CN113329732B - 乳酸菌发酵麝香草提取物为有效成分的用于改善特应性皮炎及皮肤皱纹的组合物 - Google Patents
乳酸菌发酵麝香草提取物为有效成分的用于改善特应性皮炎及皮肤皱纹的组合物 Download PDFInfo
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Abstract
本发明涉及一种用于改善特应性皮炎及皮肤皱纹的组合物,其作为有效成分含有乳酸菌发酵麝香草提取物。本发明的乳酸菌发酵麝香草提取物有效抑制特应性因子TARC及MDC蛋白质的生成,并抑制IL‑6等炎症细胞因子的生成,从而对特应性免疫或者抑制炎症反应非常有效,通过抑制MMP‑1蛋白质的生成和促进1型前胶原蛋白的生成,也对皮肤皱纹的预防及改善有效,因此能够有用地作为用于改善特应性皮炎及皮肤皱纹的化妆料、食品及药物组合物。另外,乳酸菌发酵麝香草提取物是用乳酸菌对天然物的麝香草提取物进行发酵来获得的发酵提取物,不仅对人体非常安全,而且安全性也非常卓越。
Description
技术领域
本发明涉及一种麝香草提取物为有效成分的用于改善特应性皮炎及皮肤皱纹的组合物,更具体而言,作为有效成分含有乳酸菌发酵麝香草提取物的用于改善特应性皮炎及皮肤皱纹的组合物。
背景技术
特应性皮炎(atopic dermatitis)是慢性复发的瘙痒性皮炎,常发生于婴幼儿期,是患者或家庭成员中伴有瘙痒、皮肤干燥症或者特征性湿疹的疾病。特应性皮炎的典型症状出现在手部、头皮、脸部、脖子、手肘、膝盖等,皮肤非常干燥且发生瘙痒和炎症,且鱼鳞般脱落,严重搔抓时出现皮肤变厚且皱纹加深的苔藓样变现象。
至今特应性皮炎的病因尚未明确查明,报道了IgE的异常增加、负责细胞免疫中枢作用的T细胞的数量减少和功能低下、单核细胞和巨噬细胞的浸润、肥大细胞和嗜酸性粒细胞的数量增加、CD4+T淋巴球等的数量增加等成为免疫学原因(J Invest Dermatol.,96:523-526,1991;J Invest Dermatol.,97:389-394,1991;Immunol.,11:81-88,1999;CurrDrug Targets Inflamm Allergy.,2:199-120,2003;J Allergy Clin Immunol.,107:871-877,2001;Adv Immunol.,78:57,2001;International Immunology,14(7):767-773,2002;Pediatr Allergy Immunol.,19:605-613,2008),尤其,已知因Th2细胞的数量相比Th1细胞增加导致的Th1/Th2不均衡成为重要的因素(Kor J Pharmacogn,43:59-65,2012)。
一方面,趋化因子(chemokine)作为低分子蛋白质显现出趋药性(chemoattractants),根据其结构和功能分为C、CC、CXC及CX3C等4种,最近报道这些趋化因子对免疫细胞的发生和成熟、T细胞的分化和迁徙、Th1/Th2均衡调节等发挥重要作用。
在多种趋化因子中,胸腺活化调节趋化因子(Thymus and activation-regulatedchemokine,TARC)和巨噬细胞来源趋化因子(macrophage-derived chemokine,MDC)作为作用于Th2细胞的代表性趋化因子,作用于Th2细胞的趋化因子受体4(CC chemokinereceptor4,CCR4),通过炎症病变诱导Th2细胞的移动和扩散(J Clin Invest.113(5):651-7,2004;Curr Allergy Asthma Rep.5(4):284-90,2005)。
最近报道特应性皮炎患者中TARC及MDC因子的血清浓度显著增加(J AllergyClin Immunol.,113(2):334-340),且报道特应性皮炎动物模型中TARC和MDC的皮肤表达增加,还报道对特应性皮炎患者进行用作特应性皮炎的治疗物质的环孢素A或皮质类固醇的给药时,MDC及TARC的血清浓度降低(J Dermatol Sci.,34:201-208,2004)。
