20

ORIGINAL

Extracts of citrus Sudachi peel attenuate body weight gain in
C57BL/6 mice fed a high-fat diet
Hitomi Kobayashi1, Mami Mitani1, Yuka Minatogawa1, Satoko Hayashi1, Mariko Nakamoto1, Emi Shuto1,
Yoshitaka Nii2, and Tohru Sakai1*
1
Department of Public Health and Applied Nutrition, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima,
Japan, 2Food and Biotechnology Division, Tokushima Prefectural Industrial Technology Center, Tokushima, Japan

Abstract : Citrus Sudachi is the special local product of Tokushima Prefecture, and over 98%% of Sudachi consumed
in Japan every year is produced in Tokushima Prefecture. In this study, we evaluated the function of sudachi
peel extract (SPE) using an animal model of obesity. C57BL/6 mice were fed a high-fat diet containing 1%% SPE
powder. Treatment with SPE significantly decreased body weight compared to that of mice fed a high-fat diet.
A significant difference in body weight was observed between the control and SPE groups from 7 weeks after
the start of the experiment, the significant difference continued until the end of the 14-week experiment. Reduction of blood glucose levels following insulin administration in SPE-treated mice was grater than that incontrol mice. Determination of mRNA expression in adipose tissue showed that the expression level of TNF- α
in the SPE group was significantly decreased compared to that on the control group. These results suggest that
SPE potentially has the ability to attenuate body weight gain. J. Med. Invest. 64 : 20-23, February, 2017
Keywords : Citrus sudachi, high-fat diet, inflammation, weight

INTRODUCTION

In this study, we examined the function of SPE in mice model.
We have already identify anti-obesity effect of sudachitin. However, it is difficult to apply directly to a human study because
sudachitin is a single compound as pharmacological medicine andis also expensive. A food supplement containing SPE is sold on themarket for human use. Identification of SPE function would bepromote human clinical study using a food supplement containing
SPE.

Obesity has been increasing worldwide due to changes in lifestylesuch as eating habits and a lack of exercise. In the United States,
awareness of the economic burden and health consequences associated with the growing obesity epidemic is rising (1). It is known
that visceral fat-type obesity induces type -2 diabetes mellitus,
hyperlipidaemia, and hypertension and increases the risk of cardiovascular disease (2, 3). Many researchers have investigatedanti-obesity agents as potential therapeutics for decreasing or preventing obesity.
Citrus Sudachi is the special local product in Tokushima Prefecture, and over 98% of Sudachi consumed in Japan is producedin Tokushima Prefecture. About 8,000 tons of citrus sudachi fruit
is produced every year. Half of the fruit is sold on market and half
is processed for obtaining juice. In the course of producing juice,about 200 tons of juice residual materials are generated every year.
It is known that citrus peel contains various type of functional
polyphenols. Therefore, it would be of value to evaluate the function of sudachi peel extract (SPE) for the purpose of effective utilization of citrus sudachi peel.
Sudachitin is a polymethxypolyphenol found in citrus sudachi.
We have evaluated the function of sudachitin. When mice were fed
a high-fat diet and treated with 5 mg/kg sudachitin, gain of bodyweigh was reduced compared to that in mice fed a high-fat diet
only. Fat weight including visceral and subcutaneous fat and serum
levels of tryglyceride and cholesterol were also reduced in thesudachitin-treated group. We also examined the mechanism andfound that sudachitin increases Sirt1, PGC1 - α and UCP-1 gene
expression, resulting in enhancement of energy expenditure (4).

The percentage of body fat at 12 weeks after starting SPE treatment was measured by X-ray computed tomography (CT ; LaTheta ;Aloka, Tokyo, Japan) from the first lumbar vertebra to the pubic
bone under isoflurane anesthesia. Data were analyzed using LaTheta
software.

Received for publication June 30, 2016 ; accepted August 1, 2016.

