Ginger Essential Oil Ameliorates Hepatic Injury and Lipid

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Ginger essential oil ameliorates hepatic injury and lipid accumulation in high fat diet-induced nonalcoholic fatty liver disease Yi-Syuan Lai, Wan-Ching Lee, Yu-En Lin, Chi-Tang Ho, Kuan-Hung Lu, Shih-Hang Lin, Suraphan Panyod, Yung-Lin Chu, and Lee-Yan Sheen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06159 • Publication Date (Web): 21 Feb 2016 Downloaded from http://pubs.acs.org on February 21, 2016

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Journal of Agricultural and Food Chemistry

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Ginger essential oil ameliorates hepatic injury and lipid accumulation in high fat

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diet-induced nonalcoholic fatty liver disease

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Yi-Syuan Lai,§, ⊥ Wan-Ching Lee,§ Yu-En Lin,§ Chi-Tang Ho,§,& Kuan-Hung Lu§,

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Shih-Hang Lin,§ Suraphan Panyod,§ Yung-Lin Chu,＃ and Lee-Yan Sheen＊,§,∇,○

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§

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Education and Research,

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University, Taipei 10617, Taiwan

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Institute of Food Science and Technology,

⊥

∇

National Center for Food Safety

○

Center for Food and Biomolecules, National Taiwan

Department of Hospitality Management, Yu Da University of Science and

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Technology, Miaoli 36143, Taiwan

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&

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08901, USA

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＃

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University of Science and Technology, Pingtung 91201, Taiwan

Department of Food Science, Rutgers University, New Brunswick, New Jersey

International Master’s Degree Program in Food Science, National Pingtung

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* Corresponding author: Prof. Lee-Yan Sheen, Institute of Food Science and

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Technology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei

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10617,

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[email protected]

Taiwan.

Tel:

886-2-33664129;

Fax:

886-2-2362-0849;

1

ACS Paragon Plus Environment

E-mail:


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ABSTRACT

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The objective of this study was to investigate the hepatoprotective efficacy and

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mechanism of action of ginger essential oil (GEO) against the development of

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nonalcoholic fatty liver disease (NAFLD). Mice were maintained on either a control

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diet or high-fat diet (HFD) supplemented with GEO (12.5, 62.5, and 125 mg/kg) or

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citral (2.5 and 25 mg/kg) for 12 weeks. We demonstrated that GEO and its major

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component (citral) lowered HFD-induced obesity in a dose-dependent manner,

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accompanied by antihyperlipidemic effects by reducing serum free fatty acid,

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triglyceride, and total cholesterol levels. Moreover, liver histological results showed

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that administration of 62.5 and 125 mg/kg GEO and 25 mg/kg citral significantly

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reduced hepatic lipid accumulation. Further assessment by western blotting and

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investigation of the lipid metabolism revealed that hepatic protein expression of sterol

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regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC),

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fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl-coenzyme A reductase

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(HMGCR), and cytochrome P450 2E1 (CYP2E1) were down-regulated by GEO and

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citral, indicating that GEO and citral suppressed HFD-stimulated lipid biosynthesis

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and oxidative stress. Furthermore, GEO and citral effectively enhanced the

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antioxidant capacities and reduced inflammatory response in mouse liver, which

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exerted protective effects against steatohepatitis. Collectively, GEO and citral

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exhibited potent hepatoprotective effects against NAFLD induced by HFD in obese

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mice. Thus, GEO might be an effective dietary supplement to ameliorate

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NAFLD-related metabolic diseases, and citral could play a vital role in its

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management.

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Keywords: ginger essential oil, citral, non-alcoholic fatty liver disease, lipogenesis,

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obesity

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INTRODUCTION

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Nonalcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver

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disease and its prevalence is rapidly increasing due to strong association with obesity,

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dyslipidemia, type 2 diabetes mellitus, and hypertension.1 Epidemiologic surveys

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indicate that the prevalence of NAFLD is approximately 6–35% in the general

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population, but is 30–63% in obese adults.2 Moreover, in 25% of this high-risk 2


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population, NAFLD may result in nonalcoholic steatohepatitis (NASH), and 42% of

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this group had advanced fibrosis. In severe cases, liver failure or cancer may develop.

