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J. Serb. Chem. Soc. 82 (5) 509–522 (2017) UDC 582.26:576+615.9:547.458:615.28:615.279
JSCS–4984 Original scientific paper
509
Antimicrobial, antioxidant, cytotoxic and anticholinesterase
activities of water-soluble polysaccharides extracted from
microalgae Isochrysis galbana and Nannochloropsis oculata
MHAMMED BEN HAFSA1,2*, MANEL BEN ISMAIL3, MARIEM GARRAB3,
RAIES ALY1, JONATHAN GAGNON2 and KARIM NAGHMOUCHI1
1Laboratoire des Microorganismes et Biomolécules Actives (LMBA), Faculté des Sciences de
Tunis, Université El-Manar II 2092 El-Manar-II, Tunis, Tunisia, 2Département de biologie,
chimie et géographie, Université du Québec à Rimouski, 300 allée des Ursulines, Rimouski,
Québec, G5L 3A1, Canada and 3Laboratoire de Microbiologie, Faculté de Médecine,
Université de Monastir, Monastir 5000, Tunisia
(Received 16 November 2016, revised 7 February, accepted 14 March 2017)
Abstract: The present work is carried out to evaluate potential applications of
aqueous extracts of two microalgae Isochrysis galbana (PEA) and Nanno-
chloropsis oculata (PEB) containing mainly polysaccharides. The monosac-
charide composition of microalgal extracts was determined. GC–MS analyses
after derivatization show that glucose is the major compound in both mic-
roalgae PEA (56.88 %) and PEB (68.23 %). Mannitol (38.8 %) and inositol
(20.32 %) are respectively the second major compounds in PEA and PEB.
Silylation of monosaccharides allows the determination of sorbitol that attained
3.38 % in PEB. The determination of antioxidant, antimicrobial and cytotoxic
properties were also analyzed. Antioxidant activity was evaluated from the
DPPH scavenging activity. PEA and PEB show a concentration dependent
DPPH·radical scavenging activity. At concentration of 10 mg/mL, both PEA
and PEB exhibit an antioxidant activity of 41.45 and 59.07 %, respectively.
PEB and PEA are able to inhibit the growth of Gram-negative bacteria, Gram-
positive bacteria and three Candida species. Cytotoxic activity was evaluated
on human HeLa cervical cancer cells. HeLa cell proliferation was totally
inhibited after treatment with PEA and PEB (1 mg/mL) and the inhibition
was dose dependent (from 0.031 to 1 mg/mL). Their anticholinesterase activity
was also investigated against butyrylcholinesterase enzymes. These polysac-
charides possess interesting antimicrobial, anticancer and anticholinesterase
activities that could represent an additional value for these microalgal products.
Keywords: algae; DPPH; cytotoxic activity; antimicrobial activity; polysac-
charides; GC-MS.
* Corresponding author. E-mail: mhammedbenhafsa@gmail.com
https://doi.org/10.2298/JSC161016036B
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INTRODUCTION
Algae represent a large diversity of species that are estimated from 40 000 to
10 million where the majority are microalgae.1 Microalgae are eucaryotic photo-
synthetic organisms that play a key role in aquatic ecosystems and account for
approximately 40 % of the global photosynthesis.2 They possess some different
morphological, physiological, and genetic traits that confer the ability to produce
several biologically active metabolites.3 Microalgae can yield a large pool of
biomolecules with biological activities, such as carotenoids, phycobilins, polyun-
saturated fatty acids, proteins, polysaccharides, vitamins, and sterols among other
chemicals.4 These microalgal molecules can possess several health benefits and
therefore be used in many sectors such as nutraceutical, pharmaceutical and
functional foods.
