Parasitology International 56 (2007) 135–139 www.elsevier.com/locate/parint

Leishmanicidal activity of a supercritical fluid fraction obtained from Tabernaemontana catharinensis ⁎ Deivid Costa Soares a, Camila G. Pereira b, Maria Ângela A. Meireles b, Elvira Maria Saraiva a,

a Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil b LASEFI DEA/FEA, Universidade Estadual de Campinas, Campinas, São Paulo, Cx. Postal 6121, 13001-970, Brazil Received 28 August 2006; received in revised form 11 January 2007; accepted 15 January 2007 Available online 20 January 2007

Abstract

The branches and leaves of Tabernaemontana catharinensis were extracted with supercritical fluid using a mixture of CO2 plus ethanol (SFE), and the indole enriched fraction (AF3) was selected for anti-Leishmania activity studies. We found that AF3 exhibits a potent effect against intracellular amastigotes of Leishmania amazonensis, a causative agent of New World cutaneous leishmaniasis. AF3 inhibits Leishmania survival in a dose-dependent manner, and reached 88% inhibition of amastigote growth at 100 μg/mL. The anti-parasite effect was independent of nitric oxide (NO), since AF3 was able to inhibit NO production induced by IFN-γ plus LPS. In addition, AF3 inhibited TGF-β production, which could have facilitated AF3-mediated parasite killing. The AF3 fraction obtained from SFE was nontoxic for host macrophages, as assessed by plasma membrane integrity and mitochondrial activity. We conclude that SFE is an efficient method for obtaining bioactive indole from plant extracts. Importantly, this method preserved the alkaloid properties associated with inhibition of Leishmania growth in macrophages without toxicity to host cells. © 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Leishmanicidal activity; Tabernaemontana; Leishmania; Supercritical Fluid Extraction

1. Introduction administration and high cost [3]. The novel drug miltefosine is an effective treatment for visceral leishmaniasis in India, but has Leishmaniasis is a prominent worldwide health problem. It shown limited efficiency in other countries and for other has been estimated that 12 million people are infected in the leishmaniasis forms, and is also teratogenic [2,4]. All of these tropical and subtropical areas of five continents, and that 2 and facts have given rise to the need for development of new 0.5 million cases of cutaneous and visceral leishmaniasis are approaches for leishmaniasis therapy. reported each year [1]. Recently, the overlapping geographical Plants have long been used in popular medicine for the distribution of leishmaniasis and human immunodeficiency treatment of protozoan diseases, and lately have received virus (HIV) infection has contributed to a dramatic increase in considerable attention in the search for new compounds with the number of individuals infected with Leishmania [1]. anti-leishmanial activity [5]. Tabernaemontana catharinensis The first choice treatment for leishmaniasis still relies on A. DC. (syn. Peschiera catharinensis A. DC. Miers) is an pentavalent antimonials, and amphotericin B or pentamidine, arboreal species of the Apocynaceae family, which resides in can be used as alternatives for resistant parasites [2]. All of these Southern Brazil, Argentine, Uruguay and Paraguay. This species compounds present several problems that limit their use, such as has also been denoted T. affinis, T. australis and T. hilariana [6– severe side effects, induction of parasite resistance, in-patient 10], and anti-tumor, anti-microbial, anti-inflammatory and analgesic activities have been reported for its extracts [7,11– ⁎ Corresponding author. Universidade Federal do Rio de Janeiro, Instituto de 14]. We recently demonstrated anti-leishmanial and anti-HIV-1 Microbiologia Prof. Paulo de Góes, Departamento de Imunologia, Centro de activities for an extract obtained from T. australis as well as the Ciências da Saúde, Avenida Carlos Chagas Filho, 373, Bloco I, sala i2-052, Ilha – do Fundão, CEP: 21941-902, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 6747; alkaloids purified from this extract [15 17]. fax: +55 21 2580 8344. Supercritical Fluid Extraction (SFE) is a technology that E-mail address: [email protected] (E.M. Saraiva). facilitates the removal of solvents from plant extracts,

