A thin-layer identification method of a traditional Chinese medicine composition

Abstract

The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a thin-layer identification method of a traditional Chinese medicine composition containing Cuscuta chinensis Lam and Salvia miltiorrhiza. Two kinds of test sample solutions are prepared, and Cuscuta chinensis Lam and Salvia miltiorrhiza in the traditional Chinese medicine composition are identified on a polyamide film and a silica gel G thin-layer plate respectively by using 30%-70% glacial acetic acid and a developing agent of chloroform-ethyl acetate-formic acid with a volume ratio of 2-3:1-3:1. The thin-layer identification method provides a theoretical basis for the quality control and research of the pharmacodynamic material basis of the traditional Chinese medicine composition containing Cuscuta chinensis Lam and Salvia miltiorrhiz

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and in particular relates to a thin-layer chromatography method for identifying traditional Chinese medicine compositions. Background Technology

[0002] Thin-layer chromatography (TLC) is widely used in the qualitative identification of traditional Chinese medicine and its preparations due to its dual functions of separation and analysis, high sensitivity, and strong specificity. For Cuscuta chinensis, the 2025 edition of the Chinese Pharmacopoeia uses methanol-glacial acetic acid-water (4:1:5) as the developing solvent on a polyamide film, but the spot Rf values ​​and resolution are poor, resulting in limited sensitivity. While the developing solvent systems used in Kunxian capsules and Funingkang tablets reported in related existing technologies have shown some improvement, their composition is complex and involves easily manufactured toxic reagents such as acetone or other highly toxic reagents. For Salvia miltiorrhiza, the Chinese Pharmacopoeia method uses chloroform-toluene-ethyl acetate-methanol-formic acid (6:4:8:1:4) and petroleum ether-ethyl acetate (4:1) for two-stage development, which is cumbersome and contains toxic reagents such as toluene. Related existing technologies also mainly use systems such as toluene-ethyl acetate-formic acid, which similarly suffer from high toxicity and poor environmental friendliness.

[0003] Therefore, there is an urgent need in this field to develop a thin-layer chromatography (TLC) identification method that is efficient in sample preparation, has good separation, is universally applicable, and can be used simultaneously for the identification of Cuscuta chinensis and Salvia miltiorrhiza in the herbal composition of this invention. This method should avoid using highly toxic or easily manufactured toxic reagents such as toluene and acetone as much as possible, simplify the development steps, reduce the types and amounts of organic reagents used, and achieve clear, stable, and reproducible chromatographic spots in a single development. This would ensure identification accuracy while reducing operational risks and environmental burden, thus better aligning with the development trend of green analytical chemistry. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a thin-layer chromatography method for the identification of traditional Chinese medicine compositions.

[0005] According to a first aspect of the present invention, a thin-layer chromatography (TLC) identification method for a traditional Chinese medicine composition containing Cuscuta chinensis and Salvia miltiorrhiza is provided. The TLC identification method includes a TLC identification method for Cuscuta chinensis and a TLC identification method for Salvia miltiorrhiza. The thin-layer chromatography method for identifying dodder seeds includes the following steps: The first test solution, the Cuscuta chinensis reference herb solution, and the reference solution were spotted separately on the same polyamide thin-layer plate for thin-layer chromatography analysis. The obtained chromatograms were compared. The developing solvent was glacial acetic acid with a mass percentage of 30% to 70%. The thin-layer chromatography method for identifying Salvia miltiorrhiza includes the following steps: The second test solution and the Danshen reference herb solution were spotted separately on the same silica gel G thin-layer plate for thin-layer chromatography analysis. The obtained chromatograms were compared. The developing solvent was chloroform-ethyl acetate-formic acid with a mass-volume ratio of 2~3:1~3:1.

[0006] According to some embodiments of the present invention, the traditional Chinese medicine composition contains deer antler, calcined amethyst, dodder seed, stir-fried white peony root, stir-fried yam with wheat bran, wine-processed cornus fruit, vinegar-processed Bupleurum root, and Salvia miltiorrhiza.

