using gc-ms technique for analysis of bioactive chemical
compounds of penicillium italicum and determination of its
anti-microbial activity
ghaidaa jihadi mohammed1, abeer fauzi al-rubaye2, imad hadi hameed3
1department of biology, college of science, university of al-qadisiyah, hillah city, iraq, 2department of
biology, college of science for women, university of babylon, hillah city, iraq, 3biomedical science department,
university of babylon, college of nursing, hillah city, iraq
abstract
the aims of our study were analysis of the bioactive chemical products and determination of antibacterial
activity. twenty eight bioactive compounds were identified in the methanolic extract of penicillium italicum.
the identification of bioactive chemical compounds is based on the peak area, retention time molecular
weight and molecular formula. the results of anti-fungal and anti-bacterial activity produced by penicillium
italicum showed that the volatile compounds were highly effective to suppress the growth of aspergillus
terreus 6.302±0.20 mm and proteus mirabilis 6.08±0.21 mm. in agar well diffusion method the selected
medicinal plants were effective against penicillium italicum. melissa officinalis was very highly antifungal
activity 6.70±0.25 mm.
keywords: anti-microbial, bioactive chemical compounds, gc-ms, penicillium italicum.
introduction
penicillium species are present in the air and dust
of indoor environments, such as homes and public
buildings. species of penicillium are ubiquitous soil
fungi preferring cool and moderate climates, commonly
present wherever organic material is available.
saprophytic species of penicillium and aspergillus are
among the best-known representatives of the eurotiales
and live mainly on organic biodegradable substances.
the ability of these penicillium species to grow on seeds
and other stored foods depends on their propensity to
thrive in low humidity and to colonize rapidly by aerial
dispersion while the seeds are sufficiently moist. some
penicillium species affect the fruits and bulbs of plants,
including p. expansum, apples and pears p. digitatum,
citrus fruits and p. allii, garlic. penicillium growth can
still occur indoors even if the relative humidity is low, as
long as there is sufficient moisture available on a given
surface. the objectives of our study were determination
of the bioactive products and evaluation of antibacterial
and antifungal activity.
material and method
gas chromatography – mass spectrum analysis
penicillium italicum gc–ms analysis were carried
out in a gc system (agilent 7890a series, usa). the
flow rate of the carrier gas, helium (he) was set to beat 1
ml min?1, split ratio was 1:50. the injector temperature
was adjusted at 250?c, while the detector temperature
was fixed to280?c. interpretation of mass spectrum was
conducted using the database of national institute of
standards and technology (nist, usa).
growth conditions of penicillium italicum and
determination of metabolites
penicillium italicum was isolated and maintained
in potato dextrose agar slants 19-21. spores were grown
in a liquid culture of potato dextrose broth (pdb) and
incubated at 25?c in a shaker for sixteen days at 150 rpm.
the extraction was performed by adding 50 ml methanol
to 150 ml liquid culture in an erlenmeyer flask after the
infiltration of the culture. products was separated from
the liquid culture and evaporated to dryness with a rotary
evaporator at 45?c 22,23. the residue was dissolved in 1
doi number: 10.5958/0976-5506.2018.00235.8
indian journal of public health research & development, march 2018, vol. 9, no. 3 353
ml methanol, filtered through a 0.2 ?m syringe filter, and
stored at 4?c for 24 h before being used for gc-ms.
determination of antibacterial and antifungal
activity
nine of tested bacteria were swabbed in muller
hinton agar plates. 90?l of fungal extracts was loaded
on the bored wells. the wells were bored in 0.5cm in
diameter. the plates were incubated at 37c° for 24 hr
and examined 26-28. after the incubation the diameter
of inhibition zones around the discs was measured.
penicillium italicum isolate was suspended in potato
dextrose broth and diluted to approximately 105 colony
forming unit (cfu) per ml. they were “flood inoculated
onto the surface of potato dextrose agar and then dried.
standard agar well diffusion method was followed 29-
33. five-millimeter diameter wells were cut from the
agar and 30 ?l of the plant samples 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
control. amphotericin b and fluconazole were used as
reference antifungal agent 34-41. the tests were carried
out in triplicate. the antifungal activity was evaluated
by measuring the inhibition-zone diameter observed
after 48 h of incubation. results of the study were
based on analysis of variance (anova) using statistica
software. a significance level of 0.05 was used for all
statistical tests.
table 1. major phytochemical compounds identified in methanolic extract of penicillium italicum.
