Specific chromatogram construction method and application of Xiangbai powder compound preparation

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a specific chromatogram construction method and application of a Xiangbai powder compound preparation, and the specific chromatogram construction method comprises the following steps: (1) preparing a test solution; (2) the test solution is taken and detected by adopting a high performance liquid chromatography, octadecylsilane chemically bonded silica is taken as a filler, a mobile phase comprises acetonitrile and an aqueous solution containing phosphoric acid, a gradient elution procedure comprises 0-10 min, 12 min, 32 min, 60 min, 70 min, 90 min, 93 min, 98 min, 99 min and 105 min, and the volume percentage of acetonitrile in the mobile phase is 5%-5%-7%-25%-50%-60%-80%-5%-5%. According to the method, effective separation of 12 common characteristic peaks including the boxthorn A peak can be realized, and the obtained characteristic spectrum is stable in baseline, good in peak shape of the characteristic peaks and high in separation degree.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a characteristic spectrum construction method of a Xiebaisan compound preparation and application thereof. Background Art

[0002] Xiebaisan comes from the book "Xiaoer Yaozheng Zhijue" written by Qian Yi in the Song Dynasty. The original recipe is "Rehmannia root bark (wash and remove the soil, roast), Morus alba bark (finely grind and fry until yellow) each 1 liang, licorice (roast) 1 qian. Grind the above into powder, add a pinch of polished rice, two small cups of water, boil for seven minutes, and take before meals". It has the effects of clearing the lungs and heat, relieving cough and asthma. It is mainly used to treat lung heat cough and asthma, with symptoms of "coughing, severe shortness of breath, hot skin, especially at feeding, red tongue with yellow fur, and thin and rapid pulse". This prescription has been included in the "Catalogue of Ancient Classic Prescriptions (First Batch)" by the State Administration of Traditional Chinese Medicine. In the prescription, the mulberry bark is pungent, sweet and cold. Pungent can relieve the excess of lung qi, sweet can consolidate the deficiency of vital energy, and cold can clear lung heat, so it is the main drug; the root bark of the Chinese wolfberry is cold and sweet, and its cold nature can relieve the latent fire in the lungs, and its sweet and cold taste can relieve the deficiency heat of the liver and kidneys, cool the blood and relieve steam, so it is the assistant drug; the licorice is sweet and flat, which can benefit the spleen and harmonize the various drugs. In addition, the polished rice is sweet and flat, which can benefit the spleen and stomach, eliminate thirst, and relieve heat through urination. The two are adjuvant drugs. The efficacy of Xiebaisan is closely related to the chemical composition and pharmacological effects of its constituent medicinal materials. The mulberry bark is the dried root bark of the Morus alba L. plant of the Moraceae family. It has the effects of purging the lungs and relieving asthma, promoting diuresis and reducing swelling. It is often used to treat lung heat, asthma and cough, edema, oliguria, and facial and skin edema. Studies have shown that the main active ingredients of the mulberry bark are glycoside compounds represented by morus A. Lycium bark is the dried root bark of Lycium chinense Mill. Lycium barbarum A and Lycium barbarum B are cyclic peptide components in Lycium bark, which have inhibitory effects on renin and angiotensin I converting enzyme (ACE). Glycyrrhizic acid, also known as glycyrrhizin, is the main source of sweetness in licorice. It is an oleanane-type pentacyclic triterpenoid compound, mostly in the form of potassium or calcium salts. It has multiple pharmacological effects such as antibacterial, antiviral, and immunomodulatory. It is an important active ingredient and quality evaluation indicator component of licorice.

[0003] In the prior art, there are documents disclosing the UPLC content detection method of cerebroside and morin A in Xiebaisan granules; there are also documents disclosing the method of measuring the content of cerebroside and morin A in Xiebaisan to evaluate the quality of Xiebaisan; but there are no research reports on the characteristic spectrum in Xiebaisan granules. There is also no method for constructing a characteristic spectrum for simultaneously measuring morin A, lycium barbarum A, and ammonium glycyrrhizinate, or a method for simultaneously measuring the content of morin A, lycium barbarum B, and glycyrrhizic acid. Summary of the invention

[0004] Therefore, the object of the present invention is to provide a method for constructing a characteristic spectrum of a Xiebaisan compound preparation and its application. The method establishes its characteristic spectrum based on the main active ingredients in the Xiebaisan compound preparation, and can realize the construction of the characteristic spectrum of mulberry glycoside A, lycium barbarum A, and ammonium glycyrrhizate and the analytical determination of the contents of mulberry glycoside A, lycium barbarum B, and glycyrrhizic acid under one chromatographic condition, thereby performing comprehensive quality control on the Xiebaisan compound preparation.

[0005] To this end, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing a characteristic spectrum of a Xiebaisan compound preparation, comprising the following steps: (1) preparing a test solution; (2) taking the test solution and detecting it by high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler, a mobile phase comprising acetonitrile and an aqueous solution containing phosphoric acid, a gradient elution program comprising: 0→10min→12min→32min→60min→70min→90min→93min→98min→99min→105min, and the volume percentage of acetonitrile in the mobile phase is: 5%→5%→7%→7%→25%→50%→60%→80%→80%→5%→5%.

[0007] Optionally, step (1) includes weighing the Xiebaisan compound preparation, adding a solvent for extraction to obtain an extract, separating the solid from the liquid, and taking the liquid, which is the test sample solution.

[0008] Optionally, step (2) also satisfies at least one of the following 1) to 4): 1) the detection wavelength is 210 to 220 nm, the flow rate is 0.8 to 1.2 mL / min, and the column temperature is 23 to 30° C.; optionally, the flow rate is 0.9 to 1.1 mL / min, and the column temperature is 25 to 30° C.; further optionally, the flow rate is 1.0 mL / min, and the column temperature is 27° C.; 2) a chromatographic column with a specification of 4.6 mm×150 mm and 2.7 μm is used during the detection process; 3) the injection volume is 5 to 10 μL; 4) the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.05% to 0.2%.

[0009] Optionally, the step (1) also satisfies any one or more of the following A to F: A. The mass volume ratio of the Xiebaisan compound preparation to the solvent is 0.1 to 0.5 g: 25 to 100 mL; B. The extraction method includes ultrasonic extraction; Optionally, the power of the ultrasonic extraction is 200 W to 300 W, and the frequency is 40 kHz; C. The extraction time is 15 to 45 min; D. The solid-liquid separation is selected from centrifugation or filtration; E. The solvent includes at least one of methanol aqueous solution and ethanol aqueous solution; Optionally, the volume percentage of the methanol aqueous solution is 50% to 70%; Optionally, the volume percentage of the ethanol aqueous solution is 50% to 70%; F. The Xiebaisan compound preparation includes any one of Xiebaisan granules, Xiebaisan freeze-dried powder, Xiebaisan decoction, Xiebaisan extract, Xiebaisan oral solution, and Xiebaisan tablets.

