WO2013188996A1 - 线叶旋覆花内酯a在制备治疗心肌炎药物中的应用 - Google Patents

线叶旋覆花内酯a在制备治疗心肌炎药物中的应用 Download PDF

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WO2013188996A1
WO2013188996A1 PCT/CN2012/001306 CN2012001306W WO2013188996A1 WO 2013188996 A1 WO2013188996 A1 WO 2013188996A1 CN 2012001306 W CN2012001306 W CN 2012001306W WO 2013188996 A1 WO2013188996 A1 WO 2013188996A1
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inula
lactone
extract
petroleum ether
group
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French (fr)
Inventor
张卫东
单磊
金慧子
苏娟
李慧梁
沈云亨
徐希科
柳润辉
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Second Military Medical University SMMU
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Second Military Medical University SMMU
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Priority to EP12879465.8A priority Critical patent/EP2865380A4/en
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Priority to US14/576,027 priority patent/US9303006B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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

Definitions

  • the invention relates to a traditional Chinese medicine, in particular to the application of the linear inulin a lactone A in the preparation of a medicament for treating myocarditis.
  • Myocarditis is a localized or diffuse inflammation of myocardium that may occur either in the heart muscle or as part of a systemic disease. The cause is infection, physical and chemical factors, drugs, etc. The most common is viral myositis. In recent years, due to the widespread use of antibiotics, the rheumatic fever caused by streptococcal infection has gradually decreased, the incidence of rheumatic myocarditis has decreased significantly, and the incidence of viral myocarditis has increased.
  • Viral myocarditis can be caused by a variety of viral infections, among which Coxsackie B is the most common, and chickenpox and Epstein-Barr virus can also be caused. According to the study, about 5% of people infected with the virus can affect myocarditis in the heart. It can directly invade the myocardium after viral infection, or it can be caused by autoimmune reaction after viral infection. The pathology of viral myocarditis is different. Mild and localized lesions can be asymptomatic, no abnormalities in ECG, and no increase in erythrocyte sedimentation rate or myocardial enzymes.
  • Inula lineariifolia Turcz. (syn. Inula linariaefolia) is a perennial herb of the genus Asteraceae. It is a perennial herb commonly known as the narrow-leaved invertebrate flower, the leaf-leaf invertebrate flower, and the small flowering inflorescence. Widely produced in the northeast, north, central and eastern parts of China, such as Henan, Hebei and other provinces. It is also distributed in Mongolia, North Korea, Russia (Far East) and Japan. Born on hillsides, wasteland, roadsides, and river banks, it is very common.
  • Inverted flower is a flower head of invertebrate or large flower invertebrate, and plant whole grass (gold boiled grass) is also used for medicinal purposes.
  • plant whole grass gold boiled grass
  • it was also used as a spinach flower in some areas such as East China, and as a medicine for lowering the air, water, anti-inflammatory, and softening, it was once included in the 1963 Chinese Pharmacopoeia (Part I), but after the patient took it. There are reactions such as nausea and vomiting, which have been discontinued.
  • the present inventors conducted a systematic chemical composition study on the invertebrate flower, and isolated a large number of sesquiterpenoids, and published relevant research results [Journal of Natural Products (Li-Yue Nie, Hui-Zi Jin connector et Al, J Nat Prod, 2010, 73 (6): 1117-1120). Further research found that line leaf rotation Flake-Lactone A has the effect of treating myocarditis, and it is advisable to research and develop new drugs.
  • the technical problem to be solved by the present invention is to study the design of the extract of the invertebrate, the invertebrate, for the preparation of a medicament for treating myocarditis.
  • the invention provides the application of the wire-flower inflorescence A in the preparation of a medicament for treating myocarditis.
  • the line configuration is as follows:
  • the wire inflorescence lactone A of the present invention is prepared by the following method:
  • the stalks of the leaves are smashed and dried, and the extracts are refluxed with 8 ⁇ 20 times of 80 ⁇ 95% ethanol for 1 ⁇ 3 times, each time for 2 ⁇ 3 hours, the extracts are combined, and the extract is concentrated to a flooding under reduced pressure.
  • Cream, extract is equivalent to lml containing linear leaves of 0.8-1.2g, and the extract is diluted with 1 ⁇ 3 times of water, and then extracted with petroleum ether 0.5 ⁇ 2 times V/V for 3 ⁇ 5 times. A petroleum ether fraction is obtained.
