CN1366991B - 植物提取物用于制备治疗骨吸收增加性疾病的药物的用途及其组合物 - Google Patents
植物提取物用于制备治疗骨吸收增加性疾病的药物的用途及其组合物 Download PDFInfo
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- CN1366991B CN1366991B CN021015813A CN02101581A CN1366991B CN 1366991 B CN1366991 B CN 1366991B CN 021015813 A CN021015813 A CN 021015813A CN 02101581 A CN02101581 A CN 02101581A CN 1366991 B CN1366991 B CN 1366991B
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Abstract
本发明涉及含有选自由葱属、芸苔属、石蛇床属和芝麻菜属提取物和浓缩物或其混合物构成的组中的植物提取物或浓缩物的营养或药物组合物。本发明的该组合物用于治疗或预防诸如佩吉特氏病、肿瘤诱导的骨疾病，或特别是骨质疏松症等以骨吸收增加为特征的疾病或状态。
Description
本发明涉及包含某些植物提取物或浓缩物的营养或药物组合物，以及它们作为骨吸收抑制剂的用途。
最常见的代谢性骨疾病是骨质疏松症。骨质疏松通常可定义为骨质数量减少，它是由骨形成减少或骨吸收加快引起的，无论哪种情形，结果都是骨胳组织的数量减少了。在吸收过程中，破骨细胞(骨再吸收细胞)引起部分骨的陷凹。吸收后，成骨细胞(骨形成细胞)出现，用新的骨填满被再吸收的部分。
在年轻的健康成人体内，破骨细胞和成骨细胞的形成速率以及它们的作用使骨吸收和骨形成之间维持平衡。然而，随着年龄的增长，在这种改型过程的发展中出现了不平衡，导致了骨丧失.当不平衡持续时间过长，就导致骨质减少，从而骨强度导致骨折。
在医学文献中记载了许多关于治疗骨质疏松症的组合物和方法。例如，雌激素、降钙素和双膦酸盐是已知的骨吸收的有效抑制剂。
现在，我们已令人惊奇地发现，从某些植物或蔬菜获得的产物，例如，属于百合科、伞形科和十字花科的植物，它们对骨吸收有强大的抑制作用。
因此，本发明涉及对骨吸收具有抑制作用的植物提取物或浓缩物、但不包括豆科和蛇麻花的提取物或浓缩物，在用于制备治疗或预防以骨吸收增加为特征的疾病或状态的药物或营养制剂中的用途。以骨吸收增加为特征的疾病可以是，例如佩吉特氏病、肿瘤诱导的骨疾病，或特别是骨质疏松症。
术语“豆科”是指植物分科-豆科(豌豆类)，例如，它包括大豆、菜豆、鹰嘴豆或兵豆。蛇麻花的意思是指植物种-啤酒花。
本文中所用的“骨质疏松症”包括因激素不足(例如绝经后)和老龄引起的骨质疏松，以及继发性骨质疏松，诸如继发于类固醇治疗或继发于因神经性食欲缺乏导致的营养不良的骨质疏松。
本发明还提供了治疗或预防诸如佩吉特氏病、肿瘤诱导的骨疾病、特别是骨质疏松症等以骨吸收增加为特征的疾病或状态的方法，它包括给人或其他哺乳动物服用药物或营养制剂，所述药物或营养制剂包含除来自于豆科和蛇麻花以外的蔬菜提取物或浓缩物，其量能有效地抑制骨吸收。
本发明还预见了选自葱、石蛇床、芸苔和芝麻菜提取物和浓缩物的植物提取物或浓缩物，在用于制备治疗或预防诸如佩吉特氏病、肿瘤诱导的骨疾病、特别是骨质疏松症等以骨吸收增加为特征的疾病或状态的药物或营养制剂中的用途。
本发明还提供了治疗或预防诸如佩吉特氏病、肿瘤诱导的骨疾病、特别是骨质疏松症等以骨吸收增加为特征的疾病或状态的方法，它包含给人或其他哺乳动物服用药物或营养制剂，所述药物或营养制剂包含选自葱、石蛇床、芸苔和芝麻菜提取物和浓缩物的植物提取物或浓缩物，其量能有效地抑制骨吸收。
