Ellagitannin geraniin supplementationameliorates metabolic risks in high-fatdiet-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,Malaysiab School of Science, Monash University Malaysia, 46150, Sunway City, Selangor, Malaysia

A B S T R A C T

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

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

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

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

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

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.

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

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

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

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

potential to safely mitigate obesity-induced metabolic dysfunction.

© 2014 Elsevier Ltd. All rights reserved.

A R T I C L E I N F O

Article history:

Received 23 January 2014

Received in revised form 14 March

2014

Accepted 26 March 2014

Available online

Keywords:

Beta-cell dysfunction

Geraniin

High-fat diet

Insulin sensitivity

Metabolic dysfunction

Nephelium lappaceum

1. Introduction

Excess nourishment, high fat, sugar and salt in modern diets,combined with a sedentary lifestyle, leads to metabolic over-load and consequently metabolic disturbance, which is an im-balance between energy input (EI) and energy output (EO)(Sorensen, 2009). It results in over-abundance of glucose andfatty acid accumulations within adipose tissue, skeletal muscle,hepatocytes and pancreatic cells, causing serious defects infuel partitioning and thus disrupts the total body energy ho-meostasis (Baur et al., 2006; Chen et al., 2011; Storlien et al.,

1996), finally leading to the risks associated with metabolicsyndrome (MS). Metabolic syndrome is typified by a constel-lation of metabolic risks encompassing obesity, insulin resis-tance, hyperglycaemia, dyslipidaemia and hypertension(American Diabetes Association, 2001; Christopher & Sarah,2005; National Heart, Lung and Blood Institute, 2001). Thenumber of people affected with metabolic syndrome world-wide has strikingly increased over the past two decades andthis increase is not dissociable from the worldwide pan-demic of obesity and hyperglycaemia (Zimmet, Alberti, & Shaw,2001). Many studies have shown that lifestyle interventionsincluding 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.2014.03.0291756-4646/© 2014 Elsevier Ltd. All rights reserved.

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weight management in combination with pharmacotherapyreduce the incidence of these metabolic risks (AmericanDiabetes Association, 2001; Christopher & Sarah, 2005; NationalHeart, Lung and Blood Institute, 2001). However, there are stilldifficulties in reaching the goal of normalising glucose levelsand fat contents without adverse effects, such as severe weightgain or loss, hypoglycaemic episodes, lactic acidosis,hepatoxicity, kidney damage, dyspepsia, atherogenic eventsand premature cardiovascular diseases due tohyperinsulinaemia and lipodystrophy, overstimulation and ap-petite abuse in addition to raised risk of death (Agabegi &Steven, 2008; Zimmet et al., 2001). Hence, there is a greatmedical need to develop novel drugs or alternative therapiesthat are both effective and which are free from, or with rela-tively fewer adverse effects.

Traditionally, plants have been used in the management ofa broad spectrum of metabolic dysfunction such as diabetes,cardiovascular diseases, obesity, dyslipidaemia and cancers,among others, owing to the presence of plant-derived second-ary metabolites (Aggarawal & Shishu, 2011). Plant-derived sec-ondary metabolites are a rich source of natural bioactivecomponents; the most essential of these are flavonoids andpolyphenols. Ellagitannins (ETs), the bioactive polyphenols, canbe found abundantly in fruits such as raspberries, blackber-ries, strawberries, cranberries and pomegranates; in veg-etables such as potato, tomato, lettuce and onion; in nuts andseeds (Scalbert & Williamson, 2000), traditional Chinese me-dicinal plants such as Geranium sibiricum Linne (Yang et al., 2010)and ayurvedic herbs such as Phyllanthus emblica (amla)(Krishnaveni & Mirunalini, 2010). Many studies on ellagitanninshave demonstrated positive biological actions encompassingantioxidant, antimutagenic, anticarcinogenic, antitumour, an-tiviral and antimicrobial, both in vitro and in vivo (Ito, 2011),which suggest that the consumption of ellagitannins mayprovide protective benefits on human health.

Native to Southeast Asia, Nephelium lappaceum L. (‘rambu-tan’ 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 com-mercial crop in Asia, where it can be consumed fresh, cannedor processed. Geraniin, a typical ellagitannin was recently es-tablished to be present in N. lappaceum L. rind with the highestyields of geraniin (up to 35%) among the plant studied thusfar (Palanisamy, Ling, Manaharan, & Appleton, 2011). Studieson diverse biological properties of geraniin have shown thatthe 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-L1cells have also revealed geraniin’s ability to enhance glucoseuptake (Palanisamy). Based on its manifold beneficial healthproperties, geraniin is a valuable candidate for more exten-sive study into its potential pharmaceutical applications. Thisstudy investigates the in vivo effects of supplementing geraniinpurified from the N. lappaceum L. rind on metabolic factors mim-icking MS and its clinical complications in rodents fed on ahigh-fat diet.