  • the above petroleum ether fraction was subjected to silica gel column chromatography, and eluted with a gradient of 100: 0-1:1 petroleum ether/ethyl acetate system, and thin layer chromatography was carried out to collect the indole-containing lactone A.
  • the fractions were subjected to C 18 reverse phase column chromatography and eluted with methanol/water in a weight ratio of 50:100-70:100, and thin layer chromatography was carried out to obtain a pure indica lactone A pure product. .
  • the prepared inoculum A lactone A is tested for animal efficacy, and the effects of the compound on the life protection, life extension, body weight, heart index, pathological morphology, and cardiac function of the mice are observed.
  • the serum neutralizing antibody titer and the myocardial tissue virus titer were detected.
  • Intraperitoneal injection of ZO mg/kg 'd- 1 can effectively prevent the pathogenesis of mouse autoimmune myocarditis (EAM) model; intragastric administration of lOO mg/kg A 1 to EAM also has a certain effect. Therefore, the linear inulinate A can be used for the preparation of a medicament for treating myocarditis.
  • the medicament for treating myocarditis in the present invention is a pharmaceutical composition prepared from the active ingredient of the invertebrate, as an active ingredient, and a conventional pharmaceutical carrier.
  • the pharmaceutical composition may be a tablet, a dispersible tablet, a tablet, an orally disintegrating tablet, a sustained release tablet, a capsule, a soft capsule, a dropping pellet, a granule, an injection, a powder injection or an aerosol. Has a large clinical application value.
  • the obtained compound is firstly determined by mass spectrometry, molecular weight 366, molecular formula C 19 H 28 0 7 , and then subjected to nuclear magnetic resonance analysis to obtain carbon spectrum, hydrogen spectrum and two-dimensional spectrum data, for structural analysis, and known compounds in the line of leaves The data of ester A is consistent and confirmed).
  • Test sample Line-leaf inflorescence A compound (prepared in Example 1).
  • Virus Coxsackie B virus B3 Nancy strain (CVB3).
  • Viral infection dose 10ml 5G virus 0.2ml intraperitoneal infection of mice.
  • Positive control drug recombinant human interferon a-2a, Intermec, Shenyang Sansheng Pharmaceutical Co., Ltd., product batch number 20090708.
  • Preparation and route of administration of the drug solution The test sample is weighed: the wire-leaf inflorescence A compound is ground and suspended in a 0.5% CMC (carboxymethylcellulose) solution to prepare a solution of 10 mg/ml.
  • CMC carboxymethylcellulose
  • Oral administration Once a day, 0.5 ml each time, a total of 7 days.
  • High-dose group of drug treatment lOOmg/kg, 10 rats in each group, oral administration of cyclosporine A drug in the middle dose group 50mg/kg, 10 rats in each group, oral administration of indigo lactone A drug treatment
  • Test procedure After the mouse virus infection (except the normal control group), it was administered within 2 hours, and the total administration was continued for 7 days, and observed for 14 days. The incidence and number of deaths were recorded daily. Calculate the average number of days of survival, extend the life rate, and calculate the difference by p value.
  • the mouse Coxsackie virus titer was observed on a cell model with a 10% dilution of MEM containing 10% of the third antibody (observation of myocardial isolated virus-infected cytopathic).
  • the 1:X in the table indicates the serum dilution factor. The higher the dilution factor, the stronger the neutralizing ability of the antibody. The results showed that the linear inflorescence A increased the neutralizing antibody in CVB3-infected mice, and the dose was effective at 7 days. relationship.
  • Pig myocardium myosin PCM, 1 mg/ml; Sigma, St. Louis, MO
  • FCA Freund's complete adjuvant
  • FCA human M. tuberculosis H37Ra beads
  • 0.2 ml of the emulsion was injected subcutaneously into the bilateral groin and underarm of each mouse, and the same method was used for boosting once every 7 days after the initial immunization. Animals were grouped into groups of 6 each.
  • mice were given a daily infusion of cytosine lactones from the day of immunization for 21 days.
  • mice were sacrificed by cervical dislocation on day 21, and the heart was cut, weighed, photographed, pathologically sectioned (HE (hematoxylin-eosin) staining), and cytokine TNF-a was changed.
  • HE hematoxylin-eosin
  • HW/BW Heart to body weight ratio
  • the ratio of heart/body weight after the onset of murine myocarditis should be larger than that of the model group. If the drug is effective, the specific gravity is small, similar to the normal group.
