Bioorganic & Medicinal Chemistry Letters 24 (2014) 4939–4942 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier .com/ locate/bmcl New antibacterial xanthone from the marine sponge-derived Micrococcus sp. EG45 http://dx.doi.org/10.1016/j.bmcl.2014.09.040 0960-894X/� 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +20 01092638387; fax: +20 064 3230741. E-mail address:
[email protected] (S.A. Ahmed). � Permanent address: Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, Egypt. Enas E. Eltamany a, Usama Ramadan Abdelmohsen b,�, Amany K. Ibrahim a, Hashim A. Hassanean a, Ute Hentschel b, Safwat A. Ahmed a,⇑ aDepartment of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt b Julius-von-Sachs Institute for Biological Sciences, University of Würzburg, Julius-von-Sachs-Platz 3, D-97082 Würzburg, Germany a r t i c l e i n f o a b s t r a c t Article history: Received 10 July 2014 Revised 10 September 2014 Accepted 12 September 2014 Available online 22 September 2014 Keywords: Actinomycetes Micrococcus sp. Spheciospongia vagabunda Xanthone Antimicrobial activity Microluside A [4 (19-para-hydroxy benzoyloxy-O-b-D-cellobiosyl), 5 (30-para-hydroxy benzoyloxy-O-b- D-glucopyranosyl) xanthone (1)] is a unique O-glycosylated disubstituted xanthone isolated from the broth culture of Micrococcus sp. EG45 cultivated from the Red Sea sponge Spheciospongia vagabunda. The structure of microluside A was determined by 1D- and 2D-NMR techniques as well as high resolution tandem mass spectrometry. The antimicrobial activity evaluation showed that 1 exhibited antibacterial potential against Enterococcus faecalis JH212 and Staphylococcus aureus NCTC 8325 with MIC values of 10 and 13 lM, respectively. � 2014 Elsevier Ltd. All rights reserved. Actinomycetes are prolific producers of bioactive natural prod- ucts.1 The genusMicrococcusbelongs to the suborderMicrococcineae, familyMicrococcaceae under the phylum actinobacteria. Members of the genusMicrococcus are characterized by their fast growing nature andwere cultivated fromdiverse terrestrial niches including soil and plants as well as marine sources including sea water, sediment and marine sponges.2,3 A moderate number of secondary metabolites have been isolated from this genus.4 For example, lutoside, an acyl- 1-(acyl-60-mannobiosyl)-3-glycerol was isolated from Micrococcus luteus that had been cultivated from the sponge Xestospongia sp. The known synthetic 2,4,40-trichloro-20-hydroxydiphenylether was reported from the same strain and it was active against Staphylococcus aureus, Vibrio anguillarum and Candida albicans.5 Three diketopiperazines were reported from Micrococcus sp. culti- vated from the sponge Tedania ignis.6 Another recent example is the thiazolyl peptide kocurin that was isolated from Micrococcus sp. cultivated from unidentified marine sponge, and was active against methicillin-resistant Staphylococcus aureus.7 Xanthone natural products are fluorescent dyes that are widely distributed among plants and microorganisms.8–11 They have been used in food, cosmetics, and textile industries as coloring agents.12 Moreover, they exhibit a wide array of bioactivities including anti- oxidant, antibacterial, antimalarial, antituberculosis and cytotoxic activities.13–16 Their antibacterial action was found to be through induction of photoinactivation of bacteria via formation of reactive oxygen species such as singlet oxygen, which oxidize biological molecules including lipids, proteins and nucleic acids, leading to cell death.17 Paranolin is a polycyclic xanthene that was isolated from the culture of Paraphaeosphaeria nolinae (IFB-E011), an endo- phytic fungus residing the normal stem