A method for constructing a fingerprint of sophora fruit and application thereof

Abstract

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a construction method and application of a sophora japonica L. fingerprint. The construction method comprises preparation of a test sample solution, gradient elution with acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B, and adoption of a specific gradient elution procedure. The construction method can obtain 11 common characteristic peaks, the separation degree between the common characteristic peaks is good, the characteristic peaks are uniformly distributed, and the effective components of the common characteristic peaks can be identified. The effective chemical components can be used to accurately identify sophora japonica L., overcome the defects that the quality detection and identification of sophora japonica L. cannot be comprehensively, clearly and effectively performed due to the interference caused by the complex chemical components in the sophora japonica L. medicinal materials, and the method is suitable for detection of sophora japonica L. products. Further, the construction method has the advantages of short detection time, high precision, good repeatability and stability, and can comprehensively monitor the quality of sophora japonica L.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a construction method of a sophora flower bud fingerprint and application. BACKGROUND

[0002] Sophora flower bud is the dried flower bud of Sophora japonica L. of Leguminosae. The outer surface is yellowish brown or yellowish green, slightly shriveled, the lower calyx is bell-shaped with 5 teeth at the top, and the upper part is unopened corolla with different sizes, and the outer surface is sparsely covered with white short soft hair. The quality is crisp, the smell is weak, and the taste is slightly bitter. The flower color is green and thick, the flower bud is strong, and the branch is not good. The sophora flower bud period is from July to August.

[0003] Sophora flower bud is bitter and cold in nature. Sophora flower has the effect of lowering blood pressure, improving capillary function, preventing bleeding caused by excessive capillary fragility and permeability, and preventing bleeding in patients with hypertension and diabetes. The prior art has less research on sophora flower bud. When sophora flower bud is detected, there are defects such as few index components, poor characteristic peak separation degree, and long detection time. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the defects of few index components, poor characteristic peak separation degree, and long detection time in the prior art when sophora flower bud is detected, so as to provide a construction method of sophora flower bud fingerprint.

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

[0006] The present application provides a construction method of sophora flower bud fingerprint, comprising the following steps:

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

[0008] The test sample solution is detected by high performance liquid chromatography, acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B, gradient elution, and the gradient elution program includes 0-4min, the volume percentage of mobile phase A is 10%→15%, and the volume percentage of mobile phase B is 90%→85%; 4-10min, the volume percentage of mobile phase A is 15%→16%, and the volume percentage of mobile phase B is 85%→84%; 10-16min, the volume percentage of mobile phase A is 16%→35%, and the volume percentage of mobile phase B is 84%→65%; 16-19min, the volume percentage of mobile phase A is 35%→50%, and the volume percentage of mobile phase B is 65%→50%; 19-20min, the volume percentage of mobile phase A is 50%→10%, and the volume percentage of mobile phase B is 50%→90%.

[0009] The chromatographic conditions of the high performance liquid chromatography method further include: a column temperature of 20-30 DEG C; and / or, octadecylsilane-bonded silica gel as the filler; and / or, the type and size of the chromatographic column is ACQUITY BEH C18, 1.7 μm, 2.1*100 mm; and / or, the flow rate is 0.2-0.4 ml / min; and / or, the injection volume is 0.5-2 μL; and / or, the detection wavelength is 250-265 nm; and / or, the mobile phase B is 0.05-0.3% phosphoric acid aqueous solution.

[0010] The chromatographic conditions of the high performance liquid chromatography method further include: a column temperature of 20-30 DEG C; and / or, octadecylsilane-bonded silica gel as the filler; and / or, the type and size of the chromatographic column is ACQUITY BEH C18, 1.7 μm, 2.1*100 mm; and / or, the flow rate is 0.2-0.4 ml / min; and / or, the injection volume is 0.5-2 μL; and / or, the detection wavelength is 250-265 nm; and / or, the mobile phase B is 0.05-0.3% phosphoric acid aqueous solution.

[0011] The extraction solvent includes at least one of methanol, ethanol and water when preparing the test solution.

[0012] Preferably, the extraction solvent is a 70-100% methanol solution.

[0013] Preferably, the ratio of the mass (g) of the test sample to the volume (ml) of the extraction solvent is (0.001-0.008):1 when preparing the test solution.

[0014] The fingerprint of the sophora fruit includes 11 characteristic peaks, namely, peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10 and peak 11.

[0015] Preferably, peak 1 is quercetin 3-O-beta-D-glucosyl(1→2)[alpha-L-rhamnosyl l(1→6)]-beta-D-glucoside, peak 2 is quercetin 3-O-rutinoside-7-O-rhamnoside, peak 3 is isorhamnetin 3-O-[2-O-beta-glucosyl-6-O-rhamnosyl]-beta-glucoside, peak 4 is quercetin-3-O-beta-xylosyl(1→3)-O-alpha-L-rhamnosyl-(1→6)-O-beta-D-glucoside, peak 5 is rutin, peak 6 is kaempferol-3-O-rutinoside, peak 7 is narcissin, peak 9 is quercetin, peak 10 is kaempferol and peak 11 is isorhamnetin.

[0016] The fingerprint of the sophora fruit includes 11 characteristic peaks, namely, peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10 and peak 11.

[0017] The relative retention time of peak 1, peak 2, peak 3 and peak 4 is 0.63, 0.70, 0.79 and 0.88 respectively, with peak 5 as the reference peak;

[0018] The relative retention time of peak 8, peak 10 and peak 11 is 0.93, 1.11 and 1.13 respectively, with peak 9 as the reference peak 2;

[0019] The relative retention time is within ±10% of the specified value.

[0020] The peak area ratio of peak 5 to peak 9 in the characteristic spectrum obtained by the construction method is ≤14; and / or,

[0021] The peak area ratio of peak 7 to peak 9 is >0.9.

[0022] The test sample is sophora fruit slice, sophora fruit medicinal material, sophora fruit lyophilized powder or sophora fruit formula granules.

[0023] The construction method further comprises preparation of the reference solution;

[0024] Preferably, at least one of rutin, kaempferol-3-O-rutinoside, narcissin and quercetin is used as the reference.

[0025] The application also provides a use of the above-mentioned construction method in quality detection or identification of sophora fruit products.

[0026] Further, in the quality detection or identification of sophora fruit products, the sophora fruit products can be any dosage form such as medicinal material, slice, formula granules, lyophilized powder, etc.

[0027] Further, the peak area ratio of peak 5 to peak 9 can be used for the differentiation of sophora fruit and fried sophora fruit.

[0028] The peak area ratio of peak 7 to peak 9 is used for quality control of sophora fruit samples.

