Antileishmanial Potential of Tropical Rainforest Plant Extracts

A total of 115 different plant extracts from our collection, representing 96 plant species, have been evaluated for in vitro antileishmanial activity against L. amazonensis promastigotes. In addition, the extracts were screened for cytotoxic activity against BALB/c mouse macrophages in order to assess a selectivity index. Crude extracts that showed a selectivity index (CC50 for macrophage / IC50 for promastigotes) ≥ 5 or with IC50 < 12.5 μg/mL against promastigotes, a total of 28 extracts, were further screened for anti-amastigote activity. A total of 25 extracts showed promising activity against L. amazonensis promastigotes with low cytotoxic activity. Ten of these extracts showed selectivity indices, (CC50 for macrophages / IC50 for amastigotes) greater than 10 and are considered “hits”, worthy candidates for further phytochemical exploration: Conostegia xalapensis methanol bark extract, Endiandra palmerstonii bark extract, Eugenia monteverdensis acetone bark extract, Eugenia sp. “fine leaf” acetone bark extract, Exothea paniculata chloroform bark extract, Mallotus paniculatus ethanol bark extract, Matelea pseudobarbata ethanol extract, Quercus insignis ethanol bark extract, Sassafras albidum dichloromethane bark extract, and Stemmadenia donnell-smithii acetone bark extract.


Introduction
Leishmaniasis is a collection of chronic infectious diseases caused by different species of parasitic Leishmania protozoa and are transmitted by sandflies (Phlebotomus spp. and Lutzomyia spp.) [1,2]. The disease, considered to be a "neglected disease", currently affects around 12 million people, with nearly 350 million people at risk of infection around the world. The clinical forms of leishmaniasis have been classified as cutaneous, mucocutaneous, or visceral. Additionally, global climate change will likely affect the geographical range of Leishmania infections in North America [3] and Europe [4]. Indeed, Phlebotomus sandfly distribution in Europe has shifted as far north as Germany [5] while Lutzomyia sandflies are now found as far north as Ohio [6], Maryland, and Delaware [7].
Current proven chemotherapy includes the pentavalent antimonials meglumine antimoniate (glucantime) and sodium stibogluconate (pentostam) [8], miltefosine, amphotericin B, or pentamidine [9]. However, all of these treatments are associated with undesirable side effects, induction of parasite resistance, and relatively high cost in developing countries. Due to the limitations of current chemotherapeutic regimes, along with the absence of suitable vaccines, there is a persistent need for alternative and readily available chemotherapies for treatment of leishmaniasis.
Natural products have overwhelmingly contributed to the pharmacopeia of the world and continue to provide new and effective anti-infective agents [10][11][12]. Recently, several reviews have appeared demonstrating the potential of higher plants and phytochemicals as treatment options for leishmaniasis [13][14][15][16][17][18][19]. In this current work, we present the in vitro screening of 115 different extracts from higher plants (96 species) collected from rainforests of north Queensland, Australia, Abaco Island, Bahamas, Costa Rica, southeastern U.S., and Zimbabwe, against Leishmania amazonensis.

Plant Materials and Reference Drugs
Plant materials from 96 species of plants (Table 1) were collected and extracted as previously described [20][21][22]. The crude extracts were dissolved in dimethylsulfoxide (DMSO) at 20 mg/mL. The reference drugs amphotericin B (Imefa, Havana, Cuba) and pentamidine (Richet, Buenos Aires, Argentina) were also used. diluted (1:1) down each lane of the 96-well plate with medium (removing 50 μL of test solution and diluting with 50 μL medium). Then, 50 µL of exponentially growing cells at 2 × 10 5 promastigotes/mL were added to each well to give final concentrations ranging from 12.5 to 200 μg/mL. After an incubation of 72 hours at 26 °C, 20 µL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (SIGMA, St. Louis, MO, USA) was added. MTT solutions were prepared at 5 mg/mL in PBS, filtered and sterilized just prior to use. After incubation for an additional 4 h, the formazan crystals were dissolved by addition of 100 μL of DMSO. The optical density was determined using a spectrophotometer (Sirio S Reader, 2.4-0, Italy), at a test wavelength of 560 nm and a reference wavelength of 630 nm [23,24] and median inhibitory concentrations (IC 50 ) were calculated.

