Method for identifying ginkgo formula granules and fried ginkgo formula granules based on thin-layer chromatography

Abstract

The present invention provides a method for identifying ginkgo formula granules and fried ginkgo formula granules based on thin-layer chromatography. The method comprises the following steps: coating a test sample solution on a silica gel thin-layer plate, developing according to a thin-layer chromatographic condition, adding a color developing agent after developing is finished, and then heating, and carrying out inspection. According to the present invention, trichloromethane-cyclohexane-acetone-glacial acetic acid having a volume ratio of (8-12):(3-6):(3-6):(1.5-2.5) is used as a developing agent, and it is found that in thin-layer chromatography, the obtained fried ginkgo formula granules have fluorescent spots when a retention factor Rf1 is 0.648±0.0648 and a retention factor Rf2 is 0.741±0.0741, while the ginkgo formula granules have no fluorescent spots. Therefore, the presence or absence of fluorescent spots in Rf1 and Rf2 can be used as identification points of the ginkgo formula granules and the fried ginkgo formula granules. By means of verification, the method has high specificity, good stability, and high precision.

Description

Thin layer chromatography identification method for ginkgo kernel formula granules and fried ginkgo kernel formula granules

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on October 7, 2023, with application number 202311293808.9 and invention name “A Thin Layer Chromatography Identification Method for Ginkgo Kernel Formula Granules and Fried Ginkgo Kernel Formula Granules”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention belongs to the technical field of drug detection, and particularly relates to a thin-layer chromatography identification method for ginkgo kernel formula granules and fried ginkgo kernel formula granules. Background Art

[0003] Ginkgo nuts are the dried, mature seeds of Ginkgo biloba L., a plant of the Ginkgoaceae family. They have the effects of astringing the lungs, calming asthma, and stopping leukorrhea and reducing urination. However, because raw ginkgo nuts are slightly toxic, they must be processed before clinical use. Studies have shown that stir-frying ginkgo nuts can significantly reduce their toxicity, increasing the accuracy and safety of medication. Furthermore, processed ginkgo nuts can enhance their astringent properties, primarily warming the lungs and replenishing qi, calming asthma and cough, reducing urination, and stopping leukorrhea. This suggests that the chemical composition of the two may have changed.

[0004] Li Zhuanmei et al. compared the ginkgolic acid and total ginkgolic acid content of ginkgo nuts in different processed ginkgo nuts and found that peeling, stir-frying, or steaming significantly reduced the amounts of these acids, thereby reducing toxicity. However, stir-frying, steaming, and boiling all significantly reduced the levels of these acids. Stir-frying, which requires a higher temperature and shorter cooking time, resulted in better detoxification. Li Juan et al. compared the contents of ginkgolide A and ginkgolide C in stir-fried ginkgo nuts from different origins using HPLC-UV. The results showed that the contents of ginkgolide A and ginkgolide C differed little between the two regions, corresponding to the amounts lost during processing. While the contents of ginkgolide A and ginkgolide C decreased somewhat after stir-frying, there was no qualitative difference compared to the raw product. Therefore, a more specific, reproducible, and effective method is needed to provide an effective means for the qualitative identification of stir-fried ginkgo granules.

[0005] Summary of the Invention

[0006] In view of this, the present invention aims to provide a thin-layer chromatography method for distinguishing between ginkgo kernel formula granules and stir-fried ginkgo kernel formula granules. This method uses the presence of fluorescent spots within a certain ratio shift range on the thin-layer chromatography spectrum as a basis for identification, effectively and quickly distinguishing between ginkgo kernel formula granules and stir-fried ginkgo kernel formula granules.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] The present invention provides a thin layer chromatography identification method for ginkgo kernel formula granules and fried ginkgo kernel formula granules, comprising the following steps:

[0009] Apply the test solution on a silica gel thin layer plate and develop it according to the thin layer chromatography conditions. After development, add the color developer and inspect it after heating.

[0010] Preferably, the developing solvent in the thin layer chromatography conditions is chloroform-cyclohexane-acetone-glacial acetic acid, and the volume ratio of chloroform, cyclohexane, acetone and glacial acetic acid is (8-12):(3-6):(3-6):(1.5-2.5).

[0011] More preferably, the volume ratio of chloroform, cyclohexane, acetone and glacial acetic acid is (10-12):(5-6):(5-6):2.

[0012] Preferably, the sample volume of the test solution is 15 to 25 μL.

[0013] Preferably, the unfolding environment temperature is 8-25° C., and the environment humidity is 32-75 RH%.

[0014] Preferably, in the thin layer chromatography examined, the stir-fried ginkgo kernel formula granules have fluorescent spots within the range of a ratio transfer value Rf1 of 0.648±0.0648 and a ratio transfer value Rf2 of 0.741±0.0741, while the ginkgo kernel formula granules have no fluorescent spots.

[0015] Preferably, the inspection is performed under ultraviolet light.

[0016] Preferably, the wavelength of the ultraviolet light is 365 nm.

