| China is an important center of origin for the genus Citrus L. of the family Rutaceae and is rich in wild Citrus species. The taxonomy of Citrus has been a subject of controversy for more than a half century. Characterizing the metabolite profiles of Chinese native Citrus species will facilitate further taxonomic study and use of germplasm of the genus. Citrus essential oil and their phytochemicals have been demonstrated to have antioxidant, anti-inflamation and anticancer activities. Recently, d-limonene, the major component of citrus essential oil, has been reported to alleviate fatty liver induced by high-fat diet and Nw-nitro-L-arginine methylester. However, no study has yet reported on the effect of d-limonene on high-fat diet-induced obesity or obesity-related dyslipidemia and hyperglycemia.In this study, the gas chromatography-mass spectrometry (GC-MS) was used to get the metabolite profiles of20Citrus species/varieties, among which10are the native genotypes originated in China. Based upon the metabolite profiles of citrus essential oils, a metabolomics-based classification model was then established to differentiate the three basic Citrus species, and to identify the presumed hybrid species.By using both in vitro and in vivo models, the effects of d-limonene on dyslipidemia and hyperglycemia in high-fat diet-fed mice with obesity were studied. In addition, we investigated whether d-limonene is effective in regulating peroxisome proliferator-activated receptor (PPAR) and liver X receptor (LXR) signaling. The main results obtained are as follows:1. The major components identified in all oil samples were monoterpenes, and J-limonene (55.05-91.06%), para-mentha-3,8-diene (0.04-28.04%), myrcene (3.62-11.32%), a-pinene (0.49-5.26%) and sabinene (0.07-7.54%) were the dominant compounds in the peel oils of these20Citrus species/varieties. On the basis of the GC-MS profiles of the three basic Citrus species, mandarins were distinguished from the other two species by their high relative contents of para-mentha-3,8-diene (11.39-21.83%), a-thujene (0.45-1.23%), and a-pinene (2.04-4.07%). Pummelo was differentiated by high J-limonene (85.67%) and myrcene (11.32%) contents. Citron was discriminated from others by its lowest content of<i-limonene (55.05%) and high amount of para-mentha-3,8-diene (19.18%), myrcene (7.82%) and a-pinene (3.67%).2. The three basic species of the subgenus Eucitrus (Swingle’s system), i.e., C. medica L., C. grandis Osb. and C. reticulata Blanco, were clearly discriminated based upon their metabolite profiles using hierarchical cluster analysis (HCA) and partial least square-discriminant analysis (PLS-DA). All the presumed hybrid genotypes including sweet orange (C. sinensis Osb.), sour orange (C. aurantium L.), lemon (C limon Burm.f.), rough lemon (C. jambhiri Lush.), rangpur lime (C. limonia Osb.) and grapefruit (C. paradisi Macf.) each grouped closely together with one of their suggested parent species in the HCA-dendrogram and the PLS-DA score plot. D-limonene, a-pinene, sabinene and a-terpinene were found to be the characteristic components of the essential oils of the Citrus species analyzed in this study, and these compounds contributed greatly to the metabolic classification.3. By using3T3-L1pre-adipocytes, d-limonene was found to inhibit the differentiation of the adipocytes from pre-adipocytes. In the preventive treatment, d-limonene decreased the size of white and brown adipocytes, lowered serum triglyceride (TG)(p<0.01) and fasting blood glucose levels (p<0.01), and prevented liver lipid accumulations in high-fat diet-fed C57BL/6mice. In the therapeutic treatment, d-limonene reduced serum TG (p<0.01), low-density lipoprotein cholesterol (LDL-c)(p<0.05) and fasting blood glucose levels (p<0.01) and glucose tolerance, and increased serum high-density lipoprotein cholesterol (HDL-c)(p<0.05) in obese mice.4. Using a reporter assay, we found that d-limonene activated peroxisome proliferator-activated receptor a (PPARa) signaling, and inhibited liver X receptor p (LXRp) signaling. The gene expression analysis showed that the expression of PPARa target genes including uncoupling protein-2(UCP-2), acetyl-CoAcarboxylase (ACC) and peroxisome proliferator-activated receptor y coactivator-la (PGC-1α), were markedly increased by d-limonene. In addition, d-limonene suppressed the mRNA levels of LXR target genes, including sterol regulatory element-binding protein-1 (SREBP-1), apolipo-protein E (ApoE), and3-hydroxy-3-methyl-glutaryl-CoAreductase (HMGR). |
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