Signicance of Garlic and Its Constituents in Cancer

and Cardiovascular Disease

In Vitro Interactions of Water-Soluble Garlic Components with Human

Cytochromes P45013
David J. Greenblatt,4 Richard A. Leigh-Pemberton, and Lisa L. von Moltke
Department of Pharmacology and Experimental Therapeutics, Tufts University School of
Medicine and Tufts-New England Medical Center, Boston, MA

KEY WORDS:  aged garlic extract  Cytochromes P450  in vitro metabolism  drug interaction

The increasingly extensive utilization of complementary or

alternative medical therapies over the last decade is now well
documented (17). This incorporates the use of botanical
medicines either alone or in combination with prescription
medications. With the increased prevalence of botanical use
comes the need for clinical and scientific data on the pharmacologic properties, mechanisms of action, drug interactions,

and adverse effects of these agents such that consumers and

health care providers will have the information available to
maximize therapeutic benefits of botanicals while minimizing the
likelihood of unwanted effects.
Commercial promotion of botanicals usually emphasizes that
they are safe, natural, and contain no chemicals. In fact,
plant systems have their own metabolic processes, and human
evolution has created processes to biotransform and eliminate
ingested plants. As such, plants can induce, inhibit, or be toxic
to human metabolic systems, and seemingly safe and natural
plant products may have predictable influences on human drug
metabolism. Of particular concern are the increasing numbers
of clinical and scientific reports of drug interactions involving
botanical products (819). Some of these interactions, for
example, those involving St. Johns wort, are of major clinical
importance. Mechanisms investigated to date include the
possibility that botanical medicines may induce or inhibit the
activity of human Cytochrome P450 enzymes or the activity of
transport proteins such as P-glycoprotein (20). Because the
number of botanical medicines in clinical use is very large, it is
simply not feasible to conduct clinical studies for all possible
drug interactions that need to be studied and understood.
Accordingly, there are now large gaps in knowledge, and recommendations regarding which drug combinations with botanicals are safe or unsafe are often based on incomplete data.
An extensive literature supports the existence of the therapeutically beneficial effects of garlic preparations in the prevention of atherogenesis and neoplastic disease (2131). A number
of components in garlic are postulated to act synergistically to
provide these health benefits (3239). Due to the complex
chemistry of garlic, variations in processing yield quite different
preparations. Highly unstable thiosulfinates, such as allicin,

1
Published in a supplement to The Journal of Nutrition. Presented at the
symposium Signicance of Garlic and Its Constituents in Cancer and Cardiovascular Disease held April 911, 2005 at Georgetown University, Washington, DC.
The symposium was sponsored by Strang Cancer Prevention Center, afliated
with Weill Medical College of Cornell University, and Harbor-UCLA Medical Center,
and co-sponsored by American Botanical Council, American Institute for Cancer
Research, American Society for Nutrition, Life Extension Foundation, General
Nutrition Centers, National Nutritional Foods Association, Society of Atherosclerosis Imaging, Susan Samueli Center for Integrative Medicine at the University of
California, Irvine. The symposium was supported by Alan James Group, LLC,
Agencias Motta, S.A., Antistress AG, Armal, Birger Ledin AB, Ecolandia Internacional, Essential Sterolin Products (PTY) Ltd., Grand Quality LLC, IC Vietnam,
Intervec Ltd., Jenn Health, Kernpharm BV, Laboratori Mizar SAS, Magna Trade,
Manavita B.V.B.A., MaxiPharm A/S, Natures Farm, Naturkost S. Rui a.s., Nichea
Company Limited, Nutra-Life Health & Fitness Ltd., Oy Valioravinto Ab, Panax, PT.
Nutriprima Jayasakti, Purity Life Health Products Limited, Quest Vitamins, Ltd.,
Sabinco S.A., The AIM Companies, Valosun Ltd., Wakunaga of America Co. Ltd.,
and Wakunaga Pharmaceutical Co., Ltd. Guest editors for the supplement
publication were Richard Rivlin, Matthew Budoff, and Harunobu Amagase. Guest
Editor Disclosure: R. Rivlin has been awarded research grants from Wakunaga
of America, Ltd. and received an honorarium for serving as co-chair of the
conference; M. Budoff has been awarded research grants from Wakunaga of
America, Ltd. and received an honorarium for serving as co-chair of the
conference; and Harunobu Amagase is employed by Wakunaga of America, Ltd.
2
Author disclosure: No relationships to disclose.
3
This work was supported in part by grants AT-01381, AI-55412, MH-58435,
DA-13209, DK/AI-58496, DA-13834, AG-17880, AI-58784, and RR-00054 from the
U.S. Department of Health and Human Services.
4
To whom correspondence should be addressed: E-mail: dj.greenblatt@
tufts.edu.

