CN101862350A - Active ingredients of scorpion and application thereof - Google Patents
Active ingredients of scorpion and application thereof Download PDFInfo
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Abstract
The invention discloses two active ingredients of scorpion and application thereof. The invention extracts the scorpion through a special simulated enzymatic hydrolysis method, and in addition, pepsin enzymolysis ingredients are separated by a gel filtration chromatography method. Experiments prove that compared with a traditional extraction process, the invention has the advantages that the effects of anticoagulation and thrombolysis of the active ingredients obtained by the simulated enzymatic hydrolysis method used by the invention are more obvious, the anticoagulation active ingredients obtained by the scorpion pepsin enzymolysis ingredients separation by the gel filtration chromatography method are micromolecular peptides.
Description
Technical field
The present invention relates to the active component and the extraction separation method thereof of two kinds of Scorpio anticoagulations and thrombolytic, especially, relate to the extracting method and the gel permeation chromatography isolation and purification method of bionic enzymatic.
Background technology
Scorpio (Bathus ma rtensii kansch) is the dry thing of Buthidae arthropod, the property suffering, and flavor is flat, and is poisonous, returns Liver Channel.Have effects such as removing obstruction in the collateral to relieve pain, relieving spasm by subduing liver-wind, counteracting toxic substances dissipating blood stasis, be clinical antineoplastic agent commonly used, have and studies show that the main effective ingredient of live body Scorpio is a scorpion venom, effective ingredient is still unclear in the medical material, therefore how to be used as medicine at present with full powder, it is big to exist dosage, easily causes disadvantages such as microbial contamination.In view of having only, macromolecular substances such as animal proteinoid, mucopolysaccharide are subjected to effects such as digestive enzyme, acid, alkali after arriving gastrointestinal tract, can or be hydrolyzed into micromolecular peptide, oligosaccharide and other small-molecule substances by enzymolysis, be absorbed into blood and drug effect takes place, so digestion process in the enzyme bionic extraction method analogue body, the effective ingredient therapeutic effect of extraction is better.
Gel permeation chromatography is called size exclusion, gel exclusion, molecular sieve or gel permeation chromatography again, vary in size according to protein molecule and to reach separating effect, contain a large amount of micropores in the gel filtration filler, only allow buffer and small molecular weight protein or peptide class to pass through, outside macro-molecular protein and some albumen compositions then are blocked in; High-molecular weight peptide class flows in the filler particles gap, is earlier eluted than low molecular weight peptide class, reaches the purpose of fractionated, and can reduce because of protein loss or inactivation due to the irreversible fixation.
Summary of the invention
The object of the invention provides active component and its extraction separation method and the application thereof of two kinds of Scorpio anticoagulations and thrombolytic.
The invention provides the active component of two kinds of Scorpio anticoagulations and thrombolytic, a kind of active component is to separate the product that obtains through pepsin, and another kind of active component is first through the pepsin enzymolysis, and then separates the product that obtains through trypsin.
Preferably, anticoagulant active component is a small-molecule peptide in the described pepsin hydrolysis products.
More preferably, described small-molecule peptide is that molecular weight is the small-molecule peptide of 850-5300.
The present invention also provides two kinds of extraction method of active ingredients, and it is made up of following steps:
(1) Scorpio medical material fine powder adds simulated gastric fluid, airtight, in 37 ℃ of water bath with thermostatic control effects after 30 minutes, added 3.0% pepsin hydrolysis 3 hours, enzymolysis solution is placed 85 ℃ of water bath with thermostatic control effects 15 minutes, be cooled to room temperature, centrifugal 15 minutes with 4200r/min, get supernatant, the dry Scorpio pepsin zymolyte that gets;
(2) step (1) gained precipitation is dry, continue and add 20 times of amount simulated intestinal fluids, airtight, after 30 minutes, added 5% trypsin hydrolyzing 4 hours in 37 ℃ of water bath with thermostatic control effects, with enzymolysis solution in 85 ℃ of water bath with thermostatic control effects 15 minutes, be cooled to room temperature, with 4200r/min centrifugal 15 minutes, get supernatant, dry Scorpio trypsin digestion thing.
Preferably, solid-liquid bulking value part ratio of Scorpio fine powder and simulated gastric fluid is 1: 20 in the described step (1).
