Method for detecting content of main active ingredients in ginger and liquorice decoction
The main active ingredients in ginger and licorice decoction were detected by HPLC, which solved the problem of lack of quantitative methods for ginger and licorice decoction. It enabled accurate quantification of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt, thus ensuring the quality and efficacy of ginger and licorice decoction.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SICHUAN NEO GREEN PHARMA TECH DEV
- Filing Date
- 2025-12-19
- Publication Date
- 2026-05-05
AI Technical Summary
The existing technology lacks a quantitative method for the main active ingredients in ginger and licorice decoction, making it impossible to effectively control the quality and efficacy of ginger and licorice decoction preparations.
The main active ingredients in ginger and licorice decoction were detected by HPLC. Reference solutions and test solutions were prepared, and specific chromatographic conditions were used, such as octadecylsilane-bonded silica gel as the stationary phase, acetonitrile and 0.1% phosphoric acid solution as the mobile phase, gradient elution program, detection wavelength of 230 nm, injection volume of 5 μl, and column temperature of 25 °C. This method enabled the quantification of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt.
The method achieves accurate quantification of the main active ingredients in ginger and licorice decoction, with good repeatability and strong specificity, ensuring the quality control and efficacy of ginger and licorice decoction.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of traditional Chinese medicine preparation component detection technology, specifically relating to a method for detecting the content of the main active ingredients in ginger and licorice decoction. Background Technology
[0002] The earliest record of Ginger and Licorice Decoction appears in Sun Simiao's *Qianjin Fang* (Prescriptions Worth a Thousand Gold Pieces) from the Tang Dynasty. Its formula consists of ginger, licorice, ginseng, and jujube, and is a classic Chinese medicine prescription. The entire formula uses ginger as the chief ingredient to dispel cold and resolve phlegm; ginseng to greatly replenish vital energy; licorice to soothe and regulate the body, nourishing yin and yang; and jujube to harmonize the body's defenses. Together, they replenish qi and nourish the body, strengthening the lungs and generating metal (vital energy). It is mainly used to treat symptoms such as pulmonary atrophy, persistent cough with excessive saliva, and dry throat with thirst. Its core effects are warming and tonifying the spleen and stomach, replenishing qi and generating fluids, and moistening the lungs and resolving phlegm. It is also suitable for syndromes such as spleen and stomach deficiency and cold, and insufficient lung qi.
[0003] Ginger and licorice are key ingredients in Ginger and Licorice Decoction. Ginger is pungent and warm, while licorice can moderate its pungent nature, making the medicinal effect milder and longer-lasting. These two herbs hold a unique position in Ginger and Licorice Decoction. 6-Gingerol is the representative active ingredient in ginger, the principal ingredient. Glycyrrhizin, apigenin, isoglycyrrhizin, glycyrrhizin, and glycyrrhizic acid monoammonium salt are important components of licorice. In particular, glycyrrhizin and glycyrrhizic acid monoammonium salt are relatively abundant in licorice. These components are closely related to the efficacy of Ginger and Licorice Decoction. However, there is currently no quantitative research on the active ingredients in Ginger and Licorice Decoction, and no reports on quantitative methods for these main active ingredients. Therefore, it is necessary to provide a quantitative method for the main active ingredients in Ginger and Licorice Decoction to control the quality of the preparation and ensure its efficacy. Summary of the Invention
[0004] To address the above problems, this invention establishes a method for detecting the content of the main active ingredients in ginger and licorice decoction, which includes the following steps: 1) Preparation of reference solutions: Take flavonoids, triterpenoid saponins and phenolic compounds as reference standards, add methanol solution to prepare reference solutions; 2) Preparation of the test solution: Take the ginger and licorice decoction preparation, extract it with methanol solution to obtain the test solution; 3) Detection: HPLC was used for detection. The chromatographic conditions were as follows: Column: octadecylsilane-bonded silica gel as the packing material; Mobile phase: acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; Gradient elution program was as follows: 0-60 min, 20%→45%A, 80%→55%B.
[0005] Further, in step 1), each 1 ml of the reference solution contains 50-150 μg of each reference standard.