2. Materials and methods

2.1. Materials

High-fat pellet (HFD, 60% fat by weight, AIN93G specificationspurified diet) was purchased from Specialty Feeds Inc. (GlenForrest, Western Australia). Normal rat chow (ND, 5% minimumcrude fat content) was purchased from Gold Coin (KualaLumpur, Peninsular Malaysia). Accu-Chek®Performa Glucometerwas purchased from Roche (Manheim, Germany). Diagnosticreagents 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 pur-chased from Millipore (MA, USA). BIO-RAD Benchmark PlusMicroplate Reader with Microplate Manager 5.2.1 software (CA,USA), Hitachi Model 902 Automatic Analyser (Manheim,Germany). All other chemicals and reagents were purchasedfrom Becton, Dickinson and Company (NJ, USA), Millipore (MA,USA), Sigma-Aldrich (MO, USA), Terumo (Tokyo, Japan) andVétoquinol UK Limited (Buckingham, UK) unless statedotherwise.

2.2. Preparation of geraniin from Nepheliumlappaceum L. rind by reverse phase C-18 columnchromatography

Nephelium lappaceum L. was obtained from Kuala Lumpur, Pen-insular Malaysia. Plants were authenticated by the Her-barium of the Forest Research Institute of Malaysia (FRIM). Crudeextract of N. lappaceum L. rind was prepared as described byPalanisamy et al. (2008). Geraniin was purified from the crudeextract by means of reverse-phase C-18 chromatography(Palanisamy et al., 2011). Crude extract (20 g) was dissolved ina minimum amount of water (40 mL) and loaded onto glasscolumn (250 mm × 50 mm i.d.) packed with 200 g C18 silica (par-ticle size of 50 μm, pore size of 60 Å). The column was opentubular and solvent flow rate was maintained by means ofvacuum pump attached to vacuum inlet in the column. Thecolumn was first eluted with water (300 mL) and then frac-tions were collected using a step gradient of water and ace-tonitrile. 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 se-quentially with dichloromethane (100%, 300 mL), methanol con-taining a few drops of trifluoroacetic acid (100%, 300 mL) andabsolute methanol (100%, 300 mL), allowing to dry com-pletely. This enables the column to be reused with fresh crudeextract. Purity of geraniin (> 95%) obtained was confirmed usingHPLC (Details see Perera, Appleton, Loh, Elendran, & Palanisamy,2012).

2.3. Animals and housing

Thirty-two male, post-weaning (3-week-old) outbred SpragueDawley (SD) rats (Rattus norvegicus) were obtained from theAnimal House of Monash University (Monash University, Pen-insular Malaysia). Males were used to eliminate variations in

174 j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 1 7 3 – 1 8 2

metabolic outcomes induced by sex in addition to their vul-nerability to the impacts of diet-induced obesity (DIO) (Hwang,Wang, Li, Chang, Lin, Chen et al., 2010).They were randomisedinto four groups of eight rats each and accorded a 7-dayacclimatisation period to diets and experimental conditionsprior to this in vivo study. A minimum of eight rats for eachtreatment group was required to give a significant finding atP ≤ 0.05 levels (Campbell & Machin, 1999). Normal rat chow anddistilled water (dH2O) were fed ad libitum. Body weights wererecorded at the end of the acclimatisation period, which arepresented as initial weights in this study. Throughout the ex-periment, all SD rats were housed individually in cages withstainless steel lids and with ad libitum access to food and water.They were maintained on a 12-h light–dark cycle (lights on at08:00 hours; lights off at 20:00 hours) with controlled tempera-ture (22 ± 1 °C) and humidity (55 ± 10%) in the animal housingfacility. The use and handling procedure of animals was ap-proved by the Monash University Animal Ethics Committee ac-cording to the ethics approval (Approval Code: AEC:MARP/2011/021).

