Department of Biology, College of Science, University of Al-Qadisiyah, Hillah city, Iraq;

DOI Number: 10.5958/0976-5506.2018.00217.6 Determination of Bioactive Chemical Compounds of Aspergillus Flavus Using Gc/Ms and Ftir and Evaluation of Its Anti-Microbial Activity Ghaidaa Jihadi Mohammed 1, Imad Hadi Hameed 2, Sabreen A. Kamal 3 1 Department of Biology, College of Science, University of Al-Qadisiyah, Hillah city, Iraq; 2 Biomedical Science Department, University of Babylon, College of Nursing, Hillah city, Iraq; 3 Department 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. 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 parasiticus 7-19. 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 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 anywhere on earth. It can reproduce abundantly resulting from the production of numerous airborne conidia 20-27. 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 leaves 28-35, 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 patients 36-39. 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 25ºC 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 μm syringe filter, and stored at 4ºC for 24 h before

248 Indian Journal of Public Health Research & Development, March 2018, Vol.9, No. 3 being used for GC-MS 40-43. The identification of the components was based on comparison of their mass spectra with those of NIST mass spectral library 44-49. Determination of antibacterial and antifungal activity: Bacterial pathogens were swabbed in Muller Hinton agar plates. 90μl 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 μl 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 control 50,51. 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. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Bioactive compound 1,2-cis-1,5-trans-2,5-dihydroxy-4-methyl-1-(1-htdroxy-1- isopropyl)cy 2-Furancarboxaldehyde,5-methyl 2(5H)-Furanone 6-Hydroxymethyl-5-methyl-bicyclo[3.1.0]hexan-2-one D-Glucose,6-O-α-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 α-d-glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-ß- D-fruc 1-Nitro-2-acetamido-1,2-dideoxy-d-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-γ-pyrone Cycloundecanone, oxime 6-Acetyl-ß-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 RT (min) 3.585 3.613 3.831 3.859 3.997 4.408 4.466 4.546 4.712 4.820 4.901 5.009 5.175 5.284 5.341 5.536 5.616 5.782 5.890 6.245 7.149 8.660 8.820 9.701 14.908 15.183 15.349 Formula C 10 H 18 H 6 C 4 H 4 H 12 C 12 H 22 O 11 C 9 C 11 H 17 N H 8 C 18 H 32 O 16 H 16 N 2 O 7 C 10 H 17 NO 6 S C 7 H 8 C 12 H 21 NO C 10 H 16 C 20 H 36 H 6 C 20 H 37 N C 11 H 21 NO O 7 H 6 O 8 C 7 H 5 N 5 C 5 H 4 N 4 C 14 H 22 N 2 C 38 H 68 O 8 C 20 H 32 O 10 S 2 Exact Mass 186.125594 110.036779 84.021129 140.08373 342.11621 170.094295 158.094295 195.125929 112.052429 504.169035 252.095751 279.077658 124.052429 195.162314 184.109944 308.27153 126.031694 339.277344 183.162314 222.073953 126.031694 238.068868 207.039239 168.02834 266.163042 652.49142 496.14369

