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Global Journal of Pharmacology 8 (2): 228-236, 2014
ISSN 1992-0075
© IDOSI Publications, 2014
DOI: 10.5829/idosi.gjp.2014.8.2.8369
Corresponding Author: Manal M. El-Naggar, National Institute of Oceanography and Fisheries, Alexandria, Egypt
228
Antifungal Agent from Spirulina maxima: Extraction and Characterization
Mohamed G. Battah, Hassan A.H. Ibrahim, Manal M. El-Naggar,
11 2
Fagr Kh. Abdel_Gawad and Mohamed S. Amer
32
Department of Botany, Faculty of Science, Banha University, Egypt
1
National Institute of Oceanography and Fisheries, Alexandria, Egypt
2
National Research Center, Cairo, Egypt
3
Abstract: In the current investigation the cyanobacterium; Spirulina maxima exhibited antagonistic activity
against Pencillium oxalicum. The results indicated that, the active substance produced maximally after 15 days
of incubation in aerated culture at 30°C and pH 8 in B-G 11 medium. Dimethyl was the best solvent for extracting
the active material. The antagonistic material was purified using thin layer chromatography. It was found that
the purified antagonistic agent produced had a moderately toxic level and the LC was 1027 mg/l. The partially
50
purified agent of S. maxima showed a broad spectrum of antifungal activity with an average activity of 26%
against five tested human and plant pathogenic fungi compared to the three tested commercial drugs. The most
inhibited fungus was P. oxalicum (91%) followed by F. solani (65%) and R. solani (20%) compared to the
tested antifungal drugs. The compound showed maximum absorption band at 250 nm. Infrared (IR) indicated
presence of (NH , OH, NH) groups, C-H aliphatic, carbonyl group (amide), benzene ring and ether linkage and
2
nuclear magnetic resonance (NMR) showed signals at 0.9 (t, 3H, CH ), 1.4 (t, 2H, CH ), 2.6 (s, 3H, CH ), 4.1 (s,
32 3
2H, CH ), 5.0 (s, 2H, NH ) and 7.6 (m, aromatic protons). Mass spectroscopy indicated that its molecular weight
22
is 383 Dalton.
Keywords: Spirulina maxima Cyanobacterium Antifungal Substance NMR IR UV
INTRODUCTION activity [5]. Some of cyanobacterial antifungal agents
Pencillium sp.is significant in mammalian disease fungal-induced diseases by the human pathogenic yeast;
because of its production of potent mycotoxins [1]. Candida albicans. For instance, Jaki et al. [6] isolated
Pencillium oxalicum appears to be a common pathogen two novel cyclic peptides with antifungal activity against
of greenhouse cucumbers and on animals: It has been the yeast Candida albicans from the cyanobacterium
implicated in genital diseases of water buffalo. It produces Tolypothrix byssoidea (EAWAG195). Also, Ozdemir et al.
the hepatotoxins: secalonic acid D and oxaline, together [7] obtained antifungal agents with phenolic nature
with roquefortine C [2]. Contaminated grain fed to young against Candida albicans ATCC 10239. While, Volk and
ducklings proved to be toxic [3]. Cyanobacteria have been Mundt [8] proved that the exometabolite of the
studied for producing various biologically active cyanobacterium Nodualria harveyana (9 H - pyrido (3, 4-
compounds. These include antibiotics which in laboratory b) indole) and exometabolite of the cyanobacterium
tests inhibited bacteria and fungi that incite human, plants Nostoc insulare (4.4 - dihydroxybiphenyl) showed high
and fish diseases [4]. Some of cyanobacterial antifungal sensitivity against the yeast; Candida albicans. Soltani
agents have been used in the treatment of resistant et al. [9] isolated six species of cyanobacterial that
fungal-induced diseases of domestic plants and inhibited the growth of Candida kefyr ATCC 1140 and
agricultural crops, such as; majusculamide C; a one species inhibited the growth of Candida albicans
microfilament depolymerizing agent extracted from ATCC 14053. In such manner, Shanab [10] isolated
Lyngbya majuscule, that showed potent fungicidal three Oscilatoria species: O. hamelii,O. platensis and
have been used in the treatment of resistant
Global J. Pharmacol., 8 (2): 228-236, 2014
229
O. rubescens that showed antifungal activity MATERIALS AND METHODS
against Candida albicans and Aspergillus flavous.
