CN114767600A - 含有柠檬桃金娘和绣球菌混合提取物的抗氧化、抗炎、抗污染的组合物 - Google Patents
含有柠檬桃金娘和绣球菌混合提取物的抗氧化、抗炎、抗污染的组合物 Download PDFInfo
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Abstract
本发明涉及一种抗氧化或抗炎组合物,其包括以柠檬桃金娘和绣球菌的混合提取物作为活性成分,柠檬桃金娘和绣球菌的混合提取物具有DPPH自由基清除活性及显著的脂质过氧化抑制效果,具有优异的抑制炎症的效果,且抗炎效果明显。因此,柠檬桃金娘和绣球菌的混合提取物可以用于药物、食品、化妆品等中,以达到抗氧化和/或抗炎的目的。此外,利用其抗氧化和抗炎作用,可用作抗污染材料。
Description
技术领域
本发明涉及一种抗氧化、抗炎和抗污染的组合物,其包含柠檬桃金娘和绣球菌的混合提取物。
背景技术
细尘是指粒径在10μm以下的尘埃,简称PM10(Particulate Matter 10),粒径在2.5μm以下的超细尘归类为PM2.5(Particulate Matter 2.5)。它主要是在燃烧化石燃料时产生,来自依赖煤炭度为70%的中国的细粉尘占很大一部分。此外,黄沙是指亚洲大陆中部的中国、蒙古等地的沙漠、黄土地区的细沙、黄土、尘埃漂浮在天空中,飞到空气上层并被风吹走的现象,其作为可远距离移动的空气污染物可能落入济州岛。
特别地,当细尘直接接触位于我们身体最外部的皮肤时,皮肤会在有机体和外部环境之间起屏障作用,如果频繁直接暴露在污染中,该屏障作用会下降,从而引发各种问题。特别是细尘比毛孔小20倍,很容易渗入人体皮肤。例如,与通过呼吸***吸入有毒物质如氯仿等物质一样,皮肤被认为是作为吸收环境中的污染物质的主要场所。
细粉尘在身体或皮肤中引起氧化应激和炎症。以这种方式诱发的炎症会影响全身,并增加皮肤炎症性疾病、脱发、过敏性皮炎恶化、中风、脑部炎症性疾病(如阿尔茨海默症)、呼吸***疾病和心血管疾病的风险。
目前,对由细尘等刺激引起的炎症反应和氧化应激具有保护皮肤作用的抗炎和抗氧化功能材料的需求正在迅速增加,因此,需要从具有安全性的天然材料中获得效果好和副作用小的新材料。
在先申请专利文献:(专利文献0001)大韩民国注册专利第10-1851603号。
发明内容
本发明要解决的问题是提供含有柠檬桃金娘和绣球菌的混合提取物为活性成分的抗氧化、抗炎用组合物,该组合物包括食品组合物、化妆品组合物、药品组合物和医药外品组合物。
本发明的另一个目的是提供一种用于抗污染的化妆品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
本发明的一个实施例是一种抗氧化或抗炎的食品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
上述混合提取物可以通过将柠檬桃金娘和绣球菌以1:0.1~10的重量比混合来提取。
上述混合提取物可以是柠檬桃金娘和绣球菌的热水提取物。
本发明的另一个实施例是一种抗氧化或抗炎的化妆品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
本发明的另一个实施例是用于预防或治疗炎症性疾病的药物组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
上述炎症性疾病可以是大脑或神经炎性疾病。
本发明的另一个实施例是一种抗氧化或抗炎的医药外品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
本发明的另一个实施例是一种抗污染的化妆品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
上述抗污染作用可以是抑制由沙尘或细尘引起的炎症或氧化应激。
本发明涉及一种抗氧化或抗炎组合物,其包含柠檬桃金娘和绣球菌的混合提取物。柠檬桃金娘和绣球菌的混合提取物具有优异的DPPH自由基清除活性和脂质过氧化抑制作用,并已证实能有效抑制炎症,因此可用于抗氧化或抗炎用途。
