TLC Identification Method of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules

Abstract

The present invention belongs to the technical field of traditional Chinese medicine quality detection, and particularly relates to a thin layer chromatography identification method for sanguisorba charcoal in the gynecological Duanhongyin capsules. By using the thin layer chromatography identification method of the present invention, the test samples and the solution of the sanguisorba control crude drug can both detect identification bands, and the separation degree of each band is good. The negative control does not detect characteristic bands and there is no interference. In the test sample chromatogram, at the position corresponding to the control crude drug chromatogram, spots of the same color appear; the thin layer chromatography method of the present invention has strong specificity for the detection of sanguisorba charcoal in the gynecological Duanhongyin capsules, and has good durability for different thin layer plates, temperatures and humidities; it can be quickly and accurately identified in a high humidity environment, and can be effectively used for the identification of sanguisorba charcoal in the gynecological Duanhongyin capsules.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine quality inspection, and specifically relates to a thin-layer chromatography identification method for Sanguisorbae Carbonis in Fuke Duanhongyin Capsules. Background Art

[0002] After the traditional Chinese medicine decoction pieces are carbonized, their properties and active ingredients are severely damaged, and the ingredients are not clear. The uncertainty of the material basis of carbonized drugs restricts the unification of their quality standards. Among the carbonized drugs with independent identification standards in the Chinese Pharmacopoeia 2020 Edition, there are only Cirsii Japonici Carbonis, Carbonized Human Hair, Schizonepetae Carbonis, Spica Schizonepetae Carbonis, and Dryopteridis Crassirhizomatis Carbonis. Therefore, it is currently difficult to obtain an identification method for Sanguisorbae Carbonis based on the identification of Sanguisorbae officinalis in the pharmacopoeia.

[0003] The Chinese Pharmacopoeia 2020 Edition discloses an identification method for Sanguisorbae Carbonis under Anorectal Ointment. However, when the test solution of Fuke Duanhongyin Capsules is prepared and detected by the method under Anorectal Ointment, the test samples are difficult to be effectively separated, the target spots are blurred, and no clear and available identification bands are found, so it cannot be applied to the identification of Sanguisorbae Carbonis in Fuke Duanhongyin.

[0004] Chinese invention patent CN105241997A discloses a thin-layer chromatography identification method for carbonized traditional Chinese medicine formula granules. This method uses an acid hydrolysis reduction method and uses silica gel GF 254 fluorescence quenching thin-layer plates to identify the "non-fluorescent component" part, and then combines the color development observation of characteristic components. This method is applicable to the identification of most carbonized traditional Chinese medicine formula granules containing 254nm fluorescent absorption substances. This method requires specific silica gel GF 254 fluorescence quenching thin-layer plates. The identified carbonized drugs are only carbonized traditional Chinese medicines containing 254nm fluorescent absorption substances, which cannot be applied to other different thin-layer plates, with limited durability, and it is greatly affected by environmental humidity. Therefore, it affects the quality control of Sanguisorbae Carbonis formula drugs and is not applicable to the identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules and the quality control of Fuke Duanhongyin Capsules. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the identification of Sanguisorbae Carbonis in existing Fuke Duanhongyin Capsules, and provide a thin-layer chromatography identification method for Sanguisorbae Carbonis in Fuke Duanhongyin Capsules. This method has strong specificity and good durability for the identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules, and can be used as the quality control standard for Sanguisorbae Carbonis in Fuke Duanhongyin Capsules.

[0006] The purpose of the present invention is to provide a thin-layer chromatography developing agent.

[0007] The purpose of the present invention also lies in providing the application of the thin-layer chromatography developing agent in the preparation of a thin-layer chromatography identification kit for Sanguisorbae Carbonis in Fuke Duanhongyin Capsules and / or a thin-layer chromatography identification method for Sanguisorbae Carbonis in Fuke Duanhongyin Capsules.

[0008] The present invention also aims to provide a thin layer chromatography identification method for charcoal-fried sanguisorba root in Fuke Duanhongyin Capsules.

[0009] The present invention also aims to provide a thin layer chromatography identification reagent kit for charcoal-fried sanguisorba root in Fuke Duanhongyin Capsules.

[0010] The above objects of the present invention are achieved by the following technical means:

[0011] The present invention provides a thin layer chromatography developing agent, which is a mixed solution of toluene, ethyl acetate and formic acid. The volume ratio of toluene:ethyl acetate:formic acid in the mixed solution is 15 - 30:3 - 5:0.5 - 1.5.

[0012] Preferably, the volume ratio of toluene:ethyl acetate:formic acid is 20 - 30:4 - 5:0.5 - 1.

[0013] More preferably, the volume ratio of toluene:ethyl acetate:formic acid is 20:4:0.5.

[0014] The application of the thin layer chromatography developing agent in the preparation of a thin layer chromatography identification reagent kit for charcoal-fried sanguisorba root in Fuke Duanhongyin Capsules and / or a thin layer chromatography identification method for charcoal-fried sanguisorba root in Fuke Duanhongyin Capsules is also within the protection scope of this application.

[0015] The present invention provides a thin layer chromatography identification method for charcoal-fried sanguisorba root in Fuke Duanhongyin Capsules, including the following steps:

[0016] S1. Preparation of test solution

[0017] Extract the content of Fuke Duanhongyin Capsules with water, take the water extract liquid, extract the water extract liquid with n-butanol saturated with water to obtain an n-butanol extract solution. Wash the n-butanol extract solution with ammonia test solution, remove the solvent from the fully washed n-butanol extract solution to obtain a solid sample, and dissolve the solid sample in methanol as the test solution;

[0018] S2. Preparation of control crude drug solution

[0019] Prepare a control crude drug solution of sanguisorba root according to the preparation method of the test solution in step S1.

