Method for thin-layer chromatography detection of herba epimedii in ginseng-radix puerariae kidney-tonifying preparation

Abstract

A method for thin-layer chromatography detection of herba epimedii in a ginseng-radix puerariae kidney-tonifying preparation. Raw materials of the ginseng-radix puerariae kidney-tonifying preparation comprise radix pseudostellariae, radix puerariae, and herba epimedii. The method for thin-layer chromatography detection of herba epimedii in the ginseng-radix puerariae kidney-tonifying preparation comprises the following steps: preparing a test sample solution, an icariin reference substance solution, and a herba epimedii reference medicinal material solution; spotting the test sample solution, the icariin reference substance solution, and the herba epimedii reference medicinal material solution onto a same thin-layer chromatography plate, developing with a developing solvent, adding a color visualization agent for color visualization, and visualizing; the developing solvent is a mixed solvent composed of trichloromethane, methanol, and water in a volume ratio of 7:(2.4-2.6):(0.24-0.26), and the color visualization agent is an ethanol solution of aluminium trichloride.

Description

Thin-layer chromatography method for the determination of Epimedium in Shenkui-tonifying preparation

[0001] This application claims priority to Chinese patent application No. 2023115747100, filed on November 23, 2023, entitled “Thin-layer chromatography detection method for epimedium in ginseng and kudzu kidney-tonifying preparations,” the entire text of which is hereby incorporated by reference. Technical Field

[0002] The present application relates to the technical field of traditional Chinese medicine detection, and in particular to a thin-layer chromatography detection method for epimedium in a ginseng and kudzu kidney-tonifying preparation. Background Art

[0003] Epimedium brevicornu Maxim., a perennial herb of the genus Epimedium in the Berberidaceae family, is an essential medicine for tonifying the vital energy, improving vital essence and qi, strengthening tendons and bones, and nourishing the kidneys and strengthening yang. It is commonly used clinically to treat male impotence, spermatorrhea, premature ejaculation, urinary incontinence, and female infertility. It also has the effects of lowering blood pressure and blood sugar, promoting diuresis, and relieving cough and expectoration.

[0004] The main ingredients of Shenkui Bu Shen preparations are Pseudostellaria pseudoginseng, Pueraria root, and Epimedium. They primarily benefit Qi, nourish Yin, and tonify the kidneys. They are suitable for mild to moderate depression characterized by Qi and Yin deficiency and kidney Qi deficiency according to Traditional Chinese Medicine. Symptoms include low mood, excessive worrying, decreased speech and movement, slowed eyesight, forgetfulness, poor appetite, palpitations, timidity, insomnia, frequent dreams, restlessness, a pale or reddish tongue, a white or flaky tongue coating, and a weak pulse.

[0005] Epimedium, as the main raw material in ginseng and kudzu kidney-tonifying preparations, plays an important role in the effectiveness of ginseng and kudzu kidney-tonifying preparations. However, there is currently a lack of methods that can quickly identify and specifically detect epimedium in ginseng and kudzu kidney-tonifying preparations, making it impossible to accurately and comprehensively measure or control the quality of ginseng and kudzu kidney-tonifying preparations.

[0006] Summary of the Invention

[0007] According to various embodiments of the present application, a thin layer chromatography method for detecting epimedium in a ginseng and kudzu kidney-tonifying preparation is provided.

[0008] The technical solution is as follows:

[0009] A thin layer chromatography method for detecting epimedium in a ginseng and kudzu kidney-tonifying preparation, wherein the raw materials of the ginseng and kudzu kidney-tonifying preparation include pseudoginseng, kudzu root and epimedium;

[0010] The thin layer chromatography detection method of Epimedium in the Shenkui tonifying kidney preparation comprises the following steps:

[0011] Prepare the test solution, icariin reference solution and epimedium reference medicinal material solution respectively;

[0012] Spot the test solution, the icariin reference solution and the epimedium reference medicinal material solution on the same thin layer chromatography plate, develop them in a developing agent, add a color developer, and inspect;

[0013] The developing solvent is a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7: (2.4-2.6): (0.24-0.26);

[0014] The color developer is an ethanol solution of aluminum chloride.

[0015] In some embodiments, the developing solvent is a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:2.5:(0.24-0.26).

[0016] In some embodiments, in the developer, the mass volume ratio of aluminum chloride to ethanol is 1 g: (49-199) ml.

[0017] In some embodiments, the light source used for inspection is a 365 nm ultraviolet lamp.

[0018] In some embodiments, the method for preparing the test solution comprises the following steps:

[0019] The ginseng and kudzu kidney-tonifying preparation test sample is mixed with a first alcohol-containing solvent to perform a first extraction, and an extract is obtained to prepare the test sample solution.

[0020] In some embodiments, the first alcohol-containing solvent is selected from a first alcohol or a mixed solvent of the first alcohol and water.

[0021] In some embodiments, in the test solution, the mass volume ratio of the ginseng and kudzu kidney-tonifying preparation test sample and the first alcohol-containing solvent is (1-200) mg:1 ml.

[0022] In some embodiments, the first alcohol is selected from one or more of ethanol and methanol.

[0023] In some embodiments, the first alcohol-containing solvent includes 20%-100% of the first alcohol and 0%-80% of water, calculated by volume.

[0024] In some embodiments, the first extraction method is heating extraction at a temperature of 80° C.-100° C. for 0.5 h-2 h.

[0025] In some embodiments, the preparation method of the icariin reference solution comprises the following steps:

[0026] The icariin reference substance is mixed with the second alcohol-containing solvent to prepare the icariin reference substance solution.

[0027] In some embodiments, the second alcohol-containing solvent is selected from a second alcohol or a mixed solvent of a second alcohol and water.

[0028] In some embodiments, in the icariin reference solution, the mass volume ratio of the icariin reference to the second alcohol-containing solvent is (0.1-10) mg:1 ml.

