In Vitro Schistosomicidal Activity of Some Brazilian Cerrado Species and Their Isolated Compounds
In Vitro Schistosomicidal Activity of Some Brazilian Cerrado Species and Their Isolated Compounds
Nayanne Larissa Cunha,1 Camila Jacintho de Mendonça Uchôa,1 Lucas Silva Cintra,1 Herbert Cristian de Souza,1 Juliana Andrade Peixoto,1 Claudia Peres Silva,1 Lizandra Guidi Magalhães,1 Valéria Maria Meleiro Gimenez,2 Milton Groppo,3 Vanderlei Rodrigues,4 Ademar Alves da Silva Filho,5 Márcio Luís Andrade e Silva,1 Wilson Roberto Cunha,1 Patrícia Mendonça Pauletti,1 and Ana Helena Januário1
1Universidade de Franca, Avenida. Dr. Armando Salles de Oliveira 201, 14404-600 Franca, SP, Brazil
2Centro Universitário Claretiano, Rua Dom Bosco 466, 14300-000 Batatais, SP, Brazil
3Faculdade de Filosofia, Ciências e Letras, Universidade de São Paulo, Avenida Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brazil
4Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brazil
5Departamento Farmacêutico, Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/no, 36036-900 Juiz de Fora, MG, Brazil
Received 22 April 2012; Revised 7 June 2012; Accepted 8 June 2012
Academic Editor: Jairo Kenupp Bastos
Copyright © 2012 Nayanne Larissa Cunha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Miconia langsdorffii Cogn. (Melastomataceae), Roupala montana Aubl. (Proteaceae), Struthanthus syringifolius (Mart.) (Loranthaceae), and Schefflera vinosa (Cham. & Schltdl.) Frodin (Araliaceae) are plant species from the Brazilian Cerrado whose schistosomicidal potential has not yet been described. The crude extracts, fractions, the triterpenes betulin, oleanolic acid, ursolic acid and the flavonoids quercetin 3-O-β-D-rhamnoside, quercetin 3-O-β-D-glucoside, quercetin 3-O-β-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside and isorhamnetin 3-O-β-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside were evaluated in vitro against Schistosoma mansoni adult worms and the bioactive n-hexane fractions of the mentioned species were also analyzed by GC-MS. Betulin was able to cause worm death percentage values of 25% after 120 h (at 100 μM), and 25% and 50% after 24 and 120 h (at 200 μM), respectively; besides the flavonoid quercetin 3-O-β-D-rhamnoside promoted 25% of death of the parasites at 100 μM. Farther the flavonoids quercetin 3-O-β-D-glucoside and quercetin 3-O-β-D-rhamnoside at 100 μM exhibited significantly reduction in motor activity, 75% and 87.5%, respectively. Biological results indicated that crude extracts of R. montana, S. vinosa, and M. langsdorffii and some n-hexane and EtOAc fractions of this species were able to induce worm death to some extent. The results suggest that lupane-type triterpenes and flavonoid monoglycosides should be considered for further antiparasites studies.
Schistosomiasis, caused by trematode flatworms of the genus Schistosoma, is one of the most significant, neglected tropical diseases in the world. This disease still displays significant values of prevalence and morbidity, affecting more than 200 million people worldwide and resulting in as many as 280,000 deaths each year with over 779 million people at risk of infection, despite the great advances in treatment and prevention [1–3]. Praziquantel (PZQ) and oxamniquine are the drugs that are currently available for the treatment of schistosomiasis. However, low cure rates and treatment failure following PZQ administration have been reported in patients . The Brazilian savanna, known as Cerrado, comprises a very rich and characteristic flora that covers more than 2 million square kilometers of Brazilian inland. It is a biome that is even more threatened than the Amazon rainforest [5, 6]. Cerrado is the world’s most biodiverse savanna, with high degree of endemism and very high rate of environmental loss, thus regarded as a biodiversity hotspot . This large biodiversity puts the country in a strategic position for the development of rational and sustained exploration of new metabolites of therapeutic value . The use of online techniques has assisted in the rapid identification of active compounds in Cerrado species. As an example we can cite the online identification of chlorogenic acids, sesquiterpene lactones, and flavonoids in the Brazilian Cerrado species Lychnophora ericoides by HPLC-DAD-MS and HPLC-DAD-MS/MS . Moreover, the literature lacks reports on the chemical composition of the selected species, namely Roupala montana Aubl. (Proteaceae), Struthanthus syringifolius (Mart.) (Loranthaceae), and Schefflera vinosa (Cham. & Schltdl.) Frodin (Araliaceae). Nevertheless, isolation of triterpenes, saponins, and caffeoylquinic acid derivatives from Schefflera and of terpenes, lignans, flavonoids, carbohydrates, fatty acids, amides, phenylpropanoids, tannins, and alkaloids from Struthanthus has been reported [10–14]. Miconia langsdorffii Cogn. (Melastomataceae) has been described to display antileishmanial activity, and isolation of the triterpenes ursolic acid and oleanolic acid from this plant species has been reported . As part of our continuing interest in Brazilian Cerrado species with a view to finding out schistosomicidal agents and new drugs with action against Schistosoma species [16–20], we now report on the evaluation of the schistosomicidal activity of the extracts and fractions obtained from M. langsdorffii, R. montana, S. syringifolius, and S. vinosa, as well as on the activity of the isolated compounds betulin (1), oleanolic acid (2), ursolic acid (3), quercetin 3-O-β-D-glucoside (4), quercetin 3-O-β-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside (5), isorhamnetin 3-O-β-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside (6), quercetin 3-O-β-D-rhamnoside (7).
