CN108601812B - 软骨机能改善用组合物 - Google Patents
软骨机能改善用组合物 Download PDFInfo
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- CN108601812B CN108601812B CN201780009966.3A CN201780009966A CN108601812B CN 108601812 B CN108601812 B CN 108601812B CN 201780009966 A CN201780009966 A CN 201780009966A CN 108601812 B CN108601812 B CN 108601812B
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Abstract
提供的是:软骨机能改善用组合物,其对预防和治疗各种关节疾病有用。所述软骨机能改善用组合物包含选自乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、和HMG样蛋白和/或其分解物的至少一种成分作为一种(或多种)有效成分。可以在日常的基础上长期摄取、并且发挥修正或再生变形或消失的软骨的显著效果的根据本发明的软骨机能改善用组合物,对预防或治疗各种关节疾病有用。
Description
技术领域
本发明关于一种软骨机能改善用组合物。所述组合物具有促进软骨细胞的增殖和/或抑制软骨细胞的钙化的作用,具有修正或再生变形或消失的软骨的显著效果,并且在预防和治疗各种关节疾病中有用。
背景技术
日本具有大于80岁的平均寿命,并且已达到超高龄社会,其中约四人中一人是65岁以上。在这种老龄化的情况下,运动器官失调的患病率已增加。2007年,日本整形外科学会(Japanese Orthopaedic Association Corporation)已提出新术语“运动器官综合征(locomotive syndrome)”,从而促进朝向维持和增进运动器官的健康以及护理的国民和医学从业者的意识改革。运动器官综合征表示由运动器官的机能障碍引起或增加风险的需要护理的状态。运动器官通常包括支持和移动身体的器官,例如骨、关节、韧带、脊椎、脊髓、肌肉、腱、和周围神经。在这些运动器官中观察到的代表性的疾病和机能障碍的实例包括骨质疏松症、肌肉减少症(sarcopenia)、和骨关节炎。
骨关节炎是最普遍的关节疾病,并且预期在20%至30%的50岁以上的人中发现。特别地,患者数已增加,并且具有骨关节炎的大量患者被迫在日常生活中忍受不便。认为该疾病来自由衰老和遗传因素引起的关节形成要素的变性,或由肥胖、劳动、或运动产生的对关节的负荷。在支持骨的运动的骨骺表面处发生的软骨减少导致软骨的变形或消失。这类变形或消失抑制关节的顺畅移动,导致骨关节炎。
因为软骨没有血管和神经细胞,认为软骨一旦损伤,难以自然地恢复。由于该原因,在骨关节炎的轻度情况下,使用例如热疗法或牵引等物理疗法、或使用镇痛药或抗炎药的缓和疗法(palliative therapy),在其严重情况下,进行将透明质酸注射至关节中或用人工关节手术置换。为了通过根治骨关节炎来提高患者的生活品质(QOL),由于疾病的性质,患者应日常和安全地长期摄取组合物或组合物中含有的有效成分,从而修复或再生患者的变形或消失的软骨。
软骨由从间充质干细胞分化的软骨细胞以及由软骨细胞产生的胶原蛋白、透明质酸、和蛋白多糖的软骨基质构成。通常,软骨在软骨细胞的增殖、成熟、肥大化后钙化,同时软骨基质退化从而转化为骨组织。在关节和椎间盘中,软骨细胞在整个生命中维持它们的性质;因此软骨在不转化为骨的情况下向身体提供顺畅的可动性。因此,为了修复或再生变形或消失的软骨来根治骨关节炎,重要的是促进存在于软骨中的软骨细胞的增殖和软骨细胞中软骨基质的产生,以及通过抑制软骨细胞的钙化来防止软骨细胞转化为骨。
