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RESEARCH ARTICLE
Assessment of the antioxidant and anticancer potential of different
isolated strains of cyanobacteria and microalgae from soil
and agriculture drain water
Hoda H. Senousy
1
&Sawsan Abd Ellatif
2
&Shafaqat Ali
3,4
Received: 12 September 2019 /Accepted: 5 March 2020
#Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
The potential usage of cyanobacteria and microalgae as a promising and alternative source for new and safe therapeutic
compounds is recently caught the attention, due to its versatile properties as antitumor, antioxidant, antifungal, and
antiviral agents. Primarily, the cyanobacteria and microalgae from fresh and marine water are previously studied,
however those isolated from soil and agriculture drain water were poorly investigated. Therefore, this study aimed to
screen and characterize the antioxidant profile, as well as the potential anticancer assessment of 12 species of
cyanobacteria and two species of microalgae strains isolated from soil and agriculture drain water. The data showed
that total phenol contents were highest in Anabaena oryzae and Aphanizomenon gracile (27.39 and 26.83 mg GAE/g,
respectively), followed by Leptolyngbya fragilis (22.96 mg GAE/g). Out of the 14 species identified, the cyanobacterium
Dolichospermum flos-aquae HSSASE2 exhibited the most elevated antioxidant activity in terms of NO scavenging
activity and anti-lipid peroxidation potential (IC
50
= 28.7 ± 0.1 and 11.9 ± 0.2 μg/ml, respectively) and the lowest
DPPH radical scavenging activity (467.7 μg/ml). Screening of the anticancer potential of all studied strains against four
different human cancer cell lines (Caco-2, MCF-7, PC3, and HepG-2) demonstrated that Dolichospermum crassum
HSSASE20 has the highest anticancer effect among all tested species against colon and prostate cancer cell lines
(IC
50
= 57.9 ± 0.4 and 44.1 ± 0.2 μg/ml, respectively), while Oscillatoria sancta HSSASE19 recorded the most anticancer
effect against MCF-7 (breast cancer) cell line (IC
50
=15.1±0.7μg/ml). Dolichospermum spiroides HSSASE18 obtained
the highest anticancer effect HepG-2 (hepatic cancer) cell line (IC
50
=48.8±0.7 μg/ml). Additionally, cytotoxicity
against healthy peripheral blood mononuclear cells was studied and revealed that Oscillatoria sancta was the safest
one among all studied strains. Data obtained from the sensitivity index demonstrated that Dolichospermum crassum was
the most sensitive strain against the four cancerous cell lines. Cyanobacteria and microalgae from the soil and drain
water sources are efficient free radical scavengers, containing apoptogens capable of stimulating apoptotic cascades and
overcoming chemo-resistance in cancer therapy. Thus, these novel secondary metabolites are an excellent alternative,
safe, and low-cost antioxidant and anticancer therapeutic compounds.
Keywords Blue-green algae .Bioassay .Antioxidant .Phytochemicals .Anticancer .Total phenols
Responsible editor: Diane Purchase
*Shafaqat Ali
shafaqataligill@yahoo.com; shafaqataligill@gcuf.edu.pk
1
Botany and Microbiology Department, Faculty of Science, Cairo
University, Giza 12613, Egypt
2
Bioprocess Development Department, Genetic Engineering and
Biotechnology Research Institute (GEBRI), City for Scientific
Research and Technology Applications, New Borg El-Arab City,
Universities and Research District, 21934 Alexandria, Egypt
3
Department of Environmental Sciences and Engineering,
Government College University, Allama Iqbal Road,
Faisalabad 38000, Pakistan
4
Department of Biological Sciences and Technology, China Medical
University, Taichung 40402, Taiwan
https://doi.org/10.1007/s11356-020-08332-z
Environmental Science and Pollution Research (2020) 27:18463–18474
Published online: 19 March 2020
/
Introduction
Synthetic antioxidant usage has been dramatically reduced
within the last few years. The reasons behind this are to avoid
oxidative degeneration of food and also to lessen oxidative
damage to the living cells (Shanab et al. 2011). Since the re-
lease of free radicals during oxidative damage causes signifi-
cant endogenous damage in the biological systems (Thajuddin
and Subramanian 2005). This damage is often linked to mul-
tiple degenerative diseases such as cardiovascular disease, can-
cer, declining immunity, aging, and cellular DNA damage
(Witsch 1986; Williams et al. 1999). Additionally, free radicals
are the leading cause of food decay by lipid peroxidation that
eventually impacts foods’characteristics and edibility (Huang
et al. 2007). The ideal anticancer effect should act only against
tumor cells; however, many chemotherapeutic compounds cur-
rently used in cancer therapy have significant negativeimpacts
on a healthy living cell, such as loss of hair, bleeding, diarrhea,
and immunodepression (Kranz and Dobbelstein 2012). Also,
the evolving resistance to chemotherapeutic agents is a signif-
icant obstacle during the therapy, due to the multi-drug resis-
tance after exposure to diverse anticancer agents with overall
structure and broad action mechanisms (Perez 2008).
Therefore, a recent growing interest concerning detection
of natural metabolites extracted from micro-organisms, plants,
and animals has been recently investigated, especially, for the
bioactive secondary metabolites with elevated antitumor effi-
cacy with no cellular toxicity to the healthy cells.
Cyanobacteria are a phylum of blue-green algae that obtains
their energy from sunlight through photosynthesis. These al-
gae are considered the most abundant bioactive molecules
producers (Hossain et al. 2016). Such algae have a major
appeal with a high activity of biological properties like anti-
oxidant, antiviral, anti-inflammatory, anticancer, and antimi-
crobial impacts (Ozdemir et al. 2006). Microalgae are micro-
scopic photosynthetic organisms that constitute a significant
part of fresh and marine water phytoplankton. They can ac-
commodate extreme harsh circumstances, extreme environ-
mental temperatures, and different stress circumstances
(Landsberg 2002; Caldwell 2009).
Several bioactive metabolites generated by cyanobacteria
and algae, including polysaccharides, polyphenolics, anti-
oxidant, peptide, minerals, and essential vitamins (Kim
et al. 2014; Shanura Fernando et al. 2017;Martinez
Andrade et al. 2018). These secondary metabolites demon-
strate a versatile activity of pharmacological impacts, such
as antifungal, antioxidant, antiaging, anti-inflammatory,
antibacterial, and anticancer activities (Mayer et al. 2009;
Fernando et al. 2016; Agatonovic-Kustrin and Morton
2018). The cytotoxicity of marine cyanobacteria against
different human cancer cell lines is most frequently stud-
ied, while those isolated from the soil are poorly evaluated.
Therefore, the current investigation represents an endeavor
to bridge this gap and was intended to assess and evaluate
the potential antioxidant and anticancer activity of different
strains of cyanobacteria and microalgae isolated from soil
samples at two different locations as well as agriculture
drain water (the Bahr Hadus drain brackish water station,
Egypt) as a new candidate of safe and natural antitumor
agents.
