A method for rapid detection of l-arbutin in sea buckthorn

Abstract

This invention discloses a rapid detection method for L-leucosterol in sea buckthorn, belonging to the field of detection technology. The invention includes: dissolving an L-leucosterol standard in a solvent to prepare a reference solution; adding a sea buckthorn leaf powder sample to an alcohol solvent and then performing ultrasonic extraction to obtain a test solution; spotting the test solution and the reference solution on the same silica gel thin-layer plate and developing it with a developing solvent composed of n-butanol, acetic acid, and water; spraying the developed and dried thin-layer plate with an alkaline potassium permanganate colorimetric reagent, heating until the bands are clear, and visually observing; if the sea buckthorn leaf powder sample contains L-leucosterol, it will appear as a white spot on the thin-layer plate. This invention has the advantages of high efficiency, simplicity, accuracy, and high specificity. It fills the gap in existing technologies for the specific rapid detection of L-leucosterol and can be used for the rapid detection of L-leucosterol and the efficient screening of sea buckthorn leaf raw materials with high L-leucosterol content.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a rapid detection method for L-Quebrachitol in sea buckthorn. Background Technology

[0002] In 1889, lepidocrolide was first extracted and isolated by scientists from the bark of the South American elm tree. Subsequently, in 1906, a higher concentration of lepidocrolide was discovered in the latex of Brazilian rubber. It has certain therapeutic effects on diseases such as cancer, diabetes, and stomach damage. Its structural formula is shown below:

[0003]

[0004] In 1986, a Malaysian and Japanese rubber company collaborated, using lepidocrolide, extracted from plants, as a precursor in research on the synthesis of chiral drugs. As a multifunctional structural unit with levorotatory activity, lepidocrolide can be used as a precursor to derive various compounds with different pharmacological activities. For example, lepidocrolide can be used in the synthesis of carbohydrates, chiral antibiotics, and enzyme inhibitors, showing potential application value in the production of chiral drugs for anticancer and anti-AIDS purposes. Therefore, establishing a rapid method for identifying lepidocrolide and applying it to the industrial production of lepidocrolide, including its separation, purification, and synthesis, would be beneficial for the rapid identification and detection of lepidocrolide.

[0005] Currently, leucosterol has been found in plants from 15 families, including sea buckthorn, rubber seeds and whey, *Cinnamomum camphora* leaves, *Platycladus orientalis* bark, wild hemp, *Betula*, *Coffea*, litchi, and longan. Studies have shown that sea buckthorn extract rich in leucosterol has significant blood sugar-lowering and insulin resistance-improving functions; platelet-activating factor plays an important role in the development of prostate cancer, and platelet-activating factor inhibitors can be used for prostate cancer treatment. Leucosterol has shown certain platelet-activating factor inhibitory activity, with an inhibitory IC50 value of [missing information]. 50 The concentration was 42.2 μM. Peminol exhibits good antioxidant activity; studies have shown that a concentration of 0.5 mg / mL peminol possesses a strong ability to scavenge free radicals. Peminol also has therapeutic effects on acute gastric injury and protects the gastric mucosa. Due to its numerous chiral centers, hydroxyl groups, and high polarity, peminol has a high synthesis cost. Currently, peminol is mainly extracted and prepared from rubber latex wastewater or sea buckthorn leaves. The peminol content in natural rubber latex is approximately 1.2%, while the content in sea buckthorn leaves can reach 6.5%, making them suitable raw materials for peminol extraction and preparation.

[0006] Currently, the detection of spirulina alcohol mainly employs high-performance liquid chromatography (HPLC) combined with an evaporative light scattering detector (ELSD) for qualitative and quantitative analysis. However, due to the high polarity of spirulina alcohol and the diverse and complex structure of sea buckthorn components, different compounds may exhibit similar or identical retention characteristics, easily leading to erroneous qualitative results. Furthermore, HPLC analysis can only detect one sample per injection, resulting in lengthy processing times and high organic mobile phase consumption, thus increasing costs and making it unsuitable for rapid detection and identification of spirulina alcohol. In contrast, thin-layer chromatography (TLC) analysis can detect 10-12 samples in a single step, significantly reducing the processing time for multiple samples. Therefore, developing an efficient, simple, accurate, and reliable rapid TLC identification method for spirulina alcohol is of great significance. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a rapid identification method for L-chlorophyll in sea buckthorn.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A rapid detection method for L-chlorophyll in sea buckthorn includes the following steps:

[0010] L-Baiyunol standard was dissolved in a solvent to prepare a reference solution; sea buckthorn leaf powder sample was added to an alcohol solvent and then ultrasonically extracted to obtain a test solution; the test solution and the reference solution were spotted on the same silica gel thin-layer plate and developed with a developing solvent composed of n-butanol, acetic acid and water; the developed and dried thin-layer plate was sprayed with alkaline potassium permanganate colorimetric reagent and heated until the bands were clear. If the sea buckthorn leaf powder sample contained L-Baiyunol, it would appear as a white spot on the thin-layer plate.

[0011] In some embodiments, the alcohol solvent is an aqueous solution of methanol or ethanol with a concentration of not less than 90%.

[0012] In some embodiments, the ultrasonic extraction power is 80-120 W, the frequency is 35-45 kHz, and the extraction time is 20-40 minutes.

[0013] In some embodiments, the solvent of the reference solution is a methanol aqueous solution with a volume fraction of 40%-60%.

[0014] In some embodiments, the volume ratio of n-butanol, acetic acid, and water in the developing solvent is 3:2:1.

[0015] In some embodiments, the alkaline potassium permanganate colorimetric agent is prepared by mixing equal volumes of 0.5% (w / v) potassium permanganate solution and 1 mol / L sodium hydroxide solution.

[0016] In some embodiments, during the spotting step, the spotting volume of the test solution is 1-6 μL, and the spotting volume of the reference solution is 1-5 μL.

[0017] In some embodiments, the developing agent is developed for 25-30 minutes.

[0018] A method for screening sea buckthorn leaf raw materials involves using any of the methods described above to test multiple sea buckthorn leaf samples, and screening sea buckthorn leaf raw materials with higher L-white malachite alcohol content based on the degree of color development of white spots on thin-layer plates.

[0019] A pure L-chlorophyll is prepared by purifying sea buckthorn leaf raw material with high L-chlorophyll content, wherein the sea buckthorn leaf raw material with high L-chlorophyll content is obtained by the screening method described above.

[0020] Compared with the prior art, the beneficial effects of this application are as follows:

[0021] This application provides a rapid detection method for L-malachite in sea buckthorn, based on high-performance thin-layer chromatography (HPLC). The method involves creatively screening and optimizing experimental conditions such as the chromogenic agent, developing solvent, extraction solvent and extraction method, and sample loading. This method is characterized by its high efficiency, simplicity, accuracy, and high specificity. It fills the gap in the prior art for the rapid and specific detection and identification of L-malachite, and can be used for the rapid detection of L-malachite and the efficient screening of sea buckthorn leaf raw materials with high L-malachite content. Attached Figure Description

[0022] Figure 1 These are thin-layer chromatograms of L-leucosterol in sea buckthorn leaves detected by different colorimetric reagents; among them, the colorimetric reagent in chromatogram A is iodine; the colorimetric reagent in chromatogram B is 5% sulfuric acid ethanol; and the colorimetric reagent in chromatogram C is a mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide.

[0023] Figure 2 These are thin-layer chromatograms of L-Baiyanol in sea buckthorn leaves detected by different developing solvents; among them, the developing solvent ratio of chromatogram A is n-butanol:acetic acid:water = 4:1:5; the developing solvent ratio of chromatogram B is n-butanol:acetic acid:water = 3:2:1.

[0024] Figure 3 This is a thin-layer chromatogram for detecting L-sparganol in sea buckthorn leaves extracted with different extraction solvents;

[0025] Figure 4 This is a thin-layer chromatogram for detecting L-chlorophyll in sea buckthorn leaves extracted by different extraction methods;

[0026] Figure 5 This is a graph showing the optimal experimental results for different sample sizes;

[0027] Figure 6 It is a thin-layer chromatogram used for methodological specificity validation;

[0028] Figure 7 These are thin-layer chromatograms obtained by experimenters A and B during method reproducibility verification; where chromatogram A represents the result of experimenter A's operation, and chromatogram B represents the result of experimenter B's operation.

