Thin layer chromatography identification method of Sophora japonica formula granules

Abstract

The present invention provides a thin-layer chromatography identification method for Sophora japonica formula granules, the method comprising the steps of preparing a test sample solution, preparing a control medicinal material solution, preparing a reference substance solution, and thin-layer chromatography identification; the test sample solution, the control medicinal material solution, and the reference substance solution are obtained by a simple and quick preparation method, and are spotted on the same thin-layer chromatography plate respectively. After development, the plates are inspected under ultraviolet light to obtain a multi-information thin-layer chromatography diagram of the Sophora japonica formula granules. The method uses the reference medicinal materials and rutin reference substance as reference substances, has strong specificity, is suitable for production practice, and provides a basis for establishing quality control standards for the Sophora japonica formula granules.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a thin-layer identification method for sophora japonica formula granules. Background Art

[0002] Sophora japonica (Flos Sophora Immaturus) is the dried, unopened flower bud of the Sophora japonica L. plant in the Leguminosae family. As a traditional Chinese medicinal ingredient, both a food and a medicine, Sophora japonica has a long history of use and is often used to treat hemorrhoids, bloody stools, hematuria, hematemesis, jaundice, uterine bleeding, arteriosclerosis, headaches, hypertension, dysentery, dizziness, and pyoderma. Research on the chemical composition of Sophora japonica began in the early 20th century, revealing its diverse composition, including flavonoids, phytosterols, tannins, proteins, amino acids, olefinic acids, and trace elements. These ingredients possess antioxidant, antibacterial, anti-inflammatory properties, inhibit tumor cell proliferation, and regulate glucose and lipid metabolism.

[0003] Chinese herbal formula granules are a breakthrough in the application of traditional Chinese herbal medicine slices. They are usually made from slices of medicine, and are made through standardized extraction (usually water extraction), concentration, drying and granulation. Their nature, flavor, meridians and efficacy are basically consistent with the original slices of medicine. They also have the advantages of not requiring decoction, easy adjustment, storage and convenient use, and have gradually been widely used.

[0004] In the 2020 edition of the Chinese Pharmacopoeia, under the item of Sophora japonica, a thin layer identification project is only established for Sophora japonica medicinal materials. Specifically, silica gel G thin layer plate is used, ethyl acetate-formic acid-water (8:1:1) is used as the developing agent, and aluminum chloride test solution is used as the color developer for thin layer identification; however, when this method is used for thin layer identification of Sophora japonica formula granules, there are problems such as fewer spots, darker color and poor separation; Reference 1 discloses a qualitative identification method of Sophora japonica thin layer chromatography, which is based on the thin layer chromatography method recorded in the pharmacopoeia method. After development with a developing agent, the sample on the silica gel G thin layer plate is fumigated in iodine vapor for 1 minute and then taken out. When using this method, although the yellow spots of rutin and quercetin can remain stable for 24 hours, the other spots gradually become lighter in color as the iodine evaporates, and they need to be re-fumigated with iodine before they can be colored, and this method There are also cases where there are few spots and the separation is poor; Reference 2 discloses a qualitative identification method for Sophora japonica formula granules, which uses toluene-formic acid-acetic acid-formic acid (5:2:0.5) as the developing agent on the basis of the pharmacopoeia method ethyl acetate-formic acid-water (8:1:1), and the two developing operations are relatively complicated, and the result of this method shows that rutin tailing is more serious; Reference 3 discloses a microemulsion thin-layer chromatography separation and identification method for flavonoid components in Sophora japonica seeds, which uses a microemulsion system composed of sodium dodecyl sulfate-n-butanol-n-hexane-water as the developing agent and formic acid in a certain proportion. Although this method has a good separation effect, its developing agent preparation process is cumbersome and the preparation time is long (needs to stand for 24 hours). In addition, its sample processing method is also relatively complicated (requires multiple reflux and extraction), which is not conducive to its application in actual production.

[0005] Although the prior art has explored different degrees of qualitative identification methods for the flower buds (sophora japonica flowers and sophora japonica seeds) of different forms of the legume plant Sophora japonica, most of them adopt the methods recorded in the pharmacopoeia or improve on them. Since the properties and identification characteristics of the original Chinese herbal medicine slices are lost after they are made into Chinese herbal formula granules, the methods for Sophora japonica seeds in the pharmacopoeia are no longer applicable to Sophora japonica seeds formula granules. Therefore, it is an important topic to establish a fast, simple and low-cost thin-layer identification method for Sophora japonica seeds formula granules, which provides a basis for establishing the quality control standards of Sophora japonica seeds formula granules.

[0006] References:

[0007] Reference 1: Jiang Hong et al. Qualitative identification of Sophora japonica flowers by thin layer chromatography[J]. Journal of Pharmaceutical Practice. 2003, 21(3): 185-186

[0008] Reference 2: Zhai Xufeng et al., Study on the quality standard of Sophora japonica formula granules[J]. Chinese Traditional Patent Medicine. 2004, 26(12): 1007-1009

[0009] Reference 3: Zhao Huiru et al., Separation and identification of flavonoids from Sophora japonica seeds by microemulsion thin layer chromatography[J]. Chemical Technology. 2016, 24(4): 27-30 Summary of the Invention

[0010] Problems to be solved by the invention

[0011] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to establish a thin layer identification method for Sophora japonica formula granules, which is efficient, rapid, accurate, simple and easy to operate, has obvious identification characteristics, is easy to judge the results, has good specificity, and can better reflect the overall quality of the formula granules.