另外,公开了在体外(In-vitro)实验中,将人角质细胞形成细胞株HaCaT细胞进行INF-γ或TNF-α处理时,MDC及TARC大量表达,可将能够抑制这种表达的物质用作特应性皮炎治疗剂(Int Immunol.,14(7):767-773,2002)。
目前,作为特应性皮炎治疗剂,主要使用抑制炎症反应和细胞因子生成的类固醇制剂,但长期给药导致诱发皮肤萎缩或生长迟缓可能性等多种副作用,因此最近非类固醇制剂的使用增加。然而,非类固醇制剂也存在红斑、瘙痒、浮肿、溃烂及苔藓样变等症状和降低免疫力等多种副作用,因此很难从根本上治疗特应性皮炎(Arellano FM etal.,JInvest Dermatol.2007Apr;127(4):808-16.)。
因此,倾注努力发掘对特应性皮炎的治疗效果显著的天然物质,但至今没能用单一材料研发出特应性皮炎的治疗效果高的物质,实际上作为新的尝试,需要进行对以往公知具有特应性治疗效果的稳定性高的天然物的特应性皮炎治疗效果提升方向的研究等思维转换。
为此,本发明人等进一步改善已知对特应性皮炎有治疗效果的天然物质对特应性皮炎的治疗效果,呈现出与类固醇制剂等化合物同等以上的效果,从而进一步改善其效果而努力研发,其结果,确认用作为乳酸菌的植物乳杆菌菌株对麝香草提取物进行发酵而获得的天然物质有效抑制特应性因子MDC及TARC蛋白质的生成,且抑制IL-6等炎性细胞因子的生成,从而对特应性免疫或者抑制炎症反应非常有效,而且通过抑制MMP-1蛋白质生成和促进1型前胶原蛋白生成,也对皮肤皱纹的预防及改善有效,确认到能够代替现有副作用多而难以使用的特应性皮炎治疗用化合物使用,从而完成了本发明。
发明内容
技术问题
因此,本发明的主要目的在于,提供一种用于改善特应性皮炎及皮肤皱纹的组合物,其作为有效成分含有对改善特应性皮炎及皮肤皱纹具有优异效果的乳酸菌发酵麝香草提取物。
本发明的其他目的及优点可通过具体实施方式、权利要求书及附图来明确。
解决问题的技术手段
根据本发明的一方面,本发明提供一种用于改善特应性皮炎及皮肤皱纹的化妆料组合物,其作为有效成分含有植物乳杆菌(保藏编号:KCTC 14013BP)发酵麝香草提取物。
本发明人等通过进一步改善已知对特应性皮炎的治疗有效的天然物质对特应性皮炎的治疗效果,锐意研究通过天然物发酵进一步改善其效果,从而能够呈现出与类固醇制剂等化合物同等以上的效果,确认到用乳酸菌发酵的麝香草发酵提取物对特应性皮炎的改善效果优异。
本发明人等综合调查对所述麝香草发酵最适的乳酸菌的特性,其结果,确认到对麝香草发酵具有最佳效率的菌株为植物乳杆菌菌株,并将其命名为“植物乳杆菌植物亚种(Lactobacillus plantarum subsp.plantarum)SDCM1002”,并于2019年11月4日保藏于向韩国生命工学研究院生物资源中心,保藏编号为KCTC 14013BP。所述本发明的植物乳杆菌植物亚种SDCM1002(保藏编号:KCTC 14013BP)菌株以与植物乳杆菌(保藏编号:KCTC14013BP)相同的意思使用。
本发明的“麝香草(Thymus vulgaris)”是指双子叶植物唇形目唇形科的多年生草本植物。
本发明中,术语“特应性皮炎”是指慢性、复发性的炎症皮肤疾病,是伴有瘙痒(瘙痒)和皮肤干燥症、特征性红斑或湿疹的疾病。在特应性皮炎的急性病灶上作为特征出现血清免疫球蛋白E(IgE)明显增加的现象,在此基础上,实施对病灶的组织病理学变化及皮炎病变的评价等,由此判断特应性皮炎的诊断及严重性。虽然特应性皮炎的正确原因至今尚未被完全理解,但是报道了与遗传要素同免疫学、非免疫学起因干预作用。
本发明中,术语“皮肤皱纹”是指,皮肤衰弱产生的细纹,因基因、皮肤真皮中的胶原蛋白减少或外部环境等会引发皮肤皱纹。
