Effects of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet

Transcription

journal of functional foods 10 (2014) 499–510
Available at www.sciencedirect.com
ScienceDirect
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff
Effects of Yuzu (Citrus junos Siebold ex Tanaka)
peel on the diet-induced obesity in a
zebrafish model
Liqing Zang a,*, Yasuhito Shimada b,c,d,e,f, Jumpei Kawajiri a,
Toshio Tanaka b,c,d,e,f, Norihiro Nishimura a
a
Department of Translational Medical Science, Mie University Graduate School of Medicine, 2-174 Edobashi,
Tsu, Mie 514-8507, Japan
b
Department of Systems Pharmacology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie
514-8507, Japan
c
Department of Molecular and Cellular Pharmacology, Pharmacogenomics, and Pharmacoinformatics, Mie
University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan
d
Mie University Medical Zebrafish Research Center, 2-174 Edobashi, Tsu, Mie 514-8507, Japan
e
Department of Bioinformatics, Mie University Life Science Research Center, 2-174 Edobashi, Tsu, Mie 5148507, Japan
f
Department of Omics Medicine, Mie University Industrial Technology Innovation Institute, 2-174 Edobashi,
Tsu, Mie 514-8507, Japan
A R T I C L E
I N F O
A B S T R A C T
Article history:
Effects of yuzu peel (Citrus junos Siebold ex Tanaka), yuzu pomace after hexane extraction,
Received 7 March 2014
and auraptene on metabolic disorders in zebrafish with diet-induced obesity (DIO) were evalu-
Received in revised form 31 July
ated. All materials tested exhibited anti-obesity effects. Yuzu peel significantly suppressed
2014
the rise in plasma triacylglycerol (TG) and liver lipid accumulation. The hepatic mRNA ex-
Accepted 7 August 2014
pression of pparab (peroxisome proliferator-activated receptor, alpha b) and its target genes
were significantly upregulated by yuzu peel, which suggests enhanced fatty acid β-oxidation
Keywords:
in liver. In visceral adipose tissue, yuzu peel significantly increased the mRNA expression
Anti-obesity
of pparg (peroxisome proliferator-activated receptor, gamma) and adipoqb (adiponectin, C1Q
Fatty acid oxidation
and collagen domain containing, b), which play roles in adipose differentiation and main-
PPARs
tenance. Our findings suggest that yuzu peel exerts anti-obesity effects by activating hepatic
Yuzu peel
PPARα and adipocyte PPARγ pathways. Additionally, the anti-obesity effects of yuzu pomace
Yuzu pomace
suggest a novel application to achieve complete use of yuzu instead of disposal as indus-
Zebrafish
trial waste.
© 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Obesity is one of the most challenging public health problems in developed countries and it is of growing concern in
* Corresponding author. Tel.: +81 59 231 5405; fax: +81 59 231 5405.
E-mail address: liqing@doc.medic.mie-u.ac.jp (L. Zang).
http://dx.doi.org/10.1016/j.jff.2014.08.002
1756-4646/© 2014 Elsevier Ltd. All rights reserved.
developing countries. The prevalence of obesity has increased so rapidly (worldwide obesity has nearly doubled since
1980) that it is now considered a global epidemic (WHO, 2013).
Obesity increases the likelihood of various adverse health consequences, particularly cardiovascular diseases, type 2 diabetes
500
mellitus, dyslipidaemia, nonalcoholic fatty liver, and certain
types of cancer (Haslam & James, 2005). Dieting and physical
exercise are the mainstays of treatment for obesity. If diet and
exercise are not effective, anti-obesity drugs may be taken, in
the context of a suitable diet, to reduce appetite or inhibit fat
absorption (Aronne, Powell, & Apovian, 2011; Christensen,
Kristensen, Bartels, Bliddal, & Astrup, 2007). However, most of
these drugs are associated with side effects such as high blood
pressure, restlessness, insomnia, and drug addiction (Bessesen,
2008). For this reason, a variety of natural products have been
studied for their potential to treat obesity with minimal side
effects (Hirai et al., 2010; Yun, 2010).
Yuzu originated in China, but also grows wild in Japan and
Korea. The fruit looks like a very small grapefruit with an uneven
skin, but is rarely eaten as a fruit because of its tart flavour.
Yuzu produces what has been described as a pleasant citrus
fragrance with a floral overtone, and is widely used in Japanese and Korean cuisines. Examples include Yuzu-ponzu (a
dressing made from soy sauce and yuzu juice), Yuzu-kosho (a
seasoning made from yuzu peel, chili peppers and salt) and
Yuja-cha (a traditional Korean herbal tea made from sliced yuzu
peel and combined with honey or sugar prepared as fruit preserves or marmalade). Furthermore, yuzu has been used in
traditional Chinese medicine as an aromatic stomachic and
sweating medicine. A hot yuzu bath has been reported to
improve blood circulation and prevent colds (Hirota et al., 2010).
