CN104926937A - Method for extracting hirudin from leech saliva - Google Patents
Method for extracting hirudin from leech saliva Download PDFInfo
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- CN104926937A CN104926937A CN201510343680.1A CN201510343680A CN104926937A CN 104926937 A CN104926937 A CN 104926937A CN 201510343680 A CN201510343680 A CN 201510343680A CN 104926937 A CN104926937 A CN 104926937A
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- hirudin
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- 241000545744 Hirudinea Species 0.000 title claims abstract description 70
- 229940006607 hirudin Drugs 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 26
- 210000003296 saliva Anatomy 0.000 title claims abstract description 24
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 title claims abstract description 20
- 102000007625 Hirudins Human genes 0.000 title claims abstract description 19
- 108010007267 Hirudins Proteins 0.000 title claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 35
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000012043 crude product Substances 0.000 claims abstract description 28
- 230000001939 inductive effect Effects 0.000 claims abstract description 23
- 239000000126 substance Substances 0.000 claims abstract description 23
- 238000005349 anion exchange Methods 0.000 claims abstract description 15
- 238000004440 column chromatography Methods 0.000 claims abstract description 15
- 238000001641 gel filtration chromatography Methods 0.000 claims abstract description 15
- 230000000384 rearing effect Effects 0.000 claims abstract description 8
- 238000003756 stirring Methods 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 36
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 28
- 239000011780 sodium chloride Substances 0.000 claims description 28
- 239000002895 emetic Substances 0.000 claims description 21
- 239000011259 mixed solution Substances 0.000 claims description 21
- 210000003079 salivary gland Anatomy 0.000 claims description 21
- 230000028327 secretion Effects 0.000 claims description 21
- 239000000284 extract Substances 0.000 claims description 15
- 239000012141 concentrate Substances 0.000 claims description 14
- 238000000502 dialysis Methods 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 14
- 238000010828 elution Methods 0.000 claims description 14
- 239000012467 final product Substances 0.000 claims description 14
- 235000011187 glycerol Nutrition 0.000 claims description 14
- 238000011068 loading method Methods 0.000 claims description 14
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 14
- 229960001763 zinc sulfate Drugs 0.000 claims description 14
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 14
- 241000500851 Poecilobdella manillensis Species 0.000 claims description 12
- 241001061264 Astragalus Species 0.000 claims description 10
- 235000006533 astragalus Nutrition 0.000 claims description 10
- 150000004676 glycans Chemical class 0.000 claims description 10
- 229920001282 polysaccharide Polymers 0.000 claims description 10
- 239000005017 polysaccharide Substances 0.000 claims description 10
- 210000004233 talus Anatomy 0.000 claims description 10
- 229920001661 Chitosan Polymers 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 241000361919 Metaphire sieboldi Species 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 229920005654 Sephadex Polymers 0.000 claims description 7
- 239000012507 Sephadex™ Substances 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 7
- 239000003513 alkali Substances 0.000 claims description 7
- 150000001450 anions Chemical class 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000001913 cellulose Substances 0.000 claims description 7
- 229920002678 cellulose Polymers 0.000 claims description 7
- 238000013016 damping Methods 0.000 claims description 7
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 claims description 7
- 238000007598 dipping method Methods 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 7
- 239000001509 sodium citrate Substances 0.000 claims description 7
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 7
- 238000000967 suction filtration Methods 0.000 claims description 7
- 239000000047 product Substances 0.000 abstract description 3
- 238000009313 farming Methods 0.000 abstract 1
- 230000008014 freezing Effects 0.000 abstract 1
- 238000007710 freezing Methods 0.000 abstract 1
- 238000000605 extraction Methods 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 208000025962 Crush injury Diseases 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000002434 immunopotentiative effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biodiversity & Conservation Biology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for extracting hirudin form leech saliva. The method includes the following steps of a), placing leeches in a container to allow the leeches to be hungered sufficiently, inputting inductive substances to the leeches to allow the same to absorb fully, adding vomitives to allow the leeches to spit the saliva in bodies, taking out blood-sucking leech living bodies to return to a rearing pond to feed continuously, and meanwhile, collecting hirudin crude product liquid in the container; b), freezing above hirudin crude products, adding into precooled cold acetone, stirring prior to placing in a refrigerator for setting overnight, sucking supernatant acetone the next day prior to centrifuging, adding a trichloroacetic acid solution, and centrifuging to remove residues to obtain concentrated liquid; c), eluting the above concentrated liquid with an anion exchange column chromatography method, and performing gel filtration chromatography to obtain finished products. The method has the advantages of reasonable process, convenience and practicability in operation, good quality stability of the finished hirudin products and high yield, one-time pillage on the leeches with a traditional method can be avoided, wild resources of the leeches are protected, and development of leech farming is driven.
