Curcuma longa Extract Supplementation Reduces Oxidative Stress and

Attenuates Aortic Fatty Streak Development in Rabbits

José L. Quiles, M. Dolores Mesa, César L. Ramírez-Tortosa, Concepción M. Aguilera,
Maurizio Battino, Ángel Gil and M. Carmen Ramírez-Tortosa

Arterioscler Thromb Vasc Biol 2002, 22:1225-1231: originally published online May
2, 2002
doi: 10.1161/01.ATV.0000020676.11586.F2
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.
7272 Greenville Avenue, Dallas, TX 72514
Copyright © 2002 American Heart Association. All rights reserved. Print ISSN: 1079-5642. Online
ISSN: 1524-4636

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 Curcuma longa Extract Supplementation Reduces Oxidative
Stress and Attenuates Aortic Fatty Streak
Development in Rabbits
José L. Quiles, M. Dolores Mesa, César L. Ramírez-Tortosa, Concepción M. Aguilera,
Maurizio Battino, Ángel Gil, M. Carmen Ramírez-Tortosa

Objective—This study evaluates the effect of a Curcuma longa extract on the development of experimental atherosclerosis
(fatty streak) in rabbits and its interaction with other plasmatic antioxidants.
Methods and Results—Two experimental groups of male New Zealand White rabbits, a control group and a
curcuma-extract (CU) group, were fed an atherogenic diet. Additionally, the CU group received an oral curcuma
hydroalcoholic extract. Six animals from each experimental group were killed after 10, 20, and 30 days. Compared with
the CU group, the control group showed significantly higher plasma lipid peroxide at all experimental times (10, 20, and
30 days) and significantly lower ␣-tocopherol and coenzyme Q levels at 20 and 30 days. Histological results for the fatty
streak lesions revealed damage in the thoracic and abdominal aorta that was significantly lower in the CU group than
in the control group at 30 days.
Conclusions—Supplementation with Curcuma longa reduces oxidative stress and attenuates the development of fatty
streaks in rabbits fed a high cholesterol diet. (Arterioscler Thromb Vasc Biol. 2002;22:1225-1231.)
Key Words: Curcuma longa 䡲 antioxidants 䡲 aorta 䡲 fatty streak 䡲 atherosclerosis 䡲 rabbits

A plant of Indian origin, Curcuma longa L (Zingiber-

aceae) has a rhizome of bright orange color under a fine
light brown cell layer. It is in common use as a spice in Asian
ides, molecules that play an important role in the pathogen-
esis of the disease.6 Curcumin also has different properties
that contribute to combat this disease: it reduces the suscep-
cultures, where it is considered to be a magical plant because tibility of LDL to oxidation,7 inhibits the proliferation of
of its organoleptic properties and undoubted therapeutic and vascular smooth muscle cells,8 has an antithrombotic effect,
protective effects, especially for the skin and liver.1 has a transient hypotensive effect, and inhibits platelet aggre-
Since ancient times, many properties have been ascribed to gation in vivo and ex vivo.9
extracts of Curcuma longa. The plant has been applied for the Sreejayan et al10 claimed that the presence of phenolic
prevention and cure of skin and hepatic conditions and of groups in the structure of curcumin are fundamental in
ulcers and digestive disorders. It has also been used in the explaining its ability to eliminate oxygen-derived free radi-
treatment of intestinal parasites and as a remedy for poison- cals from the medium largely responsible for the peroxidation
ing, snakebites, and various other complaints.2
of cell lipids.11 They are able mainly to eliminate the
Many studies have shown the capacity of curcumin to
hydroxyl radical,12 superoxide radical,13 singlet oxygen,14
prevent lipid peroxidation, a key process in the onset and
nitrogen dioxide,15 and NO.16 It has also been demonstrated
progression of many diseases. The capacity of curcumin to
that curcumin inhibits the generation of the superoxide
stabilize membranes has also been demonstrated.3 Our re-
search group has found that a dose of 2.4 to 9.6 ␮mol/L radical.17
curcumin inhibits the oxidation of human LDL in vitro.4 Despite all these positive effects attributed to curcumin, to
Venkatesan5 observed a protective effect of curcumin against date, there have been no time-course studies with this extract
the cardiotoxicity produced by adriamycin in rats, showing a investigating the development of fatty streak lesions in the
reduction in the parameters that indicate lipid peroxidation. atherosclerotic process. Thus, the aim of the present study
Atherosclerosis is a multifactorial disease in which a major was to evaluate the effect of our curcumin extract on the
alteration of the vascular lipid metabolism is produced. It has development of experimental atherosclerosis (fatty streaks) in
been observed that curcumin reduces plasmatic lipid perox- rabbits and its interaction with other plasmatic antioxidants.

