Baccaurea ramiflora lactone as well as pharmaceutical composition and application of baccaurea ramiflora lactone

Abstract

The invention provides baccaurea ramiflora lactone and a pharmaceutical composition and application thereof, the baccaurea ramiflora lactone is four sesquiterpenoids, and the compounds have certain antithrombotic activity, can form the pharmaceutical composition with a pharmaceutically acceptable carrier or excipient, and can be used for preparing antithrombotic drugs.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and specifically relates to ramifloraolides AD (1-4), a preparation method and application thereof, a pharmaceutical composition and application thereof. Background Art

[0002] Thrombosis is one of the main causes of morbidity and mortality in a variety of vascular diseases. Thrombotic disease is a type of cardiovascular disease, and common ones include ischemic stroke, myocardial infarction, pulmonary embolism, and disseminated intravascular coagulation, which present high morbidity and mortality, seriously endangering human life and health. Antithrombotic drugs are an important means of emergency treatment of acute thrombotic diseases and long-term treatment of chronic thrombotic diseases, mainly including anticoagulants, antiplatelet aggregation drugs, and fibrinolytic drugs. With the aging of the population, the number of patients with thrombotic diseases has increased year by year, antithrombotic drugs have been widely used, and the number of bleeding events caused by antithrombotic drugs has also increased year by year. Although commonly used antithrombotic drugs such as aspirin, clopidogrel, and warfarin can effectively reduce thrombosis, they have adverse reactions such as gastrointestinal discomfort and bleeding. For a long time, scientists have made remarkable achievements in improving pharmacokinetic characteristics and achieving oral administration by using low molecular weight heparin, synthetic pentoses, hirudin preparations, and small molecule synthetic drugs such as rivaroxaban and dabigatran. However, these drugs all act on the "common coagulation pathway" of the "coagulation cascade", and it is difficult to avoid the impact on physiological hemostasis, so no breakthrough has been made in reducing the risk of bleeding. Therefore, anti-thrombosis remains the focus of global drug research and development. Researchers are committed to developing new treatment strategies and drugs, and looking for candidate molecules with new mechanisms of action and new targets with diverse structures to overcome the limitations of existing drugs, improve treatment effects, and reduce toxic side effects.

[0003] The use of traditional Chinese medicine or natural medicine has been the main means for humans to treat various diseases for thousands of years. The effective ingredients or components contained in them have great potential for continuous exploration. Finding novel active molecules from natural products is an important way to develop new drugs. Natural molecule libraries are an important resource for screening active drugs, providing great opportunities for finding new antithrombotic lead compounds or good candidate drugs with unique antithrombotic mechanisms. Domestic and foreign scholars have conducted a lot of exploration and research, and some natural compounds with antithrombotic activity have been discovered. For example, Professor Yao Guangmin's research team at Huazhong University of Science and Technology used a variety of extraction and separation techniques under the guidance of activity to discover 6 natural product molecules with significant antithrombotic activity from the medicinal plant of Ericaceae, Piericones A and B, in particular, are a unique class of hexahydro-10H-furan[2″,3″:3′,4′]furan[2′,3′:3,4]furan[2,3-c]pyran-6(6aH)-one bridged dihydrochalcone compounds, which are nanomolar PDI inhibitors. The research of Fang Chao's team from Tongji Medical College of Huazhong University of Science and Technology and DeepKinase R&D team revealed for the first time that dihydrochalcone asebogenin has good antithrombotic effect. Asebogenin directly hinders the activation of SYK by interfering with the phosphorylation of SYK Tyr525 / 526, and can reduce platelet aggregation and fibrin production, inhibit the formation of arterial thrombus, while reducing neutrophil activation and NETs formation, reducing venous thrombosis without increasing the risk of bleeding. In addition, some components in Piper plants also show significant antiplatelet and antithrombotic activity. The study found that extracts from twenty species of Piper plants showed antiplatelet aggregation activity, and 51 antiplatelet active compounds were found from 11 species of Piper plants, mainly including alkaloids, lignans, phenylpropanoids, etc. These studies show that not only traditional antithrombotic compounds, but also other types of natural compounds may become new options for antithrombotic treatment in the future.