Insulin and glucose tolerance test

Address correspondence and reprint requests to Tohru Sakai, Department of Public Health and Applied Nutrition, Institute of Biomedical Sciences, Tokushima University Graduate School, 3 - 18 - 15 Kuramoto - cho,
Tokushima, 770 - 8563, Japan and Fax : +81 - 88 - 633 - 9427.

For insulin tolerance test, mice were intraperitoneally administered 0.75 U insulin per kg of body weight. For glucose tolerance
test, mice were orally administered 1.5 g glucose per kg of bodyweight. Blood samples were collected from the tip of the tail vein

MATERIALS AND METHODS
Mice and diets
Male C57BL/6J mice (Japan SLC, Shizuoka, Japan) were maintained under specific pathogen-free conditions with a 12 -h light :dark cycle at 25!2!
!and 55!10% relative humidity. Sudachi peel
extract was provided by Ikeda Yakusou Ltd (Tokushima, Japan).
Mice without SPE treatment (control group) were given a high-fat
diet, a purified ingredient diet with 45 kcal/g fat primarily from lard(no. D12451 ; Research Diets, New Brunswick, NJ, USA). Micein the SPE group were fed a high-fat diet containing 1% SPE. All
experimental procedures were approved by the Animal ResearchCommittee of the University of Tokushima.

Determination of body fat percentage

The Journal of Medical Investigation Vol. 64 2017

The Journal of Medical Investigation
at 0, 30, 60, 90 and 120 min after glucose administration. Blood glucose levels were measured by the FAD-glucose dehydrogenase
method with a GLUCOCARD GT-1820 device (ARKLAY, Kyoto,
Japan).

Quantitative reverse transcribed (RT)-PCR analysis
Total RNA was isolated from epididymal fat using an RNeasy
Lipid Tissue Mini kit (Qiagen Science, MD, USA). First-strand
cDNA was reverse-transcribed at 42"!for 60 min and at 95"!for5 min from 2 μg of the extracted total RNA with reverse transcriptase(Invitrogen, CA, USA) and a random primer. Real-time PCR was
performed by using specific primers and SYBR green dye (Takara
Bio, Shiga, Japan) in a StepOne PlusTM real-time PCR system(Applied Biosciences, USA) according to the manufacturer’s instructions. The primers used were 5’-ATGGCCTCCCTCTCATCAGTT-3’ (sense) and 5’- ACAGGCTTGTCACTCGAATTTTG-3’(antisense) for TNF-α, 5’-CCCAATGAGTAGGCTGGAGA-3’(sense) and 5’-TCTGGACCCATTCCTTCTTG-3’ (antisense) for
monocyte chemotactic protein (MCP)-1, 5’-GTGGACCTTCCAGGATGAGG-3’ (sense) and 5’-CGGAGCCTGTAGTGCAGTTG-3’(antisense) for interleukin (IL)-1β, 5’-ACAACCACGGCCTTCCCTACTT-3’ (sense) and 5’-CACGATTTCCCAGAGAACATGTG-3’(antisense) for IL-6, 5’-GAGCAGATACAGCAATGCCAG-3’ (sense)and 5’-GACACTGGGGCACTTTTGTT-3’ (antisense) for EGFlike module-containing mucin-like hormone receptor-like (emr)-1,
and 5’-CTTCATTGTGGGAGCAGACA-3’ (sense) and 5’-TCTCCAGAGCTGGGTTGTTC-3’ (antisense) for 36B4.

Vol. 64 February 201721

high-fat diet from 7 weeks to the end of the experiment (Fig. 1).
Both subcutaneous fat weight and visceral fat weight were determined. SPE treatment tended to reduce both fat weights, but thedifference was not significant. Data for body fat percentage weresimilar to the results for fat weight (data not shown). Next, we investigated blood glucose levels after intraperitoneal insulin andoral glucose administration. Mice treated with SPE showed significantly lower blood glucose levels at 90 and 120 min after insulin
administration (Fig. 2 A). We also evaluated reduction of glucose
levels as AUC and found that the AUC value in the SPE group was
significantly higher than that in the control group (Fig. 2 B). A significant difference was not found between the control and SPEgroups in blood glucose levels after oral glucose administration(data not shown).