优选的是，来源于葱、石蛇床、芸苔和/或芝麻菜的提取物和浓缩物是蔬菜提取物或浓缩物。
在本文中，术语“蔬菜”指的是具有可食用部分的草本植物，人以生或熟的形式消耗它们。可食用部分可以是根，诸如芜菁甘蓝、甜菜、胡萝卜、和甘薯；可以是块茎或贮藏茎，诸如马铃薯和芋头；茎，如天门冬属和苤蓝；芽，诸如抱子甘蓝；球茎，诸如洋葱和大蒜；梗或叶柄，诸如芹菜和大黄；叶，诸如卷心菜、莴苣、欧芹和菠菜；未成熟花，诸如花椰菜、花茎甘蓝和朝鲜蓟；种子；未成熟果实，如茄子、黄瓜、和甜玉米(玉蜀黍)；或成熟果实，如番茄和辣椒。
在本文中，术语“葱”指的是葱属(大蒜的拉丁文，洋葱科的一员)，它包括例如植物种-洋葱、火葱(冬葱)、大头蒜、韭葱、北葱、熊葱、蒜或大葱(叶葱)中的任何一员。优选的种是火葱(冬葱)、韭葱、洋葱和熊葱(也称作熊爪葱)，特别是后两种，而洋葱是尤为优选的。洋葱种的成员的实例有普通洋葱(红皮或白皮或黄皮的)或冬葱，红皮或白皮普通洋葱是优选的。
例如，洋葱提取物和浓缩物是用该种蔬菜的整个可食用部分制备的。合适的北葱提取物和浓缩物是从北葱草得到的。合适的熊葱提取物和浓缩物是从熊葱的球茎、新鲜的草或从整个盛开的植物获得的，优选它们是从新鲜的草得到的。
本文中所用的术语“石蛇床”是指石蛇床属(俗名欧芹)，例如，它包括植物种-皱叶欧芹的任何成员。实例是Petroselinum crispumcrispum，即具有卷曲叶片的普通欧芹，Petroselinum crispum radiosum或Petroselinum crispum var.neapolitanum，也叫作意大利欧芹，它的叶子是平的。
例如，合适的石蛇床提取物或浓缩物可以用根、果实或种子，或者特别是用草制备。
本文中所用的术语“芸苔”是指芸苔属(卷心菜的拉丁文)，例如，它包括植物种-甘蓝、欧洲油菜、芜青、芥蓝、芥菜、Brassicaperviridis、白芥和黑芥中的任何成员。
甘蓝是优选的种，该种中特别优选的成员是Brassica oleracea var.italica，即花茎甘蓝，或Brassica oleracea var.gemmifera，即抱子甘蓝。花茎甘蓝提取物和浓缩物是特别优选的芸苔属提取物或浓缩物。合适的甘蓝种的提取物和浓缩物是有利地用该种蔬菜的整个可食用部分制备的，或是用新鲜的嫩芽或枝条制备的。
本文中所用的术语“芝麻菜”是指芝麻菜属，它包括，特别是，植物种-芝麻菜(野生型)或俗名为arrugula或roquette的Erucavesicaria subsp.sativa(栽培型)的任何成员。
本发明的植物/蔬菜提取物和浓缩物优选是用该植物或蔬菜的可食用部分得到的。可食用部分是指人以生或熟的形式消耗的部分。
优选的本发明的植物/蔬菜提取物和浓缩物包含来自植物种-洋葱、火葱、熊葱、皱叶欧芹、甘蓝或芝麻菜的任何成员的浓缩物或提取物。更优选的本发明的植物/蔬菜提取物和浓缩物包含来自植物种-洋葱、皱叶欧芹(特别是Petroselinum crispum crispum和Petroselinum crispum var.neapolitanum)和甘蓝(特别是Biassicaoleracea var.italica)的任何成员的提取物和浓缩物，特别是洋葱(Allium cepa)、意大利石蛇床(Petroselinum crispum var.neapolitanium)或花茎甘蓝(Brassica oleracea var.italica)的提取物和浓缩物。使用洋葱提取物(特别是白皮洋葱提取物)是尤为优选的。
上述植物或蔬菜的提取物和浓缩物可以是液体形式或固体形式，诸如颗粒或粉末形式.
