Method for simultaneously identifying angelica sinensis, radix rehmanniae recen, scutellaria baicalensis, coptis chinensis, golden cypress and astragalus membranaceus by using thin-layer chromatography

Abstract

The invention relates to a method for simultaneously identifying angelica sinensis, radix rehmanniae recen, scutellaria baicalensis, coptis chinensis, golden cypress and astragalus membranaceus by using thin-layer chromatography. The method comprises the following steps: A, preparing a test solution; b, preparing a reference substance solution; and C, thin-layer chromatographic analysis. The method disclosed by the invention can be used for simultaneously identifying the six medicines, namely angelica sinensis, radix rehmanniae recen, scutellaria baicalensis, coptis chinensis, golden cypress and astragalus membranaceus, is simple, convenient and quick to operate, clear in spots, good in separation degree, strong in specificity, free of negative interference and strong in durability, reduces the experiment times of thin-layer identification, saves time and cost, greatly improves the detection efficiency, effectively reduces the detection cost, and is suitable for popularization and application. The method provides an important technical means for quality control of the Chinese angelica and six-yellow decoction, and is an important guarantee for realizing safety and effectiveness of the Chinese angelica and six-yellow decoction.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine component detection, and in particular to a method for simultaneously identifying angelica sinensis, raw rehmannia root, scutellaria baicalensis, coptis chinensis, phellodendron amurense and astragalus membranaceus by using thin layer chromatography. Background Art

[0002] Danggui Liuhuang Decoction was first seen in Lanshi Mizang, Volume 2, Spontaneous Sweating, and is one of the first 100 classic prescriptions announced by the State Administration of Traditional Chinese Medicine. Danggui Liuhuang Decoction is ranked 47th and is a Chinese medicine compound preparation managed according to the catalog of ancient classic prescriptions.

[0003] The original recipe of Danggui Liuhuang Decoction is composed of "Danggui, Shengdihuang, Shudihuang, Huangbai, Huangqin, Huanglian (equal parts), Huangqi (double)". The original book records the method of decoction as "the above are ground into coarse powder, take 5 qian each time, add 2 cups of water, boil until 1 cup, take before meals, children take half the dose". According to the theory of traditional Chinese medicine, Danggui, Shengdihuang, and Shudihuang in the recipe enter the liver and kidney to nourish yin and blood. When yin and blood are full, water can control fire, and they are all monarch drugs; Huanglian is used as the minister to clear the heart and purge fire, and Huangbai and Huangbai are bitter and cold to purge fire to strengthen yin; Huangqi is used twice to replenish qi and strengthen the defense to consolidate the exterior, and Danggui and Shudihuang are used to replenish qi and nourish blood, which are also minister drugs; monarch and minister are combined, nourishing yin and purging fire, replenishing qi and consolidating the exterior, taking into account both the symptoms and the root causes, and playing the role of nourishing yin and purging fire, consolidating the exterior and stopping sweating. Danggui Liuhuang Decoction is commonly used in TCM clinics to treat various sweating syndromes and patients with yin deficiency and hyperactivity of fire, such as hyperthyroidism, diabetes, and perimenopausal syndrome. It has a definite effect, and its preparation as a Chinese patent medicine has important clinical significance and value. In August 2022, the State Administration of Traditional Chinese Medicine announced the key information of Danggui Liuhuang Decoction, indicating that the research and development of new drugs for Danggui Liuhuang Decoction has entered the key implementation stage.

[0004] The thin-layer identification method is the main means of qualitative identification in the quality standards of compound Chinese medicine preparations. The "Drafting Instructions for the Quality Standards of Compound Chinese Medicine Preparations" in the "Technical Requirements for the Research of New Chinese Medicines" requires under the [Identification] item: "Corresponding identification items can be established based on the prescription composition and research data. In principle, each medicinal ingredient in the prescription should be tested and studied, and selected for inclusion in the standard based on the test results." At present, the [Identification] item of the quality standards for compound Chinese medicine preparations, including the "Chinese Pharmacopoeia", is basically set up for identification of a single medicinal ingredient. Each compound often requires a variety of test sample preparation methods, developing agents, and developing conditions to identify different medicinal ingredients separately, and the number of tests required is often large. The "Technical Guiding Principles for the Research of Quality Standards for New Chinese Medicines (Trial)" piloted in May 2020 encourages the establishment of thin-layer chromatography methods for the simultaneous identification of multiple medicinal ingredients.

[0005] CN113759065A discloses a thin layer chromatography method for simultaneously identifying the components of Coptis chinensis and Phellodendron chinense, but it does not involve the identification of other components besides Coptis chinensis and Phellodendron chinense; CN117420253A discloses a thin layer chromatography identification method for Danggui Liuhuang Decoction and its compound preparation, but it only involves the qualitative identification of the Astragalus component.

[0006] At present, there is no literature reporting the thin layer chromatography method for simultaneously identifying Angelica sinensis, Rehmannia root, Scutellaria baicalensis, Coptis chinensis, Phellodendron chinense and Astragalus membranaceus in Danggui Liuhuang Decoction. Finding a simple and fast method for detecting Chinese herbal compound preparations is an important measure for quality control of Chinese medicine. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a thin layer chromatography method for simultaneously identifying angelica, raw rehmannia, scutellaria, coptis, phellodendron and astragalus in Danggui Liuhuang Decoction. The method of the present invention can simultaneously identify the six herbs in the prescription, is simple and quick to operate, has clear spots, good separation, strong specificity, no negative interference, strong durability, reduces the number of experiments for thin layer identification, saves time and cost, greatly improves detection efficiency, effectively reduces detection cost, provides important technical means for quality control of Danggui Liuhuang Decoction, and is an important guarantee for achieving its safety and effectiveness.

[0008] In one aspect, the present invention provides a method for simultaneously identifying Chinese angelica, Radix Rehmanniae, Radix Scutellariae, Rhizoma Coptidis, Cortex Phellodendri and Radix Astragali using thin layer chromatography, comprising the following steps:

[0009] A. Preparation of test solution:

[0010] A.1. Extract the aqueous solution of the Angelica sinensis and six yellows composition with ether to obtain an ether extract and a first aqueous layer solution. Dry the ether extract to dryness, and dissolve the residue in methanol to obtain an ether layer test solution.

[0011] A.2. The first aqueous layer solution is extracted with water-saturated n-butanol to obtain an n-butanol extract and a second aqueous layer solution. The n-butanol extract is extracted with an ammonia test solution to obtain an n-butanol layer and an ammonia test solution layer. The n-butanol layer and the ammonia test solution layer are dried to dryness, respectively. The residue is dissolved with methanol to obtain an n-butanol layer test solution and an ammonia test solution layer test solution.

[0012] A.3. The second aqueous layer solution is subjected to solid-liquid separation to obtain a solution and a precipitate, which are dried to dryness respectively, and the residue is dissolved in methanol to obtain an aqueous layer solution test solution and an aqueous layer precipitate test solution;

[0013] B. Preparation of reference solution:

[0014] B.1. Add methanol to ferulic acid, ligustilide, berberine hydrochloride and palmatine hydrochloride reference substances to prepare mixed reference substance solution 1;

[0015] B.2. Take baicalin, phellodendronine hydrochloride and astragaloside IV reference substances, and add methanol to prepare baicalin reference substance solution, phellodendronine hydrochloride reference substance solution and astragaloside IV reference substance solution respectively;

[0016] B.3. Extract the Radix Rehmanniae glutinosae with methanol, and take the extract as the Radix Rehmanniae glutinosae glutinosae reference medicinal material solution;

[0017] C. Thin layer chromatography analysis:

[0018] C.1. Take the ether layer test solution prepared in step A, the n-butanol layer test solution, and the mixed reference solution 1 prepared in step B, and spot them on the same silica gel G plate respectively. Use the developing agent Z1 to develop and then dry. Place the dried silica gel G plate under ultraviolet light for inspection.

[0019] When a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the test sample in the ether layer as that of the chromatogram of the ferulic acid reference substance and the ligustilide reference substance, it is identified as the presence of Angelica sinensis;

[0020] In the chromatogram of the n-butanol layer test sample, when a fluorescent spot of the same color is shown at a position corresponding to the chromatogram of the berberine hydrochloride reference substance, it is identified as the presence of Coptis chinensis and / or Phellodendron chinense, and when a fluorescent spot of the same color is shown at a position corresponding to the chromatogram of the bamipine hydrochloride reference substance, it is identified as the presence of Coptis chinensis;

[0021] C.2. Take the n-butanol layer test solution, ammonia test solution layer test solution, water layer test solution and water layer precipitation test solution prepared in step A, and the raw rehmannia reference medicinal material solution, baicalin reference solution, phellodendron hydrochloride reference solution and astragaloside IV reference solution prepared in step B, and spot them on the same silica gel GF254 plate, develop with developing agent Z2 and then dry.

[0022] Place the dried silica gel GF254 plate under ultraviolet light for inspection. If a spot of the same color appears at the corresponding position of the chromatogram of the precipitated test sample in the water layer and the chromatogram of the baicalin reference substance, it is identified as the presence of Scutellaria baicalensis.

[0023] Place the silica gel GF254 plate after ultraviolet light inspection in an iodine jar for color development and inspect it under sunlight. If a spot of the same color appears at the corresponding position of the chromatogram of the test sample in the ammonia test solution layer and the chromatogram of the reference substance of phellodendron hydrochloride, it is identified as the presence of phellodendron;

[0024] Spray the silica gel GF254 plate after iodine color development with a color developer, heat until the spots are clearly colored, and examine under sunlight. If a spot of the same color appears at the corresponding position of the characteristic spot of the raw rehmannia reference medicinal material solution in the chromatogram of the water layer supernatant test sample, it is identified as the presence of raw rehmannia; if a spot of the same color appears at the corresponding position of the chromatogram of the astragaloside reference substance in the chromatogram of the n-butanol layer test sample, it is identified as the presence of astragalus.

[0025] The developing agent Z1 is a mixed solution of ethyl acetate, methanol, chloroform, ammonia water and triethylamine; the developing agent Z2 is a mixed solution of ethyl acetate, methanol, formic acid and water, and the color developer is ethanol sulfuric acid solution.

[0026] In some embodiments, the method of the present invention comprises the steps of:

[0027] A. Preparation of test solution:

[0028] A.1. Extract the aqueous solution of the Angelica sinensis and six yellows composition with ether twice to obtain an ether extract and a first water layer solution. Evaporate the ether extract to dryness in a water bath, and dissolve the residue in methanol to obtain an ether layer test solution.

[0029] A.2. The first aqueous layer solution is extracted twice with water-saturated n-butanol to obtain an n-butanol extract and a second aqueous layer solution. The n-butanol extract is further extracted twice with an ammonia test solution to obtain an n-butanol layer and an ammonia test solution layer. The n-butanol layer and the ammonia test solution layer are evaporated to dryness in a water bath and then dissolved with methanol to obtain an n-butanol layer test solution and an ammonia test solution layer test solution;

[0030] A.3. Centrifuge the second aqueous layer solution, evaporate the supernatant and precipitate in a water bath, and dissolve the residue in methanol to serve as the aqueous layer supernatant test solution and the aqueous layer precipitate test solution;

[0031] B. Preparation of reference solution:

[0032] B.1. Add methanol to ferulic acid, ligustilide, berberine hydrochloride and palmatine hydrochloride reference substances to prepare a mixed reference substance solution 1.

[0033] B.2. Take baicalin, phellodendronine hydrochloride and astragaloside IV reference substances, and add methanol to prepare baicalin reference substance solution, phellodendronine hydrochloride reference substance solution and astragaloside IV reference substance solution respectively;

[0034] B.3. Take the Radix Rehmanniae glutinosae reference medicinal material, add methanol and sonicate, and take the supernatant as the Radix Rehmanniae glutinosae reference medicinal material solution.

