CN116370523A - 植物精油在制备抗癫痫药物或健康产品中的应用 - Google Patents
植物精油在制备抗癫痫药物或健康产品中的应用 Download PDFInfo
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- CN116370523A CN116370523A CN202310350400.4A CN202310350400A CN116370523A CN 116370523 A CN116370523 A CN 116370523A CN 202310350400 A CN202310350400 A CN 202310350400A CN 116370523 A CN116370523 A CN 116370523A
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- essential oil
- patchouli
- plant essential
- zebra fish
- antiepileptic
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Abstract
本发明公布了植物精油在制备抗癫痫药物中的用途，属于生物医药大健康技术领域。本发明植物精油具有良好的脂溶性，易于通过血脑屏障，因此具有提高或改善的毒理学安全性(即毒性降低)、提高或改善的代谢稳定，较长的半衰期或较小的副作用的特点，同时产生类似或提高的生物活性。制备的药物可用于治疗癫痫包括惊厥等脑神经元异常放电引起反复痫性发作。在斑马鱼癫痫模型中显示显著的抗癫痫活性，广藿香精油的半数有效剂量(EC50)为2.03μg/mL，乳香精油(全成分)的半数有效剂量(EC50)为2.53μg/mL，胡萝卜籽精油的半数有效剂量(EC50)为2.16μg/mL。
Description
技术领域
本发明涉及广藿香精油、乳香精油、胡萝卜籽精油的新用途，尤其涉及几种精油在制备治疗癫痫药物或健康产品中的用途。
背景技术
癫痫是反复发作的神经系统疾病。在癫痫发作期间，身体的一部分或全身出现短暂的不自主抽搐(即部分性癫痫发作或全身性癫痫发作)，有时伴有意识丧失、尿失禁和大便失禁。癫痫发作是由一群脑细胞的异常放电引起的。大脑的不同部位均可能成为异常放电的部位。癫痫发作的范围从非常短暂的意识丧失或肌肉反射到严重和持续的抽搐。癫痫发作的频率也可能有所不同，从每年不到一次到每天几次。尽管许多潜在的疾病机制可能导致癫痫，但全世界约50％的患者的病因仍然未知，因此到目前为止，还没有理想的根治方法。目前有不少临床药物在治疗癫痫方面具有一定的效果，但是，治疗不彻底、成本高，长期服用会给患者带来沉重的经济负担。
广藿香(Pogostemon cablin(Blanco)Benth)为唇形科刺蕊草属多年生草本或灌木植物，其茎、叶均可入药。主要用于治疗食欲不振，呕吐，泄泻，外感暑湿之寒热头痛，湿温初起的发热身困，手足癣，具有芳香化湿，和胃止呕，祛暑解表的功效。广藿香精油为广藿香花通过水汽蒸馏法得到，其主要成分为倍半萜类化合物，其中百秋李醇、α-愈创木烯、α-广藿香烯、β-广藿香烯、广藿香烯是其主要成分。
