Anti-Infective Agents, 2012, 10, 111-116 111

Laboratory Studies and Clinical Trials on New Formulations from Garlic

Extract Against Cutaneous Leishmaniasis

Azam J. Samdani1,*, Samreen2, M. Iqbal Choudhary2 and Atta-ur-Rahman2

1
Jinnah Post-Graduate Medical Center, Karachi-75510, Pakistan; 2Dr. Panjwani Center for Molecular Medicine and
Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270,
Pakistan

Abstract: Leishmaniasis is a global disease, which has become a major health challenge for Pakistan and other develop-
ing countries. Infections caused by the parasitic protozoa constitute a major hindrance in socioeconomic development in
many developing countries. In this study, we developed a topical application (gel), containing active ingredients derived
from garlic, on which in vitro, in vivo, and clinical studies were conducted. Anti-leishmanial activity observed in vitro was
followed by in vivo study with the application of this active ingredient, prepared in gel form, and applied once daily on
experimental mouse model. Their lesions cured completely within 4-6 weeks. This was followed by clinical trials in hu-
mans. A total of 70 patients, 58 (82.85%) males, and 12 (17.14%) females, were selected with positive Leishmania para-
sites by smear examination. Out of 70 patients, 57 patients (81.42%) responded to the treatment, while 13 patients
(18.57%) did not. Ten patients (17.54%) showed complete clinical recovery after 06 weeks of therapy, while 40 (70.17%)
showed complete clinical recovery after 08 weeks of therapy. The results of this clinical study establish the efficacy, and
cost effectiveness of garlic-based topical gel, and pave the way for further research in its therapeutic applications.
Keywords: Leishmaniasis, cutaneous leishmaniasis, clinical trials, garlic extract, topical applications, anti-leishmanial activity,
In vitro studies.

INTRODUCTION visceral and cutaneous forms of leishmaniasis. The majority

Cutaneous leishmaniasis (CL) is an endemic disease in
many parts of the world, especially in tropical and temperate
regions of four continents. 90% cases of cutaneous leishma-
niasis are reported from Afghanistan, Algeria, Brazil Iran,
Peru, Saudi Arabia, Sudan, and Syria, while 90% cases of
visceral leishmaniasis occur in Bangladesh, Brazil, Ethiopia,
India, Nepal, and Sudan. It continues to cause serious clini-
cal & therapeutic problems. Infections by the parasitic proto-
zoa cause a physical and cosmetic disability and thus hinder
the socioeconomic development in many developing coun-
tries. Leishmaniasis, caused by Leishmania parasites, in-
cludes a broad spectrum of diseases ranging from self-
healing cutaneous leishmaniasis (CL) to nasopharyngeal
illness mucocutaneous leishmaniasis (MCL) to systemic vis-
ceral leishmaniasis (VL) [1]. According to WHO, leishmani-
asis is the second most widespread vector-born disease, after
malaria [2].
The number of people exposed to the risk of acquiring
this disease is close to 350 million. Leishmaniasis currently
infects about 12 million people in 88 countries. The WHO
estimates 1.5 to 2 million new cases of leishmaniasis each
year, including 0.5 and 1.5 million cases of visceral and cu-
taneous leishmaniasis, respectively [3]. Leishmaniasis is a
growing problem in the rural areas of all the provinces and
major cities of Pakistan. In Pakistan, mucocutaneous and
diffuse cutaneous leishmaniasis are less common than
*Address correspondence to this author at the Department of Dermatology.
Jinnah Post-Graduate Medical Centre, Karachi, Pakistan; Tel:/Fax: 00-92-
21-5242819, E-mail: azamsamdani@hotmail.com
of the skin lesions are of and wet-type and caused by
L. major, while some are dry lesions mostly in certain dis-
tricts of Sindh [4, 5]. In January 2002, a joint assessment
mission of the Ministry of Health and WHO to the North-
west Frontier Province (Khyber Pakhtoon Khuwa) identified
5000 cases of CL at Kurram Agency, most of them are
young children [6]. It is endemic in Baluchistan [7], rural
upper Sindh, and Multan (South Punjab) regions [8-10]. In
Pakistan, the authorities are facing multiple problems in
meeting this growing health problem.
Garlic (Allium sativum), belonging to family Liliaceae,
has an immense medicinal and culinary importance. It is
used for a variety of purposes based on traditional folkloric
experiences. Allicin (diallyl thiosulfinate), allyl sulfide, and
diallyl disulfide, isolated from garlic, as well as fresh and
boiled extracts of Allium sativum have also been evaluated
for antileishmanial activity [11]. Ajoene, active constituent
of a majority of garlic-based products, has anti-platelet and
in vitro anti-leishmanial activities. Recent studies indicate
that garlic extract has antimicrobial, anti-carcinogenic, insec-
ticidal, nematicidal, molluscicidal and rodenticidal activities
[12]. The extract of garlic has in vivo and in vitro effects
agai;nst leishmania parasites, like it reduces the footpad le-
sions in L. maxicana infected BALB/c mice, while in vitro it
reduces macrophage infection by inducing NO production.
Garlic extract stimulates the IFN-
production and NO syn-
thesis through the activation of T-cells as well as macro-
phages which kills the parasites [13]. Garlic extract modu-
lates the immune response, engulfment and destruction of
L. major by resident peritoneal macrophages of BALB/C

