Thin-layer chromatography identification method for rhizoma dioscoreae hypoglaucae formula granules

Abstract

Disclosed is a thin-layer chromatography identification method for Rhizoma dioscoreae hypoglaucae formula granules. The method comprises: taking a sample to be subjected to detection, and dissolving same in a solvent to obtain a test solution, wherein the sample to be subjected to detection involves Smilax glaucochina formula granules or Smilax ferox formula granules; taking resveratrol as a control, and adding a solvent to dissolve same to obtain a control solution; taking a Smilax ferox control medicinal material, and dissolving same in a solvent to obtain a control medicinal material solution; and subjecting the test solution, the control solution and the control medicinal material solution to thin-layer chromatography detection, wherein a thin-layer plate is a silica gel G thin-layer plate, and a developing solvent is toluene-ethyl acetate-formic acid. The sample is placed under an ultraviolet lamp for inspection, and if in the chromatogram of the test sample there are blue fluorescent spots at the position of an Rf value of 0.829 ± 10%, the sample is Smilax ferox, and if there are no blue fluorescent spots at the position of the Rf value of 0.829 ± 10%, the sample is Smilax glaucochina.

Description

Thin layer identification method of Radix Dichroae formula granules

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on July 4, 2023, with application number 202310812079.7 and invention name “Thin layer identification method for Radix Dichroae formula granules”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the technical field of analysis and detection, in particular to a thin layer identification method for Radix Dichroae formula granules. Background Art

[0003] Dihuang (Smilax ferox) is the dried rhizome of Smilax glauco-china Warb or Smilax ferox Wall.ex Kunth, both of the Liliaceae family. It dispels wind and dampness, detoxifies, and reduces swelling. It is used for rheumatic pain, joint discomfort, back pain, sores, carbuncles, skin rash, and dysentery.

[0004] The "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules (Draft for Comment)" published by the State Food and Drug Administration stipulates that the raw medicinal materials used in traditional Chinese medicine formula granules must be of fixed origin. Characteristic identification is an effective method for distinguishing the two. Formula granules are made from medicinal materials through a process of processing, decoction, concentration, drying, mixing, and granulation. Decoction destroys the original properties of the medicinal materials, so property identification methods are not applicable to formula granules.

[0005] Currently, there is no effective method to identify Radix Dichroae (Smilax china) and Radix Dichroae (Smilax china) formula granules, so a method to distinguish Radix Dichroae (Smilax china) and Radix Dichroae (Smilax china) formula granules is needed to be established.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a thin layer identification method for Radix Dichroae formula granules.

[0008] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0009] In this application, the term "and / or" describes the association relationship between associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0010] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0011] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the following processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0012] The present invention aims to establish a thin-layer chromatography method for identifying Radix Dichroae (Smilax china) and Radix Dichroae (Smilax china) formula granules. By observing whether fluorescent spots exist within a certain ratio shift value range in the thin-layer chromatography spectrum of the test sample, the method is used as an identification point for quickly and effectively identifying the Radix Dichroae (Smilax china) and Radix Dichroae (Smilax china) formula granules.

[0013] The present invention provides a thin layer identification method for Radix Dichroae formula granules, comprising:

[0014] A) taking a sample to be tested and dissolving it in a solvent to obtain a test solution; the sample to be tested is a granule of Smilax china or a granule of Smilax china;

[0015] B) taking a resveratrol reference substance and dissolving it in a solvent to obtain a reference substance solution;

[0016] C) taking a control medicinal material of Smilax china and dissolving it in a solvent to obtain a control medicinal material solution;

[0017] D) The test solution, reference solution, and reference medicinal material solution were subjected to thin layer chromatography using a silica gel G thin layer plate and a developing solvent of toluene-ethyl acetate-formic acid;

[0018] E) Observe under ultraviolet light. If a blue fluorescent spot is observed at an Rf value of 0.829±10% in the chromatogram of the sample, it is Smilax china. If no blue fluorescent spot is observed at an Rf value of 0.829±10%, it is Smilax china.

