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

Abstract

A method for thin-layer chromatography detection of radix pseudostellariae 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 radix pseudostellariae in the ginseng-radix puerariae kidney-tonifying preparation comprises the following steps: preparing both a test sample solution and a radix pseudostellariae contrast medicinal material solution; spotting the test sample solution and the radix pseudostellariae contrast medicinal material solution onto a same thin-layer chromatography plate, developing with a developing solvent, and adding a color visualization agent for color visualization, then visualizing and detecting; wherein the developing solvent accounts for a volume fraction of 75% and is a mixed solvent composed of isopropanol and ethanol at a volume ratio of (1-9):1, and the color visualization agent is a mixed solution of aniline, diphenylamine, phosphoric acid and acetone.

Description

TLC Detection of Pseudostellariae Radix in Shenkudzu Kidney-Tonifying Preparation

[0001] This application claims priority to Chinese patent application number 202311574695X, filed on November 23, 2023, entitled “Thin-layer chromatography detection method of Pseudostellaria baicalensis 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 Pseudostellaria baicalensis in a ginseng and kudzu kidney-tonifying preparation. Background Art

[0003] Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm., a plant of the Caryophyllaceae family, is a dried root tuber that nourishes Qi and strengthens the spleen, promotes fluid production and moistens the lungs. It is commonly used to treat spleen deficiency, fatigue, loss of appetite, weakness after illness, Qi and Yin deficiency, spontaneous sweating and thirst, and dry cough caused by lung dryness.

[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] As the main raw material in ginseng and kudzu kidney-tonifying preparations, Pseudostellaria baicalensis 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 Pseudostellaria baicalensis 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 Pseudostellariae Radix in a ginseng and kudzu kidney-tonifying preparation is provided.

[0008] The technical solution is as follows:

[0009] A thin layer chromatography method for detecting Pseudostellariae Radix in a ginseng and kudzu kidney-tonifying preparation, wherein the raw materials of the ginseng and kudzu kidney-tonifying preparation include Pseudostellariae Radix, Puerariae Radix and Epimedium;

[0010] The thin layer chromatography detection method of Pseudostellariae Radix in the ginseng and kudzu kidney-tonifying preparation comprises the following steps:

[0011] Prepare the test sample solution and Pseudostellariae Radix control medicinal material solution respectively;

[0012] The test solution and the control herbal drug solution are spotted on the same thin layer chromatography plate, developed in a developing agent, and a color developer is added for color development and inspection;

[0013] The developing agent is a mixed solvent composed of 75% by volume of isopropyl alcohol and ethanol in a volume ratio of (1 to 9):1;

[0014] The color developer is a mixed solution of aniline, diphenylamine, phosphoric acid and acetone.

[0015] In some embodiments, the developer is a mixed solution of aniline acetone solution with a volume fraction of 1% to 2%, diphenylamine acetone solution with a mass volume ratio of 1g: (49 to 99) ml, and phosphoric acid with a volume ratio of (4 to 5): (3 to 5): 1.

[0016] In some embodiments, the developer is a mixed solution of 2% aniline acetone solution by volume, 1g:49ml diphenylamine acetone solution by mass volume ratio, and phosphoric acid by volume ratio of (4-5):(3-5):1.

[0017] In some embodiments, the thin layer chromatography plate needs to be heated after adding the developer, and the temperature is 100°C to 110°C.

[0018] In some embodiments, the light source used for inspection is a fluorescent lamp.

[0019] In some embodiments, the method for preparing the test solution comprises the following steps:

[0020] The ginseng and kudzu kidney-tonifying preparation test sample is mixed with water for a first extraction to obtain an extract, and the test sample solution is prepared.

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

[0022] In some embodiments, in the test solution, the mass volume ratio of the ginseng and kudzu kidney-tonifying preparation test sample and water is (1-100) mg:1 ml.

[0023] In some embodiments, the method for preparing the Pseudostellariae Radix control medicinal material solution comprises the following steps:

[0024] The Pseudostellariae Radix control medicinal material is mixed with water for a second extraction to obtain an extract, and the Pseudostellariae Radix control medicinal material solution is prepared.

[0025] In some embodiments, the second extraction method is heating extraction at a temperature of 80° C. to 100° C. for 0.5 h to 2 h.

[0026] In some embodiments, in the Pseudostellariae Radix control medicinal material solution, the mass volume ratio of Pseudostellariae Radix and water is (1-100) mg:1 ml.

[0027] In some embodiments, 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.

