Antimalarial activity of allicin, a biologically active compound from garlic cloves

Article abstract of DOI:10.1128/AAC.50.5.1731-1737.2006

The incidence of malaria is increasing, and there is an urgent need to identify new drug targets for both prophylaxis and chemotherapy. Potential new drug targets include Plasmodium proteases that play critical roles in the parasite life cycle. We have previously shown that the major surface protein of Plasmodium sporozoites, the circumsporozoite protein (CSP), is proteolytically processed by a parasite-derived cysteine protease, and this processing event is temporally associated with sporozoite invasion of host cells. E-64, a cysteine protease inhibitor, inhibits CSP processing and prevents invasion of host cells in vitro and in vivo. Here we tested allicin, a cysteine protease inhibitor found in garlic extracts, for its ability to inhibit malaria infection. At low concentrations, allicin was not toxic to either sporozoites or mammalian cells. At these concentrations, allicin inhibited CSP processing and prevented sporozoite invasion of host cells in vitro. In vivo, mice injected with allicin had decreased Plasmodium infections compared to controls. When sporozoites were treated with allicin before injection into mice, malaria infection was completely prevented. We also tested allicin on erythrocytic stages and found that a 4-day regimen of allicin administered either orally or intravenously significantly decreased parasitemias and increased the survival of infected mice by 10 days. Together, these experiments demonstrate that the same cysteine protease inhibitor can target two different life cycle stages in the vertebrate host. Copyright

Full text of DOI:10.1128/AAC.50.5.1731-1737.2006

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2006, p. 1731–1737
0066-4804/06/$08.00ϩ0 doi:10.1128/AAC.50.5.1731–1737.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 5
Antimalarial Activity of Allicin, a Biologically
Active Compound from Garlic Cloves
Alida Coppi,1 Melissa Cabinian,1 David Mirelman,2 and Photini Sinnis1*
Department of Medical Parasitology, New York University School of Medicine, New York, New York 10010,1
and Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel2
Received 21 October 2005/Returned for modification 22 November 2005/Accepted 1 March 2006
The incidence of malaria is increasing, and there is an urgent need to identify new drug targets for both
prophylaxis and chemotherapy. Potential new drug targets include Plasmodium proteases that play critical
roles in the parasite life cycle. We have previously shown that the major surface protein of Plasmodium
sporozoites, the circumsporozoite protein (CSP), is proteolytically processed by a parasite-derived cysteine
protease, and this processing event is temporally associated with sporozoite invasion of host cells. E-64, a
cysteine protease inhibitor, inhibits CSP processing and prevents invasion of host cells in vitro and in vivo.
Here we tested allicin, a cysteine protease inhibitor found in garlic extracts, for its ability to inhibit malaria
infection. At low concentrations, allicin was not toxic to either sporozoites or mammalian cells. At these
concentrations, allicin inhibited CSP processing and prevented sporozoite invasion of host cells in vitro. In
vivo, mice injected with allicin had decreased Plasmodium infections compared to controls. When sporozoites
were treated with allicin before injection into mice, malaria infection was completely prevented. We also tested
allicin on erythrocytic stages and found that a 4-day regimen of allicin administered either orally or intrave-
nously significantly decreased parasitemias and increased the survival of infected mice by 10 days. Together,
these experiments demonstrate that the same cysteine protease inhibitor can target two different life cycle
stages in the vertebrate host.
Malaria is one of the most important infectious diseases in
the world. Each year 300 to 500 million new cases are diag-
nosed and approximately 1.5 million people die of the disease,
the majority of whom are children (11). More than 40% of the
world’s population lives in malaria-endemic areas and is at risk
of contracting the disease (11). Infection is initiated when
Plasmodium sporozoites are injected into the skin of a verte-
brate host by an infected anopheline mosquito. The sporozo-
ites enter the bloodstream and travel to the liver, where they
invade hepatocytes, differentiate, and divide asexually to pro-
duce exoerythrocytic forms. Upon maturation, exoerythrocytic
forms rupture and release merozoites that invade erythrocytes
and initiate the blood stage of infection, which is responsible
for the symptoms of the disease.
The incidence of malaria is increasing due to several factors,
including resistance of the parasite to currently available anti-
malarial drugs, and there is an urgent need to develop new
drugs for both the prophylaxis and treatment of malaria (11).
