Method for measuring component content of venenum bufonis

Abstract

The invention provides a method for measuring the component content of venenum bufonis. The method comprises the following steps: 1) providing a test solution of fresh venenum bufonis pulp and a standard reference solution of bufalin, cinobufagin and resibufogenin; 2) developing the test sample solution and the standard reference substance solution by utilizing thin layer chromatography to obtain a test sample-standard reference substance thin layer chromatography plate; (3) carrying out color development treatment on the test article thin layer-standard reference substance thin layer chromatography plate to obtain a test article-standard reference substance thin layer chromatography color development plate; and 4) calculating the contents of bufalin, cinobufagin and resibufogenin in the test solution by using the gray value of the test-standard reference substance thin layer chromatography color plate. The method provided by the invention can be used for rapidly measuring the contents of bufalin, cinobufagin and resibufogenin in the venenum bufonis.

Description

Technical Field

[0001] The invention belongs to the technical field of toad venom content determination, and specifically relates to a method for measuring the content of toad venom components, and in particular to a thin layer chromatography method capable of quickly measuring the content of index components of toad venom fresh pulp. Background Art

[0002] Toad venom is the dried secretion of the parotid glands and skin glands of the Chinese giant toad (Bufo bufo gargarizans Cantor) or the black-rimmed toad (Bufo melanostictus Schneider), which are animals of the Toad family. It has a warm taste, is toxic, and enters the heart meridian. It has the effects of detoxification, analgesia, and awakening the mind. Modern pharmacological studies have shown that toad venom has anti-tumor, cardiotonic, analgesic, and anti-inflammatory pharmacological activities.

[0003] Toad venom is a traditional precious animal medicine in my country. It has limited natural resources and is widely used in clinical practice due to its good pharmacological activity, which makes the price of toad venom relatively expensive. This has led to adulteration of toad venom on the market, and the quality is uneven. The "Pharmacopoeia of the People's Republic of China" (2020 edition) uses three toad venom glycosides, ibuprofen, cinobucoids and resintoxins, as indicator components for toad venom quality control, requiring that their total amount must not be less than 7%. The existing method for measuring the content of ibuprofen, ibuprofen and resintoxins in toad venom is mainly chromatography, which uses large-scale liquid chromatographs for measurement, which is not only costly and slow, but also inconvenient to operate. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the present invention provides a method for measuring the content of toad venom components, and the technical problem to be solved is to quickly measure the content of bufalin, cinobufolin and resifobafugin in toad venom.

[0005] In order to solve the above problems, the technical solution adopted by the present invention is: a method for measuring the content of toad venom ingredients, the method comprising the following steps:

[0006] 1) Provide the test sample solution of fresh toad venom paste, as well as the standard reference solution of bufalin, cinobufolin and resifobafugin;

[0007] 2) developing the test sample solution and the standard reference solution by thin layer chromatography to obtain a test sample-standard reference thin layer chromatography plate;

[0008] 3) performing a color development treatment on the test sample-standard reference substance thin layer chromatography plate to obtain a test sample-standard reference substance thin layer chromatography color development plate;

[0009] 4) Calculate the contents of bufalin, cinobufogenin and resifobafugin in the test solution using the gray value of the test sample-standard reference thin layer chromatography color plate.

[0010] Preferably, in step 1), the preparation step of the test solution of fresh toad venom pulp comprises: drying the fresh toad venom pulp sample, dissolving it in methanol, ultrasonically extracting it, and taking the supernatant as the test solution.

[0011] Preferably, in step 1), the preparation step of the standard reference solution of bufalin, cinobufogenin and resifobufogenin comprises: dissolving bufalin, cinobufogenin and resifobufogenin in methanol, and diluting them to different concentrations.

[0012] Preferably, in step 2), the thin layer chromatography step comprises: spotting the test sample solution and the standard reference solution of each concentration on the same silica gel thin layer plate, a total of three silica gel thin layer plates, developing with a cyclohexane-chloroform-acetone mixture as a developing agent, taking out, and drying to obtain the test sample-standard reference thin layer chromatography plate.

[0013] Preferably, the volume ratio of the components of the cyclohexane-chloroform-acetone mixture is: cyclohexane:chloroform:acetone=4:3:3.