[0028] In some embodiments, the dosage form of the Shenkui-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

[0029] 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

[0030] 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.

[0031] FIG1 shows the results of thin-layer chromatography (TLC) analysis using a mixed solvent consisting of 75% by volume isopropyl alcohol and anhydrous ethanol in a 9:1 volume ratio as a developing solvent, and a mixed solution consisting of a 2% aniline-acetone solution, a diphenylamine-acetone solution in a 1 g:49 ml mass-to-volume ratio, and phosphoric acid in a 5:5:1 volume ratio as a developer, according to an embodiment of the present application;

[0032] FIG2 is a thin layer chromatography test result using a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 8:2 as a developing solvent according to an embodiment of the present application;

[0033] FIG3 shows the thin layer chromatography test results of an embodiment of the present application using a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 7:3 as a developing solvent;

[0034] FIG4 is a thin layer chromatography test result using a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 6:4 as a developing solvent according to an embodiment of the present application;

[0035] FIG5 is a thin layer chromatography test result using a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 5:5 as a developing solvent according to an embodiment of the present application;

[0036] FIG6 shows the thin layer chromatography test results using a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 4:6 as a developing solvent according to an embodiment of the present application;

[0037] FIG7 shows the thin layer chromatography test results using a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 3:7 as a developing solvent according to an embodiment of the present application;

[0038] FIG8 is a thin layer chromatography test result using a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 2:8 as a developing solvent according to an embodiment of the present application;

[0039] FIG9 shows the thin layer chromatography test results using a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 1:9 as a developing solvent according to an embodiment of the present application;

[0040] FIG10 is a thin layer chromatography test result using a mixed solvent consisting of 50% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 9:1 as a developing solvent according to an embodiment of the present application;

[0041] FIG11 is a thin layer chromatography test result using a mixed solvent consisting of 25% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 9:1 as a developing solvent according to an embodiment of the present application;

[0042] FIG12 is a thin layer chromatography test result using a mixed solvent consisting of isopropyl alcohol and anhydrous ethanol in a volume ratio of 9:1 as a developing solvent according to an embodiment of the present application;

[0043] FIG13 shows the results of thin-layer chromatography using a mixed solvent of n-butanol, chloroform, water, and glacial acetic acid in a volume ratio of 6:2.5:2.3:3 as a developing solvent according to an embodiment of the present application;

[0044] FIG14 is a thin layer chromatography test result using a mixed solvent of chloroform and methanol in a volume ratio of 4:1 as a developing solvent according to an embodiment of the present application;

[0045] FIG15 shows the results of thin-layer chromatography using a mixed solution of a 2% aniline acetone solution by volume, a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:49 ml, and phosphoric acid in a volume ratio of 5:4:1 as a color developer according to an embodiment of the present application;

[0046] FIG16 shows the results of thin-layer chromatography using a 2% by volume aniline acetone solution, a mixed solution of a diphenylamine acetone solution with a mass volume ratio of 1 g:49 ml, and phosphoric acid with a volume ratio of 5:3:1 as a color developer according to an embodiment of the present application;

[0047] FIG17 shows the results of thin-layer chromatography using a 2% by volume aniline acetone solution, a mixed solution of a diphenylamine acetone solution with a mass volume ratio of 1 g:49 ml, and phosphoric acid with a volume ratio of 4:5:1 as a color developer according to an embodiment of the present application;

[0048] FIG18 shows the results of thin-layer chromatography using a 2% by volume aniline acetone solution, a mixed solution of a diphenylamine acetone solution with a mass volume ratio of 1 g:49 ml, and phosphoric acid with a volume ratio of 4:4:1 as a color developer according to an embodiment of the present application;

[0049] FIG19 shows the results of thin-layer chromatography using a 2% by volume aniline acetone solution, a mixed solution of a diphenylamine acetone solution with a mass volume ratio of 1 g:49 ml, and phosphoric acid with a volume ratio of 4:3:1 as a color developer according to an embodiment of the present application;

[0050] FIG20 shows the results of thin-layer chromatography using a mixed solution of a 1% aniline acetone solution by volume, a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:49 ml, and phosphoric acid in a volume ratio of 5:5:1 as a color developer according to an embodiment of the present application;

[0051] FIG21 shows the results of thin-layer chromatography using a 2% by volume aniline acetone solution, a mixed solution of a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:99 ml, and phosphoric acid in a volume ratio of 5:5:1 as a color developer according to an embodiment of the present application;

