CN103868916B - Biological test method for rapidly detecting comprehensive toxicity of traditional Chinese medicine - Google Patents

Abstract

The invention discloses a biological testing method for rapidly detecting the comprehensive toxicity of traditional Chinese medicines, which comprises the following steps: (1) preparing the bacterial liquid for testing: taking the freeze-dried powder of luminescent bacteria and resuscitating it with a solution with a chloride ion concentration of 0.1-1.0 mol/L , to obtain the test bacterial solution; (2) Detection: Take the sample of the traditional Chinese medicine to be tested, and use the test bacterial solution to detect, and determine the sample concentration with a luminous intensity inhibition rate of 50% and the concentration-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 traditional Chinese medicine

technical field

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

Background technique

Traditional Chinese medicine is a treasure of our country, and it has played a huge role in disease prevention, treatment and health care. With the expansion of the application range of traditional Chinese medicine and its preparations, clinical reports of adverse reactions caused by it are also increasing, and there are reports of fatalities. The incidence of adverse reactions of traditional Chinese medicine is second only to anti-infective drugs in chemical medicines, and there is an upward trend in recent years. . Guan Mu Tong, Guang Fang Ji, Qing Mu Xiang and other aristolochic acid drugs caused severe kidney damage, the tonic Chinese medicine Polygonum multiflorum caused severe liver damage, and the introduction of Chinese medicine injections such as Shuanghuanglian and Qingkailing into anaphylactic shock caused widespread concern about the safety of Chinese medicine .

The factors that cause adverse reactions of traditional Chinese medicine are closely related to medicinal materials, preparations, compatibility and improper use. In order to ensure the safety and effectiveness of traditional Chinese medicine, traditional Chinese medicine has been in the process of continuous standardization and normalization for thousands of years. It can be seen that improving the quality of traditional Chinese medicine is an urgent need to realize the modernization of traditional Chinese medicine. The establishment of the quality control system of traditional Chinese medicine must be based on the characteristics of traditional Chinese medicine, and the quality control method of traditional Chinese medicine must be able to control the effective components (organic components, inorganic components and complex components). Only in this way can the established quality control system truly achieve the purpose of controlling the quality of traditional Chinese medicines and ensuring the safety and effectiveness of traditional Chinese medicines.

At present, the quality control standards of traditional Chinese medicine that my country has formulated include: GLP, GMP, GCP, GSP, GAP, but these standards are far from being able to cover a series of links in the production of traditional Chinese medicine. As a quality control method of traditional Chinese medicine, fingerprinting has become the current international consensus, including chromatographic fingerprinting, spectral fingerprinting and DNA fingerprinting. However, these quality control standards and detection methods are still unable to effectively guarantee the consistency and safety of traditional Chinese medicine efficacy. 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 medicines.

The present invention rapidly detects the biological testing method of the comprehensive toxicity of traditional Chinese medicine, 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 traditional Chinese medicine to be tested, and use the test bacterial solution to detect it, and determine the concentration-effect kinetic curve and the concentration at which the luminous intensity inhibition rate is 50%.

The concentration-effect kinetic curve refers to the relationship curve between the concentration of the sample 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 Chinese medicine sample to be tested and dilute it with water into 5 concentrations of the solution to be tested. The volume percentages are: 100%, 25%, 6.25%, 1.5625%, and 0.39%. The concentration of chloride ion is 0.1-1.0 The mol/L solution 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, and it is placed for 5 to 30 minutes. , detect the luminous intensity, and calculate the luminous inhibition rate of 5 concentrations;

b. Determine the upper limit and lower limit of the detection concentration: according to the results of step a, take any concentration 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 concentration with an inhibition rate of 0 to 10% is the lower limit;

c. Test: Add 6 to 9 concentrations of the solution to be tested between the upper limit and the lower limit of the concentration determined in step b, and use a solution with a chloride ion concentration of 0.1-1.0mol/L as the standard solution in the solution to be tested Add the test bacteria solution to 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, leave it for 5~30min, measure the luminous intensity, make a concentration-effect kinetic curve, and calculate the inhibition rate 50% concentration.

The sample concentration at which the inhibition rate is 50% is the IC ₅₀ of the present invention.

If the _IC50 value is small, it means that the Chinese medicine to be tested is highly toxic, and if the _IC50 value is large, it means that the Chinese medicine to be tested is less toxic.

