Thin-layer Identification Method for Prepared Granules of Turpentine Joint

Abstract

The present invention provides a thin-layer identification method for pinecone formula granules, comprising: A) taking a test sample, and after pretreatment, obtaining a test solution; taking a pinosylvin monomethyl ether reference substance, and after pretreatment, obtaining a reference solution; B) taking a reference medicinal material of pinecone, and after pretreatment, obtaining a reference medicinal material solution; C) performing thin-layer chromatography detection on the test solution, the reference solution and the reference medicinal material solution, wherein the thin-layer plate is a silica gel G thin-layer plate; the developing agent is toluene-ethyl acetate-glacial acetic acid; D) examining, in the test chromatogram, if spots of the same color appear at the positions corresponding to the reference medicinal material and the reference substance, it is pinecone formula granules. The thin-layer identification chromatographic method for pinecone formula granules established by the present invention uses, as the identification points, the spots of the same color that appear at the positions corresponding to the reference medicinal material and the reference substance by observing the thin-layer chromatogram, so as to quickly and effectively identify pinecone formula granules.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a thin-layer identification method for pine cone formula granules. Background Art

[0002] Pine cone is the dried tuberous joint of Pinus tabulaeformis Carr. or Pinus massoniana Lamb. of the genus Pinus in the family Pinaceae. Its main effects are dispelling wind and dampness, dredging collaterals and relieving pain. It is used for wind-cold-damp arthralgia, migratory arthralgia, convulsion due to cramped tendons, and traumatic injury. Methyl pinosylvin has analgesic and anti-inflammatory effects and is the main active ingredient of pine cone. However, current research on the medicinal value of pine mainly focuses on pine pollen, rosin, pine needles and turpentine, and there is little modern research on pine cone. In the Chinese Pharmacopoeia and the Chinese herbal medicine standards and processing specifications of each province, there are no regulations on the thin-layer qualitative identification of pine cone, and there is no literature on the qualitative and quantitative research of pine cone (Pinus massoniana) formula granules, which is not conducive to the quality control of pine cone (Pinus massoniana) formula granules.

[0003] Therefore, it is necessary to develop a method with stronger specificity, stronger repeatability, effective control to provide an accurate basis for the qualitative identification of pine cone (Pinus massoniana) formula granules. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a thin-layer identification method for pine cone formula granules.

[0005] In this article, the terms "comprising", "including" and "having" can be used interchangeably, aiming to represent the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. At the same time, it should be understood that using "comprising", "including" and "having" to describe in this article also provides a solution of "consisting of...".

[0006] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural.

[0007] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expression means any combination of these items, including any combination of single item or plural items.

[0008] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the following processes do not imply the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0009] The present invention provides a thin-layer identification method for turpentine formula granules, comprising:

[0010] A) Take the test sample, and after pretreatment, obtain the test solution;

[0011] Take the pinosylvin monomethyl ether reference substance, and after pretreatment, obtain the reference solution;

[0012] B) Take the turpentine control crude drug, and after pretreatment, obtain the control crude drug solution;

[0013] C) Perform thin-layer chromatography detection on the test solution, the reference solution and the control crude drug solution. The thin-layer plate is a silica gel G thin-layer plate; the developing agent is toluene - ethyl acetate - glacial acetic acid;

[0014] D) Examine. In the test chromatogram, if spots of the same color appear at the positions corresponding to the control crude drug and the reference substance, it is turpentine formula granules.

[0015] The pretreatment in step A) of the present invention specifically is: The pretreatment specifically is: Take the test sample, dissolve it with ethyl acetate, perform ultrasonic extraction, dissolve the residue after filtration with methanol to obtain the test solution;

[0016] Specifically, the mass-volume ratio of the test sample, ethyl acetate and methanol is: 1 g : 25 mL : 1 mL.

[0017] In some embodiments, the time of ultrasonic extraction is 20 - 30 min; more preferably 30 min.

[0018] In some embodiments, the boiling point of ethyl acetate is 60 - 90 °C.

