Thin-layer chromatography method for identifying lotus root nodes and lotus root node charcoal formulation granules

Abstract

This invention provides a thin-layer chromatography (TLC) identification method for lotus root nodes and charred lotus root node formulation granules, comprising: A) pretreating lotus root node formulation granules to obtain a lotus root node test solution; pretreating lotus root node charred formulation granules to obtain a lotus root node charred test solution; B) taking lotus root node reference material, extracting by acid reflux, then extracting with chloroform, evaporating to dryness, dissolving the residue in methanol to obtain a reference material solution; C) performing TLC detection on the lotus root node test solution, the lotus root node charred test solution, and the reference material solution; D) examining under ultraviolet light. The TLC identification method for lotus root nodes and charred lotus root node formulation granules established in this invention is used for rapid, accurate, and effective identification of lotus root nodes and charred lotus root node formulation granules. Simultaneously, observing whether there are fluorescent spots in the TLC chromatogram within a certain ratio shift range serves as identification points to distinguish between lotus root nodes and charred lotus root node formulation granules.

Description

Technical Field

[0001] This invention relates to the field of drug analysis and detection technology, and in particular to a thin-layer chromatography method for identifying lotus root nodes and charred lotus root node granules. Background Technology

[0002] Lotus root nodes are the dried rhizome nodes of the lotus plant (Nelumbo nucifera Gaertn.), belonging to the Nymphaeaceae family. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), they are listed as a superior-grade herb. They are sweet and astringent in taste, and neutral in nature, possessing astringent, hemostatic, and blood-stasis-removing properties. They are used for hematemesis, hemoptysis, epistaxis, hematuria, and metrorrhagia. Modern clinical applications of lotus root nodes include both raw and charred forms. Charring weakens their blood-stasis-removing effect while enhancing their astringent properties, thus strengthening their hemostatic effect. Therefore, charred lotus root nodes are now more commonly used in medicine.

[0003] Current modern research on the charring of lotus root nodes is limited, mainly focusing on the hemostatic mechanism of the active components in charred lotus root nodes, processing techniques, and changes in the appearance and chemical composition of different processed charred lotus root nodes. For example, Zhang Shuo et al. found that lotus root nodes contain phenolic acids and tannins. Among them, 3-epiocarbamate acid and tannins are generally considered to be the main active components in lotus root nodes, but their composition changes after charring. Taking Liu Shanxin's research as an example, the characteristic peaks, extract content, and 3-epiocarbamate acid content in the characteristic spectrum of lotus root nodes changed to varying degrees after charring to different degrees, which can be used to identify charred lotus root nodes of different degrees. Hong Aixian et al. measured the tannic acid content in charred lotus root nodes before and after processing, and the results showed that the content decreased significantly after charring. However, because the component content of Chinese medicinal materials is affected by factors such as place of origin, growth years, harvesting time, and storage, the component content is not suitable for the qualitative identification of lotus root nodes and charred lotus root node granules. Furthermore, lotus root charcoal granules are made from lotus root charcoal slices through a series of processes, some of which can damage the original physical and microscopic characteristics. Physical and microscopic identification methods are also unsuitable for the identification of extracts and preparations. Therefore, to ensure accurate clinical medication, it is necessary to establish a visual identification method for qualitative differentiation between the two.

[0004] Therefore, in summary, there is currently no effective method to distinguish between lotus root nodes and charred lotus root nodes in pharmaceutical preparations. To ensure accurate clinical medication, it is necessary to establish a method for qualitatively differentiating between lotus root nodes and charred lotus root node formulation granules. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a thin-layer chromatography method for identifying lotus root nodes and lotus root node charcoal formulation particles.

[0006] The terms “comprising,” “including,” and “having” are used interchangeably in this document to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” in this document also provides for schemes “consisting of…”.

[0007] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0008] 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 refer to any combination of these items, including any combination of single or multiple items.

[0009] It should be understood that in the various embodiments of this application, the sequence number of each process below does not imply the order of execution. Some or all steps may 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 this application.

[0010] This invention discloses the construction and application of a thin-layer chromatography (TLC) method for the identification of lotus root nodes and charred lotus root node formulation granules. This TLC method is highly specific, stable, precise, convenient, and easy to master. In this invention, the TLC identification method compares the TLC chromatograms of charred lotus root node formulation granules with those of lotus root node reference material and the TLC chromatograms of lotus root node formulation granules. If the ratio shift value is within the same range of 0.486–0.602, a green fluorescent spot must appear at the same position, which is used as the identification point for the charred lotus root node formulation granules. This TLC method can rapidly and effectively identify charred lotus root node formulation granules that have lost their medicinal slice form. It is simple to operate, highly precise and sensitive, and stable, showing good application prospects and ensuring accurate medication.

