Article

pubs.acs.org/JAFC

Puried Betacyanins from Hylocereus undatus Peel Ameliorate

Obesity and Insulin Resistance in High-Fat-Diet-Fed Mice
Haizhao Song,, Qiang Chu, Dongdong Xu, Yang Xu, and Xiaodong Zheng*,,

College of Biosystems Engineering and Food Science, and Fuli Institute of Food Science, Zhejiang University, Hangzhou, Zhejiang
310058, Peoples Republic of China
*
S Supporting Information

ABSTRACT: Natural bioactive compounds in food have been shown to be benecial in preventing the development of obesity,
diabetes, and other metabolic diseases. Increasing evidence indicates that betacyanins possess free-radical-scavenging and
antioxidant activities, suggesting their benecial eects on metabolic disorders. The main objective of this study was to isolate and
identify the betaycanins from Hylocereus undatus (white-eshed pitaya) peel and evaluate their ability to ameliorate obesity,
insulin resistance, and hepatic steatosis in high-fat-diet (HFD)-induced obese mice. The puried pitaya peel betacyanins
(PPBNs) were identied by liquid chromatography/tandem mass spectrometry (LC/MS/MS), and the male C57BL/6 mice
were fed a low-fat diet, HFD, or HFD supplemented with PPBNs for 14 weeks. Our results showed that the white-eshed pitaya
peel contains 14 kinds of betacyanins and dietary PPBNs reduced HFD-induced body weight gain and ameliorated adipose tissue
hypertrophy, hepatosteatosis, glucose intolerance, and insulin resistance. Moreover, the hepatic gene expression analysis indicated
that PPBN supplementation increased the expression levels of lipid-metabolism-related genes (AdipoR2, Cpt1a, Cpt1b, Acox1,
PPAR, Insig1, and Insig2) and FGF21-related genes (-Klotho and FGFR1/2) but decreased the expression level of Fads2, Fas,
and FGF21, suggesting that the protective eect of PPBNs might be associated with the induced fatty acid oxidation, decreased
fatty acid biosynthesis, and alleviated FGF21 resistance.
KEYWORDS: Hylocereus undatus peel, betacyanins, obesity, insulin resistance, hepatic steatosis

INTRODUCTION
Obesity has been recognized as a major health problem
worldwide. As a consequence of the imbalance between energy
intake and expenditure, obesity is characterized by excessive fat
accumulation and dysregulation of lipid metabolism.1 More-
over, obesity can lead to various complications, such as type 2
diabetes, cardiovascular disease, hypertension, and non-
alcoholic fatty liver disease (NAFLD).2 Although a variety of
obesity treatment strategies were developed, such as exercise,
pharmacotherapy, and surgical interventions, many of the
approaches have a number of limitations, such as ineective-
ness, severe side eects, and high rates of secondary failure.3,4
Recently, numerous studies demonstrated that natural
bioactive compounds in food could act as important
modulators in the prevention of obesity development.5 As
water-soluble nitrogenous pigments, betalains have been
extensively used as colorants in the food industry. Increasing
evidence indicates that betalains possess free-radical-scavenging
and antioxidant activities,6,7 which are closely associated with
the protective eects of betalains against inammation, high
blood pressure, atherosclerosis, and hyperlipidemia.8,9 Betalains
consist of two distinct classes, which are redviolet betacyanins
and yelloworange betaxanthins. Betanin (betanidin 5-O--D-
glucoside) is the most abundant betacyanin, making up 75
95% of total betalains.10,11
Pitaya, commonly known as dragon fruit, has received
considerable attention not only because of its economic value
but also its health benets.12,13 Pitayas have been classied as
red (Hylocereus polyrhizus), white (Hylocereus undatus), and
yellow (Hylocereus megalanthus).14 Pitaya peel, which is usually
discarded during the processing and ends up as waste and a
source of pollution, accounts for approximately 33% of the
whole fruit weight.15 Although pitaya peel is a rich, natural, and
cost-eective source of betalains, there are very limited studies
focused on chemical composition and nutritional quality of
pitaya peel.
In this context, the objective of this study was to identify the
varieties of betalains present in H. undatus peel by liquid
chromatographymass spectrometry analysis and evaluate the
potential eects of pitaya peel betacyanins (PPBNs) on
metabolic disorders.
MATERIALS AND METHODS
Chemical Reagents and Plant Materials. Amberlite XAD7-HP
resins and betanin were obtained from Sigma-Aldrich (Sigma-Aldrich,
Steinheim, Germany). All of the other solvents and reagents were
purchased from Aladdin (Aladdin, Shanghai, China) and were of
analytical or high-performance liquid chromatography (HPLC) grade.
White-eshed pitayas were purchased from a fruit market in
Hangzhou, and the peels were collected and stored at 80 C prior
to use.
Extraction of Betacyanins from White-Fleshed Pitaya Peel.
Pitaya peels were homogenized with 90% aqueous methanol and 0.1%
triuoroacetic acid (TFA), and the homogenate was kept in 4 C for
12 h. Thereafter, the resulting extracts were centrifuged at 8000g for 40
min. Then, the supernatants were ltered through a 0.22 m pore size
Received: October 27, 2015
Revised: December 11, 2015
Accepted: December 12, 2015

