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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 4; Year 2015; Page: 250 - 256

Research Article

PHARMACOLOGICAL SCREENING OF MARINE MACROALGAE

AGAINST PATHOGENIC GRAM POSITIVE BACTERIAL ISOLATES
D. Sekar* and K. Kolanjinathan,
Division of Microbiology, Faculty of Science, Annamalai University,
Annamalai Nagar 608 002, Tamil Nadu, India.
Abstract
Many seaweeds or macroalgae are known increasing demand for therapeutic drugs from the widely
diverse biological natural resources and they are considered as an excellent source of biologically active
compounds which has a broad range of biological and pharmacological activities. The antibacterial activity
of selected marine macroalgae crude extracts was determined against pathogenic Gram positive bacteria and
The marine macroalgae crude extracts showed zone of inhibition against gram positive bacteria. The crude
methanolic extract of Padina gymnospora showed maximum zone of inhibition against gram positive
bacteria. The minimum inhibitory concentration (MIC) of marine macroalgae Padina gymnospora crude
extracts showed best against gram positive bacteria.
Key words: Seaweeds, antibacterial activity,
minimum inhibitory concentration and gram
positive bacteria.

Article History
Received : 22.04.2015
Revised : 29.04.2015
Accepted : 08.05.2015
1. Introduction

Infectious diseases caused by the

microorganisms are the major cause of morbidity
and mortality throughout the world (WHO, 2004).
Synthetic drugs are not only more expensive and
inadequate for the treatment of microbial diseases
but are also often with unwanted adulterations and
adverse side effects. (Sieradzki et al., 1999).
Among the marine organisms, the macroalgae
(otherwise called as seaweeds) occupy an
important place as a source of biomedical
compounds (Manilal et al., 2010).
Macroalgae are the most nutritious plants
on earth, since they are low in fats but contain
vitamins and bioactive compounds like terpenoids
and sulphated polysaccharides and they offer a
* Corresponding author: D. Sekar
E-mail: sekar8973@gmail.com

good source of useful chemicals like alginic acid,

mannitol, laminarin, fucoidin and iodine (Kim et
al., 1997). In folk medicine, macroalgae have been
used in eczema, gallstone, renal trouble, scabies,
psoriasis, asthma, arteriosclerosis, heart disease,
ulcers and cancer (Burtin, 2003).
Numerous substances were identified as
antimicrobial agents from algae: chlorellin
derivatives, acrylic acid, halogenated aliphatic
compounds,
terpenes,
sulphur
containing
heterocyclic compounds, phenolic inhibitors, etc.
(Espeche et al., 1984). At present, the potential
contribution of marine macroalgae on the
discovery of new biologically active molecules is
remarkably increasing day-by-day (Skulberg,
2000; Sithranga and Kathiresan, 2010). Marine
macroalgae are considered as an excellent source
of biologically active compounds which has a
broad range of biological and pharmacological

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D. Sekar / Life Science Archives (LSA), Volume 1, Issue 4, Page: 250 - 256, 2015
activities (Tuney et al., 2006; Patra et al., 2008).
Many bioactive compounds of marine macroalgae
with pharmacological activity have been isolated
and some of them are still under investigation to
protect the various infections including life style
related diseases. Some of these compounds are
sterols, terpenoids, polysaccharides, peptides,
proteins, vitamins, acrylic acid, terpenes,
chlorophyllides, phenols, heterocyclic compounds,
halo-genated ketones and alkanes and cyclic
polysulphides (Priyadharshini et al., 2011).

251

procured form Microbial type Culture Collection

(MTCC), Chandigarh.
Gram positive bacteria
a) Staphylococcus aureus (MTCC-3160)
b) Streptococcus pyogenes (MTCC- 1926)
c) Bacillus cereus (MTCC-7417)
Cultures
maintenance
preparation

and

inoculum

Maintenance of test bacterial cultures

2. Materials and Methods
Collection of seaweeds
Five different seaweeds viz., Sargassum
wightii, Caulerpa racemosa, Acanthophora
spicifera, Padina gymnospora and Turbinaria
conoids were collected from Mandapam coast of
Tamil Nadu, India, that is situated in 917N
latitude and 7907E longitude and having 9 m
MSL in Tamil Nadu. The seaweeds were
taxonomically identified at the Center for
Advanced Studies in Marine Biology, Annamalai
University, Tamil Nadu, India.

