WO2018032502A1 - 羊来源的低分子肝素及其制备方法与应用 - Google Patents

羊来源的低分子肝素及其制备方法与应用 Download PDF

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WO2018032502A1
WO2018032502A1 PCT/CN2016/096030 CN2016096030W WO2018032502A1 WO 2018032502 A1 WO2018032502 A1 WO 2018032502A1 CN 2016096030 W CN2016096030 W CN 2016096030W WO 2018032502 A1 WO2018032502 A1 WO 2018032502A1
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sodium
heparin
sheep
molecular weight
solution
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PCT/CN2016/096030
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English (en)
French (fr)
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金永生
靳彩娟
姚亦明
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苏州融析生物科技有限公司
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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
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan

Definitions

  • the invention relates to a low molecular weight heparin derived from sheep, including arginic sodium, anastrozed calcium, saponin sodium, sareparin sodium and sheepbeibei sodium, which have halality and belong to the field of medical biotechnology.
  • Low Molecular Weight Heparin is the most widely used anticoagulant and antithrombotic drug in clinical practice. Some low molecular weight heparin is also widely used in clinical treatments such as anti-inflammatory and anti-cancer. Low molecular weight heparin is prepared by depolymerization of natural macromolecular heparin. At present, the source of medical low molecular weight heparin is almost all heparin of pig intestinal mucosa.
  • heparin There are certain differences in the chemical structure of heparin from different animals or organs.
  • the molecular structure of heparin consists of glucosamine and one of two uronic acids (90% iduronic acid, 10% glucuronic acid), which can be represented by a tetrasaccharide unit, as shown in the figure below.
  • a and b, and c shows a pentasaccharide structure bound to antithrombin, which is a core structure necessary for maintaining anticoagulant activity.
  • Remarks a ordered "regular zone” structure of the trisulfated disaccharide repeating unit - ⁇ UA2S-GlcNS6S ( ⁇ IS), which is dominant in the molecular chain; b disordered "irregular zone” has a low degree of sulfation, structural changes Diverse, the frequency of occurrence in the molecular chain is small; the pentasaccharide structure in which c is combined with antithrombin, and the bold indicates that it is necessary to maintain anticoagulant activity, such as removal. The clotting activity is reduced by 95%, and italic is also important. After removal, the anticoagulant activity will decrease by 25-50%.
  • ⁇ IS, ⁇ IIS and ⁇ IIIS are 58%-66%, 9.5%-11.5% and 5.8%-7.8% in the porcine intestinal mucosa heparin, respectively.
  • the glycan chains of heparin and porcine heparin are significantly different in physical and chemical properties. There are also differences in biological activity.
  • the object of the present invention is to provide several sheep-derived low molecular weight heparins - arginous heparin sodium, anastrozed heparin calcium, sheep tamsparin sodium, sareparin sodium and sheep bemis heparin sodium, including preparation methods thereof and Anticoagulant antithrombotic anticancer and halal drugs.
  • the preparation and pretreatment of the raw material for the preparation of the low molecular weight heparin of the above-mentioned sheep comprises the following steps: using commercially available crude product heparin sodium, and purifying is carried out by a process well known in the art, that is, dissolving in water and then insulating The solution is hydrolyzed by alkali, and then hydrolyzed by protease. The pH of the hydrolyzate is adjusted by anion exchange resin and eluted, and then recovered by alcohol precipitation. The recovered heparin is used in an aqueous solution of 1% to 3% by mass of sodium chloride.
  • Dissolve to a mass concentration of 5%-10% preferably decolorize with a final volume concentration of 0.1%-5% hydrogen peroxide for more than 0.5 hours.
  • the reaction solution is finely filtered, it is preferably precipitated by ethanol or other organic solvent.
  • the volume of ethanol used is preferably the reaction liquid. 1-3 times.
  • the pretreated sheep heparin the anticoagulant activity of the whole sheep plasma method is not less than 150 units per milligram after drying; and the 3.3% mass concentration aqueous solution is clear and the color is not deeper than the standard color No. 5;
  • the heparin which does not meet the standard color may be subjected to one or more decolorization treatments until the color is acceptable.
  • the difference between the heparin and the pig heparin is mainly reflected in the molecular weight distribution, the disaccharide composition, the activity comparison and the structural difference shown by the nuclear magnetic spectrum.
  • the heparin and the porcine heparin have a molecular weight distribution and molecular weight, the porcine heparin is between 15000 Da and 19000 Da, and the heparin is between 13000 Da and 17,000 Da, and the molecular weight is smaller;
  • the composition and difference of the disaccharide of the heparin and the porcine heparin are respectively 58%-66%, 9.5%-11.5 Between % and 5.8%-7.8%, and heparin is between 66%-74%, 8%-10% and 4%-6%, with significant differences; and anti-Xa and anti-IIa activities Critical to the core pentose sugar 3-position sulfated tetrasaccharide peak - ⁇ IIA-IISglu, pig heparin between 2.1% - 2.5%, and heparparin is 1.7% - 2.1%; in addition, the overall disaccharide In terms of composition, the high sulfated components ( ⁇ IS, ⁇ IIS, and ⁇ IIIS) were more than 80%, while the porcine heparin was at least 78%, and the degree of sulf
  • the difference between the anti-coagulant activity of the goat heparin and the porcine heparin, the anti-coagulation, the anti-Xa and the anti-IIa of the whole sheep plasma, the heparin is similar to the porcine heparin, but the heparin is slightly lower; the heparin
  • the ratio of anti-Xa/anti-IIa of pig heparin was consistent, ranging from 0.95 to 1.05.
  • the structural difference between the heparin and the porcine heparin is the same as that of the porcine heparin in the nuclear magnetic resonance spectrum, but there are some differences in the details of the structure, and the nitrogen at ⁇ 2.04 ppm
  • the methyl peak of acetyl group, the heparin fraction is smaller than that of pig heparin, reflecting that the heparin has less N-acetyl modification, and correspondingly, the modification ratio of N-sulfonate is higher.
  • Daheparin sodium the current Pharmacopoeia regulations for pigs, is based on pig fine heparin sodium, the preparation method includes three steps of nitrous acid depolymerization, sodium borohydride reduction and molecular weight fractionation.
  • the preparation method of the sheep heparin sodium refers to the preparation method of the porcine heparin sodium, but the related process parameters are different, and specifically includes the following steps.
  • S11 nitrous acid depolymerization
  • S11 nitrous acid depolymerization
  • the product of S13 and arginic sodium is prepared by formulating a crude product of arginine sodium obtained in S12 into a solution, and performing molecular weight fractionation by anion exchange chromatography (or ultrafiltration), and a classification method is to use an anion exchange resin.
  • the crude salt solution of the crude heparin sodium solution was applied at a low salt concentration, washed at a slightly higher salt concentration, and finally collected at a higher concentration, and the eluted products of each part were recovered by alcohol precipitation and dried.
  • the aqueous solution of the heparparin in the S11 is between 5% and 15% by mass, more preferably at 10%; the pH is adjusted between 2 and 5, more preferably the pH is between 2.9 and 3.3; The ratio of the amount of sodium nitrate to the weight of the heparparin is 1:20-50, more preferably 1:35; the depolymerization time is between 1 hour and 6 hours; correspondingly, the solution used for the preparation of the sodium heparin sodium The poly-strength is slightly weaker than the preparation of porcine heparin sodium.
  • the above preferred sodium nitrite is less in quantity, higher in pH, and shorter in depolymerization time.
  • the temperature of reduction of sodium borohydride in said S12 is not too high, ranging from 0 °C to 40 °C, more preferably between 2 °C and 15 °C.
  • the ratio of the amount of sodium borohydride in the S12 to the weight of the heparin in the S11 is 1:5-15, more preferably 1:10; and the reduction time is not less than 0.5 hours.
  • the loading concentration is 10-100 mg/ml, and the salt concentration is controlled below 300 mmol/L, and the loading load is 5- 50 mg of arbutin per ml of resin.
  • the slightly high salt washing in the S13 is a resin which does not exceed 450 mmol per liter of the salt solution to clean and balance the adsorbed ardan heparin;
  • the higher salt in the S13 is eluted by eluting the heparin sodium with a high concentration of salt solution of 1 mole per liter or more, and the eluate is collected in successive portions.
  • the eadeparin sodium eluate collected in the fraction of S13 is separately recovered by alcohol precipitation, and the alcohol precipitation refers to slowly adding 2-4 times volume to the eluate with sufficient agitation and fine filtration.
  • Methanol which produces a precipitate of arginine sodium, which is recovered by filtration or centrifugation and dried.
  • the sodium sulphate sodium collected and recovered in the fraction in S13 is analyzed by molecular weight and molecular weight distribution, and the appropriate components are calculated and proportioned so that the molecular weight and molecular weight distribution meet the requirements of USP39 for dalteparin, and will be combined.
  • the latter component is reconstituted with purified water, sterilized by filtration at 0.22 ⁇ m, and dried and recovered, and the drying is preferably freeze-dried.
  • the ultrafiltration fractionation in the S13 is an aqueous solution of a crude crude product of arginic sodium, which is subjected to ultrafiltration of at least two rounds by an ultrafiltration membrane to analyze the molecular weight and molecular weight distribution of the ultrafiltration concentrate, in accordance with USP39.
  • the ultrafiltration concentrate is finely filtered and sterilized, directly lyophilized and recovered, and the salt concentration can be adjusted to be recovered after drying.
  • the weight average molecular weight of the finished product of the arginine sodium in the S13 is between 5600 and 6400, wherein the ratio of the molecular weight ⁇ 3000 portion is not higher than 13.0%, and the ratio of the molecular weight>8000 portion is between 15.0% and 25.0%. , in line with USP39 and other regulations for the release of dalteparin sodium
  • the finished product of arginic sodium is dried between 100-180 units per mg, and the anti-IIa activity is dried between 30-90 units per mg, anti-Xa
  • the ratio of anti-IIa is between 1.6 and 3.0, which is different from the release factor of dalteparin sodium prescribed by USP39.
  • the nataparin sodium can be further purified by molecular weight fractionation, and the anti-Xa activity and the anti-IIa activity and the proportion of the appropriate ratio are selected, so that the product meets the requirements of USP39 and the like. Line indicator.
  • the structural analysis of the finished product of the arginine sodium in the S13 is carried out by nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the carbon-hydrogen relationship linked on the sugar chain.
  • the nataparin sodium is consistent with the main structure of the dalteparin sodium from the porcine intestinal mucosa, but there are also some differences, such as the methyl peak of N-acetyl group at ⁇ 2.04 ppm, and the number of integrals on the argan heparin sodium. To be less, it is indicated that the N-acetyl modification in the heparin sugar chain is relatively small.
  • Natrafloxacin calcium EP8.0 is specified as pig source, and is based on pig fine heparin sodium.
  • the preparation method is similar to that of dalteparin sodium, including nitrous acid depolymerization, sodium borohydride reduction, anion resin exchange and calcium salting. A few steps.
  • the preparation method of the goat natriuretic calcium refers to the preparation method of the porcine nadropar calcium, and specifically includes the following steps.
  • S23 the product of the product of the natriuretic calcium, is prepared by formulating the crude product of the nadropar heparin obtained in S22 into a solution, adsorbing with an anion exchange resin, and eluting with a gradient of a low concentration to a high concentration with a calcium chloride solution, washing The liquid-removed alcohol is recovered, the precipitate is dissolved in purified water, deoxidized by adding hydrogen peroxide, the decolorizing solution is deeply filtered, calcium chloride is added and the pH is adjusted to neutral, sterile filtration, alcohol precipitation recovery and drying to obtain the nutrient calcium Finished product.
  • the aqueous solution of the heparparin in the S21 is between 5% and 15% by mass, more preferably at 10%; the pH is adjusted between 2-4, more preferably the pH is between 2.9 and 3.3; The weight ratio of sodium nitrate to the heparin is 1:10-30, more preferably 1:20; and the depolymerization time is between 1 hour and 4 hours.
  • the ratio of the amount of sodium borohydride in the S22 to the weight of the heparin in the S11 is 1:5-15, more preferably 1:10; and the reduction time is not less than 0.5 hours.
  • the loading concentration is 10-100 mg per ml, loading load 5-50 mg of sheep nadroparin per ml of resin.
  • the step of eluting from a low concentration to a high concentration of the calcium chloride solution in the S23 means washing and balancing the adsorbed goat nadropar heparin with not more than 400 millimoles per liter of the calcium chloride solution.
  • the natriuretic calcium was eluted with a high concentration of calcium chloride solution of 1 mol per liter or more, and the eluate was collected in successive portions.
  • the isoflavone calcium eluate collected in the fraction of S23 is separately recovered by alcohol precipitation, and the alcohol precipitation refers to slowly adding 2-4 times volume to the eluate with sufficient agitation and fine filtration.
  • Methanol which produces a precipitate of natriuretic calcium, which is recovered by filtration or centrifugation and dried.
  • the natriuretic calcium collected and recovered in the fraction of S23 is analyzed by molecular weight and molecular weight distribution, and the appropriate components are calculated and proportioned so that the molecular weight and molecular weight distribution meet the requirements of EP8.0 for nadropar calcium.