[0014] Preferably, in step 3), the color development step comprises: spraying a color developer onto the test sample-standard reference thin layer chromatography plate, and performing a heating treatment to obtain the test sample-standard reference thin layer chromatography color development plate; wherein the color developer is prepared by mixing ethanol, concentrated sulfuric acid and vanillin.

[0015] Preferably, the preparation step of the developer comprises: adding ethanol to water, mixing well, slowly adding concentrated sulfuric acid, and then adding vanillin after mixing well.

[0016] Preferably, in step 4), the calculation step includes: measuring the grayscale values ​​of each concentration point of the reference substance and the test sample point, making a standard curve with concentration as the horizontal axis and grayscale value as the vertical axis, and substituting the grayscale value of the test sample point into the calculated content.

[0017] Preferably, in step 4), the calculation step includes: taking a photo of the test sample-standard reference sample thin layer chromatography colorimetric plate, and measuring the grayscale values ​​of each concentration point of the reference sample and the test sample point using computer software.

[0018] Preferably, the computer software is Image J software.

[0019] The beneficial effects of the present invention are as follows: the method for measuring the content of toad venom components provided by the present invention has the advantages of being simple and rapid, does not require large-scale chromatographic instruments, and can achieve measurement conditions in outdoor environments, such as when purchasing toad venom. When purchasing fresh toad venom pulp, this method can be used to preliminarily determine whether the content of its index components meets the requirements of the pharmacopoeia.

[0020] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The thin layer spectrum and processed pictures of the fresh toad venom pulp sample and bufalin reference substance in Example 1 are shown.

[0022] Figure 2 The thin layer spectrum and processed pictures of the fresh toad venom pulp sample from a certain place in Shandong and the toad venom venom toxicity reference substance in Example 1.

[0023] Figure 3 The thin layer chromatography spectra and processed pictures of the fresh toad venom pulp sample from a certain place in Shandong and the resibufogenin reference substance in Example 1 are shown.

[0024] Figure 4 This is the HPLC spectrum of the 1 mg / mL bufalin reference substance in Example 1.

[0025] Figure 5 This is the HPLC spectrum of the 1 mg / mL cinona toad venom reference substance in Example 1.

[0026] Figure 6 This is the HPLC spectrum of the 1 mg / mL resibufogenin reference substance in Example 1.

[0027] Figure 7 This is the HPLC spectrum of the test solution of fresh toad venom paste from a certain place in Shandong in Example 1.

[0028] Figure 8 The thin layer chromatography spectra and processed pictures of the Shanghai Hengsha Toad Venom fresh paste sample and the bufalin reference substance in Example 2 are shown.

[0029] Fig. 9 The thin layer spectrum and processed pictures of the Shanghai Hengsha Toad Venom fresh paste sample and the Huachan Toad Venom toxicity reference substance in Example 2.

[0030] Fig.10 The thin layer chromatography spectra and processed pictures of the Shanghai Hengsha toad venom fresh paste sample and the resibufogenin reference substance in Example 2.

[0031] Fig.11 This is the HPLC spectrum of the Shanghai Hengsha Toad Venom fresh paste test solution in Example 2.

[0032] Fig.12 The thin layer spectrum and processed pictures of the fresh toad venom pulp sample and bufalin reference substance in Example 3 are shown.

[0033] Fig.13 The thin layer spectrum and processed pictures of the fresh toad venom pulp sample from a certain place in Jiangxi and the toad venom venom toxicity reference substance in Example 3.

[0034] Fig.14 The thin layer spectrum and processed pictures of the fresh toad venom pulp sample and the resibufogenin reference substance in Example 3 are shown.

[0035] Fig.15 This is the HPLC spectrum of the test solution of fresh toad venom paste from a certain place in Jiangxi in Example 3.

[0036] Fig.16 The thin layer spectrum and processed pictures of the fresh toad venom pulp sample and bufalin reference substance in Example 4 are shown.

[0037] Fig.17 The thin layer spectrum and processed pictures of the fresh toad venom pulp sample and the toad venom venom toxicity reference substance in Example 4 are shown.

[0038] Fig.18 The following are the thin layer chromatography spectra and processed pictures of the fresh toad venom pulp sample and the resibufogenin reference substance from a certain place in Sichuan in Example 4.

