Thin-layer identification method for Kangyanling granular lotus seeds

Abstract

The invention relates to the technical field of analysis and detection, and particularly discloses a thin-layer identification method for Kangyanling granular lotus seeds. The thin-layer identification method for the Kangyanling granular lotus seeds comprises the following steps: preparing a test solution, preparing a reference medicinal material solution and performing thin-layer chromatography. The preparation method has the advantages of reducing interference of interference spots on determination of main spots of a sample and improving specificity.

Description

Technical Field

[0001] This application relates to the technical field of analytical testing, and more specifically, it relates to a thin-layer chromatography method for identifying Kangerling granules and lotus seeds. Background Technology

[0002] Thin-layer chromatography (TLC) is commonly used for identification. During identification, a suitable stationary phase is coated onto a glass plate, plastic, or aluminum substrate to form a uniform thin layer. After spotting and development, the ratio shift value is compared with the ratio shift value of a suitable reference substance obtained using the same method. This method is used for drug identification, impurity testing, or content determination. TLC is an experimental technique for the rapid separation and qualitative analysis of small amounts of substances.

[0003] When identifying the lotus seed component in Kang'erling granules, thin-layer chromatography (TLC) is typically used. The procedure involves adding a certain amount of Kang'erling granules to chloroform, sonicating, and letting it stand overnight. After filtration, the filtrate is evaporated to dryness, and the residue is dissolved in ethyl acetate to prepare the test solution. An equal amount of lotus seed reference material is taken, and a reference material solution is prepared using the same method. An equal amount of negative samples prepared from other raw materials (excluding lotus seeds) is taken, and a negative sample solution is prepared using the same method.

[0004] According to the thin-layer chromatography method, equal amounts of the prepared test solution, negative sample, and reference herbal solution were spotted onto the same silica gel G thin-layer plate. The plate was developed using n-hexane-acetone as the developing solvent, removed, and air-dried. A 10% sulfuric acid ethanol solution was sprayed onto the dried plate, and the plate was examined under a 365 nm UV lamp. Fluorescent spots of the same color were observed at corresponding positions on the chromatograms of the test sample and the reference herbal material, thus detecting the presence of lotus seed components.

[0005] During the method validation process, when identifying lotus seeds using the thin-layer chromatography method described above, there is a possibility that the negative sample solution may show spots at the same positions as the main spot in the test sample solution, which may lead to non-compliance of method specificity and affect the accuracy of the results. Summary of the Invention

[0006] To reduce interference from interfering spots on the determination of the main spot in the sample and improve specificity, this application provides a thin-layer chromatography method for identifying lotus seeds containing Kang'erling granules.

[0007] The thin-layer chromatography method for identifying lotus seeds with Kang'erling granules provided in this application adopts the following technical solution: A thin-layer chromatography method for identifying Kang'erling granules and lotus seeds includes the following steps: S1. Preparation of test solution: Take 15±1g of Kangerling granules, grind them into a fine powder, add 30±1ml of water to dissolve them, add dilute hydrochloric acid to adjust the pH to 1-2, extract with ether 3±1 times, 30±1ml each time, discard the ether layer, adjust the pH of the aqueous layer to 9-11 with (40±1)% ammonia water, and then extract with chloroform 3±1 times, 30±1ml each time. Combine the chloroform extracts and evaporate to dryness. Dissolve the residue in 0.5ml of methanol to obtain the test solution. S2. Preparation of control medicinal material solution: Take 1±0.1g of lotus seed control medicinal material, add 30±1ml of water, heat under reflux for 30 minutes, centrifuge and take the supernatant, add dilute hydrochloric acid to adjust the pH to 1-2, extract with ether 3±1 times, 30±1ml each time, discard the ether layer, adjust the pH of the aqueous layer to 9-11 with (40±1)% ammonia water, and then extract with chloroform 3±1 times, 30±1ml each time, combine the chloroform solutions and evaporate to dryness, dissolve the residue in 0.5ml of methanol to obtain the control medicinal material solution; S3. Thin-layer chromatography analysis: The test solution prepared in S1 and the reference medicinal material solution prepared in S2 were spotted on the same silica gel G thin-layer plate to form strips. After development with the developing solvent, the plates were removed and dried. After 24±0.1h, they were examined under a 365nm ultraviolet lamp.