此外,柠檬桃金娘和绣球菌的混合提取物可用于开发抗污染用化妆品,以缓解由细尘引起的氧化应激和炎症。
具体实施方式
在下文中,将对本发明的具体实施例进行详细描述,以便本领域普通技术人员能够容易地实施本发明。然而,本发明可以以几种不同的形式实施并且不限于所描述的实施例。
在一个方面,本发明涉及一种抗氧化剂或抗炎的食品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
本发明的抗氧化或抗炎的食品组合物包括柠檬桃金娘和绣球菌的混合提取物的活性成分。
在本发明中,“柠檬桃金娘(Backhousia citriodora)”主要是指生长于澳大利亚昆士兰热带森林里的灌木。柠檬桃金娘的叶子具有柠檬香味,是澳大利亚使用最广泛的调味材料,最近被用作凉茶、糖果、饮料、营养补充剂等的原料或调味剂。此外,柠檬桃金娘精油在各种带有柠檬香味的油类中香味最强,留香时间最长,主要用于制作香皂、润唇膏或香水。柠檬桃金娘的柠檬香源自柠檬醛(citral)成分,与含有3至10%左右柠檬醛的柠檬不同,柠檬桃金娘含有约90%的柠檬醛,因此比柠檬散发更强的味道或香气,但据报告,其涩味和苦味也更强烈。柠檬醛是单萜类醛成分,已知不溶于水,主要溶于醇或醚。
在本发明中,“绣球菌(Sparassis crispa)”是属于多孔菌目(Aphyllophorales)、绣球菌科(Sparassidaceae)的菇菌,分布于韩国、日本、中国、欧洲、北美、澳大利亚等,在8~9月长于针叶树的根部附近或树桩上,子实体高10~25cm,呈肉质,底部由一个粗壮的柄上发出许多分枝,上部扁平边缘呈波浪状,呈白色花卷心菜形状,散发淡淡的香气且咀嚼质感好,具有食用价值。此外,其β-1,3-D-葡聚糖的含量明显高于其他蘑菇,已知具有多种生理活性作用。即,已知具有抗癌和抗血管增生作用、抗肿瘤作用、抗菌和抗真菌作用以及抗过敏作用,甚至对糖尿病也有效果。它还参与小鼠体内细胞因子的产生,对急性肝损伤有效。
在本发明中,“提取物”是指通过提取处理获得的提取物、提取物的稀释或浓缩液、将提取物干燥获得的干燥品、提取物的制备物或纯化物、或它们的混合物;它包括可以使用提取物本身和提取物形成的所有配方的提取物。
在本发明中,提取物的干燥植物中的有用成分没有被破坏。可以通过公知的方法,例如,可以通过在阴凉处自然干燥的方法进行。此外,可以通过破壁或粉碎成粉末,以在随后的提取过程中可以充分提取植物的有用成分。干燥、粉碎或破壁工序可以根据需要颠倒顺序或重复进行。
提取物可以按照植物提取物的常规制备方法制备,具体可以采用溶剂提取法、水浸提取法、冷浸提取法、温浸提取法或超声波提取法等,可以使用现有的提取装置、超声波粉碎提取器、分馏器等。
提取物的提取溶剂可以是水或有机溶剂。有机溶剂可以是极性溶剂、非极性溶剂或它们的混合物,例如醇、醇水溶液、己烷、二氯甲烷、丙酮、乙酸乙酯、***、氯仿或它们的混合物。醇为C1-C5的醇,例如甲醇、乙醇、丙醇、丁醇、异丙醇等。此外,可以通过将醇在水中稀释至50~99.9%(v/v)来获得醇水溶液。
此外,用溶剂提取的提取物,然后用选自己烷、二氯甲烷、丙酮、乙酸乙酯、***、氯仿、水和它们的混合物的任一种溶剂进行分馏过程。分馏过程可以使用两种以上的溶剂制备,各溶剂馏分可以根据溶剂的极性依次使用或混合使用。
然后可以将制备的提取物或通过分馏过程获得的组分进行过滤或浓缩或干燥以除去溶剂,并且可以同时进行过滤、浓缩和干燥。具体而言,例如,过滤可使用滤纸或减压过滤器,浓缩可使用减压浓缩器例如旋转蒸发器在减压下浓缩,干燥可通过冷冻干燥方法进行。
在本发明中,“混合提取物”是指将柠檬桃金娘和绣球菌提取后制备的提取物,可以分别将柠檬桃金娘及绣球菌提取后混合制成,也可以将柠檬桃金娘及绣球菌混合后提取制成。
柠檬桃金娘可以是选自茎、叶、根、花和果实中的至少一种,优选柠檬桃金娘叶。
在本发明的一个实施例中,将干燥的柠檬桃金娘和绣球菌混合后,用蒸馏水作为溶剂,在80~120℃下的加压提取1~5h。
因此,本发明的混合提取物可以是柠檬桃金娘和绣球菌的热水提取物,具体而言,可以是90~110℃的热水提取物,提取时间为2~4h。