Yuzu is effective in preventing certain diseases owing to its antiinflammatory (Hirota et al., 2010), antioxidant (Yoo, Lee, Park,
Lee, & Hwang, 2004), and anticarcinogenic (Kim et al., 2014;
Sawamura, Wu, Fujiwara, & Urushibata, 2005) properties. Recently, the ethanol extract of yuzu peel was found to exert antidiabetic effects via AMPK (AMP-activated protein kinase) and
PPARγ (peroxisome proliferator-activated receptor gamma) signalling both in vitro and in vivo (Kim et al., 2013). However, the
underlying mechanisms of the anti-obesity activity of yuzu have
not been explored.
Zebrafish (Danio rerio) species are vertebrates, and have
gained increased popularity as a model of human diseases
because their organs and genetics are similar to those of
humans (Lieschke & Currie, 2007; Shimada, Hirano, Nishimura,
& Tanaka, 2012). Zebrafish is used as a model of lipid metabolism for lipid-related diseases because of its similarities to that
in humans in terms of transport of fat and cholesterol by lipoproteins and storage as triacylglycerol (TG) in visceral,
subcutaneous, and intramuscular adipocyte depots (Shimada
et al., 2013; Stoletov et al., 2009). Moreover, a useful zebrafish
model of diet-induced obesity (DIO) has been created and was
found to be highly consistent with the obesity observed in
humans and in rodent models of DIO (Oka et al., 2010). The
overall gene expression profile of visceral adipose tissue is also
consistent with those of humans and rodents. This zebrafish
model of DIO has therefore been used to validate the antiobesity effects of natural products for their potential use in the
treatment of human obesity (Hiramitsu et al., 2014; Tainaka
et al., 2011).
In this study, we investigated the effects of yuzu peel, yuzu
pomace after hexane extraction, and auraptene (one of the
bioactive compounds present in yuzu peel) on body weight,
plasma TG, fat storage in the liver and adipose tissue, and expression of lipid metabolism-related genes in DIO zebrafish.
2.
Materials and methods
2.1.
Animals and maintenance
Zebrafish species (AB strain; the Zebrafish International Research Centre, Eugene, OR, USA) were maintained under
standard laboratory conditions at 28 °C with a light: dark cycle
of 14:10 h (Westerfield, 2007). Fish were fed twice daily with
commercial dry food (Hikari Tropical Fancy Guppy; Kyorin,
Hyogo, Japan) and once daily with live Artemia nauplii (Kitamura,
Kyoto, Japan). The zebrafish used in this study were 3-monthold females bred in our facility.
2.2.
Preparation of yuzu peel, yuzu pomace, and
auraptene-containing gluten granules
Yuzu peel and pomace were provided by Tsuji Oil Mill Co., Ltd
(Matsusaka, Mie, Japan). The fruit pulp of yuzu was removed
and the peel was sliced and stored at −20 °C for ≤ 2 months.
Hexane extraction was performed on the yuzu peel to extract
the essential yuzu oil, and the remaining pomace was heated
to 100 °C for 20 min to remove the residual hexane (1–2,000 ppm
of hexane remained at this stage. Details for this analytical
method are provided in Supplementary File S1). Samples were
then stored at −20 °C until use. Before treatment of zebrafish,
yuzu peel (containing essential oil) and pomace (not containing essential oil) were lyophilized and ground into granules
using a mortar and a 700 µm mesh sieve (AS ONE Corporation, Osaka, Japan). After lyophilization, a trace amount of
hexane (9.1 ppm) remained in pomace.
For oral administration of auraptene to adult zebrafish, we
used gluten as a carrier material. Auraptene (LKT Laboratories, St. Paul, MN, USA) was suspended in 50% ethanol and
mixed with gluten powder (Wako Pure Chemicals, Osaka, Japan).
The wet dough was kneaded, freeze-dried, and ground to granules according to methods described previously (Zang, Morikane,
Shimada, Tanaka, & Nishimura, 2011). Two kinds of gluten granules were made containing 0.1% (low-auraptene, LA) and 0.2%
(high-auraptene, HA) auraptene, respectively.
Yuzu peel, pomace, and auraptene-containing gluten granules were prepared just before use and stored at 4 °C in an
airtight container during the experimental period.
2.3.
