药学学报  2015, Vol. 50 Issue (3): 278-283   PDF    
露兜簕来源咖啡酰奎宁酸抑制HepG2肝细胞脂质积聚并调节脂代谢相关基因的表达
吴崇明1, 栾红1, 王帅1, 张小坡2, 刘海涛1, 郭鹏1     
1. 中国医学科学院药用植物研究所, 北京 100094;
2. 海南医学院药学院, 海南 海口 571101
摘要:前期研究证明, 露兜树 (Pandanus tectorius Soland.) 的果实露兜簕富含咖啡酰奎宁酸类成分并具有显著降血脂作用。为探究露兜簕发挥降脂作用的有效成分和潜在机制, 用油酸诱导的HepG2肝癌细胞脂质积聚模型对7种露兜簕来源的咖啡酰奎宁酸类成分进行了筛选。结果显示, 3-咖啡酰奎宁酸 (3-CQA)、3, 5-二咖啡酰奎宁酸 (3, 5-CQA) 和3, 4, 5-三咖啡酰奎宁酸 (3, 4, 5-CQA) 可显著降低HepG2肝细胞脂质积聚和细胞内总胆固醇和甘油三酯水平, 并且无显著细胞毒性。实时定量PCR结果表明, 这三种咖啡酰奎宁酸化合物能够显著抑制脂质合成相关基因的表达, 同时3-CQA和3, 5-CQA还可显著提高脂质氧化相关基因的表达。结果提示, 3-CQA、3, 5-CQA和3, 4, 5-CQA可能是露兜簕发挥降脂作用的主要活性成分, 它们可以通过调节脂代谢相关基因的表达发挥降脂活性。
关键词露兜簕     高脂血症     咖啡酰奎宁酸     绿原酸     脂代谢    
Pandanus tectorius derived caffeoylquinic acids inhibit lipid accumulation in HepG2 hepatoma cells through regulation of gene expression involved in lipid metabolism
WU Chong-ming1, LUAN Hong1, WANG Shuai1, ZHANG Xiao-po2, LIU Hai-tao1, GUO Peng1     
1. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100094, China;
2. School of Pharmacy, Hainan Medical University, Haikou 571101, China
Abstract: The fruit of Pandanus tectorius (PTF) has a long history of use as a folk medicine to treat hyperlipidemia in Hainan province, South China. Our previous studies have shown that the n-butanol extract of PTF is rich in caffeoylquinic acids and has an adequate therapeutic effect on dyslipidemic animals induced by high-fat diet. In this work, seven caffeoylquinic acids isolated from PTF were screened for the lipid-lowering activity in HepG2 hepatoma cells. Oil-Red O staining, microscopy and intracellular triglyceride (TG) and total cholesterol (TC) quantification showed that 3-O-caffeoylquinic acid (3-CQA), 3, 5-di-O-caffeoylquinic acid (3, 5-CQA), and 3, 4, 5-tri-O-caffeoylquinic acid (3, 4, 5-CQA) significantly inhibited lipid accumulation induced by oleic acid and decreased intracellular levels of TC and TG in a dose-dependent manner. These three caffeoylquinic acids showed no significant cytotoxicity at concentrations of 1-50 μmol·L-1 as determined by MTT assay. Realtime quantitative PCR revealed that 3-CQA and 3, 5-CQA significantly increased the expression of lipid oxidation-related genes PPARα, CPT-1 and ACOX1 while 3-CQA, 3, 5-CQA and 3, 4, 5-CQA decreased the expression of lipogenic genes SREBP-1c, SREBP-2, HMGR, ACC, FAS. Overall, 3-CQA, 3, 5-CQA and 3, 4, 5-CQA may be the principal hypolipidemic components in PTF which can decrease intracellular lipid accumulation through up-regulating the expression of lipid oxidative genes and down-regulating the expression of lipogenic genes.
Key words: Pandanus tectorius fruit     hyperlipidemia     caffeoylquinic acid     chlorogenic acid     lipid metabolism    

