CN1372920A - 应用豆类产品治疗外源性侵害 - Google Patents
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Abstract
本发明涉及一种具有胰蛋白酶抑制能力和降低了的微生物含量的豆类产品,一种净化该豆类产品的方法和含有该豆类产品的组合物,以及上述豆类产品或组合物在皮肤、指甲和毛发的局部使用。
Description
发明所属领域
本发明涉及豆类产品,包含所述豆类产品的局部用组合物,以及这些产品的制备和应用。
发明背景
豆类的果实通常含有高水平的蛋白质、脂类和糖。因此,豆类果实如大豆,以及包含豆类果实的组合物,都被认为是对人有很高营养价值的产品。豆类果实通常还含有抑制蛋白酶活性的物质。例如,两种蛋白酶抑制剂早在20世纪40年代即已从大豆中分离出来了,Kunitz型胰蛋白酶抑制剂(大豆胰蛋白酶抑制剂,STI)和Bowman-Birk胰蛋白酶抵制剂(BBI)。参考文献见Birk,Int.J.Pept.ProteinRes.25:113-131(1985)和Kennedy,Am.J.Clin.Neutr.68:1406S-1412S(1998)。
STI是通过形成稳定的化学当量的络合物抑制胰蛋白酶的蛋白水解活性的。参见Liu,K.,化学和大豆成分的营养价值。在《大豆,化学,工艺及应用》32-35页(Aspen publishers,Inc.,Gaithersburg,MD,1999)。STI含有181个氨基酸残基,通过两个二硫键桥联,并且大致为球形。参见Song等,J.Mol.Biol.275:347-63(1998)。
BBI是一个8k-Da的蛋白质,在不同的反应位点抑制胰蛋白酶和糜蛋白酶。参见Billings等,Pro.Natl.Acad.Sci.89:3120-3124(1992)。STI和BBI只在大豆种子中被发现,而在大豆植物的其它部位未发现。参见Birk,Int.J.Pept.Protein Res.25:113-131(1985)。
但是,由于其天然来源,在豆类果实如大豆的外表存在有大量的微生物。因而,净化过程如加热,用有机溶剂水提取,及高剪切力的纯化可以降低微生物的浓度使之在人应用时,比如护肤使用时,较为安全。然而已经发现这些处理过程通常会使大豆中活性物质变性,造成那些对人皮肤、毛发和指甲有美容及治疗作用的重要的生物学功效的破坏(如蛋白酶抑制活性的降低)。而且,这些处理过程还会导致大豆产品的不稳定,产生令人不愉快的气味和颜色的变化。因而便有开发在不破坏这些大豆产品的生物学功效的同时降低大豆产品的微生物数量的商业需求。
本发明的目的是提供一种大豆产品(例如,可用作皮肤、毛发或指甲的护理组合物的活性成分),该产品降低了微生物数量并且保持其蛋白酶抑制活性。本发明的另一个目的是提供包含所述大豆产品及其它任选活性成分的皮肤、毛发和指甲用护理组合物。
本发明涉及包含有降低了微生物数量且保持其有效生物学活性的豆类产品,该产品的获取方法及在化妆用组合物中的应用。
发明概述
本发明涉及具有胰蛋白酶抑制活性和降低的微生物数量的豆类产品,净化上述大豆产品的方法,以及包含上述大豆产品的组合物。在一个优选的实施方案中,该豆类产品是大豆产品。
本发明还涉及豆类产品或含有所述豆类产品的组合物在维护皮肤、毛发和指甲的健康,以及预防或治疗皮肤、毛发和指甲的疾病中局部使用,所述的预防或治疗包括但不限定为:调节皮肤、毛发和指甲的稳固性;清洁皮肤、毛发和指甲;降低或延缓毛发和指甲的生长;拉直或增亮头发;治疗或预防痤疮;调节皮肤、毛发和指甲的色泽;调节皮肤、毛发和指甲的纹理;调节皮肤的皱纹;处理皮肤、毛发和指甲的外来侵害;以及美化皮肤、毛发和指甲。
本发明的其它特征和优点将在下面的说明书详述和权利要求书中介绍。
发明详述
应当确信本领域普通技术人员能够在本申请说明书的基础上,最大限度地利用本发明。以下优选的实施方案仅用于说明和解释,并不以任何方式限定本发明。
除非另有说明,本发明所述的技术和科学术语都是按照本领域普通技术人员通常所理解的意思。而且,这里提到的所有的出版物、专利申请、专利以及其它资料都用作参考。在本文中,除非特别指出,所有百分比都是重量百分比。
这里提到的“胰蛋白酶抑制活性”指的是0.1%(w/w)浓度的豆类产品抑制胰蛋白酶活性的能力,通过以下实施例2所述测量方法测量。在一个实施方案中本发明的豆类产品具有至少约15%的胰蛋白酶活性抑制能力。进一步优选实施方案中,本发明的豆类产品至少有约25%,例如至少约50%的胰蛋白酶活性抑制能力。