例如，合适的植物或蔬菜浓缩物可以通过将刚采摘的植物或蔬菜或其根、果实或种子分别干燥或冷冻干燥，然后可选地将干燥后的材料研磨或制粒；或者通过将刚采摘的植物或蔬菜或其根、果实或种子分别压榨，将液体部分集中在一起并可选地将其干燥而制得。优选上述植物或蔬菜浓缩物以固体形式，特别是以粉末形式使用。
得到上述植物或蔬菜的提取物的适当方法是本领域中已知的。例如，植物或蔬菜提取物可以通过以下方法得到：将刚采摘的或干燥的植物或蔬菜或其根、果实或种子分别用例如水、或用一种或多种食品级溶剂、或用水和一种或多种食品级溶剂的混合物进行提取。合适的食品级溶剂包括丙烷、丁烷、乙酸丁酯、乙酸乙酯、乙醇、二氧化碳、丙酮、一氧化氮、甲醇和丙-2-醇，优选乙醇和二氧化碳；乙醇是特别优选的食品级溶剂。经过提取步骤后，可选地将液相浓缩或通过蒸发干燥或冷冻干燥。刚采摘的或干燥的植物或蔬菜原料可以加入冷水或优选热水和/或溶剂中，优选加入水或水与一种或多种溶剂的混合物中，经过规定时间，该时间可以根据植物或蔬菜原料的种类或所用的溶剂而在较宽的范围内变化，不过一般对于水提取来说，是例如1至30分钟，优选2至15分钟，最优选5至10分钟，对于乙醇/水提取来说，是例如30至90分钟，优选60分钟。对于水提取来说，温度优选在85℃至95℃的范围内，对于醇/水提取来说，温度优选在55℃至65℃的范围内。对于二氧化碳提取来说，优选在0至40℃的温度和超临界压力(例如80-200巴)下进行。经过提取后，按照已知方法将液相分离并有利地浓缩或蒸发至干。为了得到浓提取物，上述提取步骤可以联合进行两次或多次。另外，也可以通过以下方法得到植物或蔬菜提取物：将刚采摘的或干燥的植物或蔬菜加入水中，然后该混合物进行水蒸汽蒸馏，收集馏出液并有利地将其浓缩或蒸发至干。
该提取物可以液体形式，特别是以含水液体形式使用，或者以固体形式，特别是以颗粒或粉末形式使用。如果该提取物是液体形式的，例如，其固体含量为1至25％重量，优选为2至20％重量，最优选为2至15％重量。
本发明植物/蔬菜提取物或浓缩物的使用量可以在宽范围内变化，这取决于，特别是，预期的治疗、欲治疗的主体及其需要。因此，当欲治疗的主体是成人时(一般体重为约60至75公斤)，用配制成能每日给予0.1-20g、优选0.2-15g、最优选0.4-10g葱属、石蛇床属、芸苔属和/或芝麻菜属浓缩物或提取物(在没有溶剂的基础上)的组合物，大多数能得到令人满意的对骨吸收的抑制作用。
本发明的另一个目的是适当的包含上述植物/蔬菜提取物或浓缩物的营养组合物。其特征在于它们包含
(a)选自以下成员的至少一种植物/蔬菜提取物或浓缩物：葱属、石蛇床属、芸苔属和芝麻菜属提取物和浓缩物，
(b)一种钙源，和
(c)选自以下成员的至少一种能量来源：碳水化合物、脂肪和氮源，以及可选地
(d)维生素D。
关于组分(a)，适用上文中给出的有关葱属、石蛇床属、芸苔属和芝麻菜属提取物和浓缩物的定义、优选及用量.也有可能用两种或多种所述植物/蔬菜提取物和浓缩物的混合物作为组分(a).本发明的营养组合物适当地包含(以重量百分数计)，例如，约0.1-40％、优选约3-25％、最优选5-15％植物/蔬菜提取物或浓缩物组分(a).
钙源(b)可以包含任何生理学上可接受的含有钙的无机或有机化合物。实例有无机钙盐，例如氯化钙、磷酸钙、硫酸钙、氧化钙、氢氧化钙或碳酸钙，或有机钙组分，象全脂或脱脂奶粉、蛋壳钙、酪蛋白钙、或有机酸的钙盐，诸如柠檬酸钙、马来酸钙，或其混合物。优选使用有机钙化合物，特别是脱脂奶粉、蛋壳钙、酪蛋白钙或其混合物作为钙源(b)。钙组分的用量可以在宽范围内变化。一般说来，本发明组合物包含一个单位剂量为约100mg-1000mg、优选为200mg-700mg、最优选为300mg-600mg的钙(以元素为基准)。
本发明的营养组合物适当地包含(以重量百分数计)，例如，约1至60％、优选约5至50％、最优选为10至40％钙组分(b)。
合适的碳水化合物源包括，例如，麦芽糖糊精、淀粉、乳糖、葡萄糖、蔗糖、果糖、木糖醇和/或山梨糖醇。这些形式的碳水化合物即是能量供应者又是甜味剂。本发明组合物可以含有一种或多种不同的碳水化合物源。
合适的脂肪源包括ω-6多不饱和脂肪酸源、ω-3多不饱和脂肪酸源、单不饱和脂肪酸源、中链脂肪酸源(即C6-C12脂肪酸)；或其混合物。在每种情况下，上述脂肪酸可以游离酸的形式，以一、二或特别是以甘油三酸酯的形式，或以药理学上或营养上可接受的天然来源的形式使用。
合适的ω-6多不饱和脂肪酸的天然来源包括植物油，诸如红花油、向日葵油、豆油、棉油和玉米油。合适的ω-3多不饱和脂肪酸的天然来源包括亚麻子油和鱼油，诸如鲱油、鲑鱼油、鲭鱼油、金枪鱼油、鳕鱼肝油和鲚鱼油。