2211-35/12 $58.00+.00 © 2012 Bentham Science Publishers

112 Anti-Infective Agents, 2012, Vol. 10, No. 2
mice. Combination of garlic and antimonial drugs to promote
the healing effects and regulation of Th1/Th2 cytokine
patterns in highly susceptible BALB/c mice infected with
L. major has been studied [14, 15]. Methanolic extracts of
Allium sativum also have the immunomodulatory effects in
rodents, infected with L. major and L. donovani [16].
By using the “standard cage test”, garlic oil was topically
applied to five human volunteers. It showed significant pro-
tection 97% and 40% at 1% and 0.005% dilutions respec-
tively, on the skin of infected human with female Phle-
botomus papatasi [17].
The antimonial compounds, used for the treatment of this
condition, are not easily accessible to common public due to
high cost. They are not available to meet the needs of the
community at large, especially the poor population of the
rural areas. To meet this therapeutic challenge, a research
study was conducted to evaluate the effects of garlic extract
on promastigote and amastigote stages of leishmanial para-
sites and some promising and reproducible results were
obtained. We also evaluated the animal toxicity of formula-
tion and no toxic effects were noted. These results led to
in vivo studies on BALB/C mice, as well as clinical trials on
human volunteers. PLO Gel formulation was prepared by
using 25% ethyl acetate fraction of garlic, and applied topi-
cally on the lesions of infected BALB/C mice, and infected
humans as well. Promising results were obtained. Through
this study, we are introducing here a cheap and effective
topical preparation with minimal side effects and toxicity,
and maximum therapeutic benefits.
MATERIAL AND METHODS
Plant Preparation
The fresh bulbs of garlic (20 kg) were crushed, and
soaked in 20 L pet. ether, ethylacetate, chloroform, methanol
and methanol water for 3 days and followed by filtration and
evaporation to obtained 100, 200, 100, 200 and 350 g, crude
extracts respectively. These extract were stored at 4ºC and
evaluated for anti-leishmanial activity in vitro, in vivo, ani-
mal toxicity as well as for human clinical trials.
Animals
BALB/c mice, 8 to 12 week old, were used for all in vivo
studies for leishmaniasis.
Leishmania Strains and Its Maintenance
Leishmania major (DESTO-Pakistan) strain was used in
an in vitro study. L. major promastigotes were cultured in
RPMI-1640 medium (Sigma, St. Louis, USA) supplemented
with 10% fetal calf serum (PAA Laboratories GmbH,
Austria), and gentamicin (100
g/ mL) at 22-25°C.
Smear Preparation
First to clean the skin with 70% alcohol into the dermis
or effected area. Aspirate were taken from the lesions of pa-
tients and spread on a slide to make a thin smear, air dry the
smear and fix with methanol and stained with giemsa to ob-
served Leishmania major (LM) bodies.
Samdani et al.
Topical Gel Preparation
Chemotherapeutic agents with potential antileishmanial
activity have been incorporated in different ointment, and
cream formulations, and tested for their efficacy. For the
preparation of gel, we used ethyl acetate fraction of garlic in
white soft paraffin with pharmaceutical grade PLO Gel for-
mulations (Ointment or Gel used 25%). Extract prepared was
granular in nature and thus formulated into a gel formulation.
Gel preparations are generally 5-10 times more potent in
penetration. Odour of garlic was reduced by using lavender
oil.
In Vitro Leishmanicidal Assay for Promastigotes
During this in vitro study, Leishmania promastigotes
were first grown in bulk in modified NNN biphasic medium
using normal physiological saline. Leishmania parasite pro-
mastigotes were cultured with RPMI 1640 medium (Sigma,
St. Louis, USA), supplemented with 10% heat inactivated
foetal calf serum (FCS) (PAA Laboratories GmbH, Austria).
Parasites at log phase were centrifuged at 2,000 rpm for 10
minutes, and washed three times with saline at same speed
and time. Parasites were diluted with fresh culture medium
to a final density of 1x 106 cells / mL. In a 96-well micro
titer plate, medium was added in different wells, 20 μL of
the test material was added in medium, and serially diluted.
100 μL of parasite culture was added in all wells. Two rows
were left for negative and positive controls. Negative con-
trols received DMSO, while the positive controls contained
varying concentrations of standard antileishmanial com-
pounds, e.g. amphotericin B (MP Biomedical Inc. France),
pentamidine (ICN Biomedical Inc.France) and sodium stibo-
gluconate (Sigma, St. Louis, USA). The plate was incubated
at 22-25°C for 72 hrs [18]. The culture was examined micro-
scopically on an improved Neubauer counting chamber, and
IC50 values of test fractions, possessing antileishmanial activ-
ity, were calculated by Software Ezfit 5.03 Perella Scientific.
All assays were run in duplicate (Table 1).
In Vitro Leishmanicidal Assay for Amastigotes
2 mL simple RPMI-1640, and 1 mL of heparin were used
in this study. Mice were killed by cervical fracture. The skin
over the abdominal region was removed, and 3 mL of the
washing fluid was forced into the peritoneum with a hypo-
dermic needle. After light massage for a few seconds, fluid
was withdrawn from the lateral part of the peritoneal cavity.
Usually about 2 mL fluid was obtained from each mouse.
Peritoneal macrophages were obtained by a modification of
the method described [19, 20], and in vitro assay against
amastigotes was performed [21, 22]. Results were expressed
as percent reduction of parasite burden and compared with
the control wells. Percentage inhibition of parasite (PI) was
calculated by using following formula:
(Infection rate of treated culture)
% PI = 100 – ___________________________________  100
(Infection rate of the untreated culture)
Toxicity Determination (LD50)
Toxicity of soluble fraction of ethyl acetate (garlic) was
evaluated as LD50. Animals were given doses orally ranging
Laboratory Studies and Clinical Trials on New Formulations from Garlic Extract Anti-Infective Agents, 2012, Vol. 10, No. 2 113

