CN115137672A - 一种复方精油甘油体及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种复方精油甘油体及其制备方法和应用，该复方精油甘油体的质量百分比组成包括：复方精油0.5‑3.0%、磷脂2‑6%、胆固醇0.1‑0.5%、甘油10‑50%、乙醇5‑10%、余量为水，所述的复方精油的质量百分比组成为：柠檬精油50‑75%、当归精油15‑25%、乳香精油10‑25%。该复方精油甘油体可直接作为具有抗黄褐斑与焦虑症双重作用的化妆品应用，也可作为原料或中间体与其他原料复配为化妆品。本发明将柠檬精油、当归精油、乳香精油进行复配制备得到复方精油，进一步将复方精油制备为甘油体的形式，增加了精油的稳定性，促进了精油的皮肤渗透性和在皮内的滞留性，并延长了精油在皮内的作用时间，从而有效提高了精油的生物利用度。
Description
技术领域
本发明涉及一种精油产品，具体是一种具有抗黄褐斑与焦虑症双重作用的复方精油甘油体及其制备方法和应用。
背景技术
十个女人九个斑，面部色斑严重困扰着爱美女性。脸部色斑主要包括日晒斑、雀斑、黑斑、黄褐斑和老年斑等。黄褐斑为面部的黄褐色色素沉着，多对称蝶形分布于颊部，多见于女性。黄褐斑的形成主要与内分泌失调、紫外线照射、生活习惯及精神心理状态等有关。在当今社会快速发展下，女性面临来自学业、工作、生活、生育及家庭责任等诸多压力的不断增加，易伴随着不同程度的烦躁、焦虑的精神状态。面部色斑与焦虑症互相叠加、循环影响，导致色斑与焦虑在女性中的患病人数正逐年升高且有年轻化的趋向，严重影响女性患者的生活质量。
中医对黄褐斑、焦虑症的病因有许多共同观点。中医学上将黄褐斑命名为“肝斑”，认为肝郁气滞是导致黄褐斑的主要原因，中医将焦虑症归属于“郁症”范畴，二者都与情志不遂、肝失疏泄、气机郁结等因素有关。西医也认为黄褐斑和焦虑症是有一定关联的，因为精神方面的疾病会影响人体的内分泌，而内分泌紊乱是黄褐斑形成的原因之一。临床证实，很多黄褐斑患者在起病或疾病进展时有过精神创伤，存在不同程度的易怒、焦虑、抑郁、精神衰弱等负性情绪。
目前针对黄褐斑伴焦虑症这类疾病的治疗方式十分局限，一般是分别用药，通过口服维生素C和维生素E、谷胱甘肽、氨甲环酸等改善黄褐斑；通过口服三环类抗抑郁剂(如丙咪嗪、氯丙咪嗪)、苯二氮卓类(如阿普唑仑)镇静药物控制焦虑症。抗焦虑类药物普遍存在副作用较大、依赖性强等问题。
长期以来人们希望通过开发天然药物达到更安全、副作用小的预防与治疗效果。许多精油中的活性成分都能显著的抑制酪氨酸酶活性，阻止黑色素的形成，具有祛斑美白功效。精油的芳香气味通过嗅觉器官吸入触及脑部的神经系统对人的感知和情绪产生直接的作用，精油对人的皮肤色斑和心理都有一定的疗效。但目前市面上尚未有抗黄褐斑与焦虑症双重功能的精油产品。此外，精油一般都需要加入基础油稀释后使用，这就导致使用时有油腻的感觉。同时精油挥发性强，涂抹后极易挥发，发挥作用的时间短。精油稳定性差，储存放置时间短。这些都对精油的使用效果带来了一定影响。
由于皮肤黄褐斑与焦虑症往往相伴，本发明提出一款对二者有干预治疗的复方精油配方并制备成精油甘油体，进一步将精油甘油体融入到化妆品中制成各种类型的化妆品，如化妆水、乳、霜等，采用化妆品和芳香疗法中最常见的按摩、嗅闻等方式，在涂抹面部皮肤的过程中通过一定的按摩促进精油甘油体的透皮吸收和持续的芳香嗅闻，让身兼多职的职业女性在每日必用的化妆品涂抹过程中，感受到精油的功效与魅力，达到对黄褐斑和焦虑症的预防、缓解、治疗的效果。
发明内容
本发明所要解决的技术问题是，针对现有技术的不足，提供一种具有抗黄褐斑与焦虑症双重作用的复方精油甘油体及其制备方法和应用。本发明将柠檬精油、当归精油、乳香精油进行复配制备得到复方精油，进一步将复方精油制备为甘油体的形式，不仅增加了精油的稳定性，而且促进了精油的皮肤渗透性和在皮内的滞留性，能够延长精油在皮肤内的作用时间，从而有效提高精油的生物利用度。
本发明解决上述技术问题所采用的技术方案为：一种复方精油甘油体，该复方精油甘油体的质量百分比组成包括：复方精油0.5-3.0％、磷脂2-6％、胆固醇0.1-0.5％、甘油10-50％、乙醇5-10％、余量为水，所述的复方精油的质量百分比组成为：柠檬精油50-75％、当归精油15-25％、乳香精油10-25％。
柠檬精油中的柠檬烯、柠檬醛可使血清中的皮质固醇和大脑中的单胺维持在低浓度，进而降低身体和心理的压力，对于忧郁症患者可以激励副交感神经和抑制交感神经；同时柠檬精油中的柠檬烯、柠檬醛还具有保养皮肤温和美白，防止皱纹产生，增加皮肤光泽，淡化雀斑等功效。当归自古以来就是生血活血的中药，具有补血活血、调经止痛、润肠通便的功效，当归精油作用于皮肤，通过促进血红蛋白和血红细胞的生成，达到祛黄焕肤、美白祛斑的效果；当归精油具有显著的抗焦虑作用，且无苯二氮卓类药物的副作用。乳香精油散发的香气可令人感受从未有过的放松，帮助人们舒缓急躁、受挫、哀伤等负面情绪，具有抗菌、淡化疤痕、皱纹的作用，调理干燥、老化、暗沉的肌肤。