  • the results showed that the HW/BW changes significantly after intraperitoneal injection of 20 mg/kg -1 of the linear inflorescence A (XX-3), as shown in Figure 1.
  • TNF-a plays an important role in the pathogenesis of EAM.
  • Blood was taken from the posterior venous plexus 21 days after the initial immunization. The blood was collected and placed in an anticoagulation tube. After centrifugation at 3000 r / min, serum samples were taken and stored at -20 ° C for use. The test results are shown in Fig. 4.
  • the expression of TNF-a in the serum was significantly increased compared with the control group 21 days after the initial immunization ("P ⁇ 0.01".
  • Model group in the control group W P ⁇ 0 01;. Comparison of 20mg / kg group and model group, # P ⁇ 0 05..
  • mice were sacrificed by neck removal, the heart was removed, and the remaining blood was removed.
  • Each heart was fixed with 10% formaldehyde, embedded in paraffin, sectioned intermittently, and stained with conventional HE, and observed under a microscope.
  • the myocardial cells in the foci showed degeneration, cytoplasmic red staining, nuclear disappearance, partial myolysis, interstitial widening, and diffuse infiltration of various inflammatory cells; no obvious abnormalities were observed in the control group. See Figure 5
  • Preparation method the wire leaf inulin a lactone A, lactose and corn starch are mixed, uniformly wetted with water, the wet mixture is sieved (16 mesh sieve) and dried (60 ° dried for 30 min), and then sieved (14 (mesh), magnesium stearate was added, and then the mixture was tableted, each tablet weighing 400 mg, and the linear inflorescence A content was 50 mg.
  • Preparation method Dissolve the inflorescence A and glucose in an appropriate amount of water for injection, filter the solution, and put it into an infusion bottle (100 ml per bottle) under aseptic conditions, each bottle containing a wire leaf rotation Overcoat A 6 mg.
