A thin layer identification method for scutellaria baicalensis formula granules and scutellaria baicalensis charcoal formula granules

Abstract

The present invention provides a thin layer chromatography identification method for scutellaria baicalensis formula granules and scutellaria baicalensis charcoal formula granules, comprising the following steps: S1) pre-treating a sample to be tested to obtain a test solution; the sample to be tested is scutellaria baicalensis formula granules and scutellaria baicalensis charcoal formula granules; pre-treating a reference substance to obtain a reference substance solution; pre-treating a scutellaria baicalensis reference medicinal material to obtain a reference medicinal material solution; S2) performing thin layer chromatography on the test solution, the reference solution and the reference medicinal material solution, using a silica gel GF thin layer plate. 254 The thin-layer chromatography identification method for Huangqin formula granules and Huangqin charcoal formula granules established in the present invention uses the presence of spots within a specified ratio shift range on the thin-layer chromatography spectrum as identification points to quickly and effectively identify Huangqin formula granules and Huangqin charcoal formula granules, thereby ensuring the accuracy of clinical medication.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a thin layer identification method for scutellaria baicalensis formula granules and scutellaria baicalensis charcoal formula granules. Background Art

[0002] Scutellaria baicalensis, the dried root of the Lamiaceae plant Scutellaria baicalensis Georgi, has the effects of clearing heat and dampness, purging fire and detoxifying, stopping bleeding, and stabilizing pregnancy. It is used for damp-heat syndrome, summer-heat dampness, chest tightness and nausea, damp-heat fullness and distension, diarrhea, jaundice, lung-heat cough, high fever and thirst, blood-heat vomiting and epistaxis, carbuncles, sores, and fetal restlessness. Raw Scutellaria baicalensis is better at clearing damp-heat, while roasted charcoal is better at stopping bleeding. These two effects differ significantly, suggesting that their chemical composition also changes. To ensure accurate clinical use, it is necessary to establish a visual identification method for qualitatively distinguishing the two.

[0003] Current qualitative research on Scutellaria baicalensis and Scutellaria baicalensis charcoal focuses on changes in the appearance and chemical composition of different processed preparations. For example, Zhou Desheng's research demonstrated that Scutellaria baicalensis undergoes significant changes in microscopic characteristics and extract content after processing, which can be used to identify different pieces. However, since Scutellaria baicalensis charcoal granules are prepared from Scutellaria baicalensis charcoal pieces through a series of processes, some of which can destroy the original physical and microscopic characteristics, physical and microscopic identification methods are not applicable to the identification of extracts and preparations. Huang Qi's research demonstrated that the composition and ratio of volatile components change significantly after charcoalization. However, since water is used as a solvent in the preparation of Scutellaria baicalensis charcoal granules, volatile components cannot be extracted. Therefore, volatile component analysis is not suitable for the identification of Scutellaria baicalensis and Scutellaria baicalensis charcoal granules. The component content of Chinese medicinal materials is affected by factors such as origin, age, harvest time, and storage. Therefore, component content is not suitable for the qualitative identification of Scutellaria baicalensis and Scutellaria baicalensis charcoal granules.

[0004] In summary, the aforementioned methods for property identification, microscopic identification, and component quantification are not suitable for the qualitative identification of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules. Currently, there is no effective method for distinguishing Scutellaria baicalensis from Scutellaria baicalensis charcoal in preparations. Therefore, a method for distinguishing Scutellaria baicalensis, Scutellaria baicalensis charcoal extracts, and granules is needed. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a thin layer identification method for scutellaria baicalensis formula granules and scutellaria baicalensis charcoal formula granules, which has the advantages of strong specificity, good stability and high precision.

[0006] In view of this, the present application provides a thin layer identification method for Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules, comprising the following steps:

[0007] S1) pretreating a sample to be tested to obtain a test solution; the sample to be tested is a scutellaria baicalensis granule and a scutellaria baicalensis charcoal granule;

[0008] Pre-treating baicalin reference substance, baicalein reference substance and wogonin reference substance respectively to obtain reference substance solutions;

[0009] Pre-treating the scutellaria baicalensis reference medicinal material to obtain a reference medicinal material solution;

[0010] S2) The test solution, reference solution and reference medicinal material solution were subjected to thin layer chromatography (TLC) using silica gel GF 254 Thin plate, the developing solvent is chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid;

[0011] S3) Check under ultraviolet light. If there is a spot at the Rf value of 0.313±10% in the chromatogram of the test sample, it is the Scutellaria baicalensis formula granules. If there is no spot at the Rf value of 0.313±10%, it is the Scutellaria baicalensis charcoal formula granules.

[0012] Preferably, the preparation of the test solution is specifically as follows:

[0013] The sample to be tested was dissolved in water, extracted with n-butanol-ethyl acetate, and the residue obtained after evaporation was added with methanol, sonicated, and filtered to obtain the test solution;

[0014] The ratio of the sample to be tested, the n-butanol-ethyl acetate and the methanol is 1 g: (20-40) ml: (3-8) ml, the volume ratio of the n-butanol and the ethyl acetate is (1.5-2.5): 1, and the ultrasonic time is 20-40 min.

[0015] Preferably, the preparation of the reference solution is specifically as follows:

[0016] Adding baicalin reference substance, baicalein reference substance and wogonin reference substance into methanol respectively to obtain reference substance solutions;

[0017] The concentration of the baicalin reference substance in the reference solution is (0.3-0.6) mg / ml, the concentration of the baicalein reference substance in the reference solution is (0.3-0.6) mg / ml, and the concentration of the wogonin reference substance in the reference solution is (0.3-0.6) mg / ml.