本发明将柠檬精油、当归精油、乳香精油复配为复方精油，并将复方精油与磷脂、胆固醇、甘油、乙醇和水制备为复方精油甘油体。
柠檬精油、当归精油、乳香精油具有协同增效互补作用，对黄褐斑和焦虑症起到“异病同治”作用。柠檬精油、当归精油、乳香精油复配而成的复方精油含有乙酸橙花酯、柠檬醛、丁烯基苯酞和乙酸辛酯等28个有效活性成分，与黄褐斑和焦虑症疾病有26个共有靶点，包括雌激素受体(ESR1)、细胞周期蛋白D1(CCND1)、白细胞介素1β(IL-1β)、磷脂酰肌醇-3-激酶(PIK3CA)等。其中乙酸橙花酯、柠檬醛、丁烯基苯酞和乙酸辛酯均与ESR1、CCND1和PIK3CA三个核心靶点具有较好的结合性。这些靶点主要参与对激素、外来刺激反应，与雌激素、类固醇的结合等，刺激机体产生应答，引起相关蛋白环腺苷效应元件结合蛋白(CREB)、小眼畸形相关转录因子(MITF)和酪氨酸酶(TYR)等表达水平的上调或下调，进而发挥治疗黄褐斑和焦虑症共症的作用。
本发明将柠檬精油、当归精油、乳香精油进行复配制备得到复方精油，进一步将复方精油制备为甘油体的形式，增加了精油的稳定性，促进了精油的皮肤渗透性和在皮内的滞留性，并延长了精油在皮肤内的作用时间，从而有效提高了精油的生物利用度。
本发明复方精油甘油体是磷脂和甘油制备而成的囊泡体，甘油囊泡能负载精油并高效地将活性成分通过囊泡的变形性输送到皮肤内部。甘油体同时也可增强皮肤的水合作用，达到对皮肤良好的保湿效果。
作为优选，所述的柠檬精油的制备方法为：将柠檬皮干燥粉碎后，采用超临界流体萃取技术提取得到柠檬精油，萃取条件为：40-45℃、压力15-30MPa、时间60-120min、二氧化碳流速20-30kg/h。
作为优选，所述的当归精油的制备方法为：将当归根茎干燥粉碎后，采用超临界流体萃取技术提取得到当归精油，萃取条件为：温度45-48℃、压力20-25MPa、时间80-120min、二氧化碳流速25-35kg/h。
作为优选，所述的乳香精油的制备方法为：称取100g乳香碎片于蒸馏烧瓶中，加入1000mL水，室温浸泡6h后，将蒸馏烧瓶置于恒温电加热器中加热，蒸馏提取8h以上，收集馏出液，用分液漏斗进行油水分离后，用无水硫酸钠干燥，得到乳香精油。
上述复方精油甘油体的制备方法，包括以下步骤：
(1)将柠檬精油、当归精油、乳香精油混匀，得到复方精油；
(2)按质量百分比组成称取以下原料：复方精油0.5-3.0％、磷脂2-6％、胆固醇0.1-0.5％、甘油10-50％、乙醇5-10％、余量为水；
(3)采用乙醇注入法制备复方精油甘油体：将磷脂2-6g、胆固醇0.1-0.5g、复方精油0.5-3.0g混合，用5-10mL无水乙醇溶解为透明溶液；在500-1000rpm不断搅拌的条件下用注射器将透明溶液注入到含有10-50％甘油的40-50℃超纯水85-90mL中，继续搅拌5min后，室温放置30min，使其充分水化，得到甘油体；将甘油体在冰水浴中超声，最后定容到100mL，再用0.22-0.45μm的微孔滤膜整粒，即得含精油浓度为5-30mg/mL的复方精油甘油体，在4℃下储存。
作为优选，所述的磷脂为氢化磷脂、氢化大豆磷脂、蛋黄卵磷脂、大豆磷脂酰胆碱、磷脂酰胆碱、二油酰磷脂酰丝氨酸、二棕榈酰磷脂酰丝氨酸、二油酰磷脂酰甘油、二棕榈酰磷脂酰胆碱和豆磷脂中至少一种。
上述复方精油甘油体作为具有抗黄褐斑与焦虑症双重作用的化妆品的应用。
上述复方精油甘油体作为具有抗黄褐斑与焦虑症双重作用的原料或中间体，在与其他原料复配为化妆品中的应用。
与现有技术相比，本发明具有如下优点：
1)本发明将柠檬精油、当归精油、乳香精油复配为复方精油，并将复方精油与磷脂、胆固醇、甘油、乙醇和水制备为复方精油甘油体。柠檬精油、当归精油、乳香精油具有协同增效互补作用，对黄褐斑和焦虑症起到“异病同治”作用。柠檬精油、当归精油、乳香精油复配而成的复方精油含有乙酸橙花酯、柠檬醛、丁烯基苯酞和乙酸辛酯等28个有效活性成分，与黄褐斑和焦虑症疾病有26个共有靶点，包括雌激素受体(ESR1)、细胞周期蛋白D1(CCND1)、白细胞介素1β(IL-1β)、磷脂酰肌醇-3-激酶(PIK3CA)等。其中乙酸橙花酯、柠檬醛、丁烯基苯酞和乙酸辛酯均与ESR1、CCND1和PIK3CA三个核心靶点具有较好的结合性。这些靶点主要参与对激素、外来刺激反应，引起相关蛋白环腺苷效应元件结合蛋白(CREB)、小眼畸形相关转录因子(MITF)和酪氨酸酶(TYR)等表达水平的上调或下调，进而发挥治疗黄褐斑和焦虑症共症的作用。
