WO2019033869A1 - 大鲵软骨制剂 - Google Patents

大鲵软骨制剂 Download PDF

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WO2019033869A1
WO2019033869A1 PCT/CN2018/094613 CN2018094613W WO2019033869A1 WO 2019033869 A1 WO2019033869 A1 WO 2019033869A1 CN 2018094613 W CN2018094613 W CN 2018094613W WO 2019033869 A1 WO2019033869 A1 WO 2019033869A1
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cartilage
extract
supernatant
polypeptide
sputum
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French (fr)
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邢新会
朱文明
王怡
何东
张翀
苏楠
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Tsinghua University
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Tsinghua University
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Priority to US16/639,570 priority Critical patent/US20210047366A1/en
Priority to JP2020508999A priority patent/JP7250362B2/ja
Publication of WO2019033869A1 publication Critical patent/WO2019033869A1/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
    • 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
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/65—Amphibians, e.g. toads, frogs, salamanders or newts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the present invention relates to a large iliac cartilage preparation and its use.
  • Cartilage is rich in glycoproteins, peptides, small molecular proteins and polysaccharides. It has been found that certain animal-derived cartilage extracts have good anti-tumor, anti-angiogenesis, immune regulation, anti-arthritis and other activities. Among them, Shark Cartilage Preparation (SCP) is particularly rich in research and is widely used as a nutritional supplement in the world. However, with the development of the shark cartilage market, shark catches have increased. In recent decades, the number of shark populations in nature has declined drastically, and some species of sharks have entered the list of endangered species. Therefore, the use of shark cartilage is unsustainable.
  • SCP Shark Cartilage Preparation
  • Andrias davidianus As a rare and endemic animal in China, Andrias davidianus has a high edible and medicinal value and has great potential for development and utilization.
  • the wild giant salamander is a national second-class protected aquatic animal.
  • Large-scale breeding giants have been formed in many provinces and regions such as Shaanxi, Hunan, Henan, and Sichuan. Industry and market, which provides sustainable biological resources for the extraction of medical and health care products from the giant iliac cartilage, and the development of the sputum cartilage preparation is also conducive to the development of the high sputum industry.
  • the invention relates to the following:
  • a macroscopic cartilage enzymatic extract comprising:
  • CS-O non-sulfated disaccharide
  • CS-C C6 sulphated disaccharide
  • CS-A C4 sulfated disaccharide
  • the large sputum cartilage hydrolyzed extract according to any one of the items 1 to 3, wherein the weight average molecular weight (Mw) of the sorghum chondroitin sulfate is 40 kDa or more, preferably 45 kDa or more, and more preferably 48 kDa or more. Most preferably, it is 49.2 kDa.
  • Mw weight average molecular weight
  • the large sputum cartilage enzymatic extract according to any one of items 1 to 4, wherein the crude cartilage polypeptide extract comprises one or more of TGPLGPSPGP, HDLPLPLPE, DNLPPLPK, and VPSGPLGPEGPR.
  • the large sputum cartilage enzymatic extract according to any one of the items 1 to 5, wherein the large sputum chondroitin sulfate accounts for 15% by mass to 30% by mass of the total amount of the large sputum cartilage extract, the cartilage
  • the crude polypeptide extract accounts for 70% by mass to 85% by mass of the total amount of the enzymatic hydrolysate of the large sputum cartilage.
  • An alcohol-soluble component of an extract of a large sputum cartilage which is a crude extract of cartilage polypeptide, and comprises one or more of TGPLGPSPGP, HDLPLPLPE, DNLPPLPK, and VPSGPLGPEGPR.
  • the supernatant after proteolysis is obtained and dried.
  • cartilage polypeptide characterized in that the cartilage polypeptide is one or more of TGPLGPSPGP, HDLPLPLPE, DNLPPLPK, and VPSGPLGPEGPR.
  • cartilage polypeptide according to item 11 which has an activity of inhibiting XOD enzyme.
  • An alcohol precipitation component of an enzymatic extract of the giant scorpion cartilage comprising chondroitin sulfate, which contains a non-sulfated disaccharide (CS-O) and a sulfated disaccharide at the C6 position (CS- C), and the sulfated disaccharide (CS-A) at the C4 position.
  • CS-O non-sulfated disaccharide
  • CS- C C6 sulphated disaccharide
  • Mw weight average molecular weight
  • the supernatant after proteolysis is obtained and dried.
  • the precipitated fraction was obtained.
  • the cartilage polypeptide according to any of the preceding claims for use in the manufacture of a medicament for reducing uric acid or treating hyperuricemia or gout.
  • a method of obtaining an extract of a large ankle cartilage enzymatic extract comprising:
  • protease is an alkaline protease
  • protease digestion is carried out under the conditions of pH 9-11.
  • a method of obtaining an alcohol soluble component of an extract of a large ankle cartilage enzymatic extract comprising:
  • the supernatant after proteolysis is obtained and dried.
  • protease is an alkaline protease
  • protease digestion is carried out under the conditions of pH 9-11.
  • a method of obtaining an alcohol precipitation component of an extract of a large ankle cartilage enzymatic extract comprising:
  • the supernatant after proteolysis is obtained and dried.
  • the precipitated fraction was obtained.
  • protease is an alkaline protease
  • protease digestion is carried out under the conditions of pH 9-11.
  • a method of reducing uric acid or treating hyperuricemia or gout comprising:
  • the scorpion cartilage preparation according to the present invention is a polypeptide substance obtained by a method for hydrolyzing large sputum cartilage by a protease or a combination of chondroitin sulfate and a polypeptide, and has anti-tumor, anti-angiogenesis, anti-inflammatory, anti-oxidation, immune-enhancing, anti-bone. It is loose, anti-rheumatic, uric acid-lowering, anti-gout and other activities.
  • the present invention relates to an extract of the giant scorpion cartilage, which comprises: chondroitin sulfate, and a crude extract of cartilage polypeptide.
  • the large sputum chondroitin sulfate accounts for 15% by mass to 30% by mass, preferably 18% by mass to 26% by mass, based on the total amount of the extract of the large sputum cartilage, and the crude extract of the cartilage polypeptide accounts for the total amount of the extract of the sputum cartilage. 70% by mass to 85% by mass, preferably 74% by mass to 82% by mass.
  • the large sulphate chondroitin contains non-sulfated disaccharide (CS-O), C6 sulphated disaccharide (CS-C), and C4 sulphated disaccharide (CS-A), preferably by non-sulfated Disaccharide (CS-O), C6 sulfated disaccharide (CS-C), and C4 sulfated disaccharide (CS-A).
  • the preparation process of the giant iliac cartilage preparation of the present invention is as follows:
  • proteases for example, collagenase, alkaline protease, chain protease, trypsin or papain, etc., preferably an alkaline protease, to hydrolyze the sputum cartilage under suitable conditions for a period of time After heating, the temperature was raised to inactivate the protease.
  • the supernatant is obtained after the reaction solution is allowed to stand for precipitation (or centrifugation, filtration, etc.).
  • chondroitin sulfate and cartilage polypeptide which is also referred to as GSCP in the present invention.
  • the alcohol-soluble component which is composed mainly of a polypeptide, is also referred to as GSCP2 in the present invention.
  • the alcohol-soluble component includes the polypeptides TGPLGPSPGP, HDLPLPLPE, DNLPPLPK and VPSGPLGPEGPR having uric acid-lowering activity.
  • the composition of the alcohol precipitation component is dominated by chondroitin sulfate (ADCS).
  • the present invention relates to an extract of a large ankle cartilage, which is obtained by the following method, the method comprising:
  • the present invention also relates to a large ankle cartilage enzymatic extract, characterized in that it comprises:
  • the protease of the present invention is preferably an alkaline protease, and more preferably a 2709 protease.