这里提到的“硫醇保留活性”指的是0.1%(w/w)浓度的豆类产品对烟诱导的硫醇损失的抑制能力,通过以下实施例3所述测量方法测量。在一个实施方案中,本发明的豆类产品具有至少约75%的硫醇保留活性。进一步优选的实施方案中,本发明的豆类产品具有至少约90%,例如至少约95%的硫醇保留活性。
这里提到的“微生物含量”指的是存在于豆类产品的细菌、真菌和酵母菌的数量。测量微生物含量的方法的实例包括,但不限定为,在“Official Methodsof Analysis of AOAC International”,Patricia Cunniff主编,第十六版,第5次修订,1999(AOAC International)中指出的AOAC 986.23法;或在“Official Compendiaof Standards,USP 24 USP/NF 19”,美国药典协定,Inc.,2000(Board of Trustees,美国药典协定,Inc.)指出的方法。
“不允许的微生物含量”意思是指对人有害的存在于豆类产品中的细菌、真菌和酵母菌的数量,包括,但不限于,大肠菌类、大肠杆菌、沙门氏菌、嗜热孢子、杆菌、肠道球菌、葡萄球菌、粪链球菌,和那些在“Disinfection,sterilization,andpreservation”第4版,Seymour S.Block,pp.887-888(1991,Lea & Febiger,Malvern,PA)一文中所提到的微生物。
这里提到的“局部应用”指的是直接涂布或铺展于皮肤,可用手或诸如粉擦、膨胀棉、滚涂器、或喷雾器等涂抹器进行涂敷。
这里提到的“化妆可接受的”是指那些适合于与组织(如皮肤)接触的,没有不适当的毒性、不相容性、不稳定性、刺激性、过敏反应等的产品或物质。
这里提到的“局部载体”指的是一种或多种适于哺乳动物局部施用的固体或液体填充稀释剂。局部载体的例子包括,但不限于水、蜡、油、软化剂、乳化剂、增稠剂、凝胶剂,及它们的混合物。
这里提到的“稳固性调整”指的是增强稳固性或弹性,防止或治疗皮肤、毛发和指甲的松弛、疏松或下垂。皮肤的稳固性或弹性是通过表皮测定仪的应用来测定。参见Non-Invasive Methods and the Skin手册,J.Serup & G Jemec,编Chapter 14.3(1995)。皮肤稳固性或弹性的丧失是可以是多种因素造成的,包括但不限于老化、外源性侵害、或皮肤、毛发和指甲化妆品的施用。
这里提到的“色泽的调整”指的是增亮和/或变暗皮肤、毛发和指甲的外观(如增亮着色损害,加深皮肤苍白色,和/或使皮肤色泽均匀)。
这里提到的“延缓或减弱指甲的生长”指的是延缓或减弱指甲的生长速率。
这里提到的“延缓或减弱毛发的生长”指的是延缓或减弱毛发的生长速率和/或发干的宽度,包括但不限于降低毛发的可见度和外观(如手臂、腿部及面部的毛发)。
这里提到的“清洁”指的是从皮肤、毛发和指甲表面去除脏物和/或油污。
这里提到的“调节纹理”指的是消除皮肤、毛发和指甲表面的肿块和裂口,使表面光滑,包括但不限于使皮肤外观平滑均匀。
这里提到的“调节皮肤的皱纹”指的是防止、延缓或逆转皮肤细微皱纹的形成过程,包括但不限于降低表面皱纹的可见度。
这里提到的“治疗外源性侵害”指的是防止或减轻由于物理接触、化学接触或温度带来的皮肤、毛发和指甲的外源性侵害造成的损伤。外源性侵害的例子包括但不限于来自以下对皮肤、毛发和指甲的损伤:清洗剂(如含有表面活性剂的皮肤和头发的清洗剂)和化妆品的使用;修面和切割等;环境因素带来损伤如紫外线(如由阳光或如紫外灯及日光模拟器的非天然光源造成的损伤),臭氧、废气、污染、氯及含氯化合物和香烟烟雾等。外源性侵害对皮肤、毛发和指甲的影响包括但不限于对脂类、糖类、肽类、蛋白质、核酸及维生素的氧化和/或亚硝化破坏及修饰。外源性侵害的影响还包括但不限于细胞生存力的丧失、细胞功能的丧失和改变、基因和/或蛋白质表达的改变。
这里提到的“安全有效量”指的是所用的化合物或组合物(如豆类产品)足够对被调节或处理的状况产生正面修饰作用,但不会造成严重的副作用的用量。化合物或组合物的安全有效量会随着被治疗状况,最终的使用者年龄和身体状况,预防/治疗状况的严重性,治疗的持续时间,其它治疗的情况,所用物质或产品/组合物的特性,所用的化妆品可接受的载体的特性及其它一些因素的不同而改变。
豆类产品
“豆类产品”指的是从豆类果实衍生的物质。豆类植物通常来源于豆科植物,都有开裂的果实,如蚕豆、豌豆或小扁豆。豆类植物的例子包括但不限于大豆、小扁豆,豌豆和花生。
