Metabolomics of Ginger Essential Oil against Alcoholic Fatty Liver in

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Metabolomics of Ginger Essential Oil against Alcoholic Fatty Liver in Mice Chun-Ting Liu, Raghu Rajasekaran, Shu-Hsi Lin, San-Yuan Wang, Ching-Hua Kuo, Yufeng J. Tseng, and Lee-Yan Sheen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 30 Oct 2013 Downloaded from http://pubs.acs.org on November 3, 2013

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Journal of Agricultural and Food Chemistry

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Metabolomics of Ginger Essential Oil against Alcoholic Fatty Liver in Mice

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Chun-Ting Liua, Rajasekaran Raghua,b, Shu-Hsi Lina, San-Yuan Wangd, Ching-Hua

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Kuoc, Yufeng J. Tsengd, and Lee-Yan Sheena,*

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a

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National Taiwan University, Taipei 106, Taiwan

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b

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Taiwan University, Taipei 106, Taiwan (Present address)

Institute of Food Science and Technology, College of Bio-Resources and Agriculture,

Department of Horticulture, College of Bio-Resources and Agriculture, National

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c

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Taiwan

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d

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Electrical Engineering and Computer Science, National Taiwan University, Taipei 106,

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Taiwan

School of Pharmacy, College of Medicine, National Taiwan University, Taipei 106,

Graduate Institute of Biomedical Electronics and Bioinformatics, College of

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Corresponding author:

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* To whom correspondence should be addressed: No. 1, Section 4, Roosevelt Road,

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Taipei 106, Taiwan

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Tel: 886-2-33664129; Fax: 886-2-23620849; E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Fatty liver is significantly associated with hepatic cirrhosis and liver cancer.

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Excessive alcohol consumption causes alcoholic fatty liver disease (AFLD). Ginger

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has been reported to exhibit antioxidant potential and hepatoprotective activity. In the

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present study, a mouse model for AFLD was developed by employing male C57BL/6

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mice who were fed an alcohol-containing liquid diet (Lieber-DeCarli diet) ad libitum.

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In the treatment groups, ginger essential oil (GEO) and citral were orally administered

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every day for 4 weeks. Serum biochemical analysis, antioxidant enzyme activity

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analysis, and histopathological evaluation revealed that GEO and citral exhibited

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hepatoprotective activity against AFLD. Metabolites in serum samples were profiled

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by high-performance liquid chromatography coupled with quadrupole time-of-flight

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mass spectrometry (HPLC-QTOF-MS). Metabolomic data indicated the amounts of

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metabolites such as

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lithocholic acid, 2-pyrocatechuic acid, and prostaglandin E1 were increased after

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alcohol administration, but the levels were recovered in treatment groups. Our

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analysis indicated that ginger possesses hepatoprotective properties against AFLD.

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Further, these metabolites can serve as early non-invasive candidate biomarkers in the

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clinical application of AFLD for health management.

D-glucurono-6,3-lactone,

glycerol-3-phosphate, pyruvic acid,

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Keywords: AFLD, ginger, Lieber-DeCarli diet, metabolomics, biomarker

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Introduction

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The liver is the largest organ in the body, and it plays important roles in the

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metabolism. Liver diseases are mainly caused due to viruses, excessive alcohol

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consumption, and chemicals. Repeated damage-induced inflammation causes hepatitis,

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hepatic fibrosis, cirrhosis, and even death due to hepatic failure.1-3

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Fatty liver, a chronic liver disease, is a common global problem. Fatty liver can

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result in hepatitis, hepatic fibrosis, hepatic cirrhosis, liver cancer, or cardiovascular

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diseases. The pathogenesis of fatty liver is generally divided into non-alcoholic and

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alcoholic types. Excessive ethanol consumption can cause alcoholic fatty liver disease

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(AFLD).4,5 The mechanisms of AFLD are mediated by increased expression of

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cytochrome P450-2E1 (CYP2E1),6 modulation of inflammatory cytokines TNF-α,

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IL-1, and IL-6,7 and decreased hepatocyte retinoid x receptor levels.8,9

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To analyze the relationship among alcohol, lipid, and therapeutic agents, the

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Lieber-DeCarli liquid diet model is frequently used to induce AFLD in animals,

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especially in the early stages of AFLD, including liver injury, steatosis, and oxidative

