CN112370449A - 富含叶绿素的菠菜提取物在改善代谢紊乱和非酒精性脂肪肝中的用途 - Google Patents
富含叶绿素的菠菜提取物在改善代谢紊乱和非酒精性脂肪肝中的用途 Download PDFInfo
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Abstract
本发明提出了叶绿素在制备药物或食品中的用途，所述药物或食品用于预防或改善使用者的脂肪肝，所述使用者的脂肪肝与使用者肝脏中甘油三酯含量增加相关，叶绿素用于抑制肝脏中甘油三酯含量增加。叶绿素可以有效地抑制肝脏中甘油三酯含量增加，进而，通过对肝脏中甘油三酯含量增加的使用者施用叶绿素，可以有效地起到预防或改善脂肪肝的作用。由此，为叶绿素在防治脂肪肝方面奠定了科学研究和应用基础。
Description
技术领域
本发明涉及食品和医药领域。具体地，本发明涉及富含叶绿素的菠菜提取物在改善代谢紊乱和非酒精性脂肪肝中的用途。
背景技术
随着人民生活水平的提高，高脂肪、高热量的膳食结构，多坐少动的生活方式导致的疾病日益增多，其中高脂血症和脂肪肝的发病率呈逐年增高的趋势，且发病年龄日趋年轻化，正严重威胁国人的健康。脂肪肝是指由于各种原因引起的肝细胞内脂肪堆积过多的病变，是一种常见的肝脏病理改变，而非一种独立的疾病，一般分为酒精性脂肪肝和非酒精性脂肪肝(NAFLD)两大类。NAFLD通常与肥胖和某些饮食习惯有关。如有研究表明摄入大量动物蛋白的人群肝脏中可能有更多的脂肪，这类人群患NAFLD的风险也更高。NAFLD是可逆性病变，早期诊断和治疗一般可恢复正常。而饮食是NAFLD最基本、最便捷的干预和治疗措施。
叶绿素是一类广泛存在于植物中，位于植物类囊体膜上的可进行光合作用的色素，主要分为叶绿素a和叶绿素b两种，在褐藻、硅藻等藻类中还存在叶绿素c和叶绿素d。该类色素在日常膳食中容易获得。然而，目前并未发现关于膳食叶绿素摄入对于NAFLD干预方式的研究报道，若是通过叶绿素膳食干预能够降低居民高脂血症和脂肪肝的发病率，对提出有效的膳食指导，保障居民健康具有重要意义。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题。
本发明提出了叶绿素在制备药物或食品中的用途。根据本发明的实施例，所述药物或食品用于预防或改善使用者的脂肪肝，所述脂肪肝与使用者肝脏中甘油三酯含量增加相关，叶绿素用于抑制肝脏中甘油三酯含量增加。
已有研究表明并非所有的脂肪肝使用者肝脏中甘油三酯含量均增加(高于正常水平)，一些脂肪肝使用者表现为甘油三酯含量正常。本发明的发明人研究发现，叶绿素可以有效地抑制肝脏中甘油三酯含量增加，进而，通过对肝脏中甘油三酯含量增加的使用者施用叶绿素，可以有效地起到预防或改善脂肪肝的作用。由此，为叶绿素在防治脂肪肝方面奠定了科学研究和应用基础。
根据本发明的实施例，上述叶绿素在制备药物或食品中的用途还可以具有下列附加技术特征：
根据本发明的实施例，所述脂肪肝与使用者肝脏组织中脂质沉积相关，叶绿素用于抑制肝脏组织中脂质沉积。本发明的发明人发现，叶绿素可以有效地抑制肝脏组织中脂质沉积，进而，通过对肝脏组织中脂肪沉积的脂肪肝使用者服用叶绿素，可以有效地起到预防和改善脂肪肝的作用。