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Thus, the prevalence of NAFLD is much higher in patients with metabolic diseases

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caused by obesity than in non-obese populations.3

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Several factors are involved in the pathologic metabolic mechanisms that

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ultimately lead to the accumulation of liver fat and NAFLD progression. However, the

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mechanisms involved in the initiation and progression of NAFLD are yet to be fully

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clarified. Consolidated data indicate that lipid metabolism and oxidative stress may

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play a critical role in the development of obesity-triggered NAFLD. Research has

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shown that high calorie diets high in saturated fats can result in storage of excess fat

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in adipose tissue, causing adipocyte hypertrophy and adipose tissue expansion leading

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to obesity. During metabolic processing, free fatty acids (FFA) released into the liver

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stimulate the expression of the key factor controlling cholesterol synthesis,

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3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), as well as the critical fatty

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acid synthesis factors, sterol regulatory element-binding protein-1c (SREBP1-c),

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acetyl coenzyme A carboxylase (ACC), and fatty acid synthase (FAS). Together, these

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enzymes promote synthesis of liver lipids, such as cholesterol and triglycerides (TG).

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Meanwhile, fatty acid metabolism and beta-oxidation decrease due to inhibition of the

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regulatory factors peroxisome proliferator–activated receptor-α (PPARα) and

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carnitine palmitoyltransferase I (CPT-1). This results in abnormal liver lipid

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metabolism and leads to the development of hepatic steatosis, further hepatic lipid

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accumulation, and NAFLD.4 In addition, fatty acid activation by excess hepatic FFA,

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upregulates the expression of cytochrome P450 isoform 2E1 (CYP2E1) and increases

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the production of reactive oxygen species (ROS). Subsequently, acute oxidative stress

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and increased liver inflammation results in hepatocyte damage, necrosis, and

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progression of fibrosis.5 Given the high prevalence of NAFLD and the lack of

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satisfactory treatments, many studies aim to develop diet replacement therapies for

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preventing and treating the disease.6

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Ginger (Zingiber officinale Roscoe), the rhizomes of ginger, is a popular spice

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and herbal medicine for the improvement of obesity,7 cancer,8 diabetes,9,

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atherosclerosis,11 and fructose-induced fatty liver.12 Recently, studies have

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demonstrated that ginger essential oil (GEO), the volatile oil of fresh ginger, which is

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produced by steam distillation, has the potential of reducing experimental colitis 3


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symptoms,13 inhibiting gastric ulcers in the stomach,14 and protecting hepatocytes

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against acute ethanol-induced fatty liver.15 The chemical composition of GEO has

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been characterized, of which the main components are citral,α-zingiberene, camphene,

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α-farnesene, and β-sesquiphellandrene.16 Also, citral has been proven to be the major

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component and has antiadipogenic effects against energy-intense diet-induced

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obesity.17

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However, limited studies have been performed on the effect of GEO and its

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bioactive compound on the molecular biological mechanism of NAFLD induced by

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HFD.18 Therefore, the purpose of this study was to assess the efficacy of GEO or

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citral on HFD-induced NAFLD in C57BL/6J mice and to identify the possible

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mechanisms involved in preventing the progression of NAFLD. The present study can

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provide experimental evidence of GEO and citral as functional food ingredients.

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MATERIALS AND METHODS

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GEO Extraction and Mainly Ingredient Analysis

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Samples of ginger (Zingiber officinale Roscoe) were purchased from the

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Mingjian Township Farmers' Association of Nantou County (Nantou, Taiwan). The

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collected ginger was thoroughly washed under running water to remove all dirt.

