Thin-layer chromatography for scutellaria baicalensis georgi formula granules and carbonized scutellaria root formula granules

Abstract

Provided in the present invention is a thin-layer chromatography for scutellaria baicalensis georgi formula granules and carbonized scutellaria root formula granules, comprising the following steps: S1) pretreating a sample under test, so as to obtain a solution of the sample under test, said sample being scutellaria baicalensis georgi formula granules and carbonized scutellaria root formula granules; separately pretreating reference standards, so as to obtain reference standard solutions; and pretreating a scutellaria baicalensis georgi reference medicinal material so as to obtain a reference medicinal material solution; S2) carrying out a thin-layer chromatography test on the solution of the sample under test, the reference standard solutions and the reference medicinal material solution, a thin-layer plate being a silica gel GF254 thin plate, and a developing agent being chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid; and S3) carrying out inspection and identification under an ultraviolet light. By observing whether there are spots, which are used as identification points, in thin-layer chromatograms within specified retention factor value ranges, the thin-layer chromatography for scutellaria baicalensis georgi formula granules and carbonized scutellaria root formula granules established in the present invention is used for rapidly and effectively identifying scutellaria baicalensis georgi and carbonized scutellaria root formula granules, so as to ensure the accuracy of clinical medication.

Description

A thin-layer chromatography method for identifying Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules.

[0001] This application claims priority to Chinese Patent Application No. 202410500382.8, filed on April 24, 2024, entitled “A Thin-Layer Chromatography Identification Method for Scutellaria baicalensis Formula Granules and Scutellaria baicalensis Charcoal Formula Granules”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of analytical testing technology, and in particular to a thin-layer chromatography method for identifying Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules. Background Technology

[0003] Scutellaria baicalensis Georgi, a plant in the Lamiaceae family, is the dried root of a herb. It possesses properties of clearing heat and drying dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus. It is used for damp-heat syndrome, summer-heat syndrome, chest tightness and nausea, damp-heat fullness, diarrhea, jaundice, cough due to lung heat, high fever and thirst, hematemesis due to blood heat, carbuncles and boils, and threatened abortion. Raw Scutellaria baicalensis is better at clearing damp-heat, while charred Scutellaria baicalensis is better at stopping bleeding; the significant change in efficacy indicates a change in their chemical composition. To ensure accurate clinical use, it is necessary to establish a visual identification method for qualitative differentiation between the two.

[0004] Current qualitative research on Scutellaria baicalensis and charred Scutellaria baicalensis focuses on changes in appearance and chemical composition of different processed products. For example, Zhou Desheng's research shows that the microscopic characteristics and extract content of Scutellaria baicalensis undergo significant changes after processing, which can be used for the identification of different processed medicinal slices. Since charred Scutellaria baicalensis granules are made from charred Scutellaria baicalensis slices through a series of processes, some of which destroy the original morphological and microscopic characteristics, morphological and microscopic identification methods are not applicable to the identification of extracts and preparations. Huang Qi's research shows that the composition and proportion of volatile components change significantly after charring. However, the solvent used in the preparation of charred Scutellaria baicalensis granules is water, which cannot extract its volatile components. Therefore, volatile component analysis is not applicable to the identification of Scutellaria baicalensis and charred Scutellaria baicalensis granules. The component content of Chinese medicinal materials is affected by factors such as origin, growth years, harvesting time, and storage; therefore, the amount of component content is also not applicable to the qualitative identification of Scutellaria baicalensis and charred Scutellaria baicalensis granules.

[0005] In summary, the aforementioned methods for morphological identification, microscopic identification, and component quantification are not applicable to the qualitative identification of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules. Currently, there is no effective method to distinguish between Scutellaria baicalensis and Scutellaria baicalensis charcoal in formulations. Therefore, a method is needed to differentiate between Scutellaria baicalensis, Scutellaria baicalensis charcoal extract, and granules. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a thin-layer chromatography method for identifying Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, which has the advantages of strong specificity, good stability and high precision.

[0007] In view of this, this application provides a thin-layer chromatography method for identifying Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules, comprising the following steps:

[0008] S1) The sample to be tested is pretreated to obtain a test solution; the sample to be tested is Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules;

[0009] Baicalin reference standard, baicalein reference standard and wogonin reference standard were pretreated to obtain reference standard solutions;

[0010] The Scutellaria baicalensis reference material was pretreated to obtain a reference material solution;

[0011] S2) The test solution, reference solution, and reference medicinal material solution are subjected to thin-layer chromatography (TLC) detection. The TLC plate is made of silica gel GF. 254 Thin plate, the developing solvent is chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid;

[0012] S3) Examine under ultraviolet light. If there are spots in the chromatogram of the test sample at an Rf value of 0.313±10%, it is a Scutellaria baicalensis formula granule. If there are no spots at an Rf value of 0.313±10%, it is a Scutellaria baicalensis charcoal formula granule.

