Determination of paeoniflorin, liquiritin, glycyrrhizic acid, paeonol and ginsenoside in samples and its application in quality control of Wening Decoction

Abstract

The present application relates to a method for detecting the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and ginsenoside in a sample and application in quality control of Wenjing Decoction. The detection method comprises: performing first extraction treatment on the sample, collecting the first extraction liquid for first liquid chromatography detection, and determining the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid and / or paeonol in the sample based on the detection result; and / or performing second extraction treatment on the sample, collecting the second extraction liquid for second liquid chromatography detection, and determining the content of ginsenoside in the sample based on the detection result. The detection method of the present application can accurately detect the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and / or ginsenoside contained in the sample, has high accuracy, good precision, high sensitivity, good reproducibility, simple operation, short time, etc. The quality control method of Wenjing Decoction of the present application has the advantages of stability, controllability, good reproducibility, strong operability, etc., and the quality transfer traceability in the decoction process is strong.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically to a method for detecting the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and ginsenosides in samples and its application in the quality control of Wenjing Decoction. Background Technology

[0002] Wenjing Decoction, formula number 39, was originally recorded in Volume 1 of *Complete Collection of Effective Prescriptions for Women* by Chen Ziming of the Southern Song Dynasty, under the section on regulating menstruation, specifically in Chapter 12, "Treatise on Prescriptions for Abdominal Pain Due to Absence or Lack of Menstrual Flow." The original text of Wenjing Decoction states: Angelica sinensis, Ligusticum chuanxiong, Paeonia lactiflora, Cinnamomum cassia, Paeonia suffruticosa, and Curcuma zedoaria, each half a liang; Ginseng, Glycyrrhiza uralensis, and Achyranthes bidentata, each one liang. Take five qian (approximately 15 grams) of the decoction with one and a half cups of water until reduced to eight-tenths of the original volume. Strain and drink warm. This formula is also known as "Liangfang Wenjing Tang" (Good Formula for Warming the Meridians Decoction). Cinnamon bark warms the meridians, unblocks the channels, dispels cold, and relieves pain. Ginseng, licorice, angelica, and chuanxiong warm and tonify qi and blood, invigorate blood circulation, and regulate menstruation. Curcuma zedoaria, peony bark, and peony root are added to invigorate blood circulation, remove blood stasis, and relieve spasms and pain. Achyranthes bidentata guides the decoction to the meridians. The entire formula is warm without being drying, purging without causing harm, and tonifying without causing stagnation. It treats amenorrhea caused by external cold pathogens and is an essential formula for regulating menstruation in gynecology. It is effective in treating deficiency and cold of the Chong and Ren meridians with blood stasis. It is also used to treat dysmenorrhea caused by cold stagnation and blood stasis, as well as scanty menstruation, delayed menstruation, and infertility. Clinically, it is mainly used to treat dysmenorrhea, amenorrhea, scanty menstruation, infertility, endometriosis, and endocrine disorders. Wenjing Decoction is a classic formula used clinically to treat dysmenorrhea caused by cold stagnation and blood stasis. It is a widely used gynecological prescription for regulating menstruation and has significant development potential. However, the efficacy of Wenjing Decoction varies due to factors such as raw materials and processing. Therefore, establishing quality control standards for Wenjing Decoction will help improve its research and application. Summary of the Invention

[0003] This invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, this invention provides a method for detecting the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol, and ginsenosides in a sample, its application in the quality control of Wenjing Decoction, and the material standard for Wenjing Decoction. The detection method of this invention can accurately detect the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol, and / or ginsenosides in a sample, with high accuracy, good precision, high sensitivity, good reproducibility, simple operation, and short processing time. The quality control method for Wenjing Decoction of this invention has the advantages of stability, controllability, good reproducibility, and strong operability; furthermore, the quality transfer during the decoction process has strong traceability.

[0004] In one aspect of the present application, the present application provides a method for detecting the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and / or ginsenoside in a sample. According to an embodiment of the present application, the method comprises: performing a first extraction treatment on the sample, collecting a first extraction solution, performing a first liquid chromatography detection on the first extraction solution, and determining the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid and / or paeonol in the sample based on the detection result; and / or performing a second extraction treatment on the sample, collecting a second extraction solution, performing a second liquid chromatography detection on the second extraction solution, and determining the content of ginsenoside in the sample based on the detection result; wherein the first extraction solvent used in the first extraction treatment comprises a methanol solution; and the second extraction solvent used in the second extraction treatment comprises water-saturated n-butanol.

[0005] The first extraction solvent has a good extraction effect on the paeoniflorin, glycyrrhizin, glycyrrhizic acid and paeonol components in the sample, and can completely dissolve the components in the first extraction solvent. Then, the paeoniflorin, glycyrrhizin, glycyrrhizic acid and paeonol components in the first extraction solution can be separated and determined by the first liquid chromatography condition. The inventors have creatively obtained the methanol solution as the first extraction solvent through a large number of experiments, which can extract the paeoniflorin, glycyrrhizin, glycyrrhizic acid and paeonol in the sample. When multiple components need to be extracted at the same time, multiple extraction solvents do not need to be used, which improves the extraction efficiency and reduces the extraction cost and time.

[0006] The second extraction solvent has a good extraction effect on the ginsenoside in the sample, and the second liquid chromatography condition can better separate and determine the ginsenoside in the second extraction solution. The water-saturated n-butanol as the extraction solvent can efficiently extract the ginsenoside in the sample. Therefore, the detection method according to the embodiment of the present application has high accuracy, good precision, high sensitivity, good reproducibility, simple operation and short time.

[0007] According to an embodiment of the present application, the detection method can further comprise at least one of the following additional technical features:

[0008] According to an embodiment of the present application, the first extraction treatment comprises: ultrasonic mixing the sample with the first extraction solvent for 15-45 minutes. The inventors have obtained the above-mentioned preferable ultrasonic time through a large number of experiments, and the first extraction solvent has a good extraction effect on the paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and ginsenoside components, so that the components are completely dissolved in the first extraction solvent.

[0009] According to an embodiment of the present application, the ultrasonic mixing time is 25-35 minutes. Thus, the extraction efficiency is further improved.

[0010] According to the embodiment of the present application, based on 1 gram of the sample, the first extraction solvent is added in an amount of 30-50 mL, and the concentration of the methanol solution is 30-60% by volume. In this way, the extraction effect of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and ginsenoside components in the sample is good.

[0011] According to the embodiment of the present application, the first liquid chromatography detection condition is as follows: the chromatographic column is a C18 column; the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is selected from acetonitrile, and the mobile phase B is selected from 0.05-0.2% by volume of a formic acid solution; the flow rate is 0.5-1.5 mL / min; the column temperature is 20-30°C; the injection amount is 5-15 μL; when paeoniflorin and / or glycyrrhizin is detected, the detection wavelength is 230-240 nm; when glycyrrhizic acid is detected, the detection wavelength is 240-260 nm; and when paeonol is detected, the detection wavelength is 270-290 nm. The inventors obtain the above-mentioned preferable liquid chromatography detection condition through a large number of experiments, which can effectively separate paeoniflorin, glycyrrhizin, glycyrrhizic acid and paeonol, especially when the above-mentioned several substances exist simultaneously, which can achieve a good separation purpose, has a high separation degree, and prevents the peak area overlap between different components to affect the content detection result.

[0012] According to the embodiment of the present application, the second liquid chromatography detection condition is as follows: the chromatographic column is a C18 column; the mobile phase is selected from an acetonitrile aqueous solution; the flow rate is 0.5-1.5 mL / min; the column temperature is 25-35°C; the detection wavelength is 200-205 nm; and the injection amount is 5-15 μL. The inventors obtain the above-mentioned preferable liquid chromatography detection condition through a large number of experiments, which can effectively separate ginsenoside, has a high separation degree, and prevents the peak area overlap between different components to affect the content detection result.

[0013] According to the embodiment of the present application, when paeoniflorin and / or glycyrrhizin is detected, the elution mode used by the first liquid chromatography detection is as follows:

[0014] 0-8 min, the mobile phase A is 17%, and the mobile phase B is supplemented to 100% by volume;

[0015] 8-10 min, the mobile phase A is increased from 17% by volume to 19% by volume, and the mobile phase B is supplemented to 100% by volume;

[0016] 10-20 min, the mobile phase A is increased from 19% by volume to 25% by volume, and the mobile phase B is supplemented to 100% by volume;

[0017] 20-25 min, the mobile phase A is increased from 25% by volume to 70% by volume, and the mobile phase B is supplemented to 100% by volume;

[0018] 25-35 min, mobile phase A from 70% to 17%, mobile phase B to 100%.

[0019] According to the embodiment of the present application, when determining glycyrrhizic acid and / or paeonol, the elution mode of the first liquid chromatography detection is as follows:

[0020] 0-15 min, mobile phase A from 27% to 38%, mobile phase B to 100%;

[0021] 15-20 min, mobile phase A from 38% to 42%, mobile phase B to 100%;

[0022] 20-25 min, mobile phase A from 42% to 55%, mobile phase B to 100%;

[0023] 25-30 min, mobile phase A from 55% to 90%, mobile phase B to 100%;

[0024] 30-35 min, mobile phase A from 90% to 27%, mobile phase B to 100%.