[0020] S3. Identification by thin layer chromatography

[0021] Respectively spot the test solution prepared in step S1 and the control crude drug solution prepared in step S2 on the same silica gel G thin layer plate, use the mixed solution of toluene, ethyl acetate and formic acid as the developing agent, and place it under ultraviolet light at 365 nm for inspection for thin layer chromatography identification.

[0022] Preferably, the method for thin layer chromatography identification of sanguisorba charcoal in the Gynecological Duanhongyin Capsules includes the following steps:

[0023] S1. Preparation of test solution

[0024] Extract the content of the Gynecological Duanhongyin Capsules with water. The specific method is as follows: Dissolve the content of the Gynecological Duanhongyin Capsules in water, ultrasonically treat for 30 - 60 min until fully dissolved, and then filter. The mass - volume ratio of the content of the Gynecological Duanhongyin Capsules to water is 0.5 - 2 g:20 - 30 mL;

[0025] Take the water - extract liquid, and extract the water - extract liquid with n - butanol saturated with water to obtain the n - butanol extract. The specific method is as follows: Extract the filtrate with n - butanol saturated with water 3 - 6 times to obtain the n - butanol solution;

[0026] Wash the n - butanol extract with ammonia test solution. The volume ratio of water to n - butanol saturated with water is 4 - 6:11 - 14, and the volume ratio of n - butanol saturated with water to ammonia reagent is 3 - 5:1 - 3; Remove the solvent from the washed n - butanol extract by evaporation to obtain a solid sample, and dissolve the solid sample in methanol to obtain the test solution. The mass - volume ratio of the content of the Gynecological Duanhongyin Capsules to methanol is 0.5 - 2 g:0.5 - 2.5 mL;

[0027] S2. Preparation of control medicinal material solution

[0028] Prepare the sanguisorba control medicinal material solution from the sanguisorba control medicinal material according to the preparation method of the test solution in step S1. The mass - volume ratio of the sanguisorba control medicinal material to water is 0.1 - 1 g:20 - 30 mL;

[0029] S3. Identification by thin layer chromatography

[0030] Absorb 2 - 5 μL of the test solution and 2 μL of the control medicinal material solution, and spot them on the same silica gel G thin layer plate respectively. Use the mixed solution of toluene, ethyl acetate and formic acid as the developing agent. The volume ratio of toluene:ethyl acetate:formic acid is 15 - 30:3 - 5:0.5 - 1.5, and conduct thin layer chromatography identification under ultraviolet light at 365 nm for inspection.

[0031] Further preferably, in step S1, the mass - volume ratio of the content of the Gynecological Duanhongyin Capsules to water is 0.5 - 1 g:20 - 25 mL; the volume ratio of water to n - butanol saturated with water is 5 - 6:12 - 14, and the volume ratio of n - butanol saturated with water to ammonia reagent is 3 - 4:1 - 2.

[0032] Further preferably, in step S1, extract the filtrate with n - butanol saturated with water 3 times to obtain the n - butanol solution.

[0033] Further preferably, the mass-to-volume ratio of the Sanguisorba officinalis control crude drug to water in step S2 is 0.5-1 g:25-30 mL.

[0034] Further preferably, the volume ratio of toluene:ethyl acetate:formic acid in step S3 is 20-30:4-5:0.5-1.

[0035] Even more preferably, the mass-to-volume ratio of the contents of the Fuke Duanhongyin Capsule to water in step S1 is 1 g:25 mL; the volume ratio of water to water-saturated n-butanol is 5:12, and the volume ratio of water-saturated n-butanol to ammonia reagent is 3:2.

[0036] Even more preferably, the mass-to-volume ratio of the Sanguisorba officinalis control crude drug to water in step S2 is 0.5 g:25 mL.

[0037] Even more preferably, the volume ratio of toluene:ethyl acetate:formic acid in step S3 is 20:4:0.5.

[0038] Preferably, the developing temperature of the developing agent in step S3 is 4°C to 30°C.

[0039] Preferably, the developing humidity of the developing agent in step S3 is 33% to 70%.

[0040] A thin-layer chromatography identification kit for Sanguisorba charred in Fuke Duanhongyin Capsule, comprising a thin-layer chromatography developing agent with a mixed solution of toluene, ethyl acetate and formic acid as the developing agent, and the volume ratio of toluene:ethyl acetate:formic acid in the mixed solution is 15-30:3-5:0.5-1.5.

[0041] Preferably, it also contains silica gel G thin-layer plate and / or methanol. As a specific embodiment, the thin-layer chromatography identification method for Sanguisorba charred in the Fuke Duanhongyin Capsule comprises the following steps:

[0042] S1. Preparation of test solution

[0043] Dissolve 1 g of the contents of the Fuke Duanhongyin Capsule in 25 mL of water, filter after ultrasonic treatment for 30 min, extract the filtrate with water-saturated n-butanol three times, 20 mL each time, wash the extracted n-butanol solution with 40 mL of ammonia test solution, evaporate the washed n-butanol solution to dryness to obtain a solid sample, and dissolve the solid sample in 2 mL of methanol as the test solution.

[0044] S2. Preparation of control crude drug solution

[0045] Take 0.5 g of the Sanguisorba officinalis control crude drug, add 25 mL of water, and the remaining steps are the same as those for preparing the Sanguisorba officinalis control crude drug solution of the test solution.

