Open Journal of Medicinal Chemistry, 2013, 3, 60-73
http://dx.doi.org/10.4236/ojmc.2013.33009 Published Online September 2013 (http://www.scirp.org/journal/ojmc)
Assessment of Secondary Metabolites from
Marine-Derived Fungi as Antioxidant
Nihad Abdel-Monem1, Ahmed M. Abdel-Azeem2, E. S. H. El Ashry3,
Doaa A. Ghareeb1, Asmaa Nabil-Adam4*
1Department of Biochemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
2Laboratory of Systemic Mycology, Department of Botany, Faculty of Science, Suez Canal University, Ismailia, Egypt
3Department of Organic Chemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
4Marine Environmental Division, National Institute of Oceanography and Fisheries (NIOF)
Marine Biotechnology and Natural Product (MBNP), Alexandria, Egypt
Received May 2, 2013; revised June 1, 2013; accepted June 15, 2013
Copyright © 2013 Nihad Abdel-Monem et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Marine derived fungi are considered as a promising source of novel drugs due to their biodiversity and consequent
chemo-diversity. Although marine microorganisms especially fungi are not well defined taxonomically, making this a
promising frontier for the discovery of new medicines. This study focused on marine derived fungi as a model for bio-
active exploration for new entities with anti-inflammatory and antioxidant capacity. Three in-vitro assays were used to
investigate the bioactive antioxidant potentiality of fungal extracts. Thiobarbituric acid (TBARS), α,α-Diphenyl-ß-
picrylhydrazyl (DPPH) and NO assay are based on their total phenolic and flavonoid content of each extract group. Ch.
globosum recorded the highest antioxidant activity (92.82%) in TBARS assay, while G. dankaliensis came first by re-
cording 59.28% in DPPH assay in comparison with ascorbic acid (61.83%). In NO inhibition assay, N. oryzae showed
49.3% comparing with ascorbic acid (73.12%). From the preliminary result of our extracts, we can consider the marine
derived fungi extracts as promising antioxidant and anti-inflammatory drug candidate.
Keywords: DPPH; TBARS; Marine Drugs; Marine Derived Fungi; Anti-Inflammatory; Antioxidant Capacity
1. Introduction
Reactive oxygen species (ROS) or free radicals may be
designated as molecular sharks that damage molecules in
cell membranes, mitochondria, DNA and are very unsta-
ble, tend to strip electrons from the molecules in the im-
mediate surroundings in order to replace their own losses.
Reactive oxygen species (ROS) is a collective term,
which includes not only the oxygen radicals (O2•−, and
•OH) but also some non-radical derivatives of oxygen.
These include hydrogen peroxide (H2O2), hypochlorous
acid (HOCl) and ozone (O3) [1].
ROS and free radical-initiated reactions are ascer-
tained to play multiple roles in degenerative or patho-
logical events such as aging, cancer, heart dysfunction
and Alzheimer’s disease [2]. Over about 100 disorders
like rheumatoid arthritis, hemorrhagic shock, cardiovas-
cular disorders, cystic fibrosis, metabolic disorders, neu-
rodegenerative diseases, gastrointestinal ulcerogene-
sis and AIDS have been reported as ROS mediated [3].
The oxidative damage to DNA, proteins and other
cellular determinants seems inevitable when the concen-
tration of ROS exceed tolerability of cells.
The free radical nitric oxide (NO) is also a highly re-
active free radical capable of mediating a multitude of
reactions [4] NO, is an uncharged molecule containing an
unpaired electron in its outermost orbital, allowing it to
undergo several reactions functioning either as a weak
oxidant (electron donor) or as an anti-oxidant (electron
acceptor). Also, it is able to react with other inorganic
molecules (i.e. oxygen, superoxide or transition metals),
structures in DNA (pyrimidine bases), prosthetic groups
(heme) or with proteins leading to S-nitrosylation of thiol
groups, nitration of tyrosine residues or disruption of me-
tal-sulfide clusters such as zinc-finger domains or iron-
sulfide complexes [5].
Moreover, free radicals are known to take part in lipid
peroxidation in foods, which is responsible for rancid
odors and flavors, which decrease the nutritional quality.
*Corresponding author.
C
opyright © 2013 SciRes. OJMC