Received February 4, 2002; revision accepted April 18, 2002.

From the Department of Physiology (J.L.Q.) and the Department of Biochemistry and Molecular Biology (M.D.M., C.M.A., Á.G., M.C.R.-T.), Granada
University, and the Institute of Nutrition and Food Technology (J.L.Q., M.D.M., C.M.A., Á.G., M.C.R.-T.), University of Granada, Granada, Spain; Jaén
City Hospital (C.L.R.-T.), Jaén, Spain; and the Institute of Biochemistry (M.B.), Faculty of Medicine, University of Ancona, Ancona, Italy.
Correspondence to Dr M. Carmen Ramírez-Tortosa, Instituto de Nutrición y Tecnología de Alimentos, Universidad de Granada, C/Ramón y Cajal 4,
18071 Granada, Spain. E-mail [email protected]
© 2002 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000020676.11586.F2

1225
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1226 Arterioscler Thromb Vasc Biol. July 2002

cholesterol (TC) levels in plasma were measured by using a

commercially available kit (Boehringer-Mannheim).

Plasma Lipid Peroxidation

The lipid peroxide content of the plasma was determined as
thiobarbituric acid–reactive substances (TBARS). Plasma samples
were combined with a 1-mL mixture of trichloroacetic acid (15%
[wt/vol]), thiobarbituric acid (0.37%), and hydrochloric acid (25
mol/L) and vortexed, as described by Buege and Aust.19 Results
Figure 1. Main curcuminoids of Curcuma longa extract. were expressed as nanomoles of TBARS per milliliter of plasma,
with 1,1,3,3-tetramethoxypropane used as a standard.

