Thin-layer identification method and application of pokeberry root medicinal preparation

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin-layer identification method and application of a pokeberry root medicinal preparation. The invention provides a thin-layer identification method for a pokeberry root medicinal preparation. The thin-layer identification method comprises the following steps: preparing a test solution A: preparing a pokeberry root medicinal preparation test solution into the test solution A; preparing a reference medicinal material solution, namely preparing the reference medicinal material of the pokeberry root into the reference medicinal material solution of the pokeberry root; preparation of a reference substance solution: preparing the esculentoside A reference substance into the esculentoside A reference substance solution; and respectively spotting the test solution A, the pokeberry root reference medicinal material solution and the esculentoside A reference substance solution on the same silica gel thin-layer plate, and developing by taking ethyl acetate, methanol and water in a volume ratio of (8.5-9.5): 2: 0.5 as a developing solvent. The thin-layer identification method can be used for simultaneously detecting various chemical components in pokeberry root, and is high in detection efficiency and simple.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin layer identification method for a Chinese pokeweed medicinal preparation and an application thereof. Background Art

[0002] First mentioned in Shennong's Herbal Classic, pokeweed (Phytolacca acinosa) is cold in nature and bitter in taste. It enters the lung, spleen, kidney, and large intestine meridians. It has the effects of expelling water, reducing swelling, and promoting bowel movements. It is suitable for edema and constipation. It can also be used externally to detoxify and treat carbuncles and sores. Pokeweed primarily contains triterpenoid saponins, polysaccharides, flavonoids, phenolic acids, sterols, and volatile oils, with triterpenoid saponins being the primary active ingredients. Pokeweed comes from two plant sources: Phytolacca acinosa Roxb. (referred to as pokeweed) or P. americana L.

[0003] At present, the 2020 edition of the "Chinese Pharmacopoeia" and literature reports recommend the use of content determination for quality control, but only a single ingredient can be retrieved at a time, the steps are cumbersome and costly, and the detection efficiency is low; the existing technology also uses characteristic spectrum methods to distinguish between two different samples of vertical sequence pokeweed and pokeweed, but the characteristic spectrum method is cumbersome to operate, and the cost of equipment consumables such as instruments, chromatographic columns, and mobile phases is relatively high. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the cumbersome steps and high cost of distinguishing vertical order pokeweed and pokeweed in the prior art; thereby providing a thin layer identification method and application of vertical order pokeweed pharmaceutical preparations.

[0005] The thin-layer chromatography identification method for a vertical sequence pokeweed drug preparation provided by the present invention has more chromatographic spots, high detection efficiency, and a simple method. Compared with the characteristic spectrum method, the thin-layer chromatography method is more economical.

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

[0007] The pokeweed referred to in the present invention is Phytolacca acinosa Roxb.; the pokeweed referred to in the present invention is P. americana L.

[0008] The present invention provides a thin layer identification method for a vertical sequence pokeweed medicinal preparation, comprising the following steps:

[0009] Preparation of test solution A: Prepare test solution A from the Pokeweed drug preparation test sample;

[0010] Preparation of control medicinal material solution: Prepare the control medicinal material of Phytolacca sutchuenensis into the control medicinal material solution of Phytolacca sutchuenensis;

[0011] Preparation of reference solution: Prepare the pokeweed saponin A reference solution from the pokeweed saponin A reference solution;

[0012] The test solution A, the control medicinal material solution of Phytolacca americana, and the reference solution of Phytolacca saponin A were spotted on the same silica gel thin layer plate, and developed with ethyl acetate, methanol and water in a volume ratio of (8.5-9.5):2:0.5 as the developing solvent.

[0013] In an optional embodiment, in the developing solvent, the volume ratio of ethyl acetate, methanol and water is 8.5:2:0.5.

[0014] In an optional embodiment, the preparation process of the test solution A comprises: taking a test sample of a pokeweed drug preparation, extracting it with an ethanol aqueous solution, and separating it to obtain a test solution;

[0015] Optionally, the ratio of the mass of the test sample of the vertical order pokeweed drug preparation to the volume of the ethanol aqueous solution is (0.5-2.5):(10-50), the mass unit of the vertical order pokeweed drug preparation test sample is g, and the volume unit of the ethanol aqueous solution is ml;

[0016] Optionally, the volume concentration of the ethanol aqueous solution is 49.5-50.5%.

