JOURNAL OF MEDICINAL FOOD

J Med Food 14 (3) 2011, 247–256

# Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089=jmf.2009.1389

Turmeric and Laurel Aqueous Extracts Exhibit In Vitro Anti-Atherosclerotic Activity

and In Vivo Hypolipidemic Effects in a Zebrafish Model
Seori Jin,1,* Joo-Heon Hong,2,* Seung-Hyeon Jung,3 and Kyung-Hyun Cho1
1
School of Biotechnology, Yeungnam University; 2Department of Food Science and Technology, Catholic University
of Daegu, Gyeongsan; and 3Ottogi Research Center, Anyang, Republic of Korea

ABSTRACT Culinary herbs and spices have been widely used for their hypoglycemic, lipid-lowering, and anti-
inflammatory activities. This study examined the physiologic activity of hydrophilic components using extracts of turmeric or
laurel leaf powder. Aqueous extracts of turmeric and laurel showed potent inhibitory activity against fructose-mediated
glycation with antioxidant ability against low-density lipoprotein (LDL) oxidation and radical scavenging activity. The
turmeric and laurel extracts had potent cholesteryl ester transfer protein (CETP) inhibitory ability (up to 23% and 40%
inhibition, respectively) at a final concentration of 10 mg=mL. The turmeric and laurel extracts inhibited the cellular uptake of
oxidized LDL into macrophages, which is the initial step in atherogenesis. For in vivo testing, zebrafish consumed a high
cholesterol diet (HCD) (final concentration, 4% [wt=wt]) with or without turmeric or laurel powder (final concentration, 10%
[wt=wt]). The turmeric and laurel groups had a 14% and 12% decrease, respectively, in the weight and height ratios compared
to the HCD group. The plasma total cholesterol level was significantly lower in the turmeric and laurel groups (48% and 28%
less, respectively, than in the HCD group). Plasma triglycerides were more markedly reduced in the turmeric and laurel groups
than in the HCD group (68% and 56% less, respectively, than the HCD group). In conclusion, the hydrophilic extracts of
turmeric and laurel potently suppressed the incidence of atherosclerosis via a strong antioxidant potential, prevention of
apolipoprotein A-I glycation and LDL phagocytosis, and inhibition of CETP. Consumption of turmeric and laurel extracts
exhibited hypolipidemic and antioxidant activities in a hypercholesterolemic zebrafish model.

KEY WORDS:  atherosclerosis  cholesteryl ester transfer protein  laurel  turmeric  zebrafish

INTRODUCTION treatment of gastric diseases.7 Recently, Dall’Acqua et al.7

reported that laurel has antioxidant activity, and Khan et al.8
C ulinary herbs and spices have been the focus of
much attention because they contain high concentra-
tions of bioactive ingredients, which have health-promoting
showed that a 30-day consumption of bay (laurel) leaves (1
or 2 g of ground powder=day) improved glucose levels and
lipid profiles in humans with type 2 diabetes. Although
activities.1 Indeed, herbs and spices are also known as novel
laurel leaf and turmeric extract have been shown to have
antiviral2 and antidiabetic3 agents. Turmeric is a well-known
several beneficial activities in improving carbohydrate and
spice; turmeric extract contains curcuminoids and non-
lipid metabolism, as well as anticancer activity, the studies
characterized substances that have received attention for
have focused on the major ingredient of turmeric (curcu-
their strong chemopreventive ability in recent years.4 Cur-
minoids) or the lyophilized powder of laurel leaf.8 There
cumin is a major ingredient of turmeric and a hyprophobic
have been no reports on the physiologic activity of aqueous
molecule that has been extensively investigated as a potent
extracts of turmeric or laurel.
anticancer5 and cardioprotective6 agent. However, there
To determine the physiologic effect of herbs and spices in
have been no reports on the beneficial activity of curcumin
vertebrates, we used a zebrafish (Danio rerio) model in
with other minor components of turmeric extract.
which hypercholesterolemia was induced by a high cho-
Laurus nobilis L. (laurel) leaves are frequently used as a
lesterol diet (HCD) to mimic early atherogenesis and its
folk medicine in many countries. Infusions of the plant are
complications, as suggested by Stoletov et al.9 By feeding
used in stomach and carminative remedies, as well as the
HCD to adult zebrafish for several weeks, atherosclerosis
was induced, including hypercholesterolemia, lipoprotein
*The first two authors contributed equally to this work. oxidation, and fatty streak formation. We suggest that the
Manuscript received 9 February 2010. Revision accepted 11 August 2010. hypercholesterolemic zebrafish is a useful and highly in-
formative experimental model for atherosclerosis and vas-
Address correspondence to: Kyung-Hyun Cho, Ph.D., School of Biotechnology, Yeung-
nam University, 214-1 Dae-dong, Gyeongsan 712-749, Republic of Korea, E-mail:
cular inflammation.9 Although the zebrafish model system is
chok@yu.ac.kr new for lipid metabolism research compared with other