公开了促进存在于软骨中的软骨细胞的增殖和由软骨细胞产生软骨基质、或者抑制软骨细胞的钙化从而防止软骨转化为骨的组合物成分:例如,促进由软骨细胞产生软骨基质、并且抑制软骨细胞的钙化的葡糖胺盐酸盐(非专利文献1),以及源自胶原蛋白的二肽、即脯氨酰基羟脯氨酸,其促进由软骨细胞产生软骨基质并且延迟软骨细胞的钙化(非专利文献2)。
乳和乳制品是自古以来就食用、并且可以日常安全地长期摄取的食品。已知其中含有的乳碱性蛋白级分(fraction)或它们的分解物可以促进软骨基质的产生。还已知乳铁蛋白可以促进软骨细胞的增殖并且抑制软骨细胞的钙化(专利文献1、2,非专利文献3)。
现有技术文献
专利文献
专利文献1:WO2010/058679
专利文献2:WO2013/164992
非专利文献
非专利文献1:Nakatani等人,Biological&Pharmaceutical Bulletin,30:433-438(2007)
非专利文献2:Nakatani等人,Osteoarthritis and Cartilage,17:1620-1627(2009)
非专利文献3:Takayama等人,Biometals,23:477-484(2010)
发明内容
发明要解决的问题
虽然乳铁蛋白可以日常安全地长期摄取、促进软骨细胞的增殖、并且抑制软骨细胞的钙化,但其在稳定性和风味方面存在弊端。由于该原因,已需要具有改善的性质的软骨机能改善用组合物从而解决这些问题。
本发明的目的是提供软骨机能改善用组合物,其可以日常安全地长期摄取,具有促进软骨细胞的增殖的作用和/或抑制软骨细胞的钙化的作用,具有修正或再生变形或消失的软骨的显著效果,并且对预防和治疗各种关节疾病有用。
用于解决问题的方案
本发明关于以下方面:
方面(1)一种软骨机能改善用组合物,其包含选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白(cystatin)、高速泳动族(high mobility group,HMG)样蛋白、和其分解物组成的组的至少一种有效成分。
方面(2)根据方面(1)所述的软骨机能改善用组合物,其中所述乳过氧化物酶、所述抑半胱氨酸蛋白酶蛋白、和所述HMG样蛋白源自哺乳类的乳。
方面(3)根据方面(1)或(2)所述的软骨机能改善用组合物,其中所述乳过氧化物酶、所述抑半胱氨酸蛋白酶蛋白、和所述HMG样蛋白源自牛乳。
方面(4)根据方面(1)至(3)任一项所述的软骨机能改善用组合物,其中通过在蛋白酶的存在下分解所述乳过氧化物酶、所述抑半胱氨酸蛋白酶蛋白、和所述HMG样蛋白来制备所述乳过氧化物酶、所述抑半胱氨酸蛋白酶蛋白、和所述HMG样蛋白的分解物。
方面(5)根据方面(4)所述的软骨机能改善用组合物,其中所述蛋白酶是选自由胃蛋白酶、胰蛋白酶、糜蛋白酶、胰酶和木瓜蛋白酶组成的组的至少一种蛋白酶。
方面(6)一种通过每天摄取至少1mg的在根据方面(1)至(5)任一项中所述的乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和/或其分解物来改善软骨机能的方法。
发明的效果
根据本发明的软骨机能改善用组合物,由于其促进软骨细胞的增殖和/或抑制软骨细胞的钙化的作用、以及其修正或再生变形或消失的软骨的显著效果,所以在预防和治疗各种关节疾病中有用。
附图说明
[图1]图1是示出分别用含有标准水平的磷、高磷食物(phosphorus-rich food)、和实施例制品(inventive products)的标准食物饲养十周的六组小鼠中小鼠右后肢的关节软骨层的厚度的图。
[图2]图2是示出分别用含有标准水平的磷、高磷食物、和实施例制品的标准食物饲养十周的六组小鼠中小鼠右后肢的关节软骨层中存在的软骨细胞的数量的图。
