CN101778642B - 促进骨和关节的生长、修复以及维持的食品添加剂 - Google Patents
促进骨和关节的生长、修复以及维持的食品添加剂 Download PDFInfo
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- CN101778642B CN101778642B CN2008801003512A CN200880100351A CN101778642B CN 101778642 B CN101778642 B CN 101778642B CN 2008801003512 A CN2008801003512 A CN 2008801003512A CN 200880100351 A CN200880100351 A CN 200880100351A CN 101778642 B CN101778642 B CN 101778642B
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Abstract
本发明提供促进哺乳动物骨和关节结缔组织生长、修复和维持的食品添加剂。具体地说,所述食品添加剂包含至少一种金属螯合剂和至少一种软骨保护剂的组合。
Description
发明领域
本发明涉及促进骨和关节结缔组织生长、修复以及维持的食品添加剂。
发明背景
约4千万美国人患有显著水平的关节僵直和疼痛。这种僵直或疼痛是源自紧张的体育活动,例如跑步或游泳期间经历的长期机械应力的累积效应。此外,关节痛可能是外伤性伤害,例如扭伤、错位和骨折引起。关节痛还可能是关节炎疾病,例如骨关节炎和类风湿性关节炎的长期作用所致。除了关节中的疼痛,上述许多关节病症还导致畸形和丧失运动能力。约六百九十万美国人直接因关节炎而工作类型受到限制。
关节痛的治疗取决于其病因和严重程度。对于反复机械应力导致的较温和关节痛,最简单的是通过调节运动强度或持续时间来减轻应力,或者利用不同的鞋或支持性器具,但这对于运动员或表演动物(performance animal)可能不合适。对于更严重的关节痛,非处方的非甾体抗炎药(NSAID),例如布洛芬(雅维、莫特林(Motrin))和甲氧萘丙酸钠(萘普生钠)是有用的。在更严重的病例中,类固醇,例如泼尼松或可的松能起缓解作用,但伴有大量副作用,例如体重增加、高血压和面部肿胀。以慢性关节炎疾病,例如类风湿性关节炎为例,破坏免疫系统的炎性反应的其它药物,例如依那西普或阿杜里莫单抗(adulimumab)是有效的,但也伴有免疫系统破坏相关的副作用。最后,只能依靠外科手术介入,例如软骨移植或关节置换术来减轻症状。所有上述治疗可解决疼痛并限制对关节的进一步伤害,但对于促进关节组织修复几乎没用。
治疗关节痛的另一种方法是利用能刺激骨和关节结缔组织生长、修复和维持的营养添加剂。一类添加剂由关节结缔组织的组分构成:胶原、葡糖胺、透明质酸和软骨素。其它添加剂用作骨和结缔组织合成的催化剂或为之供应原材料:S-腺苷甲硫氨酸(SAM)、甲磺酰基甲烷(MSM)和其它维生素及矿物质,例如维生素C、锰、镁、锌、钙、铁和维生素B12。虽然一些所述添加剂能起一定缓解作用,但它们通常只能解决影响关节总体健康的营养问题的有限部分。因此,需要可用于改善关节总体健康的食品添加剂。
发明概述
本发明的一方面提供可一种食品添加剂。所述食品添加剂包含至少一种软骨保护剂和至少一种金属螯合剂,其中所述金属螯合剂包含至少一种金属离子和至少一种配体,其中所述配体包含式(1)所示的化合物:
式中:
n是0-2的整数;
R1是甲基或乙基;和
R2选自下组:羟基和氨基。
本发明另一方面包括促进哺乳动物对象中骨和/或关节组织的疼痛缓解、生长、修复和/或维持的至少一种的方法。该方法包括给予该哺乳动物对象包含至少一种软骨保护剂和至少一种金属螯合剂的食品添加剂。金属螯合剂包含至少一种金属离子和至少一种配体,其中所述配体包含式(1)所示的化合物:
式中:
n是0-2的整数;
R1是甲基或乙基;和
R2选自下组:羟基和氨基。
从下文将部分明白和下文将指出本发明的其它方面和特征。
发明详述
本发明提供具有化合物的平衡混合物的食品添加剂,所述化合物能促进哺乳动物骨和关节结缔组织的生长、修复和维持。因此,可以将所述食品添加剂给予哺乳动物对象以治疗或预防几种疾病或适应症,包括但不限于:骨关节炎、关节渗液(joint effusion)、关节侵蚀(joint erosion)、关节炎症和疼痛、滑膜炎、跛行、术后关节镜手术(post operative arthroscopic surgery)、包括关节运动性在内的正常关节功能衰退、软骨细胞代谢活性的降低或抑制、降解软骨的酶的减少或抑制、透明质酸产量的降低或抑制。还可将所述食品添加剂给予哺乳动物对象以减轻关节软骨的降解或关节软骨降解所致的疾病或适应症。这些疾病或适应症的例子是类风湿性关节炎、银屑病关节炎、骨关节炎和急性炎症,例如耶尔森关节炎、焦磷酸盐关节炎(pyrophosphate arthritis)和痛风关节炎(arthritis urica)。
I.食品添加剂
本发明的食品添加剂包含至少一种金属螯合剂和至少一种软骨保护剂的组合。所述食品添加剂可任选包含选自下组的至少一种额外成分:维生素、矿物质、氨基酸、抗氧化剂、酵母培养物、必需脂肪酸和药学上可接受的赋形剂。这些成分各如下所述。
(a)金属螯合剂
本发明的食品添加剂包含至少一种金属螯合剂或金属盐。在一些实施方式中,所述金属螯合剂包含金属离子和氨基酸配体或其羟基类似物。所述金属离子可选自下组:锌离子、铜离子、镁离子、锰离子、铁离子、铬离子、硒离子、钙离子和它们的组合。在优选的实施方式中,所述金属离子是锌离子、锰离子和铜离子。所述氨基酸可以选自下组:丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸或它们的羟基类似物。在某些实施方式中,所述铜和锌离子优选是二价的,即,其携带2+电荷。螯合剂分子中氨基酸与金属离子之比通常可从1∶1到3∶1不等或更高。金属螯合剂可包含1∶1、2∶1和3∶1的各类物质的混合物。螯合剂分子中氨基酸与离子之比通常优选1.5∶1到2.5∶1不等。在水性介质中,通过适用的稳定常数确定这些物质的相对比例。
如果配体的数量等于金属离子上的电荷,则该电荷通常是平衡的,因为氨基酸的羧基部分处于去质子化的形式。例如,在金属阳离子携带2+的电荷而氨基酸与金属之比是2∶1的螯合剂中,可知羟基或氨基各通过配位共价键与金属相结合,而每个羧基和金属离子之间主要是离子键。如果配体数量超过金属离子上的电荷,例如在二价金属离子的3∶1螯合剂中,超过电荷的氨基酸通常可维持质子化状态以平衡电荷。在另一方面,如果金属离子上的正电荷超过氨基酸的数量,可通过存在另一种阴离子,例如氯(离子)、溴(离子)、碘(离子)、碳酸氢根、硫酸氢根、磷酸二氢根和它们的组合来平衡该电荷。还可能存在二价阴离子。
在一示范性实施方式中,所述金属螯合剂包含金属离子和配体,其中式(1)所示化合物是配体的来源。金属盐包含金属离子和阴离子,其中式(1)所示化合物是阴离子的来源。式1所示化合物的结构如下所示:
式中:
n是0-2的整数;
R1是甲基或乙基;和
R2选自下组:羟基和氨基。
在本发明的各种实施方式中,n是2,R1是甲基并且R2是羟基(即,2-羟基-4-甲硫基-丁酸)。所述金属离子可选自下组:锌离子、铜离子、镁离子、锰离子、铁离子、铬离子、硒离子、钙离子和它们的组合。在示范性的实施方式中,所述金属离子是锌离子、镁离子和铜离子。如果金属离子是铜、锰、铬、钙和铁,其优选是二价的,即,其携带2+电荷。
