CN104215479A - Biology test method for rapidly detecting comprehensive toxicity of traditional Chinese medicine injection - Google Patents

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

A biological test method for rapid detection of comprehensive toxicity of traditional Chinese medicine injections

technical field

The invention relates to a biological testing method for rapidly detecting the comprehensive toxicity of traditional Chinese medicine injections.

Background technique

Traditional Chinese medicine injections have been used in my country for more than 70 years. They have remarkable curative effects on diseases such as cardiovascular and cerebrovascular diseases, anti-tumor diseases, and respiratory system. They have unique advantages and are widely used clinically. In recent years, as the clinical application of traditional Chinese medicine injections has become more and more extensive, the reports of adverse reactions have also increased. From the "puerarin incident" and "Houttuynia cordata incident" in 2006 to the "Acanthopanax incident" and "Yinzhihuang incident" in 2008, to the "Shuanghuanglian incident" and "Qingkailing incident" in 2009 , causing the entire Chinese medicine injection industry to fall into a crisis of confidence. After 2006, the state's attention to the safety of traditional Chinese medicine injections has reached an unprecedented height. In July 2009, the State Food and Drug Administration issued the "Notice on Doing a Good Job of Safety Re-evaluation of Traditional Chinese Medicine Injections" to comprehensively carry out risk investigations in the production and quality control links to effectively control the safety hazards of traditional Chinese medicine injections.

At present, the quality control testing items of traditional Chinese medicine injections include: traits, identification, pH value, heavy metals, burning residues, related substances (proteins, tannins, resins, oxalates, potassium ions), pyrogens or bacterial endotoxins, Sterility, loading, insoluble particles, visible foreign matter, content determination, antihypertensive substances, abnormal toxicity, allergic reaction examination, hemolysis and coagulation, osmotic pressure, harmful elements, polysorbate 80, 5-HMF, fingerprints, macromolecules Protein check, etc. However, these detection indicators still cannot effectively ensure the consistency of its drug effect and reduce the incidence of allergic reactions. Therefore, it is necessary to introduce new quality control methods.

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 traditional Chinese medicine injections.

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

(1) Preparation of bacterial solution for testing: Take the freeze-dried powder of luminescent bacteria and resuscitate it with a sodium chloride solution with a concentration of 1-5% (w/v) to obtain the bacterial solution for testing;

(2) Detection: Take the sample to be tested and test it with the test bacterial solution to determine the dilution-effect kinetic curve and the dilution rate at which the luminous intensity inhibition rate is 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 race T3, Vibrio qinghai and other non-pathogenic luminescent bacteria.

In step (1), the preparation method is: take 1 Vibrio fischeri freeze-dried powder of model CS234, add 0.2-1.0ml of sodium chloride solution with a concentration of 3% (w/v) to recover, that is Bacteria for testing.

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

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

a. Pre-test: Take the sample to be tested and dilute it with water into 5 gradients of the solution to be tested. The volume percentages are: 100%, 25%, 6.25%, 1.5625%, 0.39%, with 1~5% (w/v ) sodium chloride solution as 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, and place it for 5 to 10 minutes. 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 90-100% 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 1 to 5% (w/v) sodium chloride solution as the standard solution. Add test bacteria solution to the test solution and standard solution, the amount of bacteria solution added is 1/25 to 1/15 of the volume of the test solution or standard solution, leave it for 5 to 30 minutes, detect the luminous intensity, and make the dilution-effect Kinetic curve, calculation of inhibition rate of 50% dilution.

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

If the value of the dilution degree is small, it means that the toxicity of the Houttuynia cordata injection to be tested is high, and if the value of the dilution degree is large, it means that the toxicity of the Houttuynia cordata injection to be tested is low.

The dilution at which the inhibition rate is 50% is the EC ₅₀ of the present invention.

In step a and step c, sodium chloride is added to the test solution at a concentration of 3% (w/v); the concentration of sodium chloride in the standard solution is 3% (w/v).

In step a and step c, the pH of the test solution and the standard 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 is detected by a biotoxicity tester modeled as LUMIStox300.