高脂血症 (hyperlipidemia) 是指脂肪代谢异常或脂肪转运异常而导致血清甘油三酯(TG)、总胆固醇 (TC)、低密度脂蛋白胆固醇 (LDL-C) 及总脂质等浓度超过正常标准,是诱发心脑血管疾病的高危因素之一[1, 2]。肝脏是脂质代谢的主要器官,多种基因和蛋白参与脂质代谢的调节。固醇调节元件结合蛋白 (sterol-regulatory element binding proteins,SREBPs) 及其下游基因脂肪酸合酶 (fatty acid synthase,FAS)、乙酰辅酶A羧化酶 (ACC)、羟甲基戊二酰辅酶A 还原酶 (3-hydroxy-3-methylglutaryl-CoA reductase,HMGR) 可促进体内胆固醇、脂肪酸及甘油三酯的合成和积聚,被称作成脂基因 (lipogenic genes)[3, 4]。相反,过氧化物酶体增殖物激活受体α (peroxisone proliferators-activated receptor alpha,PPARα) 及其下游基因乙酰辅酶A氧化酶 (ACOX) 和肉毒碱棕榈酰转移酶I (CPT-1) 等可以通过促进脂肪酸β氧化而降低脂质水平[5, 6]

随着现代人们生活水平的不断提高和生活方式的改变,由于不良饮食结构及不健康的生活习惯致使高脂血症患者与日俱增,并且正朝着年轻化的趋势发展,给人类健康造成巨大威胁[1,7]。虽然目前临床上常用的降血脂药物,如他汀类和贝特类降脂药物,可以对高血脂进行良好的控制,但是它们同时也会产生一些不良反应,例如胃肠道不适、失眠、恶心、转氨酶升高、肌痛等,长期服用这些药物会增加高脂血症患者的负担,因此顺应性较差[8, 9]。为了开发疗效高、副作用少的新型降血脂药物,近年来,人们已逐渐将注意力转移到临床上已经被证明有效的传统民族药物上来。

露兜簕为露兜树科 (Pandanaceae) 露兜树属 (Pandanus) 植物露兜树 (Pandanus tectorius Soland.) 的果实[6, 10]。在中国海南省部分地区,当地黎族人民常使用它治疗高脂血症,现已成为黎族当地医院制剂,且临床疗效显著。本课题组通过高脂饲料诱导的高脂血症金黄地鼠模型和db/db自发肥胖型糖尿病 小鼠模型确证了露兜簕提取物的降血脂活性,发现该降血脂有效部位富含咖啡酰奎宁酸类成分,并可以通过上调PPARα通路的表达和活性发挥降血脂作用[6, 11]。为进一步阐明露兜簕发挥降血脂作用的物质基础及作用机制,本文利用油酸诱导的肝HepG2细胞脂质积聚模型对从露兜簕提取物中分离得到的7种咖啡酰奎宁酸类化合物的降脂活性进行了筛选和研究,并通过实时定量PCR方法分析各活性成分对肝细胞脂代谢相关基因的表达调节作用,从而初步揭示露兜簕降血脂作用的主要活性成分及潜在作用途径。

材料与方法
主要仪器与试剂

IX51倒置荧光显微镜 (OLYMPUS公司); KC junior微孔板分光光度计 (BIOTEK公司); 7500实时定量PCR仪 (ABI公司)。

7种露兜簕来源咖啡酰奎宁酸 (纯度>98%) 由中国医学科学院药用植物研究所许旭东研究员提供,如表 1所示; 洛伐他汀 (lovastatin,纯度>98%)、胰蛋白酶、四甲基氮唑蓝 (MTT)、油红O染料、油酸 (OA)、trizol试剂、青霉素、链霉素 (Sigma公司); DMEM高糖培养基 (Gibco公司); 超级小牛血清 (四季青生物研究所); 细胞胆固醇/胆固醇酯定量试剂盒、细胞甘油三酯定量试剂盒 (BioVision公司); BCA蛋白定量试剂盒、cDNA反转录试剂盒、RT-PCR试剂盒 (北京全式金生物技术有限公司); 引物由Invitrogen公司设计合成; 兔抗人SREBP-1c和ACC多克隆抗体购自Abcam公司,其余试剂为分析纯,购自于国药集团化学试剂有限公司。

Table1 List of the caffeoylquinic acid derivatives isolated from the n-butanol fraction of P. tectorius
细胞培养

人肝癌HepG2细胞购自北京协和医学院基础医学研究所细胞中心。细胞在37 ℃、5% CO2、饱和湿度条件下用含10% FBS的高糖DMEM培养基培养,同时在培养基中按100∶1的比例添加双抗 (100 u·mL-1青霉素和100 μg·mL-1链霉素),每两天更换一次新鲜培养液。