Such a study could determine the utility of the extract in Determination of LDL Oxidation Susceptibility
patients with peripheral vascular disease. LDL was isolated by a single discontinuous density-gradient ultra-
centrifugation in a vertical rotor as described previously.20 Total
Methods LDL cholesterol level was measured by using a commercially
available kit (Boehringer-Mannheim). LDL protein was measured by
Animals and Diets the Bradford method, with BSA used as a standard.21 To study the
Forty-two male New Zealand White rabbits (University of Granada susceptibility to oxidation of LDL, 2 determinations (TBARS and
Laboratory Animals Service, Granada, Spain), weighing 2500 g and conjugated dienes) were performed. Two hundred milligrams of
kept 1 per cage, were put on a 12-hour light/12-hour dark cycle with LDL protein per liter was oxidized in the presence of 25 ␮mol/L
free access to food (150 g/d) and water. All the animals were fed Cu2⫹ in PBS for 6 hours at 37°C. After incubation, oxidation was
rabbit chow for 10 days. At this point, 6 animals were killed as a terminated by cooling the samples to 4°C and adding 100 mmol/L
baseline group. The remaining animals were divided into 2 experi- EDTA and 4.5 ␮mol/L butylated hydroxytoluene.22 The lipid-
mental groups: the control group and the curcuma extract (CU) peroxide content of oxidized LDL was determined as TBARS,
group. Both groups were fed an atherogenic diet (Abbott Laborato- according to Buege and Aust19 (1978). Absorbance units were
ries SA) containing 95.7% standard chow, 3% lard, and 1.3% converted to malondialdehyde equivalents per milligram LDL pro-
cholesterol to provoke an atherosclerotic process.18 Additionally, the tein with the use of a standard curve obtained with 1,1,3,3-
CU group received an oral hydroalcoholic extract of curcuma at a tetramethoxypropane. Conjugated dienes in LDL was carried out
dosage of 1.66 mg/kg body wt, and the control group received an oral according to Puhl et al23 (1989) in a Perkin-Elmer UV-VIS Lambda
curcuma-free hydroalcoholic solution daily during the experiment. 40 spectrometer equipped with an auto-cell holder and controlled by
The atherogenic meal was kept in darkness at 4°C to avoid a Peltier element at a temperature of 37°C. Twenty milligrams of
peroxidation until use. During the study, 6 animals from each LDL protein per liter was oxidized in presence of Cu2⫹ (5 ␮mol/L)
experimental group were killed after 10, 20, and 30 days, respec- in PBS. LDL-conjugated dienes were measured every 2 minutes at
tively. The animals were clinically observed and weighed weekly. 234 nm for 60 minutes at 37°C. Results are expressed as nanomoles
The University of Granada Ethics Committee approved the present of conjugated dienes per milligram LDL protein.
study, and the animals were handled according to the guidelines of
the Spanish Society for Laboratory Animal Sciences for the care and
use of laboratory animals.
Coenzyme Q10, Retinol, and
␣-Tocopherol Determinations
Extraction and Curcuminoids Composition of Analyses of coenzyme Q10, retinol, and ␣-tocopherol in plasma were
assayed according to the method of MacCrehan24 by reversed-phase
Curcuma longa HPLC with a Spherisorb S5 ODS1 (Merck, Darmstadt, Germany)
The hydroalcoholic extract of curcuma was provided by A.S.A.C. column and ethanol/purified water (97:3 [vol/vol]) used as the
Pharmaceutical International A.I.E. For the extraction, the rhizome mobile phase. The HPLC system was a Beckman In-line Diode
of Curcuma longa was macerated with hot water (80°C) for 4 hours, Array Detector (model 168) connected to a Water 717 Plus Autosam-
and the aqueous extract was evaporated under vacuum at 60°C. The pler (Milford). Coenzyme Q10, retinol, and ␣-tocopherol were iden-
rhizome residue was reextracted with ethanol at 60°C for 2 hours, tified by predetermining the retention times of individual pure
filtered, and evaporated under vacuum. The final extract is a 1:1 standards.
mixture of both the aqueous extract and the alcoholic extract, which
are redissolved with water and alcohol, respectively. Curcuminoids
composition of the extract was carried out by high-performance
Histological Analysis of Aortic
liquid chromatography (HPLC) with a Beckman In-line Diode Array Atherosclerotic Lesions
Detector (model 168) and a Supelcosil LC-18 column Entire aortas were rapidly dissected out, and ⬇1 cm of the aortic arch
(150 mm⫻4.6 mm and 5 ␮m, Supelco). The mobile phase was 0.05 and of the thoracic and abdominal aortas was selected and standard-
mol/L sodium acetate and 55:45 (vol/vol) acetonitrile (pH 4.2 at 1 ized for all the animals. For histological examinations, buffered 4%
mL/min flow). Bis-demethoxy-curcumin, demethoxy-curcumin, and formaldehyde-fixed paraffin-embedded tissue sections were stained
curcumin, the curcuminoids with the highest biological activity with hematoxylin and eosin (H&E), Masson-Goldner trichrome, and
present in the extract, were identified by using pure curcumin as a van Gieson’s elastin stains. Samples were examined in a blinded
standard (Sigma Chemical Co) with a molar absorbance of 1607 in manner to evaluate the presence of the fatty streak. Lesions were
ethanol. Total concentration of curcuminoids in the curcuma extract scored on a 4-point–intensity semiquantitative scale for the damage
was 10% (7.34% curcumin, 1.97% demethoxy-curcumin, and 0.7% present (1, absence of damage; 2, mild damage; 3, moderate damage;
bis-demethoxy-curcumin; Figure 1). We decided to use dosages of and 4, intense damage)
1.6 mg/kg body wt because of our previous experience.7,18
Determination of Lipid Aortic Composition
Blood Sampling Similar aortic segments of ⬇2 cm for all rabbits were weighed and
At the end of the experimental period, rabbits were anesthetized with minced in a tissue homogenizer. Total lipids were extracted by using
sodium pentothal at doses of 16 mg/kg body wt and exsanguinated 1.5 mL of 0.01 mol/L HCl, 0.1 mL of 1% MgCl2, and 5 mL
through a cannulated carotid. Blood was collected into EDTA-coated hexane/isopropanol (3:2 mixture). The organic phase was separated
tubes, and plasma was separated by centrifugation at 1750g for 10 by centrifugation at 1750g for 10 minutes. Standard procedures were
minutes. All samples were stored at ⫺80°C until analyzed. Total used to measure TC and esterified cholesterol (EC) with the use of

