WO2025107495A1 - Method for thin-layer chromatography detection of herba epimedii in ginseng-radix puerariae kidney-tonifying preparation - Google Patents

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

TLC Detection of Epimedium in Shenge Kidney-tonifying Preparation

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

Technical Field

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

Epimedium brevicornu Maxim., a perennial herb of the genus Epimedium of the Berberidaceae family, is an important medicine for tonifying the life gate, replenishing the essence and Qi, strengthening the tendons and bones, and nourishing the kidney and strengthening yang. It is often 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, diuresis, and antitussive and expectorant.

The main ingredients of Shenku Bu Shen preparation are Pseudostellariae Radix, Puerariae Radix and Epimedium, and its functions and indications are to invigorate Qi, nourish Yin and tonify the kidney. It is suitable for mild and moderate depression, which belongs to Qi and Yin deficiency and kidney deficiency in TCM, with symptoms such as low mood, excessive thinking, reduced speech and movement, slow eyesight, forgetfulness, poor appetite, palpitations and timidity, insomnia and dreaminess, irritability, pale red or reddish tongue, white or peeled tongue coating, and weak pulse.

Epimedium, as the main raw material in ginseng and kudzu kidney-tonifying preparations, plays an important role in the effects 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.

Summary of the invention

According to various embodiments of the present application, a thin layer chromatography detection method for Epimedium in a Shenkui-tonifying preparation is provided.

The technical solution is as follows:

A thin layer chromatography detection method for 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;

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.

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

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

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

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

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

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

In some of the 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.

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

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

In some embodiments, the first extraction method is heating extraction, the temperature is 80° C.-100° C., and the time is 0.5 h-2 h.

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

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

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

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

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

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

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

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

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

In some of the 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.

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

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

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

In some of the 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.

In some embodiments, the dosage form of the ginseng and kudzu kidney-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

The details of one or more embodiments of the present application are set forth in the following drawings and description. Other features, objects, and advantages of the present application will become apparent from the description, drawings, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

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 drawings. The additional details or examples used to describe the drawings should not be considered as limiting the scope of any of the disclosed inventions, the embodiments and/or examples currently described, and the best modes of these inventions currently understood.

FIG1 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.25 as a developing agent and a 1% aluminum chloride ethanol solution as a color developer as shown in an embodiment of the present application;

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;

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 as shown in an embodiment of the present application;

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 as shown in an embodiment of the present application;

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 as shown in an embodiment of the present application;

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 as shown in an embodiment of the present application;

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

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 as shown in an embodiment of the present application;

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 as shown in an embodiment of the present application;

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 as shown in an embodiment of the present application;

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 as shown in an embodiment of the present application;

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 as shown in an embodiment of the present application;

FIG. 13 is a diagram showing an embodiment of the present application in which a mixed solvent consisting of chloroform, ethanol and water in a volume ratio of 7:2.5:0.25 is used as a developing solvent. Thin layer chromatography test results of the agent;

FIG14 is a thin layer chromatography test result using a mixed solvent of chloroform, ethanol and water in a volume ratio of 7:1.25:1.5 as a developing solvent as shown in an embodiment of the present application;

FIG15 is a thin layer chromatography test result using a mixed solvent of chloroform, ethanol and water in a volume ratio of 7:0.25:2.5 as a developing solvent as shown in an embodiment of the present application;

FIG16 is a thin layer chromatography test result using a mixed solvent consisting of dichloromethane, ethyl acetate, methanol and water in a volume ratio of 1:5:4 as a developing solvent as shown in an embodiment of the present application;

FIG17 is a thin layer chromatography test result using a mixed solvent consisting of methanol, butanone, chloroform and water in a volume ratio of 2:3:3:0.5 as a developing solvent as shown in an embodiment of the present application;

FIG18 is a thin layer chromatography test result using anhydrous ethanol as a solvent for dissolving a test sample as shown in an embodiment of the present application;

FIG. 19 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;

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;

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

FIG22 is a thin layer chromatography test result using anhydrous methanol as a solvent for dissolving a test sample as shown in an embodiment of the present application;

FIG. 23 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;

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;

FIG. 25 is a thin layer chromatography test result using 10% sulfuric acid ethanol solution as a color developer according to an embodiment of the present application;

FIG26 is a thin layer chromatography test result of Epimedium in different batches of Shengge Bushen Capsules;

Figure 27 shows the results of thin layer chromatography testing of the same batch of Shen Ge Bushen Capsules using different thin layer chromatography plates.

