Thin-layer identification method for perilla leaf and its preparation

Abstract

The present invention relates to the field of quality control of traditional Chinese medicines, and particularly to a thin-layer identification method for Perilla frutescens leaves and its preparations. First, a simple and rapid pretreatment method is used to obtain a test solution and a control crude drug solution, which are respectively spotted on the same polyamide film. A quaternary mixture of aromatic hydrocarbon / ester / alcohol / acid, preferably a quaternary mixture of toluene / ethyl acetate / methanol / formic acid with a volume ratio of (14-18):(3-10):1:(0.1-0.5), is used as the developing agent for development. Visual inspection is carried out under ultraviolet light to obtain a thin-layer chromatogram of the Perilla frutescens leaf sample with multiple information. Compared with the pharmacopoeia and existing literature reports, this method is simple, rapid, with clear chromatographic spots, good resolution, and low detection cost; evaluation is carried out using the consistency of the control crude drug, which is more accurate and scientific.

Description

Technical Field

[0001] The present invention relates to the field of quality control of traditional Chinese medicines, and particularly to a thin-layer identification method for Perilla leaf and its preparations. Background Art

[0002] Perilla leaf is the dried leaf (or with tender branches) of the plant Perilla frutescens (L.) Britt. of the Lamiaceae family. Its main chemical components include volatile oils, flavonoids and anthocyanins, phenolic acids, glycosides, triterpenoids and steroids, etc. Perilla leaf has the effects of relieving exterior syndrome and dispelling cold, regulating qi and harmonizing the stomach, and is clinically commonly used to treat wind-cold cold, cough and nausea, pregnancy vomiting, and fish and crab poisoning. Modern research also shows that it has effects such as anti-inflammatory, antitussive, antiemetic, sedative, analgesic, antipyretic, antibacterial, antiviral, anti-allergic, and antidepressant.

[0003] In Part I of the Chinese Pharmacopoeia (2020 Edition), two thin-layer identification methods are established under the identification item of "Perilla leaf". Among them, the first identification method uses perillaldehyde reference substance as a control to identify perillaldehyde in volatile oil components; the second identification method uses Perilla leaf reference medicinal material as a control to identify Perilla leaf powder. However, in actual operation, in the second identification method using 10% sulfuric acid ethanol solution for color development, the thin-layer chromatogram spots of the test sample (methanol extract of Perilla leaf powder) are light and blurred, and the resolution is poor. At the same time, when the developing agent (V 乙酸乙酯 :V 甲醇 :V 甲酸 :V 水 =9:0.5:1:0.5) is used for the thin-layer identification of Perilla leaf preparations, the above problems are more prominent, and it is difficult to make the thin-layer chromatogram spots of the test sample consistent with those of the reference medicinal material, resulting in inaccurate quality detection. Summary of the Invention

[0004] Problems to be Solved by the Invention

[0005] Aiming at the problem that the existing thin-layer identification methods are difficult to accurately detect the quality of Perilla leaf and its preparations in actual applications, the present invention intends to provide a new thin-layer identification method for Perilla leaf and its preparations to strengthen the quality detection of Perilla leaf samples and enrich the analysis means.

[0006] Solutions for Solving the Problems

[0007] To solve the above technical problems, the present invention provides the following technical solutions:

[0008] , A thin-layer identification method for perilla leaf samples, the method comprising preparing a test solution, preparing a control medicinal material solution, and thin-layer chromatography identification; wherein, the sample is at least one of a medicinal material sample, a cut crude drug sample, a standard decoction sample, and a formula granule sample, characterized in that,

[0009] The chromatographic conditions used in the thin-layer chromatography identification include the following:

[0010] Thin-layer plate: polyamide film;

[0011] Developing solvent: a quaternary mixture of aromatic hydrocarbon / ester / alcohol / acid, preferably a quaternary mixture of toluene / ethyl acetate / methanol / formic acid, more preferably a quaternary mixture of toluene / ethyl acetate / methanol / formic acid with a volume ratio of (14 - 18):(3 - 10):1:(0.1 - 0.5), and further preferably a quaternary mixture of toluene / ethyl acetate / methanol / formic acid with a volume ratio of 14:6:1:0.5;

[0012] Preferably, the chromatographic conditions used in the thin-layer chromatography identification further include the following:

[0013] Detection wavelength: 360 - 370 nm, preferably 365 nm;

[0014] Sample application volume: 0.5 - 2 μL, preferably 1 - 2 μL.

[0015] , The method according to [1], characterized in that,

[0016] When the sample is a medicinal material sample or a cut crude drug sample, the preparation of the test solution comprises the following steps: taking the sample, decocting with water or refluxing, filtering, evaporating the filtrate to dryness, ultrasonically treating the residue with an extraction solvent, filtering, evaporating the filtrate to dryness, and dissolving the residue with the extraction solvent to obtain the solution.

[0017] , The method according to [2], characterized in that,

[0018] In the decocting or refluxing step, the mass-volume ratio of the sample to water is 1 g:(25 - 200) mL; and / or, the time of the decocting or refluxing is 15 - 60 min;

[0019] And / or,

[0020] In the ultrasonically treating step, the mass-volume ratio of the sample to the extraction solvent is 1 g:(25 - 200) mL; and / or, the power of the ultrasonically treating is 250 - 650 W, the frequency is 40 kHz, and the time is 15 - 60 min;

[0021] And / or,

[0022] In the dissolution step, the mass-to-volume ratio of the sample to the extraction solvent is 1 g:(1 - 5) mL.

[0023] The method according to [1], characterized in that

[0024] When the sample is a standard decoction sample or a formula granule sample, the preparation of the test solution includes the following steps: taking the sample, ultrasonically treating it with an extraction solvent, filtering, evaporating the filtrate to dryness, and dissolving the residue in the extraction solvent to obtain the solution.

[0025] The method according to [4], characterized in that

[0026] In the ultrasonication step, the mass-to-volume ratio of the sample to the extraction solvent is 1 g:(25 - 200) mL; and / or, the power of the ultrasonication is 250 - 650 W, the frequency is 40 kHz, and the time is 15 - 60 min;

[0027] and / or

[0028] In the dissolution step, the mass-to-volume ratio of the sample to the extraction solvent is 1 g:(1 - 5) mL.

