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Camellia sinensis fruit peel extract inhibitsangiogenesis and ameliorates obesity inducedby high-fat diet in rats

http://dx.doi.org/10.1016/j.jff.2014.01.0081756-4646/� 2014 Elsevier Ltd. All rights reserved.

* Corresponding author at: Department of Biotechnology, Chonnam National University, San96-1, Dun-Duk Dong, Yeosu,550-749, Republic of Korea. Tel./fax: +82 61 6597305.

E-mail address: pasteur@chonnam.ac.kr (J.D. Kim).1 These authors contributed equally to this work.

Narendra Chaudharya,1, Jyoti Bhardwaja,1, Hyo Jin Seoa, Min Yong Kimb,d,Tai Sun Shinc,d, Jong Deog Kima,d,*

aDepartment of Biotechnology, Chonnam National University, San96-1, Dun-Duk Dong, Yeosu, Chonnam 550-749, Republic of KoreabDepartment of Refrigeration Engineering, Chonnam National University, San96-1, Dun-Duk Dong, Yeosu, Chonnam 550-749, Republic of

KoreacDepartment of Food Science and Nutrition, Chonnam National University, San96-1, Dun-Duk Dong, Yeosu, Chonnam 550-749, Republic of

KoreadResearch Center on Anti-Obesity and Health Care (RCAOHC), Chonnam National University, San96-1, Dun-Duk Dong, Yeosu, Chonnam

550-749, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history:

Available online 13 February 2014

Keywords:

Anti-angiogenesis

Anti-hyperlipidemic

Green tea

Leptin

White adipose tissue

Tea fruit peel is an agricultural waste of tea manufacturing industry that contains phenols

with high antioxidant activities. This study examined the effect of green tea fruit peel

extract (PE) against angiogenesis and obesity. We found that PE significantly inhibited the

tubular formation of human umbilical vein endothelial cells (HUVECs). Epigallocatechin

gallate (EGCG), epigallocatechin (EGC) and saponin were the functional components pres-

ent in PE that contributed to significant anti-angiogenesis effect. Administration of PE

(100 mg/kg/d) significantly decreased the body weight in rats fed high-fat diet (HFD)

whereas the food intakes between HFD and PE treatment groups were not significantly dif-

ferent. White adipose tissue fat-pad weights were markedly reduced in rats fed HFD plus PE

compared to those in HFD group. These results showed the potential of green tea fruit peel

extract in preventing angiogenesis and obesity.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction Christiaens & Lijnen, 2010; Hausman & Richardson, 2004;

Neoadipogenesis is accompanied by an angiogenic response

(characterized by endothelial cell proliferation, vessel sprout-

ing). Therefore, anti-angiogenic agents constitute a novel ther-

apeutic option for the prevention and treatment of human

obesity and angiogenesis-associated disorders (Cao, 2007;

Neels, Thinnes, & Loskutoff, 2004; Voros et al., 2005). Angio-

genesis and inflammation are necessary for the development

of a variety of disease conditions, such as the proliferation

and metastasis of cancer cells, rheumatism arthritis, and

diabetic blindness (Zetter, 1998). Vascular endothelial growth

factor (VEGF) is one of the most common angiogenic factors

Chonnam

480 J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 4 7 9 – 4 8 6

regulating normal physiological and tumour angiogenesis

(Ferrara, 2002). VEGFR-2 (KDR/Flk-1) controls endothelial cell

morphogenesis, and when activated, it regulates numerous

processes required for the formation of blood capillaries,

endothelial cell proliferation, migration, survival, and tube

formation (Olsson, Dimberg, Kreuger, & Claesson, 2006). The

transcription factor nuclear factor-kappa B (NF-jB) controls

the expression of a wide variety of tumour angiogenic factors,

and NF-jB p65 has been connected to multiple aspects of

oncogenesis, including the inhibition of apoptosis by increasing

the expression of survival factors (Karin, Cao, Greten, & Li, 2002).

