Recent Advances on the Nutritional Effects Associated with

the Use of Garlic as a Supplement

Suppression of LDL Oxidation by Garlic1,2
Benjamin H. S. Lau
Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University,
Loma Linda, CA 92350

KEY WORDS:

garlic

atherosclerosis

oxidized LDL

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). Lifestylerelated 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

antioxidants

cardiovascular diseases

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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. J. Nutr. 131: 985S988S, 2001.

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).
Almost two decades ago, we reviewed the world literature
on the use of garlic in modifying blood lipids and atherosclerotic diseases (Lau et al. 1983). 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 vas-

1
Presented at the conference Recent Advances on the Nutritional Benefits
Accompanying the Use of Garlic as a Supplement held November 1517, 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.

0022-3166/01 $3.00 2001 American Society for Nutritional Sciences.

985S

SUPPLEMENT

986S

TABLE 1
Risk factors in cardiovascular diseases
Lifestyle-related
Smoking
Inactivity
Stress

Uncontrollable

Diet-related

Heredity (genes)
Gender
Age

Cholesterol
Triglycerides
Homocysteine
Hypertension
Diabetes
Overweight

FIGURE 1 Diagram showing five important properties of oxidized

LDL (Ox-LDL). 1) The entry of native LDL into the subendothelial space
is followed by its oxidation by cellular oxidants produced by endothelial
cells, macrophages or smooth muscle cells. Ox-LDL then recruits
circulating monocytes to the intima and these cells are differentiated
into macrophages. 2) Ox-LDL inhibits macrophage motility. 3) Macrophages engulf Ox-LDL via scavenger receptors to become foam cells.
4) Ox-LDL injures endothelium. 5) Ox-LDL enhances proliferation of
smooth muscle and other cells.

FIGURE 2
The effect of aged garlic extract (AGE) on Cu2induced LDL oxidation. Various concentrations of AGE (0.1 mL volume)
and 0.1 mL of LDL (0.2 g protein/L) were added to 0.8 mL of 5 mol/L
CuSO4 and incubated at 37C for 24 h. After the incubation, the
reaction was stopped by adding 0.1 mL of 10 mmol/L EDTA. The extent
of lipid oxidation was determined by measuring thiobarbituric acid
reactive substances (TBARS). Bars represent means SEM of triplicate
samples. Asterisks denote significant difference (P 0.05) compared
with Cu () control without AGE.

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cular 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).
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). NAcetyl-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.
A concentration-dependent inhibition of LDL oxidation
was observed with the oil-soluble garlic compound, allixin
(Fig. 4). In this figure, the results with a known antioxidant
BHT are also presented. Allixin is of special interest because it

FIGURE 3 The effects of water-soluble constituents of aged garlic

extract (AGE) on Cu2-induced LDL oxidation. Data represent means
SEM of triplicate samples. Asterisks denote significant difference (P
0.05) compared with control without garlic compound. Abbreviations: SAC, S-allylcysteine; N-Ac-SAC, N-acetyl-S-allylcysteine;
SAMC, S-allylmercaptocysteine; TBARS, thiobarbituric acid rective
substances.

GARLIC SUPPRESSES LDL OXIDATION

987S

FIGURE 6 Lag times of LDL oxidation in subjects who consumed

aged garlic extract (AGE) and placebos. Compared with placebo, garlic
supplement significantly increased the lag time of LDL oxidation (P
0.05, paired students t test), indicating its ability to increase the
resistance of LDL to oxidation.

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.
Recently we also determined the effect of allicin, an oilsoluble organosulfur compound derived from raw garlic, on
LDL oxidation. We were surprised to find that allicin actually
enhanced Cu2-induced LDL oxidation (Fig. 5). It appears
that allicin may behave like an oxidant rather than an antioxidant.
Ox-LDLinduced 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):

FIGURE 5 The effect of allicin on Cu2-induced LDL oxidation.

Data represent means SEM of triplicate samples. BHT was used as an
antioxidant control. Asterisks denote significant difference (P 0.05)
compared with respective controls at 0 points. TBARS, thiobarbituric
acid rective substances.

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 OxLDL, 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-LDLinduced parameters of cellular injury. These
garlic compounds also inhibited Ox-LDLinduced lipid peroxidation, implicating lipids as the principal target in OxLDLmediated 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 g for 25 min)
(Beckman Instruments, Fullerton, CA). After the addition of
5 mol/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 supple-

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FIGURE 4 The effect of allixin on Cu2-induced LDL oxidation.

Data represent means SEM of triplicate samples. BHT was used as an
antioxidant control. Asterisks denote significant difference (P 0.05)
compared with respective controls at 0 points. TBARS, thiobarbituric
acid rective substances.