[0017] Preferably, the test solution is prepared according to the following method:

[0018] Mix the test sample with water, heat it, filter it, and use column chromatography to treat the filtrate. After removing impurities by eluting with water, elute it with ethanol, collect the eluate, remove the ethanol, add ethyl acetate to extract, evaporate the ethyl acetate, add methanol to the residue to obtain the test sample solution.

[0019] Preferably, the ethanol is 50 vol% ethanol.

[0020] Preferably, the thin layer chromatography identification method further comprises the step of spot-coating the reference solution and the negative solution on a silica gel thin layer plate.

[0021] Preferably, the reference substance in the reference solution includes ginkgolide B and ginkgolide C.

[0022] Preferably, the spotting volume of the reference solution is 1 to 5 μL.

[0023] Preferably, the spotting volume of the negative solution is 1 to 3 μL.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a thin layer chromatography identification method for ginkgo kernel formula granules and fried ginkgo kernel formula granules. The method involves spot-coating a test sample solution on a silica gel thin layer plate and developing it according to thin layer chromatography conditions. The developing agent used is chloroform-cyclohexane-acetone-glacial acetic acid in a volume ratio of (8-12):(3-6):(3-6):(1.5-2.5). After development, a color developer is added, and after heating, the sample is inspected. The results show that the fried ginkgo kernel formula granules in the obtained thin layer chromatography have fluorescent spots within the range of a relative transfer value Rf1 of 0.648±0.0648 and a relative transfer value Rf2 of 0.741±0.0741, while the ginkgo kernel formula granules have no fluorescent spots. Therefore, one more fluorescent spot within the above-mentioned Rf1 range can be used as identification point 1 of the fried ginkgo kernel formula granules, and one more fluorescent spot within the range of Rf2 can be used as identification point 2 of the fried ginkgo kernel formula granules. It has been verified that the thin layer chromatography identification method provided by the present invention can quickly and effectively identify the fried ginkgo kernel formula granules that have lost their medicinal material form through identification points 1 and 2 in thin layer chromatography. It is simple to operate, has high precision and sensitivity, good stability, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a thin layer chromatogram of the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution at different sample amounts;

[0027] In Figure 1, the sample volumes of 1 to 3 corresponding to the reference solution are 1 μL, 2 μL, and 5 μL, the sample volumes of 4 to 6 corresponding to the ginkgo kernel formula granule solution are 15 μL, 20 μL, and 25 μL, and the sample volumes of 7 to 9 corresponding to the stir-fried ginkgo formula granule solution are 15 μL, 20 μL, and 25 μL;

[0028] Figure 2 is a thin layer chromatogram of the negative solution, the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution;

[0029] In Figure 2, 1 corresponds to the negative solution, 2 corresponds to the reference solution; 3 corresponds to the ginkgo kernel formula granule solution; 4 corresponds to the stir-fried ginkgo kernel formula granule solution;

[0030] FIG3 is a thin layer chromatogram of a reference solution, a ginkgo formula granule solution, and a stir-fried ginkgo kernel formula granule solution using a silica gel G plate produced by Tianjin Silida Technology Co., Ltd.;

[0031] 1 to 3 in FIG3 correspond to the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution, respectively;

[0032] FIG4 is a thin layer chromatogram of a reference solution, a ginkgo formula granule solution, and a stir-fried ginkgo kernel formula granule solution using a Merck silica gel G plate;

[0033] 1 to 3 in FIG4 correspond to the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution, respectively;

[0034] Figure 5 is a thin layer chromatogram of a reference solution, a ginkgo formula granule solution, and a stir-fried ginkgo kernel formula granule solution using a silica gel G plate from Qingdao Ocean Silica Gel Reagent Factory;

[0035] 1 to 3 in FIG5 correspond to the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution, respectively;

[0036] Figure 6 is a thin layer chromatogram of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution developed at a low temperature of 8°C;

[0037] 1 to 3 in FIG6 correspond to the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution, respectively;

[0038] Figure 7 is a thin layer chromatogram of the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution obtained at room temperature 25°C;

[0039] 1 to 3 in FIG7 correspond to the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution, respectively;

[0040] FIG8 is a thin layer chromatogram of a reference solution, a ginkgo formula granule solution, and a stir-fried ginkgo kernel formula granule solution obtained by developing the solution under a humidity environment of 32 RH%;

[0041] 1 to 3 in FIG8 correspond to the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution, respectively;

[0042] FIG9 is a thin layer chromatogram of a reference solution, a ginkgo formula granule solution, and a stir-fried ginkgo kernel formula granule solution obtained by developing the solution under a humidity environment of 75 RH%;

[0043] 1 to 3 in FIG9 correspond to the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution, respectively;

[0044] Figure 10 is a thin layer chromatogram of the reference solution, as well as multiple batches of ginkgo formula granule solutions and multiple batches of stir-fried ginkgo kernel formula granule solutions for verification;

[0045] In Figure 10, 1 corresponds to the reference solution, 2 to 4 correspond to solutions of Ginkgo formula granules with batch numbers 2209001, 2209002, and 2209003, respectively, and 5 to 7 correspond to solutions of stir-fried Ginkgo formula granules with batch numbers 2304047, 2304048, and 2304049, respectively;