Statistics
Data are shown as means and standard deviation. Data were analyzed using one -way analysis of variance followed by the Scheffe
post hoc test for multiple comparisons. Data are expressed asmeans!SD. Differences were considered significant at P!0.05.

RESULTS
Mice fed a high-fat diet showed significantly greater body weight
than that of mice fed normal chow. Treatment with SPE significantly decreased body weight compared to that of mice fed only a

Figure 1. SPE attenuates body weight gain in C57BL/6 mice fed ahigh - fat diet. Mice were fed a high - fat diet and treated with (triangle)(n = 10) or without SPE (circle) (n = 9). Other mice were fed normal chow
(diamond) (n = 5). Data are shown as means!SD. *p !0.05, **p !0.01
for HFD vs SPE. #p !0.05, ##p !0.01 for NC vs HFD.

Figure 2. Treatment with SPE improves responses to insulin. Mice were administered insulin, and blood glucose was determined at 30, 60, 90 and
120 min after the treatment. Circle, high - fat diet (n = 9) ; triangle, high - fat diet plus SPE (n = 10) ; diamond, normal chow (n = 5). (A). Glucose level
was also assessed as the AUC value (mg/dl·min). Black bar, normal chow (ND) ; white bar, high - fat diet (HFD) ; hatched bar, high - fat diet plus SPE(S1). (B). Values are means!SD. *p !0.05, **p !0.01.

22

H. Kobayashi, et al. Sudachi peel extracts attenuate body weight gain

We evaluated inflammatory status in adipose tissue by quantitative RT-RCR analysis. Expression levels of emr-1 and TNF-αmRNA in mice fed a high-fat diet were significantly higher thanthose in mice fed normal chow. The expression level of TNF-α
was significantly lower and that of emr-1 was tended to be lower
in the SPE group than in the high-fat diet group (Fig. 3). Expression
levels of other inflammatory cytokines were also measured, but
no significant difference was found between the control and SPEgroups (data not shown).

