Volume 5. Issue 6. Pages 841-992. 2010 ISSN 1934-578X (printed); ISSN 1555-9475 (online) www.naturalproduct.us NPC Natural Product Communications EDITOR-IN-CHIEF DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA agrawal@naturalproduct.us HONORARY EDITOR PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT U.K. axuf64@dsl.pipex.com EDITORS PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy braca@farm.unipi.it PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China gda5958@163.com PROFESSOR J. ALBERTO MARCO Departamento de Quimica Organica, Universidade de Valencia, E-46100 Burjassot, Valencia, Spain alberto.marco@uv.es PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan mimakiy@ps.toyaku.ac.jp PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia spyne@uow.edu.au PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA m.reinecke@tcu.edu PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA wsetzer@chemistry.uah.edu PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan tezuka@inm.u-toyama.ac.jp PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK david.thurston@pharmacy.ac.uk ADVISORY BOARD Prof. Berhanu M. Abegaz Gaborone, Botswana Prof. Viqar Uddin Ahmad Karachi, Pakistan Prof. Øyvind M. Andersen Bergen, Norway Prof. Giovanni Appendino Novara, Italy Prof. Yoshinori Asakawa Tokushima, Japan Prof. Lee Banting Portsmouth, U.K. Prof. Julie Banerji Kolkata, India Prof. Anna R. Bilia Florence, Italy Prof. Maurizio Bruno Palermo, Italy Prof. Josep Coll Barcelona, Spain Prof. Geoffrey Cordell Chicago, IL, USA Prof. Cristina Gracia-Viguera Murcia, Spain Prof. Duvvuru Gunasekar Tirupati, India Prof. A.A. Leslie Gunatilaka Tucson, AZ, USA Prof. Kurt Hostettmann Lausanne, Switzerland Prof. Martin A. Iglesias Arteaga Mexico, D. 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Box 30197 (00100), Nairobi, Kenya National Center for Natural Products Research and cDepartment of Pharmacology, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677, USA b milias@olemiss.edu Received: February 4th, 2010; Accepted: March 27th, 2010 Chromatographic separation of the roots of a Kenyan medicinal plant, Clerodendrum eriophyllum, led to the isolation of ten abietane diterpenoids (1-10), one of which (1) was isolated for the first time from a natural source. Using spectroscopic data, the structure of 1 was determined to be 12-hydroxy-8,12-abietadiene-3,11,14-trione. Circular dichroism (CD) spectra showed that the stereochemistry of compounds 1, 3, and 6-8 belongs to the normal series of abietane diterpenes, which confirmed the absolute stereochemistry of the isolated compounds. Compounds 1-10 were evaluated for their in vitro antiplasmodial, antileishmanial, antifungal and antibacterial activities. Compounds 3 and 7 exhibited potent antifungal activity (IC50/MIC 0.58/1.25 and 0.96/2.5 g/mL, respectively) against C. neoformans, whereas 3, 6 and 7 showed strong antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus with IC50/MIC values between 1.33-1.75/2.5-5 and 0.961.56/2.5 g/mL, respectively. In addition, compounds 3 and 9 exhibited potent antileishmanial activity (IC50 0.08 and 0.20 g/mL, respectively) against L. donovani, while 3 and 7 displayed weak antimalarial activity against Plasmodium falciparum, but 9 was inactive. Keywords: Clerodendrum eriophyllum, Verbenaceae, abietane diterpenoids, antimicrobial, antileishmanial, antimalarial. Clerodendrum eriophyllum