[0012] Solutions for solving problems

[0013] [1]. The present invention primarily provides a thin layer chromatography identification method for Sophora japonica formula granules, wherein the method comprises the preparation of a test solution, the preparation of a reference medicinal material solution, the preparation of a reference solution and thin layer chromatography identification,

[0014] The preparation of the test solution comprises taking the Sophora japonica granules, dissolving them in water, extracting them with an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue with an alcohol solvent;

[0015] The preparation of the control medicinal material solution comprises taking a control medicinal material of Sophora japonica, adding water to reflux extraction, filtering, extracting the filtrate with an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue with an alcohol solvent;

[0016] The thin layer identification comprises taking a test sample solution, a reference medicinal material solution and a reference substance solution, spotting them on a polyamide thin layer chromatography plate respectively, developing them with n-butanol-acetone-glacial acetic acid-water as a developing agent, and inspecting them.

[0017] [2]. According to the thin layer identification method described in [1], the preparation of the reference solution includes taking a rutin reference and dissolving it in an alcohol solvent; preferably, the thin layer identification method also includes the preparation of a negative control solution, and the preparation of the negative control solution includes taking negative control formula granules without Sophora japonica seeds, and the remaining steps refer to the preparation method of the test solution.

[0018] [3] The thin layer identification method according to [1] or [2], wherein, in the preparation of the test solution, the ratio of the sophora japonica granules to water is 1 g: (150-350) mL; the organic solvent is selected from one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate; the extraction is performed twice or more, and the volume ratio of the organic solvent to water used in each extraction is (1-2): (1-1.5); the alcohol solvent is methanol or a methanol solution.

[0019] [4] The thin-layer identification method according to any one of the technical solutions [1] to [3], wherein, in the preparation of the control medicinal material solution, the ratio of the reference medicinal material Sophora japonica to water is 1 g: (200-400) mL; the reflux extraction time is 20-40 min; the organic solvent is selected from one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate; the number of extractions is two or more, and the volume ratio of the organic solvent to water used in each extraction is (1-1.5): (1-2); the alcohol solvent is methanol or a methanol solution.

[0020] [5]. The thin layer chromatography identification method according to [2], wherein, in the preparation of the reference solution, the ratio of the rutin reference substance to the alcohol solvent is (3-6) mg:1 mL; the alcohol solvent is methanol or an aqueous solution of methanol; and in the thin layer chromatography identification, the spotting volume of the reference solution is 1-3 μL.

[0021] [6]. The thin layer identification method according to any one of the technical solutions [1] to [5], wherein, in the spotting operation of the thin layer identification, the samples are spotted on the same polyamide thin layer chromatography plate, the spotting volume of the test solution is 1 to 10 μL, and the spotting volume of the control medicinal material solution is 1 to 10 μL; the inspection is performed under ultraviolet light with a wavelength of 365 nm.

[0022] [7]. The thin layer identification method according to any one of the technical solutions [1] to [6], wherein, in the thin layer identification, n-butanol-acetone-glacial acetic acid-water with a volume ratio of (1 to 4): (1 to 4): (0.5 to 2): (5 to 20) is used as the developing agent.

[0023] [8]. The thin layer identification method according to any one of the technical solutions [1] to [7], wherein the thin layer identification further comprises unfolding and drying, spraying with a color developer, heating until the spots are clearly colored, and then placing under ultraviolet light for inspection; preferably, the color developer is an aluminum chloride solution with a concentration of 1% to 5%.

[0024] [9] The thin-layer identification method according to any one of the technical solutions [1] to [8], wherein, in the thin-layer identification, the inspection includes comparing the chromatogram of the test sample with the chromatogram of the reference medicinal material and the chromatogram of the reference sample to determine whether the same fluorescent spots are displayed at one or more positions with the same Rf value.

[0025]

[10] . In addition, the present invention also provides an application of the thin layer identification method described in any one of the technical solutions [1] to [9] in identifying authentic and / or counterfeit Robinia pseudoacacia formula granules, wherein the counterfeit products include Robinia pseudoacacia formula granules and / or Albizia julibrissin formula granules.

[0026] Effects of the Invention

[0027] The thin-layer identification method for the sophora japonica formula granules provided by the present invention can well develop and separate compounds with greater polarity in the sophora japonica formula granules. After color development, the spots are rich, clear, and have high brightness, with good separation. The spots are within the Rf value range specified in the Chinese Pharmacopoeia, the identification characteristics are obvious, the results are easy to judge, and the specificity is good.

[0028] The invention can quickly identify the sophora japonica seed formula granules, improves the quality controllability of the identified objects, has high practical value, and the method is simple, fast, efficient and low in cost.

[0029] In addition, this method is easy to promote and apply. It is not only applicable to production practice, but also provides a basis for accurate clinical medication and a foundation for establishing quality control standards for Sophora japonica formula granules. This method can also be used to identify genuine and / or counterfeit Sophora japonica formula granules, among which counterfeit products include Robinia pseudoacacia formula granules, Albizia julibrissin formula granules, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art. The drawings described below are some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0031] Figure 1 The following are thin layer chromatography (TLC) patterns of the preparation methods of different test solutions and different control medicinal material solutions in Example 1 of the present invention (1-3: test solution preparation methods 1-3; S1-S2: control medicinal material solution preparation methods 1-2; T: rutin reference substance).

[0032] Figure 2 The figure shows a thin layer chromatography (TLC) spectrum using ethyl acetate-formic acid-water (8:1:1) as a developing solvent (1-3: Sophora japonica seed granules; S: Sophora japonica seed control medicinal material; T: Rutin control substance).

[0033] Figure 3 The graph shows a thin layer chromatography (TLC) spectrum using methanol-glacial acetic acid-water (8:1:1) as a developing solvent (1-3: Sophora japonica seed granules; S: Sophora japonica seed control medicinal material; T: Rutin control substance).