The present invention also provides the isolation and purification method of Scorpio pepsin enzymolysis composition, and it is made up of following steps:
(1) pretreatment of filler: take by weighing an amount of polydextran gel respectively, added the large volume water boil 1 hour, naturally cool to room temperature, topple over supernatant repeatedly to remove the suspension crushed particles;
(2) chromatographic column is installed: the chromatographic column specification is 10mm * 30cm, cleans all component, is vertically mounted on the iron stand, closes bottom piston;
(3) load: floating stuffing, use Glass rod slowly to inject filler continuously, and water fills chromatographic column along wall, open bottom piston, constant towards post to column volume, with 3 times of column volume phosphate buffer solutions towards post;
(4) go up sample: Scorpio medical material fine powder adds simulated gastric fluid, airtight, after 30 minutes, added 3.0% pepsin hydrolysis 3 hours in 37 ℃ of water bath with thermostatic control effects, enzymolysis solution is placed 85 ℃ of water bath with thermostatic control effects 15 minutes, be cooled to room temperature, with 4200r/min centrifugal 15 minutes, get supernatant, adjust concentration to 20mg/mL, last sample 2mL is no more than 10% of column volume;
(5) eluting: wash post with phosphate buffer solution, flow velocity 1.0mL/min is in charge of collection;
(6) sample detection: 1. 280 nanometer optical absorption methods: measure protein content at ultraviolet 280nm wavelength place; 2. Coomassie brilliant blue G-250 method: detecting wavelength 595nm place mensuration protein content.
Scorpio bionic enzymatic hydrolysate anticoagulation and thrombolysis activity evaluation:
The present invention adopts activated partial thrombin time method (APTT method) and fibrinogen plate assay that the anticoagulation and the thrombolytic effect of active component are estimated, and evaluation methodology is as follows:
1 material
1.1 medical material
Scorpio is Scorpio dry body (Hebei Yiling Medicine Inst. Co., Ltd provides), and it is dry below 60 ℃ to put drying baker, is ground into fine powder, standby.
1.2 reagent
Bovine fibrinogen (Sigma F8630 is available from Beijing ring Ya Taike biomedical technology company limited); Trypsin 1: 250 (Amresco0458 is available from the magnificent transduction in Beijing Science and Technology Ltd.); Pepsin 1: 10000 (Sigma P7000 is available from Beijing ring Ya Taike biomedical technology company limited); Medical injection urokinase (available from Wangjing, Beijing hospital); Thrombin (Sigma T4648 is available from Beijing ring Ya Taike biomedical technology company limited); Activated partial thrombin time (APTT) reagent 10*1.5ml (available from Shanghai sun biological reagent company); Trichloroacetic acid (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group); Ethanol (analytical pure).
(take by weighing sodium chloride 9g, adding distil water is settled to 1000ml to normal saline, shakes up, promptly); (get hydrochloric acid 9ml, adding distil water is diluted to 1000ml to simulated gastric fluid, promptly); Simulated intestinal fluid (is got 0.2mol/L potassium dihydrogen phosphate 250ml, is added 0.2mol/L sodium hydroxide solution 118ml, be diluted with water to 1000ml, shake up, promptly).
1.3 animal
Healthy Japan large ear rabbit (male), the about 2Kg of body weight (Beijing Vital River Experimental Animals Technology Co., Ltd. provides) meets national health one-level animal standard.
1.4 instrument
UV-2000 type spectrophotometer; 800B type centrifuge (Anting Scientific Instrument Factory, Shanghai); ES-120 type electronic analytical balance (the flat development in science and technology company limited in Hunan, Changsha); Thermostat water bath (Yuyao City east electric instrument factory); DZ-1BC type vacuum drying oven (Tianjin Tai Site Instr Ltd.).
2 methods
2.1 bionic enzymatic method of the present invention
(1) Scorpio medical material fine powder adds simulated gastric fluid (solid-liquid w/v 1: 20), airtight, behind 37 ℃ of water bath with thermostatic control effect 30min, add 3.0% pepsin hydrolysis 3h, enzymolysis solution is placed 85 ℃ of water bath with thermostatic control effect 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, the dry Scorpio pepsin zymolyte that gets;
(2) precipitation in the step (1) is dry, continue and add 20 times of amount simulated intestinal fluids, airtight, behind 37 ℃ of water bath with thermostatic control effect 30min, add 5% trypsin hydrolyzing 4h, with enzymolysis solution in 85 ℃ of water bath with thermostatic control effect 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, the dry Scorpio trypsin digestion thing that gets.
