Thin-layer chromatography identification method for cinnamon and ginger in vitality-preserving granules

Abstract

The invention discloses a thin-layer chromatography identification method for cinnamon and ginger in vitality-preserving granules, and belongs to the technical field of pharmaceutical analysis. Aiming at the problems of step-by-step detection, serious cinnamic acid tailing and dependence on toxic solvents in the existing method, the method comprises the following steps: dissolving vitality-preserving particles in water, carrying out ultrasonic treatment, extracting filtrate by using absolute ether, merging extract liquor, evaporating to dryness, and dissolving by adding ethyl acetate to obtain a test solution; preparing a cinnamon reference medicinal material solution and a cinnamic acid and 6-gingerol reference substance solution; a silica gel GF254 thin-layer plate is adopted, petroleum ether-normal hexane-ethyl formate-formic acid is used as a developing solvent for developing, and inspection is conducted under the ultraviolet light of 254 nm and the ultraviolet light of 365 nm respectively. Cinnamon and ginger can be synchronously identified, spots are clear and free of trailing, no toxic solvent exists, durability is high, cost is low, and an accurate and reliable basis can be provided for quality control of the vitality-preserving granules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and specifically proposes a thin-layer chromatography method for the identification of cinnamon and ginger in Baoyuan granules. Background Technology

[0002] Baoyuan Decoction, a classic Chinese medicine formula, first appeared in "Bo Ai Xin Jian" by Wei Guiyan of the Ming Dynasty. It was later systematically recorded in several authoritative medical books, including "Yi Fang Kao," "Jing Yue Quan Shu," and "Jian Ming Yi Gu," and was included in the "Catalogue of Ancient Classic Formulas (First Batch)" issued by the State Administration of Traditional Chinese Medicine. It is a representative medicine in the category of tonifying qi and warming yang in TCM clinical practice. This formula is composed of five herbs: ginseng, astragalus, licorice, cinnamon, and ginger. Its core function is to tonify qi and warm yang, comprehensively replenishing the spleen, lung, and kidney qi. Clinically, it is often used to improve symptoms such as weakness, fatigue, lethargy, and cold limbs caused by insufficient vital energy. It also has a clear effect on enhancing the body's immunity and has a long history of application in TCM health preservation and disease treatment.

[0003] With the modernization of traditional Chinese medicine preparations, Baoyuan Decoction has been developed into a granule form (i.e., Baoyuan Granules). This form has advantages such as convenient administration, accurate dosage, and good stability, making it more suitable for modern clinical medication needs. However, quality control of traditional Chinese medicine compound preparations has always been a challenge in the industry, and the identification of cinnamon and ginger in Baoyuan Granules is particularly prominent. Cinnamon is used in small amounts in the prescription (usually accounting for 5%-8% of the total weight of the prescription), and the content of target components (such as cinnamic acid) is low. Ginger is used fresh, and its characteristic components (such as 6-gingerol) are easily affected by processing techniques and storage conditions. In addition, the polysaccharides and saponins of ginseng, astragalus, and other medicinal materials in the prescription can easily interfere with identification, making the accurate identification of the two significantly more difficult than that of other medicinal ingredients.

[0004] Thin-layer chromatography (TLC) has become a core technical means for identification in the quality control of Chinese medicinal materials and prepared Chinese medicines due to its simple operation, rapid detection, high sensitivity and low cost. It is also widely used for the qualitative detection of single herbs in Chinese medicine compound prescriptions. However, in the field of simultaneous identification of multiple medicinal ingredients in traditional Chinese medicine compound prescriptions, existing technologies still have significant limitations: On the one hand, most thin-layer chromatography identification methods require the design of independent chromatographic conditions for a single medicinal ingredient. For example, identifying cinnamon requires one developing system, and identifying ginger requires another developing system. This results in the need for multiple preparations of the test solution, multiple developments, and multiple inspections during the detection process, which is cumbersome and time-consuming, making it difficult to meet the needs of rapid quality screening and batch testing in the production process of Baoyuan granules. On the other hand, a few methods that attempt to achieve simultaneous identification of multiple medicinal ingredients have key technical defects. For example, the "Simultaneous Thin-Layer Chromatographic Identification Method of Cinnamic Acid and 6-Gingerol in Baoyuan Decoction" disclosed in patent CN118362677A, although claiming to be able to detect in one go, has serious tailing phenomena in actual applications (tailing factor > 2.0), blurred spot boundaries, poor separation and identification effect, and inability to accurately determine the presence of the target component. At the same time, the developing solvent used in this method contains chloroform, which is highly toxic. Long-term exposure can damage the respiratory and nervous systems of the testing personnel, and it does not conform to the development trend of "green, environmentally friendly, low-toxicity and safe" in modern Chinese medicine testing.

[0005] Furthermore, while the identification method for Baoyuan Decoction disclosed in patent CN116068075A can detect cinnamic acid and 6-gingerol, it requires two different developing systems to complete the detection in two separate steps. The first step uses a developing solvent targeting cinnamic acid to detect cinnamon, and the second step uses a developing solvent targeting 6-gingerol to detect ginger. The entire detection process takes more than 4 hours (each developing and drying takes 1.5-2 hours), which is far less efficient than actual production and inspection needs. In addition to the aforementioned patent, existing published quality standards for Baoyuan granules often suffer from problems such as "severe component tailing, irregular spots, pale color development, and significant negative interference" in their identification methods for cinnamon and ginger, failing to provide reliable assurance for the quality stability of Baoyuan granules.

