D-半乳糖。在另一优选实施方式中，所述端基L-阿拉伯糖：1，2，4-连接的L-阿拉伯糖：端基D-木糖：1，3，4-连接的D-木糖：端基D-甘露糖：1，6-连接的D-甘露糖：1，4-连接的D-葡萄糖：1，6-连接的D-葡萄糖：1，4-连接的D-半乳糖：1，4，6-连接的D-半乳糖的摩尔比为1-10∶1-10∶1-5∶1-10∶1-5∶1-10∶15-30∶1-10∶10-25∶15-30，优选为1-5∶1-5∶1-3∶1-5∶1-3∶1-5∶20-25∶1-5∶15-20∶20-25。
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，本申请所述分离的白茅根多糖分子量为1×10 4至5×10 5Da，优选为1×10 5至3×10 5Da。
本申请在另一个方面提供一种制备分离的白茅根多糖的方法，所述方法包括以下步骤：
(1)用水一次或多次提取白茅根，得到白茅根水提取液，任选浓缩所述白茅根水提取液；
(2)向所述任选浓缩的白茅根水提取液经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白茅根多糖。
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)中水与白茅根的体积重量比为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)中用水提取白茅根的次数为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进一步的沉淀；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。
本申请所述白茅根包括商业上可获得的药材白茅根(即植物白茅根的干燥根茎)和白茅根饮片。在一种实施方式中，本申请所述白茅根是白茅根饮片。
术语“分离的白茅根多糖”是指通过人工手段(例如提取、纯化等)将白茅根多糖从其原始植物原料的自然环境中分离出来而得到的白茅根多糖。所述植物原料可以是植物形式的白茅根或药材形式的白茅根，例如植物白茅根的干燥根茎或白茅根饮片。
在一种实施方式中，本申请提供一种分离的白茅根多糖，其是根据本申请所述制备方法获得的。在一种优选的实施方式中，所述分离的白茅根多糖包含L-阿拉伯糖、D-木糖、D-甘露糖、D-葡萄糖和D-半乳糖的白茅根多糖，其中L-阿拉伯糖、D-木糖、D-甘露糖、D-葡萄糖和D-半乳糖的摩尔比为1-20∶1-15∶1-15∶15-40∶25-60，优选为5-10∶1-5∶1-5∶25-30：45-55。在一种优选的实施方式中，所述L-阿拉伯糖包含端基L-阿拉伯糖和/或1，2，4-连接的L-阿拉伯糖；所述D-木糖包含端基D-木糖和/或1，3，4-连接的D-木糖；所述D-甘露糖包含端基D-甘露糖和/或1，6-连接的D-甘露糖；所述D-葡萄糖包含1，4-连接的D-葡萄糖和/或1，6-连接的D-葡萄糖；所述D-半乳糖包含1，4-连接的D-半乳糖和/或1，4，6-连接的D-半乳糖。在一种进一步的优选实施方式中，所述端基L-阿拉伯糖：1，2，4-连接的L-阿拉伯糖：端基D-木糖：1，3，4-连接的D-木糖：端基D-甘露糖：1，6-连接的D-甘露糖：1，4-连接的D-葡萄糖：1，6-连接的D-葡萄糖：1，4-连接的D-半乳糖：1，4，6-D-连接的半乳糖的摩尔比为1-10∶1-10∶1-5∶1-10∶1-5∶1-10∶15-30∶1-10∶10-25∶15-30，优选为1-5∶1-5∶1-3∶1-5∶1-3∶1-5∶20-25∶1-5∶15-20∶20-25。
所述端基L-阿拉伯糖是指通过糖环1位上的糖苷键与相邻基团(例如相邻单糖残基)连接的L-阿拉伯糖。
所述1，2，4-连接的L-阿拉伯糖是指通过糖环1、2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-木糖是指通过糖环1位上的糖苷键与相邻基团(例 如相邻单糖残基)连接的D-木糖。
所述1，3，4连接的D-木糖是指通过糖环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D-木糖。
所述端基D-甘露糖是指通过糖环1位上的糖苷键与相邻基团(例如相邻单糖残基)连接的D-甘露糖。
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1，6-连接的D-葡萄糖是指通过糖环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-葡萄糖。
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。
所述1，4，6-连接的D-半乳糖是指通过糖环1、4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-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白茅根多糖在制备用于治疗高脂血症的药物中的用途。
在一种实施方式中，所述治疗高脂血症药物为血脂代谢调节剂。
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白茅根多糖，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白茅根多糖的药物组合物在制备用于治疗高脂血症的药物中的用途。
在一种实施方式中，所述治疗高脂血症药物为血脂代谢调节剂。
本申请在另一个方面提供一种分离的白茅根多糖，其是根据本申请所述方法获得的。
