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Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, CA 92350
ABSTRACT
It has been known for several decades that hypercholesterolemia is a major risk factor for atherosclerosis and that lowering of cholesterol can significantly reduce risk for cardiovascular diseases. More recently, oxidation of LDL has been recognized as playing an important role in the initiation and progression of atherosclerosis. Oxidized LDL, but not native LDL, promotes vascular dysfunction by exerting direct cytotoxicity toward endothelial cells, by increasing chemotactic properties for monocytes, by transforming macrophages to foam cells via scavenger-receptors and by enhancing the proliferation of various cell types, e.g., endothelial cells, monocytes and smooth muscle cells; all of these events are recognized as contributing to atherogenesis. In this paper, experimental evidence is presented that shows that several garlic compounds can effectively suppress LDL oxidation in vitro. Short-term supplementation of garlic in human subjects has demonstrated an increased resistance of LDL to oxidation. These data suggest that suppressed LDL oxidation may be one of the powerful mechanisms accounting for the antiatherosclerotic properties of garlic.
KEY WORDS: • garlic • atherosclerosis • oxidized LDL • antioxidants • cardiovascular diseases
Cardiovascular diseases are the major cause of death in the United
States and all other affluent societies in the world. There are three
main groups of risk factors, i.e., diet-related, lifestyle-related
and uncontrollable factors (Howard et al. 1998
,
Steyn et al. 1997
, Villeneuve et al. 1998
). Lifestyle-related risk factors include smoking,
inactivity and stress. The uncontrollable factors include heredity,
gender and age. Cardiovascular risk is greater for men than for
premenopausal women. As a person ages, there is a greater risk of
cardiovascular disease. Recent studies suggest that even these
so-called "uncontrollable" factors can actually be controlled
or modified (Gomez del Arco et al. 1997
, Waleh et al. 1998
). Antioxidants, for example, can regulate
transcriptional factors that are required for gene expression
(Geng et al. 1997
). Hence, dietary and lifestyle changes
may help keep the undesirable genes suppressed.
The most prominent cardiovascular disease risk factors are diet
related. It has been known for several decades that elevated blood
cholesterol and triglycerides are associated with an increased risk of
cardiovascular diseases (Kannel et al. 1971
). In the
past decade, elevated blood homocysteine has been found to increase the
incidence of cardiovascular disease (Abby et al. 1998
,
Welch et al. 1997
); this is particularly true in
individuals who suffer from such diseases yet whose blood lipids are in
the normal or lower range. Hypertension, diabetes and obesity are three
clinical conditions related to diet that also contribute to the
increased incidence of cardiovascular diseases (Table 1
).
|
). In both animal and human studies,
there was strong evidence that garlic could lower blood cholesterol and
triglycerides and thus possibly reduce the incidence of cardiovascular
diseases. Nearly all of the studies utilized raw or fresh garlic. Our
group then undertook a project to study the effect of an odorless
commercial garlic extract in human subjects with elevated blood
cholesterol and triglycerides (Lau et al. 1987
). We were
able to demonstrate the lowering of both of these lipids with the
garlic extract. We also observed a lowering of the LDL cholesterol and
a slight, yet significant elevation of the HDL cholesterol. HDL is
classified as "good" cholesterol because it does not contribute to
atherosclerosis, whereas LDL is considered the "bad" cholesterol
because it may lead to atherosclerosis. However, in this past decade,
oxidation of LDL has been recognized as playing an important role in
the initiation and progression of atherosclerosis (Steinberg et al. 1989
, Steinberg 1997
). Oxidized LDL
(Ox-LDL),3
but not native LDL may contribute to vascular dysfunction leading to
atherogenesis. When LDL is oxidized, it acquires a dozen or more of new
properties that are absent in the native or nonoxidized LDL
(Steinberg 1997
).
Ox-LDL acquires new antigenic properties that are recognized by the
host immune system as "foreign." Thus, Ox-LDL produces several
new biologic responses; some of the prominent responses include the
following: 1) a chemotactic response for monocytes, their
attractions to the intima and their differentiation into macrophages;
2) the inhibition of macrophage movement from the intima;
3) enhanced occurrence of lipid-laden foam cells,
characteristic of fatty streaks, the first sign of atherosclerosis;
4) damage to the endothelium; and 5) proliferation of
monocytes, endothelial cells and smooth muscle cells (Fig. 1
). All of these events contribute to the thickening and narrowing of
arteries, the principal event in atherosclerosis (Chan 1998
).
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Can garlic suppress LDL oxidation?
Using an in vitro model in which copper sulfate
(CuSO4) was used to oxidize LDL, we determined
the effects of aged garlic extract (AGE, the same extract we used to
study lipid-lowering effect) and its several constituents on LDL
oxidation (Ide et al. 1997
). When LDL was incubated with
CuSO4 for 24 h, a significant increase of
thiobarbituric acid reactive substances (TBARS), indicating LDL
oxidation, was noted; in the absence of CuSO4,
only a small quantity of TBARS was detected (Fig. 2A
). AGE exhibited a concentration-dependent inhibition of
Cu2+-induced LDL oxidation as manifested by the
decrease in TBARS (Fig. 2B
). The effects of the
water-soluble constituents of AGE on
Cu2+-induced oxidative modification of LDL were
studied. All four water-soluble garlic compounds significantly
inhibited the formation of TBARS to varying degrees (Fig. 3
). N-Acetyl-S-allylcysteine, a metabolite of
S-allylcysteine (SAC), inhibited the LDL oxidation at a
concentration of 10 mmol/L. SAC and alliin inhibited the oxidation of
LDL at 10 and 1 mmol/L, respectively, whereas
S-allylmercaptocysteine showed significant inhibition in
both concentrations (0.1 and 1 mmol/L) tested.
