Thin-layer qualitative identification method for synchronously identifying three medicinal materials in compound preparation containing rumex madaio and application of thin-layer qualitative identification method

Abstract

The invention discloses a thin-layer qualitative identification method for synchronously identifying three medicinal materials in a traditional Chinese medicine compound containing rumex madaio makino, which comprises the following steps: (1) pretreating the traditional Chinese medicine compound containing rumex madaio makino to obtain a test solution; the method comprises the following steps: pre-treating a traditional Chinese medicine compound lacking sanguisorba officinalis, rumex madaio or coptis chinensis to obtain a negative sample solution, and pre-treating a reference medicinal material to obtain a reference medicinal material solution; and (2) performing thin-layer chromatography identification analysis on the test solution, the negative sample solution and the reference medicinal material solution. According to the thin-layer identification method disclosed by the invention, by detecting the sanguisorba officinalis, the rumex mairei and the coptis chinensis in the traditional Chinese medicine compound containing the rumex mairei, a theoretical basis is provided for quality control of the traditional Chinese medicine compound containing the rumex mairei and research on a pharmacodynamic material basis. The method has the technical effects of few samples, simplified quality detection and improved detection efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a thin-layer qualitative identification method for synchronously identifying three medicinal materials in a compound preparation containing Radix et Rhizoma Rumicis Cripii and its application. BACKGROUND

[0002] Thin-layer qualitative identification method (Thin-Layer Chromatography, TLC) is an analysis technology widely used in the fields of chemistry, pharmacy, food science, etc., which has the advantages of simple and quick operation, low detection cost, high sensitivity, wide applicability, and intuitive visual results. TLC technology is widely used in the qualitative and semi-quantitative analysis of traditional Chinese medicines, and has the following advantages:

[0003] 1. Simple and convenient operation: simple steps, sample preparation and chromatography process do not require complex equipment, and the operation is easy to master. The analysis speed is fast, and the separation and color development can be completed in 10-30 minutes, which is suitable for batch sample screening. 2. Low cost: low equipment requirement, only thin-layer plate, development cylinder and color development tool are needed, without expensive instruments. The consumables are cheap, and the cost of thin-layer plate (such as silica gel plate) and solvent is low. 3. High sensitivity: microgram (μg) or even nanogram (ng) level substances can be detected, and the sensitivity can be enhanced by using color developing agent or fluorescence. 4. Wide applicability: compatible with various samples, suitable for flavonoids, phenolic acids, alkaloids and other compounds with different structures, multi-component synchronous analysis, single development can separate multiple components, suitable for complex mixture screening. 5. Intuitive visual detection results: combined with ultraviolet, fluorescence, visible light or chemical color development, the specificity is improved, and the spot position and color can be directly observed by color development (such as ultraviolet lamp, iodine vapor or chemical reagent), which preserves the original information and is convenient for subsequent review or recording. 6. Strong flexibility: multiple development systems, the polarity and pH value of the mobile phase can be adjusted according to the properties of the sample; 7. No complex pretreatment: low purity requirement for samples, crude extract can be directly spotted, simplifying the pretreatment steps. 8. Semi-quantitative ability: rough quantification can be performed by scanning the spot size or density (compared with standard samples). 9. Environmentally friendly: low solvent consumption (only a small amount of developing agent is needed), reducing the pressure of waste liquid treatment.

[0004] In summary, thin-layer qualitative identification is an important tool for routine analysis of traditional Chinese medicine components due to its simplicity, economy and speed, and is especially suitable for preliminary screening or rapid detection.

[0005] There is no thin-layer qualitative identification method for synchronously identifying three medicinal materials in a compound preparation containing Radix et Rhizoma Rumicis Cripii and its application in the prior art. SUMMARY

[0006] Based on this, the present application provides a kind of thin layer qualitative identification method for identifying three kinds of medicinal materials in traditional Chinese medicine compound containing Rumex madaio, which includes the following steps:

[0007] (1) the traditional Chinese medicine compound containing Rumex madaio is pretreated to obtain a test solution, the traditional Chinese medicine compound lacking Geijera japonica, Rumex madaio or Coptis chinensis is pretreated to obtain a negative sample solution, and the control medicinal material is pretreated to obtain a control medicinal material solution; and

[0008] (2) the test solution, the negative sample solution and the control medicinal material solution are subjected to thin layer chromatography identification analysis;

[0009] In the step of thin layer chromatography identification analysis, a developing agent is used for development, wherein the developing agent includes a first developing agent and a second developing agent, the first developing agent is ethyl acetate-formic acid, and the second developing agent is cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine; the volume ratio between ethyl acetate and formic acid in the first developing agent is (1-20):(0.01-1), and the volume ratio between cyclohexane, ethyl acetate, isopropyl alcohol, methanol, water and triethylamine in the second developing agent is (1-10):(1-10):(0.1-5):(0.1-5):(0.1-3):(0.1-5).

[0010] Further, the traditional Chinese medicine compound containing Rumex madaio includes Coptis chinensis, Geijera japonica, Rumex madaio and Butea frondosa root.

[0011] Further, the dosage form of the traditional Chinese medicine compound containing Rumex madaio is capsule or granule.

[0012] Further, the negative sample solution is a negative sample solution lacking Geijera japonica, a negative sample solution lacking Rumex madaio and / or a negative sample solution lacking Coptis chinensis.

[0013] Further, the control medicinal material is Geijera japonica control medicinal material, Coptis chinensis control medicinal material and / or Rumex madaio control medicinal material.

[0014] Further, the preparation method of the traditional Chinese medicine compound containing Rumex madaio includes the following steps:

[0015] (a) a suitable amount of Butea frondosa root is crushed into medium powder; and

[0016] (b) a suitable amount of Butea frondosa root, Geijera japonica, Rumex madaio and Coptis chinensis are added to ethanol of a certain concentration for warm soaking for a certain time, and reflux extraction is carried out at a certain temperature for 1-4 times, each time for 0.5-2 hours; the extraction liquid is combined, filtered, and the filtrate is concentrated to a thick paste with a relative density of about 1.1-1.45 (80-90°C) by recovering ethanol under reduced pressure; vacuum drying is carried out at 60-80°C, and the powder is crushed into fine powder; the above-mentioned medium powder and a certain amount of starch are added and mixed uniformly to obtain.

[0017] Further, the mass of the bletilla striata root in step (a) is 20-100 g, for example, about 50 g.

[0018] Further, the mass of the bletilla striata root in step (b) is 600-700 g, for example, about 650 g.

[0019] Further, the mass of the sanguisorba officinalis in step (b) is 650-750 g, for example, about 700 g.

[0020] Further, the mass of the rheum palmatum in step (b) is 200-400 g, for example, about 300 g.

[0021] Further, the mass of the coptis chinensis in step (b) is 200-400 g, for example, about 300 g.

[0022] Further, the concentration of the ethanol in step (b) is 40%-60%, for example, about 50%.

[0023] Further, the time of the warm soaking in step (b) is 0.2-1 hour, for example, about 30 minutes.

[0024] Further, the temperature of the reflux extraction in step (b) is 70℃-90℃, for example, about 80℃.

[0025] Further, the number of times of the reflux extraction in step (b) is 2 times.

[0026] Further, the time of each reflux extraction in step (b) is about 1.5 hours.

[0027] Further, the preparation method of the traditional Chinese medicine compound containing rheum palmatum comprises the following steps: taking about 50 g of bletilla striata root and crushing into medium powder; taking about 650 g of bletilla striata root, about 700 g of sanguisorba officinalis, and about 300 g of rheum palmatum and coptis chinensis, adding about 50% ethanol to warm soak for about 30 minutes, refluxing at about 80℃ for 2 times, each time for about 1.5 hours, combining the extraction liquid, filtering, recovering ethanol from the filtrate under reduced pressure and concentrating to a thick paste with a relative density of about 1.25-1.35 (about 85℃), vacuum drying at about 70℃, and crushing into fine powder, adding the above-mentioned medium powder and about 100 g of starch, and mixing uniformly to obtain.

