Schistosomicidal Activity of Alkyl-phenols from the Cashew

Article pubs.acs.org/JAFC

Schistosomicidal Activity of Alkyl-phenols from the Cashew Anacardium occidentale against Schistosoma mansoni Adult Worms Tavane A. Alvarenga,† Pollyanna F. de Oliveira,† Julia M. de Souza,† Denise C. Tavares,† Márcio L. Andrade e Silva,† Wilson R. Cunha,† Milton Groppo,§ Ana H. Januário,† Lizandra G. Magalhaẽ s,† and Patrícia M. Pauletti*,† †

Center for Research in Exact and Technological Sciences, University of Franca, Avenida Doutor Armando Salles Oliveira 201, Franca, São Paulo 14404-600, Brazil § Department of Biology, Faculdade de Filosofia, Ciências e Letras, Ribeirão Preto, University of São Paulo, Avenida Bandeirantes 3900, Ribeirão Preto, São Paulo 14040-901, Brazil S Supporting Information *

ABSTRACT: Bioassay-guided study of the ethanol extract from the cashew Anacardium occidentale furnished cardol triene (1), cardol diene (2), anacardic acid triene (3), cardol monoene (4), anacardic acid diene (5), 2-methylcardol triene (6), and 2methylcardol diene (7). 1D- and 2D-NMR experiments and HRMS analysis confirmed the structures of compounds 1−7. Compounds 2 and 7 were active against Schistosoma mansoni adult worms in vitro, with LC50 values of 32.2 and 14.5 μM and selectivity indices of 6.1 and 21.2, respectively. Scanning electron microscopy of the tegument of male worms in the presence of compound 7 at 25 μM after 24 h of incubation showed severe damage as well as peeling and reduction in the number of spine tubercles. Transmission electron microscopy analyses revealed swollen mitochondrial membrane, vacuoles, and altered tegument in worms incubated with compound 2 (25 μM after 24 h). Worms incubated with compound 7 (25 μM after 24 h) had lysed interstitial tissue, degenerated mitochondria, and drastically altered tegument. Together, the results indicated that compound 7 presents promising in vitro schistosomicidal activity. KEYWORDS: schistosomicidal activity, cashew Anacardium occidentale, alkyl-phenols

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species have identified alkyl-phenols, organic acids, flavonoids, and tannins.13 In a previous study the cashew nut shell hexane extract showed molluscicidal activity against Biomphalaria glabrata, the snail vector of schistosomiasis.14 In this study, we have decided to evaluate the ethanol extract from the nuts of A. occidentale because it displays activity against Schistosoma mansoni adult worms, which deserves further investigation. More specifically, this paper describes the bioassay-guided isolation of cardol triene (1), cardol diene (2), anacardic acid triene (3), cardol monoene (4), anacardic acid diene (5), 2-methylcardol triene (6), and 2-methylcardol diene (7) and their activities against S. mansoni and the normal human lung fibroblast cell line. Additionally, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) will help us to assess the action of compounds 2 and 7 on the tegument of S. mansoni worms.15,16

INTRODUCTION Schistosomiasisis is a water-borne ailment, and parasites of the genus Schistosoma are the etiological agent of this disease. This disease is the most prevalent human helminth infection with regard to mortality and the third most harmful tropical disease in the world.1 The World Health Organization (WHO) estimates that schistosomiasis occurs in 78 countries. In 2014, about 258 million individuals required preventive therapy. In 2010, mortality was estimated at approximately 100,000 human deaths per year.2,3 Praziquantel (PZQ) is the drug that is currently used to treat schistosomiasis. Chemotherapy with PZQ relies on only one medicine to treat over 200 million people living with schistosomiasis and 700 million at risk of being infected. This background seems alarming when one considers that the parasite may develop drug resistance.4 Phytochemical study for new antischistosomal agents can pave the way for the identification of potential compounds.5,6 The cashew of Anacardium occidentale L. (Anacardiaceae) occurs in about 30 countries worldwide, especially in tropical areas.7 In Brazil, A. occidentale is the only species from the genus Anacardium for which fruits and pseudofruits are commercially available.8 In traditional medicine, whole parts of the cashew tree are employed in the treatment of various conditions such as mouth and peptic ulcers, intestinal disorders, dyspepsia, diabetes, sore throat, asthma, bronchitis, and inflammatory diseases. Cashew nut oil is considered a folk remedy for cancerous ulcers, elephantiasis, and warts.9−11 The oil extracted from the pericarp is used to treat cracks on the feet.12 Phytochemical studies on this © 2016 American Chemical Society