[0052] FIG22 shows the results of thin-layer chromatography using a mixed solution of a 1% aniline acetone solution by volume, a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:99 ml, and phosphoric acid in a volume ratio of 5:3:1 as a color developer according to an embodiment of the present application;

[0053] FIG23 is a thin layer chromatography test result using 0.2% ninhydrin ethanol solution as a color developer according to an embodiment of the present application;

[0054] FIG24 is a thin layer chromatography test result of Pseudostellariae Radix in different batches of Shengge Bushen Capsules;

[0055] Figure 25 shows the results of thin layer chromatography testing of Pseudostellariae Radix in Shengge Bushen Capsule using different thin layer chromatography plates. DETAILED DESCRIPTION

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] Unless mentioned otherwise, terms in the singular may include plural forms and should not be construed as having one number.

[0063] 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.

[0064] 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.

[0065] In this application, no distinction is made between the terms "efficacy", "performance", "effect" and "efficacy".

[0066] 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.

[0067] 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.

[0068] In one embodiment, the normal temperature is 10°C to 35°C.

[0069] In one embodiment, the normal temperature is 20°C to 30°C.

[0070] In the present application, 75% isopropyl alcohol means that in the isopropyl alcohol aqueous solution, the volume fraction of anhydrous isopropyl alcohol is 75% and the volume fraction of water is 25%; 50% isopropyl alcohol means that in the isopropyl alcohol aqueous solution, the volume fraction of anhydrous isopropyl alcohol is 50% and the volume fraction of water is 50%; 25% isopropyl alcohol means that in the isopropyl alcohol aqueous solution, the volume fraction of anhydrous isopropyl alcohol is 25% and the volume fraction of water is 75%.

[0071] The main ingredients of Shenkui-tonifying kidney preparations are Pseudostellaria radix, Pueraria lobata, and Epimedium. Their primary functions are to invigorate Qi, nourish Yin, and tonify the kidneys. Pseudostellaria radix, as the primary ingredient in Shenkui-tonifying kidney preparations, plays a crucial role in their effectiveness. However, a method for rapidly and specifically identifying Pseudostellaria radix in Shenkui-tonifying kidney preparations is currently lacking, hindering accurate and comprehensive quality control and measurement of these preparations.

[0072] 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.

[0073] If Pseudostellariae Radix 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.

[0074] Based on this, the present application provides a thin layer chromatography detection method for Pseudostellaria baicalensis in ginseng and kudzu kidney-tonifying preparations, which can quickly and accurately detect Pseudostellaria baicalensis, 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.

[0075] The technical solution is as follows:

[0076] A thin layer chromatography method for detecting Pseudostellariae Radix in a ginseng and kudzu kidney-tonifying preparation, wherein the raw materials of the ginseng and kudzu kidney-tonifying preparation include Pseudostellariae Radix, Puerariae Radix and Epimedium;

[0077] The thin layer chromatography detection method of Pseudostellariae Radix in the ginseng and kudzu kidney-tonifying preparation comprises the following steps:

[0078] Prepare the test sample solution and Pseudostellariae Radix control medicinal material solution respectively;

[0079] The test solution and the control herbal drug solution are spotted on the same thin layer chromatography plate, developed in a developing agent, and a color developer is added for color development and inspection;

[0080] The developing agent is a mixed solvent composed of 75% by volume of isopropyl alcohol and ethanol in a volume ratio of (1 to 9):1;

[0081] The color developer is a mixed solution of aniline, diphenylamine, phosphoric acid and acetone.

[0082] In the present application, in the test sample chromatogram, on the position corresponding to the control medicinal material chromatogram, fluorescent spots of the same color are respectively shown. The method can detect the Radix Pseudostellariae in the Radix Puerariae Kidney-tonifying Preparation quickly, and the corresponding thin-layer chromatography spots are clear and the negative control is interference-free, thereby improving the accuracy and comprehensiveness of the Radix Puerariae Kidney-tonifying Preparation quality control, and is conducive to the establishment and overall evaluation of the Radix Puerariae Kidney-tonifying Preparation quality standard. In addition, the method also has the advantages of short time consumption, simple operation, high sensitivity, high resolution, low equipment cost and low cost.

[0083] 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 between S100 and S200, and the numbering is only for the convenience of description.

[0084] S100: Prepare test solution.

[0085] In some embodiments, the method for preparing the test solution comprises the following steps:

[0086] The ginseng and kudzu kidney-tonifying preparation test sample is mixed with water for a first extraction to obtain an extract, and the test sample solution is prepared.

