Abstract
The antileishmanial activity of Tunisian propolis essential oil (EO) and its combination with amphotericin B was investigated against 2 local clinical strains of Leishmania: Leishmania major and Leishmania infantum. The cytotoxic potential of this EO was evaluated against macrophage Raw264.7. Combination of propolis EO and amphotericin B was investigated using the checkerboard method. The propolis sample was collected from the region of Beni Khalled, a Tunisian city located west of Cape Bon (Nabeul). Its location is particular since it is near to sea with a steppe climate and the predominance of citrus trees. The EO was obtained by Clevenger-type apparatus. Its chemical composition was identified using gas chromatography with flame ionization detector and gas chromatography-mass spectrometry analysis. Our results demonstrate that Tunisian propolis EO exhibit good antileishmanial activity against L. major and L. infantum promastigotes (IC50 = 5.29 ± 0.31 and 3.67 ± 0.52 µg/mL, respectively) and amastigotes (IC50 = 7.38 ± 0.45 and 4.96 ± 0.24 µg/mL, respectively). Moreover, it reduced significantly the parasite proliferation on a dose-dependent response (95%) with low cytotoxicity (selectivity index = 16.18 and 23.33, respectively). Its combination with amphotericin B showed a synergistic potential (fractional inhibitory concentration = 0.37). Interestingly, the data suggest that propolis EO was involved in macrophage activation by hyperproduction of NO. A total of 51 compounds were identified in the propolis EO. The major compound identified was α-pinene (36.7% ± 2.36%) followed by α-cedrol (6.7% ± 0.10%), totarol (6.6% ± 0.09%), and dehydroabietane (5.2% ± 0.10%). Our findings suggest that Tunisian propolis might constitute a promising source for antileishmanial molecules.
Leishmaniasis is an infectious disease causing a major concern especially in immunocompromised patients and it was included in the list of neglected diseases by the World Health Organization. 1,2 It is a parasitic disease caused by an obligate intracellular parasite of genus Leishmania and spread to human by the bite of female sand flies. 3 There are several different forms of leishmaniasis. The most common ones are cutaneous and visceral. There is cutaneous form characterized by skin sores and visceral form which affects the internal organs. Currently, there are no vaccines for leishmaniasis. The available treatment is chemotherapy which is toxic, expensive, and frequently ineffective. 4 Many compounds and formulations showed antileishmanial activity in clinical uses, but the only few that have been proven worthy includes pentavalent antimonials such as amphotericin B, miltefosine, pentamidine, and aminosidine. 5 These drugs present several problems, including high cost, toxicity, and side effects leading to emergence of resistant strains and making the treatment far from suitable. 6,7
As a consequence, finding new bioactive compounds with antileishmanial activity remains crucial for the development of new perspective for leishmaniasis therapy. In fact, various natural substances with antileishmanial activity have been investigated as an alternative and could be the way for the production of new effective synthetic compounds. 8 Essential oils (EOs) of plants and their compounds are sources of bioactive molecules. 9 -15 Propolis, a natural resinous product, is widely known by its high richness in bioactive compounds and largely used in folk medicine. It is a sticky substance collected by bees from trees and used for the maintenance of their hive and as a multifunctional material for its construction. 10 It contains essentially flavonoids, EOs, resins, minerals, and vitamins. According to its chemical composition, propolis is characterized by a large spectrum of biological activities such as antioxidant, antimicrobial, anti-inflammatory, and antitumor properties. 10 -15
To the best of our knowledge, only few studies on the propolis extracts activity against the Leishmania species have been reported. 11,12 The few studies that have investigated its anti-infectious properties were performed only on its organic extracts and no studies were performed on its EO. The aim of this study was to investigate the chemical composition of the Tunisian propolis EO by gas chromatography-mass spectrometry (GC/MS) and to evaluate its in vitro antileishmanial against Leishmania major and Leishmania infantum clinical isolates and cytotoxic potential against macrophage cells (Raw264.7).
Materials and Methods
Propolis Samples
Propolis samples from Apis mellifera hives were collected by scraping during the spring of 2017 from the region of Beni Khalled in Tunisia.
The region of Beni Khalled was chosen based on its floristic character, where bees can collect resins from Citrus sinensis (L.), Citrus reticulata (L.), Citrus limon (L.), and Eucalyptus gomphocephala.
