CN115177659B - 一种短序蒲桃叶提取物及其制备方法和应用 - Google Patents
一种短序蒲桃叶提取物及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及天然药物化学技术领域，具体涉及一种短序蒲桃叶提取物及其制备方法和应用。本发明的制备方法包括如下步骤：取干燥的短序蒲桃叶，用乙醇溶液提取，过滤后合并提取液，经减压浓缩得到短序蒲桃叶醇提物总浸膏；将短序蒲桃叶醇提物总浸膏用乙酸乙酯萃取，得到短序蒲桃叶提取物。本发明通过LPS/ox‑LDL诱导的RAW264.7泡沫细胞模型，发现本发明制备的短序蒲桃叶提取物具有显著的抗炎和降脂作用，可以用于制备抗炎药物和/或降脂药物。而且，本发明制备的短序蒲桃叶提取物可以减少RAW264.7巨噬细胞源性泡沫细胞的脂质蓄积，由此提示，本发明制备的短序蒲桃叶提取物可用于制备预防和/或治疗动脉粥样硬化的药物。
Description
技术领域
本发明涉及天然药物化学技术领域，具体涉及一种短序蒲桃叶提取物及其制备方法和应用。
背景技术
动脉粥样硬化(Atherosclerosis,AS)是一种慢性、炎症性心血管疾病，可导致心脏病、中风，甚至死亡，已成为严重威胁人类健康的公共卫生问题。在AS进展的早期阶段，泡沫细胞的形成是关键因素。泡沫细胞的形成源于巨噬细胞，由于胆固醇过度积累，巨噬细胞中的氧化低密度脂蛋白(ox-LDL)的流入、酯化和流出不平衡导致巨噬细胞逐步泡沫化。同时，炎症是影响动脉粥样硬化的另一个重要因素。泡沫细胞分泌的多种细胞因子包括炎症趋化因子，激活了内皮细胞和进一步募集更多的炎症细胞进入内膜。同时，单核细胞被驱使不断分化成巨噬细胞，并吸收大量的ox-LDL形成泡沫细胞，逐渐在血管中积累，进一步加速了动脉粥样硬化的形成。
短序蒲桃(Syzygium brachythyrsum Merr.et Perry)为桃金娘科(Myrtaceae)蒲桃属(Syzygium)常绿小乔木，是中国岭南和西南地区历史悠久且非常重要的功能性草药茶和民间药材，其果及叶以野冬青果之名入药(详见《云南中草药》)。据《中国植物志》中记载：短序蒲桃具有润肺平喘的功效，主要用于治疗过敏性哮喘、肺结核和感冒性哮喘等。发明人前期证实短序蒲桃叶中含有大量化合物，包括黄酮类、单宁类、酚酸类、花色苷类、萜类等(详见Jiaqi Qiu,Xuelian Chen,Pulin Liang,et al.Integrating approach todiscover novel bergenin derivatives and phenolics with antioxidant and anti-inflammatory activities from bio-active fraction of Syzygium brachythyrsum[J].Arabian Journal of Chemistry,2021,15,103507)。流行病学研究表明，普通人群和有心血管疾病风险的患者从饮食中摄取富含多酚、三萜类等化合物的天然产品可以减少心血管事件。因此，短序蒲桃叶在减少心血管疾病方面的药用价值有待进一步挖掘。
发明内容
本发明的第一个目的在于提供一种短序蒲桃叶提取物的制备方法，本发明的第二个目的在于提供由该制备方法制得的短序蒲桃叶提取物，本发明的第三个目的在于提供该短序蒲桃叶提取物的应用。
本发明通过以下技术方案实现：
根据本发明的第一个方面，提供了一种短序蒲桃叶提取物的制备方法，包括如下步骤：
取干燥的短序蒲桃叶，用乙醇溶液提取，过滤后合并提取液，经减压浓缩得到短序蒲桃叶醇提物总浸膏；所用乙醇溶液为无水乙醇的体积分数大于50％的乙醇水溶液；
