In vitro antileishmanial, antiplasmodial and cytotoxic activities of phenolics and triterpenoids from Baccharis dracunculifolia D. C. (Asteraceae)

Fitoterapia 80 (2009) 478–482

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In vitro antileishmanial, antiplasmodial and cytotoxic activities of phenolics and triterpenoids from Baccharis dracunculifolia D. C. (Asteraceae) A.A. da Silva Filho a,⁎, D.O. Resende a, M.J. Fukui a, F.F. Santos a, P.M. Pauletti a, W.R. Cunha a, M.L.A. Silva a, L.E. Gregório b, J.K. Bastos b, N.P.D. Nanayakkara c a

Laboratório de Produtos Naturais, Núcleo de Pesquisa em Ciências Exatas e Tecnológicas, Universidade de Franca, Av. Dr. Armando Sales de Oliveira, 201, CEP 14404600, Franca, SP, Brazil b Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP 14040-903, Brazil c National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Mississippi 38677, USA

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Article history: Received 23 April 2009 Accepted in revised form 8 June 2009 Available online 18 June 2009 Keywords: Baccharis dracunculifolia Brazilian green propolis Leishmania donovani Plasmodium falciparum Ursolic acid Hautriwaic acid lactone

a b s t r a c t Baccharis dracunculifolia (Asteraceae), the most important plant source of the Brazilian green propolis (GPE), displayed in vitro activity against Leishmania donovani, with an IC50 value of 45 µg/mL, while GPE presented an IC50 value of 49 µg/mL. Among the isolated compounds of B. dracunculifolia, ursolic acid, and hautriwaic acid lactone showed IC50 values of 3.7 µg/mL and 7.0 µg/mL, respectively. Uvaol, acacetin, and ermanin displayed moderate antileishmanial activity. Regarding the antiplasmodial assay against Plasmodium falciparum, BdE and GPE gave similar IC50 values (about 20 µg/mL), while Hautriwaic acid lactone led to an IC50 value of 0.8 µg/mL (D6 clone). © 2009 Elsevier B.V. All rights reserved.

1. Introduction Leishmaniasis, a disease caused by a number of species of protozoan parasites belonging to the genus Leishmania, is regarded as a major public health problem that affects around 12 million people in 80 countries and causes morbidity and mortality mainly in Africa, Asia, and Latin America [1,2]. In Brazil alone, about 26,000 cases of leishmaniasis are registered per year [3]. Historically, the chemotherapy of leishmaniasis has been based on the use of pentavalent antimonial drugs. Other medications, such as pentamidine and amphotericin B, have been used as alternative drugs. However, these medicines are not orally active, requiring long-term parenteral administration, not to mention that they lead to serious side effects [1,2].

⁎ Corresponding author. Tel.: +55 16 3711 8871; fax: +55 16 3711 8878. E-mail addresses: [email protected], silvafi[email protected] (A.A.S. Filho). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.06.007

Malaria is another important tropical disease which has the potential to affect nearly 40% of the world's population and is responsible for 1–2 million deaths each year [2,4]. Human malaria is endemic to 90 countries and is caused by protozoan parasites of the genus Plasmodium, mainly Plasmodium falciparum. Recently, the clinical use of artemisinin, a sesquiterpene lactone produced by Artemisia annua, for the treatment of malaria has prompted interest in the discovery of new pharmaceuticals of plant origin with antiplasmodial activity [2,5]. Baccharis dracunculifolia D.C., popularly known as “alecrim do campo” and “vassoura,” is used in folk medicine as antiinflammatory and is employed in the treatment of gastrointestinal diseases [6]. Besides its use in traditional medicine, B. dracunculifolia has been described as the most important plant source of the South Eastern Brazilian propolis, which is called green propolis due to its colour [7,8]. Propolis is a natural resinous substance collected by honeybees (Apis mellifera) from buds and exudates of plants for use as a protective barrier in the beehive. It displays many

