SUPPLEMENTARY MATERIAL Characterization and bioactivity of Oosporein produced by Endophytic fungus Cochliobolus kusanoi isolated from Nerium oleander L. Ramesha Alurappaa, Madhusudhan Reddy Muthukurpalya Bojegowdab, Vijith Kumarc, Naveen kumar Mallesha and Srinivas Chowdappa a,* a Department of Microbiology and Biotechnology, Jnanabharathi Campus, Bangalore University, Bangalore, Karnataka, India, Pin code-560056. b Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India, Pin code-560012. c Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore, India, Pin code- 560012. * Corresponding author: Dr. Srinivas C., Associate professor, Department of Microbiology and Biotechnology, Jnanabharathi campus, Bangalore University, Bangalore, Karnataka, India. Telephone: +91-080-22961624 Fax: +91-080-23219295 E-mail ID: srinivasbub@gmail.com 8 Abstract Bioactive compounds comprising of secondary metabolites produced by endophytic fungi have wide applications in pharmacology and agriculture. Isolation, characterization and evaluation of biological activities of secondary metabolites were carried out from Cochliobolus kusanoi an endophytic fungus of Nerium oleander L. The fungus was identified based on18S rDNA sequence analysis. There are no reports available on the compounds of C. kusanoi hence, antimicrobial metabolite produced by this fungus was extracted and purified by fractionation using hexane, diethyl ether, dichloromethane, ethyl acetate and methanol. Out of all the solvent fractions, the methanol fraction exhibited better antimicrobial activity which was further purified and characterized as Oosporein. Oosporein from C. kusanoi exhibited broad spectrum Invitro antimicrobial, antioxidant and cytotoxic activities. The characterization and antioxidant activity of Oosporein from C. kusanoi is reported for the first time. Keywords: Nerium oleander L., Cochliobolus kusanoi, Oosporein, antimicrobial activity, antioxidant activity, anticancer activity. Experimental section Isolation and identification of endophytic fungi The endophytic fungus was isolated according to the procedure of Suryanarayan & Thennarasan (2004) with slight modification. The fungus was identified using 18S rDNA analysis. The DNA of the fungus was isolated by following the procedure of O’Donnell et al. (1997). The ITS regions were amplified using universal primers ITS1 and ITS4. The PCR product was purified using GenElute™ kit and further, sequencing was carried out in an ABI automated DNA sequencer using ABI-BigDye® Terminatorv3.1 Cycle Sequencing Kit. BLAST analysis was carried out in the NCBI database, highest homology and total score were noted and sequences were retrieved. Multiple sequence alignments of the obtained sequence and reference sequences were used to generate phylogenetic tree using server phylogeny (Dereeper et al 2008). Production and extraction of the compound The fresh mycelium of endophytic fungus was inoculated into PDB medium, incubated at 28°C, 140 rpm for 15 days. The culture was filtered and the filtrate was extracted with equal volume of ethylacetate followed by addition of 0.1N acetic acid to maintain pH 3. The ethyl acetate extract was evaporated under vacuum (Heidolph rotary evaporator) at 50°C. 9 Purification of the antimicrobial compound The crude compound was sequentially separated by dissolving with hexane, diethyl ether, dichloromethane, ethyl acetate and methanol. Each fraction was concentrated using a rotary evaporator and subjected to antimicrobial activity by agar well diffusion method (Rios & Recio 1988). Human pathogenic microorganisms (S. aureus NCIM No. 2079, B. cereus NCIM No. 2106, P. aeruginosa NCIM No. 2200, E. coli NCIM No. 2256, S. typhimurium NCIM No. 2501 and C. albicans NCIM No. 3471) were