  • Preparation method Solenoid A and mannitol are dissolved in an appropriate amount of water for injection, and the resulting solution is filtered, and placed in a vial (10 ml vial, 2 ml per bottle) under aseptic conditions, and lyophilized. Each line contains 60 mg of inflorescence A.

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Description

线叶旋覆花内酯 A在制备治疗心肌炎药物中的应用 技术领域
本发明涉及中药， 具体涉及线叶旋覆花内酯 A在制备治疗心肌炎药物中的 应用。 背景技术 心肌炎是心肌发生的局限或弥漫性炎症， 可原发于心肌， 也可是全身 性疾病的一部分。 病因有感染、 理化因素、 药物等， 最常见的是病毒性心 肌炎。 近年来， 由于抗生素的广泛应用， 因链球菌感染引起的风湿热逐渐减少， 风湿性心肌炎发病明显减少，而病毒性心肌炎发病却日益增多。病毒性心肌炎可 由多种病毒感染引起， 其中以柯萨基病毒 B最常见， 水痘、 EB病毒也可引起。 据研究， 约有 5%病毒感染者感染后可累及心脏发生心肌炎。 可为病毒感染后的 直接侵袭心肌，也可为病毒感染后的自身免疫反应所致。病毒性心肌炎的病变轻 重不一。 轻度、 局限性病变者可毫无症状， 心电图无异常表现， 血沉、 心肌酶也 无升高。重者则有明显症状， 出现心脏弥漫性扩大，心力衰竭， 以致有显著气急， 不能平卧； 有的严重心率失常， 以致发生反复晕厥， 甚至猝死。 目前， 西医对心 肌炎及心肌病的治疗效果还不理想.对病毒性心肌炎患者来说，没有特效的抗病 毒药物.对慢性心肌炎患者的心律失常疗效欠佳.中医中药在心肌炎的防治中积 累了丰富的经验， 对降低死亡率、 减少后遗症有明显的优势。
线叶旋覆花 [Inula lineariifolia Turcz. (syn. Inula linariaefolia) ] 是菊科旋覆花属植物， 多年生草本，俗名窄叶旋覆花、条叶旋覆花、小朵旋覆花。 广产于我国东北部、 北部、 中部和东部， 如河南、 河北等省。 也分布于蒙古、朝 鲜、 俄罗斯（远东地区）和日本。 生于山坡、 荒地、 路旁、 河岸， 极常见。 中药 旋覆花即为旋覆花或大花旋覆花等的头状花序， 而植物全草 (金沸草)亦供药用。 线叶旋覆花过去在华东等部分地区也作旋覆花使用，并且作为下气、行水、消炎、 软坚之药， 曾被收载于 1963年版中国药典（一部）， 但病人服后有恶心、 呕吐等 反应， 目前已停止使用。
本发明人对线叶旋覆花进行了系统的化学成分研究，分离得到大量倍半萜类 化合物， 相关研究结果发表了学术论文 [Journal of Natural Products (Li-Yue Nie, Hui-Zi Jin„ et al, J Nat Prod, 2010, 73 (6)： 1117-1120)。经进一步研究发现线叶旋 覆花内酯 A具有治疗心肌炎的功效， 宜研究开发新药。
发明内容
本发明所要解决的技术问题在于研究设计线叶旋覆花的提取物线叶旋覆花 内酯 A用于制备治疗心肌炎药物。
本发明提供了线叶旋覆花内酯 A在制备治疗心肌炎药物中的应用。 线 构式如下：
Figure imgf000004_0001
本发明所述线叶旋覆花内酯 A是通过下列方法制备得到的：
将线叶旋覆花千燥全草粉碎， 以 8~20倍重量的 80~95%乙醇回流提取 1~3 次， 每次 2~3小时， 合并提取液， 提取液减压浓缩成流浸膏， 流浸膏相当于 lml 含线叶旋覆花 0.8-1.2g， 流浸膏加 1~3倍重量的水稀释后， 以石油醚 0.5~2倍量 V/V萃取 3~5次， 得到石油醚部位。将上述石油醚部位应用硅胶柱层析， 以体积 比为 100: 0-1： 1的石油醚 /乙酸乙酯系统梯度洗脱， 薄层层析检测， 收集含线 叶旋覆花内酯 A的流分， 再经 C18反相柱层析， 以重量比为 50: 100-70： 100的 甲醇 /水进行洗脱， 薄层层析检测， 得线叶旋覆花内酯 A纯品。
