Iranian Journal of Pharmaceutical Research (2013), 12 (3): 349-355
Received: March 2012
Accepted: Jun 2012
Copyright © 2013 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services
Original Article
The Effect of Vitis vinifera L. Leaves Extract on
Leishmania infantum
Rym Mansoura*, Najoua Haouasb, Amel Ben Kahla-Nakbic,Saoussen Hammamib,
Zine Mighria, Farouk Mhennia and Hamouda Babbab
a
Unité of Recherche 12-04, Chimie Appliquée and Environnement, Faculté of Sciences of
Monastir, Monastir 5000, Tunisie. bLaboratoire of Parasitologie-Mycologie (99UR/08-05),
Faculté de Pharmacie, Département of Biologie, Clinique B, 1 Rue Avicenne, Monastir 5000,
Tunisie. cLaboratoire of Biodiversité and Biotechnologie Marine, Institut National des Sciences
and Technologies of la Mer, Annexe Monastir, BP 59, Monastir 5000, Tunisie
Abstract
Vitis vinifera L.is a traditional Asian herb widely used for different health problems. In the
present research, the ethanolic and the aqueous extracts of Vitis vinifera L. leaves collected from
shrub, grown in Tunisia, were prepared and evaluated for the antileishmanial activity against
Leishmani ainfantum promastigotes. The inhibitory concentration 50 (IC50) was determined and
the results showed that the etahnolic extract is more active than the aqueous one (IC50= 0.108
mg/mL). Microscopic observations showed that the ethanolic extract promoted the destruction
of cytoplasmic and nuclear membranes of Leishmani ainfantum promastigotes and altered the
overall shape of the cell. In order to explain the difference of antileishmanial activity between
ethanolic and aqueous extracts, anthocyanins amount was determined by spectrophotometry. It
was found that the ethanolic extract is richer in anthocyanins than the aqueous one which can
explain the higher antileishmanial activity of the ethanolic extract.
Keywords: Vitis vinifera L.; Extraction; Leishmaniasis; Antileishmanial activity;
Anthocyanins
Introduction
Leishmaniasis is a real public health problem
encountered in several countries over the world
including Tunisia. In Tunisia, incidence was
assessed to bemore than 4000 and 150 case
per year for the cutaneous and the visceral
leishmaniasis form respectively, (1, 2). Visceral
leishmaniasis form is caused by Leishmania (L.)
infantum species and is endemic in the North
and the Center of Tunisia (3, 1). The same
species may also be isolated in several cases of
* Corresponding author:
E-mail: rymmansour2004@yahoo.fr
cutaneous leishmaniasis encountered in the same
foci (4, 5).
In absence of vaccination against the parasite
Leishmania (L), the use of drugs remains the only
way for treatment. In fact, pentavalentantimony
such us Glucanthime® (meglumine antimony)
and Pentostam® (sodium stibogluconate) were
used for the treatment of both cutaneous and
visceral leishmaniasis (6). In case of resistance,
these drugs were substituted by amphotericin B
and miltefosine® (7). However, many signs of
stibio-intoxication such as cardiac conduction
disorders, hepatic cytolysis, dysfunction of renal
tubular and pancreatitis have been attributed
to these drugs (8, 9). In addition, resistance
Mansour R et al. / IJPR (2013), 12 (3): 349-355
to pentavalent antimony has been reported in
Bihar, India in 60% of treated patients (10,
11). Treatment failure was also observed in
the Mediterranean region (12, 2). Accordingly,
an urgent need to find new antileishmanial
agents,more efficient and less harmful for
patients is highly recommended.
Vitis(V.) vinifera (Vitales, Vitaceae), is an Asian
native perennial woody vine. From different parts
of this plant essentially fruits, several preparations
used in folk medicine have been derived (13). In
Ethnopharmacology, the infusion of the leaves of
red varieties has been used as haemostatic and for
diarrhea treatment. Fresh leaves have been used
externally to heal wounds and to lance abscesses
(14). Grape leaf-based medicines are traditionally
used for diarrhea, hepatitis and stomachaches
(15, 16, 13, 17). Grapes, seeds, and leaves have
been used for preventing heart and blood vessels
diseases, varicose veins, hemorrhoids, “hardening
of the arteries” (atherosclerosis), high blood
pressure, swelling after injury or surgery, heart
attack and stroke.