油红O染色

取生长状态良好的对数生长期HepG2细胞,以适宜浓度接种于96孔板中,每孔加入细胞悬液100 μL,培养24 h至细胞完全贴壁。待细胞生长汇合至70%~80%时,用含100 μmol·L-1油酸 (OA) 和10 μmol·L-1各种试剂的不完全培养基孵育细胞,每组设8个复孔,在37 ℃、5% CO2下继续培养24 h。弃掉培养基,用PBS洗一遍,4%多聚甲醛在4 ℃固定过夜。PBS清洗一遍,每孔加入油红O染液室温染色15 min,PBS漂洗干净,弃掉PBS后,每孔加入DMSO 100 μL,置于摇床上摇匀,用微孔板分光光度计在358 nm读取吸光度。

MTT实验

取生长状态良好的对数生长期的HepG2细胞,胰酶消化后接种于96孔板 (细胞数 4 000/孔)。用完全培养基配制不同浓度的待测天然产物,使药物浓度分别为100、50、10和1 μmol·L-1,空白对照组给予含溶剂的完全培养基; 每组设置8个平行复孔,在37 ℃、5% CO2下培养24 h。然后,每孔加入5 g·L-1 MTT溶液20 μL,在37 ℃、5% CO2下 继续培养4 h。弃掉板中培养液,每孔加入DMSO 150 μL,室温摇床振荡混匀,充分溶解紫色结晶。微孔板分光光度计570 nm处读取吸光度,观察给药组细胞的相对活力。

倒置显微镜观察

取状态良好的对数生长期HepG2细胞,胰酶消化后接种到6孔板中,经100 μmol·L-1 OA诱导和10 μmol·L-1各种天然产物的不完全培养基孵育细胞。500 μL油红O染色,PBS漂洗干净,在光学倒置显微镜下观察并拍照。

细胞内TC和TG定量

收集并裂解经OA诱导和药物处理的细胞,使用细胞总胆固醇和甘油三酯定量试剂盒按照制造商提供的使用说明进行细胞内TC和TG定量,并使用BCA蛋白定量试剂盒按照制造商提供的使用说明测定样品中的蛋白含量。进而计算细胞内的TC和TG含量。

实时定量PCR 总RNA提取、反转录使用试剂盒按照制造商的使用说明书进行。RT-PCR使用北京全式金生物技术有限公司生产的TransStart Top Green qPCR superMix试剂盒执行,用Sybr Green进行荧光标记。所用引物序列见表 2
Table2 Oligonucleotide primers used in this work
Western blot分析

收取经上述药物处理24 h 的细胞,用含有蛋白酶抑制剂、磷酸酶抑制剂、氟化钠和钒酸钠的加样缓冲液冰上静置裂解细胞10 min,再95 ℃变性10 min,12 000 r·min-1离心5 min,上清液即为全细胞裂解液。SDS-PAGE凝胶电泳后冰浴 湿式转印至PVDF膜,参数为恒压80 V × 3 h。用含0.5 g·L-1油酸脱脂奶粉和0.5 mL Tween-20的PBST封闭30 min后,按抗体厂家建议稀释一抗后4 ℃敷膜结合过夜,以GAPDH抗体为内参。一抗结合后PBS-T洗膜10 min × 3次,加入二抗稀释液 (1∶1 000),室温孵育2 h,再次PBS-T洗膜10 min × 3次,用ECL化学发光显色液显色,于暗室中曝光。蛋白条带用ImageJ 1.44e软件进行灰度定量,以比较蛋白水平。

数据统计

实验数据用SPSS 17.0统计分析软件处理,数据以x± s表示,采用单因素方差分析,多组之间两两相关比较进行t检验,P < 0.05为有统计学意义。

结果
1 露兜簕来源咖啡酰奎宁酸成分细胞降脂活性筛选

通过油酸诱导的HepG2肝癌细胞脂质积聚模型对从露兜簕分离得到的7种咖啡酰奎宁酸成分,即3-咖啡酰奎宁酸 (绿原酸,3-CQA)、4-咖啡酰奎宁酸 (隐绿原酸,4-CQA)、5-咖啡酰奎宁酸 (新绿原酸,5-CQA)、1,3-二咖啡酰奎尼酸 (莱蓟素,1,3 -CQA)、3,4-二咖啡酰奎尼酸 (异绿原酸B,3,4-CQA)、3,5-二咖啡酰奎尼酸 (异绿原酸A,3,5-CQA) 和3,4,5-三

