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

Abstract

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: applying a test solution on a silica gel thin layer plate, developing according to thin layer chromatography conditions, adding a color developer after the development is completed, heating, and inspecting. The present invention uses chloroform-cyclohexane-acetone-glacial acetic acid with a volume ratio of (8-12):(3-6):(3-6):(1.5-2.5) as a developing agent, and finds that the fried ginkgo kernel formula granules in the obtained thin layer chromatography have a specific transfer value Rf 1 0.648±0.0648, Rf 2 There are fluorescent spots in the range of 0.741±0.0741, while there are no fluorescent spots in the ginkgo kernel formula granules. 1 and Rf 2 The presence or absence of fluorescent spots within the range can be used as a distinguishing point for ginkgo kernel formula granules and fried ginkgo kernel formula granules. It has been verified that this method has strong specificity, good stability and high precision.

Description

Technical Field

[0001] 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

[0002] Ginkgo biloba L. is a dried mature seed of the Ginkgo biloba L. plant of the Ginkgoaceae family. It has the effects of astringing the lungs and calming asthma, stopping leukorrhea and reducing urine. However, because the raw ginkgo is slightly toxic, it needs to be processed before being used as a medicine in clinical use. Studies have shown that stir-frying ginkgo can greatly reduce its toxicity, increase the accuracy and safety of medication, and its processed products can enhance the astringent effect, mainly warming the lungs and replenishing qi, calming asthma and cough, reducing urine, and stopping leukorrhea. This shows that the chemical composition of the two may have changed.

[0003] Li Zhuanmei et al. compared the ginkgolic acid and total ginkgolic acid in different processed ginkgo products and found that peeling, frying or steaming could significantly reduce the amount of ginkgolic acid and total ginkgolic acid in ginkgo, thereby reducing toxicity. However, as for the three methods of frying, steaming and boiling, the frying method has a higher temperature, a shorter frying time and a better detoxification effect. Li Juan et al. compared the content of ginkgolide A and ginkgolide C in ginkgo from different origins after stir-frying by HPLC-UV. The results showed that the content of ginkgolide A and ginkgolide C in ginkgo from different origins after stir-frying was not much different, which was equivalent to the content lost after processing. Although the content of ginkgolide A and ginkgolide C in ginkgo after stir-frying was reduced to a certain extent, there was no qualitative difference compared with the raw product. Therefore, it is necessary to develop a more specific, repeatable, effective and controllable method to provide an effective means for the qualitative identification of stir-fried ginkgo formula granules. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a thin layer chromatography identification method for ginkgo kernel formula granules and fried ginkgo kernel formula granules. The method can effectively and quickly distinguish ginkgo kernel formula granules and fried ginkgo kernel formula granules by observing whether there are fluorescent spots in a certain ratio shift value range in the thin layer chromatography spectrum as the basis for identification.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] 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:

[0007] 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.

[0008] 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).

[0009] More preferably, the volume ratio of chloroform, cyclohexane, acetone and glacial acetic acid is (10-12):(5-6):(5-6):2.

[0010] Preferably, the spotting volume of the test solution is 15 to 25 μL.

[0011] Preferably, the unfolding environment temperature is 8-25° C., and the environment humidity is 32-75 RH%.

[0012] Preferably, in the examined thin layer chromatography, the stir-fried ginkgo kernel formula granules have fluorescent spots within the range of the ratio transfer value Rf1 of 0.648±0.0648 and the ratio transfer value Rf2 of 0.741±0.0741, while the ginkgo kernel formula granules have no fluorescent spots.

[0013] Preferably, the inspection is performed under ultraviolet light.

[0014] Preferably, the wavelength of the ultraviolet light is 365 nm.

[0015] Preferably, the test solution is prepared according to the following method:

[0016] 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.

[0017] Preferably, the ethanol is 50 vol% ethanol.

[0018] 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.

[0019] Preferably, the reference substance in the reference substance solution includes ginkgolide B and ginkgolide C.

[0020] Preferably, the spotting volume of the reference solution is 1 to 5 μL.

