CN104055800A - Ground beetle protease enzymolytic product and application thereof - Google Patents
Ground beetle protease enzymolytic product and application thereof Download PDFInfo
- Publication number
- CN104055800A CN104055800A CN201310094652.1A CN201310094652A CN104055800A CN 104055800 A CN104055800 A CN 104055800A CN 201310094652 A CN201310094652 A CN 201310094652A CN 104055800 A CN104055800 A CN 104055800A
- Authority
- CN
- China
- Prior art keywords
- pepsin
- seu steleophaga
- zymolyte
- eupolyphaga seu
- eupolyphaga
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses ground beetle protease enzymolytic products and an application thereof. The ground beetle protease enzymolytic products are obtained by the following steps particularly: A, selecting ground beetles, crushing into fine powder; B, putting the ground beetle fine powder in artificial gastric juice, adding pepsase for hydrolysis to obtain a pepsase enzymolytic liquid, then performing centrifugation; C, taking supernatant, drying to obtain a pepsase enzymolytic product; D, taking residues, transferring the residues into artificial intestinal juice, adding trypsin for hydrolysis to obtain a ground beetle trypsin enzymolytic product. The pepsase enzymolytic product obtained in step C and the trypsin enzymolytic product obtained in step D are the ground beetle protease enzymolytic products. The ground beetle protease enzymolytic products have anticoagulation or thrombolytic effect.
Description
Technical field
The present invention relates to a kind of field of traditional Chinese medicine pharmacy, be specifically related to Eupolyphaga Seu Steleophaga zymolyte and application thereof.
Background technology
Eupolyphaga Seu Steleophaga is Corydiidae insecticide eupolyphoge sinensis Eupolyphaga sinensis Walker or Ji eupolyphoge sinensis Steleophaga plancyi(Boleny) female worm dry body; Main product in Jiangsu, the ground such as Hubei, Henan, Hebei; Salty in the mouth, cold; Slightly poisonous; Return Liver Channel.Major function is removing blood stasis, reunion of fractured tendons and bones.For traumatic injury, muscles and bones fracture, blood stasis amenorrhea, postpartum stagnation stomachache ， mass in the abdomen mass in the abdomen.
The Eupolyphaga Seu Steleophaga at present main component of report is volatile oil and aminoacid.But concrete active component does not have bibliographical information, and Chinese medicine is used as medicine with Eupolyphaga Seu Steleophaga Scorpio, exists taking dose large, easily causes the disadvantages such as microbial contamination.In view of only having after arriving 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 invention discloses a kind of Eupolyphaga protein enzyme zymolyte and application thereof, there is good anticoagulation and thrombolytic effect.
The technical solution adopted in the present invention:
Eupolyphaga protein enzyme zymolyte, by following methods, prepared:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, Eupolyphaga Seu Steleophaga fine powder is placed in to simulated gastric fluid, then adds pepsin hydrolysis, obtain pepsin enzymolysis solution, centrifugal;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue and proceed to simulated intestinal fluid, add trypsin hydrolyzing, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
Eupolyphaga protein enzyme zymolyte, by following methods, prepared:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, Eupolyphaga Seu Steleophaga fine powder is placed in to simulated gastric fluid, airtight, in 37 ℃ of water bath with thermostatic control 30min, add pepsin hydrolysis 2-3 hour, obtain pepsin enzymolysis solution, pepsin enzymolysis liquid is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, centrifugal;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue and add simulated intestinal fluid, airtight, after 45 ℃ of water bath with thermostatic control 30min, add trypsin hydrolyzing 2-4 hour, enzymolysis solution, in 85 ℃ of water bath with thermostatic control 15min, is cooled to room temperature, centrifugal 15min, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
Eupolyphaga protein enzyme zymolyte, by following methods, prepared:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, to simulated gastric fluid that to add with Eupolyphaga Seu Steleophaga fine powder w/v in Eupolyphaga Seu Steleophaga fine powder be 1:20, airtight, in 37 ℃ of water bath with thermostatic control 30min, add 2% pepsin hydrolysis 2-3 hour, obtain pepsin enzymolysis solution, by pepsin enzymolysis solution, be placed in 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue add 20 times amount simulated intestinal fluids, airtight, after 45 ℃ of water bath with thermostatic control 30min, add 4% trypsin hydrolyzing 2-4h, enzymolysis solution, in 85 ℃ of water bath with thermostatic control 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
