Background

Leishmaniasis is a neglected obligatory intracellular tropical disease, caused by different species of Leishmania parasites, that is prevalent in many parts of the world [1]. Leishmaniasis is transmitted by the bites of infected female sand flies [2]. These parasitic protozoans have two distinct stages in their life cycle: promastigotes (extracellular flagellated promastigotes in the gut of the female sand fly vector which can be injected into the host dermis by vector bite), and amastigotes (transformation of promastigotes into intracellular amastigotes after internalization by host phagocytotic cells through phagocytosis) [3, 4]. At the vector bite site, the parasite in promastigote form attacks host phagocytotic cells (inflammatory monocytes, macrophages and neutrophils) and then transform into intracellular amastigotes [4, 5]. Amastigotes are able to proliferate within monocytes/macrophages and transmit the infection to other macrophages, neutrophils, monocytes, some dendritic cells and fibroblasts [4, 6, 7].

The defense reactions of host cells against Leishmania infection are based on the co-ordination of two host immune systems, innate immunity (complement-mediated lysis) and adaptive immunity (Th1-mediated response) [4, 5]. The expression of activating cytokines such as IFN-γ and TNF-α is essential for parasite proliferation control [8]. When activated by cytokines, host cells can suppress the infection by killing intracellular parasites [9]. The production of reactive oxygen species (ROS) and Nitric Oxide (NO expressed by inducible nitric oxide synthase (iNOS) gene) represent as two major effective leishmanicidal molecules for exclusion of intracellular parasites without damaging the host cell [4, 5].

Treatment of leishmaniasis has always been challenging. The absence of effective immunizations and/or emergence of treatment resistance have all contributed to the rise in prevalence of this disease [10]. Pentavalent antimonials, amphotericin B, paromomycin and pentamidine are the most regularly used drugs for leishmaniasis therapy. However, they have significant side effects, require high dosages for extended periods of time, and are supplied parenterally [11]. An effective and economical new treatment approach would be advantageous in overcoming the difficulties induced by leishmaniasis chemotherapy. In the treatment of parasitic infections, phytotherapy has recently attracted attention as a viable alternative to chemotherapy [12]. In this regard, plant studies have been expanded to discover new secondary metabolites with increased bioactivity and fewer side effects [12, 13].

Calotropis procera (Asclepiadaceae) is a common plant throughout the world (growing mainly in dry and semi-arid climates), renowned for its conventional therapeutic uses including the treatment of infectious diseases, skin and dermal illness (infections, leprosy, wounds, psoriasis), respiratory diseases (bronchial asthma and cough), gastrointestinal diseases (dysentery, constipation and nematode infections), urinary tract diseases (kidney stones and chronic renal problems), jaundice, malaria, fever, earache, neuropsychiatric disorders, liver diseases and even tumors [14,15,16,17,18,19]. Recently, C. procera extracts have been reported to exert anticancer, anti-inflammatory, antidiabetic, gastroprotective, cardiovascular, antipyretic, antioxidant, antimalarial, anthelmintic, antifungal, anti-angiogenic, hypolipidemic, antibacterial, analgesic and anticonvulsant properties [14, 18, 20,21,22,23,24,25]. C. procera leaf extract has also demonstrated effective anti-leishmanial activity against promastigotes of L. tropica, mediated via a mechanism of apoptosis induction [26].

The mechanisms and pathways that stimulate leishmania-infected macrophages are of special interest since they hold potential for the development of new treatment and prevention strategies. In the present research, we sought to evaluate the effect of C. procera extract on PBMCs infected with L. major and the expression levels of INF-γ, TNF-α and iNOS genes. It was hypothesized that treatment of L. major promastigotes and amastigotes with C. procera extract would induce ROS production and upregulation of INF-γ, TNF-α and iNOS genes which could be effective for parasite control.