[0010] Optionally, the construction method further comprises the step of preparing a reference solution using at least one solubilizing agent selected from mulberry glycoside A, lycium barbarum A, lycium barbarum B and ammonium glycyrrhizinate, and the step of detecting the reference solution by high performance liquid chromatography to obtain a reference spectrum according to the construction method described in any one of claims 1 to 4; optionally, the solvent used in the preparation of the reference solution comprises methanol; further optionally, the solvent used in the preparation of the reference solution is a 50% by volume methanol aqueous solution; optionally, each 1 mL of the reference solution contains 5 to 100 μg of the reference; further optionally, each 1 mL of the reference solution of mulberry glycoside A contains 80 to 100 μg of mulberry glycoside A reference, each 1 mL of the reference solution of lycium barbarum A contains 10 to 50 μg of lycium barbarum A reference, each 1 mL of the reference solution of lycium barbarum B contains 5 to 10 μg of lycium barbarum B reference, and each 1 mL of the reference solution of ammonium glycyrrhizinate contains 15 to 20 μg of ammonium glycyrrhizinate reference.

[0011] Optionally, the characteristic spectrum of the Xiebaisan compound preparation has 12 common characteristic peaks, peak 4 corresponds to the retention time of the mulberry glycoside A reference substance peak, peak 11 corresponds to the retention time of the wolfberry A reference substance peak, the peak corresponding to the mulberry glycoside A reference substance peak is the S1 peak, the peak corresponding to the wolfberry A reference substance peak is the S2 peak, the relative retention time of peaks 1 to 3 and the S1 peak is within the range of ±10% of the specified value, and the relative retention time of peaks 5 to 10 and peak 12 and the S2 peak is within the range of ±10% of the specified value; the specified values ​​are: 0.59 (peak 1), 0.67 (peak 2), 0.68 (peak 3), 0.74 (peak 5), 0.76 (peak 6), 0.77 (peak 7), 0.77 (peak 8), 0.85 (peak 9), 0.89 (peak 10), and 1.08 (peak 12).

[0012] The present invention also provides a method for determining the content of Lycium barbarum B, glycyrrhizic acid and Mulberry glycoside A in the Xiebaisan compound preparation, comprising the following steps: (1) preparing a test solution and a reference solution; optionally, the reference solution comprises Mulberry glycoside A reference solution, Lycium barbarum B reference solution and ammonium glycyrrhizic acid reference solution; (2) taking the test solution and detecting it by high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler, a mobile phase comprising acetonitrile and an aqueous solution containing phosphoric acid, and a gradient elution program comprising: 0→10min→12min→32min→ 60min→70min→90min→93min→98min→99min→105min, the volume percentage of acetonitrile in the mobile phase is: 5%→5%→7%→7%→25%→50%→60%→80%→80%→5%→5%, calculate the contents of lycium barbarum B, glycyrrhizic acid and mulberry glycoside A in the test solution; when the test solution is detected, the detection wavelength of lycium barbarum B is 217-220nm, the detection wavelength of glycyrrhizic acid is 250-254nm, and the detection wavelength of mulberry glycoside A is 320-324nm.

[0013] Optionally, step (2) also satisfies at least one of the following 1) to 4): 1) the flow rate is 0.8 to 1.2 mL / min and the column temperature is 23 to 30° C.; optionally, the flow rate is 0.9 to 1.1 mL / min and the column temperature is 25 to 30° C.; further optionally, the flow rate is 1.0 mL / min and the column temperature is 27° C.; 2) a chromatographic column with a specification of 4.6 mm×150 mm and 2.7 μm is used during the detection process; 3) the injection volume is 5 to 10 μL; 4) the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.05% to 0.2%.

[0014] Optionally, in step (1), the preparation of the test solution includes weighing the Xiebaisan compound preparation, adding a solvent for extraction to obtain an extract, separating the solid from the liquid, and taking the liquid, which is the test solution.

[0015] Optionally, in step (1), the solvent used in the preparation of the reference solution includes methanol; optionally, the solvent used in the preparation of the reference solution is a 50% by volume methanol aqueous solution; optionally, each 1 mL of the reference solution contains 5 to 100 μg of the reference substance; further optionally, each 1 mL of the reference solution of mulberry glycoside A contains 80 to 100 μg of the mulberry glycoside A reference substance, each 1 mL of the reference solution of lycium barbarum B contains 5 to 10 μg of the lycium barbarum B reference substance, and each 1 mL of the reference solution of ammonium glycyrrhizate contains 15 to 20 μg of the ammonium glycyrrhizate reference substance.

[0016] Optionally, the step (1) also satisfies any one or more of the following A to F: A. The mass volume ratio of the Xiebaisan compound preparation to the solvent is 0.1 to 0.5 g: 25 to 100 mL; B. The extraction method includes ultrasonic extraction; Optionally, the power of the ultrasonic extraction is 200 W to 300 W, and the frequency is 40 kHz; C. The extraction time is 15 to 45 min; D. The solid-liquid separation is selected from centrifugation or filtration; E. The solvent includes at least one of methanol aqueous solution and ethanol aqueous solution; Optionally, the volume percentage of the methanol aqueous solution is 50% to 70%; Optionally, the volume percentage of the ethanol aqueous solution is 50% to 70%; F. The Xiebaisan compound preparation includes any one of Xiebaisan granules, Xiebaisan freeze-dried powder, Xiebaisan decoction, Xiebaisan extract, Xiebaisan oral solution, and Xiebaisan tablets.

[0017] Typically, but not limiting, the raw materials of the Xiebaisan compound preparation of the present invention include, by weight: 6-11 parts of roasted Rehmannia root bark, 6-11 parts of stir-fried Morus alba bark, 0.6-1.1 parts of stir-fried licorice, and 4-8 parts of polished rice; optionally, the preparation steps of the Xiebaisan compound preparation include: taking the raw materials of the Xiebaisan compound preparation by weight, adding 6-4 times the total amount of raw materials in water and boiling for 1-3 times, each time for 20-80 minutes, filtering the decoction, concentrating to a relative density of 1.01-1.15 (temperature 50-70°C), drying into a dry paste powder, mixing evenly, and making the Xiebaisan compound preparation.

