determination of bioactive chemical compounds of
aspergillus flavus using gc/ms and ftir and evaluation of
its anti-microbial activity
ghaidaa jihadi mohammed1, imad hadi hameed2, sabreen a. kamal3
1department of biology, college of science, university of al-qadisiyah, hillah city, iraq
2biomedical science department, university of babylon, college of nursing, hillah city, iraq
3department of biology, college of science for women, university of babylon, hillah city, iraq
abstract
the objective of this study was analysis of the secondary metabolite products and evaluation antibacterial
activity. bioactives are chemical compounds often referred to as secondary metabolites. thirty one bioactive
compounds were identified in the methanolic extract of aspergillus flavus. origanum vulgare (crude)
was very highly active 6.95±0.25 mm. the results of anti-fungal and anti-bacterial activity produced by
aspergillus flavus showed that the volatile compounds were highly effective to suppress the growth of
penicillium expansum 5.33±0.21and pseudomonas eurogenosa (6.72 ± 0.23) mm.
keywords: antifungal, antibacterial, aspergillus flavus, gc-ms, medicinal plants, metabolites.
corresponding author:
imad hadi hameed
biomedical science department,
university of babylon, college of nursing,
hillah city, iraq
phone: 009647716150716
e-mail: imad_dna@yahoo.com
introduction
the genus aspergillus belongs to the deuteromycota
division of the fungi kingdom. the genus comprises
approximately 180 species, of which 33 have been
associated with human disease 1-6. a culture yielding
aspergillus spp., in addition to enabling a diagnosis of
invasive aspergillosis, may further define therapeutic
options via susceptibility testing or the isolation of
a species possessing inherent antifungal resistance
examples of the latter include a terreus and a nidulans,
which are both resistant to amphotericin b. some
species of the genus produce secondary metabolites in
food as aflatoxins (afs) which are produced mainly
by aspergillus flavus and aspergillus parasiticus7-19. a.
flavus is also an opportunistic pathogen and has been
isolated from insects, birds, mammals, and plants and
widely distributed soil-borne molds and can be found
anywhere on earth. it can reproduce abundantly resulting
from the production of numerous airborne conidia20-27.
the spores can easily disperse by air. environment has
a great impact on mould growth, with humidity being
the most important variable. it is a saprophytic fungus
that is capable of surviving on many organic nutrient
sources like plant debris, tree leaves28-35, decaying wood,
animal fodder, cotton, compost piles, dead insect and
animal carcasses, outdoor and indoor air environment
(air ventilation system), stored grains, and even human
and animal patients36-39. the aims of this study were
analysis of the secondary metabolites and evaluation
antibacterial and antifungal activity.
material and method
aspergillus flavus was isolated from dried fruit
and the pure colonies were selected, isolated and
maintained in potato dextrose agar slants. spores were
grown in a liquid culture of potato dextrose broth (pdb)
and incubated at 25?c in a shaker for eighteen days
at 130 rpm. the extraction was performed by adding
twenty five ml methanol to 150 ml liquid culture in an
erlenmeyer flask after the filtration of the culture. the
residue was dissolved in 1 ml methanol, filtered through
a 0.2 ?m syringe filter, and stored at 4?c for 24 h before
doi number: 10.5958/0976-5506.2018.00217.6
248 indian journal of public health research & development, march 2018, vol.9, no. 3
being used for gc-ms40-43. the identification of the
components was based on comparison of their mass
spectra with those of nist mass spectral library44-49.
determination of antibacterial and antifungal
activity: bacterial pathogens were swabbed in muller
hinton agar plates. 90?l of fungal extracts was loaded on
the bored wells. aspergillus flavus isolate was suspended
in potato dextrose broth and diluted to approximately
105 colony forming unit (cfu) per ml. five-millimeter
diameter wells were cut from the agar using a sterile
cork-borer, and 25 ?l of the samples plant solutions were
delivered into the wells. the plates were incubated for
48 h at room temperature. antimicrobial activity was
evaluated by measuring the zone of inhibition against
the test microorganisms. methanol was used as solvent
control50,51. amphotericin b and fluconazole were used
as reference antifungal agent. data were analyzed using
analysis of variance (anova) and differences among
the means were determined for significance at p < 0.05
using duncan’s multiple range test (by spss software)
version 9.1.