Materials and methods
Sampling location
Soil samples were collected from two different areas in El-
Sharkia Governorate: rice field soil in El-Rowad Village and
wheat field soil in Sahl El-Hussinia, while agriculture drain
water samples were collected from Bahr hadus pump station
No. 3 in El Daqhlia Governorate.
Isolation and culture conditions
The isolation of blue-green algae (cyanobacteria) from soil
was carried out according to the method of Allen and
Stanier (1968). Briefly, one or two drops of each extract were
inoculated on solid and BG11 liquid media and incubated at
25 ± 1 °C with continuous illumination with a white light of
2000 lx intensity at 150 rpm. The plates have been examined;
the well-grown colonies have been chosen, collected, and
streamed back to fresh plates. Re-streaking and sub-
culturing were repeated multiple times to obtain uni-algal cul-
tures. The purification process was discussed in details else-
where (Ferris and Hirsch 1991). After the development of
pure colonies, particular colonies were picked, replanted,
and incubated in a production culture of BG11 for
cyanobacterial strains and in bold culture for Chlorophyta
strains. The culture condition was 25 ± 1 °C illumination by
a cool white lamp with at 2000 lx intensity using photoperiod
of 12 h light/dark. A continuous 5% CO
2
airflow was supplied
via an air pump through a 0.2-μm filter at a rate of 1 L/min to
prevent culture contamination.
Experimental growth conditions
Exchanging growth between mixotrophy cultivation and pho-
toautotrophic cultivation for all algal strains were operated in
incubator shaker. A volume of 20 ml pre-algal culture was
inoculatedinto 250 ml BG11 medium in triplicate for 14 days.
The pellets were then collected by centrifugation at
10,000 rpm, and 4 °C then incubated at 55 °C for 24 h in
the oven for dryness. A 12 spieces of cyanobacteria and two
species of microalgae isolated from soil and agriculture drain
water are being listed in Table 1.
Environ Sci Pollut Res (2020) 27:18463–18474
18464
Preparation of enzyme extract for determination
of antioxidant activities
Enzymes were extracted from cyanobacteria and microalgae
by mixing 1 ml culture of each culture with 5 ml ice-cold
extraction buffer (50 mM K
3
PO
4
,pH7,and1mMEDTA).
The mixture was then centrifuged at 20,000×gfor 30 min at
4 °C. The supernatant was filtered and used to evaluate differ-
ent enzyme activities.
Catalase activity
Evaluation of the catalase activity of each extract was assessed
according to the assay described by Aebi (1984). The final
assay mixture (3 ml) contained 100 mM K
3
PO
4
buffer
(pH 7.0), 30 μL enzyme extract, and 20 μLof30%H
2
O
2
.
Catalase activity was monitored by calculating the rate of
H
2
O
2
consumption each 30 s for 3 min at 240 nm against
the blank reagents.
Peroxidase activity (POX)
Peroxidase activity was evaluated as previously mentioned
(Jiang et al. 2002). The final assay mixture (3 ml)
contained 100 mM sodium phosphate buffer (pH 7.0),
20 mM guaiacol substrate, and 1 ml of each enzyme ex-
tract. The addition of 20 μLH
2
O
2
initiated the reaction.
POX activity was assessed spectrophotometrically by ab-
sorbance increase at 470 nm. Enzyme activity was defined
as a change in the absorbance/g/min.
Phenylalanine ammonia-lyase (PAL)
PAL enzyme activity was evaluated by determination of trans-
cinnamic acid formation at 290 nm, as previously mentioned
(Whetten and Sederoff 1992). The final mixture contained
500 μl of 50 mM Tris HCI (pH 8.8), 100 μl of enzyme extract,
and 600 μl of 1 mM L-phenylalanine. The reaction was
allowed to incubate at RT for 60 min, then terminated by
adding 2 N HCI. The reaction mixture was then extracted with
1.5 ml of toluene and vortexed for 30 s. Toluene recovery was
obtained after centrifugation at 1000 rpm (CRU-5000
centrifuge ITC) for 5 min. The absorbance of trans-cinnamic
acid in the toluene phase was determined at 290 nm against
toluene blank. Enzyme activity was evaluated as nmol trans-
cinnamic acid released/min/g fresh weight.
Determination of total phenols content (TPC)
The amount of total phenols was evaluated in the methanolic
extract according to the Folin-Ciocalteu method (Saeed et al.
2012). To prepare the methanolic extract, a volume of 1 ml of
each strain culture was mixed with 5 ml 80% methanol at
25 °C for 24 h with periodic shaking at RT, then filtered. Re-
extraction has been repeated and filtered twice. All the filtrates
were then collected and used for total phenol estimation.
Briefly, 1 ml of the methanolic extract was added to 5 ml of
distilled water, and 250 μL of Folin reagent. The mixture was
allowedtoincubateat25°Cfor30min,then1mlofNa
2
CO
3
(7.5%) and distilled water were added. Incubation of the mix-
ture was done in the dark for 90 min at RT. A set of solutions of
gallic acid standard (200, 175, 150, 125, and 100 μg/mL) were
prepared. The optical density of all samples was recorded
against blank at 760 nm. The amount of total phenols was
estimated from the standard curve and expressed as mg equiv-
alent of gallic acid (GAE)/g extract (Jain et al. 2015).
DPPH radical scavenging activity
The DPPH radical scavenging effect of the cyanobacteria and
microalgae extracts was determined by the method of Braca
et al. (2001). Briefly, 100 μl of prepared DPPH (0.004% in
methanol) was added to 100 μl of the sample extract or vita-
min C (as a reference). The plate was then thoroughly mixed
before being wrapped and placed in the dark for 30 min at
25 °C. The ability of the sample to scavenge DPPH was esti-
mated by measuring absorbance decrease at 517 nm against
methanol blank. The activity was determined according to the
formula given by Yen and Duh (1994):
Antioxidant activity %ðÞ¼Ac−AtðÞ=Ac½100
Where, Ac and At are the optical density of the control
(DPPH) and tested samples, respectively.
Table 1 List of cyanobacteria and microalgae strains isolated in this
study
Strain Accession no.
