Anti-malarial activity and toxicity of Aspidosperma nitidum Benth : a plant used in traditional medicine in the Brazilian Amazon

The objective of this work was to evaluate the antiplasmodial activity and toxicity of the extract and fractions obtained from the bark of Aspidosperma nitidum. The ethanol extract obtained from the powdered bark of plants was acid-base partitioned and phytochemically analyzed. The antiplasmodial activity, in vivo antimalarial activity and in vitro cytotoxicity were acessed. Research, Society and Development, v. 9, n. 10, e5059108817, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8817 3 The selectivity index (SI) was calculated. The acute oral toxicity and pathological effects, of the ethanol extract was evaluated in mice. The major constituent of the ethanol extract was suggestive of a β-carboline chromophore. The alkaloid and neutral fractions contained compounds with an aspidospermine core as the major constituent. The ethanol extract (IC50 = 3.60 μg/mL), neutral fraction (IC50 = 3.34 μg/mL) and alkaloid fraction (IC50= 2.32 μg/mL) showed high activity against P. falciparum (W2 strain). The ethanol extract and the alkaloid fraction reduced 80% of the parasitemia of P. berghei (ANKA)-infected mice (dose of 500 mg/kg) in the 5 day, which was not sustainable at the 8 day. A similar result was obtained for chloroquine. The ethanol extract (CC50 = 410.65 μg/mL; SI = 114.07), neutral fraction (CC50 = 452.53 μg/mL; SI = 135.49), and alkaloid fraction (CC50 =346.73 μg/mL; SI 149.45) demonstrated low cytotoxicity and high SI. The ethanol extract (5000 mg/kg; gavage) presented low acute oral toxicity, with no clinical or anatomopathological modifications being observed (in comparison to the control group). In vitro studies with a chloroquine-resistant clone of P. falciparum confirmed the antiplasmodial activity of the A. nitidum ethanol extract, and its fractions had low cytotoxicity for HepG2 cells. In vivo studies with P. berghei–infected mice and acute toxicity studies corroborated these results.

The selectivity index (SI) was calculated. The acute oral toxicity and pathological effects, of the ethanol extract was evaluated in mice. The major constituent of the ethanol extract was suggestive of a β-carboline chromophore. The alkaloid and neutral fractions contained compounds with an aspidospermine core as the major constituent. The ethanol extract (IC50 = 3.60 µg/mL), neutral fraction (IC50 = 3.34 µg/mL) and alkaloid fraction (IC50= 2.32 µg/mL) showed high activity against P. falciparum (W2 strain). The ethanol extract and the alkaloid fraction reduced 80% of the parasitemia of P. berghei (ANKA)-infected mice (dose of 500 mg/kg) in the 5 th day, which was not sustainable at the 8 th day.  Research, Society and Development, v. 9, n. 10, e5059108817, 2020 (CC BY 4.

Introduction
Malaria is one of the world's leading causes of mortality, with approximately 405 000 deaths annually. The highest number of cases (94%) occurred in Africa. Every year, 292 000 African children die of malaria. However, there was an impressive 48% decrease in global mortality between 2000 and 2015 (WHO, 2019).
Parasite resistance to antimalarials remains an ever-present obstacle to the elimination of malaria (Wicht, et al., 2020). The extensive use of anti-malarial drugs imposes selective pressure on the parasites. Plasmodium falciparum resistance has been extensively described (White, 2004).
Because of this growing drug resistance, new therapeutic alternatives are necessary.
Traditional medicines can contribute to the search. In this regard, some plant species have been proven to be active against the chloroquine-resistant P. falciparum clone W2, such as, plants of the genus Aspidosperma, Apocynaceae (Dolabela, et al., 2012).
Indeed, the ethanol extract obtained from A. olivaceum bark has also been shown to be active against W2 clones (Chierrito, et al., 2014).Moreover, alkaloid extracts and alkaloid-rich fractions from A. olivaceum, A. ramiflorum, A. spruceanum, and A. parvifolium have all shown activity against both chloroquine-resistant and chloroquine-sensitive clones of P. falciparum (IC50 < 10 g/mL). The antiplasmodial activities of these extracts and fractions are related to the alkaloids they contain (Dolabela, et al., 2012).
The present study evaluated the antimalarial activity of the ethanol extract and fractions from the bark of Aspidosperma nitidum. We also describe the results of the phytochemical study, investigations into HepG2 cytotoxicity, and acute oral toxicity in Swiss mice.