[0019] The thin layer identification method of Radix Dichroae formula granules provided by the present invention first takes a sample to be tested, dissolves it with a solvent, and obtains a test solution.

[0020] The step A) of the present invention specifically comprises: grinding the sample to be tested, ultrasonically extracting with methanol, and filtering to obtain a test solution.

[0021] Specifically, the mass volume ratio of the sample to be tested and methanol is: 1 g: 5-15 mL.

[0022] The ultrasonication time is 10 to 20 minutes.

[0023] A resveratrol reference substance is taken and dissolved in a solvent to obtain a reference substance solution. The mass concentration of the resveratrol reference substance is 0.1-2.0 mg / mL.

[0024] Take a reference medicinal material of Smilax china, dissolve it in a solvent, and obtain a reference medicinal material solution.

[0025] The present invention is preferably specifically:

[0026] Take the reference medicinal material of Smilax china and boil it in water, then add methanol to extract, ultrasonicate and filter to obtain the product;

[0027] Specifically, the mass ratio of the Smilax longituba reference medicinal material, water and methanol is 1 g: 10-20 mL: 5-10 mL.

[0028] The test solution, reference solution and reference medicinal material solution were subjected to thin layer chromatography.

[0029] Preferably, the test solution, reference solution, and reference medicinal material solution are aspirated and spotted on the same silica gel G thin layer plate, developed with a developing agent, removed, dried, and inspected under ultraviolet light. The ultraviolet light wavelength is 365 nm.

[0030] According to the present invention, the developing agent is toluene-ethyl acetate-formic acid;

[0031] In some preferred embodiments of the present invention, the mass ratio of toluene-ethyl acetate-formic acid is 4:3:0.2.

[0032] The thin layer plate described in the present invention is a silica gel G thin layer plate. It can be selected from the tailorable thin layer chromatography plates of Tianjin Silida Technology Co., Ltd., Merck, and prefabricated silica gel G plates from the Qingdao Ocean Chemical Plant branch. Results show that this method is durable and can meet identification requirements. Furthermore, at the ratio shift values ​​(Rf) of 0.885, 0.800, and 0.833, the granules of Radix Dichroae (Smilax china) exhibit fluorescent spots, while the granules of Radix Dichroae (Smilax china) do not exhibit spots, which can be used to distinguish the granules of Radix Dichroae (Smilax china) from the granules of Radix Dichroae (Smilax china) .

[0033] Check under ultraviolet light. If there is a blue fluorescent spot at the Rf value of 0.829±10% in the chromatogram of the test sample, it is Smilax china; if there is no blue fluorescent spot at the Rf value of 0.829±10%, it is Smilax china.

[0034] The Rf value is 0.829±10%, preferably, the Rf value is 0.747-0.885, and specifically can be 0.747, 0.778, 0.766, 0.800, 0.833, 0.856, 0.875, 0.883, or 0.885.

[0035] The sample spotting volume of the thin layer chromatography of the present invention is 5 to 15 μL.

[0036] The inspection temperature of the present invention is 4° C. to 25° C. The method of the present invention has good adaptability to different temperatures, and at the ratio shift values ​​of 0.856 and 0.875, the granules of Radix Dichroae (Smilax china) show fluorescent spots, while the granules of Radix Dichroae (Smilax china) have no spots, which can be used to distinguish the granules of Radix Dichroae (Smilax china) from the granules of Radix Dichroae (Smilax china) .

[0037] The inspection humidity of the present invention is 32% rh to 75% rh. The method of the present invention has good adaptability to different humidity levels, and at the ratio shift values ​​(Rf) of 0.883 and 0.862, the granules of Radix Dichroae (Smilax china) show fluorescent spots, while the granules of Radix Dichroae (Smilax china) do not have spots, which can be used to distinguish the granules of Radix Dichroae (Smilax china) from the granules of Radix Dichroae (Smilax china) .

