CN112569218A - 共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用 - Google Patents
共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用 Download PDFInfo
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Abstract
本发明公开了共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用。本发明以肥胖小鼠、糖尿病小鼠和高糖高脂饲养小鼠为研究对象，采用生化指标监测、qRT‑PCR、组织学切片染色等方法检测其对脂肪代谢作用。经实验证明共轭亚油酸异构体明显促进肝脏内脂肪分解代谢体脂，降低脂肪合成和组织蓄积相关因子表达，从而有效降低动物肝脏内脂肪含量，将该单体应用于制备降脂的药物或保健品，对人体健康具有重要意义，具有良好的市场应用前景。
Description
技术领域
本发明属于医药技术领域，具体涉及共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用。
背景技术
共轭亚油酸(Conjugated Linoleic Acid CLA)是一类具有较高生理活性的脂肪酸，是亚油酸(LA)位置异构体和空间异构体的总称。CLA异构体类型丰富，自然界以及化学合成制剂中已经发现多达25种异构体的存在，其中以异构体c9，t11-CLA和t10，c12-CLA的研究最为集中，c9，t11-CLA异构体的主要作用是抗癌，t10，c12-CLA则是降低体脂、降低血脂等，其独特的生理作用使得CLA成为近年来研究的明星营养素。
近年来，肥胖等相关营养性疾病在发达和发展中国家已成为危害人类健康的重大公共卫生问题。目前我国肥胖人口呈直线上升趋势，柳叶刀最新数据显示我国平均肥胖率达到12％，肥胖人口已经超过美国位居首位。脂肪在肝脏内过度蓄积，容易诱发肝炎，肝细胞肿胀、炎细胞浸润，正常的肝小叶结构被破坏，未来可能发展为肝硬化，甚至肝癌。因此，开发降低内脏脂肪的药物显得尤其重要。
发明内容
本发明的目的是提供了共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，并研究高纯度共轭亚油酸的降脂效果，为高纯度共轭亚油酸异构体在降脂中的应用提供支持。
为实现上述发明目的，本发明的技术方案如下：
本发明提供了共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用。
进一步的：所述共轭亚油酸异构体为c9，t11-CLA和t10，c12-CLA，化学结构式分别为式1、式2；
进一步的：所述共轭亚油酸异构体促进脂肪组织内LPL基因和UCP1基因表达，抑制FAS基因和ACC1基因表达。
进一步的：所述共轭亚油酸异构体促进肝脏内PKA、Adiponectin、LPL基因表达，抑制FAS基因表达。
进一步的：所述的共轭亚油酸异构体能够减少内脏或脂肪组织脂质蓄积，减少脂肪酸合成、促进脂解和脂肪酸β氧化。
进一步的：所述共轭亚油酸异构体的有效剂量为100-400mg/Kg/d。超过400mg/Kg/d剂量的共轭亚油酸异构体使用时，虽然高剂量在降低血脂方面差异显著，但是肝脏切片显示高剂量容易形成脂肪肝，对肝的副作用影响较大。
进一步的：所述共轭亚油酸异构体对于肥胖小鼠有显著的摄食抑制作用，降低小鼠的食欲。
进一步的：所述共轭亚油酸异构体对肥胖小鼠降低体重的作用呈剂量依赖性。
进一步的：所述内脏为肝脏、肾脏、胰脏、胃和脾脏。
与现有技术相比，本发明的优点和有益效果是：本发明的共轭亚油酸异构体(t10，c12-CLA)，对正常体重小鼠摄食无显著影响，而对于代谢障碍的肥胖和高血糖模型鼠有显著的摄食抑制作用，显著促进TG分解、白色脂肪产热；肝脏中相关脂代谢基因FAS相对模型组显著降低，PKA、Adiponectin、LPL显著升高。对肝脏内的TG(甘油三酯)具有显著的降低活性，可以减少肝脏、脂肪组织脂质的蓄积，减少脂肪酸合成、促进脂解、脂肪酸β氧化；促进体重和脂肪的减少。本发明的高纯度共轭亚油酸对人体健康具有重要意义，具有良好的市场应用前景。
附图说明
图1是共轭亚油酸异构体的气相分析结果。
图2是CLA对ob/ob小鼠摄食量影响。
图3是CLA对ob/ob小鼠体重影响。
图4是CLA对ob/ob小鼠脂肪细胞大小的影响。
图5是CLA对ob/ob小鼠肝脏影响。
图6是CLA对ob/ob小鼠胰腺影响。
图7是共轭亚油酸(CLA)对ob/ob小鼠肥胖指数及脏器指数的影响。
图8是CLA对脂肪细胞脂代谢相关因子mRNA表达影响。
图9是CLA对肝脏脂代谢相关因子mRNA表达影响。
具体实施方式
