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Laboratory of Biodynamic Chemistry, Tohoku University Graduate School of Life Science and Agriculture, Sendai 981-8555, Japan
1To whom correspondence should be addressed. E-mail: miyazawa{at}biochem.tohoku.ac.jp
ABSTRACT
Curcumin and its structurally related compounds (curcuminoids), the phenolic yellowish pigments of turmeric, display antioxidative, anticarcinogenic and hypocholesterolemic activities. In this study, we investigated the effects of dietary supplemented curcuminoids [commercial grade curcumin: a mixture of curcumin (73.4%), demethoxycurcumin (16.1%) and bisdemethoxycurcumin (10.5%)] on lipid metabolism in rats. Male Sprague-Dawley rats were assigned to three diet groups (n = 6) and fed a moderately high-fat diet (15 g soybean oil/100 g diet) for 2 wk. One diet group did not receive supplements (CONT), while the others were supplemented with 0.2 g curcuminoids/100 g diet (CUR0.2) or 1.0 g curcuminoids/100 g diet (CUR1.0). Liver triacylglycerol and cholesterol concentrations were significantly lower in CUR1.0 rats than in CONT rats. Plasma triacylglycerols in the VLDL fraction were also lower in CUR1.0 rats than in CONT rats (P < 0.05). Hepatic acyl-CoA oxidase activity of both the CUR0.2 and CUR1.0 rats was significantly higher than that of CONT rats. Furthermore, epididymal adipose tissue weight was significantly reduced with curcuminoid intake in a dose-dependent manner. These results indicate that dietary curcuminoids have lipid-lowering potency in vivo, probably due to alterations in fatty acid metabolism.
KEY WORDS: • curcumin • triglyceride • liver • adipose tissue • rats
The rhizome of Curcuma longa (turmeric) has been widely
used as a spice and coloring agent in many foods. Consumption of
turmeric has been associated with various beneficial effects on human
health (1
). In tropical regions of Asia, turmeric has also
been used as a traditional remedy for the treatment of inflammation and
other diseases (1
). Curcuminoids, curcumin and its
structurally related compounds comprise the phenolic yellowish pigment
of turmeric (Fig. 1
). It has been reported that 2.71–5.18 g/100 g curcuminoids is present
in commercially available turmeric powders (2
) and that
0.34–0.47 g/100 g is present in curry powders (3
).
Dietary curcuminoids have been associated with antioxidative
(4
, 5
) and anticarcinogenic (6
, 7
) activities.
In recent years, much attention has been focused on the apoptotic
action of curcumin (8
–10
).
|
, 12
). However, there have been
few reports dealing with the effects of dietary curcuminoids on
triacylglycerol (TG)2
(2
) and fatty acid metabolism. Recently, we demonstrated
that dietary turmeric extract prevents liver TG accumulation in mice
(5
). We speculated this was attributable to the intake of
curcuminoids, the predominant constituents of the turmeric extract used
in that study. Such a TG-lowering effect was of interest to us
because TG accumulation in the liver is accompanied by biochemical
modification of the mitochondrial and endoplasmic functions
(13
) and because fatty liver disease is associated with
hyperlipidemia and obesity (14
). To evaluate the
TG-lowering effect of dietary curcuminoids, we fed rats a
moderately high-fat diet (15% soybean oil) supplemented with 0.2
or 1.0% curcuminoids for 2 wk.
MATERIALS AND METHODS
Animals and diets.
This study was conducted in conformity with the policies and procedures
detailed in the Guide for the Care and Use of Laboratory Animals
(15
). Seven-week-old male Sprague-Dawley rats were
obtained from Japan SLC (Hamamatsu, Japan) and housed individually in
stainless steel wire-mesh cages in a room kept at 23 ± 1°C
with a 12-h light:dark cycle (light period: 8:00–20:00). After
acclimatization with F-2 Standard Rodent
Chow3
(Funabashi Farm, Funabashi, Japan) and distilled water (free access)
for 1 wk, rats were randomly assigned to one of three dietary groups:
control (CONT, n = 6), 0.2% curcuminoid supplement
(CUR0.2, n = 6) and 1.0% curcuminoid supplement
(CUR1.0, n = 6). The CONT
diet4
was based on a modification of the recommendations of the American
Institute of Nutrition (16
). For the CUR0.2 and CUR1.0
diets, commercially available curcumin from Curcuma
longa (Sigma Chemical, St. Louis, MO), a mixture of three
curcuminoids (Fig. 1)
, was substituted for equivalent cornstarch in
CONT diet at 2 and 10 g/kg, respectively. Rats had free access to both
food and distilled water, which were provided fresh every day and every
other day, respectively. Food intake and body weights were recorded
every day and every other day, respectively. Rat feces were collected
in the final 3 d and stored at -30°C until analysis.
