Thin-layer identification method of heart force prescription and application of thin-layer identification method

Abstract

The invention relates to a thin-layer identification method of a heart-strengthening prescription and application thereof, and relates to the technical field of traditional Chinese medicine detection. The method comprises the following steps: preparing a test sample, preparing a reference substance, and detecting by adopting a thin layer chromatography, when the to-be-identified raw material comprises astragalus membranaceus, the developing solvent is an upper-layer solution of a first mixed solution, and the first mixed solution comprises n-butyl alcohol, methanol, water and ammonia water; when the to-be-identified raw material comprises at least one of red ginseng or pulp of dogwood fruit, the developing solvent is a lower-layer solution of a second mixed solution, and the second mixed solution comprises trichloromethane, methanol and water; when the to-be-identified raw material comprises plantain herb, the developing solvent is a third mixed solution, and the third mixed solution comprises glacial acetic acid and water. The thin-layer identification method can realize qualitative identification on multiple raw materials in the heart force prescription, reduces mutual interference among different raw materials, has relatively high specificity, multiple identification points and clear spots, and can realize high-accuracy, low-cost, easy-to-popularize and rapid qualitative identification on the heart force prescription.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, in particular to a thin-layer identification method of Xinli Fang and application thereof. BACKGROUND

[0002] Xinli Fang is a personal experience prescription for treating chronic heart failure patients formed by Academician Chen Keji according to years of clinical experience, which has achieved good therapeutic effect in clinic. The prescription is composed of Huangqi, Zhihuangqi, Hongshen, Diee Eshu, Cheqiancao and Shanyurou. Xinli Fang compound granules are prepared by using medicinal decoction pieces as raw materials, through standard decoction, concentration, drying and granulation on the basis of the prescription.

[0003] Hongshen and Huangqi in the prescription are common qi-tonifying drugs and can be found in many prescriptions. The main identification component of Hongshen is ginsenosides, and the identification component of Huangqi is astragaloside A, both of which have similar polarity and may interfere with each other. The compound is composed of multiple medicinal materials, and if sample preparation and thin-layer identification conditions are established respectively, the experimental and time costs will be increased. Therefore, it is an important task to establish a qualitative identification method with high accuracy, low cost, easy popularization and rapidness for Xinli Fang. SUMMARY

[0004] In view of the above problems, the present application provides a thin-layer identification method of Xinli Fang, which can qualitatively identify multiple raw materials in Xinli Fang, reduce the mutual interference between different raw materials, has high specificity, and has multiple identification points and clear spots, so that accurate, low-cost, easy-to-popularize and rapid qualitative identification of Xinli Fang can be achieved.

[0005] In order to achieve the above purpose, the present application provides a thin-layer identification method of Xinli Fang, comprising the following steps: preparing a test sample, preparing a control sample, and detecting by thin-layer chromatography.

[0006] When the raw material to be identified includes Huangqi, the developing agent of the thin-layer chromatography is the upper layer solution of a first mixed solution, and the first mixed solution comprises n-butanol, methanol, water and ammonia water.

[0007] When the raw material to be identified includes at least one of Hongshen or Shanyurou, the developing agent of the thin-layer chromatography is the lower layer solution of a second mixed solution, and the second mixed solution comprises chloroform, methanol and water.

[0008] When the raw material to be identified includes Cheqiancao, the developing agent of the thin-layer chromatography is a third mixed solution, and the third mixed solution comprises glacial acetic acid and water.

[0009] The inventor found that the identification method of Huangqi in Chinese Pharmacopoeia 2020 Edition Volume 1 is not applicable to the compound, and the identification point of Astragaloside A is consistent with the identification point of Ginsenoside Rf, which indicates that the identification of Huangqi is interfered by Ginsenoside Rf from Hongshen, and the identification of Huangqi in the prescription containing Hongshen or Renshen cannot be well identified. Moreover, the identification method of Cheqiancao in Chinese Pharmacopoeia 2020 Edition Volume 1 is used for the identification of Cheqiancao in the prescription, and the compound sample spot is not clear and seriously tailing without spot at the position of Plantainoside. In addition, for the compound medicine such as Xinli Fang, different sample preparation methods and developing agents are used for each medicine, which is high in time cost, reagent cost and other consumable costs. Therefore, the inventor proposes to use the above three mixed solutions as developing agents for the raw materials in Xinli Fang, which can easily identify Huangqi qualitatively under the premise of containing Hongshen or Renshen in the compound, and can also identify Shanyushu, Hongshen and Cheqiancao qualitatively, reduces the mutual interference between Huangqi and Hongshen / Renshen, and the identification result of Cheqiancao has many identification points and clear spots. The whole set of thin layer identification method has high specificity, and can accurately identify Xinli Fang with low cost, easy popularization and high speed.

[0010] In one embodiment, the volume ratio of n-butanol, methanol, water and ammonia in the first mixed solution is (18-22):4:(23-27):4.

[0011] The volume ratio of chloroform, methanol and water in the second mixed solution is (11-15):(5-9):2.

[0012] The volume ratio of glacial acetic acid and water in the third mixed solution is 2:(2.5-3.5).

[0013] The upper solution of the first mixed solution is used as the developing agent of Huangqi, which can prevent the identification point of Astragaloside A from being interfered by Ginsenoside Rf, and the spot is clear and beautiful with good separation degree. The third mixed solution is used as the developing agent of Cheqiancao, which can make the identification point of Cheqiancao clear.

[0014] In one embodiment, when the raw materials to be identified include at least one of Huangqi, Hongshen or Shanyushu, the preparation of the test sample includes the following steps: taking Xinli Fang medicine, grinding, dissolving, filtering, taking the filtrate, evaporating, dissolving, extracting with the extracting agent, taking the extract, washing, evaporating the extracting agent, dissolving, eluting with resin, discarding the eluent, gradient eluting with organic solvent, collecting the eluent, evaporating, dissolving, and obtaining the test sample.

