Thin-layer Identification Method for Canavalia Extract and Canavalia gladiata Extract

Abstract

The present invention provides a thin-layer identification method for extracts of Canavalia gladiata and Canavalia ensiformis, comprising: pretreating the extract of Canavalia gladiata to obtain a test solution; pretreating the extract of Canavalia ensiformis to obtain an extract solution of Canavalia ensiformis; taking the reference medicinal material of Canavalia gladiata, decocting with water, adding methanol after filtration to obtain a reference medicinal material solution; adding a reference substance to a methanol solution to obtain a reference substance solution; performing thin-layer chromatography detection on the test solution, the extract solution of Canavalia ensiformis, the reference substance solution and the reference medicinal material solution, spraying with ninhydrin solution, heating and examining. If the test solution and the reference medicinal material solution have fluorescent spots of the same color at the same Rf value, it is the extract of Canavalia gladiata. The thin-layer identification chromatographic method for the extract of Canavalia gladiata established by the present invention is used for quickly, accurately and effectively identifying the extract of Canavalia gladiata. At the same time, by observing whether there are fluorescent spots within a certain range of the specific migration value in the thin-layer chromatogram as the identification point, it is used to distinguish the extract of Canavalia gladiata and the extract of Canavalia ensiformis.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a thin-layer identification method for extracts of Canavalia gladiata and Canavalia ensiformis Background Art

[0002] Canavalia gladiata is widely used, but there is little research on it at present. In the Chinese Pharmacopoeia, the Chinese herbal medicine standards and processing specifications of each province, there are no regulations on its content determination and qualitative identification, and for the research on the components of Canavalia gladiata, there are only literature reports on the extraction of total flavonoids etc. (Kong Ziming, Xie Jianfeng, Li Yingchen, Wang Ying, Han Yongping. Optimization of ultrasonic extraction of total flavonoids from Tibetan medicine Canavalia gladiata and its antioxidant activity. Chinese Traditional Patent Medicine, 2016, 38(05): 1163-1167), while there are few in-depth research reports on the extracts of Canavalia gladiata.

[0003] The seeds of Canavalia ensiformis belong to the leguminous plants and are the adulterants of Canavalia gladiata, which are similar in traits to Canavalia gladiata, and the two medicinal materials can be distinguished by trait identification. However, for its extracts, after extraction, the traits disappear and trait identification cannot be used to identify the extracts.

[0004] Therefore, it is necessary to develop a method with stronger specificity, stronger repeatability, effective and controllable to provide an effective means for the qualitative identification of Canavalia gladiata extracts. The purpose of the present invention is to establish a method that can effectively identify Canavalia gladiata and Canavalia ensiformis extracts. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a thin-layer identification method for Canavalia gladiata extracts and Canavalia ensiformis extracts.

[0006] In this article, the terms "include", "comprise" and "have" can be used interchangeably, aiming to represent the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that when using "include", "comprise" and "have" to describe in this article, the solution of "consisting of..." is also provided.

[0007] In this application, the term "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural.

[0008] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following (items)" or its similar expression refers to any combination of these items, including any combination of single item (item) or plural items (items).

[0009] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the following processes do not imply the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0010] The present invention provides a method for constructing and applying a thin-layer identification method for canavalia extract. When the chromatogram of the test sample is compared with the thin-layer chromatograms of the reference substance and the reference medicinal material of canavalia, spots of the same color appear, which can be used for the identification of canavalia extract. When the thin-layer chromatograms of the test sample, the reference substance and the reference medicinal material of canavalia are compared, spots of canavalia appear at the positions with the retention factor values of 0.225 - 0.257, 0.495 - 0.605, and 0.599 - 0.732, while there are no obvious spots in the extract of its adulterant, canavalia gladiata, which can be used as an identification point to distinguish canavalia from the extract of canavalia gladiata. This thin-layer method can quickly and effectively identify the canavalia extract that has lost the form of the cut medicinal material and distinguish the canavalia extract from the extract of canavalia gladiata. It has the advantages of simple operation, high precision and sensitivity, good stability, and has good application prospects.