2.4. Induction of obesity and dietary treatments

At the end of the 7-day acclimatisation, the obesity induc-tion was carried out for 6 weeks (Andrikopoulos et al., 2005;Ikarashi et al., 2011) followed by the geraniin treatment for4 weeks. Food and water consumptions were monitored daily.Body weight and blood glucose were measured weekly. Normaldiet 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 high-fat induction, were fed with HFD and 10 mg geraniin/kg bodyweight (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 for16 h prior to being sacrificed. Tail venipuncture was per-formed to collect whole blood for the determination of bloodglucose prior to anaesthetisation. The SD rats wereanaesthetised by intraperitoneal (IP) injection of ketamine(90 mg/kg body weight) and xylazine (10 mg/kg body weight)and exsanguinated through cardiac puncture with dispos-able syringes and sterile needles. Blood samples weredivided into two parts. Blood was collected into sterileethylenediaminetetraacetic 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 andstored at −80 °C for the determination of triaylglycerol (TG), high-density lipoprotein (HDL) cholesterol, non-high-density lipo-protein (HDL) cholesterol, total cholesterol (TC), alanineaminotransferase (ALT), aspartate aminotransferase (AST),gamma-glutamyl transferase (GGT), creatinine (Cr) and cre-atine kinase (CK). Blood was also collected into sterile Red Topvacutainers which contained clot activator gel and centri-fuged at 15,000 g for 15 min at 4 °C. The serum samples ob-tained were aliquoted into sterile cryovials and stored at −80 °Cfor serum insulin measurement.

2.5.1. Measurement of fasting blood glucose and seruminsulin levelsThe fasting glucose levels were measured in the tail vein bloodwith a glucometer. The fasting serum insulin levels were mea-sured using the Rat/Mouse Insulin Sandwich ELISA kit. Absor-bance was detected at 450 and 590 nm on BIO-RAD BenchmarkPlus Microplate Reader with Microplate Manager 5.2.1software.

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) andinsulin resistance (IR) of the SD rats were analysed using theHOMA calculator based on fasting plasma glucose (mmol/L)and fasting serum insulin (μIU/mL) (Bonora et al., 2000) mea-sured using the Rat/Mouse Insulin Sandwich ELISA kit follow-ing the manufacturer’s instructions.

HOMA BCFFasting Serum Insulin IU mL

Fasting Plasma G− = ∗ ( )[ ]20 μ

llucose mmol L( ) −[ ]3 5.Equation 1

HOMA IS

Fasting Plasma Glucose mmol L Fasting Serum In

−

= ÷ ( ) ∗100

ssulin IU mLμ( ){ }22 5.

Equation 2

HOMA IR

Fasting Plasma Glucose mmol L Fasting Serum Insuli

−

= ( ) ∗ nn IU mLμ( )22 5.

Equation 3

2.5.3. Fasting plasma metabolic parameter analysisMetabolic parameters of lipid (TG, non-HDL cholesterol, HDLcholesterol and TC), liver (ALT, AST and GGT) and kidney (Crand CK) were measured using the Hitachi Model 902 Auto-matic 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. Organsof interest, namely brain, heart and aorta, pancreas, liver andkidney, and WAT (visceral and epidermal) were promptly col-lected. All the organs and WAT were washed in ice-cold phos-phate buffered saline (1 × PBS) buffer to remove excessive bloodclot and dried on absorbent paper to remove all remaining fluidsbefore weighing on an electronic balance to obtain a constantweight. The ratio of each organ to terminal body weight (rela-tive organ weight expressed as percentage of body weight) wasalso measured (modified from Khanal, Howard, Wikes, Rogers,& Prior, 2010).

% of BWAbsolute Organ Weight g

Final Body Weight g= × ( )

( )⎧⎨⎩

100⎫⎫⎬⎭

Equation 4

2.7. Statistical analysis

Data obtained were analysed using the GraphPad Prism sta-tistical software, version 5.0 (CA, USA). One-way ANOVA withTukey’s post hoc test was used for comparisons between vari-ables and t-test for pairwise comparison between the differ-

175j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 1 7 3 – 1 8 2

ent groups. All data are presented as mean ± SEM unlessotherwise 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 super-scripts a, b, c and d, respectively. Means with different super-scripts are significantly different at P-value < 0.05 betweengroups.

3. Results

3.1. Preparation of geraniin from Nepheliumlappaceum L. rind by reverse phase C-18 columnchromatography

Purification of geraniin from Nephelium lappaceum L. rind wastewas achieved in a single chromatographic method to producegeraniin of high yield and a high purity. In this method, 74.78 ggeraniin of approximately 94% purity were obtained from 362.4 gcrude extract of N. lappaceum L. rind representing a geraniinyield of approximately 21%.