[0029] In some embodiments, the second alcohol is selected from one or more of ethanol and methanol.

[0030] In some embodiments, the second alcohol-containing solvent includes 20%-100% of the second alcohol and 0%-80% of water, by volume.

[0031] In some embodiments, the method for preparing the epimedium control medicinal material solution comprises the following steps:

[0032] The epimedium is mixed with a third alcohol-containing solvent for extraction to obtain an extract, and the epimedium control medicinal material solution is prepared.

[0033] In some embodiments, the third alcohol-containing solvent is selected from a third alcohol or a mixed solvent of a third alcohol and water.

[0034] In some embodiments, in the epimedium control medicinal material solution, the mass volume ratio of the epimedium to the third alcohol-containing solvent is (5-500) mg:1 ml.

[0035] In some embodiments, the third alcohol is selected from one or more of ethanol and methanol.

[0036] In some embodiments, the third alcohol-containing solvent includes 20%-100% of the third alcohol and 0%-80% of water, calculated by volume.

[0037] In some embodiments, the second extraction method is warm immersion at a temperature of 50° C.-70° C. for 0.5 h-2 h.

[0038] In some embodiments, the raw materials of the ginseng and kudzu kidney-tonifying preparation include 1000-2000 parts of Pseudostellariae Radix, 500-1500 parts of Puerariae Radix and 100-900 parts of Epimedium.

[0039] In some embodiments, the dosage form of the Shenkui-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

[0040] The details of one or more embodiments of the present application are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the present application will become apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to better describe and illustrate the embodiments and / or examples of the inventions disclosed herein, reference may be made to one or more of the accompanying drawings. The additional details or examples used to describe the accompanying drawings should not be considered to limit the scope of the disclosed inventions, the presently described embodiments and / or examples, and any of the best modes currently understood for these inventions.

[0042] FIG1 shows the results of thin-layer chromatography using a mixed solvent of chloroform, methanol, and water in a volume ratio of 7:2.5:0.25 as a developing solvent and a 1% aluminum chloride ethanol solution as a color developer, according to an embodiment of the present application;

[0043] FIG2 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.25:0.25 as a developing solvent as shown in an embodiment of the present application;

[0044] FIG3 is a thin layer chromatography test result using a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:2:0.75 as a developing solvent according to an embodiment of the present application;

[0045] FIG4 is a thin layer chromatography test result using a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:1.75:1 as a developing solvent according to an embodiment of the present application;

[0046] FIG5 is a thin layer chromatography test result using a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:1.5:1.25 as a developing solvent according to an embodiment of the present application;

[0047] FIG6 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol, and water in a volume ratio of 7:1.25:1.5 as a developing solvent according to an embodiment of the present application;

[0048] FIG7 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol, and water in a volume ratio of 7:1:1.75 as a developing solvent according to an embodiment of the present application;

[0049] FIG8 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol, and water in a volume ratio of 7:0.75:2 as a developing solvent according to an embodiment of the present application;

[0050] FIG9 is a thin layer chromatography test result using a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:0.5:2.25 as a developing solvent according to an embodiment of the present application;

[0051] FIG10 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol, and water in a volume ratio of 7:0.25:2.5 as a developing solvent according to an embodiment of the present application;

[0052] FIG11 is a thin layer chromatography test result using a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:3:1 as a developing solvent according to an embodiment of the present application;

[0053] FIG12 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol, and water in a volume ratio of 13.5:6:2 as a developing solvent according to an embodiment of the present application;

[0054] FIG13 shows the thin layer chromatography test results using a mixed solvent consisting of chloroform, ethanol, and water in a volume ratio of 7:2.5:0.25 as a developing solvent according to an embodiment of the present application;

[0055] FIG14 shows the results of thin layer chromatography using a mixed solvent of chloroform, ethanol, and water in a volume ratio of 7:1.25:1.5 as a developing solvent according to an embodiment of the present application;

[0056] FIG15 shows the results of thin layer chromatography using a mixed solvent of chloroform, ethanol, and water in a volume ratio of 7:0.25:2.5 as a developing solvent according to an embodiment of the present application;

[0057] FIG16 shows the results of thin layer chromatography using a mixed solvent consisting of dichloromethane, ethyl acetate, methanol, and water in a volume ratio of 1:5:4 as a developing solvent according to an embodiment of the present application;

[0058] FIG17 shows the results of thin layer chromatography using a mixed solvent of methanol, butanone, chloroform, and water in a volume ratio of 2:3:3:0.5 as a developing solvent according to an embodiment of the present application;

[0059] FIG18 is a thin layer chromatography test result using anhydrous ethanol as a solvent for dissolving a test sample according to an embodiment of the present application;

[0060] FIG19 is a thin layer chromatography test result of using 60% ethanol as a solvent for dissolving a test sample according to an embodiment of the present application;

[0061] FIG20 is a thin layer chromatography test result of using 40% ethanol as a solvent for dissolving a test sample according to an embodiment of the present application;

[0062] FIG21 is a thin layer chromatography test result of using 20% ​​ethanol as a solvent for dissolving a test sample according to an embodiment of the present application;

[0063] FIG22 is a thin layer chromatography test result using anhydrous methanol as a solvent for dissolving a test sample according to an embodiment of the present application;

[0064] FIG23 is a thin layer chromatography test result of using 60% methanol as a solvent for dissolving a test sample according to an embodiment of the present application;

[0065] FIG24 is a thin layer chromatography test result of using 40% methanol as a solvent for dissolving a test sample according to an embodiment of the present application;

[0066] FIG25 shows the thin layer chromatography detection results using 10% sulfuric acid ethanol solution as a color developer according to an embodiment of the present application;

[0067] FIG26 shows the thin layer chromatography test results of Epimedium in different batches of Shengge Bushen Capsules;

[0068] FIG27 shows the results of thin layer chromatography (TLC) testing of the same batch of Shenge Bushen Capsules using different TLC plates. DETAILED DESCRIPTION

[0069] The present application will be further described in detail below with reference to specific embodiments and accompanying drawings. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the present disclosure.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0071] In the present application, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present application.