[0006] Furthermore, the flavonoids in the reference standard are glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin and / or glycyrrhizin; the triterpenoid saponins are glycyrrhizic acid monoammonium salt; and the phenolic compounds are 6-gingerol.
[0007] Further, in step 2), the mass-to-volume ratio of the ginger and licorice decoction preparation to the methanol solution is 0.2g:5~25ml.
[0008] Furthermore, the concentration of the methanol solution is 70%.
[0009] Furthermore, the ginger and licorice decoction preparation is a preparation made from ginger, licorice, ginseng and jujube as raw materials, plus pharmaceutically acceptable excipients; The mass ratio of ginger, licorice, ginseng and jujube is 65~70:52~58:40~45:32~38.
[0010] Furthermore, the mass ratio of ginger, licorice, ginseng, and jujube is 69:55.2:41.4:36.
[0011] Furthermore, the ginger and licorice decoction preparation includes ginger and licorice decoction decoction, ginger and licorice decoction granules, ginger and licorice decoction powder, ginger and licorice decoction pills, and ginger and licorice decoction material basis; Further, the chromatographic column in step 3) is a ZORBAX SB-Aq 4.6×150mm 3.5μm.
[0012] Further, the chromatographic conditions described in step 3) are as follows: column temperature 25–35°C; flow rate 0.8–1.2 mL / min; injection volume 1–15 μL; and wavelength 230–270 nm.
[0013] Furthermore, the column temperature is 25°C; the flow rate is 1.0 mL / min; the injection volume is 5 μL; and the wavelength is 230 nm.
[0014] Furthermore, it uses the standard curve method to calculate the content.
[0015] The ginger and licorice decoction described in this invention is a traditional Chinese medicine prescription, which comes from Sun Simiao's "Essential Prescriptions Worth a Thousand Gold Pieces" in the Tang Dynasty. Its composition is five liang of ginger, three liang of ginseng, four liang of licorice, and fifteen jujubes. The above four ingredients are boiled in seven liters of water until three liters remain, and then divided into three warm doses.
[0016] The ginger and licorice decoction material standard used in this invention is based on the preparation method of ginger and licorice decoction recorded in the ancient medical book "Qianjin Yaofang" (Essential Prescriptions Worth a Thousand Gold Pieces). Except for the molding process, the other preparation methods are basically consistent with those recorded in "Qianjin Yaofang". The specific preparation method is as follows: Weigh the raw materials according to the formula of ginger and licorice decoction; place the herbs and water in a clay pot, soak for 30 minutes, boil over high heat until the liquid boils, then simmer over low heat until 600 ml remains, filter while hot, remove the residue, extract the liquid, freeze dry, and pulverize to obtain the physical dry extract powder corresponding to the material basis of ginger and licorice decoction.
[0017] This invention discloses a method for detecting the content of major active ingredients in ginger and licorice decoction. Through specific sample pretreatment and chromatographic conditions, it can simultaneously and quantitatively detect the main active ingredients in ginger and licorice, including glycyrrhizin, apigenin, isoglycyrrhizin, glycyrrhizin, glycyrrhizin, glycyrrhizic acid monoammonium salt, and 6-gingerol. Methodological validation has demonstrated that this method exhibits good repeatability, high specificity, and accurate and reliable results, providing a more comprehensive approach for the quality control of ginger and licorice decoction and ensuring its clinical efficacy.
[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.
[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description
[0020] Figure 1 Mobile phase selection diagram; Figure 2 Gradient selection graph; Figure 3 Wavelength selection diagram; Figure 4 Flow velocity study results; Figure 5 Column temperature investigation results; Figure 6 Injection volume investigation; Figure 7 Identification and investigation of the ginger and licorice decoction peak; Figure 8 Existentiality of Ginger and Licorice Decoction; Figure 9 Glycyrrhizin standard curve; Figure 10 Standard curve of celery glycoside isoglycyrrhizin; Figure 11 Standard curve of isoliquiritigenin; Figure 12 Glycyrrhizin standard curve; Figure 136-Gingerol standard curve; Figure 14 Standard curve of glycyrrhizic acid monoammonium salt; Detailed Implementation Example 1: Detection Method of the Invention (1) Preparation of mixed reference standards Accurately weigh the following reference standards: glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt. Add 70% methanol to prepare a mixed solution containing 100 μg of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt per 1 ml. (2) Preparation of the test solution Accurately weigh 0.2g of the ginger and licorice decoction preparation, place it in a stoppered conical flask, accurately add 10ml of 70% methanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the final product.
