Thin-layer chromatography identification method for vigor-preserving decoction and preparation of vigor-preserving decoction

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a thin-layer chromatography identification method of vitality-preserving decoction and a preparation thereof. Comprising the following steps: S1, extracting a sample to be detected by using diethyl ether, and preparing a test solution; s2, preparing a cinnamaldehyde reference substance solution and a 6-gingerol reference substance solution; the method further comprises at least one of the steps S3 and S4; s3, dripping the test solution and the cinnamaldehyde reference solution on the same thin-layer plate, developing with a first developing solvent, and identifying the cinnamon; the first developing solvent comprises petroleum ether and ethyl acetate in a volume ratio of (15-20): 3; s4, dripping the test solution and the 6-gingerol reference substance solution on the same thin-layer plate, developing with a second developing solvent, and identifying the ginger; the second developing solvent is prepared from petroleum ether, trichloromethane and ethyl acetate according to the volume ratio of (2-4): (1-2): (1-3). The method is good in overall separation effect, clear in spots, good in specificity and durability, and capable of identifying the medicine flavors of cinnamon and ginger in the vitality-preserving decoction and the preparation thereof.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Background Technology

[0002] The *Concise Medical Treatise* (by Sun Zhihong, Ming Dynasty) records the efficacy of Baoyuan Decoction as follows: It treats weakness of vital energy, fatigue, muscle weakness, poor appetite, pale complexion, restless sleep, and other symptoms all related to weakness. The prescription for Baoyuan Decoction consists of 1 qian of ginseng, 2 qian of astragalus, 5 fen of licorice root, and 2 fen of cinnamon, with one slice of ginger added. It is decocted in water and taken orally. Baoyuan Decoction's efficacy lies in invigorating qi and warming yang, making it a general formula for treating qi and blood deficiency. It is a holy medicine for tonifying metal and earth, clearing heat and relieving irritability. This formula contains ginseng, which greatly replenishes vital energy and treats weakness and fatigue; astragalus, which tonifies the lungs and strengthens the exterior, enhancing the body's defensive functions; licorice, which tonifies the spleen and harmonizes the middle jiao, harmonizing the effects of the other herbs; and cinnamon, which warms the kidneys and invigorates yang, promoting the circulation of qi and blood. With the guidance of cinnamon, the qi-tonifying effects of ginseng and astragalus are amplified; and with the harmonizing effect of licorice, cinnamon warms yang and regulates qi and blood. The combined effects of these herbs strengthen the spleen and warm the kidneys while protecting lung qi, thus treating conditions of insufficient vital energy and yang deficiency. It has wide clinical applications in modern medicine, commonly used to treat chronic heart failure, coronary heart disease, chronic renal failure, and other common geriatric diseases.

[0003] Thin-layer chromatography (TLC) is an important chromatographic analysis technique. Based on the principle of adsorption, it utilizes the interaction between the stationary phase and the mobile phase to separate, qualitatively and quantitatively analyze the components in a mixture. It is the main means of qualitative identification in the quality standards of traditional Chinese medicine compound preparations.

[0004] Existing technologies include some methods for thin-layer chromatography identification of Baoyuan Decoction, but some methods do not identify cinnamon and ginger; some methods identify cinnamon and ginger, but do not use cinnamaldehyde, a characteristic indicator of cinnamon, or 6-gingerol, a characteristic indicator of ginger, as a control, indicating that the selection of control substances is not scientific, reasonable, or comprehensive; in some identification methods, the developing solvent system for cinnamon is too complex, and the identification methods need to be optimized. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a thin-layer chromatographic identification method for Baoyuan Decoction and its preparations. This method uses cinnamaldehyde, a characteristic indicator component of cinnamon, and 6-gingerol, a characteristic indicator component of ginger, as controls. The first developing solvent for identifying cinnamon is petroleum ether or ethyl acetate with a boiling range of 60-90℃ and a volume ratio of 15-20:3. The developing system is simple, and the overall separation effect is good, the spots are clear, the specificity is strong, and the system is durable.

[0006] Therefore, the present invention provides the following technical solution: The present invention provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations, wherein the Baoyuan Decoction and its preparations are prepared from raw materials including cinnamon and ginger, and the thin-layer chromatography identification method includes steps S1 and S2: S1: Take the sample to be tested, extract it with diethyl ether, separate the diethyl ether layer, dry it, dissolve the residue in alcohol to obtain the test solution; S2: Take at least one of cinnamaldehyde reference standard and 6-gingerol reference standard, add organic solvent to prepare cinnamaldehyde reference standard solution and 6-gingerol reference standard solution respectively; It also includes at least one of steps S3 and S4; S3: Take the test solution and cinnamaldehyde reference solution, spot them separately on the same thin-layer plate, develop with the first developing solvent, remove, dry, spray with the first color developing agent, examine and identify cinnamon; The first developing solvent is petroleum ether or ethyl acetate with a boiling range of 60-90℃ and a volume ratio of 15-20:3; S4: Take the test solution and 6-gingerol reference solution, spot them separately on the same thin-layer plate, develop with the second developing solvent, remove, air dry, spray with the second color developing solvent, heat, examine, and identify ginger. The second developing agent is petroleum ether, chloroform, or ethyl acetate with a boiling range of 60-90℃ and a volume ratio of 2-4:1-2:1-3.

