Euonymus alatus detection method based on thin-layer chromatography

Abstract

The invention discloses a method for detecting euonymus alatus based on thin-layer chromatography. According to the method, stigmas-4-ene-3-ketone is selected as a characteristic chemical marker, and a thin-layer chromatography identification system with toluene-ethyl acetate-formic acid (20: 1: 0.5) as a developing system is established. According to the method, consistent fluorescence quenching spots can be clearly detected on a silica gel GF254 thin-layer plate under an ultraviolet lamp (254nm) at a position (Rf value is 0.48 + / -0.03) corresponding to a reference substance. In addition, the invention further provides an efficient preparation process of the reference substance and a detection kit containing the key components, and the technical bottleneck that euonymus alatus medicinal materials and decoction pieces lack quality control of standardization, high specificity and good reproducibility for a long time is effectively solved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality control and testing technology, specifically to a method for identifying the authenticity of Euonymus alatus medicinal materials, processed slices and their preparations based on thin-layer chromatography (TLC). Background Technology

[0002] Euonymus alatus, or the winged young branches or wing-like parts of Euonymus alatus (Thunb.) Sieb., a plant in the Celastraceae family, is a traditional Chinese medicine. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is listed as a medium-grade herb and is used for its effects of promoting blood circulation, regulating menstruation, detoxifying, reducing swelling, and killing parasites. This herb was included in the 1963 edition of the *Chinese Pharmacopoeia*. Although it has not been included in subsequent editions, it has been included in the *Dictionary of Traditional Chinese Medicine* and in the standards and processing specifications of medicinal materials in several provinces and cities, including Hubei, Hunan, Liaoning, Beijing, and Shanghai. This reflects the clear medicinal value and the practical difficulties in its standardization process.

[0003] In recent years, with the increasing clinical application, the resource and market issues of Euonymus alatus have become increasingly prominent. Its original plant is widely distributed in my country, mainly in the wild. Because Euonymus alatus has many aliases such as Euonymus trifoliata, Euonymus scabra, Euonymus nigra, and Euonymus scabra, and its appearance is extremely similar to plants in the same family such as Euonymus trifoliata and Euonymus scabra, and even plants from different families such as Ulmus pumila, counterfeit products are prevalent in the market, seriously affecting the clinical efficacy and safety of the medicinal material.

[0004] Existing identification techniques rely heavily on the experience of testing personnel, making it difficult to meet the needs of grassroots units and distribution channels for rapid, large-scale quality inspection. Thin-layer chromatography (TLC), as a simple and rapid identification method, holds significant value in the quality control of traditional Chinese medicine. Traditional identification methods primarily depend on the observation of the original plant morphology, medicinal material characteristics, and microscopic features. These methods not only heavily depend on the personal experience of testing personnel but also become significantly more difficult after the medicinal material is broken or processed. Although TLC has been attempted for the identification and analysis of Euonymus alatus, existing methods generally suffer from insufficient selectivity of the developing system, unclear characteristic spots, or unsatisfactory reproducibility. More critically, existing TLC methods lack specific chemical markers that can clearly indicate the species specificity of Euonymus alatus, leading to discrepancies in identification results between different laboratories and hindering the establishment of unified quality control standards. This technical bottleneck prevents existing identification methods from meeting the high standards of regulations such as the Chinese Pharmacopoeia, severely restricting the standardization process of Euonymus alatus quality control.

[0005] Therefore, developing a standardized and reproducible method for identifying genuine and counterfeit Euonymus alatus based on clear chemical markers has become an urgent technical need to address market chaos, ensure medication safety, and promote the standardization of this medicinal material. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for detecting Euonymus alatus based on thin-layer chromatography.

[0007] The specific technical details are as follows:

[0008] One of the inventions provided in this application is a method for detecting Euonymus alatus based on thin-layer chromatography. Using stigmaster-4-en-3-one as a marker, this method is used to detect the authenticity of Euonymus alatus, and specifically includes the following steps:

[0009] S1. Preparation of test solution: Take the powder of the sample to be tested, add methanol for ultrasonic extraction, filter, concentrate the filtrate and redissolve to obtain the test solution;

[0010] S2. Preparation of reference solution: Take stigmaster-4-en-3-one reference standard, dissolve it in methanol to prepare a reference solution with a concentration of 0.8~1.2 mg / mL;

[0011] S3. Thin-layer chromatography spotting and development: Take the test solution and the reference solution and spot them on the same silica gel thin-layer plate. Develop the plate using a developing solvent composed of toluene, ethyl acetate and formic acid in a volume ratio of (18~22):(0.8~1.2):(0.4~0.6). Remove the plate and air dry.

