CN111346084A - 共轭亚油酸甘油酯在制备治疗高脂饮食诱导的非酒精性脂肪肝的产品中的应用 - Google Patents
共轭亚油酸甘油酯在制备治疗高脂饮食诱导的非酒精性脂肪肝的产品中的应用 Download PDFInfo
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- CN111346084A CN111346084A CN202010200976.9A CN202010200976A CN111346084A CN 111346084 A CN111346084 A CN 111346084A CN 202010200976 A CN202010200976 A CN 202010200976A CN 111346084 A CN111346084 A CN 111346084A
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- Prior art keywords
- linoleic acid
- acid glyceride
- conjugated linoleic
- fatty liver
- cla
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Abstract
本发明涉及制备一种共轭亚油酸甘油酯及其微囊粉在制备预防治疗高脂饮食诱导的非酒精性脂肪肝食品或药品的用途。所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉在制备预防、缓解、治疗高脂饮食诱导的非酒精性脂肪肝的食品、保健品或药物中的应用。本发明应用CLA‑TG预防、缓解非酒精性脂肪肝，涉及的动物疾病模型为HFD诱导的非酒精性脂肪肝模型。其功效特征为体重、脂体比降低，血液ALT、AST、肝脏TG含量降低及肝脏脂滴积聚现象的缓解；其效果较好，安全性高，产品易于获得。
Description
技术领域
本发明涉及制备一种共轭亚油酸甘油酯及其微囊粉在制备预防治疗高脂饮食诱导的非酒精性脂肪肝食品或药品的用途。
背景技术
随着社会的进步及生活水平的提高，国民饮食结构逐渐失衡。高脂食品的大量摄入，引起了多种代谢疾病，正严重威胁着国人健康。其中，脂肪性肝病由于其较高的发病率和逐年的低龄化，成为了近期研究热点。脂肪性肝病可分为酒精性脂肪性肝病与非酒精性脂肪性肝病。其中，非酒精性脂肪肝(NAFLD)作为全球范围内发病率最高的一种肝脏代谢类疾病，发病机制复杂，较经典的理论为“二次打击”学说，其中“初次打击”是由机体胰岛素抵抗首先导致肝脂肪变，包括肝脏脂肪积聚、脂代谢紊乱，使肝对外界刺激敏感性增加。“二次打击”主要是各种致病因素而引发的氧化应激使反应性氧化物和脂质过氧化物增多，进而引起细胞炎性因子释放增加，促进肝细胞发展为炎症和病变坏死。因此，脂质代谢异常、细胞炎症反应和细胞凋亡等因素在NAFLD的发展中均发挥至关重要的作用。据统计，NAFLD患者中存在5％-10％的肝脂肪变性，严重者可发展为非酒精性脂肪性肝炎、肝纤维化，最终导致肝硬化、肝癌，同时，临床上NAFLD患者可能还伴有肥胖、患有胰岛素抵抗或2型糖尿病、血脂异常、高甘油三酸酯血症和高血压等危险因素，引起代谢综合征高发病率。目前对NAFLD的预防治疗并没有明确真正有效的治疗药物上市，主要以祛除诱因、调整饮食为主，但临床研究发现，由于NAFLD发病机制的复杂性，仅通过摄入减肥降脂功效的食品药品无法显著缓解所有类型的NAFLD患者的肝损伤。因此，对具有肝保护效应且副作用小或无副作用，用于预防、控制或治疗NAFLD的功能食品或药物的研究是有必要的。
