Thin-layer identification method for compound traditional Chinese medicine preparation containing liquorice

Abstract

The invention discloses a thin-layer identification method for a compound traditional Chinese medicine preparation containing liquorice, and belongs to the technical field of thin-layer chromatography. The technical problem to be solved is to provide a thin-layer identification method capable of simultaneously identifying the white mulberry root-bark, the cortex lycii radicis and the liquorice, and the thin-layer identification method is less in sampling and high in detection efficiency. According to the technical scheme, the thin-layer identification method for the liquorice-containing compound traditional Chinese medicine preparation is characterized by comprising the following steps of: spotting a test solution and a reference solution on a thin-layer plate, respectively spotting the test solution and the reference solution on the same silica gel G thin-layer plate to form a strip shape, taking a butyl acetate-methanol-acetone-water-formic acid solution as a developing solvent, and carrying out thin-layer chromatography on the test solution and the reference solution to obtain the liquorice-containing compound traditional Chinese medicine preparation. The volume ratio of butyl acetate to methanol to acetone to water to formic acid is 5: (0.5-2): (2-3): (0.5-1): 1, expanding, taking out, airing, spraying a 10% sulfuric acid ethanol solution of 2% vanillin, heating until the color of spots is clear, and inspecting under sunlight.

Description

Technical Field

[0001] This invention relates to the field of thin-layer chromatography technology, and more specifically, to a thin-layer identification method for compound traditional Chinese medicine preparations containing licorice. Background Technology

[0002] For understanding the technical content of this invention: Xiebai Powder, a classic formula from the *Synopsis of Prescriptions of the Golden Chamber*, is mainly composed of mulberry bark, lycium bark, licorice root, and japonica rice. It has the effects of clearing heat from the internal organs, purging lung heat, relieving cough and asthma. It is primarily used to treat cough and asthma due to lung heat, characterized by wheezing and coughing, hot skin, especially in the late afternoon, red tongue with yellow coating, and a thready, rapid pulse. Clinically, it is often used to treat early-stage measles in children, pneumonia, or bronchitis, where the condition is attributed to latent heat in the lungs. Xiebai Powder is a typical decoction formula, consisting of one ounce each of stir-fried mulberry bark and lycium bark, and one mace of roasted licorice root. After being ground into powder, a pinch of japonica rice and two small cups of water are added, and the decoction is simmered until reduced to seven-tenths. The standard decoction is prepared under the guidance of traditional Chinese medicine theory, based on clinical application, using modern methods and standardized procedures, and is appropriately concentrated or dried.

[0003] Thin-layer chromatography (TLC) is currently the most common identification method in national standards for traditional Chinese medicine (TCM) preparations. It is characterized by simple equipment, easy and rapid operation, and high specificity, enabling the detection of a single component within complex compositions. It is particularly suitable for identifying a single TCM herb or component in TCM preparations. Since most TCM preparations lack quality control indicators, given the numerous components in traditional TCM preparations, research should focus on establishing TLC identification methods to identify relatively well-defined main medicinal components or known components.

[0004] Because traditional Chinese medicines have complex components, appropriate chromatographic conditions such as thin-layer plates, developing solvents, and colorimetric methods should be selected. Appropriate methods should be used to extract and purify the sample to minimize interfering components and ensure that the obtained chromatogram has high clarity, good separation, obvious spots, and good reproducibility.

[0005] Relevant patent documents retrieved: This document, published in China (CN106645545A) on October 2, 2018, discloses a thin-layer chromatography (TLC) method for identifying licorice. The method involves spotting 1-2 μL of licorice sample, reference material, and standard solution onto a TLC plate. A developing solvent, consisting of chloroform, methanol, formic acid, and ethyl acetate mixed in a volume ratio of 10:3:2:3, is added to the developing tank. After the developing tank is saturated with the solvent, the spotted samples are... A thin-layer chromatography (TLC) plate is placed in the solution and developed to 13 cm. The plate is then removed, dried, and sprayed with a 10% sulfuric acid-ethanol solution for color development. The plate is heated to 105°C until the spots are clearly visible and examined under 365 nm ultraviolet light. The preparation method for the licorice test sample is as follows: licorice is pulverized and passed through a 12-mesh sieve. The resulting powder is refluxed with methanol for 1 hour, then filtered at 18-25°C. The filtrate is passed through a neutral alumina column, eluted with methanol, and the eluent is collected and extracted with water-saturated n-butanol. Finally, the n-butanol is evaporated to dryness, and methanol is added again to obtain the licorice test sample. This invention, based on the properties of licorice, studies and improves the preparation method and developing agent of the test sample, thereby establishing a simple, rapid, specific, and reproducible TLC identification method for licorice.

