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LIST OF PUBLICATIONS
1. Siddhardha Busi, Prabhakar Peddikotla, Suryanarayana M.
Upadyayula, Venkateswarlu Yenamandra. Isolation and biological
evaluation of two bioactive metabolites from Aspergillus
gorakhpurensis. Rec. Nat. Prod. (2009), 3:3; 161-164.
2. Siddhardha Busi, Prabhakar Peddikotla, Suryanarayana M.
Upadyayula, Venkateswarlu Yenamandra. Secondary metabolites
of Curvularia oryzae MTCC 2605. Rec. Nat. Prod. (2009), 3:4; 204-
208.
3. B.Siddhardha, USN. Murty, M. Narasimhulu and Y.
Venkateswarlu. Isolation, Characterization and Biological
evaluation of secondary metabolite from Aspergillus funiculosus.
Indian journal of microbiology (In press).
4. B.Siddhardha, USN. Murty. Acute, sublethal toxicity and
antifeedent activity of fungal metabolites against Spodoptera litura
Fab. Biocontrol science and technology (Communicated).
123
Indian J Microbiol (June 2010) 50:225–228 225
SHORT COMMUNICATION
Isolation, Characterization and Biological evaluation of
secondary metabolite from Aspergillus funiculosus
B. Siddhardha · U. S. N. Murty · M. Narasimhulu · Y. Venkateswarlu
Received: 29 February 2008 / Accepted: 13 August 2008
Indian J Microbiol (June 2010) 50:225–228
DOI: 10.1007/s12088-010-0044-7
Abstract Screening of Aspergillus funiculosus for bioac-
tive secondary metabolites produced kojic acid, which is
know to have wide range of biological properties. It is very
active against Gram-negative bacteria, such as Pseudomo-
nas aeruginosa and Escherichia coli, but moderately active
against yeasts and Gram-positive bacteria except Staphylo-
coccus epidermidis .Filamentous Fungi are more sensitive
to kojic acid. When it exposed to larvicidal activity on Ae-
des aegypti third instar larvae are more sensitive than early
fourth instar larvae.
Keywords Aspergillus funiculosus · Secondary
metabolites · Kojic acid · Antibacterial · Aedes aegypti ·
Larvicidal
Introduction
Microbial natural products remain the most promising
source of novel antibiotics. The impact of microbial bio-
diversity favors the chance of isolating new antibiotics
[1–3]. Fungi are the most promising group of bioactive
compounds producer [4].The most well known examples
of natural product are antibiotics [5]. Microbial natural
products have also been developed as anti-diabetic drugs,
hormone (ion-channel or receptor) antagonists, anti-cancer
drugs, and agricultural and pharmaceutical agents [6].
Aspergillus funiculosus (NCIM 1029) was procured from
National Collection of Industrial Microorganisms (NCIM),
NCL, Pune. Aspergillus funiculosus grows rapidly on po-
tato dextrose agar medium at a temperature of 27ºC. After
three days of incubation it produced colonies ranging 1–2
cm. The colonies were powdery in texture; an early stage of
the colony was pale yellow in colour and turned to yellow
after the formation of conidia. The microscopic features of
the fungi include long hyphae bearing conidiophore and
chains of conidia on it (Fig. 2). Cultivation of the fungus
was carried out in 2000 ml of Erlenmeyer fl ask containing
1000 ml of potato dextrose medium. Culture fl asks were
incubated at optimized culture conditions (medium pH 7.0,
temperature 27ºC) for 3 days. Mycelial mat was removed
and ethyl acetate was added to the medium (1:1 v / v). After
thorough mixing, immiscible portion of the ethyl acetate
(pale yellow colored) was separated from the medium. The
mycelial mat was also washed twice with ethyl acetate. The
separated ethyl acetate portion was rotaevoparated at 45ºC.
The crude obtained was subjected to silica gel column
chromatography and the eluted pure compound was ana-
lyzed on TLC plates. Kojic acid was tentatively identifi ed
B. Siddhardha1 · U. S. N. Murty
1 (�) · M. Narasimhulu
2 ·
Y. Venkateswarlu2
1Biology Division,
2Natural Products Laboratory,
Organic Chemistry Division-I,
Indian Institute of Chemical Technology,
Hyderabad - 500 007, India
E-mail: [email protected]; [email protected]
226 Indian J Microbiol (June 2010) 50:225–228
123
by spraying the thin-layer plates with a 1% solution of
FeCl3-6H
20 in a 0.12 N HCl [7]. Further its structure was
determined by NMR spectrum (Table 2 and Fig. 1).
