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ABSTRACT
Background: Among marine organisms, seaweeds are a highly
diverse group of organisms from which many new substances have
been isolated and many of these compounds have been demonstrated
to possess a large spectrum of bioactivities.
Objective: In this study, we aim to evaluate the antibacterial,
cytotoxic and antioxidant activity of green algae, Cladophora
prolifera (Roth) Kutzing, collected from the northern Mediterranean
coast of Morocco.
Material and Method: The antibacterial activity was determined by
disk diffusion method. After fractionation by column
chromatography, the fractions from C. prolifera were tested against
Staphylococcus aureus ATCC 25923 using the broth microdilution
assay. The antitumor effect on human colon cancer cells was
investigated via sulforhodamine-B (SRB) assay. Then the Crude
extract has been tested in radical-scavenging assays to assess their
antioxidant activity.
Results: In vitro screening of methanolic extract of C. prolifera
Address for showed specific activity to inhibit the growth of five virulent strains
of pathogenic bacteria, Escherichia coli (ATCC 25922),
Correspondence
Staphylococcus aureus (ATCC 25923), Staphylococcus aureus
Department of (ATCC 29213), Enterococcus faecalis (ATCC 29212) and Klebsiella
Biology, Faculty of pneumoniae (ATCC 700603). The obtained results indicated that the
Sciences, University of extracts of C. prolifera were cytotoxic against HT29 human colon
Abdelmalek Essaâdi, cancer cells. In addition, based on the capacity of the algae to
93030 Tetouan, scavenge the ABTS radical cation, we revealed that C. prolifera
Morocco. extract presented a satisfactory antioxidant activity.
E-mail: Conclusion: These results suggest that C. prolifera has a great
zbakh.h@hotmail.com biological potential, which could be considered for future uses in
food, pharmaceutical and cosmetic industries.
INTRODUCTION
In recent years, natural products have macroalgae could be due to the fatty acids
been playing a major role in the search for constituents15. Many fatty acids isolated then
novel drugs or drug candidates against from C. prolifera showed anti-coagulant16
infectious diseases, inflammation, cancer anti-inflammatory17, antiviral18 and
19
and many other illnesses. They are an antihelmintic activities. Hence, the main
ongoing and inspiring source for researchers objective of this study was to assess the
due to their enormous structural diversity antitumoural, antibacterial and antioxidative
and complexity. potentialities of C. prolifera settled along
The marine algae represents a largely the northern Mediterranean coast of
unexplored source for the isolation of novel Morocco. The anti-proliferative effect of C.
bioactive compounds and may become even prolifera on HT-29 human colon cancer
more so as knowledge on marine natural cells was investigated. We also evaluated
products. Thus, macroalgae has been the antimicrobial activity of the macroalgae
recognized as a promising source of extract against gram-positive and gram-
bioactive secondary metabolites with negative bacterial strains using the diffusion
antitumor1,2, antibacterial3,4, antioxidant5,6, method. Finally, for assessment of
anti-inflammatory5,7 and antifouling antioxidant properties, the ABTS free
8
activities . radical decolorization assay was used.
The chemical structures of these
seaweeds derived bioactive substances are MATERIAL AND METHODS
diverse, including brominated phenols,
sterols, polysaccharides, peptides, proteins, Chemicals
acrylic acid, chlorophyllides, terpenes, Ethanol, acetone and methanol were
phenols and heterocyclic carbons9,10. Some obtained from Merck (Darmstadt,
of these bioactive compounds are Germany). 2, 2-azino-bis (3-ethylbenzothia-
antimicrobial, anticancer and antioxidant zoline-6-sulfonic acid) diammonium salt
agents. Cladophora prolifera (Roth.) (ABTS) and 6-hydroxy-2, 5, 7, 8-
Kutzing, is a green seaweed extensively tetramethylchroman-2-carboxylic acid
distributed in the Mediterranean and (Trolox) were purchased from Sigma
Atlantic seas of Morocco. Chemical Chemical Co. (St. Louis, MO, USA).