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 Quiles et al Curcumin, Antioxidants, and Atherogenesis 1227

commercially available kits (Boehringer-Mannheim). The fatty acid

pattern of aortic lipids was determined by gas-liquid chromatography
as previously described.25 The monounsaturated fatty acid (MUFA)/
polyunsaturated fatty acid (PUFA) index was calculated with the
following fatty acids: MUFA (16:1n-9, 18:1n-9, 20:1n-9, and
24:1n-9) and PUFA (18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6,
22:4n-6, 22:5n-6, 18:3n-3, 20:5n-3, 22:5n-3, and 22:6n-3).
All the chemical products and solvents, of high quality, were
acquired from Sigma and Merck.

Statistical Analyses
Before any statistical analysis, all variables were checked for
normality and homogeneous variance by using the Kolmogorov-
Smirnov and the Levene tests, respectively. When a variable was
found not to follow normality, it was logarithmically transformed
and reanalyzed. All parameters for the control and CU groups and
baseline levels were analyzed by a 1-way ANOVA; to evaluate mean
differences for groups at each time point (10, 20, and 30 days),
multiple comparison tests adjusted by Bonferroni corrections were
performed. Changes in aortic lesions were analyzed by the Kruskal-
Wallis test, and a Mann-Whitney U test was used a posteriori to
evaluate mean differences between groups. A value of P⬍0.05 was
considered significant. Data were analyzed by using a statistical
software package (SPSS for Windows, 9.0.1.,1999, SPSS Inc).