3.2. Effects of high-fat feeding and geraniin on bodyweight gain and food intake

A significant increase in body weight of the HFD-fed rats com-pared to the ND-fed rats was observed (Fig. 1) throughout theintervention study (P < 0.05). On the tenth week, the HFD-fedgroup recorded a 19% weight gain, 346 ± 5 g compared to291 ± 3 g in the ND-fed rats. Upon establishing obesity in theSD rats after 6 weeks of induction, the effect of geraniin treat-ment (10 and 50 mg/kg body weights; 4 weeks) on HFD-fed ratswas investigated. After a 4-week geraniin treatment, the twotreatment groups, HFD + 10 mg geraniin (HFD + 10 mg G) andHFD + 50 mg geraniin (HFD + 50 mg G), showed significant re-ductions in body weight compared with the HFD-fed alone rats

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

3.3. Effects of high-fat feeding and geraniin on whiteadipose tissue depositions

The total WAT depot, both visceral and epidermal, was alsoexamined after the 10-week study. The HFD-induced SD ratsdemonstrated an increase in total WAT mass whereas the

10

50

100

150

200

250

300

350

400

2 3 4 5 6 7 8 9 10Weeks

Bod

y W

eigh

t (g)

ND HFD

Fig. 1 – Body weight (g) of the SD rats throughout theintervention study. ND, normal diet control; HFD, high-fatdiet control. Means with different superscripts aresignificantly different at P-value < 0.05 between groups.The experimental groups, ND and HFD are represented bysuperscripts a and b respectively.

Fig. 2 – Body weight (g) of the SD rats throughout the treatment period (weeks 7–10). ND, normal diet control; HFD, high-fatdiet control; G, geraniin treated. Means with different superscripts are significantly different at P-value < 0.05 betweengroups. The experimental groups, ND, HFD, HFD + 10 mg G and HFD + 50 mg G, are represented by superscripts a, b, c and d,respectively.

176 j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 1 7 3 – 1 8 2

geraniin-treated groups showed a significant reduction in totalWAT deposition (Table 1). Inclusions of geraniin at 10 and 50 mgwere found to significantly reduce total WAT depositions by42 and 47%, respectively compared to the HFD-fed rats. Notably,HFD-fed rats showed a significant increase in the visceral fatmass but geraniin treatment particularly at 50 mg attenuatedthis condition. Interestingly, this treatment group also showedeven lower visceral fat mass compared to the ND-fed rats. HFDinduction did increase the epidermal and visceral masses inrats compared to those of the ND-fed rats. Although statisti-cally insignificant (P > 0.05) among all groups, the geraniin-treated groups had consistently showed a lower trend inepidermal mass.

3.4. Effects of high-fat feeding and geraniin onorgan pathology: Toxicology and relative organweight-to-body weight

Effects of 4-week geraniin treatment on SD rats organ pathol-ogy were also investigated at the end of the treatment. Mac-roscopic toxicology was examined on the pancreas, liver, heartand aorta, kidney and brain. Organ weights were also com-pared to final body weights, expressed as percent of body weight(% of BW; Table 2). The HFD-fed rats had higher ratios of pan-creas, liver, and heart and aorta over final body weight com-pared to the ND-fed rats. Geraniin treatment showed asignificant 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 relativeweights of brains and kidneys among all four groups at the endof this study.

3.5. Effects of high-fat feeding and geraniin on fastingplasma 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 endof week 10. Interestingly, HFD-fed rats did not demonstrate sig-nificant difference in PG compared to the ND rats. However, asignificant decrease in PG was observed in geraniin-treatedgroups compared to the HFD or ND controls. The HFD induc-tion did increase fasting SI concentrations of rats comparedto those of the ND-fed rats. Although statistically insignifi-cant (P > 0.05) among all groups, fasting SI levels were consis-tently lower in geraniin-treated groups throughout theintervention study. The HFD-fed rats also demonstrated sig-nificantly decreased HOMA-IS (%S) and HOMA-BCF (%B) com-pared to those of ND- and geraniin-treated. The two treatmentgroups, comparatively, demonstrated a significant increase inHOMA-IS (%S) and HOMA-BCF (%B) and correspondingly a de-crease in the HOMA-IR index. Also, worthy of mention is thatthese findings correlate well to the relative organ weight studywhere a significant increase in both liver and pancreas was seenin the HFD group compared to the ND group while a reduc-tion was seen in the geraniin-treated groups (Table 2).