Green tea [Camellia sinensis (L.) O. Kuntze] consumption has

been considered as medicine for several complicated symp-

toms like cardiovascular diseases, cancer, obesity, and diabe-

tes (Cabrera, Artacho, & Gimenez, 2006; Namal Senanayake,

2013). Green tea has many biological active compounds like

catechins, saponin, caffeine, theanine and vitamins (Cabrera,

Gimenez, & Lopez, 2003; Takeo, 1992). Epidemiological studies

have shown that green tea consumption reduces blood

cholesterol (Kono et al., 1996). Green tea fruit peel, the

by-products of tea manufacturing process which remains

un-utilized every year after harvest of seed for tea oil extrac-

tion, is usually an agricultural waste. It contains phenols with

high antioxidant activities (Wang, Huang, Shao, Qian, & Xu,

2012). However, no study has been conducted so far to evalu-

ate the chemopreventive effect of green tea fruit peel extract

(PE). Investigation of the valuable compounds of green tea

fruit peel with concern to human health may increase the uti-

lization concern. Hence, the objective of this study was to

examine the effect of PE against angiogenesis and obesity.

2. Materials and methods

2.1. Extract preparation and analysis

Green tea fruit peels were collected from Myungin Shin

GwangSu Tea Garden, Suncheon, Korea. Peels (2 kg) were

dried, ground into fine powders with particle size ranging from

10 to 200 lm and refluxed with 70% ethanol at 60 �C for 4 h.

The resulting homogenate was filtered on a filter press Super-

jet (Buon Vino, Cambridge, ON, Canada) with number 8 filters.

Filtered extract was concentrated in a rotatory vacuum evap-

orator (SB-100, Eyela, Tokyo, Japan), freeze-dried and stored

at 4 �C. Fifty grams of peel extract (PE) were dissolved in 80%

methanol and analyzed by preparative high-performance li-

quid chromatography (HPLC) (Shimadzu Co., Kyoto, Japan)

equipped with a photodiode array (PDA) detector. The PE was

separated on a Luna C-18(2) reverse phase column

(250 · 21.2 mm, 15 lm; Phenomenex, Inc., Torrance, CA, USA)

at 35 �C. Solvent A was methanol and solvent B was distilled

water containing 0.1% formic acid. The non-linear gradient

system used was initially A/B (74:26) to A/B (74.8:25.2) at

33.5 min to A/B (100:0) for 2 min and held A/B (100:0) for

10 min and then A/B (74:26) for 12 min. Components were de-

tected at 210 nm. Flow rate 7 mL/min was used. Active frac-

tions were selected based on the anti-angiogenesis assay at

each purification step. LC/TOF–MS (500–1500 m/z) was per-

formed on a LTQ Orbitrap XL instrument (Thermo Scientific,

Bremen, Germany) as described (Matsui et al., 2009) with a

change in column type. We used Zorbax Extend C18 column

(150 · 2.1 mm, 5 lm; Agilent Technologies, Palo Alto, CA, USA).

2.2. Cell culture

Human umbilical vein endothelial cell (HUVEC) and 3T3-L1

cells were obtained from the Korean Cell Line Bank, Seoul,

Korea. HUVECs were cultured in EBM-2 (Clonetics, Walkers-

ville, MD, USA) supplemented with EGM-2, using a Single

Quots Kit (Clonetics), at 37 �C in a humidified 5% CO2 incuba-

tor. 3T3-L1 cells were cultured in DMEM medium (Gibco,

Grand Island, NY, USA) supplemented with NaHCO3 (3.7 g/L),

100,000 IU/L penicillin, 100 mg/L streptomycin, and 10% (v/v)

foetal bovine serum (FBS).

2.3. In vitro angiogenesis

The formation of tubular structures by HUVECs in Matrigel

was performed as previously described (Kim, Liu, Guo, &

Meydani, 2006). In brief, twenty-four-well culture plates were

coated with 150 lL/well of Matrigel (BD Bioscience, MA, USA),

which was then allowed to solidify at 37 �C. HUVEC suspen-

sions (2.5 · 104 cells per well) in medium were added to the

Matrigel-coated wells and incubated for 4 h in 5% CO2 at

37 �C. Drugs were added to the wells and further incubated

for 4 h. Tube formation was observed and photographed using

a phase contrast inverted microscope (Nikon, Tokyo, Japan),

and the tube length of five photographs obtained from the

random field of cell cultures in each well were analyzed using

Scion Image software (NIH, Bethesda, MD, USA).