DETAILED DESCRIPTION

The present application is further described in detail below in conjunction with 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 make the understanding of the present application disclosure more thorough and comprehensive.

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

In the present application, “further”, “furthermore”, “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.

In the present application, "at least one" means more than one, such as one, two and more than two. "Multiple" or "several" means at least two, such as two, three, etc., and "multilayer" means at least two layers, such as two layers, three layers, etc., unless otherwise clearly and specifically defined. In the description of the present application, "several" means at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

When a numerical range is disclosed in this application, the above range is considered to be 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 features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

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 also 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.

Unless mentioned to the contrary, terms in the singular may include plural forms and should not be construed as being one in number.

In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features.

In this application, "includes", "comprising", "containing", "having" or other variations are intended to cover non-closed inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the final result can be added. The compositions and methods/processes of the present application include, consist of, and are essentially composed of the essential elements and limitations described herein and any additional or optional ingredients, components, steps or limitations described herein.

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

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

In this application, temperature parameters, unless otherwise specified, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control. The room temperature or normal temperature mentioned in this application refers to 0 to 40°C.

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

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

In the present 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%.

The main raw materials of Shenkui tonifying kidney preparation are Pseudostellariae Radix, Puerariae Radix and Epimedium, and the functions and indications are to invigorate Qi, nourish Yin and tonify the kidney. As the main raw material of Shenkui tonifying kidney preparation, Epimedium plays an important role in the effect of Shenkui tonifying kidney preparation, but there is currently a lack of a method that can quickly identify and specifically detect Epimedium in Shenkui tonifying kidney preparation, and it is impossible to accurately and comprehensively measure or control the quality of Shenkui tonifying kidney preparation.

Thin layer chromatography (TLC) is a method of coating a suitable stationary phase on a glass plate, plastic or aluminum substrate to form a uniform thin layer. After spotting and developing, the relative shift value (Rf) is compared with the relative shift value (Rf) of the chromatogram obtained by the same method with a suitable reference substance, and is used for drug identification, impurity inspection or content determination. As an important experimental technique for rapid separation and qualitative analysis of small amounts of substances, thin layer chromatography has the characteristics of short development time, strong separation ability, and high sensitivity. It is a very important analytical method in the quality control of traditional Chinese medicine. Compared with content determination and characteristic spectrum identification, thin layer chromatography is one of the most widely used methods in planar chromatography. It is easy to master, the equipment is cheap, and the operation is simple and flexible. It has the following characteristics: changing the developing agent and development direction at any time, derivatization, color development, observation, and detection at any time, fast method development, high sensitivity and resolution, simultaneous separation of multiple samples, 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 instrumental technologies, it has shown a trend of wider application and has become a modern, sensitive and efficient separation and analysis method.

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

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.

The technical solution is as follows:

A thin layer chromatography detection method for 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;

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.

The method can quickly separate the components in the sample solution and qualitatively analyze Epimedium, and the corresponding thin layer chromatography spots are clear and the negative control has no interference, which improves the accuracy and comprehensiveness of the quality control of the Shenkuo tonifying kidney preparation, and is conducive to the establishment and overall evaluation of the quality standard of the Shenkuo tonifying kidney preparation. In addition, the operation is simple and flexible, with high sensitivity, high resolution, low equipment cost and low cost.

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.

S100: Prepare test solution.

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

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

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

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 ethanol. A mixed solvent of alcohol and methanol.

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

In some embodiments, the first alcohol-containing solvent includes 20%-100% of the first alcohol and 0%-80% of water by volume. It is understandable 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%.

In one embodiment, the first alcohol-containing solvent consists of 80% ethanol and 20% water, measured by volume percentage.

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

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.

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

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.

In one of the 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 (10-200) mg: 1 ml.

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

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

Take 40 mg of the test sample of the ginseng and kudzu kidney-tonifying preparation, add 1 ml of 80% ethanol, heat and dissolve, filter, and use the filtrate as the test sample solution.

