Thin-layer chromatography detection method of Codonopsis pilosula in kidney-tonifying preparations

Abstract

This invention relates to a thin-layer chromatography (TLC) method for detecting *Pseudostellaria heterophylla* in a ginseng-pueraria lobata kidney-tonifying preparation. The raw materials of the preparation include *Pseudostellaria heterophylla*, kudzu root, and epimedium. The TLC method for detecting *Pseudostellaria heterophylla* in the preparation comprises the following steps: preparing a test solution and a *Pseudostellaria heterophylla* reference solution; spotting the test solution and the reference solution onto the same TLC plate, developing the solution in a developing solvent, adding a colorimetric reagent, and examining the sample; wherein the developing solvent is a mixture of 75% isopropanol and ethanol in a volume ratio of (1-9):1; and the colorimetric reagent is a mixed solution of aniline, diphenylamine, phosphoric acid, and acetone. This method can rapidly and accurately detect *Pseudostellaria heterophylla* in ginseng-pueraria lobata kidney-tonifying preparations, improving the accuracy and comprehensiveness of quality control and facilitating the establishment and overall evaluation of quality standards for these preparations.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, and in particular to a thin-layer chromatography method for detecting Codonopsis pilosula in a ginseng and kudzu kidney-tonifying preparation. Background Technology

[0002] Prince ginseng, the dried tuberous root of *Pseudostellaria heterophylla* (Miq.) Pax ex Paxet Hoffm., a plant in the Caryophyllaceae family, has the effects of invigorating qi and strengthening the spleen, promoting body fluid production and moistening the lungs. It is commonly used for spleen deficiency and fatigue, loss of appetite, weakness after illness, qi and yin deficiency, spontaneous sweating and thirst, and dry cough due to lung dryness.

[0003] The main ingredients of the Shenge Kidney-Tonifying Preparation are Codonopsis pilosula, Pueraria lobata, and Epimedium. Its functions and indications are to invigorate Qi, nourish Yin, and tonify the kidneys. It is suitable for mild to moderate depression diagnosed in Traditional Chinese Medicine as Qi and Yin deficiency with insufficient kidney Qi, characterized by symptoms such as low mood, excessive thinking and worry, reduced speech and movement, sluggish gaze, forgetfulness, poor appetite, palpitations, timidity, insomnia with excessive dreaming, irritability, pale red or reddish tongue, white or patchy tongue coating, and weak pulse.

[0004] As a major ingredient in ginseng and kudzu kidney-tonifying preparations, codonopsis plays an important role in the efficacy of these preparations. However, there is currently a lack of methods that can quickly identify and specifically detect codonopsis in these preparations, making it impossible to accurately and comprehensively determine or control their quality. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a thin-layer chromatography method for detecting Codonopsis pilosula in a ginseng and kudzu kidney-tonifying preparation. This method can rapidly and accurately detect Codonopsis pilosula, improving the accuracy and comprehensiveness of quality control in ginseng and kudzu kidney-tonifying preparations and facilitating the establishment and overall evaluation of quality standards for these preparations.

[0006] The technical solution is as follows:

[0007] A thin-layer chromatography method for detecting Codonopsis pilosula in a ginseng and kudzu kidney-tonifying preparation, wherein the raw materials of the ginseng and kudzu kidney-tonifying preparation include Codonopsis pilosula, kudzu root and Epimedium;

[0008] The thin-layer chromatography detection method for Codonopsis pilosula in the aforementioned kidney-tonifying preparation includes the following steps:

[0009] Prepare the test solution and the reference herb solution of Codonopsis pilosula separately;

[0010] Spot the test solution and the Codonopsis pilosula reference herb solution onto the same thin-layer chromatography plate, develop them in the developing solvent, add a colorimetric reagent for color development, and examine them.

[0011] The developing agent is a mixed solvent consisting of isopropanol and ethanol with a volume fraction of 75% in a volume ratio of (1-9):1.

[0012] The colorimetric reagent is a mixed solution of aniline, diphenylamine, phosphoric acid, and acetone.

[0013] In some embodiments, the color developer is a mixed solution consisting of a 1% to 2% aniline acetone solution, a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:(49 to 99) ml, and phosphoric acid in a volume ratio of (4 to 5):(3 to 5):1.

[0014] In some embodiments, the colorimetric agent is a mixed solution consisting of a 2% (v / v) aniline acetone solution, a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:49 ml, and phosphoric acid in a volume ratio of (4-5):(3-5):1.

[0015] In some embodiments, after adding the colorimetric reagent, the thin-layer chromatography plate needs to be heated to a temperature of 100°C to 110°C.

[0016] In some of these embodiments, the light source used is a fluorescent lamp.

[0017] In some embodiments, the method for preparing the test solution includes the following steps:

[0018] The test sample of the ginseng and kudzu kidney-tonifying preparation was mixed with water for the first extraction to obtain the extract, and the test sample solution was prepared.

[0019] In some embodiments, the first extraction method is room temperature shaking.

[0020] In some embodiments, the mass-to-volume ratio of the ginseng and kudzu kidney-tonifying preparation to water in the test solution is (1-100) mg: 1 ml.