Description
Technical field
The present invention relates to a kind of method extracting r-hirudin from leech saliva.
Background technology
R-hirudin is a kind of protein in leech body, containing 65 amino-acid residues and 3 pairs of disulfide linkage, has the antithrombin activity of high special, Trombin inhibiting bound substrates, therefore has blood coagulation resisting function.
The isolation and purification of natural hirudin mainly comprises separation and Extraction r-hirudin from living leech element and solid carbon dioxide leech.The acquisition of traditional r-hirudin is the disposable predation to leech, causes huge waste to the wild resource of leech, is unfavorable for the development of hirudiniculture industry.
Summary of the invention
For prior art Problems existing; the invention provides a kind of feature that quality stability is good and yield is higher with rational technology, easy to operate practicality, r-hirudin finished product; and traditional method can be avoided the disposable predation of leech; protect the wild resource of leech, drive again the development of hirudiniculture industry.
For achieving the above object, technical scheme of the present invention is as follows:
From leech saliva, extract a method for r-hirudin, comprise the following steps:
A, leech to be placed in container fully hungry, drop into inductive substance to leech inhale full after, add emetic and make its spue saliva in body and natural hirudin crude product liquid, then take out hirudinaria manillensis live body and after being rinsed well, return rearing pool and continue to raise; R-hirudin crude product liquid simultaneously in collection container, put refrigerator and cooled freeze preserve stand-by;
Described inductive substance is made up of earthworm extract 10 ~ 300ml, chitosan 5 ~ 50g, astragalus polysaccharides 1 ~ 20g, glucose powder 1 ~ 30g;
Described emetic is the mixed solution of zinc sulfate and ethanol, and its ratio is 1:1 ~ 2, and the weight ratio between the consumption of emetic and leech live body is 1:1 ~ 10;
B, thaw above-mentioned r-hirudin crude product, get 250 ~ 300ml add 4 times to put in refrigerator precooling at 4 DEG C containing in the cold acetone of 15% water, stir in rearmounted refrigerator and at 4 DEG C, allow its precipitates overnight, sucking-off next day supernatant acetone is also centrifugal, the trichoroacetic acid(TCA) 20-30ml adding precooling in gained precipitation makes it dissolve, centrifugal removing residue, obtains salivary gland secretion concentrated solution;
C, with anion-exchange column chromatography, wash-out is carried out to above-mentioned salivary gland secretion concentrated solution after, carry out gel filtration chromatography, to obtain final product.
The time that described leech is fully hungry is 15 ~ 20 days, and the time that leech inhales full inductive substance is 10 ~ 60 minutes.
Described zinc sulfate massfraction is 0.05 ~ 1%, and described ethanol massfraction is 5 ~ 10%.
In described step C, the concrete operations of anion-exchange column chromatography are as follows:
Getting appropriate diethylamino Cellulose anion exchanger adding distil water makes it expand, and is washed with distilled water to neutrality after respectively soaking 300 minutes successively with the NaOH-NaCl mixed solution of the NaOH-NaCl mixed solution of 0.5mol/L, HCl and 0.5mol/L of 0.5mol/L; Dress post 2cm x 60cm, to desired height, balances with the 0.1mol/L sodium citrate buffer solution of pH=4.6; Carry out gradient elution by the salivary gland secretion concentrated solution of 5ml or above-mentioned solution of trichloroacetic acid loading and draw out elution curve, Fractional Collections elutriant with nucleic acid-protein detector record, flow velocity is 30ml/ hour; Vigor part will be had after testing to concentrate in dialysis tubing concentrate with glycerine, to obtain final product.