[0007] In one optional embodiment, before step S3, a step of preparing a cinnamon reference material solution is further included, comprising: taking cinnamon reference material, extracting it with water, and treating the extract using the sample processing method in step S1 to obtain the cinnamon reference material solution; before development, step S3 further includes spotting the cinnamon reference material solution onto the same thin-layer plate used in step S3; in a further optional embodiment, the spotting volume of the cinnamon reference material solution is 5~15 μL.

[0008] In one optional embodiment, before step S3, a step of preparing a ginger reference herb solution is further included, comprising: taking ginger reference herb, extracting it with water, and treating the extract using the sample processing method in step S1 to obtain the ginger reference herb solution; before development, step S4 further includes spotting the ginger reference herb solution onto the same thin-layer plate used in step S4; in a further optional embodiment, the spotting volume of the ginger reference herb solution is 5~15 μL.

[0009] In one optional embodiment, the step of preparing the cinnamon reference herb solution includes: taking 0.3-0.5g of cinnamon reference herb, adding 80-120mL of water and refluxing for 30-60 minutes, filtering, taking the filtrate, and treating the filtrate using the sample processing method in S1 to obtain the cinnamon reference herb solution.

[0010] In one optional embodiment, the step of preparing the ginger reference herb solution includes: taking 1-2g of ginger reference herb, adding 80-120mL of water and refluxing for 30-60 minutes, filtering, taking the filtrate, and treating the filtrate using the sample processing method in S1 to obtain the ginger reference herb solution.

[0011] In one optional embodiment, in step S2, each 1 mL of cinnamaldehyde reference solution contains 0.5–2 μL of cinnamaldehyde.

[0012] In one optional embodiment, in step S2, each 1 mL of 6-gingerol reference solution contains 0.3 to 1 mg of 6-gingerol.

[0013] In one optional embodiment, in S2, the organic solvent includes at least one of ethanol and methanol.

[0014] In an optional embodiment, in S3, the first colorimetric agent comprises a dinitrophenylhydrazine ethanol solution.

[0015] In an optional embodiment, in step S3, the first developing agent is petroleum ether or ethyl acetate with a boiling range of 60-90°C and a volume ratio of 17:3.

[0016] In one optional embodiment, in step S3, the sample volume of the test solution and the cinnamaldehyde reference solution is 5~15 μL.

[0017] In one optional embodiment, the unfolding temperature in step S3 is ≤40°C; in a further optional embodiment, the unfolding temperature is 5~30°C.

[0018] In one alternative embodiment, in step S4, the second colorimetric agent comprises a 2% to 8% vanillin sulfuric acid solution.

[0019] In an optional embodiment, in step S4, the second developing agent is petroleum ether, chloroform, or ethyl acetate with a boiling range of 60-90°C and a volume ratio of 2:1:1.

[0020] In one optional embodiment, the heating temperature in step S4 is 100~110°C.

[0021] In one optional embodiment, in step S4, the sample volume of the test solution and the 6-gingerol reference solution is 5~15 μL.

[0022] In one optional embodiment, the Baoyuan Decoction and its preparations include solid, semi-solid, or liquid formulations of Baoyuan Decoction; in a further optional embodiment, the Baoyuan Decoction and its preparations include a decoction of Baoyuan Decoction reference sample, a concentrated extract of Baoyuan Decoction reference sample, a freeze-dried powder of Baoyuan Decoction reference sample, or Baoyuan Decoction granules.

[0023] In one optional embodiment, in step S1, when the sample to be tested is a solid or semi-solid preparation of Baoyuan Tang, the step further includes dissolving the sample to be tested in water to obtain a liquid sample to be tested; in a further optional embodiment, the mass-to-volume ratio of the sample to water is 3~5g:100mL.

[0024] In one optional embodiment, in step S1, drying includes at least one of nitrogen blowing, evaporation, and steam drying; in a further optional embodiment, drying is nitrogen blowing. Using nitrogen blowing results in clearer spots in the final product and can also shorten the pretreatment time.

[0025] In one optional embodiment, in step S1, the volume ratio of the liquid sample to diethyl ether is 100:25~50.

[0026] In one alternative implementation, in S1, extraction includes extraction.

[0027] In one optional implementation, the extraction is performed 2 to 5 times in step S1.

[0028] In one optional embodiment, in step S1, the residue is dissolved in 0.5 to 2 mL of alcohol.

[0029] In one alternative embodiment, in S1, the alcohol includes at least one of ethanol and methanol.

[0030] In one optional embodiment, step S1 includes taking 100 mL of liquid sample to be tested, extracting it 2 to 5 times with 25 to 50 mL of diethyl ether, separating the diethyl ether layers and combining the layers, drying, and dissolving the residue in 0.5 to 2 mL of alcohol to obtain the test solution.