2. Material and Methods2.1. General
1H and 13C NMR spectra were recorded in pyridine- for triterpenes and DMSO- for flavonoids using TMS as internal standard. Both analytical and preparative HPLC separation analyses were carried out on a Shimadzu LC-6AD system equipped with a degasser DGU-20A5, a UV-VIS detector SPD-20A series, a communication bus module CBM-20A, and a Reodyne manual injector. Separations of the micromolecules were accomplished on a Shimadzu Shim-pack ODS (particle diameter 5 μm, mm, and mm) columns equipped with a pre-column of the same material. The MeOH used in the experiments was HPLC grade, J. T. Baker. Ultrapure water was obtained by passing redistilled water through a Direct-Q UV3 system from Millipore.
2.2. Plant Material
The aerial parts of Roupala montana Aubl. (Proteaceae) and Schefflera vinosa (Cham. & Schltdl.) Frodin (Araliaceae) were collected in Luis Antonio, State of São Paulo, Brazil, in May 2008; Miconia langsdorffii Cogn. (Melastomataceae) was collected in Serra Azul, State of São Paulo, Brazil, in March 2009; and Struthanthus syringifolius (Mart.) (Loranthaceae) was collected in Itamarandiba, State of Minas Gerais, Brazil, in October 2009. The materials were identified by Prof. V. M. M. Gimenez and Prof. M. Groppo. Vouchers specimens (SPFR12166, SPFR12167 SPFR12288, and SPFR12171, resp.) were deposited in the Herbarium of Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, University of São Paulo, Brazil (Herbarium SPFR).
2.3. Extraction and Isolation
The aerial parts of M. langsdorffii (0.5 kg), R. montana (0.9 kg), S. syringifolius (1.5 kg), and S. vinosa (0.5 kg) were powdered and exhaustively extracted by maceration at room temperature using EtOH for the three former plants, while EtOH/H2O 8 : 2 (v/v) was employed for S. vinosa. After filtration, the solvent was removed under reduced pressure, yielding 7.8 g, 24 g, 39 g, and 31 g of crude extract from the above mentioned plants, respectively. The crude extracts of R. montana (RM, 30 g), S. syringifolius (SS, 20 g), and S. vinosa (SV, 30 g) were then dissolved in MeOH/H2O 2 : 8 (v/v) and successively partitioned with n-hexane, EtOAc, and n-BuOH. After solvent removal, each partition phase furnished 4.0 g, 5.9 g, 10.3 g, and 5.1 g of material for R. montana; 5.3 g, 2.9 g, 3.1 g, and 3.2 g for S. syringifolius; and 3.9 g, 7.0 g, 15.5 g, and 2.8 g for S. vinosa. The n-hexane fractions (200 mg) of each extract were chromatographed over silica and Florisil (1 : 1, w/w, 8 g) using CH2Cl2 as eluent, to afford three major fractions for each species, which were then analyzed by GC-MS. Besides, the n-hexane fraction of S. vinosa (SV-1) was purified by column chromatography over silica gel 60 (0.063–0.200 mm, Merck) using n-hexane and EtOAc as eluents, which yielded compound 1 from fractions 32-33 (n-hexane/EtOAc 7 : 3 (v/v); 42 mg). On the other hand the EtOAc fraction of S. vinosa (SV-2) was purified by semipreparative reverse phase HPLC using MeOH/H2O/AcOH (45 : 54.9 : 0.1, v/v/v), UV detection at 254 nm, and flow rate 9 mL/min, furnishing compound 7 (7.9 mg). In a previous study, our research group had fractioned the crude extract of M. langsdorffii (ML, 6.7 g) and obtained six fractions as follows: ML-1: n-hexane/EtOAc 75 : 25 (v/v), ML-2: n-hexane/EtOAc 50 : 50 (v/v), ML-3: EtOAc, ML-4: AcOEt/EtOH 75 : 25 (v/v), ML-5: AcOEt/EtOH 50 : 50 (v/v), and ML-6: EtOH . Fraction ML-2 (500 mg) was the most active in the schistosomicial assay, and it was chromatographed over Celite and Norit (3 : 1, w/w, 60 g) using EtOAc as eluent. The resulting solid amorphous material was dissolved in