Evaluating the in vitro anti-Leishmanial activity of essential oil extracted from Cymbopogon Citratus against Leishmania Donovani

: Leishmania is one of the protozoan parasites that are transferred to human by infected sand flies and gives rise to a range of diseases entitled as Leishmaniasis. More than 20 known species of Leishmania can infect humans and cause various clinical symptoms. Three most known clinical manifestations are Cutaneous Leishmaniasis (CL), Mucocutaneous Leishmaniasis (MCL) and Visceral Leishmaniasis (VL) (kala-azar or black fever). The difference in the clinical form dependent on several factors: species of Leishmania , type of vector that transmits the Leishmania , and the immune status of an infected individual. The current drugs which are used as anti-leishaminial treatment are characterized by enormous side effects, including their toxicity to human, long term treatment, liver problems and huge cost. Therefore, there is a necessity to find an alternative treatment marked as low cost, more specific against parasite’s stages, and metabolic pathways, and non-toxicity to human. Plants are considered one of the important sources for the remedy of the tropical diseases caused by bacteria, viruses, and parasites. Cymbopogon Citratus (lemon grass) is a herbal medicine used as anti-inflammatory, antibacterial, antifungal, anti-malarial, anti-protozoal and for gastrointestinal problems remedy. In order to detect the effects of C. Citratus against Leishmaniasis, in this study, serial dilution for the essential oil of C.citratus (1000, 500, 250, 125, 62.5 and 31.25) µg/ml were used against L. donovani Promastigotes. Viability was also evaluated at 24, 48 and 72 hours post-treatment. The results revealed that high concentrations (1000, 500 and 250) µg/ml were more effective than other concentrations during all time intervals, and IC 50 values were 640, 492 and 442 µg/ml at 24, 48 and 72 hours respectively. In conclusion, this current study is one of a persistent search to find new treatments characterized by its high activity and low adverse effects to treat protozoa parasites for instance, Leishmania , and displays the effectiveness of the essential oils as a promising alternative


Introduction
The Leishmaniasis are a category of neglected tropical diseases resulting from the genus of Leishmania parasite [1]. All genus of this parasite (Leishmania) are obligate intracellular that infect macrophage cells of the mammalian hosts [2]. Leishmania is transferred to humans through bite of a female of sand fly vectors, Phlebotomus (with old-world Leishmaniasis), and Lutzomya (with new world Leishmaniasis) [3]. More than 20 known species of Leishmania can infect humans and can cause various clinical symptoms, including three common clinical implications for example, Cutaneous Leishmaniasis(CL), Mucocutaneous Leishmaniasis (MCL), Visceral Leishmaniasis(VL) (kala-azar or black fever). The difference in the clinical form is dependent on several factors like the species of Leishmania, type of vector that transmits the Leishmania, in addition to the immune status of infected individuals [4,5,6]. The most endemic Leishmania species found in Iraq are VL and CL.L. tropica and L.major are the causative agents the CL [7]. They spread in all country except three provinces in the north of country. The VL in Iraq is caused by L .donovani and L.Infantum. VL which represents the most serious form of Leishmanisis spreads widely among children under five years (90 %). Most cases appear in central and some southeastern provinces [8,9,10]. Currently, no certified vaccine against Leishmaniasis is being used in humans. As a result, due to some difficulties relating to the vector control and other factors, all of which are contributed together to increase in the cases that lead to the failure of controlling the spread of the disease [11]. The first medicines that were used for leishmaniasis treatment are antimonial which include Pentostam (Sodium Stibogluconate) and Glucantime (Meglumine antimoniate). However, various adverse effects as a result of using these drugs, includes their toxicity in humans, prolonged treatment, increased parasite resistance, leukopenia, liver problems, cardio toxicity and cardiac arrhythmia [12]. Furthermore, alternative drugs to antimonials, like amphotericine B, miltefosine, pentamidine and paromomycin also showed side effects including toxicity, high cost and drugs resistance. All of these reasons mentioned above lead to drug resistance and therapeutic failure, making it necessary to develop alternative drugs characterized by its lower cost, toxicity, and highest efficacy, and its availability in low-income countries [13]. Natural products from medicinal plants are considered one of the strategies to produce drugs.
