A thin-layer chromatography method for identifying kelp or its formulation granules.

Abstract

This invention relates to the field of traditional Chinese medicine identification technology, specifically to a thin-layer chromatography (TLC) identification method for kelp or its preparations. The method includes the following steps: (1) preparation of the test solution and the reference herb solution; (2) spotting the test solution and the reference herb solution, developing the mixture of water, anhydrous ethanol, and n-butanol as the developing solvent, removing the sample, and then spraying a colorimetric reagent for color development. This TLC identification method has the advantages of simple test solution preparation and non-toxicity. The obtained TLC chromatogram shows rich spot information, good separation, clear color development, and accurate identification results, which is beneficial for the quality control of kelp and its preparations.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine identification technology, specifically to a thin-layer chromatography method for the identification of kelp and its preparations. Background Technology

[0002] Kelp is the dried thallus of *Laminaria japonica* Aresch. (family Laminariaceae) or *Ecklonia kurome* Okam. (family Ecklonaceae), both perennial algae. Kelp has a long history of medicinal use in my country and is a commonly used medicinal material. It is cold in nature and salty in taste, and enters the liver, stomach, and kidney meridians. It belongs to the category of traditional Chinese medicine that softens and disperses phlegm and nodules. Its functions include resolving phlegm, softening and dispersing phlegm and nodules, promoting diuresis and reducing swelling. It can be used for goiter, scrofula, testicular swelling and pain, and edema due to phlegm retention.

[0003] Kelp has a wide range of clinical applications. For example, when combined with fritillaria, green tangerine peel, and pinellia, it can be used to treat early-stage goiter, whether hard or swollen; when combined with green tangerine peel, aloe vera, and seaweed powder, it can be used to clear liver stagnation, relieve depression, and resolve phlegm; when combined with ginseng, angelica, and prepared rehmannia root, it can be used to treat long-standing goiter with qi and blood deficiency; when combined with notopterygium root, saposhnikovia root, seaweed, and forsythia, it can be used to treat chills and fever, and early-stage scrofula; when combined with ginseng, angelica, and cyperus rhizome, it can... It is used to treat scrofula caused by liver qi stagnation and qi and blood deficiency; when combined with Bupleurum, Sargassum, Gentiana, and Sparganium, it can be used to treat scrofula that is widespread on the jaw, hard and not ulcerated, with excessive heat and toxicity; when combined with Sargassum, Citrus reticulata seed, Cinnamomum cassia bark, and Corydalis yanhusuo, it can be used to treat testicular distension and pain caused by cold and dampness in the lower jiao, qi stagnation and blood stasis; when combined with Citrus reticulata peel, Pinellia ternata, and Poria cocos, it can be used to treat globus hystericus; when combined with Areca peel, Stephania tetrandra, and Plantago asiatica, it can be used to treat beriberi edema. In modern clinical practice, kelp is often used to treat retinal concussion, thyroid diseases, hypertension, chronic pelvic inflammatory disease, cancer, breast hyperplasia, phlebitis, viral anicteric hepatitis, constipation, vitreous opacity, and senile cataracts. Modern pharmacodynamic studies have shown that kelp has various pharmacological effects, including affecting the cardiovascular system, lowering blood lipids, lowering blood sugar, anticoagulation, anti-radiation, antitumor, antitussive, antiasthmatic, antimutagenic, antioxidant, antiviral, antibacterial, and affecting immune function.

[0004] The current thin-layer chromatography method for kelp is as follows: Take an appropriate amount of the product, grind it into a fine powder, take 2g, add 20ml of ethanol, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1ml of ethanol to prepare the test solution. Separately, take 2g of kelp reference material, soak for 30 minutes, decoct twice. For the first decoction, add 50ml of water and decoct for 30 minutes; for the second decoction, add 30ml of water and decoct for 20 minutes. Combine the decoctions, evaporate to dryness, and prepare the reference solution from the residue starting with "add 20ml of ethanol...". Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 General Chapter 0502). Apply 10μl of the test solution and 5μl of the reference solution separately to the same silica gel G thin-layer plate. Develop using chloroform-butanone (10:1) as the developing solvent. Remove, air dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position as in the chromatogram of the reference medicinal material. However, studies have shown that this method only produces one spot, resulting in poor specificity, making it difficult to conduct comprehensive and accurate quality testing of kelp and its preparations. Summary of the Invention

[0005] To address the problem that existing thin-layer chromatography methods for kelp (kelp) are insufficient for comprehensive and accurate quality testing of kelp (kelp) and its preparations, this invention provides a thin-layer chromatography identification method for kelp and its preparations.

