A thin layer method for identifying stone lotus seed formula granules and stone lotus meat formula granules and a preparation method of stone lotus seed formula granules and stone lotus meat formula granules

Abstract

The application provides a thin layer method for identifying formula granules of stone lily seeds and formula granules of stone lily flesh and a preparation method of the formula granules of stone lily seeds and the formula granules of stone lily flesh. The thin layer method adopts a mixed solution of ethyl acetate, formic acid and water as a developing agent, the volume ratio of the ethyl acetate, the formic acid and the water is 8.5:1:1, and after development and air drying, the thin layer chromatography of the formula granules of stone lily seeds and the formula granules of stone lily flesh is observed under 365 nm. Compared with the thin layer chromatography of the formula granules of stone lily seeds and the formula granules of stone lily flesh, the formula granules of stone lily seeds have spots in the range of specific migration values of 0.65-0.75, and the formula granules of stone lily flesh have no spots in the range of specific migration values of 0.65-0.75. The thin layer method can quickly and effectively identify the formula granules of stone lily seeds and the formula granules of stone lily flesh, has high stability and good repeatability, can effectively and comprehensively reflect the conditions of the formula granules of stone lily seeds and the formula granules of stone lily flesh, and provides a basis for the subsequent establishment of quality standards.

Description

Technical Field

[0001] This invention belongs to the field of thin-layer chromatography identification technology of traditional Chinese medicine, and specifically provides a thin-layer method for distinguishing between lotus seed formula granules and lotus pulp formula granules, as well as a method for preparing lotus seed formula granules and lotus pulp formula granules. Background Technology

[0002] Lotus is a common plant used for both food and medicine. It mainly contains chemical components such as alkaloids, flavonoids, glycosides, terpenes, steroids, fatty acids, proteins, minerals, and vitamins, and possesses pharmacological activities including lipid regulation, weight loss, antibacterial, anti-inflammatory, antioxidant, and hemostatic effects. Different parts of the lotus include the lotus seed heart, lotus seed, lotus seedpod, lotus receptacle, and lotus seed skin. A total of 385 compounds have been reported, including 86 alkaloids, 133 flavonoids, and 166 other compounds.

[0003] Historical records indicate that several plants were the origin of *Nelumbo nucifera* seeds. The dried seeds of *Nelumbo nucifera* and the dried, mature fruit of *Nelumbo nucifera* (also known as *Nelumbo nucifera*) were both recorded as *Nelumbo nucifera* seeds. Currently, *Nelumbo nucifera* seeds are considered the dried, mature fruit of *Nelumbo nucifera*, a plant in the Nymphaeaceae family. They are believed to have the effects of tonifying the spleen and stomach, clearing the heart and relieving irritability, and removing heat and toxins. They are used for conditions such as dysentery with loss of appetite, vomiting due to cold or heat, indigestion, and urinary tract infections. *Nelumbo nucifera* seeds contain alkaloids such as lotusine, isoligusine, methyl lotusine, and lotusine monoalkaloids, as well as starch and raffinose.

[0004] The processed product of lotus seeds, lotus kernels, is the dried, mature fruit pulp of the lotus plant (Chinese Pharmacopoeia 1963 edition). The processing method involves crushing the kernel, removing the skin and heart, and keeping only the pure flesh. It has the effects of clearing the heart and stimulating appetite. In the production areas, lotus seeds are processed by harvesting the lotus pods in autumn when the fruit is ripe, removing the fruit, removing the skin, and drying, or removing the lotus heart and then drying. It has the effects of tonifying the spleen and stopping diarrhea, stopping leukorrhea, benefiting the kidneys and astringing essence, nourishing the heart and calming the mind.

[0005] While morphological identification methods can differentiate between lotus seed and lotus pulp slices, their granulated formulations cannot be effectively distinguished by appearance alone. However, lotus seed and lotus pulp granules have different medicinal effects, necessitating differentiation between them after pharmaceutical manufacturing or purchase to control drug quality and ensure accurate medication. Currently, no methods for differentiating between the two have been reported; therefore, developing a method to distinguish between lotus seed and lotus pulp granules has significant practical value. Summary of the Invention

[0006] The present invention aims to at least partially solve one of the technical problems in the prior art. Therefore, one object of the present invention is to provide a thin-layer chromatography method for distinguishing between lotus seed formulation granules and lotus pulp formulation granules, as well as a method for preparing lotus seed formulation granules and lotus pulp formulation granules.

