CN104922682A - Trypsin inhibitor and application thereof - Google Patents
Trypsin inhibitor and application thereof Download PDFInfo
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- CN104922682A CN104922682A CN201410106501.8A CN201410106501A CN104922682A CN 104922682 A CN104922682 A CN 104922682A CN 201410106501 A CN201410106501 A CN 201410106501A CN 104922682 A CN104922682 A CN 104922682A
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- alginate
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Abstract
The invention relates to a trypsin inhibitor, the trypsin inhibitor is characterized by comprising the main components of alginate or derivatives thereof; compared with the traditional trypsin inhibitor, the trypsin inhibitor has the advantages of high efficiency, low cost, abundant raw materials, simple preparation, no toxic and side effects, and better bio-stability, and is more suitable for using as enzymatic hydrolysis resistant auxiliary materials in protein and polypeptide drugs.
Description
Technical field
The present invention relates to a kind of protease inhibitor, specifically a kind of based on the trypsin inhibitor of alginate or derivatives thereof.
Background technology
Trypsin is small intestinal major protein enzyme, is peptide, and then is decomposed into aminoacid by proteolysis.First it enter duodenum (small intestinal topmost) by pancreatic secretion, is the proteolytic enzyme that in small intestinal, concentration is the highest.Trypsin inhibitor refers to a class material of contestable or Noncompetition inhibition tryptic activity.At present commercial trypsin inhibitor is mainly divided into two classes, and a class derives from animal, is mainly separated from Pancreas Bovis seu Bubali, human urine and obtains, and another kind ofly derives from plant, is mainly separated from the seed of the plants such as Semen sojae atricolor and obtains.Above-mentioned two tryptase inhibitors all belong to protein, by playing non-competitive inhibition with the strong bonded in tryptic activity site, advantage is that binding constant is high, specificity is strong, defect is that process for separating and purifying is loaded down with trivial details, cost is high, the poor stability easy in inactivation of inhibitor own, enters toxicity and the side effect such as human body easily causes physiological metabolism disorderly.
Due to above-mentioned advantage and the defect of traditional trypsin inhibitor, the application of current commercialization trypsin inhibitor in field of medicaments directly plays therapeutical effect, be mainly used in the treatment of acute pancreatitis, prevent the damage of trypsin to internal organs of premature activation; Also be applied in addition in cardiac operation, suppress the enzyme spilt from myocardial cell that trauma causes, prevent the hemorrhage complication that it causes the impact of Coagulation test, research proves that heavy dose of trypsin inhibitor can reduce 40% ~ 50% of postoperative hemorrhage amount.Also trypsin inhibitor can be used for preventing and Tumor suppression transfer to have research to confirm in addition, because tumor cell destroys surrounding tissue by trypsinlike enzyme, forms passage and leaves former position.
Due to high curative effect and the low toxic and side effects of albumen and polypeptide drug, receive much concern in recent years, unfortunately because protein substance ubiquitous biological stability is poor, the bioavailability easily caused by the defect of gastrointestinal tract endoproteinase hydrolysis destruction is poor, limits the extensive use of such medicine.Add protease inhibitor administration together with proteins and peptides class medicine, it is the hands section that researcher begins one's study very early, (the A.Bernkop-Schnurch and A.Scerbe-Saiko such as such as Andreas in 1998, Synthesis and in vitro evaluation of chitosan-EDTA-protease-inhibitor conjugateswhich might be useful in oral delivery, 1998.15 (2): p.187-196), just by trypsin inhibitor, chymotrypsin inhibitor and elastase inhibitor covalence graft are on the medicinal high polymer adjuvant of one, prepare a kind of novel resistance to enzymolysis adjuvant, for the preparation of proteins and peptides class oral formulations, obtain good therapeutic effect.But after 2000, this kind of research does not have substantially, and application example does not have especially.Main because the restriction of the Cost Problems of this tryptase inhibitors, bio-toxicity problem and homeostasis sex chromosome mosaicism, traditional trypsin inhibitor is severely limited as the application of resistance to enzymolysis pharmaceutic adjuvant.