XXXX American Chemical Society A DOI: 10.1021/acs.jafc.5b05177

J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry
lter (Millipore, Bedford, MA) and concentrated using a vacuum
rotary evaporator under reduced pressure at 38 C. The concentrated
extract was subsequently extracted 3 times with ethyl acetate. Then,
the aqueous layer was collected and evaporated again. The
concentrated solution was loaded onto a XAD-7HP column and
eluted with distilled water rst to remove organic acids, sugars, and
other water-soluble compounds and then eluted with 90% methanol
(0.1% TFA). The eluate was concentrated and reconstituted with
distilled water, and the aqueous layer was lyophilized and stored at
80 C until use.
Identication of Betacyanins by HPLCElectrospray Ioniza-
tion/Tandem Mass Spectrometry (ESI/MS/MS) Analysis.
HPLCESI/MS/MS (Agilent 1290 Innity, Agilent Technologies,
Santa Clara, CA) was used to identify the betacyanin compounds from
the white-eshed pitaya peel. Conditions for HPLC were as follows:
The chromatography column was Acclaim 120 C18 (250 4.6 mm, 5
m, 100 ), and the mobile phase was solvent A (0.1% aqueous formic
acid) and solvent B (acetonitrile and 0.1% formic acid). The gradient
ow was programmed as follows: 0 min, 5% B; 3 min, 5% B; 22 min,
12% B; 42 min, 14% B; 55 min, 5% B; and 60 min, 5% B. The injection
volume and ow rate were 0.5 mL/min and 10 L, respectively. The
betacyanin compounds were monitored using a wavelength of 538 nm.
Mass spectra analyses were performed in positive mode. The settings
were as follows: mass range m/z, 1002000; ion trap temperature, 325
C; capillary voltage, 3000 V; gas ow rate, 10 L/h; and desolvation
temperature, 350 C.
Quantication of Betacyanins. The total betacyanin content was
determined in deionized water using the previously described method,
with slight modications.16 The quantication of betacyanins was
carried out by applying the equation BC = [(A DF MW V/
LW)], where BC is the betanin equivalents in mg/g, A is the
absorption value at the absorption maximum (max = 538 nm), DF is
the dilution factor, MW is the molecular weight of betanin (550 g/
mol), V is the dried powder solution volume (mL), is the molar
extinction coecient of betanin (60 000 L mol1 cm1 in H2O), L is
the path length of the cuvette (1 cm), and W is the weight of the
PPBN powder (g).
Animals and Diets. All of the protocols in this research were
approved by the Committee on the Ethics of Animal Experiments of
Zhejiang University (permission number ZJU201550501), and the
experimental procedures were conducted in accordance with the
National Institutes of Health Regulations for the Care and Use of
Animals in Research. A total of 60 male C57BL/6J mice (4 weeks old;
n = 60) were supplied by the National Breeder Center of Rodents
(Shanghai, China) and maintained, four animals per cage, in a
temperature-controlled (23 3 C), 12 h light/dark cycle
environment with free access to water and food. After 1 week for
acclimation, the mice were randomly divided into the following ve
groups (n = 12): low-fat-diet (LFD) group, high-fat-diet (HFD)
group, and HFD plus PPBNs of 50 mg/kg (HFD + L), 100 mg/kg
(HFD + M), or 200 mg/kg (HFD + H) treatment groups. The
compositions and energy densities of the puried diets were listed in
Table S1 of the Supporting Information. In the PPBN group, mice
received a low, moderate, or high dose of PPBNs by intragastric
administration for 14 weeks. The mice in LFD and HFD groups
received an equal volume of saline. The body weights and food intakes
were monitored weekly. After 14 weeks of treatment, the mice were
sacriced by decapitation after a 12 h fast period. Blood samples were
collected for serum preparation by centrifugation. The livers, hearts,
kidneys, spleens, interscapular brown fat, and epididymal and perirenal
fat pads were collected, weighted, and stored at 80 C.
Blood Chemistry and Hormone Analysis. The serum levels of
glucose, triglyceride (TG), total cholesterol (TC), aspartate amino-
transferase (AST), alanine aminotransferase (ALT), low-density
lipoprotein cholesterol (LDL-C), and high-density lipoprotein
cholesterol (HDL-C) were determined by an automatic biochemistry
analyzer (ACCUTE TBA-40FR, Toshiba Medical Systems Co.,