[0035] C. Thin layer chromatography analysis:

[0036] C.1. Take the ether layer test solution prepared in step A, the n-butanol layer test solution, and the mixed reference solution 1 prepared in step B, and spot them on the same silica gel G plate, develop them with developing agent Z1, dry them, and inspect them under ultraviolet light.

[0037] When a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the test sample in the ether layer as that of the chromatogram of the ferulic acid reference substance and the ligustilide reference substance, it is identified as the presence of Angelica sinensis;

[0038] In the n-butanol layer test sample chromatogram, when a fluorescent spot of the same color is displayed at a position corresponding to the berberine hydrochloride reference sample chromatogram, it is identified as the presence of Coptis chinensis and / or Phellodendron chinense, and when a fluorescent spot of the same color is displayed at a position corresponding to the bamipine hydrochloride reference sample chromatogram, it is identified as the presence of Coptis chinensis;

[0039] C.2. Pipette the n-butanol layer test solution, ammonia test solution layer test solution, water layer supernatant test solution and water layer precipitate test solution prepared in step A, and the Radix Rehmanniae Root reference medicinal material solution, baicalin reference solution, phellodendron hydrochloride reference solution and astragaloside IV reference solution prepared in step B, respectively, onto the same silica gel GF254 plate, develop with developing agent Z2, and air dry;

[0040] Place the dried silica gel GF254 plate under ultraviolet light for inspection. If a spot of the same color appears in the chromatogram of the aqueous layer precipitate test sample at the corresponding position of the chromatogram of the baicalin reference sample, it is identified as the presence of Scutellaria baicalensis.

[0041] After the ultraviolet light inspection, the silica gel GF254 plate is placed in an iodine jar for color development and inspected under sunlight. When a spot of the same color appears at the corresponding position of the chromatogram of the phellodendron hydrochloride reference substance in the chromatogram of the test sample in the ammonia test solution layer, it is identified as the presence of phellodendron;

[0042] The silica gel GF254 plate after color development in the iodine cylinder is sprayed with a color developer, heated until the spots are clearly colored, and inspected under sunlight. If a spot of the same color appears at the corresponding position of the characteristic spot of the raw rehmannia reference medicinal material solution in the chromatogram of the water layer supernatant test sample, it is identified as the presence of raw rehmannia; if a spot of the same color appears at the corresponding position of the chromatogram of the astragaloside reference substance in the chromatogram of the n-butanol layer test sample, it is identified as the presence of astragalus.

[0043] Wherein, the developing agents Z1, Z2 and the color developer are the same as those mentioned above.

[0044] The method of the present invention is described in more detail below.

[0045] In the present invention, the Danggui Liuhuang composition is a Chinese medicine composition, a Chinese medicine compound decoction or a compound preparation made from the Chinese medicine compound decoction, which contains Danggui, Radix Rehmanniae, Radix Rehmanniae Preparata, Radix Scutellariae, Rhizoma Coptidis, Cortex Phellodendri and Radix Astragali at the same time. In particular, the Danggui Liuhuang composition can be Danggui Liuhuang decoction or a compound preparation of Danggui Liuhuang decoction, such as Danggui Liuhuang decoction reference sample (water decoction or freeze-dried powder), Danggui Liuhuang decoction preparation (granules, etc.) and its intermediate reflux extract, concentrate, extract powder, etc.

[0046] For example, the compound preparation can be a solid preparation or a liquid preparation. Solid preparations can be tablets, powders, granules, capsules, etc., but are not limited thereto; liquid preparations can be oral liquids, decoctions, extracts, etc., but are not limited thereto.

[0047] The aqueous solution of the Angelica sinensis and six yellows composition can be a decoction obtained by boiling or heating and refluxing the pieces of each medicinal material directly or after grinding them, or it can be an aqueous solution obtained by dissolving the compound preparation in water.

[0048] A. Preparation of test solution:

[0049] The inventors found in the experiment that the preparation process of the test solution will affect the identification of the target component (see Experimental Example 1 Conditions 1 to 3). If the Angelica dahurica Liuhuang composition is directly dissolved in methanol to prepare the test sample, due to the complexity of the Chinese medicine components and the disparity in the content of the target components, it cannot be effectively separated and identified in thin layer chromatography (see Experimental Example 1 Condition 1); after the Angelica dahurica Liuhuang composition aqueous solution is extracted with water-saturated n-butanol, the n-butanol layer solution is then extracted with ammonia test solution, and the obtained n-butanol layer test solution can identify ligustilide, but not ferulic acid (see Experimental Example 1 Conditions 2 and Figure 1 ); the aqueous solution of the Angelica angelicae sutchuenensis composition was first extracted with ether to obtain an ether extract solution test sample, and then the aqueous solution of the Angelica angelicae sutchuenensis composition after ether extraction was extracted with water-saturated n-butanol, and the n-butanol layer solution was further extracted with an ammonia test solution to obtain an n-butanol layer test solution and an ammonia test solution layer test solution. However, in the above test solution, the characteristic component of baicalin could not be detected (see Experimental Example 1 Conditions 3 and Figure 2 ); Baicalin was not observed in the supernatant obtained by dissolving the aqueous layer solution after n-butanol extraction with a small amount of methanol (see Experimental Example 1 Conditions 4 and Figure 3 ); finally, baicalin was identified in the aqueous layer precipitate test solution obtained by dissolving the aqueous layer precipitate with methanol, so that the aqueous layer precipitate test solution can be used for the specific identification of Scutellaria baicalensis. Finally, the inventors finally determined the preparation process of the test solution by combining the preparation process conditions of the test solution with the selection of the developing agent and the selection of the color developing agent.

[0050] In some embodiments, in step A, the extraction is an equal volume extraction.

[0051] In some embodiments, an aqueous solution of the Danggui Liuhuang composition is prepared as follows: take 4-7 g (e.g., 7 g) of a benchmark sample or extract powder of the Danggui Liuhuang composition or an equivalent amount thereof, for example, 6-10 g (e.g., 10 g) of Danggui Liuhuang decoction granules prepared from 2 / 3 of the Danggui Liuhuang decoction extract powder and 1 / 3 of the total weight of pharmaceutical excipients, and dissolve it in 40 mL of water.

[0052] Here, for example, the Danggui Liuhuang combination benchmark sample is prepared as follows: take the medicinal materials of the Danggui Liuhuang combination (including Danggui, Raw Rehmannia, Prepared Rehmannia, Phellodendron, Coptis, Scutellaria and Astragalus) and grind them into coarse powder through a grinder that passes through a 10-mesh sieve, add water at a liquid-to-solid ratio of about 1:30, boil until the volume of the medicinal liquid is reduced by half, filter while hot, and freeze-dry the filtrate to obtain the Danggui Liuhuang decoction benchmark sample.

[0053] For example, the Danggui Liuhuang combination extract powder is prepared as follows: take the medicinal materials of the Danggui Liuhuang combination (including Danggui, Raw Rehmannia, Prepared Rehmannia, Phellodendron, Coptis, Scutellaria and Astragalus) and add water at a liquid-to-solid ratio of about 1:30. After reflux extraction for 1-1.5 hours, the extract powder is concentrated and dried in an appropriate form to obtain the Danggui Liuhuang decoction extract powder.

[0054] For example, the Danggui Liuhuang Decoction granules are prepared as follows: the Danggui Liuhuang Decoction extract powder is mixed with a single auxiliary material (dextrin, soluble starch, mannitol) or a combination of auxiliary materials (dextrin + xylitol, dextrin + mannitol, soluble starch + xylitol) in a ratio of 2:1, and 75% ethanol is used as a wetting agent to prepare a soft material. After meeting the standard of "forming a ball when held in the hand and falling apart when pressed lightly", a No. 2 sieve is used for extrusion granulation, and the prepared granules are dried in an oven at 60°C. After drying, the granules are taken out and sized, and the granules that can pass the No. 1 sieve but not the No. 5 sieve are screened out to obtain the Danggui Liuhuang Decoction granule sample.

[0055] In some embodiments, the preparation method of the test solution in step A is as follows:

[0056] A.1. Take 4-7g of Danggui Liuhuang Decoction standard sample or extract powder or its equivalent amount (for example, 6-10g of Danggui Liuhuang Decoction granules), add 40mL of water, dissolve by ultrasonic, add an equal volume of ether and shake and extract twice to obtain ether extract and the first water layer solution, evaporate the ether extract to dryness in a water bath, dissolve the residue in methanol, and use it as the ether layer test solution;

[0057] A.2. The first aqueous layer solution was extracted twice with an equal volume of water-saturated n-butanol to obtain an n-butanol extract and a second aqueous layer solution. The n-butanol extract was extracted twice with an equal volume of ammonia test solution by shaking to obtain an n-butanol layer and an ammonia test solution layer. The n-butanol layer and the ammonia test solution layer were evaporated to dryness in a water bath, and the residues were dissolved in methanol to obtain an n-butanol layer test solution and an ammonia test solution layer test solution;

[0058] A.3. Centrifuge the second aqueous layer solution, take the supernatant and evaporate it in a water bath, dissolve the residue in methanol to obtain the aqueous layer supernatant test solution; add methanol to ultrasonically dissolve the precipitate to obtain the aqueous layer precipitate test solution.

[0059] In the present invention, the ammonia test solution refers to the ammonia test solution specified in the Chinese Pharmacopoeia.

[0060] B. Preparation of reference solution:

[0061] In step B, in the mixed reference solution 1, the concentration of ferulic acid is about 0.25-1 mg / mL, for example, 0.5 mg / mL; the concentration of ligustilide is about 0.15-0.6 mg / mL, for example, 0.3 mg / mL; the concentration of berberine hydrochloride is about 13-50 μg / mL, for example, 25 μg / mL; the concentration of palmatine hydrochloride reference is about 25-100 μg / mL, for example, 50 μg / mL; in the baicalin reference solution, the concentration of baicalin reference is about 100 μg / mL, for example, 100 μg / mL; The concentration is about 0.5-2 mg / mL, for example 1 mg / mL; in the astragaloside reference solution, the concentration of the astragaloside reference is about 0.3-1 mg / mL, for example 0.5 mg / mL; in the phellodendron hydrochloride reference solution, the concentration of the phellodendron hydrochloride reference is about 0.5-2 mg / mL, for example 1 mg / mL, and in the preparation process of the raw rehmannia reference medicinal material solution, the usage ratio of the raw rehmannia reference medicinal material to methanol is about 1 g: 10 mL, but not limited thereto.

[0062] Furthermore, in step B, the preparation method of the reference solution is as follows:

[0063] Take ferulic acid, ligustilide, berberine hydrochloride and palmatine hydrochloride reference substances, add methanol to prepare a solution containing 0.5 mg ferulic acid, 0.3 mg ligustilide, 25 μg berberine hydrochloride and 50 μg palmatine hydrochloride per 1 mL, as mixed reference substance solution 1; take baicalin, astragaloside IV and phellodendronine hydrochloride reference substances, add methanol to prepare solutions with concentrations of 1, 0.5 and 1 mg / mL respectively;

[0064] The preparation method of raw Rehmannia root control medicinal material solution is as follows:

[0065] Take about 1 g of raw Rehmannia root control medicinal material, add 10 ml of methanol, ultrasonicate for 1 hour, and take the supernatant as the raw Rehmannia root control medicinal material solution.