Among the targets being explored for the development of new
drugs are the proteases of Plasmodium, which play critical
roles in the parasite’s life cycle and can be targeted with spe-
cific inhibitors (reviewed in references 3, 20–22, and 36).
We have recently found that the major surface protein of the
sporozoite, the circumsporozoite protein (CSP), is proteolyti-
cally processed by a parasite cysteine protease during invasion
and that E-64, a cysteine protease inhibitor, inhibits CSP pro-
cessing as well as sporozoite infectivity in vitro and in vivo (6).
Other groups studying the erythrocytic stages of Plasmodium
have found that parasite cysteine proteases play critical roles in
hemoglobin degradation (39, 40) and merozoite release from
erythrocytes (37). Taken together, these data suggest that cys-
teine protease inhibitors may target both preerythrocytic and
erythrocytic stages of Plasmodium and may therefore be good
drug candidates for the prevention and treatment of malaria.
The anti-infective properties of garlic have long been known
to Chinese and Indian civilizations and were first described in
Europe by Louis Pasteur (13). Garlic has an unusually high
concentration of sulfur-containing compounds, and its antibac-
terial properties are largely due to one particular class of sul-
fur-containing compounds, the thiosulfinates (18). The thio-
sulfinate structure [S(AO)S] appears to be essential for the
bactericidal, antifungal, and antiprotozoal properties of garlic,
likely reacting with SH-containing enzymes of these pathogens
(34, 45). Allicin is the most abundant thiosulfinate found in
garlic and is generated when the enzyme alliinase reacts with
its substrate alliin (18, 41). Enzyme and substrate are located in
different compartments of the clove, so that allicin is generated
only when the clove is crushed (18, 41). Many lines of evidence
indicate that allicin is primarily responsible for garlic’s anti-
infective properties (1, 5, 15, 34, 38, 43), although studies have
also found that ajoene, a metabolite of allicin found when
garlic is crushed specifically in oil, also has some antibacterial
properties (27). In fact, one study found that ajoene has an
inhibitory effect on the erythrocytic stages of Plasmodium (30).
The precise mechanism of action of the thiosulfinates has, in
many cases, not been demonstrated. However, when used at
low concentrations, allicin appears to react specifically with the
free sulfhydryl group present in the active site of cysteine
proteases (32). Experiments with the intestinal parasite Ent-
amoeba histolytica have shown that pure allicin inhibits both
* Corresponding author. Mailing address: Department of Medical
Parasitology, 341 East 25th Street, New York, NY 10010. Phone: (212)
263-6818. Fax: (212) 263-8116. E-mail: photini.sinnis@med.nyu.edu.
1731

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COPPI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
Sporozoite invasion assays. Invasion assays were performed as previously
described (31) with some modifications. P. berghei sporozoites were preincubated
with the indicated concentrations of allicin for 10 min at 28°C, diluted 12-fold
with DMEM-BSA, and added to Hepa 1-6 cells. Sporozoites were plated in each
well of semiconfluent cells (5 ϫ 104/well). After 1 h at 37°C, cells were washed
and fixed, and sporozoites were stained with a double staining assay that distin-
guishes between intracellular and extracellular sporozoites (33).
Assay for sporozoite infectivity in vivo. Female Swiss Webster mice, 5 to 6
weeks old, were injected intravenously (i.v.) with either 5 or 8 mg/kg of body
weight of allicin (in DMEM without Cys/Met) 60 min, 30 min, or immediately
before i.v. injection of 104 P. yoelii sporozoites. Forty hours later, livers were
harvested, total RNA was isolated, and malaria infection was quantified using
reverse transcription followed by real-time PCR with primers that recognize P.
yoelii-specific sequences within the 18S rRNA as previously described (4). Ten-
fold dilutions of a plasmid construct containing the P. yoelii 18S rRNA gene were
used to create a standard curve. For allicin preincubation experiments, P. yoelii
sporozoites were preincubated with or without 50 ␮M allicin (in DMEM without
Cys/Met) for 10 min at 28°C and diluted 12-fold with medium before i.v. injection
into mice. All in vivo data were analyzed using the Student t test for unpaired
samples. All experiments were performed twice with six mice per group per
experiment.
the cytopathological effects associated with infection (2) and the
growth of the parasite (23) via its inhibitory effect on the parasite’s
cysteine proteases. Because of allicin’s inhibitory activity on cys-
teine proteases and Plasmodium’s requirement for cysteine pro-
tease activity during various life cycle stages, we set out to test the
effects of allicin on the preerythrocytic and erythrocytic stages of
Plasmodium.