In described step C, the concrete operations of gel filtration chromatography are as follows:
Get appropriate sephadex ion-exchanger adding distil water and soak 4 hours, with 0.5mol/L NaOH-0.5mol/L NaCl mixed liquid dipping half an hour, suction filtration removing alkali liquid is also washed with distilled water to neutrality, heated and boiled removing bubble; Dress post 1.5cm x 50cm is to desired height, and balance with the 0.1mol/L Tris-HCl-NaCl damping fluid of pH=7.4, by the above-mentioned dialysis tubing glycerine concentrated solution loading wash-out having vigor elutriant, Fractional Collections elutriant, flow velocity is 90ml/ hour.
Beneficial effect of the present invention is as follows:
1. inductive substance of the present invention is a kind of induced liquid of pure natural, and the composition in induced liquid is nontoxic, harmless, non-corrosiveness, is conducive to the Extraction and separation purifying of natural hirudin like this; Containing earthworm extract in induced liquid, it contains various trace elements and active enzyme material, and hirudinaria manillensis can be induced to suck.
2. the chitosan in the present invention has good nutritive value and the effect such as inhibiting bacteria and diminishing inflammation is anticorrosion, effectively can improve hirudinaria manillensis survival rate in extraction natural hirudin crude product process.
3. invention increases the immunizing power of hirudinaria manillensis, astragalus polysaccharides and glucose is with the addition of in specificity induced liquid, can be hirudinaria manillensis like this and supplement nutrition needed for it and energy in time, this lays a solid foundation for repeatedly carrying out living leech extraction natural hirudin crude product, simultaneously, astragalus polysaccharides is immunopotentiating agent or conditioning agent, can enhancing body immunologic function, improve body intestinal environment, promote the growth of useful group bacterium lactobacillus, to maintain the balance of intestinal flora and to stablize, thus be conducive to preventing and treating various virus infection, bacteriosis.Effectively can prevent and treat the adding of astragalus polysaccharides and extract the opportunistic infections disease caused because of crush injury leech oral cavity in natural hirudin crude product process, thus improve hirudinaria manillensis extract its saliva and natural hirudin crude product after surviving rate, decrease mortality ratio, reduce production cost.
4. present invention process is simple, and the extraction yield of natural hirudin is stablized, and extracts total amount higher.Adopt method of the present invention, a living leech can repeatedly extract natural hirudin crude product, avoids traditional method to the disposable predation of leech, protects the wild resource of leech, driven again the development of hirudiniculture industry.
Embodiment
More being convenient to make content of the present invention understand, below in conjunction with embodiment, technical solutions according to the invention are described further, but the present invention being not limited only to this.
Embodiment 1
The method extracting r-hirudin from leech saliva of the present invention, comprises the following steps:
A, leech to be placed in container fully hungry, drop into inductive substance to leech inhale full after, add emetic and make its spue saliva in body and natural hirudin crude product liquid, then take out hirudinaria manillensis live body and after being rinsed well, return rearing pool and continue to raise; R-hirudin crude product liquid simultaneously in collection container, put refrigerator and cooled freeze preserve stand-by;
Described inductive substance is made up of earthworm extract 10ml, chitosan 5g, astragalus polysaccharides 1g, glucose powder 1g;
Described emetic is the mixed solution of zinc sulfate and ethanol, and its ratio is 1:1, and the weight ratio between the consumption of emetic and leech live body is 1:1;
B, thaw above-mentioned r-hirudin crude product, get 250ml add 4 times to put in refrigerator precooling at 4 DEG C containing in the cold acetone of 15% water, stir in rearmounted refrigerator and at 4 DEG C, allow its precipitates overnight, sucking-off next day supernatant acetone is also centrifugal, the trichoroacetic acid(TCA) 20ml adding precooling in gained precipitation makes it dissolve, centrifugal removing residue, obtains salivary gland secretion concentrated solution;
C, with anion-exchange column chromatography, wash-out is carried out to above-mentioned salivary gland secretion concentrated solution after, carry out gel filtration chromatography, to obtain final product.