[0007] This invention proposes a thin-layer chromatography method for distinguishing between lotus seed formulation granules and lotus pulp formulation granules. The developing solvent used in the thin-layer chromatography method is a mixed solution of ethyl acetate, formic acid and water, wherein the volume ratio of ethyl acetate, formic acid and water is 8.5:1:1. After development, the solution is dried and examined at 365 nm.

[0008] The thin-layer chromatography method for identifying lotus seed and lotus pulp formulations according to the present invention uses a developing solvent of ethyl acetate, formic acid, and water in a volume ratio of 8.5:1:1. The presence or absence of significant spots in the thin-layer chromatogram within the R-value range of 0.65 to 0.75 is used as the identification point. This method can be used for rapid and effective identification of lotus seed and lotus pulp formulations. Specifically, within the aforementioned R-value range, lotus seed formulations show spots, while lotus pulp formulations do not. Simultaneously, using lotus seed as a reference material, the thin-layer chromatograms of lotus seed and lotus seed formulations show little difference, good separation, and high reliability under this developing solvent. Lotus seed shows significant spots within the R-value range of 0.65 to 0.75. Furthermore, compared to the current method using provincial standards for thin-layer identification of lotus seed, which requires heating for color development, the developing plate of the present invention can be examined at room temperature at 365 nm, greatly simplifying the operation and improving the identification efficiency. Therefore, the thin-layer method of the present invention can quickly and effectively identify lotus seed formula granules and lotus pulp formula granules. Moreover, the method has high stability and good repeatability, and can effectively and comprehensively reflect the condition of lotus seed formula granules and lotus pulp formula granules, providing a basis for the subsequent formulation of quality standards.

[0009] In some embodiments of the present invention, the sample preparation process of the reference herb *Nelumbo nucifera* includes: decocting the reference herb *Nelumbo nucifera* in water and filtering it; evaporating the filtrate to dryness and extracting it with a 20-40 wt% methanol aqueous solution; then filtering it again, and the filtrate is the reference herb sample solution.

[0010] Those skilled in the art will understand that the above-mentioned formulation granules for lotus seeds and lotus pulp were prepared using lotus seed extract and lotus pulp extract, respectively. Therefore, the thin-layer chromatography method provided by this invention can also be used to identify lotus seed extract and lotus pulp extract.

[0011] In some embodiments of the present invention, the amount of the control medicinal material sample solution spotted is 15-25 μl, preferably 20 μl.

[0012] In some embodiments of the present invention, the thin-layer method uses a spreading plate comprising silicone GF. 254 Thin-layer plates, silicone G thin-layer plates.

[0013] In some embodiments of the present invention, the method for preparing spotting samples of the lotus seed formula granules includes: extracting the lotus seed formula granules with a 20-40 wt% methanol aqueous solution, and then filtering.

[0014] In some embodiments of the present invention, the mass ratio of the lotus seed formula granules to the volume ratio of the methanol aqueous solution is 1g:(20-30)ml.

[0015] In some embodiments of the present invention, the extraction is ultrasonic extraction for 20-40 minutes.

[0016] In some embodiments of the present invention, the method for preparing spotting samples of the lotus seed extract formulation granules includes: extracting the lotus seed extract formulation granules with a 20-40 wt% methanol aqueous solution, and then filtering.

[0017] In some embodiments of the present invention, the mass ratio of the lotus seed extract granules to the volume ratio of the methanol aqueous solution is 1g:(20-30)ml.

[0018] In some embodiments of the present invention, the extraction is ultrasonic extraction for 20-40 minutes.

[0019] In some embodiments of the present invention, the spotting volume of the lotus seed formula granules and the spotting volume of the lotus pulp formula granules are each independently 15~25μl, preferably 20μl.

[0020] In some embodiments of the present invention, the ambient temperature for the development of the developing agent is 4°C-25°C.