在示范性的实施方式中,式1所示化合物包括2-羟基-4-甲硫基丁酸(“HMTBA”),即,n是2,R1是甲基并且R2是羟基。在特别优选的实施方式中,所述金属离子是铜、锌或锰。如果金属离子是铜或锰,其优选是二价的,即,其携带2+电荷。锌阳离子基本上均是二价。在本发明组合物和方法中可用的其它金属螯合剂中,金属离子也优选是二价的。螯合剂分子中配体与金属离子之比通常从1∶1到3∶1不等或更高。金属螯合剂通常可包含1∶1、2∶1和3∶1的各类物质的混合物。螯合剂分子中配体与金属离子之比常优选1.5∶1到2.5∶1不等。在水性介质中,通过适用的稳定常数确定这些物质的相对比例。在n是2、R2是氨基并且R1是甲基,即式1所示化合物是甲硫氨酸的情况中,文献中有许多稳定常数可用。对于其中n是2、R2是羟基并且R1是甲基,即式1所示化合物是HMTBA的螯合剂,至少也有一些稳定常数可用。
如果配体的数量等于金属离子上的电荷,则该电荷通常是平衡的,因为配体的羧基部分处于去质子化的形式。因此,在这些螯合剂中,各配体对应于式(1A):
其中R1、R2和n如上定义,即,该方面的螯合剂还是二羧酸盐。例如,在金属阳离子携带2+的电荷并且配体与金属之比是2∶1的螯合剂中,可知羟基或氨基各通过配位共价键与金属相结合,而每个羧基和金属离子之间主要是离子键。典型的例子是Zn2+、Cu2+、Mn2+与两个2-羟基-4-甲硫基丁酸根离子形成的络合物。如果配体数量超过金属离子上的电荷,例如在二价金属离子的3∶1螯合剂中,超过电荷的配体通常可维持质子化状态以平衡电荷。在另一方面,如果金属离子上的正电荷超过配体的数量,可通过存在另一种阴离子,例如氯(离子)、溴(离子)、碘(离子)、碳酸氢根、硫酸氢根、磷酸二氢根和它们的组合来平衡该电荷。还可能存在二价阴离子。
还可利用其中的金属具有1+或2+电荷的金属盐。当金属、金属氧化物、金属氢氧化物或金属盐(例如,金属碳酸盐、金属硝酸盐或金属卤化物)与具有式1所示结构的一种或多种化合物反应从而在金属和产生的阴离子之间形成离子键时,形成这些盐。通常,可通过将金属离子源与HMTBA接触来制备这些金属盐。不想局限于特定的理论,据信Zn、Cu、Mn、Mg、Fe和Cr离子与HMTBA组合主要为螯合剂的形式。
通常可按照美国专利号4,335,257和4,579,962(各自通过引用纳入本文)所述的方法制备本发明的金属螯合剂。
(b)软骨保护剂
本发明的食品添加剂包含至少一种软骨保护剂。适用于本发明的软骨保护剂通常能改善软骨细胞功能。不想局限于特定的理论,合适的软骨保护剂可通过以下一种或多种机制改善软骨细胞功能:1)刺激软骨细胞合成胶原和蛋白聚糖,以及刺激滑膜细胞产生类透明质酸;2)抑制软骨降解;和3)防止软骨下和滑液脉管系统中形成纤维蛋白。
在一个实施方式中,软骨保护剂是葡糖胺或葡糖胺的衍生物或盐。合适的葡糖胺形式包括硫酸葡糖胺、盐酸葡糖胺、氢碘酸葡糖胺、丙酮酸葡糖胺、磷酸葡糖胺、β-葡糖胺、α-葡糖胺和N-乙酰基葡糖胺。葡糖胺的每日剂量可从约25-约3000mg,更典型是从约500到约1500mg。
在另一实施方式中,软骨保护剂是软骨素或软骨素的衍生物或盐。软骨素的合适形式包括氯化软骨素、溴化软骨素、硫酸软骨素和碘化软骨素。软骨素的每日剂量可从约25-约3000mg,更典型是从约500到约1500mg。
在还有另一实施方式中,软骨保护剂是透明质酸或透明质酸的衍生物或盐。透明质酸的合适盐包括碱金属盐以及碱土金属盐。例如,典型的盐包括透明质酸钠、透明质酸钾、透明质酸镁和透明质酸钙。透明质酸的典型剂量可以从约10-约2000mg。
在进一步的实施方式中,软骨保护剂是绿唇贻贝(例如,青边贻贝(Pernacanaliculus))的提取物。对于脂质提取物,这种提取物的每日剂量可从约100-约300mg,或者是从约1000到约1200mg的冻干粉末。
在一示范性实施方式中,软骨保护剂包括硫酸软骨素、透明质酸、葡糖胺和胶原的混合物。示范性制剂可以天然蛋壳膜(Natural Egg Shell Membrane)(密苏里州迦太基市的ETT公司(ESM Technologies,LLC,Carthage,MO)出售的为商品名而商品化购得,其包含浓缩的蛋壳膜。
(c)维生素
本发明的食品添加剂可任选包含一种或多种维生素。用于食品添加剂中的合适维生素包括维生素C、维生素A、维生素E、维生素B12、维生素K、核黄素、烟酸、维生素D、维生素B6、叶酸、吡多辛、硫胺素、泛酸和生物素。维生素的形式可包括维生素的盐、维生素的衍生物、具有与维生素相同或相似活性的化合物和维生素的代谢物。
食品添加剂可包含本文所述或本领域已知的一种或多种形式的有效量的任何维生素。示范性维生素包括维生素K、维生素D、维生素C和生物素。对于某对象,维生素的“有效量”通常定量为该特定维生素用量是美国每日供给量(“RDA”)的至少约10%。然而,考虑到某些维生素的用量超过RDA可能对某些对象有益。例如,给定维生素的用量可超过适用RDA 100%、200%、300%、400%或500%或更多。
(d)矿物质
除I(a)所述的金属螯合剂或金属盐以外,食品添加剂可包含一种或多种矿物质或矿物质源。矿物质的非限制性例子包括但不限于:钙、铁、铬、铜、碘、锌、镁、锰、钼、磷、钾和硒。任何前述矿物质的合适形式包括可溶性矿物盐、微溶性矿物盐、不溶性矿物盐、螯合的矿物质、矿物质复合物、不反应的矿物质,例如羰基矿物质、和还原的矿物质,以及它们的组合。
在一示范性实施方式中,矿物质可以是钙的形式。钙的合适形式包括α-酮戊二酸钙、乙酸钙、藻酸钙、抗坏血酸钙、天冬氨酸钙、辛酸钙、碳酸钙、钙螯合剂、氯化钙、柠檬酸钙、柠檬酸苹果酸钙、甲酸钙、葡乳醛酸钙(calciumglubionate)、葡庚糖酸钙、葡糖酸钙、戊二酸钙、甘油磷酸钙、乳酸钙、赖氨酸钙、苹果酸钙、乳清酸钙、草酸钙、氧化钙、泛酸钙、磷酸钙、焦磷酸钙、琥珀酸钙、硫酸钙、十一碳烯酸钙、珊瑚钙(coral calcium)、柠檬酸二钙、苹果酸二钙、苹果酸二羟基钙(dihydroxycalcium malate)、磷酸二钙、和磷酸三钙。
食品添加剂通常可包含一种或多种形式的有效量的本文所述或本领域已知的任何矿物质。对于某对象,矿物质的“有效量”通常定量为该特定矿物质用量是美国每日供给量(“RDA”)的至少约10%。然而,考虑到某些矿物质的用量超过RDA可能对某些对象有益。例如,给定矿物质的用量可超过适用RDA100%、200%、300%、400%或500%或更多。每份剂量的食品添加剂中矿物质的含量通常可以从约1mg到约1500mg、约5mg到约500mg,或从约50mg到约500mg。
(e)必需脂肪酸
