Thin-layer chromatography detection method for kudzu root in taizishen-kudzu kidney-nourishing formulation

Abstract

A thin-layer chromatography detection method for kudzu root in a Taizishen-kudzu kidney-nourishing formulation. The Taizishen-kudzu kidney nourishing formulation comprises the following raw materials: Taizishen, kudzu root and Epimedium. The thin-layer chromatography detection method for kudzu root in a Taizishen-kudzu kidney-nourishing formulation comprises the following steps: respectively preparing a test sample solution, a puerarin control solution and a kudzu root control medicinal material solution; putting the test solution, the puerarin control solution and the kudzu root control medicinal material solution on the same thin-layer chromatography plate, and using a mixed solution of trichloromethane, methanol and water according to a volume ratio of 7: (2.4-2.6): (0.24-0.26) as a developing agent, developing the solutions; inspecting after color development.

Description

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

[0001] This application claims priority to Chinese patent application No. 2023115747007, filed on November 23, 2023, entitled “Thin-layer chromatography detection method of Pueraria root 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 Pueraria root in a Shenkui-tonifying preparation. Background Art

[0003] Pueraria root is the dried root of the leguminous plant Pueraria lobata, commonly known as wild kudzu. It is harvested in autumn and winter, cut into thick slices or small pieces while fresh, and then dried. It has a sweet, pungent, and cooling nature. It has the properties of relieving muscle tension, reducing fever, clearing rashes, promoting fluid production and quenching thirst, promoting yang and stopping diarrhea, promoting blood circulation, and detoxifying alcohol. It is commonly used for exogenous fever, headache, stiff neck and back pain, thirst, polydipsia, measles that has not yet cleared up, dysentery, diarrhea, dizziness and headache, hemiplegia caused by stroke, chest pain, and alcohol poisoning.

[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] Pueraria root, 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 Pueraria root 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 Pueraria root in a Shenkui-tonifying preparation is provided.

[0008] The technical solution is as follows:

[0009] A thin layer chromatography method for detecting kudzu root 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 Pueraria root in the Shenkui tonifying kidney preparation comprises the following steps:

[0011] Prepare the test solution, puerarin reference solution and kudzu root reference medicinal material solution respectively;

[0012] The test solution, puerarin reference solution and kudzu root reference medicinal material solution were spotted on the same thin layer chromatography plate, developed with a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7: (2.4-2.6): (0.24-0.26) as a developing agent, and inspected after color development.

[0013] In some embodiments, the developing solvent is a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.26.

[0014] In some embodiments, the light source used for inspection is a 365 nm ultraviolet lamp.

[0015] In some embodiments, the method for preparing the test solution comprises the following steps:

[0016] 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.

[0017] In some embodiments, the first alcohol-containing solvent is selected from a first alcohol or a mixed solvent of the first alcohol and water.

[0018] 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.

[0019] In some embodiments, the first alcohol is selected from one or more of ethanol and methanol.

[0020] In some embodiments, the first alcohol-containing solvent includes 20%-100% of the first alcohol and 0%-80% of water, calculated by volume.

[0021] In some embodiments, the first extraction is performed by shaking at room temperature.

[0022] In some embodiments, the method for preparing the puerarin reference solution comprises the following steps:

[0023] The puerarin reference substance is mixed with the second alcohol-containing solvent to prepare the puerarin reference substance solution.

[0024] In some embodiments, the second alcohol-containing solvent is selected from a second alcohol or a mixed solvent of a second alcohol and water.

[0025] In some embodiments, in the puerarin reference solution, the mass volume ratio of the puerarin reference to the second alcohol-containing solvent is (0.1-10) mg:1 ml.

[0026] In some embodiments, the second alcohol is selected from one or more of ethanol and methanol.

[0027] In some embodiments, the second alcohol-containing solvent includes 20%-100% of the second alcohol and 0%-80% of water, by volume.

[0028] In some embodiments, the method for preparing the Pueraria lobata control medicinal material solution comprises the following steps:

[0029] The kudzu root is mixed with a third alcohol-containing solvent for extraction to obtain an extract, and the kudzu root control medicinal material solution is prepared.