247
248
vertebrates, there have been increasing numbers of reports
on the use of zebrafish to investigate atherosclerosis,
obesity, and diabetes.10 Those reports have raised the fea-
sibility of using zebrafish as an in vivo system to screen anti-
atherosclerotic phytochemicals.
In the current report we have compared the blood
glucose-lowering and anti-atherosclerotic activities of tur-
meric and laurel in vitro and in vivo using a hypercholes-
terolemic zebrafish model.
MATERIALS AND METHODS
Materials
Culinary turmeric and laurel powders were purchased
from Ottogi Co. (Anyang, Republic of Korea). Each herb
and spice powder (1 g) was extracted in 10 mL of water or
ethanol at ambient temperature for 2 hours and centrifuged
(3,000 g, 10 minutes), the pellet was removed, and the su-
pernatant was lyophilized. The lyophilized extract powders
were weighed and dissolved again with water to obtain a
concentration of 1 mg=mL.
Ingredient analysis
The major ingredients of each extract were analyzed by
gas chromatography=mass spectrometry using an Agilent
(Palo Alto, CA, USA) 6890 (GC=59751 MSD) instrument
equipped with an HP-INNOWAX column (cross-linked
polyethylene glycol, 60 m0.25 mm0.25 mm). In brief,
volatile gas (2 mL) was taken from the headpace sampler at
808C after a 30-minute agitation and injected into the gas
chromatograph. The injection temperature was 2508C. The
helium gas flow rate through the column was 1 mL=minute.
The column temperature was held isothermally at 408C for 2
minutes, then raised at 58C=minute to 2308C, and held
isothermally for 10 minutes. Ions were generated by a 70-kV
electron impact and recorded over the 50–650 m=z mass
range.
Ferric reducing ability of plasma assay
The ferric reducing ability (FRA) was determined using
the method described by Benzie and Strain.11 In brief, the
FRA reagents were freshly prepared by mixing 25 mL of
0.2 M acetate buffer (pH 3.6), 2.5 mL of 10 mM 2,4,6,
tripyridyl-s-triazine (Fluka Chemicals, Buchs, Switzer-
land), and 2.5 mL of 20 mM FeCl3  6H2O. The antioxidant
activities of the water extracts were then estimated by
measuring the increase in absorbance induced by the
generated ferrous ions. The freshly prepared FRA reagent
(300 mL) was mixed with equally diluted extract, which
was dialyzed extensively against phosphate-buffered sa-
line (PBS), after which the FRA was determined by
measuring the absorbance at 593 nm every 20 seconds
over a 10-minute period at 258C using a DU800 spectro-
photometer (Beckman Coulter, Fullerton, CA, USA)
equipped with a MultiTemp III thermocirculator (Amer-
sham, Uppsala, Sweden).
JIN ET AL.
Cupric ion-mediated low-density lipoprotein oxidation
In a 500-mL reaction volume, fresh human low-density
lipoprotein (LDL) (300 mg of protein) was incubated with
each extract (final concentration, 10 mg=mL) in the presence
of 10 mM (final concentration) CuSO4 for up to 2 hours.
During the incubation time, the amount of conjugated dienes
formed was monitored at 234 nm at 24.58C12 using a DU800
spectrophotometer equipped with a MultiTemp III thermo-
circulator. In order to verify the spectroscopic data, the oxi-
dized LDL samples were subjected to electrophoresis on
0.5% agarose gel for a relative electromobility comparison.13
Purification of apolipoprotein A-I
Human plasma was obtained from healthy male volun-
teers at the Blood Bank of Yeungnam University Hospital
(Daegu, Republic of Korea). Apolipoprotein (apo) A-I was
purified from the human high-density lipoprotein (HDL)
fraction (1.063 < d < 1.225) using ultracentrifugation and
column chromatography following the method described by
Brewer et al.14 The purified apoA-I was lyophilized at
808C until use.
Glycation of apoA-I
ApoA-I glycation was determined according to the
method of McPherson et al.,15 with a slight modification
using human apoA-I.16 In brief, the purified lipid-free apoA-
I (10 mg=mL) was incubated with 250 mM d-fructose
(Sigma, St. Louis, MO, USA) in 200 mM potassium phos-
phate=0.02% sodium azide buffer (pH 7.4) for up to 90
hours under gas with air containing 5% CO2. Glycated
apoA-I samples were dialyzed extensively against PBS and
sterilized by filtration. The extent of glycation and its inhi-
bition by treatment with extract were determined from
reading the fluorometric intensity (FI) at 370 nm (excitation)
and 440 nm (emission) using the following formula, as de-
scribed previously:16,17
% inhibition ¼
 
