CN105424946B - A kind of thrombin time test reagent containing rosmarinic acid - Google Patents
A kind of thrombin time test reagent containing rosmarinic acid Download PDFInfo
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- CN105424946B CN105424946B CN201511000031.8A CN201511000031A CN105424946B CN 105424946 B CN105424946 B CN 105424946B CN 201511000031 A CN201511000031 A CN 201511000031A CN 105424946 B CN105424946 B CN 105424946B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
Abstract
The present invention relates to a kind of thrombin time test reagent containing rosmarinic acid, it is made up of the Tris HCl buffer containing 25U/ml human thrombin, 2% bovine serum albumin, 1.5mM rosmarinic acid, the sodium azide of 0.05%, 3.5% methionine, 1.5%PEG, 3% mannitol, 2.5% maltose and 10mM calcium chloride, percentage ratio therein is mass volume ratio, and pH is 7.4.The preparation method of mentioned reagent is as follows: preparation Tris concentration 50mM, the buffer of pH7.4, in this buffer, add final concentration be respectively 2% BSA, 1.5mM rosmarinic acid, the sodium azide of 0.05%, the methionine of 3.5%, the PEG of 1.5%, the mannitol of 3%, the maltose of 2.5%, the calcium chloride of 10mM, wherein in addition to calcium chloride, remaining is mass body fraction；Being filtered with 0.22 micron membrane filter by above-mentioned mixed liquor, add human thrombin lyophilized formulations in filtrate, making the concentration of human thrombin in whole filtrate is 25U/ml, obtains TT reagent.The present invention uses rosmarinic acid to make stabilizer, is remarkably improved antioxygenic property and the stability of thrombin time test reagent.
Description
The application is the divisional application of Application No. 2015104204159, filing date 2015-07-16, applies for entitled " a kind of
Clotting reagent antioxidant ".
Technical field
The present invention relates to a kind of thrombin time test reagent containing rosmarinic acid.
Background technology
Blood coagulation test belongs to thrombotic disease inspection, and wherein, thrombin time (TT) is the important indicator that coagulation function checks,
For checking that plasma fibrinogen is changed into fibrin ability.
The core component of thrombin time test reagent is thrombin.Thrombin is a kind of Multi-functional wire serine protease, has and pancreas
Sequence that chrymotrypsin is similar and structure, containing two polypeptide chains of A, B, be connected by an interchain disulfide bond.B chain is functional chain,
There is typical serine protein hydrolase foldable structure, containing 1 active center between two β-pleated sheet buckets, outside 2
Binding site (Exosite I, Exosite II).Thrombin functional group and three dimensional structure, can be because of physics and the change of electrochemical conditions
And wreck.
Thrombin time inspection is carried out on manual, semi or fully automatic Blood coagulation instrument, and diagnostic reagent is mixed by it with test plasma
Close, to measure clotting time.At present, thrombin time diagnostic reagent is based on dry powder formulations, because lyophilization is protection blood coagulation
A kind of effective ways of pheron activity.Generally, powdered reagent needs to add quantitative redissolution liquid or deionized water makes it multiple
Molten, return to the solution state before lyophilizing, but, process of redissolving is time-consuming, meanwhile, and can be variant before the concentration of thrombin and lyophilizing,
If misoperation, thrombin degeneration can be caused, cause concentration of thrombin value to reduce, cruor time extending.
Compared with powdered clotting reagent, liquid thrombin reagent can overcome the defect that redissolution process or freeze-drying process bring, for user
Bring the convenient of use, also improve accuracy and the accuracy of testing result simultaneously, but, liquid reagent also brings to developer
Lot of challenges sex chromosome mosaicism.Thrombin belongs to biomacromolecule, and its activity depends on maintaining of the fine space structure of himself.
Under liquid condition, the structure comparison of protein or enzyme is fragile, acid, alkali, temperature, ionic strength and some other compound (bag
Include high price metal ion) a small amount of existence, all can cause the change of this space structure, the minor variations of partial structurtes also can cause
Protein or the forfeiture of enzymatic activity.The degeneration of thrombin or the reduction of activity, part is caused by extraneous factor, but at certain bar
Under part, protein or enzyme also can selfdecomposition, cause overall activity to decline.Accordingly, it would be desirable to elimination may promote protein or enzyme as far as possible
There is the condition of selfdecomposition.