Quantification of bioactive compounds in yuzu peel
and yuzu pomace
Levels of the primary bioactive compounds in yuzu peel and
pomace were quantified by Tsuji Oil Mill Co., Ltd (measurement of auraptene) and Japan Food Research Laboratories
(Nagoya, Aichi, Japan) (Table 1). Auraptene, hesperidin and
naringin were measured using high performance liquid chromatography (HPLC) (Ogawa et al., 2000). Limonene was
measured using gas chromatography-mass spectrometry (GCMS) (Lesjak et al., 2014). Eriocitrin was measured using liquid
chromatography-mass spectrometry (LC-MS/MS). Dietary fibre
was measured using an enzymatic-gravimetric method (AOAC
Official Methods of Analysis, 2000; Ajila & Rao, 2013). Details
for these analysis methods are provided in Supplementary
File S2. The major component of the essential oil extracted by
501
Table 1 – Contents of the primary bioactive compounds in yuzu peel and pomace.
Compound (units)
Yuzu peel
Yuzu pomace
Referencec
Auraptene (mg/g)a
Limonene (mg/100 g)b
Hesperidin (mg/100 g)b
Naringin (mg/100 g)b
Eriocitrin (mg/100 g)b
Dietary fibre (g/100 g)b
0.3 ± 0.02
150.77 ± 5.21 (83%d)
92.57 ± 0.15
52.23 ± 0.21
ND
6.4 ± 0.02
0.1 ± 0.02
9.99 ± 0.46
127 ± 3.61
68.23 ± 0.6
1.01 ± 0.07
8.35 ± 0.08
0.375
73.16%e
74.47–96.24
81.51–98.1
ND
4.4 ± 0.3
Ogawa et al., 2000
Choi, 2006
Yoo, Lee, Park, Lee, & Hwang, 2004
Yoo, Lee, Park, Lee, & Hwang, 2004
Miyake, 2006
Yoo, Hwang, Park, & Moon, 2009
ND, <0.5 mg/100 g.
a
Data are the mean ± SD (n = 3) on a dry weight basis.
b
Data are the mean ± SD (n = 3) on a fresh weight basis.
c
Contents in yuzu peel.
d
Proportion of limonene in hexane-extracted yuzu peel oil.
e
Proportion of limonene in yuzu peel oil extracted using a cold-pressing method.
hexane was limonene (83%, as measured by Tsuji Oil Mill Co.,
Ltd), with the remainder being aromatic compounds (data not
shown).
2.4.
Feeding zebrafish
The experimental protocol for induction of obesity and administration of yuzu peel, pomace, and auraptene is shown in
Supplementary Fig. S1. During the first 4 weeks, 3-month-old
female zebrafish were randomly divided with 15 fish per 2 L
tank for dietary restriction, which was performed by feeding
once daily (approximately 4 mg/fish/day) with Hikari Tropical
Fancy Guppy diet. After dietary restriction, zebrafish were assigned to one of six treatment groups (non-DIO, DIO, DIO + yuzu
peel, DIO + yuzu pomace, DIO + LA, DIO + HA) with five fish per
2 L tank. The details for feeding the zebrafish are shown in
Supplementary Table S1. Yuzu peel, pomace granules,
auraptene-containing gluten granules were fed to zebrafish
20 min before Artemia feeding. During feeding, the tank water
flow was stopped for 2 h. Leftover food was removed once daily
by vacuuming to avoid water pollution.
2.5.
Once the appropriate amount of blood was collected, suction
was stopped. The needle was removed and the blood sample
expelled from the needle onto a clean area of a piece of
parafilm. Two microlitres of blood was obtained using a pipette
set and then diluted with 6 µL of saline for determination of
plasma TG concentration. The blood samples were centrifuged for 3 min at 680 g at room temperature and the plasma
was harvested. TG was measured using a Wako L-type TG kit
(Wako Pure Chemicals) according to the manufacturer’s
protocol.
2.6.
Feeding volume assay
Feeding volume of Artemia was measured weekly during overfeeding treatment as previously described (Hasumura et al.,
2012; Tainaka et al., 2011). Briefly, freshly hatched Artemia were
uniformly suspended in the water, and then fed to zebrafish
in a 2 L fish tank. For a blank control, Artemia were placed in
a 2 L tank without zebrafish (i.e., containing only system water).
After 2 h, the number of Artemia not eaten by the zebrafish was
counted three times and subtracted from the number in the
blank tank to determine the feeding volume in each tank.
Measurement of body weight and plasma TG
2.7.
The body weight of the zebrafish was measured weekly during
the overfeeding treatment. Fish were fasted overnight and anesthetized by placing them in a tank containing 500 ppm of
2-phenoxyethanol (2-PE; Wako Pure Chemicals). Body weight
(g) was measured after the body surface was dried with soft
tissue paper (Kimwipe; Nippon Paper Crecia, Tokyo, Japan).
