Thin-layer identification method for smoked plum charcoal preparation

Abstract

The invention provides a dark plum charcoal preparation thin-layer identification method which comprises the following steps: A) taking a to-be-detected sample, and dissolving the to-be-detected sample in a solvent to obtain a test solution; the sample to be detected is a dark plum charcoal preparation; b) taking a smoked plum reference medicinal material, and dissolving with a solvent to obtain a reference medicinal material solution; c) performing thin-layer chromatography detection on the test solution and the reference medicinal material solution, wherein a thin-layer plate is a silica gel G thin-layer plate; the developing solvent is ethyl acetate-cyclohexane; and D) inspecting under an ultraviolet lamp, and if fluorescent spots with the same color are shown in the chromatogram of the sample to be tested and in the position corresponding to the chromatogram of the reference medicinal material, determining that the sample is the smoked plum charcoal formula granules. According to the established thin-layer identification chromatography method for the smoked plum charcoal formula granules, whether fluorescent spots exist at corresponding positions of a thin-layer chromatogram or not is observed to serve as identification points, and the smoked plum charcoal formula granules are rapidly and effectively identified.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a thin-layer identification method for smoked plum carbon formula granules. Background Art

[0002] Smoked plum has a long medicinal history. It was first recorded in "Shennong Ben Cao Jing" and classified as a medium-grade medicine, originally named "plum fruit". The name of smoked plum was first seen in "Ben Cao Jing Ji Zhu". Carbonized drugs are quite widely used and have functions such as astringency, drying dampness, removing stasis, stopping bleeding, and relieving diarrhea. They can be used for sores and ulcers, traumatic injuries and swelling, sore throat and oral ulcers, toothache, various bleeding disorders, dysentery with bloody and mucus stools, and chronic diarrhea. In "Zheng Lei Ben Cao" in the Song Dynasty, it was first mentioned that "burn smoked plum into powder and pound it". "Tang Ye Ben Cao" records that "to treat all kinds of malignant sores with exposed granulation tissue, burn smoked plum into powder and pound it for application". The clinical application history of smoked plum and smoked plum carbon is long. In clinical application, smoked plum has the efficacy of astringing the intestine and relieving diarrhea, and smoked plum carbon has an additional efficacy of stopping bleeding. Carbonized drugs are a type of traditional Chinese medicine with very distinctive processing characteristics and have a wide range of pharmacological effects. The common feature is their hemostatic effect. Carbonized drugs were first recorded in "Wu Shi Er Bing Fang" during the Warring States period, "To stop bleeding, burn hair". Regarding the hemostatic theory of carbonized drugs, there are many theories. It is generally believed that after traditional Chinese medicine is stir-fried into carbon, the chemical composition and content change, thus producing a hemostatic effect. The purpose of carbonizing smoked plum is mainly to facilitate preservation through heating and drying, and it is easier to decoct the medicinal effect, reduce its acidity to avoid damaging tendons and teeth when used raw, which is suitable for clinical application, and to increase the astringency through carbonization to achieve the effect of increasing astringing and stopping bleeding.

[0003] The textual research on the efficacy and clinical application in past dynasties shows that smoked plum has the functions of astringing the lung, astringing the intestine, promoting the production of body fluid, and expelling roundworms. It is used for chronic cough due to lung deficiency, chronic diarrhea and dysentery, consumptive thirst with deficiency heat, and vomiting and abdominal pain due to roundworm syncope. Smoked plum carbon is good at astringing and stopping bleeding and is commonly used for hematochezia, hematuria, metrorrhagia and metrostaxis, etc.

[0004] Since smoked plum carbon formula granules are made from smoked plum medicinal materials through a series of processes, and some of these processes will destroy the original morphological characteristics, the morphological identification method is not applicable to the identification of extracts and preparations. Therefore, it is very necessary to provide a simple thin-layer method for smoked plum carbon formula 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 identification method for smoked plum carbon formula granules.

[0006] In this article, the terms "include", "comprise" and "have" can be used interchangeably, aiming to indicate the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. At the same time, it should be understood that when using "include", "comprise" and "have" to describe in this article, the solution of "consisting of..." is also provided.