0022-3166/06 $8.00 2006 American Society for Nutrition.

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Downloaded from jn.nutrition.org by on October 19, 2010

ABSTRACT Eight water-soluble components of aged garlic extract were evaluated to assess their potential to inhibit the activity of human cytochrome-P450 (CYP) enzymes. The in vitro model consisted of human liver microsomes
with index reactions chosen to prole the activity of the following six CYP isoforms: CYP1A2, 2B6, 2C9, 2C19, 2D6,
and 3A. With only 2 exceptions, none of the 8 garlic components produced .50% inhibition even at high concentrations
(100 mmol/L). S-methyl-L-cysteine and S-allyl-L-cysteine at 100 mmol/L produced modest inhibition of CYP3A, reducing activity to 2040% of control. However available clinical evidence does not indicate CYP3A inhibition in vivo.
The ndings suggest that drug interactions involving inhibition of CYP3A enzymes by aged garlic extract are very
unlikely. J. Nutr. 136: 806S809S, 2006.

WATER-SOLUBLE GARLIC AND HUMAN CYTOCHROMES P450

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TABLE 1
Experimental conditions for inhibition studies
CYP isoform

Substrate (concentration,
mmol/L)

1A2
2B6
2C9
2C19
2D6
3A

Phenacetin (100)
Bupropion (250)
Flurbiprofen (5)
S-mephenytoin (25)
Dextromethorphan (25)
Triazolam (250)

Product(s)

Mobile phase
composition*, %

Detection
mode, nm

Acetaminophen
OH-bupropion
4-OH-urbiprofen
49-OH-mephenytoin
Dextrorphan
a-OH-triazolam, 4-OH-triazolam

CH3CN:buffer 15:85
CH3CN:buffer 21:79
CH3CN:Na acetate (10 mM) 30:70
CH3CN:buffer 42:58
CH3CN:buffer 30:70
CH3CN:CH3OH:buffer 22.5:10:67.5

U.V. 254
U.V. 214
U.V. 230
Fluorescence: excit. 260 emis. 320
Fluorescence: excit. 250 emis. 310
U.V. 220

* Aqueous buffer is 50 mmol/L KH2PO4.

The present study utilized the in vitro model to screen for

potential inhibitory metabolic effects of a number of components of aged garlic extract.
METHODS
Liver samples from individual human donors with no known
liver disease were provided by the International Institute for the
Advancement of Medicine (Exton, PA) or the Liver Tissue
Procurement and Distribution System (University of Minnesota,
Minneapolis). All samples were of the CYP2D6 and CYP2C19 normal
metabolizer phenotype based on in vitro phenotyping studies.
Microsomes were prepared by ultracentrifugation; microsomal
pellets were suspended in 0.1 mmol/L potassium phosphate buffer
containing 20% glycerol and stored at 808C until use.
Incubation mixtures contained 50 mmol/L phosphate buffer, 5
mmol/L Mg11, 0.5 mmol/L NADP1, and an isocitrate/isocitric
dehydrogenase regenerating system. Appropriate substrates (Table 1),
with and without an inhibitor in methanol solution, were added to a
series of incubation tubes. The solvent was evaporated to dryness at
408C under conditions of mild vacuum. Reactions were initiated by
addition of microsomal protein. After an appropriate incubation
duration at 378C, reactions were stopped by cooling on ice and
addition of 100 mL of acetonitrile. Internal standard was added, the
incubation mixture centrifuged, and the supernatant transferred to an
autosampling vial for HPLC analysis.
The activity of 6 human CYP isoforms were evaluated using index
reactions and methods as follows (43,46,4857) (see Table 1): CYP1A2, phenacetin (100 mmol/L) to acetaminophen; CYP-2B6, bupropion
(250 mmol/L) to hydroxybupropion; CYP-2C9, flurbiprofen (5 mmol/L)
to OH-flurbiprofen; CYP-2C19, S-mephenytoin (25 mmol/L) to
49-OH-mephenytoin; CYP-2D6, dextromethorphan (25 mmol/L) to
dextrorphan; CYP-3A, triazolam (250 mmol/L) to a-OH-triazolam and
4-OH-triazolam.

TABLE 2
Effect of aged garlic extract components on activity of human
Cytochrome P450 isoforms in vitro
Inhibitory effect vs. Cytochrome P450 isoforms
Garlic component*
Alliin
Cycloalliin
Methylin
S-Methyl-L-cysteine
SAC
N-Acetyl-SAC
S-Allomercapto-L-cysteine
Gamma-glutamyl-SAC

CYP3A

CYP2C9

CYP2C19

CYP2D6

CYP1A2

CYP2B6

1
1

* All components were tested at a concentration of 100 mmol/L. SAC, S-allyl-L-cysteine.