The antibacterial and antifungal activities of the extract
were evaluated by agar well diffusion method [8–9] against
Bacillus subtilis (MTCC 619), Bacillus sphericus (MTCC
511), Staphylococcus aureus (MTCC 737), Staphylococcus
epidermidis (MTCC 435), Escherichia coli (MTCC 1687),
Pseudomonas aeruginosa (MTCC 1688), Pseudomonas
oleovorans (MTCC 617), Klebsiella pneumoniae (MTCC
109), Candida albicans (MTCC 227), Saccharomyces ser-
viseae (MTCC 170), Aspergillus niger (MTCC 282) and
Rhizopus oryzae (MTCC 262), procured from IMTECH,
Chandigarh. The inhibition zone diameter (IZD) was mea-
sured to the nearest millimeter. The minimum inhibitory
concentration (MIC) was tested as per the NCCLS stan-
dards[10 ]against the bacteria.
Larvae of laboratory-reared strains of Aedes aegypti the
late 3rd instar and early 4th instar stages were exposed to
sub lethal concentrations of 150, 300 and 450 ppm of the
crude extracts in distilled water for 24 h at room tempera-
ture (32°C ± 2°C) according to standard WHO procedure
[11] by dissolving the compound in acetone (99.8 %). For
comparison commercial Malathion was used as positive
Fig. 2 Unstained wet mount preparation of Aspergillus funiculosus under magnifi cation of 80 × (A) and 1300 × (B, C, D) using a Polyvar
Compound Microscope attached to a CCD camera (Sony) with an aid of software (Easy-Grab; Noldus Information Technology).
123
Indian J Microbiol (June 2010) 50:225–228 227
control. 150 ppm of Malathion was prepared in 250 ml
of water. The larvae were fed with dry yeast powder by
sprinkling on the water surface. The dead larvae were
counted after 24 h and percentage mortality was reported
from the average for the two replicates taken together. A
probit analysis using a computer program [12] was em-
ployed on the results to determine LC50
values.
The invitro antimicrobial (antibacterial, antifungal) and
larvicidal results were summarized in the Table 1. The sec-
ondary metabolite isolated from Aspergillus funiculosus,
kojic acid showed prominent inhibition against Pseudomo-
nas aeruginosa and moderate activity against all other bac-
teria and fungi. The LC50
values for 3rd and 4th instar larvae
of Aedes aegypti was 204.51 and 271.64 ppm respectively.
From the LC50
values the extract was found relatively more
toxic to the 3rd instar larvae than 4th instar larvae.
The present study of screening bioactive secondary
metabolites revealed that Aspergillus funiculosus as a new
source for the production of kojic acid. Because of vari-
ous commercial applications, by using strain-improvement
techniques the rate of production of kojic acid can be im-
proved from Aspergillus funiculosus.
References
1. Fernando Pelaez (2006) The historical delivery of antibiot-
ics from microbial natural products—Can history repeat?
Biochemical Pharmacology 71–7:981–990
2. Berdy J (1985) Screening, classifi cation and identifi cation of
microbial products. In Discovery and Isolation of Microbial
Products, Ed. Verral MS. Ellis Horwood, Chichester, pp.
9–31
3. Berdy J (1988) New trends in the research of bioactive mi-
crobialmetabolites. In Chemistry and Biotechnology of Bio-
logically Active Natural Products, 4th Int. Conf.,Budapest,
1987, pp. 269–291
4. Berdy J (2005) Bioactive Microbial Metabolites. A Personal
View. J Antibiot 58(1):1–26
5. Demain A (1999) Pharmaceutically active secondary me-
tabolites of microorganisms. Appl Microbiol Biotechnol 52:
455–463
6. Grabley S and Thiericke R (1999) The impact of natural
products on drug discovery. In: Grabley S, Thiericke R (eds)
Table 1 Antibacterial, antifungal and larvicidal activity of kojic acid
Microorganism Zone of inhibition MIC μg / ml
Kojic acid
(50 μg / ml)
Kojic acid
(100 μg / ml)
Control
(30 μg / ml)
Kojic acid Control-
Nitrofurantoin
Gram +ve Bacteria Penicillin-G
Bacillus sphericus 12 15 20 200 μg 100 μg
Bacillus subtilis 12 15 20 200 μg 100 μg
Staphylococcus aureus 11 13 18 100 μg 50 μg
Staphylococcus epidermidis 16 19 18 100 μg 50 μg
Gram –ve Bacteria Streptomycin
Pseudomonas aeruginosa 19 23 34 50 μg 75 μg
Pseudomonas oleovorans 13 15 30 100 μg 75 μg
Escherichia coli 16 18 29 100 μg 50 μg
Klebsiella aerogenes 13 15 30 100 μg 50 μg
Fungi Clotrimazole Larvicidal activity LC50
Candida albicans 13 15 18 (Aedyes aegypti)
Saccharomyces serviseae 16 19 19 3rd instar Early 4th instar
Aspergillus niger 17 20 22
Rhizopus oryzae 17 21 23 204.51 271.64
Antifungal and antibacterial activity Inhibitory zone diameters are in mm.