豆类产品可以包含全部的豆类果实(如将豆类果实碾成粉末),或只用豆类的一部分(如豆类果实的提取物)。豆类产品可以是流体(如豆类果实和水的混合物)或是固体(如豆类果实的粉末)的形式。当是流体形式时,术语“豆类产品”指的是从豆类植物中衍生出的流体中的固体成分。
本发明的组合物包含安全有效量的豆类产品(如大豆产品)。在一个具体的实施方案中,组合物包含约0.001%到50%,优选约1%到30%的豆类产品(如大豆产品)。
大豆产品
“大豆产品”指的是衍生自大豆的物质。大豆产品可以只包含大豆的一部分(如大豆提取物如脂质减少的大豆粉,或滤过的豆奶),也可以包含全部的大豆(如碾碎的豆粉末)。大豆产品可以是流体(如豆奶),或是固体(如大豆粉末或大豆奶粉末)。当是流体形式时,术语“大豆产品”指的是从大豆中衍生出的流体中的固体成分。
在一个具体的实施方案中,大豆产品是大豆粉末。大豆粉末可以通过碾压干燥的大豆制得。在一个具体的实施方案中,大豆粉末具有低于10微米的平均粒径,如小于1微米。在一个具体的实施方案中,大豆粉末的水分含量小于10%,如小于5%。在一个具体的实施方案中,大豆粉末是冷冻干燥的。
在一个具体的实施方案中,大豆产品是豆奶或豆奶粉末。豆奶是从大豆中衍生出的固体和水的混合物,混合中滤除全部或部分不溶物。豆奶粉末是豆奶蒸干后得到的,在一个具体的实施方案中,是冷冻干燥或是喷雾干燥的形式。制备豆奶的过程包括但不限于以下三种方法。首先,将大豆放入水中吸收水分;膨胀的豆子随后碾压,并加入剩余的水;过滤混合物除去不溶残渣。第二,豆奶可以从大豆粉末制备。大豆粉末与水彻底混合(如至少混合一小时),接着过滤除去不溶残渣。第三,还可以通过加入水与豆奶粉末重新组合成豆奶。在一个具体的实施方案中,豆奶含1%到50%(如5%到20%)重量大豆固体。
豆类产品的抗微生物处理
如上所述,豆类果实表面通常含有较高浓度的微生物。因而,早先人们应用时,豆类产品均要经过处理减少或消除这些微生物。在一个具体的实施方案中本发明的豆类产品的微生物总数小于每克10000个菌落形成单位(cfu)。在另一个实施方案中,本发明的大豆产品的微生物总数大约小于1000cfu每克(例如小于100cfu每克)。
在一个具体的实施方案中,本发明的豆类产品中不允许的微生物总数小于300cfu每克,例如,小于150cfu每克。在进一步的实施方案中,本发明的豆类产品在至少1克(例如至少10克)产品中不允许的微生物总数达到测不出的水平。
在一个具体的实施方案中,豆类产品暴露在伽马射线的辐射中。在进一步的实施方案中,豆类产品是暴露在强度为2到30KGy的伽马射线的辐射下,例如伽马射线的辐射强度为5到10KGy。申请人意外发现这样的处理在降低了豆类产品的微生物含量的同时,保持了其生物活性(如丝氨酸蛋白酶抑制活性)。申请人还发现用伽马射线处理豆类产品可保持该产品的化妆品的优点,如保持天然色泽,且不带来明显的臭味。
其它同样保持豆类产品的蛋白酶抑制活性的微生物处理方法可以单独应用,或可以与伽马射线辐射联合使用,这些加工方法包括但不限于X-射线,高能电子束或质子束,紫外线辐射,流体静压力,和加入具有抗微生物活性的化学试剂,及它们的组合。一个降低微生物数量的方法的完整记录在“消毒,杀菌,保存”一书第4版,Seymour S.Block,pp.887-888(1991,Lea & Febiger,Malvern,PA)中已经阐明。
申请人发现使用热处理的方法会导致蛋白酶抑制活性的丧失,并且处理时要十分小心。例如,申请人发现在100℃下加热豆奶只要10分钟即可将豆奶的蛋白酶抑制活性从86%(在4℃时)降低到46%。申请人还发现加热豆奶会导致产品色泽和味道的变化。
局部用组合物
本发明所用的局部组合物包括那些适于皮肤局部应用的组合。在一个具体的实施方案中,组合物包括大豆产品和化妆品可接受的局部载体。在一个具体的实施方案中,化妆品可接受的局部载体占组合物重量的50%到99.99%(例如从80%到95%)。
组合物可以制成各种不同的产品类型,包括但不限于洗剂、霜乳剂、凝胶剂、棒状物、喷雾剂、剃须膏、软膏、清洁洗涤液和固体洗涤棒、香波、膏剂、粉剂、摩丝、剃须膏、擦剂、贴剂、指甲油、外伤敷料和粘性绷带,水凝胶、薄膜和诸如粉底、睫毛膏和唇膏等的化妆品。这些产品剂型可以含有几种化妆品可接受的局部载体,包括但不限于溶液、乳液(如微乳液和毫微乳液)、凝胶、固体和脂质体。以下是这些载体的非限定的实例。其它载体可以由本领域普通技术人员按常规方法得到。
本发明的局部组合物可以制成溶液。溶液通常包括含水溶剂(如50%-99.99%,或90%-99%的化妆品可接受的含水溶剂)。
