CN104055799A - Leech pepsin enzymolytic product and application thereof - Google Patents
Leech pepsin enzymolytic product and application thereof Download PDFInfo
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- CN104055799A CN104055799A CN201310095753.0A CN201310095753A CN104055799A CN 104055799 A CN104055799 A CN 104055799A CN 201310095753 A CN201310095753 A CN 201310095753A CN 104055799 A CN104055799 A CN 104055799A
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- hirudo
- pepsin
- zymolyte
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Abstract
The invention discloses a leech pepsin enzymolytic product and an application thereof. The leech pepsin enzymolytic product is obtained by selecting leeches, crushing into fine powder; adding the leech fine powder into artificial gastric juice, and adding pepsin for hydrolysis to obtain the leech pepsin enzymolytic product. The leech pepsin enzymolytic product has anticoagulation or thrombolytic effect.
Description
Technical field
The present invention relates to active component and the application thereof of Hirudo anticoagulation and thrombolytic.
Background technology
Hirudo (Hirudo) is the dry all of Hirudinidae animal Hirudo Whitmania pigra Whitman, Hirudo Hirudo nipponica Whitman or whitmania acranulata Whitman Whitmania acranulata Whitman.Property salty, bitter, taste is flat, slightly poisonous, returns Liver Channel.Have that removing blood stasis stimulates the menstrual flow, the effect of Zhu Yu Xiao Disorder, clinically be mainly used in preventing and treating cardiovascular and cerebrovascular disease and anticancer, main effective ingredient that there are some researches show living leech is hirudin, in medical material, effective ingredient is still unclear, therefore be used as medicine mainly with full powder at present, exist dosage large, easily cause the disadvantages such as microbial contamination.In view of only having to arrive after gastrointestinal tract, the macromolecular substances such as animal proteinoid, mucopolysaccharide are subject to the effects such as digestive enzyme, acid, alkali, can or be hydrolyzed into micromolecular peptide, oligosaccharide and other small-molecule substances by enzymolysis, absorbed into serum and drug effect occurs, therefore digestion process in enzyme bionic extraction method analogue body, the effective ingredient therapeutic effect of extraction is better.
Summary of the invention
The object of the invention is to provide Hirudo pepsin zymolyte and the application thereof of a kind of anticoagulation and thrombolytic.
A kind of Hirudo pepsin zymolyte, is made up of following steps:
A, clean Hirudo, be ground into fine powder;
B, in Hirudo fine powder, add simulated gastric fluid, then add pepsin hydrolysis, obtain Hirudo pepsin zymolyte.
Hirudo pepsin zymolyte, is made up of following steps:
A, clean Hirudo, be ground into fine powder;
B, in Hirudo fine powder, add simulated gastric fluid, Hirudo fine powder and simulated gastric fluid w/v are 1:20, then be enclosed within 43 DEG C of waters bath with thermostatic control after 30 minutes, add 2.5% pepsin hydrolysis 4 hours again, then enzymolysis solution is placed in to 85 DEG C of waters bath with thermostatic control 15 minutes, flowing water is cooled to room temperature, with 4000r/min centrifugal 15 minutes, get supernatant, dry, obtain Hirudo pepsin zymolyte.
Hirudo pepsin zymolyte, in the application of preparing in anticoagulation or thrombolytic drug.
In order to verify anticoagulation and the thrombolytic effect of Hirudo pepsin zymolyte, do following experiment:
Hirudo pepsin hydrolysis products anticoagulation and thrombolysis activity evaluation:
The present invention adopts activated partial thrombin time method (APTT method) and anticoagulation and the thrombolytic effect of fibrinogen plate assay to active component to evaluate, and evaluation methodology is as follows:
1 material
1.1 medical material
Hirudo is Scorpio dry body (Shijiazhuang Yiling Pharmaceutical Co., Ltd provides), puts 60 DEG C, drying baker and dry, pulverize into below fine powder, for subsequent use.
1.2 reagent
Bovine fibrinogen (Sigma F8630, purchased from Beijing ring Ya Taike biomedical technology company limited); Trypsin 1:250(Amresco0458, purchased from Beijing magnificent transduction Science and Technology Ltd.); Pepsin 1:10000(Sigma P7000, purchased from Beijing ring Ya Taike biomedical technology company limited); Medical injection urokinase (purchased from Wangjing, Beijing hospital); Thrombin (Sigma T4648, purchased from Beijing ring Ya Taike biomedical technology company limited); Activated partial thrombin time (APTT) reagent 10*1.5ml(is purchased from Shanghai sun biological reagent company); Trichloroacetic acid (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group); Ethanol (analytical pure).