sample FI  background FI
100 · 1 
negative control FI  background FI
where sample FI ¼ apoA-I (10 mg=ml; 82 mL) þ fructose
(final concentration, 250 mM; 8 mL) þ extract in ethanol or
water (final concentration, 10 or 100 mg=mL; 10 mL),
background FI ¼ apoA-I, and negative control FI ¼ apoA-
I þ fructose þ water or ethanol.
Purification of LDL and its oxidation
LDL (1.019 mg=dL < d < 1.063 mg=dL) was purified
from human plasma, which was obtained from the Blood
Bank of Yeungnam University Medical Center, by ultra-
centrifugation according to a standard protocol.18 Oxidized
LDL (oxLDL) was produced by incubating an LDL
(1.019 mg=dL < d < 1.063 mg=dL) fraction with CuSO4
(final concentration, 10 mM) for 4 hours at 378C. The oxLDL
 HYPOLIPIDEMIA AND 1,8-CINEOLE ENRICHMENT
was then filtered (pore size, 0.2 mm) and analyzed using a
thiobarbituric acid-reactive substances assay to determine
the extent of oxidation, as described previously.19
Cholesteryl ester transfer assay
A reconstituted HDL as a cholesteryl ester (CE) donor
containing apoA-I and cholesteryl oleate was synthesized in
accordance with the method described by our research
group20,21 with trace amounts of [3H]cholesteryl oleate
(TRK886, 3.5 mCi=mg of apoA-I; GE Healthcare, Boston,
MA, USA). Human plasma (0.05 mL) and LDL (0.05 mL,
0.25 mg=mL) were used as CE transfer protein (CETP) and
CE acceptor sources, respectively. Each equally diluted
extract (final concentration, 0.01 mL) was used as a CETP
inhibitor. CETP inhibition was calculated as follows:
 