在还有另一个方面，本申请提供一种治疗高脂血症的方法，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白茅根多糖。在一种实施方式中，所述治疗高脂血症是指调节血脂代谢。
在一种优选的实施方式中，所述的治疗高脂血症的方法，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白 茅根多糖的药物组合物。
在一个方面，本发明还提供分离的白茅根多糖，用于治疗高脂血症。在一种实施方式中，所述治疗高脂血症是指调节血脂代谢。
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，本申请所述白茅根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：白茅根多糖对高脂血症小鼠血脂水平的影响(
Figure PCTCN2019074616-appb-000001
n＝10)。与正常对照组(正常组)比较， #P＜0.05(LSD法检验)， ##P＜0.01(LSD法检验)；与高脂模型组比较， *P＜0.05(LSD法检验)， **P＜0.01(LSD法检验)。
图2：白茅根多糖对高脂血症小鼠肝TC和TG含量的影响(
Figure PCTCN2019074616-appb-000002
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(LSD法检验)， **P＜0.01 (LSD法检验)。
图3：白茅根多糖对高脂血症小鼠肝形态学的影响。图3A：正常对照组；图3B：高脂模型组；图3C：力平之40mg/kg组；图3D：白茅根多糖100mg/kg组；图3E：白茅根多糖200mg/kg组；图3F：白茅根多糖400mg/kg组。
图4：白茅根多糖对高脂血症小鼠肝脏SOD、GSH-px活性和MDA含量的影响(
Figure PCTCN2019074616-appb-000003
n＝10)。与正常对照组(正常组)比较， #P＜0.05(LSD法检验)， ##P＜0.01(LSD法检验)；与高脂模型组比较， *P＜0.05(LSD法检验)， **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
白茅根饮片，购自安徽毫州中药材市场，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-木糖(D-Xyl)、D-甘露糖(D-Man)、D-葡萄糖(D-Glc)、D-半乳糖(D-Gal)等对照品和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：白茅根多糖的制备
(1)向300g白茅根饮片中加入6L蒸馏水。在90℃下用所述蒸馏水提取白茅根饮片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以得到乙醇浓度为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，以得到白茅根多糖12g，收率4％。
实施例2：白茅根多糖的制备
(1)向300g白茅根饮片中加入9L蒸馏水。在40℃下用所述蒸馏水提取白茅根饮片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以得到甲醇浓度为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，以得到白茅根多糖9.8g，收率3.3％。
实施例3：白茅根多糖的制备
(1)向300g白茅根饮片中加入3L蒸馏水。在90℃下用所述蒸馏水提取白茅根饮片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以得到乙醇浓度为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，以得到白茅根多糖13.2g，收率4.4％。
实施例4：白茅根多糖的制备
(1)向300g白茅根饮片中加入2.4L蒸馏水。在100℃下用所述蒸馏水提取白茅根饮片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以得到丙醇浓度为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，以得到白茅根多糖10.9g，收率3.6％。
实施例5：白茅根多糖的制备
(1)向300g白茅根饮片中加入6L蒸馏水。在60℃下用所述蒸馏水提取白茅根饮片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以得到丙酮浓度为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，以得到白茅根多糖10.2g，收率3.4％。
实施例6：白茅根多糖的制备
(1)向300g白茅根饮片中加入6L蒸馏水。在80℃下用所述蒸馏水提取白茅根饮片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以得到乙醇浓度为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，以得到白茅根多糖11.8g，收率3.9％。
实施例7：白茅根多糖的制备
(1)向300g白茅根饮片中加入9L蒸馏水。在70℃下用所述蒸馏水提取白茅根饮片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以得到乙醇浓度为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，以得到白茅根多糖10.7g，收率3.6％。
实施例8：白茅根多糖的制备