具体实施方式
本发明人已进行深入研究以解决上述问题,已经发现乳和乳制品中含有的蛋白质,即乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、和高速泳动族(HMG)样蛋白以及其分解物促进软骨细胞的增殖和/或抑制软骨细胞的钙化,并且修正或再生变形或消失的软骨,并已经完成本发明。
先前未发现,乳、乳制品中含有的蛋白质,即乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物促进软骨细胞的增殖和/或抑制软骨细胞的钙化,并且修正或再生变形或消失的软骨。
本发明的特征在于将乳、乳制品中含有的蛋白质,即乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白和/或其分解物用作有效成分。本发明中使用的乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白可为从例如牛乳、人乳、山羊乳、或绵羊乳等的哺乳类的乳制备的那些,化学合成产品,通过基因工程技术生产的那些,和从血液或器官纯化的那些。也可使用纯化的乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白的商购可得的试剂。本发明中使用的乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白的分解物可从用蛋白酶处理的乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白制备。
有效成分之一的乳过氧化物酶例如通过使用具有引入其中的砜基的多糖类亲和性载体纯化乳的公知方法来制备(日本特开平3-109400号公报)。乳过氧化物酶的分解物可以在例如胰蛋白酶、胰酶、糜蛋白酶、胃蛋白酶、木瓜蛋白酶、激肽释放酶、组织蛋白酶、嗜热菌蛋白酶、和V8蛋白酶等的蛋白酶的存在下,从乳过氧化物酶制备,从而分子量为10,000以下,优选500以上。
另一有效成分抑半胱氨酸蛋白酶蛋白通过例如由柱色谱法纯化乳的公知方法来制备(日本特开2000-281587号公报)。抑半胱氨酸蛋白酶蛋白的分解物可以在例如胰蛋白酶、胰酶、糜蛋白酶、胃蛋白酶、木瓜蛋白酶、激肽释放酶、组织蛋白酶、嗜热菌蛋白酶、和V8蛋白酶等的蛋白酶的存在下,从抑半胱氨酸蛋白酶蛋白制备,从而分子量为8,000以下,优选500以上。
另一有效成分HMG样蛋白例如通过由柱色谱法纯化乳来制备(日本特开平9-227403号公报)。HMG样蛋白的分解物可以在例如胰蛋白酶、胰酶、糜蛋白酶、胃蛋白酶、木瓜蛋白酶、激肽释放酶、组织蛋白酶、嗜热菌蛋白酶、和V8蛋白酶等的蛋白酶的存在下,从HMG样蛋白制备,从而分子量为6,000以下,优选500以上。
本发明中,可将选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物组成的组的至少一种有效成分按其原样用于根据本发明的软骨机能改善用组合物。当需要时这些蛋白质和它们的分解物可以根据通常的方法配制为粉末剂、颗粒剂、片剂、胶囊剂、或液体剂形式的药物。
本发明中,有效成分可以通过任何混合或配混过程配制为药物。例如,有效成分可以以如下解决方法添加和配混:将选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物组成的组的至少一种有效成分悬浮或溶解于去离子水中,并且搅拌混合。将溶液配制为药物以供使用。
搅拌混合可以在可均匀地混合有效成分的任何条件下进行。例如,有效成分可以用超声分散器或均质混合器搅拌混合。当需要时,组合物的溶液可在通过反渗透(RO)或超滤(UF)膜脱盐或浓缩或者冷冻干燥后使用,从而组合物溶液容易配制为药物。通常用于医药品的制造的灭菌过程可以用于本发明。粉末状还可以干热灭菌。
