WO2024125540A1 - 海藻硫酸多糖及其制备方法和用途 - Google Patents

海藻硫酸多糖及其制备方法和用途 Download PDF

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WO2024125540A1
WO2024125540A1 PCT/CN2023/138342 CN2023138342W WO2024125540A1 WO 2024125540 A1 WO2024125540 A1 WO 2024125540A1 CN 2023138342 W CN2023138342 W CN 2023138342W WO 2024125540 A1 WO2024125540 A1 WO 2024125540A1
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seaweed
polysaccharide
sulfated polysaccharide
fucose
spw
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French (fr)
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丁侃
李佳
臧奕
李赛娟
朱安明
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Shanghai Institute of Materia Medica
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Shanghai Institute of Materia Medica
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Priority to KR1020257023757A priority patent/KR20250123192A/ko
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61K36/02—Algae
    • 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
    • A61K36/02—Algae
    • A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction

Definitions

  • the invention belongs to the technical field of polysaccharides, and in particular relates to a seaweed sulfate polysaccharide, a preparation method thereof and use thereof in preparing an anti-pulmonary fibrosis drug.
  • Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in the pulmonary interstitium and destruction of normal parenchymal structure, leading to progressive loss of lung function.
  • Various factors contribute to the development of pulmonary fibrosis, such as abnormal proliferation of fibroblasts, abnormal expression of myofibroblasts, damage to the alveolar epithelium or endothelium, abnormal tissue repair, dysregulation of wound healing pathways, and excessive production and accumulation of extracellular matrix.
  • the cause and pathogenesis of pulmonary fibrosis are unknown, which seriously affects the survival and quality of life of patients, and brings a heavy burden to patients' families and society. It is a chronic, progressive, and fatal lung disease. Therefore, finding a drug that can effectively treat pulmonary fibrosis is of great clinical significance.
  • the traditional Chinese medicine seaweed is the dried algae of Sargassum pallidum (Turn.) C.Ag or Sargassum fusiforme (Harv.) Setch., which belong to the Sargassum family.
  • the former is commonly known as “big leaf seaweed” and the latter is commonly known as “small leaf seaweed”.
  • Sargassum pallidum is distributed along the Yellow Sea and Bohai Sea in Liaoning and Shandong.
  • Sargassum fusiforme is distributed along the coasts of Liaoning, Shandong, Fujian, Zhejiang, Guangdong and other places.
  • Seaweed has the effects of clearing away heat and detoxifying, eliminating phlegm, softening and dispersing nodules, promoting diuresis and reducing swelling.
  • seaweed contains rich polysaccharides, which account for more than 50% of the dry weight of seaweed.
  • polysaccharides play an important role in the exertion of its biological activity.
  • Modern research shows that seaweed polysaccharides have a variety of biological activities, including antiviral, antitumor, anticoagulant, anti-atherosclerosis, anti-mutation, antioxidant and immune regulation, which has attracted the attention of many researchers.
  • the present invention adopts a simple and effective sulfated polysaccharide extraction process and method, and obtains a sulfated polysaccharide (named in this article: SPW-05-S2) using seaweed (Sargassum fusiforme) as a raw material.
  • SPW-05-S2 can improve the fibrotic phenotype of alveolar epithelial cells;
  • this polysaccharide can reduce lung damage and weaken pulmonary fibrosis in bleomycin-induced pulmonary fibrosis model mice, and is expected to be developed into an anti-pulmonary fibrosis carbohydrate drug.
  • One of the purposes of the present invention is to provide a seaweed sulfated polysaccharide.
  • the second object of the present invention is to provide a method for preparing the seaweed sulfated polysaccharide.
  • the third object of the present invention is to provide a pharmaceutical composition comprising the seaweed sulfated polysaccharide.
  • a fourth object of the present invention is to provide a use of the seaweed sulfated polysaccharide or a pharmaceutical composition comprising the seaweed sulfated polysaccharide in the preparation of a drug for preventing and/or treating pulmonary fibrosis.
  • the present invention provides a seaweed sulfated polysaccharide (SPW-05-S2), comprising 62% to 70% (e.g., 63%, 65%, 67%, 69%) polysaccharide by weight, 27% to 32% (e.g., 28%, 29%, 30%, 31%) sulfate group by weight, 0.5% to 1.5% (e.g., 0.7%, 0.9%, 1.1%, 1.3%) acetyl group by weight, and 2.0% to 4.5% (e.g., 2.5%, 3%, 3.5%, 4%) protein by weight (the weight percentages here are calculated based on the weight of the seaweed sulfated polysaccharide);
  • the polysaccharide is mainly composed of mannose, glucuronic acid, galactose, xylose and fucose.
  • the structural unit of the polysaccharide is a main chain of ⁇ -glucuronic acid linked in 1 ⁇ 4 and ⁇ -mannose linked in 1 ⁇ 2, with branches at the C-6 and C-3 positions of the ⁇ -mannose, the branches being composed of galactose (1,3,6- ⁇ -galactose, 1,6- ⁇ -galactose and terminal- ⁇ -galactose), xylose (1,4- ⁇ -xylose and terminal- ⁇ -xylose) and fucose (1,2- ⁇ -fucose, 1,3- ⁇ -fucose, 1,4- ⁇ -fucose, 1,2,4- ⁇ -fucose and terminal- ⁇ -fucose) residues;
  • the sulfate group is mainly substituted at the C-4 position of 1,2- ⁇ -fucose, the C-2 position of 1,4- ⁇ -fucose, the C-4 position of terminal- ⁇ -galactose, and the C-4 position of part of terminal- ⁇ -xylose.
  • the weight average molecular weight of the seaweed sulfated polysaccharide is in the range of 5-1000 kDa, preferably 10-500 kDa, and more preferably 15-400 kDa.
  • the molar ratio of mannose, glucuronic acid, galactose, xylose and fucose is 10-15:1.5-4:22-32:5-9:40-60, preferably 12.28:2.60:27.59:7.06:50.47.
  • the seaweed sulfated polysaccharide SPW-05-S2 contains 29.28% sulfate groups, 66.4% polysaccharides, 0.91% acetyl groups and 3.4% protein, and has a weight average molecular weight of 121.77 kDa.
  • the seaweed sulfated polysaccharide has a high performance gel permeation chromatography (HPGPC) graph substantially as shown in FIG. 1 .
  • HPGPC high performance gel permeation chromatography
  • the seaweed sulfated polysaccharide has an infrared spectrum substantially consistent with the main stretching vibration absorption peaks in Figure 2.
  • the seaweed sulfated polysaccharide has a 13 C NMR spectrum substantially consistent with the main signal values in Figure 3.
  • ⁇ 105.48- ⁇ 104.08 signals are attributed to 1,3,6- ⁇ -galactose, 1,6- ⁇ -galactose, and terminal ⁇ -galactose.
  • ⁇ 103.24- ⁇ 100.51 signals are attributed to 1,2,3- ⁇ -mannose/1,2,6- ⁇ -mannose, 1,4- ⁇ -xylose and terminal- ⁇ -xylose and 1,4- ⁇ -glucuronic acid.
  • ⁇ 99.69- ⁇ 97.44 are attributed to 1,2- ⁇ -fucose, 1,4- ⁇ -fucose, 1,2,4- ⁇ -fucose and terminal- ⁇ -fucose.
  • ⁇ 93.44 is attributed to 1,3- ⁇ -fucose.
  • the signal of ⁇ 175.02 is attributed to the signal peak of the carboxyl carbon of 1,4- ⁇ -glucuronic acid.