另外,本说明书中,“有效成分”是指单独显现出目标活性,或者会与自身不具有活性的载体一同显现活性的成分。
本发明中所使用的术语“提取物”包括通过植物提取处理获得的提取物、所述提取物的稀释液或浓缩液、通过对所述提取物进行干燥来获得的干燥物、所述提取物的粗产物或纯化产物或者它们的混合物等提取物本身及利用提取物可能形成的所有制剂形态的提取物。本发明的所述提取物优选提取后制备成干燥粉末状来使用。
在本发明的所述麝香草提取物的提取中,提取所述提取物的方法没有特别限定,可通过该技术领域中通常使用的方法来提取。作为所述提取方法的非限定性例子,可列举热水提取法、超声波提取法、过滤法、回流提取法等,可单独实施这些提取法,或者可以并用两种以上的提取方法。
本发明中用于提取所述麝香草的提取溶剂的种类没有特别限定,可使用该技术领域中公知的任意溶剂。作为所述提取溶剂的非限定性例子,可使用水、甲醇、乙醇、丙醇、丁醇等C1至C4低级醇;甘油、丁二醇、丙二醇等多元醇;以及乙酸甲酯、乙酸乙酯、丙酮、苯、己烷、乙醚、二氯甲烷等碳氢化合物溶剂或者它们的混合物,优选地,可单独或混合使用水、低级醇、1,3-丁二醇、乙酸乙酯中的两种以上。
本发明中,为了去除通过热水提取或者冷水浸提提取的提取物中的浮游固体颗粒,利用过滤,例如利用呢龙等过滤颗粒或利用冷冻过滤法等过滤后,直接使用或者冷冻干燥、热风干燥、喷雾干燥等来进行干燥后使用。
在本发明中,过滤通过热水或冷提取物提取的提取物以除去悬浮的固体颗粒,例如通过使用尼龙等过滤颗粒,或使用冷冻过滤法过滤,或直接使用它们或冻干、热风干燥,可以通过喷雾干燥等进行干燥。
本发明的化妆料组合物的特征在于,抑制胸腺活化调节趋化因子(Thymus andactivation-regulated chemokine,TARC)和巨噬细胞来源趋化因子(macrophage-derivedchemokine,MDC)的生成(参照图2及图3),且抑制IL-6等炎性细胞因子的生成(参照图4),从而对特应性免疫或者抑制炎症反应非常有效。
另外,本发明的化妆料组合物的特征在于,抑制MMP-1蛋白质的生成,促进1型前胶原蛋白的生成(参照图5及图6)。
在本发明的用于改善特应性皮炎及皮肤皱纹的化妆料组合物中,所述组合物可制备成选自由溶液、外用软膏、霜、泡沫、营养化妆水、柔软化妆水、面膜、柔软水、乳液、粉底、精华液、香皂、液体清洁剂、入浴剂、防晒霜、防晒油、悬浊液、乳浊液、糊剂、凝胶、乳液、粉末、香皂、含表面活性剂的清洁剂、油、粉末粉底,粉底乳液、粉底蜡、贴及喷雾组成的组的制剂形态,但不限定于此。
另外,本发明的化妆料组合物还可包括1种以上的调配到普通皮肤化妆料的化妆品学上可接受的载体,作为通常的成分,例如可适当调配油分、水、表面活性剂、保湿剂、低级醇、增稠剂、螯合剂、色素、防腐剂、香料等,但不限定于此。
本发明的化妆料组合物中包含的化妆品学上可接受的载体可根据制剂形态不同。
本发明的制剂形态为软膏、糊剂、霜或者凝胶时,作为载体成分,可使用动物油、植物油、蜡、石蜡、淀粉、黄蓍胶(tragacanth)、纤维素衍生物、聚乙二醇、硅、膨润土、二氧化硅、滑石粉、氧化锌或者它们的混合物。
本发明的制剂形态为粉末或者喷雾时,作为载体成分,可使用乳糖、滑石粉、二氧化硅、氢氧化铝、硅酸钙、聚酰胺粉或者它们的混合物,尤其喷雾时,还可包括氯氟烃、丙烷/丁烷或者二甲醚等推进剂。
本发明的制剂形态为溶液或者乳浊液的情况下,作为载体成分,使用溶剂、溶解剂或者浊化剂,例如可使用水、乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苄醇、苯甲酸苄酯、丙二醇、1,3-丁二醇油,尤其可使用棉籽油、花生油、玉米胚芽油、橄榄油、蓖麻油、芝麻油、甘油脂肪族酯、聚乙二醇或者脱水山梨糖醇的脂肪酸酯。
本发明的制剂形态为悬浊液的情况下,作为载体成分,可使用水、乙醇或丙二醇等液态稀释剂、乙氧基化异硬脂醇、聚氧乙烯山梨醇酯及聚氧乙烯山梨糖醇酯等悬浮剂、微晶纤维素、甲醇铝、膨润土、琼脂或者黄蓍胶等。