本发明将制备得到的线叶旋覆花内酯 A进行动物药效试验， 观察该化合物对 结果小鼠生命的保护、 生命延长、 体重、 心指数、 病理形态学等的影响， 以及对 心功能、血清中和抗体滴度、心肌组织病毒滴度的检测， 结果提示线叶旋覆花内 酯 A具有一定的抗柯萨奇病毒的活性， 且有显著的量效关系。线叶旋覆花内酯 A 腹腔注射给药 ZO mg/kg'd-1能较好的阻止小鼠自身免疫性心肌炎 （EAM) 模型的 发病过程； 灌胃给药 lOO mg/kgA1对 EAM也具有一定的疗效。 因此， 线叶旋覆花 内酯 A可用于制备治疗心肌炎的药物。
本发明所述线叶旋覆花内酯 A在制备治疗心肌炎的药物为由线叶旋覆花内 酯 A作为活性成分与常规药用载体制成的药物组合物。所述药物组合物可以是片 剂、 分散片、 含片、 口崩片、 缓释片、 胶囊剂、 软胶囊剂、 滴丸、 颗粒剂、 注射 剂、 粉针剂或气雾剂等。 有较大的临床应用价值。 附图说明
图 1 腹腔注射线叶旋覆花内酯 A后小鼠 HW/BW变化
图 2 腹腔注射线叶旋覆花内酯 A后小鼠心脏形态学
图 3 灌胃线叶旋覆花内酯 A后小鼠心脏形态学
图 4腹腔注射线叶旋覆花内酯 A(XX-3)后 TNF-a变化
图 5 腹腔注射线叶旋覆花内酯 A后心肌 HE染色
具体实施方式
实施例 1 线叶旋覆花内酯 A的制备
将线叶旋覆花干燥全草 50Kg粉碎， 以 90%乙醇 750L回流提取 2次， 每次 2小时， 合并提取液， 提取液减压浓缩成流浸膏， 流浸膏相当于 lml含线叶旋覆 花 1.0g， 流浸膏加水 50L稀释后， 以石油醚每次 50L萃取 5次， 得到石油醚部 分。 将石油醚部分应用硅胶柱层析， 以体积比为 100: 0-1： 1的石油醚 /乙酸乙 酯系统梯度洗脱， 薄层层析检测， 收集含线叶旋覆花内酯 A的流分， 再经 C18 反相柱层析， 以重量比为 50: 100-70： 100的甲醇 /水进行洗脱， 薄层层析检测， 得线叶旋覆花内酯 A纯品 45.3g。
(得到的化合物先采用质谱测定分子量 366， 分子式 C19H2807, 再进行核磁 共振分析得到碳谱、氢谱以及二维谱数据， 进行结构解析， 与已知化合物线叶旋 覆花内酯 A的数据一致而确证 )。
实施例 2 线叶旋覆花内酯 A抗柯萨奇病毒小鼠体内药效试验
一， 材料和方法
受试样品： 线叶旋覆花内酯 A化合物 （实施例 1制得）。
病 毒： 柯萨奇 B组病毒 B3型 Nancy株 （CVB3)。
病毒感染剂量： 10LD5G病毒 0.2ml经腹腔感染小鼠。
动 物： Balb/c，雄性小鼠， 13-15g。 上海西普尔 -比克实验动物有限 公司， 动物合格证号： SCXX (沪） 2008-0016。
阳性对照药： 重组人干扰素 a-2a， 因特芬， 沈阳三生制药有限责任公司， 产品批号 20090708。 药液配制和给药途径： 称取受试样品： 线叶旋覆花内酯 A化合物， 研磨后 悬浮于 0.5%CMC (羧甲基纤维素） 溶液 配制成 10mg/ml的溶液。
口服给药。 一日一次， 每次 0.5 ml， 共给药 7天。
实验分组： 病毒感染组， 每组 10只， 给予 0.5%CMC溶液。
正常对照组， 每组 10只， 给予 0.5%CMC溶液。
阳性药物对照组，每组 10只，皮下注射重组人干扰素 a-2a悬浮于 0.5%CMC 溶液， 每只小鼠 1.5万单位， 每 2天注射一次， 共注射 4 次。
药物治疗高剂量组 lOOmg/kg, 每组 10只， 口服给予线叶旋覆花内酯 A 药物治疗中剂量组 50mg/kg， 每组 10只， 口服给予线叶旋覆花内酯 A 药物治疗低剂量组 10mg/kg， 每组 10只， 口服给予线叶旋覆花内酯 A 观测指标： 生命保护、 体重、 中和抗体、 病毒滴度， 心肌组织病理切片，心 脏超声。
试验过程： 小鼠病毒感染后 （除正常对照组外） 2小时内给药， 共给药连续 7天， 观察至 14天。 每天记录其发病率、 死亡数。 计算平均生存天数、 延长生 命率， 以 p值计算差别。
另设组， 每组 10只， 感染给药， 第 7天时称重， 取 4只检测心功能， 并杀 剖小鼠，采血检测中和抗体、取心脏称重计算心指数，并研磨心脏，用含 10%三 抗的 MEM液以 1:10稀释， 在细胞模型上检测小鼠柯萨奇病毒滴度 （观察心肌 分离的病毒感染的细胞病变）。
二、 实验结果
表 1 线叶旋覆花内酯 A对 CVB3感染小鼠生命的保护
Figure imgf000006_0001
与病毒组比较， *： P<0.05, **： P<0.01
结果显示用药组 100mg/kg， 50mg/kg， 10mg/kg， 都能延长生存时间， 延长 百分率分别达到 28.77%， 23.29%, 21.92%。 干扰素也能延长生存时间， 但是与 病毒对照组相比均未见显著性差异。 表 2 线叶旋覆花内酯 A对 CVB3感染小鼠第 7天时小鼠心肌病毒滴度测定.