Methods

Preparation of plant extract

The seeds of C. procera were provided and cultivated in the greenhouse (Fig. 1) by Zarringiah Co., West Azerbaijan Province, Urmia, Iran. The growing seedlings were authenticated by a taxonomist. Voucher specimens (voucher numbers: CP/1397 433) were deposited in the Zarringiah Co. Herbarium, Urmia, Iran. The leaves of 6 weeks seedlings were carefully harvested for extraction (Fig. 1). After washing and shade drying at room temperature, the samples (500 mg) were powdered and extracted by 10 ml 80% methanol (Merck, Darmstadt, Germany) maceration method and shaking incubation at 25 ± 2  C for 72 h. The extract was paper filtered and the residue re-macerated for the second (80% methanol, 48 h) and third (80% methanol, 24 h) time. Finally, the solvent was evaporated in a vacuum rotary evaporator (Rotary Evaporator N-1110, Eyela, Tokyo, Japan). The concentrated residue was frozen at − 20  C. The dried powders were dissolved in phosphate-buffered saline (PBS, Cl2H3K2Na3O8P2, 1X, pH 7.4, Gibco, Paisley, UK) and diluted to prepare test concentrations of extract.

Fig. 1
figure 1

Seeds and seedlings of Calotropis procera

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Cultivation of Leishmania major parasite

The Iranian standard reference strain of L. major promastigotes (MRHO/IR/75/ER) was provided by the Department of Medical Parasitology and Mycology, Urmia University of Medical Sciences, Urmia, Iran. The promastigotes were cultured in RPMI-1640 culture medium (+ HEPES and L-glutamine, Gibco, Paisley, UK) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, PAN Biotech, Aidenbach, Germany) and antibiotics (100 units/ml penicillin, and 100 μg/ml streptomycin, Sigma-Aldrich, St. Louis, Missouri, USA). The cultures were placed in an incubator shaker (120 rpm) at 25 ± 1  C and grown until reaching the stationary growth phase.

MTT viability assay

In order to determine the growth-inhibitory effect of C. procera extract, the promastigotes of L. major (1 × 106 parasites/mL) at stationary phase were added (triplicate) to the 96-well plate and treated with extract concentration range of 0–400 µg/mL. Wells containing culture medium without promastigote as blank sample, glucantime-treated promastigote (Glucantime®: Sanofi, France) as positive control and PBS-treated promastigote as negative control were used. The plates were incubated for 24 and 72 h at 25 ± 1  C. Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, Missouri, USA) was dissolved in PBS (Gibco, Paisley, UK) at 5 mg/mL, added to each well (10% of the volume of well). After incubation at 25 ± 1  C for 4 h and adding dimethyl sulfoxide (DMSO, Sigma-Aldrich, St Louis, Missouri, USA) to stop the reaction, the absorbance was read by plate reader (Stat Fax 2100 ELISA Plate Reader, Awareness Technology, Palm City, Florida, USA) at 545–600 nm:

$$\% {\text{ cell viability}}\, = \,\left( {{\text{absorbance of treated wells}}{-}{\text{blank}}} \right)/{\text{ absorbance of control}}{-}{\text{blank}})\, \times \,{1}00.$$

The logarithmic regression analysis of dose–response curve was used for calculation of 50% inhibitory concentration of extract (IC50) and 50% cytotoxic concentration of extract (CC50) using GraphPad Prism 5.0.4 software (GraphPad Software, San Diego, California, USA).

Reactive oxygen species (ROS) levels

ROS production was detected using the fluorescent 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) dye according to the kit instructions (ROS assay kit: KROS96, Kiazist Life Sciences, Iran), as described by Mendoca et al. [27]. After treatment with or without extract at 100 µg/mL (< IC50), 222.44 µg/mL (IC50-72 h) and 377.28 µg/mL (IC50-24 h) concentrations for 3, 6, and 12 h, L. major promastigotes were washed with ROS buffer and incubated with DCFDA reagent for 45 min in darkness. The fluorescence intensity was immediately measured as EX / EM = 485/535 nm and analyzed by flow cytometry (PAS Particle Analysing System, Partec, Germany). Glucantime (10 μg/mL) and PBS (Gibco, Paisley, UK) were used as positive and negative controls, respectively.