Besides, polysaccharides are polymeric carbohydrates, formed by repeating
units joined together by glycosidic bonds. Recently, they have widely been
investigated due to their chemical and biological activities.5 Polysaccharides
present a large diversity of structures attributable to their variety in composition,
substitutions and glycosidic bonds. Polysaccharides isolated from plants, fungi,
yeasts and algae have attracted considerable attention for their biological acti-
vities in biochemistry and medicine.6 They exhibit a wide range of biological
activities such as anti-inflammatory, antioxidant, antitumor, anticoagulant, anti-
thrombotic, antimetastic, antiviral, antimicrobial and immunostimulatory.7–10
Laminarin and fucoidan are polysaccharides isolated from cell walls of brown
seaweeds that possess immunomodulatory, antitumor, antiviral and antioxidant
activities.11
Isochrysis galbana and Nannochloropsis oculata are two marine microalgae
that are produced industrially for aquaculture. They are important food source
and feed additive that were widely used especially in the aquaculture industry.12
N. oculata has been reported to reduce blood pressure on hypertensive rats.13 I.
galbana is well-known for its nutritional quality and to be a good source of lipids
that can be used as a substitution of fish oils in a healthy human diet.14,15 Some
promising curative effects were also reported including weight loss and reduction
of glucose, triacylglycerol and cholesterol levels in diabetic rats.16 Moreover
compounds from these microalgae exhibit interesting bioactivities like antibac-
terial, anti-inflammatory, anti-algal, antifungal, analgesic, and antioxidant acti-
vities.17–19
Herein, we report the extraction of water-soluble polysaccharides in two
microalgae pastes, I. galbana (PEA) and N. oculata (PEB), their composition in
monosaccharides, the evaluation of their cytotoxicity against a cancer cell line
and the antimicrobial activities against Gram-positive bacteria, Gram-negative
bacteria and Candida strains. Finally, this study also presents the antioxidant and
anticholinesterase activities of these polysaccharidic extracts.
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BIOLOGICAL ACTIVITY OF MICROALGAE 511
EXPERIMENTAL
Culture conditions and samples
Microalgae pastes, I. galbana (clone T-iso) and N. oculata CCMP-1325, were obtained
from NutrOcean (Rimouski, Canada). Briefly, microalgae came from NCMA-CCMP cultures
and they were produced semi-continuous during two months (partial harvests and dilutions at
24 or 48 h). Microalgae were grown in airlift cylindrical photobioreactors. The temperature
and salinity were 22±2 °C and 28±3 ‰, respectively. Irradiance was 140±20 µE (µmol m-2 s-1)
on the reactor surface. The photoperiod was always light (24 h light and 0 h dark). Culture
medium (f/2 without silicate) was sterilized (UV and ultrafiltration) before to be used.
Extraction of water soluble polysaccharides
Each freeze-dried microalgae paste (20 g each) was extracted three times with 200 mL of
methanol, the first time during 48 h and the two latter extractions during 24 h. The resulting
microalgae pastes were then extracted twice with 200 mL of deionized water during 72 h and
24 h. Microalgae aqueous extracts were combined and were freeze-dried. The yields were
determined.
Total sugar, proteins and sulfate measurement
Total sugar content in the aqueous extracts was determined by a modified phenol–
–sulfuric acid method based on literature.20 Briefly, a mixture of 0.5 mL of sample and 0.5 mL
of 5 % aqueous phenol solution was treated with 2.5 mL of concentrated sulfuric acid. The
mixture was stirred during 30 min. The absorption was measured at 490 nm and glucose was
used as external standard. Sulfate content was determined using barium chloride/gelatin
method with some modifications.21 Concisely, the aqueous extracts (0.2 mL) were treated with
trichloroacetic acid (3.8 mL) followed by the addition of 1.0 mL of barium chloride/gelatin.
The mixture was stirred during 20 min. The absorbance was read at 360 nm and potassium
sulfate was used as external standard.