[0019] Take the pinosylvin monomethyl ether reference substance, and after pretreatment, obtain the reference solution.

[0020] In some embodiments, take an appropriate amount of pinosylvin monomethyl ether reference substance, dissolve it with methanol to make a solution containing 1 mg per 1 mL, and that is the reference solution.

[0021] Take the turpentine control crude drug, and after pretreatment, obtain the control crude drug solution.

[0022] In some embodiments, the pretreatment specifically is: Take the turpentine control crude drug, decoct it with water, filter, dissolve the residue with methanol to obtain the control crude drug solution;

[0023] In some embodiments, the mass-volume ratio of the control medicinal material, water, and methanol is: 1 g : 50 mL : 1 mL;

[0024] In some embodiments, the decocting time is 20 - 30 min.

[0025] In some embodiments, the decocting time is 25 - 30 min.

[0026] Perform thin-layer chromatography detection on the test sample solution, reference substance solution, and control medicinal material solution.

[0027] Preferably: Pipette the test sample solution, reference substance solution, and control medicinal material solution, and spot them on the same silica gel G thin-layer plate respectively. Develop with the developing agent, take out, dry in the air, and examine.

[0028] The examination in the present invention is to examine under an ultraviolet lamp or spray with ninhydrin solution, heat for color development, and then examine under a fluorescent lamp.

[0029] Among them, the ultraviolet light wavelength in the present invention is 254 nm.

[0030] The thin-layer plate in the present invention is a silica gel G thin-layer plate. It can be selected from the cuttable thin-layer chromatography plate of Tianjin Sida Technology Co., Ltd. and the precast silica gel GF254 plate of Merck.

[0031] The results show that both thin-layer plates can meet the identification requirements. They are used to identify the pine cone formula granules.

[0032] In the chromatogram of the test sample, if spots of the same color appear at the positions corresponding to the control medicinal material and the reference substance, it is the pine cone formula granule.

[0033] The sample application volume of the test sample solution in the present invention is 10 - 16 μL; the sample application volume of the standard substance is 10 - 13 μL;

[0034] The sample application volume of the control medicinal material solution is 1 - 25 μL.

[0035] In some preferred embodiments of the present invention, the mass ratio of toluene - ethyl acetate - glacial acetic acid is 3:1:0.5; among them, the boiling point of ethyl acetate is 60 - 90 °C.

[0036] The examination temperature in the present invention is 8 °C - 25 °C. The method of the present invention has good durability for different temperatures.

[0037] The examination humidity in the present invention is 32% rh - 75% rh. The method of the present invention has good adaptability and durability for different humidities.

[0038] The test sample in the present invention is the pine cone (Pinus massoniana) formula granule.

[0039] The present invention provides a thin-layer identification method for pine cone formula granules, which includes: A) taking a sample to be tested, and obtaining a test solution after pretreatment; taking a pinosylvin monomethyl ether reference substance, and obtaining a reference solution after pretreatment; B) taking a reference medicinal material of pine cone, and obtaining a reference medicinal material solution after pretreatment; C) performing thin-layer chromatography detection on the test solution, the reference solution and the reference medicinal material solution, and the thin-layer plate is a silica gel G thin-layer plate; the developing agent is toluene-ethyl acetate-glacial acetic acid; D) examining, in the test chromatogram, if spots of the same color appear at the positions corresponding to the reference medicinal material and the reference substance, it is pine cone formula granules. The thin-layer identification chromatographic method for pine cone formula granules established by the present invention uses the spots of the same color appearing at the positions corresponding to the reference medicinal material and the reference substance in the thin-layer chromatogram as the identification points to quickly and effectively identify pine cone formula granules. Description of the Drawings

[0040] Figure 1 For the investigation of sample application amount;

[0041] Figure 2 For the investigation of specificity;

[0042] Figure 3 For the investigation of different thin-layer plates (examined under ultraviolet light);

[0043] Figure 4 For the investigation of different thin-layer plates (examined under daylight);

[0044] Figure 5 For different temperatures -8°C (examined under ultraviolet light);

[0045] Figure 6 For different temperatures -25°C (examined under ultraviolet light);

