The Potential of Flavonoids in the Treatment of Non-alcoholic Fatty Liver Disease

Abstract

The contemporary pathophysiological model of non-alcoholic fatty liver disease (NAFLD) consists of multiple parallel pathways with a dynamic cross talk that cumulate in steatosis and inflammation and
ultimately fibrosis, cirrhosis, liver failure and hepatocellular carcinoma. So far, no pharmacological treatment has been approved. A major impediment of drugs in general is that they are intended to act
6 on one single target in the pathology of a disease. However, the multitude of pathways involved in the
7 pathogenesis of NAFLD underpins the need for treatments that address these various pathways.
Interestingly, flavonoids have been found to have positive effects on lipid metabolism, insulin
resistance, inflammation and oxidative stress, the most important pathophysiological pathways in
NAFLD. This puts flavonoids in the spotlight for the treatment of NAFLD and prompted us to review
the existing evidence for the use of these food derived compounds in the treatment of NAFLD.

Full text

For Peer Review Only
The potential of flavonoids in the t
reatment of non
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alcoholic
fatty liver disease
Journal:
Critical Reviews in Food Science and Nutrition
Manuscript ID:
BFSN-2014-1244.R1
Manuscript Type:
Review
Date Submitted by the Author:
04-Aug-2014
Complete List of Authors:
Wier, Bregje; Maastricht University, Toxicology
Koek, Ger; Maastricht University Medical Centre, Internal Medicine,
Division of Gastroenterology/Hepatology
Bast, Aalt; Maastricht University, Department of Toxicology
Haenen, Guido; Maastricht University, Toxicology
Keywords:
non-alcoholic steatohepatitis, polyphenols, oxidative stress, antioxidants,
NAFLD
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Critical Reviews in Food Science and Nutrition

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The potential of flavonoids in the treatment of non-alcoholic fatty liver
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disease
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Bregje van de Wier
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, Ger H. Koek
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, Aalt Bast
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and Guido R.M.M. Haenen
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4
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1. Department of Toxicology, Maastricht University, The Netherlands 6
2. Department of Internal Medicine, Division of Gastroenterology/Hepatology, Maastricht University 7
Medical Centre, The Netherlands 8
9
Email: 10
b.vandewier@maastrichtuniversity.nl; gh.koek@mumc.nl; a.bast@maastrichtuniversity.nl; 11
g.haenen@maastrichtuniversity.nl 12
13
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Key words: 15
NAFLD, non-alcoholic steatohepatitis, polyphenols, oxidative stress, antioxidants. 16
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Corrsponding author: 1
B. van de Wier 2
Maastricht University, Department of Toxicology 3
P.O. Box 616, 6200 MD, Maastricht, The Netherlands 4
Tel: 043-3881097, Fax: 043-3884146 5
Email: b.vandewier@maastrichtuniversity.nl 6
7
List of abbreviations: 8
NAFLD = non-alcoholic fatty liver disease, NASH = non-alcoholic steatohepatitis, FFA = free fatty 9
acids, VLDL = very low density lipoprotein, SREBP-1c = sterol regulatory element binding protein-10
1c, ChREBP = carbohydrate response element binding protein, gk = glucokinase, FAS = fatty acid 11
synthase, ACC = acetyl-CoA carboxylase, ROS = reactive oxygen species, ER = endoplasmic 12
reticulum, GSH = glutathione, SOD = superoxide dismutase, GPx = glutathione peroxidase, PPAR = 13
peroxisome proliferator activated receptor, LXRα = liver X-receptor α, AMPK = 5' adenosine 14
monophosphate-activated protein kinase, HO-1 = heme-oxygenase 2, AREs = antioxidant response 15
elements, nrf2 = nuclear factor erythroid derived 2, IKK = IκB kinase complex, IκB = NFκB inhibitor 16
protein, PKC = protein kinase C, COX = cyclooxygenase, iNOS = nitric oxide synthase, NO = nitric 17
oxide, TNF-α = tumor necrosis factor α, ALT = alanine transaminase, AST = aspartate transaminase, 18
γGT = gamma-glutamyl transpeptidase, EGCG = epigallocatechin-gallate, GTE = green tea extract. 19
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Abstract
1
The contemporary pathophysiological model of non-alcoholic fatty liver disease (NAFLD) consists of 2
multiple parallel pathways with a dynamic cross talk that cumulate in steatosis and inflammation and 3
ultimately fibrosis, cirrhosis, liver failure and hepatocellular carcinoma. So far, no pharmacological 4
treatment has been approved. A major impediment of drugs in general is that they are intended to act 5
on one single target in the pathology of a disease. However, the multitude of pathways involved in the 6
pathogenesis of NAFLD underpins the need for treatments that address these various pathways. 7
Interestingly, flavonoids have been found to have positive effects on lipid metabolism, insulin 8
resistance, inflammation and oxidative stress, the most important pathophysiological pathways in 9
NAFLD. This puts flavonoids in the spotlight for the treatment of NAFLD and prompted us to review 10
the existing evidence for the use of these food derived compounds in the treatment of NAFLD. 11
12
13
14
15
16
17
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Introduction
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Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of liver disorders, ranging from 2
steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. The 3
development of NAFLD is associated with the metabolic syndrome. Around 20 percent of the general 4
population in Western countries has NAFLD (Bedogni et al. 2005) and 2 to 3 percent develops NASH 5
(Neuschwander-Tetri et al. 2003). The pathophysiological model that has evolved for the development 6
of NAFLD includes multiple hits that do not follow a strict sequence (fig.1) (Tilg et al. 2010). In this 7
model, metabolic disorders, oxidative stress and local and systemic inflammation are the major 8
processes involved in the progression of NAFLD. These processes appear to enforce each other and a 9
dynamic cross talk between the different processes exists (fig. 1). Some authors suggest that steatosis 10
and steatohepatitis might be two distinct disease entities (Tilg et al. 2010; Yilmaz 2012). However, 11
progression from steatosis to NASH has been documented in patients (Pais et al. 2011). 12
13
To date, no evidence based pharmacological treatment exists for NAFLD. Patients are given life style 14
advice consisting of dietary recommendations as well as encouragement to increase physical exercise 15
to lose weight. That a multitude of pathways has been implicated in the etiology of NAFLD makes the 16
treatment challenging; ideally, the treatment should address all the multiple pathways. The 17
involvement of multiple pathways explains why no effective drug has been found for the treatment of 18
NAFLD. Traditionally, drugs are designed as “silver bullets” that, according to the classical medicinal 19
chemical approach, have a well-defined, specific biologic target like a receptor (fig. 2). Since such a 20
well-defined, specific biologic target is missing in NAFLD, this traditional approach is deemed to fail. 21
22
Natural compounds, such as flavonoids, have frequently been studied in models of NAFLD and seem 23
to display beneficial effects. Flavonoids comprise a diverse group of compounds abundantly found in 24
our diet. The intake of flavonoids has been associated with several health benefits. Initially, their 25
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health benefits were attributed to their potent antioxidant activity. However, further research revealed 1
that also other activities contribute, such as anti-inflammatory and metabolic effects. Apparently, 2
flavonoids display a multitude of activities and therefore these compounds were upgraded from 3
antioxidant to bioactive (Bast et al. 2013). A bioactive produces a biological response via an array of 4
subtle effects via different targets (fig. 2). This multifarious mode of action of flavonoids seems to suit 5
seamlessly in the treatment of NAFLD, in which various pathways are involved. This concept will be 6
reviewed in the present paper. 7
8
Firstly, an inventory of the various pathways identified in the etiology of NAFLD will be made. 9
Secondly, the several biological activities of flavonoids will be presented. These activities will be 10
linked to the pathways involved in NAFLD explaining the rational for the use of flavonoids in the 11
treatment of NAFLD. Finally, the clinical studies on the efficacy of flavonoids in NAFLD will be 12
evaluated, with the focus on the effect of flavonoids on the different pathways. 13
14
To this end, a literature search was conducted on PubMed in November 2013 using the search terms: 15
flavonoids, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. To increase the number 16
of studies found, a search using the name of specific flavonoids was also included. 17
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Pathogenesis of nonalcoholic fatty liver disease
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The pathogenesis of NAFLD is complex and multifactorial, comprising multiple hits that lead to 20
steatosis and NASH (Tilg et al. 2010; Polyzos et al. 2012). Although various pathways have been 21
identified, the list is not complete as the etiology still is enigmatic and our knowledge of the disease is 22
progressing. 23
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A hallmark in the development of NAFLD is the accumulation of fat in the liver. Factors that are 1
known to contribute to this accumulation include: (1) high free fatty acids (FFA) supply due to 2
increased lipolysis from visceral and subcutaneous adipose tissue and dietary intake, (2) low FFA 3
oxidation in relation to the supply of FFAs, (3) high hepatic lipogenesis and (4) low hepatic excretion 4
of very low density lipoprotein (VLDL) (fig. 3) (Tilg et al. 2010). Another hallmark is chronic 5
inflammation causing fibrosis. Underlying processes include oxidative stress and lipid peroxidation, 6
mitochondrial dysfunction, adipocytokine/cytokine imbalance, gut-derived bacterial endotoxins, 7
hepatic stellate cell activation and genetic factors (Polyzos et al. 2012). Also activation of Kupffer 8
cells by cholesterol crystals is suggested to be a trigger for hepatic inflammation (Bieghs et al. 2013). 9
Insulin resistance plays an important role in the development of both steatosis and inflammation 10
(Polyzos et al. 2012). Due to insulin resistance, lipolysis is not inhibited by insulin. The FFA’s 11
released cause inflammation, promote ectopic fat deposition and further enhance insulin resistance, 12
creating a self-propelling feed forward process (fig. 3) (Polyzos et al. 2012). Furthermore, insulin 13
resistance stimulates gluconeogenesis in hepatocytes and reduces glycogen formation. Increased 14
glucose and insulin levels stimulate de novo lipogenesis via hepatic transcription factors such as sterol 15
regulatory element binding protein-1c (SREBP-1c) and carbohydrate response element binding protein 16
(ChREBP). This causes stimulation of lipogenic enzymes such as glucokinase (gk), fatty acid synthase 17
(FAS) and acetyl-coenzyme A carboxylase (ACC) (fig. 3) (Polyzos et al. 2012). 18
In the multifactorial etiology of NASH, oxidative stress represents a crucial process (Koek et al. 2011; 19
Rolo et al. 2012; Ucar et al. 2013): the production of reactive oxygen species (ROS) not balanced by 20
the protection against ROS by antioxidants. Various potential sources of oxidative stress have been 21
reported in NAFLD. ROS are produced during the mitochondrial and peroxisomal beta oxidation of 22
FFAs and during the metabolism of FFAs by cytochrome P450 2E1 and 4A. ROS cause endoplasmic 23
reticulum (ER) stress, which further promotes the accumulation of ROS within the cell (Ucar et al. 24
2013). Also reduction in antioxidant defenses will contribute to oxidative stress. Reduced glutathione 25
(GSH) levels and decreased superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase and 26
glutathione transferase activities are found in NASH and appear to be correlated to disease severity 27
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(Rolo et al. 2012). ROS react with biological compounds including fatty acids, proteins and DNA, 1
causing lipid peroxidation, mitochondrial dysfunction, stellate cell activation, inflammation (via NF-2
κB activation) and apoptosis (fig. 3) (Koek et al. 2011; Ucar et al. 2013). Mitochondrial dysfunction 3
and inflammation will lead to the formation of more ROS, further fueling the self-propelling feed 4
forward process. 5
6
Iron has also been implemented in the pathogenesis of NAFLD. In patients with NAFLD elevated 7
hepatic iron levels have been found (Valenti et al. 2010; Nelson et al. 2011). The precise role of iron in 8
the pathogenesis of NAFLD has not yet been established, but it is well documented that iron increases 9
oxidative stress, e.g. by its ability to generate hydroxyl radicals via the Fenton reaction (Fenton 1894). 10
Citrate, an intermediate product of lipid metabolism found to be elevated in NAFLD patients, has the 11
ability to further increase this iron-induced oxidative stress by stimulation of the Fenton reaction (van 12
de Wier et al. 2013). 13
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Flavonoids
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Flavonoids are polyphenolic compounds that are ubiquitously found in nature. They are secondary 16
metabolites in plants and are frequently bound to sugars (glycosides) (Ross et al. 2002). Flavonoids 17
also occur as aglycones (without a sugar group) (Ross et al. 2002). Most flavonoids (fig. 4) consist of 18
three rings: two aromatic rings (A and B) and one heterocyclic ring (C). Flavonoids are categorized 19
into subclasses based on variations in the C ring. The major subclasses are flavones, isoflavones, 20
flavanols, flavanones, anthocyanidins and chalcones (fig. 4) (Ross et al. 2002). 21
22
Over 5,000 different flavonoids have been identified. Because the group of flavonoids is a very 23
heterogenic group of compounds that act on multiple biological targets, various, sometimes even 24
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paradoxical, effects have been described for different classes of flavonoids. Also, the wide variation in 1
models used to study the effects of flavonoids contributes to the variation in the effects found. This 2
review addresses the potential of flavonoids and focusses on the reported positive effects of these 3
bioactives in the treatment of NAFLD. These effects are differentiated in metabolic, antioxidant and 4
anti-inflammatory effects. 5
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Metabolic effects 7
PPARs 8
The peroxisome proliferator activated receptors (PPARs) are promising targets in the treatment of 9