[0029] The technical scheme of the application has the following advantages:

[0030] 1. The present invention provides a method for constructing a fingerprint spectrum of Sophora japonica buds. The method includes the preparation of a test solution, using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B, and gradient elution. The gradient elution program includes: 0-4 min, mobile phase A volume percentage 10%→15%, mobile phase B volume percentage 90%→85%; 4-10 min, mobile phase A volume percentage 15%→16%, mobile phase B volume percentage 85%→84%; 10-16 min, mobile phase A volume percentage 16%→35%, mobile phase B volume percentage 84%→65%; 16-19 min, mobile phase A volume percentage 35%→50%, mobile phase B volume percentage 65%→50%; 19-20 min, mobile phase A volume percentage 50%→10%, mobile phase B volume percentage 50%→90%. This construction method yields 11 common characteristic peaks with good separation and uniform distribution. Furthermore, it identifies the effective components within these common characteristic peaks. These effective chemical components can be used to accurately identify Sophora japonica buds, overcoming the limitations of comprehensive, clear, and effective quality testing and identification caused by the complex chemical composition of Sophora japonica buds. This method is suitable for the testing of Sophora japonica bud products. Moreover, this construction method also offers advantages such as short detection time, high precision, good repeatability, and good stability, enabling comprehensive monitoring of Sophora japonica bud quality.

[0031] 2. The method for constructing the fingerprint spectrum of Sophora japonica buds provided by the present invention can further monitor the quality of Sophora japonica buds by controlling the relative peak areas between different characteristic peaks, and can quickly identify Sophora japonica buds and easily confused products. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 These are the chromatograms of different instruments in Section 8 of Experimental Example 1 of this invention; Figure 2 These are the characteristic and control spectra of three batches of Sophora japonica flower granules and 18 batches of standard decoctions in Experimental Example 3 of this invention; wherein, S1-S18 are the spectra corresponding to the freeze-dried powder of Sophora japonica flower standard decoction, S19-S21 are the spectra corresponding to the Sophora japonica flower granules, and R is the fitted control spectra. Figure 3 This is a comparison chromatogram of Sophora japonica flowers obtained in Example 3 of the present invention; Figure 4 This is the chromatogram of a negative sample used in the methodological verification of Embodiment 3 of the present invention. DETAILED DESCRIPTION

[0034] The following examples are provided to better enable those skilled in the art to further understand and practice the application, and are not intended to limit the scope of the application. Any product, input, or output that is obtained by any of the following examples is intended to be implicitly or explicitly included in the scope of the application. Any examples, or examples that are subsequently devised, which are based on the technical disclosure provided herein, and which are similar in purpose or function to the examples described herein, but which are not expressly shown or described herein, are intended to be expressly included in the scope of the application.

[0035] The experimental procedures or conditions not specified in the examples can be performed according to the conventional experimental procedures described in the literature. The reagents or instruments not specified by the manufacturer are conventional reagents that can be obtained commercially.

[0036] Instruments

[0037] Chromatograph 1: High performance liquid chromatograph, Agilent 1260 chromatograph system, including G1322A degasser, G1311A four-pump, G1313A automatic sampler, G1315B DAD diode array detector, G1316A column oven, Empower 3 chromatographic workstation. Chromatograph 2: High performance liquid chromatograph, Waters ACQUITY UPLC chromatograph system, including binary super high pressure solvent manager, 96-bit high performance automatic sampler (SampleManager), imported original column oven (Column Manager), photoelectric diode array detector (PDA Detector), Empower 3 chromatographic management system. Electronic balance: Sartorius scientific instruments (Beijing) Co., Ltd. (BSA124S), Sartorius scientific instruments (Beijing) Co., Ltd. (BSA224S), Shimadzu enterprise management (China) Co., Ltd. (AUW120D), Shanghai Pu Chun measurement instrument Co., Ltd. (JY2002). Ultrasonic cleaner: KQ-500DB ultrasonic instrument Co., Ltd. of Kunshan.

[0038] Column 1 : Waters CORTECS UPLC T3 1.6pm 2.1 mm x 100 mm SN: 01213123715316 Column 2: Agilent 5TC-C18(2) 5pm 4.6 mm x 250 mm SN: 593077; Column 3: CAPCELL PAK C18 2pm 2.0 mm x 100 mm SN: A58IA01020; Column 4: ACQUITY BEH 1.7pm 2.1 mm x 100 mm SN: 03763028015183; Column 5: ACQUITY BEH 1.7pm 2.1 mm x 100 mm SN: 03763029335120; Column 6: ACQUITY BEH 1.7pm 2.1 mm x 100 mm SN: 03463924135141 ; Column 7: Agilent SB-C18 RRHD, 1.8pm 2.1 mm x 100 mm SN: USDAB05821 ; Column 8: Agilent SB C18 1.8pm 2.1 mm x 100 mm SN: USDAB06286.

[0039] Reagents and reagents

[0040] Acetonitrile, methanol were chromatographic pure, water was ultrapure water, acetic acid, phosphoric acid, formic acid were analytical pure; Rutin (Batch No. 100080-201610, content for HPLC method was 91.9%, content for UV method was 92.6%, China Institute for Food and Drug Control); Rutin (Batch No. 100080-201811, content for HPLC method was 91.7%, content for UV method was 92.4%, China Institute for Food and Drug Control); Quercetin (Batch No. 100081-201610, content was 99.1%, China Institute for Food and Drug Control); Kaempferol-3-O-rutinoside (Batch No. 112007-202103, content was calculated as 94.0%, purchased from China Institute for Food and Drug Control); Zephyranthine (Batch No. 111997-201501, content was calculated as 93.1%, purchased from China Institute for Food and Drug Control); Sophora japonica L. reference drug material (Batch No. 121270-201403, China Institute for Food and Drug Control). The batch numbers of Sophora japonica L. standard decoction freeze-dried powder were 2009001Y, 2009003Y, 2009006Y, 2009007Y, 2009009Y, 2009010Y, 2009011Y, 2009012Y, 2009016Y, 2009019Y, 2009020Y, 2009022Y, 2009024Y, 2009025Y, 2009027Y, 210901Y, 210902Y, 210903Y, which were respectively recorded as S1-S18. The batch numbers of Sophora japonica L. formula granules were 2111001Y-2111003Y, which were respectively recorded as S91-S21.

[0041] Example 1

[0042] The present example provides a method for constructing a Sophora japonica L. fingerprint, comprising the following steps:

[0043] Preparation of the sample solution: 0.1 g of Sophora japonica L. standard decoction freeze-dried powder was accurately weighed and placed in a conical flask with a stopper. 50 ml of methanol was accurately added, and the weight was determined. Ultrasonic treatment (power 300 W, frequency 40 kHz) was performed for 30 min. After cooling, the weight was determined again, the lost weight was made up with methanol, and the mixture was shaken and filtered to obtain the sample solution.

