Antileishmania and immunomodulatory potential of cashew nut shell liquid and cardanol

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Conventional treatments cause serious adverse effects, poor tolerance, development of resistant strains, and are costly. Natural products have been investigated in the search for therapeutic alternatives. The cashew nut shell liquid (CNSL) is a natural source of phenolic compounds, showing antioxidant, anti-inflammatory, antimicrobials, antitumors, larvicides and insecticides, with cardanol (CN) being considered one of the most important and promising technical components. This study aimed to evaluate antileishmania, cytotoxic and immunomodulatory activities of CNSL and CN. The substances showed antileishmania potential, with values of mean inhibitory concentration (IC 50 ) of CNSL and CN against Leishmania infantum : 148.12 and 56.74 µg/mL; against Leishmania braziliensis : 85.71 and 64.28 µg/ml; against Leishmania major : 153.56 and 122.31 µg/mL, respectively. The mean cytotoxic concentrations (CC 50 ) of CNSL and CN were 37.51 and 31.44 µg/mL, respectively. CNSL and CN significantly reduced the percentage of infected macrophages, with a selectivity index (SI) > 20 for CN. CNSL and cardanol caused an increase in phagocytic capacity and lysosomal volume, however, they did not exhibit significant induction of nitric oxide synthesis. Survival rates of Zophobas morio larvae at doses of 3; 30 and 300 mg/Kg were: 85%, 75% and 60% in contact with CNSL and 85%, 60% and 40% in contact with CN, respectively. There was a significant difference between the survival curves of larvae when treated with CN, demonstrating a significant acute toxicity for this substance. Additional investigations are needed to evaluate these substances in the in vivo experimental infection model. Natural products Antileishmania activity Cytotoxicity Immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Leishmaniasis is a neglected disease caused by protozoa of the Leishmania genus, constituting a major public health problem due to its high incidence and lethality (Alvar et al. 2012 ; Benitez et al. 2018 ). In the year 2020, leishmaniasis was endemic in 98 of the 200 countries and territories that reported it to the WHO, and it is estimated that between 700,000 to 1 million new cases of cutaneous leishmaniasis and 50,000 to 90,000 new cases of visceral leishmaniasis occur worldwide each year (WHO 2021). The etiological agent of the disease is more than 20 species of the genus Leishmania , classified in the subgenus Viannia and Leishmania , transmitted by approximately 70 different species of sandflies of the genera Lutzomyia and Phlebotomus . The disease exhibits a variety of clinical manifestations, the two main ones being visceral leishmaniasis (VL) and tegumentary leishmaniasis (TL), and this, in turn, is divided into four subtypes: localized cutaneous leishmaniasis (LCL), diffuse cutaneous leishmaniasis (DCL), mucocutaneous leishmaniasis (MCL) and disseminated leishmaniasis (DL) (Akhoundi et al. 2016 ; Akhoundi et al. 2017 ). The treatment of leishmaniasis is performed with pentavalent antimonials, amphotericin B, paromomycin, miltefosine, sodium stibogluconate, meglumine antimoniate and pentamidine (Blanco e Nascimento-Júnior 2017; Kevric et al. 2015 ). However, the use of these drugs requires long periods of administration, resulting in serious adverse effects, low tolerance and development of resistant strains to the treatment, contributing to the ineffectiveness of therapeutic regimens, in addition to presenting high costs (Bapela et al. 2017 ; Souza-Silva et al. 2015 ). The investigation of the pharmaceutical potential of natural products is the main strategy for discovering new drugs that can be less costly and less toxic than conventional drugs (Tiuman et al. 2011 ). Plants are considered an important source of natural products that can be successfully exploited for the development of new drugs with antileishmania activity (Funari et al. 2016 ; González-Coloma et al. 2011 ; Machado et al. 2014 ; Mansour et al. 2013 ; Torres et al. 2014 ). Compounds such as alkaloids, phenolics, terpenoids and flavonoids have been extensively studied for their potential antileishmania activity (Silva et al. 2014 ). Among the widely used medicinal plants, cashew ( Anacardium occidentale L.) stands out and one of its main by-products, cashew nut shell liquid (CNSL), has been used for decades in traditional medicine in countries in South America, Africa and Asia (Ayyanar and Ignacimuthu 2009 ; Da Silva et al. 2018 ; Kudi et al. 1999 ). CNSL is a product with little commercial value, but with high technological potential due to its phenolic constitution and its various biological characteristics, such as anti-inflammatory, antimicrobial, antioxidant, antitumor, larvicides and insecticides, exhibiting great therapeutic potential (Kubo et al. 1993a ; Oliveira et al. 2010 ; Wu et al. 2011 ), constituting a natural source of phenolic compounds, such as anacardic acid, cardanol, cardol and 2-methylcardol (Mazzetto et al. 2009 ). CNSL derivatives are also widely explored in isolation, mainly for their properties such as antibacterial, antioxidant, antifungal, antitumor, among others (Amorati et al. 2001 ; Chen et al. 1998 ; Hemshekhar et al. 2012 ; Mazzetto et al. 2009 ; Muroi e Kubo 1996). Cardanol (CN) is the main constituent of technical CNSL and is considered one of the most important and promising components. As it is a by-product of the nut industry, any improvement in the CN, whether in concentration and/or separation, is effectively characterized as a technological innovation (Mazzetto et al. 2009 ). Thus, the CNSL demonstrates wide utility for pharmacology, and therefore further studies of its isolated metabolites, their mechanisms of action, as well as toxicological aspects are needed (Araújo et al. 2020 ). Given the above, considering the impact of leishmaniasis mainly in developing countries, the urgency for more effective compounds with fewer adverse effects, and the broad therapeutic potential of CNSL, is strategic to invest in the search for new pharmacological properties of this product and its metabolites, as a potential source of medication for the treatment of leishmaniasis. Thus, the objective of this study was to evaluate the immunomodulatory and antileishmania activities of the CNSL and its main constituent, the CN, in order to propose a search for a new therapeutic strategy. Material And Methods Substances used Schneider’s culture medium, DMEM® medium, FBS, MTT, resazurin, acridine orange dye, propidium iodide and the antibiotics Penicillin and Streptomycin from Sigma Chemical (Sigma-Aldrich Brazil). Amph B (90%), fast panoptic® was acquired from Cristália (São Paulo, SP). CNSL and CN were from the Organic Geochemistry Laboratory (LAGO/UFPI) and were diluted in DMSO at a concentration of 80 mg/mL for the experiments. Parasites and cells Strains of Leishmania infantum (MHOM / 5745), Leishmania braziliensis (10CL566) and Leishmania major (MHOM / IL / 80 / Friendlin) were obtained from the Medicinal Plants Research Center of Federal University of Piauí. Parasites were grown in supplemented Schneider’s medium (10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 26 °C) (Carneiro et al. 2012; Valadares et al. 2011). Murine macrophages were collected from the peritoneal cavities of male and female BALB/c mice (4–5 weeks old; Medicinal Plants Research Center, UFPI, Brazil), and cultivated in RPMI 1640 medium (10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO 2 ). All protocols were approved by the Animal Use Ethics Committee (CEUA-PI nº 640/2019). Invertebrates Zophobas morio larvae used to assess acute toxicity came from the Physiology Laboratory of the Department of Veterinary Morphophysiology/UFPI. The larvae were kept in plastic organizer boxes measuring 60 x 40 x 80 cm with water and feed ad libidum, at an ambient temperature of 25°C ± 2°C. Larvae weighing between 100 and 200 mg, light and uniform in color, without signs of melanization were selected for the experiment. Investigation of activity against promastigote forms of L. infantum , L. braziliensis and L. major Promastigote forms of L. infantum , L. braziliensis and L. major in the late log phase (1 × 10 6 leishmania /100 μL of medium) were plated in 96-well culture plates containing supplemented Schneider’s medium. Then, CNSL and CN (6.25, 12.5, 25, 50, 100, 200; 400 and 800 μg/mL) were added, and the plates were incubated during 48h in a BOD (biochemical oxygen demand) incubator at temperature of 26 °C. Remaining 6h to the end of this period, 20 μL of resazurin (1 × 10 −3 mol/L) was added. Afterwards, the absorbances were read in a BioTek microplate reader (model ELx800) at a wavelength of 550 nm. The results were expressed as inhibition of parasite growth (%) (Soares et al. 2007; Valadares et al. 2011). The negative control was the Schneider’s medium with promastigotes (1 × 10 6 cells/well) and for the positive control, amphotericin B (2 μg/mL). The cell viability was considered as 100% for the parasite. The blank was read for each concentration and control in order to avoid interference of absorbance of medium of other compounds (Soares et al. 2007; Valadares et al. 2011). Evaluation of cytotoxicity in macrophages Cytotoxicity evaluation was carried out in 96-well plates using the MTT assay. Macrophages (2 × 105 per well) were incubated in 100 μL of supplemented RPMI 1640 medium at 37 °C and 5% CO 2 for 4h. Non-adherent cells were removed by washing with RPMI 1640 medium. Then, CNSL and CN were diluted in supplemented RPMI 1640 medium, and added at concentrations of 6.25, 12.5, 25, 50, 100, 200; 400 and 800 μg/mL, followed by incubation at 37 °C with 5% CO 2 for 2 days. The cytotoxicity of Amph B was assessed at concentration of 0.2 μg/mL. Afterwards, cytotoxicity was assessed by adding MTT (5 mg/mL). The supernatant was discarded, and the formazan crystals were dissolved by addition of 100 μL of DMSO. Finally, absorbance at 550 nm was measured using a BioTek (ELx800) plate reader (Oliveira et al. 2017). Investigation of CNSL- and CN-induced activity on macrophages infected by Leishmania infantum and the calculation of selective index As L. infantum is a species that causes zoonotic leishmaniasis and that develops the visceral form of the disease, considered the most severe form (Steverding 2017), the activity of CNSL and CN on L. infantum promastigotes was evaluated. Macrophages (2 × 10 5 cells/mL) were harvested in 24-well plates containing sterile round coverslips at 13mm and supplemented RPMI 1640 medium (10% inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin). Culture plates were incubated at 37 °C and 5% of CO 2 for 3h. Adhered macrophages were then incubated with a new medium containing axenic amastigotes at a ratio of 10 amastigotes per 1 macrophage at 5% CO 2 and 37 °C for 4h. The medium was subsequently aspirated in order to remove non-internalized parasites, and the wells were washed with 0.01 M phosphate buffered saline (PBS). The infected macrophages were then incubated with CNSL and CN at 3.125; 6.25 and 12.5 μg/mL (non-toxic concentrations on host cells) or Amph B at 0,5; 1 and 2 μg/mL. After this period, the coverslips were removed and stained with Panoptic staining kit. For each treatment, the number of infected macrophages and the parasite load (survival index, obtained by counting the number of parasites in 100 macrophages) were counted using optical microscopy (Carneiro et al. 2012). Furthermore, the selectivity indexes of CNSL, CN, and Amph B were determined by the ratio of the mean CC 50 against macrophages to the mean IC 50 against macrophage-internalized amastigote forms of L. infantum (Oliveira et al. 2017). Evaluation of parameters related to macrophage activation Lysosomal activity Macrophages (2 × 10 5 /well) were incubated with CNSL and CN (6.25; 12.5; 25; 50 e 100 μg/mL), or Amph B (0.2 μg/mL) in a 96-well plate at 37 °C and 5% de CO 2 . After 48h, 10 μL of neutral red stock solution were added for 30 min. Then, the supernatant was discarded, the wells were washed with 0.9% saline at 37 °C, and 100 μL of extractive solution were added in order to solubilize the neutral red present within the lysosomal secretory vesicles. After 30 min on a Kline shaker, the absorbances were read in a BioTek (ELx800) plate reader at 550 nm (Bonatto et al. 2004). Phagocytic capability Macrophages (2 × 10 5 /well) were incubated with CNSL and CN (6.25; 12.5; 25; 50 e 100 μg/mL), or Amph B (0.2 μg/mL) in a 96-well plate for 48 h at 37 °C and 5% de CO 2 . After 48h, 10 μL of zymosan-stained NR solution was added for 30 min. Next, the phagocytic process was interrupted adding 100 μL of Baker’s fixative solution during 30 min. Then, the wells were washed with 0.9% saline, and 100 μL of extractive solution were added. After solubilization in a Kline shaker, the absorbances were read in a BioTek (ELx800) plate reader at 550 nm (Grando et al. 2009). Nitrite measurement Non-infected macrophages or infected by L. infantum were obtained as described previously, and then incubated with CNSL and CN (3.125; 6.25 e 12.5 μg/mL), or Amph B (0.2 μg/mL) at 37 °C and 5% de CO 2 for 24h. The lipopolysaccharide (LPS) from E. coli (2 μg/mL) was used as positive control. The standard curve was prepared with sodium nitrite in RPMI medium at varying concentrations of 1, 5, 10, 25, 50, 75, 100, and 150 μM diluted in RPMI 1640 medium. After 24h, the supernatants were transferred, and then incubated with equal parts of Griess reagent. Thereafter, the absorbances were read in a BioTek (ELx800) plate reader at 550 nm (Soares et al. 2007). Investigation of acute toxicity on Z. morio larvae To assess acute toxicity, 10 larvae of Z. morio were used for each concentration tested, in triplicate. CNSL and CN were applied to the larvae in a volume of 10 μL at doses of 3; 30 and 300 mg/kg with the aid of a Hamilton syringe (701 N, manometer 26, Capacity 100 μL) in the hemocoel, in the second or third sternite visible above the legs, in the ventral portion. Larvae were incubated at room temperature in petri dishes containing breeding diet and the number of dead larvae was evaluated after 48h. To establish the death of the larvae, a visual check of each individual was performed for the presence of myelination and/or response to physical stimuli after gentle touch (de Souza et al. 2015). Statistical analyses All tests were performed in triplicate in three independent experiments. The mean inhibitory concentration (IC 50 ) and the mean cytotoxic concentration (CC 50 ) with confidence limits of 95% were determined by regression of probits using the software SPSS 13.0. The selectivity index was calculated as the ratio between CC 50 and IC 50 of internalized amastigotes. The survival curve of Z. morio larvae was plotted by Kaplan-Meier analysis and the results were analyzed by the Log Rank test to assess the level of relationship between the substances regarding the acute toxicity parameter. Analysis of variance ANOVA followed by Bonferroni’s test was performed, taking the value of p < 0.05 as the maximum level of statistical significance. Results Antileishmania activity assay CNSL and CN demonstrated antileishmania potential, with their action dependent on concentration. It was possible to observe activity against all Leishmania species tested, where at the concentration of 800 μg/mL there was 100% growth inhibition of the promastigote forms of L. infantum , L. braziliensis and L. major in the presence of CNSL and L. infantum and L. braziliensis in the presence of CN. CN showed about 90% growth inhibition of promastigote forms of L. major at a concentration of 800 μg/mL. Amphotericin B inhibited the growth of promastigote forms of L. braziliensis by about 90% and by L. infantum and L. major by about 80%, both at a concentration of 2 μg/mL. The IC 50 values of CNSL and CN against L. infantum , L. braziliensis and L. major are shown in Table 1. Table 1 Antileishmania activity of CNSL, cardanol (CN) and amphotericin B (Amph B). Compounds L. infantum L. braziliensis L. major IC 50 µg/mL a IC 50 µg/mL a IC 50 µg/mL a CNSL 148.12±0.08 85.71±0.11 153.56±0.08 CN 56.74±0.06 64.28±0.06 122.31±0.05 Amph B 2.46±0.07 2.94±0.05 0.59±0.13 a Inhibition Concentration 50 Citotoxicity assessment When acting on macrophages, CNSL demonstrated significant cytotoxicity from a concentration of 12.5 μg/mL, with a CC50 value of 37.51 μg/mL, while CN significantly reduced macrophage viability from the concentration of 6.25 μg/mL, resulting in a CC 50 value of 31.44 μg/ml. Amph B showed high toxicity on murine macrophages, with a CC 50 of 8.75 μg/mL (Fig. 1; Table 2). Effects of CNSL and CN against infection of macrophages by L. infantum and Selectivity Index (SI) In evaluating the activity of CNSL and CN against the intracellular amastigote form, they presented IC 50 of 4.63 and 1.42 μg/ml, respectively. Amph B presented IC 50 of 0.68 μg/ml on amastigotes internalized in macrophages (Table 2). CNSL and CN were able to reduce the percentage of macrophages parasitized by L. infantum (Fig. 2). The negative control obtained approximately 87% of parasitized cells, while concentrations of 0.5; 1.0 and 2.0 μg/mL of Amph B reduced this number by about 73, 70 and 50%, respectively. The reduction of parasitized cells treated with CNSL and CN was dependent on the concentration used. Cells treated with CNSL showed parasitism around 79, 77 and 64%, and with CN the parasitism was approximately 78, 79 and 71%, after being treated at concentrations corresponding to 3.125; 6.25 and 12.5 μg/ml, respectively (Fig. 3a). Analyzing the survival index, the control obtained an average of 9.0 amastigotes/macrophage. Amph B at concentrations of 0.5; 1.0 and 2.0 μg/mL reduced this amount of parasites to approximately 4.8; 3.9 and 2.1 amastigotes/macrophage, respectively. In the treatment with CNSL, the amount of amastigotes reduced, depending on the concentration, to 5.2 amastigotes/macrophages at a concentration of 3.125 μg/mL; at the concentration of 6.25 μg/mL, this amount decreased to approximately 3.9 amastigotes/macrophages; when treated with 12.5 μg/mL, the survival index was reduced to 2.3 amastigotes/macrophages. For CN treatment, the amount of amastigotes/macrophage corresponding to concentrations of 3.125; 6.25 and 12.5 µg/ml was 3.4; 3.1 and 2.3 amastigotes/macrophage, respectively (Fig. 3b). Cytotoxicity in murine peritoneal macrophages and activity against the intracellular amastigote form of L. infantum were used to determine the selectivity index (SI), whose value represents how much the substance is more toxic to the parasite than to macrophages. CNSL, CN and Anf B showed more selectivity for L. infantum forms and promastigotes than for murine macrophages. The results obtained indicate that CNSL is approximately 8.1 times more selective for protozoa than for mammalian cells, while CN showed 22 times greater selectivity for L. infantum than for macrophages, this value being higher than that presented by Amph B, which proved to be 12.86 times more selective for the parasite (Table 2). Table 2 Cytotoxic effect on mammalian cells and calculated selectivity index values for CNSL, cardanol (CN) and amphotericin B (Amph B). Compounds Macrophages Intramacrophagic amastigotes ( L. infantum) SI m c L. infantum CC 50 µg/mL a IC 50 µg/mL b CNSL 37,51±0,04 4,63 8,10 CN 31,44±0,04 1,42 22,14 Amph B 8,75±0,02 d 0,68 12,86 a Cytotoxic concentration 50 b Inhibition concentration 50 c Selectivity index for amastigotes internalized in macrophages (CC 50 /IC 50 ) d Alves et al. (2017) Determination of lysossomal activity and phagocytic capability The results regarding macrophage activation parameters, such as lysosomal volume increase and phagocytosis were evaluated based on the retention of neutral red and Zymozan particles by macrophages. CN was able to significantly increase the lysosomal volume of macrophages at concentrations of 3.125 μg/mL, 6.25 and 12.5 μg/mL, while in CNSL there was no retention of neutral red in the secretory vesicles of macrophages (Fig. 4a). In evaluating the phagocytosis capacity of Zymozan, the tested substances significantly induced an increase in phagocytic capacity, this induction being at concentrations of 6.25 and 12.5 μg/mL for CNSL and 12.5 μg/mL for CN (Fig. 4b). Measurement of nitrite production The production of nitric oxide (NO), a macrophage activation index, was quantified by measuring nitrite concentrations by incubating macrophages with CNSL and CN in the absence and presence of promastigote forms of L. infantum . As a result, none of the substances demonstrated a significant increase in nitrite synthesis in the absence (Fig. 5a) and presence (Fig. 5b) of Leishmania at all concentrations tested. The bacterial lipopolysaccharide from Escherichia coli (LPS) was used as a positive control, demonstrating a high capacity in inducing nitrite synthesis. Acute toxicity on Z. morio larvae The survival profiles of Z. morio larvae when subjected to contact with CNSL and CN can be seen in Fig. 6, demonstrating concentration-dependent action. The results showed, after 48 h, that the larvae survival rates against CNSL were approximately 85%, 75% and 60% at doses of 3, 30 and 300 mg/kg, respectively (Fig. 6a), while the survival rates against to CN were approximately 85%, 60% and 40% at doses of 3, 30 and 300 mg/kg, respectively (Fig. 6b). After the toxicity test, it was found that there is a statistically significant difference (p < 0.05) between the CN survival curves, demonstrating a significant acute toxicity of this substance. Discussion CNSL is a natural source of phenolic compounds, which have a long aliphatic chain of fifteen carbons, in the meta position in relation to the hydroxyl, which can be saturated (C 15 H 31 ) and/or unsaturated with one (C 15 H 29 ), two (C 15 H 27 ) and three (C 15 H 25 ) unsaturations. CNSL is classified as natural (extracted by solvent), consisting of anacardic acid (60–65%), cardol (15–20%), cardanol (10%), and traces of methyl cardol, and technical (submitted to high temperatures), composed mainly of cardanol (60–65%), cardol (15–20%), polymeric material (10%), and traces of methyl cardol (Mele and Vasapollo 2008 ). CN is the main constituent of technical CNSL, and its derivatives have demonstrated antibacterial, antioxidant, antifungal and antitumor activities, in addition to hydrophobicity (Amorati et al. 2001 ; Chen et al. 1998 ; Mazzetto et al. 2009 ). CNSL and CN showed significant activity against promastigote forms of L. infantum , L. braziliensis and L. major after 48 hours of incubation. Although pharmacological data regarding the antileishmania activity of CNSL and its main constituents are scarce, other parts of A. occidentale have demonstrated antileishmania activity, corroborating our study. Results from França et al. ( 1993 ), showed in vitro activity of the hydroaucolic extract of the stem bark (at concentrations of 7.5 and 15 mg/mL) of A. occidentale on promastigote forms of L. braziliensis . In a study using ethanol extract from the leaves of A. occidentale , activity on promastigotes and amastigotes of L. amazonensis was demonstrated, showing growth inhibition of 5.4% and 32.3%, respectively, both at a concentration of 100 µg / mL (Luize et al. 2005 ). This study showed better results than those by Braga et al. ( 2007 ) using the stem bark extract of A. occidentale , where no activity against promastigote forms of L. amazonenses and L. infantum was found, resulting in IC 50 values ​​>200 µg/mL for both. In a study with compounds isolated from the leaves of Schinus terebinthifolius , a medicinal plant native to South America belonging to the Anacardiaceae family, rich in phenolic compounds, it showed efficacy against L. infantum promastigotes, with an IC 50 value of 57.82 µg/mL (Moraes et al. 2014), a result similar to the CN growth inhibition value on promastigotes of the same Leishmania species demonstrated in the present study. This study showed better results when compared to those of Dibyendu and Chakraborti (2014), in which the pentavalent antimonials, pentamidine and paramomycin, used for the treatment of CL and VL, showed a lack of response against promastigote forms of Leishmania . The performance of in vitro tests, through cell viability assays, is the first step to assess the biological compatibility of a given substance, providing important data on the analysis of biocompatibility between different materials (Rogero et al. 2003 ). For natural products to be used as alternative therapies in the treatment of leishmaniasis, cytotoxicity tests in mammalian cells are needed (Brenzan et al. 2007 ), being essential in macrophages, as they are part of the life cycle of Leishmania in the vertebrate host, seen that the differentiation of promastigote forms into amastigotes and subsequent multiplication occurs within these cells (koutsoni et al. 2014; Liu and Uzonna, 2012 ). Since CNSL and CN presented