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Taylor and Francis 2007 Pharmaceutical Biology 2007, Vol. 45, No. 5, pp. 376-380 informa healthcare Antiprotozoal Activity of Flavonoid Glycosides Isolated from Clidemia sevicea and Mosquitoxylon jamaicense Hector Montenegro1, Jose Gonzalez2, Eduardo Ortega-Barria2, and Luis Cubilla-Rios1 'Laboratory of Tropical Bioorganic Chemistry, Faculty of Natural, Exact Sciences and Technology, Apartado, University of Panama, Panama City, Republic of Panama; 2Instituto de Investigaciones Cientificas Avanzadas y Servicios de Alta Tecnologia, Secretaria Nacional de Ciencia, Tecnologia e Innovation, Clayton, Ancon, Republic of Panama c Abstract A new O-galloyl-C-glycosylflavone, 2",6"-0-digalloylvi- texin (1), along with four known glycosylflavones (2-5) have been isolated from Clidemia sericea D. Don (Melastomataceae), and four other known glycosylfla- vones (6-9) have been isolated from Mosquitoxylon jamaicense Krug & Urb. (Anacardiaceae). Compound 1, 3, and 6 showed mild antimalarial activity (24 ?1, 38 ? 2, and 44 ? 1 uM, respectively) against a chloro- quine-resistant P. falciparum strain. Additionally, tests against leishmaniasis and Trypanosoma cruzi were made. These compounds were identified by MS, UV, IR, and ID and 2D NMR data and by comparison with the literature data. Keywords: Clidemia sericea, flavonoid glycosides, ICBG, leishmania, malaria, Mosquitoxylum jamaicense, tropical diseases, Trypanosoma cruzi. Introduction Malaria is one of the tropical diseases with the greatest impact on world health, causing 300 million cases and one million deaths annually (Gelb & Hoi, 2002). Strains of Plasmodium falciparum that are resistant to the latest drugs, as well as chloroquine, have emerged and spread rapidly (Ridley, 2002a,b). Continuing with our search for antiprotozoal drugs, we report now the bioassay-guided isolation of five flavonoids from Clidemia sericea D. Don (Melastomataceae): the new O-galloyl-C-glycosylflavone 1 (Fig. 1) and the known isovitexin 2, 2"-0-galloylvitexin 3, rutin 4 (Lin et al, 2000), and vitexin 5 (Latte et al, 2000), and four known flavonoids from Mosquitoxylon jamaicense Krug & Urb. (Anacardiaceae): phloridzin 6 (Hilt et al., 2003), 4-hydroxy benzenepropanal 7 (Ishikawa & Kishi, 2000), trilobatin 8 (Tanaka et al., 1983), and quer- cetine-3-O-P-D-galactoside 9 (Zhang & Mao, 2001) as part of the ongoing ICBG program based on Panama (Coley et al., 2003). Materials and Methods General experimental procedures Optical rotations were determined on an Autopol III 6971 Automatic Polarimeter (Rudolph Research Analytical, NJ, USA). Infrared (IR) spectra were mea- sured on a Perkin-Elmer Fourier transformer infrared (FT-IR) Spectrometer Spectrum RXI (Perkin-Elmer, USA). The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 300 spectro- meter (300 MHz for proton and 75 MHz for carbon) (Bruker BioSpin, MA, USA). Low Resolution and High Resolution Mass Spectra (HRMS) were recorded on a Kratos MS50TC instrument using chemical Ioni- zation (Kratos Analytical Instruments, NJ, USA). High Pressure Liquid Chromatography (HPLC) and ultraviolet (UV) spectrum were carried out on a Waters Liquid Chromatography (LC) system, with a 600 pump and 996 phodiode array detector (Waters, MA, USA). Accepted: October 23, 2006. Address correspondence to: Luis Cubilla-Rios, Laboratory of Tropical Bioorganic Chemistry, Faculty of Natural, Exact Sciences and Technology, Apartado 0824-00159, University of Panama, Panama City, Republic of Panama. Tel: (507) 66765824; Fax: (507) 264 4450; AP: 0824.00159; E-mail: lucr@ancon.up.ac.pa DOI: 10.1080/13880200701214821 ? 