journal of functional foods 9 (2014) 173182

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Ellagitannin geraniin supplementation

ameliorates metabolic risks in high-fat
diet-induced obese Sprague Dawley rats
Alexis Panny Y.S. Chung a, So Ha Ton b, Sunil Gurtu a,
Uma D. Palanisamy a,*
a

School of Medicine and Health Sciences, Monash University Malaysia, 46150, Sunway City, Selangor,
Malaysia
b
School of Science, Monash University Malaysia, 46150, Sunway City, Selangor, Malaysia

A R T I C L E

I N F O

A B S T R A C T

Article history:

Geraniin, an ellagitannin found abundantly in many fruits, nuts, traditional Chinese medi-

Received 23 January 2014

cine (TCM) and ayurvedic herbs, has been reported to possess numerous health benefits.

Received in revised form 14 March

This is the first study that elucidates geraniin, purified from the sub-tropical fruit Nephelium

2014

lappaceum L. rind, for its therapeutic potential in ameliorating diet-induced metabolic risks

Accepted 26 March 2014

mimicking metabolic syndrome. Male post-weaning outbred Sprague Dawley rats received

Available online

a 60% high-fat diet (HFD), with and without the geraniin supplementation (10 and 50 mg/
kg body weight), while the control group (ND) was fed rat chows for 10 consecutive weeks.

Keywords:

Comparatively, HFD rats demonstrated elevated body weights, white adipose tissue depots

Beta-cell dysfunction

(WAT), organ weights, triaylglycerol, renal and hepatic dysfunction biomarkers, insulin re-

Geraniin

sistance, declined insulin sensitivity and percent of beta-cell function. A four-week in vivo

High-fat diet

geraniin treatment, particularly at 50 mg/kg body weight, exhibited significant therapeutic

Insulin sensitivity

potential to safely mitigate obesity-induced metabolic dysfunction.

2014 Elsevier Ltd. All rights reserved.

Metabolic dysfunction
Nephelium lappaceum

1.

Introduction

Excess nourishment, high fat, sugar and salt in modern diets,

combined with a sedentary lifestyle, leads to metabolic overload and consequently metabolic disturbance, which is an imbalance between energy input (EI) and energy output (EO)
(Sorensen, 2009). It results in over-abundance of glucose and
fatty acid accumulations within adipose tissue, skeletal muscle,
hepatocytes and pancreatic cells, causing serious defects in
fuel partitioning and thus disrupts the total body energy homeostasis (Baur et al., 2006; Chen et al., 2011; Storlien et al.,

1996), finally leading to the risks associated with metabolic

syndrome (MS). Metabolic syndrome is typified by a constellation of metabolic risks encompassing obesity, insulin resistance, hyperglycaemia, dyslipidaemia and hypertension
(American Diabetes Association, 2001; Christopher & Sarah,
2005; National Heart, Lung and Blood Institute, 2001). The
number of people affected with metabolic syndrome worldwide has strikingly increased over the past two decades and
this increase is not dissociable from the worldwide pandemic of obesity and hyperglycaemia (Zimmet, Alberti, & Shaw,
2001). Many studies have shown that lifestyle interventions
including increased physical activity, dietary modification and

* Corresponding author. Tel.: +603 55145840; fax: +603 55146323.

E-mail address: umadevi.palanisamy@monash.edu (U.D. Palanisamy).
http://dx.doi.org/10.1016/j.jff.2014.03.029
1756-4646/ 2014 Elsevier Ltd. All rights reserved.

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journal of functional foods 9 (2014) 173182

weight management in combination with pharmacotherapy

reduce the incidence of these metabolic risks (American
Diabetes Association, 2001; Christopher & Sarah, 2005; National
Heart, Lung and Blood Institute, 2001). However, there are still
difficulties in reaching the goal of normalising glucose levels
and fat contents without adverse effects, such as severe weight
gain or loss, hypoglycaemic episodes, lactic acidosis,
hepatoxicity, kidney damage, dyspepsia, atherogenic events
and premature cardiovascular diseases due to
hyperinsulinaemia and lipodystrophy, overstimulation and appetite abuse in addition to raised risk of death (Agabegi &
Steven, 2008; Zimmet et al., 2001). Hence, there is a great
medical need to develop novel drugs or alternative therapies
that are both effective and which are free from, or with relatively fewer adverse effects.
Traditionally, plants have been used in the management of
a broad spectrum of metabolic dysfunction such as diabetes,
cardiovascular diseases, obesity, dyslipidaemia and cancers,
among others, owing to the presence of plant-derived secondary metabolites (Aggarawal & Shishu, 2011). Plant-derived secondary metabolites are a rich source of natural bioactive
components; the most essential of these are flavonoids and
polyphenols. Ellagitannins (ETs), the bioactive polyphenols, can
be found abundantly in fruits such as raspberries, blackberries, strawberries, cranberries and pomegranates; in vegetables such as potato, tomato, lettuce and onion; in nuts and
seeds (Scalbert & Williamson, 2000), traditional Chinese medicinal plants such as Geranium sibiricum Linne (Yang et al., 2010)
and ayurvedic herbs such as Phyllanthus emblica (amla)
(Krishnaveni & Mirunalini, 2010). Many studies on ellagitannins
have demonstrated positive biological actions encompassing
antioxidant, antimutagenic, anticarcinogenic, antitumour, antiviral and antimicrobial, both in vitro and in vivo (Ito, 2011),
which suggest that the consumption of ellagitannins may
provide protective benefits on human health.
Native to Southeast Asia, Nephelium lappaceum L. (rambutan in Malaysia language) belongs to the same family
(Sapindaceae) as the sub-tropical fruits lychee (Litchi chinensis)
and longan (Dimocarpus longan). This fruit is an important commercial crop in Asia, where it can be consumed fresh, canned
or processed. Geraniin, a typical ellagitannin was recently established to be present in N. lappaceum L. rind with the highest
yields of geraniin (up to 35%) among the plant studied thus
far (Palanisamy, Ling, Manaharan, & Appleton, 2011). Studies
on diverse biological properties of geraniin have shown that
the compound exhibits antihyperglycaemic potential
(Palanisamy et al., 2008), antihypertensive activity (Lin, Wang,
Lu, Wu, & Hou, 2008), hepatoprotective action (Ambrose, Solairaj,
& Subramoniam, 2012), high antioxidant (Palanisamy et al., 2008;
Thitilertdecha, Teerawutgulrag, Kilburn, & Rakariyatham, 2010)
and nitrogen oxide (NO) scavenging capacity (Kumaran &
Karunakaran, 2006). Previous in vitro investigations on 3T3-L1
cells have also revealed geraniins ability to enhance glucose
uptake (Palanisamy). Based on its manifold beneficial health
properties, geraniin is a valuable candidate for more extensive study into its potential pharmaceutical applications. This
study investigates the in vivo effects of supplementing geraniin
purified from the N. lappaceum L. rind on metabolic factors mimicking MS and its clinical complications in rodents fed on a
high-fat diet.