[0008] By adopting the above technical solution and changing the preparation methods of the test sample solution and the control herbal solution, water-soluble components such as Atractylodes macrocephala and Poria cocos are retained during the extraction of the test sample, reducing the loss caused by direct organic extraction. At the same time, ether extraction under acidic conditions removes lipid-soluble impurities such as terpenes and oils, reducing interference, and allows alkaloids to form salts that dissolve in the aqueous phase. Extraction with chloroform under alkaline conditions promotes the release of alkaloids, thereby enabling efficient extraction of alkaloid components. Furthermore, the use of methanol as the final dissolution method can better dissolve a variety of organic compounds, facilitating subsequent analysis. When extracting the reference medicinal material, heating and reflux can fully release the water-soluble components in lotus seeds, while centrifugation removes insoluble impurities and reduces interference from subsequent organic extraction. Ether extraction effectively removes fat-soluble impurities, while alkaline chloroform extraction can efficiently enrich alkaloid components, achieving a synergistic effect of impurity removal and target component enrichment. Moreover, the amount of reference medicinal material used is less than the usual amount used in the pharmacopoeia, saving costs. By reducing the steps of spraying the color developer, experimental errors caused by uneven spraying are reduced. It also reduces the possibility that sulfuric acid and ethanol may mask the natural fluorescence or color of certain components in lotus seeds, allowing for direct observation of the original spot characteristics. At the same time, it reduces the interference of the color developer on some components that can be directly colored under ultraviolet light, thereby reducing the interference of interfering spots on the determination of the main spot of the sample and improving the specificity.

[0009] Preferably, in step S3, the amount of the test solution prepared in step S1 is 15±1 μl, and the amount of the reference medicinal material solution prepared in step S2 is 5±1 μl.

[0010] By adopting the above technical solution, the amount of sample solution used for testing is greater than that used for control medicinal material solution, thereby ensuring the detection sensitivity of trace components in the sample solution. At the same time, reducing the amount of control medicinal material solution used can reduce experimental costs and is in line with the principle of resource conservation.

[0011] Preferably, in step S3, the developing agent is prepared by mixing cyclohexane, chloroform and diethylamine, and the amount of cyclohexane added is 14±1 ml, the amount of chloroform added is 4±1 ml, and the amount of diethylamine added is 2±0.5 ml.

[0012] By adopting the above technical solution and adjusting the formulation of the developing agent and the ratio between the components, chloroform and diethylamine are added to cyclohexane to enhance the overall polarity of the developing agent, making it more suitable for the separation of moderately polar components such as alkaloids and flavonoids in lotus seeds. The addition of chloroform can improve the tailing phenomenon, and the alkaline environment of diethylamine can neutralize the acidity of silica gel, making the spots clearer. In addition, the mixed system of cyclohexane and chloroform has suitable volatility, uniform development speed, and good repeatability. When the amount of diethylamine added exceeds the above range, the alkalinity in the developing solvent system increases, which may cause a decrease in the adsorption capacity of silica gel, and thus cause the lotus seed component spots to diffuse and the RF value to be unstable. Excessive diethylamine is prone to side reactions, producing interfering spots that overlap with the target component. Furthermore, after the ratio of developing solvent is disrupted, the polar gradient of cyclohexane-trichloromethane fails, the separation effect deteriorates, and thus affects the specificity. When the amount of chloroform added to the developing solvent is insufficient, the overall polarity of the developing solvent is easily reduced. The moderately polar components in lotus seeds migrate too slowly and have a low RF value, which may prevent them from being effectively separated from interfering substances such as terpenes. In addition, the wettability of the developing solvent to silica gel decreases, and the spots are prone to tailing, thus affecting the accuracy of identification. When chloroform is excessive, it may cause the components of lotus seeds to migrate too quickly and have a high RF value, which may cause the target spot to overlap with the solvent front. At the same time, it may cause interfering substances such as terpenes to co-migrate with the target components, reducing the separation effect of the thin-layer plate.

[0013] Preferably, the developing agent further includes an additive, which accounts for (0.1-1)% of the total amount of the developing agent. The additive is prepared by mixing titanium dioxide nanoparticles and β-cyclodextrin powder in a weight ratio of 1:(1-3).