Estimates have showed that about 25% of contemporary medicines are adapted from medicinal plants instantly or indirectly. Numerous studies have shown that a large variety of plants in many regions in the world contain components that act as anti-protozoan. Various essential oils have demonstrated inhibition activity against certain human parasites such as Leishmania [14]. Cymbopogon citratus (DC) is a medicinal plant that belongs to Poaceae family which is extensively used in Southeast Asia, South American countries, and Africa. C. citratus is also as anti-inflammatory, anti-pyretic, diuretic, antispasmodic and gastrointestinal disturbances remedy in herbal medicine field. Additionally, a number of studies have revealed the effects of this plant by using it as antibacterial, antifungal, antimalarial and antiprotozoal [15,16,17,18]. According to Machado et al., (2012) [19], the essential oil of C.citratus has indicated thecytotoxicity against Leishmania parasite.

Material and method
Plant collection: C.citratus plants were obtained from the herbal center at the University of Baghdad / College of Science / Department of Biology. Plants' aerial parts (leaves) were then dried and divided into small pieces in order to extract the essential oil. The extraction process used for essential oil extraction, stated in the method described in the European Pharmacopoeia (Council of Europe, 1997) [20], depended on the hydro distillation for 3 hours by Clevenger apparatus. Parasite isolation: L.donovani isolate (MHOM/IQ/2005/MRU15) was obtained from the Laboratory of Parasitology for graduate studies, University of Baghdad / College of Science / Department of Biology. Leishmania donovani culture: Novy-Mac Neal-Nicolle (NNN) medium was used to stimulate the Leishmania donovani promastigotes, which were then transferred to RPMI-1640 media accompanied by 10% FBS (gibco®) and 1% Antibiotic (penicillin& streptomycin), and incubated at 26C° with high sterilization conditions to obtain the logarithm phase. Neubauer chamber was used to count the parasite. Finally, the promastigotes in the logarithm phase were concentrated to 2×10 6 for the purpose of the cytotoxicity assay. Dilution and preparation of concentrations from the crude essential oil extraction of C. citratus: After crude oils extraction using Clevenger apparatus, the stock was prepared in concentration 10000 µg/ml from essential oil. Desired concentrations (1000,500,250,125,62.5, and 31.25) µg/ml were then prepared from the stock solution by dissolving these concentrations in 1% Dimethysullfaxide (DMSO) stored at room temperature away from the light [21]. Anti-leishmanial assay or 3-(4, 5-dimethyl-thiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT assay): One of the techniques used to measure cellular vitality or cytotoxicity potential of drugs is MTT assay or Colorimetric assay. The MTT assay mechanism depends on converting watersoluble yellow tetrazolium MTT [3-(4, 5-dimethyl-thiazolyl-2)-2, 5-diphenyl tetrazolium bromide] (1 mg / ml) dye that uptakes viable cells into water-insoluble blue formazan. The formazan dye formed under the effect of the mitochondrial enzyme succinate-dehydrogenase within the viable cells. The amount of dye formed is proportional to the number of the viable cells. Produced formazan is dissolved in dimethylsulfoxide (DMSO) to give a purple color with a characteristic absorption at 620 nm. Although [22] mentioned that the range of wavelength ranges from 565 -630 nm, and 570 is the best wavelength. However, 620 nm is also within the range, and the reason for the use 620 nm in this study depeneded on the instrument in our lab as it was set up to measure the absorption at 620 nm only. The intensity of the purple color is directly proportional to the number of viable cells [22,23]. The experiment was designed to estimate the effect of serial dilutions for the crude essential oil of C.citratus 1000, 500,250,125, 62.5 and 31.2) µg/ml on L.donovani promastigotes proliferation by using sterile 96 well plates by following the protocol. Final volume (50 µl) of the desired crude essential oil concentrations dissolved in 1% DMSO, were added in each well. L.donovani promastigotes at 2 x 10 6 ml -1 in 50 µl of RPMI-1640 media supplemented with 10% FBS, and 1% Antibiotic (penicillin& streptomycin) were added to each well to get the final volume of 100 µl. In addition to the treated promastigotes, the control group used in this experiment were untreated promastigotes. Treated and untreated promastigotes were incubated at 26 c for 24, 48 and 72 hours. By the end of each period time, 10µl of MTT dye was added to each well according to the manufacturer's (iNtRON®). Then the plates were incubated for 4 hours at 26c before reading the results by the ELISA reader Avusturya. The result was calculated by ELISA reader in microtiter-plate., percentage of the viability was calculated from this equation: %Viability = x100 Thereafter, the inhibitory concentration (IC 50 ) was determined for each time, by using SPSS software.

