# Bio-Prospecting of a Few Brown Seaweeds for Their Cytotoxic and Antioxidant Activities

Authors: Rashmi C. Vinayak; A. S. Sabu; Anil Chatterji
Journal: Evidence-Based Complementary and Alternative Medicine
 (2011)

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## Abstract

Methanolic extracts (MEs) of seven brown seaweeds occurring in the Indian coastal waters were screened for their cytotoxic and antioxidant properties following various assays. The methanolic extracts of seaweeds in the order ofDictyopteris australis > Spatoglossum variabile > Stoechospermum marginatum > Spatoglossum aspermum showed significant cytotoxic activity. A very high DPPH radical scavenging activity was exhibited by the methanolic extracts prepared from St. marginatum, Padina tetrastromatica, Dictyopteris delicatula and S. aspermum. The total phenolic content of the MEs varied from 13.19 ± 0.32 to 25.29 ± 0.445 gallic acid equivalents (mg g−1 of methanolic extract). The reducing power assay indicated a dose dependency, at concentrations of 0.1, 0.5 and 1.0 and 2.0 mg mL−1 of MEs and decreased in the following order: Butylated hydroxy toluene > P. tetrastromatica > D. delicatula > S. aspermum > S. variabile > S. marginatum > D. australis > S. marginatum. Furthermore, D. australis, S. aspermum, S. variabile and S. marginatum demonstrated good metal ion chelating properties. All the above evidences suggest that, the antioxidant compounds found in brown seaweeds scavenge free radicals through effective intervention. This decisively promotes them as a potential source of natural antioxidants.


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## Body

## 1.Introduction

Reactive oxygen species (ROS) is a collective term used
for radicals, for example, superoxide radical, hydroxyl radical, peroxyl
radical or reactive non-radical compounds such as singlet oxygen, peroxynitrite
or hydrogen peroxide; generally produced by endogenous and exogenous factors.
These ROS are highly reactive, neutral, short lived and unstable oxygen
containing molecules with an inherent capacity to form a final stable
configuration. All such ROS possess the ability to cause far-reaching oxidative
damage to healthy cells by reacting with their nucleic acids, proteins, lipids,
enzymes and other small cellular molecules. They have been implicated in the
etiology of several degenerative disease conditions, including cancer,
cardiovascular diseases, rheumatoid arthritis, cataracts, immune system
decline, liver diseases, diabetes mellitus, renal failure, brain dysfunction
and aging [1]. Moreover, ROS-mediated oxidations
are also responsible for the rancidity of unpreserved foods rich in unsaturated
fatty acids. Synthetic antioxidants such as propyl gallate, butylated
hydroxyanisol (BHA), butylated hydroxytoluene (BHT) and
tert-butyl hydroquinone (TBHQ) are commonly used to control
lipid oxidation in foods but are suspected to be responsible for liver damage
and carcinogenesis [2, 3]. All these concerns regarding the synthetic antioxidants, together
with consumers’ preference for natural food ingredients, have reinforced the
current attention toward the development of alternative natural
antioxidants.Over the past several decades, seaweeds and their extracts
have generated an enormous amount of interest in the pharmaceutical industry as
a fresh source of bioactive compounds with immense medicinal potential [4]. Seaweeds are rich in antioxidants such as
carotenoids, pigments, polyphenols, enzymes and diverse functional
polysaccharides [5–9]. This has
been evidenced by recent investigations reporting a multitude of antioxidant
compounds; for example, phylopheophytin in Eisenia bicyclis
[10], phlorotannins in Sargassum
kjellamanianum [11], fucoxanthin in
Hijikia fusiformis [12], a
low-molecular-weight sulfated polysaccharide from Laminaria
japonica [13] and mycosporin-like amino
acids (MAAs) from red seaweeds [14]. Their
activities have been reported through a range of mechanisms, such as prevention
of chain initiation, decomposition of peroxides, prevention of continual
hydrogen abstraction, free radical scavenging, reducing power and binding of
transition metal ion catalysts [15, 16]. As a result, a lot of attention has centered on
seaweeds as alternative resources for extracting natural antioxidants.In India seaweeds are mainly exploited as a source of phycocolloids such as
agar-agar, alginate and carrageenan and not for their beneficial aspect with
respect to food and medicine [17]. Further
information on the bioutilization of Indian seaweeds is limited as not much has
been done to systemically study their therapeutic potential [18–21].The present study was undertaken to investigate cytotoxic activity of seven
brown seaweed methanolic extracts (MEs) by Brine shrimp lethality assay and
antioxidant properties by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging assay, reducing power assay and metal chelation assayin
vitro. The total content of phenolic compounds in the extract was also
determined. The results are hoped to provide an insight into the bioactive
potential of Indian seaweed extracts.


## 2. Methods

### 2.1. Collection of
Seaweeds

Seven species of seaweeds were collected from the coasts of
Goa and Maharashtra during the low tide and then transported immediately to the
Aquaculture laboratory of National Institute of Oceanography (NIO), Goa, where
they were identified (Table1). The samples
were washed thoroughly with freshwater to remove salt, sand and epiphytes,
dried at room temperature and stored at −20°C until further use.Table 1
List of brown seaweeds evaluated for the experiments.
Sr NoScientific nameFamilyCollected fromPhaeophyta(1)Dictyopteris australis LamourouxDictyotaceaeMalvan, Maharashtra(2)Dictyopteryis delicatula LamourouxDictyotaceaeAnjuna beach, Goa(3)Padina tetrastromatica HauckDictyotaceaeBaga beach, Goa(4)Sargassum
marginatum (C. Agardh) J. AgardhSargassaceaeMarvel
beach, Goa(5)Spatoglossum aspermum J.
AgardhDictyotaceaeMalvan, Maharashtra(6)Spatoglossum variabile Figari & De
NotarisDictyotaceaeAnjuna beach, Goa(7)Stoechospermum marginatum (C. Agardh)
KutzingDictyotaceaeMarvel beach,
Goa


### 2.2. Preparation of
Extract

Dried and powdered seaweed samples (20 g) were suspended in
500 ml methanol at room temperature for 24 h extraction. The extraction was
repeated twice and the total organic extracts (1.5 L) obtained were pooled,
filtered and evaporated to dryness under pressure using a rotary evaporator
(Roteva, India) to get a semi-solid residue. The product thus obtained was
designated as the ME and stored at –20°C until further analysis.