2.4. Cell viability assay

Cell viability was determined by the MTT assay. HUVECs and

3T3-L1 cells (1 · 104 cells/well) were seeded in a 96-well plate

for 24 h and treated with different concentration of drugs

for 24 h, at 37 �C in a humidified 5% CO2 incubator. 0.5%

MTT solution (Sigma, St. Lous, MO, USA) was added in the

medium and incubated for 4 h at 37 �C. MTT medium was

aspirated and dimethyl sulphoxide (DMSO) (Sigma) was

added to dissolve formazan crystals for 15 min. Absorbance

was measured at 540 nm using a microplate reader (Biochrom

Ltd., Cambridge, UK).

2.5. Oil-red O staining

Murine 3T3-L1 cells (3 · 103 cells/cm2) were seeded into six-

well plates. Two days after reaching confluence, cells were

kept for another 24 h in this state to arrest cell division. At

this point (day 0), the culture medium was changed to a dif-

ferentiation-induction medium (1 mM dexamethasone,

0.5 mM 3-isobutyl-1-methylxanthine and 10 lg/mL insulin).

After 2 days, cells were maintained in maintenance medium

(10 lg/mL insulin in culture medium). The maintenance med-

ium was changed every 2 days until day 8. To the differentia-

tion-induction medium and maintenance medium, various

concentrations of PE were added. For Oil-red O staining, cells

were washed with PBS and fixed with 10% formalin for 1 h.

Cells were washed with 60% 2-propanol to dryness and then

incubated with Oil red O working solution for 3 h. Cells were

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 4 7 9 – 4 8 6 481

washed again 4 times with distilled water, and completely

dried cells were washed with 100% 2-propanol to extract

the staining dye of the cells. The absorbance of extracted

Oil red O solution was measured at 520 nm using a 96-well

plate.

2.6. RNA isolation and RT-PCR analysis

For angiogenic gene expression analysis, HUVECs were

seeded in a 6-well plate at a density of 9.6 · 104 cells per well.

Cells were maintained in EBM-2 complete medium up to 70%

confluency, and treated with different concentration of PE.

Cells were incubated for 24 h. For adipogenic gene expression

analysis, 3T3-L1 cells were differentiated as described for the

Oil-red O staining. Total RNA was extracted from a mono-

layer of cells using TRI reagent (Sigma) according to the man-

ufacturer’s instructions. cDNA synthesis was performed with

1 lg of total RNA using a RevertAid Premium First Strand

cDNA Synthesis Kit (Thermo Scientific, Glen Burnie, MD,

USA). Reverse transcription polymerase chain reaction (RT-

PCR) was used to analyze the level of mRNA expression of

angiogenesis and adipogenesis biomarkers by using gene spe-

cific primers (Supplementary Table S1). PCR reaction was per-

formed by using Takara Ex Taq Hot Start Version (Takara,

Otsu, Japan). PCR reactions consisted of an initial denaturing

cycle at 94 �C for 3 min, followed by 30 amplification cycles:

94 �C for 30 s, 60 �C for 45 s, and at 72 �C for 1 min. One addi-

tional cycle for 7 min at 72 �C was run to allow trimming of

incomplete polymerizations. Amplified products were sepa-

rated by electrophoresis on 1.5% agarose gel and visualized

by UV transillumination. The band intensities were measured

using ImageJ software and the gene expression levels were

normalized to that of b-actin.

2.7. Animal experiment

Four week-old female Sprague–Dawley (SD) rats (Taconic,

Chungbuk, Korea) were housed under standard conditions

(12 h light/dark cycle, at 22 �C and relative humidity

50 ± 5%). After one week’s acclimatization period, animals

were divided into 4 groups (n = 5 per group): normal diet con-

trol (ND, NIH #31 M Rodent Diet, Taconic), high-fat diet control

(HFD, 45% kcal fat, D12451 Research Diets, New Brunswick, NJ,

USA), Orlistat control (OR, 50 mg/kg/d + high-fat diet) as posi-

tive control and PE treatment group (PE, 100 mg/kg/d + high-

fat diet). Animals were given food and water ad libitum. All

the drugs were administered by oral gavage once a day and

control groups (ND and HFD) were administered distilled

water without drugs at the same time. Body weight and food

intake were measured every 5 days for 50 days on a group ba-

sis. At the end of the study, the rats were anesthetized with

ether after an overnight fasting and blood was collected from

abdominal vena cava of each rat. Perirenal white adipose tis-

sue (WAT), epididymal WAT, and mesenteric WAT were ex-

cised and weighed. Serum samples were stored at �80 �Cuntil analysis. All rats in the study were treated according

to the National Institutes of Health Guide for the care and

use of Laboratory Animals and the experimental protocol

was approved by the Chonnam National University Ethical

community for Animal studies (CNU IACUC–YS–2013-2).