[0044] Preparation of the reference solution: about 0.3 g of Sophora japonica L. reference drug material was placed in a conical flask with a stopper, 50 ml of water was added, and heating reflux was performed for 45 min. The mixture was filtered, the filtrate was evaporated to dryness, 50 ml of methanol was added to the residue, ultrasonic treatment (power 300 W, frequency 40 kHz) was performed for 30 min, the mixture was cooled, shaken and filtered to obtain the reference solution of the reference drug material.

[0045] Preparation of the reference solution: take rutin, kaempferol-3-O-rutinoside, narcissus glycoside, quercetin reference substance, add methanol to prepare a mixed solution containing 50 μg of rutin, 10 μg of kaempferol-3-O-rutinoside, 20 μg of narcissus glycoside, and 5 μg of quercetin per 1 ml, as the reference solution.

[0046] Determination, respectively, 1 μL of the above solution was injected into the liquid chromatograph for determination. Among them, acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, octadecylsilane bonded silica was used as the filler, the specification was that the column length was 100 mm, the inner diameter was 2.1 mm, the particle size was 1.7 μm, the column temperature was 25°C, the flow rate was 0.3 ml / min, the detection wavelength was 257 nm, the theoretical plate number calculated according to the rutin peak should not be less than 2000, gradient elution, the gradient elution program was as follows: 0-4 min, the volume percentage of mobile phase A was 10%→15%, the volume percentage of mobile phase B was 90%→85%; 4-10 min, the volume percentage of mobile phase A was 15%→16%, the volume percentage of mobile phase B was 85%→84%; 10-16 min, the volume percentage of mobile phase A was 16%→35%, the volume percentage of mobile phase B was 84%→65%; 16-19 min, the volume percentage of mobile phase A was 35%→50%, the volume percentage of mobile phase B was 65%→50%; 19-20 min, the volume percentage of mobile phase A was 50%→10%, the volume percentage of mobile phase B was 50%→90%.

[0047] Example 2

[0048] The present embodiment provides a construction method of a sophora fruit fingerprint, comprising the following steps:

[0049] Preparation of the reference solution: take rutin, kaempferol-3-O-rutinoside, narcissus glycoside, quercetin reference substance, add methanol to prepare a mixed solution containing 50 μg of rutin, 10 μg of kaempferol-3-O-rutinoside, 20 μg of narcissus glycoside, and 5 μg of quercetin per 1 ml, as the reference solution.

[0050] Preparation of the reference solution: take rutin, kaempferol-3-O-rutinoside, narcissus glycoside, quercetin reference substance, add methanol to prepare a mixed solution containing 50 μg of rutin, 10 μg of kaempferol-3-O-rutinoside, 20 μg of narcissus glycoside, and 5 μg of quercetin per 1 ml, as the reference solution.

[0051] Preparation of the reference solution: take rutin, kaempferol-3-O-rutinoside, narcissus glycoside, quercetin reference substance, add methanol to prepare a mixed solution containing 50 μg of rutin, 10 μg of kaempferol-3-O-rutinoside, 20 μg of narcissus glycoside, and 5 μg of quercetin per 1 ml, as the reference solution.

[0052] The above solution was precisely pipetted 1 μL, injected into a liquid chromatograph, and measured. The mobile phase A was acetonitrile, the mobile phase B was 0.1% phosphoric acid aqueous solution, the filler was octadecylsilane-bonded silica gel, the specification was column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm, column temperature 25°C, flow rate 0.3 ml / min, detection wavelength 257 nm, and the theoretical plate number calculated according to rutin peak should not be less than 2000. Gradient elution was performed with the following gradient elution program: 0-4 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 4-10 min, volume percentage of mobile phase A 15%→16%, volume percentage of mobile phase B 85%→84%; 10-16 min, volume percentage of mobile phase A 16%→35%, volume percentage of mobile phase B 84%→65%; 16-19 min, volume percentage of mobile phase A 35%→50%, volume percentage of mobile phase B 65%→50%; 19-20 min, volume percentage of mobile phase A 50%→10%, volume percentage of mobile phase B 50%→90%.

[0053] Determination of chromatographic conditions

[0054] 1. Selection of detection wavelength

[0055] The test sample solution was prepared according to Example 1, and eluted according to the mobile phase and gradient elution program of Example 1. PDA full wavelength scanning was used to detect the sophora flower bud characteristic spectrum, and the maximum absorption wavelength of each characteristic peak at 257 nm was determined according to the absorption wavelength of each characteristic peak, so that 257 nm was selected as the detection wavelength.

[0056] 2. Selection of elution gradient

[0057] The test sample solution was prepared according to Example 1, and the gradient elution program was used as a variable, and other conditions were determined according to Example 1. The gradient elution program is shown in the following table, and the results are shown in the following table.

[0058] Table 1 Gradient elution program 1

[0059] Time Mobile phase A Mobile phase B 0 10 90 7 18 82 12 35 65 14 50 50 15 10 90 25 10 90

[0060] Table 2 Gradient elution program 2

[0061] Time (min) Mobile phase A (acetonitrile) Mobile phase B (0.1% phosphoric acid) 0 10 90 10 16 84 15 35 65 18 50 50 20 10 90 25 10 90

[0062] Table 3 Gradient elution program 3

[0063]

[0064]

[0065] Table 4 results of different gradient elution procedure investigation experiments

[0066]

[0067] The above results show that the chromatographic peaks in the 3-6 min time period are excessive, the separation degree is poor, and the peak 8-10 angle is greater than the threshold value, and the peak purity is poor when the gradient elution procedure 1 is used for elution. The symmetry of each characteristic peak of the gradient 2 is poor. The baseline of the chromatogram obtained by the gradient elution procedure 3 is smooth, the separation degree of each proposed characteristic peak is good, and the symmetry is good, and therefore the gradient elution procedure 3 is preferred as the elution procedure of the present application.

[0068] 3. Selection of flow rate

[0069] The test sample solution was prepared according to Example 1, and the flow rate was used as a variable, and the others were determined according to Example 1. The flow rates were 0.2 ml / min, 0.3 ml / min and 0.4 ml / min, respectively, and the results are shown in the following table.

[0070] Table 5 results of different flow rate investigation experiments

[0071]

[0072]

[0073] Suitable chromatographic parameters were selected according to the influence of the system suitability parameters of each chromatographic peak of the characteristic chromatogram, such as peak purity, peak shape, separation degree, symmetry factor, total peak area, etc. The above results show that when the flow rate is 0.3 ml / min, the separation effect of each proposed characteristic peak is optimal, and therefore the flow rate is preferably 0.3 ml / min.

[0074] 4. Selection of column temperature

[0075] The test sample solution was prepared according to Example 1, and the column temperature was used as a variable, and the others were determined according to Example 1. The column temperatures were 20℃, 25℃ and 30℃, respectively, and the results are shown in the following table.