[0021] In some embodiments, the preparation method of the Codonopsis pilosula reference herb solution includes the following steps:

[0022] The reference herb, *Codonopsis pilosula*, was mixed with water for a second extraction to obtain an extract, which was then used to prepare the *Codonopsis pilosula* reference herb solution.

[0023] In some embodiments, the second extraction method is heating extraction at a temperature of 80°C to 100°C for a time of 0.5h to 2h.

[0024] In some embodiments, the mass-to-volume ratio of the Codonopsis pilosula to water in the Codonopsis pilosula reference herb solution is (1-100) mg: 1 ml.

[0025] In some embodiments, the raw materials of the ginseng and kudzu kidney-tonifying preparation include, by weight, 1000-2000 parts of Codonopsis pilosula, 500-1500 parts of Pueraria lobata, and 100-900 parts of Epimedium.

[0026] In some embodiments, the dosage form of the ginseng and kudzu kidney-tonifying preparation is selected from granules, powders, tablets, capsules, or pills.

[0027] The present invention has at least the following beneficial effects:

[0028] Codonopsis pilosula is a major component of ginseng and kudzu kidney-tonifying preparations and is closely related to their therapeutic functions. The thin-layer chromatography method provided by this invention can rapidly detect Codonopsis pilosula in these preparations, producing clear chromatographic spots with no interference from the negative control. This improves the accuracy and comprehensiveness of quality control for ginseng and kudzu kidney-tonifying preparations, facilitating the establishment and overall evaluation of their quality standards. Furthermore, this method offers advantages such as short processing time, simple operation, high sensitivity, high resolution, low equipment cost, and low overall cost. Attached Figure Description

[0029] Figure 1 The thin-layer chromatography results shown in an embodiment of the present invention are obtained by using a mixed solvent of 75% isopropanol and anhydrous ethanol in a volume ratio of 9:1 as the developing solvent and a mixed solution of 2% aniline acetone solution, diphenylamine acetone solution in a mass-volume ratio of 1g:49ml and phosphoric acid in a volume ratio of 5:5:1 as the colorimetric reagent.

[0030] Figure 2 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (75% by volume) and anhydrous ethanol in a volume ratio of 8:2 as the developing solvent.

[0031] Figure 3 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (75% by volume) and anhydrous ethanol in a volume ratio of 7:3 as the developing solvent.

[0032] Figure 4 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (75% by volume) and anhydrous ethanol in a volume ratio of 6:4 as the developing solvent.

[0033] Figure 5 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (75% by volume) and anhydrous ethanol in a volume ratio of 5:5 as the developing solvent.

[0034] Figure 6The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (75% by volume) and anhydrous ethanol in a volume ratio of 4:6 as the developing solvent.

[0035] Figure 7 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (75% by volume) and anhydrous ethanol in a volume ratio of 3:7 as the developing solvent.

[0036] Figure 8 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (75% by volume) and anhydrous ethanol in a volume ratio of 2:8 as the developing solvent.

[0037] Figure 9 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (75% by volume) and anhydrous ethanol in a volume ratio of 1:9 as the developing solvent.

[0038] Figure 10 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (50% by volume) and anhydrous ethanol in a volume ratio of 9:1 as the developing solvent.

[0039] Figure 11 The thin-layer chromatography detection results, as shown in an embodiment of the present invention, are obtained using a mixed solvent of isopropanol (volume fraction 25%) and anhydrous ethanol in a volume ratio of 9:1 as the developing solvent.

[0040] Figure 12 The thin-layer chromatography detection results shown in an embodiment of the present invention are obtained using a mixed solvent of isopropanol and anhydrous ethanol in a volume ratio of 9:1 as the developing solvent.

[0041] Figure 13 The thin-layer chromatography detection results shown in an embodiment of the present invention are obtained 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 the developing solvent.

[0042] Figure 14 The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solvent of chloroform and methanol in a volume ratio of 4:1 as the developing solvent.

[0043] Figure 15 The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solution of 2% aniline acetone solution, diphenylamine acetone solution with a mass-to-volume ratio of 1g:49ml and phosphoric acid in a volume ratio of 5:4:1 as the colorimetric reagent.

[0044] Figure 16The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solution of 2% aniline acetone solution, diphenylamine acetone solution with a mass-to-volume ratio of 1g:49ml and phosphoric acid in a volume ratio of 5:3:1 as the colorimetric reagent.

[0045] Figure 17 The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solution of 2% aniline acetone solution, diphenylamine acetone solution with a mass-to-volume ratio of 1g:49ml and phosphoric acid in a volume ratio of 4:5:1 as the colorimetric reagent.

[0046] Figure 18 The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solution of 2% aniline acetone solution, diphenylamine acetone solution with a mass-to-volume ratio of 1g:49ml and phosphoric acid in a volume ratio of 4:4:1 as the colorimetric reagent.

[0047] Figure 19 The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solution of 2% aniline acetone solution, diphenylamine acetone solution with a mass-to-volume ratio of 1g:49ml and phosphoric acid in a volume ratio of 4:3:1 as the colorimetric reagent.

[0048] Figure 20 The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solution of 1% aniline acetone solution, diphenylamine acetone solution with a mass-to-volume ratio of 1g:49ml and phosphoric acid in a volume ratio of 5:5:1 as the colorimetric reagent.