Normal saline (take sodium chloride 9g, adding distil water is settled to 1000ml, shakes up, and to obtain final product); Simulated gastric fluid (get hydrochloric acid 9ml, adding distil water is diluted to 1000ml, to obtain final product); Simulated intestinal fluid (get 0.2mol/L potassium dihydrogen phosphate 250ml, add 0.2mol/L sodium hydroxide solution 118ml, be diluted with water to 1000ml, shake up, to obtain final product).
1.3 animal
Healthy male Japan large ear rabbit, 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 (Changsha Xiang Ping development in science and technology company limited); Thermostat water bath (Yuyao City east electric instrument factory); DZ-1BC type vacuum drying oven (Tianjin Stettlen Instrument Ltd.).
2 methods
2.1 pepsin enzymatic isolation methods
Water intaking trematodiasis fine powder, (get hydrochloric acid 9 mL, adding distil water is diluted to 1000 mL, to obtain final product to add simulated gastric fluid in 1: 20 ratio.pH1.5。), airtight, after 40 DEG C of water bath with thermostatic control effect 30 min, add 2% pepsin, after effect 4 h, enzymolysis solution is placed in to 85 DEG C of water bath with thermostatic control effect 15 min, flowing water is cooled to after room temperature, with 4000 rmin
-1centrifugal 15 min, get supernatant (pH1.5), and residue is for subsequent use.Supernatant is divided into two parts, and a part of low temperature evaporate to dryness is Hirudo pepsin zymolyte I; Another part is adjusted pH4~5 with NaOH solution, and centrifugal, supernatant low temperature evaporate to dryness, is Hirudo pepsin zymolyte II.
2.2 trypsin digestion methods
Water intaking trematodiasis fine powder, adds simulated intestinal fluid (to get 0.2 molL in 1: 20 ratio
-1potassium dihydrogen phosphate 250 mL, add 0.2 molL
-1sodium hydroxide solution 118 mL, are diluted with water to 1000 mL, to obtain final product), airtight, after 50 DEG C of water bath with thermostatic control effect 30 min, add 4% trypsin, after effect 5 h, enzymolysis solution is placed in to 85 DEG C of water bath with thermostatic control effect 15 min, and flowing water is cooled to after room temperature, with 4000 rmin
-1centrifugal 15 min, get supernatant, and low temperature evaporate to dryness obtains Hirudo trypsin digestion thing.
2.3 bionic enzyme solutions
Hirudo coarse powder is first used pepsin enzymolysis, and gained enzymolysis residue is used trypsin digestion again, has both obtained Hirudo bionic enzymatic thing.
2.5 decocting extraction
Hirudo coarse powder (40 order) 8 g 220 mL that add water, decoct 2 h, centrifugal, and precipitation 240 mL that add water continue to boil 2 h, the centrifugal precipitation of going, the supernatant of merging two times centrifugal, 100 DEG C of heating in water bath are concentrated into approximately 15 mL, recentrifuge, 100 DEG C of water-baths of supernatant volatilize, and obtain Hirudo water extract.
3 experimental results
The preparation of 3.1 sample liquid
It is extract obtained appropriate that precision takes various extracting method, adds normal saline, and making concentration is 0.2g(crude drug amount) sample of/mL.
3.2 APTT methods
3.2.1 the preparation of blood plasma
Select male Japan large ear rabbit, the anesthesia of auricular vein injection pentobarbital sodium, carotid artery is got blood, adds 3.8 % sodium citrate anticoagulants (1: 9), and after mixing, with 3000r/min, centrifugal 15min, gets supernatant, isolates platelet poor plasma (PPP).
3.2.2 determination step
In test trough, by the APTT reagent of pre-temperature 10min 100 μ l, (100 μ l PPP add sample liquid 10 μ and l) mix with the blood plasma to be measured of pre-temperature 5min, hatch the CaCl2100 μ l that adds 37 DEG C of pre-temperature after 5min, timing simultaneously, stir solution with needle point, in the time having thread concretion to be provoked by pin, writing time.
Blank group: get as stated above PPP 100 μ l, add 10 μ l normal saline and 100 μ l APTT reagent, hatch the CaCl2100 μ l that adds pre-temperature 15min after 5min, timing simultaneously, records setting time.Each sample is parallel does 6 parts.The results are shown in Table 1.
