Screening of active ingredients of Euphorbiaceae plants for hemostatic activity and its application

Abstract

The application discloses screening of hemostatic active components in Euphorbiaceae plants and application thereof, and based on ultrafiltration mass spectrometry technology and taking urokinase-type plasminogen activator as a key target enzyme, a hemostatic active component, breynol, of cyclopentanone isocoumarins in Euphorbiaceae plants is screened for the first time. The breynol shows strong specific binding to urokinase plasminogen activator, and the activity inhibition (IC 50 ) of the breynol to the urokinase plasminogen activator is 0.187±0.000 mM, which is superior to that of the positive control tranexamic acid (IC 50 =2.425±0.001 mM). The breynol is reported for the first time in the research of hemostatic activity of Euphorbiaceae plants, and can be used for preparation of daily or clinical hemostatic drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug development of lead compounds, and particularly relates to screening of active ingredients for hemostasis in Euphorbiaceae plants and application thereof. BACKGROUND

[0002] Euphorbiaceae plants are rich in plant resources in China and have unique curative effects and uses. Among them, Acalypha australis has the effect of astringing and hemostasis and is used in the Miao nationality in Guizhou to treat various bleeding syndromes (such as hemoptysis, hematuria, hematochezia, metrorrhagia and traumatic hemorrhage, etc.), and Xieyuhua tablets with Acalypha australis as the monarch drug have been used in clinical treatment. However, there are few studies on the active ingredients for hemostasis of Acalypha australis, and the mechanism of hemostasis is not clear, which hinders the full development and utilization of Acalypha australis. Urokinase-type plasminogen activator (uPA) is a kind of serine protease that can promote the activation of plasminogen and plays an important role in the process of hemostasis. Targeted inhibition of the activity of urokinase-type plasminogen activator is beneficial to the hemostatic effect in the state of bleeding or injury. In view of this, it is extremely promising to screen natural active hemostatic ingredients from Euphorbiaceae plants based on the key target enzyme urokinase-type plasminogen activator in the process of hemostasis and to develop and apply them to daily or clinical hemostatic drugs.

[0003] Investigation found that the discovery of active ingredients in Euphorbiaceae plants needs to go through complex and time-consuming steps such as component separation, structure identification and activity verification. The ultrafiltration mass spectrometry screening technology has the advantages of rapidness, high efficiency and strong specificity, and there is no application report on screening of hemostatic active ingredients from Euphorbiaceae plants by using the ultrafiltration mass spectrometry screening technology. The present application screens out, for the first time, the cyclopentanone isocoumarin components with hemostatic activity in Euphorbiaceae plants based on the ultrafiltration mass spectrometry technology and taking urokinase-type plasminogen activator as the key target enzyme, which is not only beneficial to the further development and utilization of Euphorbiaceae plants, but also provides a reference for the development of active ingredients of hemostatic traditional Chinese medicines and ethnic medicines. SUMMARY

[0004] The present application screens out, for the first time, the cyclopentanone isocoumarin components with hemostatic activity in Euphorbiaceae plants based on the ultrafiltration mass spectrometry technology and taking urokinase-type plasminogen activator as the key target enzyme, which is not only beneficial to the further development and utilization of Euphorbiaceae plants, but also provides a reference for the development of active ingredients of hemostatic traditional Chinese medicines and ethnic medicines.

[0005] The present application adopts the following technical scheme:

[0006] A method for screening hemostatic active ingredients in Euphorbiaceae plants comprises the following steps:

[0007] (1) The extract solution of Euphorbiaceae plant was co-incubated with the urokinase type plasminogen activator solution. After incubation, the solution was ultrafiltered and centrifuged. The components that did not specifically bind to the urokinase type plasminogen activator were eluted with buffer solution. After centrifugation, the components retained in the ultrafiltration membrane were dissociated. The eluent was collected, dried and reconstituted to obtain the experimental group solution. At the same time, the inactivated urokinase type plasminogen activator was used as the control group and treated in the same way as the experimental group to obtain the control group solution.

[0008] (2) Take the above extract solution, experimental group solution and control group solution for liquid chromatography-mass spectrometry analysis and collect peak data;

[0009] (3) Based on the changes in peak area of ​​different compounds in the experimental and control group solutions, the specific binding values ​​of different compounds in the extract with urokinase-type plasminogen activator were calculated. Hemostatic active compounds were screened based on the specific binding values. The formula for calculating the specific binding values ​​is as follows:

[0010] SB = A 实验 / A 对照

[0011] SB is the specific binding value of urokinase-type plasminogen activator to different compounds in the extract, A 实验 A represents the peak area of ​​the base diagram after different compounds in the experimental group solution interact with urokinase-type plasminogen activator. 对照 The peak areas are the base peaks of different compounds in the control group solution after they interact with inactivated urokinase plasminogen activator.