Cyanobacteria Dolichospermum flos-aquae HSSASE2 KT277785
Dolichospermum crassum HSSASE20 KT277803
Dolichospermum spiroides HSSASE18 KT277801
Dolichospermum circinale HSSASE14 KT277797
Oscillatoria sp. HSSASE4 KT277787
Oscillatoria nigro-viridis HSSASE15 KT277798
Oscillatoria sancta HSSASE19 KT277802
Anabaena oryzae HSSASE6 KT277789
Anabaena sp. HSSASE11 KT277794
Leptolyngbya fragilis HSSASE9 KT277792
Aphanizomenon gracile HSSASE16 KT277799
Wollea saccata HSSASE12 KT277795
Microalgae Dunaliella sp. HSSASE13 KT277796
Chlorella sorokiniana HSSASE17 KT277800
Environ Sci Pollut Res (2020) 27:18463–18474 18465
Nitric oxide radical scavenging activity
The nitric oxide (NO) scavenging potential of the extracts was
assayed, as mentioned by Ho et al. (2010). Nitric oxide radicals
interact with oxygen to produce nitrite ions, which were mea-
sured by the Griess reaction (2006). Briefly, a volume of 50 μl
of each cyanobacterium and microalgae extracts, vitamin C
(reference), or distilled water (as negative control) was pipetted
into a 96-well flat-bottomed plate. Then, 50 μlof10mMso-
dium nitroprusside solution was added to the plate. After
mixing, the plate was incubated at RT for 90 min. Finally, an
equal volume of Griess reagent (1% of sulfanilamide and 0.1%
of naphthyl ethylenediamine in 2.5% H
3
PO
4
) was added to
measure the nitrite content immediately at 490 nm. The ex-
tract’s ability to scavenge NO radical was estimated as follows:
NO scavenging activity %ðÞ¼AC−AE
ðÞ=AC
½100
Where: A
C
: The mean of absorbance of negative control,
A
E
: The mean of absorbances of extract. The antioxidant ac-
tivities of all extracts were expressed as IC
50
and determined
by the GraphPad Prism software using the data obtained from
the inhibition percentage of the tested extracts. The IC
50
value
was evaluated as the concentration of extract (μg/ml) that
inhibits the DPPH/NO radical formation by 50%.
In vitro anti-lipid peroxidation assay
Anti-lipid peroxidation assay was determined as previous-
lydescribedbyNgetal.(2000). The freshly prepared
liver homogenate was used as a source of polyunsaturated
fatty acids. The degree of lipid peroxidation was assayed
in terms of thiobarbituric acid-reactive substances
expressed as malondialdehyde. Briefly, 20% of rat liver
homogenate was freshly prepared in ice-cold 0.1 M phos-
phate buffer (pH 7.4). A volume of 3 ml homogenate was
mixed with 1 ml of each extract. Addition of 0.5 ml of
0.1 mM ascorbic acid and 0.5 ml of 2 mM FeSO4 was
added to initiate lipid peroxidation. The mixture was then
incubatedat37°Cfor1h,afterwhich0.5mlofthe
reaction mixture was transferred to a fresh tube containing
2.5 ml of 10% TCA. All tubes were then centrifuged at
10,000 rpm for 10 min. A volume of 0.5 ml 0.67% thio-
barbituric acid was mixed with 1 ml of the supernatant.
The mixture was incubated at 90 °C for 10 min, then
cooled to RT, and the absorbance of all samples was mea-
sured at 535 nm against the blank. Anti-lipid peroxidation
was evaluated as follows:
Inhibition of lipid peroxidation %ðÞ¼
A ControlðÞ−ASampleðÞ
A controlðÞx100
The IC
50
of each extract was determined, as mentioned
above.
Cytotoxicity assay against human peripheral blood
mononuclear (PBMC) cells
The cytotoxicity assay against healthy human cell growth was
determined using the microculture MTT method.
Cell preparation PBMCs were isolated, as described by
Boyum (1968). Healthy Fresh heparinized whole blood
wascarefullylayeredintoanequalvolumeoftheFicoll-
Hypaque solution (density 1.077 g/ml) and then centri-
fuged for 30 min at 600×g. After centrifugation, the buffy
coat layer containing PBMCs was isolated and washed
twice in 5 mL PBS (300×gfor 20 min at 25 °C). PBMC
were resuspended in RPMI 1640 containing 10% FBS,
counted, and viability was checked by staining with 0.5%
trypan blue then counted by a hemocytometer.
Cytotoxicity assay The cytotoxic effect of cyanobacteria and
microalgae extracts was done according to the method of
Mosmann (1983). Cultures were initiated at 1 × 10
5
mononu-
clear cells per well. Cells were treated with serial dilutions of
microalgae extract, while well containing only a complete
medium was left as blank. After incubation for 72 h in 5%
CO
2
incubator, 20 μl of MTTsolution (5 mg/ml in PBS, pH 7)
was added to each well and incubated at 37 °C for 4 h. MTT
solution was discarded after centrifugation at 2000 rpm for
10 min and the immiscible blue formazan crystals trapped in
cells were dissolved and maintained in 150 μl of DMSO. The
absorbance of each well was measured with a microplate read-
er at 570 nm. Surviving cell fraction in the presence or the
absence of synthetic compounds was calculated according to
the following equation:
Cell viability %ðÞ¼ AE−AB
ðÞ=AC−AB
ðÞ½100
Where, A
E
: The mean of absorbances of cells exposed to
compounds, A
B
: The mean of absorbances of blank, A
C
:The
mean of absorbances of control cells.
The compound concentration at which 50% of the cells do
not multiply is defined as the median inhibitory dose (IC
50
)
(Ekwall et al. 1990). The values of IC
50
and safe dose (EC100)
were determined from the cell viability equation using
GraphPad Prism software.
Evaluation of anticancer activity
Cell cultures Caco-2 (colon cancer cell line) and PC3 (prostate
cancer cell line) cells were routinely preserved as adherent cell
cultures in DMEM containing 10% fetal bovine serum (FBS)
and 2 mM L-glutamine (Gibco, USA). Whereas MCF-7
(breast cancer line) and HepG-2 (liver cancer cell line) cells
were kept as adherent cell cultures in RPMI 1640 media sup-
plemented with 10% FBS and 2 mML-glutamine at 37°C in a
Environ Sci Pollut Res (2020) 27:18463–18474
18466
humidified air incubator containing 5% CO
2
. Cells were sub-
cultured for 2 weeks before the assay. Cell number and via-
bility were determined by trypan blue exclusion assay.
MTT assay The cytotoxicity of cyanobacteria and microalgae
extracts against cancerous cell lines was evaluated as previ-
ously assessed by Mosmann (1983). Briefly, the cell suspen-
sion was collected into sterile 96-well flat-bottomed microtiter
plates (3 × 10
3
cells/well for Caco-2, MCF-7, and HepG2 cell
lines and 6 × 10
3
cells/well for PC3 cell line). After cell attach-
ment for 24 h, media were replaced with fresh culture media
(control cells) and media containing serial concentrations of
the supernatant of each extract and 5-fluorouracil (positive
control). The plates were further incubated at 37 °C in 5%
CO
2
incubator for 72 h, then MTTsolution was added to each
well and further incubated for 4 h at 37 °C, and proceeded as
described above. Each concentration of the algal extract was
assayed in triplicate. Inhibition of the growth rate of tumor
cells for each extract with different concentrations was calcu-
lated according to the following formula:
The inhibition rate %ðÞ¼100−AE−AB
ðÞ=AC−AB
ðÞ100
The effective anticancer activity was determined by calcu-
lating the IC
50
value (the concentration caused 50% cell death)
from the data obtained from the inhibition rate using
GraphPad Prism software. Additionally, the selectivity index
(SI), which was defined as the ratio of the IC
50
on healthy
colon epithelial cells versus cancerous cell lines, was also
calculated. High selectivity of the drug against cancer cells
was achieved when the SI was ≥3(Prayongetal.2008).
