A method for identifying panax quinquefolium and its application

Abstract

This invention belongs to the field of thin-layer chromatography (TLC) identification technology for traditional Chinese medicine, and specifically relates to a rapid TLC identification method and application for American ginseng and ginseng. The TLC identification method includes the following steps: taking a test sample, adding a solvent, sonicating, and taking the supernatant as the test solution; taking American ginseng and ginseng reference materials, adding solvents respectively, sonicating, and taking the supernatant as American ginseng reference material solutions and ginseng reference material solutions; taking ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, ginsenoside Rd, and pseudoginsenoside F11 reference standards, adding solvents, mixing, and obtaining reference solution; finally, performing TLC analysis and result determination on the test solution, reference material solution, and reference solution. This invention is simple to operate, highly efficient and rapid, has good repeatability and high accuracy, and is low in toxicity and durable, and can simultaneously detect adulteration, substitution, and confusion in American ginseng and ginseng.

Description

Technical Field

[0001] This invention belongs to the field of thin-layer chromatography identification technology of traditional Chinese medicine, and specifically relates to a rapid thin-layer chromatography identification method and application for identifying American ginseng and ginseng. Background Technology

[0002] American ginseng is the dried root of *Panax quinquefolium* L., while ginseng is the dried root and rhizome of *Panax ginseng* CAMey. Both originate from the same plant family, Araliaceae, and have similar appearances and chemical compositions, making identification difficult. Although both American ginseng and ginseng are tonic herbs in traditional Chinese medicine, their properties, functions, and indications differ significantly. According to the 2020 edition of the Chinese Pharmacopoeia, American ginseng is sweet and slightly bitter, cool in nature, and enters the heart, lung, and kidney meridians. It has the effects of replenishing qi and nourishing yin, clearing heat and promoting body fluids. It is used for qi and yin deficiency, fatigue due to deficiency heat, cough with phlegm and blood, internal heat and thirst, and dry mouth and throat. Ginseng is sweet and slightly bitter, slightly warm in nature, and has the effects of greatly replenishing vital energy, restoring pulse and consolidating the body, tonifying the spleen and lungs, promoting body fluids and nourishing blood, calming the mind and improving intelligence. It is used for physical weakness and impending collapse, cold limbs and weak pulse, spleen deficiency and poor appetite, lung deficiency and cough, thirst due to fluid depletion, internal heat and thirst, qi and blood deficiency, chronic illness and emaciation, palpitations and insomnia, impotence and cold uterus. In addition to treating diseases, both American ginseng and ginseng are often used in dietary therapy and health maintenance. They are commonly used bulk medicinal materials and medicinal and edible materials in my country. Product specifications include raw materials, processed slices, and powders, with the difficulty of identification increasing in that order. Therefore, rapid and objective identification of American ginseng and ginseng is of great significance.

[0003] Thin-layer chromatography (TLC) is an effective, simple, and widely used technique for verifying the authenticity of pharmaceutical products. It is characterized by its large information capacity, high specificity, and rapid, intuitive results. However, the TLC identification methods for American ginseng and ginseng included in the 2020 edition of the Chinese Pharmacopoeia, as listed in Part I, involve complex procedures, cumbersome preparation of developing solvents, testing times of nearly 24 hours, and difficulty in ensuring reproducibility. Furthermore, the methods are inconsistent and the judgment is difficult to master, significantly impacting the efficiency and accuracy of testing and hindering the high-quality development of the American ginseng and ginseng industry.

[0004] Therefore, there is an urgent need to establish a thin-layer chromatography method that can quickly and accurately distinguish between American ginseng and ginseng. This method should be simple to operate, efficient and rapid, with good repeatability and high accuracy. It should also be environmentally friendly, low in toxicity, and durable. It can simultaneously check for the possibility of adulteration, substitution, or confusion between American ginseng and ginseng, and lay the foundation for the development of on-site rapid screening and detection kits. Summary of the Invention

[0005] This invention aims to solve one or more technical problems existing in the prior art, and at least provide a beneficial alternative or create conditions. This invention provides a thin-layer chromatography method for rapidly and accurately identifying American ginseng and ginseng. It is simple to operate, highly efficient and rapid, has good repeatability and high accuracy, and is environmentally friendly, low in toxicity, and durable. It can simultaneously detect the possibility of adulteration, substitution, or confusion of American ginseng and ginseng, and lays the foundation for the development of rapid on-site screening and detection kits.

[0006] The inventive concept of this invention is as follows: In preparing the test solution and the reference herb solution, only ultrasonic extraction with a solvent is required; evaporation is unnecessary, and the supernatant can be directly used for subsequent experiments, making it simple and efficient. Simultaneously, this invention uses the reference herb solution and reference standard solution to simultaneously identify American ginseng and ginseng based on their chemical composition differences: American ginseng contains ginsenoside F11 but not ginsenoside Rf, while ginseng contains ginsenoside Rf but not ginsenoside F11. Thin-layer chromatography (TLC) provides a wealth of information, is simple, and efficient. Furthermore, this invention uses the same rapid preparation method for the test solution, the same developing solvent conditions, and the same colorimetric and inspection conditions to achieve simultaneous detection of American ginseng and ginseng. The average detection time is shorter than that of any current method used in the Chinese Pharmacopoeia for American ginseng and ginseng, achieving identification of American ginseng and ginseng samples within 60 minutes. This method is simple, efficient, has good repeatability, and high accuracy.

[0007] Therefore, the first aspect of the present invention provides a rapid thin-layer chromatography method for identifying American ginseng and ginseng.

