A thin layer chromatography method for identifying granules of southern helicida and stir-fried southern helicida

Abstract

This invention provides a thin-layer chromatography (TLC) method for identifying *Symplocos edulis* and its stir-fried formulation granules, comprising the following steps: spotting the test solution onto a silica gel TLC plate, developing the solution according to TLC conditions, and examining the sample. This invention uses toluene-diethyl ether-dichloromethane (v / v) at a ratio of (8–12):(2–5):1 as the developing solvent. Results show that within the Rf range of 0.4635–0.5665, *Symplocos edulis* formulation granules exhibit green fluorescent spots, while stir-fried *Symplocos edulis* formulation granules do not. Therefore, the presence or absence of green fluorescent spots within the aforementioned Rf range can be used as the identification point for *Symplocos edulis* and stir-fried *Symplocos edulis* formulation granules. Furthermore, this TLC method is highly specific, stable, and precise, and is convenient and easy to master, providing a more standardized quality control for the preparation of *Symplocos edulis*-related formulations and possessing broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal material detection, and in particular to a thin-layer identification chromatography method for formula granules of southern helicids and fried southern helicids. Background Art

[0002] Daucus carota L. is the dried mature fruit of the wild carrot plant Daucus carota L. of the Apiaceae family. Studies have shown that Daucus carota L. has multiple biological activities such as insecticide, antibacterial, anticancer, anti-fertility, improvement of cognitive dysfunction, and repair of liver damage.

[0003] At present, the research on the southern crane louse mainly focuses on the chemical composition and pharmacological activity. For example, Liu Na studied the extraction, purification and identification of the sesquiterpenoid components of the southern crane louse and their antioxidant activity, Fu Hongwei and others studied the guaiacane-type sesquiterpenoids and other chemical components in the fruits of the southern crane louse, and Liu Yi and others studied the lectin of the southern crane louse. In terms of the identification of the southern crane louse, Zhang Jinzhong and others identified the characteristics of the southern crane louse and the northeastern crane louse.

[0004] However, the raw product of the southern crane lice is slightly toxic and needs to be processed for clinical use. Studies have shown that stir-frying the southern crane lice (hereinafter referred to as "stir-fried southern crane lice") can greatly reduce its toxicity and increase the accuracy and safety of medication. It can be seen that the composition of the southern crane lice and stir-fried southern crane lice has changed. However, there is no report on the identification of southern crane lice and stir-fried southern crane lice. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a thin layer chromatography method for distinguishing the formula granules of southern helicids and fried southern helicids. The method observes whether there are fluorescent spots in a certain ratio shift value range in the thin layer chromatography spectrum as the basis for identification, and effectively and quickly distinguishes the formula granules of southern helicids and fried southern helicids.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] The invention provides a thin layer identification chromatography method for southern crane slugs and fried southern crane slugs formula granules, comprising the following steps:

[0008] Spot the test solution on a silica gel thin layer plate, develop it according to the thin layer chromatography conditions, and inspect it;

[0009] The developing solvent in the thin layer chromatography conditions is toluene-ethyl ether-dichloromethane, and the volume ratio of toluene, ethyl ether and dichloromethane is (8-12):(2-5):1.

[0010] Preferably, the volume ratio of toluene, ether and dichloromethane is (10-12):3:1.

[0011] Preferably, the spotting volume of the test solution is 1 to 9 μL.

[0012] Preferably, the unfolding environment temperature is 4-25° C., and the environment humidity is 32-61 RH%.

[0013] Preferably, the granules of the southern helicoids formula have no green fluorescent spots within the range of the ratio transfer value of 0.4635 to 0.5665, and the granules of the stir-fried southern helicoids formula have green fluorescent spots within the range of the ratio transfer value of 0.4635 to 0.5665.

[0014] Preferably, the test solution is prepared according to the following method:

[0015] The test sample was mixed with water and hydrochloric acid, refluxed, filtered, extracted with ether, evaporated to dryness, and ether was added to the residue to obtain the test sample solution.

[0016] Preferably, the ratio of the test sample, water, hydrochloric acid and diethyl ether is 0.5 g: (15-25) mL: (1-3) mL: (0.5-2) mL.

[0017] Preferably, the inspection is performed under ultraviolet light.

[0018] Preferably, the wavelength of the ultraviolet lamp is 365nm.

[0019] Preferably, the method further comprises the step of preparing a control medicinal material solution of the southern crane lice and a negative solution.

[0020] Preferably, the spotting volume of the control medicinal material solution of the southern crane lice is 1 to 9 μL.

[0021] Preferably, the spotting volume of the negative solution is 8 to 15 μL.