Protein contents were measured from nitrogen percentage obtained by combustion
elemental analysis. The percentage of crude protein (CP) in samples was calculated by
multiplying the nitrogen percentage (N) by a conversion factor using the following equation:22
CP= N×6.25
Hydrolysis and silylation of polysacharidic extracts
Hydrolysis of extracts was carried out according to Yang et al. with some minor
modifications.23 Freeze-dried aqueous extracts (10 mg) were hydrolyzed with 10 mL of
aqueous trifluoroacetic acid (2 M, TFA) at 120 °C during 8 h. The solution was evaporated to
dryness with a nitrogen flow. Samples were reacted with 0.2 mL of N,O-bis(trimethyl-
silyl)trifluoroacetamide containing 1% of chlorotrimethylsilane in anhydrous pyridine (0.2
mL) during 3 h at 70 °C. The resulting solution was evaporated with a nitrogen flow. The
solid was then extracted with n-hexane (2.00 mL) prior to monosaccharide analysis.24
Monosaccharide analysis
The silylated monosaccharide samples were analyzed using a Hewlett-Packard 6890 gas
chromatograph (GC) equipped with a DB-5 capillary column (30 m×0.25 mm×0.25 µm film
thickness) coupled to a mass spectrometer (MS, Micromass Platform II) operated to the
electron impact mode (70 eV). The temperature of the injector was 300 °C. The column
temperature was set at 80 °C during 5 min, then increased at a rate of 4 °C min-1 to 290 °C,
and was then maintained isothermally for 20 min. The carrier gas was helium at a constant
flow rate of 1.2 mL min-1. Arabinose was used as internal standard. A volume of 1 µL of
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512 BEN HAFSA et al.
sample was splitless injected. Chromatograms were analyzed with the MSD ChemStation
E.02.02.1431 software. Assignation of chromatographic peaks was achieved with the NIST
mass spectra search program (version 2.0d).24
DPPH assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma) radical scavenging activity was
measured according to the literature.25 Microalgae polysaccharidic extracts were dissolved in
10 mL of distilled water to a final concentration of 100 µg/mL. Two milliliters of 0.2 mM
DPPH in ethanol were added to 1 mL of each microalgal polysaccharidic solutions (PEA and
PEB). The absorbance was measured at 517 nm after 20 min of incubation at 25 °C. Distilled
water was used as the control. Percentage of inhibition was determined according to the
following formula: DPPH radical scavenging activity (%) = 100(Ac–As)/Ac, where Ac is the
absorbance value of the control group and As is the absorbance value of the group treated with
the extract.
Vitamin E (Sigma Aldrich) was used as positive control and all measurements were
performed in triplicate. The percentage inhibition of free radical activity was plotted against
the concentration of polysaccharidic extracts and the concentration for 50% of inhibition
(IC50) was determined.
Antimicrobial activity
Microorganisms and culture conditions. PEA or PEB were tested against Gram-positive
cocci (Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923) and
Gram-negative bacilli (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC
27853). The antifungal effects of polysaccharidic extracts from I. galbana or N. oculata were
also tested against a range of pathogenic reference yeasts (Candida albicans ATCC 90028, C.
glabrata ATCC 90030, C. kreusei ATCC 6258 and C. parapsilosis ATCC 22019). Bacteria or
Candida species were grown in nutrient broth and incubated aerobically without shaking for
24 h at 37 °C and sub-cultures were realized at least three times at 24 h intervals prior to tests.
All microorganisms tested were provided from the laboratory of Parasitology–Mycology and
the laboratory of Bacteriology of Monastir (Tunisia).
MIC determination. The minimum inhibitory concentration (MIC) of PEA and PEB was
determined from a microdilution assay as described in literature.26 PEA and PEB stock
solutions were prepared by dissolution of 10 mg of PEA or PEB in 2 mL of 10 % dimethyl
sulfoxide (DMSO, Sigma-Aldrich). After an overnight incubation, broth cultures were
adjusted to yield approximately to 1×106 CFU/mL of bacteria or fungus. A sample of each
extract (200 μL) was added to four wells of the first column of each plate and then diluted
with DMSO (10 %) solution (dilution factor (1:1)) up to the well number eight of first
column. Each well was then inoculated with 50 µL of bacteria or Candida species and
microplates were incubated during 24 h at 37 °C. Controls (wells inoculated with the tested
culture without polysaccharides extracts) and blanks (wells containing uninoculated broth
with polysaccharide extracts) were run on each microplate. Imipenem and vancomycin were
used as positive control for bacteria strains and fluconazole was used for Candida species. All
antibiotics and antimycotic (Sigma–Aldrich) were tested at a final concentration of about 1
mg/mL. The MIC was the lowest concentration of tested agent giving the complete inhibition
of growth (i.e., optical density equal to OD of the blank). The microplate assays were repeated
at least three times for each polysaccharide extract and the MIC was the average of three
independent experiments.
