Thin-layer chromatographic analysis model and method for Xinning tablets

Abstract

The invention discloses a thin-layer chromatographic analysis model and method for Xinning tablets, and belongs to the technical field of traditional Chinese medicine identification. According to the analysis model obtained by the establishment method, whether spots exist or not is digitally judged from double angles through an H value range of a standard hue and a color system channel judgment standard, and result deviation of manual subjective evaluation can be reduced in an objective quantitative evaluation mode.

Description

[0001] This application is a divisional application of Chinese Patent Application No. CN 202410570094X, filed on May 9, 2024, entitled "Analytical Model for Thin-Layer Chromatography, Method for Establishing the Same and its Application". The full text of the aforementioned Chinese Patent Application is incorporated herein by reference. Technical Field

[0002] This invention relates to the field of traditional Chinese medicine identification technology, and in particular to a thin-layer chromatography analysis model and method for Xinning tablets. Background Technology

[0003] Thin-layer chromatography (TLC) technology originated in the 1950s and was rapidly adopted in the 1980s. TLC is an important method for evaluating the authenticity of processed Chinese medicinal herbs and the integrity of raw materials used in prepared Chinese medicines. It boasts advantages such as ease of operation, cost-effectiveness, speed, and wide applicability, playing an irreplaceable role in the methodology system of Chinese medicine quality standards. Currently, the standard for evaluating TLC results stipulates that the test sample chromatogram should show spots of the same color at the corresponding positions as the reference herb or standard TLC chromatogram. However, in practical applications, due to the vague description of the standard and the lack of objective, quantifiable numerical values, inspectors rely on visual observation and sensory evaluation of the color and other information of the characteristic spots in the TLC chromatogram. Different inspectors have different understandings and evaluation criteria of the standard, which can easily lead to discrepancies in test conclusions. Summary of the Invention

[0004] In view of the current vague evaluation standards for thin-layer chromatography, the lack of objective and quantifiable numerical values, and the frequent discrepancies in sensory evaluations among different testing personnel, this invention provides a thin-layer chromatography analysis model and method for Xinning tablets. The analytical model obtained by this method can reduce the bias of subjective human evaluation results through objective and quantitative evaluation.

[0005] This invention discloses a thin-layer chromatography analysis model for Xinning tablets, using Panax notoginseng as an indicator herb, established through the following method:

[0006] Thin-layer chromatography detection: Take the Xinning tablet sample, grind it into a fine powder, add ether and sonicate, filter, take the residue, evaporate to dryness, add methanol and sonicate, filter, take the filtrate, evaporate the filtrate to dryness, add water to dissolve the residue, extract with water-saturated n-butanol, wash the n-butanol solution with ammonia test solution, take the n-butanol solution and evaporate to dryness, add methanol to dissolve the residue as the test solution, take 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 solvent, develop, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, take a picture, and obtain the thin-layer chromatogram;

[0007] Thin-layer chromatogram collection: collect thin-layer chromatograms of qualified and unqualified Xinning tablets. The thin-layer chromatograms of unqualified products have missing characteristic spots. The thin-layer chromatograms include those under differential experimental conditions, including at least two humidity conditions, at least two temperature conditions, and at least two batches of traditional Chinese medicine varieties. The thin-layer chromatograms of qualified Xinning tablets constitute the Xinning tablet standard chromatogram library.

[0008] Characteristic spot determination: Along the solvent spread direction, the origin and each spot are numbered sequentially from the origin to the solvent front, and the 2nd, 4th, 5th, 6th, 8th and 9th spots are designated as characteristic spots;

[0009] Establishment of a spot presence / absence judgment model: 1) Collect the H values ​​of each characteristic spot in the HSB system from the thin-layer chromatograms of all qualified products, and use the H value range of the corresponding characteristic spot in different thin-layer chromatograms as the standard H value range of the characteristic spot; 2) Collect the L channel of each characteristic spot in the CIE-Lab color space from all thin-layer chromatograms as the analysis channel, and obtain the L channel value range of the characteristic spot missing group and the L channel value range of the characteristic spot present group; 3) Determine the L channel judgment standard based on the above L channel value range of the characteristic spot missing group and the characteristic spot present group. The L channel judgment standard and the standard H value range together constitute the spot presence / absence judgment standard, which is obtained.

[0010] In some procedures for the thin-layer chromatography detection of Xinning tablets (with Panax notoginseng as the indicator herb) as described above, the preparation method of the test solution is as follows: Take 10 Xinning tablets, grind them into a fine powder, add 50 ml of ether, sonicate for 20 minutes, filter, remove the ether from the residue, add 50 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 50 ml of water to dissolve the residue; extract twice with water-saturated n-butanol, 30 ml each time, combine the n-butanol solutions, wash twice with ammonia solution, 60 ml each time, take the n-butanol solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use as the test solution.

[0011] In some procedures for the thin-layer chromatography detection of Xinning tablets (with Panax notoginseng as the indicator herb), the developing solvent is a supernatant solution of n-butanol-acetic acid-water with a volume ratio of 4:1:5.

[0012] In some procedures for the thin-layer chromatography detection of the above-mentioned sample as Xinning tablets (with Panax notoginseng as the indicator herb), the humidity conditions include relative humidity of 32%, 65%, and 88%.

[0013] In some protocols for the thin-layer chromatography detection of Xinning tablets (with Panax notoginseng as the indicator herb) as described above, the temperature conditions include 5.1±2℃ and 17.8±5℃.

[0014] In some procedures for the thin-layer chromatography detection of Xinning tablets (with Panax notoginseng as the indicator herb), the differential experimental conditions also include at least two different sizes of thin-layer plates.

[0015] In some of the above-mentioned thin-layer chromatography detection steps for Xinning tablets (with Panax notoginseng as the indicator herb), the thin-layer plate includes silica gel G thin-layer plates produced by Merck & Co., Ltd. and Yantai Chemical Industry Co., Ltd.

