Methods for evaluating the quality of sophora fruit products, pharmaceutical compositions, and uses thereof

Abstract

The application discloses a kind of material evaluation methods, pharmaceutical compositions and application of sophora fruit products.It is specific, disclose a kind of material evaluation methods of sophora fruit products, it includes the following steps: S1, using liquid chromatography, the fingerprint of the sophora fruit product of different processing time is established, and common characteristic peak is extracted;S2, the hemostatic effect of sophora fruit product of different processing time is determined;S3, according to the characteristic peak data of the sophora fruit product of different processing time and its hemostatic effect data, statistical analysis is carried out by chemometrics method, and the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect of the hemostatic effect

Description

Technical Field

[0001] This invention relates to a method for evaluating the substances in Sophora japonica fruit products, a pharmaceutical composition, 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. Summary of the Invention

[0003] The purpose of this invention is to address the current lack of effective and efficient methods for evaluating the hemostatic effect of Sophora japonica fruit products. This invention provides a material evaluation method, pharmaceutical composition, and application for Sophora japonica fruit products. The material evaluation method for the hemostatic effect of Sophora japonica fruit products based on the spectrum-effect relationship can quickly and accurately evaluate the hemostatic components of Sophora japonica fruit products and screen active ingredients, providing a scientific and effective method for the research on the pharmacodynamic material basis and quality control of Sophora japonica fruit products.

[0004] The present invention mainly solves the above-mentioned technical problems through the following technical solutions.

[0005] This invention provides a method for evaluating the material composition of Sophora japonica fruit products, which includes the following steps:

[0006] S1. Using liquid chromatography, fingerprint chromatograms of Sophora japonica fruit products with different processing times were established, and common characteristic peaks were extracted.

[0007] S2. Determine the hemostatic effect of Sophora japonica fruit products processed for different times;

[0008] S3. Based on the characteristic peak data and hemostatic effect data of Sophora japonica fruit products with different processing times, statistical analysis was conducted using chemometric methods to evaluate the pharmacodynamic material basis of hemostatic effect in Sophora japonica fruit products.

[0009] In step S1, the chromatographic conditions for the liquid chromatography method are as follows:

[0010] The chromatographic column is an octadecylsilane-bonded silica gel column;

[0011] The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a formic acid solution and mobile phase B is methanol;

[0012] A gradient elution method is used, with the total volume of the mobile phase being 100%. The gradient elution procedure is performed sequentially from top to bottom according to the table below:

[0013] Wash-off time / minute Mobile phase A Mobile phase B 8-12 90％→80％ 10％→20％ 15-20 80％→60％ 20％→40％ 2-5 Maintain 60% Maintain 40% 8-12 60％→40％ 40％→60％ 2-5 Maintain 40% Maintain 60% 5-8 40％→20％ 60％→80％ 1-4 Keep it at 20% Maintain 80% 12-18 Maintain 90% Keep it at 10%

[0014] The elution time is the difference between the elution end time and the elution start time of the elution stage.

[0015] In step S1, the liquid chromatography can be high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC).

[0016] In step S1, the Sophora japonica fruit products with different processing times may include two or more of the following: Sophora japonica fruit products with processing times of 0, 5, 9, 12, 15, 20, 30 and 50 minutes respectively.

[0017] In step S1, the Sophora japonica fruit product can be prepared by conventional methods in the art, such as by stir-frying the raw Sophora japonica fruit. Preferably, the stir-frying temperature is 200-300℃, for example, 200℃. Preferably, the stir-frying time is 5-50 minutes; for example, 5, 9, 12, 15, 20, 30, or 50 minutes.

[0018] In step S1, the chromatographic column can be a Waters ACQUITY CSH C18 column (150mm × 2.1mm, 1.7μm), a 1-ACQUITY CSH C18 column (150mm × 2.1mm, 1.7μm), or a 2-Hypersi1Gold column (100mm × 2.1mm, 1.9μm); preferably a Waters ACQUITY CSH C18 column (150mm × 2.1mm, 1.7μm).

[0019] In step S1, the gradient elution procedure can be shown in the following table:

[0020] Time / minute Mobile phase A / volume percentage Mobile phase B / volume percentage 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％

[0021] In step S1, 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.

[0022] In step S1, the chromatographic conditions may use detectors conventional in the art, such as UV-DAD detectors.

[0023] In step S1, the chromatographic conditions may use detection wavelengths that are conventional in the art, such as 210-300 nm, or 230, 254 or 280 nm, with 280 nm being the preferred wavelength.

[0024] In step S1, the flow rate in the chromatographic conditions 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.

[0025] In step S1, the column temperature in the chromatographic conditions can be 30-40℃; for example, 30℃, 35℃ or 40℃; preferably 35℃.

[0026] In step S1, the injection volume in the chromatographic conditions can be conventional in the art, for example, the injection volume is 1-5 μl, or for example, the injection volume is 1 μL.

[0027] In step S1, the injection concentration in the chromatographic conditions can be conventional in the art, for example, 0.004-0.1 g / mL, or 0.025 g / mL.

[0028] In step S1, the Sophora japonica fruit product can be pretreated before injection to meet the injection standards. The pretreatment can be a conventional pretreatment in the art; preferably, the pretreatment includes the following step: dissolving the Sophora japonica fruit product in a solvent. Further, the solvent is methanol.

