CN101216471A - Mode creature method for medicament toxicity research - Google Patents
Mode creature method for medicament toxicity research Download PDFInfo
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- CN101216471A CN101216471A CNA2008100190405A CN200810019040A CN101216471A CN 101216471 A CN101216471 A CN 101216471A CN A2008100190405 A CNA2008100190405 A CN A2008100190405A CN 200810019040 A CN200810019040 A CN 200810019040A CN 101216471 A CN101216471 A CN 101216471A
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Abstract
A method for studying toxicity of drugs by using model organism zebra fish comprises the following steps of: selecting 100 to 200 adult zebra fishes, respectively disposing in bottles containing a solution of 30 to 50 mL, keeping the temperature in the bottles substantially constant at 22 to 25 DEG C, randomly grouping, each group containing 10 to 20 fishes, wherein the solution in one of the groups is pure water (blank), the solutions in other groups are medicinal solutions with different concentrations, and 0.5% to 2% dimethylsulfoxide (DMSO) can be added to assist dissolving a drug that has poor water solubility, when an additional solvent comparison group prepared by adding 0.5% to 2% DMSO into the pure water is needed; recording the death number of zebra fishes in the medicinal solutions of different concentrations within 24 h, and calculating the death rate (%); calculating median lethal dose-LD50 by Bliss method according to the experiment result; and representing the acute toxicity by the value of LD50. According to the method, the LD50s of zebra fish of triptolide, matrine and emodin are respectively 5.39*10<-3>, 112.3 and 1.08*10<3> mug/mL. The method can objectively reflect the toxicity of drugs.
Description
Technical field:
The present invention relates to the toxicity research field of medicine, relate to the method that the biological zebra fish of application model carries out drug toxicity research.Specifically refer to carry out the toxicity research of compounds such as triptolide, matrine and archen with model organism zebra fish.This method can be used as the new method of carrying out drug toxicity research fast.
Background technology:
Medicine has safety, effectively reaches quality controllability, and the three is indispensable.If the drug safety existing problems, then its toxicity can cause damage to patient, even causes the serious toxicity reaction.It is reported that over nearest 40 years, because serious adverse effects, the U.S.'s 2.9% listing new drug is withdrawn from market by FDA, had since 1993 and cause 1000 many cases deaths after 7 new drugs listings and be forced to remove the city.We know, are three fens poison of medicine, do in the proper way with it, and poison is also rescued the people, otherwise good medicine also victimizes, and so-called " rheum officinale is rescued the people, and genseng kills a person " promptly refers to this.Though the medicine that security is very high such as the Radix Astragali, Chinese yam, white fungus etc. use extensively, generally can only be as adjuvant drug.And all poisonous persons in the Chinese medicine have special effect more.Under this prerequisite, just seem further important of research to the toxicity assessment of toxic herb.For example, rough gentian is let out the liver ball and is caused renal failure, the birthwort carcinogenesis, and the thunder godvine determined curative effect but can cause toxicity such as liver kidney.So the toxicity of research medicine has crucial meaning to guaranteeing clinical drug safety.
Drug toxicity research often comprises (animal) and external (cell, molecule etc.) test in the body.The toxicity in vivo test is normal adopts animals such as mouse, rat, dog to carry out, and main evaluation method has the acute toxicity (1～2 week) and long term toxicity (1～September) research of short-term.Wherein toxicity in vivo test generally can reflect the security of medicine more exactly, but it is long to test the general cycle, and drug dose is big, is unfavorable for the quick toxicity screening of small amount of drug.In vitro toxicity research cell, the molecules etc. of adopting more, method is simple, condition is controlled, is applicable to the quick primary dcreening operation of drug toxicity, but isolated cells, molecule etc. are difficult to accurately simulate in the body process, and illusion often appears in test findings.Therefore, set up a kind of can the simulation preferably, can predict simply fast that again the toxicity research method of drug toxicity is significant in the body process.
Zebra fish is a kind of fabulous model organism, is to replace the good test model fish as research object such as frog, fruit bat, small white mouse.The expense that zebra fish is fed and keeps is more cheap, and the requisite space place is little, is easy to indoor large-scale breeding.Long 3～the 4cm of adult fish, a mating can obtain 100～200 embryos, lays eggs weekly once, and medium scale like this fish jar is annual just can culture millions of of adult fishes, and cost is only for supporting the 0.1%-1% of mouse.Zebra fish is suitable with the similarity and the mouse of human gene, and on protein level, the homology of its key position almost is 100%, so available zebra fish simulating human disease [P.Goldsmith, Curr Opin Pharmacol, 4,504-12 (2004)].At present, what people were successful sets up many human diseases models with zebra fish, as the disease [S.Sumanas and S.Lin, DDT:TARGETS, 3,89-96 (2004) .] of aspects such as nervous system, the circulation system, the sense of hearing, vision, cancer.
