Thin layer chromatography identification method for Rehmannia root medicinal material and traditional Chinese medicine preparation containing Rehmannia root

Abstract

The invention discloses a thin-layer identification method for Rehmannia root medicinal materials and a Chinese medicine preparation containing Rehmannia root, and relates to the technical field of Chinese medicine quality control. The thin-layer identification method for the Chinese medicine preparation containing Rehmannia root of the present invention is as follows: (1) taking a sample to be tested, adding a solvent, ultrasonic treatment, filtering, evaporating the filtrate to dryness, dissolving the residue in water, extracting with an organic solvent, combining the organic solvent layers, evaporating to dryness, dissolving the residue in water, applying D101 macroporous resin to the aqueous solution, eluting with water until it is nearly colorless, then eluting with an ethanol solution of pH 2-5, collecting the eluate, evaporating to dryness, dissolving the residue in methanol, and obtaining a test solution; (2) taking a control medicinal material of Rehmannia root to prepare a control medicinal material solution; (3) taking the test solution and the control medicinal material solution and spotting them on a thin-layer plate, developing with a developing agent, drying, developing, and inspecting. The thin-layer identification method of the present invention can not only identify a single-ingredient medicinal material of Rehmannia root, but also can better identify a compound Chinese patent medicine preparation, and can detect at least three components other than catalpol in Rehmannia root, with good separation of each component and no interference.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a thin layer identification method for Rehmannia root medicinal materials and a traditional Chinese medicine preparation containing Rehmannia root. Background Art

[0002] Rehmannia glutinosa is the fresh or dried root tuber of Rehmannia glutinosa Libosch., a plant of the Scrophulariaceae family. It is harvested in autumn, the rhizome, fibrous roots, and mud removed, and used fresh or processed. Rehmannia glutinosa is generally divided into fresh and raw varieties. Rehmannia glutinosa has a cooling nature, sweet and bitter flavor, and nourishes yin and the kidneys, replenishes blood, and cools the blood. It is beneficial for those with yin, blood, and kidney deficiency. It also has cardiotonic and diuretic properties, relieves fever and inflammation, promotes blood coagulation, and lowers blood sugar.

[0003] In the prior art, the thin-layer identification method for Rehmannia glutinosa and its traditional Chinese medicine preparations mainly involves taking a sample, dissolving it in a solvent, filtering it, and evaporating it to dryness. After dissolving the filter residue, the sample is eluted with water through a macroporous adsorption resin column, and then eluted with an ethanol solution to prepare a test solution. The test solution, the control herbal solution, and the catalpol standard and reference solution are respectively spotted on the same thin-layer plate, developed with a developer, and color developed (by spraying a developer or irradiating with light). Identification is completed by inspection. Developing agents that have been publicly used include chloroform-methanol, ethyl acetate-methanol-water-glacial acetic acid, ethyl acetate-methanol-formic acid, acetone-ethanol-water, and n-butanol-pyridine-water.

[0004] CN101672834B discloses a quality testing method for a traditional Chinese medicine preparation for treating diabetic retinopathy. In this technical solution, the preparation of a test solution includes: taking 3-5 g of a solid preparation, adding 30-60 ml of ether, ultrasonically treating for 10-40 minutes, filtering, evaporating the filtrate to dryness, and adding 0.5-1.5 ml of methanol to the residue to prepare a test solution; taking another reference medicinal material of Rehmannia glutinosa and preparing a reference medicinal material solution in the same manner; or taking a reference substance of Catalpol and adding methanol to prepare a 0.5 mg / ml solution as a reference solution. Pipette 10 μl of the reference solution and the test solution, spot them separately on the same silica gel G thin layer plate, use chloroform-ethyl acetate-methanol-water = 10-20:30-50:18-25:5-15 or chloroform-methanol-concentrated ammonia = 12-20:3-6:0.5-1.510°C and place the lower layer solution for 12 hours as the developing agent, develop, take out, dry, spray with Goden's solution or anisaldehyde-concentrated sulfuric acid solution, and heat at 105°C until the spots are clearly colored.