[0007] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Here, A and B can be singular or plural.

[0008] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single items or plural items.

[0009] It should be understood that in various embodiments of this application, the magnitudes of the sequence numbers of the following processes do 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 to the implementation process of the embodiments of this application.

[0010] The present invention discloses the construction and application of a thin-layer identification method for smoked plum carbon formula granules. This thin-layer method has strong specificity, good stability, high precision, is convenient and easy to master. At the same time, through this thin-layer method, with the smoked plum control medicinal material as a reference, the identification of smoked plum medicinal materials and smoked plum carbon decoction pieces can also be carried out.

[0011] The present invention provides a thin-layer identification method for smoked plum carbon preparations, including:

[0012] A) Take the test sample, dissolve it with a solvent to obtain a test solution; the test sample is a smoked plum carbon preparation;

[0013] B) Take the smoked plum control medicinal material, dissolve it with a solvent to obtain a control medicinal material solution;

[0014] C) Perform thin-layer chromatography detection on the test solution and the control medicinal material solution. The thin-layer plate is a silica gel G thin-layer plate; the developing agent is ethyl acetate - cyclohexane;

[0015] D) Examine under an ultraviolet lamp. If in the chromatogram of the test sample, at the position corresponding to the chromatogram of the control medicinal material, there are fluorescent spots of the same color, then it is smoked plum carbon formula granules.

[0016] The thin-layer identification method for smoked plum carbon formula granules provided by the present invention first takes the test sample, dissolves it with a solvent to obtain a test solution.

[0017] Specifically, step A) of the present invention is: take the test sample, add ethanol, shake, filter and evaporate to dryness, dissolve the residue with absolute ethanol to obtain a test solution;

[0018] Specifically, the mass-volume ratio of the test sample to ethanol is: 0.2 g: 5 - 15 mL.

[0019] In a specific embodiment, the mass-to-volume ratio of the sample to be tested and ethanol is: 0.2 g: 10 mL.

[0020] The shaking time is 1.5 - 2 h; preferably 2 h.

[0021] Take the control medicinal material of smoked plum, decoct it with water, filter, evaporate the filtrate to dryness, then add ethanol for extraction, shake, filter, evaporate the filtrate to dryness, and dissolve the residue in absolute ethanol to obtain it.

[0022] Specifically, the mass ratio of the control medicinal material of smoked plum, water and ethanol is 0.2 g: 10 - 20 mL: 5 - 10 mL.

[0023] In a specific embodiment, the mass ratio of the control medicinal material of smoked plum, water and ethanol is 0.2 g: 10 mL: 10 mL.

[0024] Perform thin-layer chromatography detection on the test solution and the control medicinal material solution.

[0025] Preferably: Conduct the test according to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition). Pipette the test solution and the control medicinal material solution, and spot them on the same silica gel G thin-layer plate respectively. Develop with the developing agent, take out, dry in air, and examine under ultraviolet light. The wavelength of the ultraviolet light is 365 nm.

[0026] According to the present invention, the developing agent is ethyl acetate - cyclohexane;

[0027] In some preferred embodiments of the present invention, the mass ratio of ethyl acetate - cyclohexane is 1:1.

[0028] The thin-layer plate of the present invention is a silica gel G thin-layer plate. It can be selected from Tianjin Sida, Merck or Qingdao Yumin silica gel G plates. The results show that the method has good durability and can meet the identification requirements.

[0029] Examine under ultraviolet light. If in the chromatogram of the test solution, at the position corresponding to the chromatogram of the control medicinal material, there are fluorescent spots of the same color, it is the smoked plum carbon formula granule.

[0030] The sample application amount for thin-layer chromatography in the present invention is 5 - 7 μL; specifically, it can be 5 μL, 6 μL, 7 μL; preferably 5 μL.

[0031] That is: When the sample application amount of the control medicinal material of smoked plum is 5 μL and the sample application amount of the finished product solution of the smoked plum carbon formula granule is 5 μL, the main spots are clearly visible. Therefore, the sample application amount of the control medicinal material of smoked plum is determined to be 5 μL, and the sample application amount of the finished product solution of the smoked plum carbon formula granule is determined to be 5 μL.