[0015] The test sample preparation method can be simultaneously applied to cornus officinalis, red ginseng and astragalus, and greatly reduces the time cost of identifying different raw materials in Xinnili Fang.

[0016] In one of the embodiments, when the raw material to be identified includes at least one of astragalus, red ginseng or cornus officinalis, in the step of preparing the test sample, the extraction agent includes a water-saturated n-butanol solution, and the resin includes a macroporous resin; and the gradient elution includes: sequentially using an ethanol solution with a volume concentration of 40% and an ethanol solution with a volume concentration of 70% for elution.

[0017] In one of the embodiments, the macroporous resin is provided by a D101 type macroporous resin column.

[0018] In one of the embodiments, when the raw material to be identified includes astragalus, the sample application amount of the test sample is 1-3 μL, and the sample application amount of the control sample is 0.5-2.5 μL.

[0019] When the raw material to be identified includes at least one of red ginseng or cornus officinalis, the sample application amount of the test sample is 2-4 μL, and the sample application amount of the control sample is 1-3 μL.

[0020] In one of the embodiments, when the raw material to be identified includes plantago, the preparation of the test sample includes the following steps: taking Xinnili Fang medicine, grinding, dissolving, shaking and extracting with an extraction agent, taking the extraction liquid, drying, dissolving, and obtaining the test sample.

[0021] In one of the embodiments, when the raw material to be identified includes plantago, in the step of preparing the test sample, the extraction agent includes ethyl acetate.

[0022] In one of the embodiments, when the raw material to be identified includes plantago, the sample application amount of the test sample is 1-3 μL, and the sample application amount of the control sample is 2-4 μL.

[0023] The application further provides an application of the thin-layer identification method in quality description, evaluation and monitoring of Xinnili Fang medicine.

[0024] The application further provides a method for controlling the quality of Xinnili Fang medicine, which includes: detecting the Xinnili Fang medicine by using the thin-layer identification method.

[0025] Compared with the prior art, the application has the following beneficial effects:

[0026] The thin-layer identification method of Xinnili Fang and the application thereof can realize qualitative identification of multiple raw materials in Xinnili Fang, reduces mutual interference between different raw materials, has high specificity, and has multiple identification points and clear spots, so that the qualitative identification of Xinnili Fang is accurate, low in cost, easy to popularize and fast. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 TLC chromatogram of Astragalus membranaceus test solution preparation method, wherein, 1: method one; 2: method two; 3: method three; 4: negative control (method three); S1: astragaloside IV reference solution; S2: ginsenoside mixed reference solution (the spots from bottom to top are ginsenoside Rb1, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1);

[0028] Figure 2 TLC chromatogram of Astragalus membranaceus different developing agent, wherein, 1-3: Xinli granules (XKL240102); 4: negative control; S: astragaloside IV reference solution;

[0029] Figure 3 TLC chromatogram of Astragalus membranaceus different sample amount, wherein, 1: test product 1 μl; 2: test product 2 μl; 3: test product 3 μl; 4: test product 4 μl; 5: reference solution 0.5 μl; 6: reference solution 1 μl; 7: reference solution 1.5 μl; 8: reference solution 2.5 μl;

[0030] Figure 4 TLC chromatogram of Astragalus membranaceus different temperature, wherein, 1-3: Xinli granules (XKL240102); S: astragaloside IV reference;

[0031] Figure 5 TLC chromatogram of Astragalus membranaceus different humidity, wherein, 1-3: Xinli granules (XKL240102); S: astragaloside IV reference;

[0032] Figure 6 TLC chromatogram of Astragalus membranaceus different manufacturers, wherein, 1-3: Xinli granules (XKL240102); S: astragaloside IV reference;

[0033] Figure 7 TLC chromatogram of Astragalus membranaceus samples of multiple batches of Xinli granules, wherein, 1: Xinli granules (XKL240101); 2: Xinli granules (XKL240102); 3: Xinli granules (XKL240103); 4: Xinli granules (XKL240104); S: astragaloside IV reference;

[0034] Figure 8Different sample amount TLC chromatogram of red ginseng and Fructus Corni, wherein, 1: test sample 1 μl; 2: test sample 2 μl; 3: test sample 3 μl; 4: test sample 4 μl; 5: ginsenoside Rb1 and ginsenoside Re mixed control solution 1 μl (from bottom to top, ginsenoside Rb1 and ginsenoside Re); 6: ginsenoside Rb1 and ginsenoside Re mixed control solution 2 μl; 7: ginsenoside Rb1 and ginsenoside Re mixed control solution 3 μl; 8: red ginseng control medicinal material solution 2 μl; 9: red ginseng control medicinal material solution 3 μl; 10: red ginseng control medicinal material solution 4 μl; 11: monacolins control solution 1 μl; 12: monacolins control solution 2 μl; 13: monacolins control solution 3 μl;

[0035] Figure 9 Specificity TLC chromatogram of red ginseng and Fructus Corni, wherein, 1-3: Xinli granules (XKL240102); 4: red ginseng negative control; 5: Fructus Corni negative control; S1: ginsenoside Rb1 and ginsenoside Re mixed control (from bottom to top, ginsenoside Rb1 and ginsenoside Re); S2: red ginseng control medicinal material; S3: monacolins control;

[0036] Figure 10 Different temperature TLC chromatogram of red ginseng and Fructus Corni, wherein, 1-3: Xinli granules (XKL240102); S1: ginsenoside Rb1 and ginsenoside Re mixed control (from bottom to top, ginsenoside Rb1 and ginsenoside Re); S2: red ginseng control medicinal material; S3: monacolins control;

[0037] Figure 11 Different humidity TLC chromatogram of red ginseng and Fructus Corni, wherein, 1-3: Xinli granules (XKL240102); S1: ginsenoside Rb1 and ginsenoside Re mixed control (from bottom to top, ginsenoside Rb1 and ginsenoside Re); S2: red ginseng control medicinal material; S3: monacolins control;