Mo et al. [11] isolated Scytoscalarol (1), an antimicrobial
sesterterpene bearing a guanidino group from the
cultured cyanobacterium Scytonema sp. which shows
antifungal activities against Candida albicans and
Mycobacterium tuberculosis. The Oscilatoria sp. was
found to have antifungal activity on yeasts [12]. Other
cyanobacterial strains; Anabaena sp. and Calothrix sp.
were examined against phytopathogenic fungi, such as;
Rhizoctonia bataticola and Pythium debaryanum. The
diameter of the inhibition zone was largest when
extracellular filtrates of the two cultures incubated at
high light intensity (90-100 ìmol photons m s ) and at
21
40°C [13]. The biological control aptitude of the
cyanobacteria; Anabaena subcylindria, Nostoc
muscorum and Oscillatoria angusta filtrates was
evaluated by Abo- Shady et al. [14] on the growth of the
isolated pathogenic fungi from the different organs of
Faba bean. Their filtrates revealed high efficiency on the
control of these fungi. The reduction in fungal mat growth
diameter was greater than in that of the fungal dry weight
showing inhibited fungal spread by greater rate. The
reduction in the fungal dry weight was mostly linear and
significantly correlated with the algal filtrate
concentrations.
Biondi et al. [15] isolated Antarctic cyanobacteria
isolated from benthic mats that showed antifungal activity
against the filamentous fungus Aspergillus fumigatus or
the yeast Cryptococcus neoformans. Abedin and Taha
[16] tested cyanobacterial species; Anabaena oryza,
Tolypothrix ceytonica and Spirulina platensis for
antifungal agent production on various organisms that
incite diseases of humans and plants (Aspergillus niger,
Aspergillus flavous, Pencillium herquei, Fusarium
moniliforme, Helminthosporium sp. Allternaria
brassicae, Saccharomyces cerevisiae and Candida
albicans). They found that Spirulina platensis and
Anabaena oryza had the highest antifungal activity
towards the tested fungi. Chen and Huang [17]
demonstrated that culture filtrate of five strains of
Clitocybe nuda displayed various degrees of antifungal
activity against plant pathogenic fungus; Phytophhora
capsici.
Therefore, the current study was suggested to obtain
antifungal compounds from S. maxima and optimize the
antifungal production, purifying the antifungal agents,
study the bio-toxicity of the purified antifungal agents
and elucidate the chemical structure of the purified
antifungal agents
Isolation and Culturing of Spirulina Maxima: The blue
green alga S. maxima was isolated from soil. The dilution
culture technique adopted by Venkatarman [18] was used
for isolation and purification. The axcenic culture was
obtained using the method recommended by Bolch and
Blackburn [19]. The isolated organism was grown
autotrophically in BG-11 medium as described by Rippka
et al. [20], for determination the optimal time at which the
highest antagonistic activities of S. maxima was attained.
Microorganisms Used for Antagonistic Activity: The test
filamentous fungi used in this investigation were
Aspergillus niger, Fusarium solani, Penicillium
oxalicum, Rhizoctonia solani and Candida albicans
ATCC 14053.
The maintenance of these fungi except C. albicans
was carried out using a modified Czapek Yeast Extract
Agar (CYEA) medium, it Composed of (g/L): Sucrose, 20;
NaNO , 2.0; K HPO , 1.0; MgSO , 0.5; KCl, 0.5; FeSO ,
3 24 4 4
0.01; Yeast extract, 2.0. The pH was adjusted at 5.0 [21].
While, C.albicans was maintained using Sabouraud-
dextrose agar (SDA) medium (British Pharmacopoeia)
according to Sandven and Lassen [22].
