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Rev. bras. farmacogn. vol.18 no.2 João Pessoa Apr./June 2008
Atividade tripanocida de cumarinas e estiril-2-pironas de Polygala sabulosa A.W. Bennett (Polygalaceae)
Moacir G. PizzolattiI, *; Beatriz G. MendesI; Anildo Cunha JrI; Cristian SoldiI; Adolfo H. KogaIII; Iriane EgerII; Edmundo C. GrisardIII; Mário SteindelIII
IDepartamento de Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC, Brazil
IINúcleo de Investigações Químico-Farmacêuticas, Universidade do Vale do Itajaí, 88302-202 Itajaí-SC, Brazil
IIIDepartamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC, Brazil
Bioactivity of fractions and compounds obtained from Polygala sabulosa against Trypanosoma cruzi epimastigote, blood trypomastigote and amastigote forms were evaluated in vitro. Dichloromethane and ethyl acetate fractions showed a strong trypanocidal activity on epimastigotes (IC50 < 10.4 µg/mL). Chromatographic analysis by TLC of these fractions confirmed the presence of previously described compounds (dihydrostyryl-2-pyrones, styryl-2-pyrones and 6-methoxy-7-prenyloxycoumarin). The dichloromethane fraction was fractioned by silica gel column chromatography to afford the compound α-spinasterol and the ethyl acetate fraction yielded apigenin, quercetin and a quercetin-3-O-glucoside, being the first description for the Polygala genus. 4-Methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone, 4-methoxy-6-(11,12-dimethoxystyryl)-2-pyrone, 6-methoxy-7-prenyloxycoumarin and quercetin-3-O-glucoside showed a weak activity against blood trypomastigotes (IC50 < 1008.6 µg/mL). The prenylated coumarin was the most active compound against both epimastigote and trypomastigote forms, IC50 10.5 and 88.2 µg/mL, respectively. The hemolytic activity and cell toxicity of each active compound was also assessed. Furthermore, 4-methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone and 6-methoxy-7-prenyloxycoumarin reduced 4 times the T. cruzi infection rate for Vero cells at 100 and 50 µg/mL, respectively. These results show for the first time active compounds against T. cruzi in P. sabulosa.
Keywords: Polygala sabulosa, 6-methoxy-7-prenyloxycoumarin, styryl-2-pyrones, Trypanosoma cruzi, trypanocidal activity.
A bioatividade das frações e compostos obtidos de Polygala sabulosa contra as formas epimastigota, tripomastigota sanguínea e amastigota de Trypanosoma cruzi foram avaliadas in vitro. Frações diclorometano e acetato de etila mostraram potente atividade tripanocida sob as formas epimastigotas (IC50 < 10,4 µg/mL). Análises por cromatografia em camada delgada destas frações confirmaram a presença de compostos previamente descritos (dihidroestiril-2-pironas, estiril-2-pironas e 6-metoxi-7-preniloxicumarina). Após o fracionamento da fração diclorometano por cromatografia em coluna, obteve-se o composto α-espinasterol e da fração acetato de etila obtiveram-se os compostos apigenina, quercetina e uma quercetina-3- O-glicosídeo, todos descritos pela primeira vez para o gênero Polygala. 4-metoxi-6-(11,12-metilenodioxi-14-metoxidihidroestiril)-2-pirona, 4-metoxi-6-(11,12-dimetoxi-14-metoxiestiril)-2-pirona, 6-metoxi-7-preniloxicumarina e quercetina-3- O-glicosídeo mostraram fraca atividade contra a forma tripomastigota sanguínea (IC50 < 1008,6 µg/mL). A cumarina prenilada foi o composto mais ativo contra ambas as formas epimastigota e tripomastigota, com IC50 10,5 e 88,2 µg/mL, respectivamente. A atividade hemolítica e a toxicidade celular de cada composto foram também avaliadas. Além disso, 4-metoxi-6-(11,12-metilenodioxi-14-metoxidihidroestiril)-2-pirona e 6-metoxi-7-preniloxicumarina reduziram em quatro vezes a infecção em ratos por Vero Cells nas concentrações de 100 e 50 µg/mL respectivamente. Esses resultados mostram, pela primeira vez, a atividade de compostos de P. sabulosa contra T. cruzi.
