Construction method and detection method of traditional Chinese medicine high performance liquid fingerprint

Abstract

The invention provides a construction method and a detection method of a high performance liquid fingerprint of granules for consolidating foundation and clearing away lung-heat. According to the construction method, high performance liquid fingerprint spectrums of a test solution and a reference solution of the primordial qi-consolidating and lung-heat-clearing granules are compared, so that the main effective active substances of the primordial qi-consolidating and lung-heat-clearing granules are detected. The reference substance solution is prepared from 3 '-hydroxy puerarin, puerarin, 3'-methoxy puerarin, puerarin-6 ''-O-xyloside, puerarin apigenin, daidzin, forsythiaside A, pinoresinol-4-O-glucopyranoside, baicalin, forsythin, wogonoside, baicalein and the like. Finally, a high-performance liquid-phase fingerprint spectrum technical standard of the primordial qi-consolidating and lung-heat-clearing granules is established, the quality of the primordial qi-consolidating and lung-heat-clearing granules can be effectively and comprehensively monitored according to the characteristics of existence of common peaks in the fingerprint spectrum, and the method has the characteristics of good stability, good repeatability and strong operability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and detection, and specifically relates to a method for constructing a high-performance liquid fingerprint spectrum of Guben Qingfei granules and an application thereof. Background Art

[0002] Guben Qingfei Granule has the effects of clearing heat and drying dampness, promoting lung function and resolving phlegm, detoxifying and strengthening the body. Guben Qingfei Granule is composed of 13 Chinese herbs, including Scutellaria baicalensis, Forsythia suspensa, American ginseng, Taraxacum officinale, Patrinia scabra, Fagopyrum tiliaceum, Pinellia ternata, Bupleurum chinense, Ephedra, Angelica dahurica, Lepidium officinale, Apricot kernel, and Pueraria root. Most of the pathogenic qi of epidemic toxins are warm and hot in nature, and they invade the human body with dampness, enter through the mouth and nose, invade the lung defense, lie in the membrane, and invade the internal organs. It shows signs of dampness and heat. It is treated by clearing heat and drying dampness, promoting lung function and resolving phlegm, detoxifying and strengthening the body. Guben Qingfei Granules use scutellaria, forsythia, and American ginseng as the main drugs, with scutellaria and forsythia clearing heat, drying dampness, and detoxifying; American ginseng clears heat, promotes fluid production, replenishes qi and consolidates the foundation, and strengthens the body and expel evil; dandelion, patrinia, golden buckwheat, pinellia, and bupleurum are the assistant drugs, and cooperate with the main drugs to clear heat, detoxify, dry dampness and resolve phlegm, bupleurum disperses and reduces fever, and helps American ginseng regulate qi, replenish qi and consolidate the foundation; ephedra, angelica, scutellaria, bitter apricot, and kudzu root are the adjuvant drugs, angelica dispels wind and dampness, clears the exterior, ephedra clears the lungs and resolves the exterior, scutellaria clears the lungs and relieves asthma, bitter apricot descends qi, stops coughing and relieves asthma; kudzu root relieves muscles and reduces fever, and together with bupleurum, raises yang qi and drives evil out. Since there are many medicinal flavors in the prescription and the chemical components are complex and diverse, the quality control of this Chinese medicine formula has an important impact on the performance of its efficacy. However, at present, no relevant quality control methods have been established for Guben Qingfei Granules, which has a serious impact on the use of the product.

[0003] Therefore, it is desired to establish an evaluation method that can comprehensively reflect the quality information of Guben Qingfei Granules to ensure the uniformity and stability of the product. Summary of the invention

[0004] In view of the above-mentioned problems, the present invention provides a method for constructing an HPLC fingerprint of Guben Qingfei Granules. The fingerprint constructed by the present invention can effectively and comprehensively monitor the quality of Guben Qingfei Granules, and has the characteristics of good stability and good repeatability.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing a high-performance liquid fingerprint spectrum of Guben Qingfei granules comprises the following steps:

[0007] S1. Preparation of test solution: Take Guben Qingfei granules, crush them, accurately weigh the powder and place it in a stoppered conical flask, add solvent, weigh it, ultrasonicate it, cool it, weigh it again, make up the lost weight with solvent, shake it well, filter it, and take the filtrate to obtain the solution;

[0008] S2. Preparation of reference solution: accurately weigh 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-6"-O-xyloside, puerarin apiglycoside, daidzein, forsythiaside A, pinoresinol-4-O-pyranoglucoside, baicalin, forsythiaside, wogonin, and baicalein, respectively, and dissolve them in solvents to obtain the reference solution;

[0009] S3. Liquid chromatography analysis: Accurately pipette the test solution prepared in step S1 and the reference solution prepared in step S2 respectively, inject into ultra-high performance liquid chromatography for determination, and obtain the fingerprint of Guben Qingfei Granules with common characteristic peaks.