[0072] In this application, "at least one" means more than one, such as one, two, or more than two. "Multiple" or "several" means at least two, such as two, three, etc., and "multi-layer" means at least two layers, such as two, three, etc., unless otherwise specifically defined. In the description of this application, "several" means at least one, such as one, two, etc., unless otherwise specifically defined.

[0073] When a numerical range is disclosed in this application, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0074] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0075] Unless mentioned otherwise, terms in the singular may include plural forms and should not be construed as having one number.

[0076] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the quantity of the technical features indicated. Thus, features defined as "first" or "second" may explicitly or implicitly include at least one of these features.

[0077] As used herein, the words "comprise," "include," "contain," "have," "have," or other variations thereof are intended to encompass non-exclusive inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The compositions and methods / processes of the present application comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0078] In this application, no distinction is made between the terms "efficacy", "performance", "effect" and "efficacy".

[0079] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the weights described in the examples of this application may be mass units known in the chemical industry, such as μg, mg, g, and kg.

[0080] In this application, temperature parameters, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control. Room temperature or normal temperature as used in this application refers to 0-40°C.

[0081] In one embodiment, the normal temperature is 10°C to 35°C.

[0082] In one embodiment, the normal temperature is 20°C to 30°C.

[0083] In this application, the alcohol concentration defined in the mixture of alcohol and water is expressed as the volume percentage of alcohol in the mixture. For example, 20% ethanol means that in the ethanol-water solution, the volume percentage of anhydrous ethanol is 20% and the volume percentage of water is 80%.

[0084] The main ingredients of Shenkui-tonifying preparations are Pseudostellariae Radix, Puerariae Radix, and Epimedium. Their primary functions are to invigorate Qi, nourish Yin, and tonify the kidneys. As the primary ingredient in Shenkui-tonifying preparations, Epimedium plays a crucial role in their effectiveness. However, a rapid and specific detection method for Epimedium in Shenkui-tonifying preparations is currently lacking, hindering the accurate and comprehensive quality control of these preparations.

[0085] Thin-layer chromatography (TLC) involves coating a suitable stationary phase onto a glass, plastic, or aluminum substrate to form a uniform thin layer. After sample application and development, the relative shift (Rf) value is compared with that of a chromatogram obtained using the same method using a suitable control. This method is used for drug identification, impurity testing, or content determination. As a crucial experimental technique for the rapid separation and qualitative analysis of small amounts of substances, TLC boasts short development times, strong separation capabilities, and high sensitivity, making it a crucial analytical tool in the quality control of traditional Chinese medicines. Compared with content determination and characteristic chromatographic identification, TLC is one of the more widely used methods within planar chromatography. It is easy to master, employs inexpensive equipment, and is simple and flexible to operate. It offers the following advantages: the ability to change the developing agent and development direction at any time, as well as derivatization, color development, observation, and detection. It also allows for rapid method development, high sensitivity and resolution, the ability to separate multiple samples simultaneously, and simple sample pretreatment. In recent years, despite the rapid development of other chromatographic techniques, the application of thin-layer chromatography has not decreased significantly. On the contrary, with the introduction of new stationary phases and rapidly developing instrument technologies, it has shown a trend of wider application and has become a modern, sensitive and efficient separation and analysis method.

[0086] If epimedium in ginseng and kudzu kidney-tonifying preparations can be detected by thin-layer chromatography, the accuracy and comprehensiveness of quality control of ginseng and kudzu kidney-tonifying preparations will be significantly improved, which will be beneficial to the establishment and overall evaluation of quality standards for ginseng and kudzu kidney-tonifying preparations. However, no relevant reports have been found so far.

[0087] Based on this, the present application provides a thin layer chromatography detection method for epimedium in ginseng and kudzu kidney-tonifying preparations, which can quickly and accurately detect epimedium therein, improve the accuracy and comprehensiveness of the quality control of ginseng and kudzu kidney-tonifying preparations, and is conducive to the establishment and overall evaluation of the quality standards of ginseng and kudzu kidney-tonifying preparations.

[0088] The technical solution is as follows:

[0089] A thin layer chromatography method for detecting epimedium in a ginseng and kudzu kidney-tonifying preparation, wherein the raw materials of the ginseng and kudzu kidney-tonifying preparation include pseudoginseng, kudzu root and epimedium;

[0090] The thin layer chromatography detection method of Epimedium in the Shenkui tonifying kidney preparation comprises the following steps:

[0091] Prepare the test solution, icariin reference solution and epimedium reference medicinal material solution respectively;

[0092] Spot the test solution, the icariin reference solution and the epimedium reference medicinal material solution on the same thin layer chromatography plate, develop them in a developing agent, add a color developer, and inspect;

[0093] The developing solvent is a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7: (2.4-2.6): (0.24-0.26);

[0094] The color developer is an ethanol solution of aluminum chloride.

[0095] This method can quickly separate the components in the sample solution and qualitatively analyze Epimedium. The corresponding thin-layer chromatography spots are clear and there is no interference from the negative control. This method improves the accuracy and comprehensiveness of the quality control of ginseng and kudzu kidney-tonifying preparations and is conducive to the establishment of quality standards and overall evaluation of ginseng and kudzu kidney-tonifying preparations. In addition, the operation is simple and flexible, with high sensitivity and resolution, low equipment cost and low cost.

[0096] The present application is further described in detail below in a step-by-step manner. It should be noted that the present application does not limit the order among S100, S200 and S300, and the numbering is only for the convenience of description.

[0097] S100: Prepare test solution.