2)本发明将柠檬精油、当归精油、乳香精油进行复配制备得到复方精油，进一步将复方精油制备为甘油体的形式，增加了精油的稳定性，促进了精油的皮肤渗透性和在皮内的滞留性，延长了精油在皮肤内的作用时间，从而有效提高了精油的生物利用度。
3)本发明复方精油甘油体可作为特殊功效性化妆品直接使用，通过直接涂抹于黄褐斑的皮肤并辅以按摩作用于黄褐斑淡斑的同时，复方精油甘油体可有效渗透到皮肤内，精油愉悦的气味可达到调节人的情绪的效果。
4)本发明复方精油甘油体也可作为化妆品原料或中间体，与其他原料复配为如乳液、精华液、乳霜等多种化妆品剂型。本发明复方精油甘油体作为化妆品原料不仅有功效性，而且天然香味可以替代化妆品中的人工香精，赋予化妆品更好的使用体验，在皮肤上涂抹使用，具有涂抹舒适、不油腻的肤感。
附图说明
图1为本发明的技术路线图；
图2为复方柠檬-当归-乳香精油与黄褐斑、焦虑症的交集靶点韦恩图；
图3为复方精油-黄褐斑-焦虑症三者共有靶点构建的蛋白相互作用拓扑图；
图4为GO功能富集分析；
图5为KEGG通路富集分析；
图6为构建的“复方精油有效成分-疾病靶点-通路”网络图；
图7为精油活性成分对PI3K/Akt信号通路的调控图；
图8为复方柠檬-当归-乳香精油对DPPH自由基的清除力；
图9为复方柠檬-当归-乳香精油对ABTS自由基的清除力；
图10为复方柠檬-当归-乳香精油对酪氨酸酶的抑制率；
图11为复方柠檬-当归-乳香精油对蛋白变性的抑制率；
图12为MRT应激后与涂抹精油后生理指标差值的组间比较结果；
图13为复方精油甘油体和脂质体的TEM图；
图14为复方精油甘油体的粒径分布图；
图15为脂质体的粒径分布图；
图16为含复方精油甘油体和不含复方精油甘油体的乳液对皮肤保湿性对比测试图。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
图1为本发明的技术路线图。
一、本发明复方精油甘油体及其应用的理论基础——复方柠檬-当归-乳香精油抗黄褐斑和焦虑症的分子机制分析
1.复方柠檬-当归-乳香精油主要活性成分及对应靶点
通过GC-MS分析、文献补充并通过SwissADME和SwissTargetPrediction平台筛选和预测成分的靶点，去除无靶点的成分、再去除重复值后(α-蒎烯为柠檬精油和当归精油共有成分，芳樟醇和D-柠檬烯为柠檬精油和乳香精油的共有成分)，如表1所示，复方柠檬-当归-乳香精油共有37个活性成分(柠檬精油22个、当归精油8个、乳香精油10个)，共对应潜在靶点394个。
表1复方柠檬-当归-乳香精油的活性成分
(1)柠檬精油中的乙酸橙花酯具有柠檬、薰衣草样香气，芳香酯类物质具有抗焦虑、抑郁的作用，还具有抗自由基、抗老化的功效；柠檬精油中的柠檬醛具有抗肿瘤、抗抑郁等功效，较柠檬烯有更高的对酪氨酸酶抑制作用，动物实验与临床吸嗅实验发现柠檬醛可通过吸嗅系统作用于大脑，起到缓解人体抑郁的作用。
(2)当归精油中的丁烯基苯酞可通过活血祛瘀的方式改善黄褐斑的病症，丁烯基苯酞为有效的去甲肾上腺素和5-HT再摄取抑制剂，可透过血脑屏障，对中枢神经起到改善脑血液微循环等神经系统保护作用，丁烯基苯酞可促进精油的皮肤渗透吸收。
(3)乳香精油中的乙酸辛酯具有浓郁果香以及花香气息，是乳香发挥抗炎、活血、镇痛药效的主要成分。
推测复方柠檬-当归-乳香精油主要通过芳香酯类起到缓解心悸、温和镇静，芳香醛类的美白、抗抑郁以及苯酞类的活血化瘀、神经保护等发挥药效，起到治疗黄褐斑和焦虑症共症的作用。
2.复方精油与黄褐斑、焦虑症的交集靶点以及交集靶点的蛋白互作图
检索GeneCards、OMIM以及DrugBank数据库，将靶点合并、去重后共获得黄褐斑疾病靶点1609个、焦虑症疾病靶点1120个。通过Draw Venn Diagram网站，将复方精油活性成分对应的靶点和疾病靶点取交集绘制韦恩图，见图2，其中图2的A图为复方精油与黄褐斑和焦虑症的交集靶点韦恩图；图2的B图为柠檬、当归和乳香精油分别与黄褐斑和焦虑症的交集靶点韦恩图。
复方柠檬-当归-乳香精油与黄褐斑有105个交集靶点、与焦虑症有88个交集靶点，其中复方精油-黄褐斑-焦虑症三者共有交集靶点26个：ALDH2、BCL2、CCND1、CHRM3、CPT2、CREBBP、CYP2A6、EDNRA、ESR1、FGFR1、IL1B、KIT、MAOA、MAOB、MEN1、NOS1、NOS3、NR3C2、PDE4A、PIK3CA、PLAU、PTGS2、RORA、SHH、SLC6A3、TSPO。26个共有靶点构建的蛋白相互作用网络见图3。圆形节点的大小和颜色表示Degree值的大小，颜色越深、圆形节点越大表示Degree值越大，相关性越强；Edge颜色由深色渐变为浅色表示Combined Score由大到小。该网络包含25个节点、59条边，大于平均Dgree值4.72的核心靶点有9个，见表2。其中ESR1(雌激素受体)、CCND1(细胞周期蛋白D1)和IL1B(白细胞介素1β)靶点的Degree值分别为13、12和11，显著高于其他靶点。