[0006] Relevant non-patent literature retrieved: The journal or book title is "Master's Thesis of Changchun University of Traditional Chinese Medicine", the document title is "Research on the Preparation Process and Quality Standard of Material Reference for Xiebai Powder", and the publication date is June 2021. This document discloses the thin-layer chromatography identification of the material reference for Xiebai Powder, and establishes thin-layer chromatography identification methods for the medicinal materials of Xiebai Powder, namely, mulberry bark, lycium bark, and licorice.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: There is no existing technology that studies the thin-layer chromatography method for simultaneously analyzing the whole mulberry bark, lycium bark, and licorice root in Xiebai powder.

[0008] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: Existing technologies employ three sample processing methods to prepare test solutions for mulberry bark, lycium bark, and licorice, involving solvents such as 75% ethanol, anhydrous ethanol, diethyl ether, methanol, and n-butanol, and utilizing techniques such as ultrasonication, reflux, and extraction. This method, however, uses only n-butanol for extraction and methanol for volume adjustment to prepare the test solution. Literature studies have used 7 or 8 solvents as developing agents, including highly toxic reagents such as chloroform and toluene, and employed three thin-layer plates and two colorimetric reagents. In contrast, this invention uses only 10 mL of commonly used reagents—butyl acetate, methanol, acetone, water, and formic acid—to perform thin-layer identification of mulberry bark, lycium bark, and licorice in a single step. This achievement is based on the inventors' long-term experience and extensive experimentation. Summary of the Invention

[0009] The purpose of this invention is to provide: A thin-layer chromatography method for identifying compound traditional Chinese medicine preparations containing licorice, and related technologies, to provide a thin-layer chromatography method that can simultaneously identify mulberry bark, lycium bark, and licorice, with the advantages of small sample size and high detection efficiency.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definition of standard chemical terms can be found in the reference "Analytical Chemistry, edited by Wuhan University, Higher Education Press, 4th edition, 2004".

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as crushing, shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] In a first aspect, the present invention provides: a thin-layer chromatography method for identifying compound traditional Chinese medicine preparations containing licorice, comprising the following steps: S1: Dissolve the sample to be tested, extract it with solvent, and obtain the test solution; S2: Take Lycium chinense root bark reference material, Glycyrrhiza uralensis reference material and Morus alba root bark glycoside A reference standard respectively, and extract them by ultrasonication to obtain Lycium chinense root bark reference standard solution, Glycyrrhiza uralensis reference standard solution and Morus alba root bark glycoside A reference standard solution; S3: Spot the test solution and the reference solution onto a thin-layer plate, respectively, on the same silica gel G thin-layer plate to form strips. Use butyl acetate-methanol-acetone-water-formic acid solution as the developing solvent, wherein the volume ratio of butyl acetate-methanol-acetone-water-formic acid is 5:(0.5-2):(2-3):(0.5-1):1. Develop, remove, air dry, spray with 2% vanillin in 10% sulfuric acid ethanol solution, heat until the spots are clearly visible, and examine under sunlight.

[0016] In step S1, the preparation method of the sample to be tested is as follows: using 8.86g of Lycium chinense root bark, 8.86g of Morus alba root bark, 0.89g of Glycyrrhiza uralensis and 6g of japonica rice as a formula, take each of the medicinal pieces in the formula, crush them into the coarsest powder, add water and heat to extract, filter the decoction while hot to obtain the filtrate, and freeze-dry the filtrate to obtain the sample to be tested.

[0017] In step S1, the dissolution is performed by mixing the sample to be tested with water at a ratio of 1g:8-12mL.