[0011] The thin-layer identification chromatographic method for canavalia extract established by the present invention can quickly, accurately and effectively identify canavalia extract. At the same time, by observing whether there are spots in a certain range of retention factor values in the thin-layer chromatogram as an identification point, it can be used to quickly and effectively identify canavalia and the extract of canavalia gladiata, providing guidance for the quality control of canavalia extract and its related preparations.

[0012] The present invention provides a thin-layer identification method for canavalia extract and the extract of canavalia gladiata, including:

[0013] A) Pretreat the canavalia extract to obtain a test sample solution;

[0014] Pretreat the extract of canavalia gladiata to obtain a solution of the extract of canavalia gladiata;

[0015] B) Take the reference medicinal material of canavalia, decoct it with water, add methanol after filtration to obtain a reference medicinal material solution; add the reference substance to a methanol solution to obtain a reference substance solution;

[0016] C) Perform thin-layer chromatographic detection on the test sample solution, the solution of the extract of canavalia gladiata, the reference substance solution and the reference medicinal material solution. The thin-layer plate is a silica gel G thin-layer plate; the developing agent is ethyl acetate - methanol - glacial acetic acid;

[0017] D) Spray with ninhydrin solution, heat for examination. If the test sample and the control crude drug solution have fluorescent spots of the same color at the same Rf value, and at the same time have fluorescent spots at Rf = 0.225 - 0.257, 0.495 - 0.605, 0.599 - 0.732, it is the extract of Canavalia gladiata; if there are no fluorescent spots at Rf = 0.225 - 0.257, 0.495 - 0.605, 0.599 - 0.732, it is the extract of Canavalia ensiformis.

[0018] The present invention provides an extract of Canavalia gladiata and an extract of Canavalia ensiformis. First, the extract of Canavalia gladiata is pretreated to obtain a test sample solution.

[0019] The pretreatment of the extract of Canavalia gladiata or the extract of Canavalia ensiformis described in the present invention is specifically as follows:

[0020] a) Add methanol to the extract of Canavalia gladiata or the extract of Canavalia ensiformis, ultrasonicate, filter, evaporate to dryness, dissolve the residue in water, shake and extract with ethyl acetate to obtain an ethyl acetate layer extract.

[0021] b) Evaporate the ethyl acetate layer extract to dryness, dissolve it in water, load it onto a silica gel column, elute with methanol, collect the eluate, evaporate to dryness, and dissolve the residue in methanol.

[0022] For the pretreatment of the extract of Canavalia gladiata provided by the present invention, first add methanol to the extract of Canavalia gladiata or the extract of Canavalia ensiformis, ultrasonicate, filter, evaporate to dryness, shake and extract 3 times with ethyl acetate to obtain an ethyl acetate layer extract.

[0023] In some embodiments of the present invention, the mass - volume ratio of the extract of Canavalia gladiata / extract of Canavalia ensiformis, methanol, water, and ethyl acetate is 0.5 g: 20 mL: 10 mL: 30 mL;

[0024] In some embodiments of the present invention, the ultrasonic time is 20 - 30 min;

[0025] Evaporate the ethyl acetate layer extract to dryness, dissolve it in water, load it onto a silica gel column, elute with methanol, collect the eluate, evaporate to dryness, and dissolve the residue in methanol.

[0026] In some embodiments of the present invention, the volume ratio of water to methanol is 5:20;

[0027] In some embodiments of the present invention, the specifications of the silica gel column are: 160 - 200 mesh, 10 g, inner diameter 1.8 cm, dry - packing the column.

[0028] Take the control crude drug of Canavalia gladiata, decoct it with water, filter and add methanol to obtain a control crude drug solution;

[0029] The mass - volume ratio of the control crude drug of Canavalia gladiata and water described in the present invention is 1 g: 50 mL;

[0030] The decocting time in the present invention is 20 - 30 min; preferably 30 min.