2.2 decoction and alcohol sedimentation technique
Take by weighing 2g Scorpio medical material fine powder in the 50ml round-bottomed flask, add 8 times of amount distilled water, decoct 1h, gauze leaches decoction liquor; Medicinal residues add 6 times of amount distilled water again, decoct twice, each 30min, merge 3 times filter liquor, it is 1: 1 that water-bath is concentrated into the medicinal liquid ratio, and stirring adding 95% ethanol is 70% to containing the alcohol amount, put and place the centrifugal 20min of back 4200r/min that spends the night in the refrigerator, get supernatant and volatilize, 60 ℃ of vacuum dryings are weighed, and get Scorpio water extracting alcohol hypostasis.
2.3 normal saline extraction
Take by weighing 2g Scorpio medical material fine powder in the 50ml round-bottomed flask, add the 40ml normal saline, in 37 ℃ of thermostat water bath lixiviate 4h, the centrifugal 15min of 4200r/min gets supernatant, and 60 ℃ of vacuum dryings are weighed, and gets Scorpio normal saline extract.
2.4 compound enzyme solution
Take by weighing 2g Scorpio medical material fine powder in the 50ml round-bottomed flask, add the 40ml simulated gastric fluid, add the 60mg pepsin hydrolysis after being enclosed within 37 ℃ of water-bath 30min, regulate the pH=6.8 of enzymolysis solution behind the 3h with 1mol/mlNaOH solution, add 100mg trypsin digestion 4h, in 85 ℃ of water-bath enzyme denaturing 15min, the centrifugal 15min of 4200r/min, get supernatant, 60 ℃ of vacuum dryings are weighed, and get the dry composite zymolyte.
3 experimental results
3.1 the preparation of sample liquid
It is extract obtained an amount of that precision takes by weighing various extracting method, adds 320 μ l normal saline, and making concentration is the sample (being 0.7813mg/ μ l) of 250mg (crude drug amount)/320 μ l.
3.2APTT method
3.2.1 the preparation of blood plasma
Select male Japan large ear rabbit for use, the anesthesia of auricular vein injection pentobarbital sodium, carotid artery is got blood, adds 3.8% sodium citrate anticoagulant (1: 9), and with 3000r/min, centrifugal 15min gets supernatant behind the mixing, isolates platelet poor plasma (PPP).
3.2.2 determination step
Get PPP100 μ l in test trough, add the APTT reagent 100 μ l of sample liquid pre-temperature 5min of 10 μ l and pre-temperature 10min, hatch the CaCl that adds pre-temperature 15min behind the 5min
2100 μ l, timing is simultaneously stirred solution with needle point, when thread concretion is provoked by pin, writing time.
Blank group: get PPP 100 μ l as stated above, add 10 μ l normal saline and 100 μ l APTT reagent, hatch the CaCl that adds pre-temperature 15min behind the 5min
2100 μ l, setting time is write down in timing simultaneously.Each sample is parallel does 6 parts.The results are shown in Table 1.
The various extracting solution APTT of table 1 anticoagulant drug effect is investigated
Compare * P＜0.05 with the water extract-alcohol precipitation group
Anticoagulant experiment effect by 5 kinds of extracts of Scorpio relatively, the result shows that the enzymolysis group is better than conventional solvent extraction group, bionic enzymatic group anticoagulant effect optimum wherein, method of proof is feasible, and the effective ingredient anticoagulant effect of extracting is best.
3.3 fibrinogen plate assay
Fibrinogen has formed the fibrin plate that solidifies after thrombin adds.Contain the sample of plasminogen activator when adding after, the plasminogen that plasminogen activator activates in the flat board is converted into fibrinolysin, and fibrinolysin is hydrolysis of fibrin again, thereby forms aqueous dissolving circle in the fibrin that solidifies.
3.3.1 the preparation of test solution
Phosphate buffer (PBS): take by weighing the 3.12g sodium dihydrogen phosphate, the adding distil water standardize solution promptly gets A liquid to 100ml; Take by weighing sodium hydrogen phosphate 7.1632g, the adding distil water standardize solution promptly gets B liquid to 100ml; During use, get A liquid 760 μ l, B liquid 3240 μ l and add the 76ml normal saline, promptly get phosphate buffer.
Fibrinogen solution: take by weighing Fibrinogen 0.3g and add 60mlPBS, jolting, dissolving, promptly.
