CN104055801A - Cicada slough protease enzymolytic product and application thereof - Google Patents
Cicada slough protease enzymolytic product and application thereof Download PDFInfo
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Abstract
The invention discloses cicada slough protease enzymolytic products and an application thereof. The cicada slough protease enzymolytic products are obtained by the following steps particularly: A, selecting cicada slough, crushing into fine powder; B, putting the cicada slough fine powder in artificial gastric juice, adding pepsase for hydrolysis to obtain a pepsase enzymolytic liquid, then performing centrifugation to obtain an supernatant and residues to keep in reserve; C, taking the supernatant, drying to obtain a cicada slough pepsase enzymolytic product; D, taking the residues, transferring the residues into artificial intestinal juice, adding trypsin for hydrolysis to obtain a cicada slough trypsin enzymolytic product. The pepsase enzymolytic product obtained in step C and the trypsin enzymolytic product obtained in step D are the cicada slough protease enzymolytic products. The cicada slough 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 Periostracum Cicadae protease hydrolyzed thing and application thereof.
Background technology
Periostracum Cicadae is the nymph of cicada Cryptotympana atrata Fabr. Cryptotympana pustulata Fabricius exuviae shell while sprouting wings.Property sweet, taste is cold, returns lung, Liver Channel.There is dispelling wind and heat pathogens, sore-throat relieving, rash, improving acuity of vision and removing nebula, the effect of spasmolytic, the clinical anemopyretic cold that is mainly used in, pharyngalgia hoarseness, measles without adequate eruption, rubella pruritus, conjunctival congestion cataracta, convulsion with spasms, tetanus etc.Main component is chitin and protein, aminoacid etc., and in medical material, active component it be unclear that, and is therefore used as medicine mainly with full powder at present, exists dosage large, easily causes 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 Periostracum Cicadae protease hydrolyzed thing and the application thereof of a kind of anticoagulation and thrombolytic.
Technology contents of the present invention:
periostracum Cicadae protease hydrolyzed thing, prepared by following methods:
A, clean Periostracum Cicadae, be ground into fine powder;
B, Periostracum Cicadae fine powder is placed in to simulated gastric fluid, then adds pepsin hydrolysis, obtain pepsin enzymolysis solution, centrifugal, gained supernatant and residue are for subsequent use;
C, get supernatant, dry, obtain Periostracum Cicadae pepsin zymolyte;
D, get residue and proceed in simulated intestinal fluid, add trypsin hydrolyzing, obtain Periostracum Cicadae trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Periostracum Cicadae protease hydrolyzed thing.
Periostracum Cicadae protease hydrolyzed thing, prepared by following methods:
A, clean Periostracum Cicadae, be ground into fine powder;
B, Periostracum Cicadae fine powder is placed in to simulated gastric fluid, airtight, in 37 DEG C of waters bath with thermostatic control after 30 minutes, add 3% pepsin hydrolysis 3 hours, obtain pepsin enzymolysis solution, pepsin enzymolysis solution is placed in to 85 DEG C of waters bath with thermostatic control 15 minutes, be cooled to room temperature, with 4500r/min centrifugal 15 minutes, gained supernatant and residue were for subsequent use;
C, get supernatant, dry, obtain Periostracum Cicadae pepsin zymolyte;
D, get residue and add simulated intestinal fluid, airtight, after 30 minutes, add 5% trypsin hydrolyzing 4 hours in 45 DEG C of waters bath with thermostatic control, enzymolysis solution, in 85 DEG C of waters bath with thermostatic control 15 minutes, is cooled to room temperature, with 4500r/min centrifugal 15 minutes, get supernatant, dry, obtain Periostracum Cicadae trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Periostracum Cicadae protease hydrolyzed thing.
Periostracum Cicadae protease hydrolyzed thing, prepared by following methods:
A, clean Periostracum Cicadae, be ground into fine powder;
B, Periostracum Cicadae fine powder is placed in to simulated gastric fluid, the w/v of Periostracum Cicadae fine powder and simulated gastric fluid is 1:20, airtight, after 30 minutes, add 3% pepsin hydrolysis 3 hours in 37 DEG C of waters bath with thermostatic control, obtain pepsin enzymolysis solution, pepsin enzymolysis solution is placed in to 85 DEG C of waters bath with thermostatic control 15 minutes, be cooled to room temperature, with 4500r/min centrifugal 15 minutes, gained supernatant and residue were for subsequent use;
C, get supernatant, dry, obtain Periostracum Cicadae pepsin zymolyte;
D, get residue add 20 times amount simulated intestinal fluids, airtight, after 30 minutes, add 5% trypsin hydrolyzing 4 hours in 45 DEG C of waters bath with thermostatic control, enzymolysis solution, in 85 DEG C of waters bath with thermostatic control 15 minutes, is cooled to room temperature, with 4500r/min centrifugal 15 minutes, get supernatant, dry, obtain Periostracum Cicadae trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Periostracum Cicadae protease hydrolyzed thing.
