Antileishmanial activity of Moringa oleifera leaf extracts and potential synergy with amphotericin B

SAJB-02256; No of Pages 7 South African Journal of Botany xxx (2019) xxx

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Antileishmanial activity of Moringa oleifera leaf extracts and potential synergy with amphotericin B K.M. Hammi a,c, R. Essid b, O. Tabbene b, S. Elkahoui b, H. Majdoub c,⁎, R. Ksouri a a b c

Laboratoire des Plantes Aromatiques et Médicinales (LPAM), Centre de Biotechnologie de Borj- Cédria, BP 901, 2050 Hammam-lif, Tunisia Laboratoire des Substances Bioactives, Centre de Biotechnologie de Borj- Cédria, BP 901, 2050 Hammam-lif, Tunisia Université de Monastir, Laboratoire des Interfaces et des Matériaux Avancés (LIMA), Faculté des Sciences de Monastir, Bd. de l'environnement, 5019 Monastir, Tunisia

a r t i c l e

i n f o

Article history: Received 8 August 2018 Received in revised form 25 November 2018 Accepted 9 January 2019 Available online xxxx Edited by B Ncube Keywords: Moringa oleifera leaves Leishmania major promastigotes and amastigotes Ultrasound system Cytotoxicity Phenolic compounds Response surface methodology

a b s t r a c t Leishmaniasis is a parasitic disease affecting millions of people in Africa, Asia, and South America. It is considered as a major public health problem causing morbidity and mortality worldwide. Actually, the luck of vaccines and effective and less expensive antileishmanial therapy, have oriented pharmacological researches on natural antileishmanial drugs from medicinal plants. Hence, the purpose of this work is to optimize extraction parameters such as the percentage of aqueous ethanol mixture, the temperature and the time through the ultrasound bath equipment using a Box–Behnken Design (BBD) for maximal antileishmanial activity extraction from Moringa oleifera leaves. The cytotoxicity of the optimum extract was evaluated on RAW 264.7 macrophage cell line. The synergism between M. oleifera optimal extract and amphotericin B was also investigated using checkboard method. The optimum extract exhibiting the highest percent inhibition of Leishmania major promastigotes (99.15 ± 0.34%) was obtained using 16%v/v ethanol, at 60 °C for 20 min. Interestingly, this extract showed the best IC50 value of 6.87 ± 0.32 and 9.31 ± 0.72 μg/mL against promastigote and amastigote forms, respectively with no cytotoxicity on macrophage cells Raw 264.7 (SI = 17.53). Moreover, this extract showed a synergistic effect with amphotericin B (FIC = 0.375). HPLC analysis showed that at least six phenolic compounds were identified. Resorcinol, luteolin7-O-glucoside and syringic acid were the most active ones against L. major with IC50 values 3.788 ± 0.48, 10.70 ± 0.59 and 13.41 ± 0.59 μg/mL, respectively. Rutin and ferulic acid were further identified and showed moderate antileishmanial activity with IC50 values 78.51 ± 1.09 and 89.34 ± 1.22 μg/mL, respectively. Kaempferol 3-O-rutinoside was less active with IC50 value 204.4 ± 1.63 μg/mL. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Leishmaniasis is a parasitic disease caused by an intracellular protozoa parasites belonging to Leishmania genus (Desjeux, 2004). It is characterized by three clinical forms (cutaneous, visceral and mucocutaneous) depending on the parasite species. Pollution, immune status, deforestation and people migration to endemic areas are probably the cause of the disease spread (Patz et al., 2000; Ashford, 2000). Leishmaniasis currently affects millions of people in African, Asian and South American countries. It was estimated that about 350 million people are threatened by this disease in 88 countries around the world with 2 million new cases annually (Musuyu Muganza et al., 2012). It causes significant morbidity and mortality (About 20,000–30,000 deaths occur annually) (Amato et al., 2008). Unfortunately, there are no appropriate vaccines against leishmaniasis and the application of chemotherapy drugs including pentavalent antimonials, amphotericin B, paromomycin, miltefosin and liposomal amphotericin B is restricted ⁎ Corresponding author. E-mail address: [email protected] (H. Majdoub).