[0014] By adopting the above technical solution, the additive prepared by combining titanium dioxide nanoparticles and β-cyclodextrin powder is added to the developing solvent system according to the above addition range. This allows the β-cyclodextrin powder to encapsulate components such as isozymine and atractylodes lactone in the test solution through its own cavity structure, thereby reducing the alkaline interference of diethylamine in the developing solvent, reducing the possibility of spot diffusion caused by pH fluctuations, reducing the interaction between isozymine and the stationary phase, reducing the migration of fat-soluble components such as atractylodes lactone and components such as starch and polysaccharides, and thus making the spots of alkaloids and other components more concentrated. Titanium dioxide nanoparticles enhance the polar gradient of the eluent, optimize the migration rate of components, improve the separation, and the dispersibility of titanium dioxide helps maintain the stability of the eluent and reduce edge effects. At the same time, titanium dioxide generates active oxygen under ultraviolet light, thereby selectively degrading isozymine, breaking glycosidic bonds in starch, breaking carbon-carbon double bonds in fat-soluble components, and reducing the adverse effects on the detection of main components in lotus seeds. When there is an excessive amount of additives in the developing solvent, the polarity of the developing solvent may become unbalanced due to the excessive amount of β-cyclodextrin. This may cause alkaloids and other components in lotus seeds to migrate too quickly, affecting the separation degree from terpenoid interferences. At the same time, it may also mask natural fluorescent spots, affecting observation under ultraviolet light. Excessive titanium dioxide nanoparticles may clog the pores of the thin-layer plate, causing spot tailing or diffusion. Furthermore, excessive additives may change the viscosity of the developing solvent, which may lead to irregular development fronts or failure to climb the plate.

[0015] Preferably, the β-cyclodextrin powder is a modified β-cyclodextrin powder, which is obtained by grafting sulfonic acid groups onto β-cyclodextrin powder.

[0016] Preferably, the modified β-cyclodextrin powder is prepared as follows: β-cyclodextrin powder was dissolved in dry pyridine by weight ratio, cooled to 0-5°C in an ice bath, and reacted with sulfur trioxide gas for 2 min. The molar ratio of β-cyclodextrin to sulfur trioxide was 1:(1-3). The mixture was poured into water, and calcium carbonate was added to neutralize the unreacted acid. After filtration, washing, drying and pulverizing, modified β-cyclodextrin powder was obtained.

[0017] By employing the above technical solution and method, modified β-cyclodextrin powder is prepared and added to additives. The strong negative charge of the grafted sulfonic acid groups binds to the positively charged alkaloids in lotus seeds, forming stable inclusion complexes through electrostatic interactions. This improves the separation from terpenoids and other precipitates. The sulfonated β-cyclodextrin powder neutralizes acidic sites on the silica gel plate surface, reducing the strong adsorption of alkaloids and resulting in more concentrated spots. The sulfonic acid groups enhance the ultraviolet absorption or fluorescence signal of alkaloids, and combined with the inclusion effect of the β-cyclodextrin powder, make low-concentration components easier to detect, thereby reducing interference from other spots on the determination of the main sample spot and improving specificity.

[0018] Preferably, the titanium dioxide nanoparticles have a particle size of 15-20 nm.

[0019] By adopting the above technical solution and selecting titanium dioxide nanoparticles within the above particle size range, the interaction between the titanium dioxide nanoparticles and the sample components is enhanced by the specific surface area of ​​the titanium dioxide nanoparticles, reducing spot diffusion and improving separation resolution. In addition, the prepared developing agent has higher stability, reduces particle agglomeration on the thin-layer plate, and makes the color development more uniform. When the particle size of titanium dioxide nanoparticles is too large, the specific surface area decreases, which easily weakens the interaction with the target component, increases spot diffusion, reduces the separation effect, and the particles are prone to agglomeration due to the increase in particle size, resulting in blurred or uneven colored spots, affecting the accuracy of identification. Furthermore, large particle size is prone to uneven distribution on the plate due to sedimentation or uneven dispersion, which increases experimental error. When the particle size of titanium dioxide nanoparticles is too small, it can easily cause excessive adsorption of the target component, which can reduce the separation efficiency. Furthermore, ultra-small particles may exacerbate the free diffusion of the sample on the thin-layer plate, leading to tailing and even complete separation failure, thus affecting specificity.

[0020] Preferably, in step S3, the developing agent is developed in the following manner: Low-temperature saturation: Place the end of the thin-layer plate on the top side of the developing agent at 4±1℃; High-temperature main development: Immerse the thin-layer plate in a developing agent at 25±1℃ until development is complete.

[0021] By adopting the above technical solution, the volatilization rate of the developing agent is first reduced by low-temperature saturation, which allows volatile components such as terpenes to migrate preferentially. Then, the volatilization and adsorption of the developing agent are balanced at a developing temperature close to room temperature, so that the RF value of target components such as alkaloids and flavonoids in lotus seeds falls within the ideal range of 0.3-0.7. Moreover, the increase in temperature makes the developing agent vapor saturate faster, which can effectively separate complex components and reduce the possibility of tailing and topping.