[0007] Preferably, in the traditional Chinese medicine composition, the mass ratio of deer antler, calcined amethyst, dodder seed, stir-fried white peony root, stir-fried yam with wheat bran, wine-processed cornus fruit, vinegar-processed Bupleurum root, and Salvia miltiorrhiza is (0.1~2):(0.1~2):(0.5~3):(0.1~2):(0.5~3):(0.1~2):(0.1~1.5):(0.1~2).

[0008] More preferably, in the traditional Chinese medicine composition, the mass ratio of deer antler, calcined amethyst, dodder seed, stir-fried white peony root, stir-fried yam with wheat bran, wine-processed cornus fruit, vinegar-processed Bupleurum root, and Salvia miltiorrhiza is (0.5~1.5):(0.5~1.5):(1~2):(0.5~1.5):(1~2):(0.5~1.5):(0.3~0.9):(0.5~1.5).

[0009] More preferably, in the traditional Chinese medicine composition, the mass ratio of deer antler, calcined amethyst, dodder seed, stir-fried white peony root, bran-fried yam, wine-processed cornus fruit, vinegar-processed Bupleurum root, and Salvia miltiorrhiza is approximately 1:1:1.5:1:1.5:1:0.6:1.

[0010] According to some embodiments of the present invention, the traditional Chinese medicine composition is prepared by the following steps: Add water to deer antler and calcined amethyst at a mass-to-volume ratio of 1g:(6~10)mL, decoct for 20~50min, filter, and take the first filtrate; add the residue to the filtrate along with dodder seed, stir-fried white peony root, bran-fried yam, wine-processed cornus fruit, vinegar-processed bupleurum root, and dantian in water at a mass-to-volume ratio of 1g:(6~10)mL, soak for 25~35min, decoct for 20~50min, repeat 2~3 times, filter, and obtain the second filtrate; combine the first filtrate and the second filtrate to obtain the final product.

[0011] Preferably, the filtration step uses a 200-mesh filter.

[0012] According to some embodiments of the present invention, the developing solvent used in the thin-layer identification method for dodder contains 30% to 70% glacial acetic acid by mass, for example, any value of 35%, 40%, 45%, 50%, 55%, 60%, 65% or any range between two of these.

[0013] According to some embodiments of the present invention, the first test solution is obtained by dissolving the test sample in an alcohol solvent; preferably, the concentration of the first test solution is 10~1000 mg / mL; and / or, The dodder seed reference solution is obtained by dissolving dodder seed reference material in an alcohol solvent. Preferably, the concentration of the dodder seed reference solution is 5-500 mg / mL; and / or, The reference solution is obtained by dissolving a reference standard in an alcohol solvent. Preferably, the concentration of the reference solution is 0.1~10 mg / mL.

[0014] Preferably, the reference standard is hyperoside.

[0015] According to some embodiments of the present invention, the alcohol solvent is an 80% to 100% methanol solution.

[0016] According to some embodiments of the present invention, the thin-layer identification method for dodder further includes unfolding and drying, spraying with 1%~3% aluminum trichloride ethanol solution, drying, and developing color under a 365nm ultraviolet lamp.

[0017] According to some embodiments of the present invention, the second test solution is prepared by the following preparation steps: Take the test sample, adjust the pH to 1-3 with acid, then extract with ethyl acetate, collect the organic layer, evaporate to dryness, dissolve the residue in an alcohol solvent to obtain the final product; and / or, The Danshen reference herb solution was prepared by the following steps: Take the reference material of Salvia miltiorrhiza, add water and heat under reflux, collect the extract, add acid to adjust the pH to 1-3, then add ethyl acetate for extraction, take the organic layer, evaporate to dryness, dissolve the residue in an alcohol solvent to obtain the final product.