Bioactivity Test of S. Maxima: The supernatants of S.
maxima were applied in 5% using Czapex's agar plates.
The 5 mm discs of the tested plant and human pathogenic
fungi (Aspergillus niger, Fusarium solani, Penicillium
oxalicum and Rhizoctonia solani were placed separately
on the amended prepared agar plates (One disc/plate) and
incubated at 28°C for a week. The bioactivity of these
crude supernatants to inhibit the growth diameter of these
pathogenic fungi was estimated daily by measuring the
diameter of the fungal growth on amended agar plates
compared to the control (Fungal growth diameter on un-
amended agar plates), then the suppression percentage
was calculated as follows: (Fungal growth diameter on un-
amended agar plates in mm - fungal growth diameter on
amended agar plates in mm)/ fungal growth diameter on
un-amended agar plates in mm X100% [23]. On the other
hand, the bioactivity against C. albicans ATCC14053 was
carried out as follows: the microalgal supernatants were
amended in 5% using SDA agar plates [22]. One hundred
microliters of C. albicans TCC14053 suspension (OD ~
1.0) was added to amended and un-amended plates, the
incubation was carried out for 48 h at 30°C. The
bioactivity of the crude supernatants was estimated by
Global J. Pharmacol., 8 (2): 228-236, 2014
230
counting the colonies of C. albicans ATCC14053 on the concentration was prepared and used separately to amend
amended agar plates compared to the control the culture medium. The suppression growth % of the
(Un-amended agar plates), them the suppression tested pathogenic fungi was detected compared to the
percentage was calculated compared to the control. control (Untreated fungi).
Purification of the Antifungal Agent (s) Using TLC Partial Chemical Characterization of the Purified
Preparation of the Plates:Glass plates (20X20 cm) were Antifungal Agent of S. Maxima
cleaned and air dried. About 40 g of silica gel G60 were UV Spectrum :The UV spectrum of each obtained spot
suspended in 100ml of distilled water. The suspension
was spread over the plates with a thickness of 0.1 cm.The
plates left for air dryness then activated by heating at
120°C for one hour. The Rf of the active components was
determined using 100ml of different solvent systems
preformed in vol. /vol. as follows: Benzene: chloroform,
50:50, v/v. Benzene : acetone, 90:10,v/v. Benzene : ethyl
alchol : formic acid,75:24:1,v/v. Butanol: acetic acid :
water,50:40:10,v/v. Toluen : chloroform: acetone, 40:25:35,
v/v. Chloroform : methanol, 50:50, v/v. Dichloromethane
: methyl alcohol: water, 50:30:20, v/v. and
Dichloromethane: methyl alcohol: water, 65:32:3, v/v.
Bioactivity Test of the Developed Spots: In each case the
chromatograms were detected under a UV lamp. Each spot
was removed and dissolved in dimethylsulfooxide. Silica
gel was removed by centrifugation at 5000rpm for 15min.
The supernatant was tested for biological against the
pathogenic fungi.
Bio-toxicity of the Purified Antifungal Agent (s): The
toxicity bioassay of S. maxima was carried out according
to Meyer et al. [24] using 24h old neuplii of Artemia
salina as a bio-toxicity biomarker. Different
concentrations of the purified extract (100, 200, 1000, 1500,
2500 and 5000 mg/l) were made and distributed separately
using clean and dry glass vials (20 ml) then completed to
a total volume of 10 ml using sterile seawater. Ten live
neuplii of A. salina were transferred to each vial. The
number of the viable biomarker was counted after 24 h of
application. The mortality percentages and the half lethal
concentration (LC ) were determined using the probit
50
analysis method [25].
Comparison of the antifungal activity of the partially
purified agent of S.maxima to some commercial antifungal
agents
The bioactivity of the partially purified antifungal
component was estimated compared to three commercial
antifungal drugs Diflucan, Flocoral and Fungimycin. 10mg
of each tested agent was dissolved in 1 ml of
dimethylsulfooxide (DMSO) and then a solution of 5%
was determined using a solution of DMSO in quartz
cuvette. The wave length was ranged from 0-500 nm. The
spectrum was achieved using spectrophotometer UV-
visible Jenway 6800, The Marine chemistry lab, National
Institute of Oceanography and Fisheries, Alex. Egypt.
