A processing method of sophora fruit, fingerprint spectrum and application thereof

Abstract

The application discloses a processing method of sophora fruit, a fingerprint spectrum and application of the processing method. Specifically, the application discloses a processing method, which comprises the following steps: adopting liquid chromatography to perform gradient elution on sophora fruit to be detected; in the liquid chromatography, the chromatographic conditions are as follows: an octadecylsilane bonded silica gel chromatographic column is used as a chromatographic column; a mobile phase comprises mobile phase A and mobile phase B; the mobile phase A is a formic acid solution; and the mobile phase B is methanol. The application further discloses a fingerprint spectrum obtained by the processing method. The fingerprint spectrum provided by the application can separate and qualitatively identify multiple effective components of sophora fruit crude products or sophora fruit carbon, can quickly and accurately evaluate the quality of the sophora fruit crude products or the sophora fruit carbon, can more comprehensively characterize the chemical information in the sophora fruit and the sophora fruit carbon, is used for guiding industrial production, better guarantees product quality, and provides a scientific and effective method for research on an efficacy material basis of the sophora fruit crude products or the sophora fruit carbon and quality control.

Description

Technical Field

[0001] This invention relates to a method for processing Sophora japonica pods, fingerprint spectrum, and its application. Background Technology

[0002] Sophora japonica fruit, the dried, ripe fruit of the legume Sophora japonica L., is cold in nature and bitter in taste, and enters the liver and large intestine meridians. It has the effects of clearing heat and purging fire, cooling blood and stopping bleeding, and is used for symptoms such as intestinal heat causing hematochezia, bleeding from hemorrhoids, liver heat causing headache, dizziness, and red eyes. The main components of Sophora japonica fruit include flavonoids, isoflavones, alkaloids, triterpenoid saponins, amino acids, and stearic acid, among which isoflavones and their glycosides are the most abundant. Sophora japonica fruit has anti-cancer and estrogen-like effects, and has a good preventive and therapeutic effect on cancer, cardiovascular diseases, osteoporosis, and menopausal syndrome in women. Raw Sophora japonica fruit has a stronger effect in clearing heat and cooling blood, and is used to cool blood and stop bleeding. Charred Sophora japonica fruit has a significantly reduced cold nature and is better at astringing and stopping bleeding. The appearance and efficacy of Sophora japonica fruit change after processing, which is speculated to be related to the changes in active substances during the charring process. Sophora japonica charcoal is used as the principal ingredient in the classic formula Liangxue Dihuang Decoction, and its application is also recorded in collections of empirical prescriptions and Yang's Family Prescriptions. Currently, research on the processing of Sophora japonica charcoal is not systematic or in-depth enough. Standards for its properties are not yet unified, and judgment is based solely on subjective experience, resulting in significant differences in the quality of processed products from different regions, leading to substantial variations in clinical effects. There is also limited research on the identification of components before and after charring Sophora japonica charcoal, and no analysis of the hemostatic material basis of Sophora japonica charcoal is available.

[0003] The 2021 issue of Volume 52, Issue 21 of the Chinese Traditional and Herbal Drugs journal published a study by Li Jingfeng et al. on the establishment of HPLC fingerprint chromatograms and chemical pattern recognition of Sophora japonica fruit from different origins. The method used an Agilent Zorbax SB C18 column on an Agilent 1260 high-performance liquid chromatograph, with a detection wavelength of 260 nm, a flow rate of 0.6 mL / min, and isocratic elution using methanol-acetonitrile-0.07% phosphoric acid water (12:20:68) as the mobile phase. Nine common peaks were obtained, but only rutin, genistein, and sennain were identified, resulting in somewhat limited chemical information. Existing methods do not provide comprehensive information on the components of Sophora japonica fruit, making it difficult to objectively, accurately, and comprehensively evaluate the quality of both raw and charred Sophora japonica fruit. Therefore, establishing fingerprint chromatogram methods for raw and charred Sophora japonica fruit is of great significance for their quality control. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings of existing technologies, such as the limited number of detection methods for raw or charred Sophora japonica fruit, the limited number of detectable components, and the lack of effective and efficient methods for evaluating the quality of raw or charred Sophora japonica fruit. This invention provides a method for processing Sophora japonica fruit, a fingerprint spectrum, and its applications. The fingerprint spectrum provided by this invention can quickly and accurately evaluate the quality of raw or charred Sophora japonica fruit by separating and qualitatively identifying multiple effective components. It can comprehensively characterize the chemical information in Sophora japonica fruit and charred Sophora japonica fruit, guiding industrial production, better ensuring product quality, and providing a scientific and effective method for the research and quality control of the pharmacodynamic material basis of raw or charred Sophora japonica fruit.

[0005] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0006] This invention provides a processing method comprising the following steps:

[0007] The analytes from Sophora japonica fruit can be obtained by gradient elution using liquid chromatography.

[0008] In the liquid chromatography method, the chromatographic conditions are as follows:

[0009] The chromatographic column is an octadecylsilane-bonded silica gel column;

[0010] The mobile phase is either mobile phase A-mobile phase B or mobile phase A-mobile phase B, wherein mobile phase A is formic acid solution, mobile phase B is methanol, and mobile phase C is acetonitrile;

[0011] When the mobile phase is mobile phase A-mobile phase B, with the total volume of the mobile phase being 100%, the gradient elution procedure is performed sequentially from top to bottom according to the following table:

[0012] Wash-off time / minute Mobile phase A Mobile phase B 3-12 x1→x2 (100% - x1) → (100% - x2) 10-20 x2→x3 (100% - x2) → (100% - x3) 0-6 Keep x3 Maintain (100% - x3) 8-10 x3→x4 (100% - x3) → (100% - x4) 0-5 Keep x4 Maintain (100% - x4) 4-7 x4→x5 (100% - x4) → (100% - x5) 0-3 Keep x5 Maintain (100% x 5) 0-6 x5→x6 (100% - x5) → (100% - x6) 0-16 Keep x6 Maintain (100% x 6)

[0013] Among them, x1 is 83%-92% (85% and 90%), x2 is 68%-82% (70% and 80%), x3 is 48%-67% (50% and 60% and 65%), x4 is 38%-48% (40% and 55%), x5 is 18%-45% (20% and 40%), and x6 is 83%-92% (85% and 90%), with x1>x2>x3>x4>x5;

[0014] When the mobile phase is mobile phase A-mobile phase C, with the total volume of the mobile phase being 100%, the gradient elution procedure is performed sequentially from top to bottom according to the following table:

[0015]

[0016]

[0017] Among them, y1 is 90%-95% (93%), y2 is 88%-92% (90%), y3 is 83%-87% (85%), y4 is 78%-82% (80%), y5 is 73%-77% (75%), y6 is 68%-72% (70%), y7 is 53%-58% (55%), y8 is 38%-43% (40%), and y9 is 90%-95% (93%).

[0018] The elution time is the difference between the elution end time and the elution start time of this elution stage.

[0019] The elution time is the difference between the elution end time and the elution start time of the elution stage.

[0020] The chromatographic column can be a Waters ACQUITY CSH C18 column (150 mm × 2.1 mm, 1.7 μm), a 1-ACQUITY CSH C18 column (150 mm × 2.1 mm, 1.7 μm), or a 2-Hypersi1 Gold column (100 mm × 2.1 mm, 1.9 μm); preferably a Waters ACQUITY CSH C18 column (150 mm × 2.1 mm, 1.7 μm).

[0021] In the chromatographic conditions described, x1 can be 85% or 90%.

[0022] In the chromatographic conditions described, x2 can be 70% or 80%.

[0023] In the chromatographic conditions, x3 can be 50%, 60%, or 65%.

[0024] In the chromatographic conditions described, x4 can be 40% or 55%.

[0025] In the chromatographic conditions described, x5 can be 20% or 40%.

[0026] In the chromatographic conditions described, x6 can be 85% or 90%.

[0027] In the chromatographic conditions described, y1 can be 93%.

[0028] In the chromatographic conditions described, y2 can be 90%.

[0029] In the chromatographic conditions described, y3 can be 85%.

[0030] In the chromatographic conditions described, y4 can be 80%.

[0031] In the chromatographic conditions described, y5 can be 75%.

[0032] In the chromatographic conditions described, y6 can be 70%.

[0033] In the chromatographic conditions described, y7 can be 55%.

[0034] In the chromatographic conditions described, y8 can be 40%.

[0035] In the chromatographic conditions described, y9 can be 93%.

[0036] In the chromatographic conditions, the gradient elution program can be any of the following conditions:

[0037] Condition A:

[0038] Wash-off time / minute Mobile phase A Mobile phase B 9 90％→80％ 10％→20％ 18 80％→60％ 20％→40％ 3 Maintain 60% Maintain 40% 9 60％→40％ 40％→60％ 3 Maintain 40% Maintain 60% 6 40％→20％ 60％→80％ 2 Keep it at 20% Maintain 80% 15 Maintain 90% Keep it at 10%

[0039] Condition B:

[0040] Wash-off time / minute Mobile phase A Mobile phase B 4 85％→80％ 15％→20％ 11 80％→65％ 20％→35％ 10 65％→55％ 35％→45％ 5 60％→40％ 40％→60％ 3 Maintain 40% Maintain 60% 5 40％→85％ 60％→15％ 4 Maintain 85% Maintain 85%

[0041] Condition C:

[0042] Wash-off time / minute Mobile phase A Mobile phase B 10 90％→70％ 10％→30％ 20 70％→60％ 30％→40％ 10 60％→40％ 40％→60％ 5 40％→20％ 60％→80％ 5 20％→90％ 80％→10％

[0043] Condition D:

[0044] Wash-off time / minute Mobile phase A Mobile phase B 10 90％→80％ 10％→20％ 15 80％→60％ 20％→40％ 5 Maintain 60% Maintain 40% 10 60％→40％ 40％→60％ 5 40％→20％ 60％→80％ 5 Maintain 90% Keep it at 10%

[0045] Condition E:

[0046] Wash-off time / minute Mobile phase A Mobile phase B 10 90％→80％ 10％→20％ 15 80％→50％ 20％→50％ 5 Keep 50% Keep 50% 10 50％→40％ 50％→60％ 5 40％→20％ 60％→80％ 5 Maintain 90% Keep it at 10%

[0047] Condition F:

[0048] Wash-off time / minute Mobile phase A mobile phase C 10 93％→85％ 7％→15％ 15 85％→70％ 15％→30％ 5 Maintain 70% Keep 30% 10 70％→55％ 30％→45％ 5 55％→40％ 45％→60％ 5 Maintain 93% Maintain 7%

[0049] Condition G:

[0050] Wash-off time / minute Mobile phase A mobile phase C 10 93％→85％ 7％→15％ 15 85％→75％ 15％→25％ 5 75％→70％ 25％→30％ 10 70％→55％ 30％→45％ 5 55％→40％ 45％→60％ 5 Maintain 93% Maintain 7%

[0051] Condition H:

[0052] Wash-off time / minute Mobile phase A Mobile phase C 10 93％→90％ 7％→10％ 15 90％→80％ 10％→20％ 5 80％→70％ 20％→30％ 10 70％→55％ 30％→45％ 5 55％→40％ 45％→60％ 5 40％→93％ 60％→7％

[0053] Condition I:

[0054] Wash-off time / minute Mobile phase A mobile phase C 10 93％→90％ 7％→10％ 15 90％→85％ 10％→15％ 10 85％→80％ 15％→20％ 10 80％→75％ 20％→25％ 5 75％→55％ 25％→45％ 5 55％→40％ 45％→60％ 5 40％→93％ 60％→7％ .