根据本发明的软骨机能改善用组合物可以成形为各种形态,例如液体状、凝胶状、粉末状、和颗粒状。
根据本发明的软骨机能改善用组合物含有选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物组成的组的至少一种有效成分作为有效成分,并且可以使用例如填料、增量剂、粘合剂、崩解剂、表面活性剂、和润滑剂等的通常使用的稀释剂或填料配制为药物。填料的实例包括蔗糖、乳糖、淀粉、结晶性纤维素、甘露醇、轻质无水硅酸、铝酸镁、合成硅酸铝、偏硅酸铝镁(magnesium aluminometasilicate)、碳酸钙、碳酸氢钠、磷酸氢钙、和羧甲基纤维素钙。这些填料可单独或以组合使用。
根据本发明的软骨机能改善用组合物可以组合使用稳定剂、糖类、脂质、调味剂(flavorings)、维生素、矿物质、类黄酮、和多酚。这些可以在制备组合物中适当地配混。这些也可与改善软骨机能的其他成分例如葡糖胺盐酸盐和乳铁蛋白组合使用。
如下述实验例所示,在根据本发明的软骨机能改善用组合物中,选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物组成的组的至少一种有效成分可以以1mg/kg以上口服给予小鼠,从而促进软骨细胞的增殖和/或抑制软骨细胞的钙化。实验动物的摄取量对应于以血药浓度计的成人的摄取量("Yakkohyouka(Evaluation of DrugEfficacy)第8卷",Mitsuyoshi Nakashima(1993),Hirokawashoten,2-18页)。通常,如果成人一人一日以1mg以上摄取选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物组成的组的至少一种有效成分,可期望改善软骨的机能,特别是预防和治疗骨关节炎。
因此,将选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物组成的组的至少一种有效成分配制为药物,使得可以确保其必要量。例如,为了实现由成人一人一日1mg以上摄取选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物组成的组的至少一种有效成分,根据例如药品等的最终制品的形式,最终制品可以以相对于总质量为0.0005%至5%(重量/重量)、优选0.05至2.5%(重量/重量)的比例,含有选自由乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物组成的组的至少一种有效成分。
软骨机能改善用组合物可以含有选自由乳过氧化物酶、半胱氨酸蛋白酶抑制剂、HMG样蛋白、和其分解物组成的组的至少一种有效成分,或这些成分的任何组合。其具体实例包括所述成分的一种;所述三种成分的一种与其分解物的组合;所述三种成分的两种的组合,例如乳过氧化物酶和半胱氨酸蛋白酶抑制剂、乳过氧化物酶和HMG样蛋白、以及半胱氨酸蛋白酶抑制剂和HMG样蛋白;所述三种成分的一种与其分解物的组合;乳过氧化物酶、半胱氨酸蛋白酶抑制剂、和HMG样蛋白的组合;以及所述三种成分的组合,其中至少一种为分解物。
软骨机能改善用组合物促进软骨细胞的增殖和/或抑制软骨细胞的钙化。换言之,软骨机能改善用组合物,由于其修正或再生由于老化、外伤、或运动期间的负荷(过重)导致的变形或消失的软骨的显著效果,对改善软骨机能有用。具体地,软骨机能改善用组合物对例如骨关节炎、特别是膝关节炎等的各种关节疾病的预防和治疗有用。
以下将详细描述本发明的实施例和实验例。这些仅供用于说明目的,并且不应解释为限制本发明。
实施例
[实施例1]
(乳过氧化物酶的制备)