[0022] In summary, this application has the following beneficial effects: 1. By modifying the preparation methods of the test sample solution and the control medicinal material solution, the water-soluble components in the test sample are retained while removing lipid-soluble impurities such as terpenes and oils, promoting the release of alkaloids, thereby enabling efficient extraction of alkaloid components, better dissolving various organic compounds, and facilitating subsequent analysis; the water-soluble components in the lotus seeds of the control medicinal material are fully released, and the interference of insoluble impurities is removed, achieving a synergistic effect of impurity removal and target component enrichment; the step of spraying colorimetric reagent is reduced, experimental errors are reduced, the original spot characteristics are observed directly, and interference with some components that can be directly colored under ultraviolet light is reduced.

[0023] 2. By adjusting the formulation of the developing solvent and the ratio between its components, chloroform and diethylamine were added to cyclohexane to enhance the overall polarity of the developing solvent, making it more suitable for the separation of moderately polar components in lotus seeds, such as alkaloids and flavonoids. The addition of chloroform can improve the tailing phenomenon, and the alkaline environment of diethylamine can neutralize the acidity of silica gel, making the spots clearer. In addition, the mixture of cyclohexane and chloroform has suitable volatility, uniform development speed, and good repeatability.

[0024] 3. β-Cyclodextrin powder, through its own cavity structure, encapsulates components such as isozyzinidine and atractylodes lactone in the test solution, reducing the alkaline interference of diethylamine in the developing solvent, decreasing the possibility of spot diffusion caused by pH fluctuations, reducing the interaction between isozyzinidine and the stationary phase, and reducing the migration of fat-soluble components such as atractylodes lactone, as well as starch and polysaccharides. Titanium dioxide nanoparticles enhance the polar gradient of the developing solvent, optimize the component migration rate, improve the separation degree, and the dispersibility of titanium dioxide helps maintain the stability of the developing solvent, reduce edge effects, selectively degrade isozyzinidine, break glycosidic bonds in starch, break carbon-carbon double bonds in fat-soluble components, and reduce the adverse effects on the detection of main components in lotus seeds. Attached Figure Description

[0025] Figure 1 This is the thin-layer identification test result of Example 1 of this application; Figure 2 This is the thin-layer identification test result of the comparative example of this application.

[0026] Explanation of the reference numerals: 1. Negative sample solution; 2. Control medicinal material solution; 3. Test solution-1; 4. Test solution-2; 5. Test solution-3. Detailed Implementation

[0027] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0028] raw material

[0029] The raw materials used in the embodiments and preparation examples of this application are all commercially available. The thin-layer plates used in this application are all 10cm*20cm silica gel G thin-layer plates. Furthermore, the negative sample particles in all embodiments and comparative examples were prepared using the following steps: Take 60g of Atractylodes macrocephala and 36.4g of Acanthopanax senticosus, chop them into small pieces, and mix them with 35.5g of stir-fried Shenqu (medicated leaven), 35.5g of Poria cocos, 35.5g of stir-fried malt, 23.6g of dried tangerine peel, 30g of stir-fried immature bitter orange, 35.5g of stir-fried hawthorn (charred), 18.2g of prepared licorice root, 60g of Picrorhiza scrophulariiflora, and 48.2g of Quisqualis indica. Add water and decoct twice. For the first decoction, add 10 times the amount of water and decoct for 2 hours. For the second decoction, add 8 times the amount of water and decoct for 1 hour. Filter the decoctions separately and combine the filtrates. After standing for 24 hours, the supernatant was concentrated to a clear paste with a relative density of 1.10-1.10 (measured at 80℃). After cooling, ethanol was added to make the alcohol content reach 60%. After standing for more than 12 hours, the supernatant precipitate was washed with 50% ethanol, placed, filtered, and the washing liquid and filtrate were combined. The ethanol was recovered and concentrated to a clear paste with a relative density of 1.28-1.30 (measured at 80℃). Sucrose was added and mixed well to form granules. After drying, 1000g of granules were obtained, which were then used to obtain negative sample granules. Preparation Example

[0030] Preparation Example 1: Modified β-cyclodextrin powder Preparation Example 1.1 A modified β-cyclodextrin powder is prepared by the following steps: 0.28 g of β-cyclodextrin powder was dissolved in 4 ml of dry pyridine, cooled to 3 °C in an ice bath, and 0.02 g of sulfur trioxide gas was introduced to react for 2 min. The mixture was poured into water, and calcium carbonate was added to neutralize the unreacted acid. After filtration, washing, drying and pulverizing, modified β-cyclodextrin powder was obtained.

[0031] Preparation Example 1.2 Unlike Preparation Example 1.1, in Preparation Example 1.2, the amount of β-cyclodextrin powder added was 0.26 g, and the amount of sulfur trioxide gas added was 0.04 g.

[0032] Preparation Example 1.3 Unlike Preparation Example 1.1, in Preparation Example 1.3, the amount of β-cyclodextrin powder added was 0.25 g, and the amount of sulfur trioxide gas added was 0.05 g.

