CN113521055A - 低聚茋类单体化合物VitD在制备提高动物脂肪沉积的产品中的用途 - Google Patents
低聚茋类单体化合物VitD在制备提高动物脂肪沉积的产品中的用途 Download PDFInfo
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Abstract
本发明公开了低聚茋类单体化合物VitD在制备提高动物脂肪沉积的产品中的用途，通过对低聚茋类单体化合物VitD进行活性研究，验证了化合物VitD能够促进C2C12细胞中脂质的积累，并提高转录因子PPARγ和FABP4的表达水平。
Description
技术领域
本发明属于植物中药物活性化合物开发领域，特别是涉及低聚茋类单体化合物VitD在制备提高动物脂肪沉积的产品中的用途。
背景技术
茋类化合物是具有1,2-二苯乙烯骨架以及其组成的聚合物的一类化合物的总称。主要存在于植物的木质部，最早是从蓼科植物大黄中发现，包括二苯乙烯、二苯乙基、菲类单体及其聚合物。低聚芪类化合物属于非黄酮的多酚类化合物,存在于龙胆香科、芍药科、买麻藤科、蔷薇科、莎草科、豆科、桑科、蝶形花科、鸢尾科、葡萄科等。近年来研究报道芪类化合物具有多种生物活性，如神经保护、抗病毒、抗菌、抗炎、抗肿瘤、抗氧化，抗艾滋病及保肝等作用。
马蔺(Iris lacteal Pall.var.chinensis(Fisch.)Koidz.),别名马莲、马兰、兰花草等，是被子植物门(Angiospermae)鸢尾科(Iridaceae)鸢尾属(Iris)的多年生草本宿根植物。马蔺广泛分布于我国的西北、华北、华东和东北等地,同时中西亚、俄罗斯、蒙古及朝鲜也有分布。马蔺是一种传统的中草药,据《中华本草》中记载，马蔺为植物马蔺的全草，其性味苦、微甘,性微寒。归肾、膀胱、肝经。其具有清热解毒、利尿通淋、活血消肿的功能，用于治疗喉痹，淋浊，关节痛，痈疽恶疮等症，且马蔺的叶、花、根和种子也均可入药。马蔺花晒干后即可入药，性咸，味酸、苦，微凉，有清热凉血的功能，用于咽喉肿疼、吐血等症的治疗；马蔺根性平、味甘，具有清热解毒等功效,用于治疗牙痛、急性传染性肝炎、咽炎等症；其种子有清热解毒、利湿止血的功效，可用于黄疸、白带、泻痢、吐血等疾病的治疗。现代药理学表明马蔺具有多种药理作用，如放射增敏性、抗生物、增强免疫、抗癌、改善糖脂代谢等作用。
发明内容
在畜牧生产中，肌肉内脂肪沉积影响肉的断面大理石评分，与肉类(如猪肉、牛肉等)品质密切相关。肌肉脂肪主要包括肌内脂肪和肌间脂肪。肌间脂肪就是肌纤维束之间的脂肪；肌内脂存在于肌外膜、肌束膜、甚至肌内膜上，营养状况好的家畜，其肌纤维膜的毛细血管上也有脂肪。肌肉内脂肪含量与肉类的感官品质和食用品质密切相关，直接影响着肉的风味、多汁性、嫩度、色泽等，最终影响肉质等级与经济价值。因此，开展调控肉类中的脂肪沉积研究，对畜牧业的健康发展和满足消费者对优质肉的需求具有重大的意义。
于渤洋等(腺苷甲硫氨酸对成肌细胞成脂分化及脂肪沉积的影响，2012)以小鼠成肌细胞(C2C12)为模型探讨了蛋氨酸代谢产物腺苷甲硫氨酸(SAM)对肌肉来源的多能干细胞成脂分化及脂肪沉积的影响，经SAM处理后的C2C12细胞表现出脂肪细胞的形态特征；细胞内脂肪沉积水平上升，并且随SAM处理浓度的升高呈现出剂量依赖效应；细胞的脂肪特异性基因PPARγ、C/EBPα的mRNA及蛋白表达水平显著升高，表明了SAM可以促进C2C12细胞成脂分化及细胞内脂肪的沉积，从而增加动物肌肉中肌内脂肪的含量，明显改善肉类产品的品质。
郭红芳等(Krüppel-Like Factor 3(KLF3)基因对牛脂肪沉积的作用,2019)公开了牛肉品质主要受脂肪沉积的影响，而脂肪沉积主要与脂肪细胞分化和脂肪酸代谢密切相关，而如何提高肌内脂肪含量来满足消费者对牛肉嫩度和风味的需要，一直是牛肉脂肪沉积生物分子调控研究的一个热点，因此该文献通过确定KLF3基因在牛脂肪细胞分化和脂肪酸代谢中的作用，为进一步明确KLF3基因在调控牛脂肪沉积中的作用，改善牛肉品质提供一定的理论依据。
专利CN110590881从马蔺中分离鉴定出了多种化合物，并发现马蔺中的茋类化合物具有抑制脂质积累的作用。然而，令人意外的是，本发明研究发现，低聚茋类单体化合物VitD，具有升脂作用，促进肌内细胞脂质积累，可以提高动物肌肉脂肪沉积。
