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Leishmanicidal activity assessment of olive tree extracts.

ARTICLE INFO

Keywords:

Natural products

Oleuropein

Hydroxytyrosol

Visceral leishmaniasis

ABSTRACT

Leishmaniasis, a protozoan parasitic disease that remains a major worldwide health problem with high endemicity in developing countries, is prevalent around the Mediterranean basin. High cost, systemic toxicity, and diminished efficacy due to development of parasite resistance are the serious drawbacks of current treatment options. Thus, identifying new, effective, and safer anti-leishmanial drug(s) is of paramount importance. Here we tested the anti-promastigote and anti-amastigote activity of five natural products, including oleuropein and hydroxytyrosol, present in olive tree leaves and olive mill wastewater. These products are recognized as low-cost starting materials rich in bioactive compounds, particularly biophenols. Oleuropein and hydroxytyrosol exhibited the best inhibitory effect among the natural products tested in both stationary and middle logarithmic phase promastigotes of L. infantum, L. donovani, and L. major. Similarly, oleuropein and hydroxytyrosol demonstrated the highest selectivity index ratio against L. donovani amastigotes that parasitize J774A.1 macrophages. Moreover, oleuropein was tested in vivo in an experimental visceral leishmaniasis model. L. donovani-infected BALB/c mice received intraperitoneal oleuropein a total of 14 times at intervals of every other day. Three clays after treatment termination, the spleen parasitic burden was reduced >80%. Of interest, this effect of oleuropein persisted and was even enhanced 6 weeks after the termination of the treatment, as determined by parasite depletion of >95% in liver and spleen. These findings contribute to the potential development of natural products as effective drugs against parasites of the Leishmania genus, with low cost and diminished cytotoxicity.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Leishmaniasis is a vector-borne protozoan disease caused by species of the genus Leishmania. Leishmania spp are responsible for parasitic diseases with a wide range of symptoms and considerable impact on public health worldwide. Leishmaniasis is still one of the world's most neglected diseases, accounting for 1.5-2 million new cases and 50,000 deaths attributable to the visceral form annually (World Health Organization, Leishmaniasis Control home page). The persistence of and increase in leishmaniasis is mainly due to factors such as the AIDS epidemic, climatic changes, international travel, migration flows, lack of effective vaccines, problems with vector control, and development of drug resistance (Dujardin et al., 2008).

Treatment and control of leishmaniasis involve a limited number of drugs with various adverse reactions, effectiveness, and long duration of treatments. So far, chemotherapy includes pentavalent antimonials, numerous forms of amphotericin B, paromomycin (aminosidine), pentamidine isethionate, hexadecylphospho-choline (HePC; miltefosine), and azoles such as ketoconazole, fluconazole, and itraconazole (World Health Organization, Leishmaniasis Control home page). Despite the numerous treatments, none is fully satisfactory because of the toxicity of the treatment itself, a tendency to host relapse, and the development of resistant parasite strains (Kappagoda et al., 2011). All of these disadvantages are assisted by the fact that many people worldwide have no access to conventional pharmacological treatments (den Boer et at., 2011). These patients prefer to focus their efforts for therapy on folk remedies (alternative treatments) that consist basically of natural products that are considered non-toxic, but their safety and effectiveness require evaluation (Sen and Chatterjee, 2011). Screening natural products for potential use in the therapy of leishmaniasis is essential to satisfy the urgent need for alternative treatments (Rocha et al., 2005).

Olives and olive oil are considered the cornerstone of the Mediterranean diet and are recognized for their overall health benefits (Cicerale et al., 2009). Olive tree (Olea europaea) leaves and olive mill wastewater (OMWW), which is a high-polluting byproduct of olive oil production, are regarded as low-cost sources that are rich in phenolic compounds with strong antioxidant properties (Cardinali et al., 2010). The majority of the compounds, at least 30, are present in olive oil, and recent data have suggested that many of the constituents may have more health benefits than previously thought (Cicerale et al., 2009).