2.

Materials and methods

2.1.

Materials

High-fat pellet (HFD, 60% fat by weight, AIN93G specifications

purified diet) was purchased from Specialty Feeds Inc. (Glen
Forrest, Western Australia). Normal rat chow (ND, 5% minimum
crude fat content) was purchased from Gold Coin (Kuala
Lumpur, Peninsular Malaysia). Accu-ChekPerforma Glucometer
was purchased from Roche (Manheim, Germany). Diagnostic
reagents for metabolic parameter measurements of lipids (TG,
TC, HDL and non-HDL cholesterols), liver (ALT, AST and GGT)
and kidney (CK and Cr) were purchased from Roche (Manheim,
Germany). Rat/Mouse Insulin Sandwich ELISA kit was purchased from Millipore (MA, USA). BIO-RAD Benchmark Plus
Microplate Reader with Microplate Manager 5.2.1 software (CA,
USA), Hitachi Model 902 Automatic Analyser (Manheim,
Germany). All other chemicals and reagents were purchased
from Becton, Dickinson and Company (NJ, USA), Millipore (MA,
USA), Sigma-Aldrich (MO, USA), Terumo (Tokyo, Japan) and
Vtoquinol UK Limited (Buckingham, UK) unless stated
otherwise.

2.2.
Preparation of geraniin from Nephelium
lappaceum L. rind by reverse phase C-18 column
chromatography
Nephelium lappaceum L. was obtained from Kuala Lumpur, Peninsular Malaysia. Plants were authenticated by the Herbarium of the Forest Research Institute of Malaysia (FRIM). Crude
extract of N. lappaceum L. rind was prepared as described by
Palanisamy et al. (2008). Geraniin was purified from the crude
extract by means of reverse-phase C-18 chromatography
(Palanisamy et al., 2011). Crude extract (20 g) was dissolved in
a minimum amount of water (40 mL) and loaded onto glass
column (250 mm 50 mm i.d.) packed with 200 g C18 silica (particle size of 50 m, pore size of 60 ). The column was open
tubular and solvent flow rate was maintained by means of
vacuum pump attached to vacuum inlet in the column. The
column was first eluted with water (300 mL) and then fractions were collected using a step gradient of water and acetonitrile. Solvent system was as follows: water (100%, 400 mL),
acetonitrile/water (5:95, 350 mL), acetonitrile/water (10:90,
1000 mL). Finally the column was eluted with methanol (100%,
500 mL). The silica was cleaned by flushing the column sequentially with dichloromethane (100%, 300 mL), methanol containing a few drops of trifluoroacetic acid (100%, 300 mL) and
absolute methanol (100%, 300 mL), allowing to dry completely. This enables the column to be reused with fresh crude
extract. Purity of geraniin (> 95%) obtained was confirmed using
HPLC (Details see Perera, Appleton, Loh, Elendran, & Palanisamy,
2012).

2.3.

Animals and housing

Thirty-two male, post-weaning (3-week-old) outbred Sprague

Dawley (SD) rats (Rattus norvegicus) were obtained from the
Animal House of Monash University (Monash University, Peninsular Malaysia). Males were used to eliminate variations in

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journal of functional foods 9 (2014) 173182

metabolic outcomes induced by sex in addition to their vulnerability to the impacts of diet-induced obesity (DIO) (Hwang,
Wang, Li, Chang, Lin, Chen et al., 2010). They were randomised
into four groups of eight rats each and accorded a 7-day
acclimatisation period to diets and experimental conditions
prior to this in vivo study. A minimum of eight rats for each
treatment group was required to give a significant finding at
P 0.05 levels (Campbell & Machin, 1999). Normal rat chow and
distilled water (dH2O) were fed ad libitum. Body weights were
recorded at the end of the acclimatisation period, which are
presented as initial weights in this study. Throughout the experiment, all SD rats were housed individually in cages with
stainless steel lids and with ad libitum access to food and water.
They were maintained on a 12-h lightdark cycle (lights on at
08:00 hours; lights off at 20:00 hours) with controlled temperature (22 1 C) and humidity (55 10%) in the animal housing
facility. The use and handling procedure of animals was approved by the Monash University Animal Ethics Committee according to the ethics approval (Approval Code: AEC:MARP/
2011/021).

