Solanum nigrum Polyphenol Extracts Inhibit ... - ACS Publications

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Solanum nigrum polyphenol extracts inhibit hepatic inflammation, oxidative stress, and lipogenesis in high-fat-diet-treated mice Jia-Jen Chang, Dai-Jung Chung, Yi-Ju Li, Bo-Han Wen, and Chau-Jong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03578 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 6, 2017

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Solanum nigrum polyphenol extracts inhibit hepatic inflammation, oxidative

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stress, and lipogenesis in high-fat-diet-treated mice

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Ja-Jen Chang†, Dai-Jung Chung†#, Yi-Ju Lee‡, Bo-Han Wen†, Hsing-Yu Jao†,

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Chau-Jong Wang†§*

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†

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University, Number 110, Section 1, Jianguo North Road, Taichung 402, Taiwan.

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‡

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Jianguo North Road, Taichung 402, Taiwan.

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§

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Institute of Biochemistry, Microbiology and Immunology, Chung-Shan Medical

Institute of Medicine, Chung-Shan Medical University, Number 110, Section 1,

Department of Medical Research, Chung-Shan Medical University Hospital, Number

110, Section 1, Jianguo North Road Taichung 402, Taiwan

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#Coauthor (These authors contributed equally to this work thus sharing the first

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authorship)

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*Correspondence:

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Microbiology and Immunology, Chung-Shan Medical University, Number 110,

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Section 1, Jianguo North Road, Taichung 402, Taiwan.

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E-mail: [email protected]

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Tel: +866-4-24730022 ext 11670.

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Fax: +866-4-23248167.

Professor

Chau-Jong

Wang,

Institute

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of

Biochemistry,

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Abstract

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Patients with diabetes, obesity, and hyperlipidemia are all high-risk groups for

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fatty liver; however, the mechanism of fatty liver formation is not completely

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understood. Studies have indicated that abnormal fat metabolism, oxidative stress, and

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insulin resistance are positively correlated with peroxidation and abnormal cytokine

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production. Recent studies have revealed that Solanum nigrum extracts (SNE) possess

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anti-inflammatory, antioxidation, antihyperlipidemia, and liver protection abilities.

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Therefore, the present study investigated the in vivo and in vitro effects of an SNE on

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nonalcoholic fatty liver (NAFL)-induced hepatitis. In vivo data demonstrated that the

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SNE reduced blood triglyceride, sugar, and cholesterol levels, as well as fat

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accumulation, oxidative stress, and lipid peroxidation in high-fat-diet-treated mice.

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The results indicated that the SNE downregulated the expression of fatty acid

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synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase),

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and sterol regulatory element-binding proteins (SREBPs) through the AMP-activated

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protein kinase (AMPK) pathway and upregulated the expression of carnitine

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palmitoyltransferase 1 (CPT1) and peroxisome proliferator-activated receptor alpha.

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Furthermore, we prepared an Solanum nigrum polyphenol extract (SNPE) from the

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SNE; the SNPE reduced hepatic lipid (oleic acid) accumulation. Therefore, SNE have

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the potential to alleviate NAFL-induced hepatitis, and polyphenolic compounds are

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the main components of SNE. Moreover, SNE can be used to develop health food

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products for preventing NAFL disease.

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Keywords:

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Solanum nigrum polyphenol extract, nonalcoholic fatty liver, anti-inflammatory,

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antioxidation, antihyperlipidemia

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Introduction

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Overweight and obesity are associated with several chronic diseases including

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fatty liver, metabolic syndrome, and cardiovascular disease1. Fatty liver disease is

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related to increased abdominal or visceral fat accumulation; high triglyceride (TG),

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low high-density lipoprotein (HDL) cholesterol, and high low-density lipoprotein

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(LDL) cholesterol levels; and insulin resistance. All of these lead to impaired

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metabolic regulation in adipose tissue2-3.

