Journal of Ethnopharmacology 137 (2011) 817–827

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Phaleria macrocarpa (Scheff.) Boerl fruit aqueous extract enhances LDL receptor
and PCSK9 expression in vivo and in vitro
Soo Ching Chong a , Mohamad Aziz Dollah a,∗ , Pei Pei Chong a , Abdullah Maha b
a
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
b
Department of Pathology, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Phaleria macrocarpa (Scheff.) Boerl (Pm) has been shown to reduce
Received 8 March 2011 cholesterol level in vitro and in vivo experiment.
Received in revised form 22 June 2011 Aim of the study: This study investigated the effects of Pm fruit on weight control and mechanistic basis
Accepted 28 June 2011
of its anti-hypercholesterolemic effect in both in vivo and in vitro.
Available online 5 July 2011
Materials and method: In the in vivo study, thirty six male Sprague Dawley were randomized to six groups.
Five groups were induced into hypercholesterolemia by giving 3% cholesterol enriched-diet for 52 days
Keywords:
while one group acted as control. The rats were then treated with Pm extract (0, 20, 30 and 40 mg/ml)
Phaleria macrocarpa (Scheff.) Boerl fruit
Hypercholesterolemia
or simvastatin for 84 days. The following parameters were determined: (1) body weight, (2) blood lipid
Lipids profile (total cholesterol, triglyceride, HDL and LDL) and (3) hepatic LDL receptor (160 kDa and 120 kDa)
LDL receptor and PCSK9 proteins. In the in vitro study, HepG2 cells were cultured in serum-free RPMI supplemented
Proprotein convertase subtilisin/kexin type with 0.2% BSA with or without LDL and in the presence of Pm extract (0, 0.1, 2, 40 and 1000 ␮g/ml) or
9 simvastatin (4.60 ␮g/ml) for 24 h. The abundance of both LDL receptor and PCSK9 proteins and mRNA
Traditional medicine Asia & Oceania were investigated.
Results: Pm extract significantly (P < 0.05) reduced body weight gain, total cholesterol, triglycerides, HDL
LDL levels and upregulated hepatic LDL receptor as well as PCSK9 proteins of hypercholesterolemic rats.
These results were supported by studies in HepG2 cells whereby Pm extract also significantly upregulated
both LDL receptor and PCSK9 at protein and mRNA levels.
Conclusion: This study enhances the potential usage of Pm fruit for controlling the body weight of obese
people and for treating hypercholesterolemia.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction synthesis increases and mediates endocytosis of plasma LDL into

The prevalence of obesity and overweight has been increas-
ing lately. When the body mass index increases, total cholesterol
(TC) and LDL levels will increase while HDL level will decrease
(Liao et al., 2007; Lukeman et al., 2007). Even though cholesterol
is essential to our body, too much cholesterol termed hypercholes-
terolemia can lead to arteriosclerosis, heart disease and stroke.
LDL receptor and proprotein convertase subtilisin/kexin type 9
(PCSK9) play important roles in regulating cholesterol homeosta-
sis. When cells are deprived of cholesterol, LDL receptor protein
Abbreviations: apo-B, apolipoprotein B; BSA, bovine serum albumin; CETP,
cholesterol ester transfer protein; HDL, high density lipoprotein; HMG-CoA, 3-
hydroxy-3-methyl-glutaryl-CoA reductase; LDL, low density lipoprotein; PBS,
phosphate buffer saline; PCSK9, proprotein convertase subtilisin/kexin type 9; Pm,
Phaleria macrocarpa; RNA, ribonucleic acid; RPMI, Roswell Park Memorial Institute;
SR-B1, scavenger receptor B1; SREBP-2, sterol regulatory element binding protein-2;
TC, total cholesterol; TG, triglyceride.
∗ Corresponding author. Tel.: +60 389472309; fax: +60 389436178.
E-mail address: mohdaziz@medic.upm.edu.my (M.A. Dollah).
the cells (Brown and Goldstein, 1979). Synthesized as a 120 kDa
precursor, LDL receptor undergoes post-translational glycosyla-
tion, forming a 160 kDa mature glycoprotein that migrates to the
cell surface (Kingsley and Krieger, 1984). Besides that, PCSK9, a
protease that regulates the degradation of LDL receptor (Li et al.,
2007; Peterson et al., 2008) will also increases. However, if the cel-
lular cholesterol level is high, transcription of LDL receptor and
PCSK9 are inhibited, decreasing the intake of plasma cholesterol
into the cell. Phaleria macrocarpa (Scheff.) Boerl (Pm) is an Indone-
sian native plant under the family of Thymelaeaceae (Savolainen
et al., 2000). Studies showed that Pm fruit can prevent arteriosclero-
sis and reduced cholesterol level in Japanese quails and in primary
culture of rat hepatocytes (Adnyana et al., 2005; Armenia et al.,
2006). However, the mechanism underlying cholesterol lowering
property of Pm fruit has yet to be investigated. Therefore, studies
on in vivo and in vitro hypercholesterolemia model were conducted
to determine the effects of Pm fruit on blood lipid profile (total
cholesterol, triglyceride, high density lipoprotein and low density
lipoprotein) as well as LDL receptor and PCSK9 protein and mRNA
expression.