Table 1 Contents of flavnoides in SPE (g/100 g SPE powder)
Hesperidine

3.7

Narirutin

2.3

Naringenin

1.7

Neohesperidine

1.5

Sudachitin
Tangeretin

1.2!0.5 mg!0.5 mg

Nobiletin!0.5 mg

Rutin

DISCUSSION
We previously found that the citrus flavonoid sudachitin that ispresent in sudachi peel increases mitochondrial function, resulting
in enhancement of energy expenditure. This function is effective
for preventing obesity in an animal model. Indeed, treatment withsudachitin at 5 mg/kg body weight improved the serum lipid profile and reduced body weight in mice fed a high-fat diet (4). In thisstudy, we examined effect of SPE on body weight in mice fed ahigh-fat and found that SPE attenuate body weight gain. To ourknowledge, this is the first finding that SPE prevents obesity.
SPE contains a large number of constituents. Our analysis showedthat SPE contains many polyphenols including hesperidin, narirutin, naringenin and sudachitin etc. (Table 1). These components
have been shown to have unique physiological actions. Hesperidin
is a flavone abundantly found in citrus fruit peel. Glucosyl hesperidin has been shown to reduce serum TG in several animalmodels (5, 6) and in subjects with hypertirglyceridemia (7, 8).
Glucosyl hesperidin plus caffeine is effective for controlling obesity
mediated by the inhibition of hepatic lipogenesis (9). An anti-obestic
effect of these combinations has been shown in healthy, moderately
obese subjects (10). Naringenin is a predominant flavone in grapefruit. Naringenin has been shown to have antioxidant, anti-inflammatory and anti-proliferative activities (11). Furthermore, naringenin improves obesity-related diseases in mice (12, 13). The
results of these studies and our study using sudachitin suggest that
citrus fruit peel contains useful components for an anti-obesitic
effect. Shin et al. examined the effect of the extract of Prunus mume
fruit on obesity -related diseases. They found that the extract ofPrunus mume fruit attenuates the high-fat diet-induced increasein body weight and fat accumulation and improves the impaired
fasting glucose levels. An extract of the Prunus mume fruit contains
chlogenic acid, caffeic acid, rutin, luteolin-7 -glucoside, naringin,apigenin-7 -glucoside and hesperidin (14).
Mice were given a diet containing 1% SPE in this experiment.
We chose this dose because feeding of 1% SPE diet can given 5 mg
sudatitin per kg body weight. In our previous study, treatment
with sudachitin at 5 mg/kg body weight significantly suppressed
gain of body weight in mice fed a high-fat diet. In this study using
SPE, mice were given 5 mg sudachitin/kg body weight plus otherflavonoids including hesperidine, narirutin and naringenin. However, the beneficial effect of sudatichin on obesity is superior tothat of SPE. For example, it has been shown that sudatichin treatment alone decreases fat accumulation, serum triglyceride andNEFA levels (4). In contrast, treatment with SPE tended to decrease
fat accumulation in mice a fed high-fat diet, but the reduction was
not significant. We determined mRNA expression levels in adipose
tissue and skeletal muscle, which are affected by sudachitin treatment, but we did not find any beneficial change (data not shown).
It is possible that multiple flavonoids counteract their physiologicalactions, resulting in the appearance of phenotype observed in thisstudy.
SPE significantly attenuated body weight gain in mice fed ahigh-fat diet but did not significantly reduce subcutaneous fat
weight and visceral fat weight. However, treatment with SPE tended

Figure 3. SPE suppressed inflammatory response in adipose tissue from mice fed a high - fat diet. RNA was purified from adipose tissue of micein the control and SPE groups. Expression levels of emr - 1 (A) and TNF - α (B) mRNA were quantified by using a real - time PCR system as describedin the materials and methods section. Black bar, normal chow (ND) (n = 5) ; white bar, high - fat diet (HFD) (n = 9) ; hatched bar, high - fat diet plus
SPE (S1) (n = 10). Values are means!SD. *p !0.05, **p !0.01.

The Journal of Medical Investigation
to reduce both subcutaneous fat weight and visceral fat weight(data not shown). Reduction of weight gain might be an additive
reduction of both subcutaneous fat weight and visceral fat weight.
We did not determine the mechanism for reduction of bodyweight gain by SPE treatment. As stated previously, SPE contains
many flavonoids and some of these flavonoids have been shownto have anti-obesity action. We were not able to determine what
ingredient is effective. However, we found that SPE has a beneficial
effect in mice fed a high-fat diet. Our finding raises the possibility
that sudachi peel is applicable for a food supplement. The development of a method for effective utilization of sudachi peel wouldbe economically valuable for Tokushima Prefecture.

6.

7.

8.

CONFLICT OF INTEREST
The authors declare they have no conflict of interest.
9.

ACKNOWLEDGEMENTS
This work was supported by the Local Innovative Cluster Initiative Grant from the Ministory of Education, Culture, Sport, Scienceand Technology of Japan and Kieikai Research Foundation.

REFERENCES
1.

2.

3.

4.

5.

Finkelstein EA, Khavjou OA, Thompson H, Trogdon JG, Pan
L, Sherry B, Dietz W : Obesity and severe obesity forecasts
through 2030. Am J Prev Med 42 : 563-570, 2012
Hunt KJ, Resendez RG, Williams K, Haffner SM, Stern MP,
Study SAH : National Cholesterol Education Program versus
World Health Organization metabolic syndrome in relation
to all-cause and cardiovascular mortality in the San Antonio
Heart Study. Circulation 110 : 1251-1257, 2004
Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK,
Kumpusalo E, Tuomilehto J, Salonen JT : The metabolic syndrome and total and cardiovascular disease mortality in middleaged men. JAMA 288 : 2709 -2716, 2002
Tsutsumi R, Yoshida T, Nii Y, Okahisa N, Iwata S, Tsukayama
M, Hashimoto R, Taniguchi Y, Sakaue H, Hosaka T, Shuto E,
Sakai T : 2014. Sudachitin, a polymethoxylated flavone, improves glucose and lipid metabolism by increasing mitochondrial biogenesis in skeletal muscle. Nutr Metab (Lond) 11 :32, 2014
Akiyama S, Katsumata S, Suzuki K, Nakaya Y, Ishimi Y, Uehara
M : Hypoglycemic and hypolipidemic effects of hesperidin

