Thin-layer identification method of Xuan- yi Tong-jing medicinal preparation

Abstract

The present application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin-layer identification method of Xuanyu Tongjing pharmaceutical preparation. The present application provides a thin-layer identification method of Xuanyu Tongjing pharmaceutical preparation, comprising the following steps: preparing a test sample solution A, and based on the test sample solution A, using thin-layer chromatography to simultaneously identify gardenia, licorice, white peony root and cortex moutan in the Xuanyu Tongjing pharmaceutical preparation; according to the thin-layer chromatography: taking the test sample solution A, a control medicinal solution and a control sample solution, respectively spotting on the same silica gel thin-layer plate, and using a volume ratio of ethyl acetate, formic acid, glacial acetic acid and water as 15:(1-3):(1-3):2 as a developing agent for development. The thin-layer identification method can simultaneously detect the licorice, gardenia, white peony root and cortex moutan components in the Xuanyu Tongjing pharmaceutical preparation, and has short detection time and high efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin-layer identification method for a drug preparation for dispersing stagnation and dredging menstruation. Background Art

[0002] The origin of the Decoction for Dispersing Stagnation and Dredging Menstruation is from "Fu Qingzhu's Gynecology" by Fu Shan in the Qing Dynasty. The original text records: "White peony root, five qian (stir-fried with wine), Angelica sinensis, five qian (washed with wine), Cortex Moutan, five qian, Gardenia jasminoides Ellis, three qian (stir-fried), Semen Sinapis Alba, two qian (stir-fried and ground), Bupleurum chinense, one qian, Cyperus rotundus, one qian (stir-fried with wine), Curcuma aromatica Salisb., one qian (stir-fried with vinegar), Scutellaria baicalensis Georgi, one qian (stir-fried with wine), Licorice root, one qian". This prescription has the characteristics of nourishing liver blood, relieving liver depression, promoting liver qi, and reducing liver fire. In the prescription, white peony root nourishes blood and regulates menstruation, soothes the liver and relieves pain; Cortex Moutan clears heat and cools blood, promotes blood circulation and removes stasis; Gardenia jasminoides Ellis purges fire and relieves restlessness, clears heat and promotes diuresis, cools blood and relieves pain; Licorice root clears heat and resolves phlegm, relieves spasm and pain, and harmonizes the properties of drugs; the whole prescription makes qi flow smoothly, blood circulate smoothly, dampness and phlegm disappear.

[0003] As a classic famous prescription for gynecology, the Decoction for Dispersing Stagnation and Dredging Menstruation has great development value, and its medicinal flavors need to be detected; thin-layer chromatography detection is an important qualitative detection method in the "Chinese Pharmacopoeia", but the common thin-layer chromatography detection is carried out separately according to the medicinal flavors, that is, 1 thin-layer plate is used to identify 1 prescribed medicinal flavor, which has the problems of cumbersome methods and low detection efficiency, and there is no thin-layer identification method for multiple components of the Decoction for Dispersing Stagnation and Dredging Menstruation in the prior art. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect in the prior art that multiple components in the Decoction for Dispersing Stagnation and Dredging Menstruation cannot be detected simultaneously, resulting in cumbersome steps and low detection efficiency, so as to provide a thin-layer identification method for a drug preparation for dispersing stagnation and dredging menstruation, which can simultaneously detect the components of licorice root, gardenia jasminoides Ellis, white peony root and Cortex Moutan in the drug preparation for dispersing stagnation and dredging menstruation, with short detection time and high efficiency.

[0005] For this purpose, the present invention provides the following technical solutions.

[0006] The present invention provides a thin-layer identification method for a drug preparation for dispersing stagnation and dredging menstruation, including the following steps:

[0007] Prepare a test solution A, and based on the test solution A, simultaneously identify gardenia jasminoides Ellis, licorice root, white peony root and Cortex Moutan in the drug preparation for dispersing stagnation and dredging menstruation by thin-layer chromatography;

[0008] According to the thin-layer chromatography method:

[0009] Take the test solution A, gardenia reference solution, licorice reference solution, peony bark reference solution, white peony reference solution, geniposide reference solution, glycyrrhizin reference solution, and paeoniflorin reference solution, and spot them separately onto the same silica gel thin-layer plate. Use ethyl acetate, formic acid, glacial acetic acid, and water in a volume ratio of 15:(1-3):(1-3):2 as the developing solvent and develop.

[0010] Furthermore, in the developing solvent, the volume ratio of ethyl acetate, formic acid, glacial acetic acid, and water is 15:1:1:2.

[0011] Optionally, the preparation process of the test solution A includes: taking the test sample of the Xuanyu Tongjing drug preparation, dissolving it in methanol and then separating the solid and liquid, evaporating the liquid to dryness, dissolving the residue in water and then separating the solid and liquid, extracting the liquid with water-saturated n-butanol, washing the liquid with water saturated with n-butanol, evaporating the n-butanol washing liquid to dryness, and redissolving the residue in methanol.

[0012] For example, take 5-10g of the test sample of the anti-hypertensive and menstrual regulating drug preparation, dissolve it in 10-50ml of methanol, and then separate the solid and liquid. Evaporate the liquid to dryness, dissolve the residue in 20-40ml of water, and then separate the solid and liquid. Extract the liquid with water-saturated n-butanol 1-3 times, 20-50ml each time. Combine the extracts, wash the liquid with water saturated with n-butanol 1-3 times, 20-50ml each time, and evaporate the n-butanol washing liquid to dryness. Redissolve the residue in 1-3ml of methanol. The ratio of parts by weight to parts by volume is g / ml.

[0013] Optionally, the method further includes the steps of preparing the gardenia reference solution, the licorice reference solution, the peony bark reference solution, and the white peony reference solution according to the preparation process of the test solution A;

[0014] For example, take 0.5-2 parts by weight of each of gardenia, licorice, peony bark, and white peony root reference materials, dissolve them in 10-50 ml of methanol, and then separate the solid and liquid. Evaporate the liquid to dryness, dissolve the residue in 20-40 ml of water, and then separate the solid and liquid. Extract the liquid with water-saturated n-butanol 1-3 times, 20-50 ml each time. Combine the extracts, wash the liquid with water-saturated n-butanol 1-3 times, 20-50 ml each time, and evaporate the n-butanol washing liquid to dryness. Redissolve the residue in 1-3 ml of methanol to prepare the corresponding reference material solutions. The ratio of parts by weight to parts by volume is g / ml.