[0046] S3. Identification by thin-layer chromatography

[0047] Refer to General Principles 0502, Volume IV of Chinese Pharmacopoeia (2020 Edition). Pipette 2 - 5 μL of the test solution and 2 μL of the reference crude drug solution, and spot them on the same silica gel G thin - layer plate respectively. Use a mixture of toluene:ethyl acetate:formic acid with a volume ratio of 20:4:0.5 as the developing solvent, and examine under ultraviolet light at 365 nm.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. Identified by the thin - layer chromatography method of the present invention, the test solution and the reference crude drug solution of Sanguisorba officinalis can both detect identification bands, and the separation degree of each band is good. The negative control does not detect characteristic bands and there is no interference. In the test solution chromatogram, at the position corresponding to the reference crude drug chromatogram, spots of the same color appear.

[0050] 2. The thin - layer chromatography method of the present invention has strong specificity for the detection of Sanguisorba officinalis charcoal in Fuke Duanhongyin Capsules and has good durability for different thin - layer plates, temperatures and humidities; it can be quickly and accurately identified in a high - humidity environment and can be effectively used for the identification of Sanguisorba officinalis charcoal in Fuke Duanhongyin Capsules.

[0051] 3. It supplements the blank of the quality standard of Fuke Duanhongyin Capsules and realizes the quality control of the whole formula of Fuke Duanhongyin Capsules. Description of the Drawings

[0052] Figure 1 It is the thin - layer chromatography identification chromatogram of Sanguisorba officinalis charcoal in Fuke Duanhongyin Capsules in Example 1 of the present invention; 1 - blank solvent (methanol), 2 - reference crude drug solution of Sanguisorba officinalis, 3 - negative sample solution without Sanguisorba officinalis charcoal, 4 - 6 are test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102.

[0053] Figure 2 It is the thin - layer chromatography diagram of Fuke Duanhongyin Capsules with six different extraction methods and reference substances in Comparative Example 1; 1 - gallic acid, 2 - Method 1, 3 - Method 2, 4 - Method 3, 5 - Method 4, 6 - Method 5, 7 - Method 6.

[0054] Figure 3 It is the specificity thin - layer chromatography diagram of Fuke Duanhongyin Capsules with Methods 3, 5 and 6 and reference substances in Comparative Example 1; 1 - gallic acid, 2 - Fuke Duanhongyin Capsules of Method 3 - 20200702, 3 - negative of Method 3 without Sanguisorba officinalis charcoal, 4 - Fuke Duanhongyin Capsules of Method 5 - 20200702, 5 - negative of Method 5 without Sanguisorba officinalis charcoal, 6 - Fuke Duanhongyin Capsules of Method 6 - 20200702, 7 - negative of Method 6 without Sanguisorba officinalis charcoal.

[0055] Figure 4TLC chromatogram of Sanguisorbae Carbonis Radix in Fuke Duanhongyin Capsules identified by the method of Comparative Example 2; 1 - Sanguisorboside I, 2 - reference medicinal material of Sanguisorbae Radix DZYC-S-019, 3 - Sanguisorbae Carbonis Radix Y019-2003001, 4 - Fuke Duanhongyin Capsules of the method of Comparative Example 2 on July 2, 2020, 5 - negative control without Sanguisorbae Carbonis Radix of the method of Comparative Example 2.

[0056] Figures 5 to 12 TLC chromatograms of Sanguisorbae Carbonis Radix in Fuke Duanhongyin Capsules under different TLC conditions; in Figures 5 - 12, A is the chromatogram without spraying the developer, B is the chromatogram after spraying the developer; 1 - reference medicinal material of Sanguisorbae Radix DZYC-S-019, 2 - Sanguisorbae Carbonis Radix Y019-2003001, 3 - Fuke Duanhongyin Capsules of the method of Comparative Example 2 on July 2, 2020, 4 - negative control without Sanguisorbae Carbonis Radix of the method of Comparative Example 2.

[0057] Fig.13 TLC chromatogram of Sanguisorbae Carbonis Radix in Fuke Duanhongyin Capsules identified by the method of Comparative Example 4; 1, 7 - negative control without Sanguisorbae Carbonis Radix, 2, 6 - reference medicinal material of Sanguisorbae Radix, 3 - Fuke Duanhongyin Capsules on July 4, 2020, 4 - Fuke Duanhongyin Capsules on September 6, 2020, 5 - Fuke Duanhongyin Capsules on January 2, 2021.

[0058] Fig.14 TLC identification chromatograms of Sanguisorbae Carbonis Radix in Fuke Duanhongyin Capsules under different thin-layer plate conditions; 1 - blank solvent (methanol), 2 - solution of reference medicinal material of Sanguisorbae Radix, 3 - sample solution of negative control without Sanguisorbae Carbonis Radix, 4 - 6 are the test solution of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, 20210102; A - Qingdao Ocean Chemical G thin-layer plate, B - Qingdao Hailang silica gel G thin-layer plate, C - Merck TLC Silica gel 60G thin-layer plate, D - self-made G thin-layer plate.

[0059] Fig.15 TLC identification chromatograms of Sanguisorbae Carbonis Radix in Fuke Duanhongyin Capsules under different temperature conditions; 1 - blank solvent (methanol), 2 - solution of reference medicinal material of Sanguisorbae Radix, 3 - sample solution of negative control without Sanguisorbae Carbonis Radix, 4 - 6 are the test solution of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, 20210102; A - 20°C, B - 4°C, C - 30°C.

[0060] Fig.16 TLC identification chromatograms of Sanguisorbae Carbonis Radix in Fuke Duanhongyin Capsules under different humidity conditions; 1 - blank solvent (methanol), 2 - solution of reference medicinal material of Sanguisorbae Radix, 3 - sample solution of negative control without Sanguisorbae Carbonis Radix, 4 - 6 are the test solution of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, 20210102; A - 50%, B - 33%, C - 70%.