Oleuropein obtained from olive leaves and hydroxytyrosol obtained from OMWW have multiple biological properties (Tuck and Hayball, 2002; Waterman and Lockwood, 2007). Oleuropein, the main ingredient of olives and olive leaves, is a secoiridoid glucoside and belongs to the polyphenol group. Hydroxytyrosol is basically found in olives and in olive oil in the form of its elenolic acid ester oleuropein, and especially after hydrolysis during degradation, in its plain form. The major activity of oleu-ropein and hydroxytyrosol is to scavenge free radicals to protect cells from oxidation. Especially, the anti-oxidant ability of oleu-ropein may slow ageing and reduce the risk of coronary disease (Andreadou et al., 2006; Katsiki et al., 2007). Moreover, oleuropein enhances the response of macrophages to bacterial lipopolysaccharide via an increase in inducible nitric oxide synthase activity whereas oleuropein by itself does not enhance nitric oxide production by macrophages (Visioli et al., 1998). Finally, oleuropein and hydroxytyrosol have been reported to have antitumor, antiviral, antibacterial, and antiparasitic action (Bisignano et al., 1999: Granados-Principal et al., 2010; Jiang et at., 2008; Lee-Huang et al., 2007a, b; Tuck and Hayball, 2002; Waterman and Lockwood, 2007) whereas, in the year 2000, oleuropein was claimed in a U.S. patent (Fredrickson, Num: 6,117,844) to have potent antiviral activity against numerous viral species.

In this study, we evaluated five natural products, two pure constituents and three crude extracts, derived from olive leaves and OMWW, as potent in vitro and in vivo anti-leishmanial agents. For this purpose, they were tested against promastigotes of three Leishmania species and against L. donovani intracellular amastigotes. Finally, oleuropein was also tested in vivo against experimental visceral leishmaniasis caused by L. donovani parasites.

Materials and methods

Crude extracts and pure compounds

Air-dried and pulverized leaves (5 kg) from Olea europaea var koroneiki collected in Crete (Greece) were extracted by mechanical stirring for 12 h with acetone (2x 2.51). The extract was evaporated completely and washed with a mixture of CH2C12/MeOH:98/2 (31). The insoluble material (360g) was separated and dried, producing a yellow powder containing 60% oleuropein. Ten grams of this yellow powder was subjected to countercurrent chromatography, using a fast centrifugal partition chromatograph (FCPC) apparatus (Kromaton). The system of solvents used in this procedure was Et0Ac/Et0H/H20:10/1/10 (61). The capacity of the column was 11, the rapidity of the rotation was 900 rpm, and the flow rate was 15 ml/min. A total of 5.0 g of oleuropein (purity 90%) was isolated by the above-mentioned process.

Olive leaf water extraction (OLWE) and olive leaf decoction (OLD) were derived from the same collection of Olea europaea leaves. Olive leaf powder was extracted using the method of accelerated solvent extraction (ASE) performed on an ASE 300 system (Dionex, USA). The extraction was conducted with deionized water at 80 [degrees]C and the OLWE was finally lyophilized. For the other portion, fresh leaves were blended using a general-purpose electric blender. The blended material was added to water and boiled for 3 min. Then, the hot mixture was filtered through a filter paper, and the filtrate was extracted and evaporated to dryness under reduced pressure to yield a solid residue.

Hydroxytyrosol was extracted from OMWW resulting from olive oil production using olive fruits of Olea europaea var koroneiki. During the process, the material was filtered using filter paper. Then filtered wastewater was applied to Amberlite XAD-4 resin and the column eluted with 96% ethanol. Then ethanol was evaporated and a phenolic fraction was received. The final step aimed at the purification of hydroxytyrosol. The above polyphenolic fraction was submitted to countercurrent chromatography using the FCPC apparatus (Kromaton). The system of solvents used in this procedure was c-hexane/Et0Ac/Me0H/H20:4/6/4/6 (Agalias et al., 2007). The capacity of the column was 11, the rapidity of the rotation was 900 rpm, and the flow rate was 15 ml/min. Hydroxytyrosol (purity >90%) was isolated using the above-mentioned process.