将填充有阳离子交换树脂、即磺化CHITO PEARL(Fujibo Holdings,Inc.制)(400g)的柱(直径:5cm,高度:30cm)用去离子水充分清洗。将未杀菌脱脂乳(40L,pH:6.7)以25ml/min的流速通过柱进料。进料后,用去离子水充分清洗柱,并且用含有2.0M氯化钠的0.02M碳酸缓冲液(pH7.0)进行洗脱。将含有乳过氧化物酶的洗脱级分吸附于S-SepharoseFF柱(GE Healthcare,Inc.制)上,用去离子水充分清洗,并且用10mM磷酸缓冲液(pH:7.0)平衡。通过0至2.0M氯化钠的线性梯度洗脱吸附的级分,从而回收含有乳过氧化物酶的级分。然后将级分通过使用HiLoad 16/60Superdex 75pg(GE Healthcare,Inc.制)的凝胶过滤色谱处理,从而得到乳过氧化物酶(3.0g)。乳过氧化物酶具有94%的纯度。乳过氧化物酶可以原样用于软骨机能改善用组合物(软骨机能改善剂/实施例制品1)。
[实施例2]
(乳过氧化物酶分解物的制备)
将实施例1中制备的乳过氧化物酶(1g)溶解于水(200ml)中,添加蛋白酶胰酶(Sigma-Aldrich Corporation制)使得终浓度为0.01重量%。将溶液在37℃下进行酶处理5小时。在90℃下通过加热处理5分钟使酶失活。将溶液冷冻干燥,从而得到乳过氧化物酶分解物(0.8g)。乳过氧化物酶分解物具有10,000以下的分子量。该产品可以原样用于软骨机能改善用组合物(软骨机能改善剂/实施例制品2)。
[实施例3]
(乳过氧化物酶分解物的制备)
将实施例1中制备的乳过氧化物酶(1g)溶解于水(200ml)中,添加蛋白酶胰蛋白酶(Sigma-Aldrich Corporation制)使得终浓度为0.01重量%。将溶液在37℃下进行酶处理5小时。在90℃下通过加热处理5分钟使酶失活,并且冷冻干燥,从而得到乳过氧化物酶分解物(0.9g)。乳过氧化物酶分解物具有10,000以下的分子量。该产品可以原样用于软骨机能改善用组合物(软骨机能改善剂/实施例制品3)。
[实施例4]
(抑半胱氨酸蛋白酶蛋白的制备)
将填充有S Sepharose(3,000g)的柱用去离子水充分清洗。将脱脂乳(10,000L)通过柱进料。将柱用去离子水充分清洗,以0.1至1.0M氯化钠的线性梯度进行洗脱。将洗脱级分在90℃下进行加热处理10分钟,并且离心除去沉淀。将含有抑半胱氨酸蛋白酶蛋白的洗脱级分通过Mono S离子交换色谱再次分级。将级分在FPLC系统中通过Mono Q离子交换色谱、然后通过Superose 12凝胶过滤色谱,以及在HPLC系统中通过羟磷灰石色谱、和C4反相色谱来顺序地处理,从而得到抑半胱氨酸蛋白酶蛋白(58mg)。抑半胱氨酸蛋白酶蛋白可以原样用于软骨机能改善用组合物(软骨机能改善剂/实施例制品4)。
[实施例5]
(抑半胱氨酸蛋白酶蛋白的制备)
将5%乳清蛋白溶液(10,000L)在90℃下进行加热处理10分钟,并且离心除去沉淀。将羧甲基化的木瓜蛋白酶与Tresyl-Toyopearl(Tosoh Corporation制)结合的载体填充至柱中。将系统用0.5M氯化钠溶液平衡。将乳清蛋白溶液通过柱进料。进料后,用0.5M氯化钠溶液然后用含有0.1%吐温20的0.5M氯化钠溶液清洗柱。在下一步骤中,用20mM乙酸-0.5M氯化钠溶液洗脱半胱氨酸蛋白酶。将洗脱级分用1M氢氧化钠溶液迅速中和,通过由Mono S阴离子交换色谱以及在HPLC系统中由的羟磷灰石色谱和C4反相色谱顺序处理来分级,从而得到抑半胱氨酸蛋白酶蛋白(48mg)。抑半胱氨酸蛋白酶蛋白可以原样用于软骨机能改善用组合物(软骨机能改善剂/实施例制品5)。
[实施例6]
(抑半胱氨酸蛋白酶蛋白分解物的制备)