[0029] The method according to any one of [1] to [5], characterized in that

[0030] The preparation of the control crude drug solution includes the following steps: taking the control crude drug of Perilla leaf, decocting or refluxing it with water, filtering, evaporating the filtrate to dryness, ultrasonically treating the residue with an extraction solvent, filtering, evaporating the filtrate to dryness, and dissolving the residue in the extraction solvent to obtain the solution.

[0031] The method according to [6], characterized in that

[0032] In the decocting or refluxing step, the mass-to-volume ratio of the control crude drug to water is 1 g:(25 - 200) mL; and / or, the time of the decocting or refluxing is 15 - 60 min;

[0033] and / or

[0034] In the ultrasonication step, the mass-to-volume ratio of the control crude drug to the extraction solvent is 1 g:(25 - 200) mL; and / or, the power of the ultrasonication is 250 - 650 W, the frequency is 40 kHz, and the time is 15 - 60 min;

[0035] and / or

[0036] In the dissolution step, the mass-to-volume ratio of the control crude drug to the extraction solvent is 1 g:(1 - 5) mL.

[0037] , The method according to any one of [2] to [7], characterized in that

[0038] The extraction solvent is alcohol; preferably methanol.

[0039] , The method according to any one of [1] to [8], characterized in that

[0040] The thin-layer chromatography identification includes the following steps: respectively take the test solution and the control crude drug solution, spot them on a thin-layer plate, develop with a developing agent, and examine under an ultraviolet lamp;

[0041] Preferably, the spotting is carried out on the same thin-layer plate.

[0042] , The method according to [9], characterized in that

[0043] The examination includes observing whether the test solution chromatogram shows fluorescent spots of the same color at the position corresponding to the chromatogram of the control crude drug solution.

[0044] Effects of the Invention

[0045] The present invention provides a thin-layer identification method for perilla leaf samples. First, a simple and rapid pretreatment method is used to obtain the test solution and the control crude drug solution, which are respectively spotted on the same thin-layer plate, developed with a suitable developing agent, and examined under ultraviolet light to obtain a multi-information thin-layer chromatogram of perilla leaf samples. Compared with the pharmacopoeia and existing literature reports, this method is simple, rapid, has clear chromatographic spots, good resolution, and low detection cost; it is evaluated by the consistency of the control crude drug, which is more accurate and scientific. Description of the Drawings

[0046] Figure 1 is the thin-layer chromatogram of perilla leaf formula granules in Example 1 of the present invention; among them, 1 to 3 respectively correspond to perilla leaf formula granules with batch numbers 21030169, 21030179, and 21030189, and S corresponds to perilla leaf control crude drug.

[0047] Figure 2 is the thin-layer chromatogram of perilla leaf standard decoction in Example 1 of the present invention; among them, 1 to 3 respectively correspond to perilla leaf standard decoctions with batch numbers DG2001057, DG2001065, and DG2001013, and S corresponds to perilla leaf control crude drug.

[0048] Figure 3 is the thin-layer chromatogram of perilla leaf cut pieces in Example 1 of the present invention; among them, 1 to 3 respectively correspond to perilla leaf cut pieces with batch numbers YP2001057, YP2001065, and YP2001013, and S corresponds to perilla leaf control crude drug.

[0049] Figure 4 It is the thin-layer chromatogram with different sample application amounts in Example 2 of the present invention; among them, 1-4 correspond to 0.5 μL, 1 μL, 1.5 μL, and 2 μL of Perilla frutescens formula granules respectively, and 5-8 correspond to 0.5 μL, 1 μL, 1.5 μL, and 2 μL of Perilla frutescens control medicinal materials respectively.

[0050] Figure 5 It is the thin-layer chromatogram at different temperatures in Example 2 of the present invention; among them, 1-3 correspond to Perilla frutescens formula granules with batch numbers 21030169, 21030179, and 21030189 respectively, and S corresponds to Perilla frutescens control medicinal materials.

[0051] Figure 6 It is the thin-layer chromatogram at different humidities in Example 2 of the present invention; 1-3 correspond to Perilla frutescens formula granules with batch numbers 21030169, 21030179, and 21030189 respectively, and S corresponds to Perilla frutescens control medicinal materials.

[0052] Figure 7 It is the thin-layer chromatogram of Chinese medicinal materials in Example 3 of the present invention; among them, 1-3 correspond to Perilla frutescens medicinal materials with batch numbers YC2001057, YC2001065, and YC2001013 respectively, and 4-6 correspond to Perilla frutescens crispa medicinal materials with batch numbers YC2203060, YC2203061, and YC2203062 respectively.

[0053] Figure 8 It is the thin-layer chromatogram of the standard decoction in Example 3 of the present invention; among them, 1-3 correspond to Perilla frutescens crispa standard decoctions with batch numbers DG2203060, DG2203061, and DG2203062 respectively, and 4-6 correspond to Perilla frutescens standard decoctions with batch numbers DG2001057, DG2001065, and DG2001013 respectively.

[0054] Figure 9 It is the thin-layer chromatogram of Perilla frutescens cut pieces in Comparative Example 1 of the present invention; among them, 1-3 correspond to Perilla frutescens cut pieces with batch numbers YP2001057, YP2001065, and YP2001013 respectively, and S corresponds to Perilla frutescens control medicinal materials.

[0055] Figure 10 It is the thin-layer chromatogram obtained by spraying 10% sulfuric acid ethanol on a polyamide film in Comparative Example 1 of the present invention.

[0056] Figure 11 It is the thin-layer chromatogram in Comparative Example 2 of the present invention (developer: toluene-ethyl acetate-formic acid (6:3:1), thin-layer plate: silica gel G thin-layer plate); among them, 1 corresponds to Perilla frutescens formula granules, and 2 corresponds to Perilla frutescens control medicinal materials.

[0057] Figure 12 It is the thin-layer chromatogram in Comparative Example 2 of the present invention (developer: toluene - ethyl acetate - methanol - formic acid (8:3:2:0.2), thin-layer plate: silica gel G thin-layer plate); among them, 1 corresponds to the perilla leaf formula granules, and 2 corresponds to the control medicinal material of perilla leaf.

[0058] Figure 13 It is the thin-layer chromatogram in Comparative Example 2 of the present invention (developer: toluene - ethyl acetate - methanol - formic acid (18:3:1:0.1), thin-layer plate: polyamide film); among them, 1 corresponds to the perilla leaf formula granules, and 2 corresponds to the control medicinal material of perilla leaf.

