Antiparasitary potential and cytotoxic effect of Spondias tuberosa

The objective of this work was to evaluate the antiparasitic potential and cytotoxic effect of extracts by the leaves and roots of S. tuberosa. The results show that the extracts of S. tuberosa have low antipromastigote activity against strains of L. braziliensis, since there was no action at concentrations ≤ 500 μg/mL. While for L. infantum there was a significant action of the hydroalcoholic extract of the roots against promastigote forms, since there was 29.33 ± 1.94% of mortality for the treatment of 1000 μg/mL. The same extract showed antiepimastigote action against T. cruzi at a concentration ≥ 1000 μg/mL. Despite the low Research, Society and Development, v. 9, n. 9, e889997967, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7967 3 antiparasitic activities, it is possible to observe that the extracts of S. tuberosa have no cytotoxic action, except the hydroalcoholic extract of the roots. In this extract there was a total of 27.85 ± 2.41% of cytotoxicity against fibroblastic cells, in the highest concentration evaluated (1000 μg/mL).


Introduction
In Brazil, leishmaniasis and American trypanosomiasis are the most common examples of neglected diseases. This group of diseases is present in over 148 countries, and is characterized by affecting mainly underdeveloped and developing countries, so that they contribute to inequality and social exclusion of those affected (Menezes et al., 2019;Hotez, 2008;Who, 2018). As these diseases are not so present in developed countries, they do not represent a risk to the public health of such places, consequently these nations stopped investing in research aimed at the treatment of these pathologies. Thus, there is a therapeutic disadvantage for neglected diseases, that is, due to their absence in developed countries, the pharmaceutical industries have no interest in the search and formulation of new drugs because they sell little and reach a poor and economically excluded population (Pedra et al., 2011;Santos et al., 2017). In addition, the control of such diseases has an impact on the economy due to the high investments in medicines and patient care, as well as in the control of disease vectors .
Of the two parasitic infections highlighted above, leishmaniasis is considered zoonotic, being caused by protozoa of more than 20 species of the genus Leishmania and transmitted by females of sand flies of the genus Lutzomya and Phlebotomus during blood meal. The disease is classified into three types: cutaneous, which causes ulcers on exposed parts of the body; mucocutaneous, in which protozoa cause lesions that cause destruction of the mouth, nose, throat and surrounding mucous membranes; and finally visceral, which is the most severe form, which causes fever, weight loss, enlargement of the spleen and liver, followed by anemia (Chappuis et al., 2007). This disease is endemic in 88 countries around the world, occurring mainly in Bangladesh, India and Nepal, with an annual record of 1 million to 1.5 million cases (Chappuis et al., 2007;Sousa & Day, 2011). While for the Brazilian territory in a period of 25 years , around 60 thousand cases were recorded (Maia-Elkhoury et al., 2008).
As for American trypanosomiasis, it is known in Brazil as "Chagas disease", and it is an infection caused by flagellated protozoa of the species Trypanosoma cruzi being transmitted mainly by female triatomines belonging to the genus Triatoma, Panstrongyluse and Rhodnius during blood meal. In addition to this form of transmission, strains are transmitted through blood, vertical, oral and accidental routes (Coura, 2015;Machado et al., 2018). It is estimated that around 8 million people are chronically infected by the protozoan with an annual rate of 12 thousand deaths, and in Brazil the highest prevalence occurs in the Development, v. 9, n. 9, e889997967, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7967 5 North and Northeast regions, with around 5,000 annual deaths Martins-Melo et al., 2014;Pedra et al., 2011).
Such diseases are treated using chemotherapeutic agents, and for leishmaniasis, pentavalent antimonials, amphotericin B and pentamidine are used. However, such drugs are used in high doses to obtain the desired effects, thus, causing toxicity in the host organism, in addition to selecting resistant parasites . For Chagas disease, nifurtimoxe Benzonidazole is used to treat illness. However, nifurtimox acts in the formation of O2, which leads to oxidation of the parasite's membranes. As a consequence, the host's tissues can also be damaged since they are also eukaryotes, which explains the occurrence of several side effects such as insomnia, hyporexia, vomiting and epigastric pain, in view of this, its use was interrupted in some countries of South America (Pedra et al., 2011;Rassi et al., 2002). The second drug, Benzonidazole, has some restrictions in the treatment of disease and has low efficacy during the chronic phase of the disease and high rates of treatment interruption due to subsequent side effects. Such a drug acts by inhibiting the synthesis of proteins and RNA in extracellular and intracellular forms present in the parasitized host (Cruz et al., 2016;Ferreira, 1990;Machado et al., 2018;Pedra et al., 2011).
In view of such unwanted effects, it is necessary to search for new compounds with antiparasitic actions, such as natural products. These can be used as agents in the treatment of infections due to the variety of compounds from secondary metabolism (Costa et al., 2016;. Natural products can be found in several ecosystems, such as the A native species to the Caatinga and used medicinally for the treatment of infections, digestive disorders, diarrhea, diabetes, kidney infection and throat disorders is Spondias tuberosa, Arruda (Anacardiaceae) (Albuquerque et al., 2007;Siqueira et al., 2016). Such a species is popularly known as "imbu", "umbuzeiro", "umbu" and in popular medicine several vegetative and reproductive parts are used, which are barks, fruits, roots, resin and leaves (Lins-Neto et al., 2016;Siqueira et al., 2016). Thus, the hypothesis is raised that extracts of the species may present actions against the parasitic strains that cause leishmaniasis and American trypanosomiasis and, due to their popular use, have low toxicity. Research, Society and Development, v. 9, n. 9, e889997967, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7967 6 So, in view of the low efficacy of synthetic drugs and using the ethnopharmacological approach, this work aims to investigate whether aqueous extracts and tinctures of the leaves and roots of S. tuberosa have an effect against the agents that cause leishmaniasis and American trypanosomiasis, as well as the absence of toxicity.