[0039] Fig.19 This is the HPLC spectrum of the test solution of fresh toad venom paste from a certain place in Sichuan in Example 4. DETAILED DESCRIPTION

[0040] The following examples are used to illustrate the specific implementation of the present invention, but they cannot constitute any limitation on the scope of the present invention. In order to prove the technical effect of the present invention, the content of the index components in the fresh toad venom pulp sample was measured by HPLC external standard single point method for verification.

[0041] Example 1 Determination of the content of index components in fresh toad venom pulp produced in a certain place in Shandong

[0042] (1) Preparation of reference solution

[0043] Accurately weigh 10 mg of the reference substances bufalin, cinobufoxin, and resifobafox genin, add 2 mL of methanol to prepare a 5 mg / mL stock solution, and dilute to 4, 3, 2, and 1 mg / mL, respectively.

[0044] (2) Preparation of test solution

[0045] Use a rapid moisture meter to dry the fresh toad venom pulp sample. Take 100 mg of toad venom sample powder, add 1 mL of methanol, and ultrasonically extract for 30 minutes, and take the supernatant as the test solution.

[0046] (3) Selection of color developer

[0047] This embodiment selected iodine cylinder, phosphomolybdic acid ethanol color developer, fluorescein ethanol color developer, rhodamine B ethanol color developer, and concentrated sulfuric acid-vanillin color developer. The results showed that concentrated sulfuric acid-vanillin color developer had a better color development effect, and bufalin, cinobufolin and resifolin were displayed in different colors.

[0048] The preparation method of concentrated sulfuric acid-vanillin color developer is: add 40 mL of water to 20 mL of ethanol, mix well and slowly add 10 mL of concentrated sulfuric acid, and then add 0.7 g of vanillin.

[0049] (4) Selection of spotting amount

[0050] The present invention investigates the linearity of the standard curve when the sample spotting amount is 0.5 μL, 1 μL and 2 μL respectively, and the result shows that the linearity is best when the sample spotting amount is 1 μL.

[0051] (5) Use a 1 μL quantitative capillary to draw various concentrations of bufalin and the test solution onto the same silica gel thin layer plate, draw various concentrations of cinobucina toxin base and the test solution onto the same silica gel thin layer plate, draw various concentrations of lipobucina toxin base and the test solution onto the same silica gel thin layer plate. Develop with cyclohexane-chloroform-acetone (4:3:3) as the developing agent, take out, dry, spray with sufficient concentrated sulfuric acid-vanillin for color development, and heat at 105°C for 8 minutes.

[0052] (6) Use image J software to open the image of the thin layer of silicone plate, select Image→Type→8-bit from the menu bar to convert the image into a grayscale image; select Process→Subtract Background from the menu bar, change the default value of 50.0 to 100.0 for the Rolling ball radius item, check the Light background option to remove the background effect; select Analyze→Set Measurements from the menu bar, check the Area and Integrated density options; select Analyze→Sshiyanet Scale from the menu bar, select pixel for the Unit of length option; select the points to be calibrated one by one, and ensure that the area selected for each point is consistent; select Edit→Invert from the menu bar; select each point one by one, select Anlyze→Measure from the menu bar, and start analyzing one by one. The relevant results are shown in the attached figure. Figure 1-Figure 3In the analysis results, the IntDen column is the required gray value. The standard curve is made with the reference substance concentration as the horizontal axis and the gray value as the vertical axis. The sample gray value is substituted into the standard curve to obtain the corresponding reference substance concentration. The concentrations of bufalin, cinobufolin and resifobafugin in the test solution are 1.54 mg / mL, 4.95 mg / mL and 4.84 mg / mL, respectively, and their total content is 11.33%.

[0053] (7) HPLC was used to measure the contents of bufalin, cinobucin and resifobacillus in the test solution using the external standard single-point method in accordance with the chromatographic conditions of the 2020 edition of the Pharmacopoeia. The relevant HPLC chromatograms are shown in the attached Figure 4-7 The results are shown in Table 1 and Table 2.

[0054] Table 1 Retention time and peak area of ​​reference substances (injection concentration is 1 mg / mL)

[0055]

[0056]

[0057] Table 2 HPLC content determination results of toad venom samples from Shandong

[0058]

[0059] From the results in Table 2, it can be seen that the total content of bufalin, cinobucini and resinobufacini measured by the method provided in this embodiment is 11.33%, and the total content measured by HPLC is 11.86%, both exceeding the 7% specified in the "Pharmacopoeia of the People's Republic of China" (2020 edition). The results are consistent. When purchasing fresh toad venom pulp, the method provided in this embodiment can be used to determine whether the content of its index components meets the requirements of the pharmacopoeia.