[0046] Figure 7 For different temperatures -8°C (examined under daylight);

[0047] Figure 8 For different temperatures -25°C (examined under daylight);

[0048] Figure 9 For different humidities -32% (examined under ultraviolet light);

[0049] Figure 10 For different humidities -75% (examined under ultraviolet light);

[0050] Figure 11 For different humidities -32% (examined under daylight);

[0051] Figure 12 For different humidities -75% (examined under daylight);

[0052] Figure 13 For the verification results of different batches;

[0053] Figure 14 Result diagram of Comparative Example 1 of the present invention;

[0054] Figure 15 Result diagram of the method of Comparative Example 2;

[0055] Figure 16 Result diagram of the inspection of Comparative Example 3 under ultraviolet light;

[0056] Figure 17 Result diagram of the inspection of Comparative Example 3 under daylight lamp. Specific implementation manners

[0057] In order to further illustrate the present invention, a thin-layer identification method for a turpentine formula granule provided by the present invention will be described in detail below in combination with embodiments.

[0058] Ultrasonic machine, heating plate, mortar, thin-layer imaging system: CAMAG TLC Visualizer, silica gel GF254 thin-layer plate (Qingdao Ocean Chemical Factory, batch number: 20180527, Tianjin Silitda Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353)

[0059] Ethyl acetate, silica gel, methanol, glacial acetic acid, toluene are all of analytical grade, and water is ultrapure water (self-made in the laboratory).

[0060] Turpentine control medicinal material (Chengdu Deste Biotechnology Co., Ltd., batch number: DST230111-178), monomethyl ether of pinosylvin control (Shandong Bokang Fine Chemical Co., Ltd., batch number: CFS202201), turpentine (Pinus massoniana) formula granule (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2302028, 2302029, 2302030).

[0061] Example 1

[0062] Take about 1 g of this product, grind it finely, add 25 ml of ethyl acetate (60-90 °C), extract it by ultrasonic for 30 minutes, filter it, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol, filter it, and obtain. Separately take an appropriate amount of monomethyl ether of pinosylvin control, add methanol to make a solution containing 1 mg per 1 ml, and obtain, which is used as the control solution.

[0063] Take 1 g of turpentine control medicinal material, grind it finely, add 50 ml of water, decoct it for 30 minutes, filter it, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol, and make a control medicinal material solution.

[0064] Test according to the thin-layer chromatography method (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition). Absorb 25 μl of the test solution, 10 μl each of the reference crude drug solution and the reference substance solution, and spot them on the same silica gel GF254 thin-layer plate respectively. Use toluene-ethyl acetate-glacial acetic acid (3:1:0.5) as the developing solvent, develop, take out, dry in air, and examine under ultraviolet light (254 nm). In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference crude drug, there are main spots showing the same color; then spray with ninhydrin test solution, heat at 105 °C until the spots develop clearly, and examine under daylight. In the chromatogram of the test solution, at the positions corresponding to the chromatograms of the reference crude drug and the reference substance, there are spots showing the same color.

[0065] Methodological investigation of Example 2

[0066] 2.1 Investigation of sample application volume Under the above-mentioned established experimental conditions, respectively take 10 μl, 12 μl, 14 μl, 16 μl of the pinosylvin monomethyl ether reference substance solution, 10 μl, 11 μl, 12 μl, 13 μl of the crude drug reference solution of turpentine, and 10 μl, 15 μl, 20 μl, 25 μl of the formula granules of turpentine (Pinus massoniana Lamb.) and spot them on the same silica gel GF254 thin-layer plate. Develop according to the established thin-layer chromatography conditions and examine. The results are shown in Figure 1 , Figure 1 The above is the inspection result under ultraviolet light. Among them, 1-4 are the sample application volumes of 10 μl, 12 μl, 14 μl, 16 μl of the pinosylvin monomethyl ether reference substance; 5-8 are the sample application volumes of 10 μl, 11 μl, 12 μl, 13 μl of the crude drug reference of turpentine; 9-12 are the sample application volumes of 10 μl, 15 μl, 20 μl, 25 μl of the formula granules of turpentine (Pinus massoniana Lamb.); Figure 1 The following is the inspection result under daylight. Among them, 1-4 are the sample application volumes of 10 μl, 12 μl, 14 μl, 16 μl of the pinosylvin monomethyl ether reference substance; 5-8 are the sample application volumes of 10 μl, 11 μl, 12 μl, 13 μl of the crude drug reference of turpentine; 9-12 are the sample application volumes of 10 μl, 15 μl, 20 μl, 25 μl of the formula granules of turpentine (Pinus massoniana Lamb.);