[0029] Furthermore, in step S1, the pretreatment step may also include the following steps: soaking the Sophora japonica product in water (200mL) for 30min, decocting (boiling and decocting for 30min, then adding 160mL of water and continuing to decoct for 20min), filtering, concentrating the filtrate and freeze-drying to obtain freeze-dried powder of the Sophora japonica product, which is dissolved in the solvent in the form of freeze-dried powder and prepared fresh for use.

[0030] In step S1, common characteristic peaks can be extracted using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A" software.

[0031] In step S1, the common characteristic peaks may include characteristic peaks with the following retention times: 5.60±0.05 min, 6.56±0.01 min, 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, 41.63±0.07 min, 43.58±0.03 min, and 44.34±0.04 min. Furthermore, the common characteristic peaks may also include characteristic peaks with retention times of one or more of the following: 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, 30.14±0.05 min, 30.53±0.03 min, 32.78±0.02 min, 33.15± 0.03min, 34.06±0.03min, 34.70±0.03min, 35.96±0.06min, 38.27±0.05min, 39.89±0.06min, 40.90±0.04min, 42. 09±0.05min, 43.35±0.02min, 44.60±0.03min, 45.31±0.03min, 46.28±0.02min, 46.74±0.04min and 49.21±0.04min.

[0032] In step S2, the hemostatic effect may include one or more of the following four parameters: tail bleeding time, clotting time, and clotting.

[0033] In step S3, Pearson bivariate correlation analysis can be used to analyze the correlation between the peak area of ​​the common characteristic peak and the hemostatic effect data, and a spectrum-effect equation can be established through multiple linear regression to determine the material basis of the hemostatic effect of the Sophora japonica fruit product. Preferably, the characteristic peaks with retention times of 5.60±0.05 min, 6.56±0.01 min, 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, 41.63±0.07 min, 43.58±0.03 min, and 44.34±0.04 min show good correlation with the hemostatic effect.

[0034] Preferably, the characteristic peak with a retention time of 5.60±0.05 min is gallic acid, the characteristic peak with a retention time of 6.58 min is p-hydroxybenzyl alcohol, the characteristic peak with a retention time of 19.83±0.01 min is methyl gallate, the characteristic peak with a retention time of 28.24±0.06 min is dihydrokaempferol-3-O-glucoside, and the characteristic peak with a retention time of 33.60±0.04 min is kaempferol-3-O-(2”-O-β-D-glucoside. The characteristic peaks of kaempferol-3-O-sophorol (glycosyl)-β-D-rutin glycoside with a retention time of 34.99±0.05 min are: kaempferol-3-O-sophorol; dihydroapigenin-7-O-glucoside with a retention time of 36.28±0.05 min; rutin with a retention time of 37.43 min; kaempferol-3-O-rutin glycoside with a retention time of 41.63±0.07 min; and linalool glycoside with a retention time of 43.58±0.03 min.

[0035] In step S3, the characteristic peaks with retention times of 5.60±0.05 min, 6.56±0.01 min, 40.90±0.04 min, 42.09±0.05 min, 44.34±0.04 min, and 45.31±0.03 min are respectively introduced into the spectral effect mathematical model:

[0036] Bleeding time BT = 3.609 - 3.077 * X2;

[0037] Y clotting time CT = 3.128 + 0.688*X31 - 3.11*X26;

[0038] Y prothrombin time PT = 0.505 - 0.017*X3 - 0.012*X33;

[0039] Y-fibrinogen (FIB) = 3.644 - 0.666 * X28;

[0040] Wherein, X2 represents the peak area of ​​the chromatographic peak with a retention time of 5.60±0.05 min, X3 represents the peak area of ​​the chromatographic peak with a retention time of 6.56±0.01 min, X26 represents the peak area of ​​the chromatographic peak with a retention time of 40.90±0.04 min, X28 represents the peak area of ​​the chromatographic peak with a retention time of 42.09±0.05 min, X31 represents the peak area of ​​the chromatographic peak with a retention time of 44.34±0.04 min, and X33 represents the peak area of ​​the chromatographic peak with a retention time of 45.31±0.03 min. Y bleeding time BT, Y clotting time CT, Y prothrombin time PT, and Y fibrinogen FIB represent the hemostatic effect of Sophora japonica charcoal.

[0041] Preferably, in the mathematical model of Y bleeding time BT, P = 0.034 < 0.05, R 2 =0.556; In the mathematical model of Y clotting time CT, P=0.026<0.05, R 2 =0.905; In the mathematical model of prothrombin time (PT) for Y, P = 0.049 < 0.05, R 2 =0.858; In the mathematical model of fibrinogen (FIB), P = 0.007 < 0.05, R 2 =0.733.

[0042] In step S3, the method for evaluating the material basis of the hemostatic effect in Sophora japonica fruit products may include: characteristic peaks with a significant difference in correlation between the peak area of ​​characteristic peaks and hemostatic effect data (P<0.05) and characteristic peaks introduced into the spectral effect equation are the material basis of the hemostatic effect in Sophora japonica fruit products.

[0043] Preferably, the material basis for the hemostatic effect of Sophora japonica fruit products includes substances corresponding to characteristic peaks with retention times of one or more of the following: 5.60±0.05 min, 6.56±0.01 min, 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, 43.58±0.03 min, 44.34±0.04 min, and 45.31±0.03 min.

[0044] In step S3, preferably, the hemostatic agents in the Sophora japonica fruit product include one or more of the following: gallic acid, p-hydroxybenzyl alcohol, 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, rutin, apigenin-7-O-rutinoside, isorhamnetin-3-O-β-D-rutinoside, kaempferol-3-O-rutinoside, eleutheroside, and genistein-4′-O-(6″-acetyl)-glucoside.

