Thin-layer identification method for kelp or formula granular preparation thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine identification, in particular to a thin-layer identification method for kelp or a preparation thereof. The identification method comprises the following steps: (1) preparing a test solution and a reference medicinal material solution; and (2) taking the test sample solution and the reference medicinal material solution, spotting, developing by taking a mixed solution of water, absolute ethyl alcohol and n-butyl alcohol as a developing solvent, taking out, and spraying a color developing agent for developing. The thin-layer identification method has the advantages of simple preparation of the test solution, no toxicity and the like, the obtained thin-layer chromatography is rich in spot information, good in separation degree, clear in chromatogram color development and accurate in identification result, and the quality control of the kelp and the preparation thereof is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine identification, and in particular to a thin-layer identification method for kelp and its preparation. Background Art

[0002] Konbu is the dried thallus of Laminaria japonica Aresch. of the Laminariaceae family or Ecklonia kurome Okam. of the Pterophyceae family. The original plant belongs to perennial algae. Konbu has a long history of medicinal use in my country and is a commonly used medicinal material. Konbu is cold in nature and salty in taste. It enters the liver, stomach, and kidney meridians. It belongs to the category of Chinese medicine for softening and dispersing nodules. Its effects are to eliminate phlegm, soften and dissipate nodules, promote diuresis and reduce swelling. It can be used for goiter, scrofula, testicular swelling and pain, and edema caused by phlegm and fluid.

[0003] Kelp has a wide range of clinical applications. For example, when combined with Fritillaria, Citrus aurantium, and Pinellia, it can be used to treat the initial stage of goiter, which is either hard or swollen. When combined with Citrus aurantium, Aloe, and Sea Powder, it can be used to clear the liver, relieve depression, and resolve phlegm. When combined with ginseng, Chinese angelica, and Rehmannia glutinosa, it can be used to treat long-standing goiter and weak qi and blood. When combined with Notopterygium, Saposhnikovia, Seaweed, and Forsythia, it can be used to treat chills and fever, and the initial stage of scrofula. When combined with ginseng, Chinese angelica, and Cyperus rotundus, it can be used to treat It is used to treat liver qi stagnation and scrofula caused by qi and blood deficiency; it can be used with Bupleurum, seaweed, Gentiana, Trigonum, etc. to treat scrofula all over the jaw, which is hard but not ulcerated, and with excessive heat and toxicity; it can be used with seaweed, orange seed, cinnamon heart, Corydalis, etc. to treat testicular pain caused by cold and dampness in the lower abdomen, qi stagnation and blood stasis; it can be used with dried tangerine peel, Pinellia, Poria, etc. to treat plum pit qi; it can be used with dried tangerine peel, Stephania tetrandra, Plantago, etc. to treat beriberi edema. In modern clinical practice, kelp is often used to treat retinal concussion, thyroid disease, hypertension, chronic pelvic inflammatory disease, cancer, breast hyperplasia, phlebitis, viral non-icteric hepatitis, constipation, vitreous opacity, senile cataract and other diseases. Modern pharmacodynamics results show that kelp has multiple pharmacological effects such as affecting the cardiovascular system, lowering blood lipids, lowering blood sugar, anti-coagulation, anti-radiation, anti-tumor, antitussive, anti-asthmatic, anti-mutation, anti-oxidation, anti-virus and anti-bacterial, and affecting immune function.

[0004] At present, the thin layer method involving kelp is to take an appropriate amount of this product, grind it into powder, take 2g, add 20ml of ethanol, heat and reflux for 30 minutes, filter, evaporate the filtrate, and add 1ml of ethanol to dissolve the residue as the test solution. Take another 2g of kelp control medicinal material, soak it for 30 minutes, decoct it twice, add 50ml of water for the first time, decoct it for 30 minutes, add 30ml of water for the second time, decoct it for 20 minutes, combine the decoctions, evaporate them, and make the control medicinal material solution from "add 20ml of ethanol..." in the same way as the residue. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 10μl of the test solution and 5μl of the control medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate, use chloroform-butanone (10:1) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm). In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material. However, through research, it was found that this method only has one spot and has poor specificity, which makes it difficult to conduct comprehensive and accurate quality testing of kelp and its preparations. Summary of the invention

[0005] Aiming at the problem that the thin layer method of kelp in the prior art is difficult to carry out comprehensive and accurate quality detection of kelp (kelp) and its preparations, the present invention provides a thin layer identification method of kelp and its preparations.