[0018] The present invention also provides a quality detection method for the Xiebaisan compound preparation, comprising constructing a characteristic spectrum of the Xiebaisan compound preparation to be tested according to the above-mentioned construction method and / or comprising determining the contents of lycium barbarum B, glycyrrhizic acid and morusin A in the Xiebaisan compound preparation to be tested according to the above-mentioned content determination method. Among them, typically but not limitedly, it can specifically include the step of comparing the characteristic spectrum of the Xiebaisan compound preparation to be tested with the control characteristic spectrum of the Xiebaisan compound preparation; the characteristic spectrum of the Xiebaisan compound preparation to be tested is constructed using the Xiebaisan compound preparation to be tested according to the above-mentioned construction method, and the control characteristic spectrum of the Xiebaisan compound preparation is selected from any one of the following (1) to (3): (1) it has 12 common characteristic peaks, peak 4 corresponds to the retention time of the mulberry glycoside A reference substance peak, peak 11 corresponds to the retention time of the wolfberry A reference substance peak, the peak corresponding to the mulberry glycoside A reference substance peak is the S1 peak, the peak corresponding to the wolfberry A reference substance peak is the S2 peak, and the relative retention times of peaks 1 to 3 and S1 peak are The retention time is within the range of ±10% of the specified value, and the relative retention time of peaks 5 to 10, peak 12 and S2 peak is within the range of ±10% of the specified value; the specified values ​​are: 0.59 (peak 1), 0.67 (peak 2), 0.68 (peak 3), 0.74 (peak 5), 0.76 (peak 6), 0.77 (peak 7), 0.77 (peak 8), 0.85 (peak 9), 0.89 (peak 10), 1.08 (peak 12); (2) using a single batch or multiple batches of Xiebaisan compound preparations to obtain the Xiebaisan compound preparation characteristic spectrum according to the above construction method; (3) using multiple batches of Xiebaisan compound preparations to obtain the characteristic spectrum according to the above construction method to prepare a control characteristic spectrum by using the average value or median method.

[0019] The technical solution of the present invention has the following advantages:

[0020] 1. The characteristic spectrum construction method of the Xiebaisan compound preparation provided by the present invention uses octadecylsilane bonded silica gel as a filler, and the mobile phase includes acetonitrile and an aqueous solution containing phosphoric acid. Through a specific gradient elution program, 12 common characteristic peaks are obtained, wherein the separation of common characteristic peaks including mulberry glycoside A peak and wolfberry element A peak is achieved, and the elution procedure is simple, the obtained characteristic spectrum has a stable baseline, good characteristic peak shape, and high separation, which can provide a basis for the quality detection and control of the Xiebaisan compound preparation. The peak positions of mulberry glycoside A, wolfberry element A, wolfberry element B and ammonium glycyrrhizinate can also be accurately located, fully reflecting the integrity and characteristic of the Xiebaisan compound preparation.

[0021] 2. The characteristic spectrum construction method of the Xiebaisan compound preparation provided by the present invention is simple to prepare and process the test sample, the chromatographic conditions are easy to achieve, the operation is simple, the results are accurate, and the method has good reproducibility.

[0022] 3. The quality detection method of the Xiebaisan compound preparation provided by the present invention can comprehensively, clearly and effectively detect the quality of the Xiebaisan compound preparation by comparing the characteristic spectrum of the Xiebaisan compound preparation to be tested with the control characteristic spectrum of the Xiebaisan compound preparation.

[0023] 4. The method for determining the contents of lycium barbarum B, glycyrrhizic acid, and mulberry glycoside A in the Xiebaisan compound preparation provided by the present invention can be used to determine the contents of lycium barbarum B, glycyrrhizic acid, and mulberry glycoside A in the Xiebaisan compound preparation under the same chromatographic conditions as those for constructing the characteristic spectrum, thereby further achieving quality control of the Xiebaisan compound preparation and improving inspection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 is the chromatogram of the test solution in Example 1;

[0026] Figure 2 is a chromatogram of the mulberry glycoside A reference substance solution in Example 1;

[0027] Figure 3 is the chromatogram of the Lycium barbarum A reference solution in Example 1;

[0028] Figure 4 is the chromatogram of the test solution at a detection wavelength of 218 nm in Example 2;

[0029] Figure 5 is the chromatogram of the test solution at a detection wavelength of 252 nm in Example 2;

[0030] Figure 6 It is the chromatogram of the test solution at a detection wavelength of 324 nm in Example 2;

[0031] Figure 7 is a chromatogram of a mixed reference solution of Lycium barbarum B and ammonium glycyrrhizinate at a detection wavelength of 218 nm in Example 2;

[0032] Figure 8 is a chromatogram of a mixed reference solution of Lycium barbarum B and ammonium glycyrrhizinate at a detection wavelength of 252 nm in Example 2;

[0033] Fig. 9 is a chromatogram of the reference solution of Mulberiside A at a detection wavelength of 324 nm in Example 2;

[0034] Fig.10 This is a comparison chart of mulberry glycoside A at different flow rates in the flow rate investigation of Experimental Example 1, from top to bottom, blank solvent at a flow rate of 1.0 ml / min, reference solution of mulberry glycoside A, test solution, test solution at a flow rate of 0.8 ml / min, and test solution at a flow rate of 1.2 ml / min;

[0035] Fig.11 This is a comparison chart of Lycium barbarum B at different flow rates in the flow rate investigation of Experimental Example 1, from top to bottom, blank solvent at a flow rate of 1.0 ml / min, Lycium barbarum B, mixed reference solution of ammonium glycyrrhizinate, test solution, 0.8 ml / min test solution, and 1.2 ml / min test solution;

[0036] Fig.12 This is a comparison chart of ammonium glycyrrhizinate at different flow rates in the flow rate investigation of Experimental Example 1, from top to bottom, blank solvent at a flow rate of 1.0 ml / min, Lycium barbarum B, mixed reference solution of ammonium glycyrrhizinate, test solution, test solution at a flow rate of 0.8 ml / min, and test solution at a flow rate of 1.2 ml / min;

[0037] Fig.13 This is a comparison chart of mulberry glycoside A at different column temperatures in the column temperature investigation of Experimental Example 1, from top to bottom, blank solvent at a column temperature of 25°C, reference solution of mulberry glycoside A, and test solution, blank solvent at a column temperature of 23°C, reference solution of mulberry glycoside A, and test solution, blank solvent at a column temperature of 27°C, reference solution of mulberry glycoside A, and test solution;

[0038] Fig.14 This is a comparison chart of Lycium barbarum B at different column temperatures in the column temperature investigation of Experimental Example 1, from top to bottom, blank solvent at a column temperature of 25°C, Lycium barbarum B, mixed reference solution of ammonium glycyrrhizic acid, and test solution, blank solvent at a column temperature of 23°C, Lycium barbarum B, mixed reference solution of ammonium glycyrrhizic acid, and test solution, blank solvent at a column temperature of 27°C, Lycium barbarum B, mixed reference solution of ammonium glycyrrhizic acid, and test solution;

[0039] Fig.15 This is a comparison chart of ammonium glycyrrhizic acid at different column temperatures in the column temperature investigation of Experimental Example 1, from top to bottom, blank solvent at a column temperature of 25°C, Lycium barbarum B, mixed reference solution of ammonium glycyrrhizic acid, and test solution; blank solvent at a column temperature of 23°C, Lycium barbarum B, mixed reference solution of ammonium glycyrrhizic acid, and test solution; blank solvent at a column temperature of 27°C, Lycium barbarum B, mixed reference solution of ammonium glycyrrhizic acid, and test solution;

[0040] Fig.16The chromatograms of the blank solvent (50% methanol), the test solution, the mulberry glycoside A reference solution, and the wolfberry extract A reference solution obtained in the specificity experiment of Experimental Example 2;

