A method for preparing heparin using sheep small intestine

Abstract

The present invention provides a method for preparing heparin by using sheep small intestine, which relates to the field of biomedicine. The method specifically comprises: (1) pretreatment; (2) preparation of activated enzyme; (3) enzymolysis; (4) adsorption and elution; (5) precipitation and drying. The present invention makes full use of compound enzymes such as alkaline protease, trypsin, papain, cellulase, pectinase, amylase, xylanase, etc., and cooperates with plant extract supernatant components to effectively improve reaction efficiency, reduce enzyme usage, ensure extraction safety and save costs.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a method for preparing heparin by utilizing sheep small intestine. Background Art

[0002] Heparin is a mucopolysaccharide sulfate composed of alternating glucosamine, L-iduronide, N-acetylglucosamine and D-glucuronic acid. It exists in tissues such as the lungs, blood vessel walls, and intestinal mucosa. It is a natural anticoagulant, including heparin sodium, heparin calcium, and low molecular weight heparin, which are mainly used to prevent and treat thrombosis. The current methods for extracting heparin include a two-step oxidation method using potassium permanganate and hydrogen peroxide, a secondary oxidation method using hydrogen peroxide in batches, and an enzymatic method. Compared with the first two methods, the enzymatic method can avoid the destruction of the heparin structure and has less environmental pollution, and is widely used.

[0003] Book: Cao Xinmin and Zhang Yuehong. New Technology for Processing Animal Husbandry Products [M]. 1987. No. 176 also records a similar process for preparing heparin from sheep intestinal mucosa, including extracting heparin from sheep intestinal mucosa by alkaline hydrolysis, salt hydrolysis, filtration, ion exchange, washing and elution. Literature: Wei Zhangxiang, Guo Longze, Hao Yongqing, et al. Preparation of Heparin Sodium from Sheep Intestinal Mucosa [J]. Journal of Biochemical Pharmacy, 1984 (2): 49-50. It also records a similar process for preparing heparin.

[0004] In addition, patent CN101914170A discloses a method for producing sodium heparin using sheep small intestine, which includes the following steps: preparation of intestinal mucosa, enzymatic hydrolysis, adsorption, elution, precipitation desalination and dehydration and drying to obtain sodium heparin. The method has high yield, small loss of sodium heparin activity, stable quality, low production cost and high profit.

[0005] Patent CN106674381A discloses a method for extracting sodium heparin from sheep intestines, firstly taking fresh sheep intestines, pouring clean water into them, squeezing out the small intestinal mucosa, crushing them into particles, adding sodium hydroxide solution to adjust the pH, fermenting them, filling them with oxygen, stirring, standing, collecting the floating matter, rinsing, drying, mixing them with oxaloacetic acid, heating to boiling, keeping warm, cooling to room temperature, adding ammonium sulfate, stirring, concentrating by ultrafiltration membrane, and then precipitating, dehydrating, drying, and crushing. The final heparin sodium has a titer of 155-170u / mg and a recovery rate of more than 97%.

[0006] However, the above method requires a relatively long fermentation time as a whole, which is not conducive to efficient production applications.

[0007] In view of the problems existing in the prior art, it is necessary to find a heparin extraction process with low pollution, relatively simple process and high titer of the obtained heparin. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a method for preparing heparin by using sheep small intestine, which can effectively improve reaction efficiency, reduce enzyme usage, ensure extraction safety and save costs.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The present invention provides a method for preparing heparin by using sheep small intestine, comprising the following steps:

[0011] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0012] (2) Preparation of activated enzyme: corn cobs, cherry blossoms, hijiki, Jerusalem artichoke, rapeseed, radix sphaerocephala and foliage grass are mixed, soaked in water, boiled, filtered, and a supernatant is obtained; the supernatant and the composite enzyme are mixed and stirred to obtain an enzyme mixture, the pH is adjusted, ethanol and sodium dodecyl sulfate are added, the mixture is stirred to obtain an activated enzyme, and the mixture is allowed to stand to obtain a mixture;