10.

11.

12.

13.

14.

Vol. 64 February 201723

and cyclodextrin-clathrated hesperetin in Goto-Kakizaki rats
with type 2 diabetes. Biosci Biotechnol Biochem 73 : 27792782, 2009
Chiba H, Uehara M, Wu J, Wang X, Masuyama R, Suzuki K,
Kanazawa K, Ishimi Y : Hesperidin, a citrus flavonoid, inhibitsbone loss and decreases serum and hepatic lipids in ovariectomized mice. J Nutr 133 : 1892 -1897, 2003
Miwa Y, Mitsuzumi H, Sunayama T, Yamada M, Okada K,
Kubota M, Chaen H, Mishima Y, Kibata M : Glucosyl hesperidin lowers serum triglyceride level in hypertriglyceridemic
subjects through the improvement of very low-density lipoprotein metabolic abnormality. J Nutr Sci Vitaminol (Tokyo)
51 : 460 -470, 2005
Miwa Y, Yamada M, Sunayama T, Mitsuzumi H, Tsuzaki Y,
Chaen H, Mishima Y, Kibata M : Effects of glucosyl hesperidin
on serum lipids in hyperlipidemic subjects : preferential reduction in elevated serum triglyceride level. J Nutr Sci Vitaminol(Tokyo) 50 : 211 -218, 2004
Ohara T, Muroyama K, Yamamoto Y, Murosaki S : A combination of glucosyl hesperidin and caffeine exhibits an antiobesity effect by inhibition of hepatic lipogenesis in mice.
Phytother Res 29 : 310 -316, 2015
Ohara T, Muroyama K, Yamamoto Y, Murosaki S : Oral intake
of a combination of glucosyl hesperidin and caffeine elicits
an anti-obesity effect in healthy, moderately obese subjects :a randomized double-blind placebo-controlled trial. Nutr J
15 : 6, 2016
Yoshida H, Watanabe H, Ishida A, Watanabe W, Narumi K,
Atsumi T, Sugita C, Kurokawa M : Naringenin suppresses
macrophage infiltration into adipose tissue in an early phaseof high-fat diet-induced obesity. Biochem Biophys Res Commun 454 : 95 -101, 2014
Assini JM, Mulvihill EE, Sutherland BG, Telford DE, Sawyez
DG, Felder SL, Chhoker S, Edwards JY, Gros R, Huff MW :Naringenin prevents cholesterol-induced systemic inflammation, metabolic dysregulation, and atherosclerosis in Ldlr − / −mice. J Lipid Res 54 : 711 -724, 2013
Mulvihill EE, Assini JM, Sutherland BG, DiMattia AS, Khami
M, Koppes JB, Sawyez CG, Whitman SC, Huff MW : Naringenin decreases progression of atherosclerosis by improving
dyslipidemia in high-fat-fed low-density lipoprotein receptornull mice. Arterioscler Thromb Vasc Biol 30 : 742 -748, 2010
Shin EJ, Hur HJ, Sung MJ, Park JH, Yang HJ, Kim MS, Kwon
DY, Hwang JT : Ethanol extract of the Prunus mume fruits
stimulates glucose uptake by regulating PPAR-γ in C2C12
myotubes and ameliorates glucose intolerance and fat accumulation in mice fed a high-fat diet. Food Chem 141 : 4115 4121, 2013