[0010] Preferably, the solvent in steps S1 and S2 is one of methanol and ethanol. In the present invention, methanol or ethanol with a mass fraction of 25-100% is preferred, and 50% ethanol is more preferred.

[0011] Preferably, the ultrasonic treatment in step S1 is carried out at a power of 180 to 200 W and a frequency of 35 to 45 kHz for 30 to 60 min, more preferably 45 min.

[0012] Preferably, the ultra-high performance liquid chromatography determination conditions described in step S3 include: chromatographic column: octadecylsilane bonded chromatographic column (Waters HSS T3, specification is 5μm, 4.6×250mm); column temperature: 25-35°C; mobile phase: acetonitrile is mobile phase A, 0.08-0.12% phosphoric acid aqueous solution is mobile phase B, flow rate is 0.8-1.0mL / min; detection wavelength: 200-254nm; injection volume: 8-12μL, the injection solution is gradient eluted, analyzed, recorded, and fingerprints are constructed.

[0013] Preferably, when performing ultra-high performance liquid chromatography determination, elution is performed according to the following elution procedure:

[0014] 0~110min, A: 5%~18%, B: 95%~82%;

[0015] 110~165min, A: 18%~29%, B: 82%~71%;

[0016] 165~183min, A: 29%~49%, B: 71%~51%;

[0017] 183~190min, A: 49%~95%, B: 51%~5%.

[0018] Preferably, there are 22 common characteristic peaks described in step S3, among which peak 5, peak 6, peak 7, peak 8, peak 9, and peak 10 are attributed to Pueraria lobata medicinal material; peak 4, peak 11, peak 13, peak 14, peak 15, peak 16, and peak 18 are attributed to Forsythia suspensa medicinal material, peak 12, peak 17, peak 19, peak 20, peak 21, and peak 22 are attributed to Scutellaria baicalensis medicinal material, peak 2 and peak 3 are attributed to Ephedra medicinal material, and peak 1 is attributed to Patrinia suspensa medicinal material.

[0019] Preferably, among the above common peaks, peak 5 is 3'-hydroxypuerarin, peak 6 is puerarin, peak 7 is 3'-methoxypuerarin, peak 8 is puerarin-6"-O-xyloside, peak 9 is puerarin apiglycoside, peak 10 is daidzein, peak 14 is forsythiaside A, peak 15 is pinoresinol-4-O-pyranoglucoside, peak 17 is baicalin, peak 18 is forsythiaside, peak 21 is wogonin, and peak 22 is baicalin.

[0020] Preferably, taking peak 6 as the reference peak, the relative retention times of the remaining chromatographic peaks are 0.248±0.002, 0.287±0.001, 0.309±0.001, 0.415±0.002, 0.709±0.001, 1.085±0.002, 1.127±0.007, 1.182±0.003, 1.282±0.002, 1.61 0±0.005, 1.650±0.007, 1.795±0.007, 1.832±0.007, 1.881±0.009, 2.024±0.009, 2.412±0.011, 2.469±0.015, 2.587±0.012, 2.685±0.012, 2.782±0.015, 3.004±0.017.

[0021] The present invention also provides a method for detecting the fingerprint spectrum of Guben Qingfei granules, comprising the following process:

[0022] (1) Preparation of test solution: Take Guben Qingfei granules, crush them, accurately weigh the powder and place it in a stoppered conical flask, add solvent, weigh it, ultrasonicate it, cool it, weigh it again, make up the lost weight with solvent, shake it well, filter it, and take the filtrate to obtain the solution;

[0023] (2) Preparation of reference solution: accurately weigh 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-6"-O-xyloside, puerarin apiglycoside, daidzein, forsythiaside A, pinoresinol-4-O-pyranoglucoside, baicalin, forsythiaside, wogonin, and baicalein, respectively, and dissolve them in solvents to obtain the reference solution;

[0024] (3) Liquid chromatography analysis: Accurately aspirate the test solution prepared in step S1 and the reference solution prepared in step S2, respectively, and perform detection according to the chromatographic conditions. Compare the chromatogram of the test solution obtained by the detection with the fingerprint spectrum obtained by the established method, calculate the similarity, and evaluate the quality of the Guben Qingfei Granules.

