Antileishmanial Activity of Handroanthus serratifolius (Vahl) S. Grose (Bignoniaceae)

This study aimed to evaluate the leishmanicidal activity of ethanol extract, fractions, and isolated substance from Handroanthus serratifolius against Leishmania amazonensis. Furthermore, this activity was related to cytotoxicity, and the selectivity index was determined. The ethanol extract was obtained by maceration of the stem powder, and the extract was subjected to fractionation on chromatographic column. The lapachol was obtained by acid base extraction followed by purification in chromatographic column. The antipromastigote activity and cytotoxicity tests were carried out by the cell viability method (MTT). Modified THP-1 cells were infected with L. amazonensis promastigotes and treated for 24 h with different concentrations of the extract, fractions, and lapachol. The ethanol extract, dichloromethane, and ethyl acetate fractions were not active against promastigotes (IC50 > 200 μg/mL) or cytotoxic (CC50 > 500 μg/mL), and the selectivity index (SI) was greater than 2.5. The ethyl acetate fraction was active only in promastigotes; it is not cytotoxic (CC50 > 500 μg/mL, SI > 5). The lapachol was selectively active only against amastigote (IS > 2.5, CC50 > 500 μg/mL). In summary, lapachol and ethyl acetate fraction are promising against amastigote and promastigote forms, respectively.


Introduction
Leishmaniasis is caused by over 20 Leishmania species and it is transmitted to humans by the infected phlebotomine female sandflies. There are three main forms of the disease: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis. It is estimated that about 200,000 to 400,000 new cases of VL occur worldwide each year. Over 90% of new cases occur in 6 countries: Bangladesh, Brazil, Ethiopia, India, South Sudan, and Sudan. The cutaneous leishmaniasis is the most common form of leishmaniasis. About 95% of CL cases occur in the Americas, the Mediterranean basin, the Middle East, and Central Asia. Almost 90% of mucocutaneous leishmaniasis cases occur in Bolivia, Brazil, and Peru [1].
The leishmaniasis control is based on vector combat, extermination of infected dogs, and treatment of infected individuals [2]. The amphotericin and N-methyl glucamine antimoniate (Glucantime5) [3] are drugs currently used in the treatment of leishmaniasis. However, they have problems as severe adverse effects, and some strains have already presented increased parasitic resistance [2,4,5]. In addition, all drugs are currently available for parenteral administration [4]. As a result, many patients abandon treatment; this fact favors the appearance of resistant strains [6].
In this context, plant species are the best and greatest source of drugs for mankind. Ethnobotanical studies have demonstrated the popular use of plants in the treatment of leishmaniasis both orally and in the topical application on lesions [7,8]. Many plants present in their composition substances of the classes of alkaloids, terpenes, naphthoquinones, lignans, chalcones, flavonoids, and sesquiterpene lactones, compounds described in the literature as effective in leishmanicidal activity [9][10][11]. The search for alternative therapies for leishmaniasis is very important. Many species of the Bignoniaceae family are used in folk medicine to treat external ulcers, skin diseases, and skin disorders [7]. However, the antileishmanial activity of these species has not been tested yet.
Other studies evaluated the leishmanicidal activity of lapachol and compared its efficacy with a reference drug, sodium stibogluconate (Pentostam5). These compounds were evaluated against amastigotes of Leishmania (Viannia) braziliensis (LVb). In vitro, lapachol exhibited antiamastigote effect, whereas in vivo it did not prevent the development of LVb induced lesions at an oral dose of 300 mg/kg/day for 42 days. Pentostam5 demonstrated a significant antiamastigote effect in vitro and in vivo (60 mg/kg/day). Perhaps the lapachol inhibits the microbicide function of macrophages in vivo. Alternatively, it might be transformed into an inactive metabolite(s) or neutralized, losing its leishmanicidal activity [17].
This study aimed to evaluate the leishmanicidal activity of ethanol extract, fractions, and isolated substance obtained from Handroanthus serratifolius against Leishmania amazonensis. Furthermore, this activity is related to cytotoxicity determining the selectivity index. Plants were dried at room temperature for seven days. The material was powdered and extracted with ethanol by cold maceration. The resultant solution was concentrated in a rotary evaporator to obtain the ethanol extract. The extract was fractioned in chromatographic column (CC) with silica gel as stationary phase and increasing polarity solvents (hexane, dichloromethane, ethyl acetate, and methanol) as mobile phase ( Figure 2).

