CN107434795B - 一种预防脂肪肝的化合物及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种预防脂肪肝的化合物及其制备方法和应用。本发明通过将茵陈蒿茎和叶干燥后粉碎后乙醇提取获得茵陈蒿浸膏，然后以抗氧化生物活性(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，最终分离得到陈蒿中分离有效成分，该成分对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；其浓度依赖性能降低被LPS及ATP所提高的NO含量；该有效成分显著抑制IL‑1β的释放，以及能降低IL‑1α的释放；并能够通过抑制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
本发明涉及一种药物，具体为一种预防脂肪肝的化合物及其制备方法和应用。
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％，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。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，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
本发明要解决的技术问题是克服目前脂肪肝发病率高，缺少毒副作用小且仅作用于肝脏的药物的缺陷，提供一种预防脂肪肝的化合物及其制备方法和应用。
为了解决上述技术问题，本发明提供了如下的技术方案：
一种预防脂肪肝的化合物，其结构如下所示：
该预防脂肪肝的化合物的制备方法，其特征在于，包括以下步骤：
1)、将茵陈蒿茎和叶干燥后粉碎，用乙醇在室温下浸泡提取三次，每次24h，过滤，合并滤液，利用旋转蒸发仪减压浓缩除去乙醇，得茵陈蒿浸膏；
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；
3)、依据生物活性分析和TLC板分析结果，选取其中的一个组分Fr.9，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5个阶段性分离组分Fr.9.1～9.5；
4)、选取组分Fr.9.4，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6个组分Fr.9.4.1～9.4.6；
5)、选取组分Fr.9.4.2，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，最终得到所述预防脂肪肝的化合物。
进一步的，所述步骤2)和步骤3)中石油醚和乙酸乙酯的体积比为100:0～0:100。
进一步的，所述步骤4)中石油醚和乙酸乙酯的体积比为100:10～100:100。
进一步的，所述步骤5)中甲醇和水的体积比为60:40。
本发明的预防脂肪肝的化合物可以用于制备治疗和预防脂肪肝药物中。
本发明通过将茵陈蒿茎和叶干燥后粉碎后乙醇提取获得茵陈蒿浸膏，然后以抗氧化生物活性(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，最终分离得到陈蒿中分离有效成分，该成分对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；其浓度依赖性能降低被LPS及ATP所提高的NO含量；该有效成分显著抑制IL-1β的释放，以及能降低IL-1α的释放；并能够通过抑制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是本发明的预防脂肪肝的化合物CCK-8法实验检测细胞存活率的结果；
图2是本发明的预防脂肪肝的化合物NO测定法的结果；
图3是本发明的预防脂肪肝的化合物ELISA法分别测定对IL-1α和IL-1β的浓度影响的结果。
具体实施方式
以下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
一种预防脂肪肝的化合物，其结构如下所示：
该预防脂肪肝的化合物的制备方法，包括以下步骤：
1)、将茵陈蒿茎和叶干燥后粉碎，用乙醇在室温下浸泡提取三次，每次24h，过滤，合并滤液，利用旋转蒸发仪减压浓缩除去乙醇，得茵陈蒿浸膏；
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，石油醚和乙酸乙酯的体积比为50：80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Fr.1～12；
3)、依据生物活性分析和TLC板分析结果，选取其中的一个组分Fr.9，利用硅胶柱色谱，以不同比例的石油醚和乙酸乙酯流动相，石油醚和乙酸乙酯的体积比为40：80进行进一步的梯度洗脱分离，又得到5个阶段性分离组分Fr.9.1～9.5；
4)、选取组分Fr.9.4，经硅胶柱色谱，以不同比例的石油醚和乙酸乙酯为流动相，石油醚和乙酸乙酯的体积比为100:10，进行梯度进一步的细分，合并相似流份，得到6个组分Fr.9.4.1～9.4.6；
5)、选取组分Fr.9.4.2，利用半制备高效液相色谱，以甲醇和水做为流动相系统，甲醇和水的体积比为60:40，进行等度洗脱，最终得到所述预防脂肪肝的化合物。
生物活性实验
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CCK-8法细胞存活率实验、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.CCK-8法实验检测细胞存活率：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24h，然后用不同浓度(3.125-100μM)的本发明所述预防脂肪肝的化合物处理24h，再加入CCK-8试剂，孵育1-4h，用酶标仪测其在450nm波长处的吸光度值，计算细胞存活率，以观察该化合物物对细胞的毒性作用，如图1所示，本发明所述预防脂肪肝的化合物对RawW264.7细胞无显著细胞毒性，同时可见本发明所述预防脂肪肝的化合物的剂量越高对活化Raw 264.7巨噬细胞的抑制率越高。
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24h后，用LPS(1.0μg/mL)预处理4h，再用ATP(3.0mM)处理30min，再加入不同浓度的本发明所述预防脂肪肝的化合物(3.125-100μM)处理24h后，取其上清液，用NO检测试剂(Griess试剂)盒进行混合后，用酶标仪测其在540nm波长处的吸光度值(图2)。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2所示，本发明所述预防脂肪肝的化合物浓度依赖性能降低被LPS及ATP所提高的NO含量。
3.ELISA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24h后，用LPS(1.0μg/mL)预处理4h，再用ATP(3.0mM)处理30min，再加入不同浓度的本发明所述预防脂肪肝的化合物(25.0-100.0μM)处理24h后，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3)。结果如图3所示，本发明所述预防脂肪肝的化合物显著抑制IL-1β的释放，以及能降低IL-1α的释放。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (1)
1.一种化合物在制备治疗和预防脂肪肝药物中的应用，其结构如下所示：
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