CN114533723A - 一种调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂及其应用 - Google Patents
一种调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂及其应用,该膳食营养补充剂的活性成分包括茶黄素单体TF1,且其活性成分还包括苦瓜提取物和/或发酵桑叶提取物;还公开了该膳食营养补充剂或含有该膳食营养补充剂的制剂在制备调节骨骼肌糖代谢、降血糖和促进骨骼肌线粒体生成的药品和保健品中的应用。本发明所提供的膳食营养补充剂可以促进骨骼肌对葡萄糖的吸收,维持体内糖代谢平衡,对于糖尿病患者具有积极的作用,可以作为糖尿病患者的日常膳食补充剂,达到调节血糖的目的;同时可以促进骨骼肌线粒体的生物发生,提高肌肉能力代谢,促进肌肉力量。
Description
技术领域
本发明属于食品技术领域,更具体地,涉及一种调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂及其应用。
背景技术
据统计,二型糖尿病占全球糖尿病患者的90%以上,其发病原因与肥胖以及胰岛素作用下肝脏-脂肪组织-骨骼肌轴的糖代谢/脂肪代谢稳态具有十分密切的关系;其中,骨骼肌是胰岛素刺激的全身葡萄糖代谢与大部分脂肪酸代谢的主要场所,约占正常成年人体重的40%,也是维持人体正常生理功能,保证生命质量的重要组成部分。
现有研究表明,骨骼肌代谢功能异常和骨骼肌胰岛素抵抗是导致二型糖尿病、肥胖和骨骼肌衰老等慢性疾病发生发展的重要诱因。
目前已有多种天然产物被报道在调节人体糖脂代谢以及治疗、预防和缓解二型糖尿病中具有积极作用,且大部分天然产物被作为膳食添加剂,在糖尿病人或调节糖脂代谢的饮食中作为新型食品。
众所周知,饮茶被认为是一种调节糖脂代谢和预防二型糖尿病的有益饮食习惯。随着研究的逐步深入,结果表明,饮茶的益处主要是由茶叶中的儿茶素、茶多糖、茶黄素和聚酯儿茶素等功能成分决定的,因此,将茶叶中的功能成分进行分离,并有目的性的加入到饮食中,开发含茶叶功能成分的天然膳食补充剂成为基础研究的主要转化方向。
红茶作为世界上消费量最多的茶类,其被报道对人体具有多重有益的保护作用,包括调节人体代谢紊乱性疾病(主要是二型糖尿病和肥胖等),保护心脑血管,抵抗衰老等。茶黄素不仅是红茶中主要的品质成分,也是红茶中最具生物学活性的天然化合物,被称为茶叶中的“软黄金”。现有研究发现,红茶和茶黄素有助于预防肥胖的发生发展,在保护和调节二型糖尿病的发生中具有积极的作用。然而,有关茶黄素单体的生物学功能研究较少,尤其是茶黄素与骨骼代谢功能之间内在机制的研究鲜有报道。
申请号为20061003975.1的中国发明专利申请公开了一种苦瓜多糖降血糖组合物及其制备方法,所述组合物中的多糖类化合物分子量在15000-1000000道尔顿之间,其多糖含量为60-90%,它是一种安全有效的降血糖健康制品原料;申请号为20081017533.4的中国发明专利申请公开了一种茶多酚中药复方在制备降血脂且降血糖的药物中的应用,该配方可以显著降低高血糖小鼠的体内血脂与血糖含量;申请号为201810134624.0的中国发明专利申请公开了一种用于降血糖的茶多糖AN蛋白复合物及其应用,其可降低机体胰岛素分泌不足导致的高血糖或胰岛素不敏感所致的高血糖,抑制α葡萄糖苷酶活性和肠道葡萄糖转运蛋白的活性,维持胰岛素的正常分泌,并改善糖尿病并发症。