[0028] Further, the preparation method of the test solution comprises: weighing an appropriate amount of the traditional Chinese medicine compound containing rheum palmatum, adding a solvent, ultrasonic treatment, and filtering to obtain the test solution.

[0029] Further, the solvent is methanol.

[0030] Further, the preparation method of the test solution does not include the steps of extraction and ammonia reagent washing after extraction.

[0031] Further, the mass / volume (g / ml) ratio of the traditional Chinese medicine compound containing Radix Rumicis Cripii to the solvent is 0.01-0.1, for example, about 0.04.

[0032] Further, the mass of the traditional Chinese medicine compound containing Radix Rumicis Cripii is 0.1-5 g, for example, about 1 g.

[0033] Further, the volume of the methanol is 20-30 ml, for example, about 25 ml.

[0034] Further, the time of the ultrasonic treatment is 20-40 min, for example, about 30 min.

[0035] Further, the preparation method of the control medicinal material solution includes: weighing an appropriate amount of Fructus Arctii, Rhizoma Coptidis or Radix Rumicis Cripii control medicinal material, adding a solvent for ultrasonic extraction, filtering, and obtaining the control medicinal material solution.

[0036] Further, the solvent is methanol.

[0037] Further, the mass / volume (g / ml) ratio of the Fructus Arctii, Rhizoma Coptidis or Radix Rumicis Cripii control medicinal material to the solvent is 0.001-0.1, for example, about 0.02.

[0038] Further, the mass of the Fructus Arctii control medicinal material is 0.1-1 g, for example, about 0.5 g.

[0039] Further, the mass of the Rhizoma Coptidis control medicinal material is 0.1-1 g, for example, about 0.5 g.

[0040] Further, the mass of the Radix Rumicis Cripii control medicinal material is 0.1-1 g, for example, about 0.5 g.

[0041] Further, the volume of the methanol is 20-30 ml, for example, about 25 ml.

[0042] Further, the time of the ultrasonic extraction is 20-40 min, for example, about 30 min.

[0043] Further, the preparation method of the negative sample solution includes: weighing an appropriate amount of traditional Chinese medicine compound lacking Fructus Arctii, Radix Rumicis Cripii or Rhizoma Coptidis, respectively, and then the subsequent steps are the same as the preparation method of the test solution.

[0044] Further, the volume ratio of the ethyl acetate to the formic acid in the first developing agent is (5-15):(0.05-0.5).

[0045] Further, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is (8~12):(0.08~0.12).

[0046] Further, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is about 10:about 0.1.

[0047] Further, the volume ratio between the cyclohexane, the ethyl acetate, the isopropanol, the methanol, the water and the triethylamine in the second developing agent is (1~6):(2~6):(0.5~1.5):(1~2):(0.3~1.5):(0.5~1.5).

[0048] Further, the volume ratio between the cyclohexane, the ethyl acetate, the isopropanol, the methanol, the water and the triethylamine in the second developing agent is (2~4):(3~4):(0.8~1.2):(1.2~1.8):(0.4~1):(0.8~1.2).

[0049] Further, the volume ratio between the cyclohexane, the ethyl acetate, the isopropanol, the methanol, the water and the triethylamine in the second developing agent is about 3:about 3.5:about 1:about 1.5:about 0.5:about 1.

[0050] Further, the thin layer plate is a silica gel G thin layer plate.

[0051] Further, the silica gel G thin layer plate is a Merck silica gel G thin layer plate or a Yantai Yinlong silica gel G thin layer plate or a Qingdao Haoyang silica gel G thin layer plate.

[0052] Further, the viewing condition of Coptis chinensis and Sanguisorba officinalis in the step of thin layer chromatography identification analysis is: taking out the developed thin layer plate, drying, and viewing under the ultraviolet light lamp.

[0053] Further, the viewing condition of Rheum palmatum in the step of thin layer chromatography identification analysis is: taking out the developed thin layer plate, drying, and viewing under the ultraviolet light lamp and the visible light.

[0054] Further, the detection wavelength of the ultraviolet light lamp is 365 nm.

[0055] Further, the spotting amount of the test product solution is 1.5~12 μl, for example, about 2 μl, or about 5 μl, or about 10 μl.

[0056] Further, the spotting amount of the test product solution is about 10 μl.

[0057] Further, the spotting amount of the control medicinal material solution is 1~5 μl, for example, 1~3 μl, for example, about 2 μl.

[0058] Further, the sample solution is spotted in an amount of 1-5 μl, for example 1-3 μl, for example about 2 μl.

[0059] Further, the criteria for the thin layer identification include: under UV light, whether the thin layer plate shows the same color characteristic spots as the corresponding positions of the Coptis chinensis control drug material, the Hippophae rhamnoides control drug material and the Rumex nepalensis control drug material; under visible light, whether the thin layer plate shows the same color characteristic spots as the corresponding position of the Rumex nepalensis control drug material; and whether the negative sample is interfered; if the thin layer plate has the characteristic spots under both UV light and visible light and the negative sample is not interfered, it is determined that the traditional Chinese medicine compound containing Rumex nepalensis contains Coptis chinensis, Hippophae rhamnoides and Rumex nepalensis.

[0060] According to another aspect of the present application, there is provided a use of the above-mentioned thin layer qualitative identification method for identifying a traditional Chinese medicine compound containing Rumex nepalensis.

[0061] According to another aspect of the present application, there is provided a kit for qualitatively identifying a traditional Chinese medicine compound containing Rumex nepalensis, the kit comprising a thin layer plate and a developing agent, the developing agent comprising a first developing agent and a second developing agent, wherein the first developing agent is ethyl acetate-formic acid, the second developing agent is cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine, and the thin layer plate is a silica gel G thin layer plate.

[0062] Further, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is (1-20):(0.01-1).

[0063] Further, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is (5-15):(0.05-0.5).

[0064] Further, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is (8-12):(0.08-0.12).

[0065] Further, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is about 10:about 0.1.

[0066] Further, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water and the triethylamine in the second developing agent is (1-10):(1-10):(0.1-5):(0.1-5):(0.1-3):(0.1-5).

[0067] Further, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water and the triethylamine in the second developing agent is (1-6):(2-6):(0.5-1.5):(1-2):(0.3-1.5):(0.5-1.5).

[0068] Further, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water and the triethylamine in the second developing agent is (2~4):(3~4):(0.8~1.2):(1.2~1.8):(0.4~1):(0.8~1.2).

[0069] Further, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water and the triethylamine in the second developing agent is about 3: about 3.5: about 1: about 1.5: about 0.5: about 1.

[0070] Advantages of the present application:

[0071] The above thin layer identification method of the present application provides a theoretical basis for the quality control and the research of the pharmacodynamic material basis of the traditional Chinese medicine compound containing the Rumex nepalensis Spreng by detecting the Sanguisorba officinalis L., the Rumex nepalensis Spreng and the Coptis chinensis Franch in the traditional Chinese medicine compound containing the Rumex nepalensis Spreng. The present application has the technical effects of less sampling, simplified quality detection and improved detection efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0072] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to these drawings without exceeding the scope of the present application.

[0073] Figure 1 The schematic diagram of the investigation results of different developing agents (ethyl acetate-methanol-water (100:17:13)+cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water (3:3.5:1:1.5:0.5)) is shown. Wherein, 1. bletilla striata control drug material, 2. Coptis chinensis Franch control drug material, 3. Rumex nepalensis Spreng control drug material, 4. Sanguisorba officinalis L. control drug material, 5. test solution A.