All the concentrations are in ppm for larvicidal activity.
LC50
= Lethal concentration (ppm) at which 50 % of the larvae showed mortality.
Table 2 1H and
13C NMR spectral data for Kojic acid
S.NO H1 NMR 13C NMR
1 6.35 139.5
2. _ 145.8
3. _ 174.2
4. 8.02 110.04
5. _ 168.30
6. 4.30(-CH2) 59.6
7. -OH 9.05
(PHENOLIC)
8. -OH 5.72(-CH2)
MASS: 142.0
228 Indian J Microbiol (June 2010) 50:225–228
123
Drug discovery from nature. Springer, New York Berlin
Heidelberg, pp 3–37
7. Ciegler A, Peterson REA, Lagoda A and Hall HH
(1966) Afl atoxin production and degradation by
Aspergillus fl avus in 20-liter fermentors. Appl Microbiol 14:
826–833
8. Linday ME (1962) Practical Introduction to Microbiology. E
and F.N. Spon Ltd., United Kingdom, p. 177
9. Perez C, Paul M and Bazerque P (1990) An Antibiotic assay
by the agar well diffusion method. Acta Bio Med Exp 15:
113–115
10. NCCLS (National Committee for Clinical Laboratory Stan-
dards) (2003) Methods for Dilution Antimicrobial Suscepti-
bility Tests for Bacteria That Grow Aerobically; Approved
Standard, 6th edition. M7-A6. NCCLS, Wayne, PA
11. World Health Organization (1981) Instructions for determin-
ing susceptibility or resistance of mosquito larvae to insecti-
cides. WHO/VBC-81, pp. 807
SHORT REPORT
The article was published by Academy of Chemistry of Globe Publications www.acgpubs.org/RNP © Published 06 /07/2009 EISSN: 1307-6167
Rec. Nat. Prod. 3:3 (2009) 161-164
Isolation and Biological Evaluation of Two Bioactive Metabolites
from Aspergillus gorakhpurensis
Siddhardha Busi1, Prabhakar Peddikotla
2, Suryanarayana M.
Upadyayula1*
, Venkateswarlu Yenamandra2
1Biology Division, , Indian Institute of Chemical Technology, Tarnaka, Hubsiguda,
Hyderabad 500 007, Andhra Pradesh, India
2Organic Chemistry Division-I, Indian Institute of Chemical Technology, Tarnaka,
Hubsiguda, Hyderabad 500 007, Andhra Pradesh, India
(Received May 1, 2009; Revised June 1, 2009; Accepted June 2, 2009)
Abstract: Fungi are known to produce a vast array of secondary metabolites that are gaining importance for
their biotechnological applications. Screening of Aspergillus gorakhpurensis for the production of bioactive
secondary metabolites results in the production of 4-(N-methyl-N-phenyl amino) butan-2-one and itaconic acid.
The structure of the known compounds was established by 1H-,
13C-NMR and Mass spectral data. Biological
evaluation of the two compounds against test microorganisms showed strong inhibitory activity of 4-(N-methyl-
N-phenyl amino) butan-2-one towards bacteria and fungi. Only 4-(N-methyl-N-phenyl amino)-butan-2-one
showed a marked significant activity (LD50 = 330.69 µg/mL) in Spodoptera litura larvicidal bioassay.
Keywords: Aspergillus gorakhpurensis;4-(N-methyl-N-phenyl amino) butan-2-one; Itaconic acid; Antibacterial;
Spodoptera litura.
1. Fungal Source
Microbial natural products remain the most promising source of novel secondary metabolites.