[0087] In some embodiments, in the test solution, the mass volume ratio of the ginseng and kudzu kidney-tonifying preparation test sample and water is (1-100) 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 or 100 mg:1 ml.

[0088] In one embodiment, the mass volume ratio of the ginseng and kudzu kidney-tonifying preparation test sample and water is (10-100) mg: 1 ml.

[0089] In some embodiments, the first extraction is performed by shaking at room temperature.

[0090] In some embodiments, the method for preparing the test solution comprises the following steps:

[0091] Take 40 mg of the test sample of the ginseng and kudzu kidney-tonifying preparation, add 1 ml of water, shake to dissolve, filter, and use the filtrate as the test sample solution.

[0092] S200: preparing a Pseudostellariae Radix control medicinal material solution.

[0093] In some embodiments, the method for preparing the Pseudostellariae Radix control medicinal material solution comprises the following steps:

[0094] The Pseudostellariae Radix control medicinal material is mixed with water for a second extraction to obtain an extract, and the Pseudostellariae Radix control medicinal material solution is prepared.

[0095] In some embodiments, the second extraction method is heating extraction at a temperature of 80° C. to 100° C. for 0.5 h to 2 h.

[0096] In some embodiments, in the Pseudostellariae Radix control medicinal material solution, the mass volume ratio of Pseudostellariae Radix and water is (1-100) 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 or 100 mg:1 ml.

[0097] In one embodiment, the mass volume ratio of Pseudostellaria heterophylla and water is (10-100) mg:1 ml.

[0098] In some embodiments, the method for preparing the Pseudostellariae Radix control medicinal material solution comprises the following steps:

[0099] Take 5 g of Pseudostellariae Radix as a control medicinal material, add 50 ml of water, heat at 90°C for 1 hour, filter, and use the filtrate as the control medicinal material solution.

[0100] S300: Spot the test sample solution and Pseudostellariae Radix control medicinal material solution on the same thin layer chromatography plate, develop them in a developing agent, add a color developer, and inspect them.

[0101] It can be understood that in one example, 1 μl of the test solution and 1 μl of the Pseudostellariae Radix control medicinal material solution were respectively pipetted and spotted on the same thin layer chromatography plate.

[0102] In the present application, the developing agent is a mixed solvent composed of 75% by volume isopropanol and ethanol (understandably, usually anhydrous ethanol) in a volume ratio of (1 to 9):1. Understandably, the volume ratio of the 75% by volume isopropanol to the anhydrous ethanol includes but is not limited to 5:5, 2:1, 7:3, 3:1, 6:4, 8:2, 5:1, 6:1, 7:1, 8:1 or 9:1.

[0103] In one embodiment, the developing agent is a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 5:5, 7:3, 6:4, 8:2 or 9:1.

[0104] In one embodiment, the developing agent is a mixed solvent consisting of 75% by volume of isopropyl alcohol and anhydrous ethanol in a volume ratio of 9:1.

[0105] 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 step of adding a color developer for color development, the thin layer chromatography plate needs to be taken out of the development tank and air-dried, baked or blown to remove the developing agent.

[0106] In the present application, the developer is a mixed solution of aniline, diphenylamine, phosphoric acid and acetone.

[0107] In some embodiments, the developer is a mixed solution of aniline acetone solution with a volume fraction of 1% to 2%, diphenylamine acetone solution with a mass volume ratio of 1g: (49 to 99) ml, and phosphoric acid with a volume ratio of (4 to 5): (3 to 5): 1.

[0108] It is understood that in the aniline acetone solution, the volume fraction of aniline includes but is not limited to 1%, 1.5% or 2%. In the diphenylamine acetone solution, the mass volume ratio of diphenylamine to acetone is 1g:(49-99)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 or 1g:99ml.

[0109] In one embodiment, the color developer is a mixed solution of 1% aniline acetone solution by volume, 1g:99ml diphenylamine acetone solution by mass volume ratio, and phosphoric acid in a volume ratio of (4-5):(3-5):1. The volume ratio of 1% aniline acetone solution by volume fraction, 1g:99ml diphenylamine acetone solution by mass volume ratio, and phosphoric acid includes but is not limited to 4:3:1, 4:4:1, 4:5:1, 5:3:1, 5:4:1 or 5:5:1.