[0006] The invention discloses a thin layer identification method of kelp or its preparation, comprising the following steps:

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

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

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

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

[0011] Preferably, the developing agent further comprises 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 to 15:10 to 15:10:0.1 to 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 to 15:10 to 15:10:0.1 to 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 color developer is ninhydrin test solution.

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

[0017] Furthermore, in step (1), the method for preparing the test solution comprises mixing the test sample with a solvent, and performing extraction, solid-liquid separation, evaporating the liquid, and re-dissolving to obtain the solution.

[0018] Furthermore, 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 times is 1-3 times; and / or the volume ratio of the mass of the test sample to the solvent is 1.8-2.2 g:18-22 mL; and / or the solvent for re-dissolution is selected from one or more of ethanol and methanol; and / or the ratio of the mass of the test sample to the mass of the solvent for re-dissolution is 1.8-2.2 g:0.8-1.2 mL.

[0019] Furthermore, in step (1), the preparation method of the control medicinal material solution comprises the steps of mixing a control medicinal material extract with a solvent, extracting, separating solid from liquid, evaporating the liquid and re-dissolving the solution to obtain the control medicinal material solution.

[0020] Furthermore, 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 times is 1-3 times; and / or the mass ratio of the control medicinal material extract to the volume of the solvent is 1.8-2.2g:18-55mL; and / or the solvent for re-dissolution is selected from one or more of ethanol and methanol; and / or the mass ratio of the control medicinal material extract to the solvent for re-dissolution is 1.8-2.2g:0.8-1.2mL.

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

[0022] Take alanine and add solvent to make a solution containing 1.8-2.2 mg per 1 ml as the alanine reference solution.

[0023] Furthermore, the test sample is selected from kelp medicinal materials, kelp decoction pieces, kelp extracts or kelp formula granules.

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

[0025] 1. The thin layer identification method of kelp or its preparation provided by the present invention comprises the following steps: (1) preparing a test solution and a reference solution; the reference solution comprises a control medicinal material solution; (2) taking the test solution and the control medicinal material solution according to the thin layer chromatography method, spotting, using a mixed solution of water, anhydrous ethanol and n-butanol as a developing agent, developing, taking out, and then spraying a color developer for color development. The present invention uses a mixed solution of water, anhydrous ethanol, n-butanol and glacial acetic acid as a developing agent, and the developing agent system cooperates with the test solution to obtain a thin layer chromatogram with rich spot information, good separation, clear color development, and accurate identification results, which can comprehensively and accurately control the quality of kelp (kelp) or its preparation.

[0026] 2. The thin-layer identification method of kelp or its preparation provided by the present invention, wherein the developing agent also includes glacial acetic acid or formic acid. After adding glacial acetic acid or formic acid, the spots in the thin-layer chromatography can be further enriched in information, have good separation, and have clear color development. In particular, the developing agent having a volume ratio of water, anhydrous ethanol, n-butanol and glacial acetic acid of 10:15:10:0.1 has clear spots in the thin-layer chromatography, good separation, more spots, and richer information.

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

[0028] 4. The thin layer chromatography identification method of kelp or its preparation provided by the present invention, the preparation of the reference solution also includes the preparation of the reference solution: take alanine, add a solvent to make a solution containing 1.8-2.2 mg per 1 ml, as the alanine reference solution; compared with the prior art, the thin layer chromatography identification method of the present invention uses alanine as the reference substance and the reference medicinal material as the control indicator, and the spot information in the obtained thin layer chromatography is richer, the identification result is more accurate, and the alanine and amino acid components of the indicator components in kelp and its preparations can be identified, and the quality control is more comprehensive and accurate.

