Effects of Yuzu (Citrus junos Siebold ex Tanaka)peel on the diet-induced obesity in azebrafish 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, Japanb Department of Systems Pharmacology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie514-8507, Japanc Department of Molecular and Cellular Pharmacology, Pharmacogenomics, and Pharmacoinformatics, MieUniversity Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japand Mie University Medical Zebrafish Research Center, 2-174 Edobashi, Tsu, Mie 514-8507, Japane Department of Bioinformatics, Mie University Life Science Research Center, 2-174 Edobashi, Tsu, Mie 514-8507, Japanf 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

Article history:

Received 7 March 2014

Received in revised form 31 July

2014

Accepted 7 August 2014

A B S T R A C T

Effects of yuzu peel (Citrus junos Siebold ex Tanaka), yuzu pomace after hexane extraction,

and auraptene on metabolic disorders in zebrafish with diet-induced obesity (DIO) were evalu-

ated. All materials tested exhibited anti-obesity effects. Yuzu peel significantly suppressed

the rise in plasma triacylglycerol (TG) and liver lipid accumulation. The hepatic mRNA ex-

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

in liver. In visceral adipose tissue, yuzu peel significantly increased the mRNA expression

of pparg (peroxisome proliferator-activated receptor, gamma) and adipoqb (adiponectin, C1Q

and collagen domain containing, b), which play roles in adipose differentiation and main-

tenance. Our findings suggest that yuzu peel exerts anti-obesity effects by activating hepatic

PPARα and adipocyte PPARγ pathways. Additionally, the anti-obesity effects of yuzu pomace

suggest a novel application to achieve complete use of yuzu instead of disposal as indus-

trial waste.

© 2014 Elsevier Ltd. All rights reserved.

Keywords:

Anti-obesity

Fatty acid oxidation

PPARs

Yuzu peel

Yuzu pomace

Zebrafish

1. Introduction

Obesity is one of the most challenging public health prob-lems in developed countries and it is of growing concern in

developing countries. The prevalence of obesity has in-creased so rapidly (worldwide obesity has nearly doubled since1980) that it is now considered a global epidemic (WHO, 2013).Obesity increases the likelihood of various adverse health con-sequences, particularly cardiovascular diseases, type 2 diabetes

* 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.2014.08.0021756-4646/© 2014 Elsevier Ltd. All rights reserved.

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mellitus, dyslipidaemia, nonalcoholic fatty liver, and certaintypes of cancer (Haslam & James, 2005). Dieting and physicalexercise are the mainstays of treatment for obesity. If diet andexercise are not effective, anti-obesity drugs may be taken, inthe context of a suitable diet, to reduce appetite or inhibit fatabsorption (Aronne, Powell, & Apovian, 2011; Christensen,Kristensen, Bartels, Bliddal, & Astrup, 2007). However, most ofthese drugs are associated with side effects such as high bloodpressure, restlessness, insomnia, and drug addiction (Bessesen,2008). For this reason, a variety of natural products have beenstudied for their potential to treat obesity with minimal sideeffects (Hirai et al., 2010; Yun, 2010).

Yuzu originated in China, but also grows wild in Japan andKorea.The fruit looks like a very small grapefruit with an unevenskin, but is rarely eaten as a fruit because of its tart flavour.Yuzu produces what has been described as a pleasant citrusfragrance with a floral overtone, and is widely used in Japa-nese and Korean cuisines. Examples include Yuzu-ponzu (adressing made from soy sauce and yuzu juice), Yuzu-kosho (aseasoning made from yuzu peel, chili peppers and salt) andYuja-cha (a traditional Korean herbal tea made from sliced yuzupeel and combined with honey or sugar prepared as fruit pre-serves or marmalade). Furthermore, yuzu has been used intraditional Chinese medicine as an aromatic stomachic andsweating medicine. A hot yuzu bath has been reported toimprove blood circulation and prevent colds (Hirota et al., 2010).Yuzu is effective in preventing certain diseases owing to its anti-inflammatory (Hirota et al., 2010), antioxidant (Yoo, Lee, Park,Lee, & Hwang, 2004), and anticarcinogenic (Kim et al., 2014;Sawamura, Wu, Fujiwara, & Urushibata, 2005) properties. Re-cently, the ethanol extract of yuzu peel was found to exert anti-diabetic effects via AMPK (AMP-activated protein kinase) andPPARγ (peroxisome proliferator-activated receptor gamma) sig-nalling both in vitro and in vivo (Kim et al., 2013). However, theunderlying mechanisms of the anti-obesity activity of yuzu havenot been explored.