(1)向300g白茅根饮片中加入6L蒸馏水。在95℃下用所述蒸馏水提取白茅根饮片1小时，以得到提取液。分离提取液后，重复提取1次，每次使用6L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乙醇以得到乙醇浓度为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，以得到白茅根多糖13.1g，收率4.4％。
实施例9：白茅根多糖的结构鉴定
(1)总糖、糖醛酸、蛋白及硫酸基含量测定
根据硫酸-苯酚法(参见王海侠，白茅根多糖的提取与含量测定，中国中医药信息杂志，2010，17(2)：55-57页)测定实施例1至实施例8所得白茅根多糖总糖含量。
(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至实施例8所得白茅根多糖糖醛酸含量。
(3)根据考马斯亮蓝法(参见张婕，盐炙前后关黄柏多单糖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所得白茅根多糖蛋白含量。
(4)根据BaCl 2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所得白茅根多糖硫酸基含量。
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 76.53 未检测到 2.34 未检测到
2 73.48 未检测到 2.76 未检测到
3 77.47 未检测到 1.53 未检测到
4 75.35 未检测到 2.37 未检测到
5 74.89 未检测到 1.44 未检测到
6 76.06 未检测到 2.71 未检测到
7 75.92 未检测到 1.13 未检测到
8 76.62 未检测到 1.53 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至实施例8所得白茅根多糖的重均分子量。
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 PCTCN2019074616-appb-000004
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 2.2×10 5
2 7.4×10 4
3 1.1×10 5
4 2.4×10 4
5 8.9×10 4
6 1.6×10 5
7 4.3×10 5
8 2.4×10 5
(6)单糖组成分析
将2mg实施例1所得白茅根多糖溶解于安瓿瓶中的1mL的3mol/L的三氟乙酸(TFA)水溶液中，然后将所述安瓿瓶封口。将所述安瓿瓶中的白茅根多糖在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-Xyl∶D-Man∶D-Glc∶D-Gal)
1 8∶3∶4∶29∶45
2 11∶14∶5∶16∶43
3 7∶3∶2∶27∶50
4 18∶3∶9∶40∶55
5 10∶5∶4∶30∶47
6 9∶2∶4∶28∶45
7 5∶4∶4∶29∶46
8 8∶2∶5∶29∶46
(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白茅根多糖进行甲基化。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所获得的白茅根多糖，主要含有L-阿拉伯糖、D-木糖、D-甘露糖、D-葡萄糖和D-半乳糖5种单糖，并通过以下单糖形式连接：端基L-阿拉伯糖、1，2，4-L-阿拉 伯糖、端基D-木糖、1，3，4-D-木糖、端基D-甘露糖、1，6-D-甘露糖、1，4-D-葡萄糖、1，6-D-葡萄糖、1，4-D-半乳糖、1，4，6-D-半乳糖。甲基化分析结果见表5-12。
表4实施例1所得白茅根多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3，4-Me 3-L-Ara 端基L-阿拉伯糖 4
3-Me-L-Ara 1，2，4-L-阿拉伯糖 4
2，3，4-Me 3-D-Xyl 端基D-木糖 1
2-Me-D-Xyl 1，3，4-D-木糖 2
2，3，4，6-Me 4-D-Man 端基D-甘露糖 1
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 24
2，3，4-Me3-D-Glc 1，6-D-葡萄糖 5
2，3，6-Me 3-Gal 1，4-D-半乳糖 20
2，3-Me 2-D-Gal 1，4，6-D-半乳糖 25
表5实施例2所得白茅根多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3，4-Me 3-L-Ara 端基L-阿拉伯糖 10
3-Me-L-Ara 1，2，4-L-阿拉伯糖 1
2，3，4-Me 3-D-Xyl 端基D-木糖 4
2-Me-D-Xyl 1，3，4-D-木糖 10
2，3，4，6-Me 4-D-Man 端基D-甘露糖 2
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 15
2，3，4-Me 3-D-Glc 1，6-D-葡萄糖 1
2，3，6-Me 3-Gal 1，4-D-半乳糖 13
2，3-Me 2-D-Gal 1，4，6-D-半乳糖 30
表6实施例3所得白茅根多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3，4-Me 3-L-Ara 端基L-阿拉伯糖 2
3-Me-L-Ara 1，2，4-L-阿拉伯糖 5