[0031] The technical solution of this invention has the following advantages: The thin-layer chromatography identification method for Baoyuan Decoction and its preparations provided by this invention includes steps S1 and S2: S1: Take the sample to be tested, extract it with ether, separate the ether layer, dry it, dissolve the residue in alcohol to obtain the test solution; S2: Take at least one of cinnamaldehyde reference standard and 6-gingerol reference standard, add organic solvents respectively to prepare cinnamaldehyde reference standard solution and 6-gingerol reference standard solution; and further includes at least one of steps S3 and S4; S3: Take the test solution and cinnamaldehyde reference standard solution, spot them respectively on the same thin-layer plate, and then... Develop with a first developing solvent, remove, air dry, spray with a first colorimetric reagent, examine, and identify cinnamon; the first developing solvent is petroleum ether and ethyl acetate with a boiling range of 60-90℃ in a volume ratio of 15-20:3; S4: Take the test solution and 6-gingerol reference solution, spot them separately on the same thin-layer plate, develop with a second developing solvent, remove, air dry, spray with a second colorimetric reagent, heat, examine, and identify ginger; the second developing solvent is petroleum ether, chloroform, and ethyl acetate with a boiling range of 60-90℃ in a volume ratio of 2-4:1-2:1-3. This thin-layer chromatography identification method has good overall separation effect, clear spots, strong specificity, and good durability. It can accurately identify the medicinal flavors of cinnamon and ginger in Baoyuan Decoction and its preparations, providing a good foundation for the quality control of Baoyuan Decoction and its preparations. Using the same test solution, both cinnamon and ginger can be distinguished without the need for multiple test solution preparations; the developing system is also simpler, saving testing time and costs. Furthermore, it uses relatively less organic solvent, making it more environmentally friendly. The selection of cinnamaldehyde and 6-gingerol as reference standards further enhances the scientific validity and rationality of this thin-layer chromatography identification method. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This is the thin-layer chromatogram obtained in identification ① of Example 1 of the present invention; Figure 2 This is the thin-layer chromatogram obtained in identification ② of Example 1 of the present invention; Figure 3 This is the thin-layer chromatogram obtained in Example 2 of the present invention; Figure 4 This is the thin-layer chromatogram obtained in Example 3 of the present invention; Figure 5 This is the thin-layer chromatogram obtained in Example 4 of the present invention; Figure 6This is the thin-layer chromatogram obtained in Example 5 of the present invention; Figure 7 This is the thin-layer chromatogram obtained in identification ① of Example 6 of the present invention; Figure 8 This is the thin-layer chromatogram obtained in identification ② of Example 6 of the present invention; Figure 9 This is the thin-layer chromatogram obtained in Comparative Example 1 of the present invention; Figure 10 This is the thin-layer chromatogram obtained in Comparative Example 2 of the present invention; Figure 11 This is the thin-layer chromatogram obtained in Comparative Example 3 of the present invention; Figure 12 This is the thin-layer chromatogram obtained in Comparative Example 4 of the present invention; Figure 13 This is the thin-layer chromatogram obtained in Comparative Example 5 of the present invention; Figure 14 This is the thin-layer chromatogram obtained in Comparative Example 6 of the present invention; Figure 15 This is the thin-layer chromatogram obtained in Comparative Example 7 of the present invention; Figure 16 This is the thin-layer chromatogram obtained in Comparative Example 8 of the present invention; Figure 17 This is the thin-layer chromatogram obtained by identification ① at an developing temperature of 6°C in Experiment Example 1 of this invention; Figure 18 This is the thin-layer chromatogram obtained by identification ② at an developing temperature of 6°C in Experiment Example 1 of this invention; Figure 19 This is the thin-layer chromatogram obtained by identification ① at an developing temperature of 40°C in Experiment Example 1 of this invention; Figure 20 This is the thin-layer chromatogram obtained by identification ② at an developing temperature of 40°C in Experiment Example 1 of this invention; Figure 21 This is the thin-layer chromatogram obtained by identification ① under the developing humidity of 32% in Experiment Example 2 of the present invention; Figure 22 This is the thin-layer chromatogram obtained by identification ② under the developing humidity of 32% in Experiment Example 2 of the present invention; Figure 23 This is the thin-layer chromatogram of the corresponding test solution of the Baoyuantang reference sample obtained by identification using Qingdao Ocean brand silica gel G thin-layer plate in Experiment Example 3 of the present invention; Figure 24 This is the thin-layer chromatogram of the corresponding test solution of the Baoyuantang reference sample obtained by identification using Merck brand silica gel G thin-layer plate in Experiment Example 3 of the present invention. Figure 25This is the thin-layer chromatogram of the corresponding test solution of Baoyuantang granules obtained by identification using Merck brand silica gel G thin-layer plate in Experiment Example 3 of the present invention. Figure 26 This is the thin-layer chromatogram of the corresponding test solution of the Baoyuantang reference sample obtained by identification using Qingdao Ocean brand silica gel G thin-layer plate in Experiment Example 3 of the present invention; Figure 27 This is the thin-layer chromatogram of the corresponding test solution of the Baoyuantang reference sample obtained by identification using Merck brand silica gel G thin-layer plate in Experiment Example 3 of the present invention. Figure 28 This is the thin-layer chromatogram of the corresponding test solution of Baoyuantang granules obtained by identification using Merck brand silica gel G thin-layer plate in Experiment Example 3 of the present invention. Detailed Implementation

[0034] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the reagents used in the following examples and comparative examples are conventional reagents and can be purchased from conventional reagent manufacturers and distributors.