[0018] Any point value or any range of two points within the above range can achieve the technical effect of the present invention. For example, the material-liquid ratio includes, but is not limited to, 1g:8mL, 1g:9mL, 1g:10mL, 1g:11mL, 1g:12mL, or any range of two points within the range of 1g:8-12mL, such as 1g:9-12mL.

[0019] Preferably, in step S1, the solvent is selected from methanol, diethyl ether, ethyl acetate or n-butanol; more preferably, the solvent is selected from n-butanol.

[0020] In step S1, the ratio of the sample to solvent is 1g:10-30mL. Any point value or any range of two points within the above range can achieve the technical effect of this invention. For example, the ratio includes, but is not limited to, 1g:8mL, 1g:9mL, 1g:10mL, 1g:11mL, 1g:12mL, 1g:13mL, 1g:14mL, 1g:15mL, 1g:16mL, 1g:17mL, 1g:18mL, 1g:19mL, 1g:20mL, 1g:21mL, 1g:22mL, 1g:23mL, 1g:24mL, 1g:25mL, 1g:26mL, 1g:27mL, 1g:28mL, 1g:29mL, 1g:30mL, or any range of two points within the 1g:10-30mL range, such as 1g:11-30mL.

[0021] In step S1, the solvent extraction is performed by mixing the sample to be tested with a solvent, extracting 1-3 times, combining the extracts, washing with water and evaporating to dryness, and dissolving the residue in methanol to 1 mL to obtain the test solution.

[0022] In step S2, the preparation method of the Lycium chinense root bark reference solution is as follows: the Lycium chinense root bark reference material and methanol are mixed at a material-to-liquid ratio of 0.5g:10-30mL, ultrasonically extracted for 25-35min, the filtrate is separated, concentrated, and the Lycium chinense root bark reference solution is obtained.

[0023] Any point value or any range of two points within the above-mentioned range can achieve the technical effect of this invention. For example, the material-to-liquid ratio includes, but is not limited to, 0.5g:10mL, 0.5g:11mL, 0.5g:12mL, 0.5g:13mL, 0.5g:14mL, 0.5g:15mL, 0.5g:16mL, 0.5g:17mL, 0.5g:18mL, 0.5g:19mL, and 0.5g... : 20mL, 0.5g: 21mL, 0.5g: 22mL, 0.5g: 23mL, 0.5g: 24mL, 0.5g: 25mL, 0.5g: 26mL, 0.5g: 27mL, 0.5g: 28mL, 0.5g: 29mL, 0.5g: 30mL, or a range of values ​​consisting of any two points within the range of 10-30mL for 0.5g, such as 19-22mL for 0.5g.

[0024] The ultrasonic extraction time includes, but is not limited to, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, or a range of values ​​formed by any two points within the range of 25-35 min, such as 26-35 min.

[0025] In step S2, the licorice reference solution is prepared as follows: licorice reference material and water are mixed at a material-to-liquid ratio of 0.25g:10-30mL, and refluxed for 0.5-1.5h. The filtrate is separated, and then extracted 1-3 times with 0.6-0.8 times the volume of water in n-butanol. The extracts are combined, evaporated to dryness, and the residue is dissolved in methanol to 1mL to obtain the licorice reference solution.

[0026] Any point value or any range of two points within the above-mentioned range can achieve the technical effect of this invention. For example, the material-to-liquid ratio includes, but is not limited to, 0.25g:10mL, 0.25g:11mL, 0.25g:12mL, 0.25g:13mL, 0.25g:14mL, 0.25g:15mL, 0.25g:16mL, 0.25g:17mL, 0.25g:18mL, 0.25g:19mL, and 0.25g... : 20mL, 0.25g: 21mL, 0.25g: 22mL, 0.25g: 23mL, 0.25g: 24mL, 0.25g: 25mL, 0.25g: 26mL, 0.25g: 27mL, 0.25g: 28mL, 0.25g: 29mL, 0.25g: 30mL, or a range of values ​​consisting of any two points within the range of 10-30mL for 0.25g, such as 0.25g: 19-22mL, etc.

[0027] The reflux extraction time includes, but is not limited to, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, or a range of values ​​formed by any two points within the range of 0.5-1.5h, such as 0.6-1.5h.