[0013] Preferably, the preparation of the test solution specifically involves:

[0014] 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;

[0015] The ratio of the sample to be tested, the n-butanol-ethyl acetate and the methanol is 1g:(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 20-40min.

[0016] Preferably, the preparation of the reference solution specifically involves:

[0017] Baicalin reference standard, baicalein reference standard and wogonin reference standard were added to methanol respectively to obtain reference standard solutions;

[0018] The concentration of baicalin reference standard in the reference solution is (0.3-0.6) mg / ml, the concentration of baicalein reference standard in the reference solution is (0.3-0.6) mg / ml, and the concentration of wogonin reference standard in the reference solution is (0.3-0.6) mg / ml.

[0019] Preferably, the preparation of the reference medicinal material solution specifically involves:

[0020] Boil the Scutellaria baicalensis reference material in water, filter the filtrate, evaporate to dryness, dissolve in water, extract with n-butanol-ethyl acetate, evaporate to dryness, add methanol to the residue, sonicate and filter to obtain the reference material solution.

[0021] The ratio of the Scutellaria baicalensis reference material, boiling water, and dissolving water was 2g:(50-100)ml:(5-15)ml.

[0022] Preferably, the sample spotting volume for the thin-layer chromatography detection is 3–5 μl.

[0023] 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).

[0024] Preferably, the ultraviolet light wavelength is 254nm.

[0025] Preferably, the developing temperature during the thin-layer chromatography detection process is 4–35°C.

[0026] Preferably, during the thin-layer chromatography detection process, the developing humidity is 32% RH to 75% RH.

[0027] Preferably, the Rf value is 0.28 to 0.34.

[0028] This application provides a thin-layer chromatography (TLC) identification method for Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules. First, a test solution, a reference solution, and a reference medicinal material solution are prepared. Then, the test solution, reference solution, and reference medicinal material solution are subjected to TLC detection under ultraviolet light. If a spot is present in the chromatogram of the test solution at an Rf value of 0.313±10%, it is a Scutellaria baicalensis formula granule; if no spot is present at an Rf value of 0.313±10%, it is a Scutellaria baicalensis charcoal formula granule. In the thin-layer chromatography (TLC) identification method for Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, the introduction of TLC detection and selection of the developing solvent gives this TLC identification method advantages such as high specificity, good stability, high precision, convenience, and ease of use. Furthermore, the TLC identification method provided in this application, by comparing the TLC chromatograms of Scutellaria baicalensis charcoal formula granules with those of Scutellaria baicalensis reference material and Scutellaria baicalensis granules, shows that the ratio shift values ​​at the same positions (0.28–0.34) do not show spots, which can be used as identification points for Scutellaria baicalensis charcoal formula granules. Therefore, the TLC identification method provided in this application can quickly and effectively identify Scutellaria baicalensis charcoal formula granules that have lost their processed form. It is simple to operate, has high precision and sensitivity, good stability, and has good application prospects, ensuring accurate medication. Attached Figure Description

[0029] Figure 1 is a thin-layer chromatography pattern of the identification of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules by methods 1, 2 and 3 of the present invention;

[0030] Figure 2 shows the thin-layer chromatography (TLC) spectra of the identification of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules using developing solvent 1.

[0031] Figure 3 shows the thin-layer chromatography (TLC) spectra of the identification of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules by developing solvent 2.

[0032] Figure 4 shows the thin-layer chromatography (TLC) spectra of the identification of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules with developing solvent 3.

[0033] Figure 5 shows the thin-layer chromatography (TLC) spectra of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granule solution at different spotting amounts.

[0034] Figure 6 shows the thin-layer chromatography (TLC) spectra of the negative control solution, the reference solution, the Scutellaria baicalensis reference herb solution, the Scutellaria baicalensis formula granule solution, and the Scutellaria baicalensis charcoal formula granule solution.

[0035] Figure 7 shows the thin-layer chromatography pattern of the Tianjin Silida chromatography plate;

[0036] Figure 8 shows the thin-layer chromatography pattern of a Merck chromatography plate;

[0037] Figure 9 shows the thin-layer chromatography pattern of the Qingdao marine chromatography plate;

[0038] Figure 10 shows the thin-layer chromatography (TLC) spectra of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granules at 4℃.

[0039] Figure 11 shows the thin-layer chromatography (TLC) spectra of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granules at 35℃.