Described Periostracum Cicadae 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.
In order to verify anticoagulation and the thrombolytic effect of Periostracum Cicadae protease hydrolyzed thing, do following experiment:
Periostracum Cicadae pepsin hydrolysis products anticoagulation or 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
Periostracum Cicadae (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 Different Extraction Method
2.1.1 bionic enzymatic
Take Periostracum Cicadae medical material fine powder 8g in 250ml round-bottomed flask, add simulated gastric fluid (get hydrochloric acid 9ml, adding distil water is diluted to 1000ml, to obtain final product) by solid-to-liquid ratio 1:20, airtight, after 37 DEG C of water bath with thermostatic control effect 30min, add 3% pepsin hydrolysis 3h, enzymolysis solution is placed in to 85 DEG C of water bath with thermostatic control effect 15min enzyme denaturing, be cooled to room temperature, with the centrifugal 15min of 4500r/min, get supernatant water-bath and volatilize, obtain Periostracum Cicadae pepsin zymolyte.Residue is weighed and is added simulated intestinal fluid (to get 0.2mol/L potassium dihydrogen phosphate 250ml, add 0.2mol/L solution 118ml, be diluted with water to 1000ml by solid-to-liquid ratio 1:20, shake up, obtain), airtight, after 45 DEG C of water bath with thermostatic control effect 30min, add 5% trypsin hydrolyzing 4h, enzymolysis solution, in 85 DEG C of water bath with thermostatic control effect 15min enzyme denaturing, is cooled to room temperature, with the centrifugal 15min of 4500r/min, get supernatant, be drying to obtain Periostracum Cicadae trypsin digestion thing.
2.1.2 water extraction
Take Periostracum Cicadae medical material fine powder 8g in 250ml round-bottomed flask, by solid-to-liquid ratio 1:20 adding distil water, reflux, extract, 3 times, each 2h, merge extractive liquid,, dry, obtain Periostracum Cicadae water extract.
2.1.3 water extract-alcohol precipitation
Take Periostracum Cicadae medical material fine powder 8g in 250ml round-bottomed flask, by solid-to-liquid ratio 1:20 adding distil water, reflux, extract, 3 times, each 1h, merge extractive liquid,, is concentrated into medicinal liquid and compares 1:1, add 95% ethanol to containing alcohol amount 90%, put in refrigerator place spend the night after the centrifugal 15min of 4500r/min, get supernatant and reclaim dryly, obtain Periostracum Cicadae 90% water extracting alcohol hypostasis.Residue is dry, obtains precipitate with ethanol (slag).
2.2 anticoagulant fibrinolytic pharmacodynamics indexs
Above-mentioned appropriate Periostracum Cicadae extract is got in the preparation of need testing solution, and adding normal saline or 90% ethanol, to be mixed with concentration be 10mg/ml solution, is need testing solution.
Get healthy rabbits (male and female half and half, Beijing, the place of production) anesthesia of auricular vein injection urethane, carotid artery is got blood, 3.8% sodium citrate anticoagulant (1: 9), after mixing with 1000r/min, centrifugal 10min, gets supernatant, obtains platelet rich plasma (PRP), with 3000r/min, centrifugal 10min, gets supernatant, obtains platelet poor plasma (PPP).
2.2.1 PT test
Get PPP100 μ l in test cup, add sample liquid 10 μ l, 37 DEG C of pre-temperature 3min, add 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 the negative contrast of normal saline (6 parts of each sample parallel assays)
2.2.2 APTT test
Get PPP100 μ l in test cup, add sample liquid 10 μ l, 37 DEG C of pre-temperature 3min, add the pre-temperature 5min of people 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 the negative contrast of normal saline (6 parts of each sample parallel assays).
2.2.3 platelet aggregation rate test
After PPP zeroing, PRP 300 μ l and sample liquid 10 μ l are placed in to 37 DEG C 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 (6 parts of each sample parallel assays).