due to low efficacy, life-threatening side effects, high toxicity, induction of parasite resistance, length of treatment and high cost. Hence, in the lack of effective drugs, there is an urgent need to discover new natural drugs such as plant derivatives (flavonoids) or plant crude extracts that are non-toxic, safe, more efficient and less expensive from medicinal plants to fight this disease (Et-Touys et al., 2017; Singh et al., 2012). Recently, it was shown that the genus Moringa (Moringaceae) is indigenous to India and it has been introduced into other Asian and African countries (Osman and Abohassan, 2012). There are from 10 to 12 Moringa species. Moringa oleifera is the most widely cultivated in Asian and African countries (Vongsak et al., 2013). In Thailand, this plant is named “wonder tree” due to its potential therapeutic values against some current diseases (Chumark et al., 2008). In fact, radioprotective, antitumor and antimicrobial properties of M. oleifera leaves have been investigated (Murakami et al., 1998; Rao et al., 2001; Al_husnan and Alkahtani, 2016). Besides, it was demonstrated that the antioxidant capacity of M. oleifera leaves was related to its richness in phenolic compounds (Chumark et al., 2008; Verma et al., 2009). Surprisingly, the antiprotozoal activity of Moringa oleifera has been less studied. In fact, it has been reported that all different parts of

https://doi.org/10.1016/j.sajb.2019.01.008 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

Please cite this article as: K.M. Hammi, R. Essid, O. Tabbene, et al., Antileishmanial activity of Moringa oleifera leaf extracts and potential synergy with amphotericin B, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.008

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M. oleifera (leaf, root, bark, stem and flower) from India exhibited significant antileishmanial ability against Leishmania (L.) donovani parasite (caused visceral leishmaniasis) (Singh et al., 2015). However, no available data were reported on antiprotozoal activity of M. oleifera leaves of North Africa against L. major, the major causative agent of cutaneous leishmaniasis. Several classical methods have been adopted for the extraction of antileishmanial compounds like polyphenols from natural sources using different organic solvents pure or in mixtures (Bero et al., 2011; Al-Sokari et al., 2015; Kefi et al., 2018;). Whereas, little is known about the ultrasound extraction of antileishmanial compounds from plants with biodegradable and environmentally friendly solvents such as aqueous ethanol mixture (Nitin and Karnik, 2011). Ultrasound-assisted extraction has proven to be simple, rapid, efficient and inexpensive alternative for extracting bioactive compounds from plants due to the acoustic cavitations phenomenon generated with pressure waves (Wang et al., 2008). Hence, the present study investigates the antileishmanial properties of the edible leaf part of M. oleifera collected from Southern Tunisia (particularly in the region of Gabes) against L. major using eco-friendly solvent (aqueous-ethanol) with ultrasound-assisted extraction. The effect of extraction parameters including the percentage of solvent (water/ethanol mixture), the temperature and the time on the antiprotozoal activity of M. oleifera extracts against promastigote form of L. major was evaluated using a response surface methodology and a Box–Behnken Design (BBD). The cytotoxicity of the optimum extract was evaluated on RAW 264.7 macrophage cell line. Combination studies with amphotericin B was also investigated using checkboard method. The total phenolic and flavonoid content, as well as the characterization of active phenolic compounds in the optimized extract were also determined and discussed.

2. Materials and methods 2.1. Plant material Moringa oleifera leaves were collected from Gabes, South of Tunisia, in October 2016. Leaves were dried in lyophilizer, grounded into powder and sieved to obtain a powder with particle diameter size ranging from 400 to 500 μm. The powdered were kept in sealed containers and protected from light until the analysis.

2.4. In vitro antipromastigote activity Promastigotes form of L. major (Glc95) were grown at 26 °C in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (FBS), 100 μg/mL of streptomycin and 100 U/mL of penicillin. When the density of 106 cells/mL was reached, promastigotes were washed twice with phosphate buffered saline (PBS) and centrifuged at 2500 g for 15 min. Afterwards, parasites were plated in a 96-well culture plate at densities of 105 parasites/well for 72 h at 26 °C with different concentrations of extracts (from 15.125 μg/mL to 1 mg/mL). Moreover, all identified phenolic compounds in the optimized extract were tested with concentrations varied from 15.125 to 200 μg/mL. Amphotericin B (concentrations from 0.09 to 12.5 μg/mL) and untreated promastigotes were used as positive and negative control, respectively (Essid et al., 2015). Parasite viability was measured by adding 10 μL of MTT the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at a concentration of 10 mg/mL. After incubation for 4 h at 37 °C, the absorbance was determined at 570 nm using an ELISA plate reader (Synergy HT, Bio-TEK). The inhibition percentage of cells was calculated using the following equation (Eq. (1)): Ið%Þ ¼