MATERIALS AND METHODS
Parasites. Plasmodium berghei (NK65 and ANKA strains) and Plasmodium
yoelii (17XNL) sporozoites were grown in Anopheles stephensi mosquitoes as
previously described (26) and were obtained from infected salivary glands on the
day of the experiment.
Antibodies. Monoclonal antibody (MAb) 3D11, directed against the repeat
region of P. berghei CSP (46), was conjugated to Sepharose (14) and biotinylated
using D-biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester as outlined in
the manufacturer’s protocol (Roche Applied Science).
Allicin preparation. Pure allicin was prepared by passing the synthetic sub-
strate alliin (24) through an immobilized alliinase column (38). The concentra-
tion of allicin was determined using a spectrophotometric assay with a chromo-
genic thiol (25) and confirmed by high-performance liquid chromatography (7).
Dilute aqueous allicin solutions (1.8 mg/ml) were stored in the dark at 4°C for Ͻ3
months. When used in experiments with parasites, allicin was diluted in medium
without Cys/Met, since these amino acids react with and inactivate the drug.
Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel elec-
trophoresis analysis. P. berghei sporozoites were metabolically labeled for 1 h in
Dulbecco’s modified Eagle medium (DMEM) with L-[35S]Cys/Met as previously
described (6) and chased in the presence of 10 ␮M E-64 or the indicated
concentrations of allicin. Labeled sporozoites were lysed in 1% Triton X-100–150
mM NaCl–50 mM Tris-HCl, pH 8.0, with protease inhibitors, and lysates were
incubated with 3D11-Sepharose overnight at 4°C. CSP was eluted and run on a
7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel under nonreducing con-
ditions. The gel was fixed, enhanced with Amplify (Amersham Pharmacia),
dried, and exposed to film.
Cell contact assay. P. berghei sporozoites transgenic for green fluorescent
protein (GFP) (8) were incubated in DMEM with or without 50 ␮M allicin for
10 min at 28°C, diluted 12-fold to 4.2 ␮M allicin with DMEM, and then centri-
fuged (300 ϫ g) onto coverslips with Hepa 1-6 cells (CRL-1830; American Type
Culture Collection) at 4°C. Coverslips were then incubated at 37°C for 2 min,
fixed with 4% paraformaldehyde, and stained with polyclonal antiserum that
stains only full-length CSP (6), followed by antirabbit immunoglobulin conju-
gated to Texas Red. Sporozoites were counted using a Nikon Eclipse E600
fluorescence microscope, and each field was viewed with two filters so that GFP
sporozoites and Texas Red staining sporozoites could be enumerated.
Allicin toxicity assay. P. berghei sporozoites were incubated with the indicated
concentrations of allicin for 10 or 60 min at 28°C, washed with DMEM, and then
incubated with 1 ␮g/ml propidium iodide for 5 min at 25°C. The number of
fluorescent sporozoites in each sample was counted using a Nikon Eclipse E600
microscope. Control samples consisted of sporozoites that were incubated for 60
min at 28°C in DMEM alone to assess background level of propidium iodide
uptake and sporozoites that were heat killed at 65°C for 10 min to insure that the
assay was working.
Gliding motility assay. Glass eight-chambered Lab-Tek wells (Nalgene) were
coated with 10 ␮g/ml MAb 3D11 in phosphate-buffered saline (PBS) overnight
at 25°C and then washed three times with PBS. Precoating the wells with anti-
body captures shed CSP onto the slide for better visualization of the trails. P.
berghei (2 ϫ 104/well) sporozoites were incubated with 50 ␮M allicin in DMEM
without Cys/Met for 10 min at 28°C. The medium was removed and replaced with
DMEM–3% bovine serum albumin (BSA) containing either 50 ␮M or 4.2 ␮M
allicin before sporozoites were added to the coated Lab-Tek wells. Sporozoites
were incubated for 1 h at 37°C, the medium was removed, and the wells were
fixed with 4% paraformaldehyde, washed, and blocked with PBS–1% BSA. To
visualize the CSP-containing trails, the wells were then incubated with biotinyl-
ated MAb 3D11 followed by Streptavidin-fluorescein isothiocyanate (1:100 di-
lution; Amersham Pharmacia). All incubations were performed at 37°C for 1 h.