2.4.

2.5.1. Measurement of fasting blood glucose and serum

insulin levels
The fasting glucose levels were measured in the tail vein blood
with a glucometer. The fasting serum insulin levels were measured using the Rat/Mouse Insulin Sandwich ELISA kit. Absorbance was detected at 450 and 590 nm on BIO-RAD Benchmark
Plus Microplate Reader with Microplate Manager 5.2.1
software.

2.5.2. Homeostasis Model Assessment (HOMA)

Scores insulin sensitivity (HOMA-IS, %S), beta-cell function
(HOMA-BCF, %B) and insulin resistance (HOMA-IR)
Beta-cell function (BCF, %B), insulin sensitivity (IS, %S) and
insulin resistance (IR) of the SD rats were analysed using the
HOMA calculator based on fasting plasma glucose (mmol/L)
and fasting serum insulin (IU/mL) (Bonora et al., 2000) measured using the Rat/Mouse Insulin Sandwich ELISA kit following the manufacturers instructions.

HOMA BCF =

[20 Fasting Serum Insulin ( IU mL)]

[Fasting Plasma Glucose (mmol L) 3.5]

Equation 1

Induction of obesity and dietary treatments

HOMA IS

At the end of the 7-day acclimatisation, the obesity induction was carried out for 6 weeks (Andrikopoulos et al., 2005;
Ikarashi et al., 2011) followed by the geraniin treatment for
4 weeks. Food and water consumptions were monitored daily.
Body weight and blood glucose were measured weekly. Normal
diet control group (ND) was fed with normal rat chow and dH2O.
High-fat control rats were fed with high-fat diet pellets (HFD)
and dH2O. The two treatment groups, after the 6-week highfat induction, were fed with HFD and 10 mg geraniin/kg body
weight (HFD + 10 mg/kg G) or 50 mg geraniin/kg body weight
(HFD + 50 mg/kg G) with dH2O ad libitum.

2.5.

Experimental design and sampling

At the end of week 10, SD rats in all groups were fasted for
16 h prior to being sacrificed. Tail venipuncture was performed to collect whole blood for the determination of blood
glucose prior to anaesthetisation. The SD rats were
anaesthetised by intraperitoneal (IP) injection of ketamine
(90 mg/kg body weight) and xylazine (10 mg/kg body weight)
and exsanguinated through cardiac puncture with disposable syringes and sterile needles. Blood samples were
divided into two parts. Blood was collected into sterile
ethylenediaminetetraacetic acid (EDTA) vacutainers (Purple Top),
mixed thoroughly and centrifuged at 15,000 g for 15 min at 4 C.
The separated plasma was aliquoted into sterile cryovials and
stored at 80 C for the determination of triaylglycerol (TG), highdensity lipoprotein (HDL) cholesterol, non-high-density lipoprotein (HDL) cholesterol, total cholesterol (TC), alanine
aminotransferase (ALT), aspartate aminotransferase (AST),
gamma-glutamyl transferase (GGT), creatinine (Cr) and creatine kinase (CK). Blood was also collected into sterile Red Top
vacutainers which contained clot activator gel and centrifuged at 15,000 g for 15 min at 4 C. The serum samples obtained were aliquoted into sterile cryovials and stored at 80 C
for serum insulin measurement.

= 100

Fasting Plasma Glucose (mmol L) Fasting Serum Insulin ( IU mL)

22.5

Equation 2
HOMA IR
=

Fasting Plasma Glucose (mmol L) Fasting Serum Insulin ( IU mL)

22.5

Equation 3

2.5.3.

Fasting plasma metabolic parameter analysis

Metabolic parameters of lipid (TG, non-HDL cholesterol, HDL

cholesterol and TC), liver (ALT, AST and GGT) and kidney (Cr
and CK) were measured using the Hitachi Model 902 Automatic Analyser and Roche diagnostic GmbH reagents.

2.6.

Organ and white adipose tissue (WAT) analysis

Necropsies were also performed at the end of week 10. Organs

of interest, namely brain, heart and aorta, pancreas, liver and
kidney, and WAT (visceral and epidermal) were promptly collected. All the organs and WAT were washed in ice-cold phosphate buffered saline (1 PBS) buffer to remove excessive blood
clot and dried on absorbent paper to remove all remaining fluids
before weighing on an electronic balance to obtain a constant
weight. The ratio of each organ to terminal body weight (relative organ weight expressed as percentage of body weight) was
also measured (modified from Khanal, Howard, Wikes, Rogers,
& Prior, 2010).

Absolute Organ Weight (g )

% of BW = 100

Final Body Weight (g )

2.7.

Equation 4

Statistical analysis

Data obtained were analysed using the GraphPad Prism statistical software, version 5.0 (CA, USA). One-way ANOVA with
Tukeys post hoc test was used for comparisons between variables and t-test for pairwise comparison between the differ-

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journal of functional foods 9 (2014) 173182

3.

Results

3.1.
Preparation of geraniin from Nephelium
lappaceum L. rind by reverse phase C-18 column
chromatography
Purification of geraniin from Nephelium lappaceum L. rind waste
was achieved in a single chromatographic method to produce
geraniin of high yield and a high purity. In this method, 74.78 g
geraniin of approximately 94% purity were obtained from 362.4 g
crude extract of N. lappaceum L. rind representing a geraniin
yield of approximately 21%.