of the artemisinin-produc- ing plant Artemisia annua.11 The marine sponge-associated bacterium Micrococcus sp. EG45 was cultivated from the Red Sea sponge Spheciospongia vagabunda. Based on the acquired potent antimicrobial activity of the ethyl acetate extract of this bacterium (19 mm inhibition zone diameter against Bacillus sp. P21),18 the bioactivity-guided isolation was performed. A new antibacterial glycosylated xanthone, 4 (19-para hydroxy benzoyloxy cellobio- syl), 5 (30-para hydroxy benzoyloxy glucosyl) xanthone (1) was isolated and structurally elucidated, and it was given the trivial name microluside A (Fig. 1) To our knowledge, this is the first report of a xanthone derivative isolated from marine sponge- associated actinomycete. The actinbacteriumMicrococcus sp. EG45 isolated from the mar- ine sponge Spheciospongia vagabunda was fermented in Forty Erlenmeyer flasks (1 L), each containing 0.5 L of ISP2 medium at 30 �C with shaking at 150 rpm for 7 days.18 The culture broth was filtered and extracted with CHCl3/MeOH using the method http://crossmark.crossref.org/dialog/?doi=10.1016/j.bmcl.2014.09.040&domain=pdf http://dx.doi.org/10.1016/j.bmcl.2014.09.040 mailto:
[email protected] http://dx.doi.org/10.1016/j.bmcl.2014.09.040 http://www.sciencedirect.com/science/journal/0960894X http://www.elsevier.com/locate/bmcl Table 1 NMR spectral data for 1 in MeOD (1H: 600 MHz; 13C: 150 MHz, d in ppm) No dC dH (mult., J in Hz) HMBC 1 119.6 7.53 (d, 8.0) 4,4a,9 2 124.0 6.87 (dd, 8.0, 8.0) 4,4a 3 117.5 6.63 (d, 8.0) 4,4a,8b 4 150.7 4a 158.3 4b 158.2 5 150.8 6 117.5 6.71 (d, 8) 4b,5,8a 7 123.8 7.10 (dd, 8, 8) 4b,5 8 119.9 7.62 (d, 8) 4b,5,9 8a 120.4 8b 120.4 9 175.0 10 103.7 4.91 (d, 9.0) 4 11 71.5 3.66–3.78 (m) 12 75.4 3.48–3.56 (m) 13 78.3 3.48–3.56 (m) 14 76.3 3.48–3.56 (m) 15 62.2 3.90–3.96 (m) 16 102.3 5.06 (d, J = 9.0 Hz) 17 73.5 3.48–3.56 (m) 18 77.9 3.66–3.78 (m) 19 79.1 5.23 (m) 22 20 69.6 3.66–3.78 (m) 21 64.3 3.48–3.56 (m) 22 168.0 23 122.2 24,28 133.0 7.93 (d, 8.0) 22,26 25,27 116.0 6.82 (d, 8.0) 23,26 26 163.7 29 102.3 5.06 (d, J = 9.0 Hz) 5 30 75.4 5.12 (m) 29,35 31 76.3 3.66–3.78 (m) 32 71.5 3.48–3.56 (m) 33 78.3 3.48–3.56 (m) 34 62.4 3.66–3.78 (m) 35 167.5 36 122.2 37,41 133.0 7.95 (d, 8.0) 35,39 38,40 116.0 6.83 (d, 8.0) 36,39 39 163.7 O O O HO HO O OH O O HO OH OH O O O HO 14 4a 4b 5 8 9 10 2930 39 36 35 O HO OH OH 13 16 O O HO 19 22 26 23 Figure 1. Structure of microluside A (1). 4940 E. E. Eltamany et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4939–4942 modified by Bligh and Dyer.19 The extract was concentrated under vacuum and stored at 4 �C until analytical and isolation work. The combined crude extracts (6 g) were fractionated by vacuum liquid chromatography using silica gel with gradient elution with hexane, EtOAc, and then MeOH. The crude extract as well as its fractions were assayed for their antimicrobial activity against Bacillus sp. P21. The EtOAc–MeOH (1:1), (940 mg) fraction showed promising antimicrobial activity, so it was subjected to silica gel column chromatography using gradient elution with EtOAc commencing with 20% MeOH culminating to 100% MeOH. The bioactive semi-polar fraction eluted with 80:20 of EtOAc–MeOH (80 mg) was then further subjected to gel filtration on Sephadex LH-20 using MeOH to yield 3 sub fractions. Final purification of the active subfraction (22 mg) was performed on reversed-phase HPLC using acetonitrile (MeCN) and water as solvent mixture and was complemented by 0.05% trifluoroacetic acid: (10% MeCN/H2O to 100% MeCN over 40 min at a flow rate of 10 ml/ min), to afford compound 1 (7 mg, Rt = 13.9 min). Compound 1 was isolated as a pale yellow solid {[a]D25 +17.99 (c 0.01, MeOH)}. The IR spectrum exhibited hydroxyl, carbonyl and aromatic rings absorption bands at 3309, 1642 and (1545, 1467, 980–610 cm�1), respectively. An [M+1]+ ion in the HRESIMS at m/z 955.2511 in conjugation with the NMR data gave a molecu- lar formula of C45H47O23, representing twenty three degrees of unsaturation. The 13C NMR spectrum of 1 revealed 30 carbon resonances of which 11 resonances correspond to 4,5 dihydroxy xanthone nucleus (assigned through COSY, 2J and 3J HMBC-correlations) and 13 carbons resonances correspond to three sugar moieties. The six additional carbon resonances correspond to two carbonyl carbons at dC 168.0 (C-22) and dC 167.5 (C-35), eight methine and four quaternary carbons characteristic for two 1,4 disubsti- tuted benzene rings. The 1H NMR and COSY spectrum (Table 1) indicated four spin systems. Two spin system displayed six signals for two ABC spin system: [dH 7.53 (1H, d, J = 8 Hz, H-1), 6.87 (1H, dd, J = 8,8 Hz, H-2), 6.63 (1H, d, J = 8, H-3), 7.62 (1H, d, J = 8 Hz, H-8), 7.10 (1H, dd, J = 8,8 Hz, H-7), and 6.71 ppm (1H, d, J = 8, H-6)] indicative of 1,2,3-trisubstituted of two aromatic systems. The other two spin system displayed two signals for two A2B2-spin system at [dH 7.93 and 7.95 (4H, d, J = 8 Hz, H-24, 28, 37 and 41), 6.82 and 6.83 (4H, d, J = 8 Hz, H-25, 27, 38 and 40) which revealed the presence of 1,4-disubstituted two aromatic systems. The HMBC data analysis shows strong (3JCH) correlation for the aromatic proton doublets at dH 7.53 and 7.62 to the xanthone carbonyl group C-9 at dC 175.0 which suggest their assignment to be H-1 and H-8, respectively. In addition, the proton H-1 shows 3J and 4J E. E. Eltamany et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4939–4942 4941 HMBC-correlations to the oxyaryl carbons at 158.3 C-4a and C-4 at dC 150.7, while the proton H-8 shows 3J and 4J HMBC-correlations with the oxyaryl carbons at 158.2 C-4b and C-5at dC 150.8, respec- tively. The proton at dH 6.87 (dd, H-2) shows cross peaks in COSY spectrum with both protons at dH 7.53, H-1 and dH 6.63, H-3 and HMBC correlations to both oxyaryl carbons at dC 150.8 C-4 and 158.2 C-4a. These data support the assignment of the proton at dH 6.87 to be H-2 while the proton at dH 6.63 would be H-3 (shows 2J and 3J HMBC-correlations with C-4 at dC 150.7 and C-4a at dC 158.3). The proton H-7 at dH 7.10 (dd) shows cross peaks in COSY spectrum with both protons H-8 at dH 7.62 and H-6 at dH 6.71 and HMBC correlations to both oxyaryl carbons C-5 at dC 150.8 and C-4b dC 158.2. These data support the assignment of this pro- ton at dH 7.10 to be H-7 while the proton at dH 6.71 would be H-8 (shows 2J and 3J HMBC-correlations with C-5 at dC 150.8 and C-4b dC 158.2). Compound 1 contains three sugar moieties deduced from the presence of three anomeric protons at [dH 4.91 (H-10, d, J = 9 Hz), 5.06 (H-16, H-29 d, J = 9 Hz)], the corresponding anomeric carbon signals at [dC 103.7 (C-10) and dC 102.3 (C-16 and C-29). COSY and HMBC revealed that the two sugars are O-linked to the xanthone at C-4 (strong 3JCH correlation between dH 4.91 (H-10, d, J = 9 Hz), with C-4, dC 150.7) and the other at C-5 (strong 3JCH cor- relation between dH 5.06 (H-29, d, J = 9 Hz) with C-5, dC 150.8) (Fig. 2). By comparison of 1H and 13C NMR data with literature values,20–24 the sugar moieties were identified to be three b D-glu- cose units, the large coupling constant (J = 9.0 Hz) between the (anomeric) protons H-10 with H-11, H-16 with H-17 an H-29 and H-30 indicated the trans-diaxial relationship establishing a b-glyco- sodic linkage. The relative configuration of the sugars was deter- mined on the basis of NOESY experiment. The