[0015] For example, the paeoniflorin reference solution is prepared by dissolving paeoniflorin reference in methanol, with 1-5 mg of paeoniflorin reference dissolved in 1 mL of methanol;

[0016] For example, the geniposide reference solution is prepared by dissolving geniposide reference in methanol, with 1-5 mg of geniposide reference dissolved in each 1 mL of methanol;

[0017] For example, the glycyrrhizin reference solution is prepared by dissolving glycyrrhizin reference in methanol, with 1-5 mg of glycyrrhizin reference dissolved in 1 mL of methanol.

[0018] Optionally, the sample volume of test solution A is 3-8 μL;

[0019] Optionally, the spotting volume of the gardenia reference solution, licorice reference solution, peony bark reference solution, and white peony reference solution is 5-20 μL;

[0020] Optionally, the spotting volume of geniposide reference solution, glycyrrhizin reference solution and paeoniflorin reference solution is 3-20 μL.

[0021] Optionally, after unfolding, the process also includes removing the product, drying it, spraying it with a solution of vanillin-sulfuric acid in ethanol and / or sulfuric acid in ethanol, and then heating it until the color develops.

[0022] After color development, the process also includes inspection under fluorescent light.

[0023] Optionally, the preparation step of the sulfuric acid ethanol solution includes: mixing anhydrous ethanol and sulfuric acid, wherein the volume ratio of anhydrous ethanol to sulfuric acid is 9:1;

[0024] Optionally, the preparation steps of the vanillin-sulfuric acid ethanol solution include: mixing ethanol and sulfuric acid to obtain a sulfuric acid ethanol solution, wherein the volume ratio of ethanol to sulfuric acid is 9:1; wherein the ethanol is analytical grade ethanol, and the concentration of sulfuric acid is 9.5-10.5%; mixing the sulfuric acid ethanol solution and vanillin, wherein the weight ratio of vanillin to the volume of the sulfuric acid ethanol solution is 1:20, and the weight-to-volume ratio is g / ml.

[0025] Optionally, the heating temperature is 100-110°C;

[0026] Optionally, the silicone sheet includes a silicone G sheet or a silicone G254 sheet.

[0027] Optionally, in the inspection step, the chromatogram of the test sample is compared with the chromatograms of white peony root reference material, peony bark reference material, gardenia reference material, and licorice reference material.

[0028] Optionally, the Xuanyu Tongjing drug preparation includes a decoction of Xuanyu Tongjing Decoction reference sample, a concentrated extract of Xuanyu Tongjing Decoction reference sample, a freeze-dried powder of Xuanyu Tongjing Decoction reference sample, or a Xuanyu Tongjing Decoction preparation.

[0029] The technical solution of this invention has the following advantages:

[0030] 1. The present invention provides a thin-layer chromatography (TLC) identification method for a purgative and menstruation-regulating drug preparation, the TLC identification method comprising the following steps: preparing a test solution A, and based on the test solution A, identifying Gardenia, Licorice, White Peony Root, and Moutan Bark using TLC; the TLC identification method comprising: taking the test solution A, Gardenia reference medicinal material solution, Licorice reference medicinal material solution, Moutan Bark reference medicinal material solution, White Peony Root reference medicinal material solution, Gardenia glycoside reference solution, Licorice glycoside reference solution, and Paeoniflorin reference solution, respectively spotting them onto the same silica gel TLC plate, and developing the plate using a volume ratio of ethyl acetate, formic acid, glacial acetic acid, and water of 15:(1-3):(1-3):2. This thin-layer chromatography identification method can simultaneously identify four medicinal materials: gardenia, licorice, white peony root, and peony bark. That is, the above four medicinal materials can be identified at the same time using the same thin-layer plate and the same developing solvent system, which reduces the testing time and reagent consumption and improves the detection efficiency. The chromatograms obtained by using this thin-layer chromatography identification method have clear spots, good separation, strong specificity, no background interference, no interference from negative control standards, and good durability.

[0031] Furthermore, ethyl acetate-formic acid-glacial acetic acid-water is used as the developing solvent in a volume ratio of 15:(1-3):(1-3):2. The developing solvent prepared at this volume ratio can be used immediately without separation, so there is no need to place it at low temperature for a long time before use, thus reducing the detection time.

[0032] 2. The thin-layer chromatography (TLC) identification method for purgative and menstrual-regulating drug preparations provided by this invention involves the following steps in preparing test solution A: the residue is dissolved in water and then subjected to solid-liquid separation. Unlike existing technologies that require extraction and purification using organic solvents, this invention directly uses water as the solvent, simplifying the preparation steps of the test solution. Furthermore, the residue, after extraction with n-butanol and subsequent evaporation, can be stored for an extended period. Whether redissolved overnight or immediately after redissolving in methanol, the spots of paeoniflorin, glycyrrhizin, and geniposide in the TLC chromatogram are not significantly affected. This TLC identification method has low operational requirements. After heating and developing the TLC plate, the spots are clear, and observation within 10 minutes yields the best results.

[0033] Furthermore, the preparation of test solution for thin-layer chromatography identification of traditional Chinese medicine compound preparations is usually quite complex, with cumbersome extraction steps, which is time-consuming and labor-intensive. Moreover, the test solution for thin-layer chromatography identification of different medicinal ingredients is generally different. However, this invention only requires weighing the sample once, extracting the sample, and preparing the same test solution. Different medicinal ingredients can use the same test solution, which greatly improves the detection efficiency.