Infrared spectrum (IR): The Infrared spectrum (IR) was
obtained using Peak Find-Memory -27 spectrophotometer,
in the Microanalysis Center, Cairo University, Egypt. The
molecular structure of each antagonistic material (A) was
partially identified and compared to known antibiotics.
The method makes use of small discs made from the
mixture of about 1 mg of the tested material and 300 mg of
pure dry KBr, followed by pressing into a disc. The
measurements were carried out at infrared spectra
between 400-4000 nm.
Mass spectrum (MS): A mass spectrophotometer (DI
Analysis Shimadzu Qp-2010 plus) was used in the
Microanalysis Center, Cairo University, Egypt. The
product subjected to a steam of high energy of electrons
at elevated temperature up to 100°C cleavage fragments
were yielded which can be characterized by mass/charge
from mass spectra data.
Nuclear Magnetic Resonance Spectra (NMR): Each spot
of antagonistic agents (A) was dissolved in durated
dimethylsulfooxide. The different functional group could
be identified using NMR (JEOL ECA 500), the
Microanalysis Center and Cairo University, Egypt.
RESULTS
Effect of Different Incubation Period on Growth and
Antifungal Activity of S. Maxima: The results in
Figure (1) indicated that, the antimicrobial activity
increased as the culture age increased reaching their
maximal values at 15 day of incubation against P.
th
oxalicum about 40%. Thereafter, the antimicrobial activity
of S. maxima against P. oxalicum decreased reaching
23%.
Global J. Pharmacol., 8 (2): 228-236, 2014
231
Fig. 1: Effect of different incubation period on the growth Effect of Different PH on Growth and Antifungal Activity
and antifungal activity of S. maxima against P. of S. Maxima: The data in Figure (3) indicated that, the
oxalicum optimum pH value for maximum growth and antimicrobial
Fig. 2: Effect of different culture media on the growth and
antifungal activity of S. maxima against P.
oxalicum.
Fig. 3: Effect of different pH on the growth and
antifungal activity of S. maxima against P. oxalicum
Effect of Different Culture Media on Growth and
Antifungal Activity of S. Maxima:Four different types of
culture media (B-G 11, Zarouk, Rhode and Chu) were used.
The results indicated that the highest growth value was
obtained in BG-11 medium 3.4 g/l where the lowest value
of growth was obtained in Chu medium 2.4 g/l (Figure 2).
The results also indicated that S. maxima exhibited the
highest antimicrobial activity when being cultured in BG-
11 medium and the antimicrobial activity against P.
oxalicum was 41%, wherever S. maxima exhibited the
lowest antimicrobial activity when being cultured in Chu
medium and the antimicrobial activity against P. oxalicum
was 7.5%. As a result of this experiment, the BG-11
medium was used throughout the subsequent experiments
(Figure 2).
activity of S. maxima was 8. The growth of S. maxima was
achieved 3.5 g/l at pH 8, where higher and lower pH
values caused reduction in the growth of the organism
The antimicrobial activity of S. maxima exhibited the
highest antimicrobial activity against P. oxalicum 47%,
where the higher and lower pH values were less effective.
As a result of this experiment, cultures have been
adjusted at pH 8 throughout the subsequent experiments.
Determination of the Best Solvent for Extraction of the
Antifungal Agents (A) from S. Maxima:The results in
Figure (4) indicated that, the highest extraction of the
antagonistic material had obtained when DMSO was used
as a solvent followed by methyl alcohol, ethyl alcohol and
chloroform, while, N-butanol was less effective in
extraction of the antagonistic material
Partially purification of the antifungal agents of S.
maxima using TLC: Only one spot was developed when
the sonicated cells were eluted in a solvent system formed
of methyl alcohol: chloroform, (60: 40; v/v). Spot (A)
showed a Rf value equal 0.55.