[0029] Figure 8 These are thin-layer chromatograms for the detection of L-chlorophyll in 12 batches of sea buckthorn leaves; in Figure A, 2-5 are HL251001, HL251002, HL251003, and HL251004 respectively; in Figure B, 7-10 are HL251005, HL251006, HL251007, and HL251008 respectively; and in Figure C, 12-15 are HL251009, HL251010, HL251011, and HL251012 respectively. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described clearly and completely below in conjunction with the embodiments of this invention. It should be noted that the described embodiments are only some embodiments of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0032] Example 1

[0033] Screening test of colorimetric reagents

[0034] Option 1

[0035] Take 1 mg of L-chlorophyll alcohol reference standard in a 1 mL volumetric flask, dissolve it in 50% methanol, shake well, and dilute to volume to prepare the reference solution. Take pulverized sea buckthorn leaves, add 10 mL of 50% ethanol, and extract for 30 min under ultrasonic conditions at 100 W and 40 kHz. Use the supernatant as the test solution. Perform thin-layer chromatography, applying 5 μL each of the reference solution and the test solution to the same silica gel plate. Use n-butanol, acetone, and water as the developing solvent (volume ratio: n-butanol:acetone:water = 4:3:1). Develop, remove, dry, and place in an iodine bath for color development. Heat until the bands are clear, observe visually, and photograph. Experimental data are as follows: Figure 1As shown in Figure A.

[0036] Option 2

[0037] Take 1 mg of L-chlorophyll alcohol reference standard in a 1 mL volumetric flask, dissolve it in 50% methanol, shake well, and dilute to volume to prepare the reference solution; crush sea buckthorn leaves, add 10 mL of 50% ethanol, and extract for 30 min under ultrasonic conditions of 100 W power and 40 kHz frequency, and take the supernatant as the test solution; according to the thin-layer chromatography experiment, take 5 μL each of the above reference solution and test solution, and spot them separately on the same silica gel thin-layer plate, using n-butanol, acetone, and water as the developing solvent, with the volume ratio of n-butanol:acetone:water = 4:3:1, develop, remove, dry, spray with 5% sulfuric acid ethanol for color development, heat until the bands are clear, observe with the naked eye, and take pictures. Experimental data are as follows. Figure 1 As shown in B.

[0038] Option 3

[0039] Take 1 mg of L-chlorophyll alcohol reference standard in a 1 mL volumetric flask, dissolve it in 50% methanol, shake well, and dilute to volume to prepare the reference solution; take sea buckthorn leaves, crush them, add 10 mL of 50% ethanol, and extract them under ultrasonic conditions of 100 W and 40 kHz for 30 min, and take the supernatant as the test solution; according to the thin-layer chromatography experiment, take 5 μL each of the above reference solution and test solution, and spot them separately on the same silica gel thin-layer plate, using n-butanol, acetone, and water as the developing solvent, with the volume ratio of n-butanol:acetone:water = 4:3:1. Develop, remove, dry, and spray with a mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide for color development, with the volume ratio of 0.5% (w / v) potassium permanganate:1 mol / L sodium hydroxide = 1:1. Heat until the bands are clear, observe with the naked eye, and take pictures. Experimental data are as follows. Figure 1 As shown in C.

[0040] Analysis of test data for color developers: Figure 1 In the table, solutions 1, 3, and 5 are reference solutions; solutions 2, 4, and 6 are test solutions. When the colorimetric reagent is a mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide, with a volume ratio of 0.5% (w / v) potassium permanganate: 1 mol / L sodium hydroxide = 1:1, L-chlorophyll alcohol can be observed as a single, clear white spot.

[0041] Example 2

[0042] Screening test of developing solvent

[0043] Option 1

[0044] Take 1 mg of L-chlorophyll alcohol reference standard in a 1 mL volumetric flask, dissolve it in 50% methanol, shake well, and dilute to volume to prepare the reference solution; crush sea buckthorn leaves, add 10 mL of 50% ethanol, and extract for 30 min under ultrasonic conditions of 100 W and 40 kHz, and take the supernatant as the test solution; according to the thin-layer chromatography experiment, take 5 μL each of the above reference solution and test solution, and spot them separately on the same silica gel thin-layer plate, using n-butanol, acetic acid, and water as the developing solvent, with the volume ratio of n-butanol:acetic acid:water = 4:1:5. Develop, remove, dry, and spray with a mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide for color development, with the volume ratio of 0.5% (w / v) potassium permanganate:1 mol / L sodium hydroxide = 1:1. Heat until the bands are clear, observe with the naked eye, and take pictures. Experimental data are as follows. Figure 2 As shown in Figure A.