The purity of oleuropein, hydrotyrosol (Fig. 1) and olive leaf decoction was established by high performance liquid chromatography (HPLC) (Fig. 2) and nuclear magnetic resonance (NMR). Significant was, the presence of oleuropein and hydroxy-tyrosol alongside with other unidentified compounds in the olive leaves decoction. Prior to use, pure oleuropein, pure hydroxytyrosol, and the oleuropein 60% mixture were diluted in distilled water while the other two crude extracts, OLWE and OLD, were diluted in dimethyl sulfoxide (DMSO). All extracts were Millipore filtered with a 0.4511m filter and stored at 4 C until use.

Parasites

Three reference Leishmania strains were used in this study: Leishmania infantum (zymodeme MON-1, strain MCAN/PT/ 98/IMT 244), Leishmania donovani (zymodeme MON-2, strain MHOM/IN/1996/THAK35), and Leish mania major (zymodeme LV39, strain MRHO/SU/59/P). The promastigotes were cultured in complete medium consisting of RPM1-1640 (with low content of phenol red) (Biochrom AG, Berlin, Germany) supplemented with 2 mM L-glutamine, 10 mM HEPES, 24 mM NaHCO3, 100 Wm! penicillin, 100 [micro], g/m1 streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco, Paisley, UK) at 26C. Cultures were estimated daily for parasite growth, and promastigotes were collected at the middle of the logarithmic or at the beginning of the stationary phase.

Cell cultures

Macrophages of the J774A.1 murine macrophage cell line from ATCC (American Type Culture Collection, Rockville, MD, USA) were cultured in complete medium consisted of RPMI-1640 medium supplemented with 2 mM L-glutamine, 10 [micro]M HEPES, 24 mM NaHC[O.sub.3], 100 U/m1 penicillin, 100 [micro]g/m1 streptomycin, and 10% heat-inactivated FBS, at 37 C in a 5% C[O.sub.2] environment. Subcultures were carried out when monolayers were confluent in 25 [cm.sup.2] cell culture flasks.

Animals

Age-matched female BALB/c mice (20-25 g) were obtained from the breeding unit of the Hellenic Pasteur Institute (HPI) and reared in institutional facilities under specific pathogen-free conditions. The mice were maintained at standard temperature (25 [+ or -] 5 C) on a 12 h day/night cycle, receiving a diet of commercial food pellets and water ad libitum. Experimental protocols were approved by the Animal Bioethics Committee of the HPI (Athens, Greece) following the regulations of the EC Directive 1986/609 and the National Law 1992/2015.

Anti-promastigote activity of natural products

The leishmanicklal activity of the five natural products tested was initially established in promastigotes, with parasitic growth being evaluated by a quantitative fluorometric/colorimetric assay using the alamarBlue method (Mikus and Steverding, 2000).

Specifically, a 200111 suspension of complete medium containing promastigotes (3 x [10.sup.6] parasites/m1) obtained from stationary or mid-logarithmic promastigotes of each Leishmania strain were seeded in a 96-well flat-bottom plate. Subsequently, several concentrations (0.1,0.5, 1,5, 10,20. 50, 100, 150 and 200 [micro]g/m1) of each natural product or the equivalent volume of the solvent (distilled water or DMSO) were added separately. HePC (known as milte-fosine; Virbac S.A, France) and paromomycin (Gabbrocol, Vetem S.p.A, Italy) were also tested for their leishmanicidal efficacy as reference drugs. The plates were incubated at 26 [degrees]C for 60 h. After this incubation period, 20 [micro]l of alamarBlue was added for at least 12 h and the plates photometered at 570 nm with reference at 630 nm. Absorbance in the absence of every reference drug, natural product, or the solvent was set as the 100% control. Comparison of control with samples allowed calculation of the inhibitory concentration of the natural products or the solvents that are necessary to reduce the growth of promastigotes by 50% ([IC.sub.50] values).