[0059] Figure 14 It is the thin-layer chromatogram in Comparative Example 2 of the present invention (developer: toluene - ethyl acetate - methanol - formic acid (14:10:1:0.5), thin-layer plate: polyamide film); among them, 1 corresponds to the perilla leaf formula granules, and 2 corresponds to the control medicinal material of perilla leaf.

[0060] Figure 15 It is the thin-layer chromatogram in Comparative Example 2 of the present invention (developer: toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5), thin-layer plate: polyamide film); among them, 1 corresponds to the perilla leaf formula granules, and 2 corresponds to the control medicinal material of perilla leaf.

[0061] Figure 16 It is the thin-layer chromatogram of the perilla leaf formula granules in Comparative Example 3 of the present invention (developer: ethyl acetate - methanol - formic acid - water (9:0.5:1:0.5), thin-layer plate: silica gel G thin-layer plate).

[0062] Figure 17 It is the thin-layer chromatogram of the perilla leaf formula granules in Comparative Example 3 of the present invention (developer: toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5), thin-layer plate: polyamide film). Detailed Embodiments

[0063] The following is an explanation of the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various components described below. Various modifications can be made within the scope of the invention claimed, and the embodiments and examples obtained by appropriately combining the technical means of the respective inventions in different embodiments and examples are also included in the technical scope of the present invention.

[0064] In the present invention, the numerical range represented by "numerical value A to numerical value B" or "numerical value A - numerical value B" refers to the range including the endpoint numerical values A and B.

[0065] In the present invention, the numerical range represented by "above" or "below" refers to the numerical range including this number.

[0066] In the present invention, the meaning expressed by "may" includes both the meaning of performing a certain treatment and not performing a certain treatment. In this specification, "optional" or "optionally" means that the event or situation described next may or may not occur, and this description includes the situation where the event occurs and the situation where the event does not occur.

[0067] In the present invention, the term "a", "an", or "the" may mean "one", or may also mean "one or more", "at least one", and "one or more than one".

[0068] In the present invention, the terms "comprising", "having", "including", or "containing" may mean inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprising", "having", "including", or "containing" may also mean closed-ended, excluding additional, unrecited elements or method steps.

[0069] In the present invention, "medicinal materials" or "crude drugs" refer to the raw materials of traditional Chinese medicine that have not been processed or made into finished products.

[0070] In the present invention, "prepared slices of Chinese crude drugs" refer to the Chinese crude drugs that are formed after processing according to needs and are used for formula preparation, or the Chinese crude drugs that can be directly used in traditional Chinese medicine clinical practice.

[0071] In the present invention, "standard decoction" refers to the water decoction of single-flavor prepared slices of Chinese crude drugs prepared by a standardized process under the guidance of traditional Chinese medicine theory and based on clinical application, with reference to modern extraction methods.

[0072] In the present invention, "formula granules" are granules made from single-flavor prepared slices of Chinese crude drugs after being processed according to traditional standards and then extracted and concentrated for use in traditional Chinese medicine clinical formula preparation.

[0073] In the present invention, "test sample" refers to an experimental sample used for detection or identification.

[0074] In the present invention, "reference substance" refers to a standard substance used for identification, inspection, content determination, and calibration of the performance of inspection instruments.

[0075] In the present invention, "reference medicinal materials" refer to the standard medicinal materials that have completed species identification and are used for identifying the sample to be tested.

[0076] In the present invention, "Perilla leaf" is the dried leaf (or with tender branches) of Perilla frutescens (L.) Britt.

[0077] In the present invention, "spotting" refers to the process of adding droplets of the sample solution to be separated and identified onto a thin-layer plate.

[0078] In the present invention, "development" refers to the process in which the developer carries the sample components through the capillary action of the stationary phase on the thin-layer plate and migrates a certain distance on the thin-layer plate. The ratios between the components of the developer mentioned in the present invention are all volume ratios.

[0079] Unless otherwise defined, other technical and scientific terms used in the present invention have the same meanings as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0080] The present invention provides a thin-layer identification method for perilla leaf samples. According to the chemical structures and properties of the effective components of traditional Chinese medicines and following the extraction principle of "like dissolves like", appropriate extraction solvents are used to simply and quickly prepare the test solution and the solution of the control crude drug. Then, with an appropriate developer for development, various chemical components will, depending on their different abilities of adsorption, desorption, re-adsorption, and re-desorption with the selected developer, be well separated on the thin-layer plate. Furthermore, by virtue of various effective components with similar polarities, on the same thin-layer plate and under different inspection conditions, distinct color spots will appear, obtaining a thin-layer chromatogram with multiple information.

[0081] Specifically, a thin-layer identification method for perilla leaf samples provided by the present invention includes preparing a test solution, preparing a solution of the control crude drug, and thin-layer chromatographic identification. The identification method provided by the present invention is applicable to various perilla leaf samples, including but not limited to at least one of crude drug samples, cut crude drug samples, standard decoction samples, and formula granule samples.

[0082] <Preparation of the test solution and the solution of the control crude drug>

[0083] I. Crude drug sample / Cut crude drug sample

[0084] In some embodiments, when the sample is a crude drug sample or a cut crude drug sample, the preparation of the test solution includes the following steps: taking the sample, decocting with water or refluxing, filtering, evaporating the filtrate to dryness, ultrasonically treating the residue with the extraction solvent, filtering, evaporating the filtrate to dryness, and dissolving the residue with the extraction solvent to obtain the solution.

[0085] In order to improve the extraction efficiency of the active ingredients in the sample, in some specific embodiments, in the decocting or refluxing treatment step, the mass-volume ratio of the sample to water is 1 g:(25-200) mL, such as 1 g:25 mL, 1 g:30 mL, 1 g:35 mL, 1 g:40 mL, 1 g:45 mL, 1 g:50 mL, 1 g:55 mL, 1 g:60 mL, 1 g:65 mL, 1 g:70 mL, 1 g:75 mL, 1 g:80 mL, 1 g:85 mL, 1 g:90 mL, 1 g:95 mL, 1 g:100 mL, 1 g:150 mL or 1 g:200 mL, etc., preferably 1 g:(25-150) mL, more preferably 1 g:(25-100) mL, and even more preferably 1 g:(25-75) mL; the decocting or refluxing treatment time is 15-60 min, such as 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, etc., preferably 15-50 min, more preferably 15-40 min, and even more preferably 20-40 min.

