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Original Article
Protective Effect of Enzymatic Extracts from Microalgae
Against DNA Damage Induced by H
2
O
2
Rohan Karawita,
1
Mahinda Senevirathne,
2
Yasantha Athukorala,
1
Abu Affan,
3
Young-Jae Lee,
4
Se-Kwon Kim,
5
Joon-Baek Lee,
3
You-Jin Jeon
1,6
1
Faculty of Applied Marine Science, Cheju National University, Jeju 690–756, South Korea
2
Department of Food Science and Engineering, Cheju National University, Jeju 690–756, South Korea
3
Department of Oceanography, Cheju National University, Jeju 690–756, South Korea
4
Deparment of Veterinary Science, Cheju National University, Jeju 690–756, South Korea
5
Department of Chemistry, Pukyong National University, Busan 608–737, South Korea
6
Radiological Science Research Institute, Cheju National University, Jeju 690–756, South Korea
Received: 17 September 2006 / Accepted: 5 March 2007 / Published online: 23 May 2007
Abstract
The enzymatic extracts from seven species of micro-
algae (Pediastrum duplex, Dactylococcopsis fascicula-
ris, Halochlorococcum porphyrae, Oltmannsiellopsis
unicellularis, Achnanthes longipes, Navicula sp. and
Amphora coffeaeformis) collected from three habi-
tats (freshwater, tidal pool, and coastal benthic) at
Jeju Island in Korea were investigated for their anti-
oxidant activity. Of the extracts tested, the AMG 300
L(anexo1,4-a-D-glucosidase) extract of P. duplex, the
Viscozyme extract of Navicula sp., and the Celluclast
extract of A. longipes provided the most potential as
antioxidants. Meanwhile, the Termamyl extract of
P. duplex in an H
2
O
2
scavenging assay exhibited an
approximate 60% scavenging effect. In this study, we
report that the DNA damage inhibitory effects of
P. duplex (Termamyl extract) and D. fascicularis
(Kojizyme extract) were nearly 80% and 69% respec-
tively at a concentration of 100 mg/ml. Thus, it is
suggested that the microalgae tested in this study
yield promising DNA damage inhibitory properties on
mouse lymphoma L 5178 cells that are treated with
H
2
O
2
.Therefore,microalgaesuchasP. du ple x may be
an excellent source of naturally occurring antioxidant
compounds with potent DNA damage inhibition
potential.
Keywords: antioxidant activity — DNA damage —
enzymatic extracts — H
2
O
2
— microalgae —
scavenging activity
Introduction
Exogenous chemicals and endogenous metabolic
processes in the human body or food systems may
produce highly reactive oxygen species (ROS). As an
example, during respiration, nearly 5% of the inhaled
O
2
is converted to such ROS as superoxide anion
(O
2), hydrogen peroxide (H
2
O
2
), and the hydroxyl
radical (OH). Oxidative stresses can result even in the
presence of nonenzymatic and enzymatic protective
body mechanisms against ROS. Oxidative stresses
can create such harmful effects such as cardiovascu-
lar dysfunction, neurodegenerative disease, gastrodu-
odenal pathogenesis, and metabolic dysfunctions for
almost all vital organs, cancers, and even premature
aging. In addition, an ROS that has a very short life
span but high chemical reactivity can lead to lipid
peroxidation and the oxidation of DNA and proteins
(Mate
´sandSa
´nchez-Jime
´nez, 1999). Increased lipid
peroxidation and decreased antioxidant protection
frequently occur, after which the resulting epoxides
may spontaneously react with nucleophilic centers
within cells, thereby covalently binding to DNA,
RNA, and proteins (Mate
´setal.,1999). Single-strand
breaks, and to a lesser extent double-strand breaks,
alkali labile sites, and various species of oxidized
purines and pyrimidines are evidence that cells have
Present address: A. Affan, Korea Ocean Research and Develop-
ment Institute (KORDI), Ansan P.O. Box 29, Seoul 426–744,
Republic of Korea
Correspondence to: Joon-Baek Lee; E-mail: jblee@cheju.ac.kr;
You-Jin Jeon; E-mail: youjinj@cheju.ac.kr
DOI: 10.1007/s10126-007-9007-3 &Volume 9, 479–490 (2007) &*Springer Science + Business Media, LLC 2007 479
been exposed to oxidative insults (Singh et al., 1990;
Rueff et al., 1993).
Both natural occurring and synthetic antioxi-
dants reportedly prevent oxidative damage by free
radicals and ROS. When the antioxidant protection
mechanisms become unbalanced by such exogenous
factors as tobacco smoke, ionizing radiation, and
certain pollutants plus endogenous factors of nor-
mal aerobic respiration, stimulated polymorph nu-
clear leukocytes and macrophages and peroxisomes
may occur and may be responsible for the afore-
mentioned disease states and acceleration of aging.
Antioxidant supplements or foods containing anti-
oxidants, however, may be preferable in helping the
body reduce oxidative damage instead of the com-
mercially available antioxidants. This is especially
the case because the latter are suspected of causing
liver damage and carcinogenesis in some laboratory
animals. Therefore, the identification, development,
and utilization of more effective naturally occurring
antioxidants are desirable.
Marine algae generate 46% of the world_s net
primary production (produce carbohydrate through
the process of photosynthesis) and supporting food
webs from ponds to oceans (Field et al., 1998). Marine
microalgae have been reported to be valuable new
sources of pharmacologically active compounds.
However, their metabolites have not been studied
extensively because of the difficulties involved in the
isolation and cultivation of microalgae. Regardless,
nutritional supplements obtained from the micro-
algae have been a primary focus of microalgal
biotechnology for many years. Marine algae produce
primary and secondary metabolites that have unique
chemical structures with highly specific bioactivities
including antibiotics and toxins as secondary metab-
olites. In addition, most algae are exposed to sunlight
in aerobic conditions, an environment expected to be
highly oxidative to their metabolites, especially to
unsaturated fatty acids. Therefore, marine algae must
have evolved a protective mechanism to preserve and
protect their biomembranes from ROS.