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biological activities, such as antibacterial and antioxidant actions [9,10]. Because of its biological activities, Brazilian green propolis is currently being extensively used in foods and beverages, especially in Brazil and Japan, aiming to improve health and to prevent several diseases [11]. Baccharis dracunculifolia and Brazilian green propolis have been reported to display similar anticariogenic [11], antimicrobial [12], anti-ulcer [8], and immunomodulatory [13] activities. Recently, biological studies have demonstrated that crude extracts and isolated compounds from B. dracunculifolia and Brazilian green propolis have trypanocidal activities [6]. It has also been reported that Brazilian green propolis displays in vitro and in vivo antileishmanial activity against L. braziliensis [14]. However, it is still unknown whether B. dracunculifolia presents the antileishmanial activity reported for Brazilian green propolis. In this sense, as part of our ongoing biological studies on Brazilian green propolis and B. dracunculifolia [10–13], as well as on the antiprotozoal activities of natural products [15–17], we now report the results obtained for the in vitro evaluation of the antileishmanial and antiplasmodial activities of the crude extract and isolated compounds from B. dracunculifolia, which have not yet been described.

3 (10 mg). The dichloromethane fraction (BdE-D, 22.0 g) was chromatographed over silica gel using a VLC system and hexane/ethyl acetate mixtures in increasing proportions as eluent, giving six fractions (I–VI). Fraction II (0.28 g) was washed with cold methanol, to afford 1 (250 mg). Fractions III (2.5 g) and IV (6.5 g) were chromatographed over silica gel using a VLC system and hexane/ethyl acetate mixtures in increasing proportions as eluent. The resulting sub-fractions III.2 and IV.2 were submitted to semi-preparative reverse-phase HPLC purification using methanol/H2O (75:25) as mobile phase. Fraction III.2 furnished compounds 4 (45 mg), 5 (40 mg), 6 (30 mg), and 7 (15 mg). Fraction III.4 furnished compounds 8 (30 mg) and 11 (17 mg). Fraction IV.2 afforded compounds 9 (20 mg) and 10 (15 mg). The chemical structures of all the compounds were established by 1H (400 MHz) and 13C NMR (100 MHz) data analysis, as well as comparison of the obtained data with those of authentic compounds (Fig. 1). Purity of all the isolated compounds was estimated to be higher than 95% by both 13C NMR and HPLC analysis using different solvent systems (MeOH/MeCN/H2O, 65:5:30; MeOH/H2O 50 to 100% in 20 min.).

2. Experimental

NMR spectra were recorded on a Brucker ARX 400 spectrometer. Vacuum–liquid chromatography (VLC) was carried out with silica gel 60H (100–200 mesh ASTM, Merck), in glass columns with 5–10 cm i.d. Flash chromatography was performed with silica gel (230–400 mesh, Merck) in a 450× 25 mm glass column at 5 mL/min. Both analytical and semi-preparative HPLC separation analyses were accomplished on a Shimadzu SCL-10 AVP liquid chromatography system equipped with a SPD-M10AVP Shimadzu UV-DAD detector (the channel was set at 281 nm) and Shimadzu columns (ODS column, 250× 4.6 mm, 5 µm for analytical analyses and ODS, 250 × 20 mm, 15 µm for semi-preparative separations).

2.1. Plant and propolis materials Baccharis dracunculifolia De Candole was collected in Cajuru (São Paulo State, Brazil), in November 2001. The plant material was kindly authenticated by Jimi N. Nakagima, and a voucher specimen (SPFR 06143) was deposited in the Herbarium of the Biology Department of the University of São Paulo, Ribeirão Preto (FFCLRP-USP), SP, Brazil. Brazilian green propolis was produced and collected from Apis mellifera hives, in the same field and period as B. dracunculifolia material collection was carried out.