Distance/90 × 100% when 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%
2.2.4 blood clotting-fibrinolytic Dynamic Graph test
Get PRP 150 μ l and sample liquid 10 μ l mix, 37 DEG C of pre-temperature 10min, PPP zeroing adds 0.1mol/L CaCl simultaneously
250 μ l, stir 10s, and balance recorder records Dynamic Graph, until the continual and steady 2min of figure peak value, with normal saline or the negative contrast of 90% ethanol (6 parts of each sample parallel assays).Curve obtained is blood clotting-fibrinolytic Dynamic Graph.Observe following parameter according to Dynamic Graph:
solidify start-up time (CST): from curve starts, with the straight line line segment of transverse axis.CST reflection joins fibrin from coagulant and starts to form when needed and hear, and is the parameter that embodies blood clotting and start speed.
maximum coagulation grade (MCE): the peak value section that curve rises is also to start to be solidified to solidify the peak value that transmittance changes when complete.The height of MCE and mensuration plasma volume, fibrinogen level is relevant.It represents that liquid Fibrinogen is frozen into the fibrin of gel state substantially, and Hirschfeld-Klinger reaction completes substantially.
setting time (CT): start the time required to peak of curve from curve, CT is that blood plasma starts to be solidified to and solidifies the completely required time, is the parameter of reflection clotting of plasma speed.
average setting rate (ACR): i.e. the ratio of MCE/CT, represent the coagulation grade that form average each second, ACR is that reflection fibrin forms, i.e. the parameter of hemagglutination speed.
3 experimental results
3.1 PT and APTT Effect tests
Through SPSS17.0 software statistics, PT result shows, all Periostracum Cicadae extracts all can not obviously extend PT value, infers that its anticoagulation may be irrelevant with extrinsic coagulation system.
APTT result shows, contrasts with normal saline, and trypsin digestion thing, water extract, 90% water extract-alcohol precipitation (slag) can extend APTT, infer the activity that it can disturb the intrinsic coagulation factor, reach anticoagulation.The results are shown in Table 1 and Fig. 1.
Table 1 Periostracum Cicadae different process extract PT, APTT Effect tests (
)
Note: with the comparison of blank group, there is anticoagulant effect,
* P<0.05, * * P<0.01
3.2 platelet aggregation rate Effect tests
Result shows: through SPSS17.0 software statistics, for contrasting, Periostracum Cicadae trypsin digestion thing, 90% water extract-alcohol precipitation (slag) have the effect of remarkable anticoagulant with normal saline.
The results are shown in Table 2 and Fig. 2.
Table 2 Periostracum Cicadae different process extract platelet aggregation rate Effect tests (
)
Note: with the comparison of blank group, there is inhibition congregational rate,
* P<0.05, * * P<0.01
3.3 blood clottings-fibrinolytic Dynamic Graph Effect tests
Result shows, contrast with normal saline, Periostracum Cicadae pepsin zymolyte, trypsin digestion thing, 90% water extract-alcohol precipitation (slag) prolongation CT in various degree, reduce MCE and ACR, and only have the pepsin zymolyte can significant prolongation on the impact of CST, trypsin digestion thing extends not remarkable, illustrate that Periostracum Cicadae pepsin zymolyte, trypsin digestion thing, 90% water extracting alcohol sediment extracting method are mainly to change into fibrin by suppressing Fibrinogen, the clotting of plasma of slowing down, reaches anticoagulant effect.The results are shown in Table 3 and Fig. 3.
Table 3 Periostracum Cicadae different process extract blood clotting-fibrinolytic Dynamic Graph Effect tests (
)
Note: with the comparison of blank group, there is anticoagulant effect,
* P<0.05, * * P<0.01
3.4 Analysis of conclusion results are as table 4.
Table 4 Periostracum Cicadae Different Extraction Method anticoagulant index comprehensive result
Note: ↓ representing anticoagulation, * represents that anticoagulant effect is remarkable ,-represent to no effect or other
4 conclusions
APTT experimental result shows, trypsin digestion thing, water extract and 90% water extract-alcohol precipitation (slag) have obvious anticoagulant effect; Platelet aggregation rate is tested and is shown, trypsin digestion thing, 90% water extract-alcohol precipitation (slag) have obvious antiplatelet aggregation effect; Blood clotting-fibrinolytic Dynamic Graph drug effect shows with average setting rate (ACR) value, and trypsin digestion thing and 90% water extract-alcohol precipitation (slag) effect are the most obvious.