significant IC 50 values ​​on promastigote forms of the three Leishmania species , it was necessary to investigate the cytotoxicity of these compounds on macrophages. Both showed significant toxicity on these cells, with CNSL and CN showing similar results between them, but less toxic when compared to Amph B, used as a positive control. Despite their significant cytotoxicity, when the selectivity index was determined based on the ratio of CC 50 over IC 50 in intracellular amastigotes, it was observed that CNSL and CN were more selective for the parasite, with the CN selectivity index value being above 20, corroborating the literature data, where this index must present a value close to or greater than 20 for amastigotes internalized in macrophages (Osorio et al. 2007 ). In a study by Mesquita et al. ( 2014 ), miltefosine, used as a standard drug, had a CC 50 of 49.72 µg/mL, a result close to the values ​​of the substances tested in this study, and presented selectivity index with a value of 7 on L. infantum , a value similar to that of the CNSL, however much lower than the selectivity index of CN. This substance was also more selective for the parasite than the Amph B used as a positive control in this study. The experimental model of amastigotes internalized in macrophages is the one that best represents the way in which the infection occurs in the host (Carneiro et al. 2012 ). Thus, substances that are able to reduce the percentage of parasitized macrophages and the survival rate of amastigotes in macrophages are considerably promising to be tested in vivo (Alves et al. 2017 ). When tested against intracellular amastigotes, there was a significant reduction both in the IC 50 values ​​of CNSL and CN, in the percentage of murine macrophages experimentally infected by L. infantum and in the survival rate of amastigotes inside the macrophages, at the three concentrations tested of the compounds. This action was concentration-dependent, with better results being observed at a concentration of 12.5 µg/mL for CNSL and CN compared to Amph B. Lower results were found by Moraes et al. (2014), who tested three natural derivatives of Schinus terebinthifolius (Anacardiaceae) leaves, and found IC 50 values ​​of 66.59 µg/Ml, 64.90 µg/mL and 28.95 µg/mL, respectively, against internalized amastigotes L. infantum . Assessing the images taken by microscopy, it is observed that in the control group there is a large concentration of amastigotes around the parasitophorous vacuole, while in the groups treated with CNSL and CN there was a decrease in the agglomeration of parasites, demonstrating the reduction of the parasite load inside murine macrophages, which reinforces the antileishmania potential of these substances. This effect can be attributed to a possible activation of macrophages, which may also have promoted oxygen potentiation and an increase in cytokine regulation by them (Reimão et al. 2010 ). The activity of the compounds against promastigote and amastigote forms may differ, depending on the sites of antileishmania action, which contributes to their being selective for one of the two forms of development. The susceptibility of both evolutionary forms to the compound can be explained by the different biochemical characteristics between them, as well as by the chemical profile of the substance, such as its solubility in lipids (Athayde-Filho et al. 2000 ). The better antileishmania activity of CN when compared to CNSL can probably be due to the large amount of compounds present in it, which can have synergistic or antagonistic effects. With regard to the cellular immune response against Leishmania infections, the Th1 type is desirable, which provides cure or protection, such as increased phagocytic capacity, lysosomal volume, nitric oxide synthesis, among others, through mechanisms of activation of macrophages. In the investigation of new therapeutic alternatives for the treatment of leishmaniasis, drugs that have, in addition to activity on the parasite, immunomodulatory capacity, in order to prevail the Th1 host immune response (Islamuddin et al. 2015 ; Roy et al. 2014 ). Therefore, the activation parameters of macrophages with microbicidal capacity were evaluated. The results of the lysosomal activity and phagocytic capacity assays demonstrated that the treatment of macrophages with CNSL and CN obtained significant immunomodulation results. CN retained neutral red particles, characterized by a significant increase in lysosomal activity, which may suggest an increase in the defense of these cells. Zymosan induces stimulation of defense cells to produce a response, causing an increase in IFN production and phagocytic capacity (Wei et al. 2011 ), and this increase was demonstrated by the substances tested. These results corroborate the study carried out by Alves et al. ( 2017 ), in which gallic and ellagic acids, naturally occurring phenolic compounds found in some plants, including A. occidentale , showed immunomodulation results in the determination of phagocytic capacity and lysosomal activity at concentrations of 6.25 µg/mL and 3.125 µg/mL, similar to those in the present study. Innate immunity plays an important role in infection control through mechanisms of action such as phagocytosis and lysosomal activity, which provide for antigen activation and pathogen elimination (Harrison et al. 2003 ). The phagosome formed shortly after pathogen endocytosis fuses, followed by fusion with lysosomes to produce a phagolysosome (Niedergang and Chavrier 2004 ). The phagolysosome is a structure filled with acid hydrolases and reactive oxygen species in which most of the degradation of the contents involved takes place, and finally, the phagocytosed pathogens are killed within the phagolysosome (Lee et al. 2003 ; Lopes et al. 2006 ). Thus, macrophage activation parameters lead to conformational changes to increase the performance of their functions, such as locomotion and phagocytosis (Petropolis et al. 2014 ). A primary resistance mechanism to Leishmania infection is the production of nitric oxide (NO) by infected macrophages (Lima-Júnior et al. 2013). Inside the phagolysosome, NO reacts with O2 − 2 , forming a reactive oxygen species, peroxynitrite. From there, nitrate and nitrite are formed as the final product, which act as microbicidal agents (Bogdan; Rollinghoff 1998 ; Ueda-Nakamura et al. 2006). In the present study, it was observed that CNSL and CN did not exhibit significant induction of NO synthesis, either in infected or uninfected macrophages. Likewise, treatment with Amph B in infected macrophages did not induce significant NO production. Similar results were found by Abas et al. ( 2006 ) testing A. occidentale leaf extract, where there was no significant NO release in murine macrophages. These data reinforce that the tested compounds use another pathway to enhance their leishmanicidal activity, not depending on NO synthesis, considering that the production of this molecule is not the only way to control intracellular Leishmania infection in macrophages (Singh et al. al. 2012). As there was no significant induction of NO production, it is possible that the reduction in infected macrophages and the survival rate is due to the phagocytic capacity and lysosomal activity by macrophages stimulated by the substances. The use of Z. morio larvae for animal and human commercialization and consumption has increased in recent times, due to their nutritional composition, ease of handling and rearing and short life cycle (Han et al. 2010). In addition, given the ethical conflict and social aspects, we seek to increasingly prioritize the use of alternative models in experimental research, such as Z. morio , considering that in insects the innate immune system is evolutionarily conserved (Canteri et al. 2018 ). Due to these characteristics, interest in the use of this insect as an alternative model for in vivo studies to assess various activities such as antimicrobial (Morey et al. 2016 ), toxicity (Van Der Valk and Meijden, 2014 ) and insecticide (Wang) has increased et al. 2015) and pathogenicity (McGonigle et al. 2016 ). The main objective of toxicological studies is to predict the possible adverse effects that a substance may cause when exposed to human or animal, whether it is a candidate for a drug, pesticide, industrial chemical agent, among others (Koeter 1993 ; Stokes 2002 ; Meyer 2003). In this context, the in vivo test was carried out using Z. morio as a model organism, and the survival profiles in the larval forms of the insect were determined, in order to assess the acute toxicity of CNSL and CN. As in the cytotoxicity test on macrophages, CNSL had lower toxicity on Z. morio than CN, suggesting a synergistic effect of the compounds present in the CNSL, giving it less toxicity when compared to CN alone. These results corroborate some studies that showed that the isolated pure substances were more toxic than their source fractions (Silva et al. 2007 ; Simas et al. 2004 ). Similarly, Guissoni et al. ( 2013 ) demonstrated that CNSL was less toxic, with a higher mean lethal concentration (LC 50 ) than its fractions, in larvicidal activity against Aedes aegypti . Assessing the acute oral toxicity of CNSL in Rattus norvegicus , LD 50 > 2000 mg/kg was demonstrated, not causing any significant signs of intoxication in animals (Guissoni et al. 2013 ). According to the classification of the Organization for Economic Co-operation and Development (OECD, 2002), this value allows classifying the CNSL in class 5 (LD 50 2000–5000 mg/Kg), indicating a substance with low toxicity. Also according to the OECD, CN presents non-toxicity as one of its main characteristics. Tests carried out by this organization showed the following results regarding BC ecotoxicity: biodegradability − 96% (28 days) - (OECD-302C, 2009); solubility in water equal to 1.0 g/L; ecotoxicity (96 h): fish < 11 g/L; daphnia < 66 g/L; algae < 1 g/L - (OECD-425, 2008) and regarding genotoxicity with using the Ames Salmonella test, it was negative. According to the Safety Data Sheets (SDS, 2017), the oral LD 50 is 500 mg/kg (rats) and the dermal LD 50 is > 2,000 mg/kg (Daphnia magna). The non-toxicity of CN reported in the literature differs from the result of CC50 and acute toxicity for this substance in the present study, however, according to the OECD, this toxicity, even if low, can be improved with encapsulation and controlled release for use as a biopharmaceutical (OECD, 2002). In conclusion, CNSL and CN were able to show antileishmania potential against L. infantum , L. braziliensis and L. major , acting also through macrophage activation pathways, such as increased phagocytic capacity, increased lysosomal volume. CNSL and CN were able to reduce the percentage of murine macrophages infected by L. infantum and the survival rate of internalized amastigote forms. It was shown that Z. morio larvae are an alternative invertebrate model suitable for analyzing the acute toxicity of the tested substances. The results are promising and serve as a starting point for further research aimed at evaluating the in vivo leishmanicidal potential. Declarations Ethics approval and consent to participate All protocols were approved by the Animal Ethics Committee from Federal University of Piaui, Brazil (nº 640/2019). Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. Funding Author IMMR. has received research support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (process number 88887.489421/2020-00), as a Master's scholarship. Author Contributions Material preparation, collection and data analysis were carried out by IMMR, VCS, RCVC, MSS, JAONN, DSM, MGLC, AKSM, FAAC, MMMA and ILM. The graphics were created by VCS, MSS and MMMA, and the tables were made by IMMR. The translation of the manuscript into English was carried out by IMMR and LSAT. 