本发明的制剂形态为香皂的情况下,作为载体成分,可使用脂肪酸的碱金属盐、脂肪酸半酯盐、脂肪酸蛋白水解物、乙二酸酯、羊毛脂衍生物、脂肪醇、植物油、甘油、糖等。
根据本发明的另一方面,本发明提供一种用于改善特应性皮炎及皮肤皱纹的食品组合物,作为有效成分含有植物乳杆菌(保藏编号:KCTC 14013BP)发酵麝香草提取物。对于所述植物乳杆菌(保藏编号:KCTC 14013BP)菌株和改善特应性皮炎及皮肤皱纹,如所述化妆料组合物中所说明。
本发明的食品组合物具有如下优异的效果:抑制胸腺活化调节趋化因子(Thymusand activation-regulated chemokine,TARC)和巨噬细胞来源趋化因子(macrophage-derived chemokine,MDC)的生成,且抑制MMP-1蛋白质的生成,促进1型前胶原蛋白的生成;因此可有用地用作用于改善特应性皮炎及皮肤皱纹的食品组合物。所述食品组合物可以以健康功能食品的形式使用,但不限定于此。
本发明的食品组合物可以以植物乳杆菌(保藏编号:KCTC 14013BP)发酵麝香草提取物的馏分或者其加工物的形式被包含。另外,所述组合物除了有效成分外,可包含食品学上可接受的食品辅助添加剂。
本发明中,“食品辅助添加剂”是指可辅助添加到食品的构成要件,作为制备各制剂形态的健康功能食品时添加的添加剂,由本领域技术人员适宜地选择使用。作为食品辅助添加剂的例子,可包括各种营养剂、维生素、矿物质(电解质)、合成增味剂及天然增味剂等增味剂、着色剂及填充剂、果胶酸及其盐、藻酸及其盐、有机酸、保护性胶体增稠剂、pH调节剂、稳定剂、防腐剂、甘油、酒精、用于碳酸饮料的碳酸化剂等,但并非通过所述例子限定本发明的食品辅助添加剂的种类。
本发明的食品组合物中可包括健康功能食品。本发明中,“健康功能食品”是指使用对人体具有有用的功能性的原料或成分,以片剂、胶囊、粉末、颗粒、液态及丸等形式制备及加工的食品。这里,“功能性”是指获得对人体结构及功能调节营养素或生理学作用等保健用途有用的效果。本发明的健康功能食品可通过本领域通常使用的方法制备,在所述制备时,可通过添加本领域通常添加的原料和成分来制备。
另外,所述健康功能食品的制剂形态只要是作为健康功能食品被认可的制剂形态,则制备不受限制。本发明的食品组合物可以以多种形态的制剂形态制备,与一般药品不同,因为以食品为原料,因此没有长期服用药品会发生的副作用,且携带性优异,因此可以作为用于促进特应性皮炎及皮肤皱纹的效果的辅助剂来摄取。
本发明的健康功能食品可以采用的形态没有限制,可均包括通常意味上的食品,可以与本领域已知为功能性食品的术语混用。而且,本发明的健康功能食品可与根据本领域技术人员的选择而食品中所包括的适当的其他辅助成分和公知的添加剂混合来制备。作为可添加的食品的例子,有肉类、香肠、面包、巧克力、糖类、点心类、饼干类、披萨、方便面、其他面类、口香糖类、冰淇淋类的乳农产品、各种汤、饮料、茶、补液、酒精饮料及维生素复合剂等,可以添加到将本发明的用于改善特应性皮炎及皮肤皱纹的化妆料组合物为主要成分制备的汁、茶、果冻及果汁等来制备。另外,还包括用作动物饲料的食品。
根据本发明的另一方面,本发明提供一种用于治疗或预防特应性皮炎的药物组合物,其作为有效成分含有植物乳杆菌(保藏编号:KCTC 14013BP)发酵麝香草提取物。所述植物乳杆菌(保藏编号:KCTC 14013BP)菌株和改善特应性皮炎及皮肤皱纹,如所述化妆料组合物中所说明。
本发明的药物组合物具有抑制胸腺活化调节趋化因子(Thymus and activation-regulated chemokine,TARC)和巨噬细胞来源趋化因子(macrophage-derived chemokine,MDC)的生成,抑制IL-6蛋白质生成的优异效果,因此可有用地用作用于治疗或预防特应性皮炎的药物组合物。
本发明的术语“治疗”是指通过本发明的组合物的给药,所述特应性皮炎疾病的症状好转或有所改善的所有行为,术语“预防”是指通过本发明的组合物的给药,所述特应性皮炎相关疾病的发明得到抑制或者延迟的所有行为。
