A thin-layer identification method for Cyrtomium fortunei formula granules and carbonized Cyrtomium fortunei formula granules

Abstract

The present invention provides a thin-layer identification method for Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules, which includes: taking a sample to be tested, dissolving it with a solvent to obtain a test solution; the sample to be tested is Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules; taking Dryopteris crassirhizoma control medicinal material, dissolving it with a solvent to obtain a control medicinal material solution; taking protocatechuic acid, dissolving it with a solvent to obtain a reference substance solution; performing thin-layer chromatography detection on the test solution, the reference substance solution and the control medicinal material solution, and the thin-layer plate is silica gel GF 254 thin-layer plate; the developing agent is toluene-ethyl acetate-formic acid; visual inspection is carried out under an ultraviolet lamp. In the chromatogram of the test sample, if there are spots at an Rf value of 0.83-1.00, it is carbonized Dryopteris crassirhizoma formula granules; if there are no spots at an Rf value of 0.83-1.00, it is Dryopteris crassirhizoma formula granules. The present invention takes whether there are spots within the specified retention factor value range in the thin-layer chromatogram as the identification point to quickly and effectively identify carbonized Dryopteris crassirhizoma formula granules and Dryopteris crassirhizoma formula granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of identification, and particularly relates to a thin-layer identification method for Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules. Background Art

[0002] Dryopteris crassirhizoma is a commonly used traditional Chinese medicine in clinical practice. It was first recorded in "Shennong Ben Cao Jing" and has been recorded in all previous herbal classics. Its origin is relatively complex. The source of Dryopteris crassirhizoma used in the present invention is the dried rhizome of Woodwardia unigemmata (Makino.) Nakai, a plant of the genus Woodwardia in the family Blechnaceae. Dryopteris crassirhizoma has the effects of clearing heat and detoxifying, and stopping bleeding, and is used for preventing colds, epistaxis, dizziness, dysentery, and metrorrhagia. Carbonized Dryopteris crassirhizoma is a processed product after Dryopteris crassirhizoma is stir-fried with bran, which has astringent effects and is more effective in stopping bleeding. It can be seen that during the processing process, some internal chemical components of the two may have been transformed.

[0003] There are also reports on the identification research of Woodwardia unigemmata in the existing literature. For example, Liu Xia et al. used thin-layer chromatography to qualitatively identify Woodwardia unigemmata, but this method focuses on distinguishing medicinal materials and cut pieces from different producing areas. However, the identification points of Dryopteris crassirhizoma and its stir-fried and carbonized processed products are still blank. Dryopteris crassirhizoma and carbonized Dryopteris crassirhizoma cut pieces can be distinguished by their characters, but the extracts and preparations of both show the characteristics of yellowish-brown color, slight odor, and slightly bitter taste, which are easy to be confused, and the identification methods of the two have not been reported for the time being. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a thin-layer identification method for Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules. This method uses whether there are spots in a certain range of specific migration values by observing the thin-layer chromatography pattern as the identification point to quickly and effectively identify Dryopteris crassirhizoma and carbonized Dryopteris crassirhizoma formula granules.

[0005] The present invention provides a thin-layer identification method for Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules, including the following steps:

[0006] A) Take the sample to be tested, dissolve it with a solvent to obtain a test solution; the sample to be tested is Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules;

[0007] B) Take the Dryopteris crassirhizoma reference medicinal material, dissolve it with a solvent to obtain a reference medicinal material solution;

[0008] C) Take the protocatechuic acid reference substance, dissolve it with a solvent to obtain a reference substance solution;

[0009] D) Perform thin-layer chromatography detection on the test solution, reference substance solution, and reference medicinal material solution. The thin-layer plate is a silica gel GF 254 thin-layer plate; the developing agent is toluene - ethyl acetate - formic acid;

[0010] E) Examine under an ultraviolet lamp. If there are spots in the test sample chromatogram at an Rf value of 0.83 - 1.00, it is the Dryopteris crassirhizoma carbonisata formula granule; if there are no spots at an Rf value of 0.83 - 1.00, it is the Dryopteris crassirhizoma formula granule.