  • the present invention also relates to an extract of a large sputum cartilage, characterized in that the weight average molecular weight (Mw) of the sorghum chondroitin sulfate is 49.2 kDa.
  • Mw weight average molecular weight
  • the present invention also relates to an extract of a large ankle cartilage, characterized in that the crude cartilage polypeptide extract comprises one or more of TGPLGPSPGP, HDLPLPLPE, DNLPPLPK and VPSGPLGPEGPR.
  • the invention relates to a method for preparing an enzymatic extract of giant scorpion cartilage, characterized in that the method comprises:
  • the present invention relates to an alcohol-soluble component of an enzymatic extract of the giant scorpion cartilage, which is obtained by the following method, the method comprising:
  • the supernatant after proteolysis is obtained and dried.
  • the present invention also relates to an alcohol-soluble component of an enzymatic extract of the giant scorpion cartilage, which is characterized in that it is a crude extract of cartilage polypeptide.
  • the present invention also relates to an alcohol-soluble component of an extract of a large ankle cartilage enzymatic extract, which comprises one or more of TGPLGPSPGP, HDLPLPLPE, DNLPPLPK, and VPSGPLGPEGPR.
  • the present invention relates to a method for preparing an alcohol-soluble component of an enzymatic extract of a giant scorpion cartilage, characterized in that the method comprises:
  • the supernatant after proteolysis is obtained and dried.
  • the present invention relates to a cartilage polypeptide characterized in that the cartilage polypeptide is one or more of TGPLGPSPGP, HDLPLPLPE, DNLPPLPK, and VPSGPLGPEGPR.
  • the cartilage polypeptide of the present invention may be TGPLGPSPGP, may be HDLPLPLPE, may be DNLPPLPK, or may be VPSGPLGPEGPR, and any mixture thereof, such as a mixture of TGPLGPSPGP and HDLPLPLPE, a mixture of TGPLGPSPGP and DNLLPLPK, a mixture of TGPLGPSPGP and VPSGPLGPEGPR, HDLPLPLPE a mixture with DNLPPLPK, a mixture of HDLPLPLPE and VPSGPLGPEGPR, a mixture of DNLPPLPK and VPSGPLGPEGPR, may be a mixture of TGPLGPSPGP, HDLPLPLPE and DNLPPLPK, may be a mixture of HDLPLPLPE, DNLPPLPK and VPSGPLGPEGPR, may be a mixture of TGPLGPSPGP, HDLPLPLPE and DNLPPLPK, may be a mixture of HDLPLPLPE, DNLPPLPK and VPSG
  • the present invention also relates to a cartilage polypeptide characterized in that the cartilage polypeptide has an activity of inhibiting XOD enzyme.
  • the present invention also relates to a cartilage polypeptide characterized in that the cartilage polypeptide has uric acid-lowering activity.
  • the present invention relates to an alcohol precipitation component of an enzymatic extract of a giant scorpion cartilage, which is obtained by the following method, the method comprising:
  • the supernatant after proteolysis is obtained and dried.
  • the precipitated fraction was obtained.
  • the present invention also relates to an alcohol precipitation component of an enzymatic hydrolysis extract of the giant sputum cartilage, characterized in that it comprises chondroitin sulfate.
  • the present invention also relates to an alcohol precipitation component of an enzymatic extract of a large sputum cartilage, characterized in that the weight average molecular weight (Mw) of the sorghum chondroitin sulfate is 49.2 kDa.
  • the present invention relates to a method for preparing an alcohol precipitation component of an enzymatic extract of a giant scorpion cartilage, characterized in that the method comprises:
  • the supernatant after proteolysis is obtained and dried.
  • the precipitated fraction was obtained.
  • the alcohol-soluble component or cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR of the large sputum cartilage enzymatic extract or the sputum cartilage enzymatic extract of the present invention has an activity of inhibiting XOD enzyme.
  • the invention also relates to the use of an extract of the giant scorpion cartilage for the preparation of a medicament for reducing uric acid or treating hyperuricemia.
  • the invention also relates to the use of an extract of the giant scorpion cartilage for reducing uric acid or for treating hyperuricemia.
  • the invention also relates to the use of an extract of the giant scorpion cartilage for the preparation of a medicament for the treatment of gout.
  • the invention also relates to the use of a large ankle cartilage enzymatic extract for the treatment of gout.
  • the present invention also relates to the use of an alcohol-soluble component of a large ankle cartilage enzymatic extract for the preparation of a medicament for reducing uric acid or treating hyperuricemia.
  • the invention also relates to the use of an alcohol soluble component of a large ankle cartilage enzymatic extract for the reduction of uric acid or for the treatment of hyperuricemia.
  • the present invention also relates to the use of an alcohol-soluble component of a large ankle cartilage enzymatic extract for the preparation of a medicament for treating gout.
  • the invention also relates to the use of an alcohol soluble component of a large ankle cartilage enzymatic extract for the treatment of gout.
  • the invention further relates to the use of a cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR for the manufacture of a medicament for reducing uric acid or treating hyperuricemia.
  • a cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR for the manufacture of a medicament for reducing uric acid or treating hyperuricemia.
  • the invention also relates to the use of a cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR for reducing uric acid or treating hyperuricemia.
  • a cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR for reducing uric acid or treating hyperuricemia.
  • the invention also relates to the use of a cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR for the manufacture of a medicament for the treatment of gout.
  • a cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR
  • the invention also relates to the use of a cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR for the treatment of gout.
  • a cartilage polypeptide such as TGPLGPSPGP, HDLPLPLPE, DNLPPLPK or VPSGPLGPEGPR for the treatment of gout.
  • the large sputum cartilage enzymatic extract of the present invention and its alcohol-soluble component include a cartilage polypeptide having an effect of lowering uric acid or treating hyperuricemia or gout.
  • Figure 1 shows cellulose acetate membrane electrophoresis of GSCP components (HA: hyaluronic acid; CS: chondroitin sulfate; HP: heparin).
  • Figure 2 shows HPGPC spectra of ADCS, shark chondroitin sulfate (SCS) and bovine chondroitin sulfate (BCS).
  • Figure 3 shows the SAX-HPLC profile and disaccharide composition: (a) ADCS; (b) BCS; (c) SCS.
  • Figure 4 shows the 1 H-NMR spectrum of ADCS.
  • Figure 5 shows the total ion chromatogram.
  • Figure 6 shows the results of MALDI-TOF-MS analysis.
  • Figure 7 shows the results of XOD enzyme activity assay.
  • Figure 8 shows the effect of reducing uric acid by an animal model of high-efficiency uric acid: (a) blood uric acid content; (b) liver XOD enzyme activity.
  • the divided cartilage with large cartilage was placed in clean water, heated for 1 h, and 20 g of large cartilage was separated; the cartilage was placed in a 500 ml beaker, 200 ml of water was added, and 2 g of 2709 protease (Beijing Donghua Qiangsheng Biological Co., Ltd., source lichens spores) Bacillus), pH 9.5, hydrolysis at 50 ° C for 8 h, heating at 90 ° C for 10 minutes to inactivate the protease; centrifugation at 10000 rpm for 10 min to take the supernatant; vacuum freeze-drying for 48 h to obtain 10.6 g of solid powder, that is, the extract of the large sputum cartilage extract (ie GSCP).
  • the extract of the large sputum cartilage extract ie GSCP
  • the large sputum (3.2 kg) prepared for cartilage separation was cleaned and washed to obtain large sputum cartilage with a wet weight of 100 g.