本发明的组合物制备成药剂学组合物时,本发明的药剂学组合物包括药剂学上可接受的载体。本发明的药剂学组合物中包含的药剂学上可接受的载体是进行制剂化时通常被使用的载体,可包括乳糖、右旋糖、蔗糖、山梨糖醇、甘露醇、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、氢化甲基羟基苯甲酸酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁及矿物油等,但不限定于此。本发明的药剂学组合物除了所述成分外,还可包括润滑剂、保湿剂、甜味剂、调味剂、乳化剂、助悬剂、保存剂等。适合的药剂学上可接受的载体及制剂详细记载于雷明顿药物科学(Remington'sPharmaceutical Sciences(19thed.,1995))中。
本发明的药剂学组合物可以口服或者非口服给药,根据本发明的一实施例,以非口服的方式给药,根据本发明的另一实施例,以经皮给药的方式给药。
本发明的药剂学组合物的适宜的给药量可根据制剂化方法、给药方式、患者的年龄、体重、性别、病态、食物、给药时间、给药途径、排泄速度及反应敏感性等要素进行多种方式地处方。本发明的药剂学组合物的优选的给药量为以成人基准为0.0001-100㎎/kg范围内。
本发明的药剂学组合物通过本发明所述技术领域的普通技术人员能够容易实施的方法,使用药剂学上可接受的载体及/或者赋形剂实现制剂化,由此能够以单位容量形态制备或者加入到大容量容器内来制备。此时,制剂形态还可包括油或者水介质中的溶液、悬浊液、糖浆或者乳化液形态或者膏剂、散剂、粉剂、颗粒剂、片剂或者胶囊制剂形态,还可包括分散剂或者稳定剂。
发明的效果
本发明的特征及优点简要如下:
(a)本发明提供一种用于改善特应性皮炎及皮肤皱纹的组合物,作为有效成分含有植物乳杆菌(保藏编号:KCTC 14013BP)发酵麝香草提取物。
(b)本发明的乳酸菌发酵麝香草提取物有效抑制特应性因子TARC及MDC蛋白质的生成,且抑制IL-6等炎性细胞因子的生成,从而对特应性免疫或者抑制炎症反应非常有效,通过抑制MMP-1蛋白质生成及促进1型前胶原蛋白生成,也对预防及改善皮肤皱纹有效,因此可有用地用作用于改善特应性皮炎及皮肤皱纹的化妆料、食品及药物组合物。
(c)另外,乳酸菌发酵麝香草提取物是用乳酸菌对天然物的麝香草提取物进行发酵而获得的发酵提取物,不仅对人体非常安全,而且安全性也非常卓越。
附图说明
图1是示出本发明的乳酸菌发酵麝香草提取物的细胞毒性测定的图表。
图2是示出用乳酸菌发酵麝香草提取物处理的人角质形成细胞(HaCaT,Humanimmortalized keratinocyte)中TARC的显现变化测定的图表。
图3是示出用乳酸菌发酵麝香草提取物处理的人角质形成细胞(HaCaT,Humanimmortalized keratinocyte)中MDC的表达变化测定的图表。
图4是示出用乳酸菌发酵麝香草提取物处理的人角质形成细胞(HaCaT,Humanimmortalized keratinocyte)中MMP-1蛋白质的显现变化测定的图表。
图5是示出用乳酸菌发酵麝香草提取物处理的人体纤维芽细胞中1型前胶原蛋白的表达变化测定的图表。
图6是示出用乳酸菌发酵麝香草提取物处理的人体纤维芽细胞中IL-6的显现变化测定的图表。
具体实施方式
以下,通过实施例对本发明进行更详细的说明。这些实施例只是用于例示本发明,因此不应解释为通过这些实施例限定本发明的范围。
实施例1.乳酸菌发酵麝香草提取物的制备
用流水清洗麝香草来去除杂质。在600g的麝香草中添加6L的纯净水之后,室温下搅拌3天进行提取。所述过程使用3-批(Batch)进行。收集所获得的提取物之后,以40℃以下进行减压浓缩,来获得汁状的浓缩提取物。将浓缩的麝香草提取物以1mg比例溶于10mL的纯净水后,接种植物乳杆菌(Lactobacilus plantarum)菌株并在55℃培养基中培养24小时。