[0012] Preferably, the incubation temperature of the Euphorbiaceae plant extract solution and the urokinase plasminogen activator solution in step (1) is 37°C.

[0013] Preferably, in step (1), a methanol-water solution with a volume ratio of 7:3 is used to dissociate the components retained in the ultrafiltration membrane.

[0014] Application of bryophylloidin in the preparation of hemostatic drugs: The chemical structure of bryophylloidin is as follows Figure 4 As shown, among the screening methods described above, shortleaf hematoxylin and oleanolic acid showed the highest specific binding value to urokinase-type plasminogen activator (SB = 2.09), exhibiting strong specific binding and strong inhibitory activity against urokinase-type plasminogen activator (IC50). 50 =0.187 ± 0.000 mM).

[0015] The advantages and beneficial effects of this invention are:

[0016] The present application discloses the hemostatic activity of breyniolic acid in Euphorbiaceae plants based on the ultrafiltration mass spectrometry technology and taking urokinase-type plasminogen activator as a key target enzyme, and the specific type is cyclopentenone isocoumarin compound, which has not been reported in the hemostatic component research of Euphorbiaceae plants. The breyniolic acid as a natural active ingredient can be used in daily life or in the preparation of clinical hemostatic drugs.

[0017] The prothrombin time and activated partial thromboplastin time of the Acalypha Australis extract were 20.78 ± 0.22 seconds and 75.29 ± 5.50 seconds, which were better than the positive control Yunnan White Drug (21.12 ± 0.83 seconds; 106.76 ± 12.22 seconds). The activity inhibition (IC 50 ) of the Acalypha Australis extract on urokinase plasminogen activator was 204.50 ± 24.60 µg / mL, which was better than the positive control tranexamic acid (IC 50 = 258.80 ± 18.10 µg / mL). The activity inhibition (IC 50 ) of breyniolic acid on urokinase plasminogen activator was 0.187 ± 0.000 mM, which was better than the positive control tranexamic acid (IC 50 = 2.425 ± 0.001 mM). BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a method flowchart of Example 2 of the present application.

[0019] Figure 2 is the activity inhibition of the Acalypha Australis extract on urokinase plasminogen activator.

[0020] Figure 3 is the base peak graph of the Acalypha Australis extract after incubation with urokinase plasminogen activator.

[0021] Figure 4 is a chemical structure diagram of breyniolic acid.

[0022] Figure 5 is the activity inhibition of breyniolic acid on urokinase plasminogen activator. DETAILED DESCRIPTION

[0023] To make the purpose, technical solutions and advantages of the present application clearer, the following will further describe the embodiments of the present application in combination with the accompanying tables and drawings.

[0024] Example 1 Evaluation of Hemostatic Activity of Acalypha Australis Extract

[0025] 1. Prothrombin time evaluation of A. cordifolium: pulverized A. cordifolium was extracted with 70% ethanol by ultrasonication for 3 times with 30 min interval to obtain ethanol extract, which was suspended in warm water and extracted with n-hexane, ethyl acetate and n-butanol successively to obtain A. cordifolium extract, which was freeze-dried and stored at low temperature. 60 μL of plasma was mixed with 40 μL of A. cordifolium extract (1 mg / mL) in an EP tube, and the mixture was preheated for 3 min (water bath, 37 °C); 200 μL of freshly heated PT reagent was added to the mixture, which was incubated at 37 °C for at least 10 min; when a white clot formed in the solution at an angle, the PT time was recorded by a stopwatch. The experiment was performed in triplicate (n = 3), with blank control (0.9% saline) and coagulant Yunnan White Powder as control groups. n

[0026] Table 1. Prothrombin time and activated partial thromboplastin time results of A. cordifolium

[0027]

[0028] Note: Different superscript letters (a-c) in the same column represent statistical differences with p value < 0.05

[0029] 2. Activated partial thromboplastin time evaluation of A. cordifolium: equal volume (100 μL) of platelet-free plasma and A. cordifolium extract (1 mg / mL) were mixed in an EP tube and incubated at 37 °C for 1 min; 100 μL of aPTT reagent was added and the incubation was continued at 37 °C for 3 min; 100 μL of 25 mM calcium chloride was added to initiate coagulation; the mixture was completely coagulated at an angle, and the clotting time was recorded. The experiment was performed in triplicate (n = 3), with blank control (0.9% saline) and coagulant Yunnan White Powder as control groups. n

[0030] ​​3. Inhibition of urokinase plasminogen activator activity: 5 μL of different concentrations (31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL) of A. aspera extract was mixed with 20 μL of urokinase plasminogen activator solution (20 U / mL) and incubated at 37°C for 30 min; 150 μL of Tris buffer was added to each well of a 96-well plate and incubated for 15 min; 25 μL of different concentrations of S-2444 uPA chromogenic substrate (25 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM) was added to the buffer before the start step. The kinetics was run on a multi-mode plate reader (Infinity ProM200) at 405 nm absorbance for 120 min at 37°C (reading interval of 2 min); the resulting time response absorbance data was fitted to a polynomial to calculate the reaction rate per minute; the inhibition constant Ki was calculated using the non-competitive inhibition plot mode of Graphpad Prism software and represented using the Michaelis Menten plot.

