Thin-layer identification method for lotus rhizome node and lotus rhizome node charcoal formula granules

Abstract

The invention provides a thin-layer identification method for nodus nelumbinis rhizomatis and nodus nelumbinis rhizomatis charcoal formula granules, which comprises the following steps: A) pretreating nodus nelumbinis rhizomatis formula granules to obtain a nodus nelumbinis rhizomatis test solution; taking the nodus nelumbinis rhizomatis charcoal formula granules for pretreatment to obtain a nodus nelumbinis rhizomatis charcoal test solution; b) taking a nelumbo nucifera Gaertn reference medicinal material, adding acid for reflux extraction, extracting with trichloromethane, drying by distillation, and dissolving residues with methanol to obtain a reference medicinal material solution; c) performing thin-layer chromatography detection on the rhizoma nelumbinis test solution, the rhizoma nelumbinis charcoal test solution and the reference medicinal material solution; and D) inspecting under an ultraviolet lamp. The thin-layer identification chromatography method for the nodus nelumbinis rhizomatis and nodus nelumbinis rhizomatis charcoal formula granules established by the invention is used for rapidly, accurately and effectively identifying the nodus nelumbinis rhizomatis and the nodus nelumbinis rhizomatis charcoal formula granules. Meanwhile, whether fluorescent spots exist in a certain ratio shift value range of a thin-layer chromatogram or not is observed to serve as identification points to be used for distinguishing the rhizoma nelumbinis from the rhizoma nelumbinis charcoal formula granules.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and detection, in particular to a thin-layer identification method for lotus root nodes and lotus root node charcoal formula particles. Background Art

[0002] Lotus root nodes are the dried rhizome segments of the lotus plant Nelumbo nucifera Gaertn. First recorded in Shennong's Classic of Materia Medica, they are considered a top-grade medicinal product. They are sweet and astringent, neutral in nature, and possess astringent, hemostatic, and stasis-dissolving properties. They are used to treat hematemesis, hemoptysis, epistaxis, hematuria, and metrorrhagia. Modern clinical applications of lotus root nodes include raw and charred versions. Charring weakens the stasis-dissolving properties and enhances the astringent properties, leading to a stronger hemostatic effect. Therefore, charred lotus root nodes are often used as medicine.

[0003] Currently, there is limited research on charred lotus root nodes. This research primarily focuses on the hemostatic mechanism of the active components of charred lotus root nodes, the processing techniques, and the changes in the appearance and chemical composition of different charred lotus root node products. For example, Zhang Shuo et al. found that lotus root nodes contain chemical components such as phenolic acids and tannins. 3-Epibetulinic acid and tannins are generally considered the primary active components of lotus root nodes, but charred lotus root nodes can alter their composition. For example, Liu Shanxin's research showed that charred lotus root nodes exhibited varying degrees of changes in characteristic peaks, extract content, and 3-Epibetulinic acid content, enabling the identification of charred lotus root node pieces at different charred levels. Hong Aixian et al. measured tannic acid levels in charred lotus root nodes before and after processing, demonstrating a significant decrease in tannic acid content after charring. However, because the composition of Chinese medicinal materials is influenced by factors such as origin, age, harvest time, and storage, component content alone is not suitable for qualitative identification of lotus root nodes and charred lotus root node granules. Furthermore, Oujie charcoal granules are made from Oujie charcoal slices through a series of processes, some of which can destroy the original physical and microscopic characteristics. Physical and microscopic identification methods are therefore not suitable for the identification of extracts and preparations. Therefore, to ensure accurate clinical use, it is necessary to establish a visual identification method for qualitatively distinguishing the two.

[0004] Therefore, in summary, there is currently no effective method for distinguishing between lotus root nodes and lotus root node charcoal in preparations. In order to ensure accurate clinical medication, it is necessary to establish a method that can qualitatively distinguish between lotus root nodes and lotus root node charcoal formulation particles. 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 identification method for lotus root nodes and lotus root node charcoal formula particles.

[0006] 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.

[0007] 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.

[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 refers to any combination of these items, including any combination of single or plural items.

[0009] 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.