[0013] (3) Enzymolysis: adding saline to the sheep small intestine slurry obtained in step (1), heating for the first time, adjusting the pH, adding the activated enzyme of step (2) for the first time, stirring evenly, heating for the second time, enzymolysis, adding sodium dodecyl sulfate, continuing to stir, adding the activated enzyme of step (2) again, and enzymolysis to obtain material A;

[0014] (4) Adsorption and elution: heating the material A obtained in step (3), keeping the temperature, filtering, cooling, and then using a resin for adsorption and elution to obtain an eluent;

[0015] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted, ethanol is added thereto, precipitation is carried out, the supernatant is discarded, and a precipitate is obtained. After the alcohol evaporates, the precipitate is dried.

[0016] Furthermore, in terms of weight, the raw materials of the supernatant in step (2) include: 10-15 parts of corn cobs, 5-8 parts of Prunus guanyam, 5-10 parts of Sargassum fusiformis, 5-8 parts of Jerusalem artichoke, 3-5 parts of rapeseed, 3-5 parts of ground radish and 1-2 parts of edible leaf grass.

[0017] Preferably, in terms of weight, the raw materials of the supernatant in step (2) include: 12 parts of corn cobs, 7 parts of Prunus guanshanensis, 8 parts of Sargassum fusiformis, 7 parts of Jerusalem artichokes, 3 parts of rapeseed, 3 parts of ground radish and 1 part of edible leaf grass.

[0018] Furthermore, the complex enzyme in step (2) includes alkaline protease, trypsin, papain, cellulase, pectinase, amylase and xylanase.

[0019] Furthermore, in terms of weight, the complex enzyme in step (2) comprises 5-10 parts of alkaline protease, 1-5 parts of trypsin, 1-3 parts of papain, 18-35 parts of cellulase, 1-3 parts of pectinase, 10-20 parts of amylase, and 12-14 parts of xylanase.

[0020] Preferably, in terms of weight, the complex enzyme in step (2) comprises 8 parts of alkaline protease, 3 parts of trypsin, 2 parts of papain, 22 parts of cellulase, 2 parts of pectinase, 15 parts of amylase, and 13 parts of xylanase.

[0021] Furthermore, the weight ratio of the enzyme mixture, ethanol and sodium dodecyl sulfate in step (2) is 15-18:30-50:5-8; preferably, the weight ratio of the enzyme mixture, ethanol and sodium dodecyl sulfate in step (2) is 16:40:7.

[0022] Furthermore, in step (2), the weight ratio of the supernatant to the complex enzyme is 2:1.

[0023] Furthermore, the weight ratio of the corncob, Jerusalem artichoke and ground radish is 10-15:5-8:3-5; preferably 12:7:3.

[0024] Furthermore, in step (3), the temperature of the first heating is 37-40°C, and the temperature of the second heating is 55-60°C.

[0025] Furthermore, in step (3), the weight of the activated enzyme initially added in step (2) is consistent with the weight of the activated enzyme added again in step (2), both of which are 0.8-1‰ of the weight of the sheep small intestine slurry.

[0026] Furthermore, in step (4), the heating temperature is 85-90°C, and the cooling temperature is 55-60°C.

[0027] In some specific embodiments, the method for preparing heparin using sheep small intestine comprises the following steps:

[0028] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0029] (2) Preparation of activated enzyme: corn cobs, cherry blossoms, hijiki, Jerusalem artichoke, rapeseed, radix sphaerocephala and foliage grass are mixed, and 8-10 times the weight of water of the above raw materials are added to soak and boil for 0.5 h, and then 5-7 times the weight of water of the above raw materials are added to continue boiling for 20 min, and filtered to obtain a supernatant; the supernatant and the composite enzyme are mixed and stirred to obtain an enzyme mixture, and after adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate are added, stirred to obtain a mixture, and the mixture is allowed to stand for 3 h to obtain an activated enzyme;

