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Analysis of Secondary Metabolites Released by Pseudomonas fluorescens Using GC-MS Technique and Determination of Its Ant

Analysis of Secondary Metabolites Released by Pseudomonas fluorescens Using GC-MS Technique and Determination of Its Anti-Fungal Activity

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Analysis of Secondary Metabolites Released by Pseudomonas fluorescens Using GC-MS Technique and Determination of Its Ant

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  1. DOI Number: 10.5958/0976-5506.2018.00485.0 Analysis of Secondary Metabolites Released by Pseudomonas fluorescens Using GC-MS Technique and Determination of Its Anti-Fungal Activity Rafid Hadi Hameed1, Fatima Moeen Abbas2, Imad Hadi Hameed3 1Ministry of Public Health, Maysan Health Department, Mesan governorate, 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 Pseudomonas fluorescens is a common gram-negative, rod-shaped bacterium [1]. It belongs to the Pseudomonas genus. The objectives of this study were analysis of the secondary metabolite products and evaluation antimicrobial activity.Twenty four bioactive compounds were identified in the methanolic extract of Pseudomonas fluorescens. GC-MS analysis of Pseudomonas fluorescens revealed the existence of the cis-5,8,11,14,17-Eicosapentaenoic acid, 12,15-Octadecadiynoic acid, methyl ester, 7-epi-cis- sesquisabinene hydrate, α-D-Glucopyranoside , O-α-D-glucopyranosyl, Acetamide, N-methyl-N-[4- [2-acetoxymethyl-1- pyrrol, Acetamide, N-methyl-N-[4-[2-fluoromethyl-1- pyrrolid, Phen-1,4-diol , 2,3-dimethyl-5-trifluoromethyl, Geranyl isovalerate, Quinazoline, 4-methyl, Pentetic acid, trans-13- Octadecenoic acid, 9-Hexadecenoic acid, 13-Hexyloxacyclotridecan -2-one, 7-Methyl-Z-tetradecen-1- ol acetate, cis-13-Eicosenoic acid, Didemin B, Hexadecanoic acid ,1-(hydroxymethyl) -1,2-ethaned, and Ethyl iso –allocholate. Cassia angustifolia (Crude) was very highly active (6.007±0.20) mm against Pseudomonas fluorescens. The results of anti-fungal activity produced by Pseudomonas fluorescens showed that the volatile compounds were highly effective to suppress the growth of Trichoderma horzianum (5.019±0.18). Pseudomonas fluorescens produce many important secondary metabolites with high biological activities. Keywords: Anti-Microbial, Pseudomonas fluorescens, GC-MS, Secondary metabolites. INTRODUCTION fluorescens isolates produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG), the compound found to be responsible for antiphytopathogenic and biocontrol properties in these strains. Several strains of P. fluorescens,suchasPf-5andJL3985,havedevelopeda natural resistance to ampicillin and streptomycin. These antibiotics are regularly used in biological research as a selective pressure tool to promote plasmid expression7-11. The aims of our research were analysis of the secondary metabolite products of Pseudomonas fluorescens and evaluation antimicrobial activity. P. fluorescens has multiple flagella. It has an extremely versatile metabolism, and can be found in the soil and in water. It is an obligate aerobe, but certain strains are capable of using nitrate instead of oxygen as afinalelectronacceptorduringcellularrespiration.P. fluorescens is an unusual cause of disease in humans, and usually affects patients with compromised immune systems (e.g., patients on cancer treatment) 1-6. P. Corresponding author: Imad Hadi Hameed Biomedical Science Department, University of Babylon, College of Nursing, Hillah city, Iraq; Phone number: 009647716150716; E-mail: imad_dna@yahoo.com MATERIALS AND METHOD Detection of secondary metabolites Subcultures were obtained on the nutrient agar for 48 hrs. at 22Cº. Metabolites was separated from the liquid culture and evaporated to dryness with a rotary evaporator

