Bioactive chemical analysis of enterobacter aerogenes and test of its anti fungal and anti-bacterial activity . 2

1. Bioactive Chemical Analysis of Enterobacter aerogenes and
Test of its Anti-fungal and Anti-bacterial Activity
and Determination
Rafid Hadi Hameed1
, Fatima Moeen Abbas2
, Imad Hadi Hameed3
1
Ministry of Public Health, Maysan Health Department, Mesan Governorate, Iraq, 2
Department of Biology,
College of Science for Women, University of Babylon, Hillah City, Iraq, 3
Biomedical Science Department,
University of Babylon, College of Nursing, Hillah city, Iraq
ABSTRACT
Enterobacter is a genus of Gram-negative, rod shaped, facultative anaerobic, and non-spore forming microbes
of family Enterobacteriaceae. Enterobacter aerogenes (E. aerogenes) is well known opportunistic bacteria
emergedasnosocomialpathogeninintensivecareunitpatients.Theobjectivesofourresearchwereanalysisof
the secondary metabolite products and evaluation antimicrobial activity. Twenty seven bioactive compounds
were identified in the methanolic extract of Enterobacter aerogenes. GC-MS analysis of Enterobacter
aerogenes revealed the existence of the Butanoic acid , 3-methyl, 2,6-Lutidine-4-[benzyloxy]-3,5-dichloro,
1-Propaneamine , 3-(methylthio), Butoxyacetic acid, 3-Hydroxy-2-methylthio-3-phenylpropanoic acid,
1-Deoxy-d-arabitol, Benzeneethanamine, 2-Butanamine , (S), Thiazole , 2-amino-5-methyl, Thiophene
, 2,5-bis(1,1-dimethylethoxy), Propanedinitrile , 2-bis(3,3-dimethyl-2-oxobutylthio), Carbamic acid ,
hydroxyl-,ethyl ester, 3-Aminopiperidin-2-one, dl-Cystathionine, Ethanol , 2-(diethylamino)-,N-oxide,
2,4-Heptadien-6-yn-1-ol , (E,E), Xanthine , 1,3-dipropyl-8-[4-[β-[(benzyloxycarbonyla, Isoquinoline
,1-ethyl and Cyclohexanecarboxylic acid , 2-phenylethyl ester. Coriandrum sativum was very highly active
(6.75±0.22) mm. The results of anti-fungal activity produced by Enterobacter aerogenes showed that the
volatile compounds were highly effective to suppress the growth of Candida albicans (5.717±0.18).
Keywords: GC-MS, Secondary metabolites, Anti-fungal, Enterobacter aerogenes.
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
INTRODUCTION
Enterobacter, (genus Enterobacter), any of a group
of rod-shaped bacteria of the family Enterobacteriaceae.
Enterobacter are gram-negative bacteria that are
classified as facultative anaerobes, which means that
they are able to thrive in both aerobic and anaerobic
environments 1-3
. Enterobacter species are responsible
for high morbidity and mortality rate in recent years due
to nosocomial infections and other health care settings.
Due to extended resistance of Gram-negative bacteria
against almost all antibiotics, early initiation of drug
therapy is required, nowadays colistin, and polymyxin
antibiotic have been preferred as an alternative drugs
against Gramnegative pathogens 4
. Enterobacter are
ubiquitous in nature; their presence in the intestinal
tracts of animals results in their wide distribution in soil,
water, and sewage. They are also found in plants. In
humans, multiple Enterobacter species are known to act
as opportunistic pathogens (disease-causing organisms),
including E. cloacae, E. aerogenes, E. gergoviae, and
E. agglomerans. Pathogenic Enterobacter can cause
any of a variety of conditions, including eye and skin
infections, meningitis, bacteremia (bacterial blood
infection), pneumonia, and urinary tract infections 5-9
.
