Antileishmanial and cytotoxic activities of four Andean plant extracts from Colombia

Background and Aim: Licania salicifolia (L.S) Cuatrec., Persea ferruginea (P.F) Kunth, Oreopanax floribundus (O.F), and Psychotria buchtienii (P.B) belong to the families Chrysobalanaceae, Lauraceae, Araliaceae, and Rubiaceae, respectively, which have been used as medicines by communities in the Andes. This study evaluated the leishmanicidal and cytotoxic activities of alcohol and non-alcohol extracts from four Andean plant extracts (L.S, O.F, P.F, and P.B). Materials and Methods: Extracts were obtained by percolation with solvents of different polarities — hexane, dichloromethane, ethyl acetate, and ethanol. Phytochemical screening was conducted based on reported methods. All products were evaluated in vitro to determine the leishmanicidal activity against amastigotes of Leishmania panamensis and cytotoxicity against U937 cells. Results: Flavonoids, triterpenes, and tannins were the main secondary metabolites found. From the results, dichloromethane extracts from O.F and P.B, ethanol extract from P.B, and ethyl acetate extracts of all plants were active, with EC50 <30 μg/mL. Ethyl acetate was the most active extract, which showed EC50 values of 9.8, 14.1, 23.7, and 25.5 μg/mL, for L.S, P.B, O.F, and P.F, respectively. Hexane extracts from P.B and O.F exhibited moderate activity with EC50 values of 84.8 and 87.4 μg/mL, respectively. Hexane and ethanol extracts from O.F, ethyl acetate, and ethanol extracts from L.S, and all extracts from P.F were not toxic. Alternatively, hexane and dichloromethane extracts from L.S and P.B as well as dichloromethane and ethyl acetate extracts from O.F displayed high toxicity. Conclusion: Based on the activity we observed, ethyl acetate extract can continue in its usage in the search for new antileishmanial drugs, mainly ethyl acetate extract from L.S showed activity comparable to meglumine antimoniate and was not cytotoxic.


Introduction
Leishmaniasis is a group of diseases caused by protozoan parasites of the genus Leishmania, which infect and replicate inside macrophages of the vertebrate host. These diseases are considered a major health problem because of its presence in 98 countries, affecting mostly low-income people in rural areas of tropical and subtropical countries [1].
Leishmania (Viannia) panamensis is an important causal agent of cutaneous leishmaniasis (CL) in Central and South America [2] and approximately 0.7-1.2 million cases occur annually. Different forms of leishmaniasis demand expensive treatments, the currently used medicines pentavalent antimonials, pentamidine isethionate, and miltefosine show high toxicity; therefore, they induce severe side effects. Thus, there is an urgency to develop new drugs to treat CL [3].
In many tropical countries, the treatment of leishmaniasis usually involves the oral administration of crude plant extracts for the systemic form of the disease and topical preparations of the corresponding extracts for treating skin infections [4]. Natural products, especially those derived from plants, are considered an important source of biologically active compounds against various infectious organisms, especially parasites. Many studies related to the activity of plant extracts against leishmaniasis have been reported [5].
Based on these reports, we studied extracts of these plants to validate its traditional uses and to develop new chemotherapeutic agents against this protozoal disease.

Ethical approval
According to Colombian legislation, since this work involves only in vitro studies that do not require the use of animals or humans, the endorsement by the ethics committee is not required.

Study period and location
This study was carried out from June 2014 to December 2015 at the University of Antioquia, Medellin, Colombia.

Extraction
The plant materials were dried in an oven at 35°C for 48 h. Powdered leaves of L.S (300 g), P.F (530 g), O.F (685 g), and P.B (430 g) were successively extracted with hexane, then dichloromethane, ethyl acetate, and finally ethanol in a percolator, at room temperature after which they were concentrated in a vacuum to give the corresponding extracts.

Phytochemical screening
The phytochemical composition of the different plant extracts used in this study was analyzed. To detect steroids, triterpenoids, phenolics, flavonoids, alkaloids, saponins, anthraquinones, coumarins, and anthocyanosids, the method described by Londoño et al. was used [14].

