WO2016090800A1 - 白术内酯类化合物或其衍生物的应用及抗血小板凝聚药物 - Google Patents

白术内酯类化合物或其衍生物的应用及抗血小板凝聚药物 Download PDF

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WO2016090800A1
WO2016090800A1 PCT/CN2015/076743 CN2015076743W WO2016090800A1 WO 2016090800 A1 WO2016090800 A1 WO 2016090800A1 CN 2015076743 W CN2015076743 W CN 2015076743W WO 2016090800 A1 WO2016090800 A1 WO 2016090800A1
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platelet aggregation
atractylenolide
aggregation drug
compound
derivative
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PCT/CN2015/076743
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English (en)
French (fr)
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张俊峰
陈一竹
刘俊岭
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上海交通大学医学院附属第三人民医院
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Priority claimed from CN201410752500.0A external-priority patent/CN104490869B/zh
Priority claimed from CN201410751966.9A external-priority patent/CN104490868B/zh
Application filed by 上海交通大学医学院附属第三人民医院 filed Critical 上海交通大学医学院附属第三人民医院
Priority to US14/908,089 priority Critical patent/US9642832B2/en
Priority to CN201580067226.6A priority patent/CN107106538A/zh
Priority to EP15868066.0A priority patent/EP3231422B1/en
Priority to JP2017549568A priority patent/JP2018507907A/ja
Publication of WO2016090800A1 publication Critical patent/WO2016090800A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the invention relates to the technical field of medicine, in particular to an application of an atractylenolide compound or a derivative thereof in preparing an anti-platelet aggregation drug and an anti-platelet aggregation drug.
  • the thrombus begins with the local coagulation mechanism. After the thrombosis of the intimal surface of the artery is reached, the thrombus is covered by the proliferating endothelial cells, and the lipids and other active substances explained by the platelets and leukocyte collapse in the thrombus of the arterial wall gradually form porridge. Plaque. Later researchers thought that the disease began with arterial intimal injury, increased platelet activating factor (PAF), where platelets adhered and then aggregated, followed by fibrin deposition to form microthrombus. After platelet aggregation, some active substances are released.
  • PAF platelet activating factor
  • Thromboxane A2 can counteract the role of prostacycline (PGI2) in the synthesis of blood vessel wall to depolymerize and vasodilate platelets, and promote platelet aggregation. And vasoconstriction; platelet derived growth factor stimulates cell proliferation and contraction of smooth muscle and migrates to the intima; serotonin and fibroblast growth factor stimulate fibroblast smooth muscle cells and endothelium Cell proliferative adrenaline and adenosine diphosphate promote platelet aggregation: Factor VIII further binds platelets; platelet factor 4 causes vasoconstriction; plasminogen activator inhibitor (PAI) The dissolution of the thrombus is inhibited.
  • PKI2 prostacycline
  • LDL low-density lipoprotein
  • coagulation systems and anticoagulation systems in human blood. Under normal circumstances, the two maintain a dynamic balance to ensure the normal flow of blood in the blood vessels, that is, no thrombosis, in special circumstances, such as blood vessels have hardening, stenosis and other damage, cold weather, excessive sweating, blood pressure Under low conditions and lack of drinking water, the blood flow will be slow, the blood will be thick and viscous, and the coagulation function will be weakened or the anticoagulant function will be weakened, which will break the balance and make people "easy to plug". Thrombosis can occur anywhere in the blood vessels. The blood vessels flow in the blood vessels with the blood.
  • Thrombosis most of the symptoms will have serious symptoms, such as hemiplegic aphasia; cerebral infarction, severe precordial colic; severe chest pain caused by pulmonary infarction, dyspnea, hemoptysis, etc.; It can cause pain in both legs, or a feeling of coldness and intermittent claudication. Extremely severe heart, cerebral infarction, and pulmonary infarction can also cause sudden death. But sometimes Thrombosis can also have no obvious symptoms, such as common deep vein thrombosis of the lower extremities, only the calf is sore and discomfort, many patients think it is tired or cold, do not agree, so it is easy to miss the best treatment opportunity.