  • the signals of ⁇ 39.47 and ⁇ 21.83 indicate that the polysaccharide contains acetyl groups.
  • the signal peaks of ⁇ 17.97 and ⁇ 17.12 are attributed to the signal peaks of ⁇ -fucose C6.
  • the seaweed sulfate polysaccharide of the present invention is extracted from seaweed produced in Zhejiang (classified as Sargassum fusiformis according to its shape and origin).
  • the present invention provides a method for preparing the seaweed sulfated polysaccharide (SPW-05-S2), comprising the following steps:
  • the dried seaweed is subjected to boiling water extraction, the obtained extract is concentrated, dialyzed, re-concentrated, and centrifuged to remove the precipitate, the obtained supernatant is precipitated with alcohol, centrifuged, and the obtained precipitate is washed and dried to obtain crude sulfated seaweed polysaccharide extracted from water;
  • the step a comprises: adding about 15 to 20 times the weight of water to the dried seaweed, raising the temperature to 100°C for boiling water extraction for 3 to 5 hours, extracting 7 to 9 times in total, combining and concentrating the extracts, dialyzing, re-concentrating, centrifuging to obtain a supernatant, adding about 5 to 10 times the volume of ethanol as much as the supernatant, centrifuging to obtain a precipitate, washing the precipitate with anhydrous ethanol and acetone for 3 to 6 times, and vacuum drying or freeze-drying to obtain a water-extracted seaweed crude sulfated polysaccharide;
  • the step a comprises: adding about 20 times the weight of deionized water to the dried seaweed, raising the temperature to 100° C. to perform boiling water extraction for 4 hours, extracting 8 times in total, combining and concentrating the extracts, dialyzing, concentrating the dialyzed solution, centrifuging to obtain a supernatant, adding about 5 times the volume of ethanol of the supernatant, centrifuging to obtain a precipitate, washing the precipitate alternately with anhydrous ethanol and acetone for 3 times, drying in an oven, re-dissolving, and freeze-drying to obtain a water-extracted seaweed crude sulfated polysaccharide;
  • step b1 Dissolve the crude sulfated polysaccharide extracted from seaweed prepared in step a in water, centrifuge, and perform preliminary fractionation and purification on the supernatant by anion exchange column. Then, elute with water and 0.05-0.8 M NaCl solution in turn, collect the elution fraction of about 0.5 M NaCl solution, and obtain sulfated polysaccharide SPW-05;
  • the step b1 comprises: taking the crude sulfated polysaccharide of seaweed extracted by water prepared in step a, adding about 10 to 20 times the weight of water to dissolve, centrifuging, separating the supernatant through an anion exchange column, eluting with deionized water, 0.05M, 0.1M, 0.2M, 0.3M, 0.5M, 0.6M and 0.8M NaCl solution in sequence, detecting with sulfuric acid-phenol, collecting and merging the eluate of about 0.5M NaCl solution according to the elution curve, concentrating, centrifuging, taking the supernatant for dialysis, and freeze-drying to obtain the preliminarily purified sulfated polysaccharide SPW-05;
  • the step b1 comprises: taking the crude sulfated polysaccharide of seaweed extracted by water prepared in step a, adding about 10 times the weight of water to dissolve, centrifuging, separating the supernatant through a DEAE Sepharose Fast Flow anion exchange column, eluting with deionized water, 0.05M, 0.1M, 0.2M, 0.3M, 0.5M, 0.6M and 0.8M NaCl solution in sequence, detecting with sulfuric acid-phenol, collecting and merging the eluate of 0.5M NaCl solution according to the elution curve, concentrating, centrifuging, dialyzing the supernatant, and freeze-drying to obtain the preliminarily purified sulfated polysaccharide SPW-05;
  • step b2 Dissolve the sulfated polysaccharide SPW-05 prepared in step b1 in about 0.01 to 1 times the weight of a 0.2M NaCl solution, centrifuge, purify the supernatant by gel chromatography, elute with a 0.2M NaCl solution, collect and combine the main component of the sulfated polysaccharide SPW-05-S2 according to the elution curve, and obtain the sulfated polysaccharide SPW-05-S2;
  • the step b2 comprises: dissolving the sulfated polysaccharide SPW-05 prepared in step b1 in about 0.01 times its weight in about 0.2M NaCl solution, centrifuging, purifying the supernatant through a Sephacryl HR S-300 gel chromatography column, eluting with 0.2M NaCl solution, detecting by sulfuric acid-phenol method, collecting and combining the main component of sulfated polysaccharide SPW-05-S2 according to the elution curve, concentrating, dialyzing, and freeze-drying to obtain the sulfated polysaccharide SPW-05-S2.
  • the ethanol may be an ethanol aqueous solution of about 70% (v/v) or more, preferably an ethanol aqueous solution of about 85% (v/v) or more, and particularly an ethanol aqueous solution of about 95% (v/v) or more.
  • Structural identification of sulfated polysaccharides The structure of the seaweed sulfated polysaccharide SPW-05-S2 was determined by comprehensive analysis of its monosaccharide composition, methylation, infrared and nuclear magnetic resonance.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the seaweed sulfated polysaccharide as an active ingredient.
  • the composition may further include pharmaceutically acceptable excipients, such as carriers, excipients, adjuvants and/or diluents.
  • Another aspect of the present invention provides use of the seaweed sulfated polysaccharide or the pharmaceutical composition in preparing a drug for treating and/or preventing pulmonary fibrosis.
  • the pulmonary fibrosis is preferably bleomycin-induced pulmonary fibrosis.
  • a method for reducing fibrotic phenotype, and preventing and/or treating pulmonary fibrosis comprising administering a therapeutically effective amount of the seaweed sulfated polysaccharide or the pharmaceutical composition to a subject in need of such treatment.
  • the present invention prepares a uniform sulfated polysaccharide component (molecular weight within a certain range (normal distribution) with a certain sugar residue connection mode and sugar composition) containing a specific sugar composition, sulfate content, molecular weight and sulfate substitution position through a specific extraction and separation method.
  • the polysaccharide SPW-05-S2 can reduce bleomycin-induced pulmonary fibrosis, including restoring lung tissue morphology, reducing lung coefficient, and reducing the expression of animal lung damage and fibrosis phenotype-related proteins.
  • it has no effect on cell growth vitality in vitro, can significantly inhibit the expression of fibrosis-related proteins, and has great application prospects in the prevention and/or treatment of pulmonary fibrosis candidate drugs.
  • FIG1 is a characteristic high-performance gel permeation chromatogram of seaweed sulfate polysaccharide SPW-05-S2;
  • FIG2 is a characteristic infrared spectrum of seaweed sulfate polysaccharide SPW-05-S2;
  • FIG3 is a characteristic 13 C NMR spectrum of seaweed sulfated polysaccharide SPW-05-S2;
  • Figure 4 is a characteristic 1 H-QA (quantitative) NMR spectrum of seaweed sulfated polysaccharide SPW-05-S2;
  • Figure 5 is a bar graph (A) showing the effect of the seaweed sulfated polysaccharide SPW-05-S2 prepared in Preparation Example 1 on the mouse lung tissue morphology (A) and lung coefficient (B) in the bleomycin-induced mouse pulmonary fibrosis model (** represents P ⁇ 0.01);
  • Figure 6 is a representative image of hematoxylin-eosin staining (A) and Masson staining (B) of mouse lung tissue in a bleomycin-induced mouse pulmonary fibrosis model of seaweed sulfated polysaccharide SPW-05-S2 prepared in Preparation Example 1;
  • Figure 7 is an immunoblot of Fibronectin, Collagen I and ⁇ -smooth muscle actin proteins in mouse lung tissue in the bleomycin-induced mouse pulmonary fibrosis model by the seaweed sulfated polysaccharide SPW-05-S2 prepared in Preparation Example 1;
  • FIG8 is a diagram of the CCK-8 cell proliferation and toxicity assay for the preparation of the Bar graph showing the effect of seaweed sulfated polysaccharide SPW-05-S2 on the viability of alveolar epithelial cells A549 (+ represents addition, - represents no addition);
  • Figure 9 is an immunoblot graph of the effect of seaweed sulfated polysaccharide SPW-05-S2 prepared in Preparation Example 1 on the protein expression levels of E-cadherin, ⁇ -SMA, Collagen I and Fibronectin in TGF- ⁇ -induced alveolar epithelial cells A549 cells (+ represents addition, - represents no addition).
