Construction method of Xuanyitongjing decoction preparation fingerprint and determination method of effective component content

Abstract

The application belongs to the technical field of medicine detection, and particularly relates to a method for constructing a Xuanyu Tongjing decoction preparation fingerprint and a method for determining effective component content. The method comprises preparation of a test sample solution, mobile phase A is acetonitrile, mobile phase B comprises a phosphoric acid solution, and a specific gradient elution procedure is used for determination. The method has the advantages of good stability and reliability, good durability, high specificity and the like, and solves the problem of less research on Xuanyu Tongjing decoction preparation quality control methods in the prior art. The application has guiding significance for Xuanyu Tongjing decoction preparation quality detection and evaluation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug detection, and particularly relates to a construction method of a Xuanyu Tongjing Decoction preparation fingerprint and a determination method of effective component content. BACKGROUND

[0002] Xuanyu Tongjing Decoction is a classical prescription, which is composed of ten medicinal ingredients: wine white peony root, wine angelica, peony root, fried gardenia, fried mustard seed, bupleurum, wine rhizoma cyperi, vinegar turmeric, wine scutellaria, and licorice root. The Xuanyu Tongjing Decoction has the effects of soothing liver and purging fire, regulating qi and nourishing blood, and is used for treating premenstrual abdominal pain caused by liver depression and fire, and is used for treating premenstrual abdominal pain, especially in the lower abdomen, and dark clots in the menstrual blood. In the prescription, white peony root has the effects of nourishing blood, soothing liver, regulating menstruation, and relieving pain; angelica has the effects of tonifying blood, activating blood, regulating menstruation, and relieving pain; white peony root and angelica are combined to nourish blood and activate blood, soothe liver and relieve pain, and are the monarch drugs. Peony root has the effects of clearing heat and cooling blood, activating blood and removing blood stasis; gardenia has the effects of clearing heat and cooling blood, purging fire and relieving restlessness; both are the ministerial drugs. White mustard seed has the effects of dispersing and warming, benefiting qi, resolving phlegm, and relieving pain; scutellaria has the effects of clearing heat and purging fire, activating blood and relieving pain, regulating qi and resolving depression, and cooling blood and clearing heart; cyperus has the effects of soothing liver and regulating menstruation and relieving pain; bupleurum has the effects of soothing liver and resolving depression; licorice root has the effects of relieving pain; all of the above are the auxiliary drugs. Scutellaria and gardenia purge fire and heat of liver meridian; cyperus and peony root purge fire and heat of liver meridian, and all of the above purge fire and heat of liver meridian. Licorice root harmonizes all the drugs. In the prescription, white peony root, angelica, cyperus, and scutellaria are all fried with wine, which enhances the effects of nourishing blood, activating blood, soothing liver, and regulating qi. The combination of all the drugs tonifies blood of liver and resolves depression of liver, benefits qi of liver and purges fire of liver, and all the symptoms are relieved. Modern pharmacological research shows that the Xuanyu Tongjing Decoction has the effects of enhancing hematopoiesis, protecting liver, relieving spasm, resisting bacteria, resisting virus, and resisting inflammation.

[0003] The Xuanyu Tongjing Decoction is composed of ten medicinal ingredients, including wine white peony root, wine angelica, peony root, fried gardenia, fried mustard seed, bupleurum, wine cyperus, vinegar turmeric, wine scutellaria, and licorice root. High performance liquid chromatography is one of the most widely used methods for the determination of multiple index contents, and has the characteristics of high separation efficiency, fast analysis speed, and automatic instrument, and is a commonly used method for the determination of contents. At present, there are few reports about the control of chemical components in the Xuanyu Tongjing Decoction, and there is a lack of quantitative or qualitative control method for multiple index components in the Xuanyu Tongjing Decoction. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the lack of quantitative or qualitative detection method for the Xuanyu Tongjing Decoction in the prior art, so as to provide a construction method of Xuanyu Tongjing Decoction preparation fingerprint and a determination method of effective component content.

[0005] To this end, the present application provides the following technical solutions.

[0006] The present application provides a construction method of Xuanyu Tongjing Decoction preparation fingerprint, comprising the following steps:

[0007] Preparation of the test solution: the test sample is prepared into a test solution;

[0008] Determination: determined by high performance liquid chromatography; wherein, the chromatographic conditions include: mobile phase A is acetonitrile, mobile phase B includes phosphoric acid solution; gradient elution, the program of gradient elution includes: 0-32min, the volume percentage of mobile phase A is 11%, the volume percentage of mobile phase B is 89%; 32-45min, the volume percentage of mobile phase A is 11%→15%, the volume percentage of mobile phase B is 89%→85%; 45-63min, the volume percentage of mobile phase A is 15%→30%, the volume percentage of mobile phase B is 85%→70%; 63-75min, the volume percentage of mobile phase A is 30%, the volume percentage of mobile phase B is 70%; 75-100min, the volume percentage of mobile phase A is 30%→75%, the volume percentage of mobile phase B is 70%→25%; 100-103min, the volume percentage of mobile phase A is 75%→11%, the volume percentage of mobile phase B is 25%→89%; 103-110min, the volume percentage of mobile phase A is 11%, the volume percentage of mobile phase B is 89%.

[0009] The chromatographic conditions include: mobile phase B includes 0.05-0.1% phosphoric acid solution; and / or, the column temperature is 25-35℃; and / or, the flow rate is 0.9-1.1mL / min; and / or, the injection volume is 5-10μL.

[0010] The mobile phase B further includes potassium dihydrogen phosphate; and / or,

[0011] The concentration of potassium dihydrogen phosphate in the mobile phase B is 0.025-0.05mol / L.

[0012] The chromatographic conditions include: mobile phase B includes 0.1% phosphoric acid solution; and / or, the column temperature is 30℃; and / or, the flow rate is 1.0mL / min; and / or, the detection wavelength: 0-45min, the wavelength is 237nm, 45-110min, the wavelength is 270nm; and / or, the injection volume is 10μL; and / or, the mobile phase B further includes 0.025mol / L potassium dihydrogen phosphate.

[0013] The test sample is Xuanyutongjing Decoction lyophilized powder, Xuanyutongjing Decoction dry extract powder or Xuanyutongjing Decoction granules.

[0014] The preparation method of the test solution includes: the steps of extracting the test sample with a solvent, filtering, and taking the filtrate;

[0015] Preferably, the solvent is 70-100% methanol aqueous solution;

[0016] Preferably, the solvent used is 70% methanol aqueous solution;

[0017] Preferably, the ratio of the mass of the test sample (in g) to the volume of the extracted solvent (in ml) is (0.3-0.5):(25-50).

[0018] The preparation method also includes the preparation of a reference solution;

[0019] Preferably, at least one of geniposide, sinapine thiocyanate, paeonolactone glycoside, paeonol, ferulic acid, baicalin, wogonoside, paeonol, glycyrrhizic acid, ligustrazine lactone and 1,2,3,4,6-penta-gallic acid glycoside is used as a reference.

[0020] The Xuanyu Tongjing Decoction preparation fingerprint obtained by the preparation method comprises at least 13 characteristic peaks;

[0021] Preferably, the Xuanyu Tongjing Decoction preparation fingerprint comprises at least geniposide peaks, sinapine thiocyanate peaks, paeonolactone glycoside peaks, paeonol peaks, ferulic acid peaks, baicalin peaks, wogonoside peaks, paeonol peaks, glycyrrhizic acid peaks, ligustrazine lactone peaks and 1,2,3,4,6-penta-gallic acid glycoside peaks.

[0022] The second aspect of the present application provides a method for determining the content of effective components in Xuanyu Tongjing Decoction preparation, characterized in that the above-mentioned preparation method is used.