[0034] 3. The thin-layer chromatography method for identifying diaphoretic and menstrual regulating drug preparations provided by this invention is most effective when the colorimetric reagent, vanillin-sulfuric acid-ethanol solution, is prepared immediately before use, and it displays the most spot information. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 This is a thin-layer chromatogram obtained from the specificity verification of Embodiment 2 of the present invention;

[0037] Figure 2 These are thin-layer chromatograms obtained from different developing solvents in Example 2 of this invention;

[0038] Figure 3 This is a thin-layer chromatogram obtained from the sample quantity determination in Example 2 of the present invention;

[0039] Figure 4 These are thin-layer chromatograms obtained from different environmental temperatures in Example 2 of this invention;

[0040] Figure 5 This is a thin-layer chromatogram obtained from different environmental humidity conditions in Example 2 of the present invention;

[0041] Figure 6 This is a thin-layer chromatogram obtained from the thin-layer plate examination in Example 2 of the present invention;

[0042] Figure 7 These are thin-layer chromatograms obtained by examining different spotting methods in Example 2 of the present invention;

[0043] Figure 8 These are thin-layer chromatograms obtained from different batches of test samples in Example 2 of this invention;

[0044] Figure 9 This is a thin-layer chromatogram obtained from the comparative experiment of Example 3 of the present invention. Detailed Implementation

[0045] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0046] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0047] Reference standard:

[0048] Paeoniflorin reference standard (Source: National Institutes for Food and Drug Control, batch number: 110736-202246);

[0049] Gardenoside reference standard (Source: National Institutes for Food and Drug Control, batch number: 110749-202320);

[0050] Glycyrrhizin reference standard (Source: National Institutes for Food and Drug Control, batch number: 111610-202209);

[0051] Reference medicinal materials:

[0052] White peony reference material (Source: China National Institutes for Food and Drug Control, batch number: (120905-202011);

[0053] Peony bark reference material (Source: China National Institutes for Food and Drug Control, batch number: 121490-201603);

[0054] Licorice reference material (Source: China National Institutes for Food and Drug Control, batch number: 120904-202021);

[0055] Gardenia reference material (Source: China National Institutes for Food and Drug Control, batch number: 120986-202111);

[0056] Preparation Example

[0057] Xuanyu Tongjing Decoction Granules (240503, 240504, 240505); Xuanyu Tongjing Decoction Standard Decoction (JZ23110602); Xuanyu Tongjing Decoction Standard Sample Concentrated Extract (NS01, NS02); Xuanyu Tongjing Decoction Standard Sample Lyophilized Powder (DG01, DG02, DG03) are all prepared in-house by our company.

[0058] The preparation method can be carried out using conventional methods in this field, as detailed below:

[0059] Xuanyu Tongjing Decoction Granules: Take the ten ingredients of the prescription, stir-fry mustard seeds until crushed, and decoct them twice with the remaining nine ingredients in water. For the first decoction, add 8 times the amount of water, soak for 30 minutes, boil and keep for 30 minutes, collect the aromatic water and the decoction, and filter the decoction. For the second decoction, add 6 times the amount of water, boil and keep for 25 minutes, collect the decoction, filter the decoction, and combine the filtrates. Encapsulate the aromatic water with beta-cyclodextrin, dry, grind into a fine powder, and set aside. Concentrate the filtrate into a clear paste, dry, add an appropriate amount of excipients and the above beta-cyclodextrin inclusion complex, mix well, granulate, and make 1000g of the product.

[0060] Xuanyu Tongjing Decoction Base Decoction: Take ten ingredients from the prescription, stir-fry mustard seeds until crushed, and decoct them twice with the remaining nine ingredients in water. For the first decoction, add 8 times the amount of water, soak for 30 minutes, boil and keep for 30 minutes, then filter. For the second decoction, add 6 times the amount of water, boil and keep for 25 minutes, then filter. Combine the filtrates to obtain the final product.

[0061] Concentrated extract of Xuanyu Tongjing Decoction benchmark sample: Take ten ingredients from the prescription, stir-fry mustard seeds and crush them, add water and decoct twice with the other nine ingredients. For the first decoction, add 8 times the amount of water, soak for 30 minutes, boil and keep for 30 minutes, then filter. For the second decoction, add 6 times the amount of water, boil and keep for 25 minutes, then filter. Combine the filtrates and concentrate the filtrate into a clear extract.

[0062] Freeze-dried powder of Xuanyu Tongjing Decoction reference sample: Take ten ingredients from the prescription, crush fried mustard seeds, and decoct them twice with the remaining nine ingredients in water. For the first decoction, add 8 times the amount of water, soak for 30 minutes, boil and keep for 30 minutes, and filter. For the second decoction, add water, boil and keep for 25 minutes, filter, combine the filtrates, pre-freeze the decoction at -80℃ or below, and freeze-dry to obtain the final product.

[0063] The prescription for Xuanyu Tongjing Decoction is as follows: 18.65g of white peony root (processed with wine), 18.65g of angelica root (processed with wine), 18.65g of peony bark, 11.19g of gardenia fruit (processed with wine), 7.46g of mustard seed (processed with wine), 3.73g of bupleurum root, 3.73g of cyperus rhizome (processed with wine), 3.73g of turmeric root (processed with vinegar), 3.73g of scutellaria root (processed with wine), and 3.73g of licorice root.

[0064] Example 1

[0065] This embodiment provides a thin-layer chromatography method for identifying purgative and menstrual-regulating drug preparations, including:

[0066] 1. Preparation of test solution A: Take 7.5g of freeze-dried powder of Xuanyu Tongjing Decoction reference sample (DG01), add 30ml of methanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 30ml of water, filter, extract the filtrate twice with 30ml of water-saturated n-butanol each time, combine the n-butanol solutions, wash three times with 30ml of water saturated with n-butanol each time, evaporate the n-butanol solution to dryness, dissolve the residue in 2ml of methanol to obtain test solution A.

[0067] 2. Preparation of reference herb solutions: Take 1g each of gardenia, licorice, peony bark, and white peony root reference herbs, add 30ml of methanol to each, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 30ml of water, filter, extract the filtrate twice with 30ml of water-saturated n-butanol each time, combine the n-butanol extracts, wash three times with 30ml of water saturated with n-butanol each time, evaporate the n-butanol extract to dryness, dissolve the residue in 2ml of methanol to prepare gardenia, licorice, peony bark, and white peony root reference herb solutions.