  • numerical values should be understood to have the accuracy of the significant digits of the numerical value.
  • the number 40.0 should be understood to cover the range from 39.50 to 40.49.
  • the seaweed was purchased from Haozhou Chinese Medicine Pieces Company and was produced in Zhejiang, China.
  • HPLC High performance liquid chromatography
  • the nuclear magnetic resonance analysis was performed using a Brucker AM-500 nuclear magnetic resonance instrument (Brucker, Germany).
  • the obtained precipitate was washed alternately with anhydrous ethanol and acetone for 3 times, and the precipitate was centrifuged and vacuum dried at 50 ° C, and then redissolved in 1500 mL of deionized water, and freeze-dried to obtain 204 g of crude sulfated seaweed polysaccharide (yield 10.2%).
  • the monosaccharide composition of the completely hydrolyzed sample of sulfated polysaccharide SPW-05-S2 was measured by PMP derivatization method, and it was determined that the sulfated polysaccharide SPW-05-S2 mainly contained mannose, glucuronic acid, galactose, xylose and fucose, with a molar ratio of 12.28:2.60:27.59:7.06:50.47. Methylation analysis.
  • the raw sugar, desulfurized polysaccharide and reduced desulfurized polysaccharide were methylated and analyzed by GC-MS to determine the sugar residue connection mode and sulfate substitution position of sulfated polysaccharide SPW-05-S2, and the connection mode of glucuronic acid was determined to be 1,4-glucuronic acid.
  • the main chain of sulfated polysaccharide SPW-05-S2 was determined to be 1,4-glucuronic acid and 1,2-mannose by the sugar composition and sugar residue connection mode of the secondary polysaccharide after partial acid hydrolysis.
  • the one-dimensional and two-dimensional NMR spectra were measured at 25° C. on a Bruker AVANCE III 500M NMR instrument.
  • the structure of the sulfated polysaccharide SPW-05-S2 was confirmed by referring to the NMR spectra.
  • the 13 C NMR result of SPW-05-S2 is shown in FIG3 .
  • the ⁇ 105.48- ⁇ 104.08 signals are attributed to 1,3,6- ⁇ -galactose, 1,6- ⁇ -galactose, and terminal ⁇ -galactose.
  • the ⁇ 103.24- ⁇ 100.51 signals are attributed to 1,2,3- ⁇ -mannose/1,2,6- ⁇ -mannose, 1,4- ⁇ -xylose, terminal ⁇ -xylose, and 1,4- ⁇ -glucuronic acid.
  • ⁇ 99.69- ⁇ 97.44 are attributed to 1,2- ⁇ -fucose, 1,4- ⁇ -fucose, 1,2,4- ⁇ -fucose, and terminal ⁇ -fucose.
  • ⁇ 93.44 is attributed to 1,3- ⁇ -fucose.
  • the ⁇ 175.02 signal is attributed to the signal peak of the carboxyl carbon of 1,4- ⁇ -glucuronic acid.
  • the signals of ⁇ 39.47 and ⁇ 21.83 indicated that the polysaccharide contained acetyl groups, and the signal peaks of ⁇ 17.97 and ⁇ 17.12 were attributed to the ⁇ -fucose C6.
  • the signal at ⁇ 5.0-5.7 is the signal of the anomeric hydrogen in the sulfated polysaccharide SPW-05-S2
  • the signal at ⁇ 3.25-4.65 is the signal of the hydrogen on the sugar ring
  • the signal near ⁇ 2.28 is the typical signal of the methyl hydrogen on the acetyl group.
  • the signal near ⁇ 1.30 is the signal of fucose H6. According to the integrated area ratio of the signal near ⁇ 2.28 and the signal near ⁇ 1.30, the molar ratio of acetyl to fucose is 0.028:1. Since the molar fraction of fucose in the total sugar is 50.47%, the molar ratio of acetyl to total sugar can be converted to 0.014:1.
  • seaweed sulfated polysaccharide SPW-05-S2 may be based on 1,4- ⁇ -glucuronic acid and 1,2- ⁇ -mannose as the main chain structure, and there are branches at the C-6 and C-3 positions of ⁇ -mannose, which are composed of galactose residues (1,3,6- ⁇ -galactose, 1,6- ⁇ -galactose and terminal- ⁇ -galactose), xylose (1,4- ⁇ -xylose and terminal- ⁇ -xylose) and fucose (1,2- ⁇ -fucose, 1,3- ⁇ -fucose, 1,4- ⁇ -fucose, 1,2,4- ⁇ -fucose and terminal- ⁇ -fucose) residues.
  • Test Example 2 Anti-pulmonary fibrosis activity of seaweed sulfated polysaccharide SPW-05-S2
  • mice were administered an equal volume of 0.9% saline intratracheally as the normal control group.
  • the experimental groups were randomly divided into four groups, A, B, C, and D, with 6 mice in each group.
  • Group A was administered 25 mg/kg SPW-05-S2 polysaccharide
  • Group B was administered 50 mg/kg SPW-05-S2 polysaccharide
  • Group C was administered 100 mg/kg SPW-05-S2 polysaccharide
  • Group D was administered 100 mg/kg pirfenidone (purchased from MCE) as a positive drug control, and was administered by gavage every day for two weeks.
  • the control group and the model group were given an equal volume of normal saline.
  • the weight of the mice was recorded and the mice were sacrificed.
  • the morphology of the lung tissue of each group of mice was observed, and the lung tissue was weighed.
  • the lung tissue surface of the mice with pulmonary fibrosis after intervention with SPW-05-S2 polysaccharide was restored to a smooth and ruddy color, but the texture was not as soft and elastic as the lung tissue of the control group mice.
  • the lung coefficient (lung tissue weight/mouse weight) of the model group mice increased, and the lung coefficient of the low (25 mg/kg), medium (50 mg/kg), and high (100 mg/kg) dose groups of SPW-05-S2 decreased, which was significantly different from the model group.
  • mice Two weeks after administration, the mice were sacrificed, and the left lung lobe was fixed with 4% paraformaldehyde for 48 hours, followed by hematoxylin-eosin staining and Masson staining.
  • the results of hematoxylin-eosin staining showed that the degree of lung tissue lesions in the low, medium and high dose groups of SPW-05-S2 was significantly reduced, and only a small number of interstitial vascular dilation, congestion and mild alveolitis were observed, but the degree of alveolar wall fibrosis was greatly reduced.
  • the alveolar septum basically returned to normal, and no diffuse inflammatory infiltration was observed.
