CN107129478A - 一种半萜内酯类化合物及其制备方法和应用 - Google Patents
一种半萜内酯类化合物及其制备方法和应用 Download PDFInfo
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- 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
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Abstract
本发明公开了一种倍半萜内酯类化合物Achillin,该化合物提取自茵陈蒿,对RAW264.7细胞无显著细胞毒性,对活化RAW 264.7巨噬细胞具有浓度依赖性的抑制作用,能够提升被LPS抑制而下降的RAW 264.7细胞生长率;Achillin浓度依赖性能降低被LPS所提高的NO含量,也能够小幅度降低LPS+ATP所提高的NO含量;Achillin显著抑制IL‑1β的释放,以及能降低IL‑1α的释放。Achillin能够抑制LPS和ATP刺激的炎症反应来抑制肝星状细胞的活化,有望用于制备治疗脂肪肝疾病药物。
Description
技术领域
本发明涉及涉医药技术领域,涉及一种半萜内酯类化合物Achillin在制备保护肝脏和 预防脂肪肝疾病药物中的应用。
背景技术
脂肪肝是指由于各种原因引起的肝细胞内脂肪堆积过多的病变。它正严重威胁国人的健 康,在我国的发病率已升高到20%[Rey JW.et al.Prol2Ala polymorphism ofthe peroxisome proliferator-activated receptor y2in patients with fatty liverdiseases. Gastroenterol,2010,16(46):5830-3 7.],成为仅次于病毒性肝炎的第二大肝病。脂肪肝 是一种常见的临床现象,而非一种独立的疾病,其形成疾病的病因诸多,包括饮酒过量、高 热量饮食、肥胖,药物等。临床上将其分为酒精性脂肪肝和非酒精性脂肪肝[井源,韩婷, 吴静等。酒精性脂肪肝研究现状与进展。胃肠病学和肝病学杂志,2008,17(10):862-866.]。 一般而言,脂肪肝属可逆性疾病,早期诊断并及时治疗常可恢复正常。目前,我国的脂肪肝 患者高达八千多万人,其中酒精性脂肪肝的比例占到23.0%以上。酒精等因素引起的肝病及 相关性疾病在迅速增加,还由于酒精性肝炎、肝纤维化和肝硬化疾病的严重危害性和难治愈 性,使得酒精性脂肪肝的防治研究受到了广泛的重视。
由于酒精性脂肪肝的发病机制尚未完全明确,目前针对酒精性脂肪肝尚未找到切实有效 的靶向治疗手段及防治的特效药。水飞蓟素作为一种保肝药物已应用于临床,其对于包括酒 精性肝病在内的很多肝脏疾病都有较好的临床效果。但由于其水溶性差,口服后生物利用率 低,使得其应用受限[J.Pepping,Milk thistle:Silybum marianum,Am.J.Health-System Pharm,1999,56:1195-97.],2010年Hepatology杂志发表的美国肝病学会与美国胃肠病 学会联合制定的酒精性肝病诊断与治疗指南中指出,水飞蓟素无论是对急性还是慢性酒精性 肝病患者都没有产生令人信服的效果,仅限用于酒精性肝病的临床试验。而对于其他防治酒 精性脂肪肝的药物尚处于初级阶段。因此,对抗酒精性脂肪肝药物的研究和开发变得尤为重 要。
治疗脂肪肝疾病的理想药物应是毒副作用小且仅作用于肝脏,通过某种调控作用抑制胶 原体等,而对机体无其他不良反应。因此,为了从天然植物中寻找新型的保肝护肝药物,筛 选出朝医学中医学典籍《韩国本草图鉴》记载且民间一直传承下来的对肝胆具有良好疗效的 药用植物茵陈蒿。茵陈蒿(Artemisia capillaries.)为菊科、蒿属植物,茵陈蒿始载于《神 农本草经》茵陈蒿的嫩苗。《韩国本草图鉴》记载:茵陈蒿主治急慢性肝炎、肝硬化、肝癌、 胆囊炎、胆囊结石等。具有利胆作用,促进胆汁分泌和利尿排泄,清热解毒,保护肝细胞和 促进肝细胞的再生,降血压,血管扩张,降胆固醇,抗菌作用。