[0068] 3. Preparation of reference solutions: Take paeoniflorin reference standard, add methanol to prepare a solution containing 1 mg paeoniflorin reference standard per 1 mL; take geniposide reference standard, add methanol to prepare a solution containing 1 mg geniposide reference standard per 1 mL; take glycyrrhizin reference standard, add methanol to prepare a solution containing 1 mg glycyrrhizin reference standard per 1 mL.

[0069] 4. Chromatographic conditions: Perform thin-layer chromatography (General Rule 0502). Take 5 μL of the above-mentioned test solution A, gardenia reference material solution (5 μL), licorice reference material solution (5 μL), peony bark reference material solution (5 μL), and white peony reference material solution (5 μL); geniposide reference solution (3 μL), glycyrrhizin reference solution (3 μL), and paeoniflorin reference solution (3 μL); spot them separately onto the same silica gel G thin-layer plate (Merck plate). Use ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as the developing solvent, develop, remove, air dry, spray with 5% vanillin sulfuric acid ethanol solution (mix ethanol and 5% vanillin sulfuric acid solution, the volume ratio of ethanol to vanillin sulfuric acid solution is 9:1), and heat to 105℃ until the spots are clearly visible. Examine under fluorescent light.

[0070] In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material; fluorescent spots of the same color should also appear at the corresponding positions as in the chromatogram of the reference substance.

[0071] Example 2

[0072] This embodiment provides a thin-layer chromatography method for identifying a drug preparation for relieving stagnation and promoting menstruation, and examines it under different chromatographic conditions, including:

[0073] 1. Specificity verification

[0074] (1) Preparation of negative control solution:

[0075] The four negative test solutions (N-BMZG) lacking white peony root, peony bark, gardenia, and licorice were prepared by taking the six ingredients of the prescription (excluding white peony root, peony bark, gardenia, and licorice) and preparing them according to the preparation method of the freeze-dried powder of Xuanyu Tongjing Decoction reference sample in the preparation example.

[0076] Double negative test solution for lacking white peony root and peony bark (N-BM): Take the eight ingredients of the prescription (excluding white peony root and peony bark) and prepare them according to the preparation method of the freeze-dried powder of Xuanyu Tongjing Decoction reference sample in the preparation example.

[0077] Negative test solution for lacking gardenia and licorice (N-ZG): Take the eight ingredients of the prescription (excluding gardenia and licorice) and prepare them according to the preparation method of the freeze-dried powder of Xuanyu Tongjing Decoction reference sample in the preparation example.

[0078] Negative solution for lack of white peony root: Take the nine ingredients of the prescription (excluding white peony root) and prepare it according to the preparation method of the freeze-dried powder of Xuanyu Tongjing Decoction reference sample in the preparation example.

[0079] Negative-labeled solution for lack of peony bark (N-BM): Take the nine ingredients of the prescription (excluding peony bark) and prepare it according to the preparation method of the freeze-dried powder of Xuanyu Tongjing Decoction reference sample in the preparation example.

[0080] Gardenia-deficient negative solution (N-ZZ): Take the nine ingredients of the prescription (excluding gardenia) and prepare it according to the preparation method of the freeze-dried powder of Xuanyu Tongjing Decoction reference sample in the preparation example.

[0081] Licorice-deficient negative GC solution: Take the nine ingredients of the prescription (excluding licorice) and prepare it according to the preparation method of the freeze-dried powder of Xuanyu Tongjing Decoction reference sample in the preparation example.

[0082] The negative solutions obtained above were used to prepare four negative test solutions lacking white peony root, peony bark, gardenia, and licorice, two negative test solutions lacking white peony root and peony bark, two negative test solutions lacking gardenia and licorice, negative test solutions lacking white peony root, negative test solutions lacking peony bark, negative test solutions lacking gardenia, and negative test solutions lacking licorice, according to the preparation method of test solution A in Example 1.

[0083] (2) Chromatographic conditions: The thin-layer chromatography method (General Rule 0502) was followed. The following test solutions were prepared separately: four negative test solutions (15 μL each) lacking white peony root, peony bark, gardenia, and licorice; two negative test solutions (15 μL each) lacking white peony root and peony bark; two negative test solutions (15 μL each) lacking gardenia and licorice; a negative solution (15 μL each) lacking white peony root, peony bark, gardenia, and licorice; a negative solution (15 μL each) lacking white peony root, peony bark, gardenia, and licorice; and the white peony root reference solution (5 μL), peony bark reference solution (5 μL), gardenia reference solution (5 μL), and licorice reference solution (5 μL) from Example 1. The following solutions were spotted separately on the same silica gel G thin-layer plate (Merck plate): paeoniflorin reference solution (5 μL), geniposide reference solution (3 μL), glycyrrhizin reference solution (3 μL), and test solution A (3 μL, 5 μL, 8 μL). Developed with ethyl acetate-formic acid-glacial acetic acid-water (volume ratio 15:1:1:2), the plate was removed, dried, and sprayed with a 5% vanillin-sulfuric acid ethanol solution (ethanol and 5% vanillin-sulfuric acid solution were mixed in a volume ratio of 9:1). The plate was heated at 105°C until the spots were clearly visible and examined under fluorescent light. The results are as follows: Figure 1 As shown,

[0084] exist Figure 1In the sample, 1 is a negative solution lacking white peony root, 2 is a negative solution lacking peony bark, 3 is a negative solution lacking gardenia fruit, 4 is a negative solution lacking licorice root, 5 is a double negative test solution lacking both white peony root and peony bark, 6 is a double negative test solution lacking both gardenia fruit and licorice root, 7 is a quadruple negative test solution lacking white peony root, peony bark, gardenia fruit, and licorice root, 8 is test solution A (3 μL), 9 is test solution A (5 μL), 10 is test solution A (8 μL), 11 is paeoniflorin reference solution, 12 is geniposide reference solution, 13 is glycyrrhizin reference solution, 14 is white peony root reference medicinal material solution, 15 is peony bark reference medicinal material solution, 16 is gardenia fruit reference medicinal material solution, and 17 is licorice root reference medicinal material solution.

