CN101963589B - Method for detecting activity of antivirus medicament and application thereof - Google Patents
Method for detecting activity of antivirus medicament and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting activity of an antivirus medicament, and belongs to the technical field of the method for detecting property of the medicament. The method comprises the following steps of: (1) selecting microorganisms or cells corresponding to treatment indication of a to-be-detected medicament; (2) adding the to-be-detected medicament into the microorganisms or cells selected in the step (1) for culturing; and (3) placing the microorganisms or cells in which the to-be-detected medicament is added into a trace calorimeter to obtain a thermo-gram curve for growth and metabolism of the microorganisms or cells, and measuring the activity of the medicament according to the slope of the thermo-gram curve and the maximum heating power. The invention also comprises application of the detection method to measuring the heat clearing and detoxication activity of indigowoad root. The method of the invention can represent characteristic information during intervention of the microorganisms by the medicament, can acquire real-time online dynamic data and has the characteristics of real-time, online, high speed and accuracy.
Description
Technical field
The invention belongs to medicine property of medicine detection method technical field, be specifically related to a kind of foundation and application of antiviral drugs activity test method.
Background technology
Along with the development of field of medicaments, for safe medication is avoided drug poisoning, people more and more pay attention to for the active component and the application of these active components in the treatment disease of medicine.What present detection method to drug effective region of treating certain disease and drug dose adopted usually is that viral micro-organisms or virocyte are cultivated by elder generation; Make it produce certain macroscopical presentation at visible carrier surfaces such as nutrient culture media; And then add medicine to be measured in the living environment of viral micro-organisms or virocyte through the materialization means; Thereby certain interception is played in breeding or growth to virus; Because macroscopical presentation that the carrier surface that adds affiliation influence adding medicine of medicine to be measured produces; Produce different presentations, different drug effects and the influence of drug dose that the researchist comes the detection of drugs different parts to be had through this species diversity to treating.
The technology that detects virus at present mainly contains: (1), electron microscope observation, (2), cytopathy political reform, (3), decoration method; (4), enzyme immunoassay (EIA), the experiment of the blot hybridization of (5), virus protein, (6), round pcr; But above-mentioned technology each has defective separately, for example:
(1), electron microscope observation: though intuitively virus existence of Electronic Speculum is the important tool of diagnosis virosis.But machine costs an arm and a leg, and technical requirement is high, not too is applicable to the detection of conventional virology diagnostic test or a large amount of samples; The virus quantity positive findings that just is easy to get when big is of limited application, and can not differentiates the serum type of virus;
(2), the cytopathy political reform: through detect cytopathic effect (cytopathic effect, CPE), viral plaque forms or the output of virus is suppressed degree, reflects the BA of IFN indirectly.But be that every kind of virus can both produce cytopathy in cellular incubation, and CPE judgement subjectivity is strong, poor accuracy is inappropriate for accurate calculating virus infectivity and judges the medicine antiviral activity.
Therefore be badly in need of a kind of new active method of detection of drugs and reach purpose simple, easy-to-use, accurate detection.
Mumps is called for short the stream cheek, is respiratory infectious disease common among children and the teenager, is caused by mumps virus.Clinical symptoms swells and ache for heating and parotid gland apyetous, and can invade organs such as various glandular tissues or nervous system and liver, kidney, heart, joint.Stream cheek virus is the ribonucleic acid virus of sub-thread, belongs to the paramyxoviridae class, and qiagen rnase, protein, carbohydrates and lipoid are prone to affine to body of gland and nerve fiber.Because stream cheek virus to children and teen-age harmfulness, is therefore studied the active component of various medicine convection current cheeks viruses, has positive meaning to reducing the medicine that children and teenager take.
Summary of the invention
The growth thermodynamic argument is thought, when any physical/chemical reaction takes place, all follow the transfer and the thermal distortion of energy, and these energy transfers and all available thermodynamic (al) theory of thermal distortion and method is portrayed and analyzed.Biological heat dynamics is studied in the life entity metabolic processes energy exactly and is shifted and thermal distortion, and its basic intension characterizes the processes such as energetic supersession of life entity (body, microorganism, the cell) form with the thermokinetics function exactly.
The objective of the invention is to be the basis with the growth thermodynamic argument, disclose a kind of pharmaceutically active detection method that is changed to principle with thermodynamics in order to solve the deficiency in the above-mentioned method for detecting virus.