procured from NCIM, Pune, India. The broad spectrum antimicrobial fraction was recrystallized with methanol, rinsed with double distilled water and dried using a lyophilizer to obtain a clean powder. Further purification of the compound was carried out in Reverse Phase-HPLC (Gilson 811D Dynamic mixer separation module), with C-18 column (Luna, 100Å, 4.6 × 250µm, 5µm internal dia.), PDA detector at 25°C with the solvent system acetonitrile: 0.1% Triflouroacetic acid (60: 40 v/v). The injection volume was 10 µL, with flow rate of 1mL/min. Gradient separation at room temperature was done with detection at 254 nm. The fraction was collected and evaporated under vacuum at 50°C. Finally, the compound was crystallized in a mixture of methanol and water in the ratio of 75: 25 and allowed for slow evaporation at room temperature. After two months, fine orange red crystals developed, which were collected and washed with diethyl ether and dried. Characterization of purified compound The purified compound was characterized by TLC, UV-Vis spectrophotometry, FTIR, CHNS analysis, HRMS, NMR and single crystal XRD analysis. Analytical TLC was performed using a silica TLC plate (GF 254 60; Merck 250 mm thick) impregnated with benzene: acetic acid (9:1), then eluted with mobile phase toluene: ethylacetate: formic acid (4: 5: 1), exposed to acetic anhydride and observed under visible light. The melting point of the compound was determined by open capillary method using electric melting point operator. UV-Vis spectrum of the compound was recorded in DMSO, scanned at the wavelength range 100-1000 nm using Photodiode array detector (Shimadzu). FTIR (Perkin Elmer Spectrum GX FT-IR Version: 5.0.1) was performed by KBr pellet method. High resolution electron spray ionization mass spectroscopy (HRESI-MS) was performed using TOF spectrometer with simultaneous electron spray (MicroMass). CHNS analysis was carried out using Thermo Finnegan FLASH EA 1112 CHNS 10 analyser. 1H, 13C and HSQC were recorded on Bruker Avance 400MHz spectrometer using DMSO-d6; Solvent peak at 2.49 ppm was used as spectrum reference. The chemical shifts were expressed in ppm. HMBC was recorded on Bruker Avance 500MHz spectrometer with 13 C operating frequency being 125MHz using DMSO-d6. Analysis of X-ray Crystallography Single crystal X-ray diffraction data was collected on an Oxford Xcalibur (Mova) diffractometer (Oxford Diffraction 2008) equipped with an EOS CCD detector using MoKα radiation (λ= 0.71073 Å). The crystal was maintained at a desired temperature during data collection using the Oxford Instruments Cryojet-HT controller (Oxford instruments). All structures were solved by direct methods using SHELXS-97 and refined against F2 using SHELXL-97 (Sheldrick 2008). H-atoms were fixed geometrically and refined isotropically. The WinGX package (Farrugia 1999) was used for refinement and production of data tables and ORTEP-3 (Farrugia 1997) for structure visualization and making molecular representations. Analysis of the H-bonded interactions was carried out using PLATON (Spek 2003) Packing diagrams were generated using MERCURY (Macrae et al. 2006). Bioactivity of secondary metabolite Minimal inhibitory concentration of the compound by MTT assay Fifty percent inhibitory concentration (IC50) of the purified compound was determined by microtitre plate reader method using 3-[4,5-dimethyl-thiazol-2- yl]-2,5-diphenyltetrazolium bromide (MTT) according to previous procedure (Xu et al. 2008) with slight modifications. Test bacteria were grown in Nutrient Broth (NB) at 37° C and Candida albicans in Sabouraud Dextrose Broth (SDB) at 30° C. Sterile 96-wells microtitre plates were filled with 50 μL broth culture of test organism containing 106 Cells/mL and 50 