[0067] It can be seen from the figure that when the sample application volume of the reference substance solution is 10-16 μl, the sample application volume of the reference crude drug solution is 10-13 μl, and the sample application volume of the test solution is 10-25 μl, the fluorescence spots of the thin-layer chromatography develop clearly. It is determined that the sample application volume of the reference substance solution is 10-16 μl, the sample application volume of the reference crude drug solution is 10-13 μl, and the sample application volume of the test solution is 10-25 μl. In the chromatogram of the test solution, at the positions corresponding to the chromatograms of the reference crude drug and the reference substance, there are spots showing the same color, which can be used as the identification points of the formula granules of turpentine (Pinus massoniana Lamb.).

[0068] 2.2 Investigation of specificity

[0069] Prepare the reference substance solution, the solution of the control crude drug of Pinus massoniana Lamb., the solution of the formula granules of Pinus massoniana Lamb., and the negative solution according to the above test sample preparation method, spot them on the same thin-layer plate respectively, develop according to the proposed thin-layer chromatography conditions, and examine. The results are shown in Figure 2 , and the results show that the negative sample has no interference with the test sample of the formula granules of Pinus massoniana Lamb., and the method has good specificity. Figure 2 Note for specificity test: 1 is the negative control; 2 is the reference substance of pinosylvin monomethyl ether; 3 is the control crude drug of Pinus massoniana Lamb.; 4 is the formula granules of Pinus massoniana Lamb.

[0070] 2.3 Investigation of durability

[0071] 2.3.1 Comparison of different thin-layer plates

[0072] Select the pre-coated silica gel GF254 plates from Tianjin Sida and Merck, and conduct the tests according to the proposed test methods respectively. The results are shown in Figures 3 - 4 , and the results show that the thin-layer plates of both brands can meet the identification requirements, indicating that the method has good durability. The results are as shown in Figure 3 , Figure 3 are the results of ultraviolet lamp inspection for different thin-layer plates. Among them: 1. pinosylvin monomethyl ether; 2. the control crude drug of Pinus massoniana Lamb.; 3. the formula granules of Pinus massoniana Lamb. Figure 4 Results of inspection under daylight for different thin-layer plates; among them: 1 is pinosylvin monomethyl ether; 2 is the control crude drug of Pinus massoniana Lamb.; 3 is the formula granules of Pinus massoniana Lamb.

[0073] 2.3.2 Comparison of different temperatures

[0074] Take the spotted thin-layer plates and develop them in temperature environments of low temperature 8°C and normal temperature 25°C respectively. It can be seen from Figures 5 - 8 that the method has good durability for different temperatures.

[0075] Figure 5 are for different temperatures - 8°C (inspection under ultraviolet lamp); among them, 1. pinosylvin monomethyl ether; 2. the control crude drug of Pinus massoniana Lamb.; 3. the formula granules of Pinus massoniana; Figure 6 are for different temperatures - 25°C (inspection under ultraviolet lamp); among them, 1. pinosylvin monomethyl ether; 2. the control crude drug of Pinus massoniana Lamb.; 3. the formula granules of Pinus massoniana; Figure 7 are for different temperatures - 8°C (inspection under daylight); Figure 8 are for different temperatures - 25°C (inspection under daylight); among them, 1. pinosylvin monomethyl ether; 2. the control crude drug of Pinus massoniana Lamb.; 3. the formula granules of Pinus massoniana.

