Analysis of Methanolic extract of Secondary Metabolites
Released by Candida glabratus using GC-MS and Evaluation of
Its Antimicrobial Activity
Lena Fadhil Hamza1, Nebras M Sahi2, Imad Hadi Hameed3
1College of Pharmacy, 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 objectives of this study were analysis of the secondary metabolite products and evaluation antibacterial
and antifungal activity. Bioactives are chemical compounds often referred to as secondary metabolites. Thirty
nine bioactive compounds were identified in the methanolic extract of Candida glabratus. The identification
of bioactive chemical compounds is based on the peak area, retention time molecular weight and molecular
formula. Coriandrum sativum was very highly antifungal activity (6.80±0.25)mm. The results of anti-fungal
and anti-bacterial activity produced by Candida glabratus showed that the volatile compounds were highly
effective to suppress the growth of Penicillium expansum (5.95±0.20) and Escherichia coli (5.900±0.22).
Keywords: Candida glabratus, GC-MS, Antifungal and Antibacterial, Secondary metabolites.
INTRODUCTION
There are two widely cited potential virulence
factors that contribute to the pathogenicity of C.
glabrata. The first is a series of adhesins coded by the
EPA (epithelial adhesin) genes 1-6. These genes, located
in the subtelomeric region, can respond to environmental
cues that allow them to be expressed en masse so the
organism can adhere to biotic and abiotic surfaces in
microbial mats. This is also the suspected mechanism
by which C. glabrata forms microbial “biofilms” on
urinary catheters, and less commonly in-dwelling IV
catheters. It also causes problems with dental devices,
such as dentures. A major phenotype and potential
virulence factor that C. glabrata possesses is low-level
intrinsic resistance to the azole drugs7-15, which are the
most commonly prescribed antifungal (antimycotic)
medications. It is still highly vulnerable to polyene
Corresponding author:
Imad Hadi Hameed
Biomedical Science Department, University of
Babylon, College of Nursing, Hillah city, Iraq;
Phone number: Mob.: 009647716150716;
E-mail: imad_dna@yahoo.com
drugs such as amphotericin B and nystatin, along with
variable vulnerability to flucytosine and caspofungin.
However intravenous amphotericin B is a drug of last
resort, causing among other side effects, chronic renal
failure16-28. Amphotericin B vaginal suppositories are
used as an effective form of treatment in combination
with boric acid capsules as they are not absorbed into
the blood stream29-33. The aims of this study were
screening of the metabolite products and determination
antibacterial and antifungal activity.
MATERIAL AND METHOD
Gas chromatography – Mass Spectrum analysis
Interpretation of mass spectrum was conducted
using the database of National Institute of Standards
and Technology (NIST, USA). The database consists of
more than 62,000 patterns of known compounds34-39. The
spectrum of the extract was matched with the spectrum
of the known components stored in the NIST library.
Growth conditions of Candida glabratus and
determination of metabolites
Candida glabratus was isolated from dried fruit and
the pure colonies were selected, isolated and maintained
DOI Number: 10.5958/0976-5506.2018.00234.6
346 Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3
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 sixteen days at 150
rpm40-43. The extraction was performed by adding 50
ml methanol to 150 ml liquid culture in an Erlenmeyer
flask after the infiltration of the culture. The mixture
was incubated at 4?C 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 45?C. 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 being used for GC-MS.
Determination of antibacterial and antifungal
activity
The test pathogens (Bacillus subtilis, Pseudomonas
eurogenosa, Staphylococcus epidermidis, Escherichia
coli, Proteus mirabilis, Streptococcus pyogenes,
Staphylococcus aureus, and Klebsiella pneumonia) 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. After the incubation
the diameter of inhibition zones around the discs was
measured. Candida glabratus 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. The
plates were incubated for 48 h at room temperature.
Antimicrobial activity was evaluated by measuring the
zone of inhibition against the test microorganisms44,45.