[0055] Preferably, the chromatographic column is a Waters ACQUITY CSH C18 column, the mobile phase is mobile phase A-mobile phase B, and the gradient elution program is shown in the table below:

[0056] Time / minute Mobile phase A Mobile phase B 0～9 90％→80％ 10％→20％ 9～27 80％→60％ 20％→40％ 27～30 60％ 40％ 30～39 60％→40％ 40％→60％ 39～42 40％ 60％ 42～48 40％→20％ 60％→80％ 48～50 20％ 80％ 50～50.1 20％→90％ 80％→10％ 50.1～65 90％ 10％ .

[0057] The liquid chromatography method can be high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC).

[0058] In the chromatographic conditions, the concentration of formic acid in the mobile phase A can be conventional in the art, for example, a 0.05-0.5% formic acid solution, or, for example, a 0.05% formic acid solution.

[0059] The chromatographic conditions described may use detectors conventional in the art, such as UV-DAD detectors.

[0060] In the chromatographic conditions, conventional detection wavelengths in the art can be used, such as 210-300 nm, or 230, 254 or 280 nm, with 278 or 280 nm being preferred.

[0061] In the chromatographic conditions, the flow rate can be 0.12-0.18 mL / min; for example, 0.12 mL / min, 0.15 mL / min and 0.18 mL / min; preferably 0.12-0.15 mL / min.

[0062] In the chromatographic conditions, the column temperature can be 30-40℃; for example, 30℃, 35℃ or 40℃; preferably 35℃.

[0063] In the chromatographic conditions described, the injection volume can be conventional in the art, for example, 1-5 μL, or 1 μL.

[0064] In the chromatographic conditions, the injection concentration of the analyte from Sophora japonica can be conventional in the art, for example, 0.004-0.1 g / mL, or 0.025 g / mL.

[0065] The Sophora japonica analyte can be pretreated before elution to meet the injection standards. The pretreatment can be a conventional pretreatment in the art; preferably, the pretreatment includes the following steps: dissolving the Sophora japonica analyte in a solvent; preferably, the solvent is methanol.

[0066] The pretreatment step may further include the following steps: soaking Sophora japonica fruit in water (200 mL) for 30 min, decocting (boiling and decocting for 30 min, then adding 160 mL of water and continuing to decoct for 20 min), filtering, concentrating the filtrate and freeze-drying to obtain freeze-dried powder of Sophora japonica fruit to be tested, and dissolving it in the solvent in the form of freeze-dried powder.

[0067] The Sophora japonica fruit sample to be tested can be various Sophora japonica fruit products or other compositions containing Sophora japonica fruit components, such as raw Sophora japonica fruit and / or processed Sophora japonica fruit; the processed Sophora japonica fruit is preferably charred Sophora japonica fruit.

[0068] The charred Sophora japonica fruit can be prepared using conventional methods in the art. For example, the charred Sophora japonica fruit can be prepared by roasting raw Sophora japonica fruit. Preferably, the roasting temperature is 200-300℃, for example, 200℃. Preferably, the roasting time is 5-50 min; for example, 5, 9, 12, 15, 20, 30, or 50 min; more preferably, the roasting time is 15-20 min.

[0069] The processing method is used to detect or separate the Sophora japonica components in the Sophora japonica analyte.

[0070] Preferably, the Sophora japonica fruit component comprises one or more of the following components: methyl gallate, dihydrokaempferol-3-O-glucoside, kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutinoside, kaempferol-3-O-sophorose, dihydroapigenin-7-O-glucoside, apigenin-7-O-rutinoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-β-D-rutinoside, and linalool. More preferably, the Sophora japonica fruit component further comprises one or more of the following components: gallic acid, p-hydroxybenzyl alcohol, and genistein-4′-O-(6″-acetyl)-glucoside. Furthermore, the Sophora japonica fruit component further includes the following components: (3,4,5-trihydroxy-3-furanyl)-4-methoxybenzoic acid, p-digallic acid, genistein-7,4′-di-O-β-D-glucoside, genistein-7-O-gluco-4′-O-neohesperidin, kaempferol-3-O-(3′,4′-di-O-glucosyl)-rhamnose-7-O-rhamnose, kaempferol-3-O-sophorose-7-O-rhamnose, genistein-7-O-malonylglucoside, and genistein-7-O-malonylglucoside. One or more of the following: ′-O-glucoside, quercetin-3-O-(3′-O-glucosyl)-rutin, genistein, apigenin-7-O-(3″-acetyl)-rutin, kaempferol-7-methoxy-4′-O-glucosyl-3-O-(2-O-apio-furanyl)glucose, isorhamnetin-3-O-sophorose, naringin, rutin, sophoroside, sophoroid diglycoside, genistein-4′-O-(6″-malonyl)glucoside, camellia flavonoid A, genistein, and kaempferol.

[0071] Preferably, the components of the Sophora japonica fruit include methyl gallate, dihydrokaempferol-3-O-glucoside, kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutinoside, kaempferol-3-O-sophorose, dihydroapigenin-7-O-glucoside, apigenin-7-O-rutinoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-β-D-rutinoside, eleutheroside, gallic acid, p-hydroxybenzyl alcohol, genistein-4′-O-(6″-acetyl)-glucoside, (3,4,5-trihydroxy-3-furanyl)-4-methoxybenzoic acid, p-digallic acid, genistein-7,4′-di-O-β-D-glucoside, and genistein-7-O-glucoside-4′-O-glucoside. - Neohesperidin, kaempferol-3-O-(3′,4′-di-O-glucosyl)-rhamnose-7-O-rhamnose, kaempferol-3-O-sophorose-7-O-rhamnose, genistein-7-O-malonylglucoside-4′-O-glucoside, quercetin-3-O-(3′-O-glucosyl)-rutin, genistein, apigenin-7-O-(3″-acetyl)-rutin, kaempferol-7-methoxy-4′-O-glucosyl-3-O-(2-O-apio-furanyl)glucoside, isorhamnetin-3-O-sophorose, naringin, rutin, sophoroside, sophoroid diglycoside, genistein-4′-O-(6″-malonyl)glucoside, camellia flavonoid A, genistein, and kaempferol.

[0072] The present invention also provides a product containing a liquid chromatogram obtained using the above-described processing method.

[0073] The product may be a computer storage medium; or, a drawn or printed instruction manual.

[0074] Preferably, the liquid chromatogram has characteristic peaks at one or more of the following retention times: 19.83±0.01 min, 28.24±0.06 min, 33.60±0.04 min, 34.99±0.05 min, 36.28±0.05 min, 40.90±0.04 min, 41.63±0.07 min, 42.09±0.05 min, and 43.58±0.03 min.

[0075] Preferably, the liquid chromatogram further exhibits characteristic peaks at one or more of the following retention times: 5.60±0.05 min, 6.56±0.01 min, and 45.31±0.03 min.

[0076] Preferably, the liquid chromatogram further exhibits characteristic peaks at one or more of the following retention times: 11.31±0.02 min, 16.89±0.01 min, 22.97±0.04 min, 25.92±0.01 min, 27.44±0.02 min, 27.91±0.02 min, 30.14±0.05 min, 30.53±0.03 min, and 32.78±0.02 min. min, 33.15±0.03min, 34.06±0.03min, 34.70±0.03min, 35.96±0.06min, 37.45±0.05min, 38.27 ±0.05min, 39.89±0.06min, 43.35±0.02min, 46.28±0.02min, 46.74±0.04min and 49.21±0.04min.

[0077] Preferably, the liquid chromatogram exhibits characteristic peaks at the following retention times: 5.60±0.05 min, 6.56±0.01 min, 11.31±0.02 min, 16.89±0.01 min, 19.83±0.01 min, 22.97±0.04 min, 25.92±0.01 min, 27.44±0.02 min, 27.91±0.02 min, 28.24±0.06 min, 30.14±0.05 min, 30.53±0.03 min, 32.78±0.02 min, 33.15±0.03 min, 33.60±0.04 min, 34 .06±0.03min, 34.70±0.03min, 34.99±0.05min, 35.96±0.06min, 36.28±0.05min, 37.45±0.05min, 38.27±0.05min, 39.89±0.06min, 40.90±0. 04min, 41.63±0.07min, 42.09±0.05min, 43.35±0.02min, 43.58±0.03min, 45.31±0.03min, 46.28±0.02min, 46.74±0.04min and 49.21±0.04min.

[0078] The present invention also provides a product recording a fingerprint spectrum of raw Sophora japonica fruit, which has characteristic peaks at one or more of the following retention times: 19.83±0.01 min, 28.24±0.06 min, 33.60±0.04 min, 34.99±0.05 min, 36.28±0.05 min, 37.45±0.05 min, 40.90±0.04 min, 41.63±0.07 min, 42.09±0.05 min, and 43.58±0.03 min.

[0079] The product may be a computer storage medium; or, a drawn or printed instruction manual.