[0021] (3) HPLC chromatographic detection The chromatographic column was a ZORBAX SB-Aq 4.6×150mm 3.5μm; mobile phase A was acetonitrile, and mobile phase B was 0.1% phosphoric acid solution; the flow rate was 1.0 mL / min; the injection volume was 5 μL; the column temperature was 25℃; the detection wavelength was 230nm; and the gradient elution program was as follows: (4) The contents of glycyrrhizin, celery isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol and glycyrrhizic acid monoammonium salt in the ginger and licorice decoction were calculated using the standard curve method; The standard curve method involves plotting a standard curve using the concentration of each component in a series of concentration reference solutions as the abscissa and the peak area of each component as the ordinate, thus obtaining a linear regression equation. The content of each component in the ginger and licorice decoction preparation is then calculated based on the linear regression equation.
[0022] The following experimental examples further illustrate the beneficial effects of the present invention. Experimental Example 1: Study on the active ingredients in ginger and licorice decoction 1. Experimental instruments and materials High-performance liquid chromatograph (HPLC): Agilent 1260 HPLC system; Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instruments Ltd.); Ultrapure water system: Cellular 1810A (Shanghai Moller Scientific Instruments Co., Ltd.); Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.); Chromatographic column: ZORBAX SB-Aq4.6×150mm 3.5-Micron; Acetonitrile and phosphoric acid were chromatographic grade, water was ultrapure water, and all other reagents were analytical grade. Glycyrrhizin reference standard (China National Institutes for Food and Drug Control, batch number: 111610-201106, content calculated as 93.7%); Isoglycyrrhizin reference standard (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq20022803, content calculated as 100%); Glycyrrhizin reference standard (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 070002-202108, content calculated as 99.96%); Cinnamoxylic acid isoglycyrrhizin reference standard (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: wkq20100907, content calculated as 100%; 6-gingerol reference standard (Chengdu Desite Biotechnology Co., Ltd., batch number: 111833-201806, content calculated as 99.9%); glycyrrhizic acid monoammonium salt reference standard (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDG000802, content calculated as 98.95%); Ginger and licorice decoction material reference samples: SJGCT-01, SJGCT-02, SJGCT-03, SJGCT-04, SJGCT-05, SJGCT-6, provided by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd.: The specific steps for preparing the ginger and licorice decoction material reference are as follows: ① Processing: According to the requirements of classic prescriptions, each ingredient in the ginger and licorice decoction is processed. Remove impurities from ginger, wash it, and cut it into thick slices before use; remove impurities from licorice, wash it, moisten it thoroughly, cut it into thick slices, and dry it at a low temperature of 60℃; moisten ginseng thoroughly, cut it into thin slices, and dry it at a low temperature of 60℃; remove impurities from jujubes, wash them, sun-dry them, and remove the pits before use. ②Weighing: Weigh 69.0g of ginger, 55.2g of licorice, 41.4g of ginseng, and 36.0g of jujubes according to the prescription ratio, and add 1400ml of water; ③Decoction: Place the herbs and water in a clay pot, soak for 30 minutes, bring to a boil over high heat, then reduce to a simmer and reduce to 600ml. Filter while hot, remove the dregs, and extract the liquid. ④ Drying: Prepare the ginger and licorice decoction material standard from the aqueous extract obtained in step ③ according to the following freeze-drying parameters. The freeze-drying procedure is as follows: A: Pre-freezing: Temperature: -50℃, holding temperature: 5h; Pressure: atmospheric pressure; B: Sublimation drying: Temperature: -40℃, holding temperature: 2h; Pressure: 0.0pa; Temperature: -30℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: -20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: -10℃, temperature maintenance: 3h; Pressure: 0.0pa; Temperature: 0℃, temperature maintenance: 3h; Pressure: 0.0pa; C: Desorption drying: Temperature: 10℃, holding temperature: 3h; Pressure: 0.0pa; Temperature: 20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 30℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 40℃, temperature maintenance: 2h; pressure: 0.0pa.