[0086] In order to improve the extraction efficiency of the active ingredients in the sample, in some specific embodiments, in the ultrasonic treatment step, the mass-volume ratio of the sample to the extraction solvent is 1 g:(25-200) mL, such as 1 g:25 mL, 1 g:30 mL, 1 g:35 mL, 1 g:40 mL, 1 g:45 mL, 1 g:50 mL, 1 g:55 mL, 1 g:60 mL, 1 g:65 mL, 1 g:70 mL, 1 g:75 mL, 1 g:80 mL, 1 g:85 mL, 1 g:90 mL, 1 g:95 mL, 1 g:100 mL, 1 g:150 mL or 1 g:200 mL, etc., preferably 1 g:(25-150) mL, more preferably 1 g:(25-100) mL, and even more preferably 1 g:(25-75) mL.

[0087] In order to improve the extraction efficiency of the active ingredients in the sample, in some specific embodiments, in the ultrasonic treatment step, the power of the ultrasonic treatment is 250-650 W, such as 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, 550 W, 600 W or 650 W, etc., preferably 450-650 W, more preferably 550-650 W, the ultrasonic frequency is 40 kHz, and the ultrasonic time is 15-60 min, preferably 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, etc.

[0088] In order to improve the discriminability of the detection results, in some specific embodiments, in the dissolution step, the mass-to-volume ratio of the sample to the extraction solvent is 1 g:(1 - 5) mL, such as 1 g:1 mL, 1 g:2 mL, 1 g:3 mL, 1 g:4 mL, 1 g:5 mL, etc., and preferably 1 g:(2 - 4) mL.

[0089] In order to extract the active ingredients from the perilla leaf sample, in some embodiments, the present invention uses alcohol as the extraction solvent, such as methanol, ethanol, propanol or butanol, etc., preferably methanol or ethanol, and more preferably methanol.

[0090] II. Standard decoction sample / Formula granule sample

[0091] In some embodiments, when the sample is a standard decoction sample or a formula granule sample, the preparation of the test solution includes the following steps: taking the sample, subjecting it to ultrasonic treatment with an extraction solvent, filtering, evaporating the filtrate to dryness, and dissolving the residue in the extraction solvent to obtain the solution.

[0092] In order to improve the extraction efficiency of the active ingredients of the sample, in some specific embodiments, in the ultrasonic treatment step, the mass-to-volume ratio of the sample to the extraction solvent is 1 g:(25 - 200) mL, such as 1 g:25 mL, 1 g:30 mL, 1 g:35 mL, 1 g:40 mL, 1 g:45 mL, 1 g:50 mL, 1 g:55 mL, 1 g:60 mL, 1 g:65 mL, 1 g:70 mL, 1 g:75 mL, 1 g:80 mL, 1 g:85 mL, 1 g:90 mL, 1 g:95 mL, 1 g:100 mL, 1 g:150 mL or 1 g:200 mL, etc., preferably 1 g:(25 - 150) mL, more preferably 1 g:(25 - 100) mL, and even more preferably 1 g:(25 - 75) mL.

[0093] In order to improve the extraction efficiency of the active ingredients of the sample, in some specific embodiments, in the ultrasonic treatment step, the power of the ultrasonic treatment is 250 - 650 W, such as 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, 550 W, 600 W or 650 W, etc., preferably 450 - 650 W, more preferably 550 - 650 W, the ultrasonic frequency is 40 kHz, and the ultrasonic time is 15 - 60 min, preferably 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, etc.

[0094] In order to improve the discriminability of the detection results, in some specific embodiments, in the dissolution step, the mass-to-volume ratio of the sample to the extraction solvent is 1 g:(1 - 5) mL, such as 1 g:1 mL, 1 g:2 mL, 1 g:3 mL, 1 g:4 mL, 1 g:5 mL, etc., and preferably 1 g:(2 - 4) mL.

[0095] In order to extract the active ingredients from the perilla leaf sample, in some embodiments, the present invention uses alcohol as the extraction solvent, such as methanol, ethanol, propanol or butanol, etc., and preferably methanol.

[0096] III. Control crude drug

[0097] In some embodiments, the preparation of the control crude drug solution comprises the following steps: taking the perilla leaf control crude drug, decocting or refluxing with water, filtering, evaporating the filtrate to dryness, ultrasonically treating the residue with the extraction solvent, filtering, evaporating the filtrate to dryness, and dissolving the residue with the extraction solvent to obtain the solution.

[0098] For each condition in the preparation process of the control crude drug solution, it is the same as each condition in the preparation process of the test solution when the sample is a crude drug sample or a cut sample as described above.

[0099] In the thin-layer identification method of the perilla leaf sample provided by the present invention, the preparation operations of the test solution and the control crude drug solution are simple and rapid. The standard decoction and formula granules only need to be extracted by ultrasonic treatment with methanol for 15 - 60 min, and the crude drugs and cut samples only need an additional simple water decoction for 15 - 60 min, which provides great convenience for practical operation and application.

[0100] <Thin-layer chromatography identification>

[0101] In some embodiments, the thin-layer chromatography identification of the present invention comprises the following steps: respectively taking the test solution and the control crude drug solution, spotting on a thin-layer plate, developing with a developing agent, and examining under an ultraviolet lamp; preferably, the spotting is carried out on the same thin-layer plate.

[0102] The present invention uses a polyamide film as the stationary phase and a quaternary mixture of aromatic hydrocarbon / ester / alcohol / acid, preferably a quaternary mixture of toluene / ethyl acetate / methanol / formic acid as the developing agent, which effectively improves the resolution and clarity of each spot in the thin-layer chromatogram.

[0103] To further improve the resolvability of the thin-layer chromatogram, in some embodiments, the present invention uses a quaternary mixture of toluene / ethyl acetate / methanol / formic acid with a volume ratio of (14-18):(3-10):1:(0.1-0.5), preferably a volume ratio of (14-17):(4-10):1:(0.2-0.5), more preferably (14-16):(5-10):1:(0.3-0.5), even more preferably (14-15):(5-8):1:(0.4-0.5), and further preferably 14:6:1:0.5.

