CN112601462A - 植物抗氧化剂 - Google Patents
植物抗氧化剂 Download PDFInfo
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- CN112601462A CN112601462A CN201980055737.4A CN201980055737A CN112601462A CN 112601462 A CN112601462 A CN 112601462A CN 201980055737 A CN201980055737 A CN 201980055737A CN 112601462 A CN112601462 A CN 112601462A
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- extract
- plant extract
- plant
- cashew nut
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Abstract
一种表现出抗氧化活性的植物提取物,其中所述植物提取物至少是来自于腰果属(Anacardium)的提取物。
Description
与相关申请的交叉引用
本申请要求2018年8月31日提交的美国专利申请号62/725,435的利益,所述申请的公开内容整体通过引用并入本文。
技术领域
本发明总的来说涉及氧化诱导的DNA损伤的抑制剂,并且更具体来说涉及氧化诱导的DNA损伤的植物抑制剂和此类植物抑制剂作为抗氧化剂的用途。
背景技术
氧是高反应性原子,能够变成被称为“自由基”的潜在损伤性分子的一部分。通常被称为活性氧(“ROS”)的自由基在它们的最外层轨道中含有一个或多个未配对电子。活性氧的常见实例包括氢过氧自由基(ROO*)、超氧化物阴离子(O2*)、活性羟基(OH*)和过氧化氢(H2O2)自由基。这些自由基在活生物体中在代谢期间自发产生。由于自由基非常不稳定,它们与其附近的其他分子(例如蛋白质、脂类或DNA)反应,通过从那些分子获取电子而获得稳定性,从而引起细胞损伤并引发自由基生成的链反应。
自由基生成与细胞内抗氧化剂之间的失衡可以引起氧化胁迫。当氧原子分裂成被称为自由基的带有未配对电子的单原子时,发生氧化胁迫。由于电子偏好成对出现,因此这些自由基会清理身体以寻找与其配对的其他电子,在此期间对细胞、蛋白质和DNA造成损伤。术语氧化胁迫被用于描述当自由基生成与抗氧化防御之间的关键平衡不利时引起的氧化损伤的状况。
作为自由基产生与抗氧化防御之间失衡的结果而产生的氧化胁迫,与对包括脂类、蛋白质和核酸在内的广范围分子物质的损伤相关。过量的氧化胁迫可以导致脂类和蛋白质的氧化,这伴有它们的结构和功能的改变。在因外伤、感染、热损伤、高氧、毒素和过度运动而受损的组织中,可能发生短期氧化胁迫。这些受损组织产生增加的自由基生成酶类(例如黄嘌呤氧化酶、脂氧合酶、环氧合酶),吞噬细胞的活化,游离铁、铜离子的释放或氧化磷酸化的电子传递链的破坏,产生过量的ROS。氧化胁迫与几种变性疾病的病因有关,例如中风、帕金森氏病、阿兹海默氏病、类风湿性关节炎、糖尿病、消化性溃疡、基因突变和癌症、心脏和血液障碍以及炎性疾病。现在认为氧化胁迫对所有炎性疾病(关节炎、血管炎、肾小球肾炎、红斑狼疮、成人呼吸系统疾病综合征)、缺血性疾病(心脏病、中风、肠道局部缺血)、血色素沉着症、获得性免疫缺陷综合症、肺气肿、器官移植、胃溃疡、高血压和先兆子痫、神经障碍(阿茨海默氏病、帕金森氏病、肌营养不良症)、酗酒、与吸烟相关的疾病和许多其他疾病均具有显著贡献。
抗氧化剂能够在自由基攻击细胞之前使它们稳定或失活。使用外部来源的抗氧化剂可以帮助应对氧化胁迫。这些抗氧化剂包括合成抗氧化剂例如丁基化羟基甲苯和丁基化羟基茴香醚;然而,最近已报道这些合成抗氧化剂对人类健康有害。因此,近年来已加强对具有抗氧化活性的有效、无毒的天然化合物的搜寻。