Several microalgal species of Chlorella, Spirulina,
and Dunaliella, are grown commercially, and their
products include b-carotene and phycocyanin. The
antioxidative activity of phycocyanobilin from
Spirulina platensis was evaluated against oxidation
of methyl linoleate in a hydrophobic system or using
phosphatidylcholine liposomes (Hirata et al., 2000).
Phycocyanobilin effectively inhibited peroxidation of
the methyl linoleate, which was characterized by a
prolonged induction period. Aphanizomenon flos-
aquae (Cyanophyta) are reported to be rich in phyco-
cyanin, a photosynthetic pigment with antioxidant
and antiinflammatory properties (Benedettia et al.,
2004). Therefore, the microalgae may have promise as
future sources for the extractable biochemical com-
pounds for the pharmaceutical industry. Natural water
resources, tidal pools, and coastal areas are known to
contain a diverse microalgal community. Pediastrum
duplex (Chlorophyceae) and Dactylococcopsis
fascicularis (Cyanophyceae) are examples of what
can be found in a natural freshwater source. Halo-
chlorococcum porphyrae (synonyms: Chlorochytrium
porphyrae) and O. unicellularis, which belong to the
Chlorophyceae, are found in marine coastal habitats
(especially tidal pools) and brackish water. Benthic
diatoms such as Achnanthes longipes, Navicula sp.,
and Amphora coffeaeformis (Bacillariophyceae) are
often found in a high abundance in coastal waters.
No data on antioxidant effects, especially regarding
DNA damage in these microalgae, are available, based
on a search of the published literature. The objectives
of this work were to determine the potential antiox-
idant activity of enzymatic extracts of microalgae
obtained from several different habitats on preventing
the oxidative damage to mouse lymphoma L5178 cell
DNA induced by exposure to H
2
O
2
.
Materials and Methods
Carbohydrases such as Viscozyme L (a multienzyme
complex containing a wide range of carbohydrases,
including arabanase, cellulase, b-glucanase, hemi-
cellulase, and xylanase), Celluclast 1.5 L FG (cata-
lyzing the breakdown of cellulose into glucose,
cellobiose, and higher glucose polymers), AMG 300
L (an exo 1, 4-a-D-glucosidase), Termamyl 120 L (a
heat-stable a-amylases), Ultraflo L (a heat-stable
multiactive b-glucanase), and five different pro-
teases including Protamax (hydrolysis of food pro-
teins), Kojizyme 500 MG (to boost soy sauce
fermentation), Neutrase 0.8 L (an endoprotease),
Flavourzyme 500 MG (containing both endopepti-
dase and exopeptidase activities), and Alcalase 2.4 L
FG (an endoprotease) were obtained from the Novo
Co. (Novozyme Nordisk, Bagsvaed, Denmark).
Linoleic acid, 2-thiobarbituric acid (TBA), trichloro-
acetic acid (TCA), butylated hydroxytoluene (BHT),
a-tocopherol, 1,1,3,3-tetraethoxypropane, Na
2
EDTA,
agarose, and H
2
O
2
were purchased from the Sigma-
Aldrich (St Louis, MO). All other chemicals used for
these studies were of 99% or greater purity.
Microalgae Sample Collection and Isolation.
Freshwater microalgal samples were collected
directly into 1-L plastic bottles from different sites of
the Moo-Soo stream on Jeju Island off the (region)
Korean coast. The prevailing environmental factors of
water temperature and pH were measured and
480 ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE
recorded at the sampling site. Thereafter, the samples
were transferred to a flask containing artificial
seawater enriched with F/2 nutrients (Aquacenter,
Leland, MS). The F/2 enrichment solutions were
added at 1 ml per 7.75 ml artificial seawater for the
A and B solutions, together with 13 ml of a trace
element solution. The flasks were then transferred
immediately into plant growth chamber (Vision, VS-
3D, Korea) maintained at temperatures of 20, 25, and
30-C, where they were kept for 3 days to enable
observation of growth performance; the pH was
measured after 3 days. A 1- mL volume of the
incubated sample was transferred to the S-R
chamber for the observation of phytoplankton abun-
dance and community changes via an inverted mi-
croscope. Thereafter, single cells of P. duplex and D.
fascicularis were picked up using a clean/sterile mi-
cropipette and transferred into 12-well plates for s-
ubculture in autoclaved freshwater that had been
filtered through 0.45-mm Millipore filter paper and
enriched with the F/2 medium nutrient, the trace
metal solution and soil. The isolation process was r-
epeated until a monostrain was obtained.
The tidal pool samples were collected from
small tidal pools that were located atop volcanic
rocks at the Gangjung tidal zones of the Jeju Island
coast, Korea. The environmental factors and the
growth performance of the phytoplankton were ob-
served as mentioned in the preceding text, but the
samples were incubated for 1 week at 15, 20, and 30-C
with salinity set at 25, 30, and 35 psu for a 12 h:
12 h (light:dark cycle) to determine the optimal temper-
ature for improved growth. Phytoplankton samples
were observed under a phase-contrast microscope for
isolation and identification. The monostrain samples
from the different tidal pools were identified as Halo-
chlorococcum porphyrae and Oltamannsiellopsis
unicellularis based on the monograph descriptions by
Tom a s ( 1997).