[0031] Add the reference substance to a methanol solution to obtain a reference substance solution.

[0032] The reference substances include threonine, valine, and leucine; the concentration of each reference substance is 0.5 mg / mL. The above reference substance solution is a mixed reference substance solution.

[0033] Perform thin-layer chromatography detection on the test solution, Canavalia ensiformis extract solution, reference substance solution, and control crude drug solution. The thin-layer plate is a silica gel G thin-layer plate.

[0034] In some embodiments of the present invention, the silica gel G thin-layer plate is a prefabricated silica gel G plate from Tianjin Sida, Merck, or Qingdao Ocean. The results show that the method has good durability and can meet the identification requirements.

[0035] In some embodiments of the present invention, the developing agent is ethyl acetate - methanol - glacial acetic acid.

[0036] In some embodiments of the present invention, the mass ratio of ethyl acetate - methanol - glacial acetic acid is 2:1:1.

[0037] Spray with ninhydrin solution and heat for inspection.

[0038] The mass concentration of the ninhydrin solution is 1% - 5%; the temperature for heating and inspection is 105°C.

[0039] In some embodiments of the present invention, it is as follows:

[0040] According to the thin-layer chromatography method (General Rule 0502 of the Chinese Pharmacopoeia 2020 Edition), take the above test solution and reference substance solution, respectively spot them on the same silica gel G thin-layer plate, use ethyl acetate - methanol - glacial acetic acid (2:1:1) as the developing agent, develop, take out, dry, spray with 1% - 5% ninhydrin test solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, at the corresponding positions of the chromatograms of the reference substance and the control crude drug, spots of the same color appear.

[0041] In the preferred embodiment of the present invention, the sample application amount of the thin-layer chromatography test sample is 2 - 8 μL, the sample application amount of the control crude drug solution is 2 - 8 μL, and the sample application amount of the reference substance is 1 - 4 μL.

[0042] If the test sample and the control crude drug solution have fluorescent spots of the same color at the same Rf value, and at the same time have fluorescent spots at Rf = 0.225 - 0.257, 0.495 - 0.605, 0.599 - 0.732, then it is Canavalia ensiformis extract; if there are no fluorescent spots at Rf = 0.225 - 0.257, 0.495 - 0.605, 0.599 - 0.732, then it is Canavalia gladiata extract.

[0043] In one specific embodiment, the Rf values are 0.25, 0.55, and 0.666.

[0044] The inspection temperature of the present invention is 105 °C.

[0045] This method has good durability for different temperatures. When compared with the chromatogram of the control medicinal material, the test sample chromatogram and the control medicinal material chromatogram show spots of the same color at the corresponding positions.

[0046] The inspection humidity of the present invention is 32% rh to 75% rh.

[0047] This method has good durability for different humidities. When compared with the chromatogram of the control medicinal material, the test sample chromatogram and the control medicinal material chromatogram show spots of the same color at the corresponding positions.

[0048] The present invention provides a thin-layer identification method for Canavalia gladiata extract and Canavalia ensiformis extract, including: A thin-layer identification method for Canavalia gladiata extract and Canavalia ensiformis extract, including: A) Pretreat the Canavalia gladiata extract to obtain a test sample solution; pretreat the Canavalia ensiformis extract to obtain a Canavalia ensiformis extract solution; B) Take the control medicinal material of Canavalia gladiata, decoct with water, filter and add methanol to obtain a control medicinal material solution; add the reference substance to a methanol solution to obtain a reference substance solution; C) Perform thin-layer chromatography detection on the test sample solution, Canavalia ensiformis extract solution, reference substance solution and control medicinal material solution, and the thin-layer plate is a silica gel G thin-layer plate; the developing agent is ethyl acetate - methanol - glacial acetic acid; D) Spray with ninhydrin solution, heat and inspect. If the test sample and the control medicinal material solution have fluorescent spots of the same color at the same Rf value, and at the same time have fluorescent spots at Rf = 0.225 - 0.257, 0.495 - 0.605, 0.599 - 0.732, it is Canavalia gladiata extract; if there are no fluorescent spots at Rf = 0.225 - 0.257, 0.495 - 0.605, 0.599 - 0.732, it is Canavalia ensiformis extract. The thin-layer identification chromatographic method established by the present invention is used for quickly, accurately and effectively identifying Canavalia gladiata extract. At the same time, by observing whether there are fluorescent spots within a certain range of the specific migration value in the thin-layer chromatogram as the identification point, it is used to distinguish Canavalia gladiata extract and Canavalia ensiformis extract. Description of the Drawings

