Lactone of xylopia aethiopica and pharmaceutical composition and application thereof

Abstract

The application provides a kind of xyloaranol and a pharmaceutical composition and application thereof.Xyloaranol is four sesquiterpenes, the compound has certain antithrombotic activity, can be combined with a pharmaceutical carrier or excipient to form a pharmaceutical composition, and can be used for preparing an antithrombotic drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to ramifloraolides AD (1-4), its preparation method and application, pharmaceutical compositions and their applications. Background Technology

[0002] Thrombosis is a major cause of morbidity and mortality in various vascular diseases. Thrombotic diseases are a type of cardiovascular disease, commonly including ischemic stroke, myocardial infarction, pulmonary embolism, and disseminated intravascular coagulation (DIC), exhibiting high morbidity and mortality rates, seriously endangering human health. Antithrombotic drugs are an important means of emergency treatment for acute thrombotic diseases and long-term treatment for chronic thrombotic diseases, mainly including anticoagulants, antiplatelet aggregation drugs, and fibrinolytic drugs. With the increasing aging of the population, the number of people suffering from thrombotic diseases is rising year by year, and antithrombotic drugs are widely used. Consequently, bleeding events caused by antithrombotic drugs are also increasing annually. Currently used antithrombotic drugs such as aspirin, clopidogrel, and warfarin, although effective in reducing thrombus formation, 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," making it difficult to avoid affecting physiological hemostasis. Therefore, no breakthrough has been made in reducing the risk of bleeding. As a result, antithrombosis remains a focus of global drug development. Researchers are committed to developing new treatment strategies and drugs, and searching for structurally diverse candidate molecules with novel mechanisms of action and new targets to overcome the limitations of existing drugs, improve therapeutic efficacy, and reduce toxic side effects.

[0003] The use of traditional Chinese medicine or natural drugs has been a primary means for humans to treat various diseases for thousands of years, and the effective components or components contained therein have enormous potential for continuous exploration. Searching for novel active molecules from natural products is an important approach to new drug development. Natural molecular libraries are an important resource for screening active drugs, providing a great opportunity to find novel antithrombotic lead compounds or good candidate drugs with unique antithrombotic mechanisms. Domestic and international scholars have successively conducted extensive explorations and research, and have now discovered some natural compounds with antithrombotic activity. For example, Professor Yao Guangmin's research team at Huazhong University of Science and Technology, under activity-guided analysis, used various extraction and separation techniques to discover six natural product molecules with significant antithrombotic activity from the medicinal plant *Pieris japonica* (Eriocaulon buergerianum), a member of the Ericaceae family. In particular, *piericones* A and B are a unique class of hexahydro-10H-furan[2″,3″:3′,4′]furan[2′,3′:3,4]furan[2,3-c]pyran-6(6aH)-ketone-bridged bis(dihydrochalcone) compounds, which are nanomolar-level PDI inhibitors. A study by Fang Chao's team at Tongji Medical College of Huazhong University of Science and Technology and the DeepKinase R&D team has revealed for the first time that dihydrochalcone asebogenin possesses significant antithrombotic activity. Asebogenin directly inhibits SYK activation by interfering with the phosphorylation of SYK Tyr525 / 526, reducing platelet aggregation and fibrin production, inhibiting arterial thrombus formation, and simultaneously reducing neutrophil activation and NET formation, thus lowering venous thrombus formation without increasing bleeding risk. Furthermore, some components from Piperaceae plants also exhibited significant antiplatelet and antithrombotic activities. The study found that extracts from twenty Piperaceae plants showed antiplatelet aggregation activity, and 51 antiplatelet compounds were identified from 11 Piperaceae plants, mainly including alkaloids, lignans, and phenylpropanoids. These studies suggest that not only traditional antithrombotic compounds, but other types of natural compounds may also become new options for future antithrombotic therapy.

[0004] *Baccaurea ramiflora*, belonging to the genus *Baccaurea* in the family Euphorbiaceae, is an evergreen tree or shrub. This genus comprises approximately 80 species, mainly distributed in Southeast Asia. Only one wild species is found in my country, primarily distributed in Hainan, Guangdong, Guangxi, and Yunnan. In traditional Dai medicine, it is used to treat rheumatoid arthritis, cellulitis, abscesses, and wounds. Previous chemical studies of this plant have isolated a series of secondary metabolites, including sesquiterpenes, diterpenes, steroids, polyphenols, and aldehydes, some of which possess antifungal, antioxidant, and immunosuppressive activities. Due to its wide range of medicinal uses, novel and diverse secondary metabolites, and varied biological activities, this genus of plants is increasingly attracting widespread attention from scholars both domestically and internationally.