Cultivation of PBMCs and infection with L. major promastigotes

Peripheral blood mononuclear cells were isolated from healthy heparinized blood as described by Srivastava et al. [28] and cultured in 6-well plates (105 cells/well) containing RPMI 1640 medium (+ HEPES and L-glutamine, Gibco, Paisley, UK, 10% FBS (PAN Biotech, Aidenbach, Germany), 100 U/ml penicillin–100 µg/mL streptomycin (1% P/S, Sigma-Aldrich, Missouri, USA) as antibiotics) at 37  C – 5% CO2. The adherent cells were infected with L. major promastigotes at stationary growth phase (10:1 parasites/cell) for 4 h and washed three times with PBS (Gibco, Paisley, UK) to remove free parasites. After stabilization of infected cells (amastigote-containing cells) the treatments were performed for 24, 48 and 72 h including negative control, 377.28 μg/mL C. procera leaf extract (24 h-IC50 concentration) and 222.44 μg/mL C. procera leaf extract (72 h-IC50 concentration).

To determine the cytotoxicity effect of C. procera leaf extract on PBMCs, the concentration of extract required to reduce uninfected PBMC growth by 50% after 24–72 h was calculated using the MTT assay.

IFN-γ, TNF-α and iNOS mRNA determination by real-time PCR

Total RNA from infected PBMCs (treated or untreated with extract) was extracted using SinaClon RNXplus kit (SinaClon, Tehran, Iran). Synthesis of cDNA was performed with 1 μg of total RNA using the AccuPower® CycleScript RT PreMix Kit (Bioneer, Daejeon, South Korea) according to the manufacturer’s instructions. The specific primers targeting the genes were designed as listed in Table 1, and manufactured (Nedaye Fan Co, Tehran, Iran). The Real-time RT-PCR assays were performed by SYBR Green detection (SYBR Green qPCR Master Mix, Thermo Scientific/Fermentas, Vilnius, Lithuania) and the relative quantification (2−ΔΔCT method) was applied, using the homo-sapiens β-actin gene as the reference control. Real-time RT-PCR reactions were conducted using three-step real-time MicPCR (Bio Molecular system, Upper Coomera, Queensland, Australia) in 20 μL total volume containing 10 μL SYBR Green Master Mix, 2 μL of 1:20 diluted cDNA (50 ng), 0.5 µL of each primer (10 µM), and 7 µL nuclease-free water. The real-time PCR temperature program consisted of a hold at 95˚C for 10 min followed by 40 thermal cycles of 95˚C for 15 s, primer annealing temperature (Table 1) for 20 s, and 72  C for 30 s.

Table 1 The specific primers for β-actin, cytokine (IFN-γ and TNF-α) and iNOS genes
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Statistical analysis

Values were expressed as the mean of triplicate samples ± standard deviation (SD). The results were analyzed statistically by one-way ANOVA test followed by Duncan’s multiple range tests (p < 0.05).

Results

In vitro leishmanicidal activity of C. procera against L. major promastigotes

The effect of C. procera leaf extract on L. major promastigotes was monitored after 24 and 72 h of treatment. The extract showed a dose-dependent reduction in promastigote proliferation (Fig. 2), with 50% growth inhibition of the promastigotes at 377.28 μg/mL extract after 24 h and 222.44 μg/mL extract after 72 h of treatment. In order to ensure the selectivity of C. procera leaf extract to act only against intracellular amastigotes, the cytotoxicity against PBMCs was investigated. No cytotoxicity was observed at the concentrations analyzed (selectivity index (SI) higher than 4, Table 2).

Fig. 2
figure 2

MTT assay of L. major promastigotes viability after C. procera leaf extract treatments. Data are expressed as mean of % cell viability ± standard deviation. Significant statistical differences in relation to control are indicated as (*), (**), and (***) at the 0.05, 0.01 and 0.001 levels, respectively

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Table 2 In vitro activity of C. procera against L. major promastigotes and its cytotoxicity for PBMCs
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C. procera extract increased ROS production

Overproduction of ROS in mitochondria is one of the important defense weapons of the cell against pathological and physiological threats that lead to oxidative stress. We determined the ROS production in L. major promastigotes using fluorescent H2DCFDA detection by flow cytometry. Promastigotes treated with 222.44 and 377.28 µg/mL C. procera extract significantly enhanced ROS production by 1.65 and 4 times (p < 0.001), respectively, compared to controls (Fig. 3).

