Thin-layer detection method for Semen Lablab Album traditional Chinese medicine formula granules and its application in the identification of counterfeits

Abstract

The present application relates to a thin-layer detection method for Semen Lablab Album traditional Chinese medicine formula granules and its application in the identification of counterfeits. The thin-layer detection method for Semen Lablab Album traditional Chinese medicine formula granules comprises the following steps: extracting Semen Lablab Album traditional Chinese medicine formula granules with an aqueous methanol solution having a volume concentration of 65% to 75%, evaporating the obtained extract to dryness, dissolving the solid obtained by evaporation in water, extracting with ethyl acetate, evaporating the obtained ethyl acetate phase to dryness, dissolving the solid obtained by evaporation in methanol to prepare a test solution; preparing a solution of the control crude drug; preparing a reference substance solution by taking chikusetsusaponin IVa reference substance; spotting the test solution, the solution of the control crude drug and the reference substance solution on the same thin-layer plate, developing with a mixed solution of ethyl acetate, n-butanol, glacial acetic acid and water as the developing agent, air-drying, coloring with a coloring agent, and inspecting. This thin-layer detection method is simple, efficient and highly specific in operation.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and in particular to a thin layer detection method for white lentil traditional Chinese medicine formula granules and its application in counterfeit identification. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia defines white lentils as the dried, mature seeds of the legume plant Dolichos lablab L. It is said to invigorate the spleen and eliminate dampness, soothe the stomach and relieve summer heat. It is used for spleen and stomach deficiency, loss of appetite, loose stools, excessive vaginal discharge, vomiting and diarrhea due to summer heat and dampness, and chest tightness and abdominal distension. Current research on the chemical composition of white lentils focuses primarily on its nutritional components, such as fat, polysaccharides, protein, calcium, iron, phosphorus, and vitamins, with little attention paid to other components. Furthermore, the pharmacopoeia only specifies properties, leading to significant subjectivity among authenticators and making it difficult to accurately determine authenticity. Authentication of white lentils has been conducted using physical and chemical methods, DNA, and ultraviolet spectroscopy, but research data is limited. The most widely studied method is thin-layer chromatography, using ninhydrin, a specialized colorimetric reagent for amino acids, to detect amino acid content. However, amino acids are a major nutritional component of legumes and are present in many different types of beans and even other foods. They are not specific to white lentils. In addition, there are reports that picolinic acid (DL-homoproline) is considered to be the active ingredient in white lentils to prevent diarrhea, but this has not yet been confirmed. Therefore, the selection of amino acids as a reference is not specific.

[0003] White lentil Chinese medicine formula granules are obtained by water extraction, concentration and drying of white lentil slices. The problems faced in their identification include: Chinese medicine formula granules are granules made from single Chinese medicine slices through water extraction, separation, concentration, drying and granulation. Chinese medicine formula granules are derived from traditional Chinese medicine slices, and the active substances should be basically consistent with the decoctions of Chinese medicine slices. Traditionally, people can identify the types of medicinal materials by looking, smelling, tasting and touching. However, Chinese medicine formula granules are transformed from tangible slices through a series of processing into finished products that do not have the appearance of slices. They are difficult to identify by traditional visual inspection and smelling. Since Chinese medicine formula granules have lost the characteristics of appearance, smell and other properties that traditional slices rely on for identification, and they play an important role in prescriptions as "solid decoctions", it is particularly important to use appropriate technical means to achieve the identification of the types of medicinal materials contained in Chinese medicine formula granules.

[0004] At present, the qualitative identification of Semen Dolichoris medicated granules includes thin-layer chromatography (TLC) identification and characteristic fingerprint identification. In the standards of Semen Dolichoris medicated granules in various provinces, TLC identification and characteristic fingerprint identification methods have been established. Compared with characteristic fingerprint identification, TLC is one of the most widely used planar chromatography methods, with the advantages of being easy to master, having inexpensive equipment, and being simple and flexible in operation. In recent years, although other chromatographic techniques have developed rapidly, the application of TLC has not decreased significantly. Instead, with the introduction of new stationary phases and rapidly developing instrument technologies, it shows a more extensive application trend and has become a modern, sensitive, and efficient separation and analysis method.

[0005] In the standards of Semen Dolichoris medicated granules in various provinces, with the exception of the Hunan standard, amino acid components are used as the analysis object without exception to establish their TLC identification methods, and ninhydrin color reagent is used for color development. However, the Hunan standard only uses the control medicinal material to directly observe under ultraviolet light at 365 nm, and it is impossible to determine which type of component the detected spots are. The standard of Semen Dolichoris medicated granules issued by Shanghai uses the HPLC-ELSD method to establish a content determination method with notoginsenoside Ⅳa as the index, and a characteristic fingerprint is established for identification. However, due to the low content of saponin components in Semen Dolichoris and the fact that the maximum ultraviolet absorption wavelength of some components is at the end absorption position, this method requires column purification and enrichment, and an evaporative light scattering detector is used for determination. The pretreatment is relatively cumbersome, and its TLC identification still uses ninhydrin color reagent to study its amino acid components. According to the requirements for establishing identification methods in the "Technical Requirements for Quality Control and Standard Setting of Medicated Granules of Traditional Chinese Medicine" issued by the state in 2021, the identification method should have characteristics such as intuitiveness and strong specificity in order to be used as the main method for the identification of medicated granules of traditional Chinese medicine. Summary of the Invention

[0006] Based on this, the present application provides a simple, efficient, and highly specific TLC detection method for Semen Dolichoris medicated granules and its application in the identification of counterfeits.

[0007] In the first aspect of the present application, a TLC detection method for Semen Dolichoris medicated granules is provided, including the following steps:

[0008] Extract Semen Dolichoris medicated granules with a methanol aqueous solution with a volume concentration of 65% - 75%, evaporate the obtained extract to dryness, dissolve the solid obtained after evaporation in water, extract with ethyl acetate, evaporate the obtained ethyl acetate phase to dryness, and dissolve the solid obtained after evaporation in methanol to prepare a test solution;

[0009] Extract the control medicinal material of Semen Dolichoris with a methanol aqueous solution with a volume concentration of 65% - 75%, evaporate the obtained extract to dryness, dissolve the solid obtained after evaporation in water, extract with ethyl acetate, evaporate the obtained ethyl acetate phase to dryness, and dissolve the solid obtained after evaporation in methanol to prepare a control medicinal material solution;

[0010] Take the notoginsenoside Ⅳa reference substance, dissolve it with a solvent to obtain a reference substance solution.