Cytotoxic effect of natural products on J774A.1 macrophages

For testing the cytotoxic effect of these natural products, J774A.1 macrophages were cultured in 96-well flat-bottom tissue culture plates at a cellular density of 1 x [10.sup.5] macrophages/well. The cells were suspended in complete medium and incubated 18 h at 37 [degrees]C in a 5% C[O.sub.2] environment. All natural products tested, the equivalent volume of their solvents, and the reference drugs were assessed at various concentrations (0.1, 0.5, 1, 5, 10, 20, 50, 100, 200, 250, 500 and 100014m1) to evaluate their cytotoxic effect using the alamarBlue method as previously described (Anti-promastigote activity of natural products section). This parameter was determined by the concentration that results in 50% macrophage growth inhibition (C[C.sub.50]) in comparison to the control after 72 h incubation.

Natural product activity on in vitro intracellular L. donovani MON-2 amastigotes

Additionally, to evaluate the effect of these natural products against Leishmania amastigotes, J774A.1 macrophages and stationary-phase L. donovani MON-2 were added in a 1:15 ratio in 96-well flat-bottom plates and incubated at 37 [degrees]C in a 5% C[O.sub.2] environment for 4 h. After incubation, the excess of promastigotes was removed and the plates incubated for another 72 h under the same conditions. During this incubation, the five natural products, the equivalent volume of their solvents, and the reference drugs were added into the wells at specific concentrations (0.1, 0.5, 1, 5, 10, 20, 50, 100, 150, and 200 [micro]g/m1).

Macrophages were disrupted according to Georgopoulou et al. (2007), and the anti-amastigote activity was evaluated using the alamarBlue method as described in "Anti-promastigote activity of natural products" section.

Oleuropein leishmanicidal activity in L. donovani-infected BALB/c mice

Stationary-phase L. donovani promastigotes were used to infect 8-10-week-old female BALB/c mice by administration of 1.5 x 107 promastigotes per animal intravenously. Mice were randomly assigned to five groups 15 days post infection. Animals in the first three groups received for 28 clays, every other day, 45, 15 and 5 mg/kg of body weight (b.w.) of pure oleuropein, respectively. The fourth group received 4 mg/kg b.w. HePC by oral gavage every day for 28 consecutive days. Finally, mice infected with L. donovani and receiving no treatment served as positive control. The spleen and liver parasitic load was determined at 3 days and 6 weeks post treatment using the limited dilution method as previously described (Carrion et al., 2006).

Statistical analysis

In vitro leishmanicidal activity and cytotoxicity were expressed as the [IC.sub.50] and [CC.sub.50] by the linear regression analysis, respectively. Values are mean [+ or -] S.E. from at least three independent experiments in duplicate. Differences between means from the in vivo experiment were analyzed for significance using one-way ANOVA. When ANOVA indicated significant differences, a post-hoc analysis using the Gabriel test was conducted for multiple comparisons among groups. All data are presented as mean values [+ or -] S.E. Differences were considered significant at a 0.05 level of confidence.