Zebrafish (Danio rerio) species are vertebrates, and havegained increased popularity as a model of human diseasesbecause their organs and genetics are similar to those ofhumans (Lieschke & Currie, 2007; Shimada, Hirano, Nishimura,& Tanaka, 2012). Zebrafish is used as a model of lipid metabo-lism for lipid-related diseases because of its similarities to thatin humans in terms of transport of fat and cholesterol by li-poproteins and storage as triacylglycerol (TG) in visceral,subcutaneous, and intramuscular adipocyte depots (Shimadaet al., 2013; Stoletov et al., 2009). Moreover, a useful zebrafishmodel of diet-induced obesity (DIO) has been created and wasfound to be highly consistent with the obesity observed inhumans and in rodent models of DIO (Oka et al., 2010). Theoverall gene expression profile of visceral adipose tissue is alsoconsistent with those of humans and rodents. This zebrafishmodel of DIO has therefore been used to validate the anti-obesity effects of natural products for their potential use in thetreatment of human obesity (Hiramitsu et al., 2014; Tainakaet al., 2011).

In this study, we investigated the effects of yuzu peel, yuzupomace after hexane extraction, and auraptene (one of thebioactive compounds present in yuzu peel) on body weight,plasma TG, fat storage in the liver and adipose tissue, and ex-pression of lipid metabolism-related genes in DIO zebrafish.

2. Materials and methods

2.1. Animals and maintenance

Zebrafish species (AB strain; the Zebrafish International Re-search Centre, Eugene, OR, USA) were maintained understandard laboratory conditions at 28 °C with a light: dark cycleof 14:10 h (Westerfield, 2007). Fish were fed twice daily withcommercial 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-month-old females bred in our facility.

2.2. Preparation of yuzu peel, yuzu pomace, andauraptene-containing gluten granules

Yuzu peel and pomace were provided by Tsuji Oil Mill Co., Ltd(Matsusaka, Mie, Japan). The fruit pulp of yuzu was removedand the peel was sliced and stored at −20 °C for ≤ 2 months.Hexane extraction was performed on the yuzu peel to extractthe essential yuzu oil, and the remaining pomace was heatedto 100 °C for 20 min to remove the residual hexane (1–2,000 ppmof hexane remained at this stage. Details for this analyticalmethod are provided in Supplementary File S1). Samples werethen stored at −20 °C until use. Before treatment of zebrafish,yuzu peel (containing essential oil) and pomace (not contain-ing essential oil) were lyophilized and ground into granulesusing a mortar and a 700 µm mesh sieve (AS ONE Corpora-tion, Osaka, Japan). After lyophilization, a trace amount ofhexane (9.1 ppm) remained in pomace.

For oral administration of auraptene to adult zebrafish, weused gluten as a carrier material. Auraptene (LKT Laborato-ries, St. Paul, MN, USA) was suspended in 50% ethanol andmixed with gluten powder (Wako Pure Chemicals, Osaka, Japan).The wet dough was kneaded, freeze-dried, and ground to gran-ules according to methods described previously (Zang, Morikane,Shimada, Tanaka, & Nishimura, 2011).Two kinds of gluten gran-ules were made containing 0.1% (low-auraptene, LA) and 0.2%(high-auraptene, HA) auraptene, respectively.

Yuzu peel, pomace, and auraptene-containing gluten gran-ules were prepared just before use and stored at 4 °C in anairtight container during the experimental period.

2.3. Quantification of bioactive compounds in yuzu peeland yuzu pomace

Levels of the primary bioactive compounds in yuzu peel andpomace were quantified by Tsuji Oil Mill Co., Ltd (measure-ment of auraptene) and Japan Food Research Laboratories(Nagoya, Aichi, Japan) (Table 1). Auraptene, hesperidin andnaringin were measured using high performance liquid chro-matography (HPLC) (Ogawa et al., 2000). Limonene wasmeasured using gas chromatography-mass spectrometry (GC-MS) (Lesjak et al., 2014). Eriocitrin was measured using liquidchromatography-mass spectrometry (LC-MS/MS). Dietary fibrewas measured using an enzymatic-gravimetric method (AOACOfficial Methods of Analysis, 2000; Ajila & Rao, 2013). Detailsfor these analysis methods are provided in SupplementaryFile S2. The major component of the essential oil extracted by

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hexane was limonene (83%, as measured by Tsuji Oil Mill Co.,Ltd), with the remainder being aromatic compounds (data notshown).