2，3，4-Me 3-D-Xyl 端基D-木糖 2
2-Me-D-Xyl 1，3，4-D-木糖 1
2，3，4，6-Me 4-D-Man 端基D-甘露糖 1
2，3，4-Me 3-D-Man 1，6-D-甘露糖 1
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 22
2，3，4-Me 3-D-Glc 1，6-D-葡萄糖 5
2，3，6-Me 3-Gal 1，4-D-半乳糖 15
2，3-Me 2-D-Gal 1，4，6-D-半乳糖 25
表7实施例4所得白茅根多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3，4-Me 3-L-Ara 端基L-阿拉伯糖 8
3-Me-L-Ara 1，2，4-L-阿拉伯糖 10
2，3，4-Me 3-D-Xyl 端基D-木糖 2
2-Me-D-Xyl 1，3，4-D-木糖 1
2，3，4，6-Me 4-D-Man 端基D-甘露糖 1
2，3，4-Me 3-D-Man 1，6-D-甘露糖 8
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 30
2，3，4-Me 3-D-Glc 1，6-D-葡萄糖 10
2，3，6-Me 3-Gal 1，4-D-半乳糖 25
2，3-Me 2-D-Gal 1，4，6-D-半乳糖 30
表8实施例5获得的白茅根多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3，4-Me 3-L-Ara 端基L-阿拉伯糖 5
3-Me-L-Ara 1，2，4-L-阿拉伯糖 5
2，3，4-Me 3-D-Xyl 端基D-木糖 3
2-Me-D-Xyl 1，3，4-D-木糖 2
2，3，4，6-Me 4-D-Man 端基D-甘露糖 1
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 27
2，3，4-Me 3-D-Glc 1，6-D-葡萄糖 3
2，3，6-Me 3-Gal 1，4-D-半乳糖 19
2，3-Me 2-D-Gal 1，4，6-D-半乳糖 28
表9实施例6所得白茅根多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3，4-Me 3-L-Ara 端基L-阿拉伯糖 5
3-Me-L-Ara 1，2，4-L-阿拉伯糖 4
2，3，4-Me 3-D-Xyl 端基D-木糖 1
2-Me-D-Xyl 1，3，4-D-木糖 1
2，3，4，6-Me 4-D-Man 端基D-甘露糖 1
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 25
2，3，4-Me 3-D-Glc 1，6-D-葡萄糖 3
2，3，6-Me 3-Gal 1，4-D-半乳糖 20
2，3-Me 2-D-Gal 1，4，6-D-半乳糖 25
表10实施例7所得白茅根多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3，4-Me 3-L-Ara 端基L-阿拉伯糖 3
3-Me-L-Ara 1，2，4-L-阿拉伯糖 2
2，3，4-Me 3-D-Xyl 端基D-木糖 3
2-Me-D-Xyl 1，3，4-D-木糖 1
2，3，4，6-Me 4-D-Man 端基D-甘露糖 1
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 25
2，3，4-Me 3-D-Glc 1，6-D-葡萄糖 4
2，3，6-Me 3-Gal 1，4-D-半乳糖 16
2，3-Me 2-D-Gal 1，4，6-D-半乳糖 24
表11实施例8所得白茅根多糖的甲基化分析结果
甲基化糖残基 单糖 摩尔比
2，3，4-Me 3-L-Ara 端基L-阿拉伯糖 2
3-Me-L-Ara 1，2，4-L-阿拉伯糖 2
2，3，4-Me 3-D-Xyl 端基D-木糖 1
2-Me-D-Xyl 1，3，4-D-木糖 1
2，3，4，6-Me 4-D-Man 端基D-甘露糖 2
2，3，4-Me 3-D-Man 1，6-D-甘露糖 1
2，3，6-Me 3-D-Glc 1，4-D-葡萄糖 16
2，3，4-Me 3-D-Glc 1，6-D-葡萄糖 1
2，3，6-Me 3-Gal 1，4-D-半乳糖 15
2，3-Me 2-D-Gal 1，4，6-D-半乳糖 20
实施例10白茅根多糖降血脂作用实验
实验药物：实施例1白茅根多糖，分别以100mg/kg(低剂量)、200mg/kg(中剂量)和400mg/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6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、低剂量白茅根多糖组(ICP 100mg/kg)、中剂量白茅根多糖组(ICP 200mg/kg)和高剂量白茅根多糖组(ICP 400mg/kg)，每组10只。对于各给药组，在每天8：00-9：00按0.2ml/10g体重灌胃给予不同剂量的药物。正常对照组与高脂饮食组给予等体积的蒸馏水。给药第4天，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。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、LDL-C和HDL-C，并计算LDL-C/HDL-C比值。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)白茅根多糖对高脂血症小鼠血脂水平的影响
