A characteristic chromatogram construction method of a compound preparation of xiebai powder and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a characteristic spectrum construction method of Xiebai Powder compound preparation and application thereof, which comprises the following steps: (1) preparation of a test sample solution; (2) taking the test sample solution to be detected by high performance liquid chromatography, taking octadecylsilane bonded silica gel as a filler, and taking a mobile phase comprising acetonitrile and a phosphoric acid-containing aqueous solution, and a gradient elution procedure 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 being: 5%→5%→7%→7%→25%→50%→60%→80%→80%→5%→5%. The application can realize effective separation of 12 common characteristic peaks, including a common characteristic peak of matrimony vine A, and the obtained characteristic spectrum has a smooth baseline, good characteristic peak shape and high separation degree.

Description

TECHNICAL FIELD

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

[0002] Xiebaisan is derived from Xiaer Yaozheng Zhidu written by Qian Yi in Song Dynasty, the original formula is “Dikengpi (wash off the soil, bake), Sangbaipi (fine sanding and frying yellow) each one two, Gancao (bake) one coin. Sanding, enter a handful of Jingmi, two small bowls of water, decoct seven tenths, before eating”, which has the effects of purging lung and clearing heat, relieving cough and asthma, and is used for treating lung heat cough and asthma, and the symptoms include “cough, even gasping for breath, skin hot, day feeding is particularly severe, tongue red, yellow, pulse number”. The formula is listed in the Directory of Ancient Classical Famous Formulas (the first batch) by the State Administration of Traditional Chinese Medicine. In the formula, Sangbaipi is bitter and cold, which can clear lung heat, and is the monarch drug; Dikengpi is cold and sweet, which can clear lung heat and relieve liver and kidney heat, and is the minister drug; Gancao is sweet and flat, which can benefit the spleen and regulate various drugs, and Jingmi is sweet and flat, which can benefit the spleen and stomach, relieve thirst, and relieve heat from the small intestine, and both of them are the auxiliary drugs. The effects of Xiebaisan are closely related to the chemical components and pharmacological effects of the drugs. Sangbaipi is the dried root bark of Morus alba L. in Moraceae, which has the effects of purging lung and relieving asthma, and is often used for treating lung heat asthma, edema, and other symptoms. Research shows that the main effective components of Sangbaipi are glycosides represented by Sanggenoside A. Dikengpi is the dried root bark of Lycium chinense Mill. in Solanaceae, and lyciumin A and lyciumin B are the cyclic peptide components in Dikengpi, which can inhibit renin and angiotensin I converting enzyme (ACE). Glycyrrhizic acid, also known as glycyrrhizin, is the main source of sweetness of Glycyrrhiza, which is a pentacyclic triterpenoid compound of oleanane, and exists in the form of potassium salt or calcium salt. Glycyrrhizic acid has antibacterial, antiviral, immune regulation and other pharmacological effects, and is an important active component and quality evaluation index component of Glycyrrhiza.

[0003] In the prior art, there are documents disclosing the UPLC content detection method of Dikengpi B and Sanggenoside A in Xiebaisan granules; and there are documents disclosing the method for determining the contents of Dikengpi B and Dikengpi A in Xiebaisan to evaluate the quality of Xiebaisan; there is no report on the research of characteristic spectrum of Xiebaisan granules. There is no method for simultaneously determining the characteristic spectrum of Sanggenoside A, lyciumin A and ammonium glycyrrhizinate, or the method for simultaneously determining the contents of Sanggenoside A, lyciumin B and glycyrrhizic acid. SUMMARY

[0004] Therefore, the present application aims to provide a characteristic spectrum construction method of Xiebaisan compound preparation and application thereof, which establishes the characteristic spectrum thereof according to the main effective components in Xiebaisan compound preparation, realizes the construction of the characteristic spectrum of Morusin A, Solavernin A and ammonium glycyrrhizinate and the analysis and determination of the contents of Morusin A, Solavernin B and glycyrrhizic acid under one chromatographic condition, and can comprehensively control the quality of Xiebaisan compound preparation.

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

[0006] The present application provides a characteristic spectrum construction method of Xiebaisan compound preparation, comprising the following steps: (1) preparation of test sample solution; (2) detection of the test sample solution by high performance liquid chromatography, with octadecylsilane bonded silica gel as the filler, and the mobile phase comprising acetonitrile and phosphoric acid-containing aqueous solution, and the 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 being: 5%→5%→7%→7%→25%→50%→60%→80%→80%→5%→5%.

[0007] Optionally, step (1) comprises weighing Xiebaisan compound preparation, extracting with a solvent to obtain an extract, and performing solid-liquid separation to obtain the liquid, which is the test sample solution.

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

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

[0010] Optionally, the construction method further comprises the steps of preparing a control solution by using at least one of mulberroside A, lyciumin A, lyciumin B, and ammonium glycyrrhizinate and a solvent, and detecting the control solution by the high-performance liquid chromatography in the construction method of any one of claims 1-4 to obtain a control atlas; optionally, the solvent used in the preparation of the control solution comprises methanol; further optionally, the solvent used in the preparation of the control solution is a methanol aqueous solution with a volume percentage of 50%; optionally, 5-100 μg of the control is contained in each 1 mL of the control solution; further optionally, 80-100 μg of mulberroside A control is contained in each 1 mL of the mulberroside A control solution, 10-50 μg of lyciumin A control is contained in each 1 mL of the lyciumin A control solution, 5-10 μg of lyciumin B control is contained in each 1 mL of the lyciumin B control solution, and 15-20 μg of ammonium glycyrrhizinate control is contained in each 1 mL of the ammonium glycyrrhizinate control solution.