[0033] Preparation Example 2 Additive Preparation Example 2.1 An additive is prepared by the following steps: An additive was prepared by mixing 0.15g of titanium dioxide nanoparticles with a particle size of 15nm with 0.15g of β-cyclodextrin powder.

[0034] Preparation Example 2.2 Unlike Preparation Example 2.1, in Preparation Example 2.2, the amount of titanium dioxide nanoparticles added was 0.1 g, and the amount of β-cyclodextrin powder added was 0.2 g.

[0035] Preparation Example 2.3 Unlike Preparation Example 2.1, in Preparation Example 2.3, the amount of titanium dioxide nanoparticles added was 0.075 g, and the amount of β-cyclodextrin powder added was 0.225 g.

[0036] Preparation Example 2.4 Unlike Preparation Example 2.2, the titanium dioxide nanoparticles in Preparation Example 2.4 have a particle size of 20 nm.

[0037] Preparation Examples 2.5-2.7 Unlike Preparation Example 2.2, the β-cyclodextrin powder in Preparation Examples 2.5-2.7 was derived in equal amounts from Preparation Examples 1.1-1.3.

[0038] Preparation Example 3: Developing Solvent Preparation Example 3.1 A developing solvent is prepared by the following steps: The developing solvent was prepared by mixing 14 ml of cyclohexane, 4 ml of chloroform, and 2 ml of diethylamine.

[0039] Preparation Example 3.2 Unlike Preparation Example 3.1, the amount of diethylamine added in Preparation Example 3.2 was 1.92 ml, and the developing solvent also included 0.06 g of the additive from Preparation Example 2.1.

[0040] Preparation Example 3.3 Unlike Preparation Example 3.1, the amount of diethylamine added in Preparation Example 3.3 was 1.58 ml, and the developing solvent also included 0.3 g of the additive from Preparation Example 2.1.

[0041] Preparation Example 3.4 Unlike Preparation Example 3.1, the amount of diethylamine added in Preparation Example 3.4 was 1.22 ml, and the developing solvent also included 0.54 g of the additive from Preparation Example 2.1.

[0042] Preparation Examples 3.5-3.10 Unlike Preparation Example 3.3, the additives in Preparation Examples 3.5-3.10 were derived in equal amounts from Preparation Examples 2.2-2.7. Example 1

[0043] A thin-layer chromatography method for identifying Kang'erling granules and lotus seeds includes the following steps: S1. Preparation of test solution: Take 15g of Kang'erling granules, grind them into a fine powder, add 30ml of water to dissolve them, add dilute hydrochloric acid to adjust the pH to 1.5, extract with ether 3 times, 30ml each time, discard the ether layer, adjust the pH of the aqueous layer to 10 with 40% ammonia water, and then extract with chloroform 3 times, 30ml each time. Combine the chloroform extracts and evaporate to dryness. Dissolve the residue in 0.5ml of methanol to obtain the test solution. S2. Preparation of control herbal solution: Take 1g of lotus seed control herbal material, add 30ml of water, heat under reflux for 30 minutes, centrifuge and take the supernatant, add dilute hydrochloric acid to adjust the pH to 1.5, extract with ether 3 times, 30ml each time, discard the ether layer, adjust the pH of the aqueous layer to 10 with 40% ammonia water, and then extract with chloroform 3 times, 30ml each time, combine the chloroform extracts and evaporate to dryness, dissolve the residue in 0.5ml of methanol to obtain the control herbal solution; S3. Preparation of negative sample solution: Take 15g of negative sample particles, grind them into a fine powder, add 30ml of water to dissolve them, add dilute hydrochloric acid to adjust the pH to 1.5, extract with ether 3 times, 30ml each time, discard the ether layer, adjust the pH of the aqueous layer to 10 with 40% ammonia water, and then extract with chloroform 3 times, 30ml each time. Combine the chloroform extracts and evaporate to dryness. Dissolve the residue in 0.5ml of methanol to obtain the negative sample solution. S4. Thin-layer chromatography analysis: 15 μl of the negative sample solution prepared in S3, 5 μl of the reference herb solution prepared in S2, and three aliquots of the test solution prepared in S1 (each test solution being 15 μl) were spotted onto the same silica gel G thin-layer plate to form bands. 25 ml of the developing solvent from Preparation Example 3.1 was added to the developing tank. One end of the spotted thin-layer plate was immersed in the developing solvent for development. After development, the plate was removed and air-dried, left to stand for 24 hours, and then examined under a 365 nm UV lamp. The results are shown in [Figure number missing]. Figure 1 .