[0011] This invention provides a thin-layer chromatography method for identifying lotus root nodes and lotus root node charcoal formulation particles, comprising:

[0012] A) Take lotus root section formula granules for pretreatment to obtain lotus root section test sample solution;

[0013] The lotus root section charcoal formulation granules were pretreated to obtain a lotus root section charcoal test sample solution;

[0014] B) Take lotus root nodes as reference material, add acid and reflux to extract, then extract with chloroform, evaporate to dryness, dissolve the residue in methanol to obtain the reference material solution;

[0015] C) Thin-layer chromatography was performed on the lotus root node test solution and the lotus root node char test solution as a reference medicinal material solution. The thin-layer plate was a silica gel G thin-layer plate; the developing solvent was diethyl ether-chloroform-methanol.

[0016] D) Examine under a UV lamp. If the lotus root sample and the control medicinal material solution have fluorescent spots of the same color at the same Rf value, and no fluorescent spots at Rf = 0.486 to 0.602, then it is a lotus root formula granule; if there are fluorescent spots at Rf = 0.486 to 0.602, then it is a lotus root charcoal formula granule.

[0017] This invention provides lotus root nodes and lotus root node charcoal formulation granules. First, the lotus root node formulation granules are pretreated to obtain a lotus root node test sample solution.

[0018] The specific pretreatment of lotus root section formula particles described in this invention is as follows:

[0019] The lotus root granules were extracted by reflux with 10% hydrochloric acid, then extracted with chloroform, evaporated to dryness, and the residue was dissolved in 1 mL of methanol to obtain the lotus root test solution.

[0020] The chloroform extraction in this invention is performed 2 to 3 times; preferably 3 times.

[0021] According to the present invention, the mass-to-volume ratio of the lotus root formula granules, 10% hydrochloric acid, and chloroform is 1g:20mL:60mL; the reflux extraction time is 20-30min; preferably 30min.

[0022] The pretreatment of lotus root charcoal formulation particles according to the present invention specifically includes:

[0023] The lotus root charcoal formulation granules were extracted by reflux with 10% hydrochloric acid, followed by extraction with chloroform, evaporated to dryness, and the residue was dissolved in 1 mL of methanol to obtain the lotus root charcoal test solution.

[0024] The chloroform extraction in this invention is performed 2 to 3 times; preferably 3 times.

[0025] According to the present invention, the mass-to-volume ratio of the lotus root charcoal granules, 10% hydrochloric acid, and chloroform is 1g:20mL:60mL; the reflux extraction time is 20-30min; preferably 30min.

[0026] Take lotus root nodes as a reference material, reflux extract with acid, then extract with chloroform, evaporate to dryness, dissolve the residue in methanol to obtain a reference material solution; the number of chloroform extractions in this invention is 2 to 3 times; preferably 3 times.

[0027] According to the present invention, the mass-to-volume ratio of the lotus root reference material, 10% hydrochloric acid, and chloroform is 1g:20mL:60mL; the reflux extraction time is 20-30min; preferably 30min.

[0028] The extraction method described in this invention is preferably reflux extraction.

[0029] Through investigation of extraction methods, this invention found that only the extraction method of this invention can effectively distinguish between lotus root joint formula granules and lotus root joint charcoal formula granules.

[0030] Thin-layer chromatography was performed on the lotus root node test solution, the lotus root node char test solution, and the reference medicinal material solution. The thin-layer plate was a silica gel G thin-layer plate.

[0031] In some embodiments of the present invention, the silicone G thin-layer plate is a prefabricated silicone G plate from Tianjin Slida, Merck, or Qingdao Marine. Results show that the method has good durability and can meet the identification requirements.

[0032] In some embodiments of the present invention, the developing agent is diethyl ether-chloroform-methanol.

[0033] In some embodiments of the present invention, the mass ratio of diethyl ether-chloroform-methanol is 10:6:1.

[0034] The present invention investigated different developing agents and found that only the developing agent of the present invention can produce clear spots and can effectively distinguish between lotus root joint formula particles and lotus root joint charcoal formula particles.

[0035] When examined under a UV lamp, if the lotus root sample and the control medicinal material solution have fluorescent spots of the same color at the same Rf value, and no fluorescent spots at Rf = 0.486 to 0.602, then it is a lotus root formula granule; if there are fluorescent spots at Rf = 0.486 to 0.602, then it is a lotus root charcoal formula granule.

[0036] The ultraviolet lamp described in this invention is 365nm.

[0037] In some embodiments of the present invention, that is:

[0038] According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), the above-mentioned test solution and reference medicinal material solution were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using ether-chloroform-methanol (10:6:1) as the developing solvent. After development, the plate was removed, dried, and examined under ultraviolet light (365nm). If the test solution and reference medicinal material solution showed fluorescent spots of the same color at equal Rf values, and no fluorescent spots were observed at Rf = 0.486–0.602, then the plate was a lotus root granule formulation. If fluorescent spots were observed at Rf = 0.486–0.602, then the plate was a charred lotus root granule formulation.

[0039] In a preferred embodiment of the present invention, the amount of the thin-layer chromatographic test sample is 2 to 6 μL, specifically 2 μL, 3 μL, 4 μL, 5 μL, or 6 μL; the amount of the reference medicinal material solution is 3 to 7 μL, specifically 3 μL, 4 μL, 5 μL, 6 μL, or 7 μL.