The impact of microbial biodiversity favours the chance of isolating new antibiotics. Identification of
microorganisms that produce bioactive compounds is of great interest in the development of new
molecules to fight against many pathogens. Fungi produce a wide range of secondary metabolites with
high therapeutic value as antibiotics, cytotoxic substances, insecticides, compounds that promote or
* Corresponding author: E- Mail: [email protected].
Bioactive metabolites from Aspergillus gorakhpurensis
162
inhibit growth, attractor, repellent etc., [1]. These metabolites are being exploited in different fields of
medicine and industries [2]. Among fungi classes, Ascomycetes are reported to be active producers of
antimicrobial compounds, which have high therapeutic values [3]. Within our screening program for
antimicrobaial and larvicidal fungal secondary metabolites, we investigated an Ascomycetes fungi
Aspergillus gorakhpurensis (MTCC 547) procured from Microbial Type Culture Collection (MTCC),
IMTECH, Chandigarh, India, for chemical and biological studies. The fungus was cultivated for 7
days on potato dextrose broth medium and the culture was extracted with ethyl acetate.
2. Previous Studies
There is no previous studies on the metabolites of this fungus.
3. Present Study
In the present study Aspergillus gorakhpurensis MTCC 547 was procured from Microbial
Type Culture Collection (MTCC), IMTECH, Chandigarh India and exploited for the production of
secondary metabolites. A portion of mature agar slant was inoculated in one liter of potato dextrose
broth in 2 liter Erlenmeyer flask and incubated at 27 ± 2 º C as resting cell suspension for 7 days. The
fermented broth (8 L) was treated with ethyl acetate (V: V) and incubated overnight. The mixture
(fermented broth and solvent) was shaken vigorously for 30 min and kept in stationary condition for
another 30 min to separate the solvent from aqueous phase. The organic extract was separated, dried
over anhydrous sodium sulfate and concentrated in vacuo to yield crude (2.1 g).
Activated silica gel (60–120 mesh) was packed on to a glass column (450 mm × 40 mm) using
n-hexane solvent and 2.1 g of crude ethyl acetate extract was loaded on the top of silica gel column.
The column was eluted with the mixture of hexane and ethyl acetate (8:2). Fractions that showed
homogeneity on TLC plates were combined and concentrated together to give pure compounds.
Fraction 1 (54 mg) and Fraction 2 (31 mg) were obtained. The pure fractions were subjected to
Chemical characterization using Nuclear magnetic resonance spectroscopy (1H- & 13C-NMR) (Bruker
UXNMR at 300MHz in CDCl3) and Mass spectroscopy (Finnigan MAT 1020-B in CDCl3). The
metabolites were identified as -(N-methyl-N-phenyl amino) butan-2-one; C11H15NO; Mol.wt.177.0 (1) and
Itaconic acid; C5H6O4; Mol.wt.130.1 (2) (Figure 1).
N CH3
O
CH3
4-(N-Methyl-N-phenyl amino) butan-2-one
HO
OH
O
CH2O
Itaconic acid
Figure. 1 Chemical structures of -(N-methyl-N-phenyl amino) butan-2-one and Itaconic acid
4-(N-methyl-N-phenyl amino) butan-2-one (C11H15NO): 1H NMR (300MHz, CDCl3): δ : 2.13 (3H, s),
2.67 (2H, t, J=6.798), 2.91 (2H, s), 3.61 (2H, t, J=6.798), 6.60 (3H, m), 7.12 (2H, t, J=7.554). 13C
NMR (300MHz, CDCl3): δ : 30.54, 38.90, 40.28, 47.56, 112.74, 117.05, 129.35, 148.40, 206.29.
Itaconic acid (C13H26O): 1H NMR (300MHz, CDCl3): δ: 3.21 (2H, s), 5.65 (1H, s), 6.23 (1H, s), 11.34
(2H, s). 13
C NMR (300MHz, CDCl3): δ :36.87, 126.47, 134.47, 167.02, 171.58.
Busi et al., Rec. Nat. Prod. (2009) 3:3 161-164
163
Bioactivity Tests
Antibacterial activity evaluated against Gram-positive organisms and Gram-negative bacteria
by well diffusion method [4]. Solutions were prepared (50-150 µg/mL) by dissolving the test
compounds in dimethyl sulfoxide (DMSO) and add to appropriate well. The petri dishes with treated
and the control cups were incubated at 37°C for 24 h. The zones of inhibition diameters (mm) were
measured. Triplicates performed for each treatment. Control experiment carried out with the pure
solvent. Antifungal activity was carried out in similar manner using zone of inhibition method against
eight fungal strains according to the method of Linday.