 Absorbance untreated cells−Absorbance treated cells  100 ð1Þ Absorbance untreated cells

2.5. In vitro antiamastigote assay A parasite-macrophage ratio of 10:1 was incubated in 5% CO2 for 2 h at 37 °C for logarithmic growing phase promastigotes which infected by macrophage cells. Monolayer cells were washed with PBS then incubated for 4 h in order to remove free promastigotes. Different concentrations of active extract were added to the obtained cultures for 48 h (0.78–200 μg/mL). Furthermore, intracellular amastigotes were counted in at least 100 macrophages for each sample under light microscope (Muylder et al., 2011). The inhibition percentage of the infection rate (IR) was determined using the following equation (Eq. (2)): IRð%Þ ¼


 Absorbance untreated culture−Absorbance treated culture Absorbance untreated culture  100 ð2Þ

2.6. Nitric oxide production assay 2.2. Chemicals and reagents All reagents used for total polyphenolic contents and phenolic standards were purchased from Sigma–Aldrich (GmbH, Steinheim, Germany). Alcohol (HPLC grade) was purchased from Merck (Darmstadt, Germany). Ultrapure water was obtained from the Millipore system (Billerica, USA). For antileishmanial activity, Amphotericin B was provided from Sigma (98% purity, from Sigma–Aldrich, USA).

2.3. Ultrasound-assisted extraction The ultrasound bath equipment (Sonorex Digital 10P, Bandelin) at 35 kHz was used for the extraction of antileishmanial compounds from M. oleifera leaves. 0.5 g of powder was extracted with 25 mL of ethanol concentrations of 5 (low level) to 95%v/v (high level)) at extraction time varying from 20 (low level) to 40 min (high level) and at temperature ranging from 30 (low level) to 60 °C (high level) (Zhao et al., 2014; Mkadmini Hammi et al., 2015). Ethanol was used as extraction solvent due to its low toxicity on human cells (Moser, 1985). The crude extracts were centrifuged at 2000×g for 20 min at 4 °C and the obtained supernatants were collected for estimation of antiprotozoal capacity.

In the same time of antiamastigote assay, the production of nitrite, a stable bio-product of nitric oxide (NO), in macrophage cells Raw264.7 was measured by the Greiss reaction (Sigma–Aldrich, USA) as described previously (Kiderlen et al., 2001). Briefly, supernatant of treated and untreated cells by M. oleifera, infected and non infected by L. major parasite were mixed with the Greiss reagent and incubated for 10 min. Untreated macrophages were used as a negative control. Sodium nitrite in RPMI was used as positive control to construct a standard curve (Kolodziej et al., 2001). The reading was performed in triplicate at a wavelength 570 nm (Synergy HT, Bio-TEK) and results were expressed as the NO concentration (μM) (Ding et al., 1988; Espuelas et al., 2002). 2.7. Cytotoxicity assay Cytotoxicity assay was conducted on macrophage cell line Raw 264.7. Cells were grown in RPMI-1640 medium supplemented with 10% FBS and 50 U/mL of antimycotic and antibiotic solution. Cell viability was identified under light microscope by counting stained macrophage cells (stained by 0.1% trypan blue). Macrophages were incubated overnight at 37 °C under 5% CO2 on a 96 multi-well plate at density of 105 cells/well. After that, the medium was replaced by a fresh one containing different concentrations of the optimized extract

Please cite this article as: K.M. Hammi, R. Essid, O. Tabbene, et al., Antileishmanial activity of Moringa oleifera leaf extracts and potential synergy with amphotericin B, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.008