[0030] In some embodiments, the third alcohol-containing solvent is selected from a third alcohol or a mixed solvent of a third alcohol and water.

[0031] In some embodiments, in the Pueraria lobata control medicinal material solution, the mass volume ratio of the Pueraria lobata and the third alcohol-containing solvent is (10-500) mg:1 ml.

[0032] In some embodiments, the third alcohol is selected from one or more of ethanol and methanol.

[0033] In some embodiments, the third alcohol-containing solvent includes 20%-100% of the third alcohol and 0%-80% of water, calculated by volume.

[0034] In some embodiments, the second extraction method is warm immersion at a temperature of 50° C.-70° C. for 0.5 h-2 h.

[0035] 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.

[0036] In some embodiments, the dosage form of the Shenkui-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

[0037] 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

[0038] 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.

[0039] FIG1 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.26 as a developing solvent as shown in an embodiment of the present application;

[0040] FIG2 is a thin layer chromatography test result using a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.25:0.5 as a developing solvent as shown in an embodiment of the present application;

[0041] FIG3 is a thin layer chromatography test result using a mixed solvent 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;

[0042] FIG4 is a thin layer chromatography test result using a mixed solvent 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;

[0043] FIG5 is a thin layer chromatography test result using a mixed solvent 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;

[0044] 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;

[0045] 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;

[0046] 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;

[0047] FIG9 is a thin layer chromatography test result using a mixed solvent 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;

[0048] 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;

[0049] FIG11 is a thin layer chromatography test result using a mixed solvent of chloroform, ethanol, and water in a volume ratio of 7:2.5:0.26 as a developing solvent according to an embodiment of the present application;

[0050] FIG12 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 according to an embodiment of the present application;

[0051] FIG13 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 according to an embodiment of the present application;

[0052] FIG14 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;

[0053] FIG15 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;

[0054] FIG16 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;

[0055] FIG17 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;

[0056] FIG18 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;

[0057] FIG19 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;

[0058] FIG20 is a thin layer chromatography test result of using 20% ​​methanol as a solvent for dissolving a test sample according to an embodiment of the present application;

[0059] FIG21 is a thin layer chromatography test result of Puerariae in different batches of Shen Ge Bushen Capsules;

[0060] FIG22 shows the results of thin layer chromatography (TLC) testing of the same batch of Shenge Bushen Capsules using different TLC plates. DETAILED DESCRIPTION

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Unless mentioned otherwise, terms in the singular may include plural forms and should not be construed as having one number.

[0068] 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.

[0069] 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.

[0070] In this application, no distinction is made between the terms "efficacy", "performance", "effect" and "efficacy".

[0071] 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.

[0072] 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.

[0073] In one embodiment, the normal temperature is 10°C to 35°C.

[0074] In one embodiment, the normal temperature is 20°C to 30°C.

[0075] 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%.

[0076] The main ingredients of Shenkui-tonifying kidney preparations are Pseudostellaria suzhanensis, Pueraria root, and Epimedium. Their primary functions are to invigorate Qi, nourish Yin, and tonify the kidneys. As the primary ingredient in Shenkui-tonifying kidney preparations, Pueraria root plays a crucial role in their effectiveness. However, a rapid and specific detection method for Pueraria root in Shenkui-tonifying kidney preparations is currently lacking, hindering accurate and comprehensive quality control and measurement of these preparations.

[0077] 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.

[0078] If the Pueraria root in ginseng and kudzu kidney-tonifying preparations can be detected by thin-layer chromatography, the accuracy and comprehensiveness of the quality control of ginseng and kudzu 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 kidney-tonifying preparations. However, no relevant reports have been seen so far.

[0079] Based on this, the present application provides a thin layer chromatography detection method for Pueraria root in ginseng and kudzu kidney-tonifying preparations, which can quickly and accurately detect Pueraria root, 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.