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BIOLOGICAL ACTIVITY OF MICROALGAE 513
Cytotoxic activity. The human HeLa cervical cancer cell line was obtained from the
American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in a
humidified atmosphere at 37 °C in 5 % CO2. RPMI 1640 (Sigma-Aldrich) supplemented with
10 % fetal calf serum, 1 % (w/v) glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin
was used for HeLa cell cultures. Cell viability cytotoxicity was measured using an MTT (3-
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with slight modific-
ations.27 HeLa cells (5×103) were seeded into wells with 100 μL of growth medium and
incubated at 37 °C for 24 h. Cells were treated with polysaccharide extracts (0.031 to 1
mg/mL) and incubated for 48 h at 37 °C. After that, 10 μL of MTT (5 mg/mL) was added to
each well and microplates were incubated for an additional 2 h. Then, the medium was
dissolved with 100 μL of DMSO and the absorbance (A) was measured at 550 nm by a
BioTek microplate reader. This assay was conducted in triplicate as a cell viability index. The
percentages of cell growth were calculated as follow: Cell proliferation (%) = 100(As–Ac)/Ac,
where Ac is the absorbance value of the control group and As is the absorbance value of the
group treated with sample.
Anticholinesterase activity. The anticholinesterase activity was determined by color-
imetry using a Cholinesterase Kit (Chronolab, Spain).28 PEA and PEB stock solutions were
prepared by dissolution 20 mg of PEA or PEB in 2 mL of 10 % DMSO (Sigma–Aldrich).
Human Plasma was provided from the Biochemistry–Toxicology Laboratory, University
Hospital “Fattouma Bourguiba” of Monastir (Tunisia) and used as a source of butyr-
ylcholinesterase (BChE). PEA or PEB (500 µL, 10 mg/mL) was added to 500 µL of plasma
and the mixture was incubated at 37 °C during 5, 10, 15, 20 and 30 min. BChE activity was
measured by COBAS INTEGRA® 400 (Roche diagnostics). The control (plasma and distilled
water) was treated under the same conditions. The anticholinesterase activity was calculated
by the same formula as for DPPH radical scavenging activity. All assays were carried out in
triplicate.
RESULTS AND DISCUSSION
Total sugar, proteins, and sulfate composition
Figure 1 shows the composition in sugars, proteins and sulfate of the micro-
algae aqueous extracts. Total sugar content in aqueous extracts of I. galbana and
N. oculata were 86.9±0.8 % (22.8 % of total dried matter) and 59±0.1 % (4.1 %
of total dried matter), respectively. The sulfate groups represented respectively
7.9±1.2 % and 6.2±0.1 % of I. galbana and N. oculata extracts. Sulfate bands
were confirmed by infrared spectroscopy (data not shown). The percentage of
proteins in the microalgae aqueous extracts were respectively of 5.2±0.17 % and
21.0±0.2 % for I. galbana and N. oculata. According to literature, carbohydrates
represented around 13 % of dry matter of I. galbana.29 Brown
showed that N.
oculata is composed by 35 % of proteins and 7.8 % of carbohydrates.30 However,
Picardo et al. reported that carbohydrates represent 29.4 % when grown at 25 °C
(22 °C in our study).31 Many studies reported that chemical composition as car-
bohydrates, proteins and lipids in N. oculata and I. galbana was dependent of the
environmental growing conditions like salinity, light intensity, nitrogen content,
photoperiod, and stage of harvest.26,27,32
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Fig. 1. Total sugar, proteins and sulfate content in water-soluble polysaccharidic extracts of
Isochrysis galbana and Nannochloropsis oculata. All assays were carried out in triplicate.
Monosaccharide analysis
Polysaccharides from microalgae were hydrolyzed with TFA into monosac-
charides, which were further trimethylsilylated to obtain volatile compounds for
GC–MS analyses. Table I presents the monosaccharide composition in microal-
gal polysaccharidic extracts. Glucose was the major component in I. galbana and
N. oculata extracts with 56.9 and 68.3 %, respectively. Mannitol (38.8 %) and
inositol (20.32 %) were respectively the second major compounds in PEA and
PEB (Fig. 2).