[0017] In an optional embodiment, the preparation process of the Pokeweed control medicinal material solution comprises: taking the Pokeweed control medicinal material, extracting it with water, filtering it, evaporating the filtrate to dryness, and adding ethanol and aqueous solution to the obtained residue to obtain the control medicinal material solution;

[0018] Optionally, the ratio of the mass of the Chinese pokeweed control medicinal material to the volume of the ethanol aqueous solution is (1-5):(5-25), the mass unit of the Chinese pokeweed control medicinal material is g, and the volume unit of the ethanol aqueous solution is ml;

[0019] Optionally, the extraction method includes a heating reflux method, wherein the heating reflux time is 30-60 minutes;

[0020] Optionally, the volume concentration of the ethanol aqueous solution is 49.5-50.5%.

[0021] In an optional embodiment, the concentration of the pokeweed saponin A reference substance in the pokeweed saponin A reference substance solution is 0.1-0.3 mg / ml;

[0022] In an optional embodiment, the spotting volume of the test solution A is 10-15 μL; preferably, the spotting volume of the test solution A is 15 μL;

[0023] In an optional embodiment, the spotting volume of the Pokeweed control medicinal material solution is 10-15 μL; preferably, the spotting volume of the Pokeweed control medicinal material solution is 10 μL;

[0024] In an optional embodiment, the spotting volume of the pokeweed saponin A reference solution is 10-15 μL; preferably, the spotting volume of the pokeweed saponin A reference solution is 15 μL.

[0025] In an optional embodiment, after unfolding, the method further comprises taking out, drying, spraying with sulfuric acid ethanol solution and then heating until color is developed;

[0026] After color development, the process also includes inspecting under an ultraviolet lamp; optionally, the wavelength of the ultraviolet lamp is 365nm.

[0027] In an optional embodiment, the preparation step of the sulfuric acid ethanol solution includes: mixing ethanol and sulfuric acid; preferably, the volume ratio of ethanol to sulfuric acid is 9:1; preferably, the ethanol is analytical grade ethanol; preferably, the concentration of the sulfuric acid is 98%.

[0028] In an optional embodiment, the heating temperature is 100-110°C;

[0029] In an optional embodiment, the silica gel thin layer plate comprises a silica gel G thin layer plate;

[0030] In an optional embodiment, in the inspection step, the chromatogram of the test sample is compared with the chromatogram of the reference medicinal material Pokeweed.

[0031] In an optional embodiment, the Pokeweed pharmaceutical preparation includes a concentrated extract of a Pokeweed benchmark sample, a water decoction of a Pokeweed benchmark sample, a freeze-dried powder of a Pokeweed benchmark sample, or a Pokeweed preparation.

[0032] The present invention also provides an application of the thin-layer identification method in identifying pokeweed or its derivatives and pokeweed or its derivatives.

[0033] The technical solution of the present invention has the following advantages:

[0034] 1. The thin layer identification method of the Chinese pokeweed pharmaceutical preparation provided by the present invention comprises the following steps: preparing a test solution A: preparing a test solution A from a Chinese pokeweed pharmaceutical preparation; preparing a control medicinal material solution: preparing a Chinese pokeweed control medicinal material solution from a Chinese pokeweed control medicinal material; preparing a reference solution: preparing a Chinese pokeweed saponin A reference solution from a Chinese pokeweed saponin A reference solution; taking the test solution A, the Chinese pokeweed control medicinal material solution, and the Chinese pokeweed saponin A reference solution, respectively spotting them on the same silica gel thin layer plate, and developing them with ethyl acetate, methanol, and water in a volume ratio of (8.5-9.5):2:0.5 as a developing solvent. The thin-layer identification method can simultaneously detect multiple chemical components in pokeweed, has high detection efficiency, and is simple. That is, multiple components in pokeweed pharmaceutical preparations can be simultaneously detected on the same thin-layer plate and using the same developing agent system, thereby reducing test time and reagent usage and improving detection efficiency. The chromatogram obtained using the thin-layer identification method has clear spots, good separation, strong specificity, no background interference, no interference from negative control substances, and good durability.