在本发明的一个实施例中,将柠檬桃金娘和绣球菌以1:0.1~10的重量比混合制备提取物,根据柠檬桃金娘和绣球菌的混合比例对提取物的细胞毒性进行分析,结果显示,柠檬桃金娘和绣球菌重量比为1:1(实施例1)混合的提取物未显示出细胞毒性。
因此,在本发明中,混合提取物可以是将柠檬桃金娘和绣球菌以1:0.1~10的重量比混合提取,具体地,柠檬桃金娘和绣球菌的重量比为1:0.5~5,优选为1:1,以90~110℃的热水提取制备得到。
在本发明的一个实施例中,证实了柠檬桃金娘和绣球菌的混合提取物的浓度(实施例1,LB)与DPPH清除活性增加正相关。为了分析抗氧化作用,利用LPS诱发小鼠脑部炎症后,给予柠檬桃金娘和绣球菌的混合提取物(LB)来分析脂质过氧化物(MDA)的生成变化,该脂质过氧化物(MDA)是氧化损伤的指标,结果显示,LPS使实验动物大脑中MDA的生成量增加了170%左右,这意味着LPS引发了氧化损伤。已证实,注射高剂量(1000mg/kg)柠檬桃金娘和绣球菌的混合提取物(LB)时,因LPS增加的MDA生成量在统计上得到了显著降低。
即,证实柠檬桃金娘和绣球菌的混合提取物具有DPPH清除活性,并且通过抑制由氧化损伤引起的脂质过氧化物(MDA)而具有优异的抗氧化作用,该提取物可以用作抗氧化组合物。
在本发明中,“抗氧化”是指保护细胞免受氧化应激,具体地,抑制活性氧的产生或抑制脂质过氧化物的产生。
在本发明的另一个实施例中,分析了柠檬桃金娘和绣球菌(LB)的抗炎作用,利用LPS诱导小鼠脑部炎症后,将柠檬桃金娘和绣球菌的混合提取物(LB)口服给药7天后,分析小鼠脑组织中炎症相关细胞—小胶质细胞(microglia)和星形胶质细胞(astrocyte)的增加程度。分析结果显示,LPS在实验动物的大脑中诱导GFAP阳性细胞(一种星形胶质细胞标志物)和Iba-1阳性细胞(一种小胶质细胞标志物)的增加。经证实,当口服柠檬桃金娘和绣球菌的混合提取物(LB)时,可显著减少因LPS诱导的星形胶质细胞(GFAP阳性细胞)和小胶质细胞(Iba-1阳性细胞)的增加。
即,证实了柠檬桃金娘和绣球菌的混合提取物具有抗炎作用,本发明的柠檬桃金娘和绣球菌的混合提取物可以用作抗炎组合物。
在本发明中,“抗炎”是指抑制或预防炎症的发生,或保护细胞免受炎症损伤。
在本发明中,炎症可由微生物感染、暴露于沙尘或细尘、暴露于化学品或氧化应激引起,但不限于此。
本发明的食品组合物可以包括丸剂、散剂、颗粒剂、针剂、片剂、胶囊剂或液体的形式,对可以添加本发明的柠檬桃金娘和绣球菌混合提取物的食品种类没有特别限制,例如各种饮料、口香糖、茶、维生素复合物、健康功能性食品等。
食品组合物除柠檬桃金娘和绣球菌混合提取物外,还可以添加其他成分,其种类没有特别限制。例如,如普通食品一般,它可以包含各种草药提取物、食品中允许的食品添加剂或天然碳水化合物作为附加成分,但不限于此。
在本发明中,“食品添加剂”是指可以补充添加到食品中的成分,添加到制造各剂型的健康功能性食品中,可由本领域人员适当选择使用。食品添加剂的实例包括各种营养素、维生素、矿物质(电解质)、调味剂如合成和天然调味剂、着色剂和填充剂、果胶酸及其盐、海藻酸及其盐、有机酸、保护胶体增稠剂、pH调节剂、稳定剂、防腐剂、甘油、酒精、碳酸饮料中使用的碳酸化剂等,但上述例子不限于本发明的食品添加剂的种类。
天然碳水化合物的例子包括单糖如葡萄糖和果糖;二糖如麦芽糖和蔗糖;多糖如糊精和环糊精;以及糖醇如木糖醇、山梨糖醇和赤藓糖醇;除上述物质外,还可以合理使用天然调味剂(如奇异果甜蛋白)、甜叶菊提取物(如莱鲍迪苷A、甘草甜素等)和合成调味剂(如糖精、阿斯巴甜等)。
对上述食品的种类没有特别限制。可以添加上述物质的食品的例子包括肉类、香肠、面包、巧克力、糖果、零食、饼干、比萨饼、拉面、其他面条、口香糖、包括冰淇淋在内的乳制品、各种汤、饮料、茶、口服液、酒精饮料和维生素复合物等。
食物组合物可以是健康功能性食品组合物。
在本发明中,“健康功能性食品”是指使用对人体有用的功能性原料或成分制成片剂、胶囊剂、散剂、颗粒剂、液体剂和丸剂的形式制造及加工的食品。这里,术语“功能性”是指对人体的结构和调节营养或生理作用的功能上获得有益于健康的效果。本发明的健康功能性食品可以采用本领域常用的方法制备,在制造时加入本领域常用的原料和成分即可。此外,与一般药物不同,它以食品为原料,长期服用时不会产生副作用,携带性极佳。