Gürke (Verbenaceae), a small tree 0.5 – 2 m high, is scattered in the dry bushlands of Eastern Kenya where the plant is used by local communities for the treatment of malaria [1]. The plant has no record of previous phytochemical analysis. However, the methanol extract of C. eriophyllum root bark previously showed weak in vitro activity against Plasmodium falciparum D6 and W2 clones (IC50 9.5110.56 g/mL); while its methanol and aqueous extracts exhibited significant in vivo chemosuppression (i. e., 90.1% and 61.5%, respectively) against P. berghei infected mice treated intraperitoneally at a dose of 100 mg/kg body weight [2]. The genus Clerodendrum is known to contain iridoids [3, 4], abietane diterpenoids [5-7] and steroids [8]. In the quest for antiplasmodial compounds from Kenyan plants, we have investigated the roots of C. eriophyllum collected from Eastern Kenya. In this paper we report the isolation and structure elucidation of a new abietane diterpenoid, 12hydroxy-8,12-abietadiene-3,11,14-trione (1), obtained alongside nine other known abietane diterpenoids (2-10). OH O 15 O 11 20 16 1 10 OH HO 8 O 5 R R 3 R=O 4 R = H2 1 R=O 2 R = H2 OH HO HO O O OH HO HO R O OH 7 R=H 8 R = OH 5 R=O 6 R = H2 O HO R H H H 18 OH O OH 9 H 10 Figure 1: Chemical structures of compounds 1-10 isolated from C. eriophyllum. We also report the antiplasmodial, antileishmanial, antibacterial, and antifungal activities of the isolated compounds. 854 Natural Product Communications Vol. 5 (6) 2010 The 1:1 MeOH/CH2Cl2 extract of roots of C. eriophyllum showed moderate antiplasmodial activity with IC50 values of 8.8 g/mL against chloroquinesensitive (D6) and -resistant (W2) strains of P. falciparum. Repeated chromatographic purification of this extract gave 12-hydroxy-8,12-abietadiene-3,11,14trione (1), as well as nine known abietane diterpenoids, namely royleanone (2) [9], taxodione (3) [10], 11hydroxy-7,9(11),13-abietatrien-12-one (4) [11], sugiol (5) [12], ferruginol (6) [13], 6-hydroxysalvinolone (7) [14], 6,11,12,16-tetrahydroxy-5,8,11,13-abietatetra-en7-one (8) [15], uncinatone (9) [16], and 11-hydroxy8,11,13-abietatriene-12-O- -xylopyranoside (10) [15]. The molecular formula of compound 1 was established as C20H26O4 (m/z 331.1910 [M+H]+; calculated for m/z 331.1909) by HRESIMS. The UV absorption maxima at λmax 273 and 378 nm closely matched those of the p-quinone chromophore of royleanone 2 [9]. The IR spectrum indicated the presence of hydroxyl group(s) (νmax 3080-3430 cm-1), unconjugated carbonyl (νmax 1704 cm-1), together with olefinic and conjugated carbonyl absorptions of the p-quinone moiety (νmax 1609, 1633 and 1650 cm-1). The 13C NMR spectrum showed 20 signals, with the sp2 region displaying four olefinic quaternary carbons (δC 124.3, 144.1, 145.8, 150.6) and two conjugated carbonyls (δC 183.2, 186.9) assignable to the p-quinone moiety. The 1H NMR spectrum did not show signals in either the olefinic or aromatic regions, but did show signals for three methyl singlets at δH 1.09, 1.13 and 1.24, assignable to C-18, C-19 and C-20, respectively, of an abietane skeleton, and an isopropyl group, with two methyl doublets at δH 1.19 and 1.20 (each 3H, J = 7.0 Hz) and a methine septet at δH-15 3.15. The position of the isopropyl group was deduced from HMBC correlations (Table 1) between δH-15 3.15 and δC-12 150.6, δC-13 124.3, and δC-14 186.9. The unconjugated carbonyl at δC 216.7 was established to