Despite several new drug discoveries of broad spectrum
drugs or combination therapies, associated toxicities are
still a serious complication. Nosocomial infections are
DOI Number: 10.5958/0976-5506.2018.00484.9

2. Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5 439
the most frequent type of Enterobacter infections, but
community-acquired infections are sometimes observed
10-16
. Recently, an alternate treatment therapy approach
called biofield healing therapies or therapeutic touch
is reported with effectively inhibiting the growth of
bacterial cultures. The aims of our study were analysis
of the secondary metabolite products and evaluation
antimicrobial activity.
MATERIALS AND METHOD
Detection of secondary metabolites by Gas
chromatography – Mass Spectrum
The mixture was incubated at 4ºC for 10 min and
then shook for 10 min at 130 rpm. Metabolites was
separated from the liquid culture and evaporated to
dryness with a rotary evaporator at 45ºC. The residue
was dissolved in 1 ml methanol, filtered through a 0.2
μm syringe filter, and stored at 4ºC for 24 h before being
used for gas chromatography mass spectrometry 17-23
.
Materials of Plants Collection and Preparation
In this study, the leaves were dried at room
temperature for ten days and when properly dried the
leaves were powdered using clean pestle and mortar, and
the powdered plant was size reduced with a sieve 24-32
.
The fine powder was then packed in airtight container
to avoid the effect of humidity and then stored at room
temperature.
Spectral analysis of bioactive natural chemical
compounds of Enterobacter aerogenes 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., 0.25 um film thickness, J&W Scientific, Folsom,
CA). The oven temperature was programmed as for the
previous analysis 33-39
. Helium was used as the carrier
gas at the rate of 1.0 mL/min. Effluent of the GC column
was introduced directly into the source of the MS via a
transfer line (250oC). The components were identified
by comparing their retention times to those of authentic
samples of WILEY MASS SPECTRAL DATA BASE
Library 40-44
.
Determination of antibacterial and antifungal
activity
The studied fungi, Candida albicans, S. cerevisiae,
Fusarium sp., Mucor sp., Penicillium expansum,
Trichoderma viride, and Trichoderma horzianum 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 45-48
. The extraction was performed by
adding 25 ml methanol to 100 ml liquid culture in an
Erlenmeyer flask after the infiltration of the culture.
Methanol was used as solvent control. Amphotericin B
and fluconazole were used as reference antifungal agent.
Data analysis
All the measurements were replicated three times
for each assay and the results are presented as mean ±
SD and mean ± SE.
RESULTS AND DISCUSSION
Gas chromatography and mass spectroscopy
analysis of compounds was carried out in methanolic
extract of Enterobacter aerogenes, shown in Table
1. Peaks were determined to be Butanoic acid ,
3-methyl, 2,6-Lutidine-4-[benzyloxy]-3,5-dichloro,
1-Propaneamine , 3-(methylthio), Butoxyacetic acid,
3-Hydroxy-2-methylthio-3-phenylpropanoic acid,
1-Deoxy-d-arabitol, Benzeneethanamine, 2-Butanamine
, (S), Thiazole , 2-amino-5-methyl, Thiophene ,
2,5-bis(1,1-dimethylethoxy), Propanedinitrile ,
2-bis(3,3-dimethyl-2-oxobutylthio), Carbamic acid,
hydroxyl-,ethyl ester, 3-Aminopiperidin-2-one,
dl-Cystathionine, Ethanol , 2-(diethylamino)-,N-
oxide, 2,4-Heptadien-6-yn-1-ol , (E,E), Xanthine
, 1,3-dipropyl-8-[4-[β-[(benzyloxycarbonyla,
Isoquinoline ,1-ethyl and Cyclohexanecarboxylic
acid , 2-phenylethyl ester. Enterobacter aerogenes
produce many important secondary metabolites with
high biological activities. Based on the significance
of employing bioactive compounds in pharmacy to
produce drugs for the treatment of many diseases, the
purification of compounds produced by Enterobacter
aerogenes can be useful. Maximum zone formation
against Candida albicans (5.717±0.18) mm, Table 2. In
agar well diffusion method the selected medicinal plants
(Rosmarinus oficinalis, Citrullus colocynthis, Althaea
rosea, Coriandrum sativum, Origanum vulgare, Urtica
dioica, Foeniculum vulgare, Ocimum basilicum, Achillea
millefolia, Medicago sativa, Celosia argentea, Apium
graveolens, Brassica rapa, Cichorium endivia, Malva
sylvestris, Citrus sinensis, Ruta graveolens, Thymus

3. 440 Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5
vulgaris, Passiflora caerulea, Glycine max, Brassica oleracea, Olea europaea, Calendula officinalis, Taraxacum
officinale, Borago officinalis, Sambucus nigra, C. morifolium, Equisetum arvense, and Portulaca oleracea) were
effective against Staphylococcus aureus, Table 3. Coriandrum sativum was very highly active (6.75±0.22) mm
against Enterobacter aerogenes.