  • the cartilage was cut into small pieces and homogenized using a homogenizer.
  • the homogenized cartilage was suspended in 500 mL containing 2%.
  • Adjust the pH to 10.0 ⁇ 0.5 with 1M NaOH then add 6g of 2709 alkaline protease and stir with heating. Adjust the pH to 10.0 ⁇ 0.5 with 1M NaOH every 30min, and react for about 6h at 50°C. Until the cartilage tissue was completely digested, the reaction solution was heated to 100 ° C for 10 min to inactivate the protease.
  • the supernatant was obtained by centrifugation at 5,000 ⁇ g for 15 min to obtain a solution of the large sputum cartilage extract (GSCP).
  • the supernatant was added to 3 volumes of absolute ethanol, left at 4 ° C overnight, and centrifuged at 5,000 ⁇ g for 15 min to collect the precipitate.
  • the precipitate was dissolved in 100 mL of deionized water and the alcohol precipitation operation was repeated to obtain a precipitate, after which the precipitate was dissolved in 100 mL.
  • Deionized water was used for dialysis for 24 h using a dialysis bag with a retention of 7 kDa, followed by lyophilization to obtain 4.4 g of purified scorpion chondroitin sulfate.
  • the supernatant of the alcohol precipitation operation was collected twice, and concentrated by rotary evaporation, followed by lyophilization to obtain 19.0 g of an alcohol-soluble component mainly composed of a large cartilage polypeptide.
  • the alcohol precipitation fraction accounts for 18.8% of the mass fraction, and the alcohol soluble fraction accounts for 81.2% of the mass fraction.
  • Example 3 Cellulose Acetate Membrane Electrophoresis Analysis of Glycosaminoglycan Composition of GSCP Component Obtained in Example 1
  • Acetate film (7cm * 9cm) was fully soaked in electrode buffer (0.1mol / L pyridine, 0.47mol / L formic acid, pH 3.0) for 30 minutes, remove excess buffer, use the sampler in cellulose acetate
  • the membrane was loaded at one end (10 mg/ml GSCP), and 2 mg/ml hyaluronic acid, chondroitin sulfate, and heparin samples were used as controls.
  • Electrophoresis was performed on a DYCP-38C horizontal horizontal electrophoresis apparatus at a constant current of 6 mA for 30 minutes.
  • Figure 1 shows that there is a greater amount of chondroitin sulfate in the GSCP sample.
  • a cartilage having a wet weight of 100 g was obtained from a wet fish weighing 3.2 kg. According to the wet-dry ratio (W/D, 5.34), the dry weight of the cartilage obtained by CS extraction was 18.7 g. After extraction, separation, purification and freeze-drying, 4.4 g of ADCS samples were obtained. The purity, molecular weight distribution, disaccharide composition, and charge density of ADCS and commercially available BCS and SCS were analyzed by HPGPC and SAX-HPLC, and the results are shown in Table 1. The purity of ADCS was 98.7%, and the yield of ADCS extracted from large sputum cartilage was 23.2% (dry weight/dry weight).
  • the molecular weight distribution of CS was analyzed by HPGPC. The chromatogram is shown in Fig. 2. The dispersion coefficients of the three CS samples are all less than 1.5, and the molecular weight distribution is concentrated.
  • the weight average molecular weight (Mw) of ADCS is 49.2 kDa, which is lower than 75.8 kDa of SCS derived from aquatic animal sharks, and higher than 34.4 kDa of BCS derived from terrestrial animal cattle.
  • ADCS contains 14.6% non-sulfated disaccharide CS-O, 60.9% C6 sulfated disaccharide CS-C, and 24.5% C4 sulfated disaccharide CS-A, without disulfated II Sugar CS-D.
  • Table 1 it can be seen from Table 1 that the disaccharide composition of ADCS is significantly different from BCS and SCS.
  • the ADCS with a purity of 98% or more was obtained by performing alcohol precipitation operation and dialysis purification on the enzymatic extract of the giant salamander cartilage.
  • the molecular weight of ADCS was determined by HPGPC analysis to be 49.2 kDa.
  • the ADCS disaccharide composition was 14.6% CS-O, 60.9% CS-C and 24.5% CS-A by SAX-HPLC, which was significantly different from bovine-derived BCS and shark-derived SCS.
  • Example 5 Amino acid composition of GSCP and alcohol soluble component GSCP2 of large scorpion cartilage extract, polypeptide molecular weight distribution, protein sequencing
  • Sample type biological sample
  • Test content 50 kinds of hydrolyzed amino acid content detection
  • Amino acid kit API 45AA kit; methanol, nitrile are purchased from fisher.
  • Ion source +ESI electrospray ion source, positive ion mode
  • GSCP sample is 100mg/ml solution, others are solid samples
  • the alcohol-soluble fraction has obvious uric acid-lowering activity, and is further purified by gel size exclusion chromatography and reverse phase preparative chromatography to obtain a component having significant inhibitory effect on XOD enzyme activity, and performing molecular weight and sequence analysis thereof. And the results are as follows:
  • the molecular weight and sequence analysis of the peptide samples were commissioned by the Biomedical Testing Center of Tsinghua University.
  • the molecular weight analysis was performed using a matrix-assisted laser-resolved time-of-flight mass spectrometer MALDI-TOF-TOF 4800Plus (ABI, USA); the peptide sequence was analyzed using OrbiTrap Fusion LUMOS. Liquid chromatography/mass spectrometer (Thermo Fisher Company, USA).
  • LC-ESI-MS/MS liquid chromatography-electrospray ionization tandem mass spectrometry
  • DIA data-independent collection
  • Example 6 uric acid activity of the enzymatic extract GSCP and the alcohol soluble component GSCP2
  • XOD catalyzes the oxidation of xanthine to uric acid and superoxide anion, which is one of the main sources of active oxygen.
  • XOD is also one of the key enzymes of nucleotide metabolism, mainly distributed in the heart, lung, liver and other tissues of mammals;
  • the enzyme activity inhibition model can screen and evaluate potential urate-lowering drugs.
  • XOD enzyme Sigma, derived from milk
  • XOD enzyme activity test kit purchased from Nanjing.
  • Xanthine oxidase catalyzes the oxidation of xanthine to uric acid with specific absorption at 295 nm.
  • the product can be characterized by spectrophotometry.
  • XOD enzyme activity assay was performed according to the XOD enzyme activity test kit, and the results are shown in FIG. It can be seen from the results in Fig. 7 that GSCP has XOD activity and is dose-dependent, and the inhibitory activity is the highest when the concentration of GSCP is 40 mg/ml, and the inhibitory activity after dilution to 1/2 or 1/5 is correspondingly Reduced.
  • the ADCS having a large content in GSCP does not have XOD inhibitory activity. It is speculated that the component having the activity of inhibiting XOD enzyme is a polypeptide component in GSCP, that is, a component of GSCP2.
  • the XOD activity was 100% in the absence of inhibitor, and the XOD activity inhibition effect of the synthetic urate-lowering active peptide and the positive drug allopurinol was compared.
  • the results are shown in Table 4 (the concentration in the table is the sample in the reaction system). Final concentration).
  • the semi-inhibitory concentration (IC 50 ) was calculated by XOD enzyme inhibition assay and compared as shown in Table 5.