The minimum inhibitory concentration was determined according to the method described by
Andrews [5]. Different concentrations (200-10 µg/mL) of isolated compounds and 100 µL of the
bacterial suspension (10-5 CFU/mL) were placed aseptically in10 mL of nutrient broth separately and
incubated for 24 h at 37 °C. The growth was observed both visually and by measuring O.D. at 600 nm.
The lowest concentration of test sample showed no visible growth was recorded as the minimum
inhibitory concentration. Duplicate sets of tubes were maintained for each concentration of test
sample.
Larvicidal activity (measured as mortality after 24 h) of the compounds was determined by
topical application to early fourth instars according to Laurin et.al [6]. Lethality was estimated by
applying different concentrations (50 to 1000 µg/mL) of the metabolites. Two replicates of 10 larvae
were tested per dose. A probit analysis was carried out to calculate LD50 and LD90 [7].
The antimicrobial activity of the isolated compounds against all the test organisms had shown
in the Table. 4-(N-methyl-N-phenyl amino) butan-2-one showed strong antibacterial activity against
gram-positive bacteria i.e. Staphylococcus aureus, Staphylococcus epidermides and gram-negative
bacteria i.e. Escherichia coli with zone of inhibition between 15 to 18 mm at a concentration of 100
µg/mL. The MIC value of the 4-(N-methyl-N-phenyl amino) butan-2-one was 100 µg/mL against
Staphylococcus aureus and Staphylococcus epidermides. 4-(N-methyl-N-phenyl amino) butan-2-one
was tested for anti fungal activity against eight fungi and showed moderate activity against all fungi
except Candida albicans (15 mm). Whereas Itaconic acid showed antibacterial and antifungal activity
at very higher concentrations (150 µg/mL). 4-(N-methyl-N-phenyl amino) butan-2-one showed potent
lethality against Spodoptera litura 4th instar larvae (Table). The data was further subjected to probit
analysis and the LC50 value calculated to be 330.69 µg/mL.
In conclusion, we have reported the isolation of two known compounds from Aspergillus
gorakhpurensis. The antimicrobial and larvicidal activities of the isolated pure compounds were
reported. The isolated compounds showed moderate antimicrobial and insecticidal activities.
Microbial secondary metabolites represent a large source of compounds endowed with ingenious
structures and potent biological activities. Many of the products currently used for human or animal
therapy, in animal husbandry and in agriculture are produced by microbial fermentation, or derived
from chemical modification of a microbial product [8]. The present study of screening bioactive
secondary metabolites revealed that Aspergillus gorakhpurensis as a source for the production of two
bioactive metabolites. These metabolites can be further exploited for the biotechnological applications
in medicine and agriculture.
Bioactive metabolites from Aspergillus gorakhpurensis
164
Table 1. Antibacterial, antifungal and larvicidal bioassay of 4-(N-methyl-N- phenyl amino) butan-2-
one and Itaconic acid Zone of inhibition MIC (µg/mL)
(1) (2)
Control
30 µg (1) (2)
Control
Bacteria 50 µg 100 µg 100 µg 150 µg Penicillin-G Nitrofurantoin
Gram Positive Bacteria
S. aureus MTCC 96 15 18 - 8 18 100 >200 50 µg/mL
S. epidermides MTCC 435 14 16 - - 18 100 >200 50 µg/mL
B. subtilis MTCC 441 12 14 - - 20 150 >200 100 µg/mL
B. sphericus MTCC 511 12 14 - - 20 150 >200 100 µg/mL
Gram Negative Bacteria Streptomycin
E. coli MTCC 443 13 15 - - 29 150 >200 50 µg/mL
P. aeruginosa MTCC 741 12 14 - - 34 150 >200 75 µg/mL
P. oleovorans MTCC 617 12 14 - - 30 150 >200 75 µg/mL
K. pneumoniae MTCC 39 12 14 - - 30 150 >200 50 µg/mL
Fungi Larvicidal assay
Filamentous fungi Clotrimazole (1) A. niger MTCC 1344 10 12 - - 22 LD50 330.69 µg/mL
A. parasiticus MTCC 411 10 12 - - 22 LD90 1132.62µg/mL
R. oryzae MTCC 262 12 14 10 12 23 (2) C. cladosporides MTCC 2607 10 12 - - 20 LD50 >1000 µg/mL
Unicellular fungi LD90 >1000 µg/mL
C. albicans MTCC 227 13 15 8 10 18 Control (Pyrethrum)
C. albicans MTCC 3018 13 15 - 8 18 LD50 1.6 µg/mL
S. cerevisiae MTCC 170 12 14 - - 19 LD90 3.0 µg/mL
S. cerevisiae MTCC 171 12 14 - - 19
(1)= 4-(N-methyl-N-phenyl amino) butan-2-one; (2)= Itaconic acid. Zone of inhibition was calculated in mm.