[0030] (3) Enzymolysis: Add saline (the weight of the saline is 3-5 times that of the sheep intestine slurry, and the mass fraction is 1.5-3.5%) to the sheep intestine slurry obtained in step (1), heat it for the first time, adjust the pH to 8.5-9, add the activated enzyme of step (2) for the first time, stir evenly, heat it for the second time, perform enzymolysis for 2 hours, add sodium dodecyl sulfate in an amount of 0.5-0.8‰ of the weight of the sheep intestine slurry, continue stirring, add the activated enzyme of step (2) again, and perform enzymolysis for 2 hours to obtain material A;

[0031] (4) Adsorption and elution: After heating the material A obtained in step (3), keep the temperature for 0.5 h, filter, cool down, and use an anion exchange resin for adsorption and elution to obtain an eluate;

[0032] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0033] Furthermore, the present invention also provides heparin extracted by the above method.

[0034] The technical effects achieved by the present invention are:

[0035] The method of the present invention has low pollution and relatively simple process. Heparin is extracted from sheep small intestine as raw material. In the extraction process, a composite enzyme is used to mix the supernatant of plant extracts, which contains vitamins, oligoxylose and other effective ingredients, and can fully promote enzyme activity, reduce the requirements for reaction conditions required for enzymatic hydrolysis, and thus facilitate the maintenance of physiologically active ingredients in small intestinal tissue and promote the extraction of heparin in sheep small intestine. In addition, in the preparation process of the activated enzyme of the present invention, plants use relatively safe ingredients, such as medicinal and edible ingredients, and the effective substances in each ingredient are used to synergistically reduce the requirements for reaction conditions, which can effectively reduce the amount of enzyme used, ensure the safety of extraction and save costs. DETAILED DESCRIPTION

[0036] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0037] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0038] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0039] It is worth noting that the alkaline protease used in the present invention is 2709 alkaline protease, and the remaining raw materials are common commercial products, so their sources are not specifically limited.

[0040] Example 1

[0041] A method for preparing heparin using sheep small intestine comprises the following steps:

[0042] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0043] (2) Preparation of activated enzyme: 10 parts of corn cobs, 5 parts of cherry blossoms, 5 parts of sea lettuce, 5 parts of Jerusalem artichoke, 3 parts of rapeseed, 3 parts of ground radish and 1 part of leaf grass were mixed, 8 times the weight of water of the above raw materials were added to soak and boil for 0.5 hours, and then 7 times the weight of water of the above raw materials were added to continue boiling for 20 minutes, and filtered to obtain a supernatant; the supernatant and composite enzyme (5 parts of alkaline protease, 1 part of trypsin, 1 part of papain, 18 parts of cellulase, 1 part of pectinase, 10 parts of amylase, 12 parts of xylanase) were mixed in a weight ratio of 2:1 and stirred evenly to obtain an enzyme mixture, and after adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate (the weight ratio of the enzyme mixture, ethanol, and sodium dodecyl sulfate is 15:30:5) were added, stirred evenly, and allowed to stand for 3 hours to obtain an activated enzyme;

[0044] (3) Enzymolysis: Add saline (the weight of the saline is 3 times that of the sheep intestine slurry, and the mass fraction is 3.5%) to the sheep intestine slurry obtained in step (1), heat it to 37°C for the first time, adjust the pH to 8.5-9, add the activating enzyme of step (2) for the first time (the amount added is 0.8‰ of the weight of the sheep intestine slurry), stir evenly, heat it to 55°C for the second time, perform enzymolysis for 2h, add sodium dodecyl sulfate in an amount of 0.5‰ of the weight of the sheep intestine slurry, continue stirring, add the activating enzyme of step (2) again (the amount added is 0.8‰ of the weight of the sheep intestine slurry), and perform enzymolysis for 2h to obtain material A;

[0045] (4) Adsorption and elution: The material A obtained in step (3) was heated to 85° C., kept warm for 0.5 h, filtered, cooled to 55° C., and adsorbed and eluted using an anion exchange resin to obtain an eluate;

[0046] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0047] Example 2

[0048] A method for preparing heparin using sheep small intestine comprises the following steps:

[0049] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0050] (2) Preparation of activated enzyme: 15 parts of corn cobs, 8 parts of cherry blossoms, 10 parts of sea lettuce, 8 parts of Jerusalem artichoke, 5 parts of rapeseed, 5 parts of ground radish and 2 parts of edible grass were mixed, and 10 times the weight of water of the above raw materials was added to soak and boil for 0.5 h, and then 7 times the weight of water of the above raw materials was added, and the boiling was continued for 20 min, and filtered to obtain a supernatant; the supernatant and composite enzyme (10 parts of alkaline protease, 5 parts of trypsin, 3 parts of papain, 35 parts of cellulase, 3 parts of pectinase, 20 parts of amylase, and 14 parts of xylanase) were mixed in a weight ratio of 2:1 and stirred evenly to obtain an enzyme mixture, and after adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate (the weight ratio of the enzyme mixture, ethanol, and sodium dodecyl sulfate is 18:50:8) were added, stirred evenly, and allowed to stand for 3 h to obtain an activated enzyme;

[0051] (3) Enzymolysis: Add saline (the weight of the saline is 5 times that of the sheep intestine slurry, and the mass fraction is 1.5%) to the sheep intestine slurry obtained in step (1), heat it to 40°C for the first time, adjust the pH to 8.5-9, add the activated enzyme of step (2) for the first time (the amount added is 1‰ of the weight of the sheep intestine slurry), stir evenly, heat it to 60°C for the second time, perform enzymolysis for 2h, add sodium dodecyl sulfate in an amount of 0.8‰ of the weight of the sheep intestine slurry, continue stirring, add the activated enzyme of step (2) again (the amount added is 1‰ of the weight of the sheep intestine slurry), and perform enzymolysis for 2h to obtain material A;

[0052] (4) Adsorption and elution: The material A obtained in step (3) was heated to 90° C., kept warm for 0.5 h, filtered, cooled to 60° C., and adsorbed and eluted using an anion exchange resin to obtain an eluate;

[0053] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0054] Example 3

[0055] A method for preparing heparin using sheep small intestine comprises the following steps:

[0056] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0057] (2) Preparation of activated enzyme: 12 parts of corn cobs, 7 parts of cherry blossoms, 8 parts of sea lettuce, 7 parts of Jerusalem artichoke, 3 parts of rapeseed, 3 parts of ground radish and 1 part of leaf grass were mixed, 9 times the weight of water of the above raw materials were added to soak and boil for 0.5 hours, and then 6 times the weight of water of the above raw materials were added to continue boiling for 20 minutes, and filtered to obtain a supernatant; the supernatant and composite enzyme (8 parts of alkaline protease, 3 parts of trypsin, 2 parts of papain, 22 parts of cellulase, 2 parts of pectinase, 15 parts of amylase, and 13 parts of xylanase) were mixed in a weight ratio of 2:1 and stirred evenly to obtain an enzyme mixture, and after adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate (the weight ratio of the enzyme mixture, ethanol, and sodium dodecyl sulfate is 16:40:7) were added, stirred evenly, and allowed to stand for 3 hours to obtain an activated enzyme;

[0058] (3) Enzymolysis: Add saline (the weight of the saline is 4 times that of the sheep intestine slurry, and the mass fraction is 2%) to the sheep intestine slurry obtained in step (1), heat it to 40°C for the first time, adjust the pH to 8.5-9, add the activating enzyme of step (2) for the first time (the amount added is 0.9‰ of the weight of the sheep intestine slurry), stir evenly, heat it to 60°C for the second time, perform enzymolysis for 2h, add sodium dodecyl sulfate in an amount of 0.6‰ of the weight of the sheep intestine slurry, continue stirring, add the activating enzyme of step (2) again (the amount added is 0.9‰ of the weight of the sheep intestine slurry), and perform enzymolysis for 2h to obtain material A;

[0059] (4) Adsorption and elution: The material A obtained in step (3) was heated to 90° C., kept warm for 0.5 h, filtered, cooled to 60° C., and adsorbed and eluted using an anion exchange resin to obtain an eluate;

[0060] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0061] Comparative Example 1

[0062] A method for preparing heparin using sheep small intestine comprises the following steps:

[0063] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0064] (2) Preparation of activated enzyme: 22 parts of corn cobs, 7 parts of cherry blossoms, 8 parts of sea lettuce, 3 parts of rapeseed, and 1 part of edible grass were mixed, and 9 times the weight of water of the above raw materials were added to soak and boil for 0.5 hours, and then 6 times the weight of water of the above raw materials were added, and the boiling was continued for 20 minutes, and filtered to obtain a supernatant; the supernatant and a composite enzyme (8 parts of alkaline protease, 3 parts of trypsin, 2 parts of papain, 22 parts of cellulase, 2 parts of pectinase, 15 parts of amylase, and 13 parts of xylanase) were mixed in a weight ratio of 2:1 and stirred evenly to obtain an enzyme mixture, and after adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate (the weight ratio of the enzyme mixture, ethanol, and sodium dodecyl sulfate is 16:40:7) were added, and after stirring evenly, the mixture was allowed to stand for 3 hours to obtain an activated enzyme;

[0065] (3) Enzymolysis: Add saline (the weight of the saline is 4 times that of the sheep intestine slurry, and the mass fraction is 2%) to the sheep intestine slurry obtained in step (1), heat it to 40°C for the first time, adjust the pH to 8.5-9, add the activating enzyme of step (2) for the first time (the amount added is 0.9‰ of the weight of the sheep intestine slurry), stir evenly, heat it to 60°C for the second time, perform enzymolysis for 2h, add sodium dodecyl sulfate in an amount of 0.6‰ of the weight of the sheep intestine slurry, continue stirring, add the activating enzyme of step (2) again (the amount added is 0.9‰ of the weight of the sheep intestine slurry), and perform enzymolysis for 2h to obtain material A;

[0066] (4) Adsorption and elution: The material A obtained in step (3) was heated to 90° C., kept warm for 0.5 h, filtered, cooled to 60° C., and adsorbed and eluted using an anion exchange resin to obtain an eluate;

[0067] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0068] Comparative Example 2

[0069] A method for preparing heparin using sheep small intestine comprises the following steps:

[0070] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0071] (2) Preparation of activated enzyme: 7 parts of Sakura guanyam, 8 parts of Sargassum fusiformis, 22 parts of Jerusalem artichoke, 3 parts of rapeseed, and 1 part of edible leaf grass were mixed, and 9 times the weight of water of the above raw materials were added to soak and boil for 0.5 h, and then 6 times the weight of water of the above raw materials were added, and the boiling was continued for 20 min, and filtered to obtain a supernatant; the supernatant and composite enzyme (8 parts of alkaline protease, 3 parts of trypsin, 2 parts of papain, 22 parts of cellulase, 2 parts of pectinase, 15 parts of amylase, and 13 parts of xylanase) were mixed in a weight ratio of 2:1 and stirred evenly to obtain an enzyme mixture, and after adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate (the weight ratio of the enzyme mixture, ethanol, and sodium dodecyl sulfate is 16:40:7) were added, and after stirring evenly, the mixture was allowed to stand for 3 h to obtain an activated enzyme;

[0072] (3) Enzymolysis: Add saline (the weight of the saline is 4 times that of the sheep intestine slurry, and the mass fraction is 2%) to the sheep intestine slurry obtained in step (1), heat it to 40°C for the first time, adjust the pH to 8.5-9, add the activating enzyme of step (2) for the first time (the amount added is 0.9‰ of the weight of the sheep intestine slurry), stir evenly, heat it to 60°C for the second time, perform enzymolysis for 2h, add sodium dodecyl sulfate in an amount of 0.6‰ of the weight of the sheep intestine slurry, continue stirring, add the activating enzyme of step (2) again (the amount added is 0.9‰ of the weight of the sheep intestine slurry), and perform enzymolysis for 2h to obtain material A;

[0073] (4) Adsorption and elution: The material A obtained in step (3) was heated to 90° C., kept warm for 0.5 h, filtered, cooled to 60° C., and adsorbed and eluted using an anion exchange resin to obtain an eluate;

[0074] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0075] Comparative Example 3

[0076] A method for preparing heparin using sheep small intestine comprises the following steps:

[0077] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0078] (2) Preparation of activated enzyme: 7 parts of Sakura guanyam, 8 parts of Sargassum fusiformis, 3 parts of rapeseed, 22 parts of Melaleuca alternifolia and 1 part of foliage grass were mixed, 9 times the weight of water of the above raw materials were added to soak and boil for 0.5 h, and then 6 times the weight of water of the above raw materials were added to continue boiling for 20 min, and filtered to obtain a supernatant; the supernatant and composite enzyme (8 parts of alkaline protease, 3 parts of trypsin, 2 parts of papain, 22 parts of cellulase, 2 parts of pectinase, 15 parts of amylase, and 13 parts of xylanase) were mixed in a weight ratio of 2:1 and stirred evenly to obtain an enzyme mixture, and after adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate (the weight ratio of the enzyme mixture, ethanol, and sodium dodecyl sulfate is 16:40:7) were added, stirred evenly, and allowed to stand for 3 h to obtain an activated enzyme;

[0079] (3) Enzymolysis: Add saline (the weight of the saline is 4 times that of the sheep intestine slurry, and the mass fraction is 2%) to the sheep intestine slurry obtained in step (1), heat it to 40°C for the first time, adjust the pH to 8.5-9, add the activating enzyme of step (2) for the first time (the amount added is 0.9‰ of the weight of the sheep intestine slurry), stir evenly, heat it to 60°C for the second time, perform enzymolysis for 2h, add sodium dodecyl sulfate in an amount of 0.6‰ of the weight of the sheep intestine slurry, continue stirring, add the activating enzyme of step (2) again (the amount added is 0.9‰ of the weight of the sheep intestine slurry), and perform enzymolysis for 2h to obtain material A;

[0080] (4) Adsorption and elution: The material A obtained in step (3) was heated to 90° C., kept warm for 0.5 h, filtered, cooled to 60° C., and adsorbed and eluted using an anion exchange resin to obtain an eluate;

[0081] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0082] Comparative Example 4

[0083] A method for preparing heparin using sheep small intestine comprises the following steps:

[0084] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0085] (2) Preparation of activated enzyme: 12 parts of corn cobs, 7 parts of cherry blossoms, 8 parts of sea lettuce, 7 parts of Jerusalem artichoke, 3 parts of rapeseed, 3 parts of ground radish and 1 part of leaf grass were mixed, 9 times the weight of water of the above raw materials were added to soak and boil for 0.5 hours, and then 6 times the weight of water of the above raw materials were added to continue boiling for 20 minutes, and filtered to obtain a supernatant; the supernatant and composite enzyme (8 parts of alkaline protease, 3 parts of trypsin, 2 parts of pectinase, 15 parts of amylase) were mixed in a weight ratio of 2:1 and stirred evenly to obtain an enzyme mixture, and after adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate (the weight ratio of the enzyme mixture, ethanol, and sodium dodecyl sulfate is 16:40:7) were added, stirred evenly, and allowed to stand for 3 hours to obtain an activated enzyme;

[0086] (3) Enzymolysis: Add saline (the weight of the saline is 4 times that of the sheep intestine slurry, and the mass fraction is 2%) to the sheep intestine slurry obtained in step (1), heat it to 40°C for the first time, adjust the pH to 8.5-9, add the activating enzyme of step (2) for the first time (the amount added is 0.9‰ of the weight of the sheep intestine slurry), stir evenly, heat it to 60°C for the second time, perform enzymolysis for 2h, add sodium dodecyl sulfate in an amount of 0.6‰ of the weight of the sheep intestine slurry, continue stirring, add the activating enzyme of step (2) again (the amount added is 0.9‰ of the weight of the sheep intestine slurry), and perform enzymolysis for 2h to obtain material A;

[0087] (4) Adsorption and elution: The material A obtained in step (3) was heated to 90° C., kept warm for 0.5 h, filtered, cooled to 60° C., and adsorbed and eluted using an anion exchange resin to obtain an eluate;

[0088] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0089] Comparative Example 5

[0090] A method for preparing heparin using sheep small intestine comprises the following steps:

[0091] (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry;

[0092] (2) Preparation of composite enzyme: deionized water and composite enzyme (8 parts alkaline protease, 3 parts trypsin, 2 parts papain, 22 parts cellulase, 2 parts pectinase, 15 parts amylase, 13 parts xylanase) in a weight ratio of 2:1 were mixed and stirred to obtain an enzyme mixture. After adjusting the pH to 8.5-9, 75% by volume of ethanol and sodium dodecyl sulfate (the weight ratio of the enzyme mixture, ethanol, and sodium dodecyl sulfate was 16:40:7) were added, stirred to obtain an activated enzyme, and allowed to stand for 3 hours.

[0093] (3) Enzymolysis: Add saline (the weight of the saline is 4 times that of the sheep intestine slurry, and the mass fraction is 2%) to the sheep intestine slurry obtained in step (1), heat it to 40°C for the first time, adjust the pH to 8.5-9, add the activating enzyme of step (2) for the first time (the amount added is 0.9‰ of the weight of the sheep intestine slurry), stir evenly, heat it to 60°C for the second time, perform enzymolysis for 2h, add sodium dodecyl sulfate in an amount of 0.6‰ of the weight of the sheep intestine slurry, continue stirring, add the activating enzyme of step (2) again (the amount added is 0.9‰ of the weight of the sheep intestine slurry), and perform enzymolysis for 2h to obtain material A;

[0094] (4) Adsorption and elution: The material A obtained in step (3) was heated to 90° C., kept warm for 0.5 h, filtered, cooled to 60° C., and adsorbed and eluted using an anion exchange resin to obtain an eluate;

[0095] (5) Precipitation and drying: The pH of the eluate from step (4) is adjusted to 7-8, and 85% ethanol in a volume fraction of 2 times the volume of the eluate is added thereto, and precipitation is carried out for 24 hours. The supernatant is discarded to obtain a precipitate, and after the alcohol evaporates, the precipitate is dried.

[0096] Heparin activity and yield detection

[0097] Test 1: Heparin activity: Existing studies have shown that there is no significant difference in the heparin titer measured by the sheep plasma method and the rabbit whole blood method. At the same time, the sheep plasma method also overcomes the defects of the rabbit whole blood method, such as complex operation and poor reproducibility, and is more accurate, simple and rapid. Therefore, this test study uses the sheep plasma method. The specific steps are as follows:

[0098] (1) Add 0.8 mL of 0.25% sodium chloride solution to 1 mL of plasma and keep warm at 37°C. If the plasma coagulates within 5 minutes, the plasma can be used for subsequent tests.

[0099] (2) Preparation of standard: Take heparin sodium standard and dilute it to 100 U / mL with pure water for later use. Dilute it to 7 U / mL when needed.

[0100] (3) Preparation of test solution: Take an appropriate amount of heparin prepared in each example and dilute it with normal saline to a concentration equivalent to that of the standard solution according to the estimated potency.

[0101] (4) Plasma determination: Add gradient doses of 110 μL, 140 μL, 170 μL, 200 μL, and 230 μL of standard solution to test tubes, then add 1 mL of sheep plasma and 0.8 mL of 0.25% sodium chloride solution. After thorough mixing, keep warm at 37°C and record accurately for 1 min to determine the degree of blood coagulation in the test tube. Use the preliminary determination result as the midpoint, and use 10 μL as one level on each side. Determine the half-coagulation point (1 / 2 coagulation point) of the standard sample in the same way.

[0102] The criteria for judging the degree of plasma coagulation are shown in the following table:

[0103] Table 1 Criteria for judging the degree of plasma coagulation

[0104] Degree of solidification Judgment criteria Completely solidified The solution is completely solidified. The tube is inverted and tapped. The clot does not fall off the tube wall. 3 / 4 solidification The solution is completely solidified, but the clot can be dislodged from the tube wall by inverting the tube and tapping it. 1 / 2 solidification About half the volume of the solution solidifies 1 / 4 solidification Very little solution volume is solidified 0 Solidification The solution is completely mobile

[0105] (5) Potency determination: Add gradient doses of standard solution and test sample solution to test tubes, respectively. Take the half-gel point of the standard sample as the midpoint, and calculate the potency by counting the degree of gelation in each tube at intervals of 10 μL. The calculation formula is:

[0106]

[0107] The results are summarized in Table 2.