本发明实验中，采用小鼠成肌细胞C2C12细胞进行实验，C2C12是肌肉来源的多能干细胞的代表，是研究肌肉发育最常用的体外模型。
本发明提供的低聚茋类单体化合物VitD的用途中，所述低聚茋类单体化合物的化学结构式如下：
本发明中所述产品包括但不限于药品、饲料等。
进一步地，所述产品为促进动物肌内脂肪沉积的产品。
进一步地，所述产品为促进细胞脂质积累的产品。
目前认为脂滴是在内质网(ER)上形成，脂滴的形成包括甘油三酯，甘油和游离脂肪酸等小分子转入细胞内，在内质网上合成甘油三酯，以脂滴的形式分泌到细胞基质中。
进一步地，所述产品为PPARγ激动剂。
所述PPARγ激动剂为提高PPARγ的表达水平的产品。
进一步地，所述产品为FABP4激动剂。
所述FABP4激动剂为提高FABP4的表达水平的产品。
进一步地，所述产品为Caveolin-1激动剂。
所述Caveolin-1激动剂为提高Caveolin-1的表达水平的产品。
进一步地，所述动物选自猪、牛。
进一步地，所述产品为预防和/或治疗肌肉脂质代谢紊乱的产品。
本发明提供了一种上述低聚茋类单体化合物VitD在制备改善动物肉质的产品中的用途。
本发明还提供了上述低聚茋类单体化合物在制备PPARγ激动剂、FABP4激动剂、Caveolin-1激动剂中的任意一种的应用。
本发明的有益效果是：
(1)本发明提供的低聚茋类单体化合物能促进C2C12细胞中脂滴的积累及增加甘油三酯的含量，并提高转录因子PPARγ、FABP4的表达水平，促进脂质的积累，能显著提高肌肉内脂肪沉积。
(2)本发明更加全面挖掘了马蔺的药用价值、拓展了其应用，在生命科学领域、医学科学领域、畜牧业领域等中都具有重要的意义。
附图说明
图1低聚茋类四聚体化合物对C2C12细胞脂质积累的影响；
图2低聚茋类四聚体化合物对C2C12细胞甘油三酯含量的影响；
图3低聚茋类四聚体化合物对C2C12细胞转录因子表达水平的影响。
具体实施方式
下面通过具体实施例和具体实验对本发明所述应用做进一步说明，对所述低聚茋类单体化合物改善脂代谢紊乱相关疾病的作用进行验证和说明。
材料与试剂
本实验受试化合物是从马蔺子中分离得到的低聚茋类单体化合物，分别为ε-Viniferin(ε-vin)、VitisinA-13b-o-glucoside(VitAOG)、Vitisin A(VitA)、Vitisin D(VitD)、Hopeaphenol(Hop)和Isohopeaphenol(Isohop)。所有化合物的化学结构式如下所示：
C2C12小鼠成肌细胞购买于中国科学院上海细胞库。
C2C12小鼠成肌细胞购买于中国科学院上海细胞库。
一、C2C12细胞诱导分化
采用“鸡尾酒”法对C2C12细胞进行诱导分化，诱导分化方法如下：将细胞状态良好的细胞接种于培养板上，铺板密度5×104个/mL，用含10％FBS的高糖DMEM培养液培养至细胞密度达到85％～90％左右时细胞换液，接触抑制两天后弃去完全培养液，加入诱I培养液培养细胞2d(加诱导液时记为第0d)，随后换成诱II培养液继续培养细胞2d，随后换成正常培养液继续培养4d，隔天换液。在诱导的第8d时可观察细胞内有大量的脂滴出现。
C2C12细胞诱I培养液：10μg/mL Insulin、0.5mM IBMX、1μM Dex和10μM Rosi；
C2C12细胞诱II培养液：10μg/mL Insulin和10μM Rosi。
二、SRB法测定细胞活力
磺酰罗丹明B(Sulforhodamine B，SRB)比色法，是一种检测细胞增殖情况的方法。其具体步骤如下：(1)细胞以1×105的密度接种于96孔板，用含10％FBS的高糖DMEM培养液培养细胞，加入低聚茋类提取物孵育48h。(2)孵育结束后，弃去原液，每孔加入100μL新鲜DMEM，之后加入25μL 50％TCA(避光)，室温放置5min，4℃放置1h。(3)弃去原液，用流动的超纯水冲洗每个孔5遍，每一遍冲洗后用力甩出。在超净工作台倒置15min吹干(高风)。(4)每孔加入70μL 0.4％的SRB，振荡30min(避光)。(5)弃去SRB染液，每孔加1％的冰乙酸冲洗固定的细胞4遍，每一遍冲洗后用力甩出。在超净工作台倒置15min吹干(高风)。(6)每孔加入100μL 100mM Tribase溶解，于37℃振荡孵育20min。(7)用酶标仪于540nm波长检测OD值。按公式计算细胞活力。