Cytotoxicity Assay
The cytotoxic median concentration (CC 50 ) of the extracts was determined on mouse peritoneal macrophages, which are the host cells for the amastigote form of the parasite, aiming to investigate macrophage toxicity caused by the extracts [23]. Resident macrophages were collected from peritoneal cavities of normal BALB/c mice in ice-cold RPMI 1640 medium (SIGMA, St. Louis, MO, USA) supplemented with antibiotics, and seeded at 30,000 cells/well. The cells were incubated for 2 h at 37 °C in 5% CO 2 . Non-adherent cells were removed by washing with phosphate-buffered saline (PBS), and then 2 μL of extract/DMSO solutions were added to 98 μL medium with 10% HFBS and antibiotics. Then, five two-fold dilutions were carried out taking 50 µL each time and adding an additional 50 µL of medium to give test concentrations of the extracts ranging from 12.5 to 200 μg/mL. The cells were incubated with the test solutions for 72 h. Macrophages treated with DMSO were included as controls. The cytotoxicity was determined using the colorimetric assay with MTT as previously described; 15 μL MTT solution added to each well. After incubation for an additional 4 h the formazan crystals were dissolved by addition of 100 μL of DMSO and the optical density was determined.

Selectivity Index
The selectivity index (SI) ratio (CC 50 for macrophage / IC 50 for promastigotes) was used to compare the toxicity of the extracts for murine macrophages and the activity against Leishmania promastigotes. Extracts with a SI ≥ 5 or with IC 50 < 12.5 μg/mL against promastigotes were selected for further anti-amastigote determination.

Anti-Amastigote Assay
The peritoneal macrophages were harvested and plated at 10 6 cells/mL in 24-well Lab-Tek plates (Costar ® , Washington, DC, USA) and incubated at 37 °C under an atmosphere of 5% CO 2 for 2 h. Non-adherent cells were removed by washing with PBS. Stationary-phase of L. amazonensis promastigotes were added at a 4:1 parasite/macrophage ratio and the cultures were incubated for an additional 4 h. The cell monolayers were washed three times with PBS to remove free parasites. Then, 1990 μL of RPMI completed medium and 10 μL of the different extracts dissolved in DMSO were added to each well. Four two-fold dilutions were carried out taking 1000 µL each time to give test concentrations of the extracts ranging from 12.5 to 100 μg/mL, in duplicate cultures, incubating for an additional 48 h [25]. The parasites were then fixed in absolute methanol, stained with Giemsa, and examined under light microscopy. The number of intracellular amastigotes was determined by counting the amastigotes in 25 macrophages per sample, and the results were expressed as percent of reduction of the infection rate in comparison to that of the controls. The infection rates were obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages [26] and the IC 50 value was determined. A second SI was calculated: CC 50 for macrophages / IC 50 for intracellular amastigotes (see Table 1).

Statistical Analyses
The IC 50 of the tested extracts and reference drugs against L. amazonensis promastigotes and amastigotes and the CC 50 on peritoneal macrophages from BALB/c mice were obtained from dose-response curves fit to the data by means of the linear equation model using the STATISTICA for Windows Program (Release 4.5, StatSoft, Inc., Tulsa, OK, USA, 1993). The evaluations were performed in triplicate and the results are expressed as means ± standard deviations.