The technical scheme of pharmaceutically active detection method of the present invention is specific as follows:
A kind of antiviral drugs activity test method comprises the steps:
(1), chooses microorganism or the cell corresponding with drug therapy indication to be checked;
(2), add medicine to be checked in the microorganism in step (1), chosen or the cell, cultivate;
(3), the microorganism or the cell that will add medicine to be checked place micro-calorimeter, obtains the thermography curve of microorganism or cell growth metabolism, according to thermography slope of a curve and maximum heating power parameter evaluation pharmaceutically active size.
Antiviral drugs activity test method described in the technique scheme, wherein, the to be checked active constituents of medicine of the medicine to be checked described in the step (2) for extracting.
Antiviral drugs activity test method described in the technique scheme wherein, also comprises the toxicity test step of medicine to be checked to microorganism or cell in described step (1) and step (2) before.
Antiviral drugs activity test method described in the technique scheme; Wherein, Described medicine to be checked is to point to the medicine to be checked that adds variable concentrations in microorganism or the cell of same cell concentration to the toxicity test step of microorganism or cell; With normal microorganism or cell controlled observation cytopathy situation, press the Reed-Muench method and calculate poisonous concentration of half and maximal non-toxic concentration then.
Owing to flow cheek virus to children and teen-age harm, another object of the present invention has been to disclose above-mentioned detection method is treated the viral active component of the stream cheek in the blue radical cure of check-out console application in addition.
The technical scheme that above-mentioned detection method is treated the application of flowing cheek virus active component in the blue radical cure of check-out console is specific as follows:
The application of detection method described in the technique scheme in measuring the clearing heat and detoxicating activity of Radix Isatidis comprises the steps:
(1), selects the corresponding stream cheek virus of the clearing heat and detoxicating active indication of Radix Isatidis;
(2), get 37 ℃, 5%CO
2Hela cell behind the cultivation 24h adds 100TCID
50The stream cheek virus liquid Hela cell is carried out transfection;
(3), under 37 ℃ of constant temperature, aseptic condition, adopt the ampoule method, each ampoule bottle has accurately added the transfection that obtains in the 5ml step (2) the Hela cell of stream cheek virus, cell concentration is 1 * 10
6Cells/ml
-1, add medicine Radix Isatidis to be checked, sealing; Put into micro-calorimeter, obtain flowing cheek viral growth thermography curve, obtain the pharmaceutically active size according to thermography slope of a curve and maximum heating power parameter.
The application of detection method described in the technique scheme in measuring the clearing heat and detoxicating activity of Radix Isatidis wherein, also comprises the toxicity test of Radix Isatidis convection current cheek virus to be checked between step (1) and step (2).
The present invention is on the basis of growth thermodynamic argument; Through in life system, adding target substance; Life entity energetic supersession activity must change, and can obtain the life entity thermokinetic parameters relevant with medicine through investigating testing drug to the influence of life entity metabolism, can be fast, quantitative, sensitivity and objectively react the activity difference-microcalorimetric method of medicine; As the research method of biological heat kinetic theory, have real-time, online, characteristics fast and accurately.
Microcalorimetric method mainly is through obtaining dynamic biological heat kinetic parameter and thermography curve (P-t) characteristic information is expressed micro-thermal distortion (neither endothermic nor exothermic) rules of biosome in metabolic process such as microorganism, cell, tissue and organ.
In the growth period of virus, adopt the exponential growth model to carry out mathematics manipulation.This model is thought to be had in that exponential growth is interim:
P
t=P
0Exp (kt) (1) or ln (P
t/ P
0)=kt (1) (2)
(2) in the formula, P
0Be the heat production power of baseline starting point, P
tHeat production power during for time t.Warp is to P
tThe straight-line regression processing is carried out with t in the value of taking the logarithm back, can obtain growth rate constant k value.The k value is one of characteristic constant that characterizes the growth of microorganism metabolic process, and when adding different pharmaceutical or changing drug concentrations, the change of k value can be used as estimates the big or small parameter of pharmaceutically active effect in that condition is identical.And hot active collection of illustrative plates area under a curve is gross calorific power Q.While generation time tG=(ln2)/k.
Viral growth inhibiting rate I=(k
0-k
c)/k
cIn * 100% formula, k
0Be the growth rate constant of blank papova, kc is the growth rate constant of reagent group.