表2 Degree值前9的核心靶点
(1)雌激素受体为雌激素的作用靶点，雌激素可通过作用于核性雌激素受体ERα上调BCL2在星型胶质细胞中的表达，从而抑制神经元的凋亡。雌激素还可通过作用于皮肤中的ERβ，通过PKA途径增强cAMP水平并上调环腺苷效应元件结合蛋白(CREB)、小眼畸形相关转录因子(MITF)和酪氨酸酶蛋白(TYR，tyrosinase)表达，催化底物多巴合成黑素。
(2)细胞周期蛋白D1可促进细胞从G1期进入S期，精确调控细胞增殖和分化。CCND1的表达与黑素细胞增殖密切相关，能够致使黑素瘤的发生，而黑素瘤细胞株与人体正常黑素细胞的黑素合成功能基本一致。当神经系统受损时，CCND1在神经元中表达增多，促使神经胶质化产生。
(3)白细胞介素1β是一种多效炎性细胞因子，研究表明，通过抑制海马组织IL-1β等炎症介质的表达，可间接保护神经毒性损伤，减轻神经退行性疾病的进展。且在白细胞介素1β等炎性刺激因素的刺激下，会引起黑素细胞的活化，致使黄褐斑的生成。
(4)提示复方精油主要通过激素刺激、细胞凋亡调控以及免疫炎症等作用，双向调节黄褐斑和焦虑症，体现了中医“异病同治”的治疗原则。
3.GO功能和KEGG通路富集分析
对26个共有靶点通过Metascape数据库进行GO功能和KEGG通路富集分析。根据p值由小到大排列，列举并分析各类排名靠前条目。
(1)GO功能富集获得生物过程(BP)403条、细胞组成(CC)15条、分子功能(MF)23条，如图4所示。共有靶点主要参与的生物学过程涉及对激素、外来刺激反应；细胞组成涉及细胞器外膜、外膜和膜筏等；分子功能涉及氧化还原酶辅基、类固醇结合以及氧化还原酶活力等。其中，靶点在激素反应以及类固醇结合上富集的这一结论，进一步阐释了雌激素的重要作用，雌激素为类固醇激素可与相应受体结合，刺激机体产生应答引起相关蛋白CREB、MITF和TYR等表达水平的上调或下调，进而发挥治疗黄褐斑和焦虑症共症作用。
(2)KEGG通路富集分析获得69条信号通路，其中前20条KEGG通路富集了20个共有靶点，占26个共有靶点的77％，见图5，图5中X轴表示通路中含有的疾病对应共有靶点所占比例；Y轴为通路编号与名称的组合。KEGG通路富集排名靠前的通路多与信号以及癌症相关，如PI3K/Akt信号通路、钙信号通路以及癌症途径等。钙信号通路是维持细胞正常功能的重要通路，研究发现焦虑抑郁模型大鼠海马体脑区中Ca2+浓度偏高；且钙信号通路参与酪氨酸激活、黑素的生成乃至黑色素瘤的发生。查询癌症途径和钙信号通路的通路图显示，二者均涉及PI3K/Akt信号通路，说明可通过刺激其中一条通路，启动其他通路的级联反应。
4.“复方精油成分-疾病靶点-通路”网络
对筛选的20条通路及富集的20个共有靶点及调控靶点的28个活性成分，构建“复方精油成分-疾病靶点-通路”网络图，如图6所示，该网络中包含70个节点、215条边，平均Degree值为6.33。图6中，近V形图标代表通路，圆形图标为柠檬-当归-乳香复方精油的28个有效活性成分(NM-来自柠檬精油、DG-来自当归精油、RX-来自乳香精油)，正方形图标表示活性成分对应的20个共有靶点。
复方柠檬-当归-乳香精油的有效活性成分中Degree值≥4的有：乙酸橙花酯、乙酸薰衣草酯、3-甲基-N-苯基-4-戊酰胺、丁烯基苯酞、柠檬醛、橙花醛、洋川芎内酯A、乙酸辛酯和E-蒿苯内酯。这些活性成分作用的靶点中，Degree值≥19的有：PIK3CA、CCND1、BCL2和ESR1。靶点主要集中的通路中，Degree值≥5的有：癌症通路、前列腺癌、PI3K/Akt信号通路、神经退行性变化途径-多种疾病、蛋白多糖与癌症、MicroRNAs与癌症和钙信号通路等。
以PI3K/Akt信号通路为例，如图7所示，图中RTK、PI3K、Bcl-2、Cyclin-D1、eNOS为共有靶点所编码的蛋白。PI3K/Akt信号通路已被证实与黑色素瘤生成以及焦虑等神经系统疾病密切相关，其作用机制与磷脂酰肌醇3-激酶(PI3K)的激活和Akt的磷酸化密切相关。PI3K为PI3K/Akt信号通路中关键蛋白，被PIK3CA调控，PI3K可被上游通路的细胞因子等激活以及磷酸化下游蛋白激酶B(Akt)。Akt经磷酸化后，激活NOS3编码的eNOS，催化NO的生成，NO是角质形成细胞旁分泌作用于黑素细胞并促进其合成黑素的一种因子，研究显示，NO含量升高会刺激酪氨酸酶，从而促进黑素分泌增加，其中女性皮肤中的NO生成量和酪氨酸酶活性明显比男性的高，提示雌激素可通过刺激eNOS，促进NO的表达，并且在焦虑小鼠中，NO浓度明显升高。