抗氧化剂是还原剂,其实例包括营养物来源的抗氧化剂例如抗坏血酸(维生素C)、生育酚和生育三烯酚(维生素E)、类胡萝卜素和多酚,抗氧化酶类例如超氧化物歧化酶、谷胱甘肽过氧化物酶和谷胱甘肽还原酶,金属结合蛋白例如铁蛋白、乳铁蛋白、白蛋白和血浆铜蓝蛋白,以及痕量金属(例如锌和钼)。这些抗氧化剂可以通过向自由基提供电子来清除活性氧并抑制链反应。由膳食抗氧化剂支持的抗氧化防御系统可保护身体免受自由基的侵害。然而,在氧化胁迫期间,抗氧化剂不足以维持体内稳态。在此类情况下,抗氧化剂可以作为补充剂提供,其食用可以显著降低与自由基相关的疾病的风险。
植物药在大多数这些疾病的管理中发挥重要作用,并且植物是天然抗氧化剂的潜在来源。研究显示,食用在茶、水果和蔬菜中存在的多酚类化合物与这些疾病的低风险有关。因此,对于含有抗氧化剂的植物和作为潜在治疗剂的促进健康的植物成分的研究兴趣日益增长。药用植物为在长期暴露时可能有毒的化学抗氧化剂提供了一种安全、成本效益高的生态替代品。
腰果树(Anacardium occidentale Linn)最初来自于亚马逊地区,后来被移植到印度、东非和其他国家进行种植。这种树产生采取膨胀果梗形式的非常奇特的果或果实。在这种果梗末端的外部,腰果坚果生长在其自身的灰色肾形硬壳中。这种壳具有柔软的革质外皮和围绕果仁的被称为果壳或种皮的薄且坚硬的内皮。在这两层皮之间是一个含有腰果壳液的蜂窝状结构。这种液体包含腰果酸、腰果酚和腰果二酚以及其他成分。腰果酸是一种水杨酸,而腰果酚和腰果二酚是取代的酚。
已研究了所述果实的各个部分的用途。除了作为可食用食物之外,来自于腰果果实的汁被用于饮料中,而果实提取物在体重管理方面显示出益处。已提取了腰果壳液用于各种不同的工业和农业应用,包括摩擦衬片、涂料、层压树脂、橡胶混配树脂、腰果水泥、基于聚氨酯的聚合物、表面活性剂、环氧树脂、铸造化学品、化学中间体、杀虫剂和杀真菌剂。腰果种皮已被用于鞣料中。
作为健康生活方式和均衡健康饮食的一部分,抗氧化剂的补充被认为是改善自由基保护的重要手段。正如上文提到的,对于具有抗氧化活性的有效、无毒的天然化合物存在着需求。本发明提供了一种这样的解决方案。
发明内容
本文公开了一种包含儿茶素类的植物提取物,其中所述提取物已被标准化至以所述提取物的总重量计约15.0w/w%或更高的儿茶素含量,其中所述植物提取物表现出抗氧化活性,并且其中所述植物提取物至少包含来自于腰果属(Anacardium)的提取物。一方面,所述植物提取物可以从选自Anacardium humile、Anacardium othonianum、Anacardiumgiganteum、Anacardium nanum、Anacardium negrense和/或腰果(Anacardiumoccidentale)的植物获得。优选地,所述植物提取物至少来自于腰果(Anacardiumoccidentale L.)。在一个实施方式中,所述植物提取物至少来自于腰果(Anacardiumoccidentale L.)果实的种皮。
另一方面,本发明提供了一种含有腰果(Anacardium occidentale L.)种皮的植物提取物的组合物,其中所述植物提取物表现出抗氧化活性。所述植物提取物可以以约4.0μg/mL或更高的量存在于所述组合物中。例如,所述植物提取物可以以约4.0μg/mL至约2000.0μg/mL的量存在于所述组合物中。
一方面,所述含有腰果种皮的植物提取物的组合物抑制γ-H2AX活性。在一个实施方式中,所述植物提取物以约4.0μg/mL至约2000.0μg/mL的量存在于所述组合物中。组合物可以包括例如加入所述提取物的食品和/或饮料组合物。
本文中还提供了具有抗氧化性质的膳食补充剂,其中所述补充剂包含治疗有效量的腰果种皮提取物。例如,所述腰果种皮提取物可以以约4.0μg/mL至约2000.0μg/mL的量存在于所述膳食补充剂中。