  • Atractylenolide compounds such as atractylodesin II (Atractylodes lactone 2), Atractylodesin III (Atractylodes lactone 3), etc., are derived from the extract of dried roots of Atractylodes macrocephala Koidz.
  • atractylenolide compounds have anti-inflammatory and anti-tumor effects, and also have functions of regulating gastrointestinal motility and promoting nutrient absorption.
  • the invention solves the technical problem of the current lack of high-efficiency and safe anti-thrombotic drugs, and provides a simple composition, which is composed of an active ingredient of a natural medicinal raw material or an extract of an active ingredient of the medicinal raw material, and has anti-platelet aggregation effect.
  • Atractylenolide compound or its derivative drug the drug has good curative effect, no toxic and side effects, is not easy to produce drug resistance, and is convenient to take, and is generally suitable for preventing and treating blood viscous and thrombus caused by excessive platelet aggregation rate.
  • a drug for anti-platelet aggregation comprising an atractylenolide compound or a derivative thereof, the atractylenolide compound having the structural formula represented by the following formula (I):
  • R1 represents H or a linear or branched alkyl group of C1-C10
  • R2 represents a straight or branched alkyl group of H or C1-C10
  • R3 represents H or a hydroxyl group.
  • the atractylenolide compound has the structural formula represented by the following formula (II):
  • the structural formula represented by the formula (II) is a chemical structural formula of atractylenolide III.
  • the atractylenolide compound has a structural formula represented by the following formula (III):
  • the structural formula represented by the formula (III) is a chemical structural formula of atractylenolide I.
  • the derivative of the atractylenolide compound has a structural formula represented by the following formula (IV):
  • the structural formula represented by the formula (IV) is a chemical structural formula of atractylenolide II.
  • the pharmaceutical dosage form includes tablets, granules, capsules, patches or injections.
  • the C1-C10 alkyl group is preferably a C1-C8 alkyl group, more preferably a C1-C6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, Tert-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl.
  • alkyl group may be a linear alkyl group or a branched alkyl group, more preferably a linear alkyl group.
  • the derivative of the atractylenolide compound of the present invention refers to a type of derivatization modified with a functional group such as an ester bond, an epoxy ring or a carbon-carbon double bond contained in the chemical structural formula of atractyl alcohol lactone, and has a similar chemistry to atractylenolide.
  • a functional group such as an ester bond, an epoxy ring or a carbon-carbon double bond contained in the chemical structural formula of atractyl alcohol lactone
  • a functional group such as an ester bond, an epoxy ring or a carbon-carbon double bond contained in the chemical structural formula of atractyl alcohol lactone
  • the anti-platelet aggregation drug of the present invention further comprises a pharmaceutically acceptable adjuvant.
  • the auxiliary material comprises a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, and a helper.
  • Flowing agents flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, Surfactant, foaming agent, antifoaming agent, thickener, inclusion agent, humectant, absorbent, diluent, flocculant and deflocculant, filter aid, or release retarder or Several.
  • an atractylenolide compound represented by the formula (I) or a derivative thereof for preparing an anti-platelet aggregation drug is provided.
  • R1 represents H or a linear or branched alkyl group of C1-C10
  • R2 represents a straight or branched alkyl group of H or C1-C10
  • R3 represents H or a hydroxyl group.
  • the anti-platelet aggregation drug includes: a medicament for preventing or treating blood viscous, cerebral infarction, myocardial infarction, pulmonary embolism, lower extremity arterial thrombosis, and deep venous thrombosis of the lower extremity.
  • the anti-platelet aggregation drug of the invention has good curative effect and no toxic and side effects. Compared with the existing platelet aggregation inhibiting drug acetylsalicylic acid, the experiment shows that the atractylenolide compound or its derivative drug of the invention has very significant inhibition of platelet aggregation. The effect can be applied to prevent or treat related diseases caused by excessive platelet aggregation rate, and has broad application prospects.
  • Example 1 is a graph showing the results of an in vitro anti-platelet aggregation experiment of atractyl lactone II in Example 1;
  • Example 2 is a graph showing the results of an in vitro anti-platelet aggregation experiment of atractylenolide III in Example 2;
  • Figure 3 is an oil mirror view of the effect of atractylenolide II on platelet spreading in Example 3;
  • Figure 4 is an oil mirror view of the effect of atractyl lactone III on platelet spreading in Example 4.