Oxidation is one of major reason causing thrombin inactivation or activity reduction, i.e. some specific amino acid residue reacts,
Cause changing in thrombin function and structure, the affinity of oxide is strengthened, it is easy to be polymerized, cross-link and cause its functionally inactive.
Reactive oxygen system in protein-based tissue includes that hydroxyl radical free radical, the oxidation of general protein and lipid are all by free radical chain
Formula reaction causes, and relates to dehydrogenation, electron transmission, addition, ruptures and reset, dimerization, be disproportionated and replace, as fat oxidation,
Include equally initiateing, transmitting and terminate three phases.Therefore, the addition of suitable antioxidant, liquid thrombin reagent can be kept
Stability.
Preparation containing antioxidant clotting reagent in terms of, patent EP942284A2, US5506146, US5314695 and
US8729022B2, proposes to add aminoacid (mainly glycine) as antioxidant.
According to the documents and materials of inquiry, the antioxidant added in clotting reagent probably includes 3 classes, and one is aminoacid and derivant thereof,
With glycine as main forms；Two is acid hexose derivant, including ascorbic acid；Three be include Butylated hydroxyanisole and
Butylated hydroxytoluene is at other interior antioxidant.
In liquid thrombin time test reagent, retrieve according to domestic and international Research Literature, mostly use polyol or polysaccharide or hydrochloric acid
Salt, as the stabilizer of thrombin, adds sodium azide as antibacterial or stabilizer simultaneously, the selection of these stabilizers, substantially
Have followed the production thinking of dry powder clotting reagent, in terms of stable liquid clotting reagent, effect is the most as one wishes.As it was previously stated, it is many
Alcohol or polysaccharose substance, it is possible to promote the stability of thrombin, but the excess of this kind of material adds, and can increase the viscosity of mixed liquor,
Thus reduce the accuracy of the thrombin time test reagent using optical method to measure, and the addition of excess chlorine hydrochlorate, solution can be improved
The ionic strength of phase.Sodium azide has preferable anti-microbial property, but its toxicity is bigger.
On the basis of experimentation, the inventors discovered that, on the basis of optimizing conventional stabilizer, add rosmarinic acid, and close
Reason controls its addition concentration, can promote the stability of thrombin in liquid environment well.Its addition, can sting conjunction solution mutually in
It is unfavorable for the metal ion that thrombin is stable, thus reduces the bivalent metal ion catalytic action to thrombin oxidation reaction；Meanwhile,
Also can react with peroxy radical, terminate protein peroxidating chain reaction, thus improve the stability of reagent further.
On the basis of the studies above, present invention difference based on non-oxidizability and the impact of concentrations on product, propose with containing adjacent benzene
The phenolic acid of diphenol structure and relatively small molecular weight is as the antioxidant of liquid thrombin reagent.More particularly, rosmarinic acid is selected
As the antioxidant of different clotting reagents, to promote the antioxygenic property of different clotting reagent.
Summary of the invention
The purpose of the present invention, is on the basis of conventional clotting reagent, proposes a kind of efficient, nontoxic antioxidant, it can in case
The oxidation of protein in hemostasis-coagulation reagent, eliminates the problem that general antioxidant antioxygenic property is not enough, carries for clotting reagent (TT)
Carry out more preferable non-oxidizability.
The rosmarinic acid that the present invention proposes belongs to a class of phenols, and by the classification of WBSSH, most of phenols all have in various degree
Non-oxidizability, but not all phenols may be used to the antioxidant of clotting reagent.Studies have found that, protein is permissible
Interacting with phenols, cause protein precipitation [1] [2], on the other hand, Asano etc. [3] [4] thinks, containing proline residue
Protein easily produces precipitation when with phenols compounds complexation, because proline residue or other hydrophobic amino acid content are high
Protein is stronger to the affinity of phenols.Therefore, not all phenols may be used to clotting reagent, and especially TT surveys
Determine the antioxidant of reagent.
Mainly comprising as thrombin of TT mensuration reagent.In order to verify the phenols impact on thrombin activity, the present invention will be the denseest
The thrombin of degree mixes with different types of phenols, uses the change of luminous substrates concentration, determines that different types of phenols is to thrombin
The impact of activity.Specifically, the method proposed with Sonder et al. [5], measure the water of luminous substrates D-Phe-Pip-Arg-pNA
Solution degree, to determine the activity of human thrombin, i.e. measures the absorbance at 415nm by 96 hole microplate reader, adds and send out in every hole
Light substrates, be simultaneously introduced with phenols preculture after human thrombin.Determine maximum reacting value in each light absorption curve, and estimate according to curve
Calculate the concentration of phenols corresponding when IC50 value, i.e. thrombin amide decomposition activity suppress 50%.