[0023] The effective components are at least one of geniposide, paeonol, glycyrrhizic acid, baicalin and paeonol; and / or,

[0024] The detection wavelength of the determination method is 237 nm and / or 270 nm.

[0025] The technical scheme of the present application has the following advantages:

[0026] 1. The present application provides a preparation method for the Xuanyu Tongjing Decoction preparation fingerprint, which comprises the preparation of a test sample solution, the mobile phase A is acetonitrile, the mobile phase B comprises a phosphoric acid solution, and a specific gradient elution program is used for determination. The method has the advantages of good stability and reliability, good durability, high specificity, etc., and makes up for the problem of less research on the quality control method of Xuanyu Tongjing Decoction preparation in the prior art. The present application has guiding significance for the quality detection and evaluation of Xuanyu Tongjing Decoction preparation.

[0027] The composition of Xuanyu Tongjing Decoction preparation is complex, and there are many index components. The preparation method provided by the present application can detect a plurality of chemical components in Xuanyu Tongjing Decoction preparation and detect the content, realizing qualitative and quantitative quality control, and the method has high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the accompanying drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the accompanying drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0029] Figure 1 is the chromatogram obtained in section 1.1 of experimental example 1 of the present application by using different gradient elution procedures;

[0030] Figure 2 is the chromatogram obtained in section 1.2 of experimental example 1 of the present application by using different flow rates;

[0031] Figure 3 is the chromatogram obtained in section 1.3 of experimental example 1 of the present application by using different column temperatures;

[0032] Figure 4 is the chromatogram obtained in section 1.4 of experimental example 1 of the present application by using column A;

[0033] Figure 5 is the chromatogram obtained in section 1.4 of experimental example 1 of the present application by using column B;

[0034] Figure 6 is the chromatogram obtained in section 1.4 of experimental example 1 of the present application by using column C;

[0035] Figure 7 is the chromatogram obtained in section 1.4 of experimental example 1 of the present application by using column D;

[0036] Figure 8 is the chromatogram obtained in section 2.1 of experimental example 2 of the present application by using different extraction solvents;

[0037] Figure 9 is the chromatogram obtained in section 2.2 of experimental example 2 of the present application by using different extraction times;

[0038] Figure 10 is the chromatogram of 15 batches of Xuanyu Tongjing Decoction freeze-dried powder in experimental example 3 of the present application;

[0039] Figure 11 is the control chromatogram of Xuanyu Tongjing Decoction freeze-dried powder in experimental example 3 of the present application;

[0040] Figure 12 is the chromatogram of each control and test sample in experimental example 3 of the present application when confirming the characteristic peaks;

[0041] Figure 13 is the chromatogram of each negative test sample and test sample in experimental example 3 of the present application when studying the attribution of the characteristic peaks;

[0042] Figure 14is the spectrum of each test sample in the study of characteristic peak attribution of experimental example 3 of the present application;

[0043] Figure 15 is the spectrum of the integrity of section 4.4 in experimental example 4 of the present application;

[0044] Figure 16 is the spectrum of the specificity of jatrorrhizine, paeoniflorin and glycyrrhizic acid in section 5.1 in experimental example 5 of the present application;

[0045] Figure 17 is the spectrum of the specificity of jatrorrhizine, paeoniflorin and glycyrrhizic acid in section 5.1 in experimental example 5 of the present application. DETAILED DESCRIPTION

[0046] The following examples are provided to better further understand the present application, and are not limited to the best mode, and do not constitute a limitation on the content and scope of protection of the present application, and any person under the inspiration of the present application or the combination of the present application with other prior art features, any product identical or similar to the present application, falls within the scope of protection of the present application.

[0047] In the examples, the specific experimental steps or conditions are not specified, and can be performed according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not specified by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0048] Reagents

[0049] Xuanyutongjing Decoction Freeze-dried Powder is self-made by the present company; the batch numbers of 15 batches of Xuanyutongjing Decoction Freeze-dried Powder correspond to the numbers DG231201-DG231215. Among them, the preparation method of the standard decoction freeze-dried powder can be prepared by the conventional method in the art, and one preparation method is listed here: take the ten ingredients in the prescription, crush the fried jiezi, and add water to decoct the remaining nine ingredients twice, the first time to add water, soak, and keep boiling for 30 min, filter; the second time to add water, keep boiling for 25 min, filter, combine the filtrate, and freeze-dry the decoction under-80℃, to obtain it.

[0050] Geniposide (110749-201919), Paeoniflorin (110736-202145), Baicalin (110715-202223), Ammonium glycyrrhizinate (110731-202122), Paeonol (110708-201908), Myosmine thiocyanate (111702-202107), Ferulic acid (110773-201915), Wogonoside (112002-201702) were purchased from China Institute for Food and Drug Control; Ligustilide (PS013472), Paeonolactone (PS011455) were purchased from Chengdu Pusai Biological Technology Co., Ltd.; β-1,2,3,4,6-Penta-galloylglucose reference substance (230031-202004) was purchased from Shanghai Hongyong Biological Technology Co., Ltd.

[0051] Example 1

[0052] The present embodiment provides a method for constructing the fingerprint spectrum of Xuanyutongjing Decoction freeze-dried powder, comprising the following steps:

[0053] Preparation of test sample solution: Take about 0.3 g of Xuanyutongjing Decoction freeze-dried powder, accurately weigh, and place in a 25 ml volumetric flask. Add appropriate amount of 70% methanol, ultrasonic treat (power 280 W, frequency 40 kHz) for 15 min, cool, add 70% methanol to the mark, shake well, filter, and take the filtrate, which is obtained.

[0054] Preparation of reference substance control solution 1: Take appropriate amount of geniposide reference substance, paeoniflorin reference substance, baicalin reference substance, glycyrrhizic acid reference substance, and paeonol reference substance, accurately weigh, and add 70% methanol to prepare a mixed solution containing 100 μg of geniposide, 100 μg of paeoniflorin, 100 μg of baicalin, 15 μg of glycyrrhizic acid, and 40 μg of paeonol per 1 ml, which is obtained.(Among them, glycyrrhizic acid is added in the form of ammonium glycyrrhizinate, and the weight of glycyrrhizic acid = the weight of ammonium glycyrrhizinate / 1.0207).

[0055] Preparation of reference substance control solution 2: Take appropriate amount of myosmine thiocyanate reference substance, paeonolactone reference substance, ferulic acid reference substance, wogonoside reference substance, ligustilide reference substance, and β-1,2,3,4,6-penta-galloylglucose reference substance, accurately weigh, and add 70% methanol to prepare a mixed solution containing 15 μg of myosmine thiocyanate reference substance, 15 μg of paeonolactone, 10 μg of ferulic acid, 15 μg of wogonoside, 8 μg of ligustilide, and 15 μg of β-1,2,3,4,6-penta-galloylglucose per 1 ml, which is obtained.

[0056] Determination: 10 μL of the test solution and the control solution were respectively injected into the high performance liquid chromatograph for determination; wherein, the chromatographic conditions were as follows: octadecylsilane-bonded silica gel (CAPCELL PAK MGII, 4.6 mm x 250 mm, 5 μm) was used as the filler, acetonitrile was used as the mobile phase A, and the mobile phase B included 0.1% phosphoric acid aqueous solution and 0.025 mol / L potassium dihydrogen phosphate, gradient elution, the program of the gradient elution included: 0-32 min, the volume percentage of the mobile phase A was 11%, and the volume percentage of the mobile phase B was 89%; 32-45 min, the volume percentage of the mobile phase A was 11%→15%, and the volume percentage of the mobile phase B was 89%→85%; 45-63 min, the volume percentage of the mobile phase A was 15%→30%, and the volume percentage of the mobile phase B was 85%→70%; 63-75 min, the volume percentage of the mobile phase A was 30%, and the volume percentage of the mobile phase B was 70%; 75-100 min, the volume percentage of the mobile phase A was 30%→75%, and the volume percentage of the mobile phase B was 70%→25%; 100-103 min, the volume percentage of the mobile phase A was 75%→11%, and the volume percentage of the mobile phase B was 25%→89%; 103-110 min, the volume percentage of the mobile phase A was 11%, and the volume percentage of the mobile phase B was 89%; 0-45 min, the detection wavelength was 237 nm, 45-110 min, the detection wavelength was 270 nm; the column temperature was 30 °C, the flow rate was 1.0 mL / min, and the theoretical plate number calculated by paeoniflorin should not be less than 5000.