[0012] S4. Result Interpretation: Under 254nm ultraviolet light, if the chromatogram of the test sample shows the same fluorescent quenched spot at the corresponding position as the chromatogram of the reference sample, i.e., with an Rf value of 0.48±0.03, then the test sample is determined to be Euonymus alatus; if the spot is not visible, it is determined to be a counterfeit product.

[0013] Preferably, in step S1, the amount of the sample powder to be tested is 1.5~2.5g, and the volume of methanol added is 25~35mL; the power of the ultrasonic extraction is 200~300W, the frequency is 35~45kHz, and the time is 25~35min.

[0014] Preferably, in step S3, the sample volume of both the test solution and the reference solution is 4~6μL; the silica gel thin-layer plate is a silica gel GF254 thin-layer plate with a thickness of 0.20~0.25mm.

[0015] Preferably, this method is used for the quality control of Euonymus alatus medicinal materials and processed medicinal slices.

[0016] The second invention provided in this application is a method for preparing stigmaster-4-en-3-one, comprising the following steps:

[0017] P1. Extraction: Take dried winged young branches or wing-like powder of Euonymus alatus, add 70-80% ethanol solution, the ratio of powder to ethanol solution is 1:8-1:12 (g / mL), heat and reflux to extract 1-3 times, each time for 0.75-1.5 hours, combine the extracts and concentrate under reduced pressure to obtain extract.

[0018] P2. Enrichment and purification: The extract was mixed with silica gel and subjected to silica gel column chromatography, with isocratic elution using petroleum ether-ethyl acetate at a volume ratio of (18~22):1 and a boiling range of 60~90℃ as the eluent.

[0019] P3. Collection and preparation: Thin-layer chromatography was used to monitor the elution fractions. The eluent was developed using a solvent composed of toluene, ethyl acetate and formic acid in a volume ratio of (18~22):(0.8~1.2):(0.4~0.6). The fractions showing characteristic spots at an Rf value of 0.48±0.03 were collected, combined, concentrated and dried to obtain crude stigmaster-4-en-3-one.

[0020] Furthermore, step P4 follows step P3: 1 g of the obtained product is mixed with 80-100 mesh silica gel and loaded onto a 200-300 mesh silica gel column (0.8 cm diameter, 10 cm height). Elution with 50 mL of methanol is performed, and the methanol is recovered under reduced pressure. This yields the thin-layer chromatography identification standard, stigmaster-4-en-3-one.

[0021] The third invention provided in this application is a process design for a thin-layer chromatography detection kit for identifying the authenticity of Euonymus alatus, including:

[0022] (a) Stigmaster-4-en-3-one reference solution;

[0023] (b) A developing solvent composed of toluene, ethyl acetate and formic acid in a volume ratio of (18~22):(0.8~1.2):(0.4~0.6).

[0024] Based on the above-described invention, the technical effects achieved by this application are as follows:

[0025] This application provides a thin-layer chromatography identification method based on the specific chemical marker stigmaster-4-en-3-one, achieving accurate and reliable differentiation of Euonymus alatus and its common adulterants. This method is highly specific, consistently detecting this characteristic spot in genuine Euonymus alatus, while it is absent in adulterants such as Euonymus trifoliata, Euonymus pubescens, and Ulmus pumila, effectively eliminating false positive and false negative results.

[0026] The thin-layer chromatography method established in this application is simple to operate, has good reproducibility, and all key parameters have been systematically optimized, fully meeting the quality control requirements of high-standard regulations such as the Chinese Pharmacopoeia. Simultaneously, this application provides a process for the rapid preparation of stigmaster-4-en-3-one reference standards from Euonymus alatus itself. This process eliminates the need for expensive purification equipment, yielding thin-layer identification reference standards within 24 hours, reducing costs by more than 70%, and solving the bottleneck problem of difficult-to-obtain reference standards in standardized identification. Attached Figure Description

[0027] Figure 1 Silica gel column chromatography separation effect: Thin-layer chromatography (TLC) analysis.

[0028] (A) Fraction Monitoring: This section presents the results of TLC analysis of each fraction (F1, F2, ...) using silica gel plates. The separation of the target compound from impurities is shown under ultraviolet light.