[0035] 2. The thin-layer identification method for the Chinese pokeweed drug preparation provided by the present invention does not require the use of chloroform commonly used in the prior art. It is environmentally friendly and safe while further improving the separation of spots in the chromatogram and enriching the spot information.

[0036] 3. The identification method of Pokeweed or its derivatives provided by the present invention can effectively distinguish Pokeweed or its derivatives from Pokeweed or its derivatives. The method is convenient and has high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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.

[0038] Figure 1 This is a thin layer chromatogram obtained by investigating different developing agents in Example 2 of the present invention;

[0039] Figure 2 This is a thin layer chromatogram obtained by investigating the ratios of different developing agents in Example 2 of the present invention;

[0040] Figure 3 This is a thin layer chromatogram obtained by investigating the amount of sample spotted and the specificity in Example 2 of the present invention;

[0041] Figure 4 This is a thin layer chromatogram obtained by investigating different ambient temperatures in Example 2 of the present invention;

[0042] Figure 5 This is a thin layer chromatogram obtained by investigating different environmental humidities in Example 2 of the present invention;

[0043] Figure 6 This is a thin layer chromatogram obtained by thin layer plate inspection in Example 2 of the present invention;

[0044] Figure 7 This is a thin layer chromatogram obtained by reproducibility study by different personnel in Example 2 of the present invention;

[0045] Figure 8 This is a thin layer chromatogram obtained in the comparative experiment in Example 3 of the present invention. DETAILED DESCRIPTION

[0046] 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.

[0047] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0048] Reference substances:

[0049] Phytolaccaside A reference substance (source: China Food and Drug Inspection Institute, batch number: 111922-202403);

[0050] Control herbs:

[0051] Pokeweed control medicinal material (Source: Jiangxi Baicaoyuan Biotechnology Co., Ltd., batch number: 0912202203);

[0052] instrument:

[0053] Automatic thin layer spotter (ATS 4);

[0054] Thin layer automatic imaging instrument (TLC visualizer3);

[0055] Ultrasonic instrument KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.);

[0056] Balance XSR304 (METTLER TOLEDO, Switzerland);

[0057] Water bath HH-8 (Changzhou Aohua Instrument Co., Ltd.); development tank;

[0058] Silica gel G thin layer plate (Yantai, Merck);

[0059] Thin layer plate heater (TLC Plate heater III).

[0060] Reagents:

[0061] Ethanol (Batch No.: 2023100130; Source: Guangzhou Chemical Reagent Factory);

[0062] Methanol (batch number: 2024010105; source: Guangzhou Chemical Reagent Factory);

[0063] Ethyl acetate (Batch number: 20240607; Source: Guangdong Guanghua Technology Co., Ltd.);

[0064] Sulfuric acid (batch number: 2023090305; source: Guangzhou Chemical Reagent Factory);

[0065] The water was homemade purified water.

[0066] Preparation Example

[0067] Chuixue Phytolacca rhizome granules (2208001Y, 2208002Y, 2208003Y) and negative granules (2212001Y) were all made in-house;

[0068] The preparation method can be prepared by conventional methods in the art, as follows:

[0069] Chuixu Pyrola formula granules: Take Chuixu Pyrola slices from different batches, add water and boil them twice, filter the filtrate with a 200-mesh filter cloth, combine the filtrates, concentrate the filtrates to a fluid paste, dry, prepare, and package to obtain Chuixu Pyrola formula granules of batches 2208001Y, 2208002Y, and 2208003Y.

[0070] Negative granules: Take maltodextrin auxiliary material and prepare by dry method.

[0071] Example 1

[0072] This embodiment provides a thin layer identification method for vertical sequence pokeweed medicinal preparations, comprising:

[0073] 1. Preparation of test solution A: Take 0.5 g of Phytolacca scabra granules (2208001Y), grind it into powder, add 10 ml of dilute ethanol (volume concentration 50%), ultrasonically treat it for 30 minutes, and filter to prepare test solution A.