下述实施方式更好地说明本发明内容。但本发明不限于下述实施例。
本发明主要以肥胖小鼠、糖尿病小鼠和高糖高脂饲养小鼠为研究对象，采用生化指标监测、qRT-PCR、组织学切片染色等方法检测其对脂肪代谢作用。本发明实施例中使用的CLA为已知的技术手段制得的纯度98％的共轭亚油酸异构体，气相分析结果如图1。其中两种异构体c9，t11-CLA∶t10，c12-CLA等于1∶1；结构式如式1、式2所示。
实施例1：摄食量、体重的测量
1、实验步骤
(1)选择ob/ob小鼠作为肥胖模型鼠，以不同剂量(低、中、高剂量浓度分别为100mg/Kg/d、600mg/Kg/d、1800mg/Kg/d)CLA进行干预，分别为低剂量组(CLA-L)组、中剂量组(CLA-M)组、高剂量组(CLA-H)组，对照组为阴性对照组(CLA-C)、正常组(WT)作为阳性对照组；
(2)对体重的影响，每3天称重，反映不同处理组小鼠体重变化及比较不同受试物干预效果；
(2)对摄食量的影响，每周监测一次，反映不同处理组小鼠摄食量变化及比较不同组之间差异；
(4)对脏器重量及脏器比的影响，脏器(肝脏)，脂肪称重，计算：
①Lee’s指数＝3√m×103/L m为小鼠体质量/g；L为小鼠体长/cm；
②FC/％＝m1/m×100 m1为脂肪质量/g；m为小鼠质量/g；
③脏器系数/％＝m1/m×100 m1为脏器质量/g；m为小鼠质量/g。
2、实验结果
(1)CLA对小鼠摄食量影响
野生型小鼠处理前CLA-C、CLA-L、CLA-M、CLA-H四组摄食量分别为2.59±0.40、2.28±0.49、2.20±0.75、2.13±0.71，5周实验期内CLA低、中、高剂量处理。
ob/ob小鼠未处理前CLA-C、CLA-L、CLA-M、CLA-H组摄食量分别为3.19±0.54、3.48±1.03、4.13±0.77和4.12±1.04g/d，摄食量记录表明CLA-H组小鼠摄食量为0.87±0.22g，显著低于CLA-C组(P＜0.05)，CLA-L和CLA-M组摄食量均稍有下降(图2)。说明CLA对于肥胖模型鼠有显著的摄食抑制作用，可以减少小鼠的食欲。
(2)CLA对小鼠体重影响
ob/ob小鼠为肥胖模型鼠，肥胖为其主要特征，未处理前CLA-C、CLA-L、CLA-M、CLA-H组体重分别为47.18±1.05、46.78±1.92、46.54±1.86和46.650±0.79g，低中高剂量CLA制剂服用5周后，体重秤剂量依赖性下降，各组分别为：54.00±1.33g、51.94±1.75g、48.34±2.41g和39.22±1.58g，中、高剂量组ob小鼠体重显著降低(P＜0.05、P＜0.05 vs CLAC组，图3)。体重记录结果表明一定剂量的CLA制剂对肥胖小鼠有降低体重的作用，呈剂量依赖性。
实施例2：脂肪、肝脏、胰腺研究实验
1、试验方法
I、组织切片染色
(1)对小鼠干预一段时间后将其处死，对其肝脏和脂肪组织切片分析，观察肝内脂滴大小和白脂空泡大小的情况；
(2)取新鲜脂肪、肝脏、胰腺等组织块4％多聚甲醛固定，脱水透明，石蜡包埋后切片，一般为5-10μm厚。切片展平后二甲苯脱蜡，苏木精染色，冲洗、脱水后伊红染色，再脱水、透明，树胶封片；
(3)脂肪组织HE染色后，光学显微镜下随机选取15个高倍视野拍照(视野内脂肪细胞胞膜光滑清晰)。用画笔工具仔细圈定脂肪细胞胞膜，Image J图像软件计算出所有圈定脂肪细胞面积。
II、取新鲜肝脏组织400mg，加4ml脂质提取液(正庚烷∶异丙醇＝2∶3.5)4℃研钵匀浆，振荡提取脂质，离心沉淀后取上清液以试剂盒测定肝脏甘油三酯含量。
III、小鼠胰腺组织经多聚甲醛固定后，常规HE切片。采用Image J图像软件对组织HE切片图像进行分析。胰岛面积百分比计算如下：低倍镜下用画笔工具划定视野内胰腺区域，计算总面积，光笔仔细圈定各胰岛面积，软件得出圈定的胰岛面积。逐步测量所有胰岛和全部胰腺面积，计算胰岛占胰腺总面积的百分比。
2、实验结果
(1)CLA对脂肪组织、肝脏和胰腺组织影响
图4为各组小鼠脂肪组织切片，显示ob/ob小鼠单个脂肪细胞面积显著增大(图4A、B)，CLA处理后脂肪细胞变小，中高剂量组较为显著(P＜0.05，图4F)，提示CLA促进脂肪细胞中脂肪的分解代谢。ob/ob肥胖小鼠肝细胞呈现脂肪空泡样变(图5B)，CLA-M和CLA-H组小鼠肝脏内空泡样变程度减轻(图5D、F)，表明共轭亚油酸(CLA)可以有效减小ob/ob小鼠的脂肪空泡的大小；表明CLA也促进肝脏内脂肪分解代谢。ob/ob小鼠胰腺组织内胰岛数量较WT小鼠显著增加(图5A、B)，CLA处理后胰岛数量减少(图6C-F)，胰岛所占面积也明显降低(表1)，表明CLA影响胰腺功能。如图7，表明共轭亚油酸(CLA)可以降低ob/ob小鼠脏器指数，以肝的变化显著；中、高剂量的CLA显著降低模型鼠内脏脂含量，以肝的变化显著。