Results

Effect of natural product activity on promastigote growth

Any anti-leishmania effect was first determined using the ala-marBlue method. Pure oleuropein extract showed an effect similar to that of the oleuropein 60% mixture in all three stationary-phase Leishmania strains tested (Table 1). However, their activity against L. donovani promastigotes was markedly higher (77.2 [+ or -] 10.5 and 84 [+ or -] 13.5 [micro]g/ml, respectively). On the other hand, only pure oleuropein inhibited the growth of L. infantum and L. donovani promastigotes obtained from logarithmic phase (191.9[+ or -] 19.3 and 69.4[+ or -] 6.3 [micro]g/ml, respectively). Hydroxytyrosol also inhibited promastigote growth in the stationary and logarithmic phases in all three Leishmania strains tested. As Table 1 shows, hyclrox-ytyrosol demonstrated leishmanicidal activity similar to that of pure oleuropein against L. donovani promastigotes (60.5 [+ or -] 7.2 and 75.5 [+ or -] 0.6 [micro]g/m1 vs. 77.2 [+ or -] 10.5 and 69.4 [+ or -] 6.3 [micro]g/ml, respectively). Of interest, hydroxytyrosol exerted the highest leishmanicidal effect against stationary and logarithmic L. major promastigotes among all natural products tested in this study (50.1 [+ or -]4.8 and 105 [+ or -] 13 [micro]g/ml, respectively).

Table 1 [IC.sub.50] ([micro]g/ml) of olive tree natural products
vs Leishmania promastigotes.

                     L infantum              L donovani
                          MON-1                   MON-2

                    [IC.sub.50]             [IC.sub.50]
                   promastigote            promastigote

                          S (a)    L (b)          S (a)    L (b)

Natural product

Oleuropein            145 [+ or    191.9  77.2 [+ or -]  69.4 [+
                           -]21    [+ or            105    or -]
                                      -]                     6.3
                                    19.3

Oleuropein 60%     144 [+ or -]        -    84 [+ or -]        -
                             32                    13.5

Hydroxytyrosol    32.9 [+ or -]  71.8 [+  60.5 [+ or -]  75.5 [+
                             01    or -]            7.2    or -]
                                    12.6                     0.6

OLD (c)            113 [+ or -]        -    60 [+ or -]        -
                           25.5                     9.5

OLWE (d)         179.1 [+ or -]        -    76 [+ or -]    127.9
                           35.5                      19    [+ or
                                                          -] 3.3

Drug

Paromomycin       0.42 [+ or -]  0.72 [+  3.11 [+ or -]  1.94 [+
                           0.16    or -]            0.3    or -]
                                    0.06                    0.09

HePC              3.02 [+ or -]  3.75 [+  0.35 [+ or -]  3.27 [+
                           0.21    or -]           0,02    or -]
                                    0.74                    0.09

                  L major LV-39

                    [IC.sub.50]
                   promastigote

                          S (a)   L (b)
Natural product
Oleuropein         141 [+ or -]       -
                             23

Oleuropein 60%   151.8 [+ or -]       -
                           40.4

Hydroxytyrosol    50.1 [+ or -]  105 [+
                            4.8   or -]
                                     13

OLD (c)                       -       -

OLWE (d)                      -       -

Drug

Paromomycin       0.11 [+ or -]    0.35
                           0.05   [+ or
                                     -]
                                   0.05

HePC              0.94 [+ or -]    4.99
                           0.54   [+ or
                                     -]
                                   1.03

--. no leishmanicidal activity detected.

(a.) Stationary-phase promastigotes.

(b.) Logarithmic-phase promastigotes.

(c.) Olive leaf decoction.

(d.) Olive leaf water extraction.


OLD exhibited antiparasitic activity only against L. infant= and L. donovani promastigotes obtained from stationary phase (113 [+ or -] 25.5 and 60 [+ or -] 9.5 [micro]g/ml, respectively). OLWE had the same leishmanicidal effect as OLD against stationary L. infantum and L. donovani promastigotes (179.1 [+ or -] 35.5 and 76[+ or -] 19 lig/ml, respectively) but also inhibited logarithmic phase L donovani promastigotes (127 [+ or -] 3.3 [micro]g/ml). Overall, pure oleuropein and pure hydroxytyrosol were the most potent natural extracts tested; pure oleuropein was effective against L. donovani MON-2 promastigotes and hydroxytyrosol was more effective against L. infantum MON-1. As expected, paromomycin and HePC drastically reduced the promastigote growth of all three Leishmania strains during both cultivation phases.