[0098] In some embodiments, the method for preparing the test solution comprises the following steps:

[0099] The ginseng and kudzu kidney-tonifying preparation test sample is mixed with a first alcohol-containing solvent to perform a first extraction to obtain a test sample extract, and the test sample solution is prepared.

[0100] In some embodiments, the first alcohol-containing solvent is selected from a first alcohol or a mixed solvent of the first alcohol and water.

[0101] In some embodiments, the first alcohol is selected from one or more of ethanol and methanol, that is, the first alcohol is ethanol, methanol, or a mixed solvent of ethanol and methanol.

[0102] In one embodiment, the first alcohol is ethanol or methanol.

[0103] In some embodiments, the first alcohol-containing solvent comprises 20%-100% of the first alcohol and 0%-80% of water, measured by volume. It is understood that in the first alcohol-containing solvent, the volume percentage of the first alcohol includes, but is not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, and the volume percentage of water includes, but is not limited to, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0%.

[0104] In one embodiment, the first alcohol-containing solvent consists of 80% ethanol and 20% water, calculated by volume.

[0105] In some embodiments, the first extraction method of the ginseng and kudzu kidney-tonifying preparation sample is heating extraction.

[0106] In one embodiment, the temperature of the heating extraction is 80°C-100°C, including but not limited to 80°C, 85°C, 90°C, 95°C or 100°C.

[0107] In one embodiment, the heating extraction time is 0.5h-2h, including but not limited to 0.5h, 1h, 1.5h or 2h.

[0108] In some embodiments, in the test solution, the mass volume ratio of the ginseng and kudzu kidney-tonifying preparation test sample and the first alcohol-containing solvent is (1-200) mg:1 ml, including but not limited to 1 mg:1 ml, 5 mg:1 ml, 10 mg:1 ml, 20 mg:1 ml, 40 mg:1 ml, 50 mg:1 ml, 80 mg:1 ml, 100 mg:1 ml, 120 mg:1 ml, 140 mg:1 ml, 150 mg:1 ml, 180 mg:1 ml or 200 mg:1 ml.

[0109] In one embodiment, in the test solution, the mass volume ratio of the ginseng and kudzu kidney-tonifying preparation test sample and the first alcohol-containing solvent is (10-200) mg: 1 ml.

[0110] In some embodiments, the obtained test sample extract can be directly used as the test sample solution.

[0111] In some embodiments, the method for preparing the test solution comprises the following steps:

[0112] Take 40 mg of the test sample of the Shenkui Bu Shen preparation, add 1 ml of 80% ethanol, dissolve it in water, filter it, and use the filtrate as the test sample solution.

[0113] S200: Prepare icariin reference solution.

[0114] In some embodiments, the preparation method of the icariin reference solution comprises the following steps:

[0115] The icariin reference substance is mixed with the second alcohol-containing solvent to prepare the icariin reference substance solution.

[0116] In some embodiments, the second alcohol-containing solvent is selected from a second alcohol or a mixed solvent of a second alcohol and water.

[0117] In some embodiments, the second alcohol is selected from one or more of ethanol and methanol, that is, the second alcohol is ethanol, methanol, or a mixed solvent of ethanol and methanol.

[0118] In one embodiment, the second alcohol is methanol.

[0119] In some embodiments, the second alcohol-containing solvent comprises 20%-100% of the second alcohol and 0%-80% of water, measured by volume. It is understood that the volume percentage of the second alcohol in the second alcohol-containing solvent includes, but is not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, and the volume percentage of water includes, but is not limited to, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0%.

[0120] In one embodiment, the second alcohol-containing solvent is methanol.

[0121] In some embodiments, in the icariin reference solution, the mass volume ratio of the icariin reference and the second alcohol-containing solvent is (0.1-10) mg:1 ml, including but not limited to 0.1 mg:1 ml, 1 mg:1 ml, 5 mg:1 ml, 1 mg:1 ml, 2 mg:1 ml, 4 mg:1 ml, 5 mg:1 ml, 8 mg:1 ml or 10 mg:1 ml.

[0122] S300: preparing an Epimedium control medicinal material solution.

[0123] In some embodiments, the method for preparing the epimedium control medicinal material solution comprises the following steps:

[0124] The epimedium is mixed with a third alcohol-containing solvent for extraction to obtain an epimedium control medicinal material extract, and the epimedium control medicinal material solution is prepared.

[0125] In some embodiments, the third alcohol-containing solvent is selected from a third alcohol or a mixed solvent of a third alcohol and water.

[0126] In some embodiments, the third alcohol is selected from one or more of ethanol and methanol, that is, the third alcohol is ethanol, methanol or a mixed solvent of ethanol and methanol.

[0127] In one embodiment, the third alcohol is methanol.

[0128] In some embodiments, the third alcohol-containing solvent comprises, by volume percentage, 20%-100% of the third alcohol and 0%-80% of water. It is understood that, in the third alcohol-containing solvent, the volume percentage of the third alcohol includes, but is not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, and the volume percentage of water includes, but is not limited to, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 0%.

[0129] In one embodiment, the third alcohol-containing solvent is methanol.

[0130] In some embodiments, the extraction method of the Epimedium control medicinal material is warm immersion.

[0131] In one embodiment, the temperature of the warm soak is 50°C-70°C, including but not limited to 50°C, 55°C, 60°C, 65°C or 70°C.

[0132] In one embodiment, the warm soaking time is 0.5h-2h, including but not limited to 0.5h, 1h, 1.5h or 2h.

[0133] In some embodiments, after obtaining the epimedium control medicinal material extract, the method further includes evaporating the extract to obtain a residue, and then re-dissolving the residue in a third alcohol-containing solvent to prepare an epimedium control medicinal material solution. It is understood that the third alcohol-containing solvent used to dissolve the residue may be the same as or different from the third alcohol-containing solvent used to extract the epimedium control medicinal material.