Moreover, grape leaf has been used for
attention deficit-hyperactivity disorder (ADHD),
chronic fatiguesyndrome (CFS), diarrhea, heavy
menstrual bleeding, uterine bleeding, andcanker
sores. It has been also used as a mild laxative
forconstipation (16-24).
Few studies have been conducted on the
biological effects of leaves. Nilüfer et al have
shown that the aqueous extract from leaves of Vitis
vinifera L. possessantidiabetic and antioxidant
activities (25). It was also mentioned that the
aqueous extract of Vitis vinifera L. leaves shows
antibacterial activity against Escherichia coli,
Enterococcus feacalis, Staphylococcus aureus
and Vibrio alginolyticus (26).The aim of this study
wasto evaluate the antileishmanial activity of the
aqueous and ethanolic extract of Vitis vinifera L.
leaves and to quantify their anthocyanins amount
as anthocyanins family is well known by the
richness of its biological activities (27).
latitude is 10°25’ and its longitude is 36° 37 W.
Leaves were collected on December 8th 2010.
They were dried and reduced to fine powder (28).
Preparation of extracts
One gram of dried leaves of Vitis vinifera
L. wasextracted with 20 mL of distilled water
during 1 hour at 95°C. The mixture waspassed
through a filter paper in order to remove plant
debris. The aqueous extract was then sterilized
by filtration through a 0.22 µm membrane
filter. It was used freshly for the preparation
of different concentrations in order to evaluate
the antileishmanial activity. As for the ethanol
extract, it was obtained by soxhlet with acidified
ethanol (ethanol: HCl 99: 1 v / v: = 0.1 N HCl)
till the reflux of this mixture. (29)
Preparation of stock solution
The aqueous extract was directly used.
However, the ethanolic extract of Vitisvinifera
L. leaves was initially dissolved in dimethyl
sulfoxide (DMSO) at 1%. 10 mg of the
ethanolicextract were dissolved in 100 μL of
pure DMSO and then added to 900 μL of culture
medium suitable for a final concentration of 10
mg/mL. This solution was sterilized by passage
through a filter of 0.22 µm in a laminar flow hood.
It was then diluted to different concentrations
for the antileishmanial activity that the highest
concentration tested is 1 mg/mL. In this way the
final concentration of DMSO never exceeded
1% in the medium tested. This concentration had
no effect on the growth of Leishmania.
Maintenance and counting of parasite
The L. infantumstrain (MHOM/TN/2010/
44M) was isolated from a visceral leishmaniasis
human case and typed by isoenzyme and
molecular methods in the laboratory of
Parasitology, Faculty of Pharmacy of Monastir,
Tunisia. Promastigote Culture was maintained
at 25 °C in RPMI 1640 medium supplemented
with 10% of decomplemented fetal calf serum
(Gibco Invitrogen Corporation, New York, NY)
pH 7.0, in a cell culture dishes with weekly
subcultures. The promastigotes were counted
using Neubauer chamber and re-suspended in
fresh medium at a final concentration of 1.0 ×
106 live promastigotes/ mL. The viability of
Experimental
Plant material
The VitisviniferaL. Black Grenache leaves are
collected from “Bir Bou Ragueba”, a suburb in
the city of Nabeul, Tunisia. Bir Bou Ragueba’s
350
The Effect of Vitis vinifera L. Leaves Extract on Leishmania infantum
Figure 1. Chemical structure of cynidin-3-glucoside, Anti-leishmanialactivity of Vitis viniferaL. (Vitales, Vitaceae) leavesextracts, Rym
Mansour.
Leishmania was assessed by mobility and lack
of color in the presence of trypan blue.
was determined according to the method of
Giusti and Wrolstad. By changing the pH
different values of absorbance wereobtained.
Extracts were diluted 10 times in two buffers.