Material and Methods
This work used the experimental scientific model of comparison with positive and negative controls of biological activities in vivo and in vitro (Pereira, et al., 2018). Research, Society and Development, v. 9, n. 10, e5059108817, 2020 (CC BY 4. The bark was washed, dried in an air-vented kiln, and triturated in a knife mill. The resulting powder (500 g) was macerated with ethanol (yield 10.33 g; 2.06%). The solution was concentrated in a rotary evaporator to yield the ethanol extract. Part of the extract (2.0 g) was solubilized with hydrochloric acid (3% v/v) and subsequently dichloromethane partition. The dichloromethane solution was concentrated in a rotary evaporator (neutral fraction = 0.475 g).
The aqueous acid solution was alkalized to pH 9 with ammonium hydroxide to yield the dichloromethane fraction. This solution was concentrated in the rotary evaporator, yielding the alkaloid fraction (0.395 g; Figure 1; Henriques, et al., 2010). The ethanol extract and fractions were analyzed with high-performance liquid chromatography coupled with a diode array detector (Waters e2695 and Waters 2998). The analysis used column RP 18 (particles of 5 m, 45 × 250 mm) and, as the mobile phase, a mixture of water acidified with 0.01% trifluoroacetic acid (eluent A) and acetonitrile acidified with 0.01% trifluoroacetic acid (eluent B). A linear gradient was created using 70-30% eluent B for 15 minutes, 60-40% eluent B for 20 minutes and 50-100% eluent B for 25 minutes. The Research, Society and Development, v. 9, n. 10, e5059108817, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8817 7 oven temperature was maintained at 26°C, flow was kept at 1 mL per minute, and wavelengths from 220 to 400 nm were scanned in a methodology adapted from Coutinho, et al. (2013).
After 24 hours, samples of extract or fraction were added at different concentrations (1, 10, 100, or 1000 μg/mL), followed by 24 hours of further incubation. Then, MTT (2.0 mg/mL) was added, followed by incubation for 4 hours. Dimethyl sulfoxide (99.7 %,100 µL) was added to each well, and plaques were mixed to solubilize formazan crystals. The optical density was determined at 570 nm (Stat Fax 2100 microplate reader, Awareness Technology, USA).
Cell viability was expressed as the percentage of the absorbance in the untreated control cells. The cytotoxic concentration (CC50) was determined by linear regression.
Parasitized, untreated RBCs were the positive control. Chloroquine was used as the standard anti-malarial drug.
One hundred microliters of the antibody MPFM-55A (1.0 µg/mL in PBS) was added to each well, followed by overnight incubation at 4°C. The supernatant was discarded. Blocking solution (BSA 2% in PBS) was added (200 µL per well), and plaques were incubated at room temperature for 2 hours. The plates were washed three times with 0.5% PBS/Tween solution (200 µL per well). Thereafter, 100 µL of the hemolyzed culture was added to samples and controls (background; RBCs parasitized and frozen at time 0 h). Plaques were incubated for 1 hour at room temperature in a humidity chamber. Subsequently, plaques were washed three times with 0.5% PBS/Tween (200 µL per well ;Noedl, et al., 2002).
The chromogen o-phenylenediamine (OPD) was added to each well (100 µL). Plaques were incubated in the dark for an additional 10 minutes, and 50 µL of 1 M sulfuric acid was added. Spectrophotometric analysis was performed using an ELISA plaque reader (ELISA Stat Fax, mod. 2100) at 492 nm (Noedl, et al., 2002).
The selectivity index (SI) for the antiplasmodial activity was calculated based on the ratio between CC50 and IC50 for the in vitro activity against P. falciparum. Compounds with SI > 10 were considered nontoxic (Dolabela, et al., 2012).