[0104] In addition, in some embodiments, during the thin-layer chromatography identification process, the spotting amounts of the test solution and the reference crude drug solution are independently 0.5-2 μL, such as 0.5 μL, 1 μL, 1.5 μL, or 2 μL, etc., preferably 1-2 μL.

[0105] To better observe the results of thin-layer chromatography, after the developing agent is developed, it can be inspected using an ultraviolet lamp with a wavelength of 360-370 nm, preferably 365 nm. At the same time, in the thin-layer chromatography identification of the present invention, a color-developing agent is not required. Further, by observing whether the test solution chromatogram shows fluorescent spots of the same color at the position corresponding to the chromatogram of the reference crude drug solution, it can be determined whether the test solution contains the components of the perilla leaf sample.

[0106] Examples

[0107] To more clearly describe the technical solution of the present invention, the embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0108] The instruments and reagents used in the examples of the present invention are as follows:

[0109] Instruments: Thin-layer automatic imager (CAMAG TLC VISUALIZER), ten-thousandth balance (Mettler Toledo, Switzerland), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), electronic temperature-controlled electric furnace (Jiangyin Poly Scientific Research Instruments Co., Ltd.), GKC constant temperature water bath (Nantong Huatai Experimental Instrument Co., Ltd.), AS165W centrifuge (As One (Shanghai) Trading Co., Ltd.), polyamide film (Taizhou Luqiao Sijia Biochemical Plastics Factory, Zhejiang Province).

[0110] Test drugs: Toluene (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), formic acid (Sinopharm Chemical Reagent Co., Ltd.), methanol (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.) are all of analytical purity; water is ultrapure water.

[0111] The reference crude drug of Perilla frutescens leaves (batch number: 120914 - 201712) was purchased from the National Institutes for Food and Drug Control.

[0112] The Perilla frutescens leaves formula granules (batch numbers: 21030169, 21030179, 21030189), the Perilla frutescens leaves standard decoctions (batch numbers: DG2001057, DG2001065, DG2001013), and the Perilla frutescens leaves cut pieces (batch numbers: YP2001057, YP2001065, YP2001013) were prepared by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0113] The Perilla frutescens leaves crude drugs (batch numbers: YC2001057, YC2001065, YC2001013) and the Perilla frutescens crispa Thunb. crude drugs (YC2203060, YC2203061, YC2203062) were provided by Tianjiang Pharmaceutical Co., Ltd.

[0114] The Perilla frutescens crispa Thunb. standard decoctions (DG2203060, DG2203061, DG2203062) were prepared by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0115] For those without specific experimental steps or conditions indicated in the examples of the present invention, they were carried out according to conventional experimental steps, conditions or the conditions recommended by the manufacturer. For reagents or instruments without the indicated manufacturer, they were all conventional products that could be obtained through commercial purchase.

[0116] Example 1

[0117] Preparation of the cut pieces test solution: Take 1 g of the powder of Perilla frutescens leaves cut pieces, add 50 mL of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, and add 2 mL of methanol to dissolve the residue to obtain the test solution.

[0118] Preparation of the formula granules or standard decoctions test solution: Take 0.5 g of Perilla frutescens leaves formula granules or Perilla frutescens leaves standard decoctions, add 25 mL of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, and add 2 mL of methanol to dissolve the residue to obtain the test solution.

[0119] Preparation of the reference medicinal material solution: Take 1 g of Perilla leaf reference medicinal material, add 50 mL of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25 mL of methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 mL of methanol to obtain the reference medicinal material solution.

[0120] Thin-layer chromatography procedure: Pipette 1 - 2 μL each of the test solution and the reference medicinal material solution, and spot them respectively on the same polyamide film. Use toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) as the developing solvent, develop, take out, air dry, and examine under an ultraviolet lamp (365 nm).

[0121] Evaluation requirement: In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0122] Result analysis: Figure 1 1 - 3 correspond to the chromatograms of the test samples of Perilla leaf formula granules with batch numbers 21030169, 21030179, and 21030189 respectively, and S is the chromatogram of the Perilla leaf reference medicinal material. Figure 2 1 - 3 correspond to the chromatograms of the test samples of the standard decoction of Perilla leaf with batch numbers DG2001057, DG2001065, and DG2001013 respectively, and S is the chromatogram of the Perilla leaf reference medicinal material. Figure 3 1 - 3 correspond to the chromatograms of the test samples of Perilla leaf cut pieces with batch numbers YP2001057, YP2001065, and YP2001013 respectively, and S is the chromatogram of the Perilla leaf reference medicinal material. It can be seen that by using the thin-layer detection method provided by the present invention, in the chromatograms of multiple batches of Perilla leaf and its preparations, fluorescent spots of the same color can appear at the corresponding positions as in the chromatogram of the reference medicinal material, and each spot is bright, clear, and easy to distinguish.

[0123] Example 2

[0124] Verify the durability of the thin-layer chromatography in Example 1, specifically including:

[0125] (1) Investigation of different sample application amounts:

[0126] Take Perilla leaf formula granules (batch number: 21030179) and Perilla leaf reference medicinal material, and prepare the test solution and the reference medicinal material solution respectively according to the method described in Example 1. Then take 0.5 μL, 1 μL, 1.5 μL, 2 μL of the test solution of Perilla leaf formula granules and 0.5 μL, 1 μL, 1.5 μL, 2 μL of the reference medicinal material solution of Perilla leaf, spot them on the same polyamide film, use toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) as the developing solvent, develop, take out, air dry, and examine under an ultraviolet lamp (365 nm). The results are shown in Figure 4 ,Figure 4 1 - 4 correspond to the chromatograms of the test samples of Perilla leaf formula granules at 0.5 μL, 1 μL, 1.5 μL, and 2 μL respectively, and 5 - 8 correspond to the chromatograms of the control crude drug of Perilla leaf at 0.5 μL, 1 μL, 1.5 μL, and 2 μL respectively. It can be seen that Figure 4 when the sample application volume is 0.5 μL - 2 μL, the chromatograms of the Perilla leaf formula granules and the control crude drug chromatograms show clear spots at the corresponding positions, without other interferences.