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(from 15.125 to 500 μg/mL) then incubated for 72 h at 37 °C. The same range of concentrations was also tested for all identified phenolic compounds in this extract. Amphotericin B was used as positive control (concentrations from 0.09 to 12.5 μg/mL). Cell viability was estimated by the MTT test. The selectivity index (SI) of extracts was calculated as the ratio IC50 macrophage/IC50 parasite (Essid et al., 2015; Weniger et al., 2001). An SI value higher than 10 was considered as safe for macrophage and effective for parasites (Monzote et al., 2014). 2.8. Total phenolic and flavonoid contents Total phenolic content (TPC) was estimated using Folin–Ciocalteu reagent and gallic acid as standard (Dewanto et al., 2002). The obtained results were expressed in mg gallic acid equivalent per gram dry leaf (mgGAE/gDL). The total flavonoid content (TFC) was determined by the aluminum chloride (AlCl3) reagent using quercetin as standard (Zhishen et al., 1999). The results were expressed in mg quercetin equivalent per gram of dry leaf (mg QE/g DL). 2.9. Quantitative analysis of major active compounds by HPLC The characterization of phenolic compounds in the optimized M. oleifera leaf extract was performed using a high performance liquid chromatography system (HPLC) (Agilent technologies 1260, Germany, Japan) equipped with reverse-phase column, (100 mm × 4.6 mm id, and 3.5 μm particle size), (Zorbax Eclipse XDB C18) and UV diodearray-detector (200-400 nm). Briefly, 3 μL of sample was injected to HPLC system under flow rate of 0.4 mL/min. The mobile phase is composed of two solvents: methanol and Milli-Q water (0.1% formic acid). All chromatographic conditions were performed according to the method described previously by Mkadmini Hammi et al. (2017). For the quantitative analysis, a calibration curve was performed for each identified phenolic compound using the available standards at 280 nm. Results were expressed as milligram per gram of residue for each standard. 2.10. Synergistic activity with amphotericin B The synergism between M. oleifera optimal extract and amphotericin B was assessed as described by Johnson et al. (2004). Briefly, different concentrations of the plant extract in a serial 2-fold dilution (from 0.2 to 27.48 μg/mL) were added to different concentration of amphotericin B (from 1.94 to 0.015 μg/mL) in a checkboard assay. The combinations were incubated with two 105 parasite/ml in 96-well plates. The fractional inhibitory concentration index (FIC) was calculated for a combination of the two compounds according to the following equation: FIC index ¼ ½A=IC50 A þ ½B=IC50 B

The BBD experiments were fitted to a second-order polynomial model as given by the following equation (Eq. (4)):

Yk ¼ β0 þ

2.11. Experimental design of RSM A 3-level, 3-factors Box–Behnken Design (BBD) was used to evaluate the effect of extraction time (X1) from 20 to 40 min, extraction temperature (X2) from 30 to 60 °C and ethanol concentration (X3) from 5 to 95%v/v on the percent inhibition of L. major promastigotes of extracts prepared at the same concentration of 100 μg/mL. The range of each factor was preliminary determined based on the single-factor experiment. The BBD containing 17 experiments were applied for optimizing the extraction process.

3 X

βi Xi þ

3 X

i¼1

3 X

βii X2i þ

i¼1

βij Xi X j

ð4Þ

i≠ j¼1

Where Yk is the response variable, Xi and Xj are the levels of the independent variables and β0, βi, βii, βij, are the intercept, linear, quadratic and interactive coefficients of the model, respectively. Regression analysis and three and two dimensional response surface plots were plotted to determine the best extraction conditions using ultrasound system for obtaining an extract with potential antileishmanial activity against promastigotes form of L. major. 2.12. Data analysis Antiparasitic activity was expressed as IC50 values, the concentration of the compound that inhibits the growth by 50% of promastigotes or amastigotes forms. All assays were conducted in triplicate. For the statistical analysis of the experimental data, the Student's t-test was used to evaluate the significance of the regression coefficient and the Fischer's F-test was calculated to validate the model equation at the 5% probability level (p b 5%). Model adequacy was evaluated using the lack of fit, the coefficient of determination (R2) and the F-test value obtained from the analysis of variance (ANOVA). The data treatment of the BBD was performed using the experimental design software NEMRODW (Mathieu et al., 2000). 3. Results and discussion 3.1. Fitting the models RSM approach was employed for the determination of the extraction conditions effects including time (min) (X1), temperature (°C) (X2) and ethanol concentration (v/v,%) (X3) which were investigated on the ultrasonic-assisted extraction of bioactive compounds from M. oleifera leaves cultivated in Tunisia. As reported in Table 1, a total of 17 experiments were obtained through the combination of three coded levels (low (−1), middle (0) and high (+1)) for three independent variables Table 1 Coded levels, conditions run and measured responses according to a Box Behnken Design (BBD). Experimentsa Independent variables Time (min), X1

ð3Þ

Where IC50A and IC50B are the IC50 values of each compound tested alone and [A] and [B] are the IC50 values of the compounds A or B when treatment was carried out in combination. A FIC index less than or equal to 0.5 indicates the presence of a synergy. However, a FIC index between 0.5 and 4 indicates indifference. An index greater than 4 indicates antagonism (Odds, 2003; Fratini et al., 2017).