The benthic diatoms were collected from
National Fisheries Research and Development
Institute (NFRDI) at Jeju Island. The environmen-
tal conditions were observed and the benthic
diatom isolation procedures were performed as
described by Affan et al. (2006). Briefly stated, the
diatoms were removed from the papan using a
brush and diluted seawater. A 1-ml diluted sample
volume was transferred to an S-R counting chamber,
and single diatom cells were then micropipetted
from the counting chamber under an inverted
microscope (Olympus IX71). Thereafter, each cell
was transferred to a multiwell plate for subculture in
autoclaved seawater enriched with F/2 medium nu-
trient, trace metals, and metasilicate anhydrous
crystals (Na
2
SiO
3
). The isolation process was per-
formed until a monostrain was obtained. The
monostrain benthic diatoms were observed under a
phase-contrast Zeiss Axioplan microscope (Carl
Zeiss, Oberkochen, Germany) at 400x magnification.
The monostrain samples identified as Achnanthes
longipes, Amphora coffeaeformis,andNavicula sp.
were again streaked onto an agar plate of 2% agar
(wt/vol), 0.04% F/2 (vol/vol) nutrient medium, and
the autoclaved seawater. The monostrain colony was
subsequently transferred to a 250-ml flask containing
100 ml of F/2-enriched culture medium and various
antibiotics. Seven different dosages of an antibiotic
cocktail were used in this study (penicillin 100 to
250 U/ml, streptomycin 100 to 250 mg/ml, and
neomycin 200 to 500 mg/ml). Doses were increased
by increments of 25 U of penicillin/ml, 25 mgof
streptomycin/ml, and 50 mg neomycin/ml (P 4083;
Sigma-Aldrich). The cultured samples were again
streaked on the Bacto-agar media to observe for any
existing bacteria. This stepwise procedure enabled
the collection of an axenic strain of benthic diatoms
for further study.
Mass Culturing. The mass culture of freshwater
algal strains was achieved with F/2 nutrient media,
10% soil extract, and distilled water. The culture
conditions maintained included temperature, pH, light
intensity, and a light:dark cycle of 25-C, 8.0, 180 mmol
photons m
-2
s
-1
, and 12:12 for the P. du p lex and 30-C,
8.5, 180 mmol photons m
-2
s
-1
and 12:12 for the D.
fascicularis, respectively. The microalgal culturing
was performed in 20-L polycarbonate transparent
bottles (Nalgene, Rochester, NY). After a 2-week
culture period the biomass of the mass culture was
filtered from the medium using Advantec filter paper
(Tokyo, Japan). The subsequent collected biomass
was transferred to a Petri dish and stored in a freezer
at -70-C for 24 h. The sample was then freeze-dried
at -50-C at 5 Torr and weighed.
The mass culture of the tidal pool microalgal strain
was performed with an artificial seawater media that
was prepared with F/2 media nutrients, a trace element
solution, and with the same type of culture bottles
described previously. Mass culture conditions were
maintained with salinity, temperature, pH, a light:dark
photo cycle, and light intensity of 20-C, 35 psu, 8.20,
12:12, and 180 mmol photons m
-2
s
-1
,forH. porphyrae
and 25-C, 30 psu, 8.30, 12:12, and 180 mmol photons
m
-2
s
-1
for O. unicellularis, respectively. The standing
crop of the mass culture was separated by following
the same methods outlined previously.
The mass culture of benthic diatoms was con-
tinued for 2 weeks in the same type of bottle as
mentioned in the preceding text. The culture medi-
um that was used was the same as that used during
ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE 481
the isolation of the benthic monostrains. Mass
culture conditions were maintained with salinity,
temperature, pH, a light:dark photo cycle, and
fluorescent light intensity of 30 psu, 25-C, 8, 12:12,
and 180 mmol photons m
-2
s
-1
, respectively. The
cultured phytoplankton biomass was separated by
following the same methods as mentioned in the
preceding text.
Proximate Composition. Proximate chemical com-
position of the freeze-dried samples was determined
according to AOAC methods (1995). Crude lipid
content was determined by the Soxhlet method and
crude protein by the Kjeldahl method. Ash content
was determined by calcinations at furnace temper-
atures of 550-C and the moisture content deter-
mined after storage in a drying oven at 105-C for 24 h.
The polysaccharide content was determined using
the phenol-sulfuric method.
Preparation of Enzymatic Extracts of the Microalgae.
Freeze-dried microalgae (1 g) were ground into a fine
powder and mixed with 100 ml of distilled water. The
optimum pH of each reaction mixture was adjusted
using 1 M HCl or NaOH. The optimal pH and
temperature conditions for the respective enzymes
used were according to previous literature citations
(Heo et al., 2005). In this study, the five carbohydrase
preparations (Viscozyme, Celluclast, AMG, Termamyl,
and Ultraflo) and the five proteases (Protamax,
Kojizyme, Neutrase, Flavourzyme, and Alcalase) were
used to hydrolyze the microalgal samples and to obtain
their enzymatic extracts. The ratio of enzyme to
substrate was set at 1:100. The mixtures were placed
in a shaking incubator for 24 h. The mixtures that
resulted were filtered and the enzymatic activities of the
hydrolysates inactivated by heating (100-Cfor10min).
Finally, each hydrolysate was adjusted to pH 7 with 1 M
HCl or NaOH and a concentration of 2 mg/ml. All
enzymatic extract activities were compared to commer-
cially available antioxidants (BHT and a-tocopherol)
that were dissolved in methanol.
Hydrogen Peroxide Scavenging Assay. The
hydrogen peroxide scavenging assay was
conducted according to the method described by
Muller (1995). Enzymatic extracts of microalgae
(80 ml) and 20 ml of 10 mM hydrogen peroxide were
mixed with 100 ml of phosphate buffer (0.1 M, pH
5.0) in a 96-microwell plate and incubated at 37-C
for 5 min. Thereafter, 30 ml of a freshly prepared
1.25 mM ABTS and 30 ml of peroxidase (1 U/ml)
were mixed and incubated at 37-C for 10 min and
the absorbance was then measured via a microplate
reader at 405 nm. The scavenging ability of an
extract was expressed as a percentage of the control
(PBS instead of the extract).