[0006] This invention discloses a thin-layer chromatography method for identifying kelp or its preparations, comprising the following steps:

[0007] (1) Preparation of test solution and reference solution; the reference solution includes the control herbal material solution.

[0008] (2) According to the thin-layer chromatography method, take the test solution and reference solution, spot them, use a mixture of water, anhydrous ethanol and n-butanol as the developing solvent, develop, take out, and then spray the color developing agent to develop the color.

[0009] Furthermore, the kelp or its preparation is kelp (seaweed) or its preparation.

[0010] Further, in step (2), the developing agent is a mixture with a volume ratio of 10-15:10-15:8-12;

[0011] Preferably, the developing agent further includes glacial acetic acid or formic acid;

[0012] Preferably, the developing agent is a mixture of water, anhydrous ethanol, n-butanol and glacial acetic acid in a volume ratio of 10-15:10-15:10:0.1-0.3.

[0013] Preferably, the developing agent is a mixture of water, anhydrous ethanol, n-butanol and formic acid in a volume ratio of 10-15:10-15:10:0.1-0.3;

[0014] Preferably, the developing agent is a mixture of water, anhydrous ethanol, n-butanol and glacial acetic acid in a volume ratio of 10:15:10:0.1.

[0015] Furthermore, in step (2), the colorimetric agent is ninhydrin solution.

[0016] Further, in step (2), the amount of the test sample solution is 1-4 μL, preferably 2-3 μL; and / or, the amount of the reference solution is 1-4 μL, preferably 2-3 μL.

[0017] Further, in step (1), the preparation method of the test sample solution includes mixing the test sample with a solvent, and then obtaining the solution through extraction, solid-liquid separation, liquid evaporation and reconstitution steps.

[0018] Further, the solvent is selected from one or more of ethanol, water, and methanol; and / or, the extraction method is selected from heating reflux extraction or ultrasonic extraction; and / or, the extraction time is 25-35 min, and the number of extractions is 1-3; and / or, the mass ratio of the test sample to the volume of the solvent is 1.8-2.2 g: 18-22 mL; and / or, the solvent for reconstitution is selected from one or more of ethanol and methanol; and / or, the mass ratio of the test sample to the mass of the solvent for reconstitution is 1.8-2.2 g: 0.8-1.2 mL.

[0019] Further, in step (1), the preparation method of the reference medicinal material solution includes mixing the reference medicinal material extract with a solvent, and then obtaining the solution through extraction, solid-liquid separation, liquid evaporation and reconstitution steps.

[0020] Further, the solvent is selected from one or more of ethanol, water, and methanol; and / or, the extraction method is selected from heating reflux extraction or ultrasonic extraction; and / or, the extraction time is 25-35 min, and the number of extractions is 1-3; and / or, the mass ratio of the reference herb extract to the solvent volume is 1.8-2.2 g: 18-55 mL; and / or, the solvent for reconstitution is selected from one or more of ethanol and methanol; and / or, the mass ratio of the reference herb extract to the reconstitution solvent is 1.8-2.2 g: 0.8-1.2 mL.

[0021] Furthermore, the preparation of the reference solution also includes the preparation of a standard solution:

[0022] Take alanine and add solvent to prepare a solution containing 1.8–2.2 mg per 1 ml, which is used as alanine reference solution.

[0023] Furthermore, the test sample is selected from kelp medicinal material, kelp slices, kelp extract, or kelp formula granules.

[0024] The technical solution of this invention has the following advantages:

[0025] 1. The thin-layer chromatography method for identifying kelp or its preparations provided by the present invention includes the following steps: (1) preparation of the test solution and the reference solution; the reference solution includes a reference medicinal material solution; (2) according to the thin-layer chromatography method, taking the test solution and the reference medicinal material solution, spotting them, using a mixture of water, anhydrous ethanol, and n-butanol as the developing solvent, developing, removing, and then spraying a colorimetric reagent for color development. The present invention uses a mixture of water, anhydrous ethanol, n-butanol, and glacial acetic acid as the developing solvent. This developing solvent system, when combined with the test solution, yields rich spot information, good separation, clear color development, and accurate identification results in the thin-layer chromatography, enabling comprehensive and accurate quality control of kelp or its preparations.

