Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20

Seagrass as a potential source of natural

antioxidant and anti-inflammatory agents

N. Yuvaraj, P. Kanmani, R. Satishkumar, A. Paari, V. Pattukumar & V. Arul

To cite this article: N. Yuvaraj, P. Kanmani, R. Satishkumar, A. Paari, V. Pattukumar & V.

Arul (2012) Seagrass as a potential source of natural antioxidant and anti-inflammatory agents,
Pharmaceutical Biology, 50:4, 458-467, DOI: 10.3109/13880209.2011.611948

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Pharmaceutical Biology, 2012; 50(4): 458–467
© 2012 Informa Healthcare USA, Inc.
ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2011.611948

RESEARCH ARTICLE

Seagrass as a potential source of natural antioxidant and

anti-inflammatory agents
N. Yuvaraj, P. Kanmani, R. Satishkumar, A. Paari, V. Pattukumar, and V. Arul

Department of Biotechnology, Pondicherry University, Puducherry, India

Abstract
Context: Halophila spp. is a strong medicine against malaria and skin diseases and is found to be very effective in early
stages of leprosy. Seagrasses are nutraceutical in nature and therefore of importance as food supplements.
Objective:The antibacterial, antioxidant, and anti-inflammatory activities of Halophila ovalis R. Br. Hooke (Hydrocharitaceae)
methanol extract were investigated and the chemical constituents of purified fractions were analyzed.
Materials and methods: Plant materials were collected from Pondicherry coastal line, and antimicrobial screening of crude
extract, and purified fractions was carried out by the disc diffusion method and the minimum inhibitory concentration
(MICs) of the purified fractions and reference antibiotics were determined by microdilution method. Antioxidant and
anti-inflammatory activities were investigated in vitro. Chemical constituents of purified fractions V and VI were analyzed
by gas chromatography–mass spectrometry (GC–MS), and the phytochemicals were quantitatively determined.
Results: Methanol extract inhibited the growth of Bacillus cereus at a minimum inhibitory concentration of
50 µg/mL and other Gram-negative pathogens at 75 µg/ml, except Vibrio vulnificus. Reducing power and total
antioxidant level increased with increasing extract concentration. H. ovalis exhibited strong scavenging activity on
2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals at IC50 of 0.13 and 0.65 mg/mL, respectively. Methanol
extract of H. ovalis showed noticeable anti-inflammatory activity at IC50 of 78.72 µg/mL. The GC–MS analysis of
H. ovalis revealed the presence of triacylglycerols as major components in purified fractions. Quantitative analysis of
phytochemicals revealed that phenols are rich in seagrass H. ovalis.
Discussion and conclusion: These findings demonstrated that the methanol extract of H. ovalis exhibited appreciable
antibacterial, noticeable antioxidant, and anti-inflammatory activities, and thus could be use as a potential source for
natural health products.
Keywords:  Antibacterial, GC–MS, Halophila ovalis, MIC, triacylglycerols

Introduction
Seagrasses are submerged marine angiosperms that
grow successfully in tidal and subtidal marine environ-
ments except in polar regions. The possibility of collecting
organisms directly from the ocean with the use of SCUBA
opened a new gate to a largely untapped resource with a
wide range of unique structures and novel compounds.
Seagrasses are well documented for the presence of potent
diverse secondary metabolites (Puglisi et al., 2007).
Vibrio spp., especially luminous Vibrio harveyi
Johnson & Shunk (Vibrionaceae), and Vibrio parahaemo-
lyticus Fujino et al. (Vibrionaceae), have been implicated
as the main bacterial pathogens of shrimp in hatcheries
as well as farms (Sandip et al., 2009). Problems including
solubility, palatability, toxicity, cost, delivery, and govern-
mental restrictions have limited the available antibiotics,
especially in food fish culture (Choudhury et  al., 2005).
Decreased efficacy and increased resistance of patho-
gens to antibiotics has necessitated the development of
new alternative drugs/compounds (Smith et al., 1994).
Acinetobacter baumannii has emerged as an impor-
tant and problematic human pathogen as it is the
causative agent for several types of infections including
pneumonia, meningitis, septicemia, and urinary tract
infections. Clinical impact of A. baumannii infection
has been a matter of continuing debate. Since pathogens
gaining resistance to drugs is common due to haphazard

Address for Correspondence:  V. Arul, Department of Biotechnology, Pondicherry University, Puducherry-605014, India.
Tel: +91-413-2655994 - Extn. 429; Fax: +91-413-2655265. E-mail: varul18@yahoo.com
(Received 27 January 2011; revised 29 July 2011; accepted 06 August 2011)