[0038] The sample to be tested is the black fruit Smilax china formula granules or the longitudinal Smilax china formula granules.

[0039] The invention provides a thin layer chromatography identification method for Radix Dichroae formula granules, comprising: A) taking a sample to be tested, dissolving it with a solvent, and obtaining a test solution; the sample to be tested is the Radix Smilax chinae formula granules or the Radix Smilax chinae longipoi formula granules; B) taking a resveratrol reference substance, adding a solvent and dissolving it, and obtaining a reference substance solution; C) taking a Radix Smilax chinae longipoi reference medicinal material, dissolving it with a solvent, and obtaining a reference medicinal material solution; D) subjecting the test solution, the reference substance solution, and the reference medicinal material solution to thin layer chromatography detection, wherein the thin layer plate is a silica gel G thin layer plate and the developing solvent is toluene-ethyl acetate-formic acid; and E) inspecting under ultraviolet light, and if a blue fluorescent spot is present at an Rf value of 0.829±10% in the chromatogram of the test sample, the sample is Radix Smilax chinae longipoi; and if no blue fluorescent spot is present at an Rf value of 0.829±10%, the sample is Radix Smilax chinae longipoi. The thin-layer chromatography identification method for Radix Dichroae formula granules established by the present invention uses fluorescent spots as identification points by observing whether there are fluorescent spots within a specified ratio shift value range on the thin-layer chromatography spectrum to quickly and effectively identify the Radix Dichroae formula granules.

[0040] The thin layer identification method of the present invention compares the thin layer chromatography of the test sample with the thin layer chromatography of the control medicinal material Smilax glabra. If the spots are consistent with those of the control medicinal material, it is Dixie (Smilax glabra) granules; if there are no green fluorescent spots at the same position as the control medicinal material at the ratio shift value of 0.747 to 0.885, it is Dixie (Smilax glabra) granules.

[0041] The invention discloses the construction and application of a thin layer identification method for Radix Dichroae (Smilax china) granules and Radix Dichroae (Smilax china) granules. The method has good stability, high precision, good reproducibility, is convenient and easy to master. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Spotting quantity investigation;

[0043] Figure 2 Specificity investigation;

[0044] Figure 3 shows the inspection of different thin layer plates - Tianjin Silida;

[0045] Figure 4 Investigation of different thin layer plates - Merck;

[0046] Figure 5 Survey of different thin layer plates - Qingdao Ocean;

[0047] Figure 6 Different temperatures -4°C;

[0048] Figure 7 Different temperatures -25°C;

[0049] Figure 8 Different humidity -32%;

[0050] Figure 9 Different humidity -75%;

[0051] Figure 10 Multi-batch verification;

[0052] Figure 11 is a diagram showing the results of the method of Comparative Example 1;

[0053] Figure 12 is a diagram showing the results of the method of Comparative Example 2;

[0054] FIG13 is a diagram showing the method results of Comparative Example 3 of the present invention;

[0055] FIG14 is a diagram showing the method results of Comparative Example 4 of the present invention. DETAILED DESCRIPTION

[0056] To further illustrate the present invention, the following describes in detail a method for constructing a UPLC characteristic spectrum of a Guanzhong medicinal material, decoction pieces, standard decoction, and formula granules provided by the present invention in combination with examples.

[0057] Heating plate, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel G thin layer plate (Qingdao Ocean Chemical Plant; Tianjin Silida Technology Co., Ltd.; Merck)

[0058] Methanol, toluene, ethyl acetate, and formic acid were all of analytical grade, and water was ultrapure water (prepared in the laboratory).

