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Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082
2To whom correspondence should be addressed. E-mail: tporter{at}pop.uky.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • cholesterol biosynthesis • garlic • selenium • selenocystine • squalene epoxidase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,2)
, through meta-analyses of
clinical studies (3
4
5)
as well as in experimental studies
with animals (6
7
8)
and cultured hepatocytes
(9
10
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
(11
12
13)
. However, the mechanism underlying the inhibitory
action of these garlic compounds has not been established. Several
studies have reported that feeding garlic-supplemented 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 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 C=C double bond to yield 2,3-oxidosqualene, a
reaction more typical of cytochrome P450-type
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
(17
18
19
20)
; 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.
To address this possibility, fresh garlic extract and 16
garlic-derived 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 (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 (ß-NADPH), FAD, 2,3-dimercaptopropanol (DMP) and cytochrome c were purchased from Sigma Chemical, St. Louis, MO. Radiolabeled squalene (2.6 x 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 4°C 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 µmol/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 x g for 20 min at 25°C. The clear supernatant was divided into aliquots and stored at -80°C.
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 -80°C until use.
Squalene monooxygenase assays.
Squalene epoxidation assays were carried out as described previously
(23)
. Standard reaction mixtures (200 µL)
containing 20 mmol/L Tris-HCl (pH 7.4), 0.1% Triton X-100, 30
µmol/L FAD, 28 pmol CPR, 40 µmol/L
14C-squalene and 58 pmol squalene monooxygenase were
preincubated with or without inhibitor for 30 min at 37°C. The
reactions were then started by the addition of 1 mmol/L NADPH and
incubated for another 30 min at 37°C. Product formation was linear
for 40–60 min at 37°C, 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 µmol/L cytochrome
c and 5 pmol of reductase; reactions were initiated by
the addition of 50 µmol/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 37°C.
Statistical analysis.
Statistical analyses were carried out by one-way ANOVA with Dunnett’s or Bonferrroni’s 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 µmol/L, respectively (Fig. 2B
).
Inhibition by SC was nearly complete at 500 µmol/L,
whereas inhibition by SAC and alliin was still incomplete.
S-methylcysteine, S-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 µmol/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).
|
). Similarly, a 100 µmol/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 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.
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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
).
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). Inhibition by garlic and 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 ß-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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,14)
,
clinical trials (1
,2)
, meta-analyses
(3
4
5)
and cell culture studies (9
10
11)
have
supported the postulate that garlic extracts and garlic-derived
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 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 1–3 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 µmol/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 µmol/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 µmol/L.
Se-(methyl)selenocysteine, selenomethionine or cystine were not
inhibitory, indicating that the diseleno-bond was necessary for
inhibition.
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 garlic-derived 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 ß-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
(CH2=CHCH2SOH), 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
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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Manuscript received December 7, 2000. Initial review completed January 11, 2001. Revision accepted March 6, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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