[0045] Step S3 may further include principal component analysis of the relevant peaks of the Sophora japonica fruit products to screen for relevant peak components that have a significant impact on the differences between Sophora japonica fruit products processed at different times. Preferably, in the principal component analysis method:

[0046] The cumulative contribution rate of the retained principal components is >90%;

[0047] The retained principal components have eigenvalues ​​> 1;

[0048] Then it was tested using the KMO test and the Bartlett test.

[0049] Step S3 may also include further validation of principal component analysis using cluster analysis.

[0050] The present invention also provides a pharmaceutical composition comprising the following active ingredients: gallic acid or a pharmaceutically acceptable salt thereof, p-hydroxybenzyl alcohol or a pharmaceutically acceptable salt thereof, methyl gallate or a pharmaceutically acceptable salt thereof, dihydrokaempferol-3-O-glucoside or a pharmaceutically acceptable salt thereof, kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutin glycoside or a pharmaceutically acceptable salt thereof, and kaempferol-3-O-sophorose or a pharmaceutically acceptable salt thereof. Dihydroapigenin-7-O-glucoside or a pharmaceutically acceptable salt thereof, rutin or a pharmaceutically acceptable salt thereof, apigenin-7-O-rutinoside or a pharmaceutically acceptable salt thereof, isorhamnetin-3-O-β-D-rutinoside or a pharmaceutically acceptable salt thereof, kaempferol-3-O-rutinoside or a pharmaceutically acceptable salt thereof, linalool or a pharmaceutically acceptable salt thereof, and genistein-4′-O-(6″-acetyl)-glucoside or a pharmaceutically acceptable salt thereof.

[0051] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0052] Preferably, in the pharmaceutical composition, the active ingredient comprises the following components: gallic acid or a pharmaceutically acceptable salt thereof, p-hydroxybenzyl alcohol or a pharmaceutically acceptable salt thereof, methyl gallate or a pharmaceutically acceptable salt thereof, dihydrokaempferol-3-O-glucoside or a pharmaceutically acceptable salt thereof, kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutin glycoside or a pharmaceutically acceptable salt thereof, and kaempferol-3-O-sophorose or a pharmaceutically acceptable salt thereof. Dihydroapigenin-7-O-glucoside or a pharmaceutically acceptable salt thereof, rutin or a pharmaceutically acceptable salt thereof, apigenin-7-O-rutinoside or a pharmaceutically acceptable salt thereof, isorhamnetin-3-O-β-D-rutinoside or a pharmaceutically acceptable salt thereof, kaempferol-3-O-rutinoside or a pharmaceutically acceptable salt thereof, linalool or a pharmaceutically acceptable salt thereof, and genistein-4′-O-(6″-acetyl)-glucoside or a pharmaceutically acceptable salt thereof.

[0053] The present invention also provides the use of substance X or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described in any one of the present invention in the preparation of a hemostatic agent, wherein substance X is one or more of gallic acid, p-hydroxybenzyl alcohol, 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, rutin, apigenin-7-O-rutinoside, isorhamnetin-3-O-β-D-rutinoside, kaempferol-3-O-rutinoside, eleutheroside, and genistein-4′-O-(6″-acetyl)-glucoside.

[0054] The present invention also provides the use of substance X or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described in any one of the present invention in the quality control of Sophora japonica fruit products, wherein substance X is one or more of gallic acid, p-hydroxybenzyl alcohol, 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, rutin, apigenin-7-O-rutinoside, isorhamnetin-3-O-β-D-rutinoside, kaempferol-3-O-rutinoside, eleutheroside, and genistein-4′-O-(6″-acetyl)-glucoside.

[0055] In the application of the quality control of the Sophora japonica fruit products, substance X can be used as a reference standard for the active ingredients of the Sophora japonica fruit products.

[0056] In the application of the quality control of Sophora japonica fruit products, quality control is achieved by detecting substance X or its pharmaceutically acceptable salt in the Sophora japonica fruit products. Preferably, liquid chromatography is used to detect substance X or its pharmaceutically acceptable salt in the Sophora japonica fruit products, and the chromatographic conditions of the liquid chromatography are as described in any one of the claims of the present invention, for example, as described in any one of the steps S1.

[0057] 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.

[0058] The reagents and raw materials used in this invention are all commercially available.

[0059] The positive and progressive effects of this invention are as follows:

[0060] (1) This invention uses chemical pattern recognition to establish a correlation between the changes in chemical composition and efficacy of Sophora japonica products reflected in fingerprint spectrum, and clarifies that the pharmacological basis of the hemostatic effect of Sophora japonica products is mainly flavonoids and supplemented by small molecule phenolic acids.

[0061] (2) This invention establishes a method for evaluating the chemical components of Sophora japonica fruit products based on the spectrum-effect relationship of hemostasis, and clarifies the chemical components in Sophora japonica fruit products that are closely related to hemostatic efficacy, providing a basis for the study of the material basis of hemostatic efficacy of Sophora japonica fruit products. At the same time, the method of combining fingerprint spectrum and mathematical model avoids the interference of third-party factors and avoids individual animal bias, and can quickly and accurately evaluate the chemical components with hemostatic effect in Sophora japonica fruit products and screen the active ingredients, providing a scientific and effective method for the study of the material basis of efficacy and quality control of Sophora japonica fruit products.