[0025] The inventors obtain the above-mentioned preferable elution conditions through a large number of experiments, thereby effectively separating paeonol, glycyrrhizin, glycyrrhizic acid and paeonol, especially when the above-mentioned substances exist simultaneously, the preferable separation purpose can be achieved, the separation degree is high, and the peak area overlapping between different components is prevented to affect the content detection result.

[0026] According to the embodiment of the present application, the elution mode of the second liquid chromatography detection is as follows: 0-35 min, mobile phase A is 19%; 35-55 min, mobile phase A from 19% to 29%; 55-70 min, mobile phase A is 29%; 70-110 min, mobile phase A from 29% to 40%. The inventors obtain the above-mentioned preferable elution conditions through a large number of experiments, thereby effectively separating ginsenosides, the separation degree is high, and the peak area overlapping between different components is prevented to affect the content detection result.

[0027] According to the embodiment of the present application, the second extraction process comprises: Step 1: mixing the sample with dichloromethane, collecting the water layer after extraction; Step 2: repeatedly extracting the water layer with water-saturated n-butanol, combining the n-butanol layer obtained each time, and discarding the water layer; Step 3: repeatedly washing the n-butanol layer with ammonia solution, combining the n-butanol layer obtained each time, combining the ammonia solution layer obtained each time; Step 4: mixing the ammonia solution layer obtained in Step 3 with water-saturated n-butanol, collecting the n-butanol layer and combining it with the n-butanol layer obtained in Step 3, washing with water, drying the n-butanol layer, dissolving and constant volume of the residue collected with methanol, filtering, and passing through a membrane to obtain the test solution. The purpose of Step 1 is to remove components with small polarity and retain components with large polarity. The purpose of Step 2 is to effectively extract ginsenosides. In Steps 3 and 4, interfering components are removed by washing with ammonia solution, and water-saturated n-butanol and ammonia solution are removed by drying treatment, thereby ensuring subsequent liquid chromatography detection. Thus, the accuracy of detecting ginsenosides in the sample can be improved.

[0028] According to the embodiment of the present application, the sample is in a liquid state, and in Step 2, the volume of water-saturated n-butanol used for each extraction is 0.5-1.5 times the volume of the sample. Thus, ginsenosides in the water layer can be better extracted.

[0029] According to the embodiment of the present application, in Step 2, the volume of water-saturated n-butanol used for each extraction is 0.8-1.2 times the volume of the sample. Thus, ginsenosides in the water layer can be better extracted.

[0030] According to the embodiment of the present application, in Step 2, the number of repeated extractions is 2-4 times. Thus, ginsenosides in the water layer can be better extracted.

[0031] According to the embodiment of the present application, in Step 2, the number of repeated extractions is 3 times. Thus, ginsenosides in the water layer can be better extracted.

[0032] According to the embodiment of the present application, the sample is in a powder state, Step 1: dissolving the sample in water to obtain a sample aqueous solution, adding dichloromethane to the sample aqueous solution, and collecting the water layer after extraction; in Step 2, the volume of water-saturated n-butanol used for each extraction is 0.5-1.5 times, preferably 0.8-1.2 times, the volume of the sample aqueous solution; the number of repeated extractions is 2-4 times, preferably 3 times; in Step 3, the volume of ammonia solution used for each washing is 0.5-1.5 times, preferably 0.8-1.2 times, the volume of the sample aqueous solution; the number of repeated washings is 1-3 times, preferably 2 times. Thus, ginsenosides in the water layer can be better extracted.

[0033] According to the embodiment of the present application, the ginsenosides are selected from ginsenoside Rg1, Re, and / or Rb1. The above components have relatively high content in the Wending Decoction Material Standard and stably exist.

[0034] According to an embodiment of the present application, the sample comprises liquorice, ginseng, white peony root and cortex moutan. The paeoniflorin, liquiritin, ginsenoside, glycyrrhizic acid and paeonol in the above-mentioned raw materials are stable and have high content.

[0035] According to an embodiment of the present application, the sample is Wanjing Decoction. The Wanjing Decoction contains paeoniflorin, liquiritin, ginsenoside, glycyrrhizic acid and paeonol, and the content of paeoniflorin, liquiritin, ginsenoside, glycyrrhizic acid and paeonol in the Wanjing Decoction can be detected by the above-mentioned detection method. In some embodiments, the raw materials of the Wanjing Decoction comprise 1-3 parts of Angelica sinensis, 1-3 parts of Ligusticum chuanxiong, 1-3 parts of Radix Paeoniae Alba, 1-3 parts of Cinnamomum cassia, 1-3 parts of Cortex Moutan, 1-3 parts of Curcuma zedoary, 1-3 parts of Panax ginseng, 1-3 parts of Cyathula officinalis, and 1-3 parts of liquorice.

[0036] In another aspect of the present application, a substance reference of Wanjing Decoction is provided. According to an embodiment of the present application, the substance reference of the Wanjing Decoction comprises the following components: not less than 0.52% of paeoniflorin, not less than 0.25% of liquiritin, not less than 0.14% of ginsenoside Rg1 and Re in total, not less than 0.07% of ginsenoside Rb1, not less than 0.63% of glycyrrhizic acid, and not less than 0.09% of paeonol, which are obtained according to the above-mentioned detection method. According to the embodiment of the present application, it is established that the substance reference of the classic Wanjing Decoction can comprehensively reflect the quality information of the corresponding Wanjing Decoction sample, and meet the requirement of inheriting the classic with high quality. The Wanjing Decoction with the above-mentioned components has better curative effect, and further, the substance reference of the Wanjing Decoction with the above-mentioned components can accurately identify whether the quality of the to-be-tested Wanjing Decoction meets the standard.

[0037] According to an embodiment of the present application, the content of paeoniflorin in the substance reference of the Wanjing Decoction is 0.62%-0.86%, the content of liquiritin is 0.25%-0.76%, the content of ginsenoside Rg1 and Re is 0.14%-0.30%, the content of ginsenoside Rb1 is 0.07%-0.21%, the content of glycyrrhizic acid is 0.63%-1.16%, and the content of paeonol is 0.09%-0.25%. Thus, the substance reference of the Wanjing Decoction with the above-mentioned components can further accurately identify whether the quality of the to-be-tested Wanjing Decoction meets the standard.

[0038] In still another aspect of the present application, the present application provides a detection method for the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and / or ginsenoside in the above sample or the application of the material reference of Wending Decoction in the quality control of Wending Decoction. Thus, the detection method can accurately determine the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and ginsenoside in the Wending Decoction to be detected, and based on the determination results, it can be determined whether the quality of the Wending Decoction to be detected meets the standard, for example, by comparing the determination results with the material reference of Wending Decoction, when consistent with the material reference, it is determined that the quality meets the standard and the curative effect is good.

[0039] It should be noted that the "material reference" described in the present application, i.e. the classical famous prescription material reference, refers to the standard of traditional Chinese medicine drug substance prepared according to the preparation method of ancient classical famous prescription recorded in ancient medical books. Except for the forming process, its preparation method should be basically consistent with the record in ancient medical books. The material reference of Wending Decoction has not been disclosed in the prior art.

[0040] In still another aspect of the present application, the present application provides a quality control method of Wending Decoction. According to the embodiments of the present application, the quality control method comprises: determining the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and ginsenoside in the Wending Decoction sample according to the detection method for the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and / or ginsenoside in the above sample; and the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and ginsenoside meeting the requirements is an indication that the quality of the Wending Decoction sample meets the standard.

[0041] In the research and development of the material reference of Wending Decoction, the inventors have previously conducted a quantitative study of the effective component cinnamyl aldehyde of the monarch drug, but cinnamyl aldehyde has the disadvantages of low content, poor stability of volatile components and poor batch consistency in the material reference, and is not suitable for evaluation. Similarly, volatile oil components such as angelica and zedoary are also not suitable for evaluation indexes. The inventors have found through a large number of experiments that the four drugs of licorice, ginseng, white peony root and cortex moutan are used in large amounts in the prescription, and according to the monarch and minister positions in the field of traditional Chinese medicine, their effective components have the characteristics of high content and good stability. Further, through research and analysis of the four drugs, it is found that the five components of paeoniflorin, glycyrrhizin, ginsenoside, glycyrrhizic acid and paeonol have relatively high content, are pharmacodynamic active components, have good reproducibility, are easy to quantify, and are easy to implement in industrialization, which can better monitor the quality of Wending Decoction products and avoid the influence of unstable factors in raw materials, process or operation. Therefore, selecting them as evaluation indexes can effectively realize the quality control of Wending Decoction, has the advantages of stable and controllable, good reproducibility, strong operability, high accuracy, etc., and the quality transfer in the decoction process has strong traceability.

[0042] According to an embodiment of the present application, the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and ginsenoside in the Wanjing Decoction sample are within the ranges of the corresponding components in the material basis of the Wanjing Decoction, which is an indication that the quality of the Wanjing Decoction sample meets the requirements.