  • Figure 5 is a diagram showing the results of Western blotting of Example 5.
  • Figure 6 is a diagram showing the results of Western blotting of Example 6;
  • Example 7 is a graph showing the results of comparison of anti-platelet aggregation of atractylodesin II and acetylsalicylic acid in Example 7;
  • Figure 8 is a graph showing the results of comparison of anti-platelet aggregation of atractyl lactone III and acetylsalicylic acid in Example 8.
  • the atractylenolide compound or a derivative thereof of the present invention has an effect of inhibiting platelet aggregation by taking atractylenolide II and atractylenolide III as an example.
  • Example 1 Atractylodesin II inhibits platelet aggregation assay
  • Atractylenolide II has an inhibitory effect on platelet aggregation, and a high concentration of atractylenolide II has a significant inhibitory effect.
  • Example 2 Atractylodesin III inhibits platelet aggregation assay
  • Atractylenolide III dissolved in DMSO.
  • Atractylenolide III has an inhibitory effect on platelet aggregation, and a high concentration of atractylenolide III has a significant inhibitory effect.
  • Example 5 Western blotting was used to detect the phosphorylation level of related molecules during the inhibition of platelet aggregation by atractylenolide II
  • a platelet protein sample (2X SDS loading protein lysate) was collected and the phosphorylation level of the relevant molecule was detected by Western blotting.
  • Example 6 Western blotting was used to detect the phosphorylation level of related molecules during the inhibition of platelet aggregation by atractylenolide III
  • a platelet protein sample (2X SDS loading protein lysate) was collected and the phosphorylation level of the relevant molecule was detected by Western blotting.
  • Western blooting detects the phosphorylation level of related molecules in the signaling pathway.
  • Akt molecules are activated by phosphorylation, which activates downstream enzymes, kinases, transcription factors, etc. (such as GSK3), and eventually Platelet activation is aggregated, and if the phosphorylation level of the Akt molecule is higher, the degree of platelet aggregation is higher.
