Biochemical and Molecular Action of Nutrients

Garlic and Garlic-Derived Compounds Inhibit Human

Squalene Monooxygenase1
Nisha Gupta and Todd D. Porter2
Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky,
Lexington, KY 40536-0082

KEY WORDS:

cholesterol biosynthesis

garlic

selenium

Garlic has been reputed to possess medicinal properties

since ancient times. More recently, attention has been focused
on garlics ability to decrease blood cholesterol levels, as demonstrated in clinical trials in humans (1,2), through metaanalyses of clinical studies (35) as well as in experimental
studies with animals (6 8) and cultured hepatocytes (9 11).
Efforts have been made to isolate and identify the active
chemical components of garlic; S-allylcysteine (SAC),3 diallyl
disulfide (DADS), and allicin and its derivatives (ajoene) all
have been shown to contribute to its hypocholesterolemic
actions (1113). However, the mechanism underlying the
inhibitory action of these garlic compounds has not been
established. Several studies have reported that feeding garlicsupplemented diets to animals decreased the activity of several
cholesterolgenic enzymes, including 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (8,14 15) and acetyl-CoA synthetase (12), and it is possible that additional enzymes in the
cholesterol synthesis pathway are also inhibited.
Squalene monooxygenase (EC 1.14.99.7, earlier called

selenocystine

squalene epoxidase

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ABSTRACT Although extracts of garlic inhibit cholesterol biosynthesis in cultured hepatocytes, the inhibitory
components of garlic and the site or sites of inhibition in the cholesterol biosynthetic pathway have not been
established. To elucidate potential mechanisms of inhibition, we examined the effect of fresh garlic extract and 16
water- or lipid-soluble compounds derived from garlic on purified recombinant human squalene monooxygenase.
Squalene monooxygenase catalyzes the second and likely rate-limiting step in the downstream pathway for
cholesterol biosynthesis. A 50% inhibitory concentration (IC50) of squalene epoxidation was achieved with 1 g/L of
fresh garlic extract; of the 16 garlic compounds tested, only selenocystine (IC50 65 mol/L), S-allylcysteine (IC50
110 mol/L), alliin (IC50 120 mol/L), diallyl trisulfide (IC50 195 mol/L), and diallyl disulfide (IC50 400
mol/L) substantially inhibited the enzyme. Kinetic analysis showed that the inhibition by garlic and by these
compounds was slow and irreversible, suggestive of covalent binding to the enzyme; the ability of thiol-containing
compounds such as glutathione and 2,3-dimercaptopropanol to prevent and reverse the inhibition indicated that
the garlic compounds were reacting with sulfhydryl groups on the protein. Dithiols were better reversal agents than
monothiols, further suggesting that these inhibitors bind to the proposed vicinal sulfhydryls present on this enzyme.
These results indicate that squalene monooxygenase may be one of the target enzymes through which garlic
inhibits cholesterol biosynthesis. J. Nutr. 131: 16621667, 2001.

squalene epoxidase) is a 64-kDa FAD-containing enzyme bound

to the endoplasmic reticulum of eukaryotic cells. The enzyme
catalyzes the first oxidative step in cholesterol biosynthesis, the
epoxidation of squalene across a CC double bond to yield
2,3-oxidosqualene, a reaction more typical of cytochrome P450type chemistry. Like the cytochromes P450, this flavoprotein
monooxygenase also is dependent upon NADPH-cytochrome
P450 reductase (CPR) for reducing equivalents. Squalene monooxygenase evidently plays an important role in the overall regulation of cholesterol biosynthesis in that the addition of cholesterol to cells in culture lowers squalene monooxygenase mRNA
levels, suppresses squalene monooxygenase activity and results in
the accumulation of squalene (16).
Garlic can contain high levels of tellurium and selenium
compounds (1720); the possibility that these compounds
contribute to the overall block in cholesterol synthesis by
inhibiting squalene monooxygenase has been proposed (21)
but not yet examined. Indeed, inhibition of this enzyme by
tellurium was shown to block cholesterol synthesis in
Schwann cells and led to a peripheral demyelinating neuropathy (22). In previous work from our laboratory, tellurium,
selenium and arsenic compounds were shown to inhibit human squalene monooxygenase by binding to vicinal cysteine
sulfhydryls on this enzyme (23,24). It is therefore plausible
that tellurium and selenium compounds in garlic may inhibit
squalene monooxygenase and thereby contribute to the hypocholesterolemic actions of this plant.