[0049] Figure 21 The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solution of 2% aniline acetone solution, diphenylamine acetone solution with a mass-to-volume ratio of 1g:99ml and phosphoric acid in a volume ratio of 5:5:1 as the colorimetric reagent.

[0050] Figure 22 The thin-layer chromatography detection results are shown in an embodiment of the present invention, using a mixed solution of 1% aniline acetone solution, diphenylamine acetone solution with a mass-to-volume ratio of 1g:99ml and phosphoric acid in a volume ratio of 5:3:1 as the colorimetric reagent.

[0051] Figure 23 The thin-layer chromatography detection results using 0.2% ninhydrin ethanol solution as the colorimetric reagent are shown in an embodiment of the present invention.

[0052] Figure 24 Thin-layer chromatography results of Codonopsis pilosula in different batches of Shen Ge Bushen capsules;

[0053] Figure 25The results show the results of thin-layer chromatography detection of Codonopsis pilosula in Shenge Bushen Capsules using different thin-layer chromatography plates. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0056] In this invention, the terms "preferredly," "more preferably," "better," and "even better" refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of the invention. That is, in this invention, "preferredly," "more preferably," "better," and "even better" are merely descriptions of more effective implementations or examples, but do not constitute a limitation on the scope of protection of the invention.

[0057] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0058] In this invention, "at least one" means one or more, such as one, two, or more. "Multiple" or "several" means at least two, such as two, three, etc., and "multi-layered" means at least two layers, such as two layers, three layers, etc., unless otherwise explicitly specified. In the description of this invention, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0059] When a numerical range is disclosed in this invention, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Further, when the range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Moreover, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0060] Unless otherwise specified, all steps of this invention may be performed sequentially or randomly. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0061] Unless otherwise stated, a singular term may include a plural term and should not be understood as having a quantity of one.

[0062] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature.

[0063] In this invention, the terms "comprising," "including," "containing," "having," or other variations are intended to cover non-closed inclusion, and no distinction is made between these terms. The term "comprising" means that other steps and components may be added without affecting the final result. The compositions and methods / processes of this invention comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0064] In this invention, the terms "efficacy", "performance", "effect" and "potential" are not distinguished.

[0065] The weights of the relevant components mentioned in the embodiments of this invention can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this invention is within the scope disclosed in the embodiments of this invention. Specifically, the weights mentioned in the embodiments of this invention can be well-known units of mass in the chemical industry, such as μg, mg, g, and kg.

[0066] In this invention, the temperature parameter, unless otherwise specified, can be either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. The room temperature or ambient temperature mentioned in this invention refers to 0–40°C, preferably 10°C–35°C, and more preferably 20°C–30°C.

[0067] In this invention, 75% isopropanol means that in an aqueous isopropanol solution, the volume fraction of anhydrous isopropanol is 75% and the volume fraction of water is 25%; 50% isopropanol means that in an aqueous isopropanol solution, the volume fraction of anhydrous isopropanol is 50% and the volume fraction of water is 50%; 25% isopropanol means that in an aqueous isopropanol solution, the volume fraction of anhydrous isopropanol is 25% and the volume fraction of water is 75%.

[0068] The main ingredients of the Shen-Ge kidney-tonifying preparation are Codonopsis pilosula, Pueraria lobata, and Epimedium. Its functions and indications are to invigorate Qi, nourish Yin, and tonify the kidneys. Codonopsis pilosula, as a key ingredient in this preparation, plays a crucial role in its efficacy. However, currently, there is a lack of rapid and specific methods for identifying and detecting Codonopsis pilosula in this preparation, making it impossible to accurately and comprehensively determine or control its quality.

[0069] Thin-layer chromatography (TLC) involves coating a suitable stationary phase onto a glass plate, plastic, or aluminum substrate to form a uniform thin layer. After spotting and development, the ratio shift (Rf) is compared with the Rf of a suitable reference chromatogram obtained using the same method. This comparison is used for drug identification, impurity detection, or content determination. As a crucial experimental technique for the rapid separation and qualitative analysis of small amounts of substances, TLC is characterized by short development time, strong separation capability, and high sensitivity, making it a vital analytical tool for the quality control of traditional Chinese medicine. Compared to content determination and characteristic chromatographic identification, TLC is one of the most widely used methods in planar chromatography. It is easy to master, requires inexpensive equipment, and is simple and flexible to operate. Its advantages include: the ability to change the developing solvent and development direction at any time; immediate derivatization, color development, observation, and detection; rapid method development; high sensitivity and resolution; simultaneous separation of multiple samples; and simple sample pretreatment. In recent years, despite the rapid development of other chromatographic techniques, the application of thin-layer chromatography has not decreased significantly. On the contrary, with the introduction of new stationary phases and rapidly developing instrument technology, it has shown a trend of wider application and has become a modern, sensitive and efficient separation and analysis method.

[0070] If the Codonopsis pilosula in the Shen-Ge Tonifying Kidney Preparation could be detected by thin-layer chromatography, it would significantly improve the accuracy and comprehensiveness of the quality control of the Shen-Ge Tonifying Kidney Preparation, and would be conducive to the establishment and overall evaluation of the quality standards of the Shen-Ge Tonifying Kidney Preparation. However, there are currently no relevant reports.