[0018] Preferably, the concentration of the second test sample solution is 10~1000 mg / mL; and / or, the concentration of the Danshen reference herb solution is 5~500 mg / mL.

[0019] According to some embodiments of the present invention, the heating and reflux after adding water is to add 20 to 60 times the amount of water and then heat and reflux for 20 to 40 minutes.

[0020] Preferably, the alcohol solvent is 80% to 100% methanol.

[0021] According to some embodiments of the present invention, the thin-layer identification method for Salvia miltiorrhiza further includes unfolding and drying, spraying with 4%~6% vanillin sulfuric acid solution, drying, and developing color under sunlight.

[0022] According to some embodiments of the present invention, the ambient temperature for each unfolding is independently 4~35°C, and the ambient humidity for each unfolding is independently 30%~90%.

[0023] According to some embodiments of the present invention, the sample spotting volume for the thin-layer chromatography analysis is 1~5 μL.

[0024] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The thin-layer chromatography identification method of the present invention has the advantages of appropriate selection of developing agent, high identification efficiency and accuracy, and convenient operation. By performing thin-layer chromatography identification on Cuscuta chinensis and Salvia miltiorrhiza in traditional Chinese medicine composition, it provides a theoretical basis for the quality control and in-depth research on the pharmacodynamic material basis of the traditional Chinese medicine composition.

[0025] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the 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 based on these drawings without creative effort: Figure 1 The thin-layer chromatography (TLC) image of Cuscuta chinensis in the traditional Chinese medicine composition of Example 1 of the present invention is shown. Figure 2 The figure shows the specificity test results of the thin-layer chromatography method for identifying Cuscuta chinensis in the traditional Chinese medicine composition of Example 2 of the present invention; Figure 3 The following figures show the results of different thin-layer chromatography (TLC) studies on the identification of Cuscuta chinensis in the traditional Chinese medicine composition according to Example 2 of the present invention. Figure 4 The following diagram shows the results of different spotting methods for the thin-layer identification of Cuscuta chinensis in the traditional Chinese medicine composition of Example 2 of the present invention; Figure 5 The results of the thin-layer chromatography method for identifying Cuscuta chinensis in the traditional Chinese medicine composition according to Example 2 of the present invention are shown in the figure. Figure 6 The figure shows the results of different development temperatures of the thin-layer chromatography method for identifying Cuscuta chinensis in the traditional Chinese medicine composition of Example 2 of the present invention; Figure 7 The following figures show the results of different development humidity tests of the thin-layer identification method of Cuscuta chinensis in the traditional Chinese medicine composition of Example 2 of the present invention; Figure 8The figure shows the results of different development systems of the thin-layer chromatography method for identifying Cuscuta chinensis in the traditional Chinese medicine composition of Example 2 of the present invention; Figure 9 The thin-layer chromatography identification view of Danshen in the traditional Chinese medicine composition of Example 3 of the present invention is shown; Figure 10 The results of the specificity investigation of the thin-layer chromatography identification method of Danshen in the traditional Chinese medicine composition of Example 4 of the present invention are shown in the figure. Figure 11 The following figures show the results of different thin-layer plate examinations of the thin-layer identification method for Danshen in the traditional Chinese medicine composition of Example 4 of the present invention; Figure 12 The results of different development temperatures of the thin-layer chromatography method for identifying Danshen in the traditional Chinese medicine composition of Example 4 of the present invention are shown in the figure. Figure 13 The results of different development humidity tests of the thin-layer identification method of Danshen in the traditional Chinese medicine composition of Example 4 of the present invention are shown. Detailed Implementation

[0027] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0028] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0029] Unless otherwise specified, "about" in this invention means that the allowable error is within ±2%.

[0030] Unless otherwise specified, "room temperature" in this invention means 25±5 ℃.