MeOH/H2O 85 : 15 (v/v) and subsequently submitted to semi-preparative RP-HPLC purification using MeOH/H2O/AcOH (85 : 14.9 : 0.1, v/v/v), UV detection at 220 nm, and flow rate 9 mL/min, leading to compounds 2 (25 mg) and 3 (75 mg). The EtOAc fraction from R. montana (RM-2) followed to semi-preparative reverse phase HPLC purification, the analytical conditions were a mobile phase gradient consisting of MeOH/H2O/AcOH (47 : 52.9 : 0.1, v/v/v), UV detection at 254 nm, and a flow rate of 5 mL/min, yielding compound 4 (5.0 mg). Similarly, the n-BuOH fraction (RM-3) was submitted to semi-preparative reverse phase HPLC under the same conditions, providing compounds 5 (29.0 mg) and 6 (12.0 mg).
2.4. GC-MS Analysis
A Shimadzu QP-2010 gas chromatograph equipped with HP-1 or DB-17MS capillary columns (30 m 0.25 mm i.d. 0.25 μm film thickness) coupled to a mass spectrometer was employed. EI mass spectra were recorded at 70 eV. Conditions: for S. vinosa n-hexane fraction (SV-1): HP-1 column, injector 250°C; temperature program 100–290°C at 3°C/min, followed by 20-min isotherm; split ratio 1 : 30; carrier gas He at 1.10 mL/min flow rate. For R. montana n-hexane fraction (RM-1): DB-17MS column, injector 250°C; temperature program 100–290°C at 3°C/min, followed by 20-min isotherm; split ratio 1 : 20; carrier gas He at 1.10 mL/min flow rate. For S. syringifolius n-hexane fraction (SS-1): DB-17MS column, injector 250°C; temperature program 120–260°C at 3°C/min, followed by 5-min isotherm; then temperature program 260–280°C at 2°C/min, followed by 9-min isotherm; then temperature 280–290°C at 2°C/min, followed by 20-min isotherm; split ratio 1 : 50; carrier gas He at 1.40 mL/min flow rate. Identification of the constituents was conducted by computer search in the Wiley Mass Spectral Database 7.
2.5. In Vitro Schistosomicidal Assay
The LE (Luis Evangelista) strain of S. mansoni was maintained by passage through Biomphalaria glabrata snails and BALB/c mice. After eight weeks, S. mansoni adult worms were recovered under aseptic conditions from mice previously infected with 200 cercariae by perfusion of the livers and mesenteric veins . The worms were washed in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen), kept at pH 7.5 with HEPES 20 mM, and supplemented with penicillin (100 UI/mL), streptomycin (100 μg/mL), and 10% bovine fetal serum (Gibco). After washing, two adult worms were transferred to each well of a 24-well culture plate containing 2 mL of the same medium and incubated at 37°C in a humid atmosphere containing 5% CO2 prior to use. At 24 h after incubation, extracts, fractions, and the isolated compounds (1–7) were dissolved in dimetilsulfoxide (DMSO) and added to RPMI 1640 medium, to give final concentrations of 50, 100, and 200 μg/mL or μM. The parasites were kept for 5 days and monitored each 24 h to evaluate their general condition. The worms were considered dead when no movement was observed for at least 2 min of examination and no movement at the other observation time points was detected . Quadruplicate measurements were accomplished for each employed concentration and three independent experiments were performed. RPMI 1640 medium and RPMI 1640 with 1% DMSO (the highest concentration of drug solvent) were used as negative control groups. Praziquantel (PZQ) at 12.5 μg/mL or 12.5 μM was used as positive control group. All experiments were authorized by the Ethics Committee for Animal Care of the University of Franca and University of São Paulo, and they were in accordance with the national and international accepted principles for laboratory animal use and care.