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

20(−1) 40(1) 20(−1) 40(1) 20(−1) 40(1) 20(−1) 40(1) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0)

Temperature (°C), X2

30(−1) 30(−1) 60(1) 60(1) 45(0) 45(0) 45(0) 45(0) 30(−1) 60(1) 30(−1) 60(1) 45(0) 45(0) 45(0) 45(0) 45(0)

Response Ethanol concentration (v/v,%), X3

Observed values

Predicted values

YbPI (%)

YbPI (%)

50(0) 50(0) 50(0) 50(0) 5(−1) 5(−1) 95(1) 95(1) 5(−1) 5(−1) 95(1) 95(1) 50(0) 50(0) 50(0) 50(0) 50(0)

97.02 94.63 88.35 76.54 71.94 64.76 47.00 49.00 98.90 98.67 72.80 42.56 68.62 65.72 62.53 58.92 71.11

96.35 96.22 86.76 77.20 78.25 68.82 42.94 42.68 93.24 93.94 77.52 48.21 65.38 65.38 65.38 65.38 65.38

a

Experiments (Standard order). Percent inhibition of L. major promastigotes at a concentration of 100 μg/mL of extracts. b

Please cite this article as: K.M. Hammi, R. Essid, O. Tabbene, et al., Antileishmanial activity of Moringa oleifera leaf extracts and potential synergy with amphotericin B, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.008

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(X1, X2, X3) according to the Box–Behnken Design (BBD). The percentage of L. major inhibition of extracts at a concentration of 100 μg/mL was measured for each experiment. The experimental data of the BBD design were fitted to second order polynomial model. Table S1 illustrates the regression coefficients of the model which is considered significant (p values b.05). In fact, the statistical analysis showed that the temperature and the ethanol concentration had an important linear effect on the antipromastigote activity response. Moreover, results showed that the temperature of extraction presented a positive quadratic effect on the response whereas the ethanol concentration exhibited a negative one. It's clearly shown that there is a significant interaction effect between the temperature and the ethanol concentration parameters on the antipromastigote capacity. The analysis of variance (ANOVA) was performed on the experimental data to evaluate the full quadratic response surface model. As shown in Table S1, a satisfactory determination coefficient (R2) of 0.92 was obtained for antipromastigote activity which represented an excellent correlation between independent factors and response. 3.2. Response surface analysis of the antipromastigote activity of M. oleifera extracts The NEMRODW software generated a second-order polynomial equation (Eq. 5) for the antileishmanial activity response that revealed the functional relationship between factors regardless of their significance and the predicted response YPI: Y%PI ¼ 65:38−2:422X1 −7:154X2 −15:364X3 þ 1:849X21 þ 21:906X22 −9:054X23 −2:355X1 X2 þ 2:295X1 X3 −7:502X2 X3

ð5Þ

Concerning results of Fisher's F-test, the obtained F value of regression coefficient for the inhibition of L. major promastigotes at 100 μg/mL of extract is higher than the tabulated value (Fregression = 12.1144 N Ftabulated (9,7,0.05) = 3.68) (p b .0001), indicating that the independent factors of the regression equation had a considerable effect on the corresponding response at confidence level of 95%. Regarding the validity of model, if the ratio of the mean square of lack-of-fit and pure error is smaller than the tabulated one, the lack of fit statistic would be insignificant (p N .05). The obtained results showed that the ratio of the mean square of lack-of-fit and pure error is smaller than the tabulated value (Flack-of-fit = 3.2479 b Ftabulated (3,4,0.05) = 6.59). The