[0074] 2. Preparation of control medicinal material solution: Take 1 g of P. truncatum control medicinal material, add 25 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of dilute ethanol, and ultrasonicate for 30 minutes; filter to prepare P. truncatum control medicinal material solution.

[0075] 3. Preparation of reference solution: Take the reference substance of Phytolaccaside A, accurately weigh it, and add dilute ethanol to make a reference solution containing 0.1 mg of Phytolaccaside A per 1 mL.

[0076] 4. Chromatographic conditions: According to the thin layer chromatography method (General Rule 0502), the above-mentioned test solution A (15 μL), the vertical order Chinese herbal medicine solution (10 μL), and the Chinese herbal saponin A reference solution (15 μL) were respectively aspirated; they were spotted on the same silica gel G thin layer plate (Merck plate) and developed with ethyl acetate-methanol-water (8.5:2:0.5). The plate was removed and air-dried, and then sprayed with ethanolic sulfuric acid solution (ethanol and sulfuric acid were mixed in a volume ratio of 9:1, the ethanol was analytical grade ethanol, and the concentration of sulfuric acid was 98%), and then heated to 105°C until the spots were clearly colored and inspected under ultraviolet light (365 nm).

[0077] In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material; and in the chromatogram of the control sample, a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the control.

[0078] Experimental Example 1

[0079] This experimental example investigates the chromatographic conditions, including:

[0080] 1. Investigation of different developing agents

[0081] (1) The volume ratio of dichloromethane, methanol and water is 5:4:2;

[0082] (2) The volume ratio of isopropyl alcohol, methanol and water is 6:3:2;

[0083] (3) The volume ratio of ethyl acetate, methanol and water is 5:3:2;

[0084] The above three developing agents were developed according to Example 1 and the results were as follows. Figure 1 shown.

[0085] in, Figure 1 a in the figure is the thin layer chromatogram obtained by developing agent (1). Figure 1 In a, 1 is the test solution A (2208001Y), 2 is the control medicinal material solution of Phytolacca americana, and 3 is the reference solution of Phytolacca americana saponin A.

[0086] Figure 1 b in the figure is the thin layer chromatogram obtained by developing agent (2). Figure 1In b, 1 is the test solution A (2208001Y), 2 is the control medicinal material solution of Phytolacca americana, and 3 is the reference solution of Phytolacca americana saponin A.

[0087] Figure 1 c in the figure is the thin layer chromatogram obtained by developing agent (3). Figure 1 In c, 1 is the test solution A (2208001Y), 2 is the control medicinal material solution of Phytolacca americana, and 3 is the reference solution of Phytolacca americana saponin A.

[0088] The above Figure 1 The test solution A, reference solution, and reference medicinal material solution were all prepared according to the method of Example 1.

[0089] Depend on Figure 1 It can be seen from the ac in that when dichloromethane-methanol-water (5:4:2) and isopropanol-methanol-water (6:3:2) are used as developing solvents, the chromatographic spot separation effect is poor; when ethyl acetate-methanol-water is used as developing solvent, the chromatographic spot separation effect is good, and the developing solvent ratio is further adjusted for investigation.

[0090] 2. Investigation of volume ratio of each component of the developing agent

[0091] (1) The volume ratio of ethyl acetate, methanol and water is 7:2:1;

[0092] (2) The volume ratio of ethyl acetate, methanol and water is 8:2:1;

[0093] (3) The volume ratio of ethyl acetate, methanol and water is 8.5:2:0.5;

[0094] (4) The volume ratio of ethyl acetate, methanol and water is 9:2:0.5;

[0095] (5) The volume ratio of ethyl acetate, methanol and water is 9.5:2:0.5;

[0096] The above developing agent was developed and inspected according to the method of Example 1. The results are as follows: Figure 2 shown.

