CN109276588A - 夫妻草萃取物用于制备抑制mmp2基因表现及胶原蛋白分解的组合物的用途 - Google Patents
夫妻草萃取物用于制备抑制mmp2基因表现及胶原蛋白分解的组合物的用途 Download PDFInfo
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Abstract
本发明是关于一种夫妻草萃取物的用途，特别是关于一种夫妻草萃取物用于制备抑制基质金属蛋白酶2(MMP2)基因表现及胶原蛋白分解的组合物的用途。本发明的夫妻草萃取物是以水、醇类、或醇水混合物为溶剂对夫妻草进行萃取而制得。该夫妻草萃取物透过抑制皮肤纤维母细胞中MMP2的基因表现而降低皮肤中胶原蛋白的分解，因此有助于提升皮肤弹性与紧致度、减少皮肤细纹与皱纹、及推迟皮肤老化。
Description
技术领域
本发明是关于一种夫妻草萃取物的用途，特别是关于一种夫妻草萃取物用于制备抑制基质金属蛋白酶2(MMP2)基因表现及抑制胶原蛋白分解的组合物的用途。
背景技术
胶原蛋白(collagen)是动物体内含量高丰富的蛋白质，约占体内总蛋白质的25％。胶原蛋白主要分布于动物体内的结缔组织的细胞外基质，例如韧带、肌腱、软骨、眼睛角膜，是结缔组织中最主要的蛋白质，约占结缔组织的20％～30％。胶原蛋白具有纤维状的结构，能提供结缔组织所需的力学支撑与强度，亦与血液凝结、伤口愈合、组织修复相关，使一种非常重要的结构性蛋白质。人体内的胶原蛋白由于多胜肽组成的差异可分为不同类型，其中，与皮肤构造最相关的是第一型、第三型、与第四型胶原蛋白。真皮层中含有第一型与第三型胶原蛋白，其与皮肤的水分保持及弹性增加有关，上皮组织下方的基底膜中含有第四型胶原蛋白，其与皮肤的水分输送及养分维持有关。
随着年龄增长及紫外线长期照射，皮肤中的胶原蛋白会流失并导致皮肤含水量减少，而皮肤的干燥状态会进一步造成皮肤弹性变差、皱纹生成、及毛孔粗大。因此，设法补充皮肤胶原蛋白、提升胶原蛋白的合成、及减少胶原蛋白的分解已成为减缓皮肤老化、维持皮肤健康的研究重心。由于皮肤表皮层的阻隔作用，直接将含有胶原蛋白的保养品涂抹于皮肤表面无法有效补充皮肤深层的胶原蛋白。另一方面，若以口服方式摄取胶原蛋白，由于大分子的胶原蛋白在消化道被分解为小分子的氨基酸，人体各部位获得该些氨基酸后未必会合成所需的胶原蛋白，故口服胶原蛋白以提升皮肤胶原蛋白合成的效果有限。
有鉴于此，开发一种能有效抑制胶原蛋白分解的新颖组成物以达到减少胶原蛋白流失、避免皮肤老化的目标，实有其必要。
发明内容
缘此，本发明的一目的在提供一种夫妻草(Mimosa pudica)萃取物用于制备抑制基质金属蛋白酶2(matrix metalloproteinase 2，MMP2)基因表现的组合物的用途。
本发明的另一目的在提供一种前述夫妻草萃取物用于制备抑制胶原蛋白分解的组合物的用途，其中该夫妻草萃取物是以一溶剂萃取一夫妻草而获得。
在本发明的一实施例中，该溶剂为水、醇类、或醇水混合物，该溶剂与夫妻草的液固比为5～20∶1～5，且该萃取是在50℃～100℃进行。
在本发明的一实施例中，该夫妻草萃取物是为一夫妻草的水萃取物，其浓度为至少0.5mg/mL。
在本发明的一实施例中，该胶原蛋白为第四型胶原蛋白。
本发明以水、醇类、或醇水混合物为溶剂所萃取得的夫妻草萃取物能透过抑制皮肤纤维母细胞中MMP2的基因表现而减少细胞外基质中第四型胶原蛋白的降解，最终达到增加皮肤中胶原蛋白含量的效果，因此有助于提升皮肤弹性与紧致度、减少皮肤细纹与皱纹、及推迟皮肤老化。故本发明夫妻草萃取物可用于制备抑制MMP2基因表现及抑制胶原蛋白分解的组合物，该组合物可为粉末状、颗粒状、液状、胶状或膏状，且可制成食品、饮品、药品、试剂或营养补充剂，藉由口服、皮肤涂抹等方式给予一个体。
以下将配合图式进一步说明本发明的实施方式，下述所列举的实施例是用以阐明本发明的发明特点及应用，而非以限定本发明的范围，任何熟习此技艺者，在不脱离本发明的精神和范围内，当可做些许更动与润饰，因此本发明的保护范围当视后附的申请专利范围所界定者为准。
附图说明
图1显示经本发明夫妻草萃取物处理的人类皮肤纤维母细胞及控制组人类皮肤纤维母细胞的胶原蛋白相对产量。
图2显示经过本发明夫妻草萃取物处理的人类皮肤纤维母细胞中MMP2基因的相对表现量。
具体实施方式