Preparation of extracts
To prepare the extracts, young and healthy leaves and roots were used. Aqueous extract was obtained by the infusion process, in which initially the leaves and roots were crushed to increase their contact surface. After that, 1 L of distilled water at 100 ºC was added to each 132.2 g of vegetable part, the container was then closed and kept at rest for 15 minutes, for later filtration. While the hydroalcoholic extract (tincture) was prepared by maceration in 70% ethanol in a proportion of 500 g of fresh leaves and 400 g of dried roots for each 2.652 L of ethanol (70%). The mixture was stored in a dark place for 72 h (Matos, 2002).
The drying of the extracts was carried out using the spray drying technique (spray drying) using the Mini-spraydryer MSDi 1.0 equipment (Labmaq do Brasil), using a 1.2 mm spray nozzle, under the following operational conditions: a) control flow rate: 500 mL/h; b) inlet temperature: 130±2ºC; c) outlet temperature: 74±2 ºC; d) atomization air flow: 45 L/min; e) blower flow: 1.95 m 3 /min. The spray drying process consists of changing a product that is Research, Society and Development, v. 9, n. 9, e889997967, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7967 8 in a liquid to a solid state in powder form, through its passage in a heated medium, in a continuous operation (Masters, 1991).

Antileishmania activity
For leishmanicidal evaluation of extracts of S. tuberosa, the promastigote forms of In which, AE = absorbance of the experimental group; AEB = compound blank; AC = absorbance control group; ACB = culture medium blank.

Antitripanosome activity
Epimastigote forms of Trypanosoma cruzi (Clone CL-B5) were used to evaluate the trypanocidal effects of S. tuberosa (Buckner et al., 1996). The epimastigotes forms that were For the tests, microdilution plates were used with cultures that did not reach the stationary phase (Vega et al., 2005). T. cruzi epimastigotes were seeded at 1 x 10 5 /mL in 200 μL and the plates were incubated with extracts (250 -1000 μg/mL) at 28 °C for 72 h, with 50 μL of similar CPRG solution added to give the final concentration of 200 μM. The plates were incubated at 37 °C for an additional 6 h and then read on a spectrophotometer at 595 nm.
The LC50 was calculated and the percentage of anti-epimastigote (%AE) was calculated using the same formula as the percentalan tipromastigote (% AP).

Cytotoxic Activity
To evaluate the cytotoxic effects of the extracts of S. tuberosa, cell alignment of mammalian fibroblasts NCTC clone 929 was used. The culture medium (Roswell Park Memorial Institue -RPMI) that these cells were cultured was supplemented with 10% fetal bovine serum (SFB) inactivated by heat of 56 ºC for 30 minutes, penicillin G (100 U/mL) and streptomycin (100 mg/mL). Cell culture media were maintained at a temperature of 37 °C with a humidified atmosphere of 5% CO2.
The cytotoxic evaluation of extracts against fibroblasts was performed by the colorimetric method of Rólon et al., (2006), using resazurin as a developer. In which, NCTC 929 clone cells were seeded (3x10 4 ) in flat-bottom microdilution plates (96 wells), along with 100 µL of RPMI in each well. Cell cultivation took place at night at a temperature of 37 ° C and an atmosphere of 5% CO2. After that, the medium was replaced and extracts (250 -1000 µg/mL) were added in 200 µL of the medium for 24 h. Growth controls were also included.
After the incubation period, 20 µL of a resazurin solution (2 mM) was added to each well.
After 3 h the reduction in resazurin was determined by measuring the wavelength absorbance at 490 and 595 nm in a microplate reader. The tests were performed in triplicate. The LC50 was analyzed and for the percentage of cytotoxicity of the extracts of S. tuberosa the formula was used: in which, %C, corresponds to the percentage of cytotoxicity of natural products, A570 and A595 represent the values of optical density media at 570 and 595 nm. And the values of 80,586 and 117,216 are the molar extinction coefficients for resazurin. Development, v. 9, n. 9, e889997967, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7967

Statistical analysis
The averages with their respective standard deviations of the antiparasitic and cytotoxic activities of the extracts of S. tuberosa were calculated. Afterwards the results were investigated by bidirectional analysis of variance (Anova -Two-way) (Concentration x Extract), followed by the Tukey test at 95% reliability. All analyzes were performed using the GraphPadPrism 6.0 software.