3.2.
Effects of high-fat feeding and geraniin on body
weight gain and food intake
A significant increase in body weight of the HFD-fed rats compared to the ND-fed rats was observed (Fig. 1) throughout the
intervention study (P < 0.05). On the tenth week, the HFD-fed
group recorded a 19% weight gain, 346 5 g compared to
291 3 g in the ND-fed rats. Upon establishing obesity in the
SD rats after 6 weeks of induction, the effect of geraniin treatment (10 and 50 mg/kg body weights; 4 weeks) on HFD-fed rats
was investigated. After a 4-week geraniin treatment, the two
treatment groups, HFD + 10 mg geraniin (HFD + 10 mg G) and
HFD + 50 mg geraniin (HFD + 50 mg G), showed significant reductions in body weight compared with the HFD-fed alone rats

ND

HFD

400
350
Body Weight (g)

ent groups. All data are presented as mean SEM unless

otherwise indicated. Significance was assigned at P-value < 0.05
(confidence interval 95%). The experimental groups, ND, HFD,
HFD + 10 mg G and HFD + 50 mg G, are represented by superscripts a, b, c and d, respectively. Means with different superscripts are significantly different at P-value < 0.05 between
groups.

300
250
200
150
100
50
0
1

5
6
Weeks

10

Fig. 1 Body weight (g) of the SD rats throughout the

intervention study. ND, normal diet control; HFD, high-fat
diet control. Means with different superscripts are
significantly different at P-value < 0.05 between groups.
The experimental groups, ND and HFD are represented by
superscripts a and b respectively.

(Fig. 2). Inclusion of geraniin at 50 mg showed a more significant reduction in body weight, particularly at week 4. Throughout the experimental period there were no significant changes
observed in the daily food intake (an average of 20 g/rat/day)
and water consumptions. No animal death was observed during
the experimental period.

3.3.
Effects of high-fat feeding and geraniin on white
adipose tissue depositions
The total WAT depot, both visceral and epidermal, was also
examined after the 10-week study. The HFD-induced SD rats
demonstrated an increase in total WAT mass whereas the

Fig. 2 Body weight (g) of the SD rats throughout the treatment period (weeks 710). ND, normal diet control; HFD, high-fat
diet control; G, geraniin treated. Means with different superscripts are significantly different at P-value < 0.05 between
groups. The experimental groups, ND, HFD, HFD + 10 mg G and HFD + 50 mg G, are represented by superscripts a, b, c and d,
respectively.

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journal of functional foods 9 (2014) 173182

Table 1 Effects of HFD and geraniin feeding on WAT mass of the SD rats.
Groups

Initial BW (g)

Final BW (g)

WAT Weight (mg)

Visceral

Epidermal

Total WAT

ND
HFD
HFD + 10 mg G
HFD + 50 mg G

100 1
142 3
144 3
141 2

291 3b
346 5a,c,d
342 4bd
332 4bc

2.85 0.32
3.09 0.51d
2.12 0.62
1.45 0.22b

2.35 0.41
2.51 0.76
1.63 0.58
1.52 0.19

5.20 0.64
5.60 0.60c,d
3.75 0.63b
2.97 0.37b

ND, normal diet control; HFD, high-fat diet control; G, geraniin treated; BW, body weight; WAT, white adipose tissue.
Means with different superscripts are significantly different at P-value < 0.05 between groups.
The experimental groups, ND, HFD, HFD+10 mg G and HFD+50 mg G, are represented by superscripts a, b, c and d, respectively.

Initial BW was the weight taken after randomisation and after 1 week of acclimatisation to diet and experimental conditions (week 0).

geraniin-treated groups showed a significant reduction in total

WAT deposition (Table 1). Inclusions of geraniin at 10 and 50 mg
were found to significantly reduce total WAT depositions by
42 and 47%, respectively compared to the HFD-fed rats. Notably,
HFD-fed rats showed a significant increase in the visceral fat
mass but geraniin treatment particularly at 50 mg attenuated
this condition. Interestingly, this treatment group also showed
even lower visceral fat mass compared to the ND-fed rats. HFD
induction did increase the epidermal and visceral masses in
rats compared to those of the ND-fed rats. Although statistically insignificant (P > 0.05) among all groups, the geraniintreated groups had consistently showed a lower trend in
epidermal mass.

3.4.
Effects of high-fat feeding and geraniin on
organ pathology: Toxicology and relative organ
weight-to-body weight
Effects of 4-week geraniin treatment on SD rats organ pathology were also investigated at the end of the treatment. Macroscopic toxicology was examined on the pancreas, liver, heart
and aorta, kidney and brain. Organ weights were also compared to final body weights, expressed as percent of body weight
(% of BW; Table 2). The HFD-fed rats had higher ratios of pancreas, liver, and heart and aorta over final body weight compared to the ND-fed rats. Geraniin treatment showed a
significant reduction in the relative weights of these organs,
particularly at the dose of 50 mg compared to the HFD-fed rats.
However, there was insignificant difference in the relative
weights of brains and kidneys among all four groups at the end
of this study.