[0034] Figure 4 The figure shows the thin layer chromatography (TLC) spectrum using n-butanol-acetone-glacial acetic acid-water (2:1:1:10) as the developing solvent (1-3: Sophora japonica formula granules; S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0035] Figure 5 The figure shows the thin layer chromatography (TLC) spectrum using n-butanol-acetone-glacial acetic acid-water (2:1:1:5) as the developing solvent (1-3: Sophora japonica formula granules; S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0036] Figure 6 The figure shows the thin layer chromatography (TLC) spectrum using n-butanol-acetone-glacial acetic acid-water (2:1:1:10) as the developing solvent (1-3: Sophora japonica formula granules; S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0037] Figure 7 The figure shows the thin layer chromatography (TLC) spectrum using n-butanol-acetone-glacial acetic acid-water (2:1:1:15) as the developing solvent (1-3: Sophora japonica formula granules; S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0038] Figure 8 The thin layer chromatography (TLC) spectra using different sample spotting amounts are shown (1-4: the sample spotting amounts of the test solution are 1 μL, 2 μL, 3 μL, and 4 μL, respectively; 5-8: the sample spotting amounts of the control medicinal material solution are 1 μL, 2 μL, 3 μL, and 4 μL, respectively; T: 1 μL of rutin reference substance).

[0039] Figure 9 The thin layer chromatography (TLC) spectra at different development temperatures are shown (1-3: Sophora japonica formula granules; S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0040] Figure 10 The thin layer chromatography (TLC) spectra at different development relative humidities are shown (1-3: Sophora japonica formula granules; S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0041] Figure 11 The thin layer chromatography (TLC) patterns of the specificity test are shown (1-3: Sophora japonica formula granules; 4: negative control; S: Sophora japonica control medicinal material; T: rutin reference substance).

[0042] Figure 12 The thin layer chromatography (TLC) patterns of different batches of Sophora japonica granules in Example 2 are shown (1-3: Sophora japonica granules (batch numbers 20040109, 20040119, 20040129); S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0043] Figure 13 The TLC patterns of the Sophora japonica granules and their counterfeits in Example 3 are shown (1-3: Sophora japonica granules; 4-5: Robinia pseudoacacia granules; 6-8: Albizia julibrissin granules; S: Sophora japonica control medicinal material; T: Rutin control substance).

[0044] Figure 14 The thin layer analysis (TLC) patterns obtained by the thin layer identification method specified in the pharmacopoeia in Comparative Example 1 are shown (1 to 3: Sophora japonica formula granules (batch numbers 20040109, 20040119, 20040129); S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0045] Figure 15 The thin layer chromatography (TLC) patterns obtained in Comparative Example 2 are shown (1-3: Sophora japonica formula granules (batch numbers 20040109, 20040119, 20040129); S: Sophora japonica reference medicinal material; T1: rutin reference substance; T2: quercetin reference substance).

[0046] Figure 16 The thin layer chromatography (TLC) patterns obtained in Comparative Example 3 are shown (1-3: Sophora japonica formula granules (batch numbers 20040109, 20040119, 20040129); S: Sophora japonica reference medicinal material; T1: rutin reference substance; T2: quercetin reference substance). DETAILED DESCRIPTION

[0047] To better illustrate the present invention, numerous specific details are provided in the following detailed description. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0048] Unless defined otherwise, 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 belongs.

[0049] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0050] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0051] It will be understood that the singular article "a," "an," and "the," as used in the specification and claims of this application, includes plural referents unless the context clearly dictates otherwise.

[0052] In this specification, references to "one or some specific / preferred embodiments / solutions," "another or other specific / preferred embodiments / solutions," "one or another embodiment / solution," "one or another technical solution," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiment are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any appropriate manner.

[0053] The term "comprises" and any variations thereof in the specification and claims of the present invention and the accompanying drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements, but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0054] Unless otherwise specified, the term "herbal medicine" used in this article refers to unprocessed or unfinished Chinese medicine raw materials; "control medicinal materials" (or "reference medicinal materials") refer to standard medicinal materials that have completed species identification and are used to identify the samples to be tested.

[0055] Unless otherwise specified, the term "test article" used herein refers to an experimental sample used for detection or identification.

[0056] Unless otherwise specified, the term "thin layer chromatography" used in this article is a trace, rapid and simple separation and analysis method. A suitable stationary phase is coated on a glass plate, plastic or aluminum substrate to form a uniform thin layer. After the sample is spotted and developed, the relative shift value (Rf) is compared with the relative shift value of the chromatogram obtained by the same method with a suitable reference substance, and is used for drug identification, impurity inspection or content determination. This method is a very important experimental technique for rapid separation and qualitative analysis of small amounts of substances, and is also used to track the progress of reactions. It has the characteristics of short development time, strong separation ability and high sensitivity. Thin layer chromatography is one of the important inspection items for traditional Chinese medicines. By comparing the chromatographic behavior of the test sample and the reference medicinal materials and reference substances, the authenticity and quality of the medicinal materials can be judged.

[0057] Unless otherwise specified, the term "spotting" used herein refers to the process of adding drops of sample solution to be separated and identified onto a thin layer plate.

[0058] Unless otherwise specified, the term "development" used herein refers to the process in which the developing agent carries the sample components to migrate a certain distance on the thin layer plate by the capillary action of the stationary phase on the thin layer plate.

[0059] The present invention provides a thin-layer chromatography identification method for Sophora japonica formula granules, the method comprising the steps of preparing a test sample solution, preparing a control medicinal material solution, preparing a reference substance solution, and thin-layer chromatography identification; the test sample solution, the control medicinal material solution, and the reference substance solution are obtained by a simple and quick preparation method, and are spotted on the same thin-layer chromatography plate respectively. After development, the plates are inspected under ultraviolet light to obtain a multi-information thin-layer chromatography diagram of the Sophora japonica formula granules. The method uses the reference medicinal materials and rutin reference substance as reference substances, has strong specificity, is suitable for production practice, and provides a basis for establishing quality control standards for the Sophora japonica formula granules.

[0060] <Preparation of test solution>

[0061] The inventors investigated the preparation methods of the test solution and compared the final extraction results, and finally determined the preparation method of the test solution, which specifically includes taking the Sophora japonica formula granules, dissolving them in water, extracting them using an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue in an alcohol solvent.