乳香树(Boswellia carteri)为橄榄科乳香属植物，其树脂可入药，主治气血凝滞、心腹疼痛，痈疮肿毒，跌打损伤，痛经，产后瘀血刺痛。具有活血行气止痛，消肿生肌的功效。乳香精油为乳香树脂通过有机溶剂提取得到，其主要成分为单萜、倍半萜和脂肪族化合物，其中(+)-柠檬烯、因香酚、香茅醇等为其主要成分。
胡萝卜(Daucus carota var.sativa Hoffm)为伞形科胡萝卜属一年生或二年生草本植物，原产于亚洲西部，于12世纪经伊朗传入中国，现分布于全国各地。主要是作为食物，但也具有药用价值，据《岭南草药志》记载，胡萝卜具有健脾和中，滋肝明目，化痰止咳，清热解毒的功效。同时还具有提高机体抗癌免疫力和间接消灭癌细胞的功能。胡萝卜籽精油为胡萝卜的成熟果实，经有机溶剂提取得到，其主要成分为单萜和倍半萜，其中β-红没药胺、胡萝卜醇、胡萝卜烯、β-石竹烯、反式-β-金合欢烯、(-)-β-蒎烯、d-柠檬烯、月桂烯等是其主要成分。
发明内容
本发明的目的在于针对现有技术的不足，提供几种植物精油在制备抗癫痫药物或健康产品中的用途。
为实现上述目的，本发明采用以下技术方案完成：
本发明所述的植物精油在制备抗癫痫药物或健康产品中的应用，将植物精油作为制备治疗癫痫的有效成分，所述植物精油为广藿香精油、乳香精油和胡萝卜籽油中的一种或多种。
还可以加入一种或多种药物或健康产品上可接受的辅料，并可将药物制成滴鼻剂、喷雾剂、贴剂、膜剂、片剂、凝胶剂、糊剂、漱口剂、丸剂、散剂、颗粒剂、胶囊剂、微囊片剂、内服液体制剂中的任何一种。
所述健康产品可以为化妆品、日用品、食品、保健品、消字号产品中的任何一种。
所述广藿香精油、乳香精油、胡萝卜籽油分别是由刺蕊草属植物广藿香的花、乳香属植物的树脂、胡萝卜属植物胡萝卜的种子通过水蒸气蒸馏或有机溶剂提取制备得到的挥发性成分。
在本发明之前尚未发现上述植物精油具有治疗癫痫的作用；也尚未发现以上述植物精油为原料制备药物制剂或健康产品用于治疗癫痫疾病和病症的报道。
本发明提供了一种植物精油的新用途，首次发现几种植物精油抗癫痫活性。通过斑马鱼体内癫痫模型进行筛选，首次发现广藿香精油、乳香精油、胡萝卜籽精油在斑马鱼体内模型中能改善戊四唑诱导的斑马鱼脑神经元异常放电引起反复痫性发作。这几种精油在2μg/ml至10μg/ml的浓度范围内，抗癫痫治疗效率随着浓度的增加而增加，呈现浓度依赖性，半数有效剂量(EC50)分别为2.03μg/ml、2.53μg/ml、2.16μg/ml。
植物精油具有良好的脂溶性，易于通过血脑屏障，因此具有提高或改善的毒理学安全性(即毒性降低)、提高或改善的代谢稳定，较长的半衰期或较小的副作用的特点，同时产生类似或提高的生物活性(药效)。制备的药物可用于治疗癫痫包括惊厥等脑神经元异常放电引起反复痫性发作。
附图说明
图1是广藿香精油在斑马鱼癫痫模型中体内抗癫痫活性图。
图2是乳香精油在斑马鱼癫痫模型中体内抗癫痫活性图。
图3是胡萝卜籽精油在斑马鱼癫痫模型中体内抗癫痫活性图。
具体实施方式
许多脑功能及其障碍疾病的病理状态，例如癫痫等，发病机制复杂，单一成分或分子难以治愈或缓解。植物精油由多个活性分子组成，可能是一种潜在的抗癫痫药物。本申请发明人发现，在GABA拮抗剂戊四唑诱导癫痫斑马鱼模型中，动物体内施用本发明的几种植物精油能显著改善癫痫斑马鱼的快速运动。此外，有相关报道表明，洋甘菊精油、鼠尾草精油、迷迭香精油、薰衣草精油和玫瑰精油等在芳香疗法常被用于镇静安神，并且可以缓解焦虑。即，植物精油通过多分子协调，多靶点作用，治疗/缓解脑功能及其障碍疾病的相关症状，也没有出现毒副作用。本发明的几种植物精油以及药物配方和组合物显示出显著抗癫痫活性，其通过动物模型中癫痫的严重程度、数量或持续时间的减少得到证明。