[0028] The volume of n-butanol is a multiple of the volume of water, including but not limited to 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, or a range of values ​​formed by any two points within the range of 0.6-0.8, such as 0.61-0.8.

[0029] The number of extractions may include, but is not limited to, 1, 2, 3, or a range of values ​​formed by any two points within the range of 1-3, such as 2-3, etc.

[0030] In step S2, the preparation method of the morin A reference solution is as follows: add methanol to morin A reference standard to a concentration of 1.8-2.2 mg / mL to obtain the morin A reference solution.

[0031] Concentrations include, but are not limited to, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, or a range of values ​​consisting of any two points within the range of 1.8-2.2 mg / mL, such as 1.9-2.2 mg / mL.

[0032] In step S3, the volume ratio of butyl acetate-methanol-acetone-water-formic acid is 5:(0.5-2):(2-3):(0.5-1):1. Any point value or any range of two points within the above range can achieve the technical effect of the present invention. For example, the volume ratio includes, but is not limited to, 5:0.5:2:0.5:1, 5:2:3:1:1, or any range of two points within the range of 5:(0.5-2):(2-3):(0.5-1):1, such as 5:(0.6-2):(2.1-3):(0.6-1):1.

[0033] More preferably, in step S3, the volume ratio of butyl acetate-methanol-acetone-water-formic acid is 5:2:1:1:1.

[0034] In step S3, the sampling volume of the test solution and the reference solution is 2-4 μL. Any point value or any range of two points within the above range can achieve the technical effect of the present invention. For example, the sampling volume includes, but is not limited to, 2 μL, 3 μL, 4 μL, or any range of two points within the range of 2-4 μL, such as 2-3 μL.

[0035] In step S3, the temperature is 8-30℃ and the humidity is 20-80%. Any point value or any range of two points within the above range can achieve the technical effect of this invention. For example, temperatures include, but are not limited to, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, and 30℃, or any range of two points within the 8-30℃ range, such as 9-30℃.

[0036] Humidity values ​​include, but are not limited to, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any range of values ​​formed by any two points within the range of 20-80%, such as 21-80%.

[0037] In step S3, the preparation method of the 2% vanillin in 10% sulfuric acid ethanol solution is as follows: first, prepare a sulfuric acid ethanol solution at a ratio of 1:10, that is, dilute 1 mL of sulfuric acid with ethanol to 10 mL; then add 2 g of vanillin to 100 mL of sulfuric acid ethanol solution to dissolve it, and the solution is obtained.

[0038] The present invention has at least the following beneficial effects: This invention provides a thin-layer chromatography (TLC) identification method for compound traditional Chinese medicine preparations containing licorice. It omits the ether reflux step used in the pharmacopoeia for licorice identification, directly extracts with n-butanol, and completely changes the developing solvent, using butyl acetate-methanol-acetone-water-formic acid as the developing solvent. This allows for the simultaneous identification of mulberry bark, lycium bark, and licorice, providing a theoretical basis for the quality control and pharmacodynamic material basis research of compound traditional Chinese medicine preparations containing licorice. This invention offers the advantages of requiring less sampling, simplifying quality testing, and being highly efficient and environmentally friendly. Attached Figure Description

[0039] Figure 1 The image shows a thin-layer chromatogram of Example 1, where 1 is morin A reference solution, 2 is test solution 1, 3 is test solution 2, 4 is test solution 3, 5 is Lycium bark reference solution, and 6 is licorice reference solution.

[0040] Figure 2 The image shows a thin-layer chromatogram of Example 2, where 1 is a mulberry bark reference solution, 2 is a Lycium bark reference medicinal material solution, 3 is the test solution, and 4 is a licorice reference medicinal material solution.

[0041] Figure 3 The image shows a thin-layer chromatogram of Example 3, where 1-3 correspond to spotting volumes of 2 μL, 3 μL, and 4 μL of morin A reference solution, respectively; 4-5 correspond to spotting volumes of 2 μL, 3 μL, and 4 μL of the test solution, respectively; 7-9 correspond to spotting volumes of 2 μL, 3 μL, and 4 μL of Lycium chinense root bark reference material solution, respectively; and 10-12 correspond to spotting volumes of 2 μL, 3 μL, and 4 μL of Glycyrrhiza uralensis reference material solution, respectively.