3.6. Effects of high-fat feeding and geraniin on fastingplasma metabolites

The fasting metabolic parameters of lipids (TG, non-HDL cho-lesterol, HDL cholesterol and TC), liver (ALT, AST and GGT) andkidney (Cr and CK) in SD rats were measured at the end of week10 (Table 4). The HFD-fed rats showed a significant increase in

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 100 ± 1 291 ± 3b 2.85 ± 0.32 2.35 ± 0.41 5.20 ± 0.64HFD 142 ± 3 346 ± 5a,c,d 3.09 ± 0.51d 2.51 ± 0.76 5.60 ± 0.60c,d

HFD + 10 mg G 144 ± 3 342 ± 4bd 2.12 ± 0.62 1.63 ± 0.58 3.75 ± 0.63b

HFD + 50 mg G 141 ± 2 332 ± 4bc 1.45 ± 0.22b 1.52 ± 0.19 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).

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

Groups Final BW (g) Relative organ weight (% of BW)

Pancreas Liver Heart–aorta Kidney Brain

ND 291 ± 3b 0.31 ± 0.19b,c,d 2.16 ± 0.20b 0.35 ± 0.11b 0.66 ± 0.12 0.63 ± 0.14HFD 346 ± 5a,c,d 0.34 ± 0.10a,c,d 2.90 ± 0.15a,c,d 0.37 ± 0.07a,d 0.60 ± 0.09 0.56 ± 0.12HFD+10 mg G 342 ± 4b,d 0.29 ± 0.08a,b,d 2.74 ± 0.21b,d 0.35 ± 0.09 0.63 ± 0.08 0.55 ± 0.10HFD+50 mg G 332 ± 4b,c 0.22 ± 0.10a,b,c 2.20 ± 0.13b,c 0.31 ± 0.09b 0.65 ± 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.

177j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 1 7 3 – 1 8 2

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

4. Discussion

In this study, the development of metabolic factors mimick-ing metabolic syndrome (MS) and its clinical manifestationsinduced 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; Khanalet al., 2010; Li et al., 2008). The in vivo ability of geraniin, at 10and 50 mg per body weight (kg), to ameliorate anomalies tailingmetabolic derangements was also investigated.

Prolonged high-fat feeding resulted in the development ofmetabolic derangements mimicking MS and its clinical patho-genesis. The HFD-fed group demonstrated significantly higherbody weight gain compared to the ND-fed group (Fig. 1). Thissuggests that excess and repetitive consumption of dietary fatcontributes to the development of obesity in rodents. Similarresults have been reported by previous animal studies inducedwith high-fat rodent diets having similar source of fat and ex-perimental length (Andrikopoulos et al., 2005; Gajda et al., 2007;Ikarashi et al., 2011). Inclusion of geraniin at 50 mg showed amore significant reduction in body weight, particularly at week4.This demonstrates geraniin’s anti-obesity potential in SD rats.Similar weight reducing results have been shown with otherplant-derived compounds (Baur et al., 2006; Chen et al., 2011).Existing evidence have shown that the continual escalation inobesity rates arises from negative changes in today’s life-style, such as excess nourishment (high fat, sugar and salt indiets), reduced physical activities and poor weight manage-ment (Sorensen, 2009). Many plant-based phenolic com-pounds have been reported to be potent effectors of biologicalprocesses and have the capacity to ameliorate disease risks

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

Glycaemic indices Groups

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

FPG (mmol/L) 6.05 ± 0.32 6.70 ± 0.18c,d 5.43 ± 0.25b 5.18 ± 0.28b

FSI (μIU/mL) 4.38 ± 0.37 4.80 ± 0.50 3.77 ± 0.26 3.73 ± 0.19HOMA-BCF (%B) 44.65 ± 2.52 38.25 ± 1.52c,d 50.38 ± 3.33b 55.70 ± 5.57b

HOMA-IS (%S) 171.00 ± 16.32 154.90 ± 20.06c,d 201.80 ± 14.02b 205.20 ± 11.7b

HOMA-IR 0.60 ± 0.06 0.70 ± 0.07c,d 0.50 ± 0.04b 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.