Table 1. Major chemical compounds identified in methanolic extract of Enterobacter aerogenes.
Molecular WeightRT (min)Phytochemical compound
Serial
No.
102.06807953.396Butanoic acid , 3-methyl-1.
281.037423.6712,6-Lutidine-4-[benzyloxy]-3,5-dichloro2.
105.061223.9801-Propaneamine , 3-(methylthio)-3.
132.0786444.186Butoxyacetic acid4.
212.0507154.4093-Hydroxy-2-methylthio-3-phenylpropanoic acid5.
136.0735594.6951-Deoxy-d-arabitol6.
121.08914955.187Benzeneethanamine7.
73.08914955.3932-Butanamine , (S)-8.
114.02516945.582Thiazole , 2-amino-5-methyl-9.
228.1184016.669Thiophene , 2,5-bis(1,1-dimethylethoxy)-10.
338.112276.766Propanedinitrile , 2-bis(3,3-dimethyl-2-oxobutylthio)11.
105.0425937.115Carbamic acid , hydroxyl-,ethyl ester12.
114.0793137.5443-Aminopiperidin-2-one13.
222.0674287.905dl-Cystathionine14.
133.1102798.620Ethanol , 2-(diethylamino)-,N-oxide15.
108.05751479.0152,4-Heptadien-6-yn-1-ol , (E,E)-16.
619.2754489.456Xanthine , 1,3-dipropyl-8-[4-[β-[(benzyloxycarbonyla17.
157.08914910.674Isoquinoline ,1-ethyl-18.
265.13140910.280N-Benzyloxycarbonyl-dl-norleucine19.
177.01927110.4052-Acetyl-5-chloromethyl-isoxazolidin-3-one20.
178.14699811.0692,5-Dimethyl-3-n-pentylpyrazine21.
170.10552811.5442,5-Piperazinedione , 3-methyl-6-(1-methylethyl)-22.
164.13134911.8592,6-Dimethyl-3-sec-butylpyrazine23.
210.10044212.2083-Methyl-1,4-diazabicyclo[4.3.0]nonan-2,5-dione24.
143.09462812.5113-Pyrrolidin-2-yl-propionic acid25.
232.1463312.683Cyclohexanecarboxylic acid , 2-phenylethyl ester26.
165.07897913.930Tyramine , N-formyl-27.
Table 2. Antifungal activity of Enterobacter aerogenes metabolite products.