At the end of the experiment, blood samples were collected from individual zebrafish as described previously (Zang,
Shimada, Nishimura, Tanaka, & Nishimura, 2013). In brief, glass
microcapillary needles were prepared by pulling a glass capillary (GD-1; outer diameter, 1.0 mm; Narishige, Tokyo, Japan)
with a needle puller (PC-10; Narishige). The tips of needles were
cut obliquely and heparinized using an aspirator tube assembly (Drummond, New Bethlehem, PA, USA). Before blood
collection, the body surface of anaesthetized zebrafish was dried
with soft tissue paper. The heparinized needle was inserted
at 30–45° into the blood-collection site (along the body axis and
posterior to the anus in the region of the dorsal aorta). Once
the needle was felt to touch the spine, suction was applied to
the mouthpiece-end of the aspiration tube to collect the blood.
CT measurement of visceral adipose tissue volume
After blood collection, zebrafish were euthanized by immersion in an ice–water bath (5 parts ice/1 part water at ≤4 °C)
for ≥ 20 min (Matthews & Varga, 2012) and a 3D micro-CT scan
was performed using an in vivo System R_mCT 3D micro-CT
scanner (Rigaku, Tokyo, Japan) as described previously
(Hasumura et al., 2012). The 3D images were reconstructed and
viewed using i-View type R software (J. Morita Mfg, Kyoto, Japan),
and visualized and analysed using CTAtlas Metabolic Analysis Ver. 2.03 software (Rigaku). Measurement of visceral adipose
tissue volume was limited to the abdominal cavity, and the
initial point of the abdominal cavity was set from the cleithrum
to the anus.
2.8.
Oil red O staining
Scissors and forceps were used to strip off one side of the abdominal wall, which was then fixed in 4% formaldehyde
solution in PBS (Histo-Fresh; FALMA, Tokyo, Japan) at 4 °C for
24 h. The fixed liver tissues were isolated and placed in 30%
502
Table 2 – Primer pair sequences, accession numbers and product sizes of the studied genes.
Gene name
Zebrafish
Human
fasn
acacb
acadm
acox1
pparab
socs3b
pparg
adipoqb
18S rRNA
FASN
ACACB
ACADM
ACOX1
PPARα
SOCS3
PPARγ
ADIPOQ
18S rRNA
a
Accession
number
Forward primera
Reverse primera
Product
size (bp)
XM_005169478
XM_678989
NM_213010
NM_001005933
NM_001102567
NM_213304
NM_131467
BC165538
FJ915075
ATCTGTTCCTGTTCGATGGC
GTGGCTGGAAAACAACCTGT
TGGAGAAGGAGCTGGCTTTA
ACAGCACAGCAAGAGTAACG
CGTCGTCAGGTGTTTACGGT
CTCGGACCATCACCACTTCT
CTGCCGCATACACAAGAAGA
ACAAGAACGACAAGGCCATC
TGCATGGCCGTTCTTAGTTG
AGCATATCTCGGCTGACGTT
CCATGTGAATGATGCAGTCC
AAGACACTGCCTGGTGCTCT
TGAAGGGCATAAAGCAGAGC
AGGCACTTCTGGAATCGACA
GGCTGAGGGCATGTAATGAT
TCACGTCACTGGAGAACTCG
AAAACCGGAGAAGGTGGAGT
AGTCTCGTTCGTTATCGGAATGA
250
152
170
177
250
175
152
166
62
Sequences are given in the 5′–3′ order.
sucrose solution for 1 h at room temperature. Liver tissues were
embedded in Tissue-Tek OCT compound (Sakura Finetek Japan,
Tokyo, Japan) and rapidly frozen in liquid nitrogen-cooled
isopentane (Wako Pure Chemicals). Frozen livers were serially sectioned at 8 µm thickness with a HM-550 cryostat
(Microm, Walldorf, Germany), placed on slides, and dried at room
temperature. Sections were then stained with 0.3% oil red-O
(Wako Pure Chemicals) in 60% isopropanol solution for 15 min
at 37 °C as described previously (Tainaka et al., 2011). Sections were visualized under a BX41 microscope (Olympus, Tokyo,
Japan).
2.9.