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ment 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.
LITERATURE CITED

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Abby, S. L., Harris, I. M. & Harris, K. M. (1998) Homocysteine and cardiovascular disease. J. Am. Board Fam. Pract. 11: 391398.
Chan, A. C. (1998) Vitamin E and atherosclerosis. J. Nutr 128: 15931596.
Chung, B. H., Segrest, J. P., Ray, M. J., Brunzell, J. D., Hokanson, J. E., Krauss,
R. M., Beaudrie, K. & Cone, J. T. (1986) Single vertical spin density gradient
ultracentrifugation. Methods Enzymol. 128: 181209.
Geng, Z., Rong, Y. & Lau, B.H.S. (1997) S-Allyl cysteine inhibits activation of
nuclear factor kappa B in human T cells. Free Radic. Biol. Med. 23: 345350.
Gomez del Arco, P., Martinez-Martinez, S., Calvo, V., Armesilla, A. L & Redondo,
J. M. (1997) Antioxidants and AP-1 activation: a brief overview. Immunobiology 198: 273278.
Grisebach, H. & Ebel, J. (1978) Phytoalexins, chemical defense substances of
higher plants? Angew. Chem. Int. Ed. Engl. 17: 635 647.
Howard, G., Wagenknecht, L. E., Burke, G. L., Diez-Roux, A., Evans, G. W.,
McGovern, P., Nieto, F. J. & Tell, G. S. (1998) Cigarette smoking and
progression of atherosclerosis: the atherosclerosis risk in communities (ARIC)
study. J. Am. Med. Assoc. 279: 119 124.
Ide, N. & Lau, B.H.S. (1997) Garlic compounds protect vascular endothelial
cells from oxidized low density lipoprotein-induced injury. J. Pharm. Pharmacol. 49: 908 911.
Ide, N., Nelson, A. B. & Lau, B.H.S. (1997) Aged garlic extract and its constit-

uents inhibit Cu2-induced modification of low density lipoprotein. Planta

Med. 63: 263264.
Kannel, W. B., Castelli, W. P., Gordon, T. & McNamara, P. M. (1971) Serum
cholesterol, lipoproteins, and the risk of coronary heart disease. The Framingham study. Ann. Intern. Med. 74: 112.
Lau, B.H.S., Adetumbi, M. A. & Sanchez, A. (1983) Allium sativum (garlic) and
atherosclerosis: a review. Nutr. Res. 3: 119 128.
Lau, B.H.S., Lam, F. & Wang-Cheng, R. (1987) Effect of an odor modified garlic
preparation on blood lipids. Nutr. Res. 7: 139 149.
Phelps, S. & Harris, W. S. (1993) Garlic supplementation and lipoprotein oxidation susceptibility. Lipids 28: 475 477.
Puhl, H., Waeg, G. & Esterbauer, H. (1994) Methods to determine oxidation of
low-density lipoproteins. Methods Enzymol. 233: 425 441.
Simons, L. A., Balasubramaniam, S., von-Konigsmark, M., Parfitt, A., Simons, J.
& Peters, W. (1995) On the effect of garlic on plasma lipids and lipoproteins
in mild hypercholesterolaemia. Atherosclerosis 113: 219 225.
Steinberg, D. (1997) Low density lipoprotein oxidation and its pathobiological
significance. J. Biol. Chem. 272: 2096320966.
Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C. & Witztum, J. L.
(1989) Beyond cholesterol, modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 320: 915924.
Steiner, M. & Lin, R. S. (1998) Changes in platelet function and susceptibility of
lipoproteins to oxidation associated with administration of aged garlic extract.
J. Cardiovasc. Pharmacol. 31: 904 908.
Steyn, K., Steyn, M., Swanepoel, A. S., Jordaan, P. C., Jooste, P. L., Fourie, J. M.
& Rossouw, J. E. (1997) Twelve-year results of the coronary risk factor
study (CORIS). Int. J. Epidemiol. 26: 964 971.
Villeneuve, P. J., Morrison, H. I., Craig, C. L. & Schaubel, D. E. (1998) Physical
activity, physical fitness, and risk of dying. Epidemiology 9: 626 631.
Waleh, N. S., Calaoagan, J., Murphy, B. J., Knapp, A. M., Sutherland, R. M. &
Laderoute, K. R. (1998) The redox-sensitive human antioxidant responsive
element induces gene expression under low oxygen conditions. Carcinogenesis 19: 13331337.
Welch, G. N., Upchurch, G. R. & Loscalzo, J. (1997) Homocysteine, oxidative
stress, and vascular disease. Hosp. Pract. 32: 8192.
Yamasaki, T., Teel, R. W. & Lau, B.H.S. (1991) Effect of allixin, a phytoalexin
produced by garlic, on mutagenesis, DNA-binding and metabolism of aflatoxin B1. Cancer Lett. 59: 89 94.