[0029] 5. The thin layer identification method of kelp or its preparation 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 sample spot amounts with good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 It is the thin layer identification spectrum in Example 1, wherein, in Figure (1), 1 is the negative control solution; 2 is the reference substance solution; 3 is the reference medicinal material solution; 4-6 are the test sample solutions, and the test samples correspond to batch numbers: K402CP01, K402CP02, and K402CP03 in sequence; in Figure (2), 1 is the reference substance solution, 2 is the reference medicinal material solution, and 3 is the test sample solution prepared from the freeze-dried powder of the standard decoction of kelp.

[0032] Figure 2 This is the TLC identification spectrum in Comparative Example 1, wherein 1 is the control medicinal material solution; 2 is the test sample solution.

[0033] Figure 3 The thin layer identification spectrum in Example 2, wherein 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp formula granules (K402CP01);

[0034] Figure 4 The thin layer identification spectrum in Example 3, wherein 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp formula granules (K402CP01);

[0035] Figure 5 The thin layer identification spectrum in Example 4, wherein 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp formula granules (K402CP01);

[0036] Figure 6 The thin layer identification spectrum in Example 5, wherein 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp formula granules (K402CP01);

[0037] Figure 7The thin layer identification spectrum in Example 6, wherein 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp formula granules (K402CP01);

[0038] Figure 8 The thin layer identification spectrum in Example 7, wherein 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp formula granules (K402CP01);

[0039] Fig. 9 The thin layer identification spectrum in Example 8, wherein 1 is the reference solution, 2 is the reference medicinal material solution, and 3 is the test solution prepared from kelp formula granules (K402CP01);

[0040] Fig.10 This is the thin layer identification spectrum in Experimental Example 1, where 1 is the negative control solution; 2 is the reference solution; 3-6 are control medicinal material solutions, and the spot volumes are 1μl, 2μl, 3μl, and 4μl, respectively; 7-10 are test solution, and the spot volumes are 1μl, 2μl, 3μl, and 4μl, respectively.

[0041] Fig.11 This is the thin layer identification spectrum in Experimental Example 2 - temperature 26°C, humidity 60%, 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.

[0042] Fig.12 This is the thin layer identification spectrum in Experimental Example 2 - temperature 6°C, humidity 85%, 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.

[0043] Fig.13 This is the thin layer identification spectrum in Experimental Example 2 - temperature 26°C, humidity 88%, 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.

[0044] Fig.14 This is the thin layer identification spectrum in Experimental Example 2 - temperature 26°C, humidity 23%, 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.

[0045] Fig.15 This is the thin layer identification spectrum in Experimental Example 2 - Yantai Institute of Chemical Industry, 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.

[0046] Fig.16This is the thin layer identification spectrum in Experimental 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] Fig.17 This is the thin layer identification spectrum in Experimental Example 2 - 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 DESCRIPTION

[0048] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0049] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0050] Instruments, reagents and drugs:

[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 ZTE Weiye Instrument Co., Ltd.), silica gel G plate (Qingdao Ocean 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 formula granules: prepared by Beijing Kangrentang Pharmaceutical Co., Ltd.; the preparation method of kelp formula granules is as follows: take kelp, heat and reflux extraction twice, add 14 times the weight of water of kelp for the first time, heat and reflux extraction for 1 hour, filter, add 12 times the weight of water of kelp for the second time, heat and reflux extraction for 1 hour, filter, combine the filtrate, concentrate the filtrate to a relative density of 1.0 g / mL (measured at 60°C), spray dry, add auxiliary material maltodextrin to the dry powder obtained by spray drying (the total weight of the dry powder after adding maltodextrin accounts for 45.4% of the kelp feeding amount), mix well and then perform dry granulation to make granules.