Results
No differences were found among the experimental groups
with respect to the weight gain at the different final experi-
mental times (data not shown). Plasma and LDL cholesterol
values were significantly higher at 10, 20, and 30 days for the
control and CU groups than for the baseline group. However,
no differences were found among the periods of time for each
group (plasma cholesterol, 13.7⫾3.7 mg/dL for baseline Figure 2. Concentration of TBARS in plasma (A), conjugated
dienes in LDL (B), and TBARS in LDL (C) from rabbits with
group and mean value 2977⫾51.8 mg/dL at 10, 20, and 30 experimental atherosclerosis treated with an oral curcumin-free
days for control and CU groups; LDL cholesterol, 42.9⫾1.5 hydroalcoholic solution (control [C] group) or with a hydroalco-
mg/mg protein for baseline group and mean value 93.2⫾9.5 holic curcuma extract at dose of 1.6 mg/kg (CU group). Results
are mean⫾SEM. Six animals from each experimental group
mg/mg protein at 10, 20, and 30 days for control and CU were euthanized after 10, 20, and 30 days. Statistical signifi-
groups). TBARS have been used to achieve the degree of cance is indicated as follows: differences (P⬍0.05) among times
lipid peroxidation in the plasma of the animals (Figure 2A). for each experimental group (a indicates 10 vs 20 days; b, 10 vs
30 days; and c, 20 vs 30 days); differences from baseline
Baseline levels of TBARS were lower than those found in the (†P⬍0.05); and differences between groups at 10, 20, or 30
control group at the 3 experimental times. No differences days (*P⬍0.05).
were found between the baseline group and the CU group at
10, 20, and 30 days. Additionally, for each experimental time, Figure 3C shows plasma ␣-tocopherol levels in the studied
the control group showed significantly higher levels of animals. No significant differences were found among exper-
TBARS than did the CU group. The susceptibility of LDL to imental times in the control group compared with the baseline
oxidation was determined by conjugated dienes (Figure 2B) group. The plasma ␣-tocopherol level of the CU group at 10
and TBARS (Figure 2C). No differences were found for days was no different from that of the baseline group.
conjugated dienes between the groups. However, compared However, the CU group at 20 and 30 days reached values of
with the CU group, the control group showed a significantly ␣-tocopherol that were significantly higher than those at 10
higher level of LDL TBARS at 30 days. days and at baseline.
Figure 3A shows plasma coenzyme Q10 concentration in all The results of the histological examination for fatty streak
experimental groups. A decrease in the levels of this antiox- lesions in the aortic arch are shown in Figure 4A. No lesions
idant was found for all times (10, 20, and 30 days) in the were found in the baseline group or in the control or CU
experimental groups compared with the baseline group. This groups at 10 days. For 20 and 30 days, a dramatic and similar
decrease was higher in the control group than in the CU increase in fatty streak was found in the control and CU
group. No differences were found in either group regarding groups, with no differences between these groups. Concern-
the experimental time. Plasma retinol levels (Figure 3B) ing the presence of fatty streaks in thoracic aortas, Figure 4B
showed results similar to those described for coenzyme Q10 shows results similar to those in Figure 4A for the baseline
regarding the decrease in the levels of this antioxidant group and for the control and CU groups at 10 days. The
compared with the baseline levels. Rabbits fed curcumin control and CU groups at 20 and 30 days showed an increase
extract for 10 days reflected significantly higher levels of in fatty streaks; this increase was significantly lower in the
retinol than did the control group for this experimental time. CU group than in the control group at 30 days. The histolog-

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1228 Arterioscler Thromb Vasc Biol. July 2002