[0061] Fig.17 It is the thin-layer chromatography identification chromatogram of Sanguisorbae Carbonis in the 16th batch of Fuke Duanhongyin Capsules; 1 - blank solvent (methanol), 2 - solution of Sanguisorbae Radix control medicinal material, 3 - negative sample solution without Sanguisorbae Carbonis, 4 - 18 - batch numbers are 20200701, 20200702, 20200703, 20200704, 20200705, 20200706, 20200805, 20200806, 20200906, 20201105, 20201202, 20210102, 20210201, 20210301, 20210302 Fuke Duanhongyin Capsules. Specific implementation manners

[0062] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0063] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0064] Example 1 Thin - layer chromatography identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules

[0065] 1. Experimental method

[0066] 1.1 Preparation of test solution

[0067] Take 1 g of the content of Fuke Duanhongyin Capsules, dissolve it in 25 mL of water, sonicate for 30 min and filter. The filtrate is extracted with water - saturated n - butanol three times, 20 mL each time. The n - butanol solutions are combined, washed with 40 mL of ammonia test solution, and the washing solution is discarded. After evaporating the washed n - butanol solution to dryness, a solid sample is obtained. The solid sample is dissolved in 2 mL of methanol to obtain the test solution.

[0068] 1.2. Preparation of control solution

[0069] Take 0.5 g of Sanguisorbae Radix control medicinal material, add 25 mL of water, and prepare the solution of Sanguisorbae Radix control medicinal material by the method of 1.1; the negative control solution is prepared in the same way as the test solution.

[0070] 1.3. Identification by thin - layer chromatography

[0071] Refer to General Rules 0502, Part IV of Chinese Pharmacopoeia 2020 Edition. Absorb 2 - 5 μL of the test solution and 2 μL of the control medicinal material solution, and spot them on the same silica gel G thin - layer plate respectively. Use a mixed solution with a volume ratio of toluene: ethyl acetate: formic acid of 20:4:0.5 as the developing agent, develop, take out, dry in air, and examine under ultraviolet light (365 nm).

[0072] 2. Information on Samples and Control Medicinal Materials

[0073]

[0074]

[0075] Note: In the full text, "negative for Sanguisorbae Carbonis" means "negative for Sanguisorbae Carbonis in Fuke Duanhongyin Capsules".

[0076] 3. Results

[0077] Judgment Index: In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as those in the chromatogram of the control medicinal material. In this example, the Fuke Duanhongyin Capsules with production batch numbers 20200704, 20200906, and 20210102 produced by Zhuzhou Qianjin Pharmaceutical Co., Ltd. were identified respectively by the above method, and the identification results are as Figure 1 shown. Figure 1 Among them, 1 - blank solvent (methanol), 2 - solution of Sanguisorbae control medicinal material, 3 - negative sample solution without Sanguisorbae Carbonis, and 4 - 6 are test sample solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102.

[0078] From Figure 1 it can be seen that for the test samples of Fuke Duanhongyin Capsules of the three batch numbers and the Sanguisorbae control medicinal material, identification bands can be detected at Rf = 0.35. The resolution of each band is good, and no characteristic band can be detected in the negative sample of Fuke Duanhongyin Capsules without Sanguisorbae Carbonis, indicating that this method has strong specificity and can effectively identify Fuke Duanhongyin Capsules and the negative sample of Fuke Duanhongyin Capsules without Sanguisorbae Carbonis, and can be used for the thin - layer chromatography identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules.

[0079] The present invention also carried out thin - layer chromatography identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules under the conditions of different ratios of Fuke Duanhongyin Capsules to solvents, different concentrations of Sanguisorbae control medicinal materials, and different ratios of developing agents. The specific conditions are as follows in the table:

[0080]

[0081] Under conditions No. 1 and No. 2, for the test samples of Fuke Duanhongyin Capsules and the Sanguisorbae control medicinal material, identification bands can be detected at Rf = 0.25 - 0.55. The resolution of each band is good, and no characteristic band can be detected in the negative sample of Fuke Duanhongyin Capsules without Sanguisorbae Carbonis, indicating that this method has strong specificity and can effectively identify Fuke Duanhongyin Capsules and the negative sample of Fuke Duanhongyin Capsules without Sanguisorbae Carbonis, and can be used for the thin - layer chromatography identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules.

[0082] Comparative Example 1

[0083] 1. Influence of Six Different Extraction Methods on the Thin-Layer Chromatography Behavior of Fuke Duanhongyin Capsules and Reference Substances

[0084] 1.1 Experimental Method

[0085] Prepare corresponding solutions with the extraction methods of the test solutions of Methods 1 - 6 shown in Table 1 and reference substances, and conduct thin-layer chromatography identification. The preparation method of gallic acid solution is as follows: Take gallic acid reference substance, add methanol to make a solution containing 1.2078 mg per 1 mL as the reference substance solution; the spotting method is: strip, 8 mm; the developing agent is: toluene (saturated with water): ethyl acetate: formic acid with a volume ratio of 6:3:1; the developing distance is: 8 cm, and the developing tank is pre-saturated for 20 min; the drying method is: air-dry; the coloring method is: spray with 1% aluminum trichloride ethanol solution; the inspection method is: under daylight.

[0086] Table 1 Different Extraction Methods

[0087]

[0088] Note: In Table 1, this product refers to Fuke Duanhongyin Capsules produced by Zhuzhou Qianjin Pharmaceutical Co., Ltd., with the batch number 20200702.