0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.06.041
818 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827
2. Materials and methods
2.1. Chemicals
Enzymatic assay kits for total cholesterol, triglyceride, HDL
and LDL were purchased from Roche Diagnostic, Germany.
RPMI medium, fetal bovine serum, penicillin–streptomycin, and
trypsin–EDTA (0.25%) were purchased from Hyclone, USA. Bovine
serum albumin and low density lipoprotein were purchased from
Merck & Co., Inc., New Jersey. RevertAidTM First Strand cDNA syn-
thesis kit was purchased from Fermentes, California. GoTaq® PCR
core system I was purchased from Promega, USA. RNeasy® mini kit
was purchased from Qiagen, Germany.
2.2. Preparation of aqueous extraction of Pm fruits
Pm fruits used in this experiment were collected along Bandar
Baru Bangi, Selangor housing area and were authenticated by Mr.
Shamsul Khamis, a botanist at Institute of Bioscience (IBS), Univer-
sity Putra Malaysia (UPM). A voucher specimen (SK1929/11) was
preserved at Herbarium of IBS, UPM. Sliced and air-dried Pm fruits
were boiled, paper-filtered and freeze-dried. The yield obtained
was approximately 13%. Stock solution of Pm was prepared by
reconstituting known amount of crude extract of Pm with known
amount of distilled water.
2.3. In vivo study
2.3.1. Animals
The study was approved by Animal Care and Use Committee
(ACUC) of Medicine and Health Sciences Faculty, University Putra
Malaysia (ACUC No. UPM/FPSK/PADS/BRUUH/00273). In this study,
36 male Sprague Dawley rats, weighing approximately 350 g each
were kept in an animal house under room temperature of 20–23 ◦ C
and humidity 70–80% with 12 h of light and 12 h of dark cycle.
2.3.2. Study design
The rats were divided randomly into 6 groups (A–F). Group A
was fed with normal pellet and served as a negative control while
group B to F were fed with 3% cholesterol enriched-diet for 52 days
to induce hypercholesterolemia. Subsequently group B to E were
treated with Pm extract (0, 20, 30 and 40 mg/kg) while group F
was treated with simvastatin (Simvor 40, Ranbaxy (M) SDN. BHD. –
40 mg/kg) for 84 days. Body weight of the rats was measured before
and after the experimental period.
2.3.3. Sampling
2.3.3.1. Biochemical lipid profile analysis. Blood samples were col-
lected via retro-orbital bleeding at the end of experimental period.
Blood samples were centrifuged to isolate plasma and stored at
−80 ◦ C. Plasma concentration of total cholesterol (TC), triglyceride
(TG), HDL and LDL was analyzed and measured using Roche Hitachi
902 chemistry analyzer (Roche Diagnostic, Germany).
2.3.3.2. Western blot analysis of hepatic LDL receptor and PCSK9.
After 136 days of experimental period, all the rats were decapitated
and livers were removed and stored at −80 ◦ C. Frozen rat liver was
homogenized in ice-cold lysis buffer (Liang et al., 2005), followed
by centrifugation at 10,000 × g for 15 min at 4 ◦ C. Supernatant was
collected and protein concentration was determined using Brad-
ford protein assay (Bradford, 1976). Approximately 130 ␮g proteins
from each sample was subjected to electrophoresis in 5% stacking
and 7% resolving SDS–PAGE gel and transferred to PVDF membrane.
LDL receptor and PCSK9 were detected with anti-rat LDL recep-
tor IgG (Novus Biologicals, USA) and anti-rat PCSK9 IgG (SantaCruz
Biotechnology, California), followed by anti-rabbit IgG antibody
conjugated with horseradish peroxidase (Millipore, USA). Finally,
detection of the bands was performed using ECL enzyme substrate
kit (Pierce Biotechnology, USA). The intensity of the bands was
quantified using Alpha Imager software and normalized to internal
protein, ␤-actin (Santa Cruz Biotechnology, California).
2.4. In vitro study
2.4.1. HepG2 cell maintenance and treatment
HepG2 cells were maintained in RPMI supplemented with 10%
FBS and 1% penicillin–streptomycin in 10 cm dishes or 12-well
plates. Maintenance medium was removed when the cells reached
80–90% confluency. HepG2 cells were then cultured in serum-free
RPMI supplemented with 0.2% BSA with or without LDL and in the
presence or absence of Pm extract (0.1, 2, 40 and 1000 ␮g/ml) or
simvastatin, sodium salt, 4.60 ␮g/ml (Merck, New Jersey) for 24 h.
2.4.2. HepG2 cell viability assay
HepG2 cell viability was assessed with MTT assay. MTT was
added and incubated for 3 h. The formazan crystals were dis-
solved with 200 ␮l of DMSO by agitation. Absorbance at 570 nm
wavelength was measured using microplate reader (Dynex Tech-
nologies, USA). Cell viability was determined as percentage
compared to control.
2.4.3. Western blot analysis of LDL receptor and PCSK9 levels in
HepG2
HepG2 cells were washed twice with PBS and lysed with
ice-cold lysis buffer (Bursill et al., 2001). After centrifugation at
10,000 × g for 15 min at 4 ◦ C, supernatant was removed and stored
at −80 ◦ C. Protein concentration was determined using Bradford
protein assay (Bradford, 1976). Cell lysate (130 ␮g) was separated
on a 5% stacking and 7% resolving SDS–PAGE gel and transferred to
PVDF membrane. LDL receptor and PCSK9 were detected using anti-
LDL receptor antibody (Cayman, Belgium) and anti-PCSK9 antibody
(Santa Cruz Biotechnology, California), followed by anti-rabbit IgG
antibody conjugated with horseradish peroxidase (Millipore, USA).
The bands were detected using ECL enzyme substrate kit (Pierce
Biotechnology, USA). Intensity of the bands was quantified using
Alpha Imager software and normalized to internal protein, ␤-actin
(Santa Cruz Biotechnology, California).
2.4.4. LDL receptor and PCSK9 mRNA expression levels
Total RNA was extracted using the Qiagen RNeasy® mini kit
(Qiagen, Germany). RNA integrity was verified using nanopho-
tometer (Implen, Germany) with optical density (OD) at 260/280,
ratio between 1.8 and 2.0 and electrophoretically verified with
ethidium bromide staining. Approximately 600 ng total RNA was
reverse transcribed using RevertAidTM first strand cDNA synthesis
kit (Fermentes, California) according to the manufacturer’s instruc-
tion. After cDNA synthesis by RT, cDNA was amplified for LDL
receptor, PCSK9 and GAPDH using GoTaq® PCR core system master
mix. Each reaction had a final volume of 20 ␮l. PCR amplification
was performed with initial denaturation at 95 ◦ C for 5 min, opti-
mized cycle (GAPDH – 25 cycles and LDL receptor and PCSK9 – 30
cycles) of denaturation at 95 ◦ C for 1 min, annealing at optimized
temperature (GAPDH – 61 ◦ C, LDL receptor – 64 ◦ C and PCSK9 –
63 ◦ C) for 1 min, extension at 72 ◦ C for 20 s and final elongation
at 72 ◦ C for 7 min. Amplification of PCR products was confirmed
using 1.5% agarose gel electrophoresis. The intensity of the bands
was quantified using the Alpha Imager software and normalized to
housekeeping gene, GAPDH. Primer sequences used for PCR were
as follow (forward, reverse): GAPDH (5 -GAC CAC AGT CCA TGC
CAT CAC-3 , 5 -TCC ACC ACC CTG TTG CTG TAG-3 ), LDL receptor
(5 -CCC CGC AGA TCA ACC CCC ACT C-3 , 5 -AGA CCC CCA GGC
AAA GGA AGA CGA-3 ) and PCSK9 (5 -ATCCACGCTTCCTGCTGC-3 ,
 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827 819