[0016] In some of the steps of establishing the analytical model for the above sample as Xinning tablets (with Panax notoginseng as the indicator herb), the Rf value of the second spot was 0.068±0.01, the Rf value of the fourth spot was 0.20±0.02, the Rf value of the fifth spot was 0.22±0.02, the Rf value of the sixth spot was 0.38±0.04, the Rf value of the eighth spot was 0.58±0.06, and the Rf value of the ninth spot was 0.68±0.07.

[0017] In some of these schemes, the thin-layer chromatography analysis model for Xinning tablets also includes a spacing ratio model, which is established by the following method: Along the solvent development direction, the origin and each spot are numbered sequentially from the origin towards the solvent front. Spots 2, 4, 5, 6, 8, and 9 are designated as analytical spots. Spots 2, 4, and 5 are divided into the first group, spots 6, 8, and 9 into the last group, and spots 4, 5, and 6 into the middle group. In the first group, spot 2 is used as the positioning spot; in the middle group, spot 6 is used as the positioning spot; and in the last group, spot 6 is used as the positioning spot. In the first group, the distances L-24 and L-25 between spot 2 and spots 4 and 5 are calculated, respectively. The minimum distance L-24 and the maximum distance L-25 are also calculated. The ratio between the two spots is denoted as the spacing ratio RPf4. In the middle group, the distances L-46 and L-56 between the 6th spot and the 4th and 5th spots are calculated respectively. The ratio between the minimum distance L-56 and the maximum distance L-46 is calculated and denoted as the spacing ratio RPf5. In the last group, the distances L-68 and L-69 between the 6th spot and the 8th and 9th spots are calculated respectively. The ratio between the minimum distance L-68 and the maximum distance L-69 is calculated and denoted as the spacing ratio RPf6. Using the spacing ratios RPf4, RPf5, and RPf6 as evaluation indicators, the evaluation standard range of the characteristic ratios is established based on the spacing ratios RPf4, RPf5, and RPf6 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library.

[0018] In some of these schemes, the evaluation criteria range is as follows: RPf4 is 0.81~0.89, RPf5 is 0.64~0.76, and RPf6 is 0.74~0.91.

[0019] On the other hand, the present invention also discloses a thin-layer chromatography detection and analysis method for Xinning tablets, including the following steps: taking the thin-layer chromatogram of the sample of Shuxiong tablet to be tested obtained by the same method in the above thin-layer chromatography detection steps, comparing the H value and L channel value of its characteristic spots with the standard H value range and L channel judgment standard to obtain the result of whether the spots are present or not;

[0020] Furthermore, the corresponding spacing ratios RPf4, RPf5, and RPf6 were calculated according to the method in the spacing ratio model establishment step of the thin-layer chromatography analysis model of Xinning tablets, and compared with the evaluation standard range to obtain the spacing ratio detection results;

[0021] Preferably, the standard H value range is 280-360, the L channel judgment standard is >-2, or the RPf4 is 0.81~0.89, RPf5 is 0.64~0.76, and RPf6 is 0.74~0.91.

[0022] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0023] The reagents and raw materials used in this invention are all commercially available.

[0024] The positive and progressive effects of this invention are as follows:

[0025] The present invention provides a thin-layer chromatography analysis model for Xinning tablets. By selecting channel judgment criteria from each channel of the RGB color space, HSB color space and CIE-Lab color space that can determine the presence or absence of characteristic spots, and combining them with the standard H value range in the HSB system that can reflect hue, a spot presence or absence judgment criterion is formed. The digital model can accurately determine the spots, and reduce the result deviation of human subjective evaluation by using an objective and quantitative evaluation method.

[0026] Furthermore, by combining the interval ratio judgment model and utilizing the relative interval value theory, the interval ratio RPf can be calculated based on the relative interval value to reduce the influence of different environmental conditions on thin-layer chromatography, thereby realizing digital thin-layer chromatography detection without reference standards or without reference medicinal materials. Attached Figure Description

[0027] Figure 1 The image shows a thin-layer chromatogram of the Zhongxin Ning tablets in Example 2, with Panax notoginseng as the indicator herb.

[0028] Figure 2 The H values ​​and ranges of the six characteristic spots of Panax notoginseng in the central Ning tablets of Example 2 are shown.

[0029] Figure 3The range of RGB channel color values ​​for the strong, weak, and missing spots in the thin-layer chromatogram of Panax notoginseng tablets in Example 2 is shown.

[0030] Figure 4 The HSB channel color values ​​for the strong, weak, and missing spots in the thin-layer chromatogram of Panax notoginseng tablets in Example 2 are shown.

[0031] Figure 5 The range of Lab channel color values ​​for the clearly visible (Strong), faintly visible (weak), and missing spots in the thin-layer chromatogram of Panax notoginseng tablets in Example 2.

[0032] Figure 6 The values ​​and ranges of the spacing ratio RPf3 in different thin-layer chromatograms in Example 2 are shown.

[0033] Figure 7 The values ​​and ranges of the spacing ratio RPf4 in different thin-layer chromatograms in Example 2 are shown.

[0034] Figure 8 The interval ratio RPf5 values ​​and ranges are shown in different thin-layer chromatograms in Example 2. Detailed Implementation

[0035] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0036] Example 1

[0037] A thin-layer chromatography detection and analysis method, comprising:

[0038] I. Model Establishment

[0039] 1. Establishment of a model for determining the presence or absence of spots

[0040] 1.1 Collection of thin-layer chromatograms:

[0041] Collect thin-layer chromatograms of qualified and unqualified Chinese medicine products. The thin-layer chromatograms of unqualified products have missing characteristic spots. Record the presence and absence of characteristic spots. The thin-layer chromatograms include those under differential experimental conditions. The differential experimental conditions include at least two humidity conditions, at least two temperature conditions, at least two batches of Chinese medicine products, at least two specifications of thin-layer plates, at least two experimental dates, at least two development times, and at least two operators.