[0076] Table 6 results of different column temperature investigation experiments

[0077]

[0078]

[0079] Suitable chromatographic parameters were selected according to the influence of the system suitability parameters of each chromatographic peak of the characteristic chromatogram, such as peak purity, peak shape, separation degree, symmetry factor, total peak area, etc. The above results show that the separation effects of the chromatographic peaks of the chromatograms obtained at different column temperatures are basically consistent, and 25℃ is preferably selected as the column temperature.

[0080] 5. Selection of mobile phase type

[0081] The test sample solution was prepared according to Example 1, and the type of mobile phase B was used as a variable, and other conditions were determined according to Example 1. The mobile phase B was 0.1% formic acid, 0.1% phosphoric acid and 0.1% glacial acetic acid, respectively. The results are shown in the following table.

[0082] Table 7 Results of the experiment of different acid types

[0083]

[0084]

[0085] The above results show that when 0.1% formic acid is used as the mobile phase B, the baseline of the obtained chromatogram drifts, and there are relatively more impurity peaks. When 0.1% glacial acetic acid is used as the mobile phase B, the peak purity is poorer than that of the other two groups. Considering comprehensively, 0.1% phosphoric acid is preferably used as the mobile phase B.

[0086] 6. Investigation of the concentration of mobile phase B

[0087] The test sample solution was prepared according to Example 1, and the concentration of mobile phase B was used as a variable, and other conditions were determined according to Example 1. The concentration of mobile phase B was 0.05% phosphoric acid, 0.1% phosphoric acid and 0.15% phosphoric acid, respectively. The results are shown in the following table.

[0088] Table 8 Results of the experiment of different acid concentrations

[0089]

[0090] The above results show that when the concentration of phosphoric acid is 0.05%, the baseline of the chromatogram drifts and the angle of peak 10 is greater than the threshold value, and the peak purity is slightly poor. When the concentration of phosphoric acid is 0.1% and 0.15%, the separation effect of each proposed characteristic peak is basically consistent, and 0.1% phosphoric acid aqueous solution with a lower concentration is preferably used as the mobile phase B.

[0091] 7. Selection of the chromatographic column

[0092] The test sample solution was prepared according to Example 1, and the chromatographic column was used as a variable, and other conditions were determined according to Example 1. The chromatographic column was CORTECS T3 column (chromatographic column 1), ACQUITY BEH (chromatographic column 3) and Aiglent SB column (chromatographic column 7), respectively. The results are shown in the following table.

[0093] Table 9 Results of the experiment of different chromatographic columns

[0094]

[0095] The results show that the separation effect of each proposed characteristic peak obtained by ACQUITY BEH is good, and it is preferably used as the chromatographic column of the application.

[0096] 8. Selection of instrument

[0097] The sample solution was prepared according to Example 1, with different instruments as variables, and other conditions were determined according to Example 1. The instruments were Aiglent 1260 (chromatograph 1) and ACQUITY UPLC (chromatograph 2), respectively. The results are shown in the following table and Figure 1 .

[0098] Table 10. Results of investigation of different chromatographs

[0099]

[0100]

[0101] The above results show that the separation effects of the proposed characteristic peaks of the chromatograms obtained by different instruments are basically consistent, and different instruments are suitable for the present application.

[0102] Based on the above, the chromatographic conditions are determined as follows: according to high performance liquid chromatography, octadecylsilane-bonded silica gel is used as the filler, the column length is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.7 μm; acetonitrile is used as the mobile phase A, and 0.1% phosphoric acid aqueous solution is used as the mobile phase B, the flow rate is 0.3 ml / min, the column temperature is 25°C, the detection wavelength is 257 nm, and the theoretical plate number calculated according to the rutin peak should not be less than 2000; gradient elution is used, and the gradient elution program is as follows: 0-4 min, the volume percentage of the mobile phase A is 10%→15%, and the volume percentage of the mobile phase B is 90%→85%; 4-10 min, the volume percentage of the mobile phase A is 15%→16%, and the volume percentage of the mobile phase B is 85%→84%; 10-16 min, the volume percentage of the mobile phase A is 16%→35%, and the volume percentage of the mobile phase B is 84%→65%; 16-19 min, the volume percentage of the mobile phase A is 35%→50%, and the volume percentage of the mobile phase B is 65%→50%; 19-20 min, the volume percentage of the mobile phase A is 50%→10%, and the volume percentage of the mobile phase B is 50%→90%.

[0103] 1. Selection of extraction method

[0104] The standard flos sophorae soup freeze-dried powder was used as the sample, and the sample solution was prepared by heating reflux method and ultrasonic extraction method, respectively. The ultrasonic extraction method was the same as that in Example 1, and the heating reflux method specifically included the following steps: 0.1 g of the standard flos sophorae soup freeze-dried powder was accurately weighed, placed in a conical flask with a plug, 50 ml of methanol was accurately added, the weight was determined, heated for reflux for 30 min, cooled, the weight was determined again, the lost weight was made up with methanol, shaken uniformly, and filtered. The results are shown in the following table.

[0105] Table 11. Results of investigation of different extraction methods

[0106]

[0107]

[0108] The above results show that there is no significant difference between the chromatograms obtained by heating reflux method and ultrasonic extraction method. Considering the simplicity and operability of the method, ultrasonic extraction is preferred as the extraction method.

[0109] 2. Selection of ultrasonic power

[0110] The standard flos sophorae decoction lyophilized powder was used as the test sample, the ultrasonic power was used as the variable, the test sample solution was prepared according to Example 1, and the determination was performed; the ultrasonic power was 200 W, 300 W and 400 W, respectively. The results are shown in the following table.

[0111] Table 12 Investigation results of different ultrasonic powers

[0112]

[0113]

[0114] The above results show that when the ultrasonic power is 200-400 W, all the components can be completely extracted. Considering the durability of the method, 300 W is preferred as the ultrasonic extraction power.

[0115] 3. Selection of extraction solvent type

[0116] The standard flos sophorae decoction lyophilized powder was used as the test sample, the extraction solvent type was used as the variable, the test sample solution was prepared according to Example 1, and the determination was performed; the extraction solvent types were methanol, anhydrous ethanol and water, respectively. The results are shown in the following table.

[0117] Table 13 Investigation results of different extraction solvent types

[0118]

[0119] In the table, " / " indicates that there is no corresponding chromatographic peak. The above results show that the total peak area and the converted relative average deviation of the chromatograms obtained by the three extraction solvents are 51.19%; among them, the number of chromatographic peaks obtained by using methanol as the extraction solvent is the most and the total peak area is the largest, indicating that methanol as the extraction solvent is the most sufficient, and therefore methanol is preferred as the extraction solvent.