[0008] Specifically, a rapid thin-layer chromatography method for identifying American ginseng and ginseng includes the following steps:

[0009] (1) Preparation of test solution: Take the test sample, add solvent, sonicate, take the supernatant to obtain the test solution;

[0010] (2) Preparation of reference medicinal material solution: American ginseng and ginseng reference medicinal materials were taken, solvents were added respectively, ultrasonic treatment was performed, and the supernatant was taken to obtain American ginseng reference medicinal material solution and ginseng reference medicinal material solution;

[0011] (3) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference and pseudoginsenoside F11 reference, add solvent, mix, and obtain reference solution;

[0012] (4) Thin-layer chromatography analysis: Take the test solution, the reference medicinal material solution and the reference solution, respectively spot them on the same silica gel G thin-layer plate, add the developing solvent and color reagent, heat and examine;

[0013] (5) Result determination: Compare the thin-layer chromatography results to determine whether the test sample is American ginseng or ginseng.

[0014] Preferably, in steps (1), (2), and (3), the solvent includes at least one of methanol and ethanol.

[0015] More preferably, in steps (1) and (2), the solvent is ethanol; in step (3), the solvent is methanol.

[0016] Preferably, in steps (1) and (2), the ultrasonic treatment time is 15-16.5 min; more preferably, the ultrasonic treatment time is 15-16 min; even more preferably, the ultrasonic treatment time is 15 min.

[0017] Preferably, in step (4), the test solution and the reference medicinal material solution are both 2-4 μL; the reference solution is 1-2 μL.

[0018] Preferably, the silicone G thin film is a high-efficiency silicone G thin film, and the spots are in the form of strips.

[0019] Specifically, the test solution, the reference medicinal material solution, and the reference solution are spotted onto the same silica gel G thin-layer plate and appear as strips.

[0020] Preferably, the width of the strip is 5-10 mm; more preferably, the width of the strip is 8 mm.

[0021] Preferably, in step (4), the developing agent is a mixture of dichloromethane, anhydrous ethanol, ethyl acetate, and water.

[0022] More preferably, the volume ratio of dichloromethane, anhydrous ethanol, ethyl acetate, and water is 23:20:2:2.6.

[0023] Preferably, in step (4), the developing agent is added and the developing process is carried out under certain temperature and humidity conditions.

[0024] Preferably, the unfolding temperature is 5-30°C and the unfolding humidity is 30-90%.

[0025] More preferably, the unfolding temperature is 5-28°C; the unfolding humidity is 32-88%.

[0026] Preferably, the color developer comprises a 5-10% by mass sulfuric acid ethanol solution; more preferably, the color developer comprises a 10% by mass sulfuric acid ethanol solution.

[0027] Specifically, a 5-10% mass fraction sulfuric acid ethanol solution means that the mass fraction of sulfuric acid in the sulfuric acid ethanol solution is 5-10%.

[0028] Preferably, in step (4), the heating temperature is 95-115℃ and the heating time is 50-70s.

[0029] More preferably, the heating temperature is 100-110℃; the heating time is 55-65s.

[0030] More preferably, the heating temperature is 105°C; and the heating time is 60 seconds.

[0031] Specifically, heating makes the spots appear more clearly.

[0032] Preferably, in step (4), the inspection is performed using ultraviolet lamps and fluorescent lamps.

[0033] Preferably, the wavelength of the ultraviolet lamp is 365nm.

[0034] Specifically, in step (5), if the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of the reference ginseng and ginsenoside Rg1, ginsenoside F11, ginsenoside Re, and ginsenoside Rd, and there are no corresponding spots at the corresponding positions as those of the ginsenoside Rf reference, then the test sample is determined to be American ginseng; if the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of the reference ginseng and ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and there are no corresponding spots at the corresponding positions as those of the ginsenoside F11 reference, then the test sample is determined to be ginseng; if the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of the reference chromatogram, then the test sample is determined to be a mixture of American ginseng and ginseng.

[0035] The second aspect of the present invention provides an application of the thin-layer chromatography identification method described in the first aspect of the present invention in the field of traditional Chinese medicine identification.

[0036] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows:

[0037] (1) This invention uses the same rapid preparation method of the test solution, the same developing solvent conditions, and the same color development and inspection conditions to achieve simultaneous detection of American ginseng and ginseng. The average test time is shorter than that of any method in the current pharmacopoeia for American ginseng and ginseng. It can simultaneously, rapidly and accurately identify American ginseng and ginseng samples within 60 minutes, which greatly improves the testing efficiency. It is simple and efficient, with clear thin-layer chromatographic spots, good repeatability and high accuracy. It is also environmentally friendly and low in toxicity, with rich experimental results and easy to judge.

[0038] (2) In preparing the test solution, the present invention only requires ultrasonic extraction with methanol and / or ethanol solvents. The extraction time is about 15 minutes. The extraction time is short and there is no need to evaporate to dryness. The test solution can be obtained directly by taking the supernatant. It is simple and efficient.

[0039] (3) The developing agent selected in this invention has a fast development speed and the developing agent is easily volatilized after the thin plate is developed, which facilitates the subsequent color development operation. In addition, this invention uses a 5-10% sulfuric acid ethanol solution as a color developer. The color developer uses ethanol as a solvent, which greatly reduces the difficulty and danger of preparation. Moreover, the color developer has a clearer texture, and the immersion color development method is fast and easy to uniform. The heating time and degree are better controlled. Attached Figure Description

[0040] Figure 1 The chromatograms are those of the X1-X10 batches of American ginseng samples from Example 1 of this invention, obtained by thin-layer chromatography.