[0025] The preparation method of the Guben Qingfei granules described in the present invention is as follows:

[0026] Add 10 times the amount of water to 222.22g of Scutellaria baicalensis, 333.33g of Forsythia suspensa, 111.11g of American ginseng, 333.33g of Taraxacum mongolicum, 333.33g of Patrinia chinensis, 444.44g of Fagopyrum scabra, 111.11g of Pinellia ternata, 222.22g of Bupleurum chinense, 177.78g of Ephedra sinica, 177.78g of Apricot kernel, 222.22g of Lepidium officinale, 333.33g of Pueraria root, and 200g of Angelica dahurica, and boil twice for 1.5 hours each time. Filter the decoction, combine the filtrates, and concentrate the filtrates to a clear paste with a relative density of 1.05-1.10 (60°C), spray dry, take the dry extract powder, add an appropriate amount of dextrin, mix well, make into granules, dry, mix well, make into 1000g, and package.

[0027] The invention compares the high performance liquid fingerprints of Guben Qingfei granules with those of 12 reference substances (3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-6"-O-xyloside, puerarin apiglycoside, daidzein, forsythiaside A, pinoresinol-4-O-pyranoglucoside, baicalin, forsythin, wogonin and baicalein), and simultaneously compares the high performance liquid chromatography with the negative control medicinal materials of Guben Qingfei granules, thereby obtaining a method for simultaneously identifying five Chinese medicinal materials components in Guben Qingfei granules, namely, kudzu root, forsythia, scutellaria, ephedra and patrinia herba.

[0028] Compared with the prior art, the present invention has the following advantages: the present invention establishes the high-performance liquid fingerprint technology standard for Guben Qingfei Granules, provides rich identification information through the presence or absence characteristics of common peaks in the fingerprint spectrum, can effectively and comprehensively monitor the quality of Guben Qingfei Granules, and has the characteristics of good stability, good repeatability and strong operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The chromatograms of the Guben Qingfei granules test sample under different detection wavelength conditions;

[0030] Figure 2 Chromatograms of the Guben Qingfei granules test sample under different column temperature conditions;

[0031] Figure 3 Chromatograms of the Guben Qingfei granules test sample under different flow rate conditions;

[0032] Figure 4 Chromatograms of the Guben Qingfei granules tested under different extraction solvent conditions;

[0033] Figure 5 The chromatograms of the Guben Qingfei granules tested under different extraction conditions;

[0034] Figure 6 The chromatograms of the Guben Qingfei granules tested under different extraction time conditions;

[0035] Figure 7 This is the chromatogram of the mixed reference substance and the Guben Qingfei granules test substance;

[0036] Figure 8 The chromatograms are Guben Qingfei granules, Pueraria root control medicinal material and Pueraria root negative control;

[0037] Fig. 9 The chromatograms are Guben Qingfei granules, Forsythia suspensa control medicinal materials and Forsythia suspensa negative control;

[0038] Fig.10 The chromatograms are Guben Qingfei granules, scutellaria baicalensis reference medicinal material and scutellaria baicalensis negative control;

[0039] Fig.11 The chromatograms are for Guben Qingfei granules, ephedra control medicinal material, and ephedra negative control;

[0040] Fig.12 These are the chromatograms of Guben Qingfei Granules, Patrinia salsa control medicinal material and Patrinia salsa negative control. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. The raw materials, equipment, etc. used in the following embodiments can be purchased through conventional channels unless otherwise specified.

[0042] In the experimental process of the present invention, the specific sources and models of some raw materials, instruments and equipment involved are shown in Table 1.

[0043] Table 1 Some raw materials and instruments involved in the experimental process of the present invention

[0044]

[0045]

[0046] Example 1 Screening of Chromatographic Conditions in the Construction Method of Guben Qingfei Granule High-Performance Liquid Phase Fingerprint

[0047] 1. Detection wavelength screening: The screening process is as follows

[0048] (1) Preparation of test solution: GUBENQINGFEI granules were crushed into fine powder. 0.5 g of the powder was accurately weighed and placed in a stoppered conical flask. 25 mL of 75% methanol was added and weighed. Ultrasonic treatment (40 kHz, 200 W) was performed for 30 min. The mixture was cooled and weighed. The lost weight was supplemented with 75% methanol. The mixture was shaken and filtered. The filtrate was taken to obtain the test solution.

[0049] (2) Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature is 25° C., flow rate is 0.8 mL / min, injection volume is 10 μL, mobile phase includes A and B, A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, gradient elution is adopted, and the elution program is: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%.