[0036] 1. Experimental materials The reference sample for Baoyuan Decoction: 11.19g of ginseng, 22.38g of astragalus, 5.61g of licorice, 2.25g of cinnamon, and 9.00g of ginger were placed in a 2L clay pot, covered, and soaked for 30 minutes. For the first decoction, 12 times the total weight of the five ingredients were added to water and decocted for 45 minutes. For the second decoction, 10 times the total weight of the five ingredients were added to water and decocted for 35 minutes. The first and second decoctions were filtered through a 200-mesh filter cloth while hot, and the decoctions were combined to obtain the reference sample for Baoyuan Decoction.

[0037] Baoyuan Decoction Granules: According to the prescription ratio, take 532.86g of ginseng, 1065.71g of astragalus, 267.14g of licorice, 107.14g of cinnamon, and 428.57g of ginger (five medicinal herbs in total). Slice the ginger. Add water equal to 8 times the total weight of the medicinal herbs to the mixture and heat under reflux for 2 hours (reflux temperature is above 98℃ to boiling) for the first extraction. Collect aromatic water equal to 1 times the total weight of the medicinal herbs. Encapsulate the aromatic water with β-cyclodextrin to obtain an inclusion complex for later use. The aromatic water and β-cyclodextrin... The weight ratio was 50:3; after the first extraction, the liquid was filtered, and the residue was mixed with 8 times the total weight of the medicinal slices of water for a second extraction by heating and reflux (the heating and reflux temperature was the temperature of the liquid being heated to above 98℃ until boiling), and the extraction was carried out for 1 hour. The mixture was then filtered, and the filtrates from the two filtrations were combined and concentrated to a clear extract with a relative density of 1.05 to 1.10 (65℃ ± 5℃). The extract was dried to obtain a dry extract powder. The dry extract powder, the inclusion complex, 0.1% of magnesium stearate and an appropriate amount of maltodextrin were mixed evenly and granulated to obtain 1000g of Baoyuantang granules.

[0038] The negative samples lacking cinnamon and ginger were prepared using the same method as the Baoyuan Decoction reference sample, with the only difference being the absence of the corresponding medicinal slices.

[0039] The cinnamon-deficient and ginger-deficient granules were prepared using the same method as the Baoyuantang granules, with the only difference being the absence of the corresponding medicinal slices.

[0040] Cinnamaldehyde: China National Institutes for Food and Drug Control, batch number 110710-202223; 6-Gingerol: China National Institutes for Food and Drug Control, batch number 111833-202007; Cinnamon reference material: China National Institutes for Food and Drug Control, batch number 121363-202104; Ginger reference material: Guangdong Daxing Pharmaceutical Co., Ltd., batch number YCY23B01.

[0041] 2. Thin-layer plate The manufacturers and batch numbers of the thin-layer boards used are shown in Table 1.

[0042] Table 1

[0043] Example 1 This embodiment provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations, specifically including: Preparation of the test solution: Take 100 mL of Baoyuan Decoction reference sample and extract it three times with 30 mL of diethyl ether each time. Combine the ether layers. Dry the ether layer with nitrogen gas, and dissolve the residue in 2 mL of anhydrous ethanol to obtain the test solution. Prepare three parallel test solutions for use, and label them as Test Solution 1, Test Solution 2, and Test Solution 3.

[0044] Preparation of negative solution: Take 100 mL of cinnamon-deficient negative sample, extract with 30 mL of ether 3 times, and combine the ether layers; blow the ether layer dry with nitrogen, and dissolve the residue with 2 mL of anhydrous ethanol to obtain the cinnamon-deficient negative sample solution.

[0045] Take 100 mL of the ginger-deficient negative sample and extract it three times with 30 mL of ether each time. Combine the ether layers. Dry the ether layer with nitrogen gas and dissolve the residue in 2 mL of anhydrous ethanol to obtain the ginger-deficient negative sample solution.

[0046] Preparation of the reference herb solution: Take 0.4g of cinnamon reference herb, add 100mL of water and reflux for 45 minutes, filter, and use the filtrate to prepare the cinnamon reference herb solution in the same way as the test sample solution.

[0047] Take 1.5 g of ginger reference material, add 100 mL of water and reflux for 45 minutes, filter, and use the filtrate to prepare the ginger reference material solution using the same method as the test sample solution.

[0048] Preparation of reference solution: Take an appropriate amount of cinnamaldehyde reference standard and add anhydrous ethanol to prepare a reference solution containing 1 μL per 1 mL, which is used as the cinnamaldehyde reference solution.

