CN103868915A - Biological test method for rapidly detecting comprehensive toxicity of safflower injection - Google Patents
Biological test method for rapidly detecting comprehensive toxicity of safflower injection Download PDFInfo
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- CN103868915A CN103868915A CN201410113782.XA CN201410113782A CN103868915A CN 103868915 A CN103868915 A CN 103868915A CN 201410113782 A CN201410113782 A CN 201410113782A CN 103868915 A CN103868915 A CN 103868915A
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Abstract
The invention discloses a biological test method for rapidly detecting the comprehensive toxicity of a safflower injection. The method comprises the following steps: (1) preparing a bacterial solution for test: weighing freeze-dried luminous bacterium powder, and reviving the freeze-dried luminous bacterium powder with a solution with the chloride ion concentration of 0.1-1.0mol/L, so as to obtain the bacterial solution for test; (2) detecting: detecting a safflower injection sample to be detected with the bacterial solution for test, so as to determine a sample dilution degree at the luminous intensity inhibition rate of 50% and a dilution degree-effect power curve. The method disclosed by the invention has relatively high accuracy and sensitivity in detection and is simple in operation.
Description
Technical field
The present invention relates to a kind of biological test method of fast detecting Sofflower injection comprehensive toxicity.
Background technology
Sofflower injection forms by single medicine safflower " water extract-alcohol precipitation " is refining; carthamin yellow is its principal ingredient; there is effect of activating blood to promote menstruation, blood stasis removing analgesic; be widely used in clinically the treatment of the disease such as cardiovascular and cerebrovascular diseases, amenorrhoea, there is coronary artery dilating, protect the multiple pharmacological effect such as myocardium, anti-oxidant.When Sofflower injection clinical practice is increasingly extensive, the report that its bad reaction occurs also increases gradually.Discovery is analyzed in the bad reaction of the 32 routine Sofflower injections to 1994-2005 bibliographical information, its bad reaction generation asexuality and age differences, the time that reacts mostly concentrate on medication after 5～30min with interior (accounting for more than 50%), take allergic reaction as main (accounting for more than 50%), there is anaphylactic shock in severe patient.
At present, Sofflower injection carries out quality control detection according to standards of pharmacopoeia, but these detection indexs still cannot effectively guarantee the consistance of its drug effect and reduce its anaphylactoid incidence.Therefore, be necessary to introduce more high quality control method of new sensitivity, the abnormal conditions in monitoring production at any time occur.
Summary of the invention
In order to address the above problem, the invention provides a kind of biological test method of fast detecting Sofflower injection comprehensive toxicity.
The biological test method of fast detecting Sofflower injection comprehensive toxicity of the present invention, comprises the steps:
(1) bacterium liquid is used in preparation test: get photobacteria freeze-dried powder, the solution that is 0.1-1.0mol/L with chlorine ion concentration recovery, must test and use bacterium liquid;
(2) detect: get Sofflower injection sample to be checked, with the detection of test bacterium liquid, determine that dilutability-effect dynamic curve and luminous intensity inhibition ratio are 50% dilutability.
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 Fei Shi vibrios, photobacterium phosphoreum, Qinghai Vibrion and other non-pathogenic photobacteria.
In step (1), describedly prepare test and by the method for bacterium liquid be: get model and be 1 of the Fei Shi vibrios freeze-dried powder of CS234, the solution that the chlorine ion concentration that adds 0.2-1.0ml is 0.51mol/L, must test and use bacterium liquid.
In step (1), the pH of the solution of described chloride ion-containing is 5～9.
The method of the described detection of step (2) is as follows:
A, pretest: get Sofflower injection sample to be checked, 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%, solution take chlorine ion concentration as 0.1-1.0mol/L is titer, in described liquid to be checked and titer, adding test to 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 and detect the dilution upper limit and lower limit: according to the result of step a, the arbitrary dilutability take inhibiting rate as 90～100% is as the upper limit, if the inhibiting rate of sample stoste does not reach 90%, take sample stoste as the upper limit; Arbitrary dilutability take inhibiting rate as 0～10% is as lower limit;
C, test: between the definite dilutability upper and lower bound of step b, increase and join 6～9 evenly liquid to be checked of dilution gradient, solution take chlorine ion concentration as 0.1-1.0mol/L is titer, in described liquid to be checked and titer, add test 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, the dilutability that calculating inhibiting rate is 50%.