本发明应用的局部组合物可以制成含有润肤剂的溶液。这样的组合物优选包含约2%-50%的润肤剂。在这里,“润肤剂”是指那些可以用于防止或减轻干燥,而达到保护皮肤效果的物质。许多适合的润肤剂都是已知的,并可在此使用。Sagarin,Cosmetics,Science and Technology,第2版,Vol.1,pp.32-43(1972),和国际化妆品活性成分辞典和手册,Wenninger and McEwen编,pp.1656-61,1626,和1654-55(The Cosmetic,Toiletry,and Fragrance Assoc.,Washington,D.C.,第7版,1997)(以下简称“ICI手册”)中记载了许多合适的上述物质的例子。
洗剂可以从上述溶液制备。洗剂通常含有大约1%-20%(如,约5%-10%)的一种润肤剂以及大约50%-90%(如,约60%-80%)的水。
本发明的产品另一种可从溶液制备的剂型是乳剂。乳剂通常含有约5%-50%(如,约10%-20%)的润肤剂,和大约45%-85%(如约50%-75%)的水。
还有一种可从溶液制备的剂型是软膏。软膏通常含有一种简单的基质如动物油、植物油或半固体的烃类。软膏可以含有大约2%-10%的润肤剂,加上大约0.1%-2%的增厚剂。常用的增厚剂或增稠剂都可以在Sagarin,Cosmetics,Scienceand Technology,第2版,Vol.1,pp.72-73(1972),和ICI手册pp.1693-1697中找到。
发明的局部组合物可以制成乳液。若载体是一种乳液,应该含有约1%-10%(如,约2%-5%)的乳化剂。乳化剂可以是非离子、阴离子或阳离子的。适合的乳化剂在美国专利No.3755560,4421769,McCutcheon’s Detergents andEmulsifiers,North American Edition,pp.317-324(1986),和ICI手册,pp.1673-1686中已经公开。
洗剂和霜剂可以制成乳液。通常这样的洗剂含有大约0.5%-5%的乳化剂。这样的乳剂通常含有大约1%-20%(如约5%-10%)的润肤剂;大约20%-80%(如约30%-70%)的水;和大约1%-10%(如约2%-5%)的乳化剂。
水包油型和油包水型的单相乳液护肤制剂,如洗剂和乳剂,都是化妆品领域公知的,并在本发明中使用。多相乳液组合物,如水包油包水型,在美国专利No.4254105和4960764中公开并也在本发明中使用。通常,上述单相和多相乳化剂均含有水、润肤剂和乳化剂作为必要组分。
本发明的局部组合物还可以制成凝胶(如用适当凝胶剂制成水凝胶)。用作含水凝胶的合适的凝胶剂包括但不限于天然树胶、丙烯酸和丙烯酸酯聚合物和共聚物、纤维素衍生物(如羟甲基纤维素,羟乙基纤维素)。适合的用于油类(如矿物油)的凝胶剂包括但不限于氢化丁烯/乙烯/苯乙烯共聚物,和氢化乙烯/丙烯/苯乙烯共聚物。上述凝胶通常含有重量百分比为约0.1%-5%的上述凝胶剂。
本发明的局部用组合物还可以是固体剂型(如蜡制的条状物,肥皂组合物,粉剂,或粉擦)。
脂质体制剂也是本发明的组合物。脂质体可以是单层,多层和寡层的脂质体,可以包含或不包含磷脂。制备这样的组合物首先是将桔皮素与磷脂,如二棕榈酰磷脂酰胆碱,胆固醇和水按照Mezei & Gulasekharam,“Liposomes-ASelective Drug Delivery System for the Topical Route of Administration;Gel DosageForm”,Journal of Pharmaceutics and Pharmacology,Vol.34(1982),pp.473-474所述的方法,或改良方法进行混合。形成脂质体的适宜的组合物的表皮脂类可以替代磷脂。然后是将脂质体混入一种上述载体(如凝胶或水包油乳液),以获得脂质体剂型。局部施用脂质体的其它组合物和药用在Mezei,M.,“Liposomesas a Skin Drug Delivery System”,Topics in Pharmaceutical Sciences(D.D.Breimerand P.Speiser,eds.,)Elsevier Science Publishers B.V.,New York,N.Y.,1985,pp.345-358,PCT专利申请WO96/31194和美国专利No.5260065中有详细说明。
本发明的局部组合物除前述组分以外可以含有多种附加的油溶性物质和/或水溶性物质,它们可以以现有技术确立的水平按常规方法用于皮肤、毛发和指甲用组合物。
附加的化妆用活性剂
