Potency of Allicin in Allium sativum as a Promising Natural

Therapy for Malaria in Indonesia

Antony Halim*, Muhammad Lukman*

* Third Year Medical Student, Universitas Tanjungpura, kedokteran@untan.ac.id

*** Add your correspondent author, (telephone, address, email address)

Abstract

Keywords: The keywords consist of 4-6 words alphabetically. Use 10 pt; Italic; Times New
Roman font.

Introduction Result and Discussion

Definition of Malaria

Material and Methods Classification

A literature review was conducted from

January 2016 through February 2016. Diagnostic Criteria for Malaria
literatures were retrieved from several
Risk Factor of Malaria
journal publications and medical textbooks.
Keywords used were including
Pathophysiology of Malaria
“preeclampsia”, “heme oxygenase-1”,
“curcumin”, “HO-1 enzyme pathway as
Treatment of Malaria
treatment for preeclampsia” and “effect of
curcumin on HO-1 expression”. No Multidrug Resistance
specific keywords were required as
inclusion criteria. Allium sativum / Garlic

Three reviewers independently read each

Garlic (Allium sativum) is a cultivated food
article in full text (n = 40 articles and 2
highly regarded throughout the world.
books), evaluated the relevance of retrieved
Originally from Central Asia, garlic is one
articles and recorded the main findings of
of the earliest of cultivated plants. The
each study. Reviewers slated each article
Ebers Codex, and Egyptian medical
for “inclusion” or “exclusion” based on the
papyrus dating to about 1550 B.C.E.
article’s relevance to the topic.
mentions garlic as an effective remedy for
a variety of ailments. Early men of
medicine such as Hippocrates, Pliny and
Aristotle espoused a number of therapeutic
uses for this botanical (Murray 2005).
Today it is commonly used in many
cultures as a seasoning or spice.According
to the US Food and Drug Administration
survey of 900 people, garlic stands as the
second most utilized supplement (behind
Echinacea), with almost 17% of the
population using a garlic supplement in the
preceding 12 months (Timbo et al. 2006).
Teardrop shaped cloves are encased in dry
skin-like papers that unite to create the
bulb. The garlic bulb is the part of the plant
used most often for cooking and medicinal
uses. Garlic can be eaten raw. Most often it
is used raw after being chopped, minced,
sliced, or juiced. More often, it is cooked
where it enhances flavor as well as adds
nutritional benefit.
The taxonomy of Allium sativum are
below.
Kingdom : Plantae
Division : Magnoliophyta
Class : Liliopsida
Order : Asparagales
Family : Alliaceae
Genus : Allium
Species : Allium sativum
When crushed, Allium sativum yields
allicin, a powerful antibiotic and antifungal
compound (phytoncide). However, due to
poor bioavailability, it is of limited use for
oral consumption. It also contains alliin,
ajoene, enzymes, vitamin B, minerals, and
flavonoids. The composition of the bulbs is
approximately 84.09% water, 13.38%
organic matter, and 1.53% inorganic
matter,while the leaves are 87.14% water,
11.27% organic matter, and 1.59%
inorganic matter. The phytochemicals
responsible for the sharp flavor of garlic
are produced when the plant's cells are
damaged. When a cell is broken by
chopping, chewing, or crushing, enzymes
stored in cell vacuoles trigger the
breakdown of several sulfur-containing
compounds stored in the cell fluids.
Allium sativum or garlic is a natural herb
that is effective to treat some diseases like
atherosclerosis, high cholesterol,
respiratory infections, and triglyceride
levels. The primary ingredient found in
garlic is alliin. This chemical is similar
chemically to cysteine, an amino acid
containing sulfur, and possesses no odor.
After garlic is crushed, alliin is to
converted into allicin, the compound that
gives garlic its strong smell and numerous
health benefits. Garlic has a very long folk
history of use in a wide range of ailments,
particularly ailments such as ringworm,
candida and vaginitis where its fungicidal,
antiseptic, tonic and parasiticidal properties
have proved of benefit. The plant produces
inhibitory effects on gram-negative germs
of the typhoid-paratyphoid-enteritis group,
indeed it possesses outstanding germicidal
properties and can keep amoebic dysentery
at bay. It is also said to have anticancer
activity. It has also been shown that garlic
aids detoxification of chronic lead
poisoning. Garlic is claimed to help
prevent heart disease (including high
cholesterol, high blood pressure, and
atherosclerosis) and cancer. Garlic can be
eaten raw in order to reduce nasal
congestion and also in helping to reduce
the amount of mucous in the nasal cavities.
Garlic is also said to aid in the cleansing of
the body's blood. Eating garlic on regular
basis may provide some protection against
gastric cancer.
Extraction of Allicin
Garlic cloves were blended with HPLC
Grade water in a ratio of 5 mL of water per
1 gm of garlic (Chowdhury et al., 2008).
The blended mixture was allowed to stand
for 10 min in order to ensure a complete
enzymatic reaction of alliin with allinase.
The mixture was first filtered with filter
paper through a Buckner funnel, then
centrifuged at 3,500 rpm for 30 seconds,
Fig 1. Biosynthesis of Allicin
and finally filtered again with a syringe
filter, to ensure that no garlic particles
Mechanism of Allicin as Antimalaria
would obstruct the column. HPLC mobile
phase, in this analysis, was set with
Inhibition of CSP cleavage.
Methanol and water, at a 60:40 ratio of
Cysteine protease inhibitor E-64 prevents
water to methanol. UV wavelength was set
proteolytic cleavage of circumsporozoite
at 254 nm. Initial volume of 20 micro
protein (CSP), the major surface protein of
liters, flow of 1 microliter per min and
Plasmodium sporozoites(6). In the recent
maximum pressure of 350 Barr at ambient
study, allicin has been shown to react with
temperature was set as the conditions for
free sulfhydryl groups and reversibly
the HPLC analysis. The HPLC conditions
inhibit cysteine proteases (2). Pulsechase
were set to be as best to accommodate the
metabolic labeling experiments, in which
available chemicals/materials as well as
allicin was included in the chase, indicate
allicin’s stability.
that CSP cleavage was inhibited by 10, 25,
and 50 µM allicin (Fig. 1A). Likely this
Allicin
reflects leaky microneme secretion of the
Under the influence of alliinase, allicin is
protease in the absence of cells versus
produced by enzymatic transformation of
triggered secretion when sporozoites
alliin [(+)-(S)-allyl-L-cysteine-sulfoxide].
contact cells. Beside that, allicin also
Alliin and alliinase are found in separate
inhibits cleavage of CSP when sporozoites
parts of garlic clove [1], therefore this
are in the presence of cells, indicating that
chain reaction is initiated only after
it rapidly interacts with the CSP protease.
crushing the cells. The alliin complex with
the enzyme alliinase is then formed in the
presence of water. The unstable alliin-
alliinase complex is further subjected to
dehydration by pyridoxal phosphate and
transformed to allyl sulfenic acid, pyruvic
acid and ammonia. Allyl sulfenic acid is
unstable and very reactive at room
temperature. With the elimination of water,
two molecules of allylsulfenic acid
condense spontaneously to allicin.