[0023] 2 Experimental conditions 2.1 Chromatographic conditions and system suitability test The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 4.6 mm inner diameter, 3.5 μm particle size); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B for gradient elution; the flow rate was 1.0 mL / min; the column temperature was 25 °C; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 5000; the gradient elution program was as follows: 2.2 Preparation of reference solution Take appropriate amounts of glycyrrhizin reference standards, apigenin isoglycyrrhizin reference standards, isoglycyrrhizin reference standards, glycyrrhizin reference standards, 6-gingerol reference standards, and glycyrrhizic acid monoammonium salt reference standards, accurately weigh them, and add 70% methanol to prepare a mixed solution containing 100 μg of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt per 1 ml.
[0024] 2.3 Preparation of the test solution Take an appropriate amount of ginger and licorice decoction reference sample, about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of 70% methanol, seal tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the final product.
[0025] 2.4 Determination Method Accurately pipette 5 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.
[0026] 3. Establishment of content determination method 3.1 Chromatographic conditions and system suitability test 3.1.1 Selection of mobile phase Based on the experimental conditions outlined above, the types of mobile phases were adjusted, specifically acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% formic acid, and methanol-0.1% phosphoric acid, for further investigation. (See attached text.) Figure 1 .
[0027] Figure 1 The results showed that when acetonitrile-0.1% phosphoric acid was used as the mobile phase, the peak shape and separation effect of the target peak in the chromatogram were good. When acetonitrile-0.1% formic acid was used as the mobile phase, the elution time of the target peak in the chromatogram was generally delayed. Even after 60 minutes of gradient elution, the components in the ginger and licorice decoction sample solution could not be eluted, resulting in low detection efficiency and failing to meet the requirements of rapid and efficient detection. When methanol-0.1% phosphoric acid was used as the mobile phase, there were many interfering peaks in the chromatogram, resulting in poor separation effect of the target peak and inability to perform accurate integration. Therefore, acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase for further investigation.
[0028] 3.1.2 Gradient Selection Based on the experimental conditions outlined above, the gradient elution procedure was adjusted for further investigation, as detailed in gradients 1 through 3. Results are shown below. Figure 2 .
[0029] Gradient 1 Gradient 2 Gradient 3 (including measurement) Figure 2 The results showed that the elution patterns of the mobile phases in gradients 1 and 3 were the same, with the proportion of acetonitrile gradually increasing within 60 min. However, the change in the initial concentration meant that the target peaks in the chromatogram of gradient 1 could not be effectively separated, thus failing to meet the requirements for accurate detection. The elution pattern of the mobile phase in gradient 2 was fundamentally changed. In the first 3 min, isocratic elution occurred first, and in the following 57 min, the proportion of acetonitrile gradually increased. This resulted in peak 4 being unable to be effectively separated from the previous peak in the chromatogram, leading to integration error. This significantly reduced the quantitative accuracy of the component corresponding to peak 4, making it impossible to guarantee the reliability of the detection results. Since the peak shapes and separation effects of each target peak in gradient 3 were good, gradient 3 was selected for further investigation.
[0030] 3.1.3 Wavelength Selection Based on the above-planned experimental conditions, the test solution was scanned across the entire wavelength range using a diode array detector, and chromatograms of the test solution were extracted at wavelengths of 210 nm, 230 nm, 250 nm, 270 nm, and 300 nm. (See...) Figure 3 By comparing the chromatograms, it was found that the index peak showed greater absorption at a detection wavelength of 230 nm, and the chromatogram baseline was more stable. Therefore, the detection wavelength was determined to be 230 nm.
[0031] 3.1.4 Flow velocity assessment Under the proposed experimental conditions, the flow rates of 0.9 ml / min, 1.0 ml / min, and 1.1 ml / min were investigated. See [link / reference]. Figure 4 The results showed that when the flow rate was 0.9 ml / min, peaks 5 and 6 in the chromatogram could not be effectively separated, resulting in an integral error during quantification; when the flow rate was 1.1 ml / min, peak 1 in the chromatogram could not be effectively separated from the previous peak, resulting in an integral error during quantification; and when the flow rate was 1.0 ml / min, the peak shape and separation effect of the chromatogram were better, and the proposed flow rate was 1.0 ml / min.