Fig. 1. Effect of different concentrations of Phaleria macrocarpa (Scheff.) Boerl and simvastatin on hepatic LDL receptors 160 kDa (a), 120 kDa (b) and PCSK9 (c) levels in diet
induced hypercholesterolemic rats. Data are expressed in mean ± standard deviation (n = 3); ABCD: statistical significance at P < 0.05; Pm: Phaleria macrocarpa.
820 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827

Table 1
Initial (pre-experiment) and final (post-experiment) body weight of the rats and average daily gain.

Type of treatment Phaleria macrocarpa (Scheff.) Boerl aqueous extraction (mg extract/kg bw) Simvastatin (mg/kg)

0 0 20 30 40 40

Feeding Normal diet (control) 3% cholesterol-enriched diet

Initial body weight (pre-experiment) (g) 349.00 ± 55.13 352.71 ± 43.66 358.00 ± 58.07 354.85 ± 58.02 373.50 ± 44.99 353.67 ± 28.32
Final body weight (post-experiment) (g) 482.28 ± 40.69 514.42 ± 23.11 463.83 ± 71.64 478.71 ± 77.72 486.66 ± 80.69 477.67 ± 60.03
Changes in the body weight (final-initial) (g) 133.28 ± 33.33AB 161.71 ± 50.13A 105.83 ± 34.26B 123.85 ± 37.67AB 113.17 ± 42.78AB 124.00 ± 56.86AB
Average daily gain (g/136 days) 6.05 ± 1.51AB 7.35 ± 2.27A 4.81 ± 1.55B 5.63 ± 1.71AB 5.14 ± 1.94AB 5.63 ± 2.58AB

Note: Values are in mean ± SD (n = 6).

AB: statistical significance at P < 0.05.

Table 2
Blood lipid profiles of hypercholesterolemia diet-induced rats treated with different concentrations of Phaleria macrocarpa (Scheff.) Boerl (0, 20, 30 and 40 mg extract/kg bw)
or simvastatin.