[0108] Experiment 2: Heparin yield = heparin finished product content / fresh intestine weight. The results are summarized in Table 2.

[0109] Table 2 Heparin titer and yield

[0110]

[0111]

[0112] According to research, the heparin titer of the present invention is better, reaching more than 198U / mg, and the yield can reach more than 0.9g / kg intestine (fresh). Relatively speaking, when the process does not perform enzyme activation treatment, and when no specific enzyme or specific supernatant is added, the titer and yield of heparin finally obtained by the corresponding preparation method are significantly reduced, mainly because it is difficult to increase the enzyme activity and there is no or very little corresponding effective ingredient.

[0113] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing heparin using sheep small intestine, characterized in that: The following steps are involved: (1) Pretreatment: Pour clean water into the small intestine of sheep, squeeze out the small intestinal mucosa, and break it to obtain sheep small intestine slurry; (2) Preparation of activated enzyme: corn cobs, cherry blossoms, hijiki, Jerusalem artichoke, rapeseed, radix sphaerocephala and foliage grass are mixed, soaked in water, boiled, filtered, and a supernatant is obtained; the supernatant and the composite enzyme are mixed and stirred to obtain an enzyme mixture, the pH is adjusted, ethanol and sodium dodecyl sulfate are added, the mixture is stirred to obtain an activated enzyme, and the mixture is allowed to stand to obtain a mixture; (3) Enzymolysis: adding saline to the sheep small intestine slurry obtained in step (1), heating for the first time, adjusting the pH, adding the activated enzyme of step (2) for the first time, stirring evenly, heating for the second time, enzymolysis, adding sodium dodecyl sulfate, continuing to stir, adding the activated enzyme of step (2) again, and enzymolysis to obtain material A; (4) Adsorption and elution: heating the material A obtained in step (3), then keeping the temperature, filtering, cooling, and then using a resin for adsorption and elution to obtain an eluent; (5) precipitation and drying: adjusting the pH of the eluate from step (4), adding ethanol thereto, precipitating, discarding the supernatant to obtain a precipitate, and drying after the alcohol evaporates; The raw materials of the supernatant in step (2) are, by weight, 10-15 parts of corn cobs, 5-8 parts of cherry blossoms, 5-10 parts of sea lettuce, 5-8 parts of Jerusalem artichokes, 3-5 parts of rapeseed, 3-5 parts of radix sphaerocephala and 1-2 parts of foliage grass; the composite enzyme in step (2) is, by weight, 5-10 parts of alkaline protease, 1-5 parts of trypsin, 1-3 parts of papain, 18-35 parts of cellulase, 1-3 parts of pectinase, 10-20 parts of amylase and 12-14 parts of xylanase.

2. The method according to claim 1, characterized in that: In terms of weight, the raw materials of the supernatant in step (2) are: 12 parts of corn cobs, 7 parts of Prunus guanshanensis, 8 parts of Sargassum fusiformis, 7 parts of Jerusalem artichoke, 3 parts of rapeseed, 3 parts of Melaleuca alternifolia and 1 part of edible leaf grass.

3. The method according to claim 1, characterized in that: The weight ratio of the enzyme mixture, ethanol and sodium dodecyl sulfate in step (2) is 15-18:30-50:5-8.

4. The method according to claim 1, characterized in that: The temperature of the first heating in step (3) is 37-40°C, and the temperature of the second heating is 55-60°C.

5. The method according to claim 1, characterized in that: The weight of the activated enzyme initially added in step (2) in step (3) is consistent with the weight of the activated enzyme added again in step (2), both of which are 0.8-1‰ of the weight of the sheep small intestine slurry.

6. The method according to claim 1, characterized in that: In step (4), the heating temperature is 85-90°C, and the cooling temperature is 55-60°C.

7. Heparin extracted by the method according to any one of claims 1 to 6.

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