NAFLD. PPARs are nuclear receptors that play a role in the regulation of lipid and glucose 10
metabolism as well as inflammation (Tailleux et al. 2012). PPARα is highly expressed in the liver and 11
regulates FFA transport and stimulates enzymes involved in β-oxidation (Kallwitz et al. 2008). 12
Furthermore, it limits inflammation by inhibition of NF-κB and reduction of C-reactive protein 13
expression (Tailleux et al. 2012). Studies have found evidences for a role of PPARα in NAFLD and its 14
treatment (Macdonald et al. 2004; Kallwitz et al. 2008). PPARα
-/-
mice have increased susceptibility 15
for development of NAFLD when fed a high fat diet compared to wildtype mice (Kallwitz et al. 2008). 16
In addition, PPARα agonists, such as fibrates, reduce steatosis, inflammation and fibrosis in models of 17
NASH (Shiri-Sverdlov et al. 2006; Tailleux et al. 2012). Stimulation of PPARα is expected to decrease 18
steatosis by stimulation of β-oxidation and to mitigate inflammation by inhibition of NF-κB. Several 19
studies have demonstrated that flavonoids stimulate PPARα (Medjakovic et al. 2010; Chang et al. 20
2011; Cho et al. 2011; Goto et al. 2012; Lee et al. 2012; Jia et al. 2013; Malek et al. 2013). For this 21
stimulation various mechanisms of action have been proposed. Some studies claim that flavonoids are 22
ligands and (partial) agonists of PPARα (Medjakovic et al. 2010; Jia et al. 2013; Malek et al. 2013). 23
Other studies conclude that flavonoids upregulate PPARα gene and/or protein expression (Chang et al. 24
2011; Cho et al. 2011; Goto et al. 2012; Lee et al. 2012), possibly involving adiponectin, a stimulator 25
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of PPARα (Goto et al. 2012). As stated above, the variation in reported mechanisms might originate 1
from the variation in chemical structure between flavonoids and the multitude of targets the flavonoids 2
act on. 3
4
PPARγ is mainly expressed in adipose tissue and its activation results in adipocyte differentiation and 5
insulin sensitization (Medjakovic et al. 2010). Secretion of insulin resistance promoting factors by 6
adipose tissue is reduced and secretion of insulin sensitivity promoting factors is increased (fig. 5). By 7
upregulation of adiponectin, PPARγ also activates PPARα. Furthermore, by activation of the involved 8
genes (FABP, PEPCK, Acyl-CoA synthase, DGAT, FATP and LPL) adipogenesis and lipid storage in 9
subcutaneous adipose tissue is stimulated (fig 5). Consequently, fat from harmful visceral adipose 10
tissue is redistributed to subcutaneous fat depots (Medjakovic et al. 2010). Moreover, FFA delivery to 11
the liver is reduced (Medjakovic et al. 2010; Tailleux et al. 2012). PPARγ has also been reported to 12
increase energy expenditure by induction of uncoupling protein-2 (UCP-2) (Kallwitz et al. 2008). 13
Mutations in the PPARγ gene increase the risk of developing metabolic syndrome and NAFLD 14
(Savage et al. 2003). Additionally, glitazones, PPARγ agonists, improve insulin resistance and 15
decrease aminotransferase levels and liver fat in NASH patients, whereas positive effects on 16
histological markers of NASH are not always noted (Tailleux et al. 2012). These findings substantiate 17
the use of PPARγ agonists in the treatment of NASH. Several studies report that flavonoids stimulate 18
PPARγ (Xia et al. 2005; Chen et al. 2009; Medjakovic et al. 2010; Puhl et al. 2012; Lee et al. 2013; 19
Sharma et al. 2013). Similar as for PPARα stimulation, various mechanisms of action are described. 20
Flavonoids have been found to upregulate PPARγ gene expression (Xia et al. 2005; Chen et al. 2009; 21
Sharma et al. 2011; Lee et al. 2013). Also, some flavonoids were observed to be agonists of PPARγ 22
(Medjakovic et al. 2010; Puhl et al. 2012). An advantage of flavonoids over other drugs, like the 23
glitazones, could be that the bioactives only partially activate PPARγ. This reduces the risk of serious 24
side effects seen with the use of full agonists. For example, weight gain, an important side effect of 25
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glitazones, is not associated with intake of isoflavones that also activate PPARγ. In several studies, 1
intake of isoflavones even leads to a slight weight reduction (Medjakovic et al. 2010). 2
3
SREBP-1c 4
Another target identified in the treatment of NAFLD is SREBP-1c. SREBP-1c is a transcription factor 5
that controls de novo lipogenesis via induction of the lipogenic enzymes (FAS, ACC, gk) (Ferre et al. 6
2010), which stimulates steatosis. In liver biopsies of NAFLD patients, the expression of SREBP-1c 7
and liver X receptor α (LXRα), which controls SREBP-1c transcription, as well as the expression of 8
ACC and FAS are found to be significantly higher than in control biopsies (Higuchi et al. 2008). 9
Several flavonoids inhibit SREBP-1c (Shin et al. 2007; Hwang et al. 2011; Liu et al. 2011; Sharma et 10
al. 2011; Wu et al. 2011; Ahn et al. 2013). Multiple mechanisms of SREBP-1c inhibition have been 11
implicated. Various studies have found that flavonoids downregulate SREBP-1c protein and gene 12
expression (Hwang et al. 2011; Liu et al. 2011; Sharma et al. 2011; Wu et al. 2011; Ahn et al. 2013). 13
The isoflavone genistein reduces the expression of site-1 proteases, which are necessary for SREBP-1c 14
to act as a transcription factor (Shin et al. 2007). Furthermore, SREBP-1c is inhibited by activation of 15
5' adenosine monophosphate-activated protein kinase (AMPK) (Hwang et al. 2011; Liu et al. 2011; 16
Wu et al. 2011). Activation of AMPK inhibits LXRα, which controls SREBP-1c transcription (Yap et 17
al. 2011). Flavonoids stimulate activation of AMPK (Hwang et al. 2011; Liu et al. 2011; Wu et al. 18
2011; Lee et al. 2012). Additionally, flavonoids inhibit LXRα (Goldwasser et al. 2010; Sharma et al. 19
2011; Ahn et al. 2013). Since SREBP-1c transcription is stimulated by hyperinsulinemia, flavonoids 20
might also reduce SREBP-1c expression by improving insulin sensitivity and normalizing insulin 21
levels. Recently, activation of SREBP-1c was suggested to be one of the consequences of ER stress in 22
the steatotic liver (Ferre et al. 2010). Inhibition of ER stress is another way in which flavonoids might 23
inhibit SREBP-1c. 24
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Antioxidant effects 1
As a first line of defense, flavonoids reduce the production of radicals and other reactive species. 2
Flavonoids inhibit pro-oxidant enzymes, such as xanthine oxidase (Garcia-Lafuente et al. 2009). Also 3
inhibition of lipoxygenases and cyclooxygenases (see anti-inflammatory effects), enzymes that are 4
capable to co-oxidize molecules other than their usual substrates, reduces the production of reactive 5
species (Garcia-Lafuente et al. 2009). 6
7
Flavonoids have been found to be very effective scavengers. This is a pivotal biochemical mode of 8
action of bioactives, although the importance of radical scavenging is exaggerated as well as 9
undervalued (Bast et al. 2013). Amongst antioxidants, flavonoids are at the top of the pecking order, 10
meaning that they are the first in line to scavenge radicals. They scavenge a wide array of reactive 11
species including superoxide, hydroxyl, peroxyl and peroxynitrite radicals (Bors et al. 1997; Haenen et 12
al. 1997; Duthie et al. 2000). During this scavenging, flavonoids are oxidized by the radical, resulting 13
in a more stable, less reactive radical (Garcia-Lafuente et al. 2009). Among the various subclasses of 14
flavonoids, the flavonols that comprises quercetin and related flavonoids display superior scavenging 15
activity. This is due to a large conjugated π–system that delocalizes electrons over the entire molecule. 16
Structure-activity relationship studies reveal that two pharmacophores are present in the flavonols, (1) 17
a catechol moiety in ring B and (2) a hydroxyl (OH) group at the 3 position at a 2,3-double bound, 18
which is activated by the hydroxyl groups at the 5 and 7 position (Heijnen et al. 2002). In NAFLD, 19
radicals produced during peroxisomal and mitochondrial β-oxidation and the metabolism of FFA by 20
Cytochrome P450 2E1 and 4A can be scavenged by flavonoids, which will result in a reduction of 21
oxidative stress. 22
23
In addition to the direct radical scavenging effect, several flavonoids have the ability to chelate iron 24
and other transition metals that contribute to the formation of radicals (Pietta 2000; Ross et al. 2002). 25
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The quercetin derived semi-synthetic flavonoid monoHER can scavenge OH-radicals at an extremely 1
high apparent rate - ks = 980 X 10
8
M
-1
s
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- which is even quicker than the diffusion rate (~ 100 X 10
8
2
M
-1
s
-1
) (Lemmens et al.). This can be explained by ‘site-specific scavenging’. Essential for this activity 3
is that monoHER can chelate iron. The result of this chelation is that monoHER is present at the site of 4
the radical formation, i.e. the iron ion. This enables monoHER to immediately scavenge the newly 5
formed radical. By this site-specific scavenging, monoHER is able to prevent damage to critical 6
biomolecules such as lipids, proteins or DNA, despite the high reactivity of the radical (Lemmens et 7
al. ; Haenen et al. 1993). Since iron mediated hydroxyl radical formation has been implied in NAFLD 8
(O'Brien et al. 2011; van de Wier et al. 2013), this action of flavonoids is expected to be meaningful 9
for the treatment of NAFLD. 10
11
A third mode of action is that flavonoids can protect or enhance the endogenous antioxidant defense 12
(Ross et al. 2002; Stevenson et al. 2007). Several flavonoids induce glutathione S-transferase, heme-13
oxygenase 1 (HO-1) and other antioxidants (Ross et al. 2002; Yang et al. 2011; Sun et al. 2012; Zhang 14
et al. 2012; Huang et al. 2013). An important pathway in this response is stimulation of nuclear factor 15
erythroid derived 2 (nrf2), a transcription factor that binds to antioxidant response elements (AREs) in 16
the promoter region of genes encoding various antioxidants and phase II detoxifying enzymes. This 17
leads to the transcription of those enzymes, e.g. HO-1 and NAD(P)H-quinone oxidoreductase (Mann 18
et al. 2009). Stimulation of nrf2 by flavonoids is reported in several studies (Yang et al. 2011; Sun et 19
al. 2012; Zhang et al. 2012; Huang et al. 2013). Flavonoids increase nrf2 nuclear translocation to the 20
nucleus and the binding of nrf2 to AREs (Yang et al. 2011; Huang et al. 2013). Since depletion of 21
antioxidant defenses is seen in NASH patients, upregulation of antioxidants could be beneficial in the 22
treatment of NASH (Rolo et al. 2012). 23
24
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Anti-inflammatory effects 1
Anti-inflammatory effects of flavonoids have been mainly subscribed to inhibition of the NF-κB 2
pathway (Gonzalez et al. 2011). The NF-κB pathway consists of a canonical and non-canonical 3
pathway. The canonical pathway is involved in inflammatory responses, while the non-canonical 4
pathway regulates immunce cell differentiation and maturation and lymphoid organogenesis (Shih et 5
al. 2011). Since the canonical pathway is most important for initiation of inflammation and effects of 6
flavonoids focus on this part of the NF-κB pathway, our review is restricted to the canonical pathway. 7
The canonical pathway is activated by pro-inflammatory signals, such as cytokines and oxidative 8
stress (van den Berg et al. 2001). This causes the IKK complex to phosphorylate and designate the 9
IκBs (α, β or ε) for degradation. Degradation of the IκBs releases NF-κB into the nucleus. 10
Consequently, transcriptional activity occurs resulting in an inflammatory response (Shih et al. 2011). 11
12
NF-κB activation occurs in various inflammatory diseases. Activation of the NF-κB pathway is 13
observed in animal models of NASH as well as in NASH patients (Marra 2008), illustrating a role for 14
NF-κB activation in NASH. Flavonoids interfere with the NF-κB pathway in several ways (fig. 6) 15
(Gonzalez et al. 2011). Flavonoids inhibit the IKK complex and IκB-phosphorylation, thereby 16
preventing NF-κB translocation to the nucleus and the transcription of genes involved in the 17
inflammatory response. Flavonoids also inhibit protein kinases that control the activity of NF-κB (Kim 18
et al. 2005). In addition, flavonoids inhibit protein kinase C (PKC), mitogen activated protein kinases 19
(MAPKs), extracellular signal related kinase (ERK) and Jun N-terminal kinase (JNK) (Kim et al. 20
2005). A mechanism proposed for inhibition of protein kinases is competitive binding to nucleotide 21
binding sites (Manthey 2000). Since oxidative stress also results in NF-κB activation (van den Berg et 22
al. 2001), the antioxidant potency of flavonoids is also implicated in inhibition of the NF-κB pathway 23
(Boots et al. 2008; Gloire et al. 2009; Salamone et al. 2012). 24
25
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Besides inhibitory effects on the NF-κB pathway, flavonoids also inhibit the activity of regulatory 1
enzymes involved in the induction of the inflammatory response, such as protein tyrosine kinases, 2
PKC, phosphodiesterase, phospholipase A
2
, lipoxygenases and cyclooxygenase (COX) (Manthey 3
2000; Kim et al. 2005). These enzymes are responsible for the activation of specialized cells involved 4
in inflammation, e.g. by prostanoid biosynthesis via arachidonic acid metabolism (Manthey 2000). 5
Also, the production of various cytokines is inhibited by flavonoids, possibly involving inhibition of 6
phosphodiesterase (Manthey 2000; Gonzalez-Gallego et al. 2010). 7
8
Furthermore, several flavonoids inhibit inducible nitric oxide synthase (iNOS) expression and the 9
production of nitric oxide (NO) (Kim et al. 2005; Gonzalez-Gallego et al. 2010). NO serves as an 10
inflammatory mediator and also leads to the formation of the highly damaging peroxynitrite in 11
conditions of oxidative stress (Garcia-Lafuente et al. 2009). In addition to the inhibition of NO 12
production, flavonoids can scavenge NO (van Acker et al. 1995; Haenen et al. 1999) and peroxynitrite 13
(Haenen et al. 1997). Inhibition of iNOS and COX-2 expression is also found to be related to 14
inhibition of NF-κB and activation of PPARγ (Gonzalez-Gallego et al. 2010). 15
16
Recently, it has been reported that flavonoids are able to prevent deterioration of the anti-inflammatory 17
effect of the glucocorticoid cortisol in the presence of oxidative stress (Ruijters et al. 2014). Oxidative 18
stress extinguishes the anti-inflammatory effect of cortisol, leading to cortisol resistance. Flavonoids 19
reduce intracellular oxidative stress as well as the development of cortisol resistance. This further 20
deciphers the enigmatic mechanism of flavonoids by which these bioactives exert their biological 21
effect, and moreover shows that their anti-inflammatory and antioxidant action are intertwined. 22
23
24
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Flavonoids in the treatment of NAFLD