[0040] The present invention investigated the sampling amount and found that when 3-7 μl of the control herbal solution and 2-6 μl of the test sample solution were used, the spots were clearly visible and the separation was good. Therefore, the sampling amount of the control herbal solution was determined to be 3-7 μl and the sampling amount of the test sample solution was 2-6 μl.

[0041] In one specific embodiment, the Rf value is 0.529, 0.532, 0.533, 0.537, 0.538, 0.548, 0.552, 0.564, or 0.571.

[0042] The inspection temperature of this invention is 5℃ to 35℃; specifically, it can be 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, or 35℃.

[0043] This method is durable at different temperatures. Compared with the control medicinal material spectrum, the test sample spectrum and the control medicinal material spectrum show spots of the same color at corresponding positions.

[0044] The humidity level to be inspected in this invention ranges from 32% RH to 75% RH. Specifically, it can be: 32% RH, 33% RH, 34% RH, 35% RH, 36% RH, 37% RH, 38% RH, 39% RH, 40% RH, 41% RH, 42% RH, 43% RH, 44% RH, 45% RH, 46% RH, 47% RH, 48% RH, 49% RH, 50% RH, 51% RH, 52% RH, and 53% RH. rh, 54% rh, 55% rh, 56% rh, 57% rh, 58% rh, 59% rh, 60% rh, 61% rh, 62% rh, 63% rh, 64% r h, 65% rh, 66% rh, 67% rh, 68% rh, 69% rh, 70% rh, 71% rh, 72% rh, 73% rh, 74% rh, 75% rh.

[0045] This method is durable under varying humidity levels. Compared with the control medicinal material spectrum, the test sample spectrum and the control medicinal material spectrum show spots of the same color at corresponding positions.

[0046] This invention provides a thin-layer chromatography method for identifying lotus root nodes and charred lotus root node formulation granules, comprising: A) pre-treating lotus root node formulation granules to obtain a lotus root node test solution; pre-treating lotus root node charred formulation granules to obtain a lotus root node charred test solution; B) taking lotus root node reference material, extracting by acid reflux, then extracting with chloroform, evaporating to dryness, dissolving the residue in methanol to obtain a reference material solution; C) performing thin-layer chromatography detection on the lotus root node test solution, the lotus root node charred test solution, and the reference material solution, using a silica gel G thin-layer plate; the developing solvent is diethyl ether-chloroform-methanol; D) examining under ultraviolet light; if the lotus root node test solution and the reference material solution have fluorescent spots of the same color at equal Rf values, and no fluorescent spots at Rf = 0.486–0.602, then it is a lotus root node formulation granule; if there are fluorescent spots at Rf = 0.486–0.602, then it is a lotus root node charred formulation granule. This invention establishes a thin-layer chromatography method for the identification of lotus root nodes and charred lotus root node formulation particles, which is used for rapid, accurate, and effective identification of lotus root nodes and charred lotus root node formulation particles. Simultaneously, the presence of fluorescent spots within a certain ratio shift range in the thin-layer chromatogram is observed as identification points to distinguish between lotus root nodes and charred lotus root node formulation particles. Attached Figure Description

[0047] Figure 1 Identification results of lotus root nodes and lotus root node charcoal formulation particles by different preparation methods of test samples

[0048] Figure 2 Identification results of lotus root nodes and lotus root node charcoal granules using developing agent 1;

[0049] Figure 3 Identification results of lotus root nodes and lotus root node charcoal granules using developing agent 2;

[0050] Figure 4 Identification results of lotus root nodes and lotus root node charcoal granules using developing agent 3;

[0051] Figure 5 Sample size analysis;

[0052] Figure 6 Specificity assessment;

[0053] Figure 7 Investigation of Different Thin-Layer Plates - Tianjin Silida

[0054] Figure 8 Investigation of different thin-layer plates - Merck, Germany;

[0055] Figure 9 Investigation of different thin-layer plates - Qingdao Ocean;

[0056] Figure 10 Different temperatures -4℃;

[0057] Figure 11 Different temperatures -35℃;

[0058] Figure 12 Different humidity levels -32%;

[0059] Figure 13 Different humidity levels - 75%;

[0060] Figure 14 Multiple batch verifications;

[0061] Figure 15 The results of the identification of lotus root nodes and lotus root node charcoal granules by the pharmacopoeia identification method (1);

[0062] Figure 16 The results of the identification of lotus root nodes and lotus root node charcoal granules by the pharmacopoeia identification method (2);

[0063] Figure 17 The results of identification of lotus root nodes and lotus root node charcoal granules using Beijing's methods;

[0064] Figure 18 The identification results of lotus root nodes and lotus root node charcoal granules using methods from Anhui Province;

[0065] Figure 19 Comparative Example 4: Identification results of lotus root nodes and lotus root node charcoal granules;

[0066] Figure 20 Comparative Example 5: Identification results of lotus root nodes and lotus root node charcoal granules;

[0067] Figure 21 The identification results of lotus root nodes and lotus root node charcoal formulation particles in Comparative Example 6. Detailed Implementation

[0068] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a thin-layer chromatography method for identifying lotus root nodes and lotus root node charcoal formulation particles provided by the present invention.