Tochigi, Japan) in accordance with the instructions of the
manufacturer. The serum concentrations of insulin and neuropeptide
Y (NPY) were determined using commercial assay kits (Elabscience
B DOI: 10.1021/acs.jafc.5b05177
Article
Biotechnology, Wuhan, Hubei, China), and the serum levels of
adiponectin, leptin, and FGF-21 and were aslo measured by enzyme
linked immunosorbent assay (ELISA) using commercial assay kits
(R&D Systems, Minneapolis, MN) according to the protocols of the
manufacturer. The serum levels of glucose and insulin were used to
calculate the homeostasis model assessment-estimated insulin
resistance (HOMA-IR) and homeostasis model assessment-estimated
insulin sensitivity (HOMA-IS) according to the following formula:
HOMAIR = serum glucose (mmol/L) serum insulin (mU)
/22.5
HOMAIS = 1/[serum glucose (mmol/L)
serum insulin (mU)]
Oral Glucose Tolerance Test (OGTT). At the end of the 12th
week, the mice fasted for 12 h and the oral glucose tolerance test was
performed after gavage with glucose (2 g/kg of body weight). Blood
was collected from the tail vein at 0, 30, 60, 90, and 120 min for the
determination of glucose concentrations. The area under the curve
(AUC) of glucose was also calculated.
Histological Analysis. Standardized pieces of epididymal and
perirenal white adipose tissue, interscapular brown adipose tissue, and
livers were xed in 10% buered formalin, and then the tissue sections
were embedded in paran, sliced at 5 m thickness, and stained with
hematoxylin and eosin (H&E). For oil red O staining, the liver tissues
were embedded in optimal cutting temperature gel, then the air-dried
tissue sections of 5 m were dipped in formalin and washed with oil
red O solution. The images were captured using an Olympus CX41
camera (Olympus, Tokyo, Japan), and the hepatic steatosis scores
were graded as follows: 0 (steatosis involving <5% fatty hepatocytes),
1 (steatosis involving 533% fatty hepatocytes), 2 (steatosis involving
3466% fatty hepatocytes), and 3 (steatosis involving >66% fatty
hepatocytes).17
Determination of Hepatic Lipids. Approximately 100 mg of liver
tissues were homogenized, and the hepatic lipids were extracted with a
chloroform/methanol (2:1, v/v) solution using the Folch method.18
Then, the levels of hepatic TG and TC in the extraction solution were
determined by enzymatic methods using commercially available kits
(Elabscience Biotechnology, Wuhan, Hubei, China) according to the
instructions of the manufacturer.
Quantitative Real-Time Polymerase Chain Reaction (PCR).
Total RNA was extracted from the livers using Trizol (Invitrogen, Life
Technologies, Carlsbad, CA) according to the instructions of the
manufacturer, and the RNA concentrations were determined by
NanoDrop 2000 (Thermo Scientic, Wilmington, DE). Then, 1 g of
total RNA was converted to the rst strand of cDNA using a reverse
transcription kit (TaKaRa, Dalian, Liaoning, China).
Quantitative PCR was performed on the ABI 7500 system (Applied
Biosystems, Foster, CA). The 20 L reaction mixtures consisted of 1
L of cDNA, paired primers (300 nM), and 10 L of SYBR Green
QPCR Master Mix (Roche Diagnostics, Ltd., Lewes, U.K.). The
amplication program was set as follows: 3 min at 50 C, 10 min at 95
C, then 35 cycles of 15 s at 95 C and then 60 s at 60 C followed by
melting curve for 60 s at 95 C, then gradual decrease to 50 C, 20 s at
50 C, then gradual increase to 95 C, and 20 s at 95 C. The primer
sequences are listed in Table S2 of the Supporting Information. The
target genes were examined and normalized by -actin, and the relative
fold change was calculated using the 2CT method.19
Statistical Analysis. Values were expressed as the mean
standard error of the mean (SEM). For experiments comparing
multiple groups, the statistical signicance were analyzed by one-way
analysis of variance (ANOVA) followed by Duncans post-hoc test.
The statistical analyses were performed using SPSS 19.0 statistical
software. All of the results were considered statistically signicant at p
< 0.05.
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Figure 1. Chromatographic proles (HPLC gradient system) of puried betacyanins from white-eshed pitaya peel at 538 nm. The peak
assignments are in listed in Table S1 of the Supporting Information.

