In vitro Activity of Arbutus unedo Aqueous Extract against Leishmania infantum Promastigote

Considered as a major public health problem, leishmaniasis incidence continues to increase due to lack of vaccine. In addition, drugs routinely used for the treatment of this disease have associated side effects. This justifies a need to develop new drug treatments. It is in this perspective that our study is inscribed. In vitro antileishmanial activity of the aqueous extract of Arbutus unedo leaves was evaluated Short Communication Moualek et al.; ARRB, 12(4): 1-5, 2017; Article no.ARRB.32556 2 against promastigotes of L. infantum (MON-1/DZ/01/LIPA1227/01) by in vitro promastigote cell assay. The extract of the studied leaves showed remarkable antileishmanial in vitro activity (74% mortality at 100 μg/ml) against the promastigotes of Leishmania infantum, in a concentration dependent manner.


INTRODUCTION
Leishmaniasis constitutes a heterogeneous set of diseases all due to the infection of the host by a protozoan transmitted by a vector insect, the sandfly.
In Algeria, leishmaniasis has been marked by a considerable increase, particularly visceral leishmaniasis, due to L. infantum which infect particularly young children between one and four years of age [1,2].
The treatment, in addition to being very expensive, generates many side effects. Because of its numerous biological activities already proven [3] and for the sake of remediation we try in this study to evaluate the effect of the aqueous extract of Arbutus unedo against L. infantum.
Through the literature over 239 natural molecules that have been tested for their antileishmanial effect [4].
Arbutus unedo is known to contain many of these natural compounds [5]. This rich and varied composition is on the one hand at the origin of the numerous biological effects (antioxidant, antiinflammatory, anti-diabetic and anti-cancer) of the plant extract and on the other hand could be potentially at the origin of an antileishmanial activity.
Furthermore, there is no scientific works dealing with the antileishmanial activity of the aqueous extract of Arbutus unedo have been carried out, which motivated us for the realization of this study.

Plant Collection
Arbutus unedo L. (Ericaceae) leaves were collected in December 2014 from Tizi-Ouzou, Algeria. A voucher specimen was deposited in the herbarium of Mouloud Mammeri University of Tizi-Ouzou, Department of Vegetal Biology (FSBSA/MK/2105). The sample was dried and then ground to obtain a powder that was stored at room temperature and in the dark until extraction.

Extract Preparation
Twenty grams of powder of A. unedo leaves were dissolved in an erlenmeyer with 200 ml of distilled water. The flask was fully coated with aluminum foil and was placed on a magnetic stirrer. After 24 h of maceration at room temperature, and stirring at 40 rpm. Then, extract was filtered by using glass wool followed by Whatman № 1 filter paper. Finally the filtrate was lyophilized.

In vitro Antileishmanial Activity
L.
infantum MON-1/DZ/01/LIPA1227/01 promastigote grown on NNN medium. The cultures were centrifuged for 10 min at 2.500 rpm and then undergo 3 washes with physiological water and transferred to RPMI 1640 medium supplemented with 10% fetal calf serum for mass cultivation [6].
The screening was performed in flat-bottomed 96-well plastic tissue-cultured plates maintained at 25°C.
Promastigote forms from a logarithmic phase culture were suspended to yield 1 million of cells/ml. The test was carried out on 96 well microplates, each well was filled with 100 µl of the parasites suspension, and the plates were incubated at 25°C for 1 h before drug addition.
Finally the extract to be tested was dissolved in DMSO and added to each well in order to obtain the final concentrations of 12.5, 25, 50 and 100 µg / ml.
The incubation was carried out at 25°C for 72 h [7]. The viability of promastigotes was assed by propidium iodide colorimetric method and A. unedo activity was evaluated by comparing the mortality rates of the test wells with an untreated control.

RESULTS
The lyophilized extract was diluted in 1% DMSO and DMSO control was found to be inactive.
Results showed the antileishmal activity of the A. unedo leaves aqueous extract in a dose dependent manner at concentrations 12.5 -100 µg/ml. The maximum activity of the extract was evaluated at 74% mortality of promastigotes for the concentration of 100 µg/ml.
The collected data (Fig. 1) clearly indicate that the inhibitory concentration for the extract is lower than 12.5 µg/ml.

DISCUSSION
In recent years, the interest in scientific research for plant extracts has grown and it is aimed at the search for compounds with a powerful leishmanicidal effect.
In a recent study conducted in Turkey, in vitro testing of antileishmania activity against Leishmania. tropica promastigote was carried out. Ethanol, water and n-hexane extracts of A. unedo leaves have been tested and the ethanol extract was found to be more effective than the other extracts [8].
There are few studies in the literature that report on the activity of A. unedo aqueous extract against L.infantum, which does not allow us to compare our results.
But as a point of comparison we can cite the work of Khademvatan [9], on L. infantum promastigotes, which relate an IC 50 of 53.5 ± 2.5 µg/ml, obtained through the extract of Holothuria leucospilota.
In another study carried out by Mansour [10], on the anti-Linfantum activity of Vitis vinifera L. leaves aqueous extract, report an IC 50 of 12.53 µg / ml.
The antileishmanial action of these natural compounds has not yet been elucidated; never theless [19], report that the antileishmanial action of the flavonoids is done by inhibition of the parasitic topoisomerase II.

CONCLUSION
In the search for novel antiprotozoal agents at a time when there is an urgent need for new innovative drug leads.
The results obtained suggest that the aqueous extract of A. unedo leaves can lead to the development of an effective treatment against leishmaniasis.
The antileishmanial activity of the studied extract may be due to its composition, rich in phenolic compounds.
The perspective of applying this extract to therapeutics involves the determination of his active fraction and its evaluation by in vivo tests.