尽管现有技术中已报道了茶叶中的功能成分具有降低血糖和改善高血糖和高血脂症状的作用;但到目前为止,尚未有人研究红茶中的功能成分茶黄素在调节血糖中的应用,因此,针对TFs降血糖保护糖尿病患者的研究与产品开发具有广阔的应用前景。本发明者就此展开了长期研究,从而完成了本发明。
发明内容
本发明要解决的技术问题是研究一种具有促进骨骼肌糖吸收与线粒体生成的膳食营养补充剂及其配方研究和应用。本发明首次公开了一种用茶黄素单体TF1为主要膳食原料并加入其它天然植物提取物(包括茶叶来源的儿茶素、苦瓜提取物和发酵桑叶提取物)配制而成的可以增强骨骼肌糖吸收和线粒体生成的膳食补充剂,且该含茶黄素单体TF1的配方成分均为天然植物来源的提取物,其最主要的问题是解决配方配比与实际效果的问题。
为实现上述目的,本发明的技术方案如下:
一种调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂,其不同之处在于,其活性成分包括茶黄素单体TF1。
具体地,在上述技术方案中,TF1是红茶加工过程中由表儿茶素与表没食子儿茶素在多酚氧化酶的作用下氧化缩合而成的一种含多羟基的氧化缩合产物;其带有多个羟基结构,不仅使其具有极强的抗氧化活性,且具有多种生物学功能。
在上述技术方案中,所述调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂的活性成分还包括苦瓜提取物和/或发酵桑叶提取物。
具体地,在上述技术方案中,日常食用苦瓜和发酵桑叶可以有效的降低血糖,并且苦瓜提取物更是被称之为“植物胰岛素”;苦瓜提取物中的主要活性成分为甾体皂甙如苦瓜素,类胰岛素肽类和生物碱,其被报道具有极好的降血糖作用;发酵桑叶提取物主要包括桑叶提取物脱氧野尻霉素与葛根提取类黄酮物质,其被报道在调节人体血糖,抵抗肥胖等方面具有特殊的生物学活性。
进一步地,上述技术方案中,在所述调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂的活性成分中,所述茶黄素单体TF1、苦瓜提取物和发酵桑叶提取物的质量比为6-7∶2-3∶1-2。
具体地,在上述技术方案中,在所述苦瓜提取物中,主要的功能成分为苦瓜素,类胰岛素肽类和生物碱,均为粉碎后,热水浸泡后所得的天然提取物;其中,苦瓜皂苷的含量为20-30wt%。
具体地,在上述技术方案中,所述发酵桑叶提取物主要功能成分为脱氧野尻霉素与葛根提取类黄酮物质,是经过热水浸泡后分离所得的天然提取物;其中,桑叶多糖的含量为50-60wt%。
本发明另一方面还提供了含上述膳食营养补充剂的制剂,该制剂由膳食营养补充剂和药学上可接受的辅料组成。
具体地,在上述技术方案中,所述辅料选自淀粉、纤维素、麦芽糊精和低聚糖中的一种或多种。
在本发明的一个具体实施方式中,该膳食营养补充剂的制剂由以下重量份的原料组成:茶黄素单体TF1 2份,苦瓜提取物1份,发酵桑叶提取物1份,甜菊素C 0.2-0.8份,维生素D2 0.2-0.8份,亮氨酸0.2-0.8份,矿物盐5-10份。
具体地,在上述技术方案中,所述制剂的剂型选自片剂、胶囊剂、冲剂、颗粒剂和丸剂中的一种。
本发明又一方面还提供了上述膳食营养补充剂或上述制剂在制备调节骨骼肌糖代谢、降血糖和促进骨骼肌线粒体生成的药品和保健品中的应用。
具体地,在上述技术方案中,其剂量为控制茶黄素单体TF1的摄入量为350-450mg/d。
具体地,在上述技术方案中,所述降血糖为治疗II型糖尿病。
本发明与现有技术相比,具有以下优点:
本发明所提供的膳食营养补充剂可以促进骨骼肌对葡萄糖的吸收,维持体内糖代谢平衡,对于糖尿病患者具有积极的作用,可以作为糖尿病患者的日常膳食补充剂,达到调节血糖的目的;同时可以促进骨骼肌线粒体的生物发生,提高肌肉能力代谢,促进肌肉力量。