Biological activity assays
Extracts were subjected to in vitro evaluation to determine their cytotoxicity on U937 human cells and antileishmanial activity on intracellular amastigotes of L. (V) panamensis.

In vitro cytotoxicity
The cytotoxic activity of extracts was assessed based on the viability of the human promonocytic cell line U937 (ATCC CRL-1593.2) evaluated using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay following the methodology described previously [15]. Briefly, cells were grown in 96-well cell-culture dishes at a concentration of 100,000 cells/mL in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum and the corresponding concentrations of the extracts, starting at 200 µg/mL in duplicate. Cells were incubated at 37°C and 5% CO 2 for 72 h in the presence of the extracts and then the effect of the extracts was determined by adding 10 µL/ well of MTT solution (0.5 mg/mL) and incubating at 37°C for 3 h. The reaction was stopped by adding 100 µL/well of 50% isopropanol solution with 10% sodium dodecyl sulfate and incubating for 30 min. Cell viability was determined based on the quantity of formazan produced according to the color intensity (absorbance) -registered as optical density (O.D)obtained at 570 nm in a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, San Francisco, CA, USA). Cultured cells in the absence of the extracts were used as control of viability (negative control), while doxorubicin was used as a positive control for toxicity. The cytotoxicity of two conventional drugs (meglumine antimoniate and Amphotericin B) was also determined. Assays were conducted in duplicates with three replicates per concentration tested.

In vitro antileishmanial activity
The activity of the extracts was evaluated using intracellular amastigotes of L. (V) panamensis transfected with the green fluorescent protein gene (MHOM/CO/87/UA140pIR-GFP) [15]. The effect of each extract was determined based on infection inhibition, which is evidenced by a decrease in the number of live intracellular amastigotes. Briefly, U937 human cells at a concentration of 3×10 5 cells/mL in RPMI 1640 and 0.1 µg/mL of phorbol-12-myristate-13-acetate were infected with promastigotes in the stationary growth phase using 15:1 parasites per cell ratio and incubated at 34°C and 5% CO 2 for 3 h. Cells were washed twice with phosphate-buffered saline (PBS) to eliminate non-internalized parasites. Fresh RPMI 1640 (1 mL) was added and cells were incubated again to guarantee the multiplication of intracellular parasites.
After 24 h of infection, the culture medium was replaced by a fresh culture medium containing each extract at four concentrations (100, 25, 6.25, and 1.78 µg/mL) or lower, (based on the cytotoxicity showed previously by each extract). After 72 h, the progress of infection inhibition was determined. Cells were removed from the bottom plate with a trypsin/ ethylenediaminetetraacetic acid (250 mg) solution. Recovered cells were centrifuged at 1100 rpm at 4°C for 10 min, the supernatant was discarded and cells were washed with 1 mL of cold PBS and centrifuged at 1100 rpm at 4°C for 10 min. The supernatant was discarded and cells were suspended in 500-µL PBS and analyzed by flow cytometry (FC 500MPL, Cytomics, Brea, CA, US) with a total count of 10,000 events. The activity of tested extracts was conducted synchronously with the infection progress in culture medium alone as well as in culture medium with Amphotericin B and meglumine antimoniate as leishmanicidal drugs (positive controls). Determinations for each extract and standard drugs were conducted in triplicate in two independent experiments [15].

Statistical analysis
Cytotoxicity was determined according to cell mortality percentages obtained for each isolated experiment (extracts, AmB, Sbv, and culture medium alone). Results were expressed as median lethal concentrations (LC 50 ), and was calculated by Probit analysis [16] using the percentage of mortality, calculated using the formula in Equation 1, where the O.D of the control corresponds to 100% of viability (Cell growth).