Eupolyphaga protein enzyme zymolyte, by following methods, prepared:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, to simulated gastric fluid that to add with Eupolyphaga Seu Steleophaga fine powder w/v in Eupolyphaga Seu Steleophaga fine powder be 1:20, airtight, in 37 ℃ of water bath with thermostatic control 30min, add 3% pepsin hydrolysis 2-3 hour, obtain pepsin enzymolysis solution, with 1mol/mlNaOH solution, regulate the pH=6.8 of enzymolysis solution, pepsin enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue add 20 times amount simulated intestinal fluids, airtight, after 45 ℃ of water bath with thermostatic control 30min, add 3% trypsin hydrolyzing 2-4h, enzymolysis solution, in 85 ℃ of water bath with thermostatic control 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
Eupolyphaga protein enzyme zymolyte, by following methods, prepared:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, to simulated gastric fluid that to add with Eupolyphaga Seu Steleophaga fine powder w/v in Eupolyphaga Seu Steleophaga fine powder be 1:20, airtight, in 37 ℃ of water bath with thermostatic control 30min, add 2% pepsin hydrolysis 2-3 hour, obtain pepsin enzymolysis solution, with 1mol/mlNaOH solution, regulate the pH=6.8 of enzymolysis solution, pepsin enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue add 20 times amount simulated intestinal fluids, airtight, after 45 ℃ of water bath with thermostatic control 30min, add 2% trypsin hydrolyzing 2-4h, enzymolysis solution, in 85 ℃ of water bath with thermostatic control 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
Described Eupolyphaga Seu Steleophaga pepsin zymolyte or the application of trypsin digestion thing in the medicine of preparing anticoagulation or thrombolytic.
The dosage form of described medicine is capsule, tablet, granule, powder, oral liquid or pill.
Beneficial effect of the present invention: be used as medicine mainly with full powder due to current animal drugs, exist that dosage greatly, easily causes microbial contamination, dosage is improper and cause that quick reaction, Acute Hepatic renal function injury, hemolytic reaction, maincenter suppress and the toxic and side effects such as teratogenesis.The invention provides the isolation and purification method that Eupolyphaga Seu Steleophaga carries out anticoagulant, antithrombotic acitivity composition, can effectively overcome the above problems.
In view of only having after arriving 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 bring into play drug effect, therefore environment and the digestion process of this experimental simulation harmonization of the stomach small intestinal, with pepsin and trypsin bionic extraction Eupolyphaga Seu Steleophaga, make high molecular weight protein in medical material be hydrolyzed to small-molecule peptide and be beneficial to absorption of human body.APTT method, fibrinogen plate assay are conventional external anticoagulant, molten fine test method, and this experiment be take it as investigating index, proves the feasibility of enzymolysis process.Be specially and take degree of hydrolysis as index, single factor has been investigated the process conditions of bionic enzyme solution extraction Eupolyphaga Seu Steleophaga; Take APTT method, fibrinogen plate assay as investigating index, bionic enzyme solution is extracted to the technique of Eupolyphaga Seu Steleophaga and verified.Result confirmation, bionic enzyme solution clotting time is long, and antithrombotic effect is obvious.
Concrete grammar and result confirm by following experiment:
1 material
1.1 medical material Eupolyphaga Seu Steleophagas (being provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd)
1.2 reagent bovine serum albumin (Sigma A7906, purchased from Beijing ring Ya Taike biomedical technology company limited); 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); APTT reagent 10*1.5ml(is purchased from Shanghai sun biological reagent company); Protein Assay Dye Rgnt(Bio-red 5000006, the flat science and technology limited Company in pool, Beijing); Trichloroacetic acid (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group); Ethanol (analytical pure); Watson pure water (Guangzhou Watson food and drink company limited).
Normal saline (take sodium chloride 9g, adding distil water is settled to 1000ml, shakes up, and obtains); Simulated gastric fluid (get hydrochloric acid 9ml, adding distil water is diluted to 1000ml, obtains); 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, obtain).
1.3 instrument UV-2000 type spectrophotometers; 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.).
The male Japan large ear rabbit of 1.4 animal healths, the about 2Kg(of body weight provides by Beijing Vital River Experimental Animals Technology Co., Ltd.), meet national health one-level animal standard.
2 methods and result
Take plasma prothrombin time (PT) and activated partial thromboplastin time (APTT) is activity index, at the bottom of the enzyme of bionic enzymatic technique than and enzymolysis time investigate.
investigate index
2.1.1 plasma prothrombin time (PT) algoscopy
Sample preparation: get appropriate Eupolyphaga seu steleophaga extract, be mixed with concentration and be 10mg/ml solution, standby.