食品添加剂可任选包含必需脂肪酸源。必需脂肪酸可以是分离的,或者其可以是含有必需脂肪酸的油源或脂肪源。在一个实施方式中,必需脂肪酸可以是多不饱和脂肪酸(PUFA),其具有至少两个碳-碳双键,通常是顺式构型。PUFA可以是具有至少18个碳原子的长链脂肪酸。PUFA可以是其中第一双键位于碳链甲基端(即,与羧酸基团相对)第三个碳-碳键的ω-3脂肪酸。Ω-3脂肪酸的例子包括α-亚麻酸(18:3,ALA)、十八碳四烯酸(18:4)、二十碳四烯酸(20:4)、二十碳五烯酸(20:5;EPA)、二十二碳四烯酸(22:4)、n-3二十二碳五烯酸(22:5;n-3DPA)和二十二碳六烯酸(22:6;DHA)。PUFA还可以是其中第一双键位于甲基端第五个碳-碳键的ω-5脂肪酸。示范性ω-5脂肪酸包括肉豆蔻烯酸(14:1)、肉豆蔻烯酸酯和肉豆蔻烯酸十六烷酯。PUFA还可以是其中第一双键位于甲基端第六个碳-碳键的ω-6脂肪酸。ω-6脂肪酸的例子包括亚油酸(18:2)、γ-亚麻酸(18:3)、二十碳二烯酸(20:2)、双高-γ-亚麻酸(dihomo-gamma-linolenic acid)(20:3)、花生四烯酸(20:4)、二十二碳二烯酸(22:2)、肾上腺酸(22:4)和n-6二十二碳五烯酸(22:5)。所述脂肪酸还可以是ω-9脂肪酸,例如油酸(18:1)、二十碳烯酸(20:1)、蜜酒酸(mead acid)(20:3)、芥酸(22:1)和神经酸(24:1)。
在另一实施方式中,必需脂肪酸源可以是源自海产品的油。所述海产品可以是有脊椎的鱼或海洋生物,例如所述油可以是鱼油或海洋生物油(marineoil)。在海产品中发现长链(20C,22C)ω-3和ω-6脂肪酸。海产品中ω-3和ω-6脂肪酸之比约为8∶1到20∶1。可获得富含ω-3脂肪酸的油的海产品包括但不限于:鲍鱼(abalone scallops)、长鳍金枪鱼(albacore tuna)、凤尾鱼、鲶鱼、蛤、鳕鱼、宝石鱼(gem fish)、鲱鱼、湖红点鲑、鲭鱼、油鲱、罗非鱼(orangeroughy)、鲑鱼、沙丁鱼、海胭脂鱼、海鲈(sea perch)、鲨鱼、虾、鱿鱼、鳟鱼和鲔鱼。
在还有另一实施方式中,必需脂肪酸源可以是源自植物的油。植物和蔬菜油富含ω-6脂肪酸。一些源自植物的油,例如亚麻子油特别富含ω-3脂肪酸。植物或蔬菜油通常提取自植物种子,但还可提取自植物的其它部分。通常用于烹饪或调味的植物或蔬菜油包括但不限于:阿萨依油(acai oil)、扁桃仁油、苋菜油、杏仁油、阿干油(argan oil)、鳄梨籽油、巴西棕榈油、山萮油、黑醋栗籽油、婆罗洲油脂浆果油(Borneo tallow nut oil)、琉璃苣籽油、野生葫芦油(buffalo gourd oil)、芸苔油、角豆油、腰果油、蓖麻油、椰油、芫荽籽油、玉米油、棉籽油、月见草油、伪亚麻油(false flax oil)、亚麻籽油、葡萄籽油、榛子油、大麻籽油、木棉花籽油、拉曼油(lallemantia oil)、亚麻子油、马卡达姆坚果油、绣线菊籽油(meadowfoam seed oil)、芥菜籽油、秋葵油、橄榄油、棕榈油、棕榈仁油、花生油、山核桃油、苹果籽油(pequi oil)、紫苏籽油、松子油、阿月浑子果实油、罂粟籽油、洋李仁油(prune kernel oil)、南瓜籽油、昆诺阿藜油、黑芝麻油、米糠油、红花油、芝麻油、大豆油、葵花子油、茶油、蓟油、胡桃油、或麦胚芽油。植物来源的油还可以是氢化或部分氢化的。
在还要进一步的实施方式中,必需脂肪酸源可以是源自藻类的油。市售可得的源自藻类的油包括但不限于:隐甲藻(Crypthecodinium cohnii)和裂殖壶菌(Schizochytrium sp)。可以提取油的其它合适藻类包括但不限于:水华束丝藻(Aphanizomenon flos-aquae)、硅藻(Bacilliarophy sp.)、布朗葡萄藻(Botryococcus braunii)、绿藻(Chlorophyceae sp.)、杜氏盐藻(Dunaliellatertiolecta)、薄肌眼虫(Euglena gracilis)、球等鞭金藻(Isochrysi galbana)、微拟球藻(Nannochloropsis salina)、微小绿藻(Nannochloris sp.)、新绿藻(Neochloris oleoabundans)、三角褐指藻(Phaeodactylum tricornutum)、颗石藻(Pleurochrysis carterae)、定鞭金藻(Prymnesium parvum)、二形栅藻(Scenedesmus dimorphus)、螺旋藻(Spirulina sp.)和朱氏扁藻(Tetraselmischui)。
(f)氨基酸
食品添加剂可任选包含一种到几种氨基酸。合适的氨基酸包括丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸或它们的羟基类似物。在某些实施方式中,所述氨基酸选自必需氨基酸。必需氨基酸通常描述成生物体不能从头合成而必须在饮食中提供的氨基酸。人的必需氨基酸的非限制性例子包括:L-组氨酸、L-异亮氨酸、L-亮氨酸、L-赖氨酸、L-甲硫氨酸、L-苯丙氨酸、L-缬氨酸和L-苏氨酸。在一示范性实施方式中,所用的甲硫氨酸是对应于式(1)的甲硫氨酸的羟基类似物:
式中:
n是0-2的整数;
R1是甲基或乙基;和
R2选自下组:羟基和氨基。
在该实施方式的示范性替代方式中,n是2,R1是甲基和R2是羟基(即,2-羟基-4-甲硫基-丁酸)。
(g)抗氧化剂
食品添加剂可包含一种或多种合适的抗氧化剂。技术人员应明白,给定抗氧化剂是否适合取决于该食品添加剂所给予的物种而不同。抗氧化剂的非限制性例子包括抗坏血酸及其盐、抗坏血酸棕榈酸酯、抗坏血酸硬脂酸酯、阿诺克索默(anoxomer)、N-乙酰基半胱氨酸、异硫氰酸苄酯、邻-、间-或对-氨基苯甲酸(邻是邻氨基苯甲酸,对是PABA)、丁化羟基茴香醚(BHA)、丁羟甲苯(BHT)、咖啡酸、斑蝥黄、α-胡萝卜素、β-胡萝卜素、β-阿朴-胡萝卜素酸(beta-apo-carotenoic acid)、鼠尾草酚、香芹酚、儿茶素、没食子酸十六烷酯、氯原酸、柠檬酸及其盐、对-香豆酸、姜黄素(curcurin)、3,4-二羟基苯甲酸、N,N′-二苯基-对-亚苯基二胺(DPPD)、硫二丙酸双十二酯、硫二丙酸双十八酯、2,6-二-叔丁基苯酚、没食子酸十二烷酯、依地酸、鞣花酸、异抗坏血酸、异抗坏血酸钠、七叶甙原、七叶灵、6-乙氧基-1,2-二氢-2,2,4-三甲基喹啉、没食子酸乙酯、乙基麦芽酚、乙二胺四乙酸(EDTA)、丁香油酚、阿魏酸、黄酮类、黄酮(例如,芹菜苷配基、白杨黄素、黄色黄素)、黄酮醇(例如,橡精、杨梅黄酮、daemfero)、黄烷酮、秦皮亭、延胡索酸、没食子酸、龙胆根提取物、葡糖酸、甘氨酸、愈创木胶、陈皮素、α-羟基苄基膦酸、羟基肉桂酸(hydroxycinammic acid)、羟基戊二酸、氢醌、N-羟基琥珀酸、羟基酪醇(hydroxytryrosol)、羟基脲、乳酸及其盐、卵磷脂、柠檬酸卵磷脂、R-α-硫辛酸、叶黄素、番茄红素、苹果酸、麦芽醇、5-甲氧基色胺、没食子酸甲酯、柠檬酸单甘油酯、柠檬酸单异丙酯、桑黄素、β-萘黄酮(naphthoflavone)、去甲二氢愈创木酸(NDGA)、没食子酸辛酯、草酸、棕榈基柠檬酸酯、吩噻嗪、磷脂酰胆碱、磷酸、磷酸盐、植酸、植基泛色素(phytylubichromel)、没食子酸丙酯、聚磷酸盐、槲皮素、反式-白藜芦醇、迷迭香酸、芝麻酚、水飞蓟素、芥子酸、琥珀酸、硬脂酰柠檬酸酯、丁香酸、酒石酸、麝香草酚、生育酚(即,α-、β-、γ-和δ-生育酚)、生育三烯酚(即,α-、β-、γ-和δ-生育三烯酚)、酪醇、香草酸(vanilic acid)、2,6-二-叔丁基-4-羟基甲基苯酚(即,Ionox 100)、2,4-(三-3′,5′-二-叔丁基-4′-羟基苄基)-(即,Ionox 330)、2,4,5-三羟基丙基苯基酮、泛醌、叔丁基氢醌(TBHQ)、硫二丙酸、三羟基丙基苯基酮、色胺、酪胺、尿酸、维生素K及衍生物、维生素Q10、玉米黄素、或它们的组合。