[0043] According to an embodiment of the present application, the quality control method further comprises: providing licorice medicinal materials or decoction pieces used for preparing the Wanjing Decoction sample; determining the content of glycyrrhizin in the licorice medicinal materials or decoction pieces according to the detection method of the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and / or ginsenoside in the sample; and when the content of glycyrrhizin in the licorice medicinal materials or decoction pieces is 1-2% by mass and the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and ginsenoside in the Wanjing Decoction sample meet the requirements, it is an indication that the quality of the Wanjing Decoction meets the requirements. The inventors have found that licorice of different sources and different batches can cause great differences in the contents of glycyrrhizin and glycyrrhizic acid in the Wanjing Decoction, thereby affecting the quality and efficacy. Therefore, the inventors have found through a large number of experiments that the use of licorice with a content of glycyrrhizin of 1-2% by mass can ensure that the contents of glycyrrhizin and glycyrrhizic acid in the Wanjing Decoction prepared therefrom do not fluctuate greatly due to different sources and different batches, thereby ensuring batch consistency and the quality and efficacy of the Wanjing Decoction.

[0044] According to an embodiment of the present application, the quality control method further comprises: providing medicinal materials or decoction pieces used for preparing the Wanjing Decoction sample, which include licorice, white peony root, ginseng and tree peony bark;

[0045] determining the contents of glycyrrhizin and glycyrrhizic acid in the licorice, the content of paeoniflorin in the white peony root, the content of oxymatrine in the tree peony bark and the contents of ginsenoside Rg1, Re and / or Rb1 in the ginseng according to the detection method of the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and / or ginsenoside in the sample;

[0046] The following conditions are met:

[0047] (1) when the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and ginsenoside in the Wanjing Decoction sample meet the requirements;

[0048] (2) the transfer rate of white peony root to paeoniflorin is 14.99%-19.42%;

[0049] (3) the transfer rate of licorice to glycyrrhizin is 28.11%-40.93% and the transfer rate of licorice to glycyrrhizic acid is 23.43%-35.53%;

[0050] (4) the transfer rate of ginseng to ginsenoside Rg1 and Re is 25.92%-61.88% and the transfer rate of ginseng to ginsenoside Rb1 is 25.03%-64.06%;

[0051] (5) The transfer rate of peony bark to paeonol is 5.34% to 10.44%.

[0052] The inventor found through a large number of experiments that when the Wanjing Decoction meets the above conditions, it has good efficacy. The material basis of the Wanjing Decoction meets the good correlation between the medicinal materials or decoction pieces and the Wanjing Decoction sample, excludes the influence of medicinal material sources, preparation processes and personnel operations on the quality, adopts the values of key quality attributes such as glycyrrhizin, paeonol, glycyrrhizic acid, paeonol and ginsenosides, fully considers the difference in uniformity of medicinal materials or decoction pieces, and therefore the accuracy of quality control can be further improved.

[0053] Additional aspects and advantages of the application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0054] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0055] Figure 1 Fig. 1 is a chromatogram of the paeonol and glycyrrhizin reference solution (A) and the test solution (B) in Example 1, wherein 1 is paeonol and 2 is glycyrrhizin;

[0056] Figure 2 Fig. 2 is a chromatogram of the ginsenoside paeonol and glycyrrhizin reference solution (A) and the test solution (B) in Example 1;

[0057] Figure 3 Fig. 3 is a chromatogram of the glycyrrhizic acid and paeonol reference solution (A) and (C) and the test solution (B) and (D) in Example 1, wherein 1 is glycyrrhizic acid and 2 is paeonol;

[0058] Figure 4 Fig. 4 is a chromatogram under the elution program A condition in Example 3, wherein Fig. A is a chromatogram under the wavelength of 268 nm and Fig. B is a chromatogram under the wavelength of 289 nm;

[0059] Figure 5 Fig. 5 is a chromatogram under the elution program B condition in Example 3, wherein Fig. A is a chromatogram under the wavelength of 268 nm and Fig. B is a chromatogram under the wavelength of 289 nm;

[0060] Figure 6 Fig. 6 is a chromatogram under the elution program C condition in Example 3, wherein Fig. A is a chromatogram under the wavelength of 268 nm and Fig. B is a chromatogram under the wavelength of 289 nm;

[0061] Figure 7Chromatograms for Example 3 under elution procedure D conditions, where Figure A is the chromatogram at 268 nm wavelength and Figure B is the chromatogram at 289 nm wavelength.

[0062] Figure 8 Chromatograms for Example 3 under elution procedure E conditions, where Figure A is the chromatogram at 268 nm wavelength and Figure B is the chromatogram at 289 nm wavelength.

[0063] Figure 9 Chromatograms for Example 3 under different detection wavelength conditions, where Figure A is the chromatogram at 289 nm wavelength and Figure B is the chromatogram at 280 nm wavelength. DETAILED DESCRIPTION

[0064] The schemes of the present application will be explained below in conjunction with the examples. Those skilled in the art will understand that the examples below are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be obtained commercially.

[0065] The instruments and reagents involved in the following examples are as follows:

[0066] Waters 2695-2998 high performance liquid chromatograph (Waters, USA);

[0067] Kromasil C18 chromatographic column (4.6 mm x 250 mm, 5 μm);

[0068] Welch Ultimate C18 chromatographic column (4.6 mm x 250 mm, 5 μm);

[0069] Glycyrrhizin, ammonium glycyrrhizinate (purity: 97.4%), paeonol (purity: 97.4%), paeoniflorin, ginsenoside Rg1 (purity 92.4%), ginsenoside Re (purity 93.4%), ginsenoside Rb1 (purity 91.2%), which are all purchased from China Institute for Food and Drug Control.

[0070] Example 1: Method for detecting the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and / or ginsenoside in the freeze-dried powder of Wening Tang

[0071] 1. Preparation method of the freeze-dried powder of Wening Tang

[0072] The raw materials of the Wending Decoction include: Danggui 20.60g, Chuanxiong 20.60g, Baishao 20.60g, Rougui 20.60g, Mudanpi 20.60g, Douchi Baizhi 20.60g, Renshen 41.30g, Jiuniu'ni 41.30g, Chao'gancao 41.30g.

[0073] The above raw material mixture was crushed into coarse particles, 20g of the coarse particles and 450mL of water were taken into a sand pot, heated with an electric stove at 5 grades of high fire for 5-7 minutes, boiled, adjusted to 2.5 grades, and gently boiled for 30 minutes. The filtrate was about 240mL, which was freeze-dried to obtain the Wending Decoction freeze-dried powder.

[0074] 2. Preparation of the control solution

[0075] The paeoniflorin and glycyrrhizin control solution was prepared as follows: 19.80mg of paeoniflorin and 10.49mg of glycyrrhizin were precisely weighed and placed in a 10mL volumetric flask, dissolved with methanol and made up to the mark, and shaken to obtain a mixed control stock solution. 5mL of the above control stock solution was precisely taken and made up to 10mL in a volumetric flask to prepare a mixed control solution C1. A series of C2-C7 concentration solutions were prepared by dilution, and filtered with a 0.45μm filter to obtain the paeoniflorin and glycyrrhizin control solution.

[0076] The ginsenoside control solution was prepared as follows: 3.90mg of ginsenoside Rg1, 3.74mg of ginsenoside Re and 3.31mg of ginsenoside Rb1 were precisely weighed and placed in a 10mL volumetric flask, dissolved with methanol and made up to the mark, and shaken to obtain a ginsenoside mixed solution stock solution. 0.1, 0.25, 0.5, 1.0, 1.5 and 2.0 of the above control mixed solution stock solution were precisely taken and made up to 5mL in a volumetric flask to prepare a series of control mixed standard concentrations, and 1mL of the stock solution was made up to obtain C1-C8. The above series of concentrations were filtered with a 0.45μm filter to obtain the ginsenoside control solution.

[0077] The glycyrrhizic acid and paeonol control solution was prepared as follows: 16.55mg of ammonium glycyrrhizinate and 8.55mg of paeonol were precisely weighed and placed in a 25mL volumetric flask, dissolved with methanol and made up to the mark, and shaken to obtain a mixed solution stock solution of ammonium glycyrrhizinate and paeonol. 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0mL of the above control mixed solution stock solution were precisely taken and made up to 10mL in a volumetric flask to prepare a series of control mixed standard concentrations, which were labeled as C1-C7. 1mL of the mixed standard solution C1 was precisely taken and made up to 2mL in a volumetric flask to obtain a C0 solution. The C0-C7 were filtered with a 0.45μm filter to obtain the glycyrrhizic acid and paeonol control solution.

[0078] 3. Preparation of the test solution of Wenjing Decoction lyophilized powder

[0079] 3.1 About 0.30 g of Wenjing Decoction lyophilized powder was precisely weighed and placed in a 50 mL conical flask. 25 mL of 50% methanol solution was added, the weight was determined, and ultrasonic extraction was performed (power 250 W, frequency 45 KHZ) for 30 min. After cooling, the weight was supplemented, centrifuged for 10 min (12000 r·min -1 ), filtered, and the filtrate was taken for determination of the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, and paeonol.