[0049] Take an appropriate amount of 6-gingerol reference standard and add methanol to prepare a reference standard solution containing 0.5 mg per 1 mL, which is used as the 6-gingerol reference standard solution.

[0050] Identification: ① Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Take 10 μL each of the above-mentioned cinnamon-deficient negative sample solution, test solution 1, test solution 2, test solution 3, cinnamon reference material solution, and cinnamaldehyde reference solution, and spot them separately onto the same silica gel G thin-layer plate (Yantai brand). Use petroleum ether (60~90℃)-ethyl acetate (volume ratio 17:3) as the developing solvent. Develop (temperature 25℃, humidity RH 68%). Remove, air dry, spray with dinitrophenylhydrazine ethanol solution, air dry, and examine. The results are shown in the figure. Figure 1 . Figure 1 In the chromatogram, from left to right, are the cinnamon-deficient negative sample solution, test solution 1, test solution 2, test solution 3, cinnamaldehyde reference solution, and cinnamon reference herb solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference herb and the reference standard.

[0051] ② Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Take 10 μL each of the above-mentioned ginger-deficient negative sample solution, test solution 1, test solution 2, test solution 3, 6-gingerol reference solution, and ginger reference material solution, and spot them separately onto the same silica gel G thin-layer plate (Yantai brand). Use petroleum ether (60~90℃)-chloroform-ethyl acetate (volume ratio 2:1:1) as the developing solvent. Develop (temperature 25℃, humidity RH 68%). Remove, air dry, spray with 5% vanillin-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine the results. See below. Figure 2 . Figure 2 In the image, from left to right, are: a negative sample solution lacking ginger, test solution 1, test solution 2, test solution 3, 6-gingerol reference solution, and ginger reference herb solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0052] Example 2 This embodiment provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ① is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-ethyl acetate (20:3). The results are shown in [Figure 1]. Figure 3 . Figure 3 In the chromatogram, from left to right, are the cinnamon-deficient negative sample solution, test solution 1, test solution 2, test solution 3, cinnamaldehyde reference solution, and cinnamon reference herb solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference herb and the reference standard.

[0053] Example 3 This embodiment provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ① is performed in the identification steps, and the developing solvent used is petroleum ether (60~90℃)-ethyl acetate (15:3). The results are shown in [Figure 1]. Figure 4 . Figure 4 In the chromatogram, from left to right, are the cinnamon-deficient negative sample solution, test solution 1, test solution 2, test solution 3, cinnamaldehyde reference solution, and cinnamon reference herb solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference herb and the reference standard.

[0054] Example 4 This embodiment provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ② is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-chloroform-ethyl acetate (3:2:1). The results are shown in [Figure 1]. Figure 5 . Figure 5In the image, from left to right, are: a negative sample solution lacking ginger, test solution 1, test solution 2, test solution 3, 6-gingerol reference solution, and ginger reference herb solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0055] Example 5 This embodiment provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ② is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-chloroform-ethyl acetate (4:2:3). The results are shown in [Figure 1]. Figure 6 . Figure 6 In the image, from left to right, are: a negative sample solution lacking ginger, test solution 1, test solution 2, test solution 3, 6-gingerol reference solution, and ginger reference herb solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0056] Example 6 This embodiment provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that Baoyuan Decoction granules are used as the test sample. The preparation steps of the test sample solution are as follows: Take an appropriate amount of Baoyuan Decoction granules, grind them finely, take about 3.5g, accurately weigh it, add 100mL of water to dissolve it, and obtain a liquid sample to be tested. Extract it three times with 30mL of ether each time, and combine the ether layers. Dry the ether layer with nitrogen gas, and dissolve the residue in 2mL of anhydrous ethanol to obtain the test sample solution; prepare three parallel test sample solutions for use, and label them as test sample solution 1', test sample solution 2', test sample solution 3', and test sample solution 4' respectively. The results obtained in step ① of the identification method are shown in the figure. Figure 7 , Figure 7 In the chromatogram, 1-7 from left to right are: cinnamon-deficient negative sample solution, cinnamon reference material solution, cinnamaldehyde reference solution, test solution 1', test solution 2', test solution 3', and test solution 4'. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of the reference material and the reference standard. The results obtained in step ② of the identification procedure are shown below. Figure 8 , Figure 8 In the chromatogram, from left to right, are 6-gingerol reference solution, ginger-deficient negative sample solution, test solution 1', test solution 2', test solution 3', and test solution 4'. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions as in the chromatogram of the reference solution.

[0057] Comparative Example 1 This comparative example provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ① is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-ethyl acetate (10:3). The results are shown in [Figure 1]. Figure 9 . Figure 9 In the middle, from left to right, are the cinnamon-deficient negative sample solution, cinnamaldehyde reference solution, cinnamon reference medicinal material solution, test solution 1, test solution 2, and test solution 3.