Statistical Analysis:
The Statistical Analysis System-SAS (2012) [24] program was used in this study to observe the effects of difference factors in this study. Least significant difference -LSD test (Analysis of Variation-ANOVA) was applied to test the significance of relationship between means. Furthermore, Chi-square test was utilized to show the significant correlation between percentage at probability P ≤0.01 and 0.05) in this study. Also IC 50 values were calculated by SPSS software and excel 2010.

Results:
Anti-leishmanial activity of C.citratus crude essential oil against L.donovani promastigtes within 24, 48, 72hours. The results in general revealed the significant inhibitory effects of C.citratus crude essential oil on L. donovani promastigote proliferation during different time intervals 24, 48 and 72 hours), particularly in high concentration. The results showed that the highest concentrations (1000, 500, 250 µg/ml) had the highest inhibitory effect against parasites. However, 125 µg/ml concentration showed effects only after 72 hours. In addition, no inhibitory effects at the lower concentrations 62.5 and 31.25 µg/ml were shown in all intervals. (Figures 1, 2 and 3) (Tables 1, 2 and 3). The results demonstrated that the most effective concentrations of the crude extract oil of C. citratus in a 24-hour post-treatment were 1000, 500 and 250 µg/ml, and the viability of promastigotes were significant 46%, 51% and 54% respectively, with no inhibitory effect at concentrations 125, 62.5 and 31.25 µg/ml (Table 1) (Figure 1) with IC 50 640 µg/ml.

Discussion:
Pentamidine, Amphotericin-B, Glucantime and other drugs that are being used as antileishmanial treatment, are characterized by highl cost, enormous side effects, and could lead to the emergence of drug-resistant parasites, ineffective and variable degree of efficacy. All these reasons has increased the efforts to find alternative therapies, strongly marked with inexpensive, safe and being more specific against the pathogen, easily administrated and effective [25]. Recently, the scientific researchers are focusing on medicinal plants, and their leaves that are use in folk medicine as anti-inflammation and anti-microorganisms [21]. These plants produce essential oils (EOs) as secondary metabolites with bioactive properties and contain a wide variety of chemical components characterized by antimicrobial potential and anti-parasites. Previous studies have shown promising results in plant-derived essential oils, crude extracts, and compounds with action against pathogens, including Leishmania spp. [26,27]. Moreover, many essential oils of plants have been tested on Leishmania in amastigote and promastigote form and epimastigote forms of Trypanosoma cruzi. The results have indicated the potency of these medicinal plants including C. citratus against Leishmania and T. cruzi [28]. Furthermore, one of the studies for plant-derived components that are used as an alternative treatment against Leishmaniasis, indicated that the C. citratus essential oil is more effective in inhibiting the Leishmania growth by inducing an apoptosis process [13]. C.citratus is a herb widely used in the folk medicine in Asia, African and South American countries for the treatment of gastrointestinal disturbances for its antispasmodic, antiinflammatory, anti-pyretic, diuretic, antibacterial, antifungal, and antiprotozoa properties [18,19,30]. Crude essential oil of C.citratus has health restorative capacity, that belongs to its derivatives that work as the secondary metabolite products. In addition, it contains some active components such ascitral. It also contains fats, proteins, carbohydrates, fiber, minerals, energy and several other bioactive compounds. Some studies on herbal plants, including C.citratus, demonstrated that this plant has an anti-leishmanial activity by stimulating a programmed cell death process by citrus which is considered a major constituent of C. citratus essential oil [31]. Iraqi Journal of Science, 2022, Vol. 63, No. 4, pp: 1453-1463 1460 The results of this study represent first ever study for the effects of essential oil extracted from Cymbopogon citratus on L.donovani in promastigote stage in Iraq. It also agreed with other studies found C.citratus essential oil's proven efficacy as anti-parasites by experimented it on all three evaluative stages of Trypanosoma cruzi (trypomastigote, epimastigote and amastigotes). The results have indicated anti-proliferative effect of essential oil on these stages [18]. Besides this, another study has revealed anti-parasites action of C.citratus essential oil on the parasites that infected fish [32]. Another study used the essential oil of C.citratus against Sarcoptes mite's parasite. This study demonstrated that this oil has miticidal and ovicidal action against Sarcoptes mitess [33].