[0085] Depend on Figure 1 It can be seen that, under sunlight, the test sample chromatogram shows spots of the same color at the corresponding positions as those of the reference materials of white peony root, peony bark, and paeoniflorin; it also shows spots of the same color at the corresponding positions as those of gardenia and geniposide; and it shows spots of the same color at the corresponding positions as those of licorice and glycyrrhizin. The spots show good separation and no interference from the negative test sample solution, meeting the specificity requirements.

[0086] 2. Investigation of different developing solvents

[0087] (1) Chloroform-ethyl acetate-methanol-concentrated ammonia test solution (8:1:4:1);

[0088] (2) Ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2);

[0089] (3) Ethyl acetate-formic acid-glacial acetic acid-water (15:3:1:2);

[0090] (4) Ethyl acetate-formic acid-glacial acetic acid-water (15:1:3:2);

[0091] Compared with Example 1, the difference is that different developing solvents (1), (2), (3), and (4) were used for development, instead of ethyl acetate-formic acid-glacial acetic acid-water (15:(1-3):(1-3):2) as the developing solvent in Example 1. The results are as follows. Figure 2 As shown. Among them, Figure 2 In the figure, 'a' is the thin-layer chromatogram obtained by developing solvent (1). Figure 2 In the formula 'a', 1 is a double-negative test solution lacking white peony root and peony bark; 2 is a double-negative test solution lacking gardenia fruit and licorice root; 3 and 4 are both test solution A; 5 is paeoniflorin reference solution; 6 is geniposide reference solution; 7 is glycyrrhizin reference solution; 8 is peony bark reference medicinal material solution; 9 is white peony root reference medicinal material solution; 10 is gardenia fruit reference medicinal material solution; and 11 is licorice root reference medicinal material solution.

[0092] Figure 2 In the figure, b is the thin-layer chromatogram obtained by developing solvent (2). Figure 2 In the formula b, 1 is the test solution A, 2 is the paeoniflorin reference solution, 3 is the geniposide reference solution, 4 is the glycyrrhizin reference solution, 5 is the white peony reference herb solution, 6 is the peony bark reference herb solution, 7 is the gardenia reference herb solution, and 8 is the glycyrrhiza reference herb solution.

[0093] Figure 2 In the figure, c is the thin-layer chromatogram obtained by developing solvent (3). Figure 2 In the formula c, 1 is paeoniflorin reference solution, 2 is geniposide reference solution, 3 is glycyrrhizin reference solution, 4 is test solution A, 5 is white peony reference material solution, 6 is peony bark reference material solution, 7 is gardenia reference material solution, and 8 is glycyrrhiza reference material solution.

[0094] Figure 2 In the figure, d represents the thin-layer chromatogram obtained by developing solvent (4). Figure 2 In the d, 1 is paeoniflorin reference solution, 2 is geniposide reference solution, 3 is glycyrrhizin reference solution, 4 is white peony reference medicinal material solution, 5 is peony bark reference medicinal material solution, 6 is gardenia reference medicinal material solution, 7 is glycyrrhiza reference medicinal material solution, and 8 is test solution A.

[0095] The above Figure 2 The double-negative test solutions for Paeonia lactiflora and Paeonia suffruticosa, double-negative test solutions for Gardenia jasminoides and Glycyrrhiza uralensis, test solution A, reference solution, and reference medicinal material solution were all prepared in “1. Specificity Verification”.

[0096] Depend on Figure 2 As can be seen from the ad, when chloroform-ethyl acetate-methanol-concentrated ammonia solution (8:1:4:1) is used as the developing solvent, glycyrrhizin has a short developing distance and its color is similar to that of nearby spots, resulting in poor separation. When ethyl acetate-formic acid-glacial acetic acid-water is used as the developing solvent, the ratios of (15:1:1:2), (15:3:1:2), and (15:1:3:2) show more spot information and better separation. Therefore, this invention selects ethyl acetate-formic acid-glacial acetic acid-water as the developing solvent with a volume ratio of 15:(1-3):(1-3):2.

[0097] 3. Sample quantity investigation

[0098] Compared with Example 1, the difference is that four negative test solutions (15 μL each) lacking white peony root, peony bark, gardenia, and licorice were also prepared; two negative test solutions (15 μL each) lacking white peony root and peony bark; two negative test solutions (15 μL each) lacking gardenia and licorice; a negative solution (15 μL) lacking white peony root; a negative solution (15 μL) lacking peony bark; a negative solution (15 μL) lacking gardenia; and a negative solution (15 μL) lacking licorice were also prepared. Test solution A (3 μL, 5 μL, and 8 μL) were spotted onto the same silica gel G thin-layer plate (Merck plate). The results are as follows: Figure 3 As shown. The above negative solution was prepared in "1. Specificity Verification".

[0099] exist Figure 3 In the sample, 1 is a negative solution lacking white peony root, 2 is a negative solution lacking peony bark, 3 is a negative solution lacking gardenia fruit, 4 is a negative solution lacking licorice root, 5 is a double negative test solution lacking both white peony root and peony bark, 6 is a double negative test solution lacking both gardenia fruit and licorice root, 7 is a quadruple negative test solution lacking white peony root, peony bark, gardenia fruit, and licorice root, 8 is test solution A (3 μL), 9 is test solution A (5 μL), 10 is test solution A (8 μL), 11 is paeoniflorin reference solution, 12 is geniposide reference solution, 13 is glycyrrhizin reference solution, 14 is white peony root reference medicinal material solution, 15 is peony bark reference medicinal material solution, 16 is gardenia fruit reference medicinal material solution, and 17 is licorice root reference medicinal material solution.

[0100] 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 amounts, and the spots are clear, indicating that the method has good durability under different sample amounts.