1
Supported by a grant from the Ohio Valley Affiliate of the American Heart
Association.
2
To whom correspondence should be addressed.
E-mail: tporter@pop.uky.edu.
3
Abbreviations used: CPR, cytochrome P450 reductase; DADS, diallyl disulfide; DATS, diallyl trisulfide; DMP, 2,3-dimercaptopropanol; GSH, glutathione;
HMG, 3-hydroxy-3-methylglutaryl; IC50, 50% inhibitory concentration; SAC,
S-allylcysteine; SC, selenocystine.

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

Manuscript received 7 December 2000. Initial review completed 11 January 2001. Revision accepted 6 March 2001.
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INHIBITION OF SQUALENE MONOOXYGENASE BY GARLIC

To address this possibility, fresh garlic extract and 16 garlicderived compounds (Fig. 1) were tested for their ability to
inhibit purified human squalene monooxygenase. This work
identifies five chemicals found in garlic as inhibitors of
squalene monooxygenase and suggests that these compounds
bind to vicinal cysteine sulfhydryls on the enzyme. Although
there are several reports that selenium-containing compounds
from garlic are effective in the prevention of mammary and
other types of cancer (18,19,25), this is the first report to show
that an organoselenium compound, selenocystine, will inhibit
an enzyme involved in cholesterol synthesis.
MATERIALS AND METHODS
Materials. Garlic bulbs were purchased from a local grocery.
Alliin, S-methylcysteine, S-ethylcysteine, S-propylcysteine and SAC
were graciously provided by Wakunaga of America (Mission Viejo,
CA). DADS and diallyl sulfide were purchased from Fluka Chemika

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(St. Louis, MO); dipropyl disulfide, dipropyl sulfide, allyl mercaptan,

allyl methyl sulfide and selenomethionine were purchased from Acros
Organics (Geel, Belgium); methallyl sulfide was purchased from Aldrich (Milwaukee, WI); diallyl trisulfide (DATS) was purchased from
ICN Biomedicals; glutathione (GSH) was purchased from Boehringer
Mannheim (Indianapolis, IN), GmbH; Se-(methyl)selenocysteine,
L-selenocystine (SC), nicotinamide adenine dinucleotide phosphate,
reduced form (-NADPH), FAD, 2,3-dimercaptopropanol (DMP)
and cytochrome c were purchased from Sigma Chemical, St. Louis,
MO. Radiolabeled squalene (2.6 10 Bq) was synthesized by the
Chemical Synthesis Facility, Department of Medicinal Chemistry,
University of Utah. Precoated silica TLC plates were obtained from
Whatman (Clifton, NJ). All garlic derivatives were stored at 4C and
stocks of the water-soluble compounds were made fresh every time
before use. Lipid-soluble garlic derivatives were dissolved in dimethyl
sulfoxide to a final concentration of 200 mol/L.
Preparation of garlic extracts. Garlic cloves were peeled and
homogenized with a small amount of quartz sand in 20 mmol/L
Tris-HCl buffer (pH 7.4), and the debris was removed by centrifugation at 21,000 g for 20 min at 25C. The clear supernatant was
divided into aliquots and stored at 80C.
Purification of squalene monooxygenase. Human recombinant
squalene monooxygenase expressed from the pTYB4 vector was purified according to the protocol described by New England Biolabs
(Beverly, MA) for expression of proteins with the IMPACT T7
system (Intein-chitin binding domain fusion proteins), as previously
described (23). Protein was quantified with the Coomassie Plus Protein Assay Reagent Kit from Pierce (Rockford, IL) and was then
stored at 80C until use.
Squalene monooxygenase assays. Squalene epoxidation assays
were carried out as described previously (23). Standard reaction
mixtures (200 L) containing 20 mmol/L Tris-HCl (pH 7.4), 0.1%
Triton X-100, 30 mol/L FAD, 28 pmol CPR, 40 mol/L 14Csqualene and 58 pmol squalene monooxygenase were preincubated
with or without inhibitor for 30 min at 37C. The reactions were
then started by the addition of 1 mmol/L NADPH and incubated for
another 30 min at 37C. Product formation was linear for 40 60 min
at 37C, and yielded 1.1 nmol/L of 2,3-oxidosqualene/30-min reaction. Reactions were stopped by extraction into methylene chloride
and then fractionated on TLC plates with 5% ethyl acetate in
hexane. The developed plates were quantified by electronic autoradiography (Packard Instant Imager; Wilmington, DE).
Cytochrome P450 reductase assays. Recombinant rat CPR was
purified by affinity chromatography as described (26) and quantified
spectrally using a millimolar extinction coefficient of 21.4 at 456 nm.
Activity was determined with cytochrome c in 100 mmol/L potassium
phosphate buffer (pH 7.7) with 50 mol/L cytochrome c and 5 pmol
of reductase; reactions were initiated by the addition of 50 mol/L
NADPH and followed at 550 nm for 1 min at room temperature.
Preincubations were carried out with garlic extract or garlic compounds for 30 min at 37C.
Statistical analysis. Statistical analyses were carried out by oneway ANOVA with Dunnetts or Bonferrronis multiple comparisons
post-hoc tests, as indicated in the appropriate figure legends. Differences were considered significant at P 0.05. All graphs and statistical comparisons were prepared on Prism 3.0 (GraphPad Software,
San Diego, CA) by using values in duplicate or greater with SEM, as
shown by error bars where appropriate.