[0041] Figure 2 The chromatograms are for thin-layer chromatography identification of batches X11-X20 of American ginseng samples in Example 2 of this invention;

[0042] Figure 3 The chromatograms are those of the thin-layer chromatography identification of ginseng samples R1-R9 in Example 3 of this invention;

[0043] Figure 4 The chromatograms are for thin-layer chromatography identification of ginseng samples R10-R19 of Example 4 of the present invention;

[0044] Figure 5 This is a chromatogram of the thin-layer chromatography identification performed at a developing temperature of 6°C according to the present invention;

[0045] Figure 6 This is a chromatogram of the thin-layer chromatography identification performed at a developing temperature of 26°C according to the present invention;

[0046] Figure 7 This is a chromatogram of the thin-layer chromatography identification performed at a development temperature of 32% according to the present invention;

[0047] Figure 8This is a chromatogram of the thin-layer chromatography identification performed at an developing temperature of 88% according to the present invention;

[0048] Figure 9 This is a chromatogram of thin-layer chromatography identification performed on a MAHEREY-NAGEL high-efficiency silica gel G pre-fabricated thin-layer plate in Germany according to the present invention.

[0049] Figure 10 This is a chromatogram of thin-layer chromatography identification performed on a high-efficiency silica gel G pre-fabricated thin-layer plate from Qingdao Ocean Chemical Co., Ltd. according to the present invention.

[0050] Figure 11 This is a chromatogram of thin-layer chromatography identification performed on a high-efficiency silica gel G pre-fabricated thin-layer plate at the Yantai Chemical Industry Research Institute according to the present invention.

[0051] Figure 12 Chromatograms for thin-layer chromatography identification of the present invention and the method for preparing American ginseng test solution in the Chinese Pharmacopoeia;

[0052] Figure 13 This is a chromatogram for thin-layer chromatography identification of the ginseng test solution preparation method in this invention and the Chinese Pharmacopoeia.

[0053] Figure 14 This is a chromatogram of thin-layer chromatography identification of Comparative Example 3 of the present invention;

[0054] Figure 15 This is a chromatogram of thin-layer chromatography identification of Comparative Example 4 of the present invention;

[0055] Figure 16 This is a chromatogram of thin-layer chromatography identification of Comparative Example 5 of the present invention. Detailed Implementation

[0056] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0057] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0058] In this invention, there were 20 batches of American ginseng samples and 19 batches of ginseng samples. Detailed information is shown in Table 1.

[0059] Thirty-nine batches of samples were pulverized and passed through a 24-mesh sieve for later use.

[0060] The reference medicinal materials and reference standards were all purchased from the China National Institutes for Food and Drug Control. Detailed information is shown in Table 2.

[0061] Table 1: Detailed information on American ginseng samples and ginseng samples

[0062]

[0063]

[0064] Table 2: Detailed information on reference medicinal materials and reference standards

[0065]

[0066]

[0067] Example 1

[0068] Example 1 describes the thin-layer chromatography identification of batches 1-10 (numbered X1-X10) of American ginseng samples in Table 1.

[0069] A rapid thin-layer chromatography method for identifying American ginseng and ginseng, the method comprising the following steps:

[0070] (1) Preparation of test solution: Take 1g of each of the American ginseng sample (number: X1-X10) powders, add 3mL of ethanol, shake well, sonicate for 15min, and take the supernatant as the American ginseng test solution.

[0071] (2) Preparation of reference drug solution: Take 1g of American ginseng (D1) and ginseng reference drug (D2), add 3mL of ethanol to each, shake well, sonicate for 15min, and take the supernatant as American ginseng reference drug solution and ginseng reference drug solution.

[0072] (3) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0073] (4) Thin-layer chromatography analysis: According to the thin-layer chromatography method of General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2 μL each of the American ginseng test solution and the reference medicinal material solution obtained in steps (1)(2)(3) above, and 1 μL of the reference solution, and spot them on the same high-performance silica gel G thin-layer plate to form strips with a band width of 8 mm; use dichloromethane-anhydrous ethanol-ethyl acetate-water (23:20:2:2.6) as the developing solvent, develop to a distance of about 8 cm, air dry, immerse the plate in 10% sulfuric acid ethanol solution, air dry, heat at 105℃ for 1 min, and examine under ultraviolet light (365 nm) and sunlight respectively;

[0074] (5) Result Judgment: If the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of American ginseng reference material and ginsenoside Rg1, ginsenoside F11, ginsenoside Re, and ginsenoside Rd reference materials, and no corresponding spots at the corresponding positions as those of ginsenoside Rf reference material, then it is determined to be American ginseng. If the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of ginseng reference material, ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd reference materials, and no corresponding spots at the corresponding positions as those of ginsenoside F11 reference material, then it is determined to be ginseng. If the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of the reference materials, then the test sample is determined to be a mixture of American ginseng and ginseng.

[0075] The chromatograms for thin-layer chromatography identification of American ginseng samples (numbers: X1-X10) are as follows: Figure 1 As shown, where, Figure 1 A is the chromatogram viewed under ultraviolet light (365nm). Figure 1 B is the chromatogram viewed under sunlight. Figure 1 In the diagram, 1 represents X1, 2 represents X2, 3 represents X3, 4 represents X4, 5 represents X5, 6 represents D1, 7 represents D2, 8 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively), 9 represents X6, 10 represents X7, 11 represents X8, 12 represents X9, and 13 represents X10.

[0076] Chromatographic conditions: Merck HPTLC Silica gel 60 (batch number: HX02721141, 10×20cm); temperature: 20℃, relative humidity: 37%; examined under UV lamp (365nm) and sunlight respectively.

[0077] Depend on Figure 1 It can be seen that in the chromatograms of the above 10 batches of American ginseng, spots of the same color appear at the corresponding positions as in the chromatograms of American ginseng reference material and ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Re, and ginsenoside Rd reference materials, and there are no corresponding spots at the corresponding positions in the chromatograms of ginsenoside Rf reference material, indicating that they are American ginseng samples.