合适的单不饱和脂肪酸源的天然来源特别是ω-9单不饱和脂肪酸，例如橄榄、低芥酸菜子(canola)、红花(杂种)和向日葵(杂种)。
优选的脂肪源包含能提供预期量的ω-6多不饱和脂肪酸和ω-3多不饱和脂肪酸的甘油三酸酯油，以及富含中链脂肪酸残基(即C6-C12脂肪酸残基)和/或单不饱和脂肪酸残基的甘油三酸酯油。本发明组合物可以含有一种或多种不同的脂肪源。
本发明营养组合物的合适的氮源的实例包括含有营养上可接受的蛋白质的来源，诸如大豆蛋白；乳蛋白，如乳清蛋白或酪蛋白酸盐；和/或蛋白水解物；和/或游离氨基酸形式或盐形式的必需氨基酸混合物；和/或与多元胺的合成有关的化合物，诸如精氨酸、精氨酸前体、鸟氨酸等，为游离氨基酸的形式或盐的形式。
优选的该营养组合物的氮源是
(i)大豆蛋白，它可以大豆的形式、或以任何适当的大豆提取物或浓缩物的形式使用，例如以豆粉、干豆芽、豆奶的形式，或制成大豆的干燥水提取物；或
(ii)乳蛋白，例如乳清蛋白或酪蛋白酸盐，例如，它可以乳清粉的形式、诸如酪蛋白钙等酪蛋白酸盐的形式、和/或以全脂或优选脱脂奶粉的形式使用，和/或
(iii)必需氨基酸的混合物和/或
(iv)作为氮源的精氨酸。
乳蛋白，诸如乳清粉、酪蛋白酸盐，特别是酪蛋白钙、和/或脱脂奶粉，是该请求保护的营养组合物的特别优选的氮源。本发明组合物可以含有一种或多种不同的氮源。
该营养组合物包含(以重量百分数计)，例如，约0.1％-98.9％、优选约1-约95％、最优选为10-90％能量来源组分(c)。
氮源、碳水化合物源和脂肪源对本发明营养组合物的热量的贡献可以在宽范围内变化。例如，碳水化合物源提供该组合物总能量供给的30％至70％，氮源提供5％至45％，而脂肪源提供0.1％至15％。在本发明的优选组合物中，碳水化合物源提供该组合物总能量供给的40％至60％，氮源提供20％至35％，脂肪源提供3％至12％。还可以是，碳水化合物源提供该组合物总能量供给的30％至70％，氮源提供5％至40％，脂肪源提供0.01％至5％。
本发明组合物的优选能量来源包括
总能量供给的30％至70％由选自以下成员的一种或多种碳水化合物源提供：麦芽糖糊精、淀粉、乳糖、葡萄糖、蔗糖、果糖、木糖醇和山梨糖醇；
总能量供给的5％至45％由选自以下成员的一种或多种氮源提供：大豆蛋白、乳蛋白、必需氨基酸的混合物和精氨酸，和
总能量供给的0.1％至15％由包含ω-3和ω-6多不饱和脂肪酸的一种或多种脂肪源提供。
本发明组合物的特别优选的能量来源包括
总能量供给的40％至60％由选自以下成员的一种或多种碳水化合物源提供：麦芽糖糊精、淀粉、乳糖、葡萄糖、蔗糖、果糖、木糖醇和山梨糖醇；
总能量供给的20％至35％由选自以下成员的一种或多种氮源提供：大豆蛋白、脱脂奶粉和酪蛋白酸盐；和
总能量供给的3％至12％由包含ω-3和ω-6多不饱和脂肪酸的一种或多种脂肪源提供。
维生素D(可选组分(d))的用量可以在宽范围内变化。一般说来，本发明组合物一个单位剂量包含，为约400IU至1000IU，优选为约500IU。
本发明的营养制剂可以包含营养上可接受的其他成分，诸如维生素、矿物质、痕量元素、纤维(优选可溶性纤维)、调味剂、防腐剂、着色剂、甜味剂、乳化剂等等。
适于加入本发明组合物的维生素的实例包括药学或营养上可接受形式的维生素A、维生素D、维生素E、维生素K、维生素C、叶酸、维生素B1、维生素B2、维生素B6、维生素B12、烟酸、维生素H和泛酸(panthotenic acid)。
适于加入本发明组合物的矿物元素和痕量元素的实例包括药学或营养上可接受形式的钠、钾、磷、镁、铜、锌、铁、硒、铬和钼。
本文中所用的术语“可溶性纤维”是指能基本上在结肠中经过发酵生成短链脂肪酸的纤维。合适的可溶性纤维的实例包括琼脂、藻酸盐、卡如宾(carubin)、角叉菜胶、阿拉伯胶、瓜尔胶、刺梧桐胶、槐树豆胶、果胶、西黄蓍胶、或黄原胶。它们可以是已水解的，也可以是未水解的。
合适的调味剂包括天然或人造调味剂，例如水果调味剂，如香蕉、柑橘、桃子、凤梨或覆盆子(rasberry)；蔬菜调味剂；或香草、可可、巧克力、咖啡等等。
本发明营养组合物除了组分(a)、(b)、(c)和(d)以外的优选成分包括药学或营养上可接受形式的β-胡萝卜素(维生素A)、维生素E、维生素C、硫胺、维生素B1、B6和/或B12、钾、镁、硒、锌、磷和可溶性纤维。
该营养组合物可以包含(以重量百分数计)，例如，约从0.1％至15％、优选约从0.2％至10％、最优选从0.5％至5％的、除组分(a)、(b)、(c)和可选的(d)以外的这些附加组分.