sample cpm  blank cpm
% inhibition ¼ 100 · 1 
control cpm  blank cpm
where sample is extract treated as an inhibitor source, con-
trol is without inhibitor, and cpm is counts per minute.
Anti-atherosclerotic assay
THP-1 cells, a human monocytic cell line, were main-
tained and differentiated into macrophages as described
previously.21
The differentiated and adherent macrophages were then
rinsed with warm PBS and incubated with 400 mL of fresh
RPMI-1640 medium containing 1% fetal bovine serum,
50 mL of oxLDL (1 mg of protein=mL in PBS), and 25 mL of
distilled water or each extract (final concentration,
10 mg=mL) for 48 hours at 378C in a humidified incubator.
After incubation, the cells were washed with PBS three
times and then fixed in 4% paraformaldehyde for 10 min-
utes. Next, the fixed cells were rinsed with 100% polypro-
pylene glycol, stained with Oil red O staining solution
(0.67%), and then washed with distilled water. THP-1
macrophage-derived foam cells were then observed and
photographed using a Nikon (Tokyo, Japan) Eclipse
TE2000 microscope at 600 magnification.
In vivo test using hypercholesterolemic zebrafish
Zebrafish maintenance and procedures were approved by
the Committee of Animal Care and Use of Yeungnam
University (Gyeongsan, Republic of Korea). HCD contain-
ing 4% cholesterol (final concentration) was made by
soaking Tetrabit (47.5% crude protein, 6.5% crude fat,
2.0% crude fiber, 10.5% crude ash, 29,770 IU=kg vitamin A,
1,860 IU=kg vitamin D3, 200 mg=kg vitamin E, and
137 mg=kg vitamin C; Tetra GmbH, Melle, Germany) in a
diethyl ether solution of cholesterol (catalog number C-
3045, Sigma). After ether evaporation, the HCD was mixed
with powder of turmeric or laurel (final concentration, 10%
[wt=wt]). Each group (n ¼ 80) consumed the designated diet
(20 mg=day per fish). The zebrafish were maintained at
28  18C under a 14:10-hour light:dark cycle.
249
After feeding for 5 weeks, blood (2 mL) was drawn from
the hearts of adult fish, combined with 5 mL of PBS-EDTA
(final concentration, 1 mM), and then collected in EDTA-
treated tubes. The plasma (40 mL) was obtained by centri-
fugation after pooling the blood from 10 zebrafish. Plasma
total cholesterol (TC), HDL-cholesterol, and triglycerides
(TG) were determined using commercial assay kits (Wako
Pure Chemical, Osaka, Japan). Glutamic oxaloacetic trans-
aminase (GOT) and glutamic pyruvic transaminase (GPT)
concentrations were measured using a commercially avail-
able assay kit (Asan Pharmaceutical, Hwasung, Republic of
Korea). The CETP activity and FRA of zebrafish plasma
were compared between the groups. Aliquots of hepatic
tissue (50 mg of liver in 0.5 mL of PBS) from each group
were homogenized for 3 minutes (150 rpm) in an ice bath
using a tissue homogenizer (Euro-ST, Eurostar, IKA-
Werke, Staufen, Germany). After a brief centrifugation
(10,000 g) and protein determination using the Bradford
reagent (Bio-Rad, Hercules, CA, USA), equally diluted
supernatants (0.05 mL) were used as an antioxidant source
in the FRA assay.
Enzyme-linked immunosorbent assay
For the sandwich enzyme-linked immunosorbent assay
(ELISA) for the plasma interleukin-6 level, diluted primary
antibody (1:2,000) was coated onto a 96-well plate (Max-
isorp 439454, Nunc, Roskilde, Denmark) overnight at 48C.
As primary antibodies, mouse interleukin-6 monoclonal
antibody (sc53865, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA) was used. After incubation for 2 hours with
the primary antibody, the well was washed extensively with
PBS containing 0.1% Tween-20. Then, the well was incu-
bated with rabbit polyclonal antibody (diluted 1:2,000)
(sc7920, Santa Cruz Biotechnology) as secondary antibody
for 2 hours. Horseradish peroxidase-conjugated rabbit im-
munoglobulin G antibody (sc2004) was purchased from
Abcam (Cambridge, UK) and used as the tertiary antibody.
A substrate reagent pack (DY999, R&D Systems, Minnea-
polis, MN, USA) was used for color development. Equally
diluted (20-fold) plasma samples (20 mL) were used.
Statistical analysis
All data are expressed as mean  SD values from at least
three independent experiments with duplicate samples. The
data were evaluated via one-way analysis of variance using
SPSS version 14.0 software (SPSS, Chicago, IL, USA), and
the differences between the means were assessed using
Duncan’s multiple-range test. Statistical significance was
defined as P < .05.
RESULTS
Ingredient analysis
The major component in the aqueous extract of tumeric
was 1,8-cineole (approximately 67% of the total); minor
components were tumerone, curcumene, and cymene, as
250 JIN ET AL.