[0066] C. Thin layer chromatography analysis

[0067] The inventors found in the experiment that the developing agent would affect the identification of the target component. In the developing agent Z1, if the developing agent is not suitable, the target component cannot be effectively separated. In addition, different volume ratios of ethyl acetate, methanol, chloroform, ammonia water and triethylamine would affect the separation degree of berberine hydrochloride and phellodendron hydrochloride in the chromatogram of the test sample (see C (1) and Figure 4-5), the final experimentally determined developing agent Z1 is a mixed system of ethyl acetate-methanol-chloroform-ammonia-triethylamine. In the developing agent Z2, if the developing agent is not suitable, the target component cannot be effectively separated, and different volume ratios of ethyl acetate, methanol, formic acid and water will affect the tailing phenomenon of the spots in the chromatogram of the baicalin reference substance and the separation of baicalin in the chromatogram of the test substance; and after formic acid is replaced by glacial acetic acid in the system, the tailing phenomenon of baicalin is not improved (see C (2) and Figure 6-9 ), the final developing agent Z2 was determined to be a mixed system of ethyl acetate, methanol, formic acid and water.

[0068] In the present invention, in the developing agent Z1, the volume ratio of ethyl acetate, methanol, chloroform, aqueous ammonia and triethylamine is (5-7):(1.5-2.5):(1.5-2.5):(0.8-1.2):(0.8-1.2), preferably (5.5-6.5):(1.8-2.2):(1.8-2.2):(0.9-1.1):(0.9-1.1), more preferably (5.8-6.2):(1.9-2.1):(1.9-2.1):(0.95-1.05):(0.95-1.05), especially 6:2:2:1:1.

[0069] In the present invention, in the developing agent Z2, the volume ratio of ethyl acetate, methanol, formic acid and water is (7-9):(0.8-1.2):(0.8-1.2):(0.8-1.2), preferably (7.5-8.5):(0.9-1.1):(0.9-1.1):(0.9-1.1), more preferably (7.8-8.2):(0.95-1.05):(0.95-1.05):(0.95-1.05), especially 8:1:1:1.

[0070] In some embodiments, in step C, the spotting volume of the test solution is 3-20 μL, and the spotting method is dot spotting or strip spotting, and the preferred spotting volume is 10 μL.

[0071] In some embodiments, in step C, the development method is: placing the spotted silica gel plate, covering and sealing, and developing until the front edge of the solvent is 2-3 cm away from the upper end of the silica gel plate.

[0072] In some embodiments, in step C.1, the wavelength of the ultraviolet light examined is 366 nm.

[0073] In some embodiments, in step C.2, the wavelength of the ultraviolet light examined is 254 nm.

[0074] In some embodiments, in step C.2, the operation method of color development in the iodine cylinder is: place the iodine solution at the bottom of the glass desiccator, place the silica gel plate on the porcelain plate in the glass desiccator, cover and seal, the color development time is 30s-1min, and the color is inspected under sunlight.

[0075] In some embodiments, in step C.2, the color developing agent is a 10% sulfuric acid ethanol solution. The heating is performed at 105° C. for 2-5 min.

[0076] Beneficial Effects

[0077] Compared with the prior art, the method provided by the present invention can simultaneously identify Angelica sinensis, Rehmannia root, Scutellaria baicalensis, Coptis chinensis, Phellodendron chinense and Astragalus membranaceus in the Angelica sinensis and six-yellow composition, and has the following advantages:

[0078] (1) The method for simultaneously identifying six medicinal materials provided by the present invention is to prepare five test sample solutions from one sample, spot the samples on two thin layer plates, and respectively carry out inspection, so as to simultaneously identify the six Chinese medicines of Danggui Liuhuang Decoction; the method has clear spots, good separation, strong specificity, no background interference, no negative control interference, and good durability; one identification test can reflect the quality information of the six Chinese medicines, the identification is more comprehensive, and it is more conducive to reflecting its overall quality characteristics, and the number of thin layer identification tests is reduced, the amount of reagents used is reduced, time is saved, and the inspection efficiency is improved;

[0079] (2) The method for preparing the sample solution of the thin layer identification method of the Chinese medicine compound preparation is usually complicated, with cumbersome steps, time-consuming and labor-intensive, and the sample solutions of the thin layer identification method of different medicinal flavors are generally different. However, the present invention only needs to weigh the sample once, and prepare five kinds of sample solutions after the sample is extracted. The ether layer sample solution is used for the specific identification of Angelica sinensis; the n-butanol layer sample solution is used for the specific identification of Coptis chinensis and Astragalus membranaceus; the ammonia test solution layer sample is used for the specific identification of Phellodendron chinense; the water layer precipitate sample is used for the specific identification of Scutellaria baicalensis; the water layer supernatant sample is used for the specific identification of Rehmannia glutinosa; the five sample solutions can reflect the index component information of the six Chinese medicinal herbs in the whole recipe of Danggui Liuhuang Decoction, and the identification is comprehensive;

[0080] (3) The thin layer chromatography method provided by the present invention for simultaneously identifying angelica, raw rehmannia, scutellaria, coptis, phellodendron and astragalus in the angelica six-yellow composition only uses two thin layer plates developed in ethyl acetate-methanol-chloroform-ammonia-triethylamine system and ethyl acetate-methanol-formic acid-water, respectively. The former can be used for the identification of angelica, coptis and phellodendron after ultraviolet inspection; the latter can be used for the identification of scutellaria, phellodendron, astragalus and raw rehmannia after ultraviolet inspection, iodine cylinder color development and 10% sulfuric acid ethanol solution color development;

[0081] (4) In the thin layer chromatography method provided by the present invention for simultaneously identifying Angelica sinensis, Radix Rehmanniae, Radix Scutellariae, Rhizoma Coptidis, Cortex Phellodendri and Radix Astragali in the Angelicae Sinensis and Six Yellows composition, the sample spot separation is good, and the negative is free of interference. The method can specifically identify the six herbs of Angelica sinensis, Radix Rehmanniae, Radix Scutellariae, Rhizoma Coptidis, Cortex Phellodendri and Radix Astragali. In addition, the durability of the thin layer chromatography method of the present invention was investigated in the test examples, and it was found that the detection process of the thin layer chromatography identification method of the present invention is not easily affected by changes in external conditions such as the thin layer plate, temperature, and humidity, which proves that the method is not demanding on external conditions, has good durability, and is widely applicable. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 It is the fluorescent spot of the thin layer chromatography G plate under 366nm lamp in Experimental Example 1 Condition 2, wherein 1: ferulic acid reference solution; 2: ligustilide reference solution; 3: n-butanol layer test solution; 4: n-butanol layer test solution; 5: ammonia test solution layer test solution; 6: ammonia test solution layer test solution;

[0083] Figure 2 It is the fluorescent spot of the thin layer chromatography GF254 plate under 254nm lamp in Experimental Example 1 Condition 3, wherein: 1: baicalin reference solution; 2: phellodendronine hydrochloride reference solution; 3: astragaloside IV reference solution; 4: ether layer test solution; 5: n-butanol layer test solution; 6: ammonia test solution layer test solution;

[0084] Figure 3 It is the fluorescent spot of the thin layer chromatography GF254 plate under 254nm lamp in Experimental Example 1 Condition 4, wherein: 1: baicalin reference solution; 2: water layer test solution; 3: n-butanol layer test solution; 4: ammonia test solution layer test solution;

[0085] Figure 4 It is the fluorescent spot of the thin layer chromatography G plate in Experimental Example 2 (1) a under 366 nm lamp, wherein: 1: ferulic acid-ligustilide mixed reference solution; 2: palmatine hydrochloride reference solution; 3: berberine hydrochloride reference solution; 4: ether layer test solution; 5: n-butanol layer test solution; 6: ammonia test solution layer test solution;

[0086] Figure 5 It is the fluorescent spot of the thin layer chromatography G plate in Experimental Example 2 (1) b under 366 nm lamp, wherein: 1: ferulic acid-ligustilide mixed reference solution; 2: palmatine hydrochloride reference solution; 3: berberine hydrochloride reference solution; 4: ether layer test solution; 5: n-butanol layer test solution; 6: ammonia test solution layer test solution;

[0087] Figure 6It is the fluorescent spot of the thin layer chromatography GF254 plate under 366nm lamp in Experimental Example 2(1)c, wherein: 1: ferulic acid-ligustilide mixed reference solution; 2: n-butanol layer test solution; 3: n-butanol layer test solution; 4: ammonia test solution layer test solution; 5: ammonia test solution layer test solution; 6: baicalin reference solution; 7: phellodendron hydrochloride-palmatine hydrochloride reference solution;

[0088] Figure 7 It is the fluorescent spot of the thin layer chromatography GF254 plate under 254nm lamp in Experimental Example 2(2)a, wherein: 1: ether layer test solution; 2: ether layer test solution; 3: n-butanol layer test solution; 4: n-butanol layer test solution; 5: baicalin reference solution;

[0089] Figure 8 It is the fluorescent spot of the thin layer chromatography GF254 plate under 254nm lamp when ethyl acetate-methanol-glacial acetic acid-water (7:2:1:1, volume ratio) is used as the developing solvent in Experimental Example 2(2)b, wherein: 1: baicalin reference solution; 2: ether layer test solution; 3: n-butanol layer test solution; 4: ammonia test solution layer test solution;

[0090] Fig. 9 It is the fluorescent spot of the thin layer chromatography GF254 plate under 254nm lamp when ethyl acetate-methanol-glacial acetic acid-water (7:2:0.5:1, volume ratio) is used as the developing solvent in Experimental Example 2(2)b, wherein: 1: baicalin reference solution; 2: astragaloside I reference solution; 3: ether layer test solution; 4: n-butanol layer test solution; 5: ammonia test solution layer test solution;

[0091] Fig.10 It is the spot in the daylight after the TLC GF254 plate in Experimental Example 3 is developed, wherein: 1: ferulic acid reference solution; 2: n-butanol layer test solution; 3: n-butanol layer test solution; 4: ammonia test solution layer test solution; 5: ammonia test solution layer test solution; 6: astragaloside IV reference solution; 7: bamipine hydrochloride reference solution; 8: phellodendron hydrochloride reference solution;

[0092] Fig.11 It is the fluorescent spot of the thin layer chromatography G plate of Example 1 under 366nm lamp, wherein, 1: Angelica sinensis and Phellodendron amurense double negative test solution; 2: Angelica sinensis negative test solution; 3: Coptis chinensis and Phellodendron amurense double negative test solution; 4: Coptis chinensis negative test solution; 5: mixed reference solution 1 (A-ligustilide, B-berberine hydrochloride, C-palmatine hydrochloride, D-ferulic acid); 6: ether layer test solution; 7: n-butanol layer test solution;

[0093] Fig.12It is the fluorescent spot of the thin layer chromatography GF254 plate of Example 1 under 254nm lamp before iodine jar color development, wherein, 1: negative test solution of Radix Rehmanniae; 2: negative test solution of Radix Astragali; 3: negative test solution of Cortex Phellodendri; 4: negative test solution of Radix Scutellariae; 5: reference solution of Astragaloside IV; 6: reference solution of Phellodendri hydrochloride; 7: reference solution of Baicalin; 8: reference medicinal material solution of Radix Rehmanniae; 9: supernatant test solution of water layer; 10: precipitate test solution of water layer; 11: test solution of n-butanol layer; 12: test solution of ammonia test solution layer;

[0094] Fig.13 It is the spot of the thin layer chromatography GF254 plate of Example 1 under sunlight after color development in an iodine cylinder, wherein: 1: negative test solution of Radix Rehmanniae; 2: negative test solution of Radix Astragali; 3: negative test solution of Cortex Phellodendri; 4: negative test solution of Radix Scutellariae; 5: reference solution of Astragaloside IV; 6: reference solution of Phellodendri hydrochloride; 7: reference solution of Baicalin; 8: reference medicinal material solution of Radix Rehmanniae; 9: supernatant test solution of water layer; 10: precipitate test solution of water layer; 11: test solution of n-butanol layer; 12: test solution of ammonia test solution layer;