[0021] In some embodiments of the present invention, the ambient humidity during the unfolding of the unfolding plate in the thin-layer method is 32%-75%.

[0022] In a second aspect, the present invention provides a method for preparing the lotus seed formulation granules used in the above-mentioned thin-layer chromatography method for distinguishing between lotus seed formulation granules and lotus pulp formulation granules, comprising: Take lotus seed slices, add water and decoct, then concentrate the resulting aqueous extract under reduced pressure to obtain a concentrated solution. The concentrated solution was spray-dried to obtain spray-dried lotus seed powder. The spray-dried lotus seed powder and maltodextrin are mixed and then dry granulated.

[0023] In some embodiments of the present invention, the decoction process includes a first decoction and a second decoction. The amount of water added in the first decoction is 8-12 times that of the lotus seed slices, and the first decoction time is 40-60 minutes. The amount of water added in the second decoction is 6-10 times that of the lotus seed slices, and the second decoction time is 20-40 minutes. The decoction temperature is 90-100°C.

[0024] In some embodiments of the present invention, the vacuum concentration temperature is 60-80°C, the vacuum degree is -0.050 to -0.080 MPa, and the vacuum concentration is carried out to a density of 1.08 to 1.10.

[0025] In some embodiments of the present invention, the inlet air temperature of the spray dryer is 185-195°C and the outlet air temperature is 85-95°C.

[0026] In some embodiments of the present invention, the amount of maltodextrin added is 5-10%.

[0027] In a third aspect, the present invention provides a method for preparing the lotus seed formulation granules in the above-mentioned thin-layer chromatography method for distinguishing between lotus seed formulation granules and lotus pulp formulation granules, comprising: Take the lotus seed slices, add water and decoct. Concentrate the resulting aqueous extract under reduced pressure to obtain a concentrated solution. The concentrated solution was spray-dried to obtain spray-dried lotus seed powder. The spray-dried lotus seed powder and maltodextrin were mixed and then dry granulated.

[0028] In some embodiments of the present invention, the decoction process includes a first decoction and a second decoction. The amount of water added in the first decoction is 8-12 times that of the lotus seed slices, and the first decoction time is 40-60 minutes. The amount of water added in the second decoction is 6-10 times that of the lotus seed slices, and the second decoction time is 20-40 minutes. The decoction temperature is 90-100°C.

[0029] In some embodiments of the present invention, the vacuum concentration temperature is 60-80°C, the vacuum degree is -0.050 to -0.080 MPa, and the vacuum concentration is carried out to a density of 1.08 to 1.10.

[0030] In some embodiments of the present invention, the inlet air temperature of the spray dryer is 185-195°C and the outlet air temperature is 85-95°C.

[0031] In some embodiments of the present invention, the amount of maltodextrin added is 5-10%.

[0032] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: (1) The present invention uses an eluent with a volume ratio of ethyl acetate, formic acid and water of 8.5:1:1. The thin-layer chromatograms of the raw material and the granulated formulation of lotus seed are similar, with good separation and high reliability. In the range of 0.65 to 0.75, the granulated formulation of lotus seed has spots (the raw material of lotus seed has spots), while the granulated formulation of lotus seed pulp has no spots. The presence or absence of the above characteristic spots can quickly, accurately and effectively identify the granulated formulation of lotus seed and the granulated formulation of lotus seed pulp.

[0033] (2) The thin-layer method provided by the present invention has high stability and good repeatability, and can effectively and comprehensively reflect the condition of lotus seed formula granules and lotus pulp formula granules, providing a basis for the formulation of subsequent quality standards. Attached Figure Description

[0034] Figure 1 The thin-layer chromatogram of Example 1; Figure 2 The thin-layer chromatogram of Example 2; Figure 3 This is the chromatogram of the Qingdao marine thin-layer plate after development in Example 3; Figure 4 The chromatogram of the thin-layer plate developed by Merck in Germany in Example 3 is shown below. Figure 5 This is the chromatogram of the Tianjin Silida thin-layer plate after development in Example 3; Figure 6 The thin-layer chromatogram is shown in Example 4. Figure 7 The thin-layer chromatogram of Example 5; Figure 8 The thin-layer chromatogram of Example 6; Figure 9 This is the thin-layer chromatogram of Comparative Example 1; Figure 10 This is the thin-layer chromatogram of Comparative Example 2; Figure 11 This is the thin-layer chromatogram of Comparative Example 3; Figure 12 This is the thin-layer chromatogram of Comparative Example 4. Detailed Implementation

[0035] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way.