[0004] Baccaurea ramiflora is a plant of the Euphorbiaceae family, an evergreen tree or shrub. There are about 80 species in this genus, mainly distributed in southeastern Asia. There is only one wild species in my country, which is mainly distributed in Hainan, Guangdong, Guangxi and Yunnan. In traditional Dai medicine, it is used to treat rheumatoid arthritis, cellulitis, abscesses, trauma and other diseases. Previous chemical studies on this plant have isolated a series of secondary metabolites, including sesquiterpenes, diterpenes, steroids, polyphenols and aldehyde compounds, some of which have antifungal, antioxidant and immunosuppressive activities. This genus of plants has attracted more and more attention from scholars at home and abroad due to its wide range of medicinal value, novel and diverse secondary metabolites and diverse biological activities.

[0005] So far, there are no reports on ramifloraolides AD (1-4) in the prior art, nor are there any reports on pharmaceutical compositions containing ramifloraolides AD as active ingredients, nor are there any reports on the use of ramifloraolides AD in the preparation or treatment of thrombosis. Summary of the invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide a new class of ramifloraolides AD (ramifloraolides AD, 1-4) with medicinal value as shown in formula (I), as well as a preparation method and application thereof, a pharmaceutical composition and application thereof. This class of compounds has significant antithrombotic activity and can be used to prepare antithrombotic drugs.

[0007] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0008] As a first aspect, the present invention provides a class of ramifloraolides, which are four sesquiterpene molecules, ramifloraolides AD (ramifloraolides AD, 1-4), having the structure of any one of compounds 1 to 4 in the following formula (I):

[0009]

[0010] As a second aspect, the present invention provides a method for preparing the above-mentioned cassia lactone (compound 1-4), the dried roots of cassia twig are crushed and then extracted with 90% ethanol reflux for 3 times, concentrated to dryness, and an extract is obtained; the extract is then mixed with polyamide, chromatographed on an MCI CHP 20P column, the elution system is methanol-water, and then the same fractions are combined by TLC detection to obtain a total of 4 components Fr.M-1 to Fr.M-4; LCMS analysis speculates that component Fr.M-2 that may contain the target sesquiterpene is chromatographed on a silica gel column, and gradient eluted with methanol-dichloromethane in a volume ratio of 1:40 to 1:0 in sequence, and the obtained components are combined by TLC detection to obtain 16 components Fr.M-2-1 to Fr.M-2-16; LCMS analysis speculates that component Fr.M-2-6 containing the target sesquiterpene is chromatographed on an MCI CHP 20P column, and the same fractions are combined by TLC detection to obtain 4 components Fr.M-1 to Fr.M-4; LCMS analysis speculates that component Fr.M-2 that may contain the target sesquiterpene is chromatographed on a silica gel column, and gradient eluted with methanol-dichloromethane in a volume ratio of 1:40 to 1:0 in sequence, and the obtained components are combined by TLC detection to obtain 16 components Fr.M-2-1 to Fr.M-2-16; LCMS analysis speculates that component Fr.M-2-6 containing the target sesquiterpene is chromatographed on an MCI CHP 20P column chromatography, eluted with methanol-water with volume fractions of 50%, 60%, 70%, and 90% in sequence to obtain four components Fr.M-2-6-1 to Fr.M-2-6-4; Fr.M-2-6-2 was prepared by HPLC to obtain compound 2; the component Fr.M-2-8 containing the target sesquiterpene was eluted by gel column chromatography with methanol-dichloromethane volume ratio of 1:1 to obtain two components Fr.M-2-8a and Fr.M-2-8b. TLC detection analysis showed that the component Fr.M-2-8b containing the target sesquiterpene was fully soluble in methanol , and recrystallized to obtain compound 4; the component Fr.M-2-9 containing the target sesquiterpene was recrystallized with methanol-dichloromethane in a volume ratio of 1:9 to obtain compound 3; the component Fr.M-2-12 containing the target sesquiterpene was chromatographed on silica gel with a gradient elution of methanol-dichloromethane in a volume ratio of 1:25 to 1:15, and according to the TLC detection results, it was divided into 5 components Fr.M-2-12-1 to Fr.M-2-12-5; TLC detection and analysis of the component Fr.M-2-12-4 containing the target sesquiterpene was prepared by HPLC to obtain compound 1.

[0011] As a third aspect, the present invention provides the use of the above-mentioned pyracantha lactone (compound 1-4) in the preparation of an antithrombotic drug. The present invention has no particular limitation on the method of the application, and any method well known in the art can be used.

