Digital thin-layer chromatography detection and analysis model, method for establishing same, and application thereof

Abstract

The present invention relates to a digital thin-layer chromatography detection and analysis model, a method for establishing the same, and an application thereof, belonging to the technical field of traditional Chinese medicine quality control. The digital thin-layer chromatography detection and analysis model is established based on the relative migration distance value (RPf) theory, and can be used for digital thin-layer chromatography analysis without physical reference. Under different experimental conditions and for different experimental varieties, the relative standard deviation of the RPf value is the smallest, showing better performance than RRf, and demonstrating the application advantages of the characteristic spot migration distance method in the digital standard of traditional Chinese medicine thin-layer chromatography.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine quality control, and particularly to a digital thin-layer chromatography detection and analysis model, a method for establishing the same, and an application thereof. Background Art

[0002] Thin-layer chromatography technology began in the 1950s and was rapidly popularized and applied in the 1980s. Thin-layer chromatography is an important method for evaluating the authenticity of traditional Chinese medicine decoction pieces and the authenticity of the raw materials used in proprietary Chinese medicines. It has the advantages of simple operation, economy, rapidity, and wide application, and plays an irreplaceable role in the method system of traditional Chinese medicine quality standards.

[0003] However, in the past 40 years, the theoretical innovation research of thin-layer chromatography technology has basically stagnated and has not been able to achieve further breakthroughs. At present, the standard for evaluating thin-layer chromatography results is stipulated as follows: in the test sample chromatogram, spots of the same color appear at the corresponding positions of the thin-layer chromatogram of the control medicinal material or reference substance. However, in actual applications, due to the relatively vague description of the standard regulations and the lack of objective and quantifiable specified values, inspectors need to rely on visual observation to sensorially evaluate key information such as the number, position, color, and size of the characteristic spots of thin-layer chromatography. Since different inspectors have different understandings and evaluation scales of the standard, it is easy to cause differences in inspection conclusions. On the other hand, the reference substances or control medicinal materials required for thin-layer chromatography also greatly increase the inspection cost. At the same time, a large amount of medicinal materials and organic solvents are consumed in the preparation process of the reference substances, resulting in the waste of traditional Chinese medicine resources and the destruction of the ecological environment. Summary of the Invention

[0004] Based on this, in view of the above problems, it is necessary to provide a digital thin-layer chromatography detection method without physical reference, which can establish a theoretical model for digital characterization of traditional Chinese medicine thin-layer chromatography by formulating objective and quantifiable indicators, so as to eliminate the result deviation of manual subjective evaluation, reduce the detection cost, and protect traditional Chinese medicine resources.

[0005] On the one hand, the present invention provides a method for establishing a digital thin-layer chromatography detection and analysis model, including the following steps:

[0006] Establishment of a standard atlas library: Collect thin-layer chromatograms of qualified products of the traditional Chinese medicine varieties to be tested. The thin-layer chromatograms of the qualified products include thin-layer chromatograms under different experimental conditions, and a standard atlas library is obtained. The different experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of traditional Chinese medicine varieties;

[0007] Establishment of the analysis model: Select at least two representative spots from the above thin-layer chromatograms as analysis spots. Add or not add the origin to form characteristic spots. Group the characteristic spots to obtain a characteristic spot group with three characteristic spots in a group. Use one of the characteristic spots in each characteristic spot group as a positioning spot, calculate the distances between the other two characteristic spots and this positioning spot, calculate the ratio of the minimum distance to the maximum distance, denoted as the spacing ratio RPf. Use this spacing ratio RPf as an evaluation index, and based on the spacing ratios RPf of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library, establish the evaluation standard range of the spacing ratio RPf, and that's it.

[0008] In thin-layer chromatography, the retardation factor (Rf) is usually used to represent the relative distance of substance movement for compound identification. However, experimental temperature and humidity, the specifications and batches of silica gel G plates and polyamide films, etc. may all affect the Rf value, resulting in inaccurate comparison of the Rf values of the same component between different laboratories. The relative retardation factor (RRf) eliminates systematic errors to a certain extent and shows better repeatability and reliability than the Rf value. However, both the Rf and RRf values use the origin as the positioning point, and the results are calculated by measuring the distance between the thin-layer spots and the origin. Since the origin does not change its position with the developing agent or environmental differences during the development of thin-layer chromatography, the reproducibility of the Rf value and the relative Rf value is poor.

[0009] The inventor considered that although the thin-layer chromatography behavior is affected by various factors, such as the thickness and density of the thin-layer plate, the accuracy of the developing agent preparation, the experimental environmental temperature and humidity, the sufficiency of the spraying of the color-developing agent, the heating color-developing time, etc., which make the Rf values of the same component prone to large differences. However, since the components detected and developed in the same thin-layer system have a certain similarity, when affected by environmental factors, the degree of influence should be balanced.

[0010] Under the guidance of this concept, the inventor proposed the above digital thin-layer chromatography detection and analysis model, collected thin-layer chromatograms under different conditions, and based on his own long-term practical experience, proposed that the analysis spots can be grouped, and according to the characteristics reflected by each group of characteristic spots, for the first time proposed the theory of relative distance ratio, and calculated the spacing ratio RPf based on this relative distance ratio to reduce the influence of different environmental conditions on thin-layer chromatography, so as to achieve digital thin-layer chromatography detection without reference substances or reference medicinal materials.

[0011] In some solutions, in the step of establishing the analysis model, the judgment standard for adding or not adding the origin to form characteristic spots for grouping is:

[0012] In the first step of judgment, when the shape of the origin is irregular or the boundary is unclear, the origin is not added to form a characteristic spot. When the shape of the origin spot is regular and the boundary is clear, the second step of judgment is entered;

[0013] In the second step of judgment, when the number of the analysis spots is 2, the origin is added as a characteristic spot for grouping;

[0014] When the number of the analysis spots ≥ 3, along the solvent development direction, the origin and the first three spots of the analysis spots are sequentially numbered as A, B, C, and D from the origin to the solvent front direction. The distances L-OB and L-CA between OB and CA are calculated respectively. When L-OB ≥ L-CA, the origin is not added as a characteristic spot for grouping; when L-OB ≥ L-CA, the origin is added as a characteristic spot for grouping.

[0015] In some solutions, the analysis spots meet the following requirements:

[0016] (1) The analysis spots exist in both the sample chromatogram of this traditional Chinese medicine variety and the chromatogram of the control medicinal material or reference substance, and the positions correspond;

[0017] (2) The analysis spots have regular shapes, clear boundaries and exist stably;

[0018] (3) The Rf value of the analysis spots is 0.05 - 0.8, preferably 0.1 - 0.8, more preferably 0.2 - 0.8;

[0019] (4) The analysis spots are not interfered by the negative and have specificity.

[0020] It can be understood that the above "position corresponding" means that the Rf values of the spots in the sample chromatogram and the chromatogram of the control medicinal material or reference substance are similar, and those skilled in the art can determine that they are spots of the same component.

[0021] In some solutions, the positioning spots are determined by the following method: within each group of characteristic spots, three characteristic spots are respectively used as candidate positioning spots, the distances between the other two characteristic spots and the positioning spots are measured, the ratio of the minimum distance to the maximum distance is calculated, the relative standard deviation of each ratio is calculated, and the characteristic spot with the smallest relative standard deviation value is selected as the positioning spot. That is, different spots are taken turns as candidate positioning spots, and their standard deviations are investigated through experimental tests. The spots with small standard deviations and high stability are used as positioning spots, which can improve the accuracy and stability of this analysis model.

[0022] In some solutions, in the step of establishing the analysis model, grouping is carried out according to the following rules:

[0023] (1) Number the characteristic spots along the solvent development direction, and divide the first three spots and the last three spots into the first group and the last group respectively;

[0024] (2) There are common characteristic spots between each group and the adjacent group;

[0025] (3) Except for the first group or the last group, the characteristic spots in the remaining groups are grouped according to the principle of proximity;

[0026] (4) The grouping meets the requirements of the above items (1)-(3), and the total number of groups is the smallest.

[0027] The general idea of the above rules is to group the head and tail spots first, and then combine to obtain the middle groups. And there should be a connection between each group to reflect the overall effect. Therefore, each group should have common characteristic spots with the adjacent group. However, 1 or 2 characteristic spots can be selected as the common spots, which can be adjusted according to the number and distance of the spots to meet the requirements of items (3) and (4).

[0028] In some solutions, number the characteristic spots along the solvent development direction and group them according to the following rules:

[0029] When the total number of analyzed spots is 2, add the origin or the spotting point on the thin layer plate as a characteristic spot to form a group;

[0030] When the total number of characteristic spots is 3, form a group with these 3 characteristic spots;

[0031] When the total number of characteristic spots is 4, divide the characteristic spots numbered 1, 2, and 3 into the first group, and divide the characteristic spots numbered 2, 3, and 4 into the last group;

[0032] When the total number of characteristic spots is 5, divide the characteristic spots numbered 1, 2, and 3 into the first group, and divide the characteristic spots numbered 3, 4, and 5 into the last group;

[0033] When the total number of characteristic spots is 6, divide the characteristic spots numbered 1, 2, and 3 into the first group, and divide the characteristic spots numbered 4, 5, and 6 into the last group. Calculate the distance between the characteristic spots numbered 2 and 4 and the distance between the characteristic spots numbered 3 and 5, which are D-24 and D-35 respectively. When D-24 > D-35, use the characteristic spots numbered 3, 4, and 5 as the middle group. When D-24 < D-35, use the characteristic spots numbered 2, 3, and 4 as the middle group. When D-24 = D-35, optionally use the characteristic spots numbered 3, 4, and 5 as the middle group or the characteristic spots numbered 2, 3, and 4 as the middle group;

[0034] When the total number of characteristic spots is 7, divide the characteristic spots numbered 1, 2, and 3 into the first group, divide the characteristic spots numbered 5, 6, and 7 into the last group, and divide the characteristic spots numbered 3, 4, and 5 into the middle group;

[0035] When the total number of characteristic spots is 8, the characteristic spots numbered 1, 2, and 3 are divided into the first group, the characteristic spots numbered 6, 7, and 8 are divided into the last group, the characteristic spots numbered 3, 4, and 5 are divided into the first intermediate group, and the characteristic spots numbered 4, 5, and 6 are divided into the second intermediate group.

[0036] In some solutions, the above establishment method meets at least one of the following conditions:

[0037] (1) The traditional Chinese medicine varieties include traditional Chinese medicine decoction pieces and / or traditional Chinese medicine preparations;

[0038] (2) The differential experimental conditions also include at least one of the following conditions: thin-layer plates of at least two specifications, at least two experimental dates, at least two developing times, and at least two operators.

[0039] It can be understood that the more differential experimental conditions are selected, the more comprehensive the standard chromatograms are collected, and the more comprehensive the test samples can be analyzed and judged by the established model. However, considering different humidity, temperature, and annotated samples, the factors that have a greater impact on thin-layer chromatography have been included, and the basic judgment requirements can be met.

[0040] On the other hand, the present invention also provides a digital thin-layer chromatography analysis model, which is established by using the above method for establishing a digital thin-layer chromatography detection and analysis model.

[0041] It can be understood that the above digital thin-layer chromatography analysis model can be pre-stored chromatograms and spacing ratio RPf data, and can be judged by directly comparing the RPf data values during use. It can also be pre-programmed and encapsulated as a software program. After scanning the thin-layer chromatography map of the traditional Chinese medicine variety to be tested, spot recognition is performed using image recognition technology, and automatic judgment and result output are carried out. Its image recognition and data comparison methods can be designed according to conventional conditions.

[0042] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method. Take the thin-layer chromatography map of the traditional Chinese medicine variety to be tested, calculate the corresponding spacing ratio RPf according to the above method for establishing a digital thin-layer chromatography detection and analysis model, and compare it with the evaluation standard range to obtain the digital thin-layer chromatography detection result. For example, when the spacing ratio RPf of the traditional Chinese medicine variety to be tested falls within the evaluation standard range, it can be determined as a qualified product; on the contrary, when it exceeds the evaluation standard range, it can be determined as an unqualified product.

[0043] On the other hand, the present invention also provides a cinnamon digital thin-layer chromatography analysis model, which is established by the following method:

[0044] Thin layer chromatography detection: Take cinnamon samples, extract with ethanol as the extraction solvent to obtain the test solution. Spot the test solution on a silica gel G thin layer plate, develop with a petroleum ether - ethyl acetate mixture at 60 - 90 °C with a volume ratio of 17∶2 - 4, spray with a dinitrophenylhydrazine ethanol test solution, and take a photo at 254 ± 5 nm to obtain the thin layer chromatogram;

[0045] Standard chromatogram library establishment: Collect the thin layer chromatograms of qualified cinnamon products, where the thin layer chromatograms of the qualified products include those under different experimental conditions, to obtain a standard chromatogram library. The different experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of cinnamon samples;

[0046] Analysis model establishment: Along the solvent development direction, number the origin and each spot in sequence from the origin to the solvent front. Use the 1st, 2nd, 3rd, and 4th spots as analysis spots. Divide the 1st, 2nd, and 3rd spots into the first group, and divide the 2nd, 3rd, and 4th spots into the last group. In the first group, use the 3rd spot as the positioning spot, and in the last group, use the 2nd spot as the positioning spot. In the first group, calculate the distances L - 31 and L - 32 between the 3rd spot and the 1st spot and the 2nd spot respectively, calculate the ratio of the minimum distance L - 32 to the maximum distance L - 31, denoted as the spacing ratio RPf1. In the last group, calculate the distances L - 23 and L - 24 between the 2nd spot and the 3rd spot and the 4th spot respectively, calculate the ratio of the minimum distance L - 23 to the maximum distance L - 24, denoted as the spacing ratio RPf2. Use these spacing ratios RPf1 and RPf2 as evaluation indicators, and based on the spacing ratios RPf1 and RPf2 of each characteristic spot group in different thin layer chromatograms in the standard chromatogram library, establish the evaluation standard range of the spacing ratios RPf1 and RPf2.

[0047] In some schemes of the above thin layer chromatography detection step where the sample is cinnamon, the test solution is extracted by the following method: Take cinnamon powder, add ethanol in an amount of 20 ± 5 mL of solvent per gram of medicinal material, soak for 20 ± 10 min, filter, and the filtrate is the test solution.

[0048] In some schemes of the above thin layer chromatography detection step where the sample is cinnamon, the developing agent is a petroleum ether - ethyl acetate mixture at 60 - 90 °C with a ratio of 17∶3.

[0049] In some schemes of the above thin layer chromatography detection step where the sample is cinnamon, the humidity conditions include a relative humidity of 32 ± 5%, 59 ± 5%, and 88 ± 5%.

[0050] In some schemes of the above thin layer chromatography detection step where the sample is cinnamon, the temperature conditions include 24 ± 5 °C and 50 ± 5 °C.