Sample collection.
On completion of the experiment, all rats were weighed and blood was
collected into EDTA-treated blood collection tubes after
decapitation. The blood collection was performed between 09:30 and
10:00 h. Plasma was prepared by centrifugation of blood at 1,000
x g for 15 min at 4°C. From 3 mL of the prepared
plasma, chylomicron and VLDL fractions were isolated by sequential
ultracentrifugation (17
). The isolated lipoproteins and
residual plasma were stored at -80°C until analysis. Immediately
after blood collection, livers were perfused in situ with ice-cold
saline. Livers and epididymal adipose tissue were then removed and
weighed. Two identical portions (
1 g) of each liver were divided and
kept on ice for the enzyme preparations (described below), and the
remainder was stored at -80°C until lipid analysis.
Liver enzyme preparations were performed on the day of dissection. For
the fatty acid synthase (FAS) assay, a 105,000 x g
supernatant fraction was prepared by differential centrifugation
(18
). For the acyl-CoA oxidase (ACO) assay, a
peroxisome-rich fraction was prepared as reported by Small et al.
(19
). Both enzyme preparations were stored at -30°C
until assayed.
Analytical methods.
The total fatty acid content of rat feces was measured by
gas-liquid chromatography (GLC) using a 50-m CP-SIL 88
capillary column (Chrompack, Middelburg, the Netherlands). After adding
pentadecanoic acid as an internal standard, fecal lipid were extracted
with a mixture of chloroform/methanol (20
). For GLC
analysis, fatty acid methyl esters were prepared simultaneously from
both free and esterified fatty acids with anhydrous HCl in methanol
(21
). The fatty acid content in the soybean oil used in
the diet was also measured by GLC as described above, and the fatty
acid content in the diet was calculated from that of the soybean oil.
The apparent digestibility of dietary TGs was calculated from the
difference between the fatty acid content of diet intake and that of
feces.
Plasma phospholipids, TG and total cholesterol were measured using
Phospholipid-test, Triglyceride-E-test, and Cholesterol-E-test kits
(Wako Pure Chemical, Osaka, Japan), respectively. TG in chylomicron and
VLDL fractions were also measured using the Triglyceride-E-test. For
liver lipid analysis, total lipids were extracted with a mixture of
chloroform/methanol (20
). Liver TGs and total cholesterol
were measured enzymatically as described above after total lipids were
dissolved in Triton X-100 (22
). Phospholipid phosphorus in
total liver lipid was measured by the method of Bartlett
(23
).
FAS activity was determined spectrophotometrically from the rate
of malonyl-CoA–dependent NADPH oxidation (18
), and one
unit of activity was defined as nanomole of NADPH oxidized per minute
at 30°C. ACO activity was measured from the rate of
palmitoyl-CoA–dependent H2O2 production
coupled with dichlorofluorescein oxidation as described by Small et al.
(19
), and one unit of activity was defined as nanomole of
dichlorofluorescein oxidized per minute at 30°C. Protein
concentrations were determined using a DC Protein Assay kit (Bio-Rad
Laboratories, Hercules, CA) with bovine serum albumin as the standard.
Statistical analysis.
All data were expressed as means ± SEM. The differences among the three diet groups were analyzed by ANOVA, followed by Fisher’s protected least-significant difference (PLSD) test within the StatView version 4.5 statistical package for the Macintosh (Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05.
RESULTS AND DISCUSSION
In a preliminary study, the liver TG concentration of the rats fed a 15 g/100 g fat (soybean oil) diet for 2 wk was approximately threefold greater than that of the rats fed a 5 g/100 g fat diet (unpublished observations). Therefore, for the present study, we designed the 15 g/100g fat diet used as a model of high-fat diet–induced lipid accumulation in the liver. We then estimated the lipid-lowering efficiency of dietary curcuminoids.
The diets were well accepted over the feeding period, and neither the
body weight gain nor the relative liver weight differed among the three
diet groups (Table 1
). Epididymal adipose tissue weight was reduced significantly by
curcuminoid intake in a dose-dependent manner (Table 1)
. Hepatic TG
concentration was also reduced with curcuminoid intake to 76%
(CUR0.2, P = 0.06) and 64% (CUR1.0, P =
0.01) of CONT rats (Table 2
). Because the intestinal digestibility of TGs was not affected by
curcuminoid supplementation to the diet (Table 1)
, these effects
apparently were not due to inhibition of TG digestion or absorption. As
reported previously (5
, 11
, 12
), liver cholesterol
concentration was also reduced with curcuminoid intake (Table 2)
. Babu
and Srinivasan (12
) have suggested that such a
cholesterol-lowering effect could be mediated by the stimulation of
hepatic cholesterol-7
-hydroxylase activity. Further studies of
sterol and bile acid balance in feces would be useful to examine the
alteration in bile acid synthesis secondary to curcuminoid ingestion.