Type of treatment Phaleria macrocarpa (Scheff.) Boerl aqueous extraction (mg extract/kg bw) Simvastatin (mg/kg)

0 0 20 30 40 40

Feeding Normal diet (control) 3% cholesterol-enriched diet

TC (mmol/l) 1.43 ± 0.17C 2.4 ± 0.23A 1.54 ± 0.38BC 1.55 ± 0.16BC 1.86 ± 0.34B 1.47 ± 0.17C
TG (mmol/l) 0.69 ± 0.15B 1.13 ± 0.26A 0.38 ± 0.15C 0.33 ± 0.07C 0.89 ± 0.32AB 0.40 ± 0.09C
HDL (mmol/l) 1.03 ± 0.21A 0.95 ± 0.14AB 0.68 ± 0.12C 0.80 ± 0.13BC 0.74 ± 0.12C 0.82 ± 0.09BC
LDL (mmol/l) 0.21 ± 0.03D 1.51 ± 0.19A 0.94 ± 0.13BC 0.99 ± 0.23BC 1.03 ± 0.08BC 0.79 ± 0.19C

Note: Values are in mean ± SD (n = 6).

ABCD: statistical significance at P < 0.05.

5 -CACGGTCACCTGCTCCTG-3 (Dubuc et al., 2004; Ringseis et al.,
2006).
2.5. Statistical analysis
Data were expressed as mean ± standard deviation. Statistical
analysis was done using SAS statistical packages. Duncan’s Multiple
Range Test was used as post hoc analysis after ANOVA, differences
were considered significant at P < 0.05.
3. Results
3.1. Body weight
In this study, body weight of cholesterol-fed rats increased
by 21.33% compared to normal-diet fed rats. However, Pm treat-
ment reduced the body weight gain in rats when compared
to non-treated rats fed with cholesterol-enriched diet. Twenty
mg/kg of Pm extract significantly (P < 0.05) reduced body weight
gain in rats by 42% whereas 30 mg/kg and 40 mg/kg of Pm
extract non-significantly reduced body weight gain by 30% and
36%, respectively. Simvastatin treatment non-significantly reduced
body weight gain in rats over the period by 6%. This trend is similar
to average daily gain whereby the group treated with 20 mg/kg of
Pm extract had the lowest average daily gain, followed by 40 mg/kg
and 30 mg/kg of Pm extract and lastly simvastatin (Table 1).
3.2. Blood lipid profile
Table 2 shows the blood lipid profile of rats. Three percent
cholesterol-enriched diet significantly (P < 0.05) increased TC, TG
and LDL compared to the rats fed with normal diet. However, after
84 days of treatment, 20 mg/kg of Pm extract significantly lowered
serum TC, TG, HDL and LDL level by 36%, 66%, 28% and 38%, respec-
tively whereas 30 mg/kg of Pm extract significantly reduced TC, TG
and LDL level by 35%, 71% and 34%, respectively. Forty (40) mg/kg

Fig. 2. Effect of different concentrations of Phaleria macrocarpa (Scheff.) Boerl and simvastatin on HepG2 cell viability. Data are expressed in mean ± standard deviation
(n = 3). ABCD: statistical significance at P < 0.05; Pm: Phaleria macrocarpa; LDL: low density lipoprotein.
 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827 821

Fig. 3. Western blot analysis of LDL receptor (a) and PCSK9 (b) levels in HepG2 cells incubated in serum-free RPMI and 0.2% BSA with or without LDL (200 ␮M) and Phaleria
macrocarpa (Scheff.) Boerl (0, 0.1, 2, 40 and 1000 ␮g/ml) or simvastatin (4.60 ␮g/ml) for 24 h. Data are expressed in mean ± standard deviation (n = 3), ABC: statistical
significance at P < 0.05; Pm: Phaleria macrocarpa; LDL: low density lipoprotein.

of Pm extract significantly reduced TC, HDL and LDL level of rats by
22%, 22% and 32%, respectively compared to the non-treated rats
fed with cholesterol enriched diet. Simvastatin treatment signifi-
cantly reduced TC, TG and LDL by 39%, 65% and 48%, respectively
(Table 2).
3.3. Hepatic LDL receptor and PCSK9 levels
Hepatic LDL receptor (160 kDa and 120 kDa) and PCSK9 levels
were significantly (P < 0.05) lowered by 3% cholesterol-enriched
diet by 42%, 41% and 39%, respectively, compared to the normal-diet
fed rats. However, Pm treatment increased hepatic LDL receptor and
PCSK9 levels. Twenty mg/kg of Pm extract significantly increased
LDL receptor (160 kDa and 120 kDa) and PCSK9 levels by 115%, 97%
and 33%, respectively. Thirty mg/kg of Pm extract increased hepatic
LDL receptor levels (160 kDa and 120 kDa) by 56% and 27% but did
not increase hepatic PCSK9 level. Forty mg/kg of Pm extract had no
effect on both hepatic LDL receptor and PCSK9 levels (Fig. 1).
3.4. HepG2 cell viability
Fig. 2 shows the effect of Pm on HepG2 cell viability. Treatment
with Pm at various concentrations significantly (P < 0.05) decreased
HepG2 cell viability compared to control. Since cell viability at the
concentration of 1000 ␮g/ml of Pm extract fruit was 83%, therefore
Pm was used at the concentration of 1000 ␮g/ml and below for
further assay.
3.5. LDL receptor and PCSK9 protein levels in HepG2 cells
Effect of Pm was further analyzed on HepG2 cells. As the effect
of Pm on both 160 kDa and 120 kDa LDL receptors was similar
in vivo study, thus, only 160 kDa LDL receptor was evaluated on
822 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827