458

use of antibiotics, much attention is needed to kill or con-
trol the pathogens using bioactive substances. herefore,
seagrasses have received comparatively less bioassay
attention than seaweeds. Hence analyses of secondary
metabolites toward seagrasses possessing antimicrobial
and antifungal properties are found to be imperative to
emphasize.
Reactive oxygen species such as O2, hydroxyl radical
(OH˙), peroxide radical (ROO˙), and nitric acid radical
are involved in extensive oxidative damage to the cells
that leads to age-related degenerative diseases. Because
of the carcinogenicity of commercially available anti-
oxidants, pharmacologists have turned their attention
toward natural antioxidants. Application of bioactive
substances derived from marine organisms with respect
to functional foods, cosmetics, cosmeceuticals, and
pharmaceuticals have been reviewed (Kim et al., 2008).
Prima facie, our investigation represents a prelude
approach to characterize the antibacterial activity of
crude methanol extract of seagrass Halophila ovalis R. Br.
Hooke (Hydrocharitaceae) against marine and human
pathogens, antioxidant potential, and anti-inflammatory
effect on peripheral blood mononuclear cells (PBMCs).
Chemical constituents of purified fractions were ana-
lyzed by GC–MS and phytochemicals were quantitatively
determined.
Materials and methods
Collection of seagrass
Fresh seagrass samples of H. ovalis were collected in low
tide from Chunnambar estuary (Pondicherry), India,
during September 2009. Plant specimen was identified
by Prof. N. Parthasarathy, Salim Ali School of Ecology,
Pondicherry University. Specimen was preserved in 5%
formalin solution.
Preparation of extracts
The seagrass sample was washed with sea water three
times and then successively with tap water and distilled
water to remove the epiphytes and other wastes. Finally,
the sample material was air-dried under the shade for 2
weeks. The dried plant materials (10 g dry weight) were
ground to fine powder and extracted in 100 mL metha-
nol for 24 h using a Soxhlet extraction apparatus and
the extract was filtered through a Buchner funnel with
Whatman No. 1 filter paper. This was repeated three
times for the complete extraction of compounds, and all
the three methanol extracts were pooled. The solvent was
evaporated from the crude extract by rotatory evapora-
tion. The dried extract (1 g) was dissolved in 2 mL of
methanol and stored at 4°C until use.
Test organisms
Bacteria, Vibrio parahaemolyticus MTCC 451, V. fischeri
MTCC 1738, V. vulnificus MTCC 1145, Bacillus cereus
MTCC 430, and Escherichia coli MTCC 1687 were pro-
cured from Microbial type culture collection, IMTECH,
© 2012 Informa Healthcare USA, Inc.
Bioactive compounds from sea grass  459
Chandigarh, India. V. anguillarum was procured from
Central Institute of Brackish-water Aquaculture (CIBA),
Chennai. The multidrug resistant bacteria Acinetobacter
baumannii was obtained from Pondicherry Institute of
Medical Sciences (PIMS), Pondicherry, India, and the
strain was biochemically characterized (Prashanth et al.,
2008). The pathogenic bacteria were cultured individu-
ally on tryptic soy broth at 37°C for 18 h, before inocula-
tion for assay. Broth culture (100 µL), which contained
107–108 bacteria per milliliter was added to tryptic soy
agar medium (Hi-media, Mumbai), poured into sterile
Petri dishes and allowed to solidify.
Antibacterial assay
Growth inhibition of pathogens by seagrass extract was
assessed using the paper disc diffusion method. Briefly,
sterile filter paper discs impregnated with crude extract
(200 µg/mL), positive control (ampicillin 50 µg/mL), and
negative control (methanol) were allowed to air dry and
subsequently placed equidistantly onto the surface of the
pathogen-seeded tryptic soy agar plates. The plates were
kept in an inverted position and incubated at 37°C for
18 h. The growth inhibition was assessed as the diameter
(in mm) of the zone of inhibited microbial growth. The
experiment was carried out in triplicate. Experimental
data represent mean ± SD of each sample, unless other-
wise stated.
Minimum inhibitory concentration assay
A broth microdilution method was used to determine
the minimum inhibitory concentration (MIC) (Mazzanti
et al., 2000; Devienne & Raddi, 2002; NCCLS, 2008). All
tests were performed in Mueller–Hinton agar medium
(Hi-media).
Antioxidant assays
Reducing power
Total reducing power of seagrass extract was determined
as described by Oyaizu (1986). The crude methanol
extract at different concentrations (100–500 µg/mL) was
dissolved in minimum quantity of methanol, and vol-
ume was made up to 1 mL with phosphate buffer, and 1%
potassium ferricyanide was mixed with phosphate buffer
(0.2 M, pH 6.6), and the mixture was incubated at 50°C
for 20 min. About 2.5 mL of 10% trichloroacetic acid was
added to the reaction mixture which was centrifuged at
1000 × g for 10 min. The upper layer of solution (2.5 mL)
was mixed with distilled water (2.5 mL), FeCl3 (1.5 mL,
0.1%), and the absorbance was measured at 700 nm.
Ascorbic acid at aforementioned concentration was used
as standard. The higher the absorbance of the reaction
mixture, the greater is the reducing power.
Total antioxidant activity
Total antioxidant activity of seagrass extract was deter-
mined according to the method of Prieto et  al. (1999).
Briefly, 0.2 mL of sample at different concentrations
(0.5–2.5 mg/mL) was mixed with 0.1 mL distilled water
460  N. Yuvaraj et al.
and 3.0 mL reagent solution (0.6 M sulfuric acid, 28 mM