[0029] (B) Merging and validation: The selected fractions were merged and then analyzed by TLC again, which showed spots of the target compound with high purity;

[0030] Figure 2 Schematic diagram of the chemical structure of stigmaster-4-en-3-one;

[0031] Figure 3 Thin-layer chromatography identification diagrams of Euonymus alatus and its adulterants, including: 1. Reference sample; 2-6. Euonymus alatus G1-G5; 7-9. Euonymus truncatus S11-S13S; 10. Euonymus pubescens M14; 11. Ulmus pumila D15; 12-16. Euonymus alatus G6-G10. Detailed Implementation

[0032] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase.

[0033] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0034] In the following description of the embodiments, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of this application. However, those skilled in the art will understand that this application may also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods are omitted so as not to obscure the description of this application with unnecessary detail.

[0035] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.

[0036] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0037] As used in this application specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrase "if determined" or "if detected [the described condition or event]" may be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once detected [the described condition or event]," or "in response to detection [the described condition or event]."

[0038] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0039] References to "one embodiment" or "some embodiments" in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0040] Example 1: Screening and Validation of Characteristic Chemical Markers

[0041] 1. Analysis of the shortcomings of existing technologies For a long time, the chromatographic identification of Euonymus alatus has mainly relied on comparison with the overall fingerprint spectrum of the reference medicinal material, or on empirical judgment based on the unknown component spots at the Rf value. Due to the lack of clear chemical substances as reference standards, the existing methods have many drawbacks in practical applications: (1) Poor reproducibility: Due to the influence of environment and consumables in different laboratories, the Rf value of unknown spots drifts seriously, making it difficult to locate them accurately; (2) Lack of traceability of standards: It is impossible to control the purity or assess the content of the characteristic spots, resulting in the subjectivity of the identification results; (3) Questionable specificity: In the absence of structural confirmation, it is difficult to rule out false positive interference from other co-substitute components.

[0042] 2. Discovery and Structural Confirmation of the Marker: To establish a standardized and quantifiable quality control system, the inventors enriched a high-purity monomeric compound from Euonymus alatus. Spectroscopic analysis confirmed (specific data in Example 4) that this compound is stigmaster-4-en-3-one.

[0043] 3. Establishment and Optimization of the Identification Method: Based on the clarified chemical structure of the characteristic marker, the inventors investigated and optimized the thin-layer chromatography developing system. Multiple systems, including toluene-ethyl acetate, dichloromethane-methanol, and petroleum ether-acetone, were compared experimentally. The results showed that when toluene-ethyl acetate-formic acid (20:1:0.5) was used as the developing solvent, the stigmaster-4-en-3-one spots were round, had a moderate Rf value (0.48±0.03), and could achieve baseline separation from other background impurities.

[0044] 4. Summary This invention, through systematic chemical component screening and structural confirmation, has for the first time identified stigmaster-4-en-3-one as a characteristic chemical marker for the quality control of Euonymus alatus. The thin-layer chromatography detection system established based on this fills the long-standing gap in specific chemical reference standards for this medicinal material, effectively avoiding the poor reproducibility and false positive risks caused by unclear components or dependence on physical properties in traditional identification methods. This provides crucial technical support for the standardized quality control of Euonymus alatus.

[0045] Example 2: Summary of Main Technical Contributions and Beneficial Effects

[0046] This embodiment aims to systematically illustrate the inventive contributions of the present invention compared to the prior art and its practical application value, specifically reflected in the following three core aspects:

[0047] To address the shortcomings of existing technologies that rely on experience and lack clear chemical indicators for identifying genuine Euonymus alatus, this invention establishes stigmaster-4-en-3-one as a characteristic chemical marker for quality control of Euonymus alatus. By using a specific development system (toluene-ethyl acetate-formic acid), and leveraging the absorption characteristics of the conjugated double bonds of this compound under 254 nm ultraviolet light, sensitive detection of dark spots (fluorescence quenching) against a fluorescent background was achieved. Experiments confirmed that this marker is highly specific in genuine Euonymus alatus, but not detected in morphologically similar adulterants such as Euonymus trifoliata, Euonymus trifoliata var. truncatula, and Ulmus pumila, effectively solving the technical problems of "easily confused" in traditional morphological identification and "no standard to rely on" in traditional thin-layer chromatography identification.

[0048] To address the high cost of commercially available stigmasterone reference standards, this invention presents a novel, rapid preparation process that "comes from the medicinal material and goes back to the medicinal material." This process requires only conventional 75% ethanol extraction combined with simple silica gel column chromatography, eliminating the need for expensive equipment such as the Sephadex LH-20 or preparative HPLC. A reference standard with purity meeting identification requirements can be obtained within 24 hours. This technological breakthrough significantly reduces detection costs (by more than 70%), removing economic barriers to the widespread adoption of this method.