[0069] Instruments: Heating plate, mortar and pestle, semi-automatic thin-layer sample spotting instrument: CAMAG Lionmat-5; thin-layer imaging system: CAMAGTLC Visualizer, ultrapure water system; silica gel G thin-layer plates (Qingdao Hailang Silica Gel Desiccant Co., Ltd., batch number: 20240801; Tianjin Silida Technology Co., Ltd., batch number: 240716; Merck, Germany, batch number: HX87183353; Qingdao Haiyang Chemical Co., Ltd., batch number: 20240504)

[0070] Reagents: Methanol, n-hexane, hydrochloric acid, chloroform, diethyl ether, toluene, ethyl acetate, formic acid, n-butanol, glacial acetic acid, dichloromethane, etc. were all of analytical grade, and water was ultrapure water.

[0071] Test drugs: Lotus root node reference material (China National Institutes for Food and Drug Control, batch number: 121580-201302); Lotus root node charcoal granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch number: OJT-1); Lotus root node formula granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch number: OJ-1)

[0072] Example 1

[0073] 1.1 Examination of Extraction Methods

[0074] 1. Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them into a fine powder, add 10ml of methanol and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5ml of water to dissolve the residue, and use it as the test solution. Separately, take 2g of lotus root node reference material, add 50ml of methanol, reflux for 30 minutes, filter, and use it as the reference material solution.

[0075] 2. Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them finely, add 10ml of water to dissolve them, add water-saturated hexane and shake to extract three times, 10ml each time. Combine the hexane extracts, evaporate to dryness, add 5ml of hexane to the residue, sonicate for 30 minutes, filter, and use as the test solution. Separately, take 1g of lotus root node reference material, add 100ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to dissolve the residue, and prepare the reference material solution in the same way as in "Preparation of Test Solution".

[0076] 3. Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them finely, add 20ml of 10% hydrochloric acid and reflux for 30 minutes, then extract three times with 20ml of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 2g of lotus root node reference material, add 100ml of water, heat under reflux for 30 minutes, filter, and evaporate the filtrate to dryness. Starting from "extracting with 20ml of 10% hydrochloric acid under reflux for 30 minutes", prepare the reference material solution using the same method.

[0077] The thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was followed. 2 μl of the test solution and 3 μl of the reference herb solution were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using ether-chloroform-methanol (10:6:1) as the developing solvent. The plate was then removed, air-dried, and examined under ultraviolet light (365 nm). Results are shown below. Figure 1 .

[0078] The results showed that the test samples prepared by methods 1 and 2 could not effectively distinguish between lotus root joint formula granules and lotus root joint charcoal formula granules. The test sample preparation method was determined as follows: take 1g each of lotus root joint and lotus root joint charcoal formula granules, grind them finely, add 20ml of 10% hydrochloric acid and reflux for 30 minutes, then extract with 20ml of chloroform three times by shaking, combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test sample solution.

[0079] Figure 1 The identification results of different test sample preparation methods on lotus root nodes and lotus root node charcoal formulation granules are as follows: 1-3 are: Method 1 control medicinal material, Method 1 lotus root node formulation granules, and Method 1 lotus root node charcoal formulation granules, respectively; 4-6 are: Method 2 control medicinal material, Method 2 lotus root node formulation granules, and Method 2 lotus root node charcoal formulation granules, respectively; 7-9 are: Method 3 control medicinal material, Method 3 lotus root node formulation granules, and Method 3 lotus root node charcoal formulation granules, respectively.

[0080] Example 2

[0081] 2.3.2 Development of the developing solvent

[0082] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them finely, add 20ml of 10% hydrochloric acid and reflux for 30 minutes, then extract three times with 20ml of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 2g of lotus root node reference material, add 100ml of water, heat under reflux for 30 minutes, filter, and evaporate the filtrate to dryness. Starting from the step of "extracting with 20ml of 10% hydrochloric acid under reflux for 30 minutes", prepare the reference material solution using the same method. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 2μl of the test solution and 3μl of the reference material solution separately to the same silica gel G thin-layer plate, using toluene-ethyl acetate-methanol-formic acid (10:3:1:2) (developing solvent 1) (see...). Figure 2 Toluene-ethyl acetate-methanol-formic acid (10:1:1.5:1) (developing solvent 2) (see) Figure 3 ), ether-chloroform-methanol (10:6:1) (developing solvent 3) (see) Figure 4 Using ether-chloroform-methanol (10:6:1) as the developing solvent, the sample was developed, removed, air-dried, and examined under ultraviolet light (365 nm). The results showed that when using ether-chloroform-methanol (10:6:1) as the developing solvent, the spots were clear. The lotus root charcoal granule sample showed obvious spots at Rf=0.551, while the lotus root granule sample did not. This method can achieve qualitative identification of lotus root granule and lotus root charcoal granule (see...). Figure 4 ). Figure 2 Identification results of lotus root nodes and lotus root node charcoal granules using developing solvent 1; 1: control medicinal material; 2: lotus root node charcoal granules; 3: lotus root node granules. Figure 3 Identification results of lotus root nodes and charred lotus root node granules using developing solvent 2; 1: control medicinal material; 2: charred lotus root node granules; 3: lotus root node granules; Figure 4 Identification results of lotus root nodes and lotus root node charcoal formulation granules using developing solvent 3; 1: control medicinal material; 2: lotus root node charcoal formulation granules; 3: lotus root node formulation granules.