[0040] Figure 12 shows the thin-layer chromatography (TLC) spectra of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granules at 32% RH.

[0041] Figure 13 shows the thin-layer chromatography (TLC) spectra of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granules at 75% RH.

[0042] Figure 14 shows the thin-layer chromatography (TLC) images of different batches of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules.

[0043] Figure 15 shows the thin-layer chromatography (TLC) identification spectra of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules from Comparative Example 1. Detailed Implementation

[0044] 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, and not for limiting the scope of the claims of the present invention.

[0045] In view of the existing technology's need for qualitative differentiation between Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, this application establishes a thin-layer chromatography (TLC) identification method for Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules. This method uses the presence of spots on the TLC chromatogram within a specified ratio shift range as identification points, enabling rapid and effective identification of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules to ensure the accuracy of clinical medication. Specifically, this invention discloses a TLC identification method for Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, including the following steps:

[0046] S1) The sample to be tested is pretreated to obtain a test solution; the sample to be tested is Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules;

[0047] Baicalin reference standard, baicalein reference standard and wogonin reference standard were pretreated to obtain reference standard solutions;

[0048] The Scutellaria baicalensis reference material was pretreated to obtain a reference material solution;

[0049] S2) The test solution, reference solution, and reference medicinal material solution are subjected to thin-layer chromatography (TLC) detection. The TLC plate is made of silica gel GF. 254 Thin-layer plates, with chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid as the developing solvent;

[0050] S3) Examine under ultraviolet light. If there are spots in the chromatogram of the test sample at an Rf value of 0.313±10%, it is a Scutellaria baicalensis formula granule. If there are no spots at an Rf value of 0.313±10%, it is a Scutellaria baicalensis charcoal formula granule.

[0051] In the thin-layer chromatography identification method for Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules, step S1) involves preparing a test solution, a reference solution, and a reference herb solution, respectively. The preparation of the test solution specifically involves:

[0052] Dissolve the sample in water, then extract with n-butanol-ethyl acetate 2-4 times. Combine the n-butanol-ethyl acetate solutions, evaporate to dryness, add methanol to the residue, sonicate, and filter to obtain the test solution.

[0053] In the preparation of the test solutions described above, since the test samples are Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules, it is necessary to prepare test solutions for Scutellaria baicalensis charcoal granules and Scutellaria baicalensis granules. Both test solutions are prepared according to the above method: dissolve the test sample Scutellaria baicalensis granules in water, then extract with n-butanol-ethyl acetate 2-4 times, combine the n-butanol-ethyl acetate solutions, evaporate to dryness, add methanol to the residue, sonicate and filter to obtain test solution 1; and dissolve the test sample Scutellaria baicalensis charcoal granules in water, then extract with n-butanol-ethyl acetate 2-4 times, combine the n-butanol-ethyl acetate solutions, evaporate to dryness, add methanol to the residue, sonicate and filter to obtain test solution 2.

[0054] In the above process, the ratio of the sample to be tested, the n-butanol-ethyl acetate mixture, and the methanol is 1g:(20-40)ml:(3-8)ml, the volume ratio of n-butanol to ethyl acetate is (1.5-2.5):1, and the sonication time is 20-40min. The n-butanol-ethyl acetate extraction is performed three times, 10ml each time; specifically, the ratio of the sample to be tested, the n-butanol-ethyl acetate mixture, and the methanol is 1g:30ml:5ml, and the sonication time is 25-35min.

[0055] The preparation of the reference solution is as follows:

[0056] Baicalin reference standard, baicalein reference standard and wogonin reference standard were added to methanol to obtain reference standard solution;

[0057] The concentration of baicalin reference standard in the reference solution is (0.3–0.6) mg / ml, the concentration of baicalein reference standard in the reference solution is (0.3–0.6) mg / ml, and the concentration of wogonin reference standard in the reference solution is (0.3–0.6) mg / ml; specifically, the concentration of baicalin reference standard in the reference solution is (0.4–0.5) mg / ml, the concentration of baicalein reference standard in the reference solution is (0.4–0.5) mg / ml, and the concentration of wogonin reference standard in the reference solution is (0.4–0.5) mg / ml.

[0058] The preparation of the reference medicinal material solution is as follows:

[0059] Boil the Scutellaria baicalensis reference material in water, filter the filtrate, evaporate to dryness, dissolve in water, extract with n-butanol-ethyl acetate, evaporate to dryness, add methanol to the residue, sonicate and filter to obtain the reference material solution.