[0080] 3.2 About 0.40 g of Wenjing Decoction lyophilized powder was precisely weighed and placed in a 50 mL conical flask. 20 mL of purified water was precisely added, and the solution was dissolved by ultrasonic extraction. The solution was transferred to a separatory funnel. 20 mL of dichloromethane was precisely added, and after extraction, the dichloromethane layer was discarded. The water layer was extracted with water-saturated n-butanol for 3 times, 20 mL each time. The n-butanol layers were combined, washed with 20 mL of ammonia solution for 2 times, and then washed with 10 mL of water-saturated n-butanol for 1 time. The n-butanol layers were combined, washed with 10 mL of purified water for 1 time, and then the n-butanol layer was evaporated under reduced pressure. The residue was dissolved with 5 mL of methanol and made up to volume, shaken, filtered, and the filtrate was passed through a 0.22 μm microporous filter for determination of the contents of ginsenoside Rg1, Re, and Rb1.

[0081] 4. Chromatographic conditions

[0082] 4.1 Quantitative chromatographic conditions for paeoniflorin and glycyrrhizin

[0083] Kromasil C18 chromatographic column (4.6 mm x 250 mm, 5 μm); mobile phase: acetonitrile (A)-0.1% formic acid water (B); gradient elution (0-8 min, 17% A; 8-10 min, 17-19% A; 10-20 min, 19-25% A; 20-25 min, 25-70% A; 25-35 min, 70-17% A); flow rate 1.0 mL·min-1, column temperature 25°C, wavelength 237 nm, injection volume 10 μL. The theoretical plate number should not be less than 3000 calculated according to the paeoniflorin peak.

[0084] 4.2 Quantitative chromatographic conditions for ginsenosides

[0085] Welch Ultimate C18 chromatographic column (4.6 mm x 250 mm, 5 μm); mobile phase: acetonitrile (A)-water (B); gradient elution (0-35 min, 19% A; 35-55 min, 19-29% A; 55-70 min, 29% A; 70-110 min, 29-40% A); flow rate 1.0 mL·min-1, column temperature 30°C, detection wavelength 203 nm; injection volume 10 μL.

[0086] 4.3 Quantitative chromatographic conditions of glycyrrhizic acid and paeonol

[0087] Kromasil C18 column (4.6 mm x 250 mm, 5 μm); mobile phase acetonitrile (A) - 0.1% formic acid (B); gradient elution (0-15 min, 27-38% A; 15-20 min, 38-42% A; 20-25 min, 42-55% A; 25-30 min, 55-90% A; 30-35 min, 90-27% A); flow rate 1.0 mL min-1, column temperature 25 °C, glycyrrhizic acid detection wavelength 250 nm, paeonol detection wavelength 280 nm. The theoretical plate number should not be less than 3000 calculated by the glycyrrhizic acid peak.

[0088] 5. Inject the control solution and the test solution into the high performance liquid chromatograph, respectively, and record the chromatogram, wherein, Figure 1 is the chromatogram of the control and test samples of paeonol and glycyrrhizin in the freeze-dried powder of Wenjing decoction, Figure 2 is the chromatogram of the control and test samples of ginsenoside in the freeze-dried powder of Wenjing decoction, Figure 3 is the chromatogram of the control and test samples of glycyrrhizic acid and paeonol in the freeze-dried powder of Wenjing decoction.

[0089] 6. Linear relationship

[0090] 6.1 Paeonol and glycyrrhizin: prepare control solutions of different concentrations, the concentrations of paeonol C2-C7 are 482.1 μg mL -1 , 241.1 μg mL -1 , 120.3 μg mL -1 , 60.3 μg mL -1 , 30.1 μg mL -1 , 15.1 μg mL -1 , respectively, and the concentrations of glycyrrhizin C2-C7 are 244.2 μg mL -1 , 122.1 μg mL -1 , 61.0 μg mL -1 , 30.5 μg mL -1 , 15.3 μg mL -1 , 7.6 μg mL -1 , respectively, inject 10 μL of each sample, take the peak area as the ordinate (Y) and the control concentration as the abscissa (X) to draw the standard curve, the regression equation of paeonol is Y = 12457001.1X + 4521.0 (R 2 = 1), and the regression equation of glycyrrhizin is Y = 19926353.3X + 3926.4 (R 2 = 1), indicating that the paeonol solution is in the range of 15.1-482.1 μg mL-1 The peak area and the concentration of liquor have a good linear relationship. The liquor of glycyrrhizin has a good linear relationship with the peak area in the range of 7.6-244.2 μg·mL -1 The peak area and the concentration of liquor have a good linear relationship.

[0091] 6.2 Ginsenoside: The control solution of different concentrations was configured. The concentrations of ginsenoside Rg1 C1-C8 were 36.1 μg·mL -1 , 72.1 μg·mL -1 , 108.1 μg·mL -1 , 144.1 μg·mL -1 , 180.2 μg·mL -1 , 360.4 μg·mL -1 , 18.0 μg·mL -1 , 7.2 μg·mL -1 , respectively. The concentrations of ginsenoside Re C1-C8 were 34.9 μg·mL -1 , 69.9 μg·mL -1 , 104.8 μg·mL -1 , 139.7 μg·mL -1 , 174.7 μg·mL -1 , 349.3 μg·mL -1 , 17.5 μg·mL -1 , 7.0 μg·mL -1 , respectively. The concentrations of ginsenoside Rb1 C1-C8 were 30.2 μg·mL -1 , 60.4 μg·mL -1 , 90.6 μg·mL -1 , 120.7 μg·mL -1 , 150.9 μg·mL -1 , 301.9 μg·mL -1 , 15.1 μg·mL -1 , 6.0 μg·mL -1 , respectively. 10 μL of each sample was injected. The standard curve was drawn with the peak area as the vertical coordinate (Y) and the concentration of the control as the horizontal coordinate (X). The regression equation of ginsenoside Rg1 was Y=7083844.7X+7514.5 (R 2 =1). The regression equation of ginsenoside Re was Y=6066438.0X+4723.4 (R 2 =1). The regression equation of ginsenoside Rb1 was Y=5097842.9X+1823.9 (R 2 =1). It is shown that the liquor of ginsenoside Rg1 has a good linear relationship with the peak area in the range of 7.2-360.4 μg·mL -1 , the liquor of ginsenoside Re has a good linear relationship with the peak area in the range of 7.0-349.3 μg·mL-1 The peak area was linearly related to the concentration of ginsenoside Rbl solution in the range of 6.0-301.9 μg·mL -1 The peak area was linearly related to the concentration of ginsenoside Rbl solution in the range of 6.0-301.9 μg·mL

[0092] 6.3 Glycyrrhizic acid and paeonol: Prepare control solution with different concentrations. The concentrations of glycyrrhizic acid C1-C7 are 32.3 μg·mL -1 , 64.7 μg·mL -1 , 97.0 μg·mL -1 , 129.4 μg·mL -1 , 161.7 μg·mL -1 , 194.0 μg·mL -1 , 323.4 μg·mL -1 , respectively, and the concentrations of paeonol CO-C7 are 8.5 μg·mL -1 , 17.1 μg·mL -1 , 34.2 μg·mL -1 , 51.2 μg·mL -1 , 68.3 μg·mL -1 , 85.4 μg·mL -1 , 102.5 μg·mL -1 , respectively. Draw the standard curve with the peak area as the ordinate (Y) and the concentration of the control as the abscissa (X). The regression equation of glycyrrhizic acid is Y=8906809.3x-6438.5 (R 2 =1), and the regression equation of paeonol is Y=45740976.5x+17447.7 (R 2 =1), indicating that the glycyrrhizic acid solution is linearly related to the peak area in the range of 32.3-323.4 μg·mL -1 , and the paeonol solution is linearly related to the peak area in the range of 8.5-102.5 μg·mL -1 .

[0093] 7. Precision examination

[0094] Take the same batch of Wanjing Decoction freeze-dried powder, and prepare two test samples according to Step 3. Elute them according to the three corresponding chromatographic conditions of Step 4, and continuously inject 6 times, 10 μL each time. Measure and calculate the RSD values of the peak areas of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol, ginsenoside Rg1, Re and Rbl, which are 0.64%, 0.91%, 1.4%, 0.40%, 1.5%, 2.3%, 1.1% respectively. The RSD values are all ≤3%, indicating that the precision of the instrument is good.

[0095] 8. Stability examination

[0096] Take two kinds of test solution prepared in step 7 respectively, and sample respectively at 0, 1, 2, 4, 8, 12, 24, 48h after preparation, elute according to the three kinds of chromatographic conditions of step 4, measure and calculate the RSD values of the peak areas of paeoniflorin and glycyrrhizin, which are 0.52% and 0.91% respectively, and the RSD values are all ≤2%, indicating that the test solution is stable within 48h. Measure and calculate the RSD values of the peak areas of glycyrrhizic acid and paeonol, which are 0.68% and 0.32% respectively, and the RSD values are all ≤2%, indicating that the test solution is stable within 48h. Measure and calculate the RSD values of the peak areas of ginsenoside Rg1, Re and Rb1, which are 1.2%, 2.1% and 1.0% respectively, and the RSD values are all ≤3%, indicating that the test solution is stable within 48h.

