CN107228925A - A kind of method of the potential teratogenesis of utilization zebrafish embryo Fast Evaluation Chinese patent drug - Google Patents
A kind of method of the potential teratogenesis of utilization zebrafish embryo Fast Evaluation Chinese patent drug Download PDFInfo
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- CN107228925A CN107228925A CN201710416962.9A CN201710416962A CN107228925A CN 107228925 A CN107228925 A CN 107228925A CN 201710416962 A CN201710416962 A CN 201710416962A CN 107228925 A CN107228925 A CN 107228925A
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- chinese patent
- patent drug
- teratogenesis
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- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 44
- 239000003814 drug Substances 0.000 title claims abstract description 42
- 229940079593 drug Drugs 0.000 title claims abstract description 35
- 208000031320 Teratogenesis Diseases 0.000 title claims abstract description 31
- 241000252212 Danio rerio Species 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000011156 evaluation Methods 0.000 title claims abstract description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- 230000004720 fertilization Effects 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 231100000378 teratogenic Toxicity 0.000 claims description 9
- 230000003390 teratogenic effect Effects 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 210000002257 embryonic structure Anatomy 0.000 claims description 6
- 239000002775 capsule Substances 0.000 claims description 4
- 238000005286 illumination Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 230000008011 embryonic death Effects 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 239000003501 hydroponics Substances 0.000 claims description 2
- 238000003672 processing method Methods 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 2
- 238000007654 immersion Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 231100000419 toxicity Toxicity 0.000 abstract description 8
- 230000001988 toxicity Effects 0.000 abstract description 8
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 230000000638 stimulation Effects 0.000 abstract 1
- 238000010998 test method Methods 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 230000002159 abnormal effect Effects 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 4
- 230000034994 death Effects 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 206010013710 Drug interaction Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000009112 niuhuang Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention relates to a kind of evaluation test method of the quick potential teratogenesis of Chinese patent drug, it is characterized in that：Zebrafish embryo is used for toxic model, by embryo in the aqueous solution of Chinese patent drug, whether the development of observation zebrafish embryo produces development toxicity under the stimulation of Chinese patent drug, and the toxicity index of zebrafish embryo is judged whether with potential teratogenesis according to Chinese patent drug.This method has successfully often carried out toxicity assessment to multiple gynecological with Chinese patent drug, is effectively tested out the potential teratogenesis of Chinese patent drug, and significant instruct is provided so as to be used for Chinese patent drug clinical safety.
Description
Technical field
It is more particularly to a kind of to evaluate the potential teratogenesis of Chinese patent drug using zebrafish embryo the invention belongs to toxicity assessment field
The method of property.
Background technology
Chinese patent drug is produced according to the prescription of traditional Chinese medicine using modern technologies, may produce in process of production with
The different drug interaction of traditional concocting method, it is possible to create new toxicity.The method that tradition evaluates teratogenesis is to utilize the food in one's mouth
The embryo of the biological rat of newborn quasi-mode and mouse is evaluated, but because the growth cycle of mammal embryo and is difficult sight at length
Examine, it is difficult to carry out quick and extensive evaluation.Internal mainly water soluble ingredient can be absorbed into usual Chinese medicine, water-soluble
It is to be applicable the toxicity assessment new model with Chinese medicine that zebrafish embryo is developed in liquid.Past evaluates Chinese medicine development toxicity using zebra fish
In method, the criterion of potential teratogenesis is not established yet, does not there is the method evaluated Chinese patent drug especially, and practicality is low.
The content of the invention
In view of the above-mentioned problems, it is an object of the invention to provide one kind is simple to operate, it is adaptable in quick and extensive evaluation
The method and technology of the potential teratogenesis of patent medicine.The purpose of the present invention is extracted by Chinese patent drug water-soluble substances, Chinese patent drug is to zebra fish
Embryo Culture and teratogenesis are evaluated three steps and realized, concrete scheme is as follows：
(1) processing method of Chinese patent drug：The coating of Chinese patent drug/capsule is removed, fine powder is ground into.Chinese patent drug precise powder claims
Amount, is extracted 30 minutes in instrument is cleaned by ultrasonic with DMSO, is placed 24 hours in room temperature.Extract adds E3 nutrient solutions and is diluted to institute
Concentration (rate of embryonic death is 100% concentration) is needed, supernatant is taken after centrifugation, is diluted to multiple concentration with E3 nutrient solutions, is used for
Cultivate zebrafish embryo.
(2) culture of zebrafish embryo：The after fertilization zebrafish embryo of 3 hours is taken, hole is sub-packed according to every one piece of ground in hole
In plate, the solution of Chinese patent drug, some embryos of hydroponics of each concentration are added in hole.Orifice plate is placed in 28.5 degrees Celsius, light
According to being cultivated in the incubator of/dark cycle 12/12 (hour), a solution is changed within every 24 hours, culture to after fertilization 72 hours.
