A thin layer chromatography identification method for Danggui Liuhuang Decoction and its compound preparation

Abstract

The present invention belongs to the technical field of identification of traditional Chinese medicines, and specifically relates to a thin-layer identification method for Danggui Liuhuang Decoction and its compound preparation. The preparation method of the test solution and the control medicinal material solution includes at least one of method 1 and method 2; the developing agent includes ethyl acetate, n-propanol and water. The thin-layer identification method provided by the present invention is not only simple in sample preparation and easy to operate, but also has less loss during the sample preparation process, thus saving inspection time and cost to a great extent; and the method established by the present invention is negative and interference-free, has strong specificity, has good application prospects, and is of great significance to the thin-layer identification research of Astragalus in compound preparations. The present invention adopts active ingredient reference substances and control medicinal materials as controls at the same time, avoiding the operational inconvenience caused by separately comparing the reference substances and control medicinal materials, while improving its accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a thin layer chromatography identification method for Danggui Liuhuang Decoction and its compound preparation. Background Art

[0002] Danggui Liuhuang Decoction, from Li Dongyuan's Lanshi Mizang (Orchid Chamber Secret Collection) from the Jin and Yuan dynasties, is a classic prescription for treating night sweats, known as the "holy medicine for night sweats." It comprises angelica sinensis, raw and cooked rehmannia root, scutellaria baicalensis, coptis chinensis, phellodendron amurense, and astragalus root. Its primary effects are nourishing yin and purging fire, strengthening the exterior and arresting sweating. It is primarily used to treat night sweats caused by yin deficiency and excessive fire. Clinical symptoms include fever, night sweats, flushed face, restlessness, dry mouth and lips, dry stools, dark yellow urine, red tongue with a yellow coating, and a rapid pulse.

[0003] As public awareness of health care grows, higher standards are being placed on the quality and safety of traditional Chinese medicines. Currently, no standards exist for identifying the active ingredients in Danggui Liuhuang Decoction. To identify the astragalus root in Danggui Liuhuang Decoction, Su Ying published a thin-layer chromatography method in "A Study on the Material Standardization of Danggui Liuhuang Decoction." The sample preparation involved heating under reflux, passing through a neutral alumina column, and extraction. This complex and time-consuming sample preparation method prevented rapid confirmation of the presence of astragalus root in Danggui Liuhuang Decoction and its compound preparations. However, the recipe doubles the amount of astragalus root, achieving the goal of invigorating Qi and strengthening the exterior, and it is a key ingredient in Danggui Liuhuang Decoction.

[0004] Therefore, it is urgent to establish a method with high accuracy, time-saving and cost-effectiveness, and easy promotion to control the qualitative identification of Astragalus in Danggui Liuhuang Decoction compound preparations and material benchmarks to ensure the uniformity, stability and reliability of their quality. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problems of the prior art in thin-layer chromatography identification of Danggui Liuhuang Decoction, such as the complex sample preparation method, long time consumption, and low detection accuracy. Therefore, a thin-layer chromatography identification method for Danggui Liuhuang Decoction and its compound preparation is provided.

[0006] To this end, the present invention provides the following technical solutions:

[0007] A thin layer chromatography identification method for Danggui Liuhuang Decoction and its compound preparation comprises the following steps:

[0008] Take the test sample and the astragalus control medicinal material and prepare the test sample solution and the control medicinal material solution according to at least one of the following methods 1 and 2 respectively;

[0009] Method 1: Take the test sample and the control medicinal material of Astragalus, add alkaline solution to dissolve them respectively, perform the first solid-liquid separation, draw the liquid and add n-butanol to mix, perform the second solid-liquid separation, draw the n-butanol solution layer, add water to mix, perform the third solid-liquid separation, obtain the n-butanol solution layer, dry it, add alcohol to dissolve it, and prepare the test sample solution and control medicinal material solution respectively;

[0010] Method 2: Take the test sample and the control medicinal material of Astragalus, extract them with alcohol respectively, separate the solid and liquid, take the liquid, dry it, dissolve it in water, extract it with n-butanol saturated with water, take the n-butanol extract, wash it with alkaline solution, and then wash it with water saturated with n-butanol, take the n-butanol liquid, dry it, and dissolve it with alcohol to prepare the test sample solution and the control medicinal material solution respectively;

[0011] Identification: Spot the test sample solution, reference medicinal material solution, and astragaloside reference solution on the same thin layer plate, develop with a mixed solution of ethyl acetate, n-propanol, and water, remove, air dry, spray with a color developer, heat, and examine under visible light.

[0012] Furthermore, in the identification step, the thin layer plate used is a silica gel G thin layer plate; and / or the heating temperature is 95-105°C and the time is 3-5 minutes; and / or the spotting volume of the test solution, control medicinal material solution or astragaloside IV reference solution is 1-20 μL.

[0013] In certain preferred embodiments, the spotting volume of the test solution, control medicinal material solution or astragaloside IV reference solution is 5 to 20 μL.

[0014] In certain more preferred embodiments, the spotting volume of the test solution is 20 μL, the spotting volume of the control medicinal material solution is 20 μL, and the spotting volume of the astragaloside IV reference solution is 10 μL.

[0015] Furthermore, the color developer is sulfuric acid ethanol solution, and preferably, the concentration of the sulfuric acid ethanol solution is 5-15%.

[0016] Furthermore, method 1 also satisfies one or more of the following AH conditions:

[0017] A. The mass to volume ratio of the test sample or control medicinal material to the alkaline solution is 0.5-6:15, preferably 2-4:15, wherein the mass to volume ratio is g / mL;

[0018] B. the first solid-liquid separation, the second solid-liquid separation, or the third solid-liquid separation is centrifugation or filtration;

[0019] C. The mass-to-volume ratio of the test sample or control medicinal material to n-butanol is 0.5-6:5-30, preferably 2-4:10, wherein the mass-to-volume ratio is g / mL;

[0020] D. The volume ratio of n-butanol to water is 5-30:5-30, preferably 1:1;

[0021] E. In the dissolution step, the mass-to-volume ratio of the test sample or control medicinal material to the alcohol is 0.5-6:0.5-3, preferably 2-4:1, wherein the mass-to-volume ratio is g / mL;

[0022] F. The mass volume concentration of the alkaline solution is 0.01-52 g / 100 mL, preferably 0.01-4 g / 100 mL;

[0023] G. The alcohol includes one or more of methanol and ethanol; preferably methanol;

[0024] H. The alkaline solution includes one or more of a sodium hydroxide aqueous solution and a potassium hydroxide aqueous solution.