[0005] To date, there are no reports of ramifloraolides AD (1-4) in the prior art, nor are there any reports of pharmaceutical compositions containing them as active ingredients, nor are there any reports of their use in the preparation or treatment of thrombosis drugs. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a new class of medicinally valuable ramifloraolides AD (1-4) of formula (I), their preparation methods and applications, pharmaceutical compositions and their applications. This class of compounds has significant antithrombotic activity and can be used to prepare antithrombotic drugs.

[0007] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

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

[0009]

[0010] As a second aspect, the present invention provides a method for preparing the above-mentioned wood milk fruit lactones (compounds 1-4). The dried roots of wood milk fruit are pulverized and extracted three times by refluxing with 90% ethanol, then concentrated to dryness to obtain an extract. The extract is then mixed with polyamide and subjected to MCI CHP 20P column chromatography with methanol-water as the elution system. The fractions are then combined by TLC analysis, yielding four fractions Fr.M-1 to Fr.M-4. LCMS analysis suggests that fraction Fr.M-2 may contain the target sesquiterpene, which is then subjected to silica gel column chromatography with a gradient elution of methanol-dichloromethane at volume ratios of 1:40 to 1:0. The resulting fractions are then combined by TLC analysis, yielding 16 fractions Fr.M-2-1 to Fr.M-2-16. LCMS analysis suggests that fraction Fr.M-2-6 may contain the target sesquiterpene, which is then subjected to MCI CHP analysis. 20P column chromatography was performed, eluting sequentially with methanol-water at volume fractions of 50%, 60%, 70%, and 90% to obtain four fractions 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 fraction containing the target sesquiterpene, Fr.M-2-8, was eluted by gel column chromatography with methanol-dichloromethane at a volume ratio of 1:1 to obtain two fractions Fr.M-2-8a and Fr.M-2-8b. TLC analysis showed that the fraction containing the target sesquiterpene, Fr.M-2-8b, was fully soluble in methanol. After recrystallization, compound 4 was obtained; the fraction containing the target sesquiterpene, Fr.M-2-9, was recrystallized with methanol-dichloromethane at a volume ratio of 1:9 to obtain compound 3; the fraction containing the target sesquiterpene, Fr.M-2-12, was subjected to silica gel chromatography with a methanol-dichloromethane gradient elution of 1:25 to 1:15 (volume ratio), and according to the TLC detection results, it was separated into 5 fractions, Fr.M-2-12-1 to Fr.M-2-12-5; the fraction containing the target sesquiterpene, Fr.M-2-12-4, was prepared by HPLC after TLC analysis to obtain compound 1.

[0011] In a third aspect, the present invention provides the use of the aforementioned lactones (compounds 1-4) in the preparation of antithrombotic drugs. The present invention does not specifically limit the method of application; any method well known in the art may be used.

[0012] As a fourth aspect, the present invention also provides a pharmaceutical composition comprising at least one of the above-mentioned cinnamomea 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 this invention comprises at least one of compounds 1-4 above and a pharmaceutically acceptable carrier or excipient. In this invention, the pharmaceutically acceptable carrier or excipient is preferably a solid, semi-solid, or liquid diluent, filler, or pharmaceutical excipient. This invention does not impose any particular limitation on the pharmaceutically acceptable carrier or excipient; any pharmaceutically acceptable carrier and / or excipient well-known in the art that is non-toxic and inert to humans and animals may be selected.

[0015] The present invention does not impose any 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 does not impose any particular limitation on the mixing process. Any process well known in the art that can obtain the pharmaceutical composition can be selected.

[0016] As a fifth aspect, the present invention provides the use of the pharmaceutical composition described in the above-described technical solution in the preparation of antithrombotic drugs. The present invention does not specifically limit the method of application; any method well known in the art may be used.