1383-5769/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2007.01.004 136 D.C. Soares et al. / Parasitology International 56 (2007) 135–139 increasing selectivity and permitting the use of moderate T. catharinensis extract was added to the cultures and, after 24 h temperatures during the extraction. Considering the significant incubation as above, the cells were washed with PBS at 37 °C, advances resulting from this technology, we employed it to fixed in methanol, and stained with Giemsa. The number of produce T. catharinensis fractions [18], which were further amastigotes and the percentage of infected macrophages were tested for anti-leishmanial activity, as well as for toxicity for determined by counting at least 200 cells in triplicate cultures. vertebrate cells. Therefore, we report in this work that the Endocytic indices were obtained by multiplying the percentage selected alkaloid fraction (AF3) obtained by SFE from T. of infected macrophages by the mean number of amastigotes per catharinensis extract, presents anti-Leishmania amazonensis infected macrophage. Glucantime® (Aventis) and 18-metoxy- activity in infected macrophages independent of nitric oxide [16] were used as positive controls in these assays. production. Moreover, AF3 was nontoxic for macrophages, as Results are expressed as percentage of survival comparing judged by cell membrane integrity and mitochondrial activity endocytic indices of treated and untreated macrophages. assays. Our results suggest that SFE is a useful methodology for obtaining plant fractions with the potential for anti-leishmania 2.4. Nitric oxide production phytotherapy. Thioglycolate peritoneal mouse macrophages obtained as 2. Materials and methods above (106 cells/well in 24-well plate) were activated or not with 10% IFN-γ (4 days culture supernatant of L1210 cell line 2.1. Preparation of the extract transfected with IFN-γ gene [19]) and 100 ng/mL of lipopolysaccharide (LPS) from Escherichia coli O111:B4 The alkaloid fraction (AF3) of T. catharinensis extract was (Difco Laboratories Inc., Detroit, MI, USA). After 24 h at obtained by SFE in a previous work [18]. Briefly, SFE extract 37 °C in 5% CO2, monolayers were treated with 100 μg/mL of was obtained at 250 bar, 45 °C using supercritical CO2 plus AF3. Alternatively, macrophages were incubated with 100 μg/ ethanol (4.6%m/m) as co-solvents. A sample of the extract mL of AF3 in addition to the activators IFN-γ plus LPS, as obtained in the third hour of the process was fractionated to above. Nitrite concentrations in 48-hour culture supernatants obtain AF3, which consists of the major alkaloid compounds were determined by the Griess method [20]. The reaction was − coronaridine and voacangine (7% and 53% of AF3, respec- read at 540 nm, and the concentration of NO2 was determined tively). The remaining content of the fraction (40%), although with reference to a standard curve using sodium nitrite. Results identified (voacristine, voacristine hydroxylindolenine, voacan- are expressed as micromolar concentrations of nitrite. gine hydroxylindolenine, and 3-hydroxylcoronaridine), was not quantified [18]. Because coronaridine has been described as an 2.5. Cytotoxicity assays anti-leishmanial compound [15,16], this fraction was selected for further studies of leishmanicidal activity and to validate the Murine peritoneal macrophages were adhered to 24-well SFE methodology. plates and treated with 100 μg/mL of AF3 for 24 h at 37 °C, 5% CO2. Macrophages were then washed with PBS, incubated with 2.2. Parasites 3% Trypan blue solution and scored for viable cells in an inverted microscope. Additionally, AF3 cytotoxicity to mouse L. (L.) amazonensis, (WHOM/BR/75/Josefa) promastigotes peritoneal macrophages was determined by the reduction of 2,3- were cultured at 26 °C in Schneider Insect Medium (Sigma) bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-car- supplemented with 10% fetal calf serum (FCS - Gibco BRL, boxinilide inner salt (XTT, Sigma) assay, according to Roehm et Gaithersburg, MD, USA) and 40 μg/mL of gentamycin al., 1991 [21]. (Schering - Plough, Rio de Janeiro, Brazil). 2.6. Cytokine production 2.3. Anti-amastigote activity Thioglycolate peritoneal mouse macrophages obtained as Murine peritoneal macrophages obtained after 3 days of above were cultured in 24-well plates and activated as described stimulation with thioglycolate were harvested in RPMI 1640 above. After 24 h at 37 °C, 5% CO2, cell monolayers were medium (Biochrom KG, Berlin, Germany). Macrophages were treated with the AF3 fraction. TGF-β1 and TNF-α production plated on 13 mm2 coverslips inside 24-well plates and allowed to were evaluated by sandwich ELISA, using capture and adhere for 2 h at 37 °C in 5% CO2. Non-adherent cells were detection antibodies obtained from R&D Systems, Inc. removed by washing, and macrophages were incubated over- (Minneapolis, MN, USA) and PeproTech (Colonia Portales, night in RPMI supplemented with 10% FCS, as described above. Mexico, DF), respectively, according to the manufacturer's Adhered macrophages were infected with L. amazonensis instructions. Assays were performed in duplicate. promastigotes (stationary growth phase) at a 10:1 parasite/ macrophage ratio and incubated for 1 h at 34 °C, 5% CO2. Free 2.7. Statistical analysis parasites were washed out with 0.01 M Phosphate Buffered Saline (PBS), and cultures were maintained for 24 h at 37 °C in Data were analyzed by Student's t-test when comparing two 5% CO2 in RPMI supplemented with 10% FCS. AF3 from groups or one-way ANOVA for more than two groups, using the D.C. Soares et al. / Parasitology International 56 (2007) 135–139 137

