A high-efficiency thin-layer identification method for multi-component traditional Chinese medicine compound

Abstract

A method for high-efficiency identification of multiple components of a traditional Chinese medicine compound is characterized in that: the thin layer chromatography detection is to take a test solution, a negative control solution and a mixed control solution and point them on a silica gel HSG plate, use a developer 1 for the first development, use a developer 2 for the second development, and after drying, inspect under an ultraviolet lamp, record the results, spray a color developer and heat to develop the color, and record the results. The developer 1 is composed of n-butanol: acetic acid: water in a volume ratio of 3 to 5: 1: 1 to 3, and the developer 2 is composed of petroleum ether and acetone in a volume ratio of 4 to 5: 1. The present invention proposes for the first time a method for rapid synchronous identification of multiple components such as scutellaria saponin B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin in a traditional Chinese medicine compound. The method measures clear spots, high separation of each component, strong specificity, no interference with each other, saves detection time, and has high accuracy, effectively improving detection efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of drug quality control, and in particular to a thin layer high-efficiency identification method for multiple components of a traditional Chinese medicine compound. Background Art

[0002] Thin layer chromatography (TLC) is a method of coating a suitable stationary phase on a substrate to form a uniform thin layer, spotting and developing the sample, and comparing the relative shift value (Rf) with the relative shift value (Rf) of the chromatogram obtained by the same method with a suitable reference substance to identify drugs, check for impurities or determine the content. Thin layer chromatography is a very important experimental technique for rapid and qualitative analysis of small amounts of substances. Thin layer chromatography is an adsorption thin layer chromatography separation method that uses the different adsorption capacities of various components on the same adsorbent to continuously produce adsorption, desorption, re-adsorption, and re-desorption in the process of the mobile phase (solvent) flowing through the stationary phase (adsorbent), thereby achieving the purpose of separating the components from each other.

[0003] The establishment of quality standards for traditional Chinese medicine is a necessary link for the listing of Chinese patent medicines and an important means to ensure the safe use of drugs in clinical practice. Patent CN113181249A discloses a compound preparation formula composed of Pulsatilla scutellariae, Coptis chinensis, Gentiana scabra and Atractylodes macrocephala. In the process of establishing its quality standards, it is necessary to detect the main active ingredients, Pulsatilla scutellariae saponin B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin. At present, for the thin layer identification in the establishment of quality standards for traditional Chinese medicine, the method of identifying the single medicine in the compound preparation one by one is mostly adopted, because the sample processing methods and developing agents for detecting a single Chinese medicine component are different, which often consumes a lot of time and energy. At present, a method of one plate multiple detection has been proposed in the literature. One plate multiple detection can realize the detection of the components of different medicinal materials in the compound under the same sample processing method and developing agent conditions, which is more convenient and fast. For the above-mentioned compound powder, when using thin layer chromatography for identification, it is difficult to achieve simultaneous detection of all active ingredients due to the large difference in polarity between the true active ingredients in the formula. The existing technology uses multiple steps for thin layer chromatography analysis. Therefore, how to achieve multiple detections on one plate through thin layer chromatography is extremely important. Summary of the invention

[0004] The present invention aims to provide a thin layer efficient identification method for multiple components of a traditional Chinese medicine compound, which successfully realizes multiple detections of scutellaria saponin B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin on one plate, simplifies the identification method, and makes the identification method simple, efficient and low-cost.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A method for high-efficiency identification of multiple components of a traditional Chinese medicine compound using thin layer chromatography, wherein the traditional Chinese medicine compound comprises a compound powder composed of pulsatilla scutellariae, coptis root, gentian and atractylodes lancea, and the components to be identified by thin layer chromatography include pulsatilla scutellariae saponin B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin, and the thin layer chromatography identification sequentially comprises the preparation of a test solution, the preparation of a negative control solution, the preparation of a mixed control solution, and thin layer chromatography detection, and is characterized in that:

[0007] The preparation of the test solution is to take the compound powder, add methanol, perform ultrasonic treatment, filter, evaporate the filtrate, and then add methanol to dissolve;

[0008] The negative control solution is prepared according to the prescription ratio of the compound powder, and the compound powder lacking Pulsatilla striata, Coptis chinensis, Gentiana scabra, and Atractylodes lancea is taken respectively, and the negative control solution is prepared according to the test sample preparation method;

[0009] The preparation of the mixed reference solution is to take scutellaria saponin B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin reference substances, and add methanol to prepare a mixed reference solution;

[0010] The thin layer chromatography test specifically includes taking the test solution, the negative control solution and the mixed control solution and spotting them on the silica gel HSG plate, using the developing agent 1 for the first development, and after drying, using the developing agent 2 for the second development. After the silica gel HSG plate is dried, it is inspected under ultraviolet light, and after recording the results, the color developer is sprayed and heated for color development, and the results are recorded. The developing agent 1 is composed of n-butanol: acetic acid: water in a volume ratio of 3~5:1~2:1~3, and the developing agent 2 is composed of petroleum ether and acetone in a volume ratio of 4~5:1.

[0011] Furthermore, the developer is a 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, which is prepared by adding p-dimethylaminobenzaldehyde to a 10% sulfuric acid ethanol solution by volume, and the dosage ratio of p-dimethylaminobenzaldehyde to sulfuric acid ethanol is 1.5~2.0g:30mL.

[0012] Furthermore, the expansion cylinder for the first expansion is pre-saturated for 10-15 minutes and expanded to 6-7 cm, and the expansion cylinder for the second expansion is not pre-saturated and expanded to 9-10 cm.

[0013] Furthermore, the ultraviolet lamp is a 254mm ultraviolet lamp, and the temperature for heating and color development is 105°C.

[0014] Furthermore, in the preparation process of the test solution, the dosage ratio of the compound powder and the methanol added twice is 1-2 g: 10-15 mL: 1 mL, and the ultrasonic time is 30-50 min.

[0015] Furthermore, each 1 mL of the mixed reference solution contains 1-2 mg of scutellaria saponin B4, 0.5-1 mg of berberine hydrochloride, 0.5-1 mg of epiberberine, 1-2 mg of gentiopicroside and 0.3-0.5 mg of atractylodesin.

[0016] Further preferably, each 1 mL of the mixed reference solution contains 1 mg of scutellaria saponin B4, 0.5 mg of berberine hydrochloride, 0.5 mg of epiberberine, 1 mg of gentiopicroside and 0.3 mg of atractylodesin.

[0017] A thin layer high-efficiency identification method for multiple components of a traditional Chinese medicine compound, characterized by comprising the following steps:

[0018] (1) Preparation of test solution: Take 3-5 g of the compound powder, add 50 mL of methanol, sonicate for 30 min, cool and filter, evaporate the filtrate to dryness, and then add 3-5 mL of methanol to re-dissolve;

[0019] (2) Preparation of negative control solution: according to the prescription ratio, take the compound powders lacking Pulsatilla striata, lacking Coptis chinensis, lacking Gentiana scabra and lacking Atractylodes lancea, respectively, and prepare 4 groups of negative control solutions according to the method of step (1);

[0020] (3) Preparation of mixed reference solution: Take appropriate amount of scutellaria saponin B4, berberine hydrochloride, gentiopicroside and atractylodesin reference substances, weigh accurately, add methanol to prepare a mixed solution containing 1-2 mg of scutellaria saponin B4, 0.5-1 mg of berberine hydrochloride, 1-2 mg of gentiopicroside and 0.3-0.5 mg of atractylodesin per 1 mL as the mixed reference solution;

[0021] (4) Preparation of color developer: The color developer is a 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, which is prepared by adding p-dimethylaminobenzaldehyde to a 10% sulfuric acid ethanol solution with a volume fraction of 10%, and the dosage ratio of p-dimethylaminobenzaldehyde to sulfuric acid ethanol is 1.5-2.0 g:30 mL;