[0018] Preferably, the preparation of the control medicinal material solution is specifically as follows:

[0019] The reference medicinal material Scutellaria baicalensis was boiled in water, the filtrate obtained by filtration was evaporated to dryness, and then dissolved in water, and then extracted with n-butanol-ethyl acetate. The residue obtained after evaporation was added with methanol, and the mixture was sonicated and filtered to obtain a reference medicinal material solution;

[0020] The ratio of the scutellaria baicalensis reference medicinal material, boiling water and dissolving water is 2g: (50-100)ml: (5-15)ml.

[0021] Preferably, the sample volume for thin layer chromatography detection is 3 to 5 μl.

[0022] Preferably, the volume ratio of chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid is (9.5-10.5):(0.5-1.5):(1.0-2.0):(0.5-1.5).

[0023] Preferably, the wavelength of the ultraviolet light is 254 nm.

[0024] Preferably, during the thin layer chromatography detection process, the development temperature is 4 to 35°C.

[0025] Preferably, during the thin layer chromatography detection, the development humidity is 32% rh to 75% rh.

[0026] Preferably, the Rf value is 0.28 to 0.34.

[0027] The present application provides a thin layer chromatography identification method for Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules. The method first prepares a test solution, a reference solution and a control medicinal material solution respectively, then performs thin layer chromatography on the test solution, the reference solution and the control medicinal material solution, and inspects them under ultraviolet light. In the test sample chromatogram, if there is a spot at the Rf value of 0.313±10%, it is Scutellaria baicalensis formula granules; if there is no spot at the Rf value of 0.313±10%, it is Scutellaria baicalensis charcoal formula granules. In the thin-layer identification method of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, by introducing thin-layer chromatography detection and selecting the developing agent, the thin-layer identification method has the advantages of strong specificity, good stability, high precision, convenience and ease of mastering; further, the thin-layer identification method provided by the present application is compared with the thin-layer chromatography of Scutellaria baicalensis charcoal formula granules and the thin-layer chromatography of Scutellaria baicalensis control medicinal materials and Scutellaria baicalensis granules, and the shift value is at the same position of 0.28 to 0.34, without the appearance of spots, as the identification point of Scutellaria baicalensis charcoal formula granules. Therefore, the thin-layer identification method provided by the present application can quickly and effectively identify Scutellaria baicalensis charcoal formula granules that have lost the morphology of medicinal pieces, is simple to operate, has high precision and sensitivity, good stability, has good application prospects, and ensures accurate medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The thin layer chromatography spectra of the present invention for identifying the scutellaria baicalensis formula granules and scutellaria baicalensis charcoal formula granules by method 1, method 2 and method 3;

[0029] Figure 2 The thin layer chromatography atlas of developer 1 for identification of Huangqin formula granules and Huangqin carbon formula granules;

[0030] Figure 3 The thin layer chromatography atlas of the developer 2 for identification of Huangqin formula granules and Huangqin carbon formula granules;

[0031] Figure 4 The thin layer chromatography atlas of the developer 3 for identification of Huangqin formula granules and Huangqin carbon formula granules;

[0032] Figure 5 The following are the TLC spectra of reference solution, scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution and scutellaria baicalensis charcoal formula granule solution at different sample amounts;

[0033] Figure 6 The thin layer chromatography spectra of negative solution, reference solution, scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution and scutellaria baicalensis charcoal formula granule solution are shown;

[0034] Figure 7 This is the thin layer chromatography diagram of Tianjin Silida chromatography plate;

[0035] Figure 8 This is a thin layer chromatography diagram of a Merck chromatography plate;

[0036] Figure 9 This is a thin layer map of Qingdao marine chromatography plate;

[0037] Figure 10 The thin layer chromatography spectra of the reference solution, scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution and scutellaria baicalensis charcoal formula granule at 4°C are shown;

[0038] Figure 11 The thin layer chromatography spectra of the reference solution, the reference medicinal material solution of Scutellaria baicalensis, the Scutellaria baicalensis granule solution and the Scutellaria baicalensis charcoal granule at 35°C are shown;

[0039] Figure 12 The thin layer chromatography spectra of the reference substance solution, the reference medicinal material solution of Scutellaria baicalensis, the Scutellaria baicalensis formula granule solution and the Scutellaria baicalensis carbon formula granule under 32% rh;

[0040] Figure 13 The thin layer chromatography spectra of the reference substance solution, the reference medicinal material solution of Scutellaria baicalensis, the Scutellaria baicalensis formula granule solution and the Scutellaria baicalensis carbon formula granule under 75% rh;

[0041] Figure 14 The following are thin layer chromatography images of different batches of Huangqin formula granules and Huangqin carbon formula granules;

[0042] Figure 15 This is the thin layer identification spectrum of the Scutellaria baicalensis formula granules and the Scutellaria baicalensis carbon formula granules in comparative example 1. DETAILED DESCRIPTION

[0043] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0044] In view of the need to qualitatively distinguish between Huangqin Formula Granules and Huangqin Charcoal Formula Granules in the prior art, this application establishes a thin layer chromatography identification method for Huangqin Formula Granules and Huangqin Charcoal Formula Granules. The method uses the presence of spots within a specified ratio shift value range on the thin layer chromatography spectrum as the identification point to quickly and effectively identify Huangqin Formula Granules and Huangqin Charcoal Formula Granules to ensure the accuracy of clinical medication. Specifically, the embodiment of the present invention discloses a thin layer chromatography identification method for Huangqin Formula Granules and Huangqin Charcoal Formula Granules, comprising the following steps:

[0045] S1) pretreating a sample to be tested to obtain a test solution; the sample to be tested is a scutellaria baicalensis granule and a scutellaria baicalensis charcoal granule;

[0046] Pre-treating baicalin reference substance, baicalein reference substance and wogonin reference substance respectively to obtain reference substance solutions;

[0047] Pre-treating the scutellaria baicalensis reference medicinal material to obtain a reference medicinal material solution;

[0048] S2) The test solution, reference solution and reference medicinal material solution were subjected to thin layer chromatography (TLC) using silica gel GF 254 Thin layer plate, the developing solvent is chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid;

[0049] S3) Check under ultraviolet light. If there is a spot at the Rf value of 0.313±10% in the chromatogram of the test sample, it is the Scutellaria baicalensis formula granules. If there is no spot at the Rf value of 0.313±10%, it is the Scutellaria baicalensis charcoal formula granules.

[0050] In the thin layer chromatography identification method of Huangqin Formula Granules and Huangqin Carbon Formula Granules, step S1) prepares a test solution, a reference solution, and a reference medicinal material solution respectively; the preparation of the test solution is specifically as follows:

[0051] Dissolve the sample in water, extract it 2 to 4 times with n-butanol-ethyl acetate, combine the n-butanol-ethyl acetate solutions, evaporate to dryness, add methanol to the residue, sonicate and filter to obtain the test solution.

[0052] In the above-mentioned preparation of the test solution, since the samples to be tested are Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules, it is necessary to prepare a test solution of Scutellaria baicalensis charcoal granules and a test solution of Scutellaria baicalensis granules, and both test solutions are prepared according to the above-mentioned method; that is, the Scutellaria baicalensis granules to be tested are dissolved in water, and then extracted 2 to 4 times with n-butanol-ethyl acetate, the n-butanol-ethyl acetate solution is combined, and the residue obtained after evaporation is added with methanol, and the mixture is filtered after ultrasonication to obtain test solution 1; and the Scutellaria baicalensis charcoal granules to be tested are dissolved in water, and then extracted 2 to 4 times with n-butanol-ethyl acetate, the n-butanol-ethyl acetate solution is combined, the residue obtained after evaporation is added with methanol, and the mixture is filtered after ultrasonication to obtain test solution 2.

[0053] In the above process, the ratio of the sample to be tested, the n-butanol-ethyl acetate, and the methanol is 1 g: (20-40) ml: (3-8) ml, the volume ratio of the n-butanol to the ethyl acetate is (1.5-2.5): 1, and the ultrasonication time is 20-40 minutes. The n-butanol-ethyl acetate extraction is performed three times, each time with 10 ml. Specifically, the ratio of the sample to be tested, the n-butanol-ethyl acetate, and the methanol is 1 g: 30 ml: 5 ml, and the ultrasonication time is 25-35 minutes.

[0054] The preparation of the reference solution is as follows:

[0055] Adding baicalin reference substance, baicalein reference substance and wogonin reference substance into methanol to obtain reference substance solutions;

[0056] The concentration of the baicalin reference substance in the reference solution is (0.3-0.6) mg / ml, the concentration of the baicalein reference substance in the reference solution is (0.3-0.6) mg / ml, and the concentration of the wogonin reference substance in the reference solution is (0.3-0.6) mg / ml; specifically, the concentration of the baicalin reference substance in the reference solution is (0.4-0.5) mg / ml, the concentration of the baicalein reference substance in the reference solution is (0.4-0.5) mg / ml, and the concentration of the wogonin reference substance in the reference solution is (0.4-0.5) mg / ml.

[0057] The preparation of the control medicinal material solution is specifically as follows:

[0058] The reference medicinal material Scutellaria baicalensis was boiled in water, the filtrate obtained by filtration was evaporated to dryness, and then dissolved in water, and then extracted with n-butanol-ethyl acetate. The residue obtained after evaporation was added with methanol, and the mixture was sonicated and filtered to obtain a reference medicinal material solution;

[0059] The ratio of the scutellaria baicalensis reference medicinal material, boiling water, dissolving water, the n-butanol-ethyl acetate, and the methanol is 2g: (50-100)ml: (5-15)ml: (20-40)ml: (3-8)ml; the volume ratio of n-butanol and ethyl acetate in the n-butanol-ethyl acetate is 2:1, and the extraction is performed three times, and finally the n-butanol-ethyl acetate extracts are combined; specifically, the ratio of the scutellaria baicalensis reference medicinal material, boiling water, dissolving water, the n-butanol-ethyl acetate, and the methanol is 2g: 50ml: 10ml: 30ml: 5ml.