[0005] The traditional thin-layer chromatography (TLC) identification of catalpol, a component of Rehmannia root, still has certain limitations. For example, the detection of more than three components in Rehmannia root besides catalpol is poor, with significant interference and poor resolution. For example, the same process used to identify single-ingredient medicinal materials is not well suited for identifying compound preparations of traditional Chinese medicines. This situation makes it impossible to achieve unified, standardized identification from the source of the medicinal material to the final TCM product in production practice. Summary of the Invention

[0006] To address the above shortcomings, the present invention provides a thin-layer chromatography identification method for Rehmannia glutinosa and Chinese medicinal preparations containing Rehmannia glutinosa. This method can not only identify single-ingredient medicinal materials, but also effectively identify compound Chinese patent medicine preparations. It can also detect at least three components in Rehmannia glutinosa in addition to catalpol, with good separation of the components and no interference. The specific technical solution is as follows:

[0007] A thin layer chromatography identification method for Rehmannia glutinosa medicinal materials and traditional Chinese medicine preparations containing Rehmannia glutinosa comprises the following steps:

[0008] (1) Preparation of test solution: Take the sample to be tested, add solvent, ultrasonically treat, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with organic solvent, combine the organic solvent layers, evaporate to dryness, dissolve the residue in water, apply D101 macroporous resin to the aqueous solution, elute with water until it is almost colorless, then elute with ethanol solution, collect the eluate, evaporate to dryness, dissolve the residue in methanol, and obtain the test solution;

[0009] (2) Preparation of control medicinal material solution: Prepare control medicinal material solution by taking Rehmannia root as control medicinal material;

[0010] (3) Pipette the test sample solution and control medicinal material solution, spot them on a thin layer plate, develop them with a developing agent, dry them, develop the color, and inspect them.

[0011] Furthermore, the sample to be tested is a traditional Chinese medicine preparation containing Rehmannia glutinosa.

[0012] Preferably, the Chinese herbal preparation containing Rehmannia root is: Qinghouyan granules, Ganoderma lucidum and longan wine or Mingmu Zishen tablets. Among them, the components of Qinghouyan Granules are: Rehmannia 14.6%; Ophiopogon 13.0%; Scrophularia 21.1%; Forsythia suspensa 25.6%; Scutellaria baicalensis 25.6%; the components of Ganoderma lucidum and Longan Wine are: Ganoderma lucidum 14.3%; Longan meat 7.1%; Processed Polygonatum 14.3%; Codonopsis pilosula 7.1%; Lycium barbarum 7.1%; Honey-roasted Astragalus membranaceus 7.1%; Processed Polygonum multiflorum 14.3%; Chinese yam 3.6%; Angelica sinensis 7.1%; Rehmannia glutinosa 7.1%; Poria 3.6%; Tangerine peel 3.6%; Jujube 3.6%; the components of Mingmu Zishen Tablets are: Rehmannia glutinosa 14.3%; Ligustrum lucidum 21.4%; Lycium barbarum 28.6%; Chrysanthemum 14.3%; Cassia seed 14.3%; Achyranthes bidentata 7.1%.

[0013] Furthermore, the dosage forms of the traditional Chinese medicine preparation containing Rehmannia glutinosa are: granules, tablets, capsules, pills, tinctures, wine preparations, and syrups.

[0014] Furthermore, in step (1), the solvent is a mixture of ethanol and diethyl ether in a volume ratio of 1:2-5.

[0015] Furthermore, in step (1), the ultrasonic power is 300-320W, and the ultrasonic time is 20-40min.

[0016] Furthermore, in step (1), the organic solvent is a water-saturated n-butanol solution.

[0017] Furthermore, in step (1), the mass concentration of the ethanol solution is 10%-50%, and the pH value is 2-5.

[0018] Furthermore, in step (3), the thin layer plate is a polyamide thin layer plate.

[0019] Furthermore, in step (3), the developing agent is methanol-acetic acid-water (1-4:1-3:5-10), and the inspection condition is 365nm fluorescence.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention improves the sample collection method and forms a new thin-layer identification method together with a polyamide thin-layer plate, a developing agent (methanol-acetic acid-water), and 365nm fluorescence. It can not only identify single-ingredient medicinal materials, but also better identify compound preparations of Chinese patent medicines. It can detect at least three components in Rehmannia glutinosa in addition to catalpol, with good separation of each component and no interference. In production practice, it achieves unified and standardized identification from the source of medicinal materials to the final Chinese medicine product.