[0031] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0032] The test samples used in the following examples were warming uterus and regulating menstruation granules, produced by Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd. Baiyunshan Pharmaceutical Factory, with batch numbers 251101S, 251102S and 251103S; Cuscuta chinensis reference material, Salvia miltiorrhiza reference material and hyperoside reference standard were all purchased from the National Institutes for Food and Drug Control, with batch numbers 121232-202104, 120923-201816 and 111521-202310, respectively.

[0033] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0034] Example 1 This embodiment provides a thin-layer chromatography method for identifying dodder seed in a traditional Chinese medicine composition containing dodder seed and salvia miltiorrhiza, comprising the following steps: Preparation of the test solution of the traditional Chinese medicine composition: Take 2g of the traditional Chinese medicine composition solution, add 20mL of methanol, sonicate (power 500W, frequency 40kHz) for 15 minutes, filter, evaporate the filtrate to dryness in a water bath, add 2mL of methanol to the residue to completely dissolve it, and use it as the test solution of the traditional Chinese medicine composition.

[0035] Preparation of negative test solution of traditional Chinese medicine composition: Weigh appropriate amounts of other medicinal materials of Cuscuta chinensis to prepare negative test solution of traditional Chinese medicine composition; prepare negative test solution of traditional Chinese medicine composition of Cuscuta chinensis in the same way according to the preparation method of the above-mentioned test solution of traditional Chinese medicine composition in this embodiment.

[0036] Preparation of the reference herb solution: Take 0.5g of Cuscuta chinensis reference herb and prepare the Cuscuta chinensis reference herb solution in the same way as the preparation method of the test solution of the above-mentioned traditional Chinese medicine composition in this embodiment.

[0037] Preparation of reference solution: Take hyperoside reference standard and add methanol to prepare a solution containing 1 mg of reference standard in 1 mL, thus obtaining hyperoside reference solution.

[0038] Spotting: Perform thin-layer chromatography (Chinese Pharmacopoeia 2025, Part IV, General Chapter 0502), and spot 1 μL of the test solution of the traditional Chinese medicine composition, 1 μL of the negative test solution of the traditional Chinese medicine composition, 1 μL of the reference medicinal material solution, and 1 μL of the reference solution onto the polyamide film (produced by Luqiao Sijia Biochemical Plastics Factory, Taizhou City, Zhejiang Province) in strip form.

[0039] Development: Using glacial acetic acid with a mass percentage concentration of 50% as the developing agent, the spotted polyamide film was placed in the developing agent vapor for pre-saturation, developed, removed, and dried.

[0040] Inspection: Spray a 1% aluminum trichloride ethanol solution (i.e., the mass-volume ratio of aluminum trichloride to ethanol is 1:99) onto the dried polyamide film, let it dry, and inspect it under a UV lamp (365nm).

[0041] The results are as follows Figure 1 As shown, 1 is the hyperoside reference solution; 2 is the dodder seed reference material solution; 3 is the negative test solution of the traditional Chinese medicine composition; 4 is the test solution of the traditional Chinese medicine composition (batch number: 251101S); 5 is the test solution of the traditional Chinese medicine composition (batch number: 251102S); and 6 is the test solution of the traditional Chinese medicine composition (batch number: 251103S). It can be seen that in the chromatogram of the test solution of the traditional Chinese medicine composition, a main spot of the same color appears at the corresponding position as the reference material, and the spots are well separated and clearly distinguishable.

[0042] Example 2 This embodiment provides a methodological verification of the method in Embodiment 1.

[0043] (1) Specificity examination As described in Example 1, a test solution of the traditional Chinese medicine composition, a negative test solution of the traditional Chinese medicine composition, a control medicinal material solution, and a control solution were prepared, and the samples were spotted, developed, and examined.

[0044] The results are as follows Figure 2 As shown, 1 is the hyperoside reference solution; 2 is the dodder seed reference medicinal material solution; 3 is the negative test solution of the traditional Chinese medicine composition; and 4 is the test solution of the traditional Chinese medicine composition (batch number: 251101S). The results show that the chromatogram of the test solution of the traditional Chinese medicine composition shows a main spot of the same color at the corresponding position as the chromatogram of the reference medicinal material solution, with no negative interference and strong specificity.