[0038] Figure 12 Different manufacturer TLC chromatogram of red ginseng and Fructus Corni, wherein, S1: ginsenoside Rb1 and ginsenoside Re mixed control (from bottom to top, ginsenoside Rb1 and ginsenoside Re); S2: red ginseng control medicinal material; S3: monacolins control;

[0039] Figure 13TLC chromatograms of red ginseng and evodia fruit samples of Xingli granules in different batches, wherein 1: Xingli granules (XKL240101); 2: Xingli granules (XKL240102); 3: Xingli granules (XKL240103); 4: Xingli granules (XKL240104); S1: mixed reference substance of ginsenoside Rb1 and ginsenoside Re (from bottom to top, ginsenoside Rb1 and ginsenoside Re); S2: reference material of red ginseng; S3: reference substance of monoroside;

[0040] Figure 14 TLC chromatograms of preparation methods of test solution of plantain, wherein 1: method 1; 2-3: method 2; 4-5: plantain medicinal material (method 2);

[0041] Figure 15 TLC chromatograms of different developing agents of plantain, wherein the developing agent of the left graph is acetic acid-water (3:2), and the developing agent of the right graph is acetic acid-water (2:3), 1-3: Xingli granules (XKL240102); 4: negative control; S: reference material of plantain;

[0042] Figure 16 TLC chromatograms of different sample application amounts of plantain, wherein 1: test solution 1 μl; 2: test solution 2 μl; 3: test solution 3 μl; 4: test solution 4 μl; 5: reference material 1 μl; 6: reference material 2 μl; 7: reference material 3 μl; 8: reference material 4 μl;

[0043] Figure 17 TLC chromatograms of different temperatures of plantain, wherein 1-3: Xingli granules (XKL240102); S: reference material of plantain;

[0044] Figure 18 TLC chromatograms of different humidity of plantain, wherein 1-3: Xingli granules (XKL240102); S: reference material of plantain;

[0045] Figure 19 TLC chromatograms of astragalus membranaceus samples of Xingli granules in different batches, wherein 1: Xingli granules (XKL240101); 2: Xingli granules (XKL240102); 3: Xingli granules (XKL240103); 4: Xingli granules (XKL240104); S: reference material of plantain;

[0046] Figure 20TLC chromatogram of Huangqi and Hongshen in the examples was detected by pharmacopoeia method, wherein, Huangqi: 1-2: Xinli granules (XKL240102); 3: Huangqi negative control; S: astragaloside A control; Hongshen: 1-2: Xinli granules (XKL240102); 3: Hongshen negative control; S: mixed control solution of ginsenoside Rb1, ginsenoside Re, ginsenoside Rf and ginsenoside Rg1 (from bottom to top, ginsenoside Rb1, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1);

[0047] Figure 21 TLC chromatogram of Shanyurou in the examples was detected by pharmacopoeia method, wherein, 1-3: Xinli granules (XKL240102); S: mixed control solution of morroniside and masakioside;

[0048] Figure 22 TLC chromatogram of Cheqiancao in the examples was detected by pharmacopoeia method, wherein, 1-3: Xinli granules (XKL240102); S: macranthoidin control. DETAILED DESCRIPTION

[0049] In order to facilitate the understanding of the present application, the present application will be described in more detail below with reference to the relevant drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0051] Source:

[0052] Instruments: thin layer automatic imaging instrument (CAMAH TLC VISUALIZER), one ten thousandth balance (METTLER TOLEDO), KQ-250E ultrasonic cleaner (Kunshan ultrasonic electronics Co., Ltd.), silica gel G thin layer plate (Qingdao Haoyang Chemical Co., Ltd., Qingdao Kangyexin Medicinal Silica Gel Drier Co., Ltd., Yantai Xinceng Silica Gel Material Co., Ltd.).

[0053] Reagents: methanol (National Pharmaceutical Group Chemical Reagent Co., Ltd.), ethanol (National Pharmaceutical Group Chemical Reagent Co., Ltd.), n-butanol (National Pharmaceutical Group Chemical Reagent Co., Ltd.), ammonia (National Pharmaceutical Group Chemical Reagent Co., Ltd.), chloroform (National Pharmaceutical Group Chemical Reagent Co., Ltd.), acetic acid (National Pharmaceutical Group Chemical Reagent Co., Ltd.), ethyl acetate (National Pharmaceutical Group Chemical Reagent Co., Ltd.), formic acid (National Pharmaceutical Group Chemical Reagent Co., Ltd.), diethyl ether (National Pharmaceutical Group Chemical Reagent Co., Ltd.), macroporous adsorption resin D101 (National Pharmaceutical Group Chemical Reagent Co., Ltd.) were all of analytical purity and water; Astragaloside IV (batch number: 110781-202219), ginsenoside Rg1 (110703-202235), ginsenoside Re (110754-202129), ginsenoside Rf (111719-201813), ginsenoside Rb1 (110704-202230), monnigrin (111998-202104), plantain glycoside (111914-202105), red ginseng reference drug material (121045-201806), plantain reference drug material (121543-201302) were purchased from China Institute for Drug Control. Xinli granules (batch numbers: XKL240101, XKL240102, XKL240103 and XKL240104), plantain (20223111701) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0054] The reagents, materials and equipment used in this example were commercially available unless otherwise specified. The test methods were conventional test methods in the art unless otherwise specified.

[0055] Example

[0056] I. Radix Astragali thin layer identification

[0057] 1. Preparation of solutions.

[0058] 1.1 Preparation of test solution:

[0059] Method one: 2 g of the product was finely ground, 25 ml of methanol was added, and ultrasonic treatment was performed for 30 minutes. The filtrate was evaporated to dryness, 25 ml of water was added to the residue to dissolve it, and the water-saturated n-butanol was extracted twice, 25 ml each time. The n-butanol solution was combined, washed with ammonia solution once, 30 ml, and the ammonia solution was discarded. The n-butanol solution was evaporated to dryness, 1 ml of methanol was added to dissolve the residue, and the solution was used as the test solution.