[0078] Example 2

[0079] Example 1 describes the thin-layer chromatography identification of batches 11-20 (numbered X11-X20) of American ginseng samples in Table 1.

[0080] The only difference between Example 2 and Example 1 is that the batches of the American ginseng samples are different (i.e., different places of origin, specifications or types). See Table 1 for details. Everything else is the same as in Example 1.

[0081] The chromatograms for thin-layer chromatography identification of American ginseng samples (numbers: X11-X20) are as follows: Figure 2 As shown, where, Figure 2 A is the chromatogram viewed under ultraviolet light (365nm). Figure 2 B is the chromatogram viewed under sunlight. Figure 2 In the diagram, 1 represents X11, 2 represents X12, 3 represents X13, 4 represents X14, 5 represents X15, 6 represents D1, 7 represents D2, 8 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively), 9 represents X16, 10 represents X17, 11 represents X18, 12 represents X19, and 13 represents X20.

[0082] Depend on Figure 2 It can be seen that in the chromatograms of the 10 batches of American ginseng, spots of the same color appear at the corresponding positions as in the chromatograms of American ginseng reference material and ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Re, and ginsenoside Rd reference materials, and there are no corresponding spots at the corresponding positions in the chromatograms of ginsenoside Rf reference material, indicating that they are American ginseng samples.

[0083] Example 3

[0084] Example 3: Thin-layer chromatography was used to identify ginseng samples from batches 1-9 (numbered R1-R9) in Table 1.

[0085] A rapid thin-layer chromatography method for identifying American ginseng and ginseng, the method comprising the following steps:

[0086] (1) Preparation of test solution: Take 1g of each ginseng sample (number: R1-R9) powder, add 3mL of ethanol, shake well, sonicate for 15min, and take the supernatant as ginseng test solution.

[0087] (2) Preparation of reference drug solution: Take 1g of American ginseng (D1) and ginseng reference drug (D2), add 3mL of ethanol to each, shake well, sonicate for 15min, and take the supernatant as American ginseng reference drug solution and ginseng reference drug solution respectively.

[0088] (3) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0089] (4) Thin-layer chromatography analysis: According to the thin-layer chromatography method of General Chapter 0502 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 2 μL of each of the ginseng test solution and the reference medicinal material solution obtained in steps (1)(2)(3) above, and 1 μL of the reference solution, and spot them on the same high-performance silica gel G thin-layer plate to form strips with a band width of 8 mm; use dichloromethane-anhydrous ethanol-ethyl acetate-water (23:20:2:2.6) as the developing solvent, develop to a distance of about 8 cm, air dry, immerse the plate in 10% sulfuric acid ethanol solution, air dry, heat at 105℃ for 1 min, and examine under ultraviolet light (365 nm) and sunlight respectively;

[0090] (5) Result Judgment: If the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of American ginseng reference material and ginsenoside Rg1, ginsenoside F11, ginsenoside Re, and ginsenoside Rd reference materials, and no corresponding spots at the corresponding positions as those of ginsenoside Rf reference material, then it is determined to be American ginseng. If the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of ginseng reference material, ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd reference materials, and no corresponding spots at the corresponding positions as those of ginsenoside F11 reference material, then it is determined to be ginseng. If the chromatogram of the test sample shows spots of the same color at the corresponding positions as those of the reference materials, then the test sample is determined to be a mixture of American ginseng and ginseng.

[0091] The chromatograms for thin-layer chromatography identification of ginseng samples (numbers: R1-R9) are as follows: Figure 3 As shown, where, Figure 3 A is the chromatogram viewed under ultraviolet light (365nm). Figure 3 B is the chromatogram viewed under sunlight. Figure 3 In the diagram, 1 represents R1, 2 represents R2, 3 represents R3, 4 represents R4, 5 represents D1, 6 represents D2, 7 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively), 8 represents R5, 9 represents R6, 10 represents R7, 11 represents R8, and 12 represents R9.

[0092] Chromatographic conditions: Merck HPTLC Silica gel 60 (batch number: HX02721141, 10×20cm); temperature: 20℃, relative humidity: 39%; examined under UV lamp (365nm) and sunlight respectively.

[0093] Depend on Figure 3 It can be seen that in the chromatograms of the nine batches of ginseng, spots of the same color appear at the corresponding positions as in the chromatograms of ginseng reference material, ginsenoside Rg1 reference standard, ginsenoside Rf reference standard, ginsenoside Re reference standard, and ginsenoside Rd reference standard, and there are no corresponding spots at the corresponding positions as in the chromatograms of pseudoginsenoside F11 reference standard, which indicates that they are ginseng samples.

[0094] Example 4

[0095] Example 4 describes the thin-layer chromatography identification of ginseng samples from batches 10-19 (numbered R10-R19) in Table 1.

[0096] The difference between Example 4 and Example 3 is that the ginseng samples are from different batches (i.e., different places of origin, specifications or types). See Table 1 for details. Otherwise, they are the same as in Example 3.

[0097] The chromatograms for thin-layer chromatography identification of ginseng samples (number: R10-R19) are as follows: Figure 4 As shown, where, Figure 4 A is the chromatogram viewed under ultraviolet light (365nm). Figure 4 B is the chromatogram viewed under sunlight. Figure 4 In the diagram, 1 represents R10, 2 represents R11, 3 represents R12, 4 represents R13, 5 represents R14, 6 represents D1, 7 represents D2, 8 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively), 9 represents R15, 10 represents R16, 11 represents R17, 12 represents R18, and 13 represents R19.

[0098] Depend on Figure 4 It can be seen that in the chromatograms of the 10 batches of ginseng, spots of the same color appear at the corresponding positions as in the chromatograms of ginseng reference material, ginsenoside Rg1 reference standard, ginsenoside Rf reference standard, ginsenoside Re reference standard, and ginsenoside Rd reference standard, and there are no corresponding spots at the corresponding positions as in the chromatograms of pseudoginsenoside F11 reference standard, which indicates that they are ginseng samples.