本发明还提供了一种通过给药包含浓度为约4.0μg/mL至约2000.0μg/mL的腰果(Anacardium occidentale L.)种皮的植物提取物的组合物在对象中抑制氧化诱导的DNA损伤的方法。
附图说明
图1是腰果种皮提取物在0分钟(开始)至20分钟的保留时间内在275nm下的HPLC色谱图。
图2是腰果种皮提取物的LC/MS和LC/PDA(波长为280和350nm)色谱图。
图3是示出了腰果种皮提取物与儿茶素和soliprin标准品相比在抑制DNA损伤中的功效的图。
图4是示出了与儿茶素和soliprin标准品相比,在用各种不同剂量的腰果种皮提取物给药时细胞培养物的细胞存活率的图。
发明详述
本发明是基于下述令人吃惊的发现,即腰果(Anacardium)的种皮含有明显高的某些类黄酮。具体来说,已发现腰果种皮的提取物包含儿茶素和表儿茶素作为主要组分以及原花青素类。本文中提到的数据证实了腰果种皮提取物在保护DNA免受氧化胁迫诱导的损伤中有益。
对于本申请来说,术语“组合物”是指治疗、改善、促进、提高、管理、控制、维持、优化、修改、减轻、抑制或阻止与天然状态、生物过程或疾病或障碍相关的特定状况的产品。例如,组合物在对象中改善氧化的抑制和/或减轻炎症等。术语组合物包括但不限于包含有效量的提取物、其至少一种组分或其混合物的药品(即药物)、非处方药(OTC)、化妆品、食品、食品成分或膳食补充剂组合物。示例性的组合物包括霜剂、化妆用洗剂、面膜或粉剂,或作为乳液、洗剂、搽剂泡沫、片剂、膏药、颗粒剂或软膏。组合物还可以包括饮料,例如加入有效量的提取物的饮料或含有有效量的提取物的茶包。含有有效量的提取物的食品组合物的非限制性实例包括烘焙食品、蛋白粉、肉制品、乳制品和糖食。
当在本文中使用时,术语“提取物”或“植物提取物”是指包含至少腰果属植物(例如Anacardium humile、Anacardium othonianum、Anacardium giganteum、Anacardiumnanum、Anacardium negrense和/或腰果(Anacardium occidentale))、优选为腰果(Anacardium occidentale L.)的物质的一种或多种活性成分的固体、半流体或液体物质或制剂。优选地,所述活性成分源自于腰果种皮的提取物。所述提取物可以使用溶剂例如水、1至4个碳原子的短链醇类(例如甲醇、乙醇、丁醇等)、乙烯、丙酮、己烷、醚、氯仿、乙酸乙酯、乙酸丁酯、二氯甲烷、N,N-二甲基甲酰胺(“DMF”)、二甲基亚砜(“DMSO”)、1,3-丁二醇、丙二醇及其组合来制备,但是也可以使用所述粗提取物在此类溶剂中的级分制备。可以使用任何提取方法,只要它确保所述活性成分的提取和保护即可。
当在本文中使用时,术语纯化合物、组合物、提取物、提取物混合物、提取物组分和/或活性药剂或成分或其组合的“有效量”或“治疗有效量”,是指在剂量和时间长度上足以有效实现所需结果的量。例如,所述“有效量”或“治疗有效量”是指本发明的纯化合物、组合物、提取物、植物提取物、提取物混合物、植物提取物混合物、提取物组分和/或活性药剂或成分或其组合的在给药到对象(例如哺乳动物例如人类)时,足以在对象中实现治疗例如改善氧化的抑制和/或减轻炎症等的量。本公开的组合物、提取物、植物提取物、提取物混合物、植物提取物混合物、提取物组分和/或活性药剂或成分的构成“有效量”或“治疗有效量”的量,将随着所述活性药剂或化合物、待治疗的病症及其严重程度、给药方式、给药持续时间或待治疗对象的年龄而变,但可以由本领域普通技术人员根据自己的知识和本公开内容常规地确定。
术语“可药用”意味着那些药物、医药、提取物或惰性成分适合于与人类和较低等动物相接触使用而没有过度毒性、不相容性、不稳定性、刺激性等,并与合理的利益/风险比相称。