Antioxidant Effects in a Hemoglobin-Induced
Linoleic Acid System. The antioxidant activity was
determined via the thiocyanate method recorded
by Kuo et al. (1999) with a slight modification. The
microalgae sample (0.1 ml) was mixed with 0.025 ml
of 0.1 M linoleic acid-ethanol and 0.075 ml of 0.2 M
phosphate buffer (pH 7.2). Autoxidation was initiated
by the addition of 0.08% hemoglobin (0.05 ml). After
incubation at 37-C for 60 min, the lipid peroxidation
process was stopped by addition of 5 ml of 0.6% HCl-
ethanol. Thereafter, 0.02 ml of 20 mm FeCl
2
and
0.01 ml of 30% ammonium thiocyanate were added
and the peroxidation value of the reacted mixture
(0.2 ml) was measured at 490 nm. The antioxidant
efficacy of an extract was expressed as a percentage of
control (PBS added instead of extract).
Cell Culture. Mouse lymphoma L5178 cells were
grown in RPMI 1640 medium supplemented with 10%
(vol/vol) heat-inactivated fetal bovine serum (FBS),
penicillin (100 U/ml), and streptomycin (100 U/ml).
Cultures were maintained at 37-CinaCO
2
incubator.
Treatment of Enzymatic Extracts of Microalgae
for H
2
O
2
-induced Mouse Lymphoma L5178 Cells.
Each enzymatic extract was dissolved in PBS and
diluted to concentrations of 25.0, 50.0, and 100 mg/ml.
A50ml sample was added to the mouse lymphoma
L5178 cell suspension of 4.510
4
cell/ml and incu-
bated for 30 min at 37-C together with the untreated
control sample. The incubated cells were treated with
50 mMH
2
O
2
for 5 min on ice. The untreated control
sample, however, was resuspended in PBS without
H
2
O
2
. Cells were later centrifuged at 2000 rpm for
5 min after washing with 1.0 ml of PBS.
DNA Damage Determination. The alkaline comet
assay was performed according to the method of
Singh et al. (1990,1995) with a slight modification.
The cell suspension was mixed with 100 ml of 0.7%
low melting agarose (LMA), and added to slides
precoated with 1.0% normal melting agarose
(NMA). After agarose solidification, the slides were
covered with another 100 ml of 0.7% LMA followed
by immersion in freshly prepared lysis solution
(2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1%
sodium laurylasarcosine and 1% Triton X-100) for
90 min at 4-C to lyse the cells and their nuclear
membranes. All steps thereafter were conducted in
a darkened room to prevent DNA damage caused by
UV light. The slides were then placed into an
electrophoresis tank containing 300 mM NaOH and
482 ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE
10 mM Na
2
EDTA (pH 13.0) for 40 min for the DNA
to unwind. An electric current of 25 V/300 mA was
applied for 20 min at 4-C to electrophorese the DNA.
The slides were washed three times with a neu-
tralizing buffer (0.4M Tris, pH 7.5) for 5 min at 4-C
and dehydrated with 70% ethanol for another 5 min.
DNA was subsequently stained with 50 mlofethid-
ium bromide solution (20 mg/ml) for image analyses.
Measurements were done by image analysis (Kinetic
Imaging, Komet 5.0, UK) and fluorescence microscope
(LEICA DMLB, Germany), determining the percent-
age of fluorescence in the tail.
Statistical Analysis. Statistical analyses were con-
ducted with the SPSS 11.5 version software package
on triplicate (n=3) test data. The mean values of
each treatment were compared using one-way anal-
ysis of variance (ANOVA) followed by Tukey_s tests.
A P-value of less than 0.05 was considered to be
significant.
Results and Discussion
Mass Culture and Microalgal Yields. The algal
species selection for this study was done based on
frequently occurring and well-cultured species that
were isolated in our laboratories. As result, the
species comprised different microalgae that were
isolated from freshwater and marine habitats as
well as from a shellfish hatchery. However, the
latter microalgae are found naturally and in
abundance, as they are the dominant microalgae of
Jeju Island. Studies related to microalgae growth
performances are quite limited, especially regarding
growth characteristics as this feature has not been
evaluated systematically. In this study, critical
growth parameters of the selected species were
investigated to determine the optimum conditions
for growth (Table 1). In freshwater microalgae
collection sites, the temperature varied from 25 to
30-C with pH values of 7.6 and 8.2, respectively. An
optimal temperature for microalgae production
ranged between 25 and 30-C, but most samples
thrived at a milder temperature level (25-C) and pH
8.0, in contrast to the D. fascicularis freshwater
microalga, which thrived at 30-C and a pH of 8.5.
During the sampling period, the tidal pool area
temperature, pH, and salinity varied from 12.7 to
27.5-C, 6.59 to 8.22, and 17.4 to 35.4 psu, respec-
tively. After a 14-day growth period, the biomass
produced by the different species was evaluated and
the highest yield recorded came from P. duplex and
A. longipes (1.1 g/L, dry weight). While under cul-
ture conditions, the salinity and the light exposure
periods for the samples were also manipulated, and
nearly all samples exhibited better growth with
salinity set at 30 to 35 psu.
The monostrain of H. porphyrae and O. unicel-
lularis were obtained from their native habitats after
microscopic observation. The benthic diatoms were
found as axenic strains using a dosage of 200 U of
penicillin/ml, 200 mg of streptomycin/ml and 400 mg
of neomycin/ml among the different dosages of the
antibiotic cocktails that were prepared. Compared to
macroalgae the microalgae are quite easy to cultivate
in a large-scale bioreactors under controlled environ-
ment, and this may lead to better quality microalgae
without toxic (herbicides/pesticides) compounds.