Statistical analysis
All experimental results are presented as a mean ± standard
error from at least three independent replicates, P<0.01was
determined to represent a significant difference. Results were
processed using GraphPad Prism v5.0 (GraphPad Software,
Inc., La Jolla, CA, USA).
Results
Total phenolic content of cyanobacteria
and microalgae extracts
The total content of phenols in the methanolic extracts of the
studied species was evaluated as milligram equivalent of gal-
lic acid (GAE) per gram of the extract. Methanolic extract of
Anabaena oryzae HSSASE6 was shown to contain the highest
content of phenols among all the studied species represented
by 27.39 mg GAE, followed by Aphanizomenon gracile
HSSASE16 and Leptolyngbya fragilis HSSASE9 extracts
(26.83 and 22.96, respectively), while Dolichospermum
spiroides HSSASE18 showed the lowest phenolic content
(9.46 mg GAE) (Table 2).
Antioxidant enzyme activities of cyanobacteria
and microalgae extracts
Antioxidant enzyme activities were evaluated in the superna-
tant of the screened strains. Figure 1shows that Oscillatoria
nigro-viridis HSSASE15 contained the highest catalase activ-
ity of all studied species recording a value of 12.4 U/g, follow-
ed by Oscillatoria sancta HSSASE19 (9.4 U/g) and
Dolichospermum crassum HSSASE20 (8.6 U/g). On the op-
posite side, low catalase activity was observed in
Leptolyngbya fragilis HSSASE9 and Anabaena oryzae
HSSASE6 (0.62 and 0.93 U/g, respectively). Regarding per-
oxidase activity, the microalgae Chlorella sorokiniana
HSSASE17 demonstrated the highest activity (3.51 U/g)
followed by Aphanizomenon gracile HSSASE16 (3.32 U/g),
while Dunaliella sp. HSSASE13 reported the lowest peroxi-
dase activity (1.23 U/g) among all strains. Data obtained from
phenylalanine ammonia-lyase activity reported the highest ac-
tivity of the enzyme in Aphanizomenon gracile HSSASE16
and Oscillatoria sancta HSSASE19 (6.29 and 6.2 U/g, respec-
tively), while Dolichospermum spiroides HSSASE18 recorded
the lowest PAL activity of all studied strains (0.468 U/g)
(Fig. 1).
Table 2 List of cyanobacteria and microalgae strains isolated in this
study and their corresponding total phenolic content
Strain Accession no. Total phenols
(mg GAE/g)
Cyanobacteria
Dolichospermum flos-aquae HSSASE2 KT277785 20.1
Dolichospermum crassum HSSASE20 KT277803 11.74
Dolichospermum spiroides HSSASE18 KT277801 9.46
Dolichospermum circinale HSSASE14 KT277797 10.23
Oscillatoria sp. HSSASE4 KT277787 11.15
Oscillatoria nigro-viridis HSSASE15 KT277798 12.45
Oscillatoria sancta HSSASE19 KT277802 10.5
Anabaena oryzae HSSASE6 KT277789 27.39
Anabaena sp. HSSASE11 KT277794 12.3
Leptolyngbya fragilis HSSASE9 KT277792 22.96
Aphanizomenon gracile HSSASE16 KT277799 26.83
Wollea saccata HSSASE12 KT277795 14.1
Microalgae
Dunaliella sp. HSSASE13 KT277796 10.76
Chlorella sorokiniana HSSASE17 KT277800 11.4
Environ Sci Pollut Res (2020) 27:18463–18474 18467
0
50
100
150
200
250
300
350
400
450
500
IC50 values (µg/ ml)
Fig. 2 DPPH radical scavenging activities of the tested strains. Data are represented as half-maximal inhibitory concentration (IC
50
;μg/ml) for each
extract, compared to a positive control (vitamin C)
0
2
4
6
8
10
12
14
Enzyme Acvity (U/g)
catalase Phenyl aniline Ammonia layase Peroxidase (POD)
Fig. 1 Antioxidant enzyme activity of the studied cyanobacteria and microalgae strains. The activity of catalase, phenylalanine ammonia-lyase, and
peroxidase enzymes in all strains were expressed as U/g
Environ Sci Pollut Res (2020) 27:18463–18474
18468
DPPH radical scavenging activity
Scavenging ability of the studied cyanobacteria and
microalgae strains against DPPH radical are documented in
Fig. 2. All these samples can scavenge DPPH at varying de-
grees. The supernatants of Dolichospermum circinale
HSSASE14,Oscillatoria sp. HSSASE4,andAnabaena sp.
HSSASE11 exhibited the highest DPPH radical scavenging
potential among all studied species recording IC
50
values of
17.2 ± 0.6, 52 ± 1.9, and 53.6 ± 0.01 μg/ml, respectively,
whereas, Dolichospermum flos-aquae HSSASE2 and
Oscillatoria sancta HSSASE19 showed the minimum DPPH
radical scavenging activity (IC
50
= 467.7 ± 1.6 and 290.2 ±
3.2 μg/ml, respectively).
Nitric oxide radical scavenging activity
The scavenging ability of NO radical was evaluated as an
indicator of the antioxidant potential of the studied species.
AsshowninFig.3, the NO scavenging potential of the super-
natant of tested microalgae can be arranged in the following
order Dolichospermum flos-aquae HSSASE2 > Oscillatoria
sp. HSSASE4 > Anabaena oryzae HSSASE6 > Dunaliella sp.
HSSASE13 > Aphanizomenon gracile HSSASE16 > Chlorella
sorokiniana HSSASE17 > Dolichospermum circinale
HSSASE14 > Anabaena sp. HSSASE11 > Leptolyngbya
fragilis HSSASE9 > Dolichospermum crassum HSSASE20 >
Wollea saccata HSSASE12 > Dolichospermum spiroides
HSSASE18 > Oscillatoria sancta HSSASE19 > Oscillatoria
nigro-viridis HSSASE15. It was observed that
Dolichospermum flos-aquae HSSASE2 exhibited the highest
NO scavenging ability with the lowest IC
50
value of 28.7 ±
0.1 μg/ml as compared to that of ascorbic acid (IC
50
=45.6±
0.05 μg/ml), while Oscillatoria nigro-viridis HSSASE15 dem-
onstrated the lowest scavenging activity with the highest IC
50
value of 823.2 ± 2.5 μg/ml.