1
Several flavonoids have been studied for the treatment of NAFLD. One of the most investigated 2
flavonoids is silybin, one of the flavonoids in the flavonoid mixture silymarin. Green tea flavonoids 3
and soy isoflavones are also extensively investigated. Together with quercetin and rutin, these are the 4
most studied flavonoids for the treatment of NAFLD. These groups of flavonoids and their effects on 5
NAFLD are reviewed separately. 6
7
Animal models
8
The majority of in vivo studies investigating the use of flavonoids in NAFLD are animal studies. 9
Because NAFLD is a multifactorial disease and the patient population with NAFLD is very 10
heterogenic, it is difficult to imitate all the facets of the disease in one animal model. Furthermore, 11
NAFLD is seen as the hepatic manifestation of the metabolic syndrome: patients do not only show 12
liver abnormalities, but also have obesity, dyslipidemia and insulin resistance. Although many models 13
may succeed to mirror the liver pathology correctly, this does not always reflect the right metabolic 14
context (Larter et al. 2008). 15
Already many reviews have been devoted to animal models of NAFLD (Nanji 2004; Nagarajan et al. 16
2012; Takahashi et al. 2012). Therefore, this section will only evaluate the animal models used in the 17
studies investigating the use of flavonoids in NAFLD. Most studies are mice or rat studies. Also one 18
study investigated the development of NAFLD in gerbils. The animals used in the different studies 19
had various genetic backgrounds and some transgenic animal models were used. Furthermore, 20
different diets were used to induce NAFLD, such as high fat diets, high fructose diets and methionine- 21
and choline deficient diets. 22
To validate the different models used, it has to be examined which model pictures the disease process 23
most completely. Therefore, the models were compared regarding the development of liver damage 24
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(steatosis, inflammation and fibrosis), the presence of the most important pathogenic pathways 1
(metabolic abnormalities, oxidative stress and inflammation) and the presence of other signs of the 2
metabolic syndrome (dyslipidemia, obesity and insulin resistance). 3
Firstly, the different dietary models are compared. The methionine and choline deficient diet (MCD 4
diet) causes lipid deposition in the liver by interfering with β-oxidation and VLDL secretion. The diet 5
lacks methionine and choline, which are essential for hepatic β-oxidation and the production of VLDL. 6
In addition to liver steatosis and inflammation, oxidative stress and changes in the liver cytokines and 7
adipocytokines are found (Takahashi et al. 2012). The advantage of this model is that liver steatosis 8
and inflammation are found within ten days of the diet. Fibrosis is found after 8-10 weeks (Takahashi 9
et al. 2012). The MCD-model causes the most inflammation, oxidative stress and liver damage 10
compared to other dietary models. However, the extent of damage is dependent on species, gender and 11
strain of the animals used (Kirsch et al. 2003). C57Bl/6 mice are found to develop the most 12
inflammation and necrosis, best approximating the histological features of NASH. Male gender and 13
the strain Wistar rats are associated with the highest degree of steatosis (Kirsch et al. 2003). The 14
disadvantage of the MCD-model is that it lacks the metabolic context of human NAFLD/NASH. 15
Animals on the MCD-diet are found to lose weight, to have no insulin resistance and unchanged or 16
increased serum adiponectin levels (Kirsch et al. 2003). In some studies this is resolved by using the 17
MCD diet in genetically obese animals, such as ob/ob mice. 18
Various high fat diets have been used in animal models of NAFLD. Sprague-Dawley rats on a high fat 19
diet are found to develop steatosis, inflammation and oxidative damage in the liver. Also insulin 20
resistance is found after 3 weeks of a high fat diet (Takahashi et al. 2012). However, development of 21
steatohepatitis is dependent on the rodent species and strain used. For example, while Sprague-Dawley 22
rats do develop steatohepatitis, Wistar rats are found to be less susceptible to develop steatohepatitis 23
on a high fat diet (Romestaing et al. 2007). C57Bl/6 mice are found to develop steatosis after 10 weeks 24
of high fat diet. Also insulin resistance, increased plasma cholesterol levels and obesity are found. 25
However, slight inflammatory changes are only found after 35 weeks of high fat diet (Ito et al. 2007). 26
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The development of steatohepatitis in animals on a high fat diet is not only dependent on the rodent 1
species and strain, but also on the fat content in the diet, the composition of dietary fat and the duration 2
of the treatment (Takahashi et al. 2012). Although the use of a high fat diet seems to reproduce the 3
metabolic context of human NASH better, liver damage is less severe than with use of the MCD-diet. 4
Among the various high fat diet models, the pathological changes in the intragastric-overfeeding 5
model are found to resemble human NASH best (Ito et al. 2007). 6
Rats and mice fed a fructose rich diet have been found to be good models for the metabolic syndrome 7
(Takahashi et al. 2012). Liver damage is also found in these models. Wistar rats on a high fructose diet 8
(70%) develop liver steatosis and inflammation (Kawasaki et al. 2009). However, the distribution 9
pattern of steatosis in the liver is different from that in human NAFLD. While in human NAFLD 10
steatosis is mostly present in zone 3, the steatosis in Wistar rats on a high fructose diet is predominant 11
in zone 1 (Takahashi et al. 2012). Interestingly, inflammation does follow the same pattern as in 12
human NASH: predominantly lobular and not periportal (Kawasaki et al. 2009). Like with the use of a 13
high fat diet, the extent of liver damage developed on a high fructose diet is also dependent on the type 14
and strain of animals used and the fructose content in the diet. 15
Transgenic animal models in studies investigating flavonoids in NAFLD include db/db mice, ob/ob 16
mice, nSREBP-1c transgenic mice, obsese Zucker fa/fa rats and obese diabetic Otsuka Long-Evans 17
Tokushima Fatty (OLETF) rats. 18
In ob/ob mice, a spontaneous mutation in the leptin gene causes leptin deficiency, leading to 19
hyperphagic, inactive, extremely obese and diabetic mice that develop liver steatosis spontaneously 20
(Nagarajan et al. 2012; Takahashi et al. 2012). However, ob/ob mice do not spontaneously develop 21
steatohepatitis. Therefore, a second ‘hit’ is needed; such as a MCD diet or high fat diet (Nagarajan et 22
al. 2012; Takahashi et al. 2012). 23
Db/db mice carry a spontaneous mutation in the leptin-receptor gene. These mice have normal or 24
increased levels of leptin, but are resistant to its effects, which leads to obesity and insulin resistance 25
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(Nagarajan et al. 2012; Takahashi et al. 2012). Db/db mice also develop steatosis, but require a second 1
‘hit’ for progression to steatohepatitis, such as the MCD diet or high fat diet (Nagarajan et al. 2012; 2
Takahashi et al. 2012). The advantage of both the ob/ob and db/db mice models is that they develop 3
NAFLD in conditions resembling the metabolic syndrome. A disadvantage is that they need a second 4
hit for progression to steatohepatitis. Db/db mice fed a MCD diet, were found to have higher serum 5
ALT levels and more severe hepatic inflammation and fibrosis than ob/ob mice fed the MCD diet 6
(Takahashi et al. 2012). 7
Obese Zucker fa/fa rats also have a mutation in the leptin receptor gene leading to leptin resistance. 8
Until four weeks of age, these rats only display increased appetite (Nanji 2004). At four to five weeks 9
of age, the fat mass and the serum level of free fatty acids increase and triglycerides accumulate in 10
various organs, including the liver (Nanji 2004). Hyperinsulinemia also develops, eventually leading 11
to diabetes. Development of steatohepatitis in Zucker fa/fa rats is described after feeding a high fat 12
high cholesterol diet (Matsunami et al. 2010). 13
In SREBP-1c transgenic mice, SREBP-1c is overexpressed, causing congenital lipodystrophy and 14
severe insulin resistance. At the age of 1 week, liver steatosis is found, which progresses to 15
steatohepatitis within 20 weeks of age without the requirement of a second hit (Takahashi et al. 2012). 16
A disadvantage of this model is that in contrast with human NAFLD/NASH, visceral fat is decreased 17
in this animal model. 18
The last genetic model used to investigate flavonoids in the treatment of NAFLD is the OLETF rat. 19
This is an established model of the metabolic syndrome, characterized by insulin resistance, abdominal 20
obesity, hypertension and dyslipidemia (Song et al. 2013). Due to a gentic deletion of the 21
cholecystokinin 1 receptor, these rats lack the feeling of satiety. From 8 weeks of age, the OLETF rats 22
develop obesity and hyperinsulinemia. Also liver steatosis develops spontaneously in these rats at 18 23
weeks of age (Song et al. 2013). However, after 42 weeks of age steatosis declines and inflammation 24
and fibrosis do not develop spontaneously (Song et al. 2013). 25
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From all the studies evaluated, the models using rats/gerbils on a high fat diet (Haddad et al. 2011; 1
Yao et al. 2011; Yao et al. 2013) (table 1), (Yalniz et al. 2007; Ronis et al. 2009; Ji et al. 2011) (table 2
3), (Kuzu et al. 2008; Xiao et al. 2013) (table 4), (Ying et al. 2013) (table 5) or on a high fat diet 3
combined with high fructose or carbohydrates (Panchal et al. 2011; Panchal et al. 2012) (table 4 and 4
5), seem to approximate the human conditions of NAFLD best. Also studies using genetic models of 5
the metabolic syndrome combined with a high fat diet or MCD diet are useful (Kim et al. 2012; 6
Salamone et al. 2012; Salamone et al. 2012) (table 1). Studies using the MCD diet only, do not 7
correctly mirror the circumstances of the metabolic syndrome. Studies using mice on a high fat diet, 8
Wistar rats on only a high fat diet, or genetic models of the metabolic syndrome without a special diet 9
or second ‘hit’, do not seem to provoke sufficient liver damage compared to the human situation and 10
can only be used to evaluate the development of steatosis. 11
12
Silymarin and silybin
13
Silymarin is a flavonoid mix that originates from milk thistle extract. It was already used by doctors 14
and herbalists to treat diverse liver and gallbladder disorders in ancient history (Abenavoli et al. 2012). 15
Nowadays, 65% of the patients with liver diseases take herbal preparations, which are mainly derived 16
from milk thistle (Loguercio et al. 2012). Silymarin contains at least eight different compounds: 17
silybin (A and B), isosilybin (A and B), silichristin, silidianin, dehydrosilybin, taxifolin and others. A 18
small fraction of silymarin consists of polymerized polyphenolics that have not been identified yet 19
(Skottova et al. 2003). About 50-70% of the silymarin extract consists of the flavonolignan silybin 20
(fig. 7), also known as silibinin, which is extensively studied and is regarded as the most active 21
component of silymarin (Loguercio et al. 2011). 22
23
Silymarin flavanolignans have a limited bioavailability that was reported to be 0.45% in human 24
volunteers (Calani et al. 2012). Similarly, the bioavailability of silybin in rats was calculated to be 25
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0.95% (Wu et al. 2009). Extensive phase II metabolism, low permeability across intestinal epithelial 1
cells, low solubility in water and rapid excretion in bile and urine are the major causes of this limited 2
bioavailability (Javed et al. 2011). To improve bioavailibility, derivatives of silybin have been 3
synthesized, such as silybin-phosphatidylcholine (Javed et al. 2011; Loguercio et al. 2011). The 4
silymarin that is not absorbed in the gastrointestinal tract will be subjected to metabolism by bacteria 5
in the colon. In the biological effect of polyphenolic compounds, the effect of colonic metabolites 6
plays a key role (Mateo Anson et al. 2011). However, the contribution of the colonic metabolites to the 7
health effect of silymarin has not been examined. 8
9
In human studies daily doses up to 800 mg silymarin appeared to be safe. Only few and minor side 10
effects of silymarin, primarily on the gastrointestinal tract, have been reported (Jacobs et al. 2002; 11
Gazak et al. 2007). 12
13
The results of ten identified in vivo studies investigating the use of silymarin/silybin in NAFLD 14
models are summarized in table 1 (Serviddio et al. 2010; Shetty et al. 2010; Haddad et al. 2011; Yao et 15
al. 2011; Kim et al. 2012; Qin et al. 2012; Salamone et al. 2012; Salamone et al. 2012; Grattagliano et 16
al. 2013; Yao et al. 2013). The molecular pathways implicated in the therapeutic effect of silymarin in 17
NAFLD, are elaborated in detail in table 1, clustered in metabolic abnormalities, oxidative stress and 18
inflammation. An improvement of lipid metabolism is demonstrated by a decrease in serum and 19
hepatic lipid values in numerous studies. This is caused by a stimulation of free fatty acid oxidation 20
and positive effects on coordinating factors of lipid metabolism, such as adiponectin and PPARα. Also 21
insulin resistance is reduced in most studies. Antioxidant effects of silymarin represented by a 22
decrease in lipid peroxidation and other oxidative stress markers might be caused by stimulation of 23
endogenous antioxidants such as GSH and SOD. The anti-inflammatory effects of silymarin in 24
NAFLD models include a decrease in the pro-inflammatory cytokine TNF-α and inhibition of NF-κB. 25
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Together, the metabolic, antioxidant and anti-inflammatory effects of silymarin lead to a marked 1
improvement of steatosis and liver inflammation in in vivo models of NAFLD (table 1). 2
3
In a well-designed, randomized controlled trial (RCT), 138 patients with histologically proven 4
NAFLD were treated with a silybin-phosphatidylcholine complex or placebo for 12 months (table 2) 5
(Loguercio et al. 2012). Treatment showed positive effects on serum ALT, AST and γGT levels, 6
suggesting an improvement of hepatic damage. In addition, positive effects on insulin resistance and 7
body mass index were found. Patients of the treatment group that agreed to liver biopsy showed 8
significant improvements in steatosis, lobular inflammation, ballooning and fibrosis, while no 9
improvements were seen in the biopsies of patients in the placebo group (Loguercio et al. 2012). 10
11
Taken together, the data indicate that silymarin and silybin show inhibitory effects on NAFLD 12
progression. The influence of silymarin/silybin on NAFLD seems to be in line with the multifactorial 13
mode of action of flavonoids; not one single mechanism of action, but multiple mechanisms that 14
