柑橘精油的代谢图谱及其主要成分d-柠檬烯对糖脂代谢紊乱的防治作用研究

发布时间：2018-05-30 21:54

本文选题：柑橘精油 + 代谢图谱　；参考：《西南大学》2014年博士论文

【摘要】：柑橘(Citrus L.)属于芸香科(Rutaceae)柑橘亚科(Aurantioideae)植物。中国是柑橘属的重要起源中心之一,具有丰富的柑橘野生资源。自林奈1753年确立柑橘属以来,国内外学者从形态到分子对柑橘属植物的分类和进化问题开展了一系列研究。然而,柑橘属植物的分类、进化和重要栽培(杂)种的起源问题仍待解决。柑橘精油是广泛分布于柑橘的果实、叶片及花中的一类具芳香气味且常温挥发的油状液体的总称。柑橘精油的主要成分d-柠檬烯具有抗氧化、抗炎、抗癌等多种生物活性。特别是最新研究发现,柑橘精油的主要成分d-柠檬烯具有缓解由高脂膳食及N硝基左旋精氨酸甲酯(Nw-nitro-L-argininemethylester, L-NAME)诱导的非酒精性脂肪肝大鼠胰岛素抵抗的功效。然而,有关d-柠檬烯对由高脂膳食诱导的肥胖小鼠的脂代谢紊乱和高血糖方面的研究仍尚未见报道。本论文采用气相色谱与质谱技术对来自20种不同柑橘资源的果皮精油的挥发性成分进行了分析,其中10种为中国特有野生柑橘类型。在柑橘精油代谢图谱的基础上,建立了以代谢组学为基础的柑橘化学分类模型,并采用该分类模型对柑橘属三个基本种以及六个重要栽培(杂)种的分类鉴定和起源问题进行了分析。在此基础上,结合体外细胞模型以及体内小鼠模型,对柑橘精油的主要成分d-柠檬烯在由高脂膳食诱导的肥胖小鼠脂代谢紊乱及高血糖等代谢综合征中的防治作用进行了系统研究。最后,采用双荧光素酶报告基因和实时荧光定量PCR技术对d-柠檬烯在过氧化物酶增殖物激活受体(peroxisome proliferator-activated receptor, PPAR)以及肝X受体(liver X receptor, LXR)等核受体信号通路中的调节作用进行了探讨。有关重要研究结果如下： 1.采用气相色谱与质谱技术对20份不同柑橘资源精油代谢图谱进行分析的结果表明：柑橘精油中的主要挥发性成分为单萜类物质,其中d-柠檬烯(55.05-91.06%)、(S)-(-)-苎烯(0.04-28.04%)、月桂烯(3.62-11.32%)、α-蒎烯(0.49-5.26%)以及香桧烯(0.07-7.54%)为柑橘精油的主要化学成分。对柑橘属三个基本种果皮精油的代谢图谱的分析结果表明：枸橼(C.medica L.)精油的代谢图谱以含有相对较低浓度的d-柠檬烯(55.05%)和较高浓度的(S)-(-)-苎烯(19。18%)、月桂烯(7.82%)以及α-蒎烯(3.67%)为主要特征；柚类(C. grandisOsb.)精油的代谢图谱特征则为d-柠檬烯(85.67%)以及月桂烯(11.32%)的相对含量较高；宽皮柑橘(C. reticulata Blanco)的代谢图谱特征为(S)-(-)-苎烯(11.39-21。83%)、α-芋烯崖柏烯(0.45-1.23%)以及α-蒎烯(2.04-4.07%)含量较高。 2.对柑橘精油代谢图谱的聚类分析和偏最小二乘法辨别分析的结果显示：枸橼(C. medica L.)、柚(C. grandis Osb.)以及宽皮柑橘(C. reticulata Blanco)等柑橘属三个基本种精油的代谢图谱首先各自聚在一起,然后与其他基本种的代谢图谱清楚地分离开来。此外,甜橙(C. sinensis Osb.)、酸橙(C.aurantium L.)、柠檬(C. limon Burm.f.)、粗柠檬(C. jambhiri Lush.)、兰卜莱檬(C.limonia Osb)以及葡萄柚(C.paradisi Macf.)等重要栽培(杂)种的代谢图谱首先各自聚在一起,然后分别与其亲本或亲本之一聚在一起,且聚类结果与已有形态学、DNA分子标记和已有化学分类结果一致。D-柠檬烯、α-蒎烯、香桧烯与α-萜品烯为柑橘精油代谢图谱中的特征性成分,是本论文中基于代谢组学的柑橘化学分类模型的主要生物标记性行物质或特征性化合物。 3.利用3T3-L1前脂肪细胞模型,通过前脂肪细胞诱导分化和油红染色,我们发现,柑橘精油的主要成分d-柠檬烯具有抑制前脂肪细胞分化的功效。采用C57BL/6小鼠进行预防实验的结果表明,d-柠檬烯能够明显影响实验小鼠白色脂肪组织和褐色脂肪组织中脂肪细胞的大小,并能够有效预防由高脂膳食诱导的实验小鼠血清脂质中甘油三酯(p0.01)的上升以及空腹血糖水平(p0.01)的提高,d-柠檬烯还具有预防小鼠肝脏组织中脂肪积累的功效；治疗实验中,通过建立高脂膳食诱导的肥胖小鼠模型,d-柠檬烯能够显著降低肥胖小鼠血清脂质中甘油三酯(p0.01)、低密度脂蛋白胆固醇(p0.05)的含量以及空腹血糖水平(p0.05),并能有效提高肥胖小鼠血清中高密度脂蛋白胆固醇(p0.05)的含量以及小鼠对葡萄糖的利用率。 4.通过双荧光素酶报告基因研究,我们发现,柑橘d-柠檬烯可以提高PPARα的转录活性,并能降低LXRβ的表达水平。实时荧光定量PCR检测结果表明,d-柠檬烯能够显著提高解偶联蛋白(uncoupling protein-2, UCP-2)、乙酰辅酶A羧化酶(acetyl-CoAcarboxylase, ACC)以及过氧化物酶体增殖物激活受体γ辅激活子la (peroxisome proliferator-activated receptor y coactivator-la, PGC-1α)等PPARa靶基因的表达水平。此外,d-柠檬烯还能够抑制胆固醇调节元件结合蛋白(sterol regulatory element-binding protein-1, SREBP-1),载脂蛋白E (apolipo-protein E, ApoE),以及3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methyl-glutaryl-CoAreductase, HMGR)等LXRP靶基因的mRNA的表达水平。
[Abstract]:Citrus L . belongs to the genus Aurantioideae of Rutaceae . China is one of the most important centers of citrus , and has rich citrus wild resources . The citrus essential oil is widely distributed in citrus fruits , leaves and flowers . The citrus essential oil is widely distributed in citrus fruits , leaves and flowers .