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

[0023] The present invention provides a thin layer identification chromatography method for formula granules of southern crane slugs and stir-fried southern crane slugs. The method spots a sample solution on a silica gel thin layer plate and develops it according to thin layer chromatography conditions. The developing solvent in the thin layer chromatography conditions is toluene-ether-dichloromethane. The volume ratio of the toluene, ether and dichloromethane is (8-12):(2-5):1. After the development is completed, the thin layer chromatogram is inspected and it is found that the southern crane slug formula granules have green fluorescent spots at the ratio shift value (Rf) range of 0.4635-0.5665, while the stir-fried southern crane slug formula granules do not have green fluorescent spots. Therefore, the presence or absence of green fluorescent spots within the above Rf range can be used as a distinguishing point for the southern crane slugs and stir-fried southern crane slug formula granules. After research, the above thin layer identification chromatography method provided by the present invention has strong specificity, good stability, high precision, and is convenient to operate and easy to master. It provides more formal quality control for the preparation of southern crane slug related preparations to ensure the efficacy of the drug, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The thin layer chromatograms of the control medicinal material solution of the southern helicid, the formula granule solution of the southern helicid and the formula granule solution of the stir-fried southern helicid under different sample amounts;

[0025] in, Figure 1 1 to 5 correspond to the control medicinal material solution of the southern helicid, 6 to 10 correspond to the formula granule solution of the southern helicid, and 11 to 15 correspond to the formula granule solution of the stir-fried southern helicid;

[0026] Figure 2 The thin layer chromatograms of the negative solution, the control medicinal material solution of the southern helicid, the southern helicid formula granule solution and the stir-fried southern helicid formula granule solution;

[0027] in, Figure 2 1 corresponds to the negative solution, 2 corresponds to the Nanheli control medicinal material solution, 3 corresponds to the Nanheli formula granule solution, and 4 corresponds to the stir-fried Nanheli formula granule solution;

[0028] Figure 3 The thin layer chromatograms of the Nanheshi control medicinal material solution, the Nanheshi formula granule solution and the stir-fried Nanheshi formula granule solution using the cuttable thin layer chromatography plate of Tianjin Silida Technology Co., Ltd.

[0029] in, Figure 3 1 corresponds to the Nanheli control medicinal material solution, 2 corresponds to the Nanheli formula granule solution, and 3 corresponds to the stir-fried Nanheli formula granule solution;

[0030] Figure 4 The thin layer chromatograms of the reference medicinal material solution of the southern helicid, the southern helicid formula granule solution and the stir-fried southern helicid formula granule solution on the thin layer chromatography plate produced by Merck;

[0031] in, Figure 4 1 corresponds to the control medicinal material solution of the southern helicid, 2 corresponds to the formula granule solution of the southern helicid, and 3 corresponds to the formula granule solution of the stir-fried southern helicid;

[0032] Figure 5 The thin layer chromatograms of the reference medicinal material solution of the southern helicid, the formula granule solution of the southern helicid and the formula granule solution of the stir-fried southern helicid were prepared on the silica gel G plate produced by Qingdao Ocean Chemical Plant Branch;

[0033] in, Figure 5 1 corresponds to the control medicinal material solution of the southern helicid, 2 corresponds to the formula granule solution of the southern helicid, and 3 corresponds to the formula granule solution of the stir-fried southern helicid;

[0034] Figure 6 This is the thin layer chromatogram obtained by developing at room temperature 25°C;

[0035] in, Figure 6 1 corresponds to the control medicinal material solution of the southern helicid, 2 corresponds to the formula granule solution of the southern helicid, and 3 corresponds to the formula granule solution of the stir-fried southern helicid;

[0036] Figure 7 This is a thin layer chromatogram obtained by developing at a low temperature of 4°C;.

[0037] in, Figure 7 1 corresponds to the Nanheli control medicinal material solution, 2 corresponds to the Nanheli formula granule solution, and 3 corresponds to the stir-fried Nanheli formula granule solution;

[0038] Figure 8 This is a thin layer chromatogram obtained under an ambient humidity of 61 RH%;

[0039] in, Figure 8 1 corresponds to the control medicinal material solution of the southern helicid, 2 corresponds to the formula granule solution of the southern helicid, and 3 corresponds to the formula granule solution of the stir-fried southern helicid;

[0040] Fig. 9 This is a thin layer chromatogram obtained by developing it at an ambient humidity of 32 RH%;

[0041] in, Fig. 9 1 corresponds to the Nanheli control medicinal material solution, 2 corresponds to the Nanheli formula granule solution, and 3 corresponds to the stir-fried Nanheli formula granule solution;

[0042] Fig.10 Thin layer chromatograms for verifying multiple batches of Nanheshi control medicinal material solution, Nanheshi formula granule solution, and stir-fried Nanheshi formula granule solution;

[0043] in, Fig.101 to 3 correspond to the Nanheli control medicinal material solution, 4 to 6 correspond to the Nanheli formula granule solution, and 7 to 9 correspond to the stir-fried Nanheli formula granule solution;

[0044] Fig.11 The chromatogram obtained by using the thin layer conditions of Comparative Example 1;