N. ABDEL-MONEM ET AL. 61
Therefore, synthetic antioxidants such as butylated hy-
droxyanisole (BHA), butylated hydroxytoluene (BHT)
and tert-butyhydro-quinone (TBHQ) are widely used in
the food industry as potential inhibitors of lipid peroxida-
tion. However, previous studies have demonstrated that
BHA and BHT accumulate in the body and result in liver
damage and carcinogenesis [6-9]. An inflammatory re-
sponse implicates macrophages and neutrophils, which
secrete a number of mediators (eicosinoids, oxidants,
cytokine and lytic enzymes) responsible for initiation,
progression and persistence of acute or chronic state of
inflammation [10]. NO, along with superoxide (O2•−) and
the products of their interaction, initiates a wide range of
toxic oxidative reactions causing tissue injury [11].
The role of ROS/RNS in inflammation is clearly
demonstrated by the anti-inflammatory effects of the
antioxidants. Niric oxide synthase inhibitors are also ef-
fective as anti-inflammatory agents in carrageenan-in-
duced rat paw odema method as SOD. These may be due
to the removal of O2•− by SOD, so preventing O2•− de-
pendent formation of a factor chemotactic for neutrophils
[12].
Likewise, the neutrophils also produce oxidants and
release granular constituents comprising of lytic enzymes
performing important role in inflammatory injury [13].
Reactive oxygen intermediates (ROI) are believed to be
mediators of inflammation and responsible for the pa-
thogenesis of tissue destruction in rheumatoid arthritis
[14].
The antioxidants could attenuate this oxidative damage
of a tissue indirectly by increasing cells’ natural defenses
[15,16], and/or directly by scavenging the free radical
species [17]. In order to reduce the damage of ROS to the
human body, antioxidants are commonly used in proc-
essed foods. Antioxidants could alleviate the oxidative
damage of a tissue indirectly by increasing cells’ natural
defenses and directly by scavenging the free radical spe-
cies [9,16,17].
The potent biological activity of many marine natural
products is of relevance for their ecological function but
is also the basis of their biomedical importance [18]. As a
slightly opened reservoir of special bio-resources, some
marine microorganisms have demonstrated to be excel-
lent producers of biologically active and/or structurally
novel metabolites [19]. Marine organisms, particularly
marine fungi, are well-known for their production of uni-
que biologically active metabolites. Among marine fungi,
those living in association with algae, represent a rich
source of novel antioxidant products. The association be-
tween algae and fungi is called “Algicolous”.
Antioxidants protect cells against the damaging effects
of reactive oxygen species (singlet oxygen, superoxide,
peroxyl radicals, hydroxyl radicals and peroxynitrite) and
help us to fight diseases like artherosclerosis, dementia
and cancer. In addition, they may be useful as therapeu-
tics or food additives [20]. For that, the finding of new
antioxidative leads (marine-derived fungi), which are
capable of preventing and/or eliminating oxidative stress
and the development of suitable screening bioassays mer-
its, consequently, have high priority and would be very
welcomed [21-23].
2. Materials and Methods
2.1. Marine Sample
Samples of honeycomb sponge (Hippospongia communis)
were collected from the west coast of Alexandria to
Libya borders (Sidi Krir area to El-Salloum by dragging
ships). While, Ascidians samples (Styela canopus) and
seaweeds samples were collected monthly from Anfoshi
Bay as shown in Figure 1.
2.1.1. Isolation of Sponge-Derived Fungi
To get rid of nonspecific fungal propagules from sea-
water column on sponge and jellyfish surfaces, animal
tissues were rinsed three times with sterile seawater. The
surface of the sample was disinfected with 70% ethanol
for 2 minutes. The inner tissue was taken out with a
scalpel and forceps and then cut into small cubes approx.
0.5 cm3. A total of 15 - 20 cubes of each sample were
placed on isolation media.
All isolation and culture maintaining media for marine
taxa were prepared by sea water (SW) and isolation me-
dia basically were supplemented with Rose bengal
(1/15,000) and chloramphenicol (50 ppm) for suppres-
sion of bacterial growth. Five media were adopted for
isolation after Atlas (2004) they were: Sea Water Rose
bengal Chloramphenicol Agar (SWRCA), Sea Water
Czapeks Yeast Extract Agar (SWCYA), Sea Water Oat-
meal agar (SWOA), Sea Water Agar (SWA), and Sea
Water Potato Dextrose Agar (SWPDA).
For maintaining cultures and for proper identification,
pure cultures of isolated fungi were grown on standard
media such as Vegetable Agar (V8), Oatmeal Agar (OA),
Malt Extract Agar (MEA) Potato Dextrose Agar (PDA)
and Potato Carrot Agar (PCA).
2.1.2. Identification of Isolates
Taxonomic identification using morphology characteris-
tics of fungal isolates down to the species level on stan-
dard media was mainly based on the following identifi-
cation keys: [24] Pitt (1980) [25] for Penicilliu m (on
Czapek Yeast Extract Agar (CYA) & Malt-Extract Agar
(MEA)); Raper & Fennell (1965) [26] for Aspergillus (on
Czapek Agar (CZ)); Ellis (1971, 1976) [8,27] for dema-
tiaceous hyphomycetes (Potato Carrot Agar (PCA));
Booth (1971) [29] for Fusarium (Potato Dextrose Agar
(PDA)); Arx (1981) [30], Domsch et al. (2007) [31],