In vitro inhibition of lipid peroxidation assay
The inhibitory activity of the supernatant of cyanobacteria and
microalgae strains against lipid peroxidation was assayed in
terms of TBARS. The data obtained from the 14 tested strains
illustrated that Dolichospermum flos-aquae HSSASE2 and
Dolichospermum crassum HSSASE20 exhibited the highest
anti-lipid peroxidation activity among all species (IC
50
=
11.9 ± 0.2 and 44.4 ± 0.6 μg/ml, respectively), compared to
the positive control (IC
50
= 64.3 ± 0.7 μg/ml). On the opposite
side, Leptolyngbya fragilis HSSASE9, and Wollea saccata
HSSASE12 demonstrated the lowest activity recording IC
50
values of 2793.5 ± 62.5 and 1073.7 ± 17.9 μg/ml, respectively
(Fig. 4).
Cytotoxicity assay against normal human PBMCs
Peripheral blood is the available sourceof healthy human cells
for investigations of the toxicity of extracts. Data obtained
from the cytotoxicity of the screened strains against healthy
PBMC cells revealed that the supernatant of Oscillatoria
sancta HSSASE19 was the safest one against human PBMCs
recording IC
50
value of 991.1 ± 0.5 μg/ml, while the
0
100
200
300
400
500
600
700
800
900
IC50 values (µg/ml)
Fig. 3 Nitric oxide radical
scavenging activity of the tested
strains. Data are represented as
half-maximal inhibitory concen-
tration (IC
50
;μg/ml) for each ex-
tract, compared to a positive con-
trol (vitamin C)
Environ Sci Pollut Res (2020) 27:18463–18474 18469
Leptolyngbya fragilis HSSASE9 was the most toxic one
among all studied species (IC
50
= 27.8 ± 0.6 μg/ml).
Additionally, the supernatants of Chlorella sorokiniana
HSSASE17 and Dolichospermum flos-aquae HSSASE2 dem-
onstrated moderate cytotoxicity against PBMCs recording
IC
50
values of 498.9 ± 0.3 and 337 ± 0.9 μg/ml, respectively
(Table 3).
Evaluation of anticancer activity
The anticancer efficiency of the screened strains was in vitro
assayed against four different cancerous cell lines: colon
cancer (Caco-2), breast cancer (MCF-7), liver cancer
(HepG-2), and prostate cancer (PC3) cell lines. Table 4
Illustrates that the cyanobacteria Dolichospermum crassum
HSSASE20 had the highest anticancer potential among all
tested species against Caco-2 and PC3 cell lines, recording
the lowest IC
50
values of 57.9 ± 0.4, and 44.1 ± 0.2 μg/ml,
respectively, which was greater than that reported for positive
control; 5-fluorouracil. On the other hand, the supernatant of
Oscillatoria sancta HSSASE19 showed the most anticancer
effect against MCF-7 cell line (IC
50
=15.1±0.7 μg/ml),
followed by Wollea saccata HSSASE12 (38.4 ± 0.5 μg/ml)
and Dolichospermum crassum HSSASE20 (41.1 ±
0.3 μg/ml), compared to 5-fluorouracil (80.8 ± 1.7 μg/ml).
The supernatant of Dolichospermum spiroides HSSASE18
demonstrated a potent anticancer effect against HepG-2 cell
line, recording IC
50
value of (48.8 ± 0.7 μg/ml), followed by
Dunaliella sp. HSSASE13 (65.7 ± 0.7 μg/ml) and
Dolichospermum crassum HSSASE20 (68.6 ± 0.5 μg/ml),
compared to positive control (71.2 ± 1.6 μg/ml).
Additionally, data obtained from the sensitivity index revealed
that the supernatant of Dolichospermum crassum HSSASE20
was the most sensitive species against the four cancerous cell
lines among all the screened strains, followed by the superna-
tant of Oscillatoria sancta HSSASE19 (Fig. 5).
Discussion
Cyanobacteria are recognized as one of the most promising
organisms from which new biochemically active, antioxidant,
and anticancer natural products can be isolated. Cyanobacteria
are abundant sources of secondary metabolites with promising
0
500
1000
1500
2000
2500
3000
IC50 values (µg/ml)
Fig. 4 In vitro inhibition of lipid
peroxidation by cyanobacteria
and microalgae extracts. Data are
represented as half-maximal in-
hibitory concentration (IC
50
;
μg/ml) for each extract, compared
to a positive control (vitamin C)
Table 3 Cytotoxicity of different algal extracts against normal human
peripheral blood mononuclear cells (PBMCs)
Algal extract IC
50
values (μg/ml)
Dolichospermum flos-aquae HSSASE2 337 ± 0.9
Oscillatoria sp. HSSASE4 104.1 ± 0.3
Anabaena oryzae HSSASE6 186.1 ± 0.4
Leptolyngbya fragilis HSSASE9 27.8 ± 0.6
Anabaena sp. HSSASE11 186.7 ± 0.3
Wollea saccata HSSASE12 209 ± 0.3
Dunaliella sp. HSSASE13 266 ± 0.001
Dolichospermum circinale HSSASE14 52.8 ± 0.3
Oscillatoria nigro-viridis HSSASE15 154.2 ± 0.3
Aphanizomenon gracile HSSASE16 238 ± 0.8
Chlorella sorokiniana HSSASE17 498.9 ± 0.3
Dolichospermum spiroides HSSASE18 277.5 ± 0.9
Oscillatoria sancta HSSASE19 991.1 ± 0.5
Dolichospermum crassum HSSASE20 70.6 ± 0.8
Environ Sci Pollut Res (2020) 27:18463–18474
18470
biotechnological industries (Costa et al. 2012). Numerous dis-
tinctive chemical compounds have been isolated and exam-
ined from marine, and freshwater algae with a variety of bio-
logical activities and some remain to be investigated in the
future use for the development of pharmaceutical drugs
(Bleakley and Hayes 2017;Ercolanoetal.2019), while those
isolated from soil samples were inappropriately investigated.