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Mini Rev Med Chem 18(1): 26–41 https://doi.org/10.2174/1389557517666170425105129 Ueda-Nakamura T, Mendonça-Filho RR, Morgado-Díaz JA et al (2010) Anti-Leishmanial activity of Eugenol-rich essential oil from Ocimum gratissimum . Parasitol Int 55: 99–105. https://doi.org/10.1016/j.parint.2005.10.006 Valadares DG, Duarte MC, Oliveira JS et al (2011) Leishmanicidal activity of the Agaricus blazei Murill in different Leishmania species. Parasitol Int 60(4): 357–63. https://doi.org/10.1016/j.parint.2011.06.001 Van Der Valk T, Van Der Meijden A (2014) Toxicity of scorpion venom in chick embryo and mealworm assay depending on the use of the soluble fraction versus the whole venom. Toxicon 88: 38–43. https://doi.org/10.1016/j.toxicon.2014.06.007 Wang X, Hao Q, Chen Y, Jiang S, Yang Q, Li Q (2015) The effect of chemical composition and bioactivity of several essential oils on Tenebrio molitor (Coleoptera: Tenebrionidae). J Insect Sci 15 (1): 116. https://doi.org/10.1093/jisesa/iev093 Wei WC, Su YH, Chen SS, Sheu JH, Yang NS (2011) GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells. Cytokine 55: 79–89. https://doi.org/10.1016/j.cyto.2011.03.017 WHO - World Health Organization (2021) Leishmaniasis. https://www.who.int/en/news-room/fact-sheets/detail/Leishmaniasis . Accessed June 15, 2021. Wu K, Liu J, Tseng SF et al (2011) Anacardic Acid (6-Pentadecylsalicylic Acid) Inhibits Tumor Angiogenesis by Targeting Src/FAK/Rho GTPases Signaling Pathway. J Pharmacol Exp. Ther 339(2): 403–411. https://doi.org/10.1124/jpet.111.181891 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1588156","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":101979515,"identity":"6592efc8-1b47-464e-85cb-8b997c4bca36","order_by":0,"name":"Iuliana Marjory Martins Ribeiro","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Iuliana","middleName":"Marjory Martins","lastName":"Ribeiro","suffix":""},{"id":101979516,"identity":"2df54b1a-6ef5-488e-b0f5-b548e0e9a852","order_by":1,"name":"Valéria Carlos de Sousa","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Valéria","middleName":"Carlos","lastName":"de Sousa","suffix":""},{"id":101979517,"identity":"b9540502-e261-47b8-9d1c-098dfafc1aea","order_by":2,"name":"Rita de Cássia Viana de Carvalho","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Rita","middleName":"de Cássia Viana","lastName":"de Carvalho","suffix":""},{"id":101979519,"identity":"4a4fd196-4dd9-4731-8573-9ac95f842c6d","order_by":3,"name":"Maisa de Sousa dos Santos","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Maisa","middleName":"de Sousa dos","lastName":"Santos","suffix":""},{"id":101979520,"identity":"64bd7fd3-86bc-4838-91a9-2a6ed50a6c88","order_by":4,"name":"José Arimatéia de Oliveira Nery Neto","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Arimatéia de Oliveira Nery","lastName":"Neto","suffix":""},{"id":101979521,"identity":"392cca05-e089-457a-b24d-5e345672b4bb","order_by":5,"name":"Danielly Silva de Melo","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Danielly","middleName":"Silva","lastName":"de Melo","suffix":""},{"id":101979522,"identity":"bee6e06c-c568-463b-9942-9b00bd6d7462","order_by":6,"name":"Letícia Soares de Araújo Teixeira","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Letícia","middleName":"Soares de Araújo","lastName":"Teixeira","suffix":""},{"id":101979524,"identity":"cfe2b64a-7a48-40ba-9cb1-a91463a63c8e","order_by":7,"name":"Maria das Graças Lopes Citó","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"das Graças Lopes","lastName":"Citó","suffix":""},{"id":101979528,"identity":"d68535bc-bab4-49ae-92d1-777010b666ca","order_by":8,"name":"Arkellau Kenned Silva Moura","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Arkellau","middleName":"Kenned Silva","lastName":"Moura","suffix":""},{"id":101979529,"identity":"39644291-63cb-4727-a901-8a6987f14496","order_by":9,"name":"Fernando Aécio de Amorim Carvalho","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"Aécio de Amorim","lastName":"Carvalho","suffix":""},{"id":101979530,"identity":"a9982686-df3b-4276-ae84-79d798dac626","order_by":10,"name":"Michel Muálem Moraes Alves","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYHACxgNAgodBAsj4wCDHwAbkSTAwHMCrB6aF4eAMBmPitYCVHeYBamEgpEV3RvKDAx9ztsnwz24+cNi2zSCPj4H54G0ehjv5uLSY3UgzODhz220eiTvHEg7nthkUszGwJVvzMDyzbMCpJcHgMC9Qi4FEjgFQy5/ENgYeM2kehsMGuG1J/3D4L1hL/ofDlm0GQC383whoARrOCLGF4TAjWAsPG34tZ94UHOwF+QXkqZ5zQL8wsxlbzjF4hlvL8fSND35uu23PPyP54YMfZQZ58u3ND2+8qbiDUwsGSGBgBlHEawBpGQWjYBSMglGABgCKEVwXJluI0AAAAABJRU5ErkJggg==","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":true,"prefix":"","firstName":"Michel","middleName":"Muálem Moraes","lastName":"Alves","suffix":""},{"id":101979531,"identity":"35cd6477-6d0a-49be-87ab-4ed2152ac2bd","order_by":11,"name":"Ivete Lopes de Mendonça","email":"","orcid":"","institution":"Universidade Federal do Piauí","correspondingAuthor":false,"prefix":"","firstName":"Ivete","middleName":"Lopes","lastName":"de Mendonça","suffix":""}],"badges":[],"createdAt":"2022-04-23 18:14:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1588156/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1588156/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20992862,"identity":"cd7b9362-b6d4-4e9a-bb04-ba23bcfa78dd","added_by":"auto","created_at":"2022-05-02 17:38:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43811,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effects of CNSL (a) and CN (b) against BALB/c murine peritoneal macrophages. Macrophages and red blood cells were incubated with CNSL, CN, or Amph B for 48 h. The macrophage viability was evaluated using tetrazolium salt (MTT) test. Data are presented as mean ± SEM of three experiments performed in triplicate *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001 when compared with control (C) or Amph B\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig01.png","url":"https://assets-eu.researchsquare.com/files/rs-1588156/v1/41981d4f178493e1c3e435b2.png"},{"id":20992867,"identity":"7f3a0af4-b58a-4e09-8511-260b911ee0ee","added_by":"auto","created_at":"2022-05-02 17:38:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":480808,"visible":true,"origin":"","legend":"\u003cp\u003eMacrophages experimentally infected with \u003cem\u003eLeishmania infantum\u003c/em\u003e. Control (a). Amphotericin B was used as a positive control at a concentration of 0.5 (b); 1.0 (c) and 2.0 (d) µg/ml. For the treatment with CNSL, concentrations of 3.125 (e) were used; 6.25 (f) and 12.5 (g) µg/ml. CN was used at concentrations of 3.125 (h); 6.25 (i) and 12.5 (j) µg/ml. The arrows indicate macrophage-internalized amastigote forms of \u003cem\u003eL. infantum\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig02.png","url":"https://assets-eu.researchsquare.com/files/rs-1588156/v1/d6d5119e51f4408ea1e6b396.png"},{"id":20993168,"identity":"8cff4ce0-0166-460a-9d6f-f224f8ce8404","added_by":"auto","created_at":"2022-05-02 17:43:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71928,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CNSL, CN and amphotericin B on infected macrophages (a) and survival index (b) of BALB/c murine macrophages infected with \u003cem\u003eLeishmania infantum\u003c/em\u003e, considering the 87% infection control. Cells were treated with CNSL, CN or Amph B for 48h. Data are presented as mean ± SEM of three experiments performed in triplicate. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001 when compared with control (C)\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig03.png","url":"https://assets-eu.researchsquare.com/files/rs-1588156/v1/15334d292e84ddcc9f271d64.png"},{"id":20993362,"identity":"f0e31bab-42d8-4a12-a345-8365777610dd","added_by":"auto","created_at":"2022-05-02 17:48:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57889,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CNSL, CN, and Amph B on lysosomal activity (a) and phagocytic capability (b). Murine peritoneal macrophages were treated at ranging concentrations for 48h. Data are presented as mean ± SEM of three experiments performed in triplicate. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001 when compared with control (C)\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig04.png","url":"https://assets-eu.researchsquare.com/files/rs-1588156/v1/74386ddf1d03a5481f322317.png"},{"id":20993169,"identity":"f7eed4f6-ca8b-4b65-86bc-b338036f1cb4","added_by":"auto","created_at":"2022-05-02 17:43:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":65805,"visible":true,"origin":"","legend":"\u003cp\u003eNitrite measurement in non-infected (a) or infected (b) BALB/c murine peritoneal macrophages treated with CNSL or CN and Amph B for 24 h. The culture supernatant was mixed in equal parts with the Griess reagent. LPS (lipopolysaccharide from \u003cem\u003eEscherichia coli\u003c/em\u003e; 2 μg/mL) was used as positive control. Data are presented as mean ± SEM of three experiments performed in triplicate. p \u0026lt; 0.05 when compared with non-infected (−) or infected (+) macrophages from control group; p \u0026lt; 0.05 when compared with non-infected (−) or infected (+) macrophages from the LPS group HNS71387\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig05.png","url":"https://assets-eu.researchsquare.com/files/rs-1588156/v1/671d3b0b501ce57c32a77081.png"},{"id":20992864,"identity":"570c5e89-b2c4-43f1-ae45-54d048653902","added_by":"auto","created_at":"2022-05-02 17:38:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":34412,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of survival of \u003cem\u003eZ. morio\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003elarvae exposed to CNSL (a) and CN (b). Larvae were incubated for 48 h in the presence of different concentrations. 0.2% DMSO was used as a control and 100% DMSO was used to cause 100% larval mortality. Results represent mean ± S.E.M of treatments performed in triplicate.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig06.png","url":"https://assets-eu.researchsquare.com/files/rs-1588156/v1/702f72fa017b8c7c5e46a973.png"},{"id":20993363,"identity":"4c208416-2a00-48a3-87f7-e646bcc4144c","added_by":"auto","created_at":"2022-05-02 17:48:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1232557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1588156/v1/7009c558-e890-4247-be33-f504b511be95.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antileishmania and immunomodulatory potential of cashew nut shell liquid and cardanol","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLeishmaniasis is a neglected disease caused by protozoa of the \u003cem\u003eLeishmania\u003c/em\u003e genus, constituting a major public health problem due to its high incidence and lethality (Alvar et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Benitez et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the year 2020, leishmaniasis was endemic in 98 of the 200 countries and territories that reported it to the WHO, and it is estimated that between 700,000 to 1\u0026nbsp;million new cases of cutaneous leishmaniasis and 50,000 to 90,000 new cases of visceral leishmaniasis occur worldwide each year (WHO 2021).\u003c/p\u003e \u003cp\u003eThe etiological agent of the disease is more than 20 species of the genus \u003cem\u003eLeishmania\u003c/em\u003e, classified in the subgenus \u003cem\u003eViannia\u003c/em\u003e and \u003cem\u003eLeishmania\u003c/em\u003e, transmitted by approximately 70 different species of sandflies of the genera \u003cem\u003eLutzomyia\u003c/em\u003e and \u003cem\u003ePhlebotomus\u003c/em\u003e. The disease exhibits a variety of clinical manifestations, the two main ones being visceral leishmaniasis (VL) and tegumentary leishmaniasis (TL), and this, in turn, is divided into four subtypes: localized cutaneous leishmaniasis (LCL), diffuse cutaneous leishmaniasis (DCL), mucocutaneous leishmaniasis (MCL) and disseminated leishmaniasis (DL) (Akhoundi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Akhoundi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe treatment of leishmaniasis is performed with pentavalent antimonials, amphotericin B, paromomycin, miltefosine, sodium stibogluconate, meglumine antimoniate and pentamidine (Blanco e Nascimento-J\u0026uacute;nior 2017; Kevric et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the use of these drugs requires long periods of administration, resulting in serious adverse effects, low tolerance and development of resistant strains to the treatment, contributing to the ineffectiveness of therapeutic regimens, in addition to presenting high costs (Bapela et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Souza-Silva et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe investigation of the pharmaceutical potential of natural products is the main strategy for discovering new drugs that can be less costly and less toxic than conventional drugs (Tiuman et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Plants are considered an important source of natural products that can be successfully exploited for the development of new drugs with antileishmania activity (Funari