3.5.
Effects of high-fat feeding and geraniin on fasting
plasma glucose, serum insulin and HOMA scores
Table 3 showed the fasting concentrations of plasma glucose
(PG), serum insulin (SI) and HOMA scores measured at the end
of week 10. Interestingly, HFD-fed rats did not demonstrate significant difference in PG compared to the ND rats. However, a
significant decrease in PG was observed in geraniin-treated
groups compared to the HFD or ND controls. The HFD induction did increase fasting SI concentrations of rats compared
to those of the ND-fed rats. Although statistically insignificant (P > 0.05) among all groups, fasting SI levels were consistently lower in geraniin-treated groups throughout the
intervention study. The HFD-fed rats also demonstrated significantly decreased HOMA-IS (%S) and HOMA-BCF (%B) compared to those of ND- and geraniin-treated. The two treatment
groups, comparatively, demonstrated a significant increase in
HOMA-IS (%S) and HOMA-BCF (%B) and correspondingly a decrease in the HOMA-IR index. Also, worthy of mention is that
these findings correlate well to the relative organ weight study
where a significant increase in both liver and pancreas was seen
in the HFD group compared to the ND group while a reduction was seen in the geraniin-treated groups (Table 2).

3.6.
Effects of high-fat feeding and geraniin on fasting
plasma metabolites
The fasting metabolic parameters of lipids (TG, non-HDL cholesterol, HDL cholesterol and TC), liver (ALT, AST and GGT) and
kidney (Cr and CK) in SD rats were measured at the end of week
10 (Table 4). The HFD-fed rats showed a significant increase in

Table 2 Relative organ weight-to-body weight (% of BW) of the SD rats.

Groups

Final BW (g)

ND
HFD
HFD+10 mg G
HFD+50 mg G

291 3
346 5a,c,d
342 4b,d
332 4b,c

Relative organ weight (% of BW)

Pancreas

Liver

0.31 0.19
0.34 0.10a,c,d
0.29 0.08a,b,d
0.22 0.10a,b,c
b,c,d

2.16 0.20
2.90 0.15a,c,d
2.74 0.21b,d
2.20 0.13b,c
b

Heartaorta

Kidney

Brain

0.35 0.11
0.37 0.07a,d
0.35 0.09
0.31 0.09b

0.66 0.12
0.60 0.09
0.63 0.08
0.65 0.10

0.63 0.14
0.56 0.12
0.55 0.10
0.60 0.08

ND, normal diet control; HFD, high-fat diet control; G, geraniin treated; BW, body weight.
Means with different superscripts are significantly different at P-value < 0.05 between groups.
The experimental groups, ND, HFD, HFD + 10 mg G and HFD + 50 mg G, are represented by superscripts a, b, c and d, respectively.

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journal of functional foods 9 (2014) 173182

Table 3 Effects of HFD feeding and geraniin on glycaemic indices of the SD rats throughout the intervention study.
Glycaemic indices

FPG (mmol/L)
FSI (IU/mL)
HOMA-BCF (%B)
HOMA-IS (%S)
HOMA-IR

Groups
ND

HFD

HFD + 10 mg G

HFD + 50 mg G

6.05 0.32
4.38 0.37
44.65 2.52
171.00 16.32
0.60 0.06

6.70 0.18c,d
4.80 0.50
38.25 1.52c,d
154.90 20.06c,d
0.70 0.07c,d

5.43 0.25b
3.77 0.26
50.38 3.33b
201.80 14.02b
0.50 0.04b

5.18 0.28b
3.73 0.19
55.70 5.57b
205.20 11.7b
0.50 0.04b

ND, normal diet control; HFD, high-fat diet control; G, geraniin treated; FPG, fasting plasma glucose; FSI, fasting serum insulin; HOMA-BCF,
beta-cell function; HOMA-IS, insulin sensitivity; HOMA-IR, insulin resistance.
Means with different superscripts are significantly different at P-value < 0.05 between groups.
The experimental groups, ND, HFD, HFD + 10 mg G and HFD + 50 mg G, are represented by superscripts a, b, c and d, respectively.

TG levels and a significant decrease in HDL, non-HDL and total

cholesterols compared to the ND-fed rats. ALT, AST, CK and Cr
were also observed to be higher in the HFD group than the ND
group. Four-week geraniin treatment brought about a significant decrease in TG, non-HDL and total cholesterols, ALT, AST,
CK and Cr compared to the HFD-fed rats particularly at geraniin
inclusion of 50 mg. Interestingly, a dose-dependent increase
in HDL was observed in the geraniin-treated rats. The levels
of GGT showed insignificant difference among all four groups
and remained consistent (<3.0U/L; Table 3) throughout the study.
Also, worthy of mention is that these findings correlate well
to the significant increase in weights of pancreas, liver, and heart
and aorta in the HFD group compared to the ND group while
a reduction was observed in the geraniin-treated groups
(Table 2).

4.

Discussion

In this study, the development of metabolic factors mimicking metabolic syndrome (MS) and its clinical manifestations
induced by a high-fat diet (HFD, 60% fat by weight; 10 weeks)
in male SD rats were assessed. Similar diet-induced obesity
(DIO) rat models have been used by many researchers to study

various metabolic dysfunctions (Buettner et al., 2006; Khanal

et al., 2010; Li et al., 2008). The in vivo ability of geraniin, at 10
and 50 mg per body weight (kg), to ameliorate anomalies tailing
metabolic derangements was also investigated.
Prolonged high-fat feeding resulted in the development of
metabolic derangements mimicking MS and its clinical pathogenesis. The HFD-fed group demonstrated significantly higher
body weight gain compared to the ND-fed group (Fig. 1). This
suggests that excess and repetitive consumption of dietary fat
contributes to the development of obesity in rodents. Similar
results have been reported by previous animal studies induced
with high-fat rodent diets having similar source of fat and experimental length (Andrikopoulos et al., 2005; Gajda et al., 2007;
Ikarashi et al., 2011). Inclusion of geraniin at 50 mg showed a
more significant reduction in body weight, particularly at week
4. This demonstrates geraniins anti-obesity potential in SD rats.
Similar weight reducing results have been shown with other
plant-derived compounds (Baur et al., 2006; Chen et al., 2011).
Existing evidence have shown that the continual escalation in
obesity rates arises from negative changes in todays lifestyle, such as excess nourishment (high fat, sugar and salt in
diets), reduced physical activities and poor weight management (Sorensen, 2009). Many plant-based phenolic compounds have been reported to be potent effectors of biological
processes and have the capacity to ameliorate disease risks