[0025] Furthermore, method 2 also satisfies one or more of the following aj:

[0026] a. In the alcohol extraction step, the ratio of the mass of the test sample or control medicinal material to the volume of alcohol and water is 0.5-6:20-40:20-40, preferably 2-4:30:20, wherein the mass to volume ratio is g / mL;

[0027] b. The solid-liquid separation is performed by centrifugation or filtration;

[0028] c. The extraction method of alcohol extraction is ultrasonic extraction or hot reflux extraction, and the extraction time is at least 15 minutes (e.g. 15-60 minutes)

[0029] d. The number of water-saturated n-butanol extractions is 1-5 times, and the ratio of the mass of the test solution or the control medicinal material to the volume of the water-saturated n-butanol added in each extraction is 0.5-6:20; preferably 2-4:20, wherein the mass-to-volume ratio is g / mL;

[0030] e. The number of washes with alkaline solution or water saturated with n-butanol is 1-5 times, and the ratio of the mass of the test sample or control medicinal material to the volume of the alkaline solution or water saturated with n-butanol added during each wash is 0.5-6:20; preferably 2-4:20, wherein the mass to volume ratio is g / mL;

[0031] f. In the dissolution step, the mass-to-volume ratio of the test sample or control medicinal material to the alcohol is 0.5-6:0.5-3, preferably 2-4:1, wherein the mass-to-volume ratio is g / mL;

[0032] g. The mass volume concentration of the alkaline solution is 0.01-52 g / 100 mL, preferably 0.01-4 g / 100 mL;

[0033] h. The alcohol includes one or more of methanol and ethanol; preferably methanol;

[0034] j. The alkaline solution includes one or more of a sodium hydroxide aqueous solution and a potassium hydroxide aqueous solution.

[0035] Furthermore, the volume ratio of ethyl acetate, n-propanol and water in the developing solvent is 5-10:1-8:1-6; preferably 6-8:6:4-5; more preferably 7:5:4.

[0036] Furthermore, the solvent of the reference solution is selected from methanol or a methanol aqueous solution with a volume concentration of more than 80%; and / or each 1 mL of the reference solution contains 0.1 to 4 mg of astragaloside IV reference substance.

[0037] Furthermore, the preparation method of the test solution includes taking 0.5-6g of the test sample powder, adding 15ml of an alkaline solution with a mass volume concentration of 0.01-4g / 100mL, dissolving, centrifuging, absorbing the supernatant, adding 5-30ml of n-butanol to the supernatant, shaking, centrifuging, absorbing the n-butanol solution layer, adding 5-30ml of water, shaking, centrifuging to obtain the n-butanol solution layer, evaporating to dryness, adding 0.5-3ml of methanol to dissolve, and obtaining the test solution; or taking 0.5-6g of the test sample powder , add 20-40 ml of methanol, ultrasonically treat for 15-60 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10-30 ml of water, extract with n-butanol saturated with water 1-5 times, 20 ml each time, combine the n-butanol extracts, wash with an alkaline solution with a mass volume concentration of 0.01-4 g / 100 mL 1-5 times, 20 ml each time, then wash with water saturated with n-butanol 1-5 times, 20 ml each time, evaporate the n-butanol solution to dryness, dissolve the residue in 0.5-3 ml of methanol as the test solution;

[0038] The preparation method of the control medicinal material solution comprises: taking 0.5-6g of the control medicinal material of Astragalus, adding 15ml of an alkaline solution with a mass volume concentration of 0.01-4g / 100ml, dissolving, centrifuging, absorbing the supernatant, adding 5-30ml of n-butanol to the supernatant, shaking, centrifuging, absorbing the n-butanol solution layer, adding 5-30ml of water, shaking, centrifuging to obtain the n-butanol solution layer, evaporating to dryness, adding 0.5-3ml of methanol to dissolve, and obtaining the control medicinal material solution; or taking 0.5-6g of the control medicinal material of Astragalus, Add 20-40 ml of methanol, ultrasonically treat for 15-60 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10-30 ml of water, extract with water-saturated n-butanol 1-5 times, 20 ml each time, combine the n-butanol extracts, wash with an alkaline solution with a mass volume concentration of 0.01-4 g / 100 mL 1-5 times, 20 ml each time, then wash with water saturated with n-butanol 1-5 times, 20 ml each time, evaporate the n-butanol solution to dryness, and dissolve the residue in 0.5-3 ml of methanol as a control medicinal material solution.

[0039] Further, the preparation method of the test solution includes taking 2-4g of the test sample powder, adding 15ml of a sodium hydroxide aqueous solution with a mass volume concentration of 0.5-2g / 100mL, dissolving, centrifuging, absorbing the supernatant, adding 10ml of n-butanol to the supernatant, shaking, centrifuging, absorbing the n-butanol solution layer, adding 10ml of water, shaking, centrifuging to obtain the n-butanol solution layer, evaporating to dryness, adding 1ml of methanol to dissolve, and obtaining the test solution; or, taking 2-4g of the test sample powder, adding 30ml of methanol, ultrasonically treating for 30 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 20ml of water, extracting twice with n-butanol saturated with water, each time 20ml, combining the n-butanol extracts, washing twice with a sodium hydroxide aqueous solution with a mass volume concentration of 0.5-2g / 100mL, each time 20ml, and then washing twice with water saturated with n-butanol, each time 20ml, evaporating the n-butanol solution to dryness, and dissolving the residue in methanol 1ml to obtain the test solution;

[0040] The preparation method of the control medicinal material solution includes taking 2-4g of the control medicinal material of Astragalus, adding 15ml of a sodium hydroxide aqueous solution with a mass volume concentration of 0.5-2g / 100ml, dissolving, centrifuging, absorbing the supernatant, adding 10ml of n-butanol to the supernatant, shaking, centrifuging, absorbing the n-butanol solution layer, adding 10ml of water, shaking, centrifuging to obtain the n-butanol solution layer, evaporating to dryness, adding 1ml of methanol to dissolve, and obtaining a control medicinal material solution; or taking 2-4g of the control medicinal material of Astragalus, adding 30ml of methanol, ultrasonically treating for 30 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 20ml of water, extracting twice with n-butanol saturated with water, each time 20ml, combining the n-butanol extracts, washing twice with a sodium hydroxide aqueous solution with a mass volume concentration of 0.5-2g / 100ml, each time 20ml, and then washing twice with water saturated with n-butanol, each time 20ml, evaporating the n-butanol solution to dryness, and dissolving the residue in 1ml of methanol as the control medicinal material solution.

[0041] Furthermore, the test sample is a solid preparation, liquid preparation or semisolid preparation of Danggui Liuhuang Decoction, preferably a powder, granules, tablets, capsules, oral liquid, injection, emulsion, suspension or ointment.