[0049] Figure 1 Investigation of the sample application amount;

[0050] Figure 2 Investigation of specificity;

[0051] Figure 3 Investigation for different thin-layer plates - Tianjin Siliida;

[0052] Figure 4 Investigation for different thin-layer plates - Merck;

[0053] Figure 5 Investigation of different thin-layer plates - Qingdao Ocean;

[0054] Figure 6 Different temperature - 4°C;

[0055] Figure 7 Different temperature - 25°C;

[0056] Figure 8 Different humidity - 32%;

[0057] Figure 9 Different humidity - 75%;

[0058] Figure 10 Verification of different batches;

[0059] Figure 11 For the determination of the Rf value of the identification points of canavalia extract and jack bean extract;

[0060] Figure 12 The method result diagram of Comparative Example 1;

[0061] Figure 13 The method result diagram of Comparative Example 2. Detailed implementation mode

[0062] In order to further illustrate the present invention, the following is a detailed description of a thin-layer identification method for canavalia extract and jack bean extract provided by the present invention in combination with embodiments.

[0063] Ultrasonic machine, heating plate, water bath, mortar, thin-layer imaging system: CAMAG TLC Visualizer, silica gel G thin-layer plate (Qingdao Ocean Chemical Factory, batch number: 20180527, Tianjin Silitda Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353)

[0064] Methanol, ethyl acetate, silica gel, glacial acetic acid, ninhydrin are all of analytical grade, and water is ultrapure water (self-made in the laboratory).

[0065] Jack bean control medicinal materials (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 240072-202204), threonine (China Food and Drug Inspection Institute, batch number: 140682-201302), valine (China Food and Drug Inspection Institute, batch number: 140681-201703), leucine (China Food and Drug Inspection Institute, batch number: 140687-201905), Jack bean extract (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 230401, 230402, 230403). Jack bean extract extract (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 230301, 230302, 230303).

[0066] Example 1

[0067] Preparation of test solution

[0068] Take 0.5g of this product, grind it into powder, add 20ml of methanol, ultrasonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to dissolve the residue, add ethyl acetate and shake to extract 3 times, 10ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve in 5ml of water, add to a silica gel column (160-200 mesh, 10g, inner diameter 1.8cm, dry column), elute with 20ml of methanol, collect the eluate, evaporate to dryness, add 1ml of methanol to dissolve the residue, and use it as the test solution.

[0069] Preparation of reference medicinal materials and reference substance solutions

[0070] Take 1g of Jack Bean reference medicinal material, grind it into powder, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate, add 20ml of methanol to the residue, and prepare the reference medicinal material solution in the same way. Take threonine, valine, and leucine reference substances, add methanol to prepare a mixed solution containing 0.5mg per 1ml as the reference substance solution.

[0071] Determination method

[0072] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 General Rules 0502), 6 μl of the test solution and 3 μl of the reference solution were taken and spotted on the same silica gel G thin layer plate, respectively, with ethyl acetate-methanol-glacial acetic acid (2:1:1) as the developing agent, developed, taken out, dried, sprayed with 1% to 5% ninhydrin test solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference substance and the reference medicinal material.