[0060] The ratio of the Scutellaria baicalensis reference material, boiling water, dissolving water, n-butanol-ethyl acetate, and methanol is 2g:(50-100)ml:(5-15)ml:(20-40)ml:(3-8)ml; the volume ratio of n-butanol to ethyl acetate in the n-butanol-ethyl acetate mixture is 2:1, and the extraction is performed in three separate extractions, with the n-butanol-ethyl acetate extracts being combined at the end; specifically, the ratio of the Scutellaria baicalensis reference material, boiling water, dissolving water, n-butanol-ethyl acetate, and methanol is 2g:50ml:10ml:30ml:5ml.

[0061] In step S2), the above-mentioned test solution, reference solution, and reference medicinal material solution are subjected to thin-layer chromatography detection; in this process, the thin-layer plate is silica gel GF. 254 In a specific embodiment, the thin plate, the silicone GF 254 Thin plates can be made from materials that can be cut by Tianjin Slida Technology Co., Ltd., Merck chromatography plates, or pre-fabricated silica gel GF plates from Qingdao Ocean Chemical Plant Branch. 254 The results showed that the method was effective for the above-mentioned silica gel GF. 254 The thin plates exhibit good durability and all meet the identification requirements. Spots were observed in the Scutellaria baicalensis formulation particles at P0 values ​​of 0.313, 0.337, and 0.314, while no spots were observed in the Scutellaria baicalensis charcoal formulation particles. 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 developing temperature is 4–35℃. The method of this invention has good adaptability to different temperatures. Spots are observed in the Scutellaria baicalensis formulation particles at P0.301 and 0.306, while no spots are observed in the Scutellaria baicalensis charcoal formulation particles, thus achieving the identification of the two formulations. The developing humidity is 32% RH–75% RH. The method of this invention has good adaptability to different humidity levels. Spots are observed in the Scutellaria baicalensis formulation particles at P0.311 and 0.325, while no spots are observed in the Scutellaria baicalensis charcoal formulation particles, thus achieving the identification of the two formulations. The sample volume for the thin-layer chromatography detection is 3–5 μl, specifically 3 μl, 4 μl, or 5 μl.

[0062] The inspection is performed under a UV lamp with a wavelength of 254 nm. If there are spots at an Rf value of 0.313±10%, the product is a Scutellaria baicalensis formula granule. If there are no spots at an Rf value of 0.313±10%, the product is a Scutellaria baicalensis charcoal formula granule.

[0063] This application provides a thin-layer chromatography method for identifying Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules, comprising the following steps: S1) Pre-treating the 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 baicalin reference standard, baicalein reference standard and wogonin reference standard respectively to obtain reference solution; pre-treating Scutellaria baicalensis reference medicinal material to obtain reference medicinal material solution; S2) Performing thin-layer chromatography detection on the test solution, reference solution and reference medicinal material solution, wherein the thin-layer plate is silica gel GF. 254 Thin-layer chromatography (TLC) is used, with the developing solvent being chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid. S3) The sample is examined under UV light. If a spot is present in the chromatogram of the test sample at an Rf value of 0.313±10%, it indicates Scutellaria baicalensis formulation granules; if no spot is present at an Rf value of 0.313±10%, it indicates Scutellaria baicalensis charcoal formulation granules. In this TLC identification method for Scutellaria baicalensis formulation granules and Scutellaria baicalensis charcoal formulation granules, the introduction of TLC detection and selection of the developing solvent gives this method advantages such as high specificity, good stability, high precision, convenience, and ease of use.

[0064] The thin-layer chromatography identification method provided in this application compares the thin-layer chromatography of the test sample with that of the reference herb Scutellaria baicalensis. If the spots are consistent with those of the reference herb, the sample is Scutellaria baicalensis granules. If there are no spots at the same position as the reference herb at a ratio shift value of 0.28 to 0.34, the sample is Scutellaria baicalensis charcoal granules.

[0065] To further understand the present invention, the thin-layer chromatography identification method for Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules provided by the present invention will be described in detail below with reference to the embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0066] The instruments and reagents used in the following examples are shown below:

[0067] 1.1 Instruments

[0068] Heating plate, mortar and pestle, thin-layer imaging system: CAMAG TLC Visualizer, silicone GF 254 Thin-layer plates (Qingdao Ocean Chemical Plant, batch number: 20180527; Tianjin Slida Technology Co., Ltd., batch number: 191016; Merck, batch number: HX87183353);

[0069] 1.2 Reagents

[0070] 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;

[0071] 1.3 Drug Test

[0072] Baicalein reference standard (National Institutes for Food and Drug Control, batch number: 111595-201808, purity: 97.9%); Baicalin reference standard (National Institutes for Food and Drug Control, batch number: 110715-202223, purity: 97.2%); Baicalein reference standard (National Institutes for Food and Drug Control, batch number: 111514-202207, purity: 100%); Scutellaria baicalensis reference standard. Medicinal herbs (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: ycwkq22101407); Scutellaria baicalensis formula granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: 2210005, 2212001, 2302008); Scutellaria baicalensis charcoal formula granules (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., batch numbers: B2308010, B2308011, B2308012).