NOESY correlation observed between H-10, H-16 and H-29 indicated that they are on the same side of the molecule in a b-orientation. The configura- tion was further confirmed by GC analysis of the librated acetylated sugar after hydrolysis,25 which yielded only one peak, D-glucose, in which acetylated thiazolidine derivative of the librated sugar showed the same tR with the standard D-glucose acetylated thiazol- idine derivative (21.39 min).26 Cellobiosyl units were identified to O H O HO OH OH O O HO H HO HO HO H H H Figure 2. Selected 1H–1H COSY (double arrows) be b-D-glucopyranosyl-(1?4)-b-D-glucopyranoside (b-D-cellobiosyl moiety) deduced by HMBC and COSY correlations and comparison of 1H and 13C NMR data with literature values.20,23,24 The presence of 1,4 disubstituted aromatic ring further confirmed by 13C-chemical shifts of carbon resonances at dC 133.0 (C-24,C-28,C-37 and C-41), and dC 116.0 (C-25,C-27,C-38 and C-40) (each corresponds to four carbons). 13C-chemical shifts at dC 168.0 and 167.5 (C-22 andC-35) suggest that the compound con- tains two carboxylic groups and the 13C-chemical shifts at dC 122.2 (C-23and C-36) and dC 163.7 for the oxyaryl carbons (C-26 and C-39) together with HMBC and COSY correlations confirm the presence of two para hydroxy benzoyloxy groups one of them is located at C-30 of third glucose moiety as suggested by the downfield shifts of both C-30 at dC 75.43 and H-30 dH 5.12 (1H, m) with respect to glucose.20–22,27 This was also confirmed by HMBC correlations of H-30with C-29 at dC 102.3 and the carbonyl group C-35 at dC 167.5 and coupling between H-30 with the anomeric proton H-29 at dH 5.06 in COSY spectrum. The other hydroxy benzoyloxy group is located at C-19 of the cellobiosylmoiety, since there is only one strong 3J HMBC correlation of H-19 was observed with the car- bonyl C-22 group at dC 168.0, no coupling of this proton with any anomeric protons in COSY spectrum and the downfield shifts of both H-19 dH 5.23 (1H, t) and C-19 dC 79.1 with respect to cellobiose 1H NMR and 13C NMR data.19,23,24 Based on the detailed analysis of the NMR spectra, the structure of compound 1 was elucidated as 4 (19-para-hydroxy benzoyloxy- O-b-D-cellobiosyl), 5 (30-para-hydroxy benzoyloxy-O-b-D-gluco- pyranosyl) xanthone and the trivial name microluside A was given. On the other hand the antimicrobial activity of microluside A was tested against a panel of Gram positive and Gram negative bac- teria including Enterococcus faecalis JH212, Enterococcus faecium 6413, Staphylococcus aureus NCTC 8325, Staphylococcus epidermidis RP62A, Escherichia coli strain 536 and Pseudomonas aeruginosa strain Nr. 328 as well as Candida albicans.29 Microluside A showed antimicrobial activity against Enterococcus faecalis JH212 and Staph- ylococcus aureus NCTC 8325, with MIC values of 10 and 13 lM respectively, and without cytotoxicity against J774.1 macrophages when tested by the established method previously30 (IC50 of >200 lM). O O O H H H H HH O O H OH O OH OH H OH O O H and HMBC correlations (single arrows) of 1. 4942 E. E. Eltamany et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4939–4942 In conclusion, this study reported the isolation and structure elu- cidation of a new glycosylated xanthone, termed microluside A, from the broth culture of Micrococcus sp. EG45. The bacterium had originally been cultivated from the Red Sea sponge Spheciospongia vagabunda. Microluside A exhibited antibacterial activities against Enterococcus faecalis JH212, and Staphylococcus aureus NCTC 8325. Enterococci are common causes of nosocomial infections and are ranked second (after staphylococci) as etiological agents of hospi- tal-acquired