3. Results and Discussion
The scarcity of studies on the crude extract of M. langsdorffii, R. montana, S. syringifolius, and S. vinosa has encouraged us to accomplish biological and chemical investigations of metabolites belonging to these extracts. The chemical composition of the bioactive n-hexane fractions from EtOH or EtOH/H2O extracts of the aerial parts of R. montana (RM-1), S. syringifolius (SS-1), and S. vinosa (SV-1) was initially analyzed by GC-MS, and data are listed in Table 1. It can be noted that the identified compounds mainly belong to the following functional groups: aliphatic esters, hydrocarbons, steroids, and triterpenes. The presence of aliphatic esters is typical of all the investigated hexane fractions. RM-1 contains the largest percent amount of these esters (8.89%), followed by SV-1 (4.57%), but only trace amounts were detected in SS-1 (0.30%). Triterpenes were identified in great quantities in SS-1 (61.39%), followed by SV-1 (29.24%), and RM-1 (10.15%). Steroids were observed in SV-1 (4.67%) and SS-1 (4.91%). Hydrocarbons were detected in SS-1 (19.39) and RM-1 (8.03%). Compounds determined in minor quantities were sesquiterpenes, diterpenes, and tocopherol, which were only found in SV-1 (7.84%), RM-1 (17.91%), and RM-1 (11.71%), respectively. The spectral profiles of the isolated compounds were in agreement with previously published data, which allowed identification of betulin (1) and quercetin 3-O-β-D-rhamnoside (7) in S. vinosa [23, 24]; oleanolic (2) and ursolic (3) acids in M. langsdorffii [23, 25] in addition to quercetin 3-O-β-D-glucoside (4), quercetin 3-O-β-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside (5) and isorhamnetin 3-O-β-D-glucopyranosyl-(1-2)-α-L-rhamnopyranoside (6) in R. montana (Figure 1) [24, 26]. To the best of our knowledge, this is the first report of the presence of compound 1 in S. vinosa and the occurrence of the flavonoids 4, 5 and 6 in R. montana. In this study, the in vitro effect of the investigated extracts, fractions, and isolated compounds on S. mansoni parasite mortality was evaluated by incubation of the target microorganism with different concentrations and by evaluation of decrease in motor activity of this worm. In all the experiments, the negative control groups remained viable throughout the observation period. On the other hand, parasites belonging to the positive control group (PZQ) caused 100% parasite death on the first day of incubation. In addition no tegumental damage was observed in adult worms incubated with the evaluated crude extracts, fractions, and isolated compounds. The tegument is extremely important for parasite survival and infection success within the host, and it has been a major target for the development of drugs against Schistosoma [20, 22]. Except for SS, all the studied crude extracts displayed some effect on S. mansoni mortality (Table 2). On the first day of incubation, crude extract ML at a concentration of 100 μg/mL caused 25% adult worms mortality. In addition, on the fifth day of incubation 100% parasite mortality was achieved with extract ML at concentration of 100 μg/mL and also with extracts RM and SV at concentrations of 200 μg/mL. On the other hand extracts SS and SV displayed significant reduction in motor activity at 50, 100 and 200 μg/mL. The occurrence of lethal effect on the first treatment day was noted for fractions RM-2, SS-1, SV-2, and ML-2 at a concentration of 100 μg/mL; however, on the fifth treatment day, fraction RM-2 prompted 100% mortality at concentrations of 50 μg/mL and 100 μg/mL and fractions SV-1 and SV-2 caused 100% parasite mortality at concentrations of 100 μg/mL and 200 μg/mL. Furthermore fractions RM-1, SS-1, SV-1, and ML-2 promoted considerable reduction in motor activity at the assayed concentrations.
Table 1: CG/MS identification of the main chemical constituents of the -hexane fractions of R. montana (RM-1), S. syringifolius (SS-1), and S. vinosa (SV-1).