[0006] Therefore, in order to address the technical challenges of identifying cinnamon and ginger in Baoyuan granules, such as "difficulty in simultaneous identification of multiple medicinal ingredients, poor separation effect, dependence on toxic solvents, and low detection efficiency," it is essential to develop a thin-layer chromatography identification method that can achieve simultaneous identification of the two ingredients under the same chromatographic conditions, with good separation, safety, environmental friendliness, and rapid and efficient results. This method is of great significance for improving the quality control level of Baoyuan granules and ensuring the effectiveness and safety of clinical medication. It is also a key requirement for promoting the construction of modern quality standards for ancient classic prescriptions. Summary of the Invention

[0007] In view of this, the present invention proposes a thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules. This method can achieve simultaneous identification of cinnamon and ginger under the same chromatographic conditions, significantly improving detection efficiency. At the same time, it has good chromatographic separation effect, high specificity and safety, providing a precise and reliable technical means for the quality control of Baoyuan granules.

[0008] The technical solution of this invention is achieved as follows: This invention provides the following technical solution through systematic optimization of the test sample preparation process, developing solvent system, and inspection conditions: Preparation of the test solution: Take Baoyuan granules and add purified water at a weight-volume ratio of (0.5g-1)g:(10-20)mL. Stir until the granules are completely dissolved, then place them in an ultrasonic instrument for ultrasonic treatment for 20-40min. After ultrasonic treatment, filter while hot and collect the filtrate. Add anhydrous diethyl ether to the obtained filtrate for extraction. The extraction is performed 1-3 times. The volume of anhydrous diethyl ether used in each extraction is (20-40)mL:(0.5-1)g of Baoyuan granules. After each extraction, allow the mixture to stand and separate into layers. Collect the upper ether phase and combine all the upper ether phases. Place the combined ether phase in a rotary evaporator and evaporate to dryness to obtain a pale yellow residue. Add ethyl acetate to the residue to dissolve it. The volume of ethyl acetate used to dissolve it is (0.5-1)mL:(0.5-1)g of Baoyuan granules. Stir until the residue is completely dissolved, filter, and take the filtrate as the test solution.

[0009] Preparation of the control solution: Cinnamon reference material solution: Take 2g of cinnamon reference material (conforming to the standards of the Chinese Pharmacopoeia after thin-layer chromatography identification), add 50mL of purified water, heat to boiling for 30min, maintain a gentle boil, filter while hot after boiling, collect the filtrate and concentrate to a volume of 10mL; add anhydrous ether to the obtained filtrate for extraction, the extraction is performed 1-3 times, the volume of anhydrous ether used in each extraction is (20-40)mL:(0.5-1)g of cinnamon reference material, after each extraction the layers are allowed to stand, collect the upper ether phase, and combine all the upper ether phases; place the combined ether phase in a rotary evaporator to evaporate to dryness, and obtain the residue; add ethyl acetate to the residue to dissolve, the volume of ethyl acetate used to dissolve is (0.5-1)mL:(0.5-1)g of cinnamon reference material, stir to completely dissolve the residue, filter, and take the filtrate to obtain the cinnamon reference material solution.

[0010] Reference solutions: Take cinnamic acid reference standard and 6-gingerol reference standard (both with a purity ≥98%), add methanol as solvent, and prepare solutions containing 2 mg of cinnamic acid per 1 mL and 0.5 mg of 6-gingerol per 1 mL, respectively, as cinnamic acid reference solution and 6-gingerol reference solution.

[0011] Thin-layer chromatography development and examination: Using a silica gel GF254 thin-layer plate, 20 μL of the test solution, 8 μL of cinnamon reference material solution, 4 μL of cinnamic acid reference solution, and 4 μL of 6-gingerol reference solution were respectively applied to the same silica gel GF254 thin-layer plate at a spacing of 1.5-2 cm and a spot diameter ≤3 mm. A petroleum ether (60-90℃)-n-hexane-ethyl formate-formic acid solution with a volume ratio of (8-12):(8-30):15:1 was used as the developing solvent. The thin-layer plate was placed in a developing tank and pre-saturated for 15-20 min before development, with a development distance of 8-10 cm. After development, the thin-layer plate was removed and placed in a fume hood to air dry naturally. First, examine the dried thin-layer plate under a UV lamp (254nm) to observe the corresponding positions of the chromatogram of the test sample, the chromatogram of cinnamon reference material, and the chromatogram of cinnamic acid reference standard. Then, spray the thin-layer plate evenly with 10% sulfuric acid ethanol solution (volume fraction), place it in a 105℃ oven and heat for 5-8 minutes until the spots are clear. After cooling, examine it again under a UV lamp (365nm) to observe the corresponding positions of the chromatogram of the test sample and the chromatogram of 6-gingerol reference standard. If the chromatogram of the test sample shows spots of the same color at the corresponding positions as the chromatograms of cinnamon reference material and cinnamic acid reference material, and fluorescent spots of the same color at the corresponding positions as the chromatograms of 6-gingerol reference material, then Baoyuan granules are determined to contain cinnamon and ginger.

[0012] In some embodiments, during the preparation of the test sample, the ultrasonic power is 300-500W and the ultrasonic frequency is 40kHz.

[0013] In some implementations, the water bath temperature of the rotary evaporator is 30-40°C, and the vacuum degree is -0.08 to 0.06 MPa.