On the other hand, the whole world has found about more than 4000 kind of enzyme up to now, enzyme in organism is far longer than this quantity, and we are also constantly finding new enzyme, these enzymes overwhelming majority is being isolated, after crude separation in organism, in extracting solution except the enzyme that we are concerned about, there are other higher enzymes of abundance in many organisms toward contact, such as lipase, trypsin, amylase etc., and the enzyme that we are concerned about usually concentration is very low by contrast.Better study the lower enzyme of abundance to remove other enzymes, traditional method is by chromatographic column, utilizes object enzyme and the difference of other enzymes in the physicochemical properties such as molecular weight, charging property, chirality to carry out separation and purification, then Fractional Collections.The advantage of this method is that to obtain the purity of object enzyme high, and enzyme retentivity of living is relatively good, and defect yields poorly, and cost is high.
On the other hand, trypsin main source is the pancreas of pig or cattle, and for obtaining purer trypsin from the crude extract of these tissues, traditional handicraft comprises the methods such as salting-out separation, recrystallization separation and column chromatography.
Utilizing trypsin to prepare the enzyme reaction engineering of small peptide from high molecular weight protein, from waste liquid, reclaim the still activated trypsin of tool if wish, current method is by tryptic immobilization, improves the utilization rate of enzyme.But this method is while prolongation trypsin service time, and often cost increases, and enzyme is lived and declined to some extent.
Summary of the invention
The present invention relates to using alginate and derivant thereof as trypsin inhibitor, can be applicable to the object of the resistance to enzymolysis adjuvant as the pharmaceutical preparation of proteins and peptides class, make up the deficiency of traditional trypsin inhibitor, also can be applicable to the object removing trypsin or trypsinogen from liquid, also can be applicable to the object of collecting trypsin or trypsinogen from liquid.
Described trypsin inhibitor main component is one or two or more kinds in alginate or derivatives thereof, and alginate and derivant thereof the mass ratio in trypsin inhibitor is 10-100%.
Alginic acid (Alginic acid) is by the polysaccharide of monosaccharide aldehydic acid linear polymerization, and monomer is β-D-manna aldehydic acid (M) and α-L-guluronic acid (G).M and G unit becomes block copolymer with the compound mode of M-M, G-G or M-G by Isosorbide-5-Nitrae glycosidic bond is connected.The empirical formula of alginic acid is (C
6h
8o
6)
n.The derivant (derivative) of alginate refers to the more complicated product that hydrogen atom on alginate molecule or hydroxyl are replaced by other atoms or atomic group and formed.
In trypsin inhibitor involved in the present invention, the range of choice of effective ingredient is whole alginate or derivatives thereofs, mean molecule quantity can be restricted to further between 10kDa to 1000kDa, the potassium alginate of G/M between 0.1-10, ammonium alginate or sodium alginate; Its derivant can be restricted to acrylate alginate, acrylamide alginate or vinyl acetate alginate, can obtain and better press down enzyme effect.One or more the mixture be selected within the scope of above-described alginate or derivatives thereof can form trypsin inhibitor involved in the present invention by the mode of physical mixed or covalence graft separately or with other materials.
Sodium alginate is a kind of natural polysaecharides macromolecular material, and first its preparation with low cost is simple, and commercially producing of sodium alginate starts from nineteen twenty-seven, and the present whole world about produces 30000 tons every year, and wherein 30% for food industry; Secondly its good stability, does not substantially degrade under human physiological environment; Its good biocompatibility last, is ratified as food additive to be safe by FDA.Pressing down in enzyme efficiency also very outstanding, suppressing the trypsin of same concentrations, compared with commercial soybeans trypsin, play the inhibitor that identical Inhibiting enzyme activity only needs low several order of magnitude concentration.
Although on enzyme binding constant and enzyme specificity degree, there is larger difference in alginate and traditional protein tryptase inhibitors, but the discovery of this tryptase inhibitors, expand the application of trypsin inhibitor in field of medicaments, for the resistance to enzymolysis method of protein and polypeptide drug preparation provides a kind of new approaches.