  • the combined components are reconstituted with purified water, sterilized by filtration at 0.22 ⁇ m, and dried and recovered, and the drying is preferably freeze-dried.
  • the weight average molecular weight of the finished product of the natriloparin calcium in the S23 is between 3600 and 5000, wherein the ratio of the molecular weight ⁇ 2000 portion is not higher than 15.0%, and the ratio of the molecular weight 2000-8000 portion is 75.0%-95.0%.
  • the ratio of the molecular weight of 2000-4000 is between 35% and 55%, which is in line with the release of Natto heparin calcium as specified in EP8.0.
  • the structural analysis of the finished product of the goat natriuretic calcium in S23 is performed by nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the carbon-hydrogen relationship linked on the sugar chain.
  • the natriuretic calcium of the goat is the same as the nadroparin calcium from the intestinal mucosa of pigs, but the methyl peak of N-acetyl group at ⁇ 2.04ppm is less integrated, which reflects the cause of the calcium
  • the N-acetyl modification of the sheep source is less, and accordingly there are more N-sulfonate modifications.
  • the finished product of the goat natriuretic calcium is dried between 90-125 units per mg, and the anti-Xa/anti-IIa ratio is between 2.0 and 3.5, which is different from EP8.0.
  • the specified pig-derived nadroparin calcium is not limited to the following properties:
  • the natriloparin calcium can be refined by molecular weight classification, and the anti-Xa activity and anti-IIa activity and the proportion of the appropriate ratio are selected, so that the product meets the requirements of EP8.0 and the like. Line indicator.
  • Tinzagate sodium EP8.0 is specified as a pig source, which is based on pig fine heparin sodium.
  • the preparation method is depolymerized by heparinase I, and then refined and recovered.
  • the preparation method of the sheep sulphate sodium refer to the preparation method of the pig sulphate sodium, specifically comprising the following steps,
  • heparinase I depolymerization is to dissolve the pre-treated goat heparin, adjust the pH of the solution to neutral, then add heparinase I, stir the reaction, monitor the enzymatic reaction until the 232 nm absorbance increase of the reaction solution increases to Within the predetermined range, an enzymatic depolymerization solution of goat heparin is obtained;
  • the preparation of S32 and sheep sulphate sodium is the enzymatic depolymerization of goat heparin obtained in S31, and treated at 90 ° C for 5 minutes, the enzyme protein is removed by filtration, and the reaction solution is further added with sodium chloride to adjust the pH to neutral. After the fine filtration, the alcohol precipitation is recovered and dried to obtain the finished product of the sheep chewing heparin sodium.
  • the aqueous solution of the heparparin in the S31 is between 1% and 10% by mass, more preferably 5%; the pH is adjusted between 5-9, more preferably 6-8; the amount of heparinase I (activity) And the heparin weight ratio is 1-100:1 (unit: mass / gram), more preferably 10:1; depolymerization time between 1 hour and 24 hours; enzymatic temperature is preferably between 10 ° C -40 ° C .
  • the enzymatic depolymerization solution of the goat heparin in S31 is controlled according to different reaction solution concentrations, more preferably 5% heparin concentration concentration reaction, and the absorbance increase value is between 50 and 70.
  • the finished product of the sheep sulphate sodium in the S32 is prepared, and the enzymatic depolymerization liquid of the goat heparin obtained in S31 is rapidly heated, the heparinase I is denatured and precipitated, and then filtered to remove, more preferably, the temperature is raised to 90 ° C. Handle for 5 minutes.
  • the fine filtration and alcohol precipitation of the sodium sulphate sodium in the S32 is to add a sodium chloride having a mass concentration of 5% to 15%, more preferably 10%, to the solution of the enzyme filtered in S31;
  • the pH neutral range is between 5-9, and the pH is more preferably 5.8-7.0; after salting and pH adjustment, fine filtration is performed, preferably 0.22 micron filtration; the alcohol precipitation refers to slowly adding 2-4 to the solution with sufficient stirring.
  • the volumetric and finely filtered methanol produces a precipitate of sheep sulphate sodium, and the precipitate is recovered by filtration or centrifugation and dried.
  • the weight average molecular weight of the finished product of the saponin sodium in the S32 is between 5,500 and 7,500, wherein the ratio of the molecular weight of ⁇ 2000 is not more than 10.0%, and the proportion of the molecular weight of 2000-8000 is between 60.0% and 72.0%.
  • the ratio of molecular weight > 8000 is between 22.0% and 36.0%, which is consistent with the release rate of tinzaparin sodium as specified in EP8.0.
  • the structural analysis of the finished product of the saponin sodium in the S32 is carried out by nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the carbon-hydrogen relationship linked on the sugar chain.
  • the sheep chelated heparin sodium is consistent with the main structure of tinzaparin sodium from the intestinal mucosa of pigs. 6.0 ppm has a characteristic hydrogen peak, reflecting the 4,5-unsaturated uronic acid characteristic of the non-reducing end of the newly formed sheep chelated heparin sodium molecule when heparinase I depolymerizes.
  • the methyl peak of the N-acetyl group at ⁇ 2.04ppm is less integrated, reflecting the lower N-acetyl modification of the sheep in the fensidine sodium, which has more N-sulfonate modification.
  • the finished product of the sheep chewable heparin sodium is dried between 60-120 units per mg, and the anti-Xa/anti-IIa ratio is between 1.5 and 3.0, which is different from EP8.
  • Swine-derived tinzaparin sodium as specified in 0.
  • the sheep sulphate sodium can be fractionally refined to obtain anti-Xa activity and anti-IIa activity and a suitable proportion of the section, so that the product meets the release index of tinzaparin sodium prescribed by EP 8.0.
  • Parofan sodium is specified as the source of pig intestinal mucosa or bovine intestinal mucosa, usually based on pig fine heparin sodium, the preparation method includes copper peroxide depolymerization, chelation and cationic resin exchange to remove copper, and then Refined alcohol precipitation recovery steps.
  • the preparation method of the ergosparin sodium refers to the preparation method of porcine heparin sodium, and specifically includes the following steps: S41, copper peroxide depolymerization, dissolving the pretreated sheep heparin to adjust the pH of the solution to neutral And then separately adding copper acetate solution and hydrogen peroxide solution for depolymerization, during which pH and temperature are controlled;
  • S43 the preparation of the product of sareparin sodium, is the crude product of the saponin sodium obtained in S42, reconstituted with water, decolorized by adding hydrogen peroxide, decolorizing solution to adjust the pH to neutral, adding sodium chloride, sterilizing and filtering the alcohol The precipitate is recovered and dried to obtain the finished product of sareparin sodium.
  • the copper peroxide in the S41 is depolymerized, the concentration of the heparparin is between 1% and 15%, more preferably between 5% and 10%; the mass ratio of the copper acetate, hydrogen peroxide and the goat heparin is 0.1-1:1-3:1, more preferably 0.4:2:1; depolymerization temperature is controlled between 30 ° C and 70 ° C, more preferably 50 ° C to 55 ° C; pH is controlled between 6 and 9 during depolymerization More preferably, it is 7-8; the depolymerization time is preferably 2 to 48 hours, more preferably 18 hours.
  • the mass ratio of disodium ethylenediaminetetraacetate to heparin in the S42 is 0.5-5:1, more preferably 1:1; the salt addition refers to the addition of chlorine having a final mass concentration of 5%-15%. Sodium is more preferred, and the mass concentration is preferably 10%.
  • the mass concentration is between 1% and 20%, more preferably 10%; and the strong cation exchange resin is a food grade resin.
  • the salt-alcohol precipitation of the unreacted flow-through liquid in the S42 is carried out by adding sodium chloride to the solution to a final mass concentration of 5% to 15%, more preferably 10%, the alcohol Sinking is slowly added to the solution with 2-4 times volume and finely filtered methanol to produce a precipitate of sareparin sodium, and the crude product of sareparin sodium is recovered by filtration or centrifugation.
  • the crude product of sareparin sodium in S43 is dissolved in water to a mass concentration of between 1% and 15%, preferably 10%; the decolorization temperature of hydrogen peroxide is between 15 ° C and 40 ° C, more preferably 25 ° C; hydrogen peroxide is The final volume concentration in the solution is between 0.1% and 5%, more preferably between 1% and 2%; the decolorization time of the hydrogen peroxide is more than 10 minutes until the color of the reaction liquid is as shallow as Y5 or less.
  • the Sparta edulis sodium solution in S43 is adjusted to pH neutral with dilute hydrochloric acid before decolorization to alcohol precipitation, and is finely filtered, and the filtrate is added with sodium chloride to a concentration of 8-12% to adjust the pH to 5.0- 7.0, fine filtration.
  • the raw product of the sodium argampic sodium in the S43 is prepared, and the alcohol is precipitated, and the 2-4 times of the reaction solution is slowly added to the reaction solution under agitation, and the filtered methanol is used to produce the sodium sulphate.
  • the sodium precipitates and the precipitate is recovered by filtration or centrifugation and dried.
  • the finished product of the sodium amphetamine in S43 has a weight average molecular weight of between 4,000 and 6,000.
  • the finished product of the ergoparin sodium is between 75-110 units per milligram, and the anti-Xa/anti-IIa ratio is between 1.5 and 3.0, in accordance with EP 8.0.
  • the prescribed sodium heparin sodium standard is the prescribed sodium heparin sodium standard.
  • the structural analysis of the finished product of sareparin sodium in the S43 is carried out by nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the carbon-hydrogen relationship linked on the sugar chain.
  • the sodium heparin sodium is consistent with the main structure of the sodium heparin sodium from the intestinal mucosa of pigs, but the methyl peak of N-acetyl group at ⁇ 2.04 ppm is less integrated, reflecting the cause of haloperidin sodium.
  • the N-acetyl modification of the sheep source is less, and accordingly there are more N-sulfonate modifications.
  • Bemisparin sodium the clinical drug is derived from pigs, and is based on pig fine heparin sodium.
  • the preparation method includes the steps of preparing quaternary ammonium salt of goat heparin and refining and recovering after depolymerization of organic alkali.
  • the preparation method of the sheep bemis heparin sodium refer to the preparation method of the pig babe heparin sodium, specifically comprising the following steps,
  • the preparation of the hepatic quaternary ammonium salt is prepared by dissolving the sodium heparan sulfate solution into an aqueous solution, mixing with the aqueous solution of benzalkonium chloride, separating, washing and drying to obtain the quaternary ammonium salt of the goat heparin;
  • the crude product of S52 and sheep bemises sodium is prepared by dissolving the hepatic quaternary ammonium salt obtained by drying in S51 in dichloromethane or other organic solvent, and adding benzyltrimethylammonium hydroxide (Triton-B). Stirring to dissolve the hepatic heparin, after the depolymerization reaction is completed, the sodium acetate methanol solution is added dropwise to prepare a crude precipitate of the sheepbeibei heparin sodium;
  • S53 sheep babe heparin sodium finished product
  • S52 is the S52 in the sheep lamb rice heparin sodium crude product filtration, methanol washing and then repeated multiple reconstitution plus salt alcohol precipitation, product refining, drying, to obtain sheep babe heparin Sodium finished product.
  • the aqueous solution of the heparparin is between 5% and 15% by mass, and the aqueous solution of benzalkonium chloride is between 10% and 30% by mass, wherein the benzalkonium chloride
  • the weight ratio of solid to the heparin sodium solids is from 2 to 5:1.
  • the heparin quaternary ammonium salt, dichloromethane, Triton-B quality in the depolymerization reaction in S52 The amount ratio is 1:3-10:0.2-0.4, and more preferably the ratio is 1:5:0.25.
  • the solvent in S52 may be dichloromethane or dimethylformamide or other organic solvent.
  • the reaction temperature is between 20 ° C and 45 ° C, more preferably 30 ° C; and the reaction time is from 8 hours to 40 hours, more preferably 16 hours.
  • a sodium acetate methanol solution is added dropwise to precipitate the sodium fameparin sodium, the weight of the sodium acetate is 0.8 times that of the goat heparin quaternary ammonium salt, and the sodium acetate methanol solution is The concentration is 10%.
  • the crude precipitate of sheepbeibei sodium in the S53 is separated and washed once or several times with methanol; the precipitate after washing is reconstituted by adding 8%-12% aqueous sodium chloride solution, the chlorination
  • the weight ratio of the sodium aqueous solution to the heparin quaternary ammonium salt is 0.5-2:1, and the reconstituted solution is further subjected to alcohol precipitation crystallization with 2-5 volumes of methanol; the reconstituted alcohol precipitation can be repeated several times until The reconstituted sheep babe heparin sodium solution was clarified without turbidity.
  • the finished product of the lambami sodium heparin sodium in the S53 has a weight average molecular weight of between 3,000 and 4,200.
  • the finished product of the sheep-beibei heparin sodium is dried between 75-110 units per milligram, and the anti-Xa/anti-IIa ratio is between 1.5-3.0, and the current medical treatment.
  • Pig-derived bemiparin sodium differs in the ratio of anti-Xa/anti-IIa.