reinforce each other, emerge from the studies. The exact value of silymarin in the treatment of 15
NAFLD still has to be established, but results so far are relatively consistent and encouraging. 16
17
Soy isoflavones
18
Unlike most flavonoids, isoflavones are not commonly found in a Western diet (Messina 2010). In 19
fact, soybeans and products derived from soybeans are the only relevant sources of isoflavones 20
(Messina 2010). Isoflavone intake in Western countries does not exceed 1 mg per day, whereas 21
consumption of isoflavones in Japan and China can be as high as 40 mg/day (van Erp-Baart et al. 22
2003; Messina et al. 2006). The isoflavones in soy are genistein (fig. 7), daidzein (fig. 7) and glycitein. 23
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Generally, the content of genistein in soy is larger than the content of daidzein and glycetein (Larkin et 1
al. 2008). 2
3
Five to seven hours after intake, genistein and daidzein plasma concentrations reach their maximum 4
(Vitale et al. 2013). Plasma half-lives of genistein and daidzein aglycones are found to be 7 and 9 5
hours respectively (Larkin et al. 2008). Due to extensive first-pass metabolism, isoflavone 6
bioavailability is low (Larkin et al. 2008). Plasma genistein concentrations around 40 nM are 7
measured in people consuming a Western diet, while concentrations of approximately 4 µM are 8
measured in people consuming a traditional Japanese soybean rich diet (Mann et al. 2009). 9
10
Isoflavones are generally regarded as safe (Qin et al. 2013). Most clinical trials do not report any 11
adverse effects. Side effects that have been reported include abdominal bloating, constipation and hot 12
flushes (Qin et al. 2013). Isoflavones have estrogen-like activity in women with low endogenous 13
estrogen levels (Andres et al. 2011). Due to the estrogen-like activity of isoflavones, concerns were 14
raised that isoflavones could stimulate breast cancer development in postmenopausal women. This is 15
an issue of major concern; however, the limited studies in humans that addressed this subject did not 16
corroborate this serious side effect (Andres et al. 2011). 17
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Thirteen in vivo studies investigating the use of soy isoflavones in animal models of NAFLD were 19
found (Ae Park et al. 2006; Gudbrandsen et al. 2006; Lee et al. 2006; Davis et al. 2007; Ustundag et al. 20
2007; Yalniz et al. 2007; Gudbrandsen et al. 2009; Mohamed Salih et al. 2009; Ronis et al. 2009; Kim 21
et al. 2010; Crespillo et al. 2011; Ji et al. 2011; Kim et al. 2011). The various effects of soy 22
isoflavones on metabolic abnormalities, oxidative stress and inflammation are shown in detail in table 23
3. Improvement of lipid metabolism, evidenced by a decrease in hepatic and serum lipid values, has 24
been attributed to stimulation of free fatty acid oxidation, inhibition of lipogenesis and interaction of 25
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soy isoflavones with coordinating factors of lipid metabolism, such as adiponectin, leptin, PPARα, 1
PPARγ and others (table 3). Protection against oxidative stress is demonstrated by a decrease in lipid 2
peroxidation and protein carbonyl levels. The mechanism proposed for this protection is stimulation of 3
endogenous antioxidant levels. Inflammation is mitigated by inhibition of NF-κB activation and 4
reduced production of pro-inflammatory cytokines (table 3). All these subtle effects combined can 5
lead to a decrease in steatosis. The few studies that have investigated histological signs of 6
inflammation corroborate the anti-inflammatory effect of isoflavones. 7
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treatment of NAFLD. Although several animal studies have found positive effects of the use of soy 10
isoflavones in the treatment of NAFLD, no clinical trials have been conducted on the use of soy 11
isoflavones in NAFLD patients. 12
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Green tea flavonoids
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Green tea, similar to oolong tea and black tea, is derived from the plant Camellia Sinensis (Masterjohn 15
et al. 2012). The main flavonoids in green tea are the catechins (30-42% of solid weight). They 16
comprise epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin-17
gallate (EGCG) (Paquay et al. 2000; Masterjohn et al. 2012). The highest percentage of catechins in 18
green tea consists of EGCG (50-75%) (fig. 7). Other flavonoids that can be found in small amounts in 19
green tea are quercetin and myricitin (Masterjohn et al. 2012). 20
21
One to three hours after ingestion of green tea, maximal catechin plasma concentrations (0.1-4.4 µM) 22
are reached (Masterjohn et al. 2012). Plasma half-lives of green tea catechins range from 1.5 to 5.7 23
hours (Masterjohn et al. 2012). Similar values were obtained in studies investigating pure EGCG 24
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(Manach et al. 2005). Because of the rapid elimination of catechins from the body, intake of tea 1
catechins might be beneficial only when they are consumed several times a day (Masterjohn et al. 2
2012). 3
4
In some studies, concerns were raised regarding the possible hepatoxicity of green tea extract (GTE) 5
(Sarma et al. 2008). Nevertheless, a review of several clinical trials showed that the use of GTE/EGCG 6
up to doses of 800 mg/kg/day is safe and well tolerated in humans (Sarma et al. 2008). The only side 7
effects that were reported were mild headache and fatigue. In a recent clinical trial, daily consumption 8
of 714 mg GTE/day for 3 weeks did not lead to liver toxicity in healthy males (Frank et al. 2009). 9
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Ten studies investigating the use of GTE or EGCG in animal models of NAFLD were found (Bose et 11
al. 2008; Bruno et al. 2008; Kuzu et al. 2008; Nakamoto et al. 2009; Ueno et al. 2009; Chen et al. 12
2011; Park et al. 2011; Chung et al. 2012; Park et al. 2012; Xiao et al. 2013). The effects of 13
GTE/EGCG on metabolic abnormalities, oxidative stress and inflammation are presented in detail in 14
table 4. Improvement of lipid metabolism, represented by a decrease in serum and hepatic lipid levels, 15
might be caused by interaction of green tea catechins with coordinating factors of lipid metabolism or 16
a decrease in lipogenesis (table 4). Stimulation of antioxidants and inhibition of ROS production by 17
green tea catechins leads to an attenuation of oxidative stress, demonstrated by a decrease in lipid 18
peroxidation (table 4). Also inflammation is reduced by green tea catechins by inhibition of NF-κB 19
activity and a decrease in pro-inflammatory cytokines (table 4). In the majority of studies these effects 20
led to a decrease in steatosis. Only one study did not find a reduction of steatosis (Nakamoto et al. 21
2009). Histological inflammation was only investigated in two studies (Chung et al. 2012; Xiao et al. 22
2013). Both studies found a diminution of inflammation. 23
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Unfortunately, no studies investigating GTE/EGCG in NAFLD patients were found. In addition, most 1
of the investigated animal studies focused on steatosis only. Although in general promising effects of 2
GTE/EGCG on steatosis are reported, the effects of GTE/EGCG on inflammation in NAFLD models 3
are not conclusive. Clinical trials are lacking to substantiate the therapeutic potential of green tea 4
catechins in NAFLD. Furthermore, the rapid elimination from the body seriously questions the 5
prospect of the use of green tea in NAFLD treatment. 6
7
Quercetin
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Quercetin (fig. 7) is a flavonol that is one of the most abundant flavonoids in the human diet. It is 9
found in various fruits and vegetables, such as onions, apples and tomatoes. The average intake of 10
quercetin is estimated to be 5-40 mg/day (Hertog et al. 1995). In the diet quercetin is often bound by 11
sugars (quercetin glycosides). 12
13
Quercetin has a relatively high bioavailability compared to other flavonoids (Russo et al. 2012). The 14
bioavailability depends on the type of quercetin glycoside. It has been reported that glucosides of 15
quercetin (quercetin bound by glucose) are absorbed in the gastro-intestinal tract better than the 16
aglycon (Hollman et al. 1995; Graefe et al. 2001). Fifty-two percent of quercetin glucosides from 17
onions are absorbed in the gastro-intestinal tract, versus 24% of the quercetin aglycone (Hollman et al. 18
1995). Quercetin and its metabolites have long plasma half-lives, i.e. 11-28h (Manach et al. 2005). 19
Therefore, plasma concentrations can increase significantly upon frequent intake. Quercetin is 20
normally found in the human plasma in low nanomolar concentrations, but upon supplementation, this 21
can increase to high nanomolar or low micromolar concentrations (Hollman et al. 1996; Conquer et al. 22
1998). 23
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No adverse effects of quercetin were reported in studies investigating oral intake of quercetin or 1
quercetin glucosides in doses of 3-1000 mg/day for periods up to 12 weeks (Harwood et al. 2007). 2
Also for intravenous doses of ±10.8 mg/kg body weight no adverse effects were reported. For higher 3
intravenous doses up to 51.3 mg/kg body weight, pronounced pain at the injection site, dyspnea, 4
emesis and transient nephrotoxicity were reported (Harwood et al. 2007). Clinical symptoms lasted for 5
a period of time after each injection (Harwood et al. 2007). 6
7
Although in vitro toxicity studies have reported mutagenic effects of quercetin and two in vivo animal 8
studies reported quercetin carcinogenicity, many other animal studies fail to show increased tumor 9
incidence related to quercetin administration (Harwood et al. 2007). Quercetin toxicity is assumed to 10
be related to the formation of the quercetin-quinone, which is produced when quercetin is oxidized by 11
radicals (Boots et al. 2007; Boots et al. 2008). The quercetin-quinone binds to thiols and causes toxic 12
effects like increased membrane permeability and altered function of enzymes with a critical 13
sulfhydryl –group (Boots et al. 2007; Boots et al. 2008). 14
15
Six animal studies investigating the use of quercetin in animal models of NAFLD were identified 16
(Kobori et al. 2011; Marcolin et al. 2012; Panchal et al. 2012; Jung et al. 2013; Marcolin et al. 2013; 17
Ying et al. 2013). Effects of quercetin on lipid metabolism, oxidative stress and inflammation are 18
summarized in table 5. Quercetin improves lipid metabolism by affecting coordinating factors of lipid 19
metabolism, such as PPARα, PPARγ and adiponectin. Inhibition of lipogenesis and stimulation of 20
fatty acid oxidation by quercetin also contribute to the improvement of lipid metabolism (table 5). 21
Reduction of oxidative stress, demonstrated by a decrease in lipid peroxidation, is caused by a potent 22
direct antioxidant effect, induction of endogenous antioxidant defences and inhibition of iNOS 23
expression in the liver (table 5). In only two studies inflammatory markers, such as TNF-α and Il-6, 24
were investigated and found to be decreased by quercetin (table 5). In all studies the quercetin 25
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administration led to a decrease in steatosis. In general, histological signs of liver inflammation were 1
also reduced. Two studies that investigated fibrosis, found a reduction in fibrosis as well. 2
3
In the in vivo studies mainly positive effects of quercetin on NAFLD were demonstrated. A 4
randomized clinical trial on the use of quercetin in NAFLD patients is lacking, whereas various 5
clinical trials have investigated the use of quercetin in other diseases (Valensi et al. 2005; Edwards et 6
al. 2007; Egert et al. 2009; Heinz et al. 2010; Boots et al. 2011). To substantiate the therapeutic effect 7
of quercetin in NAFLD, clinical trials are mandatory. A concern for approval of such studies will be 8
the potential carcinogenic effect of quercetin, though the relatively high intake of quercetin in the 9
normal diet as well as the widely applied supplementation of quercetin have not pinpointed this as a 10
problem. In fact, epidemiological studies reveal the opposite: flavonoid intake, which for the 11
substantial part is quercetin, has an inverse relationship with the incidence of cancer. 12
13
Rutin
14
Rutin is quercetin with the disaccharide rutinose covalently bound to the 3-OH group (quercetin-3-O-15
β-rutinoside) (fig. 7). It is abundantly found in plants such as buckwheat (Fagopyrum Esculentum) and 16
citrus fruits such as oranges (Citrus Sinensis) and grapefruits (Citrus Paradisi) (Sharma et al. 2013). 17
The uptake of rutin in the gastro-intestinal tract is less than that of quercetin and quercetin mono-18
glucosides and lower peak plasma concentrations of rutin are reached after intake of an equivalent 19
dose compared to that of quercetin (Hollman et al. 1995; Graefe et al. 2001). The main reason for 20
rutins poor bioavailability is its poor solubility in aqueous media (Sharma et al. 2013) and the resulting 21
low bioaccessibility. However, rutin also has advantages over other flavonoids. It is relatively stable 22
and does not display prominent pro-oxidant activity. While some flavonoids are labeled as mutagenic 23
and relatively cytotoxic, rutin is neither (Sharma et al. 2013). 24
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1
Four studies examining the use of rutin in animal models of NAFLD were found (Hsu et al. 2009; 2
Ziaee et al. 2009; Panchal et al. 2011; Gao et al. 2013). The effects of rutin on metabolism, oxidative 3
stress and inflammation are presented in table 6. Hepatic and/or serum lipid values were decreased in 4
all studies, demonstrating an improvement in lipid metabolism. The mechanism of action was not 5
investigated extensively in the studies. Inhibition of leptin and SREBP-1c was suggested to play a role 6
(table 6). Only two studies examined the effects of rutin on oxidative stress. Both studies concluded 7
that rutin administration reduced oxidative stress and implied as mechanism stimulation of 8
endogenous antioxidant levels and inhibition of the production of ROS (table 6). One study reported 9
that rutin administration reduced inflammatory markers, such as TNF-α (table 6) (Gao et al. 2013). 10
The combined effects of rutin on metabolism, oxidative stress and inflammation, can explain the 11
decrease in steatosis found in all studies. Liver inflammation was investigated in two studies and 12
found to be reduced in both studies (Ziaee et al. 2009; Panchal et al. 2011). The only study that 13
examined the effect of rutin on liver fibrosis, did find a reduction in fibrosis (Panchal et al. 2011). 14
15
Rutin is not extensively investigated in animal models of NAFLD. Also no clinical trials have been 16
performed. The promising results for the use of rutin against NAFLD summarized in table 6 show the 17
potential of rutin and indicate that further studies are warranted. A disadvantage of rutin is its poor 18