[0014] In some embodiments, the filter membrane used in the preparation of the test sample has a pore size of 0.45 μm.

[0015] In some embodiments, the thickness of the silicone GF254 thin film is 0.2-0.25 mm, and the activation conditions are: activation at 105°C for 30 min.

[0016] Preferred technical parameters: To further improve the identification effect, the preferred technical parameters of this invention are as follows: the ultrasonic time in the preparation of the test sample is 30 min; the weight-to-volume ratio of Baoyuan granules to water is 0.5 g: 10 mL; the number of extractions with anhydrous diethyl ether is 2, and the volume of each extraction is 10 mL: 0.5 g of Baoyuan granules; the volume of ethyl acetate used to dissolve the residue is 0.5 mL: 0.5 g of Baoyuan granules; the volume ratio of petroleum ether (60-90℃) - n-hexane - ethyl formate - formic acid in the developing solvent is 10:10:15:1; the development environment conditions are a temperature of 5-40℃ and a relative humidity of 32%-75%; the silica gel GF254 thin-layer plate is a product produced by Yantai Chemical Industry Research Institute or Qingdao Marine Chemical Plant.

[0017] The present invention has the following advantages over the prior art: This invention optimizes the sample preparation process and developing solvent system, and for the first time achieves simultaneous identification of cinnamon and ginger in Baoyuan granules under the same chromatographic conditions. It eliminates the need for two separate development tests, and the entire detection process can be completed within 1.5-2 hours. Compared with existing stepwise detection methods, the detection efficiency is greatly improved, which can meet the needs of rapid batch sample testing and quality screening in the production process of Baoyuan granules, and significantly reduce detection time and labor costs.

[0018] This invention, through the precise formulation of the developing solvent components, wherein n-hexane can adjust the polarity of the developing solvent to match the polarity difference between cinnamic acid and 6-gingerol, and formic acid can effectively inhibit the dissociation of cinnamic acid, completely solves the problem of cinnamic acid spot tailing in the prior art; in actual detection, the cinnamic acid spot tailing factor is <1.5, the 6-gingerol spot boundary is clear, the separation degree R is ≥1.5, and the negative control has no interfering spots at the corresponding position, which has strong specificity and can accurately determine the presence or absence of cinnamon and ginger in Baoyuan granules, avoiding the risk of misjudgment due to poor separation effect.

[0019] This invention abandons the highly toxic solvent chloroform commonly used in existing technologies. The developing solvent components used are all low-toxic or slightly toxic solvents that are easily volatile and recyclable, posing no significant harm to the health of testing personnel. At the same time, it reduces the discharge of toxic waste liquid, which is in line with the technological development trend of "green, environmentally friendly, safe and low-toxic" in modern Chinese medicine testing, and reduces laboratory safety management costs and environmental governance costs. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a thin-layer identification chromatogram of Example 1 of the present invention (including the results of examination at ultraviolet 254nm and ultraviolet 365nm wavelengths). Figure 2 This is a diagram showing the specificity test results of Embodiment 2 of the present invention (including the test results at ultraviolet 254nm and ultraviolet 365nm wavelengths). Figure 3 This is a graph showing the results of the sample quantity examination in Example 3 of the present invention (including the results of the examination at ultraviolet 254nm and ultraviolet 365nm wavelengths). Figure 4 The results of the investigation of different developing solvent ratios (petroleum ether (60-90℃) - n-hexane - ethyl formate - formic acid = 10:10:15:1) in Example 4 of the present invention are shown in the figure (including the results of the examination at UV 254nm and UV 365nm wavelengths). Figure 5 The results of the investigation of different developing solvent ratios (petroleum ether (60-90℃) - n-hexane - ethyl formate - formic acid = 10:20:15:1) in Example 4 of the present invention are shown in the figure (including the results of the examination at UV 254nm and UV 365nm wavelengths). Figure 6 The results of the investigation of different developing solvent ratios (petroleum ether (60-90℃) - n-hexane - ethyl formate - formic acid = 10:30:15:1) in Example 4 of the present invention are shown in the figure (including the results of the examination at UV 254nm and UV 365nm wavelengths). Figure 7 The images show the results of the examination of thin-layer plates (Qingdao Ocean Chemical Plant) from different brands in Example 5 of the present invention (including the results of the examination at ultraviolet 254nm and ultraviolet 365nm wavelengths). Figure 8 The results of the investigation of different developing solvent ratios (petroleum ether (60-90℃) - n-hexane - ethyl formate - formic acid = 8:12:15:1) in Example 5 of the present invention are shown in the figure (including the results of the examination at UV 254nm and UV 365nm wavelengths). Figure 9 The results of the investigation of different developing solvent ratios (petroleum ether (60-90℃) - n-hexane - ethyl formate - formic acid = 12:8:15:1) in Example 5 of the present invention are shown in the figure (including the results of the examination at UV 254nm and UV 365nm wavelengths). Figure 10 This is a thin-layer identification result image under low-temperature conditions (6°C) in Example 5 of the present invention (including the inspection results at ultraviolet 254nm and ultraviolet 365nm wavelengths). Figure 11 The image shows the thin-layer identification results under high temperature conditions (40°C) in Example 5 of the present invention (including the inspection results at ultraviolet 254nm and ultraviolet 365nm wavelengths). Figure 12This is a thin-layer identification result image under low humidity conditions (RH32%) in Example 5 of the present invention (including the inspection results at ultraviolet 254nm and ultraviolet 365nm wavelengths). Figure 13 The image shows the thin-layer identification results under high humidity conditions (RH75%) in Example 5 of the present invention (including the results of the inspection at ultraviolet 254nm and ultraviolet 365nm wavelengths). Figure 14 The images show the thin-layer identification results of Comparative Example 1 of this invention (the left image shows the identification results of cinnamon under ultraviolet light at 254nm wavelength, and the right image shows the identification results of ginger under sunlight). Figure 15 The image shows the thin-layer identification results of Comparative Example 2 of the present invention (including the results of inspection under ultraviolet light at 254 nm and sunlight). Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 Experimental steps Preparation of test solution Step 1: Take 0.5g of Baoyuan Granules (Jinpai Chizhengtang Pharmaceutical Co., Ltd., batch number: 20240101), place it in a 50mL stoppered conical flask, add 10mL of purified water at a weight-volume ratio of 0.5g:10mL, and stir magnetically for 5min to completely dissolve the granules. Step 2: Place the conical flask in the ultrasonic instrument, set the ultrasonic power to 400W and the ultrasonic frequency to 40kHz, and ultrasonically treat for 30 minutes; Step 3: After sonication, while still hot, filter through quantitative filter paper (medium speed, Φ11cm) and collect the filtrate in a 25mL separatory funnel; Step 4: Add 10 mL of anhydrous diethyl ether to the separatory funnel, shake and extract for 5 min, allow to stand for separation (about 3 min), and collect the upper ether phase in another conical flask; repeat the extraction once and combine the two ether phases; Step 5: Transfer the combined ether phase to a 50mL round-bottom flask of a rotary evaporator, set the water bath temperature to 35℃ and the vacuum degree to -0.07MPa, evaporate the ether to dryness, and obtain a pale yellow residue. Step 6: Add 0.5 mL of ethyl acetate to the residue, stir with a glass rod to completely dissolve the residue, then filter through a 0.45 μm organic phase filter membrane, and take the filtrate as the test solution.