本发明涉及的几种植物精油给药方式可为鼻腔给药、口腔给药、透皮给药、口服给药等途径。鼻腔给药的常见剂型包括滴鼻剂、喷雾剂、贴剂、膜剂等；口腔黏膜给药常用剂型包括片剂、贴剂、凝胶剂、糊剂、膜剂、喷雾剂、漱口剂、丸剂等；透皮给药常用剂型为贴剂；口服剂型包括片剂、散剂、颗粒剂、丸剂、内服液体制剂。
本发明所述可药用载体包括但不限于碳酸钙、磷酸钙、硫酸钙、蔗糖、葡萄糖、乳糖、果糖、木糖醇、山梨醇、淀粉、淀粉糊、纤维素衍生物、明胶、聚乙烯吡咯烷酮、氯化钠、糊精、硬脂酸、硬脂酸镁、硬脂酸钙、植物油、聚乙二醇、无菌磷酸盐缓冲盐水、盐水、林格氏溶液以及它们的组合。
此外，本发明中的几种植物精油还可以添加到食物和饮料中，用于口服施用，如配制成口香糖，以促进口服递送和吸收。本发明中的几种植物精油或其组合物还可以添加到化妆品、洗浴用品或大健康产品中，用于香薰、嗅吸、按摩、涂抹、泡浴使用。
本发明涉及的几种植物精油也可与其他药物活性成分联用，以制备其他的新药物组合物。
上述本发明的几种植物精油或其组合的治疗作用、良好的药物代谢参数和通常无毒性使得本发明化合物成为用于治疗上述病症的理想药物。
下面将结合附图和实施例对本发明作进一步详细说明，但本发明保护范围不局限于所述内容，应当理解为可以以各种形式实现本发明而不应该被所阐述的实施例限制。相反，提供这些实施例是为了更加透彻地理解本发明。实施例中方法如无特殊说明的采用常规方法，使用的试剂如无特殊说明的使用常规市售试剂或按常规方法配制的试剂。
实施例1：采集于云南省的广藿香(Pogostemon cablin(Blanco)Benth)提取的广藿香精油(jx-66)制备
将干燥的采集于云南省的广藿香花样品用水冷式增速高效流水粉碎机粉碎，过药典标准筛3号筛。将样品放入圆底烧瓶，加入8倍量纯水，搅拌混匀，浸泡1h后采用水蒸气蒸馏法提取7小时，温度为60-70℃。收集蒸馏液，加入少量乙酸乙酯萃取3次，再加入无水硫酸钠除水，静置过夜，使乙酸乙酯挥发，分离无水硫酸钠后得到广藿香精油。用气相色谱-质谱联用仪分析，并用Nist.14谱库联合检索，确定广藿香精油中的主要成分为虎尾草醇(＞22％)、α-愈创烯(＞18％)、α-广藿香烯(＞7％)、β-广藿香烯(＞6％)、顺式-β-愈创木酚(＞4％)。
实施例2：采集于埃塞俄比亚的乳香树(Boswellia carteri)树脂提取的乳香精油(jx-83)制备
将采集于埃塞俄比亚的乳香树脂放于蒸馏烧瓶中，加十倍树脂重量的水，室温浸泡六小时。将蒸馏烧瓶置于电热恒温水浴槽中加热，蒸馏釜温度220℃，提取五小时。蒸馏时产生的蒸汽上升遇冷凝管回流，油水混合物将积于分液漏斗，下层为水，上层为精油。结束后，放出下层水，收集上层精油。用气相色谱-质谱联用仪分析，并用Nist.14谱库联合检索，确定乳香精油中的主要成分为7-氮杂吲哚(＞21％)、(2E)-4-异丙基-1，7，11-三甲基-2-氯十四烯-1-醇(＞5％)、异瑟模环烯醇(＞3％)、邻薄荷醇-1(7)(＞0.7％)、石竹烯(＞0.6％)。
实施例3：采集于印度的胡萝卜(Daucus carota)种子制作的胡萝卜籽精油(zy-pl-76)制备
