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3188
Advances in Environmental Biology, 5(10): 3188-3195, 2011
ISSN 1995-0756
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Antileishmanial Effects
Leishmaniosis In Vivo
of
Traditional
Herbal
ORIGINAL ARTICLE
Extracts
Against
Cutaneous
Bahrami Ali Mohammad
School of veterinary Medicine, Ilam University, Ilam, Iran.
Bahrami Ali Mohammad: Antileishmanial Effects of Traditional Herbal Extracts Against Cutaneous
Leishmaniosis In Vivo.
ABSTRACT
Ilam province, is located in southwest of Iran close to border with Iraq where had been engaged severely in
war for more than 8 years continuously. leishmaniasis has become an endemic disease in this area which strikes
both native and soldiers. Out of 552 referral positive patients to diagnostic centers, 150 cases of leishmaniasis
were selected for this study, group I: 110 patient under treatment with glucantime 110 patient and group II: 40
patients treated with a combination of Euphorbia milli, Aloe vera juice, animal fat and turmeric. The study was
carried out with assistance of urban and rural health center. Results indicated a significant clinical improvement
and parasite reduction in the patient treated with combination of plant extraction in comparison with those
treated with glucantime., The majority of patients were recovered of lesion less than 100 days after appearance
of disease, however 7.5% of these patient their wound remain untreated during same period of treatment. The
results of those patient who received glucantime revealed 32.7% wound healing was observed within 63-76
days, 61.8% within 90-120 days, however only 5.5% wounds of this patients did not heel during this period. The
application of current plant combination represented more efficient therapy leading to tissue softening, pinkish
color and lesion healing.
Key word: Cutaneous Leishmaniasis, Amistogotes, Glucantime, Euphorbia milli, Aloe vera.
Introduction
The study area, Ilam province, is located in
western and southwestern of Iran in the vicinity of
Iraq. The length of common Iran-Iraq borderline is
465 Km. This region had been engaged severely in
war for more than 8 years. Lieshmaniasis has become
an endemic disease in the area which strikes both
natives and military service men. The Leishmaniasis
are caused by parasitic protozoa of genus leishmania,
by a female sand fly belonging to genus
phlebotomies in the old world [12,34,40]. The
incidence of leishmaniasis is increasing in the world;
between one million and one-half million cases of
cutaneous leishmaniasis are reported annually
worldwide [32]. In World War II, there was a high
incidence of leishmaniasis in troops deployed to the
Persian Gulf region [40,7,18]. Almost 150 cases of
leishmaniasis have reportedly been diagnosed in US
soldiers serving in Iraq in 2003, and more are
expected [22]. Preliminary data on 22 cases of
cutaneous leishmaniasis contracted by American
troops in Afghanistan, Kuwait, and Iraq who treated
at Walter Reed Army Medical Center between
August 2002 and September 2003 [40].
Leishmaniasis is now an emerging zoonosis in the
United States [13,21,33] and US soldiers and
peacekeeping corps currently in the middle East are
experiencing a large outbreak of leishmaniasis with
more than 500 parasitological confirmed cases [30].
Most antileishmanial drugs are expensive, toxic and
have unacceptable side effects; dosin in also
complicated by the fact that they are given
parenterally [26,24], moreover, cases of drug
resistance are on the rise [24,10]. This has caused a
renewed interest in the study of medical plant or
traditional medicine as a source for new antiparasitic
leads. Furthermore, understanding the mode of action
and scar remain after healing of the wound with
traditional medicinal plant of these natural products
to specific target sites may be used to design potent,
novel, selective, and less toxic antileishmanial
analogous of this compound on the structural basis
[24]. Plant extracted or plant-derived compounds are
likely to provide a valuable source of new medicinal
agent [6,14] and the urgent need for potential use in
the therapy of leishmaniasis and fungi infections.
Many human cases of leishmaniotic cutaneous
ulcers were reported from different rural health
centers in all over Ilam province, it make us to
undertaken this study to collect more knowledge
regarding this disease and effectiveness of Ephorbia
milii, Aloe vera plant extracts which combined with
animal fat and turmeric to introduce a new local
Corresponding Auther
Bahrami Ali Mohammad, School of veterinary Medicine, Ilam University, Ilam, Iran.
E-mail: [email protected]
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Adv. Environ. Biol., 5(10): 3188-3195, 2011
manufactured homeopathy drug against these
chemical drug which are expensive, unavoidable the
other aim of this research were to see the acting
effect of novel local manufactured homeopathy
medicine. The classic chemical treatment are
expensive, painful with some drug resistant therefore
there is a need to investigate other targets for therapy
and control, and compare it to current synthetic
glucantime [30].