S200: Prepare icariin reference solution.

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

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

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

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.

In one embodiment, the second alcohol is methanol.

In some embodiments, the second alcohol-containing solvent includes 20%-100% of the second alcohol and 0%-80% of water by volume. It is understandable that in the second alcohol-containing solvent, the volume percentage of the second 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%.

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

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.

S300: preparing an Epimedium control medicinal material solution.

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

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

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

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.

In one embodiment, the third alcohol is methanol.

In some embodiments, the third alcohol-containing solvent includes 20%-100% of the third alcohol and 0%-80% of water by volume. It is understandable 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%.

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

In some of the embodiments, the method of extracting the Epimedium control medicinal material is warm immersion.

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.

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

In some embodiments, after obtaining the epimedium reference medicinal material extract, the step of evaporating the extract to obtain a residue, and then re-adding a third alcohol-containing solvent to dissolve the residue to prepare an epimedium reference medicinal material solution is also included. It can be understood that the third alcohol-containing solvent used to dissolve the residue can be the same as or different from the third alcohol-containing solvent used to extract the epimedium reference medicinal material.

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: : 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.

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.

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

Take 0.5 g of Epimedium reference 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 the residue to dissolve it, and use it as the reference medicinal material solution.

S400: spotting the test solution, the icariin reference solution and the epimedium reference medicinal material solution on the same thin layer chromatography plate, developing them in a developing agent, adding a color developer for color development, and inspecting.

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.

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 understandable 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.

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

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

It can be understood that the thin layer chromatography plate needs to be placed in a development 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.

In the present application, the developer is an ethanol solution of aluminum chloride.

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.

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

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

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.

In some of the 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.

In some embodiments, the dosage form of the ginseng and kudzu kidney-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

The following is further described in conjunction with specific embodiments:

Instruments and reagents

The Shen Ge Bu Shen preparation is Shen Ge Bu Shen Capsule, Shen Ge Bu Shen Capsule-1, from Xinjiang Huachun Bio-pharmaceutical Co., Ltd., batch number: 23005; Shen Ge Bu Shen Capsule-2, from Xinjiang Huachun Bio-pharmaceutical Co., Ltd., batch number: 21002; Shen Ge Bu Shen Capsule-3, from Xinjiang Huachun Bio-pharmaceutical Co., Ltd., batch number: 21003;

Icariin reference substance, from China Food and Drug Inspection Institute, batch number: 110737-202017;

Epimedium reference medicinal material, from China Food and Drug Inspection Institute, batch number: 121632-202103;

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

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

Example 1

This example investigates the separation effect of using a thin layer chromatography detection method to detect epimedium in the Shen Ge Bu Shen preparation using different mixed solvents as developing agents, and determines the developing agent suitable for this application, the steps are as follows:

Take 40mg of the contents of Shen Ge Bushen Capsule-1, add 1ml of 80% ethanol, heat dissolve at 80℃ for 2h, filter, and use the filtrate as the test solution. Take 0.5g of Epimedium reference medicinal material, add 10ml of 70% ethanol, soak at 60℃ for 2 hours, filter, evaporate the filtrate, and dissolve the residue in 1ml of methanol as the reference medicinal material solution. Take the icariin reference substance again, add methanol to make a solution containing 1mg per 1ml as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 1μl of each of the above three solutions and spot them on the same silica gel G thin layer plate-1, then take 1μl of each of the above three solutions and spot them on the same silica gel G thin layer plate-1, and each silica gel G thin layer plate is developed according to the developing agent listed in Table 1, taken out, dried, sprayed with 1% aluminum chloride ethanol solution, and placed under 365nm ultraviolet light for inspection. 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 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.

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

As can be seen from 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 fluorescent spots of the same color appear at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

As shown in Figure 2, in the test sample chromatogram, there is less information on the fluorescent spots. 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.

As shown in Figure 3, there is less information about fluorescent spots in the test sample chromatogram. Some fluorescent spots of the same color can be displayed at the corresponding positions of the reference medicinal material chromatogram. Although the samples can be separated, the separation effect is worse than that in Figure 1. There are no obvious visible fluorescent spots in the reference chromatogram.