This shows that microcalorimetric method both can receive the characteristic information in the pharmaceutical intervention process by apparent microorganism, can obtain the dynamic data of real-time online again.This method is as a kind of method of easy, quantifiable mensuration thermal distortion and be widely used in fields such as physics, chemistry and life science.
Thus, the present invention has following beneficial effect:
1, the detection method of pharmaceutically active of the present invention has real-time, online, characteristics fast and accurately.
2, the present invention has abandoned traditional detection method to pharmaceutically active, comes the active size of detection of drugs through microorganism or cell in the micro-thermal distortion that receives the life metabolism under the drug effect, has started a kind of brand-new detecting pattern.
3, pharmaceutically active detection method of the present invention both can receive the characteristic information in the pharmaceutical intervention process by apparent microorganism, can obtain the dynamic data of real-time online again.
Figure of description:
1, Fig. 1 acts on the Thermogram of mumps virus for the different extract parts of Radix Isatidis.
Embodiment:
For making technical scheme of the present invention be convenient to understand, the present invention is further described below in conjunction with embodiment.
Instrument and material in following examples are following:
1, instrument: 3115 type TAM Air isothermal micro-calorimeter (Sweden ThermometricAB companies; Have another name called the biological activity assay appearance); Can independently simultaneously measure 8 its Heat stability is goods of sample; The constant temperature working range is 5～60 ℃, and the thermal power minimum detection is limited to 2 μ w, and system's control temperature is 37 ℃ during experiment; Adopt above-mentioned micro-calorimeter in the present embodiment, but be not limited to this micro-calorimeter, any instrument that can play same function all can be used for detection method of the present invention;
The board-like ELIASA of ZS-3 (new company of giving birth); Carbon dioxide incubator (U.S. NAPCO company); The SENCOR502B type changes evaporimeter (Shensheng Science & Tech. Co., Ltd., Shanghai).
2, material:
Cell and virus: Hela clone and mumps virus (Mumpsvirus) provide by infectious disease research institute of PLA Viral Laboratory.
The LB fluid nutrient medium: NaCl 5g, yeast extract 5g, peptone 10g is dissolved in the 1L deionized water, packing behind the accent pH 7.0-7.2.121 ℃ of autoclaving 30min preserve subsequent use in the rearmounted 4 ℃ of refrigerators of cooling.
Reagent: the special-purpose dimethyl sulfoxide (DMSO) (DMSO) of cellular incubation, purity＞99.99%; Tetrazolium bromide (MTT); DMEM (H) nutrient culture media (being the Sigma Company products); Hyclone (Hyclone Company products; 96 well culture plates and 0.22 μ m filter (U.S. Corning company).
Embodiment 1:
1, implementation method:
(1), selects the corresponding stream cheek virus of the clearing heat and detoxicating active indication of Radix Isatidis;
(2), virus transfection Hela cell: get 37 ℃, 5%CO
2Hela cell behind the cultivation 24h adds 100TCID
50The stream cheek virus liquid Hela cell is carried out transfection, abandon viral liquid then;
(3), the preparation at medicinal material and chemical position: Radix Isatidis.Get Radix Isatidis meal 500g, decocting boils 2 times, merges decoction liquor and is condensed into medicinal extract; Decocting liquid total extract (the I position, 156.6g), and with the opposed polarity solvent extraction; Ligroin extraction (the II position, 0.19g), ethyl acetate extract (the III position, 0.49g), chloroform extract (IV position; 0.55g), n-butanol extract (the V position, 5.12g) and extraction back material (the opposed polarity position 116.25g) is waited in the VI position.During the cell in vitro test, the II-V extract part afterwards is formulated as the solution of variable concentrations respectively with DMEM (H) with dimethyl sulfoxide (DMSO) (DMSO) dissolving, and I, VI position are with the dissolving of DMEM (H) solution, and 0.22 μ m filter pushes away the filtering bacterium.
(4), biothermodynamics detects:
Under 37 ℃ of constant temperature, aseptic condition, adopt the ampoule method, after the Hela cell index went down to posterity with 0.2% trypsinization growth period, adding the DMEM nutrient culture media, to make final concentration of cells be 1 * 10
6Individual/ml.Each ampoule bottle has accurately added the middle transfection of 5ml step (2) and has flowed the viral Hela cell culture medium of the cheek, and cell concentration is 1 * 10 in the nutrient culture media
6Cells/ml-1 adds the soup described in the table 1, adds a cover bottle stopper, sealing.Put into this viral growth thermography curve (P-t curve) of micro-calorimeter track record figure, it is as shown in Figure 1 to obtain the thermography curve.When curve returned baseline again, experiment finished.
