CN110128569A - A kind of extracting method of heparin sodium - Google Patents

A kind of extracting method of heparin sodium Download PDF

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Publication number
CN110128569A
CN110128569A CN201910394506.8A CN201910394506A CN110128569A CN 110128569 A CN110128569 A CN 110128569A CN 201910394506 A CN201910394506 A CN 201910394506A CN 110128569 A CN110128569 A CN 110128569A
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CN
China
Prior art keywords
supercritical
extraction
heparin sodium
trypsase
intestinal mucosa
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Pending
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CN201910394506.8A
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Chinese (zh)
Inventor
刘容旭
李志如
杨红
谢智鑫
王宇聪
王子欢
刘丹怡
杨宏博
王钰堡
韩建春
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Northeast Agricultural University
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Northeast Agricultural University
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Priority to CN201910394506.8A priority Critical patent/CN110128569A/en
Publication of CN110128569A publication Critical patent/CN110128569A/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A kind of extracting method of heparin sodium, belongs to polysaccharide field.The method is as follows: chitterlings are cleaned, and are scraped intestinal mucosa and are collected to supercritical CO2In extraction equipment reaction kettle.The trypsase of quality 5% is added into reaction kettle, stirring mixes them thoroughly.Setting extracting pressure is 20MPa, separating pressure 8MPa, reacts 1.5 ~ 2h.Mixture is filtered, filtrate is removed, sediment distilled water rinses and removes filtrate.Sediment scumbling is lyophilized in freeze dryer in plate, is milled.The intestinal mucosa of powdery is packed into supercritical CO2In the feed chamber of extraction equipment, setting extracting pressure is 20MPa, separating pressure 8MPa, extracts 1.5 ~ 2h, removes extra fat to get heparin sodium.The invention has the advantages that passing through supercritical CO2The generation for promoting the enzymatic reaction of trypsase, substantially reduces reaction time, supercritical CO2Extraction also has certain bactericidal effect, to have better extraction effect compared with traditional handicraft.