[0036] The materials used in the embodiments and comparative examples of this invention are as follows: Ultrasonic machine, heating plate, mortar and pestle, thin-layer imaging system: CAMAG TLC Visualizer, silicone GF254 thin-layer plate (Tianjin Silida Technology Co., Ltd., batch number: 191026, Merck GmbH, Germany, batch number: HX87183353).

[0037] Methanol, ethyl acetate, and formic acid were all of analytical grade, and water was ultrapure water (prepared in the laboratory).

[0038] Lotus seed reference material (Lemeitian Pharmaceutical Biotechnology Co., Ltd., batch number: DSTYS003101). Lotus seed formula granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch number: 2302057). 2302058, 2302059).

[0039] Lotus seed extract granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2507001, 2507002, 2507003).

[0040] The preparation methods for lotus seed formula granules and lotus pulp formula granules are as follows: 1. Processing: Take the lotus seed medicinal material, remove impurities, crush it before use to obtain lotus seed slices.

[0041] Take the medicinal material of lotus seeds, crack them open, remove the skin and heart, and take the pure flesh to obtain lotus seed slices.

[0042] 2. Extraction and Concentration Take 1000g of lotus seed slices and lotus flesh slices. For the first decoction, add 10 times the amount of water, soak for 20 minutes, and continue decocting for 40 minutes. For the second decoction, add 8 times the amount of water and decoct for 30 minutes. During the decoction process, control the temperature at 90-100℃ to obtain the water extract.

[0043] The aqueous extract was collected and concentrated under reduced pressure to a density of 1.08-1.10 at a temperature of 60-80℃ and a vacuum degree of -0.050 to -0.080 MPa to obtain concentrated extracts of lotus seeds and lotus pulp.

[0044] 3. Drying Take the concentrated liquid and spray dry it under the conditions of inlet air temperature of 190±5℃ and outlet air temperature of 90±5℃ to obtain spray-dried powder of lotus seeds and lotus pulp.

[0045] 4. Particle preparation Take the spray-dried powder, add an appropriate amount of maltodextrin, mix well, and perform dry granulation to obtain lotus seed formula granules and lotus pulp formula granules.

[0046] Example 1 (1) Preparation of spotting samples of lotus seed reference medicinal material, lotus seed formula granules and lotus seed meat formula granules.

[0047] Preparation of spotting sample of lotus seed reference material: 1g of lotus seed reference material was added to 100ml of water and decocted for 60 minutes. The decoction was filtered, the filtrate was evaporated to dryness, the residue was added to 5ml of 30% methanol and sonicated for 30 minutes. The decoction was then filtered, and the filtrate was used as the spotting sample of lotus seed reference material.

[0048] Preparation of spotting sample of lotus seed formula granules: Grind about 0.2g of lotus seed formula granules into a fine powder, add 5ml of 30% methanol, sonicate for 30 minutes, filter, and use the filtrate as the spotting sample of lotus seed formula granules.

[0049] Preparation of spotting sample of lotus seed formula granules: Grind about 0.2g of lotus seed formula granules into a fine powder, add 5ml of 30% methanol, sonicate for 30 minutes, filter, and use the filtrate as the spotting sample of lotus seed formula granules.

[0050] (2) According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 5 μl, 10 μl, 15 μl, 20 μl, and 25 μl of the sample solution of the reference herb *Nelumbo nucifera* seed, and 10 μl, 15 μl, 20 μl, and 25 μl of the sample solution of the *Nelumbo nucifera* seed formula granules and the sample solution of the *Nelumbo nucifera* pulp formula granules were spotted onto silica gel GF. 254 On the thin-layer plate, ethyl acetate-formic acid-water (volume ratio 8.5:1:1) was used as the developing solvent. The plate was then removed, dried, and examined at 365 nm.