[0083] Thin-layer method establishment

[0084] 2.4.1 Preparation of the test solution

[0085] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them into a fine powder, add 20ml of 10% hydrochloric acid and reflux for 30 minutes, then extract with 20ml of chloroform three times by shaking. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test solution.

[0086] 2.4.2 Preparation of control herbal solution

[0087] Take 2g of lotus root section as reference material, add 100ml of water, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, and start from "add 20ml of 10% hydrochloric acid and reflux for 30 minutes" to prepare reference material solution in the same way.

[0088] 2.4.3 Determination Method

[0089] Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Take 2 μl of the test solution and 3 μl of the reference herb solution and spot them separately on the same silica gel G thin-layer plate. Use ether-chloroform-methanol (10:6:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0090] Example 3 Methodological Investigation

[0091] 3.5.1 Sampling quantity investigation

[0092] Under the proposed experimental conditions, 3 μl, 5 μl, and 7 μl of the lotus root node control solution, and 2 μl, 4 μl, and 6 μl of the lotus root node formula granule solution and the lotus root node charcoal formula granule solution were respectively spotted onto the same silica gel G thin-layer plate. (See figure) Figure 5 As shown in the figure, when 3-7 μl of the control herbal solution and 2-6 μl of the test sample solution were spotted, the spots were clearly visible and the separation was good. Therefore, the spotted amounts were determined to be 3-7 μl for the control herbal solution and 2-6 μl for the test sample solution. Furthermore, at a ratio shift value (Rf) of 0.538, the lotus root charcoal formulation granules showed fluorescent spots, while the lotus root charcoal formulation granules did not, thus distinguishing them from the lotus root charcoal formulation granules. The presence or absence of fluorescent spots at a ratio shift value (Rf) of approximately 0.54 can be used as a distinguishing point. Figure 5 The sampling volume was investigated; 1–3: 3 μl, 5 μl, and 7 μl of the control medicinal material solution; 4–6: 2 μl, 4 μl, and 6 μl of lotus root charcoal granule solution; 7–9: 2 μl, 4 μl, and 6 μl of lotus root granule solution.

[0093] 3.5.2 Specificity Examination

[0094] Negative chromatographic solution, lotus root control solution, lotus root granule solution, and lotus root charcoal granule solution were prepared according to the above preparation method. Thin-layer chromatography identification experiments were performed, and the results are shown in the figure. Figure 6 As shown in the figure, the negative sample did not interfere with the lotus root nodes and lotus root node charcoal formulation granules, indicating that the method has good specificity. Furthermore, the lotus root node charcoal formulation granules showed fluorescent spots at a ratio shift Rf = 0.529, while the lotus root node formulation granules did not. Figure 6 Specificity assessment; 1: Lotus root node control solution; 2: Lotus root node charcoal granule solution; 3: Lotus root node granule solution; 4: Negative test solution;

[0095] 3.5.3 Durability Assessment

[0096] 3.5.3.1 Comparison of different thin-layer plates

[0097] Cuttable thin-layer chromatography plates from Tianjin Slida Technology Co., Ltd., prefabricated silica gel G plates from Merck (Germany) and Qingdao Ocean Chemical Plant Branch were selected. Tests were conducted according to the planned experimental methods. (See attached table.) Figures 7-9 The results showed that the relative shift values ​​(Rf) of the fluorescent spots at a distance of 5–10 cm from the origin in the three thin-layer plates were 0.571, 0.533, and 0.571, respectively, all of which met the identification requirements. Furthermore, the lotus root charcoal formulation particles showed fluorescent spots at Rf values ​​of 0.571, 0.533, and 0.571, while the lotus root formulation particles showed no spots. Figure 7 Different Thin-Layer Plate Studies - Tianjin Silida; 1: Control Medicinal Material; 2: Lotus Root Charcoal Granules; 3: Lotus Root Granules; Figure 8 Different Thin-Layer Plate Studies - Merck, Germany; 1: Lotus root node control solution; 2: Lotus root node charcoal formulation granule solution; 3: Lotus root node formulation granule solution; Figure 9 Different Thin-Layer Plate Studies - Qingdao Ocean; 1, 2: Lotus Root Node Control Medicinal Material Solution; 3: Lotus Root Node Formula Granule Solution; 4: Lotus Root Node Charcoal Formula Granule Solution;