[0032] The inspection temperature of the present invention is 8°C to 25°C. Specifically, it can be 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; the method of the present invention has good adaptability to different temperatures and can meet the identification requirements.

[0033] 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, 54% rh, 55% rh, 56% rh, 57% rh, 58% rh, 59% rh, 60% rh, 61% rh, 62% rh, 63% rh, 64% rh, 65% rh, 66% rh, 67% rh, 68% rh, 69% rh, 70% rh, 71% rh, 72% rh, 73% rh, 74% rh, 75% rh;

[0034] The method of the present invention has good adaptability to different humidities and can meet the identification requirements

[0035] The prepared carbonized smoked plum preparation of the present invention includes carbonized smoked plum formula granules or carbonized smoked plum decoction pieces.

[0036] The present invention provides a thin-layer identification method for a prepared carbonized smoked plum preparation, including: A) taking a sample to be tested, dissolving it with a solvent to obtain a test solution; the sample to be tested is a prepared carbonized smoked plum preparation; B) taking a control medicinal material of smoked plum, dissolving it with a solvent to obtain a control medicinal material solution; C) performing thin-layer chromatography detection on the test solution and the control medicinal material solution, and the thin-layer plate is a silica gel G thin-layer plate; the developing agent is ethyl acetate - cyclohexane; D) observing under an ultraviolet lamp. If in the test solution chromatogram, at the position corresponding to the control medicinal material chromatogram, there are fluorescent spots of the same color, then it is carbonized smoked plum formula granules. The thin-layer identification chromatographic method for carbonized smoked plum formula granules established by the present invention uses the presence or absence of fluorescent spots at the corresponding positions in the thin-layer chromatogram as the identification point to quickly and effectively identify carbonized smoked plum formula granules.

[0037] The present invention discloses the construction and application of a thin-layer identification method for carbonized smoked plum granules and carbonized smoked plum decoction pieces, avoiding the use of toxic reagents such as ether and chloroform. This method has good stability, high precision, good reproducibility, is convenient and easy to master. Description of the Drawings

[0038] Figure 1 Investigation of the sample application amount;

[0039] Figure 2 Specificity investigation;

[0040] Figure 3 Investigation with different TLC plates;

[0041] Figure 4 Investigation at different temperatures (8 °C);

[0042] Figure 5 Investigation at different temperatures (25 °C);

[0043] Figure 6 Investigation at different humidities (32%);

[0044] Figure 7 Investigation at different humidities (75%);

[0045] Figure 8 For thin-layer analysis of 3 batches of finished products;

[0046] Figure 9 For the thin-layer chromatogram of the first developing solvent of Comparative Example 1;

[0047] Figure 10 For the thin-layer chromatogram of the second developing solvent of Comparative Example 1. Detailed implementation mode

[0048] To further illustrate the present invention, the following describes in detail a method for constructing a thin-layer identification method for a prepared black plum carbon formula granule provided by the present invention in combination with embodiments.

[0049] Example 1

[0050] 1 Instruments and test drugs

[0051] 1.1 Instruments

[0052] Hot plate, mortar, thin-layer imaging system: CAMAG TLC Visualizer, silica gel G thin-layer plates: Qingdao Yuminyuan, Tianjin Sida Technology Co., Ltd., Merck;

[0053] 1.2 Reagents

[0054] Chemical reagents such as ethanol, ethyl acetate, cyclohexane, butyl acetate, formic acid, and methanol are all of analytical grade, and water is ultrapure water (self-made in the laboratory).

[0055] 1.3 Test drugs

[0056] Prepared black plum reference medicinal material (National Institutes for Food and Drug Control, batch number: 121208 - 201707); batch numbers of prepared black plum carbon formula granules: WMT01, WMT02, WMT03,

[0057] 2.3 Establishment of thin-layer method

[0058] 2.3.1 Preparation of Test Solution

[0059] Take 0.2 g of smoked plum carbon formula granules, grind them finely, add 10 ml of ethanol, shake for 2 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of absolute ethanol to obtain the test solution.