The method of the present invention can effectively detect the toxicity of traditional Chinese medicine 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 its comprehensive toxicity can be evaluated ; (2) Simple operation: no need for professional techniques such as gavage, intravenous injection, etc., simple operation, convenient and easy; (3) Sensitive response; using modern sensitive photoelectric detection technology, it can detect extremely weak light intensity changes, compared with General biological cells are sensitive to several orders of magnitude; (4) can judge the magnitude of comprehensive toxicity; (5) the large sample size of bacteria overcomes the influence of small sample number 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 Fig. 6 Houttuynia cordata injection sample to Vibrio fischeri

The concentration-effect kinetic curve of Fig. 7 Houttuynia cordata injection sample to Vibrio fischeri

The concentration-effect kinetic curve of Fig. 8 safflower injection sample A to Vibrio fischeri

The concentration-effect kinetic curve of Fig. 9 safflower injection sample A to Vibrio fischeri

The concentration-effect kinetic curve of Fig. 10 safflower injection sample B to Vibrio fischeri

The concentration-effect kinetic curve of Fig. 11 safflower injection sample B to Vibrio fischeri

Detailed ways

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

Resuscitation diluent: 3% sodium chloride solution;

Osmotic adjustment solution: 20% sodium chloride solution;

Traditional Chinese medicine injection: Houttuynia cordata 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 according to 1:4 into 5 concentration 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 The method is roughly tested once 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 sample with distilled water to each concentration, 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 NaCl concentration of the sample solution to be tested is 3%.

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

Three batches of Chinese medicine injection samples (KZ-110501, KZ-110502, KZ-110503) were tested according to the method determined above, and the test was repeated 3 times for each batch of samples, and the results were evaluated.

2.6.2.2 Intermediate precision inspection

(1) Experiments with different personnel

According to the method determined above, two staff members tested the same batch of traditional Chinese medicine injection samples (KZ-110503) on the same working day, and evaluated the results.

(2) Tests on different working days

According to the method determined above, the same batch of traditional Chinese medicine injection samples (KZ-110503) were tested by the same staff on different working days, and the results were 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 traditional Chinese medicine 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 traditional Chinese medicine injection, and has good repeatability, good reproducibility and high accuracy.

Embodiment 2 application of the present invention carries out comprehensive toxicity test to two kinds of Chinese medicine injections

1 Experimental materials

With embodiment 1.

Traditional Chinese medicine injection: Houttuynia cordata injection, safflower injection, commercially available.

1.3 Main instruments and equipment

With embodiment 1.

2 Experimental methods

Both Houttuynia cordata injection and safflower injection were tested by traditional methods and were determined to be qualified products.

Using the method of Example 1, the luminous intensity of Houttuynia cordata injection and safflower injection was measured, 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 Comprehensive Toxicity Test of Houttuynia Injection

Table 7 Comprehensive toxicity results of Houttuynia cordata injection samples

It can be seen from Table 7 and Figures 6-7 that the EC ₅₀ (dilution when the luminescence intensity inhibition rate is 50%) of Houttuynia cordata injection sample A is lower than the EC ₅₀ of Houttuynia cordata injection sample B, indicating that the former The toxicity of is greater than that of the latter.

3.2 Comprehensive toxicity test of safflower injection

Table 8 Comprehensive toxicity results of safflower injection samples

It can be seen from Table 8 and Figures 8-11 that the EC ₅₀ (dilution when the luminous intensity inhibition rate is 50%) of safflower injection sample A is lower than the EC ₅₀ of safflower injection sample B, indicating the toxicity of the former more toxic than the latter.

Experiments show that, for traditional Chinese medicine injections whose quality is qualified by traditional methods, the invention can detect whether the toxicity is large or small, and can better explain the inconsistent phenomenon of adverse reactions of existing Chinese medicine injections.

In summary, the toxicity detection method of the present invention has fast detection speed, simple operation, sensitive response and high accuracy, can detect the toxicity of traditional Chinese medicine injections, provides a basis for quality control of traditional Chinese medicine injections, and has a good application prospect.

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.