2.4. Feeding zebrafish

The experimental protocol for induction of obesity and ad-ministration of yuzu peel, pomace, and auraptene is shown inSupplementary Fig. S1. During the first 4 weeks, 3-month-oldfemale zebrafish were randomly divided with 15 fish per 2 Ltank for dietary restriction, which was performed by feedingonce daily (approximately 4 mg/fish/day) with Hikari TropicalFancy Guppy diet. After dietary restriction, zebrafish were as-signed to one of six treatment groups (non-DIO, DIO, DIO + yuzupeel, DIO + yuzu pomace, DIO + LA, DIO + HA) with five fish per2 L tank. The details for feeding the zebrafish are shown inSupplementary Table S1. Yuzu peel, pomace granules,auraptene-containing gluten granules were fed to zebrafish20 min before Artemia feeding. During feeding, the tank waterflow was stopped for 2 h. Leftover food was removed once dailyby vacuuming to avoid water pollution.

2.5. Measurement of body weight and plasma TG

The body weight of the zebrafish was measured weekly duringthe overfeeding treatment. Fish were fasted overnight and anes-thetized by placing them in a tank containing 500 ppm of2-phenoxyethanol (2-PE; Wako Pure Chemicals). Body weight(g) was measured after the body surface was dried with softtissue paper (Kimwipe; Nippon Paper Crecia, Tokyo, Japan).

At the end of the experiment, blood samples were col-lected from individual zebrafish as described previously (Zang,Shimada, Nishimura, Tanaka, & Nishimura, 2013). In brief, glassmicrocapillary needles were prepared by pulling a glass cap-illary (GD-1; outer diameter, 1.0 mm; Narishige, Tokyo, Japan)with a needle puller (PC-10; Narishige).The tips of needles werecut obliquely and heparinized using an aspirator tube assem-bly (Drummond, New Bethlehem, PA, USA). Before bloodcollection, the body surface of anaesthetized zebrafish was driedwith soft tissue paper. The heparinized needle was insertedat 30–45° into the blood-collection site (along the body axis andposterior to the anus in the region of the dorsal aorta). Oncethe needle was felt to touch the spine, suction was applied tothe mouthpiece-end of the aspiration tube to collect the blood.

Once the appropriate amount of blood was collected, suctionwas stopped. The needle was removed and the blood sampleexpelled from the needle onto a clean area of a piece ofparafilm. Two microlitres of blood was obtained using a pipetteset and then diluted with 6 µL of saline for determination ofplasma TG concentration. The blood samples were centri-fuged for 3 min at 680 g at room temperature and the plasmawas harvested. TG was measured using a Wako L-type TG kit(Wako Pure Chemicals) according to the manufacturer’sprotocol.

2.6. Feeding volume assay

Feeding volume of Artemia was measured weekly during over-feeding treatment as previously described (Hasumura et al.,2012; Tainaka et al., 2011). Briefly, freshly hatched Artemia wereuniformly suspended in the water, and then fed to zebrafishin a 2 L fish tank. For a blank control, Artemia were placed ina 2 L tank without zebrafish (i.e., containing only system water).After 2 h, the number of Artemia not eaten by the zebrafish wascounted three times and subtracted from the number in theblank tank to determine the feeding volume in each tank.

2.7. CT measurement of visceral adipose tissue volume

After blood collection, zebrafish were euthanized by immer-sion 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 scanwas performed using an in vivo System R_mCT 3D micro-CTscanner (Rigaku, Tokyo, Japan) as described previously(Hasumura et al., 2012). The 3D images were reconstructed andviewed using i-View type R software (J. Morita Mfg, Kyoto, Japan),and visualized and analysed using CTAtlas Metabolic Analy-sis Ver. 2.03 software (Rigaku). Measurement of visceral adiposetissue volume was limited to the abdominal cavity, and theinitial point of the abdominal cavity was set from the cleithrumto the anus.