[0060] In step S2), the above-mentioned test solution, reference solution and reference medicinal material solution are subjected to thin layer chromatography; in this process, the thin layer plate is silica gel GF 254 Thin plate, in a specific embodiment, the silica gel GF 254 Thin plates can be made of thin layer chromatography plates, Merck chromatography plates, or prefabricated silica gel GF plates from the Qingdao Ocean Chemical Plant branch. 254 The results showed that this method can effectively 254 The thin plates have good durability and can meet the identification requirements. The scutellaria baicalensis formula particles have spots at the ratio shift values ​​of 0.313, 0.337, and 0.314, while the scutellaria baicalensis charcoal formula particles have no spots. The developing solvent is chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid. Specifically, the volume ratio of chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid is (9.5-10.5):(0.5-1.5):(1.0-2.0):(0.5-1.5). More specifically, the volume ratio of chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid is 10:1:1.5:1. The development temperature is 4 to 35°C. The method of the present invention has good adaptability to different temperatures. At the migration ratio values ​​of 0.301 and 0.306, the scutellaria baicalensis granules have spots, while the scutellaria baicalensis charcoal granules have no spots, achieving the identification of the scutellaria baicalensis granules and the scutellaria baicalensis charcoal granules. The development humidity is 32% rh to 75% rh. The method of the present invention has good adaptability to different humidity. At the migration ratio values ​​of 0.311 and 0.325, the scutellaria baicalensis granules have spots, while the scutellaria baicalensis charcoal granules have no spots, achieving the identification of the scutellaria baicalensis granules and the scutellaria baicalensis charcoal granules. The sample volume for the thin layer chromatography test is 3 to 5 μl, specifically, the sample volume for the thin layer chromatography test is 3 μl, 4 μl, or 5 μl.

[0061] The inspection is carried out under an ultraviolet lamp with a wavelength of 254 nm. If there are spots at the Rf value of 0.313±10%, it is the Scutellaria baicalensis formula granules. If there are no spots at the Rf value of 0.313±10%, it is the Scutellaria baicalensis charcoal formula granules.

[0062] The present application provides a thin layer chromatography identification method for scutellaria baicalensis granules and scutellaria baicalensis charcoal granules, comprising the following steps: S1) pre-treating a sample to be tested to obtain a test solution; the sample to be tested is scutellaria baicalensis granules and scutellaria baicalensis charcoal granules; pre-treating a baicalin reference substance, a baicalein reference substance and a wogonin reference substance respectively to obtain a reference solution; pre-treating a scutellaria baicalensis reference medicinal material to obtain a control medicinal material solution; S2) subjecting the test solution, the reference solution and the control medicinal material solution to thin layer chromatography, and the thin layer plate is a silica gel GF 254 The thin-layer plate is developed using chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid as the developing solvent; S3) the plate is inspected under ultraviolet light. If a spot is present at an Rf value of 0.313±10% in the chromatogram of the test sample, the sample is the Huangqin Formula Granules; if no spot is present at an Rf value of 0.313±10%, the sample is the Huangqin Charcoal Formula Granules. The thin-layer chromatography identification method for the Huangqin Formula Granules and Huangqin Charcoal Formula Granules, by incorporating thin-layer chromatography and selecting the developing solvent, has the advantages of strong specificity, good stability, high precision, convenience, and ease of use.

[0063] The thin layer identification method provided in this application is to compare the thin layer chromatography of the test sample with the thin layer chromatography of the Scutellaria baicalensis control medicinal material. If the spots are consistent with those of the control medicinal material, it is the Scutellaria baicalensis formula granules. If there are no spots at the same position as the control medicinal material at the ratio transfer value of 0.28 to 0.34, it is the Scutellaria baicalensis charcoal formula granules.

[0064] In order to further understand the present invention, the thin layer identification method of the Scutellaria baicalensis formula granules and the Scutellaria baicalensis charcoal formula granules provided by the present invention is described in detail below in conjunction with the examples. The scope of protection of the present invention is not limited by the following examples.

[0065] The instruments and reagents used in the following examples are specifically as follows:

[0066] 1.1 Instrument

[0067] Hot plate, mortar, thin layer imaging system: CAMAG TLC Visualizer, silica gel GF 254 Thin layer plates (Qingdao Ocean Chemical Plant, batch number: 20180527, Tianjin Silida Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353);

[0068] 1.2 Reagents

[0069] Chloroform, anhydrous ethanol, n-butanol, toluene, ethyl acetate, butyl acetate, methanol, glacial acetic acid, and formic acid were all of analytical grade, and water was ultrapure water;

[0070] 1.3 Drug testing

[0071] Baicalein reference substance (China Food and Drug Inspection Institute, batch number: 111595-201808, purity: 97.9%); baicalin reference substance (China Food and Drug Inspection Institute, batch number: 110715-202223, purity: 97.2%); wogonin reference substance (China Food and Drug Inspection Institute, batch number: 111514-202207, purity: 100%); Scutellaria baicalensis reference substance Medicinal materials (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: ycwkq22101407); Scutellaria baicalensis formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2210005, 2212001, 2302008); Scutellaria baicalensis charcoal formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: B2308010, B2308011, B2308012).

[0072] Example 1

[0073] Based on the different extraction methods, this application first determines the extraction method, and the specific process is as follows:

[0074] 1.1 Investigation of extraction methods

[0075] 1. Take 1g each of Huangqin Formula Granules and Huangqin Carbon Formula Granules and perform the following operations: grind them into powder, add 10ml of methanol and ultrasonically extract for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 5ml of methanol to prepare the test solution;

[0076] Take 2 g of Scutellaria baicalensis as a control medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of methanol to the residue and ultrasonically extract for 30 minutes, filter, evaporate the filtrate to dryness, and add 5 ml of methanol to dissolve the residue to prepare a control medicinal material solution;

[0077] Separately, take baicalin reference substance, baicalein reference substance, and wogonin reference substance, add methanol to prepare a mixed solution containing 0.5 mg of each per 1 ml, as the reference solution;