[0062] In some specific embodiments of the present invention, the sophora japonica seed granules are crushed and ground into a powder, sieved, and then dissolved in water. In the present invention, using water as the extraction solvent can maximize the extraction of water-soluble components such as flavonoids. The present invention does not particularly limit the amount of water used for extraction. To ensure extraction efficiency and better extract the components to be identified, the ratio of the sophora japonica seed granules to water can be 1g: (150-350)mL; preferably 1g: (175-300)mL; and more preferably 1g: 200mL.

[0063] After dissolving in water, an organic solvent is used for extraction. Compared with extraction methods such as ultrasonic treatment, shaking extraction is simpler and easier. The organic solvent used can be one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate. In some specific embodiments of the present invention, ethyl acetate is preferred. In order to make the extraction more complete and the extraction effect better, the number of extractions is more than two times, and the volume ratio of the organic solvent to water used in each extraction is (1-2): (1-1.5); preferably 1:1. The ethyl acetate liquid is then combined and concentrated to dryness. The present invention has no particular restrictions on the method of concentration to dryness. In some specific embodiments of the present invention, it can be carried out by vacuum distillation and drying. After evaporation, an alcohol solvent is added to the residue. In some specific embodiments of the present invention, the alcohol solvent is selected from methanol, an aqueous solution of methanol, or ethanol, an aqueous solution of ethanol, etc., preferably methanol or an aqueous solution of methanol. Considering solubility, methanol is more preferred. From the perspective of dissolution efficiency, in some specific embodiments of the present invention, the ratio of the sophora japonica rice formula granules to the alcohol solvent is (0.05-0.2) g: (1-2) mL, preferably (0.05-0.15) g: (0.8-1.5) mL, and more preferably 0.1 g: 1 mL.

[0064] <Preparation of Control Medicinal Material Solution>

[0065] The preparation of the control medicinal material solution of the present invention comprises taking a control medicinal material of Sophora japonica, adding water for reflux extraction, filtering, extracting the filtrate with an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue with an alcohol solvent.

[0066] In some specific embodiments of the present invention, the method of adding water and refluxing is selected to treat the Sophora japonica reference medicinal material because water extraction is closer to the traditional decoction method of Chinese medicine than alcohol extraction (methanol, ethanol, etc.), and can fully extract the water-soluble components in the medicinal material and better characterize the clinical quality of the medicinal material; the present invention has no particular restriction on the amount of water used for water reflux extraction. In order to ensure the efficiency of the extraction and better extract the components to be identified, the ratio of the Sophora japonica reference medicinal material to water can be 1g: (200~400)mL; preferably 1g: (220~350)mL; more preferably 1g: 250mL.

[0067] To facilitate subsequent extraction, it is preferred to add water, heat under reflux, extract, cool, and then filter. The present invention does not specifically limit the filtration method; any conventional filtration method in the art, such as filter paper or cotton filtration, can be used. After filtration, the filtrate is extracted with an organic solvent. The organic solvent used can be one or more of ether, dichloromethane, chloroform, carbon tetrachloride, or ethyl acetate. In some specific embodiments of the present invention, ethyl acetate is preferred. To ensure more complete extraction and better extraction results, the extraction is performed two or more times, and the volume ratio of organic solvent to water used in each extraction is (1-1.5):(1-2); preferably 1:1.25. The ethyl acetate solutions are then combined and concentrated to dryness. The present invention does not specifically limit the method of concentration to dryness. In some specific embodiments of the present invention, the filtration can be carried out by vacuum distillation. After evaporation to dryness, an alcoholic solvent is added to the residue. In some specific embodiments of the present invention, the alcoholic solvent is selected from methanol, an aqueous solution of methanol, or ethanol, an aqueous solution of ethanol, etc., preferably methanol or an aqueous solution of methanol. Methanol or an aqueous solution of methanol is more preferred due to solubility. From the perspective of dissolution efficiency, in some specific embodiments of the present invention, the ratio of the reference medicinal material Sophora japonica to the alcohol solvent is (0.05-0.2) g: (1-2) mL, preferably (0.05-0.15) g: (0.8-1.5) mL, and more preferably 0.1 g: 1 mL.

[0068] <Preparation of Reference Solution>

[0069] The preparation of the reference solution of the present invention comprises taking a rutin reference substance and dissolving it in an alcohol solvent. In some specific embodiments of the present invention, the amount ratio of the rutin reference substance to the alcohol solvent is (3-6) mg:1 mL, preferably (3.5-5.5) mg:1 mL, and more preferably 4 mg:1 mL. In some specific embodiments of the present invention, the alcohol solvent is selected from methanol, an aqueous solution of methanol, or ethanol, an aqueous solution of ethanol, etc., preferably methanol or an aqueous solution of methanol. Considering solubility, methanol is more preferred.

[0070] In addition, in some preferred embodiments of the present invention, the thin layer identification method of the present invention also includes the preparation of a negative control solution. The preparation of the negative control solution of the present invention includes taking negative control formula particles that do not contain Sophora japonica seeds. The remaining preparation methods refer to the above <Preparation of the test solution>.

[0071] <Thin layer identification step>

[0072] The thin layer identification step of the present invention comprises taking a test sample solution, a control medicinal material solution and a reference substance solution, spotting them on a polyamide thin layer chromatography plate respectively, developing them with n-butanol-acetone-glacial acetic acid-water as a developing agent, drying them after development, spraying them with a color developer, heating them until the spots are clearly colored, and inspecting them under ultraviolet light.