[0059] Resveratrol (China Food and Drug Inspection Institute, batch number: 111535-201703, content is 99.4%), long-tailed Smilax china control medicinal material (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: ycwkq22022805), Radix Dichroae (Smilax china) formula granules, batch numbers: 2211074, 2211075, 2211076, 2211077, Radix Dichroae (Smilax china) formula granules, batch numbers: 2212082, 2212083, 2212084, 2212085.

[0060] Example 1

[0061] Grind 1g of this product powder, add 10ml of methanol, sonicate for 20 minutes, filter, and use the filtrate as the test solution. Add 1g of the control herb to 20ml of water, boil for half an hour, filter, evaporate to dryness, add 10ml of methanol, sonicate for 20 minutes, filter, and use the filtrate as the control herb solution. Separately, take a resveratrol reference substance and add methanol to prepare a reference solution containing 0.2mg per 1ml.

[0062] According to the thin-layer chromatography method (General Method 0502), 15 μl of each solution was applied to a single silica gel G thin-layer plate. Develop the plate with toluene-ethyl acetate-formic acid (4:3:0.2). Remove the plate, air-dry it, and examine it under ultraviolet light (365 nm). A fluorescent spot of the same color should appear in the chromatogram of the test sample at the corresponding position in the chromatogram of the resveratrol reference.

[0063] Example 2 Methodological Investigation

[0064] 2.1 Sampling quantity inspection

[0065] Under the experimental conditions described above, 5μl, 10μl, and 15μl of the resveratrol reference solution and the Smilax china test solution, respectively, were spotted on the same silica gel G thin-layer plate. The results are shown in Figure 1. Ideal results were achieved when 5-15μl of the control solution and 5-15μl of the test solution were spotted. Furthermore, at a relative shift (Rf) of 0.747, the Dixie (Smilax china) granules exhibited a fluorescent spot, while the Dixie (Smilax china niger) granules did not, distinguishing the Dixie (Smilax china niger) granules from the Dixie (Smilax china niger) granules.

[0066] Figure 1. Sample size study. Note: 1-3 are resveratrol, with sample sizes of 5ul, 10ul, and 15ul, respectively; 4-6 are Radix Dichroae (Smilax china) control herbs, with sample sizes of 5ul, 10ul, and 15ul, respectively; 7-9 are Radix Dichroae (Smilax china) granules (2211074), with sample sizes of 5ul, 10ul, and 15ul, respectively; 10-12 are Radix Dichroae (Smilax china) granules (2212082), with sample sizes of 5ul, 10ul, and 15ul, respectively.

[0067] 2.2 Specificity Investigation

[0068] Following the above sample preparation method, resveratrol reference solution, reference medicinal material solution, Dixie (Smilax longituba) granule test solution, Dixie (Smilax nigra) granule test solution, and negative solution were prepared. Thin layer chromatography identification experiments were performed. The results are shown in Figure 2. The negative sample did not interfere with resveratrol, Dixie (Smilax longituba) granule test solution, or Dixie (Smilax nigra) granule test solution, indicating good specificity of the method. Furthermore, at a ratio shift (Rf) of 0.788, Dixie (Smilax longituba) granules exhibited a fluorescent spot, while Dixie (Smilax nigra) granules showed no spot, which was used to distinguish Dixie (Smilax longituba) granules from Dixie (Smilax nigra) granules.

[0069] Figure 2. Specificity investigation. Note: 1 is a negative control; 2 is resveratrol; 3 is a control medicinal material, Smilax longituba; 4 is Dixie (Smilax longituba) granules (2211074); 5 is Dixie (Smilax nigra) granules (2212082).

[0070] 2.3 Durability Investigation

[0071] 2.3.1 Comparison of different thin layer plates

[0072] Tailorable thin-layer chromatography plates from Tianjin Silida Technology Co., Ltd., and prefabricated silica gel G plates from Merck and Qingdao Ocean Chemical Plant were tested according to the proposed test method (see Figures 3-5). The results demonstrated the robustness of the method and met the identification requirements. Furthermore, fluorescent spots were observed at Rf values ​​(Rf) of 0.885, 0.800, and 0.833 in Radix Dichroae (Smilax longituba) granules, while no spots were observed in Radix Dichroae (Smilax nigra) granules, enabling differentiation between Radix Dichroae (Smilax longituba) and Radix Dichroae (Smilax nigra) granules.