将实施例4中制备的抑半胱氨酸蛋白酶蛋白(25mg)溶解于水(100ml)中,添加胰酶(Sigma-Aldrich Corporation制)使得终浓度为1%。将溶液在37℃下进行酶处理5小时。在90℃下通过加热处理5分钟使酶失活,并且冷冻干燥,从而得到抑半胱氨酸蛋白酶蛋白酶分解物(实施例制品6A)(23mg)。将实施例5中制备的抑半胱氨酸蛋白酶蛋白(25mg)如上所述处理,从而得到抑半胱氨酸蛋白酶蛋白分解物(实施例制品6B)(24mg)。抑半胱氨酸蛋白酶蛋白分解物具有8,000以下的分子量。该产品可以原样用于软骨机能改善用组合物(软骨机能改善剂/实施例制品6A和实施例制品6B)。
[实施例7]
(HMG样蛋白的制备)
将填充有磺化CHITO PEARL(3,000g)(Fujibo Holdings,Inc.制)的柱用去离子水充分清洗。将脱脂乳(300L)通过柱进料。用去离子水充分清洗柱。用0.02M碳酸缓冲液(pH:6.7)将碱性蛋白质吸附至树脂上,并且通过0.1M至1.0M NaCl的线性梯度洗脱从而回收。在下一步骤中,将含有HMG样蛋白的洗脱级分在系统用0.1M磷酸缓冲液(pH:6.5)平衡的S-Sepharose阳离子交换色谱中,通过0.1M至1.0M NaCl的线性梯度洗脱。将洗脱级分在90℃下进行加热处理10分钟,并且离心除去沉淀。将级分通过0.1M至1.0M NaCl的线性梯度在系统用0.1M磷酸缓冲液(pH:6.5)平衡的Mono Q离子交换色谱中进一步分级,然后通过系统用0.1M磷酸缓冲液(pH:6.5)平衡的Sepharose 12凝胶过滤色谱分级。最后用使用C4反相柱的高效液相色谱处理级分,从而得到HMG样蛋白(135mg)。HMG样蛋白可以原样用于软骨机能改善用组合物(软骨机能改善剂/实施例制品7)。
[实施例8]
(HMG样蛋白分解物的制备)
将实施例7中制备的HMG样蛋白(25mg)溶解于水(100ml)中,添加蛋白酶胰蛋白酶(Sigma-Aldrich Corporation制)使得终浓度为0.01重量%。将溶液在37℃下进行酶处理5小时。在90℃下通过加热处理5分钟使酶失活。将溶液冷冻干燥,从而得到HMG样蛋白分解物(24mg)。乳过氧化物酶分解物具有6,000以下的分子量。该产品可以原样用于软骨机能改善用组合物(软骨机能改善剂/实施例制品8)。
[实验例1]
(细胞实验)
评价关于实施例制品1的乳过氧化物酶、实施例制品2的乳过氧化物酶分解物、实施例制品4的抑半胱氨酸蛋白酶蛋白、实施例制品6B的抑半胱氨酸蛋白酶蛋白分解物、实施例制品7的HMG样蛋白、实施例制品8的HMG样蛋白分解物、实施例制品1和4的混合物、以及实施例制品5和7的混合物的促进软骨细胞的增殖的作用。在96孔细胞培养板中以5×103细胞/孔的密度接种源自小鼠的软骨祖细胞株(ATDC5),并且在含有5%胎牛血清的Dulbecco's改良Eagle培养基(Dulbecco's Modified Eagle medium)/Ham's营养混合物F-12(Ham'snutrient mixture F-12)中,在37℃、5%CO2气氛下用培养箱培养24小时。然后将培养基全部用新的替换。将溶解于PBS(-)中的实施例制品1、2、4、6B、7、和8,实施例制品1和4的混合物,以及实施例制品5和7的混合物各自添加至培养基,使得终浓度为1μg/mL,然后培养48小时。将用作溶剂的PBS(-)用作阴性对照。完全除去培养基。将细胞增殖试剂WST-1(RocheDiagnostics GmbH制)以培养基的1/10的含量添加至培养基,并且将样品在所得培养基中培养6小时。各样品的吸光度使用酶标仪(plate reader)在440nm下测量。因为WST-1通过活细胞的代谢活性还原为甲臜染料(formazan dye),所以甲臜染料的量与具有代谢活性的细胞的数量成比例。将反映甲臜染料的量的吸光度用作指示促进软骨细胞的增殖的指标。结果在表1中示出。
[表1]
值表示平均值±标准差(n=5)。
*表示与PBS(-)的显著差异(p<0.05)。
