CN104215479B - A kind of biological test method of quick detection Chinese medicine comprehensive toxicity - Google Patents
A kind of biological test method of quick detection Chinese medicine comprehensive toxicity Download PDFInfo
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Abstract
The invention discloses the biological test method of a kind of quick detection Chinese medicine comprehensive toxicity, comprise the steps: that (1) prepares test bacterium solution: take photobacteria lyophilized powder, be 1～5%(w/v by concentration) sodium chloride solution recovery, obtain test bacterium solution；(2) detection: take measuring samples, detects by test bacterium solution, determines dilution factor and dilution factor effect dynamic curve that luminous intensity inhibition ratio is 50%.The inventive method accuracy in detection and sensitivity are the highest, simple to operate.
Description
Technical field
The present invention relates to the biological test method of a kind of quick detection Chinese medicine comprehensive toxicity.
Background technology
Chinese medicine has had the applicating history of more than 70 year in China, to diseases such as cardiovascular and cerebrovascular vessel, antitumor, respiratory systems
Sick is evident in efficacy, has the advantage of uniqueness, uses clinically extensively.In recent years, along with Chinese medicine clinical practice
Increasingly extensive, Reporting of harms the most day by day increases.From 2006 " puerarin event ", " Herba Houttuyniae event " to 2008
" Radix Et Caulis Acanthopanacis Senticosi event ", " Yin Zhi Huang event ", then " the SHUANGHUANLIAN event " and " QINGKAILING event " by 2009, allow whole Chinese medicine note
Penetrate agent industry and be absorbed in trust crisis.After 2006, country has reached unprecedented to the concern of Chinese medicine safety problem
Height.In July, 2009, state food Drug Administration office issues and " revalues safely the logical of work about carrying out Chinese medicine
Know ", the risk investigation comprehensively carrying out Production and quality control link controls Chinese medicine potential safety hazard conscientiously.
At present, Chinese medicine quality control detection project include: character, discriminating, pH value, heavy metal, burn vehement residue,
There are related substance (protein, tannin, resin, oxalates, potassium ion), pyrogen or bacterial endotoxin, aseptic, loading amount, insoluble micro-
Grain, visible foreign matters, assay, depressor substance, undue toxicity, anaphylaxis inspection, haemolysis and cohesion, osmotic pressure, You Haiyuan
Element, polyoxyethylene sorbitan monoleate, 5-HMF, finger printing, high molecular weight protein inspection etc..But, these Testing index still cannot be effective
Ensure the concordance of its drug effect and reduce its anaphylactoid incidence rate.Therefore, it is necessary to introduce new method of quality control.
Summary of the invention
In order to solve the problems referred to above, the invention provides the biological test of a kind of quick detection Chinese medicine comprehensive toxicity
Method.
The present invention quickly detects the biological test method of Chinese medicine comprehensive toxicity, comprises the steps:
(1) prepare test bacterium solution: take photobacteria lyophilized powder, be 1～5%(w/v by concentration) sodium chloride solution multiple
Soviet Union, obtains test bacterium solution；
(2) detection: take measuring samples, detects by test bacterium solution, determines that dilution factor-effect dynamic curve and luminescence are strong
Degree suppression ratio is the dilution factor of 50%.
Dilution factor-effect dynamic curve, refers to the relation curve of solution dilution factor to be checked and luminous intensity inhibition ratio.
In step (1), described photobacteria is Fermi operator, photobacterium phosphoreum T3 microspecies, Qinghai Vibrion and other is non-
Pathogenic photobacteria.
In step (1), described preparation method is: take the Fermi operator lyophilized powder 1 that model is CS234, adds 0.2-
The concentration of 1.0ml is 3%(w/v) sodium chloride solution recovery, obtain test bacterium solution.
In step (1), the pH of described sodium chloride solution is 5～9.