RNA extraction, cDNA synthesis, and quantitative
real-time PCR
Zebrafish from which total RNA was to be extracted were given
a laparotomy, immediately transferred into tubes containing
3 mL of RNAlater (Qiagen, Hilden, Germany), and stored at 4 °C
for gene expression analysis. Liver and visceral adipose tissues
were dissected using forceps and subjected to RNA extraction. Total RNA was extracted from liver using Isogen
(Nippongene, Tokyo, Japan) combined with the cleanup protocol of the RNeasy mini kit (Qiagen). Total RNA was isolated
from visceral adipose tissue using the RNeasy Lipid tissue mini
kit (Qiagen). This kit integrates phenol/guanidine-based sample
lysis and silica-membrane purification of total RNA. Briefly, visceral adipose tissue was homogenized in 1 mL QIAzol Lysis
Reagent (provided in the kit; it is a monophasic solution of
phenol and guanidine thiocyanate that can facilitate lysis of
fatty tissues and inhibit RNases). After incubation at room temperature (RT) for 5 min, 200 µL of chloroform (Wako Pure
Chemicals) was added to the Lysis Reagent. The mixture was
incubated at RT for 3 min and then centrifuged at 12,000 g at
4 °C for 15 min. A 400-µL aliquot of the aqueous phase was separated and then the same volume of 70% ethanol added to
provide appropriate binding conditions. The mixture was trans-
ferred to an RNeasy Mini spin column, where the total RNA
bound to the membrane, and phenol and other contaminants were washed away. Total RNA was then eluted in RNasefree water. cDNA synthesis from 500 ng total RNA was
performed using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka,
Japan).
Quantitative real-time PCR (qPCR) was performed in cDNA
samples using Power SYBR Green Master Mix (Applied
Biosystems, Foster City, CA, USA) and the ABI 7300 Real-Time
PCR System (Applied Biosystems) in accordance with the manufacturer’s instructions. The sequences of the sense and
antisense primers used for amplification are shown in Table 2.
The relative mRNA expression levels were determined using
18S rRNA as an endogenous standard.
2.10.
Statistical analysis
All results are presented as means and standard errors (SE).
Differences between two groups were examined for statistical significance using Student’s t-test. For multiple comparisons,
one-way ANOVA followed by Bonferroni–Dunn multiplecomparison procedure was used. A P-value < 0.05 was
considered statistically significant.
3.
Results
3.1.
Yuzu peel and pomace exhibits high anti-obesity
effects in DIO zebrafish
Zebrafish in the DIO group exhibited a significantly higher body
weight (P < 0.001) and plasma TG (P < 0.05) compared with nonDIO zebrafish, indicating successful establishment of a DIO
model (Fig. 1A and 1B). The DIO group fed yuzu peel (6 mg/g/
Fig. 1 – Effects of yuzu peel and pomace on body weight, plasma TG, food intake, visceral adipose tissue volume, and lipid
composition in DIO zebrafish. (A) Changes in body weight in each group during 6-week overfeeding experiments (n = 15–
20). (B) Plasma TG levels in each group (n = 15–20). (C) Food intake of each group by counting Artemia numbers before and
after feeding during yuzu peel and yuzu pomace administration period. (D) Visceral adipose tissue volume in each group
(n = 15–20). (E) Oil red O staining of liver sections. Red droplets indicate neutral lipid staining. Values are means ± SE.
*P < 0.05, **P < 0.01 vs. the DIO group.
503
A
0.7
B
non-DIO
DIO
DIO + yuzu peel
DIO + yuzu pomace
0.6
*
*
800
700
600
Plasma TG (mg/dl)
Body weight (g)
0.5
*
0.4
**
0.3
**
**
**
**
**
0.2
500
400
300
200
0.1
100
0
0
0
1
2
3
4
5
6
non-DIO
DIO
DIO + yuzu peel
Week
6000
5000
D
non-DIO
DIO
DIO + yuzu peel
DIO + yuzu pomace
Artemia numbers / fish
ns
4000
ns
ns
ns
3000
2000
1000
4.0
*
**
Visceral adipose tissue volume (mm3)
C
DIO + yuzu
pomace
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
3
4
5
6
non-DIO
Week
E
non-DIO
DIO
DIO + yuzu peel
DIO + yuzu pomace
DIO
DIO + yuzu DIO + yuzu
peel
pomace
504
day) showed a trend towards reduced diet-induced body weight
gain (P = 0.13), and had significantly lower plasma TG (P < 0.05)
compared with DIO zebrafish. In addition, zebrafish that received yuzu pomace (6 mg/g/day) gained significantly less
body weight (P < 0.05) and showed a trend towards reduced
plasma TG (P = 0.09) compared with those in the DIO group.
No significant differences in feeding volume were observed
between the DIO, DIO + yuzu peel, and DIO + yuzu pomace
groups, indicating no appetite suppression in response to either
yuzu peel or pomace during the feeding experiment (Fig. 1C).