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stress.10,11

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Ginger (Zingiber officinale R.) and its extracts have been used widely as Chinese

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medicine for a long time; it has various physiological functions such as decreasing

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nausea,12 enhancing functions of the intestines and stomach,13 preventing

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cardiovascular diseases,14 promoting liver functions,15 and inhibiting cancer cell

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proliferation.16 Among a variety of applications, ginger essential oil (GEO) is

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commonly used in food, cosmetics, and beverage industries. Notably, citral, the main

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constituent of GEO, has been demonstrated to exhibit antioxidant, anti-cancer, and

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anti-candida effects.17-19

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Metabolomics studies analyze the metabolome, which are the metabolites

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synthesized by a biological system and end-products of gene expression in a

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sample.20,21 In metabolomics, blood, urine, and tissues are evaluated to generate

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individual metabolite profiles such as cholesterol and triglycerides profiles. In this

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study, we investigated the preventive effects of ginger on the formation of alcoholic

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fatty liver by using a metabolomics approach.

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Here, we evaluated the hepatoprotective efficacy for GEO and citral by using the

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Lieber-DeCarli diet-induced AFLD model. Moreover, we adopted metabolomics

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approaches to identify differentially regulated metabolites in healthy mice and mice

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with AFLD. These metabolites may serve as candidate biomarkers for non-invasive

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detection during early stages of AFLD, and this approach could be useful for humans.

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Materials and methods

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Chemicals

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Solvents used in high-performance liquid chromatography coupled with

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quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) were of MS grade.

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Water and acetonitrile were obtained from Scharlau (Sentmenat, Spain) and J. T.

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Baker (Phillipsburg, NJ, USA), respectively. Reagents were of analytical grade and

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were obtained from Sigma-Aldrich (Steinheim, Germany). Hexane, chloroform, and

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dichloromethane were procured from Merck (Darmstadt, Germany).

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Preparation of ginger essential oil extracts and citral

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GEO was prepared by the steam distillation method.22 Briefly, 1 kg of ginger

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(Zingiber officinale R.) was added to 3 L of distilled water and distilled to extract the

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essential oil. The constituents of GEO were analyzed by a Thermo Scientific Focus

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GC equipped with an AI 3000 II autosampler, a flame ionization detector, and a

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Stabilwax (crossbond Carbowax-PEG, Restek) column (60 m × 0.32 mm; 1.0 µm).

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Standard citral (purity >95%) was used. The composition of GEO analyzed by gas

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chromatography (GC) and the representative profile (Figure 1) indicates citral (neral

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and geranial) as the major components in GEO (approximately 30 %). The

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composition of GEO was similar to previous research23, which employed the same

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species of ginger used in the present study. Citral that was used for the animal

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experiments was obtained from Sigma-Aldrich.

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In vivo animal model of alcoholic fatty liver disease

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Six-week-old male C57BL/6 (B6) mice (BioLASCO Taiwan Co.) were

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maintained at the animal housing facility of the Institute of Food Science and

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Technology, National Taiwan University at a temperature of 23°C ± 2°C and relative

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humidity of 50–70% with a 12-h light/dark cycle conditions. Mice were divided into a

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normal control group, an AFLD group, and 4 treatment groups. Mice in the normal

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control group were fed with normal liquid diet, whereas those in the AFLD and

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treatment groups were fed with ethanol-containing Lieber-DeCarli Diet. Each

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treatment group received either GEO (2.5 or 12.5 mg/kg body weight [BW]) or citral

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(0.375 or 1.875 mg/kg BW), both of which were mixed with olive oil (vehicle) and

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administered orally every day for 4 weeks. Normal and AFLD groups were given the

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same volume of only olive oil (vehicle). The BW of each mouse was recorded every

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week during the entire period of the experiment. Mice were sacrificed after 4 weeks;

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serum sample were then collected and livers were sampled. Mice were handled

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according to the guidelines issued by the National Taiwan University Institutional

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Animal Care and Use Committee (Approval Number: NTU-IACUC-99-53)

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Serum biochemical and enzyme analysis

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Serum was extracted by centrifuging coagulated blood at 1000 × g for 5 min at

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4°C. The biochemical parameters of liver function indicators such as aspartate

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aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), and total

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cholesterol (TC) were estimated with commercial test strips (Commercial ALT and

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AST Spotchem™ II reagent strips, Arkray Inc., Kyoto, Japan) in an automatic blood

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analyzer (Spotchem™ EZ).