食品添加剂中可包含的天然抗氧化剂包括但不限于:苹果皮提取物、蓝莓提取物、胡萝卜汁粉、丁香提取物、咖啡豆、咖啡豆提取物、蔓越桔提取物、桉树提取物、姜提取物、葡萄籽提取物、绿茶、橄榄叶、欧芹提取物、薄荷、甘椒提取物、果渣、石榴提取物、米糠提取物、野玫瑰果、迷迭香提取物、鼠尾草提取物、酸樱桃提取物、番茄提取物、姜黄、和麦胚芽油。
(h)抗炎剂
食品添加剂可任选包含至少一种抗炎剂。在一个实施方式中,所述抗炎剂可以是合成的非甾体抗炎药(NSAID),例如乙酰水杨酸、双氯芬酸(dichlophenac)、吲哚美辛、奥沙美辛(oxamethacin)、布洛芬、吲哚洛芬、萘普生、酮洛芬、甲芬那酸(mefamanic acid)、安乃近、吡罗昔康、和塞来考昔。在另一实施方式中,抗炎剂可以是调节炎性过程的激素原。具有这种特性的合适激素原包括激素原转化酶1、阿片-促黑素细胞皮质素原、激素原B-型利钠肽、SMRl激素原,等等。在另一实施方式中,抗炎剂可以是具有抗炎作用的酶。抗炎性酶的例子包括菠罗蛋白酶、木瓜蛋白酶、血清肽酶(serrapeptidase)和蛋白水解酶,例如胰酶(胰蛋白酶、淀粉酶和脂酶的混合物)。
在还有另一实施方式中,所述抗炎剂可以是具有抗炎性作用的肽。例如,所述肽可以是磷脂酶A2的抑制剂,如抗炎素(antiflammin)-1,对应于脂皮质蛋白的氨基酸残基246-254的肽;抗炎素-2,对应于子宫珠蛋白的氨基酸残基39-47的肽;抑制白介素6与白介素6受体之间相互作用的S7肽;RP1,一种异戊二烯基蛋白抑制剂;和类似的肽。或者,所述抗炎性肽可以是皮质抑素(cortistatin),一种生长激素抑制素相关的环状神经肽;或者对应于SV-IV蛋白的N-末端片段的肽,E-、L-和P-选择蛋白的保守性区域,等等。其它合适的抗炎性制品包括胶原水解物和牛奶微量营养素浓缩物(例如,俄亥俄州辛辛那提市SMB公司(Stolle Milk Biologics,Inc.,Cincinnati,OH)的以及牛奶蛋白水解物、酪蛋白水解物、乳清蛋白水解物和植物蛋白水解物。
在进一步的实施方式中,抗炎剂可以是已显示能调节炎症的益生菌。合适的免疫调节性益生菌包括乳酸菌,例如嗜酸菌(acidophilli)、乳酸杆菌和活性乳酸菌(bifidophilli)。在还有另一实施方式中,抗炎剂可以是具有抗炎特性的植物提取物。具有抗炎益处的合适植物提取物的非限制性例子包括蓝莓、乳香(boswella)、黑儿茶和中国黄岑、芹实、甘菊、樱桃、鬼爪(devils claw)、桉树、月见草、姜、山楂果(hawthorne berry)、木贼、刺楸皮(Kalopanax pictusbark)、甘草根、姜黄、白柳(white wallow)、柳树皮、和丝兰。
(i)赋形剂
可根据与活性成分的相容性选择食品添加剂制剂中各种常规使用的赋形剂。合适赋形剂的非限制性例子包括选自下组的试剂:非泡腾崩解剂、着色剂、风味修饰剂、口服分散剂、稳定剂、防腐剂、稀释剂、压缩剂(compactionagent)、润滑剂、填充剂、粘合剂、味道遮蔽剂、泡腾崩解剂、和任何这些试剂的组合。
在一个实施方式中,所述赋形剂是粘合剂。合适的粘合剂包括淀粉、预胶化淀粉、明胶、聚乙烯吡咯烷酮、纤维素、甲基纤维素、羧甲基纤维素钠、乙基纤维素、聚丙烯酰胺、聚乙烯基噁唑烷酮(polyvinyloxoazolidone)、聚乙烯醇、C12-C18脂肪酸醇、聚乙二醇、多元醇、糖、寡糖、多肽、寡肽和它们的组合。多肽可以是约100到约300,000道尔顿的任何氨基酸排列。
在另一实施方式中,所述赋形剂可以是填充剂。合适的填充剂包括碳水化合物、无机化合物和聚乙烯吡咯烷酮。填充剂的非限制性例子可以是硫酸钙(二代或三代)、淀粉、碳酸钙、碳酸镁、微晶纤维素、二代磷酸钙、碳酸镁、氧化镁、硅酸钙、滑石粉、改性淀粉、乳糖、蔗糖、甘露醇和山梨醇。
赋形剂可以是非泡腾崩解剂。非泡腾崩解剂的合适例子包括淀粉,例如玉米淀粉、马铃薯淀粉、它们的预胶化和改性淀粉、甜味剂、粘土,例如膨润土、微晶纤维素、海藻酸盐、淀粉羟乙酸钠、树胶,例如琼脂、瓜尔胶、豆角胶、卡拉雅胶、果胶和黄蓍胶。
在另一实施方式中,赋形剂可以是泡腾崩解剂。合适的泡腾崩解剂的非限制性例子包括碳酸氢钠与柠檬酸的组合以及碳酸氢钠与酒石酸的组合。
赋形剂可包含防腐剂。防腐剂的合适例子包括抗氧化剂,例如a-生育酚或抗坏血酸,和抗微生物剂,例如对羟基苯甲酸酯、氯丁醇或苯酚。
在另一实施方式中,赋形剂可包括稀释剂。适用的稀释剂包括药学上可接受的糖,例如蔗糖、葡萄糖、乳糖、微晶纤维素、果糖、木糖和山梨醇;多元醇;淀粉;预制的直接压片稀释剂;和任何前述物质的混合物。
赋形剂可包括调味剂。掺入外层的调味剂可以选自合成的调味油和调味芳香剂和/或天然的油,植物、叶、华、果实的提取物,以及它们的组合物。例如,这些调味剂可包括肉桂油、冬青油、胡椒油、丁香油、干草油(hay oil)、茴香油、桉树(调味剂)、香草(调味剂)、柑橘油,例如柠檬油、橙油、葡萄和葡萄柚油,果实精油,包括苹果、桃子、梨、草莓、红莓、樱桃、李、菠萝和杏。
在另一实施方式中,赋形剂可包括甜味剂。甜味剂非非限制性例子选自:葡萄糖(玉米糖浆)、右旋糖、惰性糖、果糖、和它们的混合物(当不用作运载体时);糖精及其各种盐,例如钠盐;二肽甜味剂,例如阿斯巴甜;二氢查耳酮化合物、甘草皂苷;甜菊(Stevia Rebaudiana)(蛇菊苷);蔗糖的氯代衍生物,例如三氯蔗糖;糖醇,例如山梨醇、甘露醇、木糖醇,等等。
在另一实施方式中,赋形剂可以是润滑剂。润滑剂的合适非限制性例子包括硬脂酸镁、硬脂酸钙、硬脂酸锌、氢化植物油、氢化植物油、聚氧乙烯单硬脂酸酯、滑石粉、聚乙二醇、苯甲酸钠、月桂基硫酸钠、月桂基硫酸镁、和轻质液体石蜡(light mineral oil)。
赋形剂可以是分散增强剂。合适的分散剂可包括淀粉、藻酸、聚乙烯吡咯烷酮、瓜尔胶、高岭土、膨润土、纯化的木纤维素、淀粉羟乙酸钠、异无定形二氧化硅(isoamorphous silicate)和微晶纤维素,例如高HLB乳化表面活性剂。
根据具体的实施方式,在外层中提供着色剂是理想的。合适的颜色添加剂包括食物、药物和化妆品色素(FD&C)、药物和化妆品色素(D&C)或外部药物和化妆品色素(Ext.D&C)。根据具体的实施方式,这些色素或染料,连同它们相应的色淀以及某些天然和衍生的着色剂可能适用于本发明。
赋形剂可包括味道遮蔽剂。味道遮蔽物质包括,例如纤维素羟丙基醚(HPC),如Nisswo HPC和PrimaFlo HP22;低取代的羟丙基醚(L-HPC);纤维素羟丙基甲基醚(HPMC),如赛普膜(Seppifilm)-LC、Metolose SR、Opadry YS、PrimaFlo、MP3295A、Benecel MP824和Benecel MP843;甲基纤维素聚合物,如和乙基纤维素(EC)及其混合物,如E461、 -EC、苏丽丝(Surelease);聚乙烯醇(PVA),如Opadry AMB;羟乙基纤维素,如羧甲基纤维素和羧甲基纤维素的盐(CMC),如-CMC;聚乙烯醇和聚乙二醇共聚物,例如单酸甘油酯(Myverol)、甘油三酯(KLX)、聚乙二醇、改性的食品淀粉、丙烯酸聚合物和丙烯酸聚合物与纤维素醚的混合物,例如EPO、RD 100和E100;醋酸邻苯二甲酸纤维素;赛匹膜(sepifilm),例如HPMC和硬脂酸、环糊精的混合物,以及这些物质的混合物。在其它实施方式中,考虑的其它味道遮蔽物质是各自通过引用全文纳入本文的美国专利号4,851,226;5,075,114和5,876,759所述那些。