如图1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)(LSD法检验)，而且LDL-C/HDL-C比值显著升高(P＜0.05)(LSD法检验)。与高脂模型组相比，白茅根多糖能够降低血清TC、LDL-C以及LDL-C/HDL-C比值。特别地，中剂量和高剂量白茅根多糖能够显著降低血清TC含量(P＜0.05)(LSD法检验)；高剂量白茅根多糖(400mg/kg)能够显著降低血清TC、LDL-C以及LDL-C/HDL-C比值(P＜0.05或 P＜0.01)(LSD法检验)。
(2)白茅根多糖对高脂血症小鼠肝TC和TG的影响
如图2所示：与正常对照组比较，高脂模型组中小鼠肝TC和TG含量均显著升高(P＜0.01)(LSD法检验)。与高脂模型组相比，白茅根多糖能够降低肝TC和TG含量。特别地，高剂量白茅根多糖能够显著降低肝TC和TG含量(P＜0.05)(LSD法检验)。
(3)白茅根多糖对高脂血症小鼠肝形态学的影响
如图3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3A)。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3B)。白茅根多糖能明显改善肝脏的脂质空泡；高剂量白茅根多糖具有更好的效果(图3D、3E、图3F)。
(4)白茅根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，减轻脂质过氧化物对肝细胞的损伤。如图4所示：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)(LSD法检验)，同时MDA含量显著升高(P＜0.05)(LSD法检验)。白茅根多糖能够升高小鼠肝SOD和GSH-px活性(P＜0.05或P＜0.01)(LSD法检验)，降低MDA含量。特别地，低剂量、中剂量和高剂量白茅根多糖能够显著升高小鼠肝SOD和GSH-px活性(P＜0.05或P＜0.01)(LSD法检验)，中剂量和高剂量白茅根多糖能够显著降低MDA含量(P＜0.05或P＜0.01)(LSD法检验)。
实验结论：白茅根多糖可显著降低高脂饮食诱导的高脂血症小鼠血清TC和LDL-C水平以及LDL-C/HDL-C比值，同时亦可降低肝 TC和TG含量，明显减少肝中的脂质空泡。

Claims (17)

  1. 一种分离的白茅根多糖，包含L-阿拉伯糖、D-木糖、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-木糖、D-甘露糖、D-葡萄糖和D-半乳糖的摩尔比为1-20∶1-15∶1-15∶15-40∶25-60，优选为5-10∶1-5∶1-5∶25-30∶45-55。
  2. 如权利要求1所述的分离的白茅根多糖，其特征在于：
    所述L-阿拉伯糖包括端基L-阿拉伯糖和/或1，2，4-连接的L-阿拉伯糖；
    所述D-木糖包括端基D-木糖和/或1，3，4-连接的D-木糖；
    所述D-甘露糖包括端基D-甘露糖和/或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1，6-连接的D-葡萄糖；或者
    所述D-半乳糖包括1，4-连接的D-半乳糖和/或1，4，6-连接的D-半乳糖。
  3. 如权利要求2所述的分离的白茅根多糖，其特征在于，所述端基L-阿拉伯糖∶1，2，4-连接的L-阿拉伯糖∶端基D-木糖∶1，3，4-连接的D-木糖∶端基D-甘露糖∶1，6-连接的D-甘露糖∶1，4-连接的D-葡萄糖∶1，6-连接的D-葡萄糖∶1，4-连接的D-半乳糖∶1，4，6-连接的D-半乳糖的摩尔比为1-10∶1-10∶1-5∶1-10∶1-5∶1-10∶15-30∶1-10∶10-25∶15-30，优选为1-5∶1-5∶1-3∶1-5∶1-3∶1-5∶20-25∶1-5∶15-20∶20-25。
  4. 如权利要求3所述的分离的白茅根多糖，其特征在于，所述分离的白茅根多糖分子量为1×10 4至5×10 5Da，优选为1×10 5至3×10 5Da。
  5. 一种制备如权利要求1-4任一项所述的分离的白茅根多糖的方法，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白茅根，得到白茅根水提取液，任选浓缩所述白茅根水提取液；
    (2)向所述任选浓缩的白茅根水提取液经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白茅根多糖。
  6. 如权利要求5所述的方法，其特征在于，所述步骤(1)中水与白茅根的体积重量比为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)中用水提取白茅一次以上，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)中所述白茅根是白茅根饮片。
  13. 如权利要求1-4中任一项所述的分离的白茅根多糖在制备用于治疗高脂血症的药物中的用途。
  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白茅根多糖，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白茅根多糖，用于治疗高脂血症。
  17. 一种治疗高脂血症的方法，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白茅根多糖。
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