健康功能性食品以内在美食品(inner beauty)形态摄取,优点是具有更优秀的效果。内在美食品(inner beauty)是被称为“可以吃的化妆品或美容食品”的食品,是指对皮肤有益的各种成分被体内吸收,使肤质变得健康的食品,就像选择适合皮肤类型的化妆品一样,可以考虑皮肤状态和生活方式,选择适合个人的内在美食品进行摄取。更优选地,若将含有上述柠檬桃金娘和绣球菌的混合提取物的化妆品和含有上述柠檬桃金娘和绣球菌的混合提取物的内在美食品一起使用时,与只使用化妆品相比,抗氧化或抗炎效果明显提高,其特点是具有更有效的抗氧化或抗炎效果。
活性成分的混合量可以根据使用目的(预防、健康功能性或治疗)适当确定。一般来说,本发明的柠檬桃金娘和绣球菌的混合提取物在食品制备中的添加量可以为原料组合物重量的1-50%,优选5-10%,但不限于此。然而,出于健康和卫生目的,或为了健康调节而长期摄入的情况下,使用量可以低于上述范围。
另一方面,本发明涉及一种抗氧化或抗炎化妆品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
在本发明中,柠檬桃金娘、绣球菌、混合提取物、抗氧化和抗炎的描述如前所述。
除了柠檬桃金娘和绣球菌的混合提取物之外,本发明的化妆品组合物还可以包括已知具有抗氧化或抗炎作用的化合物或天然提取物,以增强柠檬桃金娘和绣球菌的抗氧化或抗炎作用。
在本发明的抗氧化或抗炎用化妆品组合物中,根据用途、制剂、制剂目的等,可以包含任意量(有效量)的活性成分,只要它可以表现出抗氧化或抗炎作用,常规有效量可以包括在基于组合物总重量的0.001wt%~99.99wt%的范围内。这里,“有效量”是指活性物质的量能够诱导抗氧化或抗炎作用的量。这种有效量可以在本领域普通技术人员的普通能力范围内通过试验确定。
本发明的化妆品组合物可以制备成各种形式,例如,本发明的化妆品组合物可以制备成本领域常规制备的任何剂型,例如溶液、悬浮液、软膏、凝胶、面霜、乳液、散粉、洗面奶、精油、粉底、乳液粉底、蜡质粉底、喷雾等,但不限于此。此外,具体来说,它可以选自润肤露、柔肤水、爽肤水、收敛剂、护肤液、乳液、保湿乳液、滋养乳液、按摩霜、滋养霜、保湿霜、护手霜、精华、滋养精华、面膜、香皂、洗发水、洁面泡沫、洁面乳、洁面霜、身体乳液、沐浴露、乳液、口红、隔离霜、粉底液、粉饼和散粉的群组中各种类型,但不限于此。
除了其活性成分外,本发明的化妆品组合物可以包含化妆品中可接受的载体。这里,“化妆品可接受的载体”是指已知和使用的可以包含在化妆品中的化合物或组合物,或未来将开发的化合物或组合物,在与皮肤接触时,其毒性、不稳定性或刺激性不超过现有技术中人体的承受量。上述载体在本发明的化妆品组合物中,约占总重量的1%~99.99%之间,优选为5%~99%。然而,由于上述比例根据本发明化妆品的配方及其具体应用部位(面部或手部)或所需的适用量而有所不同,因此上述比例不能被理解为从任何方面限制本发明的范围。
另一方面,上述载体可以为如醇、油、表面活性剂、脂肪酸、硅油、润湿剂、保湿剂、粘度调节剂、乳液、稳定剂、防晒剂、显色剂、香料等。可用作载体的醇类、油类、表面活性剂、脂肪酸、硅油、润湿剂、保湿剂、粘度调节剂、乳化剂、稳定剂、防晒剂、着色剂、香料等是本领域已知的,本领域的技术人员可以选择和使用适当的材料/组合物。
另一方面,本发明涉及用于预防或治疗炎症性疾病的药物组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
本发明中的柠檬桃金娘、绣球菌、混合提取物的描述如前所述。
在本发明中,“炎症性疾病”是由炎症(inflammation)引起的疾病,炎症可能是由微生物(细菌、病毒、真菌)感染、沙尘暴、细尘、接触化学品或氧化应激引起,但不限于此。
炎症性疾病可能是过敏性疾病、炎症性肠道疾病、动脉粥样硬化、炎症性胶原血管病、肾小球肾炎、炎症性皮肤病、结节病、视网膜炎、胃炎、肝炎、肠炎、关节炎、扁桃体炎、咽炎、支气管炎、肺炎、胰腺炎、败血症、脑或神经炎症性疾病和肾炎中的一种。
