Thin-layer identification method for sophora flower bud formula granules

Abstract

The invention provides a thin-layer identification method for sophora flower bud formula granules. The method comprises the steps of preparation of a test solution, preparation of a reference medicinal material solution, preparation of a reference substance solution and thin-layer identification, a test solution, a reference medicinal material solution and a reference substance solution are obtained by using a simple, convenient and rapid preparation method, are respectively applied on the same thin-layer chromatogram plate, and are inspected under an ultraviolet lamp after being expanded, so that a multi-information thin-layer chromatogram of the sophora flower bud formula granules can be obtained. According to the method, reference medicinal materials and rutin reference substances are adopted as reference substances, the specificity is high, the method is suitable for production practice, and a basis is provided for establishing the quality control standard of the sophora flower bud 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 bud (Flos Sophora Immaturus) is the unopened dried flower bud of the leguminous plant Sophora japonica L. As one of the traditional Chinese medicinal materials with the same origin of medicine and food, Sophora bud has a long history of medicinal use and is often used to treat hemorrhoids, blood in stool, hematuria, hematemesis, jaundice, uterine vascular bleeding, arteriosclerosis, headache, hypertension, dysentery, dizziness and pyoderma. In the early 20th century, the chemical composition of Sophora bud began to be studied, and it has been determined that it contains flavonoids, phytosterols, tannins, proteins, amino acids, enoic acids and trace elements, etc., with multiple functions such as anti-oxidation, anti-bacterial, anti-inflammatory, inhibition of tumor cell proliferation and regulation of 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 herbal medicine and are made through standardized extraction (usually mainly water extraction), concentration, drying and granulation. Their nature, flavor, meridians and efficacy are basically consistent with the original slices of herbal medicine. They also have the advantages of not requiring decocting, 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, a 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 this method is used, 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 poor separation; 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 of ethyl acetate-formic acid-water (8:1:1). The two development 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, the preparation process of the developing agent is cumbersome and the preparation time is long (needs to stand for 24 hours). In addition, the 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 flower buds (sophora japonica flowers and sophora japonica buds) of different forms of the leguminous plant Sophora japonica, most of them adopt the methods recorded in the pharmacopoeia or improve on them. Since the properties of the original Chinese medicine pieces are lost after they are made into Chinese medicine formula granules, the methods for Sophora japonica bud medicinal materials in the pharmacopoeia are no longer applicable to Sophora japonica bud formula granules. Therefore, it is an important topic to establish a fast, simple and low-cost thin layer identification method for Sophora japonica bud formula granules, which provides a basis for establishing the quality control standards of Sophora japonica bud formula granules.

[0006] References:

[0007] Reference 1: Jiang Hong et al., Qualitative identification of Sophora japonica 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 in Sophora japonica seed by microemulsion thin layer chromatography[J]. Chemical Technology. 2016, 24(4): 27-30 Summary of the invention

[0010] Problem that the invention aims to solve

[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 substance solution and thin layer chromatography identification,

[0014] The preparation of the test solution comprises taking the Sophora japonica bud granules, adding water to dissolve them, 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 for 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 comprises taking a rutin reference substance and dissolving it in an alcohol solvent; preferably, the thin layer identification method also comprises the preparation of a negative control solution, the preparation of the negative control solution comprises taking negative control formula granules that do not contain 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 amount of Sophora japonica formula 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 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.

[0019] [4]. The thin layer identification method according to any one of the technical solutions [1] to [3], wherein, in the preparation of the reference 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 extraction is performed twice 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 an aqueous solution of methanol.

[0020] [5]. The thin layer 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 identification, the spotting volume of the reference solution is 1-3 μL.

[0021] [6]. A thin layer chromatography identification method according to any one of the technical solutions [1] to [5], wherein, in the spotting operation of the thin layer chromatography 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; and the inspection is performed under ultraviolet light with a wavelength of 365 nm.

[0022] [7]. A 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 in a volume ratio of (1 to 4): (1 to 4): (0.5 to 2): (5 to 20) is used as a 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 then drying, spraying with a color developer, heating until the spots are clearly colored, and then inspecting under ultraviolet light; preferably, the color developer is an aluminum chloride solution with a concentration of 1% to 5%.

[0024] [9]. A 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 comprises Robinia pseudoacacia formula granules and / or Albizzia julibrissin formula granules.