随着化学生物学、蛋白质化学、生物技术的发展及脂肪肝疾病形成机制的认识,寻求有 效的保肝护肝等治疗脂肪肝疾病的药物将会有突破性的进展。
发明内容
本发明的目的在于提供一种半萜内酯类化合物及其制备方法和应用,其化学式如下:
本发明的倍半萜内酯类化合物提取自茵陈蒿,其提取方法如下:
一、提取
将干燥的植物茵陈蒿茎和叶,经粉碎后用乙醇在室温条件下浸泡提取三次,每次24h, 过滤,合并提取液,利用旋转蒸发仪减压浓缩除去乙醇溶剂,得茵陈蒿浸膏。
二、分离
以抗氧化生物活性(DPPH自由基清除)为指导,再以多步骤的正-反相色谱交叉分离手段 相结合,对植物茵陈蒿的化学成分进行阶段性的生物活性导向分离。上面得到的茵陈蒿乙 醇提取物,经硅胶(SiO2)柱色谱,以不同比例的石油醚(Pet)和乙酸乙酯(EA)为流动相,进行 梯度(Pet:EA=100:0~0:100)洗脱分离,利用薄层色谱(TLC)板跟踪检测,合并相似部分,得 到12个粗分离组分(Fr.1~12)。依据生物活性分析和TLC板分析结果,选取其中TLC板上 清晰以及生物活性较强的组分Fr.8,利用硅胶柱色谱,以不同比例的石油醚和乙酸乙酯流动 相,进行进一步的(梯度100:0~100:20)洗脱分离,又得到3个阶段性分离组分(Fr.8.1~8.3); 选取组分Fr.8.2,经硅胶柱色谱,以不同比例的石油醚和乙酸乙酯为流动相,进行(梯度100:0 ~100:20)进一步的细分,合并相似流份,得到4个组分(Fr.8.2.1~8.2.4);选取组分Fr.8.2.2, 利用半制备高效液相色谱(ODS-A),以甲醇和水做为流动相系统,进行等度(v/v%,甲醇:水= 80:20)洗脱,最终得到化合物Achillin。
本发明的倍半萜内酯类化合物Achillin对RAW264.7细胞无显著细胞毒性,对活化RAW 264.7巨噬细胞具有浓度依赖性的抑制作用,能够提升被LPS抑制而下降的RAW 264.7细胞 生长率;Achillin浓度依赖性能降低被LPS所提高的NO含量,也能够小幅度降低LPS+ATP 所提高的NO含量;Achillin显著抑制IL-1β的释放,以及能降低IL-1α的释放。Achillin能 够抑制LPS和ATP刺激的炎症反应来抑制肝星状细胞的活化,有望用于制备治疗脂肪肝疾 病药物。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一 起用于解释本发明,并不构成对本发明的限制。在附图中:
图1是本发明的倍半萜内酯类化合物的分离流程图。
图2是MTT细胞存活率实验结果图;
图3是NO测定法实验结果图;
图4是ELISA法实验结果图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅 用于说明和解释本发明,并不用于限定本发明。
实施例
提取分离实验
1、提取
将干燥的植物茵陈蒿茎和叶,经粉碎后用乙醇在室温条件下浸泡提取三次,每次24h, 过滤,合并提取液,利用旋转蒸发仪减压浓缩除去乙醇溶剂,得茵陈蒿浸膏。
2.分离