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

Plasma analytes Groups

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

TG (mmol/L) 0.360 ± 0.006b,c,d 0.543 ± 0.003a,c,d 0.510 ± 0.010a,b,d 0.460 ± 0.000a,b,c

HDL (mmol/L) 2.073 ± 0.035b,c,d 1.813 ± 0.027a,c,d 1.437 ± 0.012a,b,d 1.543 ± 0.003a,b,c

Non-HDL (mmol/L) 0.623 ± 0.009b,c,d 0.433 ± 0.007a,c,d 0.383 ± 0.003a,b,d 0.247 ± 0.003a,b,c

TC (mmol/L) 2.650 ± 0.021b,c,d 2.283 ± 0.113a,d 2.140 ± 0.050a,d 1.700 ± 0.012a,b,c

ALT (U/L) 49.93 ± 0.623b,d 63.53 ± 0.338a,c,d 50.53 ± 0.338b,d 42.77 ± 0.203a,b,c

AST (U/L) 213.40 ± 0.379b,c,d 233.20 ± 1.931a,c,d 146.300 ± 1.836a,b,d 121.500 ± 1.770a,b,c

GGT (U/L) <3.000 <3.000 <3.000 <3.000CK (U/L) 1459.00 ± 11.29b,c,d 1897.00 ± 19.77a,c,d 1327.00 ± 2.19a,b,c 1585.00 ± 46.09a,b,d

Cr (μmol/L) 58.67 ± 0.667b 67.00 ± 0.577a,c,d 61.67 ± 1.333b 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 lipoprotein-cholesterol; 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.

178 j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 1 7 3 – 1 8 2

such as obesity (Ikarashi et al., 2011) with lower concomitantadverse effects on humans. Geraniin, therefore, as demon-strated in this study, is a potential body weight lowering/controlling agent in the management of obesity and obesity-related health problems.

The HFD-induced SD rats showed an increase in total WATmass (visceral and epidermal), whereas the geraniin-treatedgroups demonstrated a significant reduction in total WAT mass.Geraniin supplementation had significantly reduced total WATdepositions (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, adipocytenumber, a phenomenon known as adipocyte hyperplasia; andadipocyte enlargement, a state known as adipocytehyperthrophy (Moller, 2001), thus results in ectopic expan-sion of WAT mass which ultimately disrupts the lipid storagecapacity. Ectopic expansion of WAT mass with regards to ex-cessive energy storage exacerbates physical body weight gainin obesity. Obesity, particularly abdominal adiposity in sus-ceptible human subjects, has been proven to be a key precur-sor to the increased risk of MS encompassing T2DM (prediabetesonset) and other obesity-induced metabolic sequels (Chang,Tzeng, Liou, Chang, & Liu, 2011; Guilherme, Virbasius, Puri, &Czech, 2008; McGarry, 2002). These findings hence suggest thatlong-term overnutrition also contributes to the developmentof central obesity as a result of ectopic deposition of WAT, par-ticularly visceral fat in rodents. Geraniin, in this study, also ex-hibited its adipocyte hypothrophic potential to prevent fatdepositions in an orally achievable dose in rodents.

Augmentations in fasting PG, SI and HOMA scores thatpredict the onset of diabetes were seen in the HFD-inducedSD rats whereas the geraniin-treated groups showed signifi-cant ameliorations in glycaemic and HOMA indices, compa-rable to the ND group (Table 3). Also, worthy of mention is thatthese findings correlate well to the significant increase inweights of liver and pancreas in the HFD group compared tothe ND group while a reduction was seen in the geraniin-treated groups (Table 2). Many studies have shown that pro-longed consumption of HFD causes insulin resistance (IR), whichultimately results in the disruption of glucose clearance andglucose uptake in skeletal muscle and adipose tissue, both invivo and in vitro (Baur et al., 2006; Storlien et al., 1996). Raisedlevels of circulating lipids have been shown to sufficiently induceIR in peripheral and hepatic sites, in addition to increased de-position of lipids inside adipose tissue and skeletal muscle owingto the specific increase in long-chain fatty-acyl-CoA (Buettner,Scholmerich, & Bollheimer, 2007). Lipid accumulation withinthe pancreatic islets has also been proven to contribute to theimpairment of insulin secretion. These observations supporta unified ‘lipotoxicity’ hypothesis, which states that HFD-induced diabetes can be caused by the accretion of triaylglyceroland long-chain fatty-acyl-CoA in liver, skeletal muscle andadipose tissue (leading to a reduction in insulin-mediated meta-bolic activity) and in the pancreatic islets (leading to im-paired insulin secretion) (Chang et al., 2011; Guilherme et al.,2008; McGarry, 2002; Moller, 2001). Noticeably, either insulin re-sistance or beta-cell dysfunction alone will not lead to the de-velopment of T2DM (Ostenson, 2001). Rather, to develop T2DM

both insulin resistance and beta-cell dysfunction in the pan-creas should coexist. At the end of this study, the fasting seruminsulin (FSI) levels in the HFD group did not show a signifi-cant increase compared to the ND group although changes wereobserved throughout the experimental period. Previous studieshave shown that most rodents tend to become obese on HFD(≈20–50% digestible energy from lipids) and VHFD (≥50% di-gestible energy from lipids) with variable responses in glucosetolerance, insulin resistance, triacylglycerol and other param-eters 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 fruc-tose diets for 12 weeks adapted to the nutritional interven-tion but did not develop classical signs of insulin resistanceand impaired glucose tolerance. Despite hyperglycaemia absentand lowered insulin levels as recorded, paralleling the organpathology, the HFD-fed rats were prone to be in a pre-diabeticstate. Diabetes, in fact, represents the progressive andcumulative damage caused by cellular glucose and lipidmetabolites.