Indian Journal of Public Health Research & Development, March 2018, Vol.9, No. 3 249 Contd 28. 29. 30. 31. 2-Bromotetradecanoic acid Octadecanal,2 bromo L-Ascorbic acid, 6-octadecanoate 18,19-Secoyohimban-19- oic acid,16,17,20,21-tetradehydro-16 16.694 16.860 17.084 17.186 C 14 H 27 Br C 18 H 35 BrO C 24 H 42 O 7 C 21 H 24 N 2 306.119442 346.187128 442.293055 352.178692 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 RESULTS 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-α-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, α-d-glucopyranoside, O-α-D-glucopyranosyl-(1. fwdarw.3)-ß-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-γpyrone, Cycloundecanone, oxime, D-Glucose,6- O-α-D-galactopyranosyl, 6-Acetyl-ß-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. REFERENCES 1. Hussein JH, Hameed IH, Hadi MY. Using Gas Chromatography-Mass Spectrometry (GC-MS) Technique for Analysis of Bioactive Compounds of Methanolic Leaves extract of Lepidium sativum. 2017; 10 (11): 3981-3989. 2. Hadi MY, Hameed IH. Uses of Gas Chromatography-Mass Spectrometry (GC-MS) Technique for Analysis of Bioactive Chemical Compounds of Lepidium sativum: A Review. 2017; 10 (11): 4039-4042. 3. Ubaid JM, Hadi MY, Hameed IH. Bioactive Chemical Compounds Identified in Methanolic Extract of Trogoderma granarium. Research (11): 3997-4004. 4. Hameed IH, Calixto MR, Hadi MY. Antimicrobial, Antioxidant, Hemolytic, Anti-anxiety, and Antihypertensive activity of Passiflora species. 2017; 10 (11): 4079-4084. 5. Hameed IH, Calixto MR, Hadi MY. A Review: Solanum nigrum L. Antimicrobial, Antioxidant properties, Hepatoprotective effects and Analysis of Bioactive Natural Compounds. Research (11): 4063-4068. 6. Hussein JH, Hameed IH, Hadi MY. A Review: Anti-microbial, Anti-inflammatory effect and Cardiovascular effects of Garlic: Allium sativum. 2017; 10 (11): 4069-4078. 7. Flayyih SS, Hameed IH, Fakhir FD. Road Traffic Accident Coming to Hillah Teaching Hospital: Prospective and Retrospective Study. Research (11): 3819-3825. 8. Fakhir DF, Hameed IH, Flayyih SS. Retrospective Study: Burn Injury from 2010 to 2015 in a Burn Unit-Hillah Teaching Hospital-Iraq. Research (11): 3831-3838. 9. Khudhair ME, Hameed IH, Mekhlef AK. A Prospective and Retrospective Study of Acute Bronchitis in Hillah City-Iraq. Research Journal of Pharmacy and Technology. 2017; 10 (11): 3839-3844. 10. Kamal SA, Hamza LF, Ibraheam IA. Characterization of Antifungal Metabolites Produced by Aeromonas hydrophila and Analysis of its Chemical Compounds Using GC-MS. 2017; 10 (11): 3845-3851. 11. Mohammed GJ, Kadhim MJ, Hameed IH. Proteus species: Characterization and herbal antibacterial: A review. International Journal of 2016; 8(11): 1844-1854. 12. Shireen SK, Hameed IH, Hamza LF. Acorus calamus: Parts used, insecticidal, anti-fungal, antitumour and anti-inflammatory activity: A review. International Journal of Pharmaceutical Quality Assurance. 2017; 8(3): 153-157. 13. Huda JA, Hameed IH, Hamza LF. Anethum graveolens: Physicochemical properties, medicinal uses, antimicrobial effects, antioxidant effect, anti-inflammatory and analgesic effects: A review. International Journal of Pharmaceutical Quality Assurance. 2017; 8(3): 88-91. 14. Hussein HM, Hameed IH, Ubaid JM. Analysis of the secondary metabolite products of Ammi majus and evaluation anti-insect activity. International