The method of step (2) described detection is as follows:
A, pretest: take measuring samples, be diluted with water to the liquid to be checked of 5 gradients, percent by volume respectively: 100%,
25%, 6.25%, 1.5625%, 0.39%, with 1～5%(w/v) sodium chloride solution as titer, at described liquid to be measured and titer
Middle addition test bacterium solution, bacterium solution addition is liquid to be checked or the 1/25 of titer volume～1/15, places 5～30min, detection
Luminous intensity, calculates 5 dilution luminosity suppression ratio；
B, determine the detection dilution upper limit and lower limit: according to the result of step a, with suppression ratio be 90～100% arbitrary
Dilution factor is the upper limit, if the suppression ratio of sample stock solution is not up to 90%, then with sample stock solution as the upper limit；It is 0～10% with suppression ratio
Arbitrary dilution factor be lower limit；
C, test: increase between the dilution factor upper and lower bound that step b determines and join the to be checked of 6～9 uniform dilution gradients
Liquid, with 1～5%(w/v) sodium chloride solution as titer, in described liquid to be measured and titer add test bacterium solution, bacterium solution
Addition is liquid to be checked or the 1/25 of titer volume～1/15, placement 5～30min, detection luminous intensity, making dilution factor-
Effect dynamic curve, calculating suppression ratio is the dilution factor of 50%.
Dilution factor of the present invention, refers to the degree that solution to be checked is watered down.Such as, 1ml Chinese medicine injection liquid 3ml water is dilute
Releasing, dilution factor is 25%.
If dilution value is little, illustrating that the toxicity of Herba Houttuyniae injectio to be checked is big, if dilution value is big, explanation is treated
The toxicity of inspection Herba Houttuyniae injectio is little.
Suppression ratio is the dilution factor of 50%, EC the most of the present invention50。
In step a and step c, added with sodium chloride in described liquid to be checked, its concentration is 3%(w/v)；Chlorine in described titer
The concentration changing sodium is 3%(w/v).
In step a and step c, the pH of described liquid to be checked and titer is 5～9.
In step a and step c, described bacterium solution addition is liquid to be checked or the 1/20 of titer volume.
In step a and step c, described standing time is 15min.
In aforementioned detection method, the parameter in bio kinetic model instrument detection that luminous intensity uses model to be LUMIStox300.
The inventive method can effectively detect the toxicity of Chinese medicine injection, for its clinical practice provide reliable basis, meanwhile,
Detection method has the advantage that (1) detection speed is fast: can obtain a result in 1 hour, assess it the most malicious
Property；(2) simple to operate: without the professional technique such as gavage, intravenous injection, simple to operate, convenient and easy；(3) it is quick on the draw；Utilize
Modern sensitive photoelectric detecting technology, can detect atomic weak intensity variation, be quick on the draw than general biological cell
Several orders of magnitude；(4) size of comprehensive toxicity can be judged；(5) bacteria sample amount is big, overcomes sample in animal experiment
Quantity is few and the impact such as individual variation.
Obviously, according to the foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
Under the present invention above-mentioned basic fundamental thought premise, it is also possible to make the amendment of other various ways, replace or change.
The detailed description of the invention of form by the following examples, remakes the most specifically the foregoing of the present invention
Bright.But this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below.All based on foregoing of the present invention
The technology realized belongs to the scope of the present invention.
Accompanying drawing explanation
Fig. 1 concentration 1 Fermi operator luminous intensity is schemed over time
Fig. 2 concentration 2 Fermi operator luminous intensity is schemed over time
Fig. 3 concentration 3 Fermi operator luminous intensity is schemed over time
Fig. 4 concentration 4 Fermi operator luminous intensity is schemed over time
Fig. 5 concentration 5 Fermi operator luminous intensity is schemed over time
Fig. 6 Herba Houttuyniae injectio sample concentration-effect dynamic curve to Fermi operator
Fig. 7 Herba Houttuyniae injectio sample concentration-effect dynamic curve to Fermi operator
Fig. 8 Flos Carthami injection sample A concentration-effect dynamic curve to Fermi operator
Fig. 9 Flos Carthami injection sample A concentration-effect dynamic curve to Fermi operator
Figure 10 Flos Carthami injection sample B concentration-effect dynamic curve to Fermi operator
Figure 11 Flos Carthami injection sample B concentration-effect dynamic curve to Fermi operator
Detailed description of the invention
Embodiment 1 detection method of toxicity of the present invention
1 experiment material
1.1 strain
Fermi operator lyophilized powder (CS234), purchased from Beijing Hamamatsu Technology Co., Ltd. ,-20 DEG C keep in Dark Place.
1.2 main agents
Recovery diluent: 3% sodium chloride solution；
Osmotic pressure regulation liquid: 20% sodium chloride solution；
Chinese medicine injection: houttuynia injection liquor, commercially available.