[0042] In the present invention, Danggui Liuhuang Decoction and its compound preparation are prepared from angelica sinensis, raw rehmannia root, cooked rehmannia root, scutellaria baicalensis, coptis chinensis, phellodendron amurense, and astragalus root as raw materials according to conventional processes in the art. The mass ratio of angelica sinensis, raw rehmannia root, cooked rehmannia root, scutellaria baicalensis, coptis chinensis, phellodendron amurense, and astragalus root is 2-3:2-3:2-3:2-3:2-3:2-3:4-6; preferably 2.5:2.5:2.5:2.5:2.5:2.5:5.

[0043] The technical solution of the present invention has the following advantages:

[0044] 1. The thin-layer identification method of Danggui Liuhuang Decoction and its compound preparation provided by the present invention, the preparation method of the test solution and the control medicinal material solution includes at least one of method 1 and method 2; the developing agent includes ethyl acetate, n-propanol and water. The thin-layer identification method provided by the present invention is not only simple in sample preparation and easy to operate, but also has less loss during the sample preparation process, which greatly saves inspection time and cost; and the method established by the present invention is negative and interference-free, has strong specificity, has good application prospects, and is of great significance to the thin-layer identification research of Astragalus in compound preparations. The present invention uses active ingredient reference substances and control medicinal materials as controls at the same time, avoids the operational inconvenience caused by the separate controls of the reference substances and control medicinal materials, and improves its accuracy. In particular, when method 1 is used to prepare the test solution and the control medicinal material solution, more characteristic spots can be obtained, and the accuracy is significantly improved.

[0045] 2. The thin-layer chromatography identification method for Danggui Liuhuang Decoction and its compound preparation provided by the present invention utilizes commonly used low-toxic reagents for the experiment, greatly improving the safety of the experimental operation and reducing the risk to the environment and experimenters. The identification method provided by the present invention has no special requirements for temperature and humidity, and temperature and humidity have no effect on the identification results. The identification method has good durability and reproducibility. It has excellent specificity, eliminating the possibility of interference from other ingredients causing the same spots.

[0046] 3. The thin-layer identification method provided by the present invention uses ethyl acetate, n-propanol, and water in a volume ratio of 5-10:1-8:1-6 (particularly a volume ratio of 6-8:6:4-5) as the developing solvent, resulting in clearer spots and higher resolution on the thin-layer, with the optimal volume ratio being 7:5:4. Compared with the prior art, the preparation of the test solution provided by the present invention is simple and easy to operate, without requiring complex procedures such as heating under reflux and passing through a neutral alumina column, thus saving on organic solvent usage and shortening the time required to prepare the test solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0048] Figure 1 This is the thin-layer chromatography spectrum of developing agent 1 in Example 2 of the present invention; wherein, from left to right: 1. Yin 1; 2. Supply 1; 3. Counter 1; 4. Astragaloside IV reference solution; 5. Calycosin isoflavone glucoside reference solution; 6. Counter 2; 7. Supply 2; 8. Yin 2; 9. Yin 3; 10. Supply 3; 11. Counter 3; 12. Astragaloside IV reference solution; 13. Calycosin isoflavone glucoside reference solution; 14. Counter 4; 15. Supply 4; 16. Yin 4; 17. Astragaloside IV reference solution; 18. Calycosin isoflavone glucoside reference solution; 19. Counter 5; 20. Supply 5; 21. Yin 5;

[0049] Figure 2 This is the thin-layer chromatography spectrum of developing agent 2 in Example 2 of the present invention; from left to right: 1. Yin 1; 2. Supply 1; 3. Counter 1; 4. Yin 2; 5. Supply 2; 6. Counter 2; 7. Yin 3; 8. Supply 3; 9. Counter 3; 10. Astragaloside IV reference solution; 11. Calycosin glucoside reference solution; 12. Yin 4; 13. Supply 4; 14. Counter 4; 15. Yin 5; 16. Supply 5; 17. Counter 5; 18. Astragaloside IV reference solution;

[0050] Figure 3 This is the thin-layer chromatography spectrum of developing agent 3 in Example 2 of the present invention; from left to right: 1. Negative 1; 2. Supply 1; 3. Counter 1; 4. Negative 2; 5. Supply 2; 6. Counter 2; 7. Negative 3; 8. Supply 3; 9. Counter 3; 10. Astragaloside IV reference solution; 11. Calycosin glucoside reference solution; 12. Negative 4; 13. Supply 4; 14. Counter 4; 15. Negative 5; 16. Supply 5; 17. Counter 5; 18. Astragaloside IV reference solution;

[0051] Figure 4 This is the thin-layer chromatography spectrum of developing agent 4 in Example 2 of the present invention; from left to right: 1. Yin 1; 2. Supply 1; 3. Counter 1; 4. Yin 2; 5. Supply 2; 6. Counter 2; 7. Yin 3; 8. Supply 3; 9. Counter 3; 10. Astragaloside IV reference solution; 11. Calycosin glucoside reference solution; 12. Yin 4; 13. Supply 4; 14. Counter 4; 15. Yin 5; 16. Supply 5; 17. Counter 5; 18. Astragaloside IV reference solution;

[0052] Figure 5 This is the thin-layer chromatography spectrum of developing agent 5 in Example 2 of the present invention; from left to right: 1. Negative 1; 2. Supply 1; 3. Counter 1; 4. Negative 2; 5. Supply 2; 6. Counter 2; 7. Negative 3; 8. Supply 3; 9. Counter 3; 10. Astragaloside IV reference solution; 11. Calycosin glucoside reference solution; 12. Negative 4; 13. Supply 4; 14. Counter 4; 15. Negative 5; 16. Supply 5; 17. Counter 5; 18. Astragaloside IV reference solution;

[0053] Figure 6 This is the thin-layer chromatography spectrum of developing agent 6 in Example 2 of the present invention; from left to right: 1. Negative 1; 2. Supply 1; 3. Counter 1; 4. Negative 2; 5. Supply 2; 6. Counter 2; 7. Negative 3; 8. Supply 3; 9. Counter 3; 10. Astragaloside IV reference solution; 11. Calycosin glucoside reference solution; 12. Negative 4; 13. Supply 4; 14. Counter 4; 15. Negative 5; 16. Supply 5; 17. Counter 5; 18. Astragaloside IV reference solution;

[0054] Figure 7 The thin layer chromatography of different sample amounts in Example 3 of the present invention is as follows: 1. 5 μl of test solution; 2. 10 μl of test solution; 3. 15 μl of test solution; 4. 20 μl of test solution; 5. 10 μl of astragaloside IV reference solution; 6. 10 μl of control medicinal material solution; 7. 15 μl of control medicinal material solution; 8. 20 μl of control medicinal material solution;