[0080] Preferably, the fingerprint spectrum of the raw Sophora japonica fruit further exhibits characteristic peaks at one or more of the following retention times: 22.97±0.04 min, 25.92±0.01 min, 27.44±0.02 min, 27.91±0.02 min, 30.14±0.05 min, 30.53±0.03 min, 32.78±0.02 min, 33.15±0.03 min, 34.06±0.03 min, 35.96±0.06 min, 38.27±0.05 min, 39.89±0.06 min, 44.34±0.04 min, 44.60±0.03 min, and 46.74±0.04 min.

[0081] Preferably, the fingerprint spectrum of the raw Sophora japonica fruit exhibits characteristic peaks at the following retention times: 19.83±0.01 min, 22.97±0.04 min, 25.92±0.01 min, 27.44±0.02 min, 27.91±0.02 min, 28.24±0.06 min, 30.14±0.05 min, 30.53±0.03 min, 32.78±0.02 min, 33.15±0.03 min, 33.60±0.04 min, 34.06±0.01 min. .03min, 34.99±0.05min, 35.96±0.06min, 36.28±0.05min, 37.45±0.05min, 38.27±0.05min, 39.89±0.06min, 40. 90±0.04min, 41.63±0.07min, 42.09±0.05min, 43.58±0.03min, 44.34±0.04min, 44.60±0.03min and 46.74±0.04min.

[0082] The present invention also provides a product that records the fingerprint spectrum of processed Sophora japonica fruit, which has characteristic peaks at one or more of the following retention times: 5.60±0.05 min, 6.56±0.01 min, 33.60±0.04 min, 34.99±0.05 min, 37.45±0.05 min, 40.90±0.04 min, 41.63±0.07 min, and 45.31±0.03 min.

[0083] The product may be a computer storage medium; or, a drawn or printed instruction manual.

[0084] Preferably, the fingerprint spectrum of the prepared Sophora japonica fruit further exhibits characteristic peaks at one or more of the following retention times: 5.31±0.01 min, 9.14±0.03 min, 11.31±0.02 min, 16.89±0.01 min, 22.97±0.04 min, 25.92±0.01 min, 27.44±0.02 min, 27.91±0.02 min, 30.53±0.03 min, 32.78±0.02 min, 34.70±0.03 min, 35.96±0.06 min, 38.27±0.05 min, 39.89±0.06 min, 43.35±0.02 min, 46.28±0.02 min, 46.74±0.04 min, and 49.21±0.04 min.

[0085] Preferably, the fingerprint spectrum of the prepared Sophora japonica fruit has characteristic peaks at the following retention times: 5.31±0.01 min, 5.60±0.05 min, 6.56±0.01 min, 9.14±0.03 min, 11.31±0.02 min, 16.89±0.01 min, 22.97±0.04 min, 25.92±0.01 min, 27.44±0.02 min, 27.91±0.02 min, 30.53±0.03 min, 32.78±0.02 min, 3 3.60±0.04min, 34.70±0.03min, 34.99±0.05min, 35.96±0.06min, 37.45±0.05min, 38.27±0.05min, 39.89±0.06min, 40.90±0.04min, 41.63±0.07min, 43.35±0.02min, 45.31±0.03min, 46.28±0.02min, 46.74±0.04min and 49.21±0.04min.

[0086] Preferably, in the fingerprint spectrum of the processed Sophora japonica fruit, the processed Sophora japonica fruit refers to Sophora japonica charcoal obtained by stir-frying raw Sophora japonica fruit for 20 minutes.

[0087] This invention also provides the application of the fingerprint spectrum of the above-mentioned raw Sophora japonica fruit as a standard fingerprint spectrum in the detection and quality control of raw Sophora japonica fruit.

[0088] This invention also provides the application of the fingerprint spectrum of the above-mentioned processed Sophora japonica fruit as a standard fingerprint spectrum in the detection and quality control of processed Sophora japonica fruit.

[0089] The application may also include the following steps:

[0090] a. Take the raw or processed Sophora japonica fruit to be tested and obtain the corresponding liquid chromatogram using the processing method described in any one of the present invention;

[0091] b. Evaluate the similarity between the liquid chromatogram obtained in step a and the fingerprint spectrum as described in any one of the present invention.

[0092] Preferably, in step b above, if the similarity is ≥0.90, then it is acceptable.

[0093] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0094] The reagents and raw materials used in this invention are all commercially available.

[0095] The positive and progressive effects of this invention are as follows:

[0096] (1) The fingerprint spectra of Sophora japonica pod and Sophora japonica charcoal described in this invention each include 25 common peaks, and the chemical structures of the 12 common peaks with larger peak areas have been determined. The fingerprint spectra of Sophora japonica pod and Sophora japonica charcoal provided by this invention can be reflected in a single spectrum under the same wavelength, which can more comprehensively, accurately and reliably evaluate the quality of Sophora japonica medicinal material and Sophora japonica charcoal.

[0097] (2) The fingerprint spectrum of Sophora japonica fruit and Sophora japonica fruit charcoal described in this invention can more comprehensively reflect the quality of the drug, which is more meaningful for guiding industrial production to control the quality of the drug and better ensures the quality of the product.

[0098] (3) The method for establishing fingerprint spectra of Sophora japonica fruit and Sophora japonica charcoal described in this invention employs ultra-high performance liquid chromatography (UHPLC). By rationally selecting chromatographic conditions, the fingerprint spectra of Sophora japonica fruit and Sophora japonica charcoal are constructed. This method has the advantages of being simple, stable, highly precise, and reproducible. It provides a basis for further research on the chemical composition and quality standards of Sophora japonica fruit and Sophora japonica charcoal.

[0099] (4) The fingerprint spectrum provided by this invention can separate and qualitatively identify multiple effective components of raw or charred Sophora japonica fruit, such as methyl gallate, dihydrokaempferol-3-O-glucoside, and kaempferol-3-O-(2”-O-β-D-glucosyl)-β-D-rutin, which can fully reflect the efficacy of the product and is of great significance for controlling process stability, ensuring product quality, and stabilizing product efficacy. Attached Figure Description

[0100] Figure 1 The UPLC fingerprint spectra of 10 batches of raw Sophora japonica fruit are presented in this invention.

[0101] Figure 2 UPLC fingerprint spectra of 10 batches of Sophora japonica fruit charcoal processed for 20 minutes according to the present invention.

[0102] Figure 3 This is a comparison chart of reference standards, including: 1. Gallic acid; 2. Kaempferol-3-O-β-D-sophorobiose-7-O-α-L-rhamnoside; 3. Gentian glycoside; 4. Kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutin; 5. Kaempferol-3-O-β-D-sophorose; 6. Naringin; 7. Rutin; 8. Sophoroside; 9. Sophora biglycoside; 10. Kaempferol-3-O-rutin; 11. Isorhamnetin-3-O-β-D-rutin; 12. Gentian; 13. Kaempferol;

[0103] Figure 4 UPLC fingerprints of Sophora japonica charcoal prepared at different processing times according to the present invention.

[0104] Figure 5 The graph shows the effect of the decoction of Sophora japonica fruit before and after processing on the bleeding time of mice.

[0105] Figure 6 The graph shows the effect of the decoction of Sophora japonica fruit before and after processing on the coagulation time of mice.

[0106] Figure 7 The graph shows the results of the four coagulation tests for each group of mice.

[0107] Figure 8 PCA principal component analysis chromatograms of 10 batches of raw Sophora japonica fruit and charred fruit processed for 20 minutes.

[0108] Figure 9 The validation diagram is shown in the Ward clustering analysis with principal components.

[0109] Figure 10 UPLC fingerprint obtained under chromatographic condition 1

[0110] Figure 11 UPLC fingerprint obtained under chromatographic condition 2

[0111] Figure 12 UPLC fingerprint obtained under chromatographic condition 3

[0112] Figure 13 UPLC fingerprint obtained under chromatographic condition 4

[0113] Figure 14 UPLC fingerprint obtained under chromatographic condition 5

[0114] Figure 15 UPLC fingerprint obtained under chromatographic condition 6

[0115] Figure 16 UPLC fingerprint obtained under chromatographic condition 7

[0116] Figure 17 UPLC fingerprint obtained under chromatographic condition 8

[0117] Figure 18 UPLC fingerprints obtained at different detection wavelengths.

[0118] Figure 19 UPLC fingerprints obtained from different chromatographic columns.

[0119] Figure 20 UPLC fingerprints obtained at different column temperatures.

[0120] Figure 21 UPLC fingerprints obtained at different flow rates. Detailed Implementation

[0121] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0122] 1. Test instruments and materials

[0123] 1.1 Instruments

[0124] Thremo Scientific Vanquish high-performance liquid chromatograph (HPLC); PDA full-wavelength ultraviolet detector (Thermo Fisher Scientific, Inc.); SPSS 18.0 statistical analysis software; Mettler MS105DU electronic balance; ultrasonic cleaner (KQ-250DE model); Millipore pure water system; URIT-600A semi-automatic coagulation analyzer.

[0125] 1.2 Materials

[0126] Reference standards: gallic acid (batch number: 110831-201605, 90.8%), rutin (batch number: 100080-202012, 91.6%), kaempferol (batch number: 110861-202013, 93.2%), genistein (batch number: 111704-201703, 98.8%), kaempferol-3-O-rutin (batch number: 112007-202103, 94.0%), naringin (batch number: 110722-202116, 93.5%), genistein (batch number: 111709-201702, 99.9%), and jugnanoside (batch number: 111695-201...). 703 (99.6%) was purchased from the China National Institutes for Food and Drug Control; Sophora biglycoside (batch number: ST82950105, ≥98%) was purchased from Shanghai Shidande Standard Technical Service Co., Ltd.; Kaempferol-3-O-β-D-sophorose (batch number: 21070121, ≥98%) and kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutin (batch number: 21081031, ≥98%) were purchased from Shanghai Tongtian Biotechnology Co., Ltd.; Isorhamnetin-3-O-β-D-rutin and kaempferol-3-O-β-D-sophorobiose-7-O-α-L-rhamnoside were prepared in the laboratory with a purity >98%.

[0127] Methanol and formic acid were of chromatographic grade for HPLC, water was ultrapure water, and the remaining reagents were of analytical grade.

[0128] 1.3 Laboratory Animals

[0129] ICR mice were provided by the Shanghai Laboratory Animal Center. Before and after drug administration, mice in each group were housed in separate cages, fed complete nutritional pellet feed, and given free access to water. The mice were half male and half female, and weighed 19–21g.