Figure 3. Antioxidant levels of coenzyme Q10 (A), retinol (B), and Figure 4. Histological analysis of fatty streak lesion in aortic
␣-tocopherol (3) in plasma of rabbits with experimental athero- arch (A), thoracic aorta (B), and abdominal aorta (C) from rabbit
sclerosis treated with an oral curcumin-free hydroalcoholic solu- with experimental atherosclerosis treated with an oral curcumin-
tion (group C) and with a hydroalcoholic curcuma extract at free hydroalcoholic solution (group C) and with a hydroalcoholic
dose of 1.6 mg/kg (group CU). Results are shown in nanomoles curcuma extract at dose of 1.6 mg/kg (group CU). Six animals
of retinol, nanomoles of ␣-tocopherol, and picomoles of coen- from each experimental group were euthanized after 10, 20, and
zyme Q10 per milliliter of plasma. Six animals from each experi- 30 days. Values are mean⫾SEM. Statistical significance is indi-
mental group were euthanized after 10, 20, and 30 days. Values cated as follows: differences (P⬍0.05) among times for each
are mean⫾SEM. Statistical significance is indicated as follows: experimental group (a indicates 10 vs 20 days; b, 10 vs 30 day,
differences (P⬍0.05) among times for each experimental group and c, 20 vs 30 days); differences from baseline (†P⬍0.05); and
(a indicates 10 vs 20 days; b, 10 vs 30 days; and c, 20 vs 30 differences between groups at 10, 20, or 30 days (*P⬍0.05).
days); differences from baseline (†P⬍0.05); and differences
between groups at 10, 20, or 30 days (*P⬍0.05). days. Briefly, TC, EC, and FC concentrations were signifi-
cantly different among the periods of time (at baseline and at
ical study of fatty streaks in the abdominal aorta is presented 10, 20, and 30 days) for both experimental groups in all aortic
in Figure 4C. There was an absence of fatty streaks in the fractions; the highest cholesterol level always corresponded
baseline group and in the groups studied at 10 days and 20 with the final period of time. As a marker of lipid oxidation
days. After 30 days, an increase in this lesion was found in in the aorta, the MUFA/PUFA index was determined, as
both experimental groups, although a significantly higher shown in the Table. The higher the MUFA/PUFA index, the
lesion was shown in the control group compared with the CU lower was the susceptibility to lipid oxidation. All rabbits had
group. the highest values in the abdominal aorta, followed by the
TC, EC, and free cholesterol (FC) were determined in all thoracic aorta and, finally, the aortic arch. Significant differ-
aortic fractions (Table). The aortic fraction with the highest ences were found at the various times, and the highest
concentration of cholesterol was the aortic arch, followed by susceptibility to oxidation was at 30 days. On the other hand,
thoracic and abdominal aortas. The aortic fraction with more rabbits fed curcumin showed a lipid oxidation susceptibility
significant differences between the groups and among the index that was significantly higher in all aortic fractions than
times was the aortic arch. Compared with the control group, that for the control group at 20 and 30 days.
the CU group showed significantly higher concentrations of
TC and EC in the aortic arch at 30 days. However, only TC Discussion
concentration was significantly higher in the CU group versus The best-known antioxidant mechanism of curcuma and its
the control group in thoracic and abdominal aortas at 30 days. components is their capacity to eliminate reactive oxygen
No differences were found in TC, EC, and FC between the species, such as hydroxyl radical,13 superoxide radical,14
control and CU groups in the aortic fraction at 10 and 20 singlet oxygen,15 and NO.17 The results of the present study

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 Quiles et al Curcumin, Antioxidants, and Atherogenesis 1229