The time that described leech is fully hungry is 15 days, and the time that leech inhales full inductive substance is 10 minutes.
Described zinc sulfate massfraction is 0.05%, and described ethanol massfraction is 5%.
In described step C, the concrete operations of anion-exchange column chromatography are as follows:
Getting appropriate diethylamino Cellulose anion exchanger adding distil water makes it expand, and is washed with distilled water to neutrality after respectively soaking 300 minutes successively with the NaOH-NaCl mixed solution of the NaOH-NaCl mixed solution of 0.5mol/L, HCl and 0.5mol/L of 0.5mol/L; Dress post 2cm x 60cm, to desired height, balances with the 0.1mol/L sodium citrate buffer solution of pH=4.6; Carry out gradient elution by the salivary gland secretion concentrated solution of 5ml or above-mentioned solution of trichloroacetic acid loading and draw out elution curve, Fractional Collections elutriant with nucleic acid-protein detector record, flow velocity is 30ml/ hour; Vigor part will be had after testing to concentrate in dialysis tubing concentrate with glycerine, to obtain final product.
In described step C, the concrete operations of gel filtration chromatography are as follows:
Get appropriate sephadex ion-exchanger adding distil water and soak 4 hours, with 0.5mol/L NaOH-0.5mol/L NaCl mixed liquid dipping half an hour, suction filtration removing alkali liquid is also washed with distilled water to neutrality, heated and boiled removing bubble; Dress post 1.5cm x 50cm is to desired height, and balance with the 0.1mol/L Tris-HCl-NaCl damping fluid of pH=7.4, by the above-mentioned dialysis tubing glycerine concentrated solution loading wash-out having vigor elutriant, Fractional Collections elutriant, flow velocity is 90ml/ hour.
Embodiment 2
The method extracting r-hirudin from leech saliva of the present invention, comprises the following steps:
A, leech to be placed in container fully hungry, drop into inductive substance to leech inhale full after, add emetic and make its spue saliva in body and natural hirudin crude product liquid, then take out hirudinaria manillensis live body and after being rinsed well, return rearing pool and continue to raise; R-hirudin crude product liquid simultaneously in collection container, put refrigerator and cooled freeze preserve stand-by;
Described inductive substance is made up of earthworm extract 100ml, chitosan 25g, astragalus polysaccharides 10g, glucose powder 10g;
Described emetic is the mixed solution of zinc sulfate and ethanol, and its ratio is 1:1.5, and the weight ratio between the consumption of emetic and leech live body is 1:5;
B, thaw above-mentioned r-hirudin crude product, get 280ml add 4 times to put in refrigerator precooling at 4 DEG C containing in the cold acetone of 15% water, stir in rearmounted refrigerator and at 4 DEG C, allow its precipitates overnight, sucking-off next day supernatant acetone is also centrifugal, the trichoroacetic acid(TCA) 25ml adding precooling in gained precipitation makes it dissolve, centrifugal removing residue, obtains salivary gland secretion concentrated solution;
C, with anion-exchange column chromatography, wash-out is carried out to above-mentioned salivary gland secretion concentrated solution after, carry out gel filtration chromatography, to obtain final product.
The time that described leech is fully hungry is 15 ~ 20 days, and the time that leech inhales full inductive substance is 30 minutes.
Described zinc sulfate massfraction is 0. 5%, and described ethanol massfraction is 8%.
In described step C, the concrete operations of anion-exchange column chromatography are as follows:
Getting appropriate diethylamino Cellulose anion exchanger adding distil water makes it expand, and is washed with distilled water to neutrality after respectively soaking 300 minutes successively with the NaOH-NaCl mixed solution of the NaOH-NaCl mixed solution of 0.5mol/L, HCl and 0.5mol/L of 0.5mol/L; Dress post 2cm x 60cm, to desired height, balances with the 0.1mol/L sodium citrate buffer solution of pH=4.6; Carry out gradient elution by the salivary gland secretion concentrated solution of 5ml or above-mentioned solution of trichloroacetic acid loading and draw out elution curve, Fractional Collections elutriant with nucleic acid-protein detector record, flow velocity is 30ml/ hour; Vigor part will be had after testing to concentrate in dialysis tubing concentrate with glycerine, to obtain final product.