[0073] In some specific embodiments of the present invention, the present invention has no particular restrictions on the material of the thin layer plate, and silica gel thin layer plate, polyamide thin layer plate, ordinary prefabricated plate, high-efficiency prefabricated plate, etc. can be selected. Since the sophora japonica granules of the present invention contain flavonoids and other water-soluble components with high polarity, in order to fully separate them, polyamide thin layer chromatography plates are preferred; further, in order to facilitate inspection and reduce the differences caused by the stationary phase, it is preferred to take the test solution, the control medicinal material solution and the reference substance solution, and spot them on the same polyamide thin layer chromatography plate respectively; in some specific embodiments of the present invention, Wherein, the spot amount of the test solution is 1-10 μL, preferably 2-6 μL, more preferably 3 μL; the spot amount of the control medicinal material solution is 1-10 μL, preferably 2-6 μL, more preferably 3 μL; the spot amount of the reference solution is 1-3 μL, preferably 1 μL; if the spot amount is too low, the spot separation is relatively poor, and the spots cannot be clearly displayed; the present invention uses n-butanol-acetone-glacial acetic acid-water as a developing agent for development, which is conducive to the good separation of various chemical components of the Sophora japonica formula granules and the Sophora japonica control medicinal material, especially the flavonoid components on the thin layer plate. In some preferred embodiments of the present invention, in order to obtain better separation, the developing agent of the present invention is preferably calculated by volume ratio, n-butanol: acetone: glacial acetic acid: water = (1-4): (1-4): (0.5-2): (5-20), further 2:2:1:10.

[0074] The present invention does not particularly limit the temperature during development, and can be carried out at 20-40°C or 3-8°C, and different temperatures have little effect on the separation effect. The present invention also does not particularly limit the relative humidity during development, and can be carried out at a relative humidity of 18% to 88%, and different relative humidities have little effect on the separation effect. In some specific embodiments of the present invention, the thin layer color developer is selected from aluminum chloride solution, and the concentration of the aluminum chloride solution is preferably 1% to 5%, more preferably 2% to 4%, and most preferably 3%, which can achieve better color development effect.

[0075] Afterwards, heating is performed. The present invention does not particularly limit the heating method and can be performed by conventional methods in the art, preferably by hot air drying. The heating temperature is 100-110°C, preferably 105°C. The present invention inspects under ultraviolet light (365nm). Compared with inspection under sunlight, the spots under ultraviolet light are clearer and more colorful. In some specific embodiments of the present invention, the chromatogram of the test sample is compared with the chromatogram of the reference medicinal material and the chromatogram of the reference substance to determine whether the same fluorescent spots are displayed at one or more positions with the same Rf value.

[0076] In other specific embodiments of the present invention, the thin-layer chromatography identification step of the present invention may further include spotting a test sample solution, a control medicinal material solution, a rutin reference solution, and a negative control solution onto a polyamide thin-layer chromatography plate, developing the plate with n-butanol-acetone-glacial acetic acid-water as a developing agent, drying the plate after development, spraying the plate with a color developer, heating the plate until the spots are clearly colored, and inspecting the plate under ultraviolet light. The results show that the test sample chromatogram displays the same fluorescent spots at corresponding positions in the control medicinal material chromatogram and the rutin reference chromatogram, while the negative control exhibits no interference.

[0077] By adopting the thin-layer identification method of the sophora japonica formula granules provided by the present invention, a chromatogram of the test sample with the same spots as the chromatogram of the reference medicinal material and the chromatogram of the rutin reference substance can be obtained. The separation and point characteristics in the chromatogram are good, the development time is short, the inspection is clear, and the quality of the sophora japonica formula granules can be better controlled and the authenticity thereof can be distinguished.

[0078] Example

[0079] The technical solutions of the present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to explain and illustrate the present invention, and are not intended to limit the scope of protection of the present invention.

[0080] <Instruments and reagents>

[0081] Instruments: thin layer automatic imaging instrument (CAMAH TLC VISUALIZER), one ten-thousandth balance (METTLERTOLEDO), KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Electronics Co., Ltd.), polyamide film (Luqiao Sijia Biochemical Plastic Factory, Taizhou City, Zhejiang Province).

[0082] Reagents: n-butanol (Sinopharm Chemical Reagent Co., Ltd.), acetone (Shanghai Lingfeng Chemical Reagent Co., Ltd.), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), glacial acetic acid (Sinopharm Chemical Reagent Co., Ltd.), methanol (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.) were all of analytical grade, and water;

[0083] Sophora japonica seed reference medicinal material (batch number: 121270-201403) and rutin reference substance (batch number: 100080-201610) were purchased from the China Food and Drug Inspection Institute; Sophora japonica seed formula granules (batch numbers: 20040109, 20040119, 20040129), Robinia pseudoacacia seed formula granules (batch numbers: 2205004, 2205005), and Albizia julibrissin seed formula granules (batch numbers: 2203001, 2203002, 2203003) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0084] Example 1: Construction of thin layer identification method for Sophora japonica granules

[0085] Investigation of the preparation methods of test solution and control medicinal material solution

[0086] <Preparation of test solution>

[0087] Method 1: Take 0.1 g of Sophora japonica granules (batch number: 20040119), grind it into powder, add 5 ml of methanol, ultrasonically treat it for 20 minutes, filter it, and use the filtrate as the test solution.

[0088] Method 2: Take 0.1 g of Sophora japonica granules (batch number: 20040119), grind it into powder, add 5 ml of ethanol, ultrasonically treat it for 20 minutes, filter it, and use the filtrate as the test solution.

[0089] Method 3: Take 0.1 g of Sophora japonica granules (batch number: 20040119), grind it into powder, add 20 ml of water to dissolve it, shake and extract it with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the test solution.

[0090] <Preparation of Control Medicinal Material Solution>

[0091] Method 1: Take 0.1 g of Sophora japonica seed reference medicinal material, add 5 ml of ethyl acetate, ultrasonically treat for 20 minutes, filter, and use the filtrate as the reference medicinal material solution.