共轭亚油酸甘油酯(CLA-TG)是一种天然的具有共轭双键的十八碳二烯酸异构体的新型功能性脂肪酸甘油酯衍生物，合成成本低，营养价值高，稳定性、气味较好，具有免疫调节、抗癌、抗动脉粥样硬化、抗氧化、抗过敏、影响骨形成等多方面生物学功能。研究表明，CLA-TG可以抑制脂肪酸合成酶(FAS)基因的表达，改善线粒体功能，上调脂肪动员和分解相关酶的表达，影响脂肪酸的摄取和氧化及脂质的合成代谢，进而其仅是单纯发挥减肥、降脂、降糖、提高肌内脂肪含量,改变脂肪酸组成等作用，应用于减肥产品中；并不能调节、改善脂肪性肝病的相应机能，并且在现有报道中，也并未见CLA-TG在预防治疗非酒精性脂肪肝中的应用。由此，CLA-TG在食品、药品领域中具有潜在的应用价值和广阔的市场前景。
同时，现有相关治疗脂肪性肝病的相关组合物，大多为中药混合物、不饱和脂肪酸混合物或某些天然成分提取物等。如：CN106420901公布了一种由皂角刺、生白术、茵陈蒿按一定配比制成的复方中药，一定程度上能缓解高脂饮食引起的脂肪肝；CN102698206B制备了一种由青皮、郁金、白术、山药、茯苓、泽泻等组成的组合物，该组合物在疏肝、健脾、利湿、活血中具有良好的应用，并能有效缓解非酒精性脂肪肝损伤；但中药成分服用时间长，用药依从性较低，且生物利用度存在缺陷，长期服用可能存在一定安全性问题。CN102755350阐述一种海狗油精制方法，得到的产品对缓解非酒精性脂肪肝有较好的作用，但原材料价格高昂，在使用中同样存在局限性。
发明内容
本发明目的在于一种共轭亚油酸甘油酯及其微囊粉在制备预防或治疗高脂饮食(HFD)诱导的非酒精性脂肪肝的功能食品或药物中的应用。
为实现上述目的，本发明采用技术方案为：
一种共轭亚油酸甘油酯在预防、缓解、治疗高脂饮食诱导的非酒精性脂肪肝中的应用。
所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉在制备预防、缓解、治疗高脂饮食诱导的非酒精性脂肪肝的食品、保健品或药物中的应用。
所述共轭亚油酸甘油酯由亚油酸短链醇酯在一定反应条件下以C1-4醇钠/钾为催化剂，经转位反应制得。
所述共轭亚油酸甘油酯的微囊粉由共轭亚油酸甘油酯经混合水相、油相进行剪切乳化、喷雾、干燥即得。
所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉的服用量以共轭亚油酸甘油酯含量计为0.06-0.20g/kg·BW/天。
所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉服用方式为口服。
所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉作为活性成分制成固体饮品、代餐奶昔、乳饮料、烘焙食品或营养棒的剂型。
本发明所具有的优点：
本发明应用CLA-TG预防、缓解非酒精性脂肪肝，涉及的动物疾病模型为HFD诱导的非酒精性脂肪肝模型。其功效特征为体重、脂体比降低，血液ALT、AST、肝脏TG含量降低及肝脏脂滴积聚现象的缓解；其效果较好，安全性高，产品易于获得。
本发明CLA-TG或CLA-TG微囊粉能够在不影响摄食量的前提下可以减少血液中ALT、AST含量，肝脏中TG含量及肝脏脂滴积聚，从而减轻HFD诱导的非酒精性脂肪肝。且微囊化的CLA-TG具有水溶性，便于应用具有更好的效果；为临床预防或治疗脂肪性肝病提供了一种新的选择。
附图说明
图1A为6周后基础饲料对照组、高脂饲料对照组、CLA-TG组、CLA-TG微囊粉组大鼠的体重统计图。
图1B为6周后基础饲料对照组、高脂饲料对照组、CLA-TG组、CLA-TG微囊粉组大鼠的体重增重统计图。