Therefore, further studies of the growth parameters
for the microalgae are important to maximize their
production at industrial scales. Sometimes, the
growth performances, biochemical composition,
and the nutritive value of the microalgae species
are significantly different in mixed culture condi-
tions; therefore nutritional evaluations can take
place in hatcheries where fishes are fed microalgae
(Phatarpekar et al., 2000).
Table 1. Parameters for the optimum growth of the microalgae
Name
Temperature pH Salinity Light:dark cycle Yield
a
(-C) (psu) (g/l) dry weight
Freshwaters habitat
P. duplex 25 8.0 - 12:12 1.1T0.11
D. fascicularis 30 8.5 - 12:12 0.6T0.11
Tidal pool habitat
H. porphyrae 20 8.2 35 12:12 0.8T0.11
O. unicellularis 25 8.3 30 12:12 0.8T0.22
Benthic habitat
A. longipes 25 8.0 30 12:12 1.1T0.20
Navicula sp. 25 8.0 30 12:12 0.7T0.25
A. coffeaeformis 25 8.0 30 12:12 0.9T0.31
a
The biomass production of alga was estimated after14 days of growth in 1 L of culture medium.
ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE 483
Proximate Composition of Microalgae. The con-
centrations of biochemical components of the test-
ed microalgal species of freeze dried are shown in
Table 2. Of the species tested, the major compo-
nents in most had high levels of carbohydrates and
proteins; however, A. longipes and O. unicellularis
contained relatively lower amounts of protein. The
lipid content of all tested species was notably lower
than the content of other measured constituents.
Not surprisingly, the ash content of the benthic
diatoms was higher than that of all other tested
species. Nutrient content, light intensity, tempera-
ture, photoperiod, and the salinity conditions of water
are the primary environmental factors that influence
the biochemical composition of microalgae. By ma-
nipulating nutrient components together with the
environmental factors, however, the desired com-
pounds of microalgae can be produced. Therefore,
studies related to develop and manage the biochem-
ical composition of the microalgae are important if
they are to be utilized by the food industries.
Hydrogen Peroxide Scavenging Effect of Enzymatic
Extracts of Microalgae. Hydrogen peroxide is a
nonradical reactive oxygen species derived from
normal metabolism. The H
2
O
2
crosses cellular and
organelle membranes and may oxidize a number of
biologically important compounds. Although H
2
O
2
itself is not very reactive, it can be converted into
more reactive species, for example, singlet oxygen
and hydroxyl radicals (Halliweill, 1991). Thus, the
removal of H
2
O
2
is important in the protection of
living systems. The potential H
2
O
2
scavenging
efficacies of enzymatic extracts of various microalgae
were investigated in this study and the results are
summarized in Table 3. Considerable differences in
H
2
O
2
modulating effects can be noted for the extracts
with P. du p le x and D. fascicularis in their H
2
O
2
scavenging effects. Among all tested extracts, the
Termamyl extract (approximately 60%) of P. duplex
was strongest while the Kojizyme (approximately
50%) extract of D. fascicularis followed closely. Of
the two tidal pool algae tested, the Alcalase extract of
H. porphyrae (approximately 42%) had the highest
scavenging effect, with lesser scavenging activities
exhibited by other microalgae. In general, the cell
walls of microalgae are more susceptible to
proteolytic hydrolysis because of their content of
protein, protein-lipid and protein-carbohydrate
complexes. In the results of H
2
O
2
scavenging effect
Table 2. Proximate composition (%) of microalgae
Compounds P. duplex
D. fasci-
cularis H. porphyrae
O. unicell-
ularis A. longipes Navicula sp.
A. coffeae-
formis
Moisture 6.7T0.32 5.5T0.29 8.1T0.13 3.1T0.12 8.1T0.10 3.5T0.49 5.9T0.43
Carbohydrate 34.01T0.13 33.57T0.23 34.61T0.18 30.37T0.13 16.46T0.11 13.50T0.22 15.8T0.20
Protein 46.3T0.48 47.4T0.56 27.8T0.07 4.3T0.12 6.4T0.13 16.9T0.26 15.5T0.22
Lipid 2.4T0.36 4.5T0.17 0.59T1.40 3.4T0.23 1.1T0.32 2.1T0.28 6.82T0.29
Ash 15.1T0.22 8.9T0.02 28.9T0.30 18.1T0.07 67.8T0.15 63.9T0.49 55.8T0.47
Results are means TSEM of triplicate determinations.
Table 3. H
2
O
2
scavenging activity (%) of enzymatic extracts from microalgae
Extracts
P.
duplex
D. fasci-
cularis H. porphyrae
O. unicell-
ularis A. longipes
Navicula
sp.
A. coffeae-
formis
Carbohydrases
Viscozyme 32.5T1.3 32.4T1.7 38.4T1.3 11.3T0.6 10.8T0.2 16.2T0.6 19.4T0.3
Celluclast 33.2T1.6 24.8T1.3 19.4T0.9 17.6T0.7 4.5T0.1 15.1T0.4 17.8T0.6
AMG 33.1T1.7 34.6T1.3 26.4T1.2 20.9T1.1 5.3T0.3 19.4T0.5 10.5T0.7
Termamyl 60.6T3.7 32.5T1.5 27.1T1.6 20.5T0.9 7.1T0.4 16.4T0.7 16.7T0.7
Ultraflo 30.3T1.4 31.3T1.2 22.9T1.4 12.8T0.7 10.1T0.7 24.9T0.5 15.4T0.4
Proteases
Protamex 34.1T2.1 48.6T2.2 34.8T1.7 10.1T0.1 10.9T0.7 12.4T0.2 17.3T0.8
Alcalase 48.3T1.9 27.6T0.7 42.1T2.1 27.1T0.5 8.2T0.6 15.4T0.9 21.1T1.1
Flavozyme 37.1T1.3 24.9T0.4 17.9T0.7 5.1T0.4 11.3T0.7 11.6T0.1 21.1T1.2
Neutrase 42.2T1.5 40.6T1.4 25.2T1.1 24.2T1.1 12.1T0.9 23.4T0.2 22.1T1.3
Kojizyme 27.2T0.9 50.2T2.1 16.1T0.6 12.2T0.1 7.7T0.2 18.9T0.8 13.9T0.9
BHT=60.1T4.2; a-tocopherol=62.5T4.9.