  • the synthetic polypeptides S1, S2, S3, and S4 reached a semi-inhibitory concentration of XOD at 3.2 mg/mL, 110 ⁇ g/mL, 60 ⁇ g/mL, and 60 ⁇ g/mL, respectively. It was found through experiments that GSCP2 reached a half-inhibitory concentration of XOD at 5.2 mg/mL in the same reaction system; in addition, allopurinol reached a semi-inhibitory concentration of XOD at 0.26 ⁇ g/mL, but the concentration reached 30 ⁇ g/mL. XOD still retains about 10% of enzyme activity, while X2 enzyme activity is completely inhibited when S2, S3, and S4 concentrations are three times the semi-inhibitory concentration. It is speculated that the artificially synthesized active polypeptide has XOD enzyme different from allopurinol. Live inhibition mechanism.
  • Acute hyperuricemia mouse model the experimental group was divided into normal group, model group, positive drug group (allopurinol, linglin, 2mg/ml), ADCS group (20mg/ml), GSCP group (40mg/ml), each group
  • the ml of the sigma suspension (0.5 ml) was molded in ml; the liver was taken 1 h after the modeling, and the blood uric acid level and the liver xanthine oxidase activity were measured. The results are shown in Fig. 8.
  • Hypoxanthine is a precursor of uric acid production, and potassium oxonate is an inhibitor of uricase.
  • the serum uric acid level of mice was significantly increased after intraperitoneal injection of potassium oxonate and hypoxanthine, indicating that the mouse model of hyperuricemia was successfully established.
  • the blood uric acid level of the positive drug allopurinol group was close to 0, and the blood uric acid level of the GSCP group was also at a low level.
  • the oral administration of GSCP showed prominent uric acid lowering activity.
  • liver XOD activity test showed that the XOD activity of the allopurinol group and GSCP group was lower than that of the model group, indicating that GSCP can exert uric acid lowering effect by inhibiting liver XOD activity.
  • the results of the two sets of data indicate that the active ingredient that plays a role in reducing uric acid in ADCS is not chondroitin sulfate. It is speculated that the component having uric acid-lowering activity is a polypeptide component in GSCP, namely GSCP2 component.

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Abstract

公开了一种大鲵软骨制剂。本发明的大鲵软骨酶解提取物，是通过如下方法获得的，该方法包括：从大鲵获取软骨，对获取的软骨进行蛋白酶解，以及获得蛋白酶解后的上清并对其进行干燥。本发明的大鲵软骨酶提取物及其醇溶成分包括软骨多肽，具有降低尿酸或治疗高尿酸血症的作用。

Description

大鲵软骨制剂 技术领域
本发明涉及一种大鲵软骨制剂以及其用途。
背景技术
软骨中含有丰富的糖蛋白、多肽、小分子蛋白、多糖，研究发现某些动物来源的软骨提取物具有良好的抗肿瘤、抗血管生成、免疫调节、抗关节炎等活性。其中鲨鱼软骨制剂(Shark Cartilage Preparation，SCP)相关的研究尤其丰富，并作为营养补充剂在全球广为流行。然而随着鲨鱼软骨市场的发展，鲨鱼捕捞量增多，近几十年来自然界中鲨鱼种群数量剧烈下降，部分种类的鲨鱼已进入被列入了濒危物种目录。因此鲨鱼软骨的利用具有不可持续性。
目前市场上软骨制剂主要来源于鲨鱼软骨。随着鲨鱼软骨市场的发展，鲨鱼捕捞量增多，近几十年来自然界中鲨鱼种群数量剧烈下降，部分种类的鲨鱼已进入被列入了濒危物种目录。因此鲨鱼软骨的利用具有不可持续性。
发明内容
作为中国特有的珍稀动物，大鲵(Andrias davidianus)具有极高的食用、药用价值，具有极大的开发和利用潜力。野生大鲵是国家二类保护水生动物，但近二十年来大鲵人工驯养繁殖技术逐渐成熟，大鲵产业迅速发展，在陕西、湖南、河南、四川等多个省份和地区已经形成较大规模的养殖大鲵产业和市场，这为利用大鲵软骨提取有医用和保健价值的产品提供了可持续的生物资源，同时大鲵软骨制剂的开发也有利于大鲵产业向高值化方向发展。
具体来说，本发明涉及如下内容：
1.一种大鲵软骨酶解提取物，其包括：
大鲵硫酸软骨素，以及
软骨多肽粗提物。
2.如项1所述的大鲵软骨酶解提取物，其中，所述大鲵硫酸软骨素含有非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖 (CS-A)。
3.如项1或2所述的大鲵软骨酶解提取物，其中，所述大鲵硫酸软骨素由非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)组成。
4.如项1～3中任一项所述的大鲵软骨酶解提取物，其中，所述大鲵硫酸软骨素的重均分子量(Mw)为40kDa以上，优选为45kDa以上，更优选为48kDa以上，最优选为49.2kDa。
5.如项1～4中任一项所述的大鲵软骨酶解提取物，其中，所述软骨多肽粗提物包含TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