[0011] Optionally, the characteristic atlas of the Xiebaisan compound preparation has 12 common characteristic peaks, peak 4 corresponds to the retention time of the mulberroside A control reference peak, peak 11 corresponds to the retention time of the lyciumin A control reference peak, the peak corresponding to the mulberroside A control reference peak is an S1 peak, the peak corresponding to the lyciumin A control reference peak is an S2 peak, the relative retention times of peaks 1-3 and the S1 peak are within ±10% of the specified value, the relative retention times of peaks 5-10 and peak 12 and the S2 peak are within ±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), and 1.08 (peak 12).

[0012] The application further provides a method for determining the content of loganic acid B, glycyrrhizic acid and mulberroside A in Xiebaisan compound preparation, comprising the following steps: (1) preparation of test sample solution and control sample solution; optionally, the control sample solution comprises mulberroside A control sample solution, loganic acid B control sample solution and ammonium glycyrrhizinate control sample solution; (2) taking the test sample solution and detecting by high performance liquid chromatography, using octadecylsilane-bonded silica gel as filler, and using a mobile phase comprising acetonitrile and a phosphoric acid-containing aqueous solution, and using a 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 is: 5%→5%→7%→7%→25%→50%→60%→80%→80%→5%→5%, and calculating the content of loganic acid B, glycyrrhizic acid and mulberroside A in the test sample solution; when the test sample solution is detected, the detection wavelength of loganic acid B is 217-220 nm, the detection wavelength of glycyrrhizic acid is 250-254 nm, and the detection wavelength of mulberroside A is 320-324 nm.

[0013] Optionally, step (2) further 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; 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) a chromatographic column with a specification of 4.6 mm x 150 mm and 2.7 μm is used in the detection process; 3) the injection volume is 5-10 μL; and 4) the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.05%-0.2%.

[0014] Optionally, in step (1), the preparation of the test sample solution comprises weighing Xiebaisan compound preparation, extracting with a solvent to obtain an extract, and performing solid-liquid separation to obtain a liquid, which is the test sample solution.

[0015] Optionally, the solvent used in the preparation of the control sample solution in step (1) comprises methanol; optionally, the solvent used in the preparation of the control sample solution is a 50% (volume percentage) methanol aqueous solution; optionally, each 1 mL of the control sample solution contains 5-100 μg of control sample; further optionally, each 1 mL of the mulberroside A control sample solution contains 80-100 μg of mulberroside A control sample, each 1 mL of the loganic acid B control sample solution contains 5-10 μg of loganic acid B control sample, and each 1 mL of the ammonium glycyrrhizinate control sample solution contains 15-20 μg of ammonium glycyrrhizinate control sample.

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

[0017] Typically and non-limitatively, the raw materials of the Xiebaisan compound preparation in the present application comprise, by weight: 6-11 parts of roasted Cortex Lycii Radicis, 6-11 parts of roasted Cortex Mori, 0.6-1.1 parts of roasted Radix Glycyrrhizae, and 4-8 parts of polished rice; optionally, the preparation steps of the Xiebaisan compound preparation comprise: taking the raw materials of the Xiebaisan compound preparation by weight, decocting the raw materials in water for 1-3 times, 20-80 min each time, with 6-4 times the total amount of the raw materials, filtering the decoction, concentrating to a relative density of 1.01-1.15 (temperature 50-70 °C), drying into dry extract powder, mixing uniformly, and preparing the Xiebaisan compound preparation.

[0018] The application also provides a quality detection method of the Xiebaisan compound preparation, which comprises constructing a characteristic spectrum of the Xiebaisan compound preparation to be detected according to the above construction method and / or determining the contents of the Solaviron B, glycyrrhizic acid and mulberry leaf glycoside A in the Xiebaisan compound preparation to be detected according to the above content determination method. Specifically, the method can comprise the step of comparing the characteristic spectrum of the Xiebaisan compound preparation to be detected with a control characteristic spectrum of the Xiebaisan compound preparation. The characteristic spectrum of the Xiebaisan compound preparation to be detected is constructed by using the Xiebaisan compound preparation to be detected according to the above construction method. The control characteristic spectrum of the Xiebaisan compound preparation is selected from any one of the following (1)-(3): (1) the characteristic spectrum has 12 common characteristic peaks, peak 4 corresponds to the retention time of the mulberry leaf glycoside A reference peak, peak 11 corresponds to the retention time of the Solaviron A reference peak, the peak corresponding to the mulberry leaf glycoside A reference peak is S1 peak, the peak corresponding to the Solaviron A reference peak is S2 peak, the relative retention times of peaks 1-3 and S1 peak are within ±10% of the specified value, the relative retention times of peaks 5-10 and 12 and S2 peak are within ±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); (2) the characteristic spectrum of the Xiebaisan compound preparation is obtained by using a single batch or multiple batches of the Xiebaisan compound preparation according to the above construction method; (3) the control characteristic spectrum is obtained by averaging or mediating the characteristic spectra obtained by using multiple batches of the Xiebaisan compound preparation according to the above construction method.