[0060] Example 2 Determination of the content of index components of fresh toad venom paste from Hengsha, Shanghai

[0061] (1) Preparation of reference solution

[0062] Accurately weigh 10 mg of the reference substances bufalin, cinobufoxin, and resifobafox genin, add 2 mL of methanol to prepare a 5 mg / mL stock solution, and dilute to 4, 3, 2, and 1 mg / mL, respectively.

[0063] (2) Preparation of test solution

[0064] Use a rapid moisture meter to dry the fresh toad venom pulp sample. Take 100 mg of toad venom sample powder, add 1 mL of methanol, and ultrasonically extract for 30 minutes, and take the supernatant as the test solution.

[0065] (3) Use a 1uL quantitative capillary to draw various concentrations of bufalin and the test solution onto the same silica gel thin layer plate, draw various concentrations of cinobucina toxin base and the test solution onto the same silica gel thin layer plate, draw various concentrations of lipobucina toxin base and the test solution onto the same silica gel thin layer plate. Develop with cyclohexane-chloroform-acetone (4:3:3) as the developing agent, take out, dry, spray with sufficient concentrated sulfuric acid-vanillin color developer for color development, and heat at 105°C for 8 minutes.

[0066] (4) Use image J software to open the image of the thin layer of silicone plate, select Image→Type→8-bit from the menu bar to convert the image into a grayscale image; select Process→Subtract Background from the menu bar, change the default value of 50.0 to 100.0 for the Rolling ball radius item, check the Light background option to remove the background effect; select Analyze→Set Measurements from the menu bar, check the Area and Integrated density options; select Analyze→Sshiyanet Scale from the menu bar, select pixel for the Unit of length option; select the points to be calibrated one by one, and ensure that the area selected for each point is consistent; select Edit→Invert from the menu bar; select each point one by one, select Anlyze→Measure from the menu bar, and start analyzing one by one. The relevant results are shown in the attached figure. Figure 8-Figure 10 In the analysis results, the IntDen column is the required gray value. The standard curve is made with the reference substance concentration as the horizontal axis and the gray value as the vertical axis. The sample gray value is substituted into the standard curve to obtain the corresponding reference substance concentration. The concentrations of bufalin, cinobufolin and resifobafugin in the test solution are 1.53 mg / mL, 2.75 mg / mL and 3.14 mg / mL, respectively, and their total content is 7.42%.

[0067] (5) HPLC was used to measure the contents of bufalin, cinobucin and resifugine in the test solution according to the chromatographic conditions of the 2020 edition of the Pharmacopoeia. The external standard single-point method was used to measure the contents of bufalin, cinobucin and resifugine. The relevant HPLC chromatograms are shown in the attached Fig.11 The results are shown in Table 3.

[0068] Table 3 HPLC content determination results of Shanghai Hengsha Toad Venom samples

[0069]

[0070] From the results in Table 3, it can be seen that the total content of bufalin, cinobucini and resinobufacini measured by the method provided in this embodiment is 7.42%, and the total content measured by HPLC is 8.03%, both exceeding the 7% specified in the "Pharmacopoeia of the People's Republic of China" (2020 edition). The results are consistent. When purchasing fresh toad venom pulp, the method provided in this embodiment can be used to determine whether the content of its index components meets the requirements of the pharmacopoeia.

[0071] Example 3 Determination of the content of index components of fresh toad venom pulp produced in a certain place in Jiangxi

[0072] (1) Preparation of reference solution

[0073] Accurately weigh 10 mg of the reference substances bufalin, cinobufoxin, and resifobafox genin, add 2 mL of methanol to prepare a 5 mg / mL stock solution, and dilute to 4, 3, 2, and 1 mg / mL, respectively.

[0074] (2) Preparation of test solution

[0075] Use a rapid moisture meter to dry the fresh toad venom pulp sample. Take 100 mg of toad venom sample powder, add 1 mL of methanol, and ultrasonically extract for 30 minutes, and take the supernatant as the test solution.