RESULTS

FIGURE 1 The 16 water- or lipid-soluble compounds from garlic

tested in this study for inhibition of squalene monooxygenase. Those
compounds that were found to inhibit the enzyme are underlined.

Fresh garlic extract and 16 different water- or lipid-soluble

compounds present in garlic (Fig. 1) were tested for their
ability to inhibit human squalene monooxygenase. A 50%
inhibitory concentration (IC50) was achieved using 1 g/L
garlic extract and activity was completely inhibited at 150 g/L
(Fig. 2A). Of the eight water-soluble compounds, SC, SAC
and alliin were the most potent inhibitors, with IC50 values of
65, 110 and 120 mol/L, respectively (Fig. 2B). Inhibition by
SC was nearly complete at 500 mol/L, whereas inhibition by
SAC and alliin was still incomplete. S-methylcysteine, S-

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GUPTA AND PORTER

ethylcysteine, S-propylcysteine, selenomethionine and Se(methyl)selenocysteine were not inhibitory at concentrations

up to 1 mmol/L (data not shown). Of the eight lipid-soluble
compounds tested, only DATS and DADS showed significant
inhibitory potency, with IC50 values of 195 and 400 mol/L,
respectively; at DADS concentrations as high as 1 mmol/L,
squalene monooxygenase maintained 25% of maximal activity (data not shown). The six other lipid-soluble compounds, diallyl sulfide, methallyl sulfide, dipropyl disulfide,
dipropyl sulfide, allyl mercaptan and allyl methyl sulfide were
not inhibitory at concentrations up to 1 mmol/L (data not
shown).
NADPH-CPR is the obligate electron transfer partner for
squalene monooxygenase. To determine whether inhibition of
CPR by garlic and its derivatives contributes to the inhibition
of squalene epoxidation, reductase activity was determined in
the presence of garlic extract and four of the inhibitory compounds identified above. A 5 g/L concentration of garlic extract inhibited 90% of squalene epoxidation and 70% of
cytochrome c reduction by CPR (Fig. 3). Similarly, a 100
mol/L concentration of each of the four garlic compounds
yielded similar levels of inhibition of squalene epoxidation
(Fig. 2) and cytochrome c reduction (Fig. 3). However, because the reductase concentration exceeds saturation in the
squalene monooxygenase reactions, the effect of a 70% decrease in reductase concentration, comparable to the extent of
inhibition of cytochrome c reduction by garlic extract, was
determined. A 70% decrease in reductase concentration reduced squalene monooxygenation by only 10 15%, indicating
that electron transfer from the reductase is not rate limiting in