[0032] 3.1.5 Column Temperature Investigation Based on the above-specified experimental conditions, the results were investigated at column temperatures of 20℃, 25℃, and 30℃. (See...) Figure 5 The results showed that when the column temperature was 30℃, peak 6 exhibited obvious tailing; when the column temperature was 20℃, the separation of peaks 5 and 6 was poor; and when the column temperature was 25℃, the chromatogram peak shape and separation were better, so a column temperature of 25℃ was proposed.
[0033] 3.1.6 Sample Injection Volume Examination Based on the above-established experimental conditions, the effects were investigated at injection volumes of 5 μl, 10 μl, and 15 μl. Figure 6 As shown in the figure. The results indicate that when the injection volume is 10 μl and 15 μl, the baseline of peak 1 in the chromatogram is not smooth, which affects the accurate quantification of the component corresponding to peak 1; when the injection volume is 5 μl, the resolution of each peak in the chromatogram is moderate. Therefore, the injection volume is determined to be 5 μl.
[0034] 3.1.7 Finalized Chromatographic Conditions and System Usability for the Determination of Ginger and Licorice Root Content in Ginger and Licorice Root Decoction The column was packed with octadecylsilane-bonded silica gel (ZORBAX SB-Aq, column length 150 mm, inner diameter 4.6 mm, particle size 3.5 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 25 °C; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 5000.
[0035] The assay involves precisely pipetting 5 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.
[0036] 3.2 Investigation on the preparation of test solution 3.2.1 Investigation of Extraction Solvents Take an appropriate amount (approximately 0.2 g) of the ginger and licorice decoction reference sample (batch number: SJGCT-01), weigh it, and place it in a stoppered conical flask. Accurately add 10 ml each of water, 30% methanol, 70% methanol, and methanol, weigh the flask, and sonicate (600 W, 40 kHz) for 30 minutes. Cool the flask, weigh it again, and replenish the lost weight with the appropriate solvent. Shake well, filter, and collect the filtrate. Accurately inject 5 μl of each sample solution into the liquid chromatograph. Calculate the contents of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt under different extraction solvents. The results are shown in Table 1.
[0037] Table 1 Results of the extraction solvent investigation As shown in Table 1, the extraction solvent has a significant impact on the accuracy of content determination. When the extraction solvent is 70% methanol, the content of each active ingredient is the highest and the extraction effect is the best. Therefore, the extraction solvent is determined to be 70% methanol.
[0038] 3.2.2 Examination of Extraction Methods Take an appropriate amount (approximately 0.2 g) of the ginger and licorice decoction reference sample (batch number: SJGCT-01), accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of 70% methanol, weigh it, reflux and sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, weigh it again, make up the weight loss with 70% methanol, shake well, filter, and collect the filtrate. Accurately pipette 5 μl of each test solution and inject it into the liquid chromatograph. Calculate the contents of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt under different extraction methods. The results are shown in Table 2.
[0039] Table 2. Examination of Extraction Methods The results showed that the extraction method had little effect on the contents of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt. Therefore, the ultra-fast extraction method, which is simpler to operate, was chosen.
[0040] 3.2.3 Investigation on the amount of extraction solvent added Take an appropriate amount of ginger and licorice decoction reference sample (batch number: SJGCT-01), approximately 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml, 15ml, and 25ml of 70% methanol, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate. Accurately pipette 5μl of each test solution and inject it into the liquid chromatograph. Calculate the contents of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt under different amounts of extraction solvent added. The results are shown in Table 3.
[0041] Table 3. Investigation of the amount of extraction solvent added The results showed that the amount of extraction solvent added had little effect on the component content, and the content was higher when the amount of solvent added was 10 ml. Therefore, the amount of solvent added was determined to be 10 ml.