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252 Indian Journal of Public Health Research & Development, March 2018, Vol.9, No. 3 29. Shareef HK, Muhammed HJ, Hussein HM, Hameed IH. Antibacterial effect of ginger (Zingiber officinale) roscoe and bioactive chemical analysis using gas chromatography mass spectrum. Oriental Journal of Chemistry. 2016; 32(2): 20-40. 30. Mohammed GJ, Al-Jassani MJ, Hameed IH. Anti-bacterial, Antifungal Activity and Chemical analysis of Punica grantanum (Pomegranate peel) using GC-MS and FTIR spectroscopy. International Journal of Pharmacognosy and Phytochemical Research. 2016; 8(3): 480-494. 31. Dhahir BM, Hameed IH, Jaber AR. Prospective and Retrospective Study of Fractures According to Trauma Mechanism and Type of Bone Fracture. 2017; 10(10):1827-1835. 32. Hapeep MA, Hameed IH, Jasim AA. Risk Factors, Cause and Site of Firearm Injuries: A Prospective and Retrospective Study. Research Journal of Pharmacy and Technology. 2017; 10(10): 3420-3425. 33. Jasim AA, Hameed IH, Hapeep MA. Traumatic Events in an Urban and Rural Population of Children, Adolescents and Adults in Babylon Governorate - Iraq. Research Journal of Pharmacy and Technology. 2017; 10(10): 3429-3434. 34. Altameme HJ, Hameed IH, Abu-Serag NA. Analysis of bioactive phytochemical compounds of two medicinal plants, Equisetum arvense and Alchemila valgaris seed using gas chromatography-mass spectrometry and fouriertransform infrared spectroscopy. Malays. Appl. Biol. 2015; 44(4): 47 58. 35. Hameed IH, Hamza LF, Kamal SA. Analysis of bioactive chemical compounds of Aspergillus niger by using gas chromatography-mass spectrometry and fourier-transform infrared spectroscopy. Journal of Pharmacognosy and Phytotherapy. 2015;7(8): 132-163. 36. Hussein HM, Hameed IH, Ibraheem OA. Antimicrobial Activity and spectral chemical analysis of methanolic leaves extract of Adiantum Capillus-Veneris using GC-MS and FT-IR spectroscopy. International Journal of 2016; 8(3): 369-385. 37. Hussein HJ, Hadi MY, Hameed IH. Study of chemical composition of Foeniculum vulgare using Fourier transform infrared spectrophotometer and gas chromatography - mass spectrometry. Journal of Pharmacognosy and Phytotherapy. 2016; 8(3): 60-89. 38. Kadhim MJ, Mohammed GJ, Hameed IH. In vitro antibacterial, antifungal and phytochemical analysis of methanolic fruit extract of Cassia fistula. Oriental Journal of Chemistry. 2016; 32(2): 10-30. 39. Altameme HJ, Hameed IH, Idan SA, Hadi MY. Biochemical analysis of Origanum vulgare seeds by fourier-transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Journal of Pharmacognosy and Phytotherapy. 2015; 7(9): 221-237. 40. Jaddoa HH, Hameed IH, Mohammed GJ. Analysis of volatile metabolites released by Staphylococcus aureus using gas chromatography-mass spectrometry and determination of its antifungal activity. Oriental Journal of Chemistry. 2016; 32(4): 8-24. 41. Hameed IH, Salman HD, Mohammed GJ. Evaluation of antifungal and antibacterial activity and analysis of bioactive phytochemical compounds of Cinnamomum zeylanicum (Cinnamon bark) using gas chromatography-mass spectrometry. Oriental Journal of Chemistry. 2016; 32(4): 16-25. 42. Kadhim MJ, Mohammed GJ, Hussein HM. Analysis of bioactive metabolites from Candida albicans using (GC-MS) and evaluation of antibacterial activity. International Journal of Pharmaceutical and Clinical Research. 2016; 8(7): 655-670. 43. Ubaid JM, Hussein HM, Hameed IH. Analysis of bioactive compounds of Tribolium castaneum and evaluation of anti-bacterial activity. International Journal of Pharmaceutical and Clinical Research. 2016; 8(7): 655-670. 44. Kadhim WA, Kadhim, M.J., Hameed, I.H. Antibacterial Activity of Several Plant Extracts Against Proteus Species. International Journal of Pharmaceutical and Clinical Research. 2017; 8(11): 88-94.

Indian Journal of Public Health Research & Development, March 2018, Vol.9, No. 3 253 45. Ahmed MD, Hameed IH, Abd-Ali MQ. Prospective and Retrospective Study of the Acute Heart Attack Cases in Marjan Hospital-Hillah City-Iraq. Research Journal of Pharmacy and Technology. 2017; 10(10): 3408-3416. 46. Fakhir DF, Hameed IH, Flayyih SS. Burns Injuries: A Prospective Statistical Study of 112 patients. Research Journal of Pharmacy and Technology. 2017; 10(10): 3401-3407. 47. Mekhlef AK, Hameed IH, Khudhair ME. Prevalence of Physical Injuries on the Head, Neck and Entire Body in, Hilla, Iraq. Research Journal of Pharmacy and Technology. 2017; 10(10): 3276-3282. 48. Kadhim MJ. In Vitro antifungal potential of Acinetobacter baumannii and determination of its chemical composition by gas chromatographymass spectrometry. Der Pharma Chemica. 2016; 8(19): 657-665. 49. Hameed IH, Al-Rubaye AF, Kadhim MJ. Antimicrobial Activity of Medicinal Plants and Urinary Tract Infections. International Journal of Pharmaceutical and Clinical Research. 2017; 9(1): 44-50. 50. Hussein HM, Hameed IH, Ubaid JM. Analysis of the secondary metabolite products of Ammi majus and evaluation anti-insect activity. International journal of pharmacognosy and phytochemical research. 2016; 8(8): 1192-1189. 51. Ubaid JM, Hussein HM, Hameed IH. Determination of bioactive chemical composition of Callosobruchus maculutus and investigation of its anti-fungal activity. International journal of pharmcognosy and phytochemical research. 2016; 8(8): 1293-1299.