Therefore trypsin inhibitor involved in the present invention can be applicable in the preparation of protein or polypeptide drug; avoid by trypsin degradation for the protection of protein and polypeptide, alginate and derivant thereof can mix the preparation as protein or polypeptide drug separately or with other materials jointly.Wherein, alginate or derivatives thereof and albumen or polypeptide drug can physical blending or covalence grafts; The mode that alginate or derivatives thereof and other materials mix jointly can physical blending or covalence graft;
On the other hand, also alginate or derivatives thereof can be applied to the object of collecting trypsin or trypsinogen from liquid.Because sodium alginate is with stronger elecrtonegativity, complex can be formed by the trypsin of electrostatic interaction and positively charged, this complex can be removed easily by modes such as centrifugal or filtrations or be collected, and reaches the object be separated with object enzyme by trypsin higher for abundance.Be separated the alginate-trypsin complex obtained, trypsin can be reclaimed by certain means, reach tryptic recycling object.Therefore this character can also utilize in the liquid waste processing of trypsin digestion reactor, improves tryptic utilization rate.
The range of choice utilizing trypsin inhibitor involved in the present invention can remove and collect the liquid of trypsin or trypsinogen is all liquid containing trypsin or trypsinogen, animal intestinal juice can be restricted to further, animal pancreatic juice, animal tissue's extracting solution, plant tissue extracting solution, microbial fermentation solution, enzyme reactor waste liquid and the product obtained after raw material is processed by arbitrary or two or more being mixed in above-mentioned six kinds.Can obtain and better remove or collecting effect.
Accompanying drawing explanation
Fig. 1 is insulin degradation kinetics curve.
Water all containing same volume in reaction system, the insulin of same concentrations, calcium ion and buffer salt, pH is 7.6.In figure, abscissa is enzymolysis time, and vertical coordinate is the percentage ratio of insulin concentration relative to concentration during reaction beginning that is not degraded in enzymatic hydrolysis system.
Curve from top to bottom represents respectively: blank---not containing the degradation curve () of insulin during other materials in system; Embodiment 1---the degradation curve (Δ) also containing insulin when trypsin and sodium alginate in system; Reference examples 2---the degradation curve (▽) also containing insulin when trypsin and soybean trypsin inhibitor in system; Reference examples 3---the degradation curve (zero) also containing insulin during trypsin in system.
Detailed description of the invention
Embodiment 1
(1) trypsin inhibitor is prepared: sodium alginate aqueous solution, molecular weight 500kDa, G/M=80:20, concentration 0.12mg/ml;
(2) prepare trypsin solution 5ml, component in solution is PBS, pH=7.6, and wherein contain CaCl20.2mg/ml, trypsin 0.04mg/ml(trypsin used purchased from Aladdin reagent, Rate activity is not less than 3000IU/mg);
(3) insulin stock liquid is prepared: 10mg/ml, is dissolved in 0.01M aqueous hydrochloric acid solution;
(4) in step (2) solution, add the trypsin inhibitor 0.5ml of step (1), put into preheating 15min in 37 DEG C of waters bath with thermostatic control, then the insulin stock liquid 0.5ml that step (3) is prepared is added, timing is started immediately after eddy current mixing, maintain 37 DEG C of reaction 2h, and every 30min samples 200 μ l and mixes with 100 μ l enzymolysis stop buffers (hydrochloric acid 0.25mol/L) immediately from system from 0min.All samples be stored in 4 DEG C to be measured;
(5) detection of undecomposed insulin:
In system, the content of insulin is also changed slightly with reference to Chinese Pharmacopoeia 2010 method, utilize the highly effective liquid phase chromatographic system (HPLC) of waters, chromatographic column is C18 reversed-phase column (250mm*4.6mm), detector is ultraviolet-visible spectrum detector, mobile phase is acetonitrile: aqueous phase=78:22, wherein in aqueous phase containing the sodium sulfate of 0.2mol/L, and phosphoric acid 2.7ml/L adjust pH to 2.3 with ethanolamine.Detect flow velocity 1ml/min, temperature 40 DEG C.Insulin appearance time is 8min.Final result represents with the percent value (the non-degradation rate of insulin) of current sample insulin spikes integral area and 0 moment area;
(6) testing result display, insulin joins after in simulated intestinal fluid, under trypsin acting, complete trypsin amount extends along with degradation time relative to ratio when starting and slowly declines, under the suppression of sodium alginate, the insulin of about 85% or complete in 2 little enzymatic hydrolysis systems constantly.Illustrate that sodium alginate trypsin inhibitor can suppress trypsin to the degraded of insulin, very strong protective effect is have to islets of langerhans.Enzymolysis kinetics curve is shown in accompanying drawing 1(Δ).