N. ABDEL-MONEM ET AL. 62
Figure 1. The location of sample collection.
Watanabe (2002) [32] for miscellaneous fungi (on MEA,
PDA, CYA); Arx et al. (1986) [33] and Cannon (1986)
[34] for Chaetomium (Oat Meal Agar + Lupin Stem (OA
+ LUP)). The systematic arrangement follows the latest
system of classification appearing in the 10th edition of
Ainsworth and Bisby’s Dictionary of the fungi (Kirk et al.
2008) [35] and Species Fungorum web site (http://www.
speciesfungorum.org/Names/Names.asp).
2.2. Preparation of Marine Fungus Extract
Preparative-scale production (0.5 L) was carried out in 1
L Erlenmeyer flasks contained potato dextrose extract
(Difco) for 2 weeks at 28˚C in a shaking incubator at 102
rpm. Pellets were homogenized and centrifuged by using
cooling centrifuge at 8000 rpm for 2 min at 4˚C Resul-
tant mixtures were extracted with ethyl acetate (1 × 50
ml), the organic fractions were combined, and the solvent
removed at reduced pressure and 35˚C. Residues were re-
dissolved in DMSO for further bioassay. The steps of
isolation and extraction of fungi and secondary metabo-
lites are shown in Figure 2.
2.3. Biochemical Profiling for Fungal Extract
2.3.1. Determination of Total Phenolic Content in
Different Fungal Species Extracts
Total phenolic compounds in the fungal extracts were
determined by the method of Taga et al. (1984) [36].
2.3.2. Determination of Total Flavonoid Content in
Different Fungal Species Extracts
Total flavonoid content was determined by a colorimetric
method of Zhishen et al. (1999) [37].
2.3.3. Diphenyle α-α-Picrylhydrazyl (DPPH) Radical
Scavenging Effect of Different Fungal Species
Extracts
DPPH radical scavenging assay of the total extract was
performed by using modified previously established me-
thodology by Blois (1958) [38] and Amarowicz et al.
(2000) [39].
% scavenging = [(A control − A sample]/A control ×
100
2.3.4. Determination of Thiobarbituric Acid Reactiv e
Substance M ethod Usi ng TBAR S Assay for
Different Fungal Species Extracts
The method used was adapted from Wallin et al. (1993)
[40] and modified by Fisch (year).
% scavenging = [(A control − A sample]/A control ×
100.
2.3.5. Chr omatogr aphy
20 µl sample extract analyzed with an eclipsed XDB C18