Effect of natural product activity on L. donovani amastigote growth and cytotoxic effects on J774A.1 macrophages

To investigate whether these natural products exert activity against the intracellular form of the parasite, we calculated the selectivity index (SI) ratio ([CC.sub.50] for macrophage/[IC.sub.50] for amastigotes) for every natural product tested (Table 2). The two pure extracts, oleuropein and hydroxytyrosol, exhibited the highest SI ratios, with values of 3.24 and 4.65 at 110 [+ or -] 32 and 38.7 [+ or -] 3 [micro]g/ ml, respectively, for inhibiting 50% of the L. donovani amastigote growth. The oleuropein 60% mixture and OLD showed relatively low selective activity against intracellular amastigotes. with SI values at 1.47 and 1.52, respectively. In addition, OLWE showed greater cytotoxicity against J774A.1 macrophages than an ability to inhibit amastigote growth, with an SI ratio of 0.71. Paromomycin and HePC SI values were 229.2 and 297.9, respectively.

Table 2 [IC.sub.50] and [CC.sub.50] ([micro]g/ml) of olive
tree natural products vs. Leishmania intracellular amastigotes
in J774A.1 macrophages.

                   L donovani MON-2          J774A.1  Sl (a)
                                         macrophages
                        [IC.sub.50]      [CC.sub.50]
                         amastigote

Natural Product

Oleuropein          110 [+ or -] 32  356 [+ or -] 23    3.24
Oleuropein 60%     242 [+ or -] 118  355 [+ or -] 41    1.47
Hydroxytyrosol      38.7 [+ or -] 3  180 [+ or -] 16    4.65
OLD (b)              98 [+ or -] 30  149 [+ or -] 23    1.52
OLWE (c)            203 [+ or -] 57  196 [+ or -] 11    0,71

Drug

Paromomycin       1.2 [+ or -] 0,85  275 [+ or -] 53   229.2
HePC             0,48 [+ or -] 0.23  143 [+ or -] 43   297.9

(a.) Selectivity index ratio ([CC.sub.50] macrophage/[IC.sub.50]
amastigote).

(b.) Olive leaf decoction.

(c.) Olive leaf water extraction.


Effect of natural product activity on the parasitic burden of L. donovani-infected BALB/c mice

Three doses of oleuropein (45, 15 and 5 mg/kg b.w.) were given intraperitoneally to L. donovani-infected BALB/c mice for 28 days, every second day. A control group of infected mice received oral HePC (4 mg/kg b.w.) for 28 consecutive days. During the treatment period, the mice were weighed as an indirect indicator of their overall health. Their weight had a non-significant increase ranging from 10% to 16% during the treatment period (data not shown). Experimental mice were sacrificed 3 days or 6 weeks post treatment and their spleens and livers dissected to determine their parasitic load.

Data obtained from mice sacrificed 3 days after treatment termination showed that all oleuropein doses (45, 15 and 5 mg/kg b.w.) caused a significant reduction in parasitic load in the spleen, up to 79.5% (p = 0.000), 86.8% (p = 0.000), and 57.2% (p = 0.001) compared with the positive untreated control, respectively (Fig. 3(a)). The parasitic burden in the liver of these mice was also significantly reduced by 58.1% (p = 0.003), 62% (p = 0.001), and 65.7% (p = 0.001), respectively (Fig. 3(b)). HePC, as the reference drug, exhibited a significant reduction of 98.8% (p = 0.000) and 98.2% (p = 0.000) in the parasitic load of both spleen and liver tissues compared with the positive untreated control.