Therefore, this research is conducted to screen 14 samples
of new algal species (cyanobacteria and Chlorophyta) isolated
from soil samples at two different areas in El-Sharkia
Governorate: rice field soil (El-Rowad Village) and wheat
field soil (Sahl El-Hussinia), as well as the Bahr Hadus agri-
culture drain water station in Egypt for potential antioxidant
and anticancer evaluation. In this screening, 12 cyanobacteria
and two chlorophyta species were isolated and screened for
the antioxidant profile and anticancer potential (Tables 2,3,
4). Out of the 14 strains identified, the cyanobacterium
Dolichospermum flos-aquae HSSASE2 documented the most
elevated antioxidant activity in terms of NO scavenging activ-
ity and anti-lipid peroxidation potential (28.7± 0.1 & 11.9±
0.2 μg/ml, respectively) and lowest DPPH radical scavenging
activity (467.7 ± 1.6 μg/ml). Additionally, D. flos-aquae
0
5
10
15
20
25
Sensivity Index (SI)
Caco-2
MCF-7
HepG-2
PC3
Fig. 5 Selectivity index (SI) of
different algal extracts against
colon cancer (Caco-2), breast
cancer (MCF-7), liver cancer
(HepG-2), and prostate cancer
(PC3) cell lines
Table 4 Anticancer activity of
cyanobacteria and microalgae
extracts against colon cancer
(Caco-2), breast cancer (MCF-7),
liver cancer (HepG-2), and
prostate cancer (PC3) cell lines
Sample IC
50
values (μg/ml)
Caco-2 MCF-7 HepG-2 PC3
Dolichospermum flos-aquae HSSASE2 306 ± 0.9 361.1 ± 0.5 71.8 ± 0.6 198.3 ± 0.7
Oscillatoria sp. HSSASE4 440 ± 0.8 290.8 ± 0.4 396.1 ± 0.9 286.9 ± 0.6
Anabaena oryzae HSSASE6 263 ± 0.9 223.8 ± 0.4 302.7 ± 0.5 71.1 ± 0.3
Leptolyngbya fragilis HSSASE9 167.3 ± 0.6 183.1 ± 0.6 251 ± 0.8 295.1 ± 0.8
Anabaena sp. HSSASE11 74.8 ± 0.7 197.9 ± 0.8 149 ± 0.5 143.7 ± 0.7
Wollea saccata HSSASE12 265.8 ± 0.7 38.4 ± 0.5 123 ± 0.5 202.8 ± 0.6
Dunaliella sp. HSSASE13 106.8 ± 0.3 64.7 ± 0.5 65.7 ± 0.7 162.1 ± 0.3
Dolichospermum circinale HSSASE14 640.4 ± 0.6 137.5 ± 0.5 131.2 ± 0.3 155 ± 0.3
Oscillatoria nigro-viridis HSSASE15 550.9 ± 0.1 305.6 ± 0.5 251 ± 0.2 233 ± 0.2
Aphanizomenon gracile HSSASE16 284.9 ± 0.8 97.9 ± 0.6 279.2 ± 0.7 339.1 ± 0.8
Chlorella sorokiniana HSSASE17 319 ± 0.9 115.8 ± 0.8 165.6 ± 0.5 106.5 ± 0.5
Dolichospermum spiroides HSSASE18 124.2 ± 0.5 94.7 ± 0.5 48.8 ± 0.7 293.8 ± 0.7
Oscillatoria sancta HSSASE19 96.7 ± 0.7 15.1 ± 0.7 103.8 ± 0.5 97.7 ± 0.5
Dolichospermum crassum HSSASE20 57.9 ± 0.4 41.1 ± 0.3 68.6 ± 0.5 44.1 ± 0.2
5-Fluorouracil 77.4 ± 0.8 80.8 ± 1.7 71.2 ± 1.6 79.9 ± 0.2
All values are expressed as the mean ± standard error of mean (SEM) with significance p<0.01
Environ Sci Pollut Res (2020) 27:18463–18474 18471
HSSASE2 showed a moderate anticancer potential against
Caco-2, MCF-7, and PC3 cell lines, while higher anticancer
activity was recorded against HepG-2 cell line (IC
50
=
71.8 μg/ml). The cytotoxicity of D. flos-aquae HSSASE2
could be attributed to its high content of phenols (20.1 mg
GAE/g) and antioxidant properties.
PBMCs are human lymphocytes functionally assessed by sur-
vival, proliferation, and cytotoxicity analysis (Shipkova and
Wieland 2012). The MTT assay is a suitable method used to
assess cell proliferation as a function of redox system (Molaae
et al. 2017). Screening the cytotoxicity of the studied strains
against healthy PBMC cells revealed that the cyanobacterium
Oscillatoria sancta HSSASE19 was the safest one against human
PBMCs recording IC
50
value of 991.1 ± 0.5 μg/ml and the
highest anticancer effect against MCF-7 cell line (IC
50
= 15.1 ±
0.7 μg/ml) among all strains. On the other hand, the cyanobacteria
Leptolyngbya fragilis HSSASE9 displayed the most toxic strain
among all studied species (IC
50
= 27.8 ± 0.6 μg/ml) against nor-
mal PBMCs and moderate anticancer effect against colon cancer,
breast cancer, hepatic cancer, and prostate cancer cell lines (167.3,
183.1, 251, and 295.1, respectively). This is maybe due to the
secondary metabolites produced by cyanobacteria. Secondary
metabolites extracted from cyanobacteria participate significantly
in the development of drugs with versatile biological activities,
such as anti-inflammatory, antifungal, antibacterial, immunosup-
pressive, and anticancer. Previous research documented that most
of these compounds had been extracted from the genera
Oscillatoriales, Lyngbya, Mooria, Okeania,andCaldora
(Vijayakumar et al. 2016;Mietal.2017).
Assessment of the anticancer effect of all studied strains
against different human cancer cell lines demonstrated that
the cyanobacterium Dolichospermum crassum HSSASE20
had the highest anticancer effect among all tested species
against colon and prostate cancer cell lines (IC
50
values =
57.9 ± 0.4, and 44.1 ± 0.2 μg/ml, respectively). D. crassum
HSSASE20 recorded high antioxidant enzymes activity
(Fig. 1), as well as anti-lipid peroxidation potential (44.4 ±
0.6 μg/ml), which may contribute to its cytotoxic effect
against cancerous cell lines. Additionally, D. crassum
HSSASE20 is the most sensitive species against the four can-
cerous cell lines among all the screened strains (Fig. 5). The
cytotoxicity of marine cyanobacteria compounds against hu-
man cancer cell lines are the most evaluated, and several com-
pounds have emerged for the discovery of new anticancer
drugs (Ercolano et al. 2019). Activation of the apoptotic path-
way is one of the most studied mechanisms involved in the
cyanobacteria cytotoxicity. Some drugs have been discovered
to activate caspase 3, while others have been found to cause
cell cycle arrest/mitochondrial dysfunctions or modifications
in the BCL-2 family of proteins (Costa et al. 2012). Secondary
metabolites isolated from marine cyanobacterial mainly ex-
tracted from Oscillatoriales orders followed by Nostocales
and Chroococcales (Tan 2010).