6.如项1～5中任一项所述的大鲵软骨酶解提取物，其中，所述大鲵硫酸软骨素占大鲵软骨酶解提取物总量的15质量％～30质量％，所述软骨多肽粗提物占大鲵软骨酶解提取物总量的70质量％～85质量％。
7.如项1～6任一项所述的大鲵软骨酶解提取物，其是通过如下方法获得的，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，以及
获得蛋白酶解后的上清并对其进行干燥。
8.一种大鲵软骨酶解提取物的醇溶组分，其为软骨多肽粗提物，且包含TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
9.如项8所述的醇溶组分，其是通过如下方法获得的，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，
获得蛋白酶解后的上清并对其进行干燥，
对蛋白酶解后的上清或其干燥物进行醇沉，以及
获得上清。
10.如项8或9所述的醇溶组分，其具有降尿酸活性。
11.一种软骨多肽，其特征在于，该软骨多肽为TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
12.如项11所述的软骨多肽，其有抑制XOD酶的活性。
13.如项11所述的软骨多肽，其具有降尿酸的活性。
14.一种大鲵软骨酶解提取物的醇沉组分，其包括大鲵硫酸软骨素，该大鲵硫酸软骨素含有非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)。
15.如项14所述的大鲵软骨酶解提取物的醇沉组分，其中，所述大鲵硫酸软骨素由非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)组成。
16.如项14或15所述的大鲵软骨酶解提取物的醇沉组分，其中，所述大鲵硫酸软骨素的重均分子量(Mw)为40kDa以上，优选为45kDa以上，更优选为48kDa以上，最优选为49.2kDa。
17.如项14～16中任一项所述的大鲵软骨酶解提取物的醇沉组分，其是通过如下方法获得的，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，
获得蛋白酶解后的上清并对其进行干燥，
对蛋白酶解后的上清或其干燥物进行醇沉，以及
获得沉淀部分。
18.如项1～7中任一项所述的大鲵软骨酶解提取物、如项8～10中任一项所述的大鲵软骨酶解提取物的醇溶组分或者如项11～13中任一项所述的软骨多肽用于制备降低尿酸或治疗高尿酸血症或痛风的药物的用途。
19.如项1～7中任一项所述的大鲵软骨酶解提取物、如项8～10中任一项所述的大鲵软骨酶解提取物的醇溶组分或者如项11～13中任一项所述的软骨多肽用于降低尿酸或治疗高尿酸血症或痛风的用途。
20.一种获取大鲵软骨酶解提取物的方法，其包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，以及
获得蛋白酶解后的上清并对其进行干燥。
21.根据项20所述的方法，其中，所述蛋白酶是碱性蛋白酶，在pH 9～11的条件下进行蛋白酶酶解。
22.一种获取大鲵软骨酶解提取物的醇溶组分的方法，其包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，
获得蛋白酶解后的上清并对其进行干燥，
对蛋白酶解后的上清或其干燥物进行醇沉，以及
获得上清。
23.根据项22所述的方法，其中，所述蛋白酶是碱性蛋白酶，在pH 9～11的条件下进行蛋白酶酶解。
24.一种获取大鲵软骨酶解提取物的醇沉组分的方法，其包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，
获得蛋白酶解后的上清并对其进行干燥，
对蛋白酶解后的上清或其干燥物进行醇沉，以及
获得沉淀部分。
25.根据项24所述的方法，其中，所述蛋白酶是碱性蛋白酶，在pH 9～11的条件下进行蛋白酶酶解。
26.根据项21、23或25中任一项所述的方法，其中，所述碱性蛋白酶是2709蛋白酶。
27.根据项22～26中任一项所述的方法，其中，醇沉操作中乙醇质量浓度为50％-90％。
28.根据项27所述的方法，其中，醇沉操作中乙醇质量浓度为70％。
29.一种降低尿酸或治疗高尿酸血症或痛风的方法，其包括：
向由此需要的受试者给药如项1～7中任一项所述的大鲵软骨酶解提取物、如项8～10中任一项所述的大鲵软骨酶解提取物的醇溶组分或者如项11～13中任一项所述的软骨多肽。
本发明涉及的大鲵软骨制剂是通过蛋白酶水解大鲵软骨等方法获得的多肽类物质或大鲵硫酸软骨素与多肽的组合物，具有抗肿瘤、抗血管生成、抗炎、抗氧化、增强免疫、抗骨质疏松、抗风湿、降尿酸、抗痛风等活性。
具体实施方式
本发明涉及的大鲵软骨酶解提取物，其包括：大鲵硫酸软骨素，以及软骨多肽粗提物。
所述大鲵硫酸软骨素占大鲵软骨酶解提取物总量的15质量％～30质 量％，优选为18质量％～26质量％，所述软骨多肽粗提物占大鲵软骨酶解提取物总量的70质量％～85质量％，优选为74质量％～82质量％。
其中，大鲵硫酸软骨素含有非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)，优选由非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)组成。
本发明涉及的大鲵软骨制剂其制备过程如下：
从大鲵获取软骨。
蛋白酶解：使用一种或多种蛋白酶，例如可以为胶原蛋白酶、碱性蛋白酶、链蛋白酶、胰蛋白酶或木瓜蛋白酶等，优选为碱性蛋白酶，在适宜条件下对大鲵软骨进行水解反应，一段时间后加热升温，将蛋白酶灭活。
分离：对反应后溶液静置沉淀(或离心、过滤等)后获取上清。
干燥：自然干燥(或冷冻干燥、旋蒸等)除去上清中的水分即得到所需的大鲵硫酸软骨素与软骨多肽的组合物，在本发明中也简称为GSCP。
向在分离步骤中所得上清液或干燥步骤中所得组合物中加入乙醇或乙醇溶液进行醇沉(最终溶液中乙醇含量在50％～90％之间)，获取上清并干燥，得到所需的醇溶组分，组成以多肽为主，在本发明中也称为GSCP2。
经过进一步分析发现，醇溶组分包括具有降尿酸活性的多肽TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR。醇沉组分的组成以大鲵硫酸软骨素(ADCS)为主。
本发明涉及一种大鲵软骨酶解提取物，其特征在于，其是通过如下方法获得的，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，以及
获得蛋白酶解后的上清并对其进行干燥。
本发明还涉及一种大鲵软骨酶解提取物，其特征在于，其包括：
大鲵硫酸软骨素，以及
软骨多肽粗提物。
本发明的蛋白酶优选为碱性蛋白酶，进一步优选为2709蛋白酶。
本发明还涉及一种大鲵软骨酶解提取物，其特征在于，该大鲵硫酸软骨素的重均分子量(Mw)为49.2kDa。
本发明还涉及一种大鲵软骨酶解提取物，其特征在于，该软骨多肽粗提 物包含TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
本发明涉及一种制备大鲵软骨酶解提取物的方法，其特征在于，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，以及
获得蛋白酶解后的上清并对其进行干燥。
本发明涉及一种大鲵软骨酶解提取物的醇溶组分，其特征在于，其是通过如下方法获得的，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，
获得蛋白酶解后的上清并对其进行干燥，
对蛋白酶解后的上清或其干燥物进行醇沉，以及
获得上清。
本发明还涉及一种大鲵软骨酶解提取物的醇溶组分，其特征在于，其为软骨多肽粗提物。
本发明还涉及一种大鲵软骨酶解提取物的醇溶组分，其特征在于，其包含TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