Description

A kind of extracting method of heparin sodium
Technical field
The invention belongs to polysaccharide fields, and in particular to a kind of extracting method of heparin sodium.
Background technique
Heparin sodium is a kind of mucopolysaccharide, can interfere many links of blood clotting process, there is blood coagulation resisting function in vivo and in vitro.Its Mechanism of action is more complicated, mainly by combining with antithrombin Ⅲ (AT- III), and enhances the latter to the II of activation, Ⅸ, X, Ⅺ With the inhibiting effect of Ⅻ coagulation factor, consequence is related to formation, the resistance for preventing platelet aggregation and destroying, interfering factor Ⅹ Hemostasis-coagulation proenzyme becomes fibrin ferment, inhibits fibrin ferment, to interfere fibrinogen to become fibrin, to play anticoagulant work With.Heparin sodium during the extraction process most critical the step of be trypsase hydrolytic process.
Trypsase Trypsin(Parenzyme) be protease one kind, be from ox, sheep, pig pancreas in extract one Kind serine protein hydrolase.In vertebrate, work as digestive ferment.In the precursor pancreas egg that pancreas is trypsase After white proenzyme is synthesized, as pancreatic juice ingredient and secrete, by the limitation of enterokinase or trypsase be decomposed into activation pancreas egg White enzyme is endopeptidase, it can cut off the carboxyl side in polypeptide chain in lysine and arginine residues.It not only plays digestion The effect of enzyme, and the precursor for decomposing other enzymes such as chymotrypsinogen, procarboxypeptidase, phosphatide proenzyme can also be limited, it plays activation and makees With.It is the strongest protease of specificity, in the amino acid range for determining protein, it becomes indispensable tool.Recently Some researches show that supercritical fluid has certain facilitation to the generation of enzymatic reaction.
Supercritical fluid (supercritical fluid, SCF) refers to that temperature and pressure are in critical-temperature and critical pressure Fluid more than power is the fluid during a kind of liquid gaseous state is in homogeneous.The advantages of this fluid has liquids and gases concurrently, viscosity It is small, diffusion coefficient is big, density is big, there are good dissolubility and mass transfer performances, and in Near The Critical Point fluid to pressure and temperature Variation it is very sensitive, be both a kind of good separating medium and a kind of good reaction medium.Further, since SCF has The physical property of consecutive variations can control reactivity and selectivity by influencing its local action.As a kind of non-aqueous reaction Medium, SCF is with unique property by more and more extensive concern in enzyme catalysis field.So enzyme is placed in supercriticality Under its catalytic kinetics can also be made to be improved.With supercritical CO2For, CO under high pressure2Change the pH in enzyme microenvironment Value, the free amino of covalent modification protein surface simultaneously form carbaminate, these carbaminates change lysine residue Charge, to change the activity of enzyme.
Heparin sodium extractive technique route more commonly used at present is as follows: fresh chitterlings are cleaned → are scraped intestinal mucosa → intestines and glue Film/serous coat/casing puts into reaction pot → addition quality 5% trypsase → the NaOH solution tune pH value of 3mol/L is 8 ~ 9 → and stirs Mix and be warming up to 40 DEG C or so → maintain 2.5h → be warming up to again 60 DEG C → NaCl that quality 2% is added → be warming up to 90 DEG C → adjust pH Value is 6 ~ 7, and 60 DEG C or so are filtered while hot → be cooled to → be heated to boiling and keep 10min →, with the NaOH solution tune pH of 3mol/L Value for 8 ~ 9 → by the 5% of amount of filtrate D-254 resin → stirring and adsorbing 6h is added, stand 1h, be added in sieving → resin isometric Tap water rinse for several times → with pH value be 10 3mol/L NaOH solution stirring elution resin 2h, sieving, collect filtrate → weight After backwashing is 2 times de-, and merging filtrate → isometric ethyl alcohol is added in filtrate stirs 10min, it is heavy to stand 12h or more → collection lower layer Starch obtains mucopolysaccharide with ethanol dehydration 3 times → set in 60 DEG C of baking ovens sediment and dry.This technological operation is more complicated, disappears Time-consuming is longer, also how lossy in extraction process.
Summary of the invention
The problem of the purpose of the present invention is to solve existing heparin sodium extraction technology low efficiencys, complex process, provides A kind of extracting method of heparin sodium, i.e. supercritical CO2Extraction.Chitterlings are added directly into reaction kettle by this method, are added Trypsase passes through supercritical CO2The generation for promoting the enzymatic reaction of trypsase, substantially reduces the reaction time, and surpass Critical CO2Extraction also has certain bactericidal effect, is more advantageous to the subsequent extraction to heparin sodium.
To achieve the above object, the technical solution adopted by the present invention is as follows:
A kind of extracting method of heparin sodium, specific step is as follows for the method:
Step 1: fresh chitterlings are cleaned, and are scraped intestinal mucosa and are collected to supercritical CO2In extraction equipment reaction kettle；
Step 2: according to trypsase: trypsase is added into reaction kettle for intestinal mucosa=5:100 mass ratio, and stirring fills it Divide mixing；
Step 3: setting extracting pressure is 20MPa, separating pressure 8MPa, and extraction and separation carry out simultaneously, total time for 1.5 ~ 2h；
Step 4: mixture is filtered, and removes filtrate, and sediment distilled water rinses 2 times and removes filtrate；
Step 5: sediment being coated in plate and is lyophilized in freeze dryer, with a thickness of 3 ~ 5mm, grinds after freeze-drying；
Step 6: the intestinal mucosa of above-mentioned grinds is packed into supercritical CO2In the feed chamber of extraction equipment, setting extracting pressure is 20MPa, separating pressure 8MPa, extraction and separation carry out simultaneously, and total time is 1.5 ~ 2h, and removal fat is to get heparin sodium.
The beneficial effect of the present invention compared with the existing technology is: the main step that heparin sodium is extracted from intestinal mucosa is exactly The hydrolytic process of trypsase, passes through supercritical CO2The generation for promoting the enzymatic reaction of trypsase, when substantially reducing reaction Between, it is easy to operate.And supercritical CO2Extraction also has certain bactericidal effect, has compared with traditional handicraft and preferably extracts Effect.
Specific embodiment
Further description of the technical solution of the present invention below, and however, it is not limited to this, all to the technology of the present invention Scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered in the present invention Protection scope in.
Specific embodiment 1: present embodiment record be a kind of heparin sodium extracting method, the method is by surpassing Critical CO2Promote enzymatic reaction, the supercritical CO of trypsase2Extraction removes fatty two parts composition, and the method is specific Steps are as follows:
Step 1: fresh chitterlings are cleaned, and are scraped intestinal mucosa and are collected to supercritical CO2In extraction equipment reaction kettle；
Step 2: according to trypsase: trypsase is added into reaction kettle for intestinal mucosa=5:100 mass ratio, and stirring fills it Divide mixing；
Step 3: setting extracting pressure is 20MPa, separating pressure 8MPa, and extraction and separation carry out simultaneously, total time for 1.5 ~ 2h；
Step 4: mixture is filtered, and removes filtrate, and sediment distilled water rinses 2 times and removes filtrate；
Step 5: sediment being coated in plate and is lyophilized in freeze dryer, with a thickness of 3 ~ 5mm, grinds after freeze-drying；
Step 6: the intestinal mucosa of above-mentioned grinds is packed into supercritical CO2In the feed chamber of extraction equipment, setting extracting pressure is 20MPa, separating pressure 8MPa, extraction and separation carry out simultaneously, and total time is 1.5 ~ 2h, and removal fat is to get heparin sodium.
The supercritical CO2Extraction equipment is purchased from Jiangsu Huaan Science Research Instrument Co., Ltd, model HA121-50- 01。
It is described in step 5 specific embodiment 2: a kind of extracting method of heparin sodium described in specific embodiment one Freeze-drying specifically: after -40 DEG C of pre-freeze 40min, -40 DEG C of operation 120min, -35 DEG C of operation 120min, -30 DEG C of operations 360min, -25 DEG C of operation 360min, -15 DEG C of operation 360min, 0 DEG C of operation 120min, 10 DEG C of operation 120min, 20 DEG C run 360min。