[0041] Fig.17 This is the chromatogram obtained at a detection wavelength of 218 nm in the specificity verification of Experimental Example 3, wherein from bottom to top are blank solvent, negative reference solution of lycium barbarum negative granules, test solution, and mixed reference solution of lycium barbarum B and ammonium glycyrrhizinate;

[0042] Fig.18 This is the chromatogram obtained at a detection wavelength of 252 nm in the specificity verification of Experimental Example 3, wherein from bottom to top are blank solvent, negative granule reference solution lacking licorice, test solution, and mixed reference solution of Lycium barbarum B and ammonium glycyrrhizinate;

[0043] Fig.19 This is the chromatogram obtained at a detection wavelength of 324 nm in the specificity verification of Experimental Example 3, wherein from bottom to top are blank solvent, negative granule reference solution lacking Morus alba bark, test solution, and Morus alba glycoside A reference solution;

[0044] Fig. 20 This is an image of regression analysis of the concentration and peak area of ​​Lycium barbarum B in the linearity and range verification of Experimental Example 3;

[0045] Fig.21 This is an image of regression analysis of glycyrrhizic acid concentration and peak area in the linearity and range verification of Experimental Example 3;

[0046] Fig. 22 This is an image of regression analysis of the concentration and peak area of ​​mulberry glycoside A in the linearity and range verification of Experimental Example 3. DETAILED DESCRIPTION

[0047] The following examples are provided to further understand the present invention better, are not limited to the best mode, and are not intended to limit the content and scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the enlightenment of the present invention or by combining the features of the present invention with other prior arts, all falls within the protection scope of the present invention. If no specific experimental steps or conditions are indicated in the examples, the operation or conditions of the conventional experimental steps described in the literature in this area can be carried out. If the manufacturer is not indicated for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0048] The preparation method of Xiebaisan granules used in the present invention is as follows: weigh the raw materials, 8.86g of roasted Rehmannia root bark, 8.86g of stir-fried Morus alba bark, 0.89g of stir-fried licorice, and 6.00g of polished rice; add 14 times the weight of water to all the raw materials, boil for 60 minutes, filter the decoction and set aside, then add 12 times the weight of the raw materials, boil for 40 minutes, filter the decoction, mix the two decoctions, concentrate to a relative density of 1.05 (60°C), dry to make a dry paste powder, mix well, and make Xiebaisan granules.

[0049] The preparation method of the negative granule reference substance lacking white mulberry bark used in the present invention is as follows: weigh the raw materials, 8.86g of roasted ground bone bark, 0.89g of fried licorice, and 6.00g of polished rice; add 14 times the weight of water to all the raw materials, boil for 60 minutes, filter the decoction, and set aside, then add 12 times the weight of water to the raw materials, boil for 40 minutes, filter the decoction, mix the two decoctions, concentrate to a relative density of 1.05 (60°C), dry to make a dry paste powder, mix evenly, and prepare the negative granule reference substance lacking white mulberry bark.

[0050] The preparation method of the negative granule reference substance of the Radix Glehniae used in the present invention is as follows: weigh the raw materials, stir-fry 8.86g of white mulberry bark, 0.89g of stir-fry licorice, and 6.00g of polished rice; add 14 times the weight of water to all the raw materials, boil for 60 minutes, filter the decoction, and set aside, then add 12 times the weight of water to the raw materials, boil for 40 minutes, filter the decoction, mix the two decoctions, concentrate to a relative density of 1.05 (60°C), dry to make a dry paste powder, mix well, and prepare the negative granule reference substance of the Radix Glehniae.

[0051] The preparation method of the negative granule reference substance lacking licorice used in the present invention is as follows: weighing raw materials, roasting 8.86g of Radix Rehmanniae, stir-frying 8.86g of white mulberry bark, and 6.00g of polished rice; adding 14 times of water by weight of all the raw materials, decocting for 60 minutes, filtering the decoction, and setting aside, then adding 12 times of water by weight of the raw materials, decocting for 40 minutes, filtering the decoction, mixing the two decoctions, concentrating to a relative density of 1.05 (60° C.), drying to make a dry paste powder, and mixing evenly to prepare the negative granule reference substance lacking licorice.

[0052] Example 1

[0053] This embodiment provides a method for constructing a characteristic spectrum of Xiebaisan granules, comprising the following steps:

[0054] Preparation of test solution: Take about 0.1g of Xiebaisan granules, weigh accurately, add 50ml of 50% methanol accurately, seal, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30min, take out, cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate.

[0055] Preparation of reference substance solution: Take an appropriate amount of mulberry glycoside A reference substance, weigh it accurately, and add 50% methanol to prepare a reference substance solution containing 80μg of mulberry glycoside A per 1ml; take an appropriate amount of wolfberry extract A reference substance, weigh it accurately, and add 50% methanol to prepare a reference substance solution containing 10μg of wolfberry extract A per 1ml.

[0056] Determination: Take 5 μl of the test solution and the reference solution of the above-mentioned Xiebaisan granules and inject them into an ultra performance liquid chromatograph. The chromatographic conditions are as follows: Agilent Poroshell 120SB-C18 (4.6×150 mm, 2.7 μm) is used as the chromatographic column; acetonitrile is used as the mobile phase A, and 0.1% phosphoric acid solution is used as the mobile phase B, and gradient elution is performed according to the provisions in Table 1; the flow rate is 1.0 ml / min; the column temperature is 27°C; the detection wavelength is 218 nm; the number of theoretical plates calculated based on the mulberry glycoside A peak should not be less than 3000.

[0057] A total of 3 batches of Xiebaisan granules were tested (numbered 240401, 240402, and 240403, respectively). Table 2 shows the relative retention times of the three batches of Xiebaisan granules, with mulberry glycoside A as the S1 peak, Lycium barbarum A as the S2 peak, and the retention times of peaks 1 to 3 calculated with S1 as the reference peak, and the retention times of peaks 5 to 10 and 12 calculated with S2 as the reference peak. Table 3 shows the retention time of the reference solution of the control substance. The chromatogram of the Xiebaisan granule test solution is shown in Figure 1 (From bottom to top, they are test solution 240401, test solution 240402, and test solution 240403). The chromatogram of the mulberry glycoside A reference solution is shown in Figure 2 The chromatogram of Lycium barbarum A reference solution is shown in Figure 3 .

[0058] Table 1

[0059]

[0060] Table 2

[0061]

[0062]

[0063] Table 3

[0064]

[0065] Example 2

[0066] This embodiment provides a method for determining the contents of Lycium barbarum B, glycyrrhizic acid and morusin A in Xiebaisan granules, comprising the following steps:

[0067] The same test solution as in Example 1 was used.

[0068] The same reference solution of mulberry glycoside A as in Example 1 was used. Appropriate amounts of Lycium barbarum B and ammonium glycyrrhizinate reference substances were taken separately, accurately weighed, and 50% methanol was added to prepare a mixed reference solution containing 5 μg of Lycium barbarum B and 15 μg of ammonium glycyrrhizinate per 1 ml.