[0062] (3) This invention uses Sophora japonica fruit, a blood-cooling and hemostatic drug, as a representative to study the hemostatic effect of charred drugs. This is helpful to reveal the change law of the components of charred blood-cooling and hemostatic drugs and their correlation with hemostasis, and provides a reference for the scientific explanation of the hemostatic effect of charred drugs. Attached Figure Description

[0063] Figure 1 UPLC fingerprints of Sophora japonica charcoal prepared at different processing times according to the present invention.

[0064] Figure 2 The reference standard is shown in the diagram, which includes: 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.

[0065] Figure 3 The graph shows the effect of the decoction of Sophora japonica fruit before and after processing on the bleeding time of mice.

[0066] Figure 4 The graph shows the effect of the decoction of Sophora japonica fruit before and after processing on the coagulation time of mice.

[0067] Figure 5 The graph shows the results of the four coagulation tests in each group of mice.

[0068] Figure 6 The UPLC fingerprint spectra of 10 batches of raw Sophora japonica fruit are presented in this invention.

[0069] Figure 7 UPLC fingerprint spectra of 10 batches of Sophora japonica fruit charcoal processed for 20 minutes according to the present invention.

[0070] Figure 8 PCA principal component analysis chromatograms of 10 batches of raw Sophora japonica fruit and charred fruit processed for 20 minutes.

[0071] Figure 9The validation diagram is shown in the Ward clustering analysis with principal components.

[0072] Figure 10 UPLC fingerprint obtained under chromatographic condition 1

[0073] Figure 11 UPLC fingerprint obtained under chromatographic condition 2

[0074] Figure 12 UPLC fingerprint obtained under chromatographic condition 3

[0075] Figure 13 UPLC fingerprint obtained under chromatographic condition 4

[0076] Figure 14 UPLC fingerprint obtained under chromatographic condition 5

[0077] Figure 15 UPLC fingerprint obtained under chromatographic condition 6

[0078] Figure 16 UPLC fingerprint obtained under chromatographic condition 7

[0079] Figure 17 UPLC fingerprint obtained under chromatographic condition 8

[0080] Figure 18 UPLC fingerprints obtained at different detection wavelengths.

[0081] Figure 19 UPLC fingerprints obtained from different chromatographic columns.

[0082] Figure 20 UPLC fingerprints obtained at different column temperatures.

[0083] Figure 21 UPLC fingerprints obtained at different flow rates. Detailed Implementation

[0084] 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.

[0085] Example 1

[0086] 1. Experimental instruments and materials

[0087] 1.1 Instruments

[0088] Thremo Scientific Vanquish high-performance liquid chromatograph (HPLC); 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.

[0089] 1.2 Materials

[0090] Six batches of Sophorae Fructus were from Shandong Province, sourced from Tianjin Darentang Traditional Chinese Medicine Pieces Co., Ltd., with batch numbers 20191101, 20191102, 20191103, 20191104, 20191105, and 200601 respectively; one batch was from Henan Province, sourced from Guangdong Tiancheng Traditional Chinese Medicine Pieces Co., Ltd., with batch number 200601; two batches were from Hebei Province, sourced from Kangmei Pharmaceutical Co., Ltd. (batch number 210100881) and Shanghai Kangqiao Traditional Chinese Medicine Pieces Co., Ltd. (batch number 190718) respectively; and one batch was from Sichuan Province, sourced from Sichuan Jingxin Traditional Chinese Medicine Pieces Co., Ltd., with batch number SDP201106. These 10 batches of Sophorae Fructus were used in fingerprint analysis, with batch number 200601 being used for spectral efficacy relationship analysis.

[0091] Reference standards: gallic acid (batch number: 110831-201605), rutin (batch number: 100080-202012), kaempferol (batch number: 110861-202013), genistein (batch number: 111704-201703), kaempferol-3-O-rutin (batch number: 112007-202103), isorhamnetin-3-O-β-D-neohesperidin (batch number: 111571-201806), naringin (batch number: 110722-202116), genistein (batch number: 111709- 201702) and sophoroside (batch number: 111695-201703) were purchased from the China National Institutes for Food and Drug Control; sophoroid diglycosides were purchased from Shanghai Shidander Standard Technical Service Co., Ltd.; kaempferol-3-O-sophorose was purchased from Chengdu Purifa Technology Development Co., Ltd.; kaempferol-3-O-(2″-O-β-D-glucosyl)-β-D-rutin was purchased from Chengdu Pusi 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.

[0092] Methanol and formic acid were of chromatographic grade for HPLC, water was ultrapure water, and the remaining reagents were of analytical grade.

[0093] 1.3 Experimental Animals

[0094] 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.

[0095] 2. Preparation of Sophora japonica charcoal samples

[0096] Weigh approximately 400g of cleaned raw Sophora japonica fruit and divide it into 8 portions. Put the remaining 7 portions into a pot at a temperature of about 200℃ and 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 stir-frying degrees.

[0097] 3. Establish UPLC fingerprinting of charred Sophora japonica fruit at different processing times

[0098] 3.1 Preparation of the test solution for processed Sophora japonica fruit

[0099] Grind the processed Sophora japonica fruit into powder and pass it through a No. 3 sieve. Weigh about 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 the test solution 1.

[0100] Weigh 10g of each of the different processed product coarse powders, 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 the freeze-dried powder of processed Sophora japonica fruit, prepare it with pure water as needed to obtain test solution 2 with a concentration of 0.025g / mL.