[0060] Method two: Take 2g of the product, grind finely, add 25ml of methanol, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate, add 25ml of water to the residue to dissolve, extract twice with water-saturated n-butanol, 25ml each time, combine the n-butanol solution, wash once with 30ml of ammonia solution, discard the ammonia solution, evaporate the n-butanol solution, add 1ml of methanol to the residue to dissolve, and load onto a neutral alumina column, elute with 40% methanol, 50ml, collect the eluate, evaporate, add 1ml of methanol to the residue to dissolve, and use as the test solution.

[0061] Method three: Take 2g of the product, grind finely, add 25ml of methanol, ultrasonic treatment for 30 minutes, filter, evaporate the filtrate, add 25ml of water to the residue to dissolve, extract twice with water-saturated n-butanol, 25ml each time, combine the n-butanol solution, wash once with 30ml of ammonia solution, discard the ammonia solution, evaporate the n-butanol solution, add 5ml of water to the residue to dissolve, pass through a D101 macroporous resin column, elute with 20ml of water, discard the eluate, then elute with 30ml of 40% ethanol, discard the eluate, then elute with 50ml of 70% ethanol, collect the eluate, evaporate, add 1ml of methanol to the residue to dissolve, and use as the test solution.

[0062] 1.2 Preparation of the control solution: Take the astragaloside control product, add methanol to prepare a control solution containing 1mg per 1ml. Take the ginsenoside Rbl control product, ginsenoside Re control product, ginsenoside Rf control product, and ginsenoside Rgl control product, add methanol to prepare a mixed solution containing 1mg per 1ml, as the control solution

[0063] 1.3 Preparation of the negative control solution: Take 2g of the negative sample without astragalus, and prepare the negative control solution according to the method three.

[0064] Test solution preparation method investigation: Take the Xinli granules (batch number: XKL240102), prepare the test solution according to the above three methods, and prepare the control solution, respectively, spot on the same silica gel G thin layer plate, use the lower layer solution of chloroform:methanol:water (13:7:2) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and observe under daylight and ultraviolet light (365nm) respectively.

[0065] The results are shown in Table 1. Figure 1 As shown in Table 1, the test solutions prepared by the three methods and the astragalus negative sample are all interfered. After identification by the control product, it can be seen that the interference is caused by ginsenoside Rf in red ginseng. Among the three sample preparation methods, the sample spots of method three are clear and more beautiful, and the impurity spots are reduced, so method three is selected as the sample treatment method for the thin layer identification. For the problem of astragalus negative interference, the next step is to start with the developing agent.

[0066] 2. Investigation of different developing solvents.

[0067] To solve the problem of negative interference of Huangqi, the developing solvents were investigated. The sample solution of Xinli Granules (Batch No. XKL240102) was prepared according to the above-mentioned sample solution preparation method (i.e. Method III), and was spotted on the same silica gel G thin layer plate. The upper layer solution of n-butanol-ethyl acetate-water-ammonia (volume ratio 4:0.5:5:0.5) and n-butanol-methanol-water-ammonia (volume ratio 20:4:25:4) under static condition were used as developing solvents, respectively. The plate was taken out, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under sunlight and ultraviolet light (365 nm), respectively.

[0068] The results are shown in Table 1. Figure 2 As shown in Table 1, under the developing conditions of the two solvents, the negative of Huangqi had no interference. When n-butanol-ethyl acetate-water-ammonia (volume ratio 4:0.5:5:0.5) was used as the developing solvent, the Rf value was close to 0.2; when n-butanol-methanol-water-ammonia (volume ratio 20:4:25:4) was used as the developing solvent, the Rf value was >0.2, which met the requirements. Therefore, n-butanol-methanol-water-ammonia (volume ratio 20:4:25:4) was selected as the developing solvent.

[0069] 3. Investigation of different sample application amounts.

[0070] To compare the effects of different sample application amounts on the separation degree of spots, the sample solution and the reference solution of Xinli Granules (Batch No. XKL240102) were taken, respectively, and spotted on the same silica gel G thin layer plate according to the above-mentioned thin layer chromatography conditions. The plate was taken out, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under sunlight and ultraviolet light (365 nm), respectively.

[0071] The results are shown in Table 2. Figure 3 As shown in Table 2, when the sample solution was applied in an amount of 1-3 μL and the reference solution was applied in an amount of 0.5-2.5 μL, the spots in the sample chromatogram corresponded clearly to the corresponding positions in the reference chromatogram without other interference. Therefore, the sample solution was applied in an amount of 1-3 μL and the reference solution was applied in an amount of 0.5-2.5 μL.

[0072] 4. Investigation of different temperatures.

[0073] The sample solution and the reference solution of Xinli Granules (Batch No. XKL240102) were taken, respectively, and developed at high and low temperatures, respectively, according to the above-mentioned thin layer chromatography conditions. The plate was taken out, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under sunlight and ultraviolet light (365 nm), respectively.

[0074] The results are shown in Table 3. Figure 4As shown, under different temperature conditions, the chromatograms of the Xinli Granule test sample and the reference sample showed spots of the same color at corresponding positions, and the separation effect was good. The experimental results show that temperature has no effect on the thin-layer chromatography identification of Xinli Granule, indicating that the thin-layer chromatography identification method has good durability at different temperatures.

[0075] 5. Investigation of different humidity levels.

[0076] Take the test solution and reference solution of Xinli Granules (Batch No.: XKL240102) respectively, develop them under different humidity conditions according to the above-mentioned thin layer chromatography conditions, take them out, dry them, spray them with 10% sulfuric acid ethanol solution, heat them at 105°C until the spots are clearly colored, and examine them under sunlight and ultraviolet light (365nm), respectively.

[0077] The results are as follows Figure 5 As shown, under different humidity conditions, the chromatograms of the Xinli Granule test sample and the reference sample showed spots of the same color at corresponding positions, and the separation effect was good. The experimental results show that humidity has no effect on the thin-layer chromatography identification of Xinli Granule, indicating that the thin-layer chromatography identification method has good durability under different humidity conditions.