Controls included untreated sporozoites and sporozoites added to wells in the
presence of 1 ␮M cytochalasin D. For each group, gliding motility was quantified
by counting the number of sporozoites associated with trails and, for those
sporozoites with trails, counting the number of circles in each trail.
Assay for efficacy against erythrocytic stages in vivo. The standard 4-day
suppression test (29) with some modifications was used to assess the efficacy of
allicin against malaria erythrocytic stages in vivo. Female Swiss Webster mice, 5
to 6 weeks old, were injected i.v. with 2 ϫ 105 GFP-expressing P. berghei parasites
(8), and 1 h later mice were injected i.v. with either 8 mg/kg of allicin (in DMEM
without Cys/Met) or medium alone. Mice were treated with allicin or buffer once
daily for an additional 3 days. For the experiments in which allicin was admin-
istered orally, Swiss Webster mice were infected with GFP-expressing parasites
as above, and 1 h later, allicin (diluted in water) or water alone was administered
by gavage. Total daily dosage was either 3 mg/kg/day or 9 mg/kg/day, adminis-
tered in two doses (one in the morning and one in the evening) to decrease
irritation to the gastric mucosa. Following treatment, survival of the mice was
monitored and parasitemia was determined by fluorescence-activated cell sorting
(FACS) analysis. For FACS, 2 ␮l of blood was diluted in 1 ml PBS containing 1%
fetal calf serum and 0.01% NaN3, and the number of fluorescent cells was
determined using the FACS Calibur System with CellQuest Software (Becton
Dickinson). Statistical significance was determined using the Student t test for
unpaired samples. All experiments were performed twice with five mice per
group per experiment.
RESULTS
Inhibition of CSP cleavage. We have previously shown that
the cysteine protease inhibitor E-64 prevents proteolytic cleav-
age of CSP, the major surface protein of Plasmodium sporo-
zoites (6). Because allicin has been shown to react with free
sulfhydryl groups and reversibly inhibit cysteine proteases (2),
we tested whether allicin would inhibit CSP cleavage. Pulse-
chase metabolic labeling experiments, in which allicin was in-
cluded in the chase, indicate that CSP cleavage was inhibited
by 10, 25, and 50 ␮M allicin (Fig. 1A). The degree of inhibition
was comparable to that observed with 10 ␮M E-64. In addition,
chasing with 50 ␮M allicin for 10 min followed by dilution to
4.2 ␮M for the remainder of the chase prevented CSP cleavage
to the same extent as when 50 ␮M allicin was present during
the entire chase. The pulse-chase metabolic labeling experi-
ments are performed with sporozoites in the absence of host
cells. Under these conditions, the half-life of full-length CSP is
on the order of hours (6, 46). However, when sporozoites are
added to cells, cleavage occurs in minutes (6). Likely this re-
flects leaky microneme secretion of the protease in the absence
of cells versus triggered secretion when sporozoites contact
cells. We also tested the effect of allicin on CSP cleavage in the
presence of host cells. For this, we added sporozoites to cells
for 2 min and then counted the number of sporozoites that
stained for full-length CSP. As shown in Fig. 1B, allicin also

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ANTIMALARIAL EFFECTS OF ALLICIN
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FIG. 2. Toxicity of allicin for Plasmodium sporozoites. P. berghei
sporozoites were incubated with the indicated concentrations of allicin
for 10 min (gray bars) or 60 min (black bars) before the addition of
propidium iodide. The “50 dil” bar indicates that sporozoites were
incubated with 50 ␮M allicin for 10 min, followed by a 50-min incu-
bation in 4.2 ␮M allicin. Control sporozoites were incubated in the
absence of allicin for 60 min (white bar) or were heat killed (diagonally
striped bar). For each sample, 200 sporozoites were counted and the
percentage staining with propidium iodide is shown. This experiment
was repeated three times, and a representative experiment is shown.