[0074] Figure 2 The schematic diagram of the investigation results of different developing agents (ethyl acetate-methanol-water (100:17:13)+cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1)) is shown. Wherein, 1. bletilla striata control drug material, 2. Coptis chinensis Franch control drug material, 3. Rumex nepalensis Spreng control drug material, 4. Sanguisorba officinalis L. control drug material, 5. test solution A.

[0075] Figure 3The results of the investigation for different developing agents (chloroform-methanol-water (13:7:2) lower layer + cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1)) are shown in the schematic diagram. Among them, 1. Coptis chinensis control drug material, 2. Rheum palmatum control drug material, 3. Sanguisorba officinalis control drug material, 4. Test solution A, 5. Test solution B.

[0076] Figure 4 The results of the investigation for different developing agents (ethyl acetate-methanol-water (100:17:13) + chloroform-methanol-water (13:7:2) lower layer) are shown in the schematic diagram. Among them, 1. Coptis chinensis control drug material, 2. Rheum palmatum control drug material, 3. Sanguisorba officinalis control drug material, 4. Test solution A, 5. Test solution B.

[0077] Figure 5 The results of the investigation for different developing agents (ethyl acetate-formic acid (10:0.1) + cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1)) are shown in the schematic diagram. Among them, 1. Coptis chinensis control drug material, 2. Rheum palmatum control drug material, 3. Sanguisorba officinalis control drug material, 4. Test solution A, 5. Test solution B.

[0078] Figure 6 The results of the investigation for sample preparation methods are shown in the schematic diagram. Among them, A is the investigation of whether to wash with ammonia after n-butanol extraction, 1. Coptis chinensis control drug material, 2. Rheum palmatum control drug material, 3. Sanguisorba officinalis control drug material, 4. Test solution A, 5. Test solution B; B is the investigation of extraction (extraction) method, 1. Coptis chinensis control drug material, 2. Rheum palmatum control drug material, 3. Sanguisorba officinalis control drug material, 4. Test solution C, 5. Test solution D, 6. Test solution E, 7. Test solution F, 8. Test solution A.

[0079] Figure 7 The results of the investigation for sample spotting amount are shown in the schematic diagram. Among them, 1. Coptis chinensis control drug material, 2. Rheum palmatum control drug material, 3. Sanguisorba officinalis control drug material, 4-7. Test solution 1 μL, 2 μL, 5 μL, 10 μL.

[0080] Figure 8 The results of the investigation for the specificity of the method are shown in the schematic diagram. Among them, 1. Coptis chinensis control drug material, 2. Rheum palmatum control drug material, 3. Sanguisorba officinalis control drug material, 4-6. Test solution 1-3 batches, 7. Coptis chinensis negative test solution, 8. Rheum palmatum negative test solution, 9. Sanguisorba officinalis negative test solution.

[0081] Figure 9The results of the different inspection wavelengths are shown in the schematic diagram. Among them, 1. Coptis chinensis control drug, 2. Rheum palmatum control drug, 3. Sanguisorba officinalis control drug, 4-6. Test solution 1-3 batches, 7. Coptis chinensis negative test solution, 8. Rheum palmatum negative test solution, 9. Sanguisorba officinalis negative test solution.

[0082] Figure 10 The results of the adaptability of different manufacturers of thin layer plates are shown in the schematic diagram. Among them, 1. Coptis chinensis control drug, 2. Rheum palmatum control drug, 3. Sanguisorba officinalis control drug, 4-6. Test solution 1-3 batches, 7. Coptis chinensis negative test solution, 8. Rheum palmatum negative test solution, 9. Sanguisorba officinalis negative test solution. DETAILED DESCRIPTION

[0083] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0084] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the art in the field of the application, or by one of ordinary skill in the art to which the term is applied. Although any methods, conditions, materials, or articles similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods, conditions, materials, or articles are described herein.

[0085] The present application is intended to encompass all alternatives, modifications and equivalents that can fall within the present inventive concept as defined by the claims. One skilled in the art will recognize many methods and materials as being suitable for the practice of this application. Nothing in the present application should be taken as a restriction on the scope of this application.

[0086] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0087] In the present application, the term "comprising" is synonymous with "including". The terms "comprising", "including", "having" "with" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0088] As described in the background section, there is no prior art of the claimed method for simultaneously identifying three medicinal materials in a compound preparation containing Rumex japonicus Houtt. by thin layer qualitative identification and its application. In order to solve the above problems, the present application provides a method for simultaneously identifying three medicinal materials in a compound preparation containing Rumex japonicus Houtt. by thin layer qualitative identification, which comprises the following steps:

[0089] (1) subjecting the compound preparation containing Rumex japonicus Houtt. to pretreatment to obtain a test sample solution, subjecting the compound preparation lacking Geum japonicum, Rumex japonicus Houtt. or Coptis deltoidea to pretreatment to obtain a negative sample solution, and subjecting the control medicinal materials to pretreatment to obtain a control medicinal material solution; and

[0090] (2) performing thin layer chromatography identification analysis on the test sample solution, the negative sample solution and the control medicinal material solution;

[0091] In the step of the thin layer chromatography identification analysis, a developing agent is used for development, wherein the developing agent comprises a first developing agent and a second developing agent, the first developing agent is ethyl acetate-formic acid, and the second developing agent is cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is (1-20):(0.01-1), and the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water and the triethylamine in the second developing agent is (1-10):(1-10):(0.1-5):(0.1-5):(0.1-3):(0.1-5).

[0092] In the present application, when a volume ratio, a number of times, a time, a relative density, a temperature, a volume, a mass, a concentration, a ratio, a sample application amount, or other values or parameters are expressed in a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pair of range upper limit or preferred value and range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately. For example, when the range "1-20" is disclosed, the described range should be interpreted as including the ranges "1-20", "1-16", "1-12", "1-8", "1-4", "4-20", "4-16", "4-12", "4-8", "8-20", "8-16", "8-12", "12-20", "12-16", "16-20", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include the end values and all integers and fractions within the range.

[0093] In a preferred embodiment, the compound preparation containing Rumex japonicus Houtt. comprises Coptis deltoidea, Geum japonicum, Rumex japonicus Houtt. and Honghua root.

[0094] In a preferred embodiment, the dosage form of the Chinese medicine compound comprising Rumex nepalensis is a capsule or a granule.

[0095] In a preferred embodiment, the negative sample solution is a negative sample solution lacking Sanguisorba officinalis, a negative sample solution lacking Rumex nepalensis, and / or a negative sample solution lacking Coptis chinensis.

[0096] In a preferred embodiment, the control medicinal material is a Sanguisorba officinalis control medicinal material, a Coptis chinensis control medicinal material, and / or a Rumex nepalensis control medicinal material.

[0097] In a preferred embodiment, the method of preparing the Chinese medicine compound comprising Rumex nepalensis comprises the following steps:

[0098] (a) taking an appropriate amount of Broussonetia kaempferi roots, and pulverizing the Broussonetia kaempferi roots into a medium powder; and

[0099] (b) taking an appropriate amount of Broussonetia kaempferi roots, Sanguisorba officinalis, Rumex nepalensis, and Coptis chinensis, and adding a certain concentration of ethanol to warm soak for a certain period of time, and refluxing and extracting at a certain temperature for 1-4 times, each time for 0.5-2 hours, combining the extraction solutions, filtering, recovering ethanol from the filtrate under reduced pressure, and concentrating to a thick paste with a relative density of about 1.1-1.45 (80°C-90°C), vacuum drying at 60°C-80°C, and pulverizing into a fine powder, adding the above-mentioned medium powder and a certain amount of starch, and mixing to obtain.

[0100] In a preferred embodiment, the mass of Broussonetia kaempferi roots in step (a) is 20-100 g, for example, about 50 g.

