CN105903003B - 一种改善废用性骨质疏松的组合物及其应用 - Google Patents
一种改善废用性骨质疏松的组合物及其应用 Download PDFInfo
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- CN105903003B CN105903003B CN201610290647.1A CN201610290647A CN105903003B CN 105903003 B CN105903003 B CN 105903003B CN 201610290647 A CN201610290647 A CN 201610290647A CN 105903003 B CN105903003 B CN 105903003B
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Abstract
本发明公开一种改善废用性骨质疏松的组合物及其应用,该组合物包括骨胶原蛋白肽和钙制剂。所述钙制剂选自无机钙制剂、有机钙制剂和新型钙制剂中的一种或多种。本发明的组合物能够改善废用性骨质疏松的症状,同时不会产生副作用。
Description
技术领域
本发明涉及属医疗和保健品领域,更具体地,涉及一种能够抑制废用性骨质疏松的应用。
背景技术
骨质疏松症分为原发性和继发性两大类,绝经后妇女骨密度显著降低,大多数易患原发性骨质疏松症。废用性骨质疏松症属继发性骨质疏松症,主要因骨骼机械力减少,致全身性或局部性骨量丢失引起,多见于脊髓损伤、偏瘫、脊髓灰质炎及其后遗症、骨折、长期卧床及太空飞行。常见病因为:1)运动能力受限或功能障碍;2)运动系统包括肌肉骨骼损伤或病损;3)神经系统损伤或病损。肢体的运动受到极大的限制,肌肉收缩对骨刺激应力的消失,再加上卧床的免负荷以及瘫痪后内分泌的改变,骨质疏松是不可避免的。
废用性骨质疏松常发生于老年人身上,老年人活动少,肌肉强度减弱,机械刺激少,骨量减少;同时肌肉强度的减弱以及协调障碍使老人较易摔倒,且由于骨量减少、脆性增加,易造成脊椎压缩性骨折、髋部骨折和桡骨远端骨折;又因骨折后长期卧床和肢体制动,肢体不负重或负重减少;伤肢骨矿物质丢失又会导致继发性骨质疏松,从而使已发生骨折的患者再发生骨折的可能性增高,进而形成恶性循环。
废用性骨质疏松的防治包括卧床体位、运动锻炼、物理治疗、药物治疗。在防治上应首先强调运动锻炼,就现有药物而言,对减轻症状有一定程度的帮助,但不能完全治愈。
另外,废用性骨质疏松症还可引起一系列并发症,如肾结石发病率增高、关节周围软组织骨化(异位骨化)、肢体畸形以及病理性骨折发病率增高等,已引起人们的广泛关注。然而废用性骨质疏松症具体机制仍未阐明,尚无理想的抑制药物。研究人员一直不断地探索废用性骨质疏松症具体机制和更好的治疗和改善方法。
现有研究显示,骨胶原蛋白肽是胶原或明胶经蛋白酶降解处理后制成的,富含人体需要的甘氨酸、脯氨酸、羟脯氨酸等氨基酸,具有较高的消化吸收性及安全性。细胞和动物水平研究显示骨胶原蛋白肽在促进骨形成及抑制骨丢失方面具有一定作用。如HyeKyung Kim等的研究表明,100μg/mL骨胶原蛋白肽增加成骨细胞的增殖,以剂量依赖的方式促进ALP活性,促进COL1a的合成,参与ERK/MAPK信号对COL1a表达的转录影响。每天服用150and500mg/kg骨胶原蛋白肽的去卵巢大鼠3个月后,与对照组相比,骨胶原蛋白肽阻止去卵巢引起的骨损失,改善去卵巢大鼠椎骨组成特点和生物力学强度显著增加腰椎的骨小梁数目及骨体积比,显示骨胶原蛋白肽具有骨保护效果。
综上,对已患废用性骨质疏松的患者,现有药物而言只能减轻不适的症状,而不能治愈,通过单独补充钙或维生素D不能改善废用性骨质疏松。现有骨胶原蛋白肽提示能够改善骨质疏松,然而单独使用骨胶原蛋白肽对废用性骨质疏松改善作用有限。因此,需要提供一种改善废用性骨质疏松的组合物,该组合物能够更好地改善废用性骨质疏松,同时对于运动受限,而可能导致废用性骨质疏松的人具有预防作用。