shown in Table 1. In the water extract of laurel, 1,8-cineole Antioxidant assay

(65% of the total) was also the major ingredient, and carene,
Based on the FRA assay, the water extract of laurel (final
terpinene, and cymene were detected as minor ingredients.
concentration, 10 mg=mL) had the most potent ferric ion
Based on these results, the same compound appeared as the
reducing ability (up to 150%) based on an increase in ab-
main compound in turmeric and laurel by short-term water
sorbance at 593 nm, as shown in Figure 2A. The same
extraction to mimic the physiologic digestion and absorp-
amount of water extract of turmeric had a weaker FRA
tion process in the human body.
(approximately 126% increase of absorbance at 593 nm) at
the same concentration. The laurel extract (final concen-
Inhibitory activity against glycation
tration, 10 mg=mL) showed the strongest 2,2-diphenyl-1-
The water extracts of turmeric or laurel (final concen- picrylhydrazyl radical scavenging activity (up to a 56%
tration, 10 mg=mL) showed adequate antiglycation activity decrease) in absorbance at 515 nm, while turmeric showed
(32–35% inhibition) after 48 hours of incubation based on an approximate 20% decrease (Fig. 2B) at the same con-
the fluorescence observation (excitation at 370 nm, emission centration. These results suggest that the water extract of
at 440 nm), as shown in Figure 1A. The inhibition potential laurel had a stronger activity than that of a reducing agent
was maintained at 96 hours of incubation (65% and 81% and radical scavenger from the in vitro assay.
inhibition for turmeric and laurel, respectively, at a final
concentration of 100 mg=mL). The laurel extract showed Inhibition against LDL oxidation
more potent inhibition against glycation following a longer
incubation. The electrophoretic pattern of glycated apoA-I Human LDL is sensitive to oxidative stress by treatment
(Fig. 1B) showed more multimerized bands (up to tetramers; with cupric ion, as shown in Figure 3A. Treatment with
lanes 1 and 2), while native apoA-I (lanes 3 and 4) had laurel extract (final concentration, 10 mg=mL) exhibited the
virtually no multimers. However, multimerization was de- strongest ability to suppress cupric ion-mediated LDL oxi-
creased in the presence of turmeric or laurel extract (final dation (up to 50 minutes). Monitoring of conjugated diene
concentration, 10 mg=mL), as shown in Figure 1B (lanes 5– levels (absorbance at 234 nm) was increased (up to 109%
8). The water extract of laurel-treated apoA-I had the lowest from the initial absorbance) by the addition of cupric ion,
extent of multimeric formation (lane 8). whereas native LDL had a 13% increase, as shown in Figure
3A. Laurel or turmeric extract (final concentration,
10 mg=mL) had adequate inhibitory activity (approximately
50% and 27% increase, respectively, in absorbance at
234 nm).
The electromobility of LDL on agarose gel (0.5%) sup-
Table 1. Ingredient Analysis of Water Extract of Turmeric
and Laurel Using Gas Chromatography=Mass Spectrometry
ports the results from the conjugated diene production assay.
More oxLDL moved faster to the bottom of the gel than did
Extract, component Peak area Peak % RT (minutes) the less oxLDL or native LDL. The oxLDL has an increased
Turmeric
negative charge and a reduced size resulting from apo-B
1,8-Cineole 96.5 67.7 12.7 fragmentation,22 as shown in Figure 3B. Electrophoretic
Unknown 15.6 11.0 33.6 migration revealed that the laurel extract (final concentra-
Unknown 7.7 5.4 12.8 tion, 10 mg=mL)-treated LDL had the slowest migration
Ar-tumerone 4.6 3.2 37.1 with the strongest band intensity (Fig. 3B, lane 4), whereas
Ar-curcumene 3.9 2.7 27.2 the turmeric extract-treated LDL had faster mobility with a
Unknown 2.6 1.8 33.5 weaker band intensity. This result was in good agreement
p-Cymene 2.1 1.5 29.6
b-Sesquiphellandrene 1.7 1.2 27.2 with the monitoring of conjugate diene levels with a de-
Zingiberene 1.3 0.9 26.1 crease at absorbance at 234 nm and the slowest electro-
a-Terpinolene 1.2 0.8 25.5 mobility.
Laurel
1,8-Cineole 241.7 65.4 12.7
g-Terpinene 30.8 8.2 23.3
CETP inhibitory activity
2-Carene 27.1 7.4 25.5 As shown in Figure 4A, laurel extract, in a concentration-
d-3-Carene 17.1 4.63 21.8 dependent manner, had the strongest CETP inhibitory ac-
Unknown 7.3 1.99 12.8
p-Cymene 6.1 1.66 14.3
tivity in vitro (up to 18% and 40% inhibition at final con-
g-Camphene 4.4 1.2 24.9 centrations of 5 and 10 mg=mL, respectively). The turmeric
a-Terpinene 4.0 1.0 11.5 extract also had potent inhibitory activity (14% and 23%
Unknown 3.2 0.8 12.1 inhibition at final concentrations of 5 and 10 mg=mL, re-
spectively). After feeding HCD for 5 weeks, as described
Gas chromatography=mass spectrometry was performed using an Agilent
6890 (GC=59751 MSD) instrument equipped with an HP-INNOWAX column
in Table 2, the CETP activity of zebrafish plasma was
(cross-linked polyethylene glycol, 60 m0.25 mm; particle size, 0.25 mm). The increased up to 55  4% CE transfer, while the normal diet-
first component is the largest peak. fed group had 43  3% CE transfer, suggesting that
Ar, aromatic; RT, retention time. plasma CETP activity was enhanced by HCD consumption.
 FIG. 1. Inhibitory activity against glycation of
apolipoprotein (apo) A-I by extract of turmeric
and laurel using ethanol (Et-OH) or water
(H2O). (A) Antiglycation activity determined
from fluorospectroscopic analysis (excitation at
370 nm, emission at 440 nm). Inhibition per-
centage was calculated as described in Materials
and Methods. Data are mean  SD values from
three independent experiments performed in
duplicate. (B) Electrophoretic patterns of
glycated apoA-I (12% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis [SDS-
PAGE]) in the presence of (final concentration,
10 mg=mL) either water (lanes 6 and 8) or Et-OH
(lanes 5 and 7) extracts. Lane M, molecular
weight standard (low range; Bio-Rad).