[0026] Effects of the Invention

[0027] The thin-layer identification method of 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, and after color development, the spots are abundant, clear, and have high brightness, and the separation degree is good. The spots are within the Rf value range specified in the Chinese Pharmacopoeia, the identification characteristics are obvious, the results are easy to determine, and the specificity is good.

[0028] The invention can quickly identify the bud of Sophora japonica 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 seed formula granules. This method can also be used to identify authentic and / or counterfeit Sophora japonica seed formula granules, among which counterfeit products include Robinia japonica seed formula granules, Albizzia julibrissin seed formula granules, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the following is a brief introduction to the drawings required for the specific implementation or the prior art description. The drawings described below are some implementations of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

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

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

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

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

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

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

[0038] Figure 8 The thin layer analysis (TLC) spectra using different spotting amounts are shown (1-4: the spotting amounts of the test solution are 1 μL, 2 μL, 3 μL, and 4 μL, respectively; 5-8: the 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] Fig. 9 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] Fig.10 The thin layer chromatography (TLC) spectra of different development relative humidity are shown (1-3: Sophora japonica formula granules; S: Sophora japonica reference medicinal material; T: Rutin reference substance).

[0041] Fig.11 The thin layer analysis (TLC) spectrum of the specificity test is shown (1-3: Sophora japonica formula granules; 4: negative control; S: Sophora japonica control medicinal material; T: Rutin reference substance).

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

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

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

[0045] Fig.15 The thin layer analysis (TLC) spectrum obtained in Comparative Example 2 is 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] Fig.16 The thin layer analysis (TLC) spectrum obtained in Comparative Example 3 is 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] In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter 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 numerical values ​​A and B.

[0050] In this specification, the word "may" means both performing a certain process and not performing a certain process.

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

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

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

[0054] Unless otherwise specified, the term "drug material" used in this article refers to raw materials of traditional Chinese medicine that have not been processed or made into finished products; "control drug material" (or "reference drug material") refers to standard drug materials that have completed species identification and are used to identify the samples to be tested.

[0055] Unless otherwise specified, the term "test article" as 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 spotting and development, 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 droplets 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 invention provides a thin layer identification method for Sophora japonica formula granules, which includes the steps of preparing a test solution, preparing a reference medicinal material solution, preparing a reference substance solution and thin layer identification; a test solution, a reference medicinal material solution and a reference substance solution are obtained by a simple and quick preparation method, and are spotted on the same thin layer chromatography plate respectively, and after development, a multi-information thin layer chromatogram of Sophora japonica formula granules is obtained by inspecting under ultraviolet light. The method uses reference medicinal materials and rutin reference substances as reference substances, has strong specificity, is suitable for production practice, and provides a basis for establishing a quality control standard for Sophora japonica formula granules.

[0060] <Preparation of test solution>

[0061] The inventors investigated the preparation method 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 with an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue with an alcohol solvent.

[0062] In some specific embodiments of the present invention, the granules of the sophora japonica seed formula are crushed and ground into powder, sieved, and then dissolved in water. For the present invention, water can be used as an extraction solvent to extract water-soluble components such as flavonoids to the maximum extent; the present invention has no particular restrictions on the amount of water used for extraction. In order to ensure the efficiency of the extraction and better extract the components to be identified, the ratio of the granules of the sophora japonica seed formula to water can be 1g: (150-350) mL; preferably 1g: (175-300) mL; more preferably 1g: 200 mL.