[0026] 2. The thin-layer chromatography method for identifying kelp or its preparations provided by the present invention further includes glacial acetic acid or formic acid in the developing solvent. The addition of glacial acetic acid or formic acid can further enrich the spot information, improve the separation, and enhance the color development in the thin-layer chromatography. In particular, the developing solvent with a volume ratio of water, anhydrous ethanol, n-butanol, and glacial acetic acid of 10:15:10:0.1 produces clear spots, good separation, and more spots with richer information in the thin-layer chromatography.

[0027] 3. The thin-layer identification method for kelp or its preparations provided by the present invention, in step (1), the preparation method of the test sample solution includes mixing the test sample with a solvent, and then extracting, separating solid and liquid, evaporating the liquid to dryness and reconstituted. The thin-layer identification method has the advantages of simple test sample solution preparation and non-toxicity. The obtained thin-layer chromatogram has rich spot information, good separation, clear chromatogram color development, and accurate identification results, which is beneficial to the quality control of kelp or its preparations.

[0028] 4. The thin-layer chromatography identification method for kelp or its preparations provided by the present invention further includes the preparation of a reference solution: alanine is taken and a solvent is added to prepare a solution containing 1.8-2.2 mg per 1 ml, which is used as an alanine reference solution. Compared with the prior art, the thin-layer chromatography identification method of the present invention uses alanine as a reference and reference medicinal material as control indicators, resulting in richer spot information in the obtained thin-layer chromatography, more accurate identification results, and the ability to identify alanine and amino acid components of the indicator components in kelp and its preparations, making quality control more comprehensive and accurate.

[0029] 5. The thin-layer chromatography identification method for kelp or its preparations provided by the present invention has good durability against environmental factors such as temperature and humidity, good reproducibility for thin-layer plates from different manufacturers, and clear spots in the thin-layer chromatograms at different spotting amounts, demonstrating good reproducibility. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 The thin-layer identification chromatograms in Example 1 are shown in Figure (1). In Figure (1), 1 is the negative control solution; 2 is the reference solution; 3 is the reference medicinal material solution; 4-6 are the test solutions, and the test solutions correspond to the batch numbers K402CP01, K402CP02, and K402CP03 respectively. In Figure (2), 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from the freeze-dried powder of kelp (seaweed) standard decoction.

[0032] Figure 2 The thin-layer chromatography pattern is shown in Comparative Example 1, where 1 is the reference herb solution and 2 is the test sample solution.

[0033] Figure 3 The thin-layer identification chromatogram is shown in Example 2, where 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp (seaweed) formula granules (K402CP01).

[0034] Figure 4 The thin-layer identification chromatogram is shown in Example 3, where 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp (seaweed) formula granules (K402CP01).

[0035] Figure 5 The thin-layer identification chromatogram is shown in Example 4, where 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp (seaweed) formula granules (K402CP01).

[0036] Figure 6 The thin-layer identification chromatogram is shown in Example 5, where 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp (seaweed) formula granules (K402CP01).

[0037] Figure 7The thin-layer identification chromatogram is shown in Example 6, where 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp (seaweed) formula granules (K402CP01).

[0038] Figure 8 The thin-layer identification chromatogram is shown in Example 7, where 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp (seaweed) formula granules (K402CP01).

[0039] Figure 9 The thin-layer identification chromatogram is shown in Example 8, where 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp (seaweed) formula granules (K402CP01).

[0040] Figure 10 The thin-layer chromatography identification chromatograms are shown in Example 1. In this chromatogram, 1 is the negative control solution; 2 is the reference solution; 3-6 are the reference medicinal material solutions, with spotting volumes of 1 μl, 2 μl, 3 μl, and 4 μl, respectively; and 7-10 are the test solutions, with spotting volumes of 1 μl, 2 μl, 3 μl, and 4 μl, respectively.

[0041] Figure 11 The thin-layer chromatography identification spectrum is shown in Experiment Example 2 at a temperature of 26°C and a humidity of 60%. Among them, 1 is the negative control solution; 2 is the reference solution; 3 is the reference medicinal material solution; and 4 is the test solution.

[0042] Figure 12 The thin-layer chromatography identification spectrum is shown in Experiment Example 2 at a temperature of 6°C and a humidity of 85%. Among them, 1 is the negative control solution; 2 is the reference solution; 3 is the reference medicinal material solution; and 4 is the test solution.