bioavailability. Semisynthetic analogues of rutin with increased bioavailability might be more 19
promising. In MonoHER, a hydroxyethyl group is attached to the oxygen to the 7-OH group, which 20
increases the water solubility. MonoHER is proven to be a very potent antioxidant (Haenen et al. 21
1993; van Acker et al. 1995; Haenen et al. 1997; van Acker et al. 2000) with anti-inflammatory 22
characteristics (Abou El Hassan et al. 2003). 23
24
Other flavonoids
25
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A wide spectrum of flavonoids has been investigated in animal models of NAFLD. However, the 1
number of studies on many flavonoids is limited to one or two studies. These flavonoids are presented 2
in table 7. 3
4
Naringenin, a flavanone found in grapefruit, completely reverses steatosis in LDLr
-/-
mice fed a high 5
fat diet. It also reduces dyslipidemia, hyperglycemia, hyperinsulinemia and body weight (Mulvihill et 6
al. 2009; Mulvihill et al. 2010). 7
8
Cyanidin 3-O-β-D-glucoside, an anthocyanin found in various plants and fruits that gives them a 9
purple color, reduces hepatic steatosis, oxidative stress and inflammation in diabetic db/db mice (Guo 10
et al. 2012; Zhu et al. 2012). Furthermore, it reduces hyperglycemia and insulin resistance in db/db 11
mice as well as C57Bl/6J mice fed a high fat diet (Guo et al. 2012). Cyanidin 3-O-β-D-glucoside also 12
decreases body weight and hepatic lipid content in C57Bl/6J mice (Tsuda et al. 2003). 13
14
Xanthohumol, a chalcone from the hop plant (Humulus Lupulus), decreases steatosis, inflammation 15
and fibrosis in murine models of NAFLD (Dorn et al. 2010; Doddapattar et al. 2013). Also the other 16
flavonoids noted in table 7 were found to have positive effects on NAFLD and other factors 17
contributing to NAFLD, such as dyslipidemia, body weight and insulin resistance (Guo et al. 2009; 18
Zheng et al. 2009; Lee et al. 2012; Mei et al. 2012; Lee et al. 2013). 19
20
Since the number of different flavonoids known is huge, up to more than 5,000 different chemical 21
entities, it is mandatory to make a selection in order to keep clinical research feasible form a practical 22
point of view. Although the miscellaneous flavonoids mentioned in this paragraph might have merit 23
in the treatment of NAFLD, their biochemical profile is not that different form the more extensively 24
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investigated flavonoids. Therefore, we focus on the latter group of more extensively studied flavonoids 1
in this review. 2
3
Summary and perspective
4
The contemporary pathophysiological model of NAFLD consists of multiple parallel pathways with a 5
dynamic cross talk that cumulate in steatosis and inflammation and ultimately fibrosis, cirrhosis, liver 6
failure and hepatocellular carcinoma. The multitude of pathways involved in the pathogenesis 7
underpins the need for treatments that address these various pathways. 8
9
Flavonoids are compounds derived from plants with subtle effects on multiple targets that finally 10
accumulate in a substantial health benefit. Interestingly, flavonoids have been found to have positive 11
effects on lipid metabolism, insulin resistance, inflammation and oxidative stress, the most important 12
pathological processes in the etiology of NAFLD. This puts flavonoids in the spotlight for the 13
treatment of NAFLD. In this review the existing evidence for the use of flavonoids in the treatment of 14
NAFLD is evaluated. 15
16
Flavonoids and flavonoid mixtures that have been widely investigated in animal models of NAFLD 17
include silymarin, silybin, soy isoflavones, green tea flavonoids, quercetin and rutin. The protective 18
biochemical profile of these flavonoids on lipid metabolism, insulin resistance, oxidative stress and 19
inflammation as well as their beneficial therapeutic effect on steatosis and liver inflammation in most 20
of the studies form the scientific fundament for the use of flavonoids in the treatment of NAFLD. 21
However, further clinical studies are needed to examine the exact value of flavonoids in the treatment 22
of NAFLD patients. 23
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1
Further studies examining the use of flavonoids in NAFLD should include double-blinded randomized 2
clinical trials. In designing and interpreting clinical studies, it is of importance to carefully consider the 3
heterogeneity of the NAFLD patient group. A caveat is that the high heterogeneity will negatively 4
affect the power of the study. Moreover, NAFLD patients with a very different biochemical profile 5
might benefit form flavonoids that display a biochemical profile that fits these different profiles the 6
best. This means that it is unlikely that a uniform treatment for all types of NAFLD will be found. 7
Personalized treatment with close monitoring of the therapeutic effect seems warranted to come to 8
optimal treatment. To reach this stage, the most promising flavonoids should be tested first. The 9
spectrum of flavonoids includes over 5,000 different compounds with their own unique profile, 10
illustrating that it is impossible to study all the flavonoids. Therefore, the compounds best suitable for 11
further investigation have to be identified. In identifying the most promising compound in a large 12
series, structure-activity relationships are mandatory, although these relationships should always be 13
critically evaluated (Haenen et al. 2006). Criteria that can be used to evaluate the therapeutic potential 14
and to form a rational basis for selection of flavonoids for further investigation are their molecular 15
mechanism of action and clinical evidence, bioavailability and safety. 16
17
Regarding their molecular mechanism of action, none of the flavonoids seems to protrude from the 18
animal studies. All flavonoids have been found to improve lipid metabolism, insulin resistance, 19
oxidative stress and inflammatory markers. However, in structure activity relationship studies, 20
quercetin, rutin and its derivates are found to belong to the most potent antioxidants (Heijnen et al. 21
2002). Most clinical evidence is found for silymarin and silybin because these compounds are widely 22
investigated in animal models of NAFLD and silybin was also examined in a randomized clinical trial. 23
24
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Regarding bioavailability, quercetin has the best characteristics. Bioavailability of rutin is slightly 1
lower, but can be improved by the use of semi-synthetic derivates, such as MonoHER, which is more 2
water-soluble. Bioavailability of silybin and silymarin is considerably lower and can be improved by 3
conjugation to polar and hydrophilic moieties, e.g. as in silybin-phosphatidylcholine. Due to the rapid 4
elimination from the body and consequent necessity of frequent administration, green tea flavonoids 5
are not likely to get a place in NAFLD treatment. 6
7
Although quercetin appears to possess superior antioxidant potential and bioavailability, quercetins 8
safety is still debated due to its potential carcinogenicity. Rutin, which seems to be devoid of 9
carcinogenic properties but displays similar antioxidant potential and only slightly lower 10
bioavailability as quercetin, seems to be a better option. Rutin derivates, developed to increase 11
bioavailability, seem to be even more appealing compounds for further investigation. For example 12
MonoHER, rutin with a hydroxyethyl group attached to the oxygen on the 7-position, has shown to be 13
a very potent antioxidant (Lemmens et al. ; Haenen et al. 1993). 14
15
In conclusion, with their multifaceted actions flavonoids seem to suit perfectly in the 16
pathophysiological model of NAFLD. The heterogeneity of the disease should be carefully considered 17
in the design of clinical studies investigating a treatment for NAFLD. Already, multiple in vivo 18
studies show encouraging results for the use of flavonoids in the treatment of NAFLD, which calls for 19
additional research. Silybin has the advantage that it is the most studied flavonoid. Nevertheless, rutin 20
and its derivatives, such as MonoHER, seem to be the most appealing flavonoids for further 21
investigation due to their high antioxidant potential, bioavailability and safety. 22
23
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References
3
Abenavoli, L., N. Milic, et al. (2012). Anti-oxidant therapy in non-alcoholic fatty liver disease: the role 4
of silymarin. Endocrine. 42(3): 754-755. 5
Abou El Hassan, M. A., H. M. Verheul, et al. (2003). The new cardioprotector 6
Monohydroxyethylrutoside protects against doxorubicin-induced inflammatory effects in 7
vitro. British journal of cancer. 89(2): 357-362. 8
Ae Park, S., M. S. Choi, et al. (2006). Genistein and daidzein modulate hepatic glucose and lipid 9
regulating enzyme activities in C57BL/KsJ-db/db mice. Life sciences. 79(12): 1207-1213. 10
Ahn, T. G., G. Yang, et al. (2013). Molecular mechanisms underlying the anti-obesity potential of 11
prunetin, an O-methylated isoflavone. Biochemical pharmacology. 85(10): 1525-1533. 12
Andres, S., K. Abraham, et al. (2011). Risks and benefits of dietary isoflavones for cancer. Critical 13
reviews in toxicology. 41(6): 463-506. 14
Bast, A. and G. R. Haenen (2013). Ten misconceptions about antioxidants. Trends in pharmacological 15
sciences. 34(8): 430-436. 16
Bedogni, G., L. Miglioli, et al. (2005). Prevalence of and risk factors for nonalcoholic fatty liver 17
disease: The Dionysos nutrition and liver study. Hepatology. 42(1): 44-52. 18
Bieghs, V., S. M. Walenbergh, et al. (2013). Trapping of oxidized LDL in lysosomes of Kupffer cells is a 19
trigger for hepatic inflammation. Liver international : official journal of the International 20
Association for the Study of the Liver. 33(7): 1056-1061. 21
Boots, A. W., M. Drent, et al. (2011). Quercetin reduces markers of oxidative stress and inflammation 22
in sarcoidosis. Clinical nutrition. 30(4): 506-512. 23
Boots, A. W., G. R. Haenen, et al. (2008). Health effects of quercetin: from antioxidant to 24
nutraceutical. European journal of pharmacology. 585(2-3): 325-337. 25
Boots, A. W., H. Li, et al. (2007). The quercetin paradox. Toxicology and applied pharmacology. 26
222(1): 89-96. 27
Bors, W., C. Michel, et al. (1997). Antioxidant effects of flavonoids. BioFactors. 6(4): 399-402. 28
Bose, M., J. D. Lambert, et al. (2008). The major green tea polyphenol, (-)-epigallocatechin-3-gallate, 29
inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice. The Journal 30
of nutrition. 138(9): 1677-1683. 31
Bruno, R. S., C. E. Dugan, et al. (2008). Green tea extract protects leptin-deficient, spontaneously 32
obese mice from hepatic steatosis and injury. The Journal of nutrition. 138(2): 323-331. 33
Calani, L., F. Brighenti, et al. (2012). Absorption and metabolism of milk thistle flavanolignans in 34
humans. Phytomedicine : international journal of phytotherapy and phytopharmacology. 35
20(1): 40-46. 36
Chang, C. J., T. F. Tzeng, et al. (2011). Kaempferol regulates the lipid-profile in high-fat diet-fed rats 37
through an increase in hepatic PPARalpha levels. Planta medica. 77(17): 1876-1882. 38
Chen, N., R. Bezzina, et al. (2009). Green tea, black tea, and epigallocatechin modify body 39
composition, improve glucose tolerance, and differentially alter metabolic gene expression in 40
rats fed a high-fat diet. Nutrition research. 29(11): 784-793. 41
Chen, Y. K., C. Cheung, et al. (2011). Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on 42
newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in 43
mice. Journal of agricultural and food chemistry. 59(21): 11862-11871. 44
Page 33 of 84
URL: http://mc.manuscriptcentral.com/bfsn Email: fergc@foodsci.umass.edu
Critical Reviews in Food Science and Nutrition
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49
50
51
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57
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For Peer Review Only
34
Cho, K. W., Y. O. Kim, et al. (2011). Dietary naringenin increases hepatic peroxisome proliferators-1
activated receptor alpha protein expression and decreases plasma triglyceride and adiposity 2
in rats. European journal of nutrition. 50(2): 81-88. 3
Chung, M. Y., H. J. Park, et al. (2012). Green tea extract protects against nonalcoholic steatohepatitis 4
in ob/ob mice by decreasing oxidative and nitrative stress responses induced by 5
proinflammatory enzymes. The Journal of nutritional biochemistry. 23(4): 361-367. 6
Conquer, J. A., G. Maiani, et al. (1998). Supplementation with quercetin markedly increases plasma 7
quercetin concentration without effect on selected risk factors for heart disease in healthy 8
subjects. The Journal of nutrition. 128(3): 593-597. 9
Crespillo, A., M. Alonso, et al. (2011). Reduction of body weight, liver steatosis and expression of 10
stearoyl-CoA desaturase 1 by the isoflavone daidzein in diet-induced obesity. British journal 11
of pharmacology. 164(7): 1899-1915. 12
Davis, J., A. Higginbotham, et al. (2007). Soy protein and isoflavones influence adiposity and 13
development of metabolic syndrome in the obese male ZDF rat. Annals of nutrition & 14
metabolism. 51(1): 42-52. 15
Doddapattar, P., B. Radovic, et al. (2013). Xanthohumol ameliorates atherosclerotic plaque 16
formation, hypercholesterolemia, and hepatic steatosis in ApoE-deficient mice. Molecular 17
nutrition & food research. 18
Dorn, C., B. Kraus, et al. (2010). Xanthohumol, a chalcon derived from hops, inhibits hepatic 19
inflammation and fibrosis. Molecular nutrition & food research. 54 Suppl 2: S205-213. 20
Duthie, G. and A. Crozier (2000). Plant-derived phenolic antioxidants. Current opinion in lipidology. 21
11(1): 43-47. 22
Edwards, R. L., T. Lyon, et al. (2007). Quercetin reduces blood pressure in hypertensive subjects. The 23
Journal of nutrition. 137(11): 2405-2411. 24
Egert, S., A. Bosy-Westphal, et al. (2009). Quercetin reduces systolic blood pressure and plasma 25
oxidised low-density lipoprotein concentrations in overweight subjects with a high-26
cardiovascular disease risk phenotype: a double-blinded, placebo-controlled cross-over 27
study. The British journal of nutrition. 102(7): 1065-1074. 28
Fenton, H. J. H. (1894). Oxidation of tartaric acid in presence of iron. J Chem Soc Trans. 65: 899-911. 29
Ferre, P. and F. Foufelle (2010). Hepatic steatosis: a role for de novo lipogenesis and the transcription 30
factor SREBP-1c. Diabetes, obesity & metabolism. 12 Suppl 2: 83-92. 31
Frank, J., T. W. George, et al. (2009). Daily consumption of an aqueous green tea extract supplement 32
does not impair liver function or alter cardiovascular disease risk biomarkers in healthy men. 33
The Journal of nutrition. 139(1): 58-62. 34
Gao, M., Y. Ma, et al. (2013). Rutin suppresses palmitic acids-triggered inflammation in macrophages 35
and blocks high fat diet-induced obesity and fatty liver in mice. Pharmaceutical research. 36
30(11): 2940-2950. 37
Garcia-Lafuente, A., E. Guillamon, et al. (2009). Flavonoids as anti-inflammatory agents: implications 38