[0063] After adding water to dissolve, an organic solvent is used for extraction. Compared with extraction methods such as ultrasonic treatment, shaking extraction is simpler and easier. For the organic solvent used, it 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 sufficient 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. After that, the ethyl acetate liquid is combined and concentrated to dryness. The present invention has no special restrictions on the method of concentrating 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 usage ratio of the Sophora japonica seed 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 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] In order to facilitate the operation when performing the subsequent extraction process, it is preferred to add water, heat, reflux and extract, then cool, and then perform the filtering step. The present invention does not specifically limit the filtering method, and it can be any conventional filtering means in the art, such as filter paper, cotton filtering, etc.; after filtering, the filtrate is extracted with an organic solvent, and 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 sufficient 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 (1-1.5): (1-2); preferably 1:1.25. Afterwards, the ethyl acetate solution is combined and concentrated to dryness. The present invention does not specifically limit the method for concentrating 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 usage ratio of Sophora japonica reference medicinal material to 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 usage 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 the 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, and the rest of the preparation method refers to the above-mentioned <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 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, 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 plates, polyamide thin layer plates, ordinary prefabricated plates, high-efficiency prefabricated plates, etc. can be selected. Since the sophora japonica granules of the present invention contain water-soluble components with high polarity such as flavonoids, 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 spotting amount of the test solution is 1-10 μL, preferably 2-6 μL, more preferably 3 μL; the spotting amount of the control medicinal material solution is 1-10 μL, preferably 2-6 μL, more preferably 3 μL; the spotting amount of the reference substance solution is 1-3 μL, preferably 1 μL; if the spotting 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 materials, especially the flavonoid components on the thin layer plate. In some preferred embodiments of the present invention, in order to obtain a better separation, the developing agent of the present invention is preferably measured 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 has no particular limitation on the temperature during development, and it 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 has no particular limitation on the relative humidity during development, and it can be carried out at a relative humidity of 18% to 88%, and different relative humidity has little effect on the separation effect. In some specific embodiments of the present invention, the thin layer 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 obtain a better color development effect.

[0075] After that, heating is performed. The present invention does not particularly limit the heating method. The heating method 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 is inspected under ultraviolet light (365nm). Compared with inspection under sunlight, the spots under ultraviolet light are clear and 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 some other specific embodiments of the present invention, the thin layer identification step of the present invention can also include taking the test solution, the control medicinal material solution, the rutin reference solution and the negative control solution, spotting them on a polyamide thin layer chromatography plate, 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. The results show that the same fluorescent spots appear in the test sample chromatogram at the corresponding positions of the control medicinal material chromatogram and the rutin reference chromatogram, and the negative control has no interference.

[0077] By adopting the thin-layer identification method of the sophora japonica seed formula granules provided by the present invention, a chromatogram of the test sample with the same spots as the reference medicinal material chromatogram and the chromatogram of the rutin reference substance can be obtained, the separation degree and the point property in the chromatogram are good, the development time is short, the inspection is clear, and the quality of the sophora japonica seed 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 used to limit the scope of protection of the present invention.

[0080] <Instruments and reagents>

[0081] Instruments: CAMAH TLC VISUALIZER, 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 analytical grade, and water;

[0083] Sophora japonica seed reference medicinal materials (batch number: 121270-201403) and rutin reference substance (batch number: 100080-201610) were purchased from 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 Albizzia 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 on the preparation methods of test solution and reference 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, treat it with ultrasound 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, treat it with ultrasound for 20 minutes, filter it, and use the filtrate as the test solution.

[0089] Method 3: Take 0.1 g of Sophora japonica seed formula 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 liquid, 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 seed 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 add 1 ml of methanol to dissolve the residue 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 substance 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 by 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, 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 (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 Sophora japonica control medicinal material (method two) was more consistent with the particle spots.

[0100] Investigation of thin layer identification method

[0101] <Study of the 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 the test solution and control medicinal material solution. At the same time, the control solution was prepared. 3 μL of the test solution and the control medicinal material solution and 1 μL of the control 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 film was developed at a temperature of 20.4°C and a relative humidity of 66%, 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 2 , 3 , as shown in Figure 4.

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

[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 according to the above-mentioned test solution preparation method three and control medicinal material solution preparation method two 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 developing agent was n-butanol-acetone-glacial acetic acid-water, with ratios 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%, taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under an ultraviolet lamp (365nm). 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] <Investigation of the amount of sample>

[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, and 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%. The film was taken out, dried, sprayed with 3% aluminum chloride solution, dried 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 volume of the test solution and the control medicinal material solution was 3 μL and the sample volume of the control solution was 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] <Explanation of temperature>

[0110] Sophora japonica granules (batch number: 20040119) and Sophora japonica control medicinal materials were respectively prepared into test solution and control medicinal material solution according to the above test solution preparation method 3 and control medicinal material solution preparation method 2, and the control substance solution was prepared at the same time. 3 μL of the test solution and the control medicinal material solution and 1 μL of the control substance solution were spotted on the same polyamide film, and n-butanol-acetone-glacial acetic acid-water (2:2:1:10) was used as the developing agent. The film was developed at high temperature (35°C) and low temperature (4°C) and relative humidity of 66%, respectively. 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: Fig. 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 test sample of Sophora japonica formula granules, the chromatograms of the reference medicinal materials and the chromatograms of the reference substance showed fluorescent spots of the same color at the corresponding positions, and the separation effect was good. The experimental results showed that temperature had no effect on the thin-layer identification of Sophora japonica formula granules, indicating that the thin-layer identification method had good durability at different temperatures.