[0073] Example 1

[0074] Based on the different extraction methods, this application first determines the extraction method, and the specific process is as follows:

[0075] 1.1 Examination of Extraction Methods

[0076] 1. Take 1g each of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, and perform the following operations respectively: grind finely, add 10ml of methanol for ultrasonic extraction for 30 minutes, filter, evaporate the filtrate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test solution.

[0077] Take another 2g of Scutellaria baicalensis reference material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of methanol to the residue and extract by ultrasonication for 30 minutes, filter, evaporate the filtrate to dryness, add 5ml of methanol to the residue to dissolve, and prepare the reference material solution.

[0078] Separately prepare a mixed solution containing 0.5 mg of each of baicalin, baicalein, and wogonin reference standards in methanol, and use it as the reference solution.

[0079] 2. Take 1g each of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, and perform the following operations respectively: grind finely, add 10ml of methanol and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5ml of methanol to dissolve the residue, and use it as the test solution.

[0080] Take another 2g of Scutellaria baicalensis reference material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of methanol to the residue and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 5ml of methanol to the residue to dissolve, and prepare the reference material solution.

[0081] Separately prepare reference solutions containing 0.5 mg per ml of baicalin, baicalein, and wogonin in methanol;

[0082] 3. Take 1g each of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, and perform the following operations respectively: grind finely, add 10ml of water to dissolve, 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.

[0083] Take another 2g of Scutellaria baicalensis reference material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to dissolve the residue, add n-butanol-ethyl acetate (2:1) and shake to extract 3 times, 10ml each time, combine the n-butanol-ethyl acetate extracts, evaporate to dryness, add 5ml of methanol to the residue, sonicate for 30 minutes, filter, and prepare the reference material solution;

[0084] Separately prepare reference solutions containing 0.5 mg per ml of baicalin, baicalein, and wogonin in methanol;

[0085] Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 3 μl of the test solution, 3 μl of the reference solution, and 5 μl of the reference medicinal material solution to the same silica gel GF plate. 254 On thin-layer chromatography plates, a mixture of chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid (10:1:1.5:1) was used as the developing solvent. The plates were then developed, removed, dried, and examined under ultraviolet light (254 nm). The results are shown in Figure 1. Figure 1 shows the thin-layer chromatography chromatograms of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules identified by methods 1, 2, and 3. In Figure 1, 1-4 are the thin-layer chromatography chromatograms of the reference standards of method 1 (from bottom to top: baicalin, baicalein, wogonin), the reference medicinal material of method 1, the Scutellaria baicalensis granules of method 1, and the Scutellaria baicalensis charcoal granules of method 1, respectively; 5-8 are the thin-layer chromatography chromatograms of the reference standards of method 2 (from bottom to top: baicalin, baicalein, wogonin), the reference medicinal material of method 2, the Scutellaria baicalensis granules of method 2, and the Scutellaria baicalensis charcoal granules of method 2, respectively; 9-12 are the thin-layer chromatography chromatograms of the reference standards of method 3, respectively. Thin-layer chromatography (TLC) spectra of the following samples (from bottom to top: baicalin, baicalein, wogonin), the reference medicinal material of Method 3, the Scutellaria baicalensis formula granules of Method 3, and the Scutellaria baicalensis charcoal formula granules of Method 3; the results showed that the samples prepared by Method 1 and Method 2 could not effectively distinguish between Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules. The preparation method of the sample was determined as follows: take 1g of the sample, grind it into a fine 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 extracts, evaporate to dryness, add 5ml of methanol to the residue, sonicate for 30 minutes, filter, and use as the sample solution.

[0086] 1.2 Investigation of the developing solvent

[0087] Take 1g each of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, and perform the following operations respectively: grind finely, add 10ml of water to dissolve, 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.