[0042] The selection of the above-mentioned characteristic spots should meet the following requirements:

[0043] A: The characteristic spots are present in both the chromatogram of the sample of this Chinese herbal medicine and the chromatogram of the reference herb or reference standard, and their positions correspond;

[0044] B: The characteristic spots are regular in shape, have clear boundaries, and exist stably;

[0045] C: The specific displacement value of the characteristic spot is 0.05 to 0.8, preferably 0.1 to 0.8, more preferably 0.2 to 0.8;

[0046] D: The characteristic spots are not affected by negative light and are specific.

[0047] After selecting the characteristic spot, the location of the characteristic spot is determined by the thin-layer chromatography obtained by using a reference standard or a reference medicinal material under the same conditions.

[0048] Alternatively, the characteristic spot can be determined by a preset Rf value range. When a spot falls within the preset Rf value range, it is considered a characteristic spot of this range. The preset Rf value range is defined by the Rf value range of the corresponding characteristic spot in the thin-layer chromatograms of several qualified samples. If a method without a reference standard or reference medicinal material is considered for detection and analysis, the Rf value can be used for localization.

[0049] In this embodiment, the method for determining whether the characteristic spots are missing is as follows: the thin-layer chromatogram is distributed to 5 inspectors, who independently observe whether the characteristic spots exist.

[0050] If all five inspectors determine that the characteristic spot is present, the result for that characteristic spot is considered clearly visible. If one inspector's determination differs from the others (e.g., four inspectors determine the characteristic spot is present and one determines it is not present, or four inspectors determine it is not present and one determines it is present), the result for that characteristic spot is considered faintly visible. If all five inspectors determine that the characteristic spot is completely absent, the result for that characteristic spot is considered missing.

[0051] If the final determination result is that the feature spot is clearly visible or faintly visible, it is considered to exist; if the final determination result is that the feature spot is missing, it is considered to be missing.

[0052] 1.2 Determination of Standard Hue:

[0053] Collect the H values ​​of each characteristic spot in the thin-layer chromatogram of all qualified products in the HSB system, and use the range of H values ​​of the corresponding characteristic spots in different thin-layer chromatograms as the standard H value range of the characteristic spot.

[0054] Understandably, the standard H value range for each characteristic spot can be defined based on the H value range of the corresponding characteristic spots in different thin-layer chromatograms. Therefore, if there are 3 characteristic spots in a thin-layer chromatogram, there are 3 corresponding standard H value ranges.

[0055] However, considering that thin-layer chromatograms are typically examined under the same conditions, such as under sunlight, at 254 nm, or at 365 nm, characteristic spots can be grouped according to hue. Spots exhibiting the same hue are grouped together for analysis, while spots exhibiting different hues are divided into different hue groups. Each characteristic spot within the same hue group shares a common standard H-value range, which is the range of H-values ​​for all characteristic spots in that group in the thin-layer chromatogram of a qualified sample. This method offers advantages such as ease of operation and high processing efficiency.

[0056] In this embodiment, the method for determining whether the hues are the same is as follows: the thin-layer chromatogram is distributed to at least 5 inspectors, who independently observe and judge the hues of the characteristic spots. When all inspectors judge that the hues are the same, the hues are determined to be the same. When at least one inspector's judgment is inconsistent with that of the other inspectors, the hues are determined to be different, and the groups are regrouped until all inspectors judge the characteristic spots of the same hue group to be the same.

[0057] 1.3 Determination of Color System and Channels:

[0058] The channel values ​​of each characteristic spot in all thin-layer chromatograms were collected in the R, G, and B channels of the RGB color space, the H, S, and B channels of the HSB color space, and the L, a, and b channels of the CIE-Lab color space. All thin-layer chromatograms were randomly divided into a test group and a validation group. The channel value ranges of missing characteristic spots and the channel value ranges of present characteristic spots in the test group were obtained. The minimum value of the channel value range of present characteristic spots in the test group was used as the analysis threshold. The channel values ​​of characteristic spots in the validation group were compared with the analysis threshold. When the channel value of a characteristic spot was greater than the analysis threshold, it was determined that the characteristic spot was present. This determination result was compared and verified with the records of missing and present characteristic spots. The channel with the higher determination accuracy was used as the analysis channel of the characteristic spot, and the analysis threshold of the analysis channel was used as the channel determination criterion for the characteristic spot.

[0059] Understandably, the above channel values ​​have been calibrated. The calibration method is to subtract the background value of the corresponding channel from the extracted channel value to eliminate background interference.

[0060] Specifically, the determination accuracy is calculated by dividing the number of spots in the verification group that match the determination results and the records of missing and present feature spots by the total number of feature spots in the verification group.

[0061] 1.4 Establish a model for determining the presence or absence of spots:

[0062] The channel judgment criteria and the standard H value range together constitute the spot presence or absence judgment criteria, which is obtained.

[0063] 2. Establishment of the Spacing Ratio Judgment Model

[0064] 2.1 Selecting analytical spots

[0065] Select at least two representative spots from the thin-layer chromatogram of the above-mentioned qualified products as analytical spots, and these analytical spots shall meet the following requirements:

[0066] (1) The spots were found in both the chromatogram of the sample of the Chinese herbal medicine and the chromatogram of the reference herb or reference substance, and their positions corresponded.

[0067] It is understandable that although reference standards or reference medicinal materials are not necessarily used in the establishment of the standard spectral library and in subsequent analysis and evaluation, considering the spots of index reference standards or reference medicinal materials in the establishment of the standard spectral library and selecting characteristic spots based on these can better reflect the quality of the Chinese medicine varieties being analyzed and tested.

[0068] (2) The spots are regular in shape, have clear boundaries and are stable.

[0069] This standard is consistent with the requirements for inspecting spots in the field, which can improve the stability of the method.

[0070] (3) The specific displacement value of the analytical spot is 0.05 to 0.8, preferably 0.1 to 0.8, and more preferably 0.2 to 0.8.

[0071] By limiting the specific shift value of the analytical spots to 0.05 to 0.8, preferably 0.1 to 0.8, and more preferably 0.2 to 0.8, the influence of different conditions on the specific shift value can be further reduced, thereby improving the stability of this method.