[0130] 2. Preparation of Sophora japonica charcoal samples

[0131] Ten batches of prescription medicinal materials were purchased from major medicinal material producing areas and traditional producing areas across the country. The origin information of the medicinal materials is shown in Table 1.

[0132] Table 1 Information on the Origin of Medicinal Materials

[0133]

[0134] Unless otherwise stated, the preparation method of Sophora japonica charcoal in the embodiments is as follows: Weigh about 400g of cleaned raw Sophora japonica fruit, put it into a pot at a temperature of about 200℃ and stir-fry for 20 minutes. Take it out and let it cool for later use. Grind it into powder and pass it through a No. 3 sieve to obtain Sophora japonica charcoal reference material.

[0135] The medicinal materials listed in Table 1 were used as raw materials to prepare Sophora japonica charcoal, and were numbered sequentially as t1-t10; t9 was the batch used for method validation.

[0136] The present invention will be further explained and described below with reference to specific embodiments.

[0137] Example 1

[0138] 1. Preparation of reference solution

[0139] Take appropriate amounts of naringin, sophoridine, sophoridine, rutin, kaempferol, genistein, genistein, isorhamnetin-3-O-β-D-rutinoside, kaempferol-3-O-β-D-rutinoside, kaempferol-3-O-β-D-sophora, kaempferol-3-O-β-D-sophorabiose-7-O-α-L-rhamnoside, and kaempferol-3-O-(2”-O-β-D-glucosyl)-β-D-rutinoside reference standards, dissolve them in methanol, and prepare a solution containing 10 μg of each reference standard per 1 mL as the reference solution.

[0140] 2. Preparation of the sample solution: Accurately measure about 1g of Sophora japonica fruit (s1-s10) or Sophora japonica fruit charcoal powder (t1-t10), place it in a 50mL stoppered conical flask, accurately add 30mL of 70% methanol, seal tightly, weigh, sonicate (power 250W, frequency 40KHz) for 60 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the sample solution.

[0141] When establishing the extraction method, preliminary experiments compared water extraction and alcohol extraction. Under the full wavelength scan of 200-400nm, methanol extraction yielded more peaks than water and ethanol extraction and was less expensive. Therefore, methanol extraction was chosen, and after comparing the extraction effects of different concentrations of methanol, the extraction concentration was determined to be 70% methanol.

[0142] 3. Determination of fingerprint spectra of Sophora japonica fruit and Sophora japonica charcoal by ultra-high performance liquid chromatography

[0143] Ten batches of Sophora japonica fruit samples (s1-s10), ten batches of Sophora japonica fruit charcoal samples (t1-t10), and the reference solution prepared according to steps 1 and 2 were injected into an ultra-high performance liquid chromatograph. The chromatographic conditions were as follows: Waters ACQUITY CSH C18 column (150 mm × 2.1 mm, 1.7 μm); flow rate: 0.15 ml / min; column temperature: 35℃; detection was performed using a UV detector at a wavelength of 280 nm; the theoretical plate number calculated based on the sophoroside peak was not less than 5000. The mobile phase was 0.05% formic acid water as phase A and methanol as phase B, and gradient elution was performed according to Table 2.

[0144] Table 2

[0145]

[0146] The obtained Sophora japonica fruit and Sophora japonica fruit carbon fingerprints contained a total of 35 chromatographic peaks.

[0147] Among them, the carbon fingerprint spectrum of Sophora japonica pods is shown in Figure 2 The carbon fingerprint spectrum of Sophora japonica fruit includes 25 common peaks, and the retention times of each peak are as follows:

[0148] Peak 1: 5.31 min; Peak 2: 6.56 min; Peak 3: 9.14 min; Peak 4: 11.31 min; Peak 5: 16.89 min; Peak 7: 22.97 min; Peak 8: 25.92 min; Peak 9: 27.44 min; Peak 10: 27.91 min; Peak 13: 30.53 min; Peak 14: 32.78 min; Peak 16: 33.60 min; Peak 18: 34.70 min Peak 19: 34.99 min; Peak 20: 35.96 min; Peak 22: 37.45 min; Peak 23: 38.27 min; Peak 24: 39.89 min; Peak 25: 40.90 min; Peak 26: 41.63 min; Peak 28: 43.35 min; Peak 32: 45.31 min; Peak 33: 46.28 min; Peak 34: 46.74 min; Peak 35: 49.21 min. Peak 23 is the control peak.

[0149] See fingerprint spectrum of raw Sophora japonica fruit. Figure 1 The fingerprint spectrum of Sophora japonica pods includes 25 common peaks, and the retention times of each peak are as follows:

[0150] Peak 6: 19.83 min; Peak 7: 22.97 min; Peak 8: 25.92 min; Peak 9: 27.44 min; Peak 10: 27.91 min; Peak 11: 28.24 min; Peak 12: 30.14 min; Peak 13: 30.53 min; Peak 14: 32.78 min; Peak 15: 33.15 min; Peak 16: 33.60 min; Peak 17: 34.06 min; Peak 19: 34.9 min. Peak 20: 35.96 min; Peak 21: 36.28 min; Peak 22: 37.45 min; Peak 23: 38.27 min; Peak 24: 39.89 min; Peak 25: 40.90 min; Peak 26: 41.63 min; Peak 27: 42.09 min; Peak 29: 43.58 min; Peak 30: 44.34 min; Peak 31: 44.60 min; Peak 34: 46.74 min. Peak 23 is the control peak.

[0151] The ratios of the relative retention times of each peak to the relative retention time of the control peak are as follows:

[0152] The relative retention time (RRT) for peak 1 is 0.14, for peak 2 it is 0.17, for peak 3 it is 0.24, for peak 4 it is 0.30, for peak 5 it is 0.44, for peak 6 it is 0.52, for peak 7 it is 0.60, for peak 8 it is 0.68, for peak 9 it is 0.72, and for peak 10 it is 0.72. The relative retention time (RRT) for peak 11 is 0.73, for peak 12 it is 0.74, for peak 13 it is 0.80, for peak 14 it is 0.86, for peak 15 it is 0.87, for peak 16 it is 0.88, for peak 17 it is 0.89, and for peak 18 it is... The relative retention times (RRT) for peaks 0.91, 19, 20, 21, 22, 23, 24, 25, 26, and 27 are 0.92, 0.94, 0.95, 0.98, 1.00, 1.05, 1.07, 1.09, and 1.08 respectively. The relative retention times (RRT) for peaks 28, 29, 30, 31, 32, 33, 34, and 35 were 1.28. Peak 23 was the chromatographic peak of the reference standard.

[0153] Among the peaks: peak 10 is kaempferol-3-O-β-D-sophoribio-7-O-α-L-rhamnoside; peak 14 is genistein; peak 16 is kaempferol-3-O-(2”-O-β-D-glucosyl)-β-D-rutin; peak 19 is kaempferol-3-O-sophorose; peak 20 is naringin; peak 22 is rutin; peak 23 is sophoroside; peak 24 is a sophoroid diglycoside; peak 26 is kaempferol-3-O-rutin; peak 27 is isorhamnetin-3-O-β-D-rutin; peak 34 is genistein; and peak 35 is kaempferol. See the comparison chart for each reference standard. Figure 3 .

[0154] Similarity results

[0155] The integrated signal was imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A Version" software of the Chinese Pharmacopoeia Commission. Using S1 as the reference chromatogram, the similarity result was calculated using the median method, and a standard control fingerprint chromatogram (RFP) was generated. (See...) Figure 1 and Figure 2 The similarity results are shown in Table 3. Products with a similarity of less than 0.90 are considered unqualified, and quality evaluation was conducted on 10 batches of samples.

[0156] Table 3. Similarity of fingerprint spectra between 10 batches of raw and charred Sophora japonica fruit products

[0157]

[0158] The results showed that all 10 batches of samples were qualified products.

[0159] The control fingerprint was generated using the median method with a time window of 0.1 min. The generated control fingerprint is shown below. Figure 3 Analysis revealed 25 common peaks across the 10 batches of samples.

[0160] 4. Methodological Investigation

[0161] 4.1 Precision assessment:

[0162] The sample solution for sample t9 was prepared according to the fingerprint spectroscopy determination method, and the sample was injected 6 times consecutively. The results are shown in Tables 4 and 5.

[0163] Table 4. Precision test results (retention time, min)

[0164]

[0165]

[0166] Table 5. Precision test results (peak area)

[0167]

[0168]

[0169] The results showed that the RSD values ​​of the common peak area of ​​the six consecutive injections were all less than 4.0%, and the RSD values ​​of the retention time were all less than 1.0%, indicating that the method has good precision.

[0170] 4.2 Repeatability test:

[0171] Take samples (t9) from the same batch and prepare 6 parallel test solutions according to the method under the fingerprint spectrum determination section. Determine the results according to the method. The results are shown in Tables 6 and 7.

[0172] Table 6. Repeatability Test Results (Retention Time)

[0173]

[0174]

[0175] Table 7. Repeatability Test Results (Peak Area)

[0176]

[0177] The results showed that the RSD values ​​of the peak areas of the common peaks in the six samples were all less than 5.0%, and the RSD values ​​of the retention times were all less than 1.0%, indicating that the method has good repeatability.

[0178] 4.3 Stability assessment:

[0179] The t9 sample was prepared into a test solution according to the fingerprint spectroscopy determination method. The solution was placed at room temperature for 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, 48 ​​hours and 72 hours, and the results were determined according to the method. The relative peak area and relative retention time RSD of the common peak were investigated. The results are shown in Tables 8 and 9.