[0101] In the present application, "about" refers to a value within ±5% of a particular value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0102] In a preferred embodiment, the mass of Broussonetia kaempferi roots in step (b) is 600-700 g, for example, about 650 g.

[0103] In the present application, "about" refers to a value within ±5% of a particular value. For example, "about 650" includes ±5% of 650, or from 617.5 to 682.5.

[0104] In a preferred embodiment, the mass of Sanguisorba officinalis in step (b) is 650-750 g, for example, about 700 g.

[0105] In the present application, "about" refers to a value within ±5% of a particular value. For example, "about 700" includes ±5% of 700, or from 665 to 735.

[0106] In a preferred embodiment, the mass of Rumex nepalensis in step (b) is 200-400 g, for example, about 300 g.

[0107] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 300" includes ±5% of 300, or from 285 to 315.

[0108] In a preferred embodiment, the amount of Huanglian in step (b) is 200-400 g, for example about 300 g.

[0109] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 300" includes ±5% of 300, or from 285 to 315.

[0110] In a preferred embodiment, the concentration of ethanol in step (b) is 40-60%, for example about 50%.

[0111] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 50%" includes ±5% of 50%, or from 47.5% to 52.5%.

[0112] In a preferred embodiment, the time of warm infusion in step (b) is 0.2-1 hour, for example about 30 minutes.

[0113] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0114] In a preferred embodiment, the temperature of reflux extraction in step (b) is 70-90°C, for example about 80°C.

[0115] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0116] In a preferred embodiment, the number of times of reflux extraction in step (b) is 2.

[0117] In a preferred embodiment, the time of each reflux extraction in step (b) is about 1.5 hours.

[0118] In the present application, "about" means a value that is ±5% of a specified value. For example, "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575.

[0119] In a preferred embodiment, the preparation method of the Chinese medicine compound comprising Rumex nepalensis includes the following steps: taking about 50 g of the root of Broughtonitia bicolor, crushing into medium powder; taking about 650 g of the root of Broughtonitia bicolor, about 700 g of Sanguisorba officinalis and about 300 g of Rumex nepalensis, about 300 g of Coptis chinensis, adding about 50% ethanol, warm soaking for about 30 minutes, refluxing extraction for about 2 times at about 80°C, each time for about 1.5 hours, combining the extraction liquid, filtering, recovering ethanol from the filtrate under reduced pressure and concentrating to a thick paste with a relative density of about 1.25-1.35 (about 85°C), vacuum drying at about 70°C, crushing into fine powder, adding the above-mentioned medium powder and about 100 g of starch, mixing uniformly to obtain.

[0120] In the present application, "about" refers to a value within ±5% of a particular value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 650" includes ±5% of 650, or from 617.5 to 682.5; "about 700" includes ±5% of 700, or from 665 to 735; "about 300" includes ±5% of 300, or from 285 to 315; "about 50%" includes ±5% of 50%, or from 47.5% to 52.5%; "about 30" includes ±5% of 30, or from 28.5 to 31.5; "about 80" includes ±5% of 80, or from 76 to 84; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575; "about 85" includes ±5% of 85, or from 80.75 to 89.25; "about 70" includes ±5% of 70, or from 66.5 to 73.5; "about 100" includes ±5% of 100, or from 95 to 105.

[0121] In a preferred embodiment, the preparation method of the test sample solution includes: weighing an appropriate amount of the Chinese medicine compound comprising Rumex nepalensis, adding a solvent, ultrasonic treatment, filtering to obtain the test sample solution.

[0122] In a preferred embodiment, the solvent is methanol.

[0123] In a preferred embodiment, the preparation method of the test sample solution does not include the steps of extraction and ammonia reagent washing after extraction.

[0124] In a preferred embodiment, the mass / volume (g / ml) ratio of the Chinese medicine compound comprising Rumex nepalensis to the solvent is 0.01-0.1, for example about 0.04.

[0125] In the present application, "about" refers to a value within ±5% of a particular value. For example, "about 0.04" includes ±5% of 0.04, or from 0.038 to 0.042.

[0126] In a preferred embodiment, the mass of the Chinese medicine compound comprising Rumex nepalensis is 0.1-5 g, for example about 1 g.

[0127] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0128] In a preferred embodiment, the volume of methanol is 20-30 ml, for example, about 25 ml.

[0129] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0130] In a preferred embodiment, the ultrasonic treatment time is 20 to 40 minutes, for example, about 30 minutes.

[0131] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0132] In a preferred embodiment, the preparation method of the reference medicinal material solution includes: weighing an appropriate amount of Sanguisorba officinalis, Coptis chinensis or Rheum palmatum reference medicinal materials, adding a solvent for ultrasonic extraction, filtering, and obtaining the reference medicinal material solution.

[0133] In a preferred embodiment, the solvent is methanol.

[0134] In a preferred embodiment, the mass / volume (g / ml) ratio of the reference medicinal material, such as Sanguisorba officinalis, Coptis chinensis, or Rheum palmatum, to the solvent is 0.001 to 0.1, for example, about 0.02.

[0135] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.02" includes ±5% of 0.02, or from 0.019 to 0.021.

[0136] In a preferred embodiment, the mass of the reference herb *Sanguisorba officinalis* is 0.1 to 1 g, for example, about 0.5 g.

[0137] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0138] In a preferred embodiment, the mass of the Coptis chinensis reference herb is 0.1 to 1 g, for example, about 0.5 g.

[0139] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0140] In a preferred embodiment, the quality of the Rhei Radix et Rhizoma control medicinal material is 0.1-1 g, such as about 0.5 g.

[0141] In the present application, "about" means a value within ±5% of a specified value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0142] In a preferred embodiment, the volume of the methanol is 20-30 ml, such as about 25 ml.

[0143] In the present application, "about" means a value within ±5% of a specified value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0144] In a preferred embodiment, the time of the ultrasonic extraction is 20-40 min, such as about 30 min.

[0145] In the present application, "about" means a value within ±5% of a specified value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0146] In a preferred embodiment, the preparation method of the negative sample solution comprises: separately weighing an appropriate amount of the traditional Chinese medicine compound lacking Geijiu, Rhei Radix et Rhizoma, or Coptidis Rhizoma, and then the subsequent steps are the same as the preparation method of the test solution.

[0147] In a preferred embodiment, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is (5-15):(0.05-0.5).

[0148] In a preferred embodiment, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is (8-12):(0.08-0.12).

[0149] In a preferred embodiment, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is about 10:about 0.1.

[0150] In the present application, "about" means a value within ±5% of a specified value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0151] In a preferred embodiment, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water, and the triethylamine in the second developing agent is (1-6):(2-6):(0.5-1.5):(1-2):(0.3-1.5):(0.5-1.5).

[0152] In a preferred embodiment, the ratio by volume between the cyclohexane, the ethyl acetate, the isopropanol, the methanol, the water and the triethylamine in the second developer is (2-4):(3-4):(0.8-1.2):(1.2-1.8):(0.4-1):(0.8-1.2).

[0153] In a preferred embodiment, the ratio by volume between the cyclohexane, the ethyl acetate, the isopropanol, the methanol, the water and the triethylamine in the second developer is about 3: about 3.5: about 1: about 1.5: about 0.5: about 1.

[0154] In the present application, "about" means a value within ±5% of a specified value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 3.5" includes ±5% of 3.5, or from 3.325 to 3.675; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575; "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0155] In a preferred embodiment, the thin layer plate is a silica gel G thin layer plate.

[0156] In a preferred embodiment, the silica gel G thin layer plate is a Merck silica gel G thin layer plate or a Yantai Yinlong silica gel G thin layer plate or a Qingdao Haoyang silica gel G thin layer plate.