The sample concentration was 2 mg/ml. Results are means TSEM of triplicate determinations.
484 ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE
by the enzymatic extracts of microalgae, the extracts
obtained from hydrolysis by proteases showed better
activities, but Termamyl extract (a carbohydrase) of
P. duplex indicated the highest H
2
O
2
scavenging
effect. It is noteworthy that the addition of an ROS
generator such as H
2
O
2
to the culture medium of
algae (Chlorella zofingiensis) raised the production
level of astaxanthin from 9.9 to 12.58 mg L
-1
(Ip
and Chen, 2005). Under heterotrophic conditions,
therefore, the secondary metabolites related to
microalgal antioxidant activities are elevated and
investigations to apply this technique to industry
where the goal is to elevate the yield of the desired
natural compounds (carotenoids/astaxanthin). In
response to the harsh environmental conditions im-
posed, the cellular antioxidant related metabolites
accumulate and this may explain the observed H
2
O
2
scavenging activity of the tested species.
Antioxidant Effects of the Enzymatic Extracts of
Microalgae. Oxidation of unsaturated fatty acids in
biological membranes leads to the formation and
proliferation of lipid radicals, the uptake of oxygen,
and a rearrangement of the double bonds in unsat-
urated fatty acids. Thus, during the oxidation pro-
cess, peroxide gradually decomposes into lower
molecular weight compounds. The antioxidant
effects of microalgal enzyme extracts (lipid perox-
idation) were compared with commercially antiox-
idants by determining the amount of peroxide
formed in emulsion during incubation. As seen
in Table 4, several extracts of microalgae exhibi-
ted remarkable antioxidant activities when com-
pared with the commercial products. Among the
freshwater microalgae, AMG (approximately 79%)
and Termamyl (approximately 64%) extracts of P.
duplex, and Celluclast (approximately 68%), Pro-
tamax (63.6%), and AMG extracts (approximately 6-
3%) of D. fascicularis showed high antioxidant
activities. Moreover, the Celluclast extract (approxi-
mately 72%) of A. longipes and the Viscozyme extr-
act of Navicula sp. (approximately72%), A. longipes
(approximately 69%), and A. coffeaeformis (approxi-
mately 65%) exhibited considerable antioxidant eff-
ects. This method evaluated the results with only 1
h of oxidation time, whereas other antioxidant assays
with linoleic acid require autooxidation for at least 7
days. However, there was no clear difference in ac-
tivities resulting from the carbohydrase and pro-
tease extracts, which suggests that different
antioxidant components are released from micro-
algae according to enzyme content. Lipid oxidation
is a major cause of the deterioration of many food-
stuffs that contain fats and oil. Therefore, it may be
that microalgae are a valuable potential antioxidant
source for such products owing to an ability to in-
hibit linoleic acid oxidation. As reported previously,
biliprotein phycocyanin (isolated from extracts of
Spirulina platensis) exhibited elevated microsomal
lipid peroxidation inhibition in a dose-dependent m-
anner (Estrada et al., 2001). Moreover, phenolic co-
mpounds of marine plants that accumulate as a
result of the shikimate pathway serve as effective a-
ntioxidants owing to their ability to donate a hydro-
gen and thereby block the formation of free radicals
(Duval et al., 1999). Similarly, furocoumarins and
melatonin-like compounds associated with algae are
important active principals for their high radical sc-
avenging activities (Hoppe, 1982;Balzerand
Hardeland, 1996). Therefore, the active compounds
identified in microalgae are promising in the bio-
technology field and worthy of further study.
Table 4. Antioxidant effect (%) of enzymatic extracts from microalgae
Extracts P. duplex
D. fasci-
cularis H. porphyrae
O. unicell-
ularis A. longipes
Navicula
sp.
A. coffeae-
formis
Carbohydrases
Viscozyme 59.4T3.8 54.8T3.5 45.2T2.4 49.3T2.2 69.7T4.2 72.4T5.9 65.2T3.4
Celluclast 62.4T4.9 68.0T4.6 31.1T1.6 27.3T1.2 72.2T5.3 55.8T2.4 52.3T2.3
AMG 79.2T5.7 63.1T3.2 36.5T2.1 26.2T1.4 52.3T3.4 55.9T2.1 53.5T3.4
Termamyl 64.7T4.1 56.5T3.2 45.7T3.2 31.6T1.5 36.1T1.4 45.9T3.2 59.5T3.2
Ultraflo 42.2T3.5 35.9T2.1 61.1T4.6 24.4T1.4 42.8T1.7 50.5T2.6 46.1T1.8
Proteases
Protamex 58.5T2.7 63.6T3.8 39.7T2.3 28.5T1.7 49.3T2.3 49.2T2.9 54.6T1.4
Alcalase 48.8T2.2 61.5T3.7 53.9T3.5 34.5T1.8 38.8T1.8 41.8T2.7 48.4T2.1
Flavozyme 58.8T3.2 53.1T3.4 43.6T2.4 24.9T1.3 45.2T2.4 36.9T1.3 42.8T1.7
Neutrase 49.1T2.6 47.2T2.5 47.7T2.8 33.1T1.6 46.7T1.7 34.7T2.6 44.5T2.6
Kojizyme 49.3T3.2 41.5T2.4 45.5T2.9 45.5T2.4 56.6T3.9 29.1T1.7 51.9T3.3
BHT=90.7T6.2; a-tocopherol=81.5T5.9.