et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gonz\u0026aacute;lez-Coloma et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Machado et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mansour et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Torres et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Compounds such as alkaloids, phenolics, terpenoids and flavonoids have been extensively studied for their potential antileishmania activity (Silva et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the widely used medicinal plants, cashew (\u003cem\u003eAnacardium occidentale\u003c/em\u003e L.) stands out and one of its main by-products, cashew nut shell liquid (CNSL), has been used for decades in traditional medicine in countries in South America, Africa and Asia (Ayyanar and Ignacimuthu \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Da Silva et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kudi et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCNSL is a product with little commercial value, but with high technological potential due to its phenolic constitution and its various biological characteristics, such as anti-inflammatory, antimicrobial, antioxidant, antitumor, larvicides and insecticides, exhibiting great therapeutic potential (Kubo et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1993a\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), constituting a natural source of phenolic compounds, such as anacardic acid, cardanol, cardol and 2-methylcardol (Mazzetto et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCNSL derivatives are also widely explored in isolation, mainly for their properties such as antibacterial, antioxidant, antifungal, antitumor, among others (Amorati et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Hemshekhar et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mazzetto et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Muroi e Kubo 1996). Cardanol (CN) is the main constituent of technical CNSL and is considered one of the most important and promising components. As it is a by-product of the nut industry, any improvement in the CN, whether in concentration and/or separation, is effectively characterized as a technological innovation (Mazzetto et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThus, the CNSL demonstrates wide utility for pharmacology, and therefore further studies of its isolated metabolites, their mechanisms of action, as well as toxicological aspects are needed (Ara\u0026uacute;jo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Given the above, considering the impact of leishmaniasis mainly in developing countries, the urgency for more effective compounds with fewer adverse effects, and the broad therapeutic potential of CNSL, is strategic to invest in the search for new pharmacological properties of this product and its metabolites, as a potential source of medication for the treatment of leishmaniasis. Thus, the objective of this study was to evaluate the immunomodulatory and antileishmania activities of the CNSL and its main constituent, the CN, in order to propose a search for a new therapeutic strategy.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003e\u003cstrong\u003eSubstances used\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchneider\u0026rsquo;s culture medium, DMEM\u0026reg; medium, FBS, MTT, resazurin, acridine orange dye, propidium iodide and the antibiotics Penicillin and Streptomycin from Sigma Chemical (Sigma-Aldrich Brazil). Amph B (90%), fast panoptic\u0026reg; was acquired from Crist\u0026aacute;lia (S\u0026atilde;o Paulo, SP). CNSL and CN were from the Organic Geochemistry Laboratory (LAGO/UFPI) and were diluted in DMSO at a concentration of 80 mg/mL for the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParasites and cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrains of \u003cem\u003eLeishmania infantum\u003c/em\u003e (MHOM / 5745), \u003cem\u003eLeishmania braziliensis\u003c/em\u003e (10CL566) and \u003cem\u003eLeishmania major\u003c/em\u003e (MHOM / IL / 80 / Friendlin) were obtained from the Medicinal Plants Research Center of Federal University of Piau\u0026iacute;. Parasites were grown in supplemented Schneider\u0026rsquo;s medium (10% heat-inactivated FBS, 100 U/mL penicillin, and 100 \u0026mu;g/mL streptomycin at 26 \u0026deg;C) (Carneiro et al. 2012; Valadares et al. 2011).\u003c/p\u003e\n\u003cp\u003eMurine macrophages were collected from the peritoneal cavities of male and female BALB/c mice (4\u0026ndash;5 weeks old; Medicinal Plants Research Center, UFPI, Brazil), and cultivated in RPMI 1640 medium (10% heat-inactivated FBS, 100 U/mL penicillin, and 100 \u0026mu;g/mL streptomycin at 37 \u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e). All protocols were approved by the Animal Use Ethics Committee (CEUA-PI n\u0026ordm; 640/2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvertebrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e Zophobas morio\u003c/em\u003e larvae used to assess acute toxicity came from the Physiology Laboratory of the Department of Veterinary Morphophysiology/UFPI. The larvae were kept in plastic organizer boxes measuring 60 x 40 x 80 cm with water and feed ad libidum, at an ambient temperature of 25\u0026deg;C \u0026plusmn; 2\u0026deg;C. Larvae weighing between 100 and 200 mg, light and uniform in color, without signs of melanization were selected for the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation of activity against promastigote forms of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eL. infantum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e, \u003cem\u003eL. braziliensis\u003c/em\u003e and\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eL. major\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePromastigote forms of \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eL. braziliensis\u003c/em\u003e and\u003cem\u003eL. major\u003c/em\u003e in the late log phase (1 \u0026times; 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003e\u003cem\u003eleishmania\u003c/em\u003e/100 \u0026mu;L of medium) were plated in 96-well culture plates containing supplemented Schneider\u0026rsquo;s medium. Then, CNSL and CN (6.25, 12.5, 25, 50, 100, 200; 400 and 800 \u0026mu;g/mL) were added, and the plates were incubated during 48h in a BOD (biochemical oxygen demand) incubator at temperature of 26 \u0026deg;C. Remaining 6h to the end of this period, 20 \u0026mu;L of resazurin (1 \u0026times; 10\u003csup\u003e\u0026minus;3\u003c/sup\u003e mol/L) was added. Afterwards, the absorbances were read in a BioTek microplate reader (model ELx800) at a wavelength of 550 nm. The results were expressed as inhibition of parasite growth (%) (Soares et al. 2007; Valadares et al. 2011).\u003c/p\u003e\n\u003cp\u003eThe negative control was the Schneider\u0026rsquo;s medium with promastigotes (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) and for the positive control, amphotericin B (2 \u0026mu;g/mL). The cell viability was considered as 100% for the parasite. The blank was read for each concentration and control in order to avoid interference of absorbance of medium of other compounds (Soares et al. 2007; Valadares et al. 2011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of cytotoxicity in macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytotoxicity evaluation was carried out in 96-well plates using the MTT assay. Macrophages (2 \u0026times; 105 per well) were incubated in 100 \u0026mu;L of supplemented RPMI 1640 medium at 37 \u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 4h. Non-adherent cells were removed by washing with RPMI 1640 medium. Then, CNSL and CN were diluted in supplemented RPMI 1640 medium, and added at concentrations of 6.25, 12.5, 25, 50, 100, 200; 400 and 800 \u0026mu;g/mL, followed by incubation at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 2 days. The cytotoxicity of Amph B was assessed at concentration of 0.2 \u0026mu;g/mL. Afterwards, cytotoxicity was assessed by adding MTT (5 mg/mL). The supernatant was discarded, and the formazan crystals were dissolved by addition of 100 \u0026mu;L of DMSO. Finally, absorbance at 550 nm was measured using a BioTek (ELx800) plate reader (Oliveira et al. 2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation of CNSL- and CN-induced activity on macrophages infected by \u003cem\u003eLeishmania infantum\u0026nbsp;\u003c/em\u003eand the calculation of selective index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs \u003cem\u003eL. infantum\u003c/em\u003e is a species that causes zoonotic leishmaniasis and that develops the visceral form of the disease, considered the most severe form (Steverding 2017), the activity of CNSL and CN on \u003cem\u003eL. infantum\u003c/em\u003e promastigotes was evaluated. Macrophages (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL) were harvested in 24-well plates containing sterile round coverslips at 13mm and supplemented RPMI 1640 medium (10% inactivated FBS, 100 U/mL penicillin, and 100 \u0026mu;g/mL streptomycin). Culture plates were incubated at 37 \u0026deg;C and 5% of CO\u003csub\u003e2\u003c/sub\u003e for 3h. Adhered macrophages were then incubated with a new medium containing axenic amastigotes at a ratio of 10 amastigotes per 1 macrophage at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37 \u0026deg;C for 4h. The medium was subsequently aspirated in order to remove non-internalized parasites, and the wells were washed with 0.01 M phosphate buffered saline (PBS). The infected macrophages were then incubated with CNSL and CN at 3.125; 6.25 and 12.5 \u0026mu;g/mL (non-toxic concentrations on host cells) or Amph B at 0,5; 1 and 2 \u0026mu;g/mL. After this period, the coverslips were removed and stained with Panoptic staining kit. For each treatment, the number of infected macrophages and the parasite load (survival index, obtained by counting the number of parasites in 100 macrophages) were counted using optical microscopy (Carneiro et al. 2012).\u003c/p\u003e\n\u003cp\u003eFurthermore, the selectivity indexes of CNSL, CN, and Amph B were determined by the ratio of the mean CC\u003csub\u003e50\u003c/sub\u003e against macrophages to the mean IC\u003csub\u003e50\u003c/sub\u003e against macrophage-internalized amastigote forms of \u003cem\u003eL. infantum\u0026nbsp;\u003c/em\u003e(Oliveira et al. 2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of parameters related to macrophage activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLysosomal activity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMacrophages (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/well) were incubated with CNSL and CN (6.25; 12.5; 25; 50 e 100\u0026nbsp;\u0026mu;g/mL), or Amph B (0.2 \u0026mu;g/mL) in a 96-well plate at 37 \u0026deg;C and 5% de CO\u003csub\u003e2\u003c/sub\u003e. After 48h, 10 \u0026mu;L of neutral red stock solution were added for 30 min. Then, the supernatant was discarded, the wells were washed with 0.9% saline at 37 \u0026deg;C, and 100 \u0026mu;L of extractive solution were added in order to solubilize the neutral red present within the lysosomal secretory vesicles. After 30 min on a Kline shaker, the absorbances were read in a BioTek (ELx800) plate reader at 550 nm (Bonatto et al. 2004).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhagocytic capability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMacrophages (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/well) were incubated with CNSL and CN (6.25; 12.5; 25; 50 e 100\u0026nbsp;\u0026mu;g/mL), or Amph B (0.2 \u0026mu;g/mL) in a 96-well plate for 48 h at 37 \u0026deg;C and 5% de CO\u003csub\u003e2\u003c/sub\u003e. After 48h, 10 \u0026mu;L of zymosan-stained NR solution was added for 30 min. Next, the phagocytic process was interrupted adding 100 \u0026mu;L of Baker\u0026rsquo;s fixative solution during 30 min. Then, the wells were washed with 0.9% saline, and 100 \u0026mu;L of extractive solution were added. After solubilization in a Kline shaker, the absorbances were read in a BioTek (ELx800) plate reader at 550 nm (Grando et al. 2009).