本发明的营养制剂可以任何适于肠内给药、例如口服给药，或管饲、例如鼻内给药的形式配制和给药。该制剂可方便地以含水液体的形式给药。因此，适于肠内应用的制剂优选为含水形式或粉末或颗粒形式，该粉末或颗粒可在使用前方便地加入水中。为了用于管饲，加入的水的量将尤其取决于患者的流体需要和病情。
本发明的营养组合物可以是全营养配方饮食的形式(液体或粉末形式)，这样，当它作为唯一的营养源时，本质上应含有所有日常所需的热量、氮、脂肪酸、维生素、矿物质和衡量元素。一般而言，应提供给成人的日常量范围是750至3500千卡/天，特别是1000至2000千卡/天。不过，本发明的营养组合物优选用作食品增补剂。由增补剂提供的能量不应太过量，以免不必要地抑制患者的食欲。该增补剂可方便地包含能量来源，其量可提供50至1500千卡/天、优选100至900千卡/天、最优选150至700千卡/天的能量。
本发明的营养组合物，无论是液体形式的，例如饮料形式的，或优选为固体形式的，例如颗粒或粉末形式的，都可以用本身已知的方式获得，例如，通过将各成分混合并可选地加入水中。
本发明还涉及单剂量单位形式的药物组合物，它包含
(a)选自以下成员的至少一种植物/蔬菜提取物或浓缩物：葱属、石蛇床属、芸苔属和芝麻菜属提取物和浓缩物，和
(b)药学上可接受的载体。
这些药物组合物是用于肠内给药，例如口服、鼻内或直肠给药的组合物。合适的药物组合物可以是液体形式，或优选为固体形式，其包含(以重量百分数计)，例如，约0.001％至100％、优选约0.1％至50％的活性成分(a)。
活性成分(a)是选自以下成员的一种植物/蔬菜提取物或浓缩物：葱属、石蛇床属、芸苔属和芝麻菜属提取物和浓缩物，其中适用上文所给出的定义和优选。(a)也可能是两种或多种所述植物/蔬菜提取物和浓缩物的混合物。
用于肠内给药的药物组合物是，例如，单剂量单位形式的那些，诸如糖衣丸剂、片剂、胶囊剂或扁囊剂。它们是用本身已知的方式制备的，例如，利用常规的混合、制粒、成型、溶解或冷冻干燥方法。
例如，用于口服的药物组合物可以用下述方法制得：用固体载体将活性成分结合在一起，可选地将所得混合物制粒，如果希望或需要，加入合适的赋形剂将该混合物或颗粒加工成片剂或糖衣丸芯。
合适的载体尤其是填充剂，诸如糖，如乳糖、蔗糖、甘露糖醇或山梨糖醇，纤维素制剂和/或磷酸钙盐，如磷酸三钙或磷酸氢钙，以及粘合剂，诸如用例如玉蜀黍、小麦、米或马铃薯淀粉制成的淀粉糊，明胶，西黄蓍胶，甲基纤维素和/或聚乙烯吡咯烷酮，如果需要，还可有崩解剂，如上面所提到的淀粉，以及羧甲基淀粉，交联聚乙烯吡咯烷酮，琼脂，或海藻酸或其盐，如藻酸钠.赋形剂尤其是流动调节剂和润滑剂，例如硅酸、滑石、硬脂酸或其盐，如硬脂酸镁或硬脂酸钙，和/或聚乙二醇.糖衣丸芯包覆合适的、可选的肠溶衣，特别是用浓糖溶液包衣，该溶液可含有阿拉伯胶、滑石、聚乙烯吡咯烷酮、聚乙二醇和/或二氧化钛，或用合适的有机溶剂或溶剂混合物制成的包衣溶液包衣，或者，为了制备肠溶衣，用合适的纤维素制剂，如乙酸纤维素邻苯二甲酸酯或羟丙甲基纤维素邻苯二甲酸酯的溶液包衣.片剂或糖衣丸包衣中可以加入染料或颜料，例如为了鉴别的目的或为了指示不同剂量的活性成分.