[0021] Preferably, the spotting volume of the negative solution is 1 to 3 μL.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a thin layer chromatography identification method for ginkgo kernel formula granules and fried ginkgo kernel formula granules. The method includes applying a test solution on a silica gel thin layer plate, developing according to thin layer chromatography conditions, and using a developing agent of chloroform-cyclohexane-acetone-glacial acetic acid in a volume ratio of (8-12):(3-6):(3-6):(1.5-2.5). After the development is completed, a color developer is added, and after heating, the color developer is inspected. As a result, it is found that the fried ginkgo kernel formula granules in the obtained thin layer chromatography have fluorescent spots within the range of the ratio shift value Rf1 of 0.648±0.0648 and the ratio shift 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 the identification point 1 of the fried ginkgo kernel formula granules, and one more fluorescent spot within the range of Rf2 can be used as the 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 stir-fried ginkgo kernel formula granules that have lost the medicinal material morphology through the identification points 1 and 2 in the thin layer chromatography. It is easy to operate, has high precision and sensitivity, good stability, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The thin layer chromatograms of the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution at different sample amounts are shown;

[0025] Figure 1 The spotting volume of 1 to 3 corresponding to the reference solution is 1 μL, 2 μL, and 5 μL, the spotting volume of 4 to 6 corresponding to the ginkgo kernel formula granule solution is 15 μL, 20 μL, and 25 μL, and the spotting volume of 7 to 9 corresponding to the stir-fried ginkgo formula granule solution is 15 μL, 20 μL, and 25 μL;

[0026] Figure 2 The thin layer chromatograms of negative solution, reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution are shown;

[0027] 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;

[0028] Figure 3 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution were obtained using the silica gel G plate produced by Tianjin Silida Technology Co., Ltd.

[0029] Figure 3 1 to 3 correspond to the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution, respectively;

[0030] Figure 4The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution when Merck silica gel G plate was used;

[0031] Figure 4 1 to 3 correspond to the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution, respectively;

[0032] Figure 5 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution were obtained using the silica gel G plate produced by Qingdao Ocean Silica Gel Reagent Factory.

[0033] Figure 5 1 to 3 correspond to the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution, respectively;

[0034] Figure 6 The thin layer chromatograms 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 are shown;

[0035] Figure 6 1 to 3 correspond to the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution, respectively;

[0036] Figure 7 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution developed at room temperature 25°C are shown;

[0037] Figure 7 1 to 3 correspond to the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution, respectively;

[0038] Figure 8 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution developed under a humidity environment of 32 RH% are shown;

[0039] Figure 8 1 to 3 correspond to the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution, respectively;

[0040] Fig. 9 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution developed under a humidity environment of 75 RH% are shown;

[0041] Fig. 9 1 to 3 correspond to the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution, respectively;

[0042] Fig.10The reference solution and the thin layer chromatograms for verifying multiple batches of ginkgo formula granule solutions and multiple batches of stir-fried ginkgo kernel formula granule solutions;

[0043] Fig.10 1 corresponds to the reference solution, 2-4 correspond to the solutions of the batch numbers 2209001, 2209002, and 2209003 of the ginkgo formula granules, and 5-7 correspond to the solutions of the batch numbers 2304047, 2304048, and 2304049 of the stir-fried ginkgo kernel formula granules;

[0044] Fig.11 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution obtained by using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio 8:3:3:1.5) as the developing solvent;

[0045] Fig.12 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution obtained by using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio 12:6:6:2.5) as the developing solvent;

[0046] Fig.13 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution obtained by the thin layer method of Comparative Example 1 are shown;

[0047] Fig.13 1 in the figure corresponds to the reference solution, 2-3 correspond to the sample volume of the ginkgo kernel formula granule solution of 10μL and 15μL, and 4-5 correspond to the sample volume of the fried ginkgo kernel formula granule solution of 10μL and 15μL;

[0048] Fig.14 The thin layer chromatograms of the reference solution, ginkgo formula granule solution, and stir-fried ginkgo kernel formula granule solution were obtained using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio 10:10:5:2) as the developing solvent.

[0049] Fig.14 1 to 2 correspond to the ginkgo formula granule solution and the stir-fried ginkgo kernel formula granule solution respectively;

[0050] Fig.15 The thin layer chromatograms of the reference solution, the ginkgo formula granule solution, and the stir-fried ginkgo kernel formula granule solution obtained by using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio 5:7:3:1) as the developing solvent;

[0051] Fig.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

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] In view of the problem that there is a lack of effective technical means in the prior art to qualitatively identify the stir-fried ginkgo formula granules, 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:

[0054] 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.

[0055] 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:

[0056] 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.

[0057] 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 and concentrated with ethyl acetate to increase the concentration of the test sample. Finally, the obtained solid is dissolved with methanol to obtain the corresponding test sample solution.