咖啡酰奎尼酸 (3,4,5-CQA),的降脂活性进行筛选。油红O染色定量分析结果显示,3-CQA、3,5-CQA和3,4,5-CQA在10 μmol·L-1浓度下可显著抑制油酸诱导的HepG2肝癌细胞脂质积聚,而其余咖啡酰奎宁酸类化合物则效果不明显 (图 1)。

Figure1 Effect of P. tectorius-derived caffeoylquinic acids (CQAs) on intracellular lipid accumulation in HepG2 cells. All cells except blank group were incubated with 100 μmol·L-1 oleic acid (OA) along with respective agents (10 μmol·L-1) for 24 h,then subjected to oil-red O staining and quantified by light absorptance at 358 nm. The experiment was repeated three times with n = 8 in each test. ###P < 0.001 vs blank group; P < 0.05,**P < 0.01 vs OA group
2 降脂活性咖啡酰奎宁酸成分对HepG2细胞增殖的影响

MTT实验结果表明,具有降脂活性的3-CQA、3,5-CQA和3,4,5-CQA,在10 μmol·L-1浓度下对HepG2细胞增殖没有不良影响。3-CQA在1~100 μmol·L-1内无明显的细胞毒性,3,4,5-CQA在100 μmol·L-1浓度下可抑制HepG2细胞增殖,而3,5-CQA在约50 μmol·L-1浓度下即可抑制HepG2细胞增殖 (图 2)。

Figure2 Effects of 3-CQA,3,5-CQA and 3,4,5-CQA on viability of HepG2 cells in MTT assay. Cell viability was expressed as percentage of the control group. The experiment was repeated three times with n = 8 in each test. **P < 0.01,***P < 0.001 vs control group
3 降脂活性咖啡酰奎宁酸成分剂量依赖性地抑制油酸诱导的HepG2细胞内脂质积聚

油红O染色后吸光度分析和光学显微镜观察结 果显示,具有降脂活性的咖啡酰奎宁酸成分,3-CQA、3,5-CQA和3,4,5-CQA,在1~10 μmol·L-1浓度下,均剂量依赖性地抑制油酸诱导的HepG2细胞中性脂质的积聚 (图 3A和3B)。细胞内总胆固醇 (图 4A) 和甘油三酯 (图 4B) 定量分析结果表明,3-CQA、3,5- CQA和3,4,5-CQA对细胞内胆固醇和甘油三酯的积聚均有明显的抑制作用,其作用效力依次为3-CQA > 3,5-CQA > 3,4,5-CQA。

Figure3 The dose-dependent effect of 3-CQA,3,5-CQA and 3,4,5-CQA on intracellular lipid accumulation in HepG2 cells. All cells except blank group were incubated with 100 μmol·L-1 OA along with respective agents (as indicated in μmol·L-1) for 24 h,then subjected to oil-red O staining and measured by light absorptance at 358 nm (A) or by microscopy (bar= 50 μm) (B). The concentration of lovastatin was 10 μmol·L-1. The experiment was repeated three times with n = 8 in each test. ###P < 0.001 vs blank group; P < 0.05,**P < 0.01,***P < 0.001 vs OA group

Figure4 Effect of 3-CQA,3,5-CQA and 3,4,5-CQA on intracellular concent of total cholesterol (A) and triglycerides (B) in HepG2 cells. All cells except blank group were incubated with 100 μmol·L-1 oleic acid (OA) along with respective agents (as indicated in μmol·L-1) for 24 h,then subjected to total cholesterol and triglycerides quantification by respective kit. The concentra­tion of lovastatin was 10 μmol·L-1. The experiment was repeated three times with n = 4 in each test. ##P < 0.01,###P < 0.001 vs blank group; P < 0.05,**P < 0.01,***P < 0.001 vs OA group
4 降脂活性咖啡酰奎宁酸成分对HepG2细胞中脂代谢相关基因表达水平的影响