[0097] in, Figure 2 a in the figure is the thin layer chromatogram obtained by developing agent (1). Figure 2 In a, 1 is the test solution (15 μL), 2 is the control medicinal material solution of Phytolacca japonica (10 μL), and 3 is the control medicinal material solution of Phytolacca japonica saponin A (15 μL).

[0098] Figure 2 b in the figure is the thin layer chromatogram obtained by developing agent (2). Figure 2In b, 1 is the test solution A (15 μL), 2 is the control medicinal material solution of Phytolacca japonica (10 μL), and 3 is the reference substance solution of Phytolacca japonica saponin A (15 μL).

[0099] Figure 2 c in the figure is the thin layer chromatogram obtained by developing agent (3). Figure 2 1-3 in c correspond to the test solutions (15 μL) prepared from the formula granules of Phytolacca japonica with batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively; 4 is the control medicinal material solution of Phytolacca japonica (10 μL); and 5 is the reference solution of Phytolacca japonica saponin A (15 μL).

[0100] Figure 2 The d in the figure is the thin layer chromatogram obtained by the developing agent (4). Figure 2 1-3 in d correspond to the test solutions (15 μL) prepared from the formula granules of the vertical order pokeweed of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively; 4 is the control medicinal material solution of the vertical order pokeweed (10 μL); and 5 is the reference substance solution of pokeweed saponin A (15 μL).

[0101] Figure 2 e in the figure is the thin layer chromatogram obtained by the developing agent (5), Figure 2 1-3 in e correspond to the test solutions (15 μL) prepared from the Phytolacca japonica granules of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively; 4 is the Phytolacca japonica control medicinal material solution (10 μL); and 5 is the Phytolacca japonica saponin A reference solution (15 μL).

[0102] The above Figure 2 The test solution A, reference solution, and reference medicinal material solution were all prepared according to the method of Example 1.

[0103] When ethyl acetate-methanol-water is used as the developing solvent, the ratio is (8.5:2:0.5), (9:2:0.5), and (9.5:2:0.5), the spots are clear and the separation is good. Therefore, the present invention selects ethyl acetate-methanol-water as the developing solvent with a volume ratio of (8.5-9.5):2:0.5.

[0104] 3. Inspection of sample quantity and specificity

[0105] The test solution A (10 μL, 13 μL, 15 μL), the pokeweed control medicinal material solution (10 μL, 13 μL, 15 μL), the pokeweed saponin A reference solution (10 μL, 13 μL, 15 μL), and the negative solution (15 μL) were respectively aspirated and spotted on the same silica gel G thin layer plate according to the method of Example 1, developed, and inspected; the results are as follows. Figure 3The negative solution was prepared using negative particles (2212001Y) according to "1. Preparation of Test Solution A" in Example 1;

[0106] exist Figure 3 In the table, 1 is the sample volume of 10 μL test solution A (2208001Y), 2 is the sample volume of 13 μL test solution A (2208001Y), 3 is the sample volume of 15 μL test solution A (2208001Y), 4 is the sample volume of 10 μL vertical sequence pokeweed control medicinal material solution (10 μL), 5 is the sample volume of 13 μL vertical sequence pokeweed control medicinal material solution (13 μL), 6 is the sample volume of 15 μL vertical sequence pokeweed control medicinal material solution (15 μL), 7 is the sample volume of 10 μL pokeweed saponin A reference solution, 8 is the sample volume of 13 μL pokeweed saponin A reference solution, 9 is the sample volume of 15 μL pokeweed saponin A reference solution, and 10 is the sample volume of 15 μL negative solution.

[0107] Depend on Figure 3 It can be seen that the thin layer identification method provided by the present invention can obtain good detection results under different sample sizes, the spots are clear, and the separation is good, indicating that the method has good durability for different sample sizes; preferably, the sample size of the test solution is 15 μL, the sample size of the Chinese pokeweed control medicinal material solution is 10 μL, and the sample size of the Chinese pokeweed saponin A reference solution is 15 μL.

[0108] 4. Investigation of different ambient temperatures

[0109] (1) The ambient temperature is 25°C;

[0110] (2) Ambient temperature is 4°C;

[0111] Under the conditions of (1) to (2), thin layer identification was performed according to the thin layer identification method of Example 1. The results are as follows: Figure 4 shown.