[0097] 9. Repeatability investigation

[0098] Take 6 portions of the same batch of Wanjing Decoction freeze-dried powder, prepare sample solutions according to the method of step 3.1, elute according to the chromatographic conditions of step 4.1, measure and calculate the RSD values of the contents of paeoniflorin and glycyrrhizin, which are 0.59% and 0.73% respectively, and the RSD values are all ≤2%, indicating that the method is good in repeatability.

[0099] Take 6 portions of the same batch of Wanjing Decoction freeze-dried powder, prepare sample solutions according to the method of step 3.2, elute according to the chromatographic conditions of step 4.2, measure and calculate the RSD values of the contents of ginsenoside Rg1, Re and Rb1, which are 1.5%, 4.4% and 1.2% respectively, indicating that the method is good in repeatability.

[0100] Take 6 portions of the same batch of Wanjing Decoction freeze-dried powder, prepare sample solutions according to the method of step 3.3, elute according to the chromatographic conditions of step 4.3, measure and calculate the RSD values of the contents of glycyrrhizic acid and paeonol, which are 2.48% and 0.49% respectively, and the RSD values are all ≤3%, indicating that the method is good in repeatability.

[0101] 10. Sample addition recovery test

[0102] 10.1 Paeoniflorin and glycyrrhizin recovery rate test Precisely take 9 portions of the same batch of Wanjing Decoction freeze-dried powder sample with known content about 0.18g, and divide them into 3 groups, and precisely add corresponding paeoniflorin and glycyrrhizin reference substances in the proportions of 50%, 100% and 150% respectively, prepare sample solutions according to the method of step 3.1, prepare 9 samples in parallel, and sample according to the chromatographic conditions of step 4.1, and the results are that the average recovery rates of paeoniflorin and glycyrrhizin are 96.20% and 99.10%, and the RSD values are 0.59% and 1.2%. The results are shown in Table 1.

[0103] 10.2 Paeoniflorin and glycyrrhizin recovery experiment Precisely take 6 portions of the same batch of Wanjing Decoction freeze-dried powder sample with known content about 0.20 g, and precisely add corresponding paeoniflorin and glycyrrhizin reference substances in a proportion of 1:1. Prepare sample solution according to the method of step 3.2, and prepare 6 samples in parallel. Inject sample according to the chromatographic conditions of step 4.2. The average recovery rates of paeoniflorin and glycyrrhizin are 95.60% and 94.20%, and the RSD values are 1.2% and 1.3%. The results are shown in Table 2.

[0104] 10.3 Glycyrrhizic acid and paeoniflorin recovery experiment Precisely take 9 portions of the same batch of Wanjing Decoction freeze-dried powder sample with known content about 0.15 g, and divide them into 3 groups. Each group is precisely added with corresponding glycyrrhizic acid and paeoniflorin reference substances in a proportion of 50%, 100%, and 150%, respectively. Prepare sample solution according to the method of step 3.3, and prepare 9 samples in parallel. Inject sample according to the chromatographic conditions of step 4.3. The average recovery rates of glycyrrhizic acid and paeoniflorin are 96.20% and 99.10%, and the RSD values are 2.5% and 4.4%. The results are shown in Table 3.

[0105] Table 1 Paeoniflorin and glycyrrhizin addition recovery experiment

[0106]

[0107]

[0108] Table 2 Paeoniflorin addition recovery experiment results

[0109]

[0110] Table 3 Glycyrrhizic acid and paeoniflorin addition recovery experiment

[0111]

[0112] Example 2: Detection method of paeoniflorin and glycyrrhizin content

[0113] 1. Comparison of different first extraction solvents

[0114] Precisely take 4 portions of Wanjing Decoction freeze-dried powder in Example 1, with a mass of 122.2 mg, 122.2 mg, 122.1 mg, and 122.0 mg, respectively. Dissolve them with 2 mL of water, mobile phase initial proportion of acetonitrile: water (17:83), 50% methanol, and pure methanol, respectively. Ultrasonically extract for 15 min. Centrifuge at 12000 rpm for 10 min. Filter with a 0.45 μm filter membrane to obtain the test solution. Detect according to the chromatographic conditions of Example 1, and the detection wavelength is 237 nm. The detection results are shown in Table 4.

[0115] Table 4 detection results of different first extraction solvent

[0116]

[0117] The above determination results show that when the first extraction solvent is pure water and 17% acetonitrile, the peak area of paeoniflorin and glycyrrhizin is small, and the proportion of paeoniflorin and glycyrrhizin is low; when the first extraction solvent is 50% methanol, the peak area of paeoniflorin and glycyrrhizin is the largest, and the proportion of paeoniflorin and glycyrrhizin is the highest; when the first extraction solvent is methanol, the peak area of paeoniflorin and glycyrrhizin is larger, and the proportion of paeoniflorin and glycyrrhizin is higher; therefore, 50% methanol is the best extraction solvent.

[0118] 2. Comparison of different extraction times

[0119] Three portions of the Wending decoction freeze-dried powder in Example 1 with weights of 0.3608 g, 0.3609 g and 0.3603 g, respectively, were weighed into 50 mL conical flasks, 30 mL of methanol: water = 50:50 solution was added, weighed, ultrasonic extraction was performed, the extraction time was 15 min, 30 min and 45 min, respectively, the power was 240 W, the frequency was 45 KHZ, it was cooled, the weight was supplemented, 12000 rpm centrifugation was performed for 10 min, it was shaken, 0.45 μm filter membrane filtration was performed, the filtrate was taken, the test sample solution was obtained, and detection was performed according to the chromatographic conditions of Example 1, and the detection wavelength was 237 nm. The detection results are shown in Table 5.

[0120] Table 5 detection results of different extraction times

[0121] Extraction time Paeoniflorin peak area Paeoniflorin % Glycyrrhizin peak area Glycyrrhizin % 45 min 947186 0.629 1150721 0.479 30 min 961742 0.639 1153401 0.480 15 min 944532 0.627 1142899 0.475

[0122] The above determination results show that when the extraction time is 15 min, the peak area of paeoniflorin and glycyrrhizin is the smallest, and the proportion of paeoniflorin and glycyrrhizin is the lowest; when the extraction time is 30 min, the peak area of paeoniflorin and glycyrrhizin is the largest, and the proportion of paeoniflorin and glycyrrhizin is the highest; when the extraction time is 45 min, the peak area of paeoniflorin and glycyrrhizin is larger, and the proportion of paeoniflorin and glycyrrhizin is higher; in combination with the length of time, the efficiency is improved, therefore, 30 min is the best extraction time.

[0123] 3. Comparison of different extraction solvent amounts

[0124] Three portions of the Wenjingtang lyophilized powder from Example 1, weighing 0.25029 g, 0.24999 g, and 0.25023 g respectively, were placed in 25 mL Erlenmeyer flasks. 10 mL of a methanol:water = 50:50 solution was added, and the samples were weighed. Ultrasonic extraction was performed with extraction solvent volumes of 5 mL, 10 mL, and 15 mL respectively, at a power of 240 W and a frequency of 45 kHz. The samples were cooled, weighed again, and centrifuged at 12000 rpm for 10 min. After mixing, the samples were filtered through a 0.45 μm filter membrane. The filtrate was collected to obtain the test solution, which was then analyzed under the chromatographic conditions described in Example 1 at a detection wavelength of 240 nm. The results are shown in Table 6.

[0125] Table 6. Detection results for different extraction solvent amounts

[0126] Extraction dose Paeoniflorin peak area Paeoniflorin % Glycyrrhizin peak area Glycyrrhizin % 5ml 3596859.5 0.576 5082718 0.509 10ml 1847877.5 0.592 2644630.5 0.530 15ml 1163040.5 0.558 1656916.5 0.497

[0127] The above measurements show that when the extraction dose is 5 ml, paeoniflorin has the largest peak area and glycyrrhizin has the smallest peak area, with paeoniflorin accounting for a lower proportion and glycyrrhizin accounting for the lowest proportion. When the extraction dose is 10 ml, paeoniflorin has a larger peak area and glycyrrhizin has the largest peak area, with paeoniflorin and glycyrrhizin accounting for the highest proportions. When the extraction dose is 15 ml, paeoniflorin has the smallest peak area and glycyrrhizin has a larger peak area, with paeoniflorin and glycyrrhizin accounting for the lowest proportions. Considering the peak area, the proportion of components, and cost savings, 10 ml is selected as the optimal extraction dose.

[0128] Example 3: Method for detecting glycyrrhizic acid and paeonol content

[0129] 1. Comparison of different elution procedures

[0130] The test solution from step 3.1 of Example 1 was selected and tested according to the chromatographic conditions and different gradient elution programs (elution program AE) of Example 1.

[0131] The conditions for elution program A are shown in Table 7, and the chromatogram is shown below. Figure 4 As shown. Figure 4 The results showed that at 289 nm, there was an impurity peak after the position of paeonol (20 min), indicating that paeonol and the impurity peak were not separated, and the separation effect under this condition was poor.

[0132] The conditions for elution program B are shown in Table 8, and the chromatogram is shown below. Figure 5 As shown. Figure 5 The results showed that three target peaks were visible at a wavelength of 268 nm, indicating good separation. Two chromatographic peaks of paeonol (22.9 min) were visible at a wavelength of 289 nm, and complete separation could be achieved under these conditions, indicating that the conditions were relatively ideal.