[0051] For details, please see [link / details]. Figure 1 , Figure 1 In the table, samples 1-5 represent the reference material samples of *Nelumbo nucifera* seeds (5µl, 10µl, 15µl, 20µl, 25µl, respectively); sample 6 represents the reference material sample of *Nelumbo nucifera* seeds (10µl); samples 7-10 represent the granulated formulation samples of *Nelumbo nucifera* seeds (10µl, 15µl, 20µl, 25µl, respectively); and samples 11-14 represent the granulated formulation samples of *Nelumbo nucifera* kernels (10µl, 15µl, 20µl, 25µl, respectively). Figure 1 It can be seen that when the sample volume of the test samples (lotus seed formula granules and lotus pulp formula granules) is 15~25μl, the thin-layer chromatographic spots are clearly visible, and the sample volume of the reference herb solution and the test sample solution is determined to be 20μl. Compared with the chromatogram of the reference herb, the ratio shift (R0) is significantly higher. f At a value of 0.75, the lotus seed formula granules have the same spot, while the lotus pulp formula granules have no significant spots. This spot is used as the identification point for distinguishing between lotus seed formula granules and lotus pulp formula granules.

[0052] Example 2 (Specificity Investigation) The only difference between Example 2 and Example 1 is that the sample volume of the negative control solution (30% methanol), *Nelumbo nucifera* seed reference material, *Nelumbo nucifera* seed formula granules, and *Nelumbo nucifera* pulp formula granules was 20 μl. The negative control solution, *Nelumbo nucifera* seed reference material sample solution, *Nelumbo nucifera* seed formula granule sample solution, and *Nelumbo nucifera* pulp formula granule sample solution were respectively spotted onto the same silica gel GF. 254 On the thin-layer plate, ethyl acetate-formic acid-water (volume ratio 8.5:1:1) was used as the developing solvent. The plate was then removed, dried, and examined at 365 nm.

[0053] For details, please see [link / details]. Figure 2 , Figure 2 In the table, 1 and 6 are negative controls; 2 and 7 are *Nelumbo nucifera* seed reference materials; 3-5 are *Nelumbo nucifera* seed formula granules; and 8-10 are *Nelumbo nucifera* kernel formula granules. Figure 2 It can be seen that the negative sample did not interfere with the test samples of lotus seed formula granules and lotus pulp formula granules, indicating that the method has good specificity. Compared with the chromatogram of lotus seed reference material, the ratio shift (R0) was significantly higher. f At a value of 0.73, the same spots were observed in the lotus seed formula granules, while the lotus pulp formula granules showed no significant spots.

[0054] Example 3 (Durability Study) The only difference between Example 3 and Example 1 is that the sample volume for the lotus seed control material, lotus seed formula granules, and lotus pulp formula granules is 20 μl, and pre-formulated silica gel GF from three brands—Merck (Germany), Tianjin Slida, and Qingdao Haiyang—is used. 254 Thin-layer plate.

[0055] The chromatogram of the Qingdao marine thin-layer plate after development is shown below. Figure 3 , Figure 3 In the table, 1 and 4 are reference materials of *Nelumbo nucifera* seeds; 2-3 are granulated formulations of *Nelumbo nucifera* seeds; and 5-6 are granulated formulations of *Nelumbo nucifera* kernels. The chromatograms after development on a Merck thin-layer plate are shown below. Figure 4 , Figure 4 In the table, 1 and 4 are reference materials of *Nelumbo nucifera* seeds; 2-3 are formula granules of *Nelumbo nucifera* seeds; 5-6 are formula granules of *Nelumbo nucifera* kernels; the chromatograms after thin-layer plate development by Tianjin Silida Technology Co., Ltd. are shown below. Figure 5 , Figure 5 In the table, 1 and 4 are reference materials for *Nelumbo nucifera* seeds; 2-3 are *Nelumbo nucifera* seed formula granules; and 5-6 are *Nelumbo nucifera* kernel formula granules. Figure 3-5 It can be seen that all three brands of thin-layer chromatography plates can meet the expected identification requirements, indicating that the method has good durability. Compared with the reference material chromatogram of lotus seed, the ratio shift (R0) of the Merck thin-layer chromatography plate from Germany is higher. f At a value of 0.72, the same spots were observed in the lotus seed formulation granules, while no significant spots were observed in the lotus pulp formulation granules; the specific displacement value (R0.72) of the Tianjin Silida thin-layer plate was... f At a value of 0.66, the same spots were observed in the lotus seed formulation granules, while no significant spots were observed in the lotus pulp formulation granules; the specific displacement value (R0.66) of the Qingdao marine thin-layer plate was also observed. f At a value of 0.70, the lotus seed formula granules had the same spots, while the lotus pulp formula granules had no significant spots.