[0058] 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, and the obtained 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, and the inner diameter of the polyamide column is preferably 0.8-1.2 cm, more preferably 1 cm; the mesh number of the loaded polyamide is preferably 30-60 mesh, more preferably 40-50 mesh, and the mass ratio of the loading amount of polyamide to the sample is preferably 1: (0.8-1.5), more preferably 1: 1. In the process of the column chromatography, water elution is preferably used to remove impurities, and then ethanol, preferably 50 vol% ethanol, is used for elution. 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, and the extraction is performed 2 times or more to increase the concentration of the test sample. Finally, the ethyl acetate solutions are combined, evaporated to dryness, and 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.

[0059] In a specific embodiment of the present invention, the test solution is prepared according to the following method:

[0060] 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 aqueous solution, elute with 40 mL of 50 vol% ethanol, collect the eluate, evaporate the ethanol on a water bath, extract the aqueous solution with ethyl acetate by shaking twice, 20 mL each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test sample solution.

[0061] 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 the thin layer chromatography conditions, a color developer is added after the development is completed, and the plate is inspected after heating. Among them, the silica gel thin layer plate is preferably a silica gel G thin layer plate. The present invention has been studied for durability and found that the silica gel thin layer plates produced by different manufacturers can achieve the corresponding effect, indicating that the method has good adaptability to different thin layer plates, so there is no special restriction on the source of the silica gel thin layer plate. At the same time, after the sample spotting amount is investigated, it is found that when the sample spotting amount of the test solution is 15 to 25 μL, the spots in the obtained thin layer chromatography are clearly colored and have good separation, so the sample spotting amount of the test solution is preferably 15 to 25 μL, more preferably 20 to 25 μL, and most preferably 25 μL.

[0062] 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 of 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.

[0063] The present invention performs thin layer chromatography separation on the above-mentioned test solution under development environments of different temperatures and different humidities, and 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, etc.; the environment humidity can be in the range of 32 to 75RH%, specifically 32RH%, 35RH%, 40RH%, 45RH%, 50RH%, 55RH%, 60RH%, 65RH%, 70RH% or 75RH%, etc.

[0064] After the development is completed, in order to enable fluorescent spots to appear during inspection, it is necessary to spray the silica gel thin layer plate with a color developer, preferably an acetic anhydride solution, heat at 140-160°C, preferably 150°C, for 20-40 minutes, preferably 30 minutes, and inspect under ultraviolet light (365nm).

[0065] After the inspection is completed, the thin layer chromatograms of the test samples of the ginkgo kernel formula granules or the fried ginkgo kernel formula granules obtained by observation show that the fried ginkgo kernel formula granules have one more fluorescent spot when the ratio shift value Rf1 is 0.648±0.0648 (i.e., approximately 0.58-0.71) compared with the ginkgo kernel formula granules, which is used as the identification point 1 of the fried ginkgo kernel formula granules; and one more fluorescent spot when the ratio shift value Rf2 is 0.741±0.0741 (i.e., approximately 0.67-0.82) compared with the ginkgo kernel formula granules, which is used as the identification point 2 of the fried ginkgo kernel formula granules. Therefore, whether there are more fluorescent spots within the above range of Rf1 and Rf2 can be used as the identification point of the fried ginkgo kernel formula granules.

[0066] In some embodiments of the present invention, the method further comprises the step of applying 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.

[0067] In the present invention, since the fluorescent spot of Ginkgolide A is not clear, after comprehensive consideration, the reference substance in the reference solution preferably includes Ginkgolide B and Ginkgolide C. The reference solution preferably takes Ginkgolide B reference substance and Ginkgolide C reference substance, and mixes them 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:

[0068] Take Ginkgolide B reference substance and Ginkgolide C reference substance, add methanol to prepare a mixed solution containing 1 mg of each per 1 mL, as the reference substance solution.

[0069] In the present invention, after inspection, in the chromatogram of the test sample, fluorescent spots of the same color appear at positions corresponding to those of the chromatogram of the reference sample, indicating that the fried ginkgo kernels and the fried ginkgo kernel formula granules can be effectively identified.

[0070] The negative solution can be prepared according to the following method:

[0071] Take 1 mL of methanol as the negative solution.

[0072] It should be noted that, in the present invention, after research, the spotting amount of the reference solution is preferably 1-5 μL, more preferably 2 μL; the spotting amount of the negative solution is preferably 1-3 μL, more preferably 1 μL.