[0051] In some of the above-described TLC detection procedures for cinnamon samples, the differential experimental conditions also include at least two different specifications of TLC plates.

[0052] In some of the above-described TLC detection procedures for cinnamon samples, the TLC plates include silica gel G TLC plates produced by Merck KGaA and Qingdao Ocean Chemical Co., Ltd.

[0053] In some of the above-described steps for establishing an analysis model for cinnamon samples, the Rf value of the second spot is 0.11 ± 0.01, the Rf value of the third spot is 0.27 ± 0.03, and the Rf value of the fourth spot is 0.47 ± 0.05.

[0054] On the other hand, the present invention also provides a digital TLC detection and analysis method for cinnamon, comprising the following steps: taking a TLC chromatogram of a cinnamon sample to be tested obtained by the same method as in the above-described TLC detection step, and calculating the corresponding spacing ratios RPf1 and RPf2 according to the method in the above-described analysis model establishment step, comparing with the evaluation standard range, and obtaining the digital TLC detection result of cinnamon.

[0055] In some of the above-described digital TLC detection and analysis methods for cinnamon, when the spacing ratio RPf1 is 0.54 - 0.71 and RPf2 is 0.51 - 0.59, it is determined that the cinnamon sample is qualified.

[0056] On the other hand, the present invention also provides a digital TLC analysis model for Shuxiong Tablets. Taking Chuanxiong as the index medicinal material, it is established by the following method:

[0057] TLC detection: Taking a Shuxiong Tablets sample, extracting with ether as the extraction solvent to obtain a test solution, spotting the test solution on a silica gel G TLC plate, developing with a petroleum ether - chloroform mixture at a volume ratio of 1:7 - 11 and a temperature of 30 - 60°C, and photographing at 365 ± 5 nm to obtain a TLC chromatogram;

[0058] Establishment of a standard atlas library: Collecting the TLC chromatograms of qualified Shuxiong Tablets, where the TLC chromatograms of the qualified products include TLC chromatograms under differential experimental conditions, to obtain a standard atlas library. The differential experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of Shuxiong Tablets samples;

[0059] Analysis model establishment: Along the solvent development direction, number the origin and each spot in sequence from the origin towards the solvent front. Take the 4th, 5th, and 6th spots as the analysis spots, group these three analysis spots as characteristic spot groups, take the 4th spot as the positioning spot, calculate the distances L-45 and L-46 between the 4th spot and the 5th spot and the 6th spot respectively, calculate the ratio of the minimum distance L-45 to the maximum distance L-46, denoted as the spacing ratio RPf3. Using this spacing ratio RPf3 as the evaluation index, based on the spacing ratios RPf3 of each characteristic spot group in different thin-layer chromatograms in the standard atlas library, establish the evaluation standard range of the spacing ratio RPf2.

[0060] In some of the above-mentioned thin-layer chromatography detection steps for Shuxiong Tablets (with Chuanxiong as the index medicinal material), the test solution is extracted by the following method: Take Shuxiong Tablets, remove the coating, grind them finely, add ether in an amount of 20 ± 5 mL of solvent for every 20 Shuxiong Tablets, heat under reflux for 60 ± 30 min, filter, evaporate the filtrate to dryness, and dissolve the residue in 2 ± 0.5 mL of ether to obtain the test solution.

[0061] In some of the above-mentioned thin-layer chromatography detection steps for Shuxiong Tablets (with Chuanxiong as the index medicinal material), the developing agent is petroleum ether - chloroform at 30 - 60 °C in a ratio of 1:9.

[0062] In some of the above-mentioned thin-layer chromatography detection steps for Shuxiong Tablets (with Chuanxiong as the index medicinal material), the humidity conditions include a relative humidity of 32 ± 5%, 56 ± 5%, and 88 ± 5%.

[0063] In some of the above-mentioned thin-layer chromatography detection steps for Shuxiong Tablets (with Chuanxiong as the index medicinal material), the temperature conditions include 5 ± 2 °C and 25 ± 5 °C.

[0064] In some of the above-mentioned thin-layer chromatography detection steps for Shuxiong Tablets (with Chuanxiong as the index medicinal material), the differential experimental conditions also include at least two different specifications of thin-layer plates.

[0065] In some of the above-mentioned thin-layer chromatography detection steps for Shuxiong Tablets (with Chuanxiong as the index medicinal material), the thin-layer plates include silica gel G thin-layer plates produced by Merck KGaA and Qingdao Ocean Chemical Co., Ltd.

[0066] In some of the above-mentioned analysis model establishment steps for Shuxiong Tablets (with Chuanxiong as the index medicinal material), the Rf value of the 4th spot is 0.39 ± 0.04, the Rf value of the 5th spot is 0.44 ± 0.04, and the Rf value of the 6th spot is 0.67 ± 0.06.

[0067] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method for Shuxin tablets, taking Chuanxiong in them as the index medicinal material, which comprises the following steps: taking the thin-layer chromatogram of the Shuxin tablet sample to be detected obtained by the same method as in the above-mentioned thin-layer chromatography detection step, calculating the corresponding spacing ratio RPf3 according to the method in the step of establishing the analysis model, comparing it with the evaluation standard range, and obtaining the digital thin-layer chromatography detection result of the Shuxin tablets.

[0068] In some embodiments of the step of establishing the analysis model (taking Chuanxiong as the index medicinal material) for the above sample being Shuxin tablets, when the spacing ratio RPf3 is 0.77 - 0.88, it is determined that the Shuxin tablet sample is qualified.

[0069] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method for Rhodiola rosea, which comprises the following steps:

[0070] Thin-layer chromatography detection: taking a Rhodiola rosea sample, extracting it with methanol as the extraction solvent to obtain a test solution, spotting the test solution on a silica gel G thin-layer plate, developing it with the lower layer solution of chloroform - methanol - acetone - water with a volume ratio of 5 - 7:2 - 4:0.5 - 1.5:1 as the developing agent, fumigating it in iodine vapor, and taking a photo to obtain a thin-layer chromatogram;

[0071] Establishment of a standard atlas library: collecting the thin-layer chromatograms of qualified Rhodiola rosea, wherein the thin-layer chromatograms of the qualified products include the thin-layer chromatograms under different experimental conditions, obtaining a standard atlas library, and the different experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of Rhodiola rosea samples;

[0072] Establishment of an analysis model: along the solvent development direction, numbering the origin and each spot in turn from the origin to the solvent front direction, taking the 3rd, 4th, 5th, and 6th spots as the analysis spots, dividing the 3rd, 4th, and 5th spots into the first group, dividing the 4th, 5th, and 6th spots into the last group, taking the 5th spot as the positioning spot in the first group, taking the 4th spot as the positioning spot in the last group, in the first group, calculating the distances L-35 and L-45 between the 5th spot and the 3rd spot and the 4th spot respectively, calculating the ratio between the minimum distance and the maximum distance, denoted as the spacing ratio RPf4, in the last group, calculating the distances L-45 and L-46 between the 4th spot and the 5th spot and the 6th spot respectively, calculating the ratio between the minimum distance L-45 and the maximum distance L-46, denoted as the spacing ratio RPf5, taking the spacing ratios RPf4 and RPf5 as evaluation indexes, and establishing an evaluation standard range of characteristic ratios according to the spacing ratios RPf4 and RPf5 of each characteristic spot group in different thin-layer chromatograms in the standard atlas library.

[0073] In some of the above-described schemes for the thin-layer chromatography detection step of Rhodiola rosea, the test solution is extracted by the following method: Take about 0.5 ± 0.2 g of Rhodiola rosea powder, accurately weigh it, place it in a stoppered bottle, accurately add 10 ± 3 ml of methanol, tightly stopper it, weigh it, ultrasonically treat it for 30 ± 10 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the continuous filtrate. The filtrate is the test solution.

[0074] In some of the above-described schemes for the thin-layer chromatography detection step of Rhodiola rosea, the developing agent is the lower-layer solution of chloroform - methanol - acetone - water with a volume ratio of 6:3:1:1.

[0075] In some of the above-described schemes for the thin-layer chromatography detection step of Rhodiola rosea, the humidity conditions include a relative humidity of 32 ± 5%, 63 ± 5%, and 88 ± 5%.

[0076] In some of the above-described schemes for the thin-layer chromatography detection step of Rhodiola rosea, the temperature conditions include 4 ± 2°C and 21.9 ± 5°C.

[0077] In some of the above-described schemes for the thin-layer chromatography detection step of Rhodiola rosea, the differential experimental conditions further include at least two different specifications of thin-layer plates.

[0078] In some of the above-described schemes for the thin-layer chromatography detection step of Rhodiola rosea, the thin-layer plates include silica gel G thin-layer plates produced by Merck KGaA and Qingdao Ocean Chemical Co., Ltd.

[0079] In some of the above-described schemes for the analysis model establishment step of Rhodiola rosea, the Rf value of the 3rd spot is 0.32 ± 0.03, the Rf value of the 4th spot is 0.45 ± 0.04, the Rf value of the 5th spot is 0.63 ± 0.06, and the Rf value of the 6th spot is 0.77 ± 0.08.

[0080] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method for Rhodiola rosea, including the following steps: Take the thin-layer chromatogram of the Rhodiola rosea sample to be tested obtained by the same method as in the above-described thin-layer chromatography detection step, calculate the corresponding spacing ratios RPf4 and RPf5 according to the method in the analysis model establishment step, and compare them with the evaluation standard range to obtain the digital thin-layer chromatography detection result of Rhodiola rosea.

[0081] In some of the above-described schemes for the digital thin-layer chromatography detection and analysis method of Rhodiola rosea, when the spacing ratio RPf4 is 0.54 - 0.69 and RPf5 is 0.55 - 0.85, it is determined that the Rhodiola rosea sample is qualified.

[0082] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method for cassia seeds, comprising the following steps:

[0083] Thin-layer chromatography detection: Take a cassia seed sample, impregnate it with methanol, filter to obtain the filtrate, evaporate the filtrate to dryness, dissolve the residue in water, add hydrochloric acid, heat it in a water bath, cool it, extract with ether, evaporate the ether solution to dryness, dissolve the residue in chloroform to obtain a test sample solution; Spot the test sample solution on a silica gel H thin-layer plate, use petroleum ether-acetone at 30-60°C with a volume ratio of 1.5-2.5:1 as the developing agent, develop, take out, dry in air, fumigate it in ammonia vapor, and then take a photo to obtain a thin-layer chromatogram.

[0084] Establishment of a standard spectrum library: Collect the thin-layer chromatograms of qualified cassia seeds, where the thin-layer chromatograms of the qualified products include those under different experimental conditions, to obtain a standard spectrum library. The different experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of cassia seed samples.

[0085] Establishment of an analysis model: Along the solvent development direction, number the origin and each spot in sequence from the origin to the solvent front. When the cassia seed sample is raw cassia seed, use the 1st, 3rd, and 5th spots as analysis spots, group these three analysis spots as characteristic spot groups, use the 5th spot as the positioning spot, calculate the distances L-15 and L-35 between the 5th spot and the 1st spot and the 3rd spot respectively, calculate the ratio of the minimum distance L-35 to the maximum distance L-15, denoted as the spacing ratio RPf6. Use this spacing ratio RPf6 as the evaluation index, and establish the evaluation standard range of the spacing ratio RPf6 according to the spacing ratios RPf6 of each characteristic spot group in different thin-layer chromatograms in the standard spectrum library. When the cassia seed is stir-fried cassia seed, use the 1st, 2nd, and 5th spots as analysis spots, group these three analysis spots as characteristic spot groups, use the 5th spot as the positioning spot, calculate the distances L-15 and L-25 between the 5th spot and the 1st spot and the 2nd spot respectively, calculate the ratio of the minimum distance L-25 to the maximum distance L-15, denoted as the spacing ratio RPf7. Use this spacing ratio RPf7 as the evaluation index, and establish the evaluation standard range of the spacing ratio RPf7 according to the spacing ratios RPf7 of each characteristic spot group in different thin-layer chromatograms in the standard spectrum library.

[0086] In some of the above-mentioned thin-layer chromatography detection procedures for cassia seeds as samples, the test solution is extracted by the following method: Take cassia seed samples, add 10 ± 5 ml of methanol, soak for 1 ± 0.5 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ± 5 ml of water, add 1 ± 0.2 ml of hydrochloric acid, heat in a water bath for 30 ± 10 minutes, immediately cool, extract with ether 2 - 3 times, 20 ± 10 ml each time, combine the ether solutions, evaporate to dryness, dissolve the residue in 1 ± 0.5 ml of chloroform, which is the test solution.

[0087] In some of the above-mentioned thin-layer chromatography detection procedures for cassia seeds as samples, the developing agent is petroleum ether - acetone at 30 - 60 °C with a volume ratio of 2:1.

[0088] In some of the above-mentioned thin-layer chromatography detection procedures for cassia seeds as samples, the humidity conditions include relative humidity of 32 ± 5%, 56 ± 5%, and 88 ± 5%.

[0089] In some of the above-mentioned thin-layer chromatography detection procedures for cassia seeds as samples, the temperature conditions include 5 ± 2 °C and 25 ± 5 °C.

[0090] In some of the above-mentioned thin-layer chromatography detection procedures for cassia seeds as samples, the differential experimental conditions also include at least two different specifications of thin-layer plates.

[0091] In some of the above-mentioned thin-layer chromatography detection procedures for cassia seeds as samples, the thin-layer plates include silica gel H thin-layer plates produced by Merck KGaA and Qingdao Ocean Chemical Co., Ltd.

[0092] In some of the above-mentioned procedures for establishing the analysis model of cassia seeds as samples, when the cassia seed sample is raw cassia seeds, the Rf value of the 1st spot is 0 (i.e., the origin), the Rf value of the 3rd spot is 0.63 ± 0.06, and the Rf value of the 5th spot is 0.75 ± 0.07; when the cassia seed sample is stir-fried cassia seeds, the Rf value of the 1st spot is 0 (i.e., the origin), the Rf value of the 2nd spot is 0.59 ± 0.06, and the Rf value of the 5th spot is 0.79 ± 0.08.

[0093] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method for cassia seeds, including the following steps: Take the thin-layer chromatogram of the cassia seed sample to be tested obtained by the same method in the above-mentioned thin-layer chromatography detection step, calculate the corresponding spacing ratio RPf6 or RPf7 according to the method in the above-mentioned analysis model establishment step, and compare it with the evaluation standard range to obtain the digital thin-layer chromatography detection result of cassia seeds.

[0094] In some schemes of the above-mentioned Cassia seed digital thin layer chromatography detection and analysis method, when the spacing ratio RPf6 is 0.59-0.84, or the spacing ratio RPf7 is 0.65-0.78, the raw Cassia seed or fried Cassia seed sample is judged to be qualified.