[0042] Figure 4 The image shows a thin-layer chromatogram of Example 4, where 1 is morin A reference solution, 2 is test solution 1, 3 is test solution 2, 4 is test solution 3, 5 is Lycium bark reference solution, and 6 is licorice reference solution.

[0043] Figure 5 The image shows a thin-layer chromatogram of Example 5, where 1 is morin A reference solution, 2 is test solution 1, 3 is test solution 2, 4 is test solution 3, 5 is Lycium bark reference solution, and 6 is licorice reference solution.

[0044] Figure 6 The image shows a thin-layer chromatogram of Example 6, where 1 is morin A reference solution, 2 is test solution 1, 3 is test solution 2, 4 is test solution 3, 5 is Lycium bark reference solution, and 6 is licorice reference solution.

[0045] Figure 7The image shows a thin-layer chromatogram of Example 7, where 1 is the negative test solution of mulberry bark, 2 is the reference solution of morin A, 3 is test solution 1, 4 is test solution 2, 5 is test solution 3, 6 is reference solution of lycium bark, 7 is negative test solution of lycium bark, 8 is reference solution of licorice, and 9 is negative test solution of licorice.

[0046] Figure 8 The image shows a thin-layer chromatogram of Comparative Example 1, where 1 is morin A reference solution, 2 is test solution A of Comparative Example 1-1, 3 is test solution of Comparative Example 1-2, 4 is test solution of Example 1, 5 is Lycium bark reference solution, and 6 is licorice reference solution.

[0047] Figure 9 The image shows a thin-layer chromatogram of Comparative Example 2, where 1 is morin A reference solution, 2 is mulberry bark reference solution ②, 3 is mulberry bark reference solution ①, 4 is the test solution, 5 is licorice reference material solution, and 6 is lycium bark reference material solution.

[0048] Figure 10 The image shows a thin-layer chromatogram of Comparative Example 3, where 1 is the mulberry bark reference solution, 2 is the lycium bark reference medicinal material solution, 3 is the test solution, and 4 is the licorice reference medicinal material solution.

[0049] Figure 11 The image shows a thin-layer chromatogram of Comparative Example 4, where 1 is the mulberry bark reference solution, 2 is the lycium bark reference medicinal material solution, 3 is the test solution, and 4 is the licorice reference medicinal material solution. Detailed Implementation

[0050] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0051] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0052] Reagents: The reagents used, including butyl acetate, methanol, petroleum ether, formic acid, and sulfuric acid, were all of analytical grade (Sinopharm Chemical Reagent Co., Ltd.). Precast Guijiao G thin-layer board (HX73416553 1.05553.0001, 20×20cm, Merck GmbH, Germany); Pre-fabricated silicone G thin film (Yantai Chemical Industry Research Institute, 20190307, ​​100×100mm); Precast Guijiao G thin-layer board (Qingdao Marine Chemical Plant, 20240117, 100×100mm); Reference material for Lycium chinense root bark (China National Institutes for Food and Drug Control 121087-201707); Licorice reference material (China National Institutes for Food and Drug Control 120904-201519); Mulberry bark reference material (China National Institutes for Food and Drug Control 121124-202109); Mulberry bark A reference standard (China National Institutes for Food and Drug Control 112086-202101).

[0053] Basic Example 1: Preparation of the test sample: The formula consists of 8.86g of Lycium chinense root bark, 8.86g of Morus alba root bark, 0.89g of Glycyrrhiza uralensis root, and 6g of japonica rice. Each of the ingredients in the formula is crushed into the coarsest powder, and 900mL of water is added for extraction by heating. The decoction is filtered while hot to obtain about 540mL of filtrate. The filtrate is then freeze-dried to obtain the test sample.

[0054] Preparation of Lycium chinense root bark reference solution: Take 0.5 g of Lycium chinense root bark reference material, add 20 mL of methanol, sonicate for 30 minutes, filter, and concentrate the filtrate to 2 mL as Lycium chinense root bark reference solution.