术语“给药”被定义为通过本领域中已知的途径,包括但不限于静脉内、动脉内、口服、肠胃外、颊、局部、透皮、直肠、肌肉内、皮下、骨内、透粘膜或腹膜内给药途径将组合物提供给对象。在优选实施方式中,给药组合物的口服途径是适合的。
当在本文中使用时,术语“对象”或“个体”包括可能向其给药组合物的哺乳动物。哺乳动物的非限制性实例包括人类、非人类灵长动物、犬科动物、猫科动物、马科动物、牛科动物、啮齿动物(包括转基因和非转基因小鼠)等。在某些实施方式中,所述对象是非人类哺乳动物,并且在某些实施方式中,所述对象是人。
当在本文中使用时,术语“载体”是指有助于将一种或多种植物提取物在适合于给药的形式中维持在可溶且均匀状态下的组合物,其无毒并且不以有害方式与其他组分相互作用。
除非另有指明,否则整个本公开中叙述的所有比例和百分率均以重量计。
本发明提供了一种能够抑制由氧化胁迫造成的DNA损伤的基于植物的抑制剂。更具体来说,本发明针对来自于腰果属的腰果种皮的植物提取物。已发现此类植物提取物能够通过中和自由基来抑制氧化胁迫诱导的DNA损伤,从而终止由所述自由基产生的链反应。通过终止所述链反应,阻止或抑制了由所述自由基通过它们与重要大分子例如DNA、蛋白质、脂类或细胞膜的反应造成的损伤。
根据本发明的能够抑制由氧化胁迫造成的DNA损伤的有用植物提取物包括来自于腰果属的植物提取物。更具体来说,所述基于植物的抑制剂是选自Anacardium humile、Anacardium othonianum、Anacardium giganteum、Anacardium nanum、Anacardiumnegrense和/或腰果(Anacardium occidentale)的一个或多个物种的植物提取物。优选地,所述植物提取物来自于腰果(Anacardium occidentale)物种。在一个实施方式中,所述植物提取物来自于腰果(Anacardium occidentale)物种的种皮。
根据本发明的能够抑制由氧化胁迫造成的DNA损伤的组合物可以包括能够作为活性成分起作用的一种或多种化合物。所述化合物可能是所述植物提取物的组分。例如,所述化合物可以是获得所述植物提取物的植物中存在的植物化学成分。所述化合物可能至少部分负责抑制由氧化胁迫造成的DNA损伤。所述化合物可以是能够抑制由氧化胁迫造成的DNA损伤的任何化合物。在一个实施方式中,所述化合物选自植物化学成分儿茶素类、表儿茶素类和/或原花青素(例如A、B、三聚体、四聚体)。
通常,植物的一个或多个部位可用于生产植物提取物,包括但不限于根、茎、叶、花、果实、种子和种子的种皮。在本发明中,单独地或与其他植物部位一起至少使用种子的种皮来生产所述植物提取物。来自于腰果属植物的种皮可以从各种不同来源商购。所述腰果种皮的提取物可以使用任何适合的提取技术获得。
就此而言,可以收集并粉碎所述植物的一个或多个部位,特别是所述植物的种皮。然后可以使用适合的溶剂提取所述粉碎的材料。所述溶剂可以在浓缩步骤中除去。例如,可以将所述提取过的材料过筛或过滤,以产生上清液和滤饼。所述滤饼可以被压榨以除去显著部分的液体,所述液体可以添加到所述上清液中。然后可以将所述滤饼脱水并用作纤维来源。所述上清液可以被蒸馏以除去所述溶剂或其一部分,以形成植物提取物液体浓缩物。所述被除去的溶剂可以循环利用。所述浓缩物可以被干燥(例如通过喷雾干燥),以提供干燥的植物提取物。这种干燥的植物提取物可以如本文中所述进行化验和/或标准化。优选地,所述干燥的植物提取物源自于腰果(Anacardium occidentale),特别是腰果(Anacardium occidentale)植物的种皮。
适合用于提取过程的溶剂包括水、醇或其混合物。示例性的醇性溶剂包括但不限于C1-C7醇类(例如甲醇、乙醇、丙醇、异丙醇和丁醇)、水-醇类或醇与水的混合物(例如水性乙醇)、多元醇(例如丙二醇和丁二醇)和脂肪醇。任何这些醇性溶剂可以以混合物的形式使用。在一个实施方式中,所述植物提取物使用乙醇、水或其组合(例如约70%乙醇与约30%水的混合物)来提取。在另一个实施方式中,所述植物提取物仅使用水来提取。