[0054] Freeze-dried powder of standard decoction of kelp: take 100-150g of kelp slices, put them in a decoction pot, add 16 times the amount of water to the slices for the first decoction, soak in cold water for 30 minutes, boil over high heat (500W), decoct over low heat (200W) for 30 minutes, filter with a 150-mesh filter cloth while hot, cool quickly and set aside; add 14 times the amount of water to the slices for the second decoction, boil over high heat (500W), decoct over low heat (200W) for 20 minutes, filter with a 150-mesh filter cloth while hot, cool quickly and set aside; combine the filtrate, concentrate under reduced pressure (temperature set at 65°C, vacuum degree -0.1MPa) to a solid-liquid ratio of about 1:1 (relative density of 1.05-1.10 (65°C)), freeze-dry and make into fine powder.

[0055] Kelp (Laminaria japonica) control medicinal material (Batch No. AY300005-202305, Foshan Aoyu Biotechnology Co., Ltd.);

[0056] Kelp slices were purchased from (Batch No. K402YP01, made by Beijing Kangrentang Pharmaceutical Co., Ltd.);

[0057] Alanine (Batch No.: 140680-202206, China Food and Drug Administration);

[0058] Reagents: ethanol, n-butanol, glacial acetic acid, formic acid, and ninhydrin were all of analytical grade, and water was distilled water from Watsons.

[0059] Example 1

[0060] This embodiment provides a thin layer identification method for kelp and its preparation, comprising the following steps:

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

[0062] Preparation of control medicinal solution: Take 2.0g of kelp (kelp) control medicinal material, add 50ml of water, heat and reflux twice, 30 minutes each time, centrifuge, combine the supernatants, spin dry, add 20ml of ethanol to the residue, heat and reflux for 30 minutes, then filter, take the filtrate and evaporate to dryness, then add 1ml of ethanol to the evaporated residue to reconstitute it to make a control medicinal solution.

[0063] Preparation of reference solution: Take alanine reference substance and add ethanol to make a solution containing 2 mg per 1 ml as the reference solution.

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

[0065] After conventional pre-equilibration of the thin layer plate for 15 minutes, according to the thin layer chromatography method, 2 μL of the test solution, control medicinal material solution, reference solution, and negative control solution were respectively aspirated and spotted on the same silica gel G thin layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.) and placed in a double-slot developing cylinder at 26°C and 60% humidity. A mixed solution of water, anhydrous ethanol, n-butanol, and glacial acetic acid in a volume ratio of 10:15:10:0.1 was used as the developing agent. The plate was developed in an upward direction. When the development distance was 8 cm, it was taken out, dried, sprayed with ninhydrin reagent, and inspected under sunlight.

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

[0067] Comparative Example 1

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

[0069] Preparation of test solution: Take an appropriate amount of Kelp Formula Granules (K402CP01), grind it into powder, take 2g, add 20ml of ethanol, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of ethanol to the residue to dissolve it, and use it as the test solution.

[0070] Preparation of control medicinal material solution: Take 2g of kelp (kelp) control medicinal material, soak for 30 minutes, decoct twice, add 50ml of water for the first time, decoct for 30 minutes, add 30ml of water for the second time, decoct for 20 minutes, combine the decoctions, evaporate to dryness, and prepare the control medicinal material solution with the same method as the residue starting from "add 20ml of ethanol..."

[0071] Preparation of standard decoction: Take 100-150g of kelp slices, put them in a decoction pot, add 16 times the weight of water for the first decoction, soak in cold water for 30 minutes, boil over high heat (500W), decoct over low heat (200W) for 30 minutes, filter with a 150-mesh filter cloth while hot, cool quickly and set aside; add 14 times the weight of water for the second decoction, boil over high heat (500W), decoct over low heat (200W) for 20 minutes, filter with a 150-mesh filter cloth while hot, cool quickly and set aside; combine the filtrate, concentrate under reduced pressure (temperature set at 65°C, vacuum degree -0.1MPa) to a solid-liquid ratio of about 1:1 (relative density of 1.05-1.10 (65°C)), freeze-dry and make into fine powder.