[0180] Table 8. Stability Test Results (Retention Time)

[0181] Peak 0h 1h 2h 4h 8h 12h 24h 48h Retention time RSD (%) 1 5.300 5.300 5.302 5.296 5.298 5.287 5.288 5.300 0.11 2 6.554 6.557 6.560 6.552 6.551 6.532 6.529 6.554 0.17 3 9.148 9.143 9.139 9.145 9.137 9.093 9.092 9.148 0.26 4 11.333 11.332 11.327 11.326 11.320 11.300 11.281 11.333 0.16 5 16.914 16.916 16.915 16.918 16.912 16.875 16.866 16.914 0.13 7 23.036 23.028 23.034 23.043 23.022 22.963 22.964 23.036 0.14 8 26.014 26.021 26.026 26.021 26.007 25.967 25.962 26.014 0.10 9 27.581 27.582 27.560 27.570 27.688 27.636 27.625 27.581 0.16 10 28.051 28.067 28.068 28.060 28.045 27.996 27.992 28.051 0.11 13 30.643 30.637 30.628 30.624 30.620 30.555 30.536 30.643 0.14 14 32.858 32.855 32.862 32.842 32.846 32.791 32.780 32.858 0.10 16 33.812 33.805 33.817 33.789 33.797 33.732 33.722 33.812 0.12 18 34.779 34.776 34.776 34.749 34.761 34.710 34.705 34.779 0.09 19 35.135 35.133 35.142 35.113 35.117 35.048 35.037 35.135 0.12 20 36.353 36.351 36.342 36.328 36.327 36.275 36.264 36.353 0.10 22 37.647 37.652 37.648 37.632 37.624 37.547 37.536 37.647 0.13 23 38.429 38.430 38.428 38.418 38.409 38.348 38.339 38.429 0.10 24 39.993 39.996 39.993 39.992 39.980 39.936 39.941 39.993 0.06 25 41.028 41.029 41.025 41.028 41.015 40.977 40.990 41.028 0.05 26 41.731 41.728 41.737 41.729 41.716 41.667 41.675 41.731 0.07 28 43.410 43.411 43.404 43.399 43.393 43.369 43.378 43.410 0.04 32 45.311 45.328 45.318 45.322 45.320 45.319 45.296 45.311 0.02 33 46.302 46.294 46.296 46.297 46.292 46.273 46.294 46.302 0.02 34 46.771 46.771 46.778 46.770 46.763 46.736 46.746 46.771 0.03 35 49.307 49.301 49.317 49.315 49.295 49.267 49.271 49.307 0.04

[0182] Table 9. Stability test results (peak area)

[0183]

[0184]

[0185] The results showed that the RSD values ​​of the common peak area and retention time of the test solution were all less than 5.0% at each time point within 48 hours, indicating that the test solution was stable within 48 hours.

[0186] 5. Applications of fingerprint mapping

[0187] 5.1 Preparation of Sophora japonica charcoal samples

[0188] Weigh approximately 400g of cleaned raw Sophora japonica fruit (s1-s10), divide it into 8 portions, and put the remaining 7 portions into a pot at a temperature of about 200℃. Stir-fry for 5, 9, 12, 15, 20, 30 and 50 minutes respectively. Remove and let cool for later use. Prepare 10 batches of samples for each of the different degrees of stir-frying.

[0189] 5.2. Establishing UPLC fingerprints for charred Sophora japonica fruit at different processing times

[0190] 5.2.1 Preparation of Sophora japonica charcoal test solution

[0191] Grind the charred Sophora japonica fruit into powder and pass it through a No. 3 sieve. Weigh approximately 1g of each powder and accurately add 30mL of 70% methanol. Weigh the powder, sonicate (250w, 70KHz) for 60min, cool, weigh again, add 70% methanol to make up the weight, shake well, and filter through a 0.22μm microporous membrane to obtain test solution 1.

[0192] Weigh 10g of different coarse powders of Sophora japonica charcoal, soak them in 200mL of water for 30min, boil them and decoct them for 30min, add 160mL of water and continue to decoct for 20min, filter (single layer filter cloth 300 mesh), concentrate the filtrate to 100mL, freeze dry to obtain freeze-dried powder of Sophora japonica charcoal product, prepare a test solution with a concentration of 0.025g / mL using pure water.

[0193] Based on the results of the previous investigation of sample pretreatment methods, the chromatographic peaks of test solution 1 and 2 are basically consistent.

[0194] Preparation of Yunnan Baiyao test sample (batch number ZJA2014): The clinical dosage of Yunnan Baiyao is 2g / 70kg. The dosage of Yunnan Baiyao in this experiment is twice the clinical equivalent dose, i.e., 0.52g / kg. Accurately weigh 2.6g of Yunnan Baiyao into a 100mL volumetric flask, dilute to volume with physiological saline, and shake well.

[0195] 5.2.2 Fingerprint Spectrum Establishment: The fingerprint spectrum of the above-obtained samples was determined by ultra-high performance liquid chromatography (UHPLC). Chromatographic conditions: Waters ACQUITY CSH C18 column (150 mm × 2.1 mm, 1.7 μm) was used; the mobile phases were: A was 0.05% formic acid solution, and B was methanol; gradient elution was used: 0–9 min, 10%–20% (v / v) mobile phase B; 9–27 min, 20%–40% (v / v) mobile phase B; 27–30 min, 40% (v / v) mobile phase B; 30–39 min, 40%–60% (v / v) mobile phase B; 39–42 min, ... Flow rate: 0.15 mL / min; Detector: UV-DAD detector; Detection wavelength: 280 nm; Column temperature: 35 °C; Injection volume: 1 μl. Flow rate: 0.15 mL / min; Flow rate: 0.15 mL / min; Detector: UV-DAD detector; Detection wavelength: 280 nm; Column temperature: 35 °C; Injection volume: 1 μl.

[0196] 5.2.3 The characteristic peak data were obtained through analysis using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software: Retention times were as follows: Peak 1 5.31 min, Peak 2 5.60 min, Peak 3 6.56 min, Peak 4 9.14 min, Peak 5 11.31 min, Peak 6 16.89 min, Peak 7 19.83 min, Peak 8 22.97 min, Peak 9 25.92 min, Peak 10 27.44 min, Peak 11 27.91 min, Peak 12 28.24 min, Peak 13 30.14 min, Peak 14 30.53 min, Peak 15 32.78 min, Peak 16 33.15 min, Peak 17 33.60 min. The chromatograms of characteristic peaks at different processing times are as follows: Peak 18 34.06 min, Peak 19 34.70 min, Peak 20 34.99 min, Peak 21 35.96 min, Peak 22 36.28 min, Peak 23 37.45 min, Peak 24 38.27 min, Peak 25 39.89 min, Peak 26 40.90 min, Peak 27 41.63 min, Peak 28 42.09 min, Peak 29 43.35 min, Peak 30 43.58 min, Peak 31 44.44 min, Peak 32 44.60 min, Peak 33 45.31 min, Peak 34 46.28 min, Peak 35 46.74 min, Peak 36 49.21 min. See the chromatograms of characteristic peaks at different processing times. Figure 4 After 22 minutes, the peak area of ​​flavonoid glycosides gradually decreased, while the aglycone gradually increased. Therefore, based on the characteristic of phenolic acid components first increasing and then decreasing in the first 20 minutes, it was found that they reached their maximum between 15 and 20 minutes. Thus, 15 to 20 minutes is temporarily considered to be the optimal processing time.

[0197] 5.3 Pharmacodynamic experiments were conducted on samples with different processing times.

[0198] 5.3.1 One hundred ICR mice weighing 19–21g were selected, half male and half female. After being fed normally for 3 days, they were randomly divided into 10 groups of 10 mice each, and were numbered and weighed.

[0199] 5.3.2 The samples were grouped as follows: saline group, Yunnan Baiyao group, raw Sophora japonica fruit group (fried for 0 min), Sophora japonica fruit sample group (fried for 5 min), Sophora japonica fruit sample group (fried for 9 min), Sophora japonica fruit sample group (fried for 12 min), Sophora japonica fruit sample group (fried for 15 min), Sophora japonica fruit sample group (fried for 20 min), Sophora japonica fruit sample group (fried for 30 min), and Sophora japonica fruit sample group (fried for 50 min).

[0200] 5.3.3 Bleeding Time (BT): All groups were administered the test solution 2 via gavage once daily, with a volume of 0.1 mL / 10 g. The normal control group received an equal volume of 0.9% saline via gavage for 6 consecutive days. One hour after the last gavage, the mice were placed in a restraint device and laid flat on the operating table. The tail was cut 1 mm from the tip with scissors. Timing was started when blood began to flow spontaneously. Blood droplets from the tail tip were absorbed with filter paper every 30 seconds until no more blood flowed (i.e., no bloodstains appeared on the filter paper). The time from the start of blood flow after tail amputation to the cessation of bleeding was defined as the bleeding time. Bleeding times were calculated, and inter-group comparisons were performed. Bleeding times exceeding 10 minutes were not included in the data analysis.

[0201] 5.3.4 Clotting Time (CT): Mice were administered 0.2 mL / 10 g via gavage for 6 consecutive days. One hour after the last administration, mice were fixed in a tethered dish, and blood was collected by tail clipping. Blood was dropped onto a glass slide (the first drop was discarded, two drops were added, and one drop was retested). The blood drop diameter was approximately 5 mm. A stopwatch was immediately used to time the event. Every 30 seconds, a clean No. 7 needle was used to gently prick the blood drop from the edge inwards, observing for any blood streaks. The time elapsed from blood collection to the appearance of blood streaks was the clotting time. Data are shown in Table 10 below, and the pharmacodynamic chromatogram is shown in […]. Figure 5 , 6 .

[0202] Table 10. Effects of Sophora japonica fruit decoction before and after processing on bleeding time in mice.

[0203]

[0204]

[0205] In Table 10, compared with NS * P<0.05, ** P<0.005, *** P < 0.0001; compared with 0 min, # P<0.05, ## P<0.005, ### P<0.0001.

[0206] The results above show that roasting Sophora japonica charcoal for 20 minutes can significantly shorten the hemostasis and clotting time, verifying its hemostatic effect and indicating that a roasting time of 20 minutes is appropriate.

[0207] 5.3.5 Coagulation panel

[0208] Preparation of anticoagulated blood: homemade blood collection tubes (1 hour after administration to mice, the mouse eyeballs were removed and the blood was dripped into an EP tube containing 38 g / L sodium citrate anticoagulant (blood: anticoagulant = 9:1)).

[0209] Preparation of anticoagulated plasma: Centrifuge the anticoagulated blood at 3000 r / min for 10 min. The resulting supernatant plasma is platelet-poor plasma (PPP). The obtained plasma sample needs to be tested within 4 hours.

[0210] 5.3.5.1 Determination of Activated Partial Thromboplastin Time (APTT)

[0211] Equilibrate the APTT reagent to room temperature, incubate with 0.025 mol / L CaCl2 solution at 37°C for 5 min; take 50 μL of the plasma to be tested, add 50 μL of APTT reagent equilibrated to room temperature, mix well, and incubate in a water bath at 37°C for 3 min; add 50 μL of 0.025 mol / L CaCl2 solution pre-warmed to 37°C, mix well; immediately start a stopwatch and record the coagulation time, which is the APTT value (s). Repeat the experiment twice for each sample and take the average value.