  2. 446 Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5 RESULTS AND DISCUSSION at 45Cº. The residue was dissolved in 1 ml methanol, filteredthrougha0.2μmsyringefilter,andstoredat4Cº for 24 h before being used for gas chromatography mass spectrometry 12-23.Theidentificationofthecomponents was based on comparison of their mass spectra with those of NIST mass spectral library as well as on comparison of their retention indices either with those of authentic compounds or with literature values24-35. Gas chromatography and mass spectroscopy analysis of compounds was carried out in methanolic extract of Pseudomonas fluorescens, shown in Table 1. GC-MS analysis of Pseudomonas fluorescens revealed the existence of the cis-5,8,11,14,17-Eicosapentaenoic acid, 12,15-Octadecadiynoic acid , methyl ester, 7-epi- cis-sesquisabinene hydrate, α-D-Glucopyranoside , O-α-D-glucopyranosyl,Acetamide,N-methyl-N-[4-[2- acetoxymethyl-1- pyrrol, Acetamide , N-methyl-N-[4-[2- fluoromethyl-1-pyrrolid,Phen-1,4-diol,2,3-dimethyl- 5-trifluoromethyl, Geranyl isovalerate, Quinazoline , 4-methyl, Pentetic acid, trans-13-Octadecenoic acid, 9-Hexadecenoic acid, -2-one, 7-Methyl-Z-tetradecen-1- ol acetate, cis- 13-Eicosenoic acid, Didemin B, Hexadecanoic acid ,1-(hydroxymethyl) -1,2-ethaned, and Ethyl iso – allocholate. The results of anti-fungal activity produced by Pseudomonas fluorescens showed that the volatile compounds were highly effective to suppress the growth of Trichoderma horzianum (5.019±0.18) Table 2. Pseudomonas fluorescens produce many important secondary metabolites with high biological activities. Based on the significance of employing bioactive compounds in pharmacy to produce drugs forthetreatmentofmanydiseases,thepurificationof compounds produced by Pseudomonas fluorescens can be useful. In agar well diffusion method the selected medicinal plants Nerium olender (Alkaloids), Ricinus communis (Alkaloids), Datura stramonium(Alkaloids), Linum usitatissimum (Crude), Anastatica hierochuntica (Crude), Cassia angustifolia (Crude), Althaea rosea (Crude), Coriandrum sativum (Crude), Origanum vulgare (Crude), Urtica dioica (Crude), Foeniculum vulgare (Crude), and Ocimum basilicum (Crude) were effective against Staphylococcus aureus, Table 3. Cassia angustifolia (Crude) was very highly active (6.007±0.20) mm against Pseudomonas fluorescens. Pseudomonas fluorescens was found to be sensitive to all test medicinal plants and mostly comparable to the standard reference antifungal drug Amphotericin B and fluconazoletosomeextent. Spectral analysis of bioactive natural chemical compounds of Pseudomonas fluorescens using (GC/ MS) Analysis was conducted using GC-MS (Agilent 789 A) equipped with a DB-5MS column (30 m×0.25 mm i.d.,0.25umfilmthickness,J&WScientific,Folsom, CA). The oven temperature was programmed as for the previous analysis36-39. Helium was used as the carrier gas attherateof1.0mL/min.EffluentoftheGCcolumn was introduced directly into the source of the MS via a transfer line (250 Cº) 40-42. 13-Hexyloxacyclotridecan Determination of antibacterial and antifungal activity Five-millimeter diameter wells were cut from the agarusingasterilecork-borer,and25μloftheplant 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 microorganisms43-47. The studied fungi, Microsporum canis, Aspergillus flavus, Candida albicans, S. cerevisiae, Trichoderma viride, Trichoderma horzianum, and Aspergillus terreus were 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 16 days at 130 rpm.The extraction was performed by adding 25 ml methanol to 100 ml liquid culture in anErlenmeyerflaskaftertheinfiltrationoftheculture. Methanol was used as solvent control. Amphotericin B and fluconazole were used as reference antifungal agent 48-50. Results of the study were based on analysis of variance (ANOVA) using Statistica Software. A significancelevelof0.05wasusedforallstatisticaltests.