[0133] The conditions for elution program C are shown in Table 9, and the chromatogram is shown below. Figure 6shown. Figure 6 It is shown that at 268 nm wavelength, the three target peaks can be separated well, but at 289 nm, there is an impurity peak behind the position of paeonol (20 min), indicating that paeonol and the impurity peak are not separated.

[0134] The conditions of elution procedure D are shown in Table 10, and the chromatogram is shown in Figure 7 . Figure 7 It is shown that at 289 nm, there is an impurity peak behind the position of paeonol (20 min), indicating that paeonol and the impurity peak are not separated, and the separation effect under this condition is poor.

[0135] The conditions of elution procedure E are shown in Table 11, and the chromatogram is shown in Figure 8 . Figure 8 It is shown that at 289 nm, there is an impurity peak behind the position of paeonol (18.4 min), indicating that paeonol and the impurity peak are not separated, and the separation effect under this condition is poor.

[0136] Table 7 Conditions of elution procedure A

[0137] Time (min) Acetonitrile (%) 0.1% formic acid (%) 0 25 75 25 38 62 30 90 10 32 90 10 32.1 25 75 40 25 75

[0138] Table 8 Conditions of elution procedure B

[0139] Time (min) Acetonitrile (%) 0.1% formic acid (%) 0 27 73 15 38 62 20 42 58 25 55 45 30 90 10 30.1 27 73 35 27 73

[0140] Table 9 Conditions of elution procedure C

[0141] Time (min) Acetonitrile (%) 0.1% formic acid (%) 0 27 73 10 38 62 15 42 58 20 55 45 25 90 10 25.1 27 73 30 27 73

[0142] Table 10 Conditions of elution procedure D

[0143]

[0144]

[0145] Table 11 Conditions of elution procedure E

[0146]

[0147] 2, Different detection wavelengths

[0148] The test sample solution of step 3.1 in Example 1 was selected, and the paeonol in the test sample solution was detected according to the chromatographic conditions and different detection wavelengths (289 nm and 280 nm) of Example 1. The chromatogram is shown in Figure 9 . Figure 9 It is shown that 280 nm is the compromise point of the maximum absorption of paeonol, and at 280 nm, the peak area can be considered within a reasonable range, so the detection wavelength of paeonol is determined as 280 nm.

[0149] 3. Comparison of different first extraction solvents

[0150] Take 4 portions of the Wanjing Decoction freeze-dried powder in Example 1, with a mass of 122.2 mg, 122.2 mg, 122.1 mg, and 122.0 mg, respectively, and dissolve each portion in 2 mL of pure water, mobile phase initial proportion of acetonitrile: water (17:83), 50% methanol, or pure methanol, respectively, and extract by ultrasonic for 15 min. Centrifuge at 12000 rpm for 10 min, filter through a 0.45 μm filter membrane, and take the filtrate to obtain the test sample solution. Detect according to the chromatographic conditions in Example 1. The detection results are shown in Table 12.

[0151] Table 12. Detection results of different first extraction solvents

[0152]

[0153] The detection results in Table 12 show that when the extraction solvent is pure water or 17% acetonitrile, the peak areas of glycyrrhizic acid and paeonol are small; when the extraction solvent is 50% methanol, the peak area of glycyrrhizic acid is the largest and the peak area of glycyrrhizin is large; and when the extraction solvent is methanol, the peak area of glycyrrhizic acid is the smallest and the peak area of paeonol is the largest. The inventors have considered the extraction rates and peak areas of both paeoniflorin and glycyrrhizin, and therefore selected 50% methanol as the best extraction solvent.

[0154] 4. Comparison of different extraction times

[0155] Take 3 portions of the Wanjing Decoction freeze-dried powder in Example 1, with a mass of 0.3608 g, 0.3609 g, and 0.3603 g, respectively, and place each portion in a 50 mL conical flask. Add 30 mL of a methanol: water = 50:50 solution, weigh, extract by ultrasonic, and set the extraction time to 15 min, 30 min, or 45 min, respectively. The power is 250 W and the frequency is 45 KHZ. Cool, weigh again, centrifuge at 12000 rpm for 10 min, shake well, filter through a 0.45 μm filter membrane, and take the filtrate to obtain the test sample solution. Detect according to the chromatographic conditions in Example 1. The detection results are shown in Table 13.

[0156] Table 13. Detection results of comparison of different extraction times

[0157]

[0158] The detection results in Table 13 show that when the extraction time is 15 min, the peak area of glycyrrhizic acid is the smallest and the peak area of paeonol is small; when the extraction time is 30 min, the peak areas of glycyrrhizic acid and paeonol are the largest; and when the extraction time is 45 min, the peak area of glycyrrhizic acid is large and the peak area of paeonol is the smallest. The inventors have considered the extraction rates and peak areas of both paeoniflorin and glycyrrhizin, and therefore selected 30 min as the best extraction time.

[0159] Example 4: Detection method of ginsenoside content

[0160] 1. Comparison of different water-saturated n-butanol amounts

[0161] Prepare the test sample solution according to the method of step 3.2 of Example 1, wherein the amount of water-saturated n-butanol used before the addition of the ammonia test solution is changed, and then detect the contents of ginsenosides Rg1, Re and Rb1 according to the chromatographic conditions in Example 1. The amount of water-saturated n-butanol and the detection results are shown in Table 14.

[0162] Table 14 Detection results of different water-saturated n-butanol amounts

[0163]

[0164] The detection results of Table 14 show that when the amount of water-saturated n-butanol is 10 ml each time (3 times), the contents of ginsenosides Rg1, Re and Rb1 are the lowest; when the amount of water-saturated n-butanol is 15 ml each time (3 times), the contents of ginsenosides Rg1, Re and Rb1 are relatively high; when the amount of water-saturated n-butanol is 20 ml each time (3 times), the contents of ginsenosides Rg1, Re and Rb1 are the highest. The inventors comprehensively consider the extraction rate and content of ginsenosides Rg1, Re and Rb1, and therefore the amount of water-saturated n-butanol is selected as 20 ml each time.

[0165] 2. Comparison of different extraction times of water-saturated n-butanol

[0166] Prepare the test sample solution according to the method of step 3.2 of Example 1, wherein the extraction times of the water-saturated n-butanol on the water layer are different, and then detect the contents of ginsenosides Rg1, Re and Rb1 according to the chromatographic conditions in Example 1. The extraction times of the water-saturated n-butanol and the detection results are shown in Table 15.

[0167] Table 15 Detection results of different extraction times of water-saturated n-butanol

[0168]

[0169]

[0170] The detection results of Table 15 show that when the extraction times of water-saturated n-butanol are 2 times, the contents of ginsenosides Rg1, Re and Rb1 are the lowest; when the extraction times of water-saturated n-butanol are 3 times, the contents of ginsenosides Rg1, Re and Rb1 are relatively high; when the extraction times of water-saturated n-butanol are 4 times, the contents of ginsenosides Rg1, Re and Rb1 are the highest. The inventors find that the contents of ginsenosides Rg1, Re and Rb1 have little difference between 3 times and 4 times, and therefore the extraction times of n-butanol are selected as 3 times by comprehensively considering the extraction rate, content, efficiency and cost of ginsenosides Rg1, Re and Rb1.

[0171] 3. Comparison of different amounts of ammonia test solution

[0172] The test sample solution was prepared according to the method of step 3.2 of Example 1, wherein different amounts of ammonia test solution were selected to elute the remaining water layer, and then the contents of ginsenosides Rg1, Re and Rb1 were detected according to the chromatographic conditions in Example 1. The elution times and detection results are shown in Table 16.

[0173] Table 16 Detection results of different amounts of ammonia test solution

[0174]

[0175] The detection results of Table 16 show that when the amount of ammonia test solution is 15 ml, the contents of ginsenosides Rg1, Re and Rb1 are the lowest; when the amount of ammonia test solution is 20 ml, the contents of ginsenosides Rg1, Re and Rb1 are the highest; the inventors comprehensively consider the extraction rates and contents of ginsenosides Rg1, Re and Rb1, and therefore the amount of ammonia test solution is selected as 20 ml.

[0176] 4. Comparison of different elution times of ammonia test solution

[0177] The test sample solution was prepared according to the method of step 3.2 of Example 1, wherein the elution times of ammonia test solution on the remaining water layer were different, and then the contents of ginsenosides Rg1, Re and Rb1 were detected according to the chromatographic conditions in Example 1. The elution times and detection results are shown in Table 17.

[0178] Table 17 Detection results of different elution times of ammonia test solution

[0179]

[0180] The detection results of Table 17 show that when the elution time of ammonia test solution is 1 time, the contents of ginsenosides Rg1, Re and Rb1 are the lowest; when the elution time of ammonia test solution is 2 times, the contents of ginsenosides Rg1, Re and Rb1 are the highest; the inventors comprehensively consider the extraction rates and contents of ginsenosides Rg1, Re and Rb1, and therefore the elution time of ammonia test solution is selected as 2 times.