[0078] 6. Inspection of thin layer boards from different manufacturers.

[0079] The test and reference solutions of Xinli Granules (batch number: XKL240102) were spotted on silica gel G thin layer plates from different manufacturers and developed according to the above-mentioned thin layer chromatography conditions. The plates were taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under sunlight and ultraviolet light (365nm), respectively.

[0080] The results are as follows Figure 6 As shown in the figure, the chromatograms of the Xinli Granule test sample and the reference sample showed spots of the same color at corresponding positions on the TLC plates from different manufacturers, and the separation effect was good. The experimental results show that the manufacturer of the TLC plate has no significant effect on the identification of Xinli Granule, indicating that the TLC identification method is durable for TLC plates from different manufacturers.

[0081] 7. Determination of thin layer identification method.

[0082] Based on the above research results, the thin layer chromatography identification method of Xinli Granule Astragalus is determined as follows:

[0083] Take 2g of this product, grind it, add 25ml of methanol, ultrasonically treat it for 30 minutes, filter it, evaporate the filtrate to dryness, dissolve the residue in 25ml of water, extract it twice with water-saturated n-butanol, shake it and extract it 2 times, 25ml each time, combine the n-butanol solution, wash it once with ammonia test solution, 30ml, discard the ammonia test solution, evaporate the n-butanol solution to dryness, dissolve the residue in 5ml of water, pass it through a D101 macroporous resin column, elute it with 20ml of water, discard the eluent, elute it with 30ml of 40% ethanol and discard the eluent, then elute it with 50ml of 70% ethanol, collect the eluent, evaporate it to dryness, dissolve the residue in 1ml of methanol, and use it as the test solution.

[0084] Separately, prepare a reference solution of astragaloside IV in methanol to prepare a 1 mg reference solution per 1 mL. Perform the thin-layer chromatography (TLC) test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 1-3 μL of the test solution and 0.5-2.5 μL of the reference solution to the same silica gel G thin-layer plate. Use the upper layer of a static mixture of n-butanol-methanol-water-ammonia (20:4:25:4) as the developing solvent. Develop the plate, remove the plate, air-dry it, spray it with 10% ethanolic sulfuric acid solution, heat it at 105°C until the spots are clearly visible, and examine it under sunlight and UV light (365 nm). Fluorescent spots of the same color should appear in the chromatogram of the test sample at the corresponding positions in the chromatogram of the reference sample.

[0085] 8. Thin layer chromatography identification of different batches of Xinli granules.

[0086] Take different batches of Xinli granules and prepare the test solution and reference solution according to the above test solution preparation method, spot them on the same silica gel G thin layer plate, develop them according to the above thin layer chromatography conditions, take them out, dry them, spray them with 10% sulfuric acid ethanol solution, heat them at 105°C until the spots are clearly colored, and examine them under sunlight and ultraviolet light (365nm), respectively.

[0087] The results are as follows Figure 7 As shown, the chromatogram of Xinli granules and the chromatogram of the reference substance show spots of the same color at corresponding positions.

[0088] 2. Thin layer identification of cornus fruit and red ginseng.

[0089] 1. Thin layer identification conditions.

[0090] Silica gel G thin layer plate was used, and the lower layer solution of chloroform: methanol: water (13:7:2) allowed to stand below 10°C was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and examined under ultraviolet light (365nm).

[0091] 2. Preparation of solution.

[0092] Preparation of Test Solution: The test solution was prepared according to the method described in "7. Determination of TLC method" of "I. TLC Identification of Astragalus".

[0093] Preparation of Reference Solution: 1 g of the reference material of red ginseng was prepared according to the method described in "7. Determination of TLC method" of "I. TLC Identification of Astragalus".

[0094] Preparation of Reference Solution: 1 g of the reference material of red ginseng was prepared according to the method described in "7. Determination of TLC method" of "I. TLC Identification of Astragalus".

[0095] Preparation of Reference Solution: 1 g of the reference material of red ginseng was prepared according to the method described in "7. Determination of TLC method" of "I. TLC Identification of Astragalus".

[0096] 3. Investigation of different sample application amounts.

[0097] To compare the influence of different sample application amounts on the separation degree of spots, the test solution and the reference solution of XINLI granules (batch number: XKL240102) were applied on the same silica gel G thin layer plate according to the above-mentioned TLC conditions, developed, taken out, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under ultraviolet lamp (365 nm).

[0098] The results are shown in Table 1. Figure 8 When the test solution was applied in an amount of 2-4 μL and the reference solutions of ginsenoside Rb1, ginsenoside Re, red ginseng and monacolins were applied in an amount of 1-3 μL, the spots in the test chromatogram corresponded to the spots in the reference chromatogram at the corresponding positions without other interference. Therefore, the test solution was applied in an amount of 2-4 μL and the reference solutions of ginsenoside Rb1, ginsenoside Re, red ginseng and monacolins were applied in an amount of 1-3 μL.

[0099] 4. Specificity experiment of sample.

[0100] The test solution, the reference solution, the reference material solution and the negative control solution of XINLI granules (batch number: XKL240102) were applied on the same silica gel G thin layer plate according to the above-mentioned TLC conditions, developed, taken out, air-dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under ultraviolet lamp (365 nm).

[0101] The results are shown in Table 2. Figure 9 The test chromatogram of XINLI granules showed the same color spots as the reference chromatogram at the corresponding positions without the interference of the negative control. It was indicated that the TLC identification method was specific.

[0102] 5. Investigation of different temperatures.

[0103] The test solution of Xinneli Granules (batch number: XKL240102), the solution of the control medicinal material and the solution of the control substance were taken respectively, developed and taken out under high and low temperature conditions according to the above thin layer chromatography conditions, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under ultraviolet light (365 nm).

[0104] The results are shown in Table 1. Figure 10 Under different temperature conditions, the test chromatogram of Xinneli Granules showed the same color spots as the control chromatogram at the corresponding positions. The experimental results showed that temperature had no effect on the thin layer identification of Xinneli Granules, indicating that the thin layer identification method had good durability under different temperatures.