[0024] Preparation of control solution Preparation of cinnamon reference herb solution: Step 1: Take 2g of cinnamon reference material (China National Institutes for Food and Drug Control, batch number: 121180-202306), place it in a 250mL round-bottom flask, and add 50mL of purified water; Step 2: Set up a reflux apparatus, heat to boiling, maintain a gentle boil for 30 minutes, stop heating, filter while hot using quantitative filter paper, and collect the filtrate; Step 3: Transfer the filtrate to a rotary evaporator and concentrate it at 35°C to a volume of 10 mL. After cooling, transfer it to a 25 mL separatory funnel. Step 4: Follow steps 4-6 of the test solution preparation procedure (extract twice with anhydrous diethyl ether, 10 mL each time; evaporate to dryness, add 0.5 mL of ethyl acetate to dissolve, and filter) to obtain the cinnamon reference material solution.

[0025] Preparation of reference solution: Step 1: Take 10 mg of cinnamic acid reference standard (purity ≥98%, batch number: C1020-202305), place it in a 5 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain a reference solution containing 2 mg of cinnamic acid per 1 mL; Step 2: Take 2.5 mg of 6-gingerol reference standard (purity ≥98%, batch number: G008-202304), place it in a 5 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain a reference solution containing 0.5 mg of 6-gingerol per 1 mL.

[0026] Thin-layer chromatography development and inspection Step 1: Take a silica gel GF254 thin film (Qingdao Ocean Chemical Plant, specifications: 10cm×20cm, thickness 0.25mm), place it in a 105℃ oven for 30 minutes to activate, and then remove and cool to room temperature; Step 2: Using a micro-spotting capillary tube, take 20 μL of the test solution, 8 μL of the cinnamon reference material solution, 4 μL of the cinnamic acid reference solution, and 4 μL of the 6-gingerol reference solution, and spot them onto the same activated silica gel GF254 thin-layer plate according to the requirements of a spotting interval of 1.5 cm and a spotting diameter ≤3 mm. Step 3: Measure 20 mL of developing solvent (petroleum ether (60-90℃) - n-hexane - ethyl formate - formic acid, volume ratio 10:10:15:1), pour it into the developing tank, cover and pre-saturate for 20 min; Step 4: Place the spotted thin-layer plate into the developing tank, ensuring the solvent level is below the spotting line. Seal the developing tank and wait until the solvent front rises to 8 cm. Then, remove the thin-layer plate and place it in a fume hood to air dry naturally. Step 5: Place the dried thin-layer plate under a UV lamp (254nm) for inspection and record the chromatographic spots. Then, spray the thin-layer plate evenly with a 10% sulfuric acid ethanol solution (volume fraction), place it in a 105℃ oven and heat for 6 minutes until the spots are clear. Remove it, cool it to room temperature, and then place it under a UV lamp (365nm) for inspection and record the chromatographic spots.

[0027] Performance verification The evaluation indicators of thin-layer chromatography (TLC) according to the General Chapter 0502 of the 2020 edition of the Chinese Pharmacopoeia, Part IV, were used to determine the following: (1) Rf and tailing factor (T, calculated by T=W0.05h / 2d1, where W0.05h is the peak width at 5% peak height and d1 is the distance from the peak apex to the leading edge) of cinnamic acid at 254nm. (2) The ratio shift (Rf) and resolution (R, calculated as R=2(d2-d1) / (W1+W2) of 6-gingerol (test sample and reference sample) at 365nm. d1 and d2 are the distances from the center of adjacent spots to the origin, and W1 and W2 are the peak widths of adjacent spots. (3) Observe whether interference spots appear in the corresponding positions of the negative control solutions lacking cinnamon and ginger (prepared according to the test sample preparation method using Baoyuan granules to simulate the corresponding medicinal materials).