发明内容
本发明要解决的第一个技术问题是提供一种改善废用性骨质疏松的组合物。该组合物能够更好地改善废用性骨质疏松,同时对于运动受限,而可能导致废用性骨质疏松的人具有预防作用。
为解决上述技术问题,本发明采用下述技术方案:
一种改善废用性骨质疏松的组合物,该组合物包括骨胶原蛋白肽和钙制剂。
所述钙制剂选自无机钙制剂、有机钙制剂和新型钙制剂中的一种或多种。其中所述无机钙选自磷酸钙、碳酸钙、碳酸氢钙和氧化钙中的一种或多种;所述有机钙制剂选自柠檬酸钙、乳酸钙、枸橼酸钙、醋酸钙和葡萄糖酸钙中的一种或多种;所述新型钙制剂选自有机丁酸、丙酸钙制剂、氨基酸与钙结合制成的氨基酸类钙制剂和微粉化碳酸钙中的一种或多种。
所述补钙制剂可以选自市售的补钙制剂。
所述骨胶原蛋白肽的制备方法,包括如下步骤:
1)将碱法骨明胶溶于水,配制成质量浓度为10~25%的骨明胶水溶液;
2)将步骤1)配制好的骨明胶溶液加热至45~70℃,用无机酸调节溶液pH至2~4,加酸性蛋白酶,酶解1~5小时,得到第一酶解液,其中所述的酸性蛋白酶的加入量是骨明胶重量的1~10‰;用无机碱将第一酶解液的pH值调至6~8,温度控制在45~70℃,加入中性蛋白酶,酶解1~5小时,得到第二酶解液,其中所述的中性蛋白酶的加入量是骨明胶重量的1~10‰;
3)将步骤2)得到的第二酶解液加热至95~100℃,保持5~15分钟,得到骨胶原蛋白肽溶液;
4)对步骤3)得到的可溶性骨胶原蛋白肽溶液进行超滤膜脱盐;
5)加入骨明胶重量0.5%的活性炭,升温至60℃,搅拌30min,而后通过活性炭过滤器进行过滤,除去活性炭,以脱味,脱色;
6)喷雾干燥,得到粉状骨胶原蛋白肽。
第二酶解液加热至95~100℃,并保持5~15分钟以灭活蛋白酶;
所述骨明胶选自于牛骨明胶或猪骨明胶。
所述的酸性蛋白酶是胃蛋白酶;所述的中性蛋白酶是胰蛋白酶或木瓜蛋白酶等。
所述的无机酸为盐酸或磷酸;所述的无机碱为氢氧化钙、氢氧化铵或氢氧化钠。
所述改善废用性骨质疏松的组合物用于预防废用性骨质疏松的用途。
所述组合物在抑制尾吊大鼠骨丢失实验中显示,大鼠每天使用量为750mg/kg牛骨胶原肽和75mg/kg的柠檬酸钙具有改善废用性骨质疏松的效果,该计量相当于成年人每天施用胶原肽的剂量范围为5-10克,元素钙施用范围为600-1000毫克。
本发明所述的废用性骨质疏松症的病因为长期卧床、制动、失重所导致,包括长期卧床、骨折后或骨折石膏固定后产生的骨量减少、肌肉萎缩及关节僵硬等,久之则会形成明显的废用性骨质疏松。老年人活动少,肌肉强度减弱,机械刺激少,骨量减少;同时肌肉强度的减弱以及协调障碍使老人较易摔倒,且由于骨量减少、脆性增加,易造成脊椎压缩性骨折、髋部骨折和桡骨远端骨折;又因骨折后长期卧床和肢体制动,肢体不负重或负重减少;伤肢骨矿物质丢失又会导致继发性骨质疏松,从而使已发生骨折的患者再发生骨折的可能性增高,进而形成恶性循环。
本发明的有益效果如下:
组合物能够更好地改善废用性骨质疏松,同时对于运动受限,而可能导致废用性骨质疏松的人具有预防作用。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出实验大鼠股骨密度。
图2示出Micro CT测量股骨微结构。
图3示出股骨远端相对股体积(distal femurBV/TV)。
图4示出股骨远端骨小梁数量(distal femur TR.No)。
图5示出股骨远端骨小梁厚度(distal femur Tb.Th)
图6示出股骨远端骨小梁分离度(distal femur Tb.Sp)
图7示出Masson染色观察股骨胶原纤维。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