Table 1. Identication of Betacyanins in White-Fleshed Pitaya Peela

peak betacyanin Rt (min) max (nm) m/z [M + H]+ m/z from MS/MS of [M + H]+
2022
1 betanidin-5-O--glucoside (betanin) 16.33 538 551.15 389.10, 343.09
222 17-decarboxy-betanin 18.28 505 507.16 345.11, 299.10
32022 isobetanidin-5-O--glucoside (isobetanin) 19.32 538 551.15 389.10, 343.09
422 17-decarboxy-isobetanin 21.96 505 507.16 345.11
522 betanidin-5-O-(6-O-malonyl)--glucoside (phyllocactin) 23.23 538 637.16 595.17, 389.10
620 betanidin-5-O-(6-O-3-hydroxy-butyryl)--glucoside 24.21 536 637.16 389.10
722 2-decarboxy-betanin 24.61 533 507.16 345.11, 299.10
821,22 17-decarboxy-phyllocactin 25.22 505 593.16 549.17, 507.16, 345.11
921,22 isobetanidin-5-O-(6-O-malonyl)--glucoside (isophyllocactin) 26.01 536 637.15 595.17, 389.10
1023 2-O-apiosyl-phyllocactin 26.66 536 769.19 725.19, 389.11, 683.19, 343.09, 150.06
11 unknown 27.11 656.17 612.18, 568.19, 417.13, 389.10, 345.10
1222 17-decarboxy-isophyllocactin 28.65 505 593.16 549.17, 345.11
1321,22 2-decarboxy-phyllocactin 34.7 533 593.16 549.17, 345.11
1422 2-decarboxy-isophyllocactin 37.94 533 593.16 549.17, 345.11
a
Rt = retention time. The peak number is in the order of elution (Rt) for the 14 betacyanins.

Figure 2. White-eshed PPBNs reduce weight gain but not calorie intake in HFD-fed C57BL/6 mice. (A) Body weights of mice consuming the
indicated diets for the 14 week intervention period. (B) Food intake on indicated diets of mice during the intervention period. (C) Calorie intake on
indicated diets of mice during the intervention period. LFD, mice fed a low-fat diet; HFD, mice fed a high-fat diet; and HFD + L, M, and H, mice fed
a high-fat diet supplemented with PPBNs at 50, 100, and 200 mg/kg, respectively. Data are expressed as the mean SEM (n = 12). Values not
sharing a common letter dier signicantly among groups (p < 0.05; ANOVA).