Akt亦可抑制糖原合酶激酶3β(GSK3)，激活CCND1编码的Cyclin-D1，促进细胞从G1期进入S期，进行增殖分化；以及抑制B淋巴细胞瘤-2基因相关启动子(BAD)和激活CREB促进BCL-2的表达，BCL-2为一类重要的抗凋亡蛋白，在黑素瘤细胞及阿尔茨海默病患者星状胶质细胞中常呈现高表达。因此，推测复方柠檬-当归-乳香精油可通过柠檬醛等活性成分作用于PI3K/Akt信号通路中的对应靶点并上下调活性，达到减弱eNOS分泌NO、Cyclin-D1的促细胞增殖以及BCL-2的抗凋亡，起到治疗黄褐斑和焦虑症共症的作用。并且BCL2与CCND1在黄褐斑和焦虑症疾病中的作用效果相似，均与细胞增殖、凋亡有关，提示黄褐斑、焦虑症与黑色素瘤等癌症的发病机制相似，存在细胞周期的相关性。上述GO功能和KEGG通路富集分析结果表明，复方柠檬-当归-乳香精油可以通过多成分、多靶点和多通路的协同作用治疗黄褐斑和焦虑症共症。
5.分子对接结果
筛选PPI网络中度值排名靠前的核心靶点并对照PI3K/Akt信号通路中所富集的靶点，综合选取CCND1、PIK3CA、ESR1作为对接靶点，乙酸橙花酯、丁烯基苯酞、乙酸辛酯以及柠檬醛进行分子对接验证。选择治疗黄褐斑的药物氨甲环酸和治疗焦虑症的苯二氮卓类一线药物地西泮作为阳性对照，见表3。
表3复方精油有效成分与核心靶点(PDB ID)的结合能(KJ/mol)
对复方柠檬-当归-乳香精油的乙酸橙花酯、柠檬醛、丁烯基苯酞和乙酸辛酯活性成分与ESR1、CCND1和PIK3CA三个核心靶点具有较好的结合活性(＜-5KJ/mol)，表明复方柠檬-当归-乳香精油具有治疗黄褐斑和焦虑症的良好分子基础，进一步说明复方精油可以通过多成分、多靶点治疗黄褐斑和焦虑症共症。
二、复方柠檬-当归-乳香精油的制备
1.柠檬精油、当归精油、乳香精油的制备
柠檬精油：将柠檬皮自然风干后，打粉过20目筛，一次药材量为100g装入萃取釜。萃取温度40-45℃，萃取压力15-30MPa；分离压力4-12mPa，CO2流量20-30kg/h，萃取时间为60-120min，得到柠檬精油1-1.2mL。
当归精油：将当归打粉过20目筛，一次药材量为100g装入萃取釜。萃取温度45-48℃，萃取压力20-25MPa，时间80-120min、二氧化碳流速25-35kg/h，得到当归精油1.2-1.5mL。
乳香精油：称取粉碎的乳香树脂100g于蒸馏烧瓶中，加入1000mL水，室温浸泡6h后，将蒸馏烧瓶置于恒温电加热器中加热，蒸馏提取8h以上，收集馏出液，用分液漏斗进行油水分离后，用无水硫酸钠干燥，得到乳香精油4-5mL。
2.复方精油的配制
按柠檬精油50-75％、当归精油15-25％、乳香精油10-25％的比例称取三种精油，混合均匀，即得到复方精油，低温避光保存。
3.复方柠檬-当归-乳香精油抗自由基实验
精油用乙醇稀释；1mmoL/L DPPH溶液、ABTS工作液(用乙醇稀释至A734nm为0.7±0.02范围内)均现配现用。
表4复方精油对DPPH、ABTS自由基清除实验
按表中次序由上往下依次加入各试剂后于常温避光反应30min，若反应液浑浊用0.45μm滤膜过滤，以定容样液溶剂调零，DPPH在517nm处测吸光度A1，或者ABTS在734nm处测吸光度A1。以定容样液的溶剂代替样液作为空白组，测得A0。以无水乙醇溶液代替DPPH溶液或ABTS作为本底组测得A2。以维生素C作阳性对照，试验平行操作3次。
按此公式计算清除率：
复方柠檬-当归-乳香精油对DPPH和ABTS自由基的清除力分别见图8和图9。复方精油对两种自由基的清除率随着剂量增加而增强，且同一剂量下，复方精油优于单方柠檬精油。
4.复方柠檬-当归-乳香精油体外对酪氨酸酶抑制实验
精油溶液：精确称量0.05g精油加入到10mL DMSO中得到浓度为4mg/mL溶液，用DMSO稀释为0.25、0.5、1、2、4mg/mL。
50U/mL酪氨酸酶溶液：精确称量0.004g酪氨酸酶(≥620U/mg)，溶于pH6.8磷酸缓冲溶液中，并定容至50mL，即得50U/mL酪氨酸酶溶液。
2.5mmol/L L-酪氨酸溶液：精密称量0.0453g酪氨酸，pH6.8磷酸缓冲溶液定容至100mL，配制成2.5mmol/L L-酪氨酸溶液。
表5复方精油对酪氨酸酶抑制率测定的反应液组成
按照表5向试管中依此加入酪氨酸酶溶液、样液及磷酸盐缓冲液缓冲液，充分摇匀后于37℃水浴孵化10min，立即加入酪氨酸溶液于37℃避光保温30min，冷却后，以去离子水作为调零管，测定各组样品的A475nm。以熊果苷作阳性对照。试验平行3次，计算酪氨酸酶抑制率：
复方柠檬-当归-乳香精油对酪氨酸酶的抑制率见图10。复方精油对酪氨酸酶的抑制率随着剂量增加而增强，且同一剂量下，复方精油优于单方柠檬精油，也远远优于对照熊果苷。