[0058] Comparative Example 2 This comparative example provides a thin-layer chromatographic identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ① is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-ethyl acetate (25:3). The results are shown in [Figure 1]. Figure 10 . Figure 10 In the middle, from left to right, are the cinnamon-deficient negative sample solution, cinnamaldehyde reference solution, cinnamon reference medicinal material solution, test solution 1, test solution 2, and test solution 3.

[0059] Comparative Example 3 This comparative example provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ① is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-ethyl formate (17:3). The results are shown in [Figure 1]. Figure 11 . Figure 11 In the middle, from left to right, are the cinnamon-deficient negative sample solution, cinnamaldehyde reference solution, cinnamon reference medicinal material solution, test solution 1, test solution 2, and test solution 3.

[0060] from Figures 9 to 11 As can be seen, under the developing solvent systems used in Comparative Examples 1 to 3, the separation effect of each component spot was poor and the display was unclear, resulting in poor identification effect of cinnamon flavor.

[0061] Comparative Example 4 This comparative example provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ② is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-chloroform-ethyl acetate (0.5:1:1). The results are shown in [Figure 1]. Figure 12 . Figure 12 In the middle, from left to right, are the negative sample solution lacking ginger, the 6-gingerol reference solution, the ginger reference medicinal material solution, the test solution 1, the test solution 2, and the test solution 3.

[0062] Comparative Example 5 This comparative example provides a thin-layer chromatographic identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ② is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-chloroform-ethyl acetate (5:1:1). The results are shown in [Figure 1]. Figure 13 . Figure 13In the middle, from left to right, are the negative sample solution lacking ginger, the 6-gingerol reference solution, the ginger reference medicinal material solution, the test solution 1, the test solution 2, and the test solution 3.

[0063] Comparative Example 6 This comparative example provides a thin-layer chromatographic identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ② is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-chloroform-ethyl acetate (2:0.5:4). The results are shown in [Figure 1]. Figure 14 . Figure 14 In the middle, from left to right, are the negative sample solution lacking ginger, the 6-gingerol reference solution, the ginger reference medicinal material solution, the test solution 1, the test solution 2, and the test solution 3.

[0064] Comparative Example 7 This comparative example provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ② is performed in the identification process, and the developing solvent used is petroleum ether (60~90℃)-chloroform-ethyl formate (2:1:1). The results are shown in [Figure 1]. Figure 15 . Figure 15 In the middle, from left to right, are the negative sample solution lacking ginger, the 6-gingerol reference solution, the ginger reference medicinal material solution, the test solution 1, the test solution 2, and the test solution 3.

[0065] Comparative Example 8 This comparative example provides a thin-layer chromatography identification method for Baoyuan Decoction and its preparations. Compared with Example 1, the only difference is that only step ② is performed in the identification process, and the developing solvent used is n-hexane-ethyl acetate (9:5). The results are shown in [Figure 1]. Figure 16 . Figure 16 In the middle, from left to right, are the negative sample solution lacking ginger, the 6-gingerol reference solution, the ginger reference medicinal material solution, the test solution 1, the test solution 2, and the test solution 3.

[0066] from Figures 12 to 16 As can be seen, the thin-layer chromatograms obtained using the developing solvent systems in Comparative Examples 4 to 8 exhibit problems such as poor spot shape and poor separation between spots. Specifically, in Comparative Example 5, it was almost impossible to develop each test solution; in Comparative Example 7, there was obvious overlap between the spots; in Comparative Examples 4, 6, and 8, the separation of the spots was not thorough, and the edges of the spots were unclear; the identification effect of ginger flavor was poor.

[0067] Experiment 1: Investigation of Temperature 1. The only difference between this experimental example and Example 1 is that only step ① is performed in the identification process, and the development temperature is 6℃. The results are shown in [the table below]. Figure 17 . Figure 17 In the middle, from left to right, are the cinnamon-deficient negative sample solution, test solution 1, test solution 2, test solution 3, cinnamaldehyde reference solution, and cinnamon reference medicinal material solution.

[0068] 2. The only difference between this experimental example and Example 1 is that only step ② is performed in the identification process, and the development temperature is 6℃. The results are shown in [the table below]. Figure 18 . Figure 18 In the middle, from left to right, are the negative sample solution lacking ginger, test solution 1, test solution 2, test solution 3, 6-gingerol reference solution, and ginger reference medicinal material solution.

[0069] 3. The only difference between this experimental example and Example 6 is that only step ① is performed in the identification process, and the development temperature is 40℃. The results are shown in [the table below]. Figure 19 . Figure 19 In the middle, from left to right, are the cinnamon-deficient negative sample solution, cinnamon reference medicinal material solution, cinnamaldehyde reference solution, test solution 1', test solution 2', test solution 3', and test solution 4'.

[0070] 4. The only difference between this experimental example and Example 6 is that only step ② is performed in the identification process, and the development temperature is 40℃. The results are shown in [the table below]. Figure 20 . Figure 20 In the middle, from left to right, are 6-gingerol reference solution, ginger-deficient negative sample solution, test solution 1', test solution 2', test solution 3', and test solution 4'.