[0022] (5) First development: Take 5-10 μL of the mixed reference solution, 0.5-1 μL of the test sample and 0.5-1 μL of each negative reference solution and spot them on a high-efficiency silica gel HSG plate, and perform the first development with developer 1. The developing cylinder is pre-saturated for 10-15 min, and the plate is developed to about 6-7 cm. The plate is dried naturally or blown dry with cold air. The developer 1 is a solution composed of n-butanol: acetic acid: water in a volume ratio of 3-5:1-2:1-2;

[0023] (6) Second development: The thin layer plate dried in step (5) is secondarily developed with developing agent 2 without pre-saturation of the developing cylinder, developed to about 9-10 cm, and then the thin layer plate is dried. The developing agent 2 is composed of petroleum ether and acetone in a volume ratio of 4-5:1;

[0024] (7) Record the results: After inspecting and recording the results under 254 nm ultraviolet light, spray 5% p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol solution, heat at 105°C to develop color, and record the results.

[0025] Due to the obvious differences in the polarity of different ingredients in the compound, especially the large difference in polarity between atractylodesin and other components, it is impossible to effectively separate atractylodesin from other components during a single development, resulting in obvious interference and the inability to simultaneously identify atractylodesin in the test solution.

[0026] Therefore, the present invention adopts the developing agent 1 composed of n-butanol: acetic acid: water in a volume ratio of 3~5:1~2:1~2 for the first development, and then develops with the developing agent 2 composed of petroleum ether and acetone in a volume ratio of 4~5:1. After two treatments with different developing agents, components with large polarity differences can also be colored in the same thin layer plate without interfering with each other, and it is further ensured that the point appearing at the forefront of the solvent is atractylodesin. Under the above-mentioned developing agent conditions, the simultaneous detection of scutellaria saponin B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin on a single plate is achieved.

[0027] The present invention has the following technical effects:

[0028] The present invention proposes for the first time a rapid synchronous thin-layer identification method for scutellaria baicalensis saponins, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin in a traditional Chinese medicine compound, wherein two development treatments are performed to separate substances with extremely different polarities, thereby effectively realizing multiple detections on one plate, and scutellaria baicalensis saponins B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin are detected on the same thin-layer plate. The method measures clear spots, high separation of components, strong specificity, and no mutual interference, thereby saving detection time and having high accuracy, thereby effectively improving detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : The thin layer chromatogram obtained by inspecting Example 1 of the present invention under a 254nm purple lamp.

[0030] Figure 2 : The thin layer chromatogram obtained by examining Example 1 of the present invention under color development conditions.

[0031] Figure 3 : The thin layer chromatogram obtained by inspection in Comparative Example 1. DETAILED DESCRIPTION

[0032] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0033] Example 1

[0034] A thin layer high-efficiency identification method for multiple components of a traditional Chinese medicine compound comprises the following steps:

[0035] (1) Preparation of test solution: Take 4 g of the compound powder, add 50 mL of methanol, sonicate for 30 min, cool and filter, evaporate the filtrate to dryness, and then add 4 mL of methanol to re-dissolve;

[0036] (2) Preparation of negative control solution: according to the prescription ratio, take the compound powders lacking Pulsatilla striata, lacking Coptis chinensis, lacking Gentiana scabra and lacking Atractylodes lancea, respectively, and prepare 4 groups of negative control solutions according to the method of step (1);

[0037] (3) Preparation of mixed reference solution: Take appropriate amount of scutellaria basidioside B4, berberine hydrochloride, gentiopicroside and atractylodesin reference substances, weigh accurately, add methanol to prepare a mixed solution containing 1 mg of scutellaria basidioside B4, 0.5 mg of berberine hydrochloride, 1 mg of gentiopicroside and 0.3 mg of atractylodesin per 1 mL as the mixed reference solution;