[0101] Based on the results of the previous investigation of sample pretreatment methods, the chromatographic peaks of test solution 1 and 2 are basically consistent.

[0102] 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.

[0103] 3.2 Fingerprint Spectrum Establishment: The fingerprint spectra of the above-obtained samples were determined by ultra-high performance liquid chromatography (UHPLC). Chromatographic conditions: A 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% mobile phase B (v / v); 9-27 min, 20% → 40% mobile phase B (v / v); 27-30 min, 40% mobile phase B (v / v); 30-39 min, 40% → 60% mobile phase B (v / v); 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.

[0104] 3.3 The characteristic peak data were obtained through analysis using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software: the 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, and Peak 17 33.60 min. n, 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. Chromatograms of characteristic peaks at different processing times are shown below. Figure 1After 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.

[0105] 3.4 Methodological Examination

[0106] 3.4.1 Precision test: 1g of Sophora japonica charcoal sample powder that has been stir-fried for 20min was accurately weighed and prepared according to the method of test solution 1 in "3.1". The sample was injected 6 times consecutively under the chromatographic conditions in "3.2". The peak area RSD of the 25 common peaks was less than 3.4% and the retention time RSD was less than 0.1%, indicating that the method has good precision.

[0107] 3.4.2 Repeatability test: Take 1g of each of the 6 samples of Sophora japonica charcoal powder that have been stir-fried for 20min, weigh them accurately, and prepare 6 test solutions according to the method of test solution 1 in section “3.1”. Inject them sequentially according to the chromatographic conditions in section “3.2”. The relative peak area RSD of each common peak was less than 5.0%, and the retention time RSD was less than 0.2%, indicating that the repeatability of the method is good.

[0108] 3.4.3 Stability test: 1 g of the charred Sophora japonica pod sample powder that has been stir-fried for 20 min was accurately weighed and prepared as a test solution according to the method of test solution 1 in section “3.1”. The sample was injected at 0, 3, 6, 9, 15, 18, 34 and 48 h according to the chromatographic conditions in section “3.2”. The peak area RSD of each common peak was less than 5.0% and the retention time RSD was less than 0.3%, indicating that the test solution was stable within 48 h.

[0109] 4. Pharmacodynamic experiments were conducted on samples processed at different times.

[0110] 4.1 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.

[0111] 4.2 The groups were: 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).

[0112] 4.3 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.

[0113] 4.4 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 (discarding the first drop, adding two drops, and retesting one drop). 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 1 below, and the pharmacodynamic chromatogram is shown in [reference needed]. Figure 3 , 4 .

[0114] Table 1. Effects of Sophora japonica fruit decoction before and after processing on bleeding time in mice. n=10)

[0115]

[0116] In Table 1, 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.

[0117] 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.

[0118] 4.5 Coagulation panel

[0119] 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)).

[0120] 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.

[0121] 4.5.1 Determination of Activated Partial Thromboplastin Time (APTT)

[0122] 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 pre-warmed 0.025 mol / L CaCl2 solution at 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.

[0123] 4.5.2 Measurement of prothrombin time (PT)

[0124] 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.

[0125] 4.5.3 Determination of plasma thrombin time (TT)

[0126] 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 the stopwatch to record the coagulation time, which is the TT value (s). Repeat the experiment twice for each sample and take the average value.

[0127] 4.5.4 Determination of fibrinogen (FIB)

[0128] 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.

[0129] Efficacy data are shown in Table 2, and the chromatograms are shown in [the table]. Figure 5 The results of PT and FIB experiments showed that the charred Sophora japonica fruit group was significantly different from the saline group and the raw Sophora japonica fruit group, and could shorten the PT time and increase the FIB content, indicating that the hemostatic effect of charred Sophora japonica fruit may be related to the extrinsic coagulation pathway and fibrin pathway.

[0130] Table 2. Effects of Sophora japonica fruit decoction before and after processing on plasma APTT, PT, TT, and FIB in mice. n=10)

[0131]

[0132] In Table 2, compared with NS * P<0.05, ** P<0.01, *** P<0.005; compared with 0min, # P<0.05, ## P<0.001.

[0133] 5. Spectral effect correlation analysis

[0134] 5.1 Method

[0135] 5.1.1 Bivariate Correlation Analysis

[0136] 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.

[0137] 5.1.2 Multiple Linear Regression Analysis

[0138] 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.

[0139] 5.1.3 PCA analysis and Ward cluster analysis

[0140] 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.

[0141] 5.2 Results

[0142] 5.2.1 Results of bivariate correlation analysis

[0143] As shown in Table 3-4 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:

[0144]

[0145]

[0146] Table 3 Relevant Peak Areas

[0147]

[0148]

[0149] Table 4 Results of bivariate correlation analysis

[0150]

[0151]

[0152] *P<0.05, **P<0.01

[0153] Table 5. Chromatographic peak identification results by mass spectrometry

[0154]

[0155]

[0156] *Comparison with reference standard

[0157] 5.2.2 Results of Multiple Linear Regression Analysis

[0158] As shown in Table 6 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 chromatographic peaks of Sophora japonica fruit and the hemostatic pharmacological activity.

[0159] Table 6 Results of Mathematical Model Establishment

[0160]

[0161] 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.

[0162] 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.

[0163] 5.2.3 Results of PCA and Ward cluster analysis

[0164] 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 7.