2.8. Oil red O staining

Scissors and forceps were used to strip off one side of the ab-dominal wall, which was then fixed in 4% formaldehydesolution in PBS (Histo-Fresh; FALMA, Tokyo, Japan) at 4 °C for24 h. The fixed liver tissues were isolated and placed in 30%

Table 1 – Contents of the primary bioactive compounds in yuzu peel and pomace.

Compound (units) Yuzu peel Yuzu pomace Referencec

Auraptene (mg/g)a 0.3 ± 0.02 0.1 ± 0.02 0.375 Ogawa et al., 2000Limonene (mg/100 g)b 150.77 ± 5.21 (83%d) 9.99 ± 0.46 73.16%e Choi, 2006Hesperidin (mg/100 g)b 92.57 ± 0.15 127 ± 3.61 74.47–96.24 Yoo, Lee, Park, Lee, & Hwang, 2004Naringin (mg/100 g)b 52.23 ± 0.21 68.23 ± 0.6 81.51–98.1 Yoo, Lee, Park, Lee, & Hwang, 2004Eriocitrin (mg/100 g)b ND 1.01 ± 0.07 ND Miyake, 2006Dietary fibre (g/100 g)b 6.4 ± 0.02 8.35 ± 0.08 4.4 ± 0.3 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.

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sucrose solution for 1 h at room temperature. Liver tissues wereembedded in Tissue-Tek OCT compound (Sakura Finetek Japan,Tokyo, Japan) and rapidly frozen in liquid nitrogen-cooledisopentane (Wako Pure Chemicals). Frozen livers were seri-ally sectioned at 8 µm thickness with a HM-550 cryostat(Microm,Walldorf, Germany), placed on slides, and dried at roomtemperature. Sections were then stained with 0.3% oil red-O(Wako Pure Chemicals) in 60% isopropanol solution for 15 minat 37 °C as described previously (Tainaka et al., 2011). Sec-tions were visualized under a BX41 microscope (Olympus,Tokyo,Japan).

2.9. RNA extraction, cDNA synthesis, and quantitativereal-time PCR

Zebrafish from which total RNA was to be extracted were givena laparotomy, immediately transferred into tubes containing3 mL of RNAlater (Qiagen, Hilden, Germany), and stored at 4 °Cfor gene expression analysis. Liver and visceral adipose tissueswere dissected using forceps and subjected to RNA extrac-tion. Total RNA was extracted from liver using Isogen(Nippongene, Tokyo, Japan) combined with the cleanup pro-tocol of the RNeasy mini kit (Qiagen). Total RNA was isolatedfrom visceral adipose tissue using the RNeasy Lipid tissue minikit (Qiagen).This kit integrates phenol/guanidine-based samplelysis and silica-membrane purification of total RNA. Briefly, vis-ceral adipose tissue was homogenized in 1 mL QIAzol LysisReagent (provided in the kit; it is a monophasic solution ofphenol and guanidine thiocyanate that can facilitate lysis offatty tissues and inhibit RNases). After incubation at room tem-perature (RT) for 5 min, 200 µL of chloroform (Wako PureChemicals) was added to the Lysis Reagent. The mixture wasincubated at RT for 3 min and then centrifuged at 12,000 g at4 °C for 15 min. A 400-µL aliquot of the aqueous phase was sepa-rated and then the same volume of 70% ethanol added toprovide appropriate binding conditions.The mixture was trans-

ferred to an RNeasy Mini spin column, where the total RNAbound to the membrane, and phenol and other contami-nants were washed away. Total RNA was then eluted in RNase-free water. cDNA synthesis from 500 ng total RNA wasperformed using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka,Japan).

Quantitative real-time PCR (qPCR) was performed in cDNAsamples using Power SYBR Green Master Mix (AppliedBiosystems, Foster City, CA, USA) and the ABI 7300 Real-TimePCR System (Applied Biosystems) in accordance with the manu-facturer’s instructions. The sequences of the sense andantisense primers used for amplification are shown in Table 2.The relative mRNA expression levels were determined using18S 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 statisti-cal significance using Student’s t-test. For multiple comparisons,one-way ANOVA followed by Bonferroni–Dunn multiple-comparison procedure was used. A P-value < 0.05 wasconsidered statistically significant.