  • the sheep bemiparin sodium can be fractionally refined to obtain anti-Xa activity and anti-IIa activity and a suitable ratio of the section, so that the product meets the requirements of the current medical bemiparin sodium activity.
  • the structural analysis of the finished product of the lamb rice heparin sodium in the S53 is carried out by nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the carbon-hydrogen relationship linked on the sugar chain.
  • the sheep bemiparin sodium and the bemiparin sodium from the pig intestinal mucosa have the same main structure,
  • the methyl peak of N-acetyl group at ⁇ 2.04ppm is less integrated, reflecting the lower N-acetyl modification of sheep in heparin sodium, and accordingly there are more N- Sulfonic acid group modification.
  • the structural analysis of the finished product of the lamb rice heparin sodium in the S53 is carried out by nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the carbon-hydrogen relationship linked on the sugar chain.
  • the sheep bemiparin sodium is consistent with the main structure of bemiparin sodium from the porcine intestinal mucosa.
  • 6.0ppm has a characteristic hydrogen peak, which reflects the formation of 4,5-unsaturated uronic acid in the non-reducing end of the newly formed sheepbeibei heparin sodium molecule when the ⁇ -elimination reaction of the organic base is depolymerized.
  • the methyl peak of N-acetyl group at ⁇ 2.04ppm is less integrated, reflecting the lower N-acetyl modification of sheep in heparin sodium, which has more N-sulfonate modification.
  • the alcohol precipitation referred to in the present application uses methanol as an organic solvent, and ethanol, isopropanol or acetone may be used instead of methanol unless otherwise specified.
  • the drying involved in the present application may be natural drying, vacuum drying or freeze drying, and other drying methods, in which the turning, grinding, and the like may be performed to improve the drying effect.
  • the anticoagulant effect of the several sheep low molecular weight heparin is examined in human blood in an in vitro test.
  • the effects of low molecular weight heparin on blood coagulation routines including but not limited to APTT, TT and PT, were examined according to the automatic coagulation apparatus and kit method.
  • the several low molecular weight heparins of sheep including arginic sodium, anastrozol heparin, sedative heparin sodium, sareparin sodium and sheepbeibei sodium, are shown in the anticoagulation test in vitro. Strong anticoagulant effect.
  • the sheep low molecular weight heparin including arginous heparin sodium, anastrozed heparin calcium, sheep tamsparin sodium, sareparin sodium and sheep bemiparin sodium, in the prevention and treatment of anticoagulant and antithrombotic diseases Application, and development as a halal anticoagulant antithrombotic drug.
  • the outstanding effect of the invention is as follows: several kinds of sheep low molecular weight heparin - arginous heparin sodium, anastrozed heparin calcium, sheep tamsane sodium, amphibious sodium and sheep bemis heparin sodium are provided, and a practical and stable method is adopted. be made of.
  • several sheep low molecular weight heparin and pig source The low molecular weight heparin has similar physical and chemical properties, and the molecular weight distribution is in full compliance with EP8.0 or the original release index.
  • Several sheep low molecular weight heparins have strong anticoagulant activity.
  • the invention fills the blank of the sheep heparin in the preparation of low molecular weight heparin, and can be developed into a halal drug.
  • the raw material of heparin sodium is easy to obtain and the quality is controllable, which can greatly enrich the source and yield of low molecular weight heparin and halal drugs in the market, and can also promote the effective utilization of sheep breeding and slaughtering waste (intestinal mucosa) with huge economic potential.
  • Figure 1 is a schematic diagram showing the molecular weight distribution of several sheep low molecular weight heparin samples, (1) is arginous heparin sodium, (2) is nadropar heparin calcium, (3) is sheep tibeparin sodium, and (4) is sheep.
  • Paclitaxel sodium (5) is sheep femparin sodium, (6) is a superposition of five low molecular weight heparins;
  • Example 2 is a schematic diagram showing the comparison of disaccharide profiles of several sheep low molecular weight heparin samples in Example 8;
  • FIG. 3 is a schematic diagram showing the comparison of 1H-NMR analysis of several sheep low molecular weight heparin in Example 9, wherein (7)-(11) are, in order, arginous heparin sodium, anastrozed heparin calcium, sheep tamsparin sodium, and heparaffin sodium. And sheep bemiparin sodium.
  • the embodiments of the present invention describe several kinds of sheep low molecular weight heparin - arginous heparin sodium, anastrozed heparin calcium, sheep tamsparin sodium, sareparin sodium and sheep bemis heparin sodium.
  • the specific experimental case is taken as an example below.
  • the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • Crude goat heparin sodium (manufacturer: Shandong Yulian Biotechnology Co., Ltd., batch number: 20150326), samples were sent for RT-PCR to detect the DNA content of pigs and sheep, and the pig DNA content was less than 0.1 pg per microliter, sheep DNA content It is greater than 1 ⁇ 10 5 picograms per microliter, and is determined to be crude heparin sodium according to the source of the original intestinal mucosa of the original manufacturer, and is substantially free of the pig heparin component.
  • the sample was administered to the whole sheep for plasma anticoagulant activity of 67.0 units per milligram.
  • the purification of crude goat heparin sodium is carried out by a process well known in the art, that is, dissolving and dissolving in water after hydrolysis, followed by hydrolysis by protease, and the pH of the hydrolyzate is adjusted and eluted with an anion exchange resin, and is precipitated by alcohol precipitation.
  • the whole sheep plasma anticoagulant activity of the harvested raw material heparin was 134.2 units per milligram.
  • the supernatant was discarded, the heparin precipitate was reconstituted with 2% sodium chloride solution, and the final concentration of 95% alcohol to ethanol was added at 40%, and allowed to stand for 6 hours.
  • the supernatant was discarded, the heparin precipitate was reconstituted with 2% sodium chloride solution, and 95% alcohol was added to the final concentration of ethanol at 38%, and allowed to stand overnight.
  • the supernatant was discarded and the heparin precipitate was dehydrated with 95% alcohol.
  • the dehydrated heparin is then dried by blast to remove residual water or solvent.
  • the pretreated sheep heparin weighs 3.1 kg and is 176.3 units per milligram after the anti-coagulant activity of the whole sheep plasma.
  • the method for determining the activity of the purified heparin sodium anti-Xa and anti-IIa, anti- The Xa activity was 185.1 units per milligram, the anti-IIa activity was 181.5 units per milligram, and the anti-Xa/anti-IIa ratio was 1.02.
  • Example 1 50.0 g of the pre-treated goat heparin in Example 1 was weighed, 500 ml of purified water was added, and the heparin was dissolved by stirring. The water bath was controlled at room temperature, and the stirring was continued for more than 30 minutes. The pH of the heparin hydrochloride solution was adjusted to 3.1, and 1.43 g of sodium nitrite was added to continue the agitation. The pH was then adjusted to neutrality and 4.0 grams of sodium borohydride was added and stirring continued. The pH was adjusted to neutral with dilute hydrochloric acid, and 51.5 g of sodium chloride was added thereto, and stirring was continued for 10 minutes or more at room temperature.
  • the crude heparin obtained by the reaction was precipitated by slowly adding 1250 ml of methanol and stirring for 15 minutes.
  • the reaction solution was transferred to a centrifuge bottle, and the crude heparin precipitate was collected by centrifugation.
  • the precipitate was vacuum dried and weighed to obtain 43.5 g of crude product of dalteparin sodium.
  • the precipitate was collected by centrifugation, and the precipitated methanol was dehydrated and dried. After analysis and calculation of the molecular weight distribution, the precipitates of all the above eluates were combined, dissolved in 200 ml of purified water and sterile-filtered, and lyophilized to obtain 11.2 g of the product.
  • Example 1 50.0 g of the pre-treated goat heparin in Example 1 was weighed, 500 ml of purified water was added, and the heparin was dissolved by stirring. The water bath was controlled at room temperature, and the stirring was continued for more than 30 minutes. The pH of the heparin hydrochloride solution was adjusted to 3.0, 2.5 g of sodium nitrite was added, and the mixture was kept under stirring. The pH was then adjusted to neutral and 4.5 grams of sodium borohydride was added and stirring continued. The pH was adjusted to neutral with dilute hydrochloric acid, 52.0 g of sodium chloride was added, and stirring was continued for 10 minutes at room temperature.
  • Each of the collected eluates was added to 1,250 ml of methanol, stirred for 5 minutes, allowed to stand, and finally centrifuged to collect the precipitate.
  • the precipitate was dissolved in 200 ml of purified water, deoxidized by adding hydrogen peroxide for 2 hours, added with 20 g of calcium chloride, stirred and dissolved, adjusted to pH neutral, and then sterilized and filtered.
  • the filtrate was precipitated with 500 ml of methanol, and the precipitate was vacuum dried. , obtained 12.6 grams of goat natriuretic calcium product.
  • Example 1 50.0 g of the pre-treated goat heparin in Example 1 was weighed, 500 ml of purified water was added, and the heparin was dissolved by stirring. Control the room temperature in a water bath, adjust the pH of the solution to neutrality, add 10 ml of heparinase solution, continue to incubate and stir until the A232 absorbance value of the solution increases at 60 °C, and rapidly raise the reaction solution to 95 °C for 5 minutes. Quickly cool to room temperature. Finely filter out the precipitated enzyme protein precipitate, then add 50 g of sodium chloride to the filtrate, adjust the pH to neutrality, continue stirring for more than 10 minutes at room temperature, and sterilize and filter.
  • Example 1 Weigh 100 g of heparin sodium in the above Example 1 and dissolve it in 700 ml of water; further dissolve 250 g of benzalkonium chloride in 1000 ml of water to prepare a clear aqueous solution of benzalkonium chloride; with sufficient agitation, The aqueous solution of benzalkonium chloride was added dropwise to the aqueous solution of heparin, and the addition was completed within 30 minutes, and stirring was continued for 2 hours. The mixture was centrifuged at 6000 rpm for 5 minutes in a high-speed centrifuge, and the precipitate was resuspended in 3000 ml of water, and further stirred for 5 minutes, and further centrifuged at 6000 rpm for 5 minutes. repeat.
  • Precipitated goat heparin quaternary ammonium salt wet
  • the product was transferred to a freeze-drying oven for drying for 48 hours to obtain 265 g of dry product of the hepatic heparin quaternary ammonium salt, and the loss on drying was determined to be 0.6%.
  • the crude precipitate of sheep bemislavin sodium was collected by centrifugation, reconstituted with 1000 ml of purified water, and the pH was adjusted to neutrality with hydrochloric acid. 100 g of sodium chloride was added, and 2,500 ml of methanol was added to precipitate ampicillin sodium. The above reconstituted alcohol was precipitated twice, and finally precipitated and dried to obtain 53.2 g of pure lamb rice heparin sodium.
  • Table 1 Weight average molecular weight and molecular weight distribution of arginine sodium
  • Table 2 Weight average molecular weight and molecular weight distribution of natriuretic calcium
  • Table 3 Weight average molecular weight and molecular weight distribution of saponin sodium
  • Table 4 Weight average molecular weight and molecular weight distribution of sareparin sodium
  • Table 5 Weight average molecular weight and molecular weight distribution of sheep bemises sodium
  • the low molecular weight heparin and the molecular weight distribution (if required) of the sheep low molecular weight heparin prepared in the second to sixth embodiments are in compliance with the technical indexes of the pharmacopoeia EP8.0 or the original manufacturer, that is, the sheep low molecular weight heparin and
  • the low molecular weight heparin derived from the pig intestinal mucosa is similar in molecular weight and its distribution.
  • the sheep low molecular weight heparin prepared in each example exhibits typical characteristics of sheep heparin.
  • the main disaccharide ⁇ IS is in the samples of arginous sodium, anastrozed heparin calcium, sheep tamsane sodium, and ergoparin.
  • the contents of sodium and sheep bemises sodium are as high as 70.2%, 69.2%, 68.7%, 70.6% and 71.2%, respectively, which is higher than the low molecular weight heparin in pigs.
  • the nuclear magnetic resonance spectrum analysis of several kinds of sheep low molecular weight heparin was carried out by using a 400 MHz nuclear magnetic resonance spectrometer of the Analysis and Testing Center of Suzhou University, and the zero point was determined by 3-trimethylsilylpropionate-d4 (TSP).
  • sample solution to be tested several kinds of sheep low molecular weight heparin (Examples 2 to 6), each accurately weigh about 20 mg, and weighed in water (D 2 O) to a concentration of about 20 mg per ml. 1-2 drops of TSP were added dropwise, and the mixture was shaken and mixed for 0.22 ⁇ m. The results are shown in Fig. 3. Among them, ⁇ 3.4 ppm is a methyl hydrogen peak remaining in methanol, and ⁇ 4.7 ppm is a water hydrogen peak.
  • Test results The hydrogen spectrum results of several sheep low molecular weight heparin are shown in Figure 3. Due to different processes and treatments, they exhibit a unique spectrum of low molecular weight heparin, among which, heparin sodium and anastrozal heparin calcium Both are depolymerized by sodium nitrite, and both are reduced by sodium borohydride, so the chemical structure is the same; the sheep sulphate sodium is depolymerized by enzymatic hydrolysis of heparinase.