N. ABDEL-MONEM ET AL. 63
Figure 2. Summarize the production of secondary metabo-
lites from marine deri ved fungi.
(5 µm, 4.6 × 150 mm) column using a mobile phase
consisting 1% (v/v) formic acid in aqueous solution:
acetonitrile: 2-propanol (70:22:8), pH 2.5; flow rate: 0.75
ml/min, temperature: 30˚C, UV detection at 320 nm;
Agilent technologies 1200 series [41].
3. Result
The total phenolic and flavnoids content in different fun-
gal extracts.
The highest total phenolic content goes to G. danka-
liensis while total flavnoids content the higher activity
recorded belong to Engyodontium album.
3.1. Result of Lipid Preoxidation Inhibition
Using TBARS Assay in Different Fungal
Extracts
The higher activity in the inhibition of lipid preoxidation
assay belongs to Chaetomium globosum and the lowest
activity goes to G. dankaliensis.
3.2. Chemical Composition in Different Fungal
Extract
The result of chemical copostion showed that G. danka-
liensis and Trichurus spiralis had the highest sulfur ratio
than the other three fungal extracts.
3.3. Result of Antioxidant Capacity Using DPPH
Assay in Different Fungal Extracts
Comparing to Ascorbic Acids
The result of total antioxidant by DPPH assay show that
G. dankaliensis came first by recording 59.28% in DPPH
49.3% comparing with ascorbic acid (73.12%).
3.4. Result of NO Inhibition Assay in Different
Fungal Extracts Comparing to Ascorbic
Acids
The result of total anti-inflammatory by NO assay show
that C. globosum have the highest activity from 1 mg
concentration up to 5 mg ,where at 6 mg the highest
activity goes to Nigrospora oryzae.
3.5. Determination of Total Gallic Acid Content
by HPLC
The result of total gallic content in 10 mg show that the
highest concentrations belong to Engyodontium album.
While gallic acid concentration in C. globosum at 10 mg
recorded zero content.
4. Discussion
In recent decades an increasing tendency towards the use
of natural substances instead of synthetic ones has been
obtain comparison to natural substances, it will take long
time for them to complete their natural cycles and return
to nature; thus causing a lot of environmental pollution.
Also with the increase in the price of raw materials, the
problem of cost benefits for chemical production is be-
coming more considerable. Natural antioxidants are gin-
ning importance, due to their benefits for human health,
decreasing the risk of degenerative diseases by reduction
of oxidative stress and inhibition of macromolecular
oxidation [42-44]. Their uses preserving food additives
[45].
The antioxidant field receives increased interest and
currently about 10,000 articles per year concern antioxi-
dants of various kinds (ISI Web of Knowledge). Oxida-
tion reactions are a major concern for the food industry,
and antioxidants are widely needed to prevent oxidative
changes in food. But the need for antioxidants for use in
other oxidisable goods, such as cosmetics and pharma-
ceuticals is also on the rise.
Polyphenolic compounds are widely distributed in
plant kingdom [46], reports indicate that there is a posi-
tive relation between total phenolic content and antioxi-
dant activity in many plant species [47]. Phenolic com-
pounds in plants are viewed as powerful in vitro antioxi-
dants due to their ability to donate hydrogen or electrons
and to form stable radical intermediates [48]. For small
marine antioxidants novel discoveries are increasingly
reported [49,50]. Interestingly, antioxidants found in ter-
restrial plants (catechin, gallic acid, catechol, morin, ru-
tin) were reported present, inhibit reactive oxygen spe-
cies (ROS) generation through anti-inflammatory effects
[51], and that with agreement with our result as in Fig-
ureas 3-7 using DPPH assay as a model for oxidative
stress our extracts show antioxidant activity with very
low concentration range from 1 mg to 6 mg, as well as
scavenge free radicals as in Figures 8-11 as well as lipid
preoxidation inhibition using TBARS inhibition model as
in Figure 12 [51,52].
Our study show that high inhibition activity comparing

N. ABDEL-MONEM ET AL. 64
Figure 3. Show that the gallic acid content in different fungal extract by HPLC.
Figure 4. HPLC chromatogram shows the result of gallic acid in G. dankaliensis.
Figure 5. HPLC chromatogram shows the result of gallic acid in Nigrospora oryzae.