Reference examples 2
The trypsin inhibitor used is commercialization trypsin STI) aqueous solution, STI purchased from Aladdin (article No. T113170), molecular weight 24kDa, concentration 0.12mg/ml.
According to the degraded evaluation identical with embodiment 1 and insulin detection method, result is that commercial soybeans trypsin inhibitor is when the dosage identical with sodium alginate, almost inhibitory action is not had to trypsin, only have an appointment in 2 little enzymatic hydrolysis systems constantly 13% insulin be complete, illustrate compared with inhibitor of the present invention, the commercial soybeans trypsin inhibitor of equal in quality does not almost press down enzyme effect.Enzymolysis kinetics curve is shown in accompanying drawing 1(▽).
Reference examples 3
The trypsin inhibitor used is water.
According to the degraded evaluation identical with embodiment 1 and insulin detection method, result is not having under inhibitor effect, only have an appointment in 2 little enzymatic hydrolysis systems constantly 12% insulin be complete, illustrate that most insulin degraded can destroy by trypsin when not having inhibitor.Enzymolysis kinetics curve is shown in accompanying drawing 1(zero).
Embodiment 4
The trypsin inhibitor used is the powder of potassium alginate, molecular weight 800kDa, G/M=40:60, feeds intake according to 1 milligram of every milliliter of trypsin solution.
According to the degraded evaluation identical with embodiment 1 and insulin detection method, degraded two is constantly little, and the non-degradation rate of insulin is 92%
Embodiment 5
The trypsin inhibitor used is ammonium alginate aqueous solution, molecular weight 40kDa, G/M=20:80, concentration 0.06mg/ml.
According to the degraded evaluation identical with embodiment 1 and insulin detection method, degraded two is constantly little, and the non-degradation rate of insulin is 75%
Embodiment 6
The trypsin inhibitor used is vinyl acetate sodium alginate aqueous solution, molecular weight 35kDa, G/M=60:40, concentration 1mg/ml.Sodium alginate molecule is about 5% by the ratio that the hydroxyl that vinyl acetate replaces accounts for whole hydroxyl value.
According to the degraded evaluation identical with embodiment 1 and insulin detection method, degraded two is constantly little, and the non-degradation rate of insulin is 45%
Embodiment 7
The trypsin inhibitor used is the physical mixture powder of ammonium alginate and sodium alginate, and the mass ratio of the two is 2:8.The wherein molecular weight 40kDa of ammonium alginate, G/M=20:80, the molecular weight 200kDa of sodium alginate, G/M=70:30.Trypsin inhibitor feeds intake according to 0.15 milligram of every milliliter of trypsin solution.
According to the degraded evaluation identical with embodiment 1 and insulin detection method, degraded two is constantly little, and the non-degradation rate of insulin is 75%.
Embodiment 8
The trypsin inhibitor used is potassium alginate, and potassium alginate is connected with insulin by covalence graft, and the potassium alginate molecular weight 20kDa used, G/M=10:90, mole graft ratio is potassium alginate: insulin=2.7:1.The insulin solution that product potassium alginate is derivative, feeds intake according to 0.25mg/ml trypsin solution.
According to the degraded evaluation identical with embodiment 1 and insulin detection method, degraded two is constantly little, and the non-degradation rate of potassium alginate insulin is 40%.
Embodiment 9
From the crude extract of the protease that Pancreas Bovis seu Bubali obtains, wherein containing a large amount of trypsin, also containing Chymetin and elastoser, in order to remove trypsin, study other enzymes, by adding trypsin inhibitor removing trypsin.
Trypsin minimizing technology:
Preparation trypsin inhibitor: sodium alginate aqueous solution, molecular weight 150kDa, G/M=60:40, concentration 10mg/ml;
In Pancreas Bovis seu Bubali crude extract, add trypsin inhibitor, add 1ml in every 100ml crude extract, at 4 DEG C, stirring and evenly mixing 2h(is too inviolent);
With the acceleration of 6000g centrifugal 15min at 4 DEG C;
Supernatant lyophilization can obtain the crude extract powder of Chymetin and elastoser, and a large amount of trypsin is removed, can carry out studying or further polishing purification.
Trypsin removal efficiency detects:
Tryptic enzyme is lived and is used BAEE(N-benzoyl-L-arginine ethyl ester hydrochloride) method mensuration, method is with reference to Chinese Pharmacopoeia (version in 2010).