[0105] 6. Investigation of different humidities.

[0106] The test solution of Xinneli Granules (batch number: XKL240102), the solution of the control medicinal material and the solution of the control substance were taken respectively, developed and taken out under different humidity conditions according to the above thin layer chromatography conditions, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under ultraviolet light (365 nm) respectively.

[0107] The results are shown in Table 2. Figure 11 Under different humidity conditions, the test chromatogram of Xinneli Granules showed the same color spots as the control chromatogram at the corresponding positions, and the separation effect was good. The experimental results showed that humidity had no effect on the thin layer identification of Xinneli Granules, indicating that the thin layer identification method had good durability under different humidities.

[0108] 7. Investigation of thin layer plates from different manufacturers.

[0109] The test solution of Xinneli Granules (batch number: XKL240102) and the solution of the control substance were taken respectively, spotted on thin layer plates of silica gel G from different manufacturers, developed and taken out according to the above thin layer chromatography conditions, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105°C until the spots were clearly colored, and observed under sunlight and ultraviolet light (365 nm) respectively.

[0110] The results are shown in Table 3. Figure 12 Under different manufacturer thin layer plate conditions, the test chromatogram of Xinneli Granules showed the same color spots as the control chromatogram at the corresponding positions, and the separation effect was good. The experimental results showed that the manufacturer of the thin layer plate had no significant effect on the identification of Xinneli Granules, indicating that the thin layer identification method had good durability on thin layer plates from different manufacturers.

[0111] 8. Determination of the thin layer identification method.

[0112] According to the above research results, the thin-layer identification method of heart force granules with red ginseng and cornus officinalis is determined as follows:

[0113] Take 2 g of the product, grind it finely, add 25 ml of methanol, and ultrasonically treat it for 30 minutes. Filter it, evaporate the filtrate to dryness, add 25 ml of water to the residue to dissolve it, and extract it with water-saturated n-butanol twice, 25 ml each time. Combine the n-butanol liquid, wash it with ammonia solution once, 30 ml, discard the ammonia solution, and evaporate the n-butanol liquid to dryness. Add 5 ml of water to the residue to dissolve it, pass it through a D101 macroporous resin column, elute it with 20 ml of water, discard the eluate, elute it with 40% ethanol 30 ml, discard the eluate, then elute it with 70% ethanol 50 ml, collect the eluate, evaporate it to dryness, add 1 ml of methanol to the residue to dissolve it, and use it as the test solution.

[0114] Prepare a red ginseng reference material solution by the same method. Take the monoroside reference substance, add methanol to prepare a reference substance solution containing 1 mg per 1 ml.

[0115] Take another ginsenoside Rb1 reference substance and ginsenoside Re reference substance, add methanol to prepare a mixed solution containing 1 mg of each per 1 ml, and use it as the reference substance solution.

[0116] According to the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition Part IV General Test 0502), take 2-4 μl of the above test solution, 1-3 μl of the ginsenoside Rb1 and ginsenoside Re mixed reference substance, the red ginseng reference material, and the monoroside reference substance solution, and point them on the same silica gel G thin-layer plate. Use the lower layer solution of chloroform:methanol:water (13:7:2) at 10°C below as the developing agent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and place under a UV lamp (365 nm) for inspection. In the test chromatogram, the same colored fluorescent spots appear at the corresponding positions of the reference chromatogram.

[0117] 9. Thin-layer identification of different batches of heart force granules.

[0118] Take the test solution, reference material solution, and reference substance solution prepared according to the above test solution preparation method from different batches of heart force granules, point them on the same silica gel G thin-layer plate, develop according to the above thin-layer chromatography conditions, remove, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots develop clearly, and place under a UV lamp (365 nm) for inspection.

[0119] The results are shown in Table 1. Figure 13 The chromatogram of heart force granules shows the same colored spots at the corresponding positions of the reference chromatogram.

[0120] III. Thin-layer identification of plantago.

[0121] 1. Preparation of solution.

[0122] 1.1 Preparation of test solution.

[0123] Method 1: Take 2 g of the product, grind finely, and dissolve in 25 ml of water. Extract twice with 25 ml of ethyl acetate each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution.

[0124] Method 2: Take 2 g of the product, grind finely, and dissolve in 25 ml of water. Extract twice with 25 ml of diethyl ether each time. Combine the diethyl ether extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution.

[0125] 1.2 Preparation of the same batch of plantain medicinal material solution and control medicinal material solution: Take the same batch of plantain medicinal material and prepare the medicinal material solution according to "Method 2 for preparing the test solution". Take the plantain control medicinal material and prepare the control medicinal material solution according to "Method 1 for preparing the test solution".

[0126] 1.3 Preparation of negative control solution: Take 2 g of a negative sample without plantain and prepare the negative control solution according to "Method 1 for preparing the test solution".

[0127] Investigation of test solution preparation methods: Take the Xinli granules (batch number: XKL240102) and prepare the test solution and the control solution according to the above two methods. Spot the samples on the same polyamide film, use an acetic acid-water (volume ratio 1:1) solution as the developing agent, develop, take out, air dry, and place under a UV lamp (365 nm) for inspection.

[0128] The results are shown in Table 1. Figure 14 The test solution prepared by Method 1 has more identification points and uses less toxic reagents, so Method 1 is selected as the sample processing method for the thin layer identification.

[0129] 2. Investigation of different developing agents.

[0130] Take the Xinli granules (batch number: XKL240102) and the plantain control medicinal material and prepare the test solution and the control medicinal material solution according to the above preparation methods. Spot the samples on the same polyamide film, use acetic acid-water (volume ratio 3:2) and acetic acid-water (volume ratio 2:3) as the developing agents, take out, air dry, and place under a UV lamp (365 nm) for inspection.

[0131] The results are shown in Table 2. Figure 15 When the developing agent is acetic acid-water (volume ratio 2:3), the test chromatogram and the control medicinal material chromatogram have clearer spots and better separation, so acetic acid-water (volume ratio 2:3) is selected as the developing agent.