图1C为造模饲养期间基础饲料对照组、高脂饲料对照组、CLA-TG组、CLA-TG微囊粉组大鼠的摄食量统计图。
图1D为6周后基础饲料对照组、高脂饲料对照组、CLA-TG组、CLA-TG微囊粉组大鼠的脂体比统计图。
图2A为6周后基础饲料对照组、高脂饲料对照组、CLA-TG组、CLA-TG微囊粉组大鼠血清谷丙转氨酶含量统计图。
图2B为6周后基础饲料对照组、高脂饲料对照组、CLA-TG组、CLA-TG微囊粉组大鼠血清谷草转氨酶含量统计图。
图3为6周后基础饲料对照组、高脂饲料对照组、CLA-TG组、CLA-TG微囊粉组大鼠肝脏甘油三酯含量统计图。
图4A为6周后基础饲料对照组、高脂饲料对照组、CLA-TG组、CLA-TG微囊粉组大鼠肝脏组织进行油红染色分析(40×)；
图4B为对油红O染色评分结果；
具体实施方式
以下结合实例对本发明的具体实施方式做进一步说明，应当指出的是，此处所描述的具体实施方式只是为了说明和解释本发明，并不局限于本发明。
在本专利提供的CLA-TG在预防缓解高脂饮食诱导的非酒精性脂肪肝方面的用途中，CLA-TG除对非酒精性脂肪肝显著的缓解作用外，其优势还在于其较高的安全性及服用依从性，可为非酒精性脂肪肝的预防及治疗提供较好的选择。
以下实施例中动物实验所述CLA-TG及CLA-TG微囊粉灌胃样品的获得方式为将其分散于0.2％羧甲基纤维素钠溶液中，二者剂量按CLA-TG含量计为0.375g/kg配制。
以下实施例中所述CLA-TG微囊粉均是由CLA-TG经混合水相、油相进行剪切乳化和喷雾干燥等步骤制得的CLA-TG微囊粉。[详见申请号为201810772425.2记载文献]
具体为：
(1)准备原料物CLA-TG 60-85份、淀粉或胶质5-35份、小分子填充物4-20份、抗氧化剂0.01-5份；
(2)50-60℃下将淀粉或胶质、小分子填充物溶.解于水，然后体系降温至0-30℃；
(3)将水相抗氧化剂加入到(2)的体系中，0-30℃搅拌至完全溶解，得水相；
(4)将油相抗氧化剂加入共轭亚油酸甘油酯中，10-25℃搅拌至完全溶解，得油相；
(5)将油相加入到水相中，控制体系温度0-30℃条件下，剪切乳化0.5-2h，然后40-120MPa高压均质1-3次，得乳液；
(6)将步骤(5)所制备的乳液进行喷雾干燥：进风温度90-230℃，出风温度50-110℃。
步骤(1)中所述的淀粉包括原淀粉或变性淀粉，其中，变性淀粉选自酸变性淀粉、氧化淀粉、辛烯基琥珀酸淀粉酯、辛烯基琥珀酸淀粉钠、醋酸酯淀粉、磷酸酯淀粉、交联淀粉、羟丙基淀粉、预糊化淀粉中的一种或几种的混合物；
步骤(1)中所述的胶质选自黄原胶、明胶、阿拉伯胶中的一种或几种的混合物。
步骤(1)中所述的小分子填充物选自葡萄糖浆、麦芽糊精、低聚麦芽糖、低聚果糖、抗性糊精、固体玉米糖浆、环糊精等。
步骤(1)中所述的抗氧化剂包括水相抗氧化剂和油相抗氧化剂，其中，水相抗氧化剂选自抗坏血酸钠、抗坏血酸、柠檬酸、柠檬酸钠和抗坏血酸棕榈酸酯；油相抗氧化剂选自d-α生育酚、dl-α生育酚、混合生育酚、迷迭香提取物、磷脂、丁基羟基茴香醚、抗氧剂264、特丁基对苯二酚中的一种或几种的混合物。
本发明将SD大鼠按体重随机分成4组，分别为空白对照组、高脂饲料组、CLA-TG组及CLA-TG微囊粉组。其中，空白对照组喂养基础饲料；其余各组喂养高脂饲料，并按分组要求灌胃。
其中，基础饲料能量配比为64.5％碳水化合物，13.3％脂肪，22.2％蛋白质；
高脂饲料能量配比为71％脂肪，11％碳水化合物及18％蛋白质；