Fig. 3
figure 3

ROS production in C. procera leaf extract- treated L. major. Promastigotes were treated with 100 ( < IC50), 222.44 (IC50-72 h) and 377.28 (IC50-24 h) µg/mL for 24, 48 and 72 h at 25 ± 1  C. Cells (control and treated cultures) were incubated with probe H2DCFDA (green fluorescent dye). Intracellular ROS production was analyzed by flow cytometry. Data are expressed as mean of DCF fluorescence intensity ± standard deviation. Significant statistical differences in relation to control are indicated as (*), (**), and (***) at the 0.05, 0.01 and 0.001 levels, respectively

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C. procera extract increased expression of IFN-γ and TNF-α transcripts

As shown in Fig. 4, in L. major-infected PBMCs, IFN-γ and TNF-α mRNA expression increased significantly during treatment with C. procera, depending on exposure time (p < 0.01). The highest expression of both IFN-γ and TNF-α genes was detected at 48 h treatment with 377.28 µg/mL C. procera extract, compared to control expression levels (p < 0.001). In the presence of 222.44 µg/mL C. procera extract, cytokine expression in L. major-infected PBMCs significantly increased with increasing exposure time (p < 0.001), such that 72 h treatment induced higher levels of TNF-α and IFN-γ in comparison with control, respectively (Fig. 4).

Fig. 4
figure 4

Expression level of IFN-γ (a), TNF-α (b) in L. major-infected PBMCs treated with C. procera. The highest levels of IFN-γ and TNF-α expression were detected at 48-h treatment with 377.28 µg/mL C. procera extract. Significant statistical differences in relation to control are indicated as (*), (**), and (***) at the 0.05, 0.01 and 0.001 levels, respectively

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C. procera increased iNOS mRNA expression

As hypothesized, the level of iNOS mRNA expression was significantly increased (4–16-fold, p < 0.001) by C. procera extract in L. major-infected PBMCs compared to control (Fig. 5). In L. major-infected PBMCs, induction of iNOS expression was significantly increased 4.59-fold after 72-h treatment with 222.44 μg/mL and 3.97- to 16.32-fold following 24- to 48-h treatment with 377.28 µg/mL (p < 0.001, Fig. 5).

Fig. 5
figure 5

Expression level of iNOS transcripts in L. major-infected PBMCs treated with C. procera leaf extract. The highest level of iNOS expression was detected at 48-h treatment with 377.28 µg/mL C. procera extract. Significant statistical differences in relation to control are indicated as (*), (**), and (***) at the 0.05, 0.01 and 0.001 levels, respectively

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Discussion

The current study represents the first report of C. procera and its impact on the expression of relevant genes in L. major-infected PBMCs. Our findings demonstrated that C. procera could induce ROS generation in L. major promastigotes; increase expression of IFN-γ and TNF-α cytokine genes, together with nitric oxide synthase expression, in L. major-infected PBMCs. Considered together, this indicated an inhibitory effect on the proliferation of L. major promastigotes.

