CN104215479A - Biology test method for rapidly detecting comprehensive toxicity of traditional Chinese medicine injection - Google Patents
Biology test method for rapidly detecting comprehensive toxicity of traditional Chinese medicine injection Download PDFInfo
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Abstract
The invention discloses a biology test method for rapidly detecting comprehensive toxicity of a traditional Chinese medicine injection, which comprises the following steps: 1) preparing a bacteria liquid used for test, namely, taking a photobacteria freeze-drying powder, using a sodium chloride solution with concentration of 1-5% (w/v) for recovery to obtain the bacteria liquid used for test; and 2)detecting, namely taking a sample to be detected, using the bacteria liquid for detecting, and determining the dilution with 50% of luminous intensity inhibition rate and a dilution-effect power curve. The test method has the advantages of high accuracy and sensitivity, and simple operation.
Description
Technical field
The present invention relates to a kind of biological test method of quick detection traditional Chinese medicine comprehensive toxicity.
Background technology
Traditional Chinese medicine has had the applicating history of more than 70 year in China, evident in efficacy to diseases such as cardiovascular and cerebrovascular, antitumor, respiratory systems, has unique advantage, uses extensively clinically.In recent years, increasingly extensive along with traditional Chinese medicine clinical practice, Reporting of harms also day by day increases.From 2006 " Puerarin event ", " cordate houttuynia event " to " the wilsonii event ", " Yin Zhi Huang event " of 2008, then to " the swap buffers event " and " Qing kailing event " of 2009, whole traditional Chinese medicine industry is allowed to be absorbed in trust crisis.After 2006, country reaches unprecedented height to the concern of traditional Chinese medicine safety problem.In July, 2009, state food Drug Administration office issues " notice about carrying out traditional Chinese medicine and revalue safely work ", and the risk investigation of comprehensively carrying out Production and quality control link controls traditional Chinese medicine potential safety hazard conscientiously.
At present, the project that traditional Chinese medicine quality control detects comprises: proterties, discriminating, pH value, heavy metal, burn vehement residue, related substance (protein, tannin, resin, oxalates, potassium ion), pyrogen or bacterial endotoxin, aseptic, loading amount, particulate matter, visible foreign matters, assay, depressor, undue toxicity, allergic reaction inspection, haemolysis and cohesion, osmotic pressure, harmful element, polyoxyethylene sorbitan monoleate, 5-HMF, finger-print, high molecular weight protein inspection etc.But these Testing index still effectively cannot ensure the consistance of its drug effect and reduce its anaphylactoid incidence.Therefore, be necessary to introduce new method of quality control.
Summary of the invention
In order to solve the problem, the invention provides a kind of biological test method of quick detection traditional Chinese medicine comprehensive toxicity.
The present invention detects the biological test method of traditional Chinese medicine comprehensive toxicity fast, comprises the steps:
(1) preparation test use bacterium liquid: getting photobacteria freeze-dried powder, is 1 ~ 5%(w/v by concentration) sodium chloride solution recover, must test and use bacterium liquid;
(2) detect: get measuring samples, detect with test bacterium liquid, determine that dilutability-effect dynamic curve and luminous intensity inhibition ratio are the dilutability of 50%.
Dilutability-effect dynamic curve, refers to the relation curve of solution dilution degree to be checked and luminous intensity inhibition ratio.
In step (1), described photobacteria is Fermi operator, photobacterium phosphoreum T3 microspecies, Qinghai Vibrion and other non-pathogenic photobacteria.
In step (1), described preparation method is: get the Fermi operator freeze-dried powder 1 that model is CS234, the concentration adding 0.2-1.0ml is 3%(w/v) sodium chloride solution recovery, must test and use bacterium liquid.