[0089] 1.2 Sample and Reference Substance Information

[0090] Serial number Product Name Specification source purity(%) batch number 1 gallic acid 30mg China Food and Drug Inspection Institute 91.5％ 110831-201906 2 Fuke Duanhongyin Capsule 0.4g / tablet Zhuzhou Qianjin Pharmaceutical Co., Ltd. / 20200702 3 Lack of sanguisorba officinalis negative / Zhuzhou Qianjin Pharmaceutical Co., Ltd. / /

[0091] 1.3 Results

[0092] In this comparative example, the Fuke Duanhongyin Capsules were identified by 6 methods, and the identification results are as Figure 2 . Figure 2 Among them, 1 - gallic acid, 2 - Method 1, 3 - Method 2, 4 - Method 3, 5 - Method 4, 6 - Method 5, 7 - Method 6.

[0093] From Figure 2 it can be seen that the thin-layer chromatography behaviors of the samples extracted by Methods 3, 5, and 6 are consistent with those of the gallic acid reference substance, and no clear and usable identification bands were found in the thin-layer chromatography of the samples extracted by Methods 1, 2, and 4.

[0094] 2 Specificity Detection of Extraction Methods with Consistent Thin-Layer Chromatography Behaviors between Samples and Reference Substances

[0095] 2.1 Experimental Method

[0096] Prepare test samples, reference substance solutions, and negative sample solutions with the extraction methods of Methods 3, 5, and 6 to detect the specificity of the three methods. The preparation of gallic acid solution and thin-layer chromatography conditions are the same as those in 1.1 of Comparative Example 1.

[0097] 2.2 Results

[0098] The results of specificity detection are as follows Figure 3 . Figure 3 Among them, 1-gallic acid, 2-method 3- Fuke Duohongyin Capsules on July 2, 2020, 3-method 3 is negative for the absence of Sanguisorbae Carbonis, 4-method 5- Fuke Duohongyin Capsules on July 2, 2020, 5-method 5 is negative for the absence of Sanguisorbae Carbonis, 6-method 6- Fuke Duohongyin Capsules on July 2, 2020, 7-method 6 is negative for the absence of Sanguisorbae Carbonis.

[0099] Figure 3 The results show that there are interferences in the negative samples of the extraction methods of method 3, 5 and 6, and the specificity detection is unqualified. Therefore, the extraction methods and reference substances of method 1-6 are not applicable to the TLC identification of Sanguisorbae Carbonis in Fuke Duohongyin Capsules.

[0100] Comparative Example 2 A TLC method for Sanguisorbae Carbonis in Fuke Duohongyin Capsules

[0101] 1. Experimental method

[0102] The sample solution was extracted by the extraction method of Example 1, and TLC identification was carried out using ziyu-glycoside I, Sanguisorbae Radix reference medicinal material, and Sanguisorbae Carbonis reference medicinal material as reference medicinal materials.

[0103] Preparation of Sanguisorbae Radix / Sanguisorbae Carbonis medicinal material solution: Take 0.5 g of Sanguisorbae Radix / Sanguisorbae Carbonis reference medicinal material and extract it according to the method of Example 1 as the reference medicinal material solution; Preparation of ziyu-glycoside I solution: Take ziyu-glycoside I reference substance and add methanol to make a solution containing 0.997 mg per 1 mL as the reference substance solution; The spotting method is: strip, 8 mm; The developing agent is: the lower layer solution of the mixture of chloroform, methanol and water (volume ratio of chloroform: methanol: water is 13:7:2) placed below 10 °C; The developing distance is: 8 cm, and the developing tank is pre-saturated for 20 min; The drying method is: air drying; The coloring method is: spraying with 10% sulfuric acid ethanol solution; The inspection method is: 365 nm ultraviolet light.

[0104] 2. Information of samples and reference medicinal materials

[0105]

[0106] 3. Results

[0107] The Fuke Duohongyin Capsules were identified by the method of Comparative Example 2, and the identification results are as follows Figure 4 . Figure 4 Among them, 1-ziyu-glycoside I, 2-Sanguisorbae Radix reference medicinal material DZYC-S-019, 3-Sanguisorbae Carbonis Y019-2003001, 4-Fuke Duohongyin Capsules by the method of Comparative Example 2 on July 2, 2020, 5-the method of Comparative Example 2 is negative for the absence of Sanguisorbae Carbonis.

[0108] Figure 4The results showed that there were 2 specific spots with Rf = 0.61 and Rf = 0.91 in the thin-layer chromatography of the sample extracted by this method and the control medicinal material. The specific spot with Rf = 0.61 was close to that of ziyu-glycoside I (Rf = 0.6), but the spot colors were different. It was analyzed that this was due to the hydrolysis of saponins or the change of functional groups by other methods such as decoction extraction. After trying various extraction methods, no fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of the control medicinal material; the specific spot with Rf = 0.91 was the same as that of the sanguisorba control medicinal material, but the Rf value was too high and the developing system needed to be optimized. Therefore, the extraction method of this method was applicable, while the control medicinal material was not applicable, and the developing conditions needed to be optimized.

[0109] Influence of Different Thin-Layer Chromatography Conditions on the Thin-Layer Chromatography Identification of Sanguisorba Carbonisata in Fuke Duanhongyin Capsules in Comparative Examples 3 - 10

[0110] 1. Experimental Method

[0111] The samples were extracted by the extraction method of Example 1, and the sanguisorba / sanguisorba carbonisata was used as the control medicinal material. Thin-layer chromatography identification was carried out with different developing agents. The developing agents for Comparative Examples 3 - 10 are shown in Table 3.

[0112] Preparation of the sanguisorba / sanguisorba carbonisata control medicinal material solution: Take 0.5 g of the sanguisorba / sanguisorba carbonisata control medicinal material, extract it in the manner of Example 1, and use it as the control medicinal material solution. The spotting method is: strip, 8 mm; the developing distance is: 8 cm, and the developing tank is pre-saturated for 20 min; the drying method is: air drying; the coloring method: spray with 10% sulfuric acid ethanol solution or without adding a coloring agent; the inspection method: 365 nm. The extraction method of Example 1 is also the extraction method of Comparative Example 2.