The results indicate that of the partially purified
agents (A) of S. maxima show increase of the antifungal
activities against P. oxalicum compared to the crude
product used. It was observed the spot (A) showed
increase in the antifungal activity about 25% compared to
the crude material.
Bio-toxicity Test of S. Maxima Purified Extracts Using
Artemia Salina as Biomarker: The bio-toxicity of S.
maxima purified extracts was carried out by using Artemia
salina as biomarker. As shown in (Figure 5) that different
Global J. Pharmacol., 8 (2): 228-236, 2014
232
Fig. 4: Determination of the best solvent for Fig. 5: Bio-toxicity of different concentrations of S.
extraction of the antifungal agent from maxima purified antifungal agent using Artemia
S. maxima salina as a biomarker.
(a) (b) (c) (d)
Fig. 6: UV/Vis. spectrum (A), IR spectrum (B), mass spectrum (C) and NMR (D) spectrum of the antifungal agent
produced by S. maxima
concentrations (From 100 to 5000 ppm) of S. maxima compared to the three tested commercial drugs; they
purified extract were tested and the mortality percentage showed average inhibition percentage of 48 %, 49% and
was estimated. The purified antifungal agent produced by 54%, respectively. Moreover, the most inhibited fungus
S. maxima was found to have a moderately toxicity level was P. oxalicum (91%) followed by R. solani (20%)
and the L.C of that agent (The concentration at which compared to the tested antifungal drugs; they showed
50
50% of the biomarker individuals die) was estimated from average inhibition percentage of 30% and 55%,
the best fit line obtained by using the profit analysis respectively. On the other hand, the produced marine
method, it was found to be 1027 mg/l microbial by product showed no activity against C.
Comparison of the Antifungal Activity of the Partially antifungal drugs; they showed average inhibition of 77%.
Purified Agents of S. Maxima to Some Commercial
Antifungal Drugs: The data obtained in Table (1) show
that the antifungal activities of the partially purified
antifungal agents produced by the S. maxima were presented in Figure (6) showed the UV-Vis, infra-red (IR)
estimated in comparison with three different commercial and Mass spectra of the purified antifungal agent. The UV
antifungal drugs including; Diflucan, Flocoral and spectrum of the compound (Figure 6A) resulted in a single
Fungican. It has found that S. maxima showed a broad peak appeared at 250 nm proved that the compound (B) is
spectrum of antifungal activity with an average activity of an aromatic compound. The IR spectrum showed five
26% against five tested human and plant pathogenic fungi absorption bands (Figure 6B); the first band appeared at
albicans ATCC 14053(0%) compared to the tested
Partial Characterization of the Purified Antifungal
Agent (A) of S. Maxima :The chemical characterization
H2N
HO
C
O
CH2OH
(CH2)9OCH 3
CH3
C
C
H
2
O
H
H2N
HO
C
O
CH2OH
(CH2)9
CH3
C
CH2OH
+
+
H2N
HO
C
O
CH2OH
(CH2)9C+
+
CH2OH
CH2
+
m/z = 38 3, 11%
m/z = 3 52, 11 %
m/z = 306, 13%
m/z =10 8, 100% base ion peak
m/z = 91,79%
Tropyli um cation
Global J. Pharmacol., 8 (2): 228-236, 2014
233
Fig. 7: The suggested chemical structure for the purified
antifungal agent produced from S. maxima
Table 1: Antifungal activities (%) of the partially purified antifungal of S.
maxima compared to some commercial antifungal drugs
Antifungal activity (%)
Antifungal --------------------------------------------------------------------------
Agent F. solani R. solani C. albicans A. niger P. oxalicum
S. maxima agent 20 10 0 10 91
Diflucan 55 51 78 26 28
Flucoral 57 52 80 27 29
Fungican 70 64 75 25 35
3426 cm due to (NH , OH, NH) groups, the second band
1
2
appeared at 2912 cm due to C-H aliphatic, the third band
1
appeared at 1657 cm due to carbonyl group (amide), the
1
fourth band appeared at 1413 cm due to benzene ring
1
and the last band appeared at 1029 cm due to ether
1
linkage. Moreover, the obtained Mass spectrum of this
compound (Figure 6C) showed the appearance of a
molecular ion peak at m/e = 383 which indicate the
molecular formula of (A) compound is 383 Dalton. The
proton NMR spectrum of the compound under
investigation (Figure 6D) showed signals at 0.9 (t, 3H,
CH ), 1.4 (t, 2H, CH ), 2.6 (s, 3H, CH ), 4.1 (s, 2H, CH ), 5.0
32 3 2
(s, 2H, NH ) and 7.6 (m, aromatic protons).