[0011] Spot the test sample solution, the control crude drug solution and the reference substance solution on the same thin-layer plate, develop with a mixed solution of ethyl acetate, n-butanol, glacial acetic acid and water as the developing agent, air dry, develop color with a color developing agent, and examine.

[0012] In one of the embodiments, in the developing agent, the volume ratio of ethyl acetate, n-butanol, glacial acetic acid and water is (8 - 12):(1 - 5):1:1.2; further, in the developing agent, the volume ratio of ethyl acetate, n-butanol, glacial acetic acid and water is (9 - 11):(2 - 4):1:1.2.

[0013] In one of the embodiments, the color developing agent is vanillin sulfuric acid solution or sulfuric acid ethanol solution; further, the color developing agent is sulfuric acid ethanol solution.

[0014] In one of the embodiments, the method of developing color is to heat under the temperature condition of 100°C - 110°C.

[0015] In one of the embodiments, the method of examination is to observe under an ultraviolet lamp; further, the wavelength of the ultraviolet lamp is 365 nm.

[0016] In one of the embodiments, the extraction method is ultrasonic extraction; further, the power of ultrasonic extraction is 250 W - 350 W, the frequency is from 35 kHz to 45 kHz, and the time is 20 min - 40 min.

[0017] In one of the embodiments, the spotting volume of the test sample solution and the control crude drug solution is 8 μL - 12 μL, and the spotting volume of the reference substance solution is 2 μL - 5 μL.

[0018] In the second aspect of the present application, there is provided the application of the thin-layer detection method of the Semen Lablab Album traditional Chinese medicine formula granule in the identification of Semen Lablab Album and its counterfeits.

[0019] In one of the embodiments, the counterfeits are one or more of large white kidney beans, sword beans, hyacinth beans and small white beans.

[0020] In one of the embodiments, the identification method includes: in the chromatogram of the test sample solution, whether there are the same spots at the positions corresponding to the chromatogram of the control crude drug solution and / or the chromatogram of the reference substance solution.

[0021] The above-mentioned thin-layer detection method for Semen Lablab Album traditional Chinese medicine formula granules can, on the basis of thin-layer chromatography, detect Semen Lablab Album traditional Chinese medicine formula granules that have lost their morphological characteristics by adopting a suitable preparation method for test samples and a developing agent, and can separate and detect the saponin components in Semen Lablab Album traditional Chinese medicine formula granules, filling the blank of thin-layer identification of saponin components in Semen Lablab Album traditional Chinese medicine formula granules, with strong specificity, and providing a reference for the establishment of the quality standard for Semen Lablab Album traditional Chinese medicine formula granules. At the same time, compared with methods such as high-performance liquid chromatography, the operation is simpler, the detection is faster, and the cost is low.

[0022] In addition, the above-mentioned thin-layer detection method for Semen Lablab Album traditional Chinese medicine formula granules can be used for the identification of Semen Lablab Album and its counterfeits, and can identify various counterfeits such as large white kidney beans, sword beans, hyacinth beans, and small white beans. Description of the Drawings

[0023] Figure 1 For the thin-layer chromatography of Semen Lablab Album formula granules with different sample application amounts (T: 25°C, RH: 52%; Merck silica gel G plate); among them, 1. 5 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG037); 3. 15 μl of Semen Lablab Album formula granules (CG037); 4. 5 μl of Semen Lablab Album control medicinal material; 5. 10 μl of Semen Lablab Album control medicinal material; 6. 15 μl of Semen Lablab Album control medicinal material; 7. 1 μl of chikusetsusaponin IVa; 8. 3 μl of chikusetsusaponin IVa; 9. 5 μl of chikusetsusaponin IVa;

[0024] Figure 2 For the specificity of the thin-layer chromatography of Semen Lablab Album formula granules (T: 25°C, RH: 52%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of Semen Lablab Album control medicinal material; 5. 3 μl of chikusetsusaponin IVa; 6. 10 μl of negative sample;

[0025] Figure 3 For the thin-layer identification chromatogram of Semen Lablab Album formula granules under normal temperature conditions (T: 25°C, RH: 52%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of Semen Lablab Album control medicinal material; 5. 3 μl of chikusetsusaponin IVa;

[0026] Figure 4TLC identification chromatogram of Semen Lablab Album formula granules under low temperature conditions (T: 8°C, RH: 56%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of control crude drug of Semen Lablab Album; 5. 3 μl of chikusetsusaponin IVa;

[0027] Figure 5 TLC identification chromatogram of Semen Lablab Album formula granules under high humidity conditions (T: 25°C, RH: 89%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of control crude drug of Semen Lablab Album; 5. 3 μl of chikusetsusaponin IVa;

[0028] Figure 6 TLC identification chromatogram of Semen Lablab Album formula granules under low humidity conditions (T: 25°C, RH: 19%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of control crude drug of Semen Lablab Album; 5. 3 μl of chikusetsusaponin IVa;

[0029] Figure 7 Chromatogram for investigation of silica gel G plates from different manufacturers (Yinlong) (T: 25°C, RH: 52%; Yinlong silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of control crude drug of Semen Lablab Album; 5. 3 μl of chikusetsusaponin IVa;

[0030] Figure 8 Chromatogram for investigation of silica gel G plates from different manufacturers (Qingdao) (T: 25°C, RH: 52%; Ocean silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of control crude drug of Semen Lablab Album; 5. 3 μl of chikusetsusaponin IVa;

[0031] Figure 9Chromatograms were examined for silica gel G plates from different manufacturers (Merck) (T: 25°C, RH: 52%; Merck silica gel G plates); among them, 1. 10 μl of semen lablab album formula granules (CG037); 2. 10 μl of semen lablab album formula granules (CG038); 3. 10 μl of semen lablab album formula granules (CG039); 4. 10 μl of semen lablab album control crude drug; 5. 3 μl of chikusetsusaponin Ⅳa;

[0032] Figure 10 For the thin-layer chromatography of 3 batches of semen lablab album formula granules (T: 25°C, RH: 52%; Merck silica gel G plates); among them, 1. 10 μl of semen lablab album formula granules (CG037); 2. 10 μl of semen lablab album formula granules (CG038); 3. 10 μl of semen lablab album formula granules (CG039); 4. 10 μl of semen lablab album control crude drug; 5. 3 μl of chikusetsusaponin Ⅳa;