[0044] Comparative Example A thin-layer chromatography method for identifying Kang'erling granules and lotus seeds includes the following steps: S1. Take 15g of Kangerling granules, add 50ml of chloroform, sonicate for 30min, let stand for 24h, filter, evaporate the filtrate to dryness, add 1ml of ethyl acetate to dissolve the residue, and prepare the test solution. S2. Take 5g of lotus seed reference material, add 50ml of chloroform, sonicate for 30min, let stand for 24h, filter, evaporate the filtrate to dryness, add 1ml of ethyl acetate to dissolve the residue, and prepare the reference material solution. S3. Take 15g of negative sample particles, grind them into a fine powder, add 30ml of water to dissolve them, add dilute hydrochloric acid to adjust the pH to 1.5, extract with ether 3 times, 30ml each time, discard the ether layer, adjust the pH of the aqueous layer to 10 with 40% ammonia water, and then extract with chloroform 3 times, 30ml each time. Combine the chloroform extracts and evaporate to dryness. Dissolve the residue in 0.5ml of methanol to obtain the negative sample solution. S4. Take 6 μl of the negative sample solution prepared in S3, 6 μl of the control herbal solution prepared in S2, and three aliquots of the test solution prepared in S1 (each aliquot of the test solution is 6 μl) and spot them onto the same silica gel G thin-layer plate to form bands. Place 25 ml of the developing solvent prepared by mixing 19 ml of n-hexane and 6 ml of acetone into the developing tank. Immerse one end of the spotted thin-layer plate in the developing solvent for development. Remove the plate and let it air dry. Spray it with 10% sulfuric acid ethanol solution and examine it under a 365 nm ultraviolet lamp. The test results are shown in the figure. Figure 2 .

[0045] Reference Figure 1 and Figure 2 It can be seen that, in the thin-layer chromatography identification test results of Example 1 of this application, no spots were generated at the corresponding positions of the target spots by the negative sample solvent, while spots were generated at the corresponding positions by the control medicinal material solvent, test sample solvent-1, test sample solvent-2, and test sample solvent-3. In the thin-layer chromatography identification test results of the comparative example, spots were generated at the corresponding positions of the target spots by the negative sample solvent, control medicinal material solvent, test sample solvent-1, test sample solvent-2, and test sample solvent-3. This indicates that, through improvements in the extraction methods of the test sample, control medicinal material, and negative sample in this application embodiment, the amount of sample applied to the test sample solution is greater than that applied to the control medicinal material solution. Adjusting the formulation of the developing solvent and the ratio between each component is beneficial to improving the separation degree between the target spots and the interfering spots, thereby improving the specificity.

[0046] Example 2: Adding additives Examples 2.1-2.9 Unlike Example 1, the developing solvent in Examples 2.1-2.9 was derived in equal amounts from Preparation Examples 3.2-3.10.

[0047] Rf values ​​were calculated for the detection results of Examples 2.1-2.9, and the deviations between the Rf values ​​of the target spots of the test sample and the target spots of the control medicinal material, as well as the differences in Rf values ​​between adjacent spots of the test sample solution, were calculated as follows: 1. Rf value calculation Rf=A / B in: A - Measure the distance from the center of the spot to the origin using a ruler; B - The distance from the solvent front to the origin.

[0048] 2. Deviation between the Rf value of the target spot of the test sample and the Rf value of the target spot of the control medicinal material ΔRf1 = |Rf target spot of test sample - Rf target spot of control medicinal material| × 100% 3. The difference in Rf values ​​between adjacent spots in the test solution ΔRf2=|Rf1-Rf2| in: Rf1 and Rf2 are both Rf values ​​of adjacent spots.

[0049] Calculations show that the Rf values ​​of the target spots in Examples 2.1-2.9 of this application are all within the range of 0.3-0.5, meeting the requirements for spot separation and facilitating observation and measurement; and ΔRf2 is ≥0.1, indicating that they are different substances, thus meeting the separation requirements. The calculated ΔRf1 result for Example 1 of this application is 5%, and the calculated ΔRf1 results for Examples 2.1-2.9 are shown in Table 1.

[0050] Table 1. Test data for Examples 2.1-2.9

[0051] As shown in Table 1, additives were added to the developing solvent in the thin-layer identification method of Example 2 of this application. The ΔRf1 of the thin-layer detection results was less than the ΔRf1 calculated by the thin-layer identification method of Example 1. This indicates that adding additives to the developing solvent helps to reduce the deviation between the Rf value of the target spot of the test sample and the Rf value of the target spot of the control medicinal material, thereby improving specificity.