obtained p value is 0.1424 which is superior to 5%. Hence, the model is valid (Montogomery, 2005). After validating the model and as presented in Fig. 1, a contour plot (Fig. 1a) and three-dimensional response surfaces (Fig. 1b) were plotted for the percent inhibition of the L. major promastigotes against the two significant combined factors including alcohol concentration and temperature, whereas the third factor was set constant at a low level (−1) corresponding to 20 min. As clearly shown from Fig. 1, the greater inhibition of L. major promastigotes was observed with increasing temperature of extraction and decreasing ethanol concentration and the corresponding area (red color) is so large and presents a value which can be close to 100% when the extraction time was set at 20 min. Based on response surfaces methodology, the optimum levels of independent variables with desirable response goals were successfully determined using NEMRODW software. Accordingly to response surfaces methodology (Fig. 1), the optimum point was marked by point and corresponding to the following extraction conditions: the temperature of 60 °C, hydroalcoholic percentage of 16%v/v and an extraction time of 20 min. Under these conditions, the obtained experimental value of the percent inhibition of L. major promastigotes was 99.15 ± 0.34% which is in agreement with the predicted one (99.63%). 3.3. Antileishmanial and cytotoxic activities The obtained values of IC50 for promastigotes and amastigotes forms were 6.87 ± 0.32 and 9.31 ± 0.72 μg/mL, respectively. Similar findings were reported by Singh et al. (2015) showing that the flower extracts of M. oleifera from India has a potent antileishmanial activity against L. donovani and imparted a significant reduction on infected macrophage cells (86%). Moreover, this finding corroborate with previous reports (Mbongo et al., 1998) showing the insignificant difference between activity against promastigotes and amastigotes forms of Leishmania. Compared with previous studies which have been reported on the antileishmanial activity against L. major species of different plants, our results are more significant than the ethyl acetate fraction from aerial part of Asteriscus graveolens which exhibited a potential antileishmanial activity against both promastigote and amastigote forms of Leishmania with IC50 of 33.64 ± 0.46 and 35.23 ± 0.62 μg/mL, respectively (Ramdane et al., 2017). Recently, it was demonstrated that ethyl acetate extract from Echium arenarium (Guss) exhibited a potential antileishmanial activity against L. major (IC50 = 13.91 ± 0.43 μg/mL) (Kefi et al., 2018) which is approximately two

Fig. 1. Response surface plots of percent inhibition of L. major promastigotes (YPI, %) as a function of significant interaction between extraction temperature and ethanol concentration: (a) contour plots (2D); (b) three-dimensional response surfaces (3D).

Please cite this article as: K.M. Hammi, R. Essid, O. Tabbene, et al., Antileishmanial activity of Moringa oleifera leaf extracts and potential synergy with amphotericin B, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.008

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times lower than the optimized extract of M. oleifera leaves. This difference in anti-parasitic potentiality could be explained by the variability of plants, the solvent and the method used for the extraction of bioactive compounds which are responsible for this biological activity (Ramdane et al., 2017). The optimized M. oleifera extract, showed no cytotoxicity on macrophage cells Raw 264.7 (IC50 = 120.45 ± 0.65 μg/ml) suggesting its efficiency on parasite and safety for macrophage cells (SI = 17.53) (Table 2). The investigation of nitric oxide production (NO) showed that there is no significant difference between non-infected macrophage cells treated (0.95 μM) and untreated by M. oleifera extracts (1.1 μM) suggesting its selective potential on cell death of Leishmania parasite which not related to macrophage activation. Accordingly, it was reported the high anti-inflammatory effect of M. oleifera on macrophage cells (Jaja-Chimedza et al., 2017). 3.4. Polyphenolic compounds determinations of optimized extract The total polyphenolic compounds (TPC) of the optimized extract was found to be 87.55 ± 0.25 mgGAE/gDL which is approximately 1.86 more important than those obtained in 50% aqueous ethanol extract of M. oleifera Lam leaves from Madagascar (47 ± 4 mg gallic acid equivalents (GAE)/g dry leaf) as reported by Rodríguez-Pérez et al. (2015). In addition, the total flavonoid content (TFC) was found to be 65.3 ± 0.8 mgQE/gDL which is approximately 1.4 more important than the aqueous extract from M. oleifera of South Africa as demonstrated by Moyo et al. (2012). These variability of TPC and TFC values of M. oleifera may be attributed to the geographical location, abiotic factor of this plant, the nature of solvent and the method adopted for extraction of the bioactive compounds (Ksouri et al., 2008). Phenolic compounds are known by their potential biological properties such as antimicrobial, antiviral, anti-inflammatory, anti-allergic, anti-thrombic, cardioprotective and vasodilatory effects (Garcia-Lafuente et al., 2009). However in recent years, many research papers have identified these compounds as antiparasitic agents (Ramdane et al., 2017; Kefi et al., 2018).