[0010] The present invention discloses the construction and application of a thin-layer identification method for lotus root nodes and lotus root node charcoal formula granules. The thin-layer identification method has strong specificity, good stability, high precision, convenience and is easy to master. The thin-layer identification method of the present invention compares the thin-layer chromatogram of the lotus root node charcoal formula granules with the thin-layer chromatogram of the lotus root node control medicinal material and the lotus root node formula granules. The ratio shift value must appear at the same position of 0.486 to 0.602, and a green fluorescent spot must appear as the identification point of the lotus root node charcoal formula granules. The thin-layer method can quickly and effectively identify the lotus root node charcoal formula granules that have lost the morphology of medicinal pieces. It is simple to operate, has high precision and sensitivity, good stability, has good application prospects, and ensures accurate medication.

[0011] The present invention provides a thin layer identification method for lotus root nodes and lotus root node charcoal formula particles, comprising:

[0012] A) pre-treating the lotus root node formula granules to obtain a lotus root node test solution;

[0013] The lotus root node charcoal formula particles are pretreated to obtain a lotus root node charcoal test solution;

[0014] B) extracting a reference medicinal material of lotus root node, adding acid to reflux, then extracting with chloroform, evaporating to dryness, and dissolving the residue with methanol to obtain a reference medicinal material solution;

[0015] C) The lotus root node test solution, the lotus root node charcoal test solution and the control medicinal material solution were subjected to thin layer chromatography detection using a silica gel G thin layer plate and an ether-chloroform-methanol developing solvent;

[0016] D) Place under ultraviolet light for inspection. If the lotus root node test sample and the control medicinal material solution have fluorescent spots of the same color at the same Rf, and there are no fluorescent spots at Rf = 0.486-0.602, then they are lotus root node formula granules; if there are fluorescent spots at Rf = 0.486-0.602, then they are lotus root node charcoal formula granules.

[0017] The invention provides lotus root nodes and lotus root node charcoal formula particles. First, the lotus root node formula particles are pretreated to obtain a lotus root node test solution.

[0018] The pretreatment of the lotus root node formula granules of the present invention is specifically as follows:

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

[0020] The number of chloroform extractions in the present invention is 2 to 3 times, preferably 3 times.

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

[0022] The pretreatment of the lotus root node charcoal particles of the present invention is specifically as follows:

[0023] The lotus root node charcoal granules were refluxed with 10% hydrochloric acid and then extracted with chloroform. The mixture was evaporated to dryness and the residue was dissolved with 1 mL of methanol to obtain the lotus root node charcoal test solution.

[0024] The number of chloroform extractions in the present invention is 2 to 3 times, preferably 3 times.

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

[0026] Take lotus root node reference medicinal material, add acid reflux extraction, then extract with chloroform, evaporate to dryness, dissolve the residue with methanol to obtain a reference medicinal material solution; the chloroform extraction times of the present invention is 2 to 3 times, preferably 3 times.

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

[0028] The extraction of the present invention is preferably reflux extraction.

[0029] The present invention investigates the extraction method and finds that only the extraction method of the present invention can effectively distinguish between the lotus root node formula granules and the lotus root node charcoal formula granules.

[0030] The lotus root node test solution, lotus root node charcoal test solution and control medicinal material solution were subjected to thin layer chromatography detection, and the thin layer plate was a silica gel G thin layer plate;

[0031] In some embodiments of the present invention, the silica gel G thin layer plate is a prefabricated silica gel G plate produced by Tianjin Silida, Merck or Qingdao Ocean. The results show that the method has good durability and can meet the identification requirements.

[0032] In some embodiments of the present invention, the developing solvent is 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 investigates different developing agents and finds that only the developing agent of the present invention can produce clear spots and can well identify the lotus root node formula particles and lotus root node charcoal formula particles.

[0035] Place it under ultraviolet light for inspection. If the lotus root node test sample and the control medicinal material solution have fluorescent spots of the same color at the same Rf, and there are no fluorescent spots at Rf = 0.486-0.602, then it is lotus root node formula granules; if there are fluorescent spots at Rf = 0.486-0.602, then it is lotus root node charcoal formula granules.

[0036] The ultraviolet lamp of the present invention is 365nm.

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

[0038] According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 General Chapter 0502), the test sample solution and the control herbal solution were spotted separately on the same silica gel G thin-layer plate. Eluent was ether-chloroform-methanol (10:6:1). The plate was developed, removed, air-dried, and examined under a UV lamp (365 nm). If the lotus root node test sample and the control herbal solution exhibited identical fluorescent spots of the same color at the same Rf, and no fluorescent spot was observed at Rf = 0.486-0.602, the granules were lotus root node formula. If a fluorescent spot was observed at Rf = 0.486-0.602, the granules were lotus root node charcoal formula.