[0045] in, Fig.11 1 corresponds to the Nanheli control medicinal material solution, 2 corresponds to the Nanheli formula granule solution, and 3 corresponds to the stir-fried Nanheli formula granule solution;

[0046] Fig.12 The chromatogram is obtained by using the TLC conditions of Comparative Example 1 and then spraying the TLC plate with 5% vanillin sulfuric acid solution;

[0047] in, Fig.12 1 corresponds to the Nanheli control medicinal material solution, 2 corresponds to the Nanheli formula granule solution, and 3 corresponds to the stir-fried Nanheli formula granule solution;

[0048] Fig.13 The chromatogram obtained by using the thin layer conditions of Comparative Example 2;

[0049] in, Fig.13 1 corresponds to the Nanheli control medicinal material solution, 2 corresponds to the Nanheli formula granule solution, and 3 corresponds to the stir-fried Nanheli formula granule solution;

[0050] Fig.14 The chromatogram obtained by using the thin layer conditions of Comparative Example 3;

[0051] in, Fig.14 1 corresponds to the Nanheli control medicinal material solution, 2 corresponds to the Nanheli formula granule solution, and 3 corresponds to the stir-fried Nanheli formula granule solution;

[0052] Fig.15 The chromatogram obtained by using the thin layer conditions of Comparative Example 4;

[0053] in, Fig.15 1 corresponds to the control medicinal material solution of the southern crane lice, 2 corresponds to the formula granule solution of the southern crane lice, and 3 corresponds to the formula granule solution of the stir-fried southern crane lice. DETAILED DESCRIPTION

[0054] 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.

[0055] In view of the problem that it is difficult to identify the formula granules of southern helicid and the formula granules of fried southern helicid in the prior art, the present invention provides a thin layer chromatography method for identifying the formula granules of southern helicid and fried southern helicid, comprising the following steps:

[0056] Spot the test solution on a silica gel thin layer plate, develop it according to the thin layer chromatography conditions, and inspect it.

[0057] In the present invention, the test solution refers to a solution of granules of the southern crane slug or a solution of granules of the fried southern crane slug. The test solution can preferably be prepared according to the following method:

[0058] The test sample (i.e., the southern helicid formula granules or the stir-fried southern helicid formula granules) is mixed with water and hydrochloric acid, refluxed, filtered, and extracted with ether. The ether solution is evaporated to dryness, and ether is added to the residue to obtain the test sample solution.

[0059] In some embodiments of the present invention, the dosage ratio of the test sample, water, hydrochloric acid and ether is 0.5 g: (15-25) mL: (1-3) mL: (0.5-2) mL. The (15-25) mL may be 15 mL, 17 mL, 20 mL, 22 mL or 25 mL, etc.; the (1-3) mL may be 1 mL, 1.5 mL, 2 mL, 2.5 mL or 3 mL, etc.; the (0.5-2) mL may be 0.5 mL, 1 mL, 1.5 mL or 2 mL, etc.

[0060] In a specific embodiment of the present invention, preferably take 0.5g of this product (Southern Crane or Stir-fried Southern Crane Formula Granules), grind it, add 20mL of water, add 2mL of hydrochloric acid (as an extraction reagent), reflux extract for 20min, cool, filter, add ether and shake extract 3 times, 20mL each time, combine the ether solution, evaporate to dryness, add 1mL of ether to dissolve the residue, and use it as the test solution. Wherein, the mass concentration of the hydrochloric acid is generally 35.5-37.5%.

[0061] According to the present invention, after obtaining the test solution, the test solution is spotted on a silica gel thin layer plate, developed according to the thin layer chromatography conditions, and inspected. 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 effects, so there is no special restriction on the source of the silica gel thin layer plate. At the same time, after the spotting amount is investigated, it is found that when the spotting amount of the test solution is 1 to 9 μL (can be 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL or 9 μL), the spots in the obtained thin layer chromatography are clearly colored and have good separation, so the spotting amount of the test solution is determined to be 1 to 9 μL.

[0062] It should be noted that not all developing agents can make the formula particles of southern crane lice or fried southern crane lice have a developing effect. After testing, the present invention preferably uses toluene-ethyl ether-dichloromethane as the developing agent in the thin layer chromatography conditions. In order to ensure good separation between the two and clear spot color development, the present invention preferably uses the volume ratio of toluene, ethyl ether and dichloromethane as (8-12):(2-5):1, more preferably (10-12):3:1, and most preferably 10:3:1.

[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 4 to 25°C, specifically 4°C, 8°C, 10°C, 15°C, 20°C or 25°C, etc.; the environment humidity can be in the range of 32 to 61RH%, specifically 32RH%, 35RH%, 40RH%, 45RH%, 50RH%, 55RH%, 60RH% or 61RH%, etc.

[0064] In some embodiments of the present invention, after the above development is completed, the silica gel thin layer plate is preferably taken out and dried before inspection. In the present invention, the inspection is preferably carried out under ultraviolet light, and the wavelength of the ultraviolet light is preferably 365nm.