[0098] 3.5.3.2 Comparison of different temperatures

[0099] Thin-layer plates after sampling were developed at both low (4°C) and high (35°C) temperatures. Figures 10-11 It can be seen that the method has good adaptability to different temperatures, and the lotus root charcoal formulation particles show fluorescent spots at specific values ​​of 0.548 and 0.532, while the lotus root formulation particles do not have such spots. Figure 10 Different temperatures -4℃; 1: Control medicinal material; 2: Lotus root charcoal granules; 3: Lotus root granules. Figure 11 Different temperatures -35℃; 1: Lotus root charcoal granules; 2: Lotus root granules; 3: Reference medicinal material;

[0100] 3.5.3.3 Comparison of different humidity levels

[0101] Thin-layer plates after sampling were developed under humidity conditions of 32% RH and 75% RH, respectively. (See attached figures.) Figures 12-13As can be seen from the figure, this method has good adaptability to different temperatures, and the lotus root charcoal formulation particles show fluorescent spots at specific values ​​of 0.564 and 0.537, while the lotus root formulation particles do not show spots. Figure 12 Different humidity levels -32%; 1: Control medicinal material; 2: Lotus root charcoal granules; 3: Lotus root granules; Figure 13 Different humidity levels -75%; 1: Control medicinal material; 2: Lotus root charcoal granules; 3: Lotus root granules;

[0102] 3.5.4 Verification

[0103] The solutions of lotus root node control material, lotus root node formula granules, and lotus root node charcoal formula granules were respectively spotted onto the same silica gel G thin-layer plate and analyzed according to the established method. The experimental results are shown in [Figure 1]. Figure 14 The results showed that the lotus root charcoal formulation particles had fluorescent spots at a specific gravity (Rf) of 0.552, while the lotus root formulation particles did not have such spots. These spots can be used to distinguish between the two. Figure 14 Multiple batches were verified; 1: control medicinal material; 2-4: lotus root charcoal formula granules; 5-7: lotus root formula granules.

[0104] 3.6 Determination of the identification point Rf for lotus root node formula granules and lotus root node charcoal formula granules

[0105] The ratio shift values ​​(Rf) of various parameters in the thin-layer chromatography method investigation were summarized, and the results are shown in Table 1. Based on the summarized results, the ratio shift value (Rf) of the identification spots of the lotus root charcoal formulation granules was determined to be within ±10%, with a specified value of 0.547.

[0106] Table 1 Summary of Methodological Examinations and Shift Values

[0107]

[0108]

[0109] Comparative Example 1: Pharmacopoeia Method

[0110] (1) Take 1g each of lotus root node and lotus root node charcoal powder, add 20ml of dilute ethanol, sonicate for 20 minutes, filter, and use the filtrate as the test solution. Separately, take 1g of lotus root node reference material and prepare a reference material solution using the same method. Then, take alanine reference standard, add dilute ethanol to prepare a solution containing 0.5mg per ml, and use this as the reference solution. Perform thin-layer chromatography (General Rule 0502), applying 10μl each of the test solution and reference material solution, and 2μl of the reference solution, to the same silica gel G thin-layer plate. Develop using n-butanol-glacial acetic acid-water (4:1:1) as the developing solvent. Remove, air dry, spray with ninhydrin reagent, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference material and the reference standard. See... Figure 15. Figure 15 The results of the identification of lotus root nodes and lotus root node charcoal formula granules by the pharmacopoeia identification method (1) are as follows: 1: alanine reference standard; 2: lotus root node reference medicinal material; 3: lotus root node formula granules; 4: lotus root node charcoal formula granules.

[0111] The results showed that the pharmacopoeia identification method (1) could not effectively distinguish between lotus root joint formula granules and lotus root joint charcoal formula granules.

[0112] (2) Take 1g each of lotus root node and lotus root node charcoal granule powder, add 25ml of methanol, sonicate for 30 minutes, filter, recover the solvent from the filtrate until dry, dissolve the residue in 1ml of methanol to prepare the test solution. Take another 1g of lotus root node reference material and prepare a reference material solution using the same method. Take betulinic acid reference standard and prepare a solution containing 1mg per ml using methanol to prepare a reference solution. Perform thin-layer chromatography (General Rule 0502), take 8μl of each of the above three solutions, spot them separately on the same silica gel G thin-layer plate, develop with dichloromethane-methanol (25:1) as the developing solvent, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. Examine under sunlight and ultraviolet light (365nm) respectively. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference material and the reference standard; fluorescent spots of the same color appear under ultraviolet light. See the results below. Figure 16 . Figure 16 The results of the identification of lotus root nodes and lotus root node charcoal formula granules by the pharmacopoeia identification method (2) are as follows: 1: betulinic acid reference standard; 2: lotus root node reference medicinal material; 3: lotus root node formula granules; 4: lotus root node charcoal formula granules. The results show that the above-mentioned pharmacopoeia thin-layer identification method does not show obvious differences between lotus root nodes and lotus root node charcoal formula granules, and cannot distinguish between the two. It is not applicable to the identification of lotus root node formula granules and lotus root node charcoal formula granules.