5

A total of six phenolic compounds were successfully identified in the optimized extract including two phenolic acids (ferulic acid and syringic acid), diphenol (resorcinol) and three flavonols glycosides (kaempferol 3-O-rutinoside, rutin and luteolin 7-O-glucoside) by comparing their retention time to those of phenolic standards analyzed under the same chromatographic conditions. Our results are in agreement with previous studies showing that M. oleifera leaves are rich in phenolic acids and glycone flavonoids largely known by their biological properties (Nouman et al., 2016; Singh et al., 2009). The diphenol compound (resorcinol) was also identified in M. oleifera cultivated in Iraq (Kadhim and AL-Shammaa, 2014). The quantitative results (Table S2) of the optimized extract showed that rutin was the major compound (17.69 ± 0.27 mg/g residue). Resorcinol was the second most abundant (8.07 ± 0.15 mg/g residue) followed by luteolin 7-O-glucoside (5.89 ± 0.13 mg/g residue) and kaempferol 3-O-rutinoside (2.19 ± 0.11 mg/g residue). Phenolic acids including ferulic acid (0.46 ± 0.17 mg/g residue) and syringic acid (0.34 ± 0.07 mg/g residue) are the minor compounds in the optimized extract. As reported in Table 2, all these identified metabolites were tested against promastigote form of L. major and the obtained results showed that these standards exhibited a considerable IC50 values ranging from 3.788 ± 0.48 to 204.4 ± 1.63 μg/mL. This is in agreement with previous studies which revealed the potential antiprotozoal activity of phenolics (Tasdemir et al., 2006). A significant antiprotozoal activity was clearly observed for resorcinol (IC50 = 3.788 ± 0.48 μg/mL), luteolin 7-O-glucoside (IC50 = 10.70 ± 0.59 μg/mL) and syringic acid (IC50 = 13.41 ± 0.59 μg/mL). These results suggest that the antiprotozoal activity of the optimized extract of M. oleifera leaves could be ascribed to the synergistic action of the most active metabolites regardless of their amount. In this context, it was demonstrated that the biological properties of extracts from various plants are related to the synergistic effect of the most abundant and the minor compounds of samples (Lahlou, 2013). Moreover, it was suggested that compounds with small amount in the extract could exhibit an important role in in vitro antileishmanial capacity (Peixoto et al., 2011). 3.6. Synergistic potential with conventional drug amphotericin B

3.5. RP-HPLC profiles of optimized extract As determined by the Box Behnken Design (BBD), the ultrasound assisted extraction with hydro-ethanolic extract (16%v/v) exhibited the best antileishmanial activity. This optimal extract was analyzed by HPLC. Results are illustrated in Fig. 2.

Table 2 Antipromastigote activity, cytotoxicity and selectivity index of the optimized extract and phenolic standards. Samples

Antipromastigote activity IC50 ± SD (μg/mL)

Cytotoxicity IC50 ± Selectivity SD (μg/mL) index (SI)

Optimized extract

6.87 ± 0.32

120.45

17.53

141.29 ± 1.14 285.36 ± 1.47 60.27 ± 0.98 153.21 ± 1.28

10.53 3.19 15.94 14.31

223.67 ± 1.36 368.78 ± 1.45

2.86 1.78

10.62

10.94

Phenolic standards Syringic acid 13.41 ± 0.59 Ferulic acid 89.34 ± 1.22 Resorcinol 3.78 ± 0.48 Luteolin 10.70 ± 0.59 7-O-glucoside Rutin 78.51 ± 1.09 Kaempferol 206.40 ± 1.63 3-O-rutinoside Reference drug Amphotericin B

0.97

IC50, inhibitory concentration 50 (μg/mL); IC50, lethal concentration 50 (μg/mL), SI: selectivity index expressed as the ratio IC50 macrophage/IC50 parasite; SD: standard deviation; each value was represented as mean ± SD (n = 3).