[0072] According to the thin layer chromatography method (General Rules 0502 of the 2020 edition of the Chinese Pharmacopoeia), 10 μl of the test solution, 5 μl of the control medicinal solution, and 10 μl of the standard decoction were respectively spotted on the same silica gel G thin layer plate, and chloroform-butanone (10:1, v / v) was used as the developing agent. The plate was developed, taken out, dried, and examined under ultraviolet light (365 nm).

[0073] Results Figure 2 ,From the figure, it can be seen that the spots of the control ,medicine solution and the test solution are inconsistent.

[0074] Example 2

[0075] This embodiment provides a thin layer identification method for kelp and its preparation, comprising the following steps:

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

[0077] Preparation of control medicinal solution: Take 2g of kelp (kelp) control medicinal material, add 30ml of water, heat and reflux twice, 30 minutes each time, centrifuge, combine the supernatants, spin dry, add 20ml of ethanol to the residue, heat and reflux for 30 minutes, then filter, take the filtrate and evaporate to dryness, then add 1ml of methanol to the evaporated residue to reconstitute it to make a control medicinal solution.

[0078] Preparation of reference solution: Take alanine reference substance and add methanol to make a solution containing 2 mg per 1 ml as the reference solution.

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

[0080] After conventional pre-equilibration of the thin layer plate for 15 minutes, according to the thin layer chromatography method, 4 μL of the test solution, control medicinal material solution, reference solution, and negative control solution were respectively aspirated and spotted on the same silica gel G thin layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.) and placed in a double-slot developing cylinder at 26°C and 60% humidity. A mixed solution of water, anhydrous ethanol, n-butanol, and glacial acetic acid in a volume ratio of 15:15:10:0.1 was used as the developing agent. The plate was developed in an upward direction. When the development distance was 8 cm, it was taken out, dried, sprayed with ninhydrin reagent, and inspected under sunlight.

[0081] Results Figure 3 As can be seen from the figure, the spots in the thin layer spectrum are clear, and the chromatogram of the test sample, the chromatogram of the reference medicinal material solution, and the chromatogram of the reference sample solution show spots of the same color at the same position, indicating that this method meets the requirements of thin layer identification analysis, and it can be seen from the figure that the chromatogram has richer spot information.

[0082] Example 3

[0083] This embodiment provides a thin layer identification method for kelp and its preparation, comprising the following steps:

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

[0085] Preparation of control medicinal solution: Take 2.2g of kelp (kelp) control medicinal material, add 55ml of water, heat and reflux twice, 35 minutes each time, centrifuge, combine the supernatants, spin dry, add 22ml of ethanol to the residue, heat and reflux for 35 minutes, then filter, take the filtrate and evaporate to dryness, then add 1.2ml of ethanol to the evaporated residue to reconstitute it to make a control medicinal solution.

[0086] Preparation of reference solution: Take alanine reference substance and add ethanol to make a solution containing 2.2 mg per 1 ml as the reference solution.

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

[0088] After conventional pre-equilibration of the thin layer plate for 15 minutes, according to the thin layer chromatography method, 3 μL of the test solution, control medicinal material solution, reference solution, and negative control solution were respectively aspirated and spotted on the same silica gel G thin layer plate (Yantai Jiangyou Silica Gel Development Co., Ltd.) and placed in a double-slot developing cylinder at 26°C and 60% humidity. A mixed solution of water, anhydrous ethanol, n-butanol, and glacial acetic acid in a volume ratio of 10:15:10:0.1 was used as the developing agent. The plate was developed in an upward direction. When the development distance was 8 cm, it was taken out, dried, sprayed with ninhydrin reagent, and inspected under sunlight.