[0057] Investigation of the chromatographic conditions in experimental example 1

[0058] 1.1 Gradient elution program

[0059] Preparation of the test solution: about 0.3 g of the Xuanyutongjing decoction freeze-dried powder was accurately weighed into a 25 ml volumetric flask, and then appropriate amount of 70% methanol was added, ultrasonic treatment (power 280 W, frequency 40 kHz) was carried out for 15 min, the sample was allowed to cool, 70% methanol was added to the mark, and then the sample was shaken and filtered, and the filtrate was obtained.

[0060] Take 10 μL of the test sample solution, inject into the high performance liquid chromatograph, and determine; the chromatographic conditions are as follows: take octadecylsilane-bonded silica gel as the filler (CAPCELL PAK MGII, 4.6 mm x 250 mm, 5 μm), take acetonitrile as the mobile phase A, take the phosphoric acid aqueous solution containing 0.025 mol / L potassium dihydrogen phosphate as the mobile phase B, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.1%, the flow rate is 1.0 ml / min; 0-45 min, the detection wavelength is 237 nm, 45-110 min, the detection wavelength is 270 nm, the column temperature is 30°C; the theoretical plate number calculated according to paeonol should be no less than 5000; take the gradient elution program as the variable, and the gradient elution programs are shown in Tables 1-3. The results are shown in Figure 1 .

[0061] Table 1 Gradient elution program 1

[0062] Time (min) Mobile phase A (%) Mobile phase B (%) 0～32 11 89 32～45 11→15 89→85 45～63 15→30 85→70 63～75 30 70 75～100 30→80 70→20 100～103 80→11 20→89 103～110 11 89

[0063] Table 2 Gradient elution program 2

[0064] Time (min) Mobile phase A (%) Mobile phase B (%) 0～32 11.5 88.5 32～45 11.5→15 88.5→85 45～63 15→30 85→70 63～80 30→42 70→58 80～98 42→80 58→20 98～100 80→11.5 20→88.5 100～110 11.5 88.5

[0065] Table 3 Gradient elution program 3

[0066]

[0067]

[0068] Figure 1 The graphs obtained from the bottom to the top correspond to the gradient elution programs 1-3 in sequence, and it can be seen from the graphs that the test sample chromatogram obtained by the gradient elution program 3 has a relatively uniform distribution of each larger chromatographic peak and a better separation degree, and the gradient elution program 3 is adopted in the present application.

[0069] 1.2 Flow rate

[0070] Prepare the test sample solution according to the section 1.1 of the present experimental example, take the flow rate as the variable, and the other chromatographic conditions are the same as those in the section 1.1, and determine according to the optimal gradient elution program determined in the section 1.1; wherein the flow rates are 0.9 ml / min, 1.0 ml / min and 1.1 ml / min respectively.

[0071] Figure 2 are graphs obtained at different flow rates; Figure 2 The graphs obtained from the bottom to the top correspond to the flow rates 1.1 ml / min, 0.9 ml / min and 1.0 ml / min in sequence, and it can be seen from the graphs that the separation degree of the graphs obtained at the flow rates of 0.9-1.1 ml / min is better.

[0072] 1.3 Column temperature

[0073] Prepare the test solution according to Section 1.1 of this experimental example, using column temperature as the variable. Other chromatographic conditions were the same as in Section 1.1, and the optimal gradient elution program determined in 1.1 was followed; the column temperatures were 25°C, 30°C, and 35°C, respectively.

[0074] Figure 3 The following are the spectra obtained at different column temperatures, from bottom to top, corresponding to column temperatures of 35°C, 30°C, and 25°C. As can be seen from the figure, the spectra obtained at a column temperature of 25-35°C have better resolution.

[0075] 1.4 Column inspection

[0076] The test solution was prepared according to Section 1.1 of this experimental example. The chromatographic column was used as a variable. Column A: WatersXSelect HSS T3 C18 (4.6×250 mm, 5 μm), column B: Agilent ZORBAX SB C18 (4.6×250 mm, 5 μm), column C: CAPCELL PAK ADME HR C18 (4.6×250 mm, 5 μm), and column D: CAPCELL PAK MGII (4.6×250 mm, 5 μm) were used. Other chromatographic conditions were the same as in Section 1.1.

[0077] Figure 4 This is the spectrum obtained by column A; Figure 5 is the spectrum obtained by column B; Figure 6 This is the spectrum obtained by column C; Figure 7 This is the spectrum obtained by chromatographic column D. The results show that the peak order, peak shape, and peak number vary greatly among different chromatographic columns. The chromatographic column CAPCELL PAK MGII (4.6×250mm, 5μm) has relatively good chromatographic peak separation and peak shape, a relatively flat baseline, and a high response value. Therefore, the present invention adopts the CAPCELL PAK MGII (4.6×250mm, 5μm) chromatographic column.

[0078] Based on the above content, the chromatographic conditions of the present invention were determined: gradient elution program 3 was adopted, CAPCELLPAK MGII (4.6×250 mm, 5 μm) was used as the chromatographic column, the flow rate was 0.9-1.1 ml / min, and the column temperature was 25-35°C; among them, the optimal chromatographic conditions were gradient elution program 3, CAPCELL PAK MGII (4.6×250 mm, 5 μm) was used as the chromatographic column, the flow rate was 1.0 ml / min, and the column temperature was 30°C.

[0079] Experimental Example 2 Investigation of the preparation of the test solution

[0080] 2.1 Concentration of extraction solvent

[0081] Take Queyun Tongjing Decoction freeze-dried powder 4 parts, each 0.3g, add 30% methanol, 50% methanol, 70% methanol, methanol, respectively, ultrasonic treatment (power 280W, frequency 40kHz) 15min, cool, add solvent to the scale, shake, filter, take the filtrate, get 4 test sample solution. According to the optimal chromatographic conditions determined in experimental example 1 for detection. Results see Figure 8 and table 4-5. Figure 8 From bottom to top, the extraction solvent is 30% methanol, 50% methanol, 70% methanol, methanol.