[0069] The only difference between the determination method and that in Example 1 is that the detection wavelength of the mulberry glycoside A reference solution is 324 nm, and the detection wavelengths of the mixed reference solution are 218 nm (lycium barbarum B) and 252 nm (glycyrrhizic acid). The content of mulberry glycoside A in the test solution is calculated by the peak area of ​​the mulberry glycoside A peak in the chromatogram of the test solution at 324 nm. The content of lycium barbarum B in the test solution is calculated by the peak area of ​​the lycium barbarum B peak in the chromatogram of the test solution at 218 nm. The content of glycyrrhizic acid in the test solution is calculated by the peak area of ​​the ammonium glycyrrhizate peak in the chromatogram of the test solution at 252 nm, wherein the concentration of ammonium glycyrrhizate is converted to glycyrrhizic acid (weight of glycyrrhizic acid = weight of ammonium glycyrrhizic acid / 1.0207).

[0070] A total of 3 batches of Xiebaisan granules (numbered 240401, 240402, and 240403, respectively) were tested. Table 4 shows the results of the determination of the content of Lycium barbarum B, glycyrrhizic acid, and Morusin A in the 3 batches of Xiebaisan granules. The chromatogram of the Xiebaisan granule test solution at a detection wavelength of 218 nm is shown in Table 4. Figure 4 The chromatogram of the Xiebaisan granules test solution at a detection wavelength of 252 nm is shown in Figure 5 The chromatogram of the Xiebaisan granules test solution at a detection wavelength of 324 nm is shown in Figure 6 , Figures 4 to 6 From bottom to top, they are test solution 240401, test solution 240402, and test solution 240403; the detection wavelength is 218nm, and the mixed reference solution of Lycium barbarum B and ammonium glycyrrhizinate is shown in Figure 7 , the detection wavelength of 252nm Lycium barbarum B and ammonium glycyrrhizinate mixed reference solution is shown Figure 8 The reference solution of mulberry glycoside A at a detection wavelength of 324 nm is shown in Fig. 9 .

[0071] Table 4

[0072]

[0073] Experimental Example 1 Investigation of Chromatographic Conditions

[0074] Since the content determination method of Lycium barbarum B, glycyrrhizic acid and morusin A in the Xiebaisan compound preparation of the present invention and the characteristic spectrum construction method of the Xiebaisan compound preparation share the same chromatographic conditions, and the content determination method has higher requirements on the chromatographic conditions than the characteristic spectrum construction method, only the chromatographic conditions of the content determination method are investigated, and the results obtained from the investigation can also be applied to the characteristic spectrum construction method at the same time.

[0075] 1. Investigation of flow rate

[0076] Take Xiebaisan granules (batch number: 240401), and prepare the test solution in the same way as in Example 2; prepare the reference solution of mulberry glycoside A, and the mixed reference solution of lycium barbarum B and ammonium glycyrrhizic acid in the same way as in Example 2; use 50% methanol as the blank solvent. Compare the effects of different flow rates (0.8ml / min, 1.0ml / min, 1.2ml / min) on the content determination of lycium barbarum B, glycyrrhizic acid and mulberry glycoside A in the Xiebaisan compound preparation. The other chromatographic conditions are the same as in Example 2, that is, the mulberry glycoside A reference solution, blank solvent, and test solution at different flow rates are detected at a detection wavelength of 324nm, the mixed reference solution of lycium barbarum B and ammonium glycyrrhizic acid, blank solvent, and test solution at different flow rates are detected at a detection wavelength of 252nm, and the mixed reference solution of lycium barbarum B and ammonium glycyrrhizic acid, blank solvent, and test solution at different flow rates are detected at a detection wavelength of 218nm. The results are shown in Table 2. Figures 10-12 and Tables 5 to 7. The content of the test samples was determined using different flow rates. The signal-to-noise ratio of the chromatographic peak of mulberry glycoside A was greater than 10, and the number of theoretical plates was greater than 4000, indicating that the system adaptability was good, and the separation degree was greater than 1.5 at different flow rates; the signal-to-noise ratio of the chromatographic peak of Lycium barbarum B was greater than 10, and the number of theoretical plates was greater than 4000, indicating that the system adaptability was good. When the flow rate was 1.2ml / min, the peak area of ​​the chromatographic peak of Lycium barbarum B in the test sample was only 79.477, and the peak area of ​​the chromatographic peak at other flow rates was higher, all greater than 100; the signal-to-noise ratio of the chromatographic peak of ammonium glycyrrhizinate was greater than 10, and the number of theoretical plates was greater than 4000, indicating that the system adaptability was good. When the flow rate was 1.2ml / min, the separation degree of the chromatographic peak of ammonium glycyrrhizinate in the reference sample and the test sample was less than 1.5, and the separation degree of the chromatographic peak at other flow rates was greater than 1.5. Considering comprehensively, the preferred flow rate is 1.0ml / min.

[0077] Table 5 Results of investigation of different flow rates of Mulberry glycoside A

[0078]

[0079] Table 6 Results of investigation of different flow rates of Lycium barbarum B

[0080]

[0081] Table 7 Glycyrrhizic acid different flow rate investigation results

[0082]

[0083] 2. Investigation of column temperature

[0084] Take Xiebaisan granules (batch number: 240401), and prepare a test solution in the same manner as in Example 2; prepare a reference solution of mulberry glycoside A, and a mixed reference solution of lycium barbarum B and ammonium glycyrrhizic acid in the same manner as in Example 2; use 50% methanol as a blank solvent. Compare the effects of different column temperatures (23°C, 25°C, 27°C) on the characteristic spectrum of Xiebaisan. The remaining chromatographic conditions are the same as in Example 2, i.e., the mulberry glycoside A reference solution, blank solvent, and test solutions at different column temperatures are detected at a detection wavelength of 324nm; the mixed reference solution of lycium barbarum B and ammonium glycyrrhizic acid, blank solvent, and test solutions at different column temperatures are detected at a detection wavelength of 252nm; the mixed reference solution of lycium barbarum B and ammonium glycyrrhizic acid, blank solvent, and test solutions at different column temperatures are detected at a detection wavelength of 218nm. The results are shown in Table 2. Figures 13-15 and Tables 8 to 10. The content of the test samples was determined using different column temperatures. The signal-to-noise ratio of the chromatographic peak of mulberry glycoside A was greater than 10, and the number of theoretical plates was greater than 4000, indicating that the system adaptability was good, and the separation degree was greater than 1.5 at the column temperature, but the peak purity was less than 999 when the column temperature was 23°C; the signal-to-noise ratio of the chromatographic peak of lycium barbarum B was greater than 10, and the number of theoretical plates was greater than 4000, indicating that the system adaptability of the method was good at different column temperatures, and the peak purity was greater than 999 at different column temperatures; the signal-to-noise ratio of the chromatographic peak of ammonium glycyrrhizinate was greater than 10, and the number of theoretical plates was greater than 4000, indicating that the system adaptability of the method was good at different column temperatures, and the separation degree was greater than 1.5 at different column temperatures, but the peak purity was less than 999 when the column temperature was 23°C. Considering the above, the preferred column temperature is 27°C.