[0076] (3) Use a 1uL quantitative capillary to draw various concentrations of bufalin and the test solution onto the same silica gel thin layer plate, draw various concentrations of cinobucina toxin base and the test solution onto the same silica gel thin layer plate, draw various concentrations of lipobucina toxin base and the test solution onto the same silica gel thin layer plate. Develop with cyclohexane-chloroform-acetone (4:3:3) as the developing agent, take out, dry, spray with sufficient concentrated sulfuric acid-vanillin color developer for color development, and heat at 105°C for 8 minutes.

[0077] (4) Use image J software to open the image of the thin layer of silicone plate, select Image→Type→8-bit from the menu bar to convert the image into a grayscale image; select Process→Subtract Background from the menu bar, change the default value of 50.0 to 100.0 for the Rolling ball radius item, check the Light background option to remove the background effect; select Analyze→Set Measurements from the menu bar, check the Area and Integrated density options; select Analyze→Sshiyanet Scale from the menu bar, select pixel for the Unit of length option; select the points to be calibrated one by one, and ensure that the area selected for each point is consistent; select Edit→Invert from the menu bar; select each point one by one, select Anlyze→Measure from the menu bar, and start analyzing one by one. The relevant results are shown in the attached figure. Figure 12-Figure 14 In the analysis results, the IntDen column is the required gray value. The standard curve is made with the reference substance concentration as the horizontal axis and the gray value as the vertical axis. The sample gray value is substituted into the standard curve to obtain the corresponding reference substance concentration. The concentrations of bufalin, cinobufolin and resifobafugin in the test solution are 1.16 mg / mL, 1.92 mg / mL and 2.96 mg / mL respectively, and the total content is 6.04%.

[0078] (5) HPLC was used to measure the contents of bufalin, cinobucin and resifugine in the test solution according to the chromatographic conditions of the 2020 edition of the Pharmacopoeia. The external standard single-point method was used to measure the contents of bufalin, cinobucin and resifugine. The relevant HPLC chromatograms are shown in the attached Fig.15 The results are shown in Table 4.

[0079] Table 4 HPLC content determination results of toad venom samples from Jiangxi

[0080]

[0081] From the results in Table 4, it can be seen that the total content of bufalin, cinobucoidin, and resifobacillus genistein measured by the method provided in this embodiment is 6.04%, and the total content measured by HPLC is 6.93, both of which do not reach the 7% specified in the "Pharmacopoeia of the People's Republic of China" (2020 edition). The results are consistent. When purchasing fresh toad venom pulp, the method provided in this embodiment can be used to determine whether the content of its index components meets the requirements of the pharmacopoeia.

[0082] Example 4 Determination of the content of index components in fresh toad venom paste produced in a certain place in Sichuan

[0083] (1) Preparation of reference solution

[0084] Accurately weigh 10 mg of the reference substances bufalin, cinobufoxin, and resifobafox genin, add 2 mL of methanol to prepare a 5 mg / mL stock solution, and dilute to 4, 3, 2, and 1 mg / mL, respectively.

[0085] (2) Preparation of test solution

[0086] Use a rapid moisture meter to dry the fresh toad venom pulp sample. Take 100 mg of toad venom sample powder, add 1 mL of methanol, and ultrasonically extract for 30 minutes, and take the supernatant as the test solution.

[0087] (3) Use a 1uL quantitative capillary to draw various concentrations of bufalin and the test solution and spot them on the same silica gel thin layer plate, draw various concentrations of cinobucina toxin base and the test solution and spot them on the same silica gel thin layer plate, draw various concentrations of lipobucina toxin base and the test solution and spot them on the same silica gel thin layer plate.

[0088] 3:3) as the developing agent, take out, dry, spray with sufficient concentrated sulfuric acid-vanillin for color development, and heat at 105°C for 8 minutes.

[0089] (4) Use image J software to open the thin layer of silicone plate image, and select Image→Type from the menu bar.