[0082] Table 4 relative retention time of the chromatogram obtained by different extraction solvents

[0083] Solvent Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 30% methanol 0.56 0.64 0.75 1.00 1.46 / 2.07 2.20 2.21 2.27 2.62 2.78 3.15 50% methanol 0.56 0.64 0.75 1.00 1.46 / 2.07 2.20 2.21 2.27 2.62 2.78 3.15 70% methanol 0.56 0.64 0.75 1.00 1.46 1.88 2.07 2.21 2.22 2.28 2.63 2.79 3.16 Methanol 0.56 0.64 0.75 1.00 1.46 1.89 2.08 2.22 2.23 2.29 2.64 2.80 3.18 Average 0.56 0.64 0.75 1.00 1.46 / 2.07 2.21 2.22 2.27 2.63 2.79 3.16 RSD % 0.0 0.1 0.0 0.0 0.2 / 0.4 0.4 0.4 0.4 0.4 0.4 0.4

[0084] Table 5 relative peak area of the chromatogram obtained by different extraction solvents

[0085] Solvent Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 30% methanol 1.40 0.26 0.18 1.00 0.07 / 2.00 0.17 0.03 0.55 0.48 0.02 0.01 50% methanol 1.41 0.27 0.18 1.00 0.07 / 2.20 0.18 0.04 0.59 0.56 0.02 0.02 70% methanol 1.42 0.27 0.18 1.00 0.07 0.45 2.29 0.19 0.04 0.61 0.59 0.03 0.02 Methanol 1.37 0.17 0.16 1.00 0.07 0.17 1.99 0.18 0.03 0.57 0.59 0.02 0.02 Average 1.40 0.24 0.17 1.00 0.07 / 2.12 0.18 0.04 0.58 0.55 0.02 0.02 RSD % 1.6 19.0 5.1 0.00 3.9 / 7.0 4.9 9.0 4.3 9.6 19.5 14.4

[0086] The results show that: the concentration of extraction solvent has a greater impact on peak 6, when the extraction solvent is 30% methanol, 50% methanol, peak 6 is missing. Peak 4 with stable peak time and good separation degree is selected as S peak, and the relative peak area of other characteristic peaks is calculated from S peak. From the results, it can be seen that when 70% methanol is used as the extraction solvent, more chemical components are represented and the peak response value is higher. Therefore, on the basis of considering as comprehensive as possible to represent the chemical components, 70% methanol is selected as the extraction solvent.

[0087] 2.2 extraction time

[0088] Take Queyun Tongjing Decoction freeze-dried powder 4 parts, each 0.3g, add 70% methanol, ultrasonic treatment (power 280W, frequency 40kHz) 15min, 30min, 45min, 60min, respectively, cool, add 70% methanol to the scale, shake, filter, take the filtrate, get 4 test sample solution. According to the optimal chromatographic conditions determined in experimental example 1 for detection, results see Figure 9 and table 6-7. Figure 9 From bottom to top, the ultrasonic time is 15min, 30min, 45min, 60min.

[0089] Table 6 relative retention time of the chromatogram obtained by different extraction times

[0090] Extraction time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 15 min 0.56 0.64 0.75 1.00 1.46 1.88 2.08 2.21 2.22 2.28 2.63 2.80 3.17 30 min 0.56 0.64 0.75 1.00 1.46 1.88 2.07 2.21 2.22 2.28 2.63 2.79 3.17 45 min 0.56 0.64 0.75 1.00 1.46 1.88 2.07 2.21 2.22 2.28 2.63 2.79 3.17 60 min 0.56 0.64 0.75 1.00 1.46 1.89 2.08 2.21 2.22 2.28 2.64 2.80 3.18 Average 0.56 0.64 0.75 1.00 1.46 1.89 2.08 2.21 2.22 2.28 2.63 2.80 3.17 RSD % 0.1 0.0 0.0 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2

[0091] Table 7 relative peak area of the chromatogram obtained by different extraction times

[0092] Extraction time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 15 min 1.41 0.27 0.18 1.00 0.07 0.44 2.27 0.19 0.04 0.60 0.59 0.03 0.02 30 min 1.41 0.27 0.18 1.00 0.07 0.44 2.27 0.19 0.04 0.60 0.58 0.03 0.02 45 min 1.41 0.27 0.18 1.00 0.07 0.45 2.28 0.19 0.04 0.60 0.58 0.03 0.02 60 min 1.41 0.27 0.18 1.00 0.07 0.32 2.29 0.19 0.04 0.61 0.59 0.03 0.02 Average 1.41 0.27 0.18 1.00 0.07 0.41 2.28 0.19 0.04 0.60 0.59 0.03 0.02 RSD % 0.1 0.5 0.8 0.0 0.4 15.6 0.4 0.3 1.8 0.3 0.8 0.9 1.9

[0093] The results show that in the ultrasonic time investigation, the ultrasonic time may have a slight effect on peak 6, and there is no significant effect when the ultrasonic time is 15-45 min. In order to ensure the efficiency and sufficient extraction, 15 min is selected as the extraction time of the test sample.

[0094] Determination and identification of characteristic peaks in experimental example 3

[0095] Fifteen batches of Xuanyu Tongjing Decoction freeze-dried powder were prepared according to Example 1 to prepare test sample solutions, and were determined to obtain the characteristic chromatograms of the fifteen batches of Xuanyu Tongjing Decoction freeze-dried powder. The results are shown in Table 8 and Figure 10 , Figure 10 From bottom to top, they are Xuanyu Tongjing Decoction freeze-dried powder DG231201-DG231215. The control chromatogram generated by the chromatograms of the fifteen batches of Xuanyu Tongjing Decoction freeze-dried powder is shown in Figure 11 . Taking peak No. 4 as the reference peak, the specified values of the relative retention times of each characteristic peak are as follows: peak No. 1 is 0.56, peak No. 2 is 0.65, peak No. 3 is 0.75, peak No. 5 is 1.49, peak No. 6 is 1.95, peak No. 7 is 2.14, peak No. 8 is 2.28, peak No. 9 is 2.29, peak No. 10 is 2.35, peak No. 11 is 2.72, peak No. 12 is 2.89, and peak No. 13 is 3.28. The relative retention times of each characteristic peak are within ±10% of the specified values.

[0096] Table 8 Relative retention times of the chromatograms of the fifteen batches of Xuanyu Tongjing Decoction freeze-dried powder

[0097] Sample Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 DG231201 0.56 0.65 0.75 1.00 1.49 1.95 2.14 2.28 2.29 2.35 2.72 2.89 3.29 DG231202 0.56 0.65 0.75 1.00 1.49 1.95 2.15 2.28 2.29 2.35 2.72 2.90 3.29 DG231203 0.56 0.65 0.75 1.00 1.49 1.95 2.15 2.29 2.30 2.36 2.72 2.90 3.29 DG231204 0.56 0.65 0.75 1.00 1.49 1.95 2.15 2.29 2.30 2.36 2.72 2.90 3.29 DG231205 0.56 0.65 0.75 1.00 1.49 1.95 2.14 2.28 2.29 2.35 2.72 2.89 3.29 DG231206 0.56 0.65 0.75 1.00 1.49 1.95 2.14 2.28 2.29 2.35 2.72 2.89 3.28 DG231207 0.56 0.65 0.75 1.00 1.49 1.95 2.14 2.28 2.29 2.35 2.72 2.89 3.28 DG231208 0.56 0.65 0.75 1.00 1.49 1.95 2.14 2.28 2.29 2.35 2.72 2.89 3.28 DG231209 0.56 0.65 0.75 1.00 1.49 1.95 2.15 2.28 2.29 2.35 2.72 2.89 3.29 DG231210 0.56 0.65 0.75 1.00 1.49 1.95 2.15 2.28 2.30 2.35 2.72 2.90 3.29 DG231211 0.56 0.65 0.75 1.00 1.49 1.95 2.15 2.29 2.30 2.36 2.72 2.90 3.29 DG231212 0.56 0.65 0.75 1.00 1.49 1.94 2.14 2.28 2.29 2.35 2.72 2.89 3.28 DG231213 0.56 0.65 0.75 1.00 1.49 1.94 2.14 2.28 2.29 2.35 2.71 2.88 3.28 DG231214 0.56 0.65 0.75 1.00 1.49 1.94 2.13 2.27 2.28 2.34 2.71 2.88 3.27 DG231215 0.56 0.65 0.75 1.00 1.49 1.94 2.14 2.28 2.29 2.35 2.71 2.89 3.28 Average 0.56 0.65 0.75 1.00 1.49 1.95 2.14 2.28 2.29 2.35 2.72 2.89 3.28 RSD % 0.1 0.1 0.0 0.0 0.0 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.2

[0098] The chromatogram detection AIA data was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" calculation software, and the parameters were set as follows: data cutting 0-5 min solvent peak, taking S1 as the reference chromatogram, the generation method is the average number, the time window is 0.1; multi-point correction, all peak matching for peaks with peak area accounting for more than 0% of the total peak area, and the non-common peak area accounting ratio and similarity results are shown in the table below. The results show that the matching peak number of the fifteen batches of Xuanyu Tongjing Decoction freeze-dried powder is 13 peaks, the common peak accounts for 34% of the total peak area, and the fingerprint chromatograms of the fifteen batches of Xuanyu Tongjing Decoction freeze-dried powder are greater than 0.90, and the similarity is high.