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: get measuring samples, be diluted with water to the liquid to be checked of 5 gradients, percent by volume is respectively: 100%, 25%, 6.25%, 1.5625%, 0.39%, with 1 ~ 5%(w/v) sodium chloride solution be titer, in described liquid to be measured and titer, add test use bacterium liquid, bacterium liquid addition is 1/25 ~ 1/15 of liquid to be checked or titer volume, places 5 ~ 30min, detect luminous intensity, calculate 5 dilution luminosity inhibiting rates;
B, determine to detect the dilution upper limit and lower limit: according to the result of step a, with inhibiting rate be arbitrary dilutability of 90 ~ 100% for the upper limit, if the inhibiting rate of sample stoste does not reach 90%, then with sample stoste for the upper limit; Be that arbitrary dilutability of 0 ~ 10% is for lower limit with inhibiting rate;
C, test: increase between the dilutability upper and lower bound that step b determines and join the liquid to be checked that 6 ~ 9 are evenly diluted gradient, with 1 ~ 5%(w/v) sodium chloride solution be titer, in described liquid to be measured and titer, add test use bacterium liquid, bacterium liquid addition is 1/25 ~ 1/15 of liquid to be checked or titer volume, place 5 ~ 30min, detect luminous intensity, make dilutability-effect dynamic curve, calculating inhibiting rate is the dilutability of 50%.
Dilutability of the present invention, refers to that solution to be checked is by the degree watered down.Such as, the 3ml water dilution of 1ml traditional Chinese medicine injection liquid, dilutability is 25%.
If dilution value is little, illustrates that the toxicity of houttuynia cordata injection to be checked is large, if dilution value is large, illustrate that the toxicity of houttuynia cordata injection to be checked is little.
Inhibiting rate is the dilutability of 50%, i.e. EC of the present invention
50.
In step a and step c, be added with sodium chloride in described liquid to be checked, its concentration is 3%(w/v); In described titer, the concentration of sodium chloride 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 liquid addition is 1/20 of liquid to be checked or titer volume.
In step a and step c, described standing time is 15min.
In aforementioned detection method, luminous intensity employing model is that the parameter in bio kinetic model instrument of LUMIStox300 detects.
The inventive method effectively can detect the toxicity of traditional Chinese medicine injection, and for its clinical practice provides reliable basis, meanwhile, detection method tool has the following advantages: (1) detection speed is fast: can obtain a result in 1 hour, assesses its comprehensive toxicity; (2) simple to operate: without the need to the professional technique such as gavage, intravenous injection, simple to operate, convenient and easy; (3) be quick on the draw; Utilize modern sensitive photoelectric detecting technology, can detect atomic weak intensity variation, to be quick on the draw several order of magnitude than general biological cell; (4) can judge the size of comprehensive toxicity; (5) bacteria sample amount is large, overcomes the impacts such as the few and individual difference of sample size in animal experiment.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to 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 houttuynia cordata injection sample is to the concentration-effect dynamic curve of Fermi operator
Fig. 7 houttuynia cordata injection sample is to the concentration-effect dynamic curve of Fermi operator
Fig. 8 Sofflower injection sample A is to the concentration-effect dynamic curve of Fermi operator
Fig. 9 Sofflower injection sample A is to the concentration-effect dynamic curve of Fermi operator
Figure 10 Sofflower injection sample B is to the concentration-effect dynamic curve of Fermi operator
Figure 11 Sofflower injection sample B is to the concentration-effect dynamic curve of Fermi operator
Embodiment
Embodiment 1 detection method of toxicity of the present invention
1 experiment material
1.1 bacterial classification
Fermi operator freeze-dried powder (CS234), purchased from Beijing Hamamatsu Technology Co., Ltd. ,-20 DEG C keep in Dark Place.
1.2 main agents
Recovery dilution: 3% sodium chloride solution;
Osmotic pressure regulator solution: 20% sodium chloride solution;
Traditional Chinese medicine injection: houttuynia injection liquor, commercially available.