[0089] Results Figure 4 As can be seen from the figure, the spots in the thin layer spectrum are clear, and the chromatogram of the test sample, the chromatogram of the reference medicinal material solution, and the chromatogram of the reference sample solution show spots of the same color at the same position, indicating that this method meets the requirements of thin layer identification analysis, and it can be seen from the figure that the chromatogram has richer spot information.

[0090] Example 4

[0091] The difference between this embodiment and embodiment 1 is that the developing agent 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, it can be seen that the spots in the thin layer spectrum are less clear, but the test sample chromatogram, the reference medicinal material solution chromatogram, and the reference sample solution chromatogram show spots of the same color at 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 of the chromatogram is slightly less than that of other embodiments.

[0093] Example 5

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

[0095] The results are as follows Figure 6 As shown in the figure, it can be seen that the spots in the thin layer spectrum are clear, and the chromatogram of the test sample, the chromatogram of the reference medicinal material solution, and the chromatogram of the reference sample solution show spots of the same color at the same position, indicating that this method meets the requirements of thin layer identification analysis, and it can be seen from the figure that the chromatogram has richer spot information.

[0096] Example 6

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

[0098] The results are as follows Figure 7As shown in the figure, it can be seen that the spots in the thin layer spectrum are less clear, and the chromatogram of the test sample, the chromatogram of the reference medicinal material solution, and the chromatogram of the reference sample solution show spots of the same color at the same position, indicating that this method meets the requirements of thin layer identification analysis, and it can be seen from the figure that the chromatogram has richer spot information.

[0099] Example 7

[0100] The difference between this embodiment and embodiment 1 is that 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.

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

[0102] Example 8

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

[0104] The results are as follows Fig. 9 As shown in the figure, it can be seen that the spots in the thin layer spectrum are clear, and the chromatogram of the test sample, the chromatogram of the reference medicinal material solution, and the chromatogram of the reference sample solution show spots of the same color at the same position, indicating that this method meets the requirements of thin layer identification analysis, and it can be seen from the figure that the chromatogram has richer spot information.

[0105] Experimental Example 1

[0106] The thin layer identification condition in Example 1 - the amount of sample spotting was optimized, including the following:

[0107] The test solution (batch number of kelp formula granules: K402CP01), control medicinal material solution, reference substance solution and negative control solution were prepared according to the method in Example 1. The spotting and development were basically carried out according to the method in Example 1, with the only difference that the spotting amount of the negative control solution was 5 μL, the spotting amount of the reference substance solution was 2 μL, the spotting amount of the control medicinal material solution was 1 μl, 2 μl, 3 μl, 4 μl, and the spotting amount of the test solution was 1 μl, 2 μl, 3 μl, 4 μl, respectively.

[0108] Results Fig.10As can be seen from the figure, when the sample volume of the test solution is 2μl and 3μl, the thin layer chromatography spots are clear and the separation is good. When the sample volume of the control medicinal material solution is 2μl and 3μl, the spot size and color depth are consistent with the formula granules. Therefore, it is preferred that the sample volume of the test solution prepared by the kelp formula granules is 2μl, the sample volume of the reference solution is 2μl, and the sample volume of the control medicinal material solution is 2μl.

[0109] Experimental Example 2

[0110] The method in Example 1 was subjected to a durability test, including the following contents:

[0111] 1. Investigation at different temperatures and humidity:

[0112] The test solution (Batch number of kelp formula granules: K402CP01), control medicinal material solution, control substance solution and negative control solution were prepared according to the method in Example 1. The spotting and development were basically carried out according to the method in Example 1, except that the spotting was carried out under three different temperature and humidity environments, namely: (1) temperature 26°C, humidity 60%; (2) temperature 6°C, humidity 85%; (3) temperature 26°C, humidity 88%; (4) temperature 26°C, humidity 23%.

[0113] Results Figure 11-14 As can be seen from the figure, the chromatographic separation effect of this method is good under different temperature and humidity conditions. The experimental results show that the thin layer identification method has good durability under different temperatures and humidity.