Fungi
Antibiotics / Enterobacter aerogenes metabolite products
Enterobacter aerogenes
metabolite products
Amphotericin B Fluconazol
Miconazole
nitrate
Candida albicans 5.717±0.18 3.829±0.12 2.891.±0.11 2.077±0.12
S. cerevisiae 4.002±0.17 2.071±0.11 1.887±0.10 2.896±0.13
Fusarium sp. 4.981±0.17 1.973±0.10 3.000±0.13 2.719±0.11
Mucor sp. 4.741±0.18 1.995±0.12 1.782±0.11 1.007±0.10
Penicillium expansum 4.003±0.15 3.026±0.13 3.004±0.12 2.135±0.12
Trichoderma viride 5.248±0.18 2.005±0.10 2.015±0.11 3.483±0.13
Trichoderma horzianum 3.852±0.13 1.094±0.11 2.001±0.10 3.091±0.13

4. Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5 441
ª The values (average of triplicate) are diameter of zone of inhibition at 100 mg/mL crude extract and 30 μg/mL
of (Amphotericin B; Fluconazol and Miconazole nitrate).
Table 3. Zone of inhibition (mm) of test different bioactive compounds and standard antibiotics of
medicinal plants to Enterobacter aerogenes.
Plant
Inhibition
(mm)
Plant
Inhibition
(mm)
Rosmarinus oficinalis 5.77±0.20 Citrus sinensis 6.02±0.21
Citrullus colocynthis 4.06±0.18 Ruta graveolens 4.00±0.18
Althaea rosea 5.00±0.19 Thymus vulgaris 5.67±0.20
Coriandrum sativum 6.75±0.22 Passiflora caerulea 5.982±0.18
Origanum vulgare 5.83±0.19 Glycine max 5.66±0.19
Urtica dioica 3.95±0.17 Brassica oleracea 4.14±0.18
Foeniculum vulgare 2.96±0.15 Olea europaea 2.73±0.13
Ocimum basilicum 5.03±0.18 Calendula officinalis 5.00±0.19
Achillea millefolia 5.22±0.17 Taraxacum officinale 3.22±0.16
Medicago sativa 2.84±0.16 Borago officinalis 3.58±0.16
Celosia argentea 3.25±0.16 Sambucus nigra 2.92±0.14
Apium graveolens 4.93±0.18 C. morifolium 6.00±0.20
Brassica rapa 5.98±0.20 Equisetum arvense 5.71±0.19
Cichorium endivia 5.68±0.21 Portulaca oleracea 5.86±0.20
Malva sylvestris 6.55±0.21 Control 0.00
CONCLUSION
Twenty seven bioactive chemical constituents
have been identified from methanolic extract of the
Enterobacter aerogenes. Coriandrum sativum was
very highly active. The results of anti-fungal activity
produced by Enterobacter aerogenes showed that the
volatile compounds were highly effective to suppress
the growth of Candida albicans.
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, Hillah city, Iraq.
REFERENCES
1. Chang SC, Chen YC, Hsu LY. Epidemiologic study
of pathogens causing nosocomial infections. J
Formos Med Assoc. 1990; 89: 1023-1030.
2. Stein A, Raoult D. Colistin: an antimicrobial for the
21st century? Clin Infect Dis. 2002; 35: 901-902.
3. Biswas S, Brunel JM, Dubus JC, Reynaud-Gaubert
M, Rolain JM. Colistin: An update on the antibiotic
of the 21st century. Expert Rev Anti Infect Ther
2012; 10: 917-934.
4. Lucchetti G, de Oliveira RF, Gonçalves JP, Ueda
SM, Mimica LM. Effect of Spiritist “passe”
(Spiritual healing) on growth of bacterial cultures.
Complement Ther Med. 2013; 21: 627-632.
5. Movaffaghi Z, Farsi M. Biofield therapies:
biophysical basis and biological regulations?
Complement Ther. Clin. Pract. 2009; 15: 35-37.
6. 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.
7. ShireenSK,HameedIH,HamzaLF.Acoruscalamus:
Parts used, insecticidal, anti-fungal, antitumour and
anti-inflammatory activity: A review. International
Journal of Pharmaceutical Quality Assurance. 2017;
8(3): 153-157.

5. 442 Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5
8. 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.
9. 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.
10. 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.
11. Ubaid JM, Hussein HM, Hameed IH. Determination
ofbioactivechemicalcompositionofCallosobruchus
maculutus and investigation of its anti-fungal
activity. International journal of pharmcognosy and
phytochemical research. 2016; 8(8): 1293-1299.