[0033] Figure 11 For the thin-layer identification chromatography of semen lablab album counterfeits (T: 25°C, RH: 52%; Merck silica gel G plates); among them, 1. 10 μl of semen lablab album control crude drug; 2. 10 μl of phaseolus vulgaris L. var. humilis Alef. crude drug; 3. 10 μl of dolichos lablab L. var. macrocarpus Baill. crude drug; 4. 10 μl of canavalia gladiata (Jacq.) DC. crude drug; 5. 10 μl of phaseolus acutifolius A. Gray crude drug; 6. 3 μl of chikusetsusaponin Ⅳa;

[0034] Figure 12 Chromatograms were examined for different extraction solvents (Method 1 and Method 2) (T: 25°C, RH: 52%; Merck silica gel G plates); among them, 1. 10 μl of semen lablab album formula granules (CG037, Method 1); 2. 10 μl of semen lablab album formula granules (CG038, Method 1); 3. 10 μl of semen lablab album formula granules (CG039, Method 1); 4. 10 μl of semen lablab album control crude drug (Method 1); 5. 10 μl of semen lablab album formula granules (CG037, Method 2); 6. 10 μl of semen lablab album formula granules (CG038, Method 2); 7. 10 μl of semen lablab album formula granules (CG039, Method 2); 8. 10 μl of semen lablab album control crude drug (Method 2); 9. 3 μl of chikusetsusaponin Ⅳa; 10. 3 μl of Copteroside G;

[0035] Figure 13Chromatograms were examined for different extraction solvents (Method 3 and Method 4) (T: 25 °C, RH: 52%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037, Method 3); 2. 10 μl of Semen Lablab Album formula granules (CG038, Method 3); 3. 10 μl of Semen Lablab Album formula granules (CG039, Method 3); 4. 10 μl of Semen Lablab Album control crude drug (Method 3); 5. 3 μl of Panax japonicus Torr. saponin Ⅳa; 6. 10 μl of Semen Lablab Album formula granules (CG037, Method 4); 7. 10 μl of Semen Lablab Album formula granules (CG038, Method 4); 8. 10 μl of Semen Lablab Album formula granules (CG039, Method 4); 9. 10 μl of Semen Lablab Album control crude drug (Method 4);

[0036] Figure 14 Chromatograms were examined for different extraction solvents (Method 5 and Method 6) (T: 25 °C, RH: 52%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037, Method 5); 2. 10 μl of Semen Lablab Album formula granules (CG038, Method 5); 3. 10 μl of Semen Lablab Album formula granules (CG039, Method 5); 4. 10 μl of Semen Lablab Album control crude drug (Method 5); 5. 3 μl of Panax japonicus Torr. saponin Ⅳa; 6. 10 μl of Semen Lablab Album formula granules (CG037, Method 6); 7. 10 μl of Semen Lablab Album formula granules (CG038, Method 6); 8. 10 μl of Semen Lablab Album formula granules (CG039, Method 6); 9. 10 μl of Semen Lablab Album control crude drug (Method 6);

[0037] Figure 15 Chromatograms were examined for different developing solvents (T: 25 °C, RH: 52%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of Semen Lablab Album control crude drug; 5. 3 μl of Panax japonicus Torr. saponin Ⅳa; A: Ethyl acetate - n - butanol - glacial acetic acid - water (10∶3∶1∶1.2); B: Upper layer solution of n - butanol - ethyl acetate - methanol - formic acid - water (5:10:0.5:0.3:3.5); C: n - butanol - glacial acetic acid - water (3∶1∶1); D: Ethyl acetate - n - butanol - glacial acetic acid - water (8∶1∶1∶1.2); E: Ethyl acetate - n - butanol - glacial acetic acid - water (8∶5∶1∶1.2); F: Ethyl acetate - n - butanol - glacial acetic acid - water (12∶5∶1∶1.2);

[0038] Figure 16Chromatograms were examined for different color development methods and color developers (T: 25 °C, RH: 52%; Merck silica gel G plate); among them, 1. 10 μl of Semen Lablab Album formula granules (CG037); 2. 10 μl of Semen Lablab Album formula granules (CG038); 3. 10 μl of Semen Lablab Album formula granules (CG039); 4. 10 μl of the control crude drug of Semen Lablab Album; 5. 3 μl of chikusetsusaponin Ⅳa; A: 254 nm; B: 365 nm; C: iodine fuming, daylight; D: 10% phosphomolybdic acid, daylight; E: 10% sulfuric acid ethanol, 365 nm; F: 1% vanillin sulfuric acid, daylight. Detailed implementation manners

[0039] The following further details the thin-layer detection method of the Semen Lablab Album traditional Chinese medicine formula granules of the present application and its application in the identification of counterfeits in combination with specific examples. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0041] In this application, "the first aspect", "the second aspect", etc. are only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "the first", "the second", etc. only serve the purpose of non-exhaustive enumeration and description and should be understood not to constitute a closed limitation on quantity.

[0042] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions including the listed features.

[0043] In this application, regarding numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0044] Regarding the percentage content involved in this application, unless otherwise specified, for solid-liquid mixtures and solid-solid mixtures, it refers to the mass percentage, and for liquid-liquid mixtures, it refers to the volume percentage.

[0045] In this application, the percentage concentration, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0046] In this application, the temperature parameter, unless otherwise specified, allows both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0047] Normal temperature in this application generally refers to 10°C to 30°C, preferably 25 ± 5°C.

[0048] At present, the first part of the Chinese Pharmacopoeia 2020 edition does not record the characteristic chromatogram related items of Semen Lablab Album medicinal materials. In the literature, the research on the standard characteristic chromatogram and thin-layer identification of Semen Lablab Album medicinal materials and Semen Lablab Album traditional Chinese medicine formula granules in most provinces basically focuses on amino acids and nucleoside components, and trigonelline is attributed, and trigonelline is used as the content determination index. However, there is no evidence to prove its correlation with the efficacy of "invigorating the spleen and resolving dampness, regulating the middle energizer and relieving summer heat" of Semen Lablab Album. Due to the relatively few studies on the chemical components of Semen Lablab Album in current research, it brings certain difficulties to the establishment of identification methods. In addition to amino acids and nucleoside components, only very few literatures report the saponin chemical components of Semen Lablab Album, mainly separating oleanane-type bis-saponin compounds and triterpene compounds, and the biological activity shows good immune effects, such as Copteroside G, Chikusetsusaponin IVa, etc. Another study pointed out that chikusetsusaponin IVa can significantly reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), creatinine (CREA) and cholesterol (CHO) (P < 0.05). In addition, it also has a certain repair effect on the damaged liver and pancreatic cells of diabetic mice.