[0046] Figure 11 is a thin layer chromatogram of a reference solution, a ginkgo formula granule solution, and a stir-fried ginkgo kernel formula granule solution obtained using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio 8:3:3:1.5) as a developing solvent;

[0047] Figure 12 is a thin layer chromatogram of a reference solution, a ginkgo formula granule solution, and a stir-fried ginkgo kernel formula granule solution obtained using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio 12:6:6:2.5) as a developing solvent;

[0048] Figure 13 is a thin layer chromatogram of the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution obtained by the thin layer chromatography method of Comparative Example 1;

[0049] In Figure 13, number 1 corresponds to the reference solution, numbers 2 to 3 correspond to the Ginkgo Nut Formula Granules solution with a sample volume of 10 μL and 15 μL, and numbers 4 to 5 correspond to the Roasted Ginkgo Nut Formula Granules solution with a sample volume of 10 μL and 15 μL;

[0050] Figure 14 is a thin layer chromatogram of the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution obtained using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio 10:10:5:2) as the developing solvent.

[0051] 1 to 2 in FIG14 correspond to the ginkgo formula granule solution and the stir-fried ginkgo kernel formula granule solution, respectively;

[0052] Figure 15 is a thin layer chromatogram of a reference solution, a ginkgo formula granule solution, and a stir-fried ginkgo kernel formula granule solution obtained using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio 5:7:3:1) as a developing solvent;

[0053] In Figure 15, 1 corresponds to the reference solution, 2-3 correspond to the ginkgo kernel formula granule solution, and 4-5 correspond to the stir-fried ginkgo kernel formula granule solution. DETAILED DESCRIPTION

[0054] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0055] In view of the problem that there is a lack of effective technical means for qualitatively identifying stir-fried ginkgo nut formula granules in the prior art, the present invention provides a thin layer chromatography identification method for ginkgo kernel formula granules and stir-fried ginkgo kernel formula granules, comprising the following steps:

[0056] Apply the test solution on a silica gel thin layer plate and develop it according to the thin layer chromatography conditions. After development, add the color developer and inspect it after heating.

[0057] In the present invention, the test solution refers to a ginkgo kernel formula granule solution or a fried ginkgo kernel formula granule solution, which can preferably be prepared according to the following method:

[0058] The test sample (i.e., ginkgo kernel formula granules or fried ginkgo kernel formula granules) is mixed with water, heated, filtered, and the filtrate is treated by column chromatography. After removing impurities by water elution, it is eluted with ethanol, and the eluate is collected. After removing the ethanol, ethyl acetate is added for extraction. The ethyl acetate solution is evaporated to dryness, and methanol is added to the residue to obtain the test sample solution.

[0059] According to the above method of the present invention, the aqueous solution of the test sample is first heated to dissolve the test sample, and then the insoluble matter is removed by filtration. The obtained filtrate containing the test sample is purified by column chromatography, and then the purified test sample is further extracted with ethyl acetate and concentrated to increase the concentration of the test sample. Finally, the obtained solid is dissolved in methanol to obtain the corresponding test sample solution.

[0060] In the present invention, the sample is preferably mixed with water in a ratio of 1 g: (15-25) mL, more preferably 1 g: 20 mL, and after heating, the sample is dissolved, and then the insoluble matter is removed by filtration. The filtrate containing the sample is purified by column chromatography. The column chromatography preferably adopts wet column packing, and the column used is a polyamide column. The inner diameter of the polyamide column is preferably 0.8-1.2 cm, more preferably 1 cm; the mesh number of the packed polyamide is preferably 30-60 mesh, more preferably 40-50 mesh, and the mass ratio of the polyamide loading amount to the sample is preferably 1: (0.8-1.5), more preferably 1: 1. During the column chromatography process, water elution is preferably used to remove impurities, and then elution is performed with ethanol, preferably 50 vol% ethanol. After the eluate is collected, it is preferably steamed on a water bath to remove ethanol. Then, the purified test sample is further extracted with ethyl acetate and concentrated. The extraction is performed 2 times or more to increase the concentration of the test sample. Finally, the ethyl acetate solutions are combined and evaporated to dryness. The obtained solid is dissolved in methanol. The ratio of the test sample to methanol is preferably 1 g: (0.8-3) mL, more preferably 1 g: 1 mL, to obtain the corresponding test sample solution.

[0061] In a specific embodiment of the present invention, the test solution is prepared according to the following method:

[0062] Take 1 g of the test sample (i.e., ginkgo kernel formula granules or fried ginkgo kernel formula granules), add 20 mL of water, heat to dissolve, filter, concentrate the filtrate to 5 mL, add it to a treated polyamide column (30-60 mesh, 1 g, inner diameter 1 cm, wet-packed with water), elute with 50 mL of water, discard the water and elute with 40 mL of 50 vol% ethanol, collect the eluate, place it on a water bath to evaporate the ethanol, and extract the water with ethyl acetate by shaking twice, 20 mL each time. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 mL of methanol to prepare the test solution.