[0050] Under the above-set chromatographic conditions, the test solution was injected and analyzed at detection wavelengths of 203nm, 230nm, and 254nm, and the chromatographic results obtained are shown in Figure 1 , Figure 1 In the figure, A is 203nm, B is 230nm, and C is 254nm. Figure 1 It can be seen that the absorption intensity and quantity of each chromatographic peak are 203nm>220nm>254nm, so 203nm is selected as the detection wavelength.

[0051] 2. Column temperature screening: The screening process is as follows:

[0052] (1) Preparation of test solution: GUBENQINGFEI granules were crushed into fine powder. 0.5 g of the powder was accurately weighed and placed in a stoppered conical flask. 25 mL of 75% methanol was added and weighed. Ultrasonic treatment (40 kHz, 200 W) was performed for 30 min. The mixture was cooled and weighed. The lost weight was supplemented with 75% methanol. The mixture was shaken and filtered. The filtrate was taken to obtain the test solution.

[0053] (2) Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5μm, 4.6×250mm), flow rate is 0.8mL / min, detection wavelength is 203nm, injection volume is 10μL, mobile phase includes A and B, A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, gradient elution is adopted, and the elution program is: 0-110min, A: 5%-18%, B: 95%-82%; 110-165min, A: 18%-29%, B: 82%-71%; 165-183min, A: 29%-49%, B: 71%-51%; 183-190min, A: 49%-95%, B: 51%-5%.

[0054] Under the above-mentioned chromatographic conditions, the test solution was injected and analyzed at column temperatures of 25°C, 30°C, and 35°C. The chromatographic results are shown in Figure 2 , Figure 2 In the equation, A is 25°C, B is 30°C, and C is 35°C. Figure 2 It can be seen that with the increase of column temperature, the chromatographic peaks move forward, the retention time shortens, and the separation of some chromatographic peaks is affected. Comprehensive analysis shows that when the column temperature is 25°C, the separation of each chromatographic peak is better, so 25°C is selected as the column temperature condition.

[0055] 3. Flow rate screening: The specific screening process is as follows:

[0056] (1) Preparation of test solution: GUBENQINGFEI granules were crushed into fine powder. 0.5 g of the powder was accurately weighed and placed in a stoppered conical flask. 25 mL of 75% methanol was added and weighed. Ultrasonic treatment (40 kHz, 200 W) was performed for 30 min. The mixture was cooled and weighed. The lost weight was supplemented with 75% methanol. The mixture was shaken and filtered. The filtrate was taken to obtain the test solution.

[0057] (2) Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature is 25° C., detection wavelength is 203 nm, injection volume is 10 μL, mobile phases include A and B, A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, gradient elution is adopted, and the elution program is: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%.

[0058] Under the above-mentioned chromatographic conditions, the test solution was injected and analyzed at flow rates of 0.8 mL / min, 0.9 mL / min, and 1.0 mL / min, respectively. The chromatographic results are shown in Figure 3 , Figure 3 In the equation, A is 0.8 mL / min, B is 0.9 mL / min, and C is 1.0 mL / min. Figure 3 It can be seen that when the flow rate is 0.8 mL / min, the separation effect of each chromatographic peak obtained by analysis is ideal, so 0.8 mL / min is selected as the flow rate condition.

[0059] Example 2 Screening of conditions for preparing the test solution in the method for constructing the high-performance liquid fingerprint of Guben Qingfei granules

[0060] 1. Extraction solvent screening: The specific screening process is as follows

[0061] (1) Preparation of test solution: Take Guben Qingfei granules, grind them into fine powder, accurately weigh 0.5 g of the powder, weigh 6 portions at the same time, place them in a stoppered conical flask, add 25 mL each of 50% ethanol, 75% ethanol, 25% methanol, 50% methanol, 75% methanol, and 100% methanol, weigh the weight, ultrasonically treat (40 kHz, 200 W) for 30 min, cool, weigh the weight, make up the lost weight with each solvent, shake well, filter, and take the filtrate to obtain the test solution.

[0062] (2) Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature of 25° C., flow rate of 0.8 mL / min, detection wavelength of 203 nm, injection volume of 10 μL, mobile phases including A and B, wherein A is acetonitrile, and B is 0.1% phosphoric acid aqueous solution, gradient elution is adopted, and the elution program is as follows: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%.