Dilutability of the present invention, refers to the degree that solution to be checked is watered down.For example, 3ml water dilution for 1ml Sofflower injection, dilutability is 25%.
If dilution value is little, illustrate that the toxicity of Sofflower injection to be checked is large, if dilution value is large, illustrate that the toxicity of Sofflower injection to be checked is little.
Inhibiting rate is 50% dilutability, i.e. EC50 of the present invention.
In step a and step c, be added with the solution of chloride ion-containing in described liquid to be checked, its chlorine ion concentration is 0.1-1.0mol/L; In described titer, chlorine ion concentration is 0.51mol/L.
In step a and step c, the pH of described liquid to be checked and sodium chloride solution 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, be 15min described standing time.
In aforementioned detection method, luminous intensity adopts bio-toxicity tester or other to have the instrument of luminous intensity measuring ability.
The inventive method can effectively detect the toxicity of Sofflower injection, and for its clinical practice provides reliable basis, meanwhile, detection method tool of the present invention has the following advantages: (1) detection speed is fast: in 1 hour, can obtain a result, assess its comprehensive toxicity; (2) simple to operate: without 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, than general biological cell several orders of magnitude that are quick on the draw; (4) can judge the size of comprehensive toxicity; (5) bacteria sample amount is large, has overcome in animal experiment the impacts such as the few and individual difference of sample size.
Obviously,, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
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 to the scope of the above-mentioned theme of the present invention only limits to following example.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 concentration 1 Fei Shi vibrios luminous intensity is schemed over time
Fig. 2 concentration 2 Fei Shi vibrios luminous intensities are schemed over time
Fig. 3 concentration 3 Fei Shi vibrios luminous intensities are schemed over time
Fig. 4 concentration 4 Fei Shi vibrios luminous intensities are schemed over time
Fig. 5 concentration 5 Fei Shi vibrios luminous intensities are schemed over time
Concentration-effect the dynamic curve of 10 batches of Sofflower injection samples of Fig. 6 producer first to Fei Shi vibrios
Concentration-effect the dynamic curve of 10 batches of Sofflower injection samples of Fig. 7 producer second to Fei Shi vibrios
Embodiment 1 detection method of toxicity of the present invention
1 experiment material
1.1 bacterial classification
Fei Shi vibrios freeze-dried powder (CS234), purchased from Beijing Hamamatsu Technology Co., Ltd. ,-20 ℃ keep in Dark Place.
1.2 main agents
Recovery dilution: 0.51mol/L sodium chloride solution;
Osmotic pressure regulator solution: 3.42mol/L sodium chloride solution;
Traditional Chinese medicine: commercially available
1.3 key instruments and equipment
LUMIStox300 bio-toxicity tester and the supporting pre-temperature groove of LUMIStherm and testing tube (Dr.Bruno Lange GmbH), BCD-539WF refrigerator (Haier), PB-10 acidometer (Sai Duolisi)
2 experimental techniques
The preparation of bacterium liquid for 2.1 tests
Take out 1 of Fei Shi vibrios freeze-dried powder from freezer compartment of refrigerator (20 ℃), be placed in after room temperature (20 ℃ of left and right) balance 15min, add 0.2-1.0ml recovery dilution, under room temperature, place 10min, can be used for test.
2.2 trial test
Traditional Chinese medicine is diluted to 5 gradients (being dilutability gradient) with distilled water by 1:4: 100%, 25%, 6.25%, 1.5625%, 0.39%, be prepared into testing sample solution by described in 2.3, by method bigness scale described in 2.5 one time, determine concentration (the being dilutability) scope of the upper limit and lower limit.On the concentration of sample when being limited to inhibiting rate and reaching 90～100%, if the inhibiting rate of sample stoste does not reach 90%, take sample stoste as the upper limit; Under the concentration of sample when being limited to inhibiting rate and reaching 0～10%.
2.3 testing sample solution preparations
Between upper and lower bound, increase and join 6～12 concentration more as required.Sample is arrived to each concentration with distilled water diluting, more each concentration sample is mixed with the ratio of 17:3 with osmotic pressure regulator solution, be mixed with testing sample solution, making testing sample solution chlorine ion concentration is 0.51mol/L.
2.4 blank liquid (titer) preparations
Adopt recovery dilution as blank liquid.