[0088] Take another 2g of Scutellaria baicalensis reference material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to dissolve the residue, and prepare the reference material solution in the same way as "Preparation of Test Solution";

[0089] Separately prepare reference solutions containing 0.5 mg per ml of baicalin, baicalein, and wogonin in methanol;

[0090] Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 3 μl of the test solution, 3 μl of the reference solution, and 5 μl of the reference medicinal material solution to the same silica gel GF plate. 254 On the thin-layer plates, toluene-ethyl acetate-methanol-formic acid (10:3:1:2) (developing solvent 1), toluene-ethyl acetate-methanol-formic acid (10:1:1.5:1) (developing solvent 2), and chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid (10:1:1.5:1) (developing solvent 3) were used as developing solvents. After development, the plates were removed, dried, and examined under a UV lamp (254nm). The results are shown in Figures 2-4. Figure 2 shows the thin-layer chromatography (TLC) spectra of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules identified by developing solvent 1; Figure 3 shows the TLC spectra of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules identified by developing solvent 2; and Figure 4 shows the TLC spectra of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules identified by developing solvent 3. In Figure 1, the TLC spectra of the reference standards (from bottom to top: baicalin, baicalein, and wogonin) are shown, and Figure 2 shows the TLC spectra of the Scutellaria baicalensis reference material. Figure 3 shows the thin-layer chromatography (TLC) spectrum of Scutellaria baicalensis granules, and Figure 4 shows the TLC spectrum of Scutellaria baicalensis charcoal granules. The results show that when using the developing solvent chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid (10:1:1.5:1), the spots are clear. The Scutellaria baicalensis granules sample has obvious spots at Rf=0.333, while the Scutellaria baicalensis charcoal granules sample does not have such spots. This method can achieve qualitative identification of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules.

[0091] Example 2

[0092] Thin-layer chromatography (TLC) identification of Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules was performed according to the TLC method determined in Example 1, as follows:

[0093] 2.1 Preparation of the test solution

[0094] Take 1g each of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules, and perform the following operations respectively: grind finely, add 10ml of water to dissolve, 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.

[0095] 2.2 Preparation of control herbal solution

[0096] Take 2g of Scutellaria baicalensis reference material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to dissolve the residue, 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 prepare the reference material solution;

[0097] 2.3 Preparation of reference solution

[0098] Prepare a mixed solution containing 0.5 mg of each of baicalin, baicalein, and wogonin reference standards in methanol, and use it as the reference solution.

[0099] 2.4 Determination Method

[0100] Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 3 μl of the test solution, 3 μl of the reference solution, and 5 μl of the reference medicinal material solution to the same silica gel GF plate. 254 On the thin-layer plate, chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid (10:1:1.5:1) was used as the developing solvent. The plate was then removed, dried, and examined under a UV lamp (254nm).

[0101] 2.5 Methodological Examination

[0102] 2.5.1 Sampling quantity investigation

[0103] Under the proposed experimental conditions, 1 μl, 3 μl, and 5 μl of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granule solution were spotted on the same silica gel GF. 254As shown in Figure 5, the thin-layer chromatography (TLC) spectra of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis granule solution, and Scutellaria baicalensis charcoal granule solution at different spotting volumes are as follows: 1–3 are TLC spectra of the reference solution at spotting volumes of 1, 3, and 5 μl (from bottom to top: baicalin, baicalein, and wogonin); 4–6 are TLC spectra of the Scutellaria baicalensis reference herb solution at 1, 3, and 5 μl; and 7–9 are TLC spectra of the Scutellaria baicalensis charcoal granule solution at 1, 3, and 5 μl. Figures 10-12 show the thin-layer chromatography (TLC) spectra of 1, 3, and 5 μl of Scutellaria baicalensis granule solution. As can be seen from the figures, when 3-5 μl of the reference solution, reference herb solution, and test solution were spotted, the spots were clearly visible and well-separated, confirming the spotting volume as 3-5 μl. Furthermore, at a ratio shift value of 0.304, Scutellaria baicalensis granules showed spots, while charred Scutellaria baicalensis granules did not, thus distinguishing between the two. Therefore, whether or not a spot is observed at a ratio shift value of 0.304 can be used as the identification point.

[0104] 2.5.2 Specificity Examination

[0105] Following the above preparation methods for the test samples, negative chromatograms, reference solutions, Scutellaria baicalensis reference material solutions, Scutellaria baicalensis formula granule solutions, Scutellaria baicalensis formula granule solutions, and Scutellaria baicalensis charcoal formula granule solutions were prepared respectively. Thin-layer chromatography (TLC) identification experiments were performed, and the results are shown in Figure 6. Figure 6 shows the TLC chromatograms of the negative chromatogram, reference solution, Scutellaria baicalensis reference material solution, Scutellaria baicalensis formula granule solutions, and Scutellaria baicalensis charcoal formula granule solutions. In Figure 6, 1 is the TLC chromatogram of the negative chromatogram, and 2 is the TLC chromatogram of the reference solution. Thin-layer chromatography (TLC) spectra of the solutions (from bottom to top: baicalin, baicalein, wogonin), 3 is the TLC spectra of the Scutellaria baicalensis reference material solution, 4 is the TLC spectra of the Scutellaria baicalensis granule formulation solution, and 5 is the TLC spectra of the Scutellaria baicalensis charcoal granule formulation solution. As can be seen from the figures, the negative sample did not interfere with the Scutellaria baicalensis and Scutellaria baicalensis charcoal granule test samples. The method has good specificity. Furthermore, at a ratio shift value of 0.296, the Scutellaria baicalensis granule formulation showed spots, while the Scutellaria baicalensis charcoal granule formulation did not show spots.