根据本发明的实施例，所述脂肪肝与使用者血清中甘油三酯浓度、总胆固醇浓度、低密度脂蛋白浓度和/或游离脂肪酸浓度升高相关，叶绿素用于抑制使用者血清中甘油三酯浓度、总胆固醇浓度、低密度脂蛋白浓度和游离脂肪酸浓度升高。
研究报道，有些患有脂肪肝的使用者的血清中甘油三酯浓度、总胆固醇浓度、低密度脂蛋白浓度和/或游离脂肪酸浓度高于正常水平。本发明的发明人发现，叶绿素可以有效地抑制血清中甘油三酯浓度、总胆固醇浓度、低密度脂蛋白浓度和游离脂肪酸浓度升高，进而，通过对血清中甘油三酯浓度、总胆固醇浓度、低密度脂蛋白浓度和/或游离脂肪酸浓度升高的脂肪肝使用者服用叶绿素，可以有效地起到预防和改善脂肪肝的作用。
根据本发明的实施例，所述脂肪肝与使用者肝脏组织中谷丙转氨酶和/或乳酸脱氢酶浓度升高相关，叶绿素用于抑制使用者肝脏组织中谷丙转氨酶和乳酸脱氢酶浓度升高。
研究报道，有些患有脂肪肝的使用者的肝脏组织中谷丙转氨酶和/或乳酸脱氢酶浓度高于正常水平。本发明的发明人发现，叶绿素可以有效地抑制肝脏组织中谷丙转氨酶和乳酸脱氢酶浓度升高，进而，通过对肝脏组织中谷丙转氨酶和/或乳酸脱氢酶浓度升高的脂肪肝使用者施用叶绿素，可以有效地起到预防和改善脂肪肝的作用。
根据本发明的实施例，所述使用者的FABP4基因、FATP1基因、CD36基因、FAS基因、SCD1基因、SREBP-1C基因、Dgta2基因和/或PPAR-γ基因表达量高于正常水平，叶绿素用于抑制FABP4基因、FATP1基因、CD36基因、FAS基因、SCD1基因、SREBP-1C基因、Dgta2基因和PPAR-γ基因的表达。
本发明的发明人发现，叶绿素可以有效地抑制FABP4基因、FATP1基因、CD36基因、FAS基因、SCD1基因、SREBP-1C基因、Dgta2基因和PPAR-γ基因的表达，进而，通过对FABP4基因、FATP1基因、CD36基因、FAS基因、SCD1基因、SREBP-1C基因、Dgta2基因和/或PPAR-γ基因表达量高于正常水平的脂肪肝使用者服用叶绿素，可以有效地起到预防和改善脂肪肝的作用。
根据本发明的实施例，所述脂肪肝与PPAR信号通路中EHHADH蛋白、FABP5蛋白、CYP4A蛋白、SCD1蛋白、PLIN2蛋白、ACSL4蛋白和/或PLIN5蛋白表达高于正常水平相关，叶绿素用于下调PPAR信号通路中EHHADH蛋白、FABP5蛋白、CYP4A蛋白、SCD1蛋白、PLIN2蛋白、ACSL4蛋白和PLIN5蛋白的表达。本发明的发明人发现，叶绿素可以有效地下调PPAR信号通路中EHHADH蛋白、FABP5蛋白、CYP4A蛋白、SCD1蛋白、PLIN2蛋白、ACSL4蛋白和PLIN5蛋白的表达，进而，通过对PPAR信号通路中EHHADH蛋白、FABP5蛋白、CYP4A蛋白、SCD1蛋白、PLIN2蛋白、ACSL4蛋白和/或PLIN5蛋白表达高于正常水平的脂肪肝使用者服用叶绿素，可以有效地起到预防和改善脂肪肝的作用。
根据本发明的实施例，所述脂肪肝为非酒精性脂肪肝。叶绿素对非酒精性脂肪肝具有更好的预防或改善效果。
根据本发明的实施例，叶绿素来源于绿色蔬菜和水果，优选菠菜、芹菜、油麦菜、油菜、猕猴桃。上述果蔬富含大量叶绿素，方便获得，成本低。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的小鼠肝重分析示意图；