Results and Discussion
According to Figure 2, it was demonstrated that the extracts of S. tuberosa have a low antipromastigote activity against strains of L. braziliensis, since there was no action at concentrations ≤ 500 µg/mL. Furthermore, regarding the highest concentration evaluated (1000 µg/mL), there was no activity for the hydroalcoholic extract of the leaves (HELST), while there was low activity for the other extracts, as in the case of the aqueous extract of the leaves (AELST), in which the natural product caused 10.5 ± 0.71% of mortality against L. braziliensis strains. Regarding the antipromastigote action of S. tuberosa against strains of L. infantum, it was found that there was biological action only for hydroalcoholic extracts (Figure 3). The hydroalcoholic extract of the leaves (HELST), despite having activity in concentrations ≥ 500 µg/mL, demonstrated low leishmanicidal potential. However, there was a significant action of the hydroalcoholic extract of the roots against the promastigote forms of L. infantum, since there was 29.33 ± 1.94% of mortality for the treatment of 1000 µg/mL. The extracts of the leaves and roots of S. tuberosa showed low antiparasitic activity against the epimastigote strains of T. cruzi as seen in Figure 4. The hydroalcoholic extract of the roots showed antiepimastigote action in concentration ≥ 1000 µg/mL. The aqueous extract of the leaves, on the other hand, showed activity in concentrations ≥ 250 µg/mL, with its highest concentration resulting in 23.53 ± 3.39% of mortality of the protozoa. Development, v. 9, n. 9, e889997967, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7967  According to Pedra (2011), the World Health Organization has determined some parameters to be followed for the formulation of a drug in order to treat a disease. They are: (1) Parasitological cure for acute and chronic cases; (2) Effect in one or a few doses; (3) Low cost; (4) Absence or low side effects to the sick and no teratogenic effects; (5) No need for hospitalization for treatment; and (6) No induction of organism resistance to the drug. Thus, we refute the hypothesis established in this study according to parameter 2, since there was low antiparasitic activity in the extracts of S. tuberosa. Because there was no inhibition of more than 50% of the parasites at concentrations ≤ 500 µg/mL (Rosas et al., 2007;Vandesmet et al., 2017).
However, despite this low activity, the results of this study are prosperous, because even though they did not eliminate the parasites, the extracts from the leaves and roots of S. tuberosa were able to reduce their quantity. This is advantageous for populations that do not have access to other treatments, as the reduction of parasitic strains in the body ends up  Research, Society and Development, v. 9, n. 9, e889997967, 2020 (CC BY 4. (Mokoka et al., 2013) and Spondias mombin L. (Traore et al., 2014). In this latest study, the researchers demonstrated that the aqueous extract of the stem bark has activity against strains of T. cruzi (LC50 35.8 µg/mL), Trypanosoma brucei brucei (CL50 2.3 µg/mL) and Plasmodium falciparum (LC50 59.5 µg/mL).
Despite the low antiparasitic activities, it is possible to observe that the extracts of S. tuberosa have no cytotoxic action, except the hydroalcoholic extract of the roots (HERST) ( Table 01). In this treatment, there was a total of 27.85 ± 2.41% of cytotoxicity against murine fibroblasts in the highest concentration evaluated, in addition, the action is observed in a lower concentration (500 µg/mL). Legend: Hydroalcoholic extract of leaves (HELST) and roots (HERST) and aqueous extract of leaves (AELST) and roots (AERST) of Spondias tuberosa. Average ± standard deviation. Source: The Author.
Extracts from the leaves of S. tuberosa were not able to cause toxicity, however Guedes et al. (2020), demonstrated that these organs have compounds with high toxicity, since the hexane extract of the leaves showed a high toxicity against fibroblasts. This is explained by the type of solvent used in the research.
The cytotoxic actions of the hydroalcoholic extract of the roots of S. tuberosa may be related to the chemical constituents present in such organ. The action may be the activity of the major compounds or the synergistic action . Among the secondary metabolites present in the extract, highlights (±)-Naringenin , which is a cytotoxic flavanone against fibroblasts (Stompor et al., 2017), corroborating the results of this work.
Although the study showing some results, it is worth mentioning that the study is in vitro, studies using animals (in vivo) must be carried out in order to obtain the necessary doses with the antiparasitic action, so that the research can then be submitted to a clinical study using people and obeying all ethical standards.

Conclusion
The aqueous extracts and tinctures of the leaves and roots of Spondias tuberosa have low efficacy against strains of Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi in concentrations of clinical relevance. In addition, the hydroalcoholic extract of the roots shows cytotoxicity against fibroblast-type cells.
This work opens new possibilities for the identification of compounds with antiparasitic action, so that more studies should be carried out aiming at the separation and purification of the bioactive constituents. Subsequently, in vivo and clinical studies should be performed.