N. ABDEL-MONEM ET AL. 65
Figure 6. HPLC chromatogram shows the result of gallic acid in Engyodontium album.
Figure 7. HPLC chromatogram shows the result of Gallic acid in Chaetomium globosum.
Figure 8. Show the DPPH inhibition assay by G. dankaliensis.

N. ABDEL-MONEM ET AL. 66
Figure 9. Show the DPPH inhibition assay by Nigrospora oryzae.
Figure 10. Show the DPPH inhibition assay by Engyodontium album.
with ascorbic acids and regard to low concentration used
in our study, this activity may be attributed to the high
content of total phenolic and flavnoids which act as ex-
cellent antioxidant, as shown in Figures 13 and 14. where
the best-described property of almost every group of fla-
vonoids is their capacity to act as antioxidants. Flavonoids

N. ABDEL-MONEM ET AL. 67
Figure 11. Show the DPPH inhibition assay by C. globosum.
Figure 12. The inhibition of lipid preoxidation of different fungal extracts.
are oxidized by radicals, resulting in a more stable,
less-reactive radical. In other words, flavonoids stabilize
the reactive oxygen species by reacting with the reactive
compound of the radical. Because of the high reactivity
of the hydroxyl group of the flavonoids, radicals are
made inactive, according to the following equation; Fla-
vonoid (OH) + R• > flavonoid (O•) + RH [53], and that
can explain the antioxidant apacity of our extracts, due c

N. ABDEL-MONEM ET AL. 68
Figure 13. Total phenolic content in different fungal extr act.
Figure 14. Total flavnoids content different fungal extracts.
to their flavnoids content. also the scavenger of nitric
oxide (anti-inflammatory effect) show in Figures 15-18
can be attributed to the total phenolic and flavnoids con-
tent, as The same in case of scavenger of nitric oxide as
nitric oxide reacts with free radicals, thereby producing
the highly damaging peroxynitrite. Nitric oxide injury

N. ABDEL-MONEM ET AL. 69
Figure 15. Show the inhibition of NO by G. dankaliensis fungal extract.
Figure 16. Show the inhibition of NO by Nigrospora oryzae fungal extract.
takes place for the most part through the peroxynitrite
route because peroxynitrite can directly oxidize LDLs,
resulting in irreversible damage to the cell membrane.
When flavonoids are used as antioxidants, free radicals
are scavenged and therefore can no longer react with
nitric oxide, resulting in less damage [54]. Interestingly,
nitric oxide can be viewed as a radical itself, and it is was
reported that nitric oxide molecules are directly scav-

N. ABDEL-MONEM ET AL. 70
Figure 17. Show the inhibition of NO by Engyodontium album fungal extract.
Figure 18. Show the inhibition of NO by C. globosum fungal extract.
enged by flavonoids [55].
Although we can attributed the antioxidant activity and
anti-inflammatory effect of different fungal extracts, this
may be due to the marine chemodiversty, which is also
heightened by their composition of sea water itself where,
concentration of halides in sea water of 1900 mg/L

N. ABDEL-MONEM ET AL. 71
Figure 19. Show % of C, N, H, S in different fungal extract where C: carbon, H: hydrogen, N: nitrogen, S: sulphur.
Cl−, 65 mg/L Br−, 5 × 10−4 and I/IO3− are reflected by the
number of compounds incorporating these elements and
sulfated compounds can accounted for by the relatively
high concentration of sulfur, 2700 mg /L seawater, and
that confirmed in our study as shown in Figure 19 [56] .
5. Conclusion
In conclusion, we observed beneficial influence of inves-
tigated fungal extracts as antioxidants and anti-inflam-
matory. We could assume that this extracts possess anti-
inflammatory property due to their proven antioxidant,
free radical scavenging properties, and their high sulfur
content. However, further investigations on the structures,
of the bioactivity, in the four different fungal extracts, as
well as elucidation of compounds responsible for such
activities and their effect on different antioxidant appli-
cation should be carryied on.
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