[0031] As shown in Table 1, the prothrombin time of A. aspera extract was 20.78 ± 0.22 seconds, and the activated partial thromboplastin time was 75.29 ± 5.50 seconds, which was better than the positive control Yunnan Baiyao (21.12 ± 0.83; 106.76 ± 12.22), indicating that A. aspera extract had good hemostatic effect.

[0032] As shown in Table 1, the prothrombin time of A. aspera extract was 20.78 ± 0.22 seconds, and the activated partial thromboplastin time was 75.29 ± 5.50 seconds, which was better than the positive control Yunnan Baiyao (21.12 ± 0.83; 106.76 ± 12.22), indicating that A. aspera extract had good hemostatic effect. Figure 2 As shown in Table 1, the prothrombin time of A. aspera extract was 20.78 ± 0.22 seconds, and the activated partial thromboplastin time was 75.29 ± 5.50 seconds, which was better than the positive control Yunnan Baiyao (21.12 ± 0.83; 106.76 ± 12.22), indicating that A. aspera extract had good hemostatic effect. 50 As shown in Table 1, the prothrombin time of A. aspera extract was 20.78 ± 0.22 seconds, and the activated partial thromboplastin time was 75.29 ± 5.50 seconds, which was better than the positive control Yunnan Baiyao (21.12 ± 0.83; 106.76 ± 12.22), indicating that A. aspera extract had good hemostatic effect. 50 As shown in Table 1, the prothrombin time of A. aspera extract was 20.78 ± 0.22 seconds, and the activated partial thromboplastin time was 75.29 ± 5.50 seconds, which was better than the positive control Yunnan Baiyao (21.12 ± 0.83; 106.76 ± 12.22), indicating that A. aspera extract had good hemostatic effect.

[0033] Example 2 Screening of hemostatic active ingredients of A. aspera

[0034] The target enzyme for hemostasis is urokinase plasminogen activator, and the screening of hemostatic active ingredients of A. aspera includes the following steps:

[0035] (1) Take an appropriate amount of A. aspera extract and hemostatic target enzyme—urokinase plasminogen activator and dissolve them in buffer solution to obtain A. aspera extract solution and urokinase plasminogen activator solution;

[0036] Specifically, the crushed Acalypha australis was ultrasonically extracted with 70% ethanol for 3 times, each time with an interval of 30 min, to obtain an ethanol extract. The ethanol extract was suspended in warm water and then sequentially extracted with n-hexane, ethyl acetate and n-butanol to obtain an Acalypha australis extract, which was freeze-dried and stored at low temperature. 121.1 g of tris-hydroxymethyl aminomethane was mixed with 800 mL of double-distilled water to form a solution, which was adjusted to pH 7.5 (25°C) with concentrated hydrochloric acid and diluted to 1 L to obtain the buffer solution. The volume of the Acalypha australis extract solution was 100 μL (1 mg / mL). The concentration of the urokinase-type plasminogen activator solution was 4 μM.

[0037] (2) 100 μL of the Acalypha australis extract solution was incubated with the urokinase-type plasminogen activator solution (100 μL) at 37°C. The mixed solution after incubation was ultrafiltration centrifuged for 10 min (12,000 rpm, 25°C). The buffer solution (Tris-HCL, pH 7.5) was used to elute the components not combined with the urokinase-type plasminogen activator. The elution was performed for 3 times. The methanol-water solution (7:3, v / v) was used to dissociate the ligand retained after ultrafiltration centrifugation. The eluate was collected, blown dry and redissolved to obtain the experimental group solution. Meanwhile, the inactivated urokinase-type plasminogen activator was set as the control group, and the rest was the same as the experimental group to obtain the control group solution.