[0055] Figure 8This is the thin layer chromatography used for the specificity investigation of the present invention; 1. Test solution JF-DGLH-DG2020019; 2. Astragalus reference medicinal material solution; 3. Astragaloside IV reference solution; 4. Danggui Liuhuang Decoction Astragalus single negative control solution; 5. Danggui Liuhuang Decoction Astragalus single positive control solution;

[0056] Figure 9 It is a thin layer chromatography under room temperature conditions in the development temperature investigation of the present invention; wherein, 1. test solution, 2. astragaloside IV reference solution, 3. astragalus reference medicinal material solution;

[0057] Figure 10 The thin layer chromatography under low temperature conditions in the development temperature investigation of the present invention; wherein, 1. test solution, 2. astragaloside IV reference solution, 3. astragalus reference medicinal material solution;

[0058] Figure 11 The present invention is to carry out a thin layer chromatography under low humidity conditions in the humidity investigation; wherein, 1. test solution, 2. astragaloside IV reference solution, 3. astragalus reference medicinal material solution;

[0059] Figure 12 The present invention is to carry out a thin layer chromatography under high humidity conditions in the humidity investigation; wherein, 1. test solution, 2. astragaloside IV reference solution, 3. astragalus reference medicinal material solution;

[0060] Figure 13 The thin layer chromatography of the present invention adopts Qingdao Ocean Plate (Qingdao Ocean Chemical Co., Ltd., batch number: 20200707); wherein, 1. Astragaloside IV reference solution, 2. Test solution, 3. Astragalus reference medicinal material solution;

[0061] Figure 14 The thin layer chromatography plate of the present invention adopts the Yantai Zhifu thin layer plate (Yantai Zhifu Huangwu Silica Gel Development and Experimental Factory, batch number: 20161007); wherein, 1. Astragaloside IV reference solution, 2. Test solution, 3. Astragalus reference medicinal material solution;

[0062] Figure 15 The thin layer chromatography of the present invention adopts the Yinlong thin layer plate (Yantai Chemical Industry Research Institute, batch number: 20190808) in the thin layer plate investigation; wherein, 1. Astragaloside IV reference solution, 2. Test solution, 3. Astragalus reference medicinal material solution;

[0063] Figure 16 The thin layer chromatography using ethyl acetate: n-propanol: water (6:6:5) as the developing solvent in the present invention;

[0064] Figure 17 The thin layer chromatography using ethyl acetate: n-propanol: water (8:6:4) as the developing solvent in the present invention;

[0065] Figure 18 The thin layer chromatogram of Example 4 of the present invention is from left to right: 1-4, 7-8 test solution (batch numbers are: JF-DGLH-DG2020001, JF-DGLH-DG2020002, JF-DGLH-DG2020003, JF-DGLH-DG2020004, JF-DGLH-DG2020005, JF-DGLH-DG2020006); 5. Astragaloside reference solution; 6. Astragalus control medicinal material solution; 9. Astragalus negative control solution. DETAILED DESCRIPTION

[0066] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0067] If specific experimental procedures or conditions are not specified in the examples, the procedures or conditions described in the literature in the field can be followed. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional reagents. The ratios of the solvents in the developing agent are by volume.

[0068] Instruments, reagents and test drugs

[0069] Instruments: Electronic balance: JJ500, Changshu Shuangjie Testing Instrument Factory; Electronic balance: MSA6.6S-OCE-DM, Sartorius Scientific Instrument Co., Ltd.; Thin layer imaging system: GoodLook-1000, Shanghai Kezhe Biochemical Technology Co., Ltd.; Ultrasonic cleaner: KQ-100DE, Kunshan Ultrasonic Instrument Co., Ltd. (500 W, 40 kHz); Electric constant temperature water bath: DK-S26, Zhongyi Guoke Technology Co., Ltd.; pH meter: FE28, Mettler-Toledo Instrument Co., Ltd.; Silica gel G plate: Yantai Chemical Industry Research Institute.

[0070] Reagents: Methanol, ethanol, n-butanol, water, ethyl acetate, sulfuric acid, sodium hydroxide, hydrochloric acid, anhydrous sodium sulfate, chloroform and other chemical reagents were all of analytical grade.

[0071] Test drugs: astragaloside IV (China Food and Drug Inspection Institute, batch number: 111781-201717), isoflavone glucoside (China Food and Drug Inspection Institute, batch number: 111920--201606), and astragalus control medicinal material (China Food and Drug Inspection Institute, batch number: 120904-201620).

[0072] The freeze-dried powder of Danggui Liuhuang Decoction is prepared as follows: Take the slices of each ingredient of Danggui Liuhuang Decoction and grind them separately. Weigh 2.5g each of Danggui, Rehmannia root, Rehmannia root, Phellodendron chinense, Scutellaria baicalensis, and Coptis chinensis, and 5g of Astragalus root. Place the slices in a decoction pot, add 400ml of purified water, and boil over high heat at 500w, then reduce heat to 300w, cover, and simmer for 30 minutes. Filter through 200-mesh gauze. Cool the filtrate to room temperature and dry. Different batches of raw materials were used to prepare freeze-dried powder of Danggui Liuhuang Decoction according to the above method, batch numbers: JF-DGLH-DG2020019, JF-DGLH-DG2020001, JF-DGLH-DG2020002, JF-DGLH-DG2020003, JF-DGLH-DG2020004, JF-DGLH-DG2020005, JF-DGLH-DG2020006.

[0073] Danggui Liuhuang Tang (Danggui Liuhuang Tang) freeze-dried powder of Astragalus root is prepared as follows: 2.5g each of crushed slices of Angelica sinensis, Rehmannia root, Rehmannia root, Phellodendron chinense, Scutellaria baicalensis, and Coptis chinensis are placed in a decoction pot. Add 400ml of purified water and bring to a boil over high heat at 500w. Then reduce heat to 300w, cover, and simmer for 30 minutes. Filter through 200-mesh gauze, cool the filtrate to room temperature, and dry. Batch number: JF-DGLH-DG2020026.

[0074] Danggui Liuhuang Decoction (Danggui Liuhuang Decoction) single-positive Astragalus root sample freeze-dried powder is prepared as follows: Weigh 5g of crushed Astragalus root slices into a decoction pot, add 400ml of purified water, and bring to a boil over high heat at 500W. Then reduce heat to 300W, cover, and simmer for 30 minutes. Filter through 200-mesh gauze, cool the filtrate to room temperature, and dry. Batch number: JF-DGLH-DG2020035.