[0110] In one embodiment, the color developer is a mixed solution of 1% aniline acetone solution, 2g:98ml diphenylamine acetone solution and phosphoric acid in a volume ratio of (4-5):(3-5):1. The volume ratio of 1% aniline acetone solution, 2g:98ml diphenylamine acetone solution and phosphoric acid includes but is not limited to 4:3:1, 4:4:1, 4:5:1, 5:3:1, 5:4:1 or 5:5:1.

[0111] In one embodiment, the color developer is a mixed solution of 2% aniline acetone solution, 1g:99ml diphenylamine acetone solution and phosphoric acid in a volume ratio of (4-5):(3-5):1. The volume ratio of 2% aniline acetone solution, 1g:99ml diphenylamine acetone solution and phosphoric acid includes but is not limited to 4:3:1, 4:4:1, 4:5:1, 5:3:1, 5:4:1 or 5:5:1.

[0112] In one embodiment, the color developer is a mixed solution of 2% aniline acetone solution, 2g:98ml diphenylamine acetone solution and phosphoric acid in a volume ratio of (4-5):(3-5):1. The volume ratio of 2% aniline acetone solution, 2g:98ml diphenylamine acetone solution and phosphoric acid includes but is not limited to 4:3:1, 4:4:1, 4:5:1, 5:3:1, 5:4:1 or 5:5:1.

[0113] In one embodiment, the color developer is a mixed solution of aniline acetone solution with a volume fraction of 2%, diphenylamine acetone solution with a mass volume ratio of 1g:49ml, and phosphoric acid with a volume ratio of 5:5:1.

[0114] In some embodiments, after adding the developer, the thin layer chromatography plate needs to be heated at a temperature of 100°C to 110°C, including but not limited to 100°C, 105°C or 110°C.

[0115] In some embodiments, the light source used for inspection is a fluorescent lamp.

[0116] 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.

[0117] In some embodiments, the dosage form of the Shenkui-tonifying preparation is selected from granules, powders, tablets, capsules or pills.

[0118] The following is further described with reference to specific embodiments:

[0119] Instruments and reagents

[0120] The Shen Ge Bu Shen preparations are Shen Ge Bu Shen Capsules, Shen Ge Bu Shen Capsules-1, sourced from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 23005; Shen Ge Bu Shen Capsules-2, sourced from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 21002; Shen Ge Bu Shen Capsules-3, sourced from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 21004;

[0121] Pseudostellariae pseudoginseng reference material was obtained from the China Food and Drug Administration, batch number 121004-201709;

[0122] 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;

[0123] All other reagents used were of analytical grade. The concentrations of the liquid raw material mixed solutions in the following examples are expressed in volume fractions. For example, 75% isopropyl alcohol means that the volume fraction of isopropyl alcohol is 75% and the volume fraction of water is 25% in an isopropyl alcohol aqueous solution. 1% aniline-acetone solution means that the volume fraction of aniline is 1% and the volume fraction of acetone is 99% in an aniline-acetone solution. Furthermore, 2% aniline-acetone solution means that the volume fraction of aniline is 2% and the volume fraction of acetone is 98% in an aniline-acetone solution. 1% diphenylamine-acetone solution means that the mass of diphenylamine is 1g and the volume of acetone is 99ml in a diphenylamine-acetone solution. 2% diphenylamine-acetone solution means that the mass of diphenylamine is 2g and the volume of acetone is 98ml in a diphenylamine-acetone solution. 0.2% ninhydrin-ethanol solution means that 0.2g of ninhydrin-ethanol is contained in 100ml of ninhydrin-ethanol solution.

[0124] Example 1

[0125] This example investigates the separation effect of different mixed solvents used as developing agents for detecting Pseudostellariae Radix in the Shenkudun Bushen Preparation by thin layer chromatography. The developing agent suitable for this application is determined by the following steps:

[0126] Take 40mg of the contents of Shenge Bushen Capsule, add 1ml of water, shake to dissolve, filter, and use the filtrate as the test solution. Take another 5g of Pseudostellariae Radix as the control medicinal material, add 50ml of water, heat at 90℃ for 1 hour, filter, and use the filtrate as the control medicinal material 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 two solutions and spot them on the same silica gel G thin layer plate-1, then take 1μl of each of the above two solutions and spot them on the same silica gel G thin layer plate-1, develop with the Pseudostellariae Radix identification developer listed in Table 1, take out, dry, spray with a mixed solution of 2% aniline acetone solution, 2% diphenylamine acetone solution and phosphoric acid in a volume ratio of 5:5:1 as a color developer, heat at 105℃ until the spots are clearly colored, and inspect under sunlight. In the chromatogram of the test product, observe the situation of spots appearing at the corresponding position of the chromatogram of the control medicinal material. The results are shown in Figures 1 to 14. The order of spots from left to right in Figures 1 to 14 is: 1. Shen Ge Bushen Capsule-1, dot spotting; 2. Pseudostellariae Radix control medicinal material, dot spotting; 3. Shen Ge Bushen Capsule-1, strip spotting; 4. Pseudostellariae Radix control medicinal material, strip spotting.