[0049] Based on the aforementioned core method, this invention further develops a thin-layer chromatography detection kit for Euonymus alatus (see Example 7 for details). This kit integrates standardized reference standards, pre-fabricated silica gel GF254 plates, and an optimized developing solvent system, transforming complex laboratory analysis into a standardized product solution. The introduction of this kit breaks the deadlock in quality control efforts at grassroots drug testing institutions and in the distribution market of traditional Chinese medicine materials, which were previously hampered by a lack of reference standards and standard methods. It enables point-of-care testing (POCT) and significantly improves the regulatory efficiency of this medicinal material distribution process.

[0050] The technical solution of the present invention has the following beneficial effects: (1) High specificity: 10 batches of Euonymus alatus from different places were positive, and 5 counterfeit products were negative; (2) Simple operation: TLC method only requires conventional laboratory equipment and can be completed within 30 minutes; (3) Easy to obtain reference standards: preparation cycle ≤ 24 hours, cost reduced by more than 70%; (4) High practical value: applicable to the quality control of medicinal materials and decoction pieces, and can provide technical support for the revision of the Chinese Pharmacopoeia standard.

[0051] Example 3: Pretreatment and chromatographic condition optimization for thin-layer chromatography identification of Euonymus alatus.

[0052] In order to establish a highly specific and reproducible identification method, the inventors conducted a comprehensive investigation and screening of the extraction solvent for the test sample, the selection of reference standards, the development system, and the colorimetric method.

[0053] Preparation of test sample: Take 2.0 g of the powder of this product, add 30 mL of the corresponding solvent, sonicate for 30 min, filter, evaporate the filtrate to dryness, and dissolve the residue in 1 mL of the corresponding solvent.

[0054] Sample volume: 5~10μL for both the test sample and the reference solution.

[0055] The inventors designed five different experimental schemes for screening experiments, and the results are shown in Table 1.

[0056] Table 1. Effects of different thin-layer chromatography conditions on separation efficiency

[0057] Experiment number Extraction solvent Reference standards / standard substances Stationary phase (thin-layer plate) Effervescent solvent system (v / v) Colorimetric / Inspection Methods Description of experimental phenomena and results (key columns) Overall evaluation 1 Ethyl acetate β-sitosterol, lupeol Silicone G-plate (homemade) Petroleum ether-acetone (5:1) Spray with 5% sulfuric acid ethanol, heat to 105°C, and inspect under sunlight. The spots were numerous and crowded; β-sitosterol is a common plant component and lacks species specificity, and it was detected positive in many adulterants. Not applicable (poor specificity) 2 Ethyl acetate Oleanolic acid, Tripterygium wilfordii A Silicone G-plate (homemade) Dichloromethane-methanol-formic acid (15:1:0.1) Same as above Oleanolic acid spots were clear but not specific; Tripterygium wilfordii lactone was not detected or present in very low amounts in multiple batches of Euonymus alatus, and could not be used as a stable indicator. Not applicable (indicators are unstable) 3 methanol hesperidin Silicone G-plate (homemade) Dichloromethane-methanol (10:1) Examined under ultraviolet light (365nm) The background fluorescence interference is strong, the Rf value of the target spot is unstable, the tailing phenomenon is severe, and the resolution is <1.0. Not applicable (poor separation) 4 methanol Naringin Silicone G-plate (homemade) Dichloromethane-methanol (15:2) Spray with 5% vanillin-sulfuric acid ethanol and heat at 105°C After color development, the spots fade quickly over time, and the background rapidly turns red and black, resulting in poor durability and reproducibility. Not applicable (color development is unstable) This invention methanol Reference medicinal materials Silicone GF254 board (prefabricated) Toluene-ethyl acetate-formic acid (20:1:0.5) Examined under ultraviolet light (254nm) Clear and round fluorescent quenching spots are observed at an Rf value of 0.48±0.03; the background is clean and free of impurities; genuine products are positive and counterfeit products are negative. Best (high specificity, good reproducibility)

[0058] Example 4: Preparation, structural confirmation and species distribution verification of stigmaster-4-en-3-one.

[0059] 1. Screening and optimization of extraction solvents

[0060] To achieve optimal extraction efficiency and ensure the safety and environmental friendliness of the preparation process, the inventors screened extraction solvents.