12. Ibraheam IA, Hussein HM, Hameed IH. Cyclamen
persicum: Methanolic Extract Using Gas
Chromatography-Mass Spectrometry (GC-MS)
Technique. International Journal of Pharmaceutical
Quality Assurance. 2017; 8(4); 200-213.
13. Ibraheam IA, Hadi MY, Hameed IH. Analysis of
Bioactive Compounds of Methanolic Leaves extract
of Mentha pulegium Using Gas Chromatography-
Mass Spectrometry (GC-MS) Technique.
International Journal of Pharmaceutical Quality
Assurance. 2017; 8(4); 174-182.
14. 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.
15. Hadi MY, Hameed IH, Ibraheam IA. Mentha
pulegium: Medicinal uses, Anti-Hepatic,
Antibacterial, Antioxidant effect and Analysis of
Bioactive Natural Compounds: A Review. Research
Journal of Pharmacy and Technology. 2017; 10(10):
3580-3584.
16. 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.
17. 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
18. 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.
19. 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.
20. 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.
21. Mohammed GJ, Al-Jassani MJ, Hameed IH. Anti-
bacterial,AntifungalActivity 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.
22. 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.
23. 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.
24. 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.

6. Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5 443
25. 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.
26. Hussein HM, Hameed IH, Ibraheem OA.
AntimicrobialActivityandspectralchemicalanalysis
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.
27. 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.
28. 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.
29. 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
Journal of Chemistry. 2016; 32(4): 16-25.
30. 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.
31. 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.
32. 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.
33. 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.
34. 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.
35. MekhlefAK, 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.
36. 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.
37. 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.
38. Kadhim MJ, Kaizal AF, Hameed IH. Medicinal
Plants Used for Treatment of Rheumatoid Arthritis:
A Review. International Journal of Pharmaceutical
and Clinical Research. 2016; 8(12): 1685-1694.
39. 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.
40. Ubaid JM, Hussein HM, Hameed IH. Determination
ofbioactivechemicalcompositionofCallosobruchus
maculutus and investigation of its anti-fungal
activity. International journal of pharmcognosy and
phytochemical research. 2016; 8(8): 1293-1299.
41. HadiMY,HameedIH.UsesofGasChromatography-
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.
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.

7. 444 Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5
43. Hameed IH, Calixto MR, Hadi MY. Antimicrobial,
Antioxidant, Hemolytic, Anti-anxiety, and
Antihypertensive activity of Passiflora species.
Research Journal of Pharmacy and Technology.
2017; 10 (11): 4079-4084.
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.
45. KamalSA,HamzaLF,IbraheamIA.Characterization
of Antifungal Metabolites Produced by Aeromonas
hydrophila andAnalysis of its Chemical Compounds
Using GC-MS. Research Journal of Pharmacy and
Technology. 2017; 10 (11): 3845-3851.
46. Al-Rubaye AF, Hameed IH, Kamal SA. Screening
of Metabolites Products of Fusarium oxysporum and
Determination of Its Antibacterial and Antifungal
Activity Using Medicinal Plants Extract. Indian
Journal of Public Health Research and Development.
2018; 9(3): 399-404.
47. Al-Rubaye AF, Mohammed GJ, Hameed IH.
Characterization of Antibacterial and Antifungal
Metabolites Produced by Macrophomia phaseolus
and Analysis of Its Chemical Compounds Using
GC-MS. Indian Journal of Public Health Research
and Development. 2018; 9(3): 381-387.
48. Mohammed GJ, Al-Rubaye AF, Hameed IH. Using
GC-MS Technique for Analysis of Bioactive
Chemical Compounds of Penicillium italicum
and Determination of Its Anti-Microbial Activity.
Indian Journal of Public Health Research and
Development. 2018; 9(3): 352-357.