2007 Informa Healthcare Antiprotozoal activity of flavonoids 377 Plant material Young leaves of Clidemia sericea and Mosquitoxylon jamaicense were collected in Altos de Campana National Park and Chagres National Park, respectively, in the Republic of Panama. The material was identified by Professor Mireya Correa of the University of Panama and the Smithsonian Tropical Research Institute. Vouchers have been deposited at the Herbarium of the University of Panama, numbers PMA53118 (C. sericea) and PMA53117 (M. jamaicense). Extraction and isolation The leaf material of C. sericea (300 g) and of M. jamai- cense (900 g) was homogenized and processed as pre- viously described (Torres et al., 2003). The organic extracts were concentrated as described (Montenegro et al., 2003), obtaining 15 g (IC50: 16ug/mL) and 44 g (IC50: 12ug/mL), correspondingly. All LH-20 columns were equilibrated with methanol prior to chromatography. Clidemia sericea The methanol crude extract of C. sericea (15 g) was sub- jected to liquid-liquid partition with M-hexane and meth- anol. The methanol fraction was evaporated and subjected to a second solvent partition using EtOAc and H20. The EtOAc portion (2.7 g, IC50: 5 ug/mL) was subjected to vacuum-liquid chromatography (VLC) on silica gel (7GF, VWR Scientific) using hexane- EtOAc-MeOH mixtures of increasing polarity. The fractions were combined according to TLC composition into fractions 1-7 and dried under vacuum. 950 mg of fraction 5 (IC50: 8 ug/mL), between 650-700 mL, was chromatographed on Sephadex LH-20 (2.5 x 70 cm) (25-100 run Sigma) and eluted with EtOH:H20 80:20 (800 mL), EtOH:H20 90:10 (300 mL), and 100% EtOH (500 mL). The fractions were combined according to TLC composition into Frs. 5a-5i. Fraction 5d (42 mg) was recrystallized from MeOH, yielding compound 2 (30 mg). Purification of fraction 5e used isocratic reverse phase HPLC (XTerra? 10 rim, 10 x 250 mm) with MeOH:H20 (50:50) as eluent, flow 2mL/min, yielding compounds 3 (20 mg, tR: 17 min) and 4 (7mg, tR: 32 min). Fraction 5h was recrystallized from EtOAc and purified using RP-HPLC (XTerra 10 urn, 10 x 250 mm) with MeOH:H20 (40:60), flow 2mL/min, yielding the galloylglycosylflavone 1 (6.4 mg, tR: 17.8 min). Fraction 6 (267 mg), between 700-800 mL, was redisolved in MeOH and chromatographed on Sephadex LH-20 (2.5 x 70 cm, 25-100 urn, Sigma) and eluted with EtOH:H20 80:20 (800 mL), EtOH:H20 90:10 (300 mL), and 100% EtOH (500 mL). The fractions were combined according to TLC composition into Frs. 6a-6j. Fraction 6d (45 mg) was recrystallized from MeOH, yielding 5 (5 mg). Mosquitoxylon jamaicense The methanol crude extract of M. jamaicense (44 g) was subjected to liquid-liquid partition with M-hexane and MeOH. The methanol fraction was evaporated and subjected to a second solvent partition using EtOAc and H20. The EtOAc part (6.6 g, IC50: 7 ug/mL) was subjected to vaccum-liquid chromatography (VLC) on silica