[0127] Table 1. Comparison of different developing agents in identification of Pseudostellaria heterophylla

[0128] 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 in the chromatogram of the control medicinal material.

[0129] As shown in Figure 2, in the chromatogram of the test sample, the fluorescent spots are rich in information, and fluorescent spots of the same color are displayed at the corresponding positions in the chromatogram of the control medicinal material. Although the samples can be separated, the relative shift value (Rf value) is worse than that in Figure 1.

[0130] As shown in Figure 3, in the chromatogram of the test sample, the fluorescent spots are rich in information, and fluorescent spots of the same color are displayed at the corresponding positions in the chromatogram of the control medicinal material. Although the samples can be separated, the relative shift value (Rf value) is worse than that in Figure 1.

[0131] As shown in Figure 4, in the chromatogram of the test sample, the fluorescent spots are rich in information. At the corresponding positions in the chromatogram of the control medicinal material, fluorescent spots of the same color are displayed. Although the samples can be separated, the relative shift value (Rf value) is worse than that in Figure 1.

[0132] As shown in Figure 5, in the chromatogram of the test sample, the fluorescent spots are rich in information. At the corresponding positions in the chromatogram of the control medicinal material, fluorescent spots of the same color are displayed. Although the samples can be separated, the relative shift value (Rf value) is worse than that in Figure 1.

[0133] As shown in Figure 6, there are obviously few visible spots in the chromatograms of the test sample and the control medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0134] As shown in Figure 7, there are obviously few visible spots in the chromatograms of the test sample and the control medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0135] As shown in Figure 8, there are obviously few visible spots in the chromatograms of the test sample and the control medicinal material, the separation effect of each component is very poor, and the tailing is very serious.

[0136] As shown in Figure 9, there are obviously few visible spots in the chromatograms of the test sample and the control medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0137] As shown in Figure 10, in the chromatogram of the test sample, the fluorescent spots are rich in information, and fluorescent spots of the same color are displayed at the corresponding positions in the chromatogram of the control medicinal material. Although the samples can be separated, the relative shift value (Rf value) is worse than that in Figure 1 and cannot meet the requirements.

[0138] As shown in Figure 11, there are obviously few visible spots in the chromatograms of the test sample and the control medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0139] As shown in Figure 12, there are obviously few visible spots in the chromatograms of the test sample and the control medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0140] As shown in Figure 13, there are obviously few visible spots in the chromatograms of the test sample and the control medicinal material, and the separation effect of each component is very poor.

[0141] As shown in Figure 14, there are no obvious visible spots in the chromatograms of the test sample and the control medicinal material, and the separation effect of each component is particularly poor.

[0142] 1 to 14 , a mixed solvent consisting of 75% isopropanol and anhydrous ethanol in a volume ratio of (1 to 9):1 is suitable for use as a developing agent in the thin layer chromatography method for detecting Pseudostellaria baicalensis in the ginseng and kudzu kidney-tonifying preparation of the present application.

[0143] Example 2

[0144] This example investigates the color development effect of thin layer chromatography detection method using different mixed solvents as color developing agents, and ultimately detects Pseudostellariae Radix in the Shenkudun-tonifying kidney preparation. The color developing agent suitable for this application is determined by the following steps:

[0145] Take 40mg of the contents of Shenge Bushen Capsule-1, add 1ml of water, shake to dissolve, filter, and use the filtrate as the test solution. Take another 5g of Pseudostellariae Radix as a control medicinal material, add 50ml of water, heat at 90℃ for 1 hour, filter, and use the filtrate as the control medicinal material 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 two solutions and spot them on the same silica gel G thin layer plate-1. Then take 1μl of each of the above two solutions and spot them on the same silica gel G thin layer plate-1. Repeat the spotting until 10 silica gel G thin layer plates with good spots are obtained. Each silica gel G thin layer plate is developed with a mixed solvent composed of 75% isopropanol and anhydrous ethanol in a volume ratio of 9:1 as the developing agent, taken out, dried, sprayed with the mixed solvent shown in Table 2 as the color developer, heated at 105℃ until the spots are clearly colored, and inspected under sunlight. In the chromatogram of the test sample, observe the spots that appear at the corresponding positions on the chromatogram of the control herb. The results are shown in Figures 1, 15, 23, and 24. The order of spots from left to right in Figures 1, 15, 23 is: 1. Shengge Bushen Capsule-1, dot spot; 2. Pseudostellariae Radix control herb, dot spot; 3. Shengge Bushen Capsule-1, strip spot; 4. Pseudostellariae Radix control herb, strip spot.