5.复方柠檬-当归-乳香精油的抗炎性测定
采用鸡蛋白蛋白抑制实验来评价精油的抗炎活性。
pH6.8 PBS磷酸盐缓冲溶液；
蛋清:PBS缓冲液1:1稀释配制蛋清稀释液；
用PBS缓冲液溶解稀释精油得到不同浓度的精油稀释液。
表6复方精油的抗炎实验反应体系
按照表6中次序加入各试剂样品，于37℃水浴孵化15min后，立即置于70℃水浴加热10min，冷却后，以去离子水作为调零管，测定在660nm处的吸光度A1。以PBS缓冲液代替样液作为空白组，测得A0。以PBS缓冲液代替蛋清液作为本底组，测得A2。以布洛芬(标准药物)作阳性对照。试验平行操作3次，计算抑制百分比：
抑制率计算公式：
复方柠檬-当归-乳香精油对白蛋白的变性性的抑制率见图11。复方精油对蛋白变性性的抑制率随剂量增加而增强，且同一剂量下，复方精油优于单方柠檬精油，与对照布洛芬相近。
6.复方柠檬-当归-乳香精油的抗焦虑实验
原理：采用心率变异性(heart rate variability，HRV)作为抗焦虑功效的评价指标。心率变异性是连续心跳间期的微小变异，能够单独表示交感或副交感神经活动的增强或减弱，心率变异性指标包括低频功率(Low frequency power，LF)、高频功率(Highfrequency power，HF)以及低频高频比值(LF/HF)。低频功率，频段(0.04～0.15Hz)表示交感神经系统的活性；高频功率，频段(0.15～0.4Hz)表示副交感神经系统的活性；低频高频比值表示交感和副交感活动的平衡性。以受试者接受精油治疗前后的HRV值的差值是否有显著性差异作为评价指标。急性心理应激(MAT)是影响心率变异性的一个重要因素，且应激下的生理反应是以交感神经活动调解为主的，因而选择采用MAT这一过程达到模拟致焦虑的目的，比较精油涂抹对焦虑情绪的舒缓作用强弱。
6.1实验志愿者招募入选标准
志愿者选择：选择无严重系统疾病、年龄在25～45周岁、具有焦虑情绪的人群，焦虑自评量表(Self-rating anxiety scale，SAS)得分高于40分者的志愿者；
志愿者排除标准：伴有其他器质性精神障碍、精神分裂症；非成瘾性物质所致的抑郁、抑郁性人格和双向情感性障碍，重度抑郁症者或有严重自杀倾向者；有过敏史、激素治疗史以及有其他躯体疾病接受过免疫抑制剂治疗的患者；处于妊娠期、哺乳期的女性；患者在进行测评前2周内接受过中西药抗焦虑治疗；凡不符合纳入标准，未按规定治疗，无法判定疗效或资料不全等影响疗效判断者。
根据以上标准，确定实验对象为25～45岁有焦虑倾向的女性30名，签订知情同意书。
6.2试剂与仪器
用甜杏仁油稀释制备含3％(v/v)的复方精油，课题组自制。随身型心电图记录仪(乐普PC-80D)。
6.3实验环境
等候室(25m2)、数据采集室(15m2)、涂抹室(15m2)。温度均为25±0.5℃。试验过程中保持安静。
6.4实验方法
①对志愿者进行随机分组和作息调控。要求志愿者测试当天避免酒精、尼古丁、咖啡、药物的摄入；测试前一天睡眠至少7小时。对照组使用基础油甜杏仁油，实验组为3％复方精油组。
②HRV指标测试：受试者首先静坐5min，将检测电极贴在双侧手腕背部，保持安静直到各项HRV指标完成检测。接下来给予受试者MAT刺激，MAT刺激结束后5min再测定各项HRV指标。随后给予受试者涂抹精油、对照组为基础油。涂抹结束后受试者静坐20min再次测定各项HRV指标。计算(HRV应激-HRV试样涂抹后)作为评价指标。
③快速心算应激刺激(MAT)：目的是给予短暂的精神刺激达到致焦虑效果。受试者快且准确地完成50道四位数加减的心算题目，由实验者口述题目，志愿者听到题目后快速报出答案。受试者若出现错误，立刻被告知并重新计算，直至正确。
④精油涂抹方法：受试者俯卧于按摩床上，两臂自然下垂。每位受试者涂抹量为5毫升，均匀涂抹于受试者的背部、颈部及肩部。涂抹后受试者静坐休息20min后测定HRV值。
6.5实验结果
表7受试者身体的基本参数
表8各组MRT应激后与涂抹精油后生理指标差值的组间比较
注解：RB＝LFB/HFB，RC＝LFC/HFC；*P＜0.05。
表8显示，心算MRT应激响应的志愿者，各项生理指标在组间的差异不显著，表明志愿者的虚拟焦虑水平相似。受试者在接受3％(v/v)复方精油以及甜杏仁油涂抹后，LF及LF/HF值呈现下降趋势，HF呈现上升趋势。与甜杏仁油涂抹组相比，复方精油涂抹组的LF/HF下降率显著增加(p＜0.05)。
MRT应激后与涂抹精油后，低频高频比值下降率的组间比较结果见图12。
LF/HF这一指标反映的是交感神经与副交感神经的平衡性，是评价植物神经系统紧张或舒缓程度最重要的指标。实验结果表明复方精油可以降低受试者的LF/HF值，精油对人体植物神经系统有较好的自我调节功能。具有降低受试者焦虑水平的作用。