Zebra fish becomes a kind of study of pharmacy instrument of hot topic abroad, as setting up disease model with zebra fish, carries out the high flux screening of active constituents of medicine and the research of drug metabolism.Domestic research to zebra fish mainly is limited to the phenogenetics aspect, the application of Shang Weijian aspect pharmacy, particularly toxicologic study.We intend the toxicity with compounds such as zebra fish research triptolides, thereby set up a kind of quick, simple model organism new method of studying drug toxicity.
Summary of the invention:
The objective of the invention is to set up the new method of the enough model organism zebra fish research of a kind of energy drug toxicity.
Another object of the present invention is to model organism method of the present invention is used to study the toxicity research of compounds such as triptolide, matrine, archen.
The present invention is achieved in that
Medication
Different with mammals such as mouse, rat, dogs, the volume of zebra fish is less, and is oral or the drug administration by injection mode is very difficult, and domestic do not have corresponding delivery device as yet.If can be in the water that zebra fish lived with medicine dissolution, then independently continuous from solution, absorbing the drug of zebra fish, change dosage by regulating water Chinese traditional medicine concentration, medicine will change along with the variation of drug concentration the effect of zebra fish, this method simple possible.
The toxicity assessment of medicine
The zebra fish volume is little, and the adult fish is about 3～4cm, and internal organs are littler in its body, with the naked eye is difficult to distinguish, so taking internal organ is observed the feasibility existing problems of pathology.But find in the experiment, increase along with drug concentration, zebra fish mortality ratio within a certain period of time increases, illustrate that toxigenous drug concentration is relevant with the mortality ratio of zebra fish, available lethal quantity is represented the toxicity of medicine, and the life of observing zebra fish and dead physical signs be than more obvious, objective, the easy grasp of other index, and can repeat under the same conditions to test.Can use for reference in the mammal studies on acute toxicity, with median lethal dose (LD
50) weigh the toxicity intensity of medicine.LD
50Be the important symbol of estimating a drug toxicity size, value shows that greatly drug toxicity is little, otherwise, LD
50Value is little to show that then its toxicity is big.
Experimental program
Get 100～200 of zebra fish adult fishes, place the bottle that contains 30～50mL solution respectively, solution temperature is substantially constant at 22～25 ℃ in the maintenance bottle, is divided into array at random, and every group contains 10～20.Wherein 1 group is pure water (blank), and other array is the soup of variable concentrations.If medicine is water-soluble bad, can add 0.5%～2% dimethyl sulfoxide (DMSO) (DMSO) hydrotropy, need to increase by one group of solvent control group this moment, promptly add 0.5%～2%DMSO at pure water.The dead quantity (only) of zebra fish in 24h in the record variable concentrations soup is calculated mortality ratio (%).According to experimental result, adopt the Bliss method to calculate LD
50Value.
Embodiment:
Embodiment one:
Toxicity--LD with model organism zebra fish research triptolide
50The mensuration of value.
Be subjected to the reagent thing: triptolide;
Source: consonance institute of internal medicine of medical university (Jiangsu, Nanjing) of the Chinese Academy of Medical Sciences;
Physicochemical character: white powder; Content: 94%;
Compound method: take by weighing triptolide according to desired concn and be dissolved among the 100%DMSO, again in 0.5% ratio with triptolide DMSO solution dilution in ultrapure water.
Animal: zebra fish is provided by model organism research institute of Nanjing University.
Reagent: dimethyl sulfoxide (DMSO) (DMSO) is available from Shanghai Ling Feng chemical reagent company limited, and ultrapure water is taken from the Millipore ultrapure water machine.
Experimental technique:
Get 120 of zebra fishs, place the wide-necked bottle that fills 50mL solution respectively, be divided into 8 groups at random, every group contains 15, is respectively the pure water control group, pure water+DMSO control group and contain triptolide concentration and be followed successively by 18.00 * 10
-3, 10.80 * 10
-3, 6.48 * 10
-3, 3.89 * 10
-3, 2.33 * 10
-3, 1.40 * 10
-3The administration group of μ g/mL, the death toll (only) of variant liquor strength group zebra fish is calculated mortality ratio (%) in the record 24h.Adopt the Bliss method to calculate LD
50Value.
Experimental result:
| Sample | Dosage (μ g/mL) | Dead fish number of elements (bar) | Mortality ratio (%) | LD 50(μg/mL) |
| The pure water group | - | 0 | 0 | - |
| Pure water+0.5%DMSO group | - | 0 | 0 | - |
| The triptolide group | 18.00×10 -3 10.80×10 -3 6.48×10 -3 3.89×10 -3 2.33×10 -3 1.40×10 -3 | 15 14 10 5 1 0 | 100.0 93.3 66.7 33.3 6.7 0 | 5.39×10 -3μg/mL |
Experimental result shows, (pure water and pure water+0.5%DMSO) the no zebra fish death of blank group; But the mortality ratio of triptolide group zebra fish is relevant with drug concentration, adopts the Bliss method to calculate its LD
50Value is 5.39 * 10
-3μ g/mL can objectively react the toxicity of this medicine.
