CN103868915B - A kind of biological test method of quick detection Flos Carthami injection comprehensive toxicity - Google Patents

Abstract

The invention discloses a biological test method for rapidly detecting the comprehensive toxicity of safflower injection, which comprises the following steps: (1) preparing a test bacterial liquid: taking luminescent bacteria freeze-dried powder, and using a chloride ion concentration of 0.1-1.0mol/L (2) Detection: Take the sample of safflower injection to be tested and use the test bacterial solution to detect the dilution of the sample with a luminous intensity inhibition rate of 50% and the dilution-effect kinetic curve . The detection accuracy and sensitivity of the method of the invention are high, and the operation is simple.

Description

A biological test method for rapid detection of comprehensive toxicity of safflower injection

technical field

The invention relates to a biological testing method for rapidly detecting the comprehensive toxicity of safflower injection.

Background technique

Safflower injection is refined from the single drug safflower "water extraction and alcohol precipitation". The main component is safflower yellow pigment, which has the effects of promoting blood circulation, dredging menstruation, dispelling blood stasis and relieving pain. It is widely used clinically for cardiovascular and cerebrovascular diseases , amenorrhea and other diseases, with expansion of coronary arteries, myocardial protection, anti-oxidation and other pharmacological effects. While the clinical application of safflower injection is becoming more and more extensive, the reports of its adverse reactions are gradually increasing. The analysis of 32 cases of adverse reactions of safflower injection reported in the literature from 1994 to 2005 found that there was no gender and age difference in the occurrence of adverse reactions, and most of the adverse reactions occurred within 5 to 30 minutes after administration (accounting for more than 50%). Mainly allergic reactions (accounting for more than 50%), severe cases anaphylactic shock.

At present, safflower injection is tested for quality control according to pharmacopoeia standards, but these test indicators are still unable to effectively ensure the consistency of its efficacy and reduce the incidence of allergic reactions. Therefore, it is necessary to introduce a new quality control method with higher sensitivity to monitor the occurrence of abnormal conditions in production at any time.

Contents of the invention

In order to solve the above problems, the present invention provides a biological testing method for rapidly detecting the comprehensive toxicity of safflower injection.

The present invention rapidly detects the biological testing method of safflower injection comprehensive toxicity, comprises the following steps:

(1) Preparation of bacterial solution for testing: Take the freeze-dried powder of luminescent bacteria and resuscitate with a solution with a chloride ion concentration of 0.1-1.0mol/L to obtain the bacterial solution for testing;

(2) Detection: Take the sample of safflower injection to be tested and test it with the test bacterial solution to determine the dilution-effect kinetic curve and the dilution with a luminous intensity inhibition rate of 50%.

The dilution-effect kinetic curve refers to the relationship curve between the dilution of the solution to be tested and the inhibition rate of luminous intensity.

In step (1), the luminescent bacteria are Vibrio fischeri, Photobacterium luminescens, Vibrio qinghai and other non-pathogenic luminescent bacteria.

In step (1), the method for preparing the bacterial liquid for testing is: take 1 stick of Vibrio fischeri freeze-dried powder CS234, add 0.2-1.0ml of a solution with a chloride ion concentration of 0.51mol/L, that is Bacteria for testing.

In step (1), the pH of the solution containing chloride ions is 5-9.

The detection method described in step (2) is as follows:

a. Pre-test: Take the sample of safflower injection to be tested, and dilute it with water into 5 gradients of the solution to be tested. The volume percentages are respectively: 100%, 25%, 6.25%, 1.5625%, and 0.39%. The chloride ion concentration is The solution of 0.1-1.0mol/L is the standard solution. Add the test bacteria solution to the test solution and the standard solution. The amount of the bacteria solution added is 1/25 to 1/15 of the volume of the test solution or the standard solution 5 to 30 minutes, detect the luminous intensity, and calculate the luminous inhibition rate of 5 dilutions;

b. Determine the upper limit and lower limit of the detection dilution: according to the results of step a, take any dilution with an inhibition rate of 90-100% as the upper limit, and if the inhibition rate of the sample stock solution does not reach 90%, use the sample stock solution as the upper limit ; Take any dilution with an inhibition rate of 0 to 10% as the lower limit;

c. Test: add 6 to 9 uniform dilution gradients of the solution to be tested between the upper and lower limits of the dilution determined in step b, and use the solution with a chloride ion concentration of 0.1-1.0mol/L as the standard solution. Add test bacterial liquid to the liquid to be tested and the standard liquid, the amount of bacterial liquid added is 1/25 to 1/15 of the volume of the liquid to be tested or the standard liquid, leave it for 5 to 30 minutes, detect the luminous intensity, and make the dilution-effect kinetic curve , to calculate the dilution with an inhibition rate of 50%.