[0019] The technical scheme of the application has the following advantages:

[0020] 1. The characteristic spectrum construction method of the Xiebaisan compound preparation provided by the application uses octadecylsilane-bonded silica gel as the filler, the mobile phase comprises acetonitrile and a phosphoric acid-containing aqueous solution, and 12 common characteristic peaks are obtained through a specific gradient elution program. The method realizes the separation of the common characteristic peaks including the mulberry leaf glycoside A peak and the Solaviron A peak, the elution program is simple, the baseline of the obtained characteristic spectrum is smooth, the characteristic peaks have good peak shape and high resolution, and the method can provide a basis for the quality detection and control of the Xiebaisan compound preparation. In addition, the peak positions of the mulberry leaf glycoside A, Solaviron A, Solaviron B and ammonium glycyrrhizinate can be accurately positioned, and the method fully reflects the integrity and characteristics of the Xiebaisan compound preparation.

[0021] 2. The characteristic spectrum construction method of the Xiebaisan compound preparation provided by the application is simple in sample preparation, easy to realize the chromatographic conditions, simple in operation, accurate in results and good in reproducibility.

[0022] 3. The quality detection method of the Xiebaisan compound preparation provided by the present application 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 detected with the control characteristic spectrum of the Xiebaisan compound preparation.

[0023] 4. The method for determining the contents of ginsenoside B, glycyrrhizic acid and mulberroside A in the Xiebaisan compound preparation provided by the present application can determine the contents of ginsenoside B, glycyrrhizic acid and mulberroside A in the Xiebaisan compound preparation under the same chromatographic conditions as those for constructing the characteristic spectrum, thereby further realizing the quality control of the Xiebaisan compound preparation and improving the inspection efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the following specific embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

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

[0026] Figure 2 is the chromatogram of the mulberroside A control solution in Example 1;

[0027] Figure 3 is the chromatogram of the ginsenoside A control solution in Example 1;

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

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

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

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

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

[0033] Figure 9 is the chromatogram of the control solution of mulberroside A at a detection wavelength of 324 nm in Example 2;

[0034] Figure 10 is the flow rate of the experimental example 1, mulberryin A different flow rate comparison chart, from top to bottom in turn for the flow rate 1.0 ml / min of the blank solvent, mulberryin A reference solution, test solution, 0.8 ml / min of test solution, 1.2 ml / min of test solution;

[0035] Figure 11 is the flow rate of the experimental example 1, mulberryin A different flow rate comparison chart, from top to bottom in turn for the flow rate 1.0 ml / min of the blank solvent, mulberryin A reference solution, test solution, 0.8 ml / min of test solution, 1.2 ml / min of test solution;

[0036] Figure 12 is the flow rate of the experimental example 1, mulberryin A different flow rate comparison chart, from top to bottom in turn for the flow rate 1.0 ml / min of the blank solvent, mulberryin A reference solution, test solution, 0.8 ml / min of test solution, 1.2 ml / min of test solution;

[0037] Figure 13 is the column temperature of the experimental example 1, mulberryin A different column temperature comparison chart, from top to bottom in turn for the column temperature 25 DEG C of the blank solvent, mulberryin A reference solution, test solution, column temperature 23 DEG C of the blank solvent, mulberryin A reference solution, test solution, column temperature 27 DEG C of the blank solvent, mulberryin A reference solution, test solution;

[0038] Figure 14 is the column temperature of the experimental example 1, mulberryin A different column temperature comparison chart, from top to bottom in turn for the column temperature 25 DEG C of the blank solvent, mulberryin A reference solution, test solution, column temperature 23 DEG C of the blank solvent, mulberryin A reference solution, test solution, column temperature 27 DEG C of the blank solvent, mulberryin A reference solution, test solution;

[0039] Figure 15 is the column temperature of the experimental example 1, mulberryin A different column temperature comparison chart, from top to bottom in turn for the column temperature 25 DEG C of the blank solvent, mulberryin A reference solution, test solution, column temperature 23 DEG C of the blank solvent, mulberryin A reference solution, test solution, column temperature 27 DEG C of the blank solvent, mulberryin A reference solution, test solution;

[0040] Figure 16is the chromatogram of blank solvent (50% methanol), sample solution, morusin A reference substance solution, and lyciumin A reference substance solution obtained in the specificity experiment of experimental example 2;

[0041] Figure 17 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 particle control solution without Cistanches, sample solution, lyciumin B and ammonium glycyrrhizinate mixed reference substance solution;

[0042] Figure 18 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 particle control solution without Glycyrrhiza, sample solution, lyciumin B and ammonium glycyrrhizinate mixed reference substance solution;

[0043] Figure 19 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 particle control solution without Morus alba, sample solution, morusin A reference substance solution;

[0044] Figure 20 is the image of regression analysis of the concentration and peak area of lyciumin B in the linearity and range verification of experimental example 3;

[0045] Figure 21 is the image of regression analysis of the concentration and peak area of glycyrrhizic acid in the linearity and range verification of experimental example 3;

[0046] Figure 22 is the image of regression analysis of the concentration and peak area of morusin A in the linearity and range verification of experimental example 3. DETAILED DESCRIPTION

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

[0048] The preparation method of the Xiebaisan granules used in the present application is as follows: weighing the raw materials, baking 8.86g of Cortex Lycii, frying 8.86g of Cortex Mori and 0.89g of Radix Glycyrrhizae, and 6.00g of polished rice; adding water with 14 times the weight of all the raw materials to decoct for 60min, filtering the decoction, and reserving it for use, then adding water with 12 times the weight of the raw materials to decoct for 40min, filtering the decoction, mixing the two portions of the decoction, concentrating to a relative density of 1.05 (60℃), drying to prepare dry paste powder, mixing uniformly, and preparing Xiebaisan granules.