Table 4 Effects of HFD feeding and geraniin on plasma analytes of the SD rats throughout the intervention study.
Plasma analytes

TG (mmol/L)
HDL (mmol/L)
Non-HDL (mmol/L)
TC (mmol/L)
ALT (U/L)
AST (U/L)
GGT (U/L)
CK (U/L)
Cr (mol/L)

Groups
ND

HFD

HFD + 10 mg G

HFD + 50 mg G

0.360 0.006b,c,d
2.073 0.035b,c,d
0.623 0.009b,c,d
2.650 0.021b,c,d
49.93 0.623b,d
213.40 0.379b,c,d
<3.000
1459.00 11.29b,c,d
58.67 0.667b

0.543 0.003a,c,d
1.813 0.027a,c,d
0.433 0.007a,c,d
2.283 0.113a,d
63.53 0.338a,c,d
233.20 1.931a,c,d
<3.000
1897.00 19.77a,c,d
67.00 0.577a,c,d

0.510 0.010a,b,d
1.437 0.012a,b,d
0.383 0.003a,b,d
2.140 0.050a,d
50.53 0.338b,d
146.300 1.836a,b,d
<3.000
1327.00 2.19a,b,c
61.67 1.333b

0.460 0.000a,b,c
1.543 0.003a,b,c
0.247 0.003a,b,c
1.700 0.012a,b,c
42.77 0.203a,b,c
121.500 1.770a,b,c
<3.000
1585.00 46.09a,b,d
59.67 1.453b

ND, normal diet control; HFD, high-fat diet control; G, geraniin treated; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Cr,
creatinine; CK, creatine kinase; GGT, gamma-glutamyl transferase; HDL, high-density lipoprotein-cholesterol; non-HDL, non-high-density lipoproteincholesterol; TC, total cholesterol; TG, triacylglycerol.
Means with different superscripts are significantly different at P-value < 0.05 between groups.
The experimental groups, ND, HFD, HFD + 10 mg G and HFD + 50 mg G, are represented by superscripts a, b, c and d, respectively.

journal of functional foods 9 (2014) 173182

such as obesity (Ikarashi et al., 2011) with lower concomitant

adverse effects on humans. Geraniin, therefore, as demonstrated in this study, is a potential body weight lowering/
controlling agent in the management of obesity and obesityrelated health problems.
The HFD-induced SD rats showed an increase in total WAT
mass (visceral and epidermal), whereas the geraniin-treated
groups demonstrated a significant reduction in total WAT mass.
Geraniin supplementation had significantly reduced total WAT
depositions (Table 1). When a high-caloric, energy-dense diet
(i.e. high-fat diet used in this study) is continually consumed,
a positive energy balance perpetuates a higher level of lipids,
particularly in the form of TG, is excessively deposited in WAT.
Aggravation of TG leads to increased TG input, adipocyte
number, a phenomenon known as adipocyte hyperplasia; and
adipocyte enlargement, a state known as adipocyte
hyperthrophy (Moller, 2001), thus results in ectopic expansion of WAT mass which ultimately disrupts the lipid storage
capacity. Ectopic expansion of WAT mass with regards to excessive energy storage exacerbates physical body weight gain
in obesity. Obesity, particularly abdominal adiposity in susceptible human subjects, has been proven to be a key precursor to the increased risk of MS encompassing T2DM (prediabetes
onset) and other obesity-induced metabolic sequels (Chang,
Tzeng, Liou, Chang, & Liu, 2011; Guilherme, Virbasius, Puri, &
Czech, 2008; McGarry, 2002). These findings hence suggest that
long-term overnutrition also contributes to the development
of central obesity as a result of ectopic deposition of WAT, particularly visceral fat in rodents. Geraniin, in this study, also exhibited its adipocyte hypothrophic potential to prevent fat
depositions in an orally achievable dose in rodents.
Augmentations in fasting PG, SI and HOMA scores that
predict the onset of diabetes were seen in the HFD-induced
SD rats whereas the geraniin-treated groups showed significant ameliorations in glycaemic and HOMA indices, comparable to the ND group (Table 3). Also, worthy of mention is that
these findings correlate well to the significant increase in
weights of liver and pancreas in the HFD group compared to
the ND group while a reduction was seen in the geraniintreated groups (Table 2). Many studies have shown that prolonged consumption of HFD causes insulin resistance (IR), which
ultimately results in the disruption of glucose clearance and
glucose uptake in skeletal muscle and adipose tissue, both in
vivo and in vitro (Baur et al., 2006; Storlien et al., 1996). Raised
levels of circulating lipids have been shown to sufficiently induce
IR in peripheral and hepatic sites, in addition to increased deposition of lipids inside adipose tissue and skeletal muscle owing
to the specific increase in long-chain fatty-acyl-CoA (Buettner,
Scholmerich, & Bollheimer, 2007). Lipid accumulation within
the pancreatic islets has also been proven to contribute to the
impairment of insulin secretion. These observations support
a unified lipotoxicity hypothesis, which states that HFDinduced diabetes can be caused by the accretion of triaylglycerol
and long-chain fatty-acyl-CoA in liver, skeletal muscle and
adipose tissue (leading to a reduction in insulin-mediated metabolic activity) and in the pancreatic islets (leading to impaired insulin secretion) (Chang et al., 2011; Guilherme et al.,
2008; McGarry, 2002; Moller, 2001). Noticeably, either insulin resistance or beta-cell dysfunction alone will not lead to the development of T2DM (Ostenson, 2001). Rather, to develop T2DM