[0095] Fig.14 It is the spot of the thin layer chromatography GF254 plate of Example 1 under sunlight after spraying 10% sulfuric acid ethanol for color development, wherein, 1: negative test solution of Radix Rehmanniae; 2: negative test solution of Radix Astragali; 3: negative test solution of Cortex Phellodendri; 4: negative test solution of Radix Scutellariae; 5: reference solution of Astragaloside IV; 6: reference solution of Phellodendri hydrochloride; 7: reference solution of Baicalin; 8: reference medicinal material solution of Radix Rehmanniae; 9: supernatant test solution of water layer; 10: precipitate test solution of water layer; 11: test solution of n-butanol layer; 12: test solution of ammonia test solution layer;

[0096] Fig.15 It is the thin layer chromatogram (silica gel G plate) of the reference sample of Danggui Liuhuang Decoction in Example 2, wherein: 1. Danggui Huangbai negative test solution; 2. Danggui negative test solution; 3. Coptis chinensis and Huangbai double negative test solution; 4. Coptis chinensis negative test solution; 5. Mixed reference solution 1 (the spots from top to bottom are ligustilide, berberine hydrochloride, bamipine hydrochloride, and ferulic acid); 6. Ether layer test solution; 7. n-butanol layer test solution;

[0097] Figures 16a to 16cIt is the thin layer chromatogram (silica gel GF254 plate) of the reference sample of Danggui Liuhuang Decoction in Example 2, wherein a. the thin layer chromatogram examined under ultraviolet light at 254nm; b. the thin layer chromatogram examined under daylight after color development with iodine cylinder; c. the thin layer chromatogram examined under daylight after color development with 10% sulfuric acid ethanol; wherein 1. negative test solution of Rehmannia root; 2. negative test solution of Astragalus root; 3. negative test solution of Phellodendron chinense; 4. negative test solution of Scutellaria baicalensis; 5. Rehmannia glycoside D reference solution; 6. Astragaloside IV reference solution; 7. Phellodendron hydrochloride reference solution; 8. Baicalin reference solution; 9. Rehmannia root reference medicinal material solution; 10. water layer supernatant test solution; 11. water layer precipitate test solution; 12. n-butanol layer test solution; 13. ammonia test solution layer test solution;

[0098] Fig.17 It is the thin layer chromatogram (silica gel G plate) of Danggui Liuhuang Decoction granules and Danggui Liuhuang Decoction extract in Example 3, wherein: 1. mixed control solution 1 (the spots are ligustilide, berberine hydrochloride, bamipine hydrochloride, and ferulic acid from top to bottom); 2. granule ether layer test solution; 3. granule n-butanol layer test solution; 4. extract ether layer test solution; 5. extract n-butanol layer test solution; 6. extract powder ether layer test solution; 7. extract powder n-butanol layer test solution;

[0099] Figures 18a to 18f The present invention is a thin layer chromatogram (silica gel GF254 plate) of Danggui Liuhuang Decoction granules and Danggui Liuhuang Decoction extract in Example 3, wherein: a. a thin layer chromatogram of granules and extract powder samples examined under ultraviolet lamp 254nm; b. a thin layer chromatogram of granules and extract powder samples examined under daylight after iodine jar color development; c. a thin layer chromatogram of granules and extract powder samples examined under daylight after 10% sulfuric acid ethanol color development; d. a thin layer chromatogram of extract solution samples examined under ultraviolet lamp 254nm; e. a thin layer chromatogram of extract solution samples examined under daylight after iodine jar color development; f. a thin layer chromatogram of extract solution samples examined under daylight after 10% sulfuric acid ethanol color development; wherein: 1. rehmannia glycoside D reference solution; 2. astragalus 1. Acetoglycoside reference solution; 2. Phellodendron chinense reference solution; 3. Baicalin reference solution; 4. Rehmannia glutinosa reference solution; 5. Radix Rehmanniae glutinosae reference solution; 6. Granule sample water layer supernatant test solution; 7. Extract powder water layer supernatant test solution; 8. Granule sample water layer precipitate test solution; 9. Extract powder water layer precipitate test solution; 10. Granule sample n-butanol layer test solution; 11. Extract powder n-butanol layer test solution; 12. Granule sample ammonia test solution layer test solution; 13. Extract powder ammonia test solution layer test solution; 14. Extract solution water layer supernatant test solution; 15. Extract solution water layer precipitate test solution; 16. Extract solution n-butanol layer test solution; 17. Extract solution ammonia test solution layer test solution;

[0100] Figures 19a to 19c It is a graph showing the durability test result of the silica gel G thin layer plate of Example 4, wherein: a. TLC of the German Merck silica gel G thin layer plate examined under an ultraviolet lamp at 366 nm; b. TLC of the Qingdao Ocean Silica G thin layer plate examined under an ultraviolet lamp at 366 nm; c. TLC of the Yantai Jiangyou HSG thin layer plate examined under an ultraviolet lamp at 366 nm, wherein: 1. Negative test solution of Angelica sinensis and Phellodendron chinense; 2. Negative test solution of Angelica sinensis; 3. Negative test solution of Coptis chinensis and Phellodendron chinense; 4. Negative test solution of Coptis chinensis; 5. Mixed reference solution 1 (the spots are ligustilide, berberine hydrochloride, bamipine hydrochloride, and ferulic acid from top to bottom); 6. Ether layer test solution; 7. n-butanol layer test solution;

[0101] Figures 20a to 20i The results of the durability test of the silica gel GF254 thin layer plate of Example 4 are shown in Figure 1, wherein: a. The thin layer chromatogram of the German Merck TLC Silica gel 60F254 thin layer plate examined under a UV lamp at 254nm; b. The thin layer chromatogram of the German Merck TLC Silica gel 60F254 thin layer plate examined under sunlight after color development with an iodine cylinder; c. 60F254 thin layer plate after color development with 10% sulfuric acid ethanol and examined under sunlight; d. TLC of Qingdao Ocean GF254 thin layer plate examined under UV lamp 254nm; e. TLC of Qingdao Ocean GF254 thin layer plate after color development with iodine cylinder and examined under sunlight; f. TLC of Qingdao Ocean GF254 thin layer plate after color development with 10% sulfuric acid ethanol and examined under sunlight; g. TLC of Yantai Jiangyou HSGF254 thin layer plate examined under UV lamp 254nm; h. TLC of Yantai Jiangyou HSGF254 thin layer plate examined under UV lamp 254nm after color development with iodine cylinder; i. Yantai Jiangyou HSGF25 4 Thin layer chromatograms of thin layer plates viewed under sunlight after color development with 10% sulfuric acid ethanol; wherein, 1. Radix Rehmanniae negative test solution; 2. Radix Astragali negative test solution; 3. Cortex Phellodendri negative test solution; 4. Radix Scutellariae negative test solution; 5. Rehmannia glutinosa D reference solution; 6. Astragaloside IV reference solution; 7. Cortex Phellodendri hydrochloride reference solution; 8. Baicalin reference solution; 9. Radix Rehmanniae reference medicinal material solution; 10. Water layer supernatant test solution; 11. n-butanol layer test solution; 12. Water layer precipitate test solution; 13. Ammonia test solution layer test solution; 11*. Water layer precipitate test solution; 12*. n-butanol layer test solution;

[0102] Figures 21a to 21cIt is the temperature investigation result diagram of Example 5 (silica gel G thin layer plate), wherein a. thin layer chromatogram developed at low temperature of 4°C; b. thin layer chromatogram developed at room temperature of 25°C; c. thin layer chromatogram developed at high temperature of 40°C, wherein 1. Angelica sinensis and Phellodendron amurense negative test solution; 2. Angelica sinensis negative test solution; 3. Coptis chinensis and Phellodendron amurense negative test solution; 4. Coptis chinensis negative test solution; 5. mixed reference solution 1 (the spots from top to bottom are ligustilide, berberine hydrochloride, bamipine hydrochloride, and ferulic acid); 6. ether layer test solution; 7. n-butanol layer test solution;

[0103] Figures 22a to 22i The temperature investigation result diagram of Example 5 (silica gel GF254 thin layer plate), wherein a. the thin layer chromatogram after low temperature 4°C development and inspection under ultraviolet lamp 254nm; b. the thin layer chromatogram after low temperature 4°C development and inspection under daylight after iodine jar color development; c. the thin layer chromatogram after low temperature 4°C development and inspection under daylight after 10% sulfuric acid ethanol color development; d. the thin layer chromatogram after normal temperature 25°C development and inspection under ultraviolet lamp 254nm; e. the thin layer chromatogram after normal temperature 25°C development and inspection under daylight after iodine jar color development; f. the thin layer chromatogram after normal temperature 25°C development and inspection under daylight after 10% sulfuric acid ethanol color development; g. the thin layer chromatogram after high temperature 40°C development and inspection under ultraviolet lamp 254nm Thin layer chromatogram; h. Thin layer chromatogram after high temperature 40℃ development and color development in iodine cylinder and then examined in daylight; i. Thin layer chromatogram after high temperature 40℃ development and color development in 10% sulfuric acid ethanol and then examined in daylight; Among them, 1. Radix Rehmanniae negative test solution; 2. Radix Astragali negative test solution; 3. Cortex Phellodendri negative test solution; 4. Radix Scutellariae negative test solution; 5. Rehmannia glutinosa D reference solution; 6. Astragaloside IV reference solution; 7. Cortex Phellodendri hydrochloride reference solution; 8. Baicalin reference solution; 9. Radix Rehmanniae reference medicinal material solution; 10. Water layer supernatant test solution; 11. Water layer precipitate test solution; 12. n-butanol layer test solution; 13. Ammonia test solution layer test solution;

[0104] Figures 23a to 23c It is the relative humidity investigation result diagram of Example 6 (silica gel G thin layer plate), wherein, a. thin layer chromatogram developed under a relative humidity of 40% environment; b. thin layer chromatogram developed under a relative humidity of 75% environment; c. thin layer chromatogram developed under a relative humidity of 90% environment, wherein, 1. Angelica sinensis and Phellodendron amurense negative test solution; 2. Angelica sinensis negative test solution; 3. Coptis chinensis and Phellodendron amurense negative test solution; 4. Coptis chinensis negative test solution; 5. mixed reference solution 1 (the spots are ligustilide, berberine hydrochloride, bamipine hydrochloride, and ferulic acid from top to bottom); 6. ether layer test solution; 7. n-butanol layer test solution;

[0105] Figures 24a to 24iIt is a graph of the relative humidity investigation result of Example 6 (silica gel GF254 thin layer plate), wherein a. a thin layer chromatogram obtained by developing under a relative humidity of 40% and inspecting under a UV lamp at 254 nm; b. a thin layer chromatogram obtained by developing under a relative humidity of 40% and inspecting under sunlight after being developed with an iodine cylinder; c. a thin layer chromatogram obtained by developing under a relative humidity of 40% and inspecting under sunlight after being developed with 10% sulfuric acid ethanol; d. a thin layer chromatogram obtained by developing under a relative humidity of 75% and inspecting under a UV lamp at 254 nm; e. a thin layer chromatogram obtained by developing under a relative humidity of 75% and inspecting under sunlight after being developed with an iodine cylinder; f. a thin layer chromatogram obtained by developing under a relative humidity of 75% and inspecting under sunlight after being developed with 10% sulfuric acid ethanol; g. a thin layer chromatogram obtained by developing under a relative humidity of 90% and inspecting under a UV lamp at 254 nm. h. Thin layer chromatogram after development in an environment with a relative humidity of 90% and then color development in an iodine cylinder and then inspection under sunlight; i. Thin layer chromatogram after development in an environment with a relative humidity of 90% and then color development in 10% sulfuric acid ethanol and then inspection under sunlight; Among them, 1. Radix Rehmanniae negative test solution; 2. Radix Astragali negative test solution; 3. Cortex Phellodendri negative test solution; 4. Radix Scutellariae negative test solution; 5. Rehmannia glutinosa D reference solution; 6. Astragaloside IV reference solution; 7. Cortex Phellodendri hydrochloride reference solution; 8. Baicalin reference solution; 9. Radix Rehmanniae reference medicinal material solution; 10. Water layer supernatant test solution; 11. Water layer precipitate test solution; 12. n-butanol layer test solution; 13. Ammonia test solution layer test solution; 11*. n-butanol layer test solution; 12*. Water layer precipitate test solution. DETAILED DESCRIPTION

[0106] The exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings, but it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0107] The present invention is further described in the following examples, but the examples are not intended to limit the scope of protection of the present invention.