#### 2.2.1. In Vitro Cytotoxicity
Assay

Brine Shrimp Lethality Test. The toxicity againstArtemia salina nauplii (Brine shrimp) was tested according
to the method of Sam et al. [22] with minor modifications.
Dried cysts were hatched (1 g cyst per liter) in
sterile filtered seawater (0.22 μm) at 27–30°C with strong
aeration, under a continuous light regime. Approximately
12 h after hatching, the phototrophic nauplii were collected
with a pipette and concentrated in a small vial.
Each test consisted of exposing groups of 20 nauplii to
various concentrations (50, 100, and 500 μg) of the ME of
individual seaweeds. The toxicity was determined after 6,
18, and 24 h of exposure by counting the number of survivors
and calculating the percentage of mortality.
Potassium dichromate (K2Cr2O7) and Milli-Q water
were used as a positive and negative control, respectively.
Larvae were considered dead if they did not exhibit any
internal or external movement during the observation.
Mortality below 50% was considered non-cytotoxic; mortality
higher than 50% but below 75% was considered
mildly cytotoxic; while mortality higher than 75% was
considered as highly cytotoxic.


#### 2.2.2. In Vitro
Antioxidant Assays

Total Phenolic Content. The total phenolic content
(TPC) was determined by the Folin-Ciocalteu method
as described by Sellappan and Akoh [23]. Seaweed extracts (0.5 mL) or gallic acid standard solution were
mixed with 2.5 mL of Folin-Ciocalteu’s reagent (FCR,
1:10 dilution) and left to stand for 8 min at room temperature
to facilitate the FCR to react with the oxidizable
substances or phenolates. Then, 2.0 mL of Na2CO3 (7.5%
solution in water) was added to neutralize the residual
reagent. After incubating for 2 h at room temperature,
the absorbance was measured at 760 nm. Results were
expressed as mg Gallic acid equivalents (GAE) per
gram of seaweed extract.


#### 2.2.3.
DPPH Radical Scavenging Assay

The scavenging effects of samples for
1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH*) was determined
spectrophotometrically according to the method of Duan et al. [24]. A 2 mL aliquot of test sample (in methanol) was
added to 2 mL of 0.16 mM DPPH methanolic solution. The mixture was vortexed for
1 min and then left to stand at room temperature for 30 min in the dark. The
absorbance was read at 517 nm and percentage of radical scavenging effect was
calculated using the following equation: (1)Scavenging⁢effect (%) =[1−(ASample−ASampleblank)AControl]×100, where
AControl was the absorbance of the control (DPPH
solution without sample), ASample the absorbance of
the test sample (DPPH plus test sample), and the ASample blank the absorbance of the sample only (Sample without DPPH solution).
Natural antioxidant, ascorbic acid (AA) was used as positive
control.


#### 2.2.4.
Reducing Power Assay

Total reducing power was determined as described
by Zhu et al. [25] with slight modification.
0.2 mL of the sample solution was mixed with 0.2 mL of phosphate buffer (0.2 M,
pH 7.2) and 0.2 mL of 1% potassium ferricyanide. The mixture was incubated at
50°C for 20 min. After incubation, 0.2 mL of trichloroacetic acid
(10%) was added. Finally, 0.125 mL of the mixture and 0.125 mL distilled water
was dispensed into a 96-well micro plate. To this, 0.02 mL of 0.1%
FeCl3 was added and absorbance was measured at 655 nm (Bio-Rad,
Micro plate reader, Model 680). BHT was used as a positive control for this
assay.


#### 2.2.5.
Ferrous Ion Chelating Activity

The ferrous ion chelating activity was
performed by the method of Decker and Welch [26]. A mixture of sample solution (0.1 mL), distilled water (0.1 mL)
and 0.5 mM FeCl2 (0.025 mL) was prepared and the absorbance read
immediately at 562 nm (Abs 1). Then, 2.5 mM ferrozine (0.025 mL) was added into
the mixture and incubated for 20 min at room temperature. The absorbance was
measured again (Abs 2). Ethylene diamine tetracetic acid (EDTA) was used as the
positive control. The ferrous ion chelating activity was calculated using the
following equation;(2)Ferrous ion chelating activity  [%]=[1−(SampleAbs2−SampleAbs1)(ControlAbs2−ControlAbs1)]×100.


#### 2.2.6. Statistical
Analysis

All experiments were conducted in triplicates
(n = 3) and expressed as mean ± SD. One-way ANOVA test using
STATISTICA software (Statsoft, 1999) was utilized to compare the mean values of
each treatment and P-values < .001 was considered highly
significant. The relationships between TPC and DPPH scavenging activity and TPC
and metal chelation assay were determined using regression
analysis.