[0112] <Expand the investigation of relative humidity>

[0113] Sophora japonica granules (batch number: 20040119) and Sophora japonica control medicinal materials were respectively prepared into test solution and control medicinal material solution according to the above test solution preparation method 3 and control medicinal material solution preparation method 2, and the control solution was prepared at the same time. 3 μL of the test solution and the control medicinal material solution and 1 μL of the control solution were spotted on the same polyamide film, and n-butanol-acetone-glacial acetic acid-water (2:2:1:10) was used as the developing agent, and the films were developed at 20.4°C but under different relative humidity conditions (18%, 88%), respectively. The films were taken out, dried, sprayed with 3% aluminum chloride solution, dried with hot air, and examined under ultraviolet light (365nm). The results are as follows: Fig.10 , wherein 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 test sample of Sophora japonica formula granules, the chromatograms of the reference medicinal materials and the chromatograms of the reference substance showed fluorescent spots of the same color at the corresponding positions, and the separation effect was good. The experimental results showed that relative humidity had no effect on the thin-layer identification of Sophora japonica formula granules, indicating that the thin-layer identification method had good durability under different relative humidity.

[0115] <Specificity test>

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

[0117] The results showed that the same color of fluorescent spots appeared at the corresponding positions of the chromatograms of the test sample, the reference medicinal material and the reference sample in the Sophora japonica granules, and there was no interference from the negative control, indicating that the TLC identification method had 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] Take 0.1g of Sophora japonica seed granules, grind them, add 20ml of water to dissolve them, shake and extract them with ethyl acetate twice, 20ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution. Take another 0.1g of Sophora japonica seed reference medicinal material, add 25ml of water, heat and reflux for 30 minutes, cool, filter, add 20ml of ethyl acetate to the filtrate, and prepare the reference medicinal material solution in the same way. Take the rutin reference substance, add methanol to make a solution containing 4mg per 1ml, and use it as the reference substance solution.

[0121] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 3 μL of the above test solution and control medicinal material solution, and 1 μL of the reference solution were taken and spotted on the same polyamide film, respectively, and developed with n-butanol-acetone-glacial acetic acid-water (2:2:1:10) as the developing agent at a temperature of 20.4°C and a relative humidity of 66%. Take it out, dry it, spray it with 3% aluminum chloride solution, blow it dry with hot air, and examine it under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material and the chromatogram of the reference substance.

[0122] Example 2: Validation of the Thin Layer Identification Method for Different Batches of Sophora japonica Bud Formula 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, 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, respectively, and 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: Fig.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 better 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 formula granules

[0126] Take 0.1g of each of Sophora japonica bud granules, Robinia pseudoacacia bud granules and Albizzia julibrissin granules, grind them into powder, add 20ml of water to dissolve, shake and extract with ethyl acetate twice, 20ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and use it as the test solution. Take 0.1g of Sophora japonica bud reference medicinal material, add 25ml of water, heat and reflux for 30 minutes, cool, filter, add 20ml of ethyl acetate to the filtrate, and prepare the reference medicinal material solution in the same way. Take the rutin reference substance, add methanol to make a solution containing 4mg per 1ml, and use it as the reference substance 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 drug 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 (365nm). The results were as follows: Fig.13 shown.

[0128] Compared with the granules of Sophora japonica, the granules of Robinia japonica lacked blue fluorescent spots at around Rf value 0.4, and had multiple blue-green fluorescent spots at around Rf value 0.75, so the thin-layer method can distinguish the granules of Sophora japonica from the granules of Robinia japonica. Compared with the granules of Sophora japonica, the granules of Albizzia julibrissin lacked blue fluorescent spots at around Rf value 0.25 and around Rf value 0.4, and the fluorescent spots at the corresponding positions of the rutin reference substance were lighter or absent, so the thin-layer method can distinguish the granules of Sophora japonica from the granules of Albizzia julibrissin.

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

[0130] Take 0.2g of Sophora japonica bud granules from different batches, grind them into powder, add 5ml of methanol, seal, shake for 10 minutes, filter, and take the filtrate as the test solution. Take another 0.2g of Sophora japonica bud 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 substance solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 10μL of each of the 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 Fig.14 .