Table 1. Results of In Vitro Leishmanicidal Activity of the Formulation based on extracts of the bulbs of Allium sativum

IC50
g/mL ± S.D. % Reduction of Intracellular

Extracts
(Promastigotes) Amastigotes at 25
g/mL

Pet. ether 37.5 ± 0.45 ND

Ethyl acetate 3.9 ± 0.10 85

Ethanol 10.50 ± 0.20 70

Chloroform 12.50 ± 0.45 68

Standard Drug

Amphotericin B 0.24 ± 0.02
M 73.25% at 10
M

Pentamidine 5.90 ± 0.09
M 52.50% at 12.5
M

Sodium stibogluconate >200
M 70% at 2.5
M

between 100 mg to 1,200 mg/ kg and observed for 48 hrs.
Lethality was not observed up to the dose of 1,200 mg/kg.
In Vivo Assay on BALB/c Mice
BALB/c mice 8 to 12 weeks old were used for all in vivo
studies of L. major infection. The mice were inoculated in
the base of the tail with 1x 106 to 5x 106 infective promas-
tigotes. The development of the lesion was followed macro-
scopically, while the presence of parasites in biopsy material
was monitored microscopically in both smears, and cultures.
Material aspirated with a fine glass pipette through a small
incision made at the margin of the lesion with a sterile surgi-
cal blade (B.S. 2982; Swann- Morton, Sheffield, UK). They
were stained with Giemsa and cultured as previously de-
scribed. Then topically prepared ointment was applied to the
lesions twice a day for two weeks. The size of the lesions
was measured by Vernier caliper at every 4-5 days of treat-
ment. Lesion size was measured in two dimensions (D and d)
at right angles to each other with a caliper gauge, and the
lesion size (S) was determined by the following formulation:
S = (D x d)/2
The lesions were completely cured within 4-6 weeks.
Cultures were considered negative only after 20 days without
growth. These techniques are recommended by the WHO for
detecting CL in both experimental animals and humans [23].
Recruitment of Human Volunteers for Clinical Study
This study included 70 patients (58 males, and 12 fe-
males) of confirmed cases of leishmaniasis. Only those pa-
tients were selected for study who either had the infection for
the first time or had not received any therapy or those cases
which were untreated for duration of 03 weeks to 06 months.
65 patients had multiple lesions at different sites, while 05
had solitary lesions (Table 2). Written consent was taken
from all patients. In case of minor children; the consents
were obtained their guardians. The gel preparation was
applied twice daily (in the morning, and evening) to the
lesions. The patients were examined fortnightly, and were
followed-up for a period of 12 weeks. Clinical photographs
were taken at the beginning, and termination of the therapy
with complete cure of the lesions. In some cases, photo-
graphs were taken in between the therapy also.
Control
In cases of multiple lesions, one nearby lesion was cho-
sen as control. In some cases when the nearby lesions were
not available, the nearest possible lesions were chosen as
control. All the control lesions had local application of white
soft paraffin twice daily in the same amount by weight as
they were using the gel for other lesions. None of the lesions
responded to the application of white soft paraffin. All the
control lesions were later treated with the same gel prepara-