The dilution degree of the present invention refers to the degree to which the solution to be tested is diluted. For example, 1ml of safflower injection is diluted with 3ml of water, and the dilution is 25%.

If the value of the dilution is small, it means that the safflower injection to be tested is highly toxic, and if the value of the dilution is large, it means that the safflower injection to be tested is less toxic.

The 50% dilution of the inhibition rate is the EC50 of the present invention.

In step a and step c, a solution containing chloride ions is added to the liquid to be tested, and the concentration of chloride ions is 0.1-1.0 mol/L; the concentration of chloride ions in the standard solution is 0.51 mol/L.

In step a and step c, the pH of the test solution and the sodium chloride solution is 5-9.

In step a and step c, the added amount of the bacterial solution is 1/20 of the volume of the test solution or standard solution.

In step a and step c, the standing time is 15min.

In the aforementioned detection method, the luminous intensity adopts a biotoxicity tester or other instruments with a luminous intensity detection function.

The method of the present invention can effectively detect the toxicity of safflower injection, and provide a reliable basis for its clinical application. At the same time, the detection method of the present invention has the following advantages: (1) The detection speed is fast: the result can be obtained within 1 hour, and the comprehensive evaluation of its Toxicity; (2) Simple operation: no need for gavage, intravenous injection and other professional techniques, simple operation, convenient and easy; (3) Sensitive response; using modern sensitive photoelectric detection technology, it can detect extremely weak light intensity changes, It is several orders of magnitude more sensitive than ordinary biological cells; (4) It can judge the magnitude of comprehensive toxicity; (5) The large amount of bacterial samples overcomes the influence of small sample numbers and individual differences in animal experiments.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 Concentration 1 Vibrio fischeri luminous intensity change with time

Figure 2 Concentration 2 Vibrio fischeri luminous intensity changes with time

Figure 3 Concentration 3 Vibrio fischeri luminous intensity changes with time

Fig. 4 Concentration 4 Vibrio fischeri luminous intensity changing with time

Fig. 5 Concentration 5 Vibrio fischeri luminous intensity change diagram with time

The concentration-effect kinetic curve of 10 batches of safflower injection samples of Fig. 6 manufacturer A to Vibrio fischeri

The concentration-effect kinetic curve of 10 batches of safflower injection samples of Fig. 7 manufacturer B on Vibrio fischeri

Embodiment 1 toxicity detection method of the present invention

1 Experimental materials

1.1 Strains

Freeze-dried powder of Vibrio fischeri (CS234) was purchased from Beijing Hamamatsu Photon Technology Co., Ltd., and stored at -20°C in the dark.

1.2 Main reagents

Recovery diluent: 0.51mol/L sodium chloride solution;

Osmotic adjustment solution: 3.42mol/L sodium chloride solution;

Chinese medicine injection: commercially available

1.3 Main instruments and equipment

LUMIStox300 biological toxicity tester and supporting LUMIStherm preheating tank and test tube (Dr.Bruno Lange GmbH), BCD-539WF refrigerator (Haier), PB-10 acidity meter (Sartorius)

2 Experimental methods

2.1 Preparation of bacteria solution for test

Take out 1 bottle of Vibrio fischeri freeze-dried powder from the freezer (-20°C) and place it at room temperature (about 20°C) for 15 minutes to equilibrate, then add 0.2-1.0ml recovery diluent and place it at room temperature for 10 minutes, then it can be used test.

2.2 Pre-test

Dilute the traditional Chinese medicine injection with distilled water at 1:4 into 5 gradients (i.e. dilution gradient): 100%, 25%, 6.25%, 1.5625%, 0.39%, prepare the sample solution to be tested according to 2.3, and prepare according to 2.5 Make a rough test with the above method to determine the concentration (ie dilution) range of the upper limit and the lower limit. The upper limit is the concentration of the sample when the inhibition rate reaches 90-100%. If the inhibition rate of the sample stock solution does not reach 90%, the sample stock solution is taken as the upper limit; the lower limit is the concentration of the sample when the inhibition rate reaches 0-10%.

2.3 Preparation of sample solution to be tested

Between the upper limit and the lower limit, add 6 to 12 concentrations as needed. Dilute the samples to various concentrations with distilled water, and then mix the samples of each concentration with the osmotic pressure adjustment solution at a ratio of 17:3 to prepare the sample solution to be tested, so that the chloride ion concentration of the sample solution to be tested is 0.51mol/L.

2.4 Preparation of blank control solution (standard solution)

The recovery diluent was used as the blank control solution.

