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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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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