[0132] 3. Investigation of different sample spotting amounts.

[0133] To compare the influence of different sample volumes on the separation of spots, the sample solution of Xingli granules (batch number: XKL240102) and the control medicinal material solution were respectively spotted on the same polyamide film according to the above-mentioned thin layer chromatography conditions, taken out, dried, and observed under a UV lamp (365 nm).

[0134] The results are shown in Table 1. Figure 16 As shown in Table 1, when the sample volume of the sample solution was 1-3 μl and the sample volume of the control medicinal material solution was 2-4 μl, the spots in the sample chromatogram corresponded clearly to the control medicinal material chromatogram without other interference. Therefore, the sample volume of the sample solution was selected to be 1-3 μl and the sample volume of the control medicinal material solution was selected to be 2-4 μl.

[0135] 4. Investigation of different temperatures.

[0136] The sample solution of Xingli granules (batch number: XKL240102) and the control medicinal material solution were respectively developed at high and low temperatures according to the above-mentioned thin layer chromatography conditions, taken out, dried, and observed under a UV lamp (365 nm).

[0137] The results are shown in Table 2. Figure 17 As shown in Table 2, under different temperature conditions, the sample chromatogram of Xingli granules showed the same color spots as the control medicinal material chromatogram, and the separation effect was good. The experimental results showed that temperature had no influence on the thin layer identification of Xingli granules, indicating that the thin layer identification method had good durability to different temperatures.

[0138] 5. Investigation of different humidities.

[0139] The sample solution of Xingli granules (batch number: XKL240102) and the control medicinal material solution were respectively developed under different humidity conditions according to the above-mentioned thin layer chromatography conditions, taken out, dried, and observed under a UV lamp (365 nm).

[0140] The results are shown in Table 3. Figure 18 As shown in Table 3, under different humidity conditions, the sample chromatogram of Xingli granules showed the same color spots as the control medicinal material chromatogram, and the separation effect was good. The experimental results showed that humidity had no influence on the thin layer identification of Xingli granules, indicating that the thin layer identification method had good durability to different humidities.

[0141] 6. Determination of the thin layer identification method.

[0142] According to the above-mentioned research results, the thin layer identification method of Xingli granules is determined as follows:

[0143] Take 2 g of the product, grind finely, add water 25 ml to dissolve, extract with ethyl acetate twice, 25 ml each time, combine the ethyl acetate liquid, evaporate to dryness, add methanol 1 mL to dissolve as the sample solution.

[0144] The control medicinal material solution was prepared by the same method as the plantain.

[0145] According to the thin layer chromatography (Chinese Pharmacopoeia 2020 edition four general test 0502), 1-4 μl of the above test sample solution and 2-4 μl of the control medicinal material solution were taken and spotted on the same polyamide film, an acetic acid: water (volume ratio 2:3) was used as the developing agent, and then the film was developed, taken out, dried, and placed under the ultraviolet lamp (365 nm) for inspection. In the test sample chromatogram, the same color fluorescent spots appeared at the corresponding positions of the control medicinal material chromatogram.

[0146] 7. Thin layer identification of different batches of Xinli granules.

[0147] The test sample solution and the control medicinal material solution prepared according to the preparation method of the test sample solution were spotted on the same polyamide film, and then developed according to the above thin layer chromatography conditions, taken out, dried, and placed under the ultraviolet lamp (365 nm) for inspection.

[0148] The results are shown in Table 1. Figure 19 The chromatogram of Xinli granules and the chromatogram of the control medicinal material showed the same color spots at the corresponding positions.

[0149] Four, summary.

[0150] The Xinli formula thin layer identification method of the application uses n-butanol: methanol: water: ammonia water (volume ratio 20:4:25:4) as the developing agent for astragalus, which can prevent the interference of ginsenoside Rf in the identification of astragalus saponin, and the spots are clear and beautiful, and the separation degree is good. The plantain sample is extracted by shaking with ethyl acetate, and the polyamide film is used, 40% acetic acid is used as the developing agent, and the plantain control medicinal material is used as the control substance, and the identification point is clear. Red ginseng, astragalus and dogwood are prepared by the same sample preparation method, and dogwood and red ginseng use the same developing agent.

[0151] The thin layer identification method solves the problem of interference of ginsenoside Rf in red ginseng on the identification of astragalus saponin, and can easily identify astragalus in the prescription containing red ginseng or ginseng, improve the specificity of the method, and avoid the use of chloroform which is harmful to human body and environment. The thin layer method of plantain has multiple identification points, clear spots and strong specificity. The three kinds of medicinal materials including dogwood, red ginseng and astragalus can use the same sample preparation method, which greatly reduces the time cost.

[0152] Comparative example 1

[0153] I. Astragalus and red ginseng.

[0154] Reference to the Pharmacopoeia method of Yuguang capsule prescription method: take the product 2 g, grind, add methanol 25 ml, ultrasonic treatment for 30 minutes, cool, filter, filter dry, add water 25 ml to dissolve, with water saturated n-butanol shake extraction 2 times, 25 ml each time, combined with n-butanol liquid, with ammonia test solution wash 1 times, 30 ml, discard the ammonia solution, n-butanol liquid dry, add methanol 1 ml to dissolve, as test solution.

[0155] Another astragalus negative, red ginseng negative sample preparation of negative control sample.

[0156] Take astragaloside reference substance, add methanol to prepare 1 mg / ml solution.

[0157] Again take ginsenoside Rb1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rg1 reference substance, add methanol to prepare 1 mg / ml mixed solution, as reference solution.

[0158] According to the thin layer chromatography method (China Pharmacopoeia 2020 edition four general 0502) test, 2-4 μl of the above two solutions were taken and spotted on the same silica gel G thin layer plate. The lower layer solution of chloroform-methanol-water (volume ratio 13:7:2) was used as the developing agent, and the developed solution was taken out and dried. Then 10% sulfuric acid ethanol solution was sprayed, and the spots were heated to color at 105℃. The plate was observed under ultraviolet light (365 nm) and sunlight.