Fig. 4. Effect of different concentrations on Phaleria macrocarpa (Scheff.) Boerl on LDL receptor (a) and PCSK9 (b) mRNA expression in HepG2 cells. Cells were incubated
in serum-free RPMI and 0.2% BSA with or without LDL (200 ␮M) and Phaleria macrocarpa (Scheff.) Boerl (0, 0.1, 2, 40 and 1000 ␮g/ml) or simvastatin (4.60 ␮g/ml). LDL
receptor and PCSK9 mRNA were determined using reverse transcriptase-PCR (RT-PCR) as described in Section 2. Data are expressed in mean ± standard deviation (n = 3),
ABC: statistical significance at P < 0.05, Pm: Phaleria macrocarpa, LDL: low density lipoprotein.

in vitro study. Western blot analysis shows that in the presence
of LDL, LDL receptor and PCSK9 levels in HepG2 cells were signif-
icantly (P < 0.05) reduced to 58% and 51%, respectively. However,
Pm extract significantly increased and maintained the level of LDL
receptor similar to the control despite an increase in Pm concen-
tration from 0.1 to 1000 ␮g/ml. Apart from increasing LDL receptor
level, Pm extract also increased PCSK9 level. Simvastatin treatment
significantly increased LDL receptor and PCSK9 levels in HepG2 cells
(Fig. 3).
3.6. LDL receptor and PCSK9 mRNA expression in HepG2 cells
Consistent with the changes in LDL receptor and PCSK9 pro-
tein levels in HepG2 cells, incubation of LDL reduced LDL receptor
and PCSK9 mRNA expression. Additionally, Pm extract was found
to override the LDL suppression on LDL receptor and PCSK9 mRNA
expression. Simvastatin upregulated LDL receptor and PCSK9
mRNA in HepG2 cells (Fig. 4 and Tables 3 and 4 in supplemen-
tary data).
4. Discussion
Obesity and overweight have been shown to be associated with
cardiovascular diseases (CVD) such as arteriosclerosis, stroke and
myocardial infarction (Australian Institute of Health and Welfare
and National Heart Foundation of Australia, 2004), as these con-
ditions can affect lipoprotein metabolism. Increase body weight
elevates TG and LDL levels and reduces HDL level. Conversely,
weight loss is associated with lowered levels of TG and LDL and high
level of HDL (Cabioglu and Ergene, 2005) and hence cardiovascular
benefit. Thus, it is important to maintain an ideal body weight. In
this study, it was found that Pm extract significantly reduced the
body weight of diet-induced hypercholesterolemic rats at lower
concentration. This result correlates with the reduction of blood
TC, TG and LDL level at lower concentration of Pm.
 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827
One of the mechanisms involved in the metabolism of plasma
LDL is via LDL receptor-mediated endocytosis, thus regulating the
level of apo-B containing lipoprotein in cells and plasma (Brown
and Goldstein, 1979; Goldstein et al., 1985). Both in vivo and in vitro
studies showed that Pm has anti-hypercholesterolemic property
but the mechanism involved in reducing the cholesterol level is
still unknown (Adnyana et al., 2005; Armenia et al., 2006). There-
fore, this study demonstrated that Pm reduced blood level of LDL
and suggested that the mechanism by which LDL is reduced is asso-
ciated with the increase in LDL receptor on both in vivo and in vitro
studies. This is probably due to the upregulation of its mRNA as
shown in in vitro study. The increased number of LDL receptor
would increase the binding of LDL particles in the blood to LDL
receptor followed by internalization into the peripheral cells via
receptor-mediated endocytosis, and hence decreased the circulat-
ing LDL level.
In this study, LDL receptor increase upon Pm aqueous extract
treatment accompanied the increase of PCSK9 on in vivo and in vitro
studies, probably due to the upregulation of its mRNA as shown on
in vitro study. Binding of PCSK9 to epidermal growth-factor (EGF)-
like A of cell surface LDL receptor initiates the receptor mediated
endocytosis pathway. Once inside the cell, PCSK9–LDL receptor
complex is routed to lysosomes and degraded (Kwon et al., 2008),
regulating cell surface LDL receptor. As PCSK9 is involved in choles-
terol metabolism, thus like LDL receptor, PCSK9 is encoded by sterol
regulatory element-binding protein-2 (SREBP-2), which preferen-
tially regulates genes involved in cholesterol metabolism (Kwon
et al., 2008). It is thought that PCSK9 is increases together with LDL
receptor under sterol depletion to buffer the upregulation of LDL
receptor, thereby limiting the intake of lipoprotein (Dubuc et al.,
2004; Attie and Seidah, 2005). Thus, Pm possibly upregulates LDL
receptor and PCSK9 expression through the SREBP-2 transcription
factor.
Pm treatment at low concentration not only reduced LDL level
but it also reduced HDL level, possibly due to the upregulation of
LDL receptor as LDL receptor can clear a portion of the apoE con-