Unitermos: Polygala sabulosa, 6-metoxi-7-preniloxicumarina, estiril-2-pironas, Trypanosoma cruzi, atividade tripanocida.
Trypanosoma cruzi, the etiological agent of Chagas disease, is an obligate intracellular protozoan parasite that causes acute and chronic infection in several mammalian species, including man. Transmitted by blood-sucking triatomines (Heteroptera: Reduviidae), T. cruzi occurs throughout Central and South American countries including Mexico, where it is estimated that 16 to 18 million people are nowadays infected and approximately 120 million people are living in areas under risk of infection (WHO, 2002). In urban areas of several Central and South American countries, transmission of Chagas disease by means of transfusion of infected blood is still a major health problem (Schmuñis et al., 2001).
It is well established that gentian violet (crystal violet) is an effective compound for the prevention of Chagas disease by blood transfusion. However, the use of gentian violet is limited due to the toxic and other side effects of the compound such as alteration of the skin color, mucous membranes and urine (Ramirez et al., 1995). The current chemotherapy for human Chagas disease is based on only two nitro-heterocyclic derivatives named benznidazole (Rochagan®) and nifurtimox (Lampit®). Both compounds present a limited efficacy in the chronic phase of the infection and several side effects for the patients (Croft et al., 1997).
The search for new bioactive molecules on natural products has proven to be a powerful approach for discovery and development of new drugs (Cragg et al., 1997; Soejarto, 1996; Sepúlveda-Boza & Cassels, 1996; Bezerra et al., 2006; Saúde-Guimarães & Faria, 2007; Barbosa-Filho et al., 2007; ), thus, outnumbered plant species used in traditional medicine have been screened for biological activity (Berger et al., 1998; Mafezoli et al., 2000; Muelas-Serrano et al., 2000; Weniger et al., 2001; Schinella et al., 2002). Among these, phytochemical and biological studies of species of the genus Polygala revealed the presence of a broad spectrum of secondary metabolites with promising biological activity (Pizzolatti et al., 2003). The presence of a bisanthraquinone in P. sabulosa, an coumarin-hemiterpene ether, as well as the styryl- and dihydrostyryl-2-pyrones, a new class of compounds for the genus Polygala, were recently described (Pizzolatti et al., 2000; 2004). Along with the detection of styryl-pyrones and one coumarin, already described for this species, the present work describes the isolation of a sterol (9) and 3 flavonoids (10, 11, 12) reported for the first time in P. sabulosa. Also, the evaluation of the in vitro trypanocidal activity of some compounds was carried out.
MATERIAL AND METHODS
P. sabulosa was collected in November 1999 in Rancho Queimado (Santa Catarina, Brazil) and identified by Prof. Dr. Olavo A. Guimarães. A voucher specimen was deposited under number 19640 at the Herbarium of the Department of Botany at the Universidade Federal do Paraná, Brazil.