[0063] The 6 groups of test sample solutions were injected and analyzed under the above-set chromatographic conditions. The chromatographic results obtained are shown in Figure 4 , Figure 4 In the above equation, A is 75% ethanol, B is 50% ethanol, C is 75% methanol, D is 50% methanol, E is 25% methanol, and F is 100% methanol. Figure 4 It can be seen that when 50% ethanol is used as the extraction solvent, the response values ​​of some chromatographic peaks are higher, so 50% ethanol is selected as the extraction solvent.

[0064] 2. Extraction method screening: The specific screening process is as follows:

[0065] (1) Preparation of test solution: Take Guben Qingfei granules, crush them into fine powder, accurately weigh 0.5 g of the powder, weigh 2 portions at the same time, place them in a stoppered conical flask, add 25 mL of 50% ethanol to each portion, weigh the weight, heat reflux in a 100°C water bath, and ultrasonically treat (40 kHz, 200 W) for 30 min, cool, weigh the weight, make up the lost weight with 50% ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0066] (2) Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature of 25° C., flow rate of 0.8 mL / min, detection wavelength of 203 nm, injection volume of 10 μL, mobile phases including A and B, wherein A is acetonitrile, and B is 0.1% phosphoric acid aqueous solution, gradient elution is adopted, and the elution program is as follows: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%.

[0067] The test solution was injected and analyzed under the above-mentioned chromatographic conditions. The results obtained are shown in Figure 5 , Figure 5 In the process, A is ultrasonic treatment (40kHz, 200W), and B is heating in a water bath at 100°C and reflux. Figure 5 It can be seen that the number of chromatographic peaks obtained by the two extraction methods is basically the same, and the extraction efficiency is not much different, but the ultrasonic treatment operation is simple, so the ultrasonic treatment is more efficient for Guben Qingfei Granules in the quality control process.

[0068] 3. Extraction time screening: The specific screening process is as follows

[0069] (1) Preparation of test solution: Take Guben Qingfei granules, grind them into fine powder, accurately weigh 0.5 g of the powder, weigh three portions at the same time, place them in a stoppered conical flask, add 25 mL of 50% ethanol, weigh the weight, ultrasonically treat (40 kHz, 200 W) for 30 min, 45 min, and 60 min, respectively, cool, weigh the weight, make up the lost weight with 50% ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0070] (2) Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature 25°C, flow rate 0.8 mL / min, detection wavelength 203 nm, injection volume 10 μL, mobile phases A and B, A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, gradient elution is used, elution program is: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%;

[0071] The three groups of test solution were injected and analyzed under the above-set chromatographic conditions. The chromatographic results obtained are shown in Figure 6 , Figure 6 In the above examples, A is ultrasonic treatment (40kHz, 200W) for 60min, B is ultrasonic treatment (40kHz, 200W) for 45min, and C is ultrasonic treatment (40kHz, 200W) for 30min. Figure 6 It can be seen that the number of chromatographic peaks obtained by the three extraction times is basically the same, but when the ultrasonic treatment is performed for 45 minutes, the response values ​​of some chromatographic peaks are higher, so the ultrasonic treatment for 45 minutes is selected.

[0072] Example 3 Methodological Investigation of the Construction Method of the High-Performance Liquid Phase Fingerprint of Guben Qingfei Granules

[0073] 1. Precision experiment: The specific test process is as follows

[0074] (1) Preparation of test solution: GUBENQINGFEI granules were crushed into fine powder. 0.5 g of the powder was accurately weighed and placed in a stoppered conical flask. 25 mL of 50% ethanol was added and weighed. The mixture was ultrasonically treated (40 kHz, 200 W) for 45 min. The mixture was cooled and weighed. The lost weight was supplemented with 50% ethanol. The mixture was shaken and filtered. The filtrate was taken to obtain the test solution.

[0075] (2) Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature of 25° C., flow rate of 0.8 mL / min, detection wavelength of 203 nm, injection volume of 10 μL, mobile phases including A and B, wherein A is acetonitrile, and B is 0.1% phosphoric acid aqueous solution, gradient elution is adopted, and the elution program is as follows: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%.

[0076] The test solution was injected 6 times continuously under the above-mentioned chromatographic conditions. The relative retention time of each chromatographic peak was calculated with reference to the retention time of peak 6. The results are shown in Table 2.