Before removing trypsin, in extracting solution, trypsin enzyme is lived: A0=1254BAEE units/ml.
After after removing trypsin, powder is dissolved in 100ml PBS, trypsin enzyme is lived: A
1=119BAEEunits/ml.
Removal efficiency=1-A
1/ A
0x100%=90.5%
Embodiment 10
In the commercial production of enzyme preparation, sometimes need the trypsin recycling of removing.
The tryptic recovery method removed:
The precipitation obtained centrifugal in embodiment 7 is scattered in 100ml PBS and stirs;
Add aqueous povidone solution 1ml(10mg/ml), and 1h is stirred at 4 DEG C;
With the acceleration of 6000g centrifugal 15min at 4 DEG C, supernatant with 0.22 μm of water system membrane filtration once;
Collect filtrate, be tryptic Ethylene recov.
In trypsin Ethylene recov, trypsin enzyme is lived: A
3=465BAEE units/ml.
The response rate=A
2/ (A
0-A
1) x100%=41.0%
Embodiment 11
Trypsin inhibitor involved in the present invention can make oral insulin liquid with other materials physical mixed, measures described in following ingredient lists, under stirring, add raw material successively, and stir the rear aseptic filtration of 2h mixing at 4 DEG C, low temperature is degassed and get final product.
Ingredient lists
Note: wherein sodium alginate molecular weight is 500kDa, G/M=80:20;
Evaluation methodology:
According to the enzymolysis evaluation methodology of embodiment 1, directly added in simulated intestinal fluid by insulin oral preparation 1ml, under 37 degrees Celsius, degraded 2 is constantly little, sampling, and according to the non-degradation rate of insulin in the non-degrade insulin evaluation methodology characterizing sample in embodiment 1, result is 72%.
Embodiment 12
Trypsin inhibitor involved in the present invention can covalence graft on other pharmaceutic adjuvants, and make tablet with after new resistance to enzymolysis adjuvant and protein and polypeptide drug physical mixed.
Potassium alginate (20kDa; G/M=5:5) in nitrogen protection and 60 DEG C of environment; with sodium carboxymethyl cellulose generation condensation reaction under catalyst action, the new adjuvant made is isolated by filtration also washing removing catalyst and unreacted micromolecule potassium alginate under pH=3 condition.Filter cake obtains the sodium carboxymethyl cellulose of potassium alginate covalence graft after drying.
The preparation method of Radix Ginseng extract tablet is as follows:
Measure according to described in ingredient lists, by material powder mixed grinding, cross 60 mesh sieves, tabletting.
Ingredient lists
Evaluation methodology:
According to the degraded evaluation identical with embodiment 1 and detection method, degrade two constantly little, in Radix Ginseng extract, undegradable total protein content is 52% of inventory, and the same preparation of shortage potassium alginate modified carboxy methyl cellulose sodium material in contrast, according to the degraded evaluation identical with embodiment 1 and detection method, in Radix Ginseng extract, undegradable total protein content is 12% of inventory.
Select insulin to be convenient to resistance to enzymolysis evaluation and detection as model protein in above-mentioned all embodiments, the object of protection of the present invention to trypsin inhibitor is not particularly limited, and object of protection can be protein or the polypeptides matter of the protection of any needs.
Above-mentioned exemplary embodiments of the present invention all in be exemplary; instead of it is determinate; therefore the present invention can carry out the multiple modification of exemplary embodiments according to the physicochemical property of the applied environment of trypsin inhibitor and resistance to enzymolysis object of protection; technical staff can obtain described modification from the description comprised, and all these modification are considered to fall in scope and spirit of the present invention.
Claims (9)
1. a trypsin inhibitor, is characterized in that: described trypsin inhibitor main component is one or two or more kinds in alginate and derivant thereof, and the mass fraction of alginate or derivatives thereof in trypsin inhibitor is 10-100%.
2. according to trypsin inhibitor according to claim 1, it is characterized in that: described alginate comprises one or more the salt-mixture in the sodium salt of alginic acid, ammonium salt and potassium salt;
Described alginate derivant comprises one or more the salt-mixture in acrylate alginate, Polyethylene Glycol alginate, acrylamide alginate and vinyl acetate alginate.
3. according to trypsin inhibitor according to claim 1, it is characterized in that: the mean molecule quantity of described alginate is between 10kDa to 1000kDa.