[0073] Example 2 Methodological Investigation

[0074] 2.1 Sampling quantity inspection

[0075] Under the above-mentioned experimental conditions, 1 μl, 2 μl, 3 μl, and 4 μl of the mixed reference substance solution of threonine, valine, and leucine, and 2 μl, 4 μl, 6 μl, and 8 μl of the solution of the control crude drug of Canavalia gladiata and the solution of the extract of Canavalia gladiata were respectively spotted on the same silica gel G thin-layer plate. Develop and examine according to the established thin-layer chromatography conditions. The results are shown in Figure 1 , as can be seen from the figure, when the spotted amounts of the mixed reference substance solution are 1 - 4 μl, the spotted amounts of the solution of the control crude drug are 2 - 8 μl, and the spotted amounts of the test solution are 2 - 8 μl, the fluorescent spots of the thin-layer chromatography are clearly developed. It is determined that the spotted amount of the mixed reference substance solution is 1 - 4 μl, and the spotted amounts of the solution of the control crude drug and the test solution are 2 - 8 μl. Compared with the reference substance and the control crude drug chromatograms, the test solution chromatogram shows spots of the same color at the corresponding positions as the reference substance and the control crude drug chromatograms.

[0076] Figure 1 Investigation of spotted amount. Note: 1, 4, 7, 10 are the spotted amounts of 1 μl, 2 μl, 3 μl, 4 μl of the mixed reference substance of threonine, valine, and leucine; 2, 5, 8, 11 are the spotted amounts of 2 μl, 4 μl, 6 μl, 8 μl of the control crude drug of Canavalia gladiata; 3, 6, 9, 12 are the spotted amounts of 2 μl, 4 μl, 6 μl, 8 μl of the extract of Canavalia gladiata.

[0077] 2.2 Specificity investigation

[0078] Prepare the mixed reference substance solution, the solution of the control crude drug of Canavalia gladiata, the solution of the extract of Canavalia gladiata, and the negative solution according to the above test solution preparation method, and spot them on the same thin-layer plate respectively. Develop and examine according to the established thin-layer chromatography conditions. The results are shown in Figure 2 , and the results show that the negative sample has no interference with the test solution of the extract of Canavalia gladiata, and the method has good specificity and can be used to identify the solution of the extract of Canavalia gladiata.

[0079] Figure 2 Specificity investigation. Note: 1 is the negative solution; 2 is the mixed reference substance; 3 is the control crude drug of Canavalia gladiata; 4 is the extract of Canavalia gladiata

[0080] 2.3 Investigation of durability

[0081] 2.3.1 Comparison of different thin-layer plates

[0082] Select the precast silica gel G plates of Tianjin Sida, Merck, and Qingdao Ocean, and conduct tests according to the established test methods respectively. See Figures 3 - 5 , and the results show that the thin-layer plates of all 3 brands can meet the identification requirements, indicating that the method has good durability. Compared with the reference substance and the control crude drug chromatograms, the test solution chromatogram shows spots of the same color at the corresponding positions as the reference substance and the control crude drug chromatograms.

[0083] The results are as Figure 3 , Figure 3Investigation of different thin layer plates - Tianjin Sida. Note: Note: 1 is the mixed reference substance; 2 is the control crude drug of Canavalia gladiata; 3 is the extract of Canavalia gladiata. Figure 4 Investigation of different thin layer plates - Merck. Note: 1 is the mixed reference substance; 2 is the control crude drug of Canavalia gladiata; 3 is the extract of Canavalia gladiata. Figure 5 Investigation of different thin layer plates - Qingdao Ocean. Note: Note: 1 is the mixed reference substance; 2 is the control crude drug of Canavalia gladiata; 3 is the extract of Canavalia gladiata.

[0084] 2.3.2 Comparison at different temperatures

[0085] Take the thin layer plates after spotting and develop them respectively in the temperature environments of low temperature 4°C and normal temperature 25°C. It can Figures 6 - 7 be seen that the durability of this method to different temperatures is good. Compared with the reference substance and control crude drug chromatograms, the test sample chromatogram shows spots of the same color at the corresponding positions as the reference substance and control crude drug chromatograms.