LD50 = Lethal concentration (µg/mL) at which 50 % of the larvae showed mortality.
Negative control DMSO-No activity.
References
[1] A.L. Demain (1999). Pharmaceutically active secondary metabolites of microorganisms, Appl. Microbiol.
Biotechnol. 52, 455-63.
[2] G.W. Huisman, D. Gray (2002). Towards novel processes for the fine chemical and pharmaceutical
industries, Curr. Opin. Biotechnol. 13, 352-358.
[3] D.N. Quang, T. Hashimoto, M. Tanaka, M. Baumgartner, M. Stadler, Y. Asakawa (2002). Concentriols B, C
and D, three squalene-type triterpenoids from the ascomycete Daldinia concentrica, Phytochemistry 61, 345-
353.
[4] M.E. Linday (1962). Practical introduction to microbiology. E & F. N. Spon Ltd., UK
[5] J.M. Andrews (2001). Determination of minimum inhibitory concentrations, J. Antimicrob. Chemother. 48,
5-16.
[6] Laurin A. Hummelbrunner and Murray B. Isman (2001). Acute, Sublethal, Antifeedant, and Synergistic
Effects of Monoterpenoid Essential Oil Compounds on the Tobacco Cutworm, Spodoptera litura (Lep.,
Noctuidae), J. Agric. Food Chem. 49, 715-720.
[7] D.J. Finney (1971). Probit Analysis, 3rd
ed. Cambridge University Press, New York.
[8] A. Lazzarini, L. Cavaletti, G. Toppo and F. Marinelli, (2000). Rare genera of actinomycetes as potential
producers of new antibiotics, Antonie. Leeuwenhoek. 78, 399–405.
© 2009 Reproduction is free for scientific studies
SHORT REPORT
The article was published by Academy of Chemistry of Globe Publications www.acgpubs.org/RNP © Published 10 /07/2009 EISSN: 1307-6167
Rec. Nat. Prod. 3:4 (2009) 204-208
Secondary Metabolites of Curvularia oryzae MTCC 2605
Siddhardha Busi1*
, Prabhakar Peddikotla2, Suryanarayana M.
Upadyayula1*
, Venkateswarlu Yenamandra2
1Biology Division, Indian Institute of Chemical Technology, Tarnaka, Hubsiguda, Hyderabad
500 007, Andhra Pradesh, India
2Organic Chemistry Division-I, Indian Institute of Chemical Technology, Tarnaka,
Hubsiguda, Hyderabad 500 007, Andhra Pradesh, India
(Received May 4, 2009; Revised July 10, 2009; Accepted July 13, 2009)
Abstract: Curvularia oryzae MTCC 2605 was exploited for the production of secondary metabolites. The major
compounds from the crude extract were purified by silica gel column chromatography and identified to be 11-α-
methoxycurvularin and (S)-5-ethyl-8, 8-dimethylnonanal by NMR and Mass spectral data. Bioassays showed
that 11-α-methoxycurvularin was active against bacteria, fungi and 4th
instar Spodoptera litura larvae.
Keywords: Curvularia oryzae; 11-α-methoxycurvularin; antibacterial; antifungal; antilarval.
1. Fungal Source
Curvularia oryzae is a filamentous fungus and develops black, velvet colonies with an
abundant septate mycelium. Species of Curvularia mostly occur as tropical and subtropical facultative
plant pathogens with teleomorphic states in Cochliobolus and Pseudocochliobolus. Curvularia oryzae
originally reported from rice grains and causes a fruit rot in okra (Abelmoschus esculentus). Many
varieties of C. oryzae were known to cause infection to different varieties of rice (Oryza sativa) [1, 2].
Curvularia oryzae Bugnicourt MTCC 2605 was procured from Microbial Type Culture Collection
(MTCC), IMTECH, Chandigarh, India and maintained on potato dextrose agar slants at 27 ºC prior to
cultivation.