The test bacterial isolates were subcultures and maintained on Nutrient agar slants
and stored in refrigerator at 4C.
Bacterial inoculums preparation
Bacterial inoculums was prepared by
inoculating a loopful of test organisms in 5 ml of
Nutrient broth and incubated at 37 C for 3 - 5
hours till a moderate turbidity was developed. The
turbidity was matched with 0.5 McFarland
standards and then used for the determination of
antibacterial activity.

Preparation of seaweed extracts

Disc preparation

The collected seaweeds samples were

cleaned and the necrotic parts were removed. The
seaweeds washed with tap water to remove any
associated debris and shade dried at room
temperature for 5 - 8 days or until they are brittle
easily by hand. After completely drying, the
seaweed materials (1.0 kg) were ground to a fine
powder using electrical blender. Forty gram of
powdered seaweeds was extracted successively
with 200 ml of solvents (Methanol, Acetone,
Chloroform, Hexane and Ethyl acetate) in Soxhlet
extractor until the extract was clear. The extracts
were evaporated to dryness by reduced pressure
using rotary vacuum evaporator and the resulting
pasty form extracts were stored in a refrigerator at
4 C for future use (Cheessborugh, 2000).

Preparation of algal disc for antibacterial

activity
Six mm diameter discs were prepared
using sterile Whatman No.1 filter paper. The
seaweeds crude extracts (150 mg/ml and 300
mg/ml) obtained using solvents (methanol, ethanol
acetone, ethyl acetate and chloroform) were mixed
with 1 ml of 5 % Dimethyl sulfoxide (DMSO).
The discs were impregnate with 20 l of different
solvent extracts to check their antibacterial
activity. The Ampicilin (5 g) was used as
positive control and the 5 % DMSO was used as a
blind control.
Antibacterial assay
Disc diffusion method

Collection of test bacterial culture

Eleven different bacterial cultures of Gram
positive and Gram negative bacteria were

The antibacterial activity of seaweed

extracts were determined by Disc diffusion

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D. Sekar / Life Science Archives (LSA), Volume 1, Issue 4, Page: 250 - 256, 2015
method proposed by Bauer et al. (1966). Petri
plates were prepared by pouring 20 ml Muller
Hinton agar and allowed to solidify for the use in
susceptibility test against bacteria. Plates were
dried and 0.1 ml of standardized inoculums
suspension was poured and uniformly spreaded.
The excess inoculums were drained and the plates
were allowed to dry for five minutes. After drying,
the discs with extract were placed on the surface
of the plate with sterile forceps and gently pressed
to ensure contact with the agar surface. The
Ampicilin (5 g/disc) was used as positive control
and the 5 % DMSO was used as a blind control in
these assays. The plates were incubated at 37 C
for 24 hours. The zone of inhibition was observed
and measured in millimeters. Each assay in these
experiments was repeated three times for
concordance.
Minimum inhibitory concentration for bacteria
Minimum inhibitory concentration (MIC)
of the seaweed extracts against bacterial isolates
was tested in Muller Hinton broth by Broth macro
dilution method (Ericsson and Sherri, 1971). The
seaweed extracts were dissolved in 5 % DMSO to
obtain 128 mg/ml stock solutions. A quantity of
0.5 ml of stock solution was incorporated into 0.5
ml of Mueller Hinton broth for bacteria to get a
concentration of 64, 32, 16, 8, 4, 2 and 1 mg/ml
for seaweeds extracts and 50 l of standardized
suspension of the test organism was transferred on
to each tube. The control tube contained only
organisms and devoid of seaweed extracts. The
culture tubes were incubated at 37C for 24 hours.
The lowest concentration, which did not show any
growth of tested organism after macroscopic
evaluation was determined as Minimum inhibitory
concentration (MIC).
3. Results and discussion
Antibacterial
activity
of
marine
macroalgae against Staphylococcus aureus
The antibacterial activity of selected
marine macroalgae crude extracts was determined
against pathogenic Gram positive bacteria
Staphylococcus aureus and the results were