FIG. 1. Allicin prevents cleavage of CSP. (A) P. berghei sporozoites
were metabolically labeled with [35S]Cys/Met and kept on ice (labeled
0) or chased for 2 h in the absence of protease inhibitors (labeled 2),
in the presence of 10 ␮M E-64 (labeled E-64), or in the presence of the
indicated concentrations of allicin (labeled 10, 25, and 50). The lane
labeled 50dil represents labeled sporozoites chased in the presence of
50 ␮M allicin for 10 min, which was then diluted to 4.2 ␮M for the
remainder of the chase. After 2 h, the parasites were lysed and CSP
was immunoprecipitated and analyzed by SDS-polyacrylamide gel
electrophoresis and autoradiography. (B). P. berghei sporozoites were
incubated in the presence or absence of 50 ␮M allicin for 10 min and
then added to Hepa 1-6 cells for 2 min before being fixed and stained
with antisera specific for full-length CSP. Two hundred sporozoites/
well were counted, and means Ϯ standard deviations for duplicate
samples are shown.
than 50 ␮M, allicin was toxic to the sporozoites after only a
10-min exposure.
Allicin does not inhibit gliding motility. Since uptake of
propidium iodide is a terminal event, we also tested whether
sporozoites incubated with allicin were still motile. Plasmo-
dium sporozoites exhibit a unique form of substrate-dependent
locomotion, termed gliding motility, which is required for cell
invasion (42). We reasoned that if sporozoites were motile in
the presence of allicin, then the compound was not affecting
the overall health and metabolic activity of the parasites. When
we tested allicin in motility assays, we found that preincubation
with 50 ␮M allicin for 10 min followed by dilution to 4.2 ␮M
had no effect on gliding motility (Fig. 3A). In addition, both the
percentage of sporozoites that exhibited gliding motility and
the number of circles per trail were the same in the allicin-
treated sporozoites and in controls (Fig. 3B). However, if al-
licin was not diluted and sporozoites were kept in 50 ␮M allicin
for the duration of the assay, gliding motility was completely
inhibited (Fig. 3A). This is consistent with the toxicity profile of
allicin that we observed using propidium iodide: prolonged
incubations in 50 ␮M allicin were toxic, whereas a 10-min
incubation in 50 ␮M allicin followed by an incubation in 4.2
␮M was not.
Allicin prevents cell invasion. Since allicin inhibited CSP
cleavage and our previous studies showed that cleavage is
associated with cell invasion, we next tested whether allicin
would inhibit invasion of host cells. For these experiments, P.
berghei sporozoites were pretreated with 10, 25, and 50 ␮M
allicin for 10 min, the allicin was diluted 12-fold, and then
sporozoites plus the diluted allicin were added to cells. As
shown in Fig. 4, allicin inhibited sporozoite invasion of cells in
a dose-dependent manner. At the lowest concentration tested
(10 ␮M), allicin inhibited invasion by 37%. When sporozoites
inhibits cleavage of CSP when sporozoites are in the presence
of cells, indicating that it rapidly interacts with the CSP pro-
tease.
Allicin toxicity. In order to determine if the effect of allicin
on CSP cleavage was due to a toxic effect on the sporozoites,
we incubated parasites for 10 or 60 min with different concen-
trations of allicin and then added propidium iodide, a dye that
is excluded by viable cells but penetrates the cell membranes of
dead or dying cells. When sporozoites were incubated with
either 1 or 10 ␮M allicin for up to 60 min, the percentage of
sporozoites that took up the dye was no different from that of
the untreated control (Fig. 2). A 10-min incubation with 50 ␮M
allicin also did not kill sporozoites; however, when the incuba-
tion time was increased to 60 min, the number of sporozoites
taking up the dye increased 1.5-fold, indicating that longer
exposures to 50 ␮M allicin had a toxic effect on the sporozo-
ites. Treatment of sporozoites with 50 ␮M allicin for 10 min,
followed by dilution of the allicin to 4.2 ␮M for an additional
50 min, did not increase the number of fluorescent sporozoites
compared to the untreated control. At concentrations higher

1734
COPPI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Allicin inhibits sporozoite invasion of host cells. P. berghei
sporozoites were pretreated for 10 min with the indicated concentra-
tions of allicin, which was then diluted 12-fold before sporozoite ad-
dition to cells. After 1 h, cells were fixed and stained, and the numbers
of intracellular and extracellular sporozoites were determined. “50*”
indicates that Hepa 1-6 cells were preincubated with 50 ␮M allicin for
1 h and washed, and untreated sporozoites were then added to the
cells. Each point was performed in triplicate, Ն50 fields/well were
counted, and the means Ϯ standard deviations are shown. Inhibition of
invasion was calculated based on the invasion rate for sporozoites
pretreated with buffer alone, which was 57%.