[0120] 4. Selection of extraction solvent concentration

[0121] The standard flos sophorae decoction lyophilized powder was used as the test sample, the extraction solvent concentration was used as the variable, the test sample solution was prepared according to Example 1, and the determination was performed; the extraction solvent concentrations were 30% methanol, 50% methanol, 70% methanol and methanol, respectively. The results are shown in the following table.

[0122] Table 14 Investigation results of different extraction solvent concentrations

[0123]

[0124]

[0125] The " / " in the table means that there is no corresponding chromatographic peak. The above results show that, considering the number of chromatographic peaks, the total peak area and the ease of operation, methanol with a concentration not less than 70% is preferably selected as the extraction solvent.

[0126] 5. Selection of the amount of extraction solvent

[0127] The standard decoction lyophilized powder of sophora fruit was used as the test sample, the amount of extraction solvent was used as the variable, the test sample solution was prepared according to Example 1, and the determination was performed; the sample amount of the test sample was 0.1 g, and the amount of extraction solvent methanol was 25 ml, 50 ml and 100 ml, respectively. The results are shown in the following table.

[0128] Table 15 Investigation results of different extraction solvent amounts

[0129]

[0130]

[0131] The above results show that, when the amount of extraction solvent is different, there is no significant difference in the total peak area after conversion, when the amount of extraction solvent is 50 ml, the total peak area is consistent with that of the control medicinal material, and when the amount of solvent is 100 ml, the relative response value of each peak is relatively low. Considering comprehensively, 50 ml is preferably selected as the amount of extraction solvent.

[0132] 6. Selection of extraction time

[0133] The standard decoction lyophilized powder of sophora fruit was used as the test sample, the extraction time was used as the variable, the test sample solution was prepared according to Example 1, and the determination was performed; the extraction time was 15 min, 30 min, 45 min and 60 min, respectively. The results are shown in the following table.

[0134] Table 16 Investigation results of different extraction times

[0135]

[0136]

[0137] The above results show that, the chromatograms obtained by four different extraction times have no obvious difference in the total peak area after conversion, and considering the extraction efficiency, 30 min is preferably selected as the extraction time.

[0138] 7. Selection of sample amount of test sample

[0139] The standard flos sophorae soup freeze-dried powder was used as the test sample, the sample amount was used as the variable, the test sample solution was prepared according to Example 1, and the determination was performed; the sample amounts were 0.05 g, 0.1 g, 0.2 g, and 0.4 g, respectively. The results are shown in the following table.

[0140] Table 17 Investigation results of different sample amounts

[0141]

[0142]

[0143] After conversion, there was no obvious difference, and the extraction efficiency was basically consistent. Considering the accuracy of sampling and the matching degree of the reference solution, 0.1 g was selected as the sample amount.

[0144] In summary, the extraction scheme is preferably: take 0.1 g of the test sample, accurately weigh it, place it in a conical flask with a stopper, accurately add 50 ml of methanol, weigh it, ultrasonic treat (power 300 W, frequency 40 kHz) for 30 minutes, cool, weigh it again, make up the weight loss with methanol, shake well, filter, and obtain.

[0145] Experimental Example 3 Construction and verification of the fingerprint spectrum

[0146] I. Construction of the flos sophorae fingerprint spectrum

[0147] Preparation of the test sample solution: take 0.1 g of the flos sophorae standard soup freeze-dried powder, accurately weigh it, place it in a conical flask with a stopper, accurately add 50 ml of methanol, weigh it, ultrasonic treat (power 300 W, frequency 40 kHz) for 30 minutes, cool, weigh it again, make up the weight loss with methanol, shake well, filter, and obtain.

[0148] Preparation of the reference solution: take about 0.3 g of the flos sophorae control medicinal material, place it in a conical flask with a stopper, add 50 ml of water, heat and reflux for 45 minutes, take it out, filter, evaporate the filtrate to dryness, add 50 ml of methanol to the residue, ultrasonic treat (power 300 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and obtain the control medicinal material reference solution.

[0149] Preparation of the reference solution: take appropriate amounts of rutin, kaempferol-3-O-rutinoside, zephyranthine, and quercetin reference substances, add methanol to prepare a mixed solution containing 50 μg of rutin, 10 μg of kaempferol-3-O-rutinoside, 20 μg of zephyranthine, and 5 μg of quercetin per 1 ml, as the reference solution.

[0150] Chromatographic conditions: determined by high performance liquid chromatography, octadecylsilane-bonded silica gel as the filler, column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm; acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, flow rate 0.3 ml / min, column temperature 25 °C, detection wavelength 257 nm, the theoretical plate number calculated by rutin peak should not be less than 2000; gradient elution, gradient elution program: 0-4 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 4-10 min, volume percentage of mobile phase A 15%→16%, volume percentage of mobile phase B 85%→84%; 10-16 min, volume percentage of mobile phase A 16%→35%, volume percentage of mobile phase B 84%→65%; 16-19 min, volume percentage of mobile phase A 35%→50%, volume percentage of mobile phase B 65%→50%; 19-20 min, volume percentage of mobile phase A 50%→10%, volume percentage of mobile phase B 50%→90%.

[0151] Accurately take 1 μL of the above solution respectively, inject into the liquid chromatograph, and determine, to obtain.

[0152] The characteristic spectrum of sophora flower bud has 11 characteristic peaks, which correspond to 11 characteristic peaks in the reference chromatogram of the control medicinal material, respectively, 1st peak, 2nd peak, 3rd peak, 4th peak, 5th peak, 6th peak, 7th peak, 8th peak, 9th peak, 10th peak and 11th peak. Take the 5th peak as the reference peak 1, the relative retention times of the 1st peak, 2nd peak, 3rd peak and 4th peak are 0.63, 0.70, 0.79 and 0.88 respectively; take the 9th peak as the reference peak 2, the relative retention times of the 8th peak, 10th peak and 11th peak are 0.93, 1.11 and 1.13 respectively; the relative retention time is within ±10% of the specified value.

[0153] Compared with the control, the 5th peak is rutin, the 6th peak is kaempferol-3-O-rutinoside, the 7th peak is narcissin, and the 9th peak is quercetin.

[0154] II. Control spectrum

[0155] The similarity evaluation software of chromatographic fingerprint of traditional Chinese medicines "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicines 2012 Edition" compiled by the Pharmacopoeia Commission is used to generate the control characteristic spectrum of the standard decoction pieces of sophora flower (sophora flower bud) of multiple batches with representativeness; and the chemical components of each characteristic peak of sophora flower are analyzed. The relative retention time and relative peak area of the control spectrum are shown in the following table and Figure 2 , and R in the figure is the control spectrum obtained by fitting. Figure 3 is the control spectrum of sophora flower bud.