[0042] 3.2.4 Examination of extraction time Take an appropriate amount (approximately 0.2 g) of the ginger and licorice decoction reference sample (batch number: SJGCT-01), accurately weigh it, place it in a stoppered conical flask, accurately add 10 ml of 70% methanol, weigh it, and sonicate it (power 600W, frequency 40kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. After cooling, weigh it again, and make up the weight loss with 70% methanol. Shake well, filter, and collect the filtrate. Accurately pipette 5 μl of each test solution and inject it into the liquid chromatograph. Calculate the contents of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt under different extraction solvents. The results are shown in Table 4.
[0043] Table 4. Examination of Extraction Time The results showed that extraction time had little effect on the component content; the content was higher when the extraction time was 30 minutes, therefore, 30 minutes of extraction was sufficient. Thus, the extraction time for the test sample was determined to be 30 minutes.
[0044] The preparation method for the test sample for the determination of ginger and licorice decoction content is as follows: Take 0.2g of ginger and licorice decoction accurately, place it in a stoppered conical flask, accurately add 10ml of 70% methanol, stopper tightly, weigh, sonicate (power 600W, frequency 40KHz) for 30 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test sample.
[0045] 3.2.5 Method for determining the content of ginger and licorice in decoction The column was packed with octadecylsilane-bonded silica gel (ZORBAX SB-Aq, column length 150 mm, inner diameter 4.6 mm, particle size 3.5 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B for gradient elution; the flow rate was 1.0 mL / min; the column temperature was 25 °C; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 5000; the gradient elution program was as follows: Preparation of reference solution: Take appropriate amounts of glycyrrhizin reference standard, apigenin isoglycyrrhizin reference standard, isoglycyrrhizin reference standard, glycyrrhizin reference standard, 6-gingerol reference standard, and glycyrrhizic acid monoammonium salt reference standard, accurately weigh them, and add 70% methanol to prepare a mixed solution containing 100 μg of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt per 1 ml.
[0046] Preparation of the test solution: Take an appropriate amount of ginger and licorice decoction, about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of 70% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.
[0047] The assay involves precisely pipetting 5 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.
[0048] 3.3 Methodological Examination 3.3.1 Specificity Examination Preparation of reference solution: Take appropriate amounts of glycyrrhizin reference standard, apigenin isoglycyrrhizin reference standard, isoglycyrrhizin reference standard, glycyrrhizin reference standard, 6-gingerol reference standard, and glycyrrhizic acid monoammonium salt reference standard, accurately weigh them, and add 70% methanol to prepare a mixed solution containing 100 μg of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt per 1 ml, as the reference solution.
[0049] Preparation of the test solution: Take 0.2g of the ginger and licorice decoction reference sample, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of 70% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.
[0050] Preparation of negative control solutions for single-herb deficiency: Following the above-described method for preparing test solutions, negative control solutions for ginger and licorice decoction lacking a single herb and negative control solutions for ginger and licorice decoction without ginger were prepared. Results are shown below. Figures 7-8 .
[0051] As shown in the figure, the negative solution did not interfere with the determination of the target peak, indicating that the method has good specificity; the identified peaks mainly came from licorice and ginger.
[0052] 3.3.2 Precision Examination A mixed solution of glycyrrhizin reference standard, apigenin isoglycyrrhizin reference standard, isoglycyrrhizin reference standard, glycyrrhizin reference standard, 6-gingerol reference standard, and glycyrrhizic acid monoammonium salt reference standard was injected six times consecutively. The peak area was recorded and the RSD value was calculated. The results are shown in Table 5.
[0053] Table 5 Precision test results As can be seen from the above, the peak area RSD values of each reference standard in the precision test were all less than 1.0%, indicating that the instrument precision was good.
[0054] 3.3.3 Linear Relationship Precisely pipettes were used to prepare reference solutions of different concentrations (for content determination) from the mixed mother liquor of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt. 5 μl of each solution was injected into the liquid chromatograph, and the peak area was obtained. Response curves were plotted with reference concentration (X, ng) on the x-axis and peak area (Y) on the y-axis. The results are shown in Tables 6-11. Figures 9-14 .