细胞活力(％)＝(药物组OD/空白组OD)×100％
实施例1低聚茋类单体化合物对C2C12细胞的相关指标的影响
不同诱导时间C2C12细胞胞内脂滴积累情况
1.油红O染色
将细胞状态良好的细胞接种于6孔板上，铺板密度5×104个/mL，待细胞密度达到85％～90％左右时进行诱导分化，诱导过程如2.1所述，诱导结束后，用4％中性甲醛固定细胞30min，待细胞固定结束后加入提前配制的油红O工作液染于细胞表面，避光静置60min。待染色结束后用70％乙醇洗涤细胞，弃去多余的染料，再用超纯水洗涤3～4次，最后置于显微镜下观察并拍照。其结果如图1所示。
2.TG含量的测定
将细胞以5×104个/mL的密度接种于6孔板中，待细胞密度达到85％～90％左右进行诱导分化，在诱导的第8d进行TG含量的测定，其具体方法如下：(1)细胞前处理：在诱导的第8d，将细胞培养液吸走，冷PBS洗两次后加入胰酶消化液进行消化细胞。(2)细胞收集：细胞消化后，加入PBS重悬细胞后在1000g的条件下离心5min，收集细胞沉淀团。(3)超声破碎：向收集的沉淀团中加入适量的PBS后进行超声破碎(3min)。(4)测定：在96孔板中每孔加入2μL细胞破碎悬液，空白孔中加入2μL蒸馏水，标准孔中加入2μL标准品，然后每孔中加入200μL的测定液，混匀，37℃孵育10min后，在510nm处读取吸光值。借助BCA法测定待测样品中蛋白浓度，进行校正，最后根据公式计算TG的含量。其结果如图2所示。
TG含量＝(OD样品-OD空白)/(OD校样-OD空白)×校准品浓度(mM)/待测样品蛋白浓度(gprot/L)
3.低聚茋类单体化合物对C2C12细胞成脂转录因子表达水平的影响
将生长状态良好的C2C12细胞以5×104个/mL的密度接种于6孔板上，待细胞密度达到85％～90％左右时进行诱导分化，并在诱导时加入受试化合物处理，诱导结束后收集细胞，将处理得到的细胞样品进行蛋白提取，其具体操作方法如下：
(1)将RIPA裂解液与PMSF按100:1的比例混合均匀(如分析磷酸化蛋白时，需加入磷酸酶抑制剂)，配制成细胞裂解液。细胞样品：将6孔板中的细胞用冷的PBS洗3次，每孔加入100μL细胞裂解液，放于冰上裂解5min，随后用细胞刮子刮取细胞，收集到1.5mL离心管，再次放入4℃冰箱充分裂解30min。最后在12000g，4℃，15min的条件下离心肝组织匀浆液和细胞裂解液，取上清液蛋白于新的EP管。
(2)BCA法测定蛋白质含量：取出适量蛋白样品，根据BCA蛋白定量试剂盒说明书的要求，将蛋白标准品用PBS缓冲液稀释为0.5mg/mL，按50:1的比例将试剂A：试剂B混匀配制成工作液。按表1所示将标准品和PBS加入96孔板中，每孔20μL，一般每个浓度设3个复孔，用来绘制标准曲线。蛋白样品1μL加到96孔板中，加PBS补到20μL。每孔加入200μL BCA工作液充分混匀，上述样品在微孔板孵育振荡器37℃振荡孵育30min，于酶标仪562nm处测吸光度值，根据标准曲线计算出样品的蛋白浓度。配好的蛋白样品于100℃加热10min，使蛋白完全变性。蛋白上样前，样品离心混合均匀后使用。
表1 BCA法蛋白定量表
(3)SDS-PAGE电泳：根据目标蛋白的分子量大小选择合适的分离胶浓度，配制合适的溶液体系(下层胶)，待下层胶在室温放置40min以上使至完全凝固，再配制5％的浓缩胶(上层胶)，并插入大小合适的梳子。待浓缩胶凝固后，将胶板放入电泳槽中，往两块板之间加满电泳液后拔出梳子，上蛋白样品和marker后进行跑胶。电泳条件：S1阶段，80V，30min；S2阶段，120V，大约60～90min，直至缓冲液中溴酚蓝跑到分离胶最下端时结束电泳。
(4)转膜：待到蛋白电泳结束后立即进行转膜，根据marker条带按照分子量切下目标蛋白所在的位置。取出PVDF膜剪裁成与胶相同的大小，将剪裁好的膜放置甲醇溶液活化1min后放入转膜液中。按照转印夹黑板-滤纸-胶PVDF-滤纸-转印夹白板顺序夹紧电转夹，黑板对着槽的黑面，放入电泳槽，加满转膜液至整个槽中，通电后将转膜仪置于冰水混合物中。转膜的条件为恒流250mA，时间90min。