  3. Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5447 Table 1.Major chemical compounds identified in methanolic extract of Pseudomonas fluorescens. Serial No. Phytochemical compound RT (min) Molecular Weight 1. cis-5,8,11,14,17-Eicosapentaenoic acid 3.184 302.22458 2. 12,15-Octadecadiynoic acid , methyl ester 3.293 290.22458 3. 7-epi-cis-sesquisabinene hydrate 3.659 222.198365 4. α-D-Glucopyranoside,O-α-D-glucopyranosyl 4.283 504.169035 5. Acetamide , N-methyl-N-[4-[2-acetoxymethyl-1- pyrrol 4.609 266.163042 6. Acetamide,N-methyl-N-[4-[2-fluoromethyl-1-pyrrolid 4.672 226.148142 7. Phen-1,4-diol,2,3-dimethyl-5-trifluoromethyl- 5.295 206.055464 8. Geranyl isovalerate 6.016 238.19328 9. Quinazoline , 4-methyl- 7.899 144.068748 10. Pentetic acid 8.322 393.138344 11. trans-13-Octadecenoic acid 8.551 282.25588 12. 9-Hexadecenoic acid 10.748 254.22458 13. 13-Hexyloxacyclotridecan -2-one 12.225 282.25588 14. 7-Methyl-Z-tetradecen-1- ol acetate 13.318 268.24023 15. cis-13-Eicosenoic acid 13.375 310.28718 16. 1,9-Dioxacyclohexadeca - 4,13-diene-2-10-dione , 7 13.678 308.19876 17. Z-5-Methyl-6-heneicosen-11-one 13.701 322.323566 18. Didemin B 14.525 1111.64166 19. Hexadecanoic acid ,1-(hydroxymethyl) -1,2-ethaned 14.719 568.506676 20. Pregn-4-ene-3,20-dione , 17,21-dihydroxy-, bis(O-me 18.130 404.267508 21. Androst -5,7-dien-3-ol-17-one 18.679 286.19328 22. (22S)-21-Acetoxy-6α,11β-dihydroxy-16α17α-propy 19.743 488.241018 23. Ethyl iso -allocholate 21.843 436.318874 24. 4H-Cyclopropa[5’,6’]bens[1’,2’:7,8]azuleno[5,6-b]ox 22.759 422.194067 Table 2. Antifungal activity of Pseudomonas fluorescens metabolite products. Pseudomonas fluorescens products Microorganism Fluconazol Microsporum canis 3.116±0.16 ª 3.110±0.15 Aspergillusflavus 5.000±0.19 4.309±0.17 Candida albicans 4.702±0.17 2.873.±0.12 S. cerevisiae 3.005±0.16 2.000±0.11 Trichoderma viride 4.957±0.17 1.704±0.10 Trichoderma horzianum 5.019±0.18 4.005±0.19 Aspergillus terreus 4.951±0.16 3.251±0.17 ªThevalues(averageoftriplicate)arediameterofzoneofinhibitionat100mg/mLcrudeextractand30μg/mL of (Amphotericin B; Fluconazol and Miconazole nitrate).

  4. 448 Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5 Table 3.Zone of inhibition (mm) of test different bioactive compounds and standard antibiotics of medicinal plants to Pseudomonas fluorescens. 2. Huda JA, Hameed IH, Hamza LF. Anethum graveolens: Physicochemical properties, medicinal uses, antimicrobial effects, antioxidant effect, anti-inflammatoryandanalgesiceffects:Areview. International Journal of Pharmaceutical Quality Assurance. 2017; 8(3): 88-91. Zone of inhibition (mm) S. No. Plant 3. 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. 1. 3.779±0.17 Nerium olender (Alkaloids) 2. Ricinus communis (Alkaloids) 2.639±0.15 3. 3.990±0.16 Datura stramonium(Alkaloids) 4. 4.815±0.18 Linum usitatissimum (Crude) 4. Hussein HM, Ubaid JM, Hameed IH. Inscticidal activity of methanolic seeds extract of Ricinus communis on adult of callosobruchus maculatus (coleopteran:brauchidae) and analysis of its phytochemical composition. International journal of pharmacognosy and phytochemical research. 2016; 8(8): 1385-1397. 5. Anastatica hierochuntica (Crude) 5.941±0.19 6. 6.007±0.20 Cassia angustifolia (Crude) 7. Althaea rosea (Crude) 5.000±0.18 8. 5.943±0.19 Coriandrum sativum (Crude) 9. Origanum vulgare (Crude) 5.799±0.18 10. Urtica dioica (Crude) 4.228±0.16 5. 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. 11. 3.000±0.14 Foeniculum vulgare (Crude) 12. Ocimum basilicum (Crude) 5.716±0.17 13. Control 0.00 CONCLUSION 6. Ibraheam IA, Hussein HM, Hameed IH. Cyclamen persicum: Methanolic Chromatography-Mass Spectrometry (GC-MS) Technique. International Journal of Pharmaceutical Quality Assurance. 2017; 8(4); 200-213. Twenty four bioactive chemical constituents have been identified from methanolic extract of the Pseudomonas fluorescens by GC-MS. In vitro antifungal and antibacterial evaluation of secondary metabolite products of Pseudomonas fluorescens forms a primary platform for further phytochemical and pharmacological investigation for the development of new potential antimicrobial compounds. Extract Using Gas 7. Mohammed GJ,, Kadhim MJ, Hameed IH. Proteus species: Characterization and herbal antibacterial: A review. International Journal of Pharmacognosy and Phytochemical Research. 2016; 8(11): 1844-1854. 8. Gilani AH, Janbaz KH, Akhtar MS. Selective protective effect of an extract from fumaria parvifloraonparacetamolinducedhepato-toxicity. Gen. pharmacol. 1996; 27: 979-983 Financial disclosure: There is no financial disclosure. Conflict of Interest: None to declare. 9. Ibraheam IA, Hadi MY, Hameed IH. Analysis of Bioactive Compounds of Methanolic Leaves extract of Mentha pulegium Using Gas Chromatography- Mass Spectrometry International Journal of Pharmaceutical Quality Assurance. 2017; 8(4); 174-182. Ethical Clearance: All experimental protocols were approved under the Department of Biology, College of Science, Hillah city, Iraq and all experiments were carried out in accordance with approved guidelines. (GC-MS) Technique. REFERENCES 1. 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. 10. Hadi MY, Hameed IH, Ibraheam IA. Ceratonia siliqua: Characterization, Pharmaceutical Products and Analysis of Bioactive Compounds: A Review. Research Journal of Pharmacy and Technology. 2017; 10(10): 3585-3589.