## 2.1. Collection of
Seaweeds

Seven species of seaweeds were collected from the coasts of
Goa and Maharashtra during the low tide and then transported immediately to the
Aquaculture laboratory of National Institute of Oceanography (NIO), Goa, where
they were identified (Table1). The samples
were washed thoroughly with freshwater to remove salt, sand and epiphytes,
dried at room temperature and stored at −20°C until further use.Table 1
List of brown seaweeds evaluated for the experiments.
Sr NoScientific nameFamilyCollected fromPhaeophyta(1)Dictyopteris australis LamourouxDictyotaceaeMalvan, Maharashtra(2)Dictyopteryis delicatula LamourouxDictyotaceaeAnjuna beach, Goa(3)Padina tetrastromatica HauckDictyotaceaeBaga beach, Goa(4)Sargassum
marginatum (C. Agardh) J. AgardhSargassaceaeMarvel
beach, Goa(5)Spatoglossum aspermum J.
AgardhDictyotaceaeMalvan, Maharashtra(6)Spatoglossum variabile Figari & De
NotarisDictyotaceaeAnjuna beach, Goa(7)Stoechospermum marginatum (C. Agardh)
KutzingDictyotaceaeMarvel beach,
Goa


## 2.2. Preparation of
Extract

Dried and powdered seaweed samples (20 g) were suspended in
500 ml methanol at room temperature for 24 h extraction. The extraction was
repeated twice and the total organic extracts (1.5 L) obtained were pooled,
filtered and evaporated to dryness under pressure using a rotary evaporator
(Roteva, India) to get a semi-solid residue. The product thus obtained was
designated as the ME and stored at –20°C until further analysis.


### 2.2.1. In Vitro Cytotoxicity
Assay

Brine Shrimp Lethality Test. The toxicity againstArtemia salina nauplii (Brine shrimp) was tested according
to the method of Sam et al. [22] with minor modifications.
Dried cysts were hatched (1 g cyst per liter) in
sterile filtered seawater (0.22 μm) at 27–30°C with strong
aeration, under a continuous light regime. Approximately
12 h after hatching, the phototrophic nauplii were collected
with a pipette and concentrated in a small vial.
Each test consisted of exposing groups of 20 nauplii to
various concentrations (50, 100, and 500 μg) of the ME of
individual seaweeds. The toxicity was determined after 6,
18, and 24 h of exposure by counting the number of survivors
and calculating the percentage of mortality.
Potassium dichromate (K2Cr2O7) and Milli-Q water
were used as a positive and negative control, respectively.
Larvae were considered dead if they did not exhibit any
internal or external movement during the observation.
Mortality below 50% was considered non-cytotoxic; mortality
higher than 50% but below 75% was considered
mildly cytotoxic; while mortality higher than 75% was
considered as highly cytotoxic.


### 2.2.2. In Vitro
Antioxidant Assays

Total Phenolic Content. The total phenolic content
(TPC) was determined by the Folin-Ciocalteu method
as described by Sellappan and Akoh [23]. Seaweed extracts (0.5 mL) or gallic acid standard solution were
mixed with 2.5 mL of Folin-Ciocalteu’s reagent (FCR,
1:10 dilution) and left to stand for 8 min at room temperature
to facilitate the FCR to react with the oxidizable
substances or phenolates. Then, 2.0 mL of Na2CO3 (7.5%
solution in water) was added to neutralize the residual
reagent. After incubating for 2 h at room temperature,
the absorbance was measured at 760 nm. Results were
expressed as mg Gallic acid equivalents (GAE) per
gram of seaweed extract.


### 2.2.3.
DPPH Radical Scavenging Assay

The scavenging effects of samples for
1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH*) was determined
spectrophotometrically according to the method of Duan et al. [24]. A 2 mL aliquot of test sample (in methanol) was
added to 2 mL of 0.16 mM DPPH methanolic solution. The mixture was vortexed for
1 min and then left to stand at room temperature for 30 min in the dark. The
absorbance was read at 517 nm and percentage of radical scavenging effect was
calculated using the following equation: (1)Scavenging⁢effect (%) =[1−(ASample−ASampleblank)AControl]×100, where
AControl was the absorbance of the control (DPPH
solution without sample), ASample the absorbance of
the test sample (DPPH plus test sample), and the ASample blank the absorbance of the sample only (Sample without DPPH solution).
Natural antioxidant, ascorbic acid (AA) was used as positive
control.


### 2.2.4.
Reducing Power Assay

Total reducing power was determined as described
by Zhu et al. [25] with slight modification.
0.2 mL of the sample solution was mixed with 0.2 mL of phosphate buffer (0.2 M,
pH 7.2) and 0.2 mL of 1% potassium ferricyanide. The mixture was incubated at
50°C for 20 min. After incubation, 0.2 mL of trichloroacetic acid
(10%) was added. Finally, 0.125 mL of the mixture and 0.125 mL distilled water
was dispensed into a 96-well micro plate. To this, 0.02 mL of 0.1%
FeCl3 was added and absorbance was measured at 655 nm (Bio-Rad,
Micro plate reader, Model 680). BHT was used as a positive control for this
assay.


### 2.2.5.
Ferrous Ion Chelating Activity

The ferrous ion chelating activity was
performed by the method of Decker and Welch [26]. A mixture of sample solution (0.1 mL), distilled water (0.1 mL)
and 0.5 mM FeCl2 (0.025 mL) was prepared and the absorbance read
immediately at 562 nm (Abs 1). Then, 2.5 mM ferrozine (0.025 mL) was added into
the mixture and incubated for 20 min at room temperature. The absorbance was
measured again (Abs 2). Ethylene diamine tetracetic acid (EDTA) was used as the
positive control. The ferrous ion chelating activity was calculated using the
following equation;(2)Ferrous ion chelating activity  [%]=[1−(SampleAbs2−SampleAbs1)(ControlAbs2−ControlAbs1)]×100.


### 2.2.6. Statistical
Analysis

All experiments were conducted in triplicates
(n = 3) and expressed as mean ± SD. One-way ANOVA test using
STATISTICA software (Statsoft, 1999) was utilized to compare the mean values of
each treatment and P-values < .001 was considered highly
significant. The relationships between TPC and DPPH scavenging activity and TPC
and metal chelation assay were determined using regression
analysis.