[0043] Figure 13 The thin-layer chromatography identification spectrum is shown in Experiment Example 2 at a temperature of 26°C and a humidity of 88%. Among them, 1 is the negative control solution; 2 is the reference solution; 3 is the reference medicinal material solution; and 4 is the test solution.

[0044] Figure 14 The thin-layer chromatography identification spectrum is shown in Experiment Example 2 at a temperature of 26°C and a humidity of 23%. Among them, 1 is the negative control solution; 2 is the reference solution; 3 is the reference medicinal material solution; and 4 is the test solution.

[0045] Figure 15 The thin-layer chromatography identification spectrum for Experiment Example 2 is shown by Yantai Chemical Industry Research Institute. In this spectrum, 1 is the negative control solution; 2 is the reference solution; 3 is the reference medicinal material solution; and 4 is the test solution.

[0046] Figure 16The thin-layer chromatography identification spectrum is shown in Experiment Example 2 - Qingdao Ocean Chemical Co., Ltd., where 1 is the negative control solution; 2 is the reference solution; 3 is the reference medicinal material solution; and 4 is the test solution.

[0047] Figure 17 The thin-layer chromatography identification spectrum in Experiment Example 2 is from Yantai Jiangyou Silica Gel Development Co., Ltd., where 1 is the negative control solution; 2 is the reference solution; 3 is the reference medicinal material solution; and 4 is the test solution. Detailed Implementation

[0048] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0049] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0050] Instruments, reagents and reagents:

[0051] Instruments: ME104E electronic balance (Mettler Toledo Scientific Instruments (Shanghai) Co., Ltd.), JY20002 electronic balance (Shanghai Sunny Hengping Scientific Instruments Co., Ltd.), BSA124S electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), DZKW-4 constant temperature water bath (Beijing Zhongxing Weiye Instruments Co., Ltd.), silica gel G plate (Qingdao Haiyang Chemical Co., Ltd., Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai Huayang New Material Technology Co., Ltd.).

[0052] Test drug: Kelp (kelp) formula granules (batch numbers: K402CP01, K402CP02, K402CP03);

[0053] Preparation method of kelp (kelp) formula granules: Prepared by Beijing Kangrentang Pharmaceutical Co., Ltd.; The preparation method of kelp (kelp) formula granules is as follows: Take kelp (kelp), heat and reflux extract twice. For the first extraction, add 14 times the weight of water to kelp (kelp), heat and reflux extract for 1 hour, filter. For the second extraction, add 12 times the weight of water to kelp (kelp), heat and reflux extract for 1 hour, filter, combine the filtrates, concentrate the filtrate to a relative density of 1.0 g / mL (measured at 60℃), spray dry, add maltodextrin (the total weight of the dried powder after adding maltodextrin accounts for 45.4% of the kelp (kelp) feed amount) to the spray-dried powder, mix evenly, and then dry granulate to make granules.

[0054] Freeze-dried powder of standard decoction of kelp: Take 100-150g of kelp slices and place them in a decoction pot. For the first decoction, add 16 times the amount of water as the slices, soak in cold water for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. Filter while hot through a 150-mesh filter cloth and cool quickly for later use. For the second decoction, add 14 times the amount of water as the slices, bring to a boil over high heat (500W), then simmer over low heat (200W) for 20 minutes. Filter while hot through a 150-mesh filter cloth and cool quickly for later use. Combine the filtrates and concentrate under reduced pressure (temperature set at 65℃, vacuum degree -0.1MPa) until the material-to-liquid ratio is approximately 1:1 (relative density 1.05-1.10 (65℃)). Freeze-dry to obtain a fine powder.

[0055] Kelp (kelp) reference medicinal material (batch number: AY300005-202305, Foshan Aoyu Biotechnology Co., Ltd.);

[0056] Kelp (kelp) slices were purchased from (batch number: K402YP01, manufactured by Beijing Kangrentang Pharmaceutical Co., Ltd.);

[0057] Alanine (batch number: 140680-202206, China National Institutes for Food and Drug Control);

[0058] Reagents: Ethanol, n-butanol, glacial acetic acid, formic acid, and ninhydrin were all of analytical grade, and water was Watson's distilled water.

[0059] Example 1

[0060] This embodiment provides a thin-layer chromatography method for the identification of kelp and its preparations, including the following steps:

[0061] Preparation of the test solution: Take 2.0 g of kelp (seaweed) formula granules (batch numbers: K402CP01, K402CP02, K402CP03) or lyophilized kelp (seaweed) standard decoction, add 20 ml of ethanol, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, and then add 1 ml of ethanol to the residue after evaporation to reconstitute it as the test solution.