[0045] (2) Investigation of polyamide film manufacturers Thin-layer chromatography was performed on the traditional Chinese medicine composition according to the same method as in Example 1, except that the polyamide film was purchased from Rizhao Kepu New Materials Co., Ltd.

[0046] The results are as follows Figure 3 As shown, 1 is the hyperoside reference solution; 2 is the dodder seed reference medicinal material solution; 3 is the negative test solution of the traditional Chinese medicine composition; and 4 is the test solution of the traditional Chinese medicine composition (batch number: 251101S). It can be seen that in the chromatogram of the test solution of the traditional Chinese medicine composition, a main spot of the same color appears at the corresponding position as in the chromatogram of the reference medicinal material solution, which can be clearly distinguished.

[0047] (3) Examination of sampling methods Thin-layer chromatography was performed on the traditional Chinese medicine composition according to the same method as in Example 1, except that the spotting method was spot-like.

[0048] The results are as follows Figure 4 As shown, 1 is a hyperoside reference solution; 2 is a dodder seed reference medicinal material solution; 3 is a negative test solution of the traditional Chinese medicine composition; and 4 is a test solution of the traditional Chinese medicine composition (batch number: 251101S). It can be seen that in the chromatogram of the test solution of the traditional Chinese medicine composition, a main spot of the same color appears at the corresponding position as in the chromatogram of the reference medicinal material solution, which can be clearly distinguished, indicating that the method of the present invention is also applicable to spot sampling.

[0049] (4) Investigation of glacial acetic acid concentration The dodder seed in the traditional Chinese medicine composition was identified by thin-layer chromatography using the same method as in Example 1, except that the developing solvent was glacial acetic acid with a mass percentage concentration of 40% or 60%.

[0050] The results are as follows Figure 5 As shown, 1 is the hyperoside reference solution; 2 is the dodder seed reference medicinal material solution; 3 is the negative test solution of the traditional Chinese medicine composition; and 4 is the test solution of the traditional Chinese medicine composition (batch number: 251101S). It can be seen that when the developing solvent is 40% glacial acetic acid, the chromatogram of the test solution of the traditional Chinese medicine composition shows a main spot of the same color at the corresponding position as the chromatogram of the reference medicinal material solution. However, compared with the 50% glacial acetic acid used in Example 1, the Rf value of the main spot is lower, and spot tailing is more likely to occur. When the developing solvent is 60% glacial acetic acid, the chromatogram of the test solution of the traditional Chinese medicine composition shows a main spot of the same color at the corresponding position as the chromatogram of the reference medicinal material solution. However, compared with the 50% glacial acetic acid used in Example 1, the Rf value of the main spot is higher, and the development time is longer.

[0051] (5) Conduct an investigation on temperature. Thin-layer chromatography was performed on the traditional Chinese medicine composition using the same method as in Example 1, except that the developing temperature was 4°C or 35°C.

[0052] The results are as follows Figure 6 As shown, 1 is a hyperoside reference solution; 2 is a dodder seed reference material solution; 3 is a negative test solution of the traditional Chinese medicine composition; and 4 is a test solution of the traditional Chinese medicine composition (batch number: 251101S). It can be seen that when the developing temperature is 4℃, the chromatogram of the test solution of the traditional Chinese medicine composition shows a main spot of the same color at the corresponding position as the chromatogram of the reference material solution. However, compared to the room temperature development used in Example 1, the development time is longer, but the spot is still clearly distinguishable. When the developing temperature is 35℃, the chromatogram of the test solution of the traditional Chinese medicine composition shows a main spot of the same color at the corresponding position as the chromatogram of the reference material solution, and these spots are clearly distinguishable.