这些结果表明,在含有实施例制品1的乳过氧化物酶、实施例制品2的乳过氧化物酶分解物、实施例制品4的抑半胱氨酸蛋白酶蛋白、实施例制品6B的抑半胱氨酸蛋白酶蛋白分解物、实施例制品7的HMG样蛋白、实施例制品8的HMG样蛋白分解物、实施例制品1和4的混合物、实施例制品5和7的混合物的培养基中,与含有PBS(-)的培养基相比,软骨祖细胞的数量显著增加。因此,根据本发明的乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物促进软骨细胞的增殖。
[实验例2]
(细胞实验)
评价关于实施例制品1的乳过氧化物酶、实施例制品3的乳过氧化物酶分解物、实施例制品5的抑半胱氨酸蛋白酶蛋白、实施例制品6A的抑半胱氨酸蛋白酶蛋白分解物、实施例制品7的HMG样蛋白、实施例制品8的HMG样蛋白分解物、实施例制品1和7的混合物、以及实施例制品4和8的混合物的抑制软骨细胞的钙化的作用。在96孔细胞培养板中以3×103细胞/孔的密度接种源自小鼠的软骨祖细胞株(ATDC5),并且在含有5%胎牛血清的Dulbecco's改良Eagle培养基/Ham's营养混合物F-12中,在37℃、5%CO2气氛下用培养箱培养24小时。然后将培养基全部用新的替换。将溶解于PBS(-)中的实施例制品1、3、5、6A、7、和8,实施例制品1和7的混合物,以及实施例制品4和8的混合物各自添加至培养基,使得终浓度为1μg/mL。在每天更换培养基的情况下,将细胞培养7天。将用作溶剂的PBS(-)用作阴性对照。培养结束后,将细胞用20%福尔马林溶液固定20分钟,并且用水清洗。添加水,使细胞静置15分钟。将萘酚AS-BI磷酸盐和快速红紫(Fast Red Violet)LB盐溶解于0.05M 2-氨基-2-甲基-1,3-丙二醇(AMP)溶液中,从而制备碱性磷酸酶(ALP)染色液(dyeing solution),并且将细胞用ALP染色液在37℃下染色20分钟。染色后,将细胞用水清洗5次,并且风干。从使用扫描仪取得的培养板中的染色细胞的图像,用图像处理软件计算染色细胞的面积。染色细胞的面积表示为阴性对照的值设为1的相对值。ALP活性为软骨细胞的钙化的初始标记,染色细胞的面积越小表示钙化越抑制。结果在表2中示出。
[表2]
值表示平均值±标准差(n=5)。
*表示与PBS(-)的显著差异(p<0.05)。
结果表明,在含有实施例制品1的乳过氧化物酶、实施例制品3的乳过氧化物酶分解物、实施例制品5的抑半胱氨酸蛋白酶蛋白、实施例制品6A的抑半胱氨酸蛋白酶蛋白分解物、实施例制品7的HMG样蛋白、实施例制品8的HMG样蛋白分解物、实施例制品1和7的混合物、实施例制品4和8的混合物的培养基中,ALP染色的面积显著减少。因此,与含有PBS(-)的培养基相比,根据本发明的乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、HMG样蛋白、和其分解物,抑制软骨细胞的钙化。
[实验例3]
(动物实验)
验证实施例制品1的乳过氧化物酶、实施例制品3的乳过氧化物酶分解物、实施例制品4的抑半胱氨酸蛋白酶蛋白、和实施例制品7的HMG样蛋白对于关节软骨层的初始变性的效果。将10周龄C57BL/6J雄性小鼠分成每组10只小鼠的6组,以免它们的重量在各组间变化。将摄取含有标准水平的磷(0.2g/100g组合物)而没有实施例制品的组合物(标准食物)的小鼠定义为组A,并且将摄取含有大量磷(1.2g/100g组合物)而没有实施例制品的组合物(高磷食物)的组定义为组B。将摄取实施例制品1的组定义为组C,将摄取实施例制品4的组定义为组D,将摄取实施例制品7的组定义为组E,并且将摄取实施例制品3的组定义为组F。各实施例制品配混有高磷食物,使得每天的实施例制品的摄取剂量为1mg/kg小鼠。在饲养十周后,摘取右后肢,并浸渍于10%中性缓冲福尔马林溶液中从而固定。制备膝关节的石蜡包埋部分,并且进行苏木精-曙红染色。使用染色样品,测量关节软骨层的厚度和关节软骨层中存在的软骨细胞的数量,从而评价对关节软骨层中的初始变性的作用。结果在图1和2中示出。