[0075] Example 1

[0076] This embodiment provides a method for identifying Danggui Liuhuang Decoction and its preparation, comprising the following steps:

[0077] Preparation of test solution: Use Danggui Liuhuang Decoction freeze-dried powder as the test sample, take 4 g of the test sample, add 15 ml of 1% (w / v, g / 100 mL) sodium hydroxide aqueous solution, dissolve, centrifuge, aspirate the supernatant, add 10 ml of n-butanol to the supernatant, shake well, centrifuge, aspirate the n-butanol solution layer, add 10 ml of water, shake well, centrifuge to obtain the n-butanol solution layer, evaporate to dryness, add 1 ml of methanol to dissolve, and obtain.

[0078] Preparation of control medicinal material solution: Take 4 g of Astragalus control medicinal material, add 15 ml of 1% (w / v, g / 100 mL) sodium hydroxide aqueous solution, dissolve, centrifuge, absorb the supernatant, add 10 ml of n-butanol to the supernatant, shake well, centrifuge, absorb the n-butanol solution layer, add 10 ml of water, shake well, centrifuge to obtain the n-butanol solution layer, evaporate to dryness, add 1 ml of methanol to dissolve, and obtain.

[0079] Preparation of reference solution: Take astragaloside IV reference substance and add methanol to make a solution containing 0.5 mg per 1 mL to obtain astragaloside IV reference solution.

[0080] Pipette 20 μl of test solution, 20 μl of control medicinal material solution and 10 μl of reference solution, spot them respectively on the same thin layer plate, develop them with ethyl acetate: n-propanol: water (7:5:4) as the developing agent, dry them, spray them with 10% ethanolic sulfuric acid solution, heat until the spots are colored, and examine them under visible light.

[0081] Example 2 Investigation of sample preparation method and developing agent

[0082] The same batch of Danggui Liuhuang Decoction freeze-dried powder, Astragalus control medicinal materials and Astragalus single-negative freeze-dried powder were divided into five parts, and sample solutions were prepared according to the following five methods.

[0083] Method 1: Take approximately 4g of Danggui Liuhuang Decoction freeze-dried powder (passed through a No. 4 sieve) and place it in a stoppered conical flask. Add 50ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4ml of concentrated ammonia test solution, add 80% methanol to 100ml, and shake well). Stopper the flask tightly. Heat under reflux for 1 hour. Filter and evaporate to dryness. Dissolve the residue in 10ml of 80% methanol to prepare the test solution. This is designated as "Supply 1". Separately, prepare a control solution of Astragalus membranaceus from the control herb using the same method as the test solution. This is designated as "Control 1". Prepare a negative control solution of Astragalus membranaceus from Danggui Liuhuang Decoction using the same method as the test solution. This is designated as "Negative 1".

[0084] Method 2: Take approximately 4 g of Danggui Liuhuang Decoction freeze-dried powder (passed through a No. 4 sieve) and place it in a stoppered conical flask. Add 50 ml of methanol, seal tightly, and heat under reflux for 4 hours. Filter, evaporate to dryness, and dissolve the residue in 10 ml of methanol to prepare the test solution. This is designated as "Supplement 2." Separately, prepare a control solution of Astragalus membranaceus using the same method as the test solution. This is designated as "Reference 2." Prepare a negative control solution of Astragalus membranaceus powder from Danggui Liuhuang Decoction using the same method as the test solution. This is designated as "Negative 2."

[0085] Method 3: Take 4g of Danggui Liuhuang Decoction freeze-dried powder, add 30ml of ethanol, heat under reflux for 20 minutes, filter, and evaporate the filtrate to dryness. Dissolve the residue in 15ml of 0.3% (w / v, g / 100mL) sodium hydroxide solution, filter, and adjust the pH of the filtrate to 5-6 with dilute hydrochloric acid. Extract with 15ml of ethyl acetate by shaking. Filter the ethyl acetate fraction through filter paper covered with an appropriate amount of anhydrous sodium sulfate, and evaporate the filtrate to dryness. Dissolve the residue in 1ml of ethyl acetate to prepare the test solution. This is designated as "Supplement 3." Separately, prepare a control solution of Astragalus membranaceus from the control herb using the same method as the test solution. This is designated as "Reverse 3." Prepare a negative control solution of Astragalus membranaceus from Danggui Liuhuang Decoction using the same method as the test solution. This is designated as "Negative 3."

[0086] Method 4: Take 2g of Danggui Liuhuang Decoction freeze-dried powder, add 30ml of methanol, and sonicate for 30 minutes. Filter, evaporate the filtrate to dryness, and dissolve the residue in 20ml of water. Extract twice with water-saturated n-butanol, 20ml each time. Combine the n-butanol extracts and wash twice with 1% (w / v, g / 100mL) sodium hydroxide solution, 20ml each time. Then, wash twice with n-butanol-saturated water, 20ml each time. Evaporate the n-butanol solution to dryness, and dissolve the residue in 1ml of methanol. This is the test solution. This is designated as "Supplement 4." Separately, prepare a control solution of Astragalus membranaceus from the control herb using the same method as the test solution. This is designated as "Reverse 4." Prepare a negative control solution of Astragalus membranaceus from Danggui Liuhuang Decoction using the same method as the test solution. This is designated as "Negative 4."

[0087] Method 5: Take 4g of Danggui Liuhuang Decoction freeze-dried powder, add 15ml of 1% (w / v, g / 100mL) sodium hydroxide solution, dissolve, centrifuge, and aspirate the supernatant. Add 10ml of n-butanol to the supernatant, shake well, and centrifuge. Aspirate the n-butanol layer, add 10ml of water, shake well, and centrifuge to obtain the n-butanol layer. Evaporate to dryness and dissolve in 1ml of methanol to prepare the test solution. This is designated as "Supplement 5." Separately, prepare a control solution of Astragalus membranaceus from the control herb using the same method as the test solution. This is designated as "Control 5." Prepare a negative control solution of Astragalus membranaceus from Danggui Liuhuang Decoction using the same method as the test solution. This is designated as "Negative 5."

[0088] Prepare the reference solution: take the astragaloside IV reference substance and add methanol to make a solution containing 0.5 mg per 1 mL to obtain the astragaloside IV reference solution; take the calycosin isoflavone glucoside reference substance and add methanol to make a solution containing 0.5 mg per 1 mL to obtain the calycosin isoflavone glucoside reference substance solution.

[0089] Pipette 20 μL of test solution, 20 μL of control medicinal material solution, 20 μL of negative control solution, 10 μL of astragaloside IV reference solution and 10 μL of caerulean glucoside reference solution, and spot them on the same silica gel G thin layer plate. Develop them with the following 6 solutions as developing agents, let them dry, spray them with 10% ethanolic sulfuric acid solution, heat until the spots are colored, and inspect them under visible light.