[0099] Table 9 Peak matching of the fingerprint chromatograms of the fifteen batches of Xuanyu Tongjing Decoction freeze-dried powder

[0100] Batch number Non-common peak area Total peak area Non-common peak area ratio DG231201 6169.76 17599.3 35％ DG231202 5949.69 17057.98 35％ DG231203 6267.78 18941.83 33％ DG231204 5712.1 16862.6 34％ DG231205 6410.76 19056.39 34％ DG231206 6770.02 18821.61 36％ DG231207 7109.82 20221.42 35％ DG231208 5800.06 17645.32 33％ DG231209 6972.57 19089.7 37％ DG231210 6232.1 17329.3 36％ DG231211 6127.88 18218.78 34％ DG231212 6046.38 17966.19 34％ DG231213 6099.5 17992.69 34％ DG231214 5570.93 16897.93 33％ DG231215 5900.42 16983.67 35％

[0101] Table 10 Similarity of the fingerprint chromatograms of the fifteen batches of Xuanyu Tongjing Decoction freeze-dried powder

[0102] Similarity Reference fingerprint DG231201 0.998 DG231202 0.993 DG231203 0.999 DG231204 0.996 DG231205 0.994 DG231206 0.998 DG231207 0.997 DG231208 0.999 DG231209 0.995 DG231210 0.996 DG231211 0.997 DG231212 0.999 DG231213 0.999 DG231214 0.997 DG231215 0.997

[0103] Confirmation of characteristic peaks

[0104] Figure 12From bottom to top in turn is blank solvent, gardenoside control, myronate thiocyanate control, paeonolactone glycoside control, paeonolactone glycoside control, ferulic acid control, baicalin control, hanbaikalide control, paeonolactone glycoside control, liquorice acid control, ligustrazine control, β-1,2,3,4,6-penta gallic acid glycoside control, Xuanyu Tongjing decoction freeze-dried powder test sample respectively according to the experimental example 1 preparation control solution and test sample solution, and according to the determined optimal chromatographic conditions, the obtained atlas is determined. From the figure, it can be seen that the test sample contains chromatographic peaks consistent with the retention time of the control sample, indicating that Xuanyu Tongjing decoction freeze-dried powder contains gardenoside (peak 1), myronate thiocyanate (peak 2), paeonolactone glycoside (peak 3), paeonolactone glycoside (peak 4), ferulic acid (peak 5), β-1,2,3,4,6-penta gallic acid glycoside control (peak 6), baicalin (peak 7), hanbaikalide (peak 10), paeonolactone glycoside (peak 11), liquorice acid (peak 12), ligustrazine (peak 13).

[0105] Characteristic peak attribution

[0106] To further verify the composition of the characteristic peak, according to the method of example 1, the negative test sample of fried gardenia without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain fried gardenia), the negative test sample of fried mustard without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain fried mustard), the negative test sample of wine huangqi without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain wine huangqi), the negative test sample of wine danggui without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain wine danggui), the negative test sample of wine baishao without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain wine baishao), the negative test sample of mudanpi without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain mudanpi), the negative test sample of chaihu without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain chaihu), the negative test sample of vinegar jianghuang without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain vinegar jianghuang), the negative test sample of wine xiangfu without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain wine xiangfu), the negative test sample of gancao without frying (the difference from the prescription of Xuanyu Tongjing decoction is that it does not contain gancao), and the test sample solution corresponding to the test sample of Xuanyu Tongjing decoction freeze-dried powder were prepared, and then determined to obtain the atlas of each test sample, and the results are shown in Figure 13 . Figure 13 From bottom to top in turn is blank solvent, gardenoside control, myronate thiocyanate control, paeonolactone glycoside control, paeonolactone glycoside control, ferulic acid control, baicalin control, hanbaikalide control, paeonolactone glycoside control, paeonolactone glycoside control, liquorice acid control, ligustrazine control, β-1,2,3,4,6-penta gallic acid glycoside control, Xuanyu Tongjing decoction freeze-dried powder test sample.

[0107] and the wine white peony root test sample, the wine angelica test sample, the tree peony bark test sample, the fried gardenia test sample, the fried rapeseed test sample, the wine yellow sophora test sample, the wine rhizoma cyperi test sample, the bupleurum test sample, the liquorice test sample, the Xuanyu Tongjing Decoction freeze-dried powder test sample are prepared respectively according to Example 1, and determination is performed, and the chromatograms of the various test samples are obtained, and the results are shown in Figure 14 . Figure 14 From bottom to top, the wine white peony root test sample, the wine angelica test sample, the tree peony bark test sample, the fried gardenia test sample, the fried rapeseed test sample, the wine yellow sophora test sample, the wine rhizoma cyperi test sample, the bupleurum test sample, the liquorice test sample, the freeze-dried powder test sample are prepared respectively according to Example 1.

[0108] From Figure 13-14 It can be seen that the chromatographic peaks presented in the fingerprint chromatogram of the Xuanyu Tongjing Decoction preparation mainly come from the wine white peony root (peaks 3, 4), the wine angelica (peaks 5, 13), the tree peony bark (peaks 6, 11), the fried gardenia (peak 1), the fried rapeseed (peak 2), the wine yellow sophora (peaks 7, 8, 9, 10), and the liquorice (peak 12) 7 medicinal ingredients.

[0109] Methodological verification of the construction method of the fingerprint chromatogram

[0110] 4.1 Specificity

[0111] The reference substance solution and the Xuanyu Tongjing Decoction freeze-dried powder are prepared respectively according to Example 1, and determination is performed according to Example 1, with 70% methanol as a blank solvent. The results show that the retention time of the test sample solution is consistent with that of the reference substance, and the blank solvent does not interfere, and the specificity of the fingerprint chromatogram of the present application is good.

[0112] 4.2 Reproducibility

[0113] The Xuanyu Tongjing Decoction freeze-dried powder test sample solution is prepared according to Example 1, and determination is performed, and the chromatogram is recorded. The AIA data of the chromatogram is imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" calculation software. The parameter setting is: data cutting 0-5 min solvent peak, with reproducibility-1 as the reference chromatogram, the generation method is the average number, the time window is 0.1; multi-point correction, all peak matching of the peak area accounting for more than 0% of the total peak area, and the similarity results are shown in the table below. The results show that the chromatographic similarity presented by the six test samples is greater than 0.99, and the similarity between each test sample solution and the control fingerprint chromatogram is greater than 0.99; with peak 4 as the reference peak (S peak), the relative retention time of each characteristic peak and the S peak is calculated, and the results show that the relative retention time of each characteristic peak is less than 1.0%, and the comprehensive results analysis shows that the reproducibility of the method is good.