Table 2: In vitro effects of the crude extract and fractions against S. mansoni adult worms.
Figure 1: Chemical structures of the isolated compounds.
Fractions SV-1and ML-2 fractions were selected for a further purification process, in which they were chromatographed over silica using an n-hexane/EtOAc gradient. SV-1 purification readily furnished 1. Compounds 2 and 3 were isolated after semi-preparative HPLC of ML-2 . As observed in Table 3, the isolated compounds 2 and 3 did not have lethal effects on S. mansoni adult worms, thus showing loss of activity during the phytochemical procedures. On the other hand, betulin (1) at a concentration of 200 μM caused 50% parasite mortality on the fifth day of incubation. In addition, 25% mortality was verified on the fifth treatment day at a concentration of 100 μM and demonstrated 50% of reduction in motor activity within 24 h. Thus, betulin (1) has also been demonstrated to exert an effect on S. mansoni adult worm mortality. It is noteworthy that the structures of compounds 1, 2, and 3 are quite similar, differing mainly in the presence of a five-membered ring and an alcohol moiety in 1 as compared to the existence of a six-membered ring and an acid group in 2 and 3. Therefore, bearing in mind parasite viability, it is suggested that the presence of the five-membered ring and the alcohol functional group in 1 may improve the activity of triterpenes derivatives against S. mansoni, since among compounds 1, 2, and 3 only compound 1 caused parasite death. However, the action of betulin in vitro against chloroquine resistant (K1) and sensitive (T9–96) Plasmodium falciparum strains has already been assessed, and it was found to be inactive . On the other hand the semi-preparative RP-HPLC study of fractions RM-2, RM-3, and SV-2 afforded the flavonoids 4–7. Considering the schistosomicidal activity results of the flavonoids isolated summarized in Table 3, we can observe that only quercetin 3-O-β-D-rhamnoside (7), also known as quercitrin, was able to cause 25% parasite death on the fifth day of treatment at a concentration of 100 μg/mL. Concerning the reduction of motor activity of the parasites compared with the negative control, the flavonoid monoglycosides 4 and 7 at 100 μM exhibited significantly reduction in motor activity of 75% and 87.5%, respectively. However, flavonoids 5 and 6 were inactive. Our results related the isolated flavonoids assayed suggest that the monoglycosylation at C-3 position in ring C increases the reduction in motor activity in S. mansoni adult worm. Previous investigations on the schistosomicidal activity of natural products realized by our research group reveal that aglycone quercetin was not able to kill the worms but exhibited moderately reduced motor activity . However, quercetin was identified as a selective inhibitor of the S. mansoni NAD+ catabolizing enzyme (SmNACE), localized to the outer surface (tegument) of the adult parasite and presumably involved in the parasite survival by manipulating the host’s immune regulatory pathways. These studies identified that the nature of ring C and the substitution of free hydroxyl groups in rings A, B, and C in flavonoids are key structural features for SmNACE inhibition .
Table 3: In vitro effects of isolated compounds against S. mansoni adult worms.
The mechanism by which the extracts, fractions, and betulin (1) and flavonoids 4 and 7 exert their effects remains unclear. Moreover, as suggested by our results, the lupane-type triterpene and flavonoid monoglycosides should also be considered for further antiprotozoal studies. In summary, chemical investigations of metabolites from the selected species resulted in the isolation and identification of compounds 1–7. Additionally, biological results indicated that crude extracts RM, SV, and ML; fractions RM-1, RM-2, SS-1, SV-1, SV-2, and ML-2; the triterpene betulin (1); the flavonoids quercetin 3-O-β-D-glucoside (4); quercetin 3-O-β-D-rhamnoside (7) are able to induce worm death to some extent as well as to reduce the motor activity of the parasites. Additional chemical studies are in progress in our research group to identify other natural compounds related to schistosomicidal action of the species investigated. The knowledge of chemical composition and schistosomicidal potential of R. montana, S. syringifolius, S. vinosa, and M. langsdorffii will provide insight information for the future application of these plants.
The authors are grateful to Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Coordenadoria de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for fellowships. FAPESP is also acknowledged for financial support (Grants no. 2006/60132-4, no. 2008/01268-9 and no. 2009/00604-8). The authors also thank Isabel C.C. Turatti for the GC/MS analysis.