[0095] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method for Xinning Tablets, comprising the following steps: using Panax notoginseng as an index medicinal material, establishing the method by the following method:

[0096] Thin layer chromatography detection: Take a sample of Xinning tablets, grind it into powder, add ether for ultrasonic treatment, filter, take the drug residue, evaporate it to dryness, add methanol for ultrasonic treatment, filter, take the filtrate, evaporate the filtrate to dryness, add water to dissolve the obtained residue, extract it with water-saturated n-butanol, wash the n-butanol solution with ammonia test solution, take the n-butanol solution and evaporate it to dryness, add methanol to dissolve the residue as the test solution, absorb the test solution and spot it on a silica gel G thin layer plate, use the upper layer solution of n-butanol-acetic acid-water with a volume ratio of 2-5:0.5-1.5:5 as the developing agent, develop it, spray it with 10% sulfuric acid ethanol solution, heat it at 105°C until the spots are clearly colored, take a picture, and obtain a thin layer chromatogram;

[0097] Establishment of standard atlas library: collecting thin layer chromatograms of qualified Xinning tablets, wherein the thin layer chromatograms of qualified products include thin layer chromatograms under differential experimental conditions, and obtaining a standard atlas library, wherein the differential experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two Xinning tablet sample batches;

[0098] Establishment of the analysis model: Along the solvent development direction, number the origin and each spot in sequence from the origin towards the solvent front. Take the 2nd, 4th, 5th, 6th, 8th, and 9th spots as the analysis spots. Divide the 2nd, 4th, and 5th spots into the first group, divide the 6th, 8th, and 9th spots into the last group, and the 4th, 5th, and 6th spots into the middle group. In the first group, use the 2nd spot as the positioning spot. In the middle group, use the 6th spot as the positioning spot. In the last group, use the 6th spot as the positioning spot. In the first group, calculate the distances L-24 and L-25 between the 2nd spot and the 4th spot and the 5th spot respectively, and calculate the ratio of the minimum distance L-24 to the maximum distance L-25, denoted as the spacing ratio RPf8. In the middle group, calculate the distances L-46 and L-56 between the 6th spot and the 4th spot and the 5th spot respectively, and calculate the ratio of the minimum distance L-56 to the maximum distance L-46, denoted as the spacing ratio RPf10. In the last group, calculate the distances L-68 and L-69 between the 6th spot and the 8th spot and the 9th spot respectively, and calculate the ratio of the minimum distance L-68 to the maximum distance L-69, denoted as the spacing ratio RPf9. Using these spacing ratios RPf8, RPf9, and RPf10 as evaluation indicators, establish the evaluation standard range of characteristic ratios based on the spacing ratios RPf8, RPf9, and RPf10 of each characteristic spot group in different thin-layer chromatograms in the standard atlas library.

[0099] In some of the above-mentioned thin-layer chromatography detection steps (using notoginseng as the index medicinal material) for the sample of Xinning Tablets, the test solution is extracted by the following method: Take 10 Xinning Tablets, remove the coating, grind them finely, add 50 ± 10 ml of ethyl ether, ultrasonically treat for 20 ± 10 minutes, filter, evaporate the ethyl ether from the drug residue, add 50 ± 10 ml of methanol, ultrasonically treat for 30 ± 10 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 50 ± ml of water. Shake and extract with water-saturated n-butanol 2 - 3 times, each time with 30 ± 10 ml, combine the n-butanol solutions, wash with ammonia test solution 2 - 3 times, each time with 60 ± 20 ml, take the n-butanol solution, evaporate to dryness, dissolve the residue in 1 ± 0.5 ml of methanol to obtain the test solution.

[0100] In some of the above-mentioned thin-layer chromatography detection steps (using notoginseng as the index medicinal material) for the sample of Xinning Tablets, the developing agent is the upper layer solution of n-butanol - acetic acid - water with a volume ratio of 4:1:5.

[0101] In some of the above-mentioned thin-layer chromatography detection steps (using notoginseng as the index medicinal material) for the sample of Xinning Tablets, the humidity conditions include relative humidities of 32%, 65%, and 88%.

[0102] In some of the above-mentioned thin-layer chromatography detection steps (using notoginseng as the index medicinal material) for the sample of Xinning Tablets, the temperature conditions include 5.1 ± 2 °C and 17.8 ± 5 °C.

[0103] In some of the above-described TLC detection (with notoginseng as the reference medicinal material) procedures for Xinning tablets, the differential experimental conditions also include at least two different specifications of TLC plates.

[0104] In some of the above-described TLC detection (with notoginseng as the reference medicinal material) procedures for Xinning tablets, the TLC plates include silica gel G TLC plates produced by Merck KGaA and Yantai Chemical Industry Research Institute.

[0105] In some of the above-described procedures for establishing the analysis model (with notoginseng as the reference medicinal material) for Xinning tablets, the Rf value of the second spot is 0.068 ± 0.01, the Rf value of the fourth spot is 0.20 ± 0.02, the Rf value of the fifth spot is 0.22 ± 0.02, the Rf value of the sixth spot is 0.38 ± 0.04, the Rf value of the eighth spot is 0.58 ± 0.06, and the Rf value of the ninth spot is 0.68 ± 0.07.

[0106] On the other hand, the present invention also provides a digital TLC detection and analysis method for Xinning tablets, with notoginseng as the reference medicinal material, including the following steps: taking the TLC chromatogram of the Xinning tablet sample to be tested obtained by the same method as in the above-described TLC detection step, and calculating the corresponding spacing ratios RPf8, RPf9, and RPf10 according to the method in the above-described analysis model establishment step, and comparing with the evaluation standard range to obtain the digital TLC detection result of the Xinning tablet.

[0107] In some of the above-described digital TLC detection and analysis methods for Xinning tablets with notoginseng as the reference medicinal material, when the spacing ratio RPf8 is 0.81 - 0.89, RPf9 is 0.64 - 0.76, and RPf10 is 0.74 - 0.91, it is determined that the Xinning tablet sample is qualified.

[0108] On the other hand, the present invention also provides a digital TLC detection and analysis method for Xinning tablets, including the following steps: with salvia miltiorrhiza as the reference medicinal material, established by the following method:

[0109] TLC detection: Take a Xinning tablet sample, ultrasonically extract with methanol, filter, evaporate the filtrate to dryness, dissolve the residue in water, pass through D101 macroporous adsorption resin, elute with water, discard the water extract, then use 40 ± 5% ethanol, collect the eluate, evaporate to dryness, dissolve the residue in methanol to obtain the test solution; spot the test solution on a polyamide TLC plate, use acetone - acetic acid - ammonia water with a volume ratio of 10 ± 2:25 ± 5:1 as the developing agent, develop, take out, dry in air, fumigate in ammonia vapor, and examine under ultraviolet light at 365 ± 5 nm to obtain the TLC chromatogram;

[0110] Establishment of a standard chromatogram library: Collect the thin-layer chromatograms of qualified Xinning tablets. The thin-layer chromatograms of the qualified products include those under different experimental conditions, and a standard chromatogram library is obtained. The different experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of Xinning tablet samples.

[0111] Establishment of an analysis model: Along the solvent development direction, number the origin and each spot in sequence from the origin to the solvent front. Take the 4th, 5th, and 7th spots as analysis spots, group these three analysis spots as characteristic spot groups, take the 4th spot as the positioning spot, calculate the distances L-45 and L-47 between the 4th spot and the 5th spot and the 7th spot respectively, calculate the ratio of the minimum distance L-45 to the maximum distance L-47, denoted as the spacing ratio RPf11. Using this spacing ratio RPf11 as an evaluation index, based on the spacing ratios RPf11 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library, establish the evaluation standard range of the spacing ratio RPf11.

[0112] In some protocols of the above-mentioned thin-layer chromatography detection (using salvia miltiorrhiza as the reference medicinal material) step for Xinning tablets as samples, the test solution is extracted by the following method: Take 3 ± 1 tablets of Xinning tablets, grind them finely, add 20 ± 5 ml of methanol, extract ultrasonically for 20 ± 10 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ± 2 ml of water, pass through D101 macroporous adsorption resin, elute with water for 5 - 6 column volumes (preferably 5.66 column volumes), discard the water extract, then elute with 40 ± 5% ethanol for 6 - 8 column volumes (preferably 7.08 column volumes), collect the eluate, evaporate to dryness, and dissolve the residue in 1 ± 0.5 ml of methanol to obtain the test solution.

[0113] In some protocols of the above-mentioned thin-layer chromatography detection (using salvia miltiorrhiza as the reference medicinal material) step for Xinning tablets as samples, the test solution is extracted by the following method: Take 3 tablets of Xinning tablets, grind them finely, add 20 ml of methanol, extract ultrasonically for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of water, pass through D101 macroporous adsorption resin with an inner diameter of 1.5 cm and a column height of 8 cm, elute with 80 ml of water, discard the water extract, then elute with 100 ml of 40% ethanol, collect the eluate, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution.

[0114] In some protocols of the above-mentioned thin-layer chromatography detection (using salvia miltiorrhiza as the reference medicinal material) step for Xinning tablets as samples, the developing agent is acetone - acetic acid - ammonia water with a volume ratio of 10:25:1.

[0115] In some protocols of the above-mentioned thin-layer chromatography detection (using salvia miltiorrhiza as the reference medicinal material) step for Xinning tablets as samples, the humidity conditions include a relative humidity of 32 ± 5%, 65 ± 5%, and 88 ± 5%.

[0116] In some of the above-mentioned thin-layer chromatography detection (with salvia miltiorrhiza as the reference medicinal material) procedures for Xinning tablets, the temperature conditions include 5.1 ± 2 °C and 17.8 ± 5 °C.

[0117] In some of the above-mentioned thin-layer chromatography detection (with salvia miltiorrhiza as the reference medicinal material) procedures for Xinning tablets, the differential experimental conditions also include at least two different specifications of thin-layer plates.

[0118] In some of the above-mentioned thin-layer chromatography detection (with salvia miltiorrhiza as the reference medicinal material) procedures for Xinning tablets, the thin-layer plates include polyamide films from Taizhou Luqiao Sijia Biochemical Plastic Factory in Zhejiang Province and Shanghai Jinsui Biotechnology Co., Ltd.

[0119] In some of the above-mentioned analytical model establishment (with salvia miltiorrhiza as the reference medicinal material) procedures for Xinning tablets, the Rf value of the 4th spot is 0.26 ± 0.02, the Rf value of the 5th spot is 0.42 ± 0.04, and the Rf value of the 7th spot is 0.78 ± 0.08.

[0120] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method for Xinning tablets, with salvia miltiorrhiza as the reference medicinal material, including the following steps: taking the thin-layer chromatogram of the Xinning tablet sample to be tested obtained by the same method as in the above-mentioned thin-layer chromatography detection step, and calculating the corresponding spacing ratio RPf11 according to the method in the above-mentioned analytical model establishment step, and comparing it with the evaluation standard range to obtain the digital thin-layer chromatography detection result of Xinning tablets.

[0121] In some of the above-mentioned digital thin-layer chromatography detection and analysis methods for Xinning tablets with salvia miltiorrhiza as the reference medicinal material, when the spacing ratio RPf11 is 0.65 - 0.75, it is determined that the Xinning tablet sample is qualified.

[0122] On the other hand, the present invention also provides a digital thin-layer chromatography detection and analysis method for Xinning tablets, including the following steps: with sophora japonica flower as the reference medicinal material, established by the following method:

[0123] Thin-layer chromatography detection: taking the Xinning tablet sample, ultrasonically treating it with methanol, filtering, evaporating the filtrate to dryness, dissolving the residue in water, adding it to a D101 macroporous adsorption resin column, eluting with water, discarding the eluate, eluting with 40 ± 5% ethanol, collecting the eluate, evaporating it to dryness, dissolving the residue in methanol to obtain the test solution; spotting the test solution on a silica gel G plate, using ethyl acetate - glacial acetic acid - formic acid - water with a volume ratio of 10 ± 2:1 ± 0.2:1 ± 0.2:2 as the developing agent, developing, taking out, air-drying, spraying with 10% sulfuric acid ethanol solution, heating at 105 °C until the spots are clearly visible, and examining under ultraviolet light at 365 ± 5 nm to obtain the thin-layer chromatogram;

[0124] Establishment of a standard chromatogram library: Collect the thin-layer chromatograms of qualified Xinning Tablets. The thin-layer chromatograms of the qualified products include those under different experimental conditions, to obtain a standard chromatogram library. The different experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of Xinning Tablet samples.

[0125] Establishment of an analysis model: Along the direction of solvent development, number the origin and each spot in sequence from the origin to the solvent front. Use the 5th, 6th, and 7th spots as analysis spots, and group these three analysis spots as characteristic spot groups. Use the 5th spot as the positioning spot, calculate the distances L-56 and L-57 between the 5th spot and the 6th spot and the 7th spot respectively, calculate the ratio of the minimum distance L-56 to the maximum distance L-57, denoted as the spacing ratio RPf12. Using this spacing ratio RPf12 as an evaluation index, based on the spacing ratios RPf12 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library, establish the evaluation standard range of the spacing ratio RPf12.

[0126] In some schemes of the above-mentioned thin-layer chromatography detection (using sophora flower as the index medicinal material) step for Xinning Tablets as samples, the test solution is extracted by the following method: Take 3 ± 1 tablets of Xinning Tablets, grind them finely, add 20 ± 5 ml of methanol, perform ultrasonic treatment for 20 ± 10 min, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ± 2 ml of water, add it to a D101 macroporous adsorption resin column, elute with water for 4 - 5 column volumes (preferably 4.5 column volumes), discard the eluate, elute with 40 ± 5% ethanol for 5 - 6 column volumes (preferably 5.66 column volumes), collect the eluate, evaporate it to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution.

[0127] In some schemes of the above-mentioned thin-layer chromatography detection (using sophora flower as the index medicinal material) step for Xinning Tablets as samples, the test solution is extracted by the following method: Take 3 tablets of Xinning Tablets, grind them finely, add 20 ml of methanol, perform ultrasonic treatment for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of water, add it to a D101 macroporous adsorption resin column with a specification of 1.5 cm × 10 cm, elute with 80 ml of water, discard the eluate, elute with 100 ml of 40% ethanol, collect the eluate, evaporate it to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution.

[0128] In some schemes of the above-mentioned thin-layer chromatography detection (using sophora flower as the index medicinal material) step for Xinning Tablets as samples, the developing agent is ethyl acetate - glacial acetic acid - formic acid - water with a volume ratio of 10:1:1:2.

[0129] In some schemes of the above-mentioned thin-layer chromatography detection (using sophora flower as the index medicinal material) step for Xinning Tablets as samples, the humidity conditions include a relative humidity of 32 ± 5%, 56 ± 5%, and 88 ± 5%.