2.3. General procedures

2.4. Antiplasmodial assay 2.2. Extraction and isolation The crude green propolis (3 g) was kept in a freezer for 24 h and powdered in a blender. The furnished powder was submitted to exhaustive maceration, followed by filtration, using ethanol/H2O (7:3 v/v), at room temperature. The filtered extract was concentrated under vacuum, to furnish 1.9 g of the crude hydroalcoholic green propolis extract (GPE) [11]. The rinsed leaf extract was obtained by immersing the air-dried leaves (615 g) in dichloromethane for 30 s at room temperature, and the solvent was removed under vacuum below 40 °C, affording 35 g of rinsed leaf extract (BdE). The crude BdE extract (35 g) was dissolved in methanol/H2O (7:3 v/v) and submitted to sequential partition with hexane and dichloromethane, giving 2.6 g and 22.1 g, respectively. The hexane fraction (BdE-H, 2.6 g) was chromatographed over silica gel using a vacuum–liquid chromatography (VLC) system and hexane/ ethyl acetate mixtures in increasing proportions as eluent, furnishing four fractions (I–IV). Fraction IV (0.62 g) was submitted to column chromatography over silica gel, using hexane/ethyl acetate mixtures in increasing proportions as eluent, followed by preparative TLC (hexane/ethyl acetate 75:25), affording compounds 1 (80 mg), 2 (15 mg), and

The in vitro antimalarial assay procedure employed here was an adaptation of the parasite lactate dehydrogenase (pLDH) assay described in the literature [5], using a 96-well microplate assay protocol with two P. falciparum clones [Sierra Leone D6 (chloroquine-sensitive) and Indochina W2 (chloroquine-resistant)]. The primary screening involved determination of the pLDH inhibition (percentage) of each sample tested at 15.9 and 1.59 µg/mL for extracts and pure compounds, respectively. The IC50 values were determined only for samples that inhibited parasite growth by N50% for one of the clones. The antimalarial agents chloroquine and artemisinin were used as positive controls, and DMSO was employed as the negative (vehicle) control. 2.5. Antileishmanial assay A transgenic cell line of Leishmania donovani promastigotes showing stable expression of luciferase was used as the test organism. Cells in 200 µL of growth medium (L-15 with 10% FCS) were plated at a density of 2 × 106 cells per mL in a clear 96-well microplate. Stock solutions of the standards and test compounds/extracts were prepared in DMSO. Culture

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Fig. 1. Chemical structures of isolated compounds from BdE. Ursolic acid (1); 2α-hydroxy-ursolic acid (2); uvaol (3), isosakuranetin (4); aromadendrin-4′methylether (5); acacetin (6); ermanin (7); baccharin (8); hautriwaic acid lactone (9); clerodane diterpene (10); viscidone (11).

medium without cells and the controls were incubated (at 26 °C for 72 h) simultaneously, in duplicate, at six concentrations of the test compounds. An aliquot of 50 µL was transferred from each well to a fresh opaque/black microplate, and 40 µL of Steadyglo reagent was added to each well. The plates were read immediately in a Polar Star galaxy microplate luminometer. IC50 and IC90 values were calculated from dose-response inhibition graphs. Pentamidine and amphotericin B were tested as standard antileishmanial agents [4,16]. 2.6. Cytotoxicity assay The mammalian cells Vero (African green monkey kidney fibroblast) used in this study were obtained from ATCC (Manassas, VA). Cytotoxicity was determined by the neutral red method according to a previously described procedure [5]. The IC50 value for each compound was computed from the growth inhibition curve. 3. Results and discussion The spectral data of all the isolated compounds (Fig. 1) were in agreement with the previously published data, thereby allowing for identification of ursolic acid (1) [18], 2α-hydroxy-ursolic acid (2), uvaol (3) [19], isosakuranetin (4) [20], aromadendrin-4′-methylether (5) [21], acacetin (6) [22], ermanin (7) [23], baccharin (8) [21], hautriwaic acid lactone (9) [24], clerodane diterpene (10) [25], and viscidone (11) [26]. A wide variety of chemical compounds have been identified from Brazilian green propolis, such as flavonoids, diterpenes, and mainly prenylated p-coumaric acid deriva-