[0156] Table 18 Relative retention time of different batches of Flos Sophorae Mixture Granules and standard decoction lyophilized powder characteristic chromatogram

[0157]

[0158]

[0159] Table 19 Relative peak area of different batches of Flos Sophorae Mixture Granules and standard decoction lyophilized powder characteristic chromatogram

[0160] Sample Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S1) Peak 6 Peak 7 Peak 8 Peak 9 (S2) Peak 10 Peak 11 S1 0.04 0.01 0.02 0.01 1.00 0.06 1.54 0.09 1.00 0.08 0.14 S2 0.04 0.01 0.02 0.01 1.00 0.06 1.46 0.08 1.00 0.09 0.13 S3 0.04 0.01 0.02 0.01 1.00 0.06 1.35 0.08 1.00 0.08 0.13 S4 0.04 0.01 0.02 0.01 1.00 0.06 2.25 0.16 1.00 0.11 0.17 S5 0.03 0.01 0.01 0.01 1.00 0.04 1.93 0.10 1.00 0.08 0.16 S6 0.03 0.01 0.01 0.01 1.00 0.04 1.90 0.11 1.00 0.07 0.18 S7 0.03 0.01 0.01 0.01 1.00 0.04 1.62 0.09 1.00 0.07 0.17 S8 0.03 0.01 0.02 0.01 1.00 0.06 1.88 0.08 1.00 0.10 0.16 S9 0.02 0.00 0.01 0.01 1.00 0.02 2.35 0.13 1.00 0.04 0.18 S10 0.03 0.01 0.01 0.01 1.00 0.05 1.54 0.11 1.00 0.06 0.14 S11 0.02 0.01 0.00 0.01 1.00 0.03 1.32 0.08 1.00 0.05 0.10 S12 0.02 0.02 0.01 0.02 1.00 0.04 1.35 0.10 1.00 0.05 0.11 S13 0.04 0.01 0.02 0.01 1.00 0.06 1.42 0.09 1.00 0.09 0.15 S14 0.03 0.01 0.01 0.01 1.00 0.06 1.38 0.13 1.00 0.14 0.10 S15 0.03 0.01 0.02 0.01 1.00 0.06 1.55 0.09 1.00 0.09 0.14 S16 0.03 0.01 0.02 0.01 1.00 0.05 1.89 0.06 1.00 0.06 0.13 S17 0.03 0.01 0.02 0.01 1.00 0.05 1.73 0.06 1.00 0.07 0.13 S18 0.03 0.02 0.02 0.01 1.00 0.05 1.79 0.07 1.00 0.07 0.13 S19 0.03 0.01 0.02 0.01 1.00 0.05 1.55 0.04 1.00 0.07 0.15 S20 0.03 0.01 0.02 0.01 1.00 0.05 1.54 0.04 1.00 0.07 0.15 S21 0.03 0.01 0.02 0.01 1.00 0.05 1.55 0.04 1.00 0.07 0.15 Mean 0.03 0.01 0.02 0.01 1.00 0.05 1.66 0.09 1.00 0.08 0.14 RSD% 17.90％ 22.23％ 32.19％ 24.09％ 0.00％ 20.14％ 17.29％ 36.00％ 0.00％ 29.76％ 16.62％

[0161] III. Identification of characteristic peaks

[0162] The characteristic peaks were identified and assigned by HPLC and LC / MS / MS, and it was determined that peak No. 9 was quercetin, peak No. 10 was kaempferol, and peak No. 11 was isorhamnetin, and these three components were flavonoid aglycone. Peak No. 2 was quercetin 3-O-rutinoside-7-O-rhamnoside, peak No. 5 was rutin, peak No. 6 was kaempferol-3-O-rutinoside, and peak No. 7 was tazettin, and these four components were flavonoid glycosides. Peak No. 1 was quercetin 3-O-β-D-glucosyl(l→2)[α-L-rhamnosyl l(l→6)]-β-D-glucoside, and peak No. 4 was quercetin-3-O-β-xylosyl(l→3)-O-α-L-rhamnosyl-(l→6)-O-β-D-glucoside, and these two components were flavonoid aglycone. Peak No. 8 was a benzene derivative. See the following table.

[0163] Table 20 Analysis results of Flos Sophorae by LC / MS / MS

[0164]

[0165]

[0166] IV. Methodology validation

[0167] 1. Whether the negative sample interferes

[0168] Prepare a negative sample (excipient maltodextrin) and measure it, and the results are shown in Table 18. Figure 4 The results show that the negative sample does not interfere, the chromatographic method is suitable for system suitability and specificity, and can be used as the detection method for the characteristic chromatogram of Flos Sophorae Mixture Granules.

[0169] 2. Instrument precision

[0170] The fomesone granules were used as the test sample, and the test sample solution was prepared according to Example 1. The sample was injected 6 times and determined according to Example 1. The results showed that the relative retention time RSD of peaks 1-4 to peak S2 was less than 2%, and the relative retention time RSD of peaks 8, 10 and 11 to peak S2 was less than 2%, indicating that the instrument precision was good.

[0171] 3. Reproducibility

[0172] The fomesone granules were used as the test sample, and the test sample solution was prepared according to Example 1. The sample was injected 6 times and determined according to Example 1. The results showed that the relative retention time RSD of peaks 1-4 to peak S2 was less than 2%, and the relative retention time RSD of peaks 8, 10 and 11 to peak S2 was less than 2%, indicating that the instrument precision was good.

[0173] 4. Intermediate precision

[0174] The same fomesone granules were used to prepare the test sample solution according to Example 1 by 3 different inspectors at different times, and the relative retention time and relative peak area of each common peak were determined using the same equipment. The results showed that the relative retention time RSD of peaks 1-4 to peak S1 was less than 2%, and the relative retention time RSD of peaks 8, 10 and 11 to peak S2 was less than 2%, indicating that the intermediate precision of the method was good.

[0175] 5. Stability

[0176] The same fomesone granules were used to prepare the test sample solution according to Example 1, and the sample was injected at 0, 4, 8, 12, 16 and 24 h, respectively. The relative retention time and relative peak area of each common peak were determined. The results are shown in the following table. The results showed that the relative retention time RSD of peaks 1-4 to peak S1 was less than 2%, and the relative retention time RSD of peaks 8, 10 and 11 to peak S2 was less than 2%, indicating that the test sample solution was stable within 24 h and met the determination requirements.

[0177] Table 21 Stability relative retention time results

[0178]

[0179] Table 22 Stability relative peak area results

[0180]

[0181] 6. Column temperature

[0182] Take the same portion of Flos Sophorae formula granules, with column temperature as the variable, prepare the test solution according to Example 1 and measure, the column temperature is 23℃, 25℃ and 27℃ respectively, investigate the influence of different column temperature on the relative retention time and relative peak area of each characteristic peak, the results are shown in the following table. The results show that: with the change of column temperature, the relative retention time of each characteristic chromatographic peak changes less.