[0112] in, Figure 4 a in the figure is the TLC obtained at ambient temperature (1). Figure 4 1-3 in a correspond to the test solution (15 μL) prepared from the vertical sequence pokeweed formula granules of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively, 4 is the vertical sequence pokeweed control medicinal material solution (10 μL), and 5 is the pokeweed saponin A reference solution (15 μL). The solution preparation method refers to Example 1.

[0113] Figure 4 b in the figure is the thin layer chromatogram obtained at ambient temperature (2). Figure 41-3 in b correspond to the test solution (15 μL) prepared from the vertical sequence pokeweed formula granules of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively, 4 is the vertical sequence pokeweed control medicinal material solution (10 μL), and 5 is the pokeweed saponin A reference solution (15 μL). The solution preparation method refers to Example 1.

[0114] Depend on Figure 4 It can be seen from ab in that the thin layer identification method provided by the present invention can obtain good detection effect at different temperatures, and there is no obvious difference in the separation effect and spot display in the chromatogram, indicating that the thin layer identification method provided by the present invention has good durability at an ambient temperature of 4-25°C.

[0115] 5. Investigation of humidity in different environments

[0116] (1) Ambient humidity is 88% RH;

[0117] (2) Ambient humidity is 32% RH;

[0118] Under the conditions of (1) and (2), thin layer identification was performed according to the thin layer identification method of Example 1. The results are as follows: Figure 5 shown.

[0119] in, Figure 5 a in the figure is the TLC obtained by ambient humidity (1). Figure 5 1-3 in a correspond to the test solution (15 μL) prepared from the vertical sequence pokeweed formula granules of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively, 4 is the vertical sequence pokeweed control medicinal material solution (10 μL), and 5 is the pokeweed saponin A reference solution (15 μL). The solution preparation method refers to Example 1.

[0120] Figure 5 b in the figure is the thin layer chromatogram obtained by ambient humidity (2). Figure 5 1-3 in b correspond to the test solution (15 μL) prepared from the vertical sequence pokeweed formula granules of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively, 4 is the vertical sequence pokeweed control medicinal material solution (10 μL), and 5 is the pokeweed saponin A reference solution (15 μL). The solution preparation method refers to Example 1.

[0121] Depend on Figure 5 It can be seen from ab in that the thin layer identification method provided by the present invention can obtain good detection effect under different humidity conditions, and there is no obvious difference in the separation effect and spot display in the chromatogram, indicating that the thin layer identification method provided by the present invention has good durability under an environmental humidity of 32-88% RH.

[0122] 6. Inspection of thin layer boards produced by different manufacturers

[0123] The test solution A (15 μL, corresponding to the test solution prepared from the vertical order pokeweed formula granules of 2208001Y, 2208002Y, and 2208003Y, respectively), the vertical order pokeweed control medicinal material solution (10 μL), and the pokeweed saponin A reference solution (15 μL) were respectively aspirated and spotted on different thin layer plates: Yantai plate (Yantai Linghua New Materials Co., Ltd., silica gel G plate, batch number: 20230815), Merck plate (Merck, silica gel G plate, batch number: HX45990926); and then developed according to the chromatographic conditions of Example 1. The results are as follows: Figure 6 shown.

[0124] in, Figure 6 a in the figure is the thin layer chromatogram obtained from the Yantai plate. Figure 6 1-3 in a correspond to the test solution (15 μL) prepared from the vertical sequence pokeweed formula granules of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively, 4 is the vertical sequence pokeweed control medicinal material solution (10 μL), and 5 is the pokeweed saponin A reference solution (15 μL). The solution preparation method refers to Example 1.

[0125] Figure 6 b in the figure is the thin layer chromatogram obtained from the Merck plate. Figure 6 1-3 in b correspond to the test solution (15 μL) prepared from the vertical sequence pokeweed formula granules of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively, 4 is the vertical sequence pokeweed control medicinal material solution (10 μL), and 5 is the pokeweed saponin A reference solution (15 μL). The solution preparation method refers to Example 1.