[0063] According to the present invention, after obtaining the test solution, the test solution is applied to a silica gel thin layer plate, developed according to thin layer chromatography conditions, and a color developer is added after development. After heating, the plate is inspected. The silica gel thin layer plate is preferably a silica gel G thin layer plate. The present invention has been found through durability research to achieve the corresponding effect using silica gel thin layer plates produced by different manufacturers, indicating that the method has good adaptability to different thin layer plates. Therefore, there is no particular restriction on the source of the silica gel thin layer plate. At the same time, after investigating the amount of sample applied, it was found that when the amount of the sample solution applied was 15 to 25 μL, the spots in the obtained thin layer chromatography were clearly colored and had good separation. Therefore, the amount of the sample solution applied was preferably 15 to 25 μL, more preferably 20 to 25 μL, and most preferably 25 μL.

[0064] It should be noted that not all developing agents can make the ginkgo kernel formula granules or fried ginkgo kernel formula granules have a developing effect. After research, the present invention prefers chloroform-cyclohexane-acetone-glacial acetic acid as the developing agent in the thin layer chromatography conditions. In order to ensure good separation between the two and good spot color development, the present invention prefers that the volume ratio of chloroform, cyclohexane, acetone and glacial acetic acid is (8-12):(3-6):(3-6):(1.5-2.5), more preferably (10-12):(5-6):(5-6):2, and most preferably 10:5:5:2.

[0065] The present invention performs thin layer chromatography separation on the above-mentioned test solution under development environments of different temperatures and different humidities. It is found that the method has strong adaptability to temperature and humidity. The development environment temperature can be in the range of 8 to 25°C, specifically 8°C, 10°C, 15°C, 20°C or 25°C; the environmental humidity can be in the range of 32 to 75RH%, specifically 32RH%, 35RH%, 40RH%, 45RH%, 50RH%, 55RH%, 60RH%, 65RH%, 70RH% or 75RH%.

[0066] After the development is completed, in order to enable fluorescent spots to appear during inspection, it is necessary to spray a color developer on the silica gel thin layer plate. The color developer is preferably an acetic anhydride solution, and heated at 140-160°C, preferably 150°C, for 20-40 minutes, preferably 30 minutes, and inspected under an ultraviolet lamp (365nm).

[0067] After the inspection is completed, the thin layer chromatograms of the ginkgo kernel formula granules or the roasted ginkgo kernel formula granules obtained show that the roasted ginkgo kernel formula granules have one more fluorescent spot compared to the ginkgo kernel formula granules when the ratio shift value Rf1 is 0.648±0.0648 (i.e., approximately 0.58-0.71), which serves as the roasted ginkgo kernel formula granule identification point 1; and the ratio shift value Rf2 is 0.741±0.0741 (i.e., approximately 0.67-0.82), which serves as the roasted ginkgo kernel formula granule identification point 2. Therefore, whether there is an additional fluorescent spot within the above Rf1 and Rf2 ranges can be used as the roasted ginkgo kernel formula granule identification point.

[0068] In some embodiments of the present invention, the method further comprises the step of spot-coating a reference solution and a negative solution on a silica gel thin layer plate, wherein the reference solution is used to determine whether the sample contains ginkgolide B and ginkgolide C, and the negative solution is used to eliminate interference with the sample.

[0069] In the present invention, since the fluorescent spots of Ginkgolide A are not clear, based on comprehensive considerations, the reference substances in the reference solution preferably include Ginkgolide B and Ginkgolide C. The reference solution is preferably prepared by mixing Ginkgolide B and Ginkgolide C with methanol to obtain a reference solution. In some specific embodiments of the present invention, the reference solution is preferably prepared according to the following steps:

[0070] Take the Ginkgolide B reference substance and the Ginkgolide C reference substance, add methanol to make a mixed solution containing 1 mg of each per 1 mL, which is used as the reference solution.

[0071] In the present invention, after inspection, fluorescent spots of the same color appear in the chromatogram of the test sample at the corresponding position in the chromatogram of the reference sample, indicating that the fried ginkgo kernels and the fried ginkgo kernel formula granules can be effectively identified.

[0072] The negative solution can be prepared according to the following method:

[0073] Take 1 mL of methanol as the negative solution.

[0074] It should be noted that, in the present invention, after research, the spotting volume of the reference solution is preferably 1-5 μL, more preferably 2 μL; the spotting volume of the negative solution is preferably 1-3 μL, more preferably 1 μL.

[0075] After multiple batches of verification, the thin layer chromatography identification method provided by the present invention can quickly and effectively identify the fried ginkgo kernel formula granules that have lost their medicinal material form through the identification points Rf1 and Rf2 in thin layer chromatography. It is simple to operate, has high precision and sensitivity, good stability, and has good application prospects.