The each extract A PTT measurement result of table 1 (
±
s n=6)
Group | APTT/s |
Pepsin zymolyte I | 131.18±24.04** |
Pepsin zymolyte II | 41.53±4.37* |
Trypsin digestion thing | 32.91±5.88 |
Bionic enzymatic thing | 30.06±7.24 |
Water extract | 32.33±5.07 |
Normal saline | 27.31±0.84 |
Note: with the comparison of normal saline group, *
p <0.05, * *
p <0.001;
By SAS software
tinspection, result shows, is being 0.2 gmL containing raw medicinal herbs amount
-1situation under, compared with normal saline group, pepsin zymolyte I group, II group can make APTT significant prolongation (
p< 0.001 He
p< 0.05), and other each group comparing difference not statistically significant.
3.3 fibrinogen plate assay
Fibrinogen has formed the fibrin plate solidifying after thrombin adds.When adding after the sample that contains plasminogen activator, the plasminogen that plasminogen activator activates in flat board is converted into fibrinolysin, and fibrinolysin is hydrolysis of fibrin again, thereby in the fibrin solidifying, forms aqueous dissolving circle.
3.3.1 the preparation of test solution
Phosphate buffer (PBS): take 3.12g sodium dihydrogen phosphate, adding distil water standardize solution, to 100ml, obtains A liquid; Take sodium hydrogen phosphate 7.1632g, adding distil water standardize solution, to 100ml, obtains B liquid; When use, get A liquid 760 μ l, B liquid 3240 μ l add 76ml normal saline, obtain phosphate buffer.
Fibrinogen solution: take Fibrinogen 0.3g and add 60mlPBS, jolting, dissolves, and to obtain final product.
3.3.2 the preparation of fibrin plate
In plate, add 9 mL 5 gL
-1fibrinogen solution, then add 2 × 10
3μ L
-1thrombin solution 1 mL, turn round and round immediately and mix about 10s, form the fibrin clot that 1mm is thick, add filter paper at plate upper cover, after 4h is put at level place, form fibrin plate.
3.3.3 heat the preparation of fibrin plate
Get the fibrin plate of having made, be placed in and dry 30 min in 85 DEG C of baking ovens, be heating fibrin plate after cooling.
3.3.4 determination step
By sample liquid, urokinase (6 × 10
5μ L
-1) and the 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 DEG C of baking ovens, after 4 h, observe to have or not and dissolve circle, and measure the diameter that dissolves circle, calculate and dissolve circle area, weigh the size of fibrinolytic to dissolve circle size.Dissolve circle area (mm
2)=[(major diameter+minor axis)/4]
2× π.
In table 2.
The each extract of table 2 to fibrinous dissolution (
±
s n=6)
Group | Dissolve circle area/mm 2 |
Pepsin zymolyte I | 150.55±20.85** |
Pepsin zymolyte II | 41.53±4.73 ** |
Trypsin digestion thing | — |
Bionic enzymatic thing | — |
Water extract | — |
Normal saline | — |
Note: with the comparison of normal saline group, * *
p<0.001.
Result shows, at each extract all containing 0.8 gmL
-1in the situation of raw medicinal herbs, the fibrinolysis activity of pepsin zymolyte and normal saline group relatively have utmost point significant difference (
p<0.001), other each groups all do not show obvious difference.
Table 3 Hirudo pepsin zymolyte APTT measurement result (
±
s n=5)
Group | Concentration/mgmL -1 | APTT/s |
Pepsin zymolyte | 120 | 82.18±8.01** |
Normal saline | — | 27.41±0.99 |
Note: with the comparison of normal saline group, *
p <0.05, * *
p <0.001.
Result shows, Hirudo pepsin zymolyte APTT value compared with normal saline group all have significant prolongation (
p <0.05), illustrate that Hirudo pepsin zymolyte has external anticoagulating active.
Table 4 Hirudo pepsin zymolyte to fibrinous dissolution (
±
s n=6)
Group | Concentration/mgmL -1 | Dissolve circle area/mm 2 |
Pepsin zymolyte | 480 | 150.55±20.85** |
Normal saline | — | — |
Note: with the comparison of normal saline group, * *
p<0.001.
Result shows, Hirudo pepsin zymolyte has fibrinolytic effect under above-mentioned test dose.Illustrate that Hirudo pepsin enzymolysis extracting method is comparatively suitable.