A study by Machado et al., 2010 [34] proved the activity of some essential oils, including essential oils from C.citratus, and its active compounds (myrcene, neral, and geranial) on the growth of L.infantum promastigotes. Also, essential oils of some plants, including C.citratus essential oils were experimented invitro and ex-vivo on four types of Leishmania parasite., The results demonstrated the activity of C.citratus essential oils on the four type (L. panamensis, L. braziliensis, L. major, and L. guyanensi). In-vitro experiment, however, showed toxic effects against macrophages cell line, though it may not be active against the host cells [35]. Furthermore, anti-Leishmanial activity of C.citratus essential oils was studied on L.chagasi promastigotes, indicating the effects for the essential oils of C.citratus on the parasite viability. Several appearance morphological alterations, including swelling round form and ultra-structure such as aberrant multi-septation of the cell body, were also detected [36]. In another study, major compounds of C.citratus essential oil and citral were experimented on Leishmania amazonensis in-vitro and ex-vivo. The results showed the anti-proliferative activity of essential oil on promastigotes and axenic amastigotes, with IC 50 1.7 µg/ml [14].
Another study about the anti-leishmanial activity of C.citratus by Machado et al.(2012) [19], revealed that the major components of C.citratus essential oils are mrycene and citral. This study also experimented in-vitro and ex-vivo on L. infantum, L. tropica and L. major for 24 and 48 hours, IC 50 concentrations of active components of C.citratus essential oils ranging from 25 to 52 µg/ml and IC50 of citral from 34 to 42 µg/ml. The study indicated that the major components of C.citratus essential oils showed an anti-leishmanial activity against three types of Leishmania in varying proportions, and the citral was more effective against leishmania.The study also refers to the anti-leishmanial effects of C.citratus essential oils which occurred by triggering a programmed cell death (apoptosis). In this study, high concentration of the essential oil of C. citratus particularly 1000, 500 and 250 µg/ml, and in all intervals 24, 48 and 72 hours, showed the most effectie concentrations than the others. Also, a logarithmic rise in the percentage inhibition increased with the concentration range and time period, achieving a highest inhibition approximately 27% and an IC 50 value of about 442 µg/ml in a 72 hours post-treatment could be due to the high concentration of active compounds that are found in the crude essential oil of C. citratus which increased with time period. The effects of the crude essential oil in this study against L. donovani could be due to the fact that the crude essential oil is a network composition of active substances, and a higher concentration of these active compoundlike citral plays an important role in increasing the inhibition of L. donovani promastigotes. It agreed with Machado et al., 2012 [19] who suggested that the citral, a major components that found in C. citratus crude essential oil, stimulated the anti-leishmanicidal impact in L. infantum by activating programmed cell death, in addition to its effect on the mitochondrial membrane, and cell cycle inhibition inside the parasite.
Moreover, a study by Halabi and Sheikh (2014) [37], revealed that the citral found in C. citratus has anti-proliferative effect on some cancer cells lines. This agreed with the results of this study where the inhibitory effect of crude essential oil against L. donovani also increased.
In conclusion, the results of this study demonstrated the potential therapeutic impact of the essential oil of C.citratus against L.donovani.