[0061] Screening Strategy Explanation: Although methanol is a commonly used and excellent solvent in phytochemical separation, its high neurotoxicity poses occupational health risks and environmental pressures in large-scale preparation or industrial production. To establish a green, safe, and easily applicable preparation process, this invention pre-excludes the methanol system and focuses on the safer ethanol-water system.

[0062] The inventors compared the extraction effects and impurities when water and different concentrations of ethanol (75% and 95%) were used as extraction solvents. The TLC monitoring results are shown in Table 2.

[0063] Table 2 Comparison of extraction efficiency and impurity levels of different extraction solvents for target components

[0064] Extraction solvent Target component detection status (TLC) Impurities and interference Overall evaluation water The target spot is not clear. Too many polar impurities, complex background give up 95% ethanol Able to extract target components Excessive dissolution of fat-soluble pigments such as chlorophyll leads to difficulties in subsequent separation. Not recommended 75% ethanol Target spots are clear The number of fat-soluble impurities was significantly less than that in the 95% ethanol group. Preferred

[0065] 2. Preparation and Purification

[0066] Take 50g of dried winged twigs or wing-like powder of Euonymus alatus (Thunb.) Sieb., place it in a 2000mL stoppered conical flask, add 500mL of 75% ethanol, heat and reflux for 1h, repeat the extraction once with the residue, combine the two extracts, concentrate under reduced pressure to dryness, and obtain 2.13g of extract. Dissolve the above extract in 10mL of methanol, add 3g of silica gel (200-300 mesh), stir evenly, evaporate the solvent until no alcohol odor remains, and obtain the dry loading material. Pack the material into a silica gel column (silica gel GF254, inner diameter 3cm, height 18cm), and elute isocratically with petroleum ether (60-90℃)-ethyl acetate (20:1, v / v), collecting one fraction per 10mL.

[0067] Thin-layer chromatography (TLC) was used to monitor each fraction (developing solvent: toluene-ethyl acetate-formic acid, 20:1:0.5, v / v; colorimetric assay: UV 254nm). The results are attached. Figure 1 As shown, the combined fraction exhibited a single fluorescent quenching spot at an Rf value of 0.48±0.03, with minimal interference from impurities, meeting the requirements for use as a thin-layer chromatography reference standard. The combined fraction was concentrated and dried to obtain 11.8 mg of a pale yellow oily substance.

[0068] To prepare a high-purity analytical grade standard that meets the structural confirmation requirements, the above-mentioned oily substance was further purified. The oily substance was mixed thoroughly with 1 g of silica gel (80-100 mesh) and loaded onto a silica gel column (stationary phase: 200-300 mesh silica gel; dimensions: inner diameter 0.8 cm × column height 10 cm). Elution was performed with 50 mL of methanol as the mobile phase. All eluent was collected, and the solvent was recovered under reduced pressure. After drying, 8.3 mg of a white amorphous powder was obtained. The nuclear magnetic resonance (NMR) spectra of the compound are as follows:

[0069] 1H-NMR (600 MHz, CDCl3) δ: 5.73 (1H, s, H-4), 2.24~2.45 (4H, m, H-2,6), 2.00~2.04 (2H, m, H-1a, 12a), 1.82~1.88 (2H, m, H-7a, 15a), 1.58~1.71(3H, m, H-1b, 24, 16a), 1.41~1.55 (3H, m, H-8, 11), 1.19~1.40 (5H, m, H-15b, 20, 22a, 28), 1.07~1.19 (5H, m, H-23, 17, 16b, 12b), 0.96~1.05 (3H, m,H-14, 7b, 22b), 0.90~0.95 (2H, m, H-9, 25), 1.17 (3H, s, H-19), 0.92 (3H, d,J = 6.5 Hz, H-21), 0.84 (3H, t, J = 6.8 Hz, H-29), 0.83 (3H, d, J = 6.8 Hz,H-26), 0.81 (3H, d, J = 6.8 Hz, H-27), 0.72 (3H, s, H18); 13C-NMR (150 MHz, CDCl3) δ: 199.7 (C-3), 171.7 (C-5), 123.7 (C-4), 56.0 (C-17), 55.9 (C-14), 53.8 (C9), 45.8 (C-25), 42.4 (C-13), 39.6 (C-12), 38.6 (C-10), 36.1 (C-20),35.7 (C-1), 35.6 (C-8), 34.0 (C-2), 33.9 (C-22),33.1 (C-6), 33.0 (C-7), 29.1(C-24), 28.2 (C-15), 26.1 (C-23), 24.2 (C-16), 23.1 (C-28), 21.0 (C-11), 19.8 (C-26), 19.0 (C-27), 18.7 (C-21), 17.4 (C-19), 12.0 (C18), 12.0 (C-29). The above data are basically consistent with the stigmasterols from Typha latifolia reported in the literature (Maria DG, Pietro M, Lucio P. Stigmasterols from Typha latifolia[J]. JNat Prod, 1990, 53(6): 1430-1435.), confirming the structure of this component.