[0022] 2. The present invention can improve the tailing condition by removing the extraction step in the sample treatment, and the use of an ethanol solution with a pH of 2-5 for elution can increase the number of detected components, and the separation degree of each component is good without interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0024] Figure 1 The thin layer atlas of Rehmannia root in Example 1;

[0025] Figure 2 The thin layer atlas of Rehmannia root in Example 2;

[0026] Figure 3 The thin layer atlas of Rehmannia root in Example 3;

[0027] Figure 4 The thin layer atlas of Rehmannia root in Comparative Example 1;

[0028] Figure 5 The thin layer atlas of Rehmannia root in Comparative Example 2;

[0029] Figure 6 The thin layer atlas of Rehmannia root in Comparative Example 3;

[0030] Figure 7 This is the thin layer atlas of Rehmannia root in Comparative Example 4. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0032] Example 1

[0033] Preparation of the test solution of Rehmannia compound preparation: 14 g of the test sample powder (Mingmu Zishen Tablets: Rehmannia 14.3%; Ligustrum lucidum 21.4%; Lycium barbarum 28.6%; Chrysanthemum 14.3%; Cassia seed 14.3%; Achyranthes bidentata 7.1%) was added to 30 ml of a 1:2 ethanol-ether mixture, ultrasonicated at 300 W for 30 min, filtered, evaporated to dryness, and the residue was dissolved in 20 ml of water. The mixture was extracted twice with 20 ml of saturated n-butanol solution. The n-butanol layers were combined and evaporated to dryness. The residue was dissolved in 10 ml of water and loaded onto a pretreated D101 macroporous resin column (inner diameter 15 mm, column height 3 cm); the mixture was eluted with water until nearly colorless, and then eluted with 50 ml of a 20% ethanol solution at pH 3. The eluate was collected and evaporated to dryness; the residue was dissolved in 1 ml of methanol as the test solution. Different batches of Mingmu Zishen tablets were used to prepare Rehmannia compound preparation test solution 1, Rehmannia compound preparation test solution 2, and Rehmannia compound preparation test solution 3.

[0034] Rehmannia processed product test solution: Take 2 g of Rehmannia processed product and use the same preparation method as the test sample solution to prepare Rehmannia processed product test solution.

[0035] Rehmannia root test solution: Take 2 g of Rehmannia root and prepare the Rehmannia root test solution using the same preparation method as the test solution.

[0036] Preparation of control medicinal material solution: Take 2 g of Rehmannia glutinosa control medicinal material and prepare Rehmannia glutinosa control medicinal material solution using the same preparation method as the test solution.

[0037] Preparation of negative test solution for Rehmannia glutinosa: 12 g of negative sample fine powder of Rehmannia glutinosa (formulation composition: Ligustrum lucidum 21.4%; Lycium barbarum 28.6%; Chrysanthemum Flos 14.3%; Cassia Seed 14.3%; Achyranthes Bidentata 7.1%) was prepared by the same preparation method as the test solution.

[0038] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL of the test solution, reference solution, control medicinal material solution, and negative test solution were respectively spotted on the same polyamide thin layer plate, and methanol-acetic acid-water (3:1:7) was used as the developing agent. The plate was developed, taken out, dried, and observed under 365 fluorescence.

[0039] The thin layer spectrum of this example is as follows Figure 1 As shown in the figure, the chromatogram of the test solution shows 4 spots of the same color at the corresponding positions of the chromatogram of the control medicinal material solution. The spots are well separated, do not interfere with each other, and there is no tailing phenomenon.

[0040] Example 2

[0041] Preparation of test solution: Take 14 g of test sample powder (Qinghouyan Granules: Rehmannia glutinosa 14.6%; Ophiopogon japonicus 13.0%; Scrophularia ningpoensis 21.1%; Forsythia suspensa 25.6%; Scutellaria baicalensis 25.6%), add 30 ml of ethanol-ether mixed solution with a volume ratio of 1:4, ultrasonicate at 320 W power for 20 min, filter, evaporate to dryness, dissolve the residue in 20 ml of water, extract twice with saturated n-butanol solution with water, 20 ml each time, combine the n-butanol layers, evaporate to dryness, dissolve the residue in 10 ml of water, and apply to a pretreated D101 macroporous resin column (inner diameter 15 mm, column height 3 cm); elute with water until almost colorless, then elute with 50 ml of 25% ethanol solution at pH 2, collect the eluate, and evaporate to dryness; dissolve the residue in 1 ml of methanol as the test solution.