Extraction and isolation procedures
Whole plants were air-dried (500 g), ground to a powder and extracted with ethanol 96% at room temperature for 14 days. The alcoholic extract was then dried and the crude extract (135 g) obtained was adsorbed on silica gel (250 g) and applied at the top of a silica gel short column (5 x 5 cm). Exhaustive elution with hexane (500 mL) followed by CH2Cl2 (1 L), ethyl acetate (1 L) and finally ethanol (1 L) yielded the hexane (1.6 g), dichloromethane (28 g), ethyl acetate (16.8 g) and ethanol (63 g) fractions. The dichloromethane fr. (12 g) was subjected to column chromatography on silica gel (3 x 30 cm) and was eluted with a hexane/EtOAc gradient (56 fr., 75 mL each). The fractions obtained were then monitored by TLC (Thin-layer chromatography) (viewed by spraying with sulfuric vanillin reagent followed by heating at 110 °C) and similar fractions were combined. Combined fr. 10-14 was recrystallized from acetone to give α-spinasterol 9 (15 mg). Combined fr. 19-25 were dissolved in EtOAc (50 mL) and treated under heating and shaking with active coal (1 g). After filtration, drying (anhydrous Na2SO4) and removal of the solvent, the white residue was purified by recrystallization from hexane-EtOAc (3:1) to obtain compound 8 (400 mg). The combined fr. 30-36 gave a yellow powder containing the compounds 1, 2, 3 and 4 (identified by GC-MS) that have been previously described for this species (Pizzolatti et al., 2000). The major compound 2 (350 mg) was purified from combined fr. 30-36 by silica gel (230-400 mesh) flash chromatography (2 x 25 cm) using hexane-EtOAc (3:1) as the eluent. The EtOAc-soluble fr. (10 g) was further subjected to column chromatography over silica gel (3 x 30 cm) using hexane-EtOAc and EtOAc-EtOH gradient to give 72 fr. of 50 mL each. The GC-MS analysis of the combined fr. 8-13 exhibited five peaks for compounds 2, 4, 5, 6 and 7 as previously described (Pizzolatti et al., 2000; 2004). Styryl-2-pyrone 7 (185 mg) was the major component among fr. 8-13 and was further purified by flash chromatography, as described above, using hexane-EtOAc 2:3. The combined fr. 19-21 was further crystallized from acetone to yield apigenin 10 (4 mg). The combined fr. 30-36 was further chromatographed on silica gel (3 x 25 cm) eluting with a hexane-acetone gradient from 90:10 to 0:100 (60 fr., 30 mL each) to obtain quercetin 11 (25 mg). The combined polar fr. (45-60) was further subjected to column chromatography on Sephadex LH-20 (1.5 x 10 cm) eluted with MeOH-H2O (3:1, 30 mL) to give a yellow solid that after recrystallization from acetone-MeOH, yielded quercetin-3-O-glucoside [3-O-(β-D-glycopyranosyl)-quercetin] 12 (52 mg).
Melting point, IR, NMR, GC-MS, CC, TLC and visualization were determined as previously described (Pizzolatti et al., 2000; 2004).
In vitro bioassays
The in vitro trypanocidal assays were performed as previously described (Pizzolatti et al., 2003). The activities against epimastigote and bloodstream trypomastigote forms were expressed as the percentage of reduction in the parasite number and the inhibitory concentration of 50% (IC50) and as the percentage of parasitemia reduction, respectively. Both activities were determined by one-way analysis of variance (ANOVA) using Prism® v. 2.0 software (GraphPad Software Incorporated, San Diego). Negative tests with blood trypomastigotes, i.e., tests where no parasites were observed after incubation with the compound, were inoculated into groups of 2 healthy Swiss mice to confirm the absence of parasites. One week after inoculation, fresh blood examination was performed on each mouse every two days up to 30 days of inoculation when hemoculture in LIT medium was carried out for negative animals. Cultures were maintained at 28°C and examined for the presence of parasite epimastigotes every two weeks up to 60 days. The assays involving experimentation animals were approved by the UFSC Ethics Committee (CEUA 155/2002).
Compounds proved to be active against T. cruzi epimastigote and trypomastigote forms were further evaluated against intracellular forms of the parasite. For that, culture-derived T. cruzi trypomastigotes (Y strain) were used to infect Vero cells (ATCC-CCL81) (5:1 ratio) previously cultivated on 12 mm glass cover slips in 24-well plates. After 4 hours of cell-parasite interaction in DMEM medium supplemented with 5% of FBS at 37 °C, under a 5% CO2 atmosphere, non-adhered parasites were removed by a single PBS washing and 0.5 mL of fresh medium was added to each well. The cells where then maintained as described above. Twenty-four hours after infection, compounds 8 (50 µg/mL), 7 (50 µg/mL) and 2 (100 µg/mL) were added to the cell monolayers and cultivated for 72 hours under the same conditions. Infected cells incubated with benznidazole (100 µg/mL) and in the absence of any drug or compound were used as controls. After incubation, all slides were rinsed once in cold PBS, fixed in Bouin for 15 minutes, washed in distilled water, Giemsa stained and mounted on glass slides with Entellan (Merck, Whitehouse Station). The experiments were performed in triplicate and a total of 300 randomly-chosen cells per cover slip were microscopically examined (400x) and the percentage of infected cells as well as the number of parasites/cell was determined. In addition, cell infection was followed daily for up to five days in order to evaluate the intracellular cycle of the parasite.