  • atractylenolide II affected the phosphorylation level of Akt, and the effect of its concentration on the phosphorylation level of Akt was consistent with the effect on platelet aggregation.
  • acetylsalicylic acid was not inhibited by thrombin stimulation at a concentration of up to 150 uM, while atractylodesin II inhibited platelet aggregation at low concentrations (10 uM) in vitro. .
  • Example 8 Comparative experiment of anti-platelet aggregation of atractylenolide III and acetylsalicylic acid in vitro
  • acetylsalicylic acid was stimulated by thrombin at concentrations up to 150 uM. There was still no inhibition, while atractylodesin III inhibited platelet aggregation at low concentrations (10 uM) in vitro.

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Abstract

本发明公开了一种抗血小板聚集的药物，包含白术内酯类化合物或其衍生物，所述白术内酯类化合物具有下述式(I)所示的结构式，其中，R1表示H或C1-C10的直链或带有支链的烷基，R2表示H或C1-C10的直链或带有支链的烷基，R3表示H或羟基。本发明还公开了式(I)所示白术内酯类化合物或其衍生物在制备抗血小板聚集药物中的应用。

Description

白术内酯类化合物或其衍生物的应用及抗血小板凝聚药物 技术领域
本发明涉及医药技术领域，尤其涉及一种白术内酯类化合物或其衍生物在制备抗血小板聚集药物中的应用及抗血小板聚集药物。
背景技术
现代医学认为，血栓的形成和血小板聚集率过高有一定的联系。
血栓开始于局部凝血机制，亢进动脉内膜表面血栓形成以后，血栓被增生的内皮细胞所覆盖，而并入动脉壁血栓中的血小板和白细胞崩解释出的脂质及其他活性物质，逐渐形成粥样斑块。后来的研究者认为本病开始于动脉内膜损伤，血小板活化因子(PAF)增多，血小板在该处粘附继而聚集，随后发生纤维蛋白沉积形成微血栓。血小板聚集后释出一些活性物质，其中血栓烷A2(thromboxane A2,TXA2)能对抗血管壁合成的前列环素(prostacycline,PGI2)所具有的使血小板解聚和血管扩张的作用，促使血小板进一步聚集和血管收缩；血小板源生长因子(platelet derived growth factor)可刺激平滑肌的细胞增生收缩并向内膜游移；5-羟色胺和纤维母细胞生长因子(fibroblast growth factor)可刺激纤维母细胞平滑肌细胞和内皮细胞增生肾上腺素和二磷酸腺苷，可促使血小板进一步聚集：第Ⅷ因子使血小板进一步粘附；血小板第4因子(platelet factor 4)可使血管收缩；纤溶酶原激活剂抑制物(PAI)使血栓的溶解受到抑制。这些物质使内皮细胞进一步损伤从而导致LDL(低密度脂蛋白)进入内膜和内膜下；使单核细胞聚集于内膜发展成为泡沫细胞；使平滑肌细胞增生移入内膜吞噬脂质；并使内皮细胞增殖都有利于粥样硬化的形成。
人体血液中存在着凝血***和抗凝***。在正常情况下，二者保持着动态平衡以保证血液在血管中的正常流动，即不会形成血栓，在特殊情况下，如血管有硬化，狭窄等损伤，天气寒冷，出汗过多、血压过低、缺乏饮水等情况下，会使血流缓慢、血液浓缩粘稠，致凝血功能亢进或抗凝功能削弱，则会打破这种平衡，使人处于“易栓状态”。血栓病可以发生在血管的任何地方，血栓随着血液在血管内流动，如果在脑动脉血管中停留，阻碍了脑动脉正常血液的流通就是脑血栓，从而引发缺血性中风发作，若堵塞了心脏的冠状动脉血管，则会诱发心肌梗塞，此外，还有下肢动脉血栓、下肢深静脉血栓及肺栓塞等。
血栓形成，大多数一发病就会出现严重症状，如脑梗塞的偏瘫失语；心肌梗塞的剧烈心前区绞痛；肺梗塞引起的剧烈胸痛、呼吸困难、咯血等症状；如下肢形成血栓则会引发两腿疼痛，或出现冰凉感和间歇性跛行等。极严重的心、脑梗塞和肺梗塞还可引发猝死。但有时 血栓形成也可无明显症状，如常见的下肢深静脉血栓形成，仅有小腿酸胀不适，很多患者以为是劳累或着凉，不以为然，因而容易错过最佳治疗时机。尤为遗憾的是，很多医生对此也容易误诊，待到出现典型的下肢水肿时，不仅会给治疗带来困难，也容易留下后遗症。血栓的形成常常会造成上述这些严重后果，但是至今还没有特别有效又无毒副作用的用于治疗或预防血栓形成的药物。