taining HDL (Attie and Seidah, 2005). Apart from that, scavenger
receptor B1 (SR-B1), a high affinity cell surface receptor may reg-
ulate HDL level. SR-B1 functions to mediate selective uptake of
HDL-cholesteryl ester into the liver and other steriodogenic tis-
sues in reverse cholesterol transport of HDL. Studies in human and
hamster showed that upregulation of SR-B1 by polyunsaturated
fatty acids caused a reduction in HDL level (Spady et al., 1999;
Dorfman et al., 2005), probably due to the reduction in HDL’s half-
life (Arrese and Crawford, 1997). In addition, there has been an
association between decreased HDL level due to upregulation of
SR-B1 and reduced atherosclerotic lesion development in exper-
imental animal models (Landschulz et al., 1996; Krieger, 1999;
Spady et al., 1999; Trigatti et al., 2003). Other than SR-B1, hep-
atic lipase and cholesterol ester transfer protein (CETP) can also
modulate HDL level. However, modulation of plasma HDL by these
enzymes is unlikely. This is because although plasma HDL level is
increased with downregulation of both hepatic lipase and CETP but
down-regulation of these enzymes is associated with an increase
in aortic lesion formation, which does not correlate with the
study done by Armenia et al. (2006) showing anti-atherosclerotic
effect of ethanolic extract of Pm. Thus, the possible mechanism
for Pm aqueous extract used in the present study in lowering
plasma HDL is through upregulation of SR-B1 and LDL receptor
levels.
Pm fruit is reported to have active compounds such as alkaloid,
saponin, phalerin and polyphenol such as gallic acid and lignan
823
(Sugiwati et al., 2006; Faried et al., 2007; Oshimi et al., 2008).
Studies utilizing gallic acid has found that gallic acid possesses
many therapeutic properties such as anti-cancer, anti-microbial
and antioxidant effects (Ow and Stupans, 2003). Gallic acid also
possesses anti-hypercholesterolemic property. It reduces TG and
LDL in high-fat diet mice (Jang et al., 2008) and inhibits cholesterol
biosynthesis (Lu and Hwang, 2008) which may explain the decrease
in apoB production and upregulation of LDL receptor and HMG-CoA
reductase mRNA abundance in HepG2 cell (Pal et al., 2003). When
the intracellular cholesterol level declines, synthesis of HMG-CoA
reductase, LDL receptor and PCSK9 increase but when the intracel-
lular cholesterol level increases, LDL receptor, PCSK9 and HMG-CoA
reductase synthesis decrease. This cholesterol balance is tightly
regulated by the negative feedback control via the SREBP path-
way (Brown and Goldstein, 1979, 1997; Bursill et al., 2001). Thus,
anti-hypercholesterolemic property of gallic acid may act through
SREBP pathway, the mechanism used by statins (Stancu and Sima,
2001; Dubuc et al., 2004) and that the anti-hypercholesterolemic
property of Pm may be due to the gallic acid compound found in
Pm fruit.
Even though Pm fruit possesses many therapeutic properties,
it may have toxicity effect at high concentration. Previous stud-
ies showed that Pm exhibited embryo-fetotoxicity effect in female
mice that consumed Pm at concentration of more than 27 mg/kg
(Suatma and Widyastuti, 2008) and pups were stillborn at the con-
centration of 100 mg/kg (Almahdy et al., 2008). Apart from that,
prolonged consumption of Pm by Japanese quail at the concentra-
tion of 50 mg/kg exhibited liver toxicity effect (Armenia et al., 2006).
This may explain the upregulation of LDL receptor and PCSK9 at
lower concentration but not at higher concentration of Pm in in
vivo study.
5. Conclusion
This study demonstrated that Pm fruit aqueous extract reduced
body weight and improved blood lipid profile of diet-induced
hypercholesterolemic rats. The improvement is likely to be reg-
ulated by LDL receptor and PCSK9 as observed at both the protein
and mRNA levels. Further studies are required to determine the
upstream mechanism involved in the upregulation of LDL recep-
tor and PCSK9 by Pm. In addition to that, down-regulation of HDL
due to Pm treatment and the compound in Pm that exerts the anti-
hypercholesterolemic property should also be investigated. Toxic
effects of Pm should also be studied to determine the safe dose of
Pm for human consumption.
Acknowledgements
The authors would like to thank the animal house and Chemical
Pathology Laboratory staffs of the Faculty Medicine and Health Sci-
ences, UPM for their technical assistance. This work is supported by
grants 04/01/07/00090RU from Research University Grant Scheme,
University Putra Malaysia.
Appendix A.
See Figs. A.1–A.3.
Appendix B. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jep.2011.06.041.
824 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827

Fig. A.1. GAPDH DNA sequence analysis.