[0157] In a preferred embodiment, the viewing condition of Coptis chinensis and Sanguisorba officinalis in the step of thin layer chromatography identification analysis is: taking out the developed thin layer plate, drying, and viewing under the ultraviolet light lamp.

[0158] In a preferred embodiment, the viewing condition of Rheum palmatum in the step of thin layer chromatography identification analysis is: taking out the developed thin layer plate, drying, and viewing under the ultraviolet light lamp and the visible light.

[0159] In a preferred embodiment, the detection wavelength of the ultraviolet light lamp is 365 nm.

[0160] In a preferred embodiment, the spotting amount of the test sample solution is 1.5-12 μl, for example, about 2 μl, or about 5 μl, or about 10 μl.

[0161] In the present application, "about" means a value within ±5% of a specified value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0162] In a preferred embodiment, the sample solution is applied in an amount of about 10 μl.

[0163] In the present application, "about" means a value within ±5% of a specified value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0164] In a preferred embodiment, the control drug solution is applied in an amount of 1-5 μl, such as 1-3 μl, such as about 2 μl.

[0165] In the present application, "about" means a value within ±5% of a specified value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0166] In a preferred embodiment, the negative sample solution is applied in an amount of 1-5 μl, such as 1-3 μl, such as about 2 μl.

[0167] In the present application, "about" means a value within ±5% of a specified value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0168] In a preferred embodiment, the criteria for the thin layer identification include: under UV light, whether the thin layer plate shows the same color characteristic spots as the corresponding positions of the Coptis chinensis control drug, the Hippophae rhamnoides control drug, and the Rumex nepalensis control drug; under visible light, whether the thin layer plate shows the same color characteristic spots as the corresponding position of the Rumex nepalensis control drug; and whether the negative sample is interfered; if the thin layer plate has the characteristic spots under both UV light and visible light and the negative sample is not interfered, then the detected traditional Chinese medicine compound containing Rumex nepalensis contains Coptis chinensis, Hippophae rhamnoides, and Rumex nepalensis.

[0169] According to another aspect of the present application, there is provided a use of the above-mentioned thin layer qualitative identification method for identifying a traditional Chinese medicine compound containing Rumex nepalensis.

[0170] According to another aspect of the present application, there is provided a kit for qualitatively identifying a traditional Chinese medicine compound containing Rumex nepalensis, the kit comprising a thin layer plate and a developing agent, the developing agent comprising a first developing agent and a second developing agent, wherein the first developing agent is ethyl acetate-formic acid, the second developing agent is cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine, and the thin layer plate is a silica gel G thin layer plate.

[0171] In a preferred embodiment, the volume ratio between the ethyl acetate and the formic acid in the first developing agent is (1-20):(0.01-1).

[0172] In a preferred embodiment, the volume ratio between the ethyl acetate and the formic acid in the first developer is (5-15):(0.05-0.5).

[0173] In a preferred embodiment, the volume ratio between the ethyl acetate and the formic acid in the first developer is (8-12):(0.08-0.12).

[0174] In a preferred embodiment, the volume ratio between the ethyl acetate and the formic acid in the first developer is about 10:about 0.1.

[0175] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0176] In a preferred embodiment, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water, and the triethylamine in the second developer is (1-10):(1-10):(0.1-5):(0.1-5):(0.1-3):(0.1-5).

[0177] In a preferred embodiment, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water, and the triethylamine in the second developer is (1-6):(2-6):(0.5-1.5):(1-2):(0.3-1.5):(0.5-1.5).

[0178] In a preferred embodiment, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water, and the triethylamine in the second developer is (2-4):(3-4):(0.8-1.2):(1.2-1.8):(0.4-1):(0.8-1.2).

[0179] In a preferred embodiment, the volume ratio between the cyclohexane, the ethyl acetate, the isopropyl alcohol, the methanol, the water, and the triethylamine in the second developer is about 3:about 3.5:about 1:about 1.5:about 0.5:about 1.

[0180] In the present application, "about" means a value that is within ±5% of a specified value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 3.5" includes ±5% of 3.5, or from 3.325 to 3.675; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575; "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0181] The application will be further described in connection with the following specific examples. It should be understood that the examples are only used to illustrate the application but not used to limit the scope of the application. The experimental methods not specified in the following examples are generally carried out according to the conventional conditions or the conditions suggested by the manufacturers.

[0182] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are presented by way of example only and are not intended to limit the scope of the application.

[0183] The above features mentioned in the application, or the features mentioned in the examples, can be combined arbitrarily. All features disclosed in the patent specification can be used in any combination, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0184] Examples

[0185] 1. Experimental materials

[0186] 1.1. Reagents and reagents

[0187] The medicinal materials of Sanguisorba officinalis, Rheum palmatum, Adenocaulon bicolor, and Coptis chinensis were provided by Fuhua Pharmaceutical Co., Ltd. and identified as authentic products. The products of the traditional Chinese medicine compound containing Rheum palmatum in the application were provided by Fuhua Pharmaceutical Co., Ltd.; the reagents such as ethanol, methanol, acetone, and n-butanol used were of analytical purity (National Pharmaceutical Group Chemical Reagent Co., Ltd.).

[0188] 1.2. Thin layer plate

[0189] Prepared guaiac G thin layer plate (HX73416553 1.05553.0001, 20x20cm, Merck, Germany);

[0190] Prepared silica gel G thin layer plate (Yantai Chemical Industry Research Institute, 20190307, 100x100mm);

[0191] Prepared guaiac G (Qingdao Marine Chemical Plant, 20240117, 100x100mm).

[0192] 1.3. Reference substance

[0193] Sanguisorba officinalis reference medicinal material (China Institute for Drug Control, 121286-201703);

[0194] Rheum palmatum reference medicinal material (China Institute for Drug Control, 121291-201803);

[0195] Buxus chinensis L. control drug material (Chengdu Rui Fensidan Biological Technology Co., Ltd. DZYC-Y-036);

[0196] Rhizoma Coptidis control drug material (Chengdu Rui Fensidan Biological Technology Co., Ltd. DZYC-Y-078).

[0197] 1.4, test solution extraction method

[0198] Test sample extraction method A: 4 g of traditional Chinese medicine compound sample powder containing Rumex nepalensis Spreng was weighed, 50 mL of water was added, ultrasonic treatment was performed for 30 min, filtration was performed, 25 mL of filtrate was taken, water-saturated n-butanol solution was extracted for 3 times, each time 40 mL, the extraction liquid was combined, water-saturated n-butanol solution was recovered, methanol was redissolved, filtration was performed, and the test sample solution A was obtained.

[0199] Test sample extraction method B: 4 g of traditional Chinese medicine compound sample powder containing Rumex nepalensis Spreng was weighed, 50 mL of water was added, ultrasonic treatment was performed for 30 min, filtration was performed, 25 mL of filtrate was taken, water-saturated n-butanol solution was extracted for 3 times, each time 40 mL, the extraction liquid was combined, ammonia reagent was washed for 3 times, each time 40 mL, the solution was evaporated, methanol was redissolved, filtration was performed, and the test sample solution B was obtained.

[0200] Test sample extraction method C: 1 g of traditional Chinese medicine compound sample powder containing Rumex nepalensis Spreng was weighed, 25 mL of methanol was added, ultrasonic treatment was performed for 30 min, filtration was performed, and the test sample solution C was obtained.

[0201] Test sample extraction method D: 1 g of traditional Chinese medicine compound sample powder containing Rumex nepalensis Spreng was weighed, 25 mL of ethanol was added, ultrasonic treatment was performed for 30 min, filtration was performed, and the test sample solution D was obtained.

[0202] Test sample extraction method E: 1 g of traditional Chinese medicine compound sample powder containing Rumex nepalensis Spreng was weighed, 25 mL of acetone was added, ultrasonic treatment was performed for 30 min, filtration was performed, and the test sample solution E was obtained.