Results and Discussion
A total of 115 different plant extracts from our collection, representing 96 plant species, have been screened for in vitro antileishmanial activity against L. amazonensis. In addition, the extracts were screened for cytotoxic activity against BALB/c mouse macrophages in order to assess a selectivity index. The antileishmanial and cytotoxic activities are summarized in Table 1. A total of 18 extracts showed activity against L. amazonensis promastigotes with IC 50 values < 12.5 μg/mL. An additional 25 extracts showed antileishmanial activities ranging from IC 50 17.4 μg/mL to 63.4 μg/mL. Of the plant extracts that were active against L. amazonensis promastigotes, 18 were non-toxic to BALB/c mouse macrophages (CC 50 > 200 μg/mL). In addition, 10 others were only slightly toxic to the mouse macrophages, showing selectivity indices (CC 50 for macrophage / IC 50 for promastigotes) ≥ 5, and were considered for further testing against L. amazonensis amastigotes. Additional screening against L. amazonensis amastigotes revealed ten extracts with IC 50 ≤ 21.0 μg/mL against the parasites and non-toxic to the mouse macrophages (CC 50 > 200 μg/mL). That is, the selectivity indices (CC 50 for macrophage / IC 50 for amastigotes) for these extracts were all > 10. It is generally considered that for plant extracts showing SI > 10, the antiprotozoal activity is not due to general cytotoxicity and are promising extracts for further phytochemical analysis [27][28][29].
The methanol bark extract of Conostegia xalapensis (Melastomataceae) showed good leishmanicidal activity (IC 50 < 12.5 μg/mL against both promastigotes and amastigotes), but was neither cytotoxic to BALB/c mouse macrophages nor human tumor cells [21]. Thus, this plant extract shows excellent selectivity and apparently low acute toxicity. To our knowledge, no phytochemical work has been carried out on this plant; it is not known what compounds may be responsible for the antileishmanial activity. Although the dichloromethane bark extract of Quercus insignis (Fagaceae) was inactive, the ethanol bark extract exhibited notable antileishmanial activity against L. amazonensis promastigotes and amastigotes (IC 50 = 17.8 and 21.0 μg/mL, respectively). Q. insignis ethanol bark extract showed no in vitro toxicity to mouse macrophage cells or human tumor cells [21]. The phytochemistry of this tree has not been examined.
Interestingly, both the chloroform and methanol bark extracts of Exothea paniculata (Sapindaceae) from Monteverde, Costa Rica, showed good activity against L. amazonensis with little cytotoxicity on BALB/c mouse macrophages, but neither the acetone bark extract nor the methanol bark extract of E. paniculata from Abaco Island, Bahamas was leishmanicidal. The chloroform extract of E. paniculata from Monteverde showed the most promise with IC 50 < 12.5 μg/mL against both the promastigote and amastigote forms of L. amazonensis, but non-toxic to macrophage cells. There have been no reported studies on the phytochemical composition of E. paniculata.
Two different species of Eugenia (Myrtaceae) from Monteverde, Costa Rica, showed good antileishmanial activity. Thus, the acetone bark extracts of E. monteverdensis and Eugenia sp. "fine leaf" were active against L. amazonensis promastigotes (IC 50 = 23.9 and < 12.5 μg/mL, respectively), while neither was cytotoxic to BALB/c mouse macrophages (CC 50 > 200 μg/mL). Furthermore, E. monteverdensis acetone bark extract was inactive against MCF-7 and Hs 578T human breast tumor cells (unpublished results from this laboratory), and Eugenia "fine leaf" acetone bark extracts was inactive on several human tumor cell lines (Hep-G2 MDA-MB-231, Hs 578T, and 5637) [21]. Consistent with these results, the ethanol bark extracts of E. austin-smithii and a Eugenia sp. from the Alberto Manuel Brenes Biological Reserve, Costa Rica, showed antileishmanial activity [35]. The acetone bark extract of E. austin-smithii has been found to be cytotoxic to Hs 578T, 5637, and BHK cells [21]. Both the hexane and methanol fruit extracts of E. umbelliflora were leishmanicidal to L. amazonensis and L. braziliensis promastigotes [36]. E. uniflora has been screened for antileishmanial activity. The ethanol leaf extract was marginally active against L. braziliensis promastigotes (65% inhibition at 100 μg/mL) [37], but inactive against L. amazonensis or L. chagasi promastigotes [38]. The leaf essential oil of E. uniflora showed good antileishmanial activity against both promastigotes and amastigotes of L. amazonensis (IC 50 = 3.04 and 1.92 μg/mL, respectively) [39], but E. uniflora bark essential oil was inactive against L. donovani promastigotes [40].
The bark of Endiandra palmerstonii (Lauraceae) from far north Queensland was extracted using a chloroform/ethanol mixed solvent [22]. The crude extract showed promising antileishmanial activity (IC 50 < 12.5 μg/mL on promastigotes and 18.7 μg/mL on amastigotes) and no cytotoxicity on BALB/c mouse macrophages (CC 50 > 200 μg/mL). The extract was also non-cytotoxic to Hep-G2 human liver tumor cells [22]. There have been no phytochemical investigations reported for this tree species.
Two different bark extracts of Mallotus (Euphorbiaceae) from far north Queensland, Australia, M. mollisimus (CHCl 3 /EtOH mixed solvent bark extract) and M. paniculatus (EtOH bark extract), showed promising antileishmanial activity coupled with selectivity. M. paniculatus ethanol bark extract was the more promising with IC 50 < 12.5 μg/mL on both promastigotes and amastigotes, and no cytotoxicity to macrophage cells. Previous screening for cytotoxicity against a panel of human tumor cell lines revealed M. paniculatus ethanol bark extract to be non-cytotoxic to Hep-G2, MDA-MB-231, Hs 578T, and 5673 cells [22]. Thus, this plant extract is apparently non-toxic to mammalian cells while remarkably toxic to the parasite. The triterpenoid 29-nor-3,22-hopanedione has been isolated from the stem bark of M. paniculatus, but no bioactivities were reported for this compound [41]. M. oppositifolius leaves are used in traditional medicine in the Ivory Coast to treat intestinal helminths, but the leaf extracts were inactive against L. donovani promastigotes [42].
The acetone bark extract of Acacia choriophylla (Fabaceae) from Abaco Island, Bahamas, showed a selectivity index >3. The dichloromethane bark extract of the same tree showed similar activity. Both of these crude extracts had been previously screened for in vitro cytotoxic activity against 5637 bladder tumor and Hs 578T breast tumor cells (unpublished results from our laboratory) and were non-cytotoxic, so neither seems to be acutely toxic to human cells. The phytochemistry of this tree has not been examined.