[0080] The technical solution is as follows:

[0081] A thin layer chromatography method for detecting kudzu root 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;

[0082] The thin layer chromatography detection method of Pueraria root in the Shenkui tonifying kidney preparation comprises the following steps:

[0083] Prepare the test solution, puerarin reference solution and kudzu root reference medicinal material solution respectively;

[0084] The test solution, puerarin reference solution and kudzu root reference medicinal material solution were spotted on the same thin layer chromatography plate, developed with a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7: (2.4-2.6): (0.24-0.26) as a developing agent, and inspected after color development.

[0085] This method can quickly separate the components in the sample solution and qualitatively analyze puerarin. The corresponding thin-layer chromatography spots are clear and there is no interference from the negative control. This improves 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. In addition, the operation is simple and flexible, with high sensitivity and resolution, low equipment cost and low cost.

[0086] 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.

[0087] S100: Prepare test solution.

[0088] In some embodiments, the method for preparing the test solution comprises the following steps:

[0089] 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.

[0090] In some embodiments, the first alcohol-containing solvent is selected from a first alcohol or a mixed solvent of the first alcohol and water.

[0091] 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.

[0092] In one embodiment, the first alcohol is ethanol or methanol.

[0093] 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%.

[0094] In one embodiment, the first alcohol-containing solvent consists of 80% ethanol and 20% water, calculated by volume.

[0095] In some embodiments, the first extraction is performed by shaking at room temperature.

[0096] In some embodiments, the ginseng and kudzu kidney-tonifying preparation sample is mixed with a first alcohol-containing solvent, and the ginseng and kudzu kidney-tonifying preparation sample is dissolved by shaking to extract the active ingredients.

[0097] 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, 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, 150 mg:1 ml, 180 mg:1 ml or 200 mg:1 ml.

[0098] In one embodiment, 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.

[0099] In some embodiments, the method for preparing the test solution comprises the following steps:

[0100] Take 40 mg of the test sample of the ginseng and kudzu kidney-tonifying preparation, add 1 ml of 80% ethanol, shake to dissolve, filter, and use the filtrate as the test sample solution.

[0101] S200: Prepare puerarin reference solution.

[0102] In some embodiments, the method for preparing the puerarin reference solution comprises the following steps:

[0103] The puerarin reference substance is mixed with the second alcohol-containing solvent to prepare the puerarin reference substance solution.

[0104] In some embodiments, the second alcohol-containing solvent is selected from a second alcohol or a mixed solvent of a second alcohol and water.

[0105] 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.

[0106] In one embodiment, the second alcohol is methanol.

[0107] 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%.

[0108] In one embodiment, the second alcohol-containing solvent is methanol.

[0109] In some embodiments, in the puerarin reference solution, the mass volume ratio of the puerarin reference and the second alcohol-containing solvent is (0.1-10) mg:1 ml, including but not limited to 0.1 mg:1 ml, 0.2 mg:1 ml, 0.3 mg:1 ml, 0.5 mg:1 ml, 1 mg:1 ml, 2 mg:1 ml, 3 mg:1 ml, 4 mg:1 ml, 5 mg:1 ml, 6 mg:1 ml, 7 mg:1 ml, 8 mg:1 ml, 9 mg:1 ml or 10 mg:1 ml.

[0110] In one embodiment, the mass volume ratio of the puerarin reference substance and the second alcohol-containing solvent is (0.5-10) mg:1 ml.

[0111] S300: preparing a Pueraria root control medicinal material solution.

[0112] In some embodiments, the method for preparing the Pueraria lobata control medicinal material solution comprises the following steps:

[0113] The Pueraria root is mixed with a third alcohol-containing solvent for a second extraction to obtain an extract, and the Pueraria root control medicinal material solution is prepared.

[0114] In some embodiments, the third alcohol-containing solvent is selected from a third alcohol or a mixed solvent of a third alcohol and water.

[0115] 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.

[0116] In one embodiment, the third alcohol is methanol.

[0117] 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%.

[0118] In one embodiment, the third alcohol-containing solvent is methanol.