[0073] After multiple batches of verification, the above-mentioned thin layer chromatography identification method provided by the present invention can quickly and effectively identify the stir-fried ginkgo kernel formula granules that have lost the medicinal material morphology through the identification points Rf1 and Rf2 in the thin layer chromatography. It is easy to operate, has high precision and sensitivity, good stability, and has good application prospects.

[0074] 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 spotter: 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 analytically pure, and water is ultrapure water (self-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 kernel formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2209001; 2209002; 2209003) and stir-fried Ginkgo kernel formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2304047; 2304048; 2304049).

[0075] Preparation Example 1

[0076] This preparation example provides a method for preparing a test solution of ginkgo formula granules and fried ginkgo kernel formula granules, comprising the following steps:

[0077] 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 aqueous solution, elute with 40 mL of 50 vol% ethanol, collect the eluate, evaporate the ethanol on a water bath, extract the aqueous solution with ethyl acetate (20 mL each time) by shaking twice, combine the ethyl acetate solutions, evaporate to dryness, add 1 mL of methanol to the residue to dissolve it, and use it as the test solution.

[0078] Preparation Example 2

[0079] This preparation example provides a method for preparing a reference solution, comprising the following steps:

[0080] 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 Ginkgolide C reference substance per 1 mL, as the mixed reference solution of Ginkgolide B and Ginkgolide C (referred to as "reference solution").

[0081] Preparation Example 3

[0082] This preparation example provides a method for preparing a negative solution, comprising the following steps:

[0083] Take 1 mL of methanol as the negative solution.

[0084] Example 1

[0085] This embodiment provides a thin layer chromatography method for identification of ginkgo formula granules and stir-fried ginkgo kernel formula granules, and investigates the spotting amount, comprising the following steps:

[0086] 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;

[0087] 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 Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 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).

[0088] The results are as follows Figure 1 As shown in Figures 1 to 3, the sample volume of the reference solution is 1 μL, 2 μL, and 5 μL; the sample volume of the ginkgo kernel formula granule solution is 15 μL, 20 μL, and 25 μL; the sample volume of the fried ginkgo kernel formula granule solution is 15 μL, 20 μL, and 25 μL. It can be seen that when the sample volume of the reference solution is 2 μL and the sample volume of the test solution is 20 μL, the fluorescent spots of the thin layer chromatography are clearly colored. Therefore, the sample volume of the reference solution is determined to be 2 μL and the sample volume of the test solution is 20 μL. Moreover, compared with the spectrum of the reference substance, the fried ginkgo kernel formula granules have obvious fluorescent spots at the ratio shift value (Rf1) of 0.65 and the ratio 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.

[0089] Example 2

[0090] This embodiment provides a thin layer chromatography method for identification of ginkgo formula granules and stir-fried ginkgo kernel formula granules, and investigates the specificity, comprising the following steps:

[0091] 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;

[0092] S2: Take 20 μL of the test solution of ginkgo formula granules and the test solution of stir-fried ginkgo kernel formula granules, take 1 μL of the reference solution, and take 1 μL of the negative solution, and test them according to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 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 them at 25°C, 75RH%, take them out, dry them, spray them with acetic anhydride solution, heat them at 150°C for 30 minutes, and inspect them under ultraviolet light (365nm).

[0093] The results are as follows Figure 2 As shown, it can be seen that the negative sample has no interference with the ginkgo kernel and stir-fried ginkgo kernel formula granules, and the method has good specificity. Compared with the spectrum of the reference substance, the stir-fried ginkgo kernel formula granules have obvious fluorescent spots at the ratio shift value (Rf1) of 0.7 and the ratio shift value (Rf2) of 0.771, while the ginkgo kernel formula granules have no fluorescent spots. Therefore, whether there are fluorescent spots at the ratio shift value (Rf1) of 0.7 and the ratio shift value (Rf2) of 0.771 can be used as the identification point.

[0094] Example 3

[0095] This embodiment provides a thin layer chromatography method for identification of ginkgo formula granules and stir-fried ginkgo kernel formula granules, and investigates durability (different thin layer plates), comprising the following steps:

[0096] 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;

[0097] S2: Take 20 μL of the test solution of the ginkgo formula granules and the test solution of the fried ginkgo kernel formula granules, and take 1 μL of the reference solution, and test them according to the thin layer chromatography method (General Rules 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, and use chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 10:5:5:2) as the developing agent, develop them at 25°C, 75RH%, take them out, dry them, spray them with acetic anhydride solution, heat them at 160°C for 30 minutes, and inspect them under ultraviolet light (365nm).

