Antibacterial Activity of Lipophilic and
Hydrophilic Extracts of Algae
Suneerat Ruangsomboon1*, Sakchai Choochote1, Paveena Taveekijakarn1and
Suwanna Worasing2
1
Program in Fisheries Science, Division of Animal Production Technology and Fisheries,
Faculty of Agricultural Technology, King Mongkut’s Institute of
Technology Ladkrabang, Bangkok 10520, Thailand
2
Trad Coastal Aquaculture Station, Department of Fisheries,
Ministry of Agriculture and Cooperatives
Abstract
Calothrix parietina, Gloeocapsa sp., Mastigocladus sp., Nostoc commune, Oscillatoria sp.,
Phormidium sp., Spirulina sp, were cultured in BG-11 medium and Ulva rigida were collected
during the summer from the Aou-Chaw beach, Trad province. Their metabolites were extracted
using chloroform:methanol (2:1) and water. The effect of lipophilic and hydrophilic extracts on
the antibacterial activities against five bacteria (Streptococcus agalactiae, Aeromonas hydrophila,
Pseudomonas sp., Vibrio alginolyticus, Pseudomonas fluorescent) was studied. The lipophilic
extracts of 3 algal strains Mastigocladus sp., N. commune and Oscillatoria sp. showed
antibacterial activity against S. agalactiae. The most active extract was from N. commune. The
hydrophilic extracts of 3 algal strains Mastigocladus sp., N. commune and U. rigida, showed
antibacterial activity against several pathogenic bacteria. The algal strain U. rigida provided the
most active extract. No inhibitory effect was found against the gram-negative bacterium, P.
fluorescent.
Keywords: algae, cyanobacteria, antibacterial, lipophilic extract, hydrophilic extract
1. Introduction
Research activities concerning the investigation of products of metabolism of plants and other
groups of organisms are undertaken not only for a better understanding of nature but also to
discover metabolites of possible use for humans in different fields of interest. The screening of
extracts or isolated compounds from different natural sources is a common way to discover
biological active metabolites. The current application of chemical compounds isolated from
diverse classes of algae is enormous. Recent trends in drug research from natural sources suggest
that algae are a promising group to furnish novel biochemically active substances [1].
To survive in a competitive environment, freshwater and marine algae have developed
defense strategies that result in a significant level of structural– chemical diversity, from different
*Corresponding author: Tel-Fax:66(2)-329-8517
E-mail: [email protected], [email protected]
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metabolic pathways [2-3]. The exploration of these organisms for pharmaceutical purposes has
revealed important chemical prototypes for the discovery of new agents, stimulating the new
syntheses of compounds with biomedical application.
Various group of algae especially, cyanobacteria (blue-green algae) have received
growing attention as producers of a diverse array of toxic or otherwise biologically active
compounds with potential applications in biomedicine, as well as implications for environmental
health [4-5]. A lot of active substances with antibacterial, antiviral, fungicide, enzyme inhibiting,
immunosuppressive, cytotoxic and algicide activity has been isolated from algal biomass [6-7].
Moreover, algae are promising organisms for providing essential compounds for human nutrition
[8].
The aim of the present work, was to examine the antibacterial activity of seven genera of
cyanobacteria Calothrix marchica, Gloeocapsa gelatinosa, Mastigocladus sp., Nostoc commune,
Oscillatoria jasorvensis, Phormidium angustissimum, Spirulina platensis and one genus of green
macroalga Ulva rigida.
2. Materials and Methods
2.1. Algal and growth condition
Cyanobacteria were grown in an axenic BG-11 medium under continuous illumination of 30
E/m2/s at 25 oC and with constant bubbling of air bubble. Cyanobacterial cells were harvested in
the early stationary phase. Green alga Ulva rigida was collected from Aou-Chaw beach, Trad
Province, Thailand in April and May.
2.2. Metabolite extraction
The freeze dried cyanobacterial and air-dried green algal biomass were extracted using
chloroform:methanol (2:1) and water for lipophilic and hydrophilic extracts respectively. The
solution was agitated for 3 h and after that, centrifuged at 10,000×g for 15 min and the supernatant
collected. The solvents from the supernatants were removed in vacuum and the residues were kept.