As shown in Figure 4, there are few obvious visible 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.

As shown in Figure 5, there are few obvious visible 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.

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

As shown in Figure 7, there are few obvious visible 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.

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

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

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

As can be seen from Figure 11, in the test sample chromatogram, there is less information on the fluorescent spots. 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.

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 ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

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

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

As shown in Figure 15, in the test sample chromatogram, there is little information about the fluorescent spots. At the corresponding positions of the control medicinal material 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.

As shown in Figure 16, in the test sample chromatogram, there is less information on the fluorescent spots. 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 ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

As shown in Figure 17, in the test sample chromatogram, there is less information on the fluorescent spots. 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 ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

Example 2

This example investigates the effect of using thin layer chromatography detection method, using different solvents as solvents for dissolving the test sample, to detect the epimedium in the Shenku tonifying kidney preparation, and the steps are as follows:

Take 40 mg of the contents of Shengge Bushen Capsule-1, add 1 ml of the solvent shown in Table 3, heat and dissolve at 80°C for 2 hours, filter, and use the filtrate as the test solution. Take 0.5 g of Epimedium control medicinal material, add 10 ml of 70% ethanol, soak at 60°C for 2 hours, filter, evaporate the filtrate, and add 1 ml of methanol to the residue to dissolve. Solution, as the reference medicinal material solution. Take the icariin reference substance again, add methanol to make a solution containing 1 mg per 1 ml, as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 1 μl of each of the above three solutions and spot them on the same silica gel G thin layer plate-1, then take 1 μl of each of the above three solutions and spot them on the same silica gel G thin layer plate-1, and place each silica gel G thin layer plate in a mixed solvent composed of chloroform, methanol and water in a volume ratio of 7:2.5:0.25 for development, take out, dry, spray with 1% aluminum chloride ethanol solution, and inspect under a 365nm 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 control substance. 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 control substance, dot spotting; 4. Shenge Bushen Capsule-1, strip spotting; 5. Epimedium control medicinal material, strip spotting; 6. Icariin control substance, strip spotting.

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

As can be seen from 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 fluorescent spots of the same color appear at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

As shown in Figure 18, 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 respectively displayed at the corresponding positions of the reference medicinal material chromatogram and the reference sample chromatogram.

As can be seen from Figure 19, 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.

As can be seen from Figure 20, 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 reference sample chromatogram.

As can be seen from Figure 21, in the test sample chromatogram, there is less information on the fluorescent spots. 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 ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

As can be seen from 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 reference sample chromatogram.

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

As can be seen from Figure 24, in the test sample chromatogram, there is less information on the fluorescent spots. 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 ratio shift value (Rf value) is worse than that in Figure 1, which does not meet the expected effect.

Example 3

This example investigates the color development effect of using a thin layer chromatography detection method and different mixed solvents as color developers to finally detect the epimedium in the Shen Ge tonifying kidney preparation, and determines the color developer suitable for this application, and the steps are as follows:

Take 40mg of the contents of Shen Ge Bushen Capsule-1, add 1ml of 80% ethanol, heat at 80℃ for 2h, filter, and use the filtrate as the test solution. Take 0.5g of Epimedium control medicinal material, add 10ml of 70% ethanol, soak at 60℃ for 2h, filter, evaporate the filtrate, and add 1ml of methanol to the residue to dissolve it. As a reference medicinal material solution. Take the icariin reference substance again, add methanol to make a solution containing 1 mg per 1 ml, as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 1 μl of each of the above three solutions and spot them on the same silica gel G thin layer plate-1, then take 1 μl of each of the above three solutions and spot them on the same silica gel G thin layer plate-1, and place each silica gel G thin layer plate in a mixed solvent composed of chloroform, methanol and water in a volume ratio of 7:2.5:0.25 for development, take out, dry, spray with the color developer shown in Table 3, and inspect under a 365nm ultraviolet lamp. In the chromatogram of the test sample, the spots were observed at the positions corresponding to the chromatograms of the control medicinal materials and the control 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-like spots; 2. Epimedium control medicinal material, dot-like spots; 3. Icariin control substance, dot-like spots; 4. Shenge Bushen Capsule-1, strip-like spots; 5. Epimedium control medicinal material, strip-like spots; 6. Icariin control substance, strip-like spots.