Traditional Medicine:
Traditional medicine is a comprehensive term
used to refer either to systems such as Chinese
medicine, Indian ayurveda and Arabic unani
medicine or to various forms used in indigenous and
traditional communities [5]. Traditional medicine is
characterized by the use of plants, animals and
mineral substances by archaic communities for
therapeutically purposes to treat various diseases [4].
Traditional medicines are widely disseminated and it
is estimated that 80% of the world’s population
depends on them as a primary health source [5]. In
Iran and other countries of the world’s population of
rural areas and forests rely on popular and traditional
medicine for treatment of many infectious diseases.
Some species are included in prescriptions for
therapeutic purposes such as the healing of wounds,
inflammation due to microbial or parasitic infections,
skin lesions, and ulcers [20]. In some cases, the same
plant may be used for different purposes [5].
Materials and Methods
Study Area:
Thy nature of this study was because of the
biogeographically highly epidemic outbreak of
leishmaniasis disease in the study area. Several
patients from different localities were referred for
treatment in local hospital. The area, Ilam province,
gone under study having common border line 465
Km with Iraq and this zone were involved
continuously in war for more then 8 years. There are
several military comp in the area, population 65% are
running their life with traditional husbandry. This
people grazes their animal by migrating within the
province, with two different tropical and subtropical
environment, High mountain Zagros with forest,
shepherd along with their family living in black tents
(made of goat hair), Lived and a slipped beside their
herds, dog living within a less than 50-60 m radius
from tent or house, every herdsman kept 4-6 local
breed (Kurdish) dog as a shepherd guard dogs.
Animal product kept for several hours within the
living place tent before supply to market. These
environment situation mention above were
collectively helping to this diseases be endemic in
this province.
Human Sampling:
Totally 552 suspicious patients to cutaneous
leishmaniasis (CL) were undergone study (20042008)with the help of city and rural health center (No
72) located in all over region of Ilam province,
southwest Iran,. Data were collected through
questionnaires distributed within all covering area a
detailed clinical history of each patient was
compiled. They were clinically examined, and
photographs of lesions were taken, the staffs of these
centers were trained to detect cases and send
suspected CL infected individual directly to trained
physicians in local hospital, biopsy obtained from the
lesions, all the data were shown that no one of this
patient received previous treatment against
leishmaniasis. To confirm the presence of
leishmaniasis, all suspicious patients were undergone
Smear and culture which explain below. Out of 552
patients, 150 positive persons to leishmaniasis were
selected for this study with comparison of treatment
with two different chemical (Glucantim) and local
(extraction of Ephorbia milii) drugs. Fourty patients
(19 Female and 21 Male), group 1 were treated with
local drug and group 2 (53 Female and 57 Male)
were treated with chemical drug.
Euphorbia Milli, Aloe Vera Animal Fat and
Turmeric Preparation:
Euphorbia belongs to family Euphorbiaceae and
order Euphorbials with annual and perennial plants
which have cyathium in florescence and laticifer.
The total plant milky color extract was was collected
directly by pressing stem and leaves. The same
procedure for Aloe vera extract was applied than 10
ml of both extract were mixed properly with 20 gr
of animal fat and 1 gr Turmeric (before animal fat
were dissolved in +70° by keeping it over night in
oven) cream type medicine were ready and apply
directly on wound twice daily at morning and
evening. Aloe vera, also known as the true or
medicinal aloe, is a species of succulent plant in the
genus Aloe that is believed to have originated in the
Sudan Aloe vera grows in arid climates and is widely
distributed in Africa, India, and other arid areas. The
species is frequently cited as being used in herbal
medicine. Many scientific studies of the use of Aloe
vera have been undertaken, some of them
conflicting. Despite these limitations, there is some
preliminary evidence that Aloe vera extracts may be
useful in the treatment of wound and burn healing,
minor skin infections, sebaceous cyst, diabetes, and
elevated blood lipids in humans. These positive
effects are thought to be due to the presence of
compounds such as polysaccharides, manna’s,
anthraquinones and lectins [3]. Aloe vera is now
widely used on face tissues, where it is promoted as a
moisturized and/or anti-irritant to reduce chafing of
the nose of users suffering hay-fever or cold Aloe
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Adv. Environ. Biol., 5(10): 3188-3195, 2011
vera is also used for soothing the skin, and keeping
the skin moist to help avoid flaky scalp and skin in
harsh and dry weather.
Turmeric (Curcuma longa) is a rhizomatous
herbaceous perennial plant of the ginger family,
Zingiberaceae. Turmeric is currently being
investigated for possible benefits in Alzheimer's
disease, cancer, arthritis, and other clinical disorders.