[0085] Table 8 Results of investigation of mulberry glycoside A at different column temperatures

[0086]

[0087] Table 9 Results of investigation of Lycium barbarum B at different column temperatures

[0088]

[0089] Table 10 Glycyrrhizic acid different column temperature investigation results

[0090]

[0091] Experimental Example 2 Methodological Verification of Characteristic Spectra

[0092] 1. Specificity experiment: Take the test solution of Xiebaisan granules (batch number: 240401), blank solvent (50% methanol), mulberry glycoside A reference solution, and wolfberry element A reference solution, and inject them for detection under the chromatographic conditions of Example 1. Fig.16 The results showed that the blank solvent did not emit peaks at the corresponding positions of the 12 common peaks in the test sample chromatogram, and chromatographic peaks appeared in the test sample chromatogram and the corresponding positions in the reference solution chromatogram, and the UV absorption spectra were basically consistent, indicating that this method has good specificity.

[0093] 2. Precision experiment: Take the test solution of Xiebaisan granules (batch number: 240401) and inject it continuously for 6 times according to the chromatographic conditions of Example 1. Calculate the RSD value of the relative retention time of each characteristic peak and the corresponding S1 peak and S2 peak. The results show that the RSD range of the relative retention time of each characteristic peak is between 0.0% and 0.1%, which is less than 2.0%, and is within the range of mean ±10%, indicating that the characteristic spectrum method has good precision.

[0094] 3. Method repeatability test: Prepare Xiebaisan granules (batch number: 240401) test solution by referring to the test solution preparation method in Example 1, and make 6 parallel samples. Samples were injected and measured according to the chromatographic conditions of Example 1, and the RSD values ​​of the relative retention times of each characteristic peak and the corresponding S1 peak and S2 peak were calculated. The results showed that the RSDs of the relative retention times of the characteristic peaks of the 6 test solutions were between 0.0% and 0.1%, less than 2.0%, and all within the range of mean ±10%. The characteristic spectrum method has good repeatability.

[0095] 4. Intermediate precision: Referring to the method and conditions of Example 1, the influence of different personnel and different instruments on the precision of the same batch of Xiebaisan granules (batch number: 240401) was investigated, and the relative retention time of each characteristic peak and the corresponding S1 peak and S2 peak was calculated, and its RSD was used as the evaluation index. The results showed that the RSD of the relative retention time of each characteristic peak measured by different personnel was within the range of 0.0% to 0.1%; the RSD of the relative retention time of each characteristic peak of different instruments was within the range of 0.0% to 1.1%, indicating that the intermediate precision of the characteristic spectrum was good.

[0096] 5. Stability: The test solution of Xiebai Granules (batch number: 240401) was prepared by referring to the preparation method of the test solution in Example 1. According to the chromatographic conditions of Example 1, samples were injected and tested at time points of 0h, 2h, 4h, 8h, 12h, 24h, 32h, 48h, 56h, and 72h, respectively. The relative retention time of each characteristic peak and the corresponding S1 peak and S2 peak was calculated, and its RSD was used as the evaluation index. The results showed that the RSD of the relative retention time of each characteristic peak within 0h to 72h was in the range of 0.0% to 0.3%, which was less than 2%, indicating that the test solution had good stability within 72 hours.

[0097] 6. Durability investigation at different flow rates: Take the test solution of Xiebaisan granules (batch number: 240401), and inject and detect at different flow rates (0.9ml / min, 1.0ml / min, 1.1ml / min) under the chromatographic conditions of Example 1. The relative retention time of each target peak is calculated with the corresponding S1 peak and S2 peak as reference peaks. The results show that the RSD range of the relative retention time of each characteristic peak in the test sample measured at different flow rates is between 0.0% and 1.8%, all of which are less than 2%, indicating that the characteristic spectrum method has good durability.

[0098] Experimental Example 3 Methodological Verification of the Determination Method of Lycium B, Glycyrrhizic Acid and Morin A in Xiebaisan Compound Preparation

[0099] 1. Specificity verification: Prepare the negative granule solution lacking mulberry bark, the negative granule solution lacking radix lycopersicum, and the negative granule solution lacking licorice in the same manner as the test solution preparation method in Example 1. Take the test solution of Xiebai Granule (batch number 240401), and inject and test with blank solvent (50% methanol), mulberry glycoside A reference solution, mixed reference solution of lycium barbarum B and ammonium glycyrrhizinate, negative granule solution lacking mulberry bark, negative granule solution lacking radix lycopersicum, and negative granule solution lacking licorice under the chromatographic conditions of Example 2. The test solution, blank solvent, mixed reference solution of Lycium barbarum B and ammonium glycyrrhizinate, and negative reference solution of Radix Glycyrrhizae Lack were detected at a wavelength of 218 nm; the test solution, blank solvent, mixed reference solution of Lycium barbarum B and ammonium glycyrrhizinate, and negative reference solution of Radix Glycyrrhizae Lack were detected at a wavelength of 252 nm; the test solution, blank solvent, Morusin A reference solution, and negative reference solution of Morus alba Lack were detected at a wavelength of 324 nm; the chromatograms were recorded, see Figures 17 to 19 The results showed that chromatographic peaks appeared in the chromatogram of the test sample and the reference solution at the corresponding positions, and the UV absorption spectra were basically consistent. The blank solvent did not produce peaks at the corresponding positions of the test sample chromatogram, and the blank had no interference; the negative granules lacking mulberry bark had no interference in the determination of mulberry glycoside A, the negative granules lacking radix lycii had no interference in the determination of lycium barbarum B, and the negative granules lacking licorice had no interference in the determination of ammonium glycyrrhizinate, indicating that the content determination method has good specificity.

[0100] 2. Linearity and range verification

[0101] (1) Lycium barbarum B reference substance solutions with concentrations of 0.25 μg / mL, 0.62 μg / mL, 1.24 μg / mL, 2.48 μg / mL, 4.95 μg / mL, 6.19 μg / mL and 12.38 μg / mL (corresponding to the linear numbers -1 to 7) were injected respectively, and the results were measured at 218 nm under the conditions of Example 2. The corresponding peak areas at each concentration are shown in Table 11; the concentration X of the Lycium barbarum B reference substance was regressed using the Lycium barbarum B peak area Y, as shown in Table 11. Fig. 20 The regression equation is: Y=36508X435.9, and the correlation coefficient is r=1. The results show that within the range of 0.25-12.38 μg / mL, the concentration X (μg / mL) and the peak area Y value have a good linear relationship.