在一个具体的实施方案中,局部组合物在豆类产品以外可以进一步含有另一种化妆用活性剂。这里“化妆用活性剂”指的是对皮肤、毛发和指甲具有美容或治疗作用的物质,如增亮用试剂、变暗用试剂(如自晒黑试剂)、抗痤疮试剂、消光剂、抗微生物剂、抗炎剂、抗真菌剂、抗寄生虫剂、外用镇痛剂、防晒剂、光防护剂、抗氧剂、角蛋白溶解剂、洗涤剂/表面活性剂、增湿剂、营养剂、维生素、能量增强剂、抗汗剂、收敛剂、除臭剂、毛发剥离剂、固化剂、抗硬化剂和皮肤、毛发和指甲调理剂。
在一个具体的实施方案中,这些试剂可以选自包括但不限于羟基酸、过氧苯甲酸、硫代间苯二酚、抗坏血酸、D-泛醇、氢醌、甲氧基肉桂酸辛基酯、二氧化钛、水杨酸辛基酯、水杨酸三甲环己酯、avobenzone、聚酚醛塑料、类胡萝卜素、自由基清除剂、自旋捕捉剂、类维生素A如视黄醇和视黄醇棕榈酸酯、神经酰胺、多不饱和脂肪酸、必需的脂肪酸、酶、酶抑制剂、矿物质、激素如雌激素、类固醇如氢化可的松、2-二甲基氨基乙醇、铜盐如氯化铜、含铜肽如Cu:Gly-His-Lys,辅酶Q10、如那些在PCT专利申请WO0015188中公开的肽、硫辛酸、诸如脯氨酸和酪氨酸的氨基酸、维生素、乳糖酸、乙酰辅酶A、烟酸、核黄素、维生素B1、核糖、电子转移剂如NADH和FADH2,以及其它植物提取物如芦荟、它们的衍生物及其混合物。本发明组合物中的化妆活性剂的含量通常占组合物重量的约0.001%-20%,例如约0.01%-10%,更进一步是如约0.1%-5%。
维生素的例子包括但不限于维生素A,维生素B系列如维生素B3、维生素B5、维生素B12,维生素C,维生素K,及维生素E及其衍生物。
羟基酸的例子包括但不限于乙醇酸,乳酸,苹果酸,水杨酸,柠檬酸,及酒石酸等,参见欧洲专利申请No.273202。
抗氧剂的例子包括但不限于水溶性抗氧剂如硫醇化合物及衍生物,(如偏亚硫酸氢钠和N-乙酰半胱氨酸),硫辛酸及二氢硫辛酸,白藜芦醇,乳铁蛋白,以及抗坏血酸和抗坏血酸衍生物(如维生素C棕榈酸酯和维生素C多肽)。适用于本发明的组合物的油溶性抗氧剂包括但不限于丁基化羟基甲苯,类维生素A(如视黄醇和视黄醇棕榈酸酯),维生素E(如生育酚醋酸盐),生育三烯酚,辅酶Q。适于本发明组合物的含抗氧剂的天然提取物包括但不限于含黄酮,异黄酮及衍生物(如三羟基异黄酮和diadzein)的提取物,含白藜芦醇的提取物及其它。天然提取物包括葡萄籽,绿茶,松树皮,及蜂胶。其它抗氧剂的例子参见ICI手册第1612-13页。
其它物质
其它不同的物质也可以用于本发明的组合物。这些包括保湿剂,蛋白质和多肽,防腐剂,及碱性试剂。这些试剂在ICI手册,pp.1650-1667中公开。本发明的组合物还含有螯合剂(如EDTA)和防腐剂(如对羟基苯甲酸酯类)。合适的防腐剂和螯合剂的例子已经在ICI手册,pp.1626和1654-55中公开。另外,这里的局部组合物通常含有化妆辅剂,如染料,遮光剂(如二氧化钛),颜料及香味剂。
矿质水
本发明的豆类产品(如豆奶)及组合物在制备过程中可以使用矿质水。在一个具体的实施方案中,矿质水的矿化程度至少是约200mg/L(如约300mg/L到约1000mg/L)。在一个具体的实施方案中,矿化水包含至少约10mg/L的钙和/或至少约5mg/L的镁。
含有本发明的组合物的组合物和制剂均可由本领域普通技术人员按已知的常规技术进行制备。
实施例1:豆类产品的伽马射线辐射处理
申请人发现豆奶粉在进行灭菌操作(如伽马射线辐射)前含有高水平的微生物,最多为每克50000cfu。还发现这样的产品存在可检测水平的不允许的微生物,如粪链球菌,浓度高达每克20000cfu。
申请人将不同数量的豆奶粉(如从约1克到约200公斤)置于从1KGy到16KGy的伽马射线下。所需要的使总的微生物含量低于100cfu每克的剂量大约是10KGy。使粪链球菌减少1个对数的剂量测定出是约3KGy,使10克豆奶粉样品中微生物含量保持在测不出的水平所需辐射剂量约为5KGy。不过,用于照射豆类产品的伽马射线的辐射量最终要通过微生物含量以及这样处理的大豆产品的尺寸来决定。
实施例2豆类产品的胰蛋白酶抑制活性