The U.S. National Institutes of Health currently has
registered 19 clinical trials underway to study use of
dietary turmeric and curcumin for a variety of
clinical disorders. In Ayurvedic practices, turmeric
has is used as an anti-inflammatory agent and
remedy for gastrointestinal discomfort associated
with irritable bowel syndrome and other digestive
disorders. Some may use turmeric in skin creams as
an antiseptic agent for cuts, burns andbruises.
Cosmetics:
Turmeric paste is traditionally used by Indian
women to keep them free of superfluous hair and as
an antimicrobial. Turmeric paste, as part of both
home remedies and Ayurveda, is also said to improve
the skin and is touted as an anti-aging agent.
Turmeric figures prominently in the bridal
beautification ceremonies of India, Bangladesh, and
Pakistan. Staining oneself with turmeric is believed
to improve the skin tone and tan. Turmeric is
currently used in the formulation of some sunscreens.
The government of Thailand is funding a project to
extract and isolate tetrahydrocurcuminoids (THC)
from turmeric. THCs are colorless compounds that
might have antioxidant and skin-lightening
properties, and might be used to treat skin
inflammations, making these compounds useful in
cosmetics formulations.
Glucantime:
There are two common therapies containing
antimony (known as pentavalent antimonials),
meglumine antimoniate (Glucantime) and sodium
stibogluconate (Pentostam). The patient were under
systematic treatment with this drug by specialist
doctors in local hospital for at list 45 days and they
received daily dose of 20 mg/kg body weight for 2
weeks, through intramuscular injection, patient were
followed up and their clinical improvement were
recorded.
Smear:
In brief, direct examination for amastigotes done
by performed on Giemsa-stained smear preparation,
needle aspiration of a saline-injected lesion. Also,
dermal scrapings with a scalpel obtained from an
incision made at the active border and the ulcer of
the lesion. Tissue impression smear are done by
repeatedly pressing or rubbing the button of a deep
biopsy specimen onto a glass slide. The smears are
air-dried then fixed in methyl alcohol for five
minutes prior to staining. Amastigotes could be
differentiated from other intracellular pathogens by
visualizing the nucleus and kinetoplast (a distinctive
rod-shaped structure found in the cytoplasm)
surrounded by a cell wall. Oil-immersion microscopy
was used for proper identification [2].
Culture:
In brief, specimens from a biopsy, smear, or
tissue fluid were inoculated in a biphasic medium,
such as Novy-Mac-Neal-Nicolle (NNN) in the
presence of liver infusion tryptose plus 10% fetal
bovine serum [19]. Culture was maintained at room
temperature. Promastigotes were appear after several
days or one weeks, however the culture were kept at
lest up to 4 weeks.
Experimental In Vitro Study:
To conform the results of effectiveness of plant
combination extracts on CL, cultivation of parasites
in artificial media and than inoculation in to surie
balb/c breed, including 18 divided in 3 groups, 6
mice, group: group 1 as control, group 2 as
experimental
infected with MHOM-75-ER
Leishmania major strain collected from Pasteur
Institute of Iran. Tehran Iranand group 3 were
inoculated with collected promastigotes from the
skin biopsy from the patients were experimental
performed . collection of suspension in brief: after
washing the skin biopsy with phosphate-buffered
saline (PBS) containing gentamycin 2000 IU ml-1
and gentle maceration in a tissue grinder 0.1 ml of
the suspension was seeded in tubes of blood agar
base containing 10% defibrinated rabbit blood with a
liquid phase of 1% glucose in PBS. Entire Balb/c
mice were inoculated on base of the tail with 0.1 ml
of suspension [41].
Results:
Out of the 552 suspicious patients, 150 were
selected for CL, the rate of infection in female cases
treated with local drug were 12.7% and in the cases
of male found to be 14.0%. Among ptients treated
with glucantime drug 35.3% were female and 38.0%
were male. The percentage of accumulation is given
in Table 1. The patients used plants juice medicine
40% of them were treated within 63-76 days, 52.5%
had been treated within 90-133 days, and 7.5% of
these patients remain their wound untreated within
this period of time. The result of those infected
patient who used glucantime shows that 32.7% of
their wound treated within 63-76 days, 61.8% of
them within 90-120 days, and 5.5% of this group of
patients remain untreated within these days and they
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Adv. Environ. Biol., 5(10): 3188-3195, 2011
have been advised and supervised under a GP to
continue their treatment (Table 2 and Diagram 1).