Embodiment two:
Toxicity--LD with model organism zebra fish research matrine
50The mensuration of value.
Be subjected to the reagent thing: matrine;
Source: Jiangsu Tiansheng Pharmaceutical Co., Ltd.;
Physicochemical character: pale yellow powder; Content: 98%;
Compound method: take by weighing matrine according to desired concn and be dissolved among the 100%DMSO, again in 0.5% ratio with matrine DMSO solution dilution in ultrapure water.
Animal: zebra fish is provided by model organism research institute of Nanjing University.
Reagent: dimethyl sulfoxide (DMSO) (DMSO) is available from Shanghai Ling Feng chemical reagent company limited, and ultrapure water is taken from the Millipore ultrapure water machine.
Experimental technique:
Get 105 of zebra fishs, place the wide-necked bottle that fills 50mL solution respectively, be divided into 7 groups at random, every group contains 15, be respectively the pure water control group, pure water+DMSO control group and contain matrine and be followed successively by 150.0,127.5,108.4,92.1,78.3 the administration group of μ g/mL, the death toll (only) of variant liquor strength group zebra fish is calculated mortality ratio (%) in the record 24h.Adopt the Bliss method to calculate LD
50Value.
Experimental result:
| Sample | Dosage (μ g/mL) | Dead fish number of elements (bar) | Mortality ratio (%) | LD 50(μg/mL) |
| The pure water group | - | 0 | 0 | - |
| Pure water+0.5%DMSO group | - | 0 | 0 | - |
| The matrine group | 150.0 127.5 108.4 92.1 78.3 | 15 12 6 1 0 | 100.0 80.0 40.0 6.7 0 | 112.3 |
Experimental result shows, (pure water and pure water+0.5%DMSO) the no zebra fish death of blank group; But the mortality ratio of matrine group zebra fish is relevant with drug concentration, adopts the Bliss method to calculate its LD
50Value is 112.3 μ g/mL, can objectively react the toxicity of this medicine.
Embodiment three:
Toxicity--LD with model organism zebra fish research archen
50The mensuration of value.
Be subjected to the reagent thing: archen;
Source: auspicious thunder plant preparation factory (middle inspection institute the standard items unit of providing);
Physicochemical character: yellow powder; Content: 98%;
Compound method: take by weighing archen according to desired concn and be dissolved among the 100%DMSO, again in 0.5% ratio with archen DMSO solution dilution in ultrapure water.
Animal: zebra fish is provided by model organism research institute of Nanjing University.
Reagent: dimethyl sulfoxide (DMSO) (DMSO) is available from Shanghai Ling Feng chemical reagent company limited, and ultrapure water is taken from the Millipore ultrapure water machine.
Experimental technique:
Get 105 of zebra fishs, place the wide-necked bottle that fills 50mL solution respectively, be divided into 7 groups at random, every group contains 15, is respectively the pure water control group, and pure water+DMSO control group and archen concentration are followed successively by 2.0 * 10
3, 1.4 * 10
3, 0.98 * 10
3, 0.67 * 10
3, 0.48 * 10
3The administration group of μ g/mL, the death toll (only) of variant liquor strength group zebra fish is calculated mortality ratio (%) in the record 24h.Adopt the Bliss method to calculate LD
50Value.
Experimental result:
| Sample | Dosage (μ g/mL) | Dead fish number of elements (bar) | Mortality ratio (%) | LD 50(μg/mL) |
| The pure water group | - | 0 | 0 | - |
| Pure water+0.5%DMSO group | - | 0 | 0 | - |
| The archen group | 2.00×10 3 1.40×10 3 0.98×10 3 0.67×10 3 0.48×10 3 | 15 9 4 2 0 | 100.0 60.0 26.7 13.3 0 | 1.08×10 3 |
Experimental result shows, (pure water and pure water+0.5%DMSO) the no zebra fish death of blank group; But the mortality ratio of archen group zebra fish is relevant with drug concentration, adopts the Bliss method to calculate its LD
50Value is 1.08 * 10
3μ g/mL can objectively react the toxicity of this medicine.
Embodiment result shows, measures the LD of medicine with zebra fish
50The method of value is simple, quick, objective, is a kind of new method of quick research drug toxicity.