[0099] Durability tests were conducted in Examples 5-7 of this invention, and comparative studies were carried out on factors such as different unfolding temperatures, different unfolding humidity, and different brands of thin-layer sheets.

[0100] Example 5

[0101] Example 5 investigates different developing temperatures, specifically a thin-layer chromatography identification method, including the following steps:

[0102] (1) Preparation of test solution: Take 1g each of American ginseng reference material (D1) and ginseng reference material (D2), and prepare them according to the preparation method of test solution in Example 1 of this invention, and use them as American ginseng test solution and ginseng test solution respectively.

[0103] (2) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0104] (3) Thin-layer chromatography analysis: Following the thin-layer chromatography method in Section 0502 of the 2020 edition of the Chinese Pharmacopoeia, 2 μL each of the above-mentioned American ginseng test solution and ginseng test solution, and 1 μL of the reference solution, were spotted onto the same high-performance silica gel G thin-layer plate to form strips with a band width of 8 mm. Using dichloromethane-anhydrous ethanol-ethyl acetate-water (23:20:2:2.6) as the developing solvent, the plates were developed at 6℃ and 26℃, respectively, with a development distance of approximately 8 cm in both cases. After drying, the plates were immersed in 10% sulfuric acid ethanol solution, dried again, and heated at 105℃ for approximately 1 min. The plates were then examined under ultraviolet light (365 nm) and sunlight. The chromatograms for thin-layer chromatography identification at developing temperatures of 6℃ and 26℃ are shown below. Figure 5 and Figure 6 As shown; where, Figure 5 A and 6A are chromatograms viewed under ultraviolet light (365nm). Figure 5 B and 6B are chromatograms examined under sunlight; Figure 5 and Figure 6 In the diagram, 1 represents D1, 2 represents D2, and 3 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively).

[0105] The results show that, according to the thin-layer identification method of the present invention, the HPTLC separation effect is good and meets the identification requirements when developed at two different development temperatures of 6℃ and 26℃.

[0106] Example 6

[0107] Example 6 investigates different developing humidity levels, specifically a thin-layer chromatography identification method, including the following steps:

[0108] (1) Preparation of test solution: Take 1g each of American ginseng reference material (D1) and ginseng reference material (D2), and prepare them according to the preparation method of test solution in Example 1 of this invention, and use them as American ginseng test solution and ginseng test solution respectively.

[0109] (2) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0110] (3) Thin-layer chromatography analysis: Following the thin-layer chromatography method in Section 0502 of the 2020 edition of the Chinese Pharmacopoeia, 2 μL each of the above-mentioned American ginseng test solution and ginseng test solution, and 1 μL of the reference solution, were spotted onto the same high-performance silica gel G thin-layer plate to form strips with a band width of 8 mm. Using dichloromethane-anhydrous ethanol-ethyl acetate-water (23:20:2:2.6) as the developing solvent, the plates were developed at 32% and 88% humidity, respectively, with a development distance of approximately 8 cm. After drying, the plates were immersed in 10% sulfuric acid ethanol solution, dried again, and heated at 105℃ for approximately 1 min. The plates were then examined under ultraviolet light (365 nm) and sunlight, respectively. The chromatograms for thin-layer chromatography identification at 32% and 88% developing humidity are shown below. Figure 7 and Figure 8 As shown; where, Figure 7 A and 8A are chromatograms viewed under ultraviolet light (365nm). Figure 7 B and 8B are chromatograms examined under sunlight; Figure 7 and Figure 8 In the diagram, 1 represents D1, 2 represents D2, and 3 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively).

[0111] The results show that the thin-layer identification method of the present invention, when carried out at humidity of 32% and 88%, respectively, has good HPTLC separation effect and meets the identification requirements.

[0112] Example 7

[0113] Example 7 investigates different brands of high-efficiency thin-layer laminates. Specifically, a thin-layer laminate identification method includes the following steps:

[0114] (1) Preparation of test solution: Take 1g each of American ginseng reference material (D1) and ginseng reference material (D2), and prepare them according to the preparation method of test solution in Example 1 of this invention, and use them as American ginseng test solution and ginseng test solution respectively.

[0115] (2) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0116] (3) Thin-layer chromatography analysis: Following the thin-layer chromatography method in Section 0502 of the 2020 edition of the Chinese Pharmacopoeia, 2 μL each of the above-mentioned American ginseng test solution and ginseng test solution, and 1 μL of the reference solution, were spotted onto high-efficiency silica gel G pre-prepared thin-layer plates from three brands: MACHEREY-NAGEL, Qingdao Ocean Chemical Co., Ltd., and Yantai Chemical Industry Research Institute, respectively, forming strips with a band width of 8 mm. Using dichloromethane-anhydrous ethanol-ethyl acetate-water (23:20:2:2.6) as the developing solvent, the developing distance was approximately 8 cm. After drying, the plates were immersed in 10% sulfuric acid ethanol solution, dried, and heated at 105℃ for about 1 min. The plates were then examined under ultraviolet light (365 nm) and sunlight. The chromatograms obtained using the three brands of thin-layer chromatography are shown below. Figure 9-11 As shown; where, Figure 9 A, 10A, and 11A are all chromatograms viewed under ultraviolet light (365nm). Figure 9 B, 10B, and 11B are all chromatograms examined under sunlight; Figure 9-11 In the diagram, 1 represents D1, 2 represents D2, and 3 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively).

[0117] The results show that the identification method of the present invention can achieve good separation effect and meet the identification requirements under the tests of three different brands of high-efficiency silica gel G thin-layer plates.