[0055] Preparation of licorice reference solution: Take 0.25g of licorice reference material, add 20mL of water and reflux for 1 hour, filter, hydrate the filtrate with n-butanol and extract twice, adding 15mL each time, evaporate the n-butanol layer to dryness, add methanol to dissolve to 1mL, and use as licorice reference solution.

[0056] Preparation of morin A reference solution: Take morin A reference standard and add methanol to prepare a solution containing 2 mg per 1 mL, which is used as the morin A reference solution.

[0057] Example 1 A thin-layer chromatography method for identifying compound traditional Chinese medicine preparations containing licorice includes the following steps: S1: Take 1g of the sample to be tested prepared in the basic example 1, add 10mL of water to dissolve it, extract it twice with 20mL of hydrated n-butanol, wash the n-butanol layer with 10mL of water and evaporate it to dryness, add methanol to dissolve it to 1mL, and use it as the test solution. Prepare 3 groups in parallel, namely test solution 1, test solution 2 and test solution 3. S2: Prepare Lycium chinense root bark reference solution, licorice reference solution and morin A reference solution according to Basic Example 1; S3: Perform the test according to the Thin Layer Chromatography (General Rule 0502) of Part IV of the 2020 Chinese Pharmacopoeia. Take 3 μL of each of the test solution and the reference solution and spot them on the thin layer plate. Spot them separately on the same silica gel G thin layer plate to form strips. Use butyl acetate-methanol-acetone-water-formic acid solution as the developing solvent. The volume ratio of butyl acetate-methanol-acetone-water-formic acid is 5:2:1:1:1. Develop, remove, air dry, spray with 2% vanillin in 10% sulfuric acid ethanol solution, heat until the spots are clearly visible, and examine under sunlight.

[0058] The thin-layer chromatogram of Example 1 is as follows: Figure 1 As shown.

[0059] Example 2 Investigation of the developing solvent ratio: Compared with Example 1, only the developing solvent ratio was changed to that shown in Table 1 below, while the rest remained the same as in Example 1: Table 1

[0060] Thin-layer chromatograms with different developing solvents are as follows: Figure 2 As shown, licorice, mulberry bark, and lycium bark all have corresponding spots to the test sample, which can be used for simultaneous identification of the three.

[0061] Example 3 Investigation of sample volume: Compared with Example 1, only the injection volume in step S3 (piptering the test solution and the reference solution respectively) was changed to 2 μL, 3 μL, and 4 μL. The rest was the same as in Example 1. The thin-layer chromatogram is shown below. Figure 3 As shown, when the amount of morin A reference solution is 2-4 μL, the main spot is clear, preferably 3 μL; when the amount of test solution is 2-4 μL, there are corresponding spots at the corresponding positions as in the reference solution, and the main spot is clear, preferably 3 μL. When the amount of Lycium chinense root bark reference solution is 2-4 μL, the main spot is clear, preferably 3 μL; when the amount of licorice reference solution is 2-4 μL, the main spot is clear, preferably 3 μL.

[0062] Example 4 Temperature investigation: Compared with Example 1, only the temperature in step S3 was changed to 30℃ (constant temperature incubator), 22℃ (room temperature), and 8℃ (refrigerator compartment). The rest of the experiment was the same as in Example 1. The results are as follows: Figure 4 As shown in the figure. It can be seen that under the above-mentioned different temperature conditions, the chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatograms of the reference standard and the reference medicinal material, and the separation is good. R f The value did not change significantly, indicating that the temperature range of 8-30℃ has no significant impact on the thin-layer chromatography identification of this product, and the ambient temperature does not need to be controlled.

[0063] Example 5 Effect of relative humidity environment: Compared with Example 1, only the humidity in step S3 was changed to 40% (indoor) and 80% (achieved by placing a 26.2% sulfuric acid solution in a developing tank and sealing it for 1.5 hours to saturate). The results are as follows. Figure 5 As shown.

[0064] It can be seen that under the above-mentioned different relative humidity conditions, the chromatogram of the test sample showed spots of the same color at the corresponding positions as the chromatograms of the reference standard and the reference medicinal material, and the separation was good. f The value did not change significantly, indicating that the relative humidity in the range of 20-80% has little impact on the thin-layer chromatography identification of this product, and the relative humidity of the environment does not need to be controlled.