Claims (2)
1. the biological zebra fish of the application model method of carrying out drug toxicity research: get 100～200 of zebra fish adult fishes, place the bottle that contains 30～50mL solution respectively, be divided into array at random, every group contains 10～20, wherein 1 group is the blank group of pure water, other array is the soup of variable concentrations, if medicine is water-soluble bad, can add 0.5%～2% dimethyl sulfoxide (DMSO) hydrotropy, need to increase by one group of solvent control group this moment, promptly in pure water, add 0.5%～2% dimethyl sulfoxide (DMSO), the dead number of zebra fish in 24h in the record variable concentrations soup calculated mortality ratio, represents with number percent, according to experimental result, adopt the Bliss method to calculate median lethal dose LD
50Value is used LD
50The acute toxicity of value reflection medicine.
2. the process of claim 1 wherein that medicine can be compounds such as triptolide, matrine or archen.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102023207A (en) * | 2010-11-17 | 2011-04-20 | 彭恩泽 | Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof |
| CN102288735A (en) * | 2011-06-27 | 2011-12-21 | 天津新丰制药有限公司 | Method for detecting toxicity of cefazedone sodium impurities and intermediates |
| CN102539640A (en) * | 2010-12-17 | 2012-07-04 | 中国科学院生态环境研究中心 | Method used for testing high throughput acute toxicity of early life stage of fish |
| CN103301479A (en) * | 2013-05-07 | 2013-09-18 | 南京工业大学 | Method for assessing acute toxicity of medicament by using model animal zebrafish fries |
| CN104374883A (en) * | 2014-11-25 | 2015-02-25 | 安徽省环境科学研究院 | Application of zebra fish in detection of reproduction toxicity of BBP (Butyl Benzyl Phthalate) and method for rapidly detecting reproduction toxicity of BBP |
| CN105891436A (en) * | 2016-06-30 | 2016-08-24 | 福建医科大学 | Biological testing method for comprehensively evaluating water quality of drinking water |
| CN107976495A (en) * | 2017-11-18 | 2018-05-01 | 江苏省中医药研究院 | One kind is using the micro- sample anti-osteoporosis activity of zebra fish evaluation amount and security new method |
| CN108106891A (en) * | 2017-11-15 | 2018-06-01 | 河海大学 | A kind of preparation method of colloid original solution for acting on pollutant bio-toxicity and its application |
| CN108593397A (en) * | 2018-07-02 | 2018-09-28 | 武汉轻工大学 | A kind of detection method of 2,4-DNT toxicity |
| CN113413471A (en) * | 2021-06-11 | 2021-09-21 | 浙江警察学院 | Method for evaluating toxicity of scopoletin medicaments based on zebra fish model |
-
2008
- 2008-01-10 CN CNA2008100190405A patent/CN101216471A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102023207A (en) * | 2010-11-17 | 2011-04-20 | 彭恩泽 | Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof |
| CN102023207B (en) * | 2010-11-17 | 2013-06-19 | 杭州环特生物科技有限公司 | Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof |
| CN102539640A (en) * | 2010-12-17 | 2012-07-04 | 中国科学院生态环境研究中心 | Method used for testing high throughput acute toxicity of early life stage of fish |
| CN102288735A (en) * | 2011-06-27 | 2011-12-21 | 天津新丰制药有限公司 | Method for detecting toxicity of cefazedone sodium impurities and intermediates |
| CN103301479A (en) * | 2013-05-07 | 2013-09-18 | 南京工业大学 | Method for assessing acute toxicity of medicament by using model animal zebrafish fries |
| CN103301479B (en) * | 2013-05-07 | 2014-12-31 | 南京工业大学 | Method for assessing acute toxicity of medicament by using model animal zebrafish fries |
| CN104374883A (en) * | 2014-11-25 | 2015-02-25 | 安徽省环境科学研究院 | Application of zebra fish in detection of reproduction toxicity of BBP (Butyl Benzyl Phthalate) and method for rapidly detecting reproduction toxicity of BBP |
| CN105891436A (en) * | 2016-06-30 | 2016-08-24 | 福建医科大学 | Biological testing method for comprehensively evaluating water quality of drinking water |
| CN108106891A (en) * | 2017-11-15 | 2018-06-01 | 河海大学 | A kind of preparation method of colloid original solution for acting on pollutant bio-toxicity and its application |
| CN107976495A (en) * | 2017-11-18 | 2018-05-01 | 江苏省中医药研究院 | One kind is using the micro- sample anti-osteoporosis activity of zebra fish evaluation amount and security new method |
| CN108593397A (en) * | 2018-07-02 | 2018-09-28 | 武汉轻工大学 | A kind of detection method of 2,4-DNT toxicity |
| CN113413471A (en) * | 2021-06-11 | 2021-09-21 | 浙江警察学院 | Method for evaluating toxicity of scopoletin medicaments based on zebra fish model |
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