GraphPad Program. P values of less than 0.05 were considered significant.

3. Results and discussion

We report in this paper that the AF3 fraction obtained from T. catharinensis by SFE inhibits the replication of L. amazonensis amastigotes in macrophages. The leishmanicidal activity of AF3 was evaluated in L. amazonensis-infected macrophages by adding AF3 24 h after infection. Our results show that AF3 inhibits parasite survival in a dose-dependent manner with 88, 41 and 36% inhibition of Leishmania growth at 100, 10 and 1 μg/mL, respectively (Fig. 1). This effect was mainly due to a Fig. 1. Leishmanicidal activity of AF3 on amastigote survival. L. amazonensis- decrease in the percentage of infected macrophages, since infected mouse peritoneal macrophages were treated 24 h after infection with 10 μg/mL and 100 μg/mL of AF3 increased the amount of Glucantime® (GLU, 20 μg/mL), 18-metoxycoronaridine (18-MCOR, 1 μg/mL) uninfected macrophages by 36% and 71%, respectively (data and different concentrations of AF3. Amastigote survival was assessed 24 h after not shown). The leishmanicidal activity of AF3 resulted in IC50 treatment. Results from four experiments performed in triplicate for AF3 and values of 38±5 μg/mL. AF3 and 18-metoxy-coronaridine, a one experiment done in duplicate for GLU and 18-MCOR, are shown as percentage of survival inhibition+SEM in relation to untreated control (CTRL). synthetic coronaridine analog, showed the same leishmanicidal ⁎P<0.0001. efficiency at 1 μg/mL (Fig. 1). In addition, AF3 reached the same leishmanicidal activity of Glucantime at a concentration equivalent to only 5% of the inhibitory dose of the latter compound (Fig. 1). Importantly, the use of SFE resulted in a 10-