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNitrite measurement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNon-infected macrophages or infected by \u003cem\u003eL. infantum\u003c/em\u003e were obtained as described previously, and then incubated with CNSL and CN (3.125; 6.25 e 12.5 \u0026mu;g/mL), or Amph B (0.2 \u0026mu;g/mL) at 37 \u0026deg;C and 5% de CO\u003csub\u003e2\u003c/sub\u003e for 24h. The lipopolysaccharide (LPS) from \u003cem\u003eE. coli\u003c/em\u003e (2 \u0026mu;g/mL) was used as positive control. The standard curve was prepared with sodium nitrite in RPMI medium at varying concentrations of 1, 5, 10, 25, 50, 75, 100, and 150 \u0026mu;M diluted in RPMI 1640 medium. After 24h, the supernatants were transferred, and then incubated with equal parts of Griess reagent. Thereafter, the absorbances were read in a BioTek (ELx800) plate reader at 550 nm (Soares et al. 2007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation of acute toxicity on \u003cem\u003eZ. morio\u0026nbsp;\u003c/em\u003elarvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess acute toxicity, 10 larvae of \u003cem\u003eZ. morio\u003c/em\u003ewere used for each concentration tested, in triplicate. CNSL and CN were applied to the larvae in a volume of 10 \u0026mu;L at doses of 3; 30 and 300 mg/kg with the aid of a Hamilton syringe (701 N, manometer 26, Capacity 100 \u0026mu;L) in the hemocoel, in the second or third sternite visible above the legs, in the ventral portion. Larvae were incubated at room temperature in petri dishes containing breeding diet and the number of dead larvae was evaluated after 48h. To establish the death of the larvae, a visual check of each individual was performed for the presence of myelination and/or response to physical stimuli after gentle touch (de Souza et al. 2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tests were performed in triplicate in three independent experiments. The mean inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) and the mean cytotoxic concentration (CC\u003csub\u003e50\u003c/sub\u003e) with confidence limits of 95% were determined by regression of probits using the software SPSS 13.0. The selectivity index was calculated as the ratio between CC\u003csub\u003e50\u003c/sub\u003e and IC\u003csub\u003e50\u003c/sub\u003e of internalized amastigotes. The survival curve of \u003cem\u003eZ. morio\u003c/em\u003e larvae was plotted by Kaplan-Meier analysis and the results were analyzed by the Log Rank test to assess the level of relationship between the substances regarding the acute toxicity parameter. Analysis of variance ANOVA followed by Bonferroni\u0026rsquo;s test was performed, taking the value of p \u0026lt; 0.05 as the maximum level of statistical significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAntileishmania activity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCNSL and CN demonstrated antileishmania potential, with their action dependent on concentration. It was possible to observe activity against all \u003cem\u003eLeishmania\u003c/em\u003e species tested, where at the concentration of 800 \u0026mu;g/mL there was 100% growth inhibition of the promastigote forms of \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eL. braziliensis\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e in the presence of CNSL and \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eL. braziliensis\u003c/em\u003e in the presence of CN. CN showed about 90% growth inhibition of promastigote forms of \u003cem\u003eL.\u003c/em\u003e \u003cem\u003emajor\u003c/em\u003e at a concentration of 800 \u0026mu;g/mL. Amphotericin B inhibited the growth of promastigote forms of \u003cem\u003eL. braziliensis\u003c/em\u003e by about 90% and by \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e by about 80%, both at a concentration of 2 \u0026mu;g/mL. The IC\u003csub\u003e50\u003c/sub\u003e values of CNSL and CN against \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eL. braziliensis\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e are shown in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eAntileishmania activity of CNSL, cardanol (CN) and amphotericin B (Amph B).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"26.631393298059965%\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.044091710758376%\"\u003e\n \u003cp\u003e\u003cem\u003eL. infantum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.044091710758376%\"\u003e\n \u003cp\u003e\u003cem\u003eL. braziliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003e\u003cem\u003eL. major\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.13461538461539%\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e\u0026micro;g/mL\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.13461538461539%\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e\u0026micro;g/mL\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.73076923076923%\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e\u0026micro;g/mL\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.631393298059965%\"\u003e\n \u003cp\u003eCNSL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.044091710758376%\"\u003e\n \u003cp\u003e148.12\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.044091710758376%\"\u003e\n \u003cp\u003e85.71\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003e153.56\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.631393298059965%\"\u003e\n \u003cp\u003eCN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.044091710758376%\"\u003e\n \u003cp\u003e56.74\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.044091710758376%\"\u003e\n \u003cp\u003e64.28\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003e122.31\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.631393298059965%\"\u003e\n \u003cp\u003eAmph B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.044091710758376%\"\u003e\n \u003cp\u003e2.46\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.044091710758376%\"\u003e\n \u003cp\u003e2.94\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.280423280423282%\"\u003e\n \u003cp\u003e0.59\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" width=\"100%\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Inhibition Concentration 50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eCitotoxicity assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen acting on macrophages, CNSL demonstrated significant cytotoxicity from a concentration of 12.5\u0026nbsp;\u0026mu;g/mL, with a CC50 value of 37.51\u0026nbsp;\u0026mu;g/mL, while CN significantly reduced macrophage viability from the concentration of 6.25\u0026nbsp;\u0026mu;g/mL, resulting in a CC\u003csub\u003e50\u003c/sub\u003e value of 31.44 \u0026mu;g/ml. Amph B showed high toxicity on murine macrophages, with a CC\u003csub\u003e50\u003c/sub\u003e of 8.75 \u0026mu;g/mL (Fig. 1; Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of CNSL and CN against infection of macrophages by \u003cem\u003eL. infantum\u0026nbsp;\u003c/em\u003eand Selectivity Index (SI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn evaluating the activity of CNSL and CN against the intracellular amastigote form, they presented IC\u003csub\u003e50\u003c/sub\u003e of 4.63 and 1.42 \u0026mu;g/ml, respectively. Amph B presented IC\u003csub\u003e50\u003c/sub\u003e of 0.68 \u0026mu;g/ml on amastigotes internalized in macrophages (Table 2). CNSL and CN were able to reduce the percentage of macrophages parasitized by \u003cem\u003eL. infantum\u003c/em\u003e (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe negative control obtained approximately 87% of parasitized cells, while concentrations of 0.5; 1.0 and 2.0 \u0026mu;g/mL of Amph B reduced this number by about 73, 70 and 50%, respectively. The reduction of parasitized cells treated with CNSL and CN was dependent on the concentration used. Cells treated with CNSL showed parasitism around 79, 77 and 64%, and with CN the parasitism was approximately 78, 79 and 71%, after being treated at concentrations corresponding to 3.125; 6.25 and 12.5 \u0026mu;g/ml, respectively (Fig. 3a).\u003c/p\u003e\n\u003cp\u003eAnalyzing the survival index, the control obtained an average of 9.0 amastigotes/macrophage. Amph B at concentrations of 0.5; 1.0 and 2.0 \u0026mu;g/mL reduced this amount of parasites to approximately 4.8; 3.9 and 2.1 amastigotes/macrophage, respectively. In the treatment with CNSL, the amount of amastigotes reduced, depending on the concentration, to 5.2 amastigotes/macrophages at a concentration of 3.125 \u0026mu;g/mL; at the concentration of 6.25 \u0026mu;g/mL, this amount decreased to approximately 3.9 amastigotes/macrophages; when treated with 12.5 \u0026mu;g/mL, the survival index was reduced to 2.3 amastigotes/macrophages. For CN treatment, the amount of amastigotes/macrophage corresponding to concentrations of 3.125; 6.25 and 12.5 \u0026micro;g/ml was 3.4; 3.1 and 2.3 amastigotes/macrophage, respectively (Fig. 3b).\u003c/p\u003e\n\u003cp\u003eCytotoxicity in murine peritoneal macrophages and activity against the intracellular amastigote form of \u003cem\u003eL. infantum\u003c/em\u003e were used to determine the selectivity index (SI), whose value represents how much the substance is more toxic to the parasite than to macrophages. CNSL, CN and Anf B showed more selectivity for \u003cem\u003eL. infantum\u003c/em\u003e forms and promastigotes than for murine macrophages. The results obtained indicate that CNSL is approximately 8.1 times more selective for protozoa than for mammalian cells, while CN showed 22 times greater selectivity for \u003cem\u003eL. infantum\u003c/em\u003e than for macrophages, this value being higher than that presented by Amph B, which proved to be 12.86 times more selective for the parasite (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Cytotoxic effect on mammalian cells and calculated selectivity index values for CNSL, cardanol (CN) and amphotericin B (Amph B).\u003c/p\u003e\n\u003ctable align=\"left\" border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"21.818181818181817%\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.611570247933884%\"\u003e\n \u003cp\u003eMacrophages\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.239669421487605%\"\u003e\n \u003cp\u003eIntramacrophagic amastigotes (\u003cem\u003eL. infantum)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"20.330578512396695%\"\u003e\n \u003cp\u003eSI\u003csub\u003em\u003c/sub\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eL. infantum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"46%\"\u003e\n \u003cp\u003eCC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e\u0026micro;g/mL\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54%\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e\u0026micro;g/mL\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.818181818181817%\"\u003e\n \u003cp\u003eCNSL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.611570247933884%\"\u003e\n \u003cp\u003e37,51\u0026plusmn;0,04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.239669421487605%\"\u003e\n \u003cp\u003e4,63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.330578512396695%\"\u003e\n \u003cp\u003e8,10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.818181818181817%\"\u003e\n \u003cp\u003eCN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.611570247933884%\"\u003e\n \u003cp\u003e31,44\u0026plusmn;0,04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.239669421487605%\"\u003e\n \u003cp\u003e1,42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.330578512396695%\"\u003e\n \u003cp\u003e22,14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.818181818181817%\"\u003e\n \u003cp\u003eAmph\u0026nbsp;B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.611570247933884%\"\u003e\n \u003cp\u003e8,75\u0026plusmn;0,02 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.239669421487605%\"\u003e\n \u003cp\u003e0,68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.330578512396695%\"\u003e\n \u003cp\u003e12,86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eCytotoxic concentration 50\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eInhibition concentration 50\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eSelectivity index for amastigotes internalized in macrophages (CC\u003csub\u003e50\u003c/sub\u003e/IC\u003csub\u003e50\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ed\u003c/sup\u003e Alves et al. (2017)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of lysossomal activity and phagocytic capability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results regarding macrophage activation parameters, such as lysosomal volume increase and phagocytosis were evaluated based on the retention of neutral red and \u003cem\u003eZymozan\u003c/em\u003e particles by macrophages.