C. procera is known to possess antioxidant, antipyretic, antifungal, antimicrobial, analgesic, anti-inflammatory and antinociceptive properties which have been attributed to its phytochemical composition [18, 22,23,24]. In the current study, the anti-leishmanial effects of C. procera on L. major promastigotes were evaluated by MTT assay. Our findings showed that C. procera had a dose-dependent cytotoxic effect against L. major promastigotes, as also reported against L. tropica species [26]. The IC50 value obtained from leaf extract of C. procera against L. major promastigotes in the present analysis was 377.28 μg/mL at 24-h treatment and 222.46 μg/m at 72-h treatment which was higher than the value reported (66.8 μg/mL -72 h) in Al Nasr' study [29]. It has been clear that the biosynthesis and accumulation of secondary metabolites in plants are influenced by genetic and environmental factors [30]. Therefore, it is not far from expected that the IC50s of the same plant but grown in different environmental conditions are different. C. procera comprises secondary metabolites such as phenolic compounds, flavonoids, cardiac glycosides, terpenoids, saponins and sterols [17]. The anti-leishmanial effects of these phytochemicals against Leishmania spp. have been demonstrated previously [31]. Therefore, these phytochemicals may have been responsible for the anti-leishmanial activity of C. procera in the current study. Phenolic acids such as gallic acid and ellagic acid [32], and flavonoids, for example, rutin [33] have shown growth-inhibitory effect against promastigotes and amastigotes of L. major and L. donovani, respectively.

Based our results, in a dose–time-dependent manner, L. major promastigote proliferation was inhibited after treatment with C. procera extract as a consequence of increased ROS production. It can be stated that after mitochondrial dysfunction and as a result leakage in the electron transport chain, the level of ROS in Leishmania promastigotes will exceed its basal level [34]. Phytochemicals possessing anti-leishmanial activity may therefore have been able to increase ROS levels, resulting in oxidative stress produced by ROS, thereby causing cell death [31]. Similarly, other studies have pointed out the important role of herbal products in inducing excessive production of ROS and subsequent cell death of Leishmania spp. For example, Dehydroabietic acid isolated from Pinus elliottii in the Gonçalves, et al. study [35] and total phenolic fraction from extra virgin olive oil in the Karampetsou, et al. study [34], promoted cellular ROS production in L. amazonensis and L. major parasites, respectively.

In the current study, 48 h treatment with 377.28 µg/mL C. procera induced IFN-γ and TNF-α expression and increased iNOS gene expression in L. major-infected macrophages, leading to elimination of the parasites. To success of host's defense mechanisms against Leishmania parasite, IFN-γ and TNF-αas pro-inflammatory cytokines play crucial role [36]. Evidently, FN-γ and TNF-α have synergistic effects in killing of Leishmania major by stimulating macrophages to increase ROS and reactive nitrogen species (RNS) production [4, 5, 36]. As a result of increased iNOS gene expression, host cells produce NO via activity of the iNOS enzyme [37]. NO is known to be a major leishmanicidal agent, such that deficiency or inhibition of NO production leads to parasite resistance or survival, respectively [37]. It has been demonstrated that injection of L-NG-monomethyl arginine (L-NMMA), as a NO inhibitor, into the lesions in L. major-infected CBA mice caused disease exacerbation by 10(4)-fold increasing in the number of parasites [38].

According to these findings, increased iNOS expression could represent an effective mechanism for C. procera to control Leishmania infection; supported by the RT-PCR results in the current study. It is evident that antimonials, amphotericin B and other anti-leishmanial agents have been shown to combat parasites by increasing production of ROS and NO [39, 40]. Similarly, the increased release of IFN-γ and TNF-α by C. procera leaf extract could therefore represent an underlying immune mechanism to stimulate iNOS expression and thus NO production. Considering the safety, accessibility and low cost of bioactive compounds, medicinal extracts such as C. procera can be a valuable natural source of anti-leishmanial agents.

Conclusions

In the current study, C. procera leaves extract exerted significant anti-leishmanial activity against L. major promastigotes and amastigotes. This was likely mediated by effective concentrations of C. procera extract increasing the levels of ROS in promastigotes, and increasing the expression levels of IFN-γ, TNF-α and iNOS genes in PBMCs containing amastigotes. Considering that despite the high efficacy rate, the presence of severe side effects, toxicity, some drug resistance and the high cost of chemical anti-leishmanial drugs encourage efforts to find effective, safe and cost-effective natural agents against Leishmania, C. procera can be considered as a new source of natural anti-leishmanial agents. Certainly, to better understand the effect of C. procera leaf extract on leishmaniasis infection, its active and non-toxic components should be studied further utilizing an experimental murine model.