[0078] 2. Take 1g each of Huangqin Formula Granules and Huangqin Carbon Formula Granules and perform the following operations: grind them into powder, add 10ml of methanol and reflux for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 5ml of methanol to prepare the test solution;

[0079] Take 2 g of Scutellaria baicalensis as a control medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of methanol to the residue and reflux for 30 minutes, filter, evaporate the filtrate to dryness, and add 5 ml of methanol to dissolve the residue to prepare a control medicinal material solution;

[0080] Separately, take baicalin reference substance, baicalein reference substance, and wogonin reference substance, add methanol to prepare solutions containing 0.5 mg per 1 ml, as reference solution;

[0081] 3. Take 1g each of Huangqin Formula Granules and Huangqin Carbon Formula Granules and perform the following operations: grind them into powder, add 10ml of water to dissolve them, add n-butanol-ethyl acetate (2:1) and shake to extract 3 times, 10ml each time, combine the n-butanol-ethyl acetate solutions, evaporate to dryness, add 5ml of methanol to the residue, sonicate for 30 minutes, filter, and use as the test solution;

[0082] Take 2 g of Scutellaria baicalensis as a control medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, add n-butanol-ethyl acetate (2:1) and shake and extract three times, 10 ml each time, combine the n-butanol-ethyl acetate solutions, evaporate to dryness, add 5 ml of methanol to the residue, sonicate for 30 minutes, and filter to prepare a control medicinal material solution;

[0083] Separately, take baicalin reference substance, baicalein reference substance, and wogonin reference substance, add methanol to prepare solutions containing 0.5 mg per 1 ml, as reference solution;

[0084] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition Part IV General Rules 0502), 3 μl of test solution, 3 μl of reference solution, and 5 μl of reference medicinal material solution were taken and spotted on the same silica gel GF 254 On the thin layer plate, chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid (10:1:1.5:1) was used as the developing agent, the plate was developed, taken out, dried, and examined under ultraviolet light (254nm). Figure 1 As shown, Figure 1 This is the TLC atlas for identification of Huangqin Formula Granules and Huangqin Carbon Formula Granules by methods 1, 2 and 3. Figure 1 Figures 1 to 4 are the TLC spectra of method 1 reference substance (from bottom to top: baicalin, baicalein, wogonin), method 1 reference medicinal materials, method 1 scutellaria formula granules, and method 1 scutellaria charcoal formula granules; figures 5 to 8 are the TLC spectra of method 2 reference substance (from bottom to top: baicalin, baicalein, wogonin), method 2 reference medicinal materials, method 2 scutellaria formula granules, and method 2 scutellaria charcoal formula granules; figures 9 to 12 are the TLC spectra of method 3 reference substance (from bottom to top: baicalin, baicalein, wogonin), formula Thin layer chromatography spectra of method 3 control medicinal materials, method 3 Scutellaria baicalensis formula granules and method 3 Scutellaria baicalensis charcoal formula granules; the results showed that the test samples prepared by method 1 and method 2 could not effectively distinguish between Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, and the test sample preparation method was determined as: take 1g of this product, grind it into powder, add 10ml of water to dissolve it, add n-butanol-ethyl acetate (2:1) and shake to extract 3 times, 10ml each time, combine the extracted liquids, evaporate to dryness, add 5ml of methanol to the residue, ultrasonicate for 30 minutes, filter, and use as the test sample solution.

[0085] 1.2 Development agent investigation

[0086] Take 1g each of Huangqin Formula Granules and Huangqin Carbon Formula Granules and perform the following operations: grind them into powder, add 10ml of water to dissolve them, add n-butanol-ethyl acetate (2:1) and shake to extract 3 times, 10ml each time, combine the extracts, evaporate to dryness, add 5ml of methanol to the residue, sonicate for 30 minutes, filter, and use as the test solution;

[0087] Take 2g of Scutellaria baicalensis reference medicinal material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to the residue to dissolve it, and prepare the reference medicinal material solution in the same way as "Preparation of Test Solution";

[0088] Separately, take baicalin reference substance, baicalein reference substance, and wogonin reference substance, add methanol to prepare solutions containing 0.5 mg per 1 ml, as reference solution;

[0089] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition Part IV General Rules 0502), 3 μl of test solution, 3 μl of reference solution, and 5 μl of reference medicinal material solution were taken and spotted on the same silica gel GF 254 On the thin layer plate, toluene-ethyl acetate-methanol-formic acid (10:3:1:2) (developer 1), toluene-ethyl acetate-methanol-formic acid (10:1:1.5:1) (developer 2), chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid (10:1:1.5:1) (developer 3) were used as the developing agent, and the plate was taken out, dried, and examined under ultraviolet light (254nm). The results are as follows. Figures 2-4 As shown, Figure 2 The thin layer chromatography atlas of the developer 1 for identification of Huangqin Formula Granules and Huangqin Carbon Formula Granules, Figure 3 The thin layer chromatography atlas of the developer 2 for identification of Huangqin Formula Granules and Huangqin Carbon Formula Granules, Figure 4 The figures are the thin layer chromatography (TLC) spectra of the identification of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules using developer 3, where 1 is the TLC spectra of the reference substance (baicalin, baicalein, and wogonin from bottom to top), 2 is the TLC spectra of the Scutellaria baicalensis reference medicinal material, 3 is the TLC spectra of Scutellaria baicalensis formula granules, and 4 is the TLC spectra of Scutellaria baicalensis charcoal formula granules. The results showed that when the developer chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid (10:1:1.5:1) was used, the spots were clear, the Scutellaria baicalensis formula granules sample had an obvious spot at Rf=0.333, while the Scutellaria baicalensis charcoal formula granules sample did not have this spot. This method can achieve the qualitative identification of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules.