The acetone bark extract of Tabebuia bahamensis (Bignoniaceae) was active against L. amazonensis promastigotes and amastigotes with IC 50 values of 17.4 and 23.8 μg/mL, respectively. This bark extract was screened for cytotoxic activity against a panel of human tumor cell lines, including SK-Mel-38 (melanoma), Hep-G2 (hepatocellular carcinoma), MDA-MB-231 (mammary adenocarcinoma), and 5637 (bladder carcinoma), and was found to be inactive against all cell lines screened (unpublished results from our laboratory). T. umbellata is used traditionally in Bahia, Brazil to treat cutaneous leishmaniasis (L. braziliensis) infections [48]. Likewise, T. serratifolia bark is used in Peru to treat leishmaniasis, and T. serratifolia chloroform bark extract did show in vitro activity against L. infantum promastigotes [49]. Naphthoquinones from T. avellanedae [50] and T. serratifolia [49] have shown antileishmanial activity against L. major and L. infantum, respectively. Thus, the antileishmanial activity of T. bahamensis bark extract may also be due to naphthoquinones.
Four species of Melicope (Rutaceae) from north Queensland, Australia, were screened for antileishmanial activity. M. broadbentiana and M. vitiflora bark extracts were inactive, but M. jonesii chloroform bark extract and M. rubra ethanol bark extract showed promising activity against L. amazonensis promastigotes. Further screening revealed no selectivity for L. amazonensis amastigotes over mouse macrophages, however. Both M. jonesii and M. rubra bark extracts showed in vitro cytotoxicity to human tumor cells [22]. Although the leaf essential oils of M. jonesii and M. rubra have been investigated [52], there are no reports on the bark phytochemistry. The chloroform bark extract of Polyosma alangiacea (Escalloniaceae) from far north Queensland, Australia was somewhat leishmanicidal (IC 50 = 60.9 μg/mL) against promastigotes, but non-cytotoxic to mammalian cells, either mouse macrophages or human tumor cells [22]. The phytochemistry of P. alangiacea bark has not been reported.
The chloroform bark extract of an, as yet, undescribed species of Myrcianthes "black fruit" (Myrtaceae) from Monteverde, Costa Rica, does show promise with leishmanicidal IC 50 of < 12.5 and 18.3 μg/mL, respectively, against promastigotes and amastigotes of L. amazonensis. The aerial parts of the liana Ruyschia phylladenia (Marcgraviaceae), collected from Monteverde, Costa Rica, were extracted with dichloromethane. The extract inhibited L. amazonensis promastigotes (IC 50 < 12.5 μg/mL) and amastigotes (IC 50 = 22.0 μg/mL). Although this extract was cytotoxic to human tumor cells [21] no cytotoxicity on BALB/c mouse macrophages was observed.
The dichloromethane bark extract of Cupania glabra (Sapindaceae), collected from Monteverde, Costa Rica, had shown remarkable in vitro cytotoxic activity against several human tumor cell lines, which was attributed to the fatty alcohol glycoside cupanioside [53]. The ethanol bark extract of this tree, on the other hand, was devoid of cytotoxic activity [21], and in this work, the ethanol extract was leishmanicidal (IC 50 = 17.6 and 27.0 μg/mL on promastigotes and amastigotes, respectively) and non-toxic to BALB/c mouse macrophages. Consistent with these results, the crude methanol bark extract of C. dentata from the Yucatan peninsula of Mexico showed in vitro antileishmanial activity against L. mexicana promastigotes (IC 50 = 13 μg/mL) [54]. Interestingly, the crude hexane bark extract of C. cinerea showed in vitro leishmanicidal activity against L. donovani axenic amastigotes [29], possibly attributable to the diterpene glycoside cupacinoside [55]. Similarly, the hexane leaf extract of C. vernalis was active against L. donovani promastigotes [56]. Extracts of C. macrophylla from Costa Rica, on the other hand, were inactive against Leishmania promastigotes [35].
The crude ethanol bark extract of Diospyros digyna (Ebenaceae) showed in vitro antileishmanial activity against L. amazonensis promastigotes and amastigotes with IC 50 = 25.0 and 29.2 μg/mL, respectively. Additionally, D. digyna extract was not cytotoxic to either BALB/c mouse macrophages or MCF-7, UACC-257, MDA-MB-231, or M-14 human tumor cells (unpublished results from this laboratory). Diospyrin, a bis-naphthoquinone isolated from D. montana, has shown in vitro activity against L. donovani promastigotes [57] and L. major promastigotes [58]. This compound has been shown to be a topoisomerase I inhibitor of L. donovani [59] and initiates apoptosis in L. donovani [60]. Antileishmanial bis-naphthoquinones have also been isolated and characterized from D. assimilis chloroform root extract from India (in vitro assay against L. donovani axenic amastigotes) [61] and D. burmanica methanol wood extract from Myanmar (in-vitro assay against L. major promastigotes) [62].
The crude ethanol bark extract of Erythrina lanceolata (Fabaceae) showed promising antileishmanial activity (IC 50 < 12.5 μg/mL against promastigotes) without cytotoxic activity against BALB/c mouse macrophages or a number of human tumor cell lines (Hep-G2, MDA-MB-231, MCF-7, Hs 578T, PC-3, SK-MEL-28), or baby hamster kidney (BHK) cells [21]. In contrast, the crude dichloromethane bark extract was not leishmanicidal but cytotoxic to all cells tested [21]. The hydroalcoholic extract of E. speciosa from Brazil was tested for antileishmanial activity against both L. amazonensis promastigotes and amastigotes, but was inactive [63]. Likewise, the methanol wood extract of E. suberosa from Myanmar was inactive against L. major promastigotes [64], and the ethanol bark extract of E. variegata from New Caledonia was inactive against L. donovani promastigotes [65].
The acetone bark extract of Inga sierrae (Fabaceae) from Monteverde, Costa Rica, showed leishmanicidal activity (IC 50 = 25.7 μg/mL) with reduced cytotoxic activity (CC 50 = 130.6 μg/mL). Several Inga species are used in traditional medicine to treat leishmaniasis. I. edulis leaves are used by the Kichwa in Ecuador [29] while I. edulis bark is used by the Wayãpi people of French Guiana [66] to treat leishmaniasis. The Chachi people of Ecuador also use I. oerstediana leaves and bark [29] and I. bourgoni bark is also used by the Wayãpi [66] to treat leishmaniasis.
The methanol bark extract from Pappea capensis (Sapindaceae) collected in Matabeleland, Zimbabwe, also showed promising antileishmanial activity (IC 50 < 12.5 μg/mL). Rhynchosia resinosa (Fabaceae) methanol root extract, from Matabeleland, Zimbabwe, did not exhibit cytotoxic activity toward BALB/c mouse macrophages, but was active against L. amazonensis amastigotes with an IC 50 of 27.7 μg/mL. The methanol bark extract of R. edulis from Monteverde, Costa Rica was shown to be inactive against L. amazonensis (this work), while the methanol extract of the whole plant of R. reniformis from Karak, Pakistan was also non-leishmanicidal [67].