179

both insulin resistance and beta-cell dysfunction in the pancreas should coexist. At the end of this study, the fasting serum
insulin (FSI) levels in the HFD group did not show a significant increase compared to the ND group although changes were
observed throughout the experimental period. Previous studies
have shown that most rodents tend to become obese on HFD
(2050% digestible energy from lipids) and VHFD (50% digestible energy from lipids) with variable responses in glucose
tolerance, insulin resistance, triacylglycerol and other parameters depending on the strain, gender and source of dietary fat
(Gajda et al., 2007). Similarly, Stark, Timar, and Madar (2000)
demonstrated that healthy SD rats fed high fat or high fructose diets for 12 weeks adapted to the nutritional intervention but did not develop classical signs of insulin resistance
and impaired glucose tolerance. Despite hyperglycaemia absent
and lowered insulin levels as recorded, paralleling the organ
pathology, the HFD-fed rats were prone to be in a pre-diabetic
state. Diabetes, in fact, represents the progressive and
cumulative damage caused by cellular glucose and lipid
metabolites.
The HFD-induced SD rats showed anomalies in plasma
biomarkers that mimic the onset of a constellation of metabolic risks encompassing obesity, insulin resistance,
hyperglycaemia, dyslipidaemia, hepatic steatosis (non-alcoholic
fatty liver disease, NAFLD) and progressive loss of kidney function whereas the geraniin-treated groups had significant attenuations comparable to the ND group (Table 4). Increased
adipocyte size associated with obesity increases lipolytic activity based on a correlation between the rate of lipolysis and
the size of adipocyte, a condition known as lipotoxicity (Chang
et al., 2011; Guilherme et al., 2008; McGarry, 2002; Moller, 2001).
The HFD-fed group with increased body weight also had significantly raised levels of TG but significantly lower HDL, nonHDL and total cholesterols. This finding suggests that disruption
in fuel partitioning into adipocytes results in obesity which in
turn progresses to the development of dyslipidaemia in adipose
tissue and skeletal muscle. In this study, the HFD group although showing an increased trend, did not show a significant difference in non-HDL and total cholesterols compared
to the ND group. Saturated fat (SF) has been recognised to increase serum cholesterols (Buettner et al., 2006; Hegsted,
Ausman, Johnson, & Dallal, 1993). In this study, the HFD consists of a low amount of animal SF (10% ghee), which was insufficient to significantly increase the non-HDL- and total
cholesterol levels. This observation is not odd because normal
rodents (without genetic mutations) are known to have typically very low levels of TC and LDL-cholesterol but high levels
of HDL-cholesterol. Many studies, in addition, have shown that
materials other than fatty acids and dietary cholesterols affect
serum lipid concentrations, such as dietary fibres, plant sterols,
protein sources, cholic acid and other undefined materials.
These parameters have been found to vary greatly in experimental fats and oils and/or the basal diets. Most rodents tend
to be obese but produce variable metabolic outcomes depending on the strain, gender and source of dietary fat (Buettner
et al., 2006; Gajda et al., 2007; Hegsted et al., 1993). These metabolic effects can also be altered by adjusting the feeding period.
As demonstrated in many studies using obese rodents, not all
obese subjects develop obesity and obesity-induced manifestations (Gajda et al., 2007). This association hence suggests that