C DOI: 10.1021/acs.jafc.5b05177
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Table 2. Eect of White-Fleshed PPBNs on Tissue Weight and Serum Parameters in Micea
item LFD HFD HFD + L HFD + M HFD + H
Tissue Index
heart 0.57 0.02 d 0.35 0.01 a 0.38 0.02 ab 0.4 0.01 b 0.5 0.01 c
liver 3.95 0.06 c 2.82 0.12 a 3.25 0.08 b 3.4 0.09 b 3.7 0.09 c
spleen 0.22 0.01 b 0.19 0.01 a 0.18 0.01 a 0.2 0.01 a 0.2 0.01 ab
kidney 1.23 0.05 c 0.77 0.04 a 0.9 0.04 b 0.91 0.03 b 1.15 0.04 c
perirenal white adipose tissue 0.54 0.07 a 3.31 0.11 c 2.64 0.09 b 3.77 0.09 d 2.43 0.08 b
epididymal white adipose tissue 1.44 0.14 a 5.98 0.23 d 4.98 0.15 c 4.08 0.13 b 4.51 0.09 b
total white adipose tissue 1.98 0.2 a 9.29 0.27 d 7.62 0.18 c 7.85 0.17 c 6.94 0.14 b
interscapular brown adipose tissue 0.42 0.01 d 0.29 0.01 a 0.35 0.01 b 0.37 0.01 bc 0.39 0.01 cd
Serum Parameters
ALT (U/L) 40.17 6.57 a 98.58 7.27 d 81.42 5.46 c 58.92 3.32 b 41.92 2.48 a
AST (U/L) 153.83 11.73 a 180.17 11.1 b 145.58 6.79 a 134.33 6.02 a 147.5 7.94 a
TG (mmol/L) 0.77 0.05 a 1.59 0.08 c 1.29 0.07 b 1.17 0.07 b 0.85 0.07 a
TC (mmol/L) 3.19 0.28 a 5.38 0.23 d 4.64 0.15 c 3.91 0.29 b 3.42 0.17 ab
HDL-C (mmol/L) 2.41 0.26 a 4.41 0.24 b 4.19 0.33 b 4.04 0.28 b 4.04 0.19 b
LDL-C (mmol/L) 0.29 0.03 a 0.62 0.04 c 0.54 0.03 c 0.43 0.03 b 0.31 0.04 a
adiponectin (g/L) 93.03 3.36 a 70.65 4.24 b 80.97 6.09 ab 85.32 3.68 a 92.87 3.49 a
FGF21 (ng/L) 713.83 36.04 a 1141.74 59.77 b 854.78 46.99 a 809.56 49.6 a 785.21 51.74 a
NPY (ng/L) 402.19 34.01 452.03 30.63 466.49 37.04 408.35 39.11 415.08 32.46
a
LFD, mice fed a low-fat diet; HFD, mice fed a high-fat diet; and HFD + L, M, and H, mice a fed high-fat diet supplemented with PPBNs at 50, 100,
or 200 mg/kg. Data are expressed as the mean SEM (n = 12). Means not sharing a common letter dier signicantly among groups (p < 0.05;
ANOVA).