[0130] In some of the above-mentioned TLC detection (using sophora flower as the reference medicinal material) procedures for Xinning tablets, the temperature conditions include 5.2 ± 2 °C and 25.2 ± 5 °C.

[0131] In some of the above-mentioned TLC detection (using sophora flower as the reference medicinal material) procedures for Xinning tablets, the differential experimental conditions also include at least two different specifications of TLC plates.

[0132] In some of the above-mentioned TLC detection (using sophora flower as the reference medicinal material) procedures for Xinning tablets, the TLC plates include silica gel G TLC plates produced by Merck KGaA and Yantai Chemical Industry Research Institute.

[0133] In some of the above-mentioned procedures for establishing the analysis model (using sophora flower as the reference medicinal material) for Xinning tablets, the Rf value of the 5th spot is 0.36 ± 0.03, the Rf value of the 6th spot is 0.40 ± 0.04, and the Rf value of the 7th spot is 0.44 ± 0.04.

[0134] On the other hand, the present invention also provides a digital TLC detection and analysis method for Xinning tablets, using sophora flower as the reference medicinal material, which includes the following steps: taking the TLC chromatogram of the Xinning tablet sample to be tested obtained by the same method as in the above-mentioned TLC detection step, and calculating the corresponding spacing ratio RPf12 according to the method in the above-mentioned step for establishing the analysis model, and comparing it with the evaluation standard range to obtain the digital TLC detection result of Xinning tablets.

[0135] In some of the above-mentioned digital TLC detection and analysis methods for Xinning tablets using sophora flower as the reference medicinal material, when the spacing ratio RPf12 is 0.43 - 0.64, it is determined that the Xinning tablet sample is qualified.

[0136] On the basis of conforming to the common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0137] The reagents and raw materials used in the present invention are all commercially available.

[0138] The positive and progressive effects of the present invention are as follows:

[0139] The present invention conducts research based on the concepts of Rf and RRf, and for the first time proposes the theory of relative ratio distance value (RPf), and applies it to the digital evaluation of the relative position of TLC. A method for establishing a digital TLC detection and analysis model is obtained. The digital TLC detection and analysis model established by this method can be used in digital TLC analysis without physical reference. The relative standard deviation of the RPf value is the smallest when compared under different experimental conditions and different experimental varieties, showing better performance than RRf, and demonstrating the application advantages of the characteristic spot ratio distance method in the digital standard of traditional Chinese medicine TLC. Brief Description of the Drawings

[0140] Figure 1 It is the TLC chromatogram of cinnamon in Example 2;

[0141] Figure 2 It is the diagram for explaining the ratio value in Example 2;

[0142] Figure 3 It is the value and range of the distance ratio RPf1 between different TLC chromatograms in Example 2;

[0143] Figure 4 It is the value and range of the distance ratio RPf2 between different TLC chromatograms in Example 2;

[0144] Figure 5 It is the TLC chromatogram of Shuxiong Tablets with chuanxiong as the target medicinal material in Example 3;

[0145] Figure 6 It is the value and range of the distance ratio RPf3 between different TLC chromatograms in Example 3;

[0146] Figure 7 It is the TLC chromatogram of rhodiola in Example 4;

[0147] Figure 8 It is the value and range of the distance ratio RPf4 between different TLC chromatograms in Example 4;

[0148] Figure 9 It is the value and range of the distance ratio RPf5 between different TLC chromatograms in Example 4;

[0149] Figure 10 It is the TLC chromatogram of raw semen cassiae in Example 5;

[0150] Figure 11 It is the value and range of the distance ratio RPf6 between different TLC chromatograms in Example 5;

[0151] Figure 12 It is the TLC chromatogram of fried semen cassiae in Example 6;

[0152] Figure 13 It is the value and range of the distance ratio RPf7 between different TLC chromatograms in Example 6;

[0153] Figure 14 It is the TLC chromatogram of Xinning Tablets with notoginseng as the target medicinal material in Example 7;

[0154] Figure 15 It is the value and range of the distance ratio RPf8 between different TLC chromatograms in Example 7;

[0155] Figure 16 It is the value and range of the distance ratio RPf9 between different TLC chromatograms in Example 7;

[0156] Figure 17 The spacing ratio RPf10 values ​​and ranges in different thin layer chromatograms of Example 7;

[0157] Figure 18 This is a thin layer chromatogram of the Xinning tablets in Example 8 using Danshen as the index medicinal material;

[0158] Figure 19 The values ​​and ranges of spacing ratio RPf11 in different thin layer chromatograms in Example 8;

[0159] Figure 20 This is the thin layer chromatogram of Zhongxinning Tablets in Example 9 with Sophora japonica as the index medicinal material;

[0160] Figure 21 These are the values ​​and ranges of spacing ratio RPf12 in different thin layer chromatograms in Example 9. DETAILED DESCRIPTION

[0161] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0162] Instruments and reagents

[0163] 1.1 Main instruments

[0164] XS105DU electronic balance (Mettler-Toledo Group); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu Province); AUTOMSTIC TLC Sampler 4 automatic sample spotter (CAMAG, Switzerland); TLC Visualizer thin layer digital imaging system (CAMAG, Switzerland); Milli-QAcademic ultrapure water device (Millipore, USA).

[0165] 1.2 Main reagents and medicinal materials

[0166] Cinnamaldehyde reference substance and Chuanxiong reference medicinal material were purchased from China Food and Drug Inspection Institute; petroleum ether, ethanol, ethyl acetate, and ether were all analytical grade and purchased from Guangzhou Chemical Reagent Factory; dinitrophenylhydrazine was purchased from Beijing Bailingwei Technology Co., Ltd.; Milli-Q water was homemade in the laboratory.

[0167] The cinnamon medicine on the market was identified by the chief pharmacist of the China Food and Drug Inspection Institute as the dried bark of the Lauraceae plant Cinnamomum cassia Presl. The medicine is stored in the Chinese medicine sample room of Shenzhen Institute of Drug Inspection.

[0168] Example 1

[0169] A digital thin-layer chromatography detection and analysis method.

[0170] I. Establishment of a standard spectrum library

[0171] Collect the thin-layer chromatograms of qualified products of traditional Chinese medicine varieties to be tested. The thin-layer chromatograms of the qualified products include the thin-layer chromatograms under different experimental conditions, and a standard spectrum library is obtained. The different experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of traditional Chinese medicine varieties.

[0172] The above-mentioned thin-layer chromatograms obtained from the detection of traditional Chinese medicine varieties are carried out according to the conventional detection conditions of this variety. For example, the thin-layer chromatography detection conditions recorded in the Chinese Pharmacopoeia for this variety can be referred to, or any other detection conditions that can be used to evaluate this traditional Chinese medicine variety.

[0173] It can be understood that the above-mentioned standard spectrum library needs to fully collect the thin-layer chromatograms of qualified products under different experimental conditions. The more chromatograms of different categories and conditions are included, the more comprehensive the obtained standard spectrum library is, and the higher the accuracy of the subsequent established analysis model is. Considering that there are various possibilities for unqualified products to be unqualified, it is difficult to collect them all by enumeration. Therefore, this case formulates standards based on a large number of qualified products, which not only avoids the influence of unqualified products on the overall performance, but also has high practicality and high application value.

[0174] The above-mentioned qualified products refer to traditional Chinese medicines or Chinese patent medicines that meet the relevant standards of the Chinese Pharmacopoeia. If there are traditional Chinese medicines or Chinese patent medicines that do not meet the relevant standards of the Chinese Pharmacopoeia, they are unqualified products.

[0175] The above different conditions include different humidity, temperature, batches, thin-layer plate specifications, experimental dates, developing time, operators, etc. It can be understood that factors that may affect the Rf value in actual work can be added as different conditions.

[0176] For the selection of traditional Chinese medicine varieties, it can be selected according to the actual object to be analyzed. For example, objects that need to be detected by thin-layer chromatography analysis such as traditional Chinese medicine decoction pieces and preparations can all be used.

[0177] II. Establishment of an analysis model

[0178] 1. Select analysis spots

[0179] Select at least two representative spots from the above-mentioned thin-layer chromatograms as analysis spots, and these analysis spots meet the following requirements:

[0180] (1) The analysis spots exist in both the sample chromatogram and the chromatogram of the control medicinal material or reference substance of this traditional Chinese medicine variety, and the positions correspond.

[0181] It is understandable that although reference substances or reference crude drugs are not necessarily used during the establishment of the standard chromatogram library and subsequent analysis and evaluation, considering the spots of indicative reference substances or reference crude drugs during the establishment of the standard chromatogram library and selecting characteristic spots based on this can better reflect the quality of the traditional Chinese medicine varieties being analyzed and detected.

[0182] (2) Analyze that the spot shape is regular, the boundary is clear and it exists stably.

[0183] This standard is consistent with the requirements for inspecting spots in this field, that is, it can improve the stability of the method.

[0184] (3) Analyze that the Rf value of the spot is 0.05 - 0.8, preferably 0.1 - 0.8, more preferably 0.2 - 0.8.

[0185] By limiting the Rf value of the analyzed spot to 0.05 - 0.8, preferably 0.1 - 0.8, more preferably 0.2 - 0.8, the influence of different conditions on the Rf value can be further reduced, and the stability of this solution can be improved.

[0186] (4) Analyze that the spot is not interfered by the negative and has specificity.

[0187] 2. Consider whether to add the origin

[0188] The judgment criteria for whether to add the origin to form characteristic spots for grouping are as follows:

[0189] The first step of judgment: When the spot at the origin is not clear and difficult to identify, the origin is not added to form characteristic spots. When the spot at the origin is clear and recognizable, the second step of judgment is entered;

[0190] The second step of judgment: When the number of analyzed spots is 2, the origin is added as a characteristic spot for grouping.

[0191] When the number of analyzed spots ≥ 3, along the solvent development direction, the origin and the first three spots of the analyzed spots are sequentially numbered A, B, C, D from the origin to the solvent front direction. The distances L - ac and L - bd between AC and BD. When L - ac ≥ L - bd, the origin is not added as a characteristic spot for grouping; when L - ac < L - bd, the origin is added as a characteristic spot for grouping.

[0192] 3. Grouping

[0193] Group the characteristic spots to obtain a characteristic spot group with three characteristic spots in a group. The grouping rules are as follows:

[0194] (1) Number the characteristic spots along the solvent development direction, and divide the first three spots with the earliest numbers and the last three spots with the latest numbers into the first group and the last group respectively;

[0195] (2) Each group has common characteristic spots with adjacent groups;

[0196] (3) Except for the first group or the last group, the characteristic spots in the remaining groups are grouped according to the principle of proximity;

[0197] (4) The grouping meets the requirements of the above items (1)-(3), and the total number of groups is the smallest.

[0198] The following lists the grouping methods when the number of characteristic spots is different.

[0199] Number the characteristic spots along the solvent development direction and group them according to the following rules:

[0200] When the total number of analyzed spots is 2, less than three spots, the origin or the spotting point on the thin layer plate must be added as a characteristic spot (if the origin is difficult to identify, after artificial identification through the spotting position, add the spotting point on the thin layer plate), and form a group.

[0201] When the total number of characteristic spots is 3, form a group with these 3 characteristic spots.

[0202] When the total number of characteristic spots is 4, divide the characteristic spots numbered 1, 2, and 3 into the first group, and divide the characteristic spots numbered 2, 3, and 4 into the last group.

[0203] When the total number of characteristic spots is 5, divide the characteristic spots numbered 1, 2, and 3 into the first group, and divide the characteristic spots numbered 3, 4, and 5 into the last group;

[0204] When the total number of characteristic spots is 6, divide the characteristic spots numbered 1, 2, and 3 into the first group, and divide the characteristic spots numbered 4, 5, and 6 into the last group. Calculate the distance between the characteristic spots numbered 2 and 4 and the distance between the characteristic spots numbered 3 and 5, which are D-24 and D-35 respectively. When D-24 > D-35, use the characteristic spots numbered 3, 4, and 5 as the middle group. When D-24 < D-35, use the characteristic spots numbered 2, 3, and 4 as the middle group. When D-24 = D-35, optionally use the characteristic spots numbered 3, 4, and 5 as the middle group or the characteristic spots numbered 2, 3, and 4 as the middle group.

[0205] When the total number of characteristic spots is 7, divide the characteristic spots numbered 1, 2, and 3 into the first group, divide the characteristic spots numbered 5, 6, and 7 into the last group, and divide the characteristic spots numbered 3, 4, and 5 into the middle group.

[0206] When the total number of characteristic spots is 8, divide the characteristic spots numbered 1, 2, and 3 into the first group, divide the characteristic spots numbered 6, 7, and 8 into the last group, divide the characteristic spots numbered 3, 4, and 5 into the first middle group, and divide the characteristic spots numbered 4, 5, and 6 into the second middle group.

[0207] 4. Select positioning spots

[0208] After the grouping is completed, at least one group of characteristic spots is obtained, and one of the characteristic spots in each group of characteristic spots is used as the positioning spot. The method for selecting the positioning spot is as follows: within each group of characteristic spots, three characteristic spots are respectively used as candidate positioning spots, the distances between the other two characteristic spots and the positioning spot are measured, the ratio of the minimum distance to the maximum distance is calculated, the relative standard deviation of each ratio is calculated, and the characteristic spot with the smallest relative standard deviation value is selected as the positioning spot.

[0209] 5. Calculate the spacing ratio RPf

[0210] In each group of characteristic spots, calculate the distances between the other two characteristic spots and the positioning spot, calculate the ratio between the minimum distance and the maximum distance, which is denoted as the spacing ratio RPf, and use this spacing ratio RPf as the evaluation index.

[0211] It can be understood that one corresponding spacing ratio RPf can be obtained for each group of characteristic spots. If there are two groups of characteristic spots, the corresponding spacing ratios RPf1 and RPf2 can be obtained respectively; if there are three groups of characteristic spots, the corresponding spacing ratios RPf1, RPf2, and RPf3 can be obtained respectively; and so on.

[0212] 6. Establish the evaluation standard range

[0213] According to the spacing ratio RPf of each group of characteristic spots in different thin-layer chromatograms in the standard atlas library, establish the evaluation standard range of the spacing ratio RPf, and that's it.

[0214] It can be understood that the above evaluation standard range of the spacing ratio RPf is delimited by the values that may be obtained under different experimental conditions of qualified traditional Chinese medicine varieties with a large sample size.

[0215] Specifically, it is determined according to the data of different detection requirements and different traditional Chinese medicine varieties.

[0216] III. Comparative analysis

[0217] Take the thin-layer chromatogram of the traditional Chinese medicine variety to be tested, calculate the corresponding spacing ratio RPf according to the method for establishing the above digital thin-layer chromatogram detection and analysis model, and compare it with the evaluation standard range. If it falls within the qualified range, it can be determined as qualified, and the digital thin-layer chromatogram detection result is obtained.