3. Results

3.1. Yuzu peel and pomace exhibits high anti-obesityeffects in DIO zebrafish

Zebrafish in the DIO group exhibited a significantly higher bodyweight (P < 0.001) and plasma TG (P < 0.05) compared with non-DIO zebrafish, indicating successful establishment of a DIOmodel (Fig. 1A and 1B). The DIO group fed yuzu peel (6 mg/g/

Table 2 – Primer pair sequences, accession numbers and product sizes of the studied genes.

Gene name Accessionnumber

Forward primera Reverse primera Productsize (bp)

Zebrafish Human

fasn FASN XM_005169478 ATCTGTTCCTGTTCGATGGC AGCATATCTCGGCTGACGTT 250acacb ACACB XM_678989 GTGGCTGGAAAACAACCTGT CCATGTGAATGATGCAGTCC 152acadm ACADM NM_213010 TGGAGAAGGAGCTGGCTTTA AAGACACTGCCTGGTGCTCT 170acox1 ACOX1 NM_001005933 ACAGCACAGCAAGAGTAACG TGAAGGGCATAAAGCAGAGC 177pparab PPARα NM_001102567 CGTCGTCAGGTGTTTACGGT AGGCACTTCTGGAATCGACA 250socs3b SOCS3 NM_213304 CTCGGACCATCACCACTTCT GGCTGAGGGCATGTAATGAT 175pparg PPARγ NM_131467 CTGCCGCATACACAAGAAGA TCACGTCACTGGAGAACTCG 152adipoqb ADIPOQ BC165538 ACAAGAACGACAAGGCCATC AAAACCGGAGAAGGTGGAGT 16618S rRNA 18S rRNA FJ915075 TGCATGGCCGTTCTTAGTTG AGTCTCGTTCGTTATCGGAATGA 62

a Sequences are given in the 5′–3′ order.

Fig. 1 – Effects of yuzu peel and pomace on body weight, plasma TG, food intake, visceral adipose tissue volume, and lipidcomposition 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 andafter 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.

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day) showed a trend towards reduced diet-induced body weightgain (P = 0.13), and had significantly lower plasma TG (P < 0.05)compared with DIO zebrafish. In addition, zebrafish that re-ceived yuzu pomace (6 mg/g/day) gained significantly lessbody weight (P < 0.05) and showed a trend towards reducedplasma TG (P = 0.09) compared with those in the DIO group.No significant differences in feeding volume were observedbetween the DIO, DIO + yuzu peel, and DIO + yuzu pomacegroups, indicating no appetite suppression in response to eitheryuzu peel or pomace during the feeding experiment (Fig. 1C).

3D micro-CT analysis (Fig. 1D) showed that visceral adiposetissue volume in the DIO group was significantly greater(P < 0.01) than that in the non-DIO group. The yuzu pomacegroup had significantly reduced visceral adipose tissue volume(P < 0.05) while the yuzu peel group had a trend towards a re-duction of adipose tissue volume (P = 0.16) compared with DIOzebrafish. Additionally, Oil red O staining confirmed that DIOzebrafish fed yuzu peel and yuzu pomace had much lower lipidaccumulation (numbers and size of red spots) in their liver com-pared with the DIO group (Fig. 1E).

3.2. Auraptene exhibits anti-obesity effects inDIO zebrafish

Auraptene is an abundant prenyloxycoumarin found in yuzupeel. We hypothesized that auraptene might play a role in pre-venting lipid metabolism abnormalities induced by overfeeding.Therefore, we performed the same DIO experiment and fed DIOzebrafish a low-auraptene diet (10 µg/g/day, a 5.5-fold greaterlevel than that found in yuzu peel) or a high-auraptene diet(20 µg/g/day, 11-fold greater than in yuzu peel), prepared as de-scribed in section 2.2. After a 4-week administration ofauraptene, zebrafish in the DIO + LA group showed a slight trendtowards lower body weight compared with those in the DIOgroup (Fig. 2A). No changes in plasma TG or appetite suppres-sion were observed after auraptene administration during thefeeding experiment (Fig. 2B and 2C). In addition, auraptene ad-ministration showed a dose-dependent trend towards lowervisceral adipose tissue volume (Fig. 2D). Moreover, DIO zebrafishfed auraptene had less lipid accumulation in liver than thosein the DIO group (Fig. 2E).