[0108] In the following embodiments and experimental examples, reagents not specially specified are conventional reagents and can be purchased from conventional reagent production and sales companies. The information of some raw material manufacturers is as follows:

[0109] 1. Instrument

[0110] ATS4 automatic spotter, Visualizer 2 imaging system (Kama, Switzerland); BCE223-1CCN electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); SB25-12DTD ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); DK-S24 electric constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.); 30B2 liquid heater (Chaozhou Chaoan District Kangyashun Electric Co., Ltd.); TGL-16B high-speed centrifuge (Shanghai Anting Scientific Instrument Factory); ST 16R centrifuge (Thermo Fisher Scientific (China) Co., Ltd.); DHG-9240A electric heating blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.). Silica gel GF254 thin layer plate (Qingdao Ocean Chemical Co., Ltd.); Silica gel G thin layer plate (Qingdao Ocean Chemical Co., Ltd.); TLC Silica gel 60F254 thin layer plate (Merck, Germany); TLC Silica gel60 thin layer plate (Merck, Germany); Silica gel HSGF254 thin layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.); Silica gel HSG thin layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.).

[0111] 2. Reagents

[0112] Methanol (batch number: 20240205), n-butanol (batch number: 20231011), ether (batch number: 20230814), ammonia (batch number: 20231013), ethyl acetate (batch number: 20230509), triethylamine (batch number: 20190130), chloroform (batch number: 20231013), formic acid (batch number: 20140619), anhydrous ethanol (batch number: 20240112), and sulfuric acid (batch number: 20180227) were all analytically pure and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0113] 3. Reference

[0114] Ferulic acid (batch number: 110773-201915, purity: 99.4%), ligustilide (batch number: 111737-201608, purity: 99.9%), rehmannia glutinosa (batch number: 112063-202103, purity: 94.2%), baicalin (batch number: 110715-202223, purity: 97.2%), astragaloside I (batch number: 1 10781-202219, purity: 96.2%), phellodendronine hydrochloride (batch number: 111895-201805, purity: 94.9%), berberine hydrochloride (batch number: 110713-202316, purity: 91.0%), and bamipine hydrochloride (batch number: 110732-201913, purity: 85.7%) were purchased from China Food and Drug Inspection Institutes.

[0115] 4. Medicinal materials

[0116] The decoction pieces of angelica, raw rehmannia, phellodendron, coptis root, scutellaria and astragalus were purchased from the native production area or authentic production area. It was identified that angelica was the dried root of Angelica sinensis (Oliv.) Diels of the Umbelliferae family, raw rehmannia was the fresh or dried tuber of Rehmannia glutinosa Libosch. of the Scrophulariaceae family, phellodendron was the dried bark of Phellodendron Chinense Schneid. of the Rutaceae family, coptis root was the dried rhizome of Coptis chinensis Franch. of the Ranunculaceae family, scutellaria was the dried root of Scutellaria baicalensis Georgi of the Lamiaceae family, and astragalus was the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. The above 6 medicinal materials have all passed the inspection according to the 2020 edition of the "Chinese Pharmacopoeia", and the herbal pieces have all been prepared according to the 2020 edition of the "Chinese Pharmacopoeia".

[0117] 5. Preparation of Danggui Liuhuang Decoction reference sample:

[0118] Take slices of Chinese Angelica, Radix Rehmanniae, Radix Rehmanniae Preparata, Cortex Phellodendri, Rhizoma Coptidis, Radix Scutellariae and Radix Astragali and grind them into coarse powder through a 10-mesh sieve. Except for Radix Astragali, weigh 2.58g of each medicinal ingredient and 5.16g of Radix Astragali, put them in a decoction pot, add 600mL of water, boil over high heat, then turn to low heat and boil until the volume of the liquid is 300mL, filter while hot, and freeze-dry the filtrate to obtain the Danggui Liuhuang Decoction benchmark sample.

[0119] 6. Preparation of Danggui Liuhuang Decoction Extract and Extract Powder:

[0120] Take Angelica sinensis slices, Rehmannia root slices, Rehmannia glutinosa slices, Phellodendron chinense slices, Coptis chinensis slices, Scutellaria baicalensis slices, and Astragalus membranaceus slices in a ratio of 1:1:1:1:1:1:2, add 10 times the amount of water, reflux extraction twice, filter while hot, combine the filtrate to obtain the Danggui Liuhuang Decoction extract.

[0121] The Danggui Liuhuang Decoction extract is concentrated and dried under reduced pressure to obtain the Danggui Liuhuang Decoction extract powder.

[0122] 7. Preparation of Danggui Liuhuang Decoction Granules:

[0123] Take the Danggui Liuhuang Decoction extract powder and a single auxiliary material (dextrin, soluble starch, mannitol) or a combination of auxiliary materials (dextrin + xylitol, dextrin + mannitol, soluble starch + xylitol) in a ratio of 2:1, mix them, use 75% ethanol as a wetting agent, and prepare a soft material. After meeting the standard of "forming a ball when held in the hand and falling apart when pressed lightly", use a No. 2 sieve for extrusion granulation. The prepared granules are dried in an oven at 60°C. After drying, take out and granulate, and screen out the granules that can pass the No. 1 sieve but not the No. 5 sieve to obtain the Danggui Liuhuang Decoction granule sample.

[0124] Experimental Example 1 Selection of test solution preparation conditions

[0125] Preparation of reference solution:

[0126] Take ligustilide, ferulic acid, berberine hydrochloride and palmatine hydrochloride reference substances, add methanol to prepare mixed reference substance solutions 1 with concentrations of 0.15, 0.3, 0.025, and 0.05 mg / mL, respectively. Take baicalin, astragaloside IV, and phellodendronine hydrochloride reference substances, add methanol to prepare solutions with concentrations of 1, 0.5, and 1 mg / mL, respectively.

[0127] (1) Condition 1:

[0128] Preparation of test solution: Take 1 g of Danggui Liuhuang Decoction standard sample, add 30 ml of methanol, and dissolve by ultrasonic.

[0129] Pipette 10 μL of the test solution and spot them on the same silica gel GF254 plate. Use the developing agent ethyl acetate-methanol-water (7:2:1, volume ratio) for development, dry it, examine it under a UV lamp at 254 nm, then place it in an iodine cylinder for color development for 1 minute, and examine it under sunlight; then spray it with 10% sulfuric acid ethanol solution, heat it at 105°C for 2-5 minutes, until the spots are clearly colored, and examine it under sunlight.

[0130] Results: The spot boundaries in the chromatogram were unclear and the characteristic spots were not separated, making it difficult to make effective identification.

[0131] (2) Condition 2:

[0132] Preparation of test solution: Take 1g of Danggui Liuhuang Decoction reference sample, add 30ml of water, dissolve by ultrasonic, add an equal volume of water-saturated n-butanol and shake to extract twice, combine the n-butanol liquid layer, shake and extract the n-butanol liquid layer with an equal volume of ammonia test solution twice, and combine the ammonia test solution layer. After the n-butanol liquid layer and the ammonia test solution layer are evaporated to dryness in a water bath, the residues are respectively dissolved with 1ml of methanol as the test solution for the n-butanol layer and the test solution for the ammonia test solution layer, respectively.

[0133] 10 μL of the above test solution was taken and spotted on the same silica gel GF254 plate, developed with ethyl acetate-methanol-water (7:2:1, volume ratio), dried, and examined under ultraviolet light at 254 nm.

[0134] Results Figure 1 In the chromatograms of the n-butanol layer test sample solution and the ammonia test solution layer test sample solution, the obtained n-butanol layer test sample can be used to identify ligustilide, but there is no fluorescent spot of the same color at the position corresponding to the ferulic acid reference sample, and ferulic acid is not identified.

[0135] (3) Condition 3:

[0136] Preparation of test solution: Take 1g of Danggui Liuhuang Decoction reference sample, add 30ml of water, dissolve by ultrasonic, extract twice with an equal volume of ether, combine the ether solution, add an equal volume of water-saturated n-butanol to the remaining water solution and shake and extract twice, combine the n-butanol solution, shake and extract the n-butanol solution with an equal volume of ammonia test solution twice, and combine the ammonia test solution layers. After the ether layer, n-butanol layer, and ammonia test solution layer are evaporated to dryness in a water bath, the residues are dissolved with 1ml of methanol solution, which are used as the ether layer test solution, n-butanol layer test solution, and ammonia test solution layer test solution, respectively.

[0137] 10 μL of the above test solution was taken and spotted on the same silica gel GF254 plate, developed with ethyl acetate-methanol-water-glacial acetic acid (20:5:3:2.5, volume ratio), dried, and inspected under ultraviolet light at 254 nm.

[0138] Thin layer chromatogram Figure 2 It can be seen that no identical spots were observed in the chromatograms of the ether extract test sample, the n-butanol layer test sample solution, and the ammonia test solution layer test sample solution at the positions corresponding to the chromatograms of the baicalin reference sample.

[0139] (4) Condition 4:

[0140] The test was carried out according to the above condition (3). In addition, the aqueous solution of the Angelica sinensis six yellows reference sample was extracted with ether and then with water-saturated n-butanol, and the aqueous layer was evaporated to dryness in a water bath. The residue was dissolved in 1 ml of methanol and the supernatant was taken by centrifugation as the aqueous layer supernatant for the test sample solution.

[0141] 10 μL of the above test solution was taken and spotted on the same silica gel GF254 plate, developed with ethyl acetate-methanol-water-glacial acetic acid (20:5:3:2.5, volume ratio), dried, and inspected under ultraviolet light at 254 nm.

[0142] Thin layer chromatogram Figure 3 In the chromatogram of the test solution in the aqueous supernatant, no identical spots were observed at the positions corresponding to those in the chromatogram of the baicalin reference solution.

[0143] The above centrifuged precipitate is further dissolved with methanol to obtain an aqueous precipitate test solution.

[0144] 10 μL of the test solution was taken and spotted on a silica gel GF254 plate, developed with a developing agent of ethyl acetate-methanol-formic acid-water (8:1:1:1, volume ratio), air-dried, and inspected under a UV lamp at 254 nm. The chromatogram of the obtained aqueous layer precipitate test sample shows a spot of the same color at the corresponding position of the chromatogram of the baicalin reference substance, which can be used to identify Scutellaria baicalensis.

[0145] Experimental Example 2 Selection of developing agent

[0146] A. Preparation of test solution:

[0147] Take 7g of Danggui Liuhuang Decoction reference sample, add 40mL of water, ultrasonically dissolve, add an equal volume of ether to shake and extract twice, combine the ether solution, evaporate to dryness, and dissolve the residue in 1mL of methanol as the ether layer test solution; extract the remaining water solution twice with an equal volume of water-saturated n-butanol, combine the n-butanol solution, shake and extract the n-butanol solution twice with an equal volume of ammonia test solution, combine the ammonia test solution layer, evaporate the n-butanol layer and the ammonia test solution layer in a water bath, and dissolve the residue in 2mL of methanol, respectively, as the n-butanol layer test solution and the ammonia test solution layer test solution; the water solution extracted with the n-butanol layer is centrifuged at 12000 rpm for 20 minutes, 10mL of the supernatant is evaporated in a water bath, and the residue is dissolved in 2mL of methanol to obtain the water layer supernatant test solution. After the precipitate is ultrasonically dissolved with 20mL of methanol, centrifuge at 12000 rpm for 20 minutes, take the supernatant, and obtain the water layer precipitate test solution.