[0114] Table 11 Similarity results of reproducibility test

[0115] Similarity Repeatability-1 Repeatability-2 Repeatability-3 Repeatability-4 Repeatability-5 Repeatability-6 Reference fingerprint 1.000 1.000 1.000 1.000 1.000 1.000

[0116] Table 12 Repetitive test results relative retention time

[0117] Repeatability Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 Repeatability-1 0.56 0.64 0.75 1.00 1.49 1.96 2.16 2.30 2.31 2.37 2.74 2.91 3.31 Repeatability-2 0.56 0.64 0.75 1.00 1.49 1.96 2.16 2.30 2.31 2.37 2.74 2.92 3.31 Repeatability-3 0.56 0.64 0.75 1.00 1.49 1.96 2.16 2.30 2.31 2.37 2.74 2.91 3.31 Repeatability-4 0.56 0.64 0.75 1.00 1.49 1.95 2.15 2.29 2.30 2.36 2.73 2.90 3.30 Repeatability-5 0.56 0.64 0.75 1.00 1.49 1.95 2.15 2.29 2.30 2.36 2.73 2.91 3.30 Repeatability-6 0.56 0.64 0.75 1.00 1.49 1.95 2.15 2.29 2.30 2.36 2.73 2.91 3.30 Average 0.56 0.64 0.75 1.00 1.49 1.96 2.16 2.29 2.31 2.36 2.73 2.91 3.30 RSD % 0.1 0.1 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1

[0118] 4.3 Durability

[0119] The Xuanyu Tongjing Decoction lyophilized powder test sample solution was prepared according to Example 1. 10 μl of the sample was injected at 0 h, 16 h, 20 h, 28 h, 46 h, 62 h, and 88 h, respectively, and the chromatogram was recorded. The AIA data of the chromatogram was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" calculation software. The parameters were set as follows: data cutting 0-5 min solvent peak, with stability 0 h as the reference spectrum, the generation method was average, and the time window was 0.1; multi-point correction, all peak matching of the peak area accounting for more than 0% of the total peak area, and the similarity results are shown in the table below. The results showed that the chromatographic similarity of the test sample within 88 h was greater than 0.99, and the similarity between each test sample solution and the control fingerprint was greater than 0.99; with peak 4 as the reference peak (S peak), the relative retention time of each characteristic peak and S peak was calculated, and the results showed that the relative retention time RSD% of each characteristic peak was less than 1.0%. Comprehensive analysis of the results showed that the method had good durability for 88 h.

[0120] Table 13 Durability test results

[0121] Similarity 0h 16h 20h 28h 46h 62h 88h Reference fingerprint 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0122] Table 14 Durability test relative retention time

[0123] Repeatability Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 0h 0.75 0.86 1.00 1.33 1.98 2.59 2.86 3.04 3.06 3.14 3.63 3.86 4.38 16h 0.75 0.85 1.00 1.33 1.98 2.59 2.86 3.05 3.06 3.14 3.63 3.86 4.39 20h 0.75 0.86 1.00 1.33 1.98 2.60 2.86 3.05 3.06 3.14 3.63 3.86 4.39 28h 0.75 0.86 1.00 1.33 1.98 2.60 2.86 3.05 3.06 3.14 3.63 3.87 4.39 46h 0.75 0.86 1.00 1.33 1.98 2.60 2.87 3.05 3.07 3.15 3.64 3.87 4.40 62h 0.75 0.86 1.00 1.33 1.99 2.60 2.87 3.05 3.06 3.14 3.64 3.87 4.39 88h 0.75 0.85 1.00 1.33 1.98 2.60 2.87 3.05 3.06 3.14 3.63 3.87 4.39 Average 0.75 0.86 1.00 1.33 1.98 2.60 2.86 3.05 3.06 3.14 3.63 3.86 4.39 RSD % 0.0 0.1 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

[0124] 4.4 Integrity

[0125] The Xuanyu Tongjing Decoction lyophilized powder test sample solution was prepared according to Example 1. The test was run according to the double detection time of Example 1, and the chromatogram was recorded, as shown in Figure 15 The results showed that the main chemical components were completely eluted within 0-110 min, indicating that the method could represent the complete chromatographic peaks.

[0126] Example 2

[0127] The present embodiment provides a method for determining the content of effective components in Xuanyu Tongjing Decoction lyophilized powder, comprising the following steps:

[0128] Preparation of test sample solution: Take about 0.3 g of Xuanyu Tongjing Decoction lyophilized powder, accurately weigh, and place in a 25 ml volumetric flask. Add appropriate amount of 70% methanol, ultrasonic treat (power 280 W, frequency 40 kHz) for 15 min, cool, add 70% methanol to the mark, shake well, filter, and take the filtrate, which is obtained.

[0129] Preparation of reference substance solution: Take appropriate amounts of gardeniain reference substance, paeoniflorin reference substance, baicalin reference substance, glycyrrhizic acid reference substance, and paeonol reference substance, accurately weigh them, and add 70% methanol to make a mixed solution containing 100 μg of gardeniain, 100 μg of paeoniflorin, 100 μg of baicalin, 15 μg of glycyrrhizic acid, and 40 μg of paeonol per 1 ml (wherein glycyrrhizic acid is added in the form of ammonium glycyrrhizate, weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207).

[0130] Determination: 10 μL of the test solution and the reference solution were respectively drawn and injected into a high performance liquid chromatograph for determination; wherein, the chromatographic conditions were: octadecylsilane bonded silica gel was used as the filler (CAPCELL PAK MGII, 4.6 mm × 250 mm, 5 μm), acetonitrile was used as the mobile phase A, the mobile phase B included 0.1% phosphoric acid aqueous solution and 0.025 mol / L potassium dihydrogen phosphate, gradient elution, and the gradient elution program included: 0-32 min, the volume percentage of mobile phase A was 11%, and the volume percentage of mobile phase B was 89%; 32-45 min, the volume percentage of mobile phase A was 11%→15%, and the volume percentage of mobile phase B was 89%→85%; 45-63 min, the volume percentage of mobile phase A was 15%→30%, and the volume percentage of mobile phase B was 85%→70%; 63-75 min, the volume percentage of mobile phase A was 11%→15%, and the volume percentage of mobile phase B was 85%→70% The volume percentage of mobile phase A was 30%, and the volume percentage of mobile phase B was 70%; from 75 to 100 minutes, the volume percentage of mobile phase A was 30% → 75%, and the volume percentage of mobile phase B was 70% → 25%; from 100 to 103 minutes, the volume percentage of mobile phase A was 75% → 11%, and the volume percentage of mobile phase B was 25% → 89%; from 103 to 110 minutes, the volume percentage of mobile phase A was 11%, and the volume percentage of mobile phase B was 89%; the test sample was detected simultaneously at wavelengths of 237 nm and 270 nm; the column temperature was 30°C, the flow rate was 1.0 mL / min, and the theoretical plate number calculated based on paeoniflorin should be no less than 5000. The reference substance and standard mixed solution was detected simultaneously at wavelengths of 237 nm and 270 nm.

[0131] The results of the determination of the content of each active ingredient in 15 batches of Xuanyu Tongjing Decoction freeze-dried powder are shown in Table 15. The results show that the five index components of 15 batches of Xuanyu Tongjing Decoction freeze-dried powder all meet the range of 70% to 130% of the mean, and no discrete data appear.