[0146] Table 2. Comparison of different colorants in identification of Pseudostellaria heterophylla

[0147] 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 in the chromatogram of the control medicinal material.

[0148] 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 the fluorescent spots of the same color are displayed at the corresponding positions in the control medicinal material chromatogram, but the color development effect is slightly worse than that in Figure 1.

[0149] 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 the fluorescent spots of the same color are displayed at the corresponding positions in the control medicinal material chromatogram, but the color development effect is slightly worse than that in Figure 1.

[0150] 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 in the control medicinal material chromatogram.

[0151] 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 in the control medicinal material chromatogram.

[0152] 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 in the control medicinal material chromatogram.

[0153] 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 in the control medicinal material chromatogram, but the color development effect is slightly worse than that in Figure 1.

[0154] As shown in Figure 21, 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 in the control medicinal material chromatogram.

[0155] As shown in Figure 22, in the test sample chromatogram, the fluorescent spots are rich in information, the separation effect of each component is good, and fluorescent spots of the same color are displayed at the corresponding positions in the control medicinal material chromatogram.

[0156] As can be seen from Figure 23, in the test sample chromatogram, there is little information about the fluorescent spots. Although there are obvious spots in the chromatogram, the separation effect is poor, the ratio shift value (Rf value) cannot meet the requirements, and no fluorescent spots of the same color are displayed at the corresponding position of the control medicinal material chromatogram.

[0157] Example 3 Methodological Investigation

[0158] (1) Specificity experiment

[0159] Dissolve 40 mg of each of three batches of Shengge Bushen Capsules (Shengge Bushen Capsules-1, Shengge Bushen Capsules-2, and Shengge Bushen Capsules-3) in 1 ml of water, shake to dissolve, and filter. The filtrate is used as the test solution. Separately, take 5 g of Pseudostellariae Radix as a control, add 50 ml of water, heat at 90°C for 1 hour, filter, and use the filtrate as the control solution. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μl of each of the above two solutions was aspirated and spotted on the same silica gel G thin layer plate-1, and then 1 μl of each of the above two solutions was aspirated and spotted on the same silica gel G thin layer plate-1. Repeat the spotting until 3 spotted silica gel G thin layer plates were obtained. Each silica gel G thin layer plate was developed with a mixed solvent of 75% isopropanol and anhydrous ethanol in a volume ratio of 9:1 as a developing agent. The plates were taken out, dried, and sprayed with a mixed solvent of 2% aniline acetone solution, 2% diphenylamine acetone solution and phosphoric acid in a volume ratio of 5:5:1 as a color developer. The plates were heated at 105°C until the spots were clearly colored and inspected under sunlight. The results are shown in FIG24 . The order of spots from left to right in FIG24 is: 1. Ginseng and Radix Bu Shen Capsules-1, strip spots; 2. Ginseng and Radix Bu Shen Capsules-2, strip spots; 3. Ginseng and Radix Bu Shen Capsules-3, strip spots; 4. Pseudostellariae pseudoginseng control medicinal material, strip spots.

[0160] The results show that in the test sample chromatogram shown in Figure 24, the fluorescent spots are rich in information, the separation effect of each component is good, and spots of the same color appear at the corresponding positions in the control medicinal material chromatogram, indicating that the thin layer chromatography detection method for Pseudostellaria baicalensis in the ginseng and kudzu kidney-tonifying preparation provided in this application has strong specificity.