[0218] Example 2

[0219] A digital thin-layer chromatogram detection and analysis method for cinnamon is applied to the digital thin-layer chromatogram detection and analysis of cinnamon with reference to the method of Example 1.

[0220] I. Thin-layer chromatogram detection

[0221] Take 0.5 g of cinnamon powder, add 10 mL of ethanol, soak it cold for 20 minutes, shake it constantly from time to time, filter it, and take the filtrate as the test solution. Separately take cinnamaldehyde reference substance, add ethanol to make a solution containing 1 μL per 1 mL as the reference solution. Absorb 5 μL of the test solution and 2 μL of the reference solution, spot them on a silica gel G thin layer plate, use petroleum ether (60 - 90 °C) - ethyl acetate (17∶3) as the developing agent, develop, take it out, dry it, spray it with 2,4-dinitrophenylhydrazine ethanol test solution, examine it under 254 nm, take a photo, and obtain the thin layer chromatogram (as Figure 1 ).

[0222] II. Establishment of the standard chromatogram library

[0223] Collect the thin layer chromatograms of qualified cinnamon. The thin layer chromatograms of the qualified products include the thin layer chromatograms under different experimental conditions to obtain the standard chromatogram library.

[0224] In this example, according to the above method for detection, collect the thin layer chromatograms of cinnamon on different silica gel thin layer plates (Merck KGaA, Qingdao Ocean Chemical Co., Ltd.), at different temperatures (24 °C, 50 °C), and different relative humidities (32%, 59%, 88%).

[0225] Divide the cinnamon thin layer chromatogram atlas into 54 batches of cinnamon samples for method establishment and 35 batches of cinnamon samples for method verification. Among them, the samples for method establishment evenly cover various silica gel G plates, various temperatures, and various relative humidities.

[0226] III. Establishment of the analysis model

[0227] 1. Select the analysis spots

[0228] Select the analysis spots according to the principle of Example 1. Along the direction of solvent development, number the origin and each spot in turn from the origin to the solvent front direction. According to the reference substance spot situation and the difference situation of the test sample spots in the cinnamon identification chromatogram, and comprehensively analyze in combination with the experience of manual judgment. Finally, select the relatively stable 2nd, 3rd, and 4th spots as the analysis spots. The Rf values of these spots 2, 3, and 4 are 0.11, 0.27, and 0.47 respectively.

[0229] 2. Consider whether to add the origin

[0230] There are 3 analysis spots in this example. Along the direction of solvent development, number the origin and the first three spots of the analysis spots in turn from the origin to the solvent front direction as A, B, C, D. Calculate the distances L-ac (L13) and L-bd (L24) between AC and BD, which are 3.52 and 4.69 respectively, that is, L-ac < L-bd, then add the origin as a characteristic spot for grouping.

[0231] 3. Grouping

[0232] This embodiment has a total of 4 characteristic spots. These 4 characteristic spots are divided into 2 groups. The first group (the first group) includes the 1st, 2nd, and 3rd characteristic spots, and the second group (the last group) includes the 2nd, 3rd, and 4th characteristic spots.

[0233] 4. Select positioning spots

[0234] Within the above-mentioned characteristic spot groups, three characteristic spots are respectively used as candidate positioning spots, the distances between the other two characteristic spots and the positioning spots are measured, the ratio of the minimum distance to the maximum distance is calculated, and the relative standard deviation of each ratio is calculated. The results are shown in the following table.

[0235] Table 1. Analysis results of the position data of different positioning spots in the first group

[0236]

[0237] The above results show that in the thin-layer chromatogram of cinnamon, the relative standard deviations of three different positioning points are 0.12, 0.18, and 0.07 respectively. The relative standard deviation obtained by using the 3rd spot as the positioning spot is the smallest, that is, the 3rd spot with the smallest relative standard deviation value is selected as the positioning spot.

[0238] Table 2. Analysis results of the position data of different positioning spots in the second group

[0239]

[0240] The above results show that in the thin-layer chromatogram of cinnamon, the relative standard deviations of three different positioning points are 0.04, 0.07, and 0.05 respectively. The relative standard deviation obtained by using the 2nd spot as the positioning spot is the smallest, that is, the 2nd spot with the smallest relative standard deviation value is selected as the positioning spot.

[0241] 5. Formulate the calculation rules for the positions of characteristic spots

[0242] To determine the relative position relationship of multiple target components in thin-layer chromatography, the present invention first proposes the concepts and theories of the position factor (Pf) and the relative position factor (RPf), and applies this algorithm to digital standards without physical references.

[0243] The position factor (Pf) is the ratio of the distance from the target component to the positioning component to the distance from the origin to the positioning component. Taking Figure 2 as an example, if the thin-layer spot D is used as the positioning spot, then the position factor of the thin-layer spot C The relative position factor (RPf) is the ratio of the distance from the target component to the positioning component to the distance from the reference component to the positioning component. Taking Figure 2For example, taking the thin-layer spot D as the positioning spot and the thin-layer spot B as the reference component spot, the relative specific migration value of the thin-layer spot C The relative specific migration value positioning method can effectively reflect the relative positional relationship of the three thin-layer spots, and at the same time, the solvent front and the origin do not need to be involved in the calculation.

[0244] In this embodiment, three calculation methods are simultaneously compared to evaluate the positions of the three spots.

[0245] The first calculation method: Referring to the calculation method of the relative specific migration value, the three thin-layer spots are respectively marked as the positioning points, and the other two thin-layer spots are marked as the reference spots, and the calculation formulas for the following three different situations are obtained:

[0246] ① When spot D is the positioning spot and spot B is the reference spot, the relative specific migration value of spot C

[0247] ② When spot C is the positioning spot and spot D is the reference spot, the relative specific migration value of spot B

[0248] ③ When spot B is the positioning spot and spot D is the reference spot, the relative specific migration value of spot C

[0249] The distances from different characteristic spots to the positioning spot are obtained. Defining L1 as the minimum distance and L3 as the maximum distance, the following general formula is obtained:

[0250]

[0251] The second calculation method: Taking the origin as the positioning point, measuring the distances from each characteristic spot to the positioning point, and successively dividing the smaller distance by the larger distance, the following general formula is obtained:

[0252]

[0253] The third calculation method: Calculating the distance ratio between the characteristic spots. There are two distances between the three characteristic spots. Dividing the minimum distance by the maximum distance, the following general formula is obtained:

[0254]

[0255] Calculating X1, X2, and X3 according to the above three different calculation methods, and the results are shown in the following table. In the first group, the positioning point 1 is the origin, so the calculation method 1 is the same as the calculation method 2.

[0256] Table 3 Analysis results of position data of different calculation methods in the first group

[0257]

[0258] The above results show that in the cinnamon samples, the relative standard deviations of the first group calculated by two different methods are 0.07 and 0.18 respectively. It shows that the relative standard deviation of the first calculation method is smaller, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the spot location of cinnamon thin-layer chromatography.

[0259] Table 4 Analysis results of position data for the second group with different calculation methods

[0260]

[0261] The above results show that in the cinnamon samples, the relative standard deviations of the three different calculation methods are 0.04, 0.07, and 0.05 respectively. It shows that the relative standard deviation of the first calculation method is the smallest, also indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the spot location of cinnamon thin-layer chromatography.

[0262] 6. Calculate the spacing ratio RPf

[0263] According to the above first method, in the first group, the 3rd spot is used as the positioning spot, and the distances L-32 and L-31 between the 3rd spot and the 2nd spot and the 1st spot are calculated respectively. The ratio of the minimum distance L-32 to the maximum distance L-31 is calculated and denoted as the spacing ratio RPf1. Taking this spacing ratio RPf1 as the evaluation index, according to the spacing ratios RPf1 of each characteristic spot group in different thin-layer chromatograms in the standard atlas library (54 batches of samples) (such as Figure 3 the black circled part in), the evaluation standard range of the spacing ratio RPf1 is established as 0.54 - 0.71.

[0264] In the second group, the 2nd spot is used as the positioning spot, and the distances L-23 and L-24 between the 2nd spot and the 3rd spot and the 4th spot are calculated respectively. The ratio of the minimum distance L-23 to the maximum distance L-24 is calculated and denoted as the spacing ratio RPf2. Taking this spacing ratio RPf2 as the evaluation index, according to the spacing ratios RPf2 of each characteristic spot group in different thin-layer chromatograms in the standard atlas library (54 batches of samples) (such as Figure 4 the black circled part in), the evaluation standard range of the spacing ratio RPf2 is established as 0.51 - 0.59.

[0265] IV. Comparative analysis

[0266] Take the thin-layer chromatograms of 35 batches of cinnamon samples using the above verification method, and calculate the corresponding spacing ratios RPf1 and RPf2 according to the first method determined in the above analysis model establishment steps (such as Figure 3 and Figure 4The blue circle part in the middle) was compared with the above evaluation standard range to obtain the digital thin layer chromatography test results of cinnamon.

[0267] The data results show that in the first group, the RPf values ​​of all samples are within the proposed evaluation standard range, but in the second group, the RPf1 values ​​of samples 67 and 68 are both 0.49 (e.g. Figure 4 The red circle part in the middle is outside the scope of the proposed evaluation standard. After tracing the sample maps, it was found that spot 2 was missing in the thin layer chromatograms of these two batches of samples. They were judged to be unqualified samples and needed to be re-experimented, which further proved that the digital evaluation method of cinnamon thin layer chromatography is highly feasible.

[0268] Example 3

[0269] A digital thin-layer chromatography detection and analysis method for Shuxiong tablets is applied to the digital thin-layer chromatography detection and analysis of Shuxiong tablets using Chuanxiong as an index medicinal material, referring to the method in Example 1.

[0270] 1. Thin layer chromatography detection

[0271] Take 20 pieces of Shuxiong tablets, remove the coating, grind them into powder, add 20 mL of ether, heat and reflux for 1 hour, filter, evaporate the filtrate, add 2 mL of ether to the residue to dissolve it, and use it as the test solution. Take another 1 g of Chuanxiong reference medicinal material, add 10 mL of ether, soak for 1 hour, filter, evaporate the filtrate, add 2 mL of ether to the residue to dissolve it, and use it as the reference medicinal material solution. Take 8 μL of each of the above two solutions, spot them on a silica gel G thin layer plate, use petroleum ether (30-60°C)-chloroform (1:9) as the developing agent, develop it, take it out, dry it, examine it under an ultraviolet lamp (365 nm), take a picture, and get a thin layer chromatogram (as shown in Figure 2). Figure 5 ).

[0272] 2. Establishment of Standard Spectrum Library

[0273] The thin layer chromatograms of qualified Shuxiong Tablets are collected, wherein the thin layer chromatograms of the qualified products include the thin layer chromatograms under the difference experimental conditions, and a standard spectrum library is obtained.

[0274] In this embodiment, the above method was used to detect and collect thin layer chromatograms of Shuxiong tablets at different silica gel thin layer chromatography plates (Merck KGaA, Qingdao Ocean Chemical Co., Ltd.), different temperatures (5°C, 25°C), and different relative humidity (32%, 56%, 88%).

[0275] The TLC spectra of Shuxiong Tablets were divided into 50 batches of Shuxiong Tablets samples for establishing the method and 27 batches of Shuxiong Tablets samples for verifying the method, among which the samples for establishing the method evenly covered various silica gel G plates, various temperatures and various relative humidity.

[0276] 3. Establishment of analysis model

[0277] 1. Select analysis spots

[0278] Select analysis spots according to the principle of Example 1. Along the solvent development direction, number the origin and each spot in turn from the origin to the solvent front direction. According to the control medicinal material spot situation and the difference of test sample spots in the identification chromatogram of Shuxiong Tablets, combined with the experience of manual judgment for comprehensive analysis, finally select the relatively stable 4th, 5th, and 6th spots as analysis spots. The Rf values of these spots 4, 5, and 6 are 0.39, 0.44, and 0.67 respectively.

[0279] 2. Consider whether to add the origin

[0280] There are 3 analysis spots in this example. Along the solvent development direction, number the origin and the first three spots of the analysis spots in turn from the origin to the solvent front direction as A, B, C, D (as Figure 5 shown). Calculate the distances L-ac and L-bd between AC and BD respectively. Since L-ac≥L-bd, the origin is not added as a characteristic spot for grouping.

[0281] 3. Grouping

[0282] There are 3 characteristic spots in this example, and these 3 characteristic spots are grouped into one group.

[0283] 4. Select positioning spots

[0284] Within the above-mentioned characteristic spot group, use the three characteristic spots as candidate positioning spots respectively, measure the distances between the other two characteristic spots and the positioning spot, calculate the ratio of the minimum distance to the maximum distance, and calculate the relative standard deviation of each ratio. The results are shown in the following table.

[0285] Table 5. Analysis results of the position data of different positioning spots

[0286]

[0287] The above results show that in the thin layer chromatogram of Shuxiong Tablets, the relative standard deviations of the three different positioning points are 0.04, 0.22, and 0.18 respectively. The relative standard deviation obtained by using the 4th spot as the positioning spot is the smallest, that is, select the 4th spot with the smallest relative standard deviation value as the positioning spot.

[0288] 5. Formulate the calculation rule for the position of characteristic spots

[0289] Refer to the method in Example 2. In this example, three calculation methods are simultaneously compared to evaluate the positions of the three spots in Shuxiong Tablets.

[0290] Calculate X1, X2, and X3 according to the above three different calculation methods, and the results are shown in the following table.

[0291] Data analysis results of positions with different calculation methods in Table 6

[0292]

[0293] The above results show that in the Shuxiongpian samples, the relative standard deviations of the three different calculation methods are 0.04, 0.05, and 0.21 respectively. It shows that the relative standard deviation of the first calculation method is the smallest, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the thin-layer chromatography spot location of Shuxiongpian.

[0294] 6. Calculate the spacing ratio RPf

[0295] According to the first method above, using the 4th spot as the positioning spot, calculate the distances L-45 and L-46 between the 4th spot and the 5th spot and the 6th spot respectively, and calculate the ratio of the minimum distance L-45 to the maximum distance L-46, denoted as the spacing ratio RPf3. Using this spacing ratio RPf3 as the evaluation index, according to the spacing ratios RPf3 of each characteristic spot group in different thin-layer chromatograms in the standard atlas library (50 batches of samples) (such as Figure 6 the black circled part in), establish the evaluation standard range of the spacing ratio RPf3 as 0.77 - 0.88.

[0296] IV. Comparative analysis

[0297] Take the thin-layer chromatograms of the above 27 batches of Shuxiongpian samples, and calculate the corresponding spacing ratio RPf3 according to the first method determined in the above analysis model establishment steps (such as Figure 6 the blue circled part in), and compare it with the above evaluation standard range to obtain the digital thin-layer chromatography detection results of Shuxiongpian.