[0098] Results Figures 3 to 5 , Figure 3 This is a thin layer chromatogram obtained using the silica gel G plate produced by Tianjin Slida Technology Co., Ltd. Figure 4 Thin layer chromatogram obtained using Merck silica gel G plate, Figure 5 This is a thin layer chromatogram obtained using a silica gel G plate from Qingdao Ocean Silica Gel Reagent Factory. Figures 3 to 5It can be seen that the three different silica gel G plates can meet the identification requirements, indicating that the method has good durability. In addition, compared with the reference product spectrum, when the ratio shift values ​​(Rf1) under the three different silica gel G plates are 0.7, 0.658, and 0.7, and the ratio shift values ​​(Rf2) are 0.8, 0.734, and 0.791, the stir-fried ginkgo kernel formula granules have obvious fluorescent spots, while the ginkgo kernel formula granules have no fluorescent spots. Therefore, whether there are 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.

[0099] Example 4

[0100] This embodiment provides a thin layer chromatography method for identifying ginkgo formula granules and stir-fried ginkgo kernel formula granules, and investigates durability (at different temperatures), comprising the following steps:

[0101] 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;

[0102] S2: Take 20 μL of the test solution of the ginkgo formula granules and the test solution of the fried ginkgo kernel formula granules, and 1 μL of the reference solution, and test them according to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate, all using 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).

[0103] Results Figures 6-7 , Figure 6 This is the thin layer chromatogram obtained by developing at a low temperature of 8°C. Figure 7 This is a thin layer chromatogram obtained by developing at room temperature 25°C. Figures 6-7 It can be seen that this method has good durability at different temperatures. Compared with the spectrum of the reference product, the spectrum of the test product developed at low temperature 8°C and room temperature 25°C has obvious fluorescent spots at the ratio shift value (Rf1) of 0.6 and 0.6, and the ratio shift value (Rf2) of 0.7 and 0.7, while the white nut formula granules have no fluorescent spots. Therefore, whether there are fluorescent spots at the ratio shift value (Rf1) of 0.6 and 0.6, and the ratio shift value (Rf2) of 0.7 and 0.7 can be used as the identification point.

[0104] Example 5

[0105] This embodiment provides a thin layer chromatography method for identifying ginkgo formula granules and stir-fried ginkgo kernel formula granules, and investigates durability (at different humidity levels), comprising the following steps:

[0106] 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;

[0107] S2: Take 20 μL of the test solution of the ginkgo formula granules and the test solution of the fried ginkgo kernel formula granules, and 1 μL of the reference solution, and test them according to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate, all using chloroform-cyclohexane-acetone-glacial acetic acid (volume ratio of 10:5:5:2) as the developing agent, and develop them in a humidity environment of 32RH% and 75RH% (temperature of 25°C), 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).

[0108] Results Figures 8-9 , Figure 8 This is a thin layer chromatogram obtained under a humidity environment of 32 RH%. Fig. 9 The thin layer chromatogram is obtained under a humidity environment of 75 RH%. Figures 8-9 It can be seen that the method has good durability under different humidity. Compared with the spectrum of the reference product, the spectrum of the test product developed under the humidity environment of 32RH% and 75RH% has obvious fluorescent spots at the ratio shift value (Rf1) of 0.6 and 0.6, and the ratio shift value (Rf2) of 0.7 and 0.74 for the stir-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 value (Rf1) of 0.6 and 0.6, and the ratio shift value (Rf2) of 0.7 and 0.74 can be used as the identification point.

[0109] Example 6

[0110] This example verifies the above-mentioned thin layer identification chromatography method, comprising the following steps:

[0111] S1: Take a reference substance, 3 batches of ginkgo formula granules and 3 batches of stir-fried ginkgo formula granules respectively, and provide corresponding reference substance solutions, test sample solutions of different batches of ginkgo formula granules and test sample solutions of different batches of stir-fried ginkgo kernel formula granules according to the methods of Preparation Examples 1-2;

[0112] S2: Take 20 μL of the test solution of different batches of ginkgo formula granules and 20 μL of the test solution of different batches 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 Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate, all with 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 145°C for 30 minutes, and inspect under ultraviolet light (365nm).

[0113] Results Fig.10 It can be seen that compared with the spectrum of the reference substance, the spectrum of the test solution of different batches has obvious fluorescent spots at the ratio shift value (Rf1) of 0.673 and the ratio shift value (Rf2) of 0.74 for the stir-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 value (Rf1) of 0.673 and the ratio shift value (Rf2) of 0.74 can be used as the identification point.