infections in US hospitals. The ability of enterococci to colonize thegastrointestinal tract of hospitalizedpatients is a crucial factor that influences the development of drug resistance. The emergence of resistance to the most common anti-enterococcal antibiotics has made the treatment of these infections a real challenge for clinicians.31 An important finding from this study is that microluside A might be a potential candidate for further experimental studies and for drug discovery. Acknowledgments We thank the Egyptian Environmental Affairs Agency (EEAA) for facilitating sample collection along the Red Sea coasts. We thank H. Bruhn (University of Würzburg) for coordination of the anti- infective screening assays. Financial support was provided by the DFG-SFB630-TP A5 and Egyptian Ministry of Higher Education. Supplementary data Supplementary data (1D and 2D NMR spectral data of 1) associ- ated with this article can be found, in the online version, at http:// dx.doi.org/10.1016/j.bmcl.2014.09.040. References and notes 1. Abdelmohsen, U. R.; Bayer, K.; Hentschel, U. Nat. Prod. Rep. 2014, 31, 381. 2. Prieto-Davo, A.; Villarreal-Gomez, L. J.; Forschner-Dancause, S.; Bull, A. T.; Stach, J. E.; Smith, D. C.; Rowley, D. C.; Jensen, P. R. FEMS Microbiol. Ecol. 2013, 84, 510. 3. Dastager, S. G.; Deepa, C. K.; Pandey, A. Plant Physiol. Biochem. 2010, 48, 987. 4. Wieser, M.; Denner, E. B. M.; Kampfer, P.; Schumann, P.; Tindall, B.; Steiner, U.; Vybiral, D.; Lubitx, W.; Maszenan, A. M.; Patel, B. K. C.; Seviour, R. J.; Radax, C.; Busse, H. J. Int. J. Syst. Evol. Microbiol. 2002, 52, 629. 5. Bultel-Ponce, V. V.; Debitus, C.; Berge, J. P.; Cerceau, C.; Guyot, M. J. Mar. Biotechnol. 1998, 6, 233. 6. Stierle, A. C.; Cardellina, J. H.; Singleton, F. L. Experientia 1988, 44, 1021. 7. Palomo, S.; Gonzalez, I.; De la Cruz, M.; Martin, J.; Tormo, J. R.; Anderson, M.; Hill, R. T.; Vicente, F.; Reyes, F. Mar. Drugs. 2013, 11, 1071. 8. Liu, T.; Zhang, L. M.; Li, Z. L.; Wang, Y.; Tian, L.; Pei, Y. H.; Hua, H. M. A. Chem. Nat. Compd. 2012, 48, 771. 9. Malet-Cascon, L.; Romero, F.; Espliego-Vazquez, F.; Gravalos, D.; Fernandez- Puentes, J. L. J. Antibiot. 2003, 56, 219. 10. Huang, Z.; Yang, R.; Yin, X.; She, Z.; Lin, Y. Magn. Reson. Chem. 2010, 48, 80. 11. Ge, H. M.; Song, Y. C.; Chen, J. R.; Hu, S.; Wu, J. Y.; Tan, R. X. Helv. Chim. Acta 2006, 89, 502. 12. Hashim, N. M.; Rahmani, M.; Ee, G. C.; Sukari, M. A.; Yahayu, M.; Amin, M. A.; Ali, A. M.; Go, R. Molecules 2012, 17, 6071. 13. Vennerstrom, J. L.; Makler, M. T.; Angerhofer, C. K.; Williams, J. A. Antimicrob. Agents Chemother. 1995, 39, 2671. 14. Waite, J. G.; Yousef, A. E. Adv. Appl. Microbiol. 2009, 69, 79. 15. Liu, L. L.; Xu, Y.; Han, Z.; Li, Y. X.; Lu, L.; Lai, P. Y.; Zhong, J. L.; Guo, X. R.; Zhang, X. X.; Qian, P. Y. Mar. Drugs. 2012, 10, 2571. 16. Sudta, P.; Jiarawapi, P.; Suksamrarn, A.; Hongmane, P.; Suksamrarn, S. Chem. Pharm. Bull. (Tokyo) 2013, 61, 194. 17. Kato, H.; Komagoe, K.; Nakanishi, Y.; Inoue, T.; Katsu, T. Photochem. Photobiol. 2012, 88, 423. 18. Abdelmohsen, U. R.; Pimentel-Elardo, S. M.; Hanora, A.; Radwan, M.; Abou-El- Ela, S. H.; Ahmed, S.; Hentschel, U. Mar. Drugs. 2010, 8, 399. 19. Deluca, H. F.; Fukuda, H. J. Ferment. Bioeng. 1989, 68, 174. 20. Veitch, N. C.; Grayer, R. J.; Irwin, J. L.; Takeda, K. Phytochemistry 1998, 48, 389. 21. Urbain, A.; Marston, A.; Sintra Grilo, L.; Bravo, J.; Purev, O.; Purevsuren, B.; Batsuren, D.; Reist, M.; Carrupt, P. A.; Hostettmann, K. J. Nat. Prod. 2008, 71, 895. 22. Purev, O.; Oyun, Kh.; Odontuya, G.; Tankhaeva, A. M.; Nikolaeva, G. G.; Khan, K. M.; Tasadaque, S.; Shah, A.; Voelter, W. Z. Naturforsch., B: Chem. Sci. 2002, 57, 331. 