[0049] Therefore, in combination with the current research situation, based on the blank of thin-layer identification of Semen Lablab Album saponin components, this application takes the saponin components of Semen Lablab Album as the research object, through extraction, purification and enrichment, and uses a simpler, faster and lower-cost thin-layer chromatography to establish a detection method for Semen Lablab Album traditional Chinese medicine formula granules, and can be used to distinguish its counterfeits.

[0050] An example of this application provides a thin-layer detection method for Semen Lablab Album traditional Chinese medicine formula granules, including the following steps:

[0051] Extract Semen Lablab Album traditional Chinese medicine formula granules with a methanol aqueous solution with a volume concentration of 65% to 75%, evaporate the obtained extract to dryness, dissolve the solid obtained by evaporation in water, extract with ethyl acetate, evaporate the obtained ethyl acetate phase to dryness, and dissolve the solid obtained by evaporation in methanol to prepare a test solution;

[0052] Extract the Semen Lablab Album reference medicinal material with an aqueous methanol solution with a volume concentration of 65% to 75%. Evaporate the obtained extract to dryness, dissolve the solid obtained after evaporation in water, extract with ethyl acetate, evaporate the obtained ethyl acetate phase to dryness, and dissolve the solid obtained after evaporation in methanol to prepare a reference medicinal material solution;

[0053] Take the reference substance of Panax japonicus Torr. saponin Ⅳa, dissolve it in a solvent to prepare a reference substance solution;

[0054] Spot the test sample solution, the reference medicinal material solution and the reference substance solution on the same thin layer plate, develop with a mixed solution of ethyl acetate, n-butanol, glacial acetic acid and water as the developing agent, dry in air, color with a color developing agent, and examine.

[0055] Specifically, in the process of preparing the test sample solution or the reference medicinal material solution, the volume concentration of the aqueous methanol solution includes but is not limited to: 65%, 68%, 69%, 70%, 71%, 72%, 75%.

[0056] In some of these examples, in the process of preparing the test sample solution or the reference medicinal material solution, the extraction method is ultrasonic extraction. Further, the power of ultrasonic extraction is 250W to 350W, the frequency is 35kHz to 45kHz, and the time is 20min to 40min.

[0057] In some of these examples, in the process of preparing the test sample solution or the reference medicinal material solution, the dosage of the aqueous methanol solution is 15mL to 25mL added per 1g of Semen Lablab Album traditional Chinese medicine formula granules or per 5g of Semen Lablab Album reference medicinal material.

[0058] In some of these examples, in the process of preparing the test sample solution or the reference medicinal material solution, the dosage of methanol for dissolution in methanol is 0.5mL to 1.5mL added per 1g of Semen Lablab Album traditional Chinese medicine formula granules or per 5g of Semen Lablab Album reference medicinal material.

[0059] In some of these examples, in the process of preparing the reference substance solution, the solvent is methanol.

[0060] In some of these examples, in the process of preparing the reference substance solution, the dosage of the solvent for dissolution is 0.5mL to 1.5mL added per 0.1mg of Panax japonicus Torr. saponin Ⅳa reference substance.

[0061] In some of these examples, the thin layer plate is a silica gel G thin layer plate.

[0062] In some of these examples, in the developing agent, the volume ratio of ethyl acetate, n-butanol, glacial acetic acid and water is (8-12):(1-5):1:1.2. Specifically, in the developing agent, the volume ratio of ethyl acetate, n-butanol, glacial acetic acid and water includes but is not limited to: 10:3:1:1.2, 8:2:1:1.2, 8:5:1:1.2, 12:5:1:1.2. Further, in the developing agent, the volume ratio of ethyl acetate, n-butanol, glacial acetic acid and water is (9-11):(2-4):1:1.2.

[0063] In some of these examples, the color-developing agent is vanillin sulfuric acid solution or sulfuric acid ethanol solution. Without limitation, the volume concentration of the vanillin sulfuric acid solution is 0.5%-1.5%, and the volume concentration of the sulfuric acid ethanol solution is 5%-15%.

[0064] In some of these examples, the color-developing agent is sulfuric acid ethanol solution. Further, the volume concentration of the sulfuric acid ethanol solution is 8%-12%.

[0065] In some of these examples, the method of color development is heating under the temperature condition of 100°C - 110°C.

[0066] In some of these examples, the method of inspection is observing under an ultraviolet lamp; further, the wavelength of the ultraviolet lamp is 365 nm.

[0067] In some of these examples, the spotting amount of the test solution and the control crude drug solution is 8 μL - 12 μL, and the spotting amount of the reference substance solution is 2 μL - 5 μL.

[0068] Another example of the present application provides the application of the thin-layer detection method of the Semen Lablab Album traditional Chinese medicine formula granules as described above in the identification of Semen Lablab Album and its counterfeits.

[0069] In some of these examples, the counterfeits are one or more of large white kidney beans, sword beans, hyacinth beans and small white beans. Without limitation, the forms of the Semen Lablab Album and its counterfeits can be crude drugs, cut pieces, traditional Chinese medicine formula granules or other preparation forms.

[0070] In some of these examples, the method of identification includes: in the chromatogram of the test solution, whether there are the same spots at the positions corresponding to the chromatogram of the control crude drug solution and / or the chromatogram of the reference substance solution. If there are the same spots, it is the Semen Lablab Album traditional Chinese medicine formula granules, and if not, it is a counterfeit. Additionally, it can be understood that the control crude drug is used as a reference for thin-layer identification. The identification basis has risen from a single index component to judging the authenticity with a complete thin-layer chromatogram. In the thin-layer chromatogram, it is a chromatographic image with fingerprint significance. Different varieties have their own fixed chromatograms, thereby improving the specificity of identification. Using it in combination with the reference substance for identification has higher accuracy.

[0071] The following are specific examples.

[0072] Example 1

[0073] This example is a thin-layer detection method for Semen Lablab Album traditional Chinese medicine formula granules.

[0074] 1. Instruments, reagents and test drugs

[0075] Instruments: Automatic thin-layer sampler (AUTOMATIC TLC SAMPLER4, CAMAG Switzerland), thin-layer automatic imager (TLC VISUALIZER 2, CAMAG Switzerland), ten-thousandth balance (ME204E, METTLER TOLEDO Switzerland), ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd.), double-tank developing tank, silica gel G thin-layer plate, silica gel G thin-layer plate (20 cm × 10 cm, thickness 0.20 - 0.25 mm, Yantai Chemical Industry Research Institute), silica gel G thin-layer plate (20 cm × 10 cm, thickness 0.20 - 0.25 mm, Qingdao Ocean Chemical Co., Ltd.), silica gel G thin-layer plate (20 cm × 10 cm, thickness 0.25 mm, Merck KGaA).