FIG. 2. In vitro antioxidant ability of water extracts. (A) Comparison of reduction potential determined from ferric ion reduction ability between
turmeric and laurel extracts. (B) Radical scavenging activity determined from 2,2-diphenyl-1-picrylhydrazyl removal assay. Data are mean  SD
values from three independent experiments performed in duplicate. Abs, absorbance.

251
252 JIN ET AL.

FIG. 3. Inhibition ability of cupric ion-mediated low-density lipoprotein (LDL) oxidation. (A) Continuous monitoring of conjugate diene level
by absorbance at 234 nm during copper-mediated oxidation in the presence of each extract of herbs and spices (final concentration, 10 mg=mL).
Data are mean  SD values from three independent experiments performed in duplicate. (B) Relative electrophoretic mobility profiles of the LDL
(15 mg of protein) samples from the copper-mediated oxidation in the presence of each extract (final concentration, 10 mg=mL): lane 1, native
LDL; lane 2, LDL þ Cu2þ; lane 3, LDL þ Cu2þ þ turmeric; lane 4, LDL þ Cu2þ þ laurel.

However, the turmeric- and laurel-fed groups had lower
plasma CETP activity, with 47  5% and 37  4% CE
transfer, respectively, indicating that the in vitro inhibition
ability of turmeric and laurel was consistent with decreased
CE transfer activity in vivo.
Cellular uptake of oxLDL into macrophages
To determine whether or not the spice and herb could
inhibit the cellular uptake of oxLDL into macrophages, the
THP-1 cells were stained with Oil red O to evaluate
the degree of lipid or LDL uptake into cells that occurred in
the presence of either turmeric or laurel after 48 hours of
incubation. When compared to PBS-treated cells (Fig. 5A)
as a control, oxLDL-treated cells (Fig. 5B) had a much
greater lipid-stained area with a darker red intensity. In the
presence of the same amount of oxLDL, turmeric- or laurel-
treated cells (final concentration, 5 mg=mL) had a much
smaller lipid-stained area and weaker intensity (Fig. 5C and
D, respectively). This result suggests that the putative in-
gredient in the extract could inhibit the early steps of foam
cell production, although turmeric extract-treated cells had
the lowest red intensity.
Hypolipidemic effect of turmeric in zebrafish
As shown in Table 2, the body weight of the HCD group
was increased by 11% compared to the normal group after 5
weeks of feeding 4% cholesterol in the normal diet; the
plasma TC and TG levels of the HCD group were elevated

FIG. 4. Plasma cholesteryl ester transfer protein assay. (A) Cholesteryl ester transfer from 3H-high-density lipoprotein to LDL by human plasma
was inhibited by each water extract. (B) Cholesteryl ester (CE) transfer assay with pooled zebrafish plasma (0.02 mL) from each group. Data are
mean  SD values from three independent experiments performed in duplicate. HC, high cholesterol diet group.
 HYPOLIPIDEMIA AND 1,8-CINEOLE ENRICHMENT 253

Table 2. Blood Profiles of Hypercholesterolemic Zebrafish After 5 Weeks of Feeding

Body weight Height Weight=height TC TG Glucose GOT GPT
Group* (mg) (cm) (mg=cm) (mg=dL) (mg=dL) (mg=dL) (U=L) (U=L) IL-6 (AU)
a b a a a a a a
Normal (n ¼ 80) 560  154 38.6  2.51 14.5  3.2 168  14 123  18 47  13 172  10 24  4 0.483  0.069a
HCD (n ¼ 80) 625  143b 36.9  2.75a 16.9  3.4b 586  35d 368  45c 80  2b 290  23c 52  5c NA
Turmeric (n ¼ 80) 575  147ab 38.4  2.04b 14.8  3.2a 304  25b 118  15a 31  8a 212  16b 34  5b 0.278  0.122b
Laurel (n ¼ 80) 579  156ab 37.9  2.23b 15.1  3.5a 423  29c 163  35b 46  13a 217  36b 62  15c 0.401  0.155ab
*Normal animals were fed Tetrabit (47.5% crude protein, 6.5% crude fat, 2.0% crude fiber, and 10.5% crude ash, containing vitamin A [29,770 IU=kg], vitamin D3
[1,860 IU=kg], vitamin E [200 mg=kg], and vitamin C [137 mg=kg]). HCD (high cholesterol diet) animals received 4% cholesterol (final) in the normal diet.
Turmeric and laurel animals were fed diet with the additional component as the final 10% of powder in the HCD (wt=wt).
abc
Means in the same column not sharing a common superscript are significantly different (P < .05) between groups.
AU, arbitrary units; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; IL-6, interleukin-6; NA, not available due to shortage of
samples; TC, total cholesterol; TG, triglycerides.