[0076] In order to further illustrate the present invention, the following examples are used for detailed description. The instruments used in the following examples of the present invention include a heating plate, a mortar, a semi-automatic thin layer sampler: CAMAGLionmat-5, a thin layer imaging system: CAMAGTLCVisualizer, a silica gel G thin layer plate (Qingdao Ocean Chemical Plant, batch number: 20180527, Tianjin Silida Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353); the reagents used include toluene, ethyl acetate, methanol, acetone, and acetic anhydride, all of which are analytical grade, and water is ultrapure water (made in the laboratory); The test drugs used include Ginkgolide B (China Food and Drug Inspection Institute, batch number: 110863-201611), Ginkgolide C (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq18051108), Ginkgo Nut Formula Granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2209001; 2209002; 2209003) and Fried Ginkgo Nut Formula Granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2304047; 2304048; 2304049).

[0077] Preparation Example 1

[0078] This preparation example provides a method for preparing a test solution of ginkgo formula granules and stir-fried ginkgo kernel formula granules, comprising the following steps:

[0079] Take 1 g of this product (gingko formula granules or fried ginkgo kernel formula granules), add 20 mL of water, heat to dissolve it, filter, concentrate the filtrate to 5 mL, add it to a treated polyamide column (30-60 mesh, 1 g, inner diameter 1 cm, wet-packed with water), elute with 50 mL of water, discard the water and elute with 40 mL of 50 vol% ethanol, collect the eluate, place it on a water bath to evaporate the ethanol, shake the water with ethyl acetate (20 mL each time) twice, combine the ethyl acetate, evaporate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test solution.

[0080] Preparation Example 2

[0081] This preparation example provides a method for preparing a reference solution, comprising the following steps:

[0082] Take Ginkgolide B reference substance and Ginkgolide C reference substance, add methanol to prepare a mixed solution containing 1 mg of Ginkgolide B reference substance and 1 mg of Ginkgolide C reference substance per 1 mL, which is used as the mixed reference solution of Ginkgolide B and Ginkgolide C (referred to as "reference solution").

[0083] Preparation Example 3

[0084] This preparation example provides a method for preparing a negative solution, comprising the following steps:

[0085] Take 1 mL of methanol as the negative solution.

[0086] Example 1

[0087] This embodiment provides a thin layer chromatography method for identifying ginkgo formula granules and stir-fried ginkgo kernel formula granules, and investigates the amount of sample spotted, comprising the following steps:

[0088] S1: According to the method of Preparation Examples 1-2, provide a test solution of ginkgo formula granules, a test solution of stir-fried ginkgo kernel formula granules, and a reference solution;

[0089] S2: Take 15μL, 20μL, and 25ul of the test solution of the ginkgo formula granules and the test solution of the fried ginkgo kernel formula granules, and take 1μL, 2μL, and 5μL of the reference solution, and test them according to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate. Use chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 10:5:5:2) as the developing agent, develop at 25°C, 75RH%, take out, dry, spray with acetic anhydride solution, heat at 140°C for 30 minutes, and inspect under ultraviolet light (365nm).

[0090] The results are shown in Figure 1 (1 to 3 correspond to the reference solution with a sample volume of 1 μL, 2 μL, and 5 μL; 4 to 6 correspond to the ginkgo kernel formula granule solution with a sample volume of 15 μL, 20 μL, and 25 μL; 7 to 9 correspond to the stir-fried ginkgo kernel formula granule solution with a sample volume of 15 μL, 20 μL, and 25 μL). It can be seen that when the reference solution sample volume is 2 μL and the test solution sample volume is 20 μL, the thin layer chromatography fluorescent spots are clearly colored. Therefore, the reference solution sample volume is determined to be 2 μL and the test solution sample volume is determined to be 20 μL. In addition, compared with the spectrum of the reference, the stir-fried ginkgo kernel formula granules have obvious fluorescent spots at the relative shift value (Rf1) of 0.65 and the relative shift value (Rf2) of 0.736, while the ginkgo kernel formula granules have no fluorescent spots. Therefore, whether there are fluorescent spots at the ratio shift value (Rf1) 0.65 and the ratio shift value (Rf2) 0.736 can be used as identification points.

[0091] Example 2

[0092] This embodiment provides a thin layer chromatography method for identifying ginkgo formula granules and stir-fried ginkgo kernel formula granules, and investigates the specificity, comprising the following steps:

[0093] S1: According to the methods of Preparation Examples 1 to 3, provide a test solution of ginkgo formula granules, a test solution of stir-fried ginkgo kernel formula granules, a reference solution, and a negative solution;

[0094] S2: Take 20 μL of the test solution of ginkgo formula granules and the test solution of fried ginkgo kernel formula granules, take 1 μL of the reference solution, and take 1 μL of the negative solution. Test according to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), spot them on the same silica gel G thin layer plate, and use chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 10:5:5:2) as the developing agent. Develop at 25°C and 75RH%. Take out, dry, spray with acetic anhydride solution, heat at 150°C for 30 minutes, and inspect under ultraviolet light (365nm).

[0095] The results, shown in Figure 2, indicate that the negative sample showed no interference with the ginkgo kernel and stir-fried ginkgo kernel granules, demonstrating the method's high specificity. Compared to the control, the stir-fried ginkgo kernel granules exhibited distinct fluorescent spots at Rf1 and Rf2 values ​​of 0.7 and 0.771, respectively, while the ginkgo kernel granules lacked fluorescent spots. Therefore, the presence of fluorescent spots at Rf1 and Rf2 values ​​of 0.7 and 0.771 can be used as identification criteria.