[0090] Developing solvent 1: chloroform-methanol (10:1);

[0091] Developing solvent 2: lower layer solution of chloroform-methanol-water (13:7:2);

[0092] Developing solvent 3: upper layer solution of n-butanol-ethyl acetate-water (4:1:5);

[0093] Developing solvent 4: Chloroform-ethyl acetate-methanol-water (10:20:11:5) lower layer solution placed below 10°C for 12 hours;

[0094] Developing solvent 5: ethyl acetate-butanone-formic acid-water (5:3:3:1);

[0095] Developing solvent 6: ethyl acetate: n-propanol: water (7:5:4). Figure 1-6 and shown in the table below.

[0096] Table 1 Identification results of the test samples under various sample preparation methods and developing agents

[0097]

[0098]

[0099] Summary: The results show that 1) none of the calycosin glucosides showed color, so astragaloside IV was selected as the reference standard. 2) After the developer was heated, the spots showed more information and were easier to observe, so the results listed in this experiment were all observed under white light after the developer was heated. 3) When different sample preparation methods were examined, the spots corresponding to the astragaloside IV reference standard in the test solution obtained by sample preparation methods 1, 2, and 3 were not observed, and no other negative, non-interfering spots were found. However, the spots corresponding to the astragaloside IV reference standard in the test solution obtained by sample preparation methods 4 and 5 were observable. 4) When different developing agents were examined, the astragaloside IV reference standard did not develop in developing agent 1, the astragaloside IV Rf value in developing agent 4 was too small, and the astragaloside IV Rf values ​​in developing agents 2, 3, and 5 were moderate and negative, but no other negative, non-interfering spots were found in the control herbs. 5) Compared with other combination methods, the accuracy of thin-layer identification using sample preparation method 4 or 5 combined with developing agent 6 was significantly improved. In the chromatogram of the test sample, there are spots of the same color at the corresponding positions in the chromatograms of the reference sample and the reference medicinal material, and the Rf values ​​of the spots are all moderate and negative without interference; 6) Sample preparation method 5 combined with developing agent 6 is the best combination. In the chromatogram of the test sample, there are spots of the same color at the corresponding positions in the chromatogram of the reference sample, and the positions and colors of the two spots in the reference medicinal material solution also correspond. The Rf values ​​of the spots are all moderate and negative without interference.

[0100] Example 3 Methodological Investigation

[0101] (1) Investigation of sample quantity

[0102] 10 μL of the reference solution, different spot amounts (5 μL, 10 μL, 15 μL, 20 μL) of the test solution, and different spot amounts (10 μL, 15 μL, 20 μL) of the Astragalus control medicinal material solution were respectively spotted on the same thin layer plate, and developed with ethyl acetate: n-propanol: water (volume ratio of 7:5:4), dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were colored, and inspected under visible light. The preparation method of each solution is the same as in Example 1. The results are shown in FIG. Figure 7 shown.

[0103] pass Figure 7 As can be seen, at each spotting volume, the TLC of the test solution and the reference solution displayed spots of the same color at the same location, corresponding to two spots in the control herbal solution, for a total of three characteristic spots. Spotting volumes of 10 μl of the reference solution, 20 μl of the reference herbal solution, and 20 μl of the test solution allowed for clearer and more accurate identification of Astragalus membranaceus in the Danggui Liuhuang Decoction standard. Therefore, the reference solution was preferred, with spotting volumes of 10 μl, 20 μl, and 20 μl, respectively, for the control herbal solution and the test solution.

[0104] (2) Specificity inspection

[0105] The test solution, reference medicinal material solution and reference substance solution were prepared according to the method of Example 1.

[0106] Danggui Liuhuang Decoction Astragalus single positive control solution: Take 4 g of Danggui Liuhuang Decoction Astragalus single positive freeze-dried powder instead of the test sample, and prepare the Danggui Liuhuang Decoction Astragalus single positive control solution according to the preparation method of the test sample solution in Example 1.

[0107] Danggui Liuhuang Decoction Astragalus single negative control solution: Take 4 g of Danggui Liuhuang Decoction Astragalus single negative freeze-dried powder instead of the test sample, and prepare the Danggui Liuhuang Decoction Astragalus single negative control solution according to the preparation method of the test sample solution in Example 1.

[0108] The test solution, reference solution, Astragalus control medicinal material solution, Danggui Liuhuang Tang Astragalus single negative control solution, and Danggui Liuhuang Tang Astragalus single positive control solution were aspirated and spotted on the same thin layer plate, developed with ethyl acetate: n-propanol: water (7:5:4) as the developing agent, dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were colored, and inspected under visible light. The preparation methods of the test solution, reference solution, and Astragalus control medicinal material solution were the same as those in Example 1. The results are shown in FIG. Figure 8 shown.

[0109] The results showed that the TLC of the test solution and the reference solution displayed identical spots at the same locations, corresponding to two spots in the control herbal solution. The Rf values ​​of the spots were moderate and negative with no interference. This method demonstrated good specificity.

[0110] (3) Conduct temperature and humidity surveys

[0111] A. Prepare the test solution, control medicinal material solution and reference substance solution respectively according to the method in Example 1 and spot the samples. Carry out the assay at a temperature of 15.2°C and a humidity of 20% and at a temperature of 5.0°C and a humidity of 82%, respectively. The other process conditions are the same as those in Example 1. The results show that the method has good adaptability to different temperatures. Figure 9 、 10 .

[0112] B. Prepare the test solution, control medicinal material solution and reference substance solution respectively according to the method in Example 1 and spot the samples. The humidity is 15-30% and 75-90% respectively. The other process conditions are the same as those in Example 1. The results show that the method has good adaptability to different humidity. Figure 11 、 12 .

[0113] (4) Investigation of different thin layer plates

[0114] According to the method in Example 1, the test solution, the control medicinal material solution and the reference substance solution were prepared and spotted, respectively, using different thin layer plates. The other process conditions were the same as in Example 1. The results showed that the method had good adaptability to different thin layer plates. Figure 13-15 .

[0115] (5) Investigation of different developing agents

[0116] 20 μl of the test solution, 20 μl of the reference solution, and 10 μl of the Astragalus control medicinal material solution from the same batch of Example 1 were taken and spotted on the same thin layer plate. The plates were developed with ethyl acetate: n-propanol: water (6:6:5) as the developing solvent. The plates were dried and sprayed with 10% ethanolic sulfuric acid solution. The plates were heated until the spots appeared and examined under visible light. The batch number of the test sample is JF-DGLH-DG2020001. The results are shown in the table. Figure 16 As shown, 1. test solution, 2. astragaloside IV reference solution, 3. astragalus reference medicinal material solution.