实时定量PCR结果显示,3-CQA、3,5-CQA和3,4,5-CQA对HepG2细胞中脂代谢相关基因的mRNA水平具有显著影响。3-CQA和3,5-CQA均可提高脂质氧化相关基因PPARαCPT-1ACOX-1的表达水平,同时显著抑制脂质合成相关基因SREBP-1cSREBP-2ACCHMGRFAS的表达水平,特别是3,5-CQA的抑制效果尤为明显。相对地,3,4,5-CQA对脂质氧化相关基因的表达无明显影响,但可显著抑制脂质合成相关基因SREBP-1cSREBP-2ACCHMGRFAS的表达水平 (图 5)。Western blot分析进一步印证了实时定量PCR的实验结果。三种咖啡酰奎宁酸,特别是3-CQA和3,5-CQA可显著抑制成脂重要因子SREBP-1c的蛋白表达水平,同时可降低成脂因子ACC的蛋白表达水平,但在统计学上并不显著 (图 6)。有趣的是,虽然3-CQA对SREBP-1c的转录水平没有显著影响,但却明显抑制其蛋白水平。3-CQA对SREBP-1c蛋白表达水平的影响与Murase等的实验结果一致[12]

Figure5 Effect of 3-CQA (A),3,5-CQA (B) and 3,4,5-CQA (C) on the expression of genes involved in lipid metabolism in HepG2 cells. The relative mRNA levels of respective genes were determined by realtime quantitative PCR with β-actin as internal control. n = 3. P < 0.05,**P < 0.01 vs control

Figure6 Effect of caffeoylquinic acids on protein levels of SREBP-1c and ACC in HepG2 cells. (A) Typical Western blots for ACC and SREBP-1c in HepG2 cells with GAPDH as internal control. (B) Mean expression levels of ACC and SREBP-1c obtained by quantification of the intensity of each band of the Western blot. n = 3. P < 0.05 vs NC
讨论

本课题组前期利用高脂饲料诱导的高脂血症金黄地鼠和db/db自发肥胖型糖尿病小鼠证明了露兜簕正丁醇提取物 (PTF) 具有优良的降血脂作用,并且咖啡酰奎宁酸类化合物是其主要成分[6, 11]。本文进一步利用HepG2肝癌细胞模型对露兜簕的降脂活性进行了筛选,并对其潜在的作用机制进行了研究。

比较各种咖啡酰奎宁酸化合物的结构和药效可以发现,显示降脂活性的咖啡酰奎宁酸类化合物均含有3位咖啡酰基; 同时含有3位和5位咖啡酰基的3,5-CQA显示最强的降脂活性; 而同时含有4位咖啡酰基的3,4-CQA和3,4,5-CQA则比4位不含咖啡酰基的对应化合物的降脂活性更弱。因此推测,3位取代的咖啡酰基是影响咖啡酰奎宁酸类化合物降脂活性的关键位点,5位的咖啡酰基对降脂活性有一定的协同作用,而1位和4位的咖啡酰基则对降脂活性有拮抗作用。

实验数据表明,3-CQA是其中降脂效力最高且细胞毒性最小的活性化合物。关于绿原酸具有优良降脂活性的研究也已有较多报道[13, 14, 15, 16, 17, 18]。因此,本研究结果提示,绿原酸可能是露兜簕发挥降血脂作用的重要活性成分。

脂质代谢受到多种基因和蛋白的调节。SREBP- 1c及其下游基因ACCFAS主要促进脂肪酸的合成,而SREBP2及其下游基因HMGR则主要参与胆固醇的合成[19]。另一方面,PPARα及其下游基因CPT-1ACOX1则可以通过提高脂质氧化发挥降脂作用[20]。实时定量PCR结果显示,3-CQA、3,5-CQA和3,4,5-CQA均可显著抑制脂质合成因子SREBP-1c、SREBP-2及其下游基因ACCFASHMGR的表达水平,提示它们可以通过抑制脂肪酸和胆固醇的合成发挥降脂作用。同时,3-CQA和3,5-CQA还显著提高脂质氧化相关基因PPARαCPT-1ACOX1的表达水平,表明3-CQA和3,5-CQA还可以通过促进脂肪酸β氧化实 现降低细胞内脂质积聚的作用。Western blot分析进一步证实了实时定量PCR的结果,这些结果初步揭示了露兜簕来源咖啡酰奎宁酸成分发挥降脂作用的可能途径,但还需要更进一步的实验数据加以证实,这将在后续工作中完成。