  • the newly formed low molecular weight heparin non-reducing end is characterized by 4,5-unsaturated uronic acid, 6.0 ppm is the reflection of this characteristic hydrogen, and similarly, sheepbeibei heparin sodium is the ⁇ -elimination reaction of organic base to solve Poly, also exhibits a 4,5-unsaturated uronic acid structure at the non-reducing end, similar to the sheep sulphate sodium; in addition, sareparin sodium is a depolymerization of copper peroxide, a depolymerization due to oxidation The structure on the map is different from the above.
  • the low-molecular-weight heparin reflects the N-acetyl methyl hydrogen at 2.04ppm, and the scores are similar, which is less than the low-molecular heparin derived from pigs, such as enoxaparin sodium, reflecting the low molecular weight heparin in sheep.
  • N-acetyl modification is less, and conversely, the sulfonate modification on N- is more, which is also consistent with the results of the disaccharide analysis.
  • sheep low molecular weight heparin has strong anticoagulant activity.
  • Activity measured by whole sheep plasma method In the range of 40-55 units per mg; anti-Xa activity, the highest sample of arginine sodium, up to 143.4 units per milligram, the lowest sample of argalin sodium, also in 89.7 units per milligram; anti-IIa activity, also The highest sample of arginine sodium is at 62.4 units per milligram, and the same sample of ergoparin sodium is the lowest at 34.9 units per milligram.
  • anti-Xa/anti-IIa ratio several sheep low molecular weight heparin samples are not at 1.7-2.6. Wait.
  • the experimental group is as follows: the arginine sodium sample group (described in Example 2), the anastrozine calcium sample group (described in Example 3), the spectabilin sodium sample group (described in the fourth embodiment), and the sheep pupa Heparin sodium sample group (described in Example 5), sheep Beibei heparin sodium sample group (described in Example 6) and enoxaparin sodium standard group (for clinically marketed drugs, Kesai, batch number: 24459), all The final sample concentration of the group was ⁇ 3 ⁇ g/mL, and the experimental saline was used as a blank control.
  • Sheep heparin sodium sample 2.91 ⁇ 0.51g/L 31.00 ⁇ 0.00s* Sheep nadropar calcium sample 2.66 ⁇ 0.43g/L 31.00 ⁇ 0.00s* Sheep titanate sodium sample 2.64 ⁇ 0.37g/L 31.00 ⁇ 0.00s* Parylene heparin sodium sample 2.76 ⁇ 0.53g/L 31.00 ⁇ 0.00s* Sheep bemises sodium sample 2.71 ⁇ 0.72g/L 31.00 ⁇ 0.00s* Enoxaparin sodium standard 2.89 ⁇ 0.44g/L 31.00 ⁇ 0.00s* Blank control 2.57 ⁇ 0.25g/L 9.95 ⁇ 0.40s
  • sheep low molecular weight heparin has typical sheep source characteristics, and the chemical structure (disaccharide composition and hydrogen spectrum) is different from the low molecular weight heparin of pig source.
  • the refined low molecular weight heparin and molecular weight distribution can meet the requirements of the current pharmacopoeia for low molecular weight heparin, and the biological anticoagulant activity is similar, which can be used in applications such as anticoagulation.
  • the present invention has various embodiments, and all technical solutions formed by equivalent transformation or equivalent transformation are within the scope of the present invention.

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Abstract

一种羊来源的低分子肝素，包括羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠。所述羊源的低分子肝素都有着共同的种源特征，在化学结构上，其主要二糖ΔUA2S-GlcNS6S(ΔⅠS)含量在66％-74％之间。羊低分子肝素与猪来源的低分子肝素，理化性质相似，生物学活性相近，拓展了低分子肝素类药物的来源。所述羊低分子肝素制备方法，简单易行，工艺稳定，所得产品经过精制，可完全可符合各药典对现行低分子肝素的要求。羊低分子肝素还具有猪来源低分子肝素所不具有的清真性，在广大穆斯林人群、国家和地区，有着巨大的市场，可应用于抗凝、抗栓、抗炎、抗癌及清真药物。

Description

羊来源的低分子肝素及其制备方法与应用 技术领域
本发明涉及羊来源的低分子肝素，包括羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，其具有清真性，属于医药生物技术领域。
背景技术
低分子肝素(Low Molecular Weight Heparin，LWMH)，是目前临床上应用最广泛最重要的抗凝抗栓药物，一些低分子肝素还被广泛应用于抗炎和抗癌等临床治疗。低分子肝素经天然大分子肝素解聚制备，目前医用低分子肝素的来源，几乎均是猪肠粘膜肝素。
不同动物或器官来源的肝素，化学结构上存在着一定的差异。肝素的分子结构均由氨基葡萄糖和两种糖醛酸(90％艾杜糖醛酸、10％葡萄糖醛酸)中的一种组成二糖重复单元，可以用一个四糖单元表示，如下图中的a和b，而c则显示与抗凝血酶结合的五糖结构，是维持抗凝活性所必需的核心结构。
Figure PCTCN2016096030-appb-000001
图肝素分子结构
备注：a有序“规则区”结构规整的三硫酸化二糖重复单元——ΔUA2S-GlcNS6S(ΔⅠS)，在分子链中占主要；b无序“不规则区”硫酸化程度低，结构变化多样，在分子链中出现的频率较小；c与抗凝血酶结合的五糖结构，粗体表示的是维持抗凝活性所必需的，如除去则抗 凝活性下降95％，斜体表示的也很重要，脱去后抗凝活性将下降25-50％。
本发明人在之前的专利申请(申请公布号：CN 105131153 A)中，除了公布一种抗凝抗栓的羊依诺肝素钠外，还详细描述了羊肝素和猪肝素等在分子结构、二糖组成、理化性质和生物学活性等方面的异同。羊肝素中，主要二糖ΔⅠS的含量在66％-74％之间，次要二糖ΔUA-GlcNS6S(ΔⅡS)和ΔUA2S-GlcNS(ΔⅢS)的含量分别是8％-10％和4％-6％，而ΔⅠS、ΔⅡS和ΔⅢS在猪肠粘膜肝素中则分别是58％-66％、9.5％-11.5％和5.8％-7.8％，羊肝素和猪肝素的糖链显著不同，在理化性质和生物学活性上也呈现出差异。
目前市场和临床上，没有羊来源肝素制备的低分子肝素。羊肝素和猪肝素明显不同，由其制备出的羊低分子肝素也将于猪肝素不同。另外，清真，是穆斯林在中国流行的专用名称，穆斯林教义对食品和药品等有着明确的要求，哺乳动物中只允许食用牛羊等反刍动物产品，禁食猪和狗等不反刍动物产品。全球穆斯林人口2013年突破16亿，占全球69亿人口的23％。在一些由穆斯林人口占多数的国家，如印度尼西亚、巴基斯坦、伊朗等等，符合穆斯林教义的清真药品有着无可比拟的优势。清真世界广泛缺乏清真药物，猪来源的肝素类药物不具有清真性，而羊源肝素则具有清真性，因此，开发清真的羊低分子肝素，有着极为重要的经济和社会价值。
发明内容
本发明的目的在于提供几种羊来源的低分子肝素——羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，包括它们的制备方法及在抗凝抗栓抗癌和清真药物中的应用。
本发明的目的，将通过以下技术方案得以实现：
几种羊低分子肝素——羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，是以羊肝素制备得到的。
上述羊低分子肝素制备所用的原料——羊肝素，其制备和预处理包括如下步骤：采用市售粗品羊肝素钠，提纯采取本行业所熟知的工艺，即以水溶解后保温 盐解碱解，再加蛋白酶水解，水解液调pH后以阴离子交换树脂吸附并洗脱，以醇沉沉淀回收；回收后的羊肝素，采用质量浓度为1％-3％的氯化钠水溶液溶解至质量浓度5％-10％，优选以终体积浓度为0.1％-5％双氧水进行脱色0.5小时以上，反应液精过滤后优选以乙醇或其他有机溶剂沉淀回收，所用乙醇体积优选为反应液的1-3倍。
优选地，所述经预处理得到的羊肝素，抗凝活性全绵羊血浆法折干后不少于150单位每毫克；且3.3％质量浓度的水溶液澄清且色度不深于5号标准色；所述色度不达标的羊肝素，可再进行一次或多次脱色处理，直至色度合格。
优选地，所述羊肝素与猪肝素的差异，主要体现在分子量分布、二糖组成、活性对比和核磁图谱显示的结构差异上。