## 2.2.1. In Vitro Cytotoxicity
Assay

Brine Shrimp Lethality Test. The toxicity againstArtemia salina nauplii (Brine shrimp) was tested according
to the method of Sam et al. [22] with minor modifications.
Dried cysts were hatched (1 g cyst per liter) in
sterile filtered seawater (0.22 μm) at 27–30°C with strong
aeration, under a continuous light regime. Approximately
12 h after hatching, the phototrophic nauplii were collected
with a pipette and concentrated in a small vial.
Each test consisted of exposing groups of 20 nauplii to
various concentrations (50, 100, and 500 μg) of the ME of
individual seaweeds. The toxicity was determined after 6,
18, and 24 h of exposure by counting the number of survivors
and calculating the percentage of mortality.
Potassium dichromate (K2Cr2O7) and Milli-Q water
were used as a positive and negative control, respectively.
Larvae were considered dead if they did not exhibit any
internal or external movement during the observation.
Mortality below 50% was considered non-cytotoxic; mortality
higher than 50% but below 75% was considered
mildly cytotoxic; while mortality higher than 75% was
considered as highly cytotoxic.


## 2.2.2. In Vitro
Antioxidant Assays

Total Phenolic Content. The total phenolic content
(TPC) was determined by the Folin-Ciocalteu method
as described by Sellappan and Akoh [23]. Seaweed extracts (0.5 mL) or gallic acid standard solution were
mixed with 2.5 mL of Folin-Ciocalteu’s reagent (FCR,
1:10 dilution) and left to stand for 8 min at room temperature
to facilitate the FCR to react with the oxidizable
substances or phenolates. Then, 2.0 mL of Na2CO3 (7.5%
solution in water) was added to neutralize the residual
reagent. After incubating for 2 h at room temperature,
the absorbance was measured at 760 nm. Results were
expressed as mg Gallic acid equivalents (GAE) per
gram of seaweed extract.


## 2.2.3.
DPPH Radical Scavenging Assay

The scavenging effects of samples for
1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH*) was determined
spectrophotometrically according to the method of Duan et al. [24]. A 2 mL aliquot of test sample (in methanol) was
added to 2 mL of 0.16 mM DPPH methanolic solution. The mixture was vortexed for
1 min and then left to stand at room temperature for 30 min in the dark. The
absorbance was read at 517 nm and percentage of radical scavenging effect was
calculated using the following equation: (1)Scavenging⁢effect (%) =[1−(ASample−ASampleblank)AControl]×100, where
AControl was the absorbance of the control (DPPH
solution without sample), ASample the absorbance of
the test sample (DPPH plus test sample), and the ASample blank the absorbance of the sample only (Sample without DPPH solution).
Natural antioxidant, ascorbic acid (AA) was used as positive
control.


## 2.2.4.
Reducing Power Assay

Total reducing power was determined as described
by Zhu et al. [25] with slight modification.
0.2 mL of the sample solution was mixed with 0.2 mL of phosphate buffer (0.2 M,
pH 7.2) and 0.2 mL of 1% potassium ferricyanide. The mixture was incubated at
50°C for 20 min. After incubation, 0.2 mL of trichloroacetic acid
(10%) was added. Finally, 0.125 mL of the mixture and 0.125 mL distilled water
was dispensed into a 96-well micro plate. To this, 0.02 mL of 0.1%
FeCl3 was added and absorbance was measured at 655 nm (Bio-Rad,
Micro plate reader, Model 680). BHT was used as a positive control for this
assay.


## 2.2.5.
Ferrous Ion Chelating Activity

The ferrous ion chelating activity was
performed by the method of Decker and Welch [26]. A mixture of sample solution (0.1 mL), distilled water (0.1 mL)
and 0.5 mM FeCl2 (0.025 mL) was prepared and the absorbance read
immediately at 562 nm (Abs 1). Then, 2.5 mM ferrozine (0.025 mL) was added into
the mixture and incubated for 20 min at room temperature. The absorbance was
measured again (Abs 2). Ethylene diamine tetracetic acid (EDTA) was used as the
positive control. The ferrous ion chelating activity was calculated using the
following equation;(2)Ferrous ion chelating activity  [%]=[1−(SampleAbs2−SampleAbs1)(ControlAbs2−ControlAbs1)]×100.


## 2.2.6. Statistical
Analysis

All experiments were conducted in triplicates
(n = 3) and expressed as mean ± SD. One-way ANOVA test using
STATISTICA software (Statsoft, 1999) was utilized to compare the mean values of
each treatment and P-values < .001 was considered highly
significant. The relationships between TPC and DPPH scavenging activity and TPC
and metal chelation assay were determined using regression
analysis.


## 3.
Results

The results illustrate the cytotoxic and antioxidant
properties of ME of seven brown seaweeds collected from the Indian coastal
waters.