[0076] 2.3.3 Comparison of different humidities

[0077] Take the spotted thin-layer plates and develop them in humidity environments of 32% and 75% respectively. The results are shown in Figures 9 - 12. It can be seen from the figure that the method has good durability for different humidities. Figure 9 For different humidities - 32% (examined under ultraviolet light); 1. monomethyl pinosylvin; 2. crude drug control of turpentine; 3. formula granules of turpentine.

[0078] Figure 10 For different humidities - 75% (examined under ultraviolet light); 1. monomethyl pinosylvin; 2. crude drug control of turpentine; 3. formula granules of turpentine. Figure 11 For different humidities - 32% (examined under fluorescent light); 1. monomethyl pinosylvin; 2. crude drug control of turpentine; 3. formula granules of turpentine. Figure 12 For different humidities - 75% (examined under fluorescent light); 1. monomethyl pinosylvin; 2. crude drug control of turpentine; 3. formula granules of turpentine.

[0079] 2.3.4 Verification

[0080] Three batches of formula granules of turpentine (Pinus massoniana) were used for thin-layer identification verification. The experimental results are shown in Figure 13 , in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as those in the chromatograms of the crude drug control and the reference substance.

[0081] Figure 12 For the verification results of different batches; among them, 1. monomethyl pinosylvin; 2. crude drug control of turpentine; 3 - 5 formula granules of turpentine (Pinus massoniana) (2302028, 2302029, 2302030).

[0082] Comparative Example 1

[0083] Take 1.0 g of this product, grind it finely, add 20 ml of 70% ethanol, sonicate for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Take 1 g of the crude drug control of turpentine, grind it finely, add 50 ml of water, decoct for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the crude drug control solution. According to the thin-layer chromatography method (General Rule 0502 of the Chinese Pharmacopoeia 2020 Edition), 10 μl of the above-mentioned test solution and crude drug control solution were respectively spotted on the same silica gel GF254 thin-layer plate, using chloroform - ethyl acetate - glacial acetic acid (12∶3∶1) as the developing agent, develop, take out, dry in air, and examine under ultraviolet light at 254 nm. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as those in the chromatograms of the reference substance and the crude drug control. The experimental results are shown in Figure 14 . Figure 14 Result diagram of Comparative Example 1 of the present invention; Note: 1 - 2 are the crude drug control of turpentine; 3 - 5 are the formula granules of turpentine (Pinus massoniana).

[0084] The results showed that with the above-mentioned thin-layer identification method, there were no obvious spots in the control crude drug of Pinus massoniana Lamb. twig and the formula granules of Pinus massoniana Lamb. twig, and the formula granules of Pinus massoniana Lamb. twig could not be effectively identified.

[0085] Comparative Example 2

[0086] Take 1.0 g of this product, grind it finely, add 20 ml of methanol, ultrasonicate for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Take 1 g of the control crude drug of Pinus massoniana Lamb. twig, grind it finely, add 50 ml of water, decoct for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the control crude drug solution. According to the test method of thin-layer chromatography (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition), absorb 10 μl of the above-mentioned test solution and control crude drug solution respectively, and spot them on the same silica gel GF254 thin-layer plate. Use toluene - petroleum ether - formic acid (7:2:1) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol test solution, heat at 105 °C until the spots are clearly developed, and examine under daylight. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the reference substance and the control crude drug, spots of the same color appear. The experimental results are shown in Figure 15 .

[0087] Figure 15 It is the result diagram of the method of Comparative Example 2. 1 - 3 are the control crude drug of Pinus massoniana Lamb. twig; 4 - 6 are the formula granules of Pinus massoniana Lamb. twig

[0088] The results showed that with the above-mentioned thin-layer identification method, there were no obvious spots in the control crude drug of Pinus massoniana Lamb. twig and the formula granules of Pinus massoniana Lamb. twig, and the formula granules of Pinus massoniana Lamb. twig could not be effectively identified.