μL of compound dissolved in DMSO at different concentrations was added into each well. Medium containing 1% DMSO was used as a negative control. The final concentrations of the extract were 12.5, 25, 50, 75 and 150 µM in medium. Twenty five micromoles of streptomycin sulfate and fluconazole was used as positive control for bacteria and C. albicans, respectively. The plates were incubated for 24 h at 37° C and 30° C for bacteria and C. albicans, respectively. Twenty microlitres of 3-[4,5-dimethyl-thiazol-2- yl]-2,5-diphenyltetrazolium bromide (MTT) at a concentration 5 mg/mL in phosphate-buffer saline (PBS) was added to each well as a growth indicator and the micro titre plates were incubated for an additional 4 h. After incubation, 100 µL DMSO was added to all the wells and the absorbance read at 540 nm. Antioxidant activity of the compound by DPPH assay 11 The free radical scavenging capacity of the compound was determined by using DPPH (2, 2-diphenyl1-picrylhydrazyl) modified protocol of previous report (Sultanova et al. 2001). The reaction mixture contained 1 mL of test compound and 3 mL of DPPH (300 mM) in methanol. Different concentrations (0.075-1.5 mM) of test sample and ascorbic acid (0.05-0.25 mM) were prepared and the reaction mixtures were incubated at 25±2°C for 30 min and absorbance was measured at 517 nm. The experiment was repeated thrice and the percentage of radical scavenging activity (RSA) was calculated using the formula [(Absorbance of control - Absorbance of test)/ Absorbance of control] x 100 Anti-proliferative assay The anti-proliferative effect of the compound was studied on human lung carcinoma type II epithelial cells (A549). Cell viability assay was carried out using MTT as described by (Mossman 1983) with slight modification. A549 cells (1×104 cells per well) were seeded into 96 tissue culture plates with the final volume of 200 µL of complete RPMI-1640 medium and incubated overnight at 37° C with the supply of 5% CO2. Further, the cells were treated with different concentrations (5-50 µM) of test compound and incubated for 48 h. After the removal of medium, the cells were washed with phosphate buffer saline, followed by treatment of 20 µL MTT (5 mg/mL) and incubated for 4 h at 37 °C. The formazan products were dissolved in DMSO and spectrophotometrically measured at a wavelength of 540 nm. The concentration of the compound that inhibited 50% cell growth (IC50) was determined. Statistical analysis The obtained results were statistically analysed by one way ANOVA with SPSS 19, mean values of the triplicates were compared according to Duncan Multiple Range Test (DMRT) at p < 0.05. 12 Tables Table S1: Antimicrobial activity of different solvent fractions of extract from C. kusanoi. Different solvent fraction (20µg/mL) Crude extract* Hexane Diethyl ether Dichloromethane Ethylacetate Methanol Tetracycline Fluconazole S. aureus 11.00±1c 0a 0a 0a 8±0.58b 23.67±0.58d 27.33±0.8e - Antimicrobial activity (zone of inhibition in mm) B. cereus E. coli S. typhi P. aeruginosa 9.00±00 c 15.67±1.15 c 8.67±0.58 b 10.67±0.58 c a 0 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 0a 7.33±0.58 b 10.33±0.58 b 9.33±0.58 b 9.33±0.58 b 17.33±1.52 e 21.67±1.52 d 16±1 c 15.67±0.58 d 15.33±0.58 d 16.33±1.15 c 20.33±0.58 d 10.33±0.58 c - C. albicans 8.0±00 c 0a 0a 0a 7.33±0.58 b 12.33±0.58 d 13.67±0.58 e Note: *Crude ethyl acetate extract before fractionation. Positive control- Tetracycline (bacteria), Fluconazole (yeast). In each column, mean values followed by the same letter are not significantly different according to DMRT at p < 0.05. ‘-’ Not determined. 