% Mortality = 1 -[(O.D Exposed cells)/ (O.D Control cells)×100]
(1) The antileishmanial activity was determined according to the percentage of infected cells and parasite load obtained for each experimental condition using flow cytometry. The results of antileishmanial activity were expressed as the median effective concentration (EC 50 ) determined by the probit method [16] using the percentage of viable intracellular parasites, calculated using the formula in Equation 2, where the mean fluorescence intensity (MFI) of the control-infected cells corresponds to 100% of parasitemia.
The selectivity index (SI) was calculated by dividing the cytotoxic activity and the leishmanicidal activity using the following formula: SI = LC 50 /EC 50 Experiments were repeated at least 3 times for each concentration. Statgraphics Plus program version 5.0 (Statistical Graphics Corp., Rockville, MD) was used for all regression calculations with a significance level of p<0.05.

Extraction and phytochemical screening
Extraction yield and phytochemical results for hexane, dichloromethane, ethyl acetate, and ethanol extracts for L.S, P.F, O.F, and P.B are reported in Table-2.

Leishmanicidal activity and cytotoxicity
The effects of extracts on cell growth (viability) were assessed in human macrophages (U937 cells), which are the host cells for L. (V) panamensis parasites. Alternatively, the antiparasitic activity of these extracts was tested on intracellular amastigotes of L. (V) panamensis according to their ability to reduce the number of parasites after exposure. The results are summarized in Table-3.

Discussion
Table-2 shows that the best results were recorded for ethanol extracts from L.S, P.F, and O.F with a percentage yield of 17.0, 6.2, and 8.5%, respectively, and hexane extract from P.B with a percentage yield of 15.1%. The preliminary phytochemical analysis of the extracts revealed triterpenes in hexane, dichloromethane, and ethyl acetate extracts, while, tannins, leucoanthocyanidins, and saponins were only present in ethanol extracts of all plants. Alkaloids were found only in the ethanol extract from P.F. The presence of coumarins was found in the ethanol extract from L.S, as well as in ethyl acetate and ethanol extracts from P.F and P.B.
According to the results shown in Table-2, dichloromethane extracts from O.F and P.B, ethanol extract from P.B, and ethyl acetate extracts of all plants were active with EC 50 <30 µg/mL. Ethyl acetate was the most active extract, which showed EC 50 values of 9.8, 14.1, 23.7, and 25.5 µg/mL, for L.S, P.B, O.F, and P.F, respectively. Hexane extract from P.B and O.F exhibited moderate activity with EC 50 values of 84.8 and 87.4 µg/mL, respectively. The activity of these extracts was probably due to the presence of compounds such as terpenoids, flavonoids, and coumarins (Table-1), which have long been recognized for their antiprotozoal activity [17][18][19][20].
Unfortunately, hexane extracts from L.S and P.F, dichloromethane extract from L.S, and ethanol extract from L.S, P.F, and O.F displayed leishmanicidal activity but at concentrations toxic to U937 cells, which serve as host cells of Leishmania parasites (EC 50 >20 µg/mL).
Hexane and ethanol extracts from O.F, ethyl acetate and ethanol extracts from L.S, ethanol extract from P.B, all extracts from P.F, and pentavalent antimonial (meglumine antimoniate) were not toxic. Alternatively, hexane, and dichloromethane extracts from L. salicifolia and P.B as well as dichloromethane and ethyl acetate extracts from O.F displayed high cytotoxicity in a manner similar to Amphotericin B.
Except for the hexane extract from P.B, all SIs suggest that these extracts are selective since they were more active against L. (V) panamensis than U937 cells. Leishmanicidal activity displayed by these extracts against intracellular amastigotes of L. (V) panamensis suggests that they are promising candidates in the search for new antileishmanial compounds, mainly ethyl acetate extract from L.S, exhibited high SI and activity comparable to the conventional drug -meglumine antimoniate.

Conclusion
The antileishmanial and cytotoxic screening of 16 extracts from four species of Colombian plants were reported. Based on activities observed, ethyl acetate extracts from all plants can continue in its usage in the search for new antileishmanial drugs, although further studies are required.
Different lineages of angiosperms produce useful metabolites for treating tropical diseases. In this study, it was shown that different species belonging to the Magnoliids -a basal group within the flowering plants -and the rest, which belongs to much more recent lineages, are endowed with molecules that can control tropical parasites. This is evident that the molecules of interest for controlling these diseases are widely distributed in the lineages of flowering plants.