in cancer and cardiovascular disease. Inflammation research : official journal of the European 39
Histamine Research Society ... [et al.]. 58(9): 537-552. 40
Gazak, R., D. Walterova, et al. (2007). Silybin and silymarin--new and emerging applications in 41
medicine. Current medicinal chemistry. 14(3): 315-338. 42
Gloire, G. and J. Piette (2009). Redox regulation of nuclear post-translational modifications during 43
NF-kappaB activation. Antioxidants & redox signaling. 11(9): 2209-2222. 44
Goldwasser, J., P. Y. Cohen, et al. (2010). Transcriptional regulation of human and rat hepatic lipid 45
metabolism by the grapefruit flavonoid naringenin: role of PPARalpha, PPARgamma and 46
LXRalpha. PloS one. 5(8): e12399. 47
Gonzalez-Gallego, J., M. V. Garcia-Mediavilla, et al. (2010). Fruit polyphenols, immunity and 48
inflammation. The British journal of nutrition. 104 Suppl 3: S15-27. 49
Gonzalez, R., I. Ballester, et al. (2011). Effects of flavonoids and other polyphenols on inflammation. 50
Critical reviews in food science and nutrition. 51(4): 331-362. 51
Page 34 of 84
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For Peer Review Only
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Goto, T., A. Teraminami, et al. (2012). Tiliroside, a glycosidic flavonoid, ameliorates obesity-induced 1
metabolic disorders via activation of adiponectin signaling followed by enhancement of fatty 2
acid oxidation in liver and skeletal muscle in obese–diabetic mice. The Journal of nutritional 3
biochemistry. 23(7): 768-776. 4
Graefe, E. U., J. Wittig, et al. (2001). Pharmacokinetics and bioavailability of quercetin glycosides in 5
humans. Journal of clinical pharmacology. 41(5): 492-499. 6
Grattagliano, I., C. V. Diogo, et al. (2013). A silybin-phospholipids complex counteracts rat fatty liver 7
degeneration and mitochondrial oxidative changes. World journal of gastroenterology : WJG. 8
19(20): 3007-3017. 9
Gudbrandsen, O. A., H. Wergedahl, et al. (2009). A casein diet added isoflavone-enriched soy protein 10
favorably affects biomarkers of steatohepatitis in obese Zucker rats. Nutrition. 25(5): 574-11
580. 12
Gudbrandsen, O. A., H. Wergedahl, et al. (2006). Dietary soya protein concentrate enriched with 13
isoflavones reduced fatty liver, increased hepatic fatty acid oxidation and decreased the 14
hepatic mRNA level of VLDL receptor in obese Zucker rats. The British journal of nutrition. 15
96(2): 249-257. 16
Guo, H., M. Xia, et al. (2012). Cyanidin 3-glucoside attenuates obesity-associated insulin resistance 17
and hepatic steatosis in high-fat diet-fed and db/db mice via the transcription factor FoxO1. 18
The Journal of nutritional biochemistry. 23(4): 349-360. 19
Guo, H. X., D. H. Liu, et al. (2009). Long-term baicalin administration ameliorates metabolic disorders 20
and hepatic steatosis in rats given a high-fat diet. Acta pharmacologica Sinica. 30(11): 1505-21
1512. 22
Haddad, Y., D. Vallerand, et al. (2011). Antioxidant and hepatoprotective effects of silibinin in a rat 23
model of nonalcoholic steatohepatitis. Evidence-based complementary and alternative 24
medicine : eCAM. 2011: nep164. 25
Haenen, G. R., M. J. Arts, et al. (2006). Structure and activity in assessing antioxidant activity in vitro 26
and in vivo A critical appraisal illustrated with the flavonoids. Environmental toxicology and 27
pharmacology. 21(2): 191-198. 28
Haenen, G. R. and A. Bast (1999). Nitric oxide radical scavenging of flavonoids. Methods in 29
enzymology. 301: 490-503. 30
Haenen, G. R., J. B. Paquay, et al. (1997). Peroxynitrite scavenging by flavonoids. Biochemical and 31
biophysical research communications. 236(3): 591-593. 32
Haenen, G. R. M. M., F. P. Jansen, et al. (1993). The antioxidant properties of five O-(β-hydroxyethyl)-33
rutosides of the flavonoid mixture venoruton. Phlebology. 8(suppl 1): 10-17. 34
Haenen, G. R. M. M., J. B. G. Paquay, et al. (1997). Peroxynitrite Scavenging by Flavonoids. 35
Biochemical and biophysical research communications. 236(3): 591-593. 36
Harwood, M., B. Danielewska-Nikiel, et al. (2007). A critical review of the data related to the safety of 37
quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic 38
properties. Food and chemical toxicology : an international journal published for the British 39
Industrial Biological Research Association. 45(11): 2179-2205. 40
Heijnen, C. G., G. R. Haenen, et al. (2002). Protection of flavonoids against lipid peroxidation: the 41
structure activity relationship revisited. Free radical research. 36(5): 575-581. 42
Heinz, S. A., D. A. Henson, et al. (2010). Quercetin supplementation and upper respiratory tract 43
infection: A randomized community clinical trial. Pharmacological research : the official 44
journal of the Italian Pharmacological Society. 62(3): 237-242. 45
Hertog, M. G., D. Kromhout, et al. (1995). Flavonoid intake and long-term risk of coronary heart 46
disease and cancer in the seven countries study. Archives of internal medicine. 155(4): 381-47
386. 48
Higuchi, N., M. Kato, et al. (2008). Liver X receptor in cooperation with SREBP-1c is a major lipid 49
synthesis regulator in nonalcoholic fatty liver disease. Hepatology research : the official 50
journal of the Japan Society of Hepatology. 38(11): 1122-1129. 51
Page 35 of 84
URL: http://mc.manuscriptcentral.com/bfsn Email: fergc@foodsci.umass.edu
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Hollman, P. C., J. H. de Vries, et al. (1995). Absorption of dietary quercetin glycosides and quercetin in 1
healthy ileostomy volunteers. The American journal of clinical nutrition. 62(6): 1276-1282. 2
Hollman, P. C., M. vd Gaag, et al. (1996). Absorption and disposition kinetics of the dietary 3
antioxidant quercetin in man. Free radical biology & medicine. 21(5): 703-707. 4
Hsu, C. L., C. H. Wu, et al. (2009). Phenolic compounds rutin and o-coumaric acid ameliorate obesity 5
induced by high-fat diet in rats. Journal of agricultural and food chemistry. 57(2): 425-431. 6
Huang, C. S., C. K. Lii, et al. (2013). Protection by chrysin, apigenin, and luteolin against oxidative 7
stress is mediated by the Nrf2-dependent up-regulation of heme oxygenase 1 and glutamate 8
cysteine ligase in rat primary hepatocytes. Archives of toxicology. 87(1): 167-178. 9
Hwang, Y. P., J. H. Choi, et al. (2011). Purple sweet potato anthocyanins attenuate hepatic lipid 10
accumulation through activating adenosine monophosphate-activated protein kinase in 11
human HepG2 cells and obese mice. Nutrition research. 31(12): 896-906. 12
Ito, M., J. Suzuki, et al. (2007). Longitudinal analysis of murine steatohepatitis model induced by 13
chronic exposure to high-fat diet. Hepatology research : the official journal of the Japan 14
Society of Hepatology. 37(1): 50-57. 15
Jacobs, B. P., C. Dennehy, et al. (2002). Milk thistle for the treatment of liver disease: a systematic 16
review and meta-analysis. The American journal of medicine. 113(6): 506-515. 17
Javed, S., K. Kohli, et al. (2011). Reassessing bioavailability of silymarin. Alternative medicine review : 18
a journal of clinical therapeutic. 16(3): 239-249. 19
Ji, G., Q. Yang, et al. (2011). Anti-inflammatory effect of genistein on non-alcoholic steatohepatitis 20
rats induced by high fat diet and its potential mechanisms. International 21
immunopharmacology. 11(6): 762-768. 22
Jia, Y., J. Y. Kim, et al. (2013). Cyanidin is an agonistic ligand for peroxisome proliferator-activated 23
receptor-alpha reducing hepatic lipid. Biochimica et biophysica acta. 1831(4): 698-708. 24
Jung, C. H., I. Cho, et al. (2013). Quercetin reduces high-fat diet-induced fat accumulation in the liver 25
by regulating lipid metabolism genes. Phytotherapy research : PTR. 27(1): 139-143. 26
Kallwitz, E. R., A. McLachlan, et al. (2008). Role of peroxisome proliferators-activated receptors in the 27
pathogenesis and treatment of nonalcoholic fatty liver disease. World journal of 28
gastroenterology : WJG. 14(1): 22-28. 29
Kawasaki, T., K. Igarashi, et al. (2009). Rats fed fructose-enriched diets have characteristics of 30
nonalcoholic hepatic steatosis. The Journal of nutrition. 139(11): 2067-2071. 31
Kim, H., H. Kong, et al. (2005). Metabolic and pharmacological properties of rutin, a dietary quercetin 32
glycoside, for treatment of inflammatory bowel disease. Pharmaceutical research. 22(9): 33
1499-1509. 34
Kim, K. D., H. J. Lee, et al. (2012). Silibinin regulates gene expression, production and secretion of 35
mucin from cultured airway epithelial cells. Phytotherapy research : PTR. 26(9): 1301-1307. 36
Kim, M., S. G. Yang, et al. (2012). Silymarin suppresses hepatic stellate cell activation in a dietary rat 37
model of non-alcoholic steatohepatitis: analysis of isolated hepatic stellate cells. 38
International journal of molecular medicine. 30(3): 473-479. 39
Kim, M. H., K. S. Kang, et al. (2010). The inhibitory effect of genistein on hepatic steatosis is linked to 40
visceral adipocyte metabolism in mice with diet-induced non-alcoholic fatty liver disease. The 41
British journal of nutrition. 104(9): 1333-1342. 42
Kim, M. H., J. S. Park, et al. (2011). Daidzein supplementation prevents non-alcoholic fatty liver 43
disease through alternation of hepatic gene expression profiles and adipocyte metabolism. 44
International journal of obesity. 35(8): 1019-1030. 45
Kirsch, R., V. Clarkson, et al. (2003). Rodent nutritional model of non-alcoholic steatohepatitis: 46
species, strain and sex difference studies. Journal of gastroenterology and hepatology. 47
18(11): 1272-1282. 48
Kobori, M., S. Masumoto, et al. (2011). Chronic dietary intake of quercetin alleviates hepatic fat 49
accumulation associated with consumption of a Western-style diet in C57/BL6J mice. 50
Molecular nutrition & food research. 55(4): 530-540. 51
Page 36 of 84
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Koek, G. H., P. R. Liedorp, et al. (2011). The role of oxidative stress in non-alcoholic steatohepatitis. 1
Clin Chim Acta. 412(15-16): 1297-1305. 2
Kuzu, N., I. H. Bahcecioglu, et al. (2008). Epigallocatechin gallate attenuates experimental non-3
alcoholic steatohepatitis induced by high fat diet. Journal of gastroenterology and 4
hepatology. 23(8 Pt 2): e465-470. 5
Larkin, T., W. E. Price, et al. (2008). The key importance of soy isoflavone bioavailability to 6
understanding health benefits. Critical reviews in food science and nutrition. 48(6): 538-552. 7
Larter, C. Z. and M. M. Yeh (2008). Animal models of NASH: getting both pathology and metabolic 8
context right. Journal of gastroenterology and hepatology. 23(11): 1635-1648. 9
Lee, J. W., S. S. Choe, et al. (2012). AMPK activation with glabridin ameliorates adiposity and lipid 10
dysregulation in obesity. Journal of lipid research. 53(7): 1277-1286. 11
Lee, Y.-S., B.-Y. Cha, et al. (2013). Nobiletin improves obesity and insulin resistance in high-fat diet-12
induced obese mice. The Journal of nutritional biochemistry. 24(1): 156-162. 13
Lee, Y. M., J. S. Choi, et al. (2006). Effects of dietary genistein on hepatic lipid metabolism and 14
mitochondrial function in mice fed high-fat diets. Nutrition. 22(9): 956-964. 15
Lee, Y. S., B. Y. Cha, et al. (2013). Nobiletin improves obesity and insulin resistance in high-fat diet-16
induced obese mice. The Journal of nutritional biochemistry. 24(1): 156-162. 17
Lemmens, K. J. A., B. Van de Wier, et al. The flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside is able to 18
protect endothelial cells by a direct antioxidant effect. Toxicology in vitro, in press. 19
Liu, J. F., Y. Ma, et al. (2011). Reduction of lipid accumulation in HepG2 cells by luteolin is associated 20
with activation of AMPK and mitigation of oxidative stress. Phytotherapy research : PTR. 21
25(4): 588-596. 22
Loguercio, C., P. Andreone, et al. (2012). Silybin combined with phosphatidylcholine and vitamin E in 23
patients with nonalcoholic fatty liver disease: a randomized controlled trial. Free radical 24
biology & medicine. 52(9): 1658-1665. 25
Loguercio, C. and D. Festi (2011). Silybin and the liver: from basic research to clinical practice. World 26
journal of gastroenterology : WJG. 17(18): 2288-2301. 27
Macdonald, G. A. and J. B. Prins (2004). Peroxisomal fatty acid metabolism, peroxisomal proliferator-28
activated receptors and non-alcoholic fatty liver disease. Journal of gastroenterology and 29
hepatology. 19(12): 1335-1337. 30
Malek, M. A., M. H. Hoang, et al. (2013). Ombuin-3-O-beta-D-glucopyranoside from Gynostemma 31
pentaphyllum is a dual agonistic ligand of peroxisome proliferator-activated receptors alpha 32
and delta/beta. Biochemical and biophysical research communications. 430(4): 1322-1328. 33
Manach, C., G. Williamson, et al. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. 34
Review of 97 bioavailability studies. The American journal of clinical nutrition. 81(1 Suppl): 35
230S-242S. 36
Mann, G. E., B. Bonacasa, et al. (2009). Targeting the redox sensitive Nrf2-Keap1 defense pathway in 37
cardiovascular disease: protection afforded by dietary isoflavones. Current opinion in 38
pharmacology. 9(2): 139-145. 39
Manthey, J. A. (2000). Biological properties of flavonoids pertaining to inflammation. 40
Microcirculation. 7(6 Pt 2): S29-34. 41
Marcolin, E., L. F. Forgiarini, et al. (2013). Quercetin decreases liver damage in mice with non-42
alcoholic steatohepatitis. Basic & clinical pharmacology & toxicology. 112(6): 385-391. 43
Marcolin, E., B. San-Miguel, et al. (2012). Quercetin treatment ameliorates inflammation and fibrosis 44
in mice with nonalcoholic steatohepatitis. The Journal of nutrition. 142(10): 1821-1828. 45
Marra, F. (2008). Nuclear factor-kappaB inhibition and non-alcoholic steatohepatitis: inflammation as 46
a target for therapy. Gut. 57(5): 570-572. 47
Masterjohn, C. and R. S. Bruno (2012). Therapeutic potential of green tea in nonalcoholic fatty liver 48
disease. Nutrition reviews. 70(1): 41-56. 49
Page 37 of 84
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Mateo Anson, N., A. M. Aura, et al. (2011). Bioprocessing of wheat bran in whole wheat bread 1
increases the bioavailability of phenolic acids in men and exerts antiinflammatory effects ex 2
vivo. The Journal of nutrition. 141(1): 137-143. 3
Matsunami, T., Y. Sato, et al. (2010). Regulation of oxidative stress and inflammation by hepatic 4
adiponectin receptor 2 in an animal model of nonalcoholic steatohepatitis. International 5
journal of clinical and experimental pathology. 3(5): 472-481. 6
Medjakovic, S., M. Mueller, et al. (2010). Potential health-modulating effects of isoflavones and 7
metabolites via activation of PPAR and AhR. Nutrients. 2(3): 241-279. 8
Mei, L., M. Mochizuki, et al. (2012). Hepatoprotective effects of pycnogenol in a rat model of non-9
alcoholic steatohepatitis. Phytotherapy research : PTR. 26(10): 1572-1574. 10
Messina, M. (2010). A brief historical overview of the past two decades of soy and isoflavone 11
research. The Journal of nutrition. 140(7): 1350S-1354S. 12
Messina, M., C. Nagata, et al. (2006). Estimated Asian adult soy protein and isoflavone intakes. 13
Nutrition and cancer. 55(1): 1-12. 14