[0011] The thin-layer identification method provided by the present invention can quickly and effectively identify the Dryopteris crassirhizoma and Dryopteris crassirhizoma carbonisata formula granules that have lost the form of cut herbs. The detection method is simple to operate, has good durability and sensitivity, and has good application prospects.

[0012] In the present invention, the test sample is taken and dissolved in a solvent to obtain a test sample solution; the test sample is the Dryopteris crassirhizoma formula granule and the Dryopteris crassirhizoma carbonisata formula granule. In the present invention, step A is specifically as follows: Add methanol to the Dryopteris crassirhizoma formula granule and the Dryopteris crassirhizoma carbonisata formula granule respectively, ultrasonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, then extract with ethyl acetate, evaporate the obtained ethyl acetate solution to dryness, and add methanol to the residue to obtain the Dryopteris crassirhizoma formula granule test sample solution and the Dryopteris crassirhizoma carbonisata formula granule test sample solution respectively;

[0013] The mass ratio of the Dryopteris crassirhizoma formula granule to the volume of methanol is 1.0 g : (18 - 23) mL;

[0014] The mass ratio of the Dryopteris crassirhizoma carbonisata formula granule to the volume of methanol is 1.0 g : (18 - 23) mL;

[0015] The mass ratio of the Dryopteris crassirhizoma formula granule to the volume of ethyl acetate is 1.0 g : (8 - 10) mL;

[0016] The mass ratio of the Dryopteris crassirhizoma carbonisata formula granule to the volume of ethyl acetate is 1.0 g : (8 - 10) mL;

[0017] The time of ultrasonication is 15 - 25 min; in a specific embodiment, the time of ultrasonication is 20 min.

[0018] In a specific embodiment, the test sample solution is prepared according to the following method: Take 1 g of Dryopteris crassirhizoma formula granule and 1.0 g of Dryopteris crassirhizoma carbonisata formula granule, grind them finely, add 20 ml of methanol respectively, ultrasonically treat for 20 minutes (600 W, 40 KHz), filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, then extract with ethyl acetate by shaking 2 times, 10 ml each time, combine the ethyl acetate solutions, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test sample solution.

[0019] In the present invention, the Dryopteris crassirhizoma control medicinal material is taken and dissolved in a solvent to obtain a control medicinal material solution. In the present invention, step B) is specifically as follows: Add water to decoct the Dryopteris crassirhizoma control medicinal material, filter, evaporate the filtrate to dryness, dissolve the residue in water, then extract with ethyl acetate, evaporate the obtained ethyl acetate solution to dryness, and add methanol to the residue to obtain the control medicinal material solution;

[0020] The mass ratio of the Guanzhong reference medicinal material, the volume of water and the volume ratio of methanol is 1g:(48-53)mL:(18-23)mL; the mass ratio of the Guanzhong reference medicinal material and the volume ratio of ethyl acetate is 1g:(8-10)mL; the number of ethyl acetate extractions is 2 times.

[0021] In a specific embodiment, the Guanzhong reference medicinal material solution is prepared according to the following method: take 1 g of Guanzhong reference medicinal material, add 50 ml of water, heat and boil for 30 minutes, filter, evaporate the filtrate to dryness, add 20 ml of methanol to the residue, ultrasonically treat for 20 minutes (600W, 40KHz), filter, evaporate the filtrate to dryness, add 10 ml of water to the residue to dissolve it, then add ethyl acetate and shake to extract 2 times, 10 ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1 ml of methanol to the residue to dissolve it, and obtain the Guanzhong reference medicinal material solution.

[0022] The present invention takes a protocatechuic acid reference substance and dissolves it in a solvent to obtain a reference substance solution. In the present invention, the solvent in step C) is methanol; the concentration of the reference substance solution is 0.08-0.12 mg / mL. In a specific embodiment, the concentration of the reference substance solution is 0.1 mg / mL.