[0060] 2.3.2 Preparation of Reference Substance and Reference Medicinal Material Solutions

[0061] Take 0.2 g of reference medicinal material of smoked plum, add 10 ml of water, decoct for 20 minutes, filter, evaporate the filtrate to dryness, add 10 ml of ethanol, shake for 2 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of absolute ethanol to obtain the reference medicinal material solution.

[0062] 2.3.3 Determination Method

[0063] Perform the test according to the thin-layer chromatography method (General Principles 0502 of Chinese Pharmacopoeia 2020 Edition). Respectively draw 5 μl of each of the above two solutions and spot them on the same silica gel G thin-layer plate. Use the solution of ethyl acetate - cyclohexane (1:1) as the developing agent, develop, take out, dry in air, and examine under ultraviolet light (365 nm). In the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference medicinal material, there are main fluorescent spots showing the same color.

[0064] Example 2 Methodology Investigation

[0065] 2.4.1 Investigation of Spotting Volume

[0066] Prepare the reference medicinal material of smoked plum and the test solution according to the above preparation method of the test solution, and investigate the differences in different spotting volumes. The results are shown in Figure 1 . Figure 1 Investigation of spotting volume, where 1: reference medicinal material 3 μl; 2: reference medicinal material 5 μl; 3: reference medicinal material 7 μl; 4: granules 3 μl; 5: granules 5 μl; 7: granules 7 μl;

[0067] It can be seen from the results that when the spotting volume of the reference medicinal material of smoked plum is 5 μl and the spotting volume of the finished product solution of smoked plum carbon formula granules is 5 μl, the main spots are clearly visible. Therefore, the spotting volume of the reference medicinal material of smoked plum is determined to be 5 μl, and the spotting volume of the finished product solution of smoked plum carbon formula granules is determined to be 5 μl.

[0068] 2.4.2 Investigation of Specificity

[0069] Prepare the negative control solution, reference medicinal material and test solution according to the above proposed method. The results are shown in Figure 2 .

[0070] Figure 2 Investigation of specificity, 1: negative control; 2: reference medicinal material of smoked plum; 3: smoked plum carbon granules 20040129; The results show that the specificity of this method is good.

[0071] 2.4.3 Durability Investigation

[0072] 2.4.3.1 Comparison of Different Thin Layer Plates

[0073] Select the silica gel G plates from Qingdao Yumin and the cuttable thin layer chromatography plates (silica gel G plates) from Tianjin Sida Technology Co., Ltd., and the silica gel G plates from Merck, and conduct tests according to the proposed test methods. See Figure 3 。 Figure 3 For the investigation of different thin layer plates, 1. Fructus Mume control crude drug; 2. Fructus Mume Carbonisata formula granules (20040129).

[0074] The results show that the thin layer plates from these 3 manufacturers can all meet the identification requirements.

[0075] 2.4.3.2 Investigation at Different Temperatures

[0076] Prepare the test solution and the control crude drug solution according to the selected method and spot them. Take the spotted thin layer plate and develop it in the temperature environments of 8°C and 25°C respectively. The results are shown in Figures 4-5 。

[0077] Figure 4 Investigation at different temperatures (8°C), Figure 5 Investigation at different temperatures (25°C); 1. Fructus Mume control crude drug; 2. Fructus Mume Carbonisata formula granules (20040129); The results show that both 8°C and 25°C can meet the identification requirements.

[0078] 2.4.4 Investigation at Different Humidities

[0079] Prepare the test solution and the control crude drug solution according to the selected method and spot them. Take the spotted thin layer plate and develop it in the humidity environments of 32% and 75% respectively. The results are shown in Figures 6-7 。 Figure 6 Investigation at different humidities (32%); Figure 7 Investigation at different humidities (75%); Among them, 1. Fructus Mume control crude drug; 2. Fructus Mume Carbonisata formula granules (20040129) The results show that both 32% and 75% of humidity can meet the identification requirements.