gel (7GF, VWR Scientific) using hexane-AcOEt- MeOH mixtures of increasing polarity. The fractions were combined according to TLC com- position into Frs. 1-6. Fraction 5 (2.5 g, IC50: 17 ug/mL) between 1260-1350mL was chromatographed on Sephadex LH-20 (2.5 x 70 cm, 25-100 u, Sigma) and eluted with EtOH:H20 80:20 (800 mL), EtOH:H20 90:10 (300 mL), 100% EtOH (500 mL), and MeOH 100% (500 mL). The fractions were combined according to TLC composition into Frs. 5a-5k. Fraction 5d (132mg) was filtered on a solid-phase extraction cartridge of RP- 18 (Merck) and recrystallized from MeOH. This fraction was purified using RP-HPLC (XTerra 10 rim, 10 x 250 mm) with MeOH:H20 (40:60), flow 2mL/min, yielding 6 (16 mg, tR: 19 min). Purification of fraction 5e using preparative RP-HPLC (Novapack RP-18 6urn, 25 x 200mm) with gradient MeOH:H20 (10:90- 70:30 in 40 min and 70:30-100 MeOH in 50 min) flow 4 mL/min, yielding 8 (38 mg, tR: 20 min) and 7 (6mg, tR: 40 min). Fraction 5i was recrystallized from MeOH, yielding 9 (12 mg). Galloylglycosylflavone 1 Yellow amorphous powder; [OC]D22: - 14.25 (MeOH, c 0.25); IRvma* cm"': 3352, 2924, 1624, 1598, 1516, 1452, 1262, 1206, 1174, 1076, 1046, 1024, 994, 828; UV km** (MeOH) nm 225.0, 269.8, 281.1, 339.9, 343.4; 'H NMR (MeOD; 300 MHz) 8 7.76 (2H, d, J = 8.3 Hz, H-2' and H-6'), 7.11 (2H, s, H-2"", and H-6""), 6.88 (2H, d, /= 8.3 Hz, H-3' and H-5'), 6.82 (2H, s, H-2'" and H-6'"), 6.52 (1H, s, H-3), 6.42 (1H, s, H-8), 5.50 (1H, t, 7=7.8 Hz, H-2"), 5.33 (H, m, H-3"), 5.32 (H, d, /= 7.8 Hz, H-l"), 3.67 (1H, m, H-5"), 3.89 (1H, m, H-6"), 3.83 (3H, m, H-4", H-6"); 13C NMR (MeOD; 75 MHz) 8 184.2 (C^l), 168.5 (C=0 3" ), 167.5 (C-O 2"), 166.5 (C-2), 164.9 (C-7), 163.0 (C-5 and C^'), 159.2 (C-9), 146.6 (C-3"" and C-5""), 146.5 (C-3"' and C-5'"), 140.2 (C^l"' and C^""), 129.8 (C-2' and C-6'), 123.4 (C-l'), 121.8 (C-l""), 121.3 (C-l'"), 117.3 (C-3' and C-5'), 110.8 (C-2"" and C-6""), 110.7 (C-2'" and C-6'"), 107.8 (C-10), 105.3 (C-6), 104.2 (C-3), 95.4 (C-8), 83.2 (C-5"), 79.4 (C-3"), 73.4 (C-2"), 72.0 (C^l"), 378 H. Montenegro et al. OH OH OH O OH O Figure 1. Chemical structures of compounds 1-3 and 6. 63.0 (C-6'); HRFABMS (NBA, [M]+ 736.12760 (calculated C35H28018, Assays positive mode) 736.12756). All assays were based on inhibition of growth of the parasites by added compounds or extracts, as described previously (Molinar-Toribio et al., 2006 and 2004; Cor- bett et al., 2004; Torres et al., 2003 and 2004; Williams et al., 2003). Cytotoxicity assay Vero cells adhering to 96-well plates were used to evaluate the toxicity of the compounds purified on the basis of the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma) (Torres et al., 2004). After treatment with the test compound and 4 h of incubation at 37?, cell viability was evaluated in an ELISA reader at 570 nm. Results and Discussion Clidemia sericea The ethyl acetate partition fraction of the crude extract of C. sericea showed significant antiplasmodial activity (IC50: 5 ug/mL), and was selected for bioguided fraction- ation, yielding the novel galloylglycosylflavone (1) and four known compounds (2-5). Compound 1 was obtained as a yellow powder and showed a pseudomolecular ion from its HRFABMS at m/z 737.12760 (Cs^HzgOig, calcd 736.12756), which was consistent with 35 carbons