[0165] Table 7 Information on the Origin of Medicinal Materials

[0166]

[0167]

[0168] Prepare the corresponding Sophora japonica charcoal by taking the medicinal materials listed in Table 7 as raw materials: weigh about 400g of cleaned Sophora japonica charcoal, put it into the 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 the Sophora japonica charcoal reference material, and number it sequentially as t1-t10.

[0169] (1) Preparation of reference solution

[0170] 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.

[0171] (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, stopper tightly, weigh, sonicate (power 250W, frequency 40KHz) for 60 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the sample solution.

[0172] (3) Determination of fingerprints of Sophora japonica fruit and Sophora japonica charcoal by ultra-high performance liquid chromatography

[0173] Ten batches of Sophora japonica fruit samples (s1-s10), ten batches of Sophora japonica fruit charcoal samples (t1-t10), and reference solutions prepared according to steps (1) and (2) were injected into an ultra-high performance liquid chromatograph (UPLC). The chromatographic conditions were the same as in section 3.2 above, and the UPLC fingerprints of the ten batches of raw Sophora japonica fruit were obtained (e.g., Figure 6 ) and UPLC fingerprint spectra of 10 batches of charred Sophora japonica fruit processed for 20 minutes (e.g. Figure 7 ).

[0174] (4) PCA analysis and Ward cluster analysis

[0175] The chromatograms of raw and charred Sophora japonica fruit show common peaks. Figure 6 , 7 The analysis results are shown in Table 8. Components with a cumulative contribution rate of >90%, eigenvalues ​​>1, and passing the KMO test (the closer to 1, the more suitable for PCA analysis) and Bartlett's test (P<0.01) were retained.

[0176] Table 8. Principal component analysis results

[0177]

[0178] 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 9, 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.

[0179] Table 9 Principal Component Loadings

[0180]

[0181] 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.

[0182] Example 2: Screening of chromatographic conditions for fingerprint spectroscopy

[0183] 1. Mobile phase and gradient elution

[0184] 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.

[0185] 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;

[0186] Gradient elution was performed using the procedure shown in Table 10:

[0187] Table 10

[0188] 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%

[0189] The results are shown in Table 11 and Figure 10 As shown.

[0190] Table 11

[0191]

[0192]

[0193] Chromatographic conditions 2: mobile phase 0.05% formic acid aqueous solution (A)-methanol (B) gradient elution, chromatographic column Thermo Hypersili1 Gold column (100*2.1mm, 1.9μm), flow rate 0.2mL / min, UV wavelength 278nm, column temperature 35℃, injection volume 1μL;

[0194] Gradient elution was performed using the procedure shown in Table 12:

[0195] Table 12

[0196] 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％

[0197] The results are shown in Table 13 and Figure 11 As shown.

[0198] Table 13

[0199] 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 /

[0200] 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;

[0201] Gradient elution was performed using the procedure shown in Table 14:

[0202] Table 14

[0203] 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%

[0204] The results are shown in Table 15 and Figure 12 As shown.

[0205] Table 15

[0206]

[0207]

[0208] 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;

[0209] Gradient elution was performed using the procedure shown in Table 16:

[0210] Table 16

[0211] 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%

[0212] The results are shown in Table 17 and Figure 13 As shown.

[0213] Table 17

[0214]

[0215]

[0216] 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;

[0217] Gradient elution was performed using the procedure shown in Table 18:

[0218] Table 18

[0219] 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%

[0220] The results are shown in Table 19 and Figure 14 As shown.

[0221] Table 19

[0222]

[0223]

[0224] 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;

[0225] Gradient elution was performed using the procedure shown in Table 20:

[0226] Table 20

[0227] 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%

[0228] The results are shown in Table 21 and Figure 15 As shown.

[0229] Table 21

[0230]

[0231]

[0232] 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;

[0233] Gradient elution was performed using the procedure shown in Table 22:

[0234] Table 22

[0235] 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％

[0236] The results are shown in Table 23 and Figure 16 As shown.

[0237] Table 23

[0238]

[0239]

[0240] 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;

[0241] Gradient elution was performed using the procedure shown in Table 24:

[0242] Table 24

[0243] 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％

[0244] The results are shown in Table 25 and Figure 17 As shown.

[0245] Table 25

[0246]

[0247]

[0248] 2. Wavelength selection

[0249] 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 method of test solution 1 under the fingerprint spectrum section. Record the chromatograms at detection wavelengths of 230, 254, and 280 nm respectively. Other chromatographic conditions are the same as in step 3.2 above.

[0250] 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.

[0251] 3. Selection of chromatographic column

[0252] Take an appropriate amount of Sophora japonica charcoal sample t8 (i.e., Sophora japonica charcoal sample of raw product No. 210100881, which has been stir-fried for 20 min), and prepare the test solution according to the method of test solution 1 in step 3.1. The test solution is determined sequentially 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). Other chromatographic conditions are the same as in step 3.2 above.

[0253] The test results are shown in Table 26 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.

[0254] Table 26 Peak retention time and resolution in different chromatographic columns

[0255]

[0256] 4. Column temperature selection

[0257] 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 method under test solution 1 in step 3.1. Adjust the column temperature to 30℃, 35℃ and 40℃ respectively for determination. Other chromatographic conditions are the same as in step 3.2 above.

[0258] The test results are shown in Table 27 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.

[0259] Table 27 Peak retention time and resolution under different column temperatures

[0260]

[0261]

[0262] 5. Selection of flow rate

[0263] 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 method under test solution 1 in step 3.1. 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 step 3.2 above.

