Method for detecting medicine flavor of ginger in common goldenrop granules

Abstract

The method comprises the following steps: grinding the common goldenrop particles, adding ethyl acetate, carrying out ultrasonic treatment, filtering, carrying out rotary evaporation on the filtrate to dryness, and dissolving residues with ethyl acetate to obtain a test solution; taking a 6-gingerol reference substance, and adding methanol to prepare a solution as a reference substance solution; sucking the test sample solution and the reference substance solution, respectively dripping the test sample solution and the reference substance solution on the same silica gel thin-layer plate, developing by taking cyclohexane-trichloromethane-ethyl acetate with the volume ratio of 4: 2: 3 as a developing solvent, taking out, airing, spraying a vanillic aldehyde sulfuric acid solution, heating, and inspecting. The thin-layer chromatogram obtained by the method is uniform in spot distribution, regular in shape, obvious in color contrast, stable in specific shift value and good in reproducibility. In addition, the method is excellent in durability, has good adaptability under different environment temperature and humidity and different types of thin-layer plate operation conditions, and is stable and reliable in separation effect. And a reliable method is provided for quality control of the common goldenrop particles.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine component analysis and detection technology, specifically relating to a method for detecting the ginger flavor in Xiaochaihu granules. Background Technology

[0002] Xiao Chai Hu Granules are formulated based on the classic Xiao Chai Hu Tang formula recorded in the medical work *Treatise on Febrile and Miscellaneous Diseases* by Zhang Zhongjing, a famous physician of the Eastern Han Dynasty. It is the ancestral formula of harmonizing agents, composed of seven Chinese herbs: Codonopsis pilosula (originally ginseng), Bupleurum chinense, Scutellaria baicalensis, Pinellia ternata (processed with ginger), ginger, jujube, and licorice. Xiao Chai Hu Granules are a modern dosage form of Xiao Chai Hu Tang, possessing the effects of relieving exterior syndromes, clearing heat, soothing the liver, and harmonizing the stomach. It is mainly used for exogenous diseases and Shao Yang syndrome caused by pathogenic factors. Currently, the quality standards and related research of Xiao Chai Hu Granules in the 2025 edition of the *Chinese Pharmacopoeia (Part I)* mainly involve the identification and determination of Bupleurum chinense, Scutellaria baicalensis, and licorice, but do not involve research on ginger. Furthermore, these identification methods are often complex, time-consuming, inefficient, and costly.

[0003] Ginger, as a traditional Chinese medicine, has the functions of dispelling wind-cold, warming the middle jiao and stopping vomiting, resolving phlegm and relieving cough, and detoxifying. Its main chemical components include volatile oil, gingerol, diphenylheptane derivatives, flavonoids, amino acids, polysaccharides and trace elements. In Xiaochaihu granules, ginger is used as an adjuvant and has the same effect as Pinellia ternata in relieving nausea and vomiting. Moreover, ginger can detoxify Pinellia ternata and is an indispensable Chinese medicine in this formula.

[0004] Based on this, the present invention proposes a thin-layer chromatography detection method for ginger in Xiaochaihu granules. This method is simple and rapid to operate, highly specific and durable, and further improves the quality standard of Xiaochaihu granules. Summary of the Invention

[0005] Currently, there is a lack of detection technology for ginger in the analysis of relevant components of Xiao Chai Hu granules, resulting in deficiencies in product quality control. The purpose of this invention is to provide a method for detecting the ginger flavor in Xiao Chai Hu granules, addressing the shortcomings of existing technologies. This method employs thin-layer chromatography (TLC), combined with specific sample preparation and development conditions, to qualitatively analyze the ginger flavor in Xiao Chai Hu granules. The method achieves good separation, clear and distinct spots, moderate development rate, and intuitive and easily interpretable results.

[0006] The technical solution adopted in this invention is as follows: A method for detecting the ginger flavor in Xiao Chai Hu granules, the method comprising the following steps: Preparation of test solution: Take the granules of Bupleurum chinense, grind them into a fine powder, add ethyl acetate, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in ethyl acetate to obtain the test solution; Preparation of reference solution: Take 6-gingerol reference standard, add methanol to prepare a solution, and use it as the reference solution; Test: Take the above test solution and reference solution and spot them separately on the same silica gel thin layer plate. Use cyclohexane-chloroform-ethyl acetate (volume ratio 4:2:3) as the developing solvent, develop, remove, air dry, spray with vanillin-sulfuric acid solution, heat treatment, and then examine.