[0039] In a preferred embodiment of the present invention, the spotting volume of the thin layer chromatography test sample is 2 to 6 μL, specifically 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL; the spotting volume of the control medicinal material solution is 3 to 7 μL, specifically 3 μL, 4 μL, 5 μL, 6 μL, and 7 μL.

[0040] The present invention investigated the spotting amount and found that when 3-7 μl of the control medicinal material solution and 2-6 μl of the test solution were spotted, the spots were clearly colored and the separation was good. The spotting amount was determined to be 3-7 μl of the control medicinal material solution and 2-6 μl of the test solution.

[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, and 0.571.

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

[0043] This method has good durability under different temperatures. Compared with the reference herbal medicine atlas, the test sample atlas and the reference herbal medicine atlas show spots of the same color at corresponding positions.

[0044] The inspection humidity of the present invention is 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, 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 has good durability under different humidity conditions. Compared with the reference herbal medicine atlas, the test sample atlas and the reference herbal medicine atlas show spots of the same color at corresponding positions.

[0046] The invention provides a thin layer chromatography identification method for lotus root nodes and lotus root node charcoal formula particles, comprising: A) pre-treating lotus root node formula particles to obtain a lotus root node test solution; pre-treating lotus root node charcoal formula particles to obtain a lotus root node charcoal test solution; B) taking a lotus root node control medicinal material, adding acid and refluxing for extraction, then extracting with chloroform, evaporating to dryness, and dissolving the residue with methanol to obtain a control medicinal material solution; C) performing thin layer chromatography on the lotus root node test solution and the lotus root node charcoal test solution and the control medicinal material solution, using a silica gel G thin layer plate and ether-chloroform-methanol as a developing agent; D) inspecting under an ultraviolet lamp, if the lotus root node test sample and the control medicinal material solution have fluorescent spots of the same color at equal Rf and no fluorescent spots at Rf=0.486-0.602, the lotus root node formula particles are present; if fluorescent spots are present at Rf=0.486-0.602, the lotus root node charcoal formula particles are present. The thin-layer chromatography identification method for lotus root nodes and lotus root node charcoal granules established in the present invention is used to quickly, accurately, and effectively identify lotus root nodes and lotus root node charcoal granules. Furthermore, the method distinguishes lotus root nodes and lotus root node charcoal granules by observing whether fluorescent spots within a certain ratio shift value range are present in the thin-layer chromatography spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0048] Figure 2 Identification results of lotus root node and lotus root node charcoal formula particles by developer 1;

[0049] Figure 3 Identification results of lotus root node and lotus root node charcoal formula particles by developer 2;

[0050] Figure 4 Identification results of lotus root node and lotus root node charcoal formula particles by developing agent 3;

[0051] Figure 5 Spot sampling inspection;

[0052] Figure 6 Exclusive inspection;

[0053] Figure 7 Inspection of different thin layer boards-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°C;

[0057] Figure 11 Different temperatures -35°C;

[0058] Figure 12 Different humidity - 32%;

[0059] Figure 13 Different humidity - 75%;

[0060] Figure 14 Multi-batch verification;

[0061] Figure 15 Identification results of lotus root nodes and lotus root node charcoal granules using the pharmacopoeial identification (1) method;

[0062] Figure 16 Identification results of lotus root nodes and lotus root node charcoal granules using the pharmacopoeial identification (2) method;

[0063] Figure 17 Identification results of lotus root nodes and lotus root node charcoal granules using Beijing method;

[0064] Figure 18 The results of identification of lotus root nodes and lotus root node charcoal granules by Anhui Province method;

[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 Comparative Example 6 shows the identification results of lotus root nodes and lotus root node charcoal formula particles. DETAILED DESCRIPTION

[0068] In order to further illustrate the present invention, a thin layer identification method for lotus root nodes and lotus root node charcoal formula particles provided by the present invention is described in detail below in conjunction with examples.