白术内酯类化合物如白术内酯Ⅱ(白术内酯2)、白术内酯Ⅲ(白术内酯3)等，来源于菊科植物白术Atractylodes macrocephala Koidz.干燥根的提取物。在现有技术的研究中，白术内酯类化合物具有抗炎、抗肿瘤的作用，还具有调节胃肠道蠕动和促进营养物质吸收的功能。但是至今尚无白术内酯类化合物具有抗血小板聚集作用的公开报道。
发明内容
本发明要解决目前缺乏高效安全的防治血栓药物的技术问题，提供一种组成简单，由天然药用原料有效成分或由该药用原料有效成分的提取物组合而成，具有抗血小板聚集作用的白术内酯类化合物或其衍生物药物，该药物疗效好，无毒副作用，不易产生耐药性，服用方便，普遍适用于防治由血小板聚集率过高引起的血液粘稠及血栓等症状。
在本发明的一个方面，提供了一种抗血小板聚集的药物，包含白术内酯类化合物或其衍生物，所述白术内酯类化合物具有下述式(I)所示的结构式：
Figure PCTCN2015076743-appb-000001
其中，R1表示H或C1-C10的直链或带有支链的烷基，R2表示H或C1-C10的直链或带有支链的烷基，R3表示H或羟基。
优选的，所述白术内酯类化合物具有下述式(Ⅱ)所示的结构式：
Figure PCTCN2015076743-appb-000002
该式(Ⅱ)所示结构式是白术内酯Ⅲ的化学结构式。
优选的，所述白术内酯类化合物具有下述式(Ⅲ)所示的结构式：
Figure PCTCN2015076743-appb-000003
该式(Ⅲ)所示结构式是白术内酯I的化学结构式。
优选的，所述白术内酯类化合物的衍生物具有下述式(Ⅳ)所示的结构式：
Figure PCTCN2015076743-appb-000004
该式(Ⅳ)所示结构式是白术内酯Ⅱ的化学结构式。
所述药物的剂型包括片剂、颗粒剂、胶囊剂、贴剂或注射剂。
所述C1-C10的烷基优选为C1-C8的烷基，更优选为C1-C6的烷基，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、正己基、异己基。
其中，所述烷基可以是直链烷基或支化烷基，更优选为直链烷基。
本发明的白术内酯类化合物的衍生物是指对白术内酯化学结构式中含有的酯键、环氧环、碳碳双键等官能团进行衍生化修饰得到的一类与白术内酯具有类似化学结构的物质。如白术内酯在酸性或碱性条件下发生酯水解反应，以实现对酯键的修饰，环氧环在酸性或碱性条件下也发生水解反应。
本发明所述的抗血小板聚集的药物，还包含药学上可接受的辅料。
优选的，所述辅料包括溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、 表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、或释放阻滞剂中的任意一种或几种。
在本发明的另一方面，提供了一种式(I)所示的白术内酯类化合物或其衍生物在制备抗血小板聚集药物中的应用，
Figure PCTCN2015076743-appb-000005
其中，R1表示H或C1-C10的直链或带有支链的烷基，R2表示H或C1-C10的直链或带有支链的烷基，R3表示H或羟基。
所述抗血小板聚集药物包括：用于预防或治疗血液粘稠、脑梗塞、心肌梗塞、肺栓塞、下肢动脉血栓、下肢深静脉血栓的药物。
本发明抗血小板聚集的药物，疗效好，无毒副作用，与现有抑制血小板聚集药物乙酰水杨酸对比实验显示，本发明的白术内酯类化合物或其衍生物药物具有非常显著的抑制血小板聚集作用，可适用于预防或治疗由血小板聚集率过高引起的相关疾病，应用前景广阔。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1为实施例1白术内酯Ⅱ体外抗血小板聚集实验结果图；
图2为实施例2白术内酯Ⅲ体外抗血小板聚集实验结果图；
图3为实施例3白术内酯Ⅱ对血小板铺展产生影响的油镜观察图；
图4为实施例4白术内酯Ⅲ对血小板铺展产生影响的油镜观察图；
图5为实施例5的Western blotting检测结果图；
图6为实施例6的Western blotting检测结果图；
图7为实施例7白术内酯Ⅱ与乙酰水杨酸在体外抗血小板聚集对比实验结果图；
图8为实施例8白术内酯Ⅲ与乙酰水杨酸在体外抗血小板聚集对比实验结果图。
具体实施方式
下面以白术内酯Ⅱ、白术内酯Ⅲ为例，详细阐述本发明白术内酯类化合物或其衍生物具有抑制血小板聚集的作用。
实施例1白术内酯Ⅱ抑制血小板聚集实验
一、试验材料准备
白术内酯Ⅱ，溶于DMSO中。
二、实验验证过程
血小板聚集实验
⑴准备血小板：人来源的高浓度血小板血浆，制备血小板计数为3×108/mL，置于37℃水浴中。
⑵白术内酯Ⅱ化合物的浓度梯度：在300uL血小板中，化合物的终浓度分别为0.1μm、0.5μm、1μm、5μm、10μm。
实验开始前化合物在血小板中孵育3min，实验中设置resting、DMSO作为对照。使用凝血酶(thrombin)作为刺激剂。由血小板聚集仪获得聚集曲线和聚集率。
实验技术文献：
Weng Z,Li D,Zhang L,et al.PTEN regulates collagen-induced platelet activation.Blood.2010；116(14):2579-2581.