[0038] (4) Preparation of color developer: The color developer is a 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, which is prepared by adding p-dimethylaminobenzaldehyde to a 10% sulfuric acid ethanol solution with a volume fraction of 10%, and the amount ratio of p-dimethylaminobenzaldehyde to sulfuric acid ethanol is 1.5g:30mL;

[0039] (5) First development: 7 μL of the mixed reference solution, 0.8 μL of the test sample and 0.8 μL of each negative reference solution are spotted on a high-efficiency silica gel HSG plate, and the first development is performed with developer 1. The developing cylinder is pre-saturated for 15 min and developed to about 6-7 cm. The thin layer plate is naturally dried or blown dry with cold air. The developer 1 is a solution composed of n-butanol: acetic acid: water in a volume ratio of 4:1:2;

[0040] (6) Second development: The thin layer plate dried in step (5) is secondarily developed with developing agent 2 without pre-saturation of the developing cylinder, developed to about 9-10 cm, and then the thin layer plate is dried, wherein the developing agent 2 is composed of petroleum ether and acetone in a volume ratio of 4-5:1;

[0041] (7) Record the results: After inspecting and recording the results under 254 nm ultraviolet light, spray 5% p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol solution, heat at 105°C to develop color, and record the results.

[0042] Figure 1 It is the thin layer chromatogram of the mixed reference solution, the test solution and the negative reference solution under 254nm ultraviolet light. Figure 2 It is a thin layer chromatogram of the mixed reference solution, the test solution and the negative reference solution under the action of the display agent, wherein 1 and 8 correspond to the mixed reference solution (spotting volume is 7μL), 2 and 7 correspond to the test solution (spotting volume is 0.8μL), 3 corresponds to the negative reference solution lacking Pulsatilla striata (spotting volume is 0.8μL), 4 corresponds to the negative reference solution lacking Coptis chinensis (spotting volume is 0.8μL), 5 corresponds to the negative reference solution lacking Gentiana scabra (spotting volume is 0.8μL), and 6 corresponds to the negative reference solution lacking Atractylodes macrocephala (spotting volume is 0.8μL). It can be seen from the figure that the mixed reference solution shows the corresponding spots of berberine hydrochloride and epiberberine under the ultraviolet light. Except for the negative reference solution lacking Coptis chinensis, the other groups all see spots of the same color at the corresponding positions of berberine hydrochloride and epiberberine, and the spots between berberine hydrochloride and epiberberine are unknown components not present in the mixed reference solution. After color development, the thin layer chromatogram obtained under the color development conditions of 5% p-dimethylaminobenzaldehyde and 10% sulfuric acid ethanol solution is as follows: Figure 2 As shown in the figure, the solutions corresponding to the serial numbers are Figure 1 It is consistent with the results in the chromatogram corresponding to the test solution. Spots of the same color are displayed at the corresponding positions of the mixed reference substance, while spots corresponding to the missing drugs do not appear in the negative reference solution. Two color development methods were used. First, the spots corresponding to berberine hydrochloride were observed under ultraviolet 254nm. Then, after spraying the color developer, the spots corresponding to berberine hydrochloride and epiberberine can be seen under natural light, as well as the spots corresponding to scutellariae saponin B4, gentiopicroside and atractylodesin. The two color development methods support each other.

[0043] Comparative Example 1

[0044] Compared with Example 1, the developing agents used in the first development and the second development were exchanged, that is, the developing agent used in the first development was composed of petroleum ether and acetone in a volume ratio of 4-5:1, and the developing agent used in the second development was composed of n-butanol: acetic acid: water in a volume ratio of 4:1:2. The specific steps are as follows:

[0045] (1) Preparation of test solution: Take 4 g of the compound powder, add 50 mL of methanol, sonicate for 30 min, cool and filter, evaporate the filtrate to dryness, and then add 4 mL of methanol to re-dissolve;

[0046] (2) Preparation of negative control solution: according to the prescription ratio, take the compound powder without coptis chinensis and prepare the negative control solution according to the method of step (1);