  5. Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5449 11. Hadi MY, Hameed IH, Ibraheam IA. Mentha pulegium: Medicinal Antibacterial, Antioxidant effect and Analysis of Bioactive Natural Compounds: A Review. Research Journal of Pharmacy and Technology. 2017; 10(10): 3580-3584. of Punica grantanum (Pomegranate peel) using GC- MS and FTIR spectroscopy. International Journal of Pharmacognosy and Phytochemical Research. 2016; 8(3): 480-494. uses, Anti-Hepatic, 20. Dhahir BM, Hameed IH, Jaber AR. Prospective and Retrospective Study of Fractures According to Trauma Mechanism and Type of Bone Fracture. Research Journal of Pharmacy and Technology. 2017; 10(10):1827-1835. 12. Kadhim MJ, Sosa AA, Hameed IH. Evaluation of anti-bacterial activity and bioactive chemical analysis of Ocimum basilicum using Fourier transform infrared (FT-IR) and gas chromatography-mass spectrometry (GC-MS) techniques. International Journal of Pharmacognosy and Phytochemical Research. 2016; 8(6): 127-146. 21. 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. 13. Mohammed GJ, Kadhim MJ, Hussein HM. Characterization of bioactive chemical compounds from Aspergillus terreus and evaluation of antibacterial and antifungal activity. International Journal of Pharmacognosy and Phytochemical Research. 2016; 8(6): 889-905. 22. 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. 14. Hameed IH, Altameme HJ, Idan SA. Artemisia annua: Biochemical products analysis of methanolic aerial parts extract and anti-microbial capacity. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2016; 7(2): 1843- 1868 23. 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 fourier-transform infrared spectroscopy. Malays. Appl. Biol. 2015; 44(4): 47–58. 15. Jasim H, Hussein AO, Hameed IH, Kareem MA. Characterization of alkaloid constitution and evaluation of antimicrobial activity of Solanum nigrum using gas chromatography mass spectrometry (GC-MS). Journal of Pharmacognosy and Phytotherapy. 2015; 7(4): 56-72. 24. 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 Pharmacognosy and Phytochemical Research. 2016; 8(3): 369-385. 16. Hadi MY, Mohammed GJ, Hameed IH. Analysis of bioactive chemical compounds of Nigella sativa using gas chromatography-mass spectrometry. Journal of Pharmacognosy and Phytotherapy. 2016; 8(2): 8-24. 25. Kadhim MJ, Mohammed GJ, Hameed IH. In vitro antibacterial, antifungal and phytochemical analysis ofmethanolicfruitextractofCassiafistula.Oriental Journal of Chemistry. 2016; 32(2): 10-30. 17. Shareef HK, Muhammed HJ, Hussein HM, Hameed IH. Antibacterial effect of ginger (Zingiber officinale)roscoeandbioactivechemicalanalysis using gas chromatography mass spectrum. Oriental Journal of Chemistry. 2016; 32(2): 20-40. 26. Jaddoa HH, Hameed IH, Mohammed GJ. Analysis of volatile metabolites released by Staphylococcus aureus using gas spectrometry and determination of its antifungal activity. Oriental Journal of Chemistry. 2016; 32(4): 8-24. chromatography-Mass 18. Al-Jassaci MJ, Mohammed GJ, Hameed IH. Secondary Metabolites Analysis of Saccharomyces cerievisiae and Evaluation of Antibacterial Activity. International Journal of Pharmaceutical and Clinical Research. 2016; 8(5): 304-315. 27. Hameed IH, SalmanHD, 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 19. Mohammed GJ, Al-Jassani MJ, Hameed IH. Anti- bacterial, Antifungal Activity and Chemical analysis