[0071] Accordingly, the present invention provides a thin-layer chromatography detection method for Codonopsis pilosula in ginseng and kudzu kidney-tonifying preparations, which can quickly and accurately detect Codonopsis pilosula, improve the accuracy and comprehensiveness of quality control of ginseng and kudzu kidney-tonifying preparations, and facilitate the establishment and overall evaluation of quality standards for ginseng and kudzu kidney-tonifying preparations.

[0072] The technical solution is as follows:

[0073] A thin-layer chromatography method for detecting Codonopsis pilosula in a ginseng and kudzu kidney-tonifying preparation, wherein the raw materials of the ginseng and kudzu kidney-tonifying preparation include Codonopsis pilosula, kudzu root and Epimedium;

[0074] The thin-layer chromatography detection method for Codonopsis pilosula in the aforementioned kidney-tonifying preparation includes the following steps:

[0075] Prepare the test solution and the reference herb solution of Codonopsis pilosula separately;

[0076] Spot the test solution and the Codonopsis pilosula reference herb solution onto the same thin-layer chromatography plate, develop them in the developing solvent, add a colorimetric reagent for color development, and examine them.

[0077] The developing agent is a mixed solvent consisting of isopropanol and ethanol with a volume fraction of 75% in a volume ratio of (1-9):1.

[0078] The colorimetric reagent is a mixed solution of aniline, diphenylamine, phosphoric acid, and acetone.

[0079] In this invention, the chromatogram of the test sample shows fluorescent spots of the same color at the corresponding positions as those of the reference medicinal material. This method can rapidly detect *Codonopsis pilosula* in *Shen-Ge* kidney-tonifying preparations, with clear corresponding thin-layer chromatographic spots and no interference from the negative control, improving the accuracy and comprehensiveness of quality control of *Shen-Ge* kidney-tonifying preparations, and facilitating the establishment and overall evaluation of quality standards for these preparations. Furthermore, this method also has advantages such as short processing time, simple operation, high sensitivity, high resolution, low equipment cost, and low overall cost.

[0080] The present invention will be further described in detail below by step-by-step description. It should be noted that the present invention does not limit the order between S100 and S200, and the numbering is only for the convenience of description.

[0081] S100: Prepare the test solution.

[0082] In some embodiments, the method for preparing the test solution includes the following steps:

[0083] The test sample of the ginseng and kudzu kidney-tonifying preparation was mixed with water for the first extraction to obtain the extract, and the test sample solution was prepared.

[0084] In some embodiments, the mass-to-volume ratio of the ginseng and kudzu kidney-tonifying preparation to water in the test solution 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. Preferably, the mass-to-volume ratio of the ginseng and kudzu kidney-tonifying preparation to water is (10-100) mg:1 ml.

[0085] In some embodiments, the first extraction method is room temperature shaking.

[0086] In some embodiments, the method for preparing the test solution includes the following steps:

[0087] Take 40 mg of the kidney-tonifying preparation of Ginseng and Pueraria, add 1 ml of water, shake to dissolve, filter, and use the filtrate as the test solution.

[0088] S200: Preparation of Codonopsis pilosula reference material solution.

[0089] In some embodiments, the preparation method of the Codonopsis pilosula reference herb solution includes the following steps:

[0090] The reference herb, *Codonopsis pilosula*, was mixed with water for a second extraction to obtain an extract, which was then used to prepare the *Codonopsis pilosula* reference herb solution.

[0091] In some embodiments, the second extraction method is heating extraction at a temperature of 80°C to 100°C for a time of 0.5h to 2h.

[0092] In some embodiments, the mass-to-volume ratio of *Pseudostellaria heterophylla* to water in the *Pseudostellaria heterophylla* reference herb solution 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. Preferably, the mass-to-volume ratio of *Pseudostellaria heterophylla* to water is (10-100) mg:1 ml.

[0093] In some embodiments, the preparation method of the Codonopsis pilosula reference herb solution includes the following steps:

[0094] Take 5g of Codonopsis pilosula as a reference herb, add 50ml of water, heat at 90℃ for 1 hour, filter, and use the filtrate as the reference herb solution.

[0095] S300: Spot the test solution and the Codonopsis pilosula reference solution onto the same thin-layer chromatography plate, develop in the developing solvent, add a colorimetric reagent for color development, and examine.

[0096] Understandably, in one example, 1 μl of the test solution and 1 μl of the Codonopsis pilosula reference material solution were spotted onto the same thin-layer chromatography plate.

[0097] In this invention, the developing solvent is a mixed solvent consisting of 75% isopropanol and ethanol (preferably anhydrous ethanol) in a volume ratio of 1 to 9:1. It is understood that the volume ratio of the 75% 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. Preferably, the developing solvent is a mixed solvent consisting of 75% isopropanol and anhydrous ethanol in a volume ratio of 5:5, 7:3, 6:4, 8:2, or 9:1. More preferably, the developing solvent is a mixed solvent consisting of 75% isopropanol and anhydrous ethanol in a volume ratio of 9:1.

[0098] Understandably, it is necessary to place the thin-layer chromatography plate in a developing tank containing a developing agent for development. After the development step and before the color development step of adding a colorimetric agent, the process also includes the step of removing the thin-layer chromatography plate from the developing tank and air-drying, baking, or blowing to remove the developing agent.