[0101] 4. Investigation of different ambient temperatures

[0102] (1) The ambient temperature is 6℃;

[0103] (2) The ambient temperature was 23.6℃;

[0104] (3) The ambient temperature is 45℃;

[0105] Thin-layer chromatography was performed under conditions (1) to (3) respectively, according to the thin-layer chromatography identification method of Example 1, and the results are as follows. Figure 4 As shown. Among them, Figure 4 In this context, 'a' represents the thin-layer chromatogram obtained at ambient temperature (1). Figure 4 In the formula 'a', 1-3 are all test solution A, 4 is paeoniflorin reference solution, 5 is geniposide reference solution, 6 is glycyrrhizin reference solution, 7 is white peony reference medicinal material solution, 8 is peony bark reference medicinal material solution, 9 is gardenia reference medicinal material solution, and 10 is glycyrrhiza reference medicinal material solution.

[0106] Figure 4In the figure, b represents the thin-layer chromatogram obtained at ambient temperature (2). Figure 4 In the formula b, 1 is the test solution A, 2 is the paeoniflorin reference solution, 3 is the geniposide reference solution, 4 is the glycyrrhizin reference solution, 5 is the white peony reference herb solution, 6 is the peony bark reference herb solution, 7 is the gardenia reference herb solution, and 8 is the glycyrrhiza reference herb solution.

[0107] Figure 4 In the figure, c represents the ambient temperature (3) obtained from the thin-layer chromatogram. Figure 4 In the formula b, 1 is paeoniflorin reference solution, 2 is geniposide reference solution, 3 is glycyrrhizin reference solution, 4 is test solution A, 5 is white peony reference material solution, 6 is peony bark reference material solution, 7 is gardenia reference material solution, and 8 is glycyrrhiza reference material solution.

[0108] Depend on Figure 4 As can be seen from the ac, the thin-layer identification method provided by the present invention can achieve good detection results at different temperatures. There is no significant 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 in an ambient temperature range of 6-45℃.

[0109] 5. Investigation of humidity levels in different environments

[0110] (1) The ambient humidity is 32%RH;

[0111] (2) The ambient humidity is 88%RH;

[0112] Thin-layer chromatography was performed under conditions (1) and (2) respectively, according to the thin-layer chromatography identification method of Example 1, and the results are as follows. Figure 5 As shown. Among them, Figure 5 In the figure, 'a' represents the thin-layer chromatogram obtained from the ambient humidity (1). Figure 5 In the diagram, 1 represents a negative solution lacking white peony root, 2 a negative solution lacking peony bark, 3 a negative solution lacking gardenia fruit, 4 a negative solution lacking licorice root, 5 is test solution A, 6 is paeoniflorin reference solution, 7 is geniposide reference solution, 8 is glycyrrhizin reference solution, 9 is white peony root reference solution, 10 is peony bark reference solution, 11 is gardenia fruit reference solution, and 12 is licorice root reference solution. These negative solutions were prepared according to "1. Specificity Verification".

[0113] Figure 5 b in the figure represents the thin-layer chromatogram obtained from the ambient humidity (2). Figure 5 In the formula b, 1 is paeoniflorin reference solution, 2 is geniposide reference solution, 3 is glycyrrhizin reference solution, 4 is test solution A, 5 is white peony reference material solution, 6 is peony bark reference material solution, 7 is gardenia reference material solution, and 8 is glycyrrhiza reference material solution.

[0114] Depend on Figure 5 As can be seen from section ab, the thin-layer identification method provided by this invention can achieve good detection results under different humidity levels. There is no significant difference in the separation effect and spot display in the chromatogram, indicating that the thin-layer identification method provided by this invention has good durability in an environment with humidity of 32-88%RH.

[0115] 6. Comparison of thin-layer boards produced by different manufacturers

[0116] According to the sampling amount in Example 1, test solution A, the corresponding reference solution, and the corresponding reference medicinal material solution were respectively spotted onto different thin-layer plates: Yantai plate (Yantai Huayang New Material Technology Co., Ltd., silica gel G plate, batch number 20230728), Qingdao plate (manufactured by Qingdao Haiyang Chemical Co., Ltd., silica gel G plate, batch number 20230801), and Merck plate (Merck, silica gel G plate, batch number HX28729426); then developed according to the chromatographic conditions. The results are as follows. Figure 6 As shown. Among them, Figure 6 In the figure, 'a' is the thin-layer chromatogram obtained from the Yantai plate. Figure 6 In the formula 'a', 1 is paeoniflorin reference solution, 2 is geniposide reference solution, 3 is glycyrrhizin reference solution, 4 is test solution A, 5 is white peony reference material solution, 6 is peony bark reference material solution, 7 is gardenia reference material solution, and 8 is glycyrrhiza reference material solution.

[0117] Figure 6 In the figure, b is the thin-layer chromatogram obtained from the Qingdao plate. Figure 6 In the formula b, 1 is paeoniflorin reference solution, 2 is geniposide reference solution, 3 is glycyrrhizin reference solution, 4 is test solution A, 5 is white peony reference material solution, 6 is peony bark reference material solution, 7 is gardenia reference material solution, and 8 is glycyrrhiza reference material solution.

[0118] Figure 6 In the image, 'c' represents the thin-layer chromatogram obtained from a Merck plate. Figure 6 In the formula c, 1 is the test solution A, 2 is the paeoniflorin reference solution, 3 is the geniposide reference solution, 4 is the glycyrrhizin reference solution, 5 is the white peony reference herb solution, 6 is the peony bark reference herb solution, 7 is the gardenia reference herb solution, and 8 is the glycyrrhiza reference herb solution.

[0119] Depend on Figure 6 As can be seen from the ac, the thin-layer identification method provided by the present invention can achieve good detection results when using different thin-layer plates. There is no significant 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 on thin-layer plates produced by different manufacturers.

[0120] 7. Investigation of different sampling methods

[0121] (1) The dot pattern is a strip;

[0122] (2) The dot pattern is a round dot;

[0123] Thin-layer chromatography (TLC) identification was performed using the spotting methods described in (1) and (2) respectively, following the TLC identification method of Example 1. The results are as follows: Figure 7 As shown. Among them, Figure 7 In this context, 'a' represents the thin-layer chromatogram obtained using spotting method (1). Figure 7 In the formula 'a', 1 is the test solution A, 2 is the paeoniflorin reference solution, 3 is the geniposide reference solution, 4 is the glycyrrhizin reference solution, 5 is the white peony reference herb solution, 6 is the peony bark reference herb solution, 7 is the gardenia reference herb solution, and 8 is the glycyrrhiza reference herb solution.