[0073] The results are shown in Figure 3. Figure 3 shows different thin-layer plate tests - Tianjin Silida. Note: 1 is resveratrol; 2 is the control herbal medicine, Smilax longituba; 3 is Dixie (Smilax longituba) granules (2211074); 4 is Dixie (Smilax nigra) granules (2212082). Figure 4 shows different thin-layer plate tests - Merck. Note: 1 is resveratrol; 2 is the control herbal medicine, Smilax longituba; 3 is Dixie (Smilax longituba) granules (2211074); 4 is Dixie (Smilax nigra) granules (2212082). Figure 5 Investigation of different thin layer plates - Qingdao Ocean, Note: 1 is resveratrol; 2 is the control medicinal material of Smilax glabra; 3 is Dixie (Smilax glabra) granules (2211074); 4 is Dixie (Smilax glabra) granules (2212082).

[0074] 2.3.2 Comparison of different temperatures

[0075] After spotting, the thin-layer plates were developed at both low temperatures (4°C) and room temperatures (25°C). Figures 6 and 7 demonstrate the robustness of this method across different temperatures. Fluorescent spots were observed at ratio shifts of 0.856 and 0.875 in the Dixie (Smilax longituba) granules, while no spots were observed in the Dixie (Smilax nigra) granules. This serves to distinguish the Dixie (Smilax longituba) granules from the Dixie (Smilax nigra) granules. Figure 6: Different temperatures (-4°C). Note: 1: Resveratrol; 2: Smilax longituba control medicinal material; 3: Dixie (Smilax longituba) granules (2211074); 4: Dixie (Smilax nigra) granules (2212082). Figure 7 Different temperatures -25℃, Note: 11 is resveratrol; 2 is the control medicinal material of Smilax china; 3 is Dixie (Smilax china) granules (2211074); 4 is Dixie (Smilax china) granules (2212082).

[0076] 2.3.3 Comparison of different humidity levels

[0077] After spotting, thin-layer plates were developed at 32% and 75% humidity, respectively. Figures 8 and 9 show that this method has good adaptability to different humidity levels. Furthermore, at relative shift values ​​(Rf) of 0.883 and 0.862, granules of Dixie (Smilax longituba) exhibit fluorescent spots, while granules of Dixie (Smilax nigra) do not, which can be used to distinguish granules of Dixie (Smilax longituba) from those of Dixie (Smilax nigra).

[0078] Figure 8 Different humidity -32%, Note: 1 is resveratrol; 2 is the control medicinal material of Smilax china; 3 is Dixie (Smilax china) granules (2211074); 4 is Dixie (Smilax china) granules (2212082).

[0079] Figure 9 shows different humidity levels -75%, Note: 1 is resveratrol; 2 is the control medicinal material of Smilax longituba; 3 is Radix Dichroae (Smilax longituba) granules (2211074); 4 is Radix Dichroae (Smilax nigra).

[0080] 2.3.4 Verification

[0081] Four batches of Smilax longituba and four batches of Smilax nigra were tested for TLC. The results are shown in Figure 10. The results demonstrate that the method developed can accurately distinguish between Dixie (Smilax longituba) granules and Dixie (Smilax nigra) granules. The ratio shift (Rf) was 0.776. Dixie (Smilax longituba) granules had a distinct blue spot, which could be used to distinguish the two.

[0082] Figure 10: Multi-batch validation. Note: 1 is resveratrol; 2 is the control herb Smilax longituba; 3-6 are Dixie (Smilax longituba) granules (2211074, 2211075, 2211076, 2211077); 7-10 are Dixie (Smilax nigra) granules (2212082, 2212083, 2212084, 2212085).