[0065] Example 6 Impact of thin-layer slab manufacturers: Experiments were conducted on three thin-layer precast slab manufacturers (Yantai Yinlong, Qingdao Haiyang, and Merck, Germany) according to the method described in Example 1. The results are as follows: Figure 6 As shown: Based on the development results of thin-layer plates from different manufacturers, the chromatograms of the test sample showed spots of the same color at the corresponding positions as those of the reference standard and the reference medicinal material, with good separation, no negative interference, and slight variations in Rf values. All three manufacturers' thin-layer plates can be used for the identification of the licorice-containing compound traditional Chinese medicine preparations described in this invention.

[0066] Example 7 Exclusivity Mulberry bark negative test solution: Take 1g of fine powder of mulberry bark negative extract and process it with test solution C to obtain mulberry bark negative test solution.

[0067] Lycium chinense root bark negative test solution: Take 1g of Lycium chinense root bark negative extract powder and perform the same operation as test solution A (weigh 0.5g of the sample to be tested, add 2mL of water and 4mL of ethyl acetate, shake well, centrifuge, reserve the supernatant, add 4mL of ethyl acetate to the precipitate, shake well, centrifuge, combine the supernatants, evaporate to dryness, and dissolve in methanol to 1mL) to obtain the Lycium chinense root bark negative test solution.

[0068] Licorice negative test solution: Take 1g of fine powder of licorice negative extract and process it in the same way as test solution A to prepare licorice negative test solution.

[0069] Take three batches of extract, weigh 1g of each, and perform the same operation as test solution C, namely test solution 1, test solution 2 and test solution 3; According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, 3 μL each of the test solution, negative test solution, reference medicinal material solution, and reference solution were spotted separately onto the same silica gel G thin-layer plate as bands. The plate was developed using butyl acetate-methanol-acetone-water-formic acid (5:2:1:1:1) as the developing solvent. The plate was then removed, air-dried, sprayed with 2% vanillin and 10% sulfuric acid ethanol solution, and heated until the spots were clearly visible. Figure 7 .

[0070] The chromatogram of the test sample showed spots of the same color at the corresponding positions as the chromatograms of the reference sample and the reference medicinal material, with good separation. The chromatogram of the negative control sample showed no spots at the corresponding positions as the chromatograms of the reference sample and the reference medicinal material. The results showed that the negative control sample had no interference and good specificity.

[0071] Comparative Example 1 Compared with Example 1, the preparation method of the test solution in step S1 is changed as shown in Table 2 below: Table 2

[0072] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, 3 μL each of morin A reference solution, test solutions A, B, C, Lycium chinense root bark, and licorice reference solutions were spotted onto a silica gel G thin-layer plate. The plate was developed using butyl acetate-methanol-acetone-water-formic acid (5:2:1:1:1) as the developing solvent. The plate was then removed, dried, sprayed with 2% vanillin and 10% sulfuric acid ethanol solution, and heated until the spots were clearly visible. The thin-layer chromatogram is shown below. Figure 8 As shown: The samples prepared by the three solvents and methods are quite different. Only the test solution preparation method of Example 1 of this invention can simultaneously detect Lycium bark, mulberry bark, and licorice.

[0073] Comparative Example 2 The morin A reference solution was modified to the reference solutions shown in Table 3 below: Table 3

[0074] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, 3 μL each of the following reference solutions were applied to a silica gel G plate: morin A reference solution, mulberry bark reference solutions ① and ②, the test solution prepared in Example 11, lycium bark reference solution, and licorice reference solution. The plates were developed using butyl acetate-methanol-acetone-water-formic acid (5:2:1:1:1) as the developing solvent. The plates were then removed, dried, sprayed with 2% vanillin and 10% sulfuric acid ethanol solution, and heated until the spots were clearly visible. Figure 9It can be seen that there are spots corresponding to morin A in both the mulberry bark reference solution ① and the test sample C, but no spots corresponding to morin A were found in the mulberry bark reference solution ② (China National Institutes for Food and Drug Control 121124-202109). An attempt was made to determine the content of morin A by liquid chromatography using this batch of reference medicinal materials, and the result was 0. No other spots were found that corresponded to the test sample solution. Therefore, morin A reference standard was directly used for thin-layer chromatography identification.