2
The suggested chemical structure was
presented in Figure (7) according to the obtained
molecular weight and the obtained spectrophotometric
results.
DISCUSSION
The algal extracts natural products may have potential
for the management of fungal diseases in sustainable
agriculture such as organic farming. In the past decades,
food scientists have been searching for natural
alternatives to replace synthetic antioxidants. The well-
studied carotenoids and phenolic compounds contribute
significantly to the antioxidant capacity of microalgae
[26].
The present study is an endeavor towards the
production of antifungal agents by the blue green alga
S. maxima. It has been screened for its biological
activity against different species of fungi and it was
found that it has high biological activity against
P. oxalicum.
A directed search for biologically active compounds
as well as their production by algal culture requires some
understanding of the culture conditions favoring their
production. Almost all of the biologically active
compounds of interest are secondary metabolites and
thus are usually most abundant in stationary phase or in
slow-growing cultures [27].
The algal isolate which was identified as S. Maxima
and grown on B.G- 11 medium and then screened for their
antifungal activities against some plant and human
pathogenic fungi, it was found that S. maxima, was most
potent acting against the pathogenic fungus, P. oxalicum,
the antifungal activity was 35%. The average of antifungal
activities was 7% against the used plant and human
pathogenic fungi.
Cosoveanu et al. [28] tested the biological activity of
Alaria esculenta, Fucus vesiculosus, Fucus sp. Spirulina
platensis and Ecklonia maxima was in vitro against
Fusarium roseum, F. oxysporum, Alternaria alternata, A.
dauci, A. longipes, Trichoderma viride, Botrytis cinerea,
Aspergillus niger, Penicillium expansum. Their potential
toxic effects were evaluated on mycelial growth. Results
are presented as effective concentration which inhibits
mycelial growth by 50% and 90%.
The current investigation initiated an optimization
program aimed to maximize the production of antifungal
agent produced by S. maxima and optimum conditions for
extraction. S.maxima was subjected to further
investigations in order to detect the culture conditions
under which they produced the highest antifungal
activity. Also, many investigators observed the great
relationship between the nutritional culture conditions
and antagonistic activities of microorganisms which affect
the production of the bioactive compounds [29,30].
Global J. Pharmacol., 8 (2): 228-236, 2014
234
The results indicate that S. maxima produced the The relationship between the production of antibiotic
antagonistic material optimally at the stationary phase
when it cultured under aerated conditions and reaches the
maximal values after 15 day of incubation. The results
th
obtained indicated that the antimicrobial activity was
higher in aerated than in static cultures and this results in
agreement with Piccardi et al. [31] who found high
antibacterial and antifungal activities produced from
Nostoc sp. when incubated in an orbital shaker flushed
with a mixture of (air : CO ,95 :5 V:V ).
2
Our results are in agreement with Bloor and Engl [32]
who found that the highest antimicrobial activity of N.
muscorum achieved by day 14 of the cultivation. In
addition, Gromov et al. [33] found that the antibiotic
cyanobacterium LU1 from Nostoc linckia is synthesized
throughout the growth cycle. Patterson and Bolis [34]
pointed out the production of tolytoxin in Scytonema
ocellatum is also unusual for a secondary metabolite in
that it is produced throughout the cell cycle. Oufdou et al.
[35] obtained that the extracellular and intracellular
products released by the cyanobacterium
Pseudanabaena sp. in the stationary growth phase,
reduced the survival of E. coli,Salmonella sp. S. aureus
and C. albicans.