[0089] Comparative Example 3

[0090] Take about 1 g of this product, grind it finely, add 25 ml of ethyl acetate (60 - 90 °C), ultrasonically extract for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol, filter, and obtain it. Separately take an appropriate amount of pinosylvin monomethyl ether reference substance, add methanol to make a solution containing 1 mg per 1 ml, and obtain it as the reference substance solution.

[0091] Take 1 g of the control crude drug of Pinus massoniana Lamb. twig, grind it finely, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to prepare the control crude drug solution.

[0092] Test according to the thin-layer chromatography method (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition). Pipette 25 μl of the test solution, 10 μl each of the reference crude drug solution and the reference substance solution, and spot them respectively on the same silica gel GF254 thin-layer plate. Use dichloromethane-95% ethanol (4:1) as the developing solvent, develop, take out, air dry, and examine under ultraviolet light (254 nm). In the chromatogram of the test solution, the main spots of the same color should appear at the positions corresponding to those in the chromatogram of the reference crude drug. Then spray with ninhydrin test solution, heat at 105 °C until the spots develop clearly, and examine under daylight. In the chromatogram of the test solution, the spots of the same color should appear at the positions corresponding to those in the chromatograms of the reference crude drug and the reference substance. The experimental results are shown in Figures 16 - 17 。 Figure 16 Figure showing the result of examination under ultraviolet light for Comparative Example 3; Figure 17 Figure showing the result of examination under daylight for Comparative Example 3; among which, 1 is the reference substance of pinosylvin monomethyl ether; 2 is the reference crude drug of turpentine; 3 is the formula granule of turpentine (Pinus massoniana Lamb.)

[0093] The results show that by using the above thin-layer identification method, no obvious spots are found for the reference substance of pinosylvin monomethyl ether, the reference crude drug of turpentine, and the formula granule of turpentine (Pinus massoniana Lamb.), and the formula granule of turpentine (Pinus massoniana Lamb.) cannot be effectively identified.

[0094] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A thin-layer identification method for pine cone formula granules derived from Pinus massoniana, comprising: A) Taking a test sample, after pretreatment, obtaining a test solution; The pretreatment is specifically: taking the test sample, dissolving it with ethyl acetate, ultrasonic extracting, dissolving the residue after filtration with methanol to obtain a test solution; Dissolving pinosylvin monomethyl ether reference substance with methanol to obtain a reference solution; B) Taking a reference medicinal material of pine cone, after pretreatment, obtaining a reference medicinal material solution; the pretreatment is specifically: taking the reference medicinal material of pine cone, decocting it with water, filtering, and dissolving the residue with methanol to obtain a reference medicinal material solution; C) Performing thin-layer chromatography detection on the test solution, the reference solution, and the reference medicinal material solution, using a silica gel G thin-layer plate; the developing agent is toluene - ethyl acetate - glacial acetic acid; the mass ratio of toluene - ethyl acetate - glacial acetic acid is 3:1:0.5; D) Examining, in the test chromatogram, if there are spots of the same color at the positions corresponding to the reference medicinal material and the reference substance, it is pine cone formula granules.

2. The method according to claim 1, wherein The mass-volume ratio of the test sample, ethyl acetate, and methanol is: 1 g: 25 mL: 1 mL; The time for ultrasonic extraction is 20 - 30 min; the boiling point of ethyl acetate is 60 - 90 °C.

3. The method according to claim 1, wherein In step B), the mass-volume ratio of the reference medicinal material, water, and methanol is: 1 g: 50 mL: 1 mL; The decocting time is 20 - 30 min.

4. The method according to claim 1, characterized in that, The sample application amount of the test solution is 10 - 16 μL; the sample application amount of the reference medicinal material solution is 10 - 25 μL.

5. The method according to claim 1, wherein In step D), the examination is to place it under an ultraviolet lamp for examination or spray with ninhydrin solution, heat for color development, and place it under a daylight lamp for examination; Among them, the ultraviolet light wavelength is 254 nm.

6. The method according to claim 1, wherein The examination temperature in step D) is 8 °C - 25 °C.

7. The method according to claim 6, characterized in that, The examination humidity in step D) is 32% rh - 75% rh.

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