between allicin injection and sporozoite injection. When allicin
was administered just before injection of sporozoites, it signifi-
cantly decreased sporozoite infectivity compared to untreated
controls (P Ͻ 0.001). Allicin injected 30 min prior to injection of
sporozoites also resulted in decreased infectivity compared to the
untreated mice (P Ͻ 0.001), but the protective effect was not as
great as that seen when allicin was administered just before sporo-
zoite injection. Administration of allicin 60 min prior to sporozo-
ite injection yielded little protection (P Ͻ 0.25). We also found
that protection was dose dependent, with 8 mg/kg having a larger
effect than 5 mg/kg. Both doses, however, resulted in significantly
decreased infections compared to controls (Fig. 5B) (P Ͻ 0.001).
The decrease in efficacy of allicin over time is likely a con-
sequence of its rapid decomposition in vivo (9). In order to test
the inhibitory activity of allicin in vivo, before its metabolism in
the blood, we performed a second set of experiments in which
mice were injected with P. yoelii sporozoites that had been
preincubated for 10 min with 50 ␮M allicin or buffer alone. As
shown in Fig. 5C, mice injected with the allicin-pretreated
sporozoites showed no evidence of malaria infection (P Ͻ
0.001).
Inhibition of erythrocytic stages in vivo. Last, we tested the
effect of allicin on the erythrocytic stages of P. berghei. Mice
were injected with 2 ϫ 105 erythrocytic-stage P. berghei para-
sites and then treated with allicin or buffer alone administered
intravenously once daily for 4 days, beginning on the day of
parasite injection (day 0). In the standard 4-day suppression
test, parasitemia on the day after treatment termination is an
indicator of drug potency. We found that 1 day after the last
dose of allicin, the allicin-treated mice had a 94% decrease in
parasitemia compared to controls (Fig. 6A) (P Ͻ 0.001). In
addition, allicin treatment also prolonged the average survival
time of the mice by ϳ10 days, although the drug was admin-
istered for only 4 days (Fig. 6C). We then went on to test
FIG. 3. The effect of allicin on gliding motility. P. berghei sporozo-
ites were preincubated in buffer alone, 1 ␮M cytochalasin D (CD), or
50 ␮M allicin for 10 min and then added to wells for 1 h at 37°C, after
which gliding motility was quantified. The sporozoites pretreated with
allicin were either kept in 50 ␮M allicin during the motility assay
(allicin) or diluted 12-fold so that the final concentration of allicin was
4.2 ␮M (allicin dil). Shown are (A) the percentage of sporozoites that
exhibited gliding motility and (B) the number of gliding sporozoites
exhibiting 1 (black bars), 2 to 10 (light gray bars), or Ͼ10 (dark gray
bars) circles per trail. Each point was performed in triplicate, 200
sporozoites/well were counted, and the means Ϯ standard deviations
are shown.
were pretreated with 50 ␮M allicin, invasion was inhibited by
89%, a result similar to that seen when sporozoites are pre-
treated with E-64 (6). Pretreatment of host cells with 50 ␮M
allicin had no effect on invasion (Fig. 4).
Inhibition of sporozoite infectivity in vivo. We then tested the
ability of allicin to inhibit sporozoite infectivity in vivo using the
rodent malaria parasite P. yoelii. Mice were injected with allicin or
buffer alone at different times before injection of sporozoites.
Forty hours after sporozoite injection, the parasite burden in the
liver was determined by reverse transcription followed by real
time PCR. It should be noted that only sporozoites which invade
and undergo many cycles of replication are detected in this assay,
since the small amount of rRNA present in the sporozoite inoc-
ulum is below the sensitivity of the assay. As shown in Fig. 5A,
mice injected with allicin had decreased levels of infection, and
inhibition of infection was correlated with the length of time

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ANTIMALARIAL EFFECTS OF ALLICIN
1735
last dose of drug were significantly decreased in the allicin-
treated mice compared to controls (Fig. 6B) (P Ͻ 0.001). In
addition, mice given 9 mg/kg/day by mouth survived signifi-
cantly longer than controls, whereas those given 3 mg/kg/day
had an intermediate survival curve (Fig. 6C). Overall these
data show that allicin is active against erythrocytic stages of
Plasmodium when administered either orally or intravenously.