Mohamed Salih, S., P. Nallasamy, et al. (2009). Genistein improves liver function and attenuates non-15
alcoholic fatty liver disease in a rat model of insulin resistance. Journal of diabetes. 1(4): 278-16
287. 17
Mulvihill, E. E., E. M. Allister, et al. (2009). Naringenin prevents dyslipidemia, apolipoprotein B 18
overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced insulin 19
resistance. Diabetes. 58(10): 2198-2210. 20
Mulvihill, E. E., J. M. Assini, et al. (2010). Naringenin decreases progression of atherosclerosis by 21
improving dyslipidemia in high-fat-fed low-density lipoprotein receptor-null mice. 22
Arteriosclerosis, thrombosis, and vascular biology. 30(4): 742-748. 23
Nagarajan, P., M. J. Mahesh Kumar, et al. (2012). Genetically modified mouse models for the study of 24
nonalcoholic fatty liver disease. World journal of gastroenterology : WJG. 18(11): 1141-1153. 25
Nakamoto, K., F. Takayama, et al. (2009). Beneficial Effects of Fermented Green Tea Extract in a Rat 26
Model of Non-alcoholic Steatohepatitis. Journal of clinical biochemistry and nutrition. 44(3): 27
239-246. 28
Nanji, A. A. (2004). Animal models of nonalcoholic fatty liver disease and steatohepatitis. Clinics in 29
liver disease. 8(3): 559-574, ix. 30
Nelson, J. E., L. Wilson, et al. (2011). Relationship between the pattern of hepatic iron deposition and 31
histological severity in non alcoholic fatty liver disease. Hepatology. 53(2): 448-457. 32
Neuschwander-Tetri, B. A. and S. H. Caldwell (2003). Nonalcoholic steatohepatitis: Summary of an 33
AASLD Single Topic Conference. Hepatology. 37(5): 1202-1219. 34
O'Brien, J. and L. P. Powell (2011). Non-alcoholic fatty liver disease: Is iron relevant? Hepatol Int. 6(1): 35
332-341. 36
Pais, R., A. Pascale, et al. (2011). Progression from isolated steatosis to steatohepatitis and fibrosis in 37
nonalcoholic fatty liver disease. Clinics and research in hepatology and gastroenterology. 38
35(1): 23-28. 39
Panchal, S. K., H. Poudyal, et al. (2011). Rutin attenuates metabolic changes, nonalcoholic 40
steatohepatitis, and cardiovascular remodeling in high-carbohydrate, high-fat diet-fed rats. 41
The Journal of nutrition. 141(6): 1062-1069. 42
Panchal, S. K., H. Poudyal, et al. (2012). Quercetin ameliorates cardiovascular, hepatic, and metabolic 43
changes in diet-induced metabolic syndrome in rats. The Journal of nutrition. 142(6): 1026-44
1032. 45
Paquay, J. B., G. R. Haenen, et al. (2000). Protection against nitric oxide toxicity by tea. Journal of 46
agricultural and food chemistry. 48(11): 5768-5772. 47
Park, H. J., D. A. DiNatale, et al. (2011). Green tea extract attenuates hepatic steatosis by decreasing 48
adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice. The Journal 49
of nutritional biochemistry. 22(4): 393-400. 50
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Park, H. J., J. Y. Lee, et al. (2012). Green tea extract suppresses NFkappaB activation and 1
inflammatory responses in diet-induced obese rats with nonalcoholic steatohepatitis. The 2
Journal of nutrition. 142(1): 57-63. 3
Pietta, P. G. (2000). Flavonoids as antioxidants. Journal of natural products. 63(7): 1035-1042. 4
Polyzos, S. A., J. Kountouras, et al. (2012). Nonalcoholic fatty liver disease: multimodal treatment 5
options for a pathogenetically multiple-hit disease. Journal of clinical gastroenterology. 46(4): 6
272-284. 7
Puhl, A. C., A. Bernardes, et al. (2012). Mode of peroxisome proliferator-activated receptor gamma 8
activation by luteolin. Molecular pharmacology. 81(6): 788-799. 9
Qin, R., J. Zhang, et al. (2012). Protective effects of gypenosides against fatty liver disease induced by 10
high fat and cholesterol diet and alcohol in rats. Archives of pharmacal research. 35(7): 1241-11
1250. 12
Qin, Y., K. Niu, et al. (2013). Isoflavones for hypercholesterolaemia in adults. The Cochrane database 13
of systematic reviews. 6: CD009518. 14
Rolo, A. P., J. S. Teodoro, et al. (2012). Role of oxidative stress in the pathogenesis of nonalcoholic 15
steatohepatitis. Free Radic Biol Med. 52: 59-69. 16
Romestaing, C., M. A. Piquet, et al. (2007). Long term highly saturated fat diet does not induce NASH 17
in Wistar rats. Nutrition & metabolism. 4: 4. 18
Ronis, M. J., Y. Chen, et al. (2009). Dietary soy protein isolate attenuates metabolic syndrome in rats 19
via effects on PPAR, LXR, and SREBP signaling. The Journal of nutrition. 139(8): 1431-1438. 20
Ross, J. A. and C. M. Kasum (2002). Dietary flavonoids: bioavailability, metabolic effects, and safety. 21
Annual review of nutrition. 22: 19-34. 22
Ruijters, E. J. B., G. R. M. M. Haenen, et al. (2014). The cocoa flavanol (−)-epicatechin protects the 23
cortisol response. Pharmacological Research. 79(0): 28-33. 24
Russo, M., C. Spagnuolo, et al. (2012). The flavonoid quercetin in disease prevention and therapy: 25
facts and fancies. Biochemical pharmacology. 83(1): 6-15. 26
Salamone, F., F. Galvano, et al. (2012). Silibinin modulates lipid homeostasis and inhibits nuclear 27
factor kappa B activation in experimental nonalcoholic steatohepatitis. Translational research 28
: the journal of laboratory and clinical medicine. 159(6): 477-486. 29
Salamone, F., F. Galvano, et al. (2012). Silibinin improves hepatic and myocardial injury in mice with 30
nonalcoholic steatohepatitis. Digestive and liver disease : official journal of the Italian Society 31
of Gastroenterology and the Italian Association for the Study of the Liver. 44(4): 334-342. 32
Sarma, D. N., M. L. Barrett, et al. (2008). Safety of green tea extracts : a systematic review by the US 33
Pharmacopeia. Drug safety : an international journal of medical toxicology and drug 34
experience. 31(6): 469-484. 35
Savage, D. B., G. D. Tan, et al. (2003). Human metabolic syndrome resulting from dominant-negative 36
mutations in the nuclear receptor peroxisome proliferator-activated receptor-gamma. 37
Diabetes. 52(4): 910-917. 38
Serviddio, G., F. Bellanti, et al. (2010). A silybin-phospholipid complex prevents mitochondrial 39
dysfunction in a rodent model of nonalcoholic steatohepatitis. The Journal of pharmacology 40
and experimental therapeutics. 332(3): 922-932. 41
Sharma, A. K., S. Bharti, et al. (2011). Up-regulation of PPARgamma, heat shock protein-27 and -72 by 42
naringin attenuates insulin resistance, beta-cell dysfunction, hepatic steatosis and kidney 43
damage in a rat model of type 2 diabetes. The British journal of nutrition. 106(11): 1713-44
1723. 45
Sharma, S., A. Ali, et al. (2013). Rutin : therapeutic potential and recent advances in drug delivery. 46
Expert opinion on investigational drugs. 47
Shetty, S. N., S. Mengi, et al. (2010). A study of standardized extracts of Picrorhiza kurroa Royle ex 48
Benth in experimental nonalcoholic fatty liver disease. Journal of Ayurveda and integrative 49
medicine. 1(3): 203-210. 50
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Shih, V. F., R. Tsui, et al. (2011). A single NFkappaB system for both canonical and non-canonical 1
signaling. Cell research. 21(1): 86-102. 2
Shin, E. S., H. H. Lee, et al. (2007). Genistein downregulates SREBP-1 regulated gene expression by 3
inhibiting site-1 protease expression in HepG2 cells. The Journal of nutrition. 137(5): 1127-4
1131. 5
Shiri-Sverdlov, R., K. Wouters, et al. (2006). Early diet-induced non-alcoholic steatohepatitis in APOE2 6
knock-in mice and its prevention by fibrates. Journal of Hepatology. 44(4): 732-741. 7
Skottova, N., R. Vecera, et al. (2003). Effects of polyphenolic fraction of silymarin on lipoprotein 8
profile in rats fed cholesterol-rich diets. Pharmacological research : the official journal of the 9
Italian Pharmacological Society. 47(1): 17-26. 10
Song, Y. S., C. H. Fang, et al. (2013). Time course of the development of nonalcoholic Fatty liver 11
disease in the Otsuka long-evans Tokushima Fatty rat. Gastroenterology research and 12
practice. 2013: 342648. 13
Stevenson, D. E. and R. D. Hurst (2007). Polyphenolic phytochemicals--just antioxidants or much 14
more? Cellular and molecular life sciences : CMLS. 64(22): 2900-2916. 15
Sun, G. B., X. Sun, et al. (2012). Oxidative stress suppression by luteolin-induced heme oxygenase-1 16
expression. Toxicology and applied pharmacology. 265(2): 229-240. 17
Tailleux, A., K. Wouters, et al. (2012). Roles of PPARs in NAFLD: potential therapeutic targets. 18
Biochimica et biophysica acta. 1821(5): 809-818. 19
Takahashi, Y., Y. Soejima, et al. (2012). Animal models of nonalcoholic fatty liver disease/nonalcoholic 20
steatohepatitis. World journal of gastroenterology : WJG. 18(19): 2300-2308. 21
Tilg, H. and A. R. Moschen (2010). Evolution of inflammation in nonalcoholic fatty liver disease: the 22
multiple parallel hits hypothesis. Hepatology. 52(5): 1836-1846. 23
Tsuda, T., F. Horio, et al. (2003). Dietary cyanidin 3-O-beta-D-glucoside-rich purple corn color 24
prevents obesity and ameliorates hyperglycemia in mice. The Journal of nutrition. 133(7): 25
2125-2130. 26
Ucar, F., S. Sezer, et al. (2013). The relationship between oxidative stress and nonalcoholic fatty liver 27
disease: Its effects on the development of nonalcoholic steatohepatitis. Redox report : 28
communications in free radical research. 18(4): 127-133. 29
Ueno, T., T. Torimura, et al. (2009). Epigallocatechin-3-gallate improves nonalcoholic steatohepatitis 30
model mice expressing nuclear sterol regulatory element binding protein-1c in adipose 31
tissue. International journal of molecular medicine. 24(1): 17-22. 32
Ustundag, B., I. H. Bahcecioglu, et al. (2007). Protective effect of soy isoflavones and activity levels of 33
plasma paraoxonase and arylesterase in the experimental nonalcoholic steatohepatitis 34
model. Digestive diseases and sciences. 52(8): 2006-2014. 35
Valensi, P., C. Le Devehat, et al. (2005). A multicenter, double-blind, safety study of QR-333 for the 36
treatment of symptomatic diabetic peripheral neuropathy. A preliminary report. Journal of 37
diabetes and its complications. 19(5): 247-253. 38
Valenti, L., A. L. Fracanzani, et al. (2010). HFE genotype, parenchymal iron accumulation, and liver 39
fibrosis in patients with nonalcoholic fatty liver disease. Gastroenterology. 138(3): 905-912. 40
van Acker, F. A., O. Schouten, et al. (2000). Flavonoids can replace alpha-tocopherol as an 41
antioxidant. FEBS letters. 473(2): 145-148. 42
van Acker, S. A., M. N. Tromp, et al. (1995). Flavonoids as scavengers of nitric oxide radical. 43
Biochemical and biophysical research communications. 214(3): 755-759. 44
van de Wier, B., J. M. Balk, et al. (2013). Elevated citrate levels in non-alcoholic fatty liver disease: the 45
potential of citrate to promote radical production. FEBS letters. 587(15): 2461-2466. 46
van den Berg, R., G. R. Haenen, et al. (2001). Transcription factor NF-kappaB as a potential biomarker 47
for oxidative stress. The British journal of nutrition. 86 Suppl 1: S121-127. 48
van Erp-Baart, M. A., H. A. Brants, et al. (2003). Isoflavone intake in four different European 49
countries: the VENUS approach. The British journal of nutrition. 89 Suppl 1: S25-30. 50
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Vitale, D. C., C. Piazza, et al. (2013). Isoflavones: estrogenic activity, biological effect and 1
bioavailability. European journal of drug metabolism and pharmacokinetics. 38(1): 15-25. 2
Wood, N. (2004). Hepatolipidemic effects of naringenin in high cornstarch- versus high coconut oil-3
fed rats. Journal of medicinal food. 7(3): 315-319. 4
Wu, C. H., M. C. Lin, et al. (2011). Rutin inhibits oleic acid induced lipid accumulation via reducing 5
lipogenesis and oxidative stress in hepatocarcinoma cells. Journal of Food Science. 76(2): T65-6
72. 7
Wu, J. W., L. C. Lin, et al. (2009). Drug-drug interactions of silymarin on the perspective of 8
pharmacokinetics. Journal of ethnopharmacology. 121(2): 185-193. 9
Xia, M., M. Hou, et al. (2005). Anthocyanins induce cholesterol efflux from mouse peritoneal 10
macrophages: the role of the peroxisome proliferator-activated receptor {gamma}-liver X 11
receptor {alpha}-ABCA1 pathway. The Journal of biological chemistry. 280(44): 36792-36801. 12
Xiao, J., C. T. Ho, et al. (2013). Epigallocatechin gallate attenuates fibrosis, oxidative stress, and 13
inflammation in non-alcoholic fatty liver disease rat model through TGF/SMAD, PI3 14
K/Akt/FoxO1, and NF-kappa B pathways. European journal of nutrition. 15
Yalniz, M., I. H. Bahcecioglu, et al. (2007). Preventive role of genistein in an experimental non-16
alcoholic steatohepatitis model. Journal of gastroenterology and hepatology. 22(11): 2009-17
2014. 18
Yang, Y. C., C. K. Lii, et al. (2011). Induction of glutathione synthesis and heme oxygenase 1 by the 19
flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects 20
against oxidative stress. Free radical biology & medicine. 51(11): 2073-2081. 21
Yao, J., M. Zhi, et al. (2013). Effect and the probable mechanisms of silibinin in regulating insulin 22
resistance in the liver of rats with non-alcoholic fatty liver. Brazilian journal of medical and 23
biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade 24
Brasileira de Biofisica ... [et al.]. 46(3): 270-277. 25
Yao, J., M. Zhi, et al. (2011). Effect of silybin on high-fat-induced fatty liver in rats. Brazilian journal of 26
medical and biological research = Revista brasileira de pesquisas medicas e biologicas / 27
Sociedade Brasileira de Biofisica ... [et al.]. 44(7): 652-659. 28
Yap, F., L. Craddock, et al. (2011). Mechanism of AMPK suppression of LXR-dependent Srebp-1c 29
transcription. International journal of biological sciences. 7(5): 645-650. 30
Yilmaz, Y. (2012). Review article: is non-alcoholic fatty liver disease a spectrum, or are steatosis and 31
non-alcoholic steatohepatitis distinct conditions? Alimentary pharmacology & therapeutics. 32
36(9): 815-823. 33
Ying, H. Z., Y. H. Liu, et al. (2013). Dietary quercetin ameliorates nonalcoholic steatohepatitis induced 34
by a high-fat diet in gerbils. Food and chemical toxicology : an international journal published 35
for the British Industrial Biological Research Association. 52: 53-60. 36
Zhang, Z., W. Cui, et al. (2012). Baicalein protects against 6-OHDA-induced neurotoxicity through 37
activation of Keap1/Nrf2/HO-1 and involving PKCalpha and PI3K/AKT signaling pathways. 38
Journal of agricultural and food chemistry. 60(33): 8171-8182. 39
Zheng, P., G. Ji, et al. (2009). Therapeutic effect of puerarin on non-alcoholic rat fatty liver by 40
improving leptin signal transduction through JAK2/STAT3 pathways. The American journal of 41
Chinese medicine. 37(1): 69-83. 42
Zhu, W., Q. Jia, et al. (2012). The anthocyanin cyanidin-3-O-beta-glucoside, a flavonoid, increases 43
hepatic glutathione synthesis and protects hepatocytes against reactive oxygen species 44
during hyperglycemia: Involvement of a cAMP-PKA-dependent signaling pathway. Free 45
radical biology & medicine. 52(2): 314-327. 46
Ziaee, A., F. Zamansoltani, et al. (2009). Effects of rutin on lipid profile in hypercholesterolaemic rats. 47
Basic & clinical pharmacology & toxicology. 104(3): 253-258. 48
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Table 1.