[0134] In some embodiments, in the epimedium control medicinal material solution, the mass volume ratio of the epimedium and the third alcohol-containing solvent is (5-500) mg: 1 ml, including but not limited to 5 mg: 1 ml, 10 mg: 1 ml, 20 mg: 1 ml, 40 mg: 1 ml, 50 mg: 1 ml, 80 mg: 1 ml, 100 mg: 1 ml, 120 mg: 1 ml, 140 mg: 1 ml, 150 mg: 1 ml, 180 mg: 1 ml : 1ml, 200mg: 1ml, 220mg: 1ml, 240mg: 1ml, 250mg: 1ml, 280mg: 1ml, 300mg: 1ml, 320mg: 1ml, 340mg: 1 ml, 350mg: 1ml, 380mg: 1ml, 400mg: 1ml, 420mg: 1ml, 440mg: 1ml, 450mg: 1ml, 480mg: 1ml or 500mg: 1ml.

[0135] In one embodiment, in the epimedium control medicinal material solution, the mass volume ratio of the epimedium and the third alcohol-containing solvent is (10-500) mg:1 ml.

[0136] In some embodiments, the method for preparing the epimedium control medicinal material solution comprises the following steps:

[0137] Take 0.5 g of Epimedium control medicinal material, add 10 ml of 70% ethanol, soak at warm temperature for 2 hours, filter, evaporate the filtrate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the control medicinal material solution.

[0138] S400: Spot the test solution, the icariin reference solution and the epimedium reference medicinal material solution on the same thin layer chromatography plate, develop them in a developing agent, add a color developer for color development, and inspect.

[0139] It can be understood that in one example, 1 μl of the test solution, 1 μl of the icariin reference solution and 1 μl of the epimedium reference medicinal material solution were respectively pipetted and spotted on the same thin layer chromatography plate.

[0140] In the present application, the developing agent is a mixed solvent of chloroform, methanol and water in a volume ratio of 7:(2.4-2.6):(0.24-0.26). It is understood that the volume ratio of chloroform, methanol and water includes but is not limited to 7:2.4:0.24, 7:2.4:0.25, 7:2.4:0.26, 7:2.5:0.24, 7:2.5:0.25, 7:2.5:0.26, 7:2.6:0.24, 7:2.6:0.25 or 7:2.6:0.26.

[0141] In one embodiment, the developing solvent is a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:2.5:(0.24-0.26).

[0142] In one embodiment, the developing solvent is a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:2.5:0.25.

[0143] It can be understood that the thin layer chromatography plate needs to be placed in a developing tank containing a developing agent for development. After the development step and before the color development step, the thin layer chromatography plate needs to be taken out and air-dried or baked to remove the developing agent.

[0144] In the present application, the color developer is an ethanol solution of aluminum chloride.

[0145] In one embodiment, in the developer, the mass volume ratio of aluminum chloride and ethanol is 1g:(49-199)ml, including but not limited to 1g:49ml (or 2g:98ml), 1g:50ml, 1g:60ml, 1g:65ml, 1g:70ml, 1g:80ml, 1g:85ml, 1.5g:98.5ml, 1g:99ml, 1g:100ml, 1g:150ml, 1g:180ml or 1g:199ml.

[0146] In one embodiment, in the developer, the mass volume ratio of aluminum chloride to ethanol is 1 g:99 ml.

[0147] In some embodiments, the light source used for inspection is a 365 nm ultraviolet lamp.

[0148] In the present application, in the test sample chromatogram, fluorescent spots of the same color are displayed at positions corresponding to the control medicinal material chromatogram and the control sample chromatogram.

[0149] In some embodiments, the raw materials of the ginseng and kudzu kidney-tonifying preparation include 1000-2000 parts of Pseudostellariae Radix, 500-1500 parts of Puerariae Radix and 100-900 parts of Epimedium.

[0150] In some embodiments, the dosage form of the Shenkui-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

[0151] The following is further described with reference to specific embodiments:

[0152] Instruments and reagents

[0153] The Shen Ge Bu Shen preparations are Shen Ge Bu Shen Capsules, Shen Ge Bu Shen Capsules-1, sourced from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 23005; Shen Ge Bu Shen Capsules-2, sourced from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 21002; Shen Ge Bu Shen Capsules-3, sourced from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 21003;

[0154] Icariin reference substance was obtained from the China Food and Drug Administration, batch number: 110737-202017;

[0155] Epimedium as a control herbal medicine was obtained from the China Food and Drug Administration, batch number: 121632-202103;

[0156] Silica gel G thin layer plate-1: Qingdao Ocean Chemical Co., Ltd., specifications 100 mm × 100 mm, thickness 0.20 mm-0.25 mm; Silica gel G thin layer plate-2: MACHEREY-NAGEL-Gmbh & Cak G-010303;

[0157] All other reagents used were of analytical grade. The concentrations of the alcohol-water mixed solutions in the following examples are all expressed in volume fractions. For example, 75% ethanol represents an ethanol solution with a volume fraction of 75% anhydrous ethanol and a volume fraction of 25% water; 1% aluminum chloride in ethanol represents that the mass of aluminum chloride is 1 g and the volume of ethanol is 99 ml in the aluminum chloride in ethanol solution; and 10% sulfuric acid in ethanol represents that the volume fraction of 98 wt % concentrated sulfuric acid is 10% and the volume fraction of ethanol is 90%.