23. Chencheng, Z.; Ling, J.; Nengjiang, Y.; Xuedong, Y.; Yimin, Z. Acta. Pharm. Sin. 2013, 3, 109. 24. Roslund, M. U.; Tähtinen, P.; Niemitz, M.; Sjöholm, R. Carbohydr. Res. 2008, 343, 101. 25. Acid hydrolysis and identification of sugar in 1: The compound (2 mg) was refluxed with 0.5 N HCl (3 ml) for 2 h. The reaction mixture was diluted with water and extracted with CHCl3. The water layer was dried under reduced pressure as well as under N2 to give the monosaccharide. Identification of sugars was achieved via comparing the retention time using GC with an authentic sample.24 The obtained sugars after the acid hydrolysis were dissolved in pyridine (1 ml) and 0.1 M l-cysteine methyl ester hydrochloride in pyridine (2 ml) was added. The mixture was heated at 60 �C for 1 h. An equal volume of Ac2O was added with heating continued for another 1 h. Acetylated thiazolidine derivatives were subjected to GC analysis (Conditions: Column, JW DB-5, 30 m � 0.25 mm, 0.25 lm; carrier gas He; injection temperature 280 �C, detection temperature 280 �C, column temperature; 150 �C (1 min), 10 �C/min to 250 �C (30 min). The configurations were determined by comparing their retention times with acetylated thiazolidine derivatives prepared in a similar way from standard sugars (Sigma–Aldrich) (tR D-glucose 21.39 min, tR L- glucose 22.93 min, tR L-galactose 22.82 min, tR L-galactose 22.68). 26. Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35, 501. 27. Mukhtar, N.; Abdul Malik, R. N.; Iqbal, K.; Tareen, R. B.; Khan, S. N.; Nawaz, S. A.; Siddiqui, J.; Choudhary, M. I. Helv. Chim. Acta 2004, 87, 416. 28. Antibacterial activity: 105 cells/ml of overnight cultures of Enterococcus faecalis JH212, Enterococcus faecium 6413, Staphylococcus aureus NCTC 8325, Staphylococcus epidermidis RP62A, Escherichia coli strain 536 and Pseudomonas aeruginosa strain Nr. 3 were incubated in the presence of various concentrations of the test compound in DMSO in a final volume of 200 ll in 96-well plate at 37 �C. The final concentration of DMSO was 0.8% in each well. After 18 h incubation, the optical density of the cultures was determined at 550 nm using an ELISA microplate reader with respect to the control without bacteria. The lowest concentration of the compound that inhibits bacterial or fungal growth was defined as the minimal inhibitory concentration (MIC). 29. Antifungal activity: Colony of Candida albicans 5314 (ATCC 90028) was suspended in 2 ml of 0.9% NaCl. Four microliters of this suspension was transferred to 2 ml of HR medium. Various concentrations of the test compound were diluted in 100 ll of medium in a 96-well microplate with final DMSO concentration of 0.4%. One hundred microliters of the Candida suspension was added to each well then incubated at 37 �C for 48 h. Optical density was measured at 530 nm with respect to a control well without Candida cells. The lowest concentration of the compound where no growth is detectable was used as the MIC. 30. Cytotoxicity assay: J774.1 macrophages were cultured in complete medium without phenol red in the absence or presence of increasing concentrations of the test substance (0.25–200 lM) at a cell density of 1 � 105 cells/ml for 24 h at 37 �C, 5% CO2, and 95% humidity. Following the addition of 20 ll of Alamar Blue, the plates were incubated and the optical densities were determined after 48 and 72 h in the same manner as described for the anti-trypansomal assay using a test wavelength of 540 nm and a reference wavelength of 630 nm. 31. Arias, C.; Contreras, G.; Murray, B. E. Clin. Microbiol. 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EG45 Acknowledgments Supplementary data References and notes