[0072] (4) The analysis of spots is not affected by negative interference and has specificity.

[0073] 2.2 Consider whether to add the origin.

[0074] The criteria for determining whether to include the origin to form characteristic spots for grouping are as follows:

[0075] Step 1 Judgment: When there are 2 analytical spots, the origin is added as a characteristic spot for grouping. If the origin spot is not clear and difficult to identify, the thin-layer plate spot is used as the characteristic spot.

[0076] The second step of judgment: If the number of analyzed spots is ≥3, and the original spot is not clear and difficult to identify, then the original spot is not added to form the characteristic spot. If the original spot is clear and identifiable, then proceed to the third step of judgment.

[0077] The second step involves determining the origin and the first three analytical spots along the solvent spreading direction, sequentially numbering them A, B, C, and D from the origin towards the solvent front. The distances between AC and BD are L-ac and L-bd. If L-ac ≥ L-bd, the origin is not included as a characteristic spot in the group; if L-ac < L-bd, the origin is included as a characteristic spot in the group.

[0078] 2.3 Grouping

[0079] The feature spots are grouped into groups of three, according to the following grouping rules:

[0080] (1) The characteristic spots are numbered along the solvent spreading direction, and the first three spots and the last three spots are divided into the first group and the last group, respectively;

[0081] (2) Each group shares common characteristic spots with its adjacent groups;

[0082] (3) Except for the first or last group, the feature spots in the remaining groups are grouped according to the principle of proximity;

[0083] (4) The grouping meets the requirements of (1)-(3) above, and the total number of groups is the smallest.

[0084] The following are examples of grouping methods when the number of feature spots is different.

[0085] The characteristic spots are numbered along the solvent spread direction and grouped according to the following rules:

[0086] When the total number of spots analyzed is 2, which is less than three spots, the origin or a thin-layer plate spot must be added as a characteristic spot (if the origin is difficult to identify, it can be manually identified by the spotting location and then added as a thin-layer plate spot) to form a group.

[0087] When the total number of feature spots is 3, these 3 feature spots are grouped together.

[0088] When the total number of feature spots is 4, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 2, 3, and 4 are divided into the last group.

[0089] When the total number of feature spots is 5, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 3, 4, and 5 are divided into the last group.

[0090] When the total number of feature spots is 6, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 4, 5, and 6 are divided into the last group. The distance between feature spots numbered 2 and 4 and the distance between feature spots numbered 3 and 5 are calculated, which are D-24 and D-35 respectively. When D-24 > D-35, feature spots numbered 3, 4, and 5 are selected as the middle group. When D-24 < D-35, feature spots numbered 2, 3, and 4 are selected as the middle group. When D-24 = D-35, feature spots numbered 3, 4, and 5 or feature spots numbered 2, 3, and 4 can be selected as the middle group.

[0091] When the total number of feature spots is 7, feature spots numbered 1, 2, and 3 are divided into the first group, feature spots numbered 5, 6, and 7 are divided into the last group, and feature spots numbered 3, 4, and 5 are divided into the middle group.

[0092] When the total number of feature spots is 8, feature spots numbered 1, 2, and 3 are divided into the first group, feature spots numbered 6, 7, and 8 are divided into the last group, feature spots numbered 3, 4, and 5 are divided into the first middle group, and feature spots numbered 4, 5, and 6 are divided into the second middle group.

[0093] 2.4 Selecting and Positioning Spots

[0094] After grouping, at least one feature spot group is obtained, and one feature spot in each feature spot group is selected as the localization spot. The method for selecting the localization spot is as follows: within each feature spot group, three feature spots are selected as candidate localization spots, the distances between the other two feature spots and the localization 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 feature spot with the smallest relative standard deviation value is selected as the localization spot.

[0095] 2.5 Calculate the spacing ratio RPf

[0096] In each group of feature spots, the distance between the other two feature spots and the localized spot is calculated, and the ratio between the minimum distance and the maximum distance is calculated and denoted as the spacing ratio RPf. The spacing ratio RPf is used as the evaluation index.

[0097] Understandably, each set of feature spots can yield a corresponding spacing ratio RPf. If there are two sets of feature spots, then the corresponding spacing ratios RPf1 and RPf2 can be obtained respectively; if there are three sets of feature spots, then the corresponding spacing ratios RPf1, RPf2 and RPf3 can be obtained respectively; and so on.

[0098] 2.6 Establish the scope of evaluation criteria

[0099] Based on the spacing ratio RPf of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library composed of the qualified products, the evaluation standard range of the spacing ratio RPf is established, and thus obtained.

[0100] Understandably, the evaluation criteria range for the above-mentioned interval ratio RPf is defined by the values ​​that may be obtained under different experimental conditions of a large sample size of qualified Chinese medicine varieties.

[0101] The specific requirements depend on the testing requirements and the data from different Chinese medicine varieties.

[0102] II. Analytical Methods

[0103] 1. Determining the presence or absence of spots

[0104] Take the thin-layer chromatogram of the Chinese herbal medicine to be tested, collect the H value and analytical channel value of each characteristic spot in the HSB system, substitute them into the spot presence or absence judgment model obtained above, and compare them with the spot presence or absence judgment standard. If the H value falls into the standard H value and the channel value of the characteristic spot is greater than the channel judgment standard, then the characteristic spot is judged to be present.

[0105] 2. Spacing ratio evaluation

[0106] Take the thin-layer chromatogram of the Chinese herbal medicine to be tested, calculate the corresponding interval ratio RPf according to the above-mentioned method for establishing the interval ratio judgment model, and compare it with the evaluation standard range to obtain the interval ratio evaluation result.

[0107] Example 2

[0108] A thin-layer chromatography method for detecting and analyzing Panax notoginseng in Xinning tablets, referring to the method in Example 1, is applied to the thin-layer chromatography detection and analysis of Panax notoginseng in Xinning tablets.