Result shows, in the phenols selected, different phenols is different to the activity influence of thrombin, adds epicatechin and catechu
During acid, human thrombin activity is caused to reduce interpolation concentration corresponding when 50% higher, between 130-150mM；Add tannin
During acid, even if relatively low concentration (1.42mM), also the concentration making human thrombin is reduced 50%.Salvianolic acid or rosmarinic acid pair
The impact of thrombin activity, is affected by adding concentration, when adding concentration between 0.01-2mM, is had substantially no effect on thrombin
Activity, promotes stable reagent effect simultaneously.Meanwhile, flavonoid phenols human thrombin is had maximum inhibition, this with
The result of study of Jedinak [6] and Mozzicafreddo [7] et al. is identical.This conclusion, it is also possible to according to Liu [8], Li [9]
Being verified with the Shi [10] et al. result of study in terms of molecular docking, their research shows, in the B ring of flavone structure,
-OH base is the most, and the suppression to thrombin is the biggest；The B ring of flavone structure and C ring, can respectively with the S1 in thrombin structure
Chain bag and the reaction of S2 chain bag, A ring can partly react with thrombin S3 chain bag, and 3 '-hydroxyl in B ring and 4 '-hydroxyl-functional
Group, and other 3 '-hydroxyl groups on C ring, all can anticoagulant enzyme activity.
The present invention, on the basis of above-mentioned experiment, further illustrates, although phenols all has a non-oxidizability in various degree, but not
All of phenols may be used to antioxidant or the stabilizer of clotting reagent.
On the basis of experimentation, from non-oxidizability relative to power, the antioxidation using rosmarinic acid as clotting reagent is proposed
Agent.
The clotting reagent antioxidant of the present invention, for phenolic acid, specially rosmarinic acid.
Rosmarinic acid of the present invention, containing catechol structure, catechol structure has a following advantage:
1) catechol structure has the strongest metal ion and stings conjunction ability, thus reduces bivalent metal ion to oxidation reaction
Catalytic action, especially, during preparing clotting reagent, raw materials used in a small amount of heavy metal ion, can destroy tissue because of
Son or the stablizing of thrombin.Therefore, it plays EDETATE SODIUM salt or potassium salt and the partial amino-acid chelation to metal ion.
2) catechol structure can react with peroxy radical, thus terminates protein oxidation or lipid peroxidation chain reaction,
This point is even more important to the thrombin time test reagent containing thrombin.
On the other hand, when the molecular weight of phenolic acid is bigger, can containing more phenolic hydroxyl group, so can increase phenolic acid and protein it
Between reaction probability, it is easy to form the cross-linked structure of phenolic acid (or phenols)-protein, cause protein unstable.Therefore,
The present invention, when selecting phenolic acids, selects the phenolic acids containing catechol structure of molecular weight as far as possible.Meanwhile, Binding experiment
Research, preferably rosmarinic acid.
Currently preferred rosmarinic acid, molecular formula is C18H16O8, molecular weight is 360.As the Natural antioxidant, Herba Rosmarini Officinalis
Acid has the extremely strong activity removing free radical, and its non-oxidizability is better than caffeic acid, chlorogenic acid, folic acid etc..Northwest light industry food
The evaluation test that product engineering department is carried out proves, rosmarinic acid antioxidant has the antioxidation [11] more higher than butylated hydroxytoluene.
According to the research of Zou Kun [12] et al., rosmarinic acid antioxidant mechanism includes directly reacting with Active Radicals Produced, i.e. blocks freedom
The biography chain process of base chain, thus block free chain reaction；Transition metal ions (including ferrous ion) with induced oxidation
Complexation, reaches antioxidant effect.
In the present invention, described rosmarinic acid concentration in TT measures reagent is 0.01-2mM.
The purpose of the present invention, be to provide a kind of use rosmarinic acid as the TT clotting reagent of antioxidant, by containing 25U/ml
Human thrombin, 2% bovine serum albumin, 1.5mM rosmarinic acid, the sodium azide of 0.05%, 3.5% methionine, 1.5%PEG,
The Tris-HCl buffer composition of 3% mannitol, 2.5% maltose and 10mM calcium chloride, percentage ratio therein is quality volume
Ratio, pH is 7.4.