[0045] Option 2

[0046] Take 1 mg of L-chlorophyll alcohol reference standard in a 1 mL volumetric flask, dissolve it in 50% methanol, shake well, and dilute to volume to prepare the reference solution. Take pulverized sea buckthorn leaves, add 10 mL of 50% ethanol, and extract for 30 min under ultrasonic conditions at 100 W and 40 kHz. Take the supernatant as the test solution. Perform thin-layer chromatography, applying 5 μL each of the reference solution and the test solution to the same silica gel plate. Use n-butanol, acetic acid, and water as the developing solvent (volume ratio: n-butanol:acetic acid:water = 3:2:1). Develop, remove, dry, and spray with a mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide (volume ratio: 0.5% (w / v) potassium permanganate:1 mol / L sodium hydroxide = 1:1). Heat until the bands are clear, observe visually, and photograph. Experimental data are as follows: Figure 2 As shown in B.

[0047] Analysis of experimental data on the developing solvent: Figure 2 In the diagram, 1 and 3 are reference solutions; 2 and 4 are test solutions. The developing solvent is a mixture of n-butanol, acetic acid, and water, with a volume ratio of n-butanol:acetic acid:water = 3:2:1. The colorimetric reagent is a mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide, with a volume ratio of 0.5% (w / v) potassium permanganate:1 mol / L sodium hydroxide = 1:1. L-Leymus chinensis alcohol is observed as a single, clear white spot with an Rf of 0.48-0.51.

[0048] Example 3

[0049] Screening test of extraction solvent

[0050] Take 5 portions of sea buckthorn leaf powder, each 0.2 g, and add 10 mL each of water, 30% ethanol, 60% ethanol, 95% ethanol, and 50% methanol. Under ultrasonic conditions of 100 W power and 40 kHz frequency, take the supernatant from each portion as the test solution. Separately, take 5 portions of L-chlorophyll alcohol and dissolve them in 50% methanol to prepare solutions with a concentration of 1 mg / mL, which will be used as reference solutions. Apply 6 μL of each test solution and 5 μL of each of the five reference solutions to the same silica gel thin-layer plate. Use n-butanol:acetic acid:water = 3:2:1 as the developing solvent. Develop, remove, dry, and spray with a 1:1 mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide for color development. Heat until the bands are clear, observe with the naked eye, and photograph. Experimental data are as follows: Figure 3 As shown.

[0051] Analysis of experimental data on solvent extraction: Figure 3 Table 1 is the reference solution; 2, 3, 4, 5, and 6 are the test solutions extracted with water, 30% ethanol, 60% ethanol, 95% ethanol, and 50% methanol, respectively. The Rf values ​​are 0.48-0.51. When the solvent is 95% ethanol, the white spots in the chromatogram are clearer, and the background effect is better than with other solvents.

[0052] Example 4

[0053] Screening experiment of extraction methods

[0054] Take two portions of sea buckthorn leaf powder, 0.2 g each, and add 10 mL of 95% ethanol to each. Extract one portion under ultrasonic conditions of 100 W and 40 kHz for 30 min, and use the supernatant as test solution 1. Heat the other portion under reflux for 30 min, filter, evaporate the filtrate to dryness, and dissolve the remaining residue in 2 mL of 95% ethanol to prepare test solution 2. Separately, prepare a 1 mg / mL solution of L-chlorophyll in 50% methanol as a reference solution. According to the thin-layer chromatography method in Section 0502 of the 2015 edition of the Chinese Pharmacopoeia, apply 6 μL of each test solution and 5 μL of the reference solution to the same silica gel thin-layer plate. Develop, remove, dry, and spray with 0.5% (w / v) potassium permanganate and 1... Mix mol / L sodium hydroxide solution at a 1:1 ratio for color development, heat until the bands are clear, observe with the naked eye, and take a photograph. Experimental data are as follows: Figure 4 As shown.