[0158] Example 1

[0159] This example investigates the separation effect of epimedium in the Shenkui Bushen Busheng preparation using a thin layer chromatography method with different mixed solvents as developing agents. The developing agent suitable for this application is determined by the following steps:

[0160] Take 40 mg of the contents of Shenge Bushen Capsule-1, add 1 ml of 80% ethanol, heat at 80°C for 2 hours, filter, and use the filtrate as the test solution. Separately, take 0.5 g of Epimedium reference medicinal material, add 10 ml of 70% ethanol, warm at 60°C for 2 hours, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to use as the control medicinal material solution. Take the icariin reference standard and add methanol to make a solution containing 1 mg per 1 ml, which serves as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), 1 μl of each of the above three solutions was applied to the same silica gel G thin layer plate-1. Then, 1 μl of each of the above three solutions was applied to the same silica gel G thin layer plate-1. Each silica gel G thin layer plate was developed using the developing agents listed in Table 1, removed, dried, sprayed with 1% aluminum chloride in ethanol, and inspected under a 365 nm UV lamp. In the chromatogram of the test sample, the spots displayed at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the reference substance were observed. The results are shown in Figures 1 to 17. The order of spots from left to right in Figures 1 to 17 is: 1. Shenge Bushen Capsule-1, dot spotting; 2. Epimedium control medicinal material, dot spotting; 3. Icariin reference substance, dot spotting; 4. Shenge Bushen Capsule-1, strip spotting; 5. Epimedium control medicinal material, strip spotting; 6. Icariin reference substance, strip spotting.

[0161] Table 1. Comparison of different developing agents in the identification of Epimedium

[0162] As shown in Figure 1, in the chromatogram of the test sample, the fluorescent spots are rich in information, the separation effect of each component is good, and the fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0163] As shown in Figure 2, there is less fluorescent spot information in the test sample chromatogram. At the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram, some fluorescent spots of the same color can be displayed. Although the samples can be separated, the ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

[0164] As shown in Figure 3, there is less fluorescent spot information in the test sample chromatogram. At the corresponding positions in the control herbal chromatogram, some fluorescent spots of the same color can be displayed. Although the sample can be separated, the separation effect is worse than that in Figure 1. There are no obvious visible fluorescent spots in the control chromatogram.

[0165] As shown in Figure 4, there are few obvious fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material, and the separation effect of each component is very poor.

[0166] As shown in Figure 5, there are few obvious fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material, and the separation effect of each component is very poor.

[0167] As shown in Figure 6, there are few visible fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material. The separation effect of each component is very poor, and the chromatogram of the test sample has obvious tailing.

[0168] As shown in Figure 7, there are few obvious fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material, and the separation effect of each component is very poor.

[0169] As shown in Figure 8, there are few obvious visible fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material. The separation effect of each component is very poor, and the chromatogram of the test sample has serious tailing.

[0170] As shown in Figure 9, there are few obvious fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material, and the separation effect of each component is very poor.

[0171] As shown in Figure 10, there are few obvious visible fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material. The separation effect of each component is very poor, and the chromatogram of the test sample has serious tailing.

[0172] As shown in Figure 11, there is less fluorescent spot information in the test sample chromatogram. At the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram, some fluorescent spots of the same color can be displayed. Although the samples can be separated, the ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

[0173] As shown in Figure 12, in the chromatogram of the test sample, there is little information about the fluorescent spots. Although the sample can be separated, the specific shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

[0174] As shown in Figure 13, there are few obvious visible fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material. The separation effect of each component is very poor, and the chromatogram of the test sample has serious tailing.

[0175] As shown in Figure 14, there are few obvious visible fluorescent spots in the chromatograms of the test sample, the reference sample, and the reference medicinal material. The separation effect of each component is very poor, and the chromatogram of the test sample has serious tailing.

[0176] As shown in Figure 15, there is little information about the fluorescent spots in the test sample chromatogram. At the corresponding positions in the control medicinal material chromatogram, some fluorescent spots of the same color can be displayed. Although the samples can be separated, the relative shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

[0177] As shown in Figure 16, in the chromatogram of the test sample, there is less information about the fluorescent spots. At the corresponding positions in the chromatogram of the control medicinal material, some fluorescent spots of the same color can be displayed. Although the samples can be separated, the ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

[0178] As shown in Figure 17, there is less fluorescent spot information in the test sample chromatogram. At the corresponding position of the control medicinal material chromatogram, some fluorescent spots of the same color can be displayed. Although the sample can be separated, the ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

[0179] Example 2

[0180] This example investigates the effect of thin layer chromatography detection method using different solvents as solvents for dissolving the test sample to detect epimedium in the Shenkui Bushen Bucheng preparation. The steps are as follows:

[0181] Take 40 mg of the contents of Shen Ge Bushen Capsule-1, add 1 ml of the solvent listed in Table 3, heat at 80°C for 2 hours, filter, and use the filtrate as the test solution. Separately, take 0.5 g of Epimedium control medicinal material, add 10 ml of 70% ethanol, and soak at 60°C for 2 hours. Filter, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to use as the control medicinal material solution. Also, take the icariin control standard and add methanol to a solution containing 1 mg per 1 ml. This will serve as the control solution. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μl of each of the above three solutions was aspirated and spotted on the same silica gel G thin layer plate-1. Then 1 μl of each of the above three solutions was aspirated and spotted on the same silica gel G thin layer plate-1. Each silica gel G thin layer plate was placed in a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.25 for development. The plates were taken out, dried, sprayed with 1% aluminum chloride in ethanol solution, and inspected under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, the spots displayed at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the reference substance were observed. The results are shown in Figures 1 and 18 to 24. The order of spots from left to right in Figures 1 and 17 to 23 is: 1. Shenge Bushen Capsule-1, dot spotting; 2. Epimedium control medicinal material, dot spotting; 3. Icariin reference substance, dot spotting; 4. Shenge Bushen Capsule-1, strip spotting; 5. Epimedium control medicinal material, strip spotting; 6. Icariin reference substance, strip spotting.