[0053] (6) Conduct a humidity survey The dodder seed in the traditional Chinese medicine composition was identified by thin-layer chromatography using the same method as in Example 1, except that the developing humidity was 47% or 88%.

[0054] The results are as follows Figure 7 As shown, 1 is the hyperoside reference solution; 2 is the dodder seed reference medicinal material solution; 3 is the negative test solution of the traditional Chinese medicine composition; and 4 is the test solution of the traditional Chinese medicine composition (batch number: 251101S). It can be seen that when the humidity is 47%, the chromatogram of the test solution of the traditional Chinese medicine composition shows a main spot of the same color at the corresponding position as the chromatogram of the reference medicinal material solution, which can be clearly distinguished. When the humidity is 88%, the chromatogram of the test solution of the traditional Chinese medicine composition shows a main spot of the same color at the corresponding position as the chromatogram of the reference medicinal material solution, but the development time is longer compared to the relative humidity used in Example 1, but it is still clearly distinguishable.

[0055] (7) Conduct an examination of the system The dodder seed in the traditional Chinese medicine composition was identified by thin-layer chromatography using the same method as in Example 1, except that the developing solvent system was methanol-glacial acetic acid-water with a mass-volume ratio of 4:1:5.

[0056] The results are as follows Figure 8 As shown, 1 is a hyperoside reference solution; 2 is a dodder seed reference solution; 3 is a negative test solution of the traditional Chinese medicine composition; and 4 is a test solution of the traditional Chinese medicine composition (batch number: 251101S). It can be seen that, compared to the developing solvent system used in Example 1, the Rf value of the hyperoside main spot is lower, and the spot tailing is severe, indicating that the separation effect of this developing solvent system is limited and the sensitivity is low.

[0057] Example 3 This embodiment provides a thin-layer chromatography method for identifying Salvia miltiorrhiza in a traditional Chinese medicine composition containing Cuscuta chinensis and Salvia miltiorrhiza, comprising the following steps: Preparation of test solution of traditional Chinese medicine composition: Take 2g of traditional Chinese medicine composition solution, adjust pH to 2 with 10% hydrochloric acid, extract with ethyl acetate 3 times, 30mL each time, combine the organic phase extracts, evaporate to dryness in a water bath, add 2mL of methanol to completely dissolve the residue, and use it as test solution of traditional Chinese medicine composition.

[0058] Preparation of negative test solution of traditional Chinese medicine composition: Weigh appropriate amounts of other medicinal materials lacking Salvia miltiorrhiza and prepare negative test solution of traditional Chinese medicine composition; prepare negative test solution of traditional Chinese medicine composition lacking Salvia miltiorrhiza according to the preparation method of the above-mentioned traditional Chinese medicine composition test solution in this embodiment.

[0059] Preparation of reference herb solution: Take 0.5g of Salvia miltiorrhiza reference herb, add 20mL of water, heat under reflux for 30 minutes, and prepare the Salvia miltiorrhiza reference herb solution in the same way as the test solution of the above-mentioned traditional Chinese medicine composition in this embodiment.

[0060] Spotting: Perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2025 Edition, Part IV, General Chapter 0502). Take 5 μL of the test solution of the traditional Chinese medicine composition, 5 μL of the negative test solution of the traditional Chinese medicine composition, and 5 μL of the reference medicinal material solution, and spot them separately on a silica gel G thin-layer plate.

[0061] Development: Using chloroform-ethyl acetate-formic acid in a volume ratio of 5:4:2 as the developing solvent, the spotted silica gel G thin-layer plate (purchased from Merck) was placed in the developing solvent vapor for pre-saturation, developed, removed, and dried.

[0062] Inspection: Spray a 5% vanillin-sulfuric acid solution onto the dried silica gel G thin film, heat at 105°C until the spots are clearly visible, and inspect under sunlight.