2.5 luminous intensities are measured
The each concentration of testing sample is prepared 3 testing tubes, and every testing tube adds 1ml testing sample, establishes 3 Duplicate Samples; Blank liquid is also got 3 testing tubes, and every testing tube adds 1ml recovery dilution, and 3 Duplicate Samples are set equally.With pipettor to adding successively 0.05ml test bacterium liquid in each testing tube, vibration gently, make it fully to mix, be 15 seconds the interval time that each testing tube adds bacterium liquid, place 15min in educating warm groove after, within 15 seconds, measure the luminous intensity of each testing tube with bio-toxicity tester successively interval, be calculated as follows inhibiting rate, with EC
50(concentration value of this sample when inhibiting rate equals 50%) represents the toxicity size of each sample, EC
50be worth littlely, toxicity is larger.
2.6 methodological study
2.6.1 assay method factors influencing
2.6.1.1 the impact of time on luminous intensity
In testing tube, add recovery dilution and the test bacterium liquid of different volumes, (cumulative volume is 1.05ml to be prepared into the test sample of different initial luminous intensities, sample 1 is used bacterium liquid for 1.04ml recovery dilution adds 0.01ml to test, sample 2 is used bacterium liquid for 1.02ml recovery dilution adds 0.03ml to test, sample 3 is used bacterium liquid for 1.00ml recovery dilution adds 0.05ml to test, sample 4 is used bacterium liquid for 0.95ml recovery dilution adds 0.1ml to test, sample 5 is used bacterium liquid for 0.85ml recovery dilution adds 0.2ml to test), vibration gently, make it fully to mix, every group of sample does 3 Duplicate Samples.From mixing beginning timing, within every 1 minute, measure 1 luminous intensity values, calculate the mean value of 3 Duplicate Samples, relatively the impact of time on the initial luminous intensity of difference.
2.6.1.2pH the impact of value on luminous intensity
Get recovery dilution and add NaOH or HCl, being made into respectively pH is 3,4,5,6,7,8,9,10,11 recovery dilution.Get respectively each pH value recovery dilution 1ml (every group is done 3 Duplicate Samples) in testing tube, in each testing tube, add successively 0.05ml test bacterium liquid, vibration gently, make it fully to mix, respectively at placing 5min, 10min, measures luminous intensity values with bio-toxicity tester after 15min, the relatively impact of pH value on luminous intensity.
2.6.2 precision is investigated
2.6.2.1 repeatability is investigated
By above-mentioned definite method to 3 batches of injection samples test, every batch sample repeat 3 times test, result is evaluated.
2.6.2.2 middle precision is investigated
(1) different personnel's tests
By above-mentioned definite method, same batch of injection sample tested on same working day by two staff, result is evaluated.
(2) different operating day test
By above-mentioned definite method, same batch of injection sample tested in different operating day by same staff, result is evaluated.
3 results
3.1 assay method factors influencing
3.1.1 the impact of time on luminous intensity
Experimental result is as shown in table 1 and Fig. 1:
The impact of table 1 time on the initial luminous intensity of difference
From table 1 and Fig. 1～5, the prolongation in time of the luminous intensity values of 5 samples is reduction trend, is maintained at a metastable level in 5～30min, and initial luminous intensity values is higher, and luminous intensity reduced rate is lower.Meanwhile, experiment find, when 15min luminous intensity is 500-1000 (sample 3), testing result is more accurate, cost is also handed over low, therefore, test with the addition of bacterium liquid be preferably bacteria liquid amass 1/20.
3.1.2pH be worth the impact on luminous intensity
Experimental result is as shown in table 2:
The impact of table 2pH value on luminous intensity
From table 2, when detection, pH impact on luminous intensity between 5.0-9.0 is less, and inhibiting rate is within ± 10%, and while therefore using detection method of the present invention 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 injection 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 middle 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 test
Table 6 different operating day test findings
From table 5 and table 6, the relative deviation <15% of the middle precision test of the inventive method, the repeatability of instructions the inventive method is good, and accuracy is high.
Description of test, the inventive method can effectively detect the toxicity of parenteral solution, favorable repeatability, repeatability is good, and accuracy is high.
Embodiment 2 use the inventive method detect the Sofflower injection of different manufacturers different batches
1. experiment material
With embodiment 1.
Sofflower injection: 10 batches of producer's first;
Sofflower injection: 10 batches of producer's second.
2. experimental technique
The each 10 batches of Sofflower injection finished products of producer's first and producer's second, through traditional technique in measuring, are defined as specification product.