2.5 Measurement of Luminous Intensity

Prepare 3 test tubes for each concentration of the sample to be tested, add 1ml of the sample to be tested in each test tube, and set up 3 parallel samples; take 3 test tubes for the blank control solution, add 1ml of recovery diluent to each test tube, and set the same 3 parallel samples. Use a pipette to add 0.05ml test bacteria solution to each test tube in turn, shake gently to make it fully mixed, the interval between adding bacteria solution to each test tube is 15 seconds, and place it in the incubation tank for 15 minutes Use a biological toxicity tester to measure the luminous intensity of each test tube at intervals of 15 seconds, calculate the inhibition rate according to the following formula, and express the toxicity of each sample with EC ₅₀ (the concentration value of the sample when the inhibition rate is equal to 50%), and the EC ₅₀ value The smaller it is, the more toxic it is.

2.6 Methodological investigation

2.6.1 Investigation of influencing factors of determination method

2.6.1.1 Effect of time on luminous intensity

Add different volumes of resuscitation diluent and test bacterial solution to the test tube to prepare test samples with different initial luminous intensities (total volume is 1.05ml, sample 1 is 1.04ml resuscitative diluent plus 0.01ml test bacterial solution, sample 2 Add 0.03ml test bacteria solution to 1.02ml resuscitation diluent, add 0.05ml test bacteria solution to 1.00ml resuscitation diluent for sample 3, add 0.1ml test bacteria solution to 0.95ml resuscitation diluent, sample 5 is 0.85 Add 0.2ml test bacterial solution to 0.2ml resuscitation diluent), shake gently to make it fully mixed, and make 3 parallel samples for each group of samples. Start timing from mixing, measure the luminous intensity value every 1 minute, calculate the average value of 3 parallel samples, and compare the influence of time on different initial luminous intensities.

2.6.1.2 Effect of pH value on luminous intensity

Take the recovery diluent and add NaOH or HCl to prepare the recovery diluents with pHs of 3, 4, 5, 6, 7, 8, 9, 10, and 11, respectively. Take 1ml of recovery diluent for each pH value in the test tube (3 parallel samples for each group), add 0.05ml test bacteria solution to each test tube in turn, shake gently to make it fully mixed, and place in After 5 min, 10 min, and 15 min, measure the luminous intensity value with a biotoxicity tester, and compare the influence of pH value on the luminous intensity.

2.6.2 Inspection of precision

2.6.2.1 Repeatability inspection

Test 3 batches of injection samples according to the method determined above, repeat the test 3 times for each batch of samples, and evaluate the results.

2.6.2.2 Intermediate precision inspection

(1) Experiments with different personnel

According to the method determined above, two staff members test the same batch of injection samples on the same working day, and evaluate the results.

(2) Tests on different working days

According to the method determined above, the same batch of injection samples shall be tested by the same staff member on different working days, and the results shall be evaluated.

3 results

3.1 Investigation of influencing factors of determination method

3.1.1 Effect of time on luminous intensity

The experimental results are shown in Table 1 and Figure 1:

Table 1 Effect of time on different initial luminous intensities

It can be seen from Table 1 and Figures 1 to 5 that the luminous intensity values of the five samples showed a decreasing trend with the prolongation of time, and remained at a relatively stable level within 5 to 30 minutes. Low. At the same time, the experiment found that when the luminous intensity in 15 minutes is 500-1000 (sample 3), the detection result is more accurate and the cost is also low. Therefore, the amount of bacterial liquid used for testing is preferably 1/20 of the volume of the bacterial liquid.

3.1.2 Effect of pH value on luminous intensity

The experimental results are shown in Table 2:

The influence of table 2pH value on luminous intensity

It can be seen from Table 2 that during detection, the pH value of the solution between 5.0-9.0 has little influence on the luminous intensity, and the inhibition rate is within ±10%. Therefore, when the detection method of the present invention is used for detection, the pH value of the solution is preferably 5.0-9.0 .

3.2 Inspection of precision

3.2.1 Repeatability test

The experimental results are shown in Table 3 and Table 4:

Table 3 Repeatability test results

Table 43 batches of injection samples EC ₅₀ value (%)

As seen from Table 3 and Table 4, the relative deviation<15% of the repeatability test of the inventive method shows that the inventive method has high accuracy and good repeatability.

3.2.2 Intermediate precision inspection

3.2.2.1 Different staff experiments

Table 5 Test results of different workers

3.2.2.2 Tests on different working days

Table 6 Test results on different working days

As seen from Table 5 and Table 6, the relative deviation<15% of the intermediate precision test of the inventive method shows that the reproducibility of the inventive method is good and the accuracy is high.

Experiments show that the method of the invention can effectively detect the toxicity of the injection, and has good repeatability, good reproducibility and high accuracy.

Embodiment 2 detects the safflower injection of different manufacturers' different batches with the inventive method

1. Experimental materials

With embodiment 1.

Safflower injection: 10 batches of manufacturer A;

Safflower injection: 10 batches from manufacturer B.

2. Experimental method

The 10 batches of safflower injection finished products of manufacturer A and manufacturer B were determined to be qualified products by traditional methods.