在各种实施方式中,赋形剂可包括pH调节剂。在某些实施方式中,pH调节剂可包含碳酸钠或碳酸氢钠。在其它实施方式中,利用如BHT或BHA等抗氧化剂。
食品添加剂中赋形剂或赋形剂组合的重量分数可以是药物组合物总重的约98%或更低、约95%或更低、约90%或更低、约85%或更低、约80%或更低、约75%或更低、约70%或更低、约65%或更低、约60%或更低、约55%或更低、约50%或更低、约45%或更低、约40%或更低、约35%或更低、约30%或更低、约25%或更低、约20%或更低、约15%或更低、约10%或更低、约5%或更低、约2%、或约1%或更低。
(j)示范性制剂
食品添加剂通常可包含I(a)所述的任何金属螯合剂或金属盐与I(b)所述任何软骨保护剂的组合。这些食品添加剂还可任选包含I(c)(d)(e)(f)(g)(h)或(i)中所述的任何成分。技术人员应明白,构成给定食品添加剂诸成分的类型和用量会根据哺乳动物对象而有很大不同。可根据公知的方法,将食品添加剂配制成符合几种哺乳动物对象的要求。例如,哺乳动物对象可以是人、农业动物(例如,牛、猪、绵羊或山羊)、动物园动物(例如,有蹄类动物)或陪伴动物(狗、马或猫)。
在食品添加剂的示范性实施方式中,金属螯合剂包括2-羟基-4-甲硫基丁酸的锌螯合剂、2-羟基-4-甲硫基丁酸的锰螯合剂和2-羟基-4-甲硫基丁酸的铜螯合剂的混合物;软骨保护剂包括硫酸软骨素、透明质酸、葡糖胺和胶原的混合物。在该实施方式的替代实施方式中,软骨保护剂包含在各实施方式中,食品添加剂可包含一种到几种选自下组的成分:维生素、矿物质、氨基酸、抗氧化剂、酵母培养物、抗炎剂和必需脂肪酸。实施例2非限制性地详述了为狗配制的示范性本发明食品添加剂。实施例3详述了为马配制的示范性本发明食品添加剂,而实施例6详述了为人配制的示范性本发明食品添加剂。
还考虑到如果需要,构成本发明食品添加剂的一种或多种成分可存在互变异构、几何异构或立体异构形式而不脱离本发明的范围。本发明包括所有这种化合物,包括顺式-和反式-几何异构体、E-和Z-几何异构体、R-和S-对映体、非对映体、d-异构体、1-异构体、它们的外消旋混合物以及它们的其它混合物。这种互变异构、几何异构或立体异构形式的药学上可接受的盐也属于本发明。本文所用的术语“顺式”和“反式”表示几何异构现象的某种形式,其中通过双键相连的两个碳原子各自在该双键的同侧(“顺式”)或该双键的对侧(“反式”)具有氢原子。所述的一些化合物包含烯基,表示包括顺式和反式或者“E”和“Z”几何形式。此外,所述的一些化合物包含一个或多个立构中心,表示包括存在各立构中心的R、S和R与S形式的混合物。
此外,构成本发明食品添加剂的一种或多种成分可以是游离碱或其药学上可接受的酸加成盐的形式。术语“药学上可接受的盐”是通常用于形成碱金属盐和形成游离酸或游离碱的加成盐的盐。所述盐的性质可以不同,只要其是药学上可接受的。本发明所用化合物的药学上可接受的酸加成盐可以从无机酸或有机酸制备。这种无机酸的例子是盐酸、氢溴酸、氢碘酸、硝酸、碳酸、硫酸和磷酸。合适的有机酸可以选自:脂族、环脂族、芳族、芳脂族、杂环、羧酸和磺酸类的有机酸,其例子是甲酸、乙酸、丙酸、琥珀酸、羟乙酸、葡糖酸、乳酸、苹果酸、酒石酸、柠檬酸、抗坏血酸、葡糖醛酸、马来酸、延胡索酸、丙酮酸、天冬氨酸、谷氨酸、苯甲酸、邻氨基苯甲酸、甲磺酸、4-羟基苯甲酸、苯乙酸、扁桃酸、双羟萘酸(巴莫酸)、甲磺酸、乙磺酸、苯磺酸、泛酸、2-羟基乙磺酸、甲苯磺酸、对氨基苯磺酸、环己基氨基磺酸、硬脂酸、藻酸、羟基丁酸、水杨酸、半乳糖二酸和半乳糖醛酸。本方法所用化合物的合适的药学上可接受的碱加成盐包括从铝、钙、锂、镁、钾、钠和锌制得的金属盐,或从N,N’-二苄基乙二胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、葡甲胺-(N-甲基葡糖胺)和普鲁卡因制得的有机盐。可采用常规方法,通过,例如将合适的酸或碱与本文所述一种或多种相应化合物反应而从这些相应化合物制备所有这些盐。
II.食品添加剂的剂型
可将本文所述的食品添加剂制备成一种或几种剂型。在示范性的实施方式中,所述剂型是口服剂型。合适的剂型包括片剂,包括悬液片剂、可咀嚼片剂、泡腾片或囊片;丸剂;粉末剂,例如无菌包装的粉末;可分散的粉末,和泡腾剂粉;胶囊,包括软或硬明胶胶囊或非动物衍生的聚合物,例如羟丙基甲基纤维素胶囊(即,HPMC)或支链淀粉;糖锭;囊剂;喷洒剂;可重建的粉末或摇动剂(shake);锭剂;弹丸剂;颗粒剂;液体;混悬液;乳剂;或半固体和凝胶。或者,可将食品添加剂掺入食物产品或粉末以便与液体混合,或只与非食品液体混合后口服给予。技术人员可明白,除了适合以多剂型给予外,还可采用各种剂量方案给予食品添加剂。
说明书和实施例中描述了各剂型中可用的成分(即,金属螯合剂、软骨保护剂、维生素、矿物质、氨基酸、抗氧化剂、酵母培养物和必需脂肪酸)及其它赋形剂的用量和类型。应该知道,如果描述的一种剂型包括特定用量的这些组分在内的诸成分和/或赋形剂的组合,则相同组合可用于任何其它合适的剂型。此外,应该理解,本领域技术人员借助本申请的指导,能够通过组合诸成分的用量和类型而作出上述任何剂型,所述成分可组合在一种剂型中给予或作为不同剂型而一起给予,如本申请的不同章节所述。
构成食品添加剂的活性成分的粒度可能是影响生物利用度、混合均匀性、分离和流动特性的重要因素。通常粒度较小的活性成分因表面积增加而实质性提高水溶性差的活性成分的生物吸收速度。活性成分和赋形剂的粒度也能影响食品添加剂的悬浮特性。例如,颗粒较小则不大可能沉淀,因此可形成较好的混悬液。在各种实施方式中,各种成分的干燥粉末(可以作为悬浮粉末而直接给予,或用在固体剂型中)的平均粒度的直径小于约500微米,或小于约450微米、或小于约400微米、或小于约350微米、或小于约300微米、或小于约250微米、或小于约200微米、或小于约150微米、或小于约100微米、或小于约75微米、或小于约50微米、或小于约25微米、或小于约15微米。在一些应用中,优选利用小于15微米的颗粒。在这些情况中,优选利用粒度为15微米到10纳米的胶体或纳米的颗粒。
可通过常规药理学技术制备本发明的食品添加剂。常规药理学技术包括,例如以下方法中的一种或组合:(1)干法混合,(2)直接压片,(3)研磨,(4)干法或无水制粒,(5)湿法制粒,或(6)融合。参见,例如Lachman等,《工业药学的理论与实践》(The Theory and Practice of Industrial Pharmacy)(1986)。其它方法包括,例如造粒、喷雾干燥、锅包衣、融化制粒、制粒、沃斯特包衣法、切线包衣、顶喷法(top spraying)、挤出、凝聚等。
以下实施例是为了说明本发明的优选实施方式。本领域技术人员应该知道,以下实施例揭示的技术代表了本发明人发现的能良好实施本发明的技术。然而,鉴于本文内容,本领域技术人员应该明白可在所揭示的具体实施方式中作出许多改变,但仍能获得类似或相似的结果而不脱离本发明的构思和范围,因此,所列或附图中所示的各事项均应理解成说明性而非限制性的。
实施例