Fig. 2. 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 µME-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 µM allicin for 10 min,
which was then diluted to 4.2 µM 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 µM 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 ± standard deviations
for duplicate samples are shown.
Allicin toxicity.
The same study shows that allicin have a
toxic effect on the sporozoites. parasites
were incubated for 10 or 60 min with
different concentrations 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 µM 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. 3).
Fig. 3. 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 incubation in 4.2 µM 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.
Inhibition of sporozoite infectivity in
vivo.
Allicin have an ability 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 inoculum 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 between allicin injection and
sporozoite injection. When allicin was
administered just before injection of
sporozoites, it significantly decreased
sporozoite infectivity compared to
untreated controls (P < 0.001). Allicin
injected 30 min prior to injection of
sporozoites also resulted in decreased
infectivity compared to the untreated mice
(P < 0.001), but the protective effect was
not as great as that seen when allicin was
administered just before sporozoite
injection. Administration of allicin 60 min
prior to sporozoite injection yielded little
protection (P < 0.25). Beside that, it 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 < 0.001).
Fig. 4. Allicin inhibits sporozoite invasion
of host cells. P. berghei sporozoites were
pretreated for 10 min with the indicated
concentrations of allicin, which was then
diluted 12-fold before sporozoite addition
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 µM allicin
for 1 h and washed, and untreated
sporozoites were then added to the cells.
Each point was performed in triplicate, ±50
fields/well were counted, and the means ±
standard deviations are shown. Inhibition
of invasion was calculated based on the
invasion rate for sporozoites pretreated
with buffer alone, which was 57%.
Inhibition of erythrocytic stages in vivo.
Allicin could inhibit the erythrocytic stages
of P. berghei. Recent study using mice
injected with 2 x 105 erythrocytic-stage P.
berghei parasites 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.
The result is 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 < 0.001). In addition,
allicin treatment also prolonged the
average survival time of the mice by ±10
days, although the drug was administered
for only 4 days (Fig. 6C). We then went on
to test 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 last dose of drug were
significantly decreased in the allicintreated
mice compared to controls (Fig. 6B) (P <
0.001). In addition, mice given 9
mg/kg/day by mouth survived significantly
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.