[0117] 20 μl of the test solution, 20 μl of the reference solution, and 10 μl of the Astragalus control medicinal material solution prepared in the same batch as Example 1 were taken and spotted on the same thin layer plate. Developed with ethyl acetate: n-propanol: water (8:6:4), dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots appeared, and examined under visible light. The batch number of the test sample is: JF-DGLH-DG2020001. The results are shown in Figure 17 As shown, 1. Astragaloside IV reference solution, 2. Test solution, 3. Astragalus reference medicinal material solution.

[0118] from Figure 9 、 16 , 17 It can be seen that the chromatograms of the test solution, the chromatograms of the control medicinal material solution, and the chromatograms of the reference solution with these three developing agent ratios show spots of the same color at the same position.

[0119] Example 4

[0120] This embodiment provides verification of the thin layer identification method, specifically including:

[0121] Six batches of Danggui Liuhuang Decoction freeze-dried powder were used as test samples to prepare test sample solutions according to the method of Example 1. Control medicinal material solutions and reference substance solutions were prepared respectively according to the method in Example 1. Astragalus negative control medicinal materials were used to prepare Astragalus negative control solutions according to the same method as in Example 1 "Preparation method of test sample solution". 20 μL of test sample solutions, 20 μL of control medicinal material solutions, 10 μL of reference substance solutions, and 10 μL of Astragalus negative control solutions from different batches were respectively spotted on the same thin layer plate, developed with ethyl acetate: n-propanol: water (7:5:4) as the developing agent, dried, sprayed with 10% sulfuric acid ethanol solution, heated until the spots were colored, and inspected under visible light.

[0122] according to Figure 18 The chromatogram of the test solution, the chromatogram of the control medicinal material solution, and the chromatogram of the reference solution show spots of the same color at the same position with moderate spread. There are 3 characteristic spots and no negative interference, indicating that the identification method provided by the present invention has good stability.

[0123] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A thin layer chromatography identification method for Danggui Liuhuang Decoction, characterized in that: The following steps are included: Prepare the test sample solution and the control herb solution by using at least one of the following methods 1 and 2: Method 1: Take the test sample and the control medicinal material of Astragalus, add alkaline solution to dissolve them respectively, perform the first solid-liquid separation, draw the liquid and add n-butanol to mix, perform the second solid-liquid separation, draw the n-butanol solution layer, add water to mix, perform the third solid-liquid separation, obtain the n-butanol solution layer, dry it, add alcohol to dissolve it, and prepare the test sample solution and control medicinal material solution respectively; Method 2: Take the test sample and the control medicinal material of Astragalus, extract them with alcohol respectively, separate the solid and liquid, take the liquid, dry it, dissolve it in water, extract it with n-butanol saturated with water, take the n-butanol extract, wash it with alkaline solution, and then wash it with water saturated with n-butanol, take the n-butanol liquid, dry it, and dissolve it with alcohol to prepare the test sample solution and the control medicinal material solution respectively; Identification: Spot the test sample solution, reference herbal solution, and astragaloside IV reference solution onto the same thin-layer plate and develop with a mixed solution of ethyl acetate, n-propanol, and water. Remove, air-dry, spray with a developer, heat, and examine under visible light. The volume ratio of ethyl acetate, n-propanol, and water in the developing solvent is 7:5:

4. In the identification step, the thin layer plate used was a silica gel G thin layer plate.

2. The thin layer identification method according to claim 1, characterized in that: In the identification step, the heating temperature is 95-105° C. and the time is 3-5 min; and / or the spotting volume of the test solution, the control medicinal material solution or the astragaloside IV reference solution is 1-20 μL.

3. The thin layer identification method according to claim 1 or 2, characterized in that: The color developing agent used was sulfuric acid ethanol solution.

4. The thin layer identification method according to claim 3, characterized in that: The concentration of the sulfuric acid ethanol solution is 5-15%.

5. The thin layer identification method according to claim 4, characterized in that: Method 1 also meets one or more of the following AHs: A. The mass ratio of the test sample or control medicinal material to the alkaline solution is 0.5-6:15, wherein the mass to volume ratio is g / mL; B. the first solid-liquid separation, the second solid-liquid separation, or the third solid-liquid separation is centrifugation or filtration; C. The mass-to-volume ratio of the test sample or control medicinal material to n-butanol is 0.5-6:5-30, wherein the mass-to-volume ratio is g / mL; D. The volume ratio of n-butanol to water is 5-30:5-30; E. In the dissolution step, the mass-to-volume ratio of the test sample or control medicinal material to the alcohol is 0.5-6:0.5-3, wherein the mass-to-volume ratio is g / mL; F. The mass volume concentration of the alkaline solution is 0.01-52g / 100mL; G. The alcohol includes one or more of methanol and ethanol; H. The alkaline solution includes one or more of a sodium hydroxide aqueous solution and a potassium hydroxide aqueous solution.

6. The thin layer identification method according to claim 5, characterized in that: Method 1 also satisfies one or more of the following A and F: A. The mass ratio of the test sample or control medicinal material to the alkaline solution is 2-4:15, wherein the mass to volume ratio is g / mL; B. The mass-to-volume ratio of the test sample or control medicinal material to n-butanol is 2-4:10, wherein the mass-to-volume ratio is g / mL; C. The volume ratio of the n-butanol to water is 1:1; D. In the dissolution step, the mass-to-volume ratio of the test sample or control medicinal material to the alcohol is 2-4:1, wherein the mass-to-volume ratio is g / mL; E. The mass volume concentration of the alkaline solution is 0.01-4g / 100mL; F. The alcohol is methanol.

7. The thin layer identification method according to claim 4, characterized in that: Method 2 also satisfies one or more of the following aj: a. In the alcohol extraction step, the ratio of the mass of the test sample or control medicinal material to the volume of alcohol and the volume of water is 0.5-6:20-40:10-30, wherein the mass to volume ratio is g / mL; b. The solid-liquid separation is performed by centrifugation or filtration; c. The extraction method of alcohol extraction is ultrasonic extraction or hot reflux extraction, and the extraction time is at least 15 minutes; d. The number of water-saturated n-butanol extractions is 1-5 times, and the ratio of the mass of the test sample or control medicinal material to the volume of water-saturated n-butanol added in each extraction is 0.5-6:20; wherein the mass-to-volume ratio is g / mL; e. The number of washes with alkaline solution or water saturated with n-butanol is 1-5 times, and the ratio of the mass of the test sample or control medicinal material to the volume of the alkaline solution or water saturated with n-butanol added during each wash is 0.5-6:20; wherein the mass to volume ratio is g / mL; f. In the dissolution step, the mass-to-volume ratio of the test sample or control medicinal material to the alcohol is 0.5-6:0.5-3, wherein the mass-to-volume ratio is g / mL; g. The mass volume concentration of the alkaline solution is 0.01-52 g / 100 mL; h. The alcohol includes one or more of methanol and ethanol; j. The alkaline solution includes one or more of a sodium hydroxide aqueous solution and a potassium hydroxide aqueous solution.