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

[0070] Reagents: Methanol, n-hexane, hydrochloric acid, chloroform, 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 control medicinal material (China Food and Drug Administration, batch number: 121580-201302); Lotus root node charcoal formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: OJT-1); Lotus root node formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: OJ-1)

[0072] Example 1

[0073] 1.1 Investigation of extraction methods

[0074] 1. Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them finely, add 10ml of methanol, reflux and extract for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 5ml of water to prepare the test solution. Separately, take 2g of lotus root node control medicinal material, add 50ml of methanol, reflux for 30 minutes, and filter to prepare the control medicinal 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, and shake and extract three times with 10ml of water-saturated n-hexane. Combine the n-hexane solutions, evaporate to dryness, add 5ml of n-hexane to the residue, sonicate for 30 minutes, and filter to prepare the test solution. Separately, take 1g of lotus root node control medicinal material, add 100ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 10ml of water. Prepare the control medicinal material solution in the same way as in "Preparation of Test Solution".

[0076] 3. Grind 1g each of lotus root nodes and lotus root node charcoal granules, add 20ml of 10% hydrochloric acid and reflux for 30 minutes. Extract with chloroform three times, shaking, using 20ml each time. Combine the chloroform solutions and evaporate to dryness. Dissolve the residue in 1ml of methanol to prepare the test solution. Separately, take 2g of lotus root node control medicinal material, add 100ml of water, heat and reflux for 30 minutes, filter, and evaporate the filtrate to dryness. Prepare the control medicinal material solution by the same method starting from "add 20ml of 10% hydrochloric acid and reflux for 30 minutes."

[0077] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition, Part IV, General Rules 0502), 2 μl of the test solution and 3 μl of the control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and ether-chloroform-methanol (10:6:1) was used as the developing solvent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm). The results are shown in Figure 1 .

[0078] The results showed that the samples prepared by sample preparation methods 1 and 2 could not effectively distinguish between lotus root node formula granules and lotus root node charcoal formula granules. The sample preparation method was determined as follows: take 1g of lotus root node and lotus root node charcoal formula granules, grind them into powder, add 20ml of 10% hydrochloric acid and reflux for 30 minutes, extract with chloroform by shaking 3 times, 20ml each time, combine the chloroform liquids, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test sample solution.

[0079] Figure 1 Identification results of lotus root nodes and lotus root node charcoal granules prepared by different test sample preparation methods, among which, 1 to 3 are: method 1 control medicinal material, method 1 lotus root node granules, method 1 lotus root node charcoal granules; 4 to 6 are: method 2 control medicinal material, method 2 lotus root node granules, method 2 lotus root node charcoal granules; 7 to 9 are: method 3 control medicinal material, method 3 lotus root node granules, method 3 lotus root node charcoal granules;

[0080] Example 2

[0081] 2.3.2 Developing agent investigation

[0082] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them into powder, add 20ml of 10% hydrochloric acid and reflux for 30 minutes, shake and extract with chloroform three times, 20ml each time, combine the chloroform solutions, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and use it as the test solution. Take another 2g of lotus root node control medicinal material, add 100ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the control medicinal material solution in the same way starting from "add 20ml of 10% hydrochloric acid and reflux for 30 minutes". According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), take 2μl of the test solution and 3μl of the control medicinal material solution, and spot them on the same silica gel G thin layer plate, respectively, with toluene-ethyl acetate-methanol-formic acid (10:3:1:2) (developer 1) (see Figure 2 ), toluene-ethyl acetate-methanol-formic acid (10:1:1.5:1) (developer 2) (see Figure 3 ), ether-chloroform-methanol (10:6:1) (developer 3) (see Figure 4 ) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). The results show that when using the developing agent ether-chloroform-methanol (10:6:1), the spots are clear. The lotus root node charcoal formula granules test sample has obvious spots at Rf=0.551, while the lotus root node formula granules test sample has no such spots. This method can achieve the qualitative identification of lotus root node formula granules and lotus root node charcoal formula granules (see Figure 4 ). Figure 2 Identification results of lotus root node and lotus root node charcoal granules using developer 1; 1: control medicinal material; 2: lotus root node charcoal granules; 3: lotus root node granules. Figure 3 Identification results of lotus root node and lotus root node charcoal granules by developer 2; 1: control medicinal material; 2: lotus root node charcoal granules; 3: lotus root node granules; Figure 4 Identification results of lotus root nodes and lotus root node charcoal granules using 3 developing agents; 1: control medicinal material; 2: lotus root node charcoal granules; 3: lotus root node granules.