3.3. Effects of yuzu peel, pomace, and auraptene on theexpression of lipid metabolism genes in liver and visceraladipose tissue

In the liver tissue at week 6 of the experiment, there were nosignificant differences between the five DIO groups in the mRNAexpression levels of two lipogenic enzymes, fasn or acacb, whichare key enzymes for de novo fatty acid synthesis (Fig. 3). ThemRNA expression levels of acadm (a mitochondrial β-oxidationenzyme, which catalyses the initial reaction in the β-oxidationof C4 to C12 straight-chain acyl-CoAs), acox1 (a peroxisomal

β-oxidation enzyme, which is required for β-oxidation of long-chain fatty acids), and pparab (a nuclear receptor protein thatregulates β-oxidation), were significantly higher in theDIO + yuzu peel group than in the DIO group (P < 0.05).

In the visceral adipose tissue, no significant differences wereobserved in the expression of fasn, acacb, or socs3b (a proteinthat suppresses leptin signalling) between the groups (Fig. 4).The expression level of pparab had a trend towards up-regulation 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 higherin 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 usedindustrially in sweet production, beverages, cosmetics and per-fumery. In commercial food processing, large amounts ofresidual yuzu are commonly discarded as industrial waste aftersqueezing yuzu for juice (in which case it is mainly the peelthat is discarded) or after extracting essential oil from yuzupeel (in which case pomace is discarded). This is a majorproblem that needs to be resolved; understanding how to morecompletely use yuzu fruit will contribute to the reduction ofthis industrial waste. The primary aim of this study was toevaluate the anti-obesity effects of yuzu peel and pomace inDIO zebrafish. We demonstrated here that yuzu peel andpomace treatment exhibit powerful efficacy against body weightgain, dyslipidaemia and hepatic steatosis (Fig. 1). We thus iden-tified a potential use for current waste products from theprocessing of yuzu, which may eventually enable a more com-plete use of yuzu and a reduction of the waste from itsprocessing.

Yuzu peel contains a wide variety of bioactive compo-nents, such as flavonoids (e.g. hesperidin, naringin anderiocitrin), anthocyanins, phenolic acids, carotenoids, tannins,vitamins, dietary fibres, and aromatics (e.g. limonene) (Nile &Park, 2014). In this study, we measured the contents of severalcompounds with bioactivity in yuzu peel and pomace (Table 1).Among these components, we focused on auraptene, the mostabundant prenyloxycoumarin found in plants of the genus Citrus(Epifano, Genovese, & Curini, 2008). Auraptene has variousnotable biological functions, including anti-cancer, anti-inflammatory, anti-bacterial, and angiogenic activity in vitro(Genovese & Epifano, 2011; Wang et al., 2012). Yuzu peel usedin this study contained a relatively high level of auraptene(0.3 ± 0.02 mg/g dry weight), similar to that reported in a pre-vious study (0.376 mg/g dry weight) (Ogawa et al., 2000). It hasbeen 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, visceraladipose tissue volume, and lipid composition in DIO zebrafish. (A) Changes in body weight in each group during 6-weekoverfeeding experiments (n = 10). (B) Plasma TG levels in each group (n = 10). (C) Food intake of each group during aurapteneadministration 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.

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Fig. 3 – Effects of yuzu peel, pomace, and auraptene on the expression of lipid metabolism genes in liver. fasn and acacbplay important roles in lipogenesis. acadm, acox1 and pparab are closely related to fatty acid oxidation (n = 5). Values aremeans ± SE. *P < 0.05 vs. the DIO group.

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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 aremeans ± SE. *P < 0.05 vs. the DIO group, **P < 0.01 vs. the DIO group.

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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 inplasma TG levels or adipose tissue volume between theauraptene group and the DIO group (Fig. 2B and 2D). This sug-gests that the therapeutic property of auraptene is limited toimprovement of hepatosteatosis, but not of systemic lipid me-tabolism including visceral adiposity.