在一个实施方式中,所述植物提取物可以使用有机溶剂提取技术来获得。在另一个实施方式中,可以使用溶剂顺序分离技术来获得所述植物提取物。全水-乙醇提取技术也可用于获得所述植物提取物。通常,这被称为一次性提取。
也可以使用全乙醇提取。这种技术使用乙醇作为溶剂。这种提取技术可以产生除了水溶性化合物之外还具有脂溶性和/或亲脂性化合物的植物提取物。
可用于获得所述植物提取物的提取技术的另一个实例是超临界流体二氧化碳提取(“SFE”)。在这种提取过程中,待提取的材料可以不被暴露于任何有机溶剂。相反,可以使用含有或不含改性剂的处于超临界条件(>31.3℃和>73.8巴)下的二氧化碳作为提取溶剂。本领域技术人员将会认识到,可以改变温度和压力条件以获得提取物的最佳得率。这种技术与全己烷和乙酸乙酯提取技术相似,可以产生脂溶性和/或亲脂性化合物的提取物。
在所述过程中产生的植物提取物可以包括在所述被提取的材料中存在的广泛种类的植物化学成分。所述植物化学成分可以是脂溶性或水溶性的。在收集所述提取物溶液后,可以将溶剂蒸发,得到所述提取物。
所述植物提取物可以被标准化至规定量的特定化合物。例如,所述植物提取物可以被标准化至规定量的所述提取物中存在的活性成分或植物化学成分。在一个实施方式中,所述植物提取物被标准化至以所述提取物的总重量计约15.0重量%或更高的儿茶素含量。
在所述抑制氧化胁迫诱导的DNA损伤的组合物中存在的植物提取物的量可以取决于几种因素,包括所需的氧化胁迫诱导的DNA损伤的抑制水平、特定植物提取物或其组分抑制氧化胁迫诱导的DNA损伤的水平和其他因素。优选地,所述植物提取物以所述组合物的总重量计约0.005重量%或更高、例如约0.005重量%至约50.00重量%的量存在。
所述抑制氧化胁迫诱导的DNA损伤的组合物可以包括一种或多种可接受的载体。所述载体可以帮助将所述植物提取物并入到具有适合于给药到对象的形式的抑制氧化胁迫诱导的DNA损伤的组合物中。大量可接受的载体在本领域中是已知的,并且所述载体可以是任何适合的载体。所述载体优选地适合于给药到动物包括人类,并且能够作为载体起作用而不显著影响所述植物提取物和/或任何活性成分的所需活性。所述载体可以在所需的给药途径和所述组合物的剂型的基础上选择。
适合的剂型包括液体和固体形式。在一个实施方式中,所述组合物采取凝胶、糖浆、浆液或悬液的形式。在另一个实施方式中,所述组合物采取液体剂型,例如口服液(drink shot)或液体浓缩物。在另一个实施方式中,所述组合物以固体剂型存在,例如片剂、丸剂、胶囊、糖衣丸剂或粉剂。当采取液体或固体剂型时,所述组合物可以采取适合于掺入到食品中以备递送的食品递送形式。适合用于固体形式(特别是片剂和胶囊形式)中的载体的实例包括但不限于有机和无机惰性载体材料例如明胶、淀粉、硬脂酸镁、滑石、树胶、二氧化硅、硬脂酸、纤维素等。所述载体可以是基本上惰性的。
作为实例,硅化微晶纤维素可用作载体或粘合剂。硅化微晶纤维素是微晶纤维素和胶体二氧化硅的物理混合物。一种此类适合形式的硅化微晶纤维素是可以从PenwestPharmaceutical Co.,Patterson,N.J获得的ProSolv二氧化硅除了由所述硅化微晶纤维素提供的之外,也可以作为加工助剂添加到所述组合物。例如,可以包括二氧化硅作为助流剂,以在固体剂量单元例如片剂的制造中在压片期间改善粉末的流动性。
在另一个实施方式中,所述载体至少是功能性载体例如荞麦或斯佩耳特小麦。通过在所述组合物中添加功能性载体,可以提供额外的益处,例如与例如上文提到的标准载体相比更低的血糖生成指数。此外,功能性载体可以是无过敏原的(例如荞麦),并且通过将它们添加到生产过程中,本发明的植物提取物可能从这些功能性载体的类黄酮例如芸香苷和槲皮苷获益。此外,这些功能性载体的高纤维含量也可以促进并调节肠道通过。最后,斯佩耳特小麦中存在的硒的附加的矿物质益处可能有助于代谢。