[0102] (2) The ammonium glycyrrhizinate reference substance solutions with concentrations of 0.65 μg / mL, 1.62 μg / mL, 3.24 μg / mL, 6.48 μg / mL, 12.95 μg / mL, 16.19 μg / mL and 32.38 μg / mL (corresponding to the linear numbers -1 to 7) were injected respectively, and the results were measured at 252 nm under the conditions of Example 2. The corresponding peak areas at each concentration are shown in Table 11; the ammonium glycyrrhizinate concentration was converted to glycyrrhizic acid (weight of glycyrrhizic acid = weight of ammonium glycyrrhizic acid / 1.0207), and the peak area Y was used for regression analysis on the concentration X of the reference substance, as shown in Table 11. Fig.21 The regression equation is: Y=8541.3X844.49, and the correlation coefficient is r=1. The results show that in the range of 0.63-31.72 μg / mL, the concentration X (μg / mL) and the peak area Y value of glycyrrhizic acid have a good linear relationship.

[0103] (3) Samples of mulberry glycoside A reference substance solutions with concentrations of 1.97 μg / mL, 4.92 μg / mL, 9.83 μg / mL, 19.67 μg / mL, 39.33 μg / mL, 49.16 μg / mL and 98.33 μg / mL (corresponding to the linear numbers -1 to 7) were injected respectively, and the measurement was performed at 324 nm under the conditions of Example 2. The corresponding peak area at each concentration is shown in Table 11; the mulberry glycoside A peak area Y was used to perform regression analysis on the concentration X of the mulberry glycoside A reference substance, as shown in Table 11. Fig. 22 The regression equation is: Y=26506X 1236.2, and the correlation coefficient is r=1. The results show that in the range of 1.97-98.33 μg / mL, the concentration X (μg / mL) and the peak area Y value of mulberry glycoside A have a good linear relationship.

[0104] Table 11

[0105]

[0106] 3. Precision

[0107] (1) Instrument precision: The test solution of Xiebaisan granules (batch number: 240401) was prepared by referring to the test solution preparation method of Example 2. Under the chromatographic conditions of Example 2, the injection and determination were carried out in parallel for 6 times. The retention time and peak area RSD values ​​of the 6 determinations were calculated. The results showed that the retention time RSD of Lycium barbarum B was 0.0%, and the peak area RSD was 0.2%; the retention time RSD of glycyrrhizic acid was 0.0%, and the peak area RSD was 0.5%; the retention time RSD of mulberry glycoside A was 0.1%, and the peak area RSD was 0.2%. The RSDs were all less than 2%, indicating good instrument precision.

[0108] (2) Repeatability: The test solution of Xiebaisan granules (batch number: 240401) was prepared by referring to the preparation method of the test solution in Example 2. Six samples were injected and measured under the chromatographic conditions of Example 2. The content and RSD values ​​of the six samples were calculated. The results showed that the RSD of lycium barbarum B was 0.3%; the RSD of glycyrrhizic acid was 0.5%; and the RSD of mulberry glycoside A was 0.4%. The RSDs were all less than 2%, indicating that the content determination method had good repeatability.

[0109] (3) Intermediate precision: Referring to the method and conditions of Example 2, the effects of different personnel and different instruments on the precision of the same batch of Xiebaisan granules (batch number 240401) were investigated. The results showed that the RSDs of the content of Lycium B measured by different personnel were 0.2%, the RSD of glycyrrhizic acid was 0.4%, and the RSD of mulberry glycoside A was 0.1%, which were less than 2%, indicating that the intermediate precision of the content determination method by different personnel was good; the RSDs of the content of Lycium B measured by different instruments were 0.6%, the RSD of glycyrrhizic acid was 0.4%, and the RSD of mulberry glycoside A was 0.3%, and the RSDs were all less than 2%, indicating that the intermediate precision of the content determination method by different instruments was good.

[0110] 4. Accuracy: Take 6 portions of Xiebaisan granules (batch number 240401), each portion is about 0.05g, add half the content of the sample index component content of lycium barbarum B, ammonium glycyrrhizate and mulberry glycoside A reference substances, prepare the test solution with reference to Example 2, and inject and measure according to the determined chromatographic conditions, and calculate the recovery rate. The results show that the recovery rate of lycium barbarum B ranges from 93.9% to 97.4%, and the RSD is 0.9%; the recovery rate of glycyrrhizic acid ranges from 99.8% to 104.0%, and the RSD is 1.2%; the recovery rate of mulberry glycoside A ranges from 100.4% to 101.8%, and the RSD is 0.5%, and the RSDs are all less than 2%, indicating that the content determination method has good accuracy.

[0111] 5. Stability: Prepare the test solution of Xiebai Granules (Batch No. 240401) with reference to Example 2. Under the chromatographic conditions of Example 2, samples were injected and tested at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, and 72 h, respectively. The peak area of ​​each component to be tested was recorded and the RSD value was calculated. The results showed that the RSD of the peak area of ​​lycium barbarum B within 0 to 72 h was 0.66%, the RSD of the peak area of ​​glycyrrhizic acid was 0.53%, and the RSD of the peak area of ​​mulberry glycoside A was 0.22%. The RSDs were all less than 2%, indicating that the test solution had good stability within 72 hours.

[0112] 6. Durability: The test solution of Xiebaisan granules was taken and sampled at different flow rates (0.9 ml / min, 1.0 ml / min, 1.1 ml / min) according to the chromatographic conditions of Example 2. The RSD of the lycium barbarum B content was 0.9%, the RSD of the glycyrrhizic acid content was 1.3%, and the RSD of the mulberry glycoside A content was 1.0%. The RSDs were all less than 2%, indicating that the content determination method has good durability.

[0113] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of a Xiebaisan compound preparation, characterized in that: The steps include: (1) Preparation of test solution; (2) The sample solution was detected by high performance liquid chromatography, using octadecylsilane bonded silica gel as the filler, the mobile phase comprising acetonitrile and an aqueous solution containing phosphoric acid, the gradient elution program comprising: 0→10 min→12 min→32 min→60 min→70 min→90 min→93 min→98 min→99 min→105 min, and the volume percentage of acetonitrile in the mobile phase was: 5%→5%→7%→7%→25%→50%→60%→80%→80%→5%→5%.

2. The construction method according to claim 1, characterized in that: Step (1) includes weighing the Xiebaisan compound preparation, adding a solvent for extraction to obtain an extract, separating the solid from the liquid, and taking the liquid, which is the test solution; And / or, step (2) further satisfies at least one of the following 1) to 4): 1) The detection wavelength is 210-220 nm, the flow rate is 0.8-1.2 mL / min, and the column temperature is 23-30° C.; optionally, the flow rate is 0.9-1.1 mL / min, and the column temperature is 25-30° C.; further optionally, the flow rate is 1.0 mL / min, and the column temperature is 27° C.; 2) During the detection process, a chromatographic column with a specification of 4.6 mm × 150 mm and a 2.7 μm was used; 3) The injection volume is 5-10 μL; 4) The volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.05% to 0.2%.