The thermodynamic parameter of table 1 mumps virus under the effect of Radix Isatidis different chemical position
The thermomechanical curve parameter of the mumps virus that the ampoule method is measured under the different extract part effects of Radix Isatidis under identical test condition, has good reappearance.The thermal power curve of output of mumps virus control group is compared with thermal power curve under the different extract part effects of Radix Isatidis; Can find out; Mumps virus is under I～V fraction medicine effect, and the generation time prolongs, and prolong the deadtime on the growth heating curve; Move behind the growth peak, the maximum heating power in growth period reduces.Explain that the growth of popularity mumps virus is inhibited.The VI position can make the exponential growth curve slope of mumps virus increase, and maximum heating power also increases than normal control group, explains that residue position, extraction back has the effect of the viral growth of promotion.
2, control test:
In order to verify that the present invention can detect pharmaceutically active, be provided with the control test method in this embodiment, step is following:
The transfection that in above-mentioned steps (2), obtains add respectively in the Hela cell of stream cheek virus with table 1 in medicine, every hole 100 μ L establish virus control group and normal cell control group simultaneously; Observe CPE every day; Behind the 72h, detect viral inhibiting rate with mtt assay, the result is as shown in table 2:
The inhibiting effect of table 2 Radix Isatidis different chemical position popularity mumps virus
Test findings shows that in the maximal non-toxic dosage range, the I-V position all can suppress the mumps virus biosynthesizing.Along with the increase of each fraction medicine concentration, cellular swelling, become and typical CEP characteristic such as justify, come off, be cracked and weaken gradually, viral inhibiting rate (cell survival rate) is rising obviously.It is different to suppress the synthetic inhibiting rate of the scorching viral organism of the popular cheek according to each position, can compare the not antiviral effect of extract part of Radix Isatidis.
The result that the invention of the present invention of above-mentioned usefulness detects conforms to the result of conventional pharmacodynamics method (control test); Coincideing of the trend phase of two kinds of method popularity mumps virus exercising results explained and adopted method of the present invention can the activity of medicine to be detected fully.
Embodiment 2:
Present embodiment is identical with embodiment 1 step, and difference is also to have carried out the different extract parts of Radix Isatidis before at 0～8mg/ml in the step (1) of embodiment 1
-18 concentration are carried out cytotoxicity analysis to the Hela cell in the scope, and concrete steps are:
Cell suspension is diluted to 1 * 10
6Individual/ml, be inoculated in 96 orifice plates, every hole 100 μ L put 37 ℃, 5%CO
2After cultivating 24h in the incubator, add I～VI position test sample liquid of pressing gradient dilution respectively, drug concentration is 8,4,2,1,0.5,0.25,0.125,0.063mg/ml
-1, the same terms continues to cultivate the every concentration of 48h. down and repeats 4 holes, and other establishes the normal cell contrast, and it is 4 that observation of cell pathology (CPE) situation, cytopathy are destroyed fully; 75% is 3; 50% is 2; Anosisly become 0.Calculate every batch of every concentration liquid average cell lesion degree of experiment and suppress percent.Press the Reed-Muench method and calculate the poisonous concentration (TC of half
50) and maximal non-toxic concentration (TC
0), its result sees shown in the table 3.
The toxicity of the different extract parts of table 3 Radix Isatidis pair cell in the Hela cellular incubation
The result shows that decocting liquid is 4mg/ml with the maximal non-toxic concentration of extraction rear
-1Ethyl acetate, normal butyl alcohol, chloroform, sherwood oil are 2mg/ml to the maximal non-toxic concentration of Hela cell
-1In above maximal non-toxic concentration range, the different extract parts of Radix Isatidis to transfection the Hela cytosis of mumps virus got rid of the false positive results that CDCC causes.
The above; Being merely preferred embodiment of the present invention, is not that the present invention is done any formal and substantial restriction, allly is familiar with the professional and technical personnel; In not breaking away from technical scheme scope of the present invention; The technology contents that is disclosed more than capable of using, and a little change of making, modify the equivalent variations with differentiation, be equivalent embodiment of the present invention; Simultaneously, all foundations essence technology of the present invention all still belongs in the scope of technical scheme of the present invention change, modification and the differentiation of any equivalent variations that above embodiment did.