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Many complicated biochemical reactions occur in the liver, including the

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metabolic activities of multiple nutrients such as TG oxidation and the synthesis of

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lipoproteins, cholesterol, and phospholipids. Abnormal lipid and lipoprotein

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metabolism can result in dyslipidemia, including elevated plasma cholesterol and/or

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TG levels and reduced HDL levels, which contribute to the increased prevalence of

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morbid obesity. Several proteins associated with dyslipidemia have been reported to

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play crucial roles in lipogenesis, including fatty acid synthase (FAS)4-5, sterol

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regulatory element-binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl

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-coenzyme A reductase (HMG-CoA reductase)6-8, and the expression levels of these

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enzymes are dynamic in response to cellular energy status. Acetyl-CoA carboxylase

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(ACC) catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA in a

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biotin-dependent manner9. Malonyl-CoA is a substrate of FAS for fatty acid synthesis

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and also a potent inhibitor of carnitine palmitoyltransferase 1 (CPT1) for fatty acid

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β-oxidation. FAS is mainly involved in catalyzing the transformation of malonyl-CoA

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into palmitate during the long-term process of long-chain fatty acid catalysis, which

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catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA. FAS

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regulated by multiple transcription factors. SREBPs, a transcription factor family,

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regulate the expression of enzymes for the synthesis of fatty acids, cholesterol, TG,

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and phospholipids, suggesting that this family plays a central role in energy


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homeostasis through the modulation of glycolysis, lipogenesis, and adipogenesis. In

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humans, the SREBP gene family mainly consists of SREBP-1 and SREBP-2,

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specifically responding to lipogenic and cholesterogenic metabolism, respectively10-14.

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In addition, this increase in lipid synthesis is caused by the increased activities of

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HMG-CoA reductase, a key enzyme of cholesterol synthesis. HMG-CoA reductase is

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a membrane protein located on the endoplasmic reticulum and plays a role in

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cholesterol synthesis and mevalonate production from HMG-CoA15. In mammals,

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hepatic lipolysis is mainly controlled by several enzymes, including peroxisome

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proliferator-activated receptors (PPARs), CPT1, and AMP-activated protein kinase

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(AMPK), in response to nutritional and hormonal conditions. PPARs are members of

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the steroid hormone nuclear receptor superfamily, and they have three subtypes:

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PPARα, PPARβ (PPARδ), and PPARγ; their respective tissues and regulators play

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different roles. PPARα is highly expressed in the liver and promotes lipid uptake, fatty

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acid β-oxidation, ketogenesis, and gluconeogenesis by regulating the expression of

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genes involved in fatty acid catabolism. PPARα is thus a potential target for

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alleviating lipid disorders, diabetes, and obesity16. CPT1 is localized in the

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mitochondrial outer membrane and synthesizes acylcarnitine for fatty acid transport

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across the inner mitochondrial membrane. AMPK is widely present in mammalian

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tissues. It is a serine/threonine kinase comprising a heterotrimeric complex and is

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regarded as an energy sensor in most tissues. AMPK regulates cellular lipid

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metabolism through increased fatty acid oxidation and constitutive lipoprotein

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exocytosis. In general, AMPK is a very important factor in intracellular energy

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regulation, regulating metabolism and energy in the body17-18.

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Solanum nigrum is traditionally used as food and has some pharmacological

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effects such as fever reduction, antifever diuretics, liver protection, eyesight

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improvement, blood pressure reduction, and glucose tolerance reduction19-20. Solanum



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nigrum extract (SNE) contain high amounts of polyphenols exhibiting antioxidant and

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antitumor properties. Many herbally derived polyphenolic compounds can prevent

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obesity by inhibiting hypolipidemia and reducing adipose tissue mass, thus

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suppressing the occurrence of metabolic, hepatic, and cardiovascular alterations

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associated with obesity21. Sohrabipour et al demonstrated that administration of

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Solanum nigrum reduced the blood glucose, cholesterol, and triglycerides to normal

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level in streptozotocin-induced diabetic rats22.

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On the basis of previous reports that an SNE has anti-atherogenic properties and

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hepatoprotectivee effects, the present study was conducted to examine whether an

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SNE can reduce hepatic lipid accumulation induced by a high-fat diet (HFD). In

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addition, the hepatic regulation of lipid synthesis, oxidation, inflammation, and

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clearance was investigated.