[0028] Experimental results

[0029] The embodiments fully validated the core identification process of the present invention, achieving simultaneous identification of cinnamon and ginger in Baoyuan granules. Key results showed that the cinnamic acid tailing factor was ≤1.2 and the 6-gingerol separation degree was ≥1.8, both of which met the standards of the Chinese Pharmacopoeia, and the results were negative with no interference.

[0030] Example 2 Experimental steps Preparation of negative control solution Cinnamon-deficient negative control solution: Take 0.5g of simulated granules prepared according to the Baoyuan Granules prescription (excluding cinnamon, and the remaining medicinal materials (ginseng, astragalus, licorice, and ginger) according to the process, and operate according to the test solution preparation steps (steps 1-6) in Example 1 to obtain the cinnamon-deficient negative control solution. Ginger-deficient negative control solution: Take 0.5g of simulated granules made according to the process of Baoyuan Granules (excluding ginger, ginseng, astragalus, licorice, and cinnamon) and prepare them in the same way as above to obtain the ginger-deficient negative control solution.

[0031] Thin-layer chromatography development and inspection Step 1: Take the activated silica gel GF254 thin-layer plate (same as in Example 1), and separately apply 20 μL of the test solution, 20 μL of the cinnamon-deficient negative control solution, 20 μL of the ginger-deficient negative control solution, 8 μL of the cinnamon reference herb solution, 4 μL of the cinnamic acid reference solution, and 4 μL of the 6-gingerol reference solution. Perform the spotting and development conditions (developing solvent 10:10:15:1) as described in Example 1. Step 2: Examine the chromatographic spots at 254 nm and 365 nm after heating with 10% sulfuric acid ethanol solution, and record the chromatographic spots of each solution.

[0032] Performance verification Observe whether the spots in the chromatogram of the test sample are clear at the corresponding positions as cinnamon reference material, cinnamic acid reference standard (254nm) and 6-gingerol reference standard (365nm), and at the same time confirm whether the negative control solution lacking the corresponding medicinal material has no interfering spots at the above positions.

[0033] Experimental results

[0034] This embodiment verifies the specificity of the method of the present invention by preparing negative control solutions lacking cinnamon and ginger: the test sample showed clear spots at the corresponding positions of the target components, and the negative control lacking the corresponding medicinal materials had no interference, proving that other medicinal materials such as ginseng, astragalus, and licorice in the prescription do not affect the identification of cinnamic acid and 6-gingerol.

[0035] Example 3 Experimental steps Step 1: Prepare the test solution, cinnamon reference material solution, cinnamic acid reference solution, and 6-gingerol reference solution according to Example 1; Step 2: Set the sampling volume gradient: Test solutions: 15 μL, 20 μL, 30 μL; Cinnamon reference solutions: 6 μL, 8 μL, 10 μL; Cinnamic acid reference solution: 3 μL, 4 μL, 5 μL; 6-Gingerol reference solution: 3 μL, 4 μL, 5 μL; Step 3: Take the activated silica gel GF254 thin-layer plate and spot the samples according to the above-mentioned spotting amounts. The developing solvent is 10:10:15:1 (same as in Example 1). The developing and inspection steps are the same as in Example 1.

[0036] Performance verification The clarity of the spots (evaluated on a scale of 1-5: level 1 is blurry and indistinguishable, level 5 is extremely clear), tailing factor (cinnamic acid only), and resolution (6-gingerol only) were measured under different sample amounts to determine whether they met the identification requirements (clarity ≥ level 3, T ≤ 1.5, R ≥ 1.5).

[0037] Experimental results

[0038] This embodiment verifies by setting multiple gradient sampling amounts that within the sampling amount range of claim 8 (15-30 μL for test sample, 6-10 μL for reference medicinal material, and 3-5 μL for reference standard), all sampling amounts meet the identification requirements of "spot clarity ≥ 4, tailing factor ≤ 1.3, and resolution ≥ 1.6", and the optimal sampling amounts of 20 μL / 8 μL / 4 μL yield the best results.

[0039] Example 4 Experimental steps Step 1: Prepare the test solution, cinnamon reference material solution, cinnamic acid reference solution, and 6-gingerol reference solution according to Example 1; Step 2: Set 4 sets of developing solvent ratios (all by volume): Group 1: 8:8:15:1 (lower limit of claim 1); Group 2: 10:20:15:1 (median value); Group 3: 12:30:15:1 (upper limit of claim 1); Group 4: 10:10:15:1 (optimal ratio, claim 6); Step 3: Take 4 identical activated silica gel GF254 thin-layer plates, and respectively take 20 μL of the test solution, 8 μL of cinnamon reference material solution, 4 μL of cinnamic acid reference solution, and 4 μL of 6-gingerol reference solution. Operate according to the development and inspection steps of Example 1, with each group corresponding to one ratio of developing solvent.

[0040] Performance verification The Rf value and tailing factor of cinnamic acid, the Rf value and resolution of 6-gingerol were measured under each developing solvent to determine whether the identification requirements were met (T≤1.5, R≥1.5).