Figure imgf000007_0001
-: 0%, ±:25% , +:50%, ++100%
结果显示病毒感染后的心肌可感染宿主细胞造成细胞病变，病毒组心肌中的 病毒诱导的细胞病变达到 90%。 线叶旋覆花内酯 A对柯萨奇病毒在心肌中的复 制有一定的抑制作用， 100mg/kg治疗组将细胞病变降低到 40%。 线叶旋覆花内酯 A对 CVB3病毒感染存活小鼠体重的影响
用药前 4天 8天 14天
M±SD P M±SD P M±SD P M±SD P 病毒组 14.65±0.59 1 12.84±0.92 0**
lOOmg/Kg 14.57±0.55 0.75 15.13±0.88 0.00021** 16.1±1.05 0** 18.21±0.86 Q**
50mg/Kg 14.58±0.49 0.77 15.14±0.68 0** 16.03±0.96 18.33±0.80 0** lOmg/Kg 14.59±0.71 0.84 15.23±1.01 15.59±0.89 0** 17.92±1.10 Q** 干扰素 14.53±0.59 0.65 14.80±1.50 0.0016** 15.82±0.47 0** 18.07±0.88 0** 正常组 14.65±0.56 16.77±0.75 19.06±0.77 22.65±1.03
所有组别都与正常组相比， **： P<0.01 , *： P<0.05
结果显示： 病毒感染后 4天， 小鼠体重明显下降， 在第 8天时病毒组小鼠全 部死亡， 给药组小鼠体重有所增加， 但与正常组相比仍有显著差异。 表 4 ： 线叶旋覆花内酯 A对 CVB3感染鼠血清中和抗体的作用
Figure imgf000008_0001
表中的 1:X表示血清稀释倍数， 稀释倍数越高表明抗体中和能力越强， 结 果显示：线叶旋覆花内酯 A提高 CVB3感染小鼠中和抗体， 7天时有一定的量效 关系。
实施例 3 线叶旋覆花内酯 A对小鼠自身免疫性心肌炎（EAM)模型的治疗作 用
3.1 EAM造模方法
猪心肌肌球蛋白 (PCM，1 mg/ml; Sigma, St. Louis, MO) (SIGMA),与等体积弗 氏完全佐剂 (FCA, Sigma St. Louis, MO), 充分乳化均匀， 混合成均质乳化液， 且其中人结核分枝杆菌 H37Ra珠 (Difco, Detroit, MI)终浓度为 5 mg/ml 。 于第 0 天取乳化液 0.2 ml (含抗原 200μ8) 于每只小鼠双侧腹股沟和腋下多点皮下注 射， 初次免疫后第 7天按相同方法加强免疫 1次。 动物分组， 每组 6只。
3.2给药方式
时间： 从免疫的当天开始即每天给予小鼠线叶旋覆花内酯 Α， 连续给药 21 天。
方式： 采取了两种给药方式
①腹腔注射。线叶旋覆花内酯 Α (实施例 1制得）溶于 PBS (磷酸盐缓冲液) (含有 5% DMSO (二甲基亚砜））。 设 4个浓度梯度：
• 2.5 mg/kg-d-1 (每次 2.5 mg/kg 每天一次）
參 5 mg/kg-d"1
• 10 mg/kg-d"1
參 20 mg/kg-d"1
②灌胃给药。 线叶旋覆花内酯 A悬于 0.5%的 CMC-Na (羧甲基纤维素钠） 中。 设 3个浓度梯度：
• 10 mg/kg-d"1
• 50 mg/kg-d"1
• 100 mg/kg-d"1
3.3 实验结果
小鼠于第 21天脱颈处死， 剪取心脏， 称重、 拍照、 病理切片 （HE (苏木精- 伊红） 染色）、 细胞因子 TNF-a 的变化。
( 1 ) 心脏与体重比 （HW/BW)
小鼠心肌炎发病后心脏 /体重的比值应比模型组偏大， 若药物有效， 则比重 小， 与正常组近似。 实验结果发现， 线叶旋覆花内酯 A ( XX-3 ) 腹腔注射 20 mg/kg -1后 HW/BW变化明显， 如图 1所示。
(2) 心脏形态学
小鼠 EAM发病后， 心脏由于炎症细胞的浸润， 而在形态上上表现为肿大、 苍白。腹腔注射线叶旋覆花内酯 A ( XX-3 )后各剂量给药组的动物心脏形态上， 均仍能观察到心脏的不痛程度的肿大等病变，但与模型组和溶媒对照组同病变比 较，其病变程度和波及范围均己呈现不同程度的改善，提示受试药对靶小鼠心肌 炎症具有一定疗效，其中以 SO mg/kg 1药效最佳。灌胃给药实验结果也显示线叶 旋覆花内酯 A对小鼠 EAM模型具有一定疗效，最佳药效剂量为 20 mg/kg，H。见图 2、 3。