[0099] In this invention, the colorimetric agent is a mixed solution of aniline, diphenylamine, phosphoric acid, and acetone.

[0100] In some embodiments, the color developer is a mixed solution consisting of a 1% to 2% aniline acetone solution, a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:(49 to 99) ml, and phosphoric acid in a volume ratio of (4 to 5):(3 to 5):1.

[0101] Understandably, in the aniline-acetone solution, the volume fraction of aniline is, but is not limited to, 1%, 1.5%, or 2%. In the diphenylamine-acetone solution, the mass-to-volume ratio of diphenylamine to acetone is 1 g:(49–99) ml, including but not limited to 1 g:49 ml (or 2 g:98 ml), 1 g:50 ml, 1 g:60 ml, 1 g:65 ml, 1 g:70 ml, 1 g:80 ml, 1 g:85 ml, 1.5 g:98.5 ml, or 1 g:99 ml.

[0102] In one embodiment, the color developer is a mixed solution consisting of a 1% (v / v) aniline acetone solution, a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:99 ml, and phosphoric acid in a volume ratio of (4-5):(3-5):1. The volume ratio of the 1% (v / v) aniline acetone solution, the diphenylamine acetone solution with a mass-to-volume ratio of 1 g:99 ml, 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.

[0103] In one embodiment, the color developer is a mixed solution consisting of a 1% (v / v) aniline acetone solution, a 2g:98ml (w / v) diphenylamine acetone solution, and phosphoric acid in a volume ratio of (4-5):(3-5):1. The volume ratio of the 1% (v / v) aniline acetone solution, the 2g:98ml (w / v) diphenylamine acetone solution, and the phosphoric acid is, but is not limited to, 4:3:1, 4:4:1, 4:5:1, 5:3:1, 5:4:1, or 5:5:1.

[0104] In one embodiment, the color developer is a mixed solution consisting of a 2% (v / v) aniline acetone solution, a diphenylamine acetone solution with a mass-to-volume ratio of 1 g:99 ml, and phosphoric acid in a volume ratio of (4-5):(3-5):1. The volume ratio of the 2% (v / v) aniline acetone solution, the diphenylamine acetone solution with a mass-to-volume ratio of 1 g:99 ml, 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.

[0105] In one embodiment, the color developer is a mixed solution consisting of a 2% (v / v) aniline acetone solution, a diphenylamine acetone solution with a mass-to-volume ratio of 2 g:98 ml, and phosphoric acid in a volume ratio of (4-5):(3-5):1. The volume ratio of the 2% (v / v) aniline acetone solution, the diphenylamine acetone solution with a mass-to-volume ratio of 2 g:98 ml, 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. More preferably, the color developer is a mixed solution consisting of a 2% (v / v) aniline acetone solution, 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.

[0106] In some embodiments, after adding the colorimetric agent, the thin-layer chromatography plate needs to be heated to a temperature of 100°C to 110°C, including but not limited to 100°C, 105°C or 110°C.

[0107] In some of these embodiments, the light source used is a fluorescent lamp.

[0108] In some embodiments, the raw materials of the ginseng and kudzu kidney-tonifying preparation include, by weight, 1000-2000 parts of Codonopsis pilosula, 500-1500 parts of Pueraria lobata, and 100-900 parts of Epimedium.

[0109] In some embodiments, the dosage form of the ginseng and kudzu kidney-tonifying preparation is selected from granules, powders, tablets, capsules, or pills.

[0110] The following description, in conjunction with specific embodiments, further illustrates the point:

[0111] Instruments and reagents

[0112] The kidney-tonifying preparations are Shen Ge Bu Shen Capsules. Shen Ge Bu Shen Capsules-1 are from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 23005; Shen Ge Bu Shen Capsules-2 are from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 21002; Shen Ge Bu Shen Capsules-3 are from Xinjiang Huachun Biopharmaceutical Co., Ltd., batch number: 21004.

[0113] The reference material for Codonopsis pilosula was sourced from the China National Institutes for Food and Drug Control, batch number 121004-201709;

[0114] Silicone G Thin Film Plate-1: Qingdao Ocean Chemical Co., Ltd., specifications 100mm×100mm, thickness 0.20mm-0.25mm; Silicone G Thin Film Plate-2: MACHEREY-NAGEL-Gmbh&CakG-010303;

[0115] All other reagents used were of analytical grade. The concentrations of the liquid raw material mixtures in the following examples are all volume fractions. For example, 75% isopropanol means that in an aqueous isopropanol solution, the volume fraction of isopropanol is 75% and the volume fraction of water is 25%; 1% aniline-acetone solution means that in an aniline-acetone solution, the volume fraction of aniline is 1% and the volume fraction of acetone is 99%. Similarly, 2% aniline-acetone solution means that in an aniline-acetone solution, the volume fraction of aniline is 2% and the volume fraction of acetone is 98%; 1% diphenylamine-acetone solution means that in a diphenylamine-acetone solution, the mass of diphenylamine is 1g and the volume of acetone is 99ml; 2% diphenylamine-acetone solution means that in a diphenylamine-acetone solution, the mass of diphenylamine is 2g and the volume of acetone is 98ml; 0.2% ninhydrin ethanol solution means that in 100ml of ninhydrin ethanol solution, 0.2g of ninhydrin is contained.