实施例1:制备牛骨胶原蛋白肽
1)将碱法牛骨明胶溶于水,配制成质量浓度为15%的牛骨明胶水溶液;
2)将步骤1)配制好的牛骨明胶溶液加热至55℃,用无机酸调节溶液pH值至3,加酸性蛋白酶,酶解3小时,得到第一酶解液,其中所述的酸性蛋白酶的加入量是牛骨明胶重量的5‰;用无机碱将第一酶解液的pH值调至7,温度控制在55℃,加入中性蛋白酶,酶解3小时,得到第二酶解液,其中所述的中性蛋白酶的加入量是牛骨明胶重量的5‰;
3)将步骤2)得到的第二酶解液加热至97℃,保持10分钟以灭活蛋白酶,得到牛骨胶原蛋白肽溶液;
4)对步骤3)得到的可溶性牛骨胶原蛋白肽溶液用超滤膜脱盐;
5)加入牛骨明胶重量0.5%的活性炭,升温至60℃,搅拌30min,而后通过活性炭过滤器进行过滤,除去活性炭,以脱味,脱色;
6)喷雾干燥,得到粉状骨胶原蛋白肽。
实施例2:骨胶原蛋白肽与钙复合物对尾吊大鼠骨丢失的抑制效应
大鼠尾巴悬吊法是废用性骨质疏松症的常用造模方法,这种方法采用尾部悬吊使大鼠双后肢悬空,头低位,后肢处于人工失重状态,模拟动物失重的情况,这种模型属于大鼠后肢去负荷模型。它能较真实地模拟长期卧床、骨折后或骨折石膏固定后产生的骨量减少、肌肉萎缩及关节僵硬等,久之则会形成明显的废用性骨质疏松。
1.实验设计
将购买并饲养于航天员科研训练中心(SPF级)的3月龄SPF级雄性SD大鼠48只(体重300±20g)随机均分成六组,分别为自由活动对照组(Control group,CN组)、模拟微重力组(Simulated Microgravity,SMG组)、阿伦磷酸钠(ALN)治疗组(ALN组)、钙制剂处理组(CC组)、骨胶原蛋白肽处理组(CP组)和骨胶原蛋白肽-钙制剂联合处理组(CP-CC组)。其中CN组及SMG每只每次灌服1ml双蒸水;ALN组施用1mg/kg ALN,其中阿伦磷酸钠(ALN)是临床治疗骨质疏松首选药物;CC组施用75mg/kg柠檬酸钙(Calcium citrol,CC);CP组施用750mg/kg的骨胶原蛋白肽(CP);CP-CC组施用750mg/kg的骨胶原蛋白肽和75mg/kg柠檬酸钙(CP-CC)。其中,ALN组、CC组、CP组和CP-CC组所施用的药物或化合物用双蒸水溶解,每次施用体积为1ml。
本申请所指的mg/kg为每千克大鼠体重给予的药物或化合物。
大鼠饲养条件为:CN组大鼠自由活动;SMG组、ALN组、CP组、CC组和CP-CC组都使用后肢去负荷模型大鼠。采用尾部悬吊使大鼠双后肢悬空,头低位,后肢处于人工失重状态。靠两前肢载重并在一定范围内自由活动。自由进食消毒颗粒饲料、饮消毒水,饲养温度控制在23℃,定期紫外线消毒与排风。
2.样品检测
实验持续4周,在第28天时,大鼠空腹12小时,采用水合氯醛对大鼠进行麻醉后,腹主动脉采集血液样本,分离血清。然后将大鼠解剖,剔除肌肉和筋膜,取大鼠右股骨用盐水覆盖的纱布包埋,-20℃保存。
1)血清碱性磷酸酶含量检测
采用全自动生化分析仪(Model 7060;Hitachi,Tokyo,Japan)检测所有测试大鼠的血清Ca、P和ALP,其中ALP为碱性磷酸酶,结果见表1。
2)血清骨钙素含量检测
血清骨钙素(osteocalcin,OC)含量按OC放射免疫分析操作说明检测。应用竞争原理,标准品或样品中的OC与加入的125I-OC共同与一定量的特异性抗体产生竞争性免疫反应。125I-OC与抗体的结合量与标准品或样品中OC的含量呈一定的函数关系。用免疫分离试剂(兔抗OC抗体和驴抗兔免疫分离剂)将结合部分与游离部分分离后,测定结合部分的放射性强度,并计算相应结合率。用已知标准品中OC含量与对应结合率作图,即得标准抑制曲线。从标准曲线上查知对应结合率的待测样品中OC的含量。检测所有被试大鼠的血清骨钙素含量,结果见表1。
3)血清PINP、CTX和TRAP-5b的检测