[0109] I. Thin-layer chromatography detection

[0110] Take 10 tablets of Xinning, remove the coating, grind into a fine powder, add 50ml of ether, sonicate for 20 minutes, filter, evaporate the ether from the residue, add 50ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 50ml of water. Extract twice with water-saturated n-butanol, 30ml each time, combine the n-butanol extracts, wash twice with ammonia solution, 60ml each time, take the n-butanol extract, evaporate to dryness, and dissolve the residue in 1ml of methanol to obtain the test solution. Spot the test solution onto a silica gel G thin-layer plate, using the upper layer of n-butanol-acetic acid-water (4:1:5) as the developing solvent, develop, remove, and air dry. Spray with 10% sulfuric acid ethanol, heat at 105℃ until the spots are clearly visible, and examine under sunlight to obtain the thin-layer chromatogram (e.g., ...). Figure 1 ).

[0111] II. Collection of Thin-Layer Chromatograms

[0112] Thin-layer chromatograms of qualified and unqualified Panax notoginseng tablets were collected. The unqualified products showed missing characteristic spots in their thin-layer chromatograms, and the presence and absence of characteristic spots were recorded. The thin-layer chromatograms of the qualified products were used to form a standard chromatogram library. The thin-layer chromatograms included those obtained under different experimental conditions.

[0113] Referring to the judgment criteria of Example 1, the thin-layer chromatograms were distributed to 5 inspectors who independently observed the presence of characteristic spots and determined the final results according to the criteria of Example 1.

[0114] In this embodiment, the thin-layer chromatograms of Panax notoginseng tablets in Xinning tablets were collected using the above method at different silica gel thin-layer chromatography plates (Merck Corporation and Yantai Chemical Industry Research Institute), different temperatures (5.1℃, 17.8℃), and different relative humidity (32%, 65%, 88%).

[0115] The thin-layer chromatograms of Panax notoginseng in Xinning tablets were divided into 29 batches of Xinning tablets samples used for establishing the method (test group) and 48 batches of Panax notoginseng samples used for validating the method (validation group). The samples used for establishing the method uniformly covered various silica gel G plates, various temperatures and various relative humidity.

[0116] III. Establishment of Analytical Model

[0117] 1. Establishment of a model for determining the presence or absence of spots

[0118] 1.1 Selection of Feature Spots

[0119] Based on the selection principle of characteristic spots in Example 1, select Figure 1 Spots 2, 4, 5, 6, 8, and 9 are characteristic spots, with Rf values ​​of 0.068, 0.20, 0.22, 0.38, 0.58, and 0.68 for spots 2, 4, 5, 6, 8, and 9, respectively.

[0120] 1.2 Extraction of color parameters for feature spots

[0121] The values ​​of the R, G, and B channels of the thin-layer chromatography characteristic spots in the RGB color space, the H, S, and B channels in the HSB color space, and the L, a, and b channels in the CIE-Lab color space were read using TLC Measure software and Photoshop software.

[0122] 1.3 Hue Determination

[0123] In color theory, a color wheel is a circular diagram of a rainbow, dividing hues into 12 hues according to the periodicity of colors. Each hue represents a basic color (such as red, green, blue, etc.) and a mixture of other colors. Hue (H) is a fundamental attribute of color, measuring its position on the color wheel and used to characterize its hue. The H value is a parameter describing the hue of a color, used to measure its position on the color wheel. H values ​​are usually expressed in degrees, ranging from 0 to 360 degrees. For example, 0 degrees represents red, 120 degrees represents green, and 240 degrees represents blue. Therefore, the background color of characteristic spots in thin-layer chromatography can be determined using H values, achieving an accurate description of the spot colors.

[0124] The H values ​​of all characteristic spots of Panax notoginseng in Xinning tablets were collected using TLC Measure software. The minimum and maximum values ​​were set as the range of values, such as... Figure 2 As shown in the figure, the horizontal axis represents 6 characteristic spots, and the vertical axis represents the H value. As described in Example 1, considering that the characteristic spots of Panax notoginseng in these three Xinning tablets were all examined at 254 nm, and the colors of the three characteristic spots showed the same hue, the common H value range of each characteristic spot in the thin-layer chromatography of Panax notoginseng in Xinning tablets was finally determined to be 280-360.

[0125] 1.4 Background Calibration

[0126] In each color system, the background value of each channel is extracted, and the channel color values ​​are calibrated by subtracting the background value.

[0127] 1.5 Initial Screening of Color System and Channels

[0128] The values ​​of characteristic spots in various color systems and channels were analyzed. All thin-layer chromatograms were randomly divided into a test group (29 batches) and a validation group (48 batches). The range of channel values ​​for missing characteristic spots in the test group and the range of channel values ​​for present characteristic spots in the test group were obtained. The minimum value of the channel value range for present characteristic spots in the test group was used as the analysis threshold. The channel values ​​of characteristic spots in the validation group were compared with the analysis threshold. When the channel value of a characteristic spot was greater than the analysis threshold, it was determined that the characteristic spot was present. This determination result was compared and verified with the records of missing and present characteristic spots. The channel with the higher determination accuracy was used as the analysis channel for the characteristic spot, and the analysis threshold of the analysis channel was used as the channel determination criterion for the characteristic spot.

[0129] The test group (29 batches in total) was divided into three groups according to the above method: clearly visible (strong spots), faintly visible (weak spots), and missing spots. As mentioned above, since the Panax notoginseng characteristic spots in the six Xinning tablets were all examined under sunlight, and the six characteristic spots showed the same color hue, the channel values ​​of the six characteristic spots were analyzed together without distinguishing between different characteristic spots. However, if it is confirmed that the characteristic spots to be analyzed have different hues, they need to be analyzed separately.

[0130] Using the group classification of the three data sets as the x-axis and the calibrated channel values ​​as the y-axis, a box plot is drawn. In the figure, the midline of the box plot represents the median value of the group, and the upper and lower quartiles are the upper and lower quartiles, respectively. If outliers are identified according to the Turkey's test standard, these data are removed. The resulting box plot is shown below. Figure 3-5 As shown.