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Liver enzyme activities

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Liver homogenate was prepared by homogenizing liver (0.5 g) in 10 volumes of

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ice-cold

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and 1.5% KCl, pH 7.4) at 4°C. The homogenate was then centrifuged at 10000 × g for

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30 min at 4°C. The supernatant was stored at -80°C and used to detect liver TG and

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hepatic antioxidant enzyme activity. Protein content in the homogenate was estimated

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spectrophotometrically by measuring the absorbance at 595 nm using the Bio-Rad

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protein Assay kit (Hercules, CA, USA). Commercial kits purchased from the Cayman

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Chemical Company (Ann Arbor, MI, USA) were used to examine the hepatic

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antioxidant systems of glutathione (GSH; item number 703002), glutathione

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peroxidase (GPx; item number 703102), glutathione reductase (GRd; item number

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703202), catalase (CAT; item number 707002), and superoxide dismutase (SOD; item

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number 706002).

homogenization

buffer

(8

mM

KH2PO4, 12

mM

K2HPO4,

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Liver biopsy examination

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Liver histological sections for pathological staining and semi-quantitative

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analysis were performed from the right lobe of the liver to avoid observational bias.

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For histopathological observations, formalin-fixed paraffin-embedded (FFPE) liver

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sections were observed for liver fatty accumulation, necrosis, fibrosis, and other

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changes in liver tissues, after staining with Hematoxylin and Eosin (H&E). Scoring

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and interpretation of biopsy results were carried out by an experienced pathologist,

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College of Veterinary Medicine Animal Disease Diagnostic Center, National Chung

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Hsing University, Taichung, Taiwan.

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Metabolic profiling

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(i) Sample preparation

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Aliquots of 100 µL serum were added to extraction solvents (1:4) and extracted

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by the Geno Grinder 2010 (SPEX, Pittsburg, CA, USA) at 1000 rpm for 2 min.

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Extracts were centrifuged at 15,000 × g for 5 min at 4°C. The supernatant was

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collected and evaporated by a centrifugal vaporizer (EYELA, Tokyo, Japan) for 2 h.

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The residue was reconstituted in 200 µL of 50% methanol and centrifuged at 15000 ×

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g for 5 min. The supernatant was filtered with a 0.2-µm Minisart RC 4 filter (Sartorius,

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Goettingen, Germany) and subjected to HPLC-QTOF-MS analysis. The same

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amounts of sample aliquots were pooled for the QC sample.

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(ii) Chromatographic and mass spectrometric analysis

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All serum samples were analyzed by an Agilent 1290 U-HPLC system coupled

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with 6540-QTOF (Agilent Technologies, Santa Clara, CA, USA). The sample (2 µL)

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was injected into an ACQUITY UPLC HSS T3 Column (2.1 × 100 mm; 1.8 µm)

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(Waters, Milford, MA, USA). The mobile phase consisted of 0.1% formic acid

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(solvent A) and acetonitrile (solvent B). The gradient profile used was: 0–1.5 min: 2%

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B; 1.5–9 min: linear gradient from 2% to 50% B; 9–14 min: linear gradient from 50%

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to 95% B; 14–15: 95% B, and then the column was re-equilibrated. The flow rate was

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maintained at 0.3 mL/min. The column oven was set at 40°C, and the auto-injection

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system was set at 10°C. A Jet Stream electrospray ion source with capillary voltage of

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4 kV in positive mode and 3.5 kV in negative mode were used for sample ionization.

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MS parameters were set as follows: dry gas temperature (325°C), dry gas flow (5

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L/min), nebulizer (40 psi), sheath gas temperature (325°C), sheath gas flow (10

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L/min), and fragmentor (120 V). The scan range was 50–1700 m/z.

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(iii) Data analysis

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The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to

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identify metabolite biomarkers by comparing molecular weights. The molecular

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structure of candidate compounds were retrieved by comparison, and then confirmed

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by MS scans for characteristic ions and fragmentation patterns of metabolites.

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3-Dimensional principal component analysis (3D-PCA) was performed and the values

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of PC1, PC2, and PC3 were plotted with designation of cases as control, AFLD, or

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treatment groups.

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Statistical analysis

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Statistical differences between groups were compared by one-way analysis of

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ANOVA software with the Duncan’s multiple comparison test by SPSS

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software (Statistical Package for Social Sciences, SPSS Corporation, Chicago, USA).

