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
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