Fig. 2. Monosaccharide chromatogram of polysaccharidic extracts of Isochrysis galbana
(PEA) and Nannochloropsis oculata (PEB) determined by trimethylsilylation method.
A: xylose; B: mannose; C: galactose; D: glucose; E: sorbitol; F: mannitol; G: inositol.
Mannitol represented a percentage of 5.8 % of the N. oculata extract and
inositol was absent in the I. galbana extract. Chu et al., using trimethylsilylation
method, observed 1.35 % of mannitol in I. galbana.33 Xylose, mannose and gal-
actose were minor constituents of the both PEA and PEB microalgae extracts
(< 2 %). Glucose, galactose, mannose and xylose have been reported in various
proportions in I. galbana extracts.34 Brown reported that glucose was the major
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BIOLOGICAL ACTIVITY OF MICROALGAE 515
sugar in N. oculata and I. galbana, which corresponds to 68.2 and 70.3 %, res-
pectively.30 During exponential and stationary growth phases of I. galbana, the
percentage of glucose reached 60 and 80 %, respectively.33 In our study, the
percentages of sorbitol in I. galbana and N. oculata were respectively of 1.02 and
3.38 %. Sadovskaya et al. and Brown reported that sorbitol was not observed in I.
galbana and N. oculata using the alditol acetate method.30,34 Templeton et al.
mentioned that it was impossible to distinguish between neutral sugar (glucose)
and reduced sugar (sorbitol) in the original mixture with the alditol acetate deri-
vatization method.35
TABLE I. Monosaccharides composition (%) of Isochrysis galbana and Nannochloropsis
oculata aqueous extracts determined by the trimethylsilylation method
Retention time, min
Algae
Component Isochrysis galbana Nannochloropsis oculata
28.253 0.43 0.90 Xylose
29.827 1.26 1.03 Mannose
31.049 1.73 0.96 Galactose
31.591 and 34.044 56.88 68.23 Glucose
33.044 38.74 5.78 Mannitol
32.392 1.02 3.38
D-Sorbitol
36.200 – 20.32 Inositol
DPPH radical scavenging activity
DPPH is a free radical compound that has been widely used to evaluate the
ability of antioxidant to scavenge radicals.36 Fig. 3 shows the scavenging power
of DPPH radicals by PEA and PEB. The antioxidant capacities of both PEA and
PEB were dose dependent. The inhibition percentages of PEB and PEA (at 1
mg/mL) were 24.79±0.05 % and 15.71±0.03 %, respectively. At the maximal
concentration of 10 mg/mL, PEB (59.28±0.04 %; IC50 = 4.2 mg/mL) showed a
higher antioxidant activity compared to PEA (41.45±0.03 %, IC50 > 10 mg/mL).
This difference of activity could be explained by the presence of proteins and
highly branched polymers. Vitamin E (0.12 mg/mL) had a significant higher anti-
oxidant capacity of 90.312±0.005 % (IC50 = 0.040 mg/mL), compared to PEA
(9.74±0.003 %) and PEB (16.14±0.005 %). Custódio et al. indicated that organic
extracts from N. oculata had also antioxidant properties with IC50 values between
4.93 and 7.31 %.37 Moreover, Balavigneswaran et al. reported that an ethanol
soluble polysaccharidic extract from I. galbana was active against DPPH (almost
40 %) at 10 mg/mL.38
The reducing properties are generally associated with the presence of reduc-
tones which have been shown to exert antioxidant action by breaking the free
radical chain by donating a hydrogen atom. Reductones are reported to react with
some precursors of peroxide, thus preventing peroxide formation.39 Carboxyl
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516 BEN HAFSA et al.