Table 2. Distribution of Lesions of Different Patients Involved in the Clinical Study

Sex Distribution Distribution of Lesions on Body

Age in Years
Males Females Face & Neck Upper Limbs Lower Limbs Chest Back

7-10 05 01 03 01 02 00 00

11-20 03 01 01 02 01 00 00

21-30 20 04 06 09 06 02 01

31-40 13 02 03 07 04 01 00

41-50 12 03 05 07 03 00 00

51-65 05 01 02 02 01 01 00
114 Anti-Infective Agents, 2012, Vol. 10, No. 2
tion containing pure garlic extract used in this trial. All of
them healed clinically with elimination of parasites. The
healing time being almost the same as those of the previous
lesions on the same patient, which further confirmed the
effectiveness of the gel preparation.
RESULTS
In Vitro Leishmanicidal Activity on Both Stages of Para-
sites
Ethyl acetate fraction of garlic bulb showed a potent
antileishmanial activity against the both stages of parasites
with the IC50 value 3.9 ± 0.10
g/ mL against the promas-
tigotes, and 85% reduction of amastigotes stage of parasites
at 25
g/ mL concentrations (Table 1).
Treatment of Cutaneous Leishmaniasis in BALB/c Mice
After the mice were inoculated with promastigotes, no
spontaneous healing or recovery from infection was
observed. They were then treated with the prepared gel com-
prising of 25% garlic extract in white soft paraffin, twice
daily. The mice were examined on every third day. Some of
them started to show improvement after 10 days of treat-
ment, and the lesion area started to heal. There was a com-
plete recovery in a period of 30 days in almost all experi-
mental animals.
Treatment of Cutaneous Leishmaniasis in Humans
In this study, a total of 70 patients, 58 (82.85%) males,
and 12 (17.14%) females were enrolled, who had clinical
features of the disease, and were positive for Leismania
donovani antibodies on smear examination. Fifty seven
patients (81.42%) responded to treatment, while 13 patients
(18.57%) did not respond to this treatment. Out of these, 57
patients who had recovered 47 (82.45%) were males, and 10
(17.54%) were females. Ten patients (17.54%) showed com-
plete clinical recovery after 06 weeks of therapy, 40 (70.17%)
showed complete clinical recovery after 08 weeks of therapy
Figs. (1A & B, 2A & B), while 07 (12.28%) showed complete
clinical recovery after 10 weeks of therapy Figs. (3A & B).
However an earliest response was seen in some mild lesions
as early as 03 weeks of therapy. Follow up was conducted on
fortnightly basis for 03 months after therapy with no signs of
re-infection. Out of the 13 patients who did not respond to
treatment, 09 cases had chronic, and deep seated infection,
while 04 patients showed only minimal healing.
Duration of treatment was found to be an important fac-
tor in the effectiveness of the gel. Treatment for less than 03
weeks was not sufficient to completely eliminate all the
parasites, although an improvement was noted in some
patients as early as 02 weeks. The lesions with (ulcer) started
showing a tendency of drying up, and surrounding inflamma-
tion reducing in intensity. However, treatment varying from
06 weeks to 10 weeks cured the lesions in all those cases that
responded to therapy (Table 3).
DISCUSSION AND CONCLUSION
Garlic (Allium sativum) is used all over the world for the
treatment of different diseases. Based on numerous studies, it
was concluded that garlic contained many active principles,
which were responsible for the curative action of this herb.
Samdani et al.
Allium sativum is known to have lipid and cholesterol lower-
ing, antidiabetic, antifungal, antiparasitic and other proper-
ties. Ajoene (C9H14OS) is the most important sulfur-
containing constituent of garlic. Ajoene is the standardized
active constituent of a majority of garlic based products and
known to have a potent leishmanicidal activity [24]. Besides
garlic, different plants of medicinal values are used world-
wide for the treatment of leishmaniasis [25]. Pentavalent
antimonial compounds, and amphotericin B are the medi-
cines currently in use by the dermatologists worldwide for
treatment of this condition worldwide. Due to their limited
availability in developing world with known side and toxic
effects, there are limitations in the use of these drugs.
In view of these facts, there is a need of new drugs with
new therapeutic targets to tackle this condition. This study,
therefore, was aimed to investigate the potential of anti-
leishmanial activity of natural products (garlic) in animal
model, and human in volunteers with cutaneous leishmania-
sis. A formulation with good absorption was required in or-
der to be effective. The preparation was hence developed in
gel form in white soft paraffin (petroleum) as a topical ole-
aginous ointment base, in view of its widespread use in many
pharmaceutical ointments [26]. Local gel preparation showed
two-fold benefits, clinical healing, and eradication of para-
sites from the lesion site. In addition, this preparation
showed no side effects with maximum therapeutic benefit. A
limited study of effects of garlic on five human volunteers
was patented earlier. The main problem derives from the
uncertainties regarding the stage of the disease at which drug
administration is initiated.
The results obtained from the clinical trial were very en-
couraging with complete healing of the lesions, and total
elimination of the parasite in 78.75% of the patients. The
first and foremost finding in the study was the immediate
acceptance, and willingness of the patients to apply this gel
preparation as this being a non invasive procedure, rather
than using the injectable form of therapy. Supportive treat-
ment included rest (especially in those cases with lesions of
feet with associated swelling), high-calorie diet, and antibiot-
ics for secondary infections. There was no relapse in the
monthly follow up for three months, following the comple-
tion of therapy. However a failure of treatment occurred in
those cases where the patients were non compliant or the
lesions were fairly extensive and deep with severe secondary
infections. Besides the non compliance, there could be many
other factors responsible for the failure of treatment, such as
immunological failure or the emergence of resistant strains.
Furthermore it was observed that in severely infected cases,
more time was required to clear the lesion of both the
leishmanial parasites, and the associated secondary infec-
tions (mainly bacterial), and healing was achieved in 6-8
weeks from the start of the therapy.
The limitations of this study include the lack of response
by the patients with severe and deep cutaneous infections.
The number of patients although was reasonable, but trial of
this new preparation on patients of different ages/sex needs
to be carried out to further substantiate the findings. Simi-
larly, absorption, efficacy, and cost effectiveness, although
far cheaper than to the available injectable therapy, can also
be limiting factors in the current form of proposed therapy.
Laboratory Studies and Clinical Trials on New Formulations from Garlic Extract Anti-Infective Agents, 2012, Vol. 10, No. 2 115