在本发明的上述实施方案中,柠檬桃金娘和绣球菌的混合提取物(LB)抑制了利用LPS引发实验动物大脑中与炎症相关的细胞—星状胶质细胞和小胶质细胞的增加。经证实,柠檬桃金娘和绣球菌的混合提取物可通过抑制炎症来预防或治疗炎症性疾病。
此外,证实了柠檬桃金娘和绣球菌的混合提取物抑制脑组织的炎症,因此,在本发明中,炎症性疾病具体可以是大脑或神经炎症性疾病。
大脑或神经炎症性疾病可能是由氧化应激、过度炎症反应引起的退行性脑病,具体来说,可以是阿尔茨海默症(Alzheimer's disease)、帕金森氏症(Parkinson'sdisease)、亨廷顿舞蹈症(Huntington's disease)及肌萎缩侧索硬化症(ALS;AmyotrophicLateral Sclerosis)中的一种。
在本发明中,“治疗”是指作为本发明的对象疾病的感染性疾病或炎症性疾病的减轻、抑制、镇静或根除。在本发明中,“预防”是指抑制或延缓作为本发明的目标疾病的感染性疾病或炎症性疾病的发作的任何作用,并且包括治疗意义。
本发明的药物组合物可以按照药学有效剂量给药。
在本发明中,“药学有效剂量”是指足以以适用于医疗的合理受益/风险比治疗疾病的量,有效剂量标准可由包括受试者类型和严重程度、年龄、性别、疾病类型、药物活性、对药物的敏感性、给药时间、给药途径和***率、治疗持续时间、同时使用的药物在内的因素以及其他医学领域众所周知的因素决定。本发明的组合物可以作为单独的治疗剂使用或可以与其他治疗剂组合使用,也可以与常规治疗剂依次或同时使用。并且可以单次或多次给药。重要的是,考虑到所有上述因素,在不产生副作用的情况下,给予最小的量能够获得最大的效果,并且可以由本领域的普通技术人员容易地确定。
本发明的药物组合物只要是以治疗或预防感染性疾病或炎症性疾病为目的的个体即可,没有特别限制,任何个体都可以适用。例如,非人动物如猴子、狗、猫、兔、豚鼠、大鼠、小鼠、牛、羊、猪、山羊等,人类、鸟类和鱼类等均可使用,药物组合物可用于非肠道给药、皮下、腹腔内、肺内和鼻腔内,并在必要时通过包括病灶内给药在内的任何合适方法进行局部治疗。本发明的药物组合物的优选剂量根据个体的状况和体重、疾病程度、药物形式、给药途径和周期而有所不同,但是本领域技术人员可以适当地选择。例如,可以通过口服、直肠或静脉、肌肉、皮下、子宫内隔膜或脑血管内注射给药,但不限于此。
所述药物组合物可以选自片剂、丸剂、散剂、颗粒剂、胶囊剂、混悬剂、内服液体制剂、乳剂、糖浆剂、无菌水溶液、非水溶液、混悬剂、乳剂、冻干剂和栓剂中的任一种剂型,也可以是口服或非肠道的各种剂型。制剂化时,可以用常用的如填充剂、增量剂、粘合剂、润湿剂、崩解剂和表面活性剂等稀释剂或赋形剂制备。
用于口服给药的固体制剂包括片剂、丸剂、散剂、颗粒剂、胶囊剂等,此类固体制剂包括至少一种赋形剂,它可以通过混合例如淀粉、碳酸钙、蔗糖(sucrose)或乳糖(lactose)、明胶等来制备。除了单纯的赋形剂外,还可以使用如硬脂酸镁、滑石粉等润滑剂。用于口服给药的液体制剂包括混悬剂、内服液体制剂、乳剂、糖浆剂等,除常用的简单稀释剂水和液体石蜡外,还可包括润湿剂、甜味剂、香料和防腐剂等各种赋形剂。用于肠胃外给药的制剂包括无菌水溶液、非水溶液、混悬剂、乳剂、冻干制剂和栓剂。非水溶剂和混悬剂可包括丙二醇、聚乙二醇、植物油如橄榄油和可注射酯如油酸乙酯。作为栓剂的基质,可以使用合成脂肪酸酯(witepsol)、聚乙二醇、吐温(tween)61、可可脂、月桂酸甘油酯、甘油明胶等。
适当的日总量可由主治医师在合理的医学判断范围内确定,一般为0.001~1000mg/kg,优选0.05~200mg/kg,更优选0.1~100mg/kg,可以每天一次到数次分次给药。
另一方面,本发明涉及一种抗氧化或抗炎的医药外品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
本发明中的柠檬桃金娘、绣球菌、混合提取物、抗氧化和抗炎的描述如上所述。
在本发明中,“医药外品”是指以诊断、治疗、改善、缓解、处理或预防人类或动物疾病为目的的物品中,比药品作用温和的物品。例如,根据《药师法》,医药外品是指除作为药品用途以外的产品,包括用于治疗或预防人和动物疾病的纺织品和橡胶产品,对人体的作用轻微或没有直接作用、类似非器具或机器的物品,预防它的消毒剂和杀虫剂等。