[0096] Example 3

[0097] This embodiment provides a thin layer chromatography method for identifying ginkgo nuts formula granules and stir-fried ginkgo kernel formula granules, and investigates durability (different thin layer plates), comprising the following steps:

[0098] S1: According to the method of Preparation Examples 1-2, provide a test solution of ginkgo formula granules, a test solution of stir-fried ginkgo kernel formula granules, and a reference solution;

[0099] S2: Take 20 μL of the test solution of ginkgo formula granules and the test solution of fried ginkgo kernel formula granules, and take 1 μL of the reference solution, and test them according to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), and spot them on silica gel G plate (Qingdao Ocean Silica Gel Reagent Factory), silica gel G plate (Tianjin Silida Technology Co., Ltd.), and Merck silica gel G plate, respectively. Use chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 10:5:5:2) as the developing agent, develop at 25°C, 75RH%, take out, dry, spray with acetic anhydride solution, heat at 160°C for 30 minutes, and inspect under ultraviolet light (365nm).

[0100] The results are shown in Figures 3 to 5. Figure 3 shows a thin layer chromatogram obtained using a silica gel G plate from Tianjin Silida Technology Co., Ltd., Figure 4 shows a thin layer chromatogram obtained using a Merck silica gel G plate, and Figure 5 shows a thin layer chromatogram obtained using a silica gel G plate from Qingdao Ocean Silica Gel Reagent Factory. As can be seen from Figures 3 to 5, the three different silica gel G plates all met the identification requirements, demonstrating the good durability of this method. Furthermore, compared with the reference product spectra, when the ratio shift values ​​(Rf1) under the three different silica gel G plates were 0.7, 0.658, and 0.7, and the ratio shift values ​​(Rf2) were 0.8, 0.734, and 0.791, the stir-fried ginkgo kernel formula granules had obvious fluorescent spots, while the ginkgo kernel formula granules had no fluorescent spots. Therefore, the presence of fluorescent spots at the ratio shift values ​​(Rf1) of 0.7, 0.658, and 0.7, and the ratio shift values ​​(Rf2) of 0.8, 0.734, and 0.791 can be used as identification points.

[0101] Example 4

[0102] This embodiment provides a thin layer chromatography method for identifying ginkgo nuts formula granules and stir-fried ginkgo kernel formula granules, and investigates their durability (at different temperatures), comprising the following steps:

[0103] S1: According to the method of Preparation Examples 1-2, provide a test solution of ginkgo formula granules, a test solution of stir-fried ginkgo kernel formula granules, and a reference solution;

[0104] S2: Take 20 μL of the test solution of ginkgo formula granules and the test solution of fried ginkgo kernel formula granules, and 1 μL of the reference solution, and test them according to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate. Use chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 10:5:5:2) as the developing agent, and develop them at low temperature of 8°C and room temperature of 25°C (humidity of 75RH%), respectively. Take them out, dry them, spray them with acetic anhydride solution, heat them at 145°C for 30 minutes, and inspect them under ultraviolet light (365nm).

[0105] The results are shown in Figures 6 and 7. Figure 6 is a thin layer chromatogram obtained by developing at a low temperature of 8°C, and Figure 7 is a thin layer chromatogram obtained by developing at a room temperature of 25°C. As can be seen from Figures 6 and 7, this method has good durability at different temperatures. Compared with the reference sample spectrum developed at a low temperature of 8°C and a room temperature of 25°C, the roasted ginkgo kernel formula granules have obvious fluorescent spots at the ratio shift values ​​(Rf1) of 0.6 and 0.6, and the ratio shift values ​​(Rf2) of 0.7 and 0.7, while the ginkgo kernel formula granules have no fluorescent spots. Therefore, the presence of fluorescent spots at the ratio shift values ​​(Rf1) of 0.6 and 0.6, and the ratio shift values ​​(Rf2) of 0.7 and 0.7 can be used as the identification point.

[0106] Example 5

[0107] This embodiment provides a thin layer chromatography method for identifying ginkgo nuts and stir-fried ginkgo kernels, and investigates their durability (at different humidity levels), comprising the following steps:

[0108] S1: According to the method of Preparation Examples 1-2, provide a test solution of ginkgo formula granules, a test solution of stir-fried ginkgo kernel formula granules, and a reference solution;

[0109] S2: Take 20 μL of the test solution of ginkgo formula granules and the test solution of fried ginkgo kernel formula granules, and 1 μL of the reference solution, and test them according to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate. Use chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 10:5:5:2) as the developing agent, and develop them under a humidity environment of 32RH% and 75RH% (temperature of 25°C). Take out, dry, spray with acetic anhydride solution, heat at 145°C for 30 minutes, and inspect under ultraviolet light (365nm).