[0124] Figure 7 In the image, b is the thin-layer chromatogram obtained by spotting method (2). Figure 7 In the formula b, 1 is glycyrrhizin reference solution, 2 is geniposide reference solution, 3 is paeoniflorin reference solution, 4 is white peony reference medicinal material solution, 5 is peony bark reference medicinal material solution, 6 is gardenia reference medicinal material solution, 7 is glycyrrhiza reference medicinal material solution, and 8 is test solution A.

[0125] Depend on Figure 7 As can be seen from section ab, the thin-layer identification method provided by this invention can achieve good detection results under different spotting methods. There is no significant difference in the separation effect and spot display in the chromatogram, indicating that the thin-layer identification method provided by this invention has good durability under different spotting methods.

[0126] 8. Examination of different batches of test samples

[0127] (1) The test sample was the standard decoction of Xuanyu Tongjing Decoction, batch number JZ23110602. The test sample solution was prepared according to the "Preparation of Test Sample Solution" in Example 1. The thin-layer chromatogram of the test sample was obtained according to the thin-layer identification method in Example 1. The results are shown in the figure. Figure 8 ;

[0128] (2) The test sample was a concentrated extract of Xuanyu Tongjing Decoction. Batch numbers NS01 and NS02 were used. The test sample solution was prepared according to the "Preparation of Test Sample Solution" in Example 1. The thin-layer chromatogram of the test sample was obtained according to the thin-layer identification method in Example 1. The results are shown in the figure. Figure 8 ;

[0129] (3) The test sample was the lyophilized powder of Xuanyu Tongjing Decoction reference sample. Batch numbers DG01, DG02, and DG03 were used. The test sample solution was prepared according to the "Preparation of Test Sample Solution" in Example 1. The thin-layer chromatogram of the test sample was obtained according to the thin-layer identification method in Example 1. The results are shown in […]. Figure 8;

[0130] (4) The test sample was Xuanyu Tongjing Decoction granules, batch numbers 240503, 240504, and 240505. The test sample solution was prepared according to the "Preparation of Test Sample Solution" in Example 1. The thin-layer chromatogram of the test sample was obtained according to the thin-layer identification method in Example 1. The results are shown in [Figure 1]. Figure 8 ;

[0131] in, Figure 8 S1 is paeoniflorin reference solution, S2 is geniposide reference solution, S3 is glycyrrhizin reference solution, 1 is white peony reference herb solution, 2 is peony bark reference herb solution, 3 is gardenia reference herb solution, 4 is glycyrrhiza reference herb solution, 5 is Xuanyu Tongjing Decoction reference decoction (JZ23110602), 6-7 correspond to concentrated extracts of Xuanyu Tongjing Decoction in batches NS01 and NS02 respectively, 8-10 correspond to reference samples (lyophilized powder) of Xuanyu Tongjing Decoction in batches DG01, DG02 and DG03 respectively, and 11-13 correspond to granules of Xuanyu Tongjing Decoction in batches 240503, 240504 and 240505 respectively.

[0132] Depend on Figure 8 It is understood that the thin-layer identification method provided by the present invention can be applied to Xuanyu Tongjing Decoction, Xuanyu Tongjing Decoction Concentrated Extract, Xuanyu Tongjing Decoction Reference Sample Lyophilized Powder, and Xuanyu Tongjing Decoction Granules.

[0133] Example 3

[0134] This embodiment provides a comparative experiment, as detailed below:

[0135] 1. Investigation of different methods for preparing test solutions

[0136] (1) Preparation method of test solution a: Take 7.5g of freeze-dried powder of Xuanyu Tongjing Decoction reference sample, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve the residue, and prepare test solution a.

[0137] (2) Preparation method b of test solution: Take 7.5g of freeze-dried powder of Xuanyu Tongjing Decoction reference sample, add 50ml of water, sonicate for 30 minutes, filter, add hydrochloric acid to adjust pH to 2, add ethyl acetate and shake to extract twice, 30ml each time, combine ethyl acetate, evaporate to dryness in water bath, add 2ml of methanol to dissolve the residue, and prepare test solution b.

[0138] (3) Preparation of test solution A: Take 7.5g of freeze-dried powder of Xuanyu Tongjing Decoction reference sample, add 30ml of methanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, add 30ml of water to dissolve the residue, filter, extract the filtrate twice with water-saturated n-butanol, 30ml each time, combine the n-butanol solutions, wash 3 times with water saturated with n-butanol, 30ml each time, evaporate the n-butanol solution to dryness, add 2ml of methanol to dissolve the residue, and obtain test solution A.

[0139] According to the sampling amount in Example 1, the above-mentioned test solution was taken and subjected to thin-layer chromatography identification according to the thin-layer chromatography identification method in Example 1. The results are as follows. Figure 9 As shown.

[0140] in, Figure 9 In the table, 1 is paeoniflorin reference solution, 2 is geniposide reference solution, 3 is glycyrrhizin reference solution, 4 is white peony reference medicinal material solution, 5 is peony bark reference medicinal material solution, 6 is gardenia reference medicinal material solution, 7 is glycyrrhiza reference medicinal material solution, 8 is test solution a, 9 is test solution b, and 10 is test solution A.

[0141] Depend on Figure 9 It can be seen that in the chromatogram of the preparation method of test solution A in the thin-layer identification method provided by the present invention, there is more spot information and better separation; while in the chromatogram of test solution a and b prepared by test solution preparation methods a and b, the spots are not clear and there is less spot information.

[0142] By comparing with Comparative Experiment 1, it is shown that the thin-layer chromatogram obtained by the thin-layer identification method provided by the present invention has rich spot information, good separation, clear chromatogram color development, and accurate identification results, which is beneficial for the simultaneous identification of four medicinal ingredients in the preparation of drugs for relieving depression and promoting menstruation: gardenia, white peony root, peony bark and licorice.