* Corresponding author: E- Mail: [email protected]., [email protected]
Busi et al., Rec. Nat. Prod. (2009) 3:4 204-208
205
2. Previous Studies
There has been no antimicrobial investigation of Curvularia oryzae (MTCC 2605) reported
previously. However, isolation of 11-α-methoxycurvularin was previously reported from Penicilium
citroviridae and some other Penicilium species [3-6].
3. Present Study
The fungal strain Curvularia oryzae Bugnicourt MTCC 2605 was procured from Microbial
Type Culture Collection (MTCC), IMTECH, Chandigarh, India, was cultivated on 8 L of potato
dextrose broth medium at room temperature (29 ºC) for 9 days. The cultures were then extracted with
ethyl acetate to afford 2.4 g of residue after removal of the solvent under reduced pressure. The extract
was separated into two fractions by column chromatography on silica gel, using a gradient of n-
hexane: ethyl acetate (90:10, 50:50, 0:100). Fractions that showed homogenity on TLC plates were
combined and concentrated together to give pure compounds. Fraction 1 (51 mg) and Fraction 2 (38
mg) were obtained.1H and
13C NMR were recorded on Bruker UXNMR by dissolving in CDCl3, and
Mass spectrum on Finnigan MAT 1020-B. The optical rotation was measured on a JASCO DIP-360
polarimeter. The metabolites were identified as 11-α-methoxycurvularin (fraction 1) and (S)-5-ethyl-8,
8-dimethyl nonanal (fraction 2).
11-α-methoxycurvularin (C17H22O6): [α]D
25-17.2°; 1H NMR (300 MHz, CDCl3): δ: 6.29 (1H, d,
J=2.0Hz, H-4), 6.22 (1H, d, J=1.6Hz, H-6), 4.92 (1H, t, J=6.8Hz, H-15), 3.89 (1H, d, J=15.6Hz, H-2),
3.81 (1H, d, J=3.6Hz, H-2), 3.70 (1H, dd, J=15.6Hz, 6.8Hz, H-10), 3.39 (1H, d, J=12.0Hz, H-11),
3.36 (3H, s, OMe), 3.01 (1H, dd, J=14.8Hz , 8.8Hz, H-11), 1.53-1.62 (6H, m, H-12,13 and 14), 1.19
(3H, d, J=7.2Hz,Me). 13
C NMR (300 MHz, CDCl3): δ: 172.2 (C-1), 48.8 (C-2), 135.6 (C-3), 112.4 (C-
4), 159.3 (C-5), 102.7 (C-6), 158.3 (C-7), 119.6 (C-8), 205.1 (C-9), 39.9 (C-10), 77.0 (C-11), 31.9 (C-
12), 20.9 (C-13), 32.6 (C-14), 73.9 (C-15), 20.9 (C-16), 55.9 (C-OMe). EIMS (m/z) 322 [M+].
(S)-5-Ethyl-8, 8-dimethyl nonanal (C13H26O): [α]D25-12.1°; 1H NMR (300 MHz, CDCl3): δ: 0.9 (3H,
t, C-2,C-5), 1.3 (13H, s, H-9,H-7 and H-6), 1.6 (4H, t,H-3 and H-4), 2.1 (3H, s,H-5 AND H-1), 2.4
(2H, t,H-2). 13
C NMR (300 MHz, CDCl3): δ: 178.96 (C-1), 29.66 (C-2,C-3), 29.58 (C-4), 31.92 (C-5),
29.42 (C-6), 29.35 (C-7), 33.85(C-8), 22.68(C-9), 29.23 (C-10), 14.1(C-11), 24.69(C-12), 29.05 (C-
13). EIMS (m/z) 183 [M+].
(1) (2) Figure 1. Chemical structures of 11-α-methoxycurvularin (1) and (S)-5-Ethyl-8, 8-dimethyl nonanal (2).