In addition to auraptene, some bioactive molecules in yuzupeel have been reported to induce PPAR mRNA expression withconsequent anti-obesity properties. For example, limonene pro-tected against the development of dyslipidaemia by activatingPPARα transactivation in obese mice (Jing et al., 2013). Hes-peridin and naringin are the most abundant flavonoids in yuzufruit (Nile & Park, 2014). The amounts of these flavonoids canbe affected by various factors, such as production area, mea-surement site and stage of maturation. We found thathesperidin and naringin levels were high in our Japanese yuzupeel (92.57 ± 0.15 and 52.23 ± 0.21 mg/100 g fresh weight, re-spectively); these levels are similar to those observed in Koreanyuzu peel (74.47–96.24 and 81.51–98.1 mg/100 g fresh weight,respectively) (Yoo et al., 2004). Hesperidin and naringin at-tenuated hyperlipidaemia and hepatic steatosis, partly byregulating fatty acid and cholesterol metabolism through en-hancing hepatic and adipocyte PPARγ expression in type-2diabetic animals (Jung, Lee, Park, Kang, & Choi, 2006; Sharmaet al., 2011). In this study, administration of yuzu peel in-creased the mRNA expression of markers of lipid oxidation(pparab and acadm in liver, pparg in adipose tissue, and acox1in both) and mature adipocytes (adipoqb in adipose tissue) inDIO zebrafish without affecting markers of lipogenesis (fasnand acacb in adipose tissue and liver) (Figs 3 and 4). It is wellknown that activation of PPARα (the human homolog ofzebrafish pparab) causes lipid clearance via enhancement offatty 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 conse-quently promote mitochondrial and peroxisomal fatty acidβ-oxidation pathway to increase lipolysis (Rakhshandehroo,Knoch, Muller, & Kersten, 2010). PPARγ (the human homologof zebrafish pparg) is predominantly expressed in adipose tissueand is critical for adipocyte differentiation, maintenance, andregulation of adipose tissue lipid metabolism (Spiegelman, 1998).In addition, PPARγ activates the transcription of ADIPOQ (thehuman homolog of zebrafish adipoqb) (Lee, Olson, & Evans,2003), which plays a role in energy homeostasis through theregulation of fatty acid metabolism in muscle and liver (Berg,Combs, & Scherer, 2002). These observations suggest that thesystemic anti-dyslipidaemia effect of yuzu peel is dependenton the PPARγ-ADIPOQ axis, which may be conserved in verte-brates. Our results suggest that various bioactive compoundsin yuzu peel can improve dyslipidaemia and hepatic steato-sis via activation of hepatic PPARα and adipocyte PPARγpathways.

It is notable that yuzu pomace also improved visceral adi-posity and hepatic steatosis (Fig. 1D and 1E), while the geneexpression profiles are different from those of the peel group.Yuzu contains an almost threefold greater amount of totaldietary 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, re-spectively) (Table 1). It is well known that dietary fibre cansuppress body weight gain in obese people, because of its lowdigestibility and low absorption (Fukada, Furutani, Shimizu,& Masumoto, 2013; Lattimer & Haub, 2010). In addition, whilemost of the auraptene and limonene were lost after hexaneextraction, both hesperidin and naringin remained and werefurther concentrated (Table 1). The greater amount of fibre incombination with hesperidin and naringin in pomace may havehelped to suppress the body weight increase in the DIO group,which was not seen in response to yuzu peel (Fig. 1A). Fur-thermore, we applied yuzu pomace to cultured red seabream(Pagrus major), and found that yuzu pomace-supplemented baitprevented dark muscle discoloration and decreased whiteadipose tissue weight (data not shown). Overall, our resultssuggest a possible application of yuzu peel or pomace for re-ducing obesity and related diseases, making use of materialwhich is currently disposed of as industrial waste.

5. Conclusions

Using DIO zebrafish, we clarified the anti-obesity effects of yuzupeel, pomace and the bioactive component auraptene. We es-tablished that the therapeutic mechanism of yuzu peel isthrough activation of hepatic PPARα and adipocyte PPARγ path-ways. Our findings demonstrate that yuzu peel may be an idealnatural product with actions against obesity and related dis-eases. In addition, we showed that yuzu pomace is effectivein obesity phenotypes, which suggests a novel use for citrusfruit pomace. This study is the first to identify new approachesto achieve the complete use of yuzu, and thus provides a valu-able insight into improving the processing of citrus fruit toreduce waste.

Acknowledgements

This work was supported in part by Grants-in-Aid for Scien-tific Research in Japan (KAKENHI 25860294 and 25590073). Theauthors would like to thank Tsuji Oil Mill Co., Ltd for supply-ing information, Mr. Junya Kuroyanagi and Ms. Mari Suzukifor assistance with the zebrafish feeding experiment,and Mrs. Asaka Uechi and Mrs. Eriko Nakanishi for secre-tarial assistance.

Appendix: Supplementary material

Supplementary data to this article can be found online atdoi:10.1016/j.jff.2014.08.002.

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