[0119] In some embodiments, the kudzu root is mixed with a third alcohol-containing solvent and extracted by warm infusion.

[0120] 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.

[0121] In one embodiment, the warm soaking time is 0.5h-2h, including but not limited to 0.5h, 1h, 1.5h or 2h.

[0122] In some embodiments, in the Pueraria lobata control medicinal material solution, the mass volume ratio of the Pueraria lobata and the third alcohol-containing solvent is (10-500) mg:1 ml, including but not limited to 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, 150 mg:1 ml, 180 mg:1 ml, 200 mg:1 ml, 250 mg:1 ml, 300 mg:1 ml, 350 mg:1 ml, 400 mg:1 ml, 450 mg:1 ml or 500 mg:1 ml.

[0123] In one embodiment, the mass volume ratio of the Pueraria root and the third alcohol-containing solvent is (20-300) mg:1 ml.

[0124] S400: Spot the test solution, puerarin reference solution and kudzu root reference medicinal material solution on the same thin layer chromatography plate, develop with a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7: (2.4-2.6): (0.24-0.26) as a developing agent, and inspect after color development.

[0125] It can be understood that in one example, 1 μl of the test solution, 1 μl of the puerarin reference solution and 1 μl of the kudzu root reference medicinal material solution were respectively pipetted and spotted on the same thin layer chromatography plate.

[0126] The developing agent described in the present application 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.

[0127] In one embodiment, the developing agent is a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.26.

[0128] 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.

[0129] In some embodiments, the light source used for inspection is a 365 nm ultraviolet lamp.

[0130] In the present application, in the chromatogram of the test sample, fluorescent spots of the same color are displayed at positions corresponding to those in the chromatograms of the control medicinal material and the control sample.

[0131] 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.

[0132] In some embodiments, the dosage form of the Shenkui-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

[0133] The present application is further described below with reference to specific embodiments:

[0134] Instruments and reagents

[0135] The Shen Ge Bu Shen preparations are all Shen Ge Bu Shen capsules, among which Shen Ge Bu Shen capsule-1 is from Xinjiang Huachun Bio-pharmaceutical Co., Ltd., batch number: 23005; Shen Ge Bu Shen capsule-2 is from Xinjiang Huachun Bio-pharmaceutical Co., Ltd., batch number: 21001; Shen Ge Bu Shen capsule-3 is from Xinjiang Huachun Bio-pharmaceutical Co., Ltd., batch number: 21004;

[0136] Puerarin reference substance was obtained from the China Food and Drug Administration, batch number: 110752-202217;

[0137] The control herbal material Pueraria lobata was obtained from the China Food and Drug Administration with batch number 121551-201805;

[0138] 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;

[0139] All other reagents used were of analytical grade.

[0140] Example 1

[0141] This example investigates the effect of thin layer chromatography (TLC) on the detection of Pueraria root in the Shenkui-tonifying Kidney Preparation using chloroform-methanol-water in different volume ratios as the developing solvent. The steps are as follows:

[0142] Take 40 mg of the contents of Shen Ge Bushen Capsule-1 and add 1 ml of 80% ethanol. Shake to dissolve, filter, and use the filtrate as the test solution. Separately, take 0.8 g of Pueraria root as a control herb, add 10 ml of methanol, and soak at 60°C for 2 hours. Filter, and use the filtrate as the control herb solution. Also, take the puerarin control herb 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 Rules 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, and 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 developed with a mixed solvent of chloroform-methanol-water as shown in Table 1, developed, taken out, dried, and inspected under an ultraviolet lamp (365 nm). The results are shown in Figures 1 to 10. The order of spotting from left to right in Figures 1 to 10 is: 1. Shenge Bushen Capsule-1, dot spotting; 2. Pueraria control medicinal material, dot spotting; 3. Puerarin reference substance, dot spotting; 4. Shenge Bushen Capsule-1, strip spotting; 5. Pueraria control medicinal material, strip spotting; 6. Puerarin reference substance, strip spotting.