Essential Oil Extraction
Propolis samples were cut into small pieces (40 g) and were subjected to hydrodistillation using a Clevenger-type apparatus for 3 hours and the oil was dried on anhydrous sodium sulfate and stored at 4°C until analysis.
Essential Oil Analysis by Gas Chromatography With Flame Ionization Detector (GC-FID)
Analytical gas chromatography (GC) was carried out on a Hewlett-Packard 6890 apparatus (Agilent Technologies, Palo Alto, CA, United States) equipped with a flame ionization detector (FID) and an electronic pressure control injector. An HP Innowax (polyethylene glycol) column (Hewlett-Packard; 30 m × 0.25 mm × 0.25 µm film thickness) was used at a carrier gas flow (nitrogen) of 1.6 mL/min. The split ratio was 60:1. The analysis was performed in triplicate using the following temperature program: oven temperature kept isothermally for 10 minutes at 35°C, increased to 205°C at the rate of 3°C/min, and kept isothermally at 205°C for 10 minutes. Detector and injector temperature were held at 300°C and 250°C, respectively.
Essential Oil Analysis by GC-MS
The GC/MS analysis of the EO was performed with an Agilent 7890A GC system coupled to an Agilent 5972C mass spectrometer detector with electron ionization (70 eV). A HP-5 MS capillary column (Hewlett-Packard, CA, United States) was used (30 m × 0.25 mm, coated with 5% phenyl methyl silicone, 95% dimethylpolysiloxane, 0.25 mm film thickness). The column temperature was programmed to rise from 40°C to 240°C with a 5°C/min rate; helium N60 was used as a carrier gas with a 0.9 mL/min flow rate; split ratio was 100:1. Scan time and mass range were 1 second and 50 to 550 m/z, respectively. 9
The identification of propolis volatile compounds was based on a long work with Wiley Registry 9th Edition/NIST 2011 mass spectra search library and by comparison of their retention indices relative to (C8-C22) n-alkanes with those of the literature or with those of original compounds available. The identification was considered valid if the probability was more than 90%, R. Matching over 900.
Antipromastigote Assay
In 96-well microtiter plates, propolis EO was plated at a final concentration varying from 7.81 µg/mL to 1 mg/mL, at 2-fold serial dilutions. After that, 2 × 105 promastigotes per well of L. major (LCO3) and L. infantum (LV20) were added in RPMI-1640 medium (Gibco) and incubated at 27°C for 72 hours. 9 Parasite viability was assessed by the addition of 10 µL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, United States) (10 mg/mL). After 4 hours of incubation at 37°C, the medium was removed and formazan crystals were dissolved in DMSO. Absorbance was read by ELISA reader (Synergy HT, Bio-TEK) at 570 nm. The positive control used in all tests was amphotericin B (98% purity, from Sigma-Aldrich, United States).
Results were expressed as the concentration that inhibited parasite growth by 50%, median inhibitory concentration (IC50). The IC50 was obtained by calculating from log-probit analyses using linear regression (GraphPad Prism 5). Three replicates were performed and the average was calculated from the IC50 values obtained for each separate experiment. 9
Antiamastigote Assay
Promastigote form of Leishmania, at their logarithmic growth phase, was allowed to multiply in macrophages cells Raw 264.7 already plated at 106 cells/mL (ratio parasite-macrophage 10:1 ratio) for 2 hours at 37°C. Free promastigotes were washed with PBS (pH 7, 0.01 M). In order to allow complete differentiation of promastigotes to amastigotes, infected macrophages were left in incubation for additional 24 hours. Essential oil solutions at concentrations ranging from 0.78 to 100 µg/mL were added and the samples incubated for 48 hours for all plates. 15 Cultures were marked with 10% Giemsa and studied under light microscopy (×100).
The number of amastigotes was calculated in at least 100 macrophages for each sample in triplicate cultures. 9,16 Results were expressed as infection rate (IR) that was expressed as indicated by the following formula:
%IR = 100 – [(infection rate of the treated culture/infection rate of the untreated culture × 100)]
The extract with an IC50 ≤ 50 µg/mL was considered as effective against intracellular amastigotes of Leishmania.