[0070] Example 5: Standardized thin-layer chromatography method for identifying genuine and counterfeit Euonymus alatus.

[0071] This embodiment provides a standardized detection process based on the aforementioned characteristic chemical marker—stigmaster-4-en-3-one, which is applicable to the daily quality control of medicinal material markets, processed medicinal materials factories, and drug testing institutions.

[0072] Preparation of test solution: Take 2.0 g of the sample powder to be tested, place it in a stoppered conical flask, add 30 mL of methanol, and sonicate at 250 W and 40 kHz for 30 min. Filter the solution. Evaporate the filtrate to dryness, and dissolve the residue in 1.0 mL of methanol to obtain the test solution.

[0073] Preparation of reference solution: Take the stigmaster-4-en-3-one prepared according to the method in Example 4, add methanol to prepare a 1 mg / mL solution, which is used as the reference solution.

[0074] Take 5 μL of each of the two solutions mentioned above and spot them onto the same silica gel GF254 thin-layer plate (thickness 0.20–0.25 mm). Use toluene-ethyl acetate-formic acid (20:1:0.5, v / v) as the developing solvent, place the plate in a pre-saturated developing tank for 15 minutes, and develop it upwards at a distance of about 8 cm. Remove the plate, allow it to dry, and examine it directly under a 254 nm UV lamp.

[0075] If the chromatogram of the test sample shows a fluorescent quenching spot at an Rf value of 0.48±0.03, consistent with the position of the reference sample, it is determined to be genuine Euonymus alatus or contains Euonymus alatus components; if there is no spot at this position, it is determined to be a counterfeit product.

[0076] Example 6: Verification of Method Specificity and Robustness

[0077] This embodiment systematically verifies the specificity of the method described in Embodiment 5.

[0078] Specificity verification: Ten batches of Euonymus alatus from different origins and five batches of adulterants were tested according to the method in Example 5. The results are attached. Figure 3 As shown, all 10 batches of Euonymus alatus samples exhibited clear fluorescence quenching spots at an Rf value of 0.48±0.03, resulting in a 100% positive detection rate. The 5 batches of adulterants showed no spots at the corresponding Rf value positions, resulting in a 100% negative rate. This method is highly specific, free from false positives or false negatives, and can effectively distinguish Euonymus alatus from its adulterants. Detailed sample information is shown in Table 3.

[0079] Table 3. Sample source information and specificity verification results of Euonymus alatus and its adulterants.

[0080] serial number Plant names and Latin scientific names Collection site batch number result G-1 Euonymus alatus (Thunb.) Sieb. Sijiao, Xia County, Yuncheng City, Shanxi Province G202411-1 Genuine product (detected) G-2 Euonymus alatus (Thunb.) Sieb. Tunliu District, Changzhi City, Shanxi Province G202411-2 Genuine product (detected) G-3 Euonymus alatus (Thunb.) Sieb. Shuiyu Village, Ruicheng District, Yuncheng City, Shanxi Province G202108-1 Genuine product (detected) G-4 Euonymus alatus (Thunb.) Sieb. Lingdi Village, Ruicheng District, Yuncheng City, Shanxi Province G202108-2 Genuine product (detected) G-5 Euonymus alatus (Thunb.) Sieb. Manghe Village, Yangcheng County, Jincheng City, Shanxi Province G202108-3 Genuine product (detected) G-6 Euonymus alatus (Thunb.) Sieb. Lishan East Gorge, Qinshui County, Jincheng City, Shanxi Province G202108-3 Genuine product (detected) G-7 Euonymus alatus (Thunb.) Sieb. Shangluo City, Shaanxi Province G202108-4 Genuine product (detected) G-8 Euonymus alatus (Thunb.) Sieb. Yanbian Korean Autonomous Prefecture, Jilin Province G202108-5 Genuine product (detected) G-9 Euonymus alatus (Thunb.) Sieb. Lishui City, Zhejiang Province G202108-6 Genuine product (detected) G-10 Euonymus alatus (Thunb.) Sieb. Xinxiang City, Henan Province G202108-7 Genuine product (detected) S-11 Euonymus phellomanus Loes. ex Diels. Minxian County, Dingxi City, Gansu Province S202108-1 Counterfeit product (not detected) S-12 Euonymus phellomanus Loes. ex Diels. Lingdi Village, Ruicheng District, Yuncheng City, Shanxi Province S202108-2 Counterfeit product (not detected) S-13 Euonymus phellomanus Loes. ex Diels. Lishan East Gorge, Jincheng City, Shanxi Province S202108-3 Counterfeit product (not detected) M-14 Euonymushamiltonianus Wall. Lishan East Gorge, Jincheng City, Shanxi Province M202108 Counterfeit product (not detected) D-15 Large-fruited elm (Ulmus macrocarpa) Hance. Lishan East Gorge, Jincheng City, Shanxi Province D202108 Counterfeit product (not detected)