In vivo antimalarial activity
The parasites (P. berghei ANKA) were maintained through weekly blood passages in mice intraperitoneally (i.p.), using 3.8% sodium citrate as an anticoagulant. The traditional suppressive test of Peters (1965Peters ( , 1967, as modified by Carvalho, et al. (1991), was used.
Briefly, adult Swiss albino mice (Mus musculus; 18-22 g; n= 24; 4 animals per group obtained from the Animal Facility of the Evandro Chagas Institute-IEC; Belém, Pará State, Brazil) were inoculated (i.p.) with 10 5 P. berghei-infected RBCs. Mice were divided into groups of four per cage and orally treated for three consecutive days with daily doses of the extracts. Two control Research, Society and Development, v. 9, n. 10, e5059108817, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8817 9 groups were used in each experiment, one treated with chloroquine (30 mg/kg, orally) and the other kept untreated. After 5 and 8 days of parasite inoculation, blood smears were prepared from the tail tip of all mice, fixed with methanol, stained with Giemsa, and then microscopically examined (×1000).
Parasitemia was determined from blood smears using coded and counted randomized double-blind trials, counting 10 000 erythrocytes in cases of 0% parasitemia, 5000 erythrocytes for up to 5% parasitemia, 2000 erythrocytes for 5-10% parasitemia, 1000 erythrocytes for 11-20% parasitemia, and 300 erythrocytes for parasitemia higher than 20%. Overall mortality was monitored daily in all groups for a period of 4 weeks following inoculation. The extract and alkaloid fraction were tested in three independent experiments at daily doses of 500, 250 and 125 mg/kg body weight. Samples were considered partially active when there was a reduction of parasitemia greater than 30% (Carvalho, et al., 1991).

Acute oral toxicity
Male (n = 8) and female (n = 8, nulliparous and non-pregnant) albino Swiss mice weighing 20 ± 2 g were obtained from the Animal Facility of the Evandro Chagas Institute were weighed daily, and food and water intake were recorded. At the end of the experiment, animals were anesthetized and euthanized, blood samples were drawn, and target organs were removed for anatomopathological and histopathological evaluation (brain, cerebellum, stomach, intestine, mesentery, heart, lungs, liver, spleen, pancreas and kidney; OECD, 2001).
Acute oral toxicity was measured using the Fixed Procedure Test, according to NCCLS guidelines from OECD (2001). Animals were treated with a single dose (5000 mg/kg; gavage) and evaluated with the Hippocratic test after 14 test days.
Signs of toxicity were recorded, as well as its decline and duration. Changes in the conscious state and disposition, activity, coordination, motor system, reflexes, and central nervous system and peripheral nervous system activity of the mice were evaluated (Malone & Robichaud, 1983).
On the 14th day after exposure, animals were euthanized and their brain, cerebellum, stomach, intestine, mesentery, heart, lungs, liver, spleen, pancreas and kidney were removed for anatomopathological and histopathological studies. The histological sections of excised Research, Society and Development, v. 9, n. 10, e5059108817, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8817 organs were fixed in buffered formalin (10% formaldehyde solution) and cut for histopathological processing after 24 hours. The process involved samples being dehydrated with a series of increasing alcohol concentrations (70-100%), cleared in xylene, impregnated and embedded in paraffin according to standard methods. In a microtome, tissue fragments were sectioned at a thickness of 3.0 mm and subsequently underwent hematoxylin-eosin and Masson's trichrome staining and were examined under an optical microscope (Bacha & Wood, 1990).
All procedures were carried out in accordance with the ethical principles of animal experimentation, according to the standards of the Brazilian Society of Sciences in laboratory animals and of Brazilian's National Council of Animal Experimentation Control (CONCEA).