groups may play an important role in scavenging radicals, possibly due to the
higher hydrogen donation ability of carboxyl groups than hydroxyl groups,
proteins and sulfate groups.36 The low percentage of sulfate in PEA and PEB (7.9
and 6.21 %, respectively) could explain the moderate activity against DPPH
radicals. The antioxidant activities depend on polysaccharides molecular weight,
degree of ramification, monosaccharide composition, sulfate content and config-
uration.40–42 The influence of sulfate content on the antioxidant activity depends
rather on the origin of polysaccharides. For example, the polysaccharides from
Ulva fasciata and other macro and microalgae with low sulfate content demon-
strated a strong antioxidative power, while the antioxidant activity observed in
polysaccharides from Enteromorpha linza and other seaweeds showed to be dep-
endent of sulfate content. Furthermore, highly sulfated polysaccharides were
shown to have an enhanced scavenging power, this property being also depen-
dent on the sulfate distribution pattern.43
0
20
40
60
80
100
120
0246810
DPPH Scavenging activity, %
Polysaccharidic extract concentration, mg mL
-1
PEA PEB Vitamin E
Fig. 3. DPPH scavenging power of polysaccharidic extracts of Isochrysis galbana (PEA) and
Nannochloropsis oculata (PEB). Vitamin E was tested as positive control. All assays were
carried out in triplicate.
PEA and PEB antimicrobial activity
Table II shows the MIC of polysaccharidic extracts (PEA and PEB) from
microalgae I. galbana and N. oculata. All tested bacterial strains were sensitive
to PEA and PEB. Results show that Gram-negative bacteria were more sensitive
to PEA than Gram-positive bacteria. MICs of PEA against Escherichia coli (E.
coli), Pseudomonas aeruginosa (P. aeruginosa) and Enterococcus faecalis were
1250, 1870 and 3750 µg/mL, respectively. MICs of PEB against E. coli and P.
aeruginosa were 2500 and 1870 µg/mL, respectively. On the other hand, the
same author showed that methanolic extract is not active against multiresistant
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BIOLOGICAL ACTIVITY OF MICROALGAE 517
Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative
(P. aeruginosa and Klebsiella pneumomiae) pathogens. Bruce et al. reported that
acetone extract of I. galbana was active against S. aureus and Micrococcus sp.
with corresponding inhibition zone diameters of 10 and 15 mm, respectively.44
TABLE II. Antimicrobial activity of polysaccharidic extracts of Isochrysis galbana (PEA) and
Nannochloropsis oculata (PEB) against Gram-positive bacteria, Gram-negative bacteria and
Candida strains. Imipenem, vancomycin and fluconazole were used as positive controls.
Minimum inhibitory concentration, µg mL-1, was the average of three independent replic-
ations; NA: no activity; ND: not determined
Bacterium PEAPEB Imipenem Vancomycin Fluconazole
Gram-positive bacteria
Enterococcus faecalis 37502500NA 62.5
N
D
Staphylococcus aureus 37503750NA 3.9
N
D
Gram-negative bacteria
Escherichia coli 12502500NA 1.95
N
D
Pseudomonas aeruginosa 187018701.95 NA
N
D
Candida yeasts
Candida albicans NA NA ND ND
N
A
Candida glabrata 117 100 ND ND
N
A
Candida parapsilosis 117 118 ND ND 15.62
Candida krusei 60 80 ND ND 15.62
For the antifungal activity, Candida krusei shows a higher sensitivity to PEA
(60 µg/mL) and PEB (80 µg/mL) than other Candida species (Table II). C. para-
psilosis was inhibited by PEB and fluconazole with MIC values of 118 and 15.62
µg/mL, respectively. No inhibitory activity against C. albicans for both PEA and
PEB was detected. C. glabrata was resistant to fluconazole (1 mg/mL) and
appeared to be sensitive to PEA or PEB, with respective MICs of 117 and 100
µg/mL. The mechanisms involved in antimicrobial activity of polysaccharides
extracts are worthy of further investigations.45 Polysaccharides influence the
cytoplasm permeability, the DNA decomposition after a polysaccharide/DNA
binding, and the denaturation of essential bacterial proteins.46 On the other hand,
the activity against microorganisms can be related to the bacterial membrane
composition, resistance capacity of yeasts, polysaccharides structure, degree of
ramification and degree of sulfation. Goy et al. reported that polysaccharides
inhibited the fungi growth by reacting with enzymes in hyphae.47
Cytotoxic activity
Figure 4 shows that cell proliferation decreases with increasing of PEA and
PEB content. The proliferating cells reached 42.7 and 13.8 % at PEA concentrations
of 31.25 and 500 µg/mL, respectively. After a treatment with PEB (125 and 250
µg/mL), the cell proliferation percentages were 51.5 and 38.36 %, respectively.