[0113] Table 3

[0114] Comparative Example Developing agent (volume ratio) 3 Chloroform:methanol=15:1 4 Chloroform:methanol=45:1 5 Dichloromethane: methanol = 45:1 6 Cyclohexane:ethyl acetate=3:1 7 Cyclohexane: dichloromethane: ethyl acetate: glacial acetic acid = 20:5:8:0.5 8 Toluene: ethyl acetate: formic acid = 20:4:0.5 9 n-Hexane:ethyl acetate:formic acid=20:8:0.1 10 Cyclohexane: ethyl acetate: formic acid = 20:8:0.1

[0115] 2. Information of Samples and Control Medicinal Materials

[0116] Serial number Product Name Specification source purity(%) batch number 1 Sanguisorba officinalis 1g Chengdu Raifense / DZYC-S-019 2 Sanguisorba officinalis charcoal / Zhuzhou Qianjin Pharmaceutical Co., Ltd. / Y019-2003001 3 Fuke Duanhongyin Capsule / Zhuzhou Qianjin Pharmaceutical Co., Ltd. / 20200702 4 Lack of sanguisorba officinalis negative / Zhuzhou Qianjin Pharmaceutical Co., Ltd. / /

[0117] 3 Results

[0118] The samples were extracted by the extraction method of Example 1, the sanguisorba / sanguisorba carbonisata was used as the control medicinal material, and different developing agents were used for thin-layer chromatography identification of Fuke Duanhongyin Capsules. The identification results are as Figures 5 to 12 . 1 - Sanguisorba control medicinal material DZYC - S - 019, 2 - Sanguisorba carbonisata Y019 - 2003001, 3 - The method of Comparative Example 2 for Fuke Duanhongyin Capsules on July 2, 2020, 4 - Negative without sanguisorba carbonisata in the method of Comparative Example 2.

[0119] Figure 5 The results showed that Rf = 0.79 was too high, so the developing agent of Comparative Example 3 was not applicable.

[0120] Figure 6 The results show that: Rf = 0.44 is appropriate, but chloroform is highly toxic, so the developing agent in Comparative Example 4 is not applicable.

[0121] Figure 7 The results show that: Rf = 0.48 is suitable, but it is similar to chloroform, highly toxic, and the ratio of the two-phase solvents in this developing system varies greatly, so the developing agent in Comparative Example 5 is not applicable.

[0122] Figure 8 The results show that: Rf = 0.07 / 0.08 is too low, so the developing agent in Comparative Example 6 is not applicable.

[0123] Fig. 9 The results show that: Rf = 0.34 is moderate, but the preparation of the developing agent is more complex and the safety is low, so the developing agent in Comparative Example 7 is not applicable.

[0124] Fig.10 The results show that: Rf = 0.39 / 0.38 is moderate, so the developing agent in Comparative Example 8 is applicable, that is, a mixed solution of toluene, ethyl acetate, and formic acid is used as the developing agent, and the volume ratio of toluene:ethyl acetate:formic acid is 20:4:0.5. 10% sulfuric acid ethanol solution is used as the color developing agent or not added for display, and it is inspected under ultraviolet 365 nm; preferably, no color developing agent is added, that is, a mixed solution of toluene, ethyl acetate, and formic acid is used as the developing agent, and the volume ratio of toluene:ethyl acetate:formic acid is 20:4:0.5, and it is inspected under ultraviolet 365 nm, which is also the condition of Example 1 of this application.

[0125] Fig.11 The results show that: Rf = 0.19 is too low, so the developing agent in Comparative Example 9 is not applicable.

[0126] Fig.12 The results show that: Rf = 0.21 is too low, so the developing agent in Comparative Example 10 is not applicable.

[0127] Therefore, the method of extracting samples by the extraction method of Example 1 (Comparative Example 2), using sanguisorba root as the control medicinal material, and performing thin-layer chromatography identification with the developing agent and chromatographic conditions of Example 1 is applicable to the thin-layer chromatography identification of Fuke Duanhongyin Capsules. It can be seen from the chromatogram that the preferred specific point is specific to the sanguisorba root medicinal material and is transferred to the sanguisorba charcoal medicinal material after processing, so it can be used as the specific point for qualitative identification. The sanguisorba charcoal control medicinal material is not available from the National Institutes for Food and Drug Control, and the sanguisorba root control medicinal material can be used as a reference.

[0128] Comparative Example 11

[0129] The thin-layer identification of charred sanguisorba root in Fuke Duanhongyin Capsules was carried out by the method disclosed in Patent CN105241997A. Take 1.0 g of the content of Fuke Duanhongyin Capsules, place it in a conical flask, add 15 mL of water and dissolve it by ultrasonic wave, then add 5 mL of 15% hydrochloric acid, heat it in a water bath at 98 °C for 1 hour, cool it, filter it, extract the filtrate with ether by shaking for 2 times, 20 mL each time, combine the ether solutions, evaporate to dryness, dissolve the residue in 1.5 mL of methanol to prepare the test solution. Separately take 2 g of the control medicinal material of sanguisorba root, add 80 mL of water, boil for 30 minutes, filter, concentrate the filtrate to 15 mL, add 5 mL of 15% hydrochloric acid, and prepare the control medicinal material solution in the same way as the test solution preparation method for the rest.

[0130] According to the chromatographic conditions of Example 1, the difference is only that a mixed solution with a volume ratio of toluene: ethyl formate: formic acid of 6:5:0.5 is used as the developing agent.

