Leishmaniasis is a disease caused by Leishmania species with the incidence rate of about two million cases annually ( 1 ). Currently, the increase in international travel and environmental amendments caused by war in some regions has created favorable conditions for the propagation of the parasite vectors; thus, there has been a significant increase in the incidence of leishmaniasis ( 2 ). Poverty contributes to the risk of leishmaniasis and so does the open sewerage and lack of waste management which can increase sandfly breeding sites of Leishmania vectors, as well as their access to human bodies. About 70 animal species have been found to act as natural reservoir hosts for these parasites ( 3 ). The first choice for the treatment of leishmaniasis is pentavalent antimoniate (Pa). But the Pa—because of its side effects, the emergence of resistance, and parenteral application is no longer a sufficient treatment. Therefore, it is essential to find new drugs with different mechanisms ofand greater potency ( 4 ).
Nanoparticles (NPs) can be used for therapeutic applications because of their strong antimicrobial activities ( 5 , 7 ). Such as in drug delivery, microbiology, biotechnology, and biochemistry ( 8 , 15 ). The use of metal NPs such as copper, platinum , titanium , gold, selenium, silver, zinc oxide, and palladium nanoparticles against many bacteria, viruses, and fungal pathogens has been reported ( 16 , 19 ), but the use of NPs against protozoan parasites is rather limited. Among different types of NPs, the zinc oxide (ZnO) NPs have attracted scientific attention because of their safety and high antimicrobial activity ( 20 , 21 ); they are recognized to be safe by the U.S. Food and Drug Administration (FDA). The traditional NPs synthesized by chemical methods leads to the adsorption of toxic chemical compounds onto the surface of synthesized NPs which may have adverse effects in medicine ( 22 , 23 ). Green synthesis methods using bioresources such as plants, fungi, or bacteria for the synthesis of biogenic NPs represent alternatives to conventional chemical synthesis methods ( 24 , 29 ).
The present study aims to evaluate the leishmanicidal efficiency of rod-shaped ZnO (R-ZnO) NPs on Leishmania major (L. major) cultures.
The main aim of this study was to evaluate green synthesis of parasitical zinc oxide NPs using Lilium ledebourii tuber (L. ledebourii). A greener synthesis of rod shape zinc oxide nanoparticles (R-ZnO) were studied using L. ledebourii tuber as a novel bioresource and their leishmanicidal activity has been studied against L. major.
3. Materials and Methods
All the reagents and chemicals used in the experiments were purchased from Merck, Germany.
3.2. Synthesis of Rod Shaped Zinc Oxide Nanoparticles
The L. ledebourii tuber were washed thoroughly with sterile distilled water and dried, then 10 g of Lilium tuber was ground into a powder. The powder was added to 200 mL of deionized double-distilled water, heated at 80 °C, and then filtered. To conduct green synthesis of R-ZnO NPs, 20 mL of the obtained extract was added to the 100 mL of zinc acetate dehydrate solution and stirred at ~80 °C for 1 hr. The reaction mixture (extract + zinc acetate dehydrate) was incubated at 80 °C for 2 hr and calcined at 300 °C for 1 hr.
3.3. Characterization of ZnO NRs
The synthesized ZnO nanoparticles were studied using a UV-visible spectrophotometer (Analytik Jena; Germany). XRD analysis was performed to determine the formation of ZnO crystals. The resulting powder was analyzed using an X-ray diffractometer (PANalytical X'Pert PRO; The Netherlands) at 2θ. The shape, size and size distribution of nanoparticle were investigated by TEM ( 30 ).
3.4. Leishmanicidal Assay
The L. major MRHO/IR/75/ER standard strain was cultured in RPMI1640 at 25 °C supplemented with 15% heat-inactivated FBS, streptomycin (200 mg.mL-1), and penicillin (200 IUmg.mL-1). The stationary growth phase of promastigotes was added to the macrophages to generate a parasite/macrophage ratio of 10:1. It was then incubated at 37 °C in 5% CO2 for 24 hr ( 31 ). The macrophages containing antiamastigote were treated with various concentrations of 0–500 mg.mL-1 R-ZnO NPs ( 32 ).