[0049] The preparation method of the Qiansangbaipi negative granule control used in the present application is as follows: weighing the raw materials, baking 8.86g of Cortex Lycii, frying 0.89g of Radix Glycyrrhizae, and 6.00g of polished rice; adding water with 14 times the weight of all the raw materials to decoct for 60min, filtering the decoction, and reserving it for use, then adding water with 12 times the weight of the raw materials to decoct for 40min, filtering the decoction, mixing the two portions of the decoction, concentrating to a relative density of 1.05 (60℃), drying to prepare dry paste powder, mixing uniformly, and preparing Qiansangbaipi negative granule control.

[0050] The preparation method of the Quedigupi negative granule control used in the present application is as follows: weighing the raw materials, frying 8.86g of Cortex Mori, frying 0.89g of Radix Glycyrrhizae, and 6.00g of polished rice; adding water with 14 times the weight of all the raw materials to decoct for 60min, filtering the decoction, and reserving it for use, then adding water with 12 times the weight of the raw materials to decoct for 40min, filtering the decoction, mixing the two portions of the decoction, concentrating to a relative density of 1.05 (60℃), drying to prepare dry paste powder, mixing uniformly, and preparing Quedigupi negative granule control.

[0051] The preparation method of the Qiangancan negative granule control used in the present application is as follows: weighing the raw materials, baking 8.86g of Cortex Lycii, frying 8.86g of Cortex Mori, and 6.00g of polished rice; adding water with 14 times the weight of all the raw materials to decoct for 60min, filtering the decoction, and reserving it for use, then adding water with 12 times the weight of the raw materials to decoct for 40min, filtering the decoction, mixing the two portions of the decoction, concentrating to a relative density of 1.05 (60℃), drying to prepare dry paste powder, mixing uniformly, and preparing Qiangancan negative granule control.

[0052] Example 1

[0053] The present embodiment provides a method for constructing the characteristic spectrum of Xiebaisan granules, which comprises the following steps:

[0054] Preparation of the sample solution: about 0.1g of Xiebaisan granules was accurately weighed, 50ml of 50% methanol was accurately added, the weight was determined, ultrasonic treatment (power 250W, frequency 40kHz) was performed for 30min, then the sample was taken out and cooled, the weight was determined again, the lost weight was made up with 50% methanol, and the sample was shaken and filtered to obtain the filtrate.

[0055] Preparation of reference solution: an appropriate amount of morusin A reference substance was precisely weighed, 50% methanol was added to prepare a reference solution containing 80 μg of morusin A per 1 ml; another appropriate amount of lyciumin A reference substance was precisely weighed, 50% methanol was added to prepare a reference solution containing 10 μg of lyciumin A per 1 ml, and the reference solution was obtained.

[0056] Determination: 5 μl of the test sample solution and the reference solution of the control substance described above were injected into the ultra-high performance liquid chromatograph, and the chromatographic conditions were as follows: Agilent Poroshell 120SB-C18 (4.6 x 150 mm, 2.7 μm) was used as the chromatographic column; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 1.0 ml / min; the column temperature was 27°C; the detection wavelength was 218 nm; and the theoretical plate number calculated according to the morusin A peak should not be less than 3000.

[0057] Three batches of Xiaobai San granules (numbered 240401, 240402, and 240403, respectively) were tested. Table 2 shows the relative retention times of the three batches of Xiaobai San granules, with morusin A as the S1 peak and lyciumin A as the S2 peak. The retention times of the peaks 1-3 were calculated with S1 as the reference peak, and the retention times of the peaks 5-10 and 12 were calculated with S2 as the reference peak. Table 3 shows the retention times of the reference solution of the control substance. The chromatograms of the test sample solutions of Xiaobai San granules are shown in Figure 1 (from bottom to top: test sample solution 240401, test sample solution 240402, test sample solution 240403), the chromatogram of the morusin A reference solution is shown in Figure 2 , and the chromatogram of the lyciumin 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 example provides a method for determining the contents of lyciumin B, glycyrrhizic acid, and morusin A in Xiaobai San granules, which includes the following steps:

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

[0068] The same reference solution of morin A as in Example 1 was used. Appropriate amounts of reference substances of loganic acid B and ammonium glycyrrhizate were precisely weighed, and 50% methanol was added to prepare a mixed reference solution containing 5 μg of loganic acid B and 15 μg of ammonium glycyrrhizate per 1 ml.