[0017] In this invention, when the pharmaceutical composition is used to prepare an antithrombotic drug, the content of the composition in the drug is preferably 0.1% to 99%; in the pharmaceutical composition, the content of at least one of compounds 1-4 is preferably 0.5% to 90%. The pharmaceutical composition of this invention is preferably used in the form of a dose per unit body weight. In this invention, the prepared drug is preferably administered by both 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 wood milk lactones, which have certain antithrombotic activity and can be combined with pharmaceutically acceptable carriers or excipients to form drug compositions, which can be used to prepare antithrombotic drugs and have good application prospects. Attached Figure Description

[0019] Figure 1 Here are schematic diagrams of the structural formulas of compounds 1-4 of this invention;

[0020] Figure 2 The X-ray single-crystal diffraction structure of compound 1 is shown.

[0021] Figure 3 The X-ray single-crystal diffraction structure of compound 2 is shown.

[0022] Figure 4 The X-ray single-crystal diffraction structure of compound 3 is shown.

[0023] Figure 5 The X-ray single-crystal diffraction structure of compound 4 is shown. Detailed Implementation

[0024] To better understand the essence of the present invention, the following description, in conjunction with the accompanying drawings, uses experimental examples and embodiments of the present invention to further illustrate the sesquiterpenoid compounds, ramifloraolides AD (1-4), their preparation methods, structural identification, and pharmacological effects, but these experimental examples and embodiments are not intended to limit the present invention.

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0026] Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, all percentages in the following embodiments are volume percentages.

[0028] Example 1:

[0029] Preparation of compounds 1-4:

[0030] The dried roots of *Lysimachia christinae* were pulverized and extracted three times by refluxing with 90% ethanol. The extract was then concentrated to dryness to obtain an extract. The extract was then mixed with polyamide and subjected to MCI CHP 20P column chromatography with methanol-water elution. TLC analysis and combining of identical fractions yielded four fractions, Fr.M-1 to Fr.M-4. LCMS analysis suggested that fraction Fr.M-2, which might contain the target sesquiterpene, was subjected to silica gel column chromatography with a gradient elution of methanol-dichloromethane at volume ratios of 1:40 to 1:0. The resulting fractions were analyzed and combined by TLC, yielding 16 fractions, Fr.M-2-1 to Fr.M-2-16. LCMS analysis suggested that fraction Fr.M-2-6, which might contain the target sesquiterpene, was further analyzed by MCI CHP... 20P column chromatography was performed, eluting sequentially with methanol-water at volume fractions of 50%, 60%, 70%, and 90% to obtain four fractions 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 fraction containing the target sesquiterpene, Fr.M-2-8, was subjected to gel column chromatography, eluting with methanol-dichloromethane at a volume ratio of 1:1 to obtain two fractions Fr.M-2-8a and Fr.M-2-8b. TLC analysis showed that the fraction containing the target sesquiterpene, Fr.M-2-8b, was soluble in methanol (the amount of methanol added to fraction Fr). M-2-8b was fully dissolved and recrystallized to obtain compound 4; the fraction containing the target sesquiterpene, Fr.M-2-9, was recrystallized with methanol-dichloromethane at a volume ratio of 1:9 to obtain compound 3; the fraction containing the target sesquiterpene, Fr.M-2-12, was subjected to silica gel chromatography with a methanol-dichloromethane gradient elution of 1:25 to 1:15 (v / v), and according to the TLC results, it was separated into 5 fractions, Fr.M-2-12-1 to Fr.M-2-12-5; the fraction containing the target sesquiterpene, Fr.M-2-12-4, was prepared by HPLC after TLC analysis to obtain compound 1. The structures of compounds 1-4 are as follows. Figure 1 As shown.

[0031] Spectroscopic data of compounds 1-4:

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

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

[0034]

[0035] Molecular formula: C 15 H 22 O8, molecular weight: 330.13, properties: colorless orthorhombic crystals, melting point: 170~172℃, optical rotation: +31.3 (c 0.10, methanol). HRESIMS(+) m / z: Experimental value 331.1383 [M+H] + The calculated value is 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 Crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffractions was 13352, of which 2953 were observed (R0). 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 stored in the Cambridge Crystal Data Centre, extraction number: CDCC 2162522. Website: https: / / www.ccdc.cam.ac.uk.