[0080] 2.4.5 Verification

[0081] Conduct thin layer identification on 3 batches of Fructus Mume Carbonisata finished products according to the proposed method. The results are shown in Figure 8 。 Figure 8 For the thin layer analysis of 3 batches of finished products; among them, 1. Fructus Mume control crude drug; 2. Granules (WMT01); 3. Granules (WMT02); 4. Granules (WMT03).

[0082] Comparative Example 1

[0083] Preparation of test sample: Take 0.2 g of this product, grind it finely, add 10 ml of ethanol, shake for 2 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of absolute ethanol to obtain the test sample solution.

[0084] Preparation of control crude drug: Take 0.2 g of Fructus Mume control crude drug, add 10 ml of water, decoct for 20 minutes, filter, evaporate the filtrate to dryness, add 10 ml of ethanol, shake for 2 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 2 ml of absolute ethanol to obtain the control crude drug solution. The following results were obtained by investigating with the following 2 different developing agents.

[0085] The first developing agent: methanol - water - glacial acetic acid (18:1:1). The results are as Figure 9 , and it can be seen from Figure 9 that the spots of the test sample and the control crude drug are not clearly separated, and the separation effect is also worse than that of the present invention, and the preparation of carbonized Fructus Mume cannot be well separated.

[0086] The second developing agent: cyclohexane - ethyl acetate - isopropanol - methanol - water - triethylamine (3:3.5:1:1.5:0.5:1). The results are as Figure 10 , and it can be seen from Figure 10 that the Rf value of this developing agent is relatively high, and the main spot is near the solvent front, and the separation effect is also worse than that of the present invention, and the ratio of the solvent in the developing agent used is more than that of the present invention.

[0087] Among them, 1: Fructus Mume control crude drug; 2: carbonized Fructus Mume formula granules. Through the above comparative tests, it is found that compared with the above 3 developing agents, the present invention uses less solvent in the developing agent ratio, saving time and cost. Moreover, the thin layer identification method of the present invention can effectively and quickly separate the preparation of carbonized Fructus Mume. This method is simple to operate, has high sensitivity and good stability, and has good application prospects.

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A thin layer identification method for a ebony charcoal preparation, comprising: A) Take the sample to be tested, dissolve it in solvent, and obtain the test solution; The sample to be tested is a ebony charcoal preparation; B) taking a black plum reference medicinal material, dissolving it with a solvent, and obtaining a reference medicinal material solution; C) The test solution and the control medicinal material solution are subjected to thin layer chromatography, the thin layer plate is a silica gel G thin layer plate, and the developing solvent is ethyl acetate-cyclohexane; D) Check under ultraviolet light. If the test sample chromatogram shows fluorescent spots of the same color at the corresponding positions of the control medicinal material chromatogram, it is the black plum charcoal formula granules.

2. The method according to claim 1, characterized in that The step A) is specifically as follows: adding ethanol to the sample to be tested, shaking, filtering, evaporating to dryness, and dissolving the residue in anhydrous ethanol to obtain a test solution; the shaking time is 1.5 to 2 hours; The mass volume ratio of the sample to be tested and ethanol is: 0.2g:5-15mL.

3. The method according to claim 1, characterized in that The step B) is specifically as follows: Take the black plum reference medicinal material, add water to boil, filter, evaporate the filtrate to dryness, then add ethanol to extract, shake, filter, evaporate the filtrate to dryness, and dissolve the residue in anhydrous ethanol to obtain; The mass ratio of the black plum reference medicinal material, water and ethanol is 0.2g:10-20mL:5-10mL.

4. The method according to claim 1, characterized in that The silica gel G thin layer plate is a silica gel G plate produced by Tianjin Silida, Merck or Qingdao Yumin.

5. The method according to claim 1, characterized in that The sample spotting volume of the thin layer chromatography is 5 to 7 μL.

6. The method according to claim 1, characterized in that Step C) The mass ratio of ethyl acetate to cyclohexane is 1:

1.

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

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

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

10. The method according to claim 1, characterized in that The ebony charcoal preparation includes ebony charcoal formula granules or ebony charcoal slices.

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