13 Table S2: Crystallographic data and structural refinement parameters of Oosporein. Compound Formula Formula weight CCDC number Temperature (K) Crystal form Color Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) Volume (Å3) Z Density (gcm-3) μ (mm-1) F (000) hmin, max kmin, max lmin, max Reflections collected Independent reflections R_all, R_obs wR2_all, wR2_obs min,max (e Å-3) GOOF Oosporein C14H10O8 306.22 942418 110(2) Block Red Monoclinic C2/c 11.999(2) 8.312(3) 13.792(1) 90 106.012(4) 90 1322.3(6) 4 1.538 0.129 632 -14, 13 -10, 10 -17, 17 1295 1039 0.0752, 0.0629 0.1847, 0.1757 -0.324, 0.322 1.021 14 Table S3: Intermolecular hydrogen bonds in the crystal structures of Oosporein. Compound D−H···A d(D−H)Å d(D−A)Å d(H···A) Å O(2)−H(2)···O(1) 0.98 2.120(1) 2.65 d(H···A) (deg) 112 1-x,1/2+y,1/2-z Symmetry O(2)−H(2)···O(3) 0.98 1.890(1) 2.76 146 -1/2+x,1/2+y,z Oosporein O(4)−H(4)···O(3) O(4)−H(4)···O(1) 0.98 2.130(2) 2.66 112 1-x,1/2+y,1/2-z 0.98 1.890(1) 2.79 147 -1/2+x,1/2+y,z C(7)−H(7)···O(1) 1.08 2.430(1) 2.86 102 15 -x,1/2+y,1/2-z Table S4: Minimal inhibitory concentration of Oosporein against different pathogenic microorganisms. Different concentration of oosporein (µM) 12.5 48.98±1.84e 25 S. aureus % Inhibition of pathogenic microorganisms B. cereus E. coli S. typhi P. aeruginosa C. albicans 16.76±3f 39.75±3.68d 6.80±2.19e 11.57±3.21e 85.67±2.86c 24.02±3.16e 16.67±1.47e 43.21±3.76d 11.67±3.65e 26.50±3.91d 50 94.40±2.25b 55.14±2.58c 60.67±2.58c 52.71±2.51c 31.48±2.45d 73.44±1.62b 75 95.36±0.65ab 80.42±3.18b 71.73±2.98b 76.65±3.62b 67.36±3.67b 89.33±2.56a 150 98.22±0.85a 75.32±2.63a 94.31±3.25a 0f 0f 38.21±1.49c - - 56.57±1.52c Control Streptomycin sulphate Fluconazole 7.57±3.71f 92.48±1.57a 88.93±2.61a 94.27±2.98a 0f 0g 59.79±1d 47.17±1d - - 0g 0e 49.52±1.31d 39.28±1.49d - - Note: Positive control- 25 µM Streptomycin sulphate (bacteria), 25 µM Fluconazole (yeast). In each column, mean values followed by the same letter are not significantly different according to DMRT at p < 0.05. ‘-’ Not determined. 16 Figures: Figure S1: Cochliobolus kusanoi on Potato dextrose agar (Obverse view (A) and reverse view (B) of the plate). 17 Figure S2: Phylogenetic tree constructed based on sequence of Cochliobolus kusanoi and its closest matches in the GenBank sequences. 18 Figure S3: Preparatory RP-HPLC spectrum of the Oosporein from C. kusanoi. 19 Figure S4: HRESI-MS spectrum of Oosporein from C. kusanoi showed at m/z 329.0278 [M+Na], 351.0130 [M+2Na-1H] and 635.0570 [2M+Na]. 20 Figure S5: FT-IR spectrum of Oosporein from C. kusanoi. 21 Figure S6: 1H NMR Spectrum of the Oosporein from C. kusanoi. 22 Figure S7: 13C NMR Spectrum of the Oosporein from C. kusanoi. 23 Figure S8: HSQC Spectrum of the Oosporein from C. kusanoi. 24 Figure S9: HMBC Spectrum of the Oosporein from C. kusanoi. 25 Figure S10: ORTEP diagram (drawn at 50% probability ellipsoids at 110 K) of the Oosporein. 26 Figure S11: Solid state packing; O-H···O hydrogen bonding between the molecules in compound Oosporein from C. kusanoi along C axis. 27 Figure S12: C-H··· π interactions interaction and C-H···O hydrogen bonding in the crystal structure of compound Oosporein from C. kusanoi along C axis. 28 % of DPPH scavenging activity Figure S13: Antioxidant activity of Oosporein. 120 100 a b c d 80 e 60 f 40 g 20 0 1.5 1.2 0.9 0.6 0.3 0.15 0.075 Oosporein (mM) Note: In the figure mean values followed by the different letter are significantly different according to DMRT at p < 0.05. 29 Figure S14: Cytotoxicity of Oosporein on A549 cell lines. 120 % Viability of Cells a 100 b b c 80 60 d e e 20 25 50 40 20 0 Control 5 10 15 Oosporein (μM) Note: In the figure mean values followed by the different letter are significantly different according to DMRT at p < 0.05. 30 References Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O. 2008. 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