[0023] The present invention performs thin layer chromatography on the test solution, reference solution and reference medicinal material solution. The thin layer plate is silica gel GF 254 Thin layer plate; the developing solvent is toluene-ethyl acetate-formic acid, and the volume ratio of toluene, ethyl acetate and formic acid in the developing solvent is 7: (4.9-5.1): (0.48-0.52), more preferably 7: 5: 0.5. Preferably: according to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Tong 0502) test, the above solution is taken and spotted on the same silica gel GF 254 On the thin layer plate, use toluene-ethyl acetate-formic acid as the developing agent, develop, take out, dry, and examine under ultraviolet light.

[0024] The thin layer plate of the present invention is silica gel GF 254 Thin layer plate; using Qingdao Ocean and Merck two brands of prefabricated silica gel GF 254 The results show that this method has good durability and can meet the identification requirements. And at the Rf values of 0.918 and 0.929, the Guanzhong carbon formula particles have obvious spots, while the Guanzhong formula particles have no spots of the same color, which can be used to distinguish the Guanzhong formula particles from the Guanzhong carbon formula particles.

[0025] In the present invention, during thin layer chromatography detection, the spotting volume of the reference solution and the reference medicinal material solution is 2 to 10 μL;

[0026] The sample volume of the test solution is 5 to 15 μL.

[0027] In some specific embodiments of the present invention, the volume ratio of toluene - ethyl acetate - formic acid is 7:5:0.5.

[0028] In the present invention, in step E), the wavelength of the ultraviolet light is 254 nm.

[0029] In the present invention, in step E), the inspection temperature is 4°C to 25°C. The method of the present invention has good adaptability to different temperatures, and at the retention factor (Rf) values of 0.926 and 0.918, the dryopteris rhizome carbon formula granules have obvious spots, while the dryopteris rhizome formula granules have no spots of the same color, so as to distinguish the dryopteris rhizome formula granules from the dryopteris rhizome carbon formula granules.

[0030] In the present invention, in step E), the inspection humidity is 32% rh to 75% rh. The method of the present invention has good adaptability to different humidities, and at the retention factor (Rf) values of 0.923 and 0.932, the dryopteris rhizome carbon formula granules have obvious spots, while the dryopteris rhizome formula granules have no spots of the same color, so as to distinguish the dryopteris rhizome formula granules from the dryopteris rhizome carbon formula granules.

[0031] Place it under an ultraviolet lamp for inspection. If there are spots at the Rf value of 0.83 - 1.00, it is the dryopteris rhizome carbon formula granules; if there are no spots at the Rf value of 0.83 - 1.00, it is the dryopteris rhizome formula granules. In the present invention, the Rf value is preferably 0.92 - 0.93; specifically, it can be 0.916, 0.918, 0.923, 0.924, 0.926, 0.929, 0.932.

[0032] The present invention provides a thin - layer chromatography identification method for dryopteris rhizome formula granules and dryopteris rhizome carbon formula granules, including the following steps: A) Take the test sample, dissolve it with a solvent to obtain a test solution; the test sample is dryopteris rhizome formula granules and dryopteris rhizome carbon formula granules; B) Take the dryopteris rhizome reference medicinal material, dissolve it with a solvent to obtain a reference medicinal material solution; C) Take the protocatechuic acid reference substance, dissolve it with a solvent to obtain a reference substance solution; D) Perform thin - layer chromatography detection on the test solution, reference substance solution and reference medicinal material solution. The thin - layer plate is silica gel GF 254 Thin - layer plate; the developing agent is toluene - ethyl acetate - formic acid; E) Place it under an ultraviolet lamp for inspection. If in the test sample chromatogram, if there are spots at the Rf value of 0.83 - 1.00, it is the dryopteris rhizome carbon formula granules; if there are no spots at the Rf value of 0.83 - 1.00, it is the dryopteris rhizome formula granules. The thin - layer chromatography identification method for dryopteris rhizome formula granules and dryopteris rhizome carbon formula granules established by the present invention uses whether there are spots in the specified Rf value range in the thin - layer chromatogram as the identification point to quickly and effectively identify the dryopteris rhizome carbon formula granules and dryopteris rhizome formula granules.