(3) teratogenesis evaluation：The developmental state of embryo is observed under the microscope, records under each concentration dead embryo and abnormal
The number of shape embryo, according to probit models to LC50 (LC50) and half teratogenesis concentration (EC50) solved,
By calculating LC50And EC50Ratio be worth to teratogenic index, according to the size of index judge medicine whether have potential teratogenesis.
Brief description of the drawings
Fig. 1 is the flow chart of toxicity assessment technology.
In figure：1st, Chinese patent drug, 2, Chinese patent drug powder, 3, Chinese patent drug solution, 4, zebrafish embryo, 5,96 orifice plates, 6, culture,
7th, microexamination, 8, micro- lower zebra fish developmental condition, 9, statistics and analysis
Embodiment
In order to describe the features of the present invention in detail, following instance explanation is now lifted：
Embodiment 1：
1st, take fule granules to grind to form fine powder, accurately weighed 250 milligrams, 250 microlitres of DMSO are added, in instrument is cleaned by ultrasonic
After extracting 30 minutes, 24 hours are stood at room temperature, 50 milliliters is diluted to E3 cultures, is centrifuged under 4000rpm, take supernatant,
Supernatant is 5 mg/mls.Diluted with E3 nutrient solutions, obtain a series of concentration for 0.08,0.16,0.31,0.63,
1.25th, the drug solution of 2.5 and 5 mg/mls.
2nd, the after fertilization zebrafish embryo of 3 hours is placed in 96 orifice plates, 1 piece of embryo is placed per hole, each hole adds 200
Microlitre decoction, each 30 pieces of embryos of concentration liquid culture.Orifice plate is placed in 28.5 degrees Celsius and illumination/dark cycle 12/12 is (small
When) in the environment of cultivate, update within every 24 hours a decoction, culture to after fertilization 72 hours.
3rd, the form of the zebrafish embryo of each concentration is observed under the microscope and records dead embryo and lopsided embryo is abnormal
The number of shape, calculates the death rate and abnormal rate, and is calculated with software and to obtain LC50 and half teratogenesis concentration is respectively
0.74 and 0.484 mg/ml, teratogenic index is 1.54, shows weak potential teratogenesis.
Embodiment 2：
1st, take FUYANJING JIAONANG some, remove capsule and get it filled core powder, accurately weighed 400 milligrams, add 250 microlitres
DMSO, after being extracted 30 minutes in ultrasonic cleaning instrument, stands 24 hours at room temperature, 50 milliliters is diluted to E3 cultures, every
Centrifuged 20 minutes under 4000 turns of minute, take supernatant, supernatant is 8 mg/mls.Solution is diluted with E3 nutrient solutions, is obtained
Concentration is the drug solution of 0.5,1,2,4,6 and 8 mg/mls.
2nd, the after fertilization zebrafish embryo of 3 hours is placed in 96 orifice plates, 1 piece of embryo is placed per hole, each hole adds 200
Microlitre decoction, each 30 pieces of embryos of concentration liquid culture.Orifice plate is placed in 28.5 degrees Celsius and illumination/dark cycle 12/12 is (small
When) in the environment of cultivate, update within every 24 hours a decoction, culture to after fertilization 72 hours.
3rd, the form of the zebrafish embryo of each concentration is observed under the microscope and records dead embryo and lopsided embryo is abnormal
The number of shape, calculates the death rate and abnormal rate, and is calculated with software and to obtain LC50 and half teratogenesis concentration is respectively
5.23 and 3.51 mg/mls, teratogenic index is 1.49, shows weak potential teratogenesis.
Embodiment 3：
1st, Angong Niuhuang Wan is cut into graininess, accurately weighed 250 milligrams, adds 250 microlitres of DMSO, be cleaned by ultrasonic instrument
After middle extraction 30 minutes, 24 hours are stood at room temperature, 50 milliliters is diluted to E3 cultures, centrifuges, take under 4000rpm
Clearly, supernatant is 5 mg/mls.Diluted with E3 nutrient solutions, obtain concentration for 0.25,0.38,0.5,0.75,1,1.5 and
2 mg/mls.
2nd, the after fertilization zebrafish embryo of 3 hours is placed in 96 orifice plates, 1 piece of embryo is placed per hole, each hole adds 200
Microlitre decoction, each 30 pieces of embryos of concentration liquid culture.Orifice plate is placed in 28.5 degrees Celsius and illumination/dark cycle 12/12 is (small
When) in the environment of cultivate, update within every 24 hours a decoction, culture to after fertilization 72 hours.
3rd, the form of the zebrafish embryo of each concentration is observed under the microscope and records dead embryo and lopsided embryo is abnormal
The number of shape, calculates the death rate and abnormal rate, and is calculated with software and to obtain LC50 and half teratogenesis concentration is respectively
0.633 and 0.743 mg/ml, teratogenic index is 0.85, no teratogenesis.