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FIGURE 2 Effect of garlic and its derivatives on squalene monooxygenase activity. Standard squalene monooxygenase assay components were preincubated for 30 min at 37C with increasing concentrations of (A) aqueous garlic extract as a final concentration of its fresh
weight; (B) garlic-derived compounds, S-allylcysteine (SAC), alliin, diallyl disulfide (DADS), selenocystine (SC) or diallyl trisulfide (DATS).
Reactions were started by the addition of NADPH (1 mmol/L final
concentration) and incubated for 30 min. The dashed line indicates
activity in the absence of inhibitor (1.1 nmol/L 2,3-oxidosqualene/30
min reaction). Data are expressed as means SEM, n 4 independent
experiments.

the oxygenation of squalene, a finding consistent with earlier

studies (23). Thus, it is unlikely that the inhibition of CPR by
garlic or its components in this in vitro assay contributes
significantly to the decrease in squalene monooxygenase activity.
To determine whether the garlic compounds were interfering with the binding of squalene or FAD, the extent of
inhibition by a fixed concentration of each garlic compound
was determined in the presence of several concentrations of
squalene or FAD. Doubling the concentration of squalene to
80 mol/L decreased the extent of inhibition by the garlic
compounds by 20 60%, whereas changing the FAD concentration had no effect on inhibition by these agents (data not
shown). The protection afforded by squalene suggests that
these compounds bind in the substrate-binding pocket of the
enzyme and supports the conclusion that inhibition of CPR
does not contribute significantly to the inhibition of squalene
monooxygenation.
Garlic extract, SC, SAC, alliin and DADS were slow,
time-dependent inhibitors with t1/2 values (half-life for
inactivation) of 13, 22, 36, 41 and 91 min, respectively
(Fig. 4). These results suggest that these inhibitors bind
irreversibly to squalene monooxygenase. To address this
possibility, enzyme-inhibitor dilution experiments were carried out as described previously (24). In these experiments,
enzyme was preincubated with either garlic extract or one of
its inhibitory components for 30 min and then diluted
12-fold before the addition of 1 mmol/L NADPH to start
the reaction. No recovery of activity was seen after dilution
of garlic or the four garlic derivative-containing incubations, indicating that dilution did not release these compounds from the enzyme and that these inhibitors bind
irreversibly to squalene monooxygenase (Fig. 5).
Thiol-containing compounds were tested for their ability to
protect against and reverse the inhibition of squalene monooxygenase by garlic and by four of the inhibitory compounds.
GSH, a monothiol, or DMP, a dithiol, was added before or
after the 30-min preincubation. Addition of either thiol reagent to the preincubations yielded significant protection from
inhibition by garlic and the garlic derivatives, indicating that
the garlic components were reacting with sulfhydryls on
squalene monooxygenase (Fig. 6A). Inhibition by garlic and

FIGURE 3 Effect of garlic and garlic-derived compounds on cytochrome c reduction by cytochrome P450 reductase (CPR). Standard
reductase assay components were preincubated for 30 min at 37C
with 5 g/L of garlic extract or 100 mol/L concentrations of the four
garlic compounds, S-allylcysteine (SAC), alliin, selenocystine (SC) and
diallyl disulfide (DADS), followed by the addition of NADPH to start the
reaction. The 30% CPR bar shows the effect of a 70% decrease in CPR
concentration on squalene monooxygenase activity. Data are expressed as means SEM, n 4 independent experiments. Inhibition by
garlic extract and by the garlic compounds was significantly greater
than that produced by a 70% reduction in CPR (P 0.05, one-way
ANOVA with Dunnetts multiple comparison test).