[0077] Table 2: Relative retention time of each chromatographic peak in precision test

[0078]

[0079]

[0080] 2. Stability test: The specific test process is as follows

[0081] Preparation of test solution: Take Guben Qingfei granules, crush into fine powder, accurately weigh 0.5g of powder, put into a stoppered conical flask, add 25ml of 50% ethanol, weigh the weight, ultrasonically treat (40kHz, 200W) for 45 minutes, cool, weigh the weight, make up the lost weight with 50% ethanol, shake well, filter, and take the filtrate to obtain the test solution;

[0082] Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature 25°C, flow rate 0.8 ml / min, detection wavelength 203 nm, injection volume 10 μl, mobile phases A and B, A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, gradient elution is used, elution program is: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%;

[0083] The test solution was injected at 0h, 6h, 12h, and 18h ​​under the above-mentioned chromatographic conditions, and the relative retention time of each chromatographic peak was calculated with the retention time of peak 6 as a reference. The results are shown in Table 3;

[0084] Table 3: Relative retention time of each chromatographic peak in the stability test

[0085]

[0086]

[0087] 3. Repeatability Experiment

[0088] Preparation of test solution: Take Guben Qingfei granules, crush into fine powder, accurately weigh 0.5g of powder, put into a stoppered conical flask, add 25ml of 50% ethanol, weigh the weight, ultrasonically treat (40kHz, 200W) for 45 minutes, cool, weigh the weight, make up the lost weight with 50% ethanol, shake well, filter, take the filtrate to obtain the test solution, and prepare 6 identical test solutions in parallel;

[0089] Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature 25°C, flow rate 0.8 ml / min, detection wavelength 203 nm, injection volume 10 μl, mobile phases A and B, A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, gradient elution is used, elution program is: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%;

[0090] Six portions of the test solution were injected separately under the above-set chromatographic conditions, and the relative retention time of each chromatographic peak was calculated with the retention time of peak 6 as a reference. The results are shown in Table 4.

[0091] Table 4: Relative retention time of each chromatographic peak in the repeatability test

[0092]

[0093]

[0094] From the results in Tables 2 to 4, it can be seen that the RSD of the relative retention time of each chromatographic peak is less than 3.0% after methodological investigation, indicating that the method has good adaptability.

[0095] Example 4 Construction of the High-Performance Liquid Phase Fingerprint of Guben Qingfei Granules

[0096] The specific steps of constructing the high-performance liquid fingerprint spectrum of Guben Qingfei Granules are as follows:

[0097] S1. Preparation of Guben Qingfei Granule test solution:

[0098] Take Guben Qingfei granules, crush them into fine powder, accurately weigh 0.5g of the powder, put it in a stoppered conical flask, add 25mL of 50% ethanol, weigh the weight, ultrasonically treat (40kHz, 200W) for 45min, let cool, weigh the weight, make up the lost weight with 50% ethanol, shake well, filter, and take the filtrate to obtain the Guben Qingfei granules test solution;

[0099] S2. Preparation of mixed reference solution:

[0100] 3'-Hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-6"-O-xyloside, puerarin apiglycoside, daidzein, forsythiaside A, pinoresinol-4-O-pyranoglucoside, baicalin, forsythiaside, wogonin and baicalein were weighed and dissolved in 50% methanol to prepare a solution containing 70 μg / mL 3'-hydroxypuerarin, 200 μg / mL puerarin, 100 μg / mL A mixed reference solution of 3'-methoxypuerarin, 50 μg / mL puerarin-6"-O-xyloside, 100 μg / mL puerarin apiglycoside, 100 μg / mL daidzein, 100 μg / mL forsythiaside A, 50 μg / mL pinoresinol-4-O-pyranoglucoside, 350 μg / mL baicalin, 40 μg / mL forsythiaside, 80 μg / mL wogonin, and 30 μg / mL baicalin;

[0101] S3. Liquid chromatography analysis:

[0102] Chromatographic condition setting: The chromatographic column uses Waters HSS T3 (5 μm, 4.6×250 mm), column temperature 25°C, flow rate 0.8 mL / min, detection wavelength 203 nm, injection volume 10 μL, mobile phases A and B, A is acetonitrile, B is 0.1% phosphoric acid aqueous solution, gradient elution is used, elution program is: 0-110 min, A: 5%-18%, B: 95%-82%; 110-165 min, A: 18%-29%, B: 82%-71%; 165-183 min, A: 29%-49%, B: 71%-51%; 183-190 min, A: 49%-95%, B: 51%-5%;

[0103] The test solution and the mixed reference solution prepared in step S1 were injected and analyzed under the above-set chromatographic conditions, and the obtained HPLC chromatogram of Guben Qingfei Granules was as follows: Figure 7 As shown in the figure, A is the test sample of Guben Qingfei Granules, B is the mixed reference sample, peak 5 is 3'-hydroxypuerarin, peak 6 is puerarin, peak 7 is 3'-methoxypuerarin, peak 8 is puerarin-6"-O-xyloside, peak 9 is puerarin apiglycoside, peak 10 is daidzein, peak 14 is forsythiaside A, peak 15 is pinoresinol-4-O-pyranoglucoside, peak 17 is baicalin, peak 18 is forsythiaside, peak 21 is wogonin, peak 22 is baicalin, and peak 6 (puerarin) has a moderate retention time and good separation, so peak 6 (puerarin) is selected as the reference peak.