2.8. Serum biochemistry

The level of glucose, total cholesterol (TC), triacylglycerol (TG),

alanine transaminase (ALT), and aspartate transaminase

(AST) in serum were measured according to the kit manufac-

turer’s instruction (Wako Chemicals, Osaka, Japan). Serum

leptin and insulin levels were measured by using leptin rat

ELISA kit (Abcam, ab100773) and rat insulin ELISA kit

(Shibayagi Co., Ltd., Shibukawa, Japan), respectively.

2.9. Statistical analysis

All experiments were repeated at least three times. Data are

expressed as means ± SEM. Statistical significance was deter-

mined by One-Way ANOVA, followed by the Tukey–Kramer

Multiple Comparisons test (SPSS 21). A significant value was

defined as P < 0.05.

3. Results

3.1. Identification of anti-angiogenesis component

Preparative HPLC was employed to purify the active fractions

of peal extract, which inhibits endothelial cell tube formation

on Matrigel. In these active fractions, components (–)-epigallo-

catechin gallate (EGCG), (–)-epigallocatechin (EGC), and sapo-

nin glycosides were predominantly detected. While, other

catechins such as (�)-epicatechin gallate (ECG) and (�)-epicat-

echin (EC) were detected with extreme low concentration or

absent in those active fractions. Hence, the final purified ac-

tive components of green tea fruit peel were EGCG (14.93 mg/

g of dry weight of extract), EGC (1.08 mg/g), and saponin glyco-

sides (86.41 mg/g). Components EGCG and ECG were con-

firmed against the authenticated standards. Saponins rich

fraction purified by preparative HPLC was further character-

ized by LC/TOF–MS (Fig. 1). PE showed a significant inhibitory

effect on HUVECs capillary tube formation at the concentra-

tion range of 50–100 lg/mL (Fig. 2A) and complete disruption

of capillary tubes was observed at 100 lg/mL. The minimum

angiogenesis inhibitory concentrations of EGCG and EGC from

green tea fruit peel were 10 and 25 lg/mL, respectively,

whereas the minimum angiogenesis inhibitory effect of peel

saponin was at 2.5 lg/mL (Fig. 2B). We also examined the effect

of active components on HUVECs cell viability to insure that

the anti-angiogenesis effect was not due to cell death. PE,

EGCG, and EGC were not toxic to HUVECs at 0–100 lg/mL,

whereas peel saponin showed cytotoxicity effect (0.5 LD50) at

25 lg/mL. Thus, the result suggests that the active compo-

nents from green tea fruit peel have strong anti-angiogenesis

effect without significant effect on cell viability.

3.2. Effect of PE on expression of angiogenesis markergenes

The gene expression level of major angiogenesis biomarker

genes were evaluated by semi-quantitative PCR. As shown

in Fig. 3, PE significantly inhibited the expression of VEGFR-

2, VE-Cadherin and b-Catenin in a dose-dependent manner.

The growth factors VEGFR-2, b-Catenin, and VE-Cadherin

Fig. 1 – LC/TOF–MS analysis of saponin-rich fraction of C. sinensis peel. (A) Base peak intensity chromatogram at m/z 500–

1500. (B) Base peak intensity chromatogram at m/z 1050–1300.

Fig. 2 – Green tea fruit peel extract (PE) inhibits angiogenesis. Effect of PE (A) and peel saponin (B) on endothelial tubular

structure formation. HUVECs (2.5 · 104 cells per well) were plated on Matrigel pre-coated 24-well plates and treated with

varying concentration of samples for 4 h. The network of tubular structure formation was quantified by measuring the length

of tubular network in five photomicrograph randomly obtained from each well. Data are expressed as mean ± SEM (n = 3).**P < 0.005 and ***P < 0.001 vs. control.