[0062] Preparation of the reference herb solution: Take 2.0g of kelp (seaweed) reference herb, add 50ml of water, heat under reflux twice, 30 minutes each time, centrifuge, combine the supernatants, evaporate to dryness, add 20ml of ethanol to the residue, heat under reflux for 30 minutes, filter, take the filtrate and evaporate to dryness, then add 1ml of ethanol to the evaporated residue to reconstitute and prepare the reference herb solution.

[0063] Preparation of reference solution: Take alanine reference standard and add ethanol to prepare a solution containing 2 mg per 1 ml, which is used as the reference solution.

[0064] Preparation of negative control solution: According to the proportion of excipients in the kelp (seaweed) formula granules, take an appropriate amount of maltodextrin and prepare it in the same way as the test sample solution, as a negative control solution.

[0065] After 15 minutes of routine pre-equilibration of the thin-layer plate, according to the thin-layer chromatography method, 2 μL of the test solution, reference medicinal material solution, reference solution, and negative control solution were respectively applied to the same silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.) and placed in a double-groove developing tank. The temperature was 26℃ and the humidity was 60%. A mixture of water, anhydrous ethanol, n-butanol, and glacial acetic acid with a volume ratio of 10:15:10:0.1 was used as the developing solvent. The plate was developed upwards. When the development distance was 8 cm, the plate was removed, air-dried, sprayed with ninhydrin reagent, and examined under sunlight.

[0066] See results Figure 1 As can be seen from the image, the spots in the thin-layer chromatography are clear. Figure 1 In (1) to (2), the chromatograms of the test sample (whether it is a standard decoction freeze-dried powder or a formula granule) and the chromatograms of the reference medicinal material solution and the reference solution show the same color spots at the same position, indicating that this method meets the requirements of thin-layer identification analysis, and the chromatograms show richer spot information.

[0067] Comparative Example 1

[0068] This comparative example provides a thin-layer chromatography method for the identification of kelp and its preparations, comprising the following steps:

[0069] Preparation of test solution: Take an appropriate amount of kelp formula granules (K402CP01), grind them finely, take 2g, add 20ml of ethanol, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution.

[0070] Preparation of the reference herb solution: Take 2g of kelp (seaweed) as reference herb, soak for 30 minutes, decoct twice. Add 50ml of water for the first decoction and decoct for 30 minutes. Add 30ml of water for the second decoction and decoct for 20 minutes. Combine the decoctions, evaporate to dryness, and prepare the reference herb solution from the residue starting from "add 20ml of ethanol...".

[0071] Preparation of standard decoction: Take 100-150g of kelp slices and place them in a decoction pot. For the first decoction, add 16 times the weight of water to the slices, soak in cold water for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. Filter while hot through a 150-mesh filter cloth and cool quickly for later use. For the second decoction, add 14 times the weight of water to the slices, bring to a boil over high heat (500W), then simmer over low heat (200W) for 20 minutes. Filter while hot through a 150-mesh filter cloth and cool quickly for later use. Combine the filtrates and concentrate under reduced pressure (temperature set at 65℃, vacuum degree -0.1MPa) until the material-to-liquid ratio is approximately 1:1 (relative density 1.05-1.10 (65℃)). Freeze-dry to obtain a fine powder.

[0072] Perform the thin-layer chromatography test (General Chapter 0502 of the Chinese Pharmacopoeia 2020 Edition). Take 10 μl of the test solution, 5 μl of the reference herb solution, and 10 μl of the standard decoction, and spot them separately on the same silica gel G thin-layer plate. Use chloroform-butanone (10:1, v / v) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365 nm).

[0073] See results Figure 2 As can be seen from the figure, the spots in the control herbal solution and the test sample solution are inconsistent.

[0074] Example 2

[0075] This embodiment provides a thin-layer chromatography method for the identification of kelp and its preparations, including the following steps:

[0076] Preparation of the test solution: Take 2g of kelp (seaweed) formula granules (batch number: K402CP01), add 20ml of ethanol, heat under reflux for 30 minutes and filter, take the filtrate and evaporate to dryness, then add 1ml of methanol to the residue after evaporation to reconstitute as the test solution.