sodium phosphate, and 4.0 mM ammonium molybdate).
The reaction mixture was incubated at 95°C for 90 min
under water bath. After incubation, absorbance of all the
sample mixtures was measured at 695 nm. Ascorbic acid
was used as standard. Increase in absorbance of the reac-
tion mixture indicates greater total antioxidant activity.
DPPH radical scavenging activity
The free-radical scavenging activity of seagrass extract
was measured by DPPH according to Blois method
(1958). The DPPH solution (150 µL) was added to 3 mL
methanol, and the absorbance was taken immediately
at 516 nm for control reading. Different concentration
of test samples (0.2–1.0 mg/mL) was mixed with 3 mL
of methanol, and 150 µL of DPPH (0.1 mM) solution
was added to each test tube. Absorbance was measured
at 516 nm in a UV-Visible spectrophotometer (U-2000
model, Hitachi, Japan). A low absorbance of reaction
mixture indicated a high free-radical scavenging activity.
Butylated hydroxyanisole (BHA) was used as reference
compound. The percentage DPPH scavenging effect was
calculated as follows:
DPPH Scavenging effect (%) = (Acont − Atest∕Acont) × 100,
 where, Acont was the absorbance of the control reaction,
and Atest was the absorbance in the presence of the
seagrass sample.
Superoxide anion scavenging activity
Measurement of superoxide anion scavenging activity
was based on the method described by Nishikimi et  al.
(1972). Superoxide radicals were generated in the PMS-
NADH system containing 3 mL Tris-HCl buffer (16 mM,
pH 8.0), 468 µM NADH, 300 µM NBT, 60 µM PMS, and
varying concentrations of sample (0.2–1.0 mg/mL); the
mixture prepared earlier was incubated at room tempera-
ture for 5 min, and the absorbance read at 560 nm against
the blank. In the control, the sample was substituted with
Tris-HCl buffer. Decreased absorbance of the reaction
mixture indicated increased superoxide anion scaveng-
ing activity. Gallic acid was used as reference compound.
The capability of scavenging superoxide radical was cal-
culated using the following equation.
Scavenging effect (%) = (1−Atest ∕Acont) × 100,
 where, Acont was the absorbance of the control reaction,
and Atest was the absorbance in the presence of the sea-
grass sample.
Anti-inflammatory assay
Preparation of PBMCs
Mononuclear cells were isolated from human periph-
eral blood by Histopaque-1077 (Sigma) density gradient
using standard procedures (Bignold & Ferrante, 1987).
The PBMCs from the buffy layer were finally suspended
in RPMI–1640 medium (Sigma) containing 10% fetal calf
serum (FCS), and the cells were counted and assessed for
cell viability using trypan blue exclusion test.
 Pharmaceutical Biology
Mitogen-induced lymphocyte proliferation and its inhibition
by anti-inflammatory agents
The PBMCs (2 × 105 cells/well) in a volume of 200  µL
in RPMI-1640 medium containing 10% FCS and
1 µg/mL of phytohemagglutinin (PHA) were seeded in a
96-well U bottom plate followed by the addition of anti-
inflammatory agent at various concentrations. The cul-
ture was then incubated at 37°C for 24 h in CO2 incubator
containing 5% CO2 and 90% humidity. Assays were con-
ducted in triplicate for each concentration of seagrass
extract. Experimental data represent mean ± SD of each
compound, unless otherwise stated.
Cytotoxic studies by MTT assay
To confirm the suppressive effect of crude methanol
extract on lymphocyte proliferation, 2 µL of the com-
pound was added at various concentrations (1, 10, 50,
100 µg/mL). Triton X was used as negative control and
DMSO was used as solvent control. Following the removal
of medium from the wells, 10 µL of MTT (5 mg/mL resus-
pended in PBS) to each well was added. After incubation
at 37°C for 4 h, the floating cells were carefully removed,
and 50 µL of DMSO was added to each well to lyse the
cells, and the absorbance was measured at 570 nm using
a microplate reader. Finally, the percentage of cell viabil-
ity was calculated as follows:
Cell viability (%) = (Absorbanceoftestsample∕Absorbance
of control) × 100.
Purification of fractions by thin layer chromatography
To investigate the chemical constituents, concentrated
crude methanol extract of seagrass was purified by thin
layer chromatography using ethyl acetate-hexane as
solvent systems. The crude extract was separated in a
pre-coated aluminum TLC sheet with silica gel G 60 as
stationary phase, and ethyl acetate-hexane mixture in the
ratio of 9.5:0.5 as mobile phase. The eluted spots, repre-
senting various fractions were visualized under UV transil-
luminator at 254 nm, and also in the iodine chamber. The
TLC resolved spots of methanol extract at various Rf values
were scrapped from the TLC plate, and the scrapped spots
were dissolved in methanol, mixed well, and centrifuged
at 12,000 × g for 5 min. A total of six different fractions were
collected and the supernatant (40 µL) of each fraction was
used to check the antibacterial activity against pathogens
using the disc diffusion method in triplicate. Ampicillin
was used as positive control. Experimental data represent
mean ± SD of each sample, unless otherwise stated.
Gas chromatography – mass spectrometry analysis
After the initial thin layer chromatography, the purified
fractions were analyzed using an Agilent 6890 series
high-temperature gas chromatography–mass spectrom-
eter (GC–MS), fitted with an auto injector. For GC–MS
analysis, a high-temperature column (DB-5 ht, 30 m 
× 0.25 mm id × 0.25 μm film thickness) was purchased
from Agilent Technologies (Agilent, USA). By employing
a high-temperature column, we eliminated the need for