### 3.1. Cytotoxic
Activities

The seaweed MEs were evaluated for their cytotoxicity at
different concentrations and incubation time exposures and were classified as
non-cytotoxic (NCT < 50%), mildly cytotoxic (MCT > 50% but < 75%) and
highly cytotoxic (HCT > 75%) based on their lethality to brine shrimp (Table2).Table 2
Cytotoxicity activity of seven brown seaweed extracts using brine shrimp lethality assay.
SampleConcentration (μg)6 hCytotoxicity18 hCytotoxicity24 hCytotoxicity(K2Cr2O7)501.66 ± 2.58NCT68.33 ± 6.83MCT100.00 ± 0.00HCT10010.00 ± 4.47NCT68.33 ± 2.58MCT100.00 ± 0.00HCT500100.00 ± 0.00HCT100.00 ± 0.00HCT100.00 ± 0.00HCTDictyopteryis
australis5041.66 ± 6.83NCT100.00 ± 0.00HCT100.00 ± 0.00HCTDictyopteryis delicatula5025.00 ±
4.47NCT26.66 ± 2.58NCT41.66 ± 2.58NCTPadina tetrastromatica5011.66 ± 2.58NCT20.00 ± 0.00NCT25.00 ± 4.47NCTSargassum
marginatum5010.00 ± 0.00NCT11.66 ± 2.58NCT15.00 ± 0.00NCTSpatoglossum aspermum500NCT43.33 ± 6.83NCT58.33 ± 9.31MCTSpatoglossum variable501.66 ± 2.58NCT68.33 ± 2.58MCT71.66 ± 13.66MCTStoechospermum marginatum508.33 ±
2.58NCT73.33 ± 2.58MCT86.66 ± 5.16HCTDictyopteryis australis10053.33 ±
5.16MCT100.00 ± 0.00HCT100.00 ±
0.00HCTDictyopteryis
delicatula10028.33 ± 6.83NCT30.00 ± 4.47NCT51.66 ± 2.58MCTPadina tetrastromatica10020.00 ±
0.00NCT25.00 ± 4.47NCT36.66 ± 2.58NCTSargassum marginatum10023.33 ± 2.58NCT23.33 ± 2.58NCT23.33 ± 2.58NCTSpatoglossum aspermum1006.66 ±
2.58NCT86.66 ± 2.58HCT93.33 ± 2.58HCTSpatoglossum variable10033.33 ± 5.16NCT91.66 ± 2.58HCT93.33 ± 2.58HCTStoechospermum marginatum10016.66 ±
2.58NCT90.00 ± 4.47HCT91.66 ± 9.31HCTDictyopteryis australis50090.00 ±
4.47HCT100.00 ± 0.00HCT100.00 ±
0.00HCTDictyopteryis
delicatula50030.00 ± 4.47NCT31.66 ± 2.58NCT56.66 ± 6.83MCTPadina tetrastromatica50023.33 ±
2.58NCT31.66 ± 5.16NCT41.66 ± 6.83NCTSargassum marginatum50025 ± 7.75NCT31.66 ± 2.58NCT36.66 ± 6.83NCTSpatoglossum
aspermum50030.00 ± 0.00NCT96.66 ± 5.16HCT100.00 ± 0.00HCTSpatoglossum variable500100.00 ±
0.00HCT100.00 ± 0.00HCT100.00 ±
0.00HCTStoechospermum
marginatum50060.00 ± 8.94MCT100.00 ± 0.00HCT100.00 ± 0.00HCTNCT: non-cytotoxic;
MCT: mildly cytotoxic; HCT: highly cytotoxic.


### 3.2. TPC

FCRs were
used to determine TPC of the MEs fromDictyopteris australis,
Dictyopteris delicatula, Padina tetrastromatica, Spatoglossum
variabile, Spatoglossum aspermum, Sargasssum
marginatum, Stoechospermum marginatum and the results
are shown in Table 3.Table 3
TPC of seven brown seaweeds expressed as GAE; mg g−1 of methanol extract (n = 3).
Seaweed speciesGAE; mg g−1 of total methanolic extractPhaeophyceaeDictyopteris
australis13.37 ± 0.140Dictyopteryis delicatula21.34 ± 0.428Padina tetrastromatica25.29 ±
0.445Sargassum marginatum13.19 ±
0.32Spatoglossum aspermum14.13 ±
0.046Spatoglossum variable14.85
± 0.093Stoechospermum marginatum20.04 ± 0.382All the
values are mean ± SD; SD: standard deviation significant atP
< .001.


### 3.3. DPPH Radical Scavenging
Assay

The radical-scavenging activity of the ME of seven seaweeds
assessed were expressed as percentage reduction of the initial DPPH* absorption
by the tested compound and is shown in Figure1. Stoechospermum marginatum (IC50 0.56
± 0.011 mg mL−1) displayed significantly (P <
.001) higher scavenging activity followed by P.
tetrastromatica (IC50 0.61 ± 0.005 mg mL−1), D. delicatula (IC50 0.66 ±
0.002 mg mL−1), S. aspermum (IC50 0.98 ±
0.006 mg mL−1), S. variabile (IC50 1.01
± 0.003 mg mL−1), D. australis (IC50
1.60 ± 0.013 mg mL−1) and S. marginatum
(IC50 2.87 ± 0.128 mg mL−1). Conversely,
none of the extracts showed comparable activity to the positive control, AA
(IC50 0.07 ± 0.002 mg mL−1).Figure 1
DPPH radical scavenging activity (%) of the total methanolic
extracts derived from seven species of brown seaweeds. AA: ascorbic acid.
Numbers correspond to the samples in Table1.
Values are presented as means ± SD (n = 3).


### 3.4. Reducing Power
Assay

The MEs of the seven seaweeds were able to reduce
Fe3+ to Fe2+ in a concentration-dependent manner as a
function of reducing power. Results obtained showed that the reducing power in
ME at all concentrations of 0.1, 0.5 and 1.0 and 2.0 mg mL−1
decreased in the following order: BHT > P. tetrastromatica > D.
delicatula > S. aspermum > S. variabile > S. marginatum > D.
australis > S. marginatum (Figure 2). In continuation with the antioxidant activity, the reducing
power of MEs also increased with increasing concentration.Figure 2
Reducing power of total methanolic extracts derived
from seven species of brown seaweeds. BHT: butylated hydroxy toluene. Numbers
correspond to the samples in Table1. Values
are presented as means ± SD (n = 3).


### 3.5. Ferrous Ion Chelating
Activity

A reasonably good ferrous ion-chelating efficacy was
demonstrated by most of the seaweed extracts in a dose-dependent manner (Figure3). EDTA (positive control), a strong chelator,
demonstrated the best ferrous chelating efficacy (IC50 0.042 ±
0.0008 mg mL−1). Amongst all seaweeds, the ferrous chelating
efficacy was significantly highest (P < .001) for
D. australis and decreased in the order: D.
australis (IC50 0.93 ± 0.029 mg mL−1)
> S. aspermum (IC50 1.19 ± 0.020 mg mL−1) > S. marginatum (IC50 1.30 ±
0.413 mg mL−1), > S. variabile
(IC50 1.38 ± 0.102 mg m−1) > P.
tetrastromatica (IC50 1.76 ± 0.146 mg mL−1)
> D. delicatula (IC50 2.46 ± 0.247 mg
mL−1) > S. marginatum (IC50 9.17 ±
0.413 mg mL−1).Figure 3
Ferrous
ion-chelating activity (%) of total ME from seven species of brown seaweeds.
EDTA: ethylene diamine tetra acetic acid. Numbers correspond to the samples in
Table1. Values are presented as means ± SD
(n = 3).