[0298] The data results show that the RPf3 values of the 27 batches of Shuxiongpian samples used for verification are all within the proposed range, indicating that the digital thin-layer chromatography evaluation method for Shuxiongpian based on the RPf3 value has good performance.

[0299] Example 4

[0300] A digital thin-layer chromatography detection and analysis method for Rhodiola rosea, referring to the method of Example 1, is applied to the digital thin-layer chromatography detection and analysis of Rhodiola rosea.

[0301] I. Thin-layer chromatography detection

[0302] Take about 0.5 g of Rhodiola rosea powder (sieved through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of methanol, stopper tightly, weigh, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate as the test solution. Spot the test solution on a silica gel G thin layer plate, develop with the lower layer solution of chloroform - methanol - acetone - water (6:3:1:1), expose it to iodine vapor, take a photo, and obtain a thin layer chromatogram (as Figure 7 ).

[0303] II. Establishment of Standard Atlas Library

[0304] Collect the thin layer chromatograms of qualified Rhodiola rosea products, and the thin layer chromatograms of the qualified products include those under different experimental conditions to obtain a standard atlas library.

[0305] In this example, according to the above - mentioned method for detection, collect the thin layer chromatograms of Rhodiola rosea on different silica gel thin layer plates (Merck KGaA, Yantai Yinlong Silica Gel Co., Ltd.), at different temperatures (4°C, 21.9°C), and different relative humidities (32%, 63%, 88%).

[0306] Divide the thin layer chromatograms of Rhodiola rosea into 50 batches of samples for method establishment and 39 batches of samples for method verification. Among them, the samples for method establishment evenly cover various silica gel G plates, various temperatures, and various relative humidities.

[0307] III. Establishment of Analysis Model

[0308] 1. Select analysis spots

[0309] Select analysis spots according to the principle of Example 1. Along the solvent development direction, number the origin and each spot in turn from the origin to the solvent front direction. Based on the spot conditions of the reference medicinal material for Rhodiola rosea identification chromatogram and the differences in the test sample spots, combined with the experience of manual judgment for comprehensive analysis, finally select the relatively stable No. 3, 4, 5, and 6 spots as analysis spots. The Rf values of these spots 3, 4, 5, and 6 are 0.32, 0.45, 0.63, and 0.77 respectively (as Figure 7 shown).

[0310] 2. Consider whether to add the origin

[0311] In this example, there are 4 analysis spots. Along the solvent development direction, number the origin and the first three of the analysis spots in turn from the origin to the solvent front direction as A, B, C, D (corresponding to numbers 1, 3, 4, 5), and calculate the distances L - ac (L14) and L - bd (L35) between AC and BD respectively. Since L - ac ≥ L - bd, the origin is not added as a characteristic spot for grouping.

[0312] 3. Grouping

[0313] There are a total of 4 characteristic spots in this embodiment, and these 4 characteristic spots are divided into 2 groups.

[0314] 4. Selecting and positioning spots

[0315] Within the above-mentioned characteristic spot groups, three characteristic spots are respectively used as candidate positioning spots, the distances between the other two characteristic spots and the positioning spots are measured, the ratio of the minimum distance to the maximum distance is calculated, and the relative standard deviation of each ratio is calculated. The results are shown in the following table.

[0316] Table 7 Analysis results of the position data of different positioning spots in the first group

[0317]

[0318] The above results show that in the thin-layer chromatogram of Shuxiong Tablets, the relative standard deviations of three different positioning points are 0.07, 0.12, and 0.05 respectively. The relative standard deviation obtained by using the 5th spot as the positioning spot is the smallest, that is, the 5th spot with the smallest relative standard deviation value is selected as the positioning spot of the first group.

[0319] Table 8 Analysis results of the position data of different positioning spots in the second group

[0320]

[0321] The above results show that in the thin-layer chromatogram of Shuxiong Tablets, the relative standard deviations of three different positioning points are 0.12, 0.42, and 0.30 respectively. The relative standard deviation obtained by using the 4th spot as the positioning spot is the smallest, that is, the 4th spot with the smallest relative standard deviation value is selected as the positioning spot of the second group.

[0322] 5. Formulating the calculation rules for the positions of characteristic spots

[0323] Referring to the method in Embodiment 2, in this embodiment, three calculation methods are simultaneously compared to evaluate the positions of four spots in Rhodiola rosea.

[0324] Calculate X1, X2, and X3 according to the above three different calculation methods, and the results are shown in the following table.

[0325] Table 9 Analysis results of the position data of different calculation methods in the first group

[0326]

[0327] The above results show that in the rhodiola samples, the relative standard deviations of the three different calculation methods in the first group are 0.04, 0.05, and 0.45 respectively. It shows that the relative standard deviation of the first calculation method is the smallest, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the thin-layer chromatography spot localization of rhodiola.

[0328] Table 10 Analysis Results of Position Data of Different Calculation Methods in the Second Group

[0329]

[0330] The above results show that in the rhodiola samples, the relative standard deviations of the three different calculation methods in the second group are 0.12, 0.12, and 0.45 respectively. It shows that the relative standard deviations of the first and second calculation methods are the smallest. Combining the data of the first group, the comprehensive results show that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the thin-layer chromatography spot localization of rhodiola.

[0331] 6. Calculate the spacing ratio RPf

[0332] According to the above first method, in the first group, the 5th spot is used as the localization spot, and the distances L-35 and L-45 between the 5th spot and the 3rd spot and the 4th spot are calculated respectively. The ratio of the minimum distance L-45 to the maximum distance L-35 is calculated and denoted as the spacing ratio RPf4; in the second group, the 4th spot is used as the localization spot, and the distances L-45 and L-46 between the 4th spot and the 5th spot and the 6th spot are calculated respectively. The ratio of the minimum distance L-45 to the maximum distance L-46 is calculated and denoted as the spacing ratio RPf4; taking the spacing ratios RPf4 and RPf5 as evaluation indicators, according to the spacing ratios RPf4 of each characteristic spot group in different thin-layer chromatograms in the standard atlas library (50 batches of samples) (such as Figure 8 the black circled part in), the evaluation standard range of the spacing ratio RPf4 is established as 0.54 - 0.69; the spacing ratios RPf5 of each characteristic spot group (such as Figure 9 the black circled part in), the evaluation standard range of the spacing ratio RPf5 is established as 0.55 - 0.85.

[0333] IV. Comparative Analysis

[0334] Take the thin-layer chromatograms of the above 39 batches of rhodiola samples, and calculate the corresponding spacing ratios RPf4 and RPf5 according to the first method determined in the above analysis model establishment steps (such as Figure 8 、 Figure 9 the blue circled part in), and compare with the above evaluation standard range to obtain the digital thin-layer chromatography detection results of rhodiola.

[0335] The data results show that the RPf4 and RPf5 values of 39 batches of Rhodiola samples used for verification methods are within the corresponding proposed ranges, indicating that the digital TLC evaluation method for Rhodiola based on RPf values has good performance.

[0336] Example 5

[0337] A digital TLC detection and analysis method for Cassia obtusifolia is applied to the digital TLC detection and analysis of crude Cassia obtusifolia medicinal materials by referring to the method of Example 1.

[0338] I. TLC detection

[0339] Take the Cassia obtusifolia sample, add 10 ml of methanol, soak for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, add 1 ml of hydrochloric acid, heat in a water bath for 30 minutes, cool immediately, extract with ether twice, 20 ml each time, combine the ether layers, evaporate to dryness, dissolve the residue in 1 ml of chloroform to obtain the test solution. Spot the test solution on a silica gel H TLC plate, use petroleum ether (30 - 60 °C)-acetone (2:1) as the developing agent, develop, take out, and dry in air. After fuming with ammonia vapor, take a photo to obtain the TLC chromatogram (as Figure 10 ).

[0340] II. Establishment of the standard spectrum library

[0341] Collect the TLC chromatograms of qualified Cassia obtusifolia, and the TLC chromatograms of the qualified products include the TLC chromatograms under different experimental conditions to obtain the standard spectrum library.

[0342] In this example, according to the above method for detection, collect the TLC chromatograms of Cassia obtusifolia on different silica gel TLC plates (Merck KGaA, Qingdao Ocean Chemical Co., Ltd.), at different temperatures (5 °C, 25 °C), and different relative humidities (32%, 56%, 88%).

[0343] Divide the Cassia obtusifolia TLC chromatograms into 58 batches of samples for method establishment and 30 batches of samples for method verification. Among them, the samples for method establishment evenly cover various plates, temperatures, and relative humidities.

[0344] III. Establishment of the analysis model

[0345] 1. Select the analysis spots

[0346] Select the analysis spots according to the principle of Example 1. Along the direction of solvent development, number the origin and each spot in turn from the origin to the solvent front direction. Based on the spot conditions of the reference medicinal material for Cassia obtusifolia identification chromatogram and the differences of the test sample spots, and combined with the experience of manual judgment for comprehensive analysis, finally select the relatively stable spots 3 and 5 as the analysis spots. The Rf values of these spots 3 and 5 are 0.63 and 0.75 respectively (as Figure 10as shown

[0347] 2. Consider whether to add the origin point

[0348] In this embodiment, there are only 2 analyzed spots, and the origin point is clear. The origin point is added as a characteristic spot for grouping.

[0349] 3. Grouping

[0350] There are 3 characteristic spots in this embodiment, and these 3 characteristic spots are grouped into one group.

[0351] 4. Select the positioning spots

[0352] Within the above-mentioned characteristic spot group, using the three characteristic spots as candidate positioning spots respectively, measure the distances between the other two characteristic spots and the positioning spots, calculate the ratio of the minimum distance to the maximum distance, and calculate the relative standard deviation of each ratio. The results are shown in the following table.

[0353] Table 11 Data analysis results of the positions of different positioning spots

[0354]

[0355] The above results show that in the thin-layer chromatogram of Cassia obtusifolia, the relative standard deviations of the three different positioning points are 0.25, 0.34, and 0.09 respectively. The relative standard deviation obtained by using the 5th spot as the positioning spot is the smallest. That is, the 5th spot with the smallest relative standard deviation value is selected as the positioning spot.

[0356] 5. Formulate the calculation rules for the positions of characteristic spots

[0357] Referring to the method in Embodiment 2, in this embodiment, three calculation methods are simultaneously compared to evaluate the positions of the three spots in Cassia obtusifolia. Since the origin point is added as an analyzed spot, the second calculation method is the same as the first calculation method. Calculate X1 and X3 according to the above two different calculation methods, and the results are shown in the following table.

[0358] Table 12 Data analysis results of the positions of different calculation methods

[0359]

[0360] The above results show that in the Cassia obtusifolia sample, the relative standard deviations of the two different calculation methods are 0.07 and 0.23 respectively, indicating that the relative standard deviation of the first calculation method is smaller, showing that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the positioning of thin-layer chromatographic spots of Cassia obtusifolia.

[0361] 6. Calculate the spacing ratio RPf

[0362] According to the above first method, taking the 5th spot as the positioning spot, calculate the distances L-35 and L-15 between the 5th spot and the 3rd spot and the 1st spot respectively, calculate the ratio of the minimum distance L-35 to the maximum distance L-15, denoted as the spacing ratio RPf6. Taking this spacing ratio RPf6 as the evaluation index, according to the spacing ratios RPf6 of each characteristic spot group in different thin layer chromatograms in the standard atlas library (58 batches of samples) (such as Figure 11 the black circled part in

[0363] IV. Comparative Analysis

[0364] Take the thin layer chromatograms of the above 30 batches of Cassia obtusifolia samples, and calculate the corresponding spacing ratio RPf6 according to the first method determined in the above analysis model establishment steps (such as Figure 11 the blue circled part in

[0365] Compare with the above evaluation standard range to obtain the digital thin layer chromatogram detection results of Cassia obtusifolia. The data results show that the RPf6 values of the 30 batches of Cassia obtusifolia samples used for verification are all within the proposed range, indicating that the digital evaluation method of Cassia obtusifolia thin layer chromatography based on the RPf6 value has good performance.

[0366] Example 6

[0367] A digital thin layer chromatogram detection and analysis method for stir-fried Cassia obtusifolia is applied to the digital thin layer chromatogram detection and analysis of stir-fried Cassia obtusifolia medicinal materials with reference to the method of Example 1.

[0368] I. Thin Layer Chromatography Detection

[0369] Take the stir-fried Cassia obtusifolia sample, add 10 ml of methanol, soak for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, add 1 ml of hydrochloric acid, heat in a water bath for 30 minutes, cool immediately, extract with ether twice, 20 ml each time, combine the ether solutions, evaporate to dryness, dissolve the residue in 1 ml of chloroform to obtain the test solution. Spot the test solution on a silica gel H thin layer plate, use petroleum ether (30 - 60 °C)-acetone (2:1) as the developing agent, develop, take out, and dry in air. After fumigating with ammonia vapor, take a photo to obtain the thin layer chromatogram (such as Figure 12 ).

[0370] II. Establishment of Standard Atlas Library

[0371] Collect the thin layer chromatograms of qualified stir-fried Cassia obtusifolia, and the thin layer chromatograms of the qualified products include the thin layer chromatograms under different experimental conditions to obtain the standard atlas library.

[0372] In this embodiment, according to the above method for detection, thin layer chromatograms of stir-fried Cassia obtusifolia seeds were collected on different silica gel thin layer plates (Merck KGaA, Qingdao Ocean Chemical Co., Ltd.) at different temperatures (5°C, 25°C) and different relative humidities (32%, 56%, 88%).

[0373] The thin layer chromatograms of stir-fried Cassia obtusifolia seeds were divided into 40 batches of samples for method establishment and 39 batches of samples for method verification. Among them, the samples for method establishment evenly covered various silica gel H plates, various temperatures, and various relative humidities.

[0374] III. Establishment of the analysis model

[0375] 1. Selection of analysis spots

[0376] According to the principle of Example 1, analysis spots were selected. Along the direction of solvent development, the origin and each spot were numbered in sequence from the origin to the solvent front. Based on the spot conditions of the reference medicinal material for the identification chromatogram of stir-fried Cassia obtusifolia seeds and the differences in the spots of the test sample, combined with the experience of manual judgment, a comprehensive analysis was carried out. Finally, the relatively stable No. 2 and No. 5 spots were selected as the analysis spots. The Rf values of these spots 2 and 5 were 0.59 and 0.79 respectively (as Figure 12 shown).

[0377] 2. Consider whether to add the origin

[0378] In this embodiment, there are only 2 analysis spots, and the origin is clear. The origin is added as a characteristic spot for grouping.

[0379] 3. Grouping

[0380] In this embodiment, there are a total of 3 characteristic spots, and these 3 characteristic spots are grouped into one group.