其他可口服给药的药物组合物是硬明胶胶囊和由明胶和增塑剂组成的密封软胶囊，增塑剂如甘油或山梨糖醇。硬明胶胶囊可以包含颗粒形式的活性成分，例如与诸如乳糖等填充剂混合，与诸如淀粉等粘合剂混合，和/或与诸如滑石或硬脂酸镁等润滑剂混合，以及如果需要，与稳定剂混合。在软胶囊中，活性成分优选溶解或悬浮于适当的液体中，如脂肪油、石蜡油或液态聚乙二醇，同样也可能加入稳定剂。
合适的可直肠给药的药物组合物是，例如，由活性成分与栓剂基质的混合物组成的栓剂。合适的栓剂基质是，例如，天然或合成甘油三酸酯、石蜡烃、聚乙二醇或更高级的链烷醇。也有可能使用包含活性成分与基质的混合物的明胶直肠胶囊。合适的基质是，例如，液态甘油三酸酯、聚乙二醇或石蜡烃。
本发明植物或蔬菜提取物和浓缩物对骨吸收的抑制作用可以通过测量慢性预标记大鼠的[3H]-四环素的尿排泄来评定，如R.C.Mühlbauer和H.Fleisch在《美国生理学杂志》258，R679-R689(1990)中描述的。该方法基于以下特征：(i)3H-标记的四环素在它们的形成过程中沉积于硬组织上；和(ii)当骨被吸收时，[3H]-四环素被释放，其在血液中循环并排泄到尿中，此时可通过对3H计数对其进行评定。这有可能是因为经历骨循环的[3H]-四环素是与羟磷灰石不充分结合的形式，因此，[3H]-四环素一旦从骨中释放出来，在骨更新过程中只能很少被再利用，以及有效的肾排泄的原因。该方法可如下进行：从出生后不久直至约六周龄的大鼠，每周两次，皮下注射体积逐渐升高的含有[3H]-四环素的溶液。约50天大时，将这些动物转移到单独的代谢笼中，每只大鼠供给相同量的标准化饮食，持续约3周。之后，一组大鼠供给纯食物，另一组大鼠供给还含有一定量的本发明植物或蔬菜浓缩物或提取物的纯食物。在实验过程中，动物可自由饮用软化水。当这些大鼠约60天大时，开始每天收集24小时的尿液，并通过液体闪烁计数测定尿中的3H含量，然后制图，其中两组大鼠的尿中的[3H]-四环素含量作为时间(天)的函数。
适当的实验显示，本发明的植物或蔬菜提取物和浓缩物能显著降低整体雄性大鼠和去除生殖腺的雌性大鼠的尿中蓄积的[3H]-四环素排泄物，这表明了对骨吸收的高度抑制作用。因此，本发明请求保护的营养和药物组合物可用于治疗及预防各种以骨吸收增加为特征的疾病或状态，诸如佩吉特氏病、肿瘤诱导的骨疾病、或特别是骨质疏松症。
该植物或蔬菜提取物或浓缩物对骨吸收的抑制作用还可以通过体外试验(在实施例3中描述)来评定，其中，在其上已固定了新分离的破骨细胞的牙质切片用含有要测试的提取物或浓缩物的培养基进行培养。通过对该牙质切片上的破骨细胞吸收凹坑进行计数来评定对破骨细胞的抑制作用。
在下列阐述本发明的实施例中，除非另有说明，否则％是重量百分数，所给温度为摄氏温度℃。
实施例1：
以下是一个粉末形式的、合适的本发明增补剂组合物的例子。
粉末形式的增补剂(1份)
含量 65.0g
本发明提取物/浓缩物1) 14.5g
包括碳水化合物、蛋白质和纤维
蛋白质 20.0g
包括-酪蛋白钙蛋白 8.7g
-脱脂奶粉 11.0g
脂肪 2.8g
包括-ω-6多不饱和酸 1.3g
-ω-3多不饱和酸 0.03g
碳水化合物(包括本发明提取物) 31.0g
包括-乳糖 16.5g
-麦芽糖糊精 3.5g
纤维(可溶的) 5.0g
其他成分 3.0g
包括-Na 230mg
-K 500mg
-Ca 600mg
-Mg 90mg
-P 430mg
-Cl 350mg
-Zn 150mg
-视黄醇(维生素A) 0.3mg
-麦角钙化醇(维生素D) 5.0mcg
-生育酚(维生素E) 3.0mg
-叶绿醌(维生素K1) 30.0mcg
-硫胺(维生素B1) 0.4mg
-核黄素(维生素B2) 0.5mg
-吡哆醇(维生素B6) 0.8mg
-氰钴胺(维生素B12) 0.8mcg
-抗坏血酸(维生素C) 20.0mg
-生物素 50.0mcg
-叶酸 120.0mcg
-烟酰胺 5.0mg
-泛酸 2.0mg
能量值 229kcal
1)a)提取物1，48.3g干燥花椰菜用483ml蒸馏水在89±3℃下提取10分钟，然后将该提取物蒸发至干。
b)提取物2，48.3g干燥的意大利欧芹用483ml蒸馏水在89±3℃下提取10分钟，然后将该提取物蒸发至干。
c)提取物3，26.4g干燥洋葱用264ml蒸馏水在89±3℃下提取10分钟，然后将该提取物蒸发至干.