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

[0037] The structural formula of cinnamon 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: Experimental value 299.1477 [M+H] + The calculated value is 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 Crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffractions was 10672, of which 2614 were observed (R0). 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 stored in the Cambridge Crystal Data Centre, extraction number: CDCC 2162521. Website: https: / / www.ccdc.cam.ac.uk.

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

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

[0043]

[0044] Molecular formula: C 14 H 20 O7, molecular weight: 300.30, properties: colorless orthorhombic crystals, melting point: 166~168℃, optical rotation: -6.0 (c 0.16, methanol). HRESIMS(+) m / z: experimental value 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 -1Crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffractions was 21,014, of which 2,543 were observed (R0). 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 stored in the Cambridge Crystal Data Centre, extraction number: CDCC 2162519. Website: https: / / www.ccdc.cam.ac.uk.

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

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

[0047]

[0048] Molecular formula: C 14 H 20 O6, molecular weight: 284.30, properties: colorless orthorhombic crystals, melting point: 163~165℃, optical rotation: +3.0 (c 0.05, methanol). HRESIMS(+) m / z: Experimental value 307.1888 [M+Na] + The calculated value is 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 Crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffractions was 12,824, of which 2,489 were observed (R0). 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 Crystallographic Data Centre, deposition number: CDCC 2162520. Website: https: / / www.ccdc.cam.ac.uk.

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

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

[0051]

[0052]

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

[0054]

[0055] Among them, the X-ray single crystal diffraction structure diagrams of Compounds 1 - 4 are as Figure 2-5 shown.

[0056] Example 2:

[0057] Antithrombotic activities of Compounds 1–4.

[0058] 1. Materials and methods

[0059] 1.1 Experimental animals

[0060] Zebrafish were all reared in fish culture water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L CaCO3), provided by the fish culture center of our company, and the license number for the use of experimental animals was: SYXK(Zhe)2022 - 0004. The feeding management met the requirements of international AAALAC accreditation (accreditation number: 001458), and the LACUC ethical review number was: IACUC - 2023 - 7311 - 01. Zebrafish with melanin allele gene mutation (albino) were bred by natural paired mating. Zebrafish at 3 days post-fertilization (3 dpf) were used for the evaluation of the antiplatelet aggregation thrombus efficacy of 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 automated rapid sample grinder (JXFSTPRP-24L, Shanghai Jingxin Experimental Equipment Technology Department, China); Ultrasonic cleaner (JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China); Cardiac blood flow analysis system (ZebraBlood 3.4, ViewPoint Life Sciences, France); High-speed refrigerated centrifuge (Heraeus Fresco 17, ThermoFisher, Germany).

[0063] 1.3 Reagents and Consumables

[0064] Demethylcellulose (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-anisidine (batch number MKBX3619V, Sigma, USA). Compounds 1 and 2 were both prepared as 30.0 mM stock solutions using DMSO and stored at -20°C. Enteric-coated aspirin tablets were used as a positive control (hereinafter referred to as aspirin), white tablets, batch number BJ72163, Bayer Healthcare Co., Ltd., and stored in a cool place. A 100 mg / mL stock solution was prepared using DMSO and stored at -20°C.

[0065] 1.4 Experimental Procedure

[0066] 1.4.1 Evaluation of antiplatelet aggregation thrombotic efficacy (cardiac erythrocyte staining intensity)

[0067] Zebrafish (albino) with a 3dpf melanin allele mutation were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in aqueous solution (concentrations shown in Table 1-1), with aspirin at a concentration of 60.0 μg / mL as a positive control. Normal and model control groups were also included, with a volume of 3 mL per well. After treatment at 28℃ for 4 h, except for the normal control group, all other experimental groups were administered arachidonic acid in aqueous solution to establish a zebrafish platelet aggregation thrombosis model. After further treatment at 28℃ for 135 min, o-anisidine staining was performed. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the staining intensity of zebrafish cardiac erythrocytes was analyzed. The statistical significance of this index was used to evaluate the antiplatelet aggregation thrombosis efficacy of the samples. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[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 and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (concentrations shown in Tables 1-2) were administered in water, with aspirin at a concentration of 60.0 μg / mL as a positive control. A normal control and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 4 h, except for the normal control group, all other experimental groups were given arachidonic acid in water to establish a zebrafish platelet aggregation thrombosis model. After further treatment at 28℃ for 30 min, 10 zebrafish from each experimental group were randomly selected and placed in a cardiac blood flow analysis system to record zebrafish blood flow videos, analyzing blood flow velocity and cardiac output. The antiplatelet aggregation thrombosis efficacy of the samples was evaluated based on the statistical significance of these indicators. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0070] 2. Results