所述抑制氧化胁迫诱导的DNA损伤的组合物可以包含其他惰性成分例如润滑剂和/或助流剂。润滑剂有助于制造过程中例如从模具弹出的过程中片剂的操控。助流剂在压片期间改善粉末流动性。硬脂酸是可接受的润滑剂/助流剂的实例。
所述抑制氧化胁迫诱导的DNA损伤的组合物可以被制造成固体剂型例如片剂和胶囊。这种形式提供了可以由个体容易地运输到进餐场所例如餐厅,并在进食食物之前、期间或之后服用的产品。所述组合物可以被配制成含有适量所述植物提取物和/或活性成分的剂量单元,允许个体根据适合的参数例如体重、食物量或碳水化合物(例如糖)含量来确定服用的单元的适合数量。
在一个实施方式中,所述植物提取物以治疗有效量存在于所述组合物中,例如约4μg/mL或更高、优选地约4.0μg/mL至约2000.0μg/mL、更优选地约15.0μg/mL至约1000.0μg/m、甚至更优选地约30.0μg/mL至约500.0μg/mL的量。所述组合物可以作为单剂或在多剂中给药。在一个实例中,所述化合物每天给药至多三剂。例如,所述化合物可以在餐前、餐中或餐后给药。在一个实施方式中,所述组合物是具有抗氧化性质的膳食补充剂,含有治疗有效量的腰果种皮提取物。
所述剂量可以被选择成在单个单元中提供可能对某些个体和/或某些食品有效的抑制作用水平,同时也允许相对简单的剂量增加,以提供可能对其他个体和/或其他食品有效的其他抑制作用水平。
所述抑制性组合物可以采取适合于口服摄取的形式。这种形式可以被配置成旨在提供规定剂量的所述植物提取物的单一剂型。例如,所述单一剂型可以是粉剂、丸剂、片剂、胶囊或口服液。所述单一剂型可以包含例如约4.0μg/mL至约2000.0μg/mL的所述植物提取物。
实施例
实施例-材料和化学物质剖测
实施例1-使用70%乙醇溶剂的腰果种皮提取物的制备
将干燥的腰果(Anacardium occidentale)种皮粉末(60g)装入3个100ml不锈钢试管中,并使用Thermo ScientificTM DionexTM ASE 350加速溶剂提取器或在80℃的温度和1500psi的压力下用DI水中的70%乙醇溶剂提取两次。过滤并收集所述提取物溶液。将合并的乙醇提取物溶液用旋转蒸发仪在真空下蒸发,以给出粗品腰果种皮提取物。
提取结果提供在下述表1中。
表1-腰果种皮的提取
实施例2-腰果种皮提取物的儿茶素定量
与带有光电二极管阵列检测器的日立高效液相色谱(“HPLC/PDA”)一起使用C18反相柱(5μm C18(2)LC柱250x 4.6mm,可以从Torrance,California,US获得),来确定所述腰果种皮提取物中存在的游离儿茶素类。对于流动相A来说,溶剂是水中的0.10%磷酸(“H3PO4”),并且对于流动相B来说,溶剂B是乙腈(“ACN”),其被用于以1.0ml/min的流速洗脱,并使用275nm处的UV吸收和35℃的柱温。使用的儿茶素参比标准品来自于Sigma-Aldrich Co。将所述参比标准品溶解在甲醇(“MeOH”):0.1%H3PO4(1:1比例)中,其中儿茶素(C1251)浓度为0.5mg/ml,并且表儿茶素(E1753)浓度为0.1mg/ml。测试样品在容量瓶中以2mg/ml的浓度制备在含有50%MeOH的0.1%H3PO4中,超声处理直至溶解(大约10分钟),然后冷却至室温,充分混合,并通过0.45μm尼龙注射式滤器过滤。通过将20μl样品注射到所述HPLC中进行HPLC分析。下面的表2提供了HPLC分析方法的梯度表。
表2-HPLC分析方法的梯度表
时间(min) | 流动相A | 流动相B |
0.0 | 85.0 | 15.0 |
7.0 | 85.0 | 15.0 |
12.0 | 10.0 | 90.0 |
16.5 | 10.0 | 90.0 |
16.6 | 85.0 | 15.0 |
24.0 | 85.0 | 15.0 |