[0092] Method 2: Take 0.1 g of Sophora japonica reference medicinal material, add 25 ml of water, heat and reflux for 30 minutes, cool, filter, and extract the filtrate by shaking with ethyl acetate twice, 20 ml each time, combine the ethyl acetate liquid, evaporate to dryness, and dissolve the residue in 1 ml of methanol as the reference medicinal material solution.

[0093] <Preparation of Reference Solution>

[0094] Take the rutin reference substance and add methanol to make a solution containing 4 mg per 1 ml as the reference solution.

[0095] <Preparation of Negative Control Solution>

[0096] Take 0.1 g of the negative sample lacking Sophora japonica seeds and prepare a negative control solution using the same method as in "Method 3 in <Preparation of Test Solution>".

[0097] <Thin layer identification>

[0098] The test sample solutions prepared according to the above three methods and the control medicinal material solutions and reference substance solutions prepared according to the above two methods were respectively aspirated and spotted on the same polyamide film. The film was developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) at a temperature of 20.4°C and a relative humidity of 66%. The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results were as follows: Figure 1.

[0099] The results showed that the test solution prepared by method three had clear spots and better separation effect; the control medicinal material Sophora japonica (method two) was more consistent with the granular spots.

[0100] Investigation of thin layer identification method

[0101] <Study of Developing Agent>

[0102] In order to determine the effect of different developing agents on spot separation, Sophora japonica granules (batch number: 20040119) and Sophora japonica control medicinal materials were taken respectively according to the above-mentioned test solution preparation method 3 and control medicinal material solution preparation method 2 to prepare test solution and control medicinal material solution, and the reference solution was prepared at the same time. 3 μL of the test solution and control medicinal material solution and 1 μL of the reference solution were spotted on the same polyamide film. The developing agents used were ethyl acetate-formic acid-water (8:1:1), methanol-glacial acetic acid-water (8:1:1), and n-butanol-acetone-glacial acetic acid-water (2:1:1:10), respectively. The membranes were developed at a temperature of 20.4°C and a relative humidity of 66%. The membranes were taken out, dried, sprayed with 3% aluminum chloride solution, blown dry with hot air, and examined under an ultraviolet lamp (365 nm). The results were as follows: Figure 2 、 3 , as shown in 4.

[0103] The results showed that using n-butanol-acetone-glacial acetic acid-water (2:2:1:10) as the developing agent resulted in clear spots and good separation effect.

[0104] In order to determine the effect of different ratios of developing agents on spot separation, Sophora japonica granules (batch number: 20040119) and Sophora japonica control medicinal materials were taken respectively and prepared into test solution and control medicinal material solution according to the above-mentioned test solution preparation method 3 and control medicinal material solution preparation method 2, and the reference solution was prepared at the same time. 3 μL of the test solution and the control medicinal material solution and 1 μL of the reference solution were spotted on the same polyamide film. The developing agent was n-butanol-acetone-glacial acetic acid-water with a ratio of 2:2:1:5, 2:2:1:10, and 2:2:1:15, respectively. The film was developed at a temperature of 20.4°C and a relative humidity of 66%. The film was taken out, dried, sprayed with 3% aluminum chloride solution, blown dry with hot air, and examined under an ultraviolet lamp (365 nm). The results were as follows: Figure 5 、 6 , as shown in 7.

[0105] The results showed that using n-butanol-acetone-glacial acetic acid-water (2:2:1:10) as the developing solvent, the spot Rf value was moderate and the separation effect was good.

[0106] <Inspection of Sample Quantity>

[0107] In order to compare the effect of different spot amounts on the separation of spots, Sophora japonica granules (batch number: 20040119) and Sophora japonica control medicinal materials were taken respectively according to the above-mentioned test solution preparation method 3 and control medicinal material solution preparation method 2 to prepare test solution and control medicinal material solution, and the reference solution was prepared at the same time. 1μL, 2μL, 3μL and 4μL of the test solution and the control medicinal material solution were spotted, respectively, and 1μL of the reference solution was spotted on the same polyamide film. It was developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) as the developing agent at a temperature of 20.4℃ and a relative humidity of 66%. It was taken out, dried, sprayed with 3% aluminum chloride solution, blown dry with hot air, and examined under an ultraviolet lamp (365nm). The results are as follows Figure 8 .

[0108] The results showed that when the sample solution and the control medicinal material solution were spotted in an amount of 3 μL and the control solution was spotted in an amount of 1 μL, the spots in the test sample chromatogram clearly corresponded to the spots in the control medicinal material chromatogram and the control substance chromatogram, without any other interference.

[0109] <Expansion of Temperature>

[0110] Sophora japonica granules (batch number: 20040119) and Sophora japonica control medicinal materials were respectively prepared according to the above-mentioned test solution preparation method 3 and control medicinal material solution preparation method 2 to prepare the test solution and control medicinal material solution. At the same time, the reference solution was prepared. 3 μL of the test solution and the control medicinal material solution and 1 μL of the reference solution were spotted on the same polyamide film. The film was developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) as the developing agent at high temperature (35°C) and low temperature (4°C) and relative humidity of 66%. The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results are as follows: Figure 9 , wherein the left side is the chromatogram obtained when the temperature is developed at 35°C, and the right side is the chromatogram obtained when the temperature is developed at 4°C.

[0111] The results showed that under different temperature conditions, the chromatograms of the Sophora japonica granules showed the same fluorescent spots at the corresponding positions in the chromatograms of the test herbs and the reference substances, and the separation effect was good. The experimental results showed that temperature had no effect on the thin-layer chromatography identification of Sophora japonica granules, indicating that the thin-layer chromatography identification method has good durability at different temperatures.