[0127] (2) Investigation at different temperatures:

[0128] Take Perilla leaf formula granules (batch numbers: 21030169, 21030179, 21030189) and the control crude drug of Perilla leaf, and prepare the test sample solution and the control crude drug solution respectively according to the method described in Example 1. Then take 1 μL of the test sample solution of the Perilla leaf formula granules and 1.5 μL of the control crude drug solution of the Perilla leaf, spot them on the same polyamide film, use toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) as the developing agent, and develop them respectively under low temperature (T: 4°C, RH: 35%), normal temperature (T: 20.8°C, RH: 35%), and high temperature (T: 33°C, RH: 35%) conditions. Take them out, dry them in the air, and examine them under an ultraviolet lamp (365 nm). The results are shown in Figure 5 , Figure 5 1 - 3 correspond to the chromatograms of the test samples of the Perilla leaf formula granules with batch numbers 21030169, 21030179, and 21030189 respectively, and S corresponds to the chromatogram of the control crude drug of the Perilla leaf. It can be seen that Figure 5 under different temperature conditions, the corresponding positions of the chromatograms of the test samples of the Perilla leaf formula granules and the control crude drug chromatograms show fluorescent spots of the same color, and the separation effects are all good. The experimental results show that the temperature has little influence on the thin - layer identification method of the Perilla leaf formula granules provided by the present invention, indicating that the thin - layer identification method has good durability for different temperatures.

[0129] (3) Investigation at different humidities:

[0130] Take Perilla leaf formula granules (batch numbers: 21030169, 21030179, 21030189) and the control crude drug of Perilla leaf, and prepare the test sample solution and the control crude drug solution respectively according to the method described in Example 1. Then take 1 μL of the test sample solution of the Perilla leaf formula granules and 1.5 μL of the control crude drug solution of the Perilla leaf, spot them on the same polyamide film, use toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) as the developing agent, and develop them respectively under different humidity (RH: 18%, 35%, 88%) conditions. Take them out, dry them in the air, and examine them under an ultraviolet lamp (365 nm). The results are shown in Figure 6 , Figure 6Nos. 1-3 respectively correspond to the chromatograms of the test samples of Perilla leaf formula granules with batch numbers 21030169, 21030179, and 21030189. S corresponds to the chromatogram of the control medicinal material of Perilla leaf. It can be seen from Figure 6 that under different humidity conditions, the corresponding positions of the chromatograms of the test samples of Perilla leaf formula granules and the chromatogram of the control medicinal material show fluorescent spots of the same color, and the separation effects are all good. The experimental results show that humidity has little influence on the thin-layer identification method of the Perilla leaf formula granules provided by the present invention, indicating that this thin-layer identification method has good durability for different humidities.

[0131] Example 3

[0132] Perform a specificity test on the thin-layer identification in Example 1:

[0133] Use Perilla leaf and Perilla frutescens var. crispa for comparison. Perilla frutescens var. crispa is a variety of Perilla frutescens and is widely cultivated in various parts of China. It is a type of Perilla with a large planting scale and is used as a vegetable.

[0134] Take Perilla leaf medicinal materials and Perilla frutescens var. crispa medicinal materials, and their standard decoctions, and prepare test solutions according to the preparation methods of the control medicinal material solution and the standard decoction test solution described in Example 1 respectively for thin-layer chromatography detection. The results are as shown in Figures 7 - 8 , Figure 7 where Nos. 1-3 respectively correspond to the chromatograms of the test samples of Perilla leaf medicinal materials with batch numbers: YC2001057, YC2001065, and YC2001013. Nos. 4-6 respectively correspond to the chromatograms of the test samples of Perilla frutescens var. crispa medicinal materials with batch numbers YC2203060, YC2203061, and YC2203062; Figure 8 where Nos. 1-3 respectively correspond to the chromatograms of the test samples of the standard decoctions of Perilla frutescens var. crispa with batch numbers DG2203060, DG2203061, and DG2203062. Nos. 4-6 respectively correspond to the chromatograms of the test samples of the standard decoctions of Perilla leaf with batch numbers DG2001057, DG2001065, and DG2001013. It can be seen that by using the thin-layer detection method provided by the present invention, compared with the chromatograms of Perilla leaf medicinal materials and their preparations, different fluorescent spots appear in the chromatograms of Perilla frutescens var. crispa medicinal materials and their preparations, and each spot is bright and clear, and can be clearly distinguished. The experimental results show that the thin-layer detection method provided by the present invention for Perilla leaf and its preparations has good specificity and can effectively identify Perilla leaf and its related species.

[0135] Comparative Example 1

[0136] In this comparative example, the thin-layer chromatography detection of Perilla leaf decoction pieces is carried out by the method in Part I of the Chinese Pharmacopoeia (2020 Edition). The specific process is as follows:

[0137] Preparation of the test solution: Take 0.5 g of the powder of Perilla leaf decoction pieces, add 25 mL of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, concentrate the filtrate to dryness, add 2 mL of methanol to dissolve, and use it as the test solution.

[0138] Preparation of the reference medicinal material solution: Take 0.5 g of the Perilla leaf reference medicinal material, add 25 mL of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, concentrate the filtrate to dryness, add 2 mL of methanol to dissolve, and prepare the reference medicinal material solution.

[0139] Thin-layer identification procedure: Pipette 3 μL each of the test solution and the reference medicinal material solution, and spot them on the same silica gel G thin-layer plate respectively. Use ethyl acetate - methanol - formic acid - water (9:0.5:1:0.5) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly colored, and examine under an ultraviolet lamp (365 nm).

[0140] Evaluation requirements: In the chromatogram of the test sample, at the position corresponding to the chromatogram of the reference medicinal material, fluorescent spots of the same color should appear.

[0141] The results are shown in Figure 9 , Figure 9 In it, S corresponds to the chromatogram of the Perilla leaf reference medicinal material, and 1 - 3 correspond to the chromatograms of the test samples of Perilla leaf decoction pieces (batch numbers: YP2001057, YP2001065, YP2001013). It can be seen that in the thin-layer chromatograms of Perilla leaf decoction pieces and the reference medicinal material obtained by the method in Part I of the Chinese Pharmacopoeia (2020 Edition), each spot is light and blurred, and the resolution is poor, which is not conducive to discrimination.