3. The construction method according to claim 2, characterized in that: The step (1) also satisfies any one or more of the following A to F: A. The mass volume ratio of Xiebaisan compound preparation to solvent is 0.1-0.5g:25-100mL; B. Extraction method includes ultrasonic extraction; optionally, the power of the ultrasonic extraction is 200W to 300W and the frequency is 40kHz; C. Extraction time is 15 to 45 minutes; D. The solid-liquid separation is selected from centrifugation or filtration; E. The solvent includes at least one of a methanol aqueous solution and an ethanol aqueous solution; optionally, the volume percentage of the methanol aqueous solution is 50% to 70%; optionally, the volume percentage of the ethanol aqueous solution is 50% to 70%; F. The compound preparation of Xiebaisan includes any one of Xiebaisan granules, Xiebaisan freeze-dried powder, Xiebaisan decoction, Xiebaisan extract, Xiebaisan oral liquid and Xiebaisan tablets.

4. The construction method according to any one of claims 1 to 3, characterized in that: The construction method also includes the step of preparing a reference solution using at least one solubilizing agent selected from mulberry glycoside A, lycium barbarum A, lycium barbarum B, and ammonium glycyrrhizinate, and the step of detecting the reference solution by high performance liquid chromatography to obtain a reference spectrum according to the construction method described in any one of claims 1 to 4; optionally, the solvent used in the preparation of the reference solution includes methanol; further optionally, the solvent used in the preparation of the reference solution is a 50% by volume methanol aqueous solution; optionally, each 1 mL of the reference solution contains 5 to 100 μg of the reference; further optionally, each 1 mL of the reference solution of mulberry glycoside A contains 80 to 100 μg of mulberry glycoside A reference, each 1 mL of the reference solution of lycium barbarum A contains 10 to 50 μg of lycium barbarum A reference, each 1 mL of the reference solution of lycium barbarum B contains 5 to 10 μg of lycium barbarum B reference, and each 1 mL of the reference solution of ammonium glycyrrhizinate contains 15 to 20 μg of ammonium glycyrrhizinate reference.

5. The construction method according to any one of claims 1 to 4, characterized in that: The characteristic spectrum of the Xiebaisan compound preparation has 12 common characteristic peaks, peak 4 corresponds to the retention time of the mulberry glycoside A reference substance peak, peak 11 corresponds to the retention time of the wolfberry A reference substance peak, the peak corresponding to the mulberry glycoside A reference substance peak is the S1 peak, the peak corresponding to the wolfberry A reference substance peak is the S2 peak, the relative retention time of peaks 1 to 3 and the S1 peak is within the range of ±10% of the specified value, and the relative retention time of peaks 5 to 10 and peak 12 and the S2 peak is within the range of ±10% of the specified value; the specified value is: 0.59 (peak 1), 0.67 (peak 2), 0.68 (peak 3), 0.74 (peak 5), 0.76 (peak 6), 0.77 (peak 7), 0.77 (peak 8), 0.85 (peak 9), 0.89 (peak 10), 1.08 (peak 12).

6. A method for determining the contents of Lycium barbarum B, glycyrrhizic acid and morusin A in a Xiebaisan compound preparation, characterized in that: The steps include: (1) Preparation of test solution and reference solution; Optionally, the reference solution includes mulberry glycoside A reference solution, lycium barbarum B reference solution, and ammonium glycyrrhizinate reference solution; (2) The test solution was tested by high performance liquid chromatography, with octadecylsilane bonded silica gel as the filler, the mobile phase comprising acetonitrile and an aqueous solution containing phosphoric acid, the gradient elution program comprising: 0→10min→12min→32min→60min→70min→90min→93min→98min→99min→105min, the volume percentage of acetonitrile in the mobile phase being: 5%→5%→7%→7%→25%→50%→60%→80%→80%→5%→5%, and the contents of Lycium barbarum B, glycyrrhizic acid and Morusin A in the test solution were calculated; When the test solution is tested, the detection wavelength of Lycium barbarum B is 217-220nm, the detection wavelength of glycyrrhizic acid is 250-254nm, and the detection wavelength of Mulberry glycoside A is 320-324nm.

7. The content determination method according to claim 6, characterized in that: Step (2) also satisfies at least one of the following 1) to 4): 1) The flow rate is 0.8-1.2 mL / min and the column temperature is 23-30°C; alternatively, the flow rate is 0.9-1.1 mL / min and the column temperature is 25-30°C; further alternatively, the flow rate is 1.0 mL / min and the column temperature is 27°C; 2) During the detection process, a chromatographic column with a specification of 4.6 mm × 150 mm and a 2.7 μm was used; 3) The injection volume is 5-10 μL; 4) The volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.05% to 0.2%.

8. The content determination method according to claim 6 or 7, characterized in that: In step (1), the preparation of the test solution includes weighing the Xiebaisan compound preparation, adding a solvent for extraction to obtain an extract, separating the solid from the liquid, and obtaining a liquid, which is the test solution; And / or, in the step (1), the solvent used in the preparation of the reference solution includes methanol; optionally, the solvent used in the preparation of the reference solution is a 50% by volume methanol aqueous solution; optionally, each 1 mL of the reference solution contains 5 to 100 μg of the reference substance; further optionally, each 1 mL of the reference solution of mulberry glycoside A contains 80 to 100 μg of the mulberry glycoside A reference substance, each 1 mL of the reference solution of lycium barbarum B contains 5 to 10 μg of the lycium barbarum B reference substance, and each 1 mL of the reference solution of ammonium glycyrrhizate contains 15 to 20 μg of the ammonium glycyrrhizate reference substance.

9. The content determination method according to claim 8, characterized in that: The step (1) also satisfies any one or more of the following A to F: A. The mass volume ratio of Xiebaisan compound preparation to solvent is 0.1-0.5g:25-100mL; B. Extraction method includes ultrasonic extraction; optionally, the power of the ultrasonic extraction is 200W to 300W and the frequency is 40kHz; C. Extraction time is 15 to 45 minutes; D. The solid-liquid separation is selected from centrifugation or filtration; E. The solvent includes at least one of a methanol aqueous solution and an ethanol aqueous solution; optionally, the volume percentage of the methanol aqueous solution is 50% to 70%; optionally, the volume percentage of the ethanol aqueous solution is 50% to 70%; F. The compound preparation of Xiebaisan includes any one of Xiebaisan granules, Xiebaisan freeze-dried powder, Xiebaisan decoction, Xiebaisan extract, Xiebaisan oral liquid and Xiebaisan tablets.

10. A quality detection method for Xiebaisan compound preparation, characterized in that: The method comprises constructing a characteristic spectrum of the Xiebaisan compound preparation to be tested according to the construction method described in any one of claims 1 to 5 and / or determining the content of Lycium barbarum B, glycyrrhizic acid and Morusin A of the Xiebaisan compound preparation to be tested according to the content determination method described in any one of claims 6 to 9.

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