  • mice Two weeks after administration, the mice were sacrificed, and part of the lung tissue was extracted with a tissue extraction kit (purchased from Shenggong Biotechnology Co., Ltd.). After adding 5 ⁇ loading buffer, the protein was denatured at 95°C in a metal heater for 10 minutes, cooled and stored at -80°C. Immunoblotting was used to detect the expression of Fibronectin, Collagen I and ⁇ -smooth muscle actin proteins. As shown in Figure 7, seaweed polysaccharide SPW-05-S2 can inhibit the expression of fibrosis phenotype-related proteins (Fibronectin, Collagen I and ⁇ -smooth muscle actin).
  • A549 cells (5 ⁇ 10 3 cells/well) in the logarithmic growth phase were seeded into 96-well plates, set up three replicates, and cultured in an incubator for 24 hours; the cell supernatant was aspirated, and the seaweed polysaccharide SPW-05-S2 solution with a final concentration of 0.1, 0.5 and 1 mg/mL was added for 1 hour, and then TGF- ⁇ (10 ng/mL) was added for intervention treatment. After continuing to culture for 48 hours, 10 ⁇ L of CCK-8 enhanced solution (purchased from Dalian Meilun Company) was added to each well, and the absorbance was detected at 450 nm with an enzyme marker after continuing to culture for 1 hour.
  • CCK-8 enhanced solution purchased from Dalian Meilun Company
  • Cell growth activity (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) ⁇ 100%.
  • the results are shown in Figure 8, indicating that SPW-05-S2 has no effect on cell growth activity (+ represents addition, - represents no addition).
  • Human alveolar epithelial cells A549 were cultured in RPMI1640 medium containing 10% fetal bovine serum (purchased from Gibco, USA), 100U/mL penicillin and 100 ⁇ g/mL streptomycin in a volume ratio of 1:1. A549 cells in the logarithmic growth phase were seeded in a 6-well plate at a density of 1.2 ⁇ 10 6 cells/well.
  • TGF- ⁇ recombinant protein purchased from Abclonal
  • the supernatant was discarded, the cells were rinsed with pre-cooled PBS, and the cells were lysed on ice with RIPA cell lysis buffer (purchased from Absin) for 20 minutes, and the supernatant was collected by centrifugation.
  • the protein was denatured in a metal heater for 10 minutes, cooled and stored at -80°C.
  • the protein expressions of E-cadherin, ⁇ -smooth muscle actin, Collagen I and Fibronectin were detected by immunoblotting.
  • seaweed polysaccharide SPW-05-S2 can significantly inhibit the expression of ⁇ -smooth muscle actin, Collagen I and Fibronectin proteins, and significantly increase the expression of E-cadherin protein.
  • seaweed sulfated polysaccharide SPW-05-S2 can reduce bleomycin-induced pulmonary fibrosis, including restoring lung tissue morphology, reducing lung coefficient, and reducing the expression of animal lung injury and fibrosis phenotype-related proteins. In addition, it does not affect the cell growth viability of alveolar epithelial cells A549 in vitro.
  • SPW-05-S2 can significantly inhibit the expression of fibrosis-related proteins ⁇ -SMA, Collagen I and Fibronectin, and significantly increase the expression of E-cadherin protein. Therefore, the polysaccharide component SPW-05-S2 can become a potential carbohydrate drug for preventing and/or treating pulmonary fibrosis.