RESULTS
Quantication and Identication of Puried White-
Fleshed PPBNs. The content of betacyanins expressed as
betanin equivalents per 1 g of PPBN powder was 25.32 0.23
mg/g. The betacyanin identication and peak assignment were
performed on the basis of the comparison of their retention
time, ultravioletvisible (UVvis) spectra, and mass spectral
analysis to those of standards and literature data.2023 The
absorbance peaks from HPLC were recorded at 538 nm
(Figure 1), and the data of the peak identication, retention
times, and compound identication obtained from the LC/
MS/MS analyses were summarized in Table 1.
Eect of PPBNs on Body Weight and Food Intake. At
the end of the experiment, the mice fed a HFD gained more
weight and body fat percentage (per 100 g of body weight)
compared to those fed a LFD and PPBN administration
signicantly reduced the HFD-induced increase of body weight
gain and body fat (Figure 2A and Table 2). Moreover, HFD
feeding decreased the relative weight of the heart, liver, spleen,
kidney, and interscapular brown, while PPBN administration
reversed the trend (Table 2). The mice fed a HFD consumed
more calories than those fed a LFD, but the calorie intake was
not signicantly dierent among the HFD-fed mice with or
without supplementation of betacyanins, suggesting that
PPBNs could signicantly decrease the HFD-induced body
weight gain without aecting the calorie intake (Figure 2).
Eect of PPBNs on Serum Biochemical Parameters.
The serum levels of TG, TC, HDL-C, LDL-C, ALT, and AST
were signicantly increased in mice fed a HFD compared to
those fed a LFD, and administration of PPBNs signicantly
reduced the levels of TG, TC, LDL-C, ALT, and AST. No
inuence of PPBNs on HDL-C was observed (Table 2).
Serum Levels of Leptin, Adiponectin, NPY, and
FGF21. HFD feeding induced a decrease in the serum level
of adiponectin but increased the concentration of FGF21, while
supplementation with PPBNs signicantly reversed the trend.
D DOI: 10.1021/acs.jafc.5b05177
No dierences were observed in the serum level of leptin and
NPY among all of the dietary groups (Table 2).
Impact of PPBNs on HFD-Induced Hepatic Steatosis
and Adipose Tissue Hypertrophy. Histological analysis
revealed an accumulation of lipid droplets in the livers of mice
fed a HFD, and the mice developed a high degree of hepatic
steatosis with severe cytoplasmic vacuoles and swelling of
hepatocytes, whereas no signicant abnormalities were
observed in LFD-fed mice (Figure 3A). PPBN administration
attenuated hepatic lipid accumulation, alleviated the formation
of steatosis, and decreased the hepatic steatosis grade scores in
a dose-dependent manner (panels A and B of Figure 3).
Consistent with these results, PPBN supplementation also
signicantly decreased the hepatic concentrations of TG and
TC (panels C and D of Figure 3). In addition, H&E staining of
adipose showed that PPBNs signicantly reduced the size of
white and brown adipocytes (Figure 3A), suggesting that PPBN
supplementation attenuated HFD-induced adipose hyper-
trophy.
Eect of PPBNs on Glucose Tolerance and Insulin
Resistance. As the OGTT shows, the glucose level in the mice
fed a HFD was signicantly higher at 30, 60, 90, and 120 min
after oral gavage of glucose than that in the LFD-fed mice and
the increase fasting blood glucose concentration was signi-
cantly reduced by the administration of PPBNs compared to
the HFD group (panels A and B of Figure 4). In addition, HFD
feeding induced a signicant increase in the fasting serum levels
of glucose and insulin, and the mice in the HFD + L, M, and H
groups revealed a signicant decrease in the fasting serum
glucose and insulin levels (panels C and D of Figure 4). These
data suggested that PPBN administration alleviated HFD-
induced glucose intolerance and insulin resistance in mice. This
conclusion was also supported by the PPBN-induced lower
HOMA-IR but higher HOMA-IS index (panels E and F of
Figure 4).
Inuence of PPBNs on Hepatic Gene Expression. HFD
feeding decreased the expression levels of lipid metabolism-
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Figure 3. White-eshed PPBNs attenuate hepatic steatosis and adipocyte hypertrophy. (A) Histological analysis for epididymal white adipose tissue
(eWAT; H&E), perirenal white adipose tissue (pWAT; H&E), interscapular brown adipose tissue (iBAT; H&E), and liver (H&E and oil red O
staining). (B) Steatosis grade score (n = 6). The hepatic steatosis scores were graded as follows: 0 (steatosis involving <5% fatty hepatocytes), 1
(steatosis involving 533% fatty hepatocytes), 2 (steatosis involving 3466% fatty hepatocytes), and 3 (steatosis involving >66% fatty hepatocytes).
(C) Hepatic TG levels (n = 12). (D) Hepatic TC levels (n = 12). LFD, mice fed a low-fat diet; HFD, mice fed a high-fat diet; HFD + L, M, and H,
mice fed a high-fat diet supplemented with PPBNs at 50, 100, and 200 mg/kg, respectively; and HE, hematoxylineosin. Data are expressed as the
mean SEM. Values not sharing a common letter dier signicantly among groups (p < 0.05; ANOVA).

related genes (AdipoR2 and PPAR) and the cholesterol
biosynthesis-related genes (Insig1 and Insig2), and PPBN
treatment signicantly enhanced the expression of these
genes in a dose-dependent manner (panels C and D of Figure
5). In addition, a HFD increased the expression level of
broblast growth factor 21 (FGF21) and suppressed the
expression of Klb, FGFR1, and FGFR2, while PPBN
administration reversed the trend (Figure 5E).
signicantly reduced diet-induced body weight gain and
attenuated obesity-related insulin resistance and hepatic
steatosis.
H. polyrhizus fruit and its methanol extract have previously
been shown to lower the plasma TG, TC, and LDL-C levels
and increase the HDL-C level in HFD-fed rats and type 2
diabetic subjects.25,26 However, despite the reducing eect of
pitaya fruit on the lipid prole, no changes in the body weights