The experimental protocol was approved by the Ethics in Animal Research Committee of the
Universidade Federal do Pará (report CEPAE no. BIO063-12).

Statistical analysis
Linear regression in dose-response curves was used for the cytotoxicity and antiplasmodial assays, the data regression on sigmoidal dose-response curves. Student's t-test for non-paired data was used to evaluate acute oral toxicity, with a significance level set at p < 0.05.
The chromatogram of the alkaloid fraction showed major peaks at 5.2 and 5.4 minutes (max 220.7 and 272.5 nm). The neutral fraction showed major peaks with retention times of 5.6 and 5.9 minutes for the same chromophores (max 220.7 and 272.5 nm) suggestive of indolic alkaloid with an aspidospermine core, which is the aspidospermine described for this species (Figure 3 B and C;Pereira, et al., 2007). Development, v. 9, n. 10, e5059108817, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8817 Acid-basic partitioning was not efficient for the extraction of the major alkaloid of the extract, as peaks corresponding to chromophores of β-carboline alkaloids were not found in the alkaloid and neutral fractions.
Harman alkaloids, when in contact with hydrochloric acid, tend to become their phenolharmol pairs. Phenol-harmol alkaloids can be precipitated and retained in aqueous alkaline solution (Perkin & Robinson, 1919). Thus, only indole alkaloids with an aspidospermine core are seen in the chromatograms of alkaloid and neutral fractions.

Antiplasmodial activity and cytotoxicity
The ethanol extract of A. nitidum was shown to be active against the W2 clone of P. falciparum (IC50 = 3.6 µg/mL; Table 1). Another study evaluated the antiplasmodial activity of the ethanol extract of A. nitidum using the [³H]-hypoxanthine uptake method and obtained a CI50 of 21.93 µg/mL (moderate activity). In that study, the fractioning of the extract led to the isolation of the following alkaloids: harman carboxylic acid, 3α,20β-10methoxy-18,19dihydrocorynantheol and braznitidumine (Pereira, 2005). The difference between the results of that previous study and the present study can be related to differences in methodologies used in the evaluation of antiplasmodial activity. Moreover, the fact that plants were collected in different locations for each study must be taken into account. Pereira (2005)  The neutral fraction showed high activity against the W2 P. falciparum clone (IC50 = 3.34 µg/mL; Table 1). The chromatogram of the neutral fraction showed major peaks at 5.6 and 5.9 minutes. The UV spectrum was suggestive of the indole alkaloid. Similar spectra were found in the alkaloid fraction, although there were differences in retention times. These different retention times suggest that the chromophores have different substituents, which would explain the differences in biological activity. Nevertheless, it was expected that the neutral fraction would not contain alkaloids. This was not observed, indicating incomplete separation during the extraction.
The alkaloid fraction showed high activity against P. falciparum (IC50 = 2.32 µg/mL; Table 1). In another study, the alkaloid fraction of the hydroethanolic extract of A. excelsum was moderately active against clone W2 (Gomes, 2011). These different responses may be due to different major alkaloids. In the study conducted by Gomes (2011), most of the alkaloid fraction and alkaloid extract was yohimbine. In the present study, the major alkaloid was also of indole origin but with a different maximum ultraviolet absorption, probably reflecting an alkaloid with an aspidospermine core. Aspidospermine has already been shown to be active against chloroquine-resistant P. falciparum strains (IC50 = 5.6 µg/mL; Mirtaine-Offer, et al., 2002).
The greater activity in the fraction of alkaloids is due to higher concentrations of alkaloids in this fraction, as can be observed in Figure 3.
The ethanol extract of A. nitidum and its fractions showed low cytotoxicity for HepG2 cells (Table 1). When the 50% cytotoxic concentration corresponds to the 50% inhibitory concentration, the compound is highly selective (SI > 114; Table 1).
One factor contributing to the toxicity or the antiplasmodial activity of alkaloids is the presence of the tetrahydrofuran ring. The ring decreases the antimalarial activity and contributes to cytotoxicity. The presence of aspidospermane core-derived alkaloids increases antiplasmodial activity and decreases toxicity (Mirtaine-Offer, et al., 2002). Thus, low cytotoxicity in A. nitidum may be related to the absence of alkaloids with the tetrahydrofuran ring. However, the presence of aspidospermane derivatives explains the high antiplasmodial activity displayed.