[0116] Example 1

[0117] This embodiment examines the separation effect of thin-layer chromatography (TLC) with different mixed solvents as developing solvents on the detection of Codonopsis pilosula in a kidney-tonifying preparation, and determines the developing solvent suitable for this invention, as follows:

[0118] Take 40 mg of the contents of Shenge Bushen Capsules, add 1 ml of water, shake to dissolve, filter, and use the filtrate as the test solution. Separately, take 5 g of Codonopsis pilosula reference material, add 50 ml of water, heat at 90℃ for 1 hour, filter, and use the filtrate as the reference material solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502). Apply 1 μl of each of the above two solutions to the same silica gel G thin-layer plate-1, then apply 1 μl of each solution to the same silica gel G thin-layer plate-1 again. Develop with the Codonopsis pilosula identification developing solvent listed in Table 1. Remove, air dry, and 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 the colorimetric reagent. Heat at 105℃ until the spots are clearly visible, and examine under sunlight. Observe the spots at the corresponding positions in the chromatogram of the test sample and the chromatogram of the reference material. The results are as follows: Figures 1 to 14 As shown, Figures 1 to 14 The sampling order from left to right is as follows: 1. Shen Ge Bu Shen Capsule-1, spot sampling; 2. Codonopsis pilosula reference material, spot sampling; 3. Shen Ge Bu Shen Capsule-1, strip sampling; 4. Codonopsis pilosula reference material, strip sampling.

[0119] Table 1. Comparison of different developing agents in the identification of Codonopsis pilosula

[0120]

[0121]

[0122] Depend on Figure 1 It can be seen that the chromatogram of the test sample contains abundant fluorescent spot information, the separation effect of each component is good, and fluorescent spots of the same color are displayed at the corresponding positions as those of the reference medicinal material.

[0123] Depend on Figure 2 It can be seen that the chromatogram of the test sample contains rich information about the fluorescent spots. Fluorescent spots of the same color are displayed at the corresponding positions as those in the chromatogram of the reference medicinal material. Although the samples can be separated, the specific migration value (Rf value) is relatively low. Figure 1 Difference.

[0124] Depend on Figure 3 It can be seen that the chromatogram of the test sample contains rich information about the fluorescent spots. Fluorescent spots of the same color are displayed at the corresponding positions as those in the chromatogram of the reference medicinal material. Although the samples can be separated, the specific migration value (Rf value) is relatively low. Figure 1 Difference.

[0125] Depend on Figure 4 It can be seen that the chromatogram of the test sample contains rich information about the fluorescent spots. Fluorescent spots of the same color are displayed at the corresponding positions as those in the chromatogram of the reference medicinal material. Although the samples can be separated, the specific migration value (Rf value) is relatively low. Figure 1 Difference.

[0126] Depend on Figure 5 It can be seen that the chromatogram of the test sample contains rich information about the fluorescent spots. Fluorescent spots of the same color are displayed at the corresponding positions as those in the chromatogram of the reference medicinal material. Although the samples can be separated, the specific migration value (Rf value) is relatively low. Figure 1 Difference.

[0127] Depend on Figure 6 It can be seen that there are few visible spots in the chromatograms of the test sample and the reference medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0128] Depend on Figure 7 It can be seen that there are few visible spots in the chromatograms of the test sample and the reference medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0129] Depend on Figure 8 It can be seen that there are few visible spots in the chromatograms of the test sample and the reference medicinal material, the separation effect of each component is very poor, and the tailing is very serious.

[0130] Depend on Figure 9 It can be seen that there are few visible spots in the chromatograms of the test sample and the reference medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0131] Depend on Figure 10 It can be seen that the chromatogram of the test sample contains rich information about the fluorescent spots. Fluorescent spots of the same color are displayed at the corresponding positions as those in the chromatogram of the reference medicinal material. Although the samples can be separated, the specific migration value (Rf value) is relatively low. Figure 1 Poor, cannot meet the requirements.

[0132] Depend on Figure 11 It can be seen that there are few visible spots in the chromatograms of the test sample and the reference medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0133] Depend on Figure 12 It can be seen that there are few visible spots in the chromatograms of the test sample and the reference medicinal material, the separation effect of each component is very poor, and the tailing is obvious.

[0134] Depend on Figure 13 It can be seen that there are few visible spots in the chromatograms of the test sample and the reference medicinal material, indicating that the separation effect of each component is very poor.

[0135] Depend on Figure 14 It can be seen that there are no obvious visible spots in the chromatograms of the test sample and the reference medicinal material, indicating that the separation effect of each component is particularly poor.

[0136] Combination Figures 1 to 14 It is known that a mixed solvent consisting of 75% isopropanol and anhydrous ethanol in a volume ratio of (1-9):1 is suitable as the developing solvent in the thin-layer chromatography detection method of Codonopsis pilosula in the kidney-tonifying preparation of the present invention.