1.3 key instruments and equipment
LUMIStox300 parameter in bio kinetic model instrument and the pre-temperature groove of supporting LUMIStherm and testing tube (Dr.Bruno Lange GmbH), BCD-539WF refrigerator (Haier), PB-10 acidometer (Sai Duolisi)
2 experimental techniques
The 2.1 tests preparation of bacterium liquid
Take out Fermi operator freeze-dried powder 1 from freezer compartment of refrigerator (-20 DEG C), be placed in after room temperature (about 20 DEG C) balances 15min, add 0.2-1.0ml recovery dilution, put 10min in ambient temperatare, namely can be used for test.
2.2 trial test
Traditional Chinese medicine distilled water is diluted to 5 concentration gradients (i.e. dilutability gradient) by 1:4: 100%, 25%, 6.25%, 1.5625%, 0.39%, testing sample solution is prepared into by described in 2.3, by method bigness scale described in 2.5 one time, determine concentration (i.e. dilutability) scope of the upper limit and lower limit.The upper limit is the concentration of inhibiting rate sample when reaching 90 ~ 100%, if the inhibiting rate of sample stoste does not reach 90%, then with sample stoste for the upper limit; Lower limit is the concentration of inhibiting rate sample when reaching 0 ~ 10%.
2.3 testing sample solution preparations
Increase again as required between upper and lower bound and join 6 ~ 12 concentration.By sample distilled water diluting to each concentration, more each concentration samples is mixed with the ratio of 17:3 with osmotic pressure regulator solution, be mixed with testing sample solution, make testing sample solution NaCl concentration be 3%.
2.4 blank liquid (titer) preparations
Adopt recovery dilution as blank liquid.
2.5 luminous intensities measure
The each concentration of testing sample prepares 3 testing tubes, often props up testing tube and adds 1ml testing sample, if 3 Duplicate Samples; Blank liquid also gets 3 testing tubes, often props up testing tube and adds 1ml recovery dilution, arrange 3 Duplicate Samples equally.In each testing tube, add 0.05ml test successively with pipettor and use bacterium liquid, vibrate gently, make it abundant mixing, the interval time that each testing tube adds bacterium liquid is 15 seconds, in educate place 15min in warm groove after within 15 seconds, to measure the luminous intensity of each testing tube with parameter in bio kinetic model instrument successively interval, be calculated as follows inhibiting rate, with EC
50the concentration value of this sample (when inhibiting rate equals 50%) represents the toxicity size of each sample, EC
50be worth less, toxicity is larger.
2.6 methodological study
2.6.1 assay method factors influencing
2.6.1.1 the time is on the impact of luminous intensity
Bacterium liquid is used in the recovery dilution and the test that add different volumes in testing tube, (cumulative volume is 1.05ml to be prepared into the test sample of different initial luminescence, sample 1 uses bacterium liquid for 1.04ml recovery dilution adds 0.01ml test, sample 2 uses bacterium liquid for 1.02ml recovery dilution adds 0.03ml test, sample 3 uses bacterium liquid for 1.00ml recovery dilution adds 0.05ml test, sample 4 uses bacterium liquid for 0.95ml recovery dilution adds 0.1ml test, sample 5 uses bacterium liquid for 0.85ml recovery dilution adds 0.2ml test), vibrate gently, make it abundant mixing, often organize sample and do 3 Duplicate Samples.Timing from mixing, measures 1 luminous intensity values, calculates the mean value of 3 Duplicate Samples, compare the impact of time on different initial luminescence for every 1 minute.
2.6.1.2pH the impact of value on luminous intensity
Get recovery dilution and add NaOH or HCl, being made into pH is respectively 3,4,5,6,7,8, and the recovery dilution of 9,10,11.Get each pH value recovery dilution 1ml (often group does 3 Duplicate Samples) in testing tube respectively, in each testing tube, add 0.05ml test successively use bacterium liquid, vibrate gently, make it abundant mixing, respectively at placement 5min, measure luminous intensity values with parameter in bio kinetic model instrument after 10min, 15min, compare the impact of pH value 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 traditional Chinese medicine samples (KZ-110501, KZ-110502, KZ-110503) are tested, every batch sample repeats 3 tests, result is evaluated.