图2显示了根据本发明一个实施例的肝脏中甘油三酯含量分析示意图；
图3显示了根据本发明一个实施例的肝脏HE染色图和油红O染色图；
图4显示了根据本发明一个实施例的小鼠肝脏脂代谢相关基因的相对表达水平分析示意图；
图5显示了根据本发明一个实施例的蛋白质组学分析示意图；
图6显示了根据本发明一个实施例的PPAR信号通路关键蛋白的Western Blot分析示意图。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实验方法
1、叶绿素提取及含量的测定
取新鲜菠菜，洗净沥干，加入搅拌机(Wiggens MB800，德国)中并倒入适量95％的乙醇，搅拌破碎。取6只300mL离心杯，按料液比1:3，各加入50g菠菜碎渣和150mL乙醇，于4℃、8000g的条件下离心10min，取上清液，将剩下的菠菜渣继续不断提取离心，直至离心杯内菠菜渣成灰白色。将收集的离心上清液倒入旋转瓶中，36℃进行旋转蒸发，浓缩至原体积1/4时倒入收集瓶中，置于4℃冰箱冷藏12h，直到底部为深绿色沉淀，顶部为黄色液体。取下层深绿色沉淀在4℃下使用Millipore膜(0.22μm)过滤，并保存在-20℃下直至分析和使用(全部实验在避光条件下进行)。
用移液枪吸取1mL叶绿素提取液，梯度稀释100倍或1000倍后，分别测定波长645、663nm处的吸光值，以80％丙酮为对照，全部实验在避光条件下进行。利用Arnon公式计算叶绿素含量：
叶绿素a浓度(mg/L)：Ca＝12.71×A663-2.59×A645
叶绿素b浓度(mg/L)：Cb＝22.88×A645-4.67×A663
叶绿素总浓度(mg/L)：C＝Ca+Cb＝8.04×A663+20.29×A645
等效剂量换算参照黄继汉等(2004)的方法，目标样品叶绿素浓度为9mg/mL。
2、动物分组及处理
选择3周龄C57BL/6N雄性小鼠，饲养于中国农业大学西校区实验动物平台SPF级鼠房(室温25±2℃，相对湿度45％-65％，12h昼夜明暗交替)，适应性喂养7d后随机分成4组，每组12只，4只/笼进行饲养。第一组为对照组(NC)，食用对照饲料(10％kcal,D12450B；Research Diets)，灌胃生理盐水；第二组为对照+叶绿素干预组(NCCHL)，食用对照饲料(10％kcal,D12450B；Research Diets)，灌胃叶绿素(0.18mg/10g小鼠体重，溶解于生理盐水)；第三组为高脂组(HFD)，食用高脂饲料(60％kcal，D12492；Research Diets)，灌胃生理盐水；第四组为高脂+叶绿素干预组(HFCHL)，食用高脂饲料(60％kcal,D12492；ResearchDiets)，灌胃叶绿素(0.18mg/10g小鼠体重，溶解于生理盐水)。灌胃13周后收集粪便，禁食6h后，将小鼠从笼中取出，轻放于实验台上，用消毒的镊子摘取小鼠眼球，采集血液。采用断颈法处死小鼠，采集小鼠肝脏，称重后立即投入液氮，并迅速储存于-80℃直至后续实验。
3、肝脏组织甘油三酯检测