[0047] (3) Preparation of reference solution: Take the atractylodesin reference substance, weigh it accurately, and add methanol to make a solution containing 0.3 mg of atractylodesin per 1 mL, which is used as the standard reference solution;

[0048] (4) Preparation of color developer: The color developer is a 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, which is prepared by adding p-dimethylaminobenzaldehyde to a 10% sulfuric acid ethanol solution with a volume fraction of 10%, and the amount ratio of p-dimethylaminobenzaldehyde to sulfuric acid ethanol is 1.5g:30mL;

[0049] (5) First development: 7 μL of atractylodesin standard reference solution, 0.8 μL of the test sample and 1 μL of the negative control solution were spotted on a high-efficiency silica gel HSG plate, and the first development was performed with developer 2. The developing cylinder was pre-saturated for 15 min and developed to about 6-7 cm. The thin layer plate was naturally dried or blown dry with cold air. The developer 2 was composed of petroleum ether and acetone in a volume ratio of 4-5:1;

[0050] (6) Second development: The thin layer plate dried in step (5) is secondarily developed with developer 1 without pre-saturation of the developing cylinder, developed to about 9-10 cm, and then the thin layer plate is dried. The developer 1 is a solution composed of n-butanol: acetic acid: water in a volume ratio of 4:1:2;

[0051] (7) Record the results: After inspecting and recording the results under 254 nm ultraviolet light, spray 5% p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol solution, heat at 105°C to develop color, and record the results.

[0052] The thin layer chromatogram obtained in Comparative Example 1 under 254nm ultraviolet light and the action of a developer is as follows: Figure 3 As shown, 1 and 2 are the thin layer chromatograms corresponding to the single atractylodesin standard reference solution, 3 and 4 are the thin layer chromatograms corresponding to the test solution, and 5 and 6 are the thin layer chromatograms corresponding to the negative reference solution lacking atractylodes. It can be seen that under 254nm ultraviolet light, spots corresponding to berberine hydrochloride and epiberberine appeared in the test solution and the negative reference solution lacking atractylodes. Under the action of the developer, the single atractylodesin standard reference solution showed spots corresponding to atractylodes at the front of the solvent, while the test solution and the negative reference solution lacking atractylodes did not have corresponding spots. After the developer swapped the order of the developing agents, it was found that due to the interference of other components, atractylodesin could not be separated, and ultimately atractylodesin could not be identified in the thin layer chromatography. It can be seen that the order of using the developing agents is extremely critical.

Claims

1. A method for high-efficiency identification of multiple components of a traditional Chinese medicine compound by thin layer chromatography, wherein the traditional Chinese medicine compound is a compound powder composed of pulsatilla, coptis root, gentian and atractylodes, and the components identified by thin layer chromatography include pulsatilla saponin B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin, and the chromatographic identification sequentially includes the preparation of a test solution, the preparation of a negative control solution, the preparation of a mixed control solution, and thin layer chromatography detection, characterized in that: The test solution is prepared by adding methanol to the compound powder for ultrasonic treatment, filtering, evaporating the filtrate, and then adding methanol for re-dissolution; The negative control solution is prepared according to the prescription ratio of the compound powder, and the compound powder lacking Pulsatilla striata, Coptis chinensis, Gentiana scabra, and Atractylodes lancea is taken respectively, and the negative control solution is prepared according to the test sample preparation method; The preparation of the mixed reference solution is to take scutellaria saponin B4, berberine hydrochloride, epiberberine, gentiopicroside and atractylodesin reference substances, and add methanol to prepare a mixed reference solution; The thin layer chromatography test specifically includes taking the test solution, the negative control solution and the mixed control solution and spotting them on the silica gel HSG plate, using the developing agent 1 for the first development, and after drying, using the developing agent 2 for the second development. After the silica gel HSG plate is dried, it is inspected under ultraviolet light, and after the results are recorded, the color developer is sprayed and heated for color development, and the results are recorded. The developing agent 1 is composed of n-butanol: acetic acid: water in a volume ratio of 3~5:1~2:1~3, and the developing agent 2 is composed of petroleum ether and acetone in a volume ratio of 4~5:

1.