derivatization of each sample. The injector and detector
temperatures were set at 350°C, while the initial column
temperature was set at 80°C. A 2 μL sample volume was
injected into the column and ran using split less mode.
After 2 min, the oven temperature was raised to 150°C at
a ramp rate of 10°C/min. The oven temperature was then
raised to 250°C at a ramp rate of 5°C/min, and finally the
oven temperature was raised to 280°C at a ramp rate of
20°C/min and maintained at this temperature for 40 min.
The helium carrier gas was programmed to maintain a
constant flow rate of 1 mL/min, and the mass spectra were
acquired and processed using both Agilent ChemStation
(Agilent, USA) and AMDIS32 software. The major com-
pounds were identified by comparison of their mass with
authentic standards.
Quantitative analysis of phytochemicals
The dried, powdered sample of seagrass H. ovalis was
analyzed for quantification of alkaloids (Harborne,
1973), carbohydrates (Sheifter et  al., 1950), flavanoids
(Bohm & Kocipai-Abyazan, 1974), phenols (Sadasivam &
Manickam, 1992), proteins (Lowry et al., 1951), saponins
(Obadoni & Ochuko, 2001), and tannins (Van-burden &
Robinson, 1981).
Statistical analysis
All results were expressed as mean ± standard devia-
tion. Statistical differences between the experimental
groups were determined by one-way analysis of variance
(ANOVA), and differences were considered to be statis-
tically significant, if p < 0.05. Tukey’s post hoc test was
performed for multiple group comparison between con-
centrations. All computations were done by employing
Figure 1.  Reducing power of H. ovalis methanol extract. Results are representative of three separated experiments.
© 2012 Informa Healthcare USA, Inc.
Bioactive compounds from sea grass  461
statistical software (SPSS Version 7.0, SPSS Inc., Chicago,
IL, USA).
Results
Antibacterial assay
The results of the antibacterial activity of the crude meth-
anol extract of seagrass H. ovalis against Gram-positive
and Gram-negative pathogens were summarized in
Table 1. In this study, the methanol extract of H. ovalis
(200 µg/mL) displayed a good antibacterial activity of
17 mm against Bacillus cereus followed by 14 mm against
Acinetobacter baumannii.
Minimum inhibitory concentration assay
Minimum inhibitory concentration of the methanol
extract of H. ovalis which showed maximum activity
against the pathogens was depicted (Table 1). The growth
of Bacillus cereus was inhibited at a minimum inhibitory
concentration of 50 µg/mL followed by Gram-negative
pathogens at 75 µg/mL, whereas, Vibrio vulnificus growth
was inhibited at a minimum inhibitory concentration of
100 µg/mL.
Antioxidant assays
The reducing power of H. ovalis methanol extract is por-
trayed in Figure 1. As shown in Figure 1, the reducing
power was found to be increasing in concentration depen-
dent. The absorption was increased from 0.002 ± 0.001
to 0.042 ± 0.005 with the concentration increased from
100–500 µg/mL. Similarly, the reducing power increased
in positive control (ascorbic acid) from 0.008 ± 0.003
to 0.037 ± 0.002 with increasing concentration. Also,
462  N. Yuvaraj et al.