[0071] Based on the results of the above experimental examples and Examples 1 and 6, it can be seen that different temperatures have a certain impact on the Rf values ​​of each spot, but the separation effect is not significantly different, indicating that this method has good durability in an ambient temperature range of 6~40℃. Excessively high temperatures will cause the spots to appear lighter in color, especially in the step of identifying cinnamon. In practical applications, the development should be carried out at a temperature close to room temperature as much as possible.

[0072] Experiment Example 2 investigates humidity. 1. The only difference between this experimental example and Example 1 is that only step ① is performed in the identification process, and the development humidity is RH 32%. The results are shown in [Figure 1]. Figure 21 . Figure 21 In the middle, from left to right, are the cinnamon-deficient negative sample solution, test solution 1, test solution 2, test solution 3, cinnamaldehyde reference solution, and cinnamon reference medicinal material solution.

[0073] 2. The only difference between this experimental example and Example 1 is that only step ② is performed in the identification process, and the development temperature is RH32%. The results are shown in [Figure 1]. Figure 22 . Figure 22In the middle, from left to right, are the negative sample solution lacking ginger, test solution 1, test solution 2, test solution 3, 6-gingerol reference solution, and ginger reference medicinal material solution.

[0074] Based on the results of the above experimental examples and Example 1, it can be seen that different humidity levels have a certain impact on the Rf values ​​of each spot, but there is no significant difference in the separation effect and spot display, indicating that this method has good durability under RH 32% ~ RH68% humidity.

[0075] Experiment Example 3: Investigation of Different Thin-Layer Plates 1. The only difference between this experimental example and Example 1 is that only step ① is performed in the identification process, and the thin-layer plate used is a silicone G thin-layer plate (Qingdao Ocean brand). The results are shown in [the table below]. Figure 23 . Figure 23 In the middle, from left to right, are the cinnamon-deficient negative sample solution, test solution 1, test solution 2, test solution 3, cinnamaldehyde reference solution, and cinnamon reference medicinal material solution.

[0076] 2. The only difference between this experimental example and Example 1 is that only step ① is performed in the identification process, and the thin-layer plate used is a silicone G thin-layer plate (Merck brand). The results are shown in [the table below]. Figure 24 . Figure 24 In the middle, from left to right, are the cinnamon-deficient negative sample solution, test solution 1, test solution 2, test solution 3, cinnamaldehyde reference solution, and cinnamon reference medicinal material solution.

[0077] 3. The only difference between this experimental example and Example 6 is that only step ① is performed in the identification process, and the thin-layer plate used is a silicone G thin-layer plate (Merck brand). The results are shown in [the table below]. Figure 25 . Figure 25 In the middle, 1 to 7 from left to right are, respectively, the cinnamon-deficient negative sample solution, the cinnamaldehyde reference solution, the cinnamon reference medicinal material solution, the test solution 1', the test solution 2', the test solution 3', and the test solution 4'.

[0078] 4. The only difference between this experimental example and Example 1 is that only step ② is performed in the identification process, and the thin-layer plate used is a silicone G thin-layer plate (Qingdao Ocean brand). The results are shown in [the table below]. Figure 26 . Figure 26 In the middle, from left to right, are the negative sample solution lacking ginger, test solution 1, test solution 2, test solution 3, 6-gingerol reference solution, and ginger reference medicinal material solution.

[0079] 5. Compared with Example 1, the only difference in this experimental example is that only step ② is performed in the identification process, and the thin-layer plate used is a silicone G thin-layer plate (Merck brand). The results are shown in […]. Figure 27 . Figure 27In the middle, from left to right, are the negative sample solution lacking ginger, test solution 1, test solution 2, test solution 3, 6-gingerol reference solution, and ginger reference medicinal material solution.

[0080] 6. The only difference between this experimental example and Example 6 is that only step ② is performed in the identification process, and the thin-layer plate used is a silicone G thin-layer plate (Merck brand). The results are shown in […]. Figure 28 . Figure 28 In the middle, from left to right, are 6-gingerol reference solution, ginger-deficient negative sample solution, test solution 1', test solution 2', test solution 3', and test solution 4'.

[0081] Based on the results of the above experimental examples and Examples 1 and 6, it can be seen that different thin-layer plates have a certain influence on the Rf value of each spot, but there is no significant difference in the separation effect and spot display, indicating that the method has good durability on different thin-layer plates.