[0132] Table 15 Determination results of the content of each active ingredient in 15 batches of Xuanyu Tongjing Decoction freeze-dried powder (mg / prescription)

[0133] Batch number Geniposide Paeoniflorin Baicalin Glycyrrhizic acid Catalpol DG231101 546.2 518.4 376.1 34.8 68.9 DG231102 482.7 529.0 377.4 24.7 88.6 DG231103 470.5 442.9 306.1 27.4 74.7 DG231104 502.4 581.7 327.9 31.3 74.8 DG231105 440.0 521.7 411.8 35.3 70.8 DG231106 413.4 422.5 389.3 32.1 86.8 DG231107 386.2 539.2 352.3 31.3 63.9 DG231108 457.0 575.3 339.0 27.6 85.1 DG231109 486.5 454.0 333.8 27.4 55.3 DG231110 427.4 544.3 455.3 30.5 72.7 DG231111 457.7 470.7 373.2 34.1 78.4 DG231112 460.8 431.5 402.3 33.1 61.4 DG231113 447.1 409.1 349.8 27.1 63.9 DG231114 409.5 388.3 384.0 22.8 52.5 DG231115 473.7 459.4 368.0 32.8 74.5 Mean 457.4 485.9 369.8 30.2 71.5 RSD % 8.8 12.8 10.1 12.5 15.1 Min 386.2 388.3 306.1 22.8 52.5 Max 546.2 581.7 455.3 35.3 88.6 Mean x 70% 320.2 340.1 258.8 21.1 50.0 Mean x 130% 594.6 631.6 480.7 39.2 92.9

[0134] Experimental Example 5 Methodological Verification of the Determination Method of Active Ingredient Content

[0135] 5.1 Specificity

[0136] (1) The specificity of gardenoside, paeoniflorin and glycyrrhizic acid

[0137] The double negative test sample (the difference between the prescription of Xuanyu Tongjing Decoction and the prescription of Xuanyu Tongjing Decoction is that it lacks wine white peony root and cortex moutan), the test sample lacking fried gardenia (the difference between the prescription of Xuanyu Tongjing Decoction and the prescription of Xuanyu Tongjing Decoction is that it lacks fried gardenia), the test sample lacking glycyrrhiza, the solution corresponding to the gardenoside reference substance, the paeoniflorin reference substance, the glycyrrhizic acid reference substance and the Xuanyu Tongjing Decoction freeze-dried powder test sample were prepared according to Example 2, and were determined. The detection wavelength was 237 nm, and the other chromatographic conditions were the same as in Example 2. Figure 16 From bottom to top are the double negative test sample (the difference between the prescription of Xuanyu Tongjing Decoction and the prescription of Xuanyu Tongjing Decoction is that it lacks wine white peony root and cortex moutan), the test sample lacking fried gardenia, the test sample lacking glycyrrhiza, the gardenoside reference substance, the paeoniflorin reference substance, the glycyrrhizic acid reference substance and the Xuanyu Tongjing Decoction freeze-dried powder test sample.

[0138] (2) The specificity of baicalin and peony phenol

[0139] The test sample lacking wine huangqi (the difference between the prescription of Xuanyu Tongjing Decoction and the prescription of Xuanyu Tongjing Decoction is that it lacks wine huangqi), the test sample lacking cortex moutan, the solution corresponding to the baicalin reference substance, the peony phenol reference substance and the Xuanyu Tongjing Decoction freeze-dried powder test sample were prepared according to Example 2, and were determined. The detection wavelength was 270 nm, and the other chromatographic conditions were the same as in Example 2. Figure 17 From bottom to top are the test sample lacking wine huangqi, the test sample lacking cortex moutan, the baicalin reference substance, the peony phenol reference substance and the Xuanyu Tongjing Decoction freeze-dried powder test sample.

[0140] Figures 16-17 The results show that the test sample contains chromatographic peaks with retention times consistent with those of the reference substance in the reference substance solution, and each negative control substance solution does not interfere with the detection of the content of multiple index components; each index component in the reference substance solution should correspond to the test sample, and the determination of gardenoside, paeoniflorin, baicalin, peony phenol and glycyrrhizic acid by the method has good specificity.

[0141] 5.2 Accuracy

[0142] Take the same concentration of Xuanyutongjing Decoction freeze-dried powder several times (sample amount is about 0.15 g, and the known contents of gardenoside, paeoniflorin, baicalin, glycyrrhizic acid and paeonol are 15.99 mg / g, 15.48 mg / g, 11.60 mg / g, 1.30 mg / g and 2.06 mg / g respectively), and add the gardenoside reference substance, paeoniflorin reference substance, baicalin reference substance, glycyrrhizic acid reference substance and paeonol reference substance into the Xuanyutongjing Decoction freeze-dried powder and the reference substances in a mass ratio of 1:1. The mixture of the Xuanyutongjing Decoction freeze-dried powder and the reference substances is used as the test sample, and the test sample solution is prepared according to Example 2. Six test sample solutions are prepared in parallel, and the recovery rate (%) and relative standard deviation (RSD%) are calculated.

[0143] Recovery rate calculation formula:

[0144]

[0145] Wherein, A is the amount of the measured component contained in the test sample (mg); B is the amount of the added reference substance (mg); and C is the measured value (mg).

[0146] Table 16 Accuracy test results

[0147]

[0148] The results show that according to the data requirements in the 9101 Drug Quality Standard Analysis Method Verification Guideline in the Fourth Part of the Chinese Pharmacopoeia 2020 Edition, the contents of gardenoside, paeoniflorin, baicalin, paeonol, glycyrrhizic acid, the recovery rate and the recovery rate RSD% meet the requirements, and the accuracy of the determination of gardenoside, paeoniflorin, baicalin, paeonol and glycyrrhizic acid is good.

[0149] 5.3 Reproducibility

[0150] Take 6 portions of Xuanyutongjing Decoction freeze-dried powder, prepare the test sample solution according to Example 2, and determine the content and relative standard deviation (RSD%). The results are shown in the following table.

[0151] Table 17 Reproducibility test results (content m / g)

[0152]

[0153]

[0154] The results show that according to the data requirements in the 9101 Drug Quality Standard Analysis Method Verification Guideline in the Fourth Part of the Chinese Pharmacopoeia 2020 Edition, the RSD% of the contents of gardenoside, paeoniflorin, baicalin, paeonol and glycyrrhizic acid in the test sample prepared in parallel for six times is less than 2%, which meets the requirements, indicating that the method has good reproducibility.

[0155] 5.4 Linearity and range

[0156] Take Geniposide, Paeoniflorin, Baicalin, Danpi phenol, liquorice acid control proper amount of preparation into control stock solution (equivalent to 500% control linear solution), precision transfer different volume of mother liquor in different volume of flask, add solvent dilution into control linear solution, preparation concentration see the table below. Among them, each control linear solution of different concentration was prepared, and the peak area of the control linear solution was determined according to example 2. The concentration of the control linear solution in the control linear solution was taken as the abscissa, and the peak area was taken as the ordinate, and the linear relationship between the concentration and the peak area of each control was obtained.