[0055] Analysis of experimental data on extraction methods: Figure 41 is the reference solution; 2 is the test solution after sonication for 30 min; 3 is the test solution after reflux for 30 min. The Rf values ​​are 0.48-0.51. The comparison shows that the developing marks of the reflux-extracted test solution are heavier than those obtained by sonication, indicating that sonication is more effective.

[0056] Example 5

[0057] Optimization test of sample quantity

[0058] 0.2 g of sea buckthorn leaf powder was added to 10 mL of 95% ethanol and extracted for 30 min under ultrasonic conditions at 100 W and 40 kHz. The supernatant was used as the test solution. A 0.4 mg / mL solution of L-chlorophyll was prepared by dissolving it in 50% methanol as the reference solution. 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL of both the test and reference solutions were spotted onto the same silica gel thin-layer plate. The plate was developed using n-butanol:acetic acid:water = 3:2:1 as the developing solvent. After development, the plate was removed, dried, and sprayed with a 1:1 mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide for color development. The plate was heated until the bands were clear, observed visually, and photographed. Experimental data are as follows: Figure 5 As shown.

[0059] Analysis of experimental data on sample quantity

[0060] Figure 5 Images 1-5 show thin-layer chromatograms of the reference solution with spotting volumes of 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL, respectively; images 6-10 show thin-layer chromatograms of the test solution with spotting volumes of 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL, respectively. The Rf value is 0.39. The comparison shows that the spots formed by spotting volumes of both the test solution and the reference solution are relatively clear.

[0061] Example 6

[0062] Methodological Validation

[0063] 1) Specificity verification

[0064] Take 0.2 g of sea buckthorn leaf powder, add 10 mL of 95% ethanol, and extract for 30 min under ultrasonic conditions of 100 W and 40 kHz. Use the supernatant as the test solution. Separately, prepare a 1 mg / mL solution of L-chlorophyll in 50% methanol as the reference solution. According to the thin-layer chromatography method in Section 0502 of the 2015 edition of the Chinese Pharmacopoeia, apply 6 μL of each of the six test solutions and 5 μL of the reference solution to the same silica gel thin-layer plate. Use n-butanol:acetic acid:water = 3:2:1 as the developing solvent. Develop, remove, dry, and spray with a 1:1 mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide for color development. Heat until the bands are clear, observe visually, and photograph. Experimental data are as follows: Figure 6 As shown.

[0065] Figure 6 In this solution, 1 is an L-chlorophyll alcohol reference solution; 2 is a test solution extracted with 95% ethanol; 3 is a 50% methanol solvent; and 4 is a 95% ethanol solvent. The Rf value is 0.48-0.51. Figure 6 It can be seen that the chromatogram of the sea buckthorn leaf test sample shows the same white spots at the corresponding positions as the chromatogram of the reference sample, while the solvent chromatogram does not show such spots at the corresponding positions. This indicates that the method provided in this application has strong specificity.

[0066] 2) Reproducibility verification

[0067] Experimenters A and B performed the following experimental steps respectively:

[0068] Two portions of sea buckthorn leaf powder, 0.2 g each, were extracted with 10 mL of 95% ethanol under ultrasonic conditions of 100 W and 40 kHz for 30 min. The supernatants were used as test solutions 1 and 2, respectively. A 1 mg / mL solution of L-chlorophyll was prepared in 50% methanol as a reference solution. Following the thin-layer chromatography method in Section 0502 of the 2015 edition of the Chinese Pharmacopoeia, 6 μL of each of the six test solutions and 5 μL of the reference solution were spotted onto the same silica gel thin-layer plate. The plate was developed using n-butanol:acetic acid:water = 3:2:1 as the developing solvent. After development, the plate was removed, dried, and sprayed with a 1:1 mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide for color development. The plate was heated until the bands were clear, observed visually, and photographed. Experimental data from researcher A are as follows: Figure 7 As shown in Figure A, the experimental data of experimenter B are as follows: Figure 7 As shown in B.