[0076] Reagents: Methanol (SY21812004, Tianjin Fuyu Fine Chemical Co., Ltd.), n-butanol (SY220419003, Xilong Scientific Co., Ltd.), glacial acetic acid (SY210709009, Xilong Scientific Co., Ltd.), ethyl acetate (SY220513011, Xilong Scientific Co., Ltd.), purified water (self-made in the laboratory).

[0077] Test drugs: Semen Lablab Album traditional Chinese medicine formula granules (batch numbers: CG037, CG038, CG039, provided by Guangdong Yifang Pharmaceutical Co., Ltd.); Semen Lablab Album control crude drug (batch number: 125021 - 202006A, calibrated by Gansu Institute for Drug Control), Panax japonicus Torr. saponin Ⅳa (batch number: 111861 - 201102, content: 77.6%, National Institutes for Food and Drug Control), Copteroside G (batch number: FH22A061, content: 98.0%, Chengdu Pusi Biotechnology Co., Ltd.), maltodextrin (batch number: F1710002, Nanxinyuan Biotechnology Co., Ltd.).

[0078] 2. Preparation of solutions

[0079] 2.1 Preparation of test solution

[0080] Take an appropriate amount of Semen Lablab Album traditional Chinese medicine formula granules, grind them finely, take 1 g, add 20 ml of 70% methanol, and ultrasonically treat (power: 300 W, frequency: 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with ethyl acetate by shaking, 20 ml each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0081] 2.2 Preparation of control crude drug solution

[0082] Take 5 g of Semen Lablab Album control crude drug, add 20 ml of 70% methanol, and ultrasonically treat (power: 300 W, frequency: 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with ethyl acetate by shaking, 20 ml each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to prepare the control crude drug solution.

[0083] 2.3 Preparation of reference substance solution

[0084] Take an appropriate amount of chikusetsusaponin Ⅳa reference substance, add methanol to make a solution containing 0.1 mg per 1 ml as the reference substance solution.

[0085] 2.4 Preparation of negative sample solution

[0086] Take 1 g of negative sample (maltodextrin) without Semen Lablab Album, add 20 ml of 70% methanol, and ultrasonically treat (power: 300 W, frequency: 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with ethyl acetate by shaking, 20 ml each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the negative sample solution.

[0087] 2.5 Preparation of chromogenic agent

[0088] Take 10 ml of sulfuric acid solution, dilute it to 100 ml with ethanol to obtain 10% sulfuric acid - ethanol solution.

[0089] 3. Thin - layer chromatography conditions

[0090] Thin - layer plate: Silica gel G thin - layer plate

[0091] Developing solvent: Ethyl acetate - n - butanol - glacial acetic acid - water (volume ratio: 10∶3∶1∶1.2)

[0092] Spotting method: Spray strip - like spotting.

[0093] Developing method: Use a double - trough developing tank, the pre - saturation time of the developing tank is 15 minutes, and the developing distance is about 8 cm.

[0094] Inspection: Spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly visible, and examine under ultraviolet light (365 nm).

[0095] 4. Investigation of different sample application volumes

[0096] Respectively, draw the test solution, control crude drug solution and reference substance solution of Semen Lablab Album Chinese medicine formula granules (CG037) and spot them on the same silica gel G thin layer plate. According to the above thin layer chromatography conditions, develop, take out, 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). The experimental results are shown in Figure 1 .

[0097] It can be seen from Figure 1 that when the sample application volumes of the test solution and the control crude drug solution are 10 μl and the sample application volume of the reference substance solution is 3 μl, the test chromatogram shows fluorescent spots of the same color at the corresponding positions of the control crude drug chromatogram and the reference substance chromatogram, without trailing and other interferences. Therefore, the sample application volume of the test solution and the control crude drug solution is set at 10 μl, and the sample application volume of the reference substance solution is set at 3 μl.

[0098] 5. Specificity investigation

[0099] Respectively, draw the test solution, control crude drug solution, reference substance solution and negative sample solution of Semen Lablab Album Chinese medicine formula granules (CG037) and spot them on the same silica gel G thin layer plate. According to the above thin layer chromatography conditions, develop, take out, 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). The experimental results are shown in Figure 2 .

[0100] It can be seen from Figure 2 that the test chromatogram of Semen Lablab Album Chinese medicine formula granules shows fluorescent spots of the same color at the corresponding positions of the control crude drug chromatogram and the reference substance chromatogram, and the negative sample has no interference. It shows that the thin layer method has good specificity.

[0101] 6. Investigation of different temperatures

[0102] Respectively, draw the test solution, control crude drug solution and reference substance solution of Semen Lablab Album Chinese medicine formula granules (CG037) and spot them on the same silica gel G thin layer plate. According to the above thin layer chromatography conditions, develop under normal temperature (25 °C) and low temperature (8 °C) conditions respectively, take out, 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). The experimental results are shown in Figure 3 and Figure 4 .

[0103] It can be seen from Figure 3 and Figure 4It can be seen that under normal temperature and low temperature conditions, the separation effect of the Chinese medicine formula granules of Semen Lablab Album is good. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the control crude drug and the reference substance, fluorescent spots of the same color appear. The experimental results show that temperature has no obvious effect on the thin-layer identification of the Chinese medicine formula granules of Semen Lablab Album, indicating that this thin-layer identification method has good durability for different temperatures.

[0104] 7. Investigation of different humidities

[0105] Respectively absorb the test sample solution, the solution of the control crude drug, and the reference substance solution of the Chinese medicine formula granules of Semen Lablab Album (CG037) and spot them on the same silica gel G thin-layer plate. According to the above thin-layer chromatography conditions, develop them respectively under high humidity (RH: 89%) and low humidity (RH: 19%) conditions. Take them out, dry them, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly developed, and examine them under ultraviolet light (365 nm). The experimental results are shown in Figure 5 and Figure 6 :

[0106] It can be seen from Figure 5 and Figure 6 that under high humidity and low humidity conditions, the chromatographic separation effect of the Chinese medicine formula granules of Semen Lablab Album is good. In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the control crude drug and the reference substance, fluorescent spots of the same color appear. The experimental results show that humidity has no obvious effect on the thin-layer identification of the Chinese medicine formula granules of Semen Lablab Album, and this thin-layer identification method has good durability for different humidities.