Liu J,Jackson CW,Gruppo RA,Jennings LK,Gartner TK.The beta3 suunit of the integrin alphallbbeta3 regulates alphaIIb-mediated outside-in signaling.Blood.2005；105(11):4345-4352.
⑶实验结果(见图1)：
从以上实验结果表明：白术内酯Ⅱ对血小板聚集有抑制作用，且高浓度的白术内酯Ⅱ抑制作用比较明显。
实施例2白术内酯Ⅲ抑制血小板聚集实验
一、试验材料准备
白术内酯Ⅲ，溶于DMSO中。
二、实验验证过程
血小板聚集实验
⑴准备血小板：人来源的高浓度血小板血浆，制备血小板计数为3×108/mL，置于37℃水浴中。
⑵白术内酯Ⅲ化合物的浓度梯度：在300uL血小板中，化合物的终浓度分别为0.1μm、0.5μm、1μm、5μm、10μm。
实验开始前化合物在血小板中孵育3min，实验中设置resting、DMSO作为对照。使用 凝血酶(thrombin)作为刺激剂。由血小板聚集仪获得聚集曲线和聚集率。
⑶实验结果(见图2)：
从以上实验结果表明：白术内酯Ⅲ对血小板聚集有抑制作用，且高浓度的白术内酯Ⅲ抑制作用比较明显。
实施例3白术内酯Ⅱ影响血小板铺展实验
血小板铺展实验
⑴准备血小板：同上，制备血小板为3×107/mL。
⑵白术内酯Ⅱ化合物的浓度梯度：在每100uL血小板中，化合物的终浓度分别为1μM、5μM、10μM。化合物在血小板中孵育3min。药物处理过的血小板铺在纤维蛋白上(40μg/mL)。通过荧光抗体鬼笔环肽染色，在100X油镜下观察血小板的铺展情况。
实验技术文献：
Chen X,Zhang Y,Wang Y,et al.PDK1 regulates platelet activation and arterial thrombosis.Blood.2013；121(18):3718-3726.
⑶实验结果(见图3)：说明白术内酯Ⅱ对血小板铺展有影响。
实施例4白术内酯Ⅲ影响血小板铺展实验
血小板铺展实验
⑴准备血小板：同上，制备血小板为3×107/mL。
⑵白术内酯Ⅲ化合物的浓度梯度：在每100uL血小板中，化合物的终浓度分别为1μM、5μM、10μM。化合物在血小板中孵育3min。药物处理过的血小板铺在纤维蛋白上(40μg/mL)。通过荧光抗体鬼笔环肽染色，在100X油镜下观察血小板的铺展情况。
⑶实验结果(见图4)：说明白术内酯Ⅲ对血小板铺展有影响。
实施例5用Western blotting检测在白术内酯Ⅱ抑制血小板聚集过程中相关分子的磷酸化水平
继实施例1获得聚集曲线后，收取血小板蛋白样品(2X SDS loading蛋白裂解液)，通过Western blotting检测相关分子的磷酸化水平。
实验结果(见图5)如下：
通过western blooting检测信号通路的相关分子的磷酸化水平，当PI3K/Akt信号通路被激 活，则Akt分子发生磷酸化激活，从而使下游的酶、激酶、转录因子等(如GSK3)活化，最终会使血小板活化聚集，且若Akt分子的磷酸化水平越高，则血小板聚集程度越高。实验结果显示：白术内酯Ⅱ影响Akt分子的磷酸化水平，且其浓度变化对Akt分子磷酸化水平的影响与对血小板聚集程度的影响相一致。
实施例6用Western blotting检测在白术内酯Ⅲ抑制血小板聚集过程中相关分子的磷酸化水平
继实施例2获得聚集曲线后，收取血小板蛋白样品(2X SDS loading蛋白裂解液)，通过Western blotting检测相关分子的磷酸化水平。
实验结果(见图6)如下：
通过western blooting检测信号通路的相关分子的磷酸化水平，当PI3K/Akt信号通路被激活，则Akt分子发生磷酸化激活，从而使下游的酶、激酶、转录因子等(如GSK3)活化，最终会使血小板活化聚集，且若Akt分子的磷酸化水平越高，则血小板聚集程度越高。实验结果显示：白术内酯Ⅱ影响Akt分子的磷酸化水平，且其浓度变化对Akt分子磷酸化水平的影响与对血小板聚集程度的影响相一致。