[0090] →8-bit, convert the image to grayscale; select Process→Subtract Background from the menu bar, change the default value of 50.0 to 100.0 for the Rolling ball radius item, check the Light background option to remove the background effect; select Analyze→Set Measurements from the menu bar, check the Area and Integrated density options; select Analyze→Sshiyanet Scale from the menu bar, select pixel for the Unit of length option; select the points to be calibrated one by one, and ensure that the Area selected for each point is consistent; select Edit→Invert from the menu bar; select each point one by one, select Anlyze→Measure from the menu bar, and start analyzing one by one. The relevant results are as follows Figure 16-18 In the analysis results, the IntDen column is the required gray value. The standard curve is made with the reference substance concentration as the horizontal axis and the gray value as the vertical axis. The sample gray value is substituted into the standard curve to obtain the corresponding reference substance concentration. The concentrations of bufalin, cinobufolin and resifobafugin in the test solution are 0.66 mg / mL, 2.66 mg / mL and 0 mg / mL respectively, and their total content is 3.32%.

[0091] (5) HPLC was used to measure the contents of bufalin, cinobucin and resifugine in the test solution according to the chromatographic conditions of the 2020 edition of the Pharmacopoeia. The external standard single-point method was used to measure the contents of bufalin, cinobucin and resifugine. The relevant HPLC chromatograms are shown in the attached Fig. 9 The results are shown in Table 5.

[0092] Table 5 HPLC content determination results of Sichuan toad venom samples

[0093]

[0094] From the results in Table 5, it can be seen that the total content of bufalin, cinobucoidin, and resifobacillus genistein measured by the method provided in this embodiment is 3.32%, and the total content measured by HPLC is 4.07%, both of which do not reach the 7% specified in the "Pharmacopoeia of the People's Republic of China" (2020 edition). The results are consistent. When purchasing fresh toad venom pulp, the method provided in this embodiment can be used to determine whether the content of its index components meets the requirements of the pharmacopoeia.

[0095] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for measuring the content of toad venom ingredients, characterized in that: The method comprises the following steps: 1) Provide the test sample solution of fresh toad venom paste, as well as the standard reference solution of bufalin, cinobufolin and resifobafugin; 2) developing the test sample solution and the standard reference solution by thin layer chromatography to obtain a test sample-standard reference thin layer chromatography plate; 3) performing a color development treatment on the test sample-standard reference substance thin layer chromatography plate to obtain a test sample-standard reference substance thin layer chromatography color development plate; 4) Calculate the contents of bufalin, cinobufogenin and resifobafugin in the test solution using the gray value of the test sample-standard reference thin layer chromatography color plate.

2. The method according to claim 1, characterized in that In step 1), the preparation steps of the test solution of fresh toad venom pulp include: drying the fresh toad venom pulp sample, dissolving it in methanol, ultrasonically extracting it, and taking the supernatant as the test solution.

3. The method according to claim 1, characterized in that In step 1), the preparation steps of the standard reference solution of bufalin, cinobucini and resifobacillus genin include: dissolving bufalin, cinobucini and resifobacillus genin in methanol, and diluting them to different concentrations.

4. The method according to claim 1, characterized in that In step 2), the thin layer chromatography step includes: spotting the test solution and the standard reference solution of each concentration on the same silica gel thin layer plate, a total of three silica gel thin layer plates, developing with cyclohexane-chloroform-acetone mixed solution as a developing agent, taking out, drying, and obtaining the test sample-standard reference thin layer chromatography plate.

5. The method according to claim 4, characterized in that The volume ratio of the components of the cyclohexane-chloroform-acetone mixed solution is: cyclohexane:chloroform:acetone=4:3:

3.

6. The method according to claim 1, characterized in that In step 3), the color development step includes: spraying a color developer onto the test sample-standard reference thin layer chromatography plate, and performing a heating treatment to obtain the test sample-standard reference thin layer chromatography color development plate; wherein the color developer is prepared by mixing ethanol, concentrated sulfuric acid and vanillin.

7. The method according to claim 6, characterized in that The preparation steps of the color developer include: adding ethanol to water, mixing well, slowly adding concentrated sulfuric acid, and adding vanillin after mixing well.

8. The method according to claim 1, characterized in that In step 4), the calculation step includes: measuring the grayscale values ​​of each concentration point of the reference substance and the test sample point, making a standard curve with concentration as the horizontal axis and grayscale value as the vertical axis, and substituting the grayscale value of the test sample point into the calculated content.

9. The method according to claim 8, characterized in that In step 4), the calculation step includes: taking a picture of the test sample-standard reference sample thin layer chromatography color plate, and measuring the gray value of each concentration point of the reference sample and the test sample point by computer software.

10. The method according to claim 9, characterized in that The computer software is Image J software.

Images