In described step C, the concrete operations of gel filtration chromatography are as follows:
Get appropriate sephadex ion-exchanger adding distil water and soak 4 hours, with 0.5mol/L NaOH-0.5mol/L NaCl mixed liquid dipping half an hour, suction filtration removing alkali liquid is also washed with distilled water to neutrality, heated and boiled removing bubble; Dress post 1.5cm x 50cm is to desired height, and balance with the 0.1mol/L Tris-HCl-NaCl damping fluid of pH=7.4, by the above-mentioned dialysis tubing glycerine concentrated solution loading wash-out having vigor elutriant, Fractional Collections elutriant, flow velocity is 90ml/ hour.
Embodiment 3
The method extracting r-hirudin from leech saliva of the present invention, comprises the following steps:
A, leech to be placed in container fully hungry, drop into inductive substance to leech inhale full after, add emetic and make its spue saliva in body and natural hirudin crude product liquid, then take out hirudinaria manillensis live body and after being rinsed well, return rearing pool and continue to raise; R-hirudin crude product liquid simultaneously in collection container, put refrigerator and cooled freeze preserve stand-by;
Described inductive substance is made up of earthworm extract 200ml, chitosan 40g, astragalus polysaccharides 15g, glucose powder 20g;
Described emetic is the mixed solution of zinc sulfate and ethanol, and its ratio is 1:1, and the weight ratio between the consumption of emetic and leech live body is 1:8;
B, thaw above-mentioned r-hirudin crude product, get 290ml add 4 times to put in refrigerator precooling at 4 DEG C containing in the cold acetone of 15% water, stir in rearmounted refrigerator and at 4 DEG C, allow its precipitates overnight, sucking-off next day supernatant acetone is also centrifugal, the trichoroacetic acid(TCA) 28ml adding precooling in gained precipitation makes it dissolve, centrifugal removing residue, obtains salivary gland secretion concentrated solution;
C, with anion-exchange column chromatography, wash-out is carried out to above-mentioned salivary gland secretion concentrated solution after, carry out gel filtration chromatography, to obtain final product.
The time that described leech is fully hungry is 18 days, and the time that leech inhales full inductive substance is 50 minutes.
Described zinc sulfate massfraction is 0.8%, and described ethanol massfraction is 9%.
In described step C, the concrete operations of anion-exchange column chromatography are as follows:
Getting appropriate diethylamino Cellulose anion exchanger adding distil water makes it expand, and is washed with distilled water to neutrality after respectively soaking 300 minutes successively with the NaOH-NaCl mixed solution of the NaOH-NaCl mixed solution of 0.5mol/L, HCl and 0.5mol/L of 0.5mol/L; Dress post 2cm x 60cm, to desired height, balances with the 0.1mol/L sodium citrate buffer solution of pH=4.6; Carry out gradient elution by the salivary gland secretion concentrated solution of 5ml or above-mentioned solution of trichloroacetic acid loading and draw out elution curve, Fractional Collections elutriant with nucleic acid-protein detector record, flow velocity is 30ml/ hour; Vigor part will be had after testing to concentrate in dialysis tubing concentrate with glycerine, to obtain final product.
In described step C, the concrete operations of gel filtration chromatography are as follows:
Get appropriate sephadex ion-exchanger adding distil water and soak 4 hours, with 0.5mol/L NaOH-0.5mol/L NaCl mixed liquid dipping half an hour, suction filtration removing alkali liquid is also washed with distilled water to neutrality, heated and boiled removing bubble; Dress post 1.5cm x 50cm is to desired height, and balance with the 0.1mol/L Tris-HCl-NaCl damping fluid of pH=7.4, by the above-mentioned dialysis tubing glycerine concentrated solution loading wash-out having vigor elutriant, Fractional Collections elutriant, flow velocity is 90ml/ hour.