[0042] Preparation of control medicinal material solution: Take 2 g of Rehmannia glutinosa control medicinal material and prepare the control medicinal material solution using the same preparation method as the test solution.

[0043] Preparation of negative test solution for Rehmannia glutinosa: 12 g of negative sample powder for Rehmannia glutinosa (formulation composition: Ophiopogon japonicus 13.0%; Scrophularia ningpoensis 21.1%; Forsythia suspensa 25.6%; Scutellaria baicalensis 25.6%) was prepared by the same preparation method as the test solution.

[0044] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL each of the above-mentioned test solution, catalpol reference solution, control medicinal material solution, and negative test solution were taken and spotted on the same polyamide thin layer plate, and methanol-acetic acid-water (1:3:5) was used as the developing agent. The plate was developed, taken out, dried, and observed under 365 fluorescence.

[0045] The thin layer spectrum of this example is as follows Figure 2 As shown in the figure, the chromatogram of the test solution shows 4 spots of the same color at the corresponding positions of the chromatogram of the control medicinal material solution. The spots are well separated, do not interfere with each other, and there is no tailing phenomenon.

[0046] Example 3

[0047] Preparation of test sample solution: 14 g of test sample powder (Ganoderma lucidum longan wine: Ganoderma lucidum 14.3%; Longan pulp 7.1%; Polygonatum sibiricum processed 14.3%; Codonopsis pilosula 7.1%; Lycium barbarum 7.1%; Honey-roasted Astragalus membranaceus 7.1%; Polygonum multiflorum processed 14.3%; Chinese yam 3.6%; Angelica sinensis 7.1%; Rehmannia root 7.1%; Poria cocos 3.6%; Tangerine peel 3.6%; Jujube 3.6%) was added to 30 ml of ethanol-ether mixed solution with a volume ratio of 1:5, and heated at 300 W. Ultrasonicate at low power for 40 minutes, filter, evaporate to dryness, dissolve the residue in 20 ml of water, extract twice with saturated n-butanol solution with water, 20 ml each time, combine the n-butanol layers, evaporate to dryness, dissolve the residue in 10 ml of water, and apply it to a pretreated D101 macroporous resin column (inner diameter 15 mm, column height 3 cm); elute with water until almost colorless, then elute with 50 ml of 35% ethanol solution at pH 5, collect the eluate, and evaporate to dryness; dissolve the residue in 1 ml of methanol as the test solution.

[0048] Preparation of control medicinal material solution: Take 2 g of Rehmannia glutinosa control medicinal material and prepare the control medicinal material solution using the same preparation method as the test solution.

[0049] Preparation of negative test solution for Rehmannia deficiency: 12 g of fine powder of negative sample for Rehmannia deficiency (formulation composition: Ganoderma lucidum 14.3%; Longan pulp 7.1%; Processed Polygonatum 14.3%; Codonopsis pilosula 7.1%; Lycium barbarum 7.1%; Honey-roasted Astragalus membranaceus 7.1%; Processed Polygonum multiflorum 14.3%; Dioscorea batatas 3.6%; Angelica sinensis 7.1%; Poria cocos 3.6%; Tangerine peel 3.6%; Jujube 3.6%) was prepared by the same preparation method as the test solution.

[0050] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL each of the above-mentioned test solution, catalpol reference solution, control medicinal material solution, and Rehmannia glutinosa negative test solution were taken and spotted on the same polyamide thin layer plate, and methanol-acetic acid-water (4:2:10) was used as the developing agent. The plate was developed, taken out, dried, and observed under 365 fluorescence.

[0051] The thin layer spectrum of this example is as follows Figure 3 As shown in the figure, the chromatogram of the test solution shows 4 spots of the same color at the corresponding positions of the chromatogram of the control medicinal material solution. The spots are well separated, do not interfere with each other, and there is no tailing phenomenon.