The preparation method of the above-mentioned thrombin time test reagent containing rosmarinic acid, step is as follows: preparation Tris concentration 50mM,
The buffer of pH7.4, in this buffer, add final concentration be respectively 2% BSA, 1.5mM rosmarinic acid, 0.05%
Sodium azide, the methionine of 3.5%, the PEG of 1.5%, the mannitol of 3%, the maltose of 2.5%, the calcium chloride of 10mM,
Wherein in addition to calcium chloride, remaining is mass body fraction；Above-mentioned mixed liquor is filtered with 0.22 micron membrane filter, adds in filtrate
Entering human thrombin lyophilized formulations, making the concentration of human thrombin in whole filtrate is 25U/ml, obtains TT and measures reagent.
Rosmarinic acid of the present invention, can coordinate other antioxidant to be used in conjunction with, to promote the antioxidation of clotting reagent further
Ability, improves the stability of clotting reagent.Other described antioxidant includes acids antioxidant such as ascorbic acid, aminoacid
Class antioxidant such as glycine, methionine, the addition of this kind of additive, can by addition in clotting reagent add.
Owing to phenols can suppress the activity [13] of the many enzymes including serine protease, and phenols and protein can mutually be made
With causing protein precipitation, therefore, the rarest report that aldehydes matter is used as clotting reagent stabilizer.And it is a discovery of the invention that
In clotting reagent, add one or more phenols, the stability of mixed liquor can be obviously improved.The present invention is divided by anti-oxidizing activities
Analysis, it is determined that the phenols type being suitable for is rosmarinic acid；Meanwhile, make the reaction between phenols and thrombin to analyze, and at this
On the basis of, it is proposed that the concentration value of the addition of rosmarinic acid in preparation thrombin time reagent.The present invention is by phenols kind and dense
The reasonable selection of degree, not only will not bring adverse effect to the stability of clotting reagent, can be obviously improved its stability on the contrary, especially
It is that the TT with thrombin as composition measures reagent.It addition, applicant further found that, in clotting reagent, with the addition of Vitamin C acids
On the basis of the general stabilizer such as antioxidant, amino acids antioxidant, then when adding the rosmarinic acid of the present invention, effect is obvious
It is better than general stabilizer or effect time rosmarinic acid individually makees stabilizer.
Detailed description of the invention
Below in conjunction with specific embodiment the present invention done and further describes in detail:
Embodiment 1
Rosmarinic acid measures the impact of reagent non-oxidizability to TT
The oxidation resistance test of the thrombin time test reagent of rosmarinic acid is contained under room temperature condition
Preparation Tris concentration 50mM (pH7.4) buffer, in this buffer, add final concentration be respectively 2% BSA,
The sodium azide of 0.05%, the methionine of 3.5%, the PEG of 1.5%, the mannitol of 3%, the maltose of 2.5%, 10mM
Calcium chloride, wherein in addition to calcium chloride, remaining is mass body fraction.Above-mentioned mixed liquor is filtered with 0.22 micron membrane filter,
Adding human thrombin lyophilized formulations in filtrate, making the concentration of human thrombin in whole filtrate is 25U/ml, obtains TT and measures reagent.Warp
CA1500 blood coagulation analyzer measures, and the TT value of this reagent is 15.8s.
Contrast agent: research shows, in the blood coagulation preparation containing thrombin, the addition of the rosmarinic acid of high concentration, not only do not have
Play antioxidation, promote protein degradation on the contrary, along with the reduction of concentration, they the Degradation of thrombin is weakened or
Disappear.Based on experimental study, the addition concentration of rosmarinic acid of the present invention is 0.01-2mM.In being embodied as, try at aforementioned TT
Adding the rosmarinic acid of 1.5mM in agent, the instant result measured with CA1500 coagulo meter is 15.7s.
Will preparation two kinds of TT reagent, under the conditions of 37 DEG C preserve, every day timing sampling once, detection two kinds of reagent TT value.
Result shows, within the measurement phase of altogether 5 days, TT value has the trend become larger in time.Result is as shown in table 1.
The Detection of Stability result unit of TT reagent under table 1 room temperature condition: second
The above results shows, within 5 days, only with methionine as the less stable of the TT reagent of antioxidant.?
TT reagent adds rosmarinic acid, the non-oxidizability of TT reagent can be obviously improved, and then promote the stability of reagent.