Claims (2)

1. a kind of extracting method of heparin sodium, it is characterised in that: specific step is as follows for the method:
Step 1: fresh chitterlings are cleaned, and are scraped intestinal mucosa and are collected to supercritical CO2In extraction equipment reaction kettle；
Step 2: according to trypsase: trypsase is added into reaction kettle for intestinal mucosa=5:100 mass ratio, and stirring fills it Divide mixing；
Step 3: setting extracting pressure is 20MPa, separating pressure 8MPa, and extraction and separation carry out simultaneously, total time for 1.5 ~ 2h；
Step 4: mixture is filtered, and removes filtrate, and sediment distilled water rinses 2 times and removes filtrate；
Step 5: sediment being coated in plate and is lyophilized in freeze dryer, with a thickness of 3 ~ 5mm, grinds after freeze-drying；
Step 6: the intestinal mucosa of above-mentioned grinds is packed into supercritical CO2In the feed chamber of extraction equipment, setting extracting pressure is 20MPa, separating pressure 8MPa, extraction and separation carry out simultaneously, and total time is 1.5 ~ 2h, and removal fat is to get heparin sodium.
2. a kind of extracting method of heparin sodium according to claim 1, it is characterised in that: in step 5, the freeze-drying Specifically: after -40 DEG C of pre-freeze 40min, -40 DEG C of operation 120min, -35 DEG C of operation 120min, -30 DEG C of operation 360min, -25 DEG C Run 360min, -15 DEG C of operations 360min, 0 DEG C of operation 120min, 10 DEG C of operations 120min, 20 DEG C of operation 360min.
CN201910394506.8A 2019-05-13 2019-05-13 A kind of extracting method of heparin sodium Pending CN110128569A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110787866A (en) * 2019-11-20 2020-02-14 江苏千牧生物科技股份有限公司 Vibration grinding device for heparin sodium production and production process thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1673394A (en) * 2005-03-07 2005-09-28 四川大学 Method for leather-making with Co2 supercritical fluid as medium
CN103030715A (en) * 2012-12-07 2013-04-10 青岛九龙生物医药有限公司 Method for separating purified heparin sodium
CN103689610A (en) * 2013-12-16 2014-04-02 广西大学 Method for extracting dietary fibers in fresh manioc waste by enzyme-physical process

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1673394A (en) * 2005-03-07 2005-09-28 四川大学 Method for leather-making with Co2 supercritical fluid as medium
CN103030715A (en) * 2012-12-07 2013-04-10 青岛九龙生物医药有限公司 Method for separating purified heparin sodium
CN103689610A (en) * 2013-12-16 2014-04-02 广西大学 Method for extracting dietary fibers in fresh manioc waste by enzyme-physical process

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JOANN ZAGROBELNY ET AL.: "INSITU STUDIES OF PROTEIN CONFORMATION IN SUPERCRITICAL FLUIDS-TRYPSIN IN CARBON-DIOXIDE", 《BIOTECHNOLOGY PROGRESS》 *
宋宏新等: "粗品肝素钠制备工艺中酶解条件的研究", 《陕西科技大学学报》 *
王振宇等: "《天然产物分离技术》", 31 January 2012, 中国轻工业出版社 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110787866A (en) * 2019-11-20 2020-02-14 江苏千牧生物科技股份有限公司 Vibration grinding device for heparin sodium production and production process thereof

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Application publication date: 20190816