在121℃下对培养物进行15分钟灭菌,使菌株灭活之后进行离心分离,仅收集上层清液。利用减压浓缩仪(Rotary evaporator N-1000,EYELA,Japan)在55℃下对这样获得的乳酸菌发酵麝香草提取物进行减压浓缩之后,冷冻干燥制备成粉末状。
为了比较实验,除了乳酸菌发酵处理的工序外,用如上所述的相同的方法使用未进行乳酸菌发酵处理的麝香草提取物,作为比较例。
实验例1.细胞毒性试验
利用MTT比色分析法(colorimetric assay),确认所述实施例1中制备的乳酸菌发酵麝香草提取物对人角质形成细胞(HaCaT,Human immortalized keratinocyte)生长的效果。
首先,使用包含10%胎牛血清(fetal bovine serum,FBS(培养基(Cambrex))的专用培养基即杜皮克氏改良爱哥尔氏培养基(Dulbeccos modified Eagles medium,DMEM)培养基,以1×105浓度将人角质形成细胞接种到24孔(well)细胞培养皿中后,在37℃、5%CO2的湿润条件下培养24小时。之后,将去除培养基并用无血清DMEM培养基稀释的实施例1中制备的乳酸菌发酵麝香草提取物按浓度(10及100μg/mL)进行处理并培养24小时。24小时后,用1mg/mL的MTT试剂处理,再过2小时后,用DMSO溶解通过MTT处理而细胞内生成的甲臜(formazan),并在570nm测定吸光度。作为阳性对照组,处理他克莫司(tacrolimus)(1及10μg/mL)。
其结果,确认到乳酸菌发酵麝香草提取物和麝香草提取物对人角质形成细胞的细胞生存无大影响,显示相比阳性对照组的他克莫司促进人角质形成细胞生存的结果。通过所述结果,确认到乳酸菌发酵麝香草提取物对人体无毒性(参照图1)。
实验例2.抑制特应性因子TARC表达
将人角质形成细胞(HaCaT5×105/孔)在FBS DMEM培养基中培养24小时之后,在无FBS的DMEM培养基中稀释实施例1中制备的乳酸菌发酵麝香草提取物(10及100μg/ml)和TNF-a及IFN-r(10ng/mL)进行同时处理,来追加培养。经过24小时后,使用人胸腺活化调节趋化因子酶联免疫试剂盒(human TARC ELISA kit)对各孔(well)的上层清液按生产商协议进行定量。
其结果,如图2所示,确认到通过本发明的乳酸菌发酵麝香草提取物因TNF-a及IFN-r过表达的TARC生成量得到明显抑制。
具体地,相比作为阳性对照组使用的他克莫司(tacrolimus)(10μg/mL)处理组,乳酸菌发酵麝香草提取物处理组(10μg/mL)中测定出减少63%的TARC生成量,测定出相比未进行乳酸菌发酵处理的麝香草提取物分别减少52%(10μg/mL)及49%(100μg/mL)的生成量,因此本发明的乳酸菌发酵麝香草提取物对特应性非常有效,比以往用于特应性的免疫抑制化合物他克莫司(tacrolimus)具有更优异的效果,因此能够用作替代他克莫司的天然药剂。
实验例3.抑制特应性因子MDC表达
将人角质形成细胞(HaCaT5×105/孔)在FBS DMEM培养基中培养24小时后,在无FBS的DMEM培养基中稀释实施例1中制备的乳酸菌发酵麝香草提取物(10及100μg/ml)和TNF-a及IFN-r(10ng/mL)进行同时处理,来追加培养。经过24小时后,使用人胸腺活化调节趋化因子酶联免疫试剂盒(human TARC ELISA kit)对各孔(well)的上层清液按生产商协议进行定量。
其结果,如图3所示,确认到通过本发明的乳酸菌发酵麝香草提取物因TNF-a及IFN-r过表达的MDC生成量得到明显抑制。
具体地,相比作为阳性对照组使用的他克莫司(tacrolimus)(10μg/mL)处理组,乳酸菌发酵麝香草提取物处理组(10μg/mL)中测定出减少67%的MDC生成量,测定出相比未进行乳酸菌发酵处理的麝香草提取物分别减少27%(10μg/mL)及56%(100μg/mL)的MDC生成量,因此本发明的乳酸菌发酵麝香草提取物对特应性非常有效,比以往用于特应性的免疫抑制化合物的他克莫司具有更优异的效果,因此能够用作替代他克莫司的天然药剂。