优选地，所述羊肝素与猪肝素在分子量分布与分子量，猪肝素在15000Da-19000Da之间，而羊肝素则在13000Da-17000Da之间，分子量要小一些；
优选地，所述羊肝素与猪肝素的二糖组成与差异，参照USP分析方法，猪肝素中的主要的二糖单元(ΔⅠS、ΔⅡS和ΔⅢS)分别在58％-66％、9.5％-11.5％和5.8％-7.8％之间，而羊肝素则在66％-74％、8％-10％和4％-6％之间，有显著的差异；而与抗-Ⅹa和抗-Ⅱa活性至关重要的核心五糖中3-位硫酸化的四糖峰——ΔⅡA-ⅡSglu，猪肝素在2.1％-2.5％之间，而羊肝素是1.7％-2.1％；另外，整体的二糖组成情况看，高硫酸化组份(ΔⅠS、ΔⅡS和ΔⅢS)羊肝素是在80％以上，而猪肝素则最低于78％，羊肝素中硫酸化的程度更高。
优选地，所述羊肝素与猪肝素在抗凝活性的差别，全绵羊血浆法抗凝、抗Ⅹa和抗Ⅱa三项上，羊肝素与猪肝素相近，但羊肝素要稍低一下；羊肝素与猪肝素的抗Ⅹa/抗Ⅱa比值上一致，均在0.95-1.05之间。
优选地，所述羊肝素与猪肝素在结构上的差别，核磁氢谱上，羊肝素与猪肝素主体一致，但在一些细节结构上相互间有一定的差异，在δ2.04ppm处的氮-乙酰基的甲基峰，羊肝素积分比猪肝素小，反映出羊肝素其中的N-乙酰基修饰较少，对应地，N-磺酸根的修饰比例则更高。
达肝素钠，现行各药典规定为猪来源，是以猪精品肝素钠为起始原料，制备方法包括亚硝酸解聚、硼氢化钠还原和分子量分级三个步骤。
优选地，羊达肝素钠的制备方法，参照猪达肝素钠的制备方法，但相关工艺参数不同，具体包括如下步骤，
S11、亚硝酸解聚，是将上述预处理后的羊肝素溶解，调节溶液pH至酸性，然后加入亚硝酸钠，搅拌反应，得到解聚液；
S12、硼氢化钠还原，是将S11中解聚液的pH调至中性后，加入硼氢化钠，低温下继续搅拌反应，加酸中和多余的硼氢化钠，再加盐和醇沉并干燥得到羊达肝素钠粗品；
S13、羊达肝素钠的成品制得，是将S12中得到的羊达肝素钠粗品配制成溶液，以阴离子交换层析(或超滤)进行分子量分级，一种分级方法是采用阴离子交换树脂，是将羊达肝素钠粗品溶液低盐浓度上样，以稍高的盐浓度洗杂，最后以更高的浓度分部收集洗脱，各部分的洗脱产品再以醇沉回收并干燥，均进行分子量及分子量分布分析，经计算后合并适当的组分，纯化水复溶后，0.22μm除菌过滤并冷冻干燥，收获产品；另一种分级方法是将羊达肝素钠粗品溶液以3KDa的超滤膜进行至少两个轮次以上的超滤，超滤浓缩液经0.22μm除菌过滤后直接冷冻干燥回收，也可以醇沉回收后干燥。
优选地，所述S11中羊肝素的水溶液在5％-15％质量浓度之间，更优选在10％；所述pH调节在2-5之间，更优选pH在2.9-3.3；所述亚硝酸钠的用量与所述羊肝素重量比为1:20-50，更优选在1:35；所述解聚时间1小时-6小时之间；相对应地，羊达肝素钠制备所用的解聚强度，要比猪达肝素钠制备稍弱一些，以上优选的亚硝酸钠数量更少，pH值更高，解聚时间更短。
优选地，所述S12中硼氢化钠还原的温度不能过高，范围在0℃-40℃之间，更优地是在2℃-15℃之间。
优选地，所述S12中硼氢化钠的用量与所述S11中羊肝素重量比为1:5-15，更优选为1:10；所述还原时间不低于0.5小时。
优选地，所述S13中羊达肝素钠的阴离子交换层析分级时，上样浓度在10-100毫克每毫升，上样时盐浓度控制在300毫摩尔每升以下，上样载量5-50毫克羊达肝素每毫升树脂之内。
优选地，所述S13中的稍高盐洗杂，是不高于450毫摩尔每升的盐溶液清洗和平衡已吸附羊达肝素的树脂；
优选地，所述S13中的更高盐洗脱，是用1摩尔每升以上的高浓度盐溶液洗脱出羊达肝素钠，洗脱液按先后分部收集。
优选地，所述S13中分部收集的羊达肝素钠洗脱液，分别醇沉回收，所述醇沉，指充分搅拌下向洗脱液中缓慢加入2-4倍体积并精过滤后的甲醇，产生羊达肝素钠沉淀，沉淀物以过滤或离心方式回收并干燥。
优选地，所述S13中分部收集并回收的羊达肝素钠，经分子量及分子量分布分析，计算并按比例合并适当的组分，使分子量及分子量分布符合USP39对达肝素的要求，将合并后的组分以纯化水复溶，0.22微米除菌过滤后干燥回收，所述干燥优选冷冻干燥。
优选地，所述S13中超滤分级，是将羊达肝素钠粗品的水溶液，以超滤膜进行至少两个轮次以上的超滤，分析超滤浓缩液的分子量及分子量分布，符合USP39对达肝素的要求后，将超滤浓缩液精过滤除菌，直接冷冻干燥回收，也可以调节盐浓度醇沉回收后干燥。
优选地，所述S13中羊达肝素钠成品的重均分子量在5600-6400之间，其中分子量<3000部分的比例不高于13.0％，分子量>8000部分的比例在15.0％-25.0％之间，符合USP39等规定的达肝素钠放行指标
优选地，所述S13中羊达肝素钠成品，抗-Ⅹa活性折干后在100-180单位每毫克之间，抗-Ⅱa活性折干后在30-90单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在1.6-3.0之间，与USP39等规定的达肝素钠放行指标不同。
优选地，所述羊达肝素钠，可以再经过分子量分级的精制，筛选出抗-Ⅹa活性与抗-Ⅱa活力及比例合适的区段，使产品符合USP39等规定的达肝素钠放 行指标。
优选地，所述S13中羊达肝素钠成品的结构分析，采用核磁共振氢谱(1H-NMR)，考察糖链上链接的碳-氢关系。所述羊达肝素钠与来自猪肠粘膜的达肝素钠，主体结构一致，但也存在一定的差异，如在δ2.04ppm处的N-乙酰基的甲基峰，数量积分上羊达肝素钠要更少一些，说明羊达肝素糖链中N-乙酰基修饰相对要少。
那曲肝素钙，EP8.0规定为猪来源，是以猪精品肝素钠为起始原料，制备方法与达肝素钠类似包括亚硝酸解聚、硼氢化钠还原、阴离子树脂交换并钙盐化这制备几个步骤。
优选地，羊那曲肝素钙的制备方法，参照猪那曲肝素钙的制备方法，具体包括如下步骤，
S21、亚硝酸解聚，如羊达肝素钠的制备方法中的S11所述，但解聚的强度不同；
S22、硼氢化钠还原，如羊达肝素钠的制备方法中的S12所述，但得到的是那曲肝素粗品；
S23、羊那曲肝素钙的成品制得，是将S22中得到的羊那曲肝素粗品配制成溶液后，以阴离子交换树脂吸附，并以氯化钙溶液进行低浓度到高浓度的梯度洗脱，洗脱液醇沉回收，沉淀物再以纯化水溶解，加入双氧水氧化脱色，脱色液深层过滤，加氯化钙并调节pH至中性，无菌过滤，醇沉回收和干燥，得到羊那曲肝素钙成品。
优选地，所述S21中羊肝素的水溶液在5％-15％质量浓度之间，更优选在10％；所述pH调节在2-4之间，更优选pH在2.9-3.3；所述亚硝酸钠的用量与所述羊肝素重量比为1:10-30，更优选在1:20；所述解聚时间1小时-4小时之间。
优选地，所述S22中硼氢化钠的用量与所述S11中羊肝素重量比为1:5-15，更优选为1:10；所述还原时间不低于0.5小时。
优选地，所述S23中羊那曲肝素的阴离子交换层析分级时，上样浓度在 10-100毫克每毫升，上样载量5-50毫克羊那曲肝素每毫升树脂之内。
优选地，所述S23中的以氯化钙溶液进行低浓度到高浓度的梯度洗脱，是指以不超过400毫摩尔每升的氯化钙溶液清洗和平衡已吸附羊那曲肝素的树脂，再以1摩尔每升以上的高浓度氯化钙溶液洗脱出羊那曲肝素钙，洗脱液按先后分部收集。
优选地，所述S23中分部收集的羊那曲肝素钙洗脱液，分别醇沉回收，所述醇沉，指充分搅拌下向洗脱液中缓慢加入2-4倍体积并精过滤后的甲醇，产生羊那曲肝素钙沉淀，沉淀物以过滤或离心方式回收并干燥。
优选地，所述S23中分部收集并回收的羊那曲肝素钙，经分子量及分子量分布分析，计算并按比例合并适当的组分，使分子量及分子量分布符合EP8.0对那曲肝素钙的要求，将合并后的组分以纯化水复溶，0.22微米除菌过滤后干燥回收，所述干燥优选冷冻干燥。
优选地，所述S23中羊那曲肝素钙成品的重均分子量在3600-5000之间，其中分子量<2000部分的比例不高于15.0％，分子量2000-8000部分的比例在75.0％-95.0％之间，分子量2000-4000部分的比例在35％-55％之间，符合EP8.0等规定的那曲肝素钙放行指标。
优选地，所述S23中羊那曲肝素钙成品的结构分析，采用核磁共振氢谱(1H-NMR)，考察糖链上链接的碳-氢关系。所述羊那曲肝素钙与来自猪肠粘膜的那曲肝素钙，主体结构一致，但δ2.04ppm处的N-乙酰基的甲基峰，数量积分上更少一些，体现出羊那曲肝素钙中因羊源更少的N-乙酰基修饰，相应地就有更多的N-磺酸基修饰。
优选地，所述S24中羊那曲肝素钙成品，抗-Ⅹa活性折干后在90-125单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在2.0-3.5之间，不同于EP8.0所规定的猪来源的那曲肝素钙。
优选地，所述羊那曲肝素钙，可以经过分子量分级等精制，筛选出抗-Ⅹa活性与抗-Ⅱa活力及比例合适的区段，使产品符合EP8.0等规定的那曲肝素钙放 行指标。
亭扎肝素钠，EP8.0规定为猪来源，是以猪精品肝素钠为起始原料，制备方法以肝素酶Ⅰ解聚，然后精制回收。
优选地，羊亭扎肝素钠的制备方法，参照猪亭扎肝素钠的制备方法，具体包括如下步骤，
S31、肝素酶Ⅰ解聚，是将预处理后的羊肝素溶解，调节溶液pH至中性，然后加入肝素酶Ⅰ，搅拌反应，监控酶解反应直至反应液的232nm吸光度增加值增加至预定范围内，得到羊肝素的酶解聚液；
S32、羊亭扎肝素钠的成品制得，是将S31中得到的羊肝素的酶解聚液，90℃处理5分钟，过滤除去酶蛋白，反应液再加入氯化钠，调pH至中性，精过滤后醇沉回收和干燥，得到羊亭扎肝素钠成品。
优选地，所述S31中羊肝素的水溶液在1％-10％质量浓度之间，更优选5％；pH调节在5-9之间，更优选6-8；肝素酶Ⅰ的用量(活性)与所述羊肝素重量比为1-100：1(单位：质量/克)，更优选10：1；解聚时间1小时-24小时之间；酶解温度优选10℃-40℃之间。
优选地，所述S31中羊肝素的酶解聚液，232nm的吸光度增加值根据不同反应液浓度来控制，更优选5％羊肝素质量浓度的反应液，吸光度增加值在50-70之间。
优选地，所述S32中羊亭扎肝素钠的成品制得，将S31中得到的羊肝素的酶解聚液快速升温，使肝素酶Ⅰ变性沉淀出来再过滤除去，更优选升温至90℃处理5分钟。
优选地，所述S32中羊亭扎肝素钠的精过滤和醇沉，是向S31中除酶过滤后的溶液中加入质量浓度为5％-15％的氯化钠，更优选10％；调pH中性范围为5-9之间，pH更优选5.8-7.0；加盐和调pH后进行精过滤，优选0.22微米过滤；所述醇沉，指充分搅拌下向溶液中缓慢加入2-4倍体积并精过滤后的甲醇，产生羊亭扎肝素钠沉淀，沉淀物以过滤或离心方式回收并干燥。
优选地，所述S32中羊亭扎肝素钠成品的重均分子量在5500-7500之间，其中分子量<2000部分的比例不高于10.0％，分子量2000-8000部分的比例在60.0％-72.0％之间，分子量>8000部分的比例在22.0％-36.0％之间，符合EP8.0等规定的亭扎肝素钠放行指标。
优选地，所述S32中羊亭扎肝素钠成品的结构分析，采用核磁共振氢谱(1H-NMR)，考察糖链上链接的碳-氢关系。所述羊亭扎肝素钠与来自猪肠粘膜的亭扎肝素钠，主体结构一致。6.0ppm有特征氢峰，反映的是肝素酶Ⅰ解聚时在新生成羊亭扎肝素钠分子的非还原端特征的4,5-不饱和糖醛酸。此外，但δ2.04ppm处的N-乙酰基的甲基峰，数量积分上更少一些，体现出羊亭扎肝素钠中因羊源更少的N-乙酰基修饰，相应地就有更多的N-磺酸基修饰。
优选地，所述S32中羊亭扎肝素钠成品，抗-Ⅹa活性折干后在60-120单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在1.5-3.0之间，不同于EP8.0所规定的猪来源的亭扎肝素钠。
优选地，所述羊亭扎肝素钠，可以经过分级精制，收获出抗-Ⅹa活性与抗-Ⅱa活力及比例合适的区段，使产品符合EP8.0等规定的亭扎肝素钠放行指标。
帕肝素钠，EP8.0规定为猪肠粘膜或牛肠粘膜来源，通常是以猪精品肝素钠为起始原料，制备方法包括铜过氧化物解聚、螯合和阳离子树脂交换去铜、然后精制醇沉回收等步骤。
优选地，羊帕肝素钠的制备方法，参照猪帕肝素钠的制备方法，具体包括如下步骤，S41、铜过氧化物解聚，是将预处理后的羊肝素溶解，调节溶液pH至中性，然后分别加入乙酸铜溶液和过氧化氢溶液进行解聚，期间控制pH和温度；
S42、羊帕肝素钠粗品的回收和精制，是将S41中得到的羊肝素解聚液，调节pH至9-10，加入乙二胺四乙酸二钠，继续搅拌反应，再调节pH至中性，加盐并过滤后进行醇沉回收得到羊帕肝素沉淀物，沉淀物再以纯化水溶解，以强阳离子交换树脂处理，收集未结合的羊帕肝素流穿液，加入氯化钠并醇沉回收，得到羊帕肝素钠粗品。