称取20mg新鲜肝脏组织，按照组织：生理盐水＝1:9的比例研磨组织制成肝脏组织匀浆液，低速离心收取上层液体弃去下层未破碎完全的组织，加入相同体积的氯仿甲醇的混合液(氯仿：甲醇＝2:1)，剧烈震荡后置于-20℃冰箱静置过夜，第二天高速离心后，小心收取下层样品(T1)，置于冰箱保存，上层液体部分继续加入相同体积的氯仿甲醇的混合液剧烈震荡后置于-20℃冰箱静置过夜，第三天小心收取下层样品(T2)加入到T1管得到T管。将T管中收集到的所有样品进行氮吹干燥后用相同体积氯仿甲醇的混合液以充分溶解甘油三酯，最后使用试剂盒进行甘油三脂含量检测。
4、小鼠血清中血生化参数的测定
将小鼠全血在4000rpm的速度下离心，以收取小鼠血清，随后采用全自动血生化分析仪测定血清中游离脂肪酸(FFA)、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)浓度。
5、小鼠肝脏组织的HE染色
切取部分肝组织，将其置入4％的多聚甲醛中进行固定，固定48h，随后进行石蜡包埋，切片，后续进行苏木精-伊红染色实验后采用显微镜镜检，图像采集分析。具体实验步骤为：(1)脱水。依次用浓度递增的酒精浸泡组织块：85％乙醇(30min)、95％乙醇(30min)、100％乙醇(1h)、100％乙醇(1h)；然后用二甲苯浸泡组织块直至组织块完全透明；(2)浸蜡。将已透明的组织块置于已溶化的石蜡中，放入溶蜡箱保温2h，直至石蜡完全浸入组织块；(3)包埋。在包埋盒倒入石蜡包埋组织块，待石蜡冷却凝固成块；(4)切片、展片和烤片。将蜡块固定于切片机上，切成5μm的薄片。切下的薄片放到42℃水中展平后贴到载玻片上，放37℃恒温箱中烘干过夜。然后在60℃烤片1h；(5)脱蜡、复水。用二甲苯脱去切片中的石蜡，再依次浸泡在浓度递减的酒精：100％乙醇(5min)、95％乙醇(5min)、70％乙醇(5min)、50％乙醇(5min)，最后放入蒸馏水；(6)HE染色。将切片放入苏木精水溶液中染色2min，用自来水冲洗2次，每次2min，在显微镜下观察细胞核被染成蓝色；将切片浸泡在1％盐酸分化5s脱掉多余的染色剂，用自来水冲洗切片2次，每次2min；随后将切片放在伊红染色剂2min，用自来水冲洗2次，每次2min；(7)脱水。依次用以下试剂浸泡切片：70％酒精(5s)、95％酒精(1min)、100％酒精(5min)、二甲苯(10min)；(8)封片。将已透明的切片滴上树胶，盖上盖玻片封固，晾干后观察。
6、小鼠肝脏组织油红O染色
快速采集新鲜的组织后，放入加有适量OCT包埋剂的铝箔纸小盒中，然后将特制小盒缓缓平放入液氮中迅速冻结。-20℃温度下连续切片至4-8μm后于室温放置30min，然后4℃丙酮固定5-10min，烘箱干燥20min后用PBS清洗3次，然后3％H2O2孵育5-10min。随后进行染色，图像观察。
7、基因表达检测
参照北京天根生化科技有限公司Trizol Reagent总RNA提取试剂盒提取各组织总RNA，cDNA合成后进行实时荧光定量PCR检测。
8、组织蛋白免疫印迹
组织蛋白提取及蛋白浓度测定后进行SDS-PAGE凝胶电泳，随后转膜及封闭，抗体孵育，ECL显影。
实施例1叶绿素对NAFLD小鼠肝重、肝脏中甘油三酯含量及肝脏组织病理学的影响
为了确定富含叶绿素的菠菜提取物对NAFLD的干预作用，首先测定了富含叶绿素的菠菜提取物对NAFLD小鼠肝重和肝脏中甘油三酯含量的影响。由图1和图2所示，高脂补充富含叶绿素的菠菜提取物(HFCHL)可有效缓解高脂饮食(HFD)诱导的小鼠肝脏重量增加，并对甘油三酯含量增加表现出抑制作用，而对照饮食灌胃富含叶绿素的菠菜提取物组(NCCHL)与对照组(NC)之间体重增长量无显著差别。