The aqueous solution consistedof potassium
chloride (0.025 M) with pH 1 and a second
aqueous solution consisting of sodium acetate
(0.045 M) after 15 min incubation at room
temperature, the absorbance of both extracts
weremeasured at 520 nm and 700 nm. The
total anthocyanins content wasgiven in mg
of cyanidin-3-glucoside (cy-3-glu) / liter (2),
andthe equipment used wasan UV-visible CE202 spectrophotometer ( 27).
A = (A520 nm – A 700 nm) pH 1.0 - (A520
nm - A700 nm) pH 4.5 (1)
The
concentration
of
anthocyaninswascalculated using this equation:
Anti-leishmanial activity
The trials were conducted in a liquid medium
in miroplaques of 96 round bottom wells.
Promastigotes of the logarithmic phase were resuspended to a concentration of 106Leishmania/
mL and were treated with ethanolic and
aqueous extracts of V. vinifera L. leaves. For
the ethanolic extract, the tested concentrations
were 1, 10, 100, and 1000 μg/mL. While for
the aqueous extract concentrations were 50
mg/mL, 25 mg/mL, 12.5 mg/mL and 6.25 mg/
mL. Two wells containing the negative control
cultures without extract supplemented or not
with 1% DMSO were used. A positive control
well containing culture with Glucantime®, was
also tested. The plates were incubated at 27 °C
for 72 h to assess the anti-proliferative effect of
the extracts. The number of mobile and viable
promastigotes was quantified by counting the
parasite using the Neubauer chamber. Inhibitory
concentration 50 (IC50) was determined by the
method of logarithmic regression analysis of
data obtained.
Leishmania cell morphology was also
evaluated by deposing 20 μL of culture treated
with ethanolic V. vinifera L. leaves extract on
microscopic slides. After spreading and drying
in ambient air, slides are fixed with absolute
methanol, stained with 10% Giemsa, and
examined under an oil immersion objective of
the light microscope.
C=
A×MW×DF×103
×
(2)
MW (molecular weight) = 449.2 g / mol for
cyanidin-3-glucoside (Cyd-3-glu) (Figure 1);
DF = dilution factor
l=1 cm (cuvette width)
ε = 26 900 molar coefficient of extinction
in L-1xcm xmol-1, for Cyd-3-glu, and 103 =
conversion factor from g to mg.
Statistical Analysis
In order to assure the reproducibility of
results, all experiments wererepeated thrice. The
means and standard deviation were determined.
The data werethen analyzed by SPSS version
17.0. The Student t-test was applied and
Anthocyanins quantification
The amount of anthocyanins in the extracts
351
Mansour R et al. / IJPR (2013), 12 (3): 349-355
2A
2B
2C
Figure 2. a: membrane destruction, Anti-leishmanialactivity of Vitisvinifera L. (Vitales, Vitaceae) leavesextracts, Rym Mansour. b:
Unequal division, Anti-leishmanialactivity of Vitis viniferaL. (Vitales, Vitaceae) leavesextracts, Rym Mansour. c: UntreatedLeishmania
infantumculture., Rym Mansour.
the p-value less than 0.05 wasconsidered
significant.
to the protocol of Giusti and Wrolstad. Thus,
we found that the aqueous extract and ethanolic
extract contained respectively 3.89 mg/g and
5.01 mg/g (mg anthocyanins/g of dry material).
The ethanol extract wasricher inanthocyanins
than the aqueous extract.
Results
Vine leaves extracts were tested to evaluate
their activity against promastigotes of L.
infantum. For aqueous extract, IC50= 12.55 mg/
mL and for ethanolic extract IC50=108.85 µg/
mL. In addition, the ethanol extract showed an
IC50 lower than glucanthime(IC50 = 8.504 mg/
ml). (Table1)
In order to assess the viability of promastigotes
of L. infantum in the presence of the ethanol
extract, treated cultures were stained with
May-Grunwald Giemsa and examined under
an optical microscope. The effects of ethanolic
extract on Leishmania were different from those
caused by Glucanthime. Indeed, Leishmania
incubated in the presence of the ethanolic extract
of V. vinifera L. leaves showed the following
result: destruction of cytoplasmic and nuclear
membranes and, thus, altered the overall shape
of the cell (Figure 2a, 2b). An abnormality at the
cell division: an imbalance in the nuclear division
(Figure 2b) and the division of the nucleus and
the flagellum however,without duplication of the
kinetoplast (Figure 2c).