[0161] (2) Investigation of the tolerance of thin layer plates

[0162] Take 40 mg of each of Shengge Bushen Capsule-1, Shengge Bushen Capsule-2, and Shengge Bushen Capsule-3, add 1 ml of water, shake to dissolve, filter, and use the filtrate as the test solution. Separately, take 5 g of Pseudostellaria heterophylla as a control herb, add 50 ml of water, heat at 90°C for 1 hour, filter, and use the filtrate as the control herb solution. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 1 μl of each of the above two solutions was taken and spotted on the same silica gel G thin layer plate-1, and then spotted on the same silica gel G thin layer plate-2 according to the same method. Repeat the spotting until two spotted silica gel G thin layer plates were obtained. Each silica gel G thin layer plate was developed with a mixed solvent of 75% isopropanol and anhydrous ethanol in a volume ratio of 9:1 as a developing agent. The plates were taken out, dried, and sprayed with a mixed solvent of 2% aniline acetone solution, 2% diphenylamine acetone solution, and phosphoric acid in a volume ratio of 5:5:1 as a color developer. The plates were heated at 105°C until the spots were clearly colored and inspected under sunlight. The results are shown in Figure 25. The order of spotting from left to right in Figure 25 is: 1. Ginseng and Radix Bu Shen Capsule-1, spotting; 2. Ginseng and Radix Bu Shen Capsule-2, spotting; 3. Ginseng and Radix Bu Shen Capsule-3, spotting; 4. Radix Pseudostellariae reference material, spotting; 5. Radix Pseudostellariae reference material, spotting; 6. Ginseng and Radix Bu Shen Capsule-1, spotting; 7. Ginseng and Radix Bu Shen Capsule-2, spotting; 8. Ginseng and Radix Bu Shen Capsule-3, spotting; and Figure 25 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 and 4 are spotted on silica gel G thin layer plate-1, and 5, 6, 7 and 8 are spotted on silica gel G thin layer plate-2.

[0163] The results show that in the chromatogram of the test sample shown in Figure 25, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, 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 Pseudostellaria baicalensis in the ginseng and kudzu kidney-tonifying preparation described in this application.

[0164] 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.

[0165] 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 Radix Pseudostellariae Radix in Radix Glehniae Kidney-tonifying Preparation, characterized in that: The raw materials of the Shenkui tonifying kidney preparation include Pseudostellaria heterophylla, Pueraria root and Epimedium; The thin layer chromatography detection method of Radix Pseudostellariae Radix in the Radix Ginseng and Radix Gedonis kidney-tonifying preparation comprises the following steps: Prepare the test sample solution and Pseudostellaria heterophylla control medicinal material solution respectively; The test solution and the Radix Pseudostellariae Radix control medicinal material solution are spotted on the same thin layer chromatography plate, developed in a developing agent, and a color developer is added for color development and inspection; wherein the developing agent is a mixed solvent composed of 75% by volume of isopropanol and ethanol in a volume ratio of (1-9):1; The color developer is a mixed solution of aniline, diphenylamine, phosphoric acid and acetone.

2. The thin layer chromatography detection method according to claim 1, characterized in that: The color developer is a mixed solution of aniline acetone solution with a volume fraction of 1% to 2%, diphenylamine acetone solution with a mass volume ratio of 1g:(49-99)ml, and phosphoric acid with a volume ratio of (4-5):(3-5):

1.

3. The thin layer chromatography detection method according to any one of claims 1 to 2, characterized in that: The color developer is a mixed solution of aniline acetone solution with a volume fraction of 2%, diphenylamine acetone solution with a mass volume ratio of 1g:49ml, and phosphoric acid in a volume ratio of (4-5):(3-5):

1.

4. The thin layer chromatography detection method according to any one of claims 1 to 3, characterized in that: After adding the developer, the thin layer chromatography plate needs to be heated at a temperature of 100°C to 110°C.

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

6. The thin layer chromatography detection method according to any one of claims 1 to 5, 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 water for a first extraction to obtain an extract, and the test sample solution is prepared.

7. The thin layer chromatography detection method according to claim 6, characterized in that: Satisfy one or more of the following (1) to (2): (1) The first extraction method is shaking at room temperature; (2) In the test solution, the mass volume ratio of the ginseng and kudzu kidney-tonifying preparation test sample and water is (1-100) mg: 1 ml.

8. The thin layer chromatography detection method according to any one of claims 1 to 7, characterized in that: The preparation method of the Pseudostellariae Radix control medicinal material solution comprises the following steps: The Pseudostellariae Radix control medicinal material is mixed with water for a second extraction to obtain an extract and prepare the Pseudostellariae Radix control medicinal material solution.

9. The thin layer chromatography detection method according to claim 8, characterized in that: Satisfy one or more of the following (1) to (2): (1) The second extraction method is heating extraction, the temperature is 80°C to 100°C, and the time is 0.5h to 2h; (2) In the Pseudostellariae Radix control medicinal material solution, the mass volume ratio of Pseudostellariae Radix and water is (1-100) mg:1 ml.

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) to (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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