Result shows, Periostracum Cicadae extract all without obvious effect, infers that extract is not all by exogenous cruor pathway anticoagulant to PT index, and prompting system reaches anticoagulant by intrinsic coagulation approach; APTT, platelet aggregation rate, three indexs of blood clotting-fibrinolytic show, trypsin digestion thing and water extract-alcohol precipitation (slag) all show significant anticoagulation, therefore aspect anticoagulant fibrinolysis activity, choose bionic enzymatic extraction method or decoction and alcohol sedimentation technique and have more experimental value as continuing object of study.
And decoction and alcohol sedimentation technique extraction cost is higher, and in technique, uses between the special hoolivan of ethanol needs and just can produce, and bionic enzyme solution extraction cost is cheap, produces and does not need special environment, therefore bionic enzyme solution feasible process.
Brief description of the drawings
Fig. 1 Periostracum Cicadae different process extract PT, APTT drug effect figure;
Fig. 2 Periostracum Cicadae different process extract platelet aggregation rate drug effect figure;
Fig. 3 Periostracum Cicadae different process extract blood clotting-fibrinolytic Dynamic Graph.
Specific embodiment
Embodiment 1
(1) clean Periostracum Cicadae 8g, be ground into micropowders.
(2) Periostracum Cicadae micropowders is placed in to 250ml round-bottomed flask, by solid-to-liquid ratio, 1:20 adds simulated gastric fluid, airtight, after 37 DEG C of water bath with thermostatic control effect 30min, add 3% pepsin hydrolysis 3h, obtain pepsin enzymolysis solution, pepsin enzymolysis solution is placed in to 85 DEG C of water bath with thermostatic control effect 15min enzyme denaturing, be cooled to room temperature, with the centrifugal 15min of 4500r/min, gained supernatant and residue are for subsequent use.
(3) get supernatant, dry, obtain Periostracum Cicadae pepsin zymolyte.
(4) get residue and add simulated intestinal fluid by solid-to-liquid ratio 1:20, airtight, after 45 DEG C of water bath with thermostatic control effect 30min, add 5% trypsin hydrolyzing 4h, enzymolysis solution, in 85 DEG C of water bath with thermostatic control effect 15min enzyme denaturing, is cooled to room temperature, with the centrifugal 15min of 4500r/min, get supernatant, dry, obtain Periostracum Cicadae trypsin digestion thing.
Gained pepsin enzymolysis solution and trypsin digestion thing are Periostracum Cicadae protease hydrolyzed thing.
Embodiment 2
(1) clean Periostracum Cicadae 5g, be ground into micropowders.
(2) Periostracum Cicadae micropowders is placed in to 250ml round-bottomed flask, by solid-to-liquid ratio, 1:20 adds simulated gastric fluid, airtight, after 37 DEG C of water bath with thermostatic control effect 30min, add 3% pepsin hydrolysis 3h, obtain pepsin enzymolysis solution, pepsin enzymolysis solution is placed in to 85 DEG C of water bath with thermostatic control effect 15min enzyme denaturing, be cooled to room temperature, with the centrifugal 15min of 4500r/min, gained supernatant and residue are for subsequent use.
(3) get supernatant, dry, obtain Periostracum Cicadae pepsin zymolyte.
(4) get residue and add simulated intestinal fluid by solid-to-liquid ratio 1:20, airtight, after 45 DEG C of water bath with thermostatic control effect 30min, add 5% trypsin hydrolyzing 4h, enzymolysis solution, in 85 DEG C of water bath with thermostatic control effect 15min enzyme denaturing, is cooled to room temperature, with the centrifugal 15min of 4500r/min, get supernatant, be drying to obtain Periostracum Cicadae trypsin digestion thing.
Gained pepsin enzymolysis solution and trypsin digestion thing are Periostracum Cicadae protease hydrolyzed thing.
Claims (5)
1. Periostracum Cicadae protease hydrolyzed thing, is characterized in that being prepared by following methods:
A, clean Periostracum Cicadae, be ground into fine powder;
B, Periostracum Cicadae fine powder is placed in to simulated gastric fluid, then adds pepsin hydrolysis, obtain pepsin enzymolysis solution, centrifugal, gained supernatant and residue are for subsequent use;
C, get supernatant, dry, obtain Periostracum Cicadae pepsin zymolyte;
D, get residue and proceed in simulated intestinal fluid, add trypsin hydrolyzing, obtain Periostracum Cicadae trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Periostracum Cicadae protease hydrolyzed thing.