[0381] 4. Selection of positioning spots

[0382] Within the above-mentioned group of characteristic spots, the three characteristic spots were used as candidate positioning spots respectively. The distances between the other two characteristic spots and the positioning spot were measured, the ratio of the minimum distance to the maximum distance was calculated, and the relative standard deviation of each ratio was calculated. The results are shown in the following table.

[0383] Table 13. Data analysis results of the positions of different positioning spots

[0384]

[0385] The above results show that in the thin layer chromatogram of stir-fried Cassia obtusifolia seeds, the relative standard deviations of the three different positioning points are 0.17, 0.25, and 0.07 respectively. The relative standard deviation obtained by using the No. 5 spot as the positioning spot is the smallest. That is, the No. 5 spot with the smallest relative standard deviation value is selected as the positioning spot.

[0386] 5. Establishment of Calculation Rules for Feature Spot Positions

[0387] Referring to the method in Example 2, in this example, three calculation methods are compared simultaneously to evaluate the positions of three spots in cassia seeds. Since the origin is added as an analysis spot, the second calculation method is the same as the first one. Calculate X1 and X3 according to the above two different calculation methods, and the results are shown in the following table.

[0388] Table 14 Data Analysis Results of Positions with Different Calculation Methods

[0389]

[0390]

[0391] The above results show that in the stir-fried cassia seed samples, the relative standard deviations of the three different calculation methods are 0.07 and 0.23 respectively. It shows that the relative standard deviation of the first calculation method is the smallest, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the thin-layer chromatography spot positioning of stir-fried cassia seeds.

[0392] 6. Calculate the Spacing Ratio RPf

[0393] According to the above first method, taking the 5th spot as the positioning spot, calculate the distances L-51 and L-52 between the 5th spot and the 1st spot and the 2nd spot respectively, calculate the ratio of the minimum distance L-52 to the maximum distance L-51, denoted as the spacing ratio RPf7. Taking this spacing ratio RPf7 as the evaluation index, according to the spacing ratios RPf1 of each characteristic spot group in different thin-layer chromatograms in the standard atlas library (40 batches of samples) (such as Figure 13 the black circled part in), establish the evaluation standard range of the spacing ratio RPf7 as 0.65 - 0.78.

[0394] IV. Comparative Analysis

[0395] Take the thin-layer chromatograms of the above 39 batches of stir-fried cassia seed samples, and calculate the corresponding spacing ratio RPf7 according to the first method determined in the above analysis model establishment steps (such as Figure 13 the blue circled part in), and compare it with the above evaluation standard range to obtain the digital thin-layer chromatography detection results of stir-fried cassia seeds.

[0396] The data results show that the RPf7 values of the 39 batches of stir-fried cassia seed samples used for verifying the method are all within the proposed range, indicating that the digital evaluation method for the thin-layer chromatography of stir-fried cassia seeds based on the RPf7 value has good performance.

[0397] Example 7

[0398] A digital thin-layer chromatography detection and analysis method for notoginseng in Xinning Tablets. Referring to the method of Example 1, it is applied to the digital thin-layer chromatography detection and analysis of Xinning Tablets with notoginseng as the index medicinal material.

[0399] I. Thin-layer chromatography detection

[0400] Take 10 Xinning Tablets, remove the coating, grind them finely, add 50 ml of ether, ultrasonically treat for 20 minutes, filter, evaporate the ether from the medicinal residue, add 50 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 50 ml of water. Extract twice with water-saturated n-butanol by shaking, 30 ml each time, combine the n-butanol solutions, wash twice with ammonia test solution, 60 ml each time, take the n-butanol solution, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution. Spot the test solution on a silica gel G thin-layer plate, use the upper layer solution of n-butanol - acetic acid - water (4:1:5) as the developing agent, develop, take out, and dry in air. Spray with 10% sulfuric acid ethanol, heat at 105 °C until the spots are clearly developed, and examine under daylight to obtain the thin-layer chromatogram (as Figure 14 ).

[0401] II. Establishment of the standard spectrum library

[0402] Collect the thin-layer chromatograms of qualified notoginseng in Xinning Tablets. The thin-layer chromatograms of the qualified products include the thin-layer chromatograms under different experimental conditions to obtain the standard spectrum library.

[0403] In this example, according to the above method for detection, collect the thin-layer chromatograms of notoginseng in Xinning Tablets on different silica gel thin-layer plates (Merck KGaA, Yantai Chemical Industry Research Institute), at different temperatures (5.1 °C, 17.8 °C), and different relative humidities (32%, 65%, 88%).

[0404] Divide the thin-layer chromatogram of notoginseng in Xinning Tablets into 29 batches of Xinning Tablet samples for method establishment and 48 batches of Xinning Tablet samples for method verification. Among them, the samples for method establishment evenly cover various silica gel thin-layer plates, various temperatures, and various relative humidities.

[0405] III. Establishment of the analysis model

[0406] 1. Selection of analysis spots

[0407] Select the analysis spots according to the principle of Example 1. Along the direction of solvent development, number the origin and each spot in turn from the origin to the solvent front direction. According to the spot situation of the reference medicinal material for the identification of notoginseng in Xinning Tablets and the difference situation of the test sample spots, combined with the experience of manual judgment for comprehensive analysis, finally select the relatively stable No. 2, 4, 5, 6, 8, 9 spots as the analysis spots. The Rf values of these spots 2, 4, 5, 6, 8, 9 are 0.068, 0.20, 0.22, 0.38, 0.58, 0.68 respectively (as Figure 14as shown

[0408] 2. Consider whether to add the origin point

[0409] There are 6 analysis spots in this embodiment. Along the solvent development direction, starting from the origin point and moving successively towards the solvent front direction, the origin point and the first three of the analysis spots are numbered A, B, C, D in sequence (i.e., corresponding to the 1st, 2nd, 4th, and 5th spots). Calculate the distances L-ac (L14) and L-bd (L25) between AC and BD respectively. Since L-ac > L-bd, the origin point is not added as a characteristic spot for grouping.

[0410] 3. Grouping

[0411] There are a total of 6 characteristic spots in this embodiment, and these 6 characteristic spots are divided into 3 groups. Spots 2, 4, and 5 are divided into the first group, and spots 6, 8, and 9 are divided into the second group. Calculate the distances between spot 4 and spot 6 and between spot 5 and spot 8, which are D-46 and D-58 respectively. Because D-46 < D-58, spots 4, 5, and 6 are divided into the third group.

[0412] 4. Select positioning spots

[0413] Within the above-mentioned characteristic spot groups, three characteristic spots are used as candidate positioning spots respectively. Measure the distances between the other two characteristic spots and the positioning spots, calculate the ratio of the minimum distance to the maximum distance, and calculate the relative standard deviation of each ratio. The results are shown in the following table.

[0414] Table 15. Analysis results of the position data of different positioning spots in the first group

[0415]

[0416] The above results show that in the thin-layer chromatogram of notoginseng in Xinning tablets, the relative standard deviations of three different positioning points are 0.02, 0.16, and 0.14 respectively. The relative standard deviation obtained by using the 2nd spot as the positioning spot is the smallest, that is, the 2nd spot with the smallest relative standard deviation value is selected as the positioning spot.

[0417] Table 16. Analysis results of the position data of different positioning spots in the second group

[0418]

[0419] The above results show that in the thin-layer chromatogram of notoginseng in Xinning tablets, the relative standard deviations of three different positioning points are 0.04, 0.11, and 0.08 respectively. The relative standard deviation obtained by using the 6th spot as the positioning spot is the smallest, that is, the 6th spot with the smallest relative standard deviation value is selected as the positioning spot.

[0420] Table 17. Analysis results of the position data of different positioning spots in the third group

[0421]

[0422] The above results show that in the thin-layer chromatogram of notoginseng in Xinning Tablets, the relative standard deviations of three different positioning points are 0.28, 0.34, and 0.05 respectively. The relative standard deviation obtained with the 6th spot as the positioning spot is the smallest. That is, the 6th spot with the smallest relative standard deviation value is selected as the positioning spot.

[0423] 5. Formulation of calculation rules for the position of characteristic spots

[0424] Referring to the method in Example 2, in this example, three calculation methods are simultaneously compared to evaluate the positions of 6 spots of notoginseng in Xinning Tablets. Calculate X1, X2, and X3 according to the above two different calculation methods, and the results are shown in the following table.

[0425] Table 18 Analysis results of position data of the first group with different calculation methods

[0426]

[0427] The above results show that in the notoginseng samples of Xinning Tablets, the relative standard deviations of the two different calculation methods are 0.02, 0.12, and 0.16 respectively, indicating that the relative standard deviation of the first calculation method is the smallest, suggesting that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the thin-layer chromatogram spot positioning of notoginseng in Xinning Tablets.

[0428] Table 19 Analysis results of position data of the second group with different calculation methods

[0429]

[0430] The above results show that in the Xinning Tablet samples, the relative standard deviations of the three different calculation methods in the second group are 0.04, 0.07, and 0.11 respectively, indicating that the relative standard deviation of the first calculation method is the smallest, suggesting that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the thin-layer chromatogram spot positioning of notoginseng in Xinning Tablets.

[0431] Table 20 Analysis results of position data of the third group with different calculation methods

[0432]

[0433] The above results show that in the samples of Xinning Tablets, the relative standard deviations of the three different calculation methods in the second group are 0.05, 0.14, and 0.34 respectively. It shows that the relative standard deviation of the first calculation method is the smallest, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the TLC spot localization of Notoginseng in Xinning Tablets.

[0434] 6. Calculate the spacing ratio RPf

[0435] According to the first method above, in the first group, take the second spot as the localization spot, calculate the distances L-24 and L-25 between the second spot and the fourth spot and the fifth spot respectively, calculate the ratio of the minimum distance L-24 to the maximum distance L-25, and record it as the spacing ratio RPf8; in the second group, take the sixth spot as the localization spot, calculate the distances L-68 and L-69 between the sixth spot and the eighth spot and the ninth spot respectively, calculate the ratio of the minimum distance L-68 to the maximum distance L-69, and record it as the spacing ratio RPf9; in the third group, take the sixth spot as the localization spot, calculate the distances L-65 and L-64 between the sixth spot and the fifth spot and the fourth spot respectively, calculate the ratio of the minimum distance L-65 to the maximum distance L-64, and record it as the spacing ratio RPf10; using the spacing ratios RPf8, RPf9, and RPf10 as evaluation indicators, according to the spacing ratios RPf8 of each characteristic spot group in different TLC chromatograms in the standard atlas library (29 batches of samples) (such as Figure 15 the black circled part in), establish the evaluation standard range of the spacing ratio RPf8 as 0.81 - 0.89; the spacing ratios RPf9 of each characteristic spot group (such as Figure 16 the black circled part in), establish the evaluation standard range of the spacing ratio RPf9 as 0.64 - 0.76; the spacing ratios RPf10 of each characteristic spot group (such as Figure 17 the black circled part in), establish the evaluation standard range of the spacing ratio RPf10 as 0.74 - 0.91.

[0436] IV. Comparative analysis

[0437] Take the TLC chromatograms of the Notoginseng samples in the above 48 batches of Xinning Tablets, and calculate the corresponding spacing ratios RPf8, RPf9, and RPf10 according to the first method determined in the above analysis model establishment steps (such as Figure 15 , Figure 16 , Figure 17 the blue circled part in), and compare with the above evaluation standard range to obtain the digital TLC detection results of Notoginseng in Xinning Tablets.

[0438] The data results show that the RPf8, RPf9, and RPf10 values of the notoginseng samples in 48 batches of verified Xinning Tablets using the method are all within the proposed range, indicating that the digital TLC evaluation method for notoginseng in Xinning Tablets based on RPf values has good performance.

[0439] Example 8

[0440] A digital TLC detection and analysis method for salvia miltiorrhiza in Xinning Tablets. Referring to the method of Example 1, it is applied to the digital TLC detection and analysis of Xinning Tablets with salvia miltiorrhiza as the target medicinal material.

[0441] I. TLC Detection

[0442] Take 3 tablets of Xinning Tablets, grind them finely, add 20 ml of methanol, extract ultrasonically for 20 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of water, pass through a D101 macroporous adsorption resin (inner diameter 1.5 cm, column height 8 cm), elute with 80 ml of water, discard the water extract, then elute with 100 ml of 40% ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Spot the test solution on a polyamide TLC plate, use acetone - acetic acid - ammonia water (10:25:1) as the developing agent, develop, take out, and dry in air. After fumigating in ammonia vapor, examine under ultraviolet light (365 nm) to obtain the TLC chromatogram (as Figure 18 )

[0443] II. Establishment of Standard Atlas Library

[0444] Collect the TLC chromatograms of qualified salvia miltiorrhiza in Xinning Tablets. The TLC chromatograms of the qualified products include the TLC chromatograms under different experimental conditions to obtain the standard atlas library.

[0445] In this example, according to the above method for detection, collect the TLC chromatograms of salvia miltiorrhiza in Xinning Tablets with different polyamide films (Taizhou Luqiao Sijia Biochemical Plastics Factory, Zhejiang Province; Shanghai Jinsui Biotechnology Co., Ltd.), different temperatures (5.1 °C, 17.8 °C), and different relative humidities (32%, 65%, 88%).

[0446] Divide the TLC chromatograms of salvia miltiorrhiza in Xinning Tablets into 50 batches of samples for establishing the method and 50 batches of samples for verifying the method. Among them, the samples for establishing the method evenly cover various polyamide films, various temperatures, and various relative humidities.

[0447] III. Establishment of Analysis Model

[0448] 1. Select Analysis Spots

[0449] Select analysis spots according to the principle of Example 1. Along the solvent development direction, number each spot. Based on the reference medicinal material spots in the Salvia miltiorrhiza identification chromatogram of Xinning tablets and the differences in the test sample spots, combined with the experience of manual judgment for comprehensive analysis. Finally, select the relatively stable 4th, 5th, and 7th spots as analysis spots. The Rf values of these spots 4, 5, and 7 are 0.26, 0.42, and 0.78 respectively (as Figure 18 shown).

[0450] 2. Consider whether to add the origin point

[0451] The origin point of this example is not clear, so the origin point is not added for analysis.

[0452] 3. Grouping

[0453] There are a total of 3 characteristic spots in this example, and these 3 characteristic spots are divided into 1 group.

[0454] 4. Select positioning spots

[0455] Within the above-mentioned characteristic spot group, use the three characteristic spots as candidate positioning spots respectively, measure the distances between the other two characteristic spots and the positioning spot, calculate the ratio of the minimum distance to the maximum distance, and calculate the relative standard deviation of each ratio. The results are shown in the following table.