[0182] Table 2. Comparison of solvent types used to dissolve test samples in the identification of Epimedium

[0183] As shown in Figure 1, in the chromatogram of the test sample, the fluorescent spots are rich in information, the separation effect of each component is good, and the fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0184] As shown in Figure 18, the fluorescent spots in the test sample chromatogram are rich in information, the components are well separated, and the fluorescent spots of the same color appear at the corresponding positions in the control herbal chromatogram and the control sample chromatogram.

[0185] As shown in Figure 19, in the test sample chromatogram, the fluorescent spots are rich in information, the separation effect of each component is good, and the fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

[0186] As shown in Figure 20, in the test sample chromatogram, the fluorescent spots are rich in information, the separation effect of each component is good, and the fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0187] As shown in Figure 21, there is less fluorescent spot information in the test sample chromatogram. At the corresponding position of the control medicinal material chromatogram, some fluorescent spots of the same color can be displayed. Although the sample can be separated, the ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

[0188] As shown in Figure 22, in the test sample chromatogram, the fluorescent spots are rich in information, the separation effect of each component is good, and fluorescent spots of the same color appear at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

[0189] As shown in Figure 23, the fluorescent spots are rich in information, the separation effect of each component is good, and the fluorescent spots of the same color can be displayed at the corresponding positions of the control medicinal material chromatogram and the reference product chromatogram.

[0190] As shown in Figure 24, there is less fluorescent spot information in the test sample chromatogram. At the corresponding position of the control medicinal material chromatogram, some fluorescent spots of the same color can be displayed. Although the sample can be separated, the ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

[0191] Example 3

[0192] This example investigates the color development effect of epimedium in the Shenkui tonifying kidney preparation by using a thin layer chromatography detection method and different mixed solvents as color developers, and determines the color developer suitable for this application. The steps are as follows:

[0193] Take 40 mg of the contents of Shen Ge Bushen Capsule-1 and add 1 ml of 80% ethanol. Heat at 80°C for 2 hours. Filter and use the filtrate as the test solution. Separately, take 0.5 g of Epimedium reference herb and add 10 ml of 70% ethanol. Steep at 60°C for 2 hours. Filter and evaporate the filtrate to dryness. Dissolve the residue in 1 ml of methanol to prepare the control herb solution. Also, take the icariin reference standard and add methanol to a solution containing 1 mg per 1 ml. This will serve as the control solution. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μl of each of the above three solutions was aspirated and spotted on the same silica gel G thin layer plate-1. Then 1 μl of each of the above three solutions was aspirated and spotted on the same silica gel G thin layer plate-1. Each silica gel G thin layer plate was placed in a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.25 for development. The plates were taken out, dried, sprayed with the developer shown in Table 3, and inspected under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, the spots were observed at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the reference substance. The results are shown in Figures 1 and 25. The order of spots from left to right in Figures 1 and 25 is 1. Shenge Bushen Capsule-1, dot spot; 2. Epimedium control medicinal material, dot spot; 3. Icariin reference substance, dot spot; 4. Shenge Bushen Capsule-1, strip spot; 5. Epimedium control medicinal material, strip spot; 6. Icariin reference substance, strip spot.

[0194] Table 3. Comparison of different colorants in the identification of Epimedium

[0195] As shown in Figure 1, in the chromatogram of the test sample, the fluorescent spots are rich in information, the separation effect of each component is good, and the fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0196] As shown in Figure 25, although fluorescent spots can be seen in the chromatogram of the test sample, the fluorescent spots have little information and the color development effect is poor, which does not meet the expected effect.

[0197] Example 4 Methodological Investigation

[0198] (1) Specificity experiment

[0199] Take 40 mg of the contents of three different batches of Shengge Bushen Capsules (Shengge Bushen Capsules-1, Shengge Bushen Capsules-2, and Shengge Bushen Capsules-3), add 1 ml of 80% ethanol, heat at 80°C for 2 hours, filter, and use the filtrate as the test solution. Separately, take 0.5 g of Epimedium control herb, add 10 ml of 70% ethanol, warm at 60°C for 2 hours, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 ml of methanol to use as the control herb solution. Also, take the icariin control standard and add methanol to a solution containing 1 mg per 1 ml. This is used as the control solution. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μl of each of the above three solutions was aspirated and spotted on the same silica gel G thin layer plate-1. Then 1 μl of each of the above three solutions was aspirated and spotted on the same silica gel G thin layer plate-1. Each silica gel G thin layer plate was placed in a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.25 for development. The plates were taken out, dried, sprayed with 1% aluminum chloride in ethanol solution, and inspected under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, the spots were observed at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the reference substance. The results are shown in FIG26 . The order of spots from left to right in FIG26 is: 1. Ginseng and Radix Bu Shen Capsules-1, strip spots; 1. Ginseng and Radix Bu Shen Capsules-2, strip spots; 1. Ginseng and Radix Bu Shen Capsules-3, strip spots; 4. Epimedium control medicinal material, strip spots; 5. Icariin reference substance, strip spots.

[0200] The results show that in the test sample chromatogram shown in Figure 26, the fluorescent spots are rich in information, the components are well separated, and spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