实验时间6周。每周记录给食量、撒食量、剩食量。6周实验周期结束后，称体重及解剖取肾周围脂肪、睾丸周围脂肪，称重计算脂/体比；取腹主动脉血及肝脏组织，得到血清样品，制备肝冰冻切片及肝匀浆，通过检测体重、体重增重、脂体比及摄食量；血清ALT、AST含量、肝脏TG含量及肝切片油红O染色，探究CLA-TG及CLA-TG微囊粉对HFD诱导的非酒精性脂肪肝的缓解作用。
以下实施例检测数据统计方式采用GraphPad Prism5.0统计软件进行统计学分析，计量资料以均数±标准偏差(mean±SEM)表示，组间比较用单因素方差分析(one-wayANOVA)，P<0.05视为差异有统计学意义。与正常对照组相比，**P<0.01，***P<0.001；与高脂对照组相比，#P<0.05，##P<0.01
实施例1：CLA-TG对HFD诱导的非酒精性脂肪肝大鼠体重、体重增重、脂体比及摄食量影响
(一)实验材料
(1)实验动物：
品系：SD大鼠，雄性，SPF级；
来源：北京维通利华实验动物技术有限公司；
许可证号：SCXK(京)2016-0006，实验动物质量合格证编号：No.1100111911028450
体重：160-180g
每组动物数：10只
受试物：CLA-TG及CLA-TG微囊粉
(二)实验方法
将SD大鼠按体重随机分成4组，分别为空白对照组、高脂饲料组、CLA-TG组及CLA-TG微囊粉组。其中，空白对照组喂养基础饲料；其余各组喂养高脂饲料，并按分组要求灌胃。
实验时间6周。每周记录给食量、撒食量、剩食量。6周实验周期结束后，称体重。1％戊巴比妥钠(0.5ml/100g·BW)麻醉，解剖，取腹主动脉取血5ml及肝脏待用，取肾周围脂肪、睾丸周围脂肪，并称重，计算脂体比(参见图1A-D)。
(三)实验结果
如图1A-C所示，整个试验期内各组大鼠生长良好，无腹泻及死亡现象且均未出现异常行为。在第6周末，高脂饲料饲养组与基础饲料饲养组相比，体重、体重增重显著上升(P<0.01)CLA-TG组及CLA-TG微囊粉组有效抑制了体重增长(P<0.05)。且6周内，大鼠摄食量并没有明显差异。
如图1D所示，在第6周末，高脂饲料饲养组与基础饲料饲养组相比，脂体比显著上升(P<0.001)，CLA-TG组及CLA-TG微囊粉组与高脂饲料饲养组相比脂体比下降(P<0.05)。
综上可见，CLA-TG及CLA-TG微囊粉能够在一定程度上缓解HFD诱导的非酒精性脂肪肝大鼠的体重、体脂增长。
实施例2：CLA-TG对HFD诱导的非酒精性脂肪肝大鼠的血液生化指标的影响
(一)实验材料
来自实施例1按照不同方式投喂后大鼠血液样本，谷丙转氨酶、谷草转氨酶检测试剂盒购自南京建成生物有限公司。
(二)实验方法
将得到的血液样本静置15min后，3000r/min，离心10min，得到血清样本。取20μl血清样本，分别按照南京建成生物有限公司谷丙转氨酶、谷草转氨酶试剂盒操作说明书进行检测。
(三)实验结果
如图2A-B所示，高脂饲料饲养小鼠ALT、AST与基础饲料饲养组相比有显著上升(P<0.001)，CLA-TG组及CLA-TG微囊粉组能够显著下调血清中ALT、AST含量(P<0.05)，证明CLA-TG能够缓解HFD诱导的非酒精性脂肪肝大鼠的肝损伤，且CLA-TG微囊粉组较CLA-TG组下降稍多。
实施例3：CLA-TG对HFD诱导的非酒精性脂肪肝大鼠的肝脏甘油三酯含量的影响
(一)实验材料
来自实施例1按照不同方式投喂后大鼠肝脏样本，甘油三酯试剂盒购自南京建成生物有限公司
(二)实验方法
取约50mg左右肝脏组织，制备10％肝匀浆。按照南京建成生物有限公司甘油三酯检测试剂盒操作说明书进行检测。
(三)实验结果