Fig. 5. Allicin decreases sporozoite

infectivity in vivo. Mice were injected with
allicin or buffer alone before injection of P.
yoelii sporozoites. 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 sporozoites. (C) Sporozoites
were preincubated with 50 µM 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.
Allicin enhances pro-inflammatory
immune responses
As a cysteine protease inhibitor, the
inhibitory effects of allicin on Plasmodium
parasites were attributed to the direct action
on parasites [30,32]. Because allicin also
has immunomodulatory activity, whether
improved disease outcomes by allicin
treatments could result from strengthened
host immunity against Plasmodium
infection was investigated. Previous studies
have shown that enhancement of Th1
responses during P. yoelii 17XL infection
could reduce the initial parasite load and
extend host survival time [43]. Here, the
levels of several pro-inflammatory
mediators in the sera of control and allicin-
treated mice were evaluated. Allicin
treatments increased production in
splenocytes, the in vitro synthesis of IFN-γ,
TNF, IL-12p70 and NO in cultured
splenocytes from the control and allicin-
treated mice were measured. Compared to
the control, allicin treatments at both
dosages caused significant increases in the
production of IFN-γ and TNF by
splenocytes on days 3 and 5 PI (P < 0.05)
and the effect appeared to be dose-
dependent (Figure 3A, B). More
specifically, 9 mg/kg allicin treatment led
to~ seven times higher production of IFN-γ
than 3 mg/kg allicin treatment on day 3 PI
(Figure 3A). IFN-γ can promote the
production of NO by macrophages to
reduce the parasitaemia during P. yoelii
17XL infection. Therefore, NO production
in cultured splenocytes, a hallmark
ofmacrophage activation, was further
studied. In support of earlier observation of
other Th1 cytokines, both allicin treatment
dosages increased NO production on days
3 and 5 PI (Figure 3C). Yet, the 3 mg/kg
treatment group showed slight,
insignificant increase of NO production as
compared to control (Figure 3C, P > 0.05).
Significant increase in NO2 - was evident
in the higher dose (9 mg/kg) of allicin
treatment group (Figure 3C). IL-12 is an
important stimulator of the T-cell response
and plays a critical role in resistance to
malaria [44-46]. Allicin treatments at both
dosages caused increases in the production
of IL-12p70 by splenocytes on days 3 and
5 PI and this effect also appeared to be
dose-dependent, albeit the difference was
not statistically significant (Figure 3D, P >
0.05). To evaluate whether allicin treatment
affected Th2 immune response during the
early stage of P. yoelii 17XL infection, the
amount of IL-4 in the supernatant of
cultured splenocytes was determined, and
there was no significant difference
between the experiment and control groups
(Figure 3E, P > 0.05). Altogether, these
results showed that allicin treatment
preferentially promoted the production of
pro-inflammatory mediators during acute
malaria infection in a dose-dependent
manner.

Fig. 6. Effects of allicin on pro-
inflammatory immune responses during
murine malaria infection. On day 0, 3 and 5
after infection, spleen cells were prepared
and concentrations of IFN-γ (A), TNF (B),
IL-12p70 (D) and IL-4 (E) were
determined by ELISA. The concentration
of NO2 - (C) was detected using the Griess
reaction.
Conclusion
Acknowledgement
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1. Klein S, Fabbrini E, Romijn JA.
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