8. The thin layer identification method according to claim 7, characterized in that: Method 2 also satisfies one or more of the following: a. In the alcohol extraction step, the ratio of the mass of the test sample or control medicinal material to the volume of alcohol and water is 2-4:30:20, wherein the mass to volume ratio is g / mL; b. The number of extractions with water-saturated n-butanol is 1-5 times, and the ratio of the mass of the test sample or control medicinal material to the volume of water-saturated n-butanol added in each extraction is 2-4:20, wherein the mass-to-volume ratio is g / mL; c. In the dissolution step, the mass-to-volume ratio of the test sample or control medicinal material to the alcohol is 2-4:1, wherein the mass-to-volume ratio is g / mL; d. The mass volume concentration of the alkaline solution is 0.01-4 g / 100 mL; e. The alcohol is methanol.

9. The thin layer identification method according to claim 6, characterized in that: The solvent of the reference solution is selected from methanol or a methanol aqueous solution with a volume concentration of more than 80%; and / or, each 1 mL of the reference solution contains 0.1 to 4 mg of astragaloside IV reference substance.

10. The thin layer identification method according to claim 1, characterized in that: The preparation method of the test solution includes taking 0.5-6 g of the test sample powder, adding 15 ml of an alkaline solution with a mass volume concentration of 0.01-4 g / 100 ml, dissolving, centrifuging, absorbing the supernatant, adding 5-30 ml of n-butanol to the supernatant, shaking, centrifuging, absorbing the n-butanol solution layer, adding 5-30 ml of water, shaking, centrifuging to obtain the n-butanol solution layer, evaporating to dryness, adding 0.5-3 ml of methanol to dissolve, and obtaining the test sample solution; The preparation method of the control medicinal material solution includes taking 0.5-6 g of the astragalus control medicinal material, adding 15 ml of an alkaline solution with a mass volume concentration of 0.01-4 g / 100 ml, dissolving, centrifuging, absorbing the supernatant, adding 5-30 ml of n-butanol to the supernatant, shaking, centrifuging, absorbing the n-butanol solution layer, adding 5-30 ml of water, shaking, centrifuging to obtain the n-butanol solution layer, evaporating to dryness, and adding 0.5-3 ml of methanol to dissolve to obtain the control medicinal material solution.

11. The thin layer identification method according to claim 1, characterized in that: The preparation method of the test solution includes taking 0.5-6 g of the test sample powder, adding 20-40 ml of methanol, ultrasonically treating for 15-60 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 10-30 ml of water, extracting 1-5 times with n-butanol saturated with water, each time 20 ml, combining the n-butanol extracts, washing 1-5 times with an alkaline solution having a mass volume concentration of 0.01-4 g / 100 mL, each time 20 ml, and then washing 1-5 times with water saturated with n-butanol, each time 20 ml, evaporating the n-butanol solution to dryness, and dissolving the residue in 0.5-3 ml of methanol to prepare the test solution; The preparation method of the control medicinal material solution includes: taking 0.5-6g of the astragalus control medicinal material, adding 20-40ml of methanol, ultrasonically treating for 15-60 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 10-30ml of water, extracting 1-5 times with water-saturated n-butanol, each time with 20ml, combining the n-butanol extracts, washing 1-5 times with an alkaline solution with a mass volume concentration of 0.01-4g / 100mL, each time with 20ml, and then washing 1-5 times with water saturated with n-butanol, each time with 20ml, evaporating the n-butanol solution to dryness, and dissolving the residue in 0.5-3ml of methanol to prepare the control medicinal material solution.

12. The thin layer identification method according to claim 10, characterized in that: The preparation method of the test solution includes taking 2-4 g of the test sample powder, adding 15 ml of a sodium hydroxide aqueous solution with a mass volume concentration of 0.5-2 g / 100 ml, dissolving, centrifuging, absorbing the supernatant, adding 10 ml of n-butanol to the supernatant, shaking, centrifuging, absorbing the n-butanol solution layer, adding 10 ml of water, shaking, centrifuging to obtain the n-butanol solution layer, evaporating to dryness, adding 1 ml of methanol to dissolve, and obtaining the test sample solution; The preparation method of the control medicinal material solution includes taking 2-4 g of the astragalus control medicinal material, adding 15 ml of a sodium hydroxide aqueous solution with a mass volume concentration of 0.5-2 g / 100 ml, dissolving, centrifuging, absorbing the supernatant, adding 10 ml of n-butanol to the supernatant, shaking, centrifuging, absorbing the n-butanol solution layer, adding 10 ml of water, shaking, centrifuging to obtain the n-butanol solution layer, evaporating to dryness, and adding 1 ml of methanol to dissolve to obtain the control medicinal material solution.

13. The thin layer identification method according to claim 11, characterized in that: The preparation method of the test solution includes taking 2-4 g of the test sample powder, adding 30 ml of methanol, ultrasonically treating for 30 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 20 ml of water, extracting twice with n-butanol saturated with water, each time with 20 ml, combining the n-butanol extracts, washing twice with a sodium hydroxide aqueous solution having a mass volume concentration of 0.5-2 g / 100 mL, each time with 20 ml, and then washing twice with water saturated with n-butanol, each time with 20 ml, evaporating the n-butanol solution to dryness, and dissolving the residue in 1 ml of methanol to prepare the test solution; The preparation method of the control medicinal material solution includes taking 2-4 g of the astragalus control medicinal material, adding 30 ml of methanol, ultrasonically treating for 30 minutes, filtering, evaporating the filtrate to dryness, dissolving the residue in 20 ml of water, extracting twice with water-saturated n-butanol, each time with 20 ml, combining the n-butanol extracts, washing twice with a sodium hydroxide aqueous solution with a mass volume concentration of 0.5-2 g / 100 mL, each time with 20 ml, and then washing twice with n-butanol-saturated water, each time with 20 ml, evaporating the n-butanol solution to dryness, and dissolving the residue in 1 ml of methanol to prepare the control medicinal material solution.

14. The thin layer identification method according to claim 13, characterized in that: The test sample is a solid preparation, liquid preparation or semi-solid preparation of Danggui Liuhuang Decoction.

15. The thin layer identification method according to claim 1, characterized in that: The test sample is the powder, granules, tablets, capsules, oral solution, injection, emulsion, suspension or ointment of Danggui Liuhuang Decoction.

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