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disappear during processing and are quickly and extensively

transformed (34). Efficacy and safety are also contingent upon
processing methods. The process of extraction has been assumed
to increase the potency and bioavailability of various crude herbs
and eliminate their harsh and toxic characteristics. The
irritating, acidic, and oxidizing compounds in raw garlic can be
eliminated and modified through the extraction process. In fact,
in some cultures, garlic is soaked or extracted with alcohol, wine,
milk, or vinegar before being used as a therapeutic agent. Many
adverse reactions to garlic ingestion can be attributed to the oilsoluble constituents derived from allicin. The lipid-lowering
effect attributed to oil-soluble sulfur compounds in hepatocytes
may be due to cytotoxicity, as revealed by increased lactate
dehydrogenase from cells exposed to various oil-soluble components. Acetone has been detected in the breath of subjects
consuming allicin-derived oil-soluble compounds, further suggesting the cytotoxicity of such compounds. On the other hand,
water-soluble sulfur compounds, though effective at reducing
cholesterol, are not cytotoxic. Aged garlic extract contains a
number of the water-soluble constituents, such as S-allylcysteine, that significantly reduce its toxicity, as confirmed by
various toxicological studies together with (32).
Clinical studies evaluating drug interactions with garlic
preparations are limited (4042). This is not surprising, since
design and execution of pharmacokinetic drug interaction
studies in humans are costly and time consuming. Recently, in
vitro systems, using human liver microsomal preparations, have
been increasingly utilized as approaches to screening for drug
interactions that may be probable, possible, or unlikely (4347).
Data from these in vitro models can be utilized as a guide for
targeting of clinical resources such that the most important
research priorities are addressed.

SUPPLEMENT

808S

Pure samples of water-soluble components of aged garlic extract

(Table 2) were donated by Dr. Harunobu Amagase of Wakunaga of
America. Solutions were prepared in methanol. Inhibitory effects of
100 mmol/L concentrations of each component were evaluated in each
of the in vitro systems. For studies of CYP3A activity using triazolam as
the substrate, incubations were performed both without and with
preincubation of inhibitor with triazolam. This is done to evaluate the
possibility of irreversible or mechanism-based inhibition (5860).

RESULTS
The outcome of in vitro studies is shown in Table 2. A minus
sign () indicates ,50% inhibition of the index reaction
velocity; a plus sign (1) indicates .50% inhibition. The concentrations of garlic components in the incubation mixtures
(100 mmol/L) are very high, probably exceeding by an order of
magnitude or more the in vivo exposure that would be
anticipated. Therefore the anticipated levels of clinical exposure confer a very low risk of pharmacokinetic drug interactions
including metabolic inhibition of any of the indicated CYP
isoforms.
In only 2 instances, inhibition of CYP3A exceeded 50%.
S-allyl-L-cysteine, an important component in terms of biologic
effects of aged garlic extract, reduced CYP3A activity to 40%
of the control (Fig. 1). An evaluation of the concentration
dependence of the inhibitory action did not demonstrate clear
evidence of classical concentration effect. Furthermore, inhibition of the 2 parallel pathways of triazolam hydroxylation
(a-OH- and 4-OH-triazolam formation) revealed differential
inhibition of the 2 pathways (Fig. 2). There was no evidence
that the character of inhibition was mechanism based.

DISCUSSION
Benefits and limitations of in vitro systems have been
extensively discussed (4347). Of importance is that human

microsomal systems can be used to evaluate inhibition, not

induction. Furthermore, quantitative measures of the inhibitory potency of foods and natural products derived from in vitro
studies are notoriously difficult to interpret in terms of the
predictability of clinical drug interactions. This is because processes such as glycone removal and glucuronide conjugation
may occur before natural substances reach hepatic enzymes or
the systemic circulation (6163). As an example, certain components of Ginkgo biloba (such as amentoflavone) are potent in
vitro inhibitors of human cytochrome P450 2C9 (64), but there
is no evidence to indicate that administration of ginkgo to
humans alters CYP2C9 activity in vivo (65,66).
The present study indicates that water-soluble garlic components are highly unlikely to inhibit activity of the 6 human
cytochrome P450 isoforms responsible for the majority of drug
metabolism reactions. The in vitro screen did reveal the possibility of modest inhibition of CYP3A by S-methyl-L-cysteine
and S-allyl-L-cysteine. Although available data (41) provides
no evidence that garlic inhibits CYP3A in vivo, the possibility could be confirmed or ruled out through a straightforward clinical drug-interaction study involving a suitable CYP3A
substrate such as midazolam (67).
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FIGURE 1 Effect of coaddition of S-allyl-L-cysteine (100 mM) on the

in vitro activity of human Cytochromes P450 3A, 2C9, 2C19, and IA2,
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FIGURE 2 Mean (6SE) rates of formation of triazolam metabolites

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