[0071] Compounds 1 and 2 both exhibited antiplatelet aggregation and thrombotic effects at a concentration of 30 μM, specifically by increasing the staining intensity of cardiac erythrocytes, improving blood flow velocity and cardiac output, as detailed in Tables 3 and 4.

[0072] Table 3. Results of the experiment evaluating the antiplatelet aggregation and thrombotic efficacy of the samples (n=10)

[0073]

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

[0075] Table 4. Results of the experiment evaluating the antiplatelet aggregation and thrombotic efficacy of the samples (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 *Lysimachia christinae* have antiplatelet 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 formulations are prepared according to existing conventional methods. These application examples only demonstrate that at least one of compounds 1 and 2 described in this invention can be prepared into different formulations, and no specific limitations are made on the specific reagents and operations:

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

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

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

[0085] 4. Add at least one of compounds 1 and 2 prepared in Example 1 to the excipient at a mass ratio of 5:1, and granulate and compress the mixture into tablets.

[0086] 5. Prepare an oral liquid by taking at least one of compounds 1 and 2 prepared in Example 1 according to conventional oral liquid preparation methods.

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

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

[0089] As can be seen from the above embodiments, the present invention provides ramifloraolides AD (1-4), its preparation method and application, pharmaceutical compositions and their applications. The ramifloraolides AD (A-D, 1-4) provided by the present invention possess certain antithrombotic activity and can be combined with pharmaceutically acceptable carriers or excipients to form pharmaceutical compositions, which can be used to prepare antithrombotic drugs.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A class of wood milk lactones, characterized in that, The lactone is any one of compounds 1-4 in formula (I):

2. A method for preparing the lactone of claim 1, characterized in that, Includes the following steps: The dried roots of the wood apple were crushed and extracted three times by reflux with 90% ethanol, and then concentrated to dryness to obtain an extract. The extract was then mixed with polyamide and subjected to MCI CHP 20P column chromatography with methanol-water elution. TLC analysis and combining of identical fractions yielded four fractions, Fr.M-1 to Fr.M-4. LCMS analysis suggested that fraction Fr.M-2, containing the target sesquiterpene, was subjected to silica gel column chromatography with a gradient elution of methanol-dichloromethane at volume ratios of 1:40 to 1:

0. The resulting fractions were analyzed and combined by TLC, yielding 16 fractions, Fr.M-2-1 to Fr.M-2-16. LCMS analysis suggested that fraction Fr.M-2-6, containing the target sesquiterpene, was further analyzed by MCI CHP... 20P column chromatography was performed, eluting sequentially with methanol-water at volume fractions of 50%, 60%, 70%, and 90% to obtain four fractions 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 fraction containing the target sesquiterpene, Fr.M-2-8, was eluted by gel column chromatography with methanol-dichloromethane at a volume ratio of 1:1 to obtain two fractions Fr.M-2-8a and Fr.M-2-8b. TLC analysis showed that the fraction containing the target sesquiterpene, Fr.M-2-8b, was fully soluble in methanol. After recrystallization, compound 4 was obtained; the fraction containing the target sesquiterpene, Fr.M-2-9, was recrystallized with methanol-dichloromethane at a volume ratio of 1:9 to obtain compound 3; the fraction containing the target sesquiterpene, Fr.M-2-12, was subjected to silica gel chromatography with a methanol-dichloromethane gradient elution of 1:25 to 1:15 (volume ratio), and according to the TLC detection results, it was separated into 5 fractions, Fr.M-2-12-1 to Fr.M-2-12-5; the fraction containing the target sesquiterpene, Fr.M-2-12-4, was prepared by HPLC after TLC analysis to obtain compound 1.

3. The use of the 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 lactones of claim 1 and a pharmaceutically acceptable carrier or excipient.

5. The use of the pharmaceutical composition of claim 4 in the preparation of an antithrombotic drug.

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