[0012] As a fourth aspect, the present invention also provides a pharmaceutical composition, which comprises at least one of the above-mentioned pyracantha lactones (compounds 1-4 of formula (I)) and a pharmaceutically acceptable carrier or excipient.

[0013] When at least one of the compounds 1-4 is used to prepare an antithrombotic drug, the present invention preferably uses the compounds 1-4 directly or in the form of a pharmaceutical composition.

[0014] The pharmaceutical composition provided by the present invention comprises at least one of the above-mentioned compounds 1-4 and a pharmaceutically acceptable carrier or excipient. In the present invention, the pharmaceutically acceptable carrier or excipient is preferably a solid, semisolid or liquid diluent, a filler and a pharmaceutical product adjuvant. The present invention has no particular limitation on the pharmaceutically acceptable carrier or excipient, and any pharmaceutically acceptable carrier and / or excipient well known in the art, non-toxic and inert to humans and animals can be selected.

[0015] The present invention has no particular limitation on the preparation method of the pharmaceutical composition. At least one of compounds 1-4 can be directly mixed with a pharmaceutically acceptable carrier or excipient. The present invention has no particular limitation on the mixing process. The pharmaceutical composition can be obtained by selecting a process well known in the art.

[0016] As a fifth aspect, the present invention provides the use of the pharmaceutical composition described in the above technical solution in the preparation of antithrombotic drugs. The present invention has no special limitation on the method of the application, and any method well known in the art can be selected.

[0017] In the present invention, when the pharmaceutical composition is used to prepare an antithrombotic drug, the content of the composition in the drug is preferably 0.1-99%; in the pharmaceutical composition, the content of at least one of the compounds 1-4 in the pharmaceutical composition is preferably 0.5-90%. The pharmaceutical composition of the present invention is preferably used in the form of a dosage per unit body weight. In the present invention, the prepared drug can preferably be administered in two forms: injection (intravenous injection, intramuscular injection) and oral administration.

[0018] The beneficial effects of the present invention are as follows: the present invention prepares a class of pyracantha lactones, which have certain anti-thrombotic activity, can be combined with pharmaceutically acceptable carriers or excipients to form a pharmaceutical composition, can be used to prepare anti-thrombotic drugs, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram of the structural formula of compound 1-4 of the present invention;

[0020] Figure 2 is the X-ray single crystal diffraction structure of compound 1;

[0021] Figure 3 is the X-ray single crystal diffraction structure of compound 2;

[0022] Figure 4 is the X-ray single crystal diffraction structure of compound 3;

[0023] Figure 5 This is the X-ray single crystal diffraction structure of compound 4. DETAILED DESCRIPTION

[0024] In order to better understand the essence of the present invention, the test examples and embodiments of the present invention are used below in conjunction with the accompanying drawings to further illustrate the sesquiterpenoid compounds of the present invention, ramifloraolides AD (ramifloraolides AD, 1-4) and their preparation methods, structural identification, and pharmacological effects, but the present invention is not limited to these test examples and embodiments.

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0026] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the following embodiments, unless otherwise specified, all percentages are by volume.

[0028] Embodiment 1:

[0029] Preparation of compound 1-4:

[0030] The dried roots of the fruit of the Chinese taro were crushed and extracted with 90% ethanol reflux for three times, and concentrated to dryness to obtain an extract; the extract was then mixed with polyamide and chromatographed on an MCI CHP 20P column with a methanol-water elution system, and then the same fractions were combined by TLC detection to obtain a total of 4 components Fr.M-1 to Fr.M-4; LCMS analysis speculated that the component Fr.M-2 that may contain the target sesquiterpenes was chromatographed on a silica gel column, and gradient eluted with methanol-dichloromethane in a volume ratio of 1:40 to 1:0, and the obtained components were combined by TLC detection to obtain 16 components Fr.M-2-1 to Fr.M-2-16; LCMS analysis speculated that the component Fr.M-2-6 containing the target sesquiterpenes was chromatographed on an MCI CHP 20P column with a methanol-water elution system, and then the same fractions were combined by TLC detection to obtain 4 components Fr.M-1 to Fr.M-4; LCMS analysis speculated that the component Fr.M-2 that may contain the target sesquiterpenes was chromatographed on a silica gel column, and gradient eluted with methanol-dichloromethane in a volume ratio of 1:40 to 1:0, and the obtained components were combined by TLC detection to obtain 16 components Fr.M-2-1 to Fr.M-2-16; LCMS analysis speculated that the component Fr.M-2-6 containing the target sesquiterpenes was chromatographed on an MCI CHP 20P column chromatography, eluted with methanol-water with volume fractions of 50%, 60%, 70%, and 90% in sequence to obtain four components Fr.M-2-6-1 to Fr.M-2-6-4; Fr.M-2-6-2 was prepared by HPLC to obtain compound 2; the component Fr.M-2-8 containing the target sesquiterpene was eluted by gel column chromatography with methanol-dichloromethane volume ratio of 1:1 to obtain two components Fr.M-2-8a and Fr.M-2-8b. TLC detection and analysis showed that the component Fr.M-2-8b containing the target sesquiterpene was dissolved in methanol (the amount of methanol added to the component Fr. M-2-8b was fully dissolved), and recrystallized to obtain compound 4; the target sesquiterpene-containing component Fr.M-2-9 was recrystallized with methanol-dichloromethane at a volume ratio of 1:9 to obtain compound 3; the target sesquiterpene-containing component Fr.M-2-12 was chromatographed on silica gel with a gradient elution of methanol-dichloromethane at a volume ratio of 1:25 to 1:15, and was divided into 5 components Fr.M-2-12-1 to Fr.M-2-12-5 according to the TLC detection results; TLC detection and analysis of the target sesquiterpene-containing component Fr.M-2-12-4 was prepared by HPLC to obtain compound 1. The structure of compound 1-4 is shown in Figure 1 shown.

[0031] Spectral data of compound 1-4:

[0032] The nuclear magnetic resonance spectrum was measured using an Avance III 600 (Bruker, Faellanden, Switzerland) superconducting nuclear magnetic resonance instrument, using TMS (tetramethylsilane) as an internal standard. High-resolution mass spectrometry was measured using Shimadzu LC-MS-IT-TOF (Shimadzu, Kyoto, Japan). Infrared spectra (IR) were measured using a Nicolet iS10 infrared spectrometer (Thermo Fisher Scientific, Madison, USA) using the KBr pellet method. ECD spectra were measured using a Chirascan instrument (Applied Photophysics, Surrey, UK). Optical rotation was measured using an Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, USA). Melting point was measured using X-4B micro melting point instrument was used for determination, and the melting point instrument was purchased from Shanghai Precision Scientific Instrument Co., Ltd. Thin layer chromatography silica gel plate HSGF254 was a product of Yantai Jiangyou Silica Gel Development Co., Ltd.; column chromatography silica gel (200-300 mesh) was produced by Linyi Haixiang Chemical Co., Ltd.; column chromatography dextran gel LH-20 was purchased from GE Healthcare Bio-Sciences AB. The high performance liquid chromatograph was produced by Waters, the pump model was 1525, the detector model was 2996, and the chromatographic column model used was Waters Prep OBDTM C18 column (5μm, 19×250mm) and Cosmosil C 18 -MS-Ⅱ (5μm, 10×250mm). Chromatographically pure acetonitrile was purchased from Merida, and deionized channel water was purified by MingCheTM-D 24UV Merk Millipore system. Medium pressure liquid phase (Dr Flash-Ⅱ) was a product of Shanghai Lisui Company, and MCI column was from Mitsubishi Corporation of Japan, model CHP-20P (75~150μm). Analytical pure methanol and acetonitrile were purchased from Tianjin Damao Chemical Reagent Factory. The color developer was 10% H2SO4-EtOH solution.

[0033] The structural formula of pyrifos lactone A (1) is as follows:

[0034]

[0035] Molecular formula: C 15 H 22 O8, molecular weight: 330.13, properties: colorless orthorhombic crystal, melting point: 170~172℃, optical rotation: +31.3 (c 0.10, methanol). HRESIMS (+) m / z: Found 331.1383 [M+H] + , calculated value 331.1380 [M+H] + IR(KBr)ν max :3509,3327,1748,1354,1293,1220,1069cm -1 . Crystal data: C 15 H 22 O8,M=330.32, α=90°, β=90°, γ=90°, T=100.(2)K, lattice size P212121, Z=4, μ(Cu Kα)=1.009mm -1 The crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffraction events was 13352, of which 2953 (R int =0.0401),I>2σ(I),R1=0.0286,wR(F 2 )=0.0748,F 2 =1.052, Flack parameter = 0.03 (5). The crystal parameters of compound 1 have been deposited in the Cambridge Crystallographic Data Center, accession number: CDCC 2162522. Website: https: / / www.ccdc.cam.ac.uk.