The sample concentration was 2 mg/ml. Results are means TSEM of triplicate determinations.
ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE 485
DNA Damage Inhibitory Effects of Microalgal
Enzymatic Extracts. The above positive results for
the H
2
O
2
scavenging activity prompted us to test the
extracts in the alkaline comet assay for DNA
protection. DNA is the genetic material of living
cells responsible for controlling all cell functions and
can be damaged by multiple factors including
reactive oxygen species, smoking, toxic chemicals,
and ultraviolet light, Altering the sequence of DNA
base pairs leads to errors in DNA replication,
especially if the DNA damage is not repaired by
endogenous repair mechanisms. Damage to the
DNA of cultured mouse lymphoma L5178 cells was
induced by H
2
O
2
in vitro and the ability of microalgal
extracts to inhibit such damage was extrapolated
from the fluorescence intensity of the tail extent
movement via the comet assay.
The DNA damage inhibitory properties of the
following extracts were investigated using the comet
assay based on their H
2
O
2
scavenging activities
(Table 3): Termamyl of P. duplex; Kojizyme of
D. fascicularis; Alcalase of H. porphyrae; Neutrase
of O. unicellularis, A. longipes, and A. coffeaefor-
mis;andUltrafloofNavicula sp. As shown in Figure 1,
the presence of algal extracts showed dose-dependent
DNA protecting effects against H
2
O
2
-induced cell
damage. The maximum inhibitory effect to cell
damage was approximately 80% for the Termamyl
extract of P. duplex and approximately 69% for the
Kojizyme extract of D. fascicularis at 100 mg/ml.
Because the comets were generated under alkaline
conditions, the results represent a significant increase
in single and double-strand breaks of the cellular DNA.
However, as indicated in Figure 2, the Alcalase extract
of H. porphyrae (approximately 50%) exhibited higher
DNA inhibitory effects than the Neutrase extract
of O. unicellularis (approximately 33%) when
5.2
57.1
20.6 18.2
10.6
0
10
20
30
40
50
60
70
80
90
100
Fluo rescence in tail (%)
0
10
20
30
40
50
60
70
80
90
100
Inhibitory effect of cell damage (%)
5.2
57.1
30.6
20.8 18.2
0
10
20
30
40
50
60
70
80
90
100
Fluorescence in tail (%)
0
10
20
30
40
50
60
70
80
90
100
Inhibitor
y
effect of cell dama
g
e (%)
Control 0 25 50 100
µg/ml algal extract + 50 µM H 2O2
Control 0 25 50 100
µg/ml algal extract + 50 µM H 2O2
A
B
Figure 1. DNA protecting effect of different concentrations of
(A) Termamyl extract of P. d u p l ex and (B) Kojizyme extract of
D. fascicularis on H
2
O
2
-induced L5178 cell damage.
5.2
57.1
43.6
35.2
24.4
0
10
20
30
40
50
60
70
80
90
100
Fluorescence in tail (%)
0
10
20
30
40
50
60
70
80
90
100
Inhibitor
y
effect of cell dama
g
e (%)
5.1
57.2
50.2 45.6
37.7
0
10
20
30
40
50
60
70
80
90
100
Fluorescence in tail (%)
0
10
20
30
40
50
60
70
80
90
100
Inhibitory effect of cell damage (%)
Control 0 25 50 100
µg/ml algal extract + 50 µM H2O2
B
A
Control 0 25 50 100
µg/ml algal extract + 50 µM H2O2
Figure 2. DNA protecting effect of different concentrations
of (A) Alcalase extract of H. porphyrae and (B) Neutrase
extract of O. unicellularis on H
2
O
2
-induced L5178 cell
damage.
486 ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE
used at 100 mg/ml. In Figure 3, the Neutrase
extract of A. longipes is shown to have an approx-
imately 51% inhibitory effect at 100 mg/ml, while
the Ultraflo extract of Navicula sp. and Neutrase
extract of A. coffeaeformis exhibited inhibitory
activities of approximately 43% and approximately
32%, respectively.
Photomicrographs of different DNA migration
profiles from mouse lymphoma L5178 cells when
treated with different concentrations of the Termamyl
extracts (P. duplex) are shown in Figure 4. Under this
experimental condition, as the nuclear DNA became
damaged, the content of the damaged materials mi-
grated as the tail of a comet so that tail length is
directly proportional to the extent of damage. In the
study group in which cells were treated only with,
H
2
O
2,
DNA damage was complete, as shown by the
long tail elongation. The addition of the enzymatic
extract with H
2
O
2,
ameliorated the damage success-
fully, however, as indicated by reduced tail length
and in a dose-dependent manner. Therefore, the
present study strongly suggests that H
2
O
2
-induced
DNA damage in the mouse lymphoma L5178 cells
could be repaired by a constituent(s) of some of the
microalgae species evaluated.