3.5- and threefold higher, respectively, than the normal
group. However, in contrast to the increase, the height was
significantly decreased (up to 4%) compared to the normal
group (P < .002). The weight-to-height (in mg=cm) ratio
was increased 16% in the HCD group compared to the
normal group. This result suggests that HCD might con-
tribute to an increase in body weight, rather than height. The
plasma glucose level was also elevated by the HCD (up to
1.7-fold compared to the normal group). The blood GOT
and GPT levels were elevated after the HCD diet (1.7- and
2.2-fold, respectively) compared with the normal group.
These results indicate that increased cholesterol uptake
might be connected to an increase in plasma blood glucose
and inflammatory stress in the liver.
The turmeric-supplemented group had a 14% decrease in
the weight-to-height ratio compared to the HCD group,

FIG. 5. Cellular uptake of oxidized LDL (B) alone or treated with (C) turmeric or (D) laurel extract (final concentration, 5 mg=mL) compared
with (A) phosphate-buffered saline-treated cells. The extent of cellular uptake of lipids or LDL by macrophages was then compared by Oil red O
staining, as described in the text. Color images available online at www.liebertonline.com=jmf.
254
while the laurel group had a 12% decrease, even though the
height was increased after 5 weeks of feeding (Table 2).
The plasma TC level was significantly lowered in the tur-
meric and laurel groups (48% and 28% less, respectively,
than the HCD group). The plasma TG level was more mark-
edly reduced in the turmeric group than in the laurel group
(68% and 56% less, respectively, than the HCD group).
Furthermore, the blood glucose level was 42–62% de-
creased in the turmeric and laurel groups compared to the
HCD group. The tumeric group had a 27% and 35% de-
crease in GOT and GPT, respectively. However, the laurel
group had an elevated GPT level compared to the HCD
group. Based on the ELISA determination, the plasma in-
terleukin-6 level of zebrafish (0.483 arbitrary units) was
higher than human plasma (0.186 arbitrary units). Con-
sumption of turmeric and laurel caused a decrease in the
plasma level (up to 43% and 18%), as shown in Table 2.
These improvements in plasma lipids, glucose, and inflam-
matory parameters suggest that consumption of turmeric
and=or laurel leaf powder is beneficial in treating metabolic
syndrome, which is associated with type 2 diabetes and
cardiovascular disease.
Antioxidant activity of zebrafish plasma and liver tissue
The FRA assay with pooled plasma of each group (20 mL)
revealed that 5 weeks of feeding an HCD enhanced anti-
oxidant ability with a 25% increase in absorbance at 593 nm,
whereas the normal group had an 8% decrease. Plasma from
the turmeric extract-fed group had the highest antioxidant
ability, with up to a 77% increase in absorbance at 593 nm,
whereas the laurel-fed group had an 18% increase.
Using homogenized hepatic juice (50 mL, 1.4 mg=mL),
the HCD-fed group had more enhanced antioxidant ability
with a 48% increase in absorbance at 593 nm, whereas the
normal diet-fed group had a 25% increase. The turmeric-
and laurel-fed groups had 61% and 58% increases, respec-
FIG. 6. Comparison of antioxidant ability between the treatment groups of zebrafish. (A) Ferric ion removal ability of plasma from each group.
(B) The homogenized hepatic tissue (50 mg of liver in 0.5 mL of phosphate-buffered saline [PBS]) equally diluted (1.4 mg=mL) from each group
was used as an antioxidant source. Data are mean  SD values from three independent experiments performed in duplicate.
JIN ET AL.
tively, in absorbance at 593 nm. These results suggest that
the antioxidant ability of hepatic tissue was enhanced by the
consumption of turmeric and laurel extracts, which are rich
in 1,8-cineole.
DISCUSSION
The current study was designed to compare the physio-
logic activity of hydrophilic components of extracts of
turmeric and laurel leaf powder. Herbs and spices, includ-
ing turmeric and laurel, are used widely in Ayurvedic
medicine to treat cancer and metabolic disorders. The
physiologic effects of turmeric have focused on antitumor
activity.23 Curcumin, a yellow pigment substance and
component of turmeric, has been reported to have thera-
peutic potential in cancer chemotherapy. However, the
water-extracted fraction in this study did not contain cur-