[0113] Comparative Example 2: Lotus Root Charcoal Formulation Granule Quality Standard (Beijing) Method

[0114] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them finely, add 20ml of water to dissolve them, and extract twice with ethyl acetate, 30ml each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1ml of ethyl acetate to prepare the test solution. Separately, take 1g of lotus root node reference material, add 100ml of water, heat under reflux for 30 minutes, filter, and concentrate the filtrate to approximately 20ml. Prepare the reference material solution using the same method starting from "extracted twice with ethyl acetate". Perform thin-layer chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition). Apply 2μl of the test solution and 3μl of the reference material solution separately to the same silica gel G thin-layer plate. Develop using toluene-ethyl acetate-formic acid (6:4:0.1) as the developing solvent. Remove, air dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material. See [Results omitted]. Figure 17 . Figure 17 The identification results of lotus root nodes and lotus root node charcoal formulation granules by Beijing Municipality method; among which 1: lotus root node reference material;

[0115] 2: Lotus root joint formula granules; 3: Lotus root joint charcoal formula granules; The results show that the identification method of the quality standard (Beijing) for lotus root joint charcoal formula granules cannot effectively distinguish between lotus root joint formula granules and lotus root joint charcoal formula granules.

[0116] Comparative Example 3

[0117] Quality Standard for Lotus Root Charcoal Granules (Anhui Province)

[0118] Take appropriate amounts of lotus root nodes and lotus root node charcoal granules, grind them finely, take 1g, add 10ml of ethanol, sonicate for 30 minutes, filter, and concentrate the filtrate to 1ml as the test solution. Separately, take alanine reference standard, add dilute ethanol to prepare a solution containing 0.5mg per ml, as the reference solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502). Apply 15μl of the test solution and 1μl of the reference solution separately to the same silica gel G thin-layer plate. Develop with n-butanol-glacial acetic acid-water (4:1:1) as the developing solvent. Remove, air dry, spray with ninhydrin reagent, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard. See the results below. Figure 18 . Figure 18 The identification results of lotus root nodes and lotus root node charcoal formulation granules by the method of Anhui Province; 1: Lotus root node formulation granules; 2-3: Lotus root node charcoal formulation granules; 4-6: Alanine reference standard.

[0119] Comparative Example 4

[0120] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them finely, add 50mL of ethyl acetate to each, reflux for 0.5h, filter, concentrate the filtrate to about 2mL, transfer to a 5mL volumetric flask, dilute to the mark with ethyl acetate, and shake well. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Accurately pipette 5μL of the lotus root node and lotus root node charcoal test solutions and spot them separately on the same silica gel G thin-layer plate with 0.3% sodium carboxymethyl cellulose as a binder. Develop with petroleum ether (60-90℃)-ethyl acetate (2:1) as the developing solvent. Remove, air dry, spray with 10% sulfuric acid ethanol, heat to 105℃ until the spots are clear, and examine under natural light and 365nm ultraviolet light. Results are shown below. Figure 19 . Figure 19Comparative Example 4: Identification results of lotus root nodes and lotus root node charcoal formulation particles; where 1-4 were examined under 365nm ultraviolet light, with sample amounts of 20μl lotus root node formulation particles, 20μl lotus root node charcoal formulation particles, 5μl lotus root node formulation particles, and 5μl lotus root node charcoal formulation particles respectively; 5-8 were examined under natural light, with sample amounts of 20μl lotus root node formulation particles, 20μl lotus root node charcoal formulation particles, 5μl lotus root node formulation particles, and 5μl lotus root node charcoal formulation particles respectively.

[0121] The results showed that Method 1 in Comparative Example 4, when examined at 365 nm, could not effectively distinguish between lotus root joint formulation particles and lotus root joint charcoal formulation particles. Under natural light, neither lotus root joint charcoal nor lotus root joint formulation particles showed any spots, and they could not be distinguished.

[0122] Comparative Example 5

[0123] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them finely, add 50mL of ethyl acetate to each, reflux for 0.5h, filter, concentrate the filtrate to about 2mL, transfer to a 5mL volumetric flask, dilute to the mark with ethyl acetate, and shake well. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Accurately pipette 5μL of the lotus root node and lotus root node charcoal test solutions and spot them separately on the same silica gel G thin-layer plate with 0.3% sodium carboxymethyl cellulose as a binder. Develop with petroleum ether (60-90℃)-ethyl acetate (8:5) as the developing solvent. Remove, air dry, spray with 10% sulfuric acid ethanol, heat to 105℃ until the spots are clear, and examine under natural light and 365nm ultraviolet light. Results are shown below. Figure 20 . Figure 20 Comparative Example 5: Identification results of lotus root nodes and lotus root node charcoal formulation particles. Examples 1-4 were examined under 365nm ultraviolet light, with sample amounts of 20 μl each for lotus root node formulation particles, 20 μl for lotus root node charcoal formulation particles, 5 μl for lotus root node formulation particles, and 5 μl for lotus root node charcoal formulation particles, respectively. Examples 5-8 were examined under natural light, with sample amounts of 20 μl each for lotus root node formulation particles, 20 μl for lotus root node charcoal formulation particles, 5 μl for lotus root node formulation particles, and 5 μl for lotus root node charcoal formulation particles, respectively.