[0131] Figure 3 The results are in the RGB color space system, where A, B, and C are the results for the R, G, and B channels, respectively. It can be clearly seen from the figure that the calibrated B channel has the best effect in distinguishing between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Panax notoginseng in Xinning tablets. Figure 3 In the C and B channels, the minimum channel value of the faintly visible spots is 2. This value is used as the analysis threshold for the B channel. That is, in the RGB color space system, the B value of the corrected Panax notoginseng feature spots in Xinning tablets is defined as having spots if it is greater than 2, and having spots less than or equal to 2 if it is missing.

[0132] Figure 4 The results are for the HSB color space system, where A, B, and C are the H, S, and B channel results, respectively. It can be clearly seen from the figure that the calibrated S channel has the best effect in distinguishing between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Panax notoginseng in Xinning tablets. Figure 4 In the S channel (B), the minimum channel value of the faintly visible spots is -0.3. This is used as the analysis threshold for the S channel. That is, in the HSB color space system, the S value of the Panax notoginseng characteristic spots in Xinning tablets after correction is greater than -0.3 to indicate the presence of spots, and less than or equal to -0.3 to indicate missing spots.

[0133] Figure 5 The results are from the CIE-Lab color space system, where A, B, and C are the results for channels L, a, and b, respectively. It can be clearly seen from the figure that the calibrated L channel has the best effect in distinguishing between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Panax notoginseng in Xinning tablets. Figure 5In the A and L channels, the maximum channel value of the faintly visible spots is -2. This value is used as the analysis threshold for the L channel. That is, in the CIE-Lab color space system, the L value of the Panax notoginseng characteristic spots in Xinning tablets after correction is less than -2 to indicate the presence of spots, and greater than or equal to -2 to indicate missing spots.

[0134] 1.7 Objective Validation of the Method and Further Screening of the Color System and Channels

[0135] The presence or absence of feature spots is determined according to the analysis threshold and judgment method obtained from the above test group. The judgment accuracy is calculated by dividing the number of spots in the verification group whose judgment results match the records of missing and present feature spots by the total number of feature spots in the verification group. The judgment accuracy is obtained by filtering the selected channels in each color system.

[0136] In this embodiment, 29 batches of Xining tablets were used to establish the method, with a total of 174 thin-layer chromatographic characteristic spots; 48 batches of Xining tablets were used to validate the method, with a total of 288 thin-layer chromatographic characteristic spots.

[0137] The data from 288 characteristic spots in the thin-layer chromatography verification sample of Panax notoginseng in Xinning tablets showed that, in the B channel of the RGB color system, 3 missing spots fell within the range of faintly visible spots, 8 faintly visible spots fell within the range of missing spots, and 1 clearly visible spot was in the area of ​​missing spots. A total of 12 characteristic spots were incorrectly identified, and 98 characteristic spots were correctly identified, with an accuracy rate of 276 / 288, or 95.83%.

[0138] In the B channel of the HSB color system, 5 missing spots fall within the range of faintly visible spots, and 6 faintly visible spots fall within the range of missing spots. All 277 feature spots were correctly identified, with an accuracy rate of 277 / 288, or 96.21%.

[0139] In the L channel of the CIE-Lab color system, both clearly visible spots and missing spots are within the specified range. There are 6 misjudged spots and 282 correctly judged feature spots, with an accuracy rate of 282 / 288, or 98.06%.

[0140] Therefore, the L channel of the CIE-Lab system, which has the highest accuracy, was selected to determine the color of Panax notoginseng in Xinning tablets.

[0141] The proposed digital standard for the thin-layer chromatography color of Panax notoginseng in Xinning tablets is as follows: In the CIE-Lab color system, the H value of the 6 characteristic spots should be in the range of 280-360. Using the thin-layer background as a blank to correct the characteristic spots, the L value of the 6 characteristic spots in the CIE-Lab system should be less than -2.

[0142] 2. Establishment of the Spacing Ratio Judgment Model

[0143] 1. Select and analyze spots

[0144] Following the principles of Example 1, analytical spots were selected. Along the solvent development direction, the origin and each spot were numbered sequentially from the origin towards the solvent front. Based on the characteristics of the reference herb spots in the identification chromatography of Panax notoginseng tablets, the differences in the test sample spots, and combined with experience from manual judgment, the relatively stable spots 2, 4, 5, 6, 8, and 9 were ultimately selected as analytical spots. The Rf values ​​of these spots 2, 4, 5, 6, 8, and 9 were 0.068, 0.20, 0.22, 0.38, 0.58, and 0.68, respectively (e.g., ...). Figure 1 (As shown).

[0145] 2. Consider whether to add the origin.

[0146] In this embodiment, there are 6 analytical spots. Along the solvent spread direction, the origin and the first three analytical spots are numbered A, B, C, and D (corresponding to the 1st, 2nd, 4th, and 5th spots) sequentially from the origin to the solvent front. The distances L-ac (L14) and L-bd (L25) between AC and BD are calculated respectively. Since L-ac > L-bd, the origin is not included as a feature spot for grouping.

[0147] 3. Grouping

[0148] This embodiment has a total of 6 characteristic spots, which 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. The distances between spots 4 and 6 and between spots 5 and 8 are calculated to be D-46 and D-58, respectively. Since D-46 < D-58, spots 4, 5, and 6 are divided into the third group.

[0149] 4. Select and locate spots

[0150] Within the aforementioned feature spot group, three feature spots were selected as candidate positioning spots. The distances between the other two feature spots and the positioning spots 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 table below.

[0151] Table 1. Analysis results of location data of different spots in the first group.

[0152]

[0153] The results show that in the thin-layer chromatogram of Panax notoginseng in Xinning tablets, the relative standard deviations of the three different positioning points are 0.02, 0.16 and 0.14, respectively. The second spot has the smallest relative standard deviation, so the second spot with the smallest relative standard deviation is selected as the positioning spot.

[0154] Table 2. Analysis results of location data of different spots in the second group.