The HFD-induced SD rats showed anomalies in plasmabiomarkers that mimic the onset of a constellation of meta-bolic risks encompassing obesity, insulin resistance,hyperglycaemia, dyslipidaemia, hepatic steatosis (non-alcoholicfatty liver disease, NAFLD) and progressive loss of kidney func-tion whereas the geraniin-treated groups had significant at-tenuations comparable to the ND group (Table 4). Increasedadipocyte size associated with obesity increases lipolytic ac-tivity based on a correlation between the rate of lipolysis andthe size of adipocyte, a condition known as lipotoxicity (Changet al., 2011; Guilherme et al., 2008; McGarry, 2002; Moller, 2001).The HFD-fed group with increased body weight also had sig-nificantly raised levels of TG but significantly lower HDL, non-HDL and total cholesterols.This finding suggests that disruptionin fuel partitioning into adipocytes results in obesity which inturn progresses to the development of dyslipidaemia in adiposetissue and skeletal muscle. In this study, the HFD group al-though showing an increased trend, did not show a signifi-cant difference in non-HDL and total cholesterols comparedto the ND group. Saturated fat (SF) has been recognised to in-crease serum cholesterols (Buettner et al., 2006; Hegsted,Ausman, Johnson, & Dallal, 1993). In this study, the HFD con-sists of a low amount of animal SF (10% ghee), which was in-sufficient to significantly increase the non-HDL- and totalcholesterol levels. This observation is not odd because normalrodents (without genetic mutations) are known to have typi-cally very low levels of TC and LDL-cholesterol but high levelsof HDL-cholesterol. Many studies, in addition, have shown thatmaterials other than fatty acids and dietary cholesterols affectserum 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 experi-mental fats and oils and/or the basal diets. Most rodents tendto be obese but produce variable metabolic outcomes depend-ing on the strain, gender and source of dietary fat (Buettneret al., 2006; Gajda et al., 2007; Hegsted et al., 1993). These meta-bolic effects can also be altered by adjusting the feeding period.As demonstrated in many studies using obese rodents, not allobese subjects develop obesity and obesity-induced manifes-tations (Gajda et al., 2007).This association hence suggests that

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both genetic predisposition and/or environmental factors, inpart, also play a causal role in the development of obesity. Al-though the geraniin treatments (10 and 50 mg) did not sig-nificantly decrease the lipoprotein parameters comparable tothe ND group, it had exhibited to a certain extent, a signifi-cant amount of hypolipidaemic activity. Plant-derived poly-phenols have been reported to affect lipid metabolism bysuppressing lipogenic enzymes (fatty acid synthetase, acyl-CoA synthase, glycerol-3-phosphate acyltransferase) and up-regulating signal transduction-related genes (Prior, 2012). It issuggested that geraniin’s hypolipidaemic activity may be dueto the similar mechanism. However, gene transcriptional studieswould be able to confirm this. Geraniin thus has the poten-tial as a plant-derived hypolipidaemic agent to attenuate es-calating lipids and to prevent fat accretion.