Using the method of Example 1, each 10 batches of safflower injections from manufacturer A and manufacturer B were tested for luminous intensity, the inhibition rate was calculated, and the comprehensive toxicity of each sample was compared with _EC50 .

2.1 Preparation of test bacterial solution

With embodiment 1.

2.2 Pre-test

With embodiment 1.

2.3 Preparation of sample solution to be tested

With embodiment 1.

2.4 Preparation of blank control solution

With embodiment 1.

2.5 Measurement of Luminous Intensity

With embodiment 1.

3 results

3.1 The comprehensive toxicity test of 10 batches of safflower injection from manufacturer A is shown in Table 7.

Table 7 EC ₅₀ values of 10 batches of safflower injection samples from manufacturer A (%)

Note: *p<0.05; **p<0.01 compared with 10 batches of samples from Safflower Injection Manufacturer B.

3.2 The comprehensive toxicity test of 10 batches of safflower injection from manufacturer B is shown in Table 8.

Table 8 EC ₅₀ values of 10 batches of safflower injection samples from manufacturer B (%)

It can be seen from Table 7 and Table 8 that the EC50 value (the dilution rate when the luminous intensity inhibition rate is 50%) of manufacturer A safflower injection is significantly lower than that of manufacturer B safflower injection, and there is a significant difference.

The experiment shows that for the safflower injection whose quality is qualified by the traditional method, the invention can detect whether the toxicity is large or small, and can better explain the adverse reactions of the existing safflower injection.

To sum up, the toxicity detection method of the present invention has fast detection speed, simple operation, sensitive response and high accuracy, can detect the toxicity of safflower injection, provides a basis for quality control of safflower injection, and has a good application prospect.

Claims (8)

1. a biological test method for rapid detection of safflower injection comprehensive toxicity, is characterized in that: comprise the steps: (1) Preparation of bacterial liquid for testing: take luminescent bacteria freeze-dried powder, resuscitate with a solution having a chloride ion concentration of 0.1-1.0 mol/L, and obtain bacterial liquid for testing; (2) Detection: take the safflower injection sample to be tested, detect with the bacterial liquid for testing, determine the dilution-effect kinetic curve and the dilution rate of 50% of the luminous intensity inhibition rate; In step (1), the pH of the solution containing chloride ions is 5-9; The method of detection described in step (2) is as follows: a, pre-test: take the safflower injection sample to be tested, dilute it with water into 5 gradients of the solution to be tested, the volume percentages are respectively: 100%, 25%, 6.25%, 1.5625%, 0.39%, with the chloride ion concentration as The solution of 0.51mol/L is the standard solution, and the test bacteria solution is added to the test solution and the standard solution. 30min, detect the luminous intensity, and calculate the luminous inhibition rate of 5 dilutions; b. Determine the upper limit and lower limit of the detection dilution: according to the results of step a, take any dilution with an inhibition rate of 90 to 100% as the upper limit, and if the inhibition rate of the sample stock solution does not reach 90%, take the sample stock solution as the upper limit ; Any dilution with an inhibition rate of 0 to 10% is the lower limit; c. Test: add 6 to 9 uniform dilution gradients of the solution to be tested between the upper and lower limits of the dilution determined in step b, and use a solution with a chloride ion concentration of 0.51mol/L as the standard solution. Add the bacteria solution for testing to the solution and the standard solution, the amount of the bacteria solution added is 1/25~1/15 of the volume of the solution to be tested or the standard solution, let it stand for 5~30min, detect the luminous intensity, make the dilution-effect kinetic curve, and calculate 50% inhibition rate dilution; In step a and step c, a solution containing chloride ions is added to the test solution, and the final concentration of chloride ions in the test solution is 0.51mol/L. 2. The test method according to claim 1, characterized in that: in step (1), the luminescent bacteria are non-pathogenic luminescent bacteria. 3. The test method according to claim 2, characterized in that: the non-pathogenic luminescent bacteria are Vibrio fischeri, Photobacterium luminescens, and Vibrio qinghai. 4. test method according to claim 1, is characterized in that: in step (1), the method for described preparation test bacterium liquid is: get model is 1 Vibrio fischeri freeze-dried powder of CS234, add 0.2 -1.0ml of a solution with a chloride ion concentration of 0.51mol/L to obtain the bacterial solution for testing. 5. The test method according to claim 1, characterized in that: in step a and step c, the pH of the test solution and the standard solution is 5-9. 6. The test method according to claim 1, characterized in that: in step a and step c, the added amount of the bacterial solution is 1/20 of the volume of the solution to be tested or the standard solution. 7. The test method according to claim 1, characterized in that: in step a and step c, the standing time is 15min. 8 . The test method according to any one of claims 1 to 7 , characterized in that: a biotoxicity tester model of LUMIStox 300 is used to detect the luminous intensity.