The hemolytic activity of each compound was assessed by the cyanometahemoglobin method. Briefly, 10 mL of non-infected mouse blood previously incubated with 500 µg/mL of each compound was diluted in 990 mL of a Drabkins solution in the presence of 25 mL of KCN (oxidant). The released hemoglobin is converted to cyanometahemoglobin, and the concentration was determined with a spectrophotometer (Amersham Biosciences, Piscataway) at 540 nm. The results are expressed as a percentage of hemolysis (Koga, 2003).
The cytotoxicity of the herein described compounds was determined through the MTT assay as formerly described (Sieuwerts et al., 1995). Vero cells were cultivated in 96-wells micro plates in DMEM medium supplemented with 5% of FBS, 100 UI of penicillin and 100 µg/mL of streptomycin/mL and incubated in a 5% CO2 atmosphere, 95% humidity at 37 °C. After 24 hours, a new culture medium was added and the cells were incubated for 72 hours under the same conditions in the presence of 100, 50 and 25 µg/mL of compounds 2, 7 and 8. As controls, cells were cultivated in the absence of any drug or compound, with 1% DMSO and with benznidazole (100 µg/mL). Optical density was determined in a Bio-Tek micro reader (Molecular Devices, Sunnyvale) at 540 nm and the results expressed as the percentage of cytotoxicity.
RESULTS AND DISCUSSION
During the screening test carried out with the 4 fractions obtained from P. sabulosa against T. cruzi epimastigotes in vitro, no activity was observed for the ethanol fr. and a similar activity to benznidazole was observed for the hexane, EtOAc and CH2Cl2 frs. Despite these promising results against epimastigote forms, only the CH2Cl2 fr. revealed a similar activity to gentian violet, being in accordance with our previous results (Pizzolatti et al., 2003) (Table 1).
Fractionation of the CH2Cl2 fr. allowed the isolation of a a-spinasterol 9 [α]20D -2.5º, which was characterized by physical properties (TLC, [α]20D and m.p.) and comparison of their 1H-NMR, 13C- NMR and MS with known standards (Itoh et al., 1981). Two other compounds, 6-methoxy-7-prenyloxycoumarin (8) and 4-methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone (2), were also isolated and identified by spectroscopy and comparison to authentic samples (Pizzolatti et al., 2000).
Fractionation of the EtOAc fr. on silica gel and Sephadex LH-20 revealed two major compounds, styryl-2-pyrone 7 as previously reported (Pizzolatti et al., 2000) and quercetin-3-O-glucoside 12, and also two minor flavones (apigenin 10 and quercetin 11), identified by spectral analyses and by comparison of the spectrum with literature data (Agrawal, 1989). The flavonoids 10-12 and the α-spinasterol 9 herein described are the first report of these compounds for P. sabulosa. The trypanocidal activity of compound 10 was not determined because only a little amount was isolated and compound 11 is an already known substance and its activity has already been determined (Tasdemir et al., 2006; Silveira et al., 2005).
Four isolated compounds (2, 7, 8 and 9) showed interesting trypanocidal activity against epimastigote forms (IC50 10.5 up to 33.5 µg/mL) but only compound 8 revealed to be active against trypomastigotes (IC50 88.2 µg/mL) (Table 2).