[0041] Experimental results

[0042] This embodiment verifies through four sets of experiments covering the developing solvent ratio range of claim 1 that all ratios from the lower limit (8:8:15:1) to the upper limit (12:30:15:1) can achieve the effect of "cinnamic acid tailing factor ≤1.4 and 6-gingerol separation degree ≥1.5", and the optimal ratio of 10:10:15:1 has the best separation effect.

[0043] Example 5 Experimental steps (1) Variable setting Thin-layer plate brands: Yantai Chemical Industry Research Institute (A), Qingdao Marine Chemical Plant (B), a certain imported brand (C, model: TLCSilicaGelGF254); Temperature: 5℃ (low temperature, controlled by constant temperature incubator), 25℃ (room temperature, laboratory environment), 40℃ (high temperature, controlled by constant temperature drying oven); Humidity: RH32% (low humidity, controlled by placing anhydrous calcium chloride in the desiccator), RH50% (medium humidity, laboratory environment), RH75% (high humidity, controlled by placing saturated sodium chloride solution in the desiccator).

[0044] (2) Experimental procedures Step 1: Prepare the test solution, cinnamon reference material solution, cinnamic acid reference solution, and 6-gingerol reference solution according to Example 1; Step 2: For each variable (e.g., different brands of thin-layer plates), after activation, take the corresponding thin-layer plate, spot the sample (20 μL of test sample, 8 μL of control herb, and 4 μL of control standard), develop with a developing solvent of 10:10:15:1, and follow the inspection steps in Example 1. Step 3: Perform the experiment in parallel 3 times under each variable condition and take the average value.

[0045] Performance verification The tailing factor of cinnamic acid and the separation degree of 6-gingerol were determined under different conditions, and the clarity of the spots was observed (≥3 grade is qualified) to determine whether the method is stable.

[0046] Experimental results Results of different thin-layer board brands

[0047] Results at different temperatures

[0048] Results of different humidity levels

[0049] This embodiment verifies the stability of the method under different thin-layer plate brands, temperatures (5-40℃), and humidity (32%-75%). It proves that under all variable conditions, the method meets the requirements of "cinnamic acid tailing factor ≤1.4, 6-gingerol separation ≥1.6, and spot clarity ≥4", and the parallel experiment deviation is ≤2.2%.

[0050] Example 6 Experimental steps Step 1: Prepare the mother liquor of Baoyuan granules (filtrate after ultrasonic filtration of 0.5g granules and 10mL water) according to Example 1, divide it into 3 portions, and extract each portion with the following solvents: Group 1: Anhydrous diethyl ether (in this invention), extracted twice, 10 mL each time (same as Example 1); Group 2: Ethyl acetate (common solvent), extracted twice, 10 mL each time; Group 3: Chloroform (the solvent used in CN118362677A), extracted twice, 10 mL each time; Step 2: After extraction of each group, combine the organic phases and prepare the test solution according to the steps of evaporation and dissolution in ethyl acetate as in Example 1; Step 3: Prepare the control solution according to Example 1, develop the thin layer (developing solvent 10:10:15:1), and inspect the operation steps.

[0051] Performance verification Recovery rate determination: The contents of cinnamic acid and 6-gingerol in each group of test solution were determined by high performance liquid chromatography (HPLC), and the recovery rate was calculated (recovery rate = measured content / theoretical content × 100%, the theoretical content is calculated based on the contents and extraction rates of cinnamon and ginger in the Baoyuan granule prescription). Impurity spot count: At 254 nm and 365 nm, count the number of impurity spots in the chromatogram of the test sample, excluding the target spot (a spot with an area ≥ 0.5 mm² is counted as 1 spot); Separation degree and tailing factor determination: same as in Example 1.

[0052] Experimental results

[0053] This embodiment compares the extraction effects of anhydrous diethyl ether with those of conventional solvents (ethyl acetate and chloroform), demonstrating that only anhydrous diethyl ether can simultaneously achieve the advantages of "high recovery rate (cinnamic acid 96.2%, 6-gingerol 94.5%), low impurities (2 spots), and low toxicity": ethyl acetate has a recovery rate of less than 80% and contains many impurities, while chloroform, although having a similar recovery rate, is highly toxic and contains many impurities.

[0054] Example 7 Experimental steps Step 1: Prepare the test solution, cinnamon reference material solution, cinnamic acid reference solution, and 6-gingerol reference solution according to Example 1; Step 2: Set up 3 sets of developing solvents (volume ratio): Group 1: Petroleum ether-n-hexane-ethyl formate-formic acid (10:10:15:1, this invention, all components); Group 2: Petroleum ether-ethyl formate-formic acid (10:15:1, n-hexane is missing); Group 3: Petroleum ether-n-hexane-ethyl formate (10:10:15, lacking formic acid); Step 3: Take three identical activated silica gel GF254 thin-layer plates and spot them respectively (20 μL of test sample, 8 μL of reference medicinal material, and 4 μL of reference standard), corresponding to three groups of developing solvents, and operate according to the development and inspection steps of Example 1.

[0055] Performance verification The Rf value of cinnamic acid, the tailing factor, the Rf value of 6-gingerol, and the resolution of each group were measured. The clarity of the spots (grades 1-5) was observed to determine whether the identification requirements were met.