According to previous studies, treatment of cells
with H
2
O
2
resulted in a marked decrease in cell
survival with an elevation of oxidative stresses,
characterized mostly by high MDA production and
LDH release with a reduction in antioxidant enzyme
activities. Moreover, H
2
O
2
exposure enhances Ca
2+
concentration in the extranuclear space (Wang and
Joseph, 2000). The elevation in Ca
2+
would trigger
different degradative processes such as ROS forma-
tion and activation of hydrolytic enzymes (Castillo
and Babson, 1998;Tanetal.,1998). Moreover,
caspase-3 is known to be activated by both H
2
O
2
and the disruption of intracellular Ca
2+
homeostasis
(McGinnis et al., 1999). The addition of H
2
O
2
into
the cells, therefore, may lead to critical DNA
damage. The addition of the algal extracts may
modulate H
2
O
2
-induced oxidative stresses by con-
trolling the elevation of Ca
2+
and caspase-3 produc-
tion. Well known antioxidant compounds have
shown low/high potential in influencing H
2
O
2
scav-
enging than their total antioxidant capacity. There-
fore, the structural requirements of antioxidants for
efficient quenching of hydrogen peroxide are rather
more complicated than those established for other
radical scavenging activities (Mansouri et al., 2005).
Several studies have reported that both exogenous
and endogenous factors can induce DNA damage and
likely lead to such diseases as cancer and heart disease
(Singh et al., 1995). In addition, many studies have
focused on the inhibition of DNA damage through
5.2
57.1
41.6
30.4 26.1
0
10
20
30
40
50
60
70
80
90
100
Fluorescence in tail (%)
0
10
20
30
40
50
60
70
80
90
100
Inhibitor
y
effect of cell dama
g
e (%)
5.2
57.1
43.3
37.6
31.3
0
10
20
30
40
50
60
70
80
90
100
Fluo rescence in tail (%)
0
10
20
30
40
50
60
70
80
90
100
Inhibitory effect of cell damage (%)
A
Control 0 25 50 100
µg/ml algal extract + 50 µM H 2O2
B
Control 0 25 50 100
µg/ml algal extract + 50 µM H 2O2
5.2
57.1
44.3 40.5 37.4
0
10
20
30
40
50
60
70
80
90
100
Fluorescence in tail (%)
0
10
20
30
40
50
60
70
80
90
100
Inhibitory effect of cell damage (%)
Control 0 25 50 100
µg
/ml al
g
al extract + 50
µ
M H 2O2
C
Figure 3. DNA protecting effect of different concentrations of
(A) Neutrase extracts of A. longipes, (B) Ultraflo extract of
Navicula sp., and (C) Neutrase extract of A. coffeaeformis
on H
2
O
2
-induced L5178 cell damage.
ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE 487
consumption of foodstuffs such as teas (Zhang et al.,
2002), juices (Park et al., 2003), plant extracts, and
kolaviron (Farombi et al., 2004). The microalgae could
be a source of a diverse class of bioactive compounds,
especially the carotenoids (canthaxanthin and astax-
anthin), polyunsaturated fattyacids,sulfatedpolysac-
charides, b-glucans, a-tocopherol, and vitamins E and
C, which are well documented bioactive compounds
found in the microalgae (Hoppe, 1982; Duval et al.,
1999;Murthyetal.,2005;Abeetal.,2007). Therefore,
it is speculated that these sorts of active principals
may well be associated with the results shown for the
present study.
The microalgal extracts used in our study were
prepared using food-grade enzymes to evaluate
antioxidant effects. Enzymes might be useful tools
to extract bioactive compounds from microalgae
because plants cell walls contain large amounts of
soluble polysaccharides and insoluble fibers, which
together with other cell wall materials function as a
physical barrier for the extraction of more desirable
bioactive materials. In previous studies, it was reported
that enzymatic hydrolysis yields significant quantities
of the desired bioactive compounds (Jeon et al., 2000:
Nagai and Suzuki, 2000). However, we were unable to
observe a positive relationship between the content
of protein, carbohydrate, and the extraction activity
of their respective enzymes. Brown algae with low
protein constituents digested with proteases were
shown to have high 3-[4,5-dimethylthiazole-2-yl]-2,5-
Figure 4. Comet images of L 5178 cells after treatment with H
2
O
2
and the Termamyl extract of P. duplex (A) negative control
(without sample and H
2
O
2
); (B) cells treated only with 50 mMH
2
O
2
; (C) cells treated with 25 mg/ml of extract +50 mMH
2
O
2
;
(D) cells treated with 50 mg/ml of extract +50 mMH
2
O
2
; (E) cells treated with 100 mg/ml with extract +50 mMH
2
O
2.
488 ROHAN KARAWITA ET AL.: ENZYMATIC EXTRACTS FROM MICROALGAE AGAINST H
2
O
2
DAMAGE
diphenyltetrazoliumbromide (DPPH) radical scaveng-
ing activities compared to their carbohydrase coun-
terparts. Likewise, some algae, for example, E. cava
(10.55% of protein and 68% of carbohydrate), have
been shown to have similar activities after protease
and carbohydrase digestion (Heo et al., 2003). We
interpret our results to indicate that enzymes can
release membrane-bound bioactive compounds into
the extraction media. Moreover, it has been substan-
tiated that enzymes are capable of converting insol-
uble plant material into soluble materials. It is
significant that this technique has given improved
bioactive compound yields compared to convention-
al methods (Athukorala et al., 2006). Hence, the
bioactivities of the digests depend not only on the
type of enzyme and abundance of the biomolecule
(protein/carbohydrate) but likely also on other minor
compounds and the binding strength of the bioactive
compounds within the plant cell wall. These find-
ings further suggest that the constituents of a micro-
algal enzymatic extract will have excellent
antioxidant potentials and may offer an effective
future chemotherapeutic agent(s). Such agents may
also be utilized in the field of marine biotechnology,
and therefore additional detailed studies are currently
ongoing to isolate active compounds from selected
microalgal species.
Acknowledgment
This research was supported by a grant (P-2004–03)
from the Marine Bioprocess Research Center of the
Marine Bio 21 Center, funded by the Ministry of
Maritime Affairs & Fisheries, Republic of Korea.
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O
2
DAMAGE