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Abstract

属于多糖技术领域，具体涉及一种海藻硫酸多糖及其制备方法和用途。所述海藻硫酸多糖SPW-05-S2包含多糖62％～70％、硫酸基27％～32％、乙酰基0.5％～1.5％以及蛋白2.0％～4.5％，其重均分子量为121.77kDa；所述多糖的组成主要有甘露糖，葡萄糖醛酸，半乳糖，木糖和岩藻糖。经体内实验证明，该多糖能够降低博莱霉素诱导的肺纤维化，包括恢复肺组织形态，降低肺系数，减少动物肺损伤和纤维化表型相关蛋白的表达。并且在体外对细胞生长活力没有影响，能显著抑制纤维化相关蛋白的表达。因此，所述多糖具有潜在缓解肺损伤的作用，有望成为一种治疗肺纤维化的候选糖类药物。

Description

海藻硫酸多糖及其制备方法和用途 技术领域
本发明属于多糖技术领域，具体涉及一种海藻硫酸多糖、其制备方法及在制备抗肺纤维化药物中的用途。
背景技术
肺纤维化(Pulmonary fibrosis)的特征是肺间质内细胞外基质过度沉积和正常实质结构的破坏，导致肺功能进行性丧失。各种因素促成肺纤维化的发展，如成纤维细胞的异常增殖、肌成纤维细胞的异常表达、肺泡上皮或内皮的损伤、组织的异常修复、伤口愈合途径的失调以及细胞外基质生成过多和累积。肺纤维化病因和发病机制未明，严重影响患者的生存和生活质量，为患者家庭以及社会带来沉重负担，是一种慢性进行性的、致命的肺部疾病。因此，找到一种可有效治疗肺纤维化的药物具有重要的临床意义。
中药海藻，属马尾藻科植物海嵩子Sargassum pallidum(Turn.)C.Ag或羊栖菜Sargassum fusiforme(Harv.)Setch.的干燥藻体。前者习称“大叶海藻”，后者习称“小叶海藻”。海蒿子分布于辽宁、山东的黄海和渤海沿岸。羊栖菜分布于辽宁、山东、福建、浙江、广东等地沿海。海藻具有清热解毒、消痰软坚散结，利水消肿之功效，常用于瘿瘤，瘰疬、睾丸肿痛，痰饮水肿。海藻中含有丰富的多糖，占海藻干重的50％以上。多糖作为中药中的主要活性物质，在其生物活性的发挥中起着重要的作用。现代研究表明，海藻多糖具有多种生物活性，包括抗病毒、抗肿瘤、抗凝血、抗动脉硬化、抗突变、抗氧化和调节免疫活性等，从而引起较多研究者的关注。但中药中具有抗肺纤维化活性的天然多糖鲜有报道。
发明内容
本发明采用一种简单有效的硫酸多糖提取工艺和方法，以海藻(Sargassum fusiforme)为原料获得了一种含硫酸基的多糖(文中命名：SPW-05-S2)。体外实验表明SPW-05-S2可以改善肺泡上皮细胞纤维化表型； 并通过体内实验证明，该多糖可以在博莱霉素诱导的肺纤维化模型小鼠中减轻肺部损伤并减弱肺纤维化，有望开发成为一种抗肺纤维化的糖类药物。
本发明的目的之一在于提供一种海藻硫酸多糖。
本发明的目的之二在于提供一种所述海藻硫酸多糖的制备方法。
本发明的目的之三在于提供一种包含所述海藻硫酸多糖的药物组合物。
本发明的目的之四在于提供一种所述海藻硫酸多糖或包含所述海藻硫酸多糖的药物组合物在制备预防和/或治疗肺纤维化的药物中的用途。
为了实现本发明的上述目的，特采用以下技术方案：
本发明一方面，提供了一种海藻硫酸多糖(SPW-05-S2)，包含重量百分含量为62％～70％(如63％、65％、67％、69％)的多糖，重量百分含量为27％～32％(如28％、29％、30％、31％)的硫酸基，重量百分含量为0.5％～1.5％(如0.7％、0.9％、1.1％、1.3％)的乙酰基，以及重量百分含量为2.0％～4.5％(如2.5％、3％、3.5％、4％)的蛋白(这里的重量百分含量以海藻硫酸多糖重量为基准计算)；
其中，所述多糖主要由甘露糖、葡萄糖醛酸、半乳糖、木糖和岩藻糖组成。
所述多糖的结构单元是以1→4连接的β-葡萄糖醛酸、1→2连接的α-甘露糖为主链，在α-甘露糖的C-6位和C-3位存在分支，分支由半乳糖(1,3,6-β-半乳糖、1,6-β-半乳糖和末端-β-半乳糖)、木糖(1,4-β-木糖和末端-β-木糖)和岩藻糖(1,2-α-岩藻糖、1,3-α-岩藻糖、1,4-α-岩藻糖、1,2,4-α-岩藻糖和末端-α-岩藻糖)残基组成；
所述硫酸基主要取代在1,2-α-岩藻糖的C-4位、1,4-α-岩藻糖的C-2位、末端-β-半乳糖的C-4位以及部分末端-β-木糖的C-4位。
所述海藻硫酸多糖的重均分子量的范围为5-1000kDa，优选为10-500kDa，更优选为15-400kDa。
优选地，所述甘露糖、葡萄糖醛酸、半乳糖、木糖和岩藻糖的摩尔比为10～15:1.5～4:22～32:5～9:40～60，优选为12.28:2.60:27.59:7.06:50.47。
在一些实施例中，所述海藻硫酸多糖SPW-05-S2含29.28％的硫酸基、66.4％的多糖、0.91％乙酰基及3.4％的蛋白，其重均分子量为121.77kDa。
优选地，所述海藻硫酸多糖具有基本上如图1所示的高效凝胶渗透色谱(HPGPC)图。
优选地，所述海藻硫酸多糖具有基本上与图2中的主要伸缩振动吸收峰一致的红外图谱。在所述海藻硫酸多糖SPW-05-S2的红外图谱中，3308.69cm-1为O-H伸缩振动吸收峰，2932.46cm-1为C-H伸缩振动吸收峰，1217.11cm-1处的强吸收峰为O＝S＝O的伸缩振动峰，1732.38cm-1为羧基C＝O伸缩振动，表明该多糖含有糖醛酸。
优选地，所述海藻硫酸多糖具有基本上与图3中的主要信号值一致的13C NMR谱。在所述海藻硫酸多糖的13C NMR谱中，δ105.48-δ104.08信号归属于1,3,6-β-半乳糖，1,6-β-半乳糖，末端β-半乳糖。δ103.24-δ100.51信号归属于1,2,3-α-甘露糖/1,2,6-α-甘露糖、1,4-β-木糖和末端-β-木糖及1,4-β-葡萄糖醛酸。δ99.69-δ97.44归属于1,2-α-岩藻糖，1,4-α-岩藻糖，1,2,4-α-岩藻糖和末端-α-岩藻糖。δ93.44归属于1,3-α-岩藻糖。δ175.02信号归属于1,4-β-葡萄糖醛酸羧基碳的信号峰。δ39.47及δ21.83的信号表明该多糖含有乙酰基。δ17.97和δ17.12归属于α-岩藻糖C6的信号峰。
优选地，本发明的海藻硫酸多糖是从浙江产地的海藻(按形状及产地归属为羊栖菜)中提取出的。
本发明另一方面，提供了所述海藻硫酸多糖(SPW-05-S2)的制备方法，包括以下步骤：
a.硫酸多糖提取：
干燥的海藻，进行沸水提取，将所得提取液浓缩、透析、再浓缩、离心去沉淀，所得上清液醇沉、离心分离，所得沉淀洗涤、干燥，得水提海藻粗硫酸多糖；
优选地，所述步骤a包括：干燥的海藻，加入约15～20倍重量的水，将温度升至100℃进行沸水提取3～5h，共提取7～9次，将提取液合并浓缩，透析，再浓缩，离心得上清液，加入约5倍～10倍于上清液体积的乙醇，离心得沉淀，沉淀经无水乙醇与丙酮洗涤3～6次，经真空干燥或冷冻干燥得水提海藻粗硫酸多糖；
进一步优选，所述步骤a包括：干燥的海藻，加入约20倍重量的去离子水，将温度升至100℃进行沸水提取4h，共提取8次，将提取液合并浓缩，透析，透析内液浓缩，离心，得上清液后，加入约5倍于上清液体积的乙醇，离心得沉淀，沉淀经无水乙醇与丙酮交替洗涤3次，经烘箱干燥后，复溶，再冷冻干燥得水提海藻粗硫酸多糖；
b.硫酸多糖纯化：
b1.取步骤a制备的水提海藻粗硫酸多糖用水溶解，离心，上清液经阴离子交换柱进行初步分级纯化，依次以水和0.05～0.8M的NaCl溶液洗脱，收集约0.5M NaCl溶液的洗脱组分，得硫酸多糖SPW-05；