[0456] Table 3. Analysis results of the position data of different positioning spots

[0457]

[0458] The above results show that in the thin layer chromatogram of Salvia miltiorrhiza in Xinning tablets, the relative standard deviations of the three different positioning points are 0.05, 0.13, and 0.08 respectively. The relative standard deviation obtained by using the 4th spot as the positioning spot is the smallest. That is, select the 4th spot with the smallest relative standard deviation value as the positioning spot.

[0459] 5. Formulate the calculation rules for the positions of characteristic spots

[0460] Refer to the method in Example 2. In this example, three calculation methods are simultaneously compared to evaluate the positions of the 3 spots of Salvia miltiorrhiza in Xinning tablets. Calculate X1, X2, and X3 according to the above three different calculation methods. The results are shown in the following table.

[0461] Table 6. Analysis results of the position data of different calculation methods

[0462]

[0463] The above results show that in the samples of Xinning Tablets, the relative standard deviations of the three different calculation methods in the second group are 0.03, 0.11, and 0.11 respectively. It shows that the relative standard deviation of the first calculation method is the smallest, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the TLC spot location of Salvia miltiorrhiza in Xinning Tablets.

[0464] 6. Calculate the spacing ratio RPf

[0465] According to the first method above, taking the 4th spot as the positioning spot, calculate the distances L-45 and L-47 between the 4th spot and the 5th spot and the 7th spot respectively, and calculate the ratio of the minimum distance L-45 to the maximum distance L-47, denoted as the spacing ratio RPf11; taking the spacing ratio RPf11 as the evaluation index, according to the different TLC chromatograms in the standard atlas library (50 batches of samples), establish the evaluation standard range of the spacing ratio RPf11 (such as Figure 19 the black circled part in) is 0.65 - 0.75.

[0466] IV. Comparative analysis

[0467] Take the TLC chromatograms of the Salvia miltiorrhiza samples in the above 50 batches of Xinning Tablets, and calculate the corresponding spacing ratio RPf11 (such as Figure 19 the blue circled part) according to the first method determined in the above analysis model establishment steps, and compare it with the above evaluation standard range to obtain the digital TLC detection results of Salvia miltiorrhiza in Xinning Tablets.

[0468] The data results show that the RPf11 values of the Salvia miltiorrhiza samples in the 50 batches of Xinning Tablets used for verifying the method are all within the proposed range, indicating that the digital evaluation method for the TLC of Salvia miltiorrhiza in Xinning Tablets based on the RPf value has good performance.

[0469] Example 9

[0470] A digital TLC detection and analysis method for Sophora japonica in Xinning Tablets, referring to the method of Example 1, is applied to the digital TLC detection and analysis of Xinning Tablets with Sophora japonica as the index medicinal material.

[0471] I. TLC detection

[0472] Take 3 tablets of Xinning Tablets, grind them finely, add 20 ml of methanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in 5 ml of water, add it to a D101 macroporous adsorption resin column (1.5 cm * 10 cm), elute with 80 ml of water, discard the eluate, elute with 100 ml of 40% ethanol, collect the eluate, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Spot the test solution on a silica gel G plate, use ethyl acetate - glacial acetic acid - formic acid - water (10:1:1:2) as the developing agent, develop, take out, and air dry. Spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly visible, and examine under ultraviolet light (365 nm) to obtain a thin layer chromatogram (as Figure 20 ).

[0473] II. Establishment of the standard spectrum library

[0474] Collect the thin layer chromatograms of Sophora japonica in Xinning Tablets for qualified products. The thin layer chromatograms of the qualified products include those under different experimental conditions to obtain the standard spectrum library.

[0475] In this example, according to the above method for detection, collect the thin layer chromatograms of Sophora japonica in Xinning Tablets on different silica gel thin layer plates (Merck KGaA, Yantai Chemical Industry Research Institute), at different temperatures (5.2 °C, 25.2 °C), and different relative humidities (32%, 56%, 88%).

[0476] Divide the thin layer chromatograms of Sophora japonica in Xinning Tablets into 45 batches of Xinning Tablet samples for method establishment and 45 batches of Xinning Tablet samples for method verification. Among them, the samples for method establishment evenly cover various silica gel G plates, various temperatures, and various relative humidities.

[0477] III. Establishment of the analysis model

[0478] 1. Select the analysis spots

[0479] Select the analysis spots according to the principle of Example 1. Along the solvent development direction, number the origin and each spot in turn from the origin to the solvent front direction. Based on the spot conditions of the reference medicinal material for the identification chromatogram of Sophora japonica in Xinning Tablets and the differences in the spots of the test solution, and comprehensively analyze in combination with the experience of manual judgment. Finally, select the relatively stable 5th, 6th, and 7th spots as the analysis spots. The Rf values of these spots 5, 6, and 7 are 0.36, 0.40, and 0.44 respectively (as Figure 20 shown).

[0480] 2. Consider whether to add the origin

[0481] There are 3 analysis spots in this embodiment. Along the solvent development direction, starting from the origin and moving successively towards the solvent front direction, the origin and the first three spots among the analysis spots are numbered A, B, C, and D in sequence. The distances L-ac and L-bd between AC and BD are calculated, which are 1.91 and 0.45 respectively, that is, L-ac > L-bd. Then, the origin is not added as a characteristic spot for grouping.

[0482] 3. Grouping

[0483] There are 3 characteristic spots in this embodiment, and these 3 characteristic spots are divided into 1 group.

[0484] 4. Selecting positioning spots

[0485] Within the above-mentioned characteristic spot group, using the three characteristic spots as candidate positioning spots respectively, measure the distances between the other two characteristic spots and the positioning spot, calculate the ratio of the minimum distance to the maximum distance, and calculate the relative standard deviation of each ratio. The results are shown in the following table.

[0486] Table 3. Analysis results of the position data of different positioning spots

[0487]

[0488] The above results show that in the thin-layer chromatogram of Sophora japonica in Xinning tablets, the relative standard deviations of the three different positioning points are 0.09, 0.22, and 0.11 respectively. The relative standard deviation obtained with the 5th spot as the positioning spot is the smallest. That is, the 5th spot with the smallest relative standard deviation value is selected as the positioning spot.

[0489] 5. Formulating the calculation rules for the positions of characteristic spots

[0490] Referring to the method in Embodiment 2, in this embodiment, three calculation methods are simultaneously compared to evaluate the positions of the three spots of Sophora japonica in Xinning tablets. Calculate X1, X2, and X3 according to the above two different calculation methods. The results are shown in the following table.

[0491] Table 6. Analysis results of the position data of different calculation methods

[0492]

[0493] The above results indicate that in the Xinning tablet samples, the relative standard deviations of the three different calculation methods are 0.09, 0.09, and 0.22 respectively. The relative standard deviations of the first calculation method and the second calculation method are the smallest. It shows that the marking methods of these two calculation methods are least affected by various factors. Since the second calculation method (using the origin as the positioning point) marks one more spot (the origin) than the first calculation method (using the 5th spot as the positioning point), therefore, from the analysis of operation convenience, the RPf value is selected as the evaluation method for the positioning of the thin-layer chromatogram spots of Sophora japonica in Xinning tablets.

[0494] 6. Calculate the spacing ratio RPf

[0495] According to the first method described above, using the 5th spot as the positioning spot, calculate the distances L-56 and L-57 between the 5th spot and the 6th spot and the 7th spot respectively, and calculate the ratio of the minimum distance L-56 to the maximum distance L-57, denoted as the spacing ratio RPf12; taking the spacing ratio RPf12 as the evaluation index, according to the spacing ratios RPf12 of each characteristic spot group in different thin layer chromatograms in the standard chromatogram library (45 batches of samples) (such as Figure 21 the black circled part in), establish the evaluation standard range of the spacing ratio RPf12 as 0.43 - 0.64.

[0496] IV. Comparative analysis

[0497] Take the thin layer chromatograms of Sophora japonica samples in the above 50 batches of Xinning tablets, and calculate the corresponding spacing ratio RPf12 according to the first method determined in the above analysis model establishment steps (such as Figure 21 the blue circled part in), and compare it with the above evaluation standard range to obtain the digital thin layer chromatographic detection results of Sophora japonica in Xinning tablets.

[0498] The data results show that the RPf12 values of Sophora japonica samples in the 50 batches of Xinning tablets used for verification methods are all within the proposed range, indicating that the digital thin layer chromatographic evaluation method of Sophora japonica in Xinning tablets based on the RPf value has good performance.

Claims

1. A method for establishing a digital thin layer chromatography detection and analysis model, characterized in that: The following steps are involved: Establishment of standard atlas library: collecting thin layer chromatograms of qualified products of the tested Chinese medicine varieties, wherein the thin layer chromatograms of the qualified products include thin layer chromatograms under differential experimental conditions, and obtaining a standard atlas library, wherein the differential experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of Chinese medicine varieties; Establishment of the analysis model: selecting at least two representative spots in the above-mentioned thin layer chromatogram as analysis spots, adding or not adding origins to form characteristic spots, grouping the characteristic spots to obtain characteristic spot groups with three characteristic spots as a group, taking one characteristic spot in each characteristic spot group as a positioning spot, calculating the distances between the other two characteristic spots and the positioning spot, calculating the ratio between the minimum distance and the maximum distance, recorded as the spacing ratio RPf, taking the spacing ratio RPf as an evaluation index, and establishing the evaluation standard range of the spacing ratio RPf according to the spacing ratio RPf of each characteristic spot group in different thin layer chromatograms in the standard atlas library, that is, obtaining; Among them, in the analysis model establishment step, the judgment criteria for adding or not adding origin points to form characteristic spots for grouping are: In the first step, if the origin is irregular or has unclear boundaries, the origin is not added to form the feature spot. If the origin is regular and has clear boundaries, the second step is entered. In the second step, when the number of the analysis spots is 2, the origin is added as a characteristic spot for grouping; When the number of analysis spots is ≥3, the origin and the first three spots of the analysis spots are numbered A, B, C, and D in sequence from the origin to the solvent front direction along the solvent development direction, and the distances L-ac and L-bd between AC and BD are calculated respectively. When L-ac≥L-bd, the origin is not added as a characteristic spot for grouping; when L-ac<L-bd, the origin is added as a characteristic spot for grouping; In the analysis model establishment step, the positioning spot is determined by the following method: in each characteristic spot group, three characteristic spots are respectively used as candidate positioning spots, the distance between the other two characteristic spots and the positioning spot is measured, the ratio of the minimum distance to the maximum distance is calculated, the relative standard deviation of each ratio is calculated, and the characteristic spot with the smallest relative standard deviation value is selected as the positioning spot.

2. The method for establishing a digital thin layer chromatography detection and analysis model according to claim 1, characterized in that: The analysis spots meet the following requirements: (1) The analytical spot exists in both the sample chromatogram of the Chinese medicine variety and the chromatogram of the reference medicinal material or reference substance, and the positions are corresponding; (2) The analyzed spots have regular shapes, clear boundaries, and stable existence; (3) The ratio shift value of the analyzed spots is 0.05 to 0.8; (4) The analysis spots are not affected by negative interference and are specific.

3. The method for establishing a digital thin layer chromatography detection and analysis model according to claim 2, characterized in that: The analysis spot meets the following requirements: the ratio shift value of the analysis spot is 0.1 to 0.

8.

4. The method for establishing a digital thin layer chromatography detection and analysis model according to claim 3, characterized in that: The analysis spot meets the following requirements: the ratio shift value of the analysis spot is 0.2 to 0.

8.

5. The method for establishing a digital thin layer chromatography detection and analysis model according to any one of claims 1 to 4, characterized in that: In the analysis model establishment step, grouping is performed according to the following rules: (1) numbering the characteristic spots along the solvent development direction, and dividing the first three spots and the last three spots into a first group and a last group, respectively; (2) Each group has common characteristic spots with adjacent groups; (3) Except for the first or last group, the characteristic spots in the remaining groups are grouped according to the principle of close distance; (4) The grouping meets the requirements of (1) to (3) above, and the total number of groups is the smallest.

6. The method for establishing a digital thin layer chromatography detection and analysis model according to claim 5, characterized in that: The characteristic spots are numbered along the solvent development direction and grouped according to the following rules: When the total number of analysis spots is 2, add the origin or TLC plate spot as a characteristic spot to form a group; When the total number of characteristic spots is 3, these 3 characteristic spots are grouped into one group; When the total number of characteristic spots is 4, the characteristic spots numbered 1, 2, and 3 are grouped into the first group, and the characteristic spots numbered 2, 3, and 4 are grouped into the last group; When the total number of characteristic spots is 5, the characteristic spots numbered 1, 2, and 3 are grouped into the first group, and the characteristic spots numbered 3, 4, and 5 are grouped into the last group; When the total number of characteristic spots is 6, the characteristic spots numbered 1, 2, and 3 are grouped as the first group, and the characteristic spots numbered 4, 5, and 6 are grouped as the last group. The distance between the characteristic spots numbered 2 and 4 and the distance between the characteristic spots numbered 3 and 5 are calculated, which are D-24 and D-35, respectively. When D-24>D-35, the characteristic spots numbered 3, 4, and 5 are grouped as the middle group. When D-24<D-35, the characteristic spots numbered 2, 3, and 4 are grouped as the middle group. When D-24=D-35, the characteristic spots numbered 3, 4, and 5 are grouped as the middle group or the characteristic spots numbered 2, 3, and 4 are grouped as the middle group. When the total number of characteristic spots is 7, the characteristic spots numbered 1, 2, and 3 are grouped into the first group, the characteristic spots numbered 5, 6, and 7 are grouped into the last group, and the characteristic spots numbered 3, 4, and 5 are grouped into the middle group; When the total number of characteristic spots is 8, the characteristic spots numbered 1, 2, and 3 are divided into the first group, the characteristic spots numbered 6, 7, and 8 are divided into the last group, the characteristic spots numbered 3, 4, and 5 are divided into the first intermediate group, and the characteristic spots numbered 4, 5, and 6 are divided into the second intermediate group.

7. The method for establishing a digital thin layer chromatography detection and analysis model according to claim 1, characterized in that: Meet at least one of the following conditions: (1) The Chinese medicines include Chinese herbal medicine slices and / or Chinese herbal medicine preparations; (2) The differential experimental conditions also include at least one of the following conditions: at least two specifications of thin layer plates, at least two experimental dates, at least two development times, and at least two operators.

8. A digital thin layer chromatography analysis model, characterized in that: The digital thin layer chromatography detection and analysis model is established by the method for establishing the digital thin layer chromatography detection and analysis model described in any one of claims 1 to 7.

9. A digital thin layer chromatography detection and analysis method, characterized in that: Take the thin layer chromatogram of the Chinese medicine to be tested, calculate the corresponding spacing ratio RPf according to the method for establishing the digital thin layer chromatography detection and analysis model according to any one of claims 1 to 7, compare it with the evaluation standard range, and obtain the digital thin layer chromatography detection result.

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