[0036] 1 H NMR and 13 C NMR data are shown in Tables 1 and 2.

[0037] The structural formula of pyrifos lactone B (2) is as follows:

[0038]

[0039] Molecular formula: C 15 H 22 O6, molecular weight: 299.14, properties: monoclinic crystal, melting point: 162~164℃, optical rotation: +3.9 (c 0.11, methanol). HRESIMS (+) m / z: Found 299.1477 [M+H] + , calculated value 299.1475[M+H] + IR(KBr)ν max :3452,3374,1760,1646,1461,1276,1256,1115cm -1 . Crystal data: C 15H 22 O6,M=298.32,

[0040] α=90°, β=95.186(2)°, γ=90°, T=101.(2)K, lattice size P1211, Z=2, μ(Cu Kα)=0.926mm -1 The crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffraction events was 10672, of which 2614 were observed (R int =0.0498),I>2σ(I),R1=0.0317,wR(F 2 )=0.0800,F 2 =1.067, Flack parameter = 0.11 (8). The crystal parameters of compound 2 have been deposited in the Cambridge Crystallographic Data Center, accession number: CDCC 2162521. Website: https: / / www.ccdc.cam.ac.uk.

[0041] 1 H NMR and 13 C NMR data are shown in Tables 1 and 2.

[0042] The structural formula of pyrifos lactone C(3) is as follows:

[0043]

[0044] Molecular formula: C 14 H 20 O7, molecular weight: 300.30, properties: colorless orthorhombic crystal, melting point: 166~168℃, optical rotation: -6.0 (c 0.16, methanol). HRESIMS (+) m / z: Found 339.0851 [M+K] + , calculated value 339.0846[M+K] + IR(KBr)ν max :3420,1768,1413,1356,1106,1016cm -1 . Crystal data: C 14 H 20 O7,M=300.30, α=90°, β=90°, γ=90°, T=100.(2)K, lattice size P212121, Z=4, μ(Cu Kα)=1.056mm -1The crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffraction events was 21014, of which 2543 were observed (R int =0.0399),I>2σ(I),R1=0.0292,wR(F 2 )=0.0742,F 2 =1.071, Flack parameter = 0.08 (3). The crystal parameters of compound 3 have been deposited in the Cambridge Crystal Data Center, accession number: CDCC 2162519. Website: https: / / www.ccdc.cam.ac.uk.

[0045] 1 H NMR and 13 C NMR data are shown in Tables 1 and 2.

[0046] The structural formula of pyrifos lactone D (4) is as follows:

[0047]

[0048] Molecular formula: C 14 H 20 O6, molecular weight: 284.30, properties: colorless orthorhombic crystal, melting point: 163~165℃, optical rotation: +3.0 (c 0.05, methanol). HRESIMS (+) m / z: Found 307.1888 [M+Na] + , calculated value 307.1890[M+K] + IR(KBr)ν max :3431,1756,1203,1088cm -1 . Crystal data: C 14 H 20 O6,M=284.30, α=90°, β=90°, γ=90°, T=100.(2)K, lattice size P212121, Z=4, μ(Cu Kα)=0.971mm -1 The crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffraction was 12824, of which 2489 (R int =0.0340),I>2σ(I),R1=0.0305,wR(F 2 )=0.0793,F 2=1.090, Flack parameter = 0.00 (5). The crystal parameters of compound 4 have been deposited in the Cambridge Crystal Data Center, accession number: CDCC 2162520. Website: https: / / www.ccdc.cam.ac.uk.

[0049] 1 H NMR and 13 C NMR data are shown in Tables 1 and 2.

[0050] Table 1. Compounds 1-4 1 H NMR data (600 MHz, pyridine-d6, δ in ppm, J in Hz)

[0051]

[0052]

[0053] Table 2. Compounds 1-4 13 C NMR data (150 MHz, pyridine-d6, δ in ppm)

[0054]

[0055] Among them, the X-ray single crystal diffraction structure of compound 1-4 is as follows Figure 2-5 shown.

[0056] Embodiment 2:

[0057] Antithrombotic activity of compounds 1–4.