将短序蒲桃叶醇提物总浸膏用乙酸乙酯萃取，得到短序蒲桃叶提取物。
在一些实施方式中，在用乙醇溶液提取之前，将短序蒲桃叶过4目筛进行粗粉碎。
在一些实施方式中，短序蒲桃叶与乙醇溶液的用量比为lg：5mL～1g：15mL。
在一些实施方式中，提取为加热回流提取，提取次数为4次，每次1h。
在一些实施方式中，萃取的方法为：将短序蒲桃叶醇提物总浸膏用乙酸乙酯萃取3次，合并萃取液，减压浓缩后得到短序蒲桃叶提取物，其中，乙酸乙酯与短序蒲桃叶醇提物总浸膏的用量比为1mL:1g。
根据本发明的第二个方面，提供了上述的制备方法制得的短序蒲桃叶提取物。
根据本发明的第三个方面，提供了上述的短序蒲桃叶提取物在制备具有抗炎和/或降脂作用的药物或食品中的应用。
根据本发明的第四个方面，提供了上述的短序蒲桃叶提取物在制备预防和/或治疗动脉粥样硬化的药物中的应用。
本发明的有益效果包括：
本发明通过LPS/ox-LDL诱导的RAW264.7泡沫细胞模型，发现本发明制备的短序蒲桃叶提取物具有显著的抗炎和降脂作用，可以用于制备具有抗炎和/或降脂作用的药物或食品。而且，本发明制备的短序蒲桃叶提取物可以减少RAW264.7巨噬细胞源性泡沫细胞的脂质蓄积，由此提示，本发明制备的短序蒲桃叶提取物可用于制备预防和/或治疗动脉粥样硬化的药物。
附图说明
图1为本发明的细胞实验中短序蒲桃叶提取物对RAW264.7巨噬细胞的活性影响。
图2为本发明的细胞实验中短序蒲桃叶提取物对LPS诱导的炎症因子释放的影响。
图3为本发明的细胞实验中油红O染色法检测短序蒲桃叶提取物对ox-LDL诱导的巨噬细胞脂质累积的影响。
图4为本发明的细胞实验中荧光显微镜下观察短序蒲桃叶提取物对巨噬细胞中Dil-ox-LDL荧光累积的影响。
图5为本发明的细胞实验中荧光NBD胆固醇检测短序蒲桃叶提取物对巨噬细胞脂质外排率的影响。##p<0.01是与空白对照组相比，*p<0.05和**p<0.01是与ox-LDL组相比。
图6为本发明的细胞实验中RT-PCR测定短序蒲桃叶提取物对巨噬细胞中脂质摄取受体CD36和外排受体ABCG1,SRB1的mRNA表达影响。##p<0.01是与空白对照组相比，*p<0.05和**p<0.01是与ox-LDL组相比。
具体实施方式
下面结合附图对本发明作进一步详细的说明。若无特殊说明，以下试验所用试剂均为市购，未做特殊说明的实验方法都是采用本领域已知的常规方法。
实施例1
本实施例的短序蒲桃叶提取物的制备方法，包括如下步骤：
(1)醇提步骤：称取干燥的短序蒲桃叶10kg，过4目筛进行粗粉碎，然后用10倍量(100L)的无水乙醇浸提4次，每次浸提时间为1h，过滤后合并提取液，经减压浓缩得到短序蒲桃叶醇提物总浸膏约500g。
(2)萃取步骤：将步骤(1)得到的短序蒲桃叶醇提物总浸膏用等体积(约500mL)的乙酸乙酯萃取3次，合并萃取液，减压浓缩后得到乙酸乙酯部位200g，即为短序蒲桃叶提取物(brachythyrsum extraction，简称BE)。
下面，利用实施例1制备得到的短序蒲桃叶提取物(BE)进行细胞实验，以探究短序蒲桃叶提取物的药理作用。
1、短序蒲桃叶提取物对RAW264.7巨噬细胞的活性实验
将短序蒲桃叶提取物溶解于二甲基亚砜(DMSO)中，然后用DMEM培养基稀释至各工作浓度。将密度为2×104个/孔的RAW264.7细胞接种于96孔板中，24h后弃去培养基，加入各浓度短序蒲桃叶提取物共同孵育24h，然后进行MTT实验检测细胞毒性。所有实验进行3次，数据显示平均值±SD。
结果如图1所示，25μg/mL浓度以下的短序蒲桃叶提取物对RAW264.7细胞没有毒性，因此选择了6.25，12.5，25μg/mL浓度进行后续实验。