Embodiment 4
The method extracting r-hirudin from leech saliva of the present invention, comprises the following steps:
A, leech to be placed in container fully hungry, drop into inductive substance to leech inhale full after, add emetic and make its spue saliva in body and natural hirudin crude product liquid, then take out hirudinaria manillensis live body and after being rinsed well, return rearing pool and continue to raise; R-hirudin crude product liquid simultaneously in collection container, put refrigerator and cooled freeze preserve stand-by;
Described inductive substance is made up of earthworm extract 300ml, chitosan 50g, astragalus polysaccharides 20g, glucose powder 30g;
Described emetic is the mixed solution of zinc sulfate and ethanol, and its ratio is 1:2, and the weight ratio between the consumption of emetic and leech live body is 1:10;
B, thaw above-mentioned r-hirudin crude product, get 00ml add 4 times to put in refrigerator precooling at 4 DEG C containing in the cold acetone of 15% water, stir in rearmounted refrigerator and at 4 DEG C, allow its precipitates overnight, sucking-off next day supernatant acetone is also centrifugal, the trichoroacetic acid(TCA) 30ml adding precooling in gained precipitation makes it dissolve, centrifugal removing residue, obtains salivary gland secretion concentrated solution;
C, with anion-exchange column chromatography, wash-out is carried out to above-mentioned salivary gland secretion concentrated solution after, carry out gel filtration chromatography, to obtain final product.
The time that described leech is fully hungry is 20 days, and the time that leech inhales full inductive substance is 60 minutes.
Described zinc sulfate massfraction is 1%, and described ethanol massfraction is 10%.
In described step C, the concrete operations of anion-exchange column chromatography are as follows:
Getting appropriate diethylamino Cellulose anion exchanger adding distil water makes it expand, and is washed with distilled water to neutrality after respectively soaking 300 minutes successively with the NaOH-NaCl mixed solution of the NaOH-NaCl mixed solution of 0.5mol/L, HCl and 0.5mol/L of 0.5mol/L; Dress post 2cm x 60cm, to desired height, balances with the 0.1mol/L sodium citrate buffer solution of pH=4.6; Carry out gradient elution by the salivary gland secretion concentrated solution of 5ml or above-mentioned solution of trichloroacetic acid loading and draw out elution curve, Fractional Collections elutriant with nucleic acid-protein detector record, flow velocity is 30ml/ hour; Vigor part will be had after testing to concentrate in dialysis tubing concentrate with glycerine, to obtain final product.
In described step C, the concrete operations of gel filtration chromatography are as follows:
Get appropriate sephadex ion-exchanger adding distil water and soak 4 hours, with 0.5mol/L NaOH-0.5mol/L NaCl mixed liquid dipping half an hour, suction filtration removing alkali liquid is also washed with distilled water to neutrality, heated and boiled removing bubble; Dress post 1.5cm x 50cm is to desired height, and balance with the 0.1mol/L Tris-HCl-NaCl damping fluid of pH=7.4, by the above-mentioned dialysis tubing glycerine concentrated solution loading wash-out having vigor elutriant, Fractional Collections elutriant, flow velocity is 90ml/ hour.
Embodiment 5
From leech saliva, extract the method for r-hirudin, comprise the following steps:
A, leech to be placed in container fully hungry, drop into inductive substance to leech inhale full after, add emetic and make its spue saliva in body and natural hirudin crude product liquid, then take out hirudinaria manillensis live body and after being rinsed well, return rearing pool and continue to raise; R-hirudin crude product liquid simultaneously in collection container, put refrigerator and cooled freeze preserve stand-by;
Described inductive substance is made up of earthworm extract 200ml, chitosan 10g, astragalus polysaccharides 15g, glucose powder 15g;
Described emetic is the mixed solution of zinc sulfate and ethanol, and its ratio is 1:2, and the weight ratio between the consumption of emetic and leech live body is 1:2;
B, thaw above-mentioned r-hirudin crude product, get 250 ~ 300ml add 4 times to put in refrigerator precooling at 4 DEG C containing in the cold acetone of 15% water, stir in rearmounted refrigerator and at 4 DEG C, allow its precipitates overnight, sucking-off next day supernatant acetone is also centrifugal, the trichoroacetic acid(TCA) 25ml adding precooling in gained precipitation makes it dissolve, centrifugal removing residue, obtains salivary gland secretion concentrated solution;
C, with anion-exchange column chromatography, wash-out is carried out to above-mentioned salivary gland secretion concentrated solution after, carry out gel filtration chromatography, to obtain final product.