tives, which are particularly found in South American Baccharis species [20,21]. It is well known that the chemical composition of propolis can change depending on several factors, including the collection site and the plant sources used in propolis production [13]. Therefore, the composition of propolis can be extraordinary variable because of this, creating a problem for its medical use and its quality control. Also, such variations hinder standardization of the raw material and commercialization of propolis products for medicinal purposes. Thus, the investigation of both chemical and biological properties of propolis plant sources, such as B. dracunculifolia, is important not only for its academic interest, but also for the chemical and biological standardization of propolis raw material [13]. Furthermore, it has been suggested that if B. dracunculifolia and Brazilian green propolis present comparable biological activities, B. dracunculifolia extracts could be successfully incorporated into pharmaceutical products for use in foods and beverages [11]. Previous phytochemical studies of the aerial parts of B. dracunculifolia have described the isolation of p-coumaric acid derivatives, flavonoids, diterpenes, and triterpenes [6,13,20]. It is also known that p-coumaric acid derivatives, such as artepillin C, and flavonoids are the major compounds of Brazilian green propolis [7,27]. Pentacyclic triterpenes and flavones have been isolated from some Baccharis species [12,20]. However, this is the first time that the presence of uvaol (3), acacetin (6), and ermanin (7) in B. dracunculifolia has been reported. Regarding the in vitro antileishmanial assay, the crude extract of B. dracunculifolia (BdE) presented an IC50 value of 45 µg/mL (Table 1), while GPE displayed a similar IC50 value of 49 µg/mL. Moreover, among all the isolated compounds, ursolic acid (1) and hautriwaic acid lactone (9) exhibited the

A.A.S. Filho et al. / Fitoterapia 80 (2009) 478–482 Table 1 In vitro antileishmanial, antiplasmodial and cytotoxic activities of constituents from B. dracunculifolia. Extracts/ compounds

L. donovani (µg/mL) IC50

BdE GPE 1 2 3 4 5 6 7 8 9 10 11 Chloroquine Artemisinin Pentamidine Amphotericin B

IC90

45 49 3.7 19.0 15.0 NA NA 18.0 40.0 NA 7.0 NA

93 92 6.8 34.0 33.0 NA NA 40.0 N 40 NA 28.0 NA

– – 1.9 0.7

– – 8.5 1.7

P. falciparum (µg/mL) D6 clone a c

IC50

SI

25 20 1.0 3.2 3.3 NA NA NA 2.6 NA 0.8 3.0 1.9 0.018 0.014 – –

N 1.9 N 2.4 N 4.8 N 1.5 N 1.4 – – – N 1.8 – N 5.6 N 1.6 N 2.5 N 556 N 669 – –

Cytotoxicity (Vero cells)

W2 clone b IC50

SI c

IC50 (µg/mL)

13 13 NT 3 1.9 NT NT NT 2.2 NT 2.2 2.6 2.3 0.176 0.008 – –

N 3.7 N 3.7 – N 1.6 N 2.5 – – – N 2.2 – N 2.2 N 1.8 N 2.1 N 56 N 1176 – –

NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC – –

NA = not active (concentration for pure compounds: P. falciparum 4.7 µg/ mL; L. donovani 40 µg/mL). NT = not tested. NC = not cytotoxic (up to the maximum dose tested). a Chloroquine-sensitive clone. b Chloroquine-resistant clone. c Selectivity index = IC50 Vero cells/IC50 P. falciparum.

highest antileishmanial activities, leading to IC50 values of 3.7 µg/mL and 7.0 µg/mL, respectively. In addition, 2αhydroxy-ursolic acid (2) and uvaol (3) gave IC50 values of 19.0 µg/mL and 15.0 µg/mL, respectively, while the diterpene (10) was inactive against L. donovani (Table 1). Pentamidine and amphotericin B, used as positive control, showed IC50 values of 1.9 µg/mL and 0.7 µg/mL, respectively. Hautriwaic acid lactone (9) is quite similar to the diterpene acid (10), differing mainly at the functional groups of C-3, C-4, and C-5. Taking their antileishmanial activities into account, it is suggested that the lactone ring present in compound 9 may improve the antileishmanial activity of clerodane diterpenes, since compound 10 was inactive. The antileishmanial activities of clerodane and labdane diterpenes, which are known for their antifeedant activity, have been reported in the literature [28]. Also, clerodane diterpenes from Baccharis species have been reported [20], but they are rare in propolis samples. Ursolic acid (1) and related pentacyclic triterpenes are widely found in some Baccharis species [12,20], and they display several biological activities, such as trypanocidal and antimicrobial actions [18,29]. However, compounds 1, 2, and 3 have not been previously reported from Brazilian green propolis. Comparing the antileishmanial activities of compounds 1, 2, and 3, there is evidence that the hydroxyl groups at C-2 and C-3 play an important role in the in vitro activity against L. donovani, since ursolic acid (1) and uvaol (3) were more active than 2α-hydroxy-ursolic acid (2). These findings are in accordance with the results of Torres-Santos et al. [30], who found antileishmanial activities for 1 and 2 against L. amazonensis parasites and proposed that the hydroxylation pattern of ursane triterpenoids derivatives, mainly at C-3, is important for such activity. Furthermore, a comparison of the