## 3.1. Cytotoxic
Activities

The seaweed MEs were evaluated for their cytotoxicity at
different concentrations and incubation time exposures and were classified as
non-cytotoxic (NCT < 50%), mildly cytotoxic (MCT > 50% but < 75%) and
highly cytotoxic (HCT > 75%) based on their lethality to brine shrimp (Table2).Table 2
Cytotoxicity activity of seven brown seaweed extracts using brine shrimp lethality assay.
SampleConcentration (μg)6 hCytotoxicity18 hCytotoxicity24 hCytotoxicity(K2Cr2O7)501.66 ± 2.58NCT68.33 ± 6.83MCT100.00 ± 0.00HCT10010.00 ± 4.47NCT68.33 ± 2.58MCT100.00 ± 0.00HCT500100.00 ± 0.00HCT100.00 ± 0.00HCT100.00 ± 0.00HCTDictyopteryis
australis5041.66 ± 6.83NCT100.00 ± 0.00HCT100.00 ± 0.00HCTDictyopteryis delicatula5025.00 ±
4.47NCT26.66 ± 2.58NCT41.66 ± 2.58NCTPadina tetrastromatica5011.66 ± 2.58NCT20.00 ± 0.00NCT25.00 ± 4.47NCTSargassum
marginatum5010.00 ± 0.00NCT11.66 ± 2.58NCT15.00 ± 0.00NCTSpatoglossum aspermum500NCT43.33 ± 6.83NCT58.33 ± 9.31MCTSpatoglossum variable501.66 ± 2.58NCT68.33 ± 2.58MCT71.66 ± 13.66MCTStoechospermum marginatum508.33 ±
2.58NCT73.33 ± 2.58MCT86.66 ± 5.16HCTDictyopteryis australis10053.33 ±
5.16MCT100.00 ± 0.00HCT100.00 ±
0.00HCTDictyopteryis
delicatula10028.33 ± 6.83NCT30.00 ± 4.47NCT51.66 ± 2.58MCTPadina tetrastromatica10020.00 ±
0.00NCT25.00 ± 4.47NCT36.66 ± 2.58NCTSargassum marginatum10023.33 ± 2.58NCT23.33 ± 2.58NCT23.33 ± 2.58NCTSpatoglossum aspermum1006.66 ±
2.58NCT86.66 ± 2.58HCT93.33 ± 2.58HCTSpatoglossum variable10033.33 ± 5.16NCT91.66 ± 2.58HCT93.33 ± 2.58HCTStoechospermum marginatum10016.66 ±
2.58NCT90.00 ± 4.47HCT91.66 ± 9.31HCTDictyopteryis australis50090.00 ±
4.47HCT100.00 ± 0.00HCT100.00 ±
0.00HCTDictyopteryis
delicatula50030.00 ± 4.47NCT31.66 ± 2.58NCT56.66 ± 6.83MCTPadina tetrastromatica50023.33 ±
2.58NCT31.66 ± 5.16NCT41.66 ± 6.83NCTSargassum marginatum50025 ± 7.75NCT31.66 ± 2.58NCT36.66 ± 6.83NCTSpatoglossum
aspermum50030.00 ± 0.00NCT96.66 ± 5.16HCT100.00 ± 0.00HCTSpatoglossum variable500100.00 ±
0.00HCT100.00 ± 0.00HCT100.00 ±
0.00HCTStoechospermum
marginatum50060.00 ± 8.94MCT100.00 ± 0.00HCT100.00 ± 0.00HCTNCT: non-cytotoxic;
MCT: mildly cytotoxic; HCT: highly cytotoxic.


## 3.2. TPC

FCRs were
used to determine TPC of the MEs fromDictyopteris australis,
Dictyopteris delicatula, Padina tetrastromatica, Spatoglossum
variabile, Spatoglossum aspermum, Sargasssum
marginatum, Stoechospermum marginatum and the results
are shown in Table 3.Table 3
TPC of seven brown seaweeds expressed as GAE; mg g−1 of methanol extract (n = 3).
Seaweed speciesGAE; mg g−1 of total methanolic extractPhaeophyceaeDictyopteris
australis13.37 ± 0.140Dictyopteryis delicatula21.34 ± 0.428Padina tetrastromatica25.29 ±
0.445Sargassum marginatum13.19 ±
0.32Spatoglossum aspermum14.13 ±
0.046Spatoglossum variable14.85
± 0.093Stoechospermum marginatum20.04 ± 0.382All the
values are mean ± SD; SD: standard deviation significant atP
< .001.


## 3.3. DPPH Radical Scavenging
Assay

The radical-scavenging activity of the ME of seven seaweeds
assessed were expressed as percentage reduction of the initial DPPH* absorption
by the tested compound and is shown in Figure1. Stoechospermum marginatum (IC50 0.56
± 0.011 mg mL−1) displayed significantly (P <
.001) higher scavenging activity followed by P.
tetrastromatica (IC50 0.61 ± 0.005 mg mL−1), D. delicatula (IC50 0.66 ±
0.002 mg mL−1), S. aspermum (IC50 0.98 ±
0.006 mg mL−1), S. variabile (IC50 1.01
± 0.003 mg mL−1), D. australis (IC50
1.60 ± 0.013 mg mL−1) and S. marginatum
(IC50 2.87 ± 0.128 mg mL−1). Conversely,
none of the extracts showed comparable activity to the positive control, AA
(IC50 0.07 ± 0.002 mg mL−1).Figure 1
DPPH radical scavenging activity (%) of the total methanolic
extracts derived from seven species of brown seaweeds. AA: ascorbic acid.
Numbers correspond to the samples in Table1.
Values are presented as means ± SD (n = 3).