S43、羊帕肝素钠的成品制得，是将S42中得到的羊帕肝素钠粗品，以水复溶，加入双氧水脱色，脱色液调节pH至中性，加入氯化钠，除菌过滤后醇沉回收，干燥得到羊帕肝素钠成品。
优选地，所述S41中铜过氧化物解聚，羊肝素质量浓度在1％-15％之间，更优选5％-10％；所述乙酸铜、过氧化氢与羊肝素的质量比例在0.1-1：1-3：1，更优选0.4：2：1；解聚温度控制在30℃-70℃之间，更优选50℃-55℃；解聚时pH控制在6-9之间，更优选7-8；解聚时间优选2-48小时，更优选18小时。
优选地，所述S42中乙二胺四乙酸二钠与羊肝素的质量比例在0.5-5：1，更优选1：1；所述加盐指加入终质量浓度为5％-15％的氯化钠，更优选质量浓度为10％。
优选地，所述S42中羊帕肝素沉淀物以水复溶后，质量浓度在1％-20％之间，更优选为10％；所述强阳离子交换树脂为食品级树脂。
优选地，所述S42中未结合流穿液中羊帕肝素的加盐醇沉回收，是向溶液中加入氯化钠至终质量浓度为5％-15％，更优选10％，所述醇沉指充分搅拌下向溶液中缓慢加入2-4倍体积并精过滤后的甲醇，产生羊帕肝素钠沉淀，以过滤或离心方式回收羊帕肝素钠粗品。
优选地，所述S43中羊帕肝素钠粗品以水溶解至质量浓度在1％-15％之间，优选10％；双氧水脱色温度在15℃-40℃之间，更优选25℃；双氧水在溶液中的终体积浓度在0.1％-5％之间，更优选1％-2％；双氧水脱色时间10分钟以上，直至反应液颜色浅至Y5以下。
优选地，所述S43中羊帕肝素钠溶液在脱色结束至醇沉淀前，依次用稀盐酸调pH至中性，精过滤，滤液加氯化钠至8-12％浓度，调pH至5.0-7.0，再精过滤。
优选地，所述S43中羊帕肝素钠钠成品制得，所述醇沉淀，指充分搅拌下向反应液中缓慢加入2-4倍反应溶液体积并精过滤后的甲醇，产生羊帕肝素钠钠沉淀，沉淀物以过滤或离心方式回收，并干燥。
优选地，所述S43中羊帕肝素钠成品的重均分子量在4000-6000之间。
优选地，所述S43中羊帕肝素钠成品，抗-Ⅹa活性折干后在75-110单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在1.5-3.0之间，符合EP8.0所规定的帕肝素钠标准。
优选地，所述S43中羊帕肝素钠钠成品的结构分析，采用核磁共振氢谱(1H-NMR)，考察糖链上链接的碳-氢关系。所述羊帕肝素钠与来自猪肠粘膜的帕肝素钠，主体结构一致，但δ2.04ppm处的N-乙酰基的甲基峰，数量积分上更少一些，体现出羊帕肝素钠中因羊源更少的N-乙酰基修饰，相应地就有更多的N-磺酸基修饰。
贝米肝素钠，临床上药物均是猪来源的，是以猪精品肝素钠为起始原料，制备方法包括制备羊肝素季铵盐和有机碱解聚后精制回收等步骤。
优选地，羊贝米肝素钠的制备方法，参照猪贝米肝素钠的制备方法，具体包括如下步骤，
S51、羊肝素季铵盐的制备，是将羊肝素钠溶解配制成水溶液，并与苯扎氯铵水溶液进行混合，分离、洗涤和干燥，制得羊肝素季铵盐；
S52、羊贝米肝素钠粗品的制备，是将S51中干燥得到的羊肝素季铵盐按比例溶于二氯甲烷或其他有机溶剂，加入苄基三甲基氢氧化铵(Triton-B)，搅拌使羊肝素解聚，解聚反应结束后，滴加醋酸钠甲醇溶液，制得羊贝米肝素钠粗品沉淀；
S53、羊贝米肝素钠成品制得，是将S52中的羊贝米肝素钠粗品进行过滤、甲醇洗涤和再进行多次的复溶加盐醇沉、产物精制、干燥，得到羊贝米肝素钠成品。
优选地，所述S51羊肝素季铵盐的制备中，羊肝素水溶液在5％-15％质量浓度之间，苯扎氯铵水溶液在10％-30％质量浓度之间，其中苯扎氯铵固体与所述羊肝素钠固体的重量比为2-5:1。
优选地，所述S52中解聚反应时，羊肝素季铵盐、二氯甲烷、Triton-B的质 量比为1:3-10:0.2-0.4，更优选比例为1:5:0.25。
优选地，所述S52中溶剂可以是二氯甲烷，也可以是二甲基甲酰胺或其他有机溶剂。
优选地，所述S52中有机碱解聚时，反应温度在20℃-45℃之间，更优选30℃；反应时间在8小时-40小时，更优选16小时。
优选地，所述S52中解聚反应结束时，滴加醋酸钠甲醇溶液使羊贝米肝素钠析出，所述醋酸钠的重量是羊肝素季铵盐的0.8倍，所述醋酸钠甲醇溶液的浓度为10％。
优选地，所述S53中羊贝米肝素钠粗品沉淀经分离后，以甲醇洗涤一次或数次；洗涤后的沉淀物添加8％-12％的氯化钠水溶液进行复溶，所述氯化钠水溶液与所述羊肝素季铵盐重量比为0.5-2:1，复溶的溶液再以2-5倍体积的甲醇进行醇沉结晶；所述复溶醇沉可以再重复多次，直至复溶后的羊贝米肝素钠溶液澄清无混浊。
优选地，所述S53中羊贝米肝素钠成品的重均分子量在3000-4200之间。
优选地，所述S54中羊贝米肝素钠成品，抗-Ⅹa活性折干后在75-110单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在1.5-3.0之间，与目前的医用猪来源的贝米肝素钠在抗-Ⅹa/抗-Ⅱa比例上不同。
优选地，所述羊贝米肝素钠，可以经过分级精制，收获出抗-Ⅹa活性与抗-Ⅱa活力及比例合适的区段，使产品符合目前医用的贝米肝素钠对活性的要求。
优选地，所述S53中羊贝米肝素钠成品的结构分析，采用核磁共振氢谱(1H-NMR)，考察糖链上链接的碳-氢关系。所述羊贝米肝素钠与来自猪肠粘膜的贝米肝素钠，主体结构一致，在
但δ2.04ppm处的N-乙酰基的甲基峰，数量积分上更少一些，体现出羊帕肝素钠中因羊源更少的N-乙酰基修饰，相应地就有更多的N-磺酸基修饰。
优选地，所述S53中羊贝米肝素钠成品的结构分析，采用核磁共振氢谱(1H-NMR)，考察糖链上链接的碳-氢关系。所述羊贝米肝素钠与来自猪肠粘膜 的贝米肝素钠，主体结构一致。6.0ppm有特征氢峰，反映的是有机碱的β-消除反应解聚时，在新生成羊贝米肝素钠分子的非还原端形成特征的4,5-不饱和糖醛酸。此外，δ2.04ppm处的N-乙酰基的甲基峰，数量积分上更少一些，体现出羊贝米肝素钠中因羊源更少的N-乙酰基修饰，相应地就有更多的N-磺酸基修饰。
本申请中涉及的醇沉以甲醇作为有机溶剂，除无特殊说明外，还可以用乙醇、异丙醇或丙酮代替甲醇。
本申请中涉及的干燥均可以采用自然烘干、真空烘干或冷冻干燥以及其他干燥方式，所述干燥过程中，可进行翻料、研磨打粉等，以提高干燥效果。
几种羊低分子肝素，二糖组成分析遵照USP32附录<207>“依诺肝素钠的1,6-酐衍生物检查”进行酶解和SAX-HPLC分析，羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠的主要二糖，ΔUA2S-GlcNS6S(ΔⅠS)的含量为66％-74％，次要二糖ΔUA-GlcNS6S(ΔⅡS)和ΔUA2S-GlcNS因解聚工艺的不同，含量有所差异。ΔⅠS的含量呈现出羊源的特征，在猪源低分子肝素中ΔⅠS的含量在58％-66％之间。
优选地，所述几种羊低分子肝素的抗凝作用，体外试验以人血考察。将人血液分离血浆后，按自动凝血仪和试剂盒方法，考察各低分子肝素等对血凝常规的影响，包括但不限于APTT、TT和PT等。
优选地，所述几种羊低分子肝素，包括羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，在体外抗凝试验，均表现出极强的抗凝效果。
优选地，所述羊低分子肝素，包括羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，在防治与抗凝和抗栓有关疾病中的应用，以及开发为清真抗凝抗栓药物。
本发明突出效果为：提供了几种羊低分子肝素——羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，并采用实用、稳定的方法制得。除了由种源特征带来的分子结构(二糖组成)外，几种羊低分子肝素均与猪来源 的低分子肝素理化性质相近，分子量分布等完全符合EP8.0或原研的放行指标。几种羊低分子肝素均有强力的抗凝活性，它们的抗-Ⅹa活性和抗-Ⅱa活性与猪源的类似，在人血液的体外试验中，具有类似的延长APTT和TT等抗凝生物学活性。本发明填补了羊源肝素在低分子肝素制备上的空白，可开发为清真药物。原料羊肝素钠简便易得，质量可控，可极大丰富市场中低分子肝素和清真药物的来源和产量，还可以促进羊养殖和屠宰废料(肠粘膜)的有效利用，经济潜力巨大。
以下便结合实施例附图，对本发明的具体实施方式作进一步的详述，以使本发明技术方案更易于理解、掌握。
附图说明
图1为几种羊低分子肝素样品的分子量分布比较示意图，其中(1)为羊达肝素钠，(2)为羊那曲肝素钙，(3)为羊亭扎肝素钠，(4)为羊帕肝素钠，(5)为羊贝米肝素钠，(6)为五种低分子肝素叠加比较；
图2为实施例8中几种羊低分子肝素样品的二糖谱比较示意图；
图3为实施例9中几种羊低分子肝素1H-NMR分析对比示意图，其中(7)-(11)依次为羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠。
具体实施方式
本发明实施例描述了几种羊低分子肝素——羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，下面以具体实验案例为例来说明具体实施方式，应当理解，此处所描述的具体实施例仅仅用于解释本发明，并不用于限定本发明。
实施例1
原料羊肝素钠的制备与预处理
粗品羊肝素钠(厂家：山东绅联生物科技有限公司，批号：20150326)，样品送检RT-PCR以检测其中猪与羊DNA含量，结果猪DNA含量小于0.1皮克每微升，羊DNA含量大于1×105皮克每微升，且根据原厂家初始肠粘膜的来源， 确定为粗品羊肝素钠，且基本不含猪肝素成分。此外，该样品送检全绵羊血浆法抗凝活性为67.0单位每毫克。
粗品羊肝素钠的提纯采取本行业所熟知的工艺，即以水溶解后盐解碱解，再加蛋白酶水解，水解液调pH后以阴离子交换树脂吸附并洗脱，以醇沉沉淀得到用于制备羊低分子肝素的原料羊肝素。收获的该原料羊肝素的全绵羊血浆法抗凝活性为134.2单位每毫克。
准确称取上述原料羊肝素5.0千克，倒入100升釜内，加入45升含2％氯化钠和1％碳酸钠的混合溶液，50±5℃搅拌1小时以上，取小样检查以确保溶解完全。加入终浓度2％的双氧水，搅拌均匀后静置，50±5℃保温5小时，然后自然冷却至室温，并保持过夜。将脱色液过滤至醇沉罐，加入95％酒精至乙醇的终浓度在42％，静置4小时。弃上清，肝素沉淀以2％氯化钠溶液复溶，加入95％酒精至乙醇的终浓度在40％，静置6小时。弃上清，肝素沉淀以2％氯化钠溶液复溶，加入95％酒精至乙醇的终浓度在38％，静置过夜。弃上清，肝素沉淀以95％酒精脱水。脱水后的羊肝素再以鼓风干燥脱去残余水份或溶剂。经预处理过的羊肝素，称重3.1千克，经全绵羊血浆法抗凝活性折干后为176.3单位每毫克，按USP37规定的精品肝素钠抗-Ⅹa和抗-Ⅱa活性测定方法，抗-Ⅹa活性为185.1单位每毫克，抗-Ⅱa活性为181.5单位每毫克，抗-Ⅹa/抗-Ⅱa比例为1.02。
实施例2
羊达肝素钠的制备
称取实施例一中预处理过的羊肝素50.0克，加入500毫升纯化水，搅拌使肝素溶清。水浴控室温，继续搅拌保温30分钟以上。以盐酸调肝素溶液的pH至3.1，加入1.43克亚硝酸钠，继续保温搅拌。然后调pH至中性，加入4.0克硼氢化钠，继续搅拌。再以稀盐酸调节pH至中性，加入氯化钠51.5克，室温下继续搅拌10分钟以上。缓慢加入1250毫升甲醇，搅拌15分钟使反应得到的达肝素粗品沉淀。转移反应液至离心瓶中，离心收集达肝素粗品沉淀，沉淀真空干燥后称重，得到43.5克达肝素钠粗品。
准确称取达肝素钠粗品20.0克，以1％氯化钠溶液配制成20毫克每毫升的达肝素溶液，共1000毫升，上样至已处理好的1000毫升柱体积的DEAE-FF阴 离子交换柱。上样结束后，以1％氯化钠溶液平衡柱子6个柱体积，再以6个柱体积的400毫摩尔每升氯化钠溶液洗杂。然后以3个柱体积的1.5摩尔每升氯化钠溶液洗脱，每500毫升收集一份。收集的各洗脱液均加入1250毫升甲醇，搅拌5分钟后静置，最后离心收集沉淀，沉淀甲醇脱水，再烘干。经分子量分布分析及计算后，合并上述所有洗脱液的沉淀，以200毫升纯化水溶解和无菌过滤，冷冻干燥得到羊达肝素钠产品11.2克。
实施例3
羊那曲肝素钙的制备
称取实施例一中预处理过的羊肝素50.0克，加入500毫升纯化水，搅拌使肝素溶清。水浴控室温，继续搅拌保温30分钟以上。以盐酸调肝素溶液的pH至3.0，加入2.5克亚硝酸钠，继续保温搅拌。然后调pH至中性，加入4.5克硼氢化钠，继续搅拌。再以稀盐酸调节pH至中性，加入52.0克氯化钠，室温下继续搅拌10分钟以上。缓慢加入1250毫升甲醇，搅拌15分钟使反应得到的那曲肝素粗品沉淀。转移反应液至离心瓶中，离心收集那曲肝素粗品沉淀，沉淀真空干燥后称重，得到39.5克那曲肝素粗品。