Fig. 2. Effect of AF3 on nitric oxide production by non-activated (open bars) and IFN-γ plus LPS-activated peritoneal macrophages (filled bars). (A) AF3 was added 24 h after macrophage activation or (B) macrophages were treated simultaneously with AF3 and IFN-γ plus LPS. Supernatants were harvested Fig. 3. AF3 cytotoxicity for macrophages. Adhered macrophages were treated 48 h after treatment, and nitrite concentration was estimated by the Griess (AF3) or untreated (Ctrl) with the AF3 fraction (100 μg/mL) and cytotoxicity method. AF3 was tested at 100 μg/mL. Data represent means+SEM of six was measured by XTT (A) and Trypan blue dye exclusion (B) methods. The different experiments performed in duplicate. All results were significant results of two independent experiments performed in duplicate are expressed as (⁎P <0.05). percentage of viable cells+SEM. 138 D.C. Soares et al. / Parasitology International 56 (2007) 135–139 fold higher yield of target substances than the yield obtained by conventional solvent extraction techniques [14,18]. Analysis of the T. catharinensis AF3 fraction by gas chromatography–mass spectrometry (GC–MS) detected the alkaloids voacangine and coronaridine as major compounds [18]. It has already been shown that coronaridine exhibits anti- bacterial, anti-leishmanial, anti-HIV-1 and other pharmacologi- cal activities [13–17,22]. Voacangine shares several biological activities with coronaridine but it remains to be demonstrated whether it is endowed with leishmanicidal activity. Although, the action of the AF3 fraction against Leishmania is likely due to the presence of coronaridine, we cannot exclude the potential leishmanicidal activity of voacangine and other alkaloids Fig. 4. Macrophage production of TGF-β1 by AF3. Macrophages were treated present in AF3. (gray bar) or untreated (white bar) with 100 μg/mL of AF3 fraction and cytokine The anti-leishmanial effect of AF3 could be due to its ability production was measured in culture supernatants harvested after 48 h of to activate cell killing mechanisms in host macrophages. As treatment by specific ELISA assay. Results expressed as pg/mL of TGF-β1 nitric oxide (NO) production is an important mechanism for represent the mean of two independent experiments performed in duplicate+ ⁎ Leishmania killing, we measured NO production by AF3 SEM. P<0.05. treated macrophages in order to determine if anti-amastigote activity resulted from activation of this mechanism (Fig. 2). We TNF-α activates macrophages for intracellular killing of observed that macrophages exposed to 100 μg/mL of AF3 pathogens [25,26], we analyzed the production of this cytokine produced the same amount of nitrite as untreated macrophages, by AF3. Our results show that AF3 does not induce production 4.6 μM versus 4.0 μM of nitrite, respectively (Fig. 2A, B). of TNF-α by macrophages (data not show), similar to our Similarly, the same levels of nitrite were produced by IFN-γ previous finding for coronaridine and 18-methoxy-coronaridine plus LPS activated macrophages, regardless of treatment with [16]. Although we cannot exclude other mechanisms of action, AF3 (45 μM versus 43 μM of nitrite) (Fig. 2A). However, it is possible that the main effect of AF3 leishmanicidal activity addition of AF3 simultaneously with the macrophage