[0203] Test sample extraction method F: 4 g of traditional Chinese medicine compound sample powder containing Rumex nepalensis Spreng was weighed, 50 mL of water was added, ultrasonic treatment was performed for 30 min, filtration was performed, and the test sample solution F was obtained.

[0204] 1.5, preparation method of the traditional Chinese medicine compound containing Rumex nepalensis Spreng of the present application

[0205] The preparation method comprises the following steps: taking about 50 g of bletilla striata roots, crushing into medium powder; taking about 650 g of bletilla striata roots, about 700 g of sanguisorba officinalis, about 300 g of radix rhei, about 300 g of corydalis, adding about 50% ethanol, warm soaking for about 30 minutes, refluxing extraction for about 2 times at about 80°C, each time for about 1.5 hours, combining the extraction liquid, filtering, recovering ethanol from the filtrate under reduced pressure and concentrating to a thick paste with a relative density of about 1.25-1.35 (about 85°C), vacuum drying at about 70°C, crushing into fine powder, adding the above-mentioned medium powder and about 100 g of starch, and uniformly mixing to obtain.

[0206] 2. Optimization of thin layer development conditions

[0207] 2.1. Preparation of reference medicinal material solution

[0208] 2.1.1. Preparation of corydalis reference medicinal material solution

[0209] Take 0.5 g of corydalis reference medicinal material, add 25 mL of methanol, ultrasonic extraction for 30 minutes, filter, and take the filtrate as the corydalis reference medicinal material solution.

[0210] 2.1.2. Preparation of sanguisorba officinalis reference medicinal material solution

[0211] Take 0.5 g of sanguisorba officinalis reference medicinal material, add 25 mL of methanol, ultrasonic extraction for 30 minutes, filter, and take the filtrate as the sanguisorba officinalis reference medicinal material solution.

[0212] 2.1.3. Preparation of radix rhei reference medicinal material solution

[0213] Take 0.5 g of radix rhei reference medicinal material, add 25 mL of methanol, ultrasonic extraction for 30 minutes, filter, and take the filtrate as the radix rhei reference medicinal material solution.

[0214] 2.1.4. Preparation of bletilla striata root reference medicinal material solution

[0215] Take 0.5 g of bletilla striata root reference medicinal material, add 25 mL of methanol, ultrasonic extraction for 30 minutes, filter, and take the filtrate as the bletilla striata root reference medicinal material solution.

[0216] 2.2. Preparation of negative test sample solution

[0217] Sanguisorba officinalis negative test sample solution: take 0.5 g of sanguisorba officinalis negative extract, add 25 mL of methanol, ultrasonic treatment for 30 min, filter, and take the filtrate as the sanguisorba officinalis negative test sample solution.

[0218] Radix rhei negative test sample solution: take 0.5 g of radix rhei negative extract, add 25 mL of methanol, ultrasonic treatment for 30 min, filter, and take the filtrate as the radix rhei negative test sample solution.

[0219] Coptis chinensis negative test solution: Take 0.5 g of Coptis chinensis negative extract, add 25 mL of methanol, sonicate for 30 min, filter, and use as Coptis chinensis negative test solution.

[0220] 2.3 Thin-layer development conditions and optimization

[0221] 2.3.1 Thin-layer development conditions

[0222] According to the thin-layer chromatography method (General Rule 0502) in Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2 μL of each of the above solutions and spot them separately on the same silica gel G thin-layer plate. Develop the plate with cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1), remove the plate, air dry it, and examine it under ultraviolet light (365 nm).

[0223] 2.3.2 Optimization of developing solvent types

[0224] ① Ethyl acetate-methanol-water (100:17:13) + cyclohexane-ethyl acetate-isopropanol-methanol-water (3:3.5:1:1.5:0.5), the result is as follows Figure 1 As shown.

[0225] Under these conditions, the separation of Sanguisorba officinalis, Michelia alba root, and Coptis chinensis was poor, with no corresponding spots compared to the control herbs, and the spots were trailing.

[0226] ② Ethyl acetate-methanol-water (100:17:13) + cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1) + 2 μl of sample was spotted, and the results are as follows. Figure 2 As shown.

[0227] Under these conditions, the roots of Sanguisorba officinalis and Clerodendrum trichotomum were poorly separated and showed no corresponding spots compared to the control herbs. (The content of Clerodendrum trichotomum roots in ① and ② was relatively low, so they were excluded as controls.)

[0228] ③ The lower layer of chloroform-methanol-water (13:7:2) + cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1) yielded the following results: Figure 3 As shown.

[0229] Under these development conditions, only three spots were visible with trailing patterns. The separation of *Sanguisorba officinalis* and *Coptis chinensis* was poor, and there were no corresponding spots in the control herb. Furthermore, a comparison was made between water extraction of a traditional Chinese medicine compound sample containing *Rheum palmatum*, using n-butanol extraction followed by ammonia washing and non-ammonia washing treatments. The comparison revealed no significant difference between ammonia washing and non-ammonia washing.

[0230] ④ The lower layer of ethyl acetate-methanol-water (100:17:13) + chloroform-methanol-water (13:7:2) yields the following results: Figure 4 As shown.

[0231] The development condition, the spot edge is not clear, can not find the corresponding spot with the control medicinal material. And at the same time, the water extraction of traditional Chinese medicine compound sample containing Rheum palmatum, after extraction with n-butanol, ammonia washing and non-ammonia washing treatment, comparison found that ammonia washing and non-ammonia washing difference is not significant.

[0232] ⑤Ethyl acetate-formic acid (10:0.1) + cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1), as shown in the results. Figure 5

[0233] Through the above results, compared with other developing agents, ethyl acetate-formic acid (10:0.1) + cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1) is more suitable for the thin layer identification of the product. And at the same time, the water extraction of traditional Chinese medicine compound sample containing Rheum palmatum, after extraction with n-butanol, ammonia washing and non-ammonia washing treatment, comparison found that ammonia washing and non-ammonia washing difference is not significant.

[0234] 3, the selection of test sample preparation method

[0235] The effects of different extraction solvents such as methanol, ethanol, acetone and water extraction on the separation degree and clarity of characteristic spots (components) in the test sample solution for TLC identification were investigated. The effects of extraction and non-extraction treatment, as well as ammonia washing and non-ammonia washing after extraction on the separation degree and clarity of characteristic identification spots (components) in the test sample solution were also investigated. The specific sample preparation methods are as follows:

[0236] The samples prepared by the above different solvents and after extraction were observed under a 365nm ultraviolet lamp, and the thin layer chromatography results are shown in Figure 6 It can be clearly seen that the samples prepared by different extraction solvents all show rich spot information, but the light blue spot at Rf=0.35 is not obvious for ethanol extraction, acetone extraction, water extraction, water-n-butanol extraction and water-n-butanol ammonia washing, while the spot is obvious for methanol extraction. Therefore, the methanol extraction obtained the most abundant and clear spot information, and the optimal method among the five extraction methods is methanol extraction.

[0237] 4, sample spotting amount investigation

[0238] ​According to the 2020 edition of Chinese Pharmacopoeia four thin layer chromatography (general 0502) test, different amounts of test solution, control solution, respectively, on the same silica gel G thin layer plate, with ethyl acetate-formic acid (10:0.1) + cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1) as developing agent, development, take out, dry, under UV light (365 nm) inspection, the results are shown in Figure 7 .

[0239] The test solution shows that the sample size of 1 μL, 2 μL, 5 μL, 10 μL, and the control drug in the corresponding position have corresponding spots, 2 μL, 5 μL, 10 μL, and the preferred 10 μL.