[0131] The thin-layer identification of charred sanguisorba root in Fuke Duanhongyin Capsules was carried out, and the identification results are as Fig.13 . The identification results show that the specificity of this method has not passed, and it cannot effectively distinguish the control medicinal material of sanguisorba root, the negative sample without charred sanguisorba root, and the sample of Fuke Duanhongyin Capsules.

[0132] Experimental Example 1 Influence of Different Thin-Layer Plates on the Thin-Layer Chromatographic Identification of Charred Sanguisorba Root in Fuke Duanhongyin Capsules

[0133] 1. Experimental Method

[0134] The thin-layer chromatographic identification of charred sanguisorba root in Fuke Duanhongyin Capsules was carried out by the method of Example 1 using thin-layer G plates from different manufacturers.

[0135] 2. The information of the samples and the control medicinal materials is the same as that in Example 1. The information of the thin-layer plates is as follows:

[0136]

[0137]

[0138] 3. Results

[0139] In the chromatogram of the test solution, spots of the same color appear at the positions corresponding to the chromatogram of the control medicinal material. The results of the thin-layer chromatographic identification of charred sanguisorba root in Fuke Duanhongyin Capsules using different thin-layer plates are as Fig.14 shown. In Figure 14, 1 - blank solvent (methanol), 2 - control medicinal material solution of sanguisorba root, 3 - negative sample solution without charred sanguisorba root, 4 - 6 are the test solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102; A - Qingdao Ocean Chemical G thin-layer plate, B - Qingdao Hailang Silica Gel G thin-layer plate, C - Merck TLC Silica gel 60G thin-layer plate, D - self-made G thin-layer plate.

[0140] From Fig.14 It can be seen that the Rf values of the identification bands under the thin-layer plates of different manufacturers A to D are 0.35, 0.40, 0.25, and 0.43 respectively. Although the bands identified by the C-Merck TLC Silica gel 60G thin-layer plate are shifted downward as a whole, the thin-layer chromatographic behaviors of the Fufang Duanhongyin Capsules are basically the same. This shows that the thin-layer chromatographic method of Example 1 of this application can be applied to different G thin-layer plates and has good durability; the durability and feasibility of identifying Fufang Duanhongyin Capsules and the negative sample of Fufang Duanhongyin Capsules without Radix Sanguisorbae Carbonisata by the thin-layer chromatographic method of Example 1 are good.

[0141] Experimental Example 2 Influence of Different Temperatures on the Thin-Layer Chromatographic Identification of Radix Sanguisorbae Carbonisata in Fufang Duanhongyin Capsules

[0142] 1. Experimental Method

[0143] The thin-layer chromatographic identification of Radix Sanguisorbae Carbonisata in Fufang Duanhongyin Capsules was carried out according to the method of Example 1 at 4°C, 20°C, and 30°C respectively.

[0144] 2. The information of the samples and the control medicinal materials is the same as that in Example 1.

[0145] 3. Results

[0146] In the chromatogram of the test sample, at the position corresponding to the chromatogram of the control medicinal material, spots of the same color appeared. The results of the thin-layer chromatographic identification of Radix Sanguisorbae Carbonisata in Fufang Duanhongyin Capsules at different temperatures are as Fig.15 shown. Fig.15 In which 1 - blank solvent (methanol), 2 - solution of the control medicinal material of Radix Sanguisorbae, 3 - negative control solution of Fufang Duanhongyin Capsules without Radix Sanguisorbae Carbonisata, 4 - 6 are the test sample solutions of Fufang Duanhongyin Capsules with batch numbers 20200704, 20200906, and 20210102; A - 20°C, B - 4°C, C - 30°C.

[0147] From Fig.15 It can be seen that the Rf values of the identification bands at different temperatures A to C are 0.35, 0.25, and 0.31 respectively. Although the bands identified at 4°C are shifted downward as a whole, the thin-layer chromatographic behaviors of the Fufang Duanhongyin Capsules are basically the same. This shows that the thin-layer chromatographic method of Example 1 of this application has good durability in the temperature range of 4°C to 30°C; the durability and feasibility of identifying Fufang Duanhongyin Capsules and the negative sample of Fufang Duanhongyin Capsules without Radix Sanguisorbae Carbonisata by the thin-layer chromatographic method of Example 1 are good.

[0148] Experimental Example 3 Influence of Different Humidities on the Thin-Layer Chromatographic Identification of Radix Sanguisorbae Carbonisata in Fufang Duanhongyin Capsules

[0149] 1. Experimental Method

[0150] The thin-layer chromatography identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules was carried out according to the method of Example 1 at relative humidities of 33%, 50% and 70% respectively.

[0151] The information of the samples and the control medicinal materials was the same as that in Example 1.

[0152] 3. Results

[0153] In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as the chromatogram of the control medicinal material. The results of the thin-layer chromatography identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules under different humidities are as Fig.16 shown. Fig.16 In Figure 1 - blank solvent (methanol), 2 - solution of the control medicinal material of Sanguisorbae, 3 - solution of the negative sample without Sanguisorbae Carbonis, 4 - 6 are the test sample solutions of Fuke Duanhongyin Capsules with batch numbers 20200704, 20200906, 20210102; A - 50%, B - 33%, C - 70%.

[0154] From Fig.16 it can be seen that the Rf values of the identification bands under different humidities of A - C are 0.35, 0.32, 0.33 respectively, indicating that within the range of relative humidity of 33% - 70%, the thin-layer chromatography behavior of Fuke Duanhongyin Capsules is basically consistent. It shows that the thin-layer chromatography method in Example 1 of this application has good durability within the humidity range of 33% - 70%; the durability and feasibility of identifying Fuke Duanhongyin Capsules and the negative sample of Fuke Duanhongyin Capsules without Sanguisorbae Carbonis by the thin-layer chromatography method in Example 1 are good.