Specificity (SP) value was described as the ratio between IC50 of promastigotes and IC50 of amastigotes stage. For SP more than 2, the sample was considered as being more active against the amastigote stage. For SP value under 0.4, the tested sample was considered as more active against promastigotes. While for SP values between 0.4 and 2, the sample was considered as being active against both stages. 17
Synergistic Activity With Amphotericin B
Synergistic combination of propolis EO and amphotericin B was assessed as described previously. 5,13 -15 Briefly, 2 samples were mixed at different combinations in a checkerboard assay. 13 The combinations were incubated with 2 × 105 parasite/mL in 96-well plates and the fractional inhibitory concentration (FIC) index was calculated as follows:
The IC50A and IC50B correspond to the IC50 of each compound tested alone. However, [A] and [B] are the IC50 of the sample tested in combination. For FIC index <0.5, combination was considered as synergistic. However, for FIC index situated between 0.5 and 4, combination was considered as indifferent, and for FIC index >4, combination was considered as antagonist. 15,16
Nitric Oxide (NO) Production
Nitric oxide (NO) release in supernatants of macrophage Raw264.7 cells treated by propolis EO in the absence or presence of L. major and L. infantum was evaluated by the Greiss assay after 72 hours of incubation as previously described. 14,18 A standard curve was prepared with sodium nitrite (concentrations ranging from 3.125, 6.25, 12.5, 25, 50, to 100 µM). Untreated macrophages were used as a negative control. Sodium nitrite in RPMI was used as positive control to construct a standard curve as described previously. 14 The reading was performed in triplicate at a wavelength 630 nm (Biotek, Synergy). The result was expressed as the NO concentration (µM). Each determination was performed in triplicate.
Cytotoxicity and Selectivity Index (SI)
VERO cells were maintained in RPMI-1640 medium supplemented with 10% FBS and cultured in the presence of a solution (Gibco) at 37°C in 5% CO2 atmosphere. Cells were harvested with 0.1% trypan solution and counted under light microscope. In total, 105 of macrophages were initially placed on a 96-well microtiter plate then left to incubate overnight at 37°C. The medium was aspirated off and replaced by 100 µL of various concentrations of tested samples; viability was evaluated using the MTT method after 72 hours of incubation. 19,20 The supernatant was conserved for the nitrite assay at −20°C.
The selectivity index (SI) was calculated as the ratio between (CC50) for macrophage and activity (IC50) for promastigote. An SI more than 10 indicated that samples, with no relative cytotoxicity, showed effective antileishmanial activity.
Statistical Analysis
Triplicate cultures were used in all assays. Statistical analysis was conducted using XLSTAT Software (XLSTAT, 2015, Addinsoft, New York, NY, United States). Differences between mean values obtained were tested for significance by analysis of variance. P values of 0.05 were considered significant.
Results and Discussion
Essential Oil Composition
Propolis EO from Beni Khalled obtained by hydrodistillation yielded about 0.1% (w/v). The results of chromatographic analysis by gas chromatography (GC/FID) and coupled with mass spectrometry (GC/MS) are presented in Table 1. In total, 46 components were identified which represent 97.31% of total oil listed in their elution order (Figure 1). The major component was α-pinene (36.17% ± 2.36%), α-cedrol (6.67% ± 0.10%), totarol (6.57% ± 0.09%), dehydroabietane (5.23% ± 0.10%), tricosane (3.85% ± 0.10%), 4-terpineol (3.58% ± 0.14%), longifolene (2.96% ± 0.12%), and β-elemene (2.90% ± 0.11%). The abundant compounds in Tunisian propolis EO were monoterpene hydrocarbons (54.85% ± 0.12%) followed by sesquiterpene hydrocarbons (12.53% ± 0.11%), diterpenes (9.26% ± 0.09%), and aliphatic hydrocarbons (5.27%).
Chromatogram of essential oil of propolis.
Chemical Composition of Propolis Essential Oil by Gas Chromatography-Mass Spectrometry.
GC-MS, gas chromatography-mass spectrometry; IR, infection rate.
aApolar HP-5 column.
bPolar HP Innowax column.