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Whole rhizome of ginger was cut into small pieces (5 mm of thickness) and stirred

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with 3 volumes of distilled water. The mixture was subjected to steam distillation for

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2–3 h, and the volatile extracts were then dehydrated to obtain GEO. The resulting oil

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was stored at a temperature of -20°C for further analysis. The yield of the obtained oil

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from fresh ginger was 0.12% (w/w), calculated according to the weight of ginger used

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in the extraction procedure. GEO analyses were performed using a gas chromatograph

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(GC, Thermo Scientific Focus GC) equipped with an AI 3000 II autosampler, a

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Stabilwax (Crossbond Carbowax-PEG, Restek) capillary column (60 m length, 0.32

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mm internal diameter, 1.0 µm-thick film), and a flame ionization detector. Nitrogen

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was used as the carrier gas at a flow rate of 30 mL/min with a split ratio of 80:1. The

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analysis conditions were as follows: initial column temperature 50°C; an increase of

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5°C/min to 200°C held for 10 min; injector temperature 200°C and detector

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temperature 200°C; and an injection volume of 1 µL (diluted 1:100 in acetone).

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Calibration curves were constructed with reference standards and used to determine

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the concentration of citral in GEO. Citral (purity 95%) was purchased from

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Sigma-Aldrich Corp. (St. Louis, MO, USA).

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Animals and Experimental Design

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Six-week-old male C57BL/6J mice were obtained from the Animal Center of the

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College of Medicine, National Taiwan University (Taipei, Taiwan). The animals were

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maintained in accordance with the National Taiwan University guidelines for the care

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and use of laboratory animals. The animals were housed in an alternate light/dark

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cycle (12 h) room with a temperature of 23 ± 2°C and a relative humidity of 50–70%.

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All mice were fed experimental diets ad libitum with free access to drinking water at

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all times. After two weeks of adaptive feeding, the mice were randomly assigned to

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groups of 8 animals each and fed different experimental diets as follows: mice in the

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control group were fed a normal chow diet with 13.5% kcal fat content (Laboratory

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Rodent Diet 5001, Lab Diet/PMI Nutrition International, Purina Mills LLC, Gray

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Summit, MO), while mice in the negative control group and treatment groups were

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fed an HFD with 60% kcal fat content (D12492, Research Diets, Inc., New Brunswick,

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NJ) for 12 weeks to induce a steatosis condition. Mice in the treatment groups were

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orally administered either GEO (12.5, 62.5, or 125 mg/kg) or citral (2.5 or 25 mg/kg).

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Citral was comparable amount with the current GEO sample (19.8% of GEO). The

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control and HFD groups of mice were subjected to gavage with the same volume of

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olive oil. Food intake and body weight were measured weekly for the duration of the

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experiment. At the study endpoint, mice were fasted overnight and sacrificed by

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carbon dioxide asphyxiation. Blood samples was collected by cardiac puncture and

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separated by centrifugation at 1000 × g for 10 min at 4°C to collect serum samples.

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The liver, subcutaneous adipose, epididymal adipose, and perirenal adipose were

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harvested and weighed.

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Serum Biochemical Analysis The

levels

of

serum

biochemical

parameters,

including

aspartate

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aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), and

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TG were measured colorimetrically and assayed by an automatic biochemistry

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analyzer (SPOTCHEM EZ SP-4430, ARKRAY, Inc., Kyoto, Japan) according to the

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manufacturer’s instructions. Serum levels of insulin and FFA were measured using 5



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commercial kits obtained from Mercodia AB and BioVision, respectively.

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Histological Staining

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To examine the morphology, the largest lobe of the liver tissue was fixed with

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the 10% neutral-buffered formalin solution and then embedded in paraffin serially

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sectioned at 5 µm stained with hematoxylin and eosin (H&E). Histological scoring, as

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an indicator of the degree of fatty liver, was performed by an experienced pathologist.

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Hepatic steatosis was graded as follows: grade 1 = minimal (

Ginger Essential Oil Ameliorates Hepatic Injury and Lipid

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