实施例7白术内酯Ⅱ与乙酰水杨酸在体外抗血小板聚集对比实验
(1)准备血小板：同上，制备血小板为3×108/mL；
(2)将乙酰水杨酸溶于无水乙醇，稀释至浓度为50mmol/L，用凝血酶(thrombin)刺激剂刺激，观察血小板聚集情况。
实验结果(见图7)如下：
在相同条件的体外实验中，乙酰水杨酸在凝血酶(thrombin)刺激下，浓度高达150uM时仍无抑制作用，而白术内酯Ⅱ在体外实验中低浓度(10uM)即有抑制血小板聚集作用。
实施例8白术内酯Ⅲ与乙酰水杨酸在体外抗血小板聚集对比实验
(1)准备血小板：同上，制备血小板为3×108/mL；
(2)将乙酰水杨酸溶于无水乙醇，稀释至浓度为50mmol/L，用凝血酶(thrombin)刺激剂刺激，观察血小板聚集情况。
实验结果(见图8)如下：
在相同条件的体外实验中，乙酰水杨酸在凝血酶(thrombin)刺激下，浓度高达150uM 时仍无抑制作用，而白术内酯Ⅲ在体外实验中低浓度(10uM)即有抑制血小板聚集作用。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Figure PCTCN2015076743-appb-000006
Figure PCTCN2015076743-appb-000007
Figure PCTCN2015076743-appb-000008
Figure PCTCN2015076743-appb-000009

Claims (12)

  1. 一种抗血小板聚集的药物，其特征在于，包含白术内酯类化合物或其衍生物，所述白术内酯类化合物具有下述式(I)所示的结构式：
    Figure PCTCN2015076743-appb-100001
    其中，R1表示H或C1-C10的直链或带有支链的烷基，R2表示H或C1-C10的直链或带有支链的烷基，R3表示H或羟基。
  2. 根据权利要求1所述的抗血小板聚集的药物，其特征在于，所述R1为甲基。
  3. 根据权利要求1或2所述的抗血小板聚集的药物，其特征在于，所述R2为甲基。
  4. 根据权利要求3所述的抗血小板聚集的药物，其特征在于，所述白术内酯类化合物具有下述式(Ⅱ)所示的结构式：
    Figure PCTCN2015076743-appb-100002
  5. 根据权利要求3所述的抗血小板聚集的药物，其特征在于，所述白术内酯类化合物具有下述式(Ⅲ)所示的结构式：
    Figure PCTCN2015076743-appb-100003
  6. 根据权利要求4所述的抗血小板聚集的药物，其特征在于，所述白术内酯类化合物的衍生 物具有下述式(Ⅳ)所示的结构式：
    Figure PCTCN2015076743-appb-100004
  7. 根据权利要求1所述的抗血小板聚集的药物，其特征在于，所述药物的剂型包括片剂、颗粒剂、胶囊剂、贴剂或注射剂。
  8. 根据权利要求1所述的抗血小板聚集的药物，其特征在于，还包含药学上可接受的辅料。
  9. 根据权利要求8所述的抗血小板聚集的药物，其特征在于，所述辅料包括溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、或释放阻滞剂中的任意一种或几种。
  10. 式(I)所示的白术内酯类化合物或其衍生物在制备抗血小板聚集药物中的应用，
    Figure PCTCN2015076743-appb-100005
    其中，R1表示H或C1-C10的直链或带有支链的烷基，R2表示H或C1-C10的直链或带有支链的烷基，R3表示H或羟基。
  11. 根据权利要求10所述的应用，其特征在于，所述R1和R2分别为甲基。
  12. 根据权利要求10所述的应用，其特征在于，所述抗血小板聚集药物包括：用于预防或治疗血液粘稠、脑梗塞、心肌梗塞、肺栓塞、下肢动脉血栓、下肢深静脉血栓的药物。
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