[0065] After the inspection, the thin layer chromatogram of the test sample of the formula granules of the southern helicid or stir-fried southern helicid was observed, and it was known that the formula granules of the southern helicid had no green fluorescent spots in the range of the ratio transfer value (Rf) of 0.4635 to 0.5665, and the stir-fried southern helicid formula granules had green fluorescent spots in the range of the ratio transfer value (Rf) of 0.4635 to 0.5665. Therefore, the presence or absence of green fluorescent spots in the above Rf range can be used as the identification point of the formula granules of the southern helicid and stir-fried southern helicid.

[0066] In some embodiments of the present invention, the method further comprises the steps of preparing a control medicinal solution of the southern crane louse and a negative solution for thin layer chromatography development and inspection. Among them, the control medicinal solution of the southern crane louse is often used in thin layer chromatography to prove whether the source of the sample is accurate, and the spotting amount is generally 1 to 9 μL (which can be 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL or 9 μL), and the negative solution is used to prove that the solvent used in the established method has no effect on the final presented results, and the spotting amount is 8 to 15 μL, preferably 10 μL.

[0067] Wherein, the southern crane lice control medicinal material solution can be prepared according to the following method:

[0068] The reference medicinal material of the southern crane slug is boiled in water, filtered, and hydrochloric acid is added to the filtrate. Then, the reference medicinal material solution of the southern crane slug is prepared in the same manner as in the above "Preparation of the test solution".

[0069] In some specific embodiments of the present invention, the southern crane lice control medicinal material solution is preferably prepared according to the following method:

[0070] Take 0.5 g of the southern crane slug control medicinal material, add 15-25 mL of water, preferably 20 mL, decoct for 15-40 min, preferably 30 min, filter, add 0.5-2 mL of hydrochloric acid, preferably 1 mL, to the filtrate, and then prepare the southern crane slug control medicinal material solution in the same manner as described in the "Preparation of the test solution".

[0071] The negative solution can be prepared according to the following method:

[0072] Take 0.5 g of purified water, add 20 mL of water, then add 2 mL of hydrochloric acid, reflux and extract for 20 min, cool, filter, add ether to the filtrate and shake to extract 3 times, 20 mL each time, combine the ether solutions, evaporate to dryness, add 1 mL of ether to the residue to dissolve it, and use it as the negative solution.

[0073] In the present invention, the spotting amount of the control medicinal material solution of the Southern Ligusticum aviculare is 1 to 9 μL; its function is to prove that the test sample is the formula granules prepared from the Southern Ligusticum aviculare medicinal material, or the formula granules prepared after the Southern Ligusticum aviculare medicinal material is processed; the spotting amount of the negative solution is 8 to 15 μL, which functions to prove that the established method has strong specificity.

[0074] The thin-layer identification chromatography method provided by the present invention has strong specificity, good stability, high precision, and is convenient to operate and easy to master. It provides more formal quality control for the preparation of preparations related to southern crane slugs to ensure the efficacy of the drugs, and has a very broad application prospect.

[0075] In order to further illustrate the present invention, the following examples are provided for detailed description. The instruments used in the following embodiments of the present invention include a heating plate, a mortar, and a thin layer imaging system: CAMAG TLC Visualizer, silica gel GF254 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 - cyclohexane, ethyl acetate, ether, dichloromethane, toluene, and anhydrous ethanol are all analytically pure, and water is ultrapure water (self-made in the laboratory); the test drugs used include southern helicid control medicinal materials (China Food and Drug Inspection Institute, batch number: 121339-201803), southern helicid formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2208001; 2208002; 2208003) and fried southern helicid formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: 2211001; 2211002; 2211003).

[0076] Preparation Example 1

[0077] This preparation example provides a method for preparing a test solution of a southern crane slug and a stir-fried southern crane slug formula granule, a method for preparing a corresponding control medicinal material, and a method for preparing a negative solution, comprising the following steps:

[0078] S1: Preparation of test solution

[0079] Take 0.5 g of this product (Southern Crane Lichen or Fried Southern Crane Lichen Formula Granules), grind it into powder, add 20 mL of water, then add 2 mL of hydrochloric acid, reflux and extract for 20 min, cool, filter, add ether and shake to extract 3 times, 20 mL each time, combine the ether solutions, evaporate to dryness, add 1 mL of ether to the residue to dissolve it, and use it as the test solution.

[0080] S2: Preparation of control drug solution

[0081] Take 0.5 g of the control medicinal material of South Helix, add 20 mL of water and boil for 30 min, filter, add 2 mL of hydrochloric acid to the filtrate, and prepare the control medicinal material solution in the same way as "Preparation of Test Solution".