[0142] Furthermore, in thin-layer chromatography, 10% sulfuric acid ethanol is often used for coloring flavonoid components. In order to investigate whether the coloring agent (i.e., 10% sulfuric acid ethanol) under Perilla leaf in Part I of the Chinese Pharmacopoeia (2020 Edition) is applicable to the thin-layer detection method provided by the present invention, experiments were carried out according to the method described in Example 1. After development and air drying, spray with 10% sulfuric acid ethanol solution and examine under an ultraviolet lamp (365 nm). The results are as Figure 10 , for the thin-layer plate selected by the present invention, which is a polyamide film, if sprayed with 10% sulfuric acid ethanol for coloring, it is extremely easy to be corroded, so it is not recommended to add a coloring agent.

[0143] Comparative Example 2

[0144] This comparative example investigated the influence of different developing agents on the thin-layer detection method provided by the present invention to determine the preferred developing agent. The specific process is as follows:

[0145] (1) Take the Perilla leaf formula granules (batch number: 21030169) and the Perilla leaf control medicinal material, and prepare the test solution and the control medicinal material solution respectively according to the method described in Example 1. Spot them on the same silica gel G thin layer plate respectively, and use toluene - ethyl acetate - formic acid (6:3:1) recorded in the "Taiwan Pharmacopoeia" of Taiwan, China as the developing agent. Develop, take out, dry, and examine under an ultraviolet lamp (365nm). The results are as Figure 11 , where 1 corresponds to the chromatogram of the test solution of the Perilla leaf formula granules, and 2 corresponds to the chromatogram of the Perilla leaf control medicinal material. From Figure 11 it can be seen that under this method, the pigment interference in the chromatogram of the control medicinal material sample is extremely large, affecting the spot information.

[0146] (2) Take the Perilla leaf formula granules (batch number: 21030169) and the Perilla leaf control medicinal material, and prepare the test solution and the control medicinal material solution respectively according to the method described in Example 1. Spot them on the same silica gel G thin layer plate respectively, and use toluene - ethyl acetate - methanol - formic acid (8:3:2:0.2) as the developing agent. Develop, take out, dry, and examine under an ultraviolet lamp (365nm). The results are as Figure 12 , where 1 corresponds to the chromatogram of the test solution of the Perilla leaf formula granules, and 2 corresponds to the chromatogram of the Perilla leaf control medicinal material. From Figure 12 it can be seen that under this method, although the spot resolution is improved, the pigment influence is still very serious, and the spots are blurred.

[0147] (3) Take the Perilla leaf formula granules (batch number: 21030169) and the Perilla leaf control medicinal material, and prepare the test solution and the control medicinal material solution respectively according to the method described in Example 1. Spot them on the same polyamide film respectively, and use toluene - ethyl acetate - methanol - formic acid (18:3:1:0.1) as the developing agent. Develop, take out, dry, and examine under an ultraviolet lamp (365nm). The results are as Figure 13 , where 1 corresponds to the chromatogram of the test solution of the Perilla leaf formula granules, and 2 corresponds to the chromatogram of the Perilla leaf control medicinal material. From Figure 13 it can be seen that under this method, the spot information is less, but compared with the methods shown in (1) or (2) above, the resolution is significantly improved, and the influence of the pigment in the medicinal material is greatly reduced.

[0148] (4) Take the Perilla leaf formula granules (batch number: 21030169) and the Perilla leaf control medicinal material, and prepare the test solution and the control medicinal material solution respectively according to the method described in Example 1. Spot them on the same polyamide film respectively, and use toluene - ethyl acetate - methanol - formic acid (14:10:1:0.5) as the developing agent. Develop, take out, dry, and examine under an ultraviolet lamp (365nm). The results are as Figure 14 , where 1 corresponds to the chromatogram of the test solution of the Perilla leaf formula granules, and 2 corresponds to the chromatogram of the Perilla leaf control medicinal material. From Figure 14It can be seen that compared with the method shown in (3) above, under this method, the chromatographic spot information is further increased.

[0149] (5) Take Perilla leaf formula granules (batch number: 21030169) and Perilla leaf reference medicinal materials, and prepare the test solution and reference medicinal material solution respectively according to the method described in Example 1. Spot them on the same polyamide film respectively, use toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) as the developing agent, develop, take out, dry in the air, and examine under an ultraviolet lamp (365 nm). The results are as Figure 15 , where 1 corresponds to the test chromatogram of Perilla leaf formula granules, and 2 corresponds to the chromatogram of Perilla leaf reference medicinal materials. It can be Figure 15 seen that compared with the method shown in (4) above, under this method, the specific migration value of the chromatographic spots changes slightly, the resolution is better, the positions of the spots in the test chromatogram and the chromatogram of the reference medicinal material are more consistent, and each spot is bright and clear, easy to distinguish. Therefore, toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) is used as the preferred developing agent.

[0150] Comparative Example 3

[0151] In this comparative example, the thin - layer developing agent under Perilla leaf in Part I of the Chinese Pharmacopoeia (2020 Edition): ethyl acetate - methanol - formic acid - water (9:0.5:1:0.5) and the developing agent provided by the present invention: toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) were used for development, and the preparation methods of the test solution and reference medicinal material solution and the application of the developing agent to the detection of Perilla leaf formula granules were compared and investigated. The specific process is as follows:

[0152] 1: Take 0.5 g of Perilla leaf formula granules (batch number: 21030169), grind them finely, add 25 mL of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol to obtain the test solution.

[0153] S1: Take 1 g of Perilla leaf reference medicinal material, add 25 mL of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol to obtain the reference medicinal material solution.

[0154] S2: Take 1 g of Perilla leaf reference medicinal material, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol to obtain the reference medicinal material solution.

[0155] Test according to the thin-layer chromatography method (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition). Separately absorb 3 μL of the test solution and the reference crude drug solution, spot them on the same silica gel G thin-layer plate, use ethyl acetate - methanol - formic acid - water (9:0.5:1:0.5) as the developing solvent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly visible, and examine under an ultraviolet lamp (365 nm); alternatively, absorb 1 - 2 μL of the test solution and the reference crude drug solution respectively, spot them on the same polyamide film, use toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) as the developing solvent, develop, take out, air dry, and examine under an ultraviolet lamp (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference crude drug.

[0156] The results are shown in Figures 16 - 17 , in the figure, 1 is the chromatogram of the test sample of Perilla leaf formula granules, and S1 - S2 are the chromatograms of the reference crude drugs of Perilla leaf prepared by 2 corresponding preparation methods. Among them Figure 16 the thin-layer plate is a silica gel G thin-layer plate, and the developing solvent is ethyl acetate - methanol - formic acid - water (9:0.5:1:0.5), Figure 17 the thin-layer plate is a polyamide film, and the developing solvent is toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5).