Adopt the method for embodiment 1, producer's first and the each 10 batches of Sofflower injections of producer's second are carried out to luminous intensity mensuration, calculate inhibiting rate, use EC
50the comprehensive toxicity size of more each sample.
The preparation of bacterium liquid for 2.1 tests
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 are measured
With embodiment 1.
3 results
3.1 10 batches of producer's first Sofflower injection comprehensive toxicities are tested in table 7.
10 crowdes of Sofflower injection sample EC of table 7 producer first
50value (%)
Note: with the second 10 batch sample comparisons of Sofflower injection producer, * p<0.05; * p<0.01.
3.2 10 batches of producer's second Sofflower injection comprehensive toxicities are tested in table 8.
10 crowdes of Sofflower injection sample EC of table 8 producer second
50value (%)
From table 7 and table 8, the first Sofflower injection EC50 of producer value (dilutability when luminous intensity inhibition ratio is 50%) is starkly lower than producer's second Sofflower injection, and has significant difference.
Description of test, for all qualified Sofflower injections of traditional technique in measuring quality, side of the present invention can measure its toxicity and vary, can the existing Adverse Drug Reaction of Safflower phenomenon of well explain.
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, can detect the toxicity of Sofflower injection, and for the quality control of Sofflower injection provides foundation, application prospect is good.
Claims (10)
1. a biological test method for fast detecting Sofflower injection comprehensive toxicity, is characterized in that: comprise the steps:
(1) bacterium liquid is used in preparation test: get photobacteria freeze-dried powder, the solution that is 0.1-1.0mol/L with chlorine ion concentration recovery, must test and use bacterium liquid;
(2) detect: get Sofflower injection sample to be checked, with the detection of test bacterium liquid, determine that dilutability-effect dynamic curve and luminous intensity inhibition ratio are 50% dilutability.
2. method of testing according to claim 1, is characterized in that: in step (1), described photobacteria is Fei Shi vibrios, photobacterium phosphoreum, Qinghai Vibrion and other non-pathogenic photobacteria.
3. method of testing according to claim 1, it is characterized in that: in step (1), describedly prepare test and by the method for bacterium liquid be: get model and be 1 of the Fei Shi vibrios freeze-dried powder of CS234, the solution that the chlorine ion concentration that adds 0.2-1.0ml is 0.51mol/L, must test and use bacterium liquid.
4. method of testing according to claim 1, is characterized in that: in step (1), the pH of the solution of described chloride ion-containing is 5～9.
5. method of testing according to claim 1, is characterized in that: the method for the described detection of step (2) is as follows:
A, pretest: get Sofflower injection sample to be checked, 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%, solution take chlorine ion concentration as 0.1-1.0mol/L is titer, in described liquid to be checked and titer, adding test to 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 and detect the dilution upper limit and lower limit: according to the result of step a, the arbitrary dilutability take inhibiting rate as 90～100% is as the upper limit, if the inhibiting rate of sample stoste does not reach 90%, take sample stoste as the upper limit; Arbitrary dilutability take inhibiting rate as 0～10% is as lower limit;
C, test: between the definite dilutability upper and lower bound of step b, increase and join 6～9 evenly liquid to be checked of dilution gradient, solution take chlorine ion concentration as 0.1-1.0mol/L is titer, in described liquid to be checked and titer, add test 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, the dilutability that calculating inhibiting rate is 50%.
6. method of testing according to claim 5, is characterized in that: in step a and step c, be added with the solution of chloride ion-containing in described liquid to be checked, the concentration of chlorion is 0.1-1.0mol/L; In described titer, chlorine ion concentration is 0.51mol/L.
7. method of testing according to claim 5, is characterized in that: in step a and step c, the pH of described liquid to be checked and sodium chloride solution is 5～9.
8. method of testing according to claim 5, is characterized in that: in step a and step c, 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, be 15min described standing time.
10. according to the method for testing described in claim 1～9 any one, it is characterized in that: detect luminous intensity and adopt bio-toxicity tester or other to there is the instrument of luminous intensity measuring ability.
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Address after: 610000 Chengdu South Road, Sichuan, No. 4, No. 51 Patentee after: Sichuan Academy of Chinese Medicine Sciences Patentee after: Huarun 39 (Ya'an) Pharmaceutical Co., Ltd. Address before: 610000 Chengdu South Road, Sichuan, No. 4, No. 51 Patentee before: Sichuan Academy of Chinese Medicine Sciences Patentee before: Sanjiu Pharmaceutical Industry Co., Ltd., Ya'an City |