be at C-3 from HMBC correlations between δH-18 1.13, δH-19 1.09 and the carbonyl carbon signal. Furthermore, a comparison of the 13C NMR spectral data of 1 with those of the known compound royleanone 2 showed close similarities of the carbon signals, except for the differences associated with C-2 – C-5 due to the presence of a carbonyl group at the C-3 position (δC 33.8, 216.7, 46.9 and 50.8 vs. 18.5, 41.2, 33.8 and 44.4 for 2, respectively). A complete set of 2D NMR experiments [1H-1H COSY, 1H-13C HMQC, 1H-13C HMBC (Table 1), 1H-1H NOESY] allowed the unambiguous establishment of the structure of 1 as 12-hydroxy-8,12-abietadiene-3,11,14- trione. Circular dichroism (CD) spectra showed that the stereochemistry of compounds 1, 3, and 6-8 belong to the normal series (A/B trans) of diterpenes. The Machumi et al. Table 1: 1H and 13C NMR spectroscopic data (J values in Hz, in parenthesis), and 1H-13C HMBC correlations of compound 1. H/C 1 H C 1.76, m; 2.81, m 34.5, t 2.59, ddd (15.6, 9.2, 5.4); 2.51, dt (15.6, 7.2) 1.76, m 33.8, t 6 1.46, ddd (21.8, 12.0, 4.5); 1.76,m 18.6, t 7 2.31, ddd (19.6, 12.0, 6.0); 2.84, br dd (19.6, 5.4) 26.0, t 8 9 10 11 12 13 14 15 3.15, sept (7.0) 145.8, s 144.1, s 37.3 ,s 183.2 ,s 150.6 ,s 124.3, s 186.9, s 24.1, d 16 17 18 1.20, d (7.0) 1.19, d (7.0) 1.13, s 19.9, q 19.8, q 27.7, q 2 3 4 5 19 1.09, s 20 1.24, s 12-OH 7.21, s 216.7, s 46.9, s 50.8, d 20.0, q 20.6, q - HMBC C-2, C-3, C-5, C-10, C-20 C-1, C-3, C-4, C-10 C-1, C-3, C-4, C-6, C-7, C-10, C-18, C-19, C-20 C-4, C-5, C-7, C-8, C-10 C-5, C-6, C-8, C-9. C-14 C-12, C-13, C-14, C-16, C-17 C-13, C-15, C-17 C-13, C-15, C-16 C-3, C-4, C-5, C-19 C-3, C-4, C-5, C-18 C-1, C-5, C-9, C-10 C-11, C-12, C-13 positive Cotton effect at 275 and 284 nm for compound 1 supports the β-orientation of the methyl group at C-10, i.e. (10S)-Me configuration, according to the rule for π-π* transition of an α,β-unsaturated ketone [17]. The CD spectra of compounds 3 and 6 are in agreement with those of the known taxodione analog [18] and ferruginol [19], respectively, confirming their absolute stereochemistry. CD spectra of 7 and 8, not previously reported, are similar to the related abietane diterpene cyrtophyllone A, whose absolute stereochemistry was determined by X-ray crystallography [17]. Only recently, the absolute configuration of 6hydroxysalvinolone (7) was determined as (10R)-Me by enhanced X-ray crystallography [20], thus, supporting its stereochemistry deduced from CD spectra. The antiplasmodial, antileishmanial, antifungal, antibacterial and cytotoxic activities are summarized in Tables 2-4. Compounds 3 and 9 demonstrated potent antileishmanial activities with IC50 values of 0.08 and 0.20 g/mL, respectively, against L. donovani, compared with those observed for the standard drug amphotericin B (IC50 0.13 g/mL). On the other hand, the antiplasmodial activities of compounds 3, 7 and 8 were found to be very weak, with IC50 values of 1.2 - 4.8 g/mL, when compared with the standard artemisinin (IC50 <0.026 g/mL). Strong antifungal activities were also displayed by 3 and 7, Diterpenoids from Clerodendrum eriophyllum Natural Product Communications Vol. 5 (6) 2010 855 Table 2: Antiplasmodial, antileishmanial and cytotoxic activity of compounds 2-10. Compound/extract C.eriophyllum extract 1 2 3 6 7 8 9 10 Chloroquine Artemisinin Pentamidine Amphotericin B P. falciparum D6a W2b IC50, μg/mL 8.8 8.8 1.2 1.8 3.0 <0.026 <0.026 