Santoyo et al. [36] extracted the supercritical fluid and
fractionation of Spirulina platensis in order to obtain
functional extracts with antioxidant and/or antimicrobial
activities.
Results conducted also that the BG-11 medium
proved to be the best for the production of antifungal
agent. In such manner, Bloor and England [32] found that
BG-11 medium is the best medium for antibiotic
production from N. muscorum and they elucidated and
optimized the medium constituents of BG-11medium
constituents controlling antibiotic production from the
cyanobacterium N. muscorum. Piccardi et al. [31] found
that BG-11 medium is the best medium for antibiotic
production from 38 Nostoc strains.
Moreover, Abd El-Baky et al. [37] illustrated that the
enhancing process of phenolics synthesis in Spirulina
maxima grown in Zarrouk’s medium supplemented with
different concentration of NaNO and/or combined with
3
phenylalanine (L-PA). Their results revealed that the
levels of NaNO and L-PA in growth medium had positive
3
effects on the production of biomass (34-64 mg/day), total
phenolics (4.51-16.96 mg/g d.w) and flavonoids (1.32-5.12
mg/g d.w) contents. The highest levels of these
compounds were obtained in Zarrouk’s medium
containing 3.77 g/L NaNO and 100 mg/L L-PA.
3
or antagonistic substance and the producer isolate seem
to depend largely on pH value of the culture medium. So,
the optimum pH value for productivity and growth was 7.0
for our strain. Knubel et al. [38] have reported that, the
optimum pH for production of indocarbazoles by Nostoc
sphaericum was 7.8.
In the extraction step, it was found that DMSO was
the best solvent for extraction of the antifungal agent.
Kaushik and Chauhan [39] used hexane, ethyl acetate,
dichloromethane and methanol to obtain the phenolic
extracts from Spirulina platensis and they found, on
countrary, that the methanolic extracts had the best
results. Also, Vinay et al. [40] studied the effectiveness of
Spirulina platensis in different solvent (Petroleum ether,
chloroform, acetone, methanol) extract against three
dermatophytic fungi namely Aspergillus fumigatus,
A. niger, Candida albicans. The methanolic extract
only of S. platensis showed significant activity against
A. fumigatus. While, Cosoveanu et al. [28] observed that
the ethanolic algal extracts showed almost an antifungal
activity.
On the other side Medina-Jaritz et al. [41] evaluated
the antimicrobial activity of Arthrospira maxima different
concentrations of aqueous and methanolic extracts were
tested by the agar diffusion technique against Proteus
vulgaris, Staphylococcus aureus, Bacillus subtilis,
Escherichia coli and Candida albicans. The
aqueous extracts showed antibacterial activity against
all organisms tested, except to Bacillus subtilis,
while methanol extract showed antimicrobial activity
against all microorganisms, even to Staphylococcus
aureus.
Santoyo et al. [36] found the most active fraction
against all the microorganisms tested, was the one
collected in the second fraction in the experiment
performed at 220bar and 26.7 °C with 10% of ethanol.
They tested antimicrobial activities of microalgae extracts
against four different microbial species, including a gram
positive bacterium (Staphylococcus aureus), a gram
negative bacterium (Escherichia coli), a yeast (Candida
albicans) and a fungus (Aspergillus niger). Abd El-Baky
et al. [37] explained that the HPLC-DAD profile of all
phenolic extracts of Spirulina showed the presence of
large numbers of phenolic acids and flavonoids, in
variable levels. Gallic, chlorogenic, cinnamic, pinostrobin
and p-OH-benzoic were found as the most abundant
constituents among different extracts.
Global J. Pharmacol., 8 (2): 228-236, 2014
235
ACKNOWLEDGEMENT 11. Mo, S., A. Krunic, S.D. Pegan, S.G. Franzblau and
We appreciate Prof. Abd El-Metaal M.A. -Chemistry
Department, Benha University, Egypt, for his great effort
in the elucidation of the chemical structure of the
produced antifungal agent in this study.
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