附图说明
图1为本发明实施例中膳食营养补充剂促进体外肌管对葡萄糖的吸收的结果图(其中:图1-A为TF1配方处理48h后,成熟肌管对葡萄糖的实时吸收水平;图1-B为TF1配方处理48h后,成熟肌管对荧光葡萄糖2-NBDG的实时吸收水平);
图2为本发明实施例中膳食营养补充剂调节肌管代谢相关基因的表达与糖酵解能力的结果图(其中:图2-A为TF1配方处理后,肌管中差异上调的代谢相关差异基因热图;图2-B为TF1配方处理后,成熟肌管代谢相关基因的mRNA水平图;图2-C为TF1配方处理后,肌管糖酵解能力实时检测水平;图2-D为TF1配方处理后,糖酵解能力的量化水平);
图3为本发明实施例中膳食营养补充剂促进肌管线粒体的生物发生的结果图(其中:图3-A为TF1配方处理后,流式细胞术检测肌管线粒体丰度及其量化结果图;图3-B为TF1配方处理后,高内涵荧光显微镜检测肌管线粒体丰度及其量化结果图);
图4为本发明实施例中膳食营养补充剂促进线粒体相关基因的表达与氧化磷酸化能力的结果图(其中:图4-A为TF1处理后线粒体生物发生相关基因mRNA表达水平图;图4-B为TF1处理后线粒体生物发生相关基因蛋白表达水平图;图4-C为TF1处理后,成熟肌管氧化磷酸化能力实时检测;图4-D为TF1处理后,成熟肌管氧化磷酸化能力量化水平)。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。
应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例中,如无特别说明,所用手段均为本领域常规的手段。
本文中所用的术语“包含”、“包括”或其任何其它变形,意在覆盖非排它性的包括。例如,包含所列要素的组合物、步骤、方法、制品或装置不必仅限于那些要素,而是可以包括未明确列出的其它要素或此种组合物、步骤、方法、制品或装置所固有的要素。
此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
在本发明实施例中,茶黄素单体TF1、苦瓜提取物和发酵桑叶提取物均购买自湖南艾嘉生物科技有限公司。
本发明以C2C12成肌细胞为模型,通过体外诱导分化为成熟肌管,建立TF1配方-体外骨骼肌纤维-糖代谢研究模型;通过两种不同的方式检测了体外分化成熟的肌管对葡萄糖的吸收水平,并结合分子生物学技术与Seahorse XF代谢检测系统等先进的仪器,检测了该配方对肌管线粒体生成以及一系列代谢过程和代谢方式的影响;这些结果直接用于评价TF1配方对骨骼肌葡萄糖吸收与线粒体生成的效果。
具体实施过程如下:
1、各种细胞培养用培养基的配置
细胞培养基:
①增殖培养基(GM):10%的胎牛血清+DMEM高糖培养基;
②分化培养基(DM):2%的马血清+DMEM高糖培养基;
③含TF1配方的分化培养基(DM+):固定配方+固定体积的DM;
④含TF1配方的增殖培养基(GM+):固定配方+固定体积的GM。
其中,固定配方为:茶黄素单体TF1 2份,苦瓜提取物1份,发酵桑叶提取物1份,甜菊素C 0.5份,维生素D2 0.6份,亮氨酸0.5份,矿物盐8份;固定体积为100ml。
2、体外肌管的培养方法
首先使用GM培养基将C2C12细胞在60mm培养皿中培养至90%,之后按每孔3×106/孔(六孔板),5×105/孔(12孔板)均匀接种至细胞培养板;GM继续培养至80-90%后,使用DM培养基诱导分化,每天更换新鲜培养基,直至诱导分化至第四天,成熟肌管普遍形成,使用DM+或GM+进行药物处理。