Table 1.  Antibacterial screening of crude extract and minimum inhibitory concentration of seagrass H. ovalis against pathogens
(inhibition zone was measured to nearest millimeter).
Inhibition zone in mm (mean ± SD)
Pathogens Crude extract (200 µg/mL) MIC (µg/mL) Ampicillin (50 µg/mL)
V. parahaemolyticus 10.16 ± 0.28 75 12.00 ± 0.57
V. anguillarum 10.16 ± 0.40 75 10.00 ± 0.00
V. fischeri 10.00 ± 0.00 75 13.00 ± 0.50
V. vulnificus 10.36 ± 0.75 100 12.16 ± 0.28
E. coli 08.53 ± 0.51 75 12.00 ± 0.50
B. cereus 17.16 ± 0.28 50 10.16 ± 0.28
A. baumannii 13.83 ± 1.04 75 10.16 ± 0.28
Values are means of growth inhibition of three replicates.

Figure 2.  Total antioxidant activity of H. ovalis methanol extract. Results are representative of three separated experiments.

it was observed that at given concentration (500 µg), the
methanol extract of H. ovalis had higher reducing power
than ascorbic acid. The reducing capacity of H. ovalis
the methanol extract was statistically not significant
(p > 0.05) as compared to ascorbic acid. However, both
the sample and ascorbic acid showed a significant differ-
ence between the concentrations (p < 0.05). This property
is associated with the presence of reductones that are
reported to be terminators of free radical chain reaction.
The total antioxidant activity of methanol extract of
H. ovalis by phosphomolybdenum method is depicted
in Figure 2. In this method, Mo (VI) is reduced to form
a green phosphate/Mo (V) complex. The total antioxi-
dant activity increased with increase in absorption from
0.045 ± 0.002 to 0.795 ± 0.003 at increasing concentration
(0.5–2.5 mg/mL). The sample was statistically not sig-
nificant (p > 0.05) as compared to standard but showed
a significant difference between the concentrations
(p < 0.05).
The DPPH and superoxide have been used extensively
as free radicals to evaluate reducing substances and are
useful reagents for investigating the free radical scaveng-
ing activities of compounds. The antiradical power of
an antioxidant activity by measurement of the decrease
in the absorbance of DPPH radical at 517 nm was
determined. The methanol extract of H. ovalis showed
significant DPPH radical scavenging activity (p < 0.05)
between concentrations and IC50 was observed at 0.13 mg/
mL (Figure 3). Similarly, superoxide radicals scavenged
by methanol extract of H. ovalis were found to be sta-
tistically significant (p < 0.05) and scavenging activity at
50% was observed at the concentration of 0.65 mg/mL
(Figure 4). The scavenging activities were found to be
increased with increasing concentration of extract in
DPPH as well as superoxide assays. In fact, the inhibitory
ability of the methanol extract was inferior to those of the
commercial counterparts such as butylated hydroxyani-
sole and gallic acid.

 Pharmaceutical Biology
 Bioactive compounds from sea grass  463

Figure 3.  The DPPH radical scavenging activity (%) of methanol extract obtained from seagrass H. ovalis at different concentrations. Results
were representative of three separated experiments.

Figure 4.  Superoxide radical scavenging activity (%) of methanol extract obtained from seagrass H. ovalis at different concentrations.
Results were representative of three separated experiments.

Mitogen-induced lymphocyte proliferation was a significant increase in proliferation of PBMCs on

To find out whether the crude methanol extract
obtained from seagrass H. ovalis possess anti-inflam-
matory activity, their effect at various concentrations
on the inhibition of proliferation of mitogen-induced
PBMCs was investigated and depicted in Figure 5. There
induction with PHA but this response was considerably
inhibited by crude methanol extract from H. ovalis.
A 50% inhibition (IC50) of proliferation of PBMCs was
observed at a concentration of 78.72 µg/mL of crude
methanol extract.

© 2012 Informa Healthcare USA, Inc.