结果表明,在摄取没有实施例制品的高磷食物的组B中,与摄取没有实施例制品的标准食物的组A相比,由于过量摄取磷,导致关节软骨层的厚度和存在于关节软骨层的软骨细胞的数量显著减少。相比而言,摄取实施例制品1、3、4、和7的组C至F中,关节软骨层的厚度和软骨细胞的数量的减少与组B相比受到显著抑制。因此,根据本发明的乳过氧化物酶、抑半胱氨酸蛋白酶蛋白、和HMG样蛋白、以及其分解物抑制关节软骨的初始变性。
[实施例9]
(软骨机能改善用片剂的制备)
以表3所示的比例混合原料。通过通常的方法将混合物成形为1g的软骨机能改善用片剂。片剂(1g)含有1mg的实施例制品1的乳过氧化物酶。
[表3]
[实施例10]
(软骨机能改善用组合物的制备)
将实施例制品3的乳过氧化物酶分解物(0.5g)溶解于去离子水(4,999.5g)中,并且加热至45℃。将原料用TK均质混合器(TK ROBO MICS;Tokushu Kika Kogyo Co.,Ltd.制)在6,000rpm下搅拌混合30分钟,从而制备含有0.5g/5.0kg的实施例制品3的乳过氧化物酶分解物的溶液。将表4所示的原料添加至所述溶液(5.0kg),并且进一步搅拌混合10分钟。将混合物置于200ml蒸煮袋(retort pouch)中,并且用灭菌釜(1类压力容器,型号:RCS-4CRTGN,HISAKA WORKS,LTD.制)在121℃下灭菌20分钟,从而制备50kg的根据本发明的软骨机能改善用液体组合物。所述软骨机能改善用液体组合物每100g包含1mg的实施例制品3的乳过氧化物酶分解物。
[表4]
[实施例11]
(软骨机能改善用组合物的制备)
将脱脂奶粉(140g)溶解于去离子水(569.95g)中。溶解实施例制品4的抑半胱氨酸蛋白酶蛋白(0.05g)。将系统加热至45℃,并且用超声分散器(ULTRA-TURRAX T-25;IKAJapan K.K制)在9,500rpm下搅拌混合30分钟。添加表5所示的原料,并且进一步搅拌混合15分钟。将混合物置于100ml玻璃瓶中,并且在95℃下灭菌15秒。将瓶密封,从而制备10瓶(100ml)根据本发明的软骨机能改善用组合物。该软骨机能改善用组合物每100ml含有5mg的实施例制品4的抑半胱氨酸蛋白酶蛋白。
[表5]
[实施例12]
(犬用软骨机能改善用组合物的制备)
将实施例制品7的HMG样蛋白(0.1g)溶解于去离子水(99.9g)中。将系统加热至45℃,并且用超声分散器(ULTRA-TURRAX T-25;IKA Japan K.K制)在9,500rpm下搅拌混合40分钟,从而制备含有0.1g/100g的实施例制品7的HMG样蛋白的溶液。将表6所示的原料添加至所述溶液(100g),并且进一步搅拌混合40分钟。将混合物在120℃下灭菌4分钟,从而制备1kg的根据本发明的犬用软骨机能改善用组合物。所述犬用软骨机能改善用组合物每100g含有10mg的实施例制品7的HMG样蛋白。
[表6]
Claims (3)
1.乳过氧化物酶和/或其分解物在制备用于通过每天摄取至少1mg的所述乳过氧化物酶和/或其分解物来治疗和/或预防骨关节炎中关节软骨层的初始变性和/或初始钙化的组合物中的用途,其中所述乳过氧化物酶和/或其分解物为所述组合物的有效成分,其中通过在胰蛋白酶的存在下分解所述乳过氧化物酶来制备所述乳过氧化物酶的分解物。
2.根据权利要求1所述的用途,其中所述乳过氧化物酶源自哺乳类的乳。
3.根据权利要求1或2所述的用途,其中所述乳过氧化物酶源自牛乳。
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