3、RNA提取
(1)收集目标细胞,并用1×PBS清洗细胞之后,根据细胞数目,加入适量Trizol后放置在摇床上的冰盒上进行裂解;
(2)使用1ml的枪头将培养皿中经过Trizol裂解后的细胞进行吹打,并转移至冰上预冷的1.5ml灭菌后的EP管中,静止3-5分钟后,加入预冷的三氯甲烷(Trizol∶三氯甲烷=5∶1),上下充分混匀后,放置在冰上静止5分钟,之后放入高速冷冻离心机中,12000转/分钟,离心20分钟;
(3)离心后,吸取离心管中上清,避免吸入蛋白层液体,之后加入等体积预冷后的异丙醇,放置在冰上30分钟或-20℃可永久保存;
(4)取出异丙醇与RNA混合物,至高速冷冻离心机中,经过7200转/分钟,离心半小时后,移去上清,加入75%预冷后的酒精,冲洗RNA沉淀,并使用7200转/分钟离心五分钟,重复以上步骤两次以上;
(5)倒置EP管,自然晾干7-10分钟后,清理管壁酒精后,加入适量无酶水,进行RNA浓度检测。
4、逆转录
根据诺唯赞试剂盒,配置如下逆转录体系,将目的RNA逆转录为cDNA,加入4μl 4×g DNA后,加入1000mg目的RNA,并加入无酶水补充至总体积16μl,将其放入PCR仪中,42℃反应2分钟;取出样品,加入4μl 5×Surper Mix 2,调整PCR程序为50℃15分钟,85℃5秒,下机后稀释5倍,配置成工作浓度。
5、实时荧光定量PCR
根据诺唯赞实时荧光定量PCR试剂盒程序,配置如下体系:
实时定量PCR上机设定的反应程序如下:(Cycle×39)
6、肌管葡萄糖吸收检测
使用索莱宝葡萄糖含量检测试剂盒(微量法,货号:BC2505,北京,中国)检测肌管实时葡萄糖吸收水平,实验组为茶黄素处理组,并且加入胰岛素(200ng/ml)处理组作为葡萄糖吸收阳性对照组。
具体步骤为:收集细胞,离心后弃上清,按细胞数量(104个)∶蒸馏水体积(ml)为500-1000∶1ml的比例进行处理,超声波破碎细胞(冰水浴,功率20%或200W,超声3s,间隔10s,重复30次),置沸水浴中煮沸10min(期间防止水分散失),冷却后8000g离心10min,取上清液检测。
检测原理为葡萄糖氧化酶催化葡萄糖氧化成葡萄糖酸,并产生过氧化氢;过氧化物酶催化过氧化氢氧化4-氨基安替比林偶联酚,生成有色化合物,使用酶标仪(ELX800,Beckman Coulter,USA)在505nm检测特征吸光值。
BCA蛋白分析试剂盒(Pierce,纽约州格兰德岛,纽约,美国)检测样品蛋白浓度用于结果标化。
计算公式为:葡萄糖含量(μmol/mg prot)=2×(A3-A1)÷(A2-A1)÷Cpr。
其中:Cpr为样本蛋白浓度;具体见试剂盒操作手册。
2-(N-(7-硝基苯并-2-氧杂-1,3-二唑-4-氨基)-2-脱氧葡萄糖(2-NBDG,Sigma,USA)是荧光标记的2-脱氧葡萄糖类似物,可用作评估细胞葡萄糖代谢的示踪剂。
使用10μm的2-NBDG孵育TF1配方处理后的肌管20-30min后,使用流失细胞仪(CytekAthena Dxpflow,USA)检测肌管内实时葡萄糖吸收水平,在465/540nm波长处出现最大激发/发射。
7、肌管内线粒体丰度检测
使用线粒体荧光探针Mito-Tracker-Green(货号:C1048,碧云天,中国)检测肌管内线粒体丰度。Mito-Tracker-Green是一种活细胞线粒体荧光染色剂,最大激发光波长为490nm,最大发射光波长为516nm,呈现绿色荧光。本研究使用DM培基,将Mito-Tracker-Green稀释成300nM的工作浓度,与待测细胞在细胞培养箱中共孵育40min,方法一:使用流式细胞仪(Cytek Athena Dxp)进行检测。方法二:使用激光共聚焦细胞培养板培养细胞,高内涵荧光显微镜(Thermo Scientific CellInsight CX7)观察肌管荧光强度。