[0055] Table 6. Results of Glycyrrhizin Standard Curve Analysis Table 7. Results of the standard curve analysis of apigenin isoliquiritigenin Table 8. Results of the standard curve analysis for isoliquiritigenin Table 9. Results of Glycyrrhizin Standard Curve Analysis Table 10. Results of Standard Curve Analysis of 6-Gingerol Table 11 Analytical Results of Standard Curve for Glycyrrhizic Acid Monoammonium Salt The results showed that the linear regression equation for glycyrrhizin was y = 0.4661x - 16.426, and the correlation coefficient R² = 0.9996, indicating that the concentration of glycyrrhizin and the peak area had a good linear relationship in the range of 83.9552 ng to 2098.88 ng. The linear regression equation for apigenin isoglycyrrhizin was y = 0.5343x - 0.9344, with a correlation coefficient R² = 0.9999, indicating a good linear relationship between apigenin isoglycyrrhizin concentration and peak area within the concentration range of 45.12 ng to 1128 ng. The linear regression equation for isoglycyrrhizin was y = 0.7949x - 0.9772, with a correlation coefficient R² = 0.9999, indicating a good linear relationship between isoglycyrrhizin concentration and peak area within the concentration range of 42.26 ng to 1056.5 ng. The linear regression equation for glycyrrhizin was y = 2.1116x - 4.7734, with a correlation coefficient R² = 0.9999, indicating a good linear relationship between glycyrrhizin concentration and peak area within the concentration range of 47.301072 ng ~ 1182.5268 ng. The linear regression equation for 6-gingerol was y = 0.593x - 1.2851, with a correlation coefficient R² = 0.9999, indicating a good linear relationship between 6-gingerol concentration and peak area within the concentration range of 132.74712 ng ~ 3318.678 ng. The linear regression equation for glycyrrhizic acid monoammonium salt was y = 0.1853x - 16.426, with a correlation coefficient R² = 0.9996, indicating a good linear relationship between the concentration of glycyrrhizic acid monoammonium salt and the peak area within the concentration range of 211.19888 ng ~ 5279.972 ng.
[0056] 3.3.4 Repeatability Take an appropriate amount of ginger and licorice decoction reference sample (batch number: SJGCT-01), about 0.2g, and accurately weigh 6 portions. The same operator prepares the test solution according to the determined method. Accurately pipette 5μl of each test solution and inject it into the liquid chromatograph. Calculate the contents of glycyrrhizin reference standard, apigenin isoglycyrrhizin reference standard, isoglycyrrhizin reference standard, glycyrrhizin reference standard, 6-gingerol reference standard, and glycyrrhizic acid monoammonium salt in the 6 samples. The results are shown in Tables 12-14.
[0057] Table 12 Results of Repeatability Experiments Table 13 Results of Repeatability Experiments Table 14 Results of Repeatability Experiments The results showed that the RSD values of each indicator were all less than 3%, indicating that the method had good repeatability.
[0058] 3.3.5 Intermediate Precision Based on the above-planned experimental conditions, different personnel conducted measurements at different times using different instruments. An appropriate amount (approximately 0.2g) of the ginger and licorice decoction reference material was accurately weighed and used to prepare the test sample for analysis. The calculated contents of glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, 6-gingerol, and glycyrrhizic acid monoammonium salt are shown in Tables 15-17.
[0059] Table 15 Results of Intermediate Precision Study Table 16 Results of Intermediate Precision Study Table 17 Results of Intermediate Precision Study The results showed that the RSD values of the contents were all less than 3.0%, indicating that the intermediate precision of this method was good.
[0060] 3.3.6 Recovery rate Take approximately 0.1 g of the test sample (batch number: SJGCT-01, glycyrrhizin content 5.7 mg / g, apigenin isoglycyrrhizin content 0.59 mg / g, isoglycyrrhizin content 0.78 mg / g, glycyrrhizin content 1.3 mg / g, 6-gingerol content 1.21 mg / g, glycyrrhizic acid monoammonium salt content 16.78 mg / g), in 6 portions. Accurately weigh each portion and add a specific amount of glycyrrhizin reference standard, apigenin isoglycyrrhizin reference standard, isoglycyrrhizin reference standard, glycyrrhizin reference standard, 6-gingerol reference standard, and glycyrrhizic acid monoammonium salt reference standard to each sample. Prepare and determine the test solution according to the prescribed method, calculate the recovery rate, and the results are shown in Table 18. The calculation formula is as follows: Table 18 Results of the spiking recovery experiment The results showed that the recovery rates of all indicators were between 90% and 108%. The method demonstrated good accuracy.