INHIBITION OF SQUALENE MONOOXYGENASE BY GARLIC

1665

FIGURE 4 Slow, time-dependent inhibition of squalene monooxygenase by garlic and garlic-derived compounds. Assay mixtures containing NADPH but without squalene monooxygenase were preincubated for 30 min at 37C in the presence of 5 g/L of garlic extract or 100
mol/L of S-allylcysteine (SAC), alliin, selenocystine (SC) or diallyl
disulfide (DADS). The reactions were started by the addition of
squalene monooxygenase and incubated for the indicated times at
37C. Data are expressed as means SEM , n 3 independent
experiments. Half-life (t1/2) values of inactivation for garlic, SC, SAC,
alliin and DADS were 13, 22, 36, 41 and 91 min, respectively.

DISCUSSION
Experimental data from animal studies (7,14), clinical trials
(1,2), meta-analyses (35) and cell culture studies (9 11)

FIGURE 5 Irreversible inhibition of human squalene monooxygenase by garlic and its derivatives, S-allylcysteine (SAC), alliin, selenocystine (SC) and diallyl disulfide (DADS). Assay mixtures were preincubated for 30 min at 37C in a volume of 17 L containing either fresh
garlic extract or garlic derivative. After preincubation, the volume was
increased to 200 L and inhibitor concentration was held constant at 5
g/L of garlic extract or 0.1 mmol/L garlic derivative (constant concentration), or diluted from 60 to 5 g/L of garlic extract, or from 1.2 to 0.1
mmol/L garlic derivative (diluted concentration). The concentration of
all other reaction components was held constant and NADPH was
added to start the reaction. Reactions were carried out for 30 min at
37C. Data are expressed as means SEM, n 3 independent experiments.

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the garlic compounds could also be partially reversed by the

thiol reagents (Fig. 6B). DMP was a significantly more effective reversal agent than GSH, an effect most noticeable with
DADS and SC. This difference between the dithiol and the
monothiol was significant for three of the five substances
tested, i.e., alliin, DADS and SC. Similar results were obtained
with dithiothreitol and -mercaptoethanol, confirming that
dithiols were more effective reversal agents (data not shown).
These results are similar to those obtained previously in studies
on the inhibition of squalene monooxygenase by tellurite (24)
and are consistent with the garlic compounds reacting with
vicinal cysteine sufhydryls on squalene monooxygenase.

FIGURE 6 Effect of sulfhydryls on inhibition of squalene monooxygenase by garlic and its derivatives. (A) Protection experiments:
assay mixtures were preincubated at 37C with 5 g/L of garlic extract,
65 mol/L selenocystine (SC), 110 mol/L S-allylcysteine (SAC), 120
mol/L alliin or 400 mol/L diallyl disulfide (DADS) in the absence or
presence of 1 mmol/L of either 2,3-dimercaptopropanol (DMP) or glutathione (GSH). After 30 min, the reactions were started by the addition
of NADPH and incubated for an additional 30 min. Both thiol treatments
afforded significant protection from the garlic compounds (P 0.05,
one-way ANOVA with Dunnetts multiple comparisons test). (B) Reversal experiments: after a 30-min preincubation with garlic extract or
derivative, 1 mmol/L DMP or GSH was added along with NADPH to
start the reactions. Asterisks indicate the points at which reversal by
DMP was significantly greater than that produced by GSH (P 0.05,
one-way ANOVA with Bonferronis multiple comparison test). No significant reversal by either thiol compound was obtained with the garlic
extract, and GSH did not reverse the inhibition by DADS or SC. The
formation of 2,3-oxidosqualene in the presence of DMP but no inhibitor
was 1.76 nmol/L over 30 min; in the presence of GSH and no inhibitor,
it was 1.38 nmol/L. Data are expressed as means SEM, n 3
independent experiments.