464  N. Yuvaraj et al.

Figure 5.  Inhibitory effect of crude extract from H. ovalis on mitogen-induced proliferation. Mitogen PHA (1 µg/mL)-induced PBMC
(2 × 105/well) was treated with different concentrations of crude extracts and the percentage inhibition of lymphocyte proliferation was
determined over control. Results were representative of three separated experiments.

Purification of antibacterial fractions
Purified fractions V and VI of seagrass H. ovalis showed
an inhibition zone of more than 10 mm against Vibrio
parahaemolyticus (12 mm), Acinetobacter baumannii
(12 mm), V. anguillarum (11 mm), and V. fischeri (11 mm),
whereas a diameter of less than 10 mm was observed in
other fractions against the pathogens tested (Table 2).
GC–MS analysis
The GC–MS analysis revealed the presence of saturated
fatty acids, and aromatic carboxylic acid, in the purified
fractions of H. ovalis (Tables 3 and 4). The major compo-
nent was 9-octadecenoic acid (27.01%) followed by hexa-
decanoic acid (21.63%), and octadecanoic acid (10.42%)
in fraction V, whereas, benzoic acid (11.11%) was found to
be a major chemical constituent followed by tetradecanoic
acid (6.12%), and hexadecane (3.47%) in fraction VI.
Quantitative analysis of phytochemicals
The phytochemicals of seagrass H. ovalis were quantita-
tively determined and depicted in Table 5. Phytochemical
analysis revealed that phenols were found to be rich in
H. ovalis followed by saponins, flavanoids, proteins, car-
bohydrates, and alkaloids, whereas, tannins were found
to be less than other phytochemicals.
Discussion
Natural products are considered as an important source
of new antibacterial agents. Many chemically unique
compounds of marine origin with different biological
 Pharmaceutical Biology
activities have been isolated and a number of them
are under investigation and/or are being developed as
new pharmaceuticals (Faulkner, 2000a,b; Kim et  al.,
2008). Several species of seagrass produce antimi-
crobial compounds that may act to reduce or control
microbial growth. In the present study, the methanol
extract of seagrass H. ovalis collected from Chunnambar
estuary, Pondicherry coastal line, exhibited antibacte-
rial activity against Gram-positive Bacillus cereus and
Gram-negative pathogens such as Vibrio parahaemo-
lyticus, V. fischeri, V. anguillarum, V. vulnificus, and
Acinetobacter baumannii. The results of the present
study consisted with some preceding studies of seagrass
antibacterial activity (Rengasamy et al., 2008). Similarly
extracts from Cymodocea rotundata Ehrenberg and
Hemprich ex Ascherson (Cymodoceaceae) was effec-
tive against Bacillus species (Bernard & Pesando, 1989).
It was reported that ethanol and methanol extracts of
seagrasses showed better zones of inhibition against
bacterial pathogens (Thirumaran et  al., 2009). The
methanol and diethyl methyl formamide extracts of sea-
grass sp. was found to be active against Gram-positive
pathogens (Shelat, 1979). We also observed that crude
methanol extract of seagrass H. ovalis exhibited good
antibacterial activity against Gram-positive as well as
to Gram-negative pathogens. The presence of antibac-
terial substances could be varied from algal species
to species (Lustigman & Brown, 1991). In the present
study, the methanol extract of H. ovalis controlled the
growth of B. cereus at a minimum concentration of
50 µg/mL. Similarly, crude methanol extract of H. ovalis
 Bioactive compounds from sea grass  465

Table 2.  Antibacterial screening of purified fractions of seagrass H. ovalis against pathogens (inhibition zone was measured to nearest
millimeter).
Inhibition zone in mm (mean ± SD) Ampicillin
Pathogens Fr1 Fr2 Fr3 Fr4 Fr5 Fr6 (50 µg/mL)
V. parahaemolyticus 10.00 ± 0.57 10.00 ± 0.57 09.00 ± 0.15 10.00 ± 0.57 12.00 ± 0.57 12.00 ± 0.57 12.00 ± 0.57
V. anguillarum 00.00 ± 0.00 00.00 ± 0.00 00.00 ± 0.00 00.00 ± 0.00 08.00 ± 0.57 11.00 ± 1.52 00.00 ± 0.00
V. fischeri 10.00 ± 0.57 09.00 ± 0.57 08.00 ± 0.57 10.00 ± 1.00 11.00 ± 0.66 10.00 ± 0.57 13.00 ± 0.50
V. vulnificus 08.00 ± 0.57 00.00 ± 0.00 06.00 ± 0.57 09.00 ± 0.57 08.00 ± 0.57 08.00 ± 0.57 12.16 ± 0.28
E. coli 00.00 ± 0.00 05.00 ± 0.40 00.00 ± 0.00 00.00 ± 0.00 05.00 ± 0.40 06.00 ± 0.57 12.00 ± 0.50
B. cereus 00.00 ± 0.00 07.00 ± 0.57 05.00 ± 0.57 08.00 ± 0.00 08.00 ± 0.57 06.00 ± 0.57 10.16 ± 0.28
A. baumannii 00.00 ± 0.00 00.00 ± 0.00 00.00 ± 0.00 00.00 ± 0.00 12.00 ± 0.58 10.00 ± 0.57 10.16 ± 0.28
Values are means of growth inhibition of three replicates. Fr, fraction.