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All values are expressed as the mean ± SD.

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Results and discussion

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In vivo model for alcohol-induced fatty liver

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Establishing an ideal rodent model for the study of alcoholic fatty liver is a

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prerequisite for this experiment. Since metabolic rates considerably differ among

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species, optimization and validation of the model is necessary. In our analysis,

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C57BL/6 mice were employed to develop a model for alcoholic fatty liver using the

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Leiber-DeCarli liquid diet. The ability to induce visible phenotypic symptoms of

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alcohol-induced liver disease such as steatosis, inflammation, and necrosis is a

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benchmark for the model’s success. Many studies have failed to induce hepatitis

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coupled with fatty liver.24,25 However, consumption of alcohol-containing

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Leiber-DeCarli liquid diet for 28 days has been shown to result in alcoholic fatty liver,

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inflammation, and necrosis in C57BL/6 mice.26

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Hepatoprotective activity of ginger on AFLD

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Here, we induced similar effects in C57BL/6 mice after feeding for 28 days. The

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AFLD group recorded a significant (p < 0.05) increase in the serum AST and ALT

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activity level after 28 days of feeding, whereas mice fed with control Leiber-DeCarli

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liquid diet (normal control group) exhibited no liver damage. The AFLD group

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exhibited a significant (p < 0.05) increase in TC and TG levels when compared to the

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normal control group. Moreover, AST, ALT, TC, and TG levels were recovered

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significantly in the treatment groups compared to the AFLD group (p < 0.05),

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especially in mice gavaged with high doses of GEO (Table 1). GSH is an important

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indicator of the liver antioxidant system. GSH levels were significantly decreased in

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the AFLD group compared to the normal control group. Similarly, liver antioxidant

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enzyme activity viz, GPx, GRd, catalase, and SOD were lower in the AFLD group

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than in the control group. However, a significant increase was observed in the

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antioxidant activity of these enzymes in the treatment groups (p < 0.05; Table 1)

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compared to the AFLD group. It has been reported that ginger ameliorates fatty liver

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and hypertriglyceridemia in rats.27 In addition, results of other studies have confirmed

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that ginger exerts hepatoprotective properties against liver damage by bromobenzene,

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acetaminophen, carbon tetrachloride, and ethanol.28,29 In the present study, the

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hepatoprotective activity of ginger on AFLD was found to be similar to that reported

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previously. Histopathological observation of liver sections from the control group

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exhibited a normal liver architecture, whereas fatty infiltration with macro-vesicles

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was observed in the livers of the AFLD group. However, compared to AFLD group,

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there was a significant improvement in treatment groups gavaged with a high dosage

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of GEO or citral (Table 2; Figure 2). Recently, more research has focused on herbal

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remedies for alcoholic liver diseases.30 Garlic and ginger are familiar, traditional

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Chinese herbs; it has been reported that garlic exhibits liver protection properties in

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alcohol-induced fatty liver.31 Our study results show that ginger provided

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hepatoprotection to alcohol-induced fatty liver. Hence, ginger may be a potential

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hepatoprotective candidate against AFLD.

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Metabolic profiling of serum samples of AFLD

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Metabolomes of serum samples from control, AFLD, and treatment groups were

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profiled by U-HPLC/Q-TOF MS. The metabolites with a significant difference among

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groups are listed in Table 3. We investigated the overall metabolic differences by

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using the 3D-PCA method. 3D-PCA was used as an unsupervised statistical method

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to study the metabolome differences between AFLD, treatment mice, and healthy

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controls. The score plots of 3D-PCA are displayed in Figure 3. Results revealed that

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PC1 accounted for 54% of variations in the 3D-PCA model, PC2 for 22%, and PC3

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for 15%; these extracts clearly enabled differentiation. These separation results

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indicate the

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differences among the control, AFLD, and treatment groups. A one-way ANOVA

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followed by Duncan’s multiple comparison test was used to analyze the statistically

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significant differences of metabolites among the control, AFLD, and treatment groups.

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Levels of D-glucurono-6,3-lactone, glycerol-3-phosphate, and pyruvic acid, which are

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important metabolites in many metabolic pathways, were significantly increased (p

Metabolomics of Ginger Essential Oil against Alcoholic Fatty Liver in

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