[0264] The test results are shown in Table 28 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.

[0265] Table 28 Peak retention time and resolution under different flow rates

[0266]

Claims

1. A method for evaluating the material properties of Sophora japonica fruit products, comprising the following steps: S1. Using liquid chromatography, fingerprint chromatograms of Sophora japonica fruit products with different processing times were established, and common characteristic peaks were extracted. S2. Determine the hemostatic effect of Sophora japonica fruit products processed for different times; S3. Based on the characteristic peak data and hemostatic effect data of Sophora japonica fruit products with different processing times, statistical analysis was conducted using chemometric methods to evaluate the pharmacodynamic material basis of hemostatic effect in Sophora japonica fruit products. In step S1, the Sophora japonica fruit products with different processing times include two or more of the following: products processed for 15-20 minutes; in step S1, the chromatographic conditions for the liquid chromatography method are as follows: The chromatographic column is an octadecylsilane-bonded silica gel column; The column temperature is 30-40℃; The mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a formic acid solution and mobile phase B is methanol; A gradient elution method is used, with the total volume of the mobile phase being 100%. The gradient elution procedure is shown in the table below: In step S3, the pharmacodynamic substances responsible for the hemostatic effect of Sophora japonica fruit products include gallic acid, p-hydroxybenzyl alcohol, methyl gallate, dihydrokaempferol-3-O-glucoside, and kaempferol-3-O-(2-)-glucoside. -O- β -D-glucosyl)- β -D-rutin, kaempferol-3-O-sophorol, dihydroapigenin-7-O-glucoside, rutin, apigenin-7-O-rutin, isorhamnetin-3-O- β One or more of the following: -D-rutin, kaempferol-3-O-rutin, linalool, and genistein-4′-O-(6′′-acetyl)-glucoside.

2. The material evaluation method for Sophora japonica fruit products as described in claim 1, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or more of the following conditions: (1) In step S1, the chromatographic column is a Waters ACQUITY CSH C18 column or a 2-Hypersi1 Gold column; (2) In step S1, the detection wavelength is 210-300 nm under the chromatographic conditions; (3) In step S1, the flow rate under the chromatographic conditions is 0.12-0.18 mL / min; (4) In step S1, the column temperature in the chromatographic conditions is 30℃, 35℃ or 40℃; (5) In step S1, the common characteristic peaks include the characteristic peaks with the following retention times: 5.60±0.05 min, 6.56±0.01 min, 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, 41.63±0.07 min, 43.58±0.03 min, and 44.34±0.04 min; and (6) In step S2, the hemostatic effect includes one or more of the following four parameters: tail bleeding time, clotting time, and clotting.

3. The material evaluation method for Sophora japonica fruit products as described in claim 2, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or more of the following conditions: (1) In step S1, the Waters ACQUITY CSH C18 chromatographic column has a specification of 150 mm. 2.1mm, 1.7µm; (2) In step S1, the detection wavelength in the chromatographic conditions is 230, 254 or 280 nm; (3) In step S1, the flow rates in the chromatographic conditions are 0.12 mL / min, 0.15 mL / min and 0.18 mL / min; In step S1 of (4), the column temperature is 35°C under the chromatographic conditions.

4. The material evaluation method for Sophora japonica fruit products as described in claim 2, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or two of the following conditions: (1) In step S1, the specification of the 2-Hypersi1 Gold chromatographic column is 100. 2.1mm, 1.9µm; and (2) In step S1, the flow rate under the chromatographic conditions is 0.12-0.15 mL / min.

5. The method for evaluating the material properties of Sophora japonica fruit products as described in claim 1, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or more of the following conditions: (1) In step S1, the formic acid in the mobile phase A is a 0.05-0.5% formic acid solution; (2) In step S1, the detector in the chromatographic conditions is a UV-DAD detector; (3) In step S1, the injection volume under the chromatographic conditions is 1-5 μl; (4) In step S1, the chromatographic conditions are such that the injection concentration is 0.004-0.1 g / mL; (5) In step S1, the Sophora japonica product is pretreated before injection. The pretreatment includes the following steps: dissolving the Sophora japonica product in a solvent; (6) The common characteristic peaks also include characteristic peaks with retention times of one or more of the following: 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, 30.14±0.05 min, 30.53±0.03 min, 32.78±0.02 min, 33.15±0.03 min, 34.06±0.03 min, 34.70±0.03 min, 35.96±0.06 min, 38.27±0.05 min, 39.89±0.06 min, 40.90±0.04 min, 42.09±0.05 min. min, 43.35±0.02 min, 44.60±0.03min, 45.31±0.03min, 46.28±0.02min, 46.74±0.04min and 49.21±0.04min; and (7) In step S1, the chromatographic column is a Waters ACQUITY CSH C18 column.

6. The material evaluation method for Sophora japonica fruit products as described in claim 5, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or more of the following conditions: (1) In step S1, the formic acid in the mobile phase A is a 0.05% formic acid solution; (2) In step S1, the injection volume is 1 μL under the chromatographic conditions; (3) In step S1, the chromatographic conditions are such that the injection concentration is 0.025 g / mL; (4) In step S1, the solvent in the pretreatment is methanol; In step (5) S1, the pretreatment further includes the following steps: soaking the Sophora japonica product in water, decocting, filtering, concentrating the filtrate and then freeze-drying it to obtain freeze-dried powder of the Sophora japonica product, which is then dissolved in the solvent in the form of freeze-dried powder.