[0077] Preparation of the reference herb solution: Take 2g of kelp (seaweed) reference herb, add 30ml of water, heat under reflux twice, 30 minutes each time, centrifuge, combine the supernatants, evaporate to dryness, add 20ml of ethanol to the residue, heat under reflux for 30 minutes, filter, take the filtrate and evaporate to dryness, then add 1ml of methanol to the evaporated residue to reconstitute and prepare the reference herb solution.

[0078] Preparation of reference solution: Take alanine reference standard and add methanol to prepare a solution containing 2 mg per 1 ml, which is used as the reference solution.

[0079] Preparation of negative control solution: According to the proportion of excipients in the kelp (seaweed) formula granules, take an appropriate amount of maltodextrin and prepare it in the same way as the test sample solution, as a negative control solution.

[0080] After 15 minutes of routine pre-equilibration of the thin-layer plate, according to the thin-layer chromatography method, 4 μL of the test solution, reference medicinal material solution, reference solution, and negative control solution were respectively applied to the same silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.) and placed in a double-groove developing tank. The temperature was 26℃ and the humidity was 60%. A mixture of water, anhydrous ethanol, n-butanol, and glacial acetic acid with a volume ratio of 15:15:10:0.1 was used as the developing solvent. The plate was developed upwards. When the development distance was 8 cm, the plate was removed, air-dried, sprayed with ninhydrin reagent, and examined under sunlight.

[0081] See results Figure 3 As can be seen from the figure, the spots in the thin-layer chromatogram are clear. The test sample chromatogram, the reference medicinal material solution chromatogram, and the reference solution chromatogram show the same color spots in the same position, indicating that this method meets the requirements of thin-layer identification analysis. Moreover, the chromatographic spot information is richer from the figure.

[0082] Example 3

[0083] This embodiment provides a thin-layer chromatography method for the identification of kelp and its preparations, including the following steps:

[0084] Preparation of the test solution: Take 2.2g of kelp (seaweed) formula granules (batch number: K402CP01), add 22ml of water, heat under reflux for 35 minutes and filter, take the filtrate and evaporate to dryness, then add 1.2ml of ethanol to the residue after evaporation to redissolve, as the test solution.

[0085] Preparation of the reference herb solution: Take 2.2g of kelp (seaweed) reference herb, add 55ml of water, heat under reflux twice, 35 minutes each time, centrifuge, combine the supernatants, evaporate to dryness, add 22ml of ethanol to the residue, heat under reflux for 35 minutes and filter, take the filtrate and evaporate to dryness, then add 1.2ml of ethanol to the evaporated residue to redissolve and prepare the reference herb solution.

[0086] Preparation of reference solution: Take alanine reference standard and add ethanol to prepare a solution containing 2.2 mg per ml, which is used as the reference solution.

[0087] Preparation of negative control solution: According to the proportion of excipients in the kelp (seaweed) formula granules, take an appropriate amount of maltodextrin and prepare it in the same way as the test sample solution, as a negative control solution.

[0088] After 15 minutes of routine pre-equilibration of the thin-layer plate, according to the thin-layer chromatography method, 3 μL of the test solution, reference medicinal material solution, reference solution, and negative control solution were respectively applied to the same silica gel G thin-layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.) and placed in a double-groove developing tank. The temperature was 26℃ and the humidity was 60%. A mixture of water, anhydrous ethanol, n-butanol, and glacial acetic acid with a volume ratio of 10:15:10:0.1 was used as the developing solvent. The plate was developed upwards. When the development distance was 8 cm, the plate was removed, air-dried, sprayed with ninhydrin reagent, and examined under sunlight.

[0089] See results Figure 4 As can be seen from the figure, the spots in the thin-layer chromatogram are clear. The test sample chromatogram, the reference medicinal material solution chromatogram, and the reference solution chromatogram show the same color spots in the same position, indicating that this method meets the requirements of thin-layer identification analysis. Moreover, the chromatographic spot information is richer from the figure.

[0090] Example 4

[0091] The difference between this embodiment and Embodiment 1 is that the developing solvent is a mixture of water, anhydrous ethanol, n-butanol, and glacial acetic acid in a volume ratio of 15:10:10:0.1.

[0092] The results are as follows Figure 5 As shown in the figure, the spots in the thin-layer chromatogram are slightly less clear, but the test sample chromatogram, the reference medicinal material solution chromatogram, and the reference standard solution chromatogram show the same color spots in the same position, indicating that this method meets the requirements of thin-layer identification analysis. It can be seen from the figure that the spot information in the chromatogram is slightly less than that in other examples.