优选地，所述步骤b1包括：取步骤a制备的水提海藻粗硫酸多糖，加入约10～20倍重量的水中溶解，离心，上清液经阴离交换柱分离，依次以去离子水、0.05M、0.1M、0.2M、0.3M、0.5M、0.6M及0.8M NaCl溶液洗脱，硫酸-苯酚检测，根据洗脱曲线收集合并约0.5M NaCl溶液的洗脱液，浓缩，离心，取上清液透析，冷冻干燥，得初步纯化的硫酸多糖SPW-05；
进一步优选地，所述步骤b1包括：取步骤a制备的水提海藻粗硫酸多糖，加入约10倍重量的水中溶解，离心，上清液经DEAE Sepharose Fast Flow阴离交换柱分离，依次以去离子水、0.05M、0.1M、0.2M、0.3M、0.5M、0.6M及0.8M NaCl溶液洗脱，硫酸-苯酚检测，根据洗脱曲线收集合并0.5M NaCl溶液的洗脱液，浓缩，离心，取上清液透析，冷冻干燥，得初步纯化的硫酸多糖SPW-05；
b2.将步骤b1制备的硫酸多糖SPW-05溶于约0.01～1倍重量的0.2M NaCl溶液中，离心，上清液经凝胶色谱柱纯化，以0.2M NaCl溶液洗脱，根据洗脱曲线收集合并硫酸多糖SPW-05-S2这一主要组分，得所述硫酸多糖SPW-05-S2；
优选地，所述步骤b2包括：将步骤b1制备的硫酸多糖SPW-05溶于约0.01倍重量的约0.2M NaCl溶液中，离心，上清液经Sephacryl HR S-300凝胶色谱柱纯化，以0.2M NaCl溶液洗脱，硫酸-苯酚法检测，根据洗脱曲线收集合并硫酸多糖SPW-05-S2这一主要组分，浓缩，透析，冷冻干燥，得所述硫酸多糖SPW-05-S2。
步骤a中，所述乙醇可以为约70％(v/v)以上的乙醇水溶液，优选为约85％(v/v)以上的乙醇水溶液，特别为约95％(v/v)以上的乙醇水溶液。
硫酸多糖的结构鉴定：所述海藻硫酸多糖SPW-05-S2通过对其单糖组成、甲基化、红外及核磁等进行综合解析，确定其结构。
本发明再一方面，提供了一种药物组合物，其包含治疗有效量的所述海藻硫酸多糖作为活性成分，该组合物可以进一步包括药剂学上可接受的药物辅料，例如载体、赋形剂、佐剂和/或稀释剂等。
本发明的又一方面，提供了所述海藻硫酸多糖或所述药物组合物在制备用于治疗和/或预防肺纤维化的药物中的用途。
肺纤维化优选为博莱霉素诱导的肺纤维化。
本发明的又一方面，还提供了一种减弱纤维化表型，以及预防和/或治疗肺纤维化的方法，所述方法包括向需要该治疗的对象给药治疗有效量的所述海藻硫酸多糖或所述药物组合物。
本发明的技术方案至少具有以下技术效果：
本发明通过特定提取及分离方法，制备含有特定糖组成，硫酸基含量，分子量及硫酸基取代位置等的均一硫酸多糖组分(分子量在一定范围(呈现正态分布)内的，具有确定的糖残基连接方式和糖组成)。经体内实验证明，该多糖SPW-05-S2能够降低博莱霉素诱导的肺纤维化，包括恢复肺组织形态，降低肺系数，减少动物肺损伤和纤维化表型相关蛋白的表达。并且在体外对细胞生长活力没有影响，能显著抑制纤维化相关蛋白的表达，在预防和/或治疗肺纤维化的候选药物方面具有巨大的应用前景。
在上文中已经详细地描述了本发明，但是上述实施方式本质上仅是例示性，且并不欲限制本发明。此外，本文并不受前述现有技术或发明内容或以下实施例中所描述的任何理论的限制。
附图说明
图1为海藻硫酸多糖SPW-05-S2的特征高效凝胶渗透色谱图；
图2为海藻硫酸多糖SPW-05-S2的特征红外光谱图；
图3为海藻硫酸多糖SPW-05-S2的特征13C NMR图谱；
图4为海藻硫酸多糖SPW-05-S2的特征1H-QA(定量)NMR图谱；
图5为制备实施例1中制得的海藻硫酸多糖SPW-05-S2对博莱霉素诱导小鼠肺纤维化模型中小鼠肺组织形态(A)与肺系数柱状图(B)(**代表P<0.01)；
图6为制备实施例1中制得的海藻硫酸多糖SPW-05-S2对博莱霉素诱导小鼠肺纤维化模型中小鼠肺组织苏木素-伊红染色(A)和Masson染色(B)代表图；
图7为制备实施例1中制得的海藻硫酸多糖SPW-05-S2对博莱霉素诱导小鼠肺纤维化模型中小鼠肺组织Fibronectin，Collagen I和α-smooth muscle actin蛋白免疫印迹图；
图8为使用CCK-8细胞增殖及毒性检测实验检测制备实施例1中制得的 海藻硫酸多糖SPW-05-S2对肺泡上皮细胞A549细胞活力影响的柱状图(+代表加入，—代表不加入)；
图9为制备实施例1中制得的海藻硫酸多糖SPW-05-S2对TGF-β诱导的肺泡上皮细胞A549细胞中E-cadherin、α-SMA、Collagen I和Fibronectin的蛋白水平表达量的免疫印迹图(+代表加入，—代表不加入)。
具体实施方式
在本文中，所有以数值范围或百分比范围形式界定的特征或条件仅是为了简洁及方便。据此，数值范围或百分比范围的描述应视为已涵盖且具体公开所有可能的次级范围及范围内的个别数值，特别是整数数值。举例而言，“1至8”的范围描述应视为已经具体公开如1至7、2至8、2至6、3至6、4至8、3至8等等所有次级范围，特别是由所有整数数值所界定的次级范围，且应视为已经具体公开范围内如1、2、3、4、5、6、7、8等个别数值。除非另有指明，否则前述解释方法适用于本发明全文的所有内容，不论范围广泛与否。
若数量或其他数值或参数是以范围、较佳范围或一系列上限与下限表示，则其应理解成是本文已特定公开了由任一对该范围的上限或较佳值与该范围的下限或较佳值构成的所有范围，不论这些范围是否有分别公开。此外，本文中若提到数值的范围时，除非另有说明，否则该范围应包括其端点以及范围内的所有整数与分数。
在本文中，在可实现发明目的的前提下，数值应理解成具有该数值有效位数的精确度。举例来说，数字40.0则应理解成涵盖从39.50至40.49的范围。
下面结合实施例对本发明作进一步的说明，需要说明的是，提供以下实施例仅出于说明目的并不构成对本发明要求保护范围的限制。
实施例所用的实验方法如无特殊说明，均为常规方法。
实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
材料：
海藻藻体购自毫州中药饮片公司，产地为中国浙江。
设备：
高效凝胶渗透色谱采用Shodex SUGAR KS-804(8.0mm×300mm，美国 Agilent公司)和Shodex SUGAR KS-802(8.0mm×300mm，美国Agilent公司)串联柱，以用不同分子量的T-系列标准葡聚糖(Dextran)制作标准曲线；
高效液相色谱(HPLC)采用Agilent 1260Seri高效液相系统测定(美国Agilent公司)；
红外分析采用Perkin-Elmer 599B型红外分光光度计测定(美国Perkin-Elmer公司)；
核磁共振分析采用Brucker AM-500型核磁共振仪测定(德国Brucker公司)。
制备实施例1：海藻硫酸多糖SPW-05-S2的制备
a.海藻硫酸多糖提取：
干燥的2kg海藻藻体，加入20L×2的去离子水，在100℃下沸水提取4h每次，共提取8次，将提取液合并浓缩，用流动水透析3天。将透析内液加热浓缩至2.5L，离心弃去沉淀，将上清液在搅拌下加入5倍体积(12.5L)的95％v/v乙醇，静置醇沉过夜，离心分离，所得沉淀用无水乙醇与丙酮交替洗涤3次，离心取沉淀于50℃下真空干燥，再复溶于1500mL去离子水中，进行冷冻干燥得水提海藻粗硫酸多糖204g(收率10.2％)。
b.硫酸多糖纯化：