[0056] Experimental results

[0057] This embodiment verifies the irreplaceability of hexane and formic acid in the developing solvent by setting up developing solvent control groups with "hexane deficiency" and "formic acid deficiency": hexane deficiency leads to a decrease in the separation degree of 6-gingerol to 1.2 (<1.5), which cannot meet the separation requirements; formic acid deficiency leads to an increase in the cinnamic acid tailing factor to 2.3 (>1.5), resulting in severe spot tailing; only the full-component developing solvent of this invention can achieve excellent separation.

[0058] Example 8 Experimental steps Two scheme settings Option 1 (existing technology combination): Step 1: Extraction according to CN116068075A method: Take 1g of Baoyuan granules, add 20mL of water and sonicate for 40min, extract with ethyl acetate 3 times, 20mL each time, combine the extracts, evaporate to dryness, add 5mL of methanol to dissolve, filter, and obtain the test solution. Step 2: The developing solvent is prepared according to method CN118362677A: petroleum ether-ethyl acetate-chloroform-formic acid (20:10:10:0.8, containing chloroform). Step 3: Sample volume: 20 μL of test sample, 8 μL of cinnamon reference material, 4 μL of cinnamic acid reference standard, and 4 μL of 6-gingerol reference standard. The development and inspection steps are the same as in Example 1.

[0059] Option 2 (the present invention): Follow the complete steps of Example 1.

[0060] Performance verification The identification mode (synchronous / stepwise), cinnamic acid tailing factor, 6-gingerol resolution, solvent toxicity (with chloroform / without chloroform), and detection time (total time from sample weighing to completion of inspection) of the two schemes were determined.

[0061] Experimental results

[0062] This embodiment demonstrates, through simulation of existing technology combinations, that although such combinations attempt simultaneous identification, they suffer from defects such as "severe cinnamic acid tailing (T=2.1), insufficient separation of 6-gingerol (R=1.0), presence of highly toxic chloroform, and long processing time (3.5h)," thus failing to meet the identification requirements. In contrast, the present invention's solution achieves successful simultaneous identification with excellent performance across all indicators.

[0063] Comparative Example 1 Experimental steps (1) Cinnamon identification (first test) Step 1: Take 0.5g of Baoyuan granules, add 10mL of water, sonicate for 30min, filter, extract the filtrate twice with petroleum ether (60-90℃), 10mL each time, combine the petroleum ether phases, evaporate to dryness, add 0.5mL of methanol to dissolve, and obtain the cinnamon identification test solution. Step 2: Developing solvent: petroleum ether-n-hexane-ethyl formate-glacial acetic acid (3:9:8:0.3); Step 3: Spotting (20 μL of test sample, 8 μL of cinnamon reference material, and 4 μL of cinnamic acid reference standard), developing, and examining at 254 nm.

[0064] (2) Identification of ginger (second test) Step 1: Take another 0.5g of Baoyuan granules, add 10mL of water, sonicate for 30min, filter, extract the filtrate twice with 10mL of ethyl acetate each time, combine the ethyl acetate phases, evaporate to dryness, add 0.5mL of methanol to dissolve, and obtain the ginger identification test solution; Step 2: Developing solvent: n-hexane-ethyl acetate (9:5); Step 3: Spotting (20 μL of test sample and 4 μL of 6-gingerol reference standard), developing, spraying vanillin sulfuric acid solution, and examining under sunlight.

[0065] Performance verification The total time consumed in the two tests, the cinnamic acid tailing factor, and the clarity of the ginger spots were measured and compared with the results of Example 1 of the present invention.

[0066] Comparison results

[0067] This comparative example, by replicating the stepwise detection method of CN116068075A, clarifies that it has defects such as "requiring two detections, a total time of 4 hours, the need to repeatedly prepare the test sample, and low clarity of ginger spots (level 3)"; while the present invention only requires one detection (1.5 hours), and the clarity of ginger spots reaches level 5.

[0068] Comparative Example 2 Experimental steps Step 1: Prepare the test solution and control solution according to Example 1; Step 2: Developing solvent: petroleum ether-ethyl acetate-chloroform-formic acid (20:10:10:0.8, containing chloroform); Step 3: Spotting (20 μL of test sample, 8 μL of control medicinal material, and 4 μL of control standard), the development and inspection steps are the same as in Example 1.

[0069] Performance verification The cinnamic acid tailing factor, 6-gingerol separation degree, and solvent toxicity were determined and compared with Example 1 of the present invention.

[0070] Comparison results

[0071] This comparative example, by reproducing the chloroform-containing developing solvent system of CN118362677A, demonstrates that it has defects such as "severe cinnamic acid tailing (T=2.4), insufficient separation of 6-gingerol (R=1.2), high toxicity of chloroform, and low spot clarity (level 3)," and cannot meet the identification requirements. In contrast, the chloroform-free system of this invention can achieve excellent results with "tailing factor ≤1.2, separation ≥1.8, and spot clarity level 5."

[0072] Comparative Example 3 Experimental steps The procedure for the “ethyl acetate extraction” group in Example 6 was as follows: 0.5 g of Baoyuan granules were taken, 10 mL of water was added, and the mixture was sonicated for 30 min. The mixture was filtered, and the filtrate was extracted twice with 10 mL of ethyl acetate each time. The extracts were combined, evaporated to dryness, and dissolved in 0.5 mL of ethyl acetate to obtain the test solution. The preparation and development of the control solution were the same as in Example 1.

[0073] Performance verification Same as in Example 6, the recovery rate, impurity spots, and separation effect were measured.