[0114] 2. Inspection of different thin layer plates:

[0115] The test solution (batch number of kelp formula granules: K402CP01), control medicinal material solution, reference substance solution and negative control solution were prepared according to the method in Example 1. Samples were spotted and developed on silica gel G thin layer plates of different manufacturers according to the method in Example 1. The silica gel G thin layer plates were:

[0116] (1) Thin layer board Product name: G silica gel board Manufacturer: Yantai Jiangyou Silica Gel Development Co., Ltd. Specification: 100*200㎜, Batch number: 20220725

[0117] (2) Thin layer plate trade name: G silica gel plate manufacturer: Qingdao Ocean Chemical Co., Ltd., specification: 50*100㎜, batch number: 20210807;

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

[0119] Results Figure 15-17 As can be seen from the figure, the separation effect of thin layer identification of the test solution using silica gel G thin layer plates produced by different manufacturers is good. 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 examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A thin layer identification method for kelp or its preparation, characterized in that: The following steps are involved: (1) Preparation of test solution and reference solution; Reference solutions include control medicinal material solutions; (2) According to the thin layer chromatography method, take the test sample solution and the reference solution, spot the sample, use a mixture of water, anhydrous ethanol and n-butanol as the developing solvent, develop the solution, take out the sample and develop the color.

2. The thin layer identification method according to claim 1, characterized in that: In step (2), the developing agent is a mixed solution with a volume ratio of 10-15:10-15:8-12; Preferably, the developing agent further comprises glacial acetic acid or formic acid; Preferably, the developing agent is a mixture of water, anhydrous ethanol, n-butanol and glacial acetic acid in a volume ratio of 10 to 15:10 to 15:10:0.1 to 0.3; Preferably, the developing agent is a mixture of water, anhydrous ethanol, n-butanol and formic acid in a volume ratio of 10 to 15:10 to 15:10:0.1 to 0.3; 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.

3. The thin layer identification method according to claim 1, characterized in that: In step (2), the color developing agent is ninhydrin reagent.

4. The thin layer identification method according to any one of claims 1 to 3, characterized in that: In step (2), the spotting volume is 1-4 μL; preferably, the spotting volume is 2-3 μL.

5. The thin layer identification method according to any one of claims 1 to 4, characterized in that: In step (1), the preparation method of the test solution includes mixing the test sample with a solvent, extracting, separating the solid from the liquid, evaporating the liquid, and re-dissolving the solution.

6. The thin layer identification method according to claim 5, 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 min, and the number of times is 1-3 times; And / or, the mass of the test sample to the volume ratio of the solvent is 1.8-2.2 g:18-22 mL; and / or, the solvent for re-dissolving is selected from one or more of ethanol and methanol; And / or, the ratio of the mass of the test sample to the mass of the solvent for reconstitution is 1.8-2.2 g:0.8-1.2 mL.

7. The thin layer identification method according to any one of claims 1 to 6, characterized in that: In step (1), the preparation method of the control medicinal material solution comprises the steps of mixing the control medicinal material with a solvent, extracting, separating the solid from the liquid, evaporating the liquid, and re-dissolving the solution.

8. The thin layer identification method according to claim 7, 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 min, and the number of times is 1-3 times; and / or, the mass of the control medicinal material to the volume ratio of the solvent is 1.8-2.2 g:18-55 mL; and / or, the solvent for re-dissolving 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 1.8-2.2 g:0.8-1.2 mL.

9. The thin layer identification method according to any one of claims 1 to 8, characterized in that: The preparation of the reference solution also includes the preparation of the reference substance solution: Take alanine and add solvent to make a solution containing 1.8-2.2 mg per 1 ml as the alanine reference solution.

10. The thin layer identification method according to any one of claims 1 to 9, characterized in that: The test sample is selected from kelp medicinal materials, kelp decoction pieces, kelp extracts or kelp formula granules.

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