[0052] Comparative Example 1:

[0053] In the treatment of the test sample in this comparative example, the pH of the eluent used was not 2-5. The specific identification method is as follows:

[0054] Preparation of test solution:

[0055] Take 12 g of the test sample powder (Qinghouyan Granules: Rehmannia glutinosa 14.6%; Ophiopogon japonicus 13.0%; Scrophularia ningpoensis 21.1%; Forsythia suspensa 25.6%; Scutellaria baicalensis 25.6%), add 40 ml of ethanol-ether (1:2) mixed solution, ultrasonicate at 320W for 20 min, filter, evaporate to dryness, dissolve the residue in 20 ml of water, extract twice with water-saturated n-butanol solution, 20 ml each time, combine the n-butanol layers, evaporate to dryness, dissolve the residue in 10 ml of water, and apply it to a pretreated D101 macroporous resin column (inner diameter 8 mm, column height 6 cm); elute with water until almost colorless, then elute with 50 ml of 20% ethanol, collect the eluate, and evaporate to dryness; dissolve the residue in 1 ml of methanol as the test sample solution.

[0056] Control medicinal material solution: 2 g of Rehmannia glutinosa control medicinal material was prepared using the same preparation method as the test solution.

[0057] Negative test solution for Rehmannia deficiency: 10 g of fine powder of negative sample for Rehmannia deficiency (formulation composition: Ophiopogon japonicus 13.0%; Scrophularia ningpoensis 21.1%; Forsythia suspensa 25.6%; Scutellaria baicalensis 25.6%) was prepared by the same preparation method as the test solution.

[0058] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL each of the above-mentioned test solution, catalpol reference solution, control medicinal material solution, and Rehmannia glutinosa negative test solution were taken and spotted on the same polyamide thin layer plate, and methanol-acetic acid-water (2:2:9) was used as the developing agent. The plate was developed, taken out, dried, and observed under 365 fluorescence.

[0059] The thin layer spectrum of this comparative example is as follows Figure 4 As shown in the figure, the chromatogram of the test solution shows two fluorescent spots of the same color at the corresponding position of the chromatogram of the control medicinal material solution, indicating that changing the extraction solvent and adding extraction steps can remove some impurity interference, and the tailing phenomenon of the thin layer chromatogram has improved. However, fewer components were detected in Rehmannia glutinosa, only two spots, and the separation of each component was poor.

[0060] Comparative Example 2:

[0061] In the treatment of the test sample in this comparative example, there is no extraction step, and the specific identification method is as follows:

[0062] Preparation of test solution: Take 28 g of test sample powder (Ganoderma lucidum and longan wine: Ganoderma lucidum 14.3%; Longan meat 7.1%; Processed Polygonatum 14.3%; Codonopsis pilosula 7.1%; Lycium barbarum 7.1%; Honey-roasted Astragalus 7.1%; Processed Polygonum multiflorum 14.3%; Dioscorea batatas 3.6%; Angelica sinensis 7.1%; Rehmannia root glutinosa 7.1%; Poria cocos 3.6%; Tangerine peel 3.6%; Jujube 3.6%), add 40 ml of ethanol-ether (1:2) mixed solution, ultrasonicate at 320 W for 40 min, filter, evaporate to dryness, dissolve the residue with 10 ml of water, and apply it to a pretreated D101 macroporous resin column (inner diameter 10 mm, column height 5 cm); elute with water until almost colorless, then elute with 50 ml of 20% ethanol solution at pH 2, collect the eluate, and evaporate to dryness; dissolve the residue in 1 ml of methanol as the test solution.

[0063] Control medicinal material solution: 2 g of Rehmannia glutinosa control medicinal material was prepared using the same preparation method as the test solution.

[0064] Negative test solution for Rehmannia deficiency: 26 g of fine powder of negative sample for Rehmannia deficiency (formulation composition: Ganoderma lucidum 14.3%; Longan pulp 7.1%; Processed Polygonatum 14.3%; Codonopsis pilosula 7.1%; Lycium barbarum 7.1%; Honey-roasted Astragalus membranaceus 7.1%; Processed Polygonum multiflorum 14.3%; Dioscorea opposita 3.6%; Angelica sinensis 7.1%; Poria cocos 3.6%; Tangerine peel 3.6%; Jujube 3.6%) was prepared. The negative test solution for Rehmannia deficiency was prepared by the same preparation method as the test solution.