In both sets of experiments (oral and intravenous), we contin-
ued to monitor parasitemias daily until the mice died: in the
control mice, parasitemias were 13% Ϯ 2.2% just prior to
death, whereas parasitemias in the allicin-treated mice were
70% Ϯ 5.2% just prior to death.
DISCUSSION
Here we show that allicin, a naturally occurring compound
generated when garlic cloves are crushed, can inhibit malaria
infection. Previous studies with allicin have shown that it has
inhibitory effects on a wide range of bacteria, as well as some
fungi and a few protozoans (1, 5, 12, 15, 16, 23, 38, 43). At high
doses its toxicity is likely due to a variety of thiolation reac-
tions; however, at low doses it acts more selectively as a cys-
teine protease inhibitor (2, 23, 32). We found that at low
concentrations, the effects of allicin on sporozoites parallel
those of E-64, a highly specific cysteine protease inhibitor:
allicin inhibited proteolytic cleavage of CSP as well as cell
invasion but did not affect gliding motility, suggesting that it is
an inhibitor of the sporozoite cysteine protease(s) required for
infectivity in the mammalian host.
Although the preerythrocytic stages of Plasmodium are the
focus of the work presented here, there are difficulties inherent
in solely targeting this stage with drugs. Most importantly,
inhibition of preerythrocytic stages must be 100% effective,
because a single successful sporozoite can lead to malaria. This
prompted us to also examine the effect of allicin on the eryth-
rocytic stages, and we found a significant inhibitory effect on
these stages as well. We have not investigated allicin’s mech-
anism of action in the erythrocytic stages. However, previous
studies showing that cysteine proteases are required for growth
of erythrocytic stages (39, 40) and inhibition of these proteases
can decrease parasitemias in vivo (28, 35) suggest that allicin
may be inhibiting cysteine protease function in the erythrocytic
stages. If true, these data raise the possibility that the same
cysteine protease inhibitor may be effective against different
life cycle stages of Plasmodium, thus extending the usefulness
of these potential drugs.
Interestingly, our data also show that at death, the allicin-
treated mice had much higher parasitemias than control mice.
Previous studies have shown that the ANKA strain of P.
berghei causes a rodent form of cerebral malaria and leads to
death at relatively low parasitemias (10, 19, 44). Our untreated
mice died 6 to 9 days after infection with low parasite densities,
indicating that the Swiss Webster mice we used in our exper-
iments are susceptible to cerebral malaria. In contrast, the
allicin-treated mice survived 10 to 15 days longer and their
parasitemias reached very high levels, indicating that allicin
treatment enabled them to escape the early death that is nor-
mally seen with this strain of P. berghei. One possibility is that
slower growth of the parasite in the presence of allicin changes
the nature of the immune response and thereby alters the
FIG. 5. Allicin decreases sporozoite infectivity in vivo. Mice were
injected with allicin or buffer alone before injection of P. yoelii sporo-
zoites. Forty hours later, mice were sacrificed, total liver RNA was
extracted, and malaria infection was determined by quantitative PCR.
Infection is expressed as the number of copies of P. yoelii 18S rRNA.
(A) Mice were injected i.v. with 8 mg/kg allicin 1, 30, and 60 min before
injection of sporozoites. (B) Mice were injected i.v. with 5 mg/kg
allicin, 8 mg/kg allicin, or buffer alone 1 min before injection of sporo-
zoites. (C) Sporozoites were preincubated with 50 ␮M allicin for 10
min, diluted 12-fold with buffer, and injected into mice. For all three
graphs, results represent two independent experiments with six mice
per group per experiment.
whether oral administration of allicin would also inhibit growth
of erythrocytic-stage parasites. In these experiments, allicin
was diluted in water and administered by gavage, and control
mice were given water alone. Parasitemias on the day after the

1736
COPPI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
outcome of the infection. However, it is also possible that
allicin is modulating the immune response of the host (re-
viewed in reference 34) and thereby preventing the cascade of
events that leads to early death seen with P. berghei ANKA.