The time that described leech is fully hungry is 15 days, and the time that leech inhales full inductive substance is 50 minutes.
Described zinc sulfate massfraction is 0.25%, and described ethanol massfraction is 6%.
In described step C, the concrete operations of anion-exchange column chromatography are as follows:
Getting appropriate diethylamino Cellulose anion exchanger adding distil water makes it expand, and is washed with distilled water to neutrality after respectively soaking 300 minutes successively with the NaOH-NaCl mixed solution of the NaOH-NaCl mixed solution of 0.5mol/L, HCl and 0.5mol/L of 0.5mol/L; Dress post 2cm x 60cm, to desired height, balances with the 0.1mol/L sodium citrate buffer solution of pH=4.6; Carry out gradient elution by the salivary gland secretion concentrated solution of 5ml or above-mentioned solution of trichloroacetic acid loading and draw out elution curve, Fractional Collections elutriant with nucleic acid-protein detector record, flow velocity is 30ml/ hour; Vigor part will be had after testing to concentrate in dialysis tubing concentrate with glycerine, to obtain final product.
In described step C, the concrete operations of gel filtration chromatography are as follows:
Get appropriate sephadex ion-exchanger adding distil water and soak 4 hours, with 0.5mol/L NaOH-0.5mol/L NaCl mixed liquid dipping half an hour, suction filtration removing alkali liquid is also washed with distilled water to neutrality, heated and boiled removing bubble; Dress post 1.5cm x 50cm is to desired height, and balance with the 0.1mol/L Tris-HCl-NaCl damping fluid of pH=7.4, by the above-mentioned dialysis tubing glycerine concentrated solution loading wash-out having vigor elutriant, Fractional Collections elutriant, flow velocity is 90ml/ hour.
Claims (5)
1. from leech saliva, extract a method for r-hirudin, it is characterized in that, comprise the following steps:
A, leech to be placed in container fully hungry, drop into inductive substance to leech inhale full after, add emetic and make its spue saliva in body and natural hirudin crude product liquid, then take out hirudinaria manillensis live body and after being rinsed well, return rearing pool and continue to raise; R-hirudin crude product liquid simultaneously in collection container, put refrigerator and cooled freeze preserve stand-by;
Described inductive substance is made up of earthworm extract 10 ~ 300ml, chitosan 5 ~ 50g, astragalus polysaccharides 1 ~ 20g, glucose powder 1 ~ 30g;
Described emetic is the mixed solution of zinc sulfate and ethanol, and its ratio is 1:1 ~ 2, and the weight ratio between the consumption of emetic and leech live body is 1:1 ~ 10;
B, thaw above-mentioned r-hirudin crude product, get 250 ~ 300ml add 4 times to put in refrigerator precooling at 4 DEG C containing in the cold acetone of 15% water, stir in rearmounted refrigerator and at 4 DEG C, allow its precipitates overnight, sucking-off next day supernatant acetone is also centrifugal, the trichoroacetic acid(TCA) 20-30ml adding precooling in gained precipitation makes it dissolve, centrifugal removing residue, obtains salivary gland secretion concentrated solution;
C, with anion-exchange column chromatography, wash-out is carried out to above-mentioned salivary gland secretion concentrated solution after, carry out gel filtration chromatography, to obtain final product.
2. the method extracting r-hirudin from leech saliva according to claim 1, is characterized in that: the time that described leech is fully hungry is 15 ~ 20 days, and the time that leech inhales full inductive substance is 20 ~ 50 minutes.
3. the method extracting r-hirudin from leech saliva according to claim 1, is characterized in that: described zinc sulfate massfraction is 0.05 ~ 1%, and described ethanol massfraction is 5 ~ 10%.