NT NT 1.2 2.5 4.8 0.14 <0.026 NT NT VERO TC50 μg/mL NC NC NC NC NC 4.5 NC NC NC NC NC NT NT L. donovani IC50 IC90 μg/mL μg/mL NT NT 16 NT 0.08 4 3.2 12 0.2 NT NT NT 1.4 0.13 32 NT 0.21 13 6.5 22 0.9 NT NT NT 6 0.3 a Chloroquine-sensetive clone; bChloroquine-resistant clone ;- =Not Active; NT = Not Tested; NC = Not cytotoxic (up to the maximum dose tested; 4.76 μg/mL for pure compounds and 47.6 mg/ml for crude extracts). IC50 is the concentration that affords 50% inhibition of growth. Table 3: Antifungal activities of compounds 2-10. Compound 1 2 3 6 7 8 9 10 Amphotericin B C. glabrata -/-/5.2/10 -/-/14.9/20 -/-/0.31/0.65 IC50/MIC, g/mL C. krusei C. A. C. albicans neoformans fumigatus -/-/-/-/-/-/-/-/12.0/0.58/1.25 8.9/12.5/-/-/-/-/-/0.96/2.5 11.2/-/14.5/20 5.9/20 -/-/-/-/-/-/-/-/-/-/0.95/1.25 0.44/1.25 1.29/2.50 0.43/1.25 - =Not Active; NT = Not Tested; IC50 is the concentration that affords 50% inhibition of growth; MIC is the lowest test concentration that allows no detectable growth. Table 4: Antibacterial activities of compounds 2-10. IC50/MIC, g/mL Compound S. aureus 1 2 3 6 7 8 9 10 Ciprofloxacin -/-/1.35/5 1.33/2.5 1.75/5 6.8/20 -/-/0.1/0.25 MRS E. coli P. M. aureginosa intracellulare -/-/-/-/-/-/-/-/1.47/2.5 -/-/11.9/0.96/2.5 -/-/14.5/1.56/2.5 -/-/-/8.44/20 -/-/-/-/-/-/-/-/-/-/-/0.08/0.25 0.004/0.008 0.06/0.25 0.30/1.00 =Not Active; NT = Not Tested; IC50 is the concentration that affords 50% inhibition of growth; MIC is the lowest test concentration that allows no detectable growth. showing IC50 values of 0.58 and 0.96 g/mL, respectively, against C. neoformans, as compared with 0.44 g/mL of the standard amphotericin B. Compounds 3, 6 and 7 showed strong antibacterial activity against Staphylococcus aureus and methicillinresistant S. aureus with IC50/MIC values between 1.331.75/2.5-5 and 0.96-1.56/2.5 g/mL, respectively. With regard to cytotoxicity, only 6-hydroxysalvinolone (7) showed moderate cytotoxic activity with an IC50 value of 4.5 g/mL against monkey kidney fibroblasts (VERO). Finally, compound 1 showed weak in vitro antileishmanial activity against L. donovani (IC50 16 μg/mL), but found to be inactive in antimalarial and antimicrobial assays. Experimental General: Optical rotations were measured in CHCl3 or MeOH using an AUTOPOL IV® instrument at ambient temperature. Circular dichroism (CD) spectra were recorded in MeCN using an Olis DCM 20 CD spectrometer at ambient temperature. IR spectra were taken as films on a Bruker Tensor 27 FTIR instrument. UV spectra were obtained in MeCN using a HewlettPackard 8453 spectrophotometer; 1D and 2D NMR data were acquired on a Bruker BioSpin instrument at 600 MHz (1H), 150 (13C) in CDCl3 using the residual solvent as int. standard. HRMS were obtained by direct injection using a Bruker Bioapex-FTMS with electrospray ionization (ESI). For column chromatography (CC), Merck silica gel 60 (0.063-0.200 mm) and Fluka Sephadex LH-20 were used as stationary phases; For PTLC, Merck silica gel 60 PF254+366, coated on glass plates to make 1.0 mm layers was used; Analytical TLC was carried out using factory prepared aluminum plates (0.25 mm) coated with silica gel (60 F254, Merck); The isolated compounds were visualized by observing under UV light at 254 or 365 nm, followed by spraying with 1% vanillin-H2SO4 spray reagent. Plant material: The roots of Clerodendrum eriophyllum were collected from Machakos, Eastern Kenya in November 2007 