三、复方柠檬-当归-乳香精油甘油体的制备
①按质量百分比组成称取以下原料：复方精油0.5-3.0％、磷脂2-6％、胆固醇0.1-0.5％、甘油10-50％、乙醇5-10％、余量为水。
其中，磷脂为：
80H、
90H、氢化大豆磷脂HSPC-90A、氢化大豆磷脂HSPC-70A、蛋黄卵磷脂PC-98T、蛋黄卵磷脂PL-100M、大豆磷脂酰胆碱S100、
50(P50)、磷脂S75、二油酰基磷脂酰丝氨酸DOPS、二棕榈酰磷脂酰丝氨酸DPPS、二油酰磷脂酰甘油DOPG、二棕榈酰磷脂酰胆碱、卵磷脂和豆磷脂中至少一种，优选氢化大豆磷脂HSPC-90A。
②制备：采用乙醇注入法制备复方精油甘油体。将磷脂2g、胆固醇0.2g、复方精油0.5g混合，用5mL无水乙醇溶解为透明溶液；在500-1000rpm不断搅拌和高于磷脂相转变温度(Tm)的条件下用注射器将透明溶液注入到含有40％甘油的40-50℃超纯水45mL中，至总质量为50g，继续搅拌5min后，室温放置30min，使其充分水化，得到甘油体；将甘油体在冰水浴中超声30次(90W，5s ON和2s OFF)，再用0.22-0.45μm的微孔滤膜整粒，即得含精油浓度为10mg/mL的复方精油甘油体，在4℃下储存。
采用与上述同样方法和配方(配方中不含甘油)制备脂质体。
上述制备得到的复方精油甘油体和脂质体的TEM图分别见图13a和图13b，粒径分布图分别见图14和图15。
③透射电镜观测甘油体形态，激光粒度仪测直径、多分散性指数(P.I.)、Zeta电位。
④变形性测定：挤压法测定，将甘油体通过三张孔径0.22um的滤膜挤出，测定在30s内挤出的囊泡混悬液的重量和过滤后囊泡的粒径(用DLS)。甘油体囊泡膜的弹性用公式计算：E＝J*(rv/rp)2。其中，E代表囊膜的变形性；J指在30s内挤出的囊泡混悬液的重量；rv是挤出后囊泡粒径；rp是滤膜孔径。
⑤药物包封率测定：采用Sephadex G-50微柱离心法分离甘油体和游离药物。用乙醇将甘油体破坏至透明溶液，5mL正己烷萃取，在4℃以4000r/min离心5min,取上清液测定其在273nm处的吸光度，通过标准曲线计算上清液中精油浓度。包封率％＝C包/C总×100计算。
表9精油甘油体与脂质体测定的基本参数比较
由以上可知，在所述制备工艺条件下，所得复方精油甘油体与脂质体的平均粒径相差不大，但变形性有极大差异。
⑥体外皮肤渗透实验：采用Franz垂直扩散池，有效扩散面积为2.8cm2。剔除猪皮皮下组织及脂肪，生理盐水洗净，猪皮厚度为2mm，在25℃的PBS溶液中平衡后，吸干表面水分后夹在供体室和受体室之间，角质层面向供体室。受体室中有6.5mLPBS溶液，扩散池置于37±1℃水浴、100r·min-1电磁搅拌。分别移取精油甘油体和脂质体各0.5mL于猪皮上，密封，在2，4，6，8，12h时抽取接受液1mL，并补加同温接受液1mL。抽取液用正己烷萃取后，采用UV法测定。实验结束，取下猪皮，用生理盐水将皮肤表面样品冲洗干净，用外科手术刀将表皮与真皮分离，将表皮和真皮剪碎后用正己烷提取精油，UV测定精油含量，计算累积渗透药量、稳态流速、时滞和皮内药物滞留量。Q＝(Cn×V+∑Cn-1×Vn)/A其中，Q为单位面积内药物透过量(μg/cm2)、V为接收池的体积6.5mL、A为有效透过面积2.8cm2、Cn是时间为n时的药物浓度。将所得实验结果以时间为横坐标、累积药物透过量为纵坐标画图，将渗透曲线12小时Q/A-t进行线性回归得方程：y＝ax+b，其中a＝J(渗透速度)，t时滞＝-b/a。
表10精油甘油体与其脂质体的透皮参数对比
注：经皮渗透中，精油C0均为10mg/mL，给药量为5mg。
与传统脂质体相比，甘油体处于更流体的状态。体外透皮给药实验表明，当使用30％的甘油体时，复方精油在皮肤内的沉积和渗透得到改善。
四、含复方精油甘油体的乳液制备
1.配方
表11含复方精油甘油体的乳液的配方
2.制备
①B水相配制：把B相加入到反应罐中加热到75-80℃溶解，得到透明溶液；
②油相的配制：A相倒入另一反应罐中加热到75-80℃溶解透明后，保温；
③乳化：在不断搅拌下将配制的A相倒入配制的B相中，保温75～80℃搅拌均质10～15min；
④当温度降到40～50℃，再将C相加入，在同一温度下均质2分钟后，体系降温到45℃以下加入D相搅匀，检验合格后出料。
3.对皮肤保湿性测试