Another interesting point concerning chlorophyta, they
have been used as a food source for animal feed, human con-
sumption, and aquaculture as well as for other commercial
purposes such as coloring agents, cosmetics, and others
(Plaza et al. 2009). Two chlorophyta species were isolated
from the soil samples, which are Dunaliella sp. HSSASE13
and Chlorella sorokiniana HSSASE17. Our results confirmed
a significant anticancer effect of Dunaliella sp. HSSASE13
against hepatic cancer cell line (Table 4). The cytotoxicity of
Dunaliella sp.is due to its content of phenolic compounds,
which exhibited high antioxidant activity, and antioxidant en-
zymes (Fig. 1). This result is in agreement with Murthy et al.
who reported that Dunaliella salina has a protective and anti-
oxidant effect against oxidative stress-induced in rats by CCl4
due to enhanced activity of antioxidant enzyme peroxidase,
SOD, and catalase.
Concerning the microalga Chlorella sorokiniana
HSSASE17, it showed a moderate anticancer activity against
human colon, breast, hepatic, and prostate cancer cell lines
(IC
50
= 319 ± 0.9, 115.8 ± 0.8, 165.6 ± 0.5, and 106.5 ±
0.5 μg/ml, respectively), as well as a moderate antioxidant
activity (Figs. 1,2,and3) and the highest catalase activity
(3.51 U/g) among all strains. The anticancer effect of
C. sorokiniana HSSASE17 strain may be due to activation of
the apoptotic pathway. These primary findings are consistent
with research showing that activation of the pro-apoptotic
proteins is the primary cause of the cytotoxicity of hot water
extracts of Chlorella sorokiniana (marine origin) against lung
adenocarcinoma cell lines (A549 and CL1–5) (Lin et al.
2017). Furthermore, many green algae species can produce
several secondary metabolites when subjected to stress or
sub-optimal conditions such as salinity, light intensity, nutrient
deprivation, temperature, and pH conditions, which are
health-promoting agents. Some of them also showed promis-
ing anticancer activities (Ercolano et al. 2019).
In another study, 10 strains from the microalga species
Desmococcus olivaceus,Chlorella,andScenedesmus showed
significant anticancer activity (Ördög et al. 2004;Marrezetal.
2019). Chlorella vulgaris is the best-studied, which has been
widely used as a food supplement for its health benefits and its
high nutritive value. Regarding the anticancer potential, sev-
eral studies have shown that Chlorella extracts exhibit a cyto-
toxic effect on multiple human cancer cell lines. Another an-
ticancer screening of the microalga Chlorella vulgaris was
evaluated against multiple cancerous cell lines: HepG2
(Yusof et al. 2010), human lung carcinoma A549 and NCI-
H460 cells (Zhang et al. 2017), and rat models of liver cancer
(Mohd Azamai et al. 2009). The authors linked the significant
anticancer effect of C. vulgaris to its capacity to activate apo-
ptotic signaling pathways. Also, C. vulgaris has been found to
prevent cell proliferation and to promote apoptosis cascades.
Due to the microbial diversity, secondary metabolites pro-
duced from cyanobacteria and microalgae may constitute an
Environ Sci Pollut Res (2020) 27:18463–18474
18472
efficient source for new pharmaceutical drug development.
So, further studies are needed to isolate and identify new bio-
active compounds and thoroughly analyze the in vivo biolog-
ical activity of each compound at the chemical, biochemical,
and molecular levels. In conclusion, this screening study iso-
lated 14 strains of cyanobacteria and microalgae from soil
samples with a varying degree of antioxidant and anticancer
potential against different human cancer cell lines. Results of
this study confirmed that several cyanobacteria and
microalgae are efficient free radical scavengers and preventing
cancerous cell proliferation. This influential activity might be
linked to their potent phenolic content.
Funding information This work was supported by the Ministry of
Scientific Research, Egypt under the coordination project between
Egypt and Tunisia No. 10-4-22. The project was operated at the City of
Scientific Research and Technology Application.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126
Agatonovic-Kustrin S, Morton DW (2018) Quantification of polypheno-
lic antioxidants and free radical scavengers in marine algae. J Appl
Phycol 30(1):113–120
Allen MM, Stanier RY (1968) Growth and division of some unicellular
blue-green algae. J Gen Microbiol 51(2):199–202
Bleakley S, Hayes M (2017) Algal proteins: extraction, application, and
challenges concerning production. Foods 6(5):E33. https://doi.org/
10.3390/foods6050033
Boyum A (1968) Isolation of mononuclear cells and granulocytes from
human blood. Isolation of monuclear cells by one centrifugation,
and of granulocytes by combining centrifugation and sedimentation
at 1 g. Scand J Clin Lab Investig Suppl 97:77–89
Braca A, De Tommasi N, Di Bari L, Pizza C, Politi M, Morelli I (2001)
Antioxidant principles from Bauhinia tarapotensis. J Nat Prod 64(7):
892–895
Caldwell GS (2009) The influence of bioactive oxylipins from marine
diatoms on invertebrate reproduction and development. Mar Drugs
7(3):367–400
Costa M, Costa-Rodrigues J, Fernandes MH, Barros P, Vasconcelos V,
Martins R (2012) Marine cyanobacteria compounds with anticancer
properties: a review on the implication of apoptosis. Mar Drugs
10(10):2181–2207
Ekwall B, Silano V, Paganuzzi-Stammati A, Zucco F (1990) Toxicity
tests with mammalian cell cultures. In: Short-Term Toxicity Tests
for Non-Genotoxic Effects. Wiley, New York, pp 75–97
Ercolano G, De Cicco P, Ianaro A (2019) New drugs from the sea: pro-
apoptotic activity of sponges and algae derived compounds. Mar
Drugs 17(1)
Fernando IP, Kim M, Son KT, Jeong Y, Jeon YJ (2016) Antioxidant
activity of marine algal polyphenolic compounds: a mechanistic
approach. J Med Food 19(7):615–628
Ferris MJ, Hirsch CF (1991) Method for isolation and purification of
cyanobacteria. Appl Environ Microbiol 57(5):1448–1452
Ho SC, Tang YL, Lin SM, Liew YF (2010) Evaluation of peroxynitrite-
scavenging capacities of several commonly used fresh spices. Food
Chem 119(3):1102-1107
Hossain MF, Ratnayake RR, Meerajini K, Wasantha Kumara KL (2016)
Antioxidant properties in some selected cyanobacteria isolated from
fresh water bodies of Sri Lanka. Food Sci Nutr 4(5):753–758
Huang Z, Guo BJ, Wong RNS, Jiang Y (2007) Characterization and
antioxidant activity of selenium-containing phycocyanin isolated
from Spirulina platensis. Food Chem 100(3):1137–1143
Jain S, Jain A, Jain S, Malviya N, Jain V, Kumar D (2015) Estimation of
total phenolic, tannins, and flavonoid contents and antioxidant ac-
tivity of <i>Cedrus deodara</i>heart wood extracts. Egypt Pharm J
14(1):10–14
Jiang A, Tian S, Xu Y (2002) Effect of controlled atmospheres with high
O2 or high-CO2 concentrations on postharvest physiology and
storability of “Napoleon”sweet cherry 2002. 925-930
Kim S-Y, Kim E-A, Kang M-C, Lee J-H, Yang H-W, Lee J-S, Lim TI,
Jeon Y-J (2014) Polyphenol-rich fraction from Ecklonia cava (a
brown alga) processing by-product reduces LPS-induced inflamma-
tion in vitro and in vivo in a zebrafish model. Algae 29(2):165–174
Kranz D, Dobbelstein M (2012) A killer promoting survival: p53 as a
selective means to avoid side effects of chemotherapy. Cell Cycle
(Georgetown, Tex) 11(11):2053–2054
Landsberg JH (2002) The effects of harmful algal blooms on aquatic
organisms. Rev Fish Sci 10(2):113–390
Lin P-Y, Tsai C-T, Chuang W-L, Chao Y-H, Pan IH, Chen Y-K, Lin CC,
Wang BY (2017) Chlorella sorokiniana induces mitochondrial-
mediated apoptosis in human non-small cell lung cancer cells and
inhibits xenograft tumor growth in vivo. BMC Complement Altern
Med 17(1):88
Marrez DA, Naguib MM, Sultan YY, Higazy AM (2019) Antimicrobial
and anticancer activities of Scenedesmus obliquus metabolites.