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MATERIALS AND METHODS

General Experimental Procedures. 1H and 13C NMR spectra and 2D experiments were recorded in CDCl3 on Bruker DPX 300, Bruker DPX 400, and Bruker DPX 500 NMR spectrometers; TMS was used as internal standard. Negative- and positive-ion mode HRESIMS spectra were obtained on a Bruker Daltonics HRMS ultrOTOF-Q-ESI-TOF by using electrospray ionization. HPLC analyses were performed on a Received: Revised: Accepted: Published: 8821

September 20, 2016 October 26, 2016 November 3, 2016 November 11, 2016 DOI: 10.1021/acs.jafc.6b04200 J. Agric. Food Chem. 2016, 64, 8821−8827

Article

Journal of Agricultural and Food Chemistry

130.1 (CH, C-8′), 129.9 (CH, C-9′), 128.2 (CH, C-11′), 128.0 (CH, C12′), 108.1 (CH, C-6 and C-4), 100.2 (CH, C-2), 35.8 (CH2, C-1′), 31.0 (CH2, C-2′), 29.6, 29.4, 29.3, 29.23, 29.21 (CH2, C-3′ to C-6′ and C13′), 27.2 (CH2, C-7′), 25.7 (CH2, C-10′), 22.8 (CH2, C-14′), 13.8 (CH3, C-15′); HREIMS m/z 317.2461 [M + H]+ (calcd for C21H33O2, 317.2481) and m/z 339.2286 [M + Na]+ (calcd for C21H32O2Na 339.4674). Anacardic acid triene (3): yellow oil (CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.35 (1H, t, J = 7.8 Hz, H-4), 6.86 (1H, br d, J = 7.8 Hz, H5), 6.76 (1H, br d, J = 7.8 Hz, H-3), 5.81 (1H, ddt, J = 16.8, 10.2, 6.1 Hz, H-14′), 5.40 (4H, m, H-8′, H-9′, H-11′ and H-12′), 5.04 (1H, dq, J = 16.8, 1.3 Hz, H-15′), 4.97 (1H, dq, J = 10.2, 1.3 Hz, H-15′), 2.97 (2H, t, J = 7.4 Hz, H-1′), 2.80 (4H, m, H-10′ and H-13′), 2.05 (2H, m, H-7′), 1.60 (2H, m, H-2′), 1.32 (8H, m, H-3′ to H-6′); 13C NMR (CDCl3, 100 MHz) δ 175.6 (C, COOH), 163.5 (C, C-2), 147.6 (C, C-6), 136.8 (CH, C-14′), 135.3 (CH, C-4), 130.4 (CH, C-8′), 129.3 (CH, C-9′), 127.6 (CH, C-11′), 126.8 (CH, C-12′), 122.7 (CH, C-5), 115.8 (CH, C-3), 114.7 (CH2, C-15′), 110.5 (C, C-1), 36.4 (CH2, C-1′), 32.0 (CH2, C2′), 31.5 (CH2, C-13′), 29.7, 29.6, 29.4, 29.2 (CH2, C-3′ to C-6′), 27.2 (CH2, C-7′), 25.5 (CH2, C-10′); HREIMS m/z 341.2112 [M − H]− (calcd for C22H29O3, 341.2117). Cardol monoene (4): brown oil (CHCl3); 1H NMR (CDCl3, 300 MHz) δ 6.24 (2H, d, J = 1.8 Hz, H-4 and H-6), 6.17 (br s, 1H, H-2), 5.34 (2H, m, H-8′ and H-9′), 2.47 (2H, t, J = 7.8 Hz, H-1′), 2.00 (4H, m, H-7′ and H-10′), 1.56 (2H, m, H-2′), 1.29 (16H, m, H-3′ to H-6′ and H-11′ to H-14′), 0.88 (3H, t, J = 6.8 Hz, H-15′); 13C NMR (CDCl3, 100 MHz) δ 156.5 (C, C-1 and C-3), 146.1 (C, C-5), 130.0 (CH, C-8′), 129.8 (CH, C-9′), 108.0 (CH, C-4 and C-6), 100.1 (CH, C-2), 35.8 (CH2, C-1′), 31.8 (CH2, C-13′), 31.0 (CH2, C-2′), 29.7, 29.7, 29.4, 29.3, 29.2, 29.0 (CH2, C-3′ to C-6′ and C-11′ to C-12′), 27.3 (CH2, C-7′), 27.2 (CH2, C-10′), 22.6 (CH2, C-14′), 14.1 (CH3, C-15′); EIMS m/z 319 [M + H]+ (100); HREIMS m/z 319.2612 [M + H]+ (calcd for C21H35O2, 319.2637). Anacardic acid diene (5): yellow oil (CHCl3); 1H NMR (CDCl3, 300 MHz) δ 7.32 (1H, t, J = 7.5 Hz, H-4), 6.84 (1H, d, J = 7.5 Hz, H-3), 6.75 (1H, d, J = 7.5 Hz, H-5), 5.35 (4H, m, H-8′, H-9′, H-11′, and H12′), 2.95 (2H, m, H-1′), 2.77 (2H, t, J = 5.7 Hz, H-10′), 2.04 (4H, m, H7′ and H-13′), 1.57 (2H, m, H-2′), 1.31 (10H, m, H-3′ to H-6′ and H14′), 0.90 (3H, t, J = 7.4 Hz, H-15′); 13C NMR (CDCl3, 100 MHz) δ 175.1 (C, COOH), 163.0 (C, C-2), 147.4 (C, C-6), 134.9 (CH, C-4), 130.1 (CH, C-8′), 129.9 (CH, C-9′), 128.1 (CH, C-11′), 127.9 (CH, C12′), 122.6 (CH, C-5), 115.6 (CH, C-3), 36.4 (CH2, C-1′), 32.0 (CH2, C-2′), 29.8, 29.7, 29.6, 29.4, 29.3 (CH2, C-3′ to C-6′ and C-13′), 27.2 (CH2, C-7′), 25.6 (CH2, C-10′), 22.8 (CH2, C-14′), 13.8 (CH3, C-15′); HREIMS m/z 343.2267 [M − H]− (calcd for C22H31O3, 343.2273). 2-Methylcardol triene (6): brown oil (CHCl3); 1H NMR (CDCl3, 400 MHz) δ 6.23 (2H, s, H-4 and H-6), 5.83 (1H, ddt, J = 17.1, 10.1, 6.2 Hz, H-14′), 5.39 (4H, m, H-8′, H-9′, H-11′, and H-12′), 5.05 (1H, dq, J = 17.1, 1.6 Hz, H-15′), 4.98 (1H, dq, J = 10.1, 1.6 Hz, H-15′), 2.80 (4H, m, H-10′ and H-13′), 2.44 (2H, t, J = 7.6 Hz, H-1′), 2.10 (3H, s, H-1″), 2.03 (2H, m, H-7′), 1.55 (2H, m, H-2′), 1.29 (8H, m, H-3′ to H-6′); 13C NMR (CDCl3, 125 MHz) δ 154.5 (C, C-1 and C-3), 141.9 (C, C-5), 136.8 (CH, C-14′), 130.4 (CH, C-8′), 129.3 (CH, C-9′), 127.6 (CH, C11′), 126.8 (CH, C-12′), 114.7 (CH2, C-15′), 107.8 (CH, C-4 and C-6), 107.4 (C, C-2), 35.5 (CH2, C-1′), 31.5 (CH2, C-13′), 31.1 (CH2, C-2′), 29.6, 29.3, 29.2, 29.2 (CH2, C-3′ to C-6′), 27.2 (CH2, C-7′), 25.6 (CH2, C-10′), 7.70 (CH3, C-1″); HREIMS m/z 327.2389 [M − H]− (calcd for C22H31O2, 327.2324). 2-Methylcardol diene (7): brown oil (CHCl3); 1H NMR (CDCl3, 400 MHz) δ 6.23 (2H, s, H-4 and H-6), 5.35 (4H, m, H-8′, H-9′, H-11′, and H-12′), 2.77 (2H, t, J = 6.2 Hz, H-10′), 2.44 (2H, t, J = 7.8 Hz, H-1′), 2.10 (3H, s, H-1″), 2.04 (4H, m, H-7′and H-13′), 1.55 (2H, m, H-2′), 1.30 (10H, m, H-3′ to H-6′ and H-14′), 0.90 (3H, t, J = 7.4 Hz, H-15′); 13 C NMR (CDCl3, 125 MHz) δ 154.5 (C, C-1 and C-3), 142.0 (C, C-5), 130.1 (CH, C-8′), 129.9 (CH, C-9′), 128.2 (CH, C-11′), 128.0 (CH, C12′), 107.8 (CH, C-4 and C-6), 107.4 (C, C-2), 35.5 (CH2, C-1′), 31.1 (CH2, C-2′), 29.6, 29.4, 29.3, 29.2, 29.2 (CH2, C-3′ to C-6′ and C-13′), 27.2 (CH2, C-7′), 25.6 (CH2, C-10′), 22.8 (CH2, C-14′), 13.8 (CH3, C15′), 7.69 (CH3, C-1″); HREIMS m/z 331.2616 [M + H]+ (calcd for C22H35O2, calcd for 331.2637).