[0143] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A thin-layer chromatography method for identifying a drug preparation for relieving stagnation and promoting menstruation, characterized in that, The steps include the following: Prepare test solution A, and based on test solution A, use thin-layer chromatography to simultaneously identify gardenia, licorice, white peony root and peony bark in the preparation of the drug for relieving depression and promoting menstruation; Thin-layer chromatography: Take the test solution A, gardenia reference medicinal material solution, licorice reference medicinal material solution, peony bark reference medicinal material solution, white peony reference medicinal material solution, geniposide reference solution, glycyrrhizin reference solution, and paeoniflorin reference solution, and spot them separately onto the same silica gel thin-layer plate. Use ethyl acetate, formic acid, glacial acetic acid and water in a volume ratio of 15:(1-3):(1-3):2 as the developing solvent and develop. The preparation process of the test solution A includes: taking the test sample of the Xuanyu Tongjing drug preparation, dissolving it in methanol and then separating the solid and liquid, evaporating the liquid to dryness, dissolving the residue in water and then separating the solid and liquid, extracting the liquid with water-saturated n-butanol, washing the liquid with water saturated with n-butanol, evaporating the n-butanol washing liquid to dryness, and redissolving the residue in methanol. After unfolding, the process also includes removing the product, drying it, spraying it with a vanillin-sulfuric acid ethanol solution, and then heating it until the color develops. After color development, the process also includes inspection under fluorescent light.

2. The thin-layer chromatography identification method for the purgative and menstrual-regulating drug preparation according to claim 1, characterized in that, The volume ratio of ethyl acetate, formic acid, glacial acetic acid, and water in the developing solvent is 15:1:1:

2.

3. The thin-layer chromatography identification method for the purgative and menstrual-regulating drug preparation according to claim 1, characterized in that, Take 5-10g of the test sample of the antiseptic and menstrual regulating drug preparation, dissolve it in 10-50ml of methanol, and then separate the solid and liquid. Evaporate the liquid to dryness, dissolve the residue in 20-40ml of water, and then separate the solid and liquid. Extract the liquid with 20-50ml of water-saturated n-butanol 1-3 times each time. Combine the extracts, wash the liquid with 20-50ml of water saturated with n-butanol 1-3 times each time. Evaporate the n-butanol washing liquid to dryness, and redissolve the residue in 1-3ml of methanol. The ratio of parts by weight to parts by volume is g / ml.

4. The thin-layer chromatography identification method for the diaphoretic and menstrual-regulating drug preparation according to claim 3, characterized in that, The method further includes the steps of preparing the gardenia reference medicinal material solution, the licorice reference medicinal material solution, the peony bark reference medicinal material solution, and the white peony reference medicinal material solution according to the preparation process of the test sample solution A.

5. The thin-layer chromatography identification method for the diaphoretic and menstrual-regulating drug preparation according to claim 4, characterized in that, Take 0.5-2g of each of the following reference herbs: Gardenia, Licorice, Moutan Bark, and White Peony. Dissolve each herb in 10-50ml of methanol and separate the solid and liquid. Evaporate the liquid to dryness. Dissolve the residue in 20-40ml of water and separate the solid and liquid. Extract the liquid with 20-50ml of water-saturated n-butanol 1-3 times. Combine the extracts and wash the liquid with 20-50ml of water-saturated n-butanol 1-3 times. Evaporate the n-butanol washing liquid to dryness. Redissolve the residue in 1-3ml of methanol to prepare the corresponding reference herb solutions. The ratio of parts by weight to parts by volume is expressed in g / ml.

6. The thin-layer chromatography identification method for the diaphoretic and menstrual-regulating drug preparation according to claim 5, characterized in that, The paeoniflorin reference solution is prepared by dissolving paeoniflorin reference standard in methanol, with 1-5 mg of paeoniflorin reference standard dissolved in each 1 mL of methanol. The geniposide reference solution is prepared by dissolving geniposide reference standard in methanol, with 1-5 mg of geniposide reference standard dissolved in 1 mL of methanol. The glycyrrhizin reference solution is prepared by dissolving glycyrrhizin reference in methanol, with 1-5 mg of glycyrrhizin reference dissolved in each 1 mL of methanol.

7. The thin-layer chromatography method for identifying the purgative and menstrual-regulating drug preparation according to any one of claims 1-6, characterized in that, The sample volume for test solution A is 3-8 μL; and / or, The sample volume for the gardenia reference solution, licorice reference solution, peony bark reference solution, and white peony reference solution was 5-20 μL; and / or, The spotting volume for geniposide reference solution, glycyrrhizin reference solution, and paeoniflorin reference solution is 3-20 μL.

8. The thin-layer chromatography identification method for the purgative and menstrual-regulating drug preparation according to claim 7, characterized in that, The preparation steps of the vanillin-sulfuric acid ethanol solution include: mixing ethanol and sulfuric acid to obtain a sulfuric acid ethanol solution, wherein the volume ratio of ethanol to sulfuric acid is 9:1, the ethanol is analytical grade ethanol, and the concentration of sulfuric acid is 9.5-10.5%; mixing the sulfuric acid ethanol solution with vanillin, wherein the weight ratio of vanillin to the weight-volume ratio of the sulfuric acid ethanol solution is 1:20, and the weight-volume ratio is g / ml; and / or, The heating temperature is 100-110℃; and / or, The silicone thin film includes a silicone G thin film or a silicone G254 thin film, and / or, During the inspection process, the chromatogram of the test sample was compared with the chromatograms of white peony root reference material, peony bark reference material, gardenia reference material, and licorice reference material.

9. The thin-layer chromatography identification method for the diaphoretic and menstrual-regulating drug preparation according to claim 8, characterized in that, The Xuanyu Tongjing drug preparations include the decoction of Xuanyu Tongjing Decoction reference sample, the concentrated extract of Xuanyu Tongjing Decoction reference sample, the freeze-dried powder of Xuanyu Tongjing Decoction reference sample, or Xuanyu Tongjing Decoction preparations.

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