DISCUSSION
The antiobesity eect of natural pigments from vegetables and
fruits has been demonstrated but betacyanins receive little
attention. Red- and white-eshed pitaya are rich in betacyanins,
while the consensus on the betacyanin varieties has not been
reached.14,22,24 This study was undertaken to investigate the
benecial eects of PPBNs on obesity and obesity-related
metabolic syndromes in HFD-fed C57BL/6J mice, which are
susceptible to diet-induced obesity, type 2 diabetes, and
NAFLD. Our results clearly showed that PPBN administration
E DOI: 10.1021/acs.jafc.5b05177
and fat mass were observed in these studies. We postulated that
this condition may be due to the high content of sugar and fat
in pitaya fruit, which might counteract the weight loss eect.
Consistent with these studies, our results showed that PPBN
supplementation signicantly decreased serum levels of TG,
TC, and LDL-C in HFD-fed mice. Therefore, in conjunction
with the previous studies, these observations suggested that
PPBNs might be the main bioactive compounds in pitaya that
exhibited the benecial eects on the lipid prole. Moreover,
unlike the previous studies, we found that the body weight gain
started to decrease after 1 week of supplementation of PPBNs
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Figure 4. White-eshed PPBN supplement alleviates diet-induced hyperglycemia, improves insulin resistance, and increases insulin sensitivity. (A)
Eect of PPBNs on glucose tolerance in mice fasted for 12 h as determined by the OGTT. (B) AUC for the OGTT test. Eect of PPBNs on the
fasting serum (C) glucose and (D) insulin in mice. (E) HOMA-IR index. (F) HOMA-IS index. LFD, mice fed a low-fat diet; HFD, mice fed a high-
fat diet; and HFD + L, M, and H, mice fed a high-fat diet supplemented with PPBNs at 50, 100, and 200 mg/kg, respectively. n = 12 (A), n = 10 (B,
E, and F), and n = 8 (C). Data are expressed as the mean SEM. Values not sharing a common letter dier signicantly among groups (p < 0.05;
ANOVA).

Figure 5. White eshed PPBNs alter the mRNA levels in the liver of mice. (A) Relative expression levels of Fads2 and Fas. (B) Relative expression
levels of Cpt1a, Cpt1b, and Acox1. (C) Relative expression levels of lipid metabolism genes (AdipoR2 and PPAR). (D) Relative expression levels of
cholesterol biosynthesis-related genes (Insig1 and Insig2). (E) Relative expression levels of broblast growth factor 21 (FGF21), -Klotho (Klb), and
receptors of FGF21 (FGFR1 and FGFR2). LFD, mice a fed low-fat diet; HFD, mice fed a high-fat diet; and HFD + L, M, and H, mice fed a high-fat
diet supplemented with PPBNs at 50, 100, and 200 mg/kg, respectively. Data are expressed as the mean SEM. Statistical analyses were performed
with Students t test. () p < 0.05 versus the LFD group. (#) p < 0.05 versus the HFD group.