have supported the postulate that garlic extracts and garlicderived compounds can lower cholesterol levels. Although
several studies have indicated that one site of inhibition in the
cholesterol biosynthetic pathway is HMG-CoA-reductase
(14,15), additional downstream enzymes have also been suggested as targets for garlic and its components (12). In this
work, we report the inhibition of purified recombinant human
squalene monooxygenase, a downstream enzyme in the cholesterol biosynthetic pathway, by an aqueous garlic extract and
by four organosulfur compounds and one organoselenium compound found in garlic. All four of the organosulfur compounds,
SAC, alliin, DATS and DADS, contain an S-allyl moiety.
Structurally analogous compounds that lack the allyl group,
such as S-propylcysteine and dipropyl disulfide, were not inhibitory, suggesting that the allyl group enhances the reactivity of these compounds. However, an S-allyl group is not
sufficient for reactivity because diallyl sulfide and methallyl

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GUPTA AND PORTER

The present work implicates the involvement of cysteine

sulfhydryls in the inhibition of squalene monooxygenase by
garlic and garlic-derived compounds. In previous work from
this laboratory, evidence was presented that the inhibition of
squalene monooxygenase by tellurium compounds results from
binding to critical sulfhydryl groups on the enzyme (24).
Similarly, N-ethylmaleimide, a sulfhydryl-specific reagent, was
shown to inhibit squalene monooxygenase in rat liver microsomes (22). Thus, squalene monooxygenase is highly sensitive
to inhibition by chemicals that react with sulfhydryls. The
inhibition of squalene monooxygenase by garlic and garlicderived compounds could be prevented by preincubation with
either GSH or DMP, indicating that these inhibitors react
with enzyme thiol groups. Addition of GSH or DMP after the
preincubation also partially reversed the inhibition, further
supporting the thesis that cysteine sulfhydryls on squalene
monooxygenase are the target of binding by these compounds.
The present work further suggests that there are differences
between the ability of monothiols and dithiols to reverse the
inhibition of squalene monooxygenase by garlic compounds.
DMP and dithiothreitol (dithiols) were more effective than
GSH or -mercaptoethanol (monothiols) in reversing the
inhibition, suggesting that the garlic compounds were reacting
with vicinal cysteines on the enzyme. The presence of vicinal
cysteines was proposed to explain the unusual sensitivity of
squalene monooxygenase to tellurium compounds (24). It
should be noted that heating the garlic extract or the four
garlic derivatives in a boiling water bath for 2 min completely
inactivated these compounds and prevented the inhibition,
indicating that they are relatively labile. Several other studies
have noted that raw garlic was significantly more effective
than cooked garlic in blocking cyclooxygenase activity in the
prevention of thrombosis (30,31).
Although we have identified five garlic-derived chemicals
that can inhibit squalene monooxygenase, we do not know
whether these are the only inhibitory chemicals in garlic
extract, or whether they mediate the inhibition of squalene
monooxygenase by garlic extract. Garlic contains a variety of
organosulfur and organoselenium compounds, with SAC and
alliin as the most prominent. SAC appears to be a precursor to
alliin; when garlic is crushed, alliinase is released to cleave
alliin to yield sulfenic acid (CH2CHCH2SOH), which condenses to form allicin. Allicin is also highly unstable, and
rapidly degrades to a variety of organosulfur compounds, including DADS, DATS and ajoene. These compounds give rise
to allyl mercaptan and, secondarily, allyl methyl sulfide; allyl
mercaptan has been suggested to be the ultimate active chemical species derived from garlic consumption (10). Although
allyl mercaptan is not inhibitory to squalene monooxygenase,
several of the precursors and intermediates leading to this
compound are inhibitory; most notably, SAC is one of the
more effective inhibitors found in this study. SAC is considered to be a principal active ingredient in commercial garlic
preparations; it is stable and readily absorbed from the gastrointestinal tract (32).
The present work demonstrates that the inhibition of
squalene monooxygenase by garlic is slow, irreversible and
likely to involve binding to sulfhydryls in the squalene binding
site of the enzyme. Garlic extract, as well as four of the
garlic-derived components, inhibited squalene epoxidation in
a time- and concentration-dependent manner; on the basis of
the protection and reversal by monothiols and dithiols, it is
likely that these inhibitors react with the proposed vicinal
cysteine sulfhydryls on squalene monooxygenase. The inhibition by the organoselenium compound, SC, is of particular
interest because overconsumption of selenium by pigs and