本发明的医药外品的种类或剂型没有特别限定,但优选消毒剂、沐浴露、漱口水、湿纸巾、洗衣皂、洗手液、加湿器填充剂、口罩、软膏或过滤填充剂等。
当本发明的组合物包含在用于抗氧化或抗炎目的的医药外品中时,该组合物可以直接使用,也可以与其他医药外品成分一起使用,并且可以根据常规方法适当使用,活性成分的混合量可以根据使用目的适当确定,根据本发明的医药外品组合物中,柠檬桃金娘和绣球菌的混合提取物相对组合物总重量可以占比重0.01~20%。
另一方面,本发明涉及一种用于抗污染的化妆品组合物,其包含柠檬桃金娘和绣球菌的混合提取物作为活性成分。
本发明中的柠檬桃金娘、绣球菌、混合提取物和化妆品的描述与上述相同。
在本发明中,“抗污染”是指抑制由沙尘或细尘引起的氧化应激或炎症。
在本发明的上述实施例中,证实了柠檬桃金娘和绣球菌的混合提取物具有优异的DPPH自由基清除活性,并且证实了通过抑制由LPS在小鼠中诱导的脂质过氧化物的产生而具有优异的抗氧化作用,通过抑制或减少沙尘或细尘在细胞中诱导的氧化应激,可以保护细胞免受沙尘或细尘的侵害。
此外,当用LPS诱导小鼠的脑组织中产生炎症时,口服给予小鼠柠檬桃金娘和绣球菌的混合提取物,与仅给予LPS的对照组相比,柠檬桃金娘和绣球菌的混合提取物能抑制跟炎症相关细胞—星形胶质细胞(GFAP阳性细胞)和小胶质细胞(Iba-1阳性细胞)的生成,本发明的柠檬桃金娘和绣球菌的混合提取物可以通过抑制或预防细尘引起的炎症来保护细胞。
此外,经证实,口服柠檬桃金娘和绣球菌的混合提取物可抑制脑组织炎症,这可能是为了保护细胞免受损伤。
此外,经证实,口服柠檬桃金娘和绣球菌的混合提取物抑制了脑组织中的炎症,本发明的抗污染可以特异性地保护细胞免受因细尘诱发的炎症引起的神经或脑细胞损伤。
以下,将通过实施例更详细地描述本发明的构成和效果。这些实施例只是为了举例说明本发明,本发明的范围并不限定于这些实施例。
1.柠檬桃金娘和绣球菌的混合提取物的制备
将干燥的柠檬桃金娘和绣球菌按表1所示的重量比混合后,以蒸馏水为溶剂,使用加压提取器在100℃下混合提取3小时,过滤,浓缩,-75℃冷冻干燥,干燥品用作样品。
对制备的混合提取物进行细胞毒性分析的结果显示,实施例1没有细胞毒性。
表1
2.体外(in-vitro)抗氧化作用分析
1)DPPH自由基清除活性分析
将实施例1、对比例1和2中制备的样品溶解在蒸馏水中,使其浓度为1000μg/ml并用于实验。DPPH(1,1-diphenyl-2-picrylhydrazyl)溶液是将3mg的DPPH溶解在15ml乙醇中,取1.5ml溶液在其中加入3ml乙醇和0.5mlDMSO制成。
DPPH自由基清除活性,将50μl样品和DPPH溶液混合并在室温下反应10分钟,然后使用紫外分光光度计在517nm处测量残留自由基的浓度。
DPPH自由基清除活性按照如下数学公式1计算了给电子能力(electron donatingability,EDA%)。
【数学公式1】
EDA(%)=(B-A)/B×100
A:加样组的吸光度,B:不含样品组的吸光度
表2
区分 | DPPH清除自由基能力 |
实施例(LB) | 88.91 |
对比例1(L) | 67.59 |
对比例2(B) | 71.44 |
为了测定柠檬桃金娘和绣球菌的混合提取物(实施例1,LB)的体外抗氧化效果,对DPPH自由基清除活性(%)进行分析的结果显示,柠檬桃金娘和绣球菌的混合提取物(实施例1,LB)比柠檬桃金娘(对比例1,L)或绣球菌(对比例2,B)的单独提取物表现出更优异的DPPH清除活性。
3动物模型(体内)的抗氧化和抗炎作用分析
1)实验动物的准备
雄性ICR小鼠(6周龄)购自Samtaco Bio,适应1周后使用。动物在温度为22±1℃、湿度为50±10%的个体饲养单元中饲养,其中日/夜设置为每12小时循环,水和食物作为自主饮食提供。实验组是由没有加入LPS(lipopolysaccharide)的未用药对照组(sham)、LPS对照组(control)、柠檬桃金娘和绣球菌的混合提取物(LB)与LPS一起加入的柠檬桃金娘和绣球菌的混合提取物低剂量组(100LB,100mg/kg)、柠檬桃金娘和绣球菌的混合提取物(LB)高剂量组(1000LB,1000mg/kg)、柠檬桃金娘单一提取物(L)与LPS一起加入的柠檬桃金娘单一提取物高剂量组(1000L,1000mg/kg)、绣球菌单一提取物(B)与LPS一起加入的绣球菌单一提取物高剂量组(1000B,1000mg)/kg),以及阳性对照组(阿魏酸,ferulic acid)组成。