Shimadzu LC-6AD system, with a DGU-20A5 degasser, an SPD-20A series UV−vis detector or an SPD-M20A series DAD detector, a CBM20A communication bus module, a Reodyne manual injector, and the LCsolution program. Separation of the alkyl-phenols was achieved with Shimadzu Shim-pack ODS (5 μm, 250 × 4.60 mm and 250 × 20 mm) columns and precolumns and on a Phenomenex Onyx monolithic C18 (100 × 4.60 mm and 100 × 10 mm) column with precolumns. Materials. The acetonitrile and methanol used in the experiments was of HPLC grade (J. T. Baker). A Direct-Q UV3 system (Millipore) furnished ultrapure water. Octadecyl-functionalized silica gel (SigmaAldrich, 200−400 mesh) and silica gel 60 (Sigma-Aldrich, 230−400 mesh) were used in the SPE system and in flash chromatography, respectively. TLC on silica gel 60 Alu foils with fluorescent indicator 254 nm (Sigma-Aldrich) was also used. Plant Material. Cashew nuts (A. occidentale L.) were obtained from a local market and authenticated by Prof. Milton Groppo from the Department of Biology, Faculdade de Ciências e Letras de Ribeirão Preto, University of São Paulo. A voucher specimen (SPFR 16040) was deposited in the herbarium of the Department of Biology, Laboratory of Plant Systematics, Faculdade de Ciências e Letras de Ribeirão Preto, University of São Paulo, Brazil (Herbarium SPFR). Extraction and Isolation. The air-dried powdered nuts (34.9 g) were extracted with ethanol (EEC). After filtration, the solvent was removed under reduced pressure to yield 12.0 g of the extract. Using guided fractionation of the schistosomicidal assays, aliquots of the EEC (10.85 mg/mL) were analyzed by HPLC. The mobile phase was methanol/water (+ 0.1% acetic acid) with a gradient from 70 to 100% methanol for 10 min, 5 min with 100% methanol, 1 min for return to the initial condition, and 5 min of equilibration. The injection volume was 500 μL, and the flow rate was 4.0 mL/min. This procedure yields 13 fractions. After complete evaporation of the solvent, the fractions (2 mg) were weighed in duplicates and evaluated for schistosomicidal activity and cytotoxicity at a final concentration of 100 μg/mL. After the bioactive chromatogram profile had been obtained, the ethanol extract (EEC, 5.0 g) was submitted to solid phase extraction on ODS silica gel by using methanol/water (80:20, 90:10, and 100:0 v/v). The fraction eluted with methanol/water 80:20 (v/v, 1.6 g) was subsequently fractionated by flash chromatography; hexane/EtOAc was the mobile phase. Fraction 22 (207.9 mg) was submitted to preparative RP-HPLC purification with acetonitrile/water (90:10, v/v), UV detection at 254 nm, and flow rate of 9 mL/min, which yielded 27 fractions. Fractions 3, 5, 7, 8, and 11 gave compounds 1 (63.9 mg, tR 11.8 min, approximately 97% purity), 2 (45.8 mg, tR 14.9 min, approximately 98% purity) 3 (8.9 mg, tR 18.9 min, approximately 97% purity) 4 (8.5 mg, tR 20.6 min, approximately 97% purity), and 5 (4.1 mg, tR 25.2 min, approximately 98% purity), respectively. Fractions 9−11 (98.7 mg) were also chromatographed by preparative RP-HPLC as described above. These procedures led to the isolation of compounds 6 (29.9 mg, tR 11.7 min, approximately 96% purity) and 7 (11.0 mg, tR 14.4 min, approximately 95% purity). The purity of the compounds was estimated by 1H NMR. The isolated compounds were also evaluated by HPLC-DAD. Cardol triene (1: brown oil (CHCl3); 1H NMR (CDCl3, 300 MHz) δ 6.24 (2H, d, J = 1.8 Hz, H-4 and H-6), 6.17 (1H, br s, H-2), 5.82 (1H, ddt, J = 17.1, 10.3, 6.2 Hz, H-14′), 5.37 (4H, m, H-8′, H-9′, H-11′ and H12′), 5.05 (1H, dq, J = 17.1, 1.6 Hz, H-15′), 4.98 (1H, dq, J = 10.3, 1.6 Hz, H-15′), 2.80 (4H, m, H-10′ and H-13′), 2.44 (2H, t, J = 7.7 Hz, H1′), 2.03 (2H, q, J = 6.6 Hz, H-7′), 1.53 (2H, m, H-2′), 1.28 (8H, m, H-3′ to H-6′); 13C NMR (CDCl3, 100 MHz) δ 156.4 (C, C-1 and C-3), 146.2 (C, C-5), 136.8 (CH, C-14′), 130.4 (CH, C-8′), 129.3 (CH, C-9′), 127.6 (CH, C-11′), 126.8 (CH, C-12′), 114.7 (CH2, C-15′), 108.1 (CH, C-6 and C-4), 100.2 (CH, C-2), 35.8 (CH2, C-1′), 31.5 (CH2, C-13′), 31.0 (CH2, C-2′), 29.6, 29.4, 29.3, 29.2 (CH2, C-3′ to C-6′), 27.2 (CH2, C-7′), 25.6 (CH2, C-10′); HREIMS m/z 315.2315 [M + H]+ (calcd for C21H31O2, 315.2324). Cardol diene (2): brown oil (CHCl3); 1H NMR (CDCl3, 400 MHz) δ 6.24 (2H, d, J = 1.6 Hz, H-4 and H-6), 6.17 (1H, br s, H-2), 5.35 (4H, m, H-8′, H-9′, H-11′ and H-12′), 2.77 (2H, t, J = 6.2 Hz, H-10′), 2.47 (2H, t, J = 7.8 Hz, H-1′), 2.03 (4H, m, H-7′ and H-13′), 1.55 (2H, m, H-2′), 1.30 (10H, m, H-3′ to H-6′ and H-14′), 0.90 (3H, t, J = 7.4 Hz, H-15′); 13 C NMR (CDCl3, 125 MHz) δ 156.6 (C, C-1 and C-3), 146.1 (C, C-5), 8822