To determine whether treatment with oleuropein resulted in persistent control of visceral leishmaniasis, mice were sacrificed 6 weeks after treatment completion and their parasitic burden in spleen and liver assessed. We found that all three doses of oleuropein (45, 15 and 5 mg/kg b.w.) drastically diminished the spleen parasitic burden by 99.7% (p = 0.000), 99.8% (p=0.000), and 96.9% (p = 0.000), respectively (Fig. 4(a)). Similarly, mice treated with HePC also had a significantly reduced parasitic load in both spleen and liver, by 99.9% (p = 0.000) and 99.7% (p = 0.011), respectively (Fig. 4(b)). 45 and 15 mg/kg b.w. oleuropein still had a leishmanicidal effect on liver parasitic burden, displaying a significant reduction of 99.6% (p = 0.011). In contrast, 6 weeks after treatment termination, mice treated with 5 mg/kg b.w. oleuropein did not sustain control over the multiplication of the parasite and showed a recovered parasitic load in the liver.

Discussion

Here we demonstrate for the first time that biophenols present in olive leaves and OMWW, such as oleuropein and hydroxytyrosol exhibit leishmanicidal activity in vitro. Moreover, we showed that the non-toxic secoiridoid oleuropein can abate spleen and liver parasitic burden in BALB/c mice with experimental visceral leishmaniasis. So far, the only survey that has highlighted the antiparasitic activity of oleuropein concerns its effect against Toxoplasma gondii in vitro by inhibiting proliferation of Madin-Darby bovine kidney cells infected with tachyzoites and in vivo reducing by inhibiting 55.4% the expansion of parasites in the peritoneal cavity of infected mice (Jiang et al., 2008).

Oleuropein and hydroxytyrosol, as the only pure extracts tested in this study, showed antiparasitic activity against L. infantum and L. donovani, which are responsible for visceral leishmaniasis, as well as against L major, which causes the cutaneous form of the disease. Another flavonoid in olive oil, luteolin, also exhibits antiparasitic activity against L donovani by inhibiting promastigote cell growth and inducing drastic morphological changes of the parasite as evidenced by loss of cellular integrity and degranulated nuclei of the promastgotes (Mittra et al., 2000). We showed here that oleuropein and hydroxytyrosol inhibited promastigote cell growth from both logarithmic and stationary phases of L dono-vani and L. infantum cultures. Hydroxytyrosol was also capable to abort L. major logarithmic and stationary phase promastigotes, while oleuropein inhibited only the growth of stationary phase L major promastigotes. Reduced concentrations of the active substances in the crude extracts tested (oleuropein 60% mixture, OLD, and OLWE) in combination with the admixtures reduced the efficacy of these extracts, resulting in decreased activity against these three Leishmania strains.

Nevertheless, testing the activity of the natural products against the intracellular amastigote forms of the Leishmania parasites is important since they survive and multiply within mammalian macrophages. Because L. donovani was the most sensitive among the three species examined, the efficacy of the five natural products was tested against the amastigote form of L. donovani protozoans that parasitize J774A.1 macrophages. Based on SI ratios, oleuropein and hydroxytyrosol demonstrated the highest anti-amastigote activity, taking into account the cytotoxicity that they exhibited against uninfected macrophages. Moreover, both pure oleuropein and the oleuropein 60% mixture showed identical cytotoxicity, whereas their leishmanicidal activity against L. donovani amastigotes differed significantly, being two-fold greater with oleuropein. Regarding cytotoxicity, pure oleuropein was less toxic to mammalian cells when compared with the standard drugs although paromomycin and HePC exhibited powerful activity against the amastigote forms of the parasite. In addition to luteolin, which exhibits anti-amastigote activity (Mittra et al., 2000), other bioactive compounds in olive oil and OMWW such as verbascoside, (+)-taxifolin, o-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, cinnamic acid, and gallic acid also exert in vitro leishmani-cidal activity against axenic L donovani amastigotes (Tasdemir et al., 2006). In these cases, natural products come into direct contact with axenic amastigote parasites, circumventing the difficulty inherent in having to enter a host cell to be active. However, in this study, we found that pure oleuropein and hydroxytyrosol have a direct effect on promastigotes (through programmed cell death; unpublished results) and also block proliferation of intracellular amastigotes. The latter indicates either the ability of these two bio-phenols to enter into macrophages and remain active directly or to actuate the host cell and exert indirect leishmanicidal activity.