[0086] Figure 6 Different temperature 4°C; Note: 1 is the mixed reference substance; 2 is the control crude drug of Canavalia gladiata; 3 is the extract of Canavalia gladiata. Figure 7 Different temperature 25°C; Note: Note: 1 is the mixed reference substance; 2 is the control crude drug of Canavalia gladiata; 3 is the extract of Canavalia gladiata.

[0087] 2.3.3 Comparison at different humidities

[0088] Take the thin layer plates after spotting and develop them respectively in the humidity environments of 32% and 75%, as shown Figures 8 - 9 . It can be seen from the figure that the durability of this method to different humidities is good. Compared with the reference substance and control crude drug chromatograms, the test sample chromatogram shows spots of the same color at the corresponding positions as the reference substance and control crude drug chromatograms.

[0089] Figure 8 Different humidity - 32%; Note: 1 is the mixed reference substance; 2 is the control crude drug of Canavalia gladiata; 3 is the extract of Canavalia gladiata; Figure 9 Different humidity - 75%; Note: 1 is the mixed reference substance; 2 is the control crude drug of Canavalia gladiata; 3 is the extract of Canavalia gladiata.

[0090] 2.3.4 Verification

[0091] Perform thin layer identification verification on 3 batches of Canavalia gladiata extracts. The experimental results are shown Figure 10 . The results show that compared with the reference substance and control crude drug chromatograms, the test sample chromatogram shows spots of the same color at the corresponding positions as the reference substance and control crude drug chromatograms.

[0092] Figure 10 Verification of different batches. Note: 1 Control crude drug of Canavalia gladiata; 2 - 4 are Canavalia gladiata extracts SY2211001; SY2211002; SY2211003.

[0093] 2.4 Determination of the Rf values of the identification points of the jack bean extract and the Canavalia gladiata extract

[0094] The jack bean extract and the Canavalia gladiata extract were assayed by the same method as in Example 1, and the results are as Figure 11 shown Figure 11 for the determination of the Rf values of the identification points of the jack bean extract and the Canavalia gladiata extract; Note: 1 is the mixed reference substance; 2 is the control crude drug of jack bean; 3-5 are the jack bean extracts 230401, 230402, and 230403; 6-8 are the Canavalia gladiata extracts

[0095] The results showed that at the positions of the specific migration values of 0.25, 0.55, and 0.666, fluorescent spots appeared in the jack bean extract, while there were no obvious fluorescent spots in its adulterated product, the Canavalia gladiata extract, which could be used as the identification points to distinguish between the jack bean and the Canavalia gladiata extracts

[0096] Comparative Example 1

[0097] Take 0.5 g of this product, grind it finely, add 20 ml of dilute ethanol, ultrasonicate for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of dilute ethanol to obtain the test solution. Take 1 g of the control crude drug of jack bean, grind it finely, add 50 ml of water, decoct for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of dilute ethanol to obtain the control crude drug solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition), 5 μl of the above-mentioned test solution and the control crude drug solution were respectively spotted on the same silica gel G thin-layer plate, and ethyl acetate - glacial acetic acid - methanol (9:3:1) was used as the developing agent. After development, take out, dry in air, spray with ninhydrin test solution, and heat at 105 °C until the spots are clearly developed. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference substance and the control crude drug. The experimental results are shown in Figure 12 .

[0098] Figure 12 is the method result diagram of Comparative Example 1. Note: 1-3 are the spotting amounts of 2 μl, 5 μl, and 8 μl of the control crude drug of jack bean; 4-5 are the spotting amounts of 2 μl, 5 μl, and 8 μl of the jack bean extract

[0099] The results showed that by using the above-mentioned thin-layer identification method, there were no obvious spots in the jack bean extract, and the two could not be identified

