INTRODUCTION:

Seaweeds are macroscopic, multicellular, photosynthetic marine algae that are differentiated into three divisions: Chlorophyta (green algae), Phaeophyta (brown algae), Rhodophyta (red algae) and they are the most important primary producers and ecological engineers of rocky coast of the world’s ocean¹. About ten thousand distinct species of seaweeds were reported so far². They distributed throughout the world and also found in every climatic zone, from the extremely cold northern regions are the tropical warm seas³. Seaweeds are good source of therapeutic bioactive compounds includes sulphated polysaccharides (ulvans, fucoidans and laminarans), phenolic compounds, flavonoids etc. they exhibits chemopreventive properties⁴. They have a significant biological value as they possess substances include carotenoids, protein, essential fatty acids, polysaccharides, vitamins and minerals⁵. Due to their high nutritional characteristics, they have used as fundamental foods in our daily diet but restricted to coastal areas of the world⁶.

Free radicals (peroxides, superoxide, hydroxyl radical and H₂O₂) are the chemical species, like produced by living cells from endogenous or exogenous origin⁷. These free radicals cause denaturation of enzymes, lipid peroxidation in tissue membranes that result in oxidative stress, which is responsible for various diseases. Antioxidants are the organic compounds that neutralize the body’s excess free radicals and protect the cellular structure from oxidative damage. Our body always maintains the equilibrium between free radical development and antioxidant ability to prevent the cell damage. In order to retain the oxidant-antioxidant stability in steady state, it is essential to supply the certain amount of antioxidants through diet. Natural sources of antioxidants such as ascorbic acid, polyphenols and flavonoids might be more sufficient than the synthetic antioxidants, as due to their carcinogenic potential⁸. In the recent years researchers looking for alternative natural antioxidant rich diet materials instead of synthetic ones. Seaweeds of one such source of bioactive natural compounds with antioxidant potential^9,10like that of terrestrial medicinal plants^11-19. Antioxidants from seaweeds promote the health benefits or capacity to enhance the shelf-life of food through their antioxidant potential⁵.

The chemical constituents present in the seaweeds are carotene, fucoxanthin, flavonoids and phlorotannins and they exhibit the antioxidant potential due to the presence of numerous phenolic groups²