[0056] Example 4 (Investigation at different temperatures) The only difference between Example 4 and Example 1 is that the sample volume of the lotus seed control material, lotus seed formula granules, and lotus seed pulp formula granules is 20 μl. The thin-layer plates after sampling are taken and developed at low temperature of 4℃ and normal temperature of 25℃, respectively.

[0057] For details, please see [link / details]. Figure 6 , Figure 6 In the table, 1 and 4 are reference materials for *Nelumbo nucifera* seeds; 2-3 are *Nelumbo nucifera* seed formula granules; and 5-6 are *Nelumbo nucifera* kernel formula granules. Figure 6 It can be seen that this method has good durability under different temperatures. Compared with the reference chromatogram of lotus seed, at a temperature of 4℃, the ratio shift (R0) is significantly higher. f At a value of 0.73, the same spots were observed in the lotus seed formulation granules, while no significant spots were observed in the lotus pulp formulation granules; at a temperature of 25℃, the specific displacement value (R0) was... f At a value of 0.66, the same spots were observed in the lotus seed formula granules, while the lotus pulp formula granules showed no significant spots.

[0058] Example 5 (Investigation at different humidity levels) The only difference between Example 5 and Example 1 is that the sample volume of the lotus seed control material, lotus seed formula granules, and lotus pulp formula granules is 20 μl. The thin-layer plates after sampling are developed in 32% and 75% humidity environments, respectively.

[0059] For details, please see [link / details]. Figure 7 , Figure 7 In the table, 1 and 4 are reference materials for *Nelumbo nucifera* seeds; 2-3 are *Nelumbo nucifera* seed formula granules; and 5-6 are *Nelumbo nucifera* kernel formula granules. Figure 7 It can be seen that this method has good durability under different humidity levels. Compared with the reference medicinal material atlas of lotus seeds, when the humidity is 32%, the ratio shift (R0) is significantly higher. f At a value of 0.65, the lotus seed formulation granules showed the same spots, while the lotus pulp formulation granules showed no significant spots; when the humidity was 75%, the specific displacement value (R) was... f At a value of 0.66, the same spots were observed in the lotus seed formula granules, while the lotus pulp formula granules showed no significant spots.

[0060] Example 6 The thin-layer chromatography method of Example 1 was used, with a sample size of 20 μl. Three batches of lotus seed formulation granules and three batches of lotus pulp formulation granules were used to verify the thin-layer chromatography method.

[0061] For details, please see [link / details]. Figure 8 , Figure 8 In the diagram, 1 and 5 represent the reference material of *Nelumbo nucifera* seeds; 2-4 represent the formulated granules of *Nelumbo nucifera* seeds; and 6-8 represent the formulated granules of *Nelumbo nucifera* kernels. The results showed that compared with the chromatogram of the reference material of *Nelumbo nucifera* seeds, the ratio shift (R0) was significantly higher. fAt a value of 0.72, the same spots were observed in the lotus seed formula granules, while the lotus pulp formula granules showed no significant spots.

[0062] The ratio shift values ​​of the identification spots in Examples 1, 2-6 were summarized, and the ratio shift values ​​of various data in the thin-layer chromatography method investigation were summarized. The results are shown in Table 1. Based on the summarized results, the ratio shift values ​​(R0) of the identification spots of the lotus seed and lotus pulp formulation granules were determined. f The percentage should be within 10%, with an average of 0.70.