DOI: 10.1021/acs.jafc.6b04200 J. Agric. Food Chem. 2016, 64, 8821−8827

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Figure 1. HPLC chromatogram of the ethanol extract from the fruits of A. occidentale: (a) schistosomicidal activity and (b) cytotoxicity activity against GM07492-A cells of the microfractions, at 100 μg/mL, collected during the HPLC analysis; (c) DAD detection at 201 nm. In Vitro Schistosomicidal Assays. The schistosomicidal activity was evaluated using the LE (Luis Evangelista) strain of S. mansoni as previously described.17,18 To verify the morphological alterations in S. mansoni caused by compounds 2 and 7, adult worm pairs were incubated with 2 and 7 at 25 μM or with RPMI 1640 containing 0.1% DMSO (negative control) for 24 h. After that, the worms were washed three times with PBS (0.2 M, pH 7.4) and fixed in 2.5% glutaraldehyde in the PBS buffer at room temperature for 2 h. Postfixation was performed with 1% osmium tetroxide (Sigma-Aldrich) in the same buffer at 4 °C for 2 h. For the TEM analysis, the worms were dehydrated in graded ethanol and embedded in Araldite 6005 resin (EMS). Ultrathin sections of the schistosomes were stained with 0.5% uranyl acetate (Sigma-Aldrich) and 0.3% lead citrate (Sigma-Aldrich). Ultrastructural features of the schistosome sections were examined by TEM (JEOL model JEM100CXII equipped with a Hamamatsu ORCA-HR digital camera) (Musashino, Akishima, Tokyo, Japan). For the SEM analysis, the worms were treated according to ref 15. A total of 12 adult worms were evaluated in each analysis. XTT-Based Cytotoxicity Assay. Cytotoxicity was evaluated using the in vitro Toxicology Colorimetric Assay Kit (XTT; Roche Diagnostics) and normal human lung fibroblasts (GM07492A), as previously described.19 Statistical Analysis. IC50 (inhibitory concentration at 50% cell growth inhibition) and LC50 (lethal concentration at 50% adult worm death) were obtained with GraphPad Prism 5 software. The selectivity index (SI) was calculated by dividing the IC50 value of compounds 2 and 7 obtained for GM07492A cells by the LC50 value obtained for percent of dead worms.20

worms at 24 and 48 h, respectively. Compared with previous data, these results are promising.5,6 As reported by Suffness and Pezzut,21 extracts that give IC50 values lower than 30 μg/mL in cell line assays are interesting for cytotoxic drug investigation. When assayed against GM07492-A cells, EEC provided an IC50 of 12.5 ± 1.4 μg/mL, which indicated cytotoxic action. HPLC-DAD analysis helped to investigate the constituents of the ethanol extract from the cashew and to recognize which of the peaks in the chromatogram accounted for the crude extract bioactivity. To this end, the chromatographic conditions were evaluated using an analytical monolithic column with different mobile phase gradients of methanol−water (+0.1% acetic acid) to optimize both solvent strength and selectivity. The gradient from 70 to 100% methanol for 10 min, 100% methanol for 5 min, and flow 2.0 mL/min was selected due to good resolution. Taking into account the analytical conditions, we made some adjustments to use a semipreparative HPLC monolithic column. Thus, during the chromatographic run, the eluent of the semipreparative HPLC separation of the sample (10.85 mg/ mL) was collected in falcon tubes (11 fractions). After drying, 2 mg of each fraction was weighed, in duplicate, and assayed at a final concentration of 100 μg/mL against S. mansoni and GM07492-A cells. Correlation of the bioactive fractions with time generated a bioactivity HPLC chromatogram,22 which permitted prompt identification of the retention time (tR) of the bioactive peaks in the chromatogram (Figure 1a−c). Analysis of the chromatogram at λ = 201 nm (Figure 1c) compared to the chromatogram of the schistosomicidal activity (Figure 1a) showed that the peaks at tR 4.45, 8.47, 9.15, 10.27, 10.81, and 11.45 min accounted for bioactivity. However, comparative analysis of the chromatogram at λ = 201 nm with the cytotoxic

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RESULTS AND DISCUSSION The ethanol extract from the fruits of A. occidentale (EEC) (100 and 50 μg/mL) killed 100% of adult S. mansoni worms at 24 and 48 h, respectively. EEC at 25 μg/mL killed 25 and 50% of the 8823


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Journal of Agricultural and Food Chemistry activity chromatogram (Figure 1b,c) revealed that the peaks at tR 4.45 min and, to a lesser extent (40%), 11.45 min underlay the activity. On the basis of these data, the EEC extract was fractionated in large scale, which led to the identification of the compounds cardol triene (1), cardol diene (2), anacardic acid triene (3), cardol monoene (4), anacardic acid diene (5), 2-methylcardol triene (6), and 2-methylcardol diene (7) (Figure 2) by 1D- and 2D-NMR experiments and HRMS and by comparison with the available literature data.23−27