[0038] (3) The Acalypha australis extract solution, the experimental group solution and the control group solution were subjected to chromatography-mass spectrometry analysis to collect base peak chart data. Specifically, the chromatography-mass spectrometry analysis was performed using an ultra-high performance liquid chromatograph and a high-resolution mass spectrometer. The high-performance liquid chromatograph was used to perform gradient elution using a chromatographic column with formic acid-water (A, 1:1000, v / v) and acetonitrile (B) as the mobile phase. The elution conditions were as follows: 0-10 min, 5%-15% B; 10-25 min, 15%-20% B; 25-40 min, 20%-45% B; and 40-45 min, 45%-95% B. The high-resolution mass spectrometer was used to perform molecular weight scanning in the range of 100-1500 Dalton, with electrospray ionization (ESI) in negative ion mode. Nitrogen was used as the atomization, drying and collision gas. The atomizer gas flow was maintained at 8 L / min, and the pressure was constant at 35 Psi. The ionization spray voltage was 3.5 kV, and the heating capillary temperature was 350°C.

[0039] (5) The specific binding value of the urokinase-type plasminogen activator to different compounds in the Acalypha australis extract was evaluated by the peak area change of each compound base peak chart in the experimental group solution and the control group solution, so as to screen the hemostatic active compound. The specific binding value was calculated according to the following formula:

[0040] SB = A 实验 / A 对照

[0041] SB is the specific binding value of urokinase-type plasminogen activator and different compounds, A 实验 A is the peak area of the base peak graph of the interaction between different compounds in the acacia nilotica extract and urokinase-type plasminogen activator 对照 A is the peak area of the base peak graph of the interaction between different compounds in the acacia nilotica extract and inactivated urokinase-type plasminogen activator

[0042] As shown in Figure 3 , the specific binding values (SB) of urokinase-type plasminogen activator and the compounds corresponding to peaks 1-2, 4-5, 10-11, 13 and 18 in acacia nilotica were calculated to be 1.06, 1.51, 1.30, 2.09, 1.48, 1.67, 1.95 and 1.89, respectively. Among them, the SB value (2.09) of pyrochrysene acid (peak 5, Figure 4 ) is the highest, indicating that pyrochrysene acid has better potential hemostatic activity.

[0043] The activity inhibition of pyrochrysene acid on urokinase-type plasminogen activator was determined, and the specific method is described in Example 1, as shown in Figure 5 , the activity inhibition (IC 50 ) of pyrochrysene acid on urokinase-type plasminogen activator is 0.187 ± 0.000 mM, which is better than the positive control tranexamic acid (IC 50 = 2.425 ± 0.001 mM), indicating that pyrochrysene acid has strong urokinase-type plasminogen activator inhibitory activity.

[0044] Urokinase-type plasminogen activator is closely related to the hemostatic process and is an extremely attractive therapeutic target; based on the biological affinity between urokinase-type plasminogen activator and small molecule ligands, the hemostatic active compounds are screened from acacia nilotica in the present application. The screening results show that the active compound interacting with urokinase-type plasminogen activator in acacia nilotica of euphorbiaceae is pyrochrysene acid, which is a natural source of active ingredients and has a wide application prospect in the preparation of daily or clinical hemostatic drugs.

[0045] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of screening Euphorbiaceae plants for hemostatic active ingredients, characterized in that, The method comprises the following steps: (1) incubating the Euphorbiaceae plant extract solution with the urokinase-type plasminogen activator solution, ultrafiltration centrifugation of the incubated solution, elution of the components not specifically combined with the urokinase-type plasminogen activator with a buffer solution, dissociation of the components retained on the ultrafiltration membrane after centrifugation, collection of the eluate, and drying and redissolving to obtain the experimental group solution; meanwhile, the inactivated urokinase-type plasminogen activator is used as a control group, and the same treatment as the experimental group is performed to obtain the control group solution; (2) liquid chromatography-mass spectrometry analysis is performed on the extract solution, the experimental group solution, and the control group solution, and peak data are collected; (3) the specific binding values of different compounds in the extract to the urokinase-type plasminogen activator are calculated according to the peak area difference of the base peak chromatograms of the different compounds in the experimental group and the control group solutions, and the hemostatic active compounds are screened according to the specific binding values, and the specific binding value calculation formula is as follows: SB = A 实验 / A 对照 SB is the specific binding value of urokinase-type plasminogen activator to different compounds in the extract, A 实验 A is the peak area of the base peak graph after the interaction of different compounds in the experimental group solution with urokinase-type plasminogen activator 对照 A is the peak area of the base peak graph after the interaction of different compounds in the control group solution with inactivated urokinase-type plasminogen activator The Euphorbiaceae plant is Acalypha australis, and the hemostatic active compound is breynol.

2. The method of claim 1, wherein, In step (1), the incubation temperature of the Euphorbiaceae plant extract solution and the urokinase-type plasminogen activator solution is 37 ℃.

3. The method of claim 1, wherein, In step (1), the components retained on the ultrafiltration membrane are dissociated with a methanol-water solution in a volume ratio of 7:3.

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