d)提取物4，58.9g干燥洋葱用589ml蒸馏水在89±3下提取10分钟，然后将该提取物蒸发至干，接着，该干燥水提物再用324ml 85％乙醇/15％水在60℃下进行二次提取1小时，将其冷却至室温并在-20℃下放置过夜，滗去上清液，蒸除乙醇并将该提取物冷冻干燥。
e)提取物5，45.3g干燥洋葱用453ml 85％乙醇/15％水在60℃下提取1小时，过滤，蒸除乙醇并将该提取物冷冻干燥。
f)浓缩物6，将新鲜的熊葱干燥并将其研磨成细粉。
上述增补剂可以与水混合，并以适当的浓度在两餐之间服用。
实施例2：花椰菜、皱叶欧芹(dog-parsley)和洋葱的水提取物对骨吸收的作用
本发明的植物或蔬菜提取物和浓缩物对骨吸收的作用是基于R.C.Mühlbauer和H.Fleisch在《美国生理学杂志》259，R679-R689(1990)中描述的方法。如上面所提到的参考文献中所描述的那样，利用从出生到六周龄的、用[3H]-四环素预标记的Wistar大鼠的尿中的3H排泄物来监测骨吸收。之后将这些大鼠关在单独的代谢笼中，并用含有1.0gCa、0.7g P、和80IU维生素D3/100g食物的标准实验食物(Kliba 331，Klingentalmühle，Kaiseraugst，Switzerland)喂养10天。经过该适应期后，所有大鼠供给含有1.0g Ca、1.2g P、和80IU维生素D3/100g干重的饮食。这是通过向基本低钙、低磷饮食中加入适当量的粉末形式的葡萄糖酸钙和中性磷酸盐(Sodi 2134，Klingenthalmühle，Kaiseraugst，Switzerland)而实现的，再喂养10天，并在此过程中收集尿样。然后这些大鼠“成对喂养”，每天接受28g湿食物。一组(n＝6)转换为纯食物(“食物P”，Sodi 2160，Klingenthalmühle，Kaiseraugst，Switzerland，用水调湿，水含量为45±2％，含有1.0g Ca、1.2g P、和80IU维生素D3/100g干重)，第二组(n＝5)每天给予还含有300mg花椰菜提取物(相当于1.0g干燥花椰菜用10ml蒸馏水在89±3℃下提取10分钟)的纯食物，第三组(n＝5)每天给予还含有300mg意大利欧芹提取物(相当于1.0g干燥意大利欧芹用10ml蒸馏水在89±3℃下提取10分钟)的纯食物，第四组(n＝5)每天给予还含有550mg洋葱提取物(相当于1.0g干燥洋葱用10ml蒸馏水在89±3℃下提取10分钟)的纯食物。
经过不采集尿样的10天适应期和采集尿样的10天后，将这些大鼠分配到不同的治疗组中。利用基线24小时[3H]-四环素排泄作为选择标准，经过仔细处理，每组得到了类似的起始均值。之后，这些大鼠改为用含有或不含有本发明提取物的纯食物喂养，并在14天内，每天采集24小时的尿样，然后利用液体闪烁计数测定尿中的累积[3H]-四环素排泄物。
经过14天的治疗后，每日分别食用花椰菜、意大利欧芹和洋葱的大鼠，累积骨吸收比相应的对照组低9.2％、9.5％和17.5％(p＜0.05)。
实施例3：熊葱浓缩物对骨吸收的作用
使用与实施例2中描述的相同方法，只是治疗只持续6天，且测试组(n＝5)给予还含有1g熊葱浓缩物(将新鲜的熊葱干燥并研磨而得到)的纯食物。结果发现，熊葱对雄性大鼠的骨吸收的抑制为13.5％(p＜0.05)。
实施例4：洋葱提取物对体外吸收的作用
洋葱提取物对体外吸收的作用用破骨细胞介导的吸收来研究(如Arnett TR，Spowage M，1996，“通过轻微改变与生理学范围接近的胞外pH来调节大鼠破骨细胞的吸收活性”，《骨》18：277-279)，但进行以下修正：用牙质切片代替骨薄片作为矿质底物来评估破骨细胞的吸收凹坑，经过金斑标记(gold spottering)后(VittéC，FleischH，Guenther HL，1996，“双膦酸盐诱导成骨细胞分泌破骨细胞介导的吸收的抑制剂”，《内分泌学》137：2324-2333)在切线照明下计数。在该试验中，在48孔板的每一个孔中，在5％CO2/空气气氛和37℃下，将一个其上已固定了新分离的破骨细胞的4×4毫米牙质切片用含有欲测试提取物的250μl培养基培养24小时。每种剂量使用8个切片。破骨细胞是从新生大鼠的股骨采集的，将软骨端去除后，将其劈成两半并横向割下来。此过程得到了一个富含其他细胞如成骨细胞的细胞悬液。这允许在较宽条件下检测作用效果，也就是说，对于对破骨细胞的直接作用和由其他细胞如成骨细胞介导的对破骨细胞的间接作用这两种情况都能如此。