[0112] <Expanding the investigation of relative humidity>

[0113] Sophora japonica granules (batch number: 20040119) and Sophora japonica control medicinal materials were respectively prepared according to the above-mentioned test solution preparation method 3 and control medicinal material solution preparation method 2 to prepare the test solution and control medicinal material solution. At the same time, the reference solution was prepared. 3 μL of the test solution and the control medicinal material solution and 1 μL of the reference solution were spotted on the same polyamide film. The film was developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) as the developing solvent at 20.4°C but under different relative humidity conditions (18% and 88%). The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results are as follows: Figure 10 , where the left side is the chromatogram obtained when the temperature is 20.4°C and the relative humidity is 18%, and the right side is the chromatogram obtained when the temperature is 20.4°C and the relative humidity is 88%.

[0114] The results showed that under different relative humidity conditions, the chromatograms of the Sophora japonica granules showed the same fluorescent spots at the corresponding positions in the chromatograms of the test herb and the reference herb, and the separation effect was good. The experimental results show that relative humidity has no effect on the thin-layer chromatography identification of Sophora japonica granules, indicating that the thin-layer chromatography identification method is durable under different relative humidity conditions.

[0115] <Specificity test>

[0116] Sophora japonica granules (batch number: 20040119) and Sophora japonica control medicinal materials were taken separately and prepared into test solution and control medicinal material solution according to the above-mentioned test solution preparation method 3 and control medicinal material solution preparation method 2. At the same time, the reference solution and negative control solution were prepared. 3 μL of the test solution, control medicinal material solution and negative control solution and 1 μL of the reference solution were spotted on the same polyamide film. The film was developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) as the developing agent at 20.4°C and relative humidity of 66%. The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results were as follows: Figure 11 .

[0117] The results showed that the chromatograms of the Sophora japonica granules tested, the reference medicinal materials, and the reference substance showed fluorescent spots of the same color at the corresponding positions, and there was no interference from the negative control, indicating that the thin-layer identification method has good specificity.

[0118] <Determination of Thin Layer Identification Method>

[0119] According to the above investigation results, the thin layer identification method of Sophora japonica formula granules is determined as follows:

[0120] Grind 0.1g of Sophora japonica granules into powder, dissolve in 20ml of water, and shake extract twice with 20ml of ethyl acetate. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 0.1g of Sophora japonica control medicinal material, add 25ml of water, heat under reflux for 30 minutes, cool, filter, and add 20ml of ethyl acetate to the filtrate to prepare a control medicinal material solution in the same manner. Also, take a rutin reference substance and add methanol to a solution containing 4mg per 1ml. This will serve as the reference solution.

[0121] According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0502), 3 μL each of the test sample solution and the control medicinal material solution, and 1 μL of the reference solution, were spotted onto the same polyamide film. Developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) at 20.4°C and 66% relative humidity, the film was removed, air-dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365 nm). Fluorescent spots of the same color appeared in the test sample chromatogram at corresponding positions in the control medicinal material chromatogram and the reference substance chromatogram.

[0122] Example 2: Validation of the Thin Layer Chromatography Identification Method for Different Batches of Sophora japonica Granules

[0123] Different batches of Sophora japonica granules (20040109, 20040119, 20040129) and Sophora japonica control medicinal materials were taken and prepared into test solution and control medicinal material solution according to the above-mentioned test solution preparation method 3 and control medicinal material solution preparation method 2, respectively. At the same time, the reference solution was prepared, and 3 μL of the test solution and control medicinal material solution and 1 μL of the reference solution were spotted on the same polyamide film, respectively. The film was developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) as the developing agent at 20.4°C and relative humidity of 66%. The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results are as follows: Figure 12 .

[0124] The results showed that the chromatograms of different batches of Sophora japonica formula granules, the chromatograms of the control medicinal materials, and the chromatograms of the reference substance all showed fluorescent spots of the same color at corresponding positions, indicating that the thin-layer chromatography identification method of the present invention can well develop and separate the compounds in different batches of Sophora japonica formula granules. After color development, the spots are rich, clear, and have good separation, and the identification results are easy to judge.

[0125] Example 3: Identification of counterfeit Sophora japonica granules

[0126] Take 0.1g each of Sophora japonica bud granules, Robinia pseudoacacia bud granules, and Albizia julibrissin granules, grind them finely, dissolve in 20ml of water, and shake and extract twice with 20ml of ethyl acetate. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 0.1g of Sophora japonica bud control medicinal material, add 25ml of water, heat under reflux for 30 minutes, cool, filter, and add 20ml of ethyl acetate to the filtrate to prepare the control medicinal material solution in the same manner. Also, take the rutin reference substance and add methanol to a solution containing 4mg per 1ml. This will serve as the reference solution.

[0127] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Rules 0502), 3 μl of the test solution and the control medicinal material solution, and 1 μl of the reference solution were respectively spotted on the same polyamide film, and developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) at a temperature of 20.4°C and a relative humidity of 66%. The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365 nm). The results were as follows: Figure 13 shown.

[0128] Compared with Sophora japonica rice granules, Robinia japonica rice granules lacked blue fluorescent spots at around Rf values ​​of 0.4 and had multiple blue-green fluorescent spots at around Rf values ​​of 0.75. Therefore, this thin-layer chromatography method can distinguish Sophora japonica rice granules from Robinia japonica rice granules. Compared with Sophora japonica rice granules, Albizia julibrissin rice granules lacked blue fluorescent spots at around Rf values ​​of 0.25 and 0.4, and the fluorescent spots at the corresponding positions of the rutin control were weak or absent. Therefore, this thin-layer chromatography method can distinguish Sophora japonica rice granules from Albizia julibrissin rice granules.

[0129] Comparative Example 1: Thin layer identification of Sophora japonica medicinal materials specified in the Pharmacopoeia

[0130] Take 0.2g of Sophora japonica granules from different batches, grind them, add 5ml of methanol, seal them, shake them for 10 minutes, filter them, and take the filtrate as the test solution. Take another 0.2g of Sophora japonica reference medicinal material and prepare a reference medicinal material solution in the same way. Take the rutin reference substance and add methanol to make a solution containing 4mg per 1ml as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), 10μL of each of the above three solutions were taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-formic acid-water (8:1:1) was used as the developing agent. Develop, take out, dry, spray with aluminum chloride test solution, and after the ethanol evaporates, place it under ultraviolet light (365nm) for inspection. The results are as follows Figure 14 .