[0083] Thin layer method establishment

[0084] 2.4.1 Preparation of test solution

[0085] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them into powder, add 20ml of 10% hydrochloric acid and reflux for extraction for 30 minutes, shake and extract with chloroform three times, 20ml each time, combine the chloroform liquid, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0086] 2.4.2 Preparation of control medicinal material solution

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

[0088] 2.4.3 Determination method

[0089] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0502), 2 μl of the test sample solution and 3 μl of the control medicinal material solution were respectively spotted on the same silica gel G thin layer plate, and ether-chloroform-methanol (10:6:1) was used as the developing agent. The plate was developed, taken out, dried, and inspected under ultraviolet light (365 nm).

[0090] Example 3 Methodological Investigation

[0091] 3.5.1 Sampling quantity inspection

[0092] Under the experimental conditions proposed above, 3 μl, 5 μl, and 7 μl of the lotus root node control medicinal material solution, 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 taken and spotted on the same silica gel G thin layer plate. Figure 5 As shown in the figure, when 3-7μl of the control herbal solution and 2-6μl of the test solution were applied, the spots were clearly colored and well separated. The applied sample volumes were determined to be 3-7μl of the control herbal solution and 2-6μl of the test solution. Furthermore, at the Rf = 0.538, the granules with the lotus root node charcoal formula exhibited fluorescent spots, while the granules with the lotus root node charcoal formula did not. This can be used to distinguish the granules from the lotus root node charcoal formula. The presence of fluorescent spots around the Rf = 0.54 can be used as a distinguishing factor. Figure 5 Sample volume inspection; 1-3: control medicinal material solution sample volume 3μl, 5μl, 7μl; 4-6: lotus root node charcoal granule solution 2μl, 4μl, 6μl; 7-9: lotus root node granule solution 2μl, 4μl, 6μl;

[0093] 3.5.2 Specificity Investigation

[0094] According to the above test sample preparation method, negative solution, lotus root node control medicinal material solution, lotus root node formula granule solution, and lotus root node charcoal formula granule solution were prepared respectively, and thin layer identification experiment was carried out. The results are shown in the figure. Figure 6 As can be seen from the figure, the negative sample did not interfere with the lotus root node and lotus root node charcoal granules, indicating that the method has good specificity. Furthermore, at the ratio shift value Rf = 0.529, the lotus root node charcoal granules showed a fluorescent spot, while the lotus root node granules did not. Figure 6 Specificity investigation; 1: lotus root node control medicinal material solution; 2: lotus root node charcoal formula granule solution; 3: lotus root node formula granule solution; 4; negative solution;

[0095] 3.5.3 Durability Investigation

[0096] 3.5.3.1 Comparison of different thin layer plates

[0097] The tailorable thin layer chromatography plates of Tianjin Silida Technology Co., Ltd., the prefabricated silica gel G plates of Merck, Germany, and Qingdao Ocean Chemical Plant Branch were selected and tested according to the proposed test methods. Figures 7-9 The results showed that the fluorescence spot ratio shift values (Rf) of the three thin-layer plates at 5 to 10 cm from the origin were 0.571, 0.533, and 0.571, respectively, all meeting the identification requirements. Furthermore, at Rf values of 0.571, 0.533, and 0.571, the granules formulated with the lotus root node charcoal exhibited fluorescent spots, while the granules formulated with the lotus root node charcoal exhibited no spots. Figure 7 Investigation of different thin layer plates - Tianjin Silida; 1: control medicinal materials; 2: lotus root node charcoal formula granules; 3: lotus root node formula granules; Figure 8 Different thin layer plates inspection - Merck, Germany; 1: lotus root node control medicinal material solution; 2: lotus root node charcoal formula granule solution; 3: lotus root node formula granule solution; Figure 9 Different thin layer plate inspection - 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] Take the spotted thin layer plate and develop it at a low temperature of 4°C and a high temperature of 35°C. Figures 10-11 It can be seen that this method has good adaptability to different temperatures, and the lotus root node charcoal formula particles show fluorescent spots at the ratio shift values of 0.548 and 0.532, while the lotus root node formula particles do not have such spots. Figure 10 Different temperatures -4℃; 1: control medicinal material; 2: lotus root node charcoal formula granules; 3: lotus root node formula granules; Figure 11 Different temperatures -35℃; 1: lotus root node charcoal formula granules; 2: lotus root node formula granules; 3: control medicinal material;