In order to assess the activity of prenyloxycoumarin 8 against T. cruzi blood trypomastigotes, infected mouse blood was incubated in vitro at 4 ºC for 48 h with 250 µg/mL and 500 µg/mL of the compound. After incubation, the presence of live parasites was microscopically assessed and the treated blood was inoculated into healthy mice in order to further assess the presence of parasites. As observed for the control group treated with 250 µg/mL of gentian violet, no parasites were observed by microscopy in blood treated with both concentrations of prenylcoumarin 8. However, mice inoculated with blood treated with 250 µg/mL of compound 8 presented detectable parasitemia in the second post-inoculation week and showed positive hemoculture. In contrast, all mice inoculated with infected blood treated with 500 µg/mL of compound 8 presented negative hemoculture. The hemolysis rate to mouse erythrocytes was tested using 500 µg/mL of each fraction or compound and varied from 3% - 9% and from 2% - 6%, respectively. This result allows concluding that parasite clearance is not due to unspecific lysis activity.
In the search for new chemoprophylatic agents for Chagas disease, Chiari et al. (1996) studying ethanol extracts from 47 Asteraceae species showed that 11 extracts reduced by 100% blood trypomastigotes in vitro at a concentration of 12 mg/mL. Zani et al. (1995) evaluating trypanocidal activity of ethanol extracts of 52 Asteraceae species showed that 10% of them reduced by 70% the number blood trypomastigotes in vitro at a concentration of 250 µg/mL. Mafezoli et al. (2000), studying 25 fractions from 9 species of the Rutaceae family, showed that 6 fractions produced an 80 - 100% parasitemia reduction of T. cruzi trypomastigotes in vitro at concentrations of 2 mg/mL. Scio et al. (2003a) reported that a diterpene compound from Alomia myriadenia at 250 µg/mL produced a parasitemia reduction of 100% for T. cruzi blood trypomastigotes in vitro.
Trypanocidal activity of a coumarin from Kielmeyera albopunctata, a plant species from the Brazilian Cerrado, produced an 80% mortality rate of T. cruzi blood trypomastigotes at 125 µg/mL in a 24 hour period (Scio et al., 2003b). In the present study, coumarin 8 obtained from P. sabulosa showed in vitro activity against both cultured epimastigotes and blood trypomastigotes (IC50 10.5 and 88.2 µg/mL, respectively). However, only concentrations < 500 mg/mL of the compound was able to prevent mouse infection in vivo. The other compounds 2, 7 and 12 presented a weak activity against T. cruzi blood trypomastigotes and none of them produced a 100% parasite clearance in infected blood. All active compounds described herein showed a low hemolysis rate (2 - 6%) even at high concentrations (2 mg/mL), suggesting a selective trypanocidal activity.
Based on the obtained IC50 values, we can conclude that cytotoxicity of the studied compounds toward Vero cells proved to be concentration-dependent (data not shown). Prenylcoumarin 8 and styryl-2-pyrone 7 were the most toxic compounds, whereas dihydrostyryl-2-pyrone 2 presented a similar toxicity to that of benznidazole. Infected Vero cells treated with prenylcoumarin 8 (50 µg/mL) and dihydrostyryl-2-pirone 2 (100 µg/mL) for 72 hours revealed a 3-4-fold decrease in the percentage of infected cells and a 2-fold reduction in the number of intracellular parasites/cell in comparison to the control. Compound 7 did not show activity against amastigotes at any tested concentration it was observed in cells treated with benznidazole (100 µg/mL). In vitro assays to determine the intracellular trypanocidal activity showed that compounds 8 and 2 produced a 3-4-fold reduction in cell infection by T. cruzi as well as in the number of intracellular amastigotes. However, the most active compound 8 showed a high cytotoxicity (50%) toward Vero cells even at low concentrations (~100 µg/mL) (Table 3).
Four out of five active compounds from P. sabulosa presented a significant growth inhibition of T. cruzi epimastigotes, and two of them also revealed activity against blood trypomastigotes and intracellular amastigotes in vitro, possibly constituting styryl-2-pirone and coumarin new groups of anti-T. cruzi compounds.
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