[0155] Experimental Example 4 Detection of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules of Different Batches

[0156] 1. Experimental method

[0157] Take 16 batches of Fuke Duanhongyin Capsules produced by Zhuzhou Qianjin Pharmaceutical Co., Ltd. (including 1 batch of negative sample without Sanguisorbae Carbonis), and carry out thin-layer chromatography identification according to the method of Example 1.

[0158] 2. Information of the samples and the control medicinal materials

[0159]

[0160]

[0161] 3. Results

[0162] The results of the thin-layer chromatography identification of Sanguisorbae Carbonis in Fuke Duanhongyin Capsules of different batches are as Fig.17 . Fig.171 - Blank solvent (methanol), 2 - Solution of the reference crude drug of Sanguisorba officinalis, 3 - Negative sample solution without Sanguisorbae Carbonis, 4 - 18 - Fufuke Duanhongyin capsules with batch numbers 20200701, 20200702, 20200703, 20200704, 20200705, 20200706, 20200805, 20200806, 20200906, 20201105, 20201202, 20210102, 20210201, 20210301, 20210302

[0163] Fig.17 The results showed that: when inspected under 365 nm, in the chromatograms of the test samples of 16 batches of Fufuke Duanhongyin capsules, except for 1 batch of negative sample without Sanguisorbae Carbonis, the other 15 batches of samples showed spots of the same color at the corresponding positions of the reference chromatogram, indicating that the TLC method of Example 1 can accurately and rapidly identify Sanguisorbae Carbonis in Fufuke Duanhongyin capsules.

[0164] The above - mentioned embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above - mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A thin-layer chromatography identification method for carbonized sanguisorba root in Fuke Duanhongyin Capsules, characterized in that, it includes the following steps: S1. Preparation of the test solution Extract the content of Fuke Duanhongyin Capsules with water, take the water extract liquid, extract the water extract liquid with n-butanol saturated with water to obtain an n-butanol extract, wash the n-butanol extract with ammonia test solution, remove the solvent from the fully washed n-butanol extract to obtain a solid sample, and dissolve the solid sample in methanol to serve as the test solution; S2. Preparation of the control crude drug solution Prepare the carbonized sanguisorba root control crude drug solution from the carbonized sanguisorba root control crude drug according to the preparation method of the test solution in step S1; S3. Identification by thin-layer chromatography Spot the test solution prepared in step S1 and the control crude drug solution prepared in step S2 on the same silica gel G thin-layer plate respectively, use a mixed solution of toluene, ethyl acetate and formic acid as the developing agent, and place it under ultraviolet light at 365 nm for inspection for thin-layer chromatography identification, the volume ratio of toluene: ethyl acetate: formic acid in the mixed solution is 15-30:3-5:0.5-1.

5.

2. The method according to claim 1, characterized in that, it includes the following steps: S1. Preparation of the test solution Extract the content of Fuke Duanhongyin Capsules with water. The specific method is: dissolve the content of Fuke Duanhongyin Capsules in water, filter after ultrasonic treatment for 30-60 min to fully dissolve, and the mass-volume ratio of the content of Fuke Duanhongyin Capsules to water is 0.5-2 g:20-30 mL; Take the water extract liquid, extract the water extract liquid with n-butanol saturated with water to obtain an n-butanol extract. The specific method is: extract the filtrate with n-butanol saturated with water 3-6 times to obtain an n-butanol solution; Wash the n-butanol extract with ammonia test solution. The volume ratio of water to n-butanol saturated with water is 4-6:11-14, and the volume ratio of n-butanol saturated with water to ammonia reagent is 3-5:1-3; remove the solvent from the washed n-butanol extract by evaporation to obtain a solid sample, and dissolve the solid sample in methanol to serve as the test solution. The mass-volume ratio of the content of Fuke Duanhongyin Capsules to methanol is 0.5-2 g:0.5-2.5 mL; S2. Preparation of the control crude drug solution Prepare the carbonized sanguisorba root control crude drug solution from the carbonized sanguisorba root control crude drug according to the preparation method of the test solution in step S1. The mass-volume ratio of the carbonized sanguisorba root control crude drug to water is 0.1-1 g:20-30 mL; S3. Identification by thin-layer chromatography Absorb 2-5 μL of the test solution and 2 μL of the control crude drug solution, spot them on the same silica gel G thin-layer plate respectively, use a mixed solution of toluene, ethyl acetate and formic acid as the developing agent, and place it under ultraviolet light at 365 nm for inspection for thin-layer chromatography identification. The volume ratio of toluene: ethyl acetate: formic acid in the mixed solution is 15-30:3-5:0.5-1.

5.

3. The method according to claim 1, characterized in that, the mass-volume ratio of the content of Fuke Duanhongyin Capsules to water in step S1 is 0.5-1 g:20-25 mL.

4. The method according to claim 1, characterized in that, The volume ratio of water to water-saturated n-butanol in step S1 is 5-6:12-14.

5. According to the method described in claim 1, it is characterized in that the volume ratio of water-saturated n-butanol to ammonia reagent in step S1 is 3-4:1-2.

6. According to the method described in claim 1, it is characterized in that the mass-volume ratio of the reference medicinal material of Sanguisorba officinalis to water in step S2 is 0.5-1 g: 25-30 mL.

7. According to the method described in claim 1, it is characterized in that the volume ratio of toluene: ethyl acetate: formic acid in the mixed solution is 20-30:4-5:0.5-1.

8. According to the method described in any one of claims 1-7, it is characterized in that the developing temperature of the developing agent in step 3 is 4°C to 30°C.

9. According to the method described in any one of claim 1, it is characterized in that the developing humidity of the developing agent in step 3 is 33% to 70%.

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