respectively. However, compound 4 was completely inactive at the evaluated concentrations (200−12.5 μM). In contrast, compound 7 was the most active compound with an LC50 of 14.5 ± 6.4 μM at 24 and 48 h. Compound 6 killed adult worms (100%) only at concentrations of 200 and 100 μM. The chemical structures of compounds 1−7 are closely related. They differ only in terms of the type of substituents in the benzene ring and in the number of double bonds in the aliphatic 15-carbon chain. Comparison of the benzene ring structure with the bioassay data led us to infer that the presence of 2hydroxybenzoic acid in anacardic acids (compound 5) culminated in weaker schistosomicidal activity. When 1,3benzenediol was present in the structure (compounds 1 and 2), the activity improved, and the presence of the 2-methyl-1,3benzenediol moiety (compound 7) provided the best activity against S. mansoni. However, the number of double bonds in the aliphatic 15-carbon chain also played an important role in the schistosomicidal activity. The presence of two double bonds enhanced the schistosomicidal activity of compounds 5, 2, and 7. The most potent evaluated compounds, 2 and 7, are similar in the aliphatic 15-carbon chain, both have two double bonds, although the presence of the methyl group in the 1,3-benzenediol core in compound 7 augments considerably the schistosomicidal activity. We assessed the cytotoxicity of the isolated compounds by using the normal cell line GM07492-A and the target compounds at concentrations ranging from 3.5 to 7961.8 μM (Table 1). Compounds 3 and 5 displayed IC50 values of 290.2 ± 32.4 and 286.5 ± 28.8 μM and were practically inactive. Among the evaluated compounds, cardols exhibited low cytotoxicity, with IC50 values of 192.6 ± 6.0, 196.8 ± 33.0, and 89.4 ± 19.9 μM for compounds 1, 2, and 4, respectively. Compounds 6 and 7 presented quite different cytotoxic profile with IC50 of 748.3 ± 56.4 and 309.0 ± 33.8 μM, respectively. We compared the IC50 values against GM07492 cells and the LC50 values against S. mansoni adult worms obtained for compounds 2 and 7 by using the SI. At 24 h, compounds 2 and 7 were 6.1 and 21.3 times less toxic to normal cells than to the worms (Table 1). SI values >10 indicate lack of toxicity.28 As

Figure 2. Structure of alkyl-phenols 1−7.

It was possible to determine, by posterior HPLC analysis, that compounds 1 and 6 and 2 and 7 coeluted at tR 8.47 and 9.15 min, respectively. Additionally, compounds 3, 4, and 5 showed tR at 10.27, 10.81, and 11.45 min, respectively, confirming that the alkyl-phenols were eluting in the region more active in the chromatogram of the schistosomicidal activity, which showed peaks in the chromatogram at λ = 201 nm (Figure 1a,c). Examination of the schistosomicidal activity of the isolated compounds (Table 1) demonstrated that compound 5 killed 100 and 25% of worms at 200 μM and 24 h and at 100 μM and 48 h, respectively, whereas compound 3 was inactive. Compounds 1 and 2 were more active as compared to compound 5. Compound 2 gave an LC50 of 32.2 ± 10.2 μM at 24 and 48 h, and compound 1 at 50 μM killed 25 and 75% of the worms at 24 and 48 h,

Table 1. In Vitro Effects of Compounds 1−7 against Adult Schistosoma mansonia Worms and Viability of GM07492A Cells LC50 ± SD of worms (μM)

% of dead worms compound 1 2 3 4 5 6 7 PZQ

incubation period (h) 24 48 24 48 24 48 24 48 24 48 24 48 24 48 24

200 μM 100 μM 100 100 100 100 0 0 0 0 100 100 100 100 100 100

75 100 100 100 0 0 0 0 0 25 100 100 100 100

50 μM 25 μM 25 75 100 100 0 0 0 0 0 0 0 0 100 100

0 50 50 50 0 0 0 0 0 0 0 0 100 100

12.5 μM 0 25 0 0 0 0 0 0 0 0 0 0 75 75 100d

24 h

48 h

IC50 ± SD cell viability (μM in 24 h)

SI (24 h)

192.6 ± 6.0b 32.2 ± 10.2c

32.2 ± 10.2c

196.8 ± 33.0b

6.1

290.2 ± 32.4b 89.4 ± 19.9b 286.5 ± 28.8b 748.3 ± 56.4b 14.5 ± 6.4c

14.5 ± 6.4c

309.0 ± 33.8b

21.3

RPMI 1640 + 0.1% DMSO was included as negative control and killed 0% of worms. bConcentrations of 3.5−7961.8 μM. cAverage data of two independent experiments. dConcentration tested 10 μM. a

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Figure 3. Scanning electron microscopy observations of the tegument of S. mansoni adult worms (A) untreated and after incubation with compounds (B) 2 and (C) 7 at 25 μM for 24 h. The negative control group of adult worms was incubated with RPMI 1640 containing 0.1% DMSO (A). Bars = 100 μm. tu, spine tubercles; os, oral suckers; vs, ventral suckers.