[0007] In the above technical solution, the ratio of the amount of Xiaochaihu granules, the added ethyl acetate, and the ethyl acetate used to dissolve the residue is 1g:20ml:1ml.

[0008] Furthermore, the ultrasonic treatment has a power of 360W, a frequency of 40KHz, and an ultrasonic duration of 10 minutes.

[0009] Furthermore, the filtrate is concentrated to dryness under reduced pressure at 50°C.

[0010] Furthermore, the concentration of the reference solution is 1 mg of 6-gingerol per 1 ml.

[0011] Furthermore, during the test, 5 μl of the test solution and 2 μl of the reference solution were spotted onto the same silica gel thin-layer plate.

[0012] Furthermore, the temperature of the heat treatment is 105°C.

[0013] The present invention provides a method for detecting ginger in Xiaochaihu granules. This method uses thin-layer chromatography to achieve qualitative detection of the ginger flavor in Xiaochaihu granules, improves the quality control of Xiaochaihu granules, makes the product quality controllable, and ensures the safety and effectiveness of the product.

[0014] This method, through the preparation of test and reference solutions, combined with a specific developing solvent system and colorimetric conditions, yields thin-layer chromatograms with uniformly distributed, regularly shaped spots, clear color contrast, stable specific migration values, and good reproducibility. Furthermore, this method exhibits excellent robustness, demonstrating good adaptability to different environmental temperatures and humidity levels, and under various types of thin-layer plate operating conditions, with stable and reliable separation results. In addition, the method is simple to operate, requires low-cost equipment, and is suitable for widespread use in grassroots laboratories. The method of this invention provides a scientific basis for the quality control of Xiaochaihu granules, fills the technological gap in the research of ginger medicinal materials in Xiaochaihu granules, and lays a solid foundation for ensuring product quality and medication safety. Attached Figure Description

[0015] Figure 1 The thin-layer chromatogram of Example 1 is obtained under the condition that the developing solvent is cyclohexane-chloroform-ethyl acetate (4:2:3). Figure 2 This is a thin-layer chromatogram of Example 2 under the condition that the developing solvent is petroleum ether-ethyl acetate (4:3); Figure 3This is a thin-layer chromatogram of Example 3 under the condition that the developing solvent is petroleum ether (60-90℃)-chloroform-ethyl acetate (2:1:1); Figure 4 This is a thin-layer chromatogram of Example 4 under the condition that the developing solvent is cyclohexane-chloroform-ethyl acetate (4:2:1); Figure 5 This is a thin-layer chromatogram of Example 5 under the condition that the developing solvent is cyclohexane-chloroform-ethyl acetate (2:1:2); Figure 6 This is a thin-layer chromatogram of the durability under high temperature (40°C) conditions in Example 6; Figure 7 This is a thin-layer chromatogram of the durability test under low-temperature (5°C) conditions in Example 6; Figure 8 The thin-layer chromatogram is shown under the low humidity (35%) conditions for durability in Example 6. Figure 9 This is a thin-layer chromatogram under high humidity (75%) conditions in Example 6 for durability testing; Figure 10 The following are chromatograms of silica gel H thin-layer chromatography under different thin-layer plate conditions in Example 6; Figure 11 The images show the chromatograms of silica gel G thin-layer chromatography under different thin-layer plate conditions in Example 6.

[0016] In the figure: S1 represents 6-gingerol, and 1 represents the Xiaochaihu granule sample. Detailed Implementation

[0017] All content not described in detail in this specification belongs to prior art known to those skilled in the art, and the model parameters of each device are not specifically limited; conventional equipment can be used. Experimental conditions not mentioned in this technical solution are not shown because they belong to prior art, and will not be described further here. The following descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0018] Example 1 A thin-layer chromatography method for detecting ginger in Xiaochaihu granules, the method comprising the following steps: Place 1g of Xiaochaihu granules in an Erlenmeyer flask, grind the granules into a fine powder (passing through a No. 4 sieve), add 20ml of ethyl acetate, and sonicate for 10 minutes. The sonication should be carried out at a power of 360W and a frequency of 40kHz. Filter the extract, concentrate the filtrate to dryness under reduced pressure at 50℃, and dissolve the residue in 1ml of ethyl acetate to obtain the test solution. Ethyl acetate was chosen based on its solvent polarity and excellent extraction ability, while 50℃ ensured efficient solvent evaporation while avoiding degradation of heat-sensitive components. The sonication time was optimized to ensure thorough extraction without damaging the structural integrity of the target components.