References Y. Zhang, C. MacArthur, L. Mubila, and S. Baker, “Control of neglected tropical diseases needs a long-term commitment,” BMC Medicine, vol. 8, article 67, 2010. View at Publisher · View at Google Scholar · View at ScopusP. Steinmann, J. Keiser, R. Bos, M. Tanner, and J. Utzinger, “Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk,” Lancet Infectious Diseases, vol. 6, no. 7, pp. 411–425, 2006. View at Publisher · View at Google Scholar · View at ScopusC. R. Caffrey, “Chemotherapy of schistosomiasis: present and future,” Current Opinion in Chemical Biology, vol. 11, no. 4, pp. 433–439, 2007. View at Publisher · View at Google Scholar · View at ScopusS. Botros, S. William, O. Hammam, Z. Zídek, and A. Holý, “Activity of 9-(S)-[3-Hydroxy-2-(Phosphonomethoxy)Propyl]Adenine against Schistosomiasis mansoni in Mice,” Antimicrobial Agents and Chemotherapy, vol. 47, no. 12, pp. 3853–3858, 2003. View at Publisher · View at Google Scholar · View at ScopusM. G. Ferri, A vegetação de cerrados brasileiros in E. Warming, Lagoa Santa, Universidade de São Paulo, São Paulo, Brazil, 1973. J. A. Ratter, J. F. Ribeiro, and S. Bridgewater, “The Brazilian cerrado vegetation and threats to its biodiversity,” Annals of Botany, vol. 80, no. 3, pp. 223–230, 1997. View at Publisher · View at Google Scholar · View at ScopusN. Myers, R. A. Mittermeler, C. G. Mittermeler, G. A. B. Da Fonseca, and J. Kent, “Biodiversity hotspots for conservation priorities,” Nature, vol. 403, no. 6772, pp. 853–858, 2000. View at Publisher · View at Google Scholar · View at ScopusL. A. Basso, L. H. Pereira Da Silva, A. G. Fett-Neto et al., “The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases—a review,” Memorias do Instituto Oswaldo Cruz, vol. 100, no. 6, pp. 475–506, 2005. View at Google Scholar · View at ScopusL. Gobbo-Neto and N. P. Lopes, “Online identification of chlorogenic acids, sesquiterpene lactones, and flavonoids in the Brazilian arnica Lychnophora ericoides Mart. (Asteraceae) leaves by HPLC-DAD-MS and HPLC-DAD-MS/MS and a validated HPLC-DAD method for their simultaneous analysis,” Journal of Agricultural and Food Chemistry, vol. 56, no. 4, pp. 1193–1204, 2008. View at Publisher · View at Google Scholar · View at ScopusG. Cioffi, A. Braca, G. Autore et al., “Cytotoxic saponins from Schefflera fagueti,” Planta Medica, vol. 69, no. 8, pp. 750–756, 2003. View at Publisher · View at Google Scholar · View at ScopusY. Li, P. P. H. But, and V. E. C. Ooi, “Antiviral activity and mode of action of caffeoylquinic acids from Schefflera heptaphylla (L.) Frodin,” Antiviral Research, vol. 68, no. 1, pp. 1–9, 2005. View at Publisher · View at Google Scholar · View at ScopusY. Li, R. Jiang, L. S. M. Ooi, P. P. H. But, and V. E. C. Ooi, “Antiviral triterpenoids from the medicinal plant Schefflera heptaphylla,” Phytotherapy Research, vol. 21, no. 5, pp. 466–470, 2007. View at Publisher · View at Google Scholar · View at ScopusR. Liu, Q. Q. Gu, C. B. Cui et al., “12α,13-Dihydroxyolean-3-oxo-28-oic a new triterpene, and the known oleanonic acid as a new cell cycle inhibitor from Schefflera venulosa,” Chinese Journal of Chemistry, vol. 23, no. 3, pp. 242–244, 2005. View at Publisher · View at Google Scholar · View at ScopusA. C. Guimarães, “Aspectos etnobotânicos e químicos das famílias Loranthaceae e Viscaceae: potencialidades terapêuticas das Ervas-de-passarinho parasitas,” Fitos, vol. 2, no. 1, pp. 27–47, 2006. View at Google ScholarJ. A. Peixoto, M. L. A. E. Silva, A. E. M. Crotti et al., “Antileishmanial activity of the hydroalcoholic extract of Miconia langsdorffii, isolated compounds, and