[0093] Example 5

[0094] The difference between this embodiment and Embodiment 1 is that the developing solvent is a mixture of water, anhydrous ethanol, n-butanol, and glacial acetic acid in a volume ratio of 10:15:10:0.2.

[0095] The results are as follows Figure 6 As shown in the figure, the spots in the thin-layer chromatogram are clear. The test sample chromatogram, the reference medicinal material solution chromatogram, and the reference solution chromatogram show the same color spots in the same position, indicating that this method meets the requirements of thin-layer identification analysis. Moreover, the chromatographic spot information is richer in the figure.

[0096] Example 6

[0097] The difference between this embodiment and Embodiment 1 is that the developing solvent is a mixture of water, anhydrous ethanol, n-butanol, and glacial acetic acid in a volume ratio of 10:15:10:0.3.

[0098] The results are as follows Figure 7As shown in the figure, the spots in the thin-layer chromatogram are slightly less clear. The test sample chromatogram, the reference medicinal material solution chromatogram, and the reference solution chromatogram show the same color spots at the same positions, indicating that this method meets the requirements of thin-layer identification analysis. Moreover, the chromatographic spot information is richer in the figure.

[0099] Example 7

[0100] The difference between this embodiment and Embodiment 1 is that the developing solvent is a mixture of water, anhydrous ethanol, n-butanol, and formic acid in a volume ratio of 10:15:10:0.1.

[0101] The results are as follows Figure 8 As shown in the figure, the spots in the thin-layer chromatogram are clear. The test sample chromatogram, the reference medicinal material solution chromatogram, and the reference solution chromatogram show the same color spots in the same position, indicating that this method meets the requirements of thin-layer identification analysis. Moreover, it can be seen from the figure that the chromatographic spot information is slightly less than that of other examples.

[0102] Example 8

[0103] The difference between this embodiment and Embodiment 1 is that the developing solvent is a mixture of water, anhydrous ethanol, and n-butanol in a volume ratio of 10:15:10.

[0104] The results are as follows Figure 9 As shown in the figure, the spots in the thin-layer chromatogram are clear. The test sample chromatogram, the reference medicinal material solution chromatogram, and the reference solution chromatogram show the same color spots in the same position, indicating that this method meets the requirements of thin-layer identification analysis. Moreover, the chromatographic spot information is richer in the figure.

[0105] Experimental Example 1

[0106] The thin-layer identification conditions - sample amount in Example 1 were optimized, including the following:

[0107] The test solution (batch number of kelp (kelp) formula granules: K402CP01), reference herb solution, reference standard solution, and negative control solution were prepared according to the method in Example 1. Spotting and development were performed basically according to the method in Example 1, with the only difference being that the spotting volume of the negative control solution was 5 μL, the spotting volume of the reference standard solution was 2 μL, the spotting volumes of the reference herb solution were 1 μl, 2 μl, 3 μl, and 4 μl, respectively, and the spotting volumes of the test solution were 1 μl, 2 μl, 3 μl, and 4 μl, respectively.

[0108] See results Figure 10As shown in the figure, when the spotting volume of the test sample solution was 2 μl and 3 μl, the thin-layer chromatographic spots were clear and the separation was good. When the spotting volume of the reference medicinal material solution was 2 μl and 3 μl, the spot size and color intensity were consistent with the formulation granules. Therefore, the preferred spotting volume for the test sample solution, the reference solution, and the reference medicinal material solution prepared from kelp (kelp) formulation granules is 2 μl.

[0109] Experiment Example 2

[0110] The durability of the method in Example 1 was investigated, including the following:

[0111] 1. Investigation under different temperatures and humidity levels:

[0112] The test solution (batch number of kelp (kelp) formula granules: K402CP01), reference herb solution, reference solution, and negative control solution were prepared according to the method in Example 1. The spotting and development were basically performed according to the method in Example 1, with the only difference being that spotting was conducted under three different temperature and humidity conditions: (1) temperature 26℃, humidity 60%; (2) temperature 6℃, humidity 85%; (3) temperature 26℃, humidity 88%; (4) temperature 26℃, humidity 23%.

[0113] See results Figure 11-14 As shown in the figure, the chromatographic separation effect of this method is good under different temperature and humidity conditions. The experimental results indicate that this thin-layer identification method has good durability under different temperatures and humidity conditions.