(3 ) TNF-a变化
TNF-a作为前炎症因子， 在 EAM发病过程中起了较为重要的作用。 于初次 免疫后 21天后眼眶后静脉丛取血， 采集血液放置抗凝试管中， 于 3000 r/min离 心后留取血清标本， 冻存于 -20°C备用。 测试结果如图 4所示， 模型组在初次免疫 21天后， 血清中 TNF-a的表达与 对照组相比显著性提高 （"P < 0.01)。 腹腔注射给予线叶旋覆花内酯 A与模型组 比较， 血清中 TNF-a的水平均下降， 在 20 mg/kg 组有显著性差异 （#P < 0.05)。
模型组于空白组比较， WP<0. 01 ; 20mg/kg 组与模型组比较， #P<0. 05。
(4) 病理切片 （常规 HE染色）
脱颈处死小鼠， 取出心脏， 去除余血， 将每个心脏用 10%甲醛固定， 石蜡包 埋， 间断切片， 常规 HE染色， 显微镜下观察。 模型组可见灶内心肌细胞变性坏 死， 胞质红染、核消失，伴部分肌溶解， 间质增宽， 内有多种炎症细胞弥散浸润； 对照组未见明显异常。 见图 5
各剂量给药组的动物心脏组织切片中， 均仍能观察到心肌细胞局灶性坏死、 钙化或表现有不同程度的灶片状心肌炎症等病变，但与模型组和溶媒对照组同病 变比较，其病变程度和波及范围均已呈现不同程度的改善，提示线叶旋覆花内酯 A对小鼠心肌炎症具有一定疗效， 其中以腹腔注射 SO mg/kg 和灌胃口服 100 mg/kg 1药效最佳 （均为本实验给药的最高剂量）。 实施例 4
片剂： 线叶旋覆花内酯 A 50g
乳糖 280g
玉米淀粉 62g
硬脂酸镁 8g
制备方法： 将线叶旋覆花内酯 A、 乳糖和玉米淀粉混合， 用水均匀湿润， 把湿润后的混合物过筛（16目筛）并干燥（60° 烘干 30min)， 再过筛（14目）， 加入硬脂酸镁， 然后将混合物压片， 每片重 400mg， 线叶旋覆花内酯 A含量为 50mg。
实施例 5:
注射液： 线叶旋覆花内酯 A 6g
葡萄糖 50g
制备方法： 将线叶旋覆花内酯 A和葡萄糖溶解于适量的注射用水 中， 过滤所得溶液， 在无菌条件下装入输液瓶 （每瓶 100ml) 中， 每瓶含线叶旋 覆花内酯 A 6mg。
实施例 6:
注射用冻干粉针剂： 线叶旋覆花内酯 A 60g
甘露醇 240g
制备方法： 将线叶旋覆花内酯 A和甘露醇溶解于适量的注射用水中， 过滤 所得溶液， 在无菌条件下装入西林瓶（10ml西林瓶， 每瓶 2ml) 中， 冻干， 每支 含线叶旋覆花内酯 A 60mg。

Claims

权利 要 求
1、 线叶旋覆花内酯 A在制备治疗心肌炎药物中的应用， 其特征在于所述线 叶旋覆花内酯 A的结构式如下：
Figure imgf000012_0001
2、 根据权利要求 1所述的应用， 其特征在于， 所述线叶旋覆花内酯 A通过 下列方法制备得到- 将线叶旋覆花干燥全草粉碎， 以 8~20倍重量的 80~95%乙醇回流提取 1~3 次， 每次 2~3小时， 合并提取液， 提取液减压浓缩成流浸膏， 流浸膏相当于 lml 含线叶旋覆花 0.8-1.2g， 流浸膏加 1~3倍重量的水稀释后， 以石油醚 0.5~2倍量 V/V萃取 3~5次， 得到石油醚部分； 将上述石油醚部分应用硅胶柱层析， 以体积 比为 100: 0-1： 1的石油醚 /乙酸乙酯系统梯度洗脱， 薄层层析检测， 收集含线 叶旋覆花内酯 A的流分， 再经 C18反相柱层析， 以重量比为 50: 100-70： 100的 甲醇 /水进行洗脱， 薄层层析检测， 得线叶旋覆花内酯 A纯品。
3、 根据权利要求 1所述的应用， 其特征在于所述药物为由线叶旋覆花内酯 A 作为活性成分与药用载体制成的药物组合物。
4、根据权利要求 3所述的应用，其特征在于所述药物组合物为片剂、分散片、 含片、 口崩片、 缓释片、 胶囊剂、 软胶囊剂、 滴丸、 颗粒剂、 注射剂、 粉针剂或 气雾剂。
PCT/CN2012/001306 2012-06-21 2012-09-25 线叶旋覆花内酯a在制备治疗心肌炎药物中的应用 Ceased WO2013188996A1 (zh)

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