180

journal of functional foods 9 (2014) 173182

both genetic predisposition and/or environmental factors, in

part, also play a causal role in the development of obesity. Although the geraniin treatments (10 and 50 mg) did not significantly decrease the lipoprotein parameters comparable to
the ND group, it had exhibited to a certain extent, a significant amount of hypolipidaemic activity. Plant-derived polyphenols have been reported to affect lipid metabolism by
suppressing lipogenic enzymes (fatty acid synthetase, acylCoA synthase, glycerol-3-phosphate acyltransferase) and upregulating signal transduction-related genes (Prior, 2012). It is
suggested that geraniins hypolipidaemic activity may be due
to the similar mechanism. However, gene transcriptional studies
would be able to confirm this. Geraniin thus has the potential as a plant-derived hypolipidaemic agent to attenuate escalating lipids and to prevent fat accretion.
Biomarkers of the liver dysfunction (ALT and AST) in this
study suggest that prolonged high-fat consumption causes
hepatic damage or injury in rodents. Geraniin treatment significantly decreased the circulating plasma levels of ALT and
AST in a dose-dependent manner, more so at geraniin inclusion of 50 mg. Notably, the hepatic GGT remained considerably low (<3.000 U/L) in all four groups throughout the
experiment (Table 4). These findings also correlate well to the
relative liver weight in which the HFD-fed group had significantly larger liver size compared to the ND-fed group while a
reduction was reported in the geraniin-treated groups (Table 2).
Non-alcoholic fatty liver disease (NAFLD) is associated with MSinduced hepatic insulin resistance (hepatic IR) as a critical
pathogenic factor. It has been suggested that ectopic fat accumulation in liver is most likely attributed to the increased
storage of hepatocellular lipids particularly TG, raised peripheral lipolytic activity secondary to IR, with higher fatty acid (FA)
flux to the liver and ultimately impaired insulin signalling
(Chang et al., 2011; Guilherme et al., 2008; McGarry, 2002; Moller,
2001). Hepatic IR occurs in liver leading to reduced glycogen
synthesis, storage and a failure to suppress glucose output into
the blood. Consequently, high levels of plasma glucose are perpetuated which in the face of suppressed glucose uptake and
the storage of glucose as glycogen, leads to metabolic derangements such as NAFLD. Aminotransferases and GGT are standard biochemical markers with sufficient predictive values for
non-invasive diagnosis of liver dysfunction, particularly in patients with metabolic complications of different origins unfit
for invasive liver biopsy. It gives inconclusive quantitative estimate of liver disease and its clinical manifestations. Both ALT
and AST are intracellular enzymes which catalyse steps in gluconeogenesis. ALT is present almost exclusively in the liver
whereas AST is less specific. ALT and AST are leaked into the
bloodstream when hepatocytes are damaged, even if this injury
is not severe enough to cause necrosis. GGT is found in hepatocytes as a microsomal enzyme and its activity is potentially induced by alcohols and drugs or caused by biliary
obstruction. In this study, increased levels of the liver dysfunction biomarkers, ALT and AST, hinted the increased permeability of hepatocytes, damage and/or necrosis in liver.
Outcome as such predicts the onset of NAFLD (Giannini, Testa,
& Savarino, 2005; Nelson et al., 2011; Pritchett, 2009). However,
geraniin supplementation in this in vivo experiment suggests
geraniins potential, being preventive rather than therapeutic, to inhibit ectopic lipid accumulations in the liver, attenu-

ate MS-induced liver injury and lower NAFLD risk in obese

rodents.
In this study, the HFD-fed rats showed significantly elevated circulating Cr and CK thus indicating the onset of progressive renal dysfunction as a result of prolonged high-fat
consumption. Geraniin supplementation brought about a significant reduction in the plasma levels of CK and Cr comparable to the ND group (Table 4). Cr and CK are surrogate
biomarkers with sufficient predictive values for non-invasive
diagnosis of renal disease and its clinical manifestations in patients with kidney complications of different causes and stages,
particularly when renal biopsy absent. Cr and CK are accumulated in the bloodstream when renal cells are damaged, even
if this injury is not severe enough to cause necrosis. CK is an
intracellular enzyme predominantly but non-exclusively present
in myocytes and it is a clinical indicator of renal muscle injury
to routinely screen against general renal injury or acute renal
failure whereas Cr is a degradation product from muscle mass
catalysed by CK, which is a better indicator of dysfunction in
nephrons and glomerular filtration. In the setting of MS associated with insulin resistance (IR), systemic IR often accentuates renal derangement leading to severe chronic kidney disease
(CKD) in obese individuals. Systemic IR has been recognised
as a fundamental pathogenic factor for renal disease and its
clinical manifestations (Baur et al., 2006; Chen et al., 2011;
McGarry, 2002; National Heart, Lung and Blood Institute, 2001).
Systemic IR increases intrarenal glomerular pressure, glomerular capillary permeability and renal filtration predisposing to
glomerulosclerosis; triggers mesangial cells proliferation, mediates extracellular matrix deposition, escalates endothelin synthesis and generates oxidative stress products (Sarafidis &
Ruilope, 2006). In this study, the relative kidney weight showed
insignificant differences among all four groups despite the augmentations in renal dysfunction biomarkers obtained (Table 2).
Increased Cr and CK, however, hinted the possible impaired
intrarenal filtration and damage in kidneys. These outcomes
hence predict the onset of MS-induced renal dysfunction
(Agrawal, Shah, Rice, Franklin, & McCullough, 2009; Sarafidis
& Ruilope, 2006). Geraniin supplementation in this in vivo study
proposes geraniins potential, being preventive rather than
therapeutic, to ameliorate visceral adipose tissue deposition,
attenuate MS-induced renal dysfunction, and/or damage and
lower CKD risk in obese rodents.

5.

Conclusion

Geraniin, although present in a number of plants and herbs,

has never been investigated in in vivo studies. This study is the
first, to the best of our knowledge, to show that an orally available geraniin at doses achievable in vivo can safely ameliorate many negative pathological sequels of metabolic syndrome
(MS). Geraniin therefore has the potential to be developed as
a functional food, nutraceutical or therapeutic agent targeting the metabolite derangements of lipid and glucose in MS
encompassing obesity, dyslipidaemia, type 2 diabetes mellitus (T2DM) and its associative complications. There exist
however two early clinical studies on the efficacy of Phyllanthus
species (known to have geraniin as its bioactive compound)

journal of functional foods 9 (2014) 173182

against hepatitis B (Doshi, Vaidya, Antarkar, Deolalikar, &

Antani, 1994; Wang et al., 1995).

Conflicts of interest
There are no conflicts of interest.

Acknowledgements
This research was financially supported by Monash major grant,
Major-BCHH-SM-2-02-2010 and Bioactive Compound Research Small Grant, BCHH-1-05-2009 (HDR). U.D.P., S.G and T.S.H.
collectively designed the study and contributed to interpretation of the data and drafting of the manuscript. A.P.Y.S.C. conducted the in vivo experiment, collected and analysed data,
wrote the manuscript and contributed to the design of study.
Andrew K.L. Leong of Monash Animal House Laboratory is also
thanked for excellent technical assistance.
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