以下实施例说明了本发明。具体地说,实施例1显示了检验食品添加剂在马匹中安全性的方法。实施例2显示了检验食品添加剂在狗中适口性的方法。实施例3和4提供了检验食品添加剂对成熟马匹健康关节的维持和发育益处的方法。实施例5提供了检验食品添加剂在骨关节炎大鼠模型中的治疗益处的方法。实施例5列出了为人配制的示范性食品添加剂。
实施例1.成熟马匹口服摄取食品添加剂的安全性
该项研究的目的在于确认成熟马匹口服摄取食品添加剂的安全性。所述食品添加剂包含磷酸二钙、(天然蛋膜,其包含葡糖胺、硫酸软骨素、透明质酸和胶原)、抗坏血酸、MHA(甲硫氨酸羟基类似物)、HMBTA-Mn、HMTBA-Zn、HMBTA-Cu、生物素、Zorien Se、和维生素D。利用血液样品比较含有所述食品添加剂的饲料与对照饲料对于行为观察结果和标准临床化学参数(standard clinical chemistry panel)的影响。
饲料.研究期间给所有马匹提供两种饲料。在首个7天,给所有马匹提供符合或超过NRC要求的商业马饲料(对照饲料)。在随后的14天,给予马匹含额外食品添加剂的相同商业马饲料作为追肥应用(top dress application)。给予食品添加剂的初始速度是每500kg马匹给予50g食品添加剂。在研究的最后7天中,给马匹提供先前的商业马饲料。每周收集马饲料浓缩物样品。对各批食品添加剂取样并分析矿物质含量(Cu、Mn、Zn)以及葡糖胺、软骨素和透明质酸。
动物和实验设计.两处场所的5匹马(两雄、三雌)参与该项研究。一匹雄马和两匹雌马圈养在密苏里州温菲尔德的威迈尔农场(Wehmeyer Farms,Winfield,Missouri);一匹雄马和一匹雌马圈养在密苏里州特洛伊的哈雷农场(Harrell Farms,Troy,Missouri)。每匹马指定单独的畜栏以确保摄取所需量的食品添加剂。实验结束时,放出所有每匹。
行为观察.每天至少观察马匹两次,记录观察到的任何异常现象。
血液样品.将每匹马的最多10毫升血液收集入血清分离管中,使其凝结至少30分钟。离心所有样品获得未稀释的血清,分成两等份并冷冻。一份寄给安泰兽医诊断实验室(Antech Veterinary Diagnostic Labs)并分析标准血液化学参数。研究开始时抽取所有马匹的血液样品,研究期间每7天抽取1次,研究结束时抽取1次。
数据分析和统计学.分析血液数据作配对观察(paired observation)。采用SAS的通用线性模型,比较研究前两周取得的基线饲料的血液样品与研究最后两周取得的实验性饲料的血液样品的差异,结果表示为平均值±平均值的标准偏差。
结果.发现该食品添加剂对于本项研究的所有马匹是适口的,食物摄取未因存在食品添加剂而有明显改变。此外,未在接受该食品添加剂的任何马匹中观察到不良作用。所有马匹的血液化学参数均在正常范围内,在给予该食品添加剂时未注意到任何马匹有行为改变。
实施例2.包含食品添加剂的犬食物的适口性
进行以下研究以确定包含食品添加剂的犬食物的适口性。采用最新的药品生产质量管理规范(cGMP)制造为犬配制的包含食品添加剂的两种可咀嚼犬片剂。一种片剂(食物A)用鸡肉消化物调味,第二种片剂(食物B)用牛肉和干酪帕拉坦特(palatant)调味。该食品添加剂包含MHA、磷酸三钙、抗坏血酸、HMBTA-Zn、Zorien Se、HMTBA-Cu、HMTBA-Mn、生物素和维生素D。
动物.20只雄性和雌性小猎犬饲养在按照动物福利法案(Animal WelfareAct)注册为ASDA 23-R-126号的犬舍机构中。该犬舍具有12-小时白天和12-小时黑夜周期。犬舍的温度按照动物福利法案维持在目标条件(即,50°F到80°F)。按照动物福利法案每日清洁并消毒笼子和进食碗。
实验设计.用不锈钢碗给予每只犬各种食物之一,每日一次,共两天。每天倒转碗的位置,两只碗放置最多5分钟或直至第一食物被吃完。给予食物的5小时前,给予每只犬300g标准犬饲料(俄亥俄州圣玛丽斯的欢乐宠物食品公司(Joy Pet Food,St.Marys,OH)的特别食品),30分钟。犬饲料的平均消耗率为85%。
数据分析和统计学.通过耳朵刺花和笼号鉴定每只犬。每天记录每只犬第一接近偏爱(first approach)、第一消耗偏爱(first consumption preference)和消耗第一食物的时间(以秒计)。采用κ2和t检验分析数据。
结果.表1给出了两次实验中每只犬的第一接近偏爱和第一消耗偏爱。发现这些犬接近两种食物大致相同,但它们消耗食物A的频率约是食物B的9倍。
表1.犬食物适口性总结
在所有犬中,食物A的第一接近偏爱是23次,食物B的第一接近偏爱是17次。在每只犬的水平,有3只犬(15%)对食物A显示第一接近偏爱,没有犬对食物B显示第一接近偏爱,17只犬对食物A和B接近相等(85%)。κ2分析发现第一接近偏爱没有统计学显著性。类似地,t-检验分析发现在95%的显著性水平上第一接近偏爱没有统计学显著性。P-值是0.08281。
总体上,食物A的第一消耗偏爱是36次,食物B的第一消耗偏爱是4次。在每只犬的水平,有16只犬(80%)在两天均消耗食物A,没有犬在两天均消耗食物B,4只犬(20%)一天消耗食物A,另一天消耗食物B。κ2分析发现第一消耗偏爱有统计学显著的差异。类似地,t-检验分析发现在95%的显著性水平上第一消耗偏爱有统计学显著的差异。P-值是0.00000。消耗两种食物的时间是3秒到90秒。因为消耗食物B的例子太少,不可能进行统计学分析。
本项研究揭示了犬更喜欢包含食品添加剂的鸡肉调味片剂。
实施例3.检测马匹的关节疾病和食品添加剂的治疗益处
本项研究的第一目的是监测软骨和滑膜代谢标记物以及炎症和氧化应激标记物的马匹尿液和/或血浆/血清水平,从而确定个体内和个体间的平均水平和方差并寻找可能与关节病状相对应的模式。本项研究的第二目的是检验食品添加剂作为患关节疾病的马匹的潜在替代疗法的益处。
该研究包括接受食品添加剂相同时间的两组马匹。这两组治疗前后的监测时间不同。马匹食品添加剂如以上实施例1所述。
动物.该项研究包括至少2岁的成熟驯养马(Equus cabalbus)。排除在之前120天中进行过关节手术的马匹。还排除:1)在之前30天中接受全身性氨基葡聚糖(GAGs),2)在之前14天中接受饮食维生素、矿物质或关节健康添加剂,3)在之前7天中接受全身性类固醇,或4)在之前7天中接受全身性非甾体抗炎药(NSAIDs)的马匹。马兽医利用以前的病史和体检来评估开始研究前各匹马的总体健康状况。马兽医采用美国马匹从业者协会(American Association of Equine Practitioners)(AAEP)跛行量度(参见,例如http://www.aaep.org)评估关节情况并指定跛行评分。马匹可随意饮水,并与实验前一样继续进食和训练。
实验设计.初始跛行评分后,将马匹随机分入A组或B组,使得两组的马匹数大致相同,年龄和跛行评分大致匹配。该实验包括4个时期,如表2所示。1期和4期是治疗前期和治疗后期,2期和3期是治疗期,在此期间一组进行治疗,而另一组不治疗。在表2中,“0”表示不治疗,“T”表示治疗。在治疗期间,食品添加剂的给予剂量是每匹马每天50克。添加剂可以混入每日饲料配给。
表2.实验设计
组 | 1期(4周) | 2期(8周) | 3期(8周) | 4期(4周) |
A | 0 | T | 0 | 0 |
B | 0 | 0 | T | 0 |
在1期开始前停止给予任何药物和/或添加剂,并如上所述评估各动物的总体健康状况。表3显示了该实验4期的详细时间表。
表3.实验方案.