[0201] (2) Investigation of the tolerance of thin layer plates

[0202] Take 40 mg of the contents of Shengge Bushen Capsule-2 from the same batch, add 1 ml of 80% ethanol, and heat at 80°C for 2 hours. Filter and use the filtrate as the test solution. Separately, take 0.5 g of Epimedium reference medicinal material, add 10 ml of 70% ethanol, and soak at 60°C for 2 hours. Filter and evaporate the filtrate to dryness. Dissolve the residue in 1 ml of methanol to prepare the control medicinal material solution. Also, take the icariin reference standard and add methanol to prepare a solution containing 1 mg per 1 ml. This will serve as the reference solution. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μl of each of the three solutions was taken and spotted on the same silica gel G thin layer plate-1, and then spotted on the same silica gel G thin layer plate-2 according to the same method. Each silica gel G thin layer plate was placed in a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.25 for development. The plates were taken out, dried, sprayed with 1% aluminum chloride in ethanol solution, and inspected under a 365 nm ultraviolet lamp. In the chromatogram of the test sample, the spots displayed at the positions corresponding to the chromatogram of the control medicinal material and the chromatogram of the reference substance are observed. The results are shown in Figure 27. The order of spots from left to right in Figure 27 is: 1. Epimedium control medicinal material, strip spot; 2. Ginseng and Radix Bu Shen Capsule-2, strip spot; 3. Icariin reference substance, strip spot; 4. Epimedium control medicinal material, strip spot; 5. Ginseng and Radix Bu Shen Capsule-2, strip spot; 6. Icariin reference substance, strip spot; and Figure 27 is a splicing of the chromatographic results of silica gel G thin layer plate-1 and silica gel G thin layer plate-2, 1, 2, 3 spots are on silica gel G thin layer plate-1, 4, 5, 6 spots are on silica gel G thin layer plate-2.

[0203] The results showed that in the test sample chromatogram shown in Figure 27, the fluorescent spots were rich in information and the separation effect of each component was good. Spots of the same color were displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram, indicating that both thin-layer chromatography silica gel plates can achieve good separation effects and are suitable for the thin-layer chromatography detection method of Epimedium in the Shenkui-tonifying preparation.

[0204] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0205] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A thin layer chromatography detection method for epimedium in a ginseng and kudzu kidney-tonifying preparation, characterized in that: The raw materials of the Shenkui tonifying kidney preparation include Pseudostellaria heterophylla, Pueraria root and Epimedium; The thin layer chromatography detection method of Epimedium in the Shenkui tonifying kidney preparation comprises the following steps: Prepare the test solution, icariin reference solution and epimedium reference medicinal material solution respectively; Spot the test solution, the icariin reference solution and the epimedium reference medicinal material solution on the same thin layer chromatography plate, develop them in a developing agent, add a color developer to develop the color, and inspect; Wherein, the developing agent is a mixed solvent composed of chloroform, methanol and water in a volume ratio of 7:(2.4-2.6):(0.24-0.26); The color developer is an ethanol solution of aluminum chloride.

2. The thin layer chromatography detection method according to claim 1, characterized in that: The developing agent is a mixed solvent composed of chloroform, methanol and water in a volume ratio of 7:2.5:(0.24-0.26).

3. The thin layer chromatography detection method according to any one of claims 1 to 2, characterized in that: In the developer, the mass volume ratio of aluminum chloride to ethanol is 1g:(49-199)ml.

4. The thin layer chromatography detection method according to any one of claims 1 to 3, characterized in that: The light source used for inspection is 365nm ultraviolet lamp.

5. The thin layer chromatography detection method according to any one of claims 1 to 4, characterized in that: The preparation method of the test solution comprises the following steps: Mixing the ginseng and kudzu kidney-tonifying preparation test sample with the first alcohol-containing solvent for a first extraction to obtain an extract, and preparing the test sample solution; The preparation method of the icariin reference substance solution comprises the following steps: Mixing the icariin reference substance with the second alcohol-containing solvent to prepare the icariin reference substance solution; The preparation method of the epimedium control medicinal material solution comprises the following steps: The epimedium is mixed with the third alcohol-containing solvent for a second extraction to obtain an extract, and the epimedium control medicinal material solution is prepared.

6. The thin layer chromatography detection method according to claim 5, characterized in that: In the step of preparing the test solution, one or more of the following (1)-(5) are met: (1) the first alcohol-containing solvent is selected from a first alcohol or a mixed solvent of the first alcohol and water; (2) In the test solution, the mass volume ratio of the ginseng and kudzu kidney-tonifying preparation test sample and the first alcohol-containing solvent is (1-200) mg: 1 ml; (3) the first alcohol is selected from one or more of ethanol and methanol; (4) In terms of volume percentage, the first alcohol-containing solvent comprises 20%-100% of the first alcohol and 0%-80% of water; (5) The first extraction method is heating extraction, the temperature is 80°C-100°C, and the time is 0.5h-2h.

7. The thin layer chromatography detection method according to claim 5, characterized in that: In the step of preparing the icariin reference solution, one or more of the following (1)-(4) are satisfied: (1) the second alcohol-containing solvent is selected from a second alcohol or a mixed solvent of a second alcohol and water; (2) In the icariin reference solution, the mass volume ratio of the icariin reference to the second alcohol-containing solvent is (0.1-10) mg: 1 ml; (3) the second alcohol is selected from one or more of ethanol and methanol; (4) In terms of volume percentage, the second alcohol-containing solvent comprises 20%-100% of the second alcohol and 0%-80% of water.

8. The thin layer chromatography detection method according to claim 5, characterized in that: In the step of preparing the epimedium control medicinal material solution, one or more of the following (1)-(5) are satisfied: (1) the third alcohol-containing solvent is selected from a third alcohol or a mixed solvent of a third alcohol and water; (2) In the epimedium control medicinal material solution, the mass volume ratio of the epimedium and the third alcohol-containing solvent is (5-500) mg: 1 ml; (3) the third alcohol is selected from one or more of ethanol and methanol; (4) In terms of volume percentage, the third alcohol-containing solvent comprises 20%-100% of a third alcohol and 0%-80% of water; (5) The second extraction method is warm immersion, the temperature is 50℃-70℃, and the time is 0.5h-2h.

9. The thin layer chromatography detection method according to any one of claims 1 to 8, characterized in that: In parts by weight, the raw materials of the ginseng and kudzu kidney-tonifying preparation include 1000-2000 parts of Pseudostellaria heterophylla, 500-1500 parts of Pueraria root and 100-900 parts of Epimedium.

10. The thin layer chromatography detection method according to any one of claims 1 to 9, characterized in that: The dosage form of the ginseng and kudzu kidney-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

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