Claims (2)
1. the application of antiviral drugs activity test method in measuring the clearing heat and detoxicating activity of Radix Isatidis comprises the steps:
(1), selects the corresponding stream cheek virus of the clearing heat and detoxicating active indication of Radix Isatidis;
(2), get 37 ℃, 5%CO
2Hela cell behind the cultivation 24h adds 100TCID
50The stream cheek virus liquid Hela cell is carried out transfection;
(3), under 37 ℃ of constant temperature, aseptic condition, adopt the ampoule method, each ampoule bottle has accurately added the transfection that obtains in the 5ml step (2) the Hela cell of stream cheek virus, cell concentration is 1 * 10
6Cells/ml
-1, add medicine Radix Isatidis to be checked, sealing; Put into micro-calorimeter, obtain flowing cheek viral growth thermography curve, obtain the pharmaceutically active size according to thermography slope of a curve and maximum heating power parameter.
2. the application of detection method according to claim 1 in measuring the clearing heat and detoxicating activity of Radix Isatidis is characterized in that: the toxicity test that between step (1) and step (2), also comprises Radix Isatidis convection current cheek virus to be checked.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010257186 CN101963589B (en) | 2010-08-19 | 2010-08-19 | Method for detecting activity of antivirus medicament and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010257186 CN101963589B (en) | 2010-08-19 | 2010-08-19 | Method for detecting activity of antivirus medicament and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN101963589A CN101963589A (en) | 2011-02-02 |
| CN101963589B true CN101963589B (en) | 2012-07-25 |
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| CN102520007B (en) * | 2011-11-16 | 2014-09-03 | 中国人民解放军第三〇二医院 | Method for rapidly detecting product quality of spontaneous heating preparation |
| CN103255196B (en) * | 2013-05-31 | 2014-11-26 | 中国人民解放军第三0二医院 | Method for detecting effect of Coptis chinensis alkaloid monomer on tumor cells |
| CN103499600B (en) * | 2013-09-13 | 2015-11-18 | 中国人民解放军第三0二医院 | A kind of method differentiating bacterial infection kind in ascites due to cirrhosis |
| CN104805171B (en) * | 2015-04-10 | 2018-08-28 | 中国农业科学院植物保护研究所 | A kind of activity rating new method of active material |
| CN106353469B (en) * | 2015-07-14 | 2018-12-14 | 中国人民解放军第三〇二医院 | A kind of integrated evaluating method of Quality Evaluation of Chinese Medicinal |
| CN106404831B (en) * | 2016-12-20 | 2020-01-14 | 西北大学 | Rapid detection method for sensitivity of beta-lactam antibiotics |
| CN111521631B (en) * | 2019-02-02 | 2023-07-04 | 中国人民解放军总医院第五医学中心 | Method for evaluating quality consistency of donkey-hide gelatin |
| CN111172326B (en) * | 2020-02-25 | 2020-11-10 | 芜湖天明生物技术有限公司 | Method for in vitro detection of rhTSG-6 antiviral activity and application of rhTSG-6 in antiviral drugs |
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| US6379917B1 (en) * | 1996-08-02 | 2002-04-30 | Axiom Biotechnologies | Apparatus and method for real-time measurement of cellular response |
| CN101717811A (en) * | 2009-11-09 | 2010-06-02 | 中国人民解放军第三〇二医院 | Method for evaluating biological security of solubilizing auxiliary materials |
| CN101768622A (en) * | 2009-12-30 | 2010-07-07 | 中国人民解放军第三〇二医院 | Method for evaluating efficacy of antler |
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| JP2007312738A (en) * | 2006-05-29 | 2007-12-06 | Kikkoman Corp | Method for predicting growth of microorganism in food and drink |
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| US6379917B1 (en) * | 1996-08-02 | 2002-04-30 | Axiom Biotechnologies | Apparatus and method for real-time measurement of cellular response |
| CN101717811A (en) * | 2009-11-09 | 2010-06-02 | 中国人民解放军第三〇二医院 | Method for evaluating biological security of solubilizing auxiliary materials |
| CN101768622A (en) * | 2009-12-30 | 2010-07-07 | 中国人民解放军第三〇二医院 | Method for evaluating efficacy of antler |
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