[0118] As can be seen from the analysis of Examples 5-7 above, the thin-layer chromatography identification method for rapid identification of American ginseng and ginseng established by the present invention can achieve good separation results under different developing temperatures, different relative humidity, and different brands of high-efficiency thin-layer plates, indicating that the method of the present invention has good durability.

[0119] The following is a comparative study and analysis of the effects of the preparation methods of American ginseng and ginseng test solutions in this invention and the Chinese Pharmacopoeia on the results of thin-layer chromatography.

[0120] 1. The effects of the preparation method of the American ginseng test solution of the present invention and the preparation method of the test solution of American ginseng under the thin-layer identification item of the Chinese Pharmacopoeia on the thin-layer chromatography results were compared. Among them, the thin-layer chromatography identification using the preparation method of the American ginseng test solution of the present invention was used as Example 8, and the thin-layer chromatography identification using the preparation method of the test solution of American ginseng under the thin-layer identification item of the Chinese Pharmacopoeia was used as Comparative Example 1.

[0121] A thin-layer chromatography identification method includes the following steps:

[0122] (1) Preparation of test solution: American ginseng reference material (D1) was used as the test sample with a definite source and was prepared according to the preparation method of test solution in Example 1 of this invention, as test solution 1 of Example 8; 1g of American ginseng reference material (D1) was taken and extracted according to the preparation method of test solution under the [Identification] standard item of American ginseng in the 2020 edition of the Chinese Pharmacopoeia, as test solution 2 of Comparative Example 1;

[0123] (2) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0124] (3) Thin-layer chromatography analysis: The thin-layer chromatography method was performed according to Section 0502 of the General Chapter of Part IV of the 2020 edition of the Chinese Pharmacopoeia. 2 μL each of test solution 1 and test solution 2, and 1 μL of the reference solution were spotted onto the same high-performance silica gel G thin-layer plate, forming strips with a band width of 8 mm. A dichloromethane-anhydrous ethanol-ethyl acetate-water (23:20:2:2.6) solution was used as the developing solvent, with a development distance of approximately 8 cm. The plate was dried, immersed in 10% sulfuric acid ethanol solution, dried again, and heated at 105℃ for approximately 1 min. The results were then examined under ultraviolet light (365 nm) and sunlight. Figure 12 As shown, where, Figure 12 A is the chromatogram viewed under ultraviolet light (365nm). Figure 12 B is a chromatogram examined under sunlight; Figure 12 In the diagram, 1 represents D1 of this invention, 2 represents D1 of the Chinese Pharmacopoeia, and 3 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively).

[0125] The results showed that the thin-layer chromatograms of the American ginseng test solution extracted using the method of this invention were comparable to those extracted using the current standard method (i.e., the method for preparing the test solution under the thin-layer identification section of the Chinese Pharmacopoeia). However, the present invention only requires ultrasonic extraction for 15 minutes and uses methanol as the extraction reagent, with a volume of only 3 mL. In contrast, the preparation method according to the Chinese Pharmacopoeia requires methanol and n-butanol as extraction reagents, more than 79 mL of organic reagents, and involves operations such as heating under reflux, evaporation, and extraction, taking more than 60 minutes. Therefore, the present invention is faster, less toxic, and more environmentally friendly.

[0126] 2. The effects of the preparation method of the ginseng test solution of the present invention and the preparation method of the test solution of ginseng under the thin-layer chromatography identification section of the Chinese Pharmacopoeia on the thin-layer chromatography results were compared. Among them, the thin-layer chromatography identification using the preparation method of the ginseng test solution of the present invention was used as Example 9, and the thin-layer chromatography identification using the preparation method of the test solution of ginseng under the thin-layer chromatography identification section of the Chinese Pharmacopoeia was used as Comparative Example 2.

[0127] A thin-layer chromatography identification method includes the following steps:

[0128] (1) Preparation of test solution: Ginseng reference material (D2) was used as the test sample with a definite source and was prepared according to the preparation method of test solution in Example 1 of this invention, as test solution 1 of Example 9; 1g of ginseng reference material (D2) was taken and extracted according to the method of [Identification] (2) under the ginseng standard item of the 2020 edition of the Chinese Pharmacopoeia, as test solution 2 of Comparative Example 2;

[0129] (2) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0130] (3) Thin-layer chromatography analysis: The thin-layer chromatography method was performed according to Section 0502 of the General Chapter of Part IV of the 2020 edition of the Chinese Pharmacopoeia. 2 μL each of test solution 1 and test solution 2, and 1 μL of the reference solution were spotted onto the same high-performance silica gel G thin-layer plate, forming strips with a band width of 8 mm. A dichloromethane-anhydrous ethanol-ethyl acetate-water (23:20:2:2.6) solution was used as the developing solvent, with a development distance of approximately 8 cm. The plate was dried, immersed in 10% sulfuric acid ethanol solution, dried again, and heated at 105℃ for approximately 1 min. The results were then examined under ultraviolet light (365 nm) and sunlight. Figure 13 As shown; where, Figure 13A is the chromatogram viewed under ultraviolet light (365nm). Figure 13 B is a chromatogram examined under sunlight; Figure 13 In the diagram, 1 represents D2 of this invention, 2 represents D2 of the Chinese Pharmacopoeia, and 3 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively).

[0131] The results showed that the ginseng test solution extracted using the method of this invention and the preparation method of the test solution under the thin-layer chromatography identification section of the Chinese Pharmacopoeia produced comparable thin-layer chromatographic results. However, the present invention only requires ultrasonic extraction for 15 minutes, using methanol as the extraction reagent, with a volume of only 3 mL. In contrast, the preparation method of the test solution according to the Chinese Pharmacopoeia requires more than 60 minutes, using chloroform, n-butanol, and ammonia solution as extraction reagents, with a volume of more than 50 mL, and requires operations such as heating under reflux, ultrasonic treatment, extraction, and evaporation. Therefore, the present invention is faster, less toxic, and more environmentally friendly.