[0058] 1. Materials and Methods

[0059] 1.1 Experimental animals

[0060] Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1L of reverse osmosis water, the conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), and were bred and provided by the company's fish farming center. The experimental animal use license number is: SYXK (Zhejiang) 2022-0004. The breeding management complies with the requirements of the international AAALAC certification (certification number: 001458), and the LACUC ethics review number is: IACUC-2023-7311-01. Zebrafish (albino) with melanin allele mutations were bred by natural pair mating. Zebrafish aged 3 days after fertilization (3dpf) were used to evaluate the anti-platelet aggregation thrombotic efficacy of the samples.

[0061] 1.2 Instruments

[0062] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); fully automatic sample rapid grinding machine (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China); ultrasonic cleaning machine (JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); heartbeat blood flow analysis system (ZebraBlood3.4, ViewPoint Life Sciences, France); high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany).

[0063] 1.3 Reagents and consumables

[0064] Methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, USA); arachidonic acid (batch number K2217073, Shanghai Aladdin Biochemical Technology Co., Ltd., China); o-dianisidine (batch number MKBX3619V, Sigma, USA). Compounds 1 and 2 were prepared into 30.0 mM stock solution with DMSO and stored at -20°C. Enteric-coated aspirin tablets were used as positive control (hereinafter referred to as aspirin), white tablets, batch number BJ72163, Bayer Healthcare Co., Ltd., stored in a cool place. Prepared into 100 mg / mL stock solution with DMSO and stored at -20°C.

[0065] 1.4 Experimental process

[0066] 1.4.1 Evaluation of the efficacy of antiplatelet aggregation and thrombosis (cardiac red blood cell staining intensity)

[0067] 3dpf melanin allele mutant zebrafish (albino) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were given water-soluble (concentrations are shown in Table 1-1), and the positive control aspirin concentration was 60.0μg / mL. At the same time, a normal control group and a model control group were set up, and the volume of each well was 3mL. After 4h of treatment at 28℃, except for the normal control group, all other experimental groups were given water-soluble arachidonic acid to establish a zebrafish platelet aggregation thrombosis model. After 135min of treatment at 28℃, o-dianisidine staining was performed. After staining, 10 zebrafish were randomly selected from each experimental group and placed under a dissecting microscope for photography. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the staining intensity of zebrafish heart red blood cells. The statistical significance of this indicator was used to evaluate the anti-platelet aggregation thrombosis efficacy of the sample. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0068] 1.4.2 Evaluation of antiplatelet aggregation thrombotic efficacy (blood flow velocity and cardiac output)

[0069] 3dpf melanin allele mutant zebrafish (albino) were randomly selected in 6-well plates, and 30 zebrafish were treated in each well (experimental group). The samples were given water-soluble (concentrations are shown in Table 1-2), and the positive control aspirin concentration was 60.0μg / mL. At the same time, a normal control group and a model control group were set up, and the volume of each well was 3mL. After 4h of treatment at 28℃, except for the normal control group, all other experimental groups were given arachidonic acid in water-soluble form to establish a zebrafish platelet aggregation thrombosis model. After 30min of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and placed in a heartbeat blood flow analysis system to record zebrafish blood flow videos, and analyze zebrafish blood flow velocity and cardiac output. The statistical significance of the above indicators was used to evaluate the anti-platelet aggregation thrombotic efficacy of the samples. The statistical processing results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0070] 2. Results

[0071] Compounds 1 and 2 both have anti-platelet aggregation thrombotic effects at a concentration of 30 μM, which are specifically manifested by increasing the staining intensity of cardiac red blood cells, improving blood flow velocity and cardiac output, as shown in Tables 3 and 4 for details.

[0072] Table 3. Results of the evaluation experiment on the anti-platelet aggregation thrombotic efficacy of samples (n=10)

[0073]

[0074] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001

[0075] Table 4. Sample anti-platelet aggregation thrombotic efficacy evaluation test results (n = 10)

[0076]

[0077] Compared with the model control group, *p<0.05, ***p<0.001

[0078] 3. Conclusion

[0079] The above results indicate that compounds 1 and 2 in the fruit of the genus Achyranthes have anti-platelet aggregation and thrombotic effects and can be used as drugs for thrombosis-related diseases.

[0080] Application examples 1 and 2:

[0081] In the following application examples, conventional reagents are selected and the preparation is carried out according to the conventional methods. This application example only reflects that at least one of the compounds 1 and 2 described in the present invention can be prepared into different preparations, and the specific reagents and operations are not specifically limited:

[0082] 1. Dissolve at least one of the compounds 1 and 2 prepared in Example 1 in DMSO, add water for injection, filter, and sterilize the mixture according to conventional methods to prepare an injection solution, wherein the concentration of the injection solution is 0.5 to 5 mg / mL.