[0157] When using ethyl acetate - methanol - formic acid - water (9:0.5:1:0.5) as the developing solvent, the spot resolution is not clear and the resolution is poor, and the spots at the corresponding positions of the chromatogram of the test sample and the chromatogram of the reference crude drug are inconsistent. When using toluene - ethyl acetate - methanol - formic acid (14:6:1:0.5) as the developing solvent, when the reference crude drug of Perilla leaf is prepared by method S1 (the preparation method of Perilla leaf crude drug slices and reference crude drug in the pharmacopoeia), the pigment interference in the sample chromatogram is extremely large, affecting the spot information, indicating that this preparation method of the reference crude drug solution is not suitable for the identification of Perilla leaf preparations, and the preparation method of the reference crude drug in the present invention is more suitable for the identification of Perilla leaf preparations.

[0158] Comprehensive analysis: A thin-layer identification method for Perilla leaf and its preparations provided by the present invention has simple and rapid preparation operation of the test sample. For standard decoctions and formula granules, only methanol ultrasonic extraction for 15 - 60 minutes is required, and for crude drugs and slices, only additional simple water decoction for 15 - 60 minutes is required.

[0159] The developing agent in the present invention can be used for thin-layer identification of perilla leaf medicinal slices and also for thin-layer identification of perilla leaf preparations. The chromatographic spots are richer and the resolution is good, overcoming the defects of fuzzy chromatographic spots, poor resolution and non-corresponding spots with the control medicinal materials in the existing thin-layer identification methods. The thin-layer identification method of the present invention uses "fluorescent spots of the same color at the corresponding positions in the test sample chromatogram and the control medicinal material chromatogram" for quality evaluation. Compared with the prior art, there are more corresponding spots between the sample and the control medicinal material, and the problem of inconsistency between the test sample chromatogram of the perilla leaf preparation and the control medicinal material chromatogram is eliminated, which is more comprehensive, accurate and credible.

Claims

1. A thin layer chromatography identification method for a perilla leaf sample, the method comprising preparing a test solution, preparing a reference medicinal material solution and thin layer chromatography identification; wherein, The sample is at least one of a medicinal material sample, a decoction piece sample, a standard decoction sample and a formula granule sample, characterized in that: When the sample is a medicinal material sample or a decoction piece sample, the preparation of the test solution comprises the following steps: taking the sample, decocting or refluxing the sample with water, filtering, evaporating the filtrate to dryness, ultrasonically treating the residue with an extraction solvent, filtering, evaporating the filtrate to dryness, and dissolving the residue with the extraction solvent to obtain the sample; When the sample is a standard decoction sample or a formula granule sample, the preparation of the test solution comprises the following steps: taking the sample, adding an extraction solvent for ultrasonic treatment, filtering, evaporating the filtrate to dryness, and dissolving the residue in the extraction solvent to obtain; The preparation of the control medicinal material solution comprises the following steps: taking a control medicinal material of Perilla leaf, decocting or refluxing it with water, filtering, evaporating the filtrate to dryness, ultrasonically treating the residue with an extraction solvent, filtering, evaporating the filtrate to dryness, and dissolving the residue with the extraction solvent to obtain; The extraction solvent is alcohol; The chromatographic conditions used in the thin layer chromatography identification include the following: Thin layer sheet: polyamide film; Developing solvent: a quaternary mixture of toluene / ethyl acetate / methanol / formic acid in a volume ratio of 14:6:1:0.5; Detection wavelength: 360~370nm.

2. The method according to claim 1, characterized in that The chromatographic conditions used in the thin layer chromatography identification also include the following: Sample volume: 0.5~2μL.

3. The method according to claim 2, characterized in that The detection wavelength is 365nm; The sample volume is 1~2μL.

4. The method according to claim 1, characterized in that When the sample is a medicinal material sample or a decoction piece sample, in the process of preparing the test solution, in the decoction or reflux treatment step, the mass volume ratio of the sample to water is 1g: (25-200)mL; and / or, the decoction or reflux treatment time is 15-60min; and / or, In the ultrasonic treatment step, the mass volume ratio of the sample to the extraction solvent is 1 g: (25-200) mL; and / or, the power of the ultrasonic treatment is 250-650 W, the frequency is 40 kHz, and the time is 15-60 min; and / or, In the dissolving step, the mass volume ratio of the sample to the extraction solvent is 1 g: (1-5) mL.

5. The method according to claim 1, characterized in that When the sample is a standard decoction sample or a formula granule sample, in the process of preparing the test solution, in the ultrasonic treatment step, the mass volume ratio of the sample to the extraction solvent is 1g: (25-200)mL; and / or, the power of the ultrasonic treatment is 250-650W, the frequency is 40kHz, and the time is 15-60min; and / or, In the dissolving step, the mass volume ratio of the sample to the extraction solvent is 1 g: (1-5) mL.

6. The method according to claim 1, characterized in that In the process of preparing the control medicinal material solution, in the decoction or reflux treatment step, the mass volume ratio of the control medicinal material to water is 1 g: (25-200) mL; and / or, the decoction or reflux treatment time is 15-60 min; and / or, In the ultrasonic treatment step, the mass volume ratio of the control medicinal material to the extraction solvent is 1 g: (25-200) mL; and / or, the power of the ultrasonic treatment is 250-650 W, the frequency is 40 kHz, and the time is 15-60 min; and / or, In the dissolution step, the mass volume ratio of the reference medicinal material to the extraction solvent is 1 g: (1-5) mL.

7. The method according to claim 1, characterized in that The extraction solvent is methanol.

8. The method according to claim 1, characterized in that The thin layer chromatography identification comprises the following steps: taking the test sample solution and the control medicinal material solution respectively, spotting on a thin layer plate, developing with a developing agent, and inspecting under an ultraviolet lamp.

9. The method according to claim 8, characterized in that The spotting was performed on the same thin layer plate.

10. The method according to claim 8, characterized in that The inspection includes observing whether the chromatogram of the test solution shows fluorescent spots of the same color at positions corresponding to the chromatogram of the control medicinal material solution.

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