4 conclusions
4.1 APTT result of the tests show
The various extracting method of Hirudo are extract obtained compared with normal saline group, pepsin zymolyte I group, II group can make APTT significant prolongation (
p< 0.001 He
p< 0.05), and other each group and normal saline group comparing difference not statistically significant.Therefore pepsin enzyme process is feasible.
4.2 Fibrinogen treadmill test results show
The gastric enzyme enzymolysis part of pepsin solution gained obviously increases compared with the dissolving circle of other extracts, proves that the process program of pepsin enzymolysis is feasible.
Specific embodiment
Embodiment 1
the concrete preparation process of Hirudo pepsin zymolyte is as follows:
A, clean Hirudo 5g, be ground into fine powder;
B, will in steps A gained Hirudo fine powder, be placed in 250ml round-bottomed flask, then add 100ml simulated gastric fluid to round-bottomed flask, then be enclosed within 43 DEG C of waters bath with thermostatic control after 30 minutes, add 2.5% pepsin hydrolysis 4 hours again, then enzymolysis solution is placed in to 85 DEG C of waters bath with thermostatic control 15 minutes, flowing water is cooled to room temperature, with 4000r/min centrifugal 15 minutes, get supernatant, dry, obtain Hirudo pepsin zymolyte.
Embodiment 2
The concrete preparation process of Hirudo pepsin zymolyte is as follows:
A, clean Hirudo 8g, be ground into fine powder;
B, will in steps A gained Hirudo fine powder, be placed in 250ml round-bottomed flask, then add 160ml simulated gastric fluid to round-bottomed flask, then be enclosed within 43 DEG C of waters bath with thermostatic control after 30 minutes, add 2.5% pepsin hydrolysis 4 hours again, then enzymolysis solution is placed in to 85 DEG C of waters bath with thermostatic control 15 minutes, flowing water is cooled to room temperature, with 4000r/min centrifugal 15 minutes, get supernatant, dry, obtain Hirudo pepsin zymolyte.
Claims (4)
1. a Hirudo pepsin zymolyte, it is characterized in that being made up of following steps:
A, clean Hirudo, be ground into fine powder;
B, in Hirudo fine powder, add simulated gastric fluid, then add pepsin hydrolysis, obtain Hirudo pepsin zymolyte.
2. Hirudo pepsin zymolyte according to claim 1, it is characterized in that being made up of following steps:
A, clean Hirudo, be ground into fine powder;
B, in Hirudo fine powder, add simulated gastric fluid, Hirudo fine powder and simulated gastric fluid w/v are 1:20, then be enclosed within 43 DEG C of waters bath with thermostatic control after 30 minutes, add 2.5% pepsin hydrolysis 4 hours again, then enzymolysis solution is placed in to 85 DEG C of waters bath with thermostatic control 15 minutes, flowing water is cooled to room temperature, with 4000r/min centrifugal 15 minutes, get supernatant, dry, obtain Hirudo pepsin zymolyte.
3. according to the Hirudo pepsin zymolyte described in claim 1-2 any one, it is characterized in that: in the application of preparing in anticoagulation or thrombolytic drug.
4. Hirudo pepsin zymolyte according to claim 3, is characterized in that: the dosage form of described medicine is capsule, tablet, granule, powder, oral liquid or pill.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN107287269A (en) * | 2017-06-19 | 2017-10-24 | 山东大学 | A kind of HIRULOG preparation technology for mitigating leech bleeding adverse reaction |
CN107412270A (en) * | 2017-08-24 | 2017-12-01 | 无为县慧富水蛭养殖专业合作社 | A kind of leech bionic enzymatic preparation method |
CN109907145A (en) * | 2019-04-24 | 2019-06-21 | 湖南省定生保健品经营有限公司 | The preparation method and application of tea therapy drink |
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WO1994004558A1 (en) * | 1992-08-21 | 1994-03-03 | Receptor Laboratories, Inc. | Method for generating and screening useful peptides |
CN101759775A (en) * | 2008-11-13 | 2010-06-30 | 北京和润创新医药科技发展有限公司 | Leech active small peptide and preparation method and application thereof |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107287269A (en) * | 2017-06-19 | 2017-10-24 | 山东大学 | A kind of HIRULOG preparation technology for mitigating leech bleeding adverse reaction |
CN107412270A (en) * | 2017-08-24 | 2017-12-01 | 无为县慧富水蛭养殖专业合作社 | A kind of leech bionic enzymatic preparation method |
CN109907145A (en) * | 2019-04-24 | 2019-06-21 | 湖南省定生保健品经营有限公司 | The preparation method and application of tea therapy drink |
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