[0100] Comparative Example 2

[0101] Take 0.5 g of this product, grind it finely, add 20 ml of dilute ethanol, sonicate for 20 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of dilute ethanol to obtain the test solution. Take 1 g of the control crude drug of Canavalia gladiata, grind it finely, add 50 ml of water, decoct for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of dilute ethanol to obtain the control crude drug solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition), 5 μl of the above-mentioned test solution and control crude drug solution were each taken and spotted on the same silica gel G thin-layer plate. Cyclohexane-formic acid-acetone (10:2:1) was used as the developing solvent, developed, taken out, dried, sprayed with ninhydrin test solution, and heated at 105 °C until the spots developed clearly. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatograms of the reference substance and the control crude drug. The experimental results are shown in Figure 13 . Figure 13 It is the result diagram of the method of Comparative Example 2. Note: 1-2 are the spotting amounts of 2 μl and 5 μl of the control crude drug of Canavalia gladiata; 3-4 are the spotting amounts of 2 μl and 5 μl of the Canavalia gladiata extract.

[0102] The results showed that there was no obvious difference in the Canavalia gladiata extract by the above-mentioned thin-layer identification method, and the two could not be distinguished.

[0103] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A TLC identification method for canavalia extract and jack bean extract, comprising: A) Pretreat the canavalia extract to obtain a test solution; Pretreat the jack bean extract to obtain a jack bean extract solution; The pretreatment of the canavalia extract or jack bean extract is specifically as follows: a) Add methanol to the canavalia extract or jack bean extract, ultrasonicate, filter, evaporate to dryness, dissolve the residue in water, shake and extract with ethyl acetate to obtain an ethyl acetate layer extract; b) Evaporate the ethyl acetate layer extract to dryness, dissolve in water, load onto a silica gel column, elute with methanol, collect the eluate, evaporate to dryness, and dissolve the residue in methanol; B) Take the canavalia reference crude drug, decoct with water, add methanol after filtration to obtain a reference crude drug solution; Add methanol solution to the reference substance to obtain a reference substance solution; C) Perform TLC detection on the test solution, jack bean extract solution, reference substance solution and reference crude drug solution. The TLC plate is a silica gel G TLC plate; The developing solvent is ethyl acetate - methanol - glacial acetic acid; The mass ratio of ethyl acetate - methanol - glacial acetic acid is 2:1:1; D) Spray with ninhydrin solution, heat and examine. If the test solution and the reference crude drug solution have fluorescent spots of the same color at the same Rf value, and at the same time have fluorescent spots at Rf = 0.225 - 0.257, 0.495 - 0.605, 0.599 - 0.732, it is canavalia extract; If there are no fluorescent spots at Rf = 0.225 - 0.257, 0.495 - 0.605, 0.599 - 0.732, it is jack bean extract.

2. The method according to claim 1, wherein In step a), the mass - volume ratio of the canavalia extract / jack bean extract, methanol, water, and ethyl acetate is 0.5 g:20 mL:10 mL:30 mL; The time of ultrasonication is 20 - 30 min; In step b), the volume ratio of water and methanol is 5:20; The specifications of the silica gel column are: 160 - 200 mesh, 10 g, inner diameter 1.8 cm, dry - packing the column.

3. The method according to claim 1, wherein In step B), the mass - volume ratio of the canavalia reference crude drug and water is 1 g:50 mL; The decocting time is 20 - 30 min; The reference substances include threonine, valine and leucine; The concentration of the reference substances is 0.5 mg / mL.

4. The method according to claim 1, characterized in that, The sample application amount of the test solution for TLC is 2 - 8 μL, the sample application amount of the reference crude drug solution is 2 - 8 μL, and the sample application amount of the reference substance solution is 1 - 4 μL.

5. The method according to claim 1, wherein In step D), the mass concentration of the ninhydrin solution is 1% - 5%; The temperature for heating and examination is 105 °C.

6. The method according to claim 1, characterized in that, In step D), the silica gel G TLC plate is Tianjin Sili - da, Merck or Qingdao Ocean pre - fabricated silica gel G plate.

7. The method according to claim 1, characterized in that In step D), the examination temperature is 105 °C; The humidity for examination is 32%rh - 75%rh.

8. The method according to claim 1, wherein In step D), the Rf values are 0.25, 0.55, 0.666.

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