[0137] Example 2

[0138] This embodiment examines the colorimetric effect of thin-layer chromatography (TLC) on the detection of Codonopsis pilosula in a kidney-tonifying preparation using different mixed solvents as colorimetric reagents, and determines the colorimetric reagent suitable for this invention, as detailed below:

[0139] Take 40 mg of the contents of Shenge Bushen Capsules-1, add 1 ml of water, shake to dissolve, filter, and use the filtrate as the test solution. Separately, take 5 g of Codonopsis pilosula reference material, add 50 ml of water, heat at 90℃ for 1 hour, filter, and use the filtrate as the reference material solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), take 1 μl of each of the above two solutions, and spot them separately on the same silica gel G thin-layer plate-1. Repeat the spotting process until 10 spotted silica gel G thin-layer plates are obtained. Develop each silica gel G thin-layer plate with a mixed solvent of 75% isopropanol and anhydrous ethanol in a volume ratio of 9:1 as the developing solvent. Remove the plates, air dry, spray with the mixed solvent shown in Table 2 as the colorimetric reagent, heat at 105℃ until the spots are clearly visible, and examine under sunlight. In the chromatogram of the test sample, observe the spots at the corresponding positions as in the chromatogram of the reference medicinal material. The results are as follows: Figure 1 , Figures 15 to 23 As shown, Figure 1 , Figures 15 to 23 The sampling order from left to right is as follows: 1. Shen Ge Bu Shen Capsule-1, spot sampling; 2. Codonopsis pilosula reference material, spot sampling; 3. Shen Ge Bu Shen Capsule-1, strip sampling; 4. Codonopsis pilosula reference material, strip sampling.

[0140] Table 2. Comparison of different colorimetric reagents used in the identification of Codonopsis pilosula.

[0141]

[0142]

[0143] Depend on Figure 1 It can be seen that the chromatogram of the test sample contains abundant fluorescent spot information, the separation effect of each component is good, and fluorescent spots of the same color are displayed at the corresponding positions as those of the reference medicinal material.

[0144] Depend on Figure 15 It can be seen that the chromatogram of the test sample shows rich information from the fluorescent spots, good separation of each component, and fluorescent spots of the same color at the corresponding positions as those in the chromatogram of the reference medicinal material, but the color development effect is slightly worse. Figure 1 .

[0145] Depend on Figure 16It can be seen that the chromatogram of the test sample shows rich information from the fluorescent spots, good separation of each component, and fluorescent spots of the same color at the corresponding positions as those in the chromatogram of the reference medicinal material, but the color development effect is slightly worse. Figure 1 .

[0146] Depend on Figure 17 It can be seen that the chromatogram of the test sample contains abundant fluorescent spot information, the separation effect of each component is good, and fluorescent spots of the same color are displayed at the corresponding positions as those of the reference medicinal material.

[0147] Depend on Figure 18 It can be seen that the chromatogram of the test sample contains abundant fluorescent spot information, the separation effect of each component is good, and fluorescent spots of the same color are displayed at the corresponding positions as those of the reference medicinal material.

[0148] Depend on Figure 19 It can be seen that the chromatogram of the test sample contains abundant fluorescent spot information, the separation effect of each component is good, and fluorescent spots of the same color are displayed at the corresponding positions as those of the reference medicinal material.

[0149] Depend on Figure 20 It can be seen that the chromatogram of the test sample shows rich information from the fluorescent spots, good separation of each component, and fluorescent spots of the same color at the corresponding positions as those in the chromatogram of the reference medicinal material, but the color development effect is slightly worse. Figure 1 .

[0150] Depend on Figure 21 It can be seen that the chromatogram of the test sample contains abundant fluorescent spot information, the separation effect of each component is good, and fluorescent spots of the same color are displayed at the corresponding positions as those of the reference medicinal material.

[0151] Depend on Figure 22 It can be seen that the chromatogram of the test sample contains abundant fluorescent spot information, the separation effect of each component is good, and fluorescent spots of the same color are displayed at the corresponding positions as those of the reference medicinal material.

[0152] Depend on Figure 23 It can be seen that the chromatogram of the test sample has little fluorescent spot information. Although there are obvious spots in the chromatogram, the separation effect is poor, the Rf value cannot meet the requirements, and no fluorescent spots of the same color are shown at the corresponding positions as those of the reference medicinal material.

[0153] Example 3 Methodological Investigation

[0154] (1) Specificity experiment

[0155] Take 40 mg of the contents of three different batches of Shen Ge Bu Shen Capsules-1, Shen Ge Bu Shen Capsules-2, and Shen Ge Bu Shen Capsules-3, add 1 ml of water, shake to dissolve, filter, and use the filtrate as the test solution. Separately, take 5 g of Codonopsis pilosula reference material, add 50 ml of water, heat at 90℃ for 1 hour, filter, and use the filtrate as the reference material solution. According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 1 μl of each of the two solutions mentioned above was applied separately to the same silica gel G thin-layer plate-1. This process was repeated until three silica gel G thin-layer plates were obtained. Each plate was developed using a mixed solvent of 75% isopropanol and anhydrous ethanol in a volume ratio of 9:1. After development, the plates were removed, 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 the developing agent. The plates were heated at 105°C until the spots were clearly visible and examined under sunlight. The results are as follows. Figure 24 As shown, Figure 24 Sampling order from left to right: 1. Shen Ge Bu Shen Capsules-1, strip sampling; 2. Shen Ge Bu Shen Capsules-2, strip sampling; 3. Shen Ge Bu Shen Capsules-3, strip sampling; 4. Codonopsis pilosula reference material, strip sampling.