[0106] 2.5.3 Durability Assessment

[0107] 2.5.3.1 Comparison of different thin-layer plates

[0108] The selected materials include cuttable thin-layer chromatography plates from Tianjin Slida Technology Co., Ltd., and pre-fabricated silica gel GF from Merck's Qingdao Marine Chemical Plant branch. 254The plates were tested according to the planned experimental methods, as shown in Figures 7-9. Figure 7 shows the thin-layer chromatography (TLC) spectrum of the Tianjin Silida plate, Figure 8 shows the TLC spectrum of the Merck plate, and Figure 9 shows the TLC spectrum of the Qingdao Marine plate. In these figures, 1 is the TLC spectrum of the reference solution (from bottom to top: baicalin, baicalein, wogonin), 2 is the TLC spectrum of the Scutellaria baicalensis reference herb solution, and 3 is the TLC spectrum of the Scutellaria baicalensis formula granules. The thin-layer chromatography (TLC) spectra of the granule solution are shown in Figures 7-9. As can be seen from Figures 7-9, the spot shift values ​​(Rf) at 8-10 cm from the origin in the three TLC plates are 0.313, 0.337, and 0.314, respectively, which can meet the identification requirements. Moreover, the granules of the scutellaria baicalensis formula appear as spots at Rf values ​​of 0.313, 0.337, and 0.314, while the granules of the scutellaria baicalensis charcoal formula do not have spots.

[0109] 2.5.3.2 Comparison of different temperatures

[0110] Thin-layer chromatography (TLC) plates after sampling were developed at low temperatures of 4℃ and high temperatures of 35℃, as shown in Figures 10 and 11. Figure 10 shows the TLC patterns of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granules at 4℃. Figure 11 shows the TLC patterns of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granules at 35℃. In these figures, 1 represents the reference solution... Figures 10 and 11 show the thin-layer chromatography (TLC) spectra of the solutions (from bottom to top: baicalin, baicalein, and wogonin), Figure 2 shows the TLC spectra of the Scutellaria baicalensis control herb solution, Figure 3 shows the TLC spectra of the Scutellaria baicalensis formula granule solution, and Figure 4 shows the TLC spectra of the Scutellaria baicalensis charcoal formula granule solution. As can be seen from Figures 10 and 11, this method has good adaptability to different temperatures. Furthermore, at Pt values ​​of 0.301 and 0.306, the Scutellaria baicalensis formula granules show spots, while the Scutellaria baicalensis charcoal formula granules do not show spots.

[0111] 2.5.3.3 Comparison of different humidity levels

[0112] Thin-layer chromatography (TLC) plates after sampling were developed at 32% RH and 75% RH, as shown in Figures 12 and 13. Figure 12 shows the TLC patterns of the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granules at 32% RH. Figure 13 shows the TLC patterns of the same solutions at 75% RH. Wherein, 1 represents... Thin-layer chromatography (TLC) spectra of the reference solutions (from bottom to top: baicalin, baicalein, and wogonin), 2 is the TLC spectra of the Scutellaria baicalensis reference herb solution, 3 is the TLC spectra of the Scutellaria baicalensis formula granule solution, and 4 is the TLC spectra of the Scutellaria baicalensis charcoal formula granule solution. As can be seen from Figures 12 and 13, this method has good adaptability to different humidity levels. Furthermore, at Pt values ​​of 0.311 and 0.325, the Scutellaria baicalensis formula granules show spots, while the Scutellaria baicalensis charcoal formula granules do not show spots.

[0113] 2.5.4 Verification

[0114] Take the reference solution, Scutellaria baicalensis reference herb solution, Scutellaria baicalensis formula granule solution, and Scutellaria baicalensis charcoal formula granule solution, and spot them separately on the same silica gel GF. 254 The thin-layer chromatography (TLC) plates were analyzed according to the established method. The experimental results are shown in Figure 14. Figure 14 shows the TLC spectra of different batches of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules. Among them, 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, 3-5 are the TLC spectra of different batches of Scutellaria baicalensis formula granule solutions, and 6-8 are the TLC spectra of different batches of Scutellaria baicalensis charcoal formula granule solutions. As can be seen from the figure, Scutellaria baicalensis formula granules have spots at a ratio shift (Rf) of 0.318, while Scutellaria baicalensis charcoal formula granules do not have such spots. These spots can be used to distinguish between the two.