[0126] Depend on Figure 6 It can be seen from ab in that the thin layer identification method provided by the present invention has a better chromatographic spot separation effect when using Yantai plate. Therefore, the present invention selects Yantai plate as the thin layer plate.

[0127] 7. Reproducibility study among different personnel

[0128] Two experimenters, A and B, prepared the control medicinal material solution and the reference substance solution according to Example 1, and prepared the test solution of the vertical order pokeweed formula granules with batch numbers 2208001Y, 2208002Y, and 2208003Y. The test solution (15 μL), the vertical order pokeweed control medicinal material solution (10 μL), and the pokeweed saponin A reference substance solution (15 μL) were respectively aspirated, and thin layer identification was performed according to the thin layer identification method of Example 1. The results are as follows Figure 7 shown.

[0129] in, Figure 7 a in the figure is the thin layer chromatogram obtained by person A. Figure 7 1-3 in a correspond to the test solutions (15 μL) prepared from the formula granules of the vertical order pokeweed of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively; 4 is the control medicinal material solution of the vertical order pokeweed (10 μL); and 5 is the reference substance solution of pokeweed saponin A (15 μL).

[0130] Figure 7 b is the thin layer chromatogram obtained by person A, Figure 7 1-3 in b correspond to the test solutions (15 μL) prepared from the formula granules of the vertical order pokeweed of batch numbers 2208001Y, 2208002Y, and 2208003Y, respectively; 4 is the control medicinal material solution of the vertical order pokeweed (10 μL); and 5 is the reference substance solution of pokeweed saponin A (15 μL).

[0131] Depend on Figure 7 It can be seen from ab in that the thin layer identification method provided by the present invention has good reproducibility among different people.

[0132] Example 3

[0133] This embodiment provides a method for distinguishing Phytolacca japonica and Phytolacca serrata, which is as follows:

[0134] The test sample of the Chinese pokeweed sample was the lyophilized powder of the Chinese pokeweed sample, and its preparation method was prepared according to the preparation method of the Chinese pokeweed formula granules; three batches of Chinese pokeweed sample lyophilized powder solutions were prepared according to "1. Preparation of test solution A" in Example 1 as the test solution of the Chinese pokeweed sample; the test solution of the Chinese pokeweed formula granules was prepared according to "1. Preparation of test solution A" in Example 1; the reference solution and the control medicinal material solution were both prepared according to the preparation method in Example 1.

[0135] The reference solution of Phytolaccaa saponin A (15 μL), the reference solution of Phytolacca sutchuenensis (10 μL), the test solution of Phytolacca sutchuenensis sample, and the test solution were spotted on the same silica gel G thin layer plate; the results were as follows: Figure 8 shown.

[0136] in, Figure 81 is the reference solution of pokeweed saponin A (15 μL), 2 is the reference medicinal material solution of pokeweed (10 μL), 3 is the test solution of pokeweed formula granules (batch number: 2208001Y) (13 μL), 4 is the test solution of pokeweed formula granules (batch number: 2208002Y) (13 μL), 5 is the test solution of pokeweed formula granules (batch number: 2208003Y) (13 μL), 6 is the test solution of pokeweed sample (batch number: 20250220001) (13 μL), 7 is the test solution of pokeweed sample (batch number: 20250220002) (13 μL), and 8 is the test solution of pokeweed sample (batch number: 20250220003) (13 μL).

[0137] Depend on Figure 8 It can be seen that there are significant differences in the thin layer spectra of the vertical sequence pokeweed samples and the pokeweed samples, indicating that the thin layer identification method provided by the present invention can effectively distinguish the pokeweed samples and the vertical sequence pokeweed samples, and is specific.

[0138] 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 identification method for vertical sequence pokeweed medicinal preparations, characterized in that: The steps include: Preparation of test solution A: Prepare test solution A from the Pokeweed drug preparation test sample; Preparation of control medicinal material solution: Prepare the control medicinal material of Phytolacca sutchuenensis into the control medicinal material solution of Phytolacca sutchuenensis; Preparation of reference solution: Prepare the pokeweed saponin A reference solution from the pokeweed saponin A reference solution; The test solution A, the control medicinal material solution of Phytolacca americana, and the reference solution of Phytolacca saponin A were spotted on the same silica gel thin layer plate, and developed with ethyl acetate, methanol and water in a volume ratio of (8.5-9.5):2:0.5 as the developing solvent.