[0181] Example 5: Quality control method of Wending Tang freeze-dried powder

[0182] The contents of paeonoside, glycyrrhizin, ginsenosides Rg1, Re and Rb1, glycyrrhizic acid and paeonol in 15 batches of Wending Tang freeze-dried powder (raw medicinal materials from different producing areas) were detected according to the detection method in Example 1, and the detection results are shown in Tables 18-22. The transfer rate deviation of paeonoside, glycyrrhizin, ginsenosides Rg1, Re and Rb1, glycyrrhizic acid and paeonol was calculated by the formula: transfer rate deviation / % = |single batch transfer rate-average transfer rate| / average transfer rate x 100%.

[0183] Table 18 Paeoniflorin content and transfer rate in decoction pieces and WJTS lyophilized powder

[0184]

[0185] From Table 18, the paeoniflorin content in 15 batches of WJTS lyophilized powder was in the range of 0.62% to 0.86%, and the batch-to-batch fluctuation of paeoniflorin content in 15 batches of samples was in the range of 82.42% to 113.90%, and in the range of 70% to 130% of the mean value, indicating that the paeoniflorin content in different batches was relatively stable. According to the paeoniflorin content data of the above 15 batches, 70% of the mean value (0.74%) was taken as the minimum limit, and it was suggested that the paeoniflorin content in WJTS lyophilized powder should not be less than 0.52%.

[0186] From Table 18, the paeoniflorin content in 15 batches of WJTS lyophilized powder was in the range of 0.62% to 0.86%, and the batch-to-batch fluctuation of paeoniflorin content in 15 batches of samples was in the range of 82.42% to 113.90%, and in the range of 70% to 130% of the mean value, indicating that the paeoniflorin content in different batches was relatively stable. According to the paeoniflorin content data of the above 15 batches, 70% of the mean value (0.74%) was taken as the minimum limit, and it was suggested that the paeoniflorin content in WJTS lyophilized powder should not be less than 0.52%.

[0187] Further analysis of the transfer rule of paeoniflorin from decoction pieces to material reference. In 15 batches of samples, the paeoniflorin transfer rate from decoction pieces to material reference was relatively consistent, approximately in the range of 14.99% to 19.42%, with an average transfer rate of 16.84%; the transfer rate deviation of all batches was in the range of 70% to 130% of the mean value, indicating that the paeoniflorin transfer rate in different batches was good in consistency, indicating that paeoniflorin had good stability in the preparation process, and the process was stable.

[0188] Table 19 Liquiritin content in decoction pieces and WJTS lyophilized powder and transfer rate

[0189]

[0190] The determination results of liquiritin content in 15 batches of WJTS lyophilized powder are shown in Table 19. In 15 batches of decoction pieces to material reference, the transfer rate of liquiritin was approximately in the range of 28.11% to 40.93%, with an average transfer rate of 33.75%; except for the 6th batch, the transfer rate of the other 14 batches was in the range of 70% to 130%, and the liquiritin transfer rate in different batches was good in consistency, indicating that liquiritin had good stability in the preparation process, and the process was stable.

[0191] In Table 19, the content of glycyrrhizin in 15 batches of Wenjing Decoction freeze-dried powder is in the range of 0.25% to 0.76%, and according to the glycyrrhizin content data of the above 15 batches, 70% of the average content (0.51%) is taken as the minimum limit, and it is suggested that the content of glycyrrhizin in Wenjing Decoction freeze-dried powder should not be less than 0.35%. The content of glycyrrhizin in 15 batches of samples fluctuates between 51.63% and 160.58%, and the content of 6 batches is in the range of 70% to 130% of the average, and the transfer rate of each batch of medicinal materials to the substance reference can be known. The transfer rates between batches are consistent and the deviation is small, which can exclude the batch difference caused by the decoction process; further tracing, it is found that the content of glycyrrhizin in each batch of medicinal materials is quite different, so it can be concluded that the main reason for the large batch fluctuation of the content of glycyrrhizin in the substance reference this time is the large difference in the content of glycyrrhizin in different batches of raw materials, such as batches 3, 8, 9, 11 and 15 in Table 20, the corresponding content of the medicinal materials is relatively low (the content of the above 5 batches of medicinal materials is less than 1.0%); batches 1 and 12 in the table have a relatively high content of medicinal materials (the content of the above two batches of medicinal materials is higher than 2.0%). Therefore, it is suggested that for the raw material of licorice medicinal materials, the content of glycyrrhizin in the range of 1.0% to 2.0% should be selected to ensure that the content of glycyrrhizin in the substance reference has small batch fluctuation and meets the batch consistency.

[0192] As can be seen from Table 20, the transfer rates of medicinal materials to the substance reference of 15 batches of Wenjing Decoction freeze-dried powder samples are consistent, the transfer rates of Rg1 and Re are approximately between 25.92% and 61.88%, and the transfer rates of Rb1 are approximately between 25.03% and 64.06%, and the average transfer rates are 36.64% and 35.96%, respectively; except for batch 5, the transfer rates of Rg1 and Re in all batches fluctuate between 70% and 130% of the average, and except for batches 1 and 5, the transfer rates of Rb1 in all batches fluctuate between 70% and 130% of the average; the consistency of the transfer rates of ginsenosides in different batches is good, which indicates that ginsenosides have good stability in the preparation process and the process is stable.

[0193] In Table 20, the content of ginsenosides Rg1 and Re in 15 batches of Wenjing Decoction freeze-dried powder is in the range of 0.14% to 0.30%, and the content of ginsenoside Rb1 is in the range of 0.07% to 0.21%. Taking 70% of the average content of Rg1 and Re (0.22%) and the average content of Rb1 (0.14%) as the minimum limit, it is suggested that the content of ginsenosides Rg1 and Re in Wenjing Decoction freeze-dried powder should not be less than 0.15%, and the content of ginsenoside Rb1 should not be less than 0.10%.

[0194]

[0195]

[0196] Table 21 Content and transfer rate of glycyrrhizic acid in decoction pieces and freeze-dried powder of Wenjing Decoction

[0197] Batch number Material reference content / % Decoction piece content / % Transfer rate / % Transfer rate deviation / % Wenjing Tang-1 1.13 3.88 29.18 -1.31 Wenjing Tang-2 0.63 2.67 23.43 -20.76 Wenjing Tang-3 0.64 2.11 30.18 2.07 Wenjing Tang-4 0.75 2.77 26.98 -8.75 Wenjing Tang-5 0.94 2.95 32.03 8.33 Wenjing Tang-6 0.91 3.41 26.71 -9.67 Wenjing Tang-7 0.81 3.18 25.53 -13.66 Wenjing Tang-8 0.86 3.17 27.15 -8.18 Wenjing Tang-9 0.85 2.55 33.18 12.22 Wenjing Tang-10 0.82 2.45 33.45 13.13 Wenjing Tang-11 0.87 2.51 34.85 17.86 Wenjing Tang-12 0.95 3.45 27.58 -6.72 Wenjing Tang-13 0.73 2.60 27.89 -5.68 Wenjing Tang-14 1.16 3.26 35.53 20.16 Wenjing Tang-15 0.87 2.93 29.85 0.95 Mean 0.86 2.93 29.57 - 70% Mean 0.60 2.05 20.70 - 130% Mean 1.12 3.80 38.44 - Min 0.63 2.11 23.43 - Max 1.16 3.88 35.53 - Conclusion ≥0.44％

[0198] The content determination results of glycyrrhizic acid in 15 batches of freeze-dried powder of Wenjing Decoction are shown in Table 21. The transfer rates of glycyrrhizic acid from decoction pieces to freeze-dried powder of Wenjing Decoction are relatively consistent, approximately between 23.43% and 35.53%, with an average transfer rate of 29.57%. The transfer rates of all batches fluctuate between 70% and 130% of the average value, indicating that the consistency of the transfer rates of different batches of glycyrrhizic acid is good, and that the stability of glycyrrhizic acid in the preparation process is good, and the process is stable.

[0199] In Table 21, the content of glycyrrhizic acid in 15 batches of freeze-dried powder of Wenjing Decoction ranges between 0.63% and 1.16%. Taking 70% of the average content (0.90%) as the minimum limit, it is recommended that the freeze-dried powder of Wenjing Decoction should not contain less than 0.63% of glycyrrhizic acid.

[0200] Table 22 Content and transfer rate of paeonol in decoction pieces and freeze-dried powder of Wenjing Decoction

[0201]

[0202]

[0203] The content determination results of paeonol in 15 batches of freeze-dried powder of Wenjing Decoction are shown in Table 22. The transfer rates of paeonol from decoction pieces to material reference paeonol in 15 batches of freeze-dried powder of Wenjing Decoction are relatively low, approximately between 5.34% and 10.44%, with an average transfer rate of 7.64%. The transfer rates of 3 batches exceed the fluctuation range of 70% to 130% of the average value. Paeonol is a fat-soluble component, and the water decoction rate is low, but the consistency of the transfer rates of different batches of paeonol is relatively good, indicating that the process reproducibility of paeonol in the preparation process is good.

[0204] In Table 22, the content of paeonol in 15 batches of freeze-dried powder of Wenjing Decoction ranges between 0.09% and 0.25%. Taking 70% of the average content (0.17%) as the minimum limit, it is recommended that the freeze-dried powder of Wenjing Decoction should not contain less than 0.12% of paeonol.

