Mass Spectrometry-Based Metabolomic and Lipidomic Analyses of the Effects of Dietary Platycodon grandiflorum on Liver and Serum of Obese Mice under a High-Fat Diet

nutrients Article Mass Spectrometry-Based Metabolomic and Lipidomic Analyses of the Effects of Dietary Platycodon grandiflorum on Liver and Serum of Obese Mice under a High-Fat Diet Hye Min Park 1,† , Kab-Tae Park 2,† , Edmond Changkyun Park 3,4 , Seung II Kim 3,4 , Myung Sook Choi 5 , Kwang-Hyeon Liu 2, * and Choong Hwan Lee 1, * 1 2 3 4 5 * † Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea; BK21 Plus KNU Multi-Omics Based Creative Drug Research Team, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Korea; Division of Life Science, Korea Basic Science Institute, Daejeon 34133, Korea; (E.C.P.); (S.I.K.) Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 34114, Korea Department of Food Science and Nutrition, Kyungpook National University, Daegu 41566, Korea; Correspondence: (K.-H.L.); (C.H.L.); Tel.: +82-53-950-8567 (K.-H.L.); +82-2-2049-6177 (C.H.L.) These authors contributed equally to this work. Received: 25 November 2016; Accepted: 7 January 2017; Published: 17 January 2017 Abstract: We aimed to identify metabolites involved in the anti-obesity effects of Platycodon grandiflorum (PG) in high-fat diet (HFD)-fed mice using mass spectrometry (MS)-based metabolomic techniques. C57BL/6J mice were divided into four groups: normal diet (ND)-fed mice, HFD-fed mice, HFD with 1% PG extract-fed mice (HPGL), and HFD with 5% PG extract-fed mice (HPGH). After 8 weeks, the HFD group gained more weight than the ND group, while dietary 5% PG extract attenuated this change. The partial least squares discriminant analysis (PLS-DA) score plots showed a clear distinction between experimental groups in serum and liver markers. We also identified 10 and 32 metabolites in the serum and liver, respectively, as potential biomarkers that could explain the effect of high-dose PG added to HFD-fed mice, which were strongly involved in amino acid metabolism (glycine, serine, threonine, methionine, glutamate, phenylalanine, ornithine, lysine, and tyrosine), TCA cycle (fumarate and succinate), lipid metabolism (linoleic and oleic acid methyl esters, oleamide, and cholesterol), purine/pyrimidine metabolism (uracil and hypoxanthine), carbohydrate metabolism (maltose), and glycerophospholipid metabolism (phosphatidylcholines, phosphatidylethanolamines, lysophosphatidylcholines, and lysophosphatidylethanolamines). We suggest that further studies on these metabolites could help us gain a better understanding of both HFD-induced obesity and the effects of PG. Keywords: amino acids; glycerophospholipids; high-fat diet; metabolite profiling; obesity; Platycodon grandiflorum 1. Introduction Platycodon grandiflorum (PG) is a perennial plant from the Campanulaceae family, well known as a traditional herbal medicine for the treatment of asthma, diabetes, and respiratory disorders. Nutrients 2017, 9, 71; doi:10.3390/nu9010071 www.mdpi.com/journal/nutrients Nutrients 2017, 9, 71 2 of 17 It contains diverse bioactive compounds such as triterpenoid saponins, flavonoids, polyphenols, and fibers [1–3]. PG also possesses antioxidant, anticancer, anti-inflammatory, and hepato-protective pproperties [4–6]. Furthermore, many researchers have reported on the anti-obesity effect of PG and its constituents through the reduction of total cholesterol (TC) and triglyceride (TG) levels, and the inhibition of pancreatic lipase activity [7–9]. Obesity is most commonly caused by a chronic imbalance between energy intake and energy expenditure [10]. Long-term high-fat intake induces weight gain and provokes changes in various biochemical parameters such as insulin, glucose, leptin, TC, and TG levels in the blood and liver [11,12]. Obesity can therefore be defined as a disorder characterized by an abnormal lipid metabolism. To understand the metabolic pathways and mechanisms involved in obesity more completely, high-throughput metabolomic analyses have recently been applied, using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolomics, the analysis of a huge range of small molecules in a biological system, is playing an increasingly important role in evaluating endogenous metabolite alterations in tissues and biological fluids, discovering potential biomarkers for diseases such as diabetes and obesity, or developing therapeutic applications [13–15]. Amino acids, fatty acids, carnitine, acyl-carnitines, lysophosphatidylcholines (lysoPCs), and lysophosphatidylethanolamines (lysoPEs) have been established as biomarker candidates for obesity through metabolomic analyses in obese animal models [16–18]. Based on this information, untargeted metabolite profiling has been performed on biologically active products and single compounds used to treat obesity [19,20]. In this study, we investigated the anti-obesity effect of two different concentrations of PG extract in high-fat diet (HFD)-induced obese C57BL/6J mice by profiling endogenous and exogeneous metabolites in both the serum and liver. Analyses were performed using comprehensive mass spectroscopy (MS) instruments such as ultra-performance liquid chromatography (UPLC)-quadrupole time-of-flight (Q-TOF)-MS, gas chromatography (GC)-TOF-MS, and direct infusion-MS, combined with multivariate analyses, to identify metabolites that would enable a better understanding of the beneficial effect of PG in HFD-related obesity. 2. Materials and Methods 2.1. Chemicals and Reagents Acetonitrile, water, dichloromethane, and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA) or Merck (Darmstadt, Germany). Methoxyamine hydrochloride, N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), formic acid, pyridine, ammonium acetate, chloroform, methyl-tert-buthyl ether (MTBE), and triglycerides mixture standard (16:0/18:1/18:2) were purchased from Sigma Aldrich (St. Louis, MO, USA). Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) supplied us with 2-chloro-L-phenylalanine. Phosphatidylcholine (PC) 18:0/16:0, phosphatidylethanolamine (PE) 16:0/18:1, lysoPC 18:0, and cholesteryl ester (CE) 16:0 were purchased from Avanti Polar Lipids (Alabaster, AL, USA). All chemicals and solvents were of analytical grade and commercially available. 2.2. Preparation of PG Extract PG roots were obtained from Omniherb (Daegu, Korea). PG roots (10 kg) were extracted with 70% ethanol (10 volumes) at 50 ◦ C for 6 h, concentrated under vacuum, and then lyophilized. The extract contained a variety of platycosides such as platycoside E, deapioplatycodin D3, platycodin D3, polygalacin D3, platyconic acid A, 300 -O-acetylplatyconic acid A, platycodin D2, platycodin D, 300 -O-acetylplatycodin D2, polygalacin D2, polygalacin D, 300 -O-acetylplatycodin D, platycodin V, platycodin A, 200 -O-acetylpolygalacin D2, and 200 -O-acetylpolygalacin D [21]. (...truncated)