The preparation of blood plasma: get after the anesthesia of healthy rabbits (male and female half and half, Beijing, the place of production) auricular vein injection urethane, carotid artery is got blood, 3.8% sodium citrate anticoagulant (1: 9), after mixing with 3000r/min, centrifugal 10min, get supernatant, isolate platelet poor plasma (PPP).
Get PPP100 μ l in test cup, add sample liquid 10 μ l, 37 ℃, pre-temperature 3min, adds the pre-temperature 3min of PT reagent 200 μ l(), start timing, to there being fiber protein yarn to occur, accurate recording setting time.With normal saline or the negative contrast of 90% ethanol (5 parts of each sample parallel assays).
The blank PT of prothrombin time ratio (PTR)=blood plasma PT/ to be measured
2.1.2 activated partial thromboplastin time (APTT) algoscopy
The preparation of sample and blood plasma: with PT method.
Get PPP100 μ l in test cup, add the pre-temperature 5min of sample liquid 10 μ l() and the pre-temperature 5min of APTT reagent 100 μ l(), add CaCl after hatching 5min
2the pre-temperature 15min of 100 μ l(), start timing, to there being fiber protein yarn to occur, accurate recording setting time.With normal saline or the negative contrast of 90% ethanol (5 parts of each sample parallel assays).
2.2 processing steps are as follows:
2.2.1 pepsin enzymolysis process screening
2.2.1.1 at the bottom of enzyme, ratio is investigated
Take 5g Eupolyphaga Seu Steleophaga medical material fine powder in 250ml round-bottomed flask, add simulated gastric fluid (solid-to-liquid ratio 1: 20), airtight, after 37 ℃ of water bath with thermostatic control effect 30min, add respectively at the bottom of enzyme than the pepsin hydrolysis 3h that is 2%, 3% and 4%, each enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control effect 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant water-bath and volatilize, obtain the Eupolyphaga Seu Steleophaga pepsin zymolyte of ratio at the bottom of different enzymes, parallel 5 parts of every kind of extract.
The extract of ratio at the bottom of different enzymes is carried out to PT and APTT Effect tests, the results are shown in Table 1 and Fig. 1,2.
At the bottom of the different enzymes of table 1 pepsin than extract PT and APTT investigate result (
)
Note: with the comparison of blank group, there is anticoagulant effect,
* P<0.05, * * P<0.01.
Conclusion: by SPSS17.0 statistical software analysis result, shown, at the bottom of 2% and 3% enzyme the pepsin zymolyte of ratio and matched group there were significant differences (
p<0.01).
2.2.1.2 enzymolysis time is investigated
Extract to different extraction times carries out PT and APTT Effect tests, the results are shown in Table 3 and Fig. 3,4.
The different enzymolysis time extract PT of table 2 pepsin and APTT investigation result (
)
Note: with the comparison of blank group, there is anticoagulant effect,
* P<0.05, * * P<0.01.
Conclusion: take normal saline as contrast, the extract of different extraction times is carried out to PT and APTT detection, by SPSS statistical software analysis result, shown, there were significant differences for the pepsin zymolyte of 2h and 3h and matched group (
p<0.01).
trypsin digestion craft screening
2.2.2.1 at the bottom of enzyme, ratio is investigated
Take Eupolyphaga Seu Steleophaga medical material 100g, be placed in 3000ml round-bottomed flask, add simulated gastric fluid (solid-to-liquid ratio 1: 20), airtight, after 37 ℃ of water bath with thermostatic control effect 30min, add at the bottom of enzyme than the pepsin that is 3%, hydrolysis 3h, sucking filtration, gets residue, dry for standby.
Take above-mentioned residue 1g in 250ml round-bottomed flask, add simulated intestinal fluid (solid-to-liquid ratio 1: 20), airtight, after 37 ℃ of water bath with thermostatic control effect 30min, add respectively at the bottom of enzyme than the trypsin hydrolyzing 3h that is 2%, 3% and 4%, each enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control effect 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, getting supernatant water-bath volatilizes, obtain the Eupolyphaga Seu Steleophaga trypsin digestion thing of ratio at the bottom of different enzymes, parallel 5 parts of every kind of extract, in Table 3, Fig. 5,6.
At the bottom of the different enzymes of table 3 trypsin than extract PT and APTT investigate result (
)
Note: with the comparison of blank group, there is anticoagulant effect,
* P<0.05, * * P<0.01.