1.3 key instruments and equipment
LUMIStox300 parameter in bio kinetic model instrument and supporting LUMIStherm pre-temperature groove and testing tube (Dr.Bruno
Lange GmbH), BCD-539WF electric refrigerator (Haier), PB-10 acidometer (Sai Duolisi)
2 experimental techniques
The preparation of 2.1 test bacterium solution
Take out Fermi operator lyophilized powder 1 from freezer compartment of refrigerator (-20 DEG C), be placed in room temperature (about 20 DEG C) balance 15min
After, add 0.2-1.0ml recovery diluent, place 10min at room temperature, i.e. can be used for testing.
2.2 trial test
100%, 25% Chinese medicine distilled water is diluted to 5 Concentraton gradient (i.e. dilution factor gradient) by 1:4:,
6.25%, 1.5625%, 0.39%, as described in 2.3, be prepared as testing sample solution, method bigness scale one time as described in 2.5, determine on
Limit and concentration (i.e. dilution factor) scope of lower limit.The upper limit is that suppression ratio reaches the concentration of sample when 90～100%, if sample stock solution
Suppression ratio be not up to 90%, then with sample stock solution as the upper limit；Lower limit is that suppression ratio reaches the concentration of sample when 0～10%.
Prepared by 2.3 testing sample solutions
Increase the most again between upper and lower bound and join 6～12 concentration.By sample distilled water diluting to each concentration,
Again each concentration samples is mixed with the ratio of 17:3 with osmotic pressure regulation liquid, be configured to testing sample solution, make testing sample molten
Liquid NaCl concentration is 3%.
Prepared by 2.4 blank liquid (titer)
Use recovery diluent as blank liquid.
2.5 luminous intensities measure
The each concentration of testing sample prepares 3 testing tubes, and every testing tube adds 1ml testing sample, if 3 Duplicate Samples；
Blank liquid also takes 3 testing tubes, and every testing tube adds 1ml recovery diluent, arranges 3 Duplicate Samples equally.With moving liquid
Device is sequentially added into 0.05ml test bacterium solution in each testing tube, vibrates gently, is allowed to fully mix, and each testing tube adds bacterium solution
Interval time be 15 seconds, measure each test in educating temperature groove is placed to be spaced successively 15 seconds with parameter in bio kinetic model instrument after 15min
The luminous intensity of pipe, is calculated as follows suppression ratio, with EC50(concentration value of this sample when suppression ratio is equal to 50%) represents each sample
Toxicity size, EC50Being worth the least, toxicity is the biggest.
2.6 methodological study
2.6.1 assay method factors influencing
2.6.1.1 the impact that the time is on luminous intensity
Testing tube adds recovery diluent and the test bacterium solution of different volumes, is prepared as different initial luminescence
(cumulative volume is 1.05ml to test sample, and sample 1 is that 1.04ml recovery diluent adds 0.01ml test bacterium solution, and sample 2 is
1.02ml recovery diluent adds 0.03ml test bacterium solution, and sample 3 is that 1.00ml recovery diluent adds 0.05ml test use
Bacterium solution, sample 4 is that 0.95ml recovery diluent adds 0.1ml test bacterium solution, and sample 5 is that 0.85ml recovery diluent adds
0.2ml test bacterium solution), vibrate gently, be allowed to fully mix, often group sample does 3 Duplicate Samples.Timing is started from mixing, every 1
Minute measure 1 luminous intensity values, calculate the meansigma methods of 3 Duplicate Samples, compare the time impact on different initial luminescence.
2.6.1.2pH the impact that value is on luminous intensity
Take recovery diluent and add NaOH or HCl, be made into the recovery dilution that pH is 3,4,5,6,7,8,9,10,11 respectively
Liquid.Take each pH value recovery diluent 1ml (often group does 3 Duplicate Samples) in testing tube respectively, be sequentially added in each testing tube
0.05ml test bacterium solution, vibrates gently, is allowed to fully mix, and respectively at placing 5min, uses bio-toxicity after 10min, 15min
Tester measures luminous intensity values, compares the pH value impact on luminous intensity.
2.6.2 precision is investigated
2.6.2.1 repeatability is investigated
By the above-mentioned method determined, 3 batches of Chinese medicine samples (KZ-110501, KZ-110502, KZ-110503) are entered
Row test, every batch sample are repeated 3 times test, are evaluated result.