Embodiment 2
The long-term oxidation resistance test of the thrombin time test reagent containing rosmarinic acid
By the aforementioned TT reagent without rosmarinic acid and the TT reagent containing rosmarinic acid, subpackage, preserve under the conditions of 4 DEG C, each
The moon takes out one bottle, even takes 9 months, the TT value of two kinds of reagent of detection.
Regression equation:
3.5% methionine: y=0.0352x+15.766；CV=4.2%
3.5% methionine and the rosmarinic acid of 1.5mM: y=0.0187x+15.726；CV=3.5%
This explanation, within the above-mentioned time period, due to the addition of rosmarinic acid, has been obviously improved the antioxygenic property of TT reagent.
The rosmarinic acid that emphasis of the present invention is mentioned, has potential biological activity, including its antioxygenic property.Containing thrombin
Preparation in add rosmarinic acid, the activity of thrombin can be had certain suppression, but will not on the premise of appropriate concentration exists
Affect clotting mechanism.Fan HY [14] et al. also demonstrates this point by the result of study of arteriovenous shunt model.Therefore, close
Suitable phenolic acid type and concentration, can improve the antioxygenic property of thrombin, and then promote the long-time stability of reagent.
It addition, the Main Ingredients and Appearance of FIB Yu AT-III reagent is thrombin, the rosmarinic acid additive in the present invention, it is equally applicable to
The antioxygenic property of both reagent.
The cited paper of the present patent application is as follows:
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[3]Asano,K.,Shinagawa,K.Hashmoto,Characterization of haze-forming proteins of
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[4]Asano,K.Ohtsu,K.,Shinagawa,Affinity of proantocyanidins and their oxidation
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substrates:comparison of human and bovine thrombins with and without fibrinogen clotting
activities.Clin Chem 32:934–937
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on serine proteases.Phytother Res 20:214–217
[7]Mozzicafreddo M,Cuccioloni M,Eleuteri AM,Fioretti E(2006),Flavonoids inhibit the
amidolytic activity of human thrombin.Biochimie 88:1297–1306
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flavonoids as thrombin inhibitors:structure-activity relationships.Thromb Res
126:e365–e378
[9]Li NG,Song SL,Shen MZ,Tang YP,Shi ZH,Tang H,Shi QP,Fu YF,Duan JA(2012)
Mannich bases of scutellarein as thrombininhibitors:design,synthesis,biological activity and
solubility.Bioorg Med Chem 20:6919–6923
[10]Shi ZH,Li NG,Tang YP,Wei L,Lian Y,Yang JP,Hao T,Duan JA(2012)
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analogs of quercetin as thrombin inhibitors.Eur J Med Chem 54:210–222
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Claims (2)
1. the thrombin time test reagent containing rosmarinic acid, it is characterised in that by containing 25U/ml human thrombin, 2% Sanguis Bovis seu Bubali
Pure albumen, 1.5mM rosmarinic acid, the sodium azide of 0.05%, 3.5% methionine, 1.5%PEG, 3% mannitol,
The Tris-HCl buffer composition of 2.5% maltose and 10mM calcium chloride, percentage ratio therein is mass volume ratio, pH
It is 7.4.
The preparation method of the thrombin time test reagent containing rosmarinic acid the most according to claim 1, it is characterised in that step
As follows: preparation Tris concentration 50mM, the buffer of pH7.4, in this buffer, add final concentration and be respectively 2%
BSA, 1.5mM rosmarinic acid, the sodium azide of 0.05%, the methionine of 3.5%, the PEG of 1.5%, the manna of 3%
Alcohol, the maltose of 2.5%, the calcium chloride of 10mM, wherein in addition to calcium chloride, remaining is mass body fraction；By above-mentioned
Mixed liquor filters with 0.22 micron membrane filter, adds human thrombin lyophilized formulations in filtrate, makes the dense of human thrombin in whole filtrate
Degree is 25U/ml, obtains TT and measures reagent.
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| CN107942080A (en) * | 2016-10-13 | 2018-04-20 | 北京众驰伟业科技发展有限公司 | A kind of activated partial thromboplastin time detection reagent and its detection method |
| CN108279313B (en) * | 2017-12-29 | 2021-03-02 | 广州万孚生物技术股份有限公司 | Reagent and test cup for rapidly detecting blood viscoelasticity |
| CN110208552A (en) * | 2019-05-22 | 2019-09-06 | 深圳市国赛生物技术有限公司 | Thrombin time detection reagent and preparation method thereof |
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| CN105424946A (en) | 2016-03-23 |
| CN104991079B (en) | 2016-04-06 |
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