腰果种皮提取物中HPLC儿茶素的定量结果提供了以所述提取物的总重量计,儿茶素含量为9.40%并且表儿茶素含量为6.12%,总儿茶素含量为15.52%。因此,所述腰果种皮提取物可以被标准化至以所述提取物的总重量计总儿茶素含量为约15.00重量%或更高。腰果种皮提取物在275nm波长处的HPLC色谱图提供在图1中。
实施例3-腰果种皮提取物的化学剖测
使用超高压液相色谱(“HPLC”)和质谱(UPLC I类和GS-XT-QTof系统,两者均可以从Water Corporation,Milford,Massachusetts USA获得),确定了所述腰果种皮提取物中存在的类黄酮化合物。使用的柱是UPLC HSS T32.1x100mm,1.8μm,柱温为40℃,样品温度为15℃。对于流动相来说,溶剂A是含有10%乙腈(“ACN”)的水(含有0.1%甲酸),溶剂B是ACN。采集范围是100-1500道尔顿(“Da”),采集模式是电喷雾电离(“ESI”)(-)。下面的表3提供了HPLC条件。
表3-用于分析腰果种皮提取物的HPLC条件
运行时间(min) | 进样体积(μL) | 浓度 |
20.00 | 2.00 | 1mg/mL |
峰的鉴定仅仅基于准确质量。鉴定到二没食子酰基儿茶素、儿茶素和表儿茶素是腰果种皮提取物的主要组分。在所述提取物中也检测到原花青素类,包括A和B型原花青素、原花青素四聚体和原花青素三聚体,其中B型原花青素是所述原花青素类的主要组分。除了那些刚刚提到的化合物之外,鉴定到的化合物还包括二没食子酰基儿茶素、vaccihein A、6"-对香豆酰基洋李苷和dunalianoside B等。从所述分析获得的腰果种皮提取物的LC/MS和LC/PDA色谱图示出在图2中。
实施例-生物测定法
使用食品级乙醇制备腰果种皮的提取物,然后如上所述过滤和干燥。对于测定法准备的其余部分,使用研究级试剂。将提取物溶解在二甲基亚砜(“DMSO”)中至终浓度为50mg/mL,然后在适合于每种生物测定法的缓冲液中稀释到工作浓度。
实施例4-DNA损伤测定法
当不论是内源还是外源的DNA损伤形成双链断裂(“DSB”)时,它总是跟随着组蛋白H2AX的磷酸化。H2AX是作为核小体中组蛋白八聚体的组分的H2A蛋白家族的一种变体。它被激酶例如PI3K途径中的毛细血管共济失调突变蛋白(“ATM”)和ATM-Rad3相关蛋白(“ATR”)磷酸化。蛋白γ-H2AX是召集和集中DNA修复蛋白的第一步。DSB可以被诸如电离辐射或细胞毒性剂的机制诱导,并且随后γ-H2AX灶快速形成。这些灶以1:1的方式代表了所述DSB,并且可用作损伤的生物标志物。可以针对γ-H2AX产生抗体,其因此可以通过第二抗体通过免疫荧光进行可视化。通过流式细胞术对γ-H2AX进行检测和可视化允许评估DNA损伤、相关的DNA损伤蛋白和DNA修复。
将人类皮肤成纤维细胞以8000个细胞/孔的密度接种在96孔组织培养板中。24小时后,将细胞用测试化合物处理48小时,然后将所述细胞用1mM过氧化氢处理4小时以诱导DNA损伤。过氧化氢通过在DNA中产生断裂来诱导DNA损伤。然后将细胞固定并用针对生物标志物γ-H2AX的抗体染色,所述γ-H2AX是在双链断裂处发现的一种磷酸化的组蛋白变体。在双链断裂位点处,所述组蛋白被磷酸化,表明所述DNA受损并需要修复。用于磷酸化组蛋白变体γ-H2AX的抗体可用于鉴定DNA损伤的位点。将细胞核用4',6-二脒基-2-苯基吲哚(“DAPI”)这种结合到DNA的荧光染料染色。使用Image Xpress获取图片并使用Meta Xpress进行分析,以计算每种条件下的荧光强度除以细胞总数。