2.6.2.2 Intermediate precision is investigated
(1) different personnel's test
By the above-mentioned method determined, tested with a collection of traditional Chinese medicine sample (KZ-110503) on same working day by two staff, result is evaluated.
(2) different operating day test
By the above-mentioned method determined, tested with a collection of traditional Chinese medicine sample (KZ-110503) in different operating day by same staff, result is evaluated.
3 results
3.1 assay method factors influencing
3.1.1 the time is on the impact of luminous intensity
Experimental result is as shown in table 1 and Fig. 1:
Table 1 time is on the impact of different initial luminescence
From table 1 and Fig. 1 ~ 5, the luminous intensity values prolongation in time of 5 samples is reduction trend, and in 5 ~ 30min, be maintained at a metastable level, initial luminescence value is higher, and luminous intensity reduced rate is lower.Meanwhile, experiment finds, when 15min luminous intensity is 500-1000 (sample 3), testing result is comparatively accurate, and cost is also handed over low, therefore, test be preferably that bacteria liquid amasss by the addition of bacterium liquid 1/20.
3.1.2pH the impact on luminous intensity is worth
Experimental result is as shown in table 2:
Table 2pH value is on the impact of luminous intensity
From table 2, during detection, solution ph is less on the impact of luminous intensity between 5.0-9.0, and inhibiting rate is within ± 10%, and when therefore using detection method to detect, 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
Table 43 crowd traditional Chinese medicine sample EC
50value (%)
From table 3 and table 4, the relative deviation <15% of the replica test of the inventive method, illustrates that the accuracy of the inventive method is high, favorable repeatability.
3.2.2 Intermediate precision is investigated
3.2.2.1 different operating personnel test
Table 5 different operating personnel test findings
3.2.2.2 different operating day tests
Table 6 different operating day test findings
From table 5 and table 6, the relative deviation <15% of the inventive method Intermediate precision test, illustrate that the repeatability of the inventive method is good, accuracy is high.
Description of test, the inventive method effectively can detect the toxicity of traditional Chinese medicine injection, favorable repeatability, and repeatability is good, and accuracy is high.
Embodiment 2 is applied the present invention and is carried out comprehensive toxicity test to two kinds of traditional Chinese medicine injections
1 experiment material
With embodiment 1.
Traditional Chinese medicine injection: houttuynia cordata injection, Sofflower injection, commercially available.
1.3 key instruments and equipment
With embodiment 1.
2 experimental techniques
Houttuynia cordata injection, Sofflower injection, all through traditional technique in measuring, are defined as specification product.
Adopt the method for embodiment 1, luminous intensity mensuration is carried out to houttuynia cordata injection and Sofflower injection, calculate inhibiting rate, use EC
50the relatively comprehensive toxicity size of each sample.
The 2.1 tests preparation of bacterium liquid
With embodiment 1.
2.2 trial test
With embodiment 1.
2.3 testing sample solution preparations
With embodiment 1.
2.4 blank liquid preparations
With embodiment 1.
2.5 luminous intensities measure
With embodiment 1.
3 results
3.1 houttuynia cordata injection comprehensive toxicity tests
Table 7 houttuynia cordata injection sample comprehensive toxicity result
As can be seen from table 7 and Fig. 6 ~ 7, the EC of houttuynia cordata injection sample A
50(dilutability when luminous intensity inhibition ratio is 50%) is lower than the EC of houttuynia cordata injection sample B
50, illustrate that the former toxicity is greater than the toxicity of the latter.
3.2 Sofflower injection comprehensive toxicity tests
Table 8 Sofflower injection sample comprehensive toxicity result
As can be seen from table 8 and Fig. 8 ~ 11, the EC of Sofflower injection sample A
50(dilutability when luminous intensity inhibition ratio is 50%) is lower than the EC of Sofflower injection sample B
50, illustrate that the former toxicity is greater than the toxicity of the latter.