[0107] 8. Investigation of thin-layer plates from different manufacturers

[0108] Respectively absorb the test sample solution, the solution of the control crude drug, and the reference substance solution of the Chinese medicine formula granules of Semen Lablab Album (CG037) and spot them on thin-layer plates of silica gel G from different manufacturers (Silver Dragon silica gel G plate, Ocean silica gel G plate, Merck silica gel G plate). According to the above thin-layer chromatography conditions, develop them respectively under the same temperature and humidity conditions. Take them out, dry them, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly developed, and examine them under ultraviolet light (365 nm). The experimental results are shown in Figure 7 , Figure 8 and Figure 9 :

[0109] It can be seen from Figure 7 , Figure 8 and Figure 9 that when using thin-layer plates of silica gel G from different manufacturers (Silver Dragon silica gel G plate, Ocean silica gel G plate, Merck silica gel G plate), the fluorescent spots of the test sample chromatogram, the chromatograms of the control crude drug, and the reference substance of the Chinese medicine formula granules of Semen Lablab Album are all clearly developed, the resolution is good, and the fluorescent spots of the test sample chromatogram can correspond one by one to those of the chromatograms of the control crude drug and the reference substance. It shows that this thin-layer identification method has good durability for thin-layer plates of silica gel G from different manufacturers.

[0110] TLC chromatogram of Semen Lablab Album Chinese medicinal formula granule

[0111] Respectively absorb the test solution, reference crude drug solution and reference substance solution of Semen Lablab Album Chinese medicinal formula granule (CG037, CG038 or CG039), spot them on the same silica gel G TLC plate respectively, develop according to the above TLC conditions, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly developed, and examine under ultraviolet light (365 nm). The experimental results are shown in Figure 10 :

[0112] From Figure 10 It can be seen that in the test solution chromatograms of 3 batches of large-scale production of Semen Lablab Album Chinese medicinal formula granule, at the corresponding positions of the reference crude drug chromatogram and the reference substance chromatogram, fluorescent spots of the same color are shown. Using the reference crude drug of Semen Lablab Album and chikusetsusaponin Ⅳa as references, the TLC identification of Semen Lablab Album formula granule can be effectively carried out, and the 3 batches of Semen Lablab Album Chinese medicinal formula granule samples all meet the requirements.

[0113] 10. Summary

[0114] The Rf values of each spot in the chromatogram established in this application are moderate, the overall separation effect is good, the spots are relatively clear, and the same color spots in the test solution chromatogram can correspond one by one to the corresponding positions of the reference crude drug chromatogram and the reference substance chromatogram. After methodological verification, the TLC method has good specificity, reproducibility and durability, and this method can effectively carry out qualitative identification on Semen Lablab Album Chinese medicinal formula granule that has lost the shape of the cut crude drug.

[0115] Example 2

[0116] In this example, the established TLC detection method for Semen Lablab Album Chinese medicinal formula granule was used to determine four counterfeits, namely Phaseolus lunatus Billb.ex Beurl., Canavalia gladiata (Jacq.) DC., Lablab purpureus (L.) Sweet (Dolichos lablab L.), and Phaseolus vulgaris L.f. abla Alef, and compare the differences in their TLC chromatograms ( Figure 11 ).

[0117] 1. Weigh 5 g of the reference medicinal material of Dolichos lablab, large white kidney beans, sword beans, hyacinth beans, and small white beans (passed through No. 3 sieve) respectively, add 20 ml of 70% methanol, ultrasonically treat (ultrasonic power is 300 W, frequency is 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with ethyl acetate by shaking, 20 ml each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to prepare different test solutions.

[0118] 2. Weigh an appropriate amount of chikusetsusaponin Ⅳa reference substance, add methanol to make a solution containing 0.1 mg per 1 ml as the reference substance solution.

[0119] 3. Pipette 10 μl of each test solution and 3 μl of the reference substance solution respectively, spot them on the same Merck silica gel G thin layer plate, use ethyl acetate - n - butanol - glacial acetic acid - water (volume ratio is 10∶3∶1∶1.2) as the developing agent, develop, take out, dry in air, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly visible, and examine under ultraviolet light (365 nm).

[0120] The results showed that no spots corresponding to the chikusetsusaponin Ⅳa reference substance were detected in the four samples of large white kidney beans, sword beans, hyacinth beans, and small white beans, proving that this method using saponin components (chikusetsusaponin Ⅳa) to identify Dolichos lablab has specificity.

[0121] Example 3

[0122] This example is for the investigation of extraction solvents.

[0123] 1. Method 1: Weigh an appropriate amount of the traditional Chinese medicine formula granules of Dolichos lablab (CG037), grind them finely, weigh 1 g, add 20 ml of methanol, ultrasonically treat (ultrasonic power is 300 W, frequency is 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol as the test solution.

[0124] 2. Method 2: Weigh an appropriate amount of the traditional Chinese medicine formula granules of Dolichos lablab (CG037), grind them finely, weigh 1 g, add 20 ml of 70% methanol, ultrasonically treat (ultrasonic power is 300 W, frequency is 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol as the test solution.

[0125] 3. Method 3: Weigh an appropriate amount of the traditional Chinese medicine formula granules of Dolichos lablab (CG037), grind them finely, weigh 1 g, add 20 ml of methanol, ultrasonically treat (ultrasonic power is 300 W, frequency is 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with n - butanol by shaking, 20 ml each time, combine the n - butanol extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol as the test solution.

[0126] 4. Method 4: Take an appropriate amount of Semen Lablab Album Chinese medicine formula granules (CG037), grind them finely, take 1 g, add 20 ml of methanol, and ultrasonically treat (power: 300 W, frequency: 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with ethyl acetate by shaking, 20 ml each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0127] 5. Method 5: Take an appropriate amount of Semen Lablab Album Chinese medicine formula granules (CG037), grind them finely, take 1 g, add 20 ml of 70% methanol, and ultrasonically treat (power: 300 W, frequency: 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with n-butanol by shaking, 20 ml each time. Combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0128] 6. Method 6: Take an appropriate amount of Semen Lablab Album Chinese medicine formula granules (CG037), grind them finely, take 1 g, add 20 ml of 70% methanol, and ultrasonically treat (power: 300 W, frequency: 40 kHz) for 30 minutes. Filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with ethyl acetate by shaking, 20 ml each time. Combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0129] 7. Separately take 5 g of the control medicinal material of Semen Lablab Album, and prepare the control medicinal material solution in the same method according to Methods 1 - 6.