肝脏的HE(图3(A))染色可以看出，高脂模型组小鼠肝细胞明显增大，其中充斥着脂质，形成脂肪囊肿，伴有低程度的炎症和坏死。富含叶绿素的菠菜提取物干预后表现出明显缓解作用，肝细胞几乎趋于正常状态。肝脏油红O染色结果(图3(B))也表现出富含叶绿素的菠菜提取物可显著降低肝脏中脂质沉积。综合以上结果，富含叶绿素的菠菜提取物可以有效降低肝脏组织中脂质的沉积，对肝脏脂肪变性有良好的抑制作用。
实施例2富含叶绿素的菠菜提取物对NAFLD小鼠血清的生化分析
长期的高脂饮食会诱导脂代谢紊乱，持续的血脂紊乱会加重肝脏负担，引起肝功能的损伤。通过全自动血生化分析仪对不同组别小鼠血清中与脂代谢和肝功能相关的参数进行检测。由表1可知，与对照组小鼠相比，高脂组小鼠表现为血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白(LDL)和游离脂肪酸(FFA)浓度的升高，而富含叶绿素的菠菜提取物干预可显著降低高脂饮食小鼠血清中TG、TC、LDL、FFA的浓度，说明富含叶绿素的菠菜提取物对高脂饮食导致的肝脏脂代谢异常具有缓解作用。谷丙转氨酶(ALT)和乳酸脱氢酶(AST)主要存在于肝细胞中，当肝组织受损造成肝细胞死亡时会导致其从肝细胞释放至血液中，因此可反映肝组织受损状况。高脂模型小鼠也表现出明显的肝功能损伤，表现为ALT和AST的升高。经过富含叶绿素的菠菜提取物干预后，ALT和AST浓度均表现出不同程度的降低，说明富含叶绿素的菠菜提取物对高脂饮食导致的肝功能损伤也有显著的缓解作用。
表1叶绿素补充对小鼠血生化参数的影响
数据以平均值±标准差的形式展示。*p<0.05,**p<0.01,***p<0.001,vs.正常饮食小鼠；&p<0.05,&&p<0.05,&&&p<0.001,vs.高脂膳食小鼠。
实施例3富含叶绿素的菠菜提取物对NAFLD小鼠肝脏组织中脂肪合成转运基因的变化
脂肪酸合成酶(FAS)是参与脂肪酸合成过程中的重要酶之一，其作用是催化乙酰CoA及丙二酰CoA合成长链脂肪酸。乙酰辅酶A羧化酶(ACC)属于分子间羧化转移家族，在脂代谢过程起重要作用，其中ACACA是调节长链脂肪酸合成的限速酶，可催化乙酰辅酶A转化为脂肪酸合成起始底物丙二酸单酰辅酶A，此酶是调节脂肪合成的关键酶。FAS和ACACA是调控脂肪酸从头合成的两个重要的酶。硬脂酰辅酶A去饱和酶1(SCD1)是位于内质网上的一种合成单不饱和脂肪酸的限速酶，研究表明SCD1缺陷小鼠的脂质代谢和胰岛素敏感性均得到显著改善。二脂酰甘油酰基转移酶(DGAT2)是甘油三酯合成的关键酶和限速酶，参与脂肪吸收，血浆中甘油三酯浓度的调节及肝脏中脂肪的合成与储存。固醇调节元件结合蛋白(SREBP-1C)是一种内质网蛋白，具有膜连接的作用，SREBP-1C调控ACC、FAS、ATP柠檬酸裂解酶等脂肪合成酶基因的表达，调节胆固醇和脂肪酸的合成及摄取。PPAR是过氧化物酶体增殖物激活受体，是脂肪酸的感受器，是脂肪酸氧化酶基因的主要转录调控因子，有报道指出PPAR-γ与脂质代谢紊乱具有一定的相关性。