Conclusion: by SPSS17.0 statistical software analysis result, shown, take PT during as index, at the bottom of three kinds of enzymes the extract of ratio all variant with normal saline (
p<0.05); Take APTT during as index, at the bottom of three kinds of enzymes the extract of ratio all with normal saline there were significant differences (
p<0.01), and all with 4% time drug effect best.
2.2.2.2 enzymolysis time is investigated
Take pepsin enzymolysis residue 1g in 250ml round-bottomed flask, add simulated intestinal fluid (solid-to-liquid ratio 1: 20), airtight, after 37 ℃ of water bath with thermostatic control effect 30min, than the trypsin that is 3%, be hydrolyzed respectively 2h, 3h and 4h at the bottom of adding enzyme, each enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control effect 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant water-bath and volatilize, obtain the Eupolyphaga Seu Steleophaga trypsin digestion thing of different extraction times, parallel 5 parts of every kind of extract.In Table 4, Fig. 7,8.
The different enzymolysis time extract PT of table 4 Trypsin and APTT investigation result (
)
Note: with the comparison of blank group, there is anticoagulant effect,
* P<0.05, * * P<0.01.
3
the checking of bionic enzyme solution extraction process drug effect
3.1 platelet aggregation rates (turbidimetry)
3.1.1 sample preparation:eupolyphaga Seu Steleophaga medical material fine powder adds simulated gastric fluid (solid-to-liquid ratio 1: 20), airtight, after 37 ℃ of water bath with thermostatic control effect 30min, add 2% pepsin hydrolysis 3h, enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control effect 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, be dried to obtain Eupolyphaga Seu Steleophaga pepsin zymolyte; Get the residue of pepsin enzymolysis solution after centrifugal and add 20 times of amount simulated intestinal fluids, airtight, after 45 ℃ of water bath with thermostatic control effect 30min, add 4% trypsin hydrolyzing 2h, enzymolysis solution, in 85 ℃ of water bath with thermostatic control effect 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, be dried to obtain Eupolyphaga Seu Steleophaga trypsin digestion thing.
Get appropriate Eupolyphaga seu steleophaga extract, being mixed with concentration is 10mg/ml solution, obtains.
blood plasma preparation:get healthy rabbits (male and female half and half, Beijing, the place of production) after the anesthesia of auricular vein injection urethane, carotid artery is got blood, 3.8% sodium citrate anticoagulant (1: 9), by the whole blood of anticoagulant with 1000r/min, centrifugal 10min, gets upper plasma, is platelet rich plasma (PRP), residual blood is with 3000r/min, centrifugal 10min, gets supernatant, isolates platelet poor plasma (PPP).
platelet aggregation rate algoscopy:after PPP zeroing, PRP 150 μ l and sample liquid 10 μ l are placed in to 37 ℃ of pre-temperature 10min of test cup, add derivant ADP (1mmol/L) 10 μ l, measure platelet maximum agglutination rate in 10min.With the negative contrast of normal saline (5 parts of each sample parallel assays).Computing formula is as follows:
Distance/90 * 100% during the maximum gathering of platelet maximum agglutination rate=PRP and between baseline
Platelet aggregation inhibition rate=(control tube platelet aggregation rate %-delivery tube aggregation rate %)/control tube platelet aggregation rate % * 100%
Table 5 Eupolyphaga Seu Steleophaga stomach/Trypsin thing platelet aggregation rate, suppression ratio Effect tests (
)
| Platelet maximum agglutination rate (%) | Platelet suppression ratio (%) | |
| Pepsin enzymolysis thing | 20.5±4.5 **Δ | 69.72 |
| Trypsin enzymolysis thing | 29.8±4.3 ** | 58.03 |
| Normal saline | 71.0±4.9 | -- |
Note: * compares with blank group, and there were significant differences,
* P<0.05, * * P<0.01.
Δwith the comparison of Trypsin enzymolysis thing, there were significant differences,
Δ p<0.05, Δ Δ p<0.01.
Result shows: by SPSS17.0 statistical software analysis result, shown, Eupolyphaga Seu Steleophaga stomach/trypsin digestion thing is compared with normal saline, all have significant anticoagulant effect (
p<0.01).And pepsin zymolyte inhibitory action be better than trypsin digestion thing (
p<0.05).
blood clotting-fibrinolytic Dynamic Graph
The preparation of sample and blood plasma: with PT method.