[0212] 5.3.5.2 Measurement of prothrombin time (PT)

[0213] Preheat the PT reagent to 37°C before use; take 50 μL of the plasma to be tested, mix well, incubate at 37°C for 3 min, add 100 μL of the preheated PT reagent, mix well, immediately start the stopwatch, and record the coagulation time, which is the PT value (s). Repeat the experiment twice for each sample and take the average value.

[0214] 5.3.5.3 Determination of plasma thrombin time (TT)

[0215] Equilibrate the TT reagent to room temperature; take 100 μL of the plasma to be tested, mix well, and incubate at 37°C for 3 min; add 50 μL of the TT reagent equilibrated to room temperature, mix well, and immediately start a stopwatch to record the coagulation time, which is the TT value (s). Repeat the experiment twice for each sample and take the average value.

[0216] 5.3.5.4 Determination of fibrinogen (FIB)

[0217] Dilute the plasma sample 10-fold with imidazole buffer. Take 100 μL of the diluted plasma and incubate at 37°C for 3 min. Add 50 μL of FIB assay reagent equilibrated to room temperature, mix well, and immediately start a stopwatch to record the coagulation time. Repeat the experiment twice for each sample and take the average value.

[0218] Efficacy data are shown in Table 11, and the chromatogram is shown in [the original text]. Figure 7 The results of PT and FIB experiments showed that there were significant differences between the charred Sophora japonica fruit group and the saline group and the raw Sophora japonica fruit group. The charred Sophora japonica fruit group shortened the PT time and increased the FIB content, indicating that the hemostatic effect of charred Sophora japonica fruit may be related to the extrinsic coagulation pathway and the fibrin pathway.

[0219] Table 11 Effects of Sophora japonica fruit decoction before and after processing on plasma APTT, PT, TT, and FIB in mice.

[0220]

[0221]

[0222] In Table 11, compared with NS * P<0.05, ** P<0.01, *** P<0.005; compared with 0min, # P<0.05, ## P<0.001.

[0223] 6. Spectral effect correlation analysis

[0224] 6.1 Method

[0225] 6.1.1 Bivariate Correlation Analysis

[0226] Using the quantified peak area as the independent variable and the efficacy index as the dependent variable, bivariate correlation analysis was performed using SPSS 18.0 statistical software to obtain the Pearson correlation coefficients between each relevant peak in the fingerprint spectrum and the efficacy.

[0227] 6.1.2 Multiple Linear Regression Analysis

[0228] Using the quantified peak area as the independent variable and the efficacy index as the dependent variable, linear regression analysis was performed on the data using SPSS 18.0, and the stepwise introduction method was used for model fitting.

[0229] 6.1.3 PCA Analysis and Ward Cluster Analysis

[0230] PCA analysis was performed on 35 common peaks of raw and charred Sophora japonica fruit using SPSS 18.0 to identify indicators with significant cumulative contribution rates to the differences between raw and charred Sophora japonica fruit, thus making the evaluation of spectral efficacy more accurate. The results of the PCA analysis were verified by Ward cluster analysis.

[0231] 6.2 Results

[0232] 62.1 Results of Bivariate Correlation Analysis

[0233] As shown in Table 12 below, 21 chromatographic peaks are correlated with hemostatic efficacy indicators. The chromatographic peaks with the best correlation are 2, 3, 7, 12, 17, 20, 22, 23, 27, 30 and 31. Among them, chromatographic peak 2 is strongly correlated with BT, chromatographic peaks 7, 12 and 31 are strongly correlated with CT, chromatographic peak 3 is strongly correlated with PT value, and chromatographic peak 20 is strongly correlated with FIB. Based on the comparison with reference standards and relative retention times, and the comparison of UV-Vis chromatograms and mass spectrometry data, the following chromatographic peaks were identified: peak 2 as gallic acid, peak 3 as p-hydroxybenzyl alcohol, peak 7 as methyl gallate, peak 12 as dihydrokaempferol-3-O-glucoside, peak 17 as kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutin, peak 20 as kaempferol-3-O-sophorose, peak 22 as dihydroapigenin-7-O-glucoside, peak 23 as rutin, peak 27 as kaempferol-3-O-rutin, and peak 30 as eleutheroside. The structural formulas are as follows:

[0234]

[0235]

[0236] Table 12 Relevant Peak Areas

[0237]

[0238]

[0239] Table 13 Results of bivariate correlation analysis

[0240]

[0241]

[0242] *P<0.05, **P<0.01

[0243] Table 14. Chromatographic Peak and Mass Spectrometry Identification Results

[0244]

[0245]

[0246] *Comparison with reference standard

[0247] 6.2.2 Results of Multiple Linear Regression Analysis

[0248] As shown in Table 15 below, the correlation coefficients between the chromatographic peaks related to Sophora japonica charcoal and the two hemostatic indicators CT and PT established in the model are relatively high, R 2The values ​​were all greater than 0.85, indicating that 85% of the changes in CT and PT could be explained by the independent variables. The p-values ​​were all less than 0.05, which were statistically significant, indicating that the model could effectively evaluate the relationship between the carbonized peaks of Sophora japonica fruit and the hemostatic pharmacological activity.

[0249] Table 15 Results of Mathematical Model Establishment

[0250]

[0251]

[0252] Wherein, X2 represents the peak area of ​​the chromatographic peak (chromatographic peak 2) with a retention time of 5.60 min, X3 represents the peak area of ​​the chromatographic peak (chromatographic peak 3) with a retention time of 6.56 min, X26 represents the peak area of ​​the chromatographic peak (chromatographic peak 26) with a retention time of 40.90 min, X28 represents the peak area of ​​the chromatographic peak (chromatographic peak 28) with a retention time of 42.09 min, X31 represents the peak area of ​​the chromatographic peak (chromatographic peak 31) with a retention time of 44.34 min, and X33 represents the peak area of ​​the chromatographic peak (chromatographic peak 33) with a retention time of 45.31 min.

[0253] Based on the combined results of the two analytical methods, it can be basically determined that the components responsible for the hemostatic effect of Sophora japonica charcoal are small molecule compounds of flavonoids and phenolic acids. The chromatographic peaks 2 (gallic acid), 3 (p-hydroxybenzyl alcohol), 7 (methyl gallate), 12 (dihydrokaempferol-3-O-glucoside), 17 (kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutin), 20 (kaempferol-3-O-sophorose), 22 (dihydroapigenin-7-O-glucoside), 23 (rutin), 26 (apigenin-7-O-rutin), 28 (isorhamnoside-3-O-β-D-rutin), 27 (kaempferol-3-O-rutin), 30 (e.g., linalool), and 33 (e.g., genistein-4′-O-(6″-acetyl)-glucoside) may be the material basis for the hemostatic effect of Sophora japonica charcoal.

[0254] 6.2.3 Results of PCA and Ward cluster analysis

[0255] The chromatograms of raw and charred Sophora japonica fruit show common peaks. Figure 1 , 2 The analysis results are shown in Table 16. Components with a cumulative contribution rate of >90%, eigenvalues ​​>1, and passing the KMO (the closer to 1, the more suitable for PCA analysis) and Bartlett's test (P<0.01) were retained.

[0256] Table 16 Principal Component Analysis Results

[0257]

[0258] PCA score chart of raw and charred Sophora japonica fruit ( Figure 8 As can be seen from the graph, raw Sophora japonica fruit is located on the left side of the score chart, while charred Sophora japonica fruit is located on the right side, each clustering into a separate category. This indicates that charring significantly affects the overall chemical composition of Sophora japonica fruit. From the principal component analysis above, four main components can be identified. The first principal component has a contribution rate of 73.386%, the second principal component has a contribution rate of 9.995%, the third principal component has an independent contribution rate of 5.421%, and the fourth principal component has an independent contribution rate of 3.971%. The cumulative contribution rate of the four principal components is 92.773%, which meets the requirements. Table 17, the component loading matrix, illustrates the contribution rate of each variable to the principal components. The larger the absolute value of the loading, the greater the contribution to the principal component. The first principal component has the largest information content, with an independent contribution rate of 73.386%. Peaks 27 (0.988), 16 (0.987), 30 (0.975), 19 (0.965), 6 (0.964), 21 (0.962), and 4 (0.956) have relatively large loading values. The independent contribution rate of the second principal component was 9.995%, with peaks 2 (0.748), 35 (0.735), 1 (0.680), 34 (0.583), and 33 (0.582) showing relatively large loading values. The independent contribution rate of the third principal component was 5.421%, with peaks 25 (0.820), 20 (0.353), 28 (0.337), 9 (0.330), and 3 (0.329) showing relatively large loading values. The independent contribution rate of the fourth principal component was 3.971%, with peaks 34 (0.629), 25 (0.345), 10 (0.340), 9 (0.332), and 12 (0.308) showing relatively large loading values. This indicates that multiple chemical components contribute to the quality differences in Sophora japonica fruit.

[0259] Table 17 Principal Component Loads

[0260]

[0261]

[0262] Subsequently, the principal component analysis was validated using Ward's cluster analysis method in SPSS 18.0 software, such as... Figure 9 As shown in the figure, the raw and charred Sophora japonica fruit products also clustered into separate groups. The results shown in the figure are basically consistent with the PCA analysis results, indicating that the PCA analysis results are reliable. Furthermore, the differences in the chemical fingerprint peaks between the raw and charred Sophora japonica fruit products can be identified using clustering. Combining the peak areas of each of the 36 relevant peaks and the Pearson correlation analysis results, it can be confirmed that components 1 and 3 are mainly phenolic acids and flavonoid glycosides, component 2 is mainly phenolic acids and flavonoid aglycones, and component 4 is a flavonoid glycoside.

[0263] Example 2: Screening of chromatographic conditions for fingerprint spectroscopy

[0264] 1. Mobile phase and gradient elution

[0265] Take an appropriate amount of Sophora japonica charcoal sample t1 (i.e., Sophora japonica charcoal sample of raw product with batch number 20191101 after being stir-fried for 20 minutes), and prepare the test solution according to the preparation method of the test sample solution in Example 1. Record the chromatograms under the following chromatographic conditions. Other chromatographic conditions are the same as in Example 1 above.