252

furnished in Table - 1. The marine macroalgae

crude extracts showed zone of inhibition against
Staphylococcus aureus. The crude methanolic
extract of Padina gymnospora (300 mg/ml)
showed maximum zone of inhibition against
Staphylococcus aureus (20 0.0 mm) followed by
Turbinaria conoides (19 0.4 mm), Sargassum
wightii (17 0.3 mm), Caulerpa racemosa (16
0.5 mm) and Acanthophora spicifera (16 0.0
mm). The crude hexane extract of marine
macroalgae showed minimum zone of inhibition
against Staphylococcus aureus when compared to
the other solvent extracts. No zone of inhibition
was recorded in DMSO negative control and the
positive control Ampicillin (20 g) showed 22
0.6 mm zone of inhibition against the
Staphylococcus aureus. The minimum inhibitory
concentration (MIC) of marine macroalgae crude
extracts against Staphylococcus aureus was
ranged between 20 mg/ml to 640 mg/ml and the
results were showed in Table - 2. The crude
methanolic extract of Padina gymnospora,
Turbinaria conoides, Sargassum wightii and
Caulerpa racemosa showed best MIC at 20 mg/ml
against Staphylococcus aureus. The Acanthophora
spicifera showed best MIC at 40 mg/ml.
Nowadays, there are numerous reports of
marine macroalgae derived bioactive compounds
that have a wide range of biological activities such
as
antibacterial,
antifungal,
antiviral,
antineoplastic, antifouling, antiinflammatory,
cytotoxic and antidiabetic (Mayer and Lehman,
2014).
Commercially
important
marine
macroalgae contributes numerous renewable
resources which provides the valuable thoughts for
the development of novel drugs which possesses
bioactivity against various types of cancer and
microbial diseases (Baker, 2014).
Antibacterial activity of marine macroalgae
against Streptococcus pyogenes
The antibacterial activities of selected
marine macroalgae crude extracts were evaluated
against Streptococcus pyogenes and the results
were given in Table - 3. The zone of inhibition
was more at 300 mg/ml of extracts when
compared to 150 mg/ml of marine macroalgae

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D. Sekar / Life Science Archives (LSA), Volume 1, Issue 4, Page: 250 - 256, 2015

253

Table - 1: Antibacterial activity of marine macro algae crude extract against Staphylococcus aureus
Concentration of the marine macroalgae crude extract (mg/ml) and Zone of inhibition (mm)
Name of the marine
macroalgae
Turbinaria conoides
Sargassum wightii
Padina gymnospora
Caulerpa racemosa
Acanthophora spicifera

Hexane

Chloroform

Ethyl acetate

Acetone

Positive
control*

Methanol

150
mg/ml
100.8
100.7
110.7
90.7

300
mg/ml
110.6
110.4
130.7
100.6

150
mg/ml
100.6
110.6
140.4
110.5

300
mg/ml
130.5
120.7
150.5
120.4

150
mg/ml
130.4
120.5
140.7
120.2

300
mg/ml
150.7
140.4
160.6
130.6

150
mg/ml
140.4
130.3
150.4
130.6

300
mg/ml
170.4
160.5
180.6
140.3

150
mg/ml
180.6
160.6
190.6
150.6

300
mg/ml
190.4
170.3
200.0
160.5

90.4

100.4

110.4

120.2

110.6

130.0

130.3

140.0

130.5

160.0

20

20.6

Mean SD, *Ampicillin (20 g)

Table - 2: Minimum inhibitory concentration of marine macro algae crude extract against Staphylococcus aureus
Minimum inhibitory concentration of marine macro algae crude extracts (mg/ml)
Name of the marine
macroalgae

Hexane

Chloroform

Ethyl acetate

Acetone

Methanol

Positive Control*

320
320
160
320
640

160
160
80
160
320

80
80
40
80
160

40
40
20
40
80

20
20
20
20
40

20
20
20
20
20

Turbinaria conoides
Sargassum wightii
Padina gymnospora
Caulerpa racemosa
Acanthophora spicifera
*Ampicillin (20 g)