cumin (Table 1) because it is a hydrophobic substance.
Therefore, the results of the current study provide new in-
sight into the physiologic role for hydrophilic ingredients of
turmeric and laurel.
In the water extract of turmeric and laurel, 1,8-cineole
was the major ingredient (Table 1) and showed potent in-
hibitory activity against fructose-mediated glycation (Fig.
1). These results correlate well with fluorescence observa-
tions (Fig. 1A) and suggest that both turmeric and laurel
extracts protect apoA-I from nonenzymatic glycation and
multimerization. Although there have been many reports
concerning the glycation effect of HDL from human plas-
ma,24 relatively few articles have been published regarding
the physiologic effect of apoA-I glycation. Our research
group recently reported structural and functional changes of
glycated apoA-I in lipid-free and lipid-bound states.25 By
glycation, apoA-I lost several beneficial functions, including
lipid-binding ability, ferric ion removal ability, and anti-
oxidant ability against copper-mediated LDL oxidation. The
glycated apoA-I facilitated cellular uptake of oxLDL into
 HYPOLIPIDEMIA AND 1,8-CINEOLE ENRICHMENT
macrophage cells and premature senescence in human der-
mal fibroblast cells.
The laurel extract had more potent ferric ion reducing
ability and radical scavenging activity than turmeric extract
(Fig. 2). The turmeric and laurel extracts exhibited hypolipi-
demic and anti-obesity activities in a hypercholesterolemic
zebrafish model. However, the turmeric-fed group had
stronger plasma antioxidant ability (Fig. 6A) with lower
plasma TC and TG levels (Table 2). The laurel extract
had better in vitro activities, while the turmeric group had
better in vivo activities. These differences in activity between
in vitro and in vivo assays may have originated from the
difference in water extract treatment in the in vitro assays and
powder consumption in the zebrafish model as an in vivo
assay.
An increase in FRA potential in the HCD group was
another interesting finding (Fig. 6) because humans with
hypercholesterolemia generally have decreased FRA po-
tential.26 This difference between humans and zebrafish can
be attributed to differences in the lipoprotein system. Ap-
proximately 75% of serum cholesterol is carried by LDL in
humans, whereas fish have an HDL-dominant lipoprotein
profile.27 In zebrafish, an HCD might cause an increase in
HDL-cholesterol, which is more of an increase in antioxi-
dant ability. Similarly, our group has previously reported
that FRA potential is more enhanced in hypercholester-
olemic mice than normolipidemic mice;28 mice also have an
HDL-dominant lipoprotein profile.
In agreement with our current results, 1,8-cineole from
Eucalkyptus tereticornis also exhibits potent antioxidant
activity, such as free radical (2,2-diphenyl-1-picrylhydrazyl
radical, OH  , O2) scavenging activity.29 The extraction
was carried out with a water-based solvent; 1,8-cineole was
detected as a major compound from turmeric and laurel.
Curcuminoid was not detected as a major band in the water
extract because it is lipid-soluble. In contrast to the laurel
extract, turmeric extract contained turmerone and curcu-
mene as minor ingredients (Table 1).
In conclusion, in the current study water extracts of tur-
meric and laurel, which were rich in 1,8-cineole, showed
potent antiglycation, antioxidant, and anti-atherosclerotic
activities. The laurel extract showed more potent in vitro
antioxidant ability, enhanced FRA (Fig. 2A) and radical
scavenging activity (Fig. 2B), and suppressed LDL oxida-
tion and cellular uptake of oxLDL. In vivo testing using
zebrafish showed that the turmeric-fed group had more po-
tent hypoglycemic and hypolipidemic effects (Table 2) with
stronger plasma antioxidant ability (Fig. 6). The consump-
tion of turmeric and laurel powder for 5 weeks also en-
hanced antioxidant and anti-inflammatory activities in
zebrafish plasma. Additional studies are necessary to de-
termine the in vivo activity of 1,8-cineole and curcumin to
compare their physiologic roles in the animal model.
ACKNOWLEDGMENT
This research was supported by Yeungnam University
research grants in 2009.
255
AUTHOR DISCLOSURE STATEMENT
All authors declare no competing financial interests exist.
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