## 3.4. Reducing Power
Assay

The MEs of the seven seaweeds were able to reduce
Fe3+ to Fe2+ in a concentration-dependent manner as a
function of reducing power. Results obtained showed that the reducing power in
ME at all concentrations of 0.1, 0.5 and 1.0 and 2.0 mg mL−1
decreased in the following order: BHT > P. tetrastromatica > D.
delicatula > S. aspermum > S. variabile > S. marginatum > D.
australis > S. marginatum (Figure 2). In continuation with the antioxidant activity, the reducing
power of MEs also increased with increasing concentration.Figure 2
Reducing power of total methanolic extracts derived
from seven species of brown seaweeds. BHT: butylated hydroxy toluene. Numbers
correspond to the samples in Table1. Values
are presented as means ± SD (n = 3).


## 3.5. Ferrous Ion Chelating
Activity

A reasonably good ferrous ion-chelating efficacy was
demonstrated by most of the seaweed extracts in a dose-dependent manner (Figure3). EDTA (positive control), a strong chelator,
demonstrated the best ferrous chelating efficacy (IC50 0.042 ±
0.0008 mg mL−1). Amongst all seaweeds, the ferrous chelating
efficacy was significantly highest (P < .001) for
D. australis and decreased in the order: D.
australis (IC50 0.93 ± 0.029 mg mL−1)
> S. aspermum (IC50 1.19 ± 0.020 mg mL−1) > S. marginatum (IC50 1.30 ±
0.413 mg mL−1), > S. variabile
(IC50 1.38 ± 0.102 mg m−1) > P.
tetrastromatica (IC50 1.76 ± 0.146 mg mL−1)
> D. delicatula (IC50 2.46 ± 0.247 mg
mL−1) > S. marginatum (IC50 9.17 ±
0.413 mg mL−1).Figure 3
Ferrous
ion-chelating activity (%) of total ME from seven species of brown seaweeds.
EDTA: ethylene diamine tetra acetic acid. Numbers correspond to the samples in
Table1. Values are presented as means ± SD
(n = 3).