S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827 825

Fig. A.2. LDL receptor DNA sequence analysis.

826 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827
Fig. A.3. PCSK9 DNA sequence analysis.
References
Adnyana, K., Yulinah, E., Sigit, J.I., Fitriani, D., 2005. Anticholesterol activity test of
aqueous extract of Allium sativum L. Bulbs, Eugenia polyantha wight leaves and
Phaleria macrocarpa fruit in primary culture. Acta Pharmaceutica Indonesia 30,
43–47.
Almahdy, A., Febrianti, R., Djamal, R., 2008. Effek Fetotoksisitas Ekstrak Biji Mahkota
Dewa (Phaleria macrocarpa (Scheff.) Boerl Pada Mencit. Jurnal Sains dan
Teknologi Farmasi 13, 86–88.
Armenia, E.F., Widya, R.M., Rusdi, D.J., 2006. Anti-atherosclerotic effect and liver tox-
icity of ethanolic extract of Phaleria macrocarpa (Scheff.) Boerl fruit on Japanese
Quail. In: Asian Symposium on Medicinal Plants, Spices and Other Natural Prod-
ucts XII, Padang, Indonesia.
Arrese, M.A., Crawford, J.M., 1997. Of plagues and stones: the SR-B1 (scavenger
receptor class B, type 1). Hepatology 26, 1072–1074.
Attie, A.D., Seidah, N.G., 2005. Dual regulation of the LDL receptor—some clarity and
new questions. Cell Metabolism 1, 290–292.
Australian Institute of Health and Welfare and National Heart Foundation of
Australia, 2004. The Relationship Between Overweight, Obesity and Cardiovas-
cular Disease: A Literature Review Prepared for the National Heart Foundation
of Australia. Australian Institute of Health and Welfare, Canberra.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein–dye binding.
Analytical Biochemistry 72, 248–254.
Brown, M.S., Goldstein, J.L., 1979. Receptor-mediated endocytosis: insights from the
lipoprotein receptor system. Proceedings of the National Academy of Sciences
of the United States of America 76, 3330–3337.
Brown, M.S., Goldstein, J.L., 1997. The SREBP pathway: regulation of cholesterol
metabolism by proteolysis of a membrane-bound transcription factor. Cell 89,
331–340.
Bursill, C., Roach, P.D., Bottema, C.D., Pal, S., 2001. Green tea upregulates the
low-density lipoprotein receptor through the sterol-regulated element bind-
ing protein in HepG2 liver cells. Journal of Agriculture and Food Chemistry 49,
5639–5645.
Cabioglu, M.T., Ergene, N., 2005. Electroacupuncture therapy for weight loss reduces
serum total cholesterol, triglycerides, and LDL cholesterol levels in obese
women. The American Journal of Chinese Medicine 33, 525–533.
Dorfman, S.E., Wang, S., Vega-Lopez, S., Jauhiainen, M., Lichtenstein, A.H., 2005.
Dietary fatty acids and cholesterol differentially modulate HDL cholesterol
metabolism in Golden-Syrian hamsters. Journal of Nutrition 135, 492–498.
Dubuc, G., Chamberland, A., Wassef, H., Davignon, J., Seidah, N.G., Bernier, L., Prat, A.,
2004. Statins upregulate PCSK9, the gene encoding the proprotein convertase
neural apoptosis-regulated convertase-1 implicated in familial hypercholes-
terolemia. Arteriosclerosis, Thrombosis, and Vascular Biology 24, 1454–1459.
Faried, A., Kurnia, D., Faried, L.S., Usman, N., Miyazaki, T., Kato, H., Kuwano, H., 2007.
Anticancer effects of gallic acid isolated from Indonesian herbal medicine, Phale-
ria macrocarpa (Scheff.) Boerl, on human cancer cell lines. International Journal
of Oncology 30, 605–613.
 S.C. Chong et al. / Journal of Ethnopharmacology 137 (2011) 817–827
Goldstein, J.L., Brown, M.S., Anderson, R.G., Russell, D.W., Schneider, W.J., 1985.
Receptor-mediated endocytosis: concepts emerging from the LDL receptor sys-
tem. Annual Review of Cell and Development Biology 1, 1–39.
Jang, A., Srinivasan, P., Lee, N.Y., Song, H.P., Lee, J.W., Lee, M., Jo, C., 2008. Comparison
of hypolipidemic activity of synthetic gallic acid–linoleic acid ester with mix-
ture of gallic acid and linoleic acid, gallic acid, and linoleic acid on high-fat diet
induced obesity in C57BL/6 Cr Slc mice. Chemico-Biological Interactions 174,
109–117.
Kingsley, D.M., Krieger, M., 1984. Receptor-mediated endocytosis of low density
lipoprotein: somatic cell mutants define multiple genes required for expression
of surface-receptor activity. Proceedings of the National Academy of Sciences of