[0114] 2. Investigation of different thin-layer plates:

[0115] The test solution (batch number of kelp (kelp) formula granules: K402CP01), reference herb solution, reference solution, and negative control solution were prepared according to the method in Example 1. Spotting and development were performed on silica gel G thin-layer plates from different manufacturers according to the method in Example 1. The silica gel G thin-layer plates were as follows:

[0116] (1) Thin-layer plate product name: G silicone plate Manufacturer: Yantai Jiangyou Silicone Development Co., Ltd., Specification: 100*200mm, Batch number: 20220725

[0117] (2) Thin-layer plate product name: G silicone plate Manufacturer: Qingdao Ocean Chemical Co., Ltd., Specification: 50*100mm, Batch number: 20210807;

[0118] (3) Thin-layer plate product name: G silicone plate manufacturer: Yantai Huayang New Material Technology Co., Ltd. (Yantai Chemical Industry Research Institute), specification: 200*200mm, batch number: 20220223.

[0119] See results Figure 15-17 As shown in the figure, the separation effect of the test solution was good when using silica gel G thin-layer plates produced by different manufacturers for thin-layer identification. The experimental results show that the thin-layer identification method has good durability for thin-layer plates produced by different manufacturers.

[0120] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A thin-layer chromatography method for identifying kelp or its preparations, characterized in that, Includes the following steps: (1) Preparation of the test solution and the reference solution; The reference solution includes a reference herb solution; the preparation method of the test sample solution includes mixing the test sample with a solvent, followed by extraction, solid-liquid separation, evaporation of the liquid to dryness, and reconstitution; the solvent is selected from one or more of ethanol, water, and methanol; the preparation of the reference solution also includes the preparation of a reference standard solution: alanine is taken and a solvent is added to prepare a solution containing 1.8~2.2 mg per 1 ml, which is used as an alanine reference standard solution; the test sample is selected from kelp medicinal material, kelp slices, or kelp formula granules; (2) According to the thin-layer chromatography method, take the test solution and the reference solution, spot them on the silica gel G thin-layer plate, use a mixture of water, anhydrous ethanol and n-butanol as the developing solvent, develop, take out, and develop the color; The developing agent also includes glacial acetic acid or formic acid; When the developing agent includes glacial acetic acid, the developing agent is a mixture of water, anhydrous ethanol, n-butanol and glacial acetic acid in a volume ratio of 10:15:10:0.1~0.

3. When the developing agent includes formic acid, the developing agent is a mixture of water, anhydrous ethanol, n-butanol and formic acid in a volume ratio of 10:15:10:0.1~0.

3. The colorimetric reagent is ninhydrin.

2. The thin-layer identification method according to claim 1, characterized in that, The developing agent is a mixture of water, anhydrous ethanol, n-butanol and glacial acetic acid in a volume ratio of 10:15:10:0.

1.

3. The thin-layer identification method according to claim 1, characterized in that, In step (2), the sample volume is 1-4 μL.

4. The thin-layer identification method according to claim 2, characterized in that, In step (2), the sample volume is 2-3 μL.

5. The thin-layer identification method according to claim 1, characterized in that, The extraction method is selected from either reflux extraction or ultrasonic extraction; And / or, the extraction time is 25-35 minutes, and the number of extractions is 1-3 times; And / or, the mass ratio of the test sample to the volume ratio of the solvent is 1.8-2.2 g: 18-22 mL; And / or, the solvent used for redissolution is selected from one or more of ethanol and methanol; And / or, the mass ratio of the test sample to the solvent used for reconstitution is 1.8-2.2 g: 0.8-1.2 mL.

6. The thin-layer identification method according to any one of claims 1-5, characterized in that, In step (1), the preparation method of the reference medicinal material solution includes mixing the reference medicinal material with a solvent, and then extracting, separating solid and liquid, evaporating the liquid to dryness and reconstituted.

7. The thin-layer identification method according to claim 6, characterized in that, The solvent is selected from one or more of ethanol, water, and methanol; And / or, the extraction method is selected from heating reflux extraction or ultrasonic extraction; And / or, the extraction time is 25-35 minutes, and the number of extractions is 1-3 times; And / or, the mass ratio of the reference medicinal material to the volume ratio of the solvent is 1.8-2.2 g: 18-55 mL; And / or, the solvent used for redissolution is selected from one or more of ethanol and methanol; And / or, the mass ratio of the reference medicinal material to the solvent for reconstitution is 1.8-2.2g:0.8-1.2mL.

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