Table - 3: Antibacterial activity of marine macro algae crude extracts against Streptococcus pyogenes
Concentration of the marine macro algae crude extracts (mg/ml) and Zone of inhibition (mm)
Name of the
marine
macroalgae

Hexane

Chloroform

Ethyl acetate

Acetone

Positive
control*

Methanol

150
mg/ml

300
mg/ml

150
mg/ml

300
mg/ml

150
mg/ml

300
mg/ml

150
mg/ml

300
mg/ml

150
mg/ml

300
mg/ml

100.7

120.8

120.6

130.6

130.3

150.6

140.4

160.9

170.5

190.7

100.4

110.7

110.6

130.3

120.5

140.4

130.6

150.7

160.2

180.4

110.4

130.6

130.0

140.4

140.3

160.6

150.6

170.6

180.4

210.5

90.6
110.3
Acanthophora
90.2
100.6
spicifera
Mean SD, *Ampicillin (20 g)

110.3

120.6

120.4

130.7

130.5

150.3

140.5

160.4

100.3

110.7

120.3

130.4

120.8

140.3

130.6

150.3

Turbinaria
conoides
Sargassum wightii
Padina
gymnospora
Caulerpa racemosa

20 g

220.4

Table - 4: Minimum inhibitory concentration of marine macro algae crude extracts against Streptococcus pyogenes
Minimum inhibitory concentration of marine macro algae crude extracts (mg/ml)
Name of the marine
macroalgae
Turbinaria conoides
Sargassum wightii
Padina gymnospora
Caulerpa racemosa
Acanthophora spicifera
*Ampicillin (20 g)

Hexane

Chloroform

Ethyl acetate

Acetone

Methanol

Positive Control*

320
320
160
640
640

160
160
160
320
320

80
80
80
160
160

80
80
40
80
80

40
40
20
40
40

20
20
20
20
20

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D. Sekar / Life Science Archives (LSA), Volume 1, Issue 4, Page: 250 - 256, 2015

254

Table - 5: Antibacterial activity of marine macro algae crude extracts against Bacillus cereus
Concentration of the marine macro algae crude extracts (mg/ml) and Zone of inhibition (mm)
Name of the marine
macroalgae

Hexane
150
mg/ml
110.6
100.7
120.6
90.6

Turbinaria conoides
Sargassum wightii
Padina gymnospora
Caulerpa racemosa
Acanthophora
90.2
spicifera
Mean SD, *Ampicillin (20 g)

Chloroform

Ethyl acetate

Acetone

Positive
control*

Methanol

300
mg/ml
130.4
120.4
150.4
110.7

150
mg/ml
130.7
110.7
130.7
110.9

300
mg/ml
140.5
130.6
150.8
130.3

150
mg/ml
140.2
130.4
140.7
110.7

300
mg/ml
160.6
150.5
170.6
140.4

150
mg/ml
140.8
140.4
160.4
130.3

300
mg/ml
160.9
150.8
180.9
150.4

150
mg/ml
160.3
140.6
170.5
140.2

300
mg/ml
180.3
170.6
190.4
160.4

110.3

100.9

120.6

110.2

130.6

120.7

140.5

130.5

150.5

20 g

210.6

Table - 6: Minimum inhibitory concentration of marine macro algae crude extracts against Bacillus cereus
Minimum inhibitory concentration of marine macro algae crude extracts (mg/ml)
Name of the marine
macroalgae
Turbinaria conoides
Sargassum wightii
Padina gymnospora
Caulerpa racemosa
Acanthophora spicifera
*Ampicillin (20 g)