482 J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 4 7 9 – 4 8 6

plays important role in the formation of a multi-component

receptor complex which is essential for the activation of

PI3K/AKT pathways (Carmeliet et al., 1999). The activation

of the PI3K/Akt pathways leads to downstream activation

and nuclear translocation of NF-jB (Doldi et al., 1996). Treat-

ment with PE led to significant inhibition of PI3K, AKT and

NF-jB. The result showed that PE is effective in suppressing

the angiogenic gene expression in HUVECs.

3.3. Effect of PE on adipocyte differentiation

Oil Red O staining assay provides a good measure to evaluate

adipocyte differentiation and has been a target for antiobesity

strategies. Treatment of preadipocytes with PE during differ-

entiation suppressed the accumulation of lipid in a dose-

dependent manner (Fig. 4). We also examined the effect of

PE on 3T3-L1 cell viability. 3T3-L1 cell viability was not affected

Fig. 3 – Effect of green tea fruit peel extract on angiogenic gene expression. (A) Representative RT-PCR analysis of VEGFR-2,

b-Catenin and VE-cadherin, (B) PI3K, AKT and NF-jB. HUVECs were seeded in 6-well plates at a density of 9.6 · 104 cells per

well, and 70% confluent cells were treated with different concentrations of samples for 24 h. Total RNA was extracted and the

gene expression level was analyzed by RT-PCR using gene specific primers. Data were normalized to b-actin (ACTB). Data are

expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.005 and ***P < 0.001 vs. control.

Fig. 4 – Quantitative analysis of adipocyte differentiation

measured by Oil red O staining. Green tea fruit peel extract

(PE) inhibits the lipid accumulation in 3T3-L1 adipocytes. PE

was added to differentiating cells in induction medium

(1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methyl-

xanthine and 10 lg/mL insulin) and maintenance medium

(10 lg/mL insulin) for 0–8 days. The Oil red O stained lipid

was quantified with a microplate reader at 520 nm. Data are

expressed as mean ± SEM (n = 3). **P < 0.005 and ***P < 0.001

vs. control.

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 4 7 9 – 4 8 6 483

up to a maximum concentration of 200 lg/mL. Also, we ob-

served that morphology of cells treated with 100 lg/mL were

similar to untreated non-differentiated cell, which means PE

treatment inhibited adipogenic morphology (i.e., rounded-

up appearance). These results suggest that PE inhibits the li-

pid accumulation in 3T3-L1 adipocytes due to the presence

of several bioactive compounds.

3.4. PE inhibits key adipogenesis and lipogenesisbiomarker genes

Adipogenesis is a highly regulated process requiring coordi-

nated expression and activation of key transcription factors,

which include CCAAT/enhancer binding proteins (C/EBPs),

peroxisome proliferators activated receptor gamma (PPARc),

and sterol regulatory element-binding proteins (SREBPs) (Giri

et al., 2006). The effect of PE on the expression of PPARc,

C/EBPa, and SREBP was investigated. 3T3-L1 preadipocytes

were incubated as described for 0–8 days at different concen-

tration ranges of GTFPE bioactive compounds, and total RNA

was extracted to investigate the level of gene expression by

semi-quantitative RT-PCR. As shown in Fig. 5, PE significantly

inhibited the expression of key adipogenic transcriptional

factor genes. The level of mRNA expression of PPARc and

SREBP were reduced at 25 lg/mL concentration whereas

C/EBPa was significantly inhibited at 75 lg/mL. These results

suggest that PE down-regulates early adipocyte gene

expression during differentiation.

Fig. 5 – Green tea fruit peel extract attenuated the

expression of PPARc, C/EBPa, and SREBP during 3T3-L1

preadipocyte differentiation. Total RNA was extracted at day

9 and the level of adipogenic genes were analyzed by RT-

PCR. Data were normalized to b-actin (Actb). Data are

expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.005 and***P < 0.001 vs. control.