[0082] S3: Preparation of negative solution

[0083] Take 0.5 g of purified water, add 20 mL of water, then add 2 mL of hydrochloric acid, reflux and extract for 20 min, cool, filter, add ether to the filtrate and shake to extract 3 times, 20 mL each time, combine the ether solutions, evaporate to dryness, add 1 mL of ether to the residue to dissolve it, and use it as the negative solution.

[0084] Example 1

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

[0086] S1: According to the method of Preparation Example 1, a solution of a control medicinal material of a scutellaria aviculare, a solution of a scutellaria aviculare formula granule, and a solution of a stir-fried scutellaria aviculare formula granule are provided;

[0087] S2: Each group independently took 1μL, 3μL, 5μL, 7μL, and 9μL of the southern crane sedge control medicinal material solution, the southern crane sedge formula granule solution, and the stir-fried southern crane sedge formula granule solution, and tested them according to the thin layer chromatography method (Appendix VIB of Part I of the Chinese Pharmacopoeia 2020 Edition), and spotted them on the same silica gel G thin layer plate, using toluene-ether-dichloromethane (volume ratio of 10:3:1) as the developing agent, developing, taking out, drying, and inspecting under ultraviolet light (365nm).

[0088] Results Figure 1 ,in, Figure 1 1 to 5 are the reference medicinal material solutions of South Helix sutchuenensis, 6 to 10 are the formula granule solutions of South Helix sutchuenensis, and 11 to 15 are the formula granule solutions of stir-fried South Helix sutchuenensis. Figure 1It can be seen that when the spot amount of the control medicinal material solution of the southern helicid, the southern helicid formula granule solution and the stir-fried southern helicid formula granule solution is 1 to 9 μL, the spots are clearly colored and the separation is good, and the spot amount is determined to be 1 to 9 μL. In addition, at the ratio shift value of 0.561, the southern helicid formula granule solution shows green fluorescent spots, while the stir-fried southern helicid formula granule solution has no spots, which can distinguish the southern helicid formula granules from the stir-fried southern helicid formula granules. Therefore, whether there are green fluorescent spots at the ratio shift value of 0.501 can be used as a discrimination point.

[0089] Example 2

[0090] This embodiment provides a thin layer chromatography method for identifying the formula granules of southern crane lice and stir-fried southern crane lice, and investigates the specificity, comprising the following steps:

[0091] S1: According to the method of Preparation Example 1, provide a control medicinal material solution of the southern sedge sedge, a solution of the southern sedge sedge formula granules, a solution of the stir-fried southern sedge sedge formula granules, and a negative solution;

[0092] S2: Take 5 μL of the solution of the formulated granules of southern crane scissors and the solution of the formulated granules of stir-fried southern crane scissors, 3 μL of the southern crane scissors control medicinal material solution, and 10 μL of the negative solution, and test them according to the thin layer chromatography method (Appendix VIB of Part I of the 2020 edition of the Chinese Pharmacopoeia), and spot them on the same silica gel G thin layer plate, using toluene-ether-dichloromethane (volume ratio of 10:3:1) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365 nm).

[0093] Results Figure 2 ,in, Figure 2 1 is the negative solution, 2 is the control medicinal material solution of the southern helicid, 3 is the formula granule solution of the southern helicid, and 4 is the formula granule solution of the stir-fried southern helicid. Figure 2 It can be seen that the negative sample has no interference with the test samples of the formula granules of the southern crane lice and the stir-fried southern crane lice, and the method has good specificity. In addition, the southern crane lice formula granules show green fluorescent spots at the ratio shift value of 0.523, while the stir-fried southern crane lice formula granules have no spots, which can distinguish the southern crane lice formula granules from the stir-fried southern crane lice formula granules. Therefore, whether there are green fluorescent spots at the ratio shift value of 0.523 can be used as a discrimination point.

[0094] Example 3

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

[0096] S1: According to the method of Preparation Example 1, a solution of a control medicinal material of a scutellaria aviculare, a solution of a scutellaria aviculare formula granule, and a solution of a stir-fried scutellaria aviculare formula granule are provided;

[0097] S2: Take 5 μL of the solution of the formulated granules of the southern hesperid and the solution of the formulated granules of the stir-fried southern hesperid (take 3 portions each), and 3 μL of the southern hesperid control medicinal material solution (take 3 portions), and test them according to the thin layer chromatography method (Appendix VIB of Part I of the 2020 edition of the Chinese Pharmacopoeia). Spot them on the cuttable thin layer chromatography plate of Tianjin Silida Technology Co., Ltd., the thin layer chromatography plate produced by Merck, and the prefabricated silica gel G plate of Qingdao Ocean Chemical Plant Branch, respectively, and use toluene-ether-dichloromethane (volume ratio of 10:3:1) as the developing agent. Develop, take out, dry, and inspect under ultraviolet light (365nm).