3.3.2 the preparation of fibrin plate
In plate, add 9mL 5gL
-1Fibrinogen solution, add 2 * 10 again
3μ L
-1Thrombin solution 1mL, the about 10s of mixing that turns round and round immediately promptly forms the thick fibrin clot of 1mm, adds filter paper at the plate loam cake, promptly forms fibrin plate after 4h is put at the level place.
3.3.3 the preparation of heating fibrin plate
Get the fibrin plate of having made, place baking 30min in 85 ℃ of baking ovens, be the heating fibrin plate after the cooling.
3.3.4 determination step
With sample liquid, urokinase (6 * 10
5μ L
-1) and each 10 μ L of normal saline put respectively on fibrin plate, each dot spacing is greater than 1.5cm.Put fibrin plate in wet box, put into 37 ℃ of baking ovens, observe behind the 4h to have or not and dissolve circle, and measure the diameter of dissolving circle, calculating dissolving circle area is with the big or small size of weighing fibrinolytic of dissolving circle area.See Table 2 and table 3.
Dissolving circle area (mm
2)=[(major diameter+minor axis)/4]
2* π.
The flat thrombolytic experimental result of table 2 fibrin (n=4)
Last table as can be seen, the Scorpio enzymatic isolation method is better than traditional extraction process, wherein the gastric enzyme enzymolysis of bionic enzymatic method gained part and pancreatin enzymolysis partly have obvious dissolving circle, prove that further the bionic enzymatic method is feasible.
The dull and stereotyped thrombolytic experimental result (n=4) of table 3 Scorpio bionic enzymatic fibres albumen
As can be seen, the dissolving circle of pepsin zymolyte and stock solution thereof, pancreatin zymolyte is obvious; Pepsin enzymolysis thing pH when 1.96 transfer to 5 left and right sides, is not had obvious thrombolytic effect, show the necessity of digestion process in the bionic enzymatic analogue body.
4 conclusions
4.1APTT result of the test shows
The various extracting method of Scorpio are extract obtained all to prolong APTT action time to a certain extent, wherein the gastric enzyme enzymolysis of bionic enzymatic method gained part and pancreatin enzymolysis part A PTT time the longest, so the bionic enzymatic method is feasible.
4.2 the Fibrinogen treadmill test is the result show
The gastric enzyme enzymolysis part of bionic enzymatic method gained and pancreatin enzymolysis part obviously increase than the dissolving circle of other extracts, prove that the process program of bionic enzymatic is feasible.Its reason is: the bionic enzymatic method has been simulated digestion process in the body of the oral back of animal drugs, and the animal body high molecular weight protein is degraded to small-molecule substance through gastroenteric environment, makes live part appear its active effect.Gastric enzyme enzymolysis stock solution partly also has active, shows the necessity of further using the pancreatin enzymolysis.
Gel permeation chromatography separates the active component anticoagulant active evaluation that obtains:
1 material
1.1 medical material
Scorpio pepsin enzymolysis solution
1.2 reagent and material
Bovine serum albumin (Sigma A7906 is available from Beijing ring Ya Taike biomedical technology company limited); APTT reagent 10 * 1.5ml (available from Shanghai sun biological reagent company); Ethanol (analytical pure); Watson pure water (Guangzhou Watson food and drink company limited); Sephadex g-100 (available from Beijing ring Ya Taike biomedical technology company limited).
(take by weighing sodium chloride 9g, adding distil water is settled to 1000ml to normal saline, shakes up, promptly); Phosphate buffer (PBS): take by weighing the 3.12g sodium dihydrogen phosphate, the adding distil water standardize solution promptly gets A liquid to 100ml; Take by weighing sodium hydrogen phosphate 7.1632g, the adding distil water standardize solution promptly gets B liquid to 100ml; During use, get A liquid 22.8ml, B liquid 97.2ml and add the 2280ml normal saline, promptly get phosphate buffer 2.4L, chromatographic column 10mm * 30cm (available from converging instrument Science and Technology Ltd. of extra large section in Beijing).
1.3 animal
Healthy Japan large ear rabbit (male), the about 2Kg of body weight provides by Beijing Vital River Experimental Animals Technology Co., Ltd., meets national health one-level animal standard.