[0033] The thin-layer identification method of the present invention is as follows: by comparing the thin-layer chromatography of the test sample with that of the Dryopteris rhizome control crude drug, if the spots are the same as those of the control crude drug, it is Dryopteris rhizome formula granule; if there is an additional spot compared with the control crude drug at the retention factor value of 0.83 - 1.00, it is carbonized Dryopteris rhizome formula granule.

[0034] The present invention discloses the construction and application of the thin-layer identification method for Dryopteris rhizome formula granule and carbonized Dryopteris rhizome formula granule. The present invention has good stability, high precision, good reproducibility, is convenient and easy to master. The thin-layer identification method of the present invention is not limited to the thin-layer identification of Dryopteris rhizome and carbonized Dryopteris rhizome formula granule, but also applicable to the thin-layer identification of extracts and other preparations of Dryopteris rhizome and carbonized Dryopteris rhizome. Description of the Drawings

[0035] Figure 1 For the investigation of the sample application amount, where 1 - 3 are the sample application amounts of the reference substance 2 μl, 5 μl, 10 μl, 4 - 6 are the sample application amounts of the control crude drug 2 μl, 5 μl, 10 μl, 7 - 9 are the sample application amounts of Dryopteris rhizome formula granule 5 μl, 10 μl, 15 μl, and 10 - 12 are the sample application amounts of carbonized Dryopteris rhizome formula granule 5 μl, 10 μl, 15 μl;

[0036] Figure 2 For the investigation of specificity, where 1 is the negative solution, 2 is the protocatechuic acid reference substance, 3 is the Dryopteris rhizome control crude drug, 4 is the Dryopteris rhizome formula granule, and 5 is the carbonized Dryopteris rhizome formula granule;

[0037] Figure 3 For the investigation of Qingdao Ocean thin-layer plate, where 1 is the protocatechuic acid reference substance, 2 is the Dryopteris rhizome control crude drug, 3 is the Dryopteris rhizome formula granule, and 4 is the carbonized Dryopteris rhizome formula granule;

[0038] Figure 4 For the investigation of Merck thin-layer plate, where 1 is the protocatechuic acid reference substance, 2 is the Dryopteris rhizome control crude drug, 3 is the Dryopteris rhizome formula granule, and 4 is the carbonized Dryopteris rhizome formula granule;

[0039] Figure 5 For the investigation of different developing temperatures at 4 °C, where 1 is the protocatechuic acid reference substance, 2 is the Dryopteris rhizome control crude drug, 3 is the Dryopteris rhizome formula granule, and 4 is the carbonized Dryopteris rhizome formula granule;

[0040] Figure 6 For the investigation of different developing temperatures at 25 °C, where 1 is the protocatechuic acid reference substance, 2 is the Dryopteris rhizome control crude drug, 3 is the Dryopteris rhizome formula granule, and 4 is the carbonized Dryopteris rhizome formula granule;

[0041] Figure 7 For the investigation of different developing humidities at 75%, where 1 is the protocatechuic acid reference substance, 2 is the Dryopteris rhizome control crude drug, 3 is the Dryopteris rhizome formula granule, and 4 is the carbonized Dryopteris rhizome formula granule;

[0042] Figure 8 For the investigation of different unfolding humidities of 32%, where 1 is the protocatechuic acid reference substance, 2 is the dryopteris rhizome control medicinal material, 3 is the dryopteris rhizome formula granule, and 4 is the carbonized dryopteris rhizome formula granule;

[0043] Figure 9 For the verification of different batches of samples, where 1 is the protocatechuic acid reference substance; 2 is the dryopteris rhizome control medicinal material; 3 - 5 are the dryopteris rhizome formula granules 2212016, 2212017, 2212018, and 6 - 8 are the carbonized dryopteris rhizome formula granules 2301031, 2301032, 2301033;