## 4. Discussions

ROS are
molecules or ions formed by the incomplete one-electron reduction of oxygen.
They are essentially responsible for the microbicidal activity of phagocytes,
regulation of signal transduction and gene expression. Nonetheless, excessive
production of ROS by various endogenous and exogenous factors may lead to
oxidative stress, loss of cell function and ultimately apoptosis or necrosis.
Hence, the balance between production of free radicals and the antioxidant
defenses in the body is vital for cell function, regulation and adaptation to
diverse growth conditions and has important health implications. Humans have
developed a high profile, complex antioxidant patrol including enzymes [such as
superoxide dismutases (SOD), catalases (CAT), glutathione peroxidases (GPX))
and small molecule antioxidants (such as ascorbic acid, tocopherol, uric acid
and glutathione), forming the first line of defense. The second line of defense
against free radical damage is the presence of antioxidants. Polyphenolic
antioxidants have been known to play a similar role as endogenous antioxidants
and are abundantly found in plants [27, 28]. Seaweed Polyphenols, also called phlorotannins,
are vastly different from the terrestrial plants. They are a heterogeneous
group of molecules displaying broad range of biological activities and found
abundantly in brown seaweeds, forming up to 5–15% of their dried weight [29].In our studies, we have established that
brown seaweeds are a rich source of cytotoxic and antioxidant compounds.
Seaweeds, such asS. aspermum, S. marginatum
(cytotoxic studies) and P. tetrastromatica, S. marginatum
(antioxidant studies), have been investigated earlier; while D.
australis, D. delicatula and S. variabile are first
reports in either case.Brine shrimp assay implies an easy, inexpensive
and rapid bioassay for testing cytotoxic activity of plant extracts and can be
extrapolated for cell-line toxicity and anti tumor activity. Many scientists
have reported cytotoxicity of land plants and algae using brine shrimp as a
model organism [30, 31]. Hence, in the present study seven brown seaweeds were screened
for cytotoxic activity using the brine shrimp A. salina and
the activities decreased in the following order; D. australis
> S. marginatum > Sp. variable and
Sp. aspermum > D. delicatula >
P. tetrastromatica > S. marginatum.
Seaweeds like D. australis, St. marginatum,
S. variable, S. aspermum were highly cytotoxic at 100 μg
mL−1 at 18 and 24 h and caused complete mortality of the brine
shrimp at 500 μg mL−1 at 24 h duration exposure. A dose-dependent
activity was also observed in all seaweeds. In another experiment reported by
Ara et al. [31], S. asperum was
found to be the most cytotoxic to the brine shrimp amongst the seaweeds
screened. In our studies, brine shrimp assay of seaweed extracts indicated the
existence of potent cytotoxic compounds. This may be sustained by the fact
that, several cytotoxic compounds such as fucoidans, laminarins and terpenoids
stated to possess anticancer, antitumor and antiproliferative properties are
reported to be abundant in seaweeds [4]. These
cytotoxic compounds could be further explored as novel leads in cancer
chemoprevention and complementary chemotherapy and necessitates further
investigation.Polyphenols are a class of powerful chain-breaking
antioxidants with the additional ability to scavenge ROS, inhibit lipid
peroxidation as well as chelate metal ions [32–34]. Their
radical scavenging ability has been assigned to the number of hydroxyl groups
present on them [35]. The TPC of all the
seaweeds were expressed as mg gallic acid equivalent (GAE) per gram of seaweed
extract (Table 3) ranging from 13.19 ± 0.32
to 25.29 ± 0.445. They varied significantly (P < .001) and
decreased in the following order: P. tetrastromatica >
D. delicatula > S. marginatum >
S. variabile > S. aspermum > D.
australis > S. marginatum. The major active
compounds in different seaweed extracts have been reported to be phlorotannins
and fucoxanthins [36, 37].DPPH is a stable radical with a maximum absorbance at 517 nm and is useful for investigating the free radical scavenging activities of
various compounds. The method is based on the reduction of alcoholic DPPH
solution in presence of a hydrogen donating antioxidant due to formation of a
non-radical form of DPPH-H by the reaction and this modification is visually
noticeable as a discoloration from purple to yellow [38]. This DPPH radical scavenging ability of the antioxidants has been
related to the inhibition of lipid peroxidation. In this study, it was found
that all seaweed extracts possessed the ability to scavenge DPPH radical to
various degrees in a concentration-dependent manner significantly
(P < .001). Sargasssum marginatum showed
the lowest DPPH free radical scavenging activity, while S.
marginatum had the highest. In addition to S.
marginatum; P. tetrastromatica, D. delicatula, S.
aspermum also demonstrated relatively high DPPH radical scavenging
activities. The extracts showed superior radical scavenging activity when
compared to Palmaria palmata, IC50 12.5 mg
mL−1 [39] and Kappaphycus
alvarezii extracts, IC50 4.28 mg mL−1 [20]. Further analysis revealed that, there was a
positive correlation (R2 = 0.396794,
P < .005) between the TPC and the DPPH radical scavenging
activity although not very high; suggesting that not only phenolic
constituents, but other components too may have contributed to the scavenging.
This may be also explained by the fact that, the properties of polyphenolic
compounds vary greatly depending on the number of phenolic groups and hence
react differently to the FCR [40].Reducing capacity is considered as a significant indicator of potential
antioxidant activity of a compound or sample [41]. The presence of reductants (i.e., antioxidants) causes the
reduction of the Fe3+/ferricyanide complex to the ferrous form.
Therefore, by measuring the formation of Perl’s Prussian blue at 655 nm, the
amount of Fe2+can be monitored. In the present study, there was a
steady increase in reductive potential of all seaweed MEs with increase in
concentration (Figure 2). All extracts showed
significantly (P < .001) higher activities than the
negative control but lower activities than the synthetic antioxidant BHT.
Extracts could neutralize the free radicals by donating an electron and
converting them to a more stable product, ceasing the radical chain reaction in
the process to various degrees. Higher absorbance indicated higher reducing
power. Similar results were seen in methanol extracts of higher plants as
reported by Kumaran and Karunakaran [42]. All
concentrations exhibited the OD value <1.0. This was also backed by the
findings of Kuda et al. [43].Ferrozine
forms a complex (red color) with Fe2+ ion by quantitative
interaction. This is, however, disrupted in the presence of chelating agents
resulting in a decreased red color formation of the complex. This color
reduction when measured gives an estimate of metal chelating ability of the
chelator present in the reaction mixture. In this assay, seaweed MEs obstructed
the ferrous and ferrozine complex formation implying they have chelating
properties. The chelating ability of the seaweed MEs were compared with that of
EDTA; a known metal ion chelator. The ferrous ion chelating abilities between
the extracts and EDTA are shown in Figure 3.
Both the extracts and EDTA showed statistically significant differences
(P < .001). The highest metal chelating activity was
demonstrated by D. australis (IC50 0.93 ± 0.029 mg
mL−1). The metal chelating activity was also concentration
dependent. Nevertheless, a very poor correlation of ferrous ion chelating
activity with TPC of all seaweeds (R2 = 0.077454,
P > .05) was observed, indicating that phenolic compounds
may not be the main chelator of ferrous ions. Metal-binding capacities have
been displayed by dietary fibers previously. This is supported by the various
reports on the inhibitory effects on ferrous absorption of algal dietary
fibers, such as carageenan, agar and alginate [44]. Furthermore, metal ions chelating capacity of phenolic compounds
mainly depends on the accessibility of properly oriented functional groups
[45] and can no longer bind metals when the
phenolic group is conjugated with a carbohydrate group, as in naturally
occurring phenolic glycosides [46]. Transition
metals, such as iron help superoxide anion (O·−) (Fenton reaction)
and hydrogen peroxide to convert into extremely reactive hydroxyl radical (OH.)
(Haber-Weiss reaction) that cause severe damages to membranes, proteins and DNA
[47]. They also decompose lipid hydroperoxides
into peroxy and alkoxyl radicals and accelerate lipid peroxidation [48]. In the long run, this process can bring about
cellular death, carcinogenesis and mutagenesis. An extract with higher iron
chelating ability would thus not only inhibit metal dependent oxidative events,
but would also be a combatant of ROS-mediated diseases [49].High intake of antioxidant-rich foods is inversely related
to the onset or progression of cancer as revealed by a number of
epidemiological studies [50–52]. Indeed,
a number of phytochemical antioxidants are known to confer protection against
carcinogenic assault, cytotoxic damage to normal cells wrought during cancer
therapy and acute and long-term effects of free radicals produced [53, 54]. Nevertheless,
further clinical investigations are needed to shed light on the prospective use
of antioxidants in prevention and complementary cancer therapy.The
seaweed MEs investigated in this study have revealed potent cytotoxic and
antioxidant activities. The antioxidative constituents possibly play a
complimentary role by delaying or preventing the oxidation of cellular
oxidizable substrates and selectively inhibiting the ROS cascade of events
(Figure4).  All the above data imply a
protective role for seaweeds and may prove to be of pharmacological importance,
which needs to be explored further.Figure 4
Brown seaweeds and their role in the prevention of
ROS-mediated cascade of events. SOD, along with CAT and GPX, forms the first
line of the body’s antioxidant enzyme defense mechanisms. Various endogenous
and exogenous factors give rise to oxidative burst, a phenomenon where
superoxide anion radical is the predecessor to majority of ROS and moderator of
oxidative chain reactions, which perpetuates the production of secondary ROS.
In the long term, this can lead to protein peroxidation, lipid peroxidation and
DNA damage within the cell bringing about cell death, carcinogenesis and
mutagenesis. The seaweed extracts inhibit these occurrences by preventing the
production of ROS at key stages and impeding the inception of cancer and other
diseases.



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*Source: 673083-2011-03-10.xml*