[0063] The results are as follows Figure 9 As shown, 1 is the reference medicinal material solution; 2 is the negative test solution of the traditional Chinese medicine composition; 3 is the test solution of the traditional Chinese medicine composition (batch number: 251101S); 4 is the test solution of the traditional Chinese medicine composition (batch number: 251102S); and 5 is the test solution of the traditional Chinese medicine composition (batch number: 251103S). The results show that in the chromatogram of the test solution of the traditional Chinese medicine composition, spots of the same color appear at the corresponding positions of the main spots in the chromatogram of the reference medicinal material solution. Each spot is clearly visible, and the separation is good.

[0064] Example 4 This embodiment provides a methodological verification of the method in Embodiment 3.

[0065] (1) Specificity examination As described in Example 3, a test solution of the traditional Chinese medicine composition, a negative test solution of the traditional Chinese medicine composition, and a control medicinal material solution were prepared, and the samples were spotted, developed, and examined.

[0066] The results are as follows Figure 10 As shown, 1 is the reference herb solution; 2 is the negative test solution of the traditional Chinese medicine composition; and 3 is the test solution of the traditional Chinese medicine composition (batch number: 251101S). The results show that in the chromatogram of the test solution of the traditional Chinese medicine composition, spots of the same color appear at the corresponding positions of the main spots in the chromatogram of the reference herb solution. Each spot is clearly visible, the separation is good, and there is no negative interference, indicating strong specificity.

[0067] (2) Investigation of silicone thin film The thin-layer chromatography method was used to identify Danshen in the traditional Chinese medicine composition in the same way as in Example 3, except that the silica gel thin-layer plates were purchased from Qingdao Marine Chemical Plant.

[0068] The results are as follows Figure 11As shown, 1 is the reference medicinal material solution; 2 is the negative test solution of the traditional Chinese medicine composition; and 3 is the test solution of the traditional Chinese medicine composition (batch number: 251101S). The results show that in the chromatogram of the test solution of the traditional Chinese medicine composition, spots of the same color appear at the corresponding positions of the main spots in the chromatogram of the reference medicinal material solution. Each spot is clearly visible, and the separation is good.

[0069] (3) Conduct an investigation on temperature. The thin-layer chromatography method was used to identify Danshen in the traditional Chinese medicine composition according to the same method as in Example 3, except that the developing temperatures were 4°C and 35°C, respectively.

[0070] The results are as follows Figure 12 As shown, 1 is the reference medicinal material solution; 2 is the negative test solution of the traditional Chinese medicine composition; and 3 is the test solution of the traditional Chinese medicine composition (batch number: 251101S). The results show that when the developing temperature is 4℃, the chromatogram of the test solution of the traditional Chinese medicine composition shows spots of the same color at the corresponding positions of the main spots in the chromatogram of the reference medicinal material solution. Each spot is clearly visible, and the separation is good. However, compared to the results of the examples, the Rf value of the main spot is slightly lower. When the developing temperature is 35℃, the chromatogram of the test solution of the traditional Chinese medicine composition shows spots of the same color at the corresponding positions of the main spots in the chromatogram of the reference medicinal material solution. Each spot is clearly visible, and the separation is good.

[0071] (4) Conduct a humidity survey The thin-layer chromatography method was used to identify the danshen in the traditional Chinese medicine composition in the same way as in Example 3, with the only difference being that the developing humidity was 32% and 88%, respectively.

[0072] The results are as follows Figure 13 As shown, 1 is the reference medicinal material solution; 2 is the negative test solution of the traditional Chinese medicine composition; and 3 is the test solution of the traditional Chinese medicine composition (batch number: 251101S). The results show that when the developing humidity is 32%, the chromatogram of the test solution of the traditional Chinese medicine composition shows spots of the same color at the corresponding positions of the main spots in the chromatogram of the reference medicinal material solution, and each spot is clearly visible, indicating good separation. When the developing humidity is 88%, the chromatogram of the test solution of the traditional Chinese medicine composition shows spots of the same color at the corresponding positions of the main spots in the chromatogram of the reference medicinal material solution, and each spot is clearly visible, indicating good separation.