[0100] 3.5.3.3 Comparison of different humidity levels

[0101] The thin layer plate after spotting was developed under the humidity environment of 32% rh and 75% rh respectively. Figures 12-13As can be seen from the figure, this method has good adaptability to different temperatures, and at the ratio shift values of 0.564 and 0.537, the particles of the lotus root node charcoal formula show fluorescent spots, while the particles of the lotus root node formula have no spots. Figure 12 Different humidity -32%; 1: control medicinal material; 2: lotus root node charcoal formula granules; 3: lotus root node formula granules; Figure 13 Different humidity -75%; 1: control medicinal material; 2: lotus root node charcoal formula granules; 3: lotus root node formula granules;

[0102] 3.5.4 Verification

[0103] Take lotus root node control medicinal material solution, lotus root node formula granule solution, lotus root node charcoal formula granule solution, spot them on the same silica gel G thin layer plate, and analyze them according to the determined method. The experimental results are shown in Figure 14 The results showed that the granules of lotus root node charcoal formula had a fluorescent spot at the ratio shift value (Rf) of 0.552, while the granules of lotus root node formula did not have this spot, which can be used to distinguish the two. Figure 14 Multiple batches of validation; 1: control medicinal materials; 2-4: lotus root node charcoal formula granules; 5-7: lotus root node formula granules.

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

[0105] The data of various ratio transfer values in the thin layer chromatography methodology investigation are summarized and the results are shown in Table 1. According to the summary results, the ratio transfer value (Rf) of the identification spot of the charcoal powder formula particles should be within the range of ±10%, and the specified value is 0.547.

[0106] Table 1 Summary of methodological investigation ratios

[0107]

[0108]

[0109] Comparative Example 1 Pharmacopoeia method

[0110] (1) Take 1g each of lotus root node and lotus root node charcoal granule powder, add 20ml of dilute ethanol, ultrasonically treat for 20 minutes, filter, and take the filtrate as the test solution. Take another 1g of lotus root node control medicinal material and prepare the control medicinal material solution in the same way. Take the alanine reference substance and add dilute ethanol to prepare a solution containing 0.5mg per 1ml as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 10μl of the test solution and the control medicinal material solution and 2μl of the reference solution, and spot them on the same silica gel G thin layer plate, use n-butanol-glacial acetic acid-water (4:1:1) as the developing agent, develop, take out, dry, spray with ninhydrin test solution, and heat at 105℃ until the spots are clearly colored. In the test sample chromatogram, spots of the same color appear at the corresponding positions of the control medicinal material chromatogram and the reference substance chromatogram. See Figure 15. Figure 15 Identification results of lotus root nodes and lotus root node charcoal granules using the pharmacopoeia identification (1) method; 1: alanine reference substance; 2: lotus root node reference medicinal material; 3: lotus root node granules; 4: lotus root node charcoal granules.

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

[0112] (2) Take 1g each of lotus root node and lotus root node charcoal granule powder, add 25ml of methanol, ultrasonically treat for 30 minutes, filter, recover the solvent in the filtrate until dry, add 1ml of methanol to dissolve the residue, and use it as the test solution. Take another 1g of lotus root node control medicinal material and prepare the control medicinal material solution in the same way. Take the betulinic acid reference substance and add methanol to prepare a solution containing 1mg per 1ml, which is used as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 8μl of each of the above three solutions and spot them on the same silica gel G thin layer plate, use dichloromethane-methanol (25:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly colored. Observe under sunlight and ultraviolet light (365nm) respectively. In the test sample chromatogram, at the corresponding positions of the control medicinal material chromatogram and the reference substance chromatogram, spots of the same color appear under sunlight; fluorescent spots of the same color appear under ultraviolet light. See the results. Figure 16 . Figure 16 Identification results of lotus root nodes and lotus root node charcoal granules using the pharmacopoeia identification (2) method; 1: betulinic acid reference substance; 2: lotus root node control medicinal material; 3: lotus root node granules; 4: lotus root node charcoal granules; The results showed that using the above-mentioned pharmacopoeia thin layer identification method, there were no obvious difference spots between lotus root nodes and lotus root node charcoal granules, and the two could not be distinguished, so it is not suitable for the identification of lotus root node granules and lotus root node charcoal granules.