2.6.2.2 Intermediate precision is investigated
(1) different personnel's tests
By the above-mentioned method determined, by two staff on same working day to a collection of Chinese medicine sample (KZ-
110503) test, result is evaluated.
(2) different operating day test
By the above-mentioned method determined, by same staff in different operating day to a collection of Chinese medicine sample (KZ-
110503) test, result is evaluated.
3 results
3.1 assay method factors influencing
3.1.1 the time impact on luminous intensity
Experimental result is as shown in table 1 and Fig. 1:
The impact on different initial luminescence of table 1 time
From table 1 and Fig. 1～5, the prolongation in time of the luminous intensity values of 5 samples in reducing trend, 5～
Being maintained at a metastable level in 30min, initial luminescence value is the highest, and luminous intensity reduction rate is the lowest.Meanwhile, real
Issuing after examination and approval existing, when 15min luminous intensity is 500-1000 (sample 3), testing result is relatively accurate, and cost is also handed over low, and therefore, test is used
The addition of bacterium solution is preferably the 1/20 of bacterium solution volume.
3.1.2pH the impact on luminous intensity it is worth
Experimental result is as shown in table 2:
The impact on luminous intensity of the table 2pH value
From table 2, during detection, solution ph impact on luminous intensity between 5.0-9.0 is less, suppression ratio ±
Within 10%, when therefore using detection method detection, pH value of solution is preferably 5.0～9.0.
3.2 precision are investigated
3.2.1 replica test
Experimental result is as shown in Table 3 and Table 4:
Table 3 replica test result
43 crowdes of Chinese medicine sample EC of table50Value (%)
From table 3 and table 4, and the relative deviation of the replica test of the inventive method < 15%, the inventive method is described
Accuracy is high, favorable repeatability.
3.2.2 Intermediate precision is investigated
3.2.2.1 different operating personnel test
Table 5 different operating personnel's result of the test
3.2.2.2 different operating day tests
Table 6 different operating day result of the test
From table 5 and table 6, and the relative deviation of the inventive method Intermediate precision test < 15%, the inventive method is described
Repeatability good, accuracy is high.
Description of test, the inventive method can effectively detect the toxicity of Chinese medicine injection, favorable repeatability, and repeatability is good,
Accuracy is high.
Embodiment 2 applies the present invention that two kinds of Chinese medicine injections are carried out comprehensive toxicity test
1 experiment material
With embodiment 1.
Chinese medicine injection: Herba Houttuyniae injectio, Flos Carthami injection, commercially available.
1.3 key instruments and equipment
With embodiment 1.
2 experimental techniques
Herba Houttuyniae injectio, Flos Carthami injection, all through traditional technique in measuring, are defined as qualified products.
The method using embodiment 1, carries out luminous intensity mensuration to Herba Houttuyniae injectio Flos Carthami injection, calculates suppression
Rate, uses EC50The relatively comprehensive toxicity size of each sample.
The preparation of 2.1 test bacterium solution
With embodiment 1.
2.2 trial test
With embodiment 1.
Prepared by 2.3 testing sample solutions
With embodiment 1.
Prepared by 2.4 blank liquid
With embodiment 1.
2.5 luminous intensities measure
With embodiment 1.
3 results
3.1 Herba Houttuyniae injectio comprehensive toxicity tests
Table 7 Herba Houttuyniae injectio sample comprehensive toxicity result
By table 7 and Fig. 6～7 it can be seen that the EC of Herba Houttuyniae injectio sample A50(when luminous intensity inhibition ratio is 50%
Dilution factor) less than the EC of Herba Houttuyniae injectio sample B50, the former toxicity toxicity more than the latter is described.
3.2 Flos Carthami injection comprehensive toxicity tests
Table 8 Flos Carthami injection sample comprehensive toxicity result
By table 8 and Fig. 8～11 it can be seen that the EC of Flos Carthami injection sample A50(when luminous intensity inhibition ratio is 50%
Dilution factor) less than the EC of Flos Carthami injection sample B50, the former toxicity toxicity more than the latter is described.