Table 3.  The GC – MS analysis of fraction V of H. ovalis. accepts an electron or hydrogen radical to become a
Rt (min) Compound Area (%) stable diamagnetic molecule. Therefore, DPPH is often
18.24 Hexadecanoic acid 21.63 used as a substrate to evaluate antioxidant activity of an
21.33 9, 12-Octadecadienoic acid 10.32 antioxidant. Our study showed that methanol extract
21.49 9-Octadecenoic acid 27.01 of H. ovalis exhibited 50% of scavenging activity on
21.58 10, 13-Octadecadienoic acid 07.56 DPPH radicals at 0.13 mg/mL and superoxide radicals
21.92 Octadecanoic acid 10.42 at 0.65 mg/mL, respectively, whereas seagrass Posidonia
Rt, retention time. oceanica (L.) Delile (Posidoniaceae) showed relatively a
less scavenging activity 13.9% at 2 mg/mL (Kartal et al.,
Table 4.  The GC–MS analysis of fraction VI of H. ovalis. 2009). However, in vivo studies revealed that P. oceanica
Rt (min) Compound Area (%) extract restored antioxidant enzymes and decreased
10.83 Phenol, 2,4-bis 03.81 lipid peroxidation in diabetic animals (Gokce &
(1,1-dimethylethyl) Haznedaroglu, 2008). This present finding corroborates
11.01 Benzoic acid 11.11 well with earlier reports in other higher plants including
11.99 Hexadecane 03.47 brown and red seaweeds (Kuda et al., 2005; Kumaran &
15.69 Tetradecanoic acid 06.12 Karunakaran, 2007).
Rt, retention time. In the present work, an attempt was made to study the
anti-inflammatory activity, if any, in methanol extract
Table 5.  Biochemical composition of seagrass H. ovalis (n = 3; of seagrass H. ovalis by evaluating the suppression of
means ± SD). mitogen-induced lymphocyte proliferation, respectively.
Parameters H. ovalis The ability of the methanol extract from H. ovalis on
Alkaloids (mg/g) 1.06 ± 0.03 proliferation of mononuclear cells, which are crucial for
Carbohydrates (mg/g) 4.82 ± 0.04 generation of effective immune responses, was analyzed
Flavanoids (mg/g) 5.59 ± 0.01 in the presence of the mitogen PHA. Interestingly, the
Phenols (mg/g) 9.44 ± 0.02 methanol extract of H. ovalis inhibited the PBMC pro-
Proteins (mg/g) 5.19 ± 0.02 liferation. Organic extracts from different plants were
Saponins (mg/g) 9.07 ± 0.06 reported to inhibit proliferation of PBMC (Manjula et al.,
Tannins (mg/g) 0.26 ± 0.02 2006; Gayathri et al., 2007; Wu et al., 2007). In our study,
inhibition of proliferation of mitogen-induced PBMC in
was effective in controlling the growth of Micrococcus the presence of added methanol extract was observed.
luteus at the minimum inhibitory concentration of For the first time, this finding suggests that the crude
50 µg/mL (Rengasamy et al., 2008). methanol extract of seagrass H. ovalis do contain com-
Recently, extensive studies have been conducted pounds, capable of suppressing PBMC proliferation.
on the utility of seagrasses as a source of natural anti- Further study is needed to see that the suppression of
oxidants. Hence, in the present study, we screened the proliferation of PBMC is by means of cytotoxic effects of
antioxidant activity for methanol extract of seagrass this methanol extract. Our preliminary results encourage
H. ovalis using different methods. Concentration depen- us to further investigate the immunomodulatory proper-
dency of antioxidant activity was investigated as a func- ties of the crude extract.
tion of reducing power. The reducing power increased In this study, the purified fractions of seagrass H. ovalis
with increasing concentration in methanol extract of H. displayed good antibacterial activity against Gram-positive
ovalis. The same trend has also been reported in metha- as well as Gram-negative pathogens. Fractions V and VI of
nol extracts of higher plants (Kumaran & Karunakaran, H. ovalis extract effectively inhibited V. parahaemolyticus, V.
2007). Also, the total antioxidant activity of H. ovalis vulnificus, and A. baumannii. Similarly, purified fractions of
methanol extract was found to be increased with increas- selected South African seaweeds showed broad spectrum
ing concentration. The DPPH is a stable free radical and activity against Gram-positive as well as Gram-negative

© 2012 Informa Healthcare USA, Inc.