[0074] Comparison results

[0075] This comparative example, by comparing the extraction effects of ethyl acetate and anhydrous diethyl ether, clearly shows that ethyl acetate has problems such as "low recovery rate (cinnamic acid 78.5%, 6-gingerol 75.3%), many impurity spots (4), and insufficient separation (1.3)", which cannot meet the identification requirements; while anhydrous diethyl ether has excellent extraction effect.

[0076] Comparative Example 4 Experimental steps The procedure was performed according to the steps of the "hexane-deficient" group in Example 7: the developing solvent was petroleum ether-ethyl formate-formic acid (10:15:1), and the preparation and development of the test sample and control solution were the same as in Example 1.

[0077] Performance verification Same as in Example 7, tailing factor, resolution, and spot clarity were measured.

[0078] Comparison results

[0079] This comparative example, by comparing the effects of developing solvents "lacking n-hexane" and "containing n-hexane", clearly shows that the lack of n-hexane leads to "6-gingerol Rf value being too low (0.35), resolution dropping to 1.2 (<1.5), and spot clarity only grade 3", which cannot meet the identification requirements; while the developing solvent containing n-hexane has excellent separation effect.

[0080] Comparative Example 5 Experimental steps Operate according to the steps of "Scheme 1 (Prior Art Combination)" in Example 8, namely the ethyl acetate extraction method of CN116068075A + the chloroform-containing developing agent of CN118362677A.

[0081] Performance verification Same as in Example 8, the identification effect, toxicity, and time consumption were determined.

[0082] Comparison results

[0083] This comparative example, by reproducing the existing technology combination, clarifies that the combination has defects such as "failure of simultaneous identification, severe tailing of cinnamic acid, insufficient separation of 6-gingerol, high toxicity, and long time consumption", and cannot meet the identification requirements; while the solution of this invention has excellent indicators in all aspects.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A thin-layer chromatography method for identifying cinnamon and ginger in Baoyuan granules, characterized in that, Includes the following steps: (1) Preparation of test solution: Take Baoyuan granules, add water to dissolve them at a weight-volume ratio of (0.5g-1)g:(10-20)mL, sonicate for 20-40min, and filter; take the filtrate and extract it with anhydrous ether 1-3 times, the volume of anhydrous ether used for each extraction being (20-40)mL:(0.5-1)g, and combine the upper liquids; evaporate the combined upper liquids to dryness, add ethyl acetate to dissolve the residue, the volume of ethyl acetate used for dissolution being (0.5-1)mL:(0.5-1)g, and obtain the test solution; (2) Preparation of control solution: Take cinnamon reference material, add water and boil for 30 min, filter, and evaporate the filtrate to dryness; take the residue after evaporation and dissolve it in water, and process the resulting solution in the same way as the operation in step (1) of "extracting the filtrate with anhydrous diethyl ether-combining the upper liquid-evaporating to dryness-dissolving in ethyl acetate" to obtain cinnamon reference material solution; take cinnamic acid reference standard and 6-gingerol reference standard respectively, add solvent to prepare cinnamic acid reference standard solution and 6-gingerol reference standard solution; (3) Thin-layer chromatography development: GF254 silica gel thin-layer plates were used with petroleum ether, n-hexane, ethyl formate and formic acid with a boiling range of 60-90℃ in a volume ratio of (8-12):(8-30):15:1 as the developing solvent. The plates were then removed and air-dried. (4) Inspection and identification: After drying, the thin-layer plate is examined under a 254nm ultraviolet lamp. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatograms of cinnamon reference material and cinnamic acid reference standard. Spray the thin-layer plate with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clear, and then examine it under a 365nm ultraviolet lamp. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of 6-gingerol reference standard.

2. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, The ultrasonic treatment time in step (1) is 30 minutes.

3. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, The weight-to-volume ratio of the Baoyuan granules to water in step (1) is 0.5g:10mL.

4. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, The anhydrous ether extraction in step (1) is performed twice, and the ratio of the volume of anhydrous ether used in each extraction to the weight of the Baoyuan granules is 10 mL: 0.5 g.

5. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, The volume ratio of ethyl acetate used to dissolve the residue in step (1) to the weight ratio of Baoyuan granules is 0.5 mL: 0.5 g.

6. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, In step (3), the volume ratio of petroleum ether-n-hexane-ethyl formate-formic acid with a boiling range of 60-90℃ in the developing agent is 10:10:15:

1.

7. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, The cinnamic acid reference solution mentioned in step (2) is a solution containing 2 mg of cinnamic acid per 1 mL, prepared by adding methanol to cinnamic acid reference standard, and the 6-gingerol reference solution is a solution containing 0.5 mg of 6-gingerol per 1 mL, prepared by adding methanol to 6-gingerol reference standard.

8. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, When performing thin-layer chromatography in step (3), the sample loading amounts are: 20 μL of test solution, 8 μL of cinnamon reference material solution, 4 μL of cinnamic acid reference solution, and 4 μL of 6-gingerol reference solution.

9. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, The silicone GF254 thin film mentioned in step (3) is a silicone GF254 thin film produced by Yantai Chemical Industry Research Institute or Qingdao Ocean Chemical Plant.

10. The thin-layer chromatography identification method for cinnamon and ginger in Baoyuan granules according to claim 1, characterized in that, The environmental conditions for the unfolding in step (3) are: temperature 5-40℃, relative humidity 32%-75%.

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