[0065] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 1 μL of the above-mentioned test solution, control medicinal material solution, and negative test solution of Rehmannia glutinosa were taken and spotted on the same polyamide thin layer plate, and methanol-acetic acid-water (2:1:8) was used as the developing agent. The plate was developed, taken out, dried, and observed under 365 fluorescence.

[0066] The thin layer spectrum of this comparative example is as follows Figure 5 As shown in the figure, the chromatogram of the test solution shows four fluorescent spots of the same color at the corresponding positions in the chromatogram of the control medicinal material solution. This indicates that adjusting the pH of the eluent is beneficial for elution of the identified components, but the tailing is not improved.

[0067] Comparative Example 3:

[0068] This comparative example adopts the existing existing identification method, and the specific method is as follows:

[0069] Preparation of test solution: Take sample 1 (Qinghouyan granules) and sample 2 (Ganoderma lucidum longan wine) respectively, add 40 ml of ethanol, ultrasonicate at 320W for 40 min, filter, evaporate to dryness, dissolve the residue with 10 ml of water, apply pretreated D101 macroporous resin column (inner diameter 10 mm, column height 5 cm), elute with water until almost colorless, then elute with 50 ml of 30% ethanol solution, collect the eluate, and evaporate to dryness; dissolve the residue in 1 ml of methanol to obtain test solution 1 and test solution 2.

[0070] Control medicinal material solution: 2 g of Rehmannia glutinosa control medicinal material was prepared using the same preparation method as the test solution.

[0071] Negative test solution for Rehmannia glutinosa: 26 g of fine powder of negative sample of Rehmannia glutinosa is prepared by the same preparation method as the test solution.

[0072] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 1 μL each of the above-mentioned test solution, control medicinal material solution, catalpol reference solution, and Rehmannia glutinosa negative test solution were taken and spotted on the same polyamide thin layer plate, and chloroform-methanol (7:3) was used as the developing solvent. After development, it was sprayed with 10% ethanolic sulfuric acid solution and heated at 105°C for color development.

[0073] The thin layer spectrum of this comparative example is as follows Figure 6 As shown in the figure, there is severe tailing, there is no corresponding spot for the target component catalpol, and multiple component spots in Rehmannia glutinosa cannot be detected.

[0074] Comparative Example 4

[0075] This comparative example does not use methanol-acetic acid-water (1-4:1-3:5-10) as a developing solvent, and the specific method is as follows:

[0076] Preparation of the test solution of Rehmannia compound preparation: 14 g of the test sample powder (Mingmu Zishen Tablets: Rehmannia 14.3%; Ligustrum lucidum 21.4%; Lycium barbarum 28.6%; Chrysanthemum 14.3%; Cassia seed 14.3%; Achyranthes bidentata 7.1%) was added to 30 ml of a 1:2 ethanol-ether mixture, ultrasonicated at 320 W for 30 min, filtered, evaporated to dryness, and the residue was dissolved in 20 ml of water. The mixture was extracted twice with 20 ml of saturated n-butanol solution. The n-butanol layers were combined and evaporated to dryness. The residue was dissolved in 10 ml of water and loaded onto a pretreated D101 macroporous resin column (inner diameter 15 mm, column height 3 cm); the mixture was eluted with water until nearly colorless, and then eluted with 50 ml of a 20% ethanol solution at pH 3. The eluate was collected and evaporated to dryness; the residue was dissolved in 1 ml of methanol as the test solution. Different batches of Mingmu Zishen tablets were used to prepare Rehmannia compound preparation test solution 1, Rehmannia compound preparation test solution 2, and Rehmannia compound preparation test solution 3.

[0077] Test solution of processed Rehmannia root product: Take 2 g of processed Rehmannia root product and prepare the test solution of processed Rehmannia root product using the same preparation method as the test solution.

[0078] Rehmannia root test solution: Take 2 g of Rehmannia root and prepare the Rehmannia root test solution using the same preparation method as the test solution.