[0130] 8. Take appropriate amounts of chikusetsusaponin Ⅳa and Copteroside G reference substances, and make a solution containing 0.1 mg per 1 ml with methanol as the reference substance solution.

[0131] 9. Pipette 10 μl of each of the test solution and the control medicinal material solution, and 3 μl of the reference substance solution, and spot them on the same Merck silica gel G thin layer plate respectively. Use ethyl acetate - n-butanol - glacial acetic acid - water (volume ratio: 10∶3∶1∶1.2) as the developing solvent, develop, take out, 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).

[0132] By comparing the 6 sample preparation methods, the results show that the chromatogram spots obtained by extracting with methanol alone and 70% methanol are not very different ( Figure 12 ), but during the process of dissolving the residue with methanol after evaporating the extract with 70% methanol, a considerable part of the residue cannot be completely dissolved due to solubility reasons, and it cannot truly show that the extraction effect of 70% methanol is equivalent to that of methanol extraction. Moreover, due to the incomplete dissolution, it may affect the repeatability of the results. Comparing the use of methanol ( Figure 13 ), 70% methanol ( Figure 14) After extraction and evaporation to dryness, dissolve with water, and then extract with ethyl acetate and n-butanol separately. The results show that whether using ethyl acetate or n-butanol for extraction, when extracting with 70% methanol, the spot of chikusetsusaponin Ⅳa is brighter. Therefore, 70% methanol is selected as the extraction solvent for the thin-layer identification of Semen Lablab Album Chinese medicine formula granules. Comparing the chromatograms ([ Figure 14 ) after extraction with 70% methanol and then with ethyl acetate and n-butanol respectively, there are more spots after extraction with n-butanol, but the background color is darker, and there is an adjacent spot below the spot of chikusetsusaponin Ⅳa. Developing on thin-layer plates at different laboratory temperatures, humidities or of different brands and manufacturers may have a greater impact on its resolution, affecting the resolution of chikusetsusaponin Ⅳa and thus affecting the result determination. Therefore, in order to ensure the resolution of the main spot (chikusetsusaponin Ⅳa), ethyl acetate with a smaller total number of spots is selected for extraction, which is more conducive to improving the durability of the method. In addition, by comparing with the reference substances of chikusetsusaponin Ⅳa and Copteroside G, it is found that in the chromatogram of the chikusetsusaponin Ⅳa reference substance, at the positions corresponding to the test sample chromatogram and the reference crude drug chromatogram, there are fluorescent spots showing the same color. However, due to the extremely low content of Copteroside G in the Semen Lablab Album formula granules, no obvious fluorescent spots corresponding to the Copteroside G reference substance can be observed in its chromatogram. Therefore, only chikusetsusaponin Ⅳa is used as the reference for the reference substance.

[0133] Example 4

[0134] This example is for the investigation of the developing agent and the proportion of the developing agent.

[0135] 1. Take an appropriate amount of Semen Lablab Album Chinese medicine formula granules (CG037), grind them finely, take 1 g, add 20 ml of 70% methanol, treat with ultrasound (power 300 W, frequency 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, shake and extract with ethyl acetate twice, 20 ml each time, combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test sample solution.

[0136] 2. Take another 5 g of the reference crude drug of Semen Lablab Album, add 20 ml of 70% methanol, and prepare the reference crude drug solution in the same way.

[0137] 3. Take an appropriate amount of chikusetsusaponin Ⅳa reference substance, dissolve it in methanol to make a solution containing 0.1 mg per 1 ml as the reference substance solution.

[0138] 4. Pipette 10 μl of the test solution and the control crude drug solution each, and 3 μl of the reference substance solution, and spot them separately on the same Merck silica gel G thin-layer plate. Develop with ethyl acetate - n-butanol - glacial acetic acid - water (volume ratio 10∶3∶1∶1.2), the upper layer solution of n-butanol - ethyl acetate - methanol - formic acid - water (volume ratio 5:10:0.5:0.3:3.5), n-butanol - glacial acetic acid - water (volume ratio 3∶1∶1), ethyl acetate - n-butanol - glacial acetic acid - water (volume ratio 8∶1∶1∶1.2), ethyl acetate - n-butanol - glacial acetic acid - water (volume ratio 8∶5∶1∶1.2), ethyl acetate - n-butanol - glacial acetic acid - water (volume ratio 12∶5∶1∶1.2) respectively. Take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots develop clearly, and examine under ultraviolet light (365 nm).

[0139] 5. The comparison results of different developing systems show (see Figure 15 A, B, C in it), when using the upper layer solution of n-butanol - ethyl acetate - methanol - formic acid - water (5:10:0.5:0.3:3.5) as the developing agent, the spot of japonicoside Ⅳa diffuses due to a relatively high Rf value, and there are two solvent fronts for this developing agent. When using n-butanol - glacial acetic acid - water (3∶1∶1) as the developing agent, all spots are distributed in the upper half of the thin-layer plate, with an unreasonable distribution, and there are also two solvent fronts. However, when developing with ethyl acetate - n-butanol - glacial acetic acid - water (10∶3∶1∶1.2), the spots of japonicoside Ⅳa and other components are relatively concentrated, with a smaller degree of diffusion, and all spots are evenly distributed. Therefore, ethyl acetate - n-butanol - glacial acetic acid - water (10∶3∶1∶1.2) is selected as the developing agent.

[0140] 6. The comparison results of different proportions of developing agents show (see Figure 15 A, D, E, F in it), when using ethyl acetate - n-butanol - glacial acetic acid - water as the developing system, the four proportions of 10∶3∶1∶1.2, 8:1:1:1.2, 8∶5∶1∶1.2, 12∶5∶1∶1.2 can all identify the japonicoside Ⅳa component in the Semen Lablab Album Chinese medicinal formula granule, and the main spots in the test sample can also correspond one by one to the spots of the control crude drug. However, from the comprehensive comparison of the diffusion degree of the spots and the correspondence of the spots, ethyl acetate - n-butanol - glacial acetic acid - water (10∶3∶1∶1.2) is preferentially selected as the developing agent.

[0141] Example 5

[0142] This example is for the investigation of the coloring method and the examination method.