analyses of the species, revealed its richness Potassium persulfate were procured from
in important mineral oligoelements11. The Fluka Chemical Co. (Buchs, Swizerland)
protein content is very high (24.62%)12, and sulforhodamine-B (SRB) was purchased
especially when compared to other green from Sigma-Aldrich (Taufkirchen,
algae utilized by the industry13. However, Germany).
little information about its biological active
substances is available. Therefore, numerous Algal materials
studies have demonstrated that the extracts Samples of Cladophora prolifera
of C. prolifera showed powerful (Roth) Kutzing were collected from the
14
antibacterial activities . Many authors northern Mediterranean coast of Morocco,
suggested that the antibacterial activities of during the summer of 2007 (Ksar-sghir
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flow rate of 0.5 mL/min. The purification fetal bovine serum (FBS), 100 U/mL
was archived after silica gel flash penicillin and 100 mg/mL streptomycin and
chromatography mono and bidimensional was put in a humidified incubator at 37°C
silica gel TLC. Seaweed extracts were under an atmosphere of 5% CO2. The C.
applied and the chromatogram developed prolifera extract was dissolved in DMSO
using different proportions of and added to the culture medium, so that the
acetone/hexane/methanol as solvent. TLC final concentration of DMSO was less than
plates were run in duplicate and one set was 1%.
used as the reference chromatogram. Spots
and bands were visualized by UV irradiation Sulforhodamine B assay
(254 and 366 nm) and H2SO4 spray Sulforhodamine B (SRB) (Sigma–
reagent23. Fractions of the similar TLC Aldrich, Germany) was used to test the
profile were combined to get the final effect of C. prolifera extract on cell growth
fractions, which were free from solvents, and viability based on the method described
redissolved in an appropriate solvent after by Vichai and Kirtikara24. The extract was
weighing and screened for antibacterial dissolved in dimethylsulfoxide (DMSO)
activity by disc diffusion methods as before diluting with the growth medium to a
described above (25 µL solvent/ 6 mm disc). final DMSO concentration of <0.05%. The
cancer cells were inoculated into 96 well
Broth microdilution assay plates in the growth medium at 5000
The active extract and fraction of C. cells/well. After 24 h of incubation, the cells
prolifera were tested by using the broth were exposed to various concentrations of
microdilution checkerboard technique for C. prolifera extract (6.25, 12.5, 25, 50 and
performing antimicrobial susceptibility 100 µg/mL) and 5-fluorouracil (5-FU) (5,
testing. The microtitertrays, containing 10, 25, 50 and 100 µg/mL) used as positive
various volumes (25, 6.25, 3.12, 1.56, 0.78, control. The cells were then incubated for 48
0.39, 0.19, 0.09, 0.048 and 0.024 µL) of h and 72h. The cells were fixed with TCA
methanolic extract and active fraction of C. by gently adding 50 µL TCA (50%) to each
prolifera, was prepared. Then, the bacteria well for 1 h at 4°C. The plates were then
strain was inoculated into the wells of 96- washed 5 times with deionized water and
well microtiter and incubated at 37ºC for 16 air-dried. The dried plates were stained with
to 20 h. The determination of the minimum 100 µL of 0.4% (w/v) SRB prepared in 1%
inhibitory concentration (MIC, µL/mL) was (v/v) acetic acid for 30 min at room
then made by checking whether or not S. temperature. The plates were rinsed quickly
aureus grew in the various concentrations of 5 times with 1% acetic acid to remove the
the crude extraction and fractions. Turbidity unbound dye, followed by air-drying. The
of the broth or a button of cells at the bottom bound dye was solubilized in 2 mM Tris
of the well is considered as evidence of base (100 µL/well) for 5 min. Optical
growth. densities were read on a microplate reader
(Spectrophotometer Labsystems Multiskan
Cell cultures EX at λ = 492nm).