[0266] Chromatographic conditions 1: Mobile phase: 0.05% formic acid aqueous solution (A) - methanol (B) gradient elution; chromatographic column: Thermo Hypersili1 Gold UPLC column (100*2.1mm, 1.9μm); flow rate: 0.2mL / min; UV wavelength: 278nm; column temperature: 35℃; injection volume: 1μL.

[0267] Gradient elution was performed using the procedure shown in Table 18:

[0268] Table 18

[0269] Washing time / min Mobile phase A Mobile phase B 0-4 85％→80％ 15％→20％ 4-15 80％→65％ 20％→35％ 15-25 65％→55％ 35％→45％ 25-30 60％→40％ 40％→60％ 30-33 Maintain 40% Maintain 60% 33-38 40％→85％ 60％→15％ 38-42 Maintain 85% Keep it at 15%

[0270] The results are shown in Table 19 and Figure 10 As shown.

[0271] Table 19

[0272] No. Retention time (min) Resolution 1 1.815 2.28 2 2.121 17.77 3 4.945 17.41 4 8.629 15.13 5 11.616 9.57 6 13.245 10.69 7 15.925 1.83 8 16.441 4.14 9 17.263 3.74 10 17.944 2.55 11 18.586 2.59 12 19.228 8.94 13 20.938 5.07 14 21.194 / 15 21.910 26.18 16 27.282 2.06 17 27.773 2.16 18 28.326 5.68 19 29.610 2.00 20 30.091 /

[0273] Chromatographic conditions 2: mobile phase 0.05% formic acid aqueous solution (A)-methanol (B) gradient elution, chromatographic column Thermo Hypersili 1 Gold column (100*2.1mm, 1.9μm), flow rate 0.2mL / min, UV wavelength 278nm, column temperature 35℃, injection volume 1μL;

[0274] Gradient elution was performed using the procedure shown in Table 20:

[0275] Table 20

[0276] Washing time / min Mobile phase A Mobile phase B 0-10 90％→70％ 10％→30％ 10-30 70％→60％ 30％→40％ 30-40 60％→40％ 40％→60％ 40-45 40％→20％ 60％→80％ 45-50 20％→90％ 80％→10％

[0277] The results are shown in Table 21 and Figure 11 As shown.

[0278] Table 21

[0279] No. Retention time (min) Resolution 1 2.129 2.83 2 2.583 2.51 3 3.017 20.82 4 7.185 25.35 5 11.362 13.97 6 13.144 8.28 7 14.201 10.57 8 16.333 2.00 9 16.812 3.64 10 18.112 / 11 18.247 3.21 12 19.689 / 13 20.329 4.41 14 21.734 3.85 15 22.113 / 16 22.745 / 17 23.107 / 18 31.93 / 19 35.973 / 20 36.237 /

[0280] Chromatographic conditions 3: mobile phase 0.05% formic acid aqueous solution (A)-methanol (B), chromatographic column Thermo Hypersi1 Gold column (100*2.1mm, 1.9μm), flow rate 0.2mL / min, UV wavelength 278nm, column temperature 35℃, injection volume 1μL;

[0281] Gradient elution was performed using the procedure shown in Table 22:

[0282] Table 22

[0283] Washing time / min Mobile phase A Mobile phase B 0-10 90％→80％ 10％→20％ 10-25 80％→60％ 20％→40％ 25-30 Maintain 60% Maintain 40% 30-40 60％→40％ 40％→60％ 40-45 40％→20％ 60％→80％ 45-50 Maintain 90% Keep it at 10%

[0284] The results are shown in Table 23 and Figure 12 As shown.

[0285] Table 23

[0286] No. Retention time (min) Resolution 1 2.140 2.72 2 2.592 2.51 3 3.036 20.22 4 7.439 32.92 5 14.261 19.33 6 17.553 9.99 7 18.961 / 8 19.182 14.3 9 21.756 1.20 10 21.988 2.19 11 22.456 4.21 12 23.317 3.79 13 24.031 2.44 14 24.61 2.95 15 25.312 / 16 25.597 / 17 25.755 / 18 25.98 4.31 19 27.128 3.52 20 27.375 / 21 27.835 1.21 22 28.08 23.17 23 34.13 9.72 24 36.863 1.11

[0287] Chromatographic conditions 4: mobile phase 0.05% formic acid aqueous solution (A)-methanol (B), chromatographic column Thermo Hypersi1 Gold column (100*2.1mm, 1.9μm), flow rate 0.2mL / min, UV wavelength 278nm, column temperature 35℃, injection volume 1μL;

[0288] Gradient elution was performed using the procedure shown in Table 24:

[0289] Table 24

[0290] Washing time / min Mobile phase A Mobile phase B 0-10 90％→80％ 10％→20％ 10-25 80％→50％ 20％→50％ 25-30 Keep 50% Keep 50% 30-40 50％→40％ 50％→60％ 40-45 40％→20％ 60％→80％ 45-50 Maintain 90% Keep it at 10%

[0291] The results are shown in Table 25 and Figure 13 As shown.

[0292] Table 25

[0293] No. Retention time (min) Resolution 1 2.130 2.80 2 2.579 2.52 3 3.014 20.27 4 7.368 33.05 5 14.158 18.79 6 17.235 9.13 7 18.583 11.51 8 20.690 / 9 20.773 1.93 10 21.177 3.90 11 21.829 0.99 12 21.975 2.15 13 22.338 / 14 22.881 / 15 23.547 / 16 23.760 3.52 17 24.500 2.78 18 24.711 / 19 24.957 1.26 20 25.167 22.95 21 29.022 3.37 22 29.280 / 23 29.587 4.29 24 30.372 /

[0294] Chromatographic conditions 5: mobile phase 0.05% formic acid aqueous solution (A)-acetonitrile (C), chromatographic column Thermo Hypersi1 Gold column (100*2.1mm, 1.9μm), flow rate 0.2mL / min, UV wavelength 278nm, column temperature 35℃, injection volume 1μL;

[0295] Gradient elution was performed using the procedure shown in Table 26:

[0296] Table 26

[0297] Washing time / min Mobile phase A mobile phase C 0-10 93％→85％ 7％→15％ 10-25 85％→70％ 15％→30％ 25-30 Maintain 70% Keep 30% 30-40 70％→55％ 30％→45％ 40-45 55％→40％ 45％→60％ 45-50 Maintain 93% Maintain 7%

[0298] The results are shown in Table 27 and Figure 14 As shown.

[0299] Table 27

[0300]

[0301]

[0302] Chromatographic conditions 6: Mobile phase 0.05% formic acid aqueous solution (A)-acetonitrile (C), chromatographic column Thermo Hypersi1 Gold column (100*2.1mm, 1.9μm), flow rate 0.2mL / min, UV wavelength 278nm, column temperature 35℃, injection volume 1μL;

[0303] Gradient elution was performed using the procedure shown in Table 28:

[0304] Table 28

[0305] Washing time / min Mobile phase A mobile phase C 0-10 93％→85％ 7％→15％ 10-25 85％→75％ 15％→25％ 25-30 75％→70％ 25％→30％ 30-40 70％→55％ 30％→45％ 40-45 55％→40％ 45％→60％ 45-50 Maintain 93% Maintain 7%

[0306] The results are shown in Table 29 and Figure 15 As shown.

[0307] Table 29

[0308]

[0309]

[0310] Chromatographic conditions 7: mobile phase 0.05% formic acid aqueous solution (A)-acetonitrile (C), chromatographic column Thermo Hypersi1 Gold column (100*2.1mm, 1.9μm), flow rate 0.2mL / min, UV wavelength 278nm, column temperature 35℃, injection volume 1μL;

[0311] Gradient elution was performed using the procedure shown in Table 30:

[0312] Table 30

[0313] Washing time / min Mobile phase A mobile phase C 0-10 93％→90％ 7％→10％ 10-25 90％→80％ 10％→20％ 25-30 80％→70％ 20％→30％ 30-40 70％→55％ 30％→45％ 40-45 55％→40％ 45％→60％ 45-50 40％→93％ 60％→7％

[0314] The results are shown in Table 31 and Figure 16 As shown.

[0315] Table 31

[0316]

[0317]

[0318] Chromatographic conditions 8: Mobile phase 0.05% formic acid aqueous solution (A)-acetonitrile (C), chromatographic column Thermo Hypersi1 Gold column (100*2.1mm, 1.9μm), flow rate 0.2mL / min, UV wavelength 278nm, column temperature 35℃, injection volume 1μL;

[0319] Gradient elution was performed using the procedure shown in Table 32:

[0320] Table 32

[0321] Washing time / min Mobile phase A mobile phase C 0-10 93％→90％ 7％→10％ 10-25 90％→85％ 10％→15％ 25-35 85％→80％ 15％→20％ 35-45 80％→75％ 20％→25％ 45-50 75％→55％ 25％→45％ 50-55 55％→40％ 45％→60％ 55-60 40％→93％ 60％→7％

[0322] The results are shown in Table 33 and Figure 17 As shown.

[0323] Table 33

[0324]

[0325]

[0326] 2. Wavelength selection

[0327] Take an appropriate amount of Sophora japonica charcoal sample t8 (i.e., Sophora japonica charcoal sample of raw product with batch number 210100881 that has been stir-fried for 20 minutes), and prepare the test solution according to the preparation method of the test sample solution in Example 1. Record the chromatograms at detection wavelengths of 230, 254 and 280 nm respectively. Other chromatographic conditions are the same as in Example 1 above.

[0328] Test results are shown Figure 18 In the figure, the wavelengths from bottom to top are 230, 254, and 280 nm, respectively. The chromatographic peaks are not significantly different. At a wavelength of 280 nm, the chromatographic peak response is higher. Therefore, the preferred wavelength is 280 nm.

[0329] 3. Selection of chromatographic column

[0330] Take an appropriate amount of Sophora japonica charcoal sample t8 (i.e., Sophora japonica charcoal sample of raw product roasted for 20 min with batch number 210100881), and prepare the test solution according to the preparation method of the test sample solution in Example 1. Sequentially determine the test solution using a 1-ACQUITY CSH C18 column (150 mm × 2.1 mm, 1.7 μm) and a 2-Hypersi1 Gold column (100 × 2.1 mm, 1.9 μm), with other chromatographic conditions the same as in Example 1 above.