[0155]

[0156] The results above show that in the thin-layer chromatogram of Panax notoginseng in Xinning tablets, the relative standard deviations of the 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. Therefore, the 6th spot with the smallest relative standard deviation value is selected as the positioning spot.

[0157] Table 3. Analysis results of location data of different spots in the third group.

[0158]

[0159] The results show that in the thin-layer chromatogram of Panax notoginseng in Xinning tablets, the relative standard deviations of the three different positioning points are 0.28, 0.34 and 0.05, respectively. The relative standard deviation obtained by using the 6th spot as the positioning spot is the smallest. Therefore, the 6th spot with the smallest relative standard deviation value is selected as the positioning spot.

[0160] 5. Establishment of calculation rules for the location of characteristic spots

[0161] Referring to the method in Example 2, this example simultaneously compares three calculation methods to evaluate the location of six Panax notoginseng spots in Xinning tablets. X1, X2, and X3 were calculated according to the two different calculation methods described above, and the results are shown in the table below.

[0162] Table 4. Analysis results of location data using different calculation methods in the first group.

[0163]

[0164] The above results show that the relative standard deviations of the two different calculation methods in the Panax notoginseng samples of Xinning tablets are 0.02, 0.12 and 0.16, respectively. The first calculation method has the smallest relative standard deviation, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the localization of Panax notoginseng spots in thin-layer chromatography in Xinning tablets.

[0165] Table 5. Analysis results of location data using different calculation methods in the second group.

[0166]

[0167] The above results show that the relative standard deviations of the three different calculation methods in the second group of Xinning tablets were 0.04, 0.07 and 0.11, respectively. The first calculation method has the smallest relative standard deviation, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the localization of Panax notoginseng spots in Xinning tablets using thin-layer chromatography.

[0168] Table 6. Analysis Results of Location Data Using Different Calculation Methods in the Third Group

[0169]

[0170] The above results show that the relative standard deviations of the three different calculation methods in the second group of Xinning tablets were 0.05, 0.14 and 0.34, respectively. The first calculation method has the smallest relative standard deviation, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the localization of Panax notoginseng spots in Xinning tablets using thin-layer chromatography.

[0171] 6. Calculate the spacing ratio RPf

[0172] Following the first method described above, in the first group, the second spot is used as the positioning spot. The distances L-24 and L-25 between the second spot and the fourth and fifth spots are calculated, respectively. The ratio between the minimum distance L-24 and the maximum distance L-25 is calculated and denoted as the spacing ratio RPf4. In the second group, the sixth spot is used as the positioning spot. The distances L-68 and L-69 between the sixth spot and the eighth and ninth spots are calculated, respectively. The ratio between the minimum distance L-68 and the maximum distance L-69 is calculated. The ratio between 69 and 69 is denoted as the spacing ratio RPf5; in the third group, the 6th spot is used as the positioning spot, and the distances L-65 and L-64 between the 6th spot and the 5th and 4th spots are calculated respectively. The ratio between the minimum distance L-65 and the maximum distance L-64 is calculated and denoted as the spacing ratio RPf6; using the spacing ratios RPf4, RPf5, and RPf6 as evaluation indicators, based on the spacing ratio RPf4 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library (29 batches of samples) (e.g., Figure 6 The black circle in the middle), the evaluation criteria for the spacing ratio RPf4 is set in the range of 0.81~0.89; the spacing ratio RPf5 of each feature spot group (such as Figure 7 (The black circle in the middle), the evaluation criteria for the spacing ratio RPf5 is established in the range of 0.64~0.76; the spacing ratio RPf6 of each feature spot group (such as...) Figure 8 (The black circle in the middle) The evaluation criteria for the spacing ratio RPf6 is set in the range of 0.74 to 0.91.

[0173] IV. Comparative Analysis

[0174] 1. Determining the presence or absence of spots

[0175] The thin-layer chromatograms of 48 batches of Xinning tablets were used for the above verification method. The H values ​​and analytical channel values ​​of each characteristic spot in the HSB system were collected and substituted into the spot presence and absence judgment model obtained above. The results showed that in the B channel of the RGB color system, 3 missing spots fell within the range of faintly visible spots, and 3 faintly visible spots fell within the range of missing spots. A total of 75 characteristic spots were correctly judged, and the judgment accuracy rate was 92.59%.

[0176] 2. Spacing ratio evaluation

[0177] Thin-layer chromatograms of Panax notoginseng samples from the above 48 batches of Xinning tablets were obtained, and the corresponding spacing ratios RPf4, RPf5, and RPf6 were calculated according to the first method determined in the analytical model establishment steps (e.g., ...). Figure 6 , Figure 7 , Figure 8 The results of digital thin-layer chromatography detection of Panax notoginseng in Xinning tablets were obtained by comparing the blue circled area with the above evaluation standard range.

[0178] The data results show that the RPf8, RPf9, and RPf10 values ​​of Panax notoginseng samples in Xinning tablets used for the validation method were all within the intended range, indicating that the thin-layer chromatography digital evaluation method for Panax notoginseng in Xinning tablets based on RPf values ​​has good performance.