本发明涉及一种制备大鲵软骨酶解提取物的醇溶组分的方法，其特征在于，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，
获得蛋白酶解后的上清并对其进行干燥，
对蛋白酶解后的上清或其干燥物进行醇沉，以及
获得上清。
本发明涉及一种软骨多肽，其特征在于，该软骨多肽为TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
即本发明的软骨多肽，可以是TGPLGPSPGP，可以是HDLPLPLPE，可以是DNLPPLPK，还可以是VPSGPLGPEGPR，以及它们任意的混合物，例如TGPLGPSPGP和HDLPLPLPE的混合物，TGPLGPSPGP和DNLPPLPK 的混合物，TGPLGPSPGP和VPSGPLGPEGPR的混合物，HDLPLPLPE和DNLPPLPK的混合物，HDLPLPLPE和VPSGPLGPEGPR的混合物，DNLPPLPK和VPSGPLGPEGPR的混合物，可以是TGPLGPSPGP，HDLPLPLPE和DNLPPLPK的混合物，可以是HDLPLPLPE，DNLPPLPK和VPSGPLGPEGPR的混合物，可以是TGPLGPSPGP，HDLPLPLPE和VPSGPLGPEGPR的混合物，可以是HDLPLPLPE，DNLPPLPK和VPSGPLGPEGPR的混合物，可以是TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR的混合物。
本发明还涉及一种软骨多肽，其特征在于，该软骨多肽具有抑制XOD酶的活性。
本发明还涉及一种软骨多肽，其特征在于，该软骨多肽具有降尿酸的活性。
本发明涉及一种大鲵软骨酶解提取物的醇沉组分，其特征在于，其是通过如下方法获得的，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，
获得蛋白酶解后的上清并对其进行干燥，
对蛋白酶解后的上清或其干燥物进行醇沉，以及
获得沉淀部分。
本发明还涉及一种大鲵软骨酶解提取物的醇沉组分，其特征在于，其包括大鲵硫酸软骨素。
本发明还涉及一种大鲵软骨酶解提取物的醇沉组分，其特征在于，该大鲵硫酸软骨素的重均分子量(Mw)为49.2kDa。
本发明涉及一种制备大鲵软骨酶解提取物的醇沉组分的方法，其特征在于，该方法包括：
从大鲵获取软骨，
对获取的软骨进行蛋白酶解，
获得蛋白酶解后的上清并对其进行干燥，
对蛋白酶解后的上清或其干燥物进行醇沉，以及
获得沉淀部分。
本发明涉及的大鲵软骨酶解提取物或大鲵软骨酶解提取物的醇溶组分 或软骨多肽诸如TGPLGPSPGP、HDLPLPLPE、DNLPPLPK或VPSGPLGPEGPR具有抑制XOD酶的活性。
本发明还涉及大鲵软骨酶解提取物用于制备降低尿酸或治疗高尿酸血症的药物的用途。
本发明还涉及大鲵软骨酶解提取物用于降低尿酸或治疗高尿酸血症的用途。
本发明还涉及大鲵软骨酶解提取物用于制备治疗痛风的药物的用途。
本发明还涉及大鲵软骨酶解提取物用于治疗痛风的用途。
本发明还涉及大鲵软骨酶解提取物的醇溶组分用于制备降低尿酸或治疗高尿酸血症的药物的用途。
本发明还涉及大鲵软骨酶解提取物的醇溶组分用于降低尿酸或治疗高尿酸血症的用途。
本发明还涉及大鲵软骨酶解提取物的醇溶组分用于制备治疗痛风的药物的用途。
本发明还涉及大鲵软骨酶解提取物的醇溶组分用于治疗痛风的用途。
本发明还涉及软骨多肽诸如TGPLGPSPGP、HDLPLPLPE、DNLPPLPK或VPSGPLGPEGPR用于制备降低尿酸或治疗高尿酸血症的药物的用途。
本发明还涉及软骨多肽诸如TGPLGPSPGP、HDLPLPLPE、DNLPPLPK或VPSGPLGPEGPR用于降低尿酸或治疗高尿酸血症的用途。
本发明还涉及软骨多肽诸如TGPLGPSPGP、HDLPLPLPE、DNLPPLPK或VPSGPLGPEGPR用于制备治疗痛风的药物的用途。
本发明还涉及软骨多肽诸如TGPLGPSPGP、HDLPLPLPE、DNLPPLPK或VPSGPLGPEGPR用于治疗痛风的用途。
因此，本发明的大鲵软骨酶解提取物及其醇溶组分包括软骨多肽，其具有降低尿酸或治疗高尿酸血症或痛风的作用。
附图说明
图1显示的是GSCP组分的醋酸纤维素膜电泳(HA：透明质酸；CS：硫酸软骨素；HP：肝素)。
图2显示的是ADCS、鲨鱼硫酸软骨素(SCS)和牛硫酸软骨素(BCS)的HPGPC谱图。
图3显示的是SAX-HPLC图谱和二糖组成：(a)ADCS；(b)BCS；(c)SCS。
图4显示的是ADCS的 1H-NMR谱图。
图5显示的是总离子流图。
图6显示的是MALDI-TOF-MS分析结果。
图7显示的是XOD酶活检测结果。
图8显示的是通过高效尿酸血症动物模型评价降尿酸效果：(a)血尿酸含量；(b)肝脏XOD酶活。
实施例
实施例1大鲵软骨制剂的制备
将分割的带有软骨的大鲵肉放入净水中，加热沸腾1h，分离出大鲵软骨20g；将软骨放入500ml烧杯，加入200ml水，2g 2709蛋白酶(北京东华强盛生物公司，来源地衣芽胞杆菌)，pH9.5，50℃下水解8h，90℃加热10分钟使蛋白酶灭活；10000rpm离心10min取上清；真空冷冻干燥48h得到固体粉末10.6g，即得到大鲵软骨酶解提取物(即GSCP)。取5g大鲵软骨酶解提取物，加入20ml 75％乙醇并充分混合，3000rpm离心5分钟离心取上清，置于60℃烘箱6h除醇之后冷冻干燥48h，得到醇溶组分3.5g(即GSCP2)。沉淀部分干燥得到大鲵硫酸软骨素1.2g(即ADCS)。醇沉部分占质量分数25.5％，醇溶部分占质量分数74.5％。
实施例2大鲵软骨制剂的制备
分离制备软骨的大鲵(3.2kg)经过清理洗净后得到湿重为100g的大鲵软骨，将软骨切成小块后使用匀浆机匀浆，将匀浆后的软骨悬浮在500mL含有2％的醋酸钠的溶液中，用1M的NaOH调节pH到10.0±0.5范围，之后加入6g的2709碱性蛋白酶并加热搅拌，每隔30min用1M的NaOH调节pH到10.0±0.5范围，50℃反应约6h直到软骨组织完全消化，然后将反应液加热到100℃保持10min使蛋白酶失活。反应液冷却至室温后5,000×g离心15min获取上清，得到大鲵软骨酶解提取物(GSCP)溶液。取上清加入3倍体积的无水乙醇，4℃静置过夜，5,000×g离心15min收集沉淀，将沉淀溶于100mL的去离子水后再次重复醇沉操作获取沉淀，之后将沉淀溶于 100mL去离子水中并使用截留量为7kDa的透析袋进行透析24h，然后进行冷冻干燥得到4.4g纯化的大鲵硫酸软骨素。收集两次醇沉操作的上清液，旋蒸浓缩后进行冷冻干燥，得到19.0g主要组成为大鲵软骨多肽的醇溶组分。醇沉部分占质量分数18.8％，醇溶部分占质量分数81.2％。
实施例3醋酸纤维素膜电泳分析实施例1获得的GSCP组分的糖胺聚糖组成
醋酸纤维薄膜(7cm*9cm)在电极缓冲液(0.1mol/L吡啶，0.47mol/L甲酸，pH3.0)中充分浸透30分钟，取出吸除多余缓冲液，使用加样器在醋酸纤维素薄膜一端加样(10mg/ml GSCP)，以2mg/ml透明质酸、硫酸软骨素、肝素样品为对照，在DYCP-38C型卧式水平电泳仪上以恒定电流6mA电泳30分钟，在染色液(0.5％阿利新蓝，2％醋酸)中染色30分钟，之后使用漂洗液(2％醋酸)重复漂洗3～5次，每次20分钟，将漂洗后的醋酸纤维素薄膜置于可见光下观察，结果如图1所示。图1显示GSCP样品中有较多含量的硫酸软骨素。
实施例4大鲵硫酸软骨素的表征
如前所述，从湿重为3.2kg养殖大鲵分离得到了湿重为100g的软骨，根据其湿干比(W/D，5.34)，得到CS提取得到的软骨干重为18.7g。经过提取、分离、纯化并冷冻干燥，实验获得了4.4g的ADCS样品。通过HPGPC、SAX-HPLC分析ADCS以及商品来源的BCS、SCS的纯度、分子量分布、二糖组成、电荷密度，结果如表1所示。ADCS的纯度为98.7％，从大鲵软骨中提取ADCS收率为23.2％(干重/干重)。
表1 CS的化学性质(纯度、分子量分布、二糖组成、电荷密度)
Figure PCTCN2018094613-appb-000001