[0052] In Examples 2.1-2.3 of this application, the effect of the amount of additive was investigated. The results showed that the ΔRf1 calculated by the thin-layer identification method in Example 2.2 was smaller. This indicates that the amount of additive selected in Example 2.2 of this application is beneficial to reducing the deviation between the Rf value of the target spot of the test sample and the Rf value of the target spot of the control medicinal material, thereby improving specificity.

[0053] Using Example 2.2 as a control, Examples 2.4-2.5 investigated the effect of the additive raw material ratio. The results showed that the ΔRf1 calculated by the thin-layer identification method in Example 2.4 was smaller. This indicates that the additive raw material ratio selected in Example 2.4 of this application is beneficial to reducing the deviation between the Rf value of the target spot of the test sample and the Rf value of the target spot of the control medicinal material, thereby improving specificity.

[0054] Compared with Example 2.4, Example 2.6 investigated the effect of titanium dioxide nanoparticle size. The results showed that the ΔRf1 calculated by the thin-layer identification method in Example 2.4 was smaller. This may be because the titanium dioxide nanoparticles selected in Example 2.4 had a particle size of 15 nm, which is smaller than the titanium dioxide nanoparticles selected in Example 2.6. Thus, the specific surface area of ​​the titanium dioxide nanoparticles enhances the interaction between the nanoparticles and the sample components, reduces spot diffusion, improves separation resolution, and makes the prepared developing agent more stable, reducing particle agglomeration on the thin-layer plate.

[0055] Using Example 2.4 as a control, Examples 2.7-2.9 investigated the effect of modified β-cyclodextrin powder. The results showed that in the thin-layer chromatography identification methods of Examples 2.7-2.9, modified β-cyclodextrin powder replaced the β-cyclodextrin powder in the additive of Example 2.4, and the calculated ΔRf1 was smaller than that calculated by the thin-layer chromatography identification method of Example 2.4. This indicates that the combination of modified β-cyclodextrin powder and titanium dioxide nanoparticles is beneficial to reducing the deviation between the Rf value of the target spot of the test sample and the Rf value of the target spot of the control herbal material, thereby improving specificity.

[0056] Examples 2.7-2.9 investigated the effect of the modified β-cyclodextrin powder raw material ratio. The results showed that the ΔRf1 calculated by the thin-layer identification method in Example 2.8 was smaller. This indicates that the modified β-cyclodextrin powder raw material ratio selected in Example 2.8 of this application is beneficial to reducing the deviation between the Rf value of the target spot of the test sample and the Rf value of the target spot of the control herbal material, thereby improving specificity.

[0057] Example 3: Process Improvement A thin-layer chromatography method for identifying Kang'erling granules and lotus seeds includes the following steps: S1. Preparation of test solution: Take 15g of Kang'erling granules, grind them into a fine powder, add 30ml of water to dissolve them, add dilute hydrochloric acid to adjust the pH to 1.5, extract with ether 3 times, 30ml each time, discard the ether layer, adjust the pH of the aqueous layer to 10 with 40% ammonia water, and then extract with chloroform 3 times, 30ml each time. Combine the chloroform extracts and evaporate to dryness. Dissolve the residue in 0.5ml of methanol to obtain the test solution. S2. Preparation of control herbal solution: Take 1g of lotus seed control herbal material, add 30ml of water, heat under reflux for 30 minutes, centrifuge and take the supernatant, add dilute hydrochloric acid to adjust the pH to 1.5, extract with ether 3 times, 30ml each time, discard the ether layer, adjust the pH of the aqueous layer to 10 with 40% ammonia water, and then extract with chloroform 3 times, 30ml each time, combine the chloroform extracts and evaporate to dryness, dissolve the residue in 0.5ml of methanol to obtain the control herbal solution; S3. Preparation of negative sample solution: Take 15g of negative sample particles, grind them into a fine powder, add 30ml of water to dissolve them, add dilute hydrochloric acid to adjust the pH to 1.5, extract with ether 3 times, 30ml each time, discard the ether layer, adjust the pH of the aqueous layer to 10 with 40% ammonia water, and then extract with chloroform 3 times, 30ml each time. Combine the chloroform extracts and evaporate to dryness. Dissolve the residue in 0.5ml of methanol to obtain the negative sample solution. S4. Thin-layer chromatography analysis: Take 15 μl of the negative sample solution prepared in S3, 5 μl of the reference medicinal material solution prepared in S2, and three portions of the test solution prepared in S1 (each portion of the test solution is 15 μl) and spot them on the same silica gel G thin-layer plate to form a strip. Add the developing solvent from Preparation Example 3.9 pre-cooled to 4°C to the first developing tank and add 25 ml of the developing solvent from Preparation Example 3.9 at 25°C to the second developing tank. Place one end of the spotted thin-layer plate in the first developing tank and saturate it with the developing solvent vapor in the first developing tank. Remove the plate and immerse it in the second developing tank until development is complete. Remove the plate and air dry it. Place it for 24 h and examine it under a 365 nm ultraviolet lamp.