The quantity of anthocyanins present in the
ethanolic and aqueous extracts was calculated
using the spectrophotometric method according
Discussion
This study examined the antileishmanial
activity of aqueous and ethanolic V. vinifera
L. leaves extracts. The ethanolic extract
showed a prominent activity against L.
infantumpromastigotes.
Vitisviniferaleaves are rich in tannins,
flavonoids, procyanidins and also contain
organic acids, lipids, enzymes and vitamins
(13, 30, 31, 17). Furthermore, the quantitative
analysis of compounds found in leaves has also
been evaluated by Monagas et al. (2006). They
found that V. vinifera is rich in anthocyanins and
other flavonoids (32).
We found that the difference between the
IC50 could be the result of the difference in the
anthocyanin content. The ethanol extract, having
a higher content of anthocyanins than the aqueous
extract, seems to be more effective against L.
infantum promastigotes. This couldbe due to
the number of hydroxyl groups of anthocyanins
which is supposed to be the cause of the relative
toxicity to microorganisms (33).
352
The Effect of Vitis vinifera L. Leaves Extract on Leishmania infantum
Table 1. IC50 extracts of VitisviniferaL. leaves against L.infantum.
Aqueous extract
Ethanolic extract
Glucanthime
Concentrations (mg/L)
50
25
12.5
6.25
1
0.1
0.010
0.001
200
100
50
25
Percentage of Viability
0
3.
99.9
100
31.65
46.23
71.69
99.91
10.10
42.29
52.17
81.23
IC50 (mg/L)
12.53
0.108
Anthocyanins are the anthocyanidins in their
glycoside form (linked to a sugar moiety). The
anthocyanidins are composed of an aromatic ring
attached to a heterocycle that contains oxygen,
which is also linked by a carbon-carbon to a
third aromatic ring (34). The anthocynidins are
flavonoids which are, in their turn, a subdivision
from the family of polyphenols.
Anthocyanins display a wide range of
biological activities including antioxidant,
anti-inflammatory, antimicrobial and anticarcinogenic activities. In addition, they display
a variety of effects on blood vessels, platelets and
lipoproteins able to reduce the risk of coronary
heart diseases (34).
Kolodziej et al. (2001), have reported that
proanthocyanidins possess antileishmanial
activity against the species L. donovari with IC50
between 0.8 and 10.6 nM (35).
Lewin et al., (2011) have also studied the
antileishmanial activity of the flavonoids against
L. donovani (36). Moreover, in 2010 Nour et
al., found that Ageratum conyzoides methylated
flavonoids have an antileishmanial activity
against L. donovani with IC50 = 3.4 µg/mL (37).
Flavonoids from Agaricusblazei Murill
had an antileishmanial activity against L.
amazonensis, L. chagasi and L. major (38).
Several studies have shown that many natural
compounds such as naphthoquinones, alkaloids,
lignans and triterpenes possess antileishmanial
activity against many Leishmania species (39).
Nevertheless, to our knowledge, our study
is the first to show the correlation between
anthocyanins content and IC50 of aqueous and
ethanolic extracts of V. vinifera L. leaves against
L. infantumpromastigotes.
8.504
These molecules have been used to treat all forms
of leishmaniasis. However, recently liposomal
amphotericin B has replaced the pentavalent
antimony as a treatment of choice for visceral
leishmaniasis. Nevertheless, these drugs have
dangerous side effects. In this research, we have
tried to evaluate the anti-leishmanial activity
of aqueous and ethanolic extracts of V. vinifera
L. leaves. Ethanolic extract showed interesting
activity against L. infantum promastigotes.
This extract is currently undergoing detailed
investigations with the objective of isolating
biologically active molecule(s). Based on
the results of this study, we plan to develop
bandages with V. vinifera extract inhibiting the
dermatological proliferation of leishmaniasis, by
nano-microencapsulation.
Acknowledgments
This work was supported by the finances
from the Tunisian Ministry of High Education
and Scientific Research.
Conflict of interest: This work has no
conflict of interest
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