In this paper , the volatile components of citrus essential oil from 20 different citrus resources were analyzed by gas chromatography and mass spectrometry .

The key findings are as follows :

1 . The main volatile components of citrus essential oil were analyzed by gas chromatography and mass spectrometry . The results showed that the main volatile components of citrus essential oil were monoterpene , d - monene ( 55.05 - 91.06 % ) , myrcene ( 3.62 - 11.32 % ) , 伪 - pinene ( 0.49 - 5.26 % ) and sabinene ( 0.07 - 7.54 % ) as the main chemical constituents of citrus essential oil .
Grapefruit ( C . grandisOsb . ) The metabolic profiles of essential oils were d - monene ( 85.67 % ) and myrcene ( 11.32 % ) .
The metabolic profiles of C . reticulata Blanco were ( S ) - ( - ) - Limonene ( 11.39 - 21.83 % ) , 伪 - morphene ( 0.45 - 1.23 % ) and 伪 - pinene ( 2.04 - 4.07 % ) .

2 . The metabolic profiles of three essential oils , such as C . medica L . , C . grandis Osb . and C . reticulata Blanco , were isolated from the metabolic profiles of three essential oils , such as Citrus medica L . , C . grandis Osb . and C . reticulata Blanco . The metabolic profiles of the important cultivated ( hetero ) species are firstly brought together and then together with one of their parents or parents respectively , and the clustering results are consistent with the existing morphological , DNA molecular markers and the existing chemical classification results . D - Limonene , 伪 - pinene , sabinene and 伪 - terpineene are the characteristic components in the metabolic map of the citrus essential oil , and are the main biomarkers or characteristic compounds of the metabolic group - based citrus chemical classification model in this paper .

3 . Using 3T3 - L1 preadipocytes to induce differentiation and oil - red staining , we found that d - monene of citrus essential oil has the effect of inhibiting the differentiation of preadipocytes . The results showed that d - lemonene can significantly influence the size of fat cells in white adipose tissue and brown adipose tissue of experimental mice , and can effectively prevent the increase of triglyceride ( p0.01 ) and the increase of fasting blood glucose level ( p0.01 ) in the serum lipid of experimental mice induced by high fat diet .
In the treatment experiment , by establishing a model of obesity mice induced by high - fat diet , the content of triglyceride ( p0.01 ) , low - density lipoprotein cholesterol ( p . 05 ) and fasting blood glucose level ( p . 05 ) in the serum lipids of obese mice were significantly reduced by the establishment of a high - fat diet - induced obese mouse model , and the content of high - density lipoprotein cholesterol ( p . 05 ) and the utilization rate of glucose in the serum of obese mice can be effectively improved .

4 . Through the study of luciferase reporter gene , we found that d - lemonene can improve the transcriptional activity of PPAR伪 and decrease the level of expression of LXR 尾 . The results of real - time fluorescence quantitative PCR show that d - monene can significantly improve the expression level of PPARa target genes such as uncoupling protein - 1 , acetyl - CoAscent , ACC , and peroxidase - activated receptor y coactivator - la , PGCs - 1.alpha . , and the expression level of mRNA of LXRP target gene , such as 3 - hydroxy - 3 - methyl - yl - CoAreductase , HMGR , etc .
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R151

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