[0058] The Rf value was calculated for the detection results of Example 3, and the deviation between the Rf value of the target spot of the test sample and the Rf value of the target spot of the control medicinal material and the difference between the Rf values ​​of adjacent spots of the test sample solution were calculated.

[0059] Calculations show that the Rf value of the target spot of the test sample in Example 3 of this application is in the range of 0.3-0.5, which meets the requirements for spot separation and is easy to observe and measure; and ΔRf2≥0.1, which can be judged as different substances, meeting the requirements for separation degree, and the calculated result of ΔRf1 is 1%.

[0060] Compared with Example 2.8, Example 3 investigated the effect of gradient temperature development. The results showed that the ΔRf1 calculated by the thin-layer identification method in Example 3 was smaller. This indicates that the gradient temperature development method selected in Example 3 of this application is beneficial to reduce the deviation between the Rf value of the target spot of the test sample and the Rf value of the target spot of the control medicinal material, and improve the specificity.

[0061] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for identifying lotus seeds in Kang'erling granules, characterized in that, It comprises the following steps: S1, preparing a test sample solution: take 15±1g of Kang'erling granules, grind them, add water 30±1ml to dissolve, adjust the pH to 1-2 with dilute hydrochloric acid, extract with ether 3±1 times, each time 30±1ml, discard the ether layer, adjust the pH of the water layer to 9-11 with (40±1)% ammonia water, then extract with chloroform 3±1 times, each time 30±1ml, combine the chloroform liquid and evaporate to dryness, add methanol 0.5ml to dissolve the residue to prepare the test sample solution; S2, preparing a control medicinal material solution: take 1±0.1g of lotus seed control medicinal material, add water 30±1ml, heat reflux for 30 minutes, centrifuge to take the supernatant, adjust the pH to 1-2 with dilute hydrochloric acid, extract with ether 3±1 times, each time 30±1ml, discard the ether layer, adjust the pH of the water layer to 9-11 with (40±1)% ammonia water, then extract with chloroform 3±1 times, each time 30±1ml, combine the chloroform liquid and evaporate to dryness, add methanol 0.5ml to dissolve the residue to prepare the control medicinal material solution; S3, thin layer chromatography analysis: take the test sample solution prepared in S1 and the control medicinal material solution prepared in S2 respectively, point them on the same silica gel G thin layer plate in strip shape, develop with the developing agent, take out and dry, place for 24±0.1h, and observe under a 365nm ultraviolet light.

2. The method according to claim 1, characterized in that: In S3, the sample amount of the test sample solution prepared in S1 is 15±1μl, and the sample amount of the control medicinal material solution prepared in S2 is 5±1μl.

3. The method according to claim 2, characterized in that: In S3, the developing agent is prepared by mixing cyclohexane, chloroform and diethylamine, and the addition amount of cyclohexane is 14±1ml, the addition amount of chloroform is 4±1ml, and the addition amount of diethylamine is 2±0.5ml.

4. The method according to claim 3, characterized in that: The developing agent also includes an additive, and the additive accounts for (0.1-1)% of the total amount of the developing agent, and the additive is prepared by mixing titanium dioxide nanoparticles and β-cyclodextrin powder in a weight ratio of 1:(1-3).

5. The method according to claim 4, characterized in that: The β-cyclodextrin powder is modified β-cyclodextrin powder, and the modified β-cyclodextrin powder is prepared by grafting sulfonic acid groups on β-cyclodextrin powder.

6. The method according to claim 5, characterized in that: The preparation method of the modified β-cyclodextrin powder is as follows: Dissolve β-cyclodextrin powder in dry pyridine according to a weight ratio, cool to 0-5℃ in an ice bath, pass sulfur trioxide gas for 2min, the molar ratio of β-cyclodextrin to sulfur trioxide is 1:(1-3), pour the mixture into water, add calcium carbonate to neutralize unreacted acid, filter, wash, dry and crush to prepare modified β-cyclodextrin powder.

7. The method according to claim 6, characterized in that: The particle size of the titanium dioxide nanoparticles is 15-20nm.

8. The method according to any one of claims 4-7, characterized in that: In S3, the developing agent is developed as follows: Low-temperature saturation: place the end of the thin layer plate on the upper side of the developing agent at 4±1℃; High-temperature main development: immerse the thin layer plate in the developing agent at 25±1℃ until the development is completed.

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