[0098] Results Figures 3 to 5 ,in, Figure 3 This is a thin layer chromatogram obtained using the cuttable thin layer chromatography plate of Tianjin Slida Technology Co., Ltd. Figure 4 Thin layer chromatogram obtained using Merck thin layer chromatography plates, Figure 5 This is a thin layer chromatogram obtained using the prefabricated silica gel G plate produced by Qingdao Ocean Chemical Plant Branch. Figures 3 to 5 1 in each of them is the reference medicinal solution of the southern crane lice. Figures 3 to 5 The 2 in the above are both granular solutions of Nanheli formula. Figures 3 to 5 The 3 in the above are all stir-fried southern crane lice formula granule solutions. Figures 3 to 5 It can be seen that the green fluorescence spot ratio shift value (R f ) are 0.513, 0.523, and 0.504, respectively, which can meet the identification requirements. In addition, at the ratio shift values ​​of 0.513, 0.523, and 0.504, the granules of the southern helicid formula show green fluorescent spots, while the granules of the stir-fried southern helicid formula have no spots, which can distinguish the granules of the southern helicid formula from the stir-fried southern helicid formula granules. Therefore, whether the ratio shift values ​​of 0.513, 0.523, and 0.5043 have green fluorescent spots can be used as the identification point.

[0099] Example 4

[0100] This embodiment provides a thin layer identification chromatography method for the formula granules of southern crane lice and fried southern crane lice, and investigates the durability (at different temperatures), comprising the following steps:

[0101] S1: According to the method of Preparation Example 1, a solution of a control medicinal material of a scutellaria aviculare, a solution of a scutellaria aviculare formula granule, and a solution of a stir-fried scutellaria aviculare formula granule are provided;

[0102] S2: Take 5 μL of the solution of the formulated granules of southern crane slug and the solution of the formulated granules of the stir-fried southern crane slug (take two portions each), and 3 μL of the southern crane slug control medicinal material solution (take two portions), and test them according to the thin layer chromatography method (Appendix VIB of Part I of the 2020 edition of the Chinese Pharmacopoeia). Spot them on two silica gel G thin layer plates respectively, and place the two silica gel G thin layer plates at low temperature of 4°C and room temperature of 25°C, respectively, with toluene-ether-dichloromethane (volume ratio of 10:3:1) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm).

[0103] The results are as follows Figures 6-7 As shown, Figure 6 This is the thin layer chromatogram obtained at room temperature 25°C. Figure 7 The figure is a thin layer chromatogram obtained by developing at a low temperature of 4°C. Figures 6-7 1 in each of them is the reference medicinal solution of the southern crane lice. Figures 6-7 The 2 in the above are both granular solutions of Nanheli formula. Figures 6-7 The 3 in the above are all stir-fried southern crane lice formula granule solutions. Figures 6-7 It can be seen that this method has good adaptability to different temperatures, and the granules of the southern helicid formula show green fluorescent spots at the ratio shift values ​​of 0.541 and 0.513, while the granules of the stir-fried southern helicid formula have no spots, which can distinguish the granules of the southern helicid formula from the stir-fried southern helicid formula. Therefore, whether there are green fluorescent spots at the ratio shift values ​​of 0.541 and 0.513 can be used as the identification point.

[0104] Example 5

[0105] This embodiment provides a thin layer chromatography method for identifying the granules of the southern crane lice and the stir-fried southern crane lice, and investigates the durability (at different humidity), comprising the following steps:

[0106] S1: According to the method of Preparation Example 1, a solution of a control medicinal material of a scutellaria aviculare, a solution of a scutellaria aviculare formula granule, and a solution of a stir-fried scutellaria aviculare formula granule are provided;

[0107] S2: Take 5 μL of the solution of the formulated granules of southern crane slug and the solution of the formulated granules of the stir-fried southern crane slug (take two portions each), and 3 μL of the southern crane slug control medicinal material solution (take two portions), and test them according to the thin layer chromatography method (Appendix VIB of Part I of the 2020 edition of the Chinese Pharmacopoeia). Spot them on two silica gel G thin layer plates respectively, and place the two silica gel G thin layer plates at an ambient humidity of 32RH% and 61RH%, respectively, with toluene-ether-dichloromethane (volume ratio of 10:3:1) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm).

[0108] The results are as follows Figures 8-9 As shown, Figure 8 This is the thin layer chromatogram obtained under an ambient humidity of 61RH%. Fig. 9The thin layer chromatogram was obtained by developing the sample at an ambient humidity of 32 RH%. Figures 8-9 1 in each of them is the reference medicinal solution of the southern crane lice. Figures 8-9 The 2 in the above are both granular solutions of Nanheli formula. Figures 8-9 The 3 in the above are all stir-fried southern crane lice formula granule solutions. Figures 8-9 It can be seen that this method has good adaptability to different humidity levels, and the granules of the southern helicid formula show green fluorescent spots at the ratio shift values ​​of 0.584 and 0.513, while the granules of the stir-fried southern helicid formula have no spots, which can distinguish the granules of the southern helicid formula from the stir-fried southern helicid formula. Therefore, whether there are green fluorescent spots at the ratio shift values ​​of 0.584 and 0.513 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: 3 batches of southern helicid, 3 batches of stir-fried southern helicid formula granules and 3 batches of southern helicid control medicinal materials were taken respectively, and 9 corresponding solutions were prepared according to the method of Preparation Example 1;