and nally led to a signicant decrease in the body weight of the serum level of NPY, which acts as a neurotransmitter in the
mice in a dose-dependent manner. Furthermore, histological brain and regulates food intake and storage of energy as fat. On
analysis showed that PPBN treatment signicantly reduced the the basis of these evidence, we inferred that PPBNs suppressed
size of adipocyte in the white and brown adipose tissues. In HFD-induced body weight gain without aecting the energy
addition, there was no dierence in the daily calorie intake or intake of mice. Histological analysis of liver demonstrated that
F DOI: 10.1021/acs.jafc.5b05177
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Journal of Agricultural and Food Chemistry
administration of PPBNs eectively attenuated hepatic lipid
accumulation in obese mice. In combination with the results
that PPBN supplementation decreased the hepatic steatosis
grades and reduced hepatic TG and TC and serum ALT and
AST levels, we believed that PPBNs could signicantly
alleviated HFD-induced hepatic steatosis.
Furthermore, PPBN treatment increased the hepatic
expression levels of AdipoR2, Cpt1a, Cpt1b, and Acox1 as well
as elevated the serum levels of adiponectin but decreased the
expression levels of Fas and Fads2 compared to the HFD mice.
Adiponectin, secreted by adipocytes, has been proven to
regulate the metabolism of lipids and glucose.27,28 AdipoR2
serves as a receptor for adiponectin and mediates increased
AMPK and PPAR- ligand activities as well as fatty acid
oxidation and glucose uptake by adiponectin.29 Cpt1a, Cpt1b,
and Acox1 encode the rate-controlling enzymes of the fatty acid
-oxidation pathway,3032 while Fas and Fads2 are proven to be
involved in the synthesis of fatty acids. Moreover, PPAR, the
upstream regulator of Fas and Acox1,33 was also downregulated
by PPBN treatment. Our results implied that the reduced
biosynthesis but enhanced fatty acid oxidation might contribute
to the benecial eects of PPBN. In our study, we also
observed higher serum and hepatic TC levels accompanied by
an increased expression of Insig1 and Insig2 in HFD mice, while
PPBN treatment reversed the trend. In consideration of the
previous ndings that high levels of insulin induced over-
expression of Insig1 and Insig2 in the liver, leading to
cholesterol synthesis,34 we postulated that PPBN treatment
could lower insulin levels and, consequently, decrease
expression of Insig1, Insig2, and cholesterol levels.
Obesity is often accompanied by insulin resistance, thus
increasing the risk of type 2 diabetes; therefore, the bioactive
compounds with both antiobesity and antidiabetic eects are
particularly benecial for health. Previous studies demonstrated
that red pitaya consumption signicantly decreased the blood
glucose level in type 2 diabetic subjects25 and signicantly
improved hyperinsulinemia in insulin-resistant rats.35 Consis-
tently, our results indicated that PPBN administration
signicantly decreased the fasting serum levels of glucose and
insulin. The benecial eects of PPBNs on hyperglycemia and
hyperinsulinemia were also conrmed by the OGTT and
lowered HOMA-IR index. Furthermore, the insulin-sensitizing
eect was supported by the higher HOMA-IS index of the
PPBN-treated mice compared to that fed a HFD alone. To
conrm the potential action sites for PPBNs to exert its
antidiabetic eect, we examined the expression prole of
FGF21. FGF21, mainly expressed in liver, is involved in the
regulation of glucose, lipid, and energy metabolism. Admin-
istration of exogenous FGF21 improves metabolic disorders, for
instance, increasing glucose tolerance and insulin sensitivity,
regulating lipid oxidation, improving lipid prole, attenuating
hepatic steatosis, and reducing body weights.36,37 However,
consistent studies indicate that circulating FGF21 levels are
markedly increased in obesity and impaired glucose tolerance,
insulin resistance, and hypertriglyceridemia and liver injury
states.3840 Similar to the insulin resistance, the fail of
endogenous FGF21 to suppress obesity, type 2 diabetes, and
hepatosteatosis implies a state of FGF21 resistance as a result of
the impaired FGF21 action. As expected, the fasting serum
FGF21 level was signicantly increased in the HFD-induced
obese mice, which was consistent with the previous ndings
and conrmed the speculation of compensatory FGF21
overproduction in the obese state.
G DOI: 10.1021/acs.jafc.5b05177
Article
FGF21 activity depends upon its binding to a receptor
complex consisting of a cofactor called -Klotho (Klb) and
FGFRs and then elicits the intracellular signaling cascades.41,42
To identify the potential mechanism by which obesity induced
FGF21 resistance, the hepatic gene expressions of Klb and
FGFR1/2 were evaluated. Our results indicated that HFD
feeding resulted in a signicant suppression of Klb and FGFR1/
2, while PPBN supplement signicantly increased the
expression levels of these genes. These data implied that the
impaired action of FGF21 might be due to the decreased
expression of its receptors and PPBNs may directly or indirectly
improve the expression prole of these receptors, thus
attenuating the FGF21 resistance and contributing to its
antiobesity, antidiabetic, and antihepatosteatosis eects.
Although the exact and detailed mechanisms of absorption,
metabolism, and excretion of betacyanins have not yet been
fully elucidated, there is increasing experimental evidence
proving their numerous biological activities that implied the
potential health benets of betacyanins.43,44 A total of 14
betacyanins were identied in the white-eshed pitaya peel by
LC/MS/MS analysis, but we were not clear if all of them were
the bioactive agents. Therefore, further studies are required to
identify the distinct eective compounds in PPBNs.
In summary, the present study demonstrated that PPBN
treatment protected from diet-induced obesity, hepatic
steatosis, and insulin resistance in mice and the protective
eects of PPBNs were associated with the induced fatty acid
oxidation, decreased fatty acid biosynthesis, and improved
FGF21 sensitivity. Our results suggested a potential dietary
choice of PPBNs in the management of obesity, type 2 diabetes,
and NAFLD.
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.5b05177.
Composition of puried diets (Table S1) and primer
sequences used for quantitative real-time PCR (Table
S2) (PDF)
AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-571-86098861. Fax: +86-571-86971139. E-
mail: xdzhengzju@yahoo.com.
Funding
This work was supported by the National Key Technology
R&D Program of China (Grant 2012BAD33B08), the Zhejiang
Provincial Natural Science Foundation of China (Grant
Z14C200006), and the Foundation of Fuli Institute of Food
Science, Zhejiang University (Grant KY201301).
Notes
The authors declare no competing nancial interest.
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I DOI: 10.1021/acs.jafc.5b05177
J. Agric. Food Chem. XXXX, XXX, XXXXXX