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 comprehensive toxicity of traditional Chinese medicines 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 toxicity can be evaluated; (2) Simple operation: No professional techniques such as gavage and intravenous injection are required, and the operation is simple and convenient; (3) Responsive: using modern and sensitive photoelectric detection technology, it can detect extremely weak light intensity changes, which is better than ordinary 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 size 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

Figure 6 Concentration-effect kinetic curves of 10 kinds of traditional Chinese medicines on luminescent bacteria

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;

1.3 Main instruments and equipment

LUMIStox300 biological toxicity tester and supporting LUMIStherm preheating tank and test tube (Dr.BrunoLange 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 sample with distilled water at a ratio of 1:4 to 5 concentrations: 100%, 25%, 6.25%, 1.5625%, and 0.39%, prepare the sample solution to be tested according to 2.3, and roughly test it once according to the method described in 2.5. Determine the upper and lower concentration ranges. 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 use IC ₅₀ (the concentration value of the sample when the inhibition rate is equal to 50%) to represent the toxicity of each sample, IC 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 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, the same batch of samples shall be tested by two staff members on the same working day, and the results shall be evaluated.

(2) Tests on different working days

According to the method determined above, the same batch of samples shall be tested by the same staff 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

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 comprehensive toxicity of traditional Chinese medicines, and has good repeatability, good reproducibility and high accuracy.

Embodiment 2 applies the present invention to carry out comprehensive toxicity test to 10 kinds of Chinese medicines

1. Experimental materials

With embodiment 1.

Test traditional Chinese medicines: raw tripterygium wilfordii, raw cocklebur, evodia, cnidium, vinegar kansui, vinegar pokeweed, Guanzhong, strychnium chinensis, araceae, and Chonglou, and comprehensive toxicity tests were carried out with water extracts.

2. Experimental method

Using the method of Example 1, the luminescence intensity of 10 kinds of traditional Chinese medicines was measured, the inhibition rate was calculated, and the comprehensive toxicity of each sample was compared with _IC50 .

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

The comprehensive toxicity test results of 10 traditional Chinese medicines are shown in Table 7.

Table 710 kinds of traditional Chinese medicine water extract sample IC ₅₀ value (%)

It can be seen from Table 7 that the IC ₅₀ values of different traditional Chinese medicines are quite different.

Experiments show that the inventive prescription can measure the toxicity of different traditional Chinese medicines, and can provide references for quality control and toxicity classification of traditional Chinese medicines.

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

Claims (8)

1. a biological testing method for rapidly detecting the comprehensive toxicity of Chinese medicines, is characterized in that: comprise the steps: (1) Preparation of bacterial solution for testing: take luminescent bacteria freeze-dried powder, resuscitate with a solution with a chloride ion concentration of 0.1-1.0 mol/L, and obtain bacterial solution for testing; (2) Detection: Take the sample of the traditional Chinese medicine to be tested, and use the test bacterial solution to detect it, and determine the concentration-effect kinetic curve and the concentration at which the luminous intensity inhibition rate is 50%; The traditional Chinese medicines to be tested are tripterygium wilfordii, cocklebur, evodia, cnidium, vinegar kansui, vinegar pokeweed, Guanzhong, nuxyche, araceae, and Chonglou; The detection method described in step (2) is as follows: a. Pre-test: Take the sample of traditional Chinese medicine to be tested and dilute it with water into 5 concentrations of the solution to be tested. The volume percentages are: 100%, 25%, 6.25%, 1.5625%, and 0.39%. The concentration of chloride ion is 0.51 mol/ The solution of 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 1/25~1/15 of the volume of the solution to be tested or the standard solution, and it is left for 5~30min. Luminous intensity, calculate the luminous intensity inhibition rate of 5 concentrations; b. Determine the upper limit and lower limit of the detection concentration: according to the results of step a, take any concentration 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 concentration with an inhibition rate of 0 to 10% is the lower limit; c. Test: Add 6 to 9 concentrations of the solution to be tested between the upper limit and the lower limit of the concentration determined in step b, and use a solution with a chloride ion concentration of 0.51mol/L as the standard solution. Add the test bacteria solution into the 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 a concentration-effect kinetic curve, and calculate the inhibition rate as 50 %concentration; In step a and step c, a solution containing chloride ions is added to the test solution, and the concentration of chloride ions is 0.1-1.0 mol/L. 2. The testing method according to claim 1, characterized in that: in step (1), the luminescent bacteria are Vibrio fischeri, Photobacterium luminescens or Vibrio qinghai. 3. The test method according to claim 1, characterized in that: 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 to obtain the bacterial solution for testing. 4. The test method according to claim 1, characterized in that: in step (1), the pH of the solution containing chloride ions is 5-9. 5. The test method according to claim 1, characterized in that: in step a and step c, the pH of the liquid to be tested and the standard liquid is 5 to 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 testing 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: the detection of luminous intensity adopts a biotoxicity tester modeled as LUMIStox 300 or other instruments with a luminous intensity detection function.