Heliyon 5(3):e01404
Martinez Andrade KA, Lauritano C, Romano G, Ianora A (2018) Marine
microalgae with anti-cancer properties. Mar Drugs 16(5)
Mayer AM, Rodriguez AD, Berlinck RG, Hamann MT (2009) Marine
pharmacology in 2005-6: marine compounds with anthelmintic, an-
tibacterial, anticoagulant, antifungal, anti-inflammatory, antimalari-
al, antiprotozoal, antituberculosis, and antiviral activities; affecting
the cardiovascular, immune and nervous systems, and other miscel-
laneous mechanisms of action. Biochim Biophys Acta 1790(5):
283–308
Mi Y, Zhang J, He S, Yan X (2017) New peptides isolated from marine
cyanobacteria, an overview over the past decade. Mar Drugs 15(5):
132
Mohd Azamai ES, Sulaiman S, Mohd Habib SH, Looi ML, Das S, Abdul
Hamid NA, Ngah WZW, Yusof YAM (2009) Chlorella vulgaris
triggers apoptosis in hepatocarcinogenesis-induced rats. J Zhejiang
Univ Sci B 10(1):14–21
Molaae N, Mosayebi G, Pishdadian A, Ejtehadifar M, Ganji A (2017)
Evaluating the proliferation of human peripheral blood mononuclear
cells using MTT assay. Int J Basic Sci Med 2(1):25–28
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65(1–2):55–63
Ng TB, Liu F, Wang ZT (2000) Antioxidative activity of natural products
from plants. Life Sci 66(8):709–723
Ördög V, Stirk WA, Lenobel R, Bancířová M, Strnad M, Van Staden J,
Szigeti J, Németh L (2004) Screening microalgae for some poten-
tially useful agricultural and pharmaceutical secondary metabolites.
J App Phyco 16(4):309–314
Ozdemir G, Horzum Z, Sukatar A, Karabay-Yavasoglu NU (2006)
Antimicrobial activities of volatile components and various extracts
of Dictyopteris membranaceae. and Cystoseira barbata. from the
Coast of Izmir, Turkey. Pharm Biol 44(3):183–188
Environ Sci Pollut Res (2020) 27:18463–18474 18473
Perez EA (2008) Impact, mechanisms, and novel chemotherapy strategies
for overcoming resistance to anthracyclines and taxanes in metasta-
tic breast cancer. Breast Cancer Res Treat 114(2):195
Plaza M, Herrero M, Cifuentes A, Ibanez E (2009) Innovative natural
functional ingredients from microalgae. J Agric Food Chem 57(16):
7159–7170
Prayong P, Barusrux S, Weerapreeyakul N (2008) Cytotoxic activity
screening of some indigenous Thai plants. Fitoterapia 79(7):598–
601
Saeed N, Khan MR, Shabbir M (2012) Antioxidant activity, total pheno-
lic and total flavonoid contents of whole plant extracts Torilis
leptophylla L. BMC Complement Altern Med 12:221
Shanab SMM, Shalaby EA, El-Fayoumy EA (2011) Enteromorpha
compressa exhibits potent antioxidant activity. J Biomed
Biotechnol. https://doi.org/10.1155/2011/726405
Shanura Fernando IP, Asanka Sanjeewa KK, Samarakoon KW, Lee WW,
KimH-S,KimEA,GunasekaraUKDSS,AbeytungaDTU,
Nanayakkara C, de Silva ED, Lee HS, Jeon YJ (2017) FTIR char-
acterization and antioxidant activity of water soluble crude polysac-
charides of Sri Lankan marine algae. Algae 32(1):75–86
Shipkova M, Wieland E (2012) Surface markers of lymphocyte activation
and markers of cell proliferation. Clin Chim Acta 413(17–18):1338–
1349
Tan LT (2010) Filamentous tropical marine cyanobacteria: a rich source
of natural products for anticancer drug discovery. J Appl Phycol
22(5):659–676
Thajuddin N, Subramanian G (2005) Cyanobacterial biodiversity and
potential applications in biotechnology. Curr Sci 89(1):47–57
Vijayakumar S, Manogar P, Prabhu S (2016) Potential therapeutic targets
and the role of technology in developing novel cannabinoid drugs
from cyanobacteria. Biomed Pharmacother 83:362–371
Whetten RW, Sederoff RR (1992) Phenylalanine ammonia-lyase from
loblolly pine. Plant Physiol 98(1):380–386
Williams GM, Iatropoulos MJ, Whysner J (1999) Safety assessment of
butylated hydroxyanisole and butylated hydroxytoluene as antioxi-
dant food additives. Food Chem Toxicol 37(9–10):1027–1038
Witsch HP (1986) Enhanced tumour development by butylated hydroxy-
toluene (BHT) in the liver, lung and gastro-intestinal tract. Food
Chem Toxicol 24:1127–1130
Yen GC, Duh PD (1994) Scavenging effect of methanolic extracts of
peanut hulls on free-radical and active-oxygen species. J Agric
Food Chem 42(3):629–632
Yusof YAM, Saad SM, Makpol S, Shamaan NA, Ngah WZW (2010) Hot
water extract of Chlorella vulgaris induced DNA damage and apo-
ptosis. Clinics (Sao Paulo) 65(12):1371–1377
Zhang ZD, Liang K, Li K, Wang GQ, Zhang KW, Cai L, Zhai ST, Chou
KC (2017) Chlorella vulgaris induces apoptosis of human non-small
cell lung carcinoma (NSCLC) cells. Med Chem 13(6):560–568
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