[0110] The results are shown in Figures 8 and 9. Figure 8 is a thin layer chromatogram obtained by developing in a humidity environment of 32RH%, and Figure 9 is a thin layer chromatogram obtained by developing in a humidity environment of 75RH%. It can be seen from Figures 8 and 9 that this method has good durability under different humidity conditions. Compared with the reference sample spectrum, the spectrum of the test sample developed in a humidity environment of 32RH% and 75RH% has obvious fluorescent spots at the ratio shift values ​​(Rf1) of 0.6 and 0.6, and the ratio shift values ​​(Rf2) of 0.7 and 0.74 for the fried ginkgo kernel formula granules, while the ginkgo kernel formula granules have no fluorescent spots. Therefore, whether there are fluorescent spots at the ratio shift values ​​(Rf1) of 0.6 and 0.6, and the ratio shift values ​​(Rf2) of 0.7 and 0.74 can be used as the identification point.

[0111] Example 6

[0112] This example verifies the above-mentioned thin layer differential chromatography method, comprising the following steps:

[0113] S1: Take the reference substance, 3 batches of ginkgo formula granules and 3 batches of stir-fried ginkgo formula granules respectively, and provide the corresponding reference substance solution, test solution of different batches of ginkgo formula granules and test solution of different batches of stir-fried ginkgo formula granules according to the method of Preparation Examples 1 and 2;

[0114] S2: Take 20 μL of the test solution of ginkgo formula granules from different batches and the test solution of fried ginkgo kernel formula granules from different batches, and take 1 μL of the reference solution. According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), they were spotted on the same silica gel G thin layer plate, and all were developed with chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 10:5:5:2). The plates were developed at 25°C and 75RH%. The plates were taken out, dried, sprayed with acetic anhydride solution, heated at 145°C for 30 minutes, and inspected under ultraviolet light (365nm).

[0115] The results are shown in Figure 10. Compared to the reference sample, the spectra of the test solution from different batches show distinct fluorescent spots at the Rf1 and Rf2 values ​​of 0.673 and 0.74, respectively, for the roasted ginkgo nut granules, while the ginkgo nut granules lack fluorescent spots. Therefore, the presence of fluorescent spots at Rf1 and Rf2 values ​​of 0.673 and 0.74 can be used as identification criteria.

[0116] Example 7

[0117] This embodiment provides a thin layer identification chromatography method for ginkgo formula granules and stir-fried ginkgo kernel formula granules. Compared with the method in Example 6, the only difference is that chloroform-cyclohexane-acetone-glacial acetic acid with a volume ratio of 8:3:3:1.5 is used as the developing solvent, and the other parameters and steps are consistent with Example 1.

[0118] The results are shown in Figure 11. Compared to the control, the roasted ginkgo kernel granules exhibit distinct fluorescent spots at Rf1 and Rf2 values ​​of 0.65 and 0.74, respectively, while the ginkgo kernel granules lack fluorescent spots. Therefore, the presence of fluorescent spots at Rf1 and Rf2 values ​​of 0.65 and 0.74 can be used as identification criteria.

[0119] Example 8

[0120] This example provides a thin-layer differential chromatography method for ginkgo formula granules and stir-fried ginkgo kernel formula granules. Compared with the method in Example 6, the only difference is that chloroform-cyclohexane-acetone-glacial acetic acid with a volume ratio of 12:6:6:2.5 is used as the developing solvent, and the remaining parameters and steps remain the same as in Example 1.

[0121] The results are shown in Figure 12. Compared to the control, the roasted ginkgo kernel granules exhibit distinct fluorescent spots at Rf1 of 0.65 and Rf2 of 0.77, while the ginkgo kernel granules lack fluorescent spots. Therefore, the presence of fluorescent spots at Rf1 of 0.65 and Rf2 of 0.77 can be used as a discriminant.

[0122] Determination of the identification point Rf of ginkgo formula granules and stir-fried ginkgo formula granules

[0123] The data for each ratio shift value from the thin-layer chromatography methodology investigation are summarized in Table 1. Based on the summary results, it was determined that the ratio shift value for identification spot 1 (Rf1) of the roasted ginkgo kernel formula granules should be within 10%, with a specified value of 0.648; the ratio shift value for identification spot 2 (Rf2) of the roasted ginkgo kernel formula granules should be within 10%, with a specified value of 0.741.

[0124] Table 1

[0125] Comparative Example 1

[0126] S1: Grind 1g of ginkgo kernel granules and stir-fried ginkgo kernel granules, dissolve in 30mL of water, and shake extract twice with 20mL of ethyl acetate. Combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1mL of methanol to prepare the test solution. Separately, prepare a mixed solution containing 1mg of each of the ginkgolide B and ginkgolide C reference substances in methanol to prepare a reference solution containing 1mg of each substance per mL.

[0127] S2: According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 8 μL of the test solution and 2 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate with a sodium carboxymethyl cellulose solution containing 4% sodium acetate as an adhesive, and developed with toluene-ethyl acetate-acetone-methanol (10:5:5:0.6) at 25°C and 75RH%. The plate was taken out, dried, sprayed with acetic anhydride, heated at 140°C for 30 minutes, and inspected under a UV lamp (365nm).