table 1: major bioactive chemical compounds identified in methanolic extract of aspergillus flavus
s. no. bioactive compound rt (min) formula exact mass
c 186.125594 10h18o3 1,2-cis-1,5-trans-2,5-dihydroxy-4-methyl-1-(1-htdroxy-1- 3.585
1. isopropyl)cy
c 110.036779 6h6o2 2. 2-furancarboxaldehyde,5-methyl 3.613
c 84.021129 4h4o2 3. 2(5h)-furanone 3.831
c 140.08373 8h12o2 4. 6-hydroxymethyl-5-methyl-bicyclo[3.1.0]hexan-2-one 3.859
c 342.11621 12h22o11 5. d-glucose,6-o-?-d-galactopyranosyl 3.997
c 170.094295 9h14o3 6. 2-(3-hydroxy-propyl)-cyclohexane-1,3-dione 4.408
c 158.094295 8h14o3 7. 9-oxa-bicyclo[3.3.1]nonane-1,4-diol 4.466
c 195.125929 11h17no2 8. benzenemethanol,2-(2-aminopropoxy)-3-methyl 4.546
c 112.052429 6h8o2 9. 1,2-cyclopentanedione,3-methyl 4.712
c 504.169035 18h32o16 ?-d-glucopyranoside, o-?-d-glucopyranosyl-(1.fwdarw.3)-?- 4.820
10. d-fruc
c 252.095751 8h16n2o7 11. 1-nitro-2-acetamido-1,2-dideoxy-d-mannitol 4.901
c 279.077658 10h17no612. desulphosinigrin 5.009 s
c 124.052429 7h8o2 13. orcinol 5.175
c 195.162314 12h2114. bicyclo[2.2.1]heptane-2-carboxylic acid isobutyl-amide 5.284 no
c 184.109944 10h16o3 2h-oxecin-2-one.3.4.7.8.9.10-hexahydro-4-hydroxy-10- 5.341
15. methyl-.[4
c 308.27153 20h36o2 16. 2h-pyran,tetrahydro-2-(12-pentadecynyloxy) 5.536
c 126.031694 6h6o3 17. maltol 5.616
c 339.277344 20h37no3 18. 2-tridecyl-5-(acetylamino)tetrahydro-?-pyrone 5.782
c 183.162314 11h2119. cycloundecanone , oxime 5.890 no
c 222.073953 8h14o7 20. 6-acetyl-?-d-mannose 6.245
c 126.031694 6h6o3 21. 5-hydroxymethylfurfural 7.149
c 238.068868 8h14o8 22. 1-gala-l-ido-octonic lactone 8.660
c 207.039239 7h5n5o3 23. pterin-6-carboxylic acid 8.820
c 168.02834 5h4n4o3 24. uric acid 9.701
c 266.163042 14h22n2o3 acetamide , n-methyl -n-[4-[2-acetoxymethyl-1-pyrrolidyl]- 14.908
25. 2-butynyl]-
c 652.49142 38h68o8 26. l-(+)-ascorbic acid 2,6-dihexadecanoate 15.183
c 496.14369 20h32o10s2 27. d-fructose , diethyl mercaptal , pentaacetate 15.349
indian journal of public health research & development, march 2018, vol.9, no. 3 249
contd…
c 306.119442 14h27bro2 28. 2-bromotetradecanoic acid 16.694
c 346.187128 18h3529. octadecanal ,2 –bromo 16.860 bro
c 442.293055 24h42o7 30. l-ascorbic acid , 6-octadecanoate 17.084
c 352.178692 21h24n2o3 31. 18,19-secoyohimban-19- oic acid,16,17,20,21-tetradehydro-16 17.186
table 2: zone of inhibition (mm) of test different
bioactive compounds and standard antibiotics of
medicinal plants to aspergillus flavus
s. no. plant
zone of
inhibition
(mm)
1. ricinus communis (alkaloids) 3.09 ± 0.19
2. datura stramonium
(alkaloids) 2.98 ± 0.21
3. linum usitatissimum (crude) 5.13 ± 0.23
4. anastatica hierochuntica
(crude) 6.03 ± 0.22
5. cassia angustifolia (crude) 4.90 ± 0.24
6. euphorbia lathyrus (crude) 5.99 ± 0.25
7. rosmarinus oficinalis (crude) 5.38 ± 0.23
8. citrullus colocynthis (crude) 4.76 ± 0.17
9. althaea rosea (crude) 6.01 ± 0.20
10. coriandrum sativum (crude) 6.51 ± 0.26
11. origanum vulgare (crude) 6.95 ± 0.25
12. urtica dioica (crude) 3.99 ± 0.21
13. foeniculum vulgare (crude) 3.05 ± 0.19
14. ocimum basilicum (crude) 4.94 ± 0.23
15. control 0.00
result s and discussion
gas chromatography and mass spectroscopy
analysis of compounds was carried out in methanolic
extract of a. flavus, shown in table 1. the first
set up peak were determined to be 1,2-cis-1,5-
trans-2,5-dihydroxy-4-methyl-1-(1-htdroxy-1-
isopropyl)cy. the second peak indicated to be
2-furancarboxaldehyde,5-methyl. the next peaks
considered to be 2(5h)-furanone, 6-hydroxymethyl-
5-methyl-bicyclo[3.1.0]hexan-2-one, d-glucose,6-
o-?-d-galactopyranosyl, 2-(3-hydroxy-propyl)-
cyclohexane-1,3-dione, 9-oxa-bicyclo[3.3.1]nonane-
1,4-diol, benzenemethanol,2-(2-aminopropoxy)-
3-methyl, 1,2-cyclopentanedione,3-methyl,
?-d-glucopyranoside, o-?-d-glucopyranosyl-(1.