4. the method extracting r-hirudin from leech saliva according to claim 1, is characterized in that, in described step C, the concrete operations of anion-exchange column chromatography are as follows:
Getting appropriate diethylamino Cellulose anion exchanger adding distil water makes it expand, and is washed with distilled water to neutrality after respectively soaking 300 minutes successively with the NaOH-NaCl mixed solution of the NaOH-NaCl mixed solution of 0.5mol/L, HCl and 0.5mol/L of 0.5mol/L; Dress post 2cm x 60cm, to desired height, balances with the 0.1mol/L sodium citrate buffer solution of pH=4.6; Carry out gradient elution by the salivary gland secretion concentrated solution of 5ml or above-mentioned solution of trichloroacetic acid loading and draw out elution curve, Fractional Collections elutriant with nucleic acid-protein detector record, flow velocity is 30ml/ hour; Vigor part will be had after testing to concentrate in dialysis tubing concentrate with glycerine, to obtain final product.
5. the method extracting r-hirudin from leech saliva according to claim 1, is characterized in that, in described step C, the concrete operations of gel filtration chromatography are as follows:
Get appropriate sephadex ion-exchanger adding distil water and soak 4 hours, with 0.5mol/L NaOH-0.5mol/L NaCl mixed liquid dipping half an hour, suction filtration removing alkali liquid is also washed with distilled water to neutrality, heated and boiled removing bubble; Dress post 1.5cm x 50cm is to desired height, and balance with the 0.1mol/L Tris-HCl-NaCl damping fluid of pH=7.4, by the above-mentioned dialysis tubing glycerine concentrated solution loading wash-out having vigor elutriant, Fractional Collections elutriant, flow velocity is 90ml/ hour.
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| CN105693851A (en) * | 2016-04-16 | 2016-06-22 | 徐顺青 | Hirudin extracting method |
| CN106188277A (en) * | 2016-09-30 | 2016-12-07 | 重庆志微生物技术有限公司 | A kind of blood-eating hirudo adult feeds and the sustainable extracting method of hirudin and special equipment |
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| CN105693851A (en) * | 2016-04-16 | 2016-06-22 | 徐顺青 | Hirudin extracting method |
| CN106188277A (en) * | 2016-09-30 | 2016-12-07 | 重庆志微生物技术有限公司 | A kind of blood-eating hirudo adult feeds and the sustainable extracting method of hirudin and special equipment |
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| CN107325177A (en) * | 2017-09-01 | 2017-11-07 | 荆州市民康生物科技有限公司 | A kind of method for improving Bufrudin extract yield |
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| CN109527270A (en) * | 2018-12-10 | 2019-03-29 | 苏州大学 | Leech specific nutrient induces the extracting method of liquid and natural hirudin |
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| CN110585424A (en) * | 2019-09-18 | 2019-12-20 | 周维海 | Preparation method of leech polypeptide medicinal liquor for preventing and treating cardiovascular and cerebrovascular diseases |
| CN110623275A (en) * | 2019-09-18 | 2019-12-31 | 周维海 | Composite amino acid gilt-edged leech polypeptide food with anticoagulant and antithrombotic effects and preparation method thereof |
| CN112480243A (en) * | 2021-01-06 | 2021-03-12 | 广西科康科技集团有限公司 | Large-scale hirudin separation and purification production process method and equipment |
| CN113072638A (en) * | 2021-03-16 | 2021-07-06 | 宁波博睿瀚达生物科技有限公司 | Method for removing endotoxin in recombinant hirudin protein solution |
| CN113577821A (en) * | 2021-07-07 | 2021-11-02 | 三峡大学 | Repeated extraction device and method for hirudin in living body |
| CN113577821B (en) * | 2021-07-07 | 2022-08-30 | 三峡大学 | Repeated extraction device and method for hirudin in living body |
| CN116987181A (en) * | 2023-09-27 | 2023-11-03 | 北京元延医药科技股份有限公司 | High biological activity natural hirudin and method for preparing same in high yield |
| CN116987181B (en) * | 2023-09-27 | 2023-12-08 | 北京元延医药科技股份有限公司 | High biological activity natural hirudin and method for preparing same in high yield |
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