2)药物(样本)给药和脑样本采集
柠檬桃金娘和绣球菌的混合提取物给药剂量设定为低剂量(100LB,100mg/kg)和高剂量(1000LB,1000mg/kg),柠檬桃金娘单一提取物(L)及绣球菌单一提取物(B)给药剂量设定为高剂量(1000mg/kg),阳性对照组给予阿魏酸20mg/kg。
将LPS溶解在PBS中,以1μg/3μl的剂量施用于第三脑室,将每个样品溶解在10%吐温80溶液中并在LPS施用前3天口服施用。
LPS给药后7天口服样品,7天后采集实验动物脑组织。一半的脑组织被研磨用于蛋白质印迹(western blot)和ELISA分析,另一半被固定后用于组织免疫染色。
3)抗氧化效果评价:脂质过氧化分析(MDA测定)
为了分析柠檬桃金娘和绣球菌的混合提取物(LB)、柠檬桃金娘单一提取物(L)和绣球菌单一提取物(B)的抗氧化效果,利用LPS在小鼠上诱发脑炎症后导致的氧化性损伤指标——脂质过氧化物(MDA),分析施用柠檬桃金娘和绣球菌的混合提取物(LB)、柠檬桃金娘单一提取物(L)和绣球菌单一提取物(B)后其脂质过氧化物(MDA)的生成变化。
为了测定脂质过氧化物的变化,使用磷酸盐缓冲盐水(PBS)在4℃下对小鼠海马体组织进行匀浆。然后,在10,000rpm下进行5分钟的离心分离,提取上清液使用。用0.15M氯化钾和500μl各匀浆组织进行样品处理,并向其中加入100μl的20mM氯化铁。之后,在37℃的恒温培养箱中反应30分钟后,加入500μl的含有15%TCA、0.38%TBA和0.5%BHT的0.25N冷HCl。90℃下加热60分钟后,以5,000rpm、4℃离心5分钟分离,在吸光度532nm波长处测量上清液中的硫代巴比妥酸反应物质(TBARS)。
表3
区分 | MDA(控制百分比) |
未用药对照组(sham) | 100 |
LPS对照组(control) | 170 |
100LB(100mg/kg) | 160 |
1000LB(100mg/kg) | 120 |
1000L(100mg/kg) | 140 |
1000B(100mg/kg) | 135 |
阳性对照组(FA,20mg/kg) | 115 |
分析结果表明,LPS使实验动物大脑中MDA的产生量增加了170%,这意味着LPS诱导了氧化损伤。当以高剂量(1000mg/kg)给予柠檬桃金娘和绣球菌的提取物(LB)、柠檬桃金娘单一提取物(L)及绣球菌单一提取物(B)高剂量投入时,证实经LPS增加的MDA的产生量显着降低。而柠檬桃金娘和绣球菌的混合提取物(LB)相比于柠檬桃金娘单一提取物(L)及绣球菌单一提取物(B),通过LPS增加的MDA生成量在统计学上更有显著降低。
此外,证实了用作阳性对照的阿魏酸(FA)也抑制了因LPS生成的MDA(表3)。
4)抗炎效果评价:炎症细胞的组织免疫染色分析
为了分析柠檬桃金娘和绣球菌的混合提取物(LB)、柠檬桃金娘单一提取物(L)和绣球菌单一提取物(B)的抗炎效果,用LPS诱发小鼠脑部炎症后,7天内口服柠檬桃金娘和绣球菌的混合提取物(LB)、柠檬桃金娘单一提取物(L)和绣球菌单一提取物(B)后,对其脑组织中与炎症相关细胞——小胶质细胞(microglia)和星形胶质细胞(astrocyte)增加程度进行了分析。具体而言,通过使用Iba-1抗体的小胶质细胞和使用GFAP抗体的星形胶质细胞的组织免疫染色来分析各细胞的增加程度。
选取固定的大脑组织海马体部分,用PBS清洗3次,然后用过氧化氢处理去除内源性过氧化物酶(peroxidase)。让初次抗体兔源-Iba-1抗体(1:1000稀释)或兔源-GFAP抗体(1:1000稀释)反应过夜。二次抗体使用生物素和兔抗体(1:200稀释),经过ABC反应,利用DAB进行3分钟的显色。