Claims (5)
1. the method for the potential teratogenesis of a kind of utilization zebrafish embryo Fast Evaluation Chinese patent drug, it is characterised in that including following step
Suddenly：
(1) processing method of Chinese patent drug：Chinese patent drug is configured to fine powder, takes Chinese patent drug precise powder to weigh, is being cleaned by ultrasonic instrument
It is middle to be extracted 30 minutes with DMSO, placed 24 hours in room temperature.Extract adds E3 nutrient solutions and is diluted to required concentration (embryonic death
Rate is 100% concentration), supernatant is taken after centrifugation, multiple concentration are diluted to E3 nutrient solutions, for cultivating zebrafish embryo.
(2) culture of zebrafish embryo：The after fertilization zebrafish embryo of 3 hours is taken, is sub-packed according to every one piece of ground in hole in orifice plate,
The solution of Chinese patent drug, some embryos of hydroponics of each concentration are added in hole.Orifice plate is placed in 28.5 degrees Celsius, illumination/black
Cultivated in the incubator of dark circulation 12/12 (hour), change within every 24 hours a solution, culture to after fertilization 72 hours.
(3) teratogenesis evaluation：The developmental state of embryo is observed under the microscope, records dead embryo and Embryos under each concentration
The number of tire, according to probit models to LC50 (LC50) and half teratogenesis concentration (EC50) solved, pass through meter
Calculate LC50And EC50Ratio be worth to teratogenic index, according to the size of teratogenic index index judge medicine whether have potential teratogenesis
Property.
2. the method for the potential teratogenesis of a kind of utilization zebrafish embryo Fast Evaluation Chinese patent drug according to claim 1, it is special
Levy and be, the Chinese patent drug is 2015 editions《Chinese Pharmacopoeia》In described whole Chinese patent drugs.
3. the method for the potential teratogenesis of a kind of utilization zebrafish embryo Fast Evaluation Chinese patent drug according to claim 1, it is special
Levy and be, described that Chinese patent drug is configured into fine powder, collocation method takes different Ginding process according to formulation：Coating tablet warm water
Immersion is dried after 5 minutes and grinds to form fine powder at 60 c, and capsule removes capsule and got it filled core powder, honeyed bolus operating scissors
Fine grained is cut into, other formulations are directly ground.
4. the method for the potential teratogenesis of a kind of utilization zebrafish embryo Fast Evaluation Chinese patent drug according to claim 1, it is special
Levy and be, the zebrafish embryo is produced by wild AB types Adult Zebrafish.
5. the method for the potential teratogenesis of a kind of utilization zebrafish embryo Fast Evaluation Chinese patent drug according to claim 1, it is special
Levy and be, preparation is judged according to the size of teratogenic index index, teratogenic index is less than or equal to 1 without potential teratogenesis, teratogenesis
Index is more than 1 and has weak potential teratogenesis less than or equal to 2, and teratogenic index, which is more than 2, has strong teratogenesis.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710416962.9A CN107228925A (en) | 2017-05-21 | 2017-05-21 | A kind of method of the potential teratogenesis of utilization zebrafish embryo Fast Evaluation Chinese patent drug |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710416962.9A CN107228925A (en) | 2017-05-21 | 2017-05-21 | A kind of method of the potential teratogenesis of utilization zebrafish embryo Fast Evaluation Chinese patent drug |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107941941A (en) * | 2017-11-22 | 2018-04-20 | 江苏省中医药研究院 | One kind is using zebra fish toxicity and metabolism synchronized process analysis Chinese materia medica tocixity effect new method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297222A (en) * | 2014-10-16 | 2015-01-21 | 漳州片仔癀药业股份有限公司 | Zebrafish embryo alcoholic liver detecting model and construction method and application of zebrafish embryo alcoholic liver detecting model |
| CN105651983A (en) * | 2015-12-29 | 2016-06-08 | 湖南省植物保护研究所 | Method for determining toxicity of pesticide water dispersible granule by using zebra fish |
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2017
- 2017-05-21 CN CN201710416962.9A patent/CN107228925A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297222A (en) * | 2014-10-16 | 2015-01-21 | 漳州片仔癀药业股份有限公司 | Zebrafish embryo alcoholic liver detecting model and construction method and application of zebrafish embryo alcoholic liver detecting model |
| CN105651983A (en) * | 2015-12-29 | 2016-06-08 | 湖南省植物保护研究所 | Method for determining toxicity of pesticide water dispersible granule by using zebra fish |
Non-Patent Citations (2)
| Title |
|---|
| 朱淑珍: "马兜铃水提液对斑马鱼胚胎的致畸作用和心脏毒性的研究", 《中国野生植物资源》 * |
| 陈洁烽 等: "4种孕妇禁忌中成药对斑马鱼胚胎发育毒性研究", 《中国药物警戒》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107941941A (en) * | 2017-11-22 | 2018-04-20 | 江苏省中医药研究院 | One kind is using zebra fish toxicity and metabolism synchronized process analysis Chinese materia medica tocixity effect new method |
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Application publication date: 20171003 |