取上述制备的水提海藻粗硫酸多糖6g，100mL的去离子水溶解，4000r/min离心10min除去不溶物，上清液经DEAE Sepharose Fast Flow阴离子交换柱进行分离，依次以去离子水、0.05M、0.1M、0.2M、0.3M、0.5M、0.6M及0.8M NaCl溶液梯度洗脱，硫酸-苯酚检测，并绘制洗脱曲线，根据洗脱曲线收集合并0.5M NaCl洗脱液，浓缩，离心，取上清液透析，冷冻干燥，得初步纯化的硫酸多糖SPW-05约508mg(得率8.46％)。取200mg SPW-05溶于4mL的0.2M NaCl溶液中，4000r/min离心10min，上清液经Sephacryl HR S-300凝胶色谱柱，以0.2M NaCl溶液洗脱并控制流速5mL/15min。硫酸-苯酚法检测并绘制洗脱曲线，根据洗脱曲线收集合并硫酸多糖SPW-05-S2组分，浓缩，透析，冷冻干燥，得海藻硫酸多糖SPW-05-S2约166mg(得率32.7％)。
c.海藻硫酸多糖SPW-05-S2结构鉴定及解析：
(1)经高效凝胶渗透色谱(high performance gel permeation chromatography，HPGPC)测定硫酸多糖SPW-05-S2相对分子质量为121.77 kDa，其纯度达95％(如图1，图中的小尖峰为溶剂峰)。
硫酸多糖SPW-05-S2的完全水解样品经PMP衍生法测其单糖组成，确定硫酸多糖SPW-05-S2主要含有甘露糖，葡萄糖醛酸，半乳糖，木糖和岩藻糖，其摩尔比为12.28:2.60:27.59:7.06:50.47。甲基化分析。综合对原糖、脱硫后多糖及其还原的脱硫多糖进行甲基化，通过GC-MS进行分析，确定硫酸多糖SPW-05-S2的糖残基连接方式及其硫酸基的取代位置，确定葡萄糖醛酸的连接方式为1,4-葡萄糖醛酸。通过部分酸水解后的次级多糖的糖组成和糖残基连接方式等确定硫酸多糖SPW-05-S2的主链为1,4-葡萄糖醛酸和1,2-甘露糖。此外，通过元素分析，发现硫酸多糖SPW-05-S2的含硫量为9.76，进一步确定其硫酸基含量为29.28％。以葡萄糖为标准品，通过苯酚硫酸法，进行糖含量测定，提示硫酸多糖SPW-05-S2的糖含量为66.4％(两次糖含量测定取平均值)。此外，BCA试剂盒检测出其还含有3.4％的蛋白。
(2)红外图谱(如图2)，3308.69cm-1为O-H伸缩振动吸收峰，2932.46cm-1为C-H伸缩振动吸收峰，1217.11cm-1处的强吸收峰为O＝S＝O的伸缩振动峰，1732.38cm-1为羧基C＝O伸缩振动，表明该多糖含有硫酸基和糖醛酸。
(3)NMR分析：
取硫酸多糖SPW-05-S2 35mg，加D2O 0.5mL溶解，加2.5μL丙酮为内标(δH＝2.29ppm，δC＝31.5ppm)，于Bruker AVANCE III 500M核磁共振仪上，25℃分别测定一维和二维核磁共振谱，并参考核磁图谱对硫酸多糖SPW-05-S2进行结构确证，SPW-05-S2的13C NMR结果如图3。
在硫酸多糖SPW-05-S2的13C NMR谱中，异头碳区域内，δ105.48-δ104.08信号归属于1,3,6-β-半乳糖，1,6-β-半乳糖，末端β-半乳糖。δ103.24-δ100.51信号归属于1,2,3-α-甘露糖/1,2,6-α-甘露糖、1,4-β-木糖和末端β-木糖及1,4-β-葡萄糖醛酸。δ99.69-δ97.44归属于1,2-α-岩藻糖、1,4-α-岩藻糖、1,2,4-α-岩藻糖和末端-α-岩藻糖。δ93.44归属于1,3-α-岩藻糖。δ175.02信号归属于1,4-β-葡萄糖醛酸羧基碳的信号峰。δ39.47及δ21.83的信号表明该多糖含有乙酰基。δ17.97和δ17.12归属于α-岩藻糖C6的信号峰。
1H-QA(定量)NMR图谱中(图4，未加丙酮)，位于δ5.0-5.7的信号是硫酸多糖SPW-05-S2中异头氢的信号，位于δ3.25-4.65的信号是糖环上的氢的信号，位于δ2.28附近的信号是典型的乙酰基上甲基氢的信号，位于 δ1.30附近的信号是岩藻糖H6的信号。根据δ2.28附近的信号和δ1.30附近的信号的积分面积比可知乙酰基与岩藻糖的摩尔比为0.028:1。由于岩藻糖占总糖的摩尔分数是50.47％。可以换算出乙酰基与总糖的摩尔比是0.014:1。
综合以上结果表明，海藻硫酸多糖SPW-05-S2可能以1,4-β-葡萄糖醛酸及1,2-α-甘露糖为主链结构，而在α-甘露糖的C-6位和C-3位存在分支，分支由半乳糖残基(1,3,6-β-半乳糖、1,6-β-半乳糖和末端-β-半乳糖)、木糖(1,4-β-木糖和末端-β-木糖)和岩藻糖(1,2-α-岩藻糖、1,3-α-岩藻糖、1,4-α-岩藻糖、1,2,4-α-岩藻糖和末端-α-岩藻糖)残基组成。通过对比脱硫前后的糖残基连接方式确定其硫酸基主要取代在1,2-α-岩藻糖的C-4位和1,4-α-岩藻糖的C-2位，以及末端-β-半乳糖的C-4位和部分末端-β-木糖的C-4位。
测试实施例2：海藻硫酸多糖SPW-05-S2抗肺纤维化的活性
(1)博莱霉素诱导的肺纤维化模型检测海藻多糖组分SPW-05-S2对小鼠肺组织形态和肺组织系数的影响
此次动物实验遵行实验动物管理和使用指南，获得中国科学院上海药物研究所动物伦理委员会批准。从上海斯莱克实验动物中心有限公司购得8周龄雄性C57BL/6小鼠36只。动物随机分为三组，对照组6只，模型组6只，实验组24只。自由饮食饮水，室温22±1℃，室内相对湿度65±5％，12h循环光照。模型组和实验组，气管给药博莱霉素(购自CSNpharm公司)溶液1.8mg/kg(使用0.9％生理盐水配置)。6只小鼠气管给药等体积0.9％生理盐水为正常对照组。一周后实验组随机分组给药，每组6只，分为四组A、B、C、D组。A组给药25mg/kg SPW-05-S2多糖，B组给药50mg/kg SPW-05-S2多糖，C组给药100mg/kg SPW-05-S2多糖，D组给药100mg/kg吡非尼酮(购自MCE公司)为阳性药对照，每天灌胃给药，持续两周，对照组和模型组给予等体积生理盐水。给药两周后对小鼠体重进行记录后牺牲小鼠，观察各组小鼠肺组织形态，并对肺组织进行称重。结果如图5中A和B所示，使用SPW-05-S2多糖干预后的肺纤维化小鼠，肺组织表面恢复光滑红润，色泽光亮，但质地不及对照组小鼠肺组织柔软，富有弹性。模型组小鼠肺系数(肺组织重量/小鼠体重)增高，SPW-05-S2低(25mg/kg)、中(50mg/kg)、高(100mg/kg)剂量组肺系数降低，与模型组相比具有显著差异。
(2)苏木素-伊红染色和Masson染色观察SPW-05-S2对减轻肺纤维化的作用
给药两周后小鼠牺牲，取左侧肺大叶使用4％多聚甲醛固定48小时后，进行苏木素-伊红染色和Masson染色。如图6中A和B所示，苏木素-伊红染色结果表明，SPW-05-S2低、中、高剂量组小鼠肺组织病变程度显著减轻，仅可观察到少部分间质血管扩张、充血及轻度肺泡炎，但肺泡壁纤维化程度大大减少。肺泡间隔基本恢复正常，未见弥漫性的炎性浸润。Masson染色结果表明，SPW-05-S2低、中、高剂量组小鼠肺组织中肺泡完整性变好，纤维组织团块明显减少，抑制胶原沉积现象较为明显。这说明多糖SPW-05-S2可以有效缓解肺部纤维化病灶，降低纤维增生且减缓肺纤维化进程。
(3)蛋白免疫印迹实验检测SPW-05-S2对小鼠肺中纤维化表型相关蛋白水平的影响