对过氧化氢处理做出响应的DNA损伤通过细胞中存在的γ-H2AX的量来测量。在用过氧化氢处理以诱导DNA损伤后检测到的γ-H2AX的量的基础上,测试50和100μg/mL的腰果种皮提取物的DNA损伤抑制。使用25μg/mL的儿茶素和soliprin作为阳性对照。如图3中所示,100μg/mL的腰果种皮提取物表现出显著保护DNA免于对过氧化氢处理做出响应的损伤。相对于未用提取物处理但暴露于过氧化氢的孔来计算抑制百分数。从这个实施例看出,用100mg/mL的腰果种皮提取物预处理成纤维细胞显著降低了检测到的γ-H2AX的量,显示出腰果种皮提取物保护DNA免于氧化胁迫诱导的损伤的能力。
使用细胞计数试剂盒-8(“CCK-8”)确定在每种处理中相对于未处理对照的活细胞百分率。将CCK-8试剂添加到培养基(终浓度为10%的总体积),并在37℃和5%CO2下温育一(1)小时,然后在多模式读板器上在460nm处读取吸光度,以确定相对于未处理对照孔的活细胞数目。所述腰果种皮提取物处理与所述未处理的对照没有统计显著的差异(图4),因此对人类皮肤成纤维细胞无毒性。
上述数据说明腰果(Anacardium occidentale L.)种皮的植物提取物具有一种或多种表现出抗氧化活性的化合物。更具体来说,所述腰果种皮提取物可能在改善γ-H2AX活性方面具有合理的活性。
上面的描述公开了本发明的几种方法和材料。本发明易于在材料和方法方面做出修改,并易于在制造方法和设备方面做出改变。对于本领域技术人员来说,考虑到本公开或本文公开的发明的实践,此类修改将变得显而易见。此外,除非另有定义,否则本文中使用的所有技术和科学术语均具有与本发明所属领域的普通技术人员通常理解的相同的含义。因此,本发明不打算限于本文公开的具体实施方式,而是打算覆盖落入随附的权利要求书中体现的本发明的真正范围和精神之内的所有修改和替换方案。
Claims (9)
1.一种组合物,其包含腰果(Anacardium occidentale L.)种皮的植物提取物,其中所述植物提取物抑制γ-H2AX活性。
2.根据权利要求1所述的组合物,其中所述植物提取物以约4.0μg/mL或更高的量存在。
3.根据权利要求2所述的组合物,其中所述植物提取物以约4.0μg/mL至约2000.0μg/mL的量存在。
4.一种具有抗氧化性质的膳食补充剂,其包含治疗有效量的腰果种皮提取物,其中所述植物提取物抑制γ-H2AX活性。
5.根据权利要求4所述的膳食补充剂,其中所述腰果种皮提取物以约4.0μg/mL或更高的量存在。
6.一种包含儿茶素类的植物提取物,其中所述提取物已被标准化至以所述提取物的总重量计约15.0重量/重量%或更高的儿茶素含量,其中所述植物提取物表现出抗氧化活性,并且其中所述植物提取物至少包含来自于腰果属(Anacardium)的提取物。
7.根据权利要求6所述的植物提取物,其中所述来自于腰果属的提取物至少是来自于腰果的提取物。
8.根据权利要求7所述的植物提取物,其中所述来自于腰果的提取物至少来自于腰果种子的种皮。
9.一种在对象中抑制氧化诱导的DNA损伤的方法,所述方法包括给药以约4.0μg/mL至约2000.0μg/mL的浓度包含腰果种皮的植物提取物的组合物。
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US20210346454A1 (en) | 2021-11-11 |
US20200069759A1 (en) | 2020-03-05 |
CA3110952A1 (en) | 2020-03-05 |
CN112601462B (zh) | 2023-10-31 |
EP3843557B1 (en) | 2023-09-20 |
US11590192B2 (en) | 2023-02-28 |
BR112021003813A2 (pt) | 2021-05-18 |
EP3843557A1 (en) | 2021-07-07 |
US11077160B2 (en) | 2021-08-03 |
WO2020046476A1 (en) | 2020-03-05 |
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