[0083] 2.4 Determination of the identification point Rf of Radix Dichroae (Smilax china) formula granules and Radix Dichroae (Smilax china) formula granules.

[0084] The data of various ratio shift values ​​in the thin layer chromatography methodology investigation are summarized and the results are shown in Table 1. Based on the summary results, it is determined that the ratio shift value (Rf) of the identification spot of Bixie (Smilax longituba) granules and Bixie (Smilax nigra) granules should be within 10%, and the specified value is 0.829.

[0085] Table 1 Summary of methodological investigation ratios

[0086] Comparative Example 1

[0087] Take 2g of this product granules, add 50ml of methanol, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 25ml of water, wash with 25ml of ether, discard the ether solution, add 2ml of hydrochloric acid to the aqueous solution, heat and reflux for 1.5 hours, cool, and extract twice with 25ml of ether by shaking. Combine the ether solutions, evaporate to dryness, and dissolve the residue in 1ml of chloroform to prepare the test solution. Take 5g of Radix Dichroae officinalis, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, and add 50ml of methanol to the residue to prepare the medicinal material control solution in the same way. According to the "Thin Layer Chromatography Operating Procedure" (XLS-09-OP-2897), 5 μl of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate, and chloroform-acetone (9:1) was used as the developing agent. Pre-equilibration was carried out for 30 minutes, and the plate was developed. The plate was taken out and dried in air, and sprayed with 10% phosphomolybdic acid ethanol solution. The plate was heated at 105°C until the spots were clearly colored. The plate was inspected under sunlight. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the control medicinal material.

[0088] Figure 11 shows the results of the method used in Comparative Example 1. Note: 1 and 3 are control solutions of Smilax longituba; 2 is a solution of Radix Dichroae (Smilax longituba) granules; 4 is a solution of Radix Dichroae (Smilax nigra) granules. The results show that using the thin-layer chromatography identification method described above, Radix Dichroae (Smilax nigra) and Radix Dichroae (Smilax longituba) granules do not exhibit obvious fluorescent spots, making them indistinguishable.

[0089] Comparative Example 2

[0090] Add 1g of this powder to 10ml of n-butanol, sonicate for 20 minutes, and filter. The filtrate is used as the test solution. According to the thin-layer chromatography method (General Method 0502), 15μl of each solution is applied to the same silica gel G thin-layer plate. Develop with toluene-ethyl acetate-formic acid (4:3:0.2). Remove, air-dry, and examine under ultraviolet light (365nm). The test sample chromatogram should show a fluorescent spot of the same color at the corresponding position in the control chromatogram.

[0091] Figure 12 is a graph showing the results of the method of Comparative Example 2. Note: 1 and 3 are control medicinal material solutions of Smilax china; 2 is a solution of Radix Dichroae (Smilax china) granules; 4 is a solution of Radix Dichroae (Smilax china) granules.

[0092] The results showed that using the above-mentioned thin layer identification method, there was no obvious difference between the Radix Dichroae (Smilax china) granules and the Radix Dichroae (Smilax china) granules, and the two could not be distinguished.

[0093] Comparative Example 3

[0094] Take 1g of this product powder, grind it finely, add 10ml of methanol, sonicate for 20 minutes, filter, and use the filtrate as the test solution. Take 1g of the control herb, add 20ml of water, decoct for half an hour, filter, evaporate to dryness, add 10ml of methanol, sonicate for 20 minutes, filter, and use the filtrate as the control herb solution.

[0095] According to the thin-layer chromatography method (General Method 0502), 15 μl of each solution was applied to a single silica gel G thin-layer plate. Develop the plate with petroleum ether-ethyl acetate (17:3) as the developing solvent. Remove the plate, air-dry it, and examine it under ultraviolet light (365 nm). Fluorescent spots of the same color should appear at the corresponding positions on the chromatogram of the test sample and the control.