[0148] B. Preparation of reference solution:

[0149] Take ferulic acid, ligustilide, berberine hydrochloride, and palmatine hydrochloride reference substances and add methanol to prepare a mixed reference substance solution 1.

[0150] Take baicalin, phellodendronine hydrochloride and astragaloside IV reference substances, add methanol to prepare baicalin reference substance solution, phellodendronine hydrochloride reference substance solution and astragaloside IV reference substance solution respectively.

[0151] The raw Rehmannia root control medicinal material was extracted with methanol, and the extract was used as the raw Rehmannia root control medicinal material solution.

[0152] C. Thin layer chromatography analysis:

[0153] (1) Developing agent Z1

[0154] a. Take 10 μL of each of the above test solutions and spot them on the same silica gel G plate. According to the thin layer identification method of berberine hydrochloride under the item of Coptis chinensis in the 2020 edition of the "Chinese Pharmacopoeia", use cyclohexane-ethyl acetate-methanol-isopropanol-triethylamine-water ((3:3.5:1.5:1:1:0.5, volume ratio) as the developing agent, saturated with ammonia, develop, dry, and examine under ultraviolet light at 366 nm. The thin layer chromatogram is shown in Figure 4 In the chromatogram of the test sample, the spots at the positions corresponding to the berberine hydrochloride and bamipine hydrochloride reference substances were not obvious, the separation was poor, and there was interference from impurities.

[0155] b. Developed according to the above steps using the developing agent ethyl acetate-methanol-chloroform-ammonia-triethylamine (6:2:1:1:1, volume ratio), dried, and examined under ultraviolet light at 366nm. The thin layer chromatogram is shown in Figure 5 The spots in the chromatogram of bamipine hydrochloride were tailing, and the spots in the chromatogram of the test sample corresponding to those of berberine hydrochloride and bamipine hydrochloride reference substances had poor separation and could not be effectively identified.

[0156] c. Develop with the developing agent ethyl acetate-methanol-water (7:2:1, volume ratio) according to the above steps, dry, and examine under ultraviolet light at 366nm. The thin layer chromatogram is shown in Figure 6 , the reference substances of phellodendron HCl and bamipine HCl were not separated, and the spot boundaries at the positions corresponding to the reference substance chromatograms in the test sample chromatograms were blurred and unclear, and effective identification could not be performed.

[0157] After repeated experiments, the optimized developing agent Z1 was a mixed system of ethyl acetate-methanol-chloroform-ammonia water-triethylamine with a volume ratio of 6:2:2:1:1.

[0158] (2) Developing agent Z2

[0159] a. Take 10 μL of each of the above test solution and spot them on the same silica gel GF254 plate, develop them with ethyl acetate-methanol-formic acid-water (7:2:0.3:1, volume ratio), dry them, and examine them under ultraviolet light at 254 nm. The thin layer chromatogram is shown in Figure 7 The spots of baicalin reference substance were severely tailed, which was not conducive to the result judgment.

[0160] b. Replace the formic acid in the above developing solvent with glacial acetic acid, prepare ethyl acetate-methanol-glacial acetic acid-water (7:2:1:1, volume ratio) and ethyl acetate-methanol-glacial acetic acid-water (7:2:0.5:1, volume ratio), develop according to the above steps, dry, and examine under ultraviolet light at 254nm. The thin layer chromatogram is shown in Figure 8-9 Under both conditions, the spots of baicalin reference substance showed serious tailing phenomenon, which was not conducive to the result judgment.

[0161] After repeated experiments, the optimized developing agent Z2 was a mixed system of ethyl acetate-methanol-formic acid-water with a volume ratio of 8:1:1:1.

[0162] Experimental Example 3 Selection of color developer

[0163] A. Preparation of test samples:

[0164] Take 1g of Danggui Liuhuang Decoction benchmark sample, add 30ml of water, dissolve with ultrasound, add an equal volume of water-saturated n-butanol and shake to extract twice, combine the n-butanol liquids, extract the n-butanol liquid with an equal volume of ammonia test solution by shaking twice, combine the ammonia test solution layers, evaporate the n-butanol liquid layer and the ammonia test solution layer in a water bath respectively, dissolve the residues with 1ml of methanol respectively, and use them as the test solution for the n-butanol layer and the ammonia test solution layer respectively, and perform thin layer analysis after spotting.

[0165] B. Preparation of reference solution:

[0166] Refer to Example 2.

[0167] C. Thin layer chromatography analysis:

[0168] 10 μL of each of the above test solutions was taken and spotted on the same silica gel GF254 plate, developed with ethyl acetate-methanol-water (7:2:1, volume ratio), dried, sprayed with dilute potassium bismuth iodide solution, and heated at 105°C for 5 minutes.

[0169] Thin layer chromatogram Fig.10 The color developer of the dilute potassium bismuth iodide test solution is yellow in color, and the color of the color spots of the phellodendron hydrochloride reference substance is close, which affects the identification of phellodendron in the ammonia test solution layer test sample. And under this color developer, astragaloside IV cannot be identified at the same time. If 10% sulfuric acid ethanol solution is sprayed later for the color development of raw rehmannia and astragaloside IV, the background color and impurity spots of the thin layer plate will be further deepened, affecting the subsequent identification of raw rehmannia and astragaloside IV.

[0170] After repeated experiments, the optimized developer for silica gel GF254 thin layer plate is iodine cylinder color development, which is inspected under sunlight to identify phellodendron hydrochloride; then spray with 10% sulfuric acid ethanol solution, heat at 105℃ for 2-5min, until the spots are clearly colored, and inspected under sunlight to identify Rehmannia root and astragaloside I.

[0171] Example 1 Simultaneous identification of angelica, raw rehmannia, scutellaria, coptis, phellodendron and astragalus in Danggui Liuhuang Decoction by thin layer chromatography

[0172] A. Preparation of test solution:

[0173] Take 7g of Danggui Liuhuang Decoction reference sample, add 40mL of water, ultrasonically dissolve, add an equal volume of ether to shake and extract twice, combine the ether solution, evaporate to dryness, and dissolve the residue in 1mL of methanol as the ether layer test solution; extract the remaining water solution twice with an equal volume of water-saturated n-butanol, combine the n-butanol solution, shake and extract the n-butanol solution twice with an equal volume of ammonia test solution, combine the ammonia test solution layer, evaporate the n-butanol layer and the ammonia test solution layer in a water bath, and dissolve the residue in 2mL of methanol, respectively, as the n-butanol layer test solution and the ammonia test solution layer test solution; the water solution extracted with the n-butanol layer is centrifuged at 12000 rpm for 20 minutes, 10mL of the supernatant is evaporated in a water bath, and the residue is dissolved in 2mL of methanol to obtain the water layer supernatant test solution. After the precipitate is ultrasonically dissolved with 20mL of methanol, centrifuge at 12000 rpm for 20 minutes, take the supernatant, and obtain the water layer precipitate test solution.

[0174] B. Preparation of reference solution:

[0175] Take ligustilide, ferulic acid, berberine hydrochloride and palmatine hydrochloride reference substances, add methanol to prepare mixed reference substance solutions 1 with concentrations of 0.15, 0.3, 0.025, and 0.05 mg / mL, respectively. Take baicalin, astragaloside IV, and phellodendronine hydrochloride reference substances, add methanol to prepare solutions with concentrations of 1, 0.5, and 1 mg / mL, respectively.

[0176] Take about 1 g of raw Rehmannia root control medicinal material, add 10 ml of methanol, ultrasonicate for 1 hour, and take the supernatant as the raw Rehmannia root control medicinal material solution.

[0177] C. Preparation of negative samples:

[0178] First, negative decoctions of different medicinal flavors were prepared according to the method described in the preparation of the Danggui Liuhuang Decoction benchmark sample: Danggui negative sample, Danggui and Phellodendron double negative sample, Phellodendron negative sample, Coptis chinensis negative sample, Phellodendron chinensis and Coptis chinensis double negative sample, Rehmannia glutinosa negative sample, Scutellaria baicalensis negative sample, Astragalus membranaceus negative sample, and then negative test sample solutions were prepared according to the method in step A.

[0179] D. Thin layer chromatography analysis:

[0180] (1) 10 μL of each of the ether layer test solution and the n-butanol layer test solution prepared in step A, the mixed reference solution 1 prepared in step B, and the angelica sinensis negative test solution, angelica sinensis and phellodendron double negative test solution, phellodendron negative test solution, coptis chinensis negative test solution, and phellodendron chinensis and coptis chinensis double negative test solution prepared in step C were respectively spotted on the same silica gel G plate (Merck), developed with a developing agent of ethyl acetate-methanol-chloroform-ammonia-triethylamine (6:2:2:1:1, volume ratio), air-dried, and examined under an ultraviolet lamp at 366 nm. In the ether layer test sample chromatogram, the ligustilide reference substance At the corresponding position, fluorescent spots of the same color appeared, which could identify Angelica sinensis, and there was no interference with Angelica sinensis negative samples; in the ether layer test sample chromatogram, at the corresponding position with the ferulic acid reference substance, fluorescent spots of the same color appeared, which could identify Angelica sinensis and Phellodendron amurense, and there was no interference with Angelica sinensis and Phellodendron amurense double-negative samples; in the n-butanol layer test sample chromatogram, at the corresponding position with the berberine hydrochloride reference substance chromatogram, fluorescent spots of the same color appeared, which could identify Coptis chinensis and Phellodendron amurense, and there was no interference with Phellodendron amurense and Coptis chinensis double-negative samples; in the n-butanol layer test sample chromatogram, at the corresponding position with the bamipine hydrochloride chromatogram, fluorescent spots of the same color appeared, which could identify Coptis chinensis, and there was no interference with Coptis chinensis negative samples. For details, see Fig.11 .

[0181] (2) 10 μL of each of the n-butanol layer test solution, ammonia test solution layer test solution, water layer supernatant test solution and water layer precipitate test solution prepared in step A, baicalin reference solution, phellodendron hydrochloride reference solution, astragaloside IV reference solution and rehmannia root control medicinal material solution prepared in step B, and rehmannia root negative test solution, scutellaria baicalensis negative test solution, astragalus root negative test solution and phellodendron root negative test solution prepared in step C were spotted on the same silica gel GF254 plate (Merck), developed with a developing agent of ethyl acetate-methanol-formic acid-water (8:1:1:1, volume ratio), dried, and examined under a UV lamp at 254 nm. In the chromatogram of the starch sample, a spot of the same color appears at the corresponding position of the baicalin reference substance, and Scutellaria baicalensis is identified; then placed in an iodine cylinder for color development for 1 minute, and examined under sunlight, in the chromatogram of the ammonia test solution layer, a spot of the same color appears at the corresponding position of the chromatogram of the phellodendron hydrochloride reference substance, and Phellodendron amurense is identified; then sprayed with 10% sulfuric acid ethanol solution, heated at 105℃ for 2-5min, until the spot is clearly colored, and examined under sunlight, in the chromatogram of the water layer supernatant sample, a spot of the same color appears at the corresponding position of the chromatogram of the raw rehmannia reference medicinal material, and raw rehmannia is identified; in the chromatogram of the n-butanol layer sample, a spot of the same color appears at the corresponding position of the chromatogram of the astragaloside reference substance, and Astragalus is identified. For details, see Figure 12-14 .