收集血液、尿液和热成像 | 测定AAEP跛行评分 | |
实验前 | 第0天 | |
1期(28天) | 第1、14、28天 | 第28天 |
2期(56天) | 第3、7、14、28、42、56天 | 第28、56天 |
3期(56天) | 第3、7、14、28、42、56天 | 第28、56天 |
4期(28天) | 第14、28天 | 第28天 |
采集血液.从颈静脉采集血液,置于两根不同试管中以便采集血清和血浆。-20℃冷冻样品直至分析。可检验血浆和/或血清中软骨代谢的生物学标记(例如,PIIANP、CTX-II、COMP)以及炎症的标记物(例如,IL-6)或氧化应激(例如,8-异-PGF2α、PGE2)。还可检测维生素D、维生素E、抗坏血酸、锌、铜和锰的血清水平。
采集尿液.采用自流收集采集尿液,-20℃冷冻直至分析。可分析尿液的糖基-半乳糖基-吡啶啉(pyridinoline)(滑液代谢的标记物)和CTX-II。通过尿肌酸酐浓度校正尿检获得的所有生物学标记水平。
热成像.可按照生产商的使用说明书,用华盛顿州埃弗雷特市弗鲁克公司的弗鲁克热成像系统(FLUKE thermal imager system,Fluke Corp.,Everett,WA)获得热成像。动物的全身图像可取自前面、后面,和两侧。还可取得炎症特定区域的图像。成像之时可记录环境温度和动物的体温。通常在取得图像之后立即收集其它样品。
同期的疗法.某些疗法(例如,GAG、类固醇、NSIAD和其它添加剂)在实验期间是禁止的。由兽医逐例评估其它疗法,如果相信它们不会影响所选生物标记的水平则允许。记录所有的同期疗法。此外,实验期间马匹可能不能接受针灸、按摩、脊柱推拿疗法。
数据分析.生物标记的浓度可表示为平均值±标准偏差。采用斯氏t检验测定统计学差异。可利用年龄和跛行作为因变量(dependent variable),利用1期的生物标记分析来确定个体间和个体内随时间的方差。1期检测的生物标记的模式可对应于关节病变。可将检测的生物标记模式或浓度的改变和/或跛行评分的改变和/或热成像的改变作为用食品添加剂治疗的结果。
如果食品添加剂治疗造成所选生物标记的浓度降低和/或跛行评分降低,则可以得出结论:该食品添加剂对于马匹的关节疾病的治疗能提供治疗益处。治疗益处可表现为关节疾病相关疼痛减轻,和/或关节组织的生长、修复和/或维持。
实施例4.食品添加剂在患关节疾病马匹中的治疗益处
以下研究的目的是确定用食品添加剂治疗具有跛行病史的马匹是否有助于控制关节健康相关的临床迹象(即,降低跛行评分、缓解关节疾病症状、稳定或防止关节组织进一步恶化和/或促进关节组织的生长和/或修复)。
双盲安慰剂-受控研究比较以下两组:一组喂食测试食品添加剂,第二组喂食不含活性成分的相当食品添加剂。马匹食品添加剂如以上实施例1所详述。
动物.该项研究包括具有关节健康问题的至少2岁的成熟驯养(私人拥有)马匹。关节健康问题包括对于退行性关节病或骨关节炎常见的关节渗液、跛行或其它临床迹象。所包括马匹的AAEP跛行评分等于或大于2.0并小于4.0。如果双侧跛行明显,受更严重影响的下肢称为受影响肢。排除之前120天内作过关节手术的马匹。还排除:1)在之前90天中,受影响关节中接受关节内注射,2)在之前30天中接受全身性GAG,3)在之前14天中接受饮食维生素、矿物质或关节健康添加剂,4)在之前7天中接受全身性类固醇,5)在之前7天中接受全身性非甾体抗炎药(NSAIDs),或6)在之前两周中钉马掌或修整马掌而有改变的马匹。马匹可随意饮水,并提供饲料(干草和谷物,如果需要的话)以维持体重。
实验开始前,动物经历21-天的适应期。可由马兽医对动物进行初始评估。
实验设计.该项研究包括随机化完全区组设计,根据跛行级别和受影响的关节将马匹分区。在各区(配对)中,将马匹随机分配为对照或治疗组。对照组接受安慰剂食品添加剂(即,苜蓿饲料、糖蜜和亚麻油)。治疗组可每天给予每只动物50g食品添加剂。可将食品添加剂混入饲料。如果马匹未能消耗饲料或者添加剂与饲料相分离并且未消耗完,则可用水或糖蜜润湿该饲料以提高适口性并阻止分离。临床调查人员不知治疗分配,从而确保记录的所有观察结果不偏不倚。
该项研究的持续时间可以是6周(42天)。表4给出了研究时间表。
表4.研究活动
研究日 | 活动 |
第-21天 | 兽医基线评估 |
第0天(±2天) | 病史、所有者同意、兽医评估、分区和分配治疗、滑膜液、血清收集 |
第14、28、42天(±2天) | 兽医评估,血清、滑膜液收集 |
通用兽医评估.可以不考虑同一马匹之前的评估。受影响下肢和对照-侧肢通常分别给予AAEP跛行级别,只有受影响下肢给予其余参数。然而,如果马匹在任一评估日显示双侧跛行,两下肢均给予所有参数。可利用垂直线标出直观类比标度(VAS)参数,在10cm线上标出指拨点(appropriated spot)。
跛行评分.采用AAEP跛行分级量表评估跛行。数据形式包括对评估期间所用基础的描述。在研究期间,该基础对于每匹马是不变的。
步行时的跛行(VAS).可根据10cm直观类比标度(VAS)对受影响肢在步行时的跛行评分,其中左侧符合步行完好,右侧符合非负重的。
小跑时的跛行(VAS).可根据10cm直观类比标度(VAS)对受影响下肢在小跑时的跛行评分,其中左侧符合小跑时完好,右侧符合非负重的。
步幅长度.抬起受影响下肢,清洁马蹄并喷水。可将彩色粉笔(粉末状的)涂在该马蹄底面,让该马在适于目测观察蹄印的表面(例如,混凝土或人行道)上小跑。从可见的最远马蹄印测量,可用卷尺测量步幅长度。以英尺和英寸记录最少3个,最多6个步幅。
关节弯曲.小心地,阻力很小或没有阻力地将受影响的关节弯曲至最高程度。根据10cm VAS对弯曲评分,其中0=正常,10=不能弯曲。如果受影响的关节(例如,胶骨(pastern)、蹄骨(coffin)和舟骨)本身的运动性受限,则不进行该检测,数据形式输入为“NA”,即“不适用”。
对关节弯曲的反应.根据10cm VAS给马匹对关节弯曲的反应评分,其中0=正常,10=极端反应(假定受影响的关节是能被弯曲的,见上文)。
关节弯曲后的跛行.可将受影响关节维持于弯曲位置60秒,放开弯曲的关节后让马匹立即小跑。根据10cm VAS测量跛行,其中0=正常,10=非负重的。
生活质量指数.可根据10cm VAS,利用所有以上参数以及马匹的行为评估生活质量,其中0=生活质量优秀,没有明显的DJD相关不适,10=生活质量差。
滑液.兽医评估后,可外部清洁受影响的关节(如果需要可修剪),可将滑液样品无菌收集入含EDTA的试管中。可检验滑液的WBC、总蛋白、骨钙蛋白、IL-2和TNFα。
血液.可利用肝素化注射器和试管收集血液,并离心收集血浆。可检验血浆的骨和软骨标记物(例如,骨钙蛋白、DPD、降钙素、COMP、CTX-II、PIIANP)以及炎症的量度(例如,细胞因子,如IL-1β、Il-6、TNFα、8-异-PGF2α、iNOS、COX-2)。
从研究中排除对象.如果发生严重的健康状况可从研究中排除马匹,完整记录该健康状况并给予治疗。除了逐例与主办方和研究监督人员商议后,研究人员不能排除马匹。
同期的疗法.研究期间,可能需要治疗性干预以治疗医学状况,因为检验的动物是私人拥有的。研究人员、所有者/训练师以及任何其它参与的兽医必须首先保证各马匹的健康。本项研究期间禁止对受影响关节作关节内治疗、用PSGAG或透明质酸作全身性治疗、用NSAID或皮质激素治疗、或加入关节健康食品添加剂(例如,葡糖胺、硫酸软骨素、MSM、贻贝(perna mussel)),这会将该马匹排除出该项研究。零星使用其它疾病的药物必须逐例作出评估以确定该马匹是可保留在数据组中还是必须排除。按照方案,NSAID给药的7天内不进行马匹的临床评估。在实验期间,所有者/训练师不能对研究马匹进行针灸、按摩、脊柱推拿或其它替代形式。所有同期的药物和食品添加剂将记录在案。研究期间,可对马匹进行修整/装马掌;然而,修整或装马掌的模式不允许改变(即,角度、马掌类型、衬垫的添加/去除)。研究期间,马匹的训练时间表不作大的变动。
数据分析.如果马匹的AAEP跛行评分在治疗结束时降低1或更多单位,或者跛行VAS之一降低2cm或更多cm,则将该马匹分类为有反应。比较各治疗组中有反应和无反应的数量。还测定了所有参数的平均值和标准偏差。
实施例5.食品添加剂在大鼠骨关节炎模型中的效力
本项研究的目的是确定食品添加剂是否能在骨关节炎的碘醋酸单钠(MIA)大鼠模型中减轻骨关节炎的严重程度。
实验设计.该项研究可包括三组大鼠:1)无食品添加剂的对照组,2)提供马食品添加剂(如上详述)的组,和3)添加了NEM(即,葡糖胺、硫酸软骨素、透明质酸和胶原)的组。所述大鼠通常是约250-300g的雄性Wistar大鼠,每组16只。这些大鼠可经历一个月的实验前期。对于该项研究,在各大鼠右膝注射MIA,左膝注射盐水,喂食三种饲料之一,并监测56天。表5给出了研究时间表。
表5.R大鼠研究时间表.