[0082] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A thin-layer chromatographic identification method for Baoyuan Decoction and its preparations, wherein the Baoyuan Decoction and its preparations are prepared from raw materials including cinnamon and ginger, characterized in that... The thin-layer chromatography identification method includes steps S1 and S2: S1: Take the sample to be tested, extract it with diethyl ether, separate the diethyl ether layer, dry it, dissolve the residue in alcohol to obtain the test solution; S2: Take at least one of cinnamaldehyde reference standard and 6-gingerol reference standard, add organic solvent to prepare cinnamaldehyde reference standard solution and 6-gingerol reference standard solution respectively; It also includes at least one of steps S3 and S4; S3: Take the test solution and cinnamaldehyde reference solution, spot them separately on the same thin-layer plate, develop with the first developing solvent, remove, dry, spray with the first color developing agent, examine and identify cinnamon; The first developing solvent is petroleum ether or ethyl acetate with a boiling range of 60-90℃ and a volume ratio of 15-20:3; S4: Take the test solution and 6-gingerol reference solution, spot them separately on the same thin-layer plate, develop with the second developing solvent, remove, air dry, spray with the second color developing solvent, heat, examine, and identify ginger. The second developing agent is petroleum ether, chloroform, or ethyl acetate with a boiling range of 60-90℃ and a volume ratio of 2-4:1-2:1-3.

2. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to claim 1, characterized in that, Before step S3, the method further includes preparing a cinnamon reference solution, comprising: taking cinnamon reference material, extracting it with water, and treating the extract using the sample processing method in step S1 to obtain the cinnamon reference solution; before development, step S3 further includes spotting the cinnamon reference solution onto the same thin-layer plate used in step S3; optionally, the spotting volume of the cinnamon reference solution is 5~15μL. And / or, before S3, the method further includes a step of preparing a ginger reference herb solution, including: taking ginger reference herb, extracting it with water, and treating the extract using the sample processing method in S1 to obtain a ginger reference herb solution; before development, S4 further includes spotting the ginger reference herb solution onto the same thin-layer plate used in step S4; optionally, the spotting volume of the ginger reference herb solution is 5~15μL.

3. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to claim 2, characterized in that, The steps for preparing the cinnamon reference herb solution include: taking 0.3-0.5g of cinnamon reference herb, adding 80-120mL of water and refluxing for 30-60 minutes, filtering, taking the filtrate, and treating the filtrate using the sample processing method in S1 to obtain the cinnamon reference herb solution. And / or, the step of preparing the ginger reference herb solution includes: taking 1-2g of ginger reference herb, adding 80-120mL of water and refluxing for 30-60 minutes, filtering, taking the filtrate, and treating the filtrate using the sample processing method in S1 to obtain the ginger reference herb solution.

4. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to any one of claims 1 to 3, characterized in that, In S2, each 1 mL of cinnamaldehyde reference solution contains 0.5~2 μL of cinnamaldehyde; And / or, in S2, each 1 mL of 6-gingerol reference solution contains 0.3~1 mg of 6-gingerol; And / or, in S2, the organic solvent includes at least one of ethanol and methanol.

5. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to any one of claims 1 to 3, characterized in that, In S3, the first colorimetric agent includes a dinitrophenylhydrazine ethanol solution; And / or, in S3, the first developing agent is petroleum ether or ethyl acetate with a boiling range of 60~90℃ and a volume ratio of 17:3; And / or, in S3, the spotting volume of the test solution and the cinnamaldehyde reference solution is 5~15μL.

6. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to claim 5, characterized in that, In step S3, the unfolding temperature is ≤40℃; optionally, the unfolding temperature is 5~30℃.

7. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to any one of claims 1 to 3, characterized in that, In S4, the second colorimetric agent comprises a 2% to 8% vanillin sulfuric acid solution; And / or, in S4, the second developing agent is petroleum ether, chloroform, or ethyl acetate with a boiling range of 60-90°C and a volume ratio of 2:1:1; And / or, in S4, the heating temperature is 100~110℃; And / or, in S4, the spotting volume of the test solution and the 6-gingerol reference solution is 5~15 μL.

8. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to any one of claims 1 to 7, characterized in that, The Baoyuan Decoction and its preparations include solid, semi-solid, or liquid preparations of Baoyuan Decoction; optionally, the Baoyuan Decoction and its preparations include a decoction of Baoyuan Decoction reference sample, a concentrated extract of Baoyuan Decoction reference sample, a freeze-dried powder of Baoyuan Decoction reference sample, or Baoyuan Decoction granules.

9. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to claim 8, characterized in that, In step S1, when the sample to be tested is a solid or semi-solid preparation of Baoyuan Tang, the step of dissolving the sample to be tested in water to obtain a liquid sample to be tested is further included; optionally, the mass-volume ratio of the sample to water is 3~5g:100mL. And / or, in S1, drying includes at least one of nitrogen blowing, evaporation, and drying; optionally, drying is nitrogen blowing. And / or, in S1, the volume ratio of the liquid sample to diethyl ether is 100:25~50; And / or, in S1, extraction includes extraction; And / or, in S1, the number of extractions is 2 to 5; And / or, in S1, the residue is dissolved in 0.5-2 mL of alcohol; And / or, in S1, the alcohol includes at least one of ethanol and methanol.

10. The thin-layer chromatographic identification method for Baoyuan Decoction and its preparations according to claim 9, characterized in that, S1 includes taking 100 mL of liquid sample to be tested, extracting it 2 to 5 times with 25 to 50 mL of diethyl ether, separating the diethyl ether layers and combining the layers, drying, and dissolving the residue in 0.5 to 2 mL of alcohol to obtain the test solution.

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