准确称取那曲肝素粗品20.0克，以1％氯化钠溶液配制成20毫克每毫升的那曲肝素溶液，共1000毫升，上样至已处理好的1000毫升柱体积的DEAE-FF阴离子交换柱。上样结束后，以1％氯化钙溶液平衡柱子6个柱体积，再以6个柱体积的350毫摩尔每升氯化钙溶液洗杂。然后以3个柱体积的1.2摩尔每升氯化钙溶液洗脱，每500毫升收集一份。收集的各洗脱液均加入1250毫升甲醇，搅拌5分钟后静置，最后离心收集沉淀。沉淀再以200毫升纯化水溶解，加入双氧水氧化脱色2小时，加入20克氯化钙，搅拌溶解并调pH中性后除菌过滤，滤液以500毫升甲醇醇沉，沉淀物经真空烘干后，得到羊那曲肝素钙产品12.6克。
实施例4
肝素酶解聚制备羊亭扎肝素钠
称取实施例一中预处理过的羊肝素50.0克，加入500毫升纯化水，搅拌使肝素溶清。水浴控室温，调溶液的pH至中性，加入肝素酶溶液10毫升，继续保温搅拌，至溶液的A232吸光值增加值在60时，将反应液快速升温至95℃，保持5分钟，再快速水冷至室温。精过滤出去析出的酶蛋白沉淀，然后滤液加入50克氯化钠，调pH至中性，室温下继续搅拌10分钟以上，除菌过滤。缓慢加入1200毫升甲醇，搅拌15分钟使反应得到的羊亭扎肝素粗品沉淀。转移反应液至离心瓶中，离心收集达羊亭扎肝素沉淀，沉淀真空干燥后称重，得到17.8克羊亭扎肝素钠。
实施例5
铜过氧化物解聚制备羊帕肝素钠
称取实施例一中预处理过的羊肝素100.0克，加入2000毫升纯化水，搅拌使肝素溶清，保温50℃搅拌。加入40.0克乙酸钠和40.0克乙酸铜，再加入200毫升双氧水，控制反应温度在50℃-55℃，控制pH在6-9之间，搅拌18小时。反应结束后调溶液的pH至9.5，加入100克乙二胺四乙酸二钠，继续搅拌30分钟。然后调pH至中性，加入250克氯化钠，溶解后以6000毫升乙醇以沉淀，离心收集沉淀。沉淀物以纯化水溶解，以强阳离子交换树脂处理，收集未结合的肝素流穿液，再加入氯化钠至10％浓度，调pH中性，以2.5倍体积乙醇沉淀，得到羊帕肝素钠粗品。将上述粗品再以1％氯化钠溶液溶解，加入终浓度为2％的过氧化氢，室温脱色1小时。调pH中性，无菌过滤，以3倍体积乙醇沉淀，沉淀经纯水复溶再冻干，得到羊帕肝素钠产品54.2克。
实施例6
羊贝米肝素钠的制备
称取上述实施例一的羊肝素钠100克，溶解于700毫升水中；另将250克苯扎氯铵，溶解于1000毫升水中，配制成澄清的苯扎氯铵水溶液；于充分搅拌下，将苯扎氯铵水溶液滴加至羊肝素水溶液中，30分钟内滴加完毕，继续搅拌2小时。用高速离心机6000转/分钟离心5分钟，沉淀用3000毫升水重悬，继续充分搅拌5分钟，再6000转/分钟离心5分钟。重复一次。沉淀的羊肝素季铵盐湿 品，转移冷冻干燥箱干燥48小时，得到羊肝素季铵盐干品265克，干燥失重经测定为0.6％。
取上述干燥后的羊肝素季铵盐200克于2升反应瓶中，加入800克二氯甲烷搅拌溶解，升温至30℃，加入50毫升Triton-B，继续搅拌反应解聚16小时。称量160克醋酸钠，溶解于1600毫升的甲醇中，在上述解聚反应结束后滴加至反应液中，此时产生不溶的羊贝米肝素钠粗品沉淀。
离心收集羊贝米肝素钠粗品沉淀，以1000毫升纯化水复溶，盐酸调pH至中性，加入100克氯化钠，加入2500毫升甲醇沉淀出羊贝米肝素钠。重复上述复溶醇沉两次，最终沉淀干燥后得到羊贝米肝素钠纯品53.2克。
实施例7
羊低分子肝素重均分子量及分子量分布分析
来自于实施例二至实施例六的几种羊低分子肝素，分子量及分子量分布分析参照EP8.0方法进行。
表1：羊达肝素钠的重均分子量及分子量分布
Figure PCTCN2016096030-appb-000002
表2：羊那曲肝素钙的重均分子量及分子量分布
Figure PCTCN2016096030-appb-000003
表3：羊亭扎肝素钠的重均分子量及分子量分布
Figure PCTCN2016096030-appb-000004
表4：羊帕肝素钠的重均分子量及分子量分布
Figure PCTCN2016096030-appb-000005
表5：羊贝米肝素钠的重均分子量及分子量分布
Figure PCTCN2016096030-appb-000006
结果可以看出，实施例二至实施例六制备的羊低分子肝素，其均重分子量和分子量分布(若有要求)都符合药典EP8.0或原研厂家的技术指标，即羊低分子肝素与猪肠粘膜来源的低分子肝素在分子量及其分布上是相近的。
实施例8
羊低分子肝素二糖组成分析
来自于实施例二至实施例六几种羊低分子肝素的二糖组成分析，遵照USP32附录<207>“依诺肝素钠的1,6-酐衍生物检查”进行酶解，但不进行还原，再进行SAX-HPLC分析。样品和标准品的二糖组成分析结果见图2。
从图2可以看出，各实施例制备的羊低分子肝素呈现出典型的羊源肝素特征，主要二糖ΔⅠS在样品羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠中的含量，分别高达70.2％、69.2％、68.7％、70.6％和71.2％，比通常猪源低分子肝素的含量要高。
实施例9
羊低分子肝素的核磁氢谱(1H-NMR)分析
几种羊低分子肝素的核磁氢谱分析，设备用苏州大学分析测试中心的400MHz核磁共振谱仪，以3-三甲基硅基丙酸钠-d4(TSP)定零点。
待测样品溶液配制：几种羊低分子肝素(实施例二至实施例六)，各准确各 称取20毫克左右，按重以氘水(D2O)溶解成20毫克每毫升左右的浓度，滴加1-2滴TSP，震荡混匀后0.22微米过滤送检，结果如图3所示，其中，δ3.4ppm为甲醇残留的甲基氢峰，δ4.7ppm为水氢峰。
检测结果：几种羊低分子肝素的氢谱结果如附图3所示，因不同的工艺和处理，它们呈现各低分子肝素所独有的图谱，其中，羊达肝素钠和羊那曲肝素钙都是由亚硝酸钠引起的解聚，且都进行了硼氢化钠的还原，因此在化学结构上两者是一致的；羊亭扎肝素钠是因肝素酶的酶解而解聚，在新形成的低分子肝素非还原端是特征的4,5-不饱和糖醛酸，6.0ppm即是此特征氢的反映，同样地，羊贝米肝素钠是有机碱的β-消除反应来解聚，也在非还原端呈现4,5-不饱和糖醛酸结构，与羊亭扎肝素钠类似；此外，羊帕肝素钠是铜过氧化物的解聚，一种因氧化引起的解聚，图谱上体现出结构与上述几种均有差异。共性来看，羊低分子肝素在2.04ppm处反映出的N-乙酰基的甲基氢，积分相似，比猪来源的低分子肝素如依诺肝素钠要少，反映出羊源低分子肝素中，N-乙酰基修饰较少，相反的，N-上的磺酸基修饰则更多，这也是与二糖分析的结果是一致的。
实施例11
羊低分子肝素的抗-Ⅹa、抗-Ⅱa活力和全绵羊血浆法活力对比分析
抗-Ⅹa和抗-Ⅱa的活性测定遵照EP8.0达肝素钠中的方法进行，全绵羊血浆法遵照本领域所熟知的传统方法进行，来源于实施例二至实施例六的各羊低分子肝素活性对比如下表6。
表6：羊低分子肝素样品的抗凝活力对比
Figure PCTCN2016096030-appb-000007
结果显示：羊低分子肝素均具有强力的抗凝活性。全绵羊血浆法测定的活性 在40-55单位每毫克不等；抗-Ⅹa活力上，羊达肝素钠样品最高，达143.4单位每毫克，羊帕肝素钠样品最低，也在89.7单位每毫克；抗-Ⅱa活力上，也是羊达肝素钠样品最高，在62.4单位每毫克，同样羊帕肝素钠样品最低，在34.9单位每毫克；而抗-Ⅹa/抗-Ⅱa比例上，几种羊低分子肝素样品在1.7-2.6不等。
实施例12
羊低分子肝素的人血体外抗凝试验
实验方法：每次采用5人份外周静脉血3mL，用3.8％枸橼酸钠抗凝剂1:9抗凝，3000转/分离心5分钟，分离出贫血小板血浆(PPP)。按试剂盒方法，上机(全自动血凝仪，Stago Compact)检测。实验组别如下：羊达肝素钠样品组(实施例二所述)、羊那曲肝素钙样品组(实施例三所述)、羊亭扎肝素钠样品组(实施例四所述)、羊帕肝素钠样品组(实施例五所述)、羊贝米肝素钠样品组(实施例六所述)和依诺肝素钠标准品组(为临床市售药品，克赛，批号：24459)，所有组别终样品浓度均为～3μg/mL，实验设生理盐水为空白对照。
结果与分析：
1)APTT、PT和TT
实验结果如下表格所示：
表7 体外对APTT、PT和TT的影响
组别 APTT PT TT
羊达肝素钠样品 114.1±9.5s 13.2±0.6s 157.3±47.4s
羊那曲肝素钙样品 105.3±12.1s 13.9±0.4s 145.9±43.6s
羊亭扎肝素钠样品 102.5±9.8s 13.4±0.5s 134.4±52.3s
羊帕肝素钠样品 97.3±10.3s 12.8±0.5s 124.6±49.1s
羊贝米肝素钠样品 95.8±9.8s 13.2±0.5s 132.5±44.3s
依诺肝素钠标准品 104.6±10.2s 13.8±0.3s 140.4±54.7s
空白对照 38.1±1.4s 12.7±0.6s 16.6±0.7s
由表7可知，在体外所有样品组别均可以显著延长APTT和TT，但对PT的影响较小；与依诺肝素钠标准品相比，羊低分子肝素对APTT、TT和PT的影响也是比较接近的。
2)纤维蛋白原和复钙时间：
实验结果如下表格所示：
表8 体外对纤维蛋白原和复钙时间的影响
组别 纤维蛋白原 复钙时间
羊达肝素钠样品 2.91±0.51g/L 31.00±0.00s*
羊那曲肝素钙样品 2.66±0.43g/L 31.00±0.00s*
羊亭扎肝素钠样品 2.64±0.37g/L 31.00±0.00s*
羊帕肝素钠样品 2.76±0.53g/L 31.00±0.00s*
羊贝米肝素钠样品 2.71±0.72g/L 31.00±0.00s*
依诺肝素钠标准品 2.89±0.44g/L 31.00±0.00s*
空白对照 2.57±0.25g/L 9.95±0.40s
*：均已超出检测范围
由表8可知，与依诺肝素钠标准品相比，几种羊低分子肝素样品对纤维蛋白原并没有显著的影响，复钙时间均大大延长，超出检测范围。
所有以上数据揭示，羊低分子肝素在体外有着良好的抗凝效果，且与依诺肝素钠标准品作用相近。
综合以上所有测试的结果，羊低分子肝素呈现典型的羊源特征，化学结构(二糖组成和氢谱)与猪源的低分子肝素有一定的差异。经精制制备的羊低分子肝素，分子量分布等指标均可以符合现行药典对猪低分子肝素的要求，生物学抗凝活性相近，可用于抗凝等领域的应用。
本发明尚有多种实施方式，凡采用等同变换或者等效变换而形成的所有技术方案，均落在本发明的保护范围之内。

Claims (32)

  1. 羊来源的低分子肝素，包括羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，其特征在于，以上低分子肝素由羊肝素制备得到，以上低分子肝素主要二糖ΔUA2S-GlcNS6S(ΔⅠS)的含量为66％-74％。
  2. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，所述羊肝素的抗凝活性全绵羊血浆法折干后不少于150单位每毫克，且3.3％质量浓度的水溶液澄清且色度不深于5号标准色。
  3. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊达肝素钠抗-Ⅹa活性折干后在100-180单位每毫克之间，抗-Ⅱa活性折干后在30-90单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在1.6-3.0之间。
  4. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊达肝素钠重均分子量在5600-6400之间，其中分子量<3000部分的比例不高于13.0％，分子量>8000部分的比例在15.0％-25.0％之间。
  5. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊达肝素钠羊达肝素钠中羊达肝素糖链中N-乙酰基修饰要少于猪肠粘膜达肝素钠。
  6. 根据权利要求3-5任一项所述的羊来源的低分子肝素，其特征在于，羊达肝素钠制备方法包括如下步骤：
    S11、亚硝酸解聚，是将权利要求2中的羊肝素用水溶解，调节溶液pH至酸性，然后加入亚硝酸钠，搅拌反应，得到解聚液；
    S12、硼氢化钠还原，是将S11中解聚液的pH调至中性后，加入硼氢化钠，低温下继续搅拌反应，加酸中和多余的硼氢化钠，再加盐和醇沉并干燥得到羊达肝素钠粗品；