为探究富含叶绿素的菠菜提取物缓解NAFLD发生的机制，本发明对小鼠肝脏脂代谢相关基因的相对表达水平进行测定。由图4可知，HFD组小鼠肝脏的脂肪转运合成代谢相关基因如FABP4、FATP1、CD36、FAS、SCD1、SREBP-1C、Dgta2和PPAR-γ显著高于NC和NCCHL组，而HFCHL组中相关基因表达趋于正常。综上所述，富含叶绿素的菠菜提取物可通过调控小鼠肝脏组织中脂肪转运合成和代谢基因表达，进而改善NAFLD。
实施例4蛋白质组学揭示富含叶绿素的菠菜提取物调控PPAR信号通路缓解小鼠非酒精性脂肪肝
为进一步探究富含叶绿素的菠菜提取物如何调控肝脏的脂代谢过程缓解NAFLD，本发明进行了肝脏蛋白质组学分析。差异蛋白分析结果发现，富含叶绿素的菠菜提取物可以调控脂代谢过程，主要介导PPAR信号通路(图5)。蛋白质组学结果发现富含叶绿素的菠菜提取物可显著下调PPAR信号通路中EHHADH、FABP5、CYP4A、SCD1、PLIN2、ACSL4、PLIN5关键蛋白起到缓解NAFLD的作用(图6)。因此，富含叶绿素的菠菜提取物可有效调控肝脏脂质代谢从而预防性地减轻肝脂肪化程度，预防性缓解NAFLD的发生发展。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (8)
1.叶绿素在制备药物或食品中的用途，其特征在于，所述药物或食品用于预防或改善使用者的脂肪肝，所述脂肪肝与使用者肝脏中甘油三酯含量增加相关，叶绿素用于抑制肝脏中甘油三酯含量增加。
2.根据权利要求1所述的用途，其特征在于，所述脂肪肝与使用者肝脏组织中脂质沉积相关，叶绿素用于抑制肝脏组织中脂质沉积。
3.根据权利要求1所述的用途，其特征在于，所述脂肪肝与使用者血清中甘油三酯浓度、总胆固醇浓度、低密度脂蛋白浓度和/或游离脂肪酸浓度升高相关，叶绿素用于抑制使用者血清中甘油三酯浓度、总胆固醇浓度、低密度脂蛋白浓度和游离脂肪酸浓度升高。
4.根据权利要求1所述的用途，其特征在于，所述脂肪肝与使用者肝脏组织中谷丙转氨酶和/或乳酸脱氢酶浓度升高相关，叶绿素用于抑制使用者肝脏组织中谷丙转氨酶和乳酸脱氢酶浓度升高。
5.根据权利要求1所述的用途，其特征在于，所述使用者的FABP4基因、FATP1基因、CD36基因、FAS基因、SCD1基因、SREBP-1C基因、Dgta2基因和/或PPAR-γ基因表达量高于正常水平，叶绿素用于抑制FABP4基因、FATP1基因、CD36基因、FAS基因、SCD1基因、SREBP-1C基因、Dgta2基因和PPAR-γ基因的表达。
6.根据权利要求1所述的用途，其特征在于，所述脂肪肝与PPAR信号通路中EHHADH蛋白、FABP5蛋白、CYP4A蛋白、SCD1蛋白、PLIN2蛋白、ACSL4蛋白和/或PLIN5蛋白表达高于正常水平相关，叶绿素用于下调PPAR信号通路中EHHADH蛋白、FABP5蛋白、CYP4A蛋白、SCD1蛋白、PLIN2蛋白、ACSL4蛋白和PLIN5蛋白的表达。
7.根据权利要求1所述的用途，其特征在于，所述脂肪肝为非酒精性脂肪肝。
8.根据权利要求1所述的用途，其特征在于，叶绿素来源于绿色蔬菜和水果，优选菠菜、芹菜、油麦菜、油菜、猕猴桃。
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