[0143] Table 1. Comparison of the first group of developing agents in identification of Pueraria root

[0144] 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 fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0145] As shown in Figure 2, in the chromatogram of the test sample, there is little information about the fluorescent spots. Although the sample can be separated, the separation effect of the components is poor (the specific shift value (Rf value) is not good) and the tailing is serious.

[0146] As shown in Figure 3, in the chromatogram of the test sample, there is little information about the fluorescent spots and the separation effect of the components is very poor.

[0147] As shown in Figure 4, in the chromatogram of the test sample, there is little information about the fluorescent spots, the separation effect of each component is very poor, and there is a tailing phenomenon.

[0148] As shown in Figure 5, in the chromatogram of the test sample, there is little information about the fluorescent spots, the separation effect of the components is very poor, and the tailing is serious.

[0149] As shown in Figure 6, in the chromatogram of the test sample, there is little information about the fluorescent spots, the separation effect of the components is very poor, and the tailing is serious.

[0150] As shown in Figure 7, in the chromatogram of the test sample, there is little information about the fluorescent spots, the separation effect of the components is very poor, and the tailing is serious.

[0151] As shown in Figure 8, in the chromatogram of the test sample, there is little information about the fluorescent spots, the separation effect of the components is very poor, the tailing is very serious, and the fluorescent spots in the chromatogram of the reference sample are not obvious.

[0152] As shown in Figure 9, in the chromatogram of the test sample, there is little information about the fluorescent spots, the separation effect of the components is very poor, the tailing is very serious, and the fluorescent spots in the chromatogram of the reference sample are not obvious.

[0153] As shown in Figure 10, in the test sample chromatogram, the fluorescent spots are not obvious, the fluorescent spot information is small, the tailing is very serious, the separation effect of each component is very poor, and no fluorescent spots are observed at the corresponding positions in the control sample chromatogram.

[0154] Example 2

[0155] This example investigates the effect of using thin layer chromatography (TLC) to detect kudzu root in a ginseng-kudzu kidney-tonifying preparation using a mixed solvent of different types of solvents as a developing solvent. The steps are as follows:

[0156] Take 40 mg of the contents of Shen Ge Bushen Capsule-1 and add 1 ml of 80% ethanol. Shake to dissolve, filter, and use the filtrate as the test solution. Separately, take 0.8 g of Pueraria root as a control herb, add 10 ml of methanol, and soak at 60°C for 2 hours. Filter, and use the filtrate as the control herb solution. Also, take the puerarin control herb 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 Rules 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, and 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-1 was developed with the mixed solvent shown in Table 2 as the developing agent, developed, taken out, dried, and inspected under an ultraviolet lamp (365 nm). The results are shown in Figures 1 and 11 to 13. The order of spotting from left to right in Figures 1 and 11 to 13 is: 1. Shenge Bushen Capsule-1, dot spotting; 2. Pueraria control medicinal material, dot spotting; 3. Puerarin reference substance, dot spotting; 4. Shenge Bushen Capsule-1, strip spotting; 5. Pueraria control medicinal material, strip spotting; 6. Puerarin reference substance, strip spotting.

[0157] Table 2. Comparison of the second group of developing agents in identification of Pueraria root

[0158] 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 fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0159] As shown in Figure 11, in the test sample chromatogram, although the sample can be separated, the specific shift value (Rf value) is poor, indicating that the separation effect is poor, and the fluorescent spot of the reference sample chromatogram is not obvious, which does not meet the expected effect.

[0160] As shown in Figure 12, in the test sample chromatogram, there is little information about the fluorescent spots, and the separation effect of each component is very poor. The fluorescent spots in the test sample chromatogram and the control medicinal material chromatogram are not distributed sequentially on the vertical line from the origin to the development front, and the fluorescent spots in the control sample chromatogram are not obvious.

[0161] As shown in Figure 13, in the test sample chromatogram, there is little fluorescent spot information, the separation effect of each component is very poor, and the tailing is very serious. There are no fluorescent spots of the same color at the corresponding positions of the control medicinal material chromatogram, and the fluorescent spots of the control sample chromatogram are not obvious.