Blood clotting-fibrinolytic Dynamic Graph test: get PRP 200 μ l and sample liquid 10 μ l mix, 37 ℃ of pre-temperature 10min, PPP zeroing, add 0.1mol/L CaCl2 50 μ l simultaneously, stir 10s, balance recorder records Dynamic Graph, until the continual and steady 2min of figure peak value, with the negative contrast of normal saline (5 parts of each sample parallel assays).Curve obtained is blood clotting-fibrinolytic Dynamic Graph.According to Dynamic Graph, observe following three parameters:
solidify start-up time (CST);
setting time (CT);
relative coagulation grade (RCE);
Table 6 Eupolyphaga Seu Steleophaga stomach/Trypsin thing blood clotting-fibrinolytic Effect tests (
)
| CST(s) | CT(s) | RCE(%) | |
| Pepsin enzymolysis thing | 233.31±18.31 **ΔΔ | 35.10±4.65 **ΔΔ | 42.10±3.56 ΔΔ |
| Trypsin enzymolysis thing | 322.8±19.53 ** | 12.90±2.27 ** | 15.86±4.16 |
| Normal saline | 163.5±23.95 | 49.50±4.11 | 100 |
Note: * compares with blank group, and there were significant differences,
* P<0.05, * * P<0.01.
Δwith the comparison of Trypsin enzymolysis thing, there were significant differences,
Δ p<0.05, Δ Δ p<0.01.
Result shows: by SPSS17.0 statistical software analysis result, shown, Eupolyphaga Seu Steleophaga stomach/trypsin digestion thing is compared with normal saline, all can extend CST, and significantly reduces maximum coagulation grade (RCE<50%).But the two also makes CT reduce simultaneously.Prompting Eupolyphaga Seu Steleophaga stomach/trypsin digestion thing may be converted into thrombin by proenzyme anticoagulant and reach anticoagulant effect, and RCE minimizing shows that it has the effect of solution fibrin proenzyme.Trypsin digestion thing is compared with pepsin zymolyte, can extend more significantly CST, and reduce RCE(
p<0.01).
The interpretation of result of table 7 Eupolyphaga Seu Steleophaga stomach/Trypsin thing anticoagulant fibrinolytic index comprehensive
| PT | APTT | Platelet aggregation rate % | CST | CT | RCE | |
| Pepsin enzymolysis thing | ↑** | ↑** | ↓** | ↑** | ↓** | ↓** |
| Trypsin enzymolysis thing thing | ↑** | ↑** | ↓** | ↑** | ↓** | ↓** |
Note: ↑ represent to extend, ↓ representing to reduce, * represents effect significantly,
* P<0.05, * * P<0.01.
PT/APTT experimental result shows, Eupolyphaga Seu Steleophaga stomach/trypsin digestion thing has significant anticoagulant effect; Platelet aggregation rate result of the test shows that two kinds of zymolytes all have the effect of anticoagulant, above-mentioned three kinds of indexs relatively in, pepsin zymolyte is all better than trypsin digestion thing; The test of blood clotting-fibrinolytic Dynamic Graph shows that stomach/trypsin digestion thing all has the effect of solution fibrin proenzyme, and trypsin digestion thing activity is stronger.
This research is all positive with PT/APTT experimental result, shows that Eupolyphaga Seu Steleophaga stomach/trypsin digestion thing may reach anticoagulant object by endogenous and exogenous two kinds of coagulation pathways, determines thus Eupolyphaga Seu Steleophaga enzymolysis process parameter; By indexs such as platelet aggregation rate, blood clotting-fibrinolytic Dynamic Graphs, evaluate the effect of this technique extract, result is also all positive, shows to adopt bionic enzyme solution to extract the feasible process of Eupolyphaga Seu Steleophaga.In coagulation-fibrinolysis system Dynamic Graph, CST prolongation, CT shorten result, prompting Eupolyphaga Seu Steleophaga stomach/trypsin digestion thing may be converted into thrombin by proenzyme anticoagulant and extend blood coagulation start-up time, and after blood coagulation startup, two kinds of zymolytes all cannot suppress plasminogen and be converted into fibrin, but can reduce the maximum coagulation grade (RCE<100%) of blood plasma by solution fibrin proenzyme.
conclusion:by SPSS statistical software analysis result, shown, take PT during as index, the extract of three kinds of enzymolysis times all variant with normal saline (
p<0.05); Take APTT during as index, the extract of three kinds of enzymolysis times all with normal saline there were significant differences (
p<0.01), for cost-saving, determine enzymolysis 2h.