荧光酪蛋白肽对胰蛋白酶诱导裂解的抑制作用,是使用EnzChexTM蛋白酶测定试剂盒,按照厂商指导的方法进行测量(EnzChexTM蛋白酶测定试剂盒产品信息,3/15/99修订;Molecular Probes,Eugene OR)。简单地说,不同的大豆制品首先在1X消化缓冲液(在试剂盒中预备)中稀释,并在不同的浓度下与溶解在1X消化缓冲液中的1000单位胰蛋白酶(Sigma,St.Louis,MO)一起培养。纯的丝氨酸蛋白酶抑制剂(大豆胰蛋白酶抑制剂,来自Sigma,St.Louis,MO)在0.1%,0.01%和0.001%w/v浓度用作阳性对照。接着,1.0mg/ml BODIPYFL酪蛋白储备液通过向提供底物(在试剂盒中预备)的小瓶加入0.2ml去离子水获得,然后,在消化缓冲液中制得最终浓度为10微克/毫升。加入或不加入测试物质的情况下,胰蛋白酶与BODIPY荧光酪蛋白底物室温下培养一小时后,荧光测定(激发波长485nm,发射波长530nm)是通过SpectraMaxGemini微量滴定平板读数器(Molecular Devices Corporation,Sunnyvale,CA),使用SoftmaxPro3.0软件(Molecular Devices Corporation)完成的。每个试验均一式三份,每次做两遍。
该测量方法可用于用七种不同方法处理的大豆产品的测量。实例A是碾成粉末的大豆(阳光食品公司,台湾台北,R.O.C)。实例B是用约8-15KGy的伽马射线辐射处理的实例A的粉末。实例C是油脂被提取出的大豆粉末(来自CentralSoya Company Inc.,Fort Weyne,IN的Soyafluff,)。实例D是豆奶粉末,该粉末由去皮大豆和水通过过滤、加热、喷雾干燥(Devansoy Farms,Carroll,Iowa)并用7-9KGy的伽马射线辐射处理。实例E是豆奶粉末,该粉末是通过将大豆与水混合,将混合物加热过夜,再加入1,3-丁二醇后混合得到(来自日本IchimaruPharcos CO.,Ltd,Gifu的FlavosteroneSB)。实例F是豆奶粉末,该粉末是通过将大豆与水混合,将混合物加热过夜,再加入乙醇后得到(来自日本IchimaruPharcos CO.,Ltd,Gifu的FlavosteroneSE)。实例G是大豆蛋白提取物(来自法国Laboratories Serobiologiques S.A.,Pulnoy的Vegeseryl HGP LS 8572)。所有这些大豆产品均与大豆胰蛋白酶抑制剂(STI)(Sigma)进行对比。
不同大豆制品对底物的胰蛋白酶裂解抑制作用的百分数通过MicrosoftExcelTM计算并记录在表1。
表1
测试产品 | 浓度 | %胰蛋白酶抑制能力 |
STI | 0.01 | 43.0 |
STI | 0.1 | 76.1 |
实例A | 0.01 | 32.8 |
实例A | 0.1 | 67.1 |
实例B | 0.01 | 31.5 |
实例B | 0.1 | 67.2 |
实例C | 0.01 | 22.7 |
实例C | 0.1 | 36.2 |
实例D | 0.01 | 8.92 |
实例D | 0.1 | 17.4 |
实例E | 0.01 | 7.83 |
实例E | 0.1 | 10.8 |
实例F | 0.01 | 4.87 |
实例F | 0.1 | 5.99 |
实例G | 0.1 | 6.85 |
如表1所示,STI可以以剂量反应方式抑制胰蛋白酶诱导的裂解。实例A,大豆粉末,同样具有显著的胰蛋白酶抑制活性。其它大豆粉末的伽马辐射(如实例B)在减少微生物数量的同时令人意想不到地未显著影响其胰蛋白酶抑制活性。加热和/或提取处理的如实例C-G的产品,却明显降低了大豆粉末的胰蛋白酶抑制活性。
实施例3豆类产品的硫醇保留活性
大豆粉末防止由烟尘诱导的硫醇损失的能力是在人体正常皮肤纤维母细胞进行评价的(Clonetics,San Diego,CA)。硫醇,主要是谷胱甘肽,是内源性细胞抗氧化防御系统的一部分。谷胱甘肽是氧化还原的缓冲物,因而能保持氧化剂和抗氧剂的平衡。谷胱甘肽还是一些酶优选的底物,如谷胱甘肽过氧化物酶(分解过氧化氢)和谷胱甘肽S转移酶(一类重要的解毒酶)。参见A.Meister,Cancer Res.54:1969s-1975s(1994)。
包括谷胱甘肽在内的皮肤抗氧化剂(酶促和非酶促的)在紫外线或臭氧存在下即会失去作用。参见M.J.Connor和L.A.Wheeler,Photochem.Photobiol.46:239-246(1987),和R.M.Tyrrell和M.Pidoux,Photochem.Photobiol.47:405-412(1988)。在细胞培养模型中,细胞内谷胱甘肽(GSH)的低水平导致较高的对紫外线辐射的敏感性。在大鼠皮肤表面局部施用半胱氨酸衍生物可防止紫外线对皮肤的光损伤;该作用与谷胱甘肽合成增加有关。参见L.T.van den Broeke和G.M.J.Beijersbergen van Henegouwen,J.Photochem.Photobiol.B Biol.27:61-65(1995);K.Hanada,等,J.Invest.Dermatol.108:727-730(1997);以及D.P.T.Steenvoorden,等,Photochem.Photobiol.67:651-656(1998)。因此,谷胱甘肽是主要的内源性抗氧化剂,具有对环境变化的较高的反应能力,能够调节皮肤色调和皱纹,可用于治疗外源性侵害。