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antileishmanial activity of the pentacyclic triterpenes 1 and 3 suggested that the presence of the carboxyl group at C-28 may be important for their antileishmanial activities, as reported by Cunha et al. [18], which described the trypanocidal activity of ursolic acid derivatives. Moreover, as previously suggested, this class of compounds should also be considered for further antiprotozoal studies [18]. Additionally, isosakuranetin (4) and aromadendrin-4′methylether (5) are major flavonoids found in both green propolis and B. dracunculifolia [7]. However, both compounds 4 and 5 were inactive against promastigote forms of L. donovani. On the other hand, acacetin (6) and ermanin (7) exhibited antileishmanial activities, displaying IC50 values of 18.0 µg/mL and 40.0 µg/mL, respectively. The structureantileishmanial activity relationship of flavonones has been described in the literature, and it has been shown that this class of flavonoids, mainly dihydroxyflavone derivatives, consists in potential antiprotozoal agents [31]. As for baccharin (8) and viscidone (11), these compounds have frequently been isolated from B. dracunculifolia [6,7,12,21,27], but they were inactive in the antiprotozoal assays. Besides, our results showed that the BdE, which is similar to the GPE, displays antileishmanial activity, which may be related to the effect of several compounds present in the crude extract, mainly triterpenes, diterpenes, and flavonoids. Concerning the antiplasmodial assay, BdE led to an IC50 value of 25 µg/mL (D6 clone) (Table 1), while GPE displayed a similar IC50 value of 20 µg/mL against P. falciparum D6 clone. Among the evaluated compounds, only hautriwaic acid lactone (9) was moderately active, with IC50 values of 0.8 µg/mL (D6 clone) and 2.2 µg/mL (W2 clone), while chloroquine and artemisinin, used as positive controls, gave IC50 values of 0.018 µg/mL and 0.014 µg/mL, respectively, against P. falciparum (D6 clone). Moreover, it is important to point out that all evaluated samples showed no cytotoxicity against Vero cells in the maximum dose tested (Table 1). In conclusion, the present study provided biological evidence that B. dracunculifolia, like Brazilian green propolis, displays in vitro antileishmanial activity. Finally, since B. dracunculifolia is the main botanical source of the Brazilian green propolis, further studies are in progress to disclose other important biological effects of this medicinal plant, including the in vivo antileishmanial activity. Acknowledgments The authors are grateful to FAPESP (grants # 06/60132-4; 01/14209-7) for financial support and CAPES for fellowships (grant # PDEE/BEX 0387/04-5). We are thankful to J. Nakagima (Instituto de Biologia, Universidade Federal de Uberlândia, Brazil) for plant identification. References [1] Rocha LG, Almeida JRGS, Macedo RO, Barbosa-Filho JM. Phytomedicine 2005;12:514. [2] Kayser O, Kiderlen AF, Croft SL. Stud Nat Prod Chem 2002;26:779. [3] Neto AG, Da Silva Filho AA, Costa JMLC, Vinholis AHC, Souza GHB, Cunha WR, et al. Phytomedicine 2004;11:662. [4] Andrade SF, Da Silva Filho AA, Resende DO, Silva MLA, Cunha WR, Nanayakkara NPD, et al. Z Naturforsch C 2008;63:889.

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