[0240] 5. Specificity

[0241] The test solution, the lack of flavor negative test solution is the same as "test solution preparation", the control drug solution is the same as "control drug solution preparation"; according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general 0502) test, 2 μL of the above solution was taken and spotted on the ethyl acetate-formic acid (10:0.1) + cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1) as developing agent, development, take out, dry, under UV light (365 nm) inspection, the results are shown in Figure 8 .

[0242] Figure 8 The test chromatogram shows that the test chromatogram has the same color spots in the corresponding position of the control and control drug chromatogram, and the separation degree is good, and the negative control chromatogram has no spot in the corresponding position of the control and control drug chromatogram. From the results, the negative has no interference, and the specificity is good.

[0243] 6. Investigation of different wavelengths

[0244] 2 μL of the control drug solution of 3.1.1, 3.1.2, 3.1.3 and 3.2 of the test solution was taken, according to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general 0502) test, respectively, on the ethyl acetate-formic acid (10:0.1) + cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1) as developing agent, development, take out, dry, respectively, under UV light (365 nm) and visible light inspection, the results are shown in Figure 9 .

[0245] Figure 9The test sample chromatogram and the reference material chromatogram showed rich spot information under ultraviolet light (365 nm). The typical characteristics of Coptis chinensis and Sanguisorba officinalis in the TCM compound granules containing Rumex nepalensis were clearly visible. An orange-red spot was visible in Rumex nepalensis, but since the spot was at the solvent front, it was not sufficient as a basis for identification. Under visible light, Rumex nepalensis reference material and the three batches of samples all showed a clear pink spot (as shown in the black box), which can be used as a typical component identification characteristic of Rumex nepalensis in TCM compound granules containing Rumex nepalensis. Therefore, it is recommended to use ultraviolet light (365 nm) + visible light to observe comprehensively. Figure 9

[0246] 7. Adaptability of thin layer plates from different manufacturers

[0247] Three thin layer pre-plates (Merk, Yantai Yinlong, Qingdao Haoyang) were selected for thin layer testing. The results are shown in Table 7. Figure 10

[0248] The results showed that the test sample chromatogram had the same color spots at the corresponding positions of the control and reference material chromatograms, with good separation and no interference from the negative control. The Rf values varied slightly. The thin layer plates from the three manufacturers could all be used for the identification of Coptis chinensis, Sanguisorba officinalis, and Rumex nepalensis.

[0249] 8. TLC analysis of three batches of TCM compound granules containing Rumex nepalensis

[0250] Through optimization of the developing conditions, investigation of sample preparation methods, investigation of sample spotting volume, investigation of the specificity of the method, investigation of different observation wavelengths, and investigation of the adaptability of thin layer plates from different manufacturers, the optimal developing agent composition and ratio were determined to be: ethyl acetate-formic acid (10:0.1) + cyclohexane-ethyl acetate-isopropyl alcohol-methanol-water-triethylamine (3:3.5:1:1.5:0.5:1). The optimal sample preparation method was methanol extraction. The optimal spotting volume was 10 μL. The specificity investigation found that there was no interference at the characteristic spot, indicating that the method had good specificity. The optimal observation wavelength was ultraviolet light (365 nm), under which the characteristic identification spots of Coptis chinensis and Sanguisorba officinalis, Rumex nepalensis were visible, combined with the observation of the characteristic identification spot of Rumex nepalensis under visible light. The identification characteristics were clear on thin layer plates from different manufacturers, with good separation of the characteristic spots. The three types of thin layer plates were all suitable for the identification. Based on the above optimization conditions, three batches of TCM compound samples containing Rumex nepalensis were prepared and analyzed, and the test results are shown in Table 8. The three batches of samples all showed the characteristic spots of Coptis chinensis, Rumex nepalensis, and Sanguisorba officinalis, with good reproducibility between batches, indicating that the method can be used for the qualitative identification of the three medicinal materials in TCM compound granules containing Rumex nepalensis. Figure 10

[0251] ​​​The above has carried out the detailed introduction to the embodiment of the application, the principle and implementation mode of the application have been described by applying specific examples in this paper, the above embodiment description is only used for helping understanding the method of the application and its core idea. At the same time, the changes or deformations made by the person skilled in the art according to the idea of the application, based on the specific implementation mode and application range of the application, all belong to the protection scope of the application. In summary, the content of the specification should not be understood as the limitation of the application.

Claims

1. A method for simultaneous identification of three medicinal materials in a traditional Chinese medicine compound containing rhubarb, characterized in that, The thin-layer qualitative identification method includes the following steps: (1) The traditional Chinese medicine compound containing Rheum palmatum was pretreated to obtain the test solution, the traditional Chinese medicine compound lacking Sanguisorba officinalis, Rheum palmatum or Coptis chinensis was pretreated to obtain the negative sample solution, and the control medicinal material was pretreated to obtain the control medicinal material solution. as well as (2) Thin-layer chromatography was performed to identify the test solution, the negative sample solution and the reference medicinal material solution; In the thin-layer chromatography identification analysis step, a developing solvent is used for development. The developing solvent includes a first developing solvent and a second developing solvent. The first developing solvent is ethyl acetate-formic acid, and the second developing solvent is cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine. The volume ratio of ethyl acetate to formic acid in the first developing solvent is (1~20):(0.01~1). The volume ratio of cyclohexane, ethyl acetate, isopropanol, methanol, water and triethylamine in the second developing solvent is (1~10):(1~10):(0.1~5):(0.1~5):(0.1~3):(0.1~5).

2. The thin-layer qualitative identification method according to claim 1, characterized in that, The traditional Chinese medicine compound containing rhubarb includes Coptis chinensis, Sanguisorba officinalis, rhubarb, and the root of Rosa rugosa. Preferably, the dosage form of the traditional Chinese medicine compound containing rhubarb is capsules or granules; Preferably, the negative sample solution is a negative sample solution lacking Sanguisorba officinalis, a negative sample solution lacking Rheum palmatum, and / or a negative sample solution lacking Coptis chinensis; Preferably, the reference medicinal material is Sanguisorba officinalis reference medicinal material, Coptis chinensis reference medicinal material and / or Rheum palmatum reference medicinal material; More preferably, the preparation method of the traditional Chinese medicine compound containing rhubarb includes the following steps: (a) Take an appropriate amount of rouge flower root and grind it into a medium powder; and (b) Take an appropriate amount of rouge flower root, burnet root, rhubarb root and coptis root, add a certain concentration of ethanol and soak for a certain time, reflux extract at a certain temperature 1 to 4 times, each time for 0.5 to 2 hours, combine the extracts, filter, recover ethanol from the filtrate under reduced pressure and concentrate to a thick paste with a relative density of about 1.1 to 1.45 (80℃ to 90℃), vacuum dry at 60℃ to 80℃, pulverize into fine powder, add the above medium powder and a certain amount of starch, mix well to obtain the product; More preferably, the mass of the rouge flower root in step (a) is 20 to 100 g, for example, about 50 g; More preferably, the mass of the rouge root in step (b) is 600-700g, for example, about 650g; More preferably, the mass of Sanguisorba officinalis in step (b) is 650-750g, for example, about 700g; More preferably, the mass of rhubarb in step (b) is 200-400g, for example, about 300g; More preferably, the mass of Coptis chinensis in step (b) is 200-400g, for example, about 300g; More preferably, the ethanol concentration in step (b) is 40% to 60%, for example, about 50%; More preferably, the soaking time in step (b) is 0.2 to 1 hour, for example, about 30 minutes; More preferably, the reflux extraction temperature in step (b) is 70°C to 90°C, for example, about 80°C; More preferably, the reflux extraction in step (b) is performed twice; More preferably, the time for each reflux extraction in step (b) is approximately 1.5 hours; More preferably, the preparation method of the traditional Chinese medicine compound containing rhubarb includes the following steps: take about 50g of *Rhizoma Cimicifugae* root and crush it into medium powder; take about 650g of *Rhizoma Cimicifugae* root, about 700g of *Radix Sanguisorbae*, about 300g of rhubarb, and about 300g of *Coptis chinensis*, add about 50% ethanol and soak at a warm temperature for about 30 minutes, reflux extract twice at about 80°C, each time for about 1.5 hours, combine the extracts, filter, recover ethanol from the filtrate under reduced pressure and concentrate to a thick paste with a relative density of about 1.25-1.35 (about 85°C), vacuum dry at about 70°C, crush into fine powder, add the above medium powder and about 100g of starch, and mix well to obtain the final product.