[0131] The results showed that in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material and the chromatogram of the reference sample, but there were fewer spots, the color was darker, and the separation was poor.

[0132] Comparative Example 2: The method in the prior art "Qualitative identification of Sophora japonica thin layer chromatography"

[0133] Take 0.2g of Sophora japonica granules from different batches, grind them into powder, add 10ml of methanol, ultrasonically treat for 10 minutes, filter, and take the filtrate as the test solution. Take another 0.2g of Sophora japonica reference medicinal material and prepare a reference medicinal material solution in the same way. Then take rutin reference substance and quercetin reference substance, add methanol to make solutions containing 2mg of each per 1ml, as reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), 2-10μL of each of the above three solutions were taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-formic acid-water (8:1:1) was used as the developing agent. Develop, take out, dry, fumigate in iodine vapor for 1 minute, and inspect under sunlight. The results are as follows: Figure 15 .

[0134] The results showed that in the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material and the chromatogram of the reference sample, but there were fewer spots and the separation was poor.

[0135] Comparative Example 3: Method in the prior art "Study on Quality Standards of Sophora japonica Formula Granules"

[0136] Grind 1g of Sophora japonica granules from different batches, add 20ml of methanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2ml of methanol to prepare the test solution. Separately, take 1g of Sophora japonica control medicinal material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, and add 20ml of methanol to the residue to prepare the control medicinal material solution in the same manner. Also, take the rutin and quercetin reference substances and add methanol to prepare solutions containing 1mg of each per 1ml to prepare the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0502), 2 μL of each of the three solutions was taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-formic acid-water (8:1:1) was used as the developing solvent, and the plate was extended to about 4 cm, taken out, and dried; then toluene-ethyl formate-formic acid (5:2:0.5) was used as the developing solvent, and the plate was extended to about 8 cm, taken out, and dried, sprayed with aluminum chloride ethanol solution, dried with hot air, and examined under ultraviolet light (365 nm). The results are as follows Figure 16 .

[0137] The results showed that in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the reference medicinal material and the reference substance, but the spots of the rutin reference substance had more serious tailing and poor separation.

[0138] The above examples are merely intended to illustrate several embodiments of the present invention. Although the descriptions thereof are relatively specific and detailed, they are not to be construed as limiting the scope of the present invention. It should be understood that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A thin layer detection method for Sophora japonica formula granules, characterized in that: The method includes the preparation of a test solution, the preparation of a reference medicinal material solution, the preparation of a reference substance solution and thin layer detection. The preparation of the test solution comprises taking the Sophora japonica granules, dissolving them in water, extracting them with ethyl acetate, separating the organic phase and concentrating it to dryness, and dissolving the residue in an alcohol solvent; The preparation of the control medicinal material solution comprises taking a control medicinal material of Sophora japonica, adding water to reflux extraction, filtering, extracting the filtrate with ethyl acetate, separating the organic phase and concentrating to dryness, and dissolving the residue with an alcohol solvent; The preparation of the reference solution comprises taking a rutin reference and dissolving it in an alcohol solvent; The thin layer chromatography test comprises taking a test sample solution, a control medicinal material solution and a reference substance solution, spotting them on a polyamide thin layer chromatography plate, developing them with n-butanol-acetone-glacial acetic acid-water in a volume ratio of 2:2:1:10 as a developing agent, and inspecting them. The thin layer detection further includes drying after unfolding, spraying with a color developer, heating until the spots are clearly colored, and then inspecting under ultraviolet light; the color developer is an aluminum chloride solution with a concentration of 1% to 5%.

2. The thin layer detection method according to claim 1, characterized in that: The thin layer detection method also includes the preparation of a negative control solution, which includes taking negative control formula particles that do not contain Sophora japonica seeds, and the remaining steps refer to the preparation method of the test solution.

3. The thin layer detection method according to claim 1 or 2, characterized in that: In the preparation of the test solution, the dosage ratio of the Sophora japonica granules to water is 1 g: (150~350) mL; the number of extractions is more than two times, and the volume ratio of the organic solvent to water used in each extraction is (1~2): (1~1.5); the alcohol solvent is methanol or an aqueous solution of methanol.

4. The thin layer detection method according to claim 1 or 2, characterized in that: In the preparation of the control medicinal material solution, the dosage ratio of the Sophora japonica control medicinal material to water is 1 g: (200~400) mL; the reflux extraction time is 20~40 min; the number of extractions is more than two times, and the volume ratio of the organic solvent to water used in each extraction is (1~1.5): (1~2); the alcohol solvent is methanol or an aqueous solution of methanol.

5. The thin layer detection method according to claim 1 or 2, characterized in that: In the preparation of the reference solution, the usage ratio of the rutin reference substance to the alcohol solvent is (3-6) mg:1 mL; the alcohol solvent is methanol or a methanol aqueous solution; in the thin layer detection, the spotting volume of the reference solution is 1-3 µL.

6. The thin layer detection method according to claim 1 or 2, characterized in that: In the spotting operation of the thin layer detection, the samples are spotted on the same polyamide thin layer chromatography plate, the spotting volume of the test solution is 1~10μL, and the spotting volume of the control medicinal material solution is 1~10μL; the inspection is carried out under an ultraviolet lamp with a wavelength of 365nm.

7. The thin layer detection method according to claim 1 or 2, characterized in that: In the thin layer test, the inspection includes comparing the chromatogram of the test sample with the chromatogram of the reference medicinal material and the chromatogram of the reference sample to determine whether the same fluorescent spots are displayed at one or more positions with the same Rf value.

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