Methanol was used as solvent control. Fluconazole were
used as reference antifungal agent. The tests were carried
out in triplicate. The antifungal activity was evaluated
by measuring the inhibition-zone diameter observed
after 48 h of incubation.
Table 1. Major phytochemical compounds identified in methanolic extract of Candida glabratus.
Molecular
Weight
RT
(min)
Phytochemical
compound
Molecular
Phytochemical compound RT (min) Weight
5.753 365.166079
5-Oxazolidinone,3-
benzoyl-2-(1,1-
dimethylethyl)-4-
Methanesulfonic acid , 3.465 218.06128
6-oxobicyclo[2.2.1]hept-2-yl
5.868 222.073953
6-Acetyl-?-d-mannose
Cyclobut[c]inden-2-ol , decahydro-2- 3.493 180.151415
methyl-
5.954 251.100502
Muramic acid
3,3’-Methylenebis(1,5,8,11- 3.522 392.241018
tetraoxacyclotridecane)
6.017 142.09938
Cyclohexanone ,
4-ethoxy-
N-[2-[[2-Pyridylmethyl]amino]ethyl] 3.613 177.126597
aziridine
1-Nitro-2-acetamido-1,2- 6.360 252.095751
Benzeneacetaldehyde 3.699 120.0575147 dideoxy-d-mannitol
8-Methylenecyclooctene- 6.692 154.09938
3.928 261.023533 3,4-diol 3-Benzylsulfanyl-3-fluoro-2-
trifluoromethyl-acrylonitril
7.270 174.100442
1-Methyl-4-
[nitromethyl]-4-
piperidinol
4.191 92.047344
Glycerin
7.350 443.17646
Glucopyranuronamide ,
1-(4-amino-2-oxo-1(2H)-
pyri
Propane , 2-fluoro-2-methyl- 4.231 76.0688286
2-Bromotetradecanoic aid 7.579 306.119442 Cytidine , 5-methyl- 13.312 257.101171
Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3 347
14.285 210.136827
Pyrrolo[1,2-a]pyrazine-
1,4-dione , hexahydro-3-
(2-me
8.345 252.183778
Tertbutyloxyformamide , N-methyl-N-
[4-(1-pyrrolidinyl
14.628 256.24023
n-Hexadecanoic acid
12-Hydroxy-14-methyl-oxa- 8.511 240.1725445
cyclotetradec-6-en-2-on
14.949 186.107836
?-Thionodecalactone
1-Methyl-4-[nitromethyl]-4- 9.084 174.100442
piperidinol
15.263 410.166414
3-Oxa-16-
demethoxycarbonyl-16-
(2-methyl-sulphiny
9.312 324.227615
3-Trifluoroacetoxypentadecane
15.240 210.173213
1-Propyl-3,6-
diazahomoadamantan-
9-ol
2H-Oxecin-2-one , 9.524 184.109944
3,4,7,8,9,10-hexahydro-4-hydrox
12,15-Octadecadiynoic 15.990 290.22458
10.686 156.060886 acid , methyl ester
9-Thiabicyclo[3.3.1]non-7-en-2-ol
Nitrosothymol 10.926 179.094628 Octadecanoic acid 16.516 284.27153
16.591 255.064391
Pyridazine-3-carboxylic
acid , 5-cyano-4-methyl-
6-ox
11.716 180.063388
d-Mannose
Table 2. Zone of inhibition (mm) of test different bioactive compounds and standard antibiotics of
medicinal plants to Candida glabratus.