在该实验中,在曝露烟尘环境(1支香烟,BASIC Full Flavor100’s烟,PhilipMorris,Richmond,VA)或使用安慰剂模拟环境10分钟之前,接种在24-孔格式的Transwell插入物(Corning Costar,Cambridge,MA)中的人体正常新生皮肤纤维细胞在包含不同浓度的豆奶粉末(用大约5KGy的伽马射线辐射处理)中培养24小时。在烟尘环境接触之前,除去大豆产品的插入物上的培养基,用Dulbecco’s磷酸缓冲盐溶液(Life Technologies,Gaithersbury,MD)洗三遍,随后只使插入物下的培养基与烟接触。处理后,立刻将细胞用先前的培养基再培养24小时,细胞再用Dulbecco’s磷酸缓冲盐溶液洗五遍,通过向细胞中加入60μM的monobromobimane(Molecular Probes,Eugene,OR,USA)并在37℃下培养30分钟后读取荧光以测量细胞内的硫醇。若硫醇存在,则monobromobimane显示荧光。荧光测量是通过使用CytoFluor荧光板读取器(PerSeptive Biosystems,Framingham,MA,USA)在以下滤光条件下测量:激发波长360nm,发射波长460nm。
该实验结果见表2。
表2
豆奶粉末浓度(重量%) | 硫醇(占无烟组所含的硫醇的1百分数;均数±S.E.M) | |
没有烟 | 0 | 100±6.71 |
有烟(10分钟) | 00.512 | 65.38±7.1691.24±14.2595.39±4.52106.92±17.06 |
结果表明用伽马射线辐射处理的豆奶粉末令人意想不到地具有对抗烟造成的硫醇损失的保护作用(数据表示为三个独立重复实验的平均值均值±标准差)。
可以理解本发明的详细的说明书是用于说明本发明,但不限定本发明的范围,本发明的范围是由附加的权利要求书的范围所确定。其它的特征,优点,以及修饰均在权利要求书的范围内。
Claims (31)
1、一种局部用组合物,包括:
(a)具有至少约15%的胰蛋白酶抑制活性和小于每克约1000cfu微生物数量的大豆产品;和
(b)化妆品可接受的局部载体。
2、权利要求1的组合物,其中所述大豆产品含有每克小于约150cfu的不允许的微生物。
3、权利要求1的组合物,其中所述大豆产品含有每克小于约100cfu的微生物。
4、一种局部用组合物,包括:
(a)具有至少约50%的胰蛋白酶抑制活性和小于每克约10000cfu微生物数量的大豆产品;和
(b)化妆品可接受的局部载体。
5、权利要求4的组合物,其中所述大豆产品含有每克小于约150cfu的不允许的微生物。
6、权利要求4的组合物,其中所述大豆产品含有每克不超过1000cfu的微生物。
7、权利要求4的组合物,其中所述大豆产品含有每克检测不出的不允许的微生物。
8、一种局部用组合物,包括:
(a)一种经伽马射线辐射接触的大豆产品;和
(b)化妆品可接受的局部载体。
9、权利要求1的组合物,其中所述大豆产品具有至少约75%的硫醇保留活性。
10、权利要求4的组合物,其中所述大豆产品具有至少约75%的硫醇保留活性。
11、权利要求8的组合物,其中所述大豆产品具有至少约75%的硫醇保留活性。
12、权利要求1的组合物,其中所述大豆产品已用伽马射线辐射处理。
13、权利要求4的组合物,其中所述大豆产品已用伽马射线辐射处理。
14、权利要求1的组合物,其中所述大豆产品是豆奶粉末。
15、权利要求1的组合物,其中所述大豆产品是大豆粉末。
16、权利要求4的组合物,其中所述大豆产品是豆奶粉末。
17、权利要求4的组合物,其中所述大豆产品是大豆粉末。
18、权利要求8的组合物,其中所述大豆产品是豆奶粉末。
19、权利要求8的组合物,其中所述大豆产品是大豆粉末。
20、权利要求1的组合物,其中所述组合物进一步含有维生素A、维生素B3、维生素B5、维生素B12、维生素C、维生素K、维生素E及它们的衍生物。
21、权利要求4的组合物,其中所述组合物进一步含有维生素A、维生素B3、维生素B5、维生素B12、维生素C、维生素K、维生素E及它们的衍生物。
22、权利要求8的组合物,其中所述组合物进一步含有维生素A、维生素B3、维生素B5、维生素B12、维生素C、维生素K、维生素E及它们的衍生物。