Material and Methods
The powder of H. serratifolius was treated with 2.5% sodium carbonate solution for 24 h for lapachol isolation. The solution was filtered, and the precipitate was solubilized in aqueous hydrochloric acid. After 30 minutes, it was centrifuged (3,000 rpm/10 minutes) and a yellow solid precipitate was separated (Figure 2). The precipitate was dried and submitted to fractionation on chromatographic column. Nuclear magnetic resonance was used to identify the isolated compounds.  The L. amazonensis promastigotes were cultivated at 26 ∘ C in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco5, Grand Island, NY, USA), penicillin (100 U/mL), and streptomycin (100 g/mL) [18].
Culture of promastigote forms in logarithm phase was adjusted to 5 × 10 6 parasites/100 L. The susceptibility testing was performed in 96-well plates. The extract, fraction, and lapachol were tested in triplicate in a concentration gradient (200 to 3.125 g/mL). Negative control was performed with parasites and incubation medium. The positive control was made with amphotericin B (25-0.3906 g/mL). After 24 h of incubation at 26 ∘ C in 5% de CO 2 , 10 L of tetrazolium salt (5 mg/mL) was added to each well, and the parasites were quantified in enzyme-linked immunosorbent-assay plate reader. The IC 50 was determined by linear regression (Graph Pad Prism version 5.04). The results were classified as follows: IC 50 ≤ 100 g/mL were considered active, IC 50 between 101 and 200 g/mL were considered moderate active, and IC 50 ≥ 200 g/mL were considered to be inactive [18].

Results and Discussion
In this study, lapachol (C 15 H 14 O 3 ) was isolated from stem powder of H. serratifolius. However, other studies isolated ethanol extract of H. serratifolius lapachol (2.9% yield) [21]. The antipromastigote activity of lapachol has been described in posterior study [16].
To verify if H. serratifolius has other substances with antileishmanial activity, the ethanol extract of the stem was obtained (13% yield). This extract was fractioned resulting in four fractions: hexane (3.68% yield), dichloromethane (8.02% yield), ethyl acetate (28.64% yield), and methanol (58.02% yield) (Figure 3).  Lapachol, ethanol extract, and fractions were tested against L. amazonensis promastigotes. Unlike a previous study [16], lapachol was not active in L. amazonensis promastigotes (IC 50 > 200 g/mL; Table 1). The L. amazonensis strain used in this assay was isolated from a patient who had previously not responded to conventional therapy. This may explain the divergent response.
We assessed the cytotoxicity of all samples for modified THP-1 cell line. Extract, fractions, and lapachol showed no toxicity for this cell (CC 50 > 500 g/mL; Table 1). Similarly, another study showed that the ethanol extracts of leaves and flowers from H. aureus were not cytotoxic for macrophages rats (CC 50 > 1000 g/mL) [24]. Unlike this study, several studies describe the cytotoxicity of lapachol [25][26][27]. The most active fraction against promastigotes showed higher selectivity index (SI > 5). Lapachol showed selectivity index greater than 2.5 (Table 1). Lapachol reduced the infection of macrophages, with greater effect observed at 250 g/mL ( Figure 4; Table 2). Antiamastigote activity of lapachol against Leishmania (Viannia) braziliensis was described [17]. This effect has been linked to stabilization of the complex and DNA topoisomerase [28]. Some have antiparasitic effect as time-dependent [29,30]. Thus, increased exposure time can contribute to the inhibitory effect.

Conclusion
The ethanol extract, hexane, dichloromethane, and methanol fractions from H. serratifolius showed no antipromastigote and antiamastigote activities. It was also not cytotoxic. The ethyl acetate fraction showed selective effect for promastigotes, while lapachol was active for amastigotes.