在每个过程之间用PBS清洗3次。染色反应完成后,用封片液覆盖具有海马体组织的盖玻片并干燥,然后保存。将脑组织放置在涂有明胶的载玻片上后,经过70~100%乙醇和二甲苯的处理,然后用盖玻片覆盖组织并保存。使用光学显微镜(Olympus MicroscopeSystem BX51;Olympus,Tokyo,Japan)观察染色的海马体切片。各抗体阳性区域的面积与总海马体面积的比值即可显示出来。
表4
区分 | CA3区GFAP阳性面积(%) |
未用药对照组(sham) | 0.021 |
LPS对照组(control) | 0.053 |
100LB(100mg/kg) | 0.045 |
1000LB(100mg/kg) | 0.029 |
1000L(100mg/kg) | 0.038 |
1000B(100mg/kg) | 0.041 |
阳性对照组(FA,20mg/kg) | 0.027 |
表5
区分 | CA3区Iba-1阳性面积(%) |
未用药对照组(sham) | 0.012 |
LPS对照组(control) | 0.059 |
100LB(100mg/kg) | 0.039 |
1000LB(100mg/kg) | 0.019 |
1000L(100mg/kg) | 0.034 |
1000B(100mg/kg) | 0.037 |
阳性对照组(FA,20mg/kg) | 0.017 |
分析的结果显示,LPS在实验动物的大脑中诱导了星形胶质细胞标志物GFAP阳性细胞和小胶质细胞标志物Iba-1阳性细胞的增加。口服给予柠檬桃金娘和绣球菌的混合提取物(LB)、柠檬桃金娘单一提取物(L)及绣球菌单一提取物(B)时,可以减少由LPS引发的星形胶质细胞(GFAP阳性细胞)和小胶质细胞(Iba-1阳性细胞)的增加,柠檬桃金娘和绣球菌的混合提取物(LB)相比于柠檬桃金娘单一提取物(L)和绣球菌单一提取物(B),显著减少了LPS引发的星形胶质细胞(GFAP阳性细胞)及小胶质细胞(Iba-1阳性细胞)的增加。
阳性对照组(阿魏酸,FA)也抑制了由LPS引起的星形胶质细胞的增加(表4、表5)。
即,在动物试验中确认了经口给予柠檬桃金娘和绣球菌的混合提取物(LB)具有抗炎效果,并且确认了其比柠檬桃金娘单一提取物(L)和绣球菌单一提取物(B)具有更优异的抗炎效果。
如上所述,根据本发明结果可以看出,相比于单独使用柠檬桃金娘和绣球菌的情况,柠檬桃金娘和绣球菌的混合提取物具有更优异的协同作用。
以上详细说明了本发明的优选实施例,本发明所属领域的普通技术人员将理解,本发明只是举例说明,在不改变本发明的技术思想或者必要特征的情况下,可以很容易变形成其他具体的形式。
因此,本发明的范围由所附权利要求书而不是由说明书来表示,并且从权利要求书的含义、范围和等同概念衍生的所有变化或修改均被解释为包含在本发明的范围内。
Claims (9)
1.一种抗氧化或抗炎食品组合物,其特征在于,所述组合物是包括柠檬桃金娘和绣球菌的混合提取物作为活性成分的抗氧化或抗炎食品组合物。
2.根据权利要求1所述的抗氧化或抗炎食品组合物,其特征在于,所述混合提取物是通过将柠檬桃金娘和绣球菌以1:0.1~10的重量比混合提取得到。
3.根据权利要求1所述的抗氧化或抗炎食品组合物,其特征在于,所述混合提取物是柠檬桃金娘和绣球菌的热水提取物。
4.一种抗氧化或抗炎化妆品组合物,其特征在于,所述组合物是包括柠檬桃金娘和绣球菌的混合提取物作为活性成分的抗氧化或抗炎化妆品组合物。
5.一种用于预防或治疗炎症性疾病的药物组合物,其特征在于,所述组合物包括以柠檬桃金娘和绣球菌的混合提取物作为活性成分的预防或治疗炎症性疾病的药物组合物。
6.根据权利要求5所述的药物组合物,其特征在于,所述炎症性疾病是脑或神经炎性疾病。
7.一种抗氧化或抗炎的医药外品组合物,其特征在于,所述组合物是包括以柠檬桃金娘和绣球菌的混合提取物作为活性成分的抗氧化或抗炎的医药外品组合物。
8.一种用于抗污染的化妆品组合物,其特征在于,所述组合物是包括柠檬桃金娘和绣球菌的混合提取物作为活性成分的抗污染的化妆品组合物。
9.根据权利要求8所述的抗污染的化妆品组合物,其特征在于,所述抗污染能抑制由沙尘或细尘引起的炎症或氧化应激。
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