Description of test, for all qualified traditional Chinese medicine injection of traditional technique in measuring quality, side of the present invention can measure its toxicity and vary, can the inconsistent phenomenon of well explain existing traditional Chinese medicine injection bad reaction.
To sum up, the detection speed of detection method of toxicity of the present invention is fast, and simple to operate, be quick on the draw, accuracy is high, can detect the toxicity of traditional Chinese medicine injection, and for the quality control of traditional Chinese medicine injection provides foundation, application prospect is good.
Claims (10)
1. detect a biological test method for traditional Chinese medicine comprehensive toxicity fast, it is characterized in that: comprise the steps:
(1) preparation test use bacterium liquid: getting photobacteria freeze-dried powder, is 1 ~ 5%(w/v by concentration) sodium chloride solution recover, must test and use bacterium liquid;
(2) detect: get measuring samples, detect with test bacterium liquid, determine that dilutability-effect dynamic curve and luminous intensity inhibition ratio are the dilutability of 50%.
2. method of testing according to claim 1, is characterized in that: in step (1), and described photobacteria is Fermi operator, photobacterium phosphoreum T3 microspecies, Qinghai Vibrion and other non-pathogenic photobacteria.
3. method of testing according to claim 1, it is characterized in that: in step (1), described preparation method is: get the Fermi operator freeze-dried powder 1 that model is CS234, the concentration adding 0.2-1.0ml is 3%(w/v) sodium chloride solution recovery, must test and use bacterium liquid.
4. method of testing according to claim 1, is characterized in that: in step (1), and the pH of described sodium chloride solution is 5 ~ 9.
5. method of testing according to claim 1, is characterized in that: the method for step (2) described detection is as follows:
A, pretest: get measuring samples, be diluted with water to the liquid to be checked of 5 gradients, percent by volume is respectively: 100%, 25%, 6.25%, 1.5625%, 0.39%, with 1 ~ 5%(w/v) sodium chloride solution be titer, in described liquid to be measured and titer, add test use bacterium liquid, bacterium liquid addition is 1/25 ~ 1/15 of liquid to be checked or titer volume, places 5 ~ 30min, detect luminous intensity, calculate 5 dilution luminosity inhibiting rates;
B, determine to detect the dilution upper limit and lower limit: according to the result of step a, with inhibiting rate be arbitrary dilutability of 90 ~ 100% for the upper limit, if the inhibiting rate of sample stoste does not reach 90%, then with sample stoste for the upper limit; Be that arbitrary dilutability of 0 ~ 10% is for lower limit with inhibiting rate;
C, test: increase between the dilutability upper and lower bound that step b determines and join the liquid to be checked that 6 ~ 9 are evenly diluted gradient, with 1 ~ 5%(w/v) sodium chloride solution be titer, in described liquid to be measured and titer, add test use bacterium liquid, bacterium liquid addition is 1/25 ~ 1/15 of liquid to be checked or titer volume, place 5 ~ 30min, detect luminous intensity, make dilutability-effect dynamic curve, calculating inhibiting rate is the dilutability of 50%.
6. method of testing according to claim 5, is characterized in that: in step a and step c, is added with sodium chloride in described liquid to be checked, and its concentration is 3%(w/v); In described titer, the concentration of sodium chloride is 3%(w/v).
7. method of testing according to claim 5, is characterized in that: in step a and step c, and the pH of described liquid to be checked and titer is 5 ~ 9.
8. method of testing according to claim 5, is characterized in that: in step a and step c, and described bacterium liquid addition is 1/20 of liquid to be checked or titer volume.
9. method of testing according to claim 5, is characterized in that: in step a and step c, and described standing time is 15min.
10. the method for testing according to claim 1 ~ 9 any one, is characterized in that: detect the parameter in bio kinetic model instrument detection that luminous intensity employing model is LUMIStox300.
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