|
|
). In this figure, the results with a known antioxidant BHT are also
presented. Allixin is of special interest because it is a phytoalexin
(phyto = plant, alexin = to ward off), the major weapon for
plant defense. Phytoalexins have been described as "stress
compounds" because their synthesis is induced by exposure of a plant
to certain kinds of stress, such as contact with bacteria, viruses,
fungi, insects and chemicals (Grisebach and Ebel 1978
).
Allixin was previously shown to inhibit the metabolism of the chemical
carcinogen aflatoxin B1 and its binding to DNA
(Yamasaki et al. 1991
). In this study, allixin was shown
to suppress LDL oxidation.
|
). It appears that allicin may behave like an oxidant rather than an
antioxidant.
|
Ox-LDL–induced damage of endothelial cells.
We used the following three in vitro assays to determine the effects of
Ox-LDL on vascular endothelial cells (Ide and Lau 1997
): lactate dehydrogenase (LDH) release as an index of
membrane damage, methylthiazol tetrazolium (MTT) absorbance for
mitochondrial function and TBARS, indicating lipid peroxidation. When
vascular endothelial cells were exposed to Ox-LDL, there was a
significant increase of LDH release, indicating cell membrane damage,
and a decrease of MTT absorbance, indicating mitochondrial injury.
Pretreatment of vascular endothelial cells with AGE and SAC minimized
these Ox-LDL–induced parameters of cellular injury. These garlic
compounds also inhibited Ox-LDL–induced lipid peroxidation,
implicating lipids as the principal target in Ox-LDL–mediated cellular
injury.
Human studies.
Two recent small-scale human studies suggest that commercial garlic
preparations may increase the resistance of plasma LDL to oxidation. In
one study, subjects consumed 600-mg tablets of a commercial garlic
powder daily for 2 wk (Phelps and Harris 1993
). Garlic
supplementation decreased TBARS formation, indicating that it decreased
susceptibility to LDL oxidation. In another study, subjects consumed
7.2 g AGE/d for 3 mo (Steiner and Lin 1998
).
Compared with the placebo group, those taking garlic had lower TBARS,
again indicating an increased resistance to LDL oxidation. Another
study found that garlic powder containing allicin had no demonstrable
effect on either the susceptibility or resistance of LDL to oxidation
(Simons et al. 1995
).
We have conducted a small-scale preliminary study. This was a
double-blind, placebo-controlled, crossover study involving eight
subjects (4 men and 4 women; mean age, 68 y). Four subjects took
1.2 g AGE three times a day for 2 wk, then 2 wk of no garlic
(washout period), followed by 2 wk of placebo. The remaining four
subjects took a placebo for the first 2 wk, followed by a 2-wk washout
and 2 wk of 1.2 g AGE three times a day. Blood was drawn at the
beginning of the experiment, and at 2, 4 and 6 wk when the experiment
was completed. Plasma LDL was isolated by a 30-min single vertical spin
density ultracentrifugation (Chung et al. 1986
) using a
TL-100 tabletop ultracentrifuge (543,000 x g for 25 min)
(Beckman Instruments, Fullerton, CA). After the addition of 5
µmol/L CuSO4, the absorbance at 234
nm was measured in a DU650 spectrophotometer (Beckman Instruments)
every 2 min for 3 h. Resistance of LDL to oxidation was determined
by continuous measurement of the formation of conjugated dienes
(Puhl et al. 1994
). The lag time of LDL oxidation was
estimated from the intercept of the tangents to the slow and fast
increase of diene absorption. The use of the garlic supplement was
found to significantly increase the resistance of LDL to oxidation
(Fig. 6
).
|
Summary
Several in vitro studies have demonstrated that garlic compounds can suppress LDL oxidation. A few small-scale human studies support the ability of garlic supplementation to increase the resistance of plasma LDL to copper-induced oxidation. Suppressed LDL oxidation may be one of the effective mechanisms that account for the beneficial effects of garlic.
FOOTNOTES
1 Presented at the conference "Recent Advances on the Nutritional Benefits Accompanying the Use of Garlic as a Supplement" held November 15–17, 1998 in Newport Beach, CA. The conference was supported by educational grants from Pennsylvania State University, Wakunaga of America, Ltd. and the National Cancer Institute. The proceedings of this conference are published as a supplement to The Journal of Nutrition. Guest editors: John Milner, The Pennsylvania State University, University Park, PA and Richard Rivlin, Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer Center, New York, NY. 
2 Supported by the Chan Shun International Foundation, San Francisco, CA, and Wakunaga Pharmaceutical Company, Osaka, Japan.
3 Abbreviations used: AGE, aged garlic extract; LDH, lactate dehydrogenase; MTT, methylthiazol tetrazolium; Ox-LDL, oxidized LDL; SAC, S-allylcysteine; TBARS, thiobarbituric acid reactive substances.
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