[0162] 1 to 13 , a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.26 is suitable for use as a developing agent in the thin layer chromatography method for detecting Pueraria root in the Shenkui-tonifying preparation of the present application.

[0163] Example 3

[0164] This example investigates the effect of thin layer chromatography (TLC) on the detection of Pueraria root in the Shenkui-tonifying Kidney Preparation using different solvents as solvents for dissolving the test sample. The steps are as follows:

[0165] Take 40 mg of the contents of Shen Ge Bushen Capsule-1 and add 1 ml of the solvent listed in Table 3. Shake to dissolve. Filter and use the filtrate as the test solution. Separately, take 0.8 g of Pueraria root as a control herb and add 10 ml of methanol. Steep at 60°C for 2 hours. Filter and use the filtrate as the control herb solution. Also, take the puerarin control herb 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 Rules 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, and 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-1 was developed with a mixed solvent of chloroform-methanol-water with a volume ratio of 7:2.5:0.26. The plates were developed, taken out, dried, and inspected under an ultraviolet lamp (365 nm). The results are shown in Figures 1 and 14 to 20. The order of spotting from left to right in Figures 1 and 14 to 20 is: 1. Shenge Bushen Capsule-1, dot spotting; 2. Pueraria control medicinal material, dot spotting; 3. Puerarin reference substance, dot spotting; 4. Shenge Bushen Capsule-1, strip spotting; 5. Pueraria control medicinal material, strip spotting; 6. Puerarin reference substance, strip spotting.

[0166] Table 3. Comparison of solvent types for dissolving test samples in Pueraria identification

[0167] 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 fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0168] As shown in Figure 14, 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 are displayed at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

[0169] As shown in Figure 15, 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 are displayed at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

[0170] As shown in Figure 16, 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 are displayed at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

[0171] As shown in Figure 17, 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 are displayed at the corresponding positions of the control medicinal material chromatogram and the control sample chromatogram.

[0172] 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 fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0173] 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 fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0174] 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 fluorescent spots of the same color are displayed at the corresponding positions of the control medicinal material chromatogram and the reference sample chromatogram.

[0175] As can be seen from Figures 1 and 14 to 20, ethanol, ethanol aqueous solution, methanol or methanol aqueous solution are all suitable for use as reagents for dissolving the ginseng and kudzu kidney-tonifying preparation in the thin layer chromatography detection method of kudzu root in the ginseng and kudzu kidney-tonifying preparation of the present application.

[0176] Example 4 Methodological Investigation

[0177] (1) Specificity experiment

[0178] Take 40 mg of the contents of Shengge Bushen Capsules-1, Shengge Bushen Capsules-2, and Shengge Bushen Capsules-3 from different batches, add 1 ml of 80% ethanol, shake to dissolve, filter, and use the filtrate as the test solution. Separately, take 0.8 g of Pueraria root as a control herb, add 10 ml of methanol, and soak at 60°C for 2 hours. Filter, and use the filtrate as the control herb solution. Also, take the puerarin control herb 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 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. The silica gel G thin layer plate-1 was developed with a mixed solvent of chloroform-methanol-water with a volume ratio of 7:2.5:0.26. The plate was developed, taken out, dried, and inspected under an ultraviolet lamp (365 nm). The results are shown in Figure 21. The order of spotting from left to right in Figure 21 is: 1. Shenkui Bushen Capsule-1, strip spotting; 2. Shenkui Bushen Capsule-2, strip spotting; 3. Shenkui Bushen Capsule-3, strip spotting; 4. Pueraria reference medicinal material, strip spotting; 5. Puerarin reference substance, strip spotting.

[0179] The results showed that in the chromatogram of the test sample, the fluorescent spots were rich in information and the separation effect of each component was good. Spots of the same color appeared at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference sample.