[0112] S2: The 9 corresponding solutions prepared were spotted on the same silica gel G thin layer plate according to the thin layer chromatography method (Appendix VIB of Part I of the 2020 edition of the Chinese Pharmacopoeia) according to the spotting volume of 5 μL of the formula granule solution of the southern crane louse and the formula granule solution of the stir-fried southern crane louse, 3 μL of the southern crane louse control medicinal material solution, and 10 μL of the negative solution. Toluene-ether-dichloromethane (volume ratio of 10:3:1) was used as the developing solvent. The plate was developed, taken out, dried, and inspected under ultraviolet light (365 nm).

[0113] The results are as follows Fig.10 As shown, Fig.10 1 to 3 are the reference medicinal material solutions of South Helix sutchuenensis, 4 to 6 are the formula granule solutions of South Helix sutchuenensis, and 7 to 9 are the formula granule solutions of stir-fried South Helix sutchuenensis. Fig.10 It can be seen that the granules of the formula of southern helicid have green fluorescent spots at the ratio transfer value (Rf) of 0.499, while the granules of the formula of fried southern helicid do not have such spots. The fluorescent spots can be used to distinguish the two.

[0114] Determination of the identification point Rf of Nanheshi formula granules and stir-fried Nanheshi formula granules

[0115] The data of various ratio shift values ​​in the thin layer identification chromatography methodology investigation are summarized, and the results are shown in the following Table 1. According to the summary results, it is determined that the range of the ratio shift value (Rf) of the identification spot of the Nanheshi formula granule should be within 10%, and the specified value is 0.515.

[0116] Table 1

[0117]

[0118] Comparative Example 1

[0119] Take 1g of the powder of this product (Southern Heshi Formula Granules, Stir-fried Southern Heshi Formula Granules), add 20mL of ether, soak overnight, filter, evaporate the filtrate, add 1mL of ether to the residue to dissolve it, and use it as the test solution. Take another 1g of Southern Heshi reference medicinal material, and prepare the reference medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 1~2μL of each of the above two solutions, and spot them on the same silica gel G thin layer plate, use toluene-ethyl acetate-formic acid (8:1:1) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm).

[0120] The results are as follows Fig.11 As shown, Fig.11 1 is the reference medicinal material solution of South Helix sutchuenensis, 2-6 are the formula granule solutions of South Helix sutchuenensis, and 7-11 are the formula granule solutions of stir-fried South Helix sutchuenensis. Fig.11 It can be seen that in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0121] The thin layer plate was sprayed with 5% vanillin sulfuric acid solution and heated until the spots were clearly colored. Fig.12 As shown, Fig.12 1 is the reference medicinal material solution of South Helix sutchuenensis, 2-6 are the formula granule solutions of South Helix sutchuenensis, and 7-11 are the formula granule solutions of stir-fried South Helix sutchuenensis. Fig.12 It can be seen that in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0122] The above results show that using the above thin layer identification method, there are no obvious spots on the granules of the southern crane lice and the fried southern crane lice formula granules, and the two cannot be identified.

[0123] Comparative Example 2

[0124] Take 1g of the powder of this product (Southern Heshi Formula Granules, Stir-fried Southern Heshi Formula Granules), grind it, add 20mL of anhydrous ethanol, ultrasonically treat it for 20min, filter it, evaporate the filtrate to dryness, add 1mL of anhydrous ethanol to dissolve the residue, and use it as the test solution. Take another 1g of Southern Heshi reference medicinal material, and prepare the reference medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 8μL of the above test solution and reference medicinal material solution, respectively, and spot them on the same silica gel G thin layer plate, use cyclohexane-ether-ethyl acetate (20:5.5:2.5) as the developing solvent, develop it, take it out, dry it, spray it with 10% sulfuric acid ethanol solution, and heat it until the spots are clearly colored.

[0125] The results are as follows Fig.13 As shown, Fig.131 is the reference medicinal material solution of southern helicidae, 2 is the formula granule solution of southern helicidae, and 3 is the formula granule solution of stir-fried southern helicidae. Fig.13 It can be seen that in the test sample chromatogram, spots of the same color appear at the corresponding positions in the control medicinal material chromatogram and the control sample chromatogram.

[0126] The above results show that using the above thin layer identification method, there are no obvious spots on the granules of the southern crane lice and the fried southern crane lice formula granules, and the two cannot be identified.