如图3所示，高脂饲料饲养大鼠肝脏甘油三酯水平与基础饲料饲养组相比有显著上升(P<0.001)，在CLA-TG组及CLA-TG微囊粉组，肝脏甘油三酯含量显著下调(P<0.05)，证明CLA-TG能够降低HFD诱导的非酒精性脂肪肝大鼠肝脏甘油三酯的含量，且CLA-TG微囊粉组较CLA-TG组下降稍多。
实施例4：肝切片油红O染色实验
(一)实验材料
来自实施例1按照不同方式投喂后大鼠肝脏组织；油红O染液试剂盒购自南京建成生物有限公司。
(二)实验方法
取少量肝组织制作冰冻切片，按油红O染液试剂盒说明书进行染色，光镜下观察切片肝细胞脂滴积聚情况。
(三)实验结果
如图4A-B所示，HFD诱导的非酒精性脂肪肝大鼠肝脏细胞中出现大量的脂滴积聚现象(P<0.001)，在CLA-TG微囊粉组，肝脏细胞中脂滴堆积情况有一定减轻(P<0.05)，证明CLA-TG微囊粉能够减轻脂肪肝细胞中脂滴堆积。
由上述各实施例可见，CLA-TG及CLA-TG微囊粉能够缓解HFD诱导的非酒精性脂肪肝损伤。
实施例5：CLA-TG对非酒精性脂肪肝人群治疗作用
(一)实验人群
招募了50例诊断为不同程度的脂肪肝患者志愿者进行产品试食实验，志愿者年龄为20-50岁。其中，有6人因不同原因退出实验。44名志愿者中有6名重度脂肪肝患者，18名中度脂肪肝患者，20名轻度脂肪肝患者。
受试物为CLA-TG微囊粉。
(二)实验方法
所有志愿者试食CLA-TG微囊粉，剂量为0.06g/kg·BW/天，服用时间为6周。6周实验周期结束后，对所有志愿者进行腹部超声检查，观察脂肪肝恢复情况。
(三)实验结果
在6例重度脂肪肝患者中，有3例变为中度脂肪肝，3例无明显改善，治愈率为50％；
在18例中度脂肪肝患者中，有12例变为轻度脂肪肝，6例无明显改善，治愈率为66.7％；
在20例轻度脂肪肝患者中，有18例变为正常，2例无明显改善，治愈率为90％。
以上数据证明CLA-TG微囊粉对脂肪肝有一定的治疗作用。
由上述数据可见本发明利用共轭亚油酸甘油酯及其微囊粉能够显著改善NAFLD大鼠肝脏脂质代谢、缓解肝脂肪变、减轻肝脏损伤，同时对各种程度的脂肪肝患者均有一定治疗作用，因此，可以认为本发明提供的共轭亚油酸甘油酯及其微囊粉对NAFLD有较好的预防治疗作用，可用于相关功能食品及药物的开发。
Claims (7)
1.一种共轭亚油酸甘油酯在预防、缓解、治疗高脂饮食诱导的非酒精性脂肪肝中的应用。
2.按权利要求1所述的应用，其特征在于：所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉在制备预防、缓解、治疗高脂饮食诱导的非酒精性脂肪肝的食品、保健品或药物中的应用。
3.按权利要求2所述的应用，其特征在于：所述共轭亚油酸甘油酯由亚油酸短链醇酯在一定反应条件下以C1-4醇钠/钾为催化剂，经转位反应制得。
4.按权利要求2或3所述的应用，其特征在于：所述共轭亚油酸甘油酯的微囊粉由共轭亚油酸甘油酯经混合水相、油相进行剪切乳化、喷雾、干燥即得。
5.按权利要求1或2所述的应用，其特征在于：所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉的服用量以共轭亚油酸甘油酯含量计为0.06-0.20g/kg·BW/天。
6.按权利要求1或5所述的应用，其特征在于：所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉服用方式为口服。
7.按权利要求6所述的应用，其特征在于：所述共轭亚油酸甘油酯或共轭亚油酸甘油酯的微囊粉作为活性成分制成固体饮品、代餐奶昔、乳饮料、烘焙食品或营养棒的剂型。
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