2. Periostracum Cicadae protease hydrolyzed thing according to claim 1, is characterized in that being prepared by following methods:
A, clean Periostracum Cicadae, be ground into fine powder;
B, Periostracum Cicadae fine powder is placed in to simulated gastric fluid, airtight, in 37 DEG C of waters bath with thermostatic control after 30 minutes, add 3% pepsin hydrolysis 3 hours, obtain pepsin enzymolysis solution, pepsin enzymolysis solution is placed in to 85 DEG C of waters bath with thermostatic control 15 minutes, be cooled to room temperature, with 4500r/min centrifugal 15 minutes, gained supernatant and residue were for subsequent use;
C, get supernatant, dry, obtain Periostracum Cicadae pepsin zymolyte;
D, get residue and add simulated intestinal fluid, airtight, after 30 minutes, add 5% trypsin hydrolyzing 4 hours in 45 DEG C of waters bath with thermostatic control, enzymolysis solution, in 85 DEG C of waters bath with thermostatic control 15 minutes, is cooled to room temperature, with 4500r/min centrifugal 15 minutes, get supernatant, dry, obtain Periostracum Cicadae trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Periostracum Cicadae protease hydrolyzed thing.
3. Periostracum Cicadae protease hydrolyzed thing according to claim 1, is characterized in that being prepared by following methods:
A, clean Periostracum Cicadae, be ground into fine powder;
B, Periostracum Cicadae fine powder is placed in to simulated gastric fluid, the w/v of Periostracum Cicadae fine powder and simulated gastric fluid is 1:20, airtight, after 30 minutes, add 3% pepsin hydrolysis 3 hours in 37 DEG C of waters bath with thermostatic control, obtain pepsin enzymolysis solution, pepsin enzymolysis solution is placed in to 85 DEG C of waters bath with thermostatic control 15 minutes, be cooled to room temperature, with 4500r/min centrifugal 15 minutes, gained supernatant and residue were for subsequent use;
C, get supernatant, dry, obtain Periostracum Cicadae pepsin zymolyte;
D, get residue add 20 times amount simulated intestinal fluids, airtight, after 30 minutes, add 5% trypsin hydrolyzing 4 hours in 45 DEG C of waters bath with thermostatic control, enzymolysis solution, in 85 DEG C of waters bath with thermostatic control 15 minutes, is cooled to room temperature, with 4500r/min centrifugal 15 minutes, get supernatant, dry, obtain Periostracum Cicadae trypsin digestion thing;
Step C gained pepsin zymolyte and step D gained trypsin digestion thing are Periostracum Cicadae protease hydrolyzed thing.
4. according to the application of the Periostracum Cicadae protease hydrolyzed thing described in claim 1-3 any one, it is characterized in that: described Periostracum Cicadae pepsin zymolyte or the application of trypsin digestion thing in the medicine of preparing anticoagulation or thrombolytic.
5. application according to claim 4, 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 (4)
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| CN105168255A (en) * | 2015-09-08 | 2015-12-23 | 浙江海洋学院 | Zymolytic tuna blood oral liquid for antithrombotic and thrombolytic therapies and preparation method thereof |
| CN106676068A (en) * | 2017-03-04 | 2017-05-17 | 南京九寿堂医药科技有限公司 | Biologically active peptide and method for proliferating CIK cell in vitro |
| CN109907358A (en) * | 2019-03-29 | 2019-06-21 | 蔡树成 | A kind of chrysanthemum cigarette production method and equipment |
| CN116473926A (en) * | 2023-05-12 | 2023-07-25 | 河北地邦动物保健科技有限公司 | Preparation method of seven-ingredient stagnation-eliminating granule |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105168255A (en) * | 2015-09-08 | 2015-12-23 | 浙江海洋学院 | Zymolytic tuna blood oral liquid for antithrombotic and thrombolytic therapies and preparation method thereof |
| CN106676068A (en) * | 2017-03-04 | 2017-05-17 | 南京九寿堂医药科技有限公司 | Biologically active peptide and method for proliferating CIK cell in vitro |
| CN106676068B (en) * | 2017-03-04 | 2019-01-01 | 绍兴市逸晨医疗科技有限公司 | A kind of method of biologically active peptide and amplification in vitro CIK cell |
| CN109907358A (en) * | 2019-03-29 | 2019-06-21 | 蔡树成 | A kind of chrysanthemum cigarette production method and equipment |
| CN116473926A (en) * | 2023-05-12 | 2023-07-25 | 河北地邦动物保健科技有限公司 | Preparation method of seven-ingredient stagnation-eliminating granule |
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