[0069] The determination method was the same as in Example 1, except that the detection wavelength of the reference solution of morin A was 324 nm, and the detection wavelengths of the mixed reference solution were 218 nm (loganic acid B) and 252 nm (glycyrrhizic acid), respectively. The content of morin A in the test solution was calculated from the peak area of the morin A peak in the chromatogram of the test solution at 324 nm. The content of loganic acid B in the test solution was calculated from the peak area of the loganic acid B peak in the chromatogram of the test solution at 218 nm. The content of glycyrrhizic acid in the test solution was calculated from the peak area of the ammonium glycyrrhizate peak in the chromatogram of the test solution at 252 nm, wherein the concentration of ammonium glycyrrhizate was converted to glycyrrhizic acid (the weight of glycyrrhizic acid = the weight of ammonium glycyrrhizate / 1.0207).

[0070] Three batches of Xiaobai San granules (labeled as 240401, 240402, and 240403, respectively) were tested. Table 4 shows the determination results of the contents of loganic acid B, glycyrrhizic acid, and morin A in the three batches of Xiaobai San granules. The chromatogram of the test solution of Xiaobai San granules at a detection wavelength of 218 nm is shown in Figure 4 , the chromatogram of the test solution of Xiaobai San granules at a detection wavelength of 252 nm is shown in Figure 5 , and the chromatogram of the test solution of Xiaobai San granules at a detection wavelength of 324 nm is shown in Figure 6 , Figures 4-6 All of them are, from bottom to top, test solution 240401, test solution 240402, and test solution 240403; the mixed reference solution of loganic acid B and ammonium glycyrrhizate at a detection wavelength of 218 nm is shown in Figure 7 , the mixed reference solution of loganic acid B and ammonium glycyrrhizate at a detection wavelength of 252 nm is shown in Figure 8 , and the reference solution of morin A at a detection wavelength of 324 nm is shown in Figure 9 .

[0071] Table 4

[0072]

[0073] Investigation of chromatographic conditions in Experimental Example 1

[0074] The content determination method of lyciumin B, glycyrrhizic acid and mulberroside A in the compound preparation of Xiaobai San and the characteristic spectrum construction method of the compound preparation of Xiaobai San share the same chromatographic conditions, the content determination method has higher requirement on the chromatographic conditions than the characteristic spectrum construction method, therefore, only the chromatographic conditions for the content determination method are investigated, and the results obtained by the investigation can also be applied to the characteristic spectrum construction method.

[0075] 1. Investigation of flow rate

[0076] Take Xiaobai San granules (batch number: 240401), and prepare the sample solution according to the same method as in Example 2; prepare the reference substance solution of mulberroside A, the mixed reference substance solution of lyciumin B and ammonium glycyrrhizinate according to the same method as in Example 2; and use 50% methanol as the blank solvent. Compare the influence of different flow rates (0.8 ml / min, 1.0 ml / min, 1.2 ml / min) on the content determination of lyciumin B, glycyrrhizic acid and mulberroside A in the compound preparation of Xiaobai San, and the rest of the chromatographic conditions are the same as in Example 2, that is, detect the reference substance solution of mulberroside A, the blank solvent, the sample solution with different flow rates at a detection wavelength of 324 nm, detect the mixed reference substance solution of lyciumin B and ammonium glycyrrhizinate, the blank solvent, the sample solution with different flow rates at a detection wavelength of 252 nm, and detect the mixed reference substance solution of lyciumin B and ammonium glycyrrhizinate, the blank solvent, the sample solution with different flow rates at a detection wavelength of 218 nm, and the results are shown in Table 4 and Tables 5-7. Figures 10-12 The signal-to-noise ratio of the chromatographic peak of mulberroside A is greater than 10, the theoretical plate number is greater than 4000, and the system suitability is good, and the resolution is greater than 1.5 under different flow rates; the signal-to-noise ratio of the chromatographic peak of lyciumin B is greater than 10, the theoretical plate number is greater than 4000, and the system suitability is good. When the flow rate is 1.2 ml / min, the peak area of the chromatographic peak of lyciumin B in the sample is only 79.477, and the peak area of the chromatographic peak under other flow rates is higher, all greater than 100; the signal-to-noise ratio of the chromatographic peak of ammonium glycyrrhizinate is greater than 10, the theoretical plate number is greater than 4000, and the system suitability is good, and when the flow rate is 1.2 ml / min, the resolution of the chromatographic peak of ammonium glycyrrhizinate in the reference substance and the sample is less than 1.5, and the resolution of the chromatographic peak under other flow rates is greater than 1.5. In combination, the preferred flow rate is 1.0 ml / min.

[0077] Table 5 Investigation results of different flow rates of mulberroside A

[0078]

[0079] Table 6 Investigation results of different flow rates of lyciumin B

[0080]

[0081] Table 7 Investigation results of different flow rates of glycyrrhizic acid

[0082]