[0019] The preparation of the reference solution followed these steps: 6-Gingerol reference standard was dissolved in methanol to prepare a solution containing 1 mg per ml, which was then filtered through a 0.45 μm microporous membrane for later use as the reference solution. In this step, 6-gingerol was chosen based on its representativeness in ginger, effectively reflecting the key component characteristics of the herb. The use of the microporous membrane ensured the purity of the solution, preventing impurities from interfering with subsequent detection.

[0020] The detection process follows these steps. Perform thin-layer chromatography (Chinese Pharmacopoeia 2025 Edition, Part IV, General Chapter 0502). Place an appropriate amount of developing solvent in the developing tank. The developing solvent is prepared as cyclohexane-chloroform-ethyl acetate (4:2:3). Place the silica gel G thin-layer plate in the developing tank. Apply 5 μl of the test solution and 2 μl of the reference solution to the same silica gel G thin-layer plate, ensuring even distribution of the spots. During development, the developing solvent moves upwards along the thin-layer plate. When the plate reaches an appropriate height, remove it, allow it to dry, and then spray it with vanillin-sulfuric acid solution. Heat at 105℃ until the spots are clearly visible, and examine again under sunlight.

[0021] In this step, compared with Examples 2-5, the type and ratio of the developing agent have been optimized through multiple experiments to achieve a balance between separation effect and development rate, ensuring that the spots of the target component are clear and easy to identify.

[0022] The colorimetric results showed that the spots of the test sample and the reference sample, developed under the same conditions, were clearly visible in terms of position and color contrast. In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatogram of the reference sample. This result indicates that the components in the test sample solution have a good correspondence with the reference sample, enabling effective qualitative analysis of ginger in Xiaochaihu granules. The consistency in the color and position of the spots reflects good separation of the target components.

[0023] Example 2 Place 1g of Xiaochaihu granules in an Erlenmeyer flask, grind the granules into a fine powder (pass through a No. 4 sieve), add 20ml of ethyl acetate, and sonicate (power 360W, frequency 40KHz) for 10 minutes; filter the extract, concentrate the filtrate under reduced pressure at 50℃ to dryness, add 1ml of ethyl acetate to dissolve the residue, and use it as the test solution.

[0024] Preparation of reference solution: Take 6-gingerol reference standard, add methanol to prepare a solution containing 1 mg per 1 ml, filter through a 0.45 μm microporous membrane and use it as reference solution.

[0025] The thin-layer chromatography method (Chinese Pharmacopoeia 2025 Edition, Part IV, General Chapter 0502) was followed. An appropriate amount of developing solvent (petroleum ether-ethyl acetate (4:3)) was placed in the developing tank. A silica gel G thin-layer plate was placed in the developing tank, and 5 μl of the test solution and 2 μl of the reference solution were spotted separately onto the same silica gel G thin-layer plate. During development, the developing solvent moved upwards along the thin-layer plate. When the plate reached an appropriate height, it was removed, dried, and then sprayed with vanillin-sulfuric acid solution. The plate was heated at 105℃ until the spots were clearly visible, and then examined again under sunlight.

[0026] The colorimetric results showed that no clear spots were observed at the corresponding positions in the chromatogram of the test sample compared to the reference sample. This result indicates that the thin-layer colorimetric results obtained in Example 1 using the three-phase developing solvent cyclohexane-chloroform-ethyl acetate (4:2:3) were better than those obtained using the two-phase developing solvent petroleum ether-ethyl acetate (4:3).

[0027] Example 3 Place 1g of Xiaochaihu granules in an Erlenmeyer flask, grind the granules into a fine powder (pass through a No. 4 sieve), add 20ml of ethyl acetate, and sonicate for 10 minutes. The sonication should be carried out at a power of 360W and a frequency of 40KHz. Filter the extract, concentrate the filtrate under reduced pressure at 50℃ to dryness, and dissolve the residue in 1ml of ethyl acetate to obtain the test solution.