484 J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 4 7 9 – 4 8 6

3.5. Effect of PE on body weight, food intake and fat-padweights

To determine the effect of PE on obesity, SD rats were fed with

high-fat diet and supplemented with PE (100 mg/kg/day). The

body weight of rats in the HFD group was significantly higher

than ND group rats. Treatment with HFD + PE significantly

(P < 0.05) reduced the body weight gain by 16.53% as com-

pared to HFD group (Fig. 6A) whereas no significant difference

Fig. 6 – The anti-obesity effect of green tea fruit peel extract (PE)

gain after 50 days of experimental diet feeding. (B) Effect of PE o

adipose tissue (WAT) fat-pad. All values are expressed as mean

in food intake was observed (Table 1). Decreased body weight

in rats of PE treatment group was statistically significant dur-

ing 25–50 days compared to HFD group. Total white adipose

tissue (Perirenal WAT, epididymal WAT, and mesenteric

WAT) weights were decreased in the PE group by 79% in com-

pare to HFD group (Fig 6B).

3.6. Serum biochemical parameters

The serum glucose, TC, TG, ALT, AST, insulin and leptin level

of HFD group were significantly higher than that of ND group

(P < 0.05). PE treatment group shows significant decline in ser-

um glucose (by 49.8%), TC (by 47.2%), TG (by 42.3%), ALT (by

52.4%), AST (by 53.7%), insulin (by 40.9%) and leptin (by

40.16%) (Table 1). Orlisat supplemented group (OR) showed

higher hypolipidemic and fat-pad lowering effect. However,

gastro-intestinal side effect such as oily, loose stools and

abnormal intestine color was also observed.

4. Discussion

Many studies have investigated the effect of tea against vari-

ous physiological or pathological conditions. EGCG has been

considers to be a major constituent of tea and has been

widely studied. The pharmacological effect of green tea by-

products has never been studied. By-products of plants such

as peels or hulls have been known to be enriched with bioac-

tive compounds (Albishi, John, Al-Khalifa, & Shahidi, 2013; Li

et al., 2013; Makris, Boskou, & Andrikopoulos, 2007). Thus,

such compounds exhibit pharmaceutical value. In the present

study, we first evaluated the anti-angiogenesis effect of green

tea fruit peel extract (PE). PE significantly inhibited HUVEC

tubular formation on Matrigel. We then fractionated PE by

preparative HPLC to investigate the bioactive constituents of

PE on the basis of angiogenesis assay. Three major active

components were purified from green tea fruit peel on the ba-

sis of anti-angiogenesis effect. Two active constituents were

EGCG and EGC which were identified on the basis of authentic

in rats fed high-fat diet (HFD). (A) Effect of PE on body weight

n white adipose tissue weight. (C) A comparison of white

s ± SEM (n = 5; *P < 0.05 vs. HFD).

Table 1 – Effect of green tea fruit peel extract (PE) on food intake, serum glucose, total cholesterol (TC), triacylglycerol (TG),alanine transaminase (ALT), aspartate transaminase (AST), insulin, and leptin in rats fed high-fat diet.

ND HFD OS PE

Food intake (g/d) 21.07 ± 0.60 20.41 ± 1.25 23.50 ± 3.50 19.05 ± 1.81

Glucose (mg/dL) 271.38 ± 31.79b 535.91 ± 29.26a 215.24 ± 5.48b 269 ± 6.50b

TC (mg/dL) 239.86 ± 6.95b 481.7 ± 21.42a 236.91 ± 4.38b 254.3 ± 7.43b

TG (mg/dL) 143.91 ± 2.51b 264.55 ± 4.81a 139.77 ± 69b 152.58 ± 3.58b

ALT (IU/L) 23.09 ± 1.41b 54.68 ± 2.57a 21.54 ± 1.60b 25.98 ± 2.68b

AST (IU/L) 32.27 ± 7.34b 79.27 ± 4.25a 35.06 ± 5.37b 36.68 ± 9.26b

Insulin (ng/mL) 1.67 ± 0.02b 2.49 ± 0.62a 1.70 ± 0.58b 1.47 ± 0.15b

Leptin (ng/mL) 2.56 ± 0.55b 4.98 ± 0.29a 2.29 ± 0.22b 2.98 ± 0.73b

Data are expressed as means ± SEM (n = 5). abDifferent superscript letters in a row show significant differences, P < 0.05. ND, control group rats

fed normal diet; HFD, control group rats fed high-fat diet (HFD); OR, positive control group rats fed HFD plus orlistat (50 mg/kg/d); PE, treatment

group rats fed HFD plus peel extract (100 mg/kg/d).