Total, Free, and Esterified Cholesterol and MUFA/PUFA Index

Ten Days Twenty Days Thirty Days

Baseline Group C Group CU Group C Group CU Group C Group CU

Total cholesterol
Aortic arch 1.28⫾0.04 1.03⫾0.25 2.62⫾0.35 5.58⫾0.61†‡ 6.34⫾1.23†‡ 7.26⫾1.96†§ 13.77⫾0.34*†§㛳
Thoracic aorta 1.48⫾0.39 2.9⫾0.37 2.3⫾0.1 4.25⫾0.81† 3.42⫾0.39† 3.42⫾0.49 5.28⫾0.288†§㛳
Abdominal aorta 1.43⫾0.07 3.19⫾0.34† 3.3⫾0.27† 4.67⫾0.67† 5.1⫾0.33†‡ 4.52⫾0.49† 7.43⫾0.68*†
Free cholesterol
Aortic arch 1.23⫾0.05 2.01⫾0.18† 2.3⫾0.24† 3.29⫾0.24† 3.59⫾0.56† 4.17⫾0.65†§ 5.34⫾0.78†§
Thoracic aorta 0.92⫾0.04 2.06⫾0.38† 1.88⫾0.07† 2.36⫾0.19† 2.1⫾0.19† 1.99⫾0.26† 2.57⫾0.09†§†
Abdominal aorta 0.97⫾0.14 2.25⫾0.24† 2.4⫾0.12† 3.37⫾0.75† 2.91⫾0.19† 2.68⫾0.27† 4.19⫾0.2*†§㛳
Esterified cholesterol
Aortic arch 0.05⫾0.01 0.49⫾0.1 0.32⫾0.13 1.65⫾0.47 2.75⫾0.73†‡ 3.09⫾1.35†§ 8.43⫾0.44*†§㛳
Thoracic aorta 0.2⫾0.08 0.85⫾0.07 0.42⫾0.09 2.1⫾0.73† 1.33⫾0.22†‡ 1.43⫾0.5 2.71⫾0.24†§㛳
Abdominal aorta 0.27⫾0.1 0.94⫾0.21 0.9⫾0.16 1.3⫾0.4† 2.19⫾0.38† 1.84⫾0.24† 3.24⫾0.28†§
MUFA/PUFA index
Aortic arch 0.87⫾0.04 0.97⫾0.02 1.19⫾0.04*† 0.88⫾0.03 1.01⫾0.02* 0.47⫾0.08†§㛳 0.77⫾0.07*§㛳
Thoracic aorta 1.54⫾0.06 1.05⫾0.09† 1.20⫾0.02† 0.93⫾0.04† 1.13⫾0.02*† 0.86⫾0.03† 1.10⫾0.04*†
Abdominal aorta 2.21⫾0.09 1.12⫾0.05† 1.32⫾0.09† 1.07⫾0.03† 1.21⫾0.03*† 1.09⫾0.02† 1.23⫾0.04*†
Six rabbits, with experimental atherosclerosis treated orally with a curcuma-free hydroalcoholic solution (group C) or with a
hydroalcoholic curcuma extract (1.6 mg/kg, group CU), from each experimental group were sacrificed after 10, 20, and 30 days.
Results are mean⫾SEM for n⫽6 rabbits. Total, esterified, and free cholesterol are expressed as mg/g of tissue.
*P⬍0.05, control vs curcuma groups in the same period of time (10, 20, or 30 days).
†P⬍0.05, with the baseline.
‡P⬍0.05, 20 days vs 10 days within the same group of treatment (group C or group CU).
§P⬍0.05, 30 days vs 10 days within the same group of treatment (group C or group CU).
㛳P⬍0.05, 30 days vs 20 days within a same group of treatment (group C or group CU).
MUFA/PUFA indicates monounsaturated fatty acids/polyunsaturated fatty acids.

show other points of view regarding the antioxidant mecha- The situation found in the plasma of the control rabbits
nism of curcuma extract by protection of the endogenous suggests that even if ␣-tocopherol does not change, a great
antioxidants from oxidative damage. deal of oxidative damage still occurs. In fact, plasma retinol
Lipid peroxidation is a fundamental process in atherogen- and the essential co-antioxidant coenzyme Q10 were lost, and
esis. LDL is modified by free radical–mediated reactions, their concentrations were drastically reduced within the first
both in their lipid and protein moieties, leading to the 10 days of treatment. A concomitant increase in peroxidation
alterations that are the starting point for the first events in the products was also found in the control group, confirming that
outcome and development of the atherogenic process.26 coenzyme Q10 (as well as retinol) is essential for antioxidant
Because plasma contains various antioxidants, the extent to activity by ␣-tocopherol to attenuate plasma peroxidation.28
which LDL oxidation occurs in the circulation was thought to Curcuma treatment (CU group) significantly buffered such
be limited until (at least) a sufficiently active amount of such impaired oxidant/antioxidant unbalance, leading to levels of
antioxidants was available. In the LDL in the arterial wall, the
TBARS similar to baseline levels and to less reduced coen-
lipid peroxidation proceeds as a chain reaction, which may be
zyme Q10 levels. Moreover, similar (for 10 days) or higher
terminated only by suitable antioxidants within the LDLs
plasma levels of ␣-tocopherol (20 and 30 days), compared
themselves. Moreover, the presence of several co-
with baseline levels, were found. Different reasons may
antioxidants is also required for effectively scavenging free
radicals, because some antioxidant molecules, per se, would account for the higher levels of ␣-tocopherol found in the CU
act paradoxically as pro-oxidant once activated in the lipid group at 20 and 30 days: (1) It has been estimated that ⬎90%
moiety. This is the case for ␣-tocopherol: its interaction with of the body pool of ␣-tocopherol is located in the adipose
a peroxyl radical converts it to an ␣-tocopheroxyl radical, tissue. This tissue does provide a source of vitamin E for the
which, in turn, is alternatively regenerated to ␣-tocopherol by rest of the body.29 Maybe a mobilization of ␣-tocopherol
specific co-antioxidants (such as coenzyme Q10); if the from the adipose tissue of rabbits fed curcuma occurs, thus
co-antioxidants fail, or are limited, a so-called tocopherol- protecting the body against the oxidative damage produced
mediated peroxidation has been identified27 in which the during the development of atherosclerosis. (2) Plasma ␣-to-
␣-tocopherol itself does not act as a chain-breaking antioxi- copherol concentration is also maintained by the secretion of
dant but rather facilitates the transfer of radical reactions from nascent VLDL by the liver that is due to the hepatic
the aqueous phase inside the lipophilic environment, thus ␣-tocopherol transfer protein that incorporates ␣-tocopherol
mediating radical chain reactions within the lipid moieties. into VLDL. Rabbits fed curcuma extract could transport more
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1230 Arterioscler Thromb Vasc Biol. July 2002