实验例4.抑制炎性细胞因子生成
特应性皮炎因炎症反应进一步扩大,通过皮肤发作、皮肤过敏反应或者光过敏反应临床上呈现出特应性皮炎症状更加恶化的倾向。因此,为了有效改善特应性皮炎的皮肤状态,炎症反应的早期阻断是痤疮症状的治疗上非常重要的因素。
通过如下的方法验证乳酸菌发酵麝香草提取物抑制炎症反应和免疫活性的效果。
因特应性皮炎产生的炎症反应机制中,会有IL-6等炎症介质物质干预,因炎症介质物质的作用导致炎症反应扩大,抑制IL-6的表达时,能够有效地阻断炎症反应。
为了验证特应性皮炎对IL-6蛋白质的显现的影响,将人角质形成细胞(HaCaT)作为对象实施实验。将HaCaT细胞以5.0×105的浓度接种到96孔板(well plate)之后,以144mJ/cm2条件照射UVB,在无FBS的DMEM中对乳酸菌发酵麝香草提取物(分别为1、10及100μg/ml)进行处理,追加培养24小时。培养结束后,取上澄液使用酶联免疫吸附测定法(Enzyme-Linked Immunosorbent Assay,ELISA)试剂盒分析IL-6蛋白质生成量。
参照图4,进行乳酸菌发酵麝香草提取物处理时,显示出通过UVB诱导的IL-6蛋白质的生成得到抑制,这种IL-6蛋白质生成抑制能与浓度相关。
具体地,未进行乳酸菌发酵处理的麝香草提取物相比紫外线照射对照组,分别在1、10及100μg/ml容量中测定出减少44%、61%及77%的IL-6生成量,本发明的乳酸菌发酵麝香草提取物相比紫外线照射对照组分别在1、10及100μg/ml容量中测定出进一步减少51%、70%及84%的IL-6生成量。通过如上所述的实验结果,本发明的乳酸菌发酵麝香草提取物对特应性免疫或者炎症反应的抑制效果明显,能够确认到麝香草提取物通过乳酸菌发酵以往所具有的免疫或者炎症反应得到巨大改善,因此能够使用。
实验例5.抑制MMP-1生成
在40mm细胞培养皿中放入2ml的DMEM培养液,以约1.2×105的浓度接种人体纤维芽细胞后,在37℃、5%CO2环境下培养24小时。之后,以144mJ/cm2条件照射UVB后,更换到包含实施例1制备的乳酸菌发酵麝香草提取物(分别1、10及100μg/ml)的培养基,培养3天。之后,回收(harvest)培养液,在4℃、7,500rpm环境下进行5分钟离心分离(centrifuge),利用ELISA方法确认MMP-1(HumanTotalMMP-1kit,R&DSystems,Inc.,Minneapolis,MN,USA)蛋白质的表达量。
参照图5,以144mJ/cm2容量进行紫外线照射处理的人体纤维芽细胞相比无紫外线照射处理组,MMP-1蛋白质的生成增加3倍左右。这样通过紫外线照射处理的MMP-1蛋白质的生成增加显示出因麝香草提取物的处理而减少的现象,相比紫外线照射对比组,显示出分别在1、10及100μg/ml容量下减少2%、14%及25%的MMP-1蛋白质的生成阻碍效果。
一方面,如上所述的麝香草的MMP-1蛋白质的生成抑制效果呈现出通过乳酸菌发酵处理进一步增加的趋势,本发明的乳酸菌发酵麝香草提取物处理组中相比紫外线照射对比组分别在1、10及100μg/ml容量进一步减少9%、37%及54%的MMP-1蛋白质的生成阻碍效果。
通过如上所述的结果,可知本发明的乳酸菌发酵麝香草提取物有效抑制成为皱纹产生原因的MMP-1蛋白质的生成,从而能够有效地用于预防及改善皮肤皱纹。
实验例6.促进1型前胶原蛋白的合成
在40mm细胞培养皿加入2ml的DMEM培养液,以约1.2×105的浓度接种人体纤维芽细胞后,在37℃、5%CO2环境下培养24小时。之后,以144mJ/cm2条件下照射UVB后,更换到包含实施例1中制备的乳酸菌发酵麝香草提取物(分别10及100μg/ml)的培养基进行培养。之后,回收(harvest)培养液,在4℃、7,500rpm环境下进行5分钟离心分离(centrifuge),利用ELISA方法确认1型前胶原(ProcollagenTypeICPeptideEIAKit,Takara,Shiga,Japan)的蛋白质显现量变化。