O
O
O
OH
OH OMe
1
2 3
4
5
6
78
910
11 12
13
14
15
O
H12
34
5
6
78
9
12
8I8II
Secondary metabolites of Curvularia oryzae
206
Bioactivity Tests
The cytotoxic activity of the compound 11-α-methoxycurvularin was previously reported
against NCI-H460, MCF-7, and SF-268 cell lines [7]. This is the first report for antibacterial,
antifungal and larvicidal activity of 11-α-methoxycurvularin. The antibacterial and antifungal
Table 1. Antibacterial, antifungal and larvicidal activities of 11-α-methoxycurvularin
(1)= 11-α-methoxycurvularin; LD50 = Lethal concentration (µg/mL) at which 50 % of the larvae showed
mortality; Negative control DMSO-No activity.
activities of the compounds were determined according to Linday [8]. The tested compounds were
dissolved in dimethylsulfoxide (DMSO) at a concentration of 1 mg/mL. 50 µL and 100 µL of the
solutions were pipetted into agar wells which were bored on appropriate growth medium (PDA and
NA) spreader with respective test organism. The radius of zone of inhibition was measured in mm.
The minimum inhibitory concentration was determined according to the method described by
Andrews [9]. Spodoptera litura is an economically important polyphagous pest in India, China and
Japan, causing considerable economic loss to many vegetable and field crops. Crop loss due to insect
pests varies between 10% and 30% for major crops [10]. Larvicidal activity (measured as mortality
after 24 h) of the compounds was determined by topical application to early fourth instars according
to Luria et.al [11]. Lethality was estimated by applying different concentrations (100 to 1000 µg/mL)
of the metabolites. Two replicates of 10 larvae were tested per dose. A probit analysis was carried out
to calculate LD50 and LD90 [12].
Zone of inhibition MIC (µg/mL)
(1) Control
30 µg (1) Control
Bacteria 50 µg 100 µg Penicillin-G Nitrofurantoin
Gram Positive Bacteria
S. aureus MTCC 96 12 14 18 100 50 µg/mL
S. epidermides MTCC 435 10 12 18 200 50 µg/mL
B. subtilis MTCC 441 10 12 20 >200 100 µg/mL
B. sphericus MTCC 511 14 16 20 100 100 µg/mL
Gram Negative Bacteria Streptomycin
E. coli MTCC 443 10 12 29 >200 50 µg/mL
P. aeruginosa MTCC 741 12 14 34 200 75 µg/mL
P. oleovorans MTCC 617 12 14 30 200 75 µg/mL
K. pneumoniae MTCC 39 10 12 30 >200 50 µg/mL
Fungi Larvicidal assay
Filamentous fungi Clotrimazole (1)
A. niger MTCC 1344 12 14 22 LD50 205.59 µg/mL
A. parasiticus MTCC 411 12 14 22 LD90 645.33 µg/mL
R. oryzae MTCC 262 10 12 23 Control (Pyrethrum)
C. cladosporides MTCC 2607 10 12 20 LD50 1.6 µg/mL
Unicellular fungi LD90 3.0 µg/mL
C. albicans MTCC 227 12 14 18
C. albicans MTCC 3018 12 14 18
S. cerevisiae MTCC 170 12 14 19
S. cerevisiae MTCC 171 12 14 19
Busi et al., Rec. Nat. Prod. (2009) 3:4 204-208
207
The antimicrobial activity of the isolated compounds against all the test organisms is given in
the Table 1. 11-α-methoxycurvularin showed strong antibacterial activity against gram-positive
bacteria i.e. Staphylococcus aureus, Bacillus sphericus and gram-negative bacteria i.e. Pseudomonas
aeruginosa, Pseudomonas oleovorans with zone of inhibition between 12 to 16 mm. The MIC value
of the 11-α-methoxycurvularin was 100µg/mL against Staphylococcus aureus and Bacillus sphericus.
11-α-methoxycurvularin was tested for antifungal activity against eight fungi and showed moderate
activity against all fungi except Rhizopus oryzae and Cladosporium cladosporides. Against
Spodoptera litura 4th instar larvae LD50 was determined to be 205.59 µg/mL while (S)-5-Ethyl-8, 8-
dimethyl nonanal doesn’t showed any biological activity.
The present study of screening bioactive secondary metabolites from fungi revealed that
Curvularia oryzae as a source for the production of two secondary metabolites. Fungi are remarkable
organisms that readily produce a wide range of natural products called secondary metabolites.
Microbial secondary metabolites form an immense reservoir of natural chemical diversity, providing
us with an enormous diversity of unique carbon skeletons and functional group modifications. The
significance of these compounds is considerable, as many natural products are of medical, industrial
and/or agricultural importance [13]. Two compounds were isolated from Curvularia oryzae and their
antimicrobial and larvicidal activities reported. 11-α-Methoxycurvularin showed potent antibacterial,
antifungal and larvicidal activities. These compounds can be further exploited for biotechnological
applications.
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