[0113] Comparative Example 2 Quality Standard for Lotus Root Node Charcoal Formula Granules (Beijing)

[0114] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them into powder, add 20ml of water to dissolve them, shake and extract with ethyl acetate twice, 30ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of ethyl acetate to dissolve the residue, and use it as the test solution. Take another 1g of lotus root node control medicinal material, add 100ml of water, heat and reflux for 30 minutes, filter, concentrate the filtrate to about 20ml, and prepare the control medicinal material solution in the same way starting from "shake and extract twice with ethyl acetate". According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 2μl of the test solution and 3μl of the control medicinal material solution were taken and spotted on the same silica gel G thin layer plate, respectively, with toluene-ethyl acetate-formic acid (6:4:0.1) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm). In the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions of the control medicinal material chromatogram. See the results. Figure 17 . Figure 17 The identification results of lotus root nodes and lotus root node charcoal granules by Beijing method; 1: lotus root node control medicinal material;

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

[0116] Comparative Example 3

[0117] Quality standard for lotus root charcoal granules (Anhui Province)

[0118] Take an appropriate amount of lotus root nodes and lotus root node charcoal granules, grind them finely, take 1g, add 10ml of ethanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to 1ml as the test solution. Take another alanine reference substance, add dilute ethanol to make a solution containing 0.5mg per 1ml, as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 15μl of the test solution and 1μl of the reference solution were taken and spotted on the same silica gel G thin layer plate, and developed with n-butanol-glacial acetic acid-water (4:1:1) as the developing agent. Develop, take out, dry, spray with ninhydrin test solution, and heat at 105°C until the spots are clearly colored. In the test sample chromatogram, spots of the same color appear at the corresponding position of the reference chromatogram. See the results. Figure 18 . Figure 18 Identification results of lotus root node and lotus root node charcoal granules using the Anhui Province method; 1: lotus root node granules; 2-3: lotus root node charcoal granules; 4-6: alanine reference substance.

[0119] Comparative Example 4

[0120] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them into powder, add 50mL of ethyl acetate respectively, reflux extraction for 0.5h, filter, concentrate the filtrate to about 2mL, transfer to a 5mL volumetric flask, dilute to the scale with ethyl acetate, shake well, and obtain. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), accurately aspirate 5μL of the test solution of lotus root nodes and lotus root node charcoal, respectively, and spot them on the same silica gel G thin layer plate with 0.3% sodium carboxymethyl cellulose as the adhesive, and use petroleum ether (60-90℃)-ethyl acetate (2:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol, heat at 105℃ to develop the color until the spots are clear, and inspect under natural light and 365nm ultraviolet light. The results are shown in Figure 19 . Figure 19Comparative Example 4 shows the identification results of lotus root nodes and lotus root node charcoal formula particles; among them, 1 to 4 were inspected under 365nm ultraviolet light, and the results were: 20μl of lotus root node formula particles, 20μl of lotus root node charcoal formula particles, 5μl of lotus root node formula particles, and 5μl of lotus root node charcoal formula particles; 5 to 8 were inspected under natural light, and the results were: 1. 20μl of lotus root node formula particles, 20μl of lotus root node charcoal formula particles, 5μl of lotus root node formula particles, and 5μl of lotus root node charcoal formula particles.

[0121] The results showed that Method 1 of Comparative Example 4 could not effectively distinguish between the lotus root node formula particles and the lotus root node charcoal formula particles when inspected at 365nm. When inspected under natural light, neither the lotus root node charcoal nor the lotus root node formula particles showed any spots, and the lotus root node formula particles and the lotus root node charcoal formula particles could be distinguished.

[0122] Comparative Example 5

[0123] Take 1g each of lotus root nodes and lotus root node charcoal granules, grind them into powder, add 50mL of ethyl acetate respectively, reflux extraction for 0.5h, filter, concentrate the filtrate to about 2mL, transfer to a 5mL volumetric flask, dilute to the scale with ethyl acetate, shake well, and obtain. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), accurately aspirate 5μL of the test solution of lotus root nodes and lotus root node charcoal, respectively, and spot them on the same silica gel G thin layer plate with 0.3% sodium carboxymethyl cellulose as the adhesive, and use petroleum ether (60-90℃)-ethyl acetate (8:5) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol, heat at 105℃ to develop the color until the spots are clear, and inspect under natural light and 365nm ultraviolet light. The results are shown in Figure 20 . Figure 20 Comparative Example 5 shows the identification results of lotus root nodes and lotus root node charcoal granules. Samples 1-4 were inspected under 365nm UV light, with a sample volume of 20μl for the lotus root node granules, 20μl for the lotus root node charcoal granules, 5μl for the lotus root node granules, and 5μl for the lotus root node charcoal granules. Samples 5-8 were inspected under natural light, with a sample volume of 20μl for the lotus root node granules, 20μl for the lotus root node charcoal granules, 5μl for the lotus root node granules, and 5μl for the lotus root node charcoal granules.