1.4 instrument
UV-2000 type spectrophotometer; 800B type centrifuge (Anting Scientific Instrument Factory, Shanghai); ES-120 type electronic analytical balance (the flat development in science and technology company limited in Hunan, Changsha); Thermostat water bath (Yuyao City east electric instrument factory); DZ-1BC type vacuum drying oven (Tianjin Tai Site Instr Ltd.), MALDI-TOF-MASS mass spectrograph (measuring) in the Department Of Medicine, Peking University.
2. method and result
2.1 sephadex chromatography
2.1.1 the pretreatment of filler
Take by weighing an amount of sephadex g-100 respectively, add large volume water boil 1h.Naturally cool to room temperature, topple over supernatant repeatedly to remove the suspension crushed particles.
2.1.2 installation chromatographic column
Select 10mm * 30cm chromatographic column, clean all component.Be vertically mounted on the iron stand, close bottom piston
2.1.3 load
Floating stuffing uses Glass rod slowly to inject filler continuously along wall, and water fills chromatographic column, opens bottom piston, and is constant towards post to column volume, with 3 times of column volume phosphate buffer solutions towards post.
2.1.4 last sample
Scorpio medical material fine powder adds simulated gastric fluid, airtight, after 30 minutes, added 3.0% pepsin hydrolysis 3 hours in 37 ℃ of water bath with thermostatic control effects, enzymolysis solution is placed 85 ℃ of water bath with thermostatic control effects 15 minutes, be cooled to room temperature, with 4200r/min centrifugal 15 minutes, get supernatant, adjust concentration to 20mg/mL, last sample 2mL is no more than 10% of column volume;
2.1.5 eluting
Wash post with phosphate buffer, flow velocity 1.0ml/min is in charge of collection.
2.1.6 sample detection
2.1.6.1280 nanometer (A280) optical absorption method
Owing to often contain benzene ring structures such as tyrosine, tryptophan, phenylalanine in the protein molecule, maximum absorption band is arranged at ultraviolet 280nm wavelength place, its absorption value is directly proportional with protein concentration, so measure protein content with this method
2.1.6.2 Coomassie brilliant blue G-250 method
The form that red, blue two kinds of different colours are arranged based on Coomassie brilliant blue G-250.Under certain density ethanol and acid condition, can be made into pink solution, when with protein bound after, produce blue chemical compound, be swift in response and stablize.Compound of reaction has maximum absorbance value at 465～595mn place, the depth of chemical compound color and the height of protein concentration are proportional, therefore can detect the size of the absorbance value of 595nm and measure protein content.
2.1.6.3 result and conclusion
Sephadex g-100 chromatography collection of illustrative plates has 2 peaks, and it is yellow that peak 1 is, and peak 2 is a colorless chromogenic.First eluting peak is the big part of molecular weight, and the peak is lower slightly, and content is less; Second eluting peak is the medium part of molecular weight, peak height and wide, and content is many, specifically sees Fig. 1.The separating ranges of sephadex g-100 is 200-12 * 10
5
2.2APTT anticoagulation drug effect experiment
2.2.1 the preparation of sample
The eluting flow point merges enrichment by the ultraviolet determination result, vacuum drying, and it is an amount of that precision takes by weighing each sample drying thing, adds corresponding normal saline, and making concentration is the sample (being 0.7813mg/ μ l) of 250mg (crude drug amount)/320 μ l.
2.2.2 the preparation of blood plasma
Select male Japan large ear rabbit for use, the anesthesia of auricular vein injection pentobarbital sodium, carotid artery is got blood, adds 3.8% sodium citrate anticoagulant (1: 9), and with 3000r/min, centrifugal 15min gets supernatant behind the mixing, isolates platelet poor plasma (PPP).
2.2.3 determination step
Get PPP100 μ l in test trough, add the APTT reagent 100 μ l of sample liquid pre-temperature 5min of 10 μ l and pre-temperature 10min, hatch the CaCl that adds pre-temperature 15min behind the 5min
2100 μ l, timing is simultaneously stirred solution with needle point, when thread concretion is provoked by pin, writing time.
Blank group: get PPP 100 μ l as stated above, add 10 μ l normal saline and 100 μ l APTT reagent, hatch the CaCl that adds pre-temperature 15min behind the 5min
2100 μ l, setting time is write down in timing simultaneously.Each sample is parallel does 6 parts.
2.2.4 experimental result
The anticoagulant effect experiment the results are shown in Table 4.
Table 4 Scorpio protein purification APTT anticoagulant drug effect is investigated (n=6)
Compare * P＜0.05 with the normal saline group.