Today, combinatory therapy has shown promising incidence in anti-infectious therapies. It aims to decrease side effects, toxicity, treatment dose and duration of conventional drugs. Moreover, it is an effective procedure to prevent drug resistance (Corral et al., 2014). Interestingly, when combined with amphotericin B, M. oleifera extracts showed high synergistic effect with FIC value of 0.375 and lead to 99.5% of parasitic growth inhibition. In addition, the synergistic concentration of M. oleifera was fourfold lower than its MIC when used alone. However, the synergistic MIC of amphotericin B was decreased by eightfold. Therefore, M. oleifera extracts may constitute novel promising molecules for the development of new leishmaniasis combination therapies. It is well known that the mechanism of action of amphotericin B is mainly related to its binding to ergosterol, which impairs cell death by causing membranes permeability (Larabi et al., 2003). Concerning the mechanism of action of the flavonoid-rich M. oleifera extract, its strong antileishmanial capacities might be attributed to phenolic compounds (Ramdane et al., 2017). Particularly, it was demonstrated that flavonoid compounds possess a good antileishmanial activity in vitro and they are able to modify immunological response of parasites (Kiderlen et al., 2001; Kolodziej et al., 2001). In fact, these compounds could affect the growth of Leishmania parasite by inhibiting various types of proteins and parasite enzymes. In addition, they are able to cleave a specific DNA of parasite by blocking DNA topoisomerases (Mittra et al., 2000). They are also able to inhibit arginase enzyme which plays a central role in the biosynthesis of polyamine essential for

Please cite this article as: K.M. Hammi, R. Essid, O. Tabbene, et al., Antileishmanial activity of Moringa oleifera leaf extracts and potential synergy with amphotericin B, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.008

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Fig. 2. Chromatographic profiles acquired at 280 nm of: (a) Moringa oleifera aqueous extract; (b) Standards mixture: The identified compounds are: Peak N°1: resorcinol (Rt = 11.19 min), Peak N°2: syringic acid (Rt = 17.78 min), Peak N°3: ferulic acid (Rt = 20.4 min), Peak N°4: luteolin 7-O-glucoside (Rt = 21.09 min), Peak N°5: rutin (Rt = 21.29 min) and Peak N°6: kaempefrol 3-O-rutinoside (Rt = 22.45 min).

protecting the parasite against reactive oxygen species (ROS) generated by the host's defense system (Oryan, 2015). Thus, the synergistic effect between amphotericin B and M. oleifera extract could be related to their different antileishmanial mechanisms. 4. Conclusion These findings showed that the optimized extract obtained from M. oleifera using ultrasound system at optimal conditions could represent a promising source of bioactive metabolites and might have potential application in pharmaceutical industries as antiprotozoal drugs against cutaneous leishmaniasis. This work provides a significant basis for further investigation into the separation of these effective natural substances and in vivo experiments. Conflict of interest statement There is no conflict of interest.

Acknowledgements This work was supported by a grant from Ministry of Higher Education and Scientific Research, Tunisia. The authors would like to thank Pr. Abdurrahman SANHOURI from university of Nouakchott of Mauritania for English improvement of the manuscript. References Al_husnan, L.A., Alkahtani, M.D., 2016. Impact of Moringa aqueous extract on pathogenic bacteria and fungi in vitro. Annals of Agricultural Sciences 61, 247–250. Al-Sokari, S.S., Ali, N.A.A., Monzote, L., Al-Fatimi, M.A., 2015. Evaluation of antileishmanial activity of albaha medicinal plants against Leishmania amazonensis. BioMed Research International 2015, 938747. Amato, V.S., Tuon, F.F., Bacha, H.A., Neto, V.A., Nicodemo, A.C., 2008. Mucosal leishmaniasis. Current scenario and prospects for treatment. Acta Tropica 105, 1–9. Ashford, R.W., 2000. The leishmaniases as emerging and re-emerging zoonoses. International Journal for Parasitology 30, 1269–1281. Bero, J., Hannaert, V., Chataigné, G., Hérent, M.F., Quetin-Leclercq, J., 2011. In vitro antitrypanosomal and antileishmanial activity of plants used in Benin in traditional medicine and bio-guided fractionation of the most active extract. Journal of Ethnopharmacology 137, 998–1002.

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Please cite this article as: K.M. Hammi, R. Essid, O. Tabbene, et al., Antileishmanial activity of Moringa oleifera leaf extracts and potential synergy with amphotericin B, South African Journal of Botany, https://doi.org/10.1016/j.sajb.2019.01.008