|
|
. In general, blood from animals with
free access to food has high levels of chylomicrons and VLDL, both of
which are rich in TGs. Therefore, no significant difference in TG
concentration in whole plasma (Table 2)
should be caused by the higher
levels of chylomicrons, which are synthesized in intestinal epithelial
cells and secreted into blood circulation via lymph ducts. On the other
hand, VLDLs are synthesized in hepatic mesenchymal cells and secreted
into the blood circulation. Thus, this hypotriacylglycerolemic action
in VLDLs, but not in chylomicrons, also suggests that dietary
curcuminoids alter TG metabolism in the liver and/or the VLDL clearance
in the peripheral tissues without affecting intestinal absorption of
TGs.
Curcumin (11
, 12
) and turmeric extract (24
, 25
)
exhibit hypocholesterolemic effects, particularly in
cholesterol-fed animals. In contrast with these reports, in this
study without cholesterol supplementation, the plasma cholesterol
concentration was not affected by the curcuminoid supplementation
(Table 2)
. Several previous reports (5
, 11
, 12
) also
indicated that the plasma cholesterol levels of animals fed a
cholesterol-free diet were not affected by curcumin
supplementation. Therefore, the hypocholesterolemic effect of
curcuminoids seems to be limited in cholesterol-fed,
hypercholesterolemic animals.
Hepatic ACO activity of curcuminoid-fed rats was significantly
stimulated to 1.7-fold (CUR0.2) and 2.2-fold (CUR1.0) of that of CONT
rats, whereas FAS activity was not affected (Fig. 2
), suggesting that dietary curcuminoids affect fatty acid catabolism in
the liver rather than de novo synthesis of fatty acids. ACO performs
the first catalytic step enzyme of peroxisomal fatty acid
ß-oxidation, and its gene expression is regulated by the peroxisome
proliferator–activated receptors (PPARs) (26
). PPARs are
ligand-activated transcriptional factors that regulate gene
expression of a variety of lipid metabolizing proteins, such as ACO,
enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase,
medium-chain acyl-CoA dehydrogenase, acyl-CoA synthase, the
cytochrome P-450 4A family, fatty acid binding proteins, lipoprotein
lipase and apolipoproteins (26
). Therefore, PPARs are
proposed to play a central role in a signaling system that controls
lipid homeostasis. Hypolipidemic fibrates (27
),
antidiabetic thiazolidinediones (28
, 29
), polyunsaturated
fatty acids (PUFAs) (27
, 30
) and some eicosanoids
(27
, 30
) are ligands for PPAR. Although we cannot explain
entirely how curcuminoids lower the TG levels in rats, one possible
mechanism may be through multiple inductions of intra- and
extracellular fatty acid catabolism and utilization pathways (e. g.,
induction of fatty acid ß-oxidation and TG hydrolysis), with
metabolites of absorbed curcuminoids serving as ligands that can
activate PPAR. Recent reports have indicated that some of the
curcuminoids administered orally to rodents is absorbed and present in
the blood circulation conjugated with glucuronic acid and sulfuric acid
(31
–33
). The formation of further degradation products of
curcumin has also been proposed (34
). Thus, some of these
degradation products may function as hypolipidemic drugs by serving as
ligands for PPAR. However, until now, the distribution of the
metabolites of orally administered curcuminoids in liver and other
tissues, the interaction of the metabolites and PPAR, and the
modulation of a variety of the PPAR-regulated lipid metabolizing
proteins have not been revealed. To evaluate our hypothesis, further
elucidation of these issues is necessary.
|
FOOTNOTES
2 Abbreviations used: ACO, acyl-CoA
oxidase; CONT, control diet; CUR0.2, 0.2% curcuminoid diet; CUR1.0,
1.0% curcuminoid diet; FAS, fatty acid synthase; GLC, gas-liquid
chromatography; PPAR, peroxisome proliferator–activated receptor; TG,
triacylglycerol; VLDL-TG, TG concentration in the VLDL fraction. 
3 Approximate composition of F-2 (g/kg diet):
carbohydrate, 586; protein, 208; fat, 45; fiber, 34.
4 Ingredient of the CONT diet (g/kg diet):
cornstarch, 449.5; sucrose, 100; casein, 200; soybean oil, 150;
cellulose, 50; AIN-93G mineral mix, 35; AIN-93 vitamin mix, 10;
L-cystine, 3; choline bitartrate, 2.5.
Manuscript received 14 May 2001. Revision accepted 11 August 2001.
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