[0183] Table 23 Relative retention time at different column temperatures

[0184]

[0185] Table 24 Relative peak area at different column temperatures

[0186]

[0187] 7, Flow rate

[0188] Take the same portion of Flos Sophorae formula granules, with flow rate as the variable, prepare the test solution according to Example 1 and measure, the flow rate is 0.28ml / min, 0.30ml / min and 0.32ml / min respectively, investigate the influence of the test solution on the relative retention time and relative peak area of each characteristic common peak when the flow rate changes slightly, the results are shown in the following table. The results show that: the flow rate has less influence on the relative retention time of each characteristic peak when the flow rate changes slightly.

[0189] Table 25 Relative retention time at different flow rates

[0190]

[0191] Table 26 Relative peak area at different flow rates

[0192]

[0193] 8, Investigation of different chromatographic columns

[0194] Take the same portion of Flos Sophorae formula granules, with chromatographic column as the variable, prepare the test solution according to Example 1 and measure, the chromatographic column is ① ACQUITY BEH C18, 2.1mm×100mm, 1.7μm (chromatographic column 3); ② Agilent SB-C18 RRHD, 2.1mm×100mm, 1.8μm (chromatographic column 6); ③ CAPCELL PAK C18, 2.0mm×100mm, 2μm (chromatographic column 2), the results are shown in the following table. The results show that: combined with the separation effect of each chromatographic column, the chromatographic column 1 is preferred for the determination of Flos Sophorae formula granules.

[0195] Table 27 Relative retention time at different chromatographic columns

[0196]

[0197] Table 28 Relative peak area of different chromatographic columns

[0198]

[0199]

[0200] 9. Investigation of chromatographic column batch

[0201] Take the same batch of sophora fruit formula granules, with 3 batches of different batch numbers of chromatographic column ACQUITY BEH C18, 2.1 mm x 100 mm, 1.7 μm (chromatographic columns 3-5) as variables, prepare the test solution according to Example 1 and determine, investigate the influence of the change of chromatographic column batch number on the relative retention time and relative peak area of each characteristic peak, the results are shown in the table below. The results show that the deviation of the relative retention time of each characteristic peak is less than 2%, and the relative retention time of each characteristic peak will not change with the change of chromatographic column batch number.

[0202] Table 29 Relative retention time results of different batches of chromatographic columns

[0203]

[0204] Table 30 Relative peak area results of different batches of chromatographic columns

[0205]

[0206] Example 3

[0207] The present embodiment provides a method for identifying sophora fruit and fried sophora fruit, comprising the following steps:

[0208] Preparation of test solution: Take 0.1 g of sophora fruit standard decoction lyophilized powder and fried sophora fruit standard decoction lyophilized powder respectively, accurately weigh, place in a conical flask with a plug, accurately add 50 ml of methanol, weigh, ultrasonic treat (power 300 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the weight loss with methanol, shake well, filter to obtain sophora fruit test solution and fried sophora fruit test solution respectively.

[0209] Determination, respectively, 1 μL of the above solution was precisely pipetted, injected into a liquid chromatograph, and determined. Among them, acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, octadecylsilane-bonded silica gel was used as filler, the specification was that the column length was 100 mm, the inner diameter was 2.1 mm, the particle size was 1.7 μm, the column temperature was 25°C, the flow rate was 0.3 ml / min, the detection wavelength was 257 nm, the theoretical plate number calculated according to the rutin peak should not be less than 2000, gradient elution, the gradient elution program was as follows: 0-4 min, the volume percentage of mobile phase A was 10%→15%, the volume percentage of mobile phase B was 90%→85%; 4-10 min, the volume percentage of mobile phase A was 15%→16%, the volume percentage of mobile phase B was 85%→84%; 10-16 min, the volume percentage of mobile phase A was 16%→35%, the volume percentage of mobile phase B was 84%→65%; 16-19 min, the volume percentage of mobile phase A was 35%→50%, the volume percentage of mobile phase B was 65%→50%; 19-20 min, the volume percentage of mobile phase A was 50%→10%, the volume percentage of mobile phase B was 50%→90%.

[0210] The peak area ratio of peaks No. 5 and No. 9 in the characteristic spectrum of sophora fruit is ≤14; when the ratio is greater than 14, the test product is fried sophora fruit.

[0211] The peak area ratio of peaks No. 5 and No. 9 in the characteristic spectrum of sophora fruit is ≤14; when the ratio is greater than 14, the test product is fried sophora fruit.

[0212] Table 31 Peak area ratio of peaks No. 5 and No. 9 in different batches of sophora fruit

[0213]

[0214] From the above results, it can be seen that the relative peak area of peak No. 5 in the characteristic spectrum of sophora fruit is actually measured in the range of 8.59-13.31, which is significantly lower than the actually measured range of fried sophora fruit, i.e. 15.40-38.16. The lower limit of the actually measured range of fried sophora fruit is 15.4 x 0.95 = 14.6, and the peak area ratio of peaks No. 5 and No. 9 in the characteristic spectrum of fried sophora fruit should not be lower than this value; the upper limit of the actually measured range of sophora fruit is 13.31 x 1.05 = 14, and the peak area ratio of peaks No. 5 and No. 9 in the characteristic spectrum of sophora fruit should not be higher than this value. Therefore, taking the peak area ratio of peaks No. 5 and No. 9 not higher than 14 as the basis for identifying fried sophora fruit and sophora fruit.

[0215] Example 4

[0216] The present embodiment provides a method for identifying sophora fruit and sophora flower, comprising the following steps:

[0217] Preparation of test solution: 0.1 g of flos sophorae tonkinensis standard infusion lyophilized powder of different batches was precisely weighed and placed in a conical flask with a stopper. 50 ml of methanol was precisely added, the weight was determined, and the mixture was ultrasonically treated (power 300 W, frequency 40 kHz) for 30 min. The mixture was allowed to cool, the weight was determined again, the lost weight was made up with methanol, and the mixture was shaken and filtered to obtain flos sophorae tonkinensis test solution.

[0218] Determination: 1 μL of each of the above solutions was precisely taken and injected into a liquid chromatograph for determination. The mobile phase A was acetonitrile, the mobile phase B was 0.1% phosphoric acid aqueous solution, the filler was octadecylsilane-bonded silica gel, the specification was that the column length was 100 mm, the inner diameter was 2.1 mm, the particle size was 1.7 μm, the column temperature was 25°C, the flow rate was 0.3 ml / min, the detection wavelength was 257 nm, the theoretical plate number calculated according to the rutin peak should not be less than 2000, and gradient elution was performed. The gradient elution program was as follows: 0-4 min, volume percentage of mobile phase A 10%→15%, volume percentage of mobile phase B 90%→85%; 4-10 min, volume percentage of mobile phase A 15%→16%, volume percentage of mobile phase B 85%→84%; 10-16 min, volume percentage of mobile phase A 16%→35%, volume percentage of mobile phase B 84%→65%; 16-19 min, volume percentage of mobile phase A 35%→50%, volume percentage of mobile phase B 65%→50%; 19-20 min, volume percentage of mobile phase A 50%→10%, volume percentage of mobile phase B 50%→90%.