466  N. Yuvaraj et al.
pathogens than crude extracts of the same seaweeds (Vlachos
et al., 1997). The antibacterial activity of marine algae and
mangrove plants against fish pathogens was observed, and
the fractions of methanol extract of red seaweed Gracilaria
corticata J. Agardh (Gracilariaceae) showed good activity
against fish pathogens Pseudomonas aeruginosa and V. algi-
nolyticus (Sree et al., 2005).
There are numerous reports of compounds derived
from macro algae with a broad range of biological
activities such as antibiotics (Scheuer, 1990), and tria-
cylglycerols in model diatoms (Eizadora et  al., 2009).
The GC–MS analysis of active fractions of H. ovalis in this
study revealed the presence of triacylglycerols such as
hexadecanoic acid, tetradecanoic acid, 9-octadecenoic
acid, octadecanoic acid, and hydrocarbon, hexade-
cane. Similar group of triacylglycerols were reported in
Zosteraceae species Zostera japonica Aschers. & Graeben
(Hua et al., 2006), and Zostera marina L. (Sanina et al.,
2004), respectively. The presence of hydrocarbon, hexa-
decane in the H. ovalis extract corroborated well with
earlier reports in brown algae Cystoseira barbata (Good
et Woodw.) J. Agardh (Cystoseiraceae), and Dictyotaceae
species Dictyopteris membranaceae (Stackhouse) batters
(Ozdemir et al., 2006).
In this study, a wide range of phytochemicals were
analyzed quantitatively. The protein content in the pres-
ent investigation was found to be less than the level of
protein reported in an earlier study on seagrass H. ovalis
(Athiperumalsami et al., 2008). The amount of carbohy-
drate was found to be more in H. ovalis. The amount of
phenol in the seagrass H. ovalis investigated in the present
study appears to be more than reported in pulses (Vadivel
& Janarthanan, 2000). Soluble phenolic acids have been
shown to be abundant in a variety of seagrasses (Zapata
& McMillan, 1979; Buchsbaum et al., 1991). Phenol rich
seagrass indicated that extracts may have antioxidant
activity and antimicrobial activity which may help in pre-
venting a number of diseases through free-radical scav-
enging activity (Harrison & Chan, 1980; Anggadiredja
et al., 1997; Ruberto et al., 2001; Vasanthi et al., 2006). The
amount of tannin in the seagrass was found in the aver-
age level (2.940–9.800 mg/g) corroborated with the ear-
lier study on seagrasses (Athiperumalsami et  al., 2008).
In addition, we quantitatively determined the saponins,
flavanoids, and alkaloids content of H. ovalis, which so
far has not been reported by any authors. In the pres-
ent study, phytochemical analysis showed that phenols
and saponins were found to be in high concentration in
H. ovalis than other phytochemicals.
In conclusion, our study showed that the crude and
purified fractions of H. ovalis showed appreciable anti-
bacterial activity against Gram-positive as well as Gram-
negative human and fish pathogens. The methanol
extract of H. ovalis was found to be a promising candidate
as antioxidant and anti-inflammatory agent. The GC–MS
analysis of purified fractions revealed the presence of
oleic acid, palmitic acid, and benzoic acid as major
constituents. Further study is underway to elucidate the
 Pharmaceutical Biology
structure of compound, and the mechanism of inhibition
of pathogens. However, the abundant availability of sea-
grasses along the Indian coastline opens up a new avenue
for the entry of pharmaceutical industries in developing
new drugs for aquaculture remedies.
Acknowledgments
The authors thank Prof. N. Parthasarathy, Salim Ali
School of Ecology, Pondicherry University, Pondicherry
for the identification of seagrasses. We also thank Dr.
Babu Rajendar, Dept. of Environmental Biotechnology,
Bharathidasan University, Tiruchirappalli for providing
the GC–MS facility.
Declaration of interest
This work was supported by a grant from Department
of Biotechnology (DBT), New Delhi, India. The authors
declare that there are no other conflicts of interest.
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