将干燥的采集于印度的胡萝卜种子样品用水冷式增速高效流水粉碎机粉碎，过药典标准筛3号筛。将样品放入圆底烧瓶，加入8倍量纯水，搅拌混匀，浸泡1h后采用水蒸气蒸馏法提取7小时，温度为60-70℃。收集蒸馏液，加入少量乙酸乙酯萃取3次，再加入无水硫酸钠除水，静置过夜，使乙酸乙酯挥发，分离无水硫酸钠后得到胡萝卜籽精油。用气相色谱-质谱联用仪分析，并用Nist.14谱库联合检索，确定胡萝卜籽精油中的主要成分为2-异丙基-5-甲基-9-亚甲基双环[4.4.0]十烯-1-烯(＞25％)、胡罗卜醇(＞2％)、胡萝卜烯(＞2％)、β-石竹烯(＞1％)、异胡萝卜烯(＞1％)。
实施例4：斑马鱼癫痫模型建立及优化
选取成年的野生型AB品系的斑马鱼，按雄鱼：雌鱼2：1的比例进行胚胎繁殖，收集受精卵在28℃条件下用养鱼用E3水孵育胚胎。在受精后6小时(即6hpf)和24hpf对胚胎进行清理(移除已死亡胚胎)，每24hpf更换养殖用水。因为胚胎可以从自身的卵黄囊中获取营养物质，所以在受精后9天内(9dpf)不需要喂食。实验完成后，用三卡因甲磺酸对各个发育阶段的斑马鱼进行过度暴露处理，从而将斑马鱼麻醉处死。麻醉处死的操作步骤符合美国兽医协会(AVMA)对动物麻醉处死的规范要求。
选取7dpf的斑马鱼进行实验，此时的斑马鱼器官发育完善，且具有自主行动能力。采用PTZ(戊四唑)诱导斑马鱼癫痫，设置PTZ浓度为1.25、2.5、5、10、20mM，10mM的PTZ能诱导斑马鱼使其呈现惊厥状态且实验数据稳定。精油所用溶剂为DMSO，对DMSO进行安全性评价，选择0.1vol％，1vol％，3vol％的DMSO水溶液进行实验，结果显示0.1vol％DMSO水溶液与空白对照组无显著性差异，故选择0.1vol％DMSO进行实验。阳性药选用苯妥英钠，设置100、200、300、400、500、600、800μM的苯妥英钠进行实验，发现600μM时斑马鱼惊厥状态得到显著改善，故选择600μM的苯妥英钠作为阳性对照。因此，模型条件为，溶剂DMSO水溶液浓度为0.1vol％，戊四唑浓度为10mM，阳性药苯妥英钠浓度为600μM。
对斑马鱼的快速运动(V>20mm/sec)距离进行定量分析，通过计算得到以下指标：①斑马鱼快速运动距离；②精油的抗癫痫治疗效率；③精油对斑马鱼快速运动影响的浓度变化曲线。对斑马鱼的运动距离进行定量分析，计算精油的抗癫痫治疗效率，并根据待测物癫痫治疗效率绘制剂量-效应曲线，计算化合物抗癫痫作用的EC50；用方差分析和T-检验进行统计学分析，p<0.05为显著性差异。抗癫痫治疗效率的计算公式如下：
式中，Dmodel表示模型组平均运动距离，DCompounds表示实验组平均运动距离，Dcontrol表示溶剂对照组平均运动距离。
实施例5：以实施例1-3制备得到的几种植物精油进行斑马鱼体内抗癫痫作用的评价
采用实施例4构建斑马鱼癫痫模型进行实验，本研究共设置8组实验，每组实验包括1个溶剂对照组、1个癫痫模型组、1个阳性对照组(苯妥英钠)、4个实验组。采用实施例4方法进行实验，E3水处理的斑马鱼为空白对照组；含0.1vol％的DMSO的E3水处理的斑马鱼为阴性对照组(溶剂对照组)；含0.1vol％的DMSO与戊四唑的E3水共处理的斑马鱼为癫痫模型组；含0.1vol％的DMSO、苯妥英钠与戊四唑的E3水共处理的斑马鱼为阳性对照组；含0.1vol％的DMSO、实施例1-3制备的植物精油与戊四唑的E3水共处理的斑马鱼为实验组，精油浓度为2、4、6、8和10μg/mL。