[0075] The reference medicinal materials, Lycium chinense root bark and Glycyrrhiza uralensis, showed corresponding spots to the test sample solution. Therefore, Lycium chinense root bark and Glycyrrhiza uralensis were chosen as reference medicinal materials for identification.

[0076] Comparative Example 3 Investigation of the developing solvent ratio: Compared with Example 1, only the developing solvent ratio was changed to that shown in Table 4 below, while the rest remained the same as in Example 1: Table 4

[0077] Thin-layer chromatogram as shown Figure 10 As shown, only with the specific developing agent and its proportion of the present invention can the mulberry bark, lycium bark, and licorice be clearly identified simultaneously.

[0078] Comparative Example 4 Investigation of the developing solvent: Compared with Example 1, only the developing solvent was changed to that listed in Table 5 below; the rest was the same as in Example 1: Table 5

[0079] Thin-layer chromatogram as shown Figure 11 As shown: Only with the specific developing agent and its proportions of this invention can the mulberry bark, lycium bark, and licorice be clearly identified simultaneously.

[0080] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for identifying licorice-containing traditional Chinese medicine preparation by thin layer chromatography, characterized in that, It comprises the following steps: S1: dissolving the sample to be tested, adding solvent to extract, and obtaining a test solution; S2: separately taking the cortex periplocae control medicinal material, the licorice control medicinal material, and the morusin A control, ultrasonic extraction, and obtaining a cortex periplocae control solution, a licorice control solution, and a morusin A control solution; S3: taking the test solution and the control solution and spotting them on a thin layer plate, respectively, in the form of strips on the same silica gel G thin layer plate, using butyl acetate-methanol-acetone-water-formic acid as a developing agent, the volume ratio of the butyl acetate-methanol-acetone-water-formic acid being 5: (0.5-2): (2-3): (0.5-1): 1, developing, taking out, air-drying, spraying with 2% vanillin in 10% sulfuric acid ethanol solution, heating until the spots develop clearly, and observing under daylight.

2. The thin layer discrimination method according to claim 1, characterized in that, In step S1, the solvent is selected from methanol, diethyl ether, ethyl acetate, or n-butanol.

3. The thin layer discrimination method of claim 1, wherein, In step S1, the ratio of the sample to be tested to the solvent is 1g: 10-30mL.

4. The thin layer discrimination method of claim 1, wherein In step S1, the solvent extraction is performed by mixing the sample to be tested with the solvent, extracting 1-3 times, combining the extract, washing with water, and evaporating to dryness, dissolving the obtained residue in methanol to 1mL to obtain the test solution.

5. The thin layer discrimination method of claim 1, wherein In step S2, the preparation method of the cortex periplocae control solution is as follows: mixing the cortex periplocae control medicinal material with methanol at a ratio of 0.5g: 10-30mL, ultrasonic extraction for 25-35min, separating to obtain a filtrate, and concentrating to obtain the cortex periplocae control solution.

6. The thin layer discrimination method of claim 1, wherein In step S2, the preparation method of the licorice control solution is as follows: mixing the licorice control medicinal material with water at a ratio of 0.25g: 10-30mL, reflux extraction for 0.5-1.5h, separating to obtain a filtrate, adding n-butanol to 0.6-0.8 times the volume of water, extracting 1-3 times, combining the extract, evaporating to dryness, and dissolving the obtained residue in methanol to 1mL to obtain the licorice control solution.

7. The thin layer discrimination method of claim 1, wherein In step S2, the preparation method of the morusin A control solution is as follows: adding methanol to the morusin A control to a concentration of 1.8-2.2mg / mL to obtain the morusin A control solution.

8. The thin layer discrimination method of claim 1, wherein, In step S3, the volume ratio of the butyl acetate-methanol-acetone-water-formic acid is 5:2:1:1:

1.

9. The thin layer discrimination method of claim 8, wherein, In step S3, the sampling volume of the test solution and the control solution is 2-4μL.

10. The thin layer discrimination method of claim 1, wherein, The temperature of step S3 is 8-30℃, and the humidity is 20-80%.

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