observed in the 13C NMR spectra, sorted by DEPT experiments into 1-CH2 oxygenate, 15-CH (5 oxygenate), and 19 quaternary. The UV spectra of compound 1 exhibited three absorp- tion bands at 222, 272, and 337 nm, consistent with a flavone derivative (Latte et al., 2000). The IR spectrum showed bands consistent with the presence of one or more hydroxyl groups (3352 cm-1), an ester carbonyl (1624cm-1), a conjugated carbonyl (1598cm-1), and a phenyl group (1516, 1452cm-1). The lH NMR spectrum showed signals due to two galloyl groups 8 6.82 (2H, s) and 7.11 (2H, s) and 13C NMR signals at 5, 110.7, 121.3, 140.2, 146.5, 167.5 (COO), due to one galloyl group, and signals at 5 110.8, 121.8, 140.2, 146.6, 168.5 (COO), due to a second galloyl group. The :H NMR spectrum revealed the presence of two protons at 8 6.42 (H-8) and 8 6.52 (H-3) belonging to the flavone skeleton, assigned from HMQC and HMBC correlations. A ^^-substituted phenol was charac- terized by aromatic A2B2-spin system of the B-ring at 8 7.76 and 8 6.88 (each 2H, d, /= 8.3 Hz). The anomeric proton of the P-D-glucopyranosyl moiety (8 5.32, d, /= 7.8 Hz) had correlations with C-2" and C-3" and a long-range correlation with C-6". A methylene proton at 8 5.50 (t, / = 7.89 Hz) correlated with C-3" and had a long range correlation with the carbonyl (167.5) of a galloyl group, indicating that one of the galloyl group Antiprotozoal activity of flavonoids 379 Table 1. Antimalarial" activity and cytotoxicities of flavonoid glycosides. Compound 1 2 3 4 5 6 7 8 9 Activity IC50 pg/mL (uM) cytooxicity* IC50 ug/mL 18 24 ? 1 96 >50 >116 >100 23 38?2 > 151 33 76 ?3 NDC >50 > 116 ND 19 44 ?1 ND 32 218 ?1 ND >50 > 115 >100 23 50 ?2 >100 "Chloroquine-resistant strain of Plasmodium fakiparum (IC50 0.06 pM). ^Experiments performed with Vero cells. 'ND = not determined. is attached to C-2" as in 3. A second methines proton at 8 5.33 (m) correlated with C-2" and another carbonyl group (168.5) of a second galloyl group, indicating that this second galloyl group was attached to C-3." Mosquitoxylon jamaicense The EtOAc liquid-liquid partition fraction of the crude MeOH-EtOAc extract of M. jamaicense with an activity IC50: 12ug/mL against P. fakiparum was selected to bioassay-guided fractionation; yielding four known compounds (6-9). From the nine compounds reported here, three (i.e., 1, 3, and 6) showed moderate activity against W2, a Plasmodium fakiparum strain (chloroquine-resistant), while a further six isolated compounds were with activi- ties that exceeded 50 ug/mL (Table 1). Compounds 1 and 3 are more active than 2, and this suggests that the galloyl moiety is necessary for the activity as pre- viously reported by our group (Corbett et al., 2004). Likewise, none of the isolated compounds demonstrated activity against Leishmania and T. cruzi parasites at concentrations of 40 and 50 (ig/mL, respectively, or sig- nificant cytotoxic activity when tested against Vero cells (lOOug/mL). Acknowledgments The authors extend special thanks to Drs. Phyllis D. Coley and Thomas A. Kursar from the University of Utah, who were instrumental in establishing the present bioprospecting program in Panama and for the technical assistance from R. Aizprua, N. Flores, and B. Arauz. 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