Table 3. Comparison of different colorants in the identification of Epimedium

As can be seen from 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 fluorescent spots of the same color appear at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

As can be seen from 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.

Example 4 Methodological Investigation

(1) Specificity experiment

Take 40mg of the contents of three different batches of Shengge Bushen Capsule-1, Shengge Bushen Capsule-2 and Shengge Bushen Capsule-3, add 1ml of 80% ethanol, heat at 80℃ for 2h, filter, and use the filtrate as the test solution. Take 0.5g of Epimedium reference medicinal material, add 10ml of 70% ethanol, soak at 60℃ for 2h, filter, evaporate the filtrate, and add 1ml of methanol to the residue to dissolve it as the reference medicinal material solution. Take the icariin reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference substance solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μl of each of the three solutions was aspirated and spotted on the same silica gel G thin layer plate-1, and then 1 μl of each of the 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, taken out, dried, sprayed with 1% aluminum chloride ethanol solution, and inspected under a 365nm 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 control substance. The results are shown in Figure 26. The order of spots from left to right in Figure 26 is: 1. Ginseng and Radix Bu Shen Capsule-1, strip spot; 1. Ginseng and Radix Bu Shen Capsule-2, strip spot; 1. Ginseng and Radix Bu Shen Capsule-3, strip spot; 4. Epimedium control medicinal material, strip spot; 5. Icariin reference substance, strip spot.

The results show that in the test sample chromatogram shown in Figure 26, the fluorescent spots are rich in information, the separation effect of each component is good, and spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

(2) Investigation of the tolerance of thin layer plates

Take 40mg of the contents of Shengge Bushen Capsule-2 from the same batch, add 1ml of 80% ethanol, heat dissolve at 80℃ for 2h, filter, and use the filtrate as the test solution. Take 0.5g of Epimedium reference medicinal material, add 10ml of 70% ethanol, soak at 60℃ for 2h, filter, evaporate the filtrate to dryness, add 1ml of methanol to the residue to dissolve, and use it as the reference medicinal material solution. Take the icariin reference substance, add methanol to make a solution containing 1mg per 1ml, and use it as the reference substance solution. According to the thin layer chromatography method (General Rules 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 in the same way. 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, taken out, dried, sprayed with 1% aluminum chloride ethanol solution, and inspected under a 365nm ultraviolet lamp. In the test sample chromatogram, observe the spots displayed at the positions corresponding to the control medicinal material chromatogram and the control substance chromatogram. 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.

The results show that in the test sample chromatogram shown in Figure 27, the fluorescent spots are rich in information and the separation effect of each component is good. Spots of the same color are displayed at the corresponding positions of the reference medicinal material chromatogram and the reference sample chromatogram, indicating that both thin layer chromatography silica gel plates can achieve good separation effects. Both are applicable to the thin layer chromatography detection method of Epimedium in the ginseng and kudzu kidney-tonifying preparation.

The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims (10)

A thin layer chromatography detection method for epimedium in a ginseng and kudzu kidney-tonifying preparation, characterized in that the raw materials of the ginseng and kudzu kidney-tonifying 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. The thin layer chromatography detection method according to claim 1 is characterized in that the developing solvent is a mixed solvent composed of chloroform, methanol and water in a volume ratio of 7:2.5:(0.24-0.26). The thin layer chromatography detection method according to any one of claims 1 to 2 is characterized in that, in the developer, the mass volume ratio of aluminum chloride and ethanol is 1g:(49-199)ml. The thin layer chromatography detection method according to any one of claims 1 to 3 is characterized in that the light source used for inspection is a 365nm ultraviolet lamp. The thin layer chromatography detection method according to any one of claims 1 to 4, characterized in that the method for preparing 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. 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 satisfied: (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. 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 and 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. 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. The thin layer chromatography detection method according to any one of claims 1 to 8 is 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. The thin layer chromatography detection method according to any one of claims 1 to 9 is characterized in that the dosage form of the ginseng and kudzu kidney-tonifying preparation is selected from granules, powders, tablets, capsules or pills.