These dry extracts were used for antibacterial activity analysis.
2.3. Antibacterial bioassays
A disk diffusion assay was used to determine qualitatively the ability of the compounds to inhibit
growth of bacteria. Strains of the following bacteria were tested: Gram-positive bacteria
Streptococcus agalactiae, Gram-negative bacteria Aeromonas hydrophila, Vibrio alginolyticus,
Pseudomonas sp., Pseudomonas fluorescent. Strains of bacteria were obtained from the collection
of the Program in Fisheries Science, Faculty of Agricultural Technology, King Mongkut’s
Institute of Technology Ladkrabang. Bacteria were grown on beef extract medium at 30 °C.
Sterilized paper disks (Ø 6 mm) impregnated with concentrated antibiotic were placed on
the surface of the inoculated medium. After 24 h, the plates were examined for the presence of a
clear inhibition zone around the disks and results were reported as diameter of inhibition zone,
including the paper disk diameter. All the measurements were done in triplicates and the value
averaged.
2.4. Statistical analysis
Average values of the results of three replications are presented in the table. The significance of
difference was tested using ANOVA.
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3. Results and Discussion
3.1. Algal extract production
Productions of the lipophilic and hydrophilic extracts of algae are presented in Table 1. The
highest yields of lipophilic and hydrophilic extracts were 0.18 and 0.10 g/g dry wt, which were
obtained from O. jasorvensis and N. commune respectively.
Table 1 The lipophilic and hydrophilic extracts production from algae.
Algae
lipophilic extract
(g/g dry wt)
Calothrix marchica
0.01
Gloeocapsa gelatinosa
0.01
Mastigocladus sp.
0.07
Nostoc commune
0.12
Oscillatoria jasorvensis
0.18
Phormidium angustissimum
0.02
Spirulina platensis
0.13
Ulva rigida
0.04
hydrophilic extract
(g/g dry wt)
0.03
0.03
0.01
0.10
0.01
0.01
0.00
0.02
3.2. Antibacterial activity
The effects of algal extracts on the growth of bacteria are shown in Tables 2-3. Several
hydrophilic extracts showed bioactivity against bacteria S. agalactiae, A. hydrophila, V.
alginolyticus and Pseudomonas sp. (Table 2). No activity against the P. fluorescent was found.
Mastigocladus sp., N. commune and U. rigida were the strain that showed antibacterial activity.
Two hydrophilic extracts of the U. rigida inhibited notably the growth of two of the tested Gramnegative bacteria and one Gram-positive bacterium. The hydrophilic extracts of U. rigida gave the
widest inhibition zones (16.8 mm) against S. agalactiae. The extracts of N. commune and U.
rigida at all concentrations (1-5 mg/disc) inhibited the growth of S. agalactiae and the inhibition
zones were increased with increase in extract concentration (Table 4).
Table 2 Antibacterial activities of hydrophilic extract (5mg/disc) from algae.
Algae
Inhibition zone (mm)
S.
A.
V.
Pseudomonas
P.
agalactiae
hydrophila alginolyticus
sp.
fluorescent
C. marchica
G. gelatinosa
Mastigocladus sp.
10.5± 0.3a
7.7±0.4
N. commune
14.7±0.2b
O. jasorvensis
P. angustissimum
S. platensis
U. rigida
16.8±0.6c
10.2±0.4
12.0±0.5
The same superscript letter in each column is not significantly different at 95 % confident level.
Bioactivity analysis using lipophilic extracts showed bioactivity against only S. agalactiae
(Table 3). Mastigocladus sp., N. commune and O. jasorvensis were the strains that showed
antibacterial activity. None of the lipophilic extracts showed activity against Gram-negative
bacteria. The lipophilic extracts of N. commune gave the widest inhibition zones (21.67 mm)
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against S. agalactiae and the inhibition zones were increased with increase in extract concentration
(Table 5).
Table 3 Comparison of antibacterial activities of lipophilic extract (5mg/disc) from algae.