[0044] Figure 10 For the thin - layer chromatogram of Comparative Example 1, where 1 is the protocatechuic acid reference substance, 2 is the dryopteris rhizome control medicinal material, 3 is the dryopteris rhizome formula granule, and 4 is the carbonized dryopteris rhizome formula granule;

[0045] Figure 11 For the thin - layer chromatogram of Comparative Example 2, where 1 is the dryopteris rhizome formula granule and 2 is the carbonized dryopteris rhizome formula granule. Detailed implementation method

[0046] To further illustrate the present invention, the following describes in detail a thin - layer identification method for dryopteris rhizome formula granules and carbonized dryopteris rhizome formula granules provided by the present invention in combination with examples, but they cannot be understood as limiting the protection scope of the present invention.

[0047] Example 1

[0048] 2.2.1 Instruments and reagents

[0049] 2.1.1.1 Instruments

[0050] Hot plate, mortar, thin - layer imaging system: CAMAG TLC Visualizer, silica gel GF 254 Thin - layer plates (Qingdao Ocean Chemical Factory, batch number: 20180527, Tianjin Sida Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353)

[0051] 2.1.1.2 Reagents

[0052] Toluene, ethyl acetate, and formic acid are all of analytical grade, and water is ultrapure water (self - made in the laboratory).

[0053] 2.1.1.3 Reagents

[0054] Dryopteris rhizome reference drug (Sichuan Institute for Drug Control, batch number: NO.SCZD003-201905), protocatechuic acid reference substance (National Institutes for Food and Drug Control, batch number: 110809-201205), Dryopteris rhizome formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2212016; 2212017; 2212018), carbonized Dryopteris rhizome formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 2301031; 2301032; 2301033).

[0055] 2.2.2 Establishment of TLC method for formula granules

[0056] Take 1 g of Dryopteris rhizome formula granules and 1 g of carbonized Dryopteris rhizome formula granules, grind them finely, add 20 ml of methanol to each, ultrasonically treat for 20 minutes (600 W, 40 KHz), filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, then extract with ethyl acetate by shaking 2 times, 10 ml each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution.

[0057] Take 1 g of Dryopteris rhizome reference drug, add 50 ml of water, heat to boiling and boil for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of methanol, ultrasonically treat for 20 minutes (600 W, 40 KHz), filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, then extract with ethyl acetate by shaking 2 times, 10 ml each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the reference drug solution. Additionally, take protocatechuic acid reference substance and prepare a reference substance solution with methanol at a concentration of 0.1 mg / ml.

[0058] According to the test of thin-layer chromatography (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition), absorb 5 μl of each of the above solutions and spot them on the same silica gel GF 254 thin-layer plate. Use toluene-ethyl acetate-formic acid (7:5:0.5) as the developing solvent, develop, take out, dry in air, and examine under ultraviolet light (254 nm). In the chromatogram of the test solution of Dryopteris rhizome formula granules, at the positions corresponding to the chromatograms of the reference substance and the reference drug, there are spots showing the same color. In the chromatogram of the test solution of carbonized Dryopteris rhizome formula granules, at the positions corresponding to the chromatograms of the reference substance and the reference drug, there are main spots showing the same color.

[0059] 2.3.1 Methodology investigation

[0060] 2.2.3.1 Investigation of spotting volume

[0061] Under the above-determined experimental conditions, respectively take 2 μl, 5 μl, and 10 μl of the reference substance solution and the reference drug solution; take 5 μl, 10 μl, and 15 μl of the test solution and spot them on the same silica gel GF 254 thin-layer plate. Develop according to the determined thin-layer chromatography conditions, examine, and the results are shown inFigure 1 As can be seen from the figure, when 2 - 10 μl of the reference substance and reference crude drug solutions are spotted, and 5 - 15 μl of the test solution is spotted, the spots on the thin - layer chromatography are clearly colored. It is determined that the spotting volumes of the reference substance and reference crude drug solutions are 2 - 10 μl, and the spotting volume of the test solution is 5 - 15 μl. Compared with the chromatogram of the reference crude drug of Dryopteris crassirhizoma, there are obvious spots at the Rf value of 0.924 for the Dryopteris crassirhizoma carbonized formula granules, while there are no spots of the same color for the Dryopteris crassirhizoma formula granules.