[0069] Figure 7 In the table, 1 and 6 are L-chlorophyll alcohol reference solutions; 2 and 7 are 50% methanol solvent; 3 and 8 are 95% ethanol solvent; 4 and 5 are test solutions 1 and 2 extracted with 95% ethanol, respectively. Figure 7 The Rf of A is 0.48-0.51. Figure 7 The Rf values ​​for B were 0.48-0.51, showing no significant difference. Comparison revealed that, under different operator conditions, the thin-layer chromatograms of the sea buckthorn leaf test sample showed the same white spots at the corresponding positions as the reference sample, indicating that the method provided in this application has good reproducibility.

[0070] Example 6

[0071] Thin-layer chromatography detection of L-chlorophyll in sea buckthorn leaves at different stages

[0072] Twelve batches of sea buckthorn leaves from three different periods were collected. The batch numbers are shown in Table 1. The procedure is as follows:

[0073] Take 0.2 g of each sea buckthorn leaf powder, add 10 mL of 95% ethanol, and extract for 30 min under ultrasonic conditions of 100 W and 40 kHz. Use the supernatant as the test solution. Separately, prepare a 1 mg / mL solution of L-chlorophyll in 50% methanol as the reference solution. According to the thin-layer chromatography method in Section 0502 of the 2015 edition of the Chinese Pharmacopoeia, apply 6 μL of each test solution and 5 μL of the reference solution to the same silica gel thin-layer plate. Use n-butanol:acetic acid:water = 3:2:1 as the developing solvent. Develop, remove, dry, and spray with a 1:1 mixture of 0.5% (w / v) potassium permanganate and 1 mol / L sodium hydroxide for color development. Heat until the bands are clear, observe visually, and photograph. The time from spotting and development to color development for 15 samples and the reference solution is approximately 30 minutes. Experimental data are as follows: Figure 8 As shown;

[0074] Figure 8 2-5, 7-10, and 12-15 are test solutions of sea buckthorn leaves from batches HL251001, HL251002, HL251003, HL251004, HL251005, HL251006, HL251007, HL251008, HL251009, HL251010, HL251011, and HL251012, respectively; 1, 6, and 11 are L-chlorophyll alcohol reference solutions.

[0075] Figure 8 The Rf is 0.48-0.51, and spots of the same color appear at the corresponding positions on the chromatogram of the reference standard.

[0076] Table 1. Sampling data of sea buckthorn leaves

[0077]

[0078] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A rapid detection method for L-chlorophyll in sea buckthorn, characterized in that, Includes the following steps: L-Baiyanol standard was dissolved in a solvent to prepare a reference solution; The sea buckthorn leaf powder sample was added to an alcohol solvent and then subjected to ultrasonic extraction to obtain the test solution. The test solution and the reference solution were spotted on the same silica gel thin-layer plate and developed with a developing solvent composed of n-butanol, acetic acid and water, wherein the volume ratio of n-butanol, acetic acid and water in the developing solvent was 3:2:

1. Spray the unfolded and dried thin-layer plate with alkaline potassium permanganate colorimetric agent, heat until the bands are clear, and observe with the naked eye. If the sea buckthorn leaf powder sample contains L-leucosterol, it will appear as a white spot on the thin-layer plate. The alkaline potassium permanganate colorimetric agent is prepared by mixing equal volumes of 0.5% potassium permanganate solution and 1 mol / L sodium hydroxide solution.

2. The method as described in claim 1, characterized in that, The alcohol solvent is an aqueous solution of methanol or ethanol with a concentration of not less than 90%.

3. The method as described in claim 1, characterized in that, The ultrasonic extraction power is 80-120 W, the frequency is 35-45 kHz, and the extraction time is 20-40 minutes.

4. The method as described in claim 1, characterized in that, The solvent for the reference solution is a methanol aqueous solution with a volume fraction of 40%-60%.

5. The method as described in claim 1, characterized in that, In the spotting step, the spotting volume of the test solution is 1-6 μL, and the spotting volume of the reference solution is 1-5 μL.

6. The method as described in claim 1, characterized in that, The developing agent is developed for 25-30 minutes.

7. A method for screening sea buckthorn leaf raw materials, characterized in that, Multiple sea buckthorn leaf samples were tested using the method described in any one of claims 1 to 6, and sea buckthorn leaf raw materials with higher L-white malol content were screened based on the degree of color development of white spots on thin-layer plates.

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