通过Elisa法测定大鼠血清骨代谢指标PINP、CTX和TRAP-5b含量,检测结果见表1。
其中CTX为血清I型胶原交联羧基末端肽,检测试剂盒为RatLaps ELISA(IDS,UK)。
PINP为总骨I型前胶原氨基端肽,检测试剂盒为Rat PINP EIA(IDS,UK)。
TRACP-5b为血清抗酒石酸酸性磷酸酶5b,检测试剂盒为RatTRAP(IDS,UK)。
表1血清骨代谢指标
每组n=8*代表P<0.05,**代表P<0.01与SMG组相比;#代表P<0.05,##代表P<0.01与CN相比。
结果表明:大鼠尾吊28天后,SMG组大鼠血清Ca、ALP和OC含量显著低于CN组大鼠(P<0.05),表明SMG条件下大鼠的骨代谢显著降低。施用ALN(ALN组)、柠檬酸钙(CC组)或胶原蛋白肽(CP组)对上述指标无显著改善。施用柠檬酸钙和胶原蛋白肽后(CP-CC组),CP-CC组大鼠血清ALP含量与SMG组大鼠相比无显著差异(P>0.05),OC含量与SMG组大鼠相比有显著升高(P<0.05)。施用ALN或柠檬酸钙能够显著抑制SMG大鼠血清PINP含量的升高,施用柠檬酸钙和骨胶原蛋白肽(CP-CC组)对大鼠血清PINP的含量无显著影响,但CP组大鼠血清中PINP含量显著高于CN组(P<0.05),可能是由于钙离子降低了大鼠血清中PINP含量。各组大鼠血清中的CTX与TRAP-5b没有显著差异。
骨钙素是骨基质中最丰富的蛋白质,是骨基质矿化的必需物质,是反映骨转换的一个特异、灵敏的生化指标。通过骨钙素可以了解成骨细胞,特别是新形成的成骨细胞的活动状态。上述研究结果表明CP-CC能够显著增加大鼠血清OC含量,表明CP-CC对维持骨的正常矿化速率具有积极的作用。
4)股骨骨密度(BMD)分析
对于骨质疏松症而言,骨折的风险可以通过测量腰椎和股骨的骨密度预测。各组大鼠右股骨BMD的检测采用美国通用公司的Lunar PIXImus BMD测量仪,用于实验室小动物的BMD评估。测量时,由同一操作者测量并将所有大鼠置于同一方向。测量结果见图1,其中femoral BMD表示股骨骨密度。
结果显示,股骨BMD的变化如图1所示,图中cc代表cc组、cp代表cp组、cp-cc代表cp-cc组、ALN代表ALN组、SMG代表SMG组(下面表示相同)。SMG组大鼠股骨的股骨骨密度与CN组大鼠相比显著降低(P<0.01),ALN处理(ALN组)能够显著改善后肢去负荷导致的股骨骨密度降低。单独施用柠檬酸钙对SMG诱导的股骨BMD降低无任何改善作用。CP-CC及CP的处理尽管对后肢去负荷导致的股骨骨密度降低没有达到显著抑制的水平,但仍显示出抑制的趋势,而且CP-CC的抑制效果优于CP。
5)micro-CT成像分析
用小动物微型CT影像系统成像(μCT40,SCANCO MEDICAL,Switzerland)进行分析各组大鼠右侧股骨,分析区域选择在骨骺线中间1mm处往下2mm的区域。利用显微CT系统软件对所选区域进行3D重建,并利用该软件进行骨微结构分析,得出3D重建效果图,见图2,最大剖面图及骨形态参数,所述骨形态参数包括相对骨体积(bone volume/tissue volume,BV/TV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨小梁数量(trabecular number,Tb.No)、骨小梁分离度(trabecular spacing,Tb/Sp)。
从Micro CT三维结构重建图可以看出,SMG组大鼠股骨骨小梁结构破坏严重,中间有较大空洞;CN组大鼠股骨骨小梁结构致密完整,破坏不明显;与SMG组相比,ALN组、CP-CC组和CP组,股骨具有较厚的骨小梁,增加骨小梁的完整性,而且CP-CC处理的效果优于CP处理,表明上述处理对尾吊失重引起的骨丢失具有预防作用。CC的处理对骨小梁结构破坏没有明显改善效果。