[0028] Preparation of reference solution: Take 6-gingerol reference standard, add methanol to prepare a solution containing 1 mg per 1 ml, filter through a 0.45 μm microporous membrane and use it as reference solution.

[0029] Perform thin-layer chromatography (Chinese Pharmacopoeia 2025 Edition, Part IV, General Chapter 0502). Place an appropriate amount of developing solvent in the developing tank. The developing solvent is petroleum ether (60-90℃)-chloroform-ethyl acetate (2:1:1). Place the silica gel G thin-layer plate in the developing tank. Take 5 μl of the test solution and 2 μl of the reference solution and spot them separately on the same silica gel G thin-layer plate. During development, the developing solvent moves upward along the thin-layer plate. When it reaches an appropriate height, remove the thin-layer plate, let it dry, and then spray it with vanillin-sulfuric acid solution. Heat at 105℃ until the spots are clearly visible, and examine them again under sunlight.

[0030] The colorimetric results showed that the test sample chromatogram did not produce clear spots at the corresponding positions as the reference sample chromatogram, and the spots diffused. This result indicates that the thin-layer colorimetric results are better when using the three-phase developing solvent cyclohexane-chloroform-ethyl acetate (4:2:3) compared to petroleum ether (60-90℃)-chloroform-ethyl acetate (2:1:1).

[0031] Example 4 Place 1g of Xiaochaihu granules in an Erlenmeyer flask, grind the granules into a fine powder (pass through a No. 4 sieve), add 20ml of ethyl acetate, and sonicate (power 360W, frequency 40KHz) for 10 minutes; filter the extract, concentrate the filtrate under reduced pressure at 50℃ to dryness, add 1ml of ethyl acetate to dissolve the residue, and use it as the test solution.

[0032] Preparation of reference solution: Take 6-gingerol reference standard, add methanol to prepare a solution containing 1 mg per 1 ml, filter through a 0.45 μm microporous membrane and use it as reference solution.

[0033] Perform thin-layer chromatography (Chinese Pharmacopoeia 2025 Edition, Part IV, General Chapter 0502). Place an appropriate amount of developing solvent (cyclohexane-chloroform-ethyl acetate, 4:2:1) in the developing tank. Place a silica gel G thin-layer plate in the developing tank. Apply 5 μl of the test solution and 2 μl of the reference solution separately to the same silica gel G thin-layer plate. During development, the developing solvent moves upwards along the plate. Remove the plate when it reaches an appropriate height, allow it to dry, and then spray it with vanillin-sulfuric acid solution. Heat at 105℃ until the spots are clearly visible, and examine again under sunlight.

[0034] The colorimetric results showed that in the chromatogram of the test sample, the spots at the corresponding positions as in the chromatogram of the reference sample were too close to the origin and could not be effectively separated. This result indicates that the thin-layer colorimetric separation effect is better when the developing solvent ratio is cyclohexane-chloroform-ethyl acetate (4:2:1) compared to petroleum ether (60-90℃)-chloroform-ethyl acetate (4:2:1).

[0035] Example 5 Place 1g of Xiaochaihu granules in an Erlenmeyer flask, grind the granules into a fine powder (pass through a No. 4 sieve), add 20ml of ethyl acetate, and sonicate (power 360W, frequency 40KHz) for 10 minutes; filter the extract, concentrate the filtrate under reduced pressure at 50℃ to dryness, add 1ml of ethyl acetate to dissolve the residue, and use it as the test solution.

[0036] Preparation of reference solution: Take 6-gingerol reference standard, add methanol to prepare a solution containing 1 mg per 1 ml, filter through a 0.45 μm microporous membrane and use it as reference solution.

[0037] Perform thin-layer chromatography (Chinese Pharmacopoeia 2025 Edition, Part IV, General Chapter 0502). Place an appropriate amount of developing solvent in the developing tank (cyclohexane-chloroform-ethyl acetate (2:1:2)). Place the silica gel G thin-layer plate in the developing tank. Apply 5 μl of the test solution and 2 μl of the reference solution separately to the same silica gel G thin-layer plate. During development, the developing solvent moves upward along the thin-layer plate. When the plate reaches an appropriate height, remove it, air dry it, and then spray it with vanillin-sulfuric acid solution. Heat at 105℃ until the spots are clearly visible, and examine again under sunlight.