serial no. phytochemical compound rt (min) molecular weight
1. dl-arabinose 3.173 150.052823
2. dihydroxyacetone 3.504 90.031694
3. 4-decene , 2,2-dimethyl - ,(z)- 4.025 168.1878
4. 2,5-dimethyl-4-hydroxy-3(2h)-furanone 4.271 128.047344
5. pentanoic acid , octyl ester 4.849 214.19328
6. : 9-oxabicyclo[6.1.0]nonan-4-ol 5.204 142.09938
7. 4h-pyran-4-one , 2,3-dihydro-3,5-dihydroxy-6-methyl 5.450 144.042258
8. d-gala-l-ido-octonic amide 5.971 255.095416
9. 4h,5h-pyrano[4,3-d]-1,3-dioxin , tetrahydro-8a-meth 6.383 158.094295
10. 9-thiabicyclo[3.3.1]non-7-en-2-ol 7.001 156.060886
11. pyrrolizidine-3-one-5-ol , ethyl ether 7.241 169.110279
12. 7-methyl-z-tetradecen-1-ol acetate 7.785 268.24023
13. dodecanoic acid , 3-hydroxy- 8.305 216.1725445
14. z-8-methyl-9-tetradecenoic acid 8.923 240.20893
15. tertbutyloxyformamide , n-methyl-n-[4-(1-pyrrolidinyl 9.816 252.183778
16. 1h-purin-2-amine , 6-methoxy-n-methyl- 10.926 179.080709
17. benzocycloheptano[2,3,4-lj]isoquinoline , 4,5,6,6a- 11.098 341.162708
18. 1-oxaspiro[4.5]decan-3-carboxylic acid ,2-oxo-4-cy 11.469 251.115758
19. 1-(3-methyl-2-butenyl)-3,6-diazahomoadamantan-9- 13.329 236.188864
20. n-(o-nitrophenylthio)-l-leucine 13.644 284.083078
21. benzenemethanol ,3-hydroxy-5-methoxy- 14.296 154.062994
22. benzenepropanoic acid , 3,5-bis(1,1-dimethylethyl)- 14.548 292.203844
23. n-hexadecanoic acid 14.634 256.24023
24. 10-methyl-8-tetradecen-1-ol acetate 14.828 268.24023
25. 2(1h)-naphthalenone , 4a,5,6,7,8,8a-hexahydro-6- 15.646 234.16198
26. 2-[5-(2,2-dimethyl-6-methylene-cyclohexyl)-3-methyl 16.133 312.208931
27. cis- vaccenic acid 16.322 282.25588
28. octadecanoic acid 16.493 284,27153
354 indian journal of public health research & development, march 2018, vol. 9, no. 3
table 2. zone of inhibition (mm) of test different bioactive compounds and standard antibiotics of
medicinal plants to penicillium italicum.
s.
no. plant
inhibition
(mm)
s.
no. plant
inhibition
(mm)
1. datura stramonium 3.39±0.20 16. malva parviflora 3.38±0.21
2. linum usitatissimum 5.01±0.22 17. mentha pulegium 5.05±0.22
3. diplotaxis cespitosa 6.05±0.20 18. daucus carota 6.00±0.23
4. cassia angustifolia 5.73±0.23 19. vitex agnus-castus 5.61±0.23
5. citrullus colocynthis 3.96±0.18 20. ruta graveolens 3.99±0.18
6. ocimum basilicum 4.82±0.22 21. calendula officinalis 4.95±0.22
7. achillea millefolia 5.58±0.23 22. taraxacum officinale 3.09±0.17
8. medicago sativa 3.06±0.18 23. borago officinalis 3.33±0.20
9. celosia argentea 3.37±0.20 24. sambucus nigra 4.08±0.22
10. apium graveolens 5.04±0.22 25. c. morifolium 6.01±0.21
11. brassica rapa 6.07±0.21 26. equisetum arvense 5.53±0.23
12. cichorium endivia 5.60±0.23 27. portulaca oleracea 5.86±0.24
13. linum usitatissimum 3.93±0.18 28. althaea officinalis 5.84±0.21
14. a. esculentus 4.92±0.21 29. melissa officinalis 6.70±0.25
15. malva sylvestris 6.57±0.25 30. control 0.00
results and discussion
analysis of compounds was carried out in
methanolic extract of penicillium italicum, shown in
table 1. escherichia coli, bacillus subtilis, pseudomonas
eurogenosa, staphylococcus aureus, staphylococcus
epidermidis, proteus mirabilis, streptococcus pyogenes,
and klebsiella pneumonia selected for antibacterial
activity, and we found proteus mirabilis 6.08±0.21 mm
recorded maximum zone formation against penicillium
italicum. methanolic extraction of penicillium italicum
showed notable antifungal activities against m. canis,
aspergillus flavus, aspergillus terreus, aspergillus
fumigatus, candida albicans, penicillium expansum,
trichoderma viride, and saccharomyces cerevisiae.
aspergillus terreus was very highly active against
penicillium italicum 6.302±0.20. in agar well diffusion
method the selected medicinal plants were effective
against penicillium italicum table 2. five-millimeter
diameter wells were cut from the agar using a sterile
cork-borer, and 25 ?l of the samples solutions datura
stramonium(alkaloids), linum usitatissimum (crude),
anastatica hierochuntica (crude), cassia angustifolia
(crude), citrullus colocynthis (crude), ocimum
basilicum (crude), achillea millefolia, medicago sativa,
celosia argentea, apium graveolens, brassica rapa,
cichorium endivia, linum usitatissimum, a. esculentus,
malva sylvestris, malva parviflora, daucus carota, vitex
agnus-castus, ruta graveolens, taraxacum officinale,
borago officinalis, sambucus nigra, c. morifolium,
equisetum arvense, portulaca oleracea, portulaca
oleracea, althaea officinalis, and melissa officinalis
were delivered into the wells. melissa officinalis was
very highly antifungal activity 6.70±0.25 mm.
conclusion
twenty eight bioactive chemical constituents
have been identified from methanolic extract of the
penicillium italicum by (gc-ms). in vitro antimicrobial
determination of bioactive chemical products of
penicillium italicum forms a primary platform for further
phytochemical and pharmacological investigation for
the development of new potential antibacterial and
antifungal compounds.
financial disclosure: there is no financial
disclosure.
conflict of interest:none to declare.
ethical clearance: these experiments were
carried out in accordance with approved guidelines
and all protocols were approved under the department
of biology, college of science for women, hillah city,
iraq.
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