3.5. Statistical Analysis
The differences between the groups were determined using one-way analysis of variance (ANOVA) to analyse the obtained results. A p-value of < 0.05 was considered significant.
4.1. Biosynthesis and Characterization of ZnO NRs
The XRD pattern shows dispersion peaks at 31, 35, 37, 48, 57, 62, 66, 68, and 69, thus confirming the presence of ZnO NRs in the sample (Fig. 2). which corroborated previous with previous findings ( 33 , 34 ).
TEM images confirm that biogenic ZnO NPs have rod-shaped morphology; however, spherical NPs were observed in the TEM images. R-ZnO NPs are below 100 nm in size (Fig. 3).
4.2. Antiamastigote Assay
The R-ZnO NPs were found to inhibit the multiplication rate of amastigotes in a dose-dependent manner (). The IC50 values were about 10 mg.mL-1 for both R-ZnO NPs and Meglumine antimoniate (glucantime) as positive control. The concentration of 0.5 mg.mL-1 of biogenic R-ZnO NPs revealed a higher toxicity effect on L. major (amastigotes). The results disclose that by increasing the concentration of R-ZnO NPs, the viability of the tested parasites significantly decreases.
In this study, for the first time, the rod shaped ZnO NPs were biosynthesized as a safe therapeutic nanomaterial. A novel protocol for the greener production of the rod shaped ZnO NPs is presented in this study. The traditional NPs synthesized by chemical methods leads to the adsorption of toxic chemical compounds onto the surface of synthesized NPs which may have adverse effects in medicine. The nanoparticles that are produced by plant extract have lower environmental risk due to lack of harsh chemicals often in their synthesis process. Therefore, they can be applied in medical programs such as drug delivery. The salient advantages of producing plant nanoparticles via greener methods such as using bacterial or fungal extracts is the safety and high availability of medicinal plants that are more reliable and healthier than bacterial or fungal extracts mediated for the production of nanoparticles. Green synthesis methods using bioresources such as plants, fungi, or bacteria for the synthesis of biogenic NPs represent alternatives to conventional chemical synthesis methods.
The use of factors present in the plant and fungus residue not only are responsible in the synthesis of nanoparticles, but by stacking around the nanoparticles, they cause stability and prevent their agglomoration. Plants, in addition to being non-toxic, have different types of metabolites, which can be effective in the synthesis of nanoparticles which can include terpenoids, flavonoids, carbonyls, amides, and carboxylic acids, which directly contribute to the formation of nanoparticles.
The result of a study conducted against L. major showed that rod shaped ZnO NPs have good leishmanicidal activity against L. major and this outcome could help in the development of formulations as efficient means to synthesize R-ZnO NPs from natural products in our fight against the resistant microorganisms ( 35 , 36 ).
In our study, the rod shaped ZnO nanoparticles displayed strong leishmanicidal efficiency (IC50 about 0.012 mg.mL-1). There are no reports on the leishmanicidal activity of biogenic rod shaped ZnO NPs nor on the cytotoxic effect of chemically synthesized Rod shaped ZnO NPs on living cells. The study by Delavari et al. 2014 ( 37 ) have reported leishmanicidal activity of chemicaly synthesized spherial shaped ZnO NPs on L. major, the IC50 ZnO NPs being 0.0378 mg.mL-1 on promastigotes of L. major. Also Sumaira et al. ( 38 ) have reported leishmanicidal activity od spherical greener sythsized ZnO NPs, the IC50 ZnO NPs being 0.25 mg.mL-1 against Leishmania tropica. But in our present study, the biogenic greener synthesized NPs rod shaped ZnO NPs displayed much stronger leishmanicidal activity (IC50 about 0.012 mg.mL-1); they are more effective in leishmanicidal activity compared to chemically assembled spherical shape ZnO NPs or greener spherial shaped ZnO nanoarticles. Additionally, biogenic R-ZnO NPs can also be used in vivo.
These reslts show that it is possible to prepare a safe and ecofriendly synthsized NPs with leishmancidal potential. The greener synthesized rod shaped ZnO NPs, displayed stronger leishmanicidal activity (IC50 about 0.012 mg.mL-1); compared to chemically assembled spherical shape ZnO NPs and greener spherical shaped ZnO nanoparticles( 37 ).
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