Biomarkers of the liver dysfunction (ALT and AST) in thisstudy suggest that prolonged high-fat consumption causeshepatic damage or injury in rodents. Geraniin treatment sig-nificantly decreased the circulating plasma levels of ALT andAST in a dose-dependent manner, more so at geraniin inclu-sion of 50 mg. Notably, the hepatic GGT remained consider-ably low (<3.000 U/L) in all four groups throughout theexperiment (Table 4). These findings also correlate well to therelative liver weight in which the HFD-fed group had signifi-cantly larger liver size compared to the ND-fed group while areduction was reported in the geraniin-treated groups (Table 2).Non-alcoholic fatty liver disease (NAFLD) is associated with MS-induced hepatic insulin resistance (hepatic IR) as a criticalpathogenic factor. It has been suggested that ectopic fat ac-cumulation in liver is most likely attributed to the increasedstorage of hepatocellular lipids particularly TG, raised periph-eral 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 glycogensynthesis, storage and a failure to suppress glucose output intothe blood. Consequently, high levels of plasma glucose are per-petuated which in the face of suppressed glucose uptake andthe storage of glucose as glycogen, leads to metabolic derange-ments such as NAFLD. Aminotransferases and GGT are stan-dard biochemical markers with sufficient predictive values fornon-invasive diagnosis of liver dysfunction, particularly in pa-tients with metabolic complications of different origins unfitfor invasive liver biopsy. It gives inconclusive quantitative es-timate of liver disease and its clinical manifestations. Both ALTand AST are intracellular enzymes which catalyse steps in glu-coneogenesis. ALT is present almost exclusively in the liverwhereas AST is less specific. ALT and AST are leaked into thebloodstream when hepatocytes are damaged, even if this injuryis not severe enough to cause necrosis. GGT is found in he-patocytes as a microsomal enzyme and its activity is poten-tially induced by alcohols and drugs or caused by biliaryobstruction. In this study, increased levels of the liver dys-function biomarkers, ALT and AST, hinted the increased per-meability 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 suggestsgeraniin’s potential, being preventive rather than therapeu-tic, to inhibit ectopic lipid accumulations in the liver, attenu-

ate MS-induced liver injury and lower NAFLD risk in obeserodents.

In this study, the HFD-fed rats showed significantly el-evated circulating Cr and CK thus indicating the onset of pro-gressive renal dysfunction as a result of prolonged high-fatconsumption. Geraniin supplementation brought about a sig-nificant reduction in the plasma levels of CK and Cr compa-rable to the ND group (Table 4). Cr and CK are surrogatebiomarkers with sufficient predictive values for non-invasivediagnosis of renal disease and its clinical manifestations in pa-tients with kidney complications of different causes and stages,particularly when renal biopsy absent. Cr and CK are accu-mulated in the bloodstream when renal cells are damaged, evenif this injury is not severe enough to cause necrosis. CK is anintracellular enzyme predominantly but non-exclusively presentin myocytes and it is a clinical indicator of renal muscle injuryto routinely screen against general renal injury or acute renalfailure whereas Cr is a degradation product from muscle masscatalysed by CK, which is a better indicator of dysfunction innephrons and glomerular filtration. In the setting of MS asso-ciated with insulin resistance (IR), systemic IR often accentu-ates renal derangement leading to severe chronic kidney disease(CKD) in obese individuals. Systemic IR has been recognisedas a fundamental pathogenic factor for renal disease and itsclinical manifestations (Baur et al., 2006; Chen et al., 2011;McGarry, 2002; National Heart, Lung and Blood Institute, 2001).Systemic IR increases intrarenal glomerular pressure, glomeru-lar capillary permeability and renal filtration predisposing toglomerulosclerosis; triggers mesangial cells proliferation, me-diates extracellular matrix deposition, escalates endothelin syn-thesis and generates oxidative stress products (Sarafidis &Ruilope, 2006). In this study, the relative kidney weight showedinsignificant differences among all four groups despite the aug-mentations in renal dysfunction biomarkers obtained (Table 2).Increased Cr and CK, however, hinted the possible impairedintrarenal filtration and damage in kidneys. These outcomeshence predict the onset of MS-induced renal dysfunction(Agrawal, Shah, Rice, Franklin, & McCullough, 2009; Sarafidis& Ruilope, 2006). Geraniin supplementation in this in vivo studyproposes geraniin’s potential, being preventive rather thantherapeutic, to ameliorate visceral adipose tissue deposition,attenuate MS-induced renal dysfunction, and/or damage andlower 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 thefirst, to the best of our knowledge, to show that an orally avail-able geraniin at doses achievable in vivo can safely amelio-rate many negative pathological sequels of metabolic syndrome(MS). Geraniin therefore has the potential to be developed asa functional food, nutraceutical or therapeutic agent target-ing the metabolite derangements of lipid and glucose in MSencompassing obesity, dyslipidaemia, type 2 diabetes melli-tus (T2DM) and its associative complications. There existhowever two early clinical studies on the efficacy of Phyllanthusspecies (known to have geraniin as its bioactive compound)

180 j o u rna l o f f un c t i ona l f o od s 9 ( 2 0 1 4 ) 1 7 3 – 1 8 2

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 Re-search Small Grant, BCHH-1-05-2009 (HDR). U.D.P., S.G and T.S.H.collectively designed the study and contributed to interpre-tation of the data and drafting of the manuscript. A.P.Y.S.C. con-ducted 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 alsothanked for excellent technical assistance.

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