[0038] The colorimetric results showed that no clear spots were observed at the corresponding positions in the chromatogram of the test sample compared to the reference sample. This result indicates that a thin-layer chromatography (TLC) colorimetric result is better when using a cyclohexane-chloroform-ethyl acetate ratio (4:2:3) compared to a cyclohexane-chloroform-ethyl acetate ratio (2:1:2).

[0039] Example 6 The testing method disclosed in Example 1 was verified in terms of durability and type of thin-layer plate, as follows: 1. Durability Developing solvent: cyclohexane-chloroform-ethyl acetate (4:2:3) Color developer: vanillin sulfuric acid Color development conditions: Heat at 105℃ until color develops. Ambient temperature and humidity: Low temperature (5℃), High temperature (40℃), Low humidity (35%), High humidity (75%) Durability tests were conducted on this product, investigating the effects of different relative temperatures (5°C, 40°C) and relative humidity (35%, 75%). The results are as follows: Figure 6-9 .

[0040] As shown in the figure, under different relative temperatures and humidity conditions, the test sample chromatogram shows spots of the same color at the corresponding positions as the reference sample chromatogram. The separation effect of each spot in the test sample chromatogram is not significantly different, and the method is robust under different temperatures and humidity conditions.

[0041] 2. Types of thin-layer boards Developing solvent: cyclohexane-chloroform-ethyl acetate (4:2:3) Color developer: vanillin sulfuric acid Color development conditions: Heat at 105℃ until color develops. Thin-layer laminates: High-efficiency silicone G-plate, Qingdao Haiyang Chemical Co., Ltd.; High-efficiency silicone H-plate, Qingdao Haiyang Chemical Co., Ltd. Different types of thin-layer plates were tested to investigate the effects of this product. The results are as follows: Figure 10-11 .

[0042] As shown in the figure, compared to the silica gel plate H, the test sample chromatogram using the silica gel plate G exhibits clearer spots and better separation at the corresponding positions as the reference sample chromatogram. Therefore, the silica gel plate G is the preferred choice for thin-layer chromatography.

[0043] The embodiments described above are merely some preferred embodiments of the present invention, and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for detecting the ginger flavor in Xiaochaihu granules, characterized in that, include: Preparation of test solution: Take the granules of Bupleurum chinense, grind them into a fine powder, add ethyl acetate, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in ethyl acetate to obtain the test solution; Preparation of reference solution: Take 6-gingerol reference standard, add methanol to prepare a solution, and use it as the reference solution; Test: Take the above test solution and reference solution and spot them separately on the same silica gel thin layer plate. Use cyclohexane-chloroform-ethyl acetate (volume ratio 4:2:3) as the developing solvent, develop, remove, air dry, spray with vanillin-sulfuric acid solution, heat treatment, and then examine.

2. The method for detecting the ginger flavor in Xiaochaihu granules according to claim 1, characterized in that, The ratio of the amount of Xiaochaihu granules, the added ethyl acetate, and the ethyl acetate used to dissolve the residue is 1g:20ml:1ml.

3. The method for detecting the ginger flavor in Xiaochaihu granules according to claim 1, characterized in that, The ultrasonic treatment has a power of 360W, a frequency of 40KHz, and an ultrasonic duration of 10 minutes.

4. The method for detecting the ginger flavor in Xiaochaihu granules according to claim 1, characterized in that, The filtrate was concentrated to dryness by rotary evaporation under reduced pressure in a 50°C water bath.

5. The method for detecting the ginger flavor in Xiaochaihu granules according to claim 1, characterized in that, The concentration of the reference solution is 1 mg of 6-gingerol per 1 ml.

6. The method for detecting the ginger flavor in Xiaochaihu granules according to claim 1, characterized in that, During testing, 5 μl of the test solution and 2 μl of the reference solution were spotted onto the same silica gel thin-layer plate.

7. The method for detecting the ginger flavor in Xiaochaihu granules according to claim 1, characterized in that, The temperature for the heat treatment is 105°C.

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