[0132] 3. To compare the effects of the developing solvent of this invention and the developing solvent under the TLC identification section of the Chinese Pharmacopoeia on the results of thin-layer chromatography, the developing solvent under the TLC identification section of the Chinese Pharmacopoeia for ginseng and American ginseng was used for TLC identification as Comparative Example 3. The specific TLC identification method of Comparative Example 3 is as follows:

[0133] A thin-layer chromatography identification method includes the following steps:

[0134] (1) Preparation of test solution: Using American ginseng reference material (D1) and ginseng reference material (D2) as test samples with definite sources, test solution was prepared according to the preparation method of test solution in Example 1 of this invention to obtain American ginseng test solution (referred to as test solution 1) and ginseng test solution (referred to as test solution 2).

[0135] (2) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0136] (3) Thin-layer chromatography analysis: Following the thin-layer chromatography method in Section 0502 of the 2020 edition of the Chinese Pharmacopoeia, 2 μL each of test solution 1 and test solution 2, and 1 μL of reference solution, were spotted onto the same high-performance silica gel G thin-layer plate to form strips with a band width of 8 mm. The developing solvent was the lower layer of the ginseng and American ginseng standard thin-layer identification section of the 2020 edition of the Chinese Pharmacopoeia, prepared by chloroform-ethyl acetate-methanol-water (15:40:22:10) at 8℃ for 12 hours. The developing distance was approximately 8 cm. After drying, the plates were sprayed with 10% sulfuric acid ethanol solution, dried again, and heated at 105℃ until the spots were clearly visible. The spots were then examined under ultraviolet light (365 nm) and sunlight. The results are as follows: Figure 14 As shown; where, Figure 14 A is the chromatogram viewed under ultraviolet light (365nm). Figure 14 B is a chromatogram examined under sunlight; Figure 14 In the diagram, 1 represents D2, 2 represents D1, and 3 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively).

[0137] The results show that the developing solvent of the present invention has a good separation effect on the mixed reference standard (e.g., Figure 1 (As shown) and the separation effect of the developing solvent for ginseng and American ginseng under the thin-layer identification section of the current edition of the Chinese Pharmacopoeia on the mixed reference standards (e.g.) Figure 14 As shown in the figure, the effects are comparable, but the developing solvent of this invention is prepared by dichloromethane, anhydrous ethanol, ethyl acetate and water, and the amount of organic reagent used is only 45 mL. It does not require the hepatotoxic reagent chloroform and the preparation time is 5 min. In contrast, the developing solvent for the thin-layer identification item of ginseng and American ginseng in the Chinese Pharmacopoeia is prepared by chloroform, ethyl acetate, methanol and water, and the amount of organic reagent used is 77 mL. The developing solvent contains the hepatotoxic reagent chloroform and the preparation time is more than 12 hours. Therefore, this invention is faster, less toxic and more environmentally friendly.

[0138] 4. To compare the effects of the developing solvent of this invention and the developing solvent under the ginseng inspection item in the Chinese Pharmacopoeia of American ginseng on the thin-layer chromatography results, the developing solvent under the ginseng inspection item in the Chinese Pharmacopoeia of American ginseng was used for thin-layer chromatography identification as Comparative Example 4. The specific thin-layer identification method of Comparative Example 4 is as follows:

[0139] A thin-layer chromatography identification method includes the following steps:

[0140] (1) Preparation of test solution: American ginseng reference material (D1) and ginseng reference material (D2) were used as test samples with definite sources. The test solution was prepared according to the preparation method of test solution in Example 1 of this invention to obtain American ginseng test solution (referred to as test solution 1) and ginseng test solution (referred to as test solution 2).

[0141] (2) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0142] (3) Thin-layer chromatography analysis: The thin-layer chromatography method was performed according to Section 0502 of the General Chapter of Part IV of the 2020 edition of the Chinese Pharmacopoeia. 2 μL each of the above-mentioned test solution 1 and test solution 2, and 1 μL of the reference solution were spotted onto the same high-performance silica gel G thin-layer plate, forming strips with a band width of 8 mm. The developing solvent was the lower layer of chloroform-methanol-water (13:7:2) that had been left at 8℃ for 12 hours, as specified in the 2020 edition of the Chinese Pharmacopoeia for American ginseng. The developing distance was approximately 8 cm. After drying, the plates were sprayed with 10% sulfuric acid ethanol solution, dried again, and heated at 105℃ until the spots were clearly visible. The spots were then examined under ultraviolet light (365 nm) and sunlight. The results are as follows: Figure 15 As shown; where, Figure 15 A is the chromatogram viewed under ultraviolet light (365nm). Figure 15 B is a chromatogram examined under sunlight; Figure 15 In the diagram, 1 represents D2, 2 represents D1, and 3 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, respectively).

[0143] The results show that the developing solvent of the present invention has a good separation effect on the mixed reference standard (e.g., Figure 1 (As shown) and the separation effect of the developing solvent on the mixed reference standard under the ginseng test item in the current edition of the Chinese Pharmacopoeia (e.g., Figure 15 Compared to the previous method, the developing solvent of this invention can clearly separate the five ginsenoside components in the mixed reference solution, resulting in better performance. Furthermore, the developing solvent of this invention is prepared from dichloromethane, anhydrous ethanol, ethyl acetate, and water, requiring only 45 mL of organic reagent. It eliminates the need for the hepatotoxic reagent chloroform and takes only 5 minutes to prepare. In contrast, the developing solvent for ginseng testing in the Chinese Pharmacopoeia is prepared from chloroform, methanol, and water, requiring 20 mL of organic reagent and containing the hepatotoxic reagent chloroform, taking over 12 hours to prepare. Therefore, this invention is faster, less toxic, and more environmentally friendly.