Fig. (1A). Lesion before treatment (1B) Healed lesion after 08 weeks.

Fig. (2A). Crusted lesion on the elbow (2B) Healed lesion after 08 weeks.

Fig. (3A). Deep ulcerated lesion on the ankle (3B) Healed lesion after 10 weeks.

Table 3. Duration of Treatment with an Ointment Containing 25% Garlic Extracts and Its Therapeutic Response on Different
Patients

Number of Patients
Duration of Treatment in Weeks Males Females Therapeutic Response
Total 47 Total 10

02 00 00 Decrease in inflammation

04 00 00 Scar formation initiated

06 08 02 Clinically clear. Smear -ve

08 33 07 Clinically clear. Smear -ve

10 06 01 Clinically clear. Smear -ve

116 Anti-Infective Agents, 2012, Vol. 10, No. 2
In brief, this new gel formulation, containing garlic
extracts, is capable of eliminating the parasites and healing
the wound when applied directly to the leishmanial lesion
during a clinical study. This preparation is non invasive, and
easy to comply with. It has good absorption with no notable
local / systemic effects. The encouraging results of this study
suggest that this can be developed as a cost effective and
efficient local therapy (gel formulation) for the treatment of
cutaneous leishmaniasis.
ACKNOWLEDGEMENTS
This research project was jointly supported by the Higher
Education Commission (HEC), and Pakistan Academy of
Sciences (PAS). We would like to thank Nigehbon Com-
pounding Pharmacy (Pakistan) for their cooperation in
preparation of the topical gel for the clinical study.
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