[0079] Preparation of control medicinal material solution: Take 2 g of Rehmannia glutinosa control medicinal material and prepare Rehmannia glutinosa control medicinal material solution using the same preparation method as the test solution.

[0080] Preparation of negative test solution for Rehmannia glutinosa: 12 g of negative sample fine powder of Rehmannia glutinosa (formulation composition: Ligustrum lucidum 21.4%; Lycium barbarum 28.6%; Chrysanthemum Flos 14.3%; Cassia Seed 14.3%; Achyranthes Bidentata 7.1%) was prepared by the same preparation method as the test solution.

[0081] According to the thin layer chromatography method (General Rules 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), 2 μL of the test solution, reference solution, control medicinal material solution, and negative test solution of Rehmannia glutinosa were taken and spotted on the same silica gel G254 thin layer plate with sodium carboxymethyl cellulose as the adhesive, and ethyl acetate-methanol-formic acid-water (18:3:1.5:1) was used as the developing agent. The plate was developed, taken out, dried, and observed under 254 fluorescence.

[0082] The thin layer spectrum of this comparative example is as follows Figure 7 As shown in the figure, the chromatogram of the test solution shows three spots of the same color at the corresponding positions of the chromatogram of the control medicinal material solution. The separation of each spot is not ideal and they interfere with each other.

[0083] In summary, the present invention improves tailing by adding an extraction step to the sample processing; using an ethanol solution with a pH of 2-5 for elution can increase the number of detected components, and the separation of the components is good without interference; and using methanol-acetic acid-water (1-4:1-3:5-10) as a developing solvent can improve the separation of each spot, making it convenient for application in production practice.

[0084] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A thin layer chromatography identification method for Rehmannia glutinosa and a Chinese medicine preparation containing Rehmannia glutinosa, characterized in that: The steps include: (1) Preparation of test solution: Take the sample to be tested, add a solvent, which is a mixture of ethanol and ether in a volume ratio of 1:2-5, ultrasonically treat, filter, evaporate the filtrate to dryness, dissolve the residue in water, and extract with an organic solvent, which is a water-saturated n-butanol solution. Combine the organic solvent layers, evaporate to dryness, dissolve the residue in water, apply D101 macroporous resin to the aqueous solution, elute with water until it is almost colorless, and then elute with an ethanol solution with a mass concentration of 10%-50% and a pH value of 2-5. Collect the eluate, evaporate to dryness, and dissolve the residue in methanol to obtain the test solution. (2) Preparation of control medicinal material solution: Prepare the control medicinal material solution by taking Rehmannia root as control medicinal material; (3) The test solution and control medicinal material solution are aspirated, spotted on a thin layer plate, developed with a developing agent, dried, developed, and inspected; the developing agent is methanol-acetic acid-water (1-4:1-3:5-10), and the inspection condition is 365nm fluorescence.

2. The thin layer chromatography identification method for Rehmannia glutinosa and Chinese medicinal preparations containing Rehmannia glutinosa according to claim 1, characterized in that: In step (1), the sample to be tested is a traditional Chinese medicine preparation containing Rehmannia root.

3. The thin layer chromatography identification method for Rehmannia glutinosa and Chinese medicinal preparations containing Rehmannia glutinosa according to claim 2, characterized in that: The Chinese medicinal preparation containing Rehmannia root is: Qinghouyan granules, Ganoderma lucidum and longan wine or Mingmu Zishen tablets.

4. The thin layer chromatography identification method for Rehmannia glutinosa and Rehmannia glutinosa-containing Chinese medicine preparations according to claim 2, characterized in that: The dosage forms of the traditional Chinese medicine preparation containing Rehmannia root are granules, tablets, capsules, pills, tinctures, wine preparations and syrups.

5. The thin layer chromatography identification method for Rehmannia glutinosa and Chinese medicinal preparations containing Rehmannia glutinosa according to claim 1, characterized in that: In step (1), the ultrasonic power is 300-320W, and the ultrasonic time is 20-40min.

6. The thin layer chromatography identification method for Rehmannia glutinosa and Chinese medicinal preparations containing Rehmannia glutinosa according to claim 1, characterized in that: In step (3), the thin layer plate is a polyamide thin layer plate.

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