[0182] Example 2 Simultaneous identification of Angelica sinensis, Radix Rehmanniae, Radix Scutellariae, Rhizoma Coptidis, Cortex Phellodendri and Radix Astragali in Danggui Liuhuang Decoction by thin layer chromatography

[0183] A. Preparation of test solution:

[0184] Take 4g of Danggui Liuhuang Decoction reference sample, add 40mL of water, dissolve by ultrasonic, add an equal volume of ether and shake to extract twice, combine the ether solution, evaporate to dryness, and dissolve the residue in 1mL of methanol as the ether layer test solution;

[0185] The remaining aqueous solution was extracted twice with an equal volume of water-saturated n-butanol, and the n-butanol solution was combined. The n-butanol solution was extracted twice with an equal volume of ammonia test solution by shaking, and the ammonia test solution layers were combined. After the n-butanol layer and the ammonia test solution layer were evaporated to dryness in a water bath, the residues were dissolved in 2 mL of methanol, and used as the test solution for the n-butanol layer and the test solution for the ammonia test solution layer, respectively;

[0186] The aqueous solution after extraction with the n-butanol layer was centrifuged at 12,000 rpm for 20 minutes, 10 mL of the supernatant was evaporated in a water bath, and the residue was dissolved in 2 mL of methanol to obtain the aqueous supernatant test solution. The precipitate was dissolved in 20 mL of methanol by ultrasonication, centrifuged at 12,000 rpm for 20 minutes, and the supernatant was taken to obtain the aqueous precipitate test solution.

[0187] B. Thin layer chromatography analysis:

[0188] The test solution prepared in Example 2 was used for thin layer chromatography analysis according to the method of Example 1. The results are shown in Figure 15-16c .

[0189] The results showed that in the test solution prepared from a 4g sampling amount of the Danggui Liuhuang Decoction benchmark sample, spots of the same color appeared at the corresponding positions on the chromatograms of the reference samples of ligustilide, ferulic acid, berberine hydrochloride, bamipine hydrochloride, baicalin, astragaloside I, and phellodendron chinense, and spots of the same color appeared at the corresponding positions on the chromatograms of the reference medicinal material of Rehmannia glutinosa. The six medicinal herbs of Angelica sinensis, Rehmannia glutinosa, Scutellaria baicalensis, Coptis chinensis, Phellodendron chinense, and Astragalus membranaceus in the Danggui Liuhuang Decoction benchmark sample can be identified.

[0190] Example 3 Simultaneous identification of angelica, raw rehmannia, scutellaria, coptis, phellodendron, and astragalus in Danggui Liuhuang Decoction granules and extracts by thin layer chromatography

[0191] A. Preparation of test solution:

[0192] Take 50mL of Danggui Liuhuang Decoction extract, 7g of Danggui Liuhuang Decoction extract powder, and 10g of Danggui Liuhuang Decoction granules respectively, add 40mL of water to the extract powder and granules respectively, and dissolve them by ultrasound. The extract, extract powder solution, and granule solution were shaken and extracted twice with equal volumes of ether, and the ether solution was combined and evaporated to dryness. The residue was dissolved in 1mL of methanol as the ether layer test solution; the remaining water solution was extracted twice with an equal volume of water-saturated n-butanol, and the n-butanol solution was combined. The n-butanol solution was shaken and extracted twice with an equal volume of ammonia test solution, and the ammonia test solution layer was combined. After the n-butanol layer and the ammonia test solution layer were evaporated to dryness in a water bath, the residue was dissolved in 2mL of methanol respectively, and used as the n-butanol layer test solution and the ammonia test solution layer test solution respectively; the water solution after the n-butanol layer extraction was centrifuged at 12,000 rpm for 20 minutes, 10mL of the supernatant was taken and evaporated to dryness in a water bath, and the residue was dissolved in 2mL of methanol to obtain the water layer supernatant test solution. After adding 20 mL of methanol to dissolve the precipitate by ultrasonication, centrifuge at 12,000 rpm for 20 min, take the supernatant, and obtain the aqueous precipitate test solution.

[0193] B. Thin layer chromatography analysis:

[0194] The test solution prepared in Example 3 was used for thin layer chromatography analysis according to the method of Example 1. The results are shown in Figure 17-18f .

[0195] The results showed that in the test solution of Danggui Liuhuang Decoction granules and extracts, spots of the same color appeared at the corresponding positions on the chromatograms of ligustilide, ferulic acid, berberine hydrochloride, bamipine hydrochloride, baicalin, astragaloside I, and phellodendronine reference substances, and spots of the same color appeared at the corresponding positions on the chromatograms of Rehmannia glutinosa reference medicinal materials. The six herbs of Angelica sinensis, Rehmannia glutinosa, Scutellaria baicalensis, Coptis chinensis, Phellodendron chinense, and Astragalus membranaceus in Danggui Liuhuang Decoction granules and extracts could be identified.

[0196] Example 4 Investigation of thin layer plates

[0197] The reference solution, negative solution and test solution of Example 1 were taken and spotted on three brands of thin layer plates according to the spotting method of Example 1: German Merck TLC Silica gel 60 thin layer plate, Qingdao Ocean Silica G thin layer plate, Yantai Jiangyou HSG thin layer plate, and developed with a developing agent of ethyl acetate-methanol-chloroform-ammonia-triethylamine (6:2:2:1:1, volume ratio), dried, and examined under an ultraviolet lamp at 366nm to identify Angelica sinensis, Phellodendron amurense and Coptis chinensis. For details, see Figures 19a to 19c .

[0198] The reference solution, negative solution and test solution of Example 1 were taken and spotted on three brands of thin layer plates according to the spotting method of Example 1: German Merck TLC Silica gel 60F254 thin layer plate, Qingdao Ocean Silica Gel GF254 thin layer plate, Yantai Jiangyou HSGF254 thin layer plate, developed with a developing agent of ethyl acetate-methanol-formic acid-water (8:1:1:1, volume ratio), air-dried, and inspected under a 254nm ultraviolet lamp to identify Scutellaria baicalensis; then placed in an iodine tank for color development for 1 minute, inspected under sunlight to identify Phellodendron chinense; then sprayed with 10% sulfuric acid ethanol solution, heated at 105°C for 2-5 minutes, until the spots were clearly colored, inspected under sunlight to identify Radix Rehmanniae and Radix Astragali. For details, see Figures 20a to 20i .

[0199] The results showed that the three brands of thin layer plates were all able to perform exclusive identification of Angelica sinensis, Radix Rehmanniae, Rhizoma Coptidis, Cortex Phellodendri, Radix Scutellariae and Radix Astragali in the product, indicating that the present invention has good durability and wide applicability.

[0200] Example 5 Temperature Investigation

[0201] The reference solution, negative solution and test solution of Example 1 were taken and spotted on the same German Merck TLC Silica gel 60 thin layer plate according to the spotting method of Example 1. The developing agent was ethyl acetate-methanol-chloroform-ammonia-triethylamine (6:2:2:1:1, volume ratio), and the plates were developed under different temperature environments (low temperature 4°C, room temperature 25°C, and high temperature 40°C). The plates were dried and examined under an ultraviolet lamp at 366nm to identify Angelica sinensis, Phellodendron amurense and Coptis chinensis. For details, see Figures 21a to 21c .

[0202] The reference solution, negative solution and test solution of Example 1 were taken and spotted on the same German Merck TLC Silica gel 60F254 thin layer plate according to the spotting method of Example 1. The developing agent was ethyl acetate-methanol-formic acid-water (8:1:1:1, volume ratio). The plates were developed under different temperature environments (low temperature 4°C, room temperature 25°C, and high temperature 40°C), dried, and inspected under a 254nm ultraviolet lamp to identify Scutellaria baicalensis. The plates were then placed in an iodine jar for color development for 1 minute and inspected under sunlight to identify Phellodendron chinense. The plates were then sprayed with 10% ethanolic sulfuric acid solution and heated at 105°C for 2-5 minutes until the spots were clearly colored. The plates were inspected under sunlight to identify Rehmannia glutinosa and Astragalus membranaceus. For details, see Figures 22a to 22i .

[0203] The results show that the spot inspection effects of the chromatograms of the test samples at different temperatures are not much different, which indicates that the thin layer chromatography identification method provided by the present invention is less affected by temperature changes and has good durability.

[0204] Example 6 Investigation of relative humidity

[0205] The reference solution, negative solution and test solution of Example 1 were taken and spotted on the same German Merck TLC Silica gel 60 thin layer plate according to the spotting method of Example 1. The developing agent was ethyl acetate-methanol-chloroform-ammonia-triethylamine (6:2:2:1:1, volume ratio). The plates were developed under different relative humidity environments (40%, 75%, 90%), dried, and examined under ultraviolet light at 366 nm to identify Angelica sinensis, Phellodendron amurense and Coptis chinensis. For details, see Figures 23a to 23c .

[0206] The reference solution, negative solution and test solution of Example 1 were taken and spotted on the same German Merck TLC Silica gel 60F254 thin layer plate according to the spotting method of Example 1. The developing agent was ethyl acetate-methanol-formic acid-water (8:1:1:1, volume ratio). The plates were developed under different relative humidity (40%, 75%, 90%), dried, and inspected under a 254nm ultraviolet lamp to identify Scutellaria baicalensis. The plates were then placed in an iodine jar for color development for 1 minute and inspected under sunlight to identify Phellodendron chinense. The plates were then sprayed with 10% sulfuric acid ethanol solution and heated at 105°C for 2-5 minutes until the spots were clearly colored. The plates were inspected under sunlight to identify Radix Rehmanniae and Radix Astragali. Figures 24a to 24i .

[0207] The results show that the spot inspection effect of the chromatogram of the test sample under different relative humidity is not much different, which indicates that the thin layer chromatography identification method provided by the present invention is less affected by humidity changes and has good durability.

[0208] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously identifying Chinese angelica, Radix Rehmanniae, Radix Scutellariae, Rhizoma Coptidis, Cortex Phellodendri and Radix Astragali using thin layer chromatography, comprising the following steps: A. Preparation of test solution: A.

1. Extract the aqueous solution of the Angelica sinensis and six yellows composition with ether to obtain an ether extract and a first aqueous layer solution. Dry the ether extract to dryness, and dissolve the residue in methanol to obtain an ether layer test solution. A.

2. The first aqueous layer solution is extracted with water-saturated n-butanol to obtain an n-butanol extract and a second aqueous layer solution. The n-butanol extract is extracted with an ammonia test solution to obtain an n-butanol layer and an ammonia test solution layer. The n-butanol layer and the ammonia test solution layer are dried to dryness, respectively. The residue is dissolved with methanol to obtain an n-butanol layer test solution and an ammonia test solution layer test solution. A.

3. The second aqueous layer solution is subjected to solid-liquid separation to obtain a solution and a precipitate, which are dried to dryness respectively, and the residue is dissolved in methanol to obtain an aqueous layer solution test solution and an aqueous layer precipitate test solution; B. Preparation of reference solution: B.

1. Add methanol to ferulic acid, ligustilide, berberine hydrochloride and palmatine hydrochloride reference substances to prepare mixed reference substance solution 1; B.

2. Take baicalin, phellodendronine hydrochloride and astragaloside IV reference substances, and add methanol to prepare baicalin reference substance solution, phellodendronine hydrochloride reference substance solution and astragaloside IV reference substance solution respectively; B.

3. Extract the Radix Rehmanniae glutinosae with methanol, and take the extract as the Radix Rehmanniae glutinosae glutinosae reference medicinal material solution; C. Thin layer chromatography analysis: C.

1. Take the ether layer test solution prepared in step A, the n-butanol layer test solution, and the mixed reference solution 1 prepared in step B, and spot them on the same silica gel G plate respectively. Use the developing agent Z1 to develop and then dry. Place the dried silica gel G plate under ultraviolet light for inspection. When a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the test sample in the ether layer as that of the chromatogram of the ferulic acid reference substance and the ligustilide reference substance, it is identified as the presence of Angelica sinensis; In the chromatogram of the n-butanol layer test sample, when a fluorescent spot of the same color is shown at a position corresponding to the chromatogram of the berberine hydrochloride reference substance, it is identified as the presence of Coptis chinensis and / or Phellodendron chinense, and when a fluorescent spot of the same color is shown at a position corresponding to the chromatogram of the bamipine hydrochloride reference substance, it is identified as the presence of Coptis chinensis; C.