8、肌管糖酵解压力与线粒体压力检测
使用Seahorse XF(Agilent,美国)实时活细胞代谢分析系统检测肌管氧气消耗率(OCR)和细胞外酸化率(ECAR)来指示肌管代谢能力变化。具体步骤如下:将C2C12成肌细胞以1×105个/孔,均匀接种至XF 8孔微孔板中,GM培养基培养至细胞密度90%左右,更换DM培养基诱导分化4天后,实验组更换含DM+培养基培养48h,备检。检测探针浸泡在无菌水中,至37℃无CO2培箱过夜,使用前1h,将无菌水更换为校准液(PH.7.4),至37℃无CO2培箱活化。糖酵解压力试验前,使用置换液(XF分析培养基+1%谷氨酰胺)对待检细胞进行两次置换后加入180μl置换液至无CO2培箱置换1h。检测前A仓:20μl10mM葡萄糖;B仓:22μl 20mMoligomycin;C仓:25μl 5mM 2-DG;线粒体压力检测,置换液为XF分析培养基+1%谷氨酰胺+1%丙酮酸,A仓:20μl 20mM oligomycin;B仓:22μl 20mM FCCP;C仓:25μl 10mM Rot/AA。检测后,用BCA试剂盒定量细胞蛋白含量,并相应地对OCR和ECAR进行标化。
结果
为了验证TF1配方是否对成熟肌管的代谢具有调节作用,我们检测了TF1配方处理成熟肌管48h后,肌管内葡萄糖含量;其中:以胰岛素处理组作为阳性对照组。
结果显示,TF1配方处理后的成熟肌管内,葡萄糖的吸收量显著高于对照组,与胰岛素处理组结果一致,但是TF1配方与胰岛素共同处理下的肌管,并没有显示出对葡萄糖具有更突出的吸收作用(图1A);同时,我们使用荧光葡萄糖(2-NBDG)吸收实验,通过流式细胞术,实时检测了TF1配方处理48h,肌管内荧光葡萄糖的含量,TF1配方处理组与胰岛素处理组同样可以显著促进肌管对荧光葡萄糖的吸收水平,但是两者之间并没有表现出显著的协同效应(图1B)。
通过两种不同的实验方法,均证明TF1配方对成熟肌管的葡萄糖吸收具有显著促进作用。
之后,通过Q-PCR进一步检测了差异基因中调节肌管代谢功能的基因,包括Myh7(氧化型慢肌纤维)、Myh1(酵解型快肌纤维)、以及Myl2和Tnnt1的mRNA表达水平;与转录组数据一致的是,慢肌纤维(Myh7)的表达水平较快肌纤维(Myh1)更高,且慢肌纤维特异性基因(Myl2,Tnnt1)同样高表达(图2A-2B)。
为进一步的验证TF1配方是否促进肌纤维偏向慢肌纤维转变,改变肌管代谢功能,我们通过Agilent Seahorse XF分析仪检测TF1配方处理后,成熟肌管ECAR值的实时变化,表征TF1处理后肌管的代谢类型是否发生转变,图2C为TF1配方处理组与对照组实时ECAR水平。量化结果显示,对照组和TF1处理组的非糖酵解酸化率保持一致(图2D,左),说明实验组与对照组数量一致;在加入葡萄糖之后,对照组的糖酵解速率显著高于TF1配方处理组(图2D,中,P=0.0064),并且对照组的糖酵解能力也显著高于TF1配方处理组(图2D,右,P=0.004)。
线粒体丰度的差异是慢肌纤维与快肌纤维代谢特征差异的主要原因;本发明使用Mito-Tracker-Green对肌管内得线粒体进行染色,之后使用流式细胞术与高内涵荧光显微镜检测肌管内线粒体丰度。