[0144] This invention also compares the effect of the chromogenic reagent used in this invention with other commonly used glycoside chromogenic reagents on the thin-layer chromatography results. A thin-layer identification method using 5% vanillin-sulfuric acid solution as the chromogenic reagent is presented as Comparative Example 5. The specific thin-layer identification method of Comparative Example 5 is as follows:

[0145] A thin-layer chromatography identification method includes the following steps:

[0146] (1) Preparation of test solution: American ginseng reference material (D1) and ginseng reference material (D2) were used as test samples with definite sources. The test solution was prepared according to the preparation method of test solution in Example 1 of this invention to obtain American ginseng test solution (referred to as test solution 1) and ginseng test solution (referred to as test solution 2).

[0147] (2) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference, and pseudoginsenoside F11 reference, add methanol to prepare a mixed solution containing 1 mg of each of ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd, and 0.5 mg of pseudoginsenoside F11 per 1 mL, as the reference solution (D3);

[0148] (3) Thin-layer chromatography analysis: The thin-layer chromatography method was performed according to Section 0502 of the General Chapter of Part IV of the 2020 edition of the Chinese Pharmacopoeia. 2 μL each of test solution 1 and test solution 2, and 1 μL of the reference solution were spotted onto the same high-performance silica gel G thin-layer plate, forming strips with a band width of 8 mm. A dichloromethane-anhydrous ethanol-ethyl acetate-water (23:20:2:2.6) solution was used as the developing solvent, with a development distance of approximately 8 cm. After drying, the plates were sprayed with 5% vanillin-sulfuric acid solution, dried again, and heated at 105℃ until the spots were clearly visible. The results were examined under sunlight and ultraviolet light (365 nm). The results are as follows: Figure 16 As shown; where, Figure 16 A is a chromatogram examined under sunlight; Figure 16 B is the chromatogram viewed under ultraviolet light (365nm); Figure 16 In the diagram, 1 represents D2, 2 represents D1, and 3 represents D3 (the spots corresponding to the Rf values ​​from largest to smallest are ginsenoside Rg1, pseudoginsenoside F11, ginsenoside Rf, ginsenoside Re, and ginsenoside Rd).

[0149] The results show that the color development effect of the color developer according to the present invention (e.g.) Figure 1 The color development effect of (as shown) and 5% vanillin sulfuric acid color developer (as shown) Figure 16Compared to the method shown, the colorimetric reagent of the present invention can display the spot color under sunlight and the fluorescence characteristics under ultraviolet light (365nm) of each component in the test sample and mixed reference solution, providing rich thin-layer information. In contrast, the 5% vanillin sulfuric acid solution colorimetric reagent cannot provide the fluorescence characteristics of each spot under ultraviolet light, and the spot color under sunlight is not as rich as that of the colorimetric reagent used in the present invention. Furthermore, the 5% vanillin sulfuric acid solution is prepared using sulfuric acid as a solvent, and the risk level during preparation and use is higher than that of the colorimetric reagent used in the method of the present invention. Therefore, the present invention is safer and has a better colorimetric effect.

[0150] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A thin-layer chromatography identification method, characterized in that, Includes the following steps: (1) Preparation of test solution: Take the test sample, add solvent, sonicate, take the supernatant to obtain the test solution; (2) Preparation of reference medicinal material solution: American ginseng and ginseng reference medicinal materials were taken, solvents were added respectively, ultrasonic treatment was performed, and the supernatant was taken to obtain American ginseng reference medicinal material solution and ginseng reference medicinal material solution; (3) Preparation of reference solution: Take ginsenoside Rg1 reference, ginsenoside Rf reference, ginsenoside Re reference, ginsenoside Rd reference and pseudoginsenoside F11 reference, add solvent, mix, and obtain reference solution; (4) Thin-layer chromatography analysis: Take the test solution, the American ginseng reference material solution, the ginseng reference material solution and the reference solution, spot them on the same silica gel G thin-layer plate, add the developing solvent and color developing agent, heat and examine; (5) Result determination: Compare the thin-layer chromatography results to determine whether the test sample is American ginseng or ginseng; In steps (1), (2), and (3), the solvent includes at least one of methanol and ethanol. In step (4), the developing agent is a mixture of dichloromethane, anhydrous ethanol, ethyl acetate, and water; the volume ratio of dichloromethane, anhydrous ethanol, ethyl acetate, and water is 23:20:2:2.

6. In step (4), the colorimetric agent comprises a 5-10% by mass sulfuric acid ethanol solution.

2. The thin-layer chromatography identification method according to claim 1, characterized in that, In steps (1) and (2), the ultrasonic treatment time is 15-16.5 min.

3. The thin-layer chromatography identification method according to claim 1, characterized in that, In step (4), the silicone G thin film is a high-efficiency silicone G thin film.

4. The thin-layer chromatography identification method according to claim 1, characterized in that, In step (4), the developing agent is added and the development is carried out under certain temperature and humidity conditions; the development temperature is 5-30℃ and the development humidity is 30-90%.

5. The thin-layer chromatography identification method according to claim 1, characterized in that, In step (4), the heating temperature is 95-115℃; the heating time is 50-70s.

6. The thin-layer chromatography identification method according to claim 1, characterized in that, In step (4), the inspection is performed using ultraviolet lamps and fluorescent lamps, respectively.

7. The application of the thin-layer chromatography identification method according to any one of claims 1-6 in the field of identification of American ginseng or ginseng.

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