Importantly, at the doses used to inhibit Plasmodium, we saw
no toxic effects of allicin on the mammalian host: mice treated
with allicin had activity levels and weight gain similar to those
of the untreated controls. Although allicin likely reacts with
host cysteine proteases, there are several reasons why mam-
malian cells may be more resistant to the effects of protease
inhibitors than single-celled protozoan parasites (20, 21). Com-
plex animals have a redundancy in protease function that does
not exist in protozoan parasites, which are of necessity more
genetically streamlined organisms (20, 21). In addition, para-
site proteases may be more accessible to inhibitors than host
enzymes, which are found within intracellular compartments.
This may be, in part, because parasites actively import small
compounds from the extracellular environment, as occurs with
the erythrocytic stages of Plasmodium (21), and in the case of
the sporozoite, it may be because the parasite protease acts
extracellularly to cleave surface proteins during invasion (6).
Higher doses of allicin would have eliminated this relative
selectivity for the pathogen. In fact, previous studies in mice
have shown that at high doses allicin is toxic, with a 50% lethal
dose of 60 mg/kg after i.v. injection and 120 mg/kg after sub-
cutaneous administration (17). In our study, a daily dose of 8
mg/kg i.v. was found to be toxic to the parasite with no obvious
effects on the host.
Although allicin had a dramatic effect on both the sporozoite
stage and erythrocytic stages, in experiments where mice were
treated with the drug, neither stage was completely eradicated.
Likely this is because in the blood circulation, allicin is rapidly
metabolized to allyl-mercaptoglutathion, diallyl disulfide, diallyl
trisulfide, and other various thiosulfinate products (9, 18). This is
supported by our finding that the interval between allicin admin-
istration and sporozoite injection correlated well with allicin’s
inhibitory activity against sporozoites. These metabolites have not
been found to be active against other pathogens, and our data
suggest that they also are not active against Plasmodium. The
doses we used in vivo were greater than what was required to see
an effect in vitro, and this likely reflects the rapid metabolism of
allicin discussed above, as well as the possibility that it is reacting
with other free sulfhydryl groups present in a variety of serum
proteins. Importantly, we found that allicin is also active after oral
administration, supporting previous findings that allicin is not
altered by passage through the digestive tract (9). We do not
know what levels of allicin can be achieved after ingestion of
garlic; however, we are currently testing whether frequent inges-
tion of garlic could lead to blood levels of allicin that are inhibi-
tory to Plasmodium.
FIG. 6. Allicin increases survival of mice infected with erythrocytic
stages of Plasmodium. Mice were injected i.v. with GFP-expressing
erythrocytic-stage P. berghei and 1 h later treated with allicin delivered
intravenously (A and C) or orally (B and C). Treatment was continued
once daily for an additional 3 days. (A) Parasitemias in control (buffer
alone administered intravenously) and allicin-treated (8 mg/kg admin-
istered intravenously) groups on day 4. (B) Parasitemias in control
(water administered orally) and allicin-treated (3 mg/kg and 9 mg/kg
administered orally) groups on day 4. (C) Survival curves of mice
receiving buffer administered intravenously, filled circles; water admin-
istered orally, filled triangles; 3-mg/kg/day allicin administered orally,
unfilled triangles; 9-mg/kg/day allicin administered orally, filled
squares; 8-mg/kg/day allicin administered intravenously, unfilled cir-
cles. Arrows indicate the days that mice were treated. Results repre-
sent two independent experiments with five to seven mice per group
per experiment.

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In conclusion, we have shown that allicin, a cysteine protease
inhibitor present in freshly crushed garlic cloves, significantly in-
hibits sporozoite infectivity in vivo and decreases parasite loads in
mice with blood-stage infections. These experiments demonstrate
the feasibility of using the same cysteine protease inhibitor to
target two different life cycle stages in the vertebrate host and
support the idea that cysteine protease inhibitors may be useful
drugs for the prophylaxis and treatment of malaria.
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ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health, RO1
AI056840 (P.S.) and Training Grant 5T32 AI07180 (A.C.), and by
grants from the Drake Family Foundation and by Yeda Co at the
Weizmann Institute of Science (D.M.).
We thank Dabeiba Bernal and Jean Noonan for their expert assis-
tance with mosquito rearing and infection and Daniel Eichinger for his
critical reading of the manuscript.
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