洋葱的细粉用蒸馏水(100g/l)在90℃下提取10分钟，过滤并将滤液冷冻干燥，接着，该干燥水提物在60℃下用85％乙醇/15％水进行二次提取1小时，冷却至室温，在-20℃下放置过夜，使不需要的物质沉淀出来，滗析上清液，蒸除乙醇并将由此得到的残余物冷冻干燥，从而由每g干燥整洋葱得到250mg冻干的洋葱提取物。
该洋葱提取物(0.017mg、0.17mg、1.7mg洋葱提取物/ml培养基)以剂量依赖方式抑制了牙质的由破骨细胞介导的吸收，同时，耐酒石酸的碱性磷酸酶阳离子(TRAP+)多核细胞(MNC)没有显著减少。因此，凹坑/TRAP+MNC的比值显著降低(p＜0.001)。由此可得出结论，存在于这些培养物中的大量TRAP+MNC与洋葱提取物的加入无关，这表明，洋葱提取物对于这些细胞是无毒的，而是仅抑制破骨细胞的活性。
Claims (18)
1.意大利欧芹提取物或浓缩物在用于制备治疗或预防以骨吸收增加为特征的疾病或状态的药物或营养制剂中的用途，所述提取物是用水或者是水和乙醇的混合物提取得到的。
2.按照权利要求1的用途，其中意大利欧芹提取物或浓缩物以固体形式应用。
3.按照权利要求1的用途，其中该药物或营养制剂配制成，在无溶剂的基础上，每日提供0.1至20克意大利欧芹提取物或浓缩物。
4.按照权利要求1的用途，其中所述疾病为佩吉特氏病、肿瘤诱导的骨疾病或骨质疏松症。
5.按照权利要求1的用途，其中所述疾病为骨质疏松症。
6.一种用于治疗或预防以骨吸收增加为特征的疾病或状态的营养组合物，它包含
(a)意大利欧芹植物提取物或浓缩物，所述提取物是用水或者是水和乙醇的混合物提取得到的，
(b)一种钙源，和
(c)选自以下成员的至少一种能量来源：碳水化合物、脂肪和氮源，以及可选地
(d)维生素D，
其中所述营养组合物包含，以重量％计，3至25％的组分(a)、5至50％的组分(b)，和1至95％的组分(c)，且满足各组分之和为100％。
7.按照权利要求6所述的营养组合物，其中钙源(b)是有机钙盐。
8.按照权利要求6所述的营养组合物，其中组分(c)中的碳水化合物源选自以下成员：麦芽糖糊精、淀粉、乳糖、葡萄糖、蔗糖、果糖、木糖醇和山梨糖醇。
9.按照权利要求6所述的营养组合物，其中组分(c)中的脂肪源选自以下成员：ω-6多不饱和脂肪酸源、ω-3多不饱和脂肪酸源、单不饱和脂肪酸源、C6-C12脂肪酸源，及其混合物。
10.按照权利要求6所述的营养组合物，其中组分(c)中的氮源选自以下成员中的一种或多种：大豆蛋白，乳蛋白，蛋白水解物，必需氨基酸的混合物，和精氨酸。
11.按照权利要求6所述的营养组合物，其中碳水化合物源提供了该组合物总能量供给的30％至70％，氮源提供5％至45％，脂肪源提供0.1％至15％，且满足各组分之和为100％。
12.按照权利要求6所述的营养组合物，它还包含，以重量％计，0.2至10％的选自以下成员的营养上可接受的其他组分：维生素、矿物质、痕量元素、纤维、调味剂、防腐剂、着色剂和乳化剂。
13.按照权利要求6所述的营养组合物，它是具有50至1500千卡/天的食品增补剂的形式。
14.按照权利要求6所述的营养组合物，它是液体形式。
15.按照权利要求6所述的营养组合物，它是固体形式。
16.按照权利要求15所述的营养组合物，它是颗粒或粉末形式。
17.一种用于治疗或预防以骨吸收增加为特征的疾病或状态的单剂量单位形式的药物组合物，它包含
(a)意大利欧芹提取物或浓缩物，所述提取物是用水或者是水和乙醇的混合物提取得到的，和
(b)药学上可接受的载体。
18.按照权利要求17的药物组合物，用于胃肠内给药，它是糖衣丸剂、片剂、胶囊剂、扁囊剂或栓剂的形式。
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| PL (1) | PL190370B1 (zh) |
| RU (1) | RU2196601C2 (zh) |
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| CA2546180A1 (en) * | 2003-11-26 | 2005-06-09 | Universitaet Bern | Plant extracts for the treatment of increased bone resorption |
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| CN117180328B (zh) * | 2023-03-01 | 2024-03-22 | 再造再生健康科技(杭州)有限公司 | 一种促进胫骨生长发育的贴剂及其制备方法 |
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