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Experimental method

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Preparation of SNE and Solanum nigrum polyphenol extract

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In brief, Solanum nigrum was collected from the central part of Taiwan. Whole plants

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were cut into small pieces, vacuum freeze-dried, and stored at room temperature until

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further use. Dried Solanum nigrum was mixed with water for 30 min and subjected to

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continuous heat extraction at 100°C for 40 min. The resulting water extract was

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filtered and subsequently concentrated in a water bath at 90°C until it became creamy

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and was dried in an oven at 70°C. For the preparation of the Solanum nigrum

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polyphenol extract (SNPE), we mixed 100 g of dried Solanum nigrum powders in 300

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mL of ethanol, followed by heating at 50°C for 3 h. The extracts were filtered,

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followed by lyophilization under reduced pressure at room temperature. The powders

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were resuspended in 500 mL distilled water, extracted with 180 mL of ethyl acetate

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three times, resolved in 250 mL of distilled water, stored at -70°C overnight, and


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lyophilized. Furthermore, the identities of the 18 components were established from

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recorded mass spectra. The powders were resuspended in distilled water and passed

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through a 0.22-μm filter for use in cell culture.

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HPLC-UV analysis

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The SNE was separated through HPLC-UV; this process was using a Waters system

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(the in-line 4-channel Degasser AF, Waters 600 HPLC Controller and pump, 2998

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PDA detector). Separating column was by a Mightysil RP-18 GP (5 μm particle size,

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250 x 4.6 mm I.D) at room temperature. The mobile phase contained of formic acid in

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water (solution A, pH = 2.5), acetonitrile (solution B) and methanol (solution C).

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HPLC method was flowing with gradient elution program: 0 min, 100% A; 5 min,

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90% A, 0% B; 15 min, 85% A, 3% B; 20 min, 85% A, 5% B; 30 min, 85% A, 8% B;

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50 min, 85% A, 15% B; 65 min, 75% A, 25% B; 70 min, 70% A, 30% B; 80 min,

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50% A, 50% B; 85 min, 100% B; 100 min, 100% B. Flow rate was 1.0 mL/min and

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injection volume was 20 μL. The UV detection range was on 210 ~ 400 nm. Methanol

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and acetonitrile (liquid chromatography grade, 99.9% and 99+ %, respectively) were

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purchased from Merck Millipore (Germany). Formic acid (reagent grade, 96%) was

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purchased from Tedia (USA). The standers of various phenolic compounds included

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peltatoside, isoquercitrin and miquelianin (liquid chromatography grade, 99%, 95%

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and 98%, respectively). These were obtained from Extrasynthese (French).

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Animal model design

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Male C57BL/6 mice (25–30 g; age, 42 days) were purchased from Lasco Laboratory

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Animal Co. (Yilian County, Taiwan), housed in an environmentally controlled

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laboratory, and acclimatized for 7 days. All mice housed under standard laboratory

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conditions (18–23C, humidity 55–60%, and 12 h light/dark cycle) in agreement with



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animal protocols approved by the Institutional Animal Care and Use Committee of the

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Chung Shan Medical University (IACUC approval No. 740), Taichung, Taiwan. We

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used an HFD to induce nonalcoholic fatty liver (NAFL) in the animal model. The feed

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was pulverized to prepare high-lipid feed by using lard and cholesterol in the

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preparation of high-lipid formulations. In the HFD group, the contents of lard and

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cholesterol were 20% and 2%, respectively. Lard and cholesterol were mixed

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uniformly after the addition of water. The formulation for the high-lipid feed was

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based on a reference. The animals were divided into five dietary groups (n =

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12/group) : (A) control diet group, (B) HFD group, and(C, D, E) diet containing HFD

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group (0.5% SNE ;1% SNE, and 2% SNE group). After the completion of the

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experiment, the mice were killed, and liver and blood samples were collected for

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further examination. Plasma was separated by centrifugation at 5000 g for 10 min at

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4°C and was then maintained at −20°C for additional analyses (Commission Permit

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No. 1235).

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Histological examination of the liver

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After the mice were killed, the liver was immediately fixed in 10% buffered

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formaldehyde for 1 day and then maintained in a decalcification solution. After 3 days,

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the specimens were reimmersed in formaldehyde, and hematoxylin and eosin (H&E)

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staining was performed for histological examination. The liver section was observed

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under high-power fields (× 200).

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Immunohistochemical staining

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A formalin-fixed paraffin-embedded liver section was analyzed to determine the

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expression of inflammation markers in the liver. The bone section was incubated in 10

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mmol/L citrate-Tween buffer at 100°C and assessed using the Thermo-HRP system.