and identified at the Department of Botany, University of Nairobi, Kenya, where a voucher specimen No. JMFM/2007/11 has been deposited. Extraction and isolation: The roots of C. eriophyllum were air dried and pulverized to give 1.8 kg of material. This was extracted by cold percolation at room temperature using 1:1 MeOH/ CH2Cl2 (3×4 L, 24 h each), followed by 100% methanol (1×4 L, 24 h) to give 65 g of brown gummy extract, of which 35 g was adsorbed onto 40 g of silica gel and subjected to CC on a silica gel column (300 g, 5×35 cm), eluted with nhexane/CH2Cl2 (95:5, 3.5 L; 9:1, 1.25 L; 3:1, 2 L; 1:1, 3 L; 1:3, 1 L; 100% CH2Cl2, 1.5 L) followed by CH2Cl2/MeOH (99:1, 1.25 L; 98:2, 1 L; 95:5, 1 L). Sixty-two fractions of eluents, collected in 250 mL aliquots, were concentrated using a rotary evaporator and similar fractions were combined on the basis of TLC analysis. Combination of fraction 5-9 crystallized in n-hexane/CH2Cl2 (95:5) gave 2 (260 mg). Fractions 11-19 (640 mg) were rechromatographed over silica gel (50 g, 2.0×30 cm) and eluted with n-hexane/CH2Cl2 (95:5) to give 4 (8.7 mg) after 0.6 L of elution, and 6 (65 mg) after 1.4 L of elution. The latter was further 856 Natural Product Communications Vol. 5 (6) 2010 purified by PTLC developed with n-hexane/CH2Cl2 (8:2). Fraction 22-28 (160 mg) was purified on a Sephadex LH 20 column (100 g, 2.5×30 cm), eluted with MeOH/ CH2Cl2 1:1 (0.3 L), followed by PTLC developed with n-hexane/CH2Cl2 7:3 to give 3 (28 mg). Fraction 34-45 (600 mg) was subjected to silica gel CC (50 g, 2.0×30 cm), eluted with CH2Cl2/ n-hexane (8:2) to give 1 (4.0 mg) after 0.28 L of elution, 9 (6.7 mg) after 0.35 L of elution, and 5 (10.3 g) after 0.8 L of elution. Fractions 52-57 (580 mg) were rechromatographed over silica gel (50 g, 2.0×30 cm) and eluted with CH2Cl2 to yield 7 (6.5 mg) and 8 (64.2 mg), both of which crystallized from CH2Cl2, after 0.52 L and 1.3 L of elution, respectively. Compound 10 (15 mg) was obtained after purifying fractions 58-60 on a Sephadex LH 20 column (100 g, 2.5×30 cm) eluted with MeOH/ CH2Cl2 1:1 (0.3 L). Royleanone (2) [9], taxodione (3) [10], 11-hydroxy7,9(11),13-abietatrien-12-one (4) [11], sugiol (5) [12], ferruginol (6) [13], 6-hydroxysalvinolone (7) [14], 6,11,12,16-tetrahydroxy-5,8,11,13-abietatetra-en-7-one (8) [15], uncinatone (9) [16] and 11-hydroxy-8,11,13abietatriene-12-O- -xylopyranoside (10) [15] were identified by comparison of their full physical (mp and optical rotation) and spectral data (UV, IR, 1H and 13C NMR, and MS) with those reported in the literatures. 12-Hydroxy-8,12-abietadiene-3,11,14-trione (1) Yellow solid Rf 0.5 (n-hexane/CH2Cl2/MeOH 60:39:1) [α]D25: +184 (c 0.16, CHCl3) IR (film) νmax, cm-1: 3430-3080 (OH), 2965 (C-H), 2934 (C-H), 2873 (C-H), 1704 (C=O), 1650 (C=O), 1633 (C=O), 1609 (C=C), 1462 (C-H), 1271 (C-O). UV (MeCN) λmax (log ε), nm: 201 (3.94), 205 (3.86), 273 (3.98), 378 (2.78) CD (MeCN) λmax ([θ], deg·cm2/dmol), nm: 208 (+6.4·103), 275 (+18.9·103), 284 (+20.2·103), 330 (-2.3·103). 1 H and 13C NMR: Table 1. HRESIMS: m/z 331.1910 [M+H]+ (calcd. for C20H27O4, 331.1909), 329.1758 [M-H]- (calcd. for C20H25O4, 329.1753). Taxodione (3) UV (MeCN) λmax (log ε), nm: 211 (3.81), 315 (4.13), 325 (4.13), 334 (4.12), 337 (4.11), 394 (3.38). CD (MeCN) λmax ([θ], deg·cm2/dmol), nm: 205 (20.2·103), 261 (-12.7·103), 321 (-18.7·103), 337 (19.2·103), 445 (+13.6·103). Ferruginol (6) UV (MeCN) λmax (log ε), nm: 211 (3.90), 219 (3.87), 281 (3.58). Machumi et al. CD (MeCN) λmax ([θ], deg·cm2/dmol), nm: 206 (-2.8·103), 211 (+12.9·103), 227 (+9.5·103), 265 (-0.9·103), 301 (-2.3·103). 