结果显示,流式细胞术检测下的肌管线粒体荧光强度,TF1配方处理组显著高于对照组(图3A,P=0.0044),高内涵实时成像系统的结果同样如此,TF1配方处理后的肌管中,Mito-Tracker-Green的平均荧光强度同样高于对照组(图3B,P=0.023)。
为了进一步探索TF1配方调节体外肌管葡萄糖吸收和线粒体丰度的作用,本发明使用qPCR检测了TF1处理后肌管中CaMMK2,AMPK,PGC-1α和SIRT1的mRNA表达水平。
结果显示,在TF1处理48小时后,肌管中CaMMK2,AMPK,PGC-1α和SIRT1的mRNA水平显着高于对照组(图4A);然后,使用蛋白质印迹法测定这些基因的蛋白质水平,结果表明,CaMMK2、AMPK、PGC-1α和SIRT1的蛋白质表达水平上调(图4B),支持在mRNA水平获得的结果。在分子水平上的结果表明,TF1上调了肌管中的钙离子丰度,激活了CaMMK2-AMPK信号转导轴,并协调了PGC-1α/SIRT1表达的变化。
肌管线粒体丰度的变化,是决定肌管代谢方式和肌管类型转变的关键因子。线粒体的代谢过程主要由氧化磷酸化作用,OCR是线粒体代谢能力的指标。TF1处理后检测到线粒体压力,结果表明,TF1处理组的线粒体代谢活性显着高于对照组(图4C)。定量了线粒体代谢的关键过程,包括基础呼吸,ATP产生,备用呼吸能力和最大呼吸。结果显示,实验组和对照组之间的基本呼吸能力没有差异,但TF1治疗组的ATP产量高于对照组,但无统计学差异(图4D,P=0.0581)。在备用呼吸能力和最大呼吸的统计数据中,TF1治疗组显着高于对照组(图4D)。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。
应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂,其特征在于,
其活性成分包括茶黄素单体TF1。
2.根据权利要求1所述的调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂,其特征在于,
其活性成分还包括苦瓜提取物和/或发酵桑叶提取物。
3.根据权利要求2所述的调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂,其特征在于,
其活性成分中,所述茶黄素单体TF1、苦瓜提取物和发酵桑叶提取物的质量比为6-7∶2-3∶1-2。
4.根据权利要求2或3所述的调节骨骼肌糖代谢与线粒体生成的膳食营养补充剂,其特征在于,
在所述苦瓜提取物中,苦瓜皂苷的含量为20-30wt%;
和/或,在所述发酵桑叶提取物中,桑叶多糖的含量为50-60wt%。
5.一种含权利要求1-4任一项所述的膳食营养补充剂的制剂,其特征在于,
所述制剂由膳食营养补充剂和药学上可接受的辅料组成。
6.根据权利要求5所述的制剂,其特征在于,
所述辅料选自淀粉、纤维素、麦芽糊精和低聚糖中的一种或多种。
7.根据权利要求5所述的制剂,其特征在于,
其剂型选自片剂、胶囊剂、冲剂、颗粒剂和丸剂中的一种。
8.权利要求1-4任一项所述的膳食营养补充剂或权利要求5-7任一项所述的制剂在制备调节骨骼肌糖代谢、降血糖和促进骨骼肌线粒体生成的药品和保健品中的应用。
9.根据权利要求8所述的应用,其特征在于,
其剂量为控制茶黄素单体TF1的摄入量为350-450mg/d。
10.根据权利要求8所述的应用,其特征在于,
所述降血糖为治疗II型糖尿病。
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