(5)封闭：转膜结束后，将PVDF膜快速从转膜板中用镊子轻轻拿出，放于TBST缓冲液中漂洗5min，随后立刻用含5％脱脂奶粉的TBST在室温摇床上封闭1h。
(6)一抗孵育：封闭结束后，将PVDF膜放入稀释好的一抗溶液中，在4℃条件下置于水平摇床上慢摇孵育过夜。
(7)二抗孵育：一抗孵育结束后，TBST洗膜三次，一次8min。随后将PVDF膜置于含有HRP标记的鼠二抗或兔二抗溶液中室温慢摇孵育1h。
(8)显影：二抗孵育结束后，TBST洗膜三次，一次8min。洗膜结束后，将混合好的ECL显影液滴加在PVDF膜上，并用凝胶成像系统进行拍照。
(9)结果分析：目标蛋白和内参蛋白的灰度值用Image J软件进行定量分析，蛋白的相对表达量为目标蛋白与内参蛋白的比值(磷酸化与非磷酸化蛋白的比值)。
统计分析
使用GraphPad Prism7软件进行作图和统计学分析，两组间均数的比较采用独立样本t检验，多组数据之间均数的相互比较采用单因素方差分析(one-way ANOVA)，统计数据以平均值±标准差(x±s)表示，*或者#表示P<0.05说明具有统计学意义，**或者##表示P<0.01说明其差异具有极显著统计学意义。
结果
如图1所示，相比未分化的细胞undiff组，诱导分化的细胞diff组中有大量的脂滴积累(P<0.01)。化合物VitAOG、VitA和Hop处理后能显著性地降低胞内脂滴的积累(P<0.05)，而化合物VitD和Isohop能显著性地增加脂滴的积累(P<0.01)。染色结束后用异丙醇溶出着色的脂滴定量分析脂滴的相对含量，实验结果发现脂滴相对定量的结果和油红O染色的结果一致。
其结果如图2所示，相比未分化的细胞undiff组，诱导分化的细胞diff组的细胞内TG含量显著升高(P<0.01)。四聚体化合物处理后细胞内TG含量表现出不同的变化趋势，其中化合物VitAOG能显著降低胞内TG的含量(P<0.01)，而VitD、Isohop处理后细胞中TG含量升高，其中Isohop处理后细胞中TG含量显著性升高(P<0.01)。
结果如图3所示，相比未分化的细胞undiff组，诱导分化的细胞diff组中转录因子PPARγ、FABP4表达水平显著升高(P<0.01)。四聚体化合物VitAOG、VitA和Hop能显著降低PPARγ、FABP4表达水平(P<0.01)，而化合物VitD和Isohop能显著增加PPARγ、FABP4表达水平(P<0.01)。
综上所述，低聚茋类单体化合物VitAOG、VitA、Hop和ε-Vin能抑制细胞脂滴的积累和TG的含量及降低PPARγ和FABP4等蛋白的表达水平，而化合物VitD和Isohop却能促进细胞中脂滴的积累，并提高转录因子PPARγ和FABP4的表达水平，显著增加Caveolin-1的表达水平。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
Claims (10)
1.低聚茋类单体化合物VitD在制备提高动物脂肪沉积的产品中的用途；所述低聚茋类单体化合物的化学结构式为：
2.根据权利要求1所述的用途，其特征在于，所述产品为促进动物肌内脂肪沉积的产品。
3.根据权利要求1所述的用途，其特征在于，所述产品为促进细胞脂质积累的产品。
4.根据权利要求3所述的用途，其特征在于，所述产品为PPARγ激动剂。
5.根据权利要求3所述的用途，其特征在于，所述产品为FABP4激动剂。
6.根据权利要求3所述的用途，其特征在于，所述产品为Caveolin-1激动剂。
7.根据权利要求1所述的用途，其特征在于，所述动物选自猪、牛。
8.根据权利要求2所述的用途，其特征在于，所述产品为预防和/或治疗肌肉脂质代谢紊乱的产品。
9.低聚茋类单体化合物VitD在制备改善动物肉质的产品中的用途。
10.低聚茋类单体化合物在制备PPARγ激动剂、FABP4激动剂、C/EBPβ激动剂、Caveolin-1激动剂中的任意一种的应用，所述低聚茋类单体化合物的结构式为：
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