[0124] The results show that the method of Comparative Example 5 cannot effectively distinguish between lotus root node formula granules and lotus root node charcoal formula granules.

[0125] Comparative Example 6

[0126] Take 5g each of lotus root nodes and lotus root node charcoal granules, grind them into powder, add 100mL of 50% methanol and heat in a water bath for 40min, filter, recover the solvent in the filtrate until dry, and then use 150mL of ethyl acetate to heat in a water bath for 20min, filter, recover ethyl acetate, and adjust the volume of the residue to 5mL as the test solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0502), accurately aspirate 10μL of the test solution of lotus root nodes and lotus root node charcoal, respectively, and spot them on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as the adhesive, and use chloroform-ethyl acetate-formic acid (8:8:0.5) as the developing agent. Develop, take out, dry, and inspect under ultraviolet light at 365nm. The results are shown in Figure 21 . Figure 21 Comparative Example 6 shows the identification results of lotus root nodes and lotus root node charcoal formula particles; 1 to 2 are inspected under 365nm ultraviolet light, and are respectively: lotus root node formula particles and lotus root node charcoal formula particles.

[0127] The results show that the method in Comparative Example 6 cannot effectively distinguish between lotus root node formula granules and lotus root node charcoal formula granules.

[0128] 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 lotus root nodes and lotus root node charcoal particles, comprising: A) pre-treating the lotus root node formula granules to obtain a lotus root node test solution; The lotus root node charcoal formula particles are pretreated to obtain a lotus root node charcoal test solution; B) extracting a reference medicinal material of lotus root node, adding acid to reflux, then extracting with chloroform, evaporating to dryness, and dissolving the residue with methanol to obtain a reference medicinal material solution; C) The lotus root node test solution, the lotus root node charcoal test solution and the control medicinal material solution were subjected to thin layer chromatography detection using a silica gel G thin layer plate and an ether-chloroform-methanol developing solvent; D) Place the sample under ultraviolet light for inspection. If the lotus root node test sample and the control medicinal material solution have fluorescent spots of the same color at the same Rf, and no fluorescent spots are found at Rf = 0.486-0.602, then the sample is the lotus root node formula granule. If there are fluorescent spots at Rf=0.486~0.602, it is lotus root node charcoal formula particles.

2. The method according to claim 1, characterized in that The step A) of pre-treating the lotus root node formula particles is specifically as follows: The lotus root node formula granules are refluxed with 10% hydrochloric acid for extraction, and then extracted with chloroform, evaporated to dryness, and the residue is dissolved with methanol to obtain a lotus root node test solution; the chloroform extraction is performed 2 to 3 times.

3. The method according to claim 1, characterized in that Step A) The mass volume ratio of the lotus root node formula particles, 10% hydrochloric acid, and chloroform is 1g:20mL:60mL; and the reflux extraction time is 20 to 30 minutes.

4. The method according to claim 1, wherein The pretreatment of the lotus root node charcoal particles is specifically as follows: The lotus root charcoal granules are refluxed with 10% hydrochloric acid and then extracted with chloroform. The mixture is evaporated to dryness and the residue is dissolved in methanol to obtain a lotus root charcoal test solution. The chloroform extraction is repeated 2 to 3 times. The mass volume ratio of the lotus root node charcoal particles, 10% hydrochloric acid and chloroform is 1g:20mL:60mL; and the reflux extraction time is 20 to 30 minutes.

5. The method according to claim 1, wherein The sample spotting volume of the thin layer chromatography test sample is 2 to 6 μL, and the sample spotting volume of the control medicinal material solution is 3 to 7 μL.

6. The method according to claim 1, characterized in that Step C) The mass ratio of ether-chloroform-methanol is 10:6:

1.

7. The method according to claim 1, characterized in that The UV lamp in step D) is 365 nm.

8. The method according to claim 1, characterized in that The silica gel G thin layer plate in step D) is a prefabricated silica gel G plate produced by Tianjin Silida, Merck or Qingdao Ocean.

9. The method according to claim 8, characterized in that Step D) checking the temperature to be 5°C to 35°C; checking the humidity to be 32% rh to 75% rh.

10. 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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