Accompanying drawing explanation
Fig. 1 is than pepsin zymolyte PT drug effect figure at the bottom of different enzymes;
Fig. 2 is than pepsin zymolyte APTT drug effect figure at the bottom of different enzymes;
Fig. 3 is different extraction time pepsin zymolyte PT drug effect figure;
Fig. 4 is different extraction time pepsin zymolyte APTT drug effect figure;
Fig. 5 is than trypsin digestion thing PT drug effect figure at the bottom of different enzymes;
Fig. 6 is than trypsin digestion thing APTT drug effect figure at the bottom of different enzymes;
Fig. 7 is different extraction time trypsin digestion thing PT drug effect figure;
Fig. 8 is different extraction time trypsin digestion thing APTT drug effect figure.
The specific embodiment
embodiment 1:
(1): take 2g Eupolyphaga Seu Steleophaga medical material, be ground into fine powder.
(2): Eupolyphaga Seu Steleophaga fine powder is placed in to 50ml round-bottomed flask, then to round-bottomed flask, add 40ml simulated gastric fluid, be enclosed within the 2% pepsin hydrolysis 3h that adds 40mg after 37 ℃ of water-bath 30min, obtain pepsin enzymolysis solution, enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min.
(3) get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte.
(4) get the simulated intestinal fluid that residue adds 40ml, be enclosed within 45 ℃ of water bath with thermostatic control 30min, the 4% trypsin digestion 2h that adds again 100mg, in 85 ℃ of water-bath enzyme denaturing 15min, be cooled to room temperature, the centrifugal 15min of 4200r/min, gets supernatant, 60 ℃ of vacuum dryings are weighed, and obtain Eupolyphaga Seu Steleophaga trypsin digestion thing.Gained pepsin zymolyte and trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
Embodiment 2
(1): take 5g Eupolyphaga Seu Steleophaga medical material, be ground into fine powder.
(2): Eupolyphaga Seu Steleophaga fine powder is placed in to 250ml round-bottomed flask, then to round-bottomed flask, add 100ml simulated gastric fluid, be enclosed within the 3% pepsin hydrolysis 2h that adds 40mg after 37 ℃ of water-bath 30min, obtain pepsin enzymolysis solution, enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min.
(3) get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte.
(4) get residue and add 100ml simulated intestinal fluid, be enclosed within 45 ℃ of water bath with thermostatic control 30min, the 3% trypsin digestion 3h that adds again 100mg, in 85 ℃ of water-bath enzyme denaturing 15min, be cooled to room temperature, the centrifugal 15min of 4200r/min, gets supernatant, 60 ℃ of vacuum dryings are weighed, and obtain Eupolyphaga Seu Steleophaga trypsin digestion thing.Gained pepsin zymolyte and trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
Embodiment 3
(1): take 5g Eupolyphaga Seu Steleophaga medical material, be ground into fine powder.
(2): Eupolyphaga Seu Steleophaga fine powder is placed in to 250ml round-bottomed flask, then to round-bottomed flask, add 100ml simulated gastric fluid, be enclosed within the 2% pepsin hydrolysis 2h that adds 40mg after 37 ℃ of water-bath 30min, obtain pepsin enzymolysis solution, enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min
(3) get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte.
(4) get residue and add 100ml simulated intestinal fluid, be enclosed within 45 ℃ of water bath with thermostatic control 30min, the 2% trypsin digestion 4h that adds again 100mg, in 85 ℃ of water-bath enzyme denaturing 15min, be cooled to room temperature, the centrifugal 15min of 4200r/min, gets supernatant, 60 ℃ of vacuum dryings are weighed, and obtain Eupolyphaga Seu Steleophaga trypsin digestion thing.Gained pepsin zymolyte and trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
Claims (7)
1. Eupolyphaga protein enzyme zymolyte, is characterized in that being prepared by following methods:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, Eupolyphaga Seu Steleophaga fine powder is placed in to simulated gastric fluid, then adds pepsin hydrolysis, obtain pepsin enzymolysis solution, centrifugal;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue and proceed to simulated intestinal fluid, add trypsin hydrolyzing, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