As demonstrated by several studies, the propolis EO chemical composition has been well explored. 18,21 -23 In most regions, terpenoid compounds are the major constituents identified. However, its composition is quite variable depending on the environmental factors, the geographic region of production, the season of harvest, and the concentration of metabolites in plants visited by bees. 22
In line with the other study, our findings showed the richness of Tunisian propolis EO by monoterpene hydrocarbons (54.85%) and specially the α-pinene (36.17%). These results were consistent with those found in the Iranian propolis EO which was rich in α-pinene (43.9%), 1,8-cineole (11.1%), and camphene (8.6%). 22 Kerman’s propolis EO also contained a similar composition revealing high amounts of α-pinene (46.1%), 1,8-cineole (11.1%), camphene (9.6%), and camphor (5.3%) but also other important compounds such as sabinene (4.6%), borneol (3.4%), bornyl acetate (2.8%), verbenone (2.3%), and linalool (2.1%). 22,23 Similar results have been found in the previous work on Greek propolis 21 in which α-pinene was the major compound and its content varied between 7.9% and 45.8%, trans-β-terpineol (2.2%-6.6%), junipene (1.5%-11.7%), and δ-cadinene (0.3%-8.4%), as well as Brazilian propolis that contained α-pinene (18.3%), β-pinene (6.5%), and δ-cadinene (7.0%). 22 In addition, in other regions of Brazil, the most abundant components in EO were β-caryophyllene (12.7%), acetophenone (12.3%), farnesene (9.2%), and linalool (6.47%). 21 -23
Antileishmanial Activity of Propolis EO
Propolis EO exhibited strong antileishmanial activity against promastigotes of L. major and L. infantum with IC50 values of 5.29 ± 0.31 and 3.67 ± 0.52 µg/mL, respectively (Table 2). Moreover, a high activity was found against amastigote forms of L. major and L. infantum (IC50 = 7.38 ± 0.45 and 4.96 ± 0.24 µg/mL, respectively) with significant (P < 0.05) reduction of the parasite proliferation (more than 95%).
Antileishmanial and Cytotoxic Activities of Propolis Essential Oil.
IC50, the half-maximal inhibitory concentration (µg/mL); CC50, the median lethal concentration (µg/mL); SD, standard deviation.
Selectivity index is calculated based on the CC50/IC50 ratios.
Moreover, this EO showed low toxicity against macrophage cells Raw264.7 (CC50 = 85.63 ± 0.28 µg/mL) with a selective activity against L. major and L. infantum (SI = 16.18 and 23.33, respectively). Interestingly, the propolis EO was considered as active against both stages of Leishmania (SP = 1.39 and 1.35 for L. major and L. infantum, respectively) and this result reinforces its antileishmanial potential (Table 3). To the best of our knowledge, this is the first study assessing the antileishmanial activities of Tunisian propolis EO on promastigotes and amastigotes form of L. major and L. infantum.
Antiamastigote Activity of Propolis Essential Oil, Selectivity, and Specificity.
EO, essential oil; IC50, the half-maximal inhibitory concentration (µg/mL); SD, standard deviation.
Specificity is the ratio between promastigote IC50 and amastigote IC50.
Previously, good antileishmanial activity was reported in propolis extracts. 12 In fact, Bulgarian propolis extracts have been associated with high antileishmanial activity against numerous leishmaniasis species. 24,25 In fact, it was reported that ethanolic and ketonic extracts showed similar activities on promastigote form of L. major (IC50 = 7.2 and 2.8 µg/mL, respectively), Leishmania amazonensis (IC50 = 29.3 ± 7.6 and 26.9 ± 6.4 µg/mL, respectively), and Leishmania chagasi (IC50 = 53.9 ± 7.3 and 41.3 ± 0.9 µg/mL, respectively).