给药两周后小鼠牺牲，部分肺组织用组织提取试剂盒(购自生工生物工程有限公司)提取蛋白，加入5×上样缓冲液后将蛋白于金属加热器中95℃变性10min，冷却后储存于-80℃。免疫印迹检测Fibronectin、Collagen I和α-smooth muscle actin蛋白表达。结果如图7所示，海藻多糖SPW-05-S2能够抑制纤维化表型相关蛋白(Fibronectin、Collagen I和α-smooth muscle actin)的表达。
(4)CCK-8实验检测海藻多糖SPW-05-S2对肺泡上皮细胞A549生长活力的影响
对数生长期的A549细胞(5×103个/孔)种入96孔板中，设三复孔，于培养箱中培养24h；吸去细胞上清液，加入终浓度分别为0.1、0.5及1mg/mL的海藻多糖SPW-05-S2溶液培养1小时后，在加入TGF-β(10ng/mL)干预处理，继续培养48h后，每孔加入10μL CCK-8增强型溶液(购自大连美仑公司)，继续培养1h后用酶标仪在450nm下检测吸光度。细胞生长活力按照以下公式进行计算：细胞活力＝(实验组OD值－空白组OD值)/(对照组OD值－空白组OD值)×100％。结果如图8所示，说明SPW-05-S2对细胞生长活力没有影响(+代表加入，—代表不加入)。
(5)蛋白免疫印迹实验检测海藻多糖SPW-05-S2对肺泡上皮细胞A549细胞中纤维化表型相关蛋白水平表达的影响
人肺泡上皮细胞A549培养于含10％胎牛血清(购自美国Gibco公司)、100U/mL青霉素和100μg/mL链霉素的以体积比1:1混合的RPMI1640培养基中。取对数生长期的A549细胞，以1.2×106个/孔的密度种于6孔板中，培养24h后，用0.1、0.5及1mg/mL的海藻多糖SPW-05-S2和阳性药1mg/mL吡非尼酮处理1h后，以0、10ng/ml的浓度加入TGF-β重组蛋白(购自Abclonal公司)刺激。培养24小时后，弃上清，用预冷的PBS漂洗细胞，RIPA细胞裂解液(购自Absin公司)冰上裂解细胞20min，离心收集上清液。加入5×上样缓冲液后将蛋白于金属加热器中变性10min，冷却后储存于-80℃。免疫印迹检测E-cadherin、α-smooth muscle actin、Collagen I和Fibronectin的蛋白表达情况。结果如图9所示，海藻多糖SPW-05-S2可以显著地抑制α-smooth muscle actin、Collagen I和Fibronectin蛋白的表达，显著地增加E-cadherin蛋白的表达。
综上，通过测试实施例结果可知，海藻硫酸多糖SPW-05-S2能够降低博莱霉素诱导的肺纤维化，包括恢复肺组织形态，降低肺系数，减少动物肺损伤和纤维化表型相关蛋白的表达。并且在体外不影响肺泡上皮细胞A549的细胞生长活力，SPW-05-S2能明显抑制纤维化相关蛋白α-SMA、Collagen I和Fibronectin的表达，显著地增加E-cadherin蛋白的表达。因此该多糖组分SPW-05-S2可成为预防和/或治疗肺纤维化的潜在的糖类药物。
以上各实施例仅用以举例说明本发明的技术方案，而非对其限制。尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：在没有脱离本发明权利要求所限定的精神和实质的范围内，可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换仍然在本发明权利要求所限定的范围内。

Claims (10)

  1. 一种海藻硫酸多糖，其特征在于，以重量百分含量计，所述海藻硫酸多糖包含多糖62％～70％、硫酸基27％～32％、乙酰基0.5％～1.5％以及蛋白2.0％～4.5％；
    所述多糖主要由甘露糖、葡萄糖醛酸、半乳糖、木糖和岩藻糖组成。
  2. 根据权利要求1所述的海藻硫酸多糖，其特征在于，所述多糖的结构单元是以1→4连接的β-葡萄糖醛酸、1→2连接的α-甘露糖为主链，在α-甘露糖的C-6位和C-3位存在分支，分支由1,3,6-β-半乳糖、1,6-β-半乳糖、末端-β-半乳糖、1,4-β-木糖、末端-β-木糖、1,2-α-岩藻糖、1,3-α-岩藻糖、1,4-α-岩藻糖、1,2,4-α-岩藻糖和末端-α-岩藻糖残基组成；
    所述硫酸基主要取代在1,2-α-岩藻糖的C-4位、1,4-α-岩藻糖的C-2位、末端-β-半乳糖的C-4位以及部分末端-β-木糖的C-4位。
  3. 根据权利要求1所述的海藻硫酸多糖，其特征在于，所述海藻硫酸多糖的重均分子量的范围为5-1000kDa，优选为10-500kDa，更优选为15-400kDa。
  4. 根据权利要求1所述的海藻硫酸多糖，其特征在于，所述甘露糖、葡萄糖醛酸、半乳糖、木糖和岩藻糖的摩尔比为10～15:1.5～4:22～32:5～9:40～60，优选为12.28:2.60:27.59:7.06:50.47。
  5. 如权利要求1-4任一项所述的海藻硫酸多糖的制备方法，其特征在于，包括以下步骤：
    a.硫酸多糖提取：
    干燥的海藻用沸水提取，将所得提取液浓缩、透析、再浓缩、离心去沉淀，所得上清液醇沉、离心分离，所得沉淀洗涤、干燥，得水提海藻粗硫酸多糖；
    b.硫酸多糖纯化：
    b1.取步骤a制备的水提海藻粗硫酸多糖用水溶解，离心，上清液经阴离子交换柱进行初步分级纯化，依次以水和0.05～0.8M的NaCl溶液洗脱，收集0.5M NaCl溶液的洗脱组分，得硫酸多糖SPW-05；
    b2.将步骤b1制备的硫酸多糖SPW-05溶于0.01～1倍重量的0.2M NaCl溶液中，离心，上清液经凝胶色谱柱纯化，以0.2M NaCl溶液洗脱，收集合并硫酸多糖组分。
  6. 根据权利要求5所述的制备方法，其特征在于，
    所述步骤a包括：在干燥的海藻中加入15～20倍重量的水，浸泡过夜后，沸水提取3～5小时，每天3次，提取至苯酚硫酸法检测至无明显颜色变化；将提取液合并，浓缩，透析，再浓缩，离心得上清液，加入5倍～10倍于上清液体积的乙醇，离心得沉淀，沉淀经无水乙醇与丙酮洗涤3～6次，经干燥得水提海藻粗硫酸多糖；
    所述步骤b包括：b1.取步骤a制备的水提海藻粗硫酸多糖，加入10～20倍重量的水中溶解，离心；上清液经阴离子交换柱分离，依次以水、0.05M、0.1M、0.2M、0.3M、0.5M、0.6M及0.8M NaCl溶液洗脱，硫酸-苯酚检测，根据洗脱曲线收集合并0.5M NaCl溶液的洗脱液，浓缩，离心，取上清液透析，冷冻干燥，得硫酸多糖SPW-05；
    b2.将步骤b1制备的硫酸多糖SPW-05溶于0.01～1倍重量的0.2M NaCl溶液中，离心，上清液经凝胶色谱柱纯化，以0.2M NaCl溶液洗脱，硫酸-苯酚检测，根据洗脱曲线收集合并硫酸多糖这一组分，浓缩，透析，冷冻干燥，得所述硫酸多糖。
  7. 根据权利要求6所述的制备方法，其特征在于，步骤a中，所述乙醇为70％(v/v)以上的乙醇水溶液，优选为85％(v/v)以上的乙醇水溶液，特别为95％(v/v)以上的乙醇水溶液。
  8. 一种药物组合物，其包含权利要求1-4任一项所述的海藻硫酸多糖以及任选的药学上可接受的辅料。
  9. 权利要求1-4任一项所述的海藻硫酸多糖或权利要求8所述的药物组合物在制备用于预防和/或治疗肺纤维化的药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述肺纤维化为博莱霉素诱导的肺纤维化。
PCT/CN2023/138342 2022-12-16 2023-12-13 海藻硫酸多糖及其制备方法和用途 Ceased WO2024125540A1 (zh)

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