3. The thin-layer qualitative identification method according to claim 1, characterized in that, The preparation method of the test solution includes: weighing an appropriate amount of traditional Chinese medicine compound containing rhubarb, adding solvent, sonicating, filtering, and obtaining the test solution; Preferably, the solvent is methanol; Preferably, the method for preparing the test solution does not include the steps of extraction and washing with ammonia reagent after extraction; More preferably, the mass / volume (g / ml) ratio of the traditional Chinese medicine compound containing rhubarb to the solvent is 0.01 to 0.1, for example, about 0.04; More preferably, the mass of the traditional Chinese medicine compound containing rhubarb is 0.1-5 g, for example, about 1 g; More preferably, the volume of the methanol is 20-30 ml, for example, about 25 ml; More preferably, the ultrasonic treatment time is 20 to 40 minutes, for example, about 30 minutes.

4. The thin-layer qualitative identification method according to claim 1, characterized in that, The preparation method of the reference medicinal material solution includes: weighing an appropriate amount of Sanguisorba officinalis, Coptis chinensis or Rheum palmatum reference medicinal materials, adding solvent for ultrasonic extraction, filtering, and obtaining the reference medicinal material solution; Preferably, the solvent is methanol; Preferably, the mass / volume (g / ml) ratio of the reference medicinal materials, such as Sanguisorba officinalis, Coptis chinensis, or Rheum palmatum, to the solvent is 0.001 to 0.1, for example, about 0.02; More preferably, the mass of the *Sanguisorba officinalis* reference material is 0.1-1 g, for example, about 0.5 g; More preferably, the mass of the Coptis chinensis reference material is 0.1~1 g, for example, about 0.5 g; More preferably, the mass of the rhubarb reference material is 0.1~1 g, for example, about 0.5 g; More preferably, the volume of the methanol is 20-30 ml, for example, about 25 ml; Preferably, the ultrasonic extraction time is 20-40 minutes, for example, about 30 minutes.

5. The thin-layer qualitative identification method according to claim 3, characterized in that, The preparation method of the negative sample solution includes: weighing appropriate amounts of traditional Chinese medicine compound lacking Sanguisorba officinalis, Rheum palmatum, or Coptis chinensis, and the subsequent steps are the same as the preparation method of the test sample solution.

6. The thin-layer qualitative identification method according to claim 1, characterized in that, The volume ratio of ethyl acetate to formic acid in the first developing solvent is (5~15):(0.05~0.5). Preferably, the volume ratio of ethyl acetate to formic acid in the first developing solvent is (8~12):(0.08~0.12). More preferably, the volume ratio of ethyl acetate to formic acid in the first developing solvent is about 10: about 0.1; Preferably, the volume ratio of cyclohexane, ethyl acetate, isopropanol, methanol, water, and triethylamine in the second developing agent is (1~6):(2~6):(0.5~1.5):(1~2):(0.3~1.5):(0.5~1.5). More preferably, the volume ratio of cyclohexane, ethyl acetate, isopropanol, methanol, water and triethylamine in the second developing agent is (2~4):(3~4):(0.8~1.2):(1.2~1.8):(0.4~1):(0.8~1.2). Particularly preferably, the volume ratio of the cyclohexane, ethyl acetate, isopropanol, methanol, water and triethylamine in the second developing agent is about 3: about 3.5: about 1: about 1.5: about 0.5: about 1.

7. The thin-layer qualitative identification method according to claim 1, characterized in that, The thin-layer plate is a silicone G thin-layer plate; Preferably, the silicone G thin film is a Merck silicone G thin film from Germany, a Yantai Yinlong silicone G thin film, or a Qingdao Haiyang silicone G thin film. More preferably, the inspection conditions for Coptis chinensis and Sanguisorba officinalis in the thin-layer chromatography identification analysis step are as follows: after the thin-layer plate has been developed, it is taken out, dried, and inspected under ultraviolet light. More preferably, the inspection conditions for rhubarb in the thin-layer chromatography identification and analysis step are as follows: take out the developed thin-layer plate, dry it, and inspect it under ultraviolet light and visible light. Preferably, the detection wavelength of the ultraviolet lamp is 365 nm; More preferably, the sample volume of the test solution is 1.5 to 12 μl, for example, about 2 μl, or about 5 μl, or about 10 μl; Particularly preferably, the sample volume of the test solution is about 10 μl; Particularly preferably, the amount of the reference medicinal material solution applied is 1~5 μl, for example 1~3 μl, for example about 2 μl; Particularly preferably, the amount of the negative sample solution applied is 1 to 5 μl, for example 1 to 3 μl, for example about 2 μl.

8. The thin-layer qualitative identification method according to claim 1, characterized in that, The criteria for thin-layer chromatography identification include: examining under ultraviolet light whether characteristic spots of the same color are displayed at corresponding positions on the thin-layer plate and the corresponding positions on the control herbs Coptis chinensis, Sanguisorba officinalis, and Rheum palmatum; examining under visible light whether characteristic spots of the same color are displayed at corresponding positions on the thin-layer plate and the control herbs on the Rheum palmatum; and whether there is interference from negative samples. If the thin-layer plate has the characteristic spots under both ultraviolet and visible light and the negative samples do not interfere, then the detected traditional Chinese medicine compound containing Rheum palmatum contains Coptis chinensis, Sanguisorba officinalis, and Rheum palmatum.

9. The use of the thin-layer qualitative identification method according to any one of claims 1 to 8 in identifying traditional Chinese medicine compound containing rhubarb.

10. A kit for qualitative identification of traditional Chinese medicine compound containing rhubarb, characterized in that, The kit includes a thin-layer plate and a developing solvent, wherein the developing solvent includes a first developing solvent and a second developing solvent, wherein the first developing solvent is ethyl acetate-formic acid, the second developing solvent is cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine, and the thin-layer plate is a silica gel G thin-layer plate. Preferably, the volume ratio of ethyl acetate to formic acid in the first developing solvent is (1~20):(0.01~1). More preferably, the volume ratio of ethyl acetate to formic acid in the first developing solvent is (5~15):(0.05~0.5). Preferably, the volume ratio of ethyl acetate to formic acid in the first developing solvent is (8~12):(0.08~0.12). More preferably, the volume ratio of ethyl acetate to formic acid in the first developing solvent is about 10: about 0.1; Particularly preferably, the volume ratio of cyclohexane, ethyl acetate, isopropanol, methanol, water and triethylamine in the second developing agent is (1~10):(1~10):(0.1~5):(0.1~5):(0.1~3):(0.1~5). Particularly preferably, the volume ratio of cyclohexane, ethyl acetate, isopropanol, methanol, water, and triethylamine in the second developing agent is (1~6):(2~6):(0.5~1.5):(1~2):(0.3~1.5):(0.5~1.5). Particularly preferred is that the volume ratio of cyclohexane, ethyl acetate, isopropanol, methanol, water, and triethylamine in the second developing agent is (2~4):(3~4):(0.8~1.2):(1.2~1.8):(0.4~1):(0.8~1.2). Most preferably, the volume ratio of the cyclohexane, ethyl acetate, isopropanol, methanol, water and triethylamine in the second developing solvent is about 3: about 3.5: about 1: about 1.5: about 0.5: about 1.

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