[0205] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0206] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method for detecting the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and / or ginsenoside in a sample, characterized in that, The method comprises: performing first extraction on the sample, collecting a first extraction solution, performing first liquid chromatography detection on the first extraction solution, and determining the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid and / or paeonol in the sample based on the detection result; and / or performing second extraction on the sample, collecting a second extraction solution, performing second liquid chromatography detection on the second extraction solution, and determining the content of ginsenoside in the sample based on the detection result; wherein the first extraction solvent used in the first extraction process comprises a methanol solution; the second extraction solvent used in the second extraction process comprises water-saturated n-butanol; the second extraction process comprises: Step 1: mixing the sample with dichloromethane, collecting the water layer after extraction; Step 2: repeatedly extracting the water layer with water-saturated n-butanol, combining the n-butanol layers obtained each time, and discarding the water layer; Step 3: repeatedly washing the n-butanol layer with ammonia test solution, combining the n-butanol layers obtained each time, and combining the ammonia test solution layers obtained each time; Step 4: mixing the ammonia test solution layer obtained in Step 3 with water-saturated n-butanol, collecting the n-butanol layer and combining it with the n-butanol layer obtained in Step 3, washing with water, drying the n-butanol layer, dissolving and constant volume of the residue collected with methanol, filtering, and passing through a membrane to obtain a test solution; The conditions of the second liquid chromatography detection are as follows: The chromatographic column is a C18 column; The mobile phase is selected from mobile phase A and mobile phase B, and the mobile phase A is acetonitrile and the mobile phase B is water; The elution mode used in the second liquid chromatography detection is as follows: 0~35 min, 19% (by volume) of mobile phase A; 35~55 min, 19% (by volume) of mobile phase A is increased to 29% (by volume); 55~70 min, 29% (by volume) of mobile phase A; 70~110 min, 29% (by volume) of mobile phase A is increased to 40% (by volume); The ginsenoside is selected from ginsenoside Rg1, Re and / or Rb1; The sample is Wending Decoction.

2. The method of claim 1, wherein, The first extraction process comprises: ultrasonically mixing the sample with the first extraction solvent for 15~45 minutes.

3. The method of claim 1, wherein, The first extraction process comprises: ultrasonically mixing the sample with the first extraction solvent for 25~35 minutes.

4. The method of claim 1, wherein, Based on 1 gram of the sample, the addition amount of the first extraction solvent is 30~50 milliliters, and the concentration of the methanol solution is 30~60% (by volume).

5. The method of claim 1, wherein, The conditions of the first liquid chromatography detection are as follows: The chromatographic column is a C18 column; The mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is selected from acetonitrile, and the mobile phase B is selected from 0.05~0.2% (by volume) formic acid solution; The flow rate is 0.5~1.5 mL / min; The column temperature is 20~30℃; The injection amount is 5~15 μL; When paeoniflorin and / or glycyrrhizin is determined, the detection wavelength is 230~240 nm; When glycyrrhizic acid is determined, the detection wavelength is 240~260 nm; When paeonol is determined, the detection wavelength is 270~290 nm.

6. The method of claim 1, wherein, The conditions of the second liquid chromatography detection are as follows: The flow rate is 0.5~1.5 mL / min; The column temperature is 25~35℃; The detection wavelength is 200~205 nm; The injection amount is 5~15 μL.

7. The method of claim 1, wherein, When paeoniflorin and / or glycyrrhizin is determined, the elution mode used in the first liquid chromatography detection is as follows: 0~8 min, mobile phase A is 17 %, make up mobile phase B to 100 %; 8~10 min, mobile phase A is raised from 17 % to 19 %, make up mobile phase B to 100 %; 10~20 min, mobile phase A is raised from 19 % to 25 %, make up mobile phase B to 100 %; 20~25 min, mobile phase A is raised from 25 % to 70 %, make up mobile phase B to 100 %; 25~35 min, mobile phase A is lowered from 70 % to 17 %, make up mobile phase B to 100 %; When determining glycyrrhizic acid and / or paeonol, the elution mode used in the first liquid chromatography detection is as follows: 0~15 min, mobile phase A is raised from 27 % to 38 %, make up mobile phase B to 100 %; 15~20 min, mobile phase A is raised from 38 % to 42 %, make up mobile phase B to 100 %; 20~25 min, mobile phase A is raised from 42 % to 55 %, make up mobile phase B to 100 %; 25~30 min, mobile phase A is raised from 55 % to 90 %, make up mobile phase B to 100 %; 30~35 min, mobile phase A is lowered from 90 % to 27 %, make up mobile phase B to 100 %.

8. The method of claim 1, wherein, The sample is in liquid state, In step 2, the volume of water-saturated n-butanol used in each extraction is 0.5~1.5 times the volume of the sample; the number of repeated extractions is 2~4 times; In step 3, the volume of ammonia test solution used in each washing is 0.5~1.5 times the volume of the sample; the number of repeated washings is 1~3 times.

9. The method of claim 8, wherein, In step 2, the volume of water-saturated n-butanol used in each extraction is 0.8~1.2 times the volume of the sample; the number of repeated extractions is 3 times; In step 3, the volume of ammonia test solution used in each washing is 0.8~1.2 times the volume of the sample; the number of repeated washings is 2 times.

10. The method of claim 1, wherein, The sample is in powder state, Step 1: dissolve the sample in water to obtain a sample aqueous solution, add dichloromethane to the aqueous solution, and collect the water layer after extraction; In step 2, the volume of water-saturated n-butanol used in each extraction is 0.5~1.5 times the volume of the sample aqueous solution; the number of repeated extractions is 2~4 times; In step 3, the volume of ammonia test solution used in each washing is 0.5~1.5 times the volume of the sample aqueous solution; the number of repeated washings is 1~3 times.

11. The method of claim 10, wherein, In step 2, the volume of water-saturated n-butanol used in each extraction is 0.8~1.2 times the volume of the sample aqueous solution; the number of repeated extractions is 3 times; In step 3, the volume of ammonia test solution used in each washing is 0.8~1.2 times the volume of the sample aqueous solution; the number of repeated washings is 2 times.

12. The use of the method for detecting the content of paeoniflorin, glycyrrhizin, glycyrrhizic acid, paeonol and / or ginsenoside in the sample according to any one of claims 1~11 in the quality control of Wening Tang.

13. A quality control method of Wenjing Decoction, characterized in that, It comprises: The method for detecting the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and / or ginsenoside in the sample according to any one of claims 1-11 is used to determine the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and ginsenoside in the Wanjing Decoction sample. The contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and ginsenoside meet the requirements, which is an indication that the quality of the Wanjing Decoction sample meets the standards.

14. The method of claim 13, wherein, The contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and ginsenoside in the Wanjing Decoction sample are within the ranges of the corresponding components in the substance reference of the Wanjing Decoction, which is an indication that the quality of the Wanjing Decoction sample meets the standards. The substance reference of the Wanjing Decoction comprises the following components: not less than 0.52% of paeoniflorin by mass, not less than 0.25% of glycyrrhizin by mass, not less than 0.14% of ginsenoside Rg1 and Re by mass in total, not less than 0.07% of ginsenoside Rb1 by mass, not less than 0.63% of glycyrrhizic acid by mass and not less than 0.09% of oxymatrine by mass.

15. The method according to claim 13 or 14, characterized in that, Further comprising: providing licorice medicinal materials or decoction pieces used for preparing the Wanjing Decoction sample; the method for detecting the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and / or ginsenoside in the sample according to any one of claims 1-11 is used to determine the content of glycyrrhizin in the licorice medicinal materials or decoction pieces; when the content of glycyrrhizin in the licorice medicinal materials or decoction pieces is 1-2% by mass and the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and ginsenoside in the Wanjing Decoction sample meet the requirements, it is an indication that the quality of the Wanjing Decoction meets the standards.

16. The method according to claim 13 or 14, characterized in that Further comprising: providing medicinal materials or decoction pieces used for preparing the Wanjing Decoction sample, which comprise licorice, white peony root, ginseng and monkshood root; the method for detecting the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and / or ginsenoside in the sample according to any one of claims 1-11 is used to determine the contents of glycyrrhizin and glycyrrhizic acid in the licorice, the content of paeoniflorin in the white peony root, the content of oxymatrine in the monkshood root and the contents of ginsenoside Rg1, Re and / or Rb1 in the ginseng; the following conditions are met, which is an indication that the quality of the Wanjing Decoction meets the standards: (1) when the contents of paeoniflorin, glycyrrhizin, glycyrrhizic acid, oxymatrine and ginsenoside in the Wanjing Decoction sample meet the requirements; (2) the transfer rate of white peony root to paeoniflorin is 14.99%-19.42%; (3) the transfer rate of licorice to glycyrrhizin is 28.11%-40.93% and the transfer rate of licorice to glycyrrhizic acid is 23.43%-35.53%; (4) the transfer rate of ginseng to ginsenoside Rg1 and Re is 25.92%-61.88% and the transfer rate of ginseng to ginsenoside Rb1 is 25.03%-64.06%; (5) the transfer rate of monkshood root to oxymatrine is 5.34%-10.44%.

Images