[0143] 1. Take an appropriate amount of Semen Lablab Album traditional Chinese medicine formula granule (CG037), grind it finely, take 1 g, add 20 ml of 70% methanol, ultrasonically treat (power: 300 W, frequency: 40 kHz) for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract twice with ethyl acetate by shaking, 20 ml each time, combine the ethyl acetate extracts, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0144] 2. Take another 5 g of the control crude drug of Semen Lablab Album, add 20 ml of 70% methanol, and prepare the control crude drug solution in the same way.

[0145] 3. Take an appropriate amount of notoginsenoside Ⅳa reference substance, add methanol to make a solution containing 0.1 mg per 1 ml as the reference substance solution.

[0146] 4. Pipette 10 μl each of the test solution and the control crude drug solution, and 3 μl of the reference substance solution, respectively spot them on the same Merck silica gel GF254 thin layer plate, use ethyl acetate - n - butanol - glacial acetic acid - water (10∶3∶1∶1.2) as the developing solvent, develop, take out, air dry, and reserve.

[0147] 5. Observe directly under an ultraviolet lamp (365 nm) without using a developer.

[0148] 6. Observe directly under an ultraviolet lamp (254 nm) without using a developer.

[0149] 7. Spray with 10% sulfuric acid ethanol solution, heat at 105 °C until the spots are clearly developed, and observe under ultraviolet light (365 nm).

[0150] 8. Spray with 10% phosphomolybdic acid ethanol solution, heat at 105 °C until the spots are clearly developed, and observe under ultraviolet light (365 nm).

[0151] 9. Spray with 1% vanillin sulfuric acid solution, heat at 105 °C until the spots are clearly developed, and observe under ultraviolet light (365 nm).

[0152] 10. Fumigate in iodine vapor until the spots are clearly developed, and observe under daylight.

[0153] According to the chromatogram ( Figure 16)Results showed that without using a chromogenic agent, no spots were observed under an ultraviolet lamp (254 nm), and 3 common spots were observed between the test sample and the control crude drug under an ultraviolet lamp (365 nm), but the spot of chikusetsusaponin Ⅳa was not observed. For saponin components, common thin-layer chromogenic agents generally include sulfuric acid ethanol, phosphomolybdic acid, vanillin sulfuric acid, and iodine fuming. After comparison, it was found that there were no spots in the chromatograms of phosphomolybdic acid and iodine fuming, the spots of vanillin sulfuric acid were relatively faint, while the spots of sulfuric acid ethanol were clear, and the spots of the test sample, the control crude drug, and the chikusetsusaponin Ⅳa reference substance could all be clearly shown. Therefore, sulfuric acid ethanol was selected as the chromogenic agent for the thin-layer identification of saponin components in the Semen Lablab Album formula granules.

[0154] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification.

[0155] The above-described embodiments merely represent several implementation manners of this application, which are convenient for understanding the technical solutions of this application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of this application. It should be noted that for those of ordinary skill in the art, without departing from the concept of this application, several modifications and improvements can still be made, and these all belong to the protection scope of this application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided in this application are all within the protection scope of the appended claims of this application. Therefore, the protection scope of the patent of this application should be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A thin-layer detection method for traditional Chinese medicine formula granules of Dolichos lablab, characterized in that, It includes the following steps: Extract the Semen Lablab Album Chinese medicine formula granules with a methanol aqueous solution with a volume concentration of 65% - 75%, evaporate the obtained extract to dryness, dissolve the solid obtained by evaporation in water, extract with ethyl acetate, evaporate the obtained ethyl acetate phase to dryness, dissolve the solid obtained by evaporation in methanol to prepare a test solution; Extract the Semen Lablab Album reference medicinal material with a methanol aqueous solution with a volume concentration of 65% - 75%, evaporate the obtained extract to dryness, dissolve the solid obtained by evaporation in water, extract with ethyl acetate, evaporate the obtained ethyl acetate phase to dryness, dissolve the solid obtained by evaporation in methanol to prepare a reference medicinal material solution; Take the reference substance of chikusetsusaponin Ⅳa, dissolve it in a solvent to prepare a reference substance solution; Spot the test solution, the reference medicinal material solution and the reference substance solution on the same silica gel G thin layer plate, develop with a mixed solution of ethyl acetate, n-butanol, glacial acetic acid and water as the developing agent, air dry, develop color with a color developing agent, and examine; the examination method is to observe under an ultraviolet lamp; In the developing agent, the volume ratio of ethyl acetate, n-butanol, glacial acetic acid and water is (8 - 12):(1 - 5):1:1.2; The color developing agent is vanillin sulfuric acid solution or sulfuric acid ethanol solution.

2. The thin layer detection method of the Semen Dolichoris medicated formula granule according to claim 1, characterized in that In the developing agent, the volume ratio of ethyl acetate, n-butanol, glacial acetic acid and water is (9 - 11):(2 - 4):1:1.

2.

3. The thin-layer detection method of the semen lablab album traditional Chinese medicine formula granule according to claim 1, characterized in that, The color developing agent is sulfuric acid ethanol solution.

4. The thin-layer detection method of the semen lablab album traditional Chinese medicine formula granule according to claim 1, characterized in that, The color developing method is to heat under the temperature condition of 100°C - 110°C.

5. The thin-layer detection method of the Semen Lablab Album traditional Chinese medicine formula granule according to claim 1, characterized in that, The wavelength of the ultraviolet lamp is 365 nm.

6. The thin-layer detection method for the semen lablab album traditional Chinese medicine formula granule according to any one of claims 1 to 5, characterized in that, The extraction method is ultrasonic extraction.

7. The TLC detection method of the Semen Lablab Album traditional Chinese medicine formula granule according to claim 6, characterized in that, The power of ultrasonic extraction is 250 W - 350 W, the frequency is 35 kHz - 45 kHz, and the time is 20 min - 40 min.

8. The thin-layer detection method for the semen lablab album traditional Chinese medicine formula granule according to any one of claims 1 to 5, characterized in that, The spotting amount of the test solution and the reference medicinal material solution is 8 μL - 12 μL, and the spotting amount of the reference substance solution is 2 μL - 5 μL.

9. Application of the thin layer detection method of the Semen Lablab Album Chinese medicine formula granules described in any one of claims 1 - 8 in the identification of Semen Lablab Album and its counterfeits, and the counterfeits are one or more of large white kidney beans, sword beans, hyacinth beans and small white beans.

10. The application according to claim 9, characterized in that The identification method includes: in the chromatogram of the test solution, whether there are the same spots at the positions corresponding to the chromatogram of the reference medicinal material solution and / or the chromatogram of the reference substance solution.

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