[0083] 2. Investigation of column temperature

[0084] Take Xiebaisan granules (batch number: 240401) and prepare the test sample solution according to the same method as in Example 2; prepare the control sample solution of morusin A, the mixed control sample solution of physcioscosponin B and ammonium glycyrrhizinate according to the same method as in Example 2; and use 50% methanol as the blank solvent. Compare the effects of different column temperatures (23°C, 25°C, 27°C) on the characteristic chromatogram of Xiebaisan, and the rest of the chromatographic conditions are the same as in Example 2, i.e., detect the control sample solution of morusin A, the blank solvent, and the test sample solution at different column temperatures at a detection wavelength of 324 nm, detect the mixed control sample solution of physcioscosponin B and ammonium glycyrrhizinate, the blank solvent, and the test sample solution at different column temperatures at a detection wavelength of 252 nm, and detect the mixed control sample solution of physcioscosponin B and ammonium glycyrrhizinate, the blank solvent, and the test sample solution at different column temperatures at a detection wavelength of 218 nm. The results are shown in Table 7 and Tables 8-10. Figures 13-15 The content of the test sample was determined using different column temperatures, the signal-to-noise ratio of the chromatographic peak of morusin A was greater than 10, the theoretical plate number was greater than 4000, the system suitability was good, and the resolution at different column temperatures was greater than 1.5, but the peak purity at a column temperature of 23°C was less than 999; the signal-to-noise ratio of the chromatographic peak of physcioscosponin B was greater than 10, the theoretical plate number was greater than 4000, the system suitability was good at different column temperatures, the peak purity at different column temperatures was greater than 999; the signal-to-noise ratio of the chromatographic peak of ammonium glycyrrhizinate was greater than 10, the theoretical plate number was greater than 4000, the system suitability was good at different column temperatures, the resolution at different column temperatures was greater than 1.5, but the peak purity at a column temperature of 23°C was less than 999. In view of the above, the preferred column temperature is 27°C.

[0085] Table 8 Investigation results of different column temperatures of morusin A

[0086]

[0087] Table 9 Investigation results of different column temperatures of physcioscosponin B

[0088]

[0089] Table 10 Investigation results of different column temperatures of glycyrrhizic acid

[0090]

[0091] Methodology verification of the characteristic chromatogram in Experimental Example 2

[0092] 1. Specificity experiment: Take Xiebaisan granules (batch number: 240401) test sample solution and blank solvent (50% methanol), morusin A control sample solution, lycium A control sample solution, respectively, under the chromatographic conditions of Example 1, sample detection, see Figure 16 , the results show that the blank solvent does not peak in the corresponding position of the 12 common peaks of the test sample chromatogram, and the chromatographic peaks in the test sample chromatogram and the control sample solution chromatogram appear at the corresponding position, and the ultraviolet absorption spectrum is basically the same, indicating that the method has good specificity.

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

[0094] 3. Method repeatability test: Refer to the test sample solution preparation method of Example 1 to prepare Xiebaisan granules (batch number: 240401) test sample solution, 6 parallel, according to the chromatographic conditions of Example 1, sample determination, calculate the RSD value of the relative retention time of each characteristic peak and the corresponding S1 peak, S2 peak, the results show that the relative retention time RSD of 6 test sample solutions is in the range of 0.0% ~ 0.1%, less than 2.0%, all within the range of mean ± 10%, the characteristic spectrum method has good repeatability.

[0095] 4. Intermediate precision: According to the method and condition 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, S2 peak was calculated, and the RSD was taken as the evaluation index. The results show that the relative retention time RSD of each characteristic peak measured by different personnel is in the range of 0.0% ~ 0.1%; the relative retention time RSD of each characteristic peak of different instruments is in the range of 0.0% ~ 1.1%, indicating that the intermediate precision of the characteristic spectrum is good.

[0096] 5. Stability: Refer to the preparation method of test sample solution of Example 1 to prepare Xiebaisan granules (batch number: 240401) test sample solution, according to the chromatographic conditions of Example 1, sample detection at 0h, 2h, 4h, 8h, 12h, 24h, 32h, 48h, 56h, 72h time point, calculate the relative retention time of each characteristic peak and the corresponding S1 peak, S2 peak, take its RSD as the evaluation index, the results show that the relative retention time RSD of each characteristic peak is in the range of 0.0% ~ 0.3% within 72h, less than 2%, indicating that the test sample solution is stable within 72 hours.

[0097] 6. Different flow rate durability investigation: Take the Xiaobai San granules (batch number: 240401) test sample solution, and inject under the chromatographic conditions of Example 1 at different flow rates (0.9 ml / min, 1.0 ml / min, 1.1 ml / min) for detection, and calculate the relative retention time of each target peak with the corresponding S1 peak, S2 peak reference peak. The results show that the relative retention time of each characteristic peak in the test sample measured at different flow rates is in the range of 0.0% to 1.8%, all less than 2%, indicating that the characteristic spectrum method has good durability.

[0098] Methodology verification of the determination method of matrimony vine B, glycyrrhizic acid, and mulberry leaf glycoside A in Xiaobai San compound preparation