通过HPGPC分析CS的分子量分布，色谱图如图2所示，三种CS样品的分散系数均小于1.5，分子量分布较为集中。ADCS的重均分子量(Mw)为 49.2kDa，低于水生动物鲨鱼来源的SCS的75.8kDa，高于陆地动物牛来源的BCS的34.4kDa。
经ChonABC彻底降解后通过SAX-HPLC分析CS的二糖组成，结果如图3所示。ADCS中含有14.6％的非硫酸化的二糖CS-O，60.9％的C6位硫酸化二糖CS-C，以及24.5％的C4位硫酸化二糖CS-A，不含双硫酸化的二糖CS-D。同时从表1可以看出，ADCS的二糖组成和BCS、SCS有明显的差异。因为鲨鱼来源的CS由于CS-C的含量较高，通常称SCS为CSC，而我们的实验结果表明ADCS中CS-C的含量高达60％以上，比鲨鱼来源的SCS更高。同时ADCS中非硫酸化的二糖单位CS-O的含量也比BCS和SCS中CS-O的含量高，且和SCS相比，ADCS中不含双硫酸化的二糖单位，因此ADCS的电荷密度(0.85)也低于BCS(0.95)和SCS(1.17)。此外，同样是从两栖动物软骨中提取的CS，ADCS的二糖组成也和已有文献报道(Mathews M B,Hinds L D.Acid mucopolysaccharides of frog cartilage in thyroxine-induced metamorphosis[J].Biochimica Et Biophysica Acta,1963,74(2):198)的蛙来源的CS(40％CS-O，40％CS-A，20％CS-C)有明显差异。
ADCS的 1H-NMR图谱分析结果如图4所示。结合CS核磁分析的文献报道(Mucci A,Schenetti L,Volpi N.H and 13C nuclear magnetic resonance identification and characterization of components of chondroitin sulfates of various origin[J].2000,41:37–45)对核磁共振图谱进行分析，在1.9-2.1ppm的一组交叠的出峰归属于未硫酸化和硫酸化的GalNAc结构甲基中H原子的信号峰，4.18ppm和4.70ppm的信号峰分别归属于CS-C中GalNAc6S的C6位H原子和CS-A中GalNAc4S的C4位H原子，4.46ppm的信号峰归属于GlcUA和GalNAc中的C1位H原子，3.32ppm、3.54ppm和3.69ppm的信号峰分别归属于GlcUA的C2、C3和C4/C5位的H原子。
可见，经过分析发现，通过对大鲵软骨酶解提取物进行醇沉操作和透析纯化得到纯度98％以上的ADCS。通过HPGPC分析确定ADCS的分子量为49.2kDa。经ChonABC酶彻底降解后通过SAX-HPLC分析ADCS二糖组成为14.6％CS-O，60.9％CS-C和24.5％CS-A，和牛来源的BCS以及鲨鱼来源的SCS有显著差异。
实施例5大鲵软骨酶解提取物GSCP及醇溶组分GSCP2的氨基酸组 成、多肽分子量分布、蛋白测序
分析1：GSCP氨基酸组成分析委托北京农业生物检测中心进行。
一、样本信息
样本类型：生物样本
储存条件：-20度
样本数量：7个
检测内容：50种水解氨基酸含量检测
二、仪器试剂信息
采用技术：质谱-同位素内标
使用仪器：高效液相色谱-四级杆离子阱串联质谱仪HPLC-MS/MS Q-TRAP
仪器型号：HPLC-MS/MS(Ultimate3000-API 3200Q TRAP)
氨基酸试剂盒：API 45AA试剂盒；甲醇、已腈均购自fisher。
三、样本前处理
酸水解处理(固体样：浸泡—匀浆—离心)
(1)取100ul样，加400ul水，加500ul浓盐酸，混匀冲氮气保护，110度密封，高温消化21小时。
(2)取出消化液，离心取上清，取50ul氮气吹干，加1ml水复溶。
衍生化处理
(1)取40μL复溶液，置于试管内，加10μL磺基水杨酸，漩涡30秒，13200转，离心4分钟。
(2)取10μL上层液体置于另一试管。加入40μL标记缓冲液，漩涡混匀，旋转离心。
(3)取10μL上层液体置于另一试管。每样品管中加入5μL稀释的aTRAQ试剂，漩涡混匀，旋转离心。
(4)室温下孵化至少30分钟。管中加入5μL羟胺，漩涡混匀，旋转离心。
(5)每支管中加入32μL内标，漩涡混匀，旋转离心。保存待检。
四、实验参数
(1)液相条件：
色谱柱：MSLab HP-C18(150*4.6mm 5um)
流动相：A:水+0.1％甲酸  B:乙腈+0.1％甲酸
流速：0.8ml/min
柱温：50℃
进样量：3ul
液相分析梯度：
Figure PCTCN2018094613-appb-000002
(2)质谱条件：
离子源:+ESI电喷雾离子源，正离子方式
扫描方式：MRM多反应监测
CUR:20(气帘气)
CAD:Medium(碰撞气)
IS:+5500V(喷雾电压)
TEM:500℃(雾化温度)
GS1:55psi(雾化气)
GS2:60psi(辅助气)
DP:35v(去族电压)
EP:10(射入电压)
CE:30(碰撞能量)
CXP:5.0(碰撞室射出电压)
五、实验数据
(1)总离子流图
总离子流图如图5所示。
(2)检测结果，见表2。
表2氨基酸组成检测结果
Figure PCTCN2018094613-appb-000003
Figure PCTCN2018094613-appb-000004
说明：GSCP送样为100mg/ml溶液，其他为固体样品
分析2：醇溶部分GSCP2多肽分子量分布、蛋白测序
醇溶部分具有明显的降尿酸活性，通过凝胶尺寸排阻色谱和反相制备色谱对其进一步纯化，得到对XOD酶活具有明显抑制作用的组分，对其进行多肽分子量及序列分析，方法及结果如下：
多肽样品的分子量和序列分析委托清华大学生物医学测试中心进行，其中分子量的分析使用基质辅助激光解析飞行时间质谱仪MALDI-TOF-TOF 4800Plus(美国ABI公司)进行；多肽序列的分析使用OrbiTrap Fusion LUMOS液质联用仪(美国Thermo Fisher公司)进行。
MALDI-TOF-MS分析结果如图6所示。分析结果表明样品多肽质荷比(m/z)在100-3000范围，且两个样品均在m/z为739和1162的位置有较强的峰。
通过液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)分析样品，并通过数据非依赖采集(DIA)的方案分析多肽的氨基酸序列，得到主要的四个多肽序列如表3所示，分别命名S1、S2、S3、S4并进行人工合成。
表3主要的四个多肽序列分析
Figure PCTCN2018094613-appb-000005
实施例6酶解提取物GSCP和醇溶组分GSCP2的降尿酸活性
分析1：
XOD催化黄嘌呤氧化生成尿酸和超氧阴离子，是活性氧主要来源之一，同时XOD也是核苷酸代谢的关键酶之一，主要分布于哺乳动物的心、肺、 肝脏等组织中；通过XOD酶活抑制模型可以对潜在的降尿酸药物进行筛选和评价。
试剂：XOD酶(Sigma，来源于牛奶)，XOD酶活检测试剂盒(购买自南京建成)。
黄嘌呤氧化酶(XOD)催化黄嘌呤氧化生成尿酸在295nm处有特异吸收，通过分光光度法测定产物可表征酶活。按照XOD酶活检测试剂盒说明进行XOD酶活检测，结果如图7所示。从图7的结果可以看出，GSCP具有抑制XOD活性且有剂量依赖性，GSCP的浓度为40mg/ml时的抑制活性最大，而稀释到1/2或1/5浓度后的抑制活性相应地减小。
此外，从图7中可以看出，在GSCP中含量较大的ADCS不具有XOD抑制活性。推测具有抑制XOD酶活性的成分为GSCP中的多肽成分，即GSCP2组分。
以无抑制剂存在时XOD酶活为100％，比较人工合成的降尿酸活性肽以及阳性药物别嘌醇的XOD酶活抑制效果，结果如表4所示(表格中的浓度为样品在反应体系的终浓度)。
表4 GSCP2、人工合成多肽及别嘌醇的XOD抑制活性
Figure PCTCN2018094613-appb-000006
Figure PCTCN2018094613-appb-000007