[0063] Table 1

[0064] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is as follows: Provincial standard method: Take 1g of this product, grind it finely, add 10ml of 70% ethanol, sonicate for 30 minutes, filter, and use the filtrate as the test solution. Separately, take 1g of *Nelumbo nucifera* seed reference material, add 100ml of water, boil, maintain a gentle boil for 1 hour, cool, filter, evaporate the filtrate to dryness, add 10ml of 70% ethanol to the residue, and prepare the reference material solution using the same method. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, General Chapter 0502), taking 5μl of the test solution and 10μl of the reference material solution, and spotting them separately on the same silica gel GF254 thin-layer plate. Use n-butanol-isopropanol-water-glacial acetic acid (7:5:1:1) as the developing solvent, develop, remove, air dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0065] For details, please see [link / details]. Figure 9 , Figure 9 In the table, 1 represents the reference medicinal material of lotus seed, 2 represents the formula granules of lotus seed, and 3 represents the formula granules of lotus pulp. Figure 9 It can be seen that, compared with the reference medicinal material atlas of lotus seeds, there are no obvious differences in the spots between the lotus seed formula granules and the lotus seed pulp formula granules, and the two cannot be distinguished.

[0066] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the sample amount of the lotus seed control material, lotus seed formula granules, and lotus pulp formula granules was 20 μl, and the developing solvent was n-hexane-acetone (7:2).

[0067] For details, please see [link / details]. Figure 10 , Figure 10 In the table, 1 represents the reference medicinal material of lotus seed, 2 represents the formula granules of lotus seed, and 3 represents the formula granules of lotus pulp. Figure 10 It can be seen that no obvious dots are found in the atlas, so the two cannot be distinguished.

[0068] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that the sample amount of the lotus seed control material, lotus seed formula granules, and lotus pulp formula granules was 20 μl, and the developing solvent was chloroform-ethyl acetate-diethylamine (5:4:1).

[0069] For details, please see [link / details]. Figure 11 , Figure 11 In the table, 1 represents the reference medicinal material of lotus seed, 2 represents the formula granules of lotus seed, and 3 represents the formula granules of lotus pulp. Figure 11 It can be seen that, compared with the reference medicinal material atlas of lotus seeds, there are no obvious differences in the spots between the lotus seed formula granules and the lotus seed pulp formula granules, and the two cannot be distinguished.

[0070] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that the sample amount of the reference herb *Nelumbo nucifera*, the formula granules of *Nelumbo nucifera*, and the formula granules of *Nelumbo nucifera* kernel was 20 μl, and the developing solvent was ethyl acetate-acetic acid-water (8.5:1:1).

[0071] For details, please see [link / details]. Figure 12 , Figure 12 In the table, 1 represents the reference medicinal material of lotus seed, 2 represents the formula granules of lotus seed, and 3 represents the formula granules of lotus pulp. Figure 12 It can be seen that, compared with the reference medicinal material atlas of lotus seeds, there are no obvious differences in the spots between the lotus seed formula granules and the lotus seed pulp formula granules, and the two cannot be distinguished.

[0072] Any numerical value mentioned in this invention, if there is only a two-unit interval between any minimum and any maximum value, includes all values ​​that increase by one unit each time from the minimum to the maximum value. For example, if the amount of a component, or the value of a process variable such as temperature, pressure, or time, is stated as 50-90, in this specification it means specifically listing values ​​such as 51-89, 52-88… and 69-71 and 70-71, etc. For non-integer values, it may be appropriately considered that a unit is 0.1, 0.01, 0.001, or 0.0001. These are merely some specifically specified examples. In this application, in a similar manner, all possible combinations of numerical values ​​between the listed minimum and maximum values ​​are considered to have been disclosed.

[0073] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A thin-layer chromatography method for distinguishing between lotus seed formulation granules and lotus pulp formulation granules, characterized in that, The thin-layer method uses a silicone GF spreading plate. 254 Thin-layer chromatography (TLC) was performed using a mixed solution of ethyl acetate, formic acid, and water as the developing solvent, with a volume ratio of 8.5:1:

1. After development and drying, the TLC was examined at 365 nm. Compared with the reference chromatogram of *Nelumbo nucifera* seed, the TLC of *Nelumbo nucifera* seed formulation granules showed that *Nelumbo nucifera* seed formulation granules had spots in the range of 0.65-0.75, while *Nelumbo nucifera* seed formulation granules had no spots in the range of 0.65-0.