[0127] Comparative Example 3

[0128] Take 0.5g of the powder of this product (Southern Heshi Formula Granules, Stir-fried Southern Heshi Formula Granules), grind it, add 20mL of water, then add 2mL of hydrochloric acid, reflux and extract for 20min, cool, filter, add ether and shake to extract 3 times, 20mL each time, combine the ether solution, evaporate to dryness, add 1mL of ether to the residue to dissolve it as the test solution. Take 0.5g of the Southern Heshi reference medicinal material, add 20mL of water and boil for 30min, filter, add 2mL of hydrochloric acid to the filtrate, and prepare the reference medicinal material solution in the same way as "Preparation of Test Solution". According to the thin layer chromatography method (Appendix VIB of Part I of the 2020 Edition of the Chinese Pharmacopoeia), take 5μL of the test solution and 3μL of the reference medicinal material solution, and spot them on the same silica gel G thin layer plate, use toluene-ether-dichloromethane (volume ratio of 5:7:1) as the developing solvent, develop, take out, dry, and inspect under ultraviolet light (365nm).

[0129] The results are as follows Fig.14 As shown, Fig.14 1 is the reference medicinal material solution of southern helicidae, 2 is the formula granule solution of southern helicidae, and 3 is the formula granule solution of stir-fried southern helicidae. Fig.14 It can be seen that in the test sample chromatogram, spots of the same color appear at the corresponding positions in the control medicinal material chromatogram and the control sample chromatogram.

[0130] 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 southern crane slug and stir-fried southern crane slug formula granules.

[0131] Comparative Example 4

[0132] Take 0.5g of this product (Southern Heshi Formula Granules, Stir-fried Southern Heshi Formula Granules), grind it, add 20mL of water, then add 2mL of hydrochloric acid, reflux and extract for 20min, cool, filter, add ether and shake to extract 3 times, 20mL each time, combine the ether solution, evaporate to dryness, add 1mL of ether to the residue to dissolve it as the test solution. Take 0.5g of Southern Heshi reference medicinal material, add 20mL of water and boil for 30min, filter, add 2mL of hydrochloric acid to the filtrate, and prepare the reference medicinal material solution in the same way as "Preparation of Test Solution". According to the thin layer chromatography method (Appendix VIB of Part I of the 2020 Edition of the Chinese Pharmacopoeia), take 5μL of the test solution and 3μL of the reference medicinal material solution, and spot them on the same silica gel G thin layer plate, use acetone-petroleum ether (60℃~90℃)-formic acid (15:8:4) as the developing agent, develop, take out, dry, and inspect under ultraviolet light (365nm).

[0133] The results are as follows Fig.15 As shown, Fig.15 1 is the reference medicinal material solution of southern helicidae, 2 is the formula granule solution of southern helicidae, and 3 is the formula granule solution of stir-fried southern helicidae. Fig.15 It can be seen that in the test sample chromatogram, spots of the same color appear at the corresponding positions in the control medicinal material chromatogram and the control sample chromatogram.

[0134] The above results show that the thin layer chromatography obtained by using a developing agent other than that provided by the present invention cannot identify the southern helicid and stir-fried southern helicid formula granules.

[0135] 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 identification chromatography method for the formula granules of southern helicid and stir-fried southern helicid, characterized in that: The following steps are involved: Spot the test solution on a silica gel thin layer plate, develop it according to the thin layer chromatography conditions, and inspect it; The developing solvent in the thin layer chromatography conditions is toluene-ether-dichloromethane, and the volume ratio of toluene, ether and dichloromethane is (8-12):(2-5):1; The test solution was prepared according to the following method: The test sample was mixed with water and hydrochloric acid, refluxed, filtered, extracted with ether, evaporated to dryness, and ether was added to the residue to obtain the test sample solution; After the inspection, there are no green fluorescent spots in the Rf range of 0.4635~0.5665, which means the particles are the formula of southern helix; there are green fluorescent spots in the Rf range of 0.4635~0.5665, which means the particles are the formula of fried southern helix.

2. The method according to claim 1, characterized in that: The volume ratio of toluene, ether and dichloromethane is (10-12):3:

1.

3. The method according to claim 1, characterized in that The sample volume of the test solution is 1~9 μL.

4. The method according to claim 1, characterized in that: The ambient temperature of the development is 4-25° C., and the ambient humidity is 32-61 RH%.

5. The method according to claim 1, characterized in that The formula granules of southern crane lice have no green fluorescent spots in the ratio transfer value range of 0.4635~0.5665, while the formula granules of stir-fried southern crane lice have green fluorescent spots in the ratio transfer value range of 0.4635~0.5665.

6. The method according to claim 1, characterized in that The ratio of the test sample, water, hydrochloric acid and ether is 0.5g:(15~25)mL:(1~3)mL:(0.5~2)mL.

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

8. The method according to claim 1, characterized in that The method also includes the steps of preparing a control medicinal material solution of the southern crane lice and a negative solution.

9. The method according to claim 8, characterized in that The spotting volume of the southern crane lice control medicinal material solution is 1~9μL; The spotting volume of the negative solution is 8-15 μL.

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