[0104] The chromatographic peaks of the fingerprint identification results of Guben Qingfei Granules obtained above are further assigned in the following steps:

[0105] Referring to the methods of steps S1 and S2 above, the test solution of Guben Qingfei granules and the control medicinal material solutions (i.e., the control medicinal material solution of Pueraria lobata, the control medicinal material solution of Forsythia suspensa, the control medicinal material solution of Scutellaria baicalensis, the control medicinal material solution of Ephedra sinica, and the control medicinal material solution of Patrinia chinensis) were prepared respectively. The preparation of the negative control in Guben Qingfei granules is as follows:

[0106] Pueraria root negative control: that is, without Pueraria root, the composition, content and preparation method of the other 12 herbs are the same as those of Guben Qingfei Granules. The preparation method of Pueraria root negative control solution is the same as that of Guben Qingfei Granules test sample solution;

[0107] Forsythia negative control: that is, without Forsythia, the composition, content and preparation method of the other 12 herbs are the same as those of Guben Qingfei Granules. The preparation method of Forsythia negative control solution is the same as that of Guben Qingfei Granules test sample solution;

[0108] Scutellariae baicalensis negative control: that is, excluding Scutellariae baicalensis, the composition, content and preparation method of the other 12 herbs are the same as those of Guben Qingfei Granules, and the preparation method of Scutellariae baicalensis negative control solution is the same as that of Guben Qingfei Granules test sample solution;

[0109] Ephedra negative control: that is, without ephedra, the composition, content and preparation method of the other 12 herbs are the same as those of Guben Qingfei Granules. The preparation method of ephedra negative control solution is the same as that of Guben Qingfei Granules test sample solution;

[0110] Patrinia salsa negative control: that is, without Patrinia salsa, the composition, content and preparation method of the other 12 herbs are the same as those of Guben Qingfei Granules. The preparation method of Patrinia salsa negative control solution is the same as that of the test sample solution of Guben Qingfei Granules.

[0111] The prepared Guben Qingfei granule test solution, each control medicinal material solution and 5 groups of negative control solutions were injected and analyzed under the above-set chromatographic conditions (same as step S3), and the retention time and ultraviolet absorption were used for comparison analysis. The specific results are shown in Figures 8 to 12 , Figure 8 Among them, A is the negative control of Pueraria root, B is the test product of Guben Qingfei granules, and C is the control medicinal material of Pueraria root; Fig. 9 Among them, A is the negative control of Forsythia suspensa, B is the test product of Guben Qingfei granules, and C is the control medicinal material of Forsythia suspensa; Fig.10 In the figure, A is the negative control of Scutellaria baicalensis, B is the test product of Guben Qingfei Granule, and C is the control medicinal material of Scutellaria baicalensis; Fig.11 Among them, A is the negative control of Ephedra, B is the test product of Guben Qingfei Granule, and C is the control medicinal material of Ephedra; Fig.12 In the figure, A is the negative control of Patrinia chinensis, B is the test product of Guben Qingfei Granule, and C is the control medicinal material of Patrinia chinensis.

[0112] It can be seen that among the 22 common peaks marked in the fingerprint of Guben Qingfei Granules, peak 5, peak 6, peak 7, peak 8, peak 9, and peak 10 are attributed to Pueraria lobata; peak 4, peak 11, peak 13, peak 14, peak 15, peak 16, and peak 18 are attributed to Forsythia suspensa, peak 12, peak 17, peak 19, peak 20, peak 21, and peak 22 are attributed to Scutellaria baicalensis, peak 2 and peak 3 are attributed to Ephedra, and peak 1 is attributed to Patrinia salsa.

[0113] Finally, it should be noted that the above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for constructing a high-performance liquid fingerprint of Guben Qingfei granules, characterized in that: The steps include: S1. Preparation of test solution: Take Guben Qingfei granules, crush them, accurately weigh the powder and place it in a stoppered conical flask, add solvent, weigh it, ultrasonicate it, cool it, weigh it again, make up the lost weight with solvent, shake it well, filter it, and take the filtrate to obtain the solution; S2. Preparation of reference solution: accurately weigh 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-6"-O-xyloside, puerarin apiglycoside, daidzein, forsythiaside A, pinoresinol-4-O-pyranoglucoside, baicalin, forsythiaside, wogonin, and baicalein, respectively, and dissolve them in solvents to obtain the reference solution; S3. Liquid chromatography analysis: Accurately pipette the test solution prepared in step S1 and the reference solution prepared in step S2 respectively, inject into ultra-high performance liquid chromatography for determination, and obtain the fingerprint of Guben Qingfei Granules with common characteristic peaks.