Study
Species (n/group)
Model
Intervention Dose
(mg/kg/day) or (% of
diet)
Effects Silybin/Silymarin
Conclusion Effect
Lipid Metabolism
Oxidative Stress
Inflammation
Other
ALT
Histology
Weight
IR
Lipids
liver/serum
Coordinating
factors
Markers FA
oxidaton
Markers
Lipogenesis
Antioxidants
O.S. markers
Lipid
Peroxidation
Anti
Pro
Steatosis
Inflammation
Fibrosis
Intraperitoneal administration
Salamo
ne et al.
2012
Mice
(n=8)
Db/db mice +
MCD diet
20
Sb
dihydrogen
succinate
4 wks
↑
↑↑
↑
1
↑
↑↑
↑
2, 3
↓
↓↓
↓
1,2,3
↓
↓↓
↓
↓
↓↓
↓
1
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
↔
↔↔
↔
bw
↓
↓↓
↓
+++
Salamo
ne et al.
2012
(2)
Mice
(n=6)
Db/db mice +
MCD diet
20
Sb
dihydrogen
succinate
4 wks
↓
↓↓
↓
4
↑
↑↑
↑
4
↓
↓↓
↓
5,6
↓
↓↓
↓
↑
↑↑
↑
1
↓
↓↓
↓
2
↑
↑↑
↑
2
↓
↓↓
↓
3
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
↔
↔↔
↔
bw
↓
↓↓
↓
+++
Intragastrical administration
Yao et
al.
2013
R
ats
(n=15)
HFD
26.25 Sb
6 wks
↓
↓↓
↓
5, 6
↓
↓↓
↓
4, 5, 6
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
bw
↓
↓↓
↓
vf
↓
↓↓
↓
vf/bw
↔
↔↔
↔
scf
↓
↓↓
↓
+++
Yao et
al.
2011
Rats
(n=15)
HFD
26.25 Sb
6 wks
↓
↓↓
↓
5
↔
↔↔
↔
6, 7
↑
↑↑
↑
8
↓
↓↓
↓
9
↑
↑↑
↑
4
↑
↑↑
↑
7
↓
↓↓
↓
↓
↓↓
↓
7
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
+++
Qin et
al.
2012*
Rats
(n=13)
HFD +
alcohol i.g.
23 Sm
10 wks
↓
↓↓
↓
7, 10-12
↑
↑↑
↑
13
↔
↔↔
↔
4-6
↑
↑↑
↑
14
↓
↓↓
↓
↓
↓↓
↓
8
↔
↔↔
↔
9
↓
↓↓
↓
↔
↔↔
↔
bw
↓
↓↓
↓
lw/bw
+
Oral administration
Shetty
et al.
2010*
Rats
(n=6)
HFD 6 wks
50 Sm
last 4 wks
of diet
↓
↓↓
↓
5, 6, 15
↔
↔↔
↔
13
↓
↓↓
↓
+
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Grattag
liano et
al.
2013
Rats
(n=5)
CD diet 47 mg
Sb/day
7/14/30
days
↑
↑↑
↑
4, 12
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
↔
↔↔
↔
bw
↔
↔↔
↔
lw
+
HFD 47 mg
Sb/day
14/30/60
days
↔
↔↔
↔
4,
12
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
↔
↔↔
↔
bw
↓
↓↓
↓
lw
+
Dietary intervention
Haddad
et al.
2011
Rats
(n=6-
8)
HFD 12 wks
0.02% Sb
-
phosphatid
ylcholine
last 5 wks of
diet
↓
↓↓
↓
7
↓
↓↓
↓
13
↔
↔↔
↔
5
↔
↔↔
↔
14
↓
↓↓
↓
4$
↓
↓↓
↓
8
↓
↓↓
↓
↓
↓↓
↓
2§
↓
↓↓
↓
10,11
↔
↔↔
↔
9,12
↔
↔↔
↔
↓
↓↓
↓
↓
↓↓
↓
↔
↔↔
↔
bw
↓
↓↓
↓
lw
↓
↓↓
↓
lw/bw
↓
↓↓
↓
++
Servidd
io et al.
2010
Rats
(n=5)
HF MCD diet
0.04% Sb
-
phospholipi
d complex
7/14 wks
↔
↔↔
↔
4
↓
↓↓
↓
9-11
↓
↓↓
↓
↓
↓↓
↓
13
↑
↑↑
↑
14, 15
↓
↓↓
↓
↓
↓↓
↓
+
Kim et
al.
2012
Rats
(n=10)
OLEFTs +
MCD diet
0.5% Sm
8 wks
↔
↔↔
↔
16
↓
↓↓
↓
2
↑
↑↑
↑
16
↓
↓↓
↓
17-19
↔
↔↔
↔
↓
↓↓
↓
↔
↔↔
↔
bw
↔
↔↔
↔
lw
↔
↔↔
↔
lw/bw
+
Effects of silymarin/silybin are observed in the treatment group compared to the model group. Green = inhibiting effect on the development of NAFLD, blue = unknown or no
effect on development of NAFLD, orange = stimulating effect on development NAFLD. $ = theoretically a stimulating effect on the development/progression of NAFLD,
however the effect is opposite from the effect induced by the NAFLD model. *= silymarin used as control group for comparison other compounds, therefore limited
description of effects silymarin.
Specific markers related to lipid metabolism are categorized in four categories: lipids in liver or serum, general coordinating factors of lipid metabolism, markers involved in
fatty acids oxidation, and markers involved in lipogenesis. Markers related to oxidative stress are divided in antioxidants, oxidative stress markers (O.S. markers) and lipid
peroxidation. Specific markers related to inflammation are categorized in 2 categories: pro = pro-inflammatory factors, anti = anti-inflammatory factors. ALT = alanine
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aminotransferase levels serum. Histology is divided in steatosis, inflammation and fibrosis. IR = insulin resistance. Markers of inflammation and oxidative stress are measured
in the liver, unless mentioned by § = measured in serum.
Abbreviations: MCD diet = methionine choline deficient diet, CD diet = choline deficient diet, HFD = high fat diet, HF MCD diet = high fat MCD diet, bw = body weight, vf
= visceral fat weight, bw/vf = body weight/visceral fat weight-ratio, scf = subcutaneous fat weight, lw = liver weight, lw/bw = liver weight/body weight-ratio, OLEFTs=
Otsuka Long-Evans Tokushima Fatty rats with type 2 diabetes.
Markers:
Lipid metabolism: 1 = SCD-1 (g.ex + act), 2= Acyl-CoA oxidase (g.ex + act), 3 = L-FABP (g.ex), 4 = TG liver, 5 = TG serum, 6 = TC serum, 7 = LDL-C serum, 8 =
Adiponectin (p.ex + g.ex), 9= Resistin (p.ex + g.ex), 10 = FFA serum, 11= FFA liver, 12 = TC liver, 13= HDL-C serum , 14 = PPARα (p.ex + g.ex), 15 = total lipid content
liver, 16 = SREBP-1c (g.ex)
Oxidative stress: 1 = ROS, 2 = iNOS (p.ex), 3 = protein nitration, 4 = GSH, 5 = DNA-damage, 6 = liver nitrites/nitrates, 7 = SOD, 8= superoxide, 9 = GSSG, 10 =
GSSG/GSH-ratio, 11 = H
2
O
2
production , 12 = GPx activity
Inflammation: 1 = Il-6, 2 = tnf-α
Other: 1 = NF-κB binding activity, 2 = MRC-complexes activity, 3 = phosphorylated JNK (p.ex), 4 = FoxO1 (g.ex), 5 = PEPCK (g.ex), 6 = G-6-Pase (g.ex), 7 =
mitochondrial microviscosity, 8 = hepatocyte apoptosis, 9=cyp2e1 (p.ex), 10 = LDH serum, 11 = serum insulin, 12 = serum glucose, 13 = mitochondrial proton leakage, 14 =
mitochondrial respiration, 15 = ATP liver, 16 = nuclear nrf2 (p.ex), 17 = α1-procollagen (g.ex), 18 = α-SMA (g.ex), 19 = pERK1/2 in HSCs
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Table 2.
Study Model Intervention Effects of Silymarin found on Effect
Concl
Histology
Weight
IR
Liver enzymes
Other
S I F
Oral administration
Loguercio
et al. 2012
Double-blind RCT:
 Placebo (n=69)
 Realsil. (n=69)
All patients received recommendations
for life style modifications and an
individually designed diet.
Realsil = 94 mg silybin
complexed with 194 mg
phosphatidylcholine + 89.28
mg vitamin E acetate, 2x/day
p.o
↓
↓↓
↓
#
↓
↓↓
↓
#
↓
↓↓
↓
# Normalisati
on BMI*
↓
↓↓
↓
Normalisation ALT, AST,
γGT*
↓
↓↓
↓
Blood
glucose +
insulin
++
Effects of Realsil (silybin-phosphatidylcholine complex with vitamin E) in a randomized controlled clinical trial. *= Higher percentage of patients with normalization of ALT,
AST, γGT and BMI than in placebo group. #= Only 35 patients of the study agreed to liver biopsy at the end of the study. In the silymarin group there was an improvement in
liver biopsy, while in the placebo group there was no change.
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Table 3.
Study
Species (n/group)
Model
Intervention Dose
(mg/kg/day) or (% of
diet)
Effects soy isoflavones
Conclusion Effect
Lipid Metabolism
Oxidative Stress
Inflammation
Other
ALT
Histology
Weight
IR
Lipids
liver/serum
Coordinatin
g factors
Markers FA
oxidaton
Markers
Lipogenesis
Antioxidants
O.S.
markers
Lipid
Peroxidation
Anti
Pro
Steatosis
Inflammtion
Fibrosis
Subcutaneous administration
Yalniz
et al.
2007
Rats
(n=12)
HFD
0.2 G
6 wks
↓
↓↓
↓
1
↔
↔↔
↔
2
↓
↓↓
↓
↓
↓↓
↓
§
↔
↔↔
↔
3§
↓
↓↓
↓
1§
↔
↔↔
↔
1*
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
↔
↔↔
↔
*
↔
↔↔
↔
bw*,
lw*, bw/lw
+
Intraperitoneal administration
Crespill
o et al.
2011
Rats
(n=8)
HFD 12 wks
50 D
Last 2 wks
of diet
↔
↔↔
↔
1*,
2*,9*
↓
↓↓
↓
11
↓
↓↓
↓
3,17$,
18$, 20#
↑
↑↑
↑
4
↓
↓↓
↓
24
↔
↔↔
↔
16,
16#, 18# 23,
23#
↓
↓↓
↓
21#
↔
↔↔
↔
27
↓
↓↓
↓
27#$
↔
↔↔
↔
29
↑
↑↑
↑
30$
↑
↑↑
↑
1
*
↔
↔↔
↔
2
*
↔
↔↔
↔
*
↓
↓↓
↓
↓
↓↓
↓
bw
+/-
Intragastrical administration
Ji et al.
2011
Rats
(n=9)
HFD
4/8 G
12 wks
↓
↓↓
↓
↓
↓↓
↓
§
↔
↔↔
↔
3
↔
↔↔
↔
3§
↓
↓↓
↓
1, 2
↓
↓↓
↓
1, 2§
↓
↓↓
↓
3-5
↑
↑↑
↑
6
↔
↔↔
↔
7, 8
↓
↓↓
↓
↓
↓↓
↓
+
Oral administration
Salih et
al.
2009
Rats
(n=12)
HFrD
1 G
8.6 wks
↓
↓↓
↓
1,2,5-
8,12-14
↑
↑↑
↑
9
↑
↑↑
↑
15
↑
↑↑
↑
1,2,5-
7
↑
↑↑
↑
5-7§
↓
↓↓
↓
3, 4,
8
↓
↓↓
↓
↓
↓↓
↓
§
↓
↓↓
↓
1,2§
↓
↓↓
↓
1, 2, 9
↑
↑↑
↑
10-12
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
bw
↓
↓↓
↓
lw
↓
↓↓
↓
lw/bw
↓
↓↓
↓
++
Dietary intervention
Kim et
al.
2010
Mice
(n=18)
HFD
0.1/0.2/0.4
% G
12 wks
↓
↓↓
↓
1,2,5,7,
11-13
↑
↑↑
↑
9
↑
↑↑
↑
16#, 20#,
31#
↓
↓↓
↓
18#$
↓
↓↓
↓
21#
↑
↑↑
↑
32#
↓
↓↓
↓
25#,
33#, 35-
37#
↓
↓↓
↓
1#
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
bw
↓
↓↓
↓
vfw
+
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Lee et
al.
2006
Mice
(n=15)
HFD
0.1/0.2/0.4
% G
12 wks
↓
↓↓
↓
1, 2, 12,
13
↔
↔↔
↔
16, 18,
22
↑
↑↑
↑
38
↔
↔↔
↔
39
↑
↑↑
↑
40
↑
↑↑
↑
7
↔
↔↔
↔
9-11
↔
↔↔
↔
↓
↓↓
↓
↓
↓↓
↓
bw
↓
↓↓
↓
lw
↓
↓↓
↓
efw
+
Park et
al.
2006
Mice
(n=10)
Db/db mice
0.2% G/D
6 wks
↓
↓↓
↓
1, 2, 5,
12
↑
↑↑
↑
9
↔
↔↔
↔
13
↔
↔↔
↔
4
↓
↓↓
↓
42, 43
↑
↑↑
↑
41$
↓
↓↓
↓
30
↓
↓↓
↓
1, 15, 16,
20, 21,
↔
↔↔
↔
2
↑
↑↑
↑
13
↑
↑↑
↑
14
↔
↔↔
↔
b
w
↓
↓↓
↓
+/-
Kim et
al.
2011
Mice
(n=18)
HFD
0.01/0.05/0.
1/0.2% D
12 wks
↓
↓↓
↓
1,2, 10-
13
↑
↑↑
↑
4,49#
↓
↓↓
↓
3
↑
↑↑
↑
18, 50#,
31#
↓
↓↓
↓
46
↓
↓↓
↓
47
↔
↔↔
↔
16,18#
↑
↑↑
↑
51#,52
#
↑
↑↑
↑
33
↔
↔↔
↔
29
↓
↓↓
↓
48
↑
↑↑
↑
12
↑
↑↑
↑
13
↓
↓↓
↓
1
↓
↓↓
↓
1#
↓
↓↓
↓
1,2,18
↑
↑↑
↑
17
↓
↓↓
↓
↓
↓↓
↓
↓
↓↓
↓
bw
↓
↓↓
↓
efw
↔
↔↔
↔
vfw
↓
↓↓
↓
+/-
Gudbra
ndsen
et al.
2006
Rats
(n=6)
Obese Zucker
fa/fa rats
(0.045% D
+ 0.04% G)
/ (0.45% D
+ 0.4% G)
†
6 wks
↑
↑↑
↑
1, 5
↓
↓↓