通过XOD酶活抑制实验计算半抑制浓度(IC 50)并比较，如表5所示。
表5别嘌醇及人工合成多肽的半抑制浓度(IC 50)
Figure PCTCN2018094613-appb-000008
合成多肽S1、S2、S3、S4分别在3.2mg/mL、110μg/mL、60μg/mL、60μg/mL时达到对XOD的半抑制浓度。而经过实验发现，同样的反应体系下GSCP2在5.2mg/mL时达到对XOD的半抑制浓度；此外，别嘌醇在0.26μg/mL时达到对XOD的半抑制浓度，但是浓度达到30μg/mL时XOD仍保留有约10％的酶活，而S2、S3、S4浓度为半抑制浓度的三倍时XOD酶活即被完全抑制，推测人工合成的活性多肽具有和别嘌醇不同的XOD酶活抑制机理。
分析2：
急性高尿酸血症小鼠模型：实验分正常组、模型组、阳性药物组(别嘌醇，百灵威，2mg/ml)、ADCS组(20mg/ml)、GSCP组(40mg/ml)，每组10只小鼠，连续7天灌胃(20ml/kg，正常组和模型组给超纯水)；第7天灌胃后1h腹腔注射15mg/ml氧嗪酸钾(生工生物)和10mg/ml次黄嘌呤(sigma)悬液0.5ml造模；造模后1h取血取肝脏，测定血尿酸水平和肝脏黄嘌呤氧化酶活性，结果如图8所示。
次黄嘌呤是尿酸生成的前体物质，氧嗪酸钾是尿酸酶的抑制剂。腹腔注射氧嗪酸钾和次黄嘌呤后小鼠血尿酸水平显著升高，表明高尿酸血症小鼠模型建立成功。和模型组相比，阳性药物别嘌醇组小鼠的血尿酸水平接近于0，GSCP组血尿酸水平也处于较低水平，GSCP经口给药表现出突出的降尿酸 活性。肝脏XOD酶活检测结果中别嘌醇组和GSCP组XOD活性低于模型组，表明GSCP能够通过抑制肝脏XOD活性发挥降尿酸作用。两组数据的结果表明ADCS中发挥降尿酸作用的有效成分不是硫酸软骨素，推测具有降尿酸活性的成分为GSCP中的多肽成分，即GSCP2组分。
前面仅仅示出了本发明的原理，应理解，本发明的范围不预期限制在本文所述的示例性方面，而应包括所有当前已知的和未来开发的等同物。另外，应当指出，在不脱离本发明技术原理的前提下，还可以作出若干改进和修改，这些改进和修改也应被视为本发明的范围。

Claims (29)

  1. 一种大鲵软骨酶解提取物，其包括：
    大鲵硫酸软骨素，以及
    软骨多肽粗提物。
  2. 如权利要求1所述的大鲵软骨酶解提取物，其中，所述大鲵硫酸软骨素含有非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)。
  3. 如权利要求1或2所述的大鲵软骨酶解提取物，其中，所述大鲵硫酸软骨素由非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)组成。
  4. 如权利要求1～3中任一项所述的大鲵软骨酶解提取物，其中，所述大鲵硫酸软骨素的重均分子量(Mw)为40kDa以上，优选为45kDa以上，更优选为48kDa以上，最优选为49.2kDa。
  5. 如权利要求1～4中任一项所述的大鲵软骨酶解提取物，其中，所述软骨多肽粗提物包含TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
  6. 如权利要求1～5中任一项所述的大鲵软骨酶解提取物，其中，所述大鲵硫酸软骨素占大鲵软骨酶解提取物总量的15质量％～30质量％，所述软骨多肽粗提物占大鲵软骨酶解提取物总量的70质量％～85质量％。
  7. 如权利要求1～6任一项所述的大鲵软骨酶解提取物，其是通过如下方法获得的，该方法包括：
    从大鲵获取软骨，
    对获取的软骨进行蛋白酶解，以及
    获得蛋白酶解后的上清并对其进行干燥。
  8. 一种大鲵软骨酶解提取物的醇溶组分，其为软骨多肽粗提物，且包含TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
  9. 如权利要求8所述的醇溶组分，其是通过如下方法获得的，该方法包括：
    从大鲵获取软骨，
    对获取的软骨进行蛋白酶解，
    获得蛋白酶解后的上清并对其进行干燥，
    对蛋白酶解后的上清或其干燥物进行醇沉，以及
    获得上清。
  10. 如权利要求8或9所述的醇溶组分，其具有降尿酸活性。
  11. 一种软骨多肽，其特征在于，该软骨多肽为TGPLGPSPGP、HDLPLPLPE、DNLPPLPK和VPSGPLGPEGPR中的一种或多种。
  12. 如权利要求11所述的软骨多肽，其有抑制XOD酶的活性。
  13. 如权利要求11所述的软骨多肽，其具有降尿酸的活性。
  14. 一种大鲵软骨酶解提取物的醇沉组分，其包括大鲵硫酸软骨素，该大鲵硫酸软骨素含有非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)。
  15. 如权利要求14所述的大鲵软骨酶解提取物的醇沉组分，其中，所述大鲵硫酸软骨素由非硫酸化的二糖(CS-O)，C6位硫酸化二糖(CS-C)，以及C4位硫酸化二糖(CS-A)组成。
  16. 如权利要求14或15所述的大鲵软骨酶解提取物的醇沉组分，其中，所述大鲵硫酸软骨素的重均分子量(Mw)为40kDa以上，优选为45kDa以上，更优选为48kDa以上，最优选为49.2kDa。
  17. 如权利要求14～16中任一项所述的大鲵软骨酶解提取物的醇沉组分，其是通过如下方法获得的，该方法包括：
    从大鲵获取软骨，
    对获取的软骨进行蛋白酶解，
    获得蛋白酶解后的上清并对其进行干燥，
    对蛋白酶解后的上清或其干燥物进行醇沉，以及
    获得沉淀部分。
  18. 如权利要求1～7中任一项所述的大鲵软骨酶解提取物、如权利要求8～10中任一项所述的大鲵软骨酶解提取物的醇溶组分或者如权利要求11～13中任一项所述的软骨多肽用于制备降低尿酸或治疗高尿酸血症或痛风的药物的用途。
  19. 如权利要求1～7中任一项所述的大鲵软骨酶解提取物、如权利要求 8～10中任一项所述的大鲵软骨酶解提取物的醇溶组分或者如权利要求11～13中任一项所述的软骨多肽用于降低尿酸或治疗高尿酸血症或痛风的用途。
  20. 一种获取大鲵软骨酶解提取物的方法，其包括：
    从大鲵获取软骨，
    对获取的软骨进行蛋白酶解，以及
    获得蛋白酶解后的上清并对其进行干燥。
  21. 根据权利要求20所述的方法，其中，所述蛋白酶是碱性蛋白酶，在pH 9～11的条件下进行蛋白酶酶解。
  22. 一种获取大鲵软骨酶解提取物的醇溶组分的方法，其包括：
    从大鲵获取软骨，
    对获取的软骨进行蛋白酶解，
    获得蛋白酶解后的上清并对其进行干燥，
    对蛋白酶解后的上清或其干燥物进行醇沉，以及
    获得上清。
  23. 根据权利要求22所述的方法，其中，所述蛋白酶是碱性蛋白酶，在pH 9～11的条件下进行蛋白酶酶解。
  24. 一种获取大鲵软骨酶解提取物的醇沉组分的方法，其包括：
    从大鲵获取软骨，
    对获取的软骨进行蛋白酶解，
    获得蛋白酶解后的上清并对其进行干燥，
    对蛋白酶解后的上清或其干燥物进行醇沉，以及
    获得沉淀部分。
  25. 根据权利要求24所述的方法，其中，所述蛋白酶是碱性蛋白酶，在pH 9～11的条件下进行蛋白酶酶解。
  26. 根据权利要求21、23或25中任一项所述的方法，其中，所述碱性蛋白酶是2709蛋白酶。
  27. 根据权利要求22～26中任一项所述的方法，其中，醇沉操作中乙醇质量浓度为50％-90％。
  28. 根据权利要求27所述的方法，其中，醇沉操作中乙醇质量浓度为70％。
  29. 一种降低尿酸或治疗高尿酸血症或痛风的方法，其包括：
    向由此需要的受试者给药如权利要求1～7中任一项所述的大鲵软骨酶解提取物、如权利要求8～10中任一项所述的大鲵软骨酶解提取物的醇溶组分或者如权利要求11～13中任一项所述的软骨多肽。
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