CN107129478B - 一种半萜内酯类化合物及其制备方法和应用 - Google Patents
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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 (1)
1.一种倍半萜内酯类化合物在制备治疗和预防脂肪肝疾病药物中的应用，所述倍半萜内酯类化合物的结构如式为：
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。
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