[0062] 2.2.3.2 Specificity investigation

[0063] Prepare the solutions of the reference crude drug of Dryopteris crassirhizoma, Dryopteris crassirhizoma formula granules, Dryopteris crassirhizoma carbonized formula granules, and negative solution according to the above test solution preparation method, and spot them on the same silica gel GF 254 thin - layer plate (manufacturer: Tianjin Sida), develop according to the established thin - layer chromatography conditions, and examine. The results are shown in Figure 2 The results show that the negative samples do not interfere with the test samples of Dryopteris crassirhizoma and Dryopteris crassirhizoma carbonized formula granules, and the method has good specificity. Compared with the chromatogram of the reference crude drug of Dryopteris crassirhizoma, there are obvious spots at the Rf value of 0.916 for the Dryopteris crassirhizoma carbonized formula granules, while there are no spots of the same color for the Dryopteris crassirhizoma formula granules.

[0064] 2.2.3.3 Durability investigation

[0065] 2.2.3.3.1 Comparison of different thin - layer plates

[0066] Use pre - made silica gel GF plates of two brands, Qingdao Ocean and Merck, and conduct tests according to the established test methods respectively. See 254 The results show that both brands of thin - layer plates can meet the expected identification requirements, indicating good durability of the method. Compared with the chromatogram of the reference crude drug of Dryopteris crassirhizoma, there are obvious spots at the Rf values of 0.918 and 0.929 for the Dryopteris crassirhizoma carbonized formula granules, while there are no spots of the same color for the Dryopteris crassirhizoma formula granules. Figures 3 - 4

[0067] 2.2.3.3.1 Comparison of different developing temperatures

[0068] Figures 5 - 6 Take the spotted thin - layer plates and develop them in temperature environments of low temperature 4 °C and normal temperature 25 °C respectively. As can be seen, the method has good durability for different temperatures. Compared with the chromatogram of the reference crude drug of Dryopteris crassirhizoma, there are obvious spots at the Rf values of 0.926 and 0.918 for the Dryopteris crassirhizoma carbonized formula granules, while there are no spots of the same color for the Dryopteris crassirhizoma formula granules.

[0069] 2.2.3.3.2 Comparison of different developing humidities

[0070] Take the spotted thin - layer plates and develop them in humidity environments of 32% and 75% respectively. AsFigures 7 - 8 It can be seen that this method has good durability for different humidities. Compared with the chromatogram of Dryopteris crassirhizoma Nakai control medicinal material, Dryopteris crassirhizoma Nakai carbonized formula granules have obvious spots at the Rf values of 0.923 and 0.932, while Dryopteris crassirhizoma Nakai formula granules do not have the same color spots.

[0071] 2.3.3.4 Verification

[0072] Three batches of Dryopteris crassirhizoma Nakai formula granules and three batches of Dryopteris crassirhizoma Nakai carbonized formula granules were used for thin-layer method verification, and the experimental results are shown in Figure 9 , and the results show that, compared with the chromatogram of Dryopteris crassirhizoma Nakai control medicinal material, Dryopteris crassirhizoma Nakai carbonized formula granules have obvious spots at the Rf value of 0.924, while Dryopteris crassirhizoma Nakai formula granules do not have the same color spots.