3D micro-CT analysis (Fig. 1D) showed that visceral adipose
tissue volume in the DIO group was significantly greater
(P < 0.01) than that in the non-DIO group. The yuzu pomace
group had significantly reduced visceral adipose tissue volume
(P < 0.05) while the yuzu peel group had a trend towards a reduction of adipose tissue volume (P = 0.16) compared with DIO
zebrafish. Additionally, Oil red O staining confirmed that DIO
zebrafish fed yuzu peel and yuzu pomace had much lower lipid
accumulation (numbers and size of red spots) in their liver compared with the DIO group (Fig. 1E).
3.2.
Auraptene exhibits anti-obesity effects in
DIO zebrafish
Auraptene is an abundant prenyloxycoumarin found in yuzu
peel. We hypothesized that auraptene might play a role in preventing lipid metabolism abnormalities induced by overfeeding.
Therefore, we performed the same DIO experiment and fed DIO
zebrafish a low-auraptene diet (10 µg/g/day, a 5.5-fold greater
level than that found in yuzu peel) or a high-auraptene diet
(20 µg/g/day, 11-fold greater than in yuzu peel), prepared as described in section 2.2. After a 4-week administration of
auraptene, zebrafish in the DIO + LA group showed a slight trend
towards lower body weight compared with those in the DIO
group (Fig. 2A). No changes in plasma TG or appetite suppression were observed after auraptene administration during the
feeding experiment (Fig. 2B and 2C). In addition, auraptene administration showed a dose-dependent trend towards lower
visceral adipose tissue volume (Fig. 2D). Moreover, DIO zebrafish
fed auraptene had less lipid accumulation in liver than those
in the DIO group (Fig. 2E).
3.3.
Effects of yuzu peel, pomace, and auraptene on the
expression of lipid metabolism genes in liver and visceral
adipose tissue
In the liver tissue at week 6 of the experiment, there were no
significant differences between the five DIO groups in the mRNA
expression levels of two lipogenic enzymes, fasn or acacb, which
are key enzymes for de novo fatty acid synthesis (Fig. 3). The
mRNA expression levels of acadm (a mitochondrial β-oxidation
enzyme, which catalyses the initial reaction in the β-oxidation
of C4 to C12 straight-chain acyl-CoAs), acox1 (a peroxisomal
β-oxidation enzyme, which is required for β-oxidation of longchain fatty acids), and pparab (a nuclear receptor protein that
regulates β-oxidation), were significantly higher in the
DIO + yuzu peel group than in the DIO group (P < 0.05).
In the visceral adipose tissue, no significant differences were
observed in the expression of fasn, acacb, or socs3b (a protein
that suppresses leptin signalling) between the groups (Fig. 4).
The expression level of pparab had a trend towards upregulation by yuzu peel but this was not statistically significant
(data not shown). However, the gene expression of acox1, pparg
(a regulator of adipocyte differentiation), and adipoqb
(adiponectin, an antilipogenic protein) were significantly higher
in the DIO + yuzu peel group compared with the DIO group
(P < 0.05).
4.
Discussion
Yuzu has traditionally been used in cuisines, and is also used
industrially in sweet production, beverages, cosmetics and perfumery. In commercial food processing, large amounts of
residual yuzu are commonly discarded as industrial waste after
squeezing yuzu for juice (in which case it is mainly the peel
that is discarded) or after extracting essential oil from yuzu
peel (in which case pomace is discarded). This is a major
problem that needs to be resolved; understanding how to more
completely use yuzu fruit will contribute to the reduction of
this industrial waste. The primary aim of this study was to
evaluate the anti-obesity effects of yuzu peel and pomace in
DIO zebrafish. We demonstrated here that yuzu peel and
pomace treatment exhibit powerful efficacy against body weight
gain, dyslipidaemia and hepatic steatosis (Fig. 1). We thus identified a potential use for current waste products from the
processing of yuzu, which may eventually enable a more complete use of yuzu and a reduction of the waste from its
processing.
Yuzu peel contains a wide variety of bioactive components, such as flavonoids (e.g. hesperidin, naringin and
eriocitrin), anthocyanins, phenolic acids, carotenoids, tannins,
vitamins, dietary fibres, and aromatics (e.g. limonene) (Nile &
Park, 2014). In this study, we measured the contents of several
compounds with bioactivity in yuzu peel and pomace (Table 1).