Figure 4. Transmission electron microscopy images of adult S. mansoni worms (A) in the group control and after incubation with compounds (B) 2 and (C) 7 at 25 μM for 24 h. The negative control group of adult worms was incubated with RPMI 1640 containing 0.1% DMSO (A). T, tegument; m, mitochondria; s, spine.

reported previously,29 compounds with LC50 ranging from 1.4 to 9.5 μM and SI >1 are promising compounds regarding antischistosomal activity. We selected the most active com-

pounds (2 and 7) to evaluate whether they prompted any morphological alterations in S. mansoni adult worms. We employed SEM (Figure 3) and TEM (Figure 4) to verify 8825


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to M.L.A.S., D.C.T., W.R.C., A.H.J., and P.M.P. T.A.A. was supported by FAPESP (2013/20280-8) scholarships.

whether changes occurred in the tegument, which is an essential structure for the survival and maintenance of Schistosoma worms.30 In the control group (Figure 3A), the male worms had the tegument covered with a large number of spine tubercles (tu). Besides, the area between the oral (os) and ventral suckers (vs) did not display any morphological alterations. After 24 h of treatment with compound 2 at 25 μM, there were no drastic differences in the tegument of the experimental and control group worms (Figure 3B). On the other hand, male adult worms treated with compound 7 at 25 μM for 24 h (Figure 3C) presented severely damaged tegument. Peeling of the tegument took place, and the number of spine tubercles decreased. Moreover, peeling took place in the area between the oral and ventral suckers. To analyze the ultrastructural alterations, adult worm pairs were incubated with compound 2 or 7 at a concentration of 25 μM for 24 h and analyzed by TEM. Analysis of S. mansoni adult worms in the control group (RPMI 1640 with 0.1% DMSO) showed intact tegument. The muscular layer exhibited preserved fibers throughout the body, and mitochondria and cells had normal morphology (Figure 4A). After incubation for 24 h with compound 2 (Figure 4B), the mitochondrial membrane swelled, vacuoles emerged, and the tegument changed in adult S. mansoni worms. Additionally, analysis of the tegument indicated that vacuoles of different sizes arose. The worms incubated with compound 7 (Figure 4C) presented lysed interstitial tissue and degenerated mitochondria. Analysis of the tegument indicated drastic alterations. The isolated compounds exhibit a wide range of biological properties including antioxidant action; larvicidal control of Aedes aegypti; antiacetylcholinesterase31 and antityrosinase32 activities; moderate cytotoxicity to murine B16-F10 melanoma cells,33 BT-20 breast, and HeLa epithelioid cervix carcinoma;34 inhibition of the enzymes α-glucosidase, aldose reductase, invertase,35 15-lipoxygenase,36 and xanthine oxidase;37 antibacterial action;38 and molluscicidal activity against B. glabratus.39 In summary, chemical investigations into the ethanol extract from the fruits of A. occidentale resulted in the isolation and identification of compounds 1−7. Additionally, biological results indicated that compound 7 showed promising activity against S. mansoni adult worms with good selectivity. The present study highlights the value of alkyl-phenols in the search for novel therapeutic options to treat patients with schistosomiasis.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Electron Microscopy Laboratory in Ribeirão Preto, University of São Paulo, Brazil, is acknowledged for support with the transmission electron microscopy examination.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04200.

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REFERENCES

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NMR and HRMS data of compounds 1−7 (PDF)

AUTHOR INFORMATION

Corresponding Author

*(P.M.P.) E-mail: [email protected]. Phone: +5516-3711-8871. Fax: +55-16-3711-8886. Funding

FAPESP (São Paulo Research Foundation) is acknowledged for financial support (Grant 2013/09280-6). CNPq (The National Council for Scientific and Technological Development) is acknowledged for Research Productivity Fellowships granted 8826


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Schistosomicidal Activity of Alkyl-phenols from the Cashew

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