6-Hydroxysalvinolone (7) UV (MeCN) λmax (log ε), nm: 219 (3.97), 250 (3.98), 284 (3.94), 335 (4.00). CD (MeCN) λmax ([θ], deg·cm2/dmol), nm: 213 (+36.3·103), 281 (+30.4·103), 338 (-19.7·103). 6,11,12,16-Tetrahydroxy-5,8,11,13-abietatetra-en-7one (8) UV (MeCN) λmax (log ε), nm: 218 (3.98), 251 (4.01), 285 (3.89), 339 (3.97), 407 (2.90). CD (MeCN) λmax ([θ], deg·cm2/dmol), nm: 212 (+48.9·103), 284 (+29.7·103), 339 (-19.7·103). Uncinatone (9) UV (MeCN) λmax (log ε), nm: 220 (4.06), 228 (4.08), 232 (4.09), 283 (4.17), 298 (4.17), 334 (4.01), 376 (3.95). CD (MeCN) λmax ([θ], deg·cm2/dmol), nm: 205 (-18.8·103), 213 (-18.9·103), 234 (+24.5·103), 241 (+23.9·103), 271 (sh) (-4.3·103), 297 (-17.1·103), 323 (+12.7·103), 350 (-2.7·103), 381 (-5.6·103). Antimicrobial assay: All organisms were obtained from the American Type Culture Collection (Manassas, VA) and included the fungi Candida albicans ATCC 90028, C. glabrata ATCC 90030, C. krusei ATCC 6258, Cryptococcus neoformans ATCC 90113 and Aspergillus fumigatus ATCC 90906; the bacteria Staphylococcus aureus ATCC 29213, methicillinresistant S. aureus ATCC 43300 (MRS), Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853 and Mycobacterium intracellulare ATCC 23068. Susceptibility testing was performed using a modified version of the CLSI methods [21,22], as described by Samoylenko et al [23]. Drug controls, ciprofloxacin (ICN Biomedicals, Ohio) for bacteria and amphotericin B (ICN Biomedicals, Ohio) for fungi, were included in each assay. Antimalarial/ parasite LDH assay: The in vitro antimalarial activity was measured by a colorimetric assay that determines the parasitic lactate dehydrogenase (pLDH) activity [23, 24]. The assay was performed in a 96-well microplate and included two P. falciparum strains [Sierra Leone D6 (chloroquinesensitive) and Indochina W2 (chloroquine-resistant)]. The IC50 values were computed from the dose response curves generated by plotting percent growth against test concentrations. DMSO, artemisinin and chloroquine were included in each assay as vehicle and drug controls, respectively. Diterpenoids from Clerodendrum eriophyllum Antileishmanial assay: Antileishmanial activity of the compounds was tested in vitro on a culture of Leishmania donovani promastigotes. In a 96 well microplate assay, compounds with appropriate dilution were added to the Leishmania promastigotes culture (2×106 cells/mL). The plates were incubated at 26°C for 72 h and growth of Leishmania promastigotes was determined by Alamar blue assay [25]. Pentamidine and amphotericin B were used as standard antileishmanial agents. IC50 values for each compound were computed from the growth inhibition curve. Cytotoxicity assay: The in vitro cytotoxic activity was determined against monkey kidney fibroblasts (VERO) Natural Product Communications Vol. 5 (6) 2010 857 following the method described by Samoylenko et al [23]. Doxorubicin was used as the positive and DMSO as the negative (vehicle) control. Acknowledgments - The authors sincerely thank Mr John P. Hester for database management and technical assistance, Mr John Trott and Ms Marsha Wright for assistance in biological work and Dr Bharathi Avula for recording mass spectra. 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