2. Eupolyphaga protein enzyme zymolyte according to claim 1, is characterized in that being prepared by following methods:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, Eupolyphaga Seu Steleophaga fine powder is placed in to simulated gastric fluid, airtight, in 37 ℃ of water bath with thermostatic control 30min, add pepsin hydrolysis 2-3 hour, obtain pepsin enzymolysis solution, pepsin enzymolysis liquid is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, centrifugal;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue and add simulated intestinal fluid, airtight, after 45 ℃ of water bath with thermostatic control 30min, add trypsin hydrolyzing 2-4 hour, enzymolysis solution, in 85 ℃ of water bath with thermostatic control 15min, is cooled to room temperature, centrifugal 15min, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
3. Eupolyphaga protein enzyme zymolyte according to claim 1, is characterized in that being prepared by following methods:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, to simulated gastric fluid that to add with Eupolyphaga Seu Steleophaga fine powder w/v in Eupolyphaga Seu Steleophaga fine powder be 1:20, airtight, in 37 ℃ of water bath with thermostatic control 30min, add 2% pepsin hydrolysis 2-3 hour, obtain pepsin enzymolysis solution, by pepsin enzymolysis solution, be placed in 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue add 20 times amount simulated intestinal fluids, airtight, after 45 ℃ of water bath with thermostatic control 30min, add 4% trypsin hydrolyzing 2-4h, enzymolysis solution, in 85 ℃ of water bath with thermostatic control 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
4. Eupolyphaga protein enzyme zymolyte according to claim 1, is characterized in that being prepared by following methods:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, to simulated gastric fluid that to add with Eupolyphaga Seu Steleophaga fine powder w/v in Eupolyphaga Seu Steleophaga fine powder be 1:20, airtight, in 37 ℃ of water bath with thermostatic control 30min, add 3% pepsin hydrolysis 2-3 hour, obtain pepsin enzymolysis solution, with 1mol/mlNaOH solution, regulate the pH=6.8 of enzymolysis solution, pepsin enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue add 20 times amount simulated intestinal fluids, airtight, after 45 ℃ of water bath with thermostatic control 30min, add 3% trypsin hydrolyzing 2-4h, enzymolysis solution, in 85 ℃ of water bath with thermostatic control 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
5. Eupolyphaga protein enzyme zymolyte according to claim 1, is characterized in that being prepared by following methods:
A, clean Eupolyphaga Seu Steleophaga, be ground into fine powder;
B, to simulated gastric fluid that to add with Eupolyphaga Seu Steleophaga fine powder w/v in Eupolyphaga Seu Steleophaga fine powder be 1:20, airtight, in 37 ℃ of water bath with thermostatic control 30min, add 2% pepsin hydrolysis 2-3 hour, obtain pepsin enzymolysis solution, with 1mol/mlNaOH solution, regulate the pH=6.8 of enzymolysis solution, pepsin enzymolysis solution is placed in to 85 ℃ of water bath with thermostatic control 15min, be cooled to room temperature, with the centrifugal 15min of 4200r/min;
C, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga pepsin zymolyte;
D, get residue add 20 times amount simulated intestinal fluids, airtight, after 45 ℃ of water bath with thermostatic control 30min, add 2% trypsin hydrolyzing 2-4h, enzymolysis solution, in 85 ℃ of water bath with thermostatic control 15min, is cooled to room temperature, with the centrifugal 15min of 4200r/min, get supernatant, dry, obtain Eupolyphaga Seu Steleophaga trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Eupolyphaga protein enzyme zymolyte.
6. according to the application of the Eupolyphaga protein enzyme zymolyte described in claim 1-5 any one, it is characterized in that: described Eupolyphaga Seu Steleophaga pepsin zymolyte or the application of trypsin digestion thing in the medicine of preparing anticoagulation or thrombolytic.