以抗氧化生物活性(DPPH自由基清除)为指导,再以多步骤的正-反相色谱交叉分离手段 相结合,对植物茵陈蒿的化学成分进行阶段性的生物活性导向分离。上面得到的茵陈蒿乙 醇提取物,经硅胶(SiO2)柱色谱,以不同比例的石油醚(Pet)和乙酸乙酯(EA)为流动相,进行 梯度(Pet:EA=100:0~0:100)洗脱分离,利用薄层色谱(TLC)板跟踪检测,合并相似部分,得 到12个粗分离组分(Fr.1~12)。依据生物活性分析和TLC板分析结果,选取其中的组分Fr.8, 利用硅胶柱色谱,以不同比例的石油醚和乙酸乙酯流动相,进行进一步的(梯度100:0~100:20) 洗脱分离,又得到3个阶段性分离组分(Fr.8.1~8.3);选取组分Fr.8.2,经硅胶柱色谱,以不 同比例的石油醚和乙酸乙酯为流动相,进行(梯度100:0~100:20)进一步的细分,合并相似流 份,得到4个组分(Fr.8.2.1~8.2.4);选取组分Fr.8.2.2,利用半制备高效液相色谱(ODS-A), 以甲醇和水做为流动相系统,进行等度(v/v%,甲醇:水=80:20)洗脱,最终得到化合物 Achillin(分离流程如图1所示)。
生物活性实验
有研究表明,ATP介导的P2X7受体在肝纤维化的治疗过程当中,是一种新型的治疗靶 点,脂多糖(LPS)和促炎因子的作用可以与之相关。本发明从MTT细胞存活率实验、NO测定 法、ELISA法分别测定了与炎症小体相关的细胞毒性、NO含量和IL-1β和IL-1α的变化。
1.MTT细胞存活率实验:取小鼠巨噬细胞株RAW 264.7巨噬细胞以1.0×105/孔的密 度接种于96孔板上,孵育24小时,然后用不同浓度(0.78-100μM)的Achillin处理1小时,再加入LPS(1.0μg/mL)继续孵育24小时。同时,加入用PBS配制成5.0mg/mL浓度的MTT 溶液10.0μL,孵育3小时,吸走溶液,加入DMSO处理后,在酶标仪上测定540nm波长处 的吸光度值,以观察药物对细胞的毒性作用(图2)。图2中所示,Achillin对RAW264.7细 胞无显著细胞毒性,观察不同浓度(0.78-100μM)的Achillin对活化RAW 264.7巨噬细胞的 抑制率,可见Achillin的剂量越高呈现出越高的对活化RAW 264.7巨噬细胞的抑制率。同 时,能够提升被LPS抑制而下降的RAW 264.7细胞生长率。
2.NO测定法:取小鼠巨噬细胞株RAW 264.7巨噬细胞以1.0×105/孔的密度接种于96孔板上,孵育24小时。然后以不同浓度的Achillin(0.78-100μM)处理1小时,再加入LPS(1.0μg/mL)继续孵育24小时。或以不同浓度的Achillin(0.78-100μM)处理1小时, 然后再用LPS(1.0μg/mL)处理后孵育4小时,最后用ATP处理30分钟。培养基中的一氧化 氮含量采用格里斯(Griess)试剂测定。100.0μL上清液与100.0μL格里斯试剂混合后, 在室温孵育15分钟,再在540nm波长处测定吸光度(图3)。每个样品中的一氧化氮浓度, 用亚硝酸钠的梯度浓度标准溶液计算回归方程来求得。如图3所示,Achillin浓度依赖性能 降低被LPS所提高的NO含量,并且也能够小幅度降低LPS+ATP所提高的NO含量。
3.ELISA法:将小鼠巨噬细胞株RAW264.7巨噬细胞,用不同浓度的 Achillin(0.78-100μM)处理1小时,然后用LPS(1.0μg/mL)处理后孵育4小 时,最后用ATP处理30分钟。取处理后的上清液,采用IL-1αELISA试剂盒和 IL-1βELISA试剂盒分别测定其IL-1α和IL-1β的浓度(图4)。结果如图4 所示,Achillin显著抑制IL-1β的释放,以及能降低IL-1α的释放。
结果表明,Achillin对RAW264.7细胞无显著细胞毒性,对活化RAW 264.7巨噬细胞具 有浓度依赖性的抑制作用,能够提升被LPS抑制而下降的RAW 264.7细胞生长率;Achillin 浓度依赖性能降低被LPS所提高的NO含量,也能够小幅度降低LPS+ATP所提高的NO含 量;Achillin显著抑制IL-1β的释放,以及能降低IL-1α的释放。Achillin能够抑制LPS和ATP 刺激的炎症反应来抑制肝星状细胞的活化,有望用于制备治疗脂肪肝疾病药物。
Claims (4)
1.一种倍半萜内酯类化合物,其特征在于,其结构如式为:
2.如权利要求1所述的倍半萜内酯类化合物,其特征在于,所述的倍半萜内酯类化合物提取自茵陈蒿。
3.一种如权利要求1所述的倍半萜内酯类化合物的制备方法,其特征在于,包括以下步骤:
1)、将干燥的茵陈蒿茎和叶,粉碎后用乙醇在室温条件下浸泡提取三次,每次24h,过滤,合并提取液,利用旋转蒸发仪减压浓缩除去乙醇溶剂,得茵陈蒿浸膏;
2)、以DPPH自由基清除为指导,再以多步骤的正-反相色谱交叉分离手段相结合,对植物茵陈蒿的化学成分进行阶段性的生物活性导向分离,得到茵陈蒿乙醇提取物;
3)、经硅胶(SiO2)柱色谱,以不同比例的石油醚(Pet)和乙酸乙酯(EA)为流动相,进行梯度洗脱分离,利用薄层色谱(TLC)板跟踪检测,合并相似部分得到12个粗分离组分;
4)、依据生物活性分析和TLC板分析结果,选取其中的一个组分,利用硅胶柱色谱,以不同比例的石油醚和乙酸乙酯流动相,进行进一步的梯度洗脱分离,又得到3个阶段性分离组分;
5)、选取其中的一个组分,经硅胶柱色谱,以不同比例的石油醚和乙酸乙酯为流动相,进行梯度进一步的细分,合并相似流份,得到4个组分;
6)、选取其中的一个组分,利用半制备高效液相色谱,以甲醇和水做为流动相系统,进行等度洗脱,最终得到所述化合物。
4.如权利要求1所述的倍半萜内酯类化合物,在制备保护和预防脂肪肝疾病药物中的应用。
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CN115010720A (zh) * | 2022-06-02 | 2022-09-06 | 中国科学院昆明植物研究所 | 中甸艾中倍半萜二聚体及其药物组合物与其制备方法和应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101279964A (zh) * | 2008-02-29 | 2008-10-08 | 中国药科大学 | 愈创木烷型倍半萜、其制备方法及其医药用途 |
WO2011080721A2 (en) * | 2010-01-01 | 2011-07-07 | Himalaya Global Holdings Ltd. | A herbal composition as hepatoprotective and treatment for liver disorders |
CN103804334A (zh) * | 2014-01-08 | 2014-05-21 | 石河子大学 | 从毛菊苣中提取c15h18o5方法及其用途 |
CN106344558A (zh) * | 2015-07-17 | 2017-01-25 | 中山大学 | 含野菊花提取物的降血脂药物及野菊花提取物的制备方法 |
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WO2011080721A2 (en) * | 2010-01-01 | 2011-07-07 | Himalaya Global Holdings Ltd. | A herbal composition as hepatoprotective and treatment for liver disorders |
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CN115010720A (zh) * | 2022-06-02 | 2022-09-06 | 中国科学院昆明植物研究所 | 中甸艾中倍半萜二聚体及其药物组合物与其制备方法和应用 |
CN115010720B (zh) * | 2022-06-02 | 2023-08-11 | 中国科学院昆明植物研究所 | 中甸艾中倍半萜二聚体及其药物组合物与其制备方法和应用 |
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