23、权利要求1的组合物,其中所述组合物进一步含有2-二甲基氨基乙醇。
24、权利要求4的组合物,其中所述组合物进一步含有2-二甲基氨基乙醇。
25、权利要求8的组合物,其中所述组合物进一步含有2-二甲基氨基乙醇。
26、权利要求1的组合物,其中所述组合物进一步含有水杨酸、乳酸或乙醇酸。
27、权利要求4的组合物,其中所述组合物进一步含有水杨酸、乳酸或乙醇酸。
28、权利要求8的组合物,其中所述组合物进一步含有水杨酸、乳酸或乙醇酸。
29、权利要求1的组合物,其中所述组合物进一步含有N-乙酰半胱氨酸。
30、权利要求4的组合物,其中所述组合物进一步含有N-乙酰半胱氨酸。
31、权利要求8的组合物,其中所述组合物进一步含有N-乙酰半胱氨酸。
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EP1586336A1 (en) | 2004-04-06 | 2005-10-19 | JOHNSON & JOHNSON GmbH | Compositions containing copper salts and soy products |
AU2005203059B2 (en) | 2004-07-16 | 2011-11-24 | Johnson & Johnson Consumer Companies, Inc. | Use of legume products for the treatment and prevention of radiotherapy-induced skin damage |
-
2001
- 2001-02-28 US US09/796,054 patent/US7192615B2/en not_active Expired - Lifetime
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2002
- 2002-02-22 AU AU18050/02A patent/AU784856B2/en not_active Expired
- 2002-02-27 JP JP2002052088A patent/JP4740511B2/ja not_active Expired - Fee Related
- 2002-02-27 KR KR1020020010550A patent/KR100905398B1/ko active IP Right Grant
- 2002-02-27 TW TW091103506A patent/TWI319323B/zh not_active IP Right Cessation
- 2002-02-27 MX MXPA02002118A patent/MXPA02002118A/es active IP Right Grant
- 2002-02-28 BR BR0200923-4A patent/BR0200923A/pt not_active Application Discontinuation
- 2002-02-28 CN CN02108046A patent/CN1372920A/zh active Pending
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TWI319323B (en) | 2010-01-11 |
US20020160062A1 (en) | 2002-10-31 |
BR0200923A (pt) | 2002-10-29 |
KR20020070836A (ko) | 2002-09-11 |
KR100905398B1 (ko) | 2009-06-30 |
AU1805002A (en) | 2002-08-29 |
US20070160564A1 (en) | 2007-07-12 |
MXPA02002118A (es) | 2004-04-21 |
JP2002348227A (ja) | 2002-12-04 |
AU784856B2 (en) | 2006-07-06 |
US7897144B2 (en) | 2011-03-01 |
HK1049627A1 (zh) | 2003-05-23 |
US7192615B2 (en) | 2007-03-20 |
JP4740511B2 (ja) | 2011-08-03 |
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