Hexane

Chloroform

Ethyl acetate

Acetone

Methanol

160
160
160
320
640

80
160
80
160
320

80
80
40
160
320

20
40
20
80
80

20
20
20
40
40

crude extracts. The crude methanolic extract of

Padina gymnospora (300 mg/ml) showed
maximum mean zone of inhibition against
Streptococcus pyogenes (21 0.5 mm) followed
by Turbinaria conoides (19 0.7 mm), Sargassum
wightii (18 0.4 mm) Caulerpa racemosa (16
0.4 mm) and Acanthophora spicifera (15 0.3
mm). The crude hexane extract of marine
macroalgae crude extracts showed minimum zone
of inhibition against Streptococcus pyogenes when
compared to the other solvent extracts. No zone of
inhibition was observed in DMSO negative
control and the positive control Ampicillin (20 g)
showed 22 0.4 mm zone of inhibition against the
Streptococcus pyogenes. The minimum inhibitory
concentration (MIC) of marine macroalgae crude
extracts against Streptococcus pyogenes was
ranged between 20 mg/ml to 640 mg/ml and the
results were showed in Table - 4. The crude
methanolic extract of Padina gymnospora showed
best MIC at 20 mg/ml against Streptococcus
pyogenes. Turbinaria conoides, Sargassum
wightii, Caulerpa racemosa and Acanthophora
spicifera showed best MIC at 40 mg/ml.

Positive
Control*
20
20
20
20
20

Vijayabaskar and Shiyamala (2011)

determined the antibacterial activity of Sargassum
wightii which was collected from the Gulf of
Mannar region. The methanol extract of both
seaweeds were tested against various Gram
positive and Gram negative human pathogenic
microbes. Their finding envisages that methanol
extracts of Sargassum wightii could be utilized as
a good source of antimicrobial agent in
pharmaceutical industry. The findings of the
present study also showed the methanol extract of
Sargassum wightii as a good source of
antimicrobial agent.
Antibacterial activity of marine macro algae
against Bacillus cereus
The antibacterial activity of selected
marine macroalgae crude extracts was tested
against pathogenic bacteria Bacillus cereus and
the results were tabulated in Table - 5. The zone of
inhibition was more at 300 mg/ml of extracts
when compared to 150 mg/ml of extracts. The
crude methanolic extract of Padina gymnospora
(300 mg/ml) showed maximum mean zone of
inhibition against Bacillus cereus (19 0.4 mm)

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D. Sekar / Life Science Archives (LSA), Volume 1, Issue 4, Page: 250 - 256, 2015
followed by Turbinaria conoides (18 0.3 mm),
Sargassum wightii (17 0.6 mm), Caulerpa
racemosa (16 0.4 mm) and Acanthophora
spicifera (15 0.5 mm). The crude hexane extract
of marine macroalgae showed minimum zone of
inhibition against Bacillus cereus when compared
to the other solvent extracts. No zone of inhibition
was observed in DMSO negative control and the
positive control Ampicillin (20 g) showed 21
0.6 mm zone of inhibition against the Bacillus
cereus. The minimum inhibitory concentration
(MIC) of marine macroalgae crude extracts
against Bacillus cereus was ranged between 20
mg/ml to 640 mg/ml and the results were showed
in Table - 6. The crude methanolic extract of
Padina gymnospora, Turbinaria conoides and
Sargassum wightii showed best MIC at 20 mg/ml
against Bacillus cereus. The extract of
Acanthophora spicifera and Caulerpa racemosa
recorded best MIC at 40 mg/ml.
The results of the present research showed
that seaweed extract of Padina gymnospora
showed maximum inhibitory activity against
Gram positive bacteria and the findings obtained
from the study of Salvador et al. (2007) was well
matched with the present study.
In the study carried by Salvador et al.
(2007), the extracts of Padina gymnospora were
prepared and screened for its antibacterial activity.
Padina gymnospora extract that was prepared and
it showed highest inhibitor activity against Gram
positive bacteria when compared to Gram negative
bacteria. The results of the present research
showed that seaweed extract of Padina
gymnospora showed maximum inhibitory activity
against Gram positive bacteria and the findings
obtained from the study of Salvador et al. (2007)
was well matched with the present study.
4. References
1. Baker, J.T. 2014. Seaweeds in Pharmaceutical
studies and applications. Hydrobiology, 116
(117): 29-40.
2. Bauer, A. W., W. M. M. Kirby, J. C. Sherris
and M. Turck. 1966. Antibiotic susceptibility

255

testing by a standardized single disk method.