Among these components, we focused on auraptene, the most
abundant prenyloxycoumarin found in plants of the genus Citrus
(Epifano, Genovese, & Curini, 2008). Auraptene has various
notable biological functions, including anti-cancer, antiinflammatory, anti-bacterial, and angiogenic activity in vitro
(Genovese & Epifano, 2011; Wang et al., 2012). Yuzu peel used
in this study contained a relatively high level of auraptene
(0.3 ± 0.02 mg/g dry weight), similar to that reported in a previous study (0.376 mg/g dry weight) (Ogawa et al., 2000). It has
been reported that auraptene administration ameliorates
Fig. 2 – Effects of low-dose auraptene (LA) and high-dose auraptene (HA) on body weight, plasma TG, food intake, visceral
adipose tissue volume, and lipid composition in DIO zebrafish. (A) Changes in body weight in each group during 6-week
overfeeding experiments (n = 10). (B) Plasma TG levels in each group (n = 10). (C) Food intake of each group during auraptene
administration period. (D) Visceral adipose tissue volume in each group (n = 10). (E) Oil red O staining of liver sections.
Values are means ± SE. *P < 0.05, **P < 0.01 vs. the DIO group.
505
A
0.7
B
non-DIO
DIO
DIO + LA
DIO + HA
0.6
ns
*
800
700
**
0.4
**
0.3
**
**
**
Plasma TG (mg/dl)
Body weight (g)
600
0.5
**
0.2
500
400
300
200
0.1
100
0.0
0
1
2
3
4
5
0
6
non-DIO
DIO
DIO + LA
DIO + HA
Week
6000
Artemia nembers / fish
5000
4.0
D
non-DIO
DIO
DIO + LA
DIO + HA
ns
ns
ns
4000
ns
3000
2000
1000
ns
**
Visceral adipose tissue volume (mm3)
C
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
3
4
5
6
non-DIO
Week
E
non-DIO
DIO
DIO + LA
DIO + HA
DIO
DIO + LA
DIO + HA
506
fasn
acacb
3
Relative mRNA expression level
3
2.5
2
1.5
1
0.5
0
2.5
2
1.5
1
0.5
0
non-DIO
DIO
DIO + LA DIO + HA DIO + yuzu DIO + yuzu
peel
pomace
non-DIO
DIO
acox1
acadm
14
*
10
8
6
4
2
12
peel
pomace
*
12
10
8
6
4
2
0
0
non-DIO
DIO
peel
pomace
non-DIO
DIO
peel
pomace
pparab
16
*
14
12
10
8
6
4
2
0
non-DIO
DIO
peel
pomace
Fig. 3 – Effects of yuzu peel, pomace, and auraptene on the expression of lipid metabolism genes in liver. fasn and acacb
play important roles in lipogenesis. acadm, acox1 and pparab are closely related to fatty acid oxidation (n = 5). Values are
means ± SE. *P < 0.05 vs. the DIO group.
507
fasn
acacb
3
3
2.5
2
1.5
1
0.5
0
2.5
2
1.5
1
0.5
0
non-DIO
DIO
peel
pomace
non-DIO
DIO
acox1
socs3b
7
1.6
1.4
1.2
1
0.8
0.6
0.4
peel
pomace
*
6
5
4
3
2
1
0.2
0
0
non-DIO
DIO
non-DIO
peel
pomace
DIO
adipoqb
pparg
7
30
6
**
5
4
3
2
1
peel
pomace
*
25
20
15
10
5
0
0
non-DIO
DIO
peel
pomace
non-DIO
DIO
peel
pomace
Fig. 4 – Effects of yuzu peel, pomace, and auraptene on the expression of lipid metabolism genes in visceral adipose tissue.
socs3b is a negative regulator of leptin signalling. pparg and adipoqb play roles in adipose differentiation (n = 5). Values are
means ± SE. *P < 0.05 vs. the DIO group, **P < 0.01 vs. the DIO group.
508
hepatic TG accumulation in the livers of obese rats (Nagao et al.,
2010), which is consistent with our results in zebrafish livers
(Fig. 2E). However, there were no significant differences in
plasma TG levels or adipose tissue volume between the
auraptene group and the DIO group (Fig. 2B and 2D). This suggests that the therapeutic property of auraptene is limited to
improvement of hepatosteatosis, but not of systemic lipid metabolism including visceral adiposity.