[0131] The results showed that in the test sample chromatogram, fluorescent spots of the same color appeared at the corresponding positions in the control medicinal material chromatogram and the control sample chromatogram, 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 flower by thin layer chromatography"

[0133] Take 0.2g of Sophora japonica bud 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 bud reference medicinal material, and prepare the reference medicinal material solution in the same way. Then take the rutin reference substance and the quercetin reference substance, add methanol to make solutions containing 2mg of each per 1ml, as the reference substance solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), 2-10μL of each of the 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 it, take it out, dry it, fumigate it in iodine vapor for 1 minute, and inspect it under sunlight. The results are as follows: Fig.15 .

[0134] The results showed that in the test sample chromatogram, spots of the same color appeared at the corresponding positions in the control medicinal material chromatogram and the control sample chromatogram, 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] Take 1g of Sophora japonica granules from different batches, grind them, add 20ml of methanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to the residue to dissolve it as the test solution. Take 1g of Sophora japonica reference medicinal material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 20ml of methanol to the residue, and prepare the reference medicinal material solution in the same way. Take the rutin reference substance and the quercetin reference substance, add methanol to make a solution containing 1mg of each per 1ml, as the reference substance solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 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, dried, sprayed with aluminum chloride ethanol solution, dried with hot air, and examined under ultraviolet light (365 nm). The results are as follows Fig.16 .

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

[0138] The above embodiments are only used to illustrate several embodiments of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood that any limitation is imposed on the scope of the present invention. It should be clear that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A thin layer identification method for Sophora japonica granules, characterized in that: The method comprises the preparation of a test solution, the preparation of a reference medicinal material solution, the preparation of a reference substance solution and thin layer identification. The preparation of the test solution comprises taking the Sophora japonica granules, adding water to dissolve them, extracting them with an organic solvent, separating the organic phase and concentrating it to dryness, and dissolving the residue with an alcohol solvent; The preparation of the control medicinal material solution comprises taking a control medicinal material of Sophora japonica, adding water to reflux for 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; 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.

2. The thin layer identification method according to claim 1, characterized in that: The preparation of the reference solution includes taking a rutin reference substance 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 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 identification method according to claim 1 or 2, characterized in that: In the preparation of the test solution, the dosage ratio of the Sophora japonica seed formula granules to water is 1g:(150-350)mL; the organic solvent is selected from one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate; 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 identification method according to any one of claims 1 to 3, characterized in that: In the preparation of the control medicinal material solution, the dosage ratio of Sophora japonica reference medicinal material to water is 1g:(200-400)mL; the reflux extraction time is 20-40min; the organic solvent is selected from one or more of ether, dichloromethane, chloroform, carbon tetrachloride or ethyl acetate; 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 identification method according to claim 2, characterized in that: In the preparation of the reference solution, the usage ratio of the rutin reference to the alcohol solvent is (3-6) mg:1 mL; the alcohol solvent is methanol or a methanol aqueous solution; in the thin layer identification, the spotting amount of the reference solution is 1-3 μL.

6. The thin layer identification method according to any one of claims 1 to 5, characterized in that: In the spotting operation of the thin layer identification, the samples are spotted on the same polyamide thin layer chromatography plate, the spotting amount of the test solution is 1 to 10 μL, and the spotting amount of the control medicinal material solution is 1 to 10 μL; the inspection is performed under an ultraviolet lamp with a wavelength of 365 nm.

7. The thin layer identification method according to any one of claims 1 to 6, characterized in that: In the thin layer identification, n-butanol-acetone-glacial acetic acid-water with a volume ratio of (1-4):(1-4):(0.5-2):(5-20) is used as a developing solvent.

8. The thin layer identification method according to any one of claims 1 to 7, characterized in that: The thin layer identification further comprises drying after unfolding, spraying with a color developer, heating until the spots are clearly colored and then inspecting under ultraviolet light; preferably, the color developer is an aluminum chloride solution with a concentration of 1% to 5%.

9. The thin layer identification method according to any one of claims 1 to 8, characterized in that: 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.

10. Use of the thin layer identification method according to any one of claims 1 to 9 in identifying authentic and / or counterfeit Sophora japonica formula granules, wherein: The counterfeits include Robinia pseudoacacia rice formula granules and / or Albizzia julibrissin rice formula granules.

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