[0114] Example 7

[0115] The present 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 a developing solvent, and the other parameters and steps are consistent with Example 1.

[0116] Results Fig.11 It can be seen that compared with the spectrum of the reference substance, the stir-fried ginkgo kernel formula granules have obvious fluorescent spots at the ratio shift value (Rf1) of 0.65 and the ratio shift value (Rf2) of 0.74, while the ginkgo kernel formula granules have no fluorescent spots. Therefore, whether there are fluorescent spots at the ratio shift value (Rf1) of 0.65 and the ratio shift value (Rf2) of 0.74 can be used as the identification point.

[0117] Example 8

[0118] This embodiment provides a thin layer chromatography method for identification of 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 other parameters and steps are consistent with Example 1. Results are shown in Fig.12 It can be seen that compared with the spectrum of the reference substance, the stir-fried ginkgo kernel formula granules have obvious fluorescent spots at the ratio shift value (Rf1) of 0.65 and the ratio shift value (Rf2) of 0.77, while the ginkgo kernel formula granules have no fluorescent spots. Therefore, whether there are fluorescent spots at the ratio shift value (Rf1) of 0.65 and the ratio shift value (Rf2) of 0.77 can be used as the identification point.

[0119] Determination of the identification point Rf of ginkgo formula granules and stir-fried ginkgo formula granules

[0120] The data of various ratio shift values ​​in the thin layer chromatography methodology investigation are summarized, and the results are shown in Table 1. According to the summary results, it is determined that the ratio shift value (Rf1) of the identification spot 1 of the stir-fried ginkgo kernel formula granules should be within the range of 10%, and the specified value is 0.648; the ratio shift value (Rf2) of the identification spot 2 of the stir-fried ginkgo kernel formula granules should be within the range of 10%, and the specified value is 0.741.

[0121] Table 1

[0122]

[0123] Comparative Example 1

[0124] S1: Take 1g of ginkgo kernel formula granules and stir-fried ginkgo kernel formula granules, grind them into powder, add 30mL of water to dissolve, shake and extract with ethyl acetate twice, 20mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as the test solution. Separately, take ginkgolide B reference substance and ginkgolide C reference substance, add methanol to make a mixed solution containing 1mg of each in 1mL, and use it as the reference substance solution;

[0125] S2: According to the thin layer chromatography method (General Rules 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 4% sodium acetate sodium carboxymethyl cellulose solution as an adhesive, and developed with toluene-ethyl acetate-acetone-methanol (10:5:5:0.6) at 25°C and 75RH%. After development, the plate was taken out, dried, sprayed with acetic anhydride, heated at 140°C for 30 minutes, and inspected under ultraviolet light (365nm).

[0126] Results Fig.13 Using the above-mentioned thin layer identification method, there was no obvious difference in the spots of ginkgo kernel and fried ginkgo kernel formula granules, so it was impossible to effectively distinguish the ginkgo kernel formula granules from the fried ginkgo kernel formula granules.

[0127] The above results show that the thin layer chromatogram obtained by using a thin layer method other than that provided by the present invention cannot identify the ginkgo kernel formula granules and the fried ginkgo kernel formula granules.

[0128] Comparative Example 2

[0129] 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;

[0130] S2: According to the thin layer chromatography method (General Rules 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 75RH%. The plate was taken out, dried, sprayed with acetic anhydride solution, heated at 140-160°C for 30 minutes, and inspected under ultraviolet light (365nm).

[0131] Results Fig.14 It can be seen that the separation degree of ginkgo kernel 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.

[0132] 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.

[0133] Comparative Example 3

[0134] 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;

[0135] S2: Take 20 μL of the test solution of the ginkgo formula granules and the test solution of the fried ginkgo kernel formula granules, and take 1 μL of the reference solution, and test them according to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate, all with 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).

[0136] Results Fig.15 It can be seen that the separation degree of ginkgo kernel 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.

[0137] 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.

[0138] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those 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 will not be limited to the embodiments shown herein, but rather to the widest scope 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); The test solution was prepared according to the following method: 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; 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.

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 15~25 μL.

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

5. 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.

6. The method according to claim 1, characterized in that The ethanol is 50 vol% ethanol.

7. 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.

8. The method according to claim 7, characterized in that The reference substance in the reference substance solution includes ginkgolide B and ginkgolide C; The sample volume of the reference solution is 1~5 μL; The spotting volume of the negative solution is 1~3 μL.

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