J O U R N A L O F F U N C T I O N A L F O O D S 7 ( 2 0 1 4 ) 4 7 9 – 4 8 6 485

standards. It has been reported that EGCG and EC were the

major catechins in ethanol extract of green tea fruit peel

(Wang et al., 2012). By contrast, in the present study only

EGCG and EGC were detected. This suggests that EGCG and

EGC were the main active catechins of PE. Molecular mass

of all the reported tea saponins falls between m/z 1050 and

m/z 1300 (Kitagawa, Hori, Motozawa, Murakami, & Yoshikawa,

1998). Hence, we screened the molecular mass of saponin rich

fraction (Fig. 1A) using m/z 1050–1300 (Fig. 1B). Thus, the third

component was saponin glycosides.

Anti-angiogenesis effect of PE was further confirmed by

the reversal of expression of angiogenesis biomarker genes

(Fig. 3). PE reduced the mRNA expression of potential signal

molecules VEGFR-2, b-Catenin, and VE-Cadherin. These mol-

ecules are responsible for the formation of a multi-compo-

nent receptor complex which is the basic step that is

essential for the activation of Akt and subsequent activation

of endothelial cell migration and tube formation (Jeong,

Cynthia, Michael, Hyeon, & John, 2005). Importantly, PI3K,

AKT and NF-jB genes were down-regulated. Akt regulates

NF-jB, and NF-jB regulates the expression of a battery of

genes involved in tumour cell survival, proliferation, metasta-

sis, invasion, and angiogensis (Aggarwal & Gehlot, 2009).

The differentiating 3T3-L1 cells treated with various con-

centrations of PE had a visible decrease in lipid droplet forma-

tion measured by Oil Red O staining. PPARc, C/EBPa, and

SREBP are key regulators of the adipogenesis and lipogenesis

pathways. It can be anticipated that the inhibition of these

key regulators could prevent obesity. Development of obesity

is characterized by the accumulation of white adipose tissue

mass (Baboota et al., 2013). Green tea peel extract supplemen-

tation caused significant decrease in the body weight gain

and fat-pad weights. In rat, epididymal adipose tissue is con-

sidered as main white adipose tissue (WAT) with a character-

istic structure and function (Loncar, Afzelius, & Cannon,

1998). The weight of epididymal adipose tissue and mesen-

teric adipose tissue in the treatment group was significantly

decreased in compared to HFD group (Fig. 1C). Furthermore,

we found that PE decreases the serum glucose, total choles-

terol, triacylglycerol, leptin and insulin level which resembles

the hypolipidemic effect of PE (Table 1). Importantly, leptin

levels were significantly decreased by the consumption of

PE (by 54%) compared to HFD group. Leptins are synthesized

primarily in the adipocytes of white adipose tissues and the

level of circulating leptin in the blood is proportional to the to-

tal amount of fat in the body (Margetic, Gazzola, Pegg, & Hill,

2002). Epidemiological and in vitro studies indicate that phyto-

chemicals possess strong protective effects against major dis-

ease including cancer, diabetes and cardiovascular diseases

due to their antioxidant potential (Jin et al., 2013; Vertolli

et al., 2013; Willett, 2002).

In conclusion, results of this study suggested that green

tea fruit peel extract may prevent angiogenesis by inhibiting

endothelial cell tubular formation. PE exerts significant anti-

obesity effect by decreasing the blood lipid levels and white

adipose tissue accumulation in rat. Reduced serum choles-

terol and triacylglycerol may be due to the reduction of leptin

levels by green tea fruit peel extract. The active components

were identified here as EGCG, EGC and saponin. Hence, it

might help in preventing angiogenesis and obesity related

complications and may be a potential source of functional

compounds with medicinal and industrial value.

Acknowledgements

The research reported in this manuscript was funded by the

Korean Institute of Planning and Evaluation for Technology

in Food, Agriculture, Forestry, and Fisheries (112075-3).

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at http://dx.doi.org/10.1016/j.jff.

2014.01.008.

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