In addition to auraptene, some bioactive molecules in yuzu
peel have been reported to induce PPAR mRNA expression with
consequent anti-obesity properties. For example, limonene protected against the development of dyslipidaemia by activating
PPARα transactivation in obese mice (Jing et al., 2013). Hesperidin and naringin are the most abundant flavonoids in yuzu
fruit (Nile & Park, 2014). The amounts of these flavonoids can
be affected by various factors, such as production area, measurement site and stage of maturation. We found that
hesperidin and naringin levels were high in our Japanese yuzu
peel (92.57 ± 0.15 and 52.23 ± 0.21 mg/100 g fresh weight, respectively); these levels are similar to those observed in Korean
yuzu peel (74.47–96.24 and 81.51–98.1 mg/100 g fresh weight,
respectively) (Yoo et al., 2004). Hesperidin and naringin attenuated hyperlipidaemia and hepatic steatosis, partly by
regulating fatty acid and cholesterol metabolism through enhancing hepatic and adipocyte PPARγ expression in type-2
diabetic animals (Jung, Lee, Park, Kang, & Choi, 2006; Sharma
et al., 2011). In this study, administration of yuzu peel increased the mRNA expression of markers of lipid oxidation
(pparab and acadm in liver, pparg in adipose tissue, and acox1
in both) and mature adipocytes (adipoqb in adipose tissue) in
DIO zebrafish without affecting markers of lipogenesis (fasn
and acacb in adipose tissue and liver) (Figs 3 and 4). It is well
known that activation of PPARα (the human homolog of
zebrafish pparab) causes lipid clearance via enhancement of
fatty acid β-oxidation in liver (Berger, Akiyama, & Meinke, 2005;
Kersten, 2002). The expression levels of PPARα target genes
(ACOX1 and ACADM) were also enhanced which consequently promote mitochondrial and peroxisomal fatty acid
β-oxidation pathway to increase lipolysis (Rakhshandehroo,
Knoch, Muller, & Kersten, 2010). PPARγ (the human homolog
of zebrafish pparg) is predominantly expressed in adipose tissue
and is critical for adipocyte differentiation, maintenance, and
regulation of adipose tissue lipid metabolism (Spiegelman, 1998).
In addition, PPARγ activates the transcription of ADIPOQ (the
human homolog of zebrafish adipoqb) (Lee, Olson, & Evans,
2003), which plays a role in energy homeostasis through the
regulation of fatty acid metabolism in muscle and liver (Berg,
Combs, & Scherer, 2002). These observations suggest that the
systemic anti-dyslipidaemia effect of yuzu peel is dependent
on the PPARγ-ADIPOQ axis, which may be conserved in vertebrates. Our results suggest that various bioactive compounds
in yuzu peel can improve dyslipidaemia and hepatic steatosis via activation of hepatic PPARα and adipocyte PPARγ
pathways.
It is notable that yuzu pomace also improved visceral adiposity and hepatic steatosis (Fig. 1D and 1E), while the gene
expression profiles are different from those of the peel group.
Yuzu contains an almost threefold greater amount of total
dietary fibre than other citrus fruits (Yoo, Hwang, Park, & Moon,
2009). The content of dietary fibre in yuzu pomace was higher
than in peel (8.35 ± 0.08 and 6.4 ± 0.02 g/100 g fresh weight, respectively) (Table 1). It is well known that dietary fibre can
suppress body weight gain in obese people, because of its low
digestibility and low absorption (Fukada, Furutani, Shimizu,
& Masumoto, 2013; Lattimer & Haub, 2010). In addition, while
most of the auraptene and limonene were lost after hexane
extraction, both hesperidin and naringin remained and were
further concentrated (Table 1). The greater amount of fibre in
combination with hesperidin and naringin in pomace may have
helped to suppress the body weight increase in the DIO group,
which was not seen in response to yuzu peel (Fig. 1A). Furthermore, we applied yuzu pomace to cultured red seabream
(Pagrus major), and found that yuzu pomace-supplemented bait
prevented dark muscle discoloration and decreased white
adipose tissue weight (data not shown). Overall, our results
suggest a possible application of yuzu peel or pomace for reducing obesity and related diseases, making use of material
which is currently disposed of as industrial waste.
5.
Conclusions
Using DIO zebrafish, we clarified the anti-obesity effects of yuzu
peel, pomace and the bioactive component auraptene. We established that the therapeutic mechanism of yuzu peel is
through activation of hepatic PPARα and adipocyte PPARγ pathways. Our findings demonstrate that yuzu peel may be an ideal
natural product with actions against obesity and related diseases. In addition, we showed that yuzu pomace is effective
in obesity phenotypes, which suggests a novel use for citrus
fruit pomace. This study is the first to identify new approaches
to achieve the complete use of yuzu, and thus provides a valuable insight into improving the processing of citrus fruit to
reduce waste.
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific Research in Japan (KAKENHI 25860294 and 25590073). The
authors would like to thank Tsuji Oil Mill Co., Ltd for supplying information, Mr. Junya Kuroyanagi and Ms. Mari Suzuki
for assistance with the zebrafish feeding experiment,
and Mrs. Asaka Uechi and Mrs. Eriko Nakanishi for secretarial assistance.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.jff.2014.08.002.
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Effects of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet

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