[0180] (2) Investigation of the tolerance of thin layer plates

[0181] Take 40 mg of the contents of Shen Ge Bushen Capsule-1, add 1 ml of 80% ethanol, shake to dissolve, filter, and use the filtrate as the test solution. Take another 0.8 g of Pueraria root reference medicinal material, add 10 ml of methanol, soak at 60°C for 2 hours, filter, and use the filtrate as the control medicinal material solution. Take the puerarin reference substance again, add methanol to make a solution containing 1 mg per 1 ml, as the reference 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 above 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 developed with a mixed solvent of chloroform-methanol-water with a volume ratio of 7:2.5:0.26 as the developing agent, taken out, dried, and inspected under ultraviolet light (365 nm). The results are shown in Figure 22. The order of spotting from left to right in Figure 22 is: 1 .1. Ginseng and Kudzu Kidney-Nourishing Capsule-1, strip spot; 2. Kudzu root reference medicinal material, strip spot; 3. Puerarin reference substance, strip spot; 4. Ginseng and Kudzu Kidney-Nourishing Capsule-1, strip spot; 5. Kudzu root reference medicinal material, strip spot; 6. Puerarin reference substance, strip spot; 7. Ginseng and Kudzu Kidney-Nourishing Capsule-1, strip spot; and, Figure 22 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 are spotted on silica gel G thin layer plate-1, 4, 5, 6, 7 are spotted on silica gel G thin layer plate-2.

[0182] The results showed that in the chromatograms of the two test samples, 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 chromatograms of the control medicinal materials and the reference sample, 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 Pueraria root in the ginseng and kudzu kidney-tonifying preparation.

[0183] 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.

[0184] 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 Pueraria root in a Shenku tonifying kidney 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 Pueraria root in the ginseng and kudzu kidney-tonifying preparation comprises the following steps: Prepare the test solution, puerarin reference solution and pueraria reference medicinal material solution respectively; The test solution, puerarin reference solution and pueraria reference medicinal material solution were spotted on the same thin layer chromatography plate, developed with a mixed solvent consisting of chloroform, methanol and water in a volume ratio of 7:(2.4-2.6):(0.24-0.26) as a developing agent, and inspected after color development.

2. The thin layer chromatography detection method according to claim 1, characterized in that: The developing agent is a mixed solvent of chloroform, methanol and water in a volume ratio of 7:2.5:0.

26.

3. The thin layer chromatography detection method according to any one of claims 1 to 2, characterized in that: The light source used for inspection is 365nm ultraviolet lamp.

4. The thin layer chromatography detection method according to any one of claims 1 to 3, characterized in that: The preparation method of 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.

5. The thin layer chromatography detection method according to claim 4, characterized in that: Satisfy one or more of the following (1)-(5): (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 shaking at room temperature.

6. The thin layer chromatography detection method according to any one of claims 1 to 5, characterized in that: The preparation method of the puerarin reference substance solution comprises the following steps: The puerarin reference substance is mixed with the second alcohol-containing solvent to prepare the puerarin reference substance solution.

7. The thin layer chromatography detection method according to claim 6, characterized in that: Satisfy one or more of the following (1)-(4): (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 puerarin reference solution, the mass volume ratio of the puerarin 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 any one of claims 1 to 7, characterized in that: The preparation method of the Pueraria lobata reference medicinal material solution comprises the following steps: The Pueraria root is mixed with the third alcohol-containing solvent for a second extraction to obtain an extract, and the Pueraria root control medicinal material solution is prepared.

9. The thin layer chromatography detection method according to claim 8, characterized in that: Satisfy one or more of the following (1)-(5): (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 Pueraria root control medicinal material solution, the mass volume ratio of the Pueraria root and the third alcohol-containing solvent is (10-500) 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.

10. The thin layer chromatography detection method according to any one of claims 1 to 9, characterized in that: Satisfy one or more of the following (1)-(2): (1) In parts by weight, the raw materials of the Shenkui tonifying kidney preparation include 1000-2000 parts of Pseudostellariae Radix, 500-1500 parts of Puerariae Radix and 100-900 parts of Epimedium; (2) The dosage form of the ginseng and kudzu kidney-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

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