By the comparison of above anticoagulant experiment drug effect, the collected two parts of Scorpio pepsin zymolyte gel chromatography are suitable with gastric enzyme enzymolysis part anticoagulant effect as can be seen, are defined as active site.
2.3 the mass spectrometry results of sephadex g-100 obtained component
Adopt MALDI-TOF-MASS (substance assistant laser desorpted ionized flight time mass spectrum) that Scorpio protein active position is detected, to determine the range of molecular weight distributions of its active site, by analyzing mass spectrum, the result shows the active site molecular weight distribution between 850～5300, and is small-molecule peptide.
3 conclusions
3.1 the collected two parts of Scorpio pepsin zymolyte gel chromatography are suitable with gastric enzyme enzymolysis part anticoagulant effect as can be seen, are defined as active site.
3.2MAILDIA-TOF-MASS mass spectrum is the result show, Scorpio anticoagulant active position molecular weight distribution is small-molecule peptide 850～5300, and anticoagulant effect obvious (P＜0.05).
Description of drawings
Fig. 1: Sephadex G-100 chromatography collection of illustrative plates: 2 peaks are arranged, and it is yellow that peak 1 is, and peak 2 is a colorless chromogenic.First eluting peak is the big part of molecular weight, and the peak is lower slightly, and content is less; Second eluting peak is the medium part of molecular weight, peak height and wide, and content is many.
Specific embodiment
Embodiment
1, Scorpio anticoagulation and thrombolytic extraction process of effective component:
(1) Scorpio medical material fine powder adds simulated gastric fluid (solid-liquid w/v 1: 20), airtight, behind 37 ℃ of water bath with thermostatic control effect 30min, add 3.0% pepsin hydrolysis 3h, enzymolysis solution is placed 85 ℃ of water bath with thermostatic control effect 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, the dry Scorpio pepsin zymolyte that gets;
(2) precipitation in the step (1) is dry, continue and add 20 times of amount simulated intestinal fluids, airtight, behind 37 ℃ of water bath with thermostatic control effect 30min, add 5% trypsin hydrolyzing 4h, with enzymolysis solution in 85 ℃ of water bath with thermostatic control effect 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, the dry Scorpio trypsin digestion thing that gets.
2, gel-filtration chromatography separation and purification Scorpio anticoagulant active composition
(1) pretreatment of filler: take by weighing an amount of sephadex g-100, added the large volume water boil 1 hour.Naturally cool to room temperature, topple over supernatant repeatedly to remove the suspension crushed particles.
(2) chromatographic column is installed: select 10mm * 30cm chromatographic column, clean all component, be vertically mounted on the iron stand, close bottom piston
(3) load: floating stuffing, use Glass rod slowly to inject filler continuously, and water fills chromatographic column along wall, open bottom piston, constant towards post to column volume, with 3 times of column volume phosphate buffer solutions towards post.
(4) go up sample: Scorpio medical material fine powder adds simulated gastric fluid (solid-liquid w/v 1: 20), airtight, behind 37 ℃ of water bath with thermostatic control effect 30min, add 3.0% pepsin hydrolysis 3h, enzymolysis solution is placed 85 ℃ of water bath with thermostatic control effect 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, adjust concentration to 20mg/mL, last sample 2mL is no more than 10% of column volume.
(5) eluting: wash post with phosphate buffer, flow velocity 1.0ml/min is in charge of collection.
(6) sample detection: 1. 280 nanometer optical absorption methods: measure protein content at ultraviolet 280nm wavelength place; 2. Coomassie brilliant blue G-250 method: detecting wavelength 595nm place mensuration protein content.Evaluation result:
(1), compare other traditional extraction techniques, the gastric enzyme enzymolysis part of bionic enzymatic method gained and the pancreatin enzymolysis effective ingredient in partly, anticoagulation and thrombolytic effect have significant difference.
(2), gel-filtration chromatography separation and purification Scorpio anticoagulant active composition is feasible, its active site molecular weight distribution be small-molecule peptide, and anticoagulant effect is obvious 850～5300.
Claims (6)
1. the active component of two kinds of Scorpio anticoagulations and thrombolytic, it is characterized in that: a kind of active component is to separate the product that obtains through pepsin, another kind of active component is earlier through the pepsin enzymolysis, and then separates the product that obtains through trypsin.