semi-synthetic derivatives,” Molecules, vol. 16, no. 2, pp. 1825–1833, 2011. View at Publisher · View at Google Scholar · View at ScopusN. I. De Melo, L. G. Magalhaes, C. E. De Carvalho et al., “Schistosomicidal activity of the essential oil of Ageratum conyzoides L. (Asteraceae) against adult Schistosoma mansoni worms,” Molecules, vol. 16, no. 1, pp. 762–773, 2011. View at Publisher · View at Google Scholar · View at ScopusN. A. Parreira, L. G. Magalhães, D. R. Morais et al., “Antiprotozoal, schistosomicidal, and antimicrobial activities of the essential oil from the leaves of baccharis dracunculifolia,” Chemistry and Biodiversity, vol. 7, no. 4, pp. 993–1001, 2010. View at Publisher · View at Google Scholar · View at ScopusC. G. Braguine, E. S. Costa, L. G. Magalhães et al., “Schistosomicidal evaluation of Zanthoxylum naranjillo and its isolated compounds against Schistosoma mansoni adult worms,” Zeitschrift fur Naturforschung, vol. 64, no. 11-12, pp. 793–797, 2010. View at Google Scholar · View at ScopusL. G. Magalhães, C. B. Machado, E. R. Morais et al., “In vitro schistosomicidal activity of curcumin against Schistosoma mansoni adult worms,” Parasitology Research, vol. 104, no. 5, pp. 1197–1201, 2009. View at Publisher · View at Google Scholar · View at ScopusJ. D. Moraes, C. Nascimento, P. O. M. V. Lopes et al., “Schistosoma mansoni: in vitro schistosomicidal activity of piplartine,” Experimental Parasitology, vol. 127, no. 2, pp. 357–364, 2011. View at Publisher · View at Google Scholar · View at ScopusS. R. Smithers and R. J. Terry, “The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms,” Parasitology, vol. 55, no. 4, pp. 695–700, 1965. View at Google Scholar · View at ScopusT. Manneck, Y. Haggenmüller, and J. Keiser, “Morphological effects and tegumental alterations induced by mefloquine on schistosomula and adult flukes of Schistosoma mansoni,” Parasitology, vol. 137, no. 1, pp. 85–98, 2010. View at Publisher · View at Google Scholar · View at ScopusS. B. Mahato and A. P. Kundu, “13C NMR spectra of pentacyclic triterpenoids,” Phytochemistry, vol. 37, no. 6, pp. 1517–1575, 1994. View at Publisher · View at Google Scholar · View at ScopusY. L. Li, J. Li, N. L. Wang, and X. S. Yao, “Flavonoids and a new polyacetylene from Bidens parviflora willd,” Molecules, vol. 13, no. 8, pp. 1931–1941, 2008. View at Publisher · View at Google Scholar · View at ScopusW. R. Cunha, C. Martins, D. Da Silva Ferreira, A. E. Miller Crotti, N. P. Lopes, and S. Albuquerque, “In vitro trypanocidal activity of triterpenes from Miconia species,” Planta Medica, vol. 69, no. 5, pp. 470–472, 2003. View at Publisher · View at Google Scholar · View at ScopusA. Hasler, G. A. Gross, B. Meier, and O. Sticher, “Complex flavonol glycosides from the leaves of Ginkgo biloba,” Phytochemistry, vol. 31, no. 4, pp. 1391–1394, 1992. View at Google Scholar · View at ScopusJ. C. Steele, D. C. Warhurst, G. C. Kirby, and M. S. Simmonds, “In vitro and in vivo evaluation of betulinic acid as an antimalarial,” Phytochemistry, vol. 31, no. 4, pp. 1391–1394, 1992. View at Google ScholarC. G. Braguine, C. S. Bertanha, U. O. Gonçalves et al., “Schistosomicidal evaluation of flavonoids from two species of Styrax against Schistosoma mansoni adult worms,” Pharmaceutical Biology, vol. 50, no. 7, pp. 925–929, 2012. View at Google ScholarI. Kuhn, E. Kellenberger, F. Said-Hassane et al., “Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD+ catabolizing enzyme,” Bioorganic and Medicinal Chemistry, vol. 18, no. 22, pp. 7900–7910, 2010. View at Publisher · View at Google Scholar · View at Scopus