在28℃条件下共处理60min，利用行为分析仪记录60min内斑马鱼的运动轨迹，然后对斑马鱼的快速运动(seizure，V>20mm/sec)距离进行定量分析，计算广藿香精油的抗癫痫治疗效率，并根据广藿香精油癫痫治疗效率绘制剂量-效应曲线，计算广藿香精油抗癫痫作用的EC50；用方差分析和T-检验进行统计学分析，p<0.05为显著性差异。实验过程中各组斑马鱼均未出现死亡现象，且广藿香精油各浓度组与癫痫模型组相比，斑马鱼快速运动轨迹明显减少。广藿香精油各浓度组与溶剂对照组相比均具有统计学意义(p<0.001)，广藿香精油与抗癫痫药物苯妥英钠比较，广藿香精油抗癫痫活性强于苯妥英钠(600μM)。图1为广藿香精油抗癫痫作用的剂量-效应曲线，由图1可知，广藿香精油在2μg/ml至10μg/ml的浓度范围内，抗癫痫治疗效率随着浓度的增加而增加，呈现浓度依赖性，EC50为2.03μg/ml。
(图1，表1)
乳香精油(全成分)YR020(样品)各浓度组与溶剂对照组相比均具有统计学意义(p<0.001)，乳香精油(全成分)YR020(样品)与抗癫痫药物苯妥英钠比较，乳香精油(全成分)YR020(样品)抗癫痫活性强于苯妥英钠(600μM)。图2为乳香精油(全成分)YR020(样品)抗癫痫作用的剂量-效应曲线，由图2可知，乳香精油(全成分)YR020(样品)在2μg/ml至10μg/ml的浓度范围内，抗癫痫治疗效率随着浓度的增加而增加，呈现浓度依赖性，EC50为2.53μg/ml。(图2，表1)
胡萝卜籽油各浓度组与癫痫模型组相比，斑马鱼快速运动轨迹明显减少，胡萝卜籽油各浓度组与溶剂对照组相比均具有统计学意义(p<0.001)。胡萝卜籽油与抗癫痫药物苯妥英钠比较，胡萝卜籽油抗癫痫活性强于苯妥英钠(600μM)。图3为胡萝卜籽油抗癫痫作用的剂量-效应曲线，由图3可知，胡萝卜籽油在2μg/ml至10μg/ml的浓度范围内，抗癫痫治疗效率随着浓度的增加而增加，呈现浓度依赖性，EC50为2.16μg/ml。(图3，表1)
表1.几种植物精油对斑马鱼癫痫模型的半数有效浓度(EC50)
表2为几种植物精油对斑马鱼癫痫模型的惊厥改善效果，从表2中可以看出，广藿香精油、乳香精油、胡萝卜籽油均具有良好的癫痫治疗效果，其中广藿香精油效果最好。
表2.几种植物精油对斑马鱼癫痫模型的惊厥改善效率
上述实施例用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明做出的任何修改和改变，都落入本发明的保护范围。
Claims (4)
1.植物精油在制备抗癫痫药物或健康产品中的应用，其特征在于，所述植物精油为广藿香精油、乳香精油和胡萝卜籽油中的一种或多种。
2.根据权利要求1所述的应用，其特征在于，所述广藿香精油、乳香精油、胡萝卜籽油分别是由刺蕊草属植物广藿香的花、乳香属植物的树脂、胡萝卜属植物胡萝卜的种子通过水蒸气蒸馏或有机溶剂提取制备得到的挥发性成分。
3.根据权利要求1所述的应用，其特征在于，所述抗癫痫药物为片剂、丸剂、胶囊剂、颗粒剂、微囊片剂、混悬剂、滴丸、口服液体、注射剂、气雾剂、栓剂、皮下给药剂型。
4.根据权利要求1所述的应用，其特征在于，所述健康产品为化妆品、日用品、食品、保健品、消字号产品中的任何一种。
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