Plant Inhibition (mm) Plant Inhibition (mm)
Ricinus communis 3.02±0.18 Cordia myxa 3.04±0.19
Datura stramonium 3.51±0.22 Malva parviflora 3.60±0.23
Linum usitatissimum 5.08±0.21 Mentha pulegium 5.19±0.21
Diplotaxis cespitosa 6.05±0.24 Daucus carota 6.00±0.23
Cassia angustifolia 5.69±0.25 Vitex agnus-castus 5.71±0.25
Euphorbia lathyrus 5.94±0.23 Cressa cretica 5.96±0.26
Rosmarinus oficinalis 5.68±0.25 Citrus sinensis 5.81±0.21
Citrullus colocynthis 3.90±0.16 Ruta graveolens 3.90±0.18
Althaea rosea 4.99±0.21 Thymus vulgaris 5.88±0.24
Coriandrum sativum 6.80±0.25 Passiflora caerulea 6.09±0.24
Origanum vulgare 5.71±0.23 Glycine max 5.73±0.23
Urtica dioica 4.14±0.24 Brassica oleracea 4.08±0.21
Foeniculum vulgare 3.19±0.19 Olea europaea 3.00±0.19
Ocimum basilicum 4.98±0.25 Calendula officinalis 4.93±0.24
Achillea millefolia 5.38±0.26 Taraxacum officinale 3.19±0.19
Medicago sativa 3.09±0.19 Borago officinalis 3.63±0.21
Celosia argentea 3.35±0.22 Sambucus nigra 3.07±0.24
Apium graveolens 5.08±0.24 C. morifolium 6.08±0.21
Brassica rapa 6.00±0.21 Equisetum arvense 5.81±0.23
Cichorium endivia 5.71±0.25 Portulaca oleracea 5.90±0.25
Anethum graveolens 5.88±0.22 Malva neglecta 5.49±0.22
Plantago major 5.39±0.24 L. angustifolia 3.10±0.18
Linum usitatissimum 3.84±0.18 Althaea Officinalis 6.01±0.21
A. esculentus 6.07±0.22 Melissa officinalis 6.51±0.27
Malva sylvestris 6.39±0.24 Control 0.00
Cont... Table 1. Major phytochemical compounds identified in methanolic extract of Candida glabratus.
348 Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3
RESULTS AND DISCUSSION
Identification of biochemical compounds
Analysis of compounds was carried out in
methanolic extract of Salvadora persica, shown in Table
1. Clinical pathogens selected for antibacterial activity
namely, Bacillus subtilis, Pseudomonas eurogenosa,
Staphylococcus epidermidis, Escherichia coli, Proteus
mirabilis, Streptococcus pyogenes, Staphylococcus
aureus, and Klebsiella pneumonia maximum zone
formation against Proteus mirabilis (6.19±0.20)
mm. Methanolic extraction of Candida glabratus
showed notable antifungal activities against M. canis,
Aspergillus flavus, Aspergillus fumigatus, Candida
albicans, Saccharomyces cerevisiae, Penicillium
expansum, Trichoderma viride, and Aspergillus terreus.
Penicillium expansum was very highly active against
Candida glabratus (5.95±0.20). In agar well diffusion
method the selected medicinal plants were effective
against Candida albicans Table 2. Five-millimeter
diameter wells were cut from the agar using a sterile
cork-borer, and 25 ?l of the samples solutions (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), Achillea
millefolia, Medicago sativa, Celosia argentea, Apium
graveolens, Brassica rapa, Cichorium endivia, Anethum
graveolens, Plantago major, Linum usitatissimum,
A. esculentus, Malva sylvestris, Cordia myxa, Malva
parviflora, Daucus carota, Vitex agnus-castus, Cressa
cretica, Citrus sinensis, Ruta graveolens, Thymus
vulgaris, Passiflora caerulea, Glycine max, Brassica
oleracea, Olea europaea, Taraxacum officinale, Borago
officinalis, Sambucus nigra, C. morifolium, Equisetum
arvense, Portulaca oleracea, Portulaca oleracea, Malva
neglecta, L. angustifolia, Althaea Officinalis, and Melissa
officinalis) were delivered into the wells. Coriandrum
sativum was very highly antifungal activity (6.80±0.25)
mm.
CONCLUSION
Candida glabratus 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
Candida glabratus 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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Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3 351