HT-29 human colon carcinoma cells
were obtained from the American Type ABTS radical scavenging activity
Culture Collection (ATCC, USA). The cells Antioxidant activity was determined
were maintained in McCoy’s medium by ABTS free radical decolorization assay
supplemented with 10% heat-inactivated developed by Arnao et al.25 with a slight
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positive bacteria were more effectively showed higher efficiency compared to the
controlled by the majority of Australian crude extracts (Figure 2).
algal extracted. The identity of the peaks was
confirmed by TLC with known standards.
HPLC analysis and fractionation of the Organic acids and phenolic compounds,
methanolic extract especially polyphenols or tannins have been
Active methanolic extract of the C. shown to have antimicrobial activities34,35.
prolifera (Rothpletz) Kutzing was analyzed Rosell and Srivastava15 detected small
by HPLC to identify the nature and the amounts of free organic acids and
number of major constituents of the active polyphenols in the algal extracts and
extract. The obtained chromatogram is consequently pointed out that these
shown in the Figure 1. The chromatogram substances play only a minor role in the
revealed the presence of a major compound antibacterial activities recorded in their
that corresponds to the peak C (retention study. We think that the antimicrobial
time, RT = 29 min). The other peaks activity shown by C. prolifera is attributed
correspond to minor compounds, A (RT = to the presence of fatty acids in the fractions
27 min), B (RT = 28 min) and D (RT = 37 obtained from this alga. The noteworthy
min) (Figure 1). capability of fatty acid to produce
The methanolic extract was then antimicrobial activities has been also
separated using chromatography column. reported by Kabara36, McCracken et al. 37
Twelve fractions were further obtained after and Rosell and Srivastava15 as well as the
silica gel flash chromatography; they were antibiotic activity from ten Xantophyta was
eluted using a gradient polarity (hexane, associated with the presence of organic
ethyl acetate and methanol). Each fraction acids, unsaturated fatty acids and phenolic
displayed a major spot in TLC using compounds. While, Vaskovsky38 and
different dyes and UV light. Zhukova et al.39 reported the presence of
The obtained fractions were then fatty acids, in the algae Cladophora
tested against Staphylococcus aureus 1 rudolphiana and also in some species of the
(Table 2). Tree fractions (CPF3, CPF4 and genera Rhodomela, Gracilaria, Sargassum,
CPF7) of petroleum ether extract of C. Ulva, Enteromorpha and Zostera, Dunaliella
prolifera showed high growth inhibitory and Chlorella, ranging from 14:0 to 20:5
activity against Staphylococcus aureus 1. with predominating unsaturated fatty acids.
However, only one fraction (CPF5) showed
moderate activity and seven showed no Cell proliferation inhibition
activity against the tested bacteria (Table 2). To identify the anti-cancer
Comparative data of the minimal phytochemicals potential, we examined the
inhibitory concentrations (MIC) of cytotoxicity of the seaweed C. prolifera
methanolic extract and active fractions of C. extract on the HT-29 human colon cancer
prolifera are presented in Figure 2. The cells using the SRB method with 5-FU as a
fractions of C. prolifera extract, CPF3, positive control. The result of the cytotoxic
CPF4, CPF5 and CPF7 (eluted with ethyl activity is shown in Figure 3. The
acetate and hexane) showed MIC values of percentages of growth inhibition of HT-29
3.12, 6.25, 1.56 and 3.12 µL/mL, cells by the C. prolifera extract at various
respectively. In addition, the inhibitory concentrations were determined as the
effect of fractions of microdilution method percentage of viable treated cells in
comparison with viable cells of untreated
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Toluene-ethanol
- + - - -
(1:1)
Ethanol - - - - -
Ampicillin + + nt ++ ++
nt: Not tested
CPF1 -
CPF2 -
CPF3 +++
CPF4 +++
CPF5 ++
CPF6 -
CPF7 +++
CPF8 +
CPF9 -
CPF10 -
CPF11 -
CPF12 -
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Figure 1. HPLC chromatogram of the analysis of the methanolic crude extract of C. prolifera
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120
C. prolifera 48 h
C. prolifera 72 h
100
5-FU 48 h
5-FU 72 h
Cell viability (%) 80
60
40
20
0
0 20 40 60 80 100
Concentration (µg/mL)
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