2. A thin layer high-efficiency identification method for multi-component Chinese medicine compound according to claim 1, characterized in that: The color developer is a 10% sulfuric acid ethanol solution of 5% p-dimethylaminobenzaldehyde, which is prepared by adding p-dimethylaminobenzaldehyde to a 10% sulfuric acid ethanol solution with a volume fraction.

3. A thin layer high-efficiency identification method for multiple components of a traditional Chinese medicine compound as claimed in claim 1 or 2, characterized in that: The expansion cylinder for the first expansion is pre-saturated for 10-15 minutes and expanded to 6-7 cm. The expansion cylinder for the second expansion is not pre-saturated and expanded to 9-10 cm.

4. A thin layer high-efficiency identification method for multiple components of a traditional Chinese medicine compound as claimed in claim 3, characterized in that: During the preparation of the test solution, the dosage ratio of the compound powder, the methanol added for the first time, and the methanol added for the second time is 1-2 g:10-15 mL:1 mL, and the ultrasonic time is 30-50 min.

5. A thin layer high-efficiency identification method for multiple components of a traditional Chinese medicine compound as claimed in claim 4, characterized in that: Each 1 mL of the mixed reference solution contains 1-2 mg of scutellaria saponin B4, 0.5-1 mg of berberine hydrochloride, 0.5-1 mg of epiberberine, 1-2 mg of gentiopicroside, and 0.3-0.5 mg of atractylodesin.

6. A thin layer high-efficiency identification method for multiple components of a traditional Chinese medicine compound, characterized in that: The steps include: (1) Preparation of test solution: Take 3-5 g of the compound powder, add 50 mL of methanol, sonicate for 30-50 min, cool and filter, evaporate the filtrate to dryness, and then add 3-5 mL of methanol to dissolve; (2) Preparation of negative control solution: according to the prescription ratio, take the compound powders lacking Pulsatilla striata, lacking Coptis chinensis, lacking Gentiana scabra and lacking Atractylodes lancea, respectively, and prepare 4 groups of negative control solutions according to the method of step (1); (3) Preparation of mixed reference solution: Take appropriate amount of scutellaria saponin B4, berberine hydrochloride, gentiopicroside and atractylodesin reference substances, weigh accurately, add methanol to prepare a mixed solution containing 1-2 mg of scutellaria saponin B4, 0.5-1 mg of berberine hydrochloride, 1-2 mg of gentiopicroside and 0.3-0.5 mg of atractylodesin per 1 mL as the mixed reference solution; (4) Preparation of color developer: The color developer is a 10% sulfuric acid ethanol solution of 5% p-dimethylaminobenzaldehyde, which is prepared by adding p-dimethylaminobenzaldehyde to a 10% sulfuric acid ethanol solution with a volume fraction; (5) First development: 5-10 μL of the mixed reference solution, 0.5-1 μL of the test sample and 0.5-10 μL of each negative reference solution are spotted on a high-efficiency silica gel HSG plate, and the first development is performed with developer 1. The developing cylinder is pre-saturated for 10-15 min, and the plate is developed to 6-7 cm. The plate is dried naturally or blown dry with cold air. The developer 1 is a solution composed of n-butanol: acetic acid: water in a volume ratio of 3-5:1-2:1-2; (6) Second development: The thin layer plate dried in step (5) is secondarily developed with developing agent 2 without pre-saturation of the developing cylinder, developed to 9-10 cm, and then the thin layer plate is dried. The developing agent 2 is composed of petroleum ether and acetone in a volume ratio of 4-5:1; (7) Record the results: After inspecting and recording the results under 254 nm ultraviolet light, spray 5% p-dimethylaminobenzaldehyde in 10% sulfuric acid ethanol solution, heat at 105°C to develop color, and record the results.

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