[0090] Example 2

[0091] The thin-layer chromatography method determined in Example 1 was used to perform thin-layer chromatography identification of the Radix Scutellariae Formula Granules and the Radix Scutellariae Carbon Formula Granules, as follows:

[0092] 2.1 Preparation of test solution

[0093] Take 1g each of Huangqin Formula Granules and Huangqin Carbon Formula Granules and perform the following operations: grind them into powder, add 10ml of water to dissolve them, add n-butanol-ethyl acetate (2:1) and shake to extract 3 times, 10ml each time, combine the n-butanol-ethyl acetate solutions, evaporate to dryness, add 5ml of methanol to the residue, sonicate for 30 minutes, filter, and use as the test solution;

[0094] 2.2 Preparation of control medicinal material solution

[0095] Take 2 g of Scutellaria baicalensis reference medicinal material, add 50 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, add n-butanol-ethyl acetate (2:1) and shake and extract three times, 10 ml each time, combine the n-butanol-ethyl acetate solutions, evaporate to dryness, add 5 ml of methanol to the residue, sonicate for 30 minutes, and filter to prepare the reference medicinal material solution;

[0096] 2.3 Preparation of reference solution

[0097] Take baicalin reference substance, baicalein reference substance, and wogonin reference substance, add methanol to prepare a mixed solution containing 0.5 mg of each per 1 ml, which is used as the reference solution.

[0098] 2.4 Assay

[0099] According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 edition Part IV General Rules 0502), 3 μl of test solution, 3 μl of reference solution, and 5 μl of reference medicinal material solution were taken and spotted on the same silica gel GF 254 On the thin layer plate, use chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid (10:1:1.5:1) as the developing agent, develop, take out, dry, and examine under ultraviolet light (254nm).

[0100] 2.5 Methodological Investigation

[0101] 2.5.1 Sampling quantity inspection

[0102] Under the experimental conditions proposed above, 1 μl, 3 μl and 5 μl of reference solution, scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution and scutellaria baicalensis carbon formula granule solution were respectively taken and spotted on the same silica gel GF 254 On thin layer plates, such as Figure 5 As shown, Figure 5Figures 1 to 3 are TLC spectra of reference solution, scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution and scutellaria baicalensis carbonized formula granule solution at different sample volumes. Figures 1 to 3 are TLC spectra of reference solution sample volumes of 1, 3, and 5 μl (from bottom to top: baicalin, baicalein, and wogonin); Figures 4 to 6 are TLC spectra of scutellaria baicalensis reference medicinal material solution of 1, 3, and 5 μl; Figures 7 to 9 are TLC spectra of scutellaria baicalensis carbonized formula granule solution of 1, 3, and 5 μl; Figures 10 to 12 are TLC spectra of reference solution sample volumes of 1, 3, and 5 μl (from bottom to top: baicalin, baicalein, and wogonin); Figures 11 to 12 are TLC spectra of reference solution sample volumes of 1, 3, and 5 μl; Figures 13 to 14 are TLC spectra of reference solution sample volumes of 1, 3, and 5 μl; Figures 15 to 16 are TLC spectra of reference solution sample volumes of 1, 3, and 5 μl; Figures 17 to 18 are TLC spectra of reference solution sample volumes of 1, 3, and 5 μl; Figures 19 to 20 are TLC spectra of reference solution sample volumes of 1, 3, and 5 μl; Figures 10 to 12 Thin layer chromatography of 1, 3, and 5 μl of Scutellaria baicalensis formula granules solution; it can be seen from the figure that when 3-5 μl of the reference solution, the control medicinal material solution, and the test solution are spotted, the spots are clearly colored and the separation is good, and the spot volume is determined to be 3-5 μl. Moreover, at the shift ratio value of 0.304, the Scutellaria baicalensis formula granules show spots, while the Scutellaria baicalensis charcoal formula granules have no spots, which is used to distinguish the Scutellaria baicalensis formula granules from the Scutellaria baicalensis charcoal formula granules. Therefore, whether the shift ratio value of 0.304 shows spots can be used as the identification point.

[0103] 2.5.2 Specificity Investigation

[0104] According to the above test sample preparation method, negative solution, reference solution, scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution, scutellaria baicalensis formula granule solution, and scutellaria baicalensis charcoal formula granule solution were prepared respectively, and thin layer identification experiments were carried out. The results are as follows Figure 6 As shown, Figure 6 These are the thin layer chromatography spectra of negative solution, reference solution, scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution and scutellaria baicalensis charcoal formula granule solution. Among them, 1 is the thin layer chromatography spectra of negative solution, 2 is the thin layer chromatography spectra of reference solution (from bottom to top: baicalin, baicalein, wogonin), 3 is the thin layer chromatography spectra of scutellaria baicalensis reference medicinal material solution, 4 is the thin layer chromatography spectra of scutellaria baicalensis formula granule solution, and 5 is the thin layer chromatography spectra of scutellaria baicalensis charcoal formula granule solution. It can be seen from the figure that the negative sample has no interference with the scutellaria baicalensis and scutellaria baicalensis charcoal formula granule test samples. The method has good specificity, and the scutellaria baicalensis formula granules show spots at the ratio shift value of 0.296, while the scutellaria baicalensis charcoal formula granules have no spots.