[0219] The peak area ratio of peaks 7 and 9 in the characteristic spectrum of flos sophorae tonkinensis was >0.9. The peak area ratio of peaks 7 and 9 in the characteristic spectra of flos sophorae tonkinensis of multiple batches was verified, and the results are shown in the following table.

[0220] Table 32 Peak area of peaks 7 and 9 in flos sophorae tonkinensis of multiple batches

[0221] Sample Peak area ratio of peak 7 to peak 9 2009001Y 1.54 2009003Y 1.46 2009006Y 1.35 2009007Y 2.25 2009009Y 1.93 2009010Y 1.90 2009011Y 1.62 2009012Y 1.88 2009016Y 2.35 2009019Y 1.54 2009020Y 1.32 2009022Y 1.35 2009024Y 1.42 2009025Y 1.38 2009027Y 1.55 210901Y 1.89 210902Y 1.73 210903Y 1.79 2111001Y 1.55 2111002Y 1.54 2111003Y 1.55 Mean 1.66 RSD% 17.29％ Maximum 2.35 Minimum 1.32

[0222] The above results show that the relative peak area of peak 7 in the characteristic spectrum of flos sophorae tonkinensis is 1.32-2.35. The lower limit of the measured range of flos sophorae tonkinensis is 1.32 x 0.7 = 0.92, which is rounded to 0.9. Therefore, the relative peak area of peaks 7 and 9 not less than 0.9 can be used as a basis for identification of flos sophorae tonkinensis.

[0223] Obviously, the above examples are only examples for the purpose of clear illustration, and are not intended to limit the embodiments. Based on the above description, those skilled in the art can also make other different forms of changes or modifications. It is not necessary or possible to exhaust all embodiments. The obvious changes or modifications derived therefrom are still within the protection scope of the present application.

Claims

1. A method for constructing a fingerprint of sophora fruit, characterized in that, The method comprises the following steps: Preparation of the test sample solution: the test sample is prepared into a test sample solution; The test sample solution is detected by using the ultra-high performance liquid chromatography, acetonitrile is used as the mobile phase A, and the aqueous phosphoric acid solution is used as the mobile phase B, gradient elution, and the gradient elution program comprises 0-4 min, the volume percentage of the mobile phase A is 10%→15%, and the volume percentage of the mobile phase B is 90%→85%; 4-10 min, the volume percentage of the mobile phase A is 15%→16%, and the volume percentage of the mobile phase B is 85%→84%; 10-16 min, the volume percentage of the mobile phase A is 16%→35%, and the volume percentage of the mobile phase B is 84%→65%; 16-19 min, the volume percentage of the mobile phase A is 35%→50%, and the volume percentage of the mobile phase B is 65%→50%; 19-20 min, the volume percentage of the mobile phase A is 50%→10%, and the volume percentage of the mobile phase B is 50%→90%; The model and specification of the chromatographic column are ACQUITY BEH C18, 1.7 µm, 2.1×100 mm; The detection wavelength is 250-265 nm; The test sample is a standard decoction of sophora fruitling medicinal material, sophora fruitling crude drug, sophora fruitling freeze-dried powder or sophora fruitling formula granules; When the test sample is the standard decoction of sophora fruitling medicinal material, the extraction solvent used for extraction of the sophora fruitling freeze-dried powder or the sophora fruitling formula granules is a 70-100% methanol solution; When the test sample is the sophora fruitling crude drug, the extraction method is as follows: 0.3 g of sophora fruitling crude drug powder is placed in a conical flask with a plug, 50 ml of water is added, heating reflux is carried out for 45 min, the solution is taken out, filtered, the filtrate is evaporated to dryness, 50 ml of methanol is added to the residue, ultrasonic treatment is carried out for 30 min, the power of the ultrasonic treatment is 300 W, the frequency is 40 kHz, the solution is cooled, shaken uniformly, filtered, and the filtrate is taken, thus obtaining the test sample solution; The fingerprint of the sophora fruitling comprises 11 characteristic peaks, which are peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10 and peak 11; The peak 1 is quercetin 3-O-β-D-glucosyl(1→2)[α-L-rhamnosyl l(1→6)]-β-D-glucoside, the peak 2 is quercetin 3-O-rutinoside-7-O-rhamnoside, the peak 3 is isorhamnetin 3-O-[2-O-β-glucosyl-6-O-rhamnosyl]-β-glucoside, the peak 4 is quercetin-3-O-β-xylosyl(1→3)-O-α-L-rhamnosyl-(1→6)-O-β-D-glucoside, the peak 5 is rutin, the peak 6 is kaempferol-3-O-rutinoside, the peak 7 is zephyranthine, the peak 9 is quercetin, the peak 10 is kaempferol, and the peak 11 is isorhamnetin.

2. The construction method of claim 1, wherein, The chromatographic conditions of the ultra-high performance liquid chromatography further comprise: the column temperature is 20-30 ℃; and / or, the flow rate is 0.2-0.4 ml / min; and / or, the injection volume is 0.5-2 µL; and / or, the mobile phase B is an aqueous phosphoric acid solution with a concentration of 0.05-0.3%.

3. The construction method of claim 1, wherein, The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: the column temperature is 25 DEG C, the flow rate is 0.3 ml / min, the injection volume is 1 mu L, the detection wavelength is 257 nm, and the mobile phase B is 0.1% phosphoric acid aqueous solution.

4. The construction method of claim 1, wherein, When the test sample solution is prepared, the ratio of the mass of the test sample to the volume of the extraction solvent is (0.001-0.008):1, the unit of the mass is g, and the unit of the volume is ml.

5. The construction method of claim 1, wherein, The fingerprint spectrum of the sophora fruit includes 11 characteristic peaks, namely, peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10 and peak 11. Taking peak 5 as the reference peak 1, the relative retention times of peak 1, peak 2, peak 3 and peak 4 are 0.63, 0.70, 0.79 and 0.88 respectively. Taking peak 9 as the reference peak 2, the relative retention times of peak 8, peak 10 and peak 11 are 0.93, 1.11 and 1.13 respectively. The relative retention time is within ±10% of the specified value.

6. The construction method of claim 1, wherein, The peak area ratio of peak 5 to peak 9 in the characteristic spectrum obtained by the construction method is ≤14; and / or the peak area ratio of peak 7 to peak 9 is >0.

9.

7. The construction method according to any one of claims 1 to 6, characterized in that, The preparation of the control solution is also included. At least one of rutin, kaempferol-3-O-rutinoside, narcissin and quercetin is used as the control.

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