75. The preparation method of the spotting sample of the reference medicinal material of lotus seed includes: 1g of lotus seed reference medicinal material, add 100ml of water, decoct for 60 minutes, filter, evaporate the filtrate to dryness, add 5ml of 30% methanol to the residue, sonicate for 30 minutes, filter, and use the filtrate as the spotting sample of lotus seed reference medicinal material. The preparation method of the spotting sample of the lotus seed formula granules includes: extracting the lotus seed formula granules with a 20-40 wt% methanol aqueous solution, and then filtering; The preparation method of the spotting sample of the lotus seed formula granules includes: extracting the lotus seed formula granules with a 20-40 wt% methanol aqueous solution, and then filtering.

2. The method according to claim 1, characterized in that, The spotting volume of the lotus seed formula granules and the spotting volume of the lotus pulp formula granules are each 15~25μl independently; And / or, the ambient temperature for the development of the developing agent is 4℃-25℃; And / or, the ambient humidity during the unfolding of the unfolding plate in the thin-layer method is 32%-75%.

3. The method according to claim 2, characterized in that, The sample amounts of the lotus seed formula granules and the lotus pulp formula granules were 20 μl each.

4. The method according to any one of claims 1-3, characterized in that, The mass ratio of the lotus seed formula granules to the volume ratio of the methanol aqueous solution is 1g:(20-30)ml; And / or, the extraction is ultrasonic extraction for 20-40 minutes.

5. The method according to any one of claims 1-3, characterized in that, The mass ratio of the lotus seed extract granules to the volume ratio of the methanol aqueous solution is 1g:(20-30)ml; And / or, the extraction is ultrasonic extraction for 20-40 minutes.

6. The method according to any one of claims 1-3, characterized in that, The preparation method of the lotus seed formula granules includes: Take lotus seed slices, add water and decoct, then concentrate the resulting aqueous extract under reduced pressure to obtain a concentrated solution. The concentrated solution was spray-dried to obtain spray-dried lotus seed powder. The spray-dried lotus seed powder and maltodextrin are mixed and then dry granulated.

7. The method according to claim 6, characterized in that, The decoction process includes a first decoction and a second decoction. The amount of water added in the first decoction is 8-12 times that of the lotus seed slices, and the first decoction time is 40-60 minutes. The amount of water added in the second decoction is 6-10 times that of the lotus seed slices, and the second decoction time is 20-40 minutes. The decoction temperature is 90-100℃. And / or, the vacuum concentration temperature is 60-80℃, the vacuum degree is -0.050~-0.080Mpa, and the vacuum concentration is carried out to a density of 1.08~1.10; And / or, the inlet air temperature of the spray dryer is 185-195℃, and the outlet air temperature is 85-95℃; And / or, the amount of maltodextrin added is 5-10%.

8. The method according to any one of claims 1-3, characterized in that, The preparation method of the lotus seed extract granules includes: Take the lotus seed slices, add water and decoct. Concentrate the resulting aqueous extract under reduced pressure to obtain a concentrated solution. The concentrated solution was spray-dried to obtain spray-dried lotus seed powder. The spray-dried lotus seed powder and maltodextrin were mixed and then dry granulated.

9. The method according to claim 8, characterized in that, The decoction process includes a first decoction and a second decoction. The amount of water added in the first decoction is 8-12 times that of the lotus seed slices, and the first decoction time is 40-60 minutes. The amount of water added in the second decoction is 6-10 times that of the lotus seed slices, and the second decoction time is 20-40 minutes. The decoction temperature is 90-100℃. And / or, the vacuum concentration temperature is 60-80℃, the vacuum degree is -0.050~-0.080Mpa, and the vacuum concentration is carried out to a density of 1.08~1.10; And / or, the inlet air temperature of the spray dryer is 185-195℃, and the outlet air temperature is 85-95℃; And / or, the amount of maltodextrin added is 5-10%.

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