[0331] The test results are shown in Table 34 and Figure 19In the figure, from bottom to top, are the ACQUITY CSH C18 column and the Hypersi1 Gold column. The results show that the chromatographic peaks with retention times of 25.000 min and 31.508 min on the Hypersi1 Gold column (100 × 2.1 mm, 1.9 μm) have poor resolution; therefore, the ACQUITY CSH C18 column (150 mm × 2.1 mm, 1.7 μm) was used.

[0332] Table 34 Peak retention time and resolution in different chromatographic columns

[0333]

[0334]

[0335] 4. Column temperature selection

[0336] Take an appropriate amount of Sophora japonica charcoal sample t8 (i.e., Sophora japonica charcoal sample of raw product roasted for 20 min with batch number 210100881), and prepare the test solution according to the preparation method of the test sample solution in Example 1. Adjust the column temperature to 30℃, 35℃ and 40℃ respectively for determination. Other chromatographic conditions are the same as in Example 1 above.

[0337] The test results are shown in Table 35 and Figure 20 In the figure, the column temperatures from bottom to top are 30℃, 35℃, and 40℃. A column temperature of 30℃ is suitable for flavonoids such as rutin (t...). R =39.357min), isorhamnetin-3-O-rutin (t R =41.159min) The resolution was not as good as at 35℃, and the small peaks after the seventh peak (t R =34.622min), none; the flavonoid separation was not as good at 40℃ as at 35℃, so 35℃ was chosen in summary.

[0338] Table 35 Peak retention time and resolution under different column temperatures

[0339]

[0340] 5. Selection of flow rate

[0341] Take an appropriate amount of Sophora japonica charcoal sample t8 (i.e., Sophora japonica charcoal sample of raw product roasted for 20 min with batch number 210100881), and prepare the test solution according to the preparation method of the test sample solution in Example 1. Adjust the flow rate to 0.12 mL / min, 0.15 mL / min and 0.18 mL / min respectively and measure them sequentially. Other chromatographic conditions are the same as in Example 1 above.

[0342] The test results are shown in Table 36 and Figure 21In the figure, the flow rates from bottom to top are 0.12 mL / min, 0.15 mL / min, and 0.18 mL / min. The overall chromatographic peaks show little difference, except that the small peak disappears at a flow rate of 0.18 mL / min (t...). R =31.59min, 32.289min), therefore, 0.12~0.15mL / min is selected.

[0343] Table 36 Peak retention time and resolution under different flow rates

[0344]

Claims

1. A method for constructing a fingerprint spectrum of Sophora japonica pods, comprising the following steps: The analytes from Sophora japonica fruit can be obtained by gradient elution using liquid chromatography. The liquid chromatography method is ultra-high performance liquid chromatography; The extract of the Sophora japonica fruit analyte was water or methanol. In the liquid chromatography method, the chromatographic conditions are as follows: The chromatographic column is an octadecylsilane-bonded silica gel column; The detection wavelength is 278-300 nm; the column temperature is 30-40℃; the mobile phase is mobile phase A-mobile phase B, wherein mobile phase A is formic acid solution and mobile phase B is methanol; The gradient elution procedure is shown in the table below: 。 2. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 1, characterized in that, It meets one or more of the following conditions: (1) The chromatographic column is a Waters ACQUITY CSH C18 column; (2) In the chromatographic conditions, the mobile phase A is a 0.05-0.5% formic acid solution; (3) In the chromatographic conditions described, the detector is a UV-DAD detector; (4) Under the chromatographic conditions described, the flow rate is 0.12-0.18 mL / min; (5) In the chromatographic conditions, the column temperature is 30℃, 35℃ or 40℃; (6) Under the chromatographic conditions described, the injection volume is 1-5 μl; (7) In the chromatographic conditions, the injection concentration of the analyte from Sophora japonica is 0.004-0.1 g / mL; (8) The Sophora japonica fruit to be tested is raw Sophora japonica fruit and / or processed Sophora japonica fruit; (9) In the extract of the Sophora japonica fruit to be tested, the methanol is 70% methanol.

3. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 2, characterized in that, It meets one or more of the following conditions: (1) The specifications of the Waters ACQUITY CSH C18 chromatographic column are 150 mm. 2.1mm, 1.7µm; (2) In the chromatographic conditions, the mobile phase A is a 0.05% formic acid solution; (3) The flow rate in the chromatographic conditions is 0.12 mL / min, 0.15 mL / min or 0.18 mL / min; (4) In the chromatographic conditions described, the column temperature is 35℃; (5) Under the chromatographic conditions described, the injection volume is 1 μL; (6) In the chromatographic conditions, the injection concentration of the analyte from Sophora japonica is 0.025 g / mL; (7) In the chromatographic conditions, the Sophora japonica analyte is pretreated before elution. The pretreatment further includes the following steps: soaking Sophora japonica in water, decocting, filtering, concentrating the filtrate and then freeze-drying it to obtain freeze-dried powder of Sophora japonica analyte dissolved in a solvent in the form of freeze-dried powder. (8) The processed product of the Sophora japonica fruit is Sophora japonica fruit charcoal.

4. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 1, characterized in that, It meets one or two of the following conditions: (1) In the chromatographic conditions, the detection wavelength is 278 or 280 nm; (2) The flow rate under the chromatographic conditions is 0.12-0.15 mL / min.

5. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 3, characterized in that, It meets one or two of the following conditions: (1) In the pretreatment, the solvent is methanol; (2) The charred Sophora japonica fruit can be prepared by frying raw Sophora japonica fruit.

6. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 5, characterized in that, It meets one or two of the following conditions: (1) The frying temperature is 200-300℃. (2) The stir-frying time is 5-50 min.

7. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 6, characterized in that, It meets one or two of the following conditions: (1) The frying temperature is 200℃. (2) The stir-frying time is 5, 9, 12, 15, 20, 30 or 50 min.

8. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 7, characterized in that, The frying time is 15 - 20 min.

9. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 1, characterized in that, The construction method is used to detect or isolate the Sophora japonica components in the Sophora japonica test substance to be detected.

10. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 9, characterized in that, The components of the sophora japonica fruit include the following: methyl gallate, dihydrokaempferol-3- O - Glucoside, Kaempferol-3- O -(2 - O - β - D -glucosyl)- β - D - Rutin, Kaempferol-3-O - Sophoroside, dihydroapigenin-7- O -Glucoside, Apigenin-7- O -Rutin, Kaempferol-3- O -Rutin, Isorhamnetin-3- O - β - D - One or more of rutin and linalool.

11. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 10, characterized in that, The components of the sophora japonica fruit include the following: gallic acid, p-hydroxybenzyl alcohol, and genistein-4′- O One or more of the following: (6′′-acetyl)-glucosides.

12. The method for constructing a fingerprint spectrum of Sophora japonica as described in claim 11, characterized in that, The sophora japonica fruit components include the following components: (3,4,5-trihydroxy-3-furanyl)-4-methoxybenzoic acid, p-digallic acid, genistein-7,4′-di- O-β-D -glucoside, genistein-7- O -glucose-4′- O -neohesperidoside, kaempferol-3- O -(3′,4′-di- O -glucosyl)-rhamnose-7- O -rhamnoside, kaempferol-3- O -sophoroside-7- O -rhamnoside, genistein-7- O -malonylglucose-4′- O [[ID=...]]-glucoside, quercetin-3- O -(3′- O -glucosyl)-rutinoside, genistin, apigenin-7- O -(3′′-acetyl)-rutinoside, kaempferol-7-methoxy-4′- O -glucosyl-3- O -(2- O-apio -furoyl)glucoside, isorhamnetin-3- O -sophoroside, naringin, rutin, sophoricoside, sophoraflavanone glycoside, genistein-4′- O -(6′′-malonyl)glucoside, camelliaflavone glycoside A, genistein and kaempferol, one or more of them. It should be noted that there are some incomplete or seemingly incorrect tags in the original text (such as the ellipsis in the middle of the translation). If possible, it is recommended to check and correct the original text for a more accurate translation.

13. The method for constructing a Sophora japonica fingerprint spectrum as described in claim 12, characterized in that, The sophora japonica fruit components include methyl gallate, dihydrokaempferol-3- O -glucoside, kaempferol-3- O -(2 - O - β - D -glucosyl)- β - D -rutinoside, kaempferol-3- O- sophoroside, dihydroapigenin-7- O -glucoside, apigenin-7- O -rutinoside, kaempferol-3- O -rutinoside, isorhamnetin-3- O - β - D -rutinoside, eriodictyol glycoside, gallic acid, p-hydroxybenzyl alcohol, genistein-4′- O -(6′′-acetyl)-glucoside, (3,4,5-trihydroxy-3-furanyl)-4-methoxybenzoic acid, p-bigallic acid, genistein-7,4′-di- O-β-D -glucoside, genistein-7-[[ID=3s]] O -glucose-4′- O -neohesperidoside, kaempferol-3- O -(3′,4′-di- O -glucosyl)-rhamnose-7- O -rhamnoside, kaempferol-3- O -sophorose-7- O -rhamnoside, genistein-7- O -malonylglucose-4′- O -glucoside, quercetin-3- O -(3′- O -glucosyl)-rutinoside, genistin, apigenin-7- O -(3′′-acetyl)-rutinoside, kaempferol-7-methoxy-4′- O -glucosyl-3- O -(2- O-apio -furanyl)glucoside, isorhamnetin-3- O -sophoroside, naringin, rutin, sophoricoside, sophorabioside, genistein-4′- O -(6′′-malonyl)glucoside, camelliaflavone glycoside A, genistein and kaempferol.

14. An application of a fingerprint obtained by the method for constructing a fingerprint of Sophora japonica as claimed in any one of claims 1 - 13 as a standard fingerprint, wherein the application is for the detection and quality control of raw Sophora japonica or for the detection and quality control of processed Sophora japonica.

15. The application as described in claim 14, characterized in that, The said application comprises the following steps: a. Take the raw Sophora japonica or processed Sophora japonica to be detected, and obtain the corresponding liquid chromatogram by using the method for constructing a fingerprint of Sophora japonica as claimed in any one of claims 1 - 13; b. Evaluate the similarity between the liquid chromatogram obtained in step a and the standard fingerprint.

16. The application as described in claim 15, characterized in that, In the said step b, if the similarity ≥ 0.90, it is qualified.

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