股骨远端的微结构指标分析结果显示见图3-6,SMG组与CN组比较,BV/TV、Tb.Th、Tb.N值显著降低(P<0.05),Tb.Sp值显著升高。与SMG组比较,ALN组、CP-CC组、CP组的BV/TV、Tb.No值升高,Tb.Sp值降低。特别是CP-CC处理,与CP处理相比,能够显著增加BV/TV,从而呈现出一定的优越性。ALN是目前临床治疗骨质疏松的首选药物,由于ALN在临床使用的过程中具有一些副作用,例如导致下颌骨溃烂,因此并不是对所有的患者都适用。
6)Masson染色观察骨胶原纤维
具体实验操作为:右侧胫骨骨标本固定于4%多聚甲醛(pH 7.26)7天,在10%的EDTA脱钙14~30天,然后石蜡包埋,选取胫骨近端切成5微米厚的组织切片,采用Masson三色染色。染色后,胶原纤维、粘液、软骨呈蓝色;胞浆、肌肉、纤维素、红细胞呈红色;胞核呈蓝黑色。使用徕卡显微镜进行图像采集,分析,结果见图7。
Masson染色观察大各组鼠右侧胫骨近端骨胶原纤维,与CN组相比,SMG组和CC组骨胶原纤维数目少,排列稀疏,连接性差。CN、CP-CC组、CP组和ALN组大鼠股骨骨小梁较粗大,数目多,厚度较一致,骨小梁连接呈网状,排列较有序,表明CP-CC处理和CP处理能够抑制后肢去负荷所致的骨胶原纤维的减少。
显然,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定,对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动,这里无法对所有的实施方式予以穷举,凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
Claims (8)
1.一种改善废用性骨质疏松的组合物,其特征在于,该组合物包括骨胶原蛋白肽和钙制剂;
所述骨胶原蛋白肽的制备方法包括如下步骤:
1) 将碱法骨明胶溶于水,配制成质量浓度为10~25%的骨明胶水溶液;
2) 将步骤1)配制好的骨明胶溶液加热至45~70℃,用无机酸调节溶液的pH至2~4,加酸性蛋白酶,酶解1~5小时,得到第一酶解液,其中所述的酸性蛋白酶的加入量是骨明胶重量的1~10‰;用无机碱将第一酶解液的pH值调至6~8,温度控制在45~70℃,加入中性蛋白酶,酶解1~5小时,得到第二酶解液,其中所述的中性蛋白酶的加入量是骨明胶重量的1~10‰;
其中,所述酸性蛋白酶是胃蛋白酶;所述的中性蛋白酶是胰蛋白酶或木瓜蛋白酶;
3) 将步骤2)得到的第二酶解液加热至95~100℃,保持5~15分钟,得到骨胶原蛋白肽溶液;
4) 对步骤3)得到的可溶性骨胶原蛋白肽溶液用超滤膜脱盐;
5)加入骨明胶重量0.5%的活性炭,升温至60℃,搅拌30min,而后通过活性炭过滤器进行过滤,除去活性炭;
6)喷雾干燥,得到粉状骨胶原蛋白肽。
2.如权利要求1所述的组合物,其特征在于,所述钙制剂选自无机钙制剂、有机钙制剂和新型钙制剂中的一种或多种。
3.如权利要求2所述的组合物,其特征在于,所述无机钙选自磷酸钙、碳酸钙、碳酸氢钙和氧化钙中的一种或多种。
4.如权利要求2所述的组合物,其特征在于,所述有机钙制剂选自柠檬酸钙、乳酸钙、枸橼酸钙、醋酸钙和葡萄糖酸钙中的一种或多种。
5.如权利要求2所述的组合物,其特征在于,所述新型钙制剂选自有机丁酸、丙酸钙制剂、氨基酸与钙结合制成的氨基酸类钙制剂和微粉化碳酸钙中的一种或多种。
6.如权利要求5所述的组合物,其特征在于,所述骨明胶选自牛骨明胶或猪骨明胶。
7.如权利要求5所述的组合物,其特征在于,所述的无机酸为盐酸或磷酸;所述的无机碱为氢氧化钙、氢氧化铵或氢氧化钠。
8.如权利要求1所述的组合物用于制备预防废用性骨质疏松的药物的用途。
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