4. according to trypsin inhibitor according to claim 1, it is characterized in that: the guluronic acid (G) in described alginate and the molar average ratio of mannuronic acid (M) two kinds of monomers, namely G/M is between 0.1-10.
5. an application for trypsin inhibitor described in claim 1,2,3,4 or 5, is characterized in that:
Inhibitor described in claim 1,2,3,4 or 5 is made an addition in the preparation of protein and/or polypeptide drug, avoids by trypsin degradation for the protection of protein and polypeptide.
6., according to application according to claim 5, it is characterized in that:
Described alginate or derivatives thereof can separately or mix as the preparation of adjuvant for the preparation of protein and/or polypeptide drug with other materials jointly;
Wherein, other described component is made up of one or more in water, sodium chloride, sodium dihydrogen phosphate, sodium hydroxide, hydrochloric acid, mannitol, sucrose, lactose, dextrin, sodium stearate, xylitol, amylum pregelatinisatum, microcrystalline Cellulose, poly-hydroxymethylacrylate Arrcostab, chitosan, polyvinylpyrrolidone, Polyethylene Glycol, Carboxymethyl cellulose sodium, soybean trypsin inhibitor, elastase inhibitor and chymotrypsin inhibitor;
Alginate or derivatives thereof and protein and/or polypeptide drug can physical blending or covalence grafts;
The mode that alginate or derivatives thereof and other materials mix jointly can physical blending or covalence graft.
7., according to application according to claim 5, it is characterized in that:
Inhibitor described in claim 1,2,3 or 4 is applied to the object removing trypsin or trypsinogen from liquid;
Its detailed process implemented is: the aqueous solution adding the inhibitor described in 0.01 to 2 part of volume in the described liquid of 1 part of volume, wherein the mass fraction of inhibitor is between 0.0000001% to 10%, under 0 DEG C to 15 DEG C environment, 15min is hatched in concussion, last high speed centrifugation (acceleration of gravity is between 1000g to 15000g) discards precipitation, and supernatant is the liquid after process.
8., according to application according to claim 5, it is characterized in that:
Inhibitor described in claim 1,2,3 or 4 is applied to the object of collecting trypsin or trypsinogen from liquid;
Its detailed process implemented is: the aqueous solution adding the inhibitor described in 0.01 to 2 part of volume in the described liquid of 1 part of volume, wherein the mass fraction of inhibitor is between 0.0000001% to 10%, under 0 DEG C to 15 DEG C environment, 15min is hatched in concussion, then high speed centrifugation (acceleration of gravity is between 200g to 12000g) abandoning supernatant, precipitation concussion is scattered in 0.01 part in the separating medium of 2 parts of volumes, and described separating medium is NaCl, KCl, CaCl
2, Na
2cO3, Na
2sO
4, Na
3pO
4, the aqueous solution of one or more mixture in polymine and sodium carboxymethyl cellulose, the concentration of described separating medium, between 0.1% to 3%, finally uses the membrane filtration of 0.22 μm to 1.2 μm, collects filtrate and is gained.
9., according to the application described in claim 7 or 8, it is characterized in that:
Described liquid comprises animal intestinal juice, animal pancreatic juice, animal tissue's extracting solution, plant tissue extracting solution, microbial fermentation solution, enzyme reactor waste liquid and the product obtained after raw material carries out pretreatment by arbitrary or two or more being mixed in above-mentioned six kinds, wherein said pretreatment comprise in mechanical activation comminution, centrifugal and filter operation one or more.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643754A (en) * | 2019-09-29 | 2020-01-03 | 四川大学 | Method for regulating protease catalytic activity in tanning process |
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2014
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| CN1430517A (en) * | 2000-05-19 | 2003-07-16 | 雷克特本克斯尔保健(英国)有限公司 | Pepsin inhabition by alginates |
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| CN101092494A (en) * | 2007-06-15 | 2007-12-26 | 天津大学 | Hybridized microballons of calcium polyacrylate / calcium alginate of macro molecule engram |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643754A (en) * | 2019-09-29 | 2020-01-03 | 四川大学 | Method for regulating protease catalytic activity in tanning process |
| CN110643754B (en) * | 2019-09-29 | 2021-09-14 | 四川大学 | Method for regulating protease catalytic activity in tanning process |
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Application publication date: 20150923 |