[0081] The results show that the proposed method is highly specific, reproducible, and stable, and meets the technical requirements for inclusion in drug standards.

[0082] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0083] 2. Applicability and Stability Assessment of the Method The establishment and validation experiments of this method spanned different seasons, covering the temperature and humidity fluctuations under normal laboratory conditions. Long-term experimental observations showed that although seasonal changes in environmental temperature and humidity may cause slight fluctuations in solvent evaporation rate and spot Rf values, the use of a reference standard as a parallel control (i.e., the reference standard and the test sample are spotted on the same plate) eliminated systematic errors caused by environmental factors. In repeated, long-term experiments, the genuine Euonymus alatus test sample consistently showed clear fluorescence quenching spots at the corresponding positions as the reference standard, with good separation performance. This confirms that this method has no particularly demanding environmental requirements, possesses good anti-interference capabilities, and has broad laboratory applicability.

[0084] Example 7: Composition and Application of the Euonymus alatus Thin-Layer Chromatography Detection Kit

[0085] The present invention also provides a test kit that is easy to carry and use on-site, serving as a rapid testing method for medicinal material markets and grassroots drug testing institutions.

[0086] 1. Reagent Kit Composition and Structure: This reagent kit is designed in individually packaged form and contains the following core components:

[0087] Component A (Standard Control Solution): Contains stigmaster-4-en-3-one reference standard (dry powder, 1 mg / vial) prepared according to the method of Example 4, or a prepared methanol solution of the reference standard (1 mg / mL, 1 mL / vial, sealed and protected from light).

[0088] Component B (Premixed Developing Agent): A developing agent premixed in a preferred ratio (toluene-ethyl acetate-formic acid = 20:1:0.5, v / v). Given the volatility and corrosiveness of formic acid, this component is packaged in a corrosion-resistant high-density glass bottle or designed as a single-use ampoule (10 mL / ampoule).

[0089] Component C (Stationary Phase): Prefabricated high-efficiency silica gel GF254 thin-layer board, cut to 2.5cm × 10cm (or 5cm × 10cm) dimensions. Packaged in an aluminum foil vacuum bag with a desiccant for moisture protection.

[0090] Component D (Auxiliary Consumables): Includes disposable micro-volume spotting capillary tubes (2μL), simple cylindrical glass development tank (compatible with thin-layer plate size), and portable 254nm UV lamp pen.

[0091] 2. On-site testing procedure: When using this kit to identify the authenticity of Euonymus alatus, users should follow these steps:

[0092] Simple extraction: Take about 0.5g of the powdered medicinal material to be tested (finely ground through a No. 3 sieve), place it in a stoppered conical flask or centrifuge tube, and add 10 mL of methanol. Seal tightly and place in an ultrasonic cleaner (power recommended ≥200W, frequency 40kHz), sonicate for 20-30 minutes. Remove, let stand or centrifuge, and take the supernatant as the test solution.

[0093] Spotting: Using the capillary tube in component D, draw up the test solution and component A (reference solution) respectively, and spot them on the baseline of component C (thin-layer plate). The spotting volume is about 2 to 5 μL.

[0094] Development: Pour component B (developing solvent) into the development tank (to a depth of about 0.5 cm), place the spotted thin-layer plate in the tank, carefully place the spotted thin-layer plate into the tank, ensuring that the baseline is above the liquid surface, and cover the tank for upward development.

[0095] Inspection: Once the leading edge of the developing solvent reaches approximately 1 cm from the end of the plate, remove it and allow the solvent to evaporate. Irradiate the plate using a portable UV lamp (254 nm) from component D.