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Finally, the results were developed by reacting the liver section with 3,3ʹ-

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diaminobenzidine (DakoCytomation, Carpinteria, CA, USA) and subjecting it to H&E

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staining and subsequent microscopic examination. Furthermore, the section was

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conjugated with a mouse monoclonal antibody against interleukin (IL)-6 (1:200;

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Abcam Co, Cambridge Science Park, Cambridge, UK) and tumor necrosis

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factor-alpha (TNF-α; 1:200). The intensity of IL-6- and TNF-α-positive cells in a

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cross section was observed by examining five randomly selected areas of the section

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under high-power fields (×200). Image Pro Plus 4.0 was used to quantify the

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integrated optical density within the positive areas.

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Cell Line and Cell Culture

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Human hepatoma (HepG2) cells were obtained from the American Type Culture

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Collection and grown in Dulbecco’s modified Eagle’s medium supplemented with

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10% fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, and 2 mM

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L-glutamine (HyClone R, Thermo Scientific, Logan, UT, USA). The cells were

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cultured at 37°C in a humidified atmosphere of 95% air to 5% CO2.

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Cytotoxicity assay

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HepG2 cells were seeded at a density of 1 × 10 5 cells/well into a 24-well plate and

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treated with oleic acid (OA) and the SNPE at various concentrations for 24 h. The

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MTT reagent (0.5 mg/mL) was added to each well and incubated for 4 h. The medium

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was removed, and isopropanol was added to dissolve the MTT–formazan complex.

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The absorbance was measured at 563 nm with a spectrophotometer (Hatachi 3210,

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Hitachi, Tokyo, Japan).

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Oil red staining

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HepG2 cells were seeded in a six-well plate (3 × 106 cells/well) and treated with 500

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mM OA and indicated concentrations of the SNPE for 24 h. The cells were washed

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two times with phosphate-buffered saline (PBS), fixed with 4% formaldehyde in PBS

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for 1 h, and then stained with 1 μg/mL oil red for 30 min at room temperature. After

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the staining process, the lipid distribution in cells was immediately analyzed using a

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FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). In addition,

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lipid-bound Nile red fluorescence was detected through inverted fluorescence

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microscopy.

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Western blot analysis

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HepG2 cells were seeded in a 10-cm dish (3 × 106 cells/well) and treated with 500

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mM OA at indicated concentrations of the SNPE for 24 h. The proteins were

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harvested from the cells in a cold radioimmunoprecipitation assay buffer (1% NP-40,

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50 mM Tris-base, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholic acid, 150

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mM NaCl, pH 7.5) containing leupeptin (1.7 μg/mL) and sodium orthovanadate (10

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μg/mL). The cell mixture was vortexed at 4°C for 4 h. All mixtures were then

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centrifuged at 12000 rpm and 4°C for 10 min, and the protein contents of the

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supernatants were determined with the Coomassie Brilliant Blue total protein reagent

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(Kenlor Industries, Costa Mesa, CA, USA) by using bovine serum albumin (BSA) as

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the standard. Equal amounts of protein samples were subjected to SDS–

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polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes

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(Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk

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powder with 0.1% Tween-20 in tris-buffered saline and then incubated with the first

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antibody at 4°C overnight. Thereafter, the membranes were washed three times with

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0.1% Tween-20 in PBS and incubated with the secondary antibody to antimouse

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horseradish peroxidase (GE Healthcare, Little Chalfont, Buckinghamshire, UK).


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Antibodies against AMPK and phospho-AMPK (AMPK-P) were purchased from Cell

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Signaling Technology (Beverly, MA, USA). FAS, SREBP-1c, and CPT1 antibodies

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were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody

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against

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(Charlottesville, VA, USA). Band detection was revealed through enhanced

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chemiluminescence (ECL) by using ECL Western blotting detection reagents and

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exposed ECL hyper film in FUJIFILM LAS-3000 (Tokyo, Japan). The proteins were

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quantified through densitometry by using FUJIFILM-Multi Gauge V2.2 software

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(FUJIFILM, Stockholm, Sweden).

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Statistical analysis

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Data are reported as the mean ± standard deviation of three independent experiments

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and were evaluated using the Student t test. A P value of

Solanum nigrum Polyphenol Extracts Inhibit ... - ACS Publications

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