[0105] 2.5.3 Durability Investigation

[0106] 2.5.3.1 Comparison of different thin layer plates

[0107] Select the tailorable thin layer chromatography plate of Tianjin Silida Technology Co., Ltd., Merck, and Qingdao Ocean Chemical Plant Branch prefabricated silica gel GF 254 The boards were tested according to the proposed test methods, such as Figures 7-9 As shown, Figure 7 This is the thin layer spectrum of Tianjin Silida chromatography plate. Figure 8 This is a thin layer chromatography chart of Merck chromatography plate. Figure 9The TLC spectra of Qingdao Ocean Chromatography Plate, 1 is the TLC spectra of the reference solution (from bottom to top: baicalin, baicalein, wogonin), 2 is the TLC spectra of the Scutellaria baicalensis reference medicinal material solution, 3 is the TLC spectra of the Scutellaria baicalensis formula granule solution, 4 is the TLC spectra of the Scutellaria baicalensis charcoal formula granule solution, Figures 7-9 It can be seen that the spot ratio shift values ​​(Rf) of the three thin layer plates at 8 to 10 cm away from the origin are 0.313, 0.337, and 0.314, respectively, which can meet the identification requirements. The Scutellaria baicalensis formula granules show spots at the ratio shift values ​​of 0.313, 0.337, and 0.314, while the Scutellaria baicalensis charcoal formula granules have no spots.

[0108] 2.5.3.2 Comparison of different temperatures

[0109] Take the spotted thin layer plate and develop it at low temperature of 4℃ and high temperature of 35℃ respectively. Figures 10-11 As shown, Figure 10 The thin layer chromatography spectra of the reference solution, scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution and scutellaria baicalensis carbon formula granule at 4°C are shown. Figure 11 The thin layer chromatography spectra of the reference solution, the scutellaria baicalensis reference medicinal material solution, the scutellaria baicalensis formula granule solution and the scutellaria baicalensis charcoal formula granule at 35°C, wherein 1 is the thin layer chromatography spectra of the reference solution (from bottom to top: baicalin, baicalein, wogonin), 2 is the thin layer chromatography spectra of the scutellaria baicalensis reference medicinal material solution, 3 is the thin layer chromatography spectra of the scutellaria baicalensis formula granule solution, and 4 is the thin layer chromatography spectra of the scutellaria baicalensis charcoal formula granule solution. Figure 10 and 11 It can be seen that this method has good adaptability to different temperatures, and the scutellaria baicalensis formula granules show spots at the ratio shift values ​​of 0.301 and 0.306, while the scutellaria baicalensis charcoal formula granules have no spots.

[0110] 2.5.3.3 Comparison of different humidity levels

[0111] Take the spotted thin layer plate and develop it in a humidity environment of 32% rh and 75% rh, respectively. Figures 12-13 As shown, Figure 12 The thin layer chromatography spectra of the reference solution, the reference medicinal material solution of Scutellaria baicalensis, the Scutellaria baicalensis formula granule solution and the Scutellaria baicalensis carbon formula granule under 32% rh are shown. Figure 13 The thin layer chromatography spectra of the reference solution, the scutellaria baicalensis reference medicinal material solution, the scutellaria baicalensis formula granule solution and the scutellaria baicalensis charcoal formula granule under 75% rh, wherein 1 is the thin layer chromatography spectra of the reference solution (from bottom to top: baicalin, baicalein, wogonin), 2 is the thin layer chromatography spectra of the scutellaria baicalensis reference medicinal material solution, 3 is the thin layer chromatography spectra of the scutellaria baicalensis formula granule solution, 4 is the thin layer chromatography spectra of the scutellaria baicalensis charcoal formula granule solution, Figure 12 and 13It can be seen that this method has good adaptability to different humidity levels, and the granules of the scutellaria formula show spots at the ratio transfer values ​​of 0.311 and 0.325, while the granules of the scutellaria baicalensis charcoal formula have no spots.

[0112] 2.5.4 Verification

[0113] Take the reference solution and the scutellaria baicalensis reference medicinal material solution, scutellaria baicalensis formula granule solution, and scutellaria baicalensis carbon formula granule solution respectively, and apply them to the same silica gel GF 254 On the thin layer plate, the analysis was carried out according to the determined method. The experimental results are as follows Figure 14 As shown, Figure 14 These are the thin layer chromatography spectra of different batches of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, among which 1 is the thin layer chromatography spectra of the reference solution (baicalin, baicalein, and wogonin from bottom to top), 2 is the thin layer chromatography spectra of the Scutellaria baicalensis reference medicinal material solution, 3 to 5 are the thin layer chromatography spectra of different batches of Scutellaria baicalensis formula granule solutions, and 6 to 8 are the thin layer chromatography spectra of different batches of Scutellaria baicalensis charcoal formula granule solutions. It can be seen from the figure that the Scutellaria baicalensis formula granules have a spot at the ratio transfer value (Rf) of 0.318, while the Scutellaria baicalensis charcoal formula granules do not have this spot. This spot can be used to distinguish the two.

[0114] 2.6 Determination of the identification point Rf for Huangqin Formula Granules and Huangqin Carbon Formula Granules

[0115] The data of various ratio transfer values ​​in the thin layer chromatography methodology investigation are summarized and the results are shown in Table 1.