[0073] 2.2.4 Determination of the Rf values of the identification points of Dryopteris crassirhizoma Nakai and Dryopteris crassirhizoma Nakai carbonized formula granules

[0074] Summarize the Rf value data in the thin-layer chromatography methodology investigation, and the results are shown in Table 1. According to the summary results, it is determined that the Rf values of the identification spots of Dryopteris crassirhizoma Nakai and Dryopteris crassirhizoma Nakai carbonized formula granules should be within the range of 10%, and the specified value is 0.923.

[0075] Table 1 Summary of Rf values in methodology investigation

[0076]

[0077] Comparative Example 1

[0078] Take 1 g of Dryopteris crassirhizoma Nakai formula granules and 1 g of Dryopteris crassirhizoma Nakai carbonized formula granules, grind them finely, add 20 ml of methanol to each respectively, ultrasonically treat for 20 minutes (600 W, 40 KHz), filter, evaporate to dryness, dissolve the residue in 10 ml of water, add 10 ml of chloroform and shake for extraction, take the chloroform layer and evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution. Another 1 g of Dryopteris crassirhizoma Nakai control medicinal material was taken, added with 50 ml of water, heated and boiled for 30 minutes, filtered, the filtrate was evaporated to dryness, the residue was added with 20 ml of methanol, ultrasonically treated for 20 minutes (600 W, 40 KHz), filtered, evaporated to dryness, the residue was dissolved in 10 ml of water, added with 10 ml of chloroform and shaken for extraction, take the chloroform layer and evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the control medicinal material solution. Another protocatechuic acid reference substance was taken and made into a reference substance solution of 0.1 mg / ml with methanol. According to the thin-layer chromatography method (General Rule 0502 of the Chinese Pharmacopoeia 2020 Edition) for testing, 5 μl of each of the above solutions was respectively spotted on the same silica gel GF 254 thin-layer plate, using toluene-dichloromethane-formic acid (5:3:0.5) as the developing agent, develop, take out, air dry, and examine under ultraviolet light (254 nm). The results are as Figure 10 shown, and the results show that by using the above thin-layer identification method, the spots of Dryopteris crassirhizoma Nakai formula granules and Dryopteris crassirhizoma Nakai carbonized formula granules are not obvious, and the two cannot be identified.

[0079] Comparative Example 2

[0080] Take 1 g of Dryopteris crassirhizoma formula granules and 1 g of carbonized Dryopteris crassirhizoma formula granules, grind them finely, add 20 ml of diethyl ether respectively, ultrasonically treat for 20 minutes (600 W, 40 KHz), filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of chloroform to obtain the test solution. According to the thin-layer chromatography method (General Principles 0502 of the Chinese Pharmacopoeia 2020 Edition) test, absorb 10 μl of each of the above solutions, spot them on the same silica gel G thin-layer plate respectively, use chloroform-methanol (20:1) as the developing agent, develop, take out, dry in air, and examine under ultraviolet light (365 nm). The results are as Figure 11 shown. The results show that by using the above thin-layer identification method, there are no obvious spots in the Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules, and the two cannot be identified.

[0081] It can be seen from the above examples that the present invention provides a thin-layer identification method for Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules, which includes the following steps: A) Take the test sample, dissolve it with a solvent to obtain the test solution; the test sample is Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules; B) Take Dryopteris crassirhizoma control medicinal material, dissolve it with a solvent to obtain the control medicinal material solution; C) Take protocatechuic acid reference substance, dissolve it with a solvent to obtain the reference substance solution; D) Perform thin-layer chromatography detection on the test solution, reference substance solution and control medicinal material solution, and the thin-layer plate is silica gel GF 254 thin-layer plate; the developing agent is toluene-ethyl acetate-formic acid; E) Examine under ultraviolet light. If there are spots at the Rf value of 0.83 - 1.00 in the test sample chromatogram, it is carbonized Dryopteris crassirhizoma formula granules; if there are no spots at the Rf value of 0.83 - 1.00, it is Dryopteris crassirhizoma formula granules. The thin-layer identification method for Dryopteris crassirhizoma formula granules and carbonized Dryopteris crassirhizoma formula granules established by the present invention uses the presence or absence of spots in the specified specific migration value range of the thin-layer chromatogram as the identification point to quickly and effectively identify carbonized Dryopteris crassirhizoma formula granules and Dryopteris crassirhizoma formula granules.