[0099] 1. Specificity verification: Prepare the negative granule control solution of mulberry leaf without bark, the negative granule control solution of cortex canthi, and the negative granule control solution of glycyrrhiza by the same method as the preparation of the test sample solution in Example 1. Take the Xiaobai granules (batch number 240401) test sample solution, and inject under the chromatographic conditions of Example 2 with the blank solvent (50% methanol), the mulberry leaf glycoside A control solution, the matrimony vine B and ammonium glycyrrhizinate mixed control solution, the negative granule control solution of mulberry leaf without bark, the negative granule control solution of cortex canthi, and the negative granule control solution of glycyrrhiza for detection. Detect the test sample solution, the blank solvent, the matrimony vine B and ammonium glycyrrhizinate mixed control solution, and the negative granule control solution of cortex canthi at a wavelength of 218 nm; detect the test sample solution, the blank solvent, the matrimony vine B and ammonium glycyrrhizinate mixed control solution, and the negative granule control solution of glycyrrhiza at a wavelength of 252 nm; and detect the test sample solution, the blank solvent, the mulberry leaf glycoside A control solution, and the negative granule control solution of mulberry leaf without bark at a wavelength of 324 nm. Record the chromatogram, as shown in Figures 17-19 . The results show that the chromatographic peaks appear at the corresponding positions of the control solution chromatogram in the test sample chromatogram, and the ultraviolet absorption spectrum is basically consistent. The blank solvent does not produce peaks at the corresponding positions of the test sample chromatogram, and there is no interference. The negative granule of mulberry leaf without bark does not interfere with the determination of mulberry leaf glycoside A, the negative granule of cortex canthi does not interfere with the determination of matrimony vine B, and the negative granule of glycyrrhiza does not interfere with the determination of ammonium glycyrrhizinate, indicating that the content determination method has good specificity.

[0100] 2. Linearity and range verification

[0101] (1) Inject solutions of lycopene B reference standard at 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 numbers are linear-1 to 7) into the solutions respectively, and measure them at 218 nm according to the conditions of Example 2. The peak areas at each concentration are detailed in Table 11. Regression analysis was performed on the peak area Y of lycopene B against the concentration X of lycopene B reference standard, as detailed in Table 11. Figure 20 The regression equation was Y = 36508X435.9, with a correlation coefficient r = 1. The results showed that within the range of 0.25–12.38 μg / mL, the concentration X (μg / mL) of Lycium barbarum B had a good linear relationship with the peak area Y.

[0102] (2) Glycyrrhizic acid ammonium reference 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 linearity -1 to 7) were injected separately and measured at 252 nm according to the conditions in Example 2. The peak areas for each concentration are detailed in Table 11. The concentration of glycyrrhizic acid ammonium was converted to glycyrrhizic acid (glycyrrhizic acid weight = glycyrrhizic acid ammonium weight / 1.0207), and regression analysis was performed on the peak area Y against the concentration X of the reference standard. See Table 11 for details. Figure 21 The regression equation was: Y = 8541.3X844.49, with a correlation coefficient r = 1. The results showed that in the range of 0.63 to 31.72 μg / mL, the concentration X (μg / mL) of glycyrrhizic acid had a good linear relationship with the peak area Y.

[0103] (3) Morin A reference 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 numbers are linear-1 to 7) were injected and measured at 324 nm according to the conditions in Example 2. The peak areas at each concentration are detailed in Table 11. Regression analysis was performed using the peak area Y of morin A against the concentration X of morin A reference standard, as detailed in Table 11. Figure 22 The regression equation was: Y = 26506X 1236.2, with a correlation coefficient r = 1. The results showed that in the range of 1.97 to 98.33 μg / mL, the concentration X (μg / mL) of morin A had a good linear relationship with the peak area Y.

[0104] Table 11

[0105]

[0106] 3. Precision

[0107] (1) Instrument precision: The test sample solution of Xiaobai San Granules (Batch No. 240401) was prepared according to the test sample solution preparation method of Example 2. The sample was injected for determination 6 times under the chromatographic conditions of Example 2, and the RSD values of the retention time and peak area of 6 determinations were calculated. The results showed that the RSD of the retention time of Gynostemma B was 0.0%, the RSD of the peak area was 0.2%; the RSD of the retention time of glycyrrhizic acid was 0.0%, the RSD of the peak area was 0.5%; the RSD of the retention time of Mulberry Bark Glycoside A was 0.1%, the RSD of the peak area was 0.2%, and the RSD was less than 2%, indicating that the instrument precision was good.

[0108] (2) Reproducibility: The test sample solution of Xiaobai San Granules (Batch No. 240401) was prepared according to the test sample solution preparation method of Example 2. Six samples were prepared in parallel, and the sample was injected for determination under the chromatographic conditions of Example 2. The content and RSD value of the 6 samples were calculated. The results showed that the RSD of Gynostemma B was 0.3%; the RSD of glycyrrhizic acid was 0.5%; the RSD of Mulberry Bark Glycoside A was 0.4%, and the RSD was less than 2%, indicating that the content determination method had good reproducibility.

[0109] (3) Intermediate precision: The same batch of Xiaobai San Granules (Batch No. 240401) was investigated by different personnel and different instruments according to the method and conditions of Example 2 to investigate the influence of different personnel and different instruments on precision. The results showed that the RSD of the content of Gynostemma B measured by different personnel was 0.2%, the RSD of glycyrrhizic acid was 0.4%, and the RSD of Mulberry Bark Glycoside A was 0.1%, which was less than 2%, indicating that the content determination method had good intermediate precision of different personnel; the RSD of the content of Gynostemma B measured by different instruments was 0.6%; the RSD of glycyrrhizic acid was 0.4%; the RSD of Mulberry Bark Glycoside A was 0.3%, and the RSD was less than 2%, indicating that the method had good intermediate precision of different instruments.