Conclusions
Of the 115 extracts screened, 25 (21.7%) showed promising activity against L. amazonensis promastigotes with low cytotoxic activity. Additional antileishmanial screening against L. amazonensis amastigotes revealed ten of these extracts (8.7%) to be considered "hits", with selectivity indices, CC 50 (macrophage)/IC 50 (amastigotes) greater than 10 that are worthy candidates for further phytochemical exploration: Conostegia xalapensis methanol bark extract, Endiandra palmerstonii bark extract, Eugenia monteverdensis acetone bark extract, Eugenia sp. "fine leaf" acetone bark extract, Exothea paniculata chloroform bark extract, Mallotus paniculatus ethanol bark extract, Matelea pseudobarbata ethanol extract, Quercus insignis ethanol bark extract, Sassafras albidum dichloromethane bark extract, and Stemmadenia donnell-smithii acetone bark extract. The good antileishmanial activity coupled with low cytotoxicity to mammalian cells indicates promise in terms of therapeutic index. Phytochemical analyses are currently underway in our laboratories. The results of this study demonstrate the medicinal potential of tropical rainforests and may provide complementary, safe, and affordable therapeutics for treatment of leishmaniasis. A total of 85 extracts were inactive or unspecific, which constitute 74% of tested samples, and reinforces the need to screen large series of natural products to find promising ones.