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sulfide were not inhibitory; both contain an S-allyl group, but,

in contrast to DADS, these compounds contain only a single
sulfur atom. It should be noted that alliin and SAC, both of
which contain an S-allyl group, also contain only a single
sulfur atom; evidently the peptide moiety of these compounds
promotes their reactivity. Although the thiol protection experiments indicate that these compounds react with sulfhydryls on squalene monooxygenase, the reactive groups on
these inhibitors and the products of the reaction remain to be
determined.
Of the four inhibitory organosulfur compounds, three have
been shown to be potent inhibitors of cholesterol synthesis in
cultured hepatocytes (SAC, DATS and DADS), whereas alliin was ineffective (11,27). Similarly, Liu and Yeh (11) found
that S-ethylcysteine and S-propylcysteine were equipotent
with SAC in decreasing cholesterol synthesis in hepatocytes,
but these compounds are not inhibitors of squalene monooxygenase. Thus, there is not an exact correspondence between
inhibitors of cholesterol synthesis in hepatocytes and inhibitors of squalene monooxygenase, and it may be that these
compounds can act at different steps in the cholesterol biosynthetic pathway, as has been suggested (27).
It is difficult to compare our studies with isolated enzyme to
in vivo studies on the reduction in blood cholesterol by garlic
consumption because the amount of garlic consumed and its
form (raw, cooked or as a garlic pill supplement) differ greatly
among studies. Most importantly, the concentration of garlic
compounds in the blood, and, more importantly, in the liver,
is not known. Study participants typically consume 13 g of
garlic per day (5,28,29), which presumably yields reasonable
levels of organosulfur compounds in the portal bloodstream
and contributes partially or wholly to the established ability of
garlic to decrease blood cholesterol levels. Because these organosulfur compounds are irreversible inhibitors of squalene
monooxygenase, it is conceivable, but not yet shown, that
even relatively low levels of these compounds in the blood
might be sufficient to inhibit squalene monooxygenase activity
and thereby decrease cholesterol synthesis.
Studies in vitro with hepatocyte culture are more amenable
to comparison. Inhibition of cholesterol synthesis in cultured
primary rat hepatocytes by SAC was evident at 100 mol/L
(11), a concentration that yielded 50% inhibition of squalene
monooxygenase in the present study. Similarly, DATS and
DADS were effective inhibitors of cholesterol synthesis at
concentrations of 100 200 mol/L (10), similar to their IC50
values for inhibition of squalene monooxygenase, although
higher concentrations were associated with significant cytotoxicity (11). Thus, inhibitory concentrations of several of
these garlic compounds with isolated hepatocytes correspond
to concentrations that inhibit purified squalene monooxygenase. Whether inhibition of squalene monooxygenase contributes to the inhibition of cholesterol synthesis by garlic in cell
culture and in vivo remains to be determined.
Garlic has been reported to contain high levels of selenium
and tellurium (17,18), especially if grown in soils containing
these elements, and it has been proposed that tellurium- and
selenium-containing compounds present in garlic contribute
to the block in cholesterol synthesis by inhibiting squalene
monooxygenase (21). In previous work from our laboratory, it
was shown that micromolar concentrations of selenite and
selenium dioxide inhibit purified human squalene monooxygenase (23). Here, we showed that SC, an organoselenium
compound present in garlic (19), inhibits squalene monooxygenase with an IC50 value of 65 mol/L. Se-(methyl)selenocysteine, selenomethionine or cystine were not inhibitory,
indicating that the diseleno-bond was necessary for inhibition.

INHIBITION OF SQUALENE MONOOXYGENASE BY GARLIC

cattle is associated with a variety of pathologies suggestive of

disruption of lipid metabolism in neural tissue (33). The effect
of garlic and its components on the intracellular flux of cholesterol intermediates in vivo is the subject of current investigations.
ACKNOWLEDGMENT
We thank Brian Laden for advice and assistance.

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