2. Take the n-butanol layer test solution, ammonia test solution layer test solution, water layer test solution and water layer precipitation test solution prepared in step A, and the raw rehmannia reference medicinal material solution, baicalin reference solution, phellodendron hydrochloride reference solution and astragaloside IV reference solution prepared in step B, and spot them on the same silica gel GF254 plate, develop with developing agent Z2 and then dry. Place the dried silica gel GF254 plate under ultraviolet light for inspection. If a spot of the same color appears at the corresponding position of the chromatogram of the precipitated test sample in the water layer and the chromatogram of the baicalin reference substance, it is identified as the presence of Scutellaria baicalensis. Place the silica gel GF254 plate after ultraviolet light inspection in an iodine jar for color development and inspect it under sunlight. If a spot of the same color appears at the corresponding position of the chromatogram of the phellodendron hydrochloride reference substance in the chromatogram of the ammonia test solution test sample, it is identified as the presence of phellodendron; Spray the silica gel GF254 plate after color development with the iodine cylinder with a color developer, heat until the spots are clearly colored, and inspect under sunlight. If a spot of the same color appears at a position corresponding to the characteristic spot of the raw rehmannia reference medicinal material solution in the chromatogram of the water layer supernatant test sample, it is identified as the presence of raw rehmannia; if a spot of the same color appears at a position corresponding to the chromatogram of the astragaloside IV reference medicinal material in the chromatogram of the n-butanol layer test sample, it is identified as the presence of astragalus; in, The developing agent Z1 is a mixed solution of ethyl acetate, methanol, chloroform, ammonia water and triethylamine; the developing agent Z2 is a mixed solution of ethyl acetate, methanol, formic acid and water, and the color developer is ethanol sulfuric acid solution.

2. The method according to claim 1, in, The method comprises the following steps: A. Preparation of test solution: A.

1. Extract the aqueous solution of the Angelica sinensis and six yellows composition with ether twice to obtain an ether extract and a first water layer solution. Evaporate the ether extract to dryness in a water bath, and dissolve the residue in methanol to obtain an ether layer test solution. A.

2. The first aqueous layer solution is extracted twice with water-saturated n-butanol to obtain an n-butanol extract and a second aqueous layer solution. The n-butanol extract is further extracted twice with an ammonia test solution to obtain an n-butanol layer and an ammonia test solution layer. The n-butanol layer and the ammonia test solution layer are evaporated to dryness in a water bath and then dissolved with methanol to obtain an n-butanol layer test solution and an ammonia test solution layer test solution; A.

3. Centrifuge the second aqueous layer solution, evaporate the supernatant and precipitate in a water bath, and dissolve the residue in methanol to serve as the aqueous layer supernatant test solution and the aqueous layer precipitate test solution; B. Preparation of reference solution: B.

1. Add methanol to ferulic acid, ligustilide, berberine hydrochloride and palmatine hydrochloride reference substances to prepare mixed reference substance solution 1; B.

2. Take baicalin, phellodendronine hydrochloride and astragaloside IV reference substances, and add methanol to prepare baicalin reference substance solution, phellodendronine hydrochloride reference substance solution and astragaloside IV reference substance solution respectively; B.

3. Take the Radix Rehmanniae glutinosae reference medicinal material, add methanol and sonicate, and take the supernatant as the Radix Rehmanniae glutinosae reference medicinal material solution. C. Thin layer chromatography analysis: C.

1. Take the ether layer test solution prepared in step A, the n-butanol layer test solution, and the mixed reference solution 1 prepared in step B, and spot them on the same silica gel G plate, develop them with developing agent Z1, dry them, and inspect them under ultraviolet light. When a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the test sample in the ether layer as that of the chromatogram of the ferulic acid reference substance and the ligustilide reference substance, it is identified as the presence of Angelica sinensis; In the n-butanol layer test sample chromatogram, when a fluorescent spot of the same color is displayed at a position corresponding to the berberine hydrochloride reference sample chromatogram, it is identified as the presence of Coptis chinensis and / or Phellodendron chinense, and when a fluorescent spot of the same color is displayed at a position corresponding to the bamipine hydrochloride reference sample chromatogram, it is identified as the presence of Coptis chinensis; C.

2. Take the n-butanol layer test solution, ammonia test solution layer test solution, water layer supernatant test solution and water layer precipitate test solution prepared in step A, and the Radix Rehmanniae Root reference medicinal material solution, baicalin reference solution, phellodendron hydrochloride reference solution and astragaloside IV reference solution prepared in step B, and spot them on the same silica gel GF254 plate, develop with developing agent Z2, and air dry. Place the dried silica gel GF254 plate under ultraviolet light for inspection. If a spot of the same color appears in the chromatogram of the aqueous layer precipitate test sample at the corresponding position of the chromatogram of the baicalin reference sample, it is identified as the presence of Scutellaria baicalensis. After the ultraviolet light inspection, the silica gel GF254 plate is placed in an iodine jar for color development and inspected under sunlight. When a spot of the same color appears at the corresponding position of the chromatogram of the phellodendron hydrochloride reference substance in the chromatogram of the test sample in the ammonia test solution layer, it is identified as the presence of phellodendron; The silica gel GF254 plate developed with iodine cylinder is sprayed with developer again, heated until the spots are clearly colored, and inspected under sunlight. When spots of the same color appear at the corresponding positions of the characteristic spots of the raw rehmannia reference medicinal material solution in the chromatogram of the water layer supernatant test sample, raw rehmannia is identified as present; when spots of the same color appear at the corresponding positions of the chromatogram of the astragaloside reference material in the chromatogram of the n-butanol layer test sample, astragalus is identified as present. Wherein, the developing agents Z1, Z2 and the developer are the same as those in claim 1.

3. The method according to claim 1 or 2, in, The Angelica and Six Yellows composition is a traditional Chinese medicine composition, a traditional Chinese medicine compound decoction, or a compound preparation made from the traditional Chinese medicine compound decoction, which contains Angelica, Rehmannia root, Rehmannia root, Scutellaria baicalensis, Coptis chinensis, Phellodendron chinense and Astragalus membranaceus at the same time; in particular, the Angelica and Six Yellows composition is Angelica and Six Yellows decoction or a compound preparation of Angelica and Six Yellows decoction.

4. The method according to claim 1 or 2, in, In step A, the extraction is an equal volume extraction; and / or, the aqueous solution of the Danggui Liuhuang composition is prepared as follows: 4-7 g of Danggui Liuhuang decoction reference sample or extract powder or its equivalent amount is taken and dissolved in 40 mL of water; and / or In step B, in the mixed reference solution 1, the concentration of ferulic acid is 0.25-1 mg / mL, for example, 0.5 mg / mL; the concentration of ligustilide is 0.15-0.6 mg / mL, for example, 0.3 mg / mL; the concentration of berberine hydrochloride is 13-50 μg / mL, for example, 25 μg / mL; the concentration of palmatine hydrochloride reference is 25-100 μg / mL, for example, 50 μg / mL; in the baicalin reference solution, the concentration of baicalin is 0.15-0.6 mg / mL, for example, 0.3 mg / mL; the concentration of berberine hydrochloride is 13-50 μg / mL, for example, 25 μg / mL; the concentration of palmatine hydrochloride reference is 25-100 μg / mL, for example, 50 μg / mL; The concentration of the reference substance is 0.5-2 mg / mL, for example 1 mg / mL; in the astragaloside reference substance solution, the concentration of the astragaloside reference substance is 0.3-1 mg / mL, for example 0.5 mg / mL; in the phellodendron hydrochloride reference substance solution, the concentration of the phellodendron hydrochloride reference substance is 0.5-2 mg / mL, for example 1 mg / mL, and in the preparation process of the raw rehmannia reference medicinal material solution, the usage ratio of the raw rehmannia reference medicinal material to methanol is 1g:10mL.

5. The method according to claim 1 or 2, in, The preparation method of the test solution in step A is as follows: A.

1. Take 4-7 g of the standard sample or extract powder of the Angelica sinensis six yellows composite material or its equivalent amount, add 40 mL of water, ultrasonically dissolve, add an equal volume of ether and shake to extract twice to obtain an ether extract and a first aqueous layer solution, evaporate the ether extract to dryness in a water bath, dissolve the residue in methanol, and use it as the ether layer test solution; A.

2. The first aqueous layer solution was extracted twice with an equal volume of water-saturated n-butanol to obtain an n-butanol extract and a second aqueous layer solution. The n-butanol extract was extracted twice with an equal volume of ammonia test solution by shaking to obtain an n-butanol layer and an ammonia test solution layer. The n-butanol layer and the ammonia test solution were evaporated to dryness in a water bath, and the residues were dissolved in methanol to obtain an n-butanol layer test solution and an ammonia test solution layer test solution; A.

3. Centrifuge the second aqueous layer solution, take the supernatant and evaporate it in a water bath, dissolve the residue in methanol to obtain the aqueous layer supernatant test solution; add methanol to ultrasonically dissolve the precipitate to obtain the aqueous layer precipitate test solution; and / or In step B, the preparation method of the reference solution is as follows: Take ferulic acid, ligustilide, berberine hydrochloride and palmatine hydrochloride reference substances, add methanol to prepare a solution containing 0.5 mg ferulic acid, 0.3 mg ligustilide, 25 μg berberine hydrochloride and 50 μg palmatine hydrochloride per 1 mL, as mixed reference substance solution 1; take baicalin, astragaloside IV and phellodendronine hydrochloride reference substances, add methanol to prepare solutions with concentrations of 1, 0.5 and 1 mg / mL respectively; The preparation method of raw Rehmannia root control medicinal material solution is as follows: Take about 1 g of raw Rehmannia root control medicinal material, add 10 ml of methanol, ultrasonicate for 1 hour, and take the supernatant as the raw Rehmannia root control medicinal material solution.

6. The method according to claim 1 or 2, in, In the developing agent Z1, the volume ratio of ethyl acetate, methanol, chloroform, aqueous ammonia and triethylamine is (5-7):(1.5-2.5):(1.5-2.5):(0.8-1.2):(0.8-1.2), preferably (5.5-6.5):(1.8-2.2):(1.8-2.2):(0.9-1.1):(0.9-1.1), more preferably (5.8-6.2):(1.9-2.1):(1.9-2.1):(0.95-1.05):(0.95-1.05), especially 6:2:2:1:1; and / or In the developing agent Z2, the volume ratio of ethyl acetate, methanol, formic acid and water is (7-9):(0.8-1.2):(0.8-1.2):(0.8-1.2), preferably (7.5-8.5):(0.9-1.1):(0.9-1.1):(0.9-1.1), more preferably (7.8-8.2):(0.95-1.05):(0.95-1.05):(0.95-1.05), especially 8:1:1:

1.

7. The method according to claim 1 or 2, in, In the step C, the sample solution is spotted in an amount of 3-20 μL, and the spotting method is spot spotting or strip spotting, and the preferred spotting amount is 10 μL; and / or The unfolding method is: put in the spotted silica gel plate, cover and seal, and unfold until the front edge of the solvent is 2-3 cm away from the upper end of the silica gel plate.

8. The method according to claim 1 or 2, in, In step C.1, the wavelength of the ultraviolet light is checked to be 366 nm.

9. The method according to claim 1 or 2, in, In step C.2, The UV wavelength is 254nm; and / or The operation method of iodine cylinder color development is: place iodine liquid at the bottom of a glass desiccator, place a silica gel plate on the porcelain plate in the glass desiccator, cover and seal, the color development time is 30s-1min, and inspect under sunlight.

10. The method according to claim 1 or 2, in, In step C.2, The color developing agent is 10% sulfuric acid ethanol solution; and / or The heating is performed at 105° C. for 2-5 min.

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