  6. 450 Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5 Journal of Chemistry. 2016; 32(4): 16-25. Plants Used for Treatment of Rheumatoid Arthritis: A Review. International Journal of Pharmaceutical and Clinical Research. 2016; 8(12): 1685-1694. 28. 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. 38. 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. 29. 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. 39. 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. 30. Hameed, I.H., Al-Rubaye A.F. and Kadhim, M.J. Antimicrobial Activity of Medicinal Plants and Urinary Tract Infections. International Journal of Pharmaceutical and Clinical Research. 2017; 8(11): 44-54. 40. 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. Research Journal of Pharmacy and Technology. 2017; 10 (11): 3981-3989. 31. 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. 41. Hadi MY, Hameed IH. Uses of Gas Chromatography- Mass Spectrometry (GC-MS) Technique for Analysis of Bioactive Chemical Compounds of Lepidium sativum: A Review. Research Journal of Pharmacy and Technology. 2017; 10 (11): 4039- 4042. 32. 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. 42. Ubaid JM, Hadi MY, Hameed IH. Bioactive Chemical Compounds Identified in Methanolic Extract of Trogoderma granarium. Research Journal of Pharmacy and Technology. 2017; 10 (11): 3997- 4004. 33. 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. 43. Hameed IH, Calixto MR, Hadi MY. Antimicrobial, Antioxidant, Hemolytic, Antihypertensive activity of Passiflora species. Research Journal of Pharmacy and Technology. 2017; 10 (11): 4079-4084. 34. 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. Anti-anxiety, and 35. 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. 44. Hameed IH, Calixto MR, Hadi MY. A Review: Solanum nigrum L. Antimicrobial, Antioxidant properties, Hepatoprotective effects and Analysis of Bioactive Natural Compounds. Research Journal of Pharmacy and Technology. 2017; 10 (11): 4063- 4068. 36. Al-Rubaye AF, Hameed IH, Kadhim MJ. A Review: Uses of Gas Chromatography-Mass Spectrometry (GC-MS) Technique for Analysis of Bioactive Natural Compounds of Some Plants. International Journal of Toxicological and Pharmacological Research. 2017; 9(1); 81-85. 45. Kamal SA, Hamza LF, Ibraheam IA. Characterization of Antifungal Metabolites Produced by Aeromonas hydrophila and Analysis of its Chemical Compounds Using GC-MS. Research Journal of Pharmacy and Technology. 2017; 10 (11): 3845-3851. 37. Kadhim MJ, Kaizal AF, Hameed IH. Medicinal 46. Sahi NM, Mohammed GJ, Hameed IH. Detection

  7. Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5451 of Bioactive Compounds of Raphanus sativus Using GC-MS and FT-IR Technical Analysis and Determination of its Anti-Bacterial and Anti-Fungal Activity. Indian Journal of Public Health Research and Development. 2018; 9(3): 235-240. Evaluation of Its Anti-Microbial activity. Indian Journal of Public Health Research and Development. 2018; 9(3): 247-253. 49. Kamal SA, Mohammed GJ, Hameed IH. Antimicrobial, Anti-inflammatory, Potential and Cytotoxic Activity of Salvadora persica: A review. Indian Journal of Public Health Research and Development. 2018; 9(3): 393-398. Analgesic 47. Hameed RH, Mohammed GJ, Hameed IH. Characterization of Antimicrobial Metabolites Produced by Salvadora persica and Analysis of Its Chemical Compounds Using GC-MS and FTIR. Indian Journal of Public Health Research and Development. 2018; 9(3): 241-246. 50. Hamza LF, Sahi NM, Hameed IH. Analysis of Methanolic extract of Secondary Metabolites Released by Candida glabratus Using GC-MS and Evaluation of its Antimicrobial Activity. Indian Journal of Public Health Research and Development. 2018; 9(3): 345-351. 48. Mohammed GJ, Hameed IH, Kamal SA. Determination of Bioactive chemical Compounds ofAspergillusflavusUsingGC/MSandFTIRand

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