Figure 5. Histological photomicrographs

of aortic segments from atherosclerotic
rabbits. Photomicrograph A, No lesion
(score 1) on luminal surface of aortic
arch (H&E stain). Bar⫽1000 ␮m. This
was observed in the baseline group and
in the control [C] and CU groups at 10
days for all aortic segments. Photomicro-
graph B, High subintimal deposits of
foam cells and extracellular lipids show-
ing the extension and thickness of the
aortic arch (H&E stain, score 4). Bar⫽100
␮m. This type of lesion was observed in
both treatment groups (C and CU),
mainly at 30 days. Photomicrograph C,
Subintimal deposits of foam cells (fatty
streak), which have a moderated exten-
sion with a medium thickness of thoracic
aorta (H&E stain, score 2). Bar⫽100 ␮m.
This type of lesion was more observed in
group C at 20 days, when the score was
mainly 2 in the thoracic aorta. Photomi-
crograph D, Development of fatty streak
in the intima of abdominal aortoiliac
bifurcation (H&E stain, score 2). Bar⫽100
␮m. This lesion (score 2) in the abdomi-
nal aorta at 30 days was more frequent
in the CU group than in group C, for
which the lesion was scored 3.

VLDL in plasma, increasing the levels of ␣-tocopherol. (3) macrophage uptake of these LDL particles, thus generating
Both these hypotheses plus a better plasma antioxidant foam cells26 and attenuating the development of atheroscle-
defense of the CU group, which recycles the ␣-tocopherol rosis. This is also supported by the fact that rabbits fed
radical, could also help us to understand the high plasma curcumin extract had a significantly higher aortic MUFA/
levels of ␣-tocopherol at 20 and 30 days. PUFA index than did the control group, inasmuch as their
Concerning the histological analysis, the intensity and fatty acids were less susceptible to oxidation in the vessel
extension of the lesions in the aortas of the control and CU wall.
groups are in accordance with the study of Wöjicicki et al,30 In conclusion, supplementation with Curcuma longa ex-
in the sense that the highest grade of lesion was found in the tract reduces oxidative stress and attenuates the development
aortic arch, followed by the thoracic aorta and, finally, by the of fatty streaks in rabbits fed a high cholesterol diet. Thus,
abdominal aorta (Figures 4 and 5). This finding could indicate this extract could be taken as a preventive by patients with
a major susceptibility of the aortic arch to LDL permeability, peripheral vascular disease.
leading to the conclusion that the antioxidant mechanisms are
not capable of protecting the high levels of modified LDL Acknowledgment
deposited in the intima because of the rapid and intense This study was supported by a grant from A.S.A.C. Pharmaceutical
development of the fatty streak in both groups (control and International A.I.E., Alicante, Spain.
CU) at 20 and especially at 30 days (Figures 4 and 5), when
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