2. The thin layer identification method of the vertical sequence pokeweed pharmaceutical preparation according to claim 1, characterized in that: In the developing solvent, the volume ratio of ethyl acetate, methanol and water is 8.5:2:0.

5.

3. The thin layer identification method of the vertical sequence pokeweed pharmaceutical preparation according to claim 1 or 2, characterized in that: The preparation process of the test solution A comprises: taking a test sample of a pokeweed drug preparation, adding an ethanol aqueous solution for extraction, and separating to obtain a test solution; Optionally, the ratio of the mass of the test sample of the vertical order pokeweed drug preparation to the volume of the ethanol aqueous solution is (0.5-2.5):(10-50), the mass unit of the vertical order pokeweed drug preparation test sample is g, and the volume unit of the ethanol aqueous solution is ml; Optionally, the volume concentration of the ethanol aqueous solution is 49.5-50.5%.

4. The thin layer identification method of the vertical sequence pokeweed pharmaceutical preparation according to claim 3, characterized in that: The preparation process of the Chinese pokeweed control medicinal material solution comprises: taking the Chinese pokeweed control medicinal material, extracting it with water, filtering it, evaporating the filtrate to dryness, and adding ethanol water solution to the obtained residue to obtain the control medicinal material solution; Optionally, the ratio of the mass of the Chinese pokeweed control medicinal material to the volume of the ethanol aqueous solution is (1-5):(5-25), the mass unit of the Chinese pokeweed control medicinal material is g, and the volume unit of the ethanol aqueous solution is ml; Optionally, the extraction method includes a heating reflux method, wherein the heating reflux time is 30-60 minutes; Optionally, the volume concentration of the ethanol aqueous solution is 49.5-50.5%.

5. The thin layer identification method of the vertical order pokeweed medicinal preparation according to any one of claims 1 to 4, characterized in that: The concentration of the pokeweed saponin A reference substance in the pokeweed saponin A reference substance solution is 0.1-0.3 mg / ml.

6. The thin layer identification method of the vertical order pokeweed medicinal preparation according to any one of claims 1 to 5, characterized in that: The sample volume of the test solution A is 10-15 μL; preferably, the sample volume of the test solution A is 15 μL; and / or, The spotting volume of the control medicinal material solution of Phytolacca versicolor is 10-15 μL; preferably, the spotting volume of the control medicinal material solution of Phytolacca versicolor is 10 μL; and / or, The spotting volume of the pokeweed saponin A reference solution is 10-15 μL; preferably, the spotting volume of the pokeweed saponin A reference solution is 15 μL.

7. The thin layer identification method for the vertical order pokeweed medicinal preparation according to any one of claims 1 to 6, characterized in that: After unfolding, it also includes taking out, drying, spraying with sulfuric acid ethanol solution and heating until color develops; After color development, the process also includes inspecting under an ultraviolet lamp; optionally, the wavelength of the ultraviolet lamp is 365nm.

8. The thin layer identification method of the vertical sequence pokeweed pharmaceutical preparation according to claim 7, characterized in that: The heating temperature is 100-110° C.; and / or, The silica gel thin layer plate includes a silica gel G thin layer plate; and / or, In the inspection step, the chromatogram of the test sample is compared with the chromatogram of the reference medicinal material Pokeweed.

9. The thin layer identification method for the vertical sequence pokeweed medicinal preparation according to any one of claims 1 to 8, characterized in that: The pokeweed pharmaceutical preparation includes a pokeweed benchmark sample concentrated extract, a pokeweed benchmark sample water decoction, a pokeweed benchmark sample freeze-dried powder or a pokeweed preparation.

10. Use of the thin layer identification method according to any one of claims 1 to 9 in identifying Pokeweed or its derivatives and Pokeweed or its derivatives.

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