Luteolin, which is more active than oleuropein and hydroxytyrosol in vitro, seems to have little effect in L. donovani-infected BALB/c mice (Tasdemir et al., 2006). On the other hand, luteolin can diminish the spleen parasitic load of golden hamsters infected by the same Leishmania species (Mittra et al., 2000). In our study, oleuropein in an in vivo experimental model not only managed to block the spread of organ parasitosis but also was able to maintain an enhanced durable control of the visceral form of the disease. All three doses of pure oleuropein almost depleted the parasitic load of the spleen 42 days after treatment termination, demonstrating an effect similar to the standard drug HePC used in clinical practice against human and canine visceral leishmaniasis. Analogous depletion of parasites occurred in the liver with 45 and 15 mg/kg b.w. of oleuropein and HePC, while in contrast oleuropein at a dose of 5 mg/kg b.w. failed to inhibit the progression of the disease. An explanation for this finding may be the faster metabolism of oleuropein in the liver during detoxification relative to its persistence in the plasma, which is correlated with spleen absorption because of the close relationship of the spleen with blood circulation (Serra et al., 2012). Sustained oleuropein levels in plasma may exert an anti-inflammatory effect via IL-lb decrease since has been reported that in human whole blood cultures stimulated with LPS, oleuropein inhibited the production of IL-lb by 80% (Miles et al., 2005). The capacity of oleuropein to reduce IL-1 molecules suggests a possible switching of the immunological response toward the Th-1 type. This later hypothesis is supported by the presence of IL-12 and IL-18 in spleen cell culture of IL-1 knockout BALB/c mice that selectively induce IFN--y production that determines resistance to visceral leishmaniasis (Voronov et al., 2010).

It is important to investigate the exact mechanism by which oleuropein inhibits visceral leishmaniasis caused by L. donovani. The low cost of oleuropein and its high efficacy are important because of the great need for new antiparasitic compounds accessible for low-income patients. Of note, oleuropein might constitute an ideal chemotherapy for patients who are co-infected with visceral leishmaniasis and HIV because oleuropein can inhibit both viral entry and integration (Lee-Huang et al., 2007a, b). Finally, oleuropein could serve as a forerunner for the rational design of synthetic analogs with higher in vitro and in vivo activities.

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Acknowledgments

This work was funded by an internal grant from the HPI.I.D.K. received scholarships from the General Secretariat of Research and Technology, Ministry of Development, Competitiveness, Infrastructure, Transport and Networks.

We thank Dr. K. Soteriadou for the introduction of our laboratories, helpful discussions and for providing us L. donovani MON-2 strain. Also, we thank Dr. E. Karagouni for critical discussions.

* Corresponding author. Tel.: +30 210 6478 828; fax: +30 210 6478 828. E-mail address: e.dotsika@pasteur.gr (E. Dotsika).

0944-7113/$--see front matter [c] Elsevier GmbH. All rights reserved hattp://dx.doi.org/10.1016/j.phymed.2012.11.0103

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Joannis D. Kyriazis (a), Nektarios Aligiannis (b), Panagiotis Polychronopoulos (b), Alexios-Leandros Skaltsounis (b), Eleni Dotsika (a), *

(a.) Laboratory of Cellular Immunology. Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, 11521 Athens, Greece

(b.) Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, 15777 Athens, Greece
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Author:Kyriazis, Joannis D.; Aligiannis, Nektarios; Polychronopoulos, Panagiotis; Skaltsounis, Alexios-Lean
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGR
Date:Feb 15, 2013
Words:5644
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