参照化妆品保湿功效评价指南QB/T 4256-2011方法测定。选取符合条件的志愿者年龄在18岁～65岁26人(妊娠或哺乳期妇女除外)参加测试。测试部位为前臂内侧。样品及空白对照测试区域分布于志愿者前臂内侧，每个区域大小为3.0cm×3.0cm，样品用量为(2.0±0.1)mg/cm2。测试人员在产品使用前、后进行皮肤表面水分含量测试。测试环境温度∶20℃～25℃，湿度：40％～60％。含精油甘油体的乳液(实施例1)和不含精油甘油体的乳液(实施例2)对皮肤保湿性对比测试结果见图16。可见，含甘油体的乳液保湿效果更平稳，这是因为甘油体在表皮层和真皮层中易滞留所致。
4.抗黄褐斑的效果评价
试验对象：治疗组-实施例1、对照组-实施例2。
试验方法：
(1)方案：采用治疗组(实施例1)与对照组(实施例2)进行随机对照试验研究。
(2)分组：黄褐斑患者30～40岁，30例，女性。随机分为治疗组和对照组进行实验。组间年龄、病情、病程评分等一般资料经统计学检验要求无差异性(p＞0.05)，具有齐同可比性。
(3)治疗方法：测试对象每日晨、晚在面部涂抹实施例1～2样品，每次涂抹时间2～5min，涂抹量以见涂抹层即可。期间不用其他化妆品，4周为1疗程，共3个疗程后评价效果。
(4)疗效评定：
基本治愈：①肉眼察色的消退斑面积＞90％，皮损的颜色能够基本消失；②评分法计算，治疗后的下降指数≥0.8；
显效：①肉眼察色的消退斑面积＞60％，皮损的颜色明显能够变淡；②评分法计算，治疗后的下降指数≥0.5；
有效：①肉眼察色的消退斑面积＞30％，皮损的颜色能够变淡；②评分法计算，治疗后的下降指数≥0.3；
无效：①肉眼察色的消退斑面积＜30％，皮损的颜色变化不怎么明显；②评分法的计算，治疗后的下降指数≥0；
统计学方法：运用Spsstor Windows软件包，采用t检验，p＜0.05有显著性差异。
表12两组病例疗效比较
| 治愈 | 显效 | 有效 | 无效 | 总有效数 | |
| 治疗组 | 5 | 7 | 2 | 1 | 14 |
| 对照组 | / | 1 | 3 | 13 | 4 |
结果分析：治疗组与对照组之间经秩和检验p＜0.05，表明两组治疗后疗效差异有显著性意义。
Claims (8)
1.一种复方精油甘油体，其特征在于，该复方精油甘油体的质量百分比组成包括：复方精油0.5-3.0％、磷脂2-6％、胆固醇0.1-0.5％、甘油10-50％、乙醇5-10％、余量为水，所述的复方精油的质量百分比组成为：柠檬精油50-75％、当归精油15-25％、乳香精油10-25％。
2.根据权利要求1所述的一种复方精油甘油体，其特征在于，所述的柠檬精油的制备方法为：将柠檬皮干燥粉碎后，采用超临界流体萃取技术提取得到柠檬精油，萃取条件为：40-45℃、压力15-30MPa、时间60-120min、二氧化碳流速20-30kg/h。
3.根据权利要求1所述的一种复方精油甘油体，其特征在于，所述的当归精油的制备方法为：将当归根茎干燥粉碎后，采用超临界流体萃取技术提取得到当归精油，萃取条件为：温度45-48℃、压力20-25MPa、时间80-120min、二氧化碳流速25-35kg/h。
4.根据权利要求1所述的一种复方精油甘油体，其特征在于，所述的乳香精油的制备方法为：称取100g乳香碎片于蒸馏烧瓶中，加入1000mL水，室温浸泡6h后，将蒸馏烧瓶置于恒温电加热器中加热，蒸馏提取8h以上，收集馏出液，用分液漏斗进行油水分离后，用无水硫酸钠干燥，得到乳香精油。
5.权利要求1-4中任一项所述的复方精油甘油体的制备方法，其特征在于，包括以下步骤：
(1)将柠檬精油、当归精油、乳香精油混匀，得到复方精油；
(2)按质量百分比组成称取以下原料：复方精油0.5-3.0％、磷脂2-6％、胆固醇0.1-0.5％、甘油10-50％、乙醇5-10％、余量为水；
(3)采用乙醇注入法制备复方精油甘油体：将磷脂2-6g、胆固醇0.1-0.5g、复方精油0.5-3.0g混合，用5-10mL无水乙醇溶解为透明溶液；在500-1000rpm不断搅拌的条件下用注射器将透明溶液注入到含有10-50％甘油的40-50℃超纯水85-90mL中，继续搅拌5min后，室温放置30min，使其充分水化，得到甘油体；将甘油体在冰水浴中超声，最后定容到100mL，再用0.22-0.45μm的微孔滤膜整粒，即得含精油浓度为5-30mg/mL的复方精油甘油体，在4℃下储存。
6.根据权利要求5所述的一种复方精油甘油体的制备方法，其特征在于，所述的磷脂为氢化磷脂、氢化大豆磷脂、蛋黄卵磷脂、大豆磷脂酰胆碱、磷脂酰胆碱、二油酰磷脂酰丝氨酸、二棕榈酰磷脂酰丝氨酸、二油酰磷脂酰甘油、二棕榈酰磷脂酰胆碱和豆磷脂中至少一种。
7.权利要求1-4中任一项所述的复方精油甘油体作为具有抗黄褐斑与焦虑症双重作用的化妆品的应用。
8.权利要求1-4中任一项所述的复方精油甘油体作为具有抗黄褐斑与焦虑症双重作用的原料或中间体，在与其他原料复配为化妆品中的应用。
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