[0073] The above description, in conjunction with specific embodiments, provides a detailed explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A thin-layer chromatography method for identifying a traditional Chinese medicine composition, characterized in that, The traditional Chinese medicine composition contains Cuscuta chinensis and Salvia miltiorrhiza, and the thin-layer chromatography identification method includes a thin-layer chromatography identification method for Cuscuta chinensis and a thin-layer chromatography identification method for Salvia miltiorrhiza; wherein, The thin-layer chromatography method for identifying dodder seeds includes the following steps: The first test solution, the Cuscuta chinensis reference herb solution, and the reference solution were spotted separately on the same polyamide thin-layer plate for thin-layer chromatography analysis. The obtained chromatograms were compared. The developing solvent was glacial acetic acid with a mass percentage of 30% to 70%. The thin-layer chromatography method for identifying Salvia miltiorrhiza includes the following steps: The second test solution and the Danshen reference herb solution were spotted separately on the same silica gel G thin-layer plate for thin-layer chromatography analysis. The obtained chromatograms were compared. The developing solvent was chloroform-ethyl acetate-formic acid with a mass-volume ratio of 2~3:1~3:

1.

2. The thin-layer identification method according to claim 1, characterized in that, The herbal composition contains deer antler, calcined amethyst, dodder seed, stir-fried white peony root, stir-fried yam with wheat bran, wine-processed cornus fruit, vinegar-processed Bupleurum root, and Salvia miltiorrhiza.

3. The thin-layer identification method according to claim 2, characterized in that, In the aforementioned traditional Chinese medicine composition, the mass ratio of deer antler, calcined amethyst, dodder seed, stir-fried white peony root, stir-fried yam with wheat bran, wine-processed cornus fruit, vinegar-processed Bupleurum root, and Salvia miltiorrhiza is (0.1~2):(0.1~2):(0.5~3):(0.1~2):(0.5~3):(0.1~2):(0.1~1.5):(0.1~2).

4. The thin-layer identification method according to claim 1, characterized in that, The first test solution is obtained by dissolving the test sample in an alcohol solvent; and / or, The dodder seed reference solution was obtained by dissolving dodder seed reference material in an alcoholic solvent; and / or, The reference solution is obtained by dissolving the reference standard in an alcohol solvent.

5. The thin-layer identification method according to claim 1, characterized in that, The reference standard was hyperoside.

6. The thin-layer identification method according to claim 1, characterized in that, The thin-layer identification method for dodder also includes unfolding and drying, spraying with 1%~3% aluminum trichloride ethanol solution, drying, and developing color under a 365nm ultraviolet lamp.

7. The thin-layer identification method according to claim 1, characterized in that, The second test solution was prepared by the following steps: Take the test sample, adjust the pH to 1-3 with acid, then extract with ethyl acetate, collect the organic layer, evaporate to dryness, dissolve the residue in an alcohol solvent to obtain the final product; and / or, The Danshen reference herb solution was prepared by the following steps: Take the reference material of Salvia miltiorrhiza, add water and heat under reflux, collect the extract, add acid to adjust the pH to 1-3, then add ethyl acetate for extraction, take the organic layer, evaporate to dryness, dissolve the residue in an alcohol solvent to obtain the final product.

8. The thin-layer identification method according to claim 1, characterized in that, The thin-layer identification method for Salvia miltiorrhiza also includes unfolding and drying, spraying with 4%~6% vanillin sulfuric acid solution, drying, and developing color under sunlight.

9. The thin-layer identification method according to claim 6 or 8, characterized in that, The ambient temperature for each of the unfolding operations is independently 4~35°C, and / or the ambient humidity for each of the unfolding operations is independently 30%~90%.

10. The thin-layer identification method according to claim 1, characterized in that, The sample spotting volume for the thin-layer chromatography analysis is 1~5 μL.

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