2. The construction method according to claim 1, characterized in that: The solvent in steps S1 and S2 is one of methanol and ethanol.

3. The construction method according to claim 1, characterized in that: The ultrasonic treatment conditions in step S1 are 180-200 W power and 35-45 kHz frequency for 30-60 min.

4. The construction method according to claim 1, wherein: The ultra-high performance liquid chromatography determination conditions described in step S3 include: chromatographic column: octadecylsilane bonded chromatographic column; column temperature: 25-35°C; mobile phase: acetonitrile as mobile phase A, 0.08-0.12% phosphoric acid aqueous solution as mobile phase B, flow rate of 0.8-1.0 mL / min; detection wavelength: 200-254 nm; injection volume: 8-12 μL, the injection solution is gradient eluted, analyzed, recorded, and the fingerprint is constructed.

5. The construction method according to claim 4, characterized in that: When conducting ultra-high performance liquid chromatography determination, elution is performed according to the following elution program: 0-110 min, A: 5%-18%, B: 95%-82%; 110~165min, A: 18%~29%, B: 82%~71%; 165~183min, A: 29%~49%, B: 71%~51%; 183~190min, A: 49%~95%, B: 51%~5%.

6. The construction method according to claim 1, characterized in that: There are 22 common characteristic peaks described in step S3, among which peak 5, peak 6, peak 7, peak 8, peak 9, and peak 10 are attributed to Pueraria lobata medicinal material; peak 4, peak 11, peak 13, peak 14, peak 15, peak 16, and peak 18 are attributed to Forsythia suspensa medicinal material, peak 12, peak 17, peak 19, peak 20, peak 21, and peak 22 are attributed to Scutellaria baicalensis medicinal material, peak 2 and peak 3 are attributed to Ephedra medicinal material, and peak 1 is attributed to Patrinia suspensa medicinal material.

7. The construction method according to claim 6, characterized in that: Among the above common peaks, peak 5 is 3'-hydroxypuerarin, peak 6 is puerarin, peak 7 is 3'-methoxypuerarin, peak 8 is puerarin-6"-O-xyloside, peak 9 is puerarin apiglycoside, peak 10 is daidzein, peak 14 is forsythiaside A, peak 15 is pinoresinol-4-O-pyranoglucoside, peak 17 is baicalin, peak 18 is forsythiaside, peak 21 is wogonin, and peak 22 is baicalin.

8. The construction method according to claim 5, characterized in that: Taking peak 6 as the reference peak, the relative retention times of the other chromatographic peaks were 0.248±0.002, 0.287±0.001, 0.309±0.001, 0.415±0.002, 0.709±0.001, 1.085±0.002, 1.127±0.007, 1.182±0.003, 1.282±0.002, 1.610± 0.005, 1.650±0.007, 1.795±0.007, 1.832±0.007, 1.881±0.009, 2.024±0.009, 2.412±0.011, 2.469±0.015, 2.587±0.012, 2.685±0.012, 2.782±0.015, 3.004±0.

017.

9. A method for detecting the fingerprint of Guben Qingfei granules, characterized in that: The process includes the following: (1) Preparation of test solution: Take Guben Qingfei granules, crush them, accurately weigh the powder and place it in a stoppered conical flask, add solvent, weigh it, ultrasonicate it, cool it, weigh it again, make up the lost weight with solvent, shake it well, filter it, and take the filtrate to obtain the solution; (2) Preparation of reference solution: accurately weigh 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, puerarin-6"-O-xyloside, puerarin apiglycoside, daidzein, forsythiaside A, pinoresinol-4-O-pyranoglucoside, baicalin, forsythiaside, wogonin, and baicalein, respectively, and dissolve them in solvents to obtain the reference solution; (3) Liquid chromatography analysis: Accurately aspirate the test solution prepared in step S1 and the reference solution prepared in step S2, respectively, and perform detection according to the chromatographic conditions of claim 1, compare the chromatogram of the test solution obtained by the detection with the fingerprint obtained by the method of claim 1, calculate the similarity, and evaluate the quality of the Guben Qingfei Granules.

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