[0110] 4. Accuracy: Six samples of Xiaobai San Granules (Batch No. 240401), each about 0.05 g, were taken, and the index component contents of Gynostemma B, ammonium glycyrrhizinate and Mulberry Bark Glycoside A reference substances were added. The test sample solution was prepared according to Example 2, and the sample was injected for determination under the determined chromatographic conditions. The recovery rate was calculated. The results showed that the recovery rate of Gynostemma B was in the range of 93.9% to 97.4%, the RSD was 0.9%; the recovery rate of glycyrrhizic acid was in the range of 99.8% to 104.0%, the RSD was 1.2%; the recovery rate of Mulberry Bark Glycoside A was in the range of 100.4% to 101.8%, the RSD was 0.5%, and the RSD was less than 2%; indicating that the content determination method had good accuracy.

[0111] 5. Stability: The test solution of the Gouteng granules (batch No. 240401) prepared according to Example 2 was injected for detection under the chromatographic conditions of Example 2 at time points of 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, and 72 h, respectively, the peak areas of each component to be detected were recorded, and the RSD values were calculated. The results showed that the RSD of the peak area of Gynostemma B was 0.66%, the RSD of the peak area of glycyrrhizic acid was 0.53%, and the RSD of the peak area of Morusin A was 0.22% within 0-72 h, all of which were less than 2%, indicating that the test solution was stable within 72 h.

[0112] 6. Durability: The test solution of the Gouteng granules was injected for detection under the chromatographic conditions of Example 2 at different flow rates (0.9 ml / min, 1.0 ml / min, and 1.1 ml / min), respectively, and the RSD of the content of Gynostemma B was 0.9%, the RSD of the content of glycyrrhizic acid was 1.3%, and the RSD of the content of Morusin A was 1.0%, all of which were less than 2%, indicating that the content determination method was durable.

[0113] Obviously, the above examples are merely examples for the purpose of clarity, and are not limitations on the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. All embodiments do not need to be exhaustively listed here. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for constructing a characteristic chromatogram of a compound preparation of Xiebai Powder, characterized in that, The method comprises the following steps: (1) Preparation of the test sample solution, 0.1-0.5 g of the Xiebaisan compound preparation is added into 25-100 mL of a solvent and ultrasonically extracted for 15-45 min to obtain an extract, and then the liquid is separated from the solid to obtain the test sample solution; The solvent comprises at least one of a 50%-70% (by volume) methanol aqueous solution and a 50%-70% (by volume) ethanol aqueous solution; Preparation of the reference solution of the control sample, a 50% (by volume) methanol aqueous solution is added into morroniside A and lycium A to prepare the reference solution of the control sample; (2) The test sample solution and the reference solution of the control sample are detected by high performance liquid chromatography, octadecylsilane-bonded silica gel is used as the filler, the mobile phase comprises acetonitrile and a phosphoric acid-containing aqueous solution, and the gradient elution program comprises: 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 is: 5%→5%→7%→7%→25%→50%→60%→80%→80%→5%→5%; The detection wavelength of the high performance liquid chromatography detection is 210-220 nm, the flow rate is 0.8-1.2 mL / min, and the column temperature is 23-30 ℃; a chromatographic column with a specification of 4.6 mm×150 mm, 2.7 μm is used; the injection volume is 5-10 μL; and the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.05%-0.2%. The characteristic spectrum of the Xiebaisan compound preparation has 12 common characteristic peaks, peak 4 corresponds to the retention time of the morroniside A control sample reference peak, peak 11 corresponds to the retention time of the lycium A control sample reference peak, the peak corresponding to the morroniside A control sample reference peak is an S1 peak, the peak corresponding to the lycium A control sample reference peak is an S2 peak, the relative retention times of peaks 1-3 and the S1 peak are within ±10% of the specified value, the relative retention times of peaks 5-10 and peak 12 and the S2 peak are within ±10% of the specified value; and the specified values are 0.59, 0.67, 0.68, 0.74, 0.76, 0.77, 0.77, 0.85, 0.89, and 1.08, respectively.

2. The construction method of claim 1, wherein, The flow rate is 0.9-1.1 mL / min, and the column temperature is 25-30 ℃.

3. The construction method of claim 2, wherein, The flow rate is 1.0 mL / min, and the column temperature is 27 ℃.

4. The construction method of claim 1, wherein, The step (1) further satisfies any one or more of the following A-C: A. The ultrasonic extraction power is 200-300 W, and the frequency is 40 kHz; B. The solid-liquid separation is selected from centrifugation or filtration; C. The Xiebaisan compound preparation comprises any one of Xiebaisan granules, Xiebaisan freeze-dried powder, Xiebaisan soup, Xiebaisan extract, Xiebaisan oral liquid, and Xiebaisan tablets.

5. The construction method according to any one of claims 1 to 4, characterized in that, Each 1 mL of the reference solution of the control sample contains 5-100 μg of the control sample.

6. The construction method of claim 5, wherein, Each 1 mL of the reference solution of morroniside A contains 80-100 μg of morroniside A control sample, and each 1 mL of the reference solution of lycium A contains 10-50 μg of lycium A control sample.

7. A method for detecting the quality of a compound preparation of Xiebai San, characterized in that, The method comprises the steps of constructing a characteristic spectrum of the to-be-tested Xiebaisan compound preparation according to the construction method in any one of claims 1 to 6, and comparing the obtained characteristic spectrum with a control characteristic spectrum of the Xiebaisan compound preparation.

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