Present of Amastigote in infected, mice were
microscopicaly observed of stained material from
lesions of both experimental groups of animal. The
healing
period after applying plants juice
combination on wound the average time of healing
development were between 68 to 78 days but there
were no any sign of hair growing in the wounded
place of some animals. The effects of combination
plant medicine on the healing of the wounds and
scar as it shows in the picture No 1-2-6 and7 in
comparison to glucantime was better, the patients
were using plant medicine during treatment were
more relax and skin of around their wound were soft
and pinkish color ,they were not had feeling to
scratch their wound area but those patient were
receive chemical injection they were not relax and
feel more like to scratching their wound, skin around
their wound were dried and peeling.
Table 1: Comparison of the two tested groups according to sex, number, percent, accumulation percent and the type of drug.
Title
Frequency
Percent
Cumulative percent
♀
19
12.7
12.7
Plants juice
combination
♂
21
14.0
26.7
♀
53
35.3
62.0
Glucantim
♂
57
38
100
Total
150
100
Table 2: Comparison of the two different tested groups according to treatment days, number, percentage, accumulation percentage and the
types of the drug.
Comparison
Treatment days
Frequency
Percentage
Cumulative percentage
Untreated
3
7.5
7.5
63-76 days
16
40
47.5
Plants juice combination
90-133 days
21
52.5
100
Total
40
100
Untreated
6
5.5
5.5
63-76 days
36
32.7
38.2
Glucantim
90-120 days
68
61.8
100
Total
110
100
Diagram 1: Comparison of the two tested groups according to treatment days, percent, and the type of drug.
Table 3: Comparison of the two different tested group according to number, standard deviation, and standard error of mean.
Comparison
Frequency
Mean
Std. Deviation
Std. Error Mean
Group 1
40
95.1
43.32809
6.850772
Group 2
110
102.8909
40.77491
3.887735
Table 4: Comparison of the two different groups according to T, degree of freedom, significance level, mean differences, and lower and
upper limit of treatment days.
Mean
95% Confidence Interval of the Difference
Sig. (2-tailed)
Comparison
T
DF
Difference
Significance level
Lower
Upper
Group 1
13.88165
39
0/…
95.1
81.24301
108.957
Group 2
26.46551
109
0/…
102.8909
95.18554
110.5963
X1 is the effect of local drug on the time of treatment.
X2 is the effect of chemical drug Glucantim on the time of treatment.
So Zero hypothesis can be explained as such: mean differences between the two groups is no significant. The obtained table shows that T
and significance level (Sig = 0.000) imply that mean difference between the two groups is significant and so the local drug can be used
instead of chemical one.
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Adv. Environ. Biol., 5(10): 3188-3195, 2011
Fig. 1: Cutaneous leishmaniasis before treatment (left hand).
Fig. 2: Cutaneous leishmaniasis in the same patient picture No 1 and 2 after treatments with plants combination
medicine.
Fig. 3: Amastigote in tissue.
Fig. 4 and 5: Appearance of Cutaneous leishmaniasis before and after treatment with experimental plant
medicine in laboratory rats.
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Adv. Environ. Biol., 5(10): 3188-3195, 2011
T-Test and Means:
Zero hypotheses with approach to T are:
H0=MX!-MX2
Mean differences is not significant in Zero
hypothesis and the difference results from chance.
Rejection of Zero hypotheses shows the significant
difference between tested variables.
Aim of the aforementioned test is to answer to
this question that if Mean differences results from
chance or from actual difference between the means
of society which the sample has been chosen from.
After calculating T and df (degree of freedom)
and comparison with Students T-test of Table, we
can propose that either mean difference between the
two groups is not significant, that is the two groups
are similar, or vice versa, the Zero hypothesis rejects
and mean differences is significant.
compounds present an enormous structural diversity.
Currently, several alkaloids [30,23,5] and other
isolated molecules [1,15,17] have biological activity
against important parasites including leishmania sp
[5]. Other purified molecules and extracts from the
genera Annona (Annonaceae) [9], Cassia (Fabaceae)
[36],
Copaifera
(Fabaceae)
[35],
Croton
(Euphorbiaceae) [33], Cuatteria (Annonaceae) [8],
Jacaranda (Bignoniaceae) [31], Piper (Piperaceae)
[27], Stachytrarpheta (Verbenaceae) [25] and
pourouma (Moraceaea) [39], That also have
Amazonian species, present effective antileishmanial
activity.
We can conclude that combination of E. milli,
Aloe vera juice, animal fat and turmeric has effective
influences on treatment of CL in mice model.
Determining of how this combination bringing
amastigotes under control and acts on the wounds
needs further studies.
Discussion:
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