7. The method for evaluating the material properties of Sophora japonica fruit products as described in claim 1, characterized in that, In step S3, Pearson bivariate correlation analysis is used to analyze the correlation between the peak area of ​​the common characteristic peak and the hemostatic effect data, and a spectrum-effect equation is established through multiple linear regression to determine the material basis of the hemostatic effect of Sophora japonica fruit products.

8. The method for evaluating the material properties of Sophora japonica fruit products as described in claim 7, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or more of the following conditions: (1) The characteristic peaks with retention times of 5.60±0.05 min, 6.56±0.01 min, 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, 41.63±0.07 min, 43.58±0.03 min and 44.34±0.04 min showed a good correlation with the hemostatic effect; (2) In step S3, the characteristic peaks with retention times of 5.60±0.05 min, 6.56±0.01 min, 40.90±0.04 min, 42.09±0.05 min, 44.34±0.04 min, and 45.31±0.03 min are respectively introduced into the spectral effect mathematical model: Bleeding time (BT) = 3.609 - 3.077 seconds X2; Y clotting time CT = 3.128 + 0.688 X31-3.11 X26; Y prothrombin time (PT) = 0.505 - 0.017 X3-0.012 X33; Y-fibrinogen (FIB) = 3.644 - 0.666 X28; Wherein, X2 represents the peak area of ​​the chromatographic peak with a retention time of 5.60±0.05 min, X3 represents the peak area of ​​the chromatographic peak with a retention time of 6.56±0.01 min, X26 represents the peak area of ​​the chromatographic peak with a retention time of 40.90±0.04 min, X28 represents the peak area of ​​the chromatographic peak with a retention time of 42.09±0.05 min, X31 represents the peak area of ​​the chromatographic peak with a retention time of 44.34±0.04 min, and X33 represents the peak area of ​​the chromatographic peak with a retention time of 45.31±0.03 min. Y bleeding time BT, Y clotting time CT, Y prothrombin time PT, and Y fibrinogen FIB represent the hemostatic effect of Sophora japonica charcoal. In step S3 of (3), the methods for evaluating the material basis of hemostasis in Sophora japonica fruit products include: characteristic peaks with significant differences in correlation between the peak area of ​​characteristic peaks and hemostasis effect data (P<0.05) and characteristic peaks introduced into the spectral effect equation are the material basis of hemostasis in Sophora japonica fruit products.

9. The method for evaluating the material properties of Sophora japonica fruit products as described in claim 8, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or more of the following conditions: (1) The characteristic peak with a retention time of 5.60±0.05 min is gallic acid, the characteristic peak with a retention time of 6.58 min is p-hydroxybenzyl alcohol, the characteristic peak with a retention time of 19.83±0.01 min is methyl gallate, the characteristic peak with a retention time of 28.24±0.06 min is dihydrokaempferol-3-O-glucoside, the characteristic peak with a retention time of 33.60±0.04 min is kaempferol-3-O-(2''-O-β-D-glucosyl)-β-D-rutin, the characteristic peak with a retention time of 34.99±0.05 min is kaempferol-3-O-sophorose, the characteristic peak with a retention time of 36.28±0.05 min is dihydroapigenin-7-O-glucoside, the characteristic peak with a retention time of 37.43 min is rutin, and the characteristic peak with a retention time of 41.63±0.07 min is... The characteristic peak at min is kaempferol-3-O-rutin, and the characteristic peak at retention time of 43.58±0.03 min is eleutheroside. (2) In step S3, in the mathematical model of Y bleeding time BT, P = 0.034 < 0.05, R 2 =0.556; In the mathematical model of Y clotting time CT, P = 0.026 < 0.05, R 2 =0.905; In the mathematical model of prothrombin time (PT) for Y, P = 0.049 < 0.05, R 2 =0.858; In the mathematical model of fibrinogen (FIB), P = 0.007 < 0.05, R 2 =0.733; In step S3 of (3), the material basis for the hemostatic effect of Sophora japonica fruit products includes substances corresponding to the characteristic peaks of the following retention times: 5.60±0.05 min, 6.56±0.01 min, 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, 43.58±0.03 min, 44.34±0.04 min, and 45.31±0.03 min.

10. The method for evaluating the material composition of Sophora japonica fruit products as described in claim 1, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or more of the following conditions: (1) In step S1, the Sophora japonica fruit product is prepared by the following method: stir-frying the raw Sophora japonica fruit; (2) In step S1, the liquid chromatography is high performance liquid chromatography or ultra-high performance liquid chromatography; (3) In step S1, common characteristic peaks are extracted using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012A" software; and (4) Step S3 also includes principal component analysis of the relevant peaks of Sophora japonica products to screen the relevant peak components that have a greater impact on the differences between Sophora japonica products with different processing times.

11. The method for evaluating the material properties of Sophora japonica fruit products as described in claim 10, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or more of the following conditions: (1) In step S1, the frying temperature is 200-300℃; (2) In step S1, the frying time is 15-20 min; In step (3) S3, the principal component analysis includes: The cumulative contribution rate of the retained principal components is >90%; The retained principal components have eigenvalues ​​> 1; Then it was tested using the KMO test and the Bartlett test.

12. The method for evaluating the material composition of Sophora japonica fruit products as described in claim 11, characterized in that, The material evaluation method for the aforementioned Sophora japonica fruit products meets one or two of the following conditions: (1) In step S1, the frying temperature is 200℃; In step (2) S3, the principal component analysis is also validated using cluster analysis.

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