Claims

1. A thin-layer chromatographic analysis model for Xinning tablets, characterized in that, Using Panax notoginseng as an indicator herb, the following method was used to establish: Thin-layer chromatography detection: Take the Xinning tablet sample, grind it into a fine powder, add ether and sonicate, filter, take the residue, evaporate to dryness, add methanol and sonicate, filter, take the filtrate, evaporate the filtrate to dryness, add water to dissolve the residue, extract with water-saturated n-butanol, wash the n-butanol solution with ammonia test solution, take the n-butanol solution and evaporate to dryness, add methanol to dissolve the residue as the test solution, take 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 solvent, develop, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, take a picture, and obtain the thin-layer chromatogram; Thin-layer chromatogram collection: collect thin-layer chromatograms of qualified and unqualified Xinning tablets. The thin-layer chromatograms of unqualified products have missing characteristic spots. The thin-layer chromatograms include those under differential experimental conditions, including at least two humidity conditions, at least two temperature conditions, and at least two batches of traditional Chinese medicine varieties. The thin-layer chromatograms of qualified Xinning tablets constitute the Xinning tablet standard chromatogram library. Characteristic spot determination: Along the solvent spread direction, the origin and each spot are numbered sequentially from the origin to the solvent front, and the 2nd, 4th, 5th, 6th, 8th and 9th spots are designated as characteristic spots; Establishment of a spot presence / absence judgment model: 1) Collect the H values ​​of each characteristic spot in the HSB system from the thin-layer chromatograms of all qualified products, and use the H value range of the corresponding characteristic spot in different thin-layer chromatograms as the standard H value range of the characteristic spot; 2) Collect the L channel of each characteristic spot in the CIE-Lab color space from all thin-layer chromatograms as the analysis channel, and obtain the L channel value range of the characteristic spot missing group and the L channel value range of the characteristic spot present group; 3) Determine the L channel judgment standard based on the above L channel value range of the characteristic spot missing group and the characteristic spot present group. The L channel judgment standard and the standard H value range together constitute the spot presence / absence judgment standard, which is obtained.

2. The thin-layer chromatography analysis model for Xinning tablets as described in claim 1, characterized in that, In the thin-layer chromatography detection step, the preparation method of the test solution is as follows: Take 10 tablets of Xinning tablets, grind them into a fine powder, add 50 ml of ether, sonicate for 20 minutes, filter, remove the ether from the residue, add 50 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 50 ml of water to dissolve the residue; extract twice with water-saturated n-butanol, 30 ml each time, combine the n-butanol solutions, wash twice with ammonia solution, 60 ml each time, take the n-butanol solution, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use as the test solution.

3. The thin-layer chromatography analysis model for Xinning tablets as described in claim 1, characterized in that, The thin-layer chromatography detection meets one or more of the following conditions: (1) The developing solvent is the upper layer solution of n-butanol-acetic acid-water with a volume ratio of 4:1:5; (2) The humidity conditions include relative humidity of 32%, 65% and 88%; (3) The temperature conditions include 5.1±2℃ and 17.8±5℃; (4) The differential experimental conditions also include at least two different sizes of thin-layer plates; (5) The thin-layer plate includes the silicone G thin-layer plate produced by Merck AG and Yantai Chemical Industry.

4. The thin-layer chromatography analysis model for Xinning tablets as described in claim 1, characterized in that, The Rf value of the second spot was 0.068±0.01, the Rf value of the fourth spot was 0.20±0.02, the Rf value of the fifth spot was 0.22±0.02, the Rf value of the sixth spot was 0.38±0.04, the Rf value of the eighth spot was 0.58±0.06, and the Rf value of the ninth spot was 0.68±0.

07.

5. The thin-layer chromatography analysis model for Xinning tablets according to any one of claims 1-4, characterized in that, The system also includes a spacing ratio model, which is established as follows: Along the solvent spread direction, the origin and each spot are numbered sequentially from the origin towards the solvent front. Spots 2, 4, 5, 6, 8, and 9 are designated as analytical spots. Spots 2, 4, and 5 are divided into the first group; spots 6, 8, and 9 are divided into the last group; and spots 4, 5, and 6 are divided into the middle group. In the first group, spot 2 is used as the positioning spot; in the middle group, spot 6 is used as the positioning spot; and in the last group, spot 6 is used as the positioning spot. In the first group, the distances L-24 and L-25 between spot 2 and spots 4 and 5 are calculated, respectively. The ratio between the minimum distance L-24 and the maximum distance L-25 is calculated and denoted as the spacing. In the intermediate group, the distances L-46 and L-56 between the 6th spot and the 4th and 5th spots, respectively, are calculated. The ratio between the minimum distance L-56 and the maximum distance L-46 is calculated and denoted as the spacing ratio RPf5. In the final group, the distances L-68 and L-69 between the 6th spot and the 8th and 9th spots, respectively, are calculated. The ratio between the minimum distance L-68 and the maximum distance L-69 is calculated and denoted as the spacing ratio RPf6. Using the spacing ratios RPf4, RPf5, and RPf6 as evaluation indicators, an evaluation standard range for the characteristic ratios is established based on the spacing ratios RPf4, RPf5, and RPf6 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library.

6. The thin-layer chromatography analysis model for Xinning tablets as described in claim 5, characterized in that, The evaluation criteria range is as follows: RPf4 is 0.81~0.89, RPf5 is 0.64~0.76, and RPf6 is 0.74~0.

91.

7. A thin-layer chromatography detection and analysis method for Xinning tablets, characterized in that, The method includes the following steps: taking the thin-layer chromatogram of the sample of the chest-soothing tablet to be tested, obtained by the same method in the thin-layer chromatography detection step according to any one of claims 1-6, and comparing the H value and L channel value of its characteristic spots with the standard H value range and L channel judgment standard to obtain the result of whether the spots are present or not.

8. The thin-layer chromatography detection and analysis method for Xinning tablets as described in claim 7, characterized in that, The corresponding spacing ratios RPf4, RPf5, and RPf6 are calculated according to the method in the spacing ratio model establishment step of the thin-layer chromatography analysis model of Xinning tablets as described in claim 4, and compared with the evaluation standard range to obtain the spacing ratio detection results.

9. The thin-layer chromatography detection and analysis method for Xinning tablets as described in claim 7, characterized in that, The standard H value range is 280-360, and the L channel judgment standard is >-2.

10. The thin-layer chromatography detection and analysis method for Xinning tablets as described in claim 7, characterized in that, The corresponding spacing ratios RPf4, RPf5, and RPf6 are calculated according to the method in the spacing ratio model establishment step of the thin-layer chromatography analysis model of Xinning tablets as described in claim 4, and compared with the evaluation standard range to obtain the spacing ratio detection results; and the evaluation standard range is: RPf4 is 0.81~0.89, RPf5 is 0.64~0.76, and RPf6 is 0.74~0.91.

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