Both PEA and PEB inhibited HeLa cell proliferation at a final concentration of 1
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518 BEN HAFSA et al.
mg/mL. Remarkably, HeLa cell proliferations were more abundant with N. ocul-
ata aqueous extract (PEB) than with I. galbana extract (PEA) at different con-
centrations of extracts. The cell proliferation percentages of PEA and PEB at the
minimal studied concentration (31 µg/mL) were respectively of 44 and 59 %.
Sadovskaya et al. showed that the polysaccharide extracts from I. galbana
inhibited U937 human leukemic monocyte lymphoma cell proliferation (30 % at
100 µg/mL) and consequently have potential antitumor activity.34 Atasever-
Arslan et al. reported that essential oils from N. oculata extract (at 500 µg/mL)
caused K562 cell lines cytotoxicity (human chronic myeloid leukemia cell line)
of 45.64 %.48 Polysaccharides, especially sulfate polysaccharides, could affect
the proliferation, differentiation, apoptosis and metastasis of tumor cells.49 They
bound the proteins like growth factors and inhibit the growth of tumors.4,50
Inhibition of the cell proliferation may be mediated by the chemical properties of
sulfated polysaccharides and the species of tumor cells.49 Another mechanism of
antiproliferative effect is to block the G1 phase.49 Sulfated polysaccharides iso-
lated from the filtrate of marine Pseudomonas sp. culture induced the apoptosis
of human leukaemic cells.50 Fucoidan induced apoptosis in human lymphoma
HS-Sultan cell lines, which is accompanied by the activation of caspase-3 and
down-regulation of extracellular signal-regulated kinase pathway.51 The sulfated
heteropolysaccharides isolated from red alga Schizymenia dubyi can induce the
terminal maturation of non-small bronchopulmonary carcinoma cells and arrest
cells in the G1 phase.49
Fig. 4. Percentage of cell proliferation in presence of polysaccharidic extracts of Isochrysis
galbana (PEA) and Nannochloropsis oculata (PEB). Error bars represent the standard
deviation calculated from duplicate experiments.
Anticholinesterase activity
Alzheimer’s disease is a deadly neurodegenerative disease with progressive
character and has become a major health problem especially in industrialized
countries where the life expectancy is higher. It is also a common form of dem-
entia especially among the elder population in which irreversible neuronal loss and
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BIOLOGICAL ACTIVITY OF MICROALGAE 519
abnormal behavioral changes are evident in this disease.52 Antioxidant extracts
from plants play an important role in the prevention of Alzheimer’s disease.53 In
addition, reports indicated a correlation between antioxidant power and the
anticholinesterase activity.54 The use of antioxidants may reduce the Alzheimer’s
disease progression and minimize the neuronal degeneration by inhibition of the
acetylcholinesterase enzyme.55 Treatments of the Alzheimer’s disease include
disease-modifying treatments, psychotropic agents and especially the
cholinesterase inhibitors, which block the hydrolysis of two chemical
neurotransmitters, i.e., acetylcholine and butyrylcholine (by butylcholineesterase,
BChE).52 However, most of these drugs have side effects such as liver damage
and bradycardia. Synthetic antioxidants also caused liver damage and carcino-
genesis in rats, that stimulated scientists to find new natural and harmless anti-
oxidants, as well as anticholinesterase compounds.55 Figure 5 shows the effects
of PEA or PEB (10 mg/mL) on anticholinesterase activity at different incubation
times (5 to 30 min). Remarkably, time-dependent inhibition of butyrylcholine-
sterase was observed after PEA and PEB treatments. PEB was more active than
PEA (Fig. 5). For PEA, the percentage of BChE inhibition after 5 and 30 min
were respectively of 1.25±0.25 % and 7.30±0.48 %. After 30 min of PEB treat-
ment, the BChE inhibition reached a maximum of 11.53±0.12 %. Anticholine-
sterase activities of polysaccharides were not well studied. No significant evid-
ence has been proven that they were specifically active toward the Alzheimer’s
disease. But many polysaccharides could have regenerative properties and func-
tions as memory and learning enhancers.56 Asker et al. suggested that polysac-
charides isolated from Bacillus sp. may be a good natural source for Alzheimer’s
disease therapy.56 On the other hand, Custódio et al. evaluated the BChE activity
of N. oculata organics and water extracts.37 Maximum inhibition (21 %) was
Fig. 5. Effect of polysaccharidic extracts from Isochrysis galbana (PEA) and Nannochloropsis
oculata (PEB) on anticholinesterase activity. The results were expressed as butyrylcholine-
sterase inhibition percentage (%). Error bars represent the standard deviation calculated
from triplicate experiments.