2、短序蒲桃叶提取物对LPS诱导的RAW264.7细胞炎症反应实验
按照2×105个/孔的细胞密度将RAW264.7细胞接种于24孔板中，于37℃细胞培养箱中培养24h后，给药组加入含有不同浓度的短序蒲桃叶提取物培养基，阳性对照组加入1μg/mL的地塞米松，空白对照组加入等量新鲜的细胞完全培养基，除空白对照组外，给药组和阳性对照组均加入2μg/mL的LPS。24h后吸取上清液，用Griess法测定NO含量。所有实验进行3次，数据显示平均值±SD。
结果如图2所示，6.25，12.5，25μg/mL的短序蒲桃叶提取物能剂量依赖性地降低泡沫细胞中NO释放。
3、短序蒲桃叶提取物对泡沫细胞脂质堆积的影响实验
(1)以1.2×105个/mL的密度将巨噬细胞RAW264.7加入24孔板，37℃、5％CO2条件下放在细胞培养箱中24h培养；给药组分别加入梯度浓度的药物，同时除空白组外，每孔分别加入终浓度为80μg/mL的ox-LDL，继续在细胞培养箱中培养24h；用多聚甲醛固定细胞三十分钟，加入配好的油红染液染色20-30min，弃去染液，用PBS洗涤数次，置于倒置显微镜下观察脂质积累情况并拍照。
结果如图3所示，短序蒲桃叶提取物(6.25，12.5，25μg/mL)能不同程度地减少细胞中的红色脂滴。
(2)将RAW264.7细胞按1×105个/孔的细胞密度接种于24孔板中，24h后给药组加入含有不同浓度的短序蒲桃叶提取物和20μg/mL Dil-ox-LDL，模型组加入20μg/mL Dil-ox-LDL，空白组加入等量新鲜的细胞完全培养基，培养3h后弃去培养液，PBS洗三遍，于荧光显微镜下观察。同样操作步骤，PBS洗三遍后避光收集细胞在流式细胞仪下检测Dil-ox-LDL的荧光强度。
结果如图4所示，Dil-ox-LDL实验更直观地显示短序蒲桃叶提取物(6.25，12.5，25μg/mL)能够减少RAW264.7细胞摄取ox-LDL。
由此可见，短序蒲桃叶提取物可以减少巨噬细胞对ox-LDL的摄取，抑制巨噬细胞形成泡沫细胞。
4、短序蒲桃叶提取物对泡沫细胞胆固醇外排的影响实验
将RAW264.7细胞按5×105个/孔的细胞密度接种于12孔板中，24小时后加入80μg/mL ox-LDL和5μg/mL的25-NBD荧光胆固醇孵育24h，然后按各给药组加入含有不同浓度的短序蒲桃叶提取物和50μg/mL HDL的无酚红培养基继续培养24小时。吸上清液，用PBS冲洗细胞三遍，用RIPA裂解细胞，收集细胞裂解液，于激发波长469nm，发射波长538nm处检测上清液和裂解液荧光强度，NBD-胆固醇外流率＝FI上清液/(FI上清液+FI细胞裂解液)×100％。所有实验进行3次，数据显示平均值±SD。
结果如图5所示，短序蒲桃叶提取物(6.25，12.5，25μg/mL)可以促进泡沫细胞内胆固醇外排率。
5、RT-PCR测定短序蒲桃叶提取物对巨噬细胞中脂质摄取受体CD36和外排受体ABCG1,SRB1的mRNA表达
将RAW264.7细胞按照1×106个/孔的细胞密度将对数生长期的RAW264.7细胞接种于6孔板中，于37℃细胞培养箱中培养24h后，按分组给药。24h后用TRIzol试剂从巨噬细胞中提取总RNA。测定完RNA纯度及浓度后使用罗氏cDNA逆转录试剂盒合成cDNA，然后使用Roche FastStart Universal SYBR Green Master在real-time PCR System 7500(Applied Biosystems)中进行PCR实验(引物序列见表1)。检测各组细胞中CD36,SRB1和ABCG1的表达。所有实验进行3次，数据显示平均值±SD。
表1目的基因的引物序列