参考文献
[1] Palinski W, Yamashita T, Freigang S, et al. Developmental programming: maternal hypercholesterolemia and immunity influence susceptibility to atherosclerosis [J]. Nutr Rev, 2007, 65: S182-187.
[2] Wu C, Guo Y, Su Y, et al. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit [J]. J Cell Mol Med, 2014, 18: 293-304.
[3] Wu C, Luan H, Wang S, et al. Modulation of lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes by sophoricoside [J]. Molecules, 2013, 18: 15624-15635.
[4] Sundqvist A, Bengoechea-Alonso MT, Ye X, et al. Control of lipid metabolism by phosphorylation-dependent degradation of the SREBP family of transcription factors by SCF (Fbw7) [J]. Cell Metab, 2005, 1: 379-391.
[5] Serviddio G, Bellanti F, Vendemiale G. Free radical biology for medicine: learning from nonalcoholic fatty liver disease [J]. Free Radic Biol Med, 2013, 65: 952-968.
[6] Wu C, Zhang X, Zhang X, et al. The caffeoylquinic acid-rich Pandanus tectorius fruit extract increases insulin sensitivity and regulates hepatic glucose and lipid metabolism in diabetic db/db mice [J]. J Nutr Biochem, 2014, 25: 412-419.
[7] Dou XB, Wo XD, Fan CL. Progress of research in treatment of hyperlipidemia by monomer or compound recipe of Chinese herbal medicine [J]. Chin J Integr Med, 2008, 14: 71-75.
[8] Kashyap ML, McGovern ME, Berra K, et al. Long-term safety and efficacy of a once-daily niacin/lovastatin formulation for patients with dyslipidemia [J]. Am J Cardiol, 2002, 89: 672-678.
[9] Scarpini F, Cappellone R, Auteri A, et al. Role of genetic factors in statins side-effects [J]. Cardiovasc Hematol Disord Drug Targets, 2012, 12: 35-43.
[10] Englberger L, Aalbersberg W, Dolodolotawake U, et al. Carotenoid content of pandanus fruit cultivars and other foods of the Republic of Kiribati [J]. Public Health Nutr, 2006, 9: 631-643.
[11] Zhang X, Wu C, Wu H, et al. Anti-hyperlipidemic effects and potential mechanisms of action of the caffeoylquinic acid-rich Pandanus tectorius fruit extract in hamsters fed a high fat-diet [J]. PLoS One, 2013, 8: e61922.
[12] Murase T, Misawa K, Minegishi Y, et al. Coffee polyphenols suppress diet-induced body fat accumulation by downregulating SREBP-1c and related molecules in C57BL/6J mice [J]. Am J Physiol Endocrinol Metab, 2011, 300: E122-133.
[13] Meng S, Cao J, Feng Q, et al. Roles of chlorogenic Acid on regulating glucose and lipids metabolism: a review [J]. Evid Based Complement Alternat Med, 2013, 2013: 801457.
[14] Ong KW, Hsu A, Tan BK. Anti-diabetic and anti-lipidemic effects of chlorogenic acid are mediated by ampk activation [J]. Biochem Pharmacol, 2013, 85: 1341-1351.
[15] Wan CW, Wong CN, Pin WK, et al. Chlorogenic acid exhibits cholesterol lowering and fatty liver attenuating properties by up-regulating the gene expression of PPAR-α in hypercholesterolemic rats induced with a high-cholesterol diet [J]. Phytother Res, 2013, 27: 545-551.
[16] Karthikesan K, Pari L, Menon VP. Antihyperlipidemic effect of chlorogenic acid and tetrahydrocurcumin in rats subjected to diabetogenic agents [J]. Chem Biol Interact, 2010, 188: 643-650.
[17] Cho AS, Jeon SM, Kim MJ, et al. Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high- fat diet-induced-obese mice [J]. Food Chem Toxicol, 2010, 48: 937-943.
[18] Rodriguez de Sotillo DV, Hadley M. Chlorogenic acid modifies plasma and liver concentrations of: cholesterol, triacylglycerol, and minerals in (fa/fa) Zucker rats [J]. J Nutr Biochem, 2002, 13: 717-726.
[19] Assaf S, Lagarrigue S, Daval S, et al. Genetic linkage and expression analysis of SREBP and lipogenic genes in fat and lean chicken [J]. Comp Biochem Physiol B Biochem Mol Biol, 2004, 137: 433-441.
[20] Hsu WH, Chen TH, Lee BH, et al. Monascin and ankaflavin act as natural AMPK activators with PPARα agonist activity to down-regulate nonalcoholic steatohepatitis in high-fat diet-fed C57BL/6 mice [J]. Food Chem Toxicol, 2014, 64: 94-103.