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520 BEN HAFSA et al.
observed after treatment with N. oculata aqueous extract at the maximal concen-
tration of 0.5 mg/mL. Custódio et al. indicated that aqueous extract from Iso-
chrysis galbana possessed an anticholinesterase activity (IC50 of 0.11 mg/mL).57
CONCLUSIONS
I. galbana and N. oculata are used widely in aquaculture for feeding and
pathogens prevention. PEA and PEB possessed important functional properties
such as antioxidant, antimicrobial, anticholinesterase and antiproliferation acti-
vities, demonstrating the important value of these microalgae. I. galbana and N.
oculata can be further tested for their nutritional and medical human applications.
The mode of action of polysaccharidic extracts on pathogenic bacteria or fungi
constitutes also an important field of study for future works.
Acknowledgment. We thank NutrOcean for the grateful donation of microalgae pastes.
ИЗВОД
АНТИМИКРОБНА, АНТИОКСИДАТИВНА, ЦИТОТОКСИЧНА И
АНТИХОЛИНЕСТЕРАЗНА АКТИВНОСТ ПОЛИСАХАРИДА МИКРОАЛГИ Isochrysis
galbana И Nannochloropsis oculata РАСТВОРНИХ У ВОДИ
MHAMMED BEN HAFSA1,2, MANEL BEN ISMAIL3, MARIEM GARRAB3, RAIES ALY1,
JONATHAN GAGNON2 и KARIM NAGHMOUCHI1
1Laboratoire des Microorganismes et Biomolécules Actives (LMBA), Faculté des Sciences de Tunis, Université
El-Manar II 2092 El-Manar-II, Tunis, Tunisia, 2Département de biologie, chimie et géographie, Université du
Québec à Rimouski, 300 allée des Ursulines, Rimouski, Québec, G5L 3A1, Canada и 3Laboratoire de
Microbiologie, Faculté de Médecine, Université de Monastir, Monastir 5000, Tunisia
У овом раду је испитана могућност примене водених екстраката микроалги Iso-
chrysis galbana (PEA) и Nannochloropsis oculata (PEB), који садрже претежно полисаха-
риде. Одређен је садржај моносахарида у екстрактима. GC–MS анализа након дерива-
тизације је показала да је главни састојак обе микроалге глукоза: у PEA 56,88 % и у PEB
68,23 %. Манитол (38,80 %) и инозитол (20,32 %) су следећи по заступљености у PEA
односно PEB. Силиловањем моносахарида је утврђено да сорбитола има 3,38 % у PEB.
Даље су анализиране антиоксидативне, антимикробне и цитотоксичне особине
екстраката. Антиоксидативна активност је утврђивана DPPH методом и зависила је од
концентрације. При концентрацији екстракта од 10 mg/mL, антиоксидативна активност
PEA и PEB је била 41,45 %, односно 59,07 %. Екстракти су били способни да инхибирају
раст Грам негативних и Грам позитивних бактерија, као и три врсте гљиве Candida.
Цитотоксична активност је процењена на хуманим HeLa ћелијама тумора грлића
материце. Пролиферација HeLa ћелија је била потпуно инхибирана третманом PEA и
PEB екстрактима у концентрацији 1 mg/mL, а инхибиција је зависила од дозе у опсегу
0,03 до 1 mg/mL. Антихолинестеразна активност је потврђена спрам бутирилхоли-
нестеразе. Због својих активности, полисахариди наведених микроалги могу имати
додатну примену осим нутритивне.
(Примљено 16. новембра 2016, ревидирано 7. фебруара, прихваћено 14. марта 2017)
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