[0124] The results showed that the method in Comparative Example 5 could not effectively distinguish between lotus root joint formula granules and lotus root joint charcoal formula granules.

[0125] Comparative Example 6

[0126] Take 5g each of lotus root nodes and lotus root node charcoal granules, grind them finely, add 100mL of 50% methanol and heat in a water bath for 40min, filter, recover the solvent from the filtrate to dryness, then extract with 150mL of ethyl acetate in a water bath for 20min, filter, recover the ethyl acetate, and dilute the residue to 5mL as the test solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), accurately pipette 10μL of the lotus root node and lotus root node charcoal test solutions, and spot them separately on the same silica gel G thin-layer plate with sodium carboxymethyl cellulose as the binder. Use chloroform-ethyl acetate-formic acid (8:8:0.5) as the developing solvent, develop, remove, air dry, and examine under a UV lamp at 365nm. The results are shown below. Figure 21 . Figure 21 Comparative Example 6: Identification results of lotus root nodes and lotus root node charcoal formulation granules; Examples 1 and 2 were examined under 365nm ultraviolet light, and were lotus root node formulation granules and lotus root node charcoal formulation granules, respectively.

[0127] The results showed that the method in Comparative Example 6 could not effectively distinguish between lotus root joint formula granules and lotus root joint charcoal formula granules.

[0128] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A thin-layer chromatography method for identifying lotus root nodes and lotus root node charcoal formulation particles, comprising: A) Take lotus root nodes and pre-treat them with the formula granules to obtain lotus root node test solution; The pretreatment of lotus root node formula granules specifically involves: extracting the lotus root node formula granules by reflux with 10% hydrochloric acid, then extracting with chloroform, evaporating to dryness, and dissolving the residue in methanol to obtain a lotus root node test solution; pretreatment of lotus root node char formula granules specifically involves: extracting the lotus root node char formula granules by reflux with 10% hydrochloric acid, then extracting with chloroform, evaporating to dryness, and dissolving the residue in methanol to obtain a lotus root node char test solution; B) Take lotus root nodes as reference material, add acid and reflux to extract, then extract with chloroform, evaporate to dryness, dissolve the residue in methanol to obtain the reference material solution; C) Thin-layer chromatography was performed on the lotus root node test solution and the lotus root node char test solution as a reference medicinal material solution. The thin-layer plate was a silica gel G thin-layer plate; the developing solvent was diethyl ether-chloroform-methanol; the mass ratio of diethyl ether-chloroform-methanol was 10:6:

1. D) Examine under a UV lamp. If the lotus root sample and the control medicinal material solution have fluorescent spots of the same color at the same Rf, and no fluorescent spots at Rf=0.486~0.602, then it is a lotus root formula granule. If fluorescent spots are present at Rf = 0.486~0.602, then it is a lotus root charcoal formulation particle.

2. The method according to claim 1, characterized in that, The number of times the trichloromethane extraction is performed in step A) is 2 to 3.

3. The method according to claim 1, characterized in that, In step A), the mass-to-volume ratio of the lotus root granules, 10% hydrochloric acid, and chloroform is 1g:20mL:60mL; the reflux extraction time is 20-30min.

4. The method according to claim 1, characterized in that, The specific pretreatment of the lotus root charcoal formulation particles is as follows: The lotus root charcoal granules were extracted by reflux with 10% hydrochloric acid, followed by extraction with chloroform, evaporated to dryness, and the residue was dissolved in methanol to obtain the lotus root charcoal test solution; the chloroform extraction was performed 2 to 3 times. The mass-to-volume ratio of the lotus root charcoal granules, 10% hydrochloric acid, and chloroform is 1g:20mL:60mL; the reflux extraction time is 20-30min.

5. The method according to claim 1, characterized in that, The sample volume for thin-layer chromatography is 2-6 μL, and the sample volume for the reference medicinal material solution is 3-7 μL.

6. The method according to claim 1, characterized in that, Step D) The ultraviolet lamp is 365nm.

7. The method according to claim 1, characterized in that, Step D) The silicone G thin film is a prefabricated silicone G film from Tianjin Slida, Merck, or Qingdao Ocean.

8. The method according to claim 7, characterized in that, Step D) The temperature to be inspected is 5℃~35℃; the humidity to be inspected is 32%rh~75%rh.

9. The method according to claim 1, characterized in that, The Rf values ​​in step D) are 0.529, 0.532, 0.533, 0.537, 0.538, 0.548, 0.552, 0.564, and 0.571.

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