Algae
Inhibition zone (mm)
S.
A.
V.
Pseudomonas
P.
agalactiae
hydrophila
alginolyticus
sp.
fluorescent
C. marchica
G. gelatinosa
Mastigocladus sp.
13.6±0.8a
N. commune
21.7±0.9b
O. jasorvensis
14.2±0.2a
P. angustissimum
S. platensis
U. rigida
The same superscript letter in each column is not significantly different at 95 % confident level.
Table 4 Effect of hydrophilic extract concentrations on antibacterial activity in algae.
Algae /
Inhibition zone (mm)
concentration
S.
A.
V.
Pseudomonas
P.
(mg/disc)
agalactiae
hydrophila
alginolyticus
sp.
fluorescent
Mastigocladus sp.
0
1
2
3
4
9.7±0.1
5
10.5± 0.3
7.7±0.4
N. commune
0
0
1
10.67±0.1
2
11.67±0.1
3
12.67±0.1
4
13.67±0.2
5
14.67±0.2
U. rigida
0
1
13.7±0.3
9.0±0.6
2
14.5±0.3
7.5±0.0
9.8±0.4
3
15.3±0.3
8.0±0.0
10.5±0.5
4
16.2±0.4
9.0±0.0
11.3±0.2
5
16.8±0.4
10.2±0.4
12.0±0.5
The antibiotic activities are not only the interesting form of algal researches, but are also
commercially important. This study demonstrated that the extracts from 3 genera of cyanobacteria
and one genus of green algae were active against Gram-positive and Gram-negative bacteria.
There have been a number of reports on antibacterial activities from algae [10-15]. Bloor
and England [11] reported that the antibiotic produced by cyanobacterium Nostoc muscorum
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inhibit the growth of bacteria, notably multiple-resistant Staphylococcus aureus, and a biocide
resistant Pseudomonas aeruginosa. The product of cyanobacteria Oscillatoria angustissima and
Calothrix parietina inhibit the growth of bacteria and some fungi [12]. Metabolites produced and
excreted by cyanobacterium Nostoc insulare during growth showed antibacterial activities against
Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Gram-positive bacteria
Bacillus cereus, Bacillus subtilis, Staphylococcus aureus [15].
Table 5 Effect of lipophilic extract concentrations on antibacterial activity in algae.
Algae
Inhibition zone (mm)
/concentration
S.
A.
V.
Pseudomonas
P.
(mg/disc)
agalactiae
hydrophila
alginolyticus
sp.
fluorescent
Mastigocladus sp.
0
1
2
3
12±0.8
4
13.5±0.9
5
13.6±0.8
N. commune
0
1
2
3
4
5
O. jasorvensis
0
1
2
3
4
5
0
14.67±1.0
17.00±0.8
19.00±0.8
20.00±0.7
21.67±0.6
-
-
-
-
12.5±0.3
14.2±0.2
-
-
-
-
This study showed that antibacterial activity of cyanobacteria was especially found in the
more lipophilic extracts, the hydrophilic extracts were less effective. However, the hydrophilic
extracts of green algae, U. rigida was more effective, the lipophilic extract was ineffective. Only
the growth of Gram positive bacteria was inhibited by lipophilic extract of cyanobacteria, a
phenomenon that has already been described [16]. A reason could be that cyanobacteria are also
Gram negative bacteria.
However, in this study, the Gram negative bacteria A. hydrophila, was inhibited by the
hydrophilic extract of Mastigocladus sp. Similarly, Jaki et al. [7] showed that the bioactive
compounds isolated from Nostoc commune are also active against Gram negative bacteria.
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4. Conclusions
The present work was reported on the antibacterial activity of hydrophilic and lipophilic extract of
algae. In this study, hydrophilic and lipophilic extracts of N. commune and hydrophilic extract of
U. rigida showed the most remarkable activities. Their ability to inhibit bacterial growth, leads us
to conclude that both N. commune and U. rigida are a potential source for bioactive compound
production.
5. Acknowledgements
This study was granted by the National Research Council of Thailand.
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