\u003c/p\u003e\n\u003cp\u003eCN was able to significantly increase the lysosomal volume of macrophages at concentrations of 3.125 \u0026mu;g/mL, 6.25 and 12.5 \u0026mu;g/mL, while in CNSL there was no retention of neutral red in the secretory vesicles of macrophages (Fig. 4a). In evaluating the phagocytosis capacity of Zymozan, the tested substances significantly induced an increase in phagocytic capacity, this induction being at concentrations of 6.25 and 12.5 \u0026mu;g/mL for CNSL and 12.5 \u0026mu;g/mL for CN (Fig. 4b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of nitrite production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe production of nitric oxide (NO), a macrophage activation index, was quantified by measuring nitrite concentrations by incubating macrophages with CNSL and CN in the absence and presence of promastigote forms of \u003cem\u003eL. infantum\u003c/em\u003e. As a result, none of the substances demonstrated a significant increase in nitrite synthesis in the absence (Fig. 5a) and presence (Fig. 5b) of \u003cem\u003eLeishmania\u003c/em\u003e at all concentrations tested. The bacterial lipopolysaccharide from \u003cem\u003eEscherichia coli\u003c/em\u003e (LPS) was used as a positive control, demonstrating a high capacity in inducing nitrite synthesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcute toxicity on \u003cem\u003eZ. morio\u0026nbsp;\u003c/em\u003elarvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival profiles of \u003cem\u003eZ. morio\u0026nbsp;\u003c/em\u003elarvae when subjected to contact with CNSL and CN can be seen in Fig. 6, demonstrating concentration-dependent action. The results showed, after 48 h, that the larvae survival rates against CNSL were approximately 85%, 75% and 60% at doses of 3, 30 and 300 mg/kg, respectively (Fig. 6a), while the survival rates against to CN were approximately 85%, 60% and 40% at doses of 3, 30 and 300 mg/kg, respectively (Fig. 6b). After the toxicity test, it was found that there is a statistically significant difference (p \u0026lt; 0.05) between the CN survival curves, demonstrating a significant acute toxicity of this substance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCNSL is a natural source of phenolic compounds, which have a long aliphatic chain of fifteen carbons, in the meta position in relation to the hydroxyl, which can be saturated (C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003e) and/or unsaturated with one (C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e29\u003c/sub\u003e), two (C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003e) and three (C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003e) unsaturations. CNSL is classified as natural (extracted by solvent), consisting of anacardic acid (60\u0026ndash;65%), cardol (15\u0026ndash;20%), cardanol (10%), and traces of methyl cardol, and technical (submitted to high temperatures), composed mainly of cardanol (60\u0026ndash;65%), cardol (15\u0026ndash;20%), polymeric material (10%), and traces of methyl cardol (Mele and Vasapollo \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). CN is the main constituent of technical CNSL, and its derivatives have demonstrated antibacterial, antioxidant, antifungal and antitumor activities, in addition to hydrophobicity (Amorati et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Mazzetto et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCNSL and CN showed significant activity against promastigote forms of \u003cem\u003eL. infantum\u003c/em\u003e, L. braziliensis and \u003cem\u003eL. major\u003c/em\u003e after 48 hours of incubation. Although pharmacological data regarding the antileishmania activity of CNSL and its main constituents are scarce, other parts of \u003cem\u003eA. occidentale\u003c/em\u003e have demonstrated antileishmania activity, corroborating our study. Results from Fran\u0026ccedil;a et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), showed in vitro activity of the hydroaucolic extract of the stem bark (at concentrations of 7.5 and 15 mg/mL) of \u003cem\u003eA. occidentale\u003c/em\u003e on promastigote forms of \u003cem\u003eL. braziliensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn a study using ethanol extract from the leaves of \u003cem\u003eA. occidentale\u003c/em\u003e, activity on promastigotes and amastigotes of \u003cem\u003eL. amazonensis\u003c/em\u003e was demonstrated, showing growth inhibition of 5.4% and 32.3%, respectively, both at a concentration of 100 \u0026micro;g / mL (Luize et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This study showed better results than those by Braga et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) using the stem bark extract of \u003cem\u003eA. occidentale\u003c/em\u003e, where no activity against promastigote forms of \u003cem\u003eL. amazonenses\u003c/em\u003e and \u003cem\u003eL. infantum\u003c/em\u003e was found, resulting in IC\u003csub\u003e50\u003c/sub\u003e values ​​\u0026gt;200 \u0026micro;g/mL for both.\u003c/p\u003e \u003cp\u003eIn a study with compounds isolated from the leaves of \u003cem\u003eSchinus terebinthifolius\u003c/em\u003e, a medicinal plant native to South America belonging to the Anacardiaceae family, rich in phenolic compounds, it showed efficacy against \u003cem\u003eL. infantum\u003c/em\u003e promastigotes, with an IC\u003csub\u003e50\u003c/sub\u003e value of 57.82 \u0026micro;g/mL (Moraes et al. 2014), a result similar to the CN growth inhibition value on promastigotes of the same \u003cem\u003eLeishmania\u003c/em\u003e species demonstrated in the present study. This study showed better results when compared to those of Dibyendu and Chakraborti (2014), in which the pentavalent antimonials, pentamidine and paramomycin, used for the treatment of CL and VL, showed a lack of response against promastigote forms of \u003cem\u003eLeishmania\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe performance of in vitro tests, through cell viability assays, is the first step to assess the biological compatibility of a given substance, providing important data on the analysis of biocompatibility between different materials (Rogero et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). For natural products to be used as alternative therapies in the treatment of leishmaniasis, cytotoxicity tests in mammalian cells are needed (Brenzan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), being essential in macrophages, as they are part of the life cycle of \u003cem\u003eLeishmania\u003c/em\u003e in the vertebrate host, seen that the differentiation of promastigote forms into amastigotes and subsequent multiplication occurs within these cells (koutsoni et al. 2014; Liu and Uzonna, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince CNSL and CN presented significant IC\u003csub\u003e50\u003c/sub\u003e values ​​on promastigote forms of the three \u003cem\u003eLeishmania species\u003c/em\u003e, it was necessary to investigate the cytotoxicity of these compounds on macrophages. Both showed significant toxicity on these cells, with CNSL and CN showing similar results between them, but less toxic when compared to Amph B, used as a positive control. Despite their significant cytotoxicity, when the selectivity index was determined based on the ratio of CC\u003csub\u003e50\u003c/sub\u003e over IC\u003csub\u003e50\u003c/sub\u003e in intracellular amastigotes, it was observed that CNSL and CN were more selective for the parasite, with the CN selectivity index value being above 20, corroborating the literature data, where this index must present a value close to or greater than 20 for amastigotes internalized in macrophages (Osorio et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a study by Mesquita et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), miltefosine, used as a standard drug, had a CC\u003csub\u003e50\u003c/sub\u003e of 49.72 \u0026micro;g/mL, a result close to the values ​​of the substances tested in this study, and presented selectivity index with a value of 7 on \u003cem\u003eL. infantum\u003c/em\u003e, a value similar to that of the CNSL, however much lower than the selectivity index of CN. This substance was also more selective for the parasite than the Amph B used as a positive control in this study.\u003c/p\u003e \u003cp\u003eThe experimental model of amastigotes internalized in macrophages is the one that best represents the way in which the infection occurs in the host (Carneiro et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thus, substances that are able to reduce the percentage of parasitized macrophages and the survival rate of amastigotes in macrophages are considerably promising to be tested \u003cem\u003ein vivo\u003c/em\u003e (Alves et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen tested against intracellular amastigotes, there was a significant reduction both in the IC\u003csub\u003e50\u003c/sub\u003e values ​​of CNSL and CN, in the percentage of murine macrophages experimentally infected by \u003cem\u003eL. infantum\u003c/em\u003e and in the survival rate of amastigotes inside the macrophages, at the three concentrations tested of the compounds. This action was concentration-dependent, with better results being observed at a concentration of 12.5 \u0026micro;g/mL for CNSL and CN compared to Amph B. Lower results were found by Moraes et al. (2014), who tested three natural derivatives of \u003cem\u003eSchinus terebinthifolius\u003c/em\u003e (Anacardiaceae) leaves, and found IC\u003csub\u003e50\u003c/sub\u003e values ​​of 66.59 \u0026micro;g/Ml, 64.90 \u0026micro;g/mL and 28.95 \u0026micro;g/mL, respectively, against internalized amastigotes \u003cem\u003eL. infantum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAssessing the images taken by microscopy, it is observed that in the control group there is a large concentration of amastigotes around the parasitophorous vacuole, while in the groups treated with CNSL and CN there was a decrease in the agglomeration of parasites, demonstrating the reduction of the parasite load inside murine macrophages, which reinforces the antileishmania potential of these substances. This effect can be attributed to a possible activation of macrophages, which may also have promoted oxygen potentiation and an increase in cytokine regulation by them (Reim\u0026atilde;o et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe activity of the compounds against promastigote and amastigote forms may differ, depending on the sites of antileishmania action, which contributes to their being selective for one of the two forms of development. The susceptibility of both evolutionary forms to the compound can be explained by the different biochemical characteristics between them, as well as by the chemical profile of the substance, such as its solubility in lipids (Athayde-Filho et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The better antileishmania activity of CN when compared to CNSL can probably be due to the large amount of compounds present in it, which can have synergistic or antagonistic effects.\u003c/p\u003e \u003cp\u003eWith regard to the cellular immune response against \u003cem\u003eLeishmania\u003c/em\u003e infections, the Th1 type is desirable, which provides cure or protection, such as increased phagocytic capacity, lysosomal volume, nitric oxide synthesis, among others, through mechanisms of activation of macrophages. In the investigation of new therapeutic alternatives for the treatment of leishmaniasis, drugs that have, in addition to activity on the parasite, immunomodulatory capacity, in order to prevail the Th1 host immune response (Islamuddin et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Roy et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, the activation parameters of macrophages with microbicidal capacity were evaluated.\u003c/p\u003e \u003cp\u003eThe results of the lysosomal activity and phagocytic capacity assays demonstrated that the treatment of macrophages with CNSL and CN obtained significant immunomodulation results. CN retained neutral red particles, characterized by a significant increase in lysosomal activity, which may suggest an increase in the defense of these cells. Zymosan induces stimulation of defense cells to produce a response, causing an increase in IFN production and phagocytic capacity (Wei et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and this increase was demonstrated by the substances tested. These results corroborate the study carried out by Alves et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), in which gallic and ellagic acids, naturally occurring phenolic compounds found in some plants, including \u003cem\u003eA. occidentale\u003c/em\u003e, showed immunomodulation results in the determination of phagocytic capacity and lysosomal activity at concentrations of 6.25 \u0026micro;g/mL and 3.125 \u0026micro;g/mL, similar to those in the present study.