Chemical composition of Tunisian propolis showed that α-pinene was the major compound identified (36.71%). The individual antileishmanial activity of this compound has been demonstrated. 9,26 -28 In fact, α-pinene exhibits good antileishmanial activity against promastigote of L. major and L. infantum (IC50 = 19.8 ± 0.23 and 17.6 ± 88 µg/mL, respectively) with low toxicity (SI = 11.63 and 13.08, respectively). 9 Similar findings were reported previously. 26 According to Rodrigues et al, α-pinene showed effective inhibition against promastigotes (IC50 = 19.7 µg/mL) and amastigotes (IC50 = 15.6 µg/mL) of L. amazonensis. Our result suggested that propolis EO activity may be attributed to major compounds but also to the synergistic activity of minor compounds. In fact, it was reported previously that α-cedrol and totarol may exhibit significant antileishmanial activity (IC50 = 1.5 and 12.2 µM, respectively). 27,28 The antileishmanial properties of EOs were mainly attributed to monoterpenes which may diffuse into parasite membrane structure and cause its damage. 28 These compounds could also cross cell membrane and interact with intracellular organisms. 29
Synergistic Combination of Propolis EO With Amphotericin B
To date, combination therapy has shown a great potential for infectious disease treatments such as malaria, candidiasis, AIDS, and tuberculosis. It was ever more and more applied particularly in highly endemic regions. It aimed to find a new alternative to develop more potent drugs and to minimize side effect, toxicity, duration, and cost of conventional drugs and to prevent the development of drug resistance as well as to enlarge their antimicrobial spectra. 30 Our data showed that Tunisian propolis EO exhibited a synergistic combination with conventional drug “amphotericin B” with a FIC value of 0.37 and more than 98% of Leishmania growth inhibition. Moreover, the synergistic MIC of amphotericin B and propolis EO were reduced by 8- and 4-fold, respectively. Therefore, Tunisian propolis EO could be used as a potential promising agent to increase treatment efficiency of conventional antileishmanial drugs.
Several studies have reported the synergistic potential of propolis extract with conventional treatment. 5,30 -32 Oksuz et al showed a synergistic potential of ciprofloxacin and propolis for the treatment of Staphylococcus aureus. 31 Moreover, the high effect of propolis in increasing bacterial resistance to antibiotics (ampicillin, cefalexin, and amoxicillin) showing a synergistic potential with antibiotics such as tetracycline, chloramphenicol, and neomycin was reported. 32 However, to the best of our knowledge, this is the first study investigating the synergistic potential of propolis EO with amphotericin B.
NO Production
The evaluation of NO production was conducted in the same time of the antiamastigote assay. We collected the supernatant of macrophages infected by Leishmania and treated or not by several concentration of propolis EO (Figure 2). Results revealed that the infected macrophage produces 36.8% more NO than the noninfected macrophage. Although, no significant difference was observed between L. major and L. infantum in NO production. Moreover, the noninfected macrophage treated by 14.76, 7.38, and 3.69 µg/mL of propolis EO produced, respectively, 50.4%, 38.1%, and 25% more NO than control cells (uninfected macrophage and not treated by propolis EO). However, the infected macrophage Raw264.7 treated with propolis EO produced significantly (P < 0.05) higher NO levels (230%, 190%, and 175% higher, respectively) than infected macrophages untreated by propolis EO. Although amphotericin B did not induce NO production, 30 its synergistic combination with propolis EO showed an overproduction of NO (180%) and was therefore involved in the activation of macrophage cells by the hyperproduction of nitric oxide.
Effect of propolis essential oil on nitric oxide production by macrophage cells Raw264.7.
Macrophage cells are the major immunologic pathway implicated in the immune system for the elimination of microorganisms. The activation of macrophages induces the release of several cytotoxic metabolites mainly H2O2 and NO. 33 Thus, we investigated if the antileishmanial properties of propolis EO could be associated with the activation of macrophages cells by the NO production. The result obtained showed that significant high NO production (P < 0.05) was obtained on macrophage cells treated by high concentration of propolis EO (14.76 µg/mL) compared to untreated cells. This is in complete agreement with data reported previously. 34 In fact, nitric oxide production is a potent cytotoxin largely implicated in the inhibition of a variety of intracellular pathogens including Leishmania. This is another mechanism involved in Leishmania death. 32,35 So, we suggested that propolis EO may activate macrophagic cells and makes them more sensitive to stimuli such as interferon-γ implicated in the immune response. 33
Conclusion
It is very important to characterize EOs of natural products which are used in folk medicine in the prevention of diseases. This study showed that Tunisian propolis EO exhibits high antileishmanial potential with a potential synergism with conventional treatment “amphotericin B” with an activation of macrophage cells. Moreover, it was characterized by the presence of monoterpene hydrocarbons (54.85%) and the predominance of α-pinene as the major compound (36.71%). However, further investigations are therefore needed to evaluate its in vivo properties and clinical responses.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