[0096] Figure 13 is a graph showing the results of the method of Comparative Example 3 of the present invention, wherein 1 and 3 are control medicinal material solutions of Smilax china; 2 is a solution of Radix Dichroae (Smilax china) granules; and 4 is a solution of Radix Dichroae (Smilax china) granules.

[0097] The results showed that using the above-mentioned thin layer identification method, there was no obvious difference in the spots of Dixie (Smilax china) granules and Dixie (Smilax china) granules, and the two could not be identified.

[0098] Comparative Example 4

[0099] Take 1g of this product powder, grind it finely, add 10ml of methanol, sonicate for 20 minutes, filter, and use the filtrate as the test solution. Take 1g of the control herb, add 20ml of water, decoct for half an hour, filter, evaporate to dryness, add 10ml of methanol, sonicate for 20 minutes, filter, and use the filtrate as the control herb solution.

[0100] According to the thin-layer chromatography method (General Method 0502), 15 μl of each solution was applied to a single silica gel G thin-layer plate. Develop the plate using chloroform-acetone (10:0.3) as the developing solvent. Remove the plate, allow it to air dry, and examine it under ultraviolet light (365 nm). Fluorescent spots of the same color should appear at the corresponding positions on the chromatogram of the test sample and the control.

[0101] Figure 14 is a graph showing the results of the method of Comparative Example 4 of the present invention. 1 and 3 are control medicinal material solutions of Smilax china; 2 is a solution of Radix Dichroae (Smilax china) granules; 4 is a solution of Radix Dichroae (Smilax china) granules.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A thin layer identification method for Radix Dichroae formula granules, comprising: A) Take the sample to be tested, dissolve it in solvent, and obtain the test solution; The sample to be tested is a granule of Smilax china or a granule of Smilax china; B) taking a resveratrol reference substance, adding a solvent to dissolve it, and obtaining a reference substance solution; C) taking a control medicinal material of Smilax china, dissolving it with a solvent to obtain a control medicinal material solution; D) The test solution, the reference solution and the reference medicinal material solution are subjected to thin layer chromatography, the thin layer plate is a silica gel G thin layer plate, and the developing solvent is toluene-ethyl acetate-formic acid; E) Check under ultraviolet light. If there is a blue fluorescent spot at the Rf value of 0.829±10% in the chromatogram of the test sample, it is Smilax glabra. If there is no blue fluorescent spot at the Rf value of 0.829±10%, it is Smilax glabra.

2. The method according to claim 1, characterized in that The step A) specifically comprises: adding methanol to the sample to be tested, and ultrasonicating to obtain a test solution; the ultrasonication time is 10 to 20 minutes; The mass volume ratio of the sample to be tested and methanol is: 1g:5-15mL.

3. The method according to claim 1, characterized in that Step B) The solvent is methanol; the mass concentration of the resveratrol reference substance is 0.1-2.0 mg / mL.

4. The method according to claim 1, characterized in that: The step C) is specifically: Take the reference medicinal material of Smilax china and boil it in water, then add methanol to extract, ultrasonicate and filter to obtain; The mass ratio of the Smilax china reference medicinal material, water and methanol is 1 g: 10-20 mL: 5-10 mL.

5. The method according to claim 1, characterized in that The sample spotting volume of the thin layer chromatography is 5 to 15 μL.

6. The method according to claim 1, characterized in that Step D) The mass ratio of toluene-ethyl acetate-formic acid is 4:3:0.

2.

7. The method according to claim 1, characterized in that The wavelength of the ultraviolet light in step E) is 365 nm.

8. The method according to claim 1, characterized in that Step E) Check the temperature to be between 4°C and 25°C.

9. The method according to claim 8, characterized in that Step E) The humidity is checked to be 32% rh to 75% rh.

10. The method according to claim 1, characterized in that The Rf value is 0.747 to 0.885.

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