\u003c/p\u003e \u003cp\u003eInnate immunity plays an important role in infection control through mechanisms of action such as phagocytosis and lysosomal activity, which provide for antigen activation and pathogen elimination (Harrison et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The phagosome formed shortly after pathogen endocytosis fuses, followed by fusion with lysosomes to produce a phagolysosome (Niedergang and Chavrier \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The phagolysosome is a structure filled with acid hydrolases and reactive oxygen species in which most of the degradation of the contents involved takes place, and finally, the phagocytosed pathogens are killed within the phagolysosome (Lee et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lopes et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Thus, macrophage activation parameters lead to conformational changes to increase the performance of their functions, such as locomotion and phagocytosis (Petropolis et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA primary resistance mechanism to \u003cem\u003eLeishmania\u003c/em\u003e infection is the production of nitric oxide (NO) by infected macrophages (Lima-J\u0026uacute;nior et al. 2013). Inside the phagolysosome, NO reacts with O2\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, forming a reactive oxygen species, peroxynitrite. From there, nitrate and nitrite are formed as the final product, which act as microbicidal agents (Bogdan; Rollinghoff \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Ueda-Nakamura et al. 2006).\u003c/p\u003e \u003cp\u003eIn the present study, it was observed that CNSL and CN did not exhibit significant induction of NO synthesis, either in infected or uninfected macrophages. Likewise, treatment with Amph B in infected macrophages did not induce significant NO production. Similar results were found by Abas et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) testing \u003cem\u003eA. occidentale\u003c/em\u003e leaf extract, where there was no significant NO release in murine macrophages. These data reinforce that the tested compounds use another pathway to enhance their leishmanicidal activity, not depending on NO synthesis, considering that the production of this molecule is not the only way to control intracellular \u003cem\u003eLeishmania\u003c/em\u003e infection in macrophages (Singh et al. al. 2012). As there was no significant induction of NO production, it is possible that the reduction in infected macrophages and the survival rate is due to the phagocytic capacity and lysosomal activity by macrophages stimulated by the substances.\u003c/p\u003e \u003cp\u003eThe use of \u003cem\u003eZ. morio\u003c/em\u003e larvae for animal and human commercialization and consumption has increased in recent times, due to their nutritional composition, ease of handling and rearing and short life cycle (Han et al. 2010). In addition, given the ethical conflict and social aspects, we seek to increasingly prioritize the use of alternative models in experimental research, such as \u003cem\u003eZ. morio\u003c/em\u003e, considering that in insects the innate immune system is evolutionarily conserved (Canteri et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Due to these characteristics, interest in the use of this insect as an alternative model for \u003cem\u003ein vivo\u003c/em\u003e studies to assess various activities such as antimicrobial (Morey et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), toxicity (Van Der Valk and Meijden, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and insecticide (Wang) has increased et al. 2015) and pathogenicity (McGonigle et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main objective of toxicological studies is to predict the possible adverse effects that a substance may cause when exposed to human or animal, whether it is a candidate for a drug, pesticide, industrial chemical agent, among others (Koeter \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Stokes \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Meyer 2003). In this context, the \u003cem\u003ein vivo\u003c/em\u003e test was carried out using \u003cem\u003eZ. morio\u003c/em\u003e as a model organism, and the survival profiles in the larval forms of the insect were determined, in order to assess the acute toxicity of CNSL and CN. As in the cytotoxicity test on macrophages, CNSL had lower toxicity on \u003cem\u003eZ. morio\u003c/em\u003e than CN, suggesting a synergistic effect of the compounds present in the CNSL, giving it less toxicity when compared to CN alone.\u003c/p\u003e \u003cp\u003eThese results corroborate some studies that showed that the isolated pure substances were more toxic than their source fractions (Silva et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Simas et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Similarly, Guissoni et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) demonstrated that CNSL was less toxic, with a higher mean lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) than its fractions, in larvicidal activity against \u003cem\u003eAedes aegypti\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAssessing the acute oral toxicity of CNSL in \u003cem\u003eRattus norvegicus\u003c/em\u003e, LD\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;2000 mg/kg was demonstrated, not causing any significant signs of intoxication in animals (Guissoni et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). According to the classification of the Organization for Economic Co-operation and Development (OECD, 2002), this value allows classifying the CNSL in class 5 (LD\u003csub\u003e50\u003c/sub\u003e 2000\u0026ndash;5000 mg/Kg), indicating a substance with low toxicity.\u003c/p\u003e \u003cp\u003e Also according to the OECD, CN presents non-toxicity as one of its main characteristics. Tests carried out by this organization showed the following results regarding BC ecotoxicity: biodegradability \u0026minus;\u0026thinsp;96% (28 days) - (OECD-302C, 2009); solubility in water equal to 1.0 g/L; ecotoxicity (96 h): fish\u0026thinsp;\u0026lt;\u0026thinsp;11 g/L; daphnia\u0026thinsp;\u0026lt;\u0026thinsp;66 g/L; algae\u0026thinsp;\u0026lt;\u0026thinsp;1 g/L - (OECD-425, 2008) and regarding genotoxicity with using the Ames Salmonella test, it was negative. According to the Safety Data Sheets (SDS, 2017), the oral LD\u003csub\u003e50\u003c/sub\u003e is 500 mg/kg (rats) and the dermal LD\u003csub\u003e50\u003c/sub\u003e is \u0026gt;\u0026thinsp;2,000 mg/kg (Daphnia magna).\u003c/p\u003e \u003cp\u003eThe non-toxicity of CN reported in the literature differs from the result of CC50 and acute toxicity for this substance in the present study, however, according to the OECD, this toxicity, even if low, can be improved with encapsulation and controlled release for use as a biopharmaceutical (OECD, 2002).\u003c/p\u003e \u003cp\u003eIn conclusion, CNSL and CN were able to show antileishmania potential against \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eL. braziliensis\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e, acting also through macrophage activation pathways, such as increased phagocytic capacity, increased lysosomal volume. CNSL and CN were able to reduce the percentage of murine macrophages infected by \u003cem\u003eL. infantum\u003c/em\u003e and the survival rate of internalized amastigote forms. It was shown that \u003cem\u003eZ. morio\u003c/em\u003e larvae are an alternative invertebrate model suitable for analyzing the acute toxicity of the tested substances. The results are promising and serve as a starting point for further research aimed at evaluating the \u003cem\u003ein vivo\u003c/em\u003e leishmanicidal potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll protocols were approved by the Animal Ethics Committee from Federal University of Piaui, Brazil (n\u0026ordm; 640/2019).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAuthor IMMR. has received research support from Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; CAPES (process number 88887.489421/2020-00), as a Master\u0026apos;s scholarship.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMaterial preparation, collection and data analysis were carried out by IMMR, VCS, RCVC, MSS, JAONN, DSM, MGLC, AKSM, FAAC, MMMA and ILM. The graphics were created by VCS, MSS and MMMA, and the tables were made by IMMR. The translation of the manuscript into English was carried out by IMMR and LSAT. The first draft of the manuscript was written by IMMR and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbas F, Lajis NH, Israf DA, Khozirah S, Kalsom YU (2006) Antioxidant and nitric oxide inhibition activities of selected Malay traditional vegetables. 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Ther 339(2): 403\u0026ndash;411. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1124/jpet.111.181891\u003c/span\u003e\u003cspan address=\"10.1124/jpet.111.181891\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Natural products, Antileishmania activity, Cytotoxicity, Immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-1588156/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1588156/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLeishmaniasis is a neglected disease caused by protozoa of the genus \u003cem\u003eLeishmania\u003c/em\u003e, endemic in 98 countries, constituting a major public health problem. Conventional treatments cause serious adverse effects, poor tolerance, development of resistant strains, and are costly. Natural products have been investigated in the search for therapeutic alternatives. The cashew nut shell liquid (CNSL) is a natural source of phenolic compounds, showing antioxidant, anti-inflammatory, antimicrobials, antitumors, larvicides and insecticides, with cardanol (CN) being considered one of the most important and promising technical components. This study aimed to evaluate antileishmania, cytotoxic and immunomodulatory activities of CNSL and CN. The substances showed antileishmania potential, with values of mean inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of CNSL and CN against \u003cem\u003eLeishmania infantum\u003c/em\u003e: 148.12 and 56.74 \u0026micro;g/mL; against \u003cem\u003eLeishmania braziliensis\u003c/em\u003e: 85.71 and 64.28 \u0026micro;g/ml; against \u003cem\u003eLeishmania major\u003c/em\u003e: 153.56 and 122.31 \u0026micro;g/mL, respectively. The mean cytotoxic concentrations (CC\u003csub\u003e50\u003c/sub\u003e) of CNSL and CN were 37.51 and 31.44 \u0026micro;g/mL, respectively. CNSL and CN significantly reduced the percentage of infected macrophages, with a selectivity index (SI)\u0026thinsp;\u0026gt;\u0026thinsp;20 for CN. CNSL and cardanol caused an increase in phagocytic capacity and lysosomal volume, however, they did not exhibit significant induction of nitric oxide synthesis. Survival rates of \u003cem\u003eZophobas morio\u003c/em\u003e larvae at doses of 3; 30 and 300 mg/Kg were: 85%, 75% and 60% in contact with CNSL and 85%, 60% and 40% in contact with CN, respectively. There was a significant difference between the survival curves of larvae when treated with CN, demonstrating a significant acute toxicity for this substance. Additional investigations are needed to evaluate these substances in the \u003cem\u003ein vivo\u003c/em\u003e experimental infection model.\u003c/p\u003e","manuscriptTitle":"Antileishmania and immunomodulatory potential of cashew nut shell liquid and cardanol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-02 17:38:49","doi":"10.21203/rs.3.rs-1588156/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae37113b-fd72-4ebd-8f40-06d3e118631b","owner":[],"postedDate":"May 2nd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-02T17:38:51+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-02 17:38:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1588156","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1588156","identity":"rs-1588156","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}