[0061] 3.3.7 Stability Test According to the experimental conditions proposed above, a test solution was prepared, and the peak area of each index was measured at 0h, 2h, 4h, 8h, 12h and 24h respectively. The results are shown in Table 19.
[0062] Table 19 Stability test results The results showed that under the experimental conditions, the RSD% of each index was below 3%, and the test solution had good stability within 24 hours.
[0063] 3.3.8 Verification of Sample Content Determination The six batches of reference samples of ginger and licorice decoction were tested according to the proposed method, and the results are shown in Table 20.
[0064] Table 20. Results of content determination in six batches of ginger and licorice decoction reference samples As can be seen from the above, the method for determining the content of ginger and licorice decoction can effectively detect the reference sample of ginger and licorice decoction, and the method is stable and feasible.
[0065] In summary, the content detection method of this invention can simultaneously and quantitatively detect the main active ingredients in ginger and licorice in ginger and licorice decoction, including glycyrrhizin, apigenin isoglycyrrhizin, isoglycyrrhizin, glycyrrhizin, glycyrrhizic acid monoammonium salt and 6-gingerol. The method has good repeatability and specificity, and the results are accurate and reliable, providing a comprehensive method for the quality control of ginger and licorice decoction.
Claims
1. A method for detecting the content of the main active ingredients in ginger and licorice decoction, characterized in that: It includes the following steps: 1) Preparation of reference solutions: Take flavonoids, triterpenoid saponins and phenolic compounds as reference standards, add methanol solution to prepare reference solutions; 2) Preparation of the test solution: Take the ginger and licorice decoction preparation, extract it with methanol solution to obtain the test solution; 3) Detection: HPLC was used for detection. The chromatographic conditions were as follows: Column: octadecylsilane-bonded silica gel as the packing material; Mobile phase: acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; Gradient elution program was as follows: 0-60 min, 20%→45%A, 80%→55%B.
2. The content detection method according to claim 1, characterized in that: Step 1) Each 1 ml of the reference solution contains 50-150 μg of each reference standard.
3. The content detection method according to claim 1 or 2, characterized in that: The reference standard contains flavonoids such as glycyrrhizin, apigenin, isoglycyrrhizin, and / or glycyrrhizin; triterpenoid saponins such as glycyrrhizic acid monoammonium salt; and phenolic compounds such as 6-gingerol.
4. The content detection method according to claim 1, characterized in that: Step 2) The mass-to-volume ratio of the ginger and licorice decoction preparation to the methanol solution is 0.2g: 5~25ml; the concentration of the methanol solution is 70%.
5. The content detection method according to claim 1 or 4, characterized in that: The ginger and licorice decoction preparation is made from ginger, licorice, ginseng and jujube as raw materials, plus pharmaceutically acceptable excipients.
6. The content detection method according to claim 5, characterized in that: The ginger and licorice decoction preparations include ginger and licorice decoction decoction, ginger and licorice decoction granules, ginger and licorice decoction powder, ginger and licorice decoction pills, and ginger and licorice decoction material basis.
7. The content detection method according to claim 1, characterized in that: Step 3) The chromatographic column is a ZORBAX SB-Aq 4.6×150mm 3.5μm.
8. The content detection method according to claim 1, characterized in that: The chromatographic conditions described in step 3) are as follows: column temperature 25–35℃; flow rate 0.8–1.2 mL / min; injection volume 1–15 μL; and wavelength 230–270 nm.
9. The content detection method according to claim 1, characterized in that: The column temperature was 25℃; the flow rate was 1.0 mL / min; the injection volume was 5 μL; and the wavelength was 230 nm.
10. The content detection method according to any one of claims 1 to 9, characterized in that: It uses the standard curve method to calculate the content.