参照图6,以144mJ/cm2容量进行紫外线照射处理的人体纤维芽细胞相比无紫外线照射处理组,1型前胶原蛋白的生成减少2.3倍左右。这样通过紫外线照射处理的1型前胶原蛋白的生成减少显示出因麝香草提取物的处理再次增加的现象。此时,麝香草提取物处理组相比紫外线照射对比组,显示出分别在10及100μg/ml容量中改善109%及138%的1型前胶原蛋白的生成增加效果。
一方面,如上所述的麝香草的1型前胶原蛋白的生成增加效果呈现出通过乳酸菌发酵处理进一步增加的趋势,在本发明的乳酸菌发酵麝香草提取物处理组中分别在10及100μg/ml容量中相比无处理组进一步增加153%及234%的1型前胶原蛋白的生成增加效果。
通过如上所述的结果,可知本发明的乳酸菌发酵麝香草提取物有效促进抑制产生皱纹的1型前胶原蛋白的生成,从而能够有效地用于预防及改善皮肤皱纹。
通过如上所述的结果,可知本发明的乳酸菌发酵麝香草提取物作为有效成分含有组合物有效抑制特应性因子TARC及MDC蛋白质的生成,且抑制IL-6等炎性细胞因子生成,特应性免疫或者炎症反应的抑制效果显著,通过抑制MMP-1蛋白质生成及促进1型前胶原蛋白生成,也对预防及改善皮肤皱纹有效。
以上,对本发明的特定部分进行详细说明,本领域普通技术人员应明确这些具体的说明只是优先实施例,本发明的范围并非限定于此。因此,本发明的实质范围应通过权利要求书及其等价物来定义。
[委托编号]
保藏机关名:韩国生命工学研究院
委托编号:KCTC14013
委托日期:20191104
[微生物保藏证明]
国际承认的布达佩斯条约
国际样式
委托证明
To.SD生命工学 以下是国际保藏机关根据规定7.1发行的委托证书原件
SD生命工学韩国首尔市江西区
麻姑中央8路1街
样式BP/4(KTCT样式17)
[支援该发明的国家研究开发事业]
-课题固有编号:10076337
-部名:产业通商资源部
-研究管理专门机关:韩国产业技术评价管理院
-研究事业名:创意产业专门技术开发事业(脑力优秀专门技术开发事业)
-研究课题名:通过调节NFAT的特应性改善及皱纹改善的双重复合效能新功能性化妆品原料开发及事业化
-贡献率:1/1
-主管机关:SD生命工学
-研究期间:2017.04.01~2020.02.29
Claims (7)
1.一种用于改善特应性皮炎及皮肤皱纹的化妆料组合物,其作为有效成分含有植物乳杆菌发酵麝香草提取物,所述植物乳杆菌的保藏编号为KCTC 14013BP。
2.根据权利要求1所述的用于改善特应性皮炎及皮肤皱纹的化妆料组合物,其特征在于,
所述组合物抑制胸腺活化调节趋化因子(TARC)及巨噬细胞来源趋化因子(MDC)的生成。
3.根据权利要求1所述的用于改善特应性皮炎及皮肤皱纹的化妆料组合物,其特征在于,
所述组合物抑制MMP-1蛋白质的生成,并且促进1型前胶原蛋白的生成。
4.一种用于改善特应性皮炎及皮肤皱纹的食品组合物,其作为有效成分含有植物乳杆菌发酵麝香草提取物,所述植物乳杆菌的保藏编号为KCTC 14013BP。
5.根据权利要求4所述的用于改善特应性皮炎及皮肤皱纹的食品组合物,其特征在于,
所述组合物抑制胸腺活化调节趋化因子(TARC)及巨噬细胞来源趋化因子(MDC)的生成。
6.一种用于治疗或预防特应性皮炎的药物组合物,其作为有效成分含有植物乳杆菌发酵麝香草提取物,所述植物乳杆菌的保藏编号为KCTC 14013BP。
7.根据权利要求6所述的用于治疗或预防特应性皮炎的药物组合物,其特征在于,
所述组合物抑制胸腺活化调节趋化因子(TARC)及巨噬细胞来源趋化因子(MDC)的生成。
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