fwdarw.3)-?-d-fruc, 1-nitro-2-acetamido-1,2-dideoxyd-
mannitol, desulphosinigrin, orcinol, bicyclo[2.2.1]
heptane-2-carboxylic acid isobutyl-amide, 2h-oxecin-
2-one.3.4.7.8.9.10-hexahydro-4-hydroxy-10-methyl-.
[4, 2h-pyran,tetrahydro-2-(12-pentadecynyloxy),
maltol, 2-tridecyl-5-(acetylamino)tetrahydro-?-
pyrone, cycloundecanone, oxime, d-glucose,6-
o-?-d-galactopyranosyl, 6-acetyl-?-d-mannose,
5-hydroxymethylfurfural, 1-gala-l-ido-octonic
lactone, pterin-6-carboxylic acid, uric acid, acetamide,
n-methyl -n-[4-[2-acetoxymethyl-1-pyrrolidyl]-2-
butynyl], l-(+)-ascorbic acid 2,6-dihexadecanoate,
d-fructose, diethyl mercaptal, pentaacetate,
2-bromotetradecanoic acid, octadecanal, 2–bromo,
l-ascorbic acid, 6-octadecanoate, 18,19-secoyohimban-
19-oic acid,16,17,20,21-tetradehydro-16. clinical
pathogens selected for antibacterial activity namely,
(streptococcus pneumonia, pseudomonas eurogenosa,
staphylococcus epidermidis, escherichia coli, proteus
mirabilis, streptococcus pyogenes, staphylococcus
aureus, and klebsiella pneumonia, maximum zone
formation against pseudomonas eurogenosa (6.72
± 0.23) mm. methanolic extraction of candida
glabratus showed notable antifungal activities against
microsporum canis, aspergillus terreus, aspergillus
5.902fumigatus, candida albicans, saccharomyces
cerevisiae, penicillium expansum and trichoderma
viride with high activity against penicillium expansum
5.33±0.21. in agar well diffusion method the selected
medicinal plants (ricinus communis (alkaloids),
datura stramonium(alkaloids), linum usitatissimum
(crude), anastatica hierochuntica (crude), cassia
angustifolia (crude), euphorbia lathyrus (crude),
rosmarinus oficinalis (crude), citrullus colocynthis
(crude), althaea rosea (crude), coriandrum sativum
(crude), origanum vulgare (crude), urtica dioica
(crude), foeniculum vulgare (crude), and ocimum
basilicum (crude), table 2. origanum vulgare (crude)
was very highly active 6.95 ± 0.25 mm against a.
flavus. aspergillus flavus was found to be sensitive to
all test medicinal plants and mostly comparable to the
standard reference antifungal drug amphotericin b and
fluconazole to some extent.
250 indian journal of public health research & development, march 2018, vol.9, no. 3
conclusion
the results of this study showed that a. flavus
species produce many important secondary metabolites
with high biological activities. the purification of
compounds produced by a. flavus species can be useful.
financial disclosure: there is no financial disclosure.
conflict of interest: none to declare.
ethical clearance: in this research, all experimental
protocols were approved under the department of
biology, college of science for women, university
of babylon, hillah city, iraq and all experiments were
carried out in accordance with approved guidelines.
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