结果如图6所示，短序蒲桃叶提取物(6.25，12.5，25μg/mL)减少了ox-LDL诱导的CD36的mRNA表达，促进了SRB1/ABCG1的mRNA表达，说明短序蒲桃叶提取物可以通过抑制CD36 mRNA表达来减少细胞脂质摄取，以及通过促进ABCG1/SRB1的mRNA表达来促进泡沫细胞外排胆固醇。
综上，本发明通过LPS/ox-LDL诱导的RAW264.7泡沫细胞模型，发现本发明制备的短序蒲桃叶提取物具有显著的抗炎和降脂作用，可以用于制备具有抗炎和/或降脂作用的药物或食品。而且，本发明制备的短序蒲桃叶提取物可以减少RAW264.7巨噬细胞源性泡沫细胞的脂质蓄积，由此提示，本发明制备的短序蒲桃叶提取物可用于制备预防和/或治疗动脉粥样硬化的药物。
以上所述的仅是本发明的一些实施方式。对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，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。
Claims (5)
1.短序蒲桃叶提取物在制备具有降脂作用的药物或食品中的应用，其中，短序蒲桃叶提取物的制备方法，包括如下步骤：
取干燥的短序蒲桃叶，用无水乙醇提取，提取次数为4次，每次1h，过滤后合并提取液，经减压浓缩得到短序蒲桃叶醇提物总浸膏；
将短序蒲桃叶醇提物总浸膏用乙酸乙酯萃取，得到短序蒲桃叶提取物。
2.根据权利要求1所述的应用，其特征在于，在用无水乙醇提取之前，将短序蒲桃叶过4目筛进行粗粉碎。
3.根据权利要求1或2所述的应用，其特征在于，所述短序蒲桃叶与无水乙醇的用量比为lg：5mL~1g：15mL。
4.根据权利要求1或2所述的应用，其特征在于，所述的提取为加热回流提取。
5.根据权利要求1或2所述的应用，其特征在于，所述萃取的方法为：将短序蒲桃叶醇提物总浸膏用乙酸乙酯萃取3次，合并萃取液，减压浓缩后得到短序蒲桃叶提取物，所述乙酸乙酯与短序蒲桃叶醇提物总浸膏的用量比为1mL:1g。
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| Integrating approach to discover novel bergenin derivatives and phenolics with antioxidant and anti-inflammatory activities from bio-active fraction of Syzygium brachythyrsum;Jiaqi Qiu等;Arabian Journal of Chemistry;第15卷;1-11 * |
| Jiaqi Qiu等.Integrating approach to discover novel bergenin derivatives and phenolics with antioxidant and anti-inflammatory activities from bio-active fraction of Syzygium brachythyrsum.Arabian Journal of Chemistry.2021,第15卷1-11. * |
| 基于UHPLC-Q-Exactive-MS技术的短序蒲桃叶岩白菜素新衍生物的快速发现;涂海声等;分析测试学报;第41卷(第7期);988-997 * |
| 药物治疗动脉粥样硬化新途径——抗炎作用;林蓉等;中国药理学通报;第20卷(第5期);499-503 * |
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