[0116] Table 1 Summary of the above mentioned methodological investigation ratio transfer value data

[0117]

[0118] According to Table 1, the identification spot ratio shift value (Rf) of the Scutellaria baicalensis formula granules should be within the range of 10%, and the specified value is 0.313.

[0119] Comparative Example 1

[0120] Take 1g of the powder of this product, add 30ml of a mixed solution of ethyl acetate and methanol (3:1), heat and reflux for 30 minutes, cool, filter, evaporate the filtrate, add 5ml of methanol to dissolve the residue, and take the supernatant as the test solution; take another 1g of the reference medicinal material of Scutellaria baicalensis, and prepare the reference medicinal material solution in the same way; then take the reference material of baicalin, baicalein, and wogonin, add methanol to prepare solutions containing 1mg, 0.5mg, and 0.5mg per ml respectively, as the reference solution; according to the thin layer chromatography method (General Rules 0502), take 2μl of the above test solution, reference medicinal material solution, and 1μl of the above three reference solution, respectively, and spot them on the same polyamide film, use toluene-ethyl acetate-methanol-formic acid (10:3:1:2) as the developing solvent, pre-saturate for 30 minutes, develop, take out, dry, and examine under ultraviolet light (365nm). The results are as follows Figure 15 As shown, Figure 15 This is the thin layer spectrum of the Scutellaria baicalensis formula granules and the Scutellaria baicalensis charcoal formula granules in comparative example 1, where 1 is the thin layer spectrum of the baicalin reference solution, 2 is the thin layer spectrum of wogonin, 3 is the thin layer spectrum of baicalein, 4 is the thin layer spectrum of the Scutellaria baicalensis reference medicinal material solution, 5 is the thin layer spectrum of the Scutellaria baicalensis formula granule solution, and 6 is the thin layer spectrum of the Scutellaria baicalensis charcoal formula granule solution. It can be seen from the figure that using the above-mentioned thin layer identification method, there are no obvious difference spots between the Scutellaria baicalensis and the Scutellaria baicalensis charcoal formula granules, and the two cannot be distinguished. Moreover, since a large amount of formic acid is used in the developing agent, it is easy to corrode the polyamide thin layer plate, which is not suitable for the identification of the Scutellaria baicalensis formula granules and the Scutellaria baicalensis charcoal formula granules.

[0121] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0122] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A thin layer chromatography identification method for Huangqin formula granules and Huangqin carbon formula granules, comprising the following steps: S1) Dissolve the sample in water, extract with n-butanol-ethyl acetate, evaporate to dryness, add methanol to the residue, sonicate, and filter to obtain the test solution; The samples to be tested are scutellaria baicalensis granules and scutellaria baicalensis charcoal granules; Pre-treating baicalin reference substance, baicalein reference substance and wogonin reference substance respectively to obtain reference substance solutions; Pre-treating the scutellaria baicalensis reference medicinal material to obtain a reference medicinal material solution; S2) The test solution, reference solution and reference medicinal material solution are subjected to thin layer chromatography, and the thin layer plate is silica gel GF 254 Thin plate, the developing agent is chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid, and the volume ratio of chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid is (9.5-10.5): (0.5-1.5): (1.0-2.0): (0.5-1.5); S3) Check under ultraviolet light. If there is a spot at the Rf value of 0.313±10% in the chromatogram of the test sample, it is the Scutellaria baicalensis formula granules. If there is no spot at the Rf value of 0.313±10%, it is the Scutellaria baicalensis charcoal formula granules.

2. The thin layer identification method according to claim 1, characterized in that: The ratio of the sample to be tested, the n-butanol-ethyl acetate and the methanol is 1 g: (20-40) ml: (3-8) ml, the volume ratio of the n-butanol and the ethyl acetate is (1.5-2.5):1, and the ultrasonic time is 20-40 min.

3. The thin layer identification method according to claim 1, characterized in that: The preparation of the reference solution is specifically as follows: Adding baicalin reference substance, baicalein reference substance and wogonin reference substance into methanol respectively to obtain reference substance solutions; The concentration of the baicalin reference substance in the reference solution is (0.3-0.6) mg / ml, the concentration of the baicalein reference substance in the reference solution is (0.3-0.6) mg / ml, and the concentration of the wogonin reference substance in the reference solution is (0.3-0.6) mg / ml.

4. The thin layer identification method according to claim 1, characterized in that: The preparation of the control medicinal material solution is specifically as follows: The reference medicinal material Scutellaria baicalensis was boiled in water, the filtrate obtained by filtration was evaporated to dryness, and then dissolved in water, and then extracted with n-butanol-ethyl acetate. The residue obtained after evaporation was added with methanol, and the mixture was sonicated and filtered to obtain a reference medicinal material solution; The ratio of the scutellaria baicalensis reference medicinal material, boiling water and dissolving water is 2g: (50-100)ml: (5-15)ml.

5. The thin layer identification method according to claim 1, characterized in that: The sample volume for thin layer chromatography detection is 3-5 μl.

6. The thin layer identification method according to claim 1, characterized in that: The wavelength of the ultraviolet lamp is 254nm.

7. The thin layer identification method according to claim 1, characterized in that: During the thin layer chromatography detection process, the development temperature is 4-35°C.

8. The thin layer identification method according to claim 1, characterized in that: During the thin layer chromatography detection process, the development humidity is 32% rh~75% rh.

9. The thin layer identification method according to claim 1, characterized in that: The Rf value is 0.28~0.34.

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