the United States of America 81, 5454–5458.
Krieger, M., 1999. Charting the fate of the “good cholesterol”: identification and
characterization of the high-density lipoprotein receptor SR-BI. Annual Review
Biochemistry 68, 523–558.
Kwon, H.J., Lagace, T.A., McNutt, M.C., Horton, J.D., Deisenhofer, J., 2008.
Molecular basis for LDL receptor recognition by PCSK9. Proceedings of
the National Academy of Sciences of the United States of America 105,
1820–1825.
Landschulz, K.T., Pathak, R.K., Rigotti, A., Krieger, M., Hobbs, H.H., 1996. Regulation
of scavenger receptor, class B, type I, a high density lipoprotein receptor, in
liver and steroidogenic tissues of the rat. The Journal of Clinical Investigation
98, 984–995.
Li, J., Tumanut, C., Gavigan, J.A., Huang, W.J., Hampton, E.N., Tumanut, R., Suen, K.F.,
Trauger, J.W., Spraggon, G., Lesley, S.A., Liau, G., Yowe, D., Harris, J.L., 2007.
Secreted PCSK9 promotes LDL receptor degradation independently of prote-
olytic activity. Biochemical Journal 406, 203–207.
Liang, K., Kim, C.H., Vaziri, N.D., 2005. HMG-CoA reductase inhibition reverses LCAT
and LDL receptor deficiencies and improves HDL in rats with chronic renal fail-
ure. American Journal of Physiology - Renal Physiology 288, F539–F544.
Liao, F.H., Shieh, M.J., Yang, S.C., Lin, S.H., Chien, Y.W., 2007. Effectiveness of a soy-
based compared with a traditional low-calorie diet on weight loss and lipid
levels in overweight adults. Nutrition 23, 551–556.
Lu, C., Hwang, L.S., 2008. Polyphenol contents of Pu-Erh teas and their abilities
to inhibit cholesterol biosynthesis in Hep G2 cell line. Food Chemistry 111,
67–71.
827
Lukeman, A.J.S., Munir, A.B., Olufemi, A.O., Falade, A.K., Remilekun, K.S., Marian, N.B.,
Titilade, A.A., Tayo, A.O., Oladapo, A.A., 2007. Weight reduction with improve-
ment of serum lipid profile of Sesamum radiatum leaves diet in a non-obese
Sprague Dawley rats. African Journal of Biotechnology 6, 2428–2433.
Oshimi, S., Zaima, K., Matsuno, Y., Hirasawa, Y., Iizuka, T., Studiawan, H., Indrayanto,
G., Zaini, N.C., Morita, H., 2008. Studies on the constituents from the fruits of
Phaleria macrocarpa. Journal of Natural Medicine 62, 207–210.
Ow, Y.Y., Stupans, I., 2003. Gallic acid and gallic acid derivatives: effects on drug
metabolizing enzymes. Current Drug Metabolism 4, 241–248.
Pal, S., Ho, N., Santos, C., Dubois, P., Mamo, J., Croft, K., Allister, E., 2003. Red
wine polyphenolics increase LDL receptor expression and activity and suppress
the secretion of ApoB100 from human HepG2 cells. Journal of Nutrition 133,
700–706.
Peterson, A.S., Fong, L.G., Young, S.G., 2008. PCSK9 function and physiology. The
Journal of Lipid Research 49, 1152–1156.
Ringseis, R., Konig, B., Leuner, B., Schubert, S., Nass, N., Stangl, G., Eder, K., 2006.
LDL receptor gene transcription is selectively induced by t10c12-CLA but not by
c9t11-CLA in the human hepatoma cell line HepG2. Biochimica et Biophysica
Acta 1761, 1235–1243.
Savolainen, V., Chase, M.W., Hoot, S.B., Morton, C.M., Soltis, D.E., Bayer, C., Fay, M.F.,
de Bruijn, A.Y., Sullivan, S., Qiu, Y.L., 2000. Phylogenetics of flowering plants
based on combined analysis of plastid atpB and rbcL gene sequences. Systematic
Biology 49, 306–362.
Spady, D.K., Kearney, D.M., Hobbs, H.H., 1999. Polyunsaturated fatty acids up-
regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl
ester uptake in the hamster. The Journal of Lipid Research 40, 1384–1394.
Stancu, C., Sima, A., 2001. Statins: mechanism of action and effects. Journal of Cellular
and Molecular Medicine 5, 378–387.
Suatma, H.A., Widyastuti, N., 2008. Effek toksik buah mahkota dewa (Phaleria macro-
carpa) pada mencit (Mus musculus) swiss webster. Jurnal Biotika 5, 42–48.
Sugiwati, S., Leonardus, B.S.K., Bintang, M., 2006. ␣-Glucosidase inhibitory activity
and hypoglycemic effect of Phaleria macrocarpa fruit pericarp extracts by oral
administration to rats. Journal of Applied Sciences 6, 2312–2316.
Trigatti, B.L., Krieger, M., Rigotti, A., 2003. Influence of the HDL receptor SR-BI on
lipoprotein metabolism and atherosclerosis. Arteriosclerosis, Thrombosis, and
Vascular Biology 23, 1732–1738.