2. two kinds of active component according to claim 1 is characterized in that their extracting method is made up of following steps:
(1) Scorpio medical material fine powder adds simulated gastric fluid, airtight, in 37 ℃ of water bath with thermostatic control effects after 30 minutes, added 3.0% pepsin hydrolysis 3 hours, enzymolysis solution is placed 85 ℃ of water bath with thermostatic control effects 15 minutes, be cooled to room temperature, centrifugal 15 minutes with 4200r/min, get supernatant, the dry Scorpio pepsin zymolyte that gets;
(2) step (1) gained precipitation is dry, continue and add 20 times of amount simulated intestinal fluids, airtight, after 30 minutes, added 5% trypsin hydrolyzing 4 hours in 37 ℃ of water bath with thermostatic control effects, with enzymolysis solution in 85 ℃ of water bath with thermostatic control effects 15 minutes, be cooled to room temperature, with 4200r/min centrifugal 15 minutes, get supernatant, dry Scorpio trypsin digestion thing.
3. two kinds of active component according to claim 2 is characterized in that, the solid-liquid w/v of Scorpio fine powder and simulated gastric fluid is 1: 20 in the described extracting method step (1).
4. two kinds of active component according to claim 1 is characterized in that their isolation and purification method is made up of following steps:
(1) pretreatment of filler: take by weighing an amount of polydextran gel respectively, added the large volume water boil 1 hour, naturally cool to room temperature, topple over supernatant repeatedly to remove the suspension crushed particles;
(2) chromatographic column is installed: the chromatographic column specification is 10mm * 30cm, cleans all component, is vertically mounted on the iron stand, closes bottom piston;
(3) load: floating stuffing, use Glass rod slowly to inject filler continuously, and water fills chromatographic column along wall, open bottom piston, constant towards post to column volume, with 3 times of column volume phosphate buffer solutions towards post;
(4) go up sample: Scorpio medical material fine powder adds simulated gastric fluid, airtight, after 30 minutes, added 3.0% pepsin hydrolysis 3 hours in 37 ℃ of water bath with thermostatic control effects, enzymolysis solution is placed 85 ℃ of water bath with thermostatic control effects 15 minutes, be cooled to room temperature, with 4200r/min centrifugal 15 minutes, get supernatant, adjust concentration to 20mg/mL, last sample 2mL is no more than 10% of column volume;
(5) eluting: wash post with phosphate buffer solution, flow velocity 1.0mL/min is in charge of collection;
(6) sample detection: 1. 280 nanometer optical absorption methods: measure protein content at ultraviolet 280nm wavelength place; 2. Coomassie brilliant blue G-250 method: detecting wavelength 595nm place mensuration protein content.
5. active component according to claim 1 is characterized in that, anticoagulant active component is a small-molecule peptide in the described pepsin hydrolysis products.
6. active component according to claim 5 is characterized in that, described small-molecule peptide is that molecular weight is the small-molecule peptide of 850-5300.
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| CN103396472A (en) * | 2013-06-24 | 2013-11-20 | 陕西步长制药有限公司 | Scorpion extract with thrombolytic activity |
| CN104004808A (en) * | 2014-05-12 | 2014-08-27 | 华南理工大学 | Buthus martensii karsch polypeptide having anticoagulant and thrombolytic effects, and enzymatic hydrolysis preparation method and application thereof |
| CN104055800A (en) * | 2013-03-23 | 2014-09-24 | 河北以岭医药研究院有限公司 | Ground beetle protease enzymolytic product and application thereof |
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| CN104055800A (en) * | 2013-03-23 | 2014-09-24 | 河北以岭医药研究院有限公司 | Ground beetle protease enzymolytic product and application thereof |
| CN103330924A (en) * | 2013-06-07 | 2013-10-02 | 朱民生 | Biological extraction method of traditional Chinese medicine activin |
| CN103330924B (en) * | 2013-06-07 | 2015-07-29 | 朱民生 | A kind of biological extraction process of active Chinese drug component element |
| CN103396472A (en) * | 2013-06-24 | 2013-11-20 | 陕西步长制药有限公司 | Scorpion extract with thrombolytic activity |
| CN103396472B (en) * | 2013-06-24 | 2015-04-29 | 陕西步长制药有限公司 | Scorpion extract with thrombolytic activity |
| CN104004808A (en) * | 2014-05-12 | 2014-08-27 | 华南理工大学 | Buthus martensii karsch polypeptide having anticoagulant and thrombolytic effects, and enzymatic hydrolysis preparation method and application thereof |
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