表1 CLA对ob/ob小鼠胰腺组织学变化
Note：##：P＜0.01 VS WT；*：p＜0.05 VS CLA-C，**：p＜0.01 VS CLA-C。
实施例3：mRNA表达测定
1、实验方法
脂肪、肝脏、肌肉等组织采用Trizol法提取总RNA，进行反转录，得到cDNA调整浓度为100ng/ul。qRT-PCR反应体系为20uL：100ng cDNA，10ul 2XFaststart Universal SYBRGreenMaster、0.6uL Primer F/R、8.4ul RNase Free Water。反应条件为：95℃10min，95℃15s，57℃1min，40个循环。实验对每个样品进行3次重复，运用2-ΔΔCt的方法计算目的基因相对表达量，β-actin为内参。引物序列见表2。
表2引物序列汇总
2、实验结果
(1)CLA对脂肪组织和肝脏内脂肪代谢因子mRNA表达影响
ob/ob小鼠脂肪组织内脂肪合成酶(FAS)和乙酰辅酶A羧化酶1(ACC1)基因表达显著增多(图8)，UCP1表达减少，提示模型鼠体内脂肪合成代谢增强，而分解代谢降低。CLA灌胃后，模型鼠脂肪内LPL和UCP1 mRNA表达升高，表明脂肪分解代谢增强；FAS和ACC1在中低剂量CLA组mRNA表达收到抑制，高剂量组有升高趋势，但仍明显低于模型对照组(图8)，表明CLA处理后模型鼠脂肪合成和组织蓄积相关因子表达较未处理组显著下降，利于动物体内脂肪含量下降。
ob/ob小鼠肝脏组织脂肪代谢相关基因表达变化与脂肪组织类似。肝脏中相关脂合成基因FAS相对模型组显著降低，PKA、Adiponectin、LPL显著升高(图9)，表明CLA也可抑制肝脏细胞内脂肪合成，增强脂肪分解代谢，促进肥胖小鼠体重下降。
本发明如图8、图9所示，进一步结合肝脏和脂肪的基因分析，白色脂肪中相关脂代谢基因LPL、Plin5、UCP1、AP2的表达，相对模型组，CLA组显著升高，说明CLA显著促进TG分解、白色脂肪产热；肝脏中相关脂代谢基因FAS相对模型组显著降低，PKA、Adiponectin、LPL显著升高，说明CLA显著促进肝脏脂质氧化分解，减少脂质蓄积。采用不同剂量的CLA干预ob/ob小鼠，可显著降低小鼠体重，减少肝脏、脂肪组织脂质蓄积，减少脂肪酸合成、促进脂解、脂肪酸β氧化。不同剂量的CLA呈现出剂量效应。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (9)
1.共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用。
2.根据权利要求1所述的共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，其特征在于：所述共轭亚油酸异构体为c9，t11-CLA和t10，c12-CLA，化学结构式分别为式1、式2：
3.根据权利要求2所述的共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，其特征在于：所述共轭亚油酸异构体促进脂肪组织内LPL基因和UCP1基因表达，抑制FAS基因和ACC1基因表达。
4.根据权利要求2所述的共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，其特征在于：所述共轭亚油酸异构体促进肝脏内PKA、Adiponectin和LPL基因表达，抑制FAS基因表达。
5.根据权利要求2所述的共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，其特征在于：所述共轭亚油酸异构体能够减少内脏或脂肪组织脂质蓄积，减少脂肪酸合成、促进脂解和脂肪酸β氧化。
6.根据权利要求2所述的共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，其特征在于：所述共轭亚油酸异构体的有效使用剂量为100-400mg/Kg/d。
7.根据权利要求2所述的共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，其特征在于：所述共轭亚油酸异构体对于肥胖小鼠有显著的摄食抑制作用，降低小鼠的食欲。
8.根据权利要求6所述的共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，其特征在于：所述共轭亚油酸异构体对肥胖小鼠降低体重的作用呈剂量依赖性。
9.根据权利要求1所述的共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用，其特征在于：所述内脏为肝脏、肾脏、胰脏、胃和脾脏。
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