7. application according to claim 6, is characterized in that: the dosage form of described medicine is capsule, tablet, granule, powder, oral liquid or pill.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310094652.1A CN104055800A (en) | 2013-03-23 | 2013-03-23 | Ground beetle protease enzymolytic product and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310094652.1A CN104055800A (en) | 2013-03-23 | 2013-03-23 | Ground beetle protease enzymolytic product and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104055800A true CN104055800A (en) | 2014-09-24 |
Family
ID=51543919
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310094652.1A Pending CN104055800A (en) | 2013-03-23 | 2013-03-23 | Ground beetle protease enzymolytic product and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104055800A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101647813A (en) * | 2008-08-13 | 2010-02-17 | 北京凯瑞创新医药科技有限公司 | Bionic enzymatic hydrolysate for animal medicaments and application thereof |
| CN101647822A (en) * | 2008-08-13 | 2010-02-17 | 北京凯瑞创新医药科技有限公司 | Bionic enzymatic hydrolysate for ground beeltle and application thereof |
| CN101862349A (en) * | 2009-04-14 | 2010-10-20 | 河北以岭医药研究院有限公司 | Effective part of centipede and application thereof |
| CN101862350A (en) * | 2009-04-14 | 2010-10-20 | 河北以岭医药研究院有限公司 | Active ingredients of scorpion and application thereof |
| CN102114047A (en) * | 2010-01-06 | 2011-07-06 | 浙江医药股份有限公司新昌制药厂 | Woodlouse lyophilized powders 1 and 2 prepared by combined enzymolysis of pepsin and trypsinase and application thereof |
-
2013
- 2013-03-23 CN CN201310094652.1A patent/CN104055800A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101647813A (en) * | 2008-08-13 | 2010-02-17 | 北京凯瑞创新医药科技有限公司 | Bionic enzymatic hydrolysate for animal medicaments and application thereof |
| CN101647822A (en) * | 2008-08-13 | 2010-02-17 | 北京凯瑞创新医药科技有限公司 | Bionic enzymatic hydrolysate for ground beeltle and application thereof |
| CN101862349A (en) * | 2009-04-14 | 2010-10-20 | 河北以岭医药研究院有限公司 | Effective part of centipede and application thereof |
| CN101862350A (en) * | 2009-04-14 | 2010-10-20 | 河北以岭医药研究院有限公司 | Active ingredients of scorpion and application thereof |
| CN102114047A (en) * | 2010-01-06 | 2011-07-06 | 浙江医药股份有限公司新昌制药厂 | Woodlouse lyophilized powders 1 and 2 prepared by combined enzymolysis of pepsin and trypsinase and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 王三根等: "《植物生理生化》", 31 January 2013, 中国林业出版社 * |
| 秦仲君等: "土鳖虫多肽的制备工艺及抗凝血的作用研究", 《安徽农业科学》 * |
| 高鹏: "土鳖虫不同工艺提取物抗凝作用的比较研究", 《时珍国医国药》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| HENDRIX et al. | Whipple's intestinal lipodystrophy: report of four cases and discussion of possible pathogenic factors | |
| TWI777606B (en) | Traditional chinese medicine formula flora capsule, preparation method and use thereof in preparing medicine for treating type 2 diabetes | |
| Tjandrawinata et al. | The safety and tolerability of lumbrokinase DLBS1033 in healthy adult subjects | |
| CN101647822A (en) | Bionic enzymatic hydrolysate for ground beeltle and application thereof | |
| Yoo et al. | The effects of nattokinase supplementation on collagen–epinephrine closure time, prothrombin time and activated partial thromboplastin time in nondiabetic and hypercholesterolemic subjects | |
| CN101780107A (en) | Hippocampus extract and preparation method thereof | |
| CN104055801A (en) | Cicada slough protease enzymolytic product and application thereof | |
| CN101862350B (en) | Active ingredients of scorpion and application thereof | |
| Fu et al. | Tubiechong: A review on ethnomedicinal uses, bioactive chemical constituents and pharmacological activities | |
| CN104055799A (en) | Leech pepsin enzymolytic product and application thereof | |
| CN101862349B (en) | Effective part of centipede and application thereof | |
| CN104055800A (en) | Ground beetle protease enzymolytic product and application thereof | |
| KR20150070515A (en) | Composition comprising the extract of complex herbs as an active ingredient for preventing and treating allergy | |
| JP2017119683A (en) | Blood coagulation inhibitor | |
| CN106552255A (en) | A kind of preparation method of Sanguis cervi peptidoliposome | |
| WO2016141774A1 (en) | Uses of jilin ginseng oligopeptide in preparing food product or healthcare food product for improving and enhancing sexual function | |
| CN106668131B (en) | Earthworm composition and preparation method thereof | |
| Olaniyan et al. | Raw Cucumber (Cucumis sativus) Fruit Juice as Possible First-Aid Antidote in Drug-Induced Toxicity | |
| Reynoso et al. | The contribution of the intestinal mucosa to the total serum alkaline phosphatase activity | |
| Errasfa | Blood hemostasis dysfunction and inflammation in COVID-19 patients: Viral and host active molecules as therapeutic targets | |
| Arirudran et al. | Alteration in levels of minerals in DEN induced hepatocellular carcinoma in wistar albino rats | |
| CN106890189A (en) | Application of the chonglou saponin in antineoplastic sensitizer is prepared | |
| CN110354250A (en) | A kind of composite plant Gly-His-Lys and its preparation method and application | |
| CN110468175A (en) | A kind of composite plant Gly-His-Lys and its preparation method and application | |
| CN102327274B (en) | Adrenal cortical hormone compound preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140924 |
|
| RJ01 | Rejection of invention patent application after publication |