[0082] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A thin-layer identification method for Dryopteris crassirhizoma formula granules and charred Dryopteris crassirhizoma formula granules, comprising the following steps: A) Take the test sample, dissolve it with a solvent to obtain a test solution; the test sample is Dryopteris crassirhizoma formula granules and charred Dryopteris crassirhizoma formula granules; The specific operation of step A is as follows: Take Dryopteris crassirhizoma formula granules and charred Dryopteris crassirhizoma formula granules respectively, add methanol, ultrasonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, then extract with ethyl acetate, evaporate the obtained ethyl acetate solution to dryness, dissolve the residue in methanol to obtain the test solution of Dryopteris crassirhizoma formula granules and the test solution of charred Dryopteris crassirhizoma formula granules respectively; The mass ratio of the Dryopteris crassirhizoma formula granules to the volume of methanol is 1.0 g:(18 - 23) mL; The mass ratio of the charred Dryopteris crassirhizoma formula granules to the volume of methanol is 1.0 g:(18 - 23) mL; The mass ratio of the Dryopteris crassirhizoma formula granules to the volume of ethyl acetate is 1.0 g:(8 - 10) mL; The mass ratio of the charred Dryopteris crassirhizoma formula granules to the volume of ethyl acetate is 1.0 g:(8 - 10) mL; B) Take Dryopteris crassirhizoma control medicinal material, dissolve it with a solvent to obtain a control medicinal material solution; C) Take protocatechuic acid reference substance, dissolve it with a solvent to obtain a reference substance solution; D) Perform thin-layer chromatography detection on the test sample solution, reference substance solution, and reference crude drug solution. The thin-layer plate is silica gel GF 254 thin-layer plate; the developing agent is toluene-ethyl acetate-formic acid; the volume ratio of the toluene-ethyl acetate-formic acid is 7:(4.9~5.1):(0.48~0.52); E) Examine under an ultraviolet lamp. If there is a spot at an Rf value of 0.83 - 1.00 in the chromatogram of the test solution, it is charred Dryopteris crassirhizoma formula granules; If there is no spot at an Rf value of 0.83 - 1.00, it is Dryopteris crassirhizoma formula granules.

2. The authentication method according to claim 1, characterized in that The specific operation of step B) is as follows: Take Dryopteris crassirhizoma control medicinal material, decoct it with water, filter, evaporate the filtrate to dryness, dissolve the residue in water, then extract with ethyl acetate, evaporate the obtained ethyl acetate solution to dryness, dissolve the residue in methanol to obtain a control medicinal material solution; The mass ratio of the Dryopteris crassirhizoma control medicinal material, the volume of water and the volume of methanol is 1 g:(48 - 53) mL:(18 - 23) mL; The mass ratio of the Dryopteris crassirhizoma control medicinal material to the volume of ethyl acetate is 1 g:(8 - 10) mL.

3. The identification method according to claim 1, characterized in that, The solvent in step C) is methanol; the concentration of the reference substance solution is 0.08 - 0.12 mg / mL.

4. The identification method according to claim 1, wherein When performing thin-layer chromatography detection, the spotting volume of the reference substance solution and the control medicinal material solution is 2 - 10 μL; The spotting volume of the test solution is 5 - 15 μL.

5. The authentication method according to claim 1, characterized in that, The volume ratio of toluene - ethyl acetate - formic acid is 7:5:0.

5.

6. The authentication method according to claim 1, wherein The ultraviolet light wavelength in step E) is 254 nm.

7. The authentication method according to claim 1, wherein The examination temperature in step E) is 4°C - 25°C.

8. The authentication method according to claim 1, characterized in that The examination humidity in step E) is 32%rh - 75%rh.

9. The identification method according to claim 1, characterized in that The Rf value is 0.92 - 0.93.

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