研究天数 | 活动 |
第-28天 | 进食前一个月。大鼠喂食典型的大鼠饲料;治疗组2和3喂食相同饲料,但在饲料中掺入测试添加剂。 |
第0天 | 将所有大鼠分配入3组之一。将碘醋酸单钠(MIA)注射入右髌下膝韧带;将盐水注射入左膝。 |
第1天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。 |
第3天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。 |
第5天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。 |
第7天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。每组麻醉4只大鼠(总共12只);收集血液,检测关节肿胀和收集数据以供组织学分析。(剩余36只) |
第10天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。 |
第14天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。每组麻醉4只大鼠(总共12只);收集血液,检测关节肿胀和收集数据以供组织病理学分析。(剩余24只) |
第21天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。 |
第28天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。每组麻醉4只大鼠(总共12只)。收集血液,检测关节肿胀和收集数据以供组织病理学分析。(剩余12只) |
第56天 | 获取各大鼠左右膝的热成像。评估后爪腿分布。每组其余的大鼠(12只)。收集血液,检测关节肿胀和收集数据以供组织 |
病理学学分析。 |
关节肿胀.可借助卡尺检测关节肿胀程度。或者,还可利用热成像定量测定肿胀程度。先用异氟烷轻微麻醉大鼠再检测。
血液.可利用肝素化的注射器和试管,通过心脏穿刺收集用异氟烷麻醉的大鼠的血液,离心收集血浆并冷冻直至分析。可分析样品的软骨和骨代谢的生物标记(PIIANP、CTX-II、COMP、骨钙蛋白)和炎症的量度(IL-6、IL-1β)。
热成像.可用弗鲁克热成像系统在指定日获得大鼠的图像。评估同一图像中的左右膝。还记录环境温度。
组织学.可用H&E、番红O和甲苯胺蓝染色剂染色样品以检测骨组织病理学情况以及软骨和骨生物标记。
失能测试.失能测试仪(incapacitance tester)对后爪重量分布分析作出估计。该测量值可定量测定运动性和疼痛。注射MIA的膝盖应更痛,因此,更多的重量应分布在注射盐水的膝盖上。
数据分析.各种指标可以平均值与标准偏差表示。估计所有动物可在早期时间点(注射后不久)显示炎症和肿胀的迹象,而骨降解与合成可在较晚时间点发生。治疗组与对照组中反应的差异可表明该治疗缓解了骨关节炎相关症状和/或防止了骨关节炎的发生。
实施例6.人制剂
Claims (28)
1.一种食品添加剂,其包含至少一种软骨保护剂和至少一种金属螯合剂;所述金属螯合剂包含至少一种金属离子和至少一种配体,所述配体包含式(1)所示化合物:
式中:
n是0-2的整数;
R1是甲基或乙基;和
R2选自下组:羟基和氨基。
2.如权利要求1所述的食品添加剂,其特征在于,还包含选自下组的至少一种成分:维生素、矿物质、氨基酸、抗氧化剂、酵母培养物、抗炎剂和必需脂肪酸。
3.如权利要求1所述的食品添加剂,其特征在于,配体与金属离子的平均比例是1∶1到3∶1。
4.如权利要求1所述的食品添加剂,其特征在于,所述金属离子选自下组:锌离子、铜离子、镁离子、锰离子、铁离子、铬离子、钙离子和它们的组合。
5.如权利要求3所述的食品添加剂,其特征在于,所述金属离子选自下组:锰离子、锌离子和铜离子,所述配体是2-羟基-4-甲硫基-丁酸。
6.如权利要求5所述的食品添加剂,其特征在于,所述软骨保护剂选自下组:葡糖胺、软骨素、透明质酸、胶原、浓缩的蛋壳膜和绿唇贻贝。
7.如权利要求1所述的食品添加剂,其特征在于,所述金属螯合剂包括2-羟基-4-甲硫基丁酸的锌螯合剂、2-羟基-4-甲硫基丁酸的锰螯合剂和2-羟基-4-甲硫基丁酸的铜螯合剂的混合物;所述软骨保护剂包括浓缩的蛋壳膜。
8.如权利要求7所述的食品添加物,其特征在于,还包含选自下组的至少一种成分:维生素、矿物质、氨基酸、抗氧化剂、酵母培养物、抗炎剂和必需脂肪酸。
9.如权利要求7所述的食品添加物,其特征在于,还包含维生素C、生物素、维生素D、硒、钙、磷酸盐和2-羟基-4-甲硫基丁酸。
10.如权利要求9所述的食品添加物,其特征在于,还包含干燥的糖蜜、亚麻油、酵母培养物和苜蓿饲料。
11.如权利要求7所述的食品添加物,其特征在于,还包含镁、维生素C、维生素K、硒、钙和维生素D。
12.如权利要求7所述的食品添加物,其特征在于,所述食品添加剂具有2重量%到20重量%的金属螯合剂和5重量%到40重量%的软骨保护剂。
14.如权利要求13所述的用途,其特征在于,所述食品添加剂还包含选自下组的至少一种成分:维生素、矿物质、氨基酸、抗氧化剂、酵母培养物、抗炎剂和必需脂肪酸。
15.如权利要求13所述的用途,其特征在于,配体与金属离子的平均比例是1∶1到3∶1。
16.如权利要求13所述的用途,其特征在于,所述金属离子选自下组:锌离子、铜离子、镁离子、锰离子、铁离子、铬离子、钙离子和它们的组合。
17.如权利要求15所述的用途,其特征在于,所述金属离子选自下组:锰离子、锌离子和铜离子,所述配体是2-羟基-4-甲硫基-丁酸。
18.如权利要求17所述的用途,其特征在于,所述软骨保护剂选自下组:葡糖胺、软骨素、透明质酸、胶原、浓缩的蛋壳膜和绿唇贻贝。
19.如权利要求13所述的用途,其特征在于,所述金属螯合剂包括2-羟基-4-甲硫基丁酸的锌螯合剂、2-羟基-4-甲硫基丁酸的锰螯合剂和2-羟基-4-甲硫基丁酸的铜螯合剂的混合物;所述软骨保护剂包括浓缩的蛋壳膜。
20.如权利要求19所述的用途,其特征在于,所述食品添加剂还包含选自下组的至少一种成分:维生素、矿物质、氨基酸、抗氧化剂、酵母培养物、抗炎剂和必需脂肪酸。
21.如权利要求19所述的用途,其特征在于,所述哺乳动物对象是马或陪伴动物,并且所述食品添加剂还包含维生素C、生物素、维生素D、硒、钙、磷酸盐和2-羟基-4-甲硫基丁酸。
22.如权利要求21所述的用途,其特征在于,所述哺乳动物对象是马,并且所述食品添加剂还包含干燥的糖蜜、亚麻油、酵母培养物和苜蓿饲料。
23.如权利要求21所述的用途,其特征在于,所述哺乳动物对象是狗,并且所述食品添加剂还包含酿酒酵母和调味赋形剂。
24.如权利要求19所述的用途,其特征在于,所述哺乳动物对象是人;并且所述食品添加剂还包含镁、维生素C、维生素K、硒、钙和维生素D。
25.如权利要求13所述的用途,其特征在于,所述食品添加剂具有2重量%到20重量%的金属螯合剂和5重量%到40重量%的软骨保护剂。
26.如权利要求13所述的用途,其特征在于,所述哺乳动物对象选自下组:陪伴动物、动物园动物和人。
27.如权利要求13所述的用途,其特征在于,所述哺乳动物对象具有关节相关适应症。
28.如权利要求27所述的用途,其特征在于,所述关节相关适应症选自下组:骨关节炎、类风湿性关节炎、银屑病关节炎、关节渗液、关节炎症和/或疼痛、滑膜炎、跛行、术后关节镜手术、正常关节功能衰退、软骨细胞代谢活性受抑制和透明质酸产生受抑制。
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AR066877A1 (es) | 2009-09-16 |
CL2008001619A1 (es) | 2009-03-06 |
BRPI0812774A2 (pt) | 2014-12-02 |
EP2162153A1 (en) | 2010-03-17 |
MX2009013265A (es) | 2010-01-25 |
US8968791B2 (en) | 2015-03-03 |
TWI432147B (zh) | 2014-04-01 |
WO2008154178A1 (en) | 2008-12-18 |
PE20090327A1 (es) | 2009-03-27 |
US20110171187A1 (en) | 2011-07-14 |
TW200906316A (en) | 2009-02-16 |
US20150132402A1 (en) | 2015-05-14 |
EP2162153B1 (en) | 2016-03-30 |
EP2162153A4 (en) | 2014-10-22 |
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