[0115] 2.6 Determination of the identification point Rf for Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules

[0116] The data from the thin-layer chromatography methodology investigation are summarized in Table 1.

[0117] Table 1. Summary of the shift data for the different methodologies mentioned above.

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

[0119] Comparative Example 1

[0120] Take 1g of the powder, add 30ml of a mixed solution of ethyl acetate and methanol (3:1), heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 5ml of methanol, and take the supernatant as the test solution; take 1g of Scutellaria baicalensis reference material and prepare a reference material solution in the same way; take baicalin reference standard, baicalein reference standard, and wogonin reference standard, and prepare solutions containing 1mg, 0.5mg, and 0.5mg per ml respectively in methanol as reference solutions; perform thin-layer chromatography (General Rule 0502), take 2μl of each of the above test solution and reference material solution and 1μl of each of the above three reference solutions, and spot them separately 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, remove, air dry, and examine under ultraviolet light (365nm). The results are shown in Figure 15. Figure 15 shows the thin-layer chromatography (TLC) spectra of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules in Comparative Example 1. 1 is the TLC spectrum of baicalin reference solution, 2 is the TLC spectrum of baicalein, 3 is the TLC spectrum of baicalein, 4 is the TLC spectrum of Scutellaria baicalensis reference medicinal material solution, 5 is the TLC spectrum of Scutellaria baicalensis formula granule solution, and 6 is the TLC spectrum of Scutellaria baicalensis charcoal formula granule solution. As can be seen from the figure, using the above TLC identification method, there are no obvious differences in spots between Scutellaria baicalensis and Scutellaria baicalensis charcoal formula granules, making it impossible to distinguish between the two. Furthermore, because a large amount of formic acid is used in the developing solvent, it easily corrodes the polyamide TLC plate, making it unsuitable for the identification of Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules.

[0121] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0122] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A thin-layer chromatography method for identifying Scutellaria baicalensis granules and Scutellaria baicalensis charcoal granules, comprising the following steps: S1) Pre-treat the sample to be tested to obtain the test solution; The samples to be tested were Scutellaria baicalensis formula granules and Scutellaria baicalensis charcoal formula granules; Baicalin reference standard, baicalein reference standard and wogonin reference standard were pretreated to obtain reference standard solutions; The Scutellaria baicalensis reference material was pretreated to obtain a reference material solution; S2) The test solution, reference solution, and reference medicinal material solution are subjected to thin-layer chromatography (TLC) detection. The TLC plate is made of silica gel GF. 254 Thin plate, the developing solvent is chloroform-butyl acetate-anhydrous ethanol-glacial acetic acid; S3) Examine under ultraviolet light. If there are spots in the chromatogram of the test sample at an Rf value of 0.313±10%, it is a Scutellaria baicalensis formula granule. If there are no spots at an Rf value of 0.313±10%, it is a Scutellaria baicalensis charcoal formula granule.

2. The thin-layer identification method according to claim 1, characterized in that, The preparation of the test solution is specifically as follows: 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 ratio of the sample to be tested, the n-butanol-ethyl acetate and the methanol is 1g:(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 20-40min.

3. The thin-layer identification method according to claim 1, characterized in that, The preparation of the reference solution is as follows: Baicalin reference standard, baicalein reference standard and wogonin reference standard were added to methanol respectively to obtain reference standard solutions; The concentration of baicalin reference standard in the reference solution is (0.3-0.6) mg / ml, the concentration of baicalein reference standard in the reference solution is (0.3-0.6) mg / ml, and the concentration of wogonin reference standard 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 reference medicinal material solution is specifically as follows: Boil the Scutellaria baicalensis reference material in water, filter the filtrate, evaporate to dryness, dissolve in water, extract with n-butanol-ethyl acetate, evaporate to dryness, add methanol to the residue, sonicate and filter to obtain the reference material solution. The ratio of the Scutellaria baicalensis reference material, boiling water, and dissolving water was 2g:(50-100)ml:(5-15)ml.

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

6. The thin-layer identification method according to claim 1, characterized in that, 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).

7. The thin-layer identification method according to claim 1, characterized in that, The ultraviolet light wavelength is 254 nm.

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

9. The thin-layer identification method according to claim 1, characterized in that, During the thin-layer chromatography detection process, the developing humidity is 32% RH to 75% RH.

10. The thin-layer identification method according to claim 1, characterized in that, The Rf value is 0.28 to 0.34.

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