[0128] The results are shown in Figure 13. Using the above-mentioned thin layer identification method, there is no obvious difference in the spots of ginkgo nuts and fried ginkgo nuts formula granules, so it is impossible to effectively distinguish the ginkgo nuts formula granules from the fried ginkgo nuts formula granules.

[0129] The above results show that the thin layer chromatography obtained by using a thin layer method other than the one provided by the present invention cannot identify the ginkgo kernel formula granules and the fried ginkgo kernel formula granules.

[0130] Comparative Example 2

[0131] S1: According to the method of Preparation Examples 1-2, provide a test solution of ginkgo formula granules, a test solution of stir-fried ginkgo kernel formula granules, and a reference solution;

[0132] S2: According to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), 8 μL of the test solution and 2 μL of the reference solution were respectively spotted on the same silica gel G thin layer plate, and chloroform-cyclohexane-acetone-glacial acetic acid (10:10:5:2) was used as the developing agent. The plate was developed at 25°C and 75% RH. The plate was taken out, dried, sprayed with acetic anhydride solution, heated at 140-160°C for 30 minutes, and inspected under ultraviolet light (365 nm).

[0133] The results are shown in Figure 14. It can be seen that using the above-mentioned thin layer identification method, the separation degree of ginkgo kernels and fried ginkgo kernel formula granules is poor, so it is impossible to effectively distinguish the ginkgo kernel formula granules from the fried ginkgo kernel formula granules.

[0134] The above results show that the thin layer chromatography obtained by using a developing solvent that is not within the ratio range of the present invention cannot identify the ginkgo kernel formula granules and the fried ginkgo kernel formula granules.

[0135] Comparative Example 3

[0136] S1: According to the method of Preparation Examples 1-2, provide a test solution of ginkgo formula granules, a test solution of stir-fried ginkgo kernel formula granules, and a reference solution;

[0137] S2: Take 20 μL of the test solution of ginkgo formula granules and the test solution of fried ginkgo kernel formula granules, and take 1 μL of the reference solution, and test them according to the thin layer chromatography method (General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate. Use chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 5:7:3:1) as the developing agent, develop at 25°C, 75RH%, take out, dry, spray with acetic anhydride solution, heat at 140-160°C for 30 minutes, and inspect under ultraviolet light (365nm).

[0138] The results are shown in Figure 15. It can be seen that the separation degree of ginkgo kernels and fried ginkgo kernel formula granules is poor using the above-mentioned thin layer identification method, so it is impossible to effectively distinguish the ginkgo kernel formula granules from the fried ginkgo kernel formula granules.

[0139] The above results show that the thin layer chromatography obtained by using a developing solvent that is not within the ratio range of the present invention cannot distinguish the ginkgo kernel formula granules and the fried ginkgo kernel formula granules.

[0140] The above description of the disclosed embodiments will enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. A thin layer chromatography identification method for ginkgo kernel formula granules and fried ginkgo kernel formula granules, characterized in that: The following steps are involved: Apply the test solution on a silica gel thin layer plate and develop it according to the thin layer chromatography conditions. After the development is completed, add a color developer, heat it, and inspect it. The developing solvent in the thin layer chromatography conditions is chloroform-cyclohexane-acetone-glacial acetic acid, and the volume ratio of chloroform, cyclohexane, acetone and glacial acetic acid is (8-12):(3-6):(3-6):(1.5-2.5).

2. The method according to claim 1, characterized in that The volume ratio of chloroform, cyclohexane, acetone and glacial acetic acid is (10-12):(5-6):(5-6):

2.

3. The method according to claim 1, characterized in that The sample volume of the test solution is 15 to 25 μL.

4. The method according to claim 1, characterized in that The unfolding environment temperature is 8-25° C., and the environment humidity is 32-75 RH%.

5. The method according to claim 1, characterized in that In the thin layer chromatogram examined, the stir-fried ginkgo kernel formula granules had fluorescent spots in the range of Rf1 of 0.648±0.0648 and Rf2 of 0.741±0.0741, while the ginkgo kernel formula granules had no fluorescent spots.

6. The method according to claim 1, characterized in that The inspection is carried out under ultraviolet light; The wavelength of the ultraviolet rays is 365 nm.

7. The method according to claim 1, characterized in that The test solution was prepared according to the following method: Mix the test sample with water, heat it, filter it, and treat the filtrate with column chromatography. After removing impurities by eluting with water, elute it with ethanol, collect the eluate, remove the ethanol, add ethyl acetate to extract, evaporate the ethyl acetate, add methanol to the residue to obtain the test sample solution.

8. The method according to claim 7, characterized in that The ethanol is 50 vol% ethanol.

9. The method according to claim 1, characterized in that: The method also includes the step of spot coating the reference solution and the negative solution on the silica gel thin layer plate.

10. The method according to claim 9, characterized in that The reference substance in the reference substance solution includes ginkgolide B and ginkgolide C; The spotting volume of the reference solution is 1 to 5 μL; The spotting volume of the negative solution is 1 to 3 μL.

Images