[0159] The results are shown in Figure 20 If the Pharmacopoeia method is used directly, the astragalus negative has interference, the red ginseng identification point ginsenoside Rf is interfered by astragaloside, and the chromatogram has many impurities and is not clear.

[0160] Two, Shanyu meat.

[0161] Reference to the Pharmacopoeia method of Liuwei Dihuang pill prescription method: take the product 2 g, grind, add methanol 25 ml, ultrasonic treatment for 30 minutes, filter, filter dry, add water 20 ml to dissolve, with n-butanol-ethyl acetate (volume ratio 1:1) mixed solution shake extraction 2 times, 20 ml each time, combined with extraction liquid, with ammonia solution (1→10) 30 ml wash, discard the ammonia solution, n-butanol liquid dry, add methanol 1 ml to dissolve, as test solution.

[0162] Another morroniside reference substance, malonoside reference substance, add methanol to prepare 1 mg / ml mixed solution, as reference solution.

[0163] The above two solutions were 1 μl, respectively, spotted on the same silica gel G thin layer plate, chloroform-methanol (volume ratio 3:1) was used as the developing agent, developed, taken out, dried, and observed under ultraviolet light (365 nm).

[0164] The results are shown in Table 1. Figure 21 As shown in Table 1, the pharmacopoeia method can also identify cornus officinalis, but considering the time cost, cornus officinalis and astragalus root and red ginseng use the same sample preparation method, and red ginseng uses the same thin layer developing condition.

[0165] 3, Plantago.

[0166] Pharmacopoeia method: 5 g of the product was finely ground, 15 ml of methanol was added, ultrasonic treatment was performed for 30 minutes, filtration was performed, and the filtrate was used as the test solution.

[0167] Another plantain glycoside reference substance was prepared into a reference solution containing 1 mg per 1 ml by adding methanol.

[0168] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition four general rules 0502) test, 10 μl of the above two solutions was respectively spotted on the same silica gel G thin layer plate, ethyl acetate-methanol-formic acid-water (volume ratio 18:3:1.5:1) was used as the developing agent, developed, taken out, dried, and observed under ultraviolet light (365 nm).

[0169] The results are shown in Table 1. Figure 22 As shown in Table 1, if the pharmacopoeia method is directly used for the test sample, there is no corresponding spot.

[0170] The technical features of the above-described embodiments can be combined in any way. In order to make the description simple, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not exist, they should be considered as the scope of the description.

[0171] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. A thin layer identification method of Xinlifang, characterized in that: The following steps are involved: Prepare test samples and reference substances, and perform testing using thin layer chromatography; When the raw material to be identified includes Astragalus, the developing solvent of the thin layer chromatography is the upper layer solution of the first mixed solution, and the first mixed solution includes n-butanol, methanol, water and ammonia water; When the raw material to be identified includes at least one of red ginseng or cornus fruit, the developing solvent of the thin layer chromatography is the lower layer solution of the second mixed solution, and the second mixed solution includes chloroform, methanol, and water; When the raw material to be identified includes plantain, the developing solvent of the thin layer chromatography is a third mixed liquid, and the third mixed liquid includes glacial acetic acid and water.

2. The thin layer identification method according to claim 1, characterized in that: The volume ratio of n-butanol:methanol:water:ammonia solution in the first mixed solution is (18-22):4:(23-27):4; The volume ratio of chloroform, methanol and water in the second mixed liquid is (11-15): (5-9): 2; The volume ratio of glacial acetic acid to water in the third mixed solution is 2:(2.5-3.5).

3. The thin layer identification method according to any one of claims 1 to 2, characterized in that: When the raw materials to be identified include at least one of astragalus, red ginseng or cornus fruit, the preparation of the test sample includes the following steps: taking the Xinlifang medicine, grinding it, dissolving it, filtering it, taking the filtrate, evaporating it to dryness, dissolving it, shaking it with an extractant, taking the extract, washing it, evaporating the extractant to dryness, dissolving it, eluting it with a resin, discarding the eluate, performing gradient elution with an organic solvent, collecting the eluate, evaporating it to dryness, dissolving it, and obtaining the test sample.

4. The thin layer identification method according to claim 3, characterized in that: When the raw materials to be identified include at least one of astragalus, red ginseng or cornus fruit, in the step of preparing the test sample, the extractant includes a water-saturated n-butanol solution, the resin includes a macroporous resin; and the gradient elution includes: eluting with ethanol solutions with a volume concentration of 40% and 70% in sequence.

5. The thin layer identification method according to claim 4, characterized in that: When the raw material to be identified includes Astragalus, the sample volume of the test sample is 1-3 μL, and the sample volume of the reference sample is 0.5-2.5 μL; When the raw materials to be identified include at least one of red ginseng or cornus fruit, the spotting volume of the test sample is 2-4 μL, and the spotting volume of the reference sample is 1-3 μL.

6. The thin layer identification method according to any one of claims 1-2, characterized in that: When the raw material to be identified includes plantain, the preparation of the test sample includes the following steps: taking the Xinlifang medicine, grinding it, dissolving it, shaking it with an extractant, taking the extract, evaporating it, and dissolving it to obtain the test sample.

7. The thin layer identification method according to claim 6, characterized in that: When the raw material to be identified includes plantain, in the step of preparing the sample, the extractant includes ethyl acetate.

8. The thin layer identification method according to claim 7, characterized in that: When the raw material to be identified includes plantain, the spotting volume of the test sample is 1-3 μL, and the spotting volume of the reference sample is 2-4 μL.

9. Use of the thin-layer chromatography identification method according to any one of claims 1 to 8 in the description, evaluation and monitoring of the quality of Xinlifang medicines.

10. A method for quality control of Xinlifang medicine, characterized in that: include: The Xinlifang medicine is detected using the thin layer identification method described in any one of claims 1 to 8.

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