[0096] 3. Judgment Criteria and Technical Advantages

[0097] Judgment: If the chromatogram of the test sample shows a fluorescent quenched spot of the same color at the corresponding position (Rf value 0.48±0.03) as the chromatogram of the reference sample, it is judged as genuine; otherwise, it is counterfeit.

[0098] Technical advantages: This kit integrates standard laboratory methods, eliminating the tedious process of preparing developing solvents on-site (especially measuring trace amounts of formic acid). The entire testing process requires no large instruments, greatly improving the efficiency of market supervision.

[0099] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for detecting Euonymus alatus based on thin-layer chromatography, characterized in that, This method uses stigmaster-4-en-3-one as a characteristic chemical marker and thin-layer chromatography to identify genuine and counterfeit Euonymus alatus.

2. The method for identifying Euonymus alatus based on thin-layer chromatography according to claim 1, characterized in that, Includes the following steps: S1. Preparation of test solution: Take the powder of the sample to be tested, add methanol for ultrasonic extraction, filter, concentrate the filtrate and redissolve to obtain the test solution; S2. Preparation of reference solution: Take stigmaster-4-en-3-one reference standard, dissolve it in methanol to prepare a reference solution with a concentration of 0.8~1.2 mg / mL; S3. Thin-layer chromatography spotting and development: Take the test solution and reference solution and spot them separately on the same silica gel GF254 thin-layer plate. Use a mixed solvent of toluene-ethyl acetate-formic acid in a volume ratio of (18~22):(0.8~1.2):(0.4~0.6) as the developing solvent for upward development. Remove the plate and air dry. S4. Result Interpretation: Examine under 254nm ultraviolet light. If the test sample chromatogram shows the same fluorescent quenching spot at the corresponding position as the reference chromatogram, i.e., an Rf value of 0.48±0.03, the test sample is determined to contain Euonymus alatus; if no such spot is shown, it is determined to be a negative or adulterant.

3. The method for detecting Euonymus alatus based on thin-layer chromatography according to claim 2, characterized in that, In step S1, the amount of the sample powder to be tested is 1.5~2.5g, and the volume of methanol added is 25~35mL; the power of the ultrasonic extraction is 200~300W, the frequency is 35~45kHz, and the time is 25~35min.

4. The method for detecting Euonymus alatus based on thin-layer chromatography according to claim 2, characterized in that, In step S3, the sample volume of both the test solution and the reference solution is 4~6μL; the thickness of the silica gel GF254 thin-layer plate is 0.20~0.25mm.

5. The method for detecting Euonymus alatus based on thin-layer chromatography according to claim 1, characterized in that, The method is used for the quality control of Euonymus alatus medicinal materials, processed slices, or traditional Chinese medicines containing Euonymus alatus.

6. A method for preparing stigmaster-4-en-3-one, characterized in that, Includes the following steps: P1. Extraction: Take dried winged young branches or wing-like powder of Euonymus alatus, add 70-80% ethanol solution, the ratio of powder to ethanol solution is 1:8-1:12 (g / mL), heat and reflux to extract 1-3 times, each time for 0.75-1.5 hours, combine the extracts and concentrate under reduced pressure to obtain extract; P2. Enrichment and purification: The extract was mixed with silica gel and loaded onto a column for silica gel column chromatography. Isocratic elution was performed using petroleum ether-ethyl acetate with a volume ratio of (18~22):1 and a boiling range of 60~90℃ as the eluent. P3. Collection and preparation: Thin-layer chromatography was used to monitor the elution fractions. The developing solvent was toluene-ethyl acetate-formic acid at a volume ratio of (18~22):(0.8~1.2):(0.4~0.6). The fractions showing characteristic spots at Rf values ​​of 0.48±0.03 were collected, combined, concentrated and dried to obtain crude stigmaster-4-en-3-one.

7. The method for preparing stigmaster-4-en-3-one according to claim 6, characterized in that, Step P3 is followed by step P4: the crude product is purified by silica gel column chromatography to obtain the thin-layer identification reference standard stigmaster-4-ene-3-one.

8. A thin-layer chromatography detection kit for identifying genuine and counterfeit Euonymus alatus, characterized in that, include: (a) Stigmaster-4-en-3-one reference standard or solution thereof; (b) Silica gel GF254 thin-layer plate; (c) Developing solvent components: toluene, ethyl acetate and formic acid.

9. Application of stigmaster-4-en-3-one in the preparation of test reagents for identifying the authenticity of Euonymus alatus or for quality control.

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