activators is direct damage of amastigotes, similar to coronaridine action IFN-γ plus LPS, resulted in a marked reduction (82%) in NO [15]. production (Fig. 2B). These results demonstrate that AF3 SFE could be considered to be an efficient method for overrides the ability of LPS plus IFN-γ to induce macrophage fractionation of plant extracts. The main advantages of this production of NO when added simultaneously, but did not methodology are easy solvent removal from the final fraction, change nitrite production in pre-activated macrophages. These moderate temperature, which avoids thermal degradation, results strongly suggest that Leishmania killing mediated by associated with high selectivity, rapidity, as well as environ- AF3 is independent of NO production. In fact, AF3 was able to mental safety [27]. SFE has already been used for alkaloid inhibit NO production induced by a powerful stimulus, such as fractionation, however, it remains poorly explored for indole IFN-γ plus LPS, similar to the phenomena we have described alkaloids [14,18]. In conclusion, our results suggest that SFE for coronaridine and 18-methoxy-coronaridine [15,16]. is an efficient method for the extraction of bioactive indole In order to test the safety of AF3 for mammalian cells, alkaloids from plant extracts, while retaining the alkaloids' macrophages were treated with AF3 and cell viability was properties associated with inhibiting L. amazonensis amasti- assessed using the XTT assay. We found that mitochondrial gote replication in macrophages without incurring host cell activity as measured by this method was preserved in AF3- toxicity. treated cells. In damaged cells, mitochondrial dehydrogenases are unable to reduce the XTT reagent to a water-soluble Acknowledgements formazan dye [23]. Macrophage treatment with the highest concentration of AF3 examined resulted in mild toxicity, since We thank Antonio Carlos Siani (FarManguinhos, Fundação only 5.6% of the cells did not metabolize the XTT reagent (Fig. Oswaldo Cruz, Rio de Janeiro, Brazil) for the helpful discussions 3A). The safety of AF3 to macrophages was further evaluated and Dumith Chequer Bou-Habib (Instituto Oswaldo Cruz, Rio using the Trypan blue dye exclusion assay. Cell toxicity was not de Janeiro, Brazil) for the critical review of the manuscript. We observed after treating macrophages with 100 μg/mL of AF3, are grateful for funds provided by CNPq, CAPES and FAPERJ. the same concentration that induced a potent leishmanicidal effect (Fig. 3B). References Interestingly, treatment of macrophages with AF3 reduced the basal production of TGF-β1(Fig. 4), a cytokine that is [1] Desjeux P, Alvar J. Leishmania/HIV co-infection: epidemiology in – clearly involved in Leishmania survival and disease suscept- Europe. Ann Trop Med Parasitol 2003;97:4 15. [2] Croft SL, Coombs GH. Leishmaniasis — current chemotherapy and recent ibility [24], by 49%. Thus, parasite destruction mediated by advances in the search for novel drugs. Trends Parasitol 2003;19:502–8. AF3 could have been facilitated by TGF-β1 inhibition, as we [3] Berman JD. Current treatment approaches to leishmaniasis. Curr Opin have previously demonstrated for coronaridine [15]. Because Infect Dis 2003;16:397–401. D.C. Soares et al. / Parasitology International 56 (2007) 135–139 139