[0157] Table 18 linear solution preparation

[0158] Equivalent to control concentration Aliquot volume (ml) Vial volume (ml) 500％ / 50 250％ 5 10 100％ 1 5 50％ 1 10 40％ 2 25 20％ 1 25 10％ 1 50

[0159] Table 19 linear solution concentration (μg / ml)

[0160] Linear Geniposide Paeoniflorin Baicalin Glycyrrhizic acid Paeonol Linear 10% 7.75 9.90 8.75 0.68 1.46 Linear 20% 15.50 19.80 17.50 1.37 2.91 Linear 40% 31.00 39.61 34.99 2.74 5.83 Linear 50% 38.75 49.51 43.74 3.42 7.29 Linear 100% 77.50 99.02 87.48 6.84 14.57 Linear 250% 193.75 247.55 218.70 17.11 36.43 Linear 500% 387.50 495.10 437.40 34.22 72.85

[0161] Table 20 linear solution peak area

[0162] Linear Geniposide Paeoniflorin Baicalin Glycyrrhizic acid Paeonol Linear 10% 2.3123 1.4205 3.979 0.0769 1.0574 Linear 20% 4.6531 2.8816 7.9423 0.1502 2.0818 Linear 40% 9.2462 5.8506 15.75 0.2953 4.0668 Linear 50% 11.6649 7.3402 19.8371 0.371 5.0764 Linear 100% 23.1198 14.5884 39.3218 0.732 10.2778 Linear 250% 57.8136 36.7358 98.0935 1.8276 25.6551 Linear 500% 115.7504 73.6831 194.7335 3.645 51.3371

[0163] The results show that: the linear regression equation of Geniposide is y = 0.2986x + 0.0112, R2 = 1.000, the linear relationship is good; the linear regression equation of Paeoniflorin is y = 0.1489x - 0.0746, R2 = 1.000, the linear relationship is good; the linear regression equation of Baicalin is y = 0.4451x + 0.2761, R2 = 1.000, the linear relationship is good; the linear regression equation of liquorice acid is y = 0.1064x + 0.0049, R2 = 1.000, the linear relationship is good; the linear regression equation of Danpi phenol is y = 0.7047x - 0.0058, R2 = 1.000, the linear relationship is good; according to the standard curve, the linear range of Geniposide concentration is 7.75 ~ 387.50 μg / ml, the linear range of Paeoniflorin concentration is 9.90 ~ 495.10 μg / ml, the linear range of Baicalin concentration is 8.75 ~ 437.40 μg / ml, the linear range of liquorice acid concentration is 0.68 ~ 34.22 μg / ml, the linear range of Danpi phenol concentration is 1.46 ~ 72.85 μg / ml, the concentration of the control solution and the test solution used in the product is within this range.

[0164] 5.5 Durability

[0165] (1) Solution stability

[0166] Take Xuanyu Tongjing Decoction freeze-dried powder and prepare a test solution according to Example 2. Inject 10 μl of the sample at 0 h, 16 h, 20 h, 28 h, 46 h, 62 h, and 88 h after the test solution is prepared. Determine and record the chromatographic peak area. Calculate the content and relative standard deviation (RSD%).

[0167] Table 21 Solution stability test results (content mg / g)

[0168]

[0169]

[0170] The results showed that the RSDs of the contents of various multi-index components (garcinia jasminoides, paeoniflorin, baicalin, paeonol, and glycyrrhizic acid) in the stability test were all less than 2.0%, indicating that the test solution had good stability within 88 hours at room temperature.

[0171] (2) Temperature resistance

[0172] Take the lyophilized powder of Xuanyu Tongjing Decoction and prepare the test solution according to Example 2. The test solution was measured at different column temperatures; wherein the column temperatures were 25°C, 28°C, 30°C, 32°C, and 35°C, respectively. The chromatographic peak area was recorded, and the content and its relative standard deviation (RSD%) were calculated.

[0173] Table 22 Results obtained at different column temperatures

[0174] Column temperature Geniposide Paeoniflorin Baicalin Glycyrrhizic acid Paeonol 30℃ 16.080 15.692 11.778 1.368 2.130 25℃ 16.601 15.581 11.835 1.363 2.099 28℃ 16.094 15.443 11.751 1.412 2.137 32℃ 15.817 15.488 11.776 1.330 2.127 35℃ 15.852 14.942 11.755 1.387 2.158 Mean 16.089 15.429 11.779 1.372 2.130 RSD % 1.9 1.9 0.3 2.2 1.0 Standard provision ≤2 ≤2 ≤2 ≤3 ≤3

[0175] The results showed that the RSD% of the contents of various multi-index components (gardena glycoside, paeoniflorin, baicalin, paeonol, glycyrrhizic acid, etc.) of column temperature durability all met the requirements, indicating that the test solution had good detection durability at a column temperature of 25℃~35℃.

[0176] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint of Xuanyu Tongjing Decoction preparation, characterized in that: The following steps are involved: Preparation of the test solution: ultrasonically extract the test sample with 70-100% methanol aqueous solution for 15-45 minutes, filter, and collect the filtrate; Determination: Determination was performed by high performance liquid chromatography; the chromatographic conditions included: octadecylsilane bonded silica gel as the filler, CAPCELL PAK MGII, 4.6 mm × 250 mm, 5 μm, mobile phase A was acetonitrile, mobile phase B included 0.05-0.1% phosphoric acid solution, the mobile phase B also included potassium dihydrogen phosphate, and the concentration of potassium dihydrogen phosphate in the mobile phase B was 0.025-0.05 mol / L; the column temperature was 25-35°C; the flow rate was 0.9-1.1 mL / min; the injection volume was 5-10 μL; the detection wavelength was 237 nm for 0-45 min and 270 nm for 45-110 min; the gradient elution procedure included: 11% by volume of mobile phase A and 89% by volume of mobile phase B for 0-32 min; 1% by volume of mobile phase A and 1% by volume of mobile phase B for 32-45 min; 1%→15%, the volume percentage of mobile phase B is 89%→85%; 45~63min, the volume percentage of mobile phase A is 15%→30%, and the volume percentage of mobile phase B is 85%→70%; 63~75min, the volume percentage of mobile phase A is 30%, and the volume percentage of mobile phase B is 70%; 75~100min, the volume percentage of mobile phase A is 30%→75%, and the volume percentage of mobile phase B is 70%→25%; 100~103min, the volume percentage of mobile phase A is 75%→11%, and the volume percentage of mobile phase B is 25%→89%; 103~110min, the volume percentage of mobile phase A is 11%, and the volume percentage of mobile phase B is 89%.

2. The construction method according to claim 1, characterized in that The chromatographic conditions include: mobile phase B includes 0.1% phosphoric acid solution; and / or, the column temperature is 30°C; and / or, the flow rate is 1.0 mL / min; and / or, the injection volume is 10 µL; and / or, mobile phase B also includes 0.025 mol / L potassium dihydrogen phosphate.

3. The construction method according to claim 1, characterized in that The test samples are Xuanyu Tongjing Decoction freeze-dried powder, Xuanyu Tongjing Decoction dry paste powder or Xuanyu Tongjing Decoction granules.

4. The construction method according to any one of claims 1 to 3, characterized in that The ratio of the mass of the test sample to the volume of the extracted solvent is (0.3~0.5):(25~50), the unit of the mass of the test sample is g, and the unit of the volume of the extracted solvent is ml.

5. The construction method according to any one of claims 1 to 3, characterized in that It also includes the preparation of reference solution.

6. The construction method according to claim 5, characterized in that: At least one of gardenia glycoside, sinapines thiocyanate, paeoniflorin, paeoniflorin, ferulic acid, baicalin, wogonin, paeonol, glycyrrhizic acid, ligustilide and 1,2,3,4,6-pentagalloside was used as a reference substance.

7. The construction method according to any one of claims 1 to 3, characterized in that The fingerprint spectrum of the Xuanyu Tongjing Decoction preparation obtained by the construction method includes at least 13 characteristic peaks.

8. The construction method according to claim 7, characterized in that: The fingerprint of the Xuanyu Tongjing Decoction preparation at least includes a gardenoside peak, a sinapinic acid thiocyanate peak, a paeoniflorin peak, a paeoniflorin peak, a ferulic acid peak, a baicalin peak, a wogonin peak, a paeonol peak, a glycyrrhizic acid peak, a ligustilide peak, and a 1,2,3,4,6-pentagalloside peak.

9. A method for determining the content of active ingredients in a Xuanyu Tongjing Decoction preparation, characterized in that: The construction method according to any one of claims 1 to 8 is adopted.

10. The measuring method according to claim 9, characterized in that The active ingredient is at least one of geniposide, paeoniflorin, glycyrrhizic acid, baicalin and paeonol; and / or, The detection wavelengths of the assay are 237 nm and 270 nm.

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