[0156] The results show that: Figure 24 The chromatogram of the test sample shows abundant fluorescent spot information, good separation of each component, and spots of the same color at the corresponding positions as those of the reference medicinal material, indicating that the thin-layer chromatography detection method of Codonopsis pilosula in the ginseng and kudzu kidney-tonifying preparation provided by the present invention has strong specificity.

[0157] (2) Test of the durability of thin-layer plates

[0158] Take 40 mg of the contents of each of the following capsules (Shen Ge Bu Shen Capsules-1, Shen Ge Bu Shen Capsules-2, and Shen Ge Bu Shen Capsules-3), add 1 ml of water, shake to dissolve, filter, and use the filtrate as the test solution. Separately, take 5 g of Codonopsis pilosula (Taizishen) reference material, add 50 ml of water, heat at 90℃ for 1 hour, filter, and use the filtrate as the reference material solution. According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 1 μl of each of the above two solutions was applied separately to the same silica gel G thin-layer plate-1, and then applied to the same silica gel G thin-layer plate-2 in the same manner. This process was repeated until two silica gel G thin-layer plates were obtained. Each silica gel G thin-layer plate was developed using a mixed solvent of 75% isopropanol and anhydrous ethanol in a volume ratio of 9:1 as the developing solvent. The plates were removed, 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 the colorimetric reagent. The plates were heated at 105°C until the spots were clearly visible and examined under sunlight. The results are as follows: Figure 25 As shown, Figure 25The sampling order from left to right is as follows: 1. Shen Ge Bu Shen Capsules-1, dotted sampling; 2. Shen Ge Bu Shen Capsules-2, dotted sampling; 3. Shen Ge Bu Shen Capsules-3, dotted sampling; 4. Codonopsis pilosula reference material, dotted sampling; 5. Codonopsis pilosula reference material, dotted sampling; 6. Shen Ge Bu Shen Capsules-1, dotted sampling; 7. Shen Ge Bu Shen Capsules-2, dotted sampling; 8. Shen Ge Bu Shen Capsules-3, dotted sampling; and... Figure 25 The chromatographic results of silica gel G thin-layer plate-1 and silica gel G thin-layer plate-2 were spliced ​​together. Spots 1, 2, 3 and 4 were on silica gel G thin-layer plate-1, and spots 5, 6, 7 and 8 were on silica gel G thin-layer plate-2.

[0159] The results show that: Figure 25 In the chromatogram of the test sample shown, spots of the same color appear at the corresponding positions as in the chromatogram of the reference 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 Codonopsis pilosula in the ginseng and kudzu kidney-tonifying preparation of the present invention.

[0160] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0161] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A thin-layer chromatography method for detecting Codonopsis pilosula in a kidney-tonifying preparation, characterized in that, The raw materials of the kidney-tonifying preparation include Codonopsis pilosula, Pueraria lobata and Epimedium; The thin-layer chromatography detection method for Codonopsis pilosula in the aforementioned kidney-tonifying preparation includes the following steps: Prepare the test solution and the reference herb solution of Codonopsis pilosula separately; Spot the test solution and the Codonopsis pilosula reference herb solution onto the same thin-layer chromatography plate, develop them in the developing solvent, add a colorimetric reagent for color development, and examine them. The developing agent is a mixed solvent consisting of isopropanol and ethanol with a volume fraction of 75% in a volume ratio of (1-9):1; The colorimetric reagent is a mixed solution of aniline, diphenylamine, phosphoric acid, and acetone; The preparation method of the test solution includes the following steps: The test sample of the ginseng and kudzu kidney-tonifying preparation was mixed with water for the first extraction to obtain the extract, and the test sample solution was prepared.

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

1.

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

1.

4. The thin-layer chromatography detection method according to claim 1, characterized in that, After adding the colorimetric reagent, the thin-layer chromatography plate needs to be heated at a temperature of 100℃~110℃.

5. The thin-layer chromatography detection method according to claim 1, characterized in that, The light source used was determined to be a fluorescent lamp.

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

7. The thin-layer chromatography detection method according to any one of claims 1 to 5, characterized in that, The preparation method of the Codonopsis pilosula reference herb solution includes the following steps: The reference herb, *Codonopsis pilosula*, was mixed with water for a second extraction to obtain an extract, which was then used to prepare the *Codonopsis pilosula* reference herb solution.

8. The thin-layer chromatography detection method according to claim 7, characterized in that, Satisfy one or more of the following (1) to (2): (1) The second extraction method is heating extraction, with a temperature of 80℃~100℃ and a time of 0.5h~2h; (2) In the above-mentioned Codonopsis pilosula reference medicinal material solution, the mass-volume ratio of Codonopsis pilosula to water is (1-100) mg: 1 ml.

9. The thin-layer chromatography detection method according to any one of claims 1 to 4, characterized in that, Satisfy one or more of the following (1) to (2): (1) By weight, the raw materials of the ginseng and kudzu kidney-tonifying preparation include 1,000 to 2,000 parts of Codonopsis pilosula, 500 to 1,500 parts of kudzu root and 100 to 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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