本发明提供一种夫妻草萃取物用于制备抑制胶原蛋白分解的组合物的用途。本发明的夫妻草萃取物是以一溶剂萃取一夫妻草的植株而获得，其中，该溶剂为水、醇类、或醇水混合物，该溶剂与该夫妻草的液固比为5～20∶1～5，且该萃取是在50℃～100℃进行。透过胶原蛋白含量试验及胶原蛋白分解相关基因的表现量分析，本发明夫妻草萃取物被证实能提高人类皮肤纤维母细胞的胶原蛋白产量，且该产量的增加与MMP2基因的表现量减少有关。因此，本发明的夫妻草萃取物亦可用于制备抑制MMP2基因表现的组合物。
定义
本文中所使用数值为近似值，所有实验数据皆表示在20％的范围内，较佳为在10％的范围内，最佳为在5％的范围内。
材料与方法
材料
自Gibco购买含Earle’s平衡盐溶液的Eagle’s最低基本培养基(Eagle’s minimumessential medium，简称MEM培养基)，胎牛血清(fetal bovine serum，简称FBS)，非必需氨基酸，碳酸氢钠，丙酮酸钠，及磷酸缓冲盐溶液(phosphate buffered saline，简称PBS溶液)。
细胞培养
以下实施例使用人类皮肤纤维母细胞(Human skin fibroblast)CCD-966SK(BCRC60153)进行实验。人类皮肤纤维母细胞在37℃、5％二氧化碳的条件下培养于添加10％FBS、0.1mM非必需氨基酸、1.5g/L碳酸氢钠、1mM丙酮酸钠的MEM培养基，以下称细胞培养基。
胶原蛋白含量试验
本发明利用可溶性胶原蛋白分析套组(Sircol Soluble Collagen Assay Kit，Bicolor)测定细胞所分泌胶原蛋白的含量，其步骤简述如下。取1000μL细胞培养基与200μL胶原蛋白分离/浓缩试剂混合，于4℃下翻转培养一段时间后，以12000rpm的转速离心该混合液10分钟，移除上清液。其次，将余下沉淀与1000μL Sircol染剂混合30分钟，再以12000rpm的转速离心该染剂混合液10分钟以形成胶原蛋白沉淀物。待移除上清液，先以750μL酸性盐溶液清洗该胶原蛋白沉淀物并移除多余液体，添加250μL碱性试剂以溶解该胶原蛋白沉淀物，再使用酵素免疫分析仪(ELISA reader，BioTek)测量该胶原蛋白溶液在波长555nm的吸光值。对照水溶性胶原蛋白标准品的标准曲线，可计算得待测的细胞培养基中的胶原蛋白含量。统计分析是使用Excel软件的学生t检定进行判定。
基因表现量分析
本发明以定量聚合酶链锁反应(quantitative polymerase chain reaction，简称Q-PCR)测定细胞中与胶原蛋白分解有关的酵素的基因表现量，其步骤简述如下。依据厂商使用说明，利用核糖核酸萃取套组(RNA Extraction Kit，Geneaid)自细胞分离出RNA后，于37℃下以反转录酶III Reverse Transcriptase(Invitrogen)将RNA反转录为互补脱氧核醣核酸(cDNA)，其次，利用Q-PCR套组(KAPA CYBR FAST qPCR Kit(2X)，KAPA Biosystems)、针对MMP2基因的正向引子5’-GATACCCCTTTGACGGTAAGGA-3’(SEQ IDNO：1)与反向引子5’-CCTTCTCCCAAGGTCCATAGC-3’(SEQ ID NO：2)、及聚合酶链锁反应仪(StepOnePlusTM Real-Time PCR Systems)对该cDNA进行PCR，以取得基因表现量数据。
实施例1
夫妻草萃取物的制备
首先，将夫妻草洗净晾干，以均质机将夫妻草磨碎。其次，以水、醇类、或醇水混合物为溶剂对夫妻草均质物进行萃取，该溶剂与夫妻草均质物混合的液固比为5～20∶1～5。萃取温度为介于50℃～100℃，较佳为75℃～95℃。本实施例中萃取时间为0.5～2小时。
经上述萃取步骤所得夫妻草萃取物冷却至室温后，可以400目(mesh)的滤网过滤，以移除残余固体物。该过滤后的夫妻草萃取物可进一步在45℃～70℃进行减压浓缩而获得一浓缩产物。为获得固态的夫妻草萃取物，可将前述经减压浓缩的夫妻草萃取物以喷雾干燥方式去除溶剂，因此获得夫妻草萃取物粉末。
实施例2
夫妻草萃取物提升纤维母细胞的胶原蛋白产量