[4] Seifert K, Matu S, Pérez-Victoria FJ, Castanys S, Gamarro F, Croft SL. and 18-methoxycoronaridine against Leishmania amazonensis. Antimicrob Characterization of Leishmania donovani promastigotites resistant to Agents Chemother 2002;46:2111–5. hexadecylphosphocholine (miltefosine). Int J Antimicrob Agents 2003; [17] Silva EM, Cirne-Santos CC, Frugulhetti ICPP, Galvão-Castro B, Saraiva 22:380–7. EMB, Kuehne ME, et al. Anti-HIV-1 activity of the iboga alkaloid [5] Kaiser O, Kiderlen AF, Croft SL. Natural products as antiparasitic drugs. congener 18-methoxycoronaridine. Planta Med 2004;70:808–12. Parasitol Res 2003;90:S55–62. [18] Pereira CG, Marques MOM, Barreto AS, Siani AC, Fernandes EC, [6] Okuyama E, Gao LH, Yamazaki M. Analgesic components from Bornean Meireles MAA. Extraction of indole alkaloids from Tabernamontana

medicinal plants, Tabernaemontana pauciflora Blume and Tabernae- catharinensis using supercritical CO2 +ethanol: an evaluation of the montana pandacaqui Poir. Chem Pharm Bull Tokyo 1992;40:2075–9. process variables and the raw material origin. J Supercrit Fluids 2004; [7] Rates SMK, Schapoval EES, Souza IA, Henriques AT. Chemical 30:51–61. constituents and pharmacological activities of Peschiera australis. Int J [19] Reis DS, Souza MA, Mineo JR, Espindola FS. Myosin V and iNOS Pharmacogn 1993;31:288–94. expression is enhanced in J774 murine macrophages treated with IFN-γ. [8] Leeuwenberg AJM. Tabernaemontana. In: Lock JM, editor. Revision of Braz J Med Biol Res 2001;34:221–6. Tabernaemontana. II. The New World Species and Stemmadenia. Kew, [20] Green SJ, Meltzer MS, Hibbs Jr JB, Nacy CA. Activated macrophages UK: The Trustees of the Royal Botanic Gardens; 1994. p. 441. destroy intracellular Leishmania major amastigotes by an L-arginine- [9] Spitzer V, Rates SMK, Henriques AT, Marx F. The fatty acid composition dependent killing mechanism. J Immunol 1990;144:278–83. of the seed oil of Peschiera australis. Fett (Germany) 1995;97:334–5. [21] Roehm NW, Rodgers GH, Hatfield SM, Glasebrook AL. An improved [10] Cardoso CAL, Vilegas W, Pozetti GL. Gas chromatographic analysis of colorimetric assay for cell proliferation and viability utilizing the indole alkaloids from Tabernaemontana hilariana. J Chromatogr A tetrazolium salt XTT. J Immunol Methods 1991;142:257–65. 1997;788:204–6. [22] Andrade MT, Lima JA, Pinto AC, Rezende CM, Carvalho MP, Epifanio [11] Pereira PS, Dias DA, Sampaio SV, França SC, Toldo MPA, Albuquerque RA. Indole alkaloids from Tabernaemontana australis (Muell. Arg) Miers S. Trypanocidal activity from Tabernaemontana catharinensis A. DC. In: that inhibit acetylcholinesterase enzyme. Bioorg Med Chem 2005;13: Martino V, Caffini N, Lappa A, editors. In ISHS Acta Horticulturae 501: II 4092–5. WOCMAP Congress Medicinal and Aromatic Plants, Part 2: Pharma- [23] Bou-Habib DC, Roderiquez G, Oravecz T, Berman PW, Lusso P, Norcross cognosy, Pharmacology, Phytomedicine, Toxicology [CD-ROM]. Men- MA. Cryptic nature of envelope V3 region epitopes protects primary donza, Argentinapp: International Society for Horticultural Science; 1999. monocytotropic human immunodeficiency virus type 1 from antibody p. 165–70. neutralization. J Virol 1994;68:6006–13. [12] Henriques AT, Melo AA, Moreno PR, Ene LL, Henriques JA, Schapoval [24] Sacks DL, Anderson C. Re-examination of the immunosuppressive EE. Ervatamia coronaria: chemical constituents and some pharmacolo- mechanisms mediating non-cure of Leishmania infection in mice. gical activities. J Ethnopharmacol 1996;50:19–25. Immunol Rev 2004;201:225–38. [13] Rastogi N, Abaul J, Goh SK, Devallois A, Philogène E, Bourgeois P. [25] Afonso LCC, Scott P. Immune responses associated with susceptibility of Antimycobacterial activity of chemically defined natural substances from C57BL/10 mice to Leishmania amazonensis. Infect Immun 1993;61: the Caribbean flora in Guadeloupe. FEMS Microbiol Lett 1998;20: 2952–9. 267–73. [26] Maioli TU, Takane E, Arantes RME, Fietto JLR, Afonso LCC. Immune [14] Pereira CG, Leal PF, Sato DN, Meireles MAA. Antioxidant and response induced by New World Leishmania species in C57BL/6 mice. antimycobacterial activities of Tabernaemontana catharinensis extracts Parasitol Res 2004;94:207–12.

obtained by supercritical CO2 +cosolvent. J Med Food 2005;8:533–8. [27] Ellington E, Bastida J, Viladomat F, Codina C. Supercritical carbon [15] Delorenzi JC, Attias M, Gattass CR, Andrade M, Rezende C, Da Cunha dioxide extraction of colchicine and related alkaloids from seeds of Col- Pinto A, et al. Leishmanial activity of an indole alkaloid from Peschiera chicum autumnale L. Phytochem Anal 2003;14:164–9. australis. Antimicrob Agents Chemother 2001;45:1349–54. [16] Delorenzi JC, Freire-De-Lima L, Gattass CR, De Andrade Costa D, He L, Kuehne ME, et al. In vitro activities of iboga alkaloid congeners coronaridine