Description of test, for the Chinese medicine injection the most qualified by traditional technique in measuring quality, side of the present invention can measure it
Toxicity varies, it is possible to preferably explain the phenomenon that existing Chinese medicine injection untoward reaction is inconsistent.
To sum up, the detection speed of detection method of toxicity of the present invention is fast, simple to operate, is quick on the draw, and accuracy is high, Ke Yijian
Surveying the toxicity of Chinese medicine injection, the quality control for Chinese medicine injection provides foundation, and application prospect is good.
Claims (9)
1. the biological test method of a quick detection Chinese medicine comprehensive toxicity, it is characterised in that: comprise the steps:
(1) prepare test bacterium solution: take photobacteria lyophilized powder, with the sodium chloride solution recovery that concentration is 1～5%w/v, obtain survey
Bacterium solution on probation；
(2) detection: take measuring samples, detects by test bacterium solution, determines that dilution factor-effect dynamic curve and luminous intensity press down
Rate processed is the dilution factor of 50%；
The method of step (2) described detection is as follows:
A, pretest: take measuring samples, be diluted with water to the liquid to be checked of 5 gradients, percent by volume respectively: 100%,
25%, 6.25%, 1.5625%, 0.39%, with the sodium chloride solution of 3%w/v as titer, at described liquid to be measured and titer
Middle addition test bacterium solution, bacterium solution addition is liquid to be checked or the 1/25 of titer volume～1/15, places 5～30min, detection
Luminous intensity, calculates 5 dilution luminosity suppression ratio；
B, determine the detection dilution upper limit and lower limit: according to the result of step a, with suppression ratio be 90～100% arbitrary dilute
Degree of releasing is the upper limit, if the suppression ratio of sample stock solution is not up to 90%, then with sample stock solution as the upper limit；It is 0～10% with suppression ratio
Arbitrary dilution factor be lower limit；
C, test: between the dilution factor upper and lower bound that step b determines, increase the liquid to be checked joining 6～9 uniform dilution gradients, with
The sodium chloride solution of 3%w/v is titer, adds test bacterium solution in described liquid to be checked and titer, and bacterium solution addition is
Liquid to be checked or the 1/25 of titer volume～1/15, places 5～30min, detects luminous intensity, makes dilution factor-effect power
Curve, calculating suppression ratio is the dilution factor of 50%；
In step a and step c, added with sodium chloride in described liquid to be checked, the sodium chloride concentration of liquid to be checked is 3%w/v.
Method of testing the most according to claim 1, it is characterised in that: in step (1), described photobacteria is that non-causing a disease is sent out
Photobacteria.
Method of testing the most according to claim 2, it is characterised in that: described photobacteria is Fermi operator, bright burn
Bacillus T3 microspecies or Qinghai Vibrion.
Method of testing the most according to claim 1, it is characterised in that: in step (1), described preparation method is: take model
For the Fermi operator lyophilized powder 1 of CS234, add the sodium chloride solution that concentration the is 3%w/v recovery of 0.2-1.0ml, obtain survey
Bacterium solution on probation.
Method of testing the most according to claim 1, it is characterised in that: in step (1), the pH of described sodium chloride solution is 5
～9.
Method of testing the most according to claim 1, it is characterised in that: in step a and step c, described liquid to be checked and standard
The pH of liquid is 5～9.
Method of testing the most according to claim 1, it is characterised in that: in step a and step c, described bacterium solution addition is
Liquid to be checked or the 1/20 of titer volume.
Method of testing the most according to claim 1, it is characterised in that: in step a and step c, described standing time is
15min。
9. according to the method for testing described in claim 1～8 any one, it is characterised in that: detection luminous intensity uses model
Parameter in bio kinetic model instrument for LUMIStox 300 detects.
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| CN105973875B (en) * | 2016-04-27 | 2021-06-25 | 四川省中医药科学院 | Quality control method of drug micro-toxicity test system |
| CN107091833A (en) * | 2017-05-02 | 2017-08-25 | 北京大学 | A kind of method of Fast Evaluation petroleum polluted soil ecology toxicity |
| CN108627503B (en) * | 2017-05-05 | 2021-03-02 | 四川省中医药科学院 | Method for detecting quality of ginkgolide injection |
| CN107643371B (en) * | 2017-09-13 | 2020-08-07 | 广州瑞森生物科技股份有限公司 | Method for establishing edible agricultural product quality safety risk assessment model |
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