[0083] 2. Dissolve at least one of the compounds 1 and 2 prepared in Example 1 in DMSO, dissolve it in sterile water for injection, stir to dissolve it, filter it with a sterile suction funnel, filter it with sterile fine filtration, pack it into ampoules, freeze-dry it at low temperature, and seal it with sterile sealing to obtain a powder injection.

[0084] 3. At least one of the compounds 1 and 2 prepared in Example 1 was added to the excipient at a mass ratio of 9:1 to the excipient to prepare a powder.

[0085] 4. At least one of the compounds 1 and 2 prepared in Example 1 was added to the excipient at a mass ratio of 5:1, and granulated and tableted.

[0086] 5. Prepare at least one of the compounds 1 and 2 prepared in Example 1 into an oral liquid according to a conventional oral liquid preparation method.

[0087] 6. At least one of the compounds 1 and 2 prepared in Example 1 was added to the excipient at a mass ratio of 5:1 to the excipient to prepare capsules.

[0088] 7. At least one of the compounds 1 and 2 prepared in Example 1 was added to the excipient at a mass ratio of 5:1 to the excipient to prepare granules.

[0089] From the above examples, it can be seen that the present invention provides ramifloraolides AD (ramifloraolides AD, 1-4) and its preparation method and application, pharmaceutical composition and application. The ramifloraolides AD (ramifloraolides A-D, 1-4) provided by the present invention has certain antithrombotic activity, can be combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition, and can be used to prepare an antithrombotic drug.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A type of pyrifos lactone, characterized in that: The pyracantha lactone is any one of compound 1 to compound 4 in the following formula (I):

2. A method for preparing the pyracantha lactone according to claim 1, characterized in that: The following steps are involved: The dried roots of the cassia twig were crushed and then refluxed and extracted three times with 90% ethanol, and concentrated to dryness to obtain an extract; Subsequently, the extract was mixed with polyamide and chromatographed on an MCI CHP 20P column with a methanol-water elution system. The same fractions were then combined by TLC detection to obtain a total of 4 components Fr.M-1 to Fr.M-4. LCMS analysis speculated that the component Fr.M-2 containing the target sesquiterpene was subjected to silica gel column chromatography and gradient eluted with methanol-dichloromethane in a volume ratio of 1:40 to 1:

0. The obtained components were combined by TLC detection to obtain 16 components Fr.M-2-1 to Fr.M-2-16. LCMS analysis speculated that the component Fr.M-2-6 containing the target sesquiterpene was subjected to MCI CHP 20P column chromatography with a methanol-water elution system. The same fractions were combined by TLC detection to obtain 4 components Fr.M-1 to Fr.M-4. 20P column chromatography, eluted with methanol-water with volume fractions of 50%, 60%, 70%, and 90% in sequence to obtain four components Fr.M-2-6-1 to Fr.M-2-6-4; Fr.M-2-6-2 was prepared by HPLC to obtain compound 2; the component Fr.M-2-8 containing the target sesquiterpene was eluted by gel column chromatography with methanol-dichloromethane volume ratio of 1:1 to obtain two components Fr.M-2-8a and Fr.M-2-8b. TLC detection analysis showed that the component Fr.M-2-8b containing the target sesquiterpene was fully soluble in methanol , and recrystallized to obtain compound 4; the component Fr.M-2-9 containing the target sesquiterpene was recrystallized with methanol-dichloromethane in a volume ratio of 1:9 to obtain compound 3; the component Fr.M-2-12 containing the target sesquiterpene was chromatographed on silica gel with a gradient elution of methanol-dichloromethane in a volume ratio of 1:25 to 1:15, and according to the TLC detection results, it was divided into 5 components Fr.M-2-12-1 to Fr.M-2-12-5; TLC detection and analysis of the component Fr.M-2-12-4 containing the target sesquiterpene was prepared by HPLC to obtain compound 1.

3. Use of the cassia bark lactone described in claim 1 in the preparation of antithrombotic drugs.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises at least one of the cassia bark lactones according to claim 1 and a pharmaceutically acceptable carrier or excipient.

5. Use of the pharmaceutical composition according to claim 4 in the preparation of antithrombotic drugs.

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