为检验本发明夫妻草萃取物对皮肤细胞的胶原蛋白产量的影响，本实施例以胶原蛋白含量试验分析人类皮肤纤维母细胞CCD-966SK经夫妻草的水萃取物处理后，其胶原蛋白产出量的变化。首先，将人类皮肤纤维母细胞依2×104细胞/孔接种于含有500μL细胞培养基的24孔盘的各孔，在37℃下培养24小时后，移除细胞培养基并以PBS溶液清洗细胞。其次，将500μL、1mg/mL夫妻草的水萃取物与500μL不含FBS的细胞培养基添加至各孔细胞，或仅以500μL不含FBS的细胞培养基处理细胞以作为控制组。前述二组细胞在37℃下培养48小时后，自各孔收集细胞培养基，测定其中胶原蛋白含量。
图1显示经本发明夫妻草萃取物处理的人类皮肤纤维母细胞及控制组细胞的胶原蛋白相对产量。依据图1，相较于控制组人类皮肤纤维母细胞，对人类皮肤纤维母细胞施予夫妻草萃取物明显提升细胞培养基中的胶原蛋白含量约8.3倍。此结果显示本发明夫妻草萃取物具有增加皮肤纤维母细胞的胶原蛋白产量的功效。
实施例3
夫妻草萃取物抑制胶原蛋白分解相关基因的表现
为进一步探讨本发明夫妻草萃取物对胶原蛋白分解酵素的基因表现调节作用，本实施例以Q-PCR测定人类皮肤纤维母细胞CCD-966SK(BCRC 60153)经夫妻草的水萃取物处理后，其胶原蛋白分解相关基因的表现量变化。首先，将人类皮肤纤维母细胞依1.5×105个细胞/孔接种于含有2mL细胞培养基的6孔盘的各孔，置于37℃下培养。其次，以0.5mg/mL或1mg/mL夫妻草的水萃取物处理细胞24小时，再收集经处理细胞以进行Q-PCR。控制组的人类皮肤纤维母细胞是经不含夫妻草水萃取物的细胞培养基处理。
上述人类皮肤纤维母细胞中基质金属蛋白酶2(MMP2)基因的相对表现量如图2所示，图中纵坐标的基因相对表现量表示相对于控制组细胞中MMP2基因的RNA表现量的倍数。MMP2又称第四型胶原蛋白水解酶(type IV collagenase)，由细胞合成后被分泌至细胞外基质，其功能包括分解上皮组织下方基底膜中的第四型胶原蛋白。依据图2，对人类皮肤纤维母细胞施予浓度0.5mg/mL以上的夫妻草萃取物明显降低MMP2的基因表现。当前述细胞以0.5mg/mL夫妻草萃取物处理，其MMP2基因表现量约为控制组细胞的46％。此结果说明本发明夫妻草萃取物能抑制皮肤纤维母细胞中MMP2的基因表现，因此对皮肤中胶原蛋白的降解有抑制效果。
综上所述，本发明以水、醇类、或醇水混合物为溶剂所萃取得的夫妻草萃取物能经由抑制皮肤纤维母细胞中MMP2的基因表现，减少细胞外基质中第四型胶原蛋白的降解，最终达到增加皮肤中胶原蛋白含量的效果。因此，本发明夫妻草萃取物可用于制备抑制MMP2基因表现及抑制胶原蛋白分解的组合物，该组合物可为粉末状、颗粒状、液状、胶状或膏状，且可制成食品、饮品、药品、试剂或营养补充剂，藉由口服、皮肤涂抹等方式给予一个体。
Claims (13)
1.一种夫妻草萃取物用于制备抑制基质金属蛋白酶2(MMP2)基因表现的组合物的用途，其中该夫妻草萃取物是以一溶剂萃取一夫妻草而获得。
2.根据权利要求1所述的用途，其中该溶剂为水、醇类、或醇水混合物。
3.根据权利要求1所述的用途，其特征在于，所述溶剂与该夫妻草的液固比为5～20∶1～5。
4.根据权利要求1所述的用途，其特征在于，所述萃取是在50℃～100℃进行。
5.根据权利要求1所述的用途，其特征在于，所述夫妻草萃取物是为一夫妻草的水萃取物，其浓度为至少0.5mg/mL。
6.根据权利要求1所述的用途，其特征在于，所述夫妻草萃取物是以口服或皮肤涂抹的方式给予。
7.一种夫妻草萃取物用于制备抑制胶原蛋白分解的组合物的用途，其中该夫妻草萃取物是以一溶剂萃取一夫妻草而获得。
8.根据权利要求7所述的用途，其特征在于，所述溶剂为水、醇类、或醇水混合物。
9.根据权利要求7所述的用途，其特征在于，所述溶剂与该夫妻草的液固比为5～20∶1～5。
10.根据权利要求7所述的用途，其特征在于，所述萃取是在50℃～100℃进行。
11.根据权利要求7所述的用途，其特征在于，所述夫妻草萃取物是为一夫妻草的水萃取物，其浓度为至少0.5mg/mL。
12.根据权利要求7所述的用途，其特征在于，所述胶原蛋白为第四型胶原蛋白。
13.根据权利要求7所述的用途，其特征在于，所述夫妻草萃取物是以口服或皮肤涂抹的方式给予。
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