Acute oral toxicity and in vivo anti-malarial activity
Doses of 125, 250 and 500 mg/kg of the extract and fractions were used to assess antimalarial activity. The ethanol extract and the alkaloid fraction were active in all doses tested at the 5th day of analysis. The anti-malarial activities for both samples were similar to the chloroquine standard (30 mg/kg). The extract and its fraction did not interfere with parasitemia on the 8th day. These results suggest that the anti-malarial response is not sustained (Table 2). Research, Society and Development, v. 9, n. 10, e5059108817, 2020 (CC BY 4. To minimize the number of animals in the experiment, the neutral fraction was not tested. This fraction presented a profile alike the alkaloid fraction. Similar to the in vitro test, fractioning of the extract did not increase anti-malarial activity. The in vivo study showed no significant differences in parasitemia (p> 0.05, Student's t test) between doses (Table 2), but the in vitro study showed a direct concentration-response relation. In another study, mice infected with P. berghei and treated with ethanol extract of A. nitidum (doses= 250 and 125 mg/kg) showed a similar reduction in parasitemia (Coutinho, et al., 2013).
In the present study and Coutinho et al. (2013), the greatest reduction in parasitemia was observed at the 5th day (Table 2), and the presence of alkaloids was probably responsible for A. nitidum activity. Notwithstanding, the alkaloid Manzamine F caused a rapid and unsustained reduction in parasitemia of mice infected with P. berghei (Ang, et al., 2000).
Likewise, only the fixed dose of 5000 mg/kg of the ethanol extract was used for the oral acute toxicity test. The animals were divided into two groups (test and control), again minimizing the number of animals used (Brito, 1994).
During the first 4 hours of testing, no signs of toxicity were observed, except that the mice were resistant to handling during the first 15 minutes. There were no animal deaths during the 14 days of the experiment. Based on this, the ethanol extract was classified as toxicity V (2000 mg/kg < LD50 < 5000 mg/kg; OECD, 2001).
There were no significant changes in weight, and the consumption of water and feed was similar between groups (Table 3). Moreover, there were no anatomo-histopathological alterations in the organs analyzed (brain, cerebellum, stomach, intestine, mesentery, heart, lungs, liver, spleen, pancreas, and kidney) compared to the control group. This result confirms the low toxicity of the ethanol extract. The in vitro and in vivo results suggested low toxicity of the ethanol extract from A. nitidum. The acute oral toxicity of the hydroethanolic extract of A. excelsum showed low toxicity in mice (5000 mg/kg).
Additionally, changes in weight, feed intake and water were not observed (Gomes, 2011).

Conclusion
The ethanol extract and fractions proved highly active against resistant strains of P.
falciparum. The fractioning of the extract did not significantly increase its antiplasmodial activity. In the in vivo anti-malarial activity test, both the extract and the alkaloid fraction reduced parasitemia with values of reduction equal to the control. The cytotoxicity assay showed low cytotoxicity. The correlation of these results to the results of the in vitro antiplasmodial tests indicates a high selectivity index for all samples tested, implying that the effect is pharmacological and not toxicological. The acute oral and fixed-dose toxicity tests confirmed the low toxicity of the extract. Thus, A. nitidum has high antiplasmodial potential and is worth studying for the development of new drugs for the treatment of this disease.