Amer. J. Clin. Pathol., 45 (4): 493 - 496.
3. Burtin, P. 2003. Nutritional value of
seaweeds. Electron. J. Environ. Agric. Food
Chem., 2: 498503.
4. Chessbrough, M. 2000. Medical laboratory
manual for Tropical countries, Linacre House,
Jordan Hill, Oxford.
5. Ericsson, H. M. and J.C. Sherris. 1971.
Antibiotic sensitivity testing. Report of an
International Collaborative Study. Acta. path.
Microbiol. Scand., Sec. B, Suppl. No.217.
6. Espeche, M.E., E.R. Fraile and A.M.S.
Mayer. 1984. Screening of Argentine marine
algae
from
antimicrobial
activity.
Hydrobiologia. 116/117: 525-528.
7. Kim, J.B, A.M. Hudson, K. Huang, A.
Bannistes, T.J. Jin and G.H.N. Choi. 1997.
Biological activity of seaweed extracts from
British, Colombia, Canada and Korea. I.
Antiviral activity. Can. J. Bot. Rev., 75:
165660.
8. Manilal, A., S. Sujith, B. Sabarathnam, G.S.
Kiran, J. Selvin, C. Shakir and A.P Lipton.
2010. Bioactivity of the red alga
Asparagopsis taxiformis collected from the
south-western coast of India. Braz. J.
Oceonography, 58(2): 93-100.
9. Mayer, A.M.S and V.K.B. Lehman. 2014.
Marine Pharmacology, Marine compounds
with antibacterial, anticoagulant, antifungal,
antiinflammatory, antihelmentic, antiplatelet,
antiprozoal and antiviral activities; with
actions on the cardiovascular endocrine,
immune and nervous system; and other
miscellaneous mechanisms of action.
Pharmacologist, 42-62.
10. Patra, J.K., S.K. Rath, K. Jena, V.K. Rathod
and H. Thatoi. 2008. Evaluation of
antioxidant and antimicrobial ac-tivity of
seaweed (Sargassum spp.). Extract: A study
on inhibition of glutathione-S-transferase
activity. Turkish Journal of Biology, 32: 119125.
11. Priyadharshini, S., S. Bragadeeswaran, K.
Prabhu and S.S. Ran. 2011. Antimicrobial and
hemolytic activity of seaweed extracts Ulva
fasciata (Delile 1813) from Man-dapam,

2015 Published by JPS Scientific Publications Ltd. All rights reserved

D. Sekar / Life Science Archives (LSA), Volume 1, Issue 4, Page: 250 - 256, 2015

256

Southeast coast of India. Asian Pacific

Journal of Tropical Biomedicine, 1: S38-S39.
12. Salvador, N., A.G. Garreta, L. Lavelli and
M.A. Ribera. 2007. Antimicrobial activity of
Iberian macroalgae. Scientia Marina, 71: 101113.
13. Sieradzki, K., S.W. Wu and A. Tomasz. 1999.
Inactivation of the methicillin resistance gene
mecA in vancomycin resistant Staphylococcus
aureus. Micro Drug Resist., 5: 253-257.
14. Sithranga, N and K. Kathiresan. 2010.
Anticancer drugs from marine flora: An
overview. Journal of Oncology, 21: 41-86.
15. Skulberg, O.M. 2000. Microalgal as a source
of bioac- tive molecules-experience from
Cyanophyte research. Journal of Applied
Phycology, 12: 341-348.
16. Tuney, I., B.H. Cadirci, D. Unal and A.
Sukatar. 2006. Antimicrobial activities of the
extracts of marine algae from the coast of
Urla (Izmir, Turkey). Turkish Journal of
Biology, 30: 171-175.
17. Vijayabaskar P. and V. Shiyamala. 2011.
Antibacterial activities of Brown marine algae
(Sargassum wightii and Turbinaria ornata)
from the Gulf of Mannar. Biosphere Reserve
Advances in Biological Research, 5 (2): 99102.
1) WHO (World Health Organization). 2004. The
world health report. Changing history.
Statistical annex. Death by cause, sex and
mortality stratum in WHO regions, estimates
for 2002. Geneva, Switzerland WHO; 120121.

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