    S13、羊达肝素钠的成品制得，是将S12中得到的羊达肝素钠粗品配制成溶液，以阴离子交换层析或超滤进行分子量分级，再精制、回收和干燥，得到羊达肝素钠成品。
  7. 根据权利要求6所述的羊来源的低分子肝素，其特征在于，羊达肝素钠制备方法S11中羊肝素用水溶解后的质量比浓度为5-15％，优选为10％；调节溶液pH在2-5之间，优选为2.9-3.3之间；亚硝酸钠的用量与所述羊肝素重量比为1:20-50，优选为1:35；搅拌反应的反应时间1-6小时。
  8. 根据权利要求6所述的羊来源的低分子肝素，其特征在于，羊达肝素钠制备方法S12中低温下继续搅拌反应的反应温度为0℃-40℃，优选为2℃-15℃；硼氢化钠的用量与S11中羊肝素重量比为1:5-15，优选为1:10；硼氢化钠还原的还原时间不低于0.5小时。
  9. 根据权利要求6所述的羊来源的低分子肝素，其特征在于，羊达肝素钠制备方法S13中阴离子交换层析分级时，上样浓度在10-100毫克每毫升，上样时盐浓度控制在300毫摩尔每升以下，上样载量5-50毫克羊达肝素每毫升树脂之内。
  10. 根据权利要求6所述的羊来源的低分子肝素，其特征在于，羊达肝素钠制备方法S13中分子量分级方法为采用阴离子交换树脂，是将羊达肝素钠粗品溶液低盐浓度上样，以稍高的盐浓度洗杂，最后以更高的浓度分部收集洗脱，各部分的洗脱产品再以醇沉回收并干燥，均进行分子量及分子量分布分析，经计算后合并适当的组分，纯化水复溶后，0.22μm除菌过滤并冷冻干燥，收获产品；另一种分级方法是将羊达肝素钠粗品溶液以3KDa的超滤膜进行至少两个轮次以上的超滤，超滤浓缩液经0.22μm除菌过滤后直接冷冻干燥回收，也可以醇沉回收后干燥。
  11. 根据权利要求6所述的羊来源的低分子肝素，其特征在于，羊达肝素钠制备方法S13中采用阴离子交换树脂层析分级时，低盐浓度上样的浓度为300毫摩尔每升以下；以稍高的盐浓度洗杂是不高于450毫摩尔每升的盐溶液清洗和平衡已吸附羊达肝素的树脂；以更高的浓度分部收集洗脱是用1摩尔每升以上的高浓度盐溶液洗脱出羊达肝素钠，洗脱液按先后分部收集。
  12. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊那曲肝素钙重均分子量在3600-5000之间，其中分子量<2000部分的比例不高于15.0％，分子量2000-8000部分的比例在75.0％-95.0％之间，分子量2000-4000部分的比例在35％-55％之间。
  13. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊那曲肝素钙抗-Ⅹa活性折干后在90-125单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在2.0-3.5之间。
  14. 根据权利要求12或13所述的羊来源的低分子肝素，其特征在于，羊那曲肝素钙的制备方法包括如下步骤：
    S21、亚硝酸解聚，如权利要求6中的S11所述；
    S22、硼氢化钠还原，如权利要求6中的S12所述，但得到的是那曲肝素粗品；
    S23、羊那曲肝素钙的成品制得，是将S22中得到的羊那曲肝素粗品配制成溶液后，以阴离子交换树脂吸附，并以氯化钙溶液进行低浓度到高浓度的梯度洗脱，洗脱液醇沉回收，沉淀物再以纯化水溶解，加入双氧水氧化脱色，脱色液深层过滤，加氯化钙并调节pH至中性，无菌过滤，醇沉回收和干燥，得到羊那曲肝素钙成品。
  15. 根据权利要求14所述的羊来源的低分子肝素，其特征在于，羊那曲肝素钙的制备方法 S21中羊肝素的水溶液在5％-15％质量浓度之间，更优选在10％；所述pH调节在2-4之间，更优选pH在2.9-3.3；所述亚硝酸钠的用量与所述羊肝素重量比为1:10-30，更优选在1:20；所述解聚时间1小时-4小时之间。
  16. 根据权利要求14所述的羊来源的低分子肝素，其特征在于，羊那曲肝素钙的制备方法S23中羊那曲肝素的阴离子交换树脂吸附时，上样浓度在10-100毫克每毫升，上样载量5-50毫克羊那曲肝素每毫升树脂之内；以氯化钙溶液进行低浓度到高浓度的梯度洗脱，是指以不超过400毫摩尔每升的氯化钙溶液清洗和平衡已吸附羊那曲肝素的树脂，再以1摩尔每升以上的高浓度氯化钙溶液洗脱出羊那曲肝素钙，洗脱液按先后分部收集。
  17. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊亭扎肝素钠抗-Ⅹa活性折干后在60-120单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在1.5-3.0之间。
  18. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊亭扎肝素钠重均分子量在5500-7500之间，其中分子量<2000部分的比例不高于10.0％，分子量2000-8000部分的比例在60.0％-72.0％之间，分子量>8000部分的比例在22.0％-36.0％之间。
  19. 根据权利要求17或18所述的羊来源的低分子肝素，其特征在于，羊亭扎肝素钠制备方法包括如下步骤：
    S31、肝素酶Ⅰ解聚，是将权利要求2所述的羊肝素溶于水中得到水溶液，调节溶液pH至中性，然后加入肝素酶Ⅰ，搅拌反应，监控酶解反应直至反应液的232nm吸光度增加值增加至预定范围内，得到羊肝素的酶解聚液；
    S32、羊亭扎肝素钠的成品制得，是将S31中得到的羊肝素的酶解聚液，90℃处理5分钟，过滤除去酶蛋白，反应液再加入氯化钠，调pH至中性，精过滤后醇沉回收和干燥，得到羊亭扎肝素钠成品。
  20. 根据权利要求19所述的羊来源的低分子肝素，其特征在于，羊亭扎肝素钠制备方法S31中羊肝素的水溶液在质量比浓度在1％-10％之间，优选为5％；pH调节在5-9之间，优选为6-8；肝素酶Ⅰ的用量与所述羊肝素重量比为1-100：1，优选为10：1；解聚反应时间1小时-24小时之间；酶解温度优选10℃-40℃之间；羊肝素的酶解聚液，232nm的吸光度增加值根据不同反应液浓度来控制，更优选5％羊肝素质量浓度的反应液，吸光度增加值在50-70之间。
  21. 根据权利要求19所述的羊来源的低分子肝素，其特征在于，羊亭扎肝素钠制备方法S32中羊亭扎肝素钠的成品制得，将S31中得到的羊肝素的酶解聚液快速升温，使肝素酶Ⅰ变性 沉淀出来再过滤除去，更优选升温至90℃处理5分钟；羊亭扎肝素钠的精过滤和醇沉，是向S31中除酶过滤后的溶液中加入质量浓度为5％-15％的氯化钠，更优选10％；调pH中性范围为5-9之间，pH更优选5.8-7.0；加盐和调pH后进行精过滤，优选0.22微米过滤；所述醇沉，指充分搅拌下向溶液中缓慢加入2-4倍体积并精过滤后的甲醇，产生羊亭扎肝素钠沉淀，沉淀物以过滤或离心方式回收并干燥。
  22. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊帕肝素钠重均分子量在4000-6000之间；抗-Ⅹa活性折干后在75-110单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在1.5-3.0之间。
  23. 根据权利要求22所述的羊来源的低分子肝素，其特征在于，羊帕肝素钠的制备方法包括如下步骤：
    S41、铜过氧化物解聚，是将预处理后的羊肝素溶解，调节溶液pH至中性，然后分别加入乙酸铜溶液和过氧化氢溶液进行解聚，期间控制pH和温度；
    S42、羊帕肝素钠粗品的回收和精制，是将S41中得到的羊肝素解聚液，调节pH至9-10，加入乙二胺四乙酸二钠，继续搅拌反应，再调节pH至中性，加盐并过滤后进行醇沉回收得到羊帕肝素沉淀物，沉淀物再以纯化水溶解，以强阳离子交换树脂处理，收集未结合的羊帕肝素流穿液，加入氯化钠并醇沉回收，得到羊帕肝素钠粗品；
    S43、羊帕肝素钠的成品制得，是将S42中得到的羊帕肝素钠粗品，以氯化钠水溶液复溶，加入双氧水脱色，脱色液调节pH至中性，加入氯化钠，除菌过滤后醇沉回收，干燥得到羊帕肝素钠成品。
  24. 根据权利要求23所述的羊来源的低分子肝素，其特征在于，羊帕肝素钠的制备方法S41中铜过氧化物解聚，羊肝素质量浓度在1％-15％之间，更优选5％-10％；所述乙酸铜、过氧化氢与羊肝素的质量比例在0.1-1：1-3：1，更优选0.4：2：1；解聚温度控制在30℃-70℃之间，更优选50℃-55℃；解聚时pH控制在6-9之间，更优选7-8；解聚时间优选2-48小时，更优选18小时。
  25. 根据权利要求23所述的羊来源的低分子肝素，其特征在于，羊帕肝素钠的制备方法S42中乙二胺四乙酸二钠与羊肝素的质量比例在0.5-5：1，更优选1：1；所述加盐指加入终质量浓度为5％-15％的氯化钠，更优选质量浓度为10％；羊帕肝素沉淀物以水复溶后，质量浓度在1％-20％之间，更优选为10％；所述强阳离子交换树脂为食品级树脂；未结合流穿液中羊帕肝素的加盐醇沉回收，是向溶液中加入氯化钠至终质量浓度为5％-15％，更优选10％，所 述醇沉指充分搅拌下向溶液中缓慢加入2-4倍体积并精过滤后的甲醇，产生羊帕肝素钠沉淀，以过滤或离心方式回收羊帕肝素钠粗品。
  26. 根据权利要求23所述的羊来源的低分子肝素，其特征在于，羊帕肝素钠的制备方法S43中羊帕肝素钠粗品以水溶解至质量浓度在1％-15％之间，优选10％；双氧水脱色温度在15℃-40℃之间，更优选25℃；双氧水在溶液中的终体积浓度在0.1％-5％之间，更优选1％-2％；双氧水脱色时间10分钟以上，直至反应液颜色浅至Y5以下；羊帕肝素钠溶液在脱色结束至醇沉淀前，依次用稀盐酸调pH至中性，精过滤，滤液加氯化钠至8-12％浓度，调pH至5.0-7.0，再精过滤；羊帕肝素钠钠成品制得，所述醇沉淀，指充分搅拌下向反应液中加入2-4倍反应溶液体积并精过滤后的甲醇，产生羊帕肝素钠钠沉淀，沉淀物以过滤或离心方式回收，并干燥。
  27. 根据权利要求1所述的羊来源的低分子肝素，其特征在于，羊贝米肝素钠重均分子量在3000-4200之间；抗-Ⅹa活性折干后在75-110单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在1.5-3.0之间。
  28. 根据权利要求27所述的羊来源的低分子肝素，其特征在于，羊贝米肝素钠的制备方法包括如下步骤：
    S51、羊肝素季铵盐的制备，是将羊肝素钠溶解配制成水溶液，并与苯扎氯铵水溶液进行混合，分离、洗涤和干燥，制得羊肝素季铵盐；
    S52、羊贝米肝素钠粗品的制备，是将S51中干燥得到的羊肝素季铵盐按比例溶于二氯甲烷或其他有机溶剂，加入苄基三甲基氢氧化铵，搅拌使羊肝素解聚，解聚反应结束后，滴加醋酸钠甲醇溶液，制得羊贝米肝素钠粗品沉淀；
    S53、羊贝米肝素钠成品制得，是将S52中的羊贝米肝素钠粗品进行过滤、甲醇洗涤和再进行多次的复溶加盐醇沉、产物精制、干燥，得到羊贝米肝素钠成品。
  29. 根据权利要求28所述的羊来源的低分子肝素，其特征在于，羊贝米肝素钠的制备方法S51羊肝素季铵盐的制备中，羊肝素水溶液在5％-15％质量浓度之间，苯扎氯铵水溶液在10％-30％质量浓度之间，其中苯扎氯铵固体与所述羊肝素钠固体的重量比为2-5:1。
  30. 根据权利要求28所述的羊来源的低分子肝素，其特征在于，羊贝米肝素钠的制备方法S52中解聚反应时，羊肝素季铵盐、二氯甲烷、苄基三甲基氢氧化铵的质量比为1:3-10:0.2-0.4，优选为1:5:0.25；其他有机溶剂为二甲基甲酰胺；解聚反应温度在20℃-45℃之间，优选30℃；反应时间在8小时-40小时，优选16小时；解聚反应结束时，滴加醋酸钠甲醇溶 液使羊贝米肝素钠析出，所述醋酸钠的重量是羊肝素季铵盐的0.8倍，所述醋酸钠甲醇溶液的浓度为10％。
  31. 根据权利要求28所述的羊来源的低分子肝素，其特征在于，羊贝米肝素钠的制备方法S53中羊贝米肝素钠粗品沉淀经分离后，以甲醇洗涤；洗涤后的沉淀物添加质量比浓度8％-12％的氯化钠水溶液进行复溶，所述氯化钠水溶液与所述羊肝素季铵盐重量比为0.5-2:1，复溶的溶液再以2-5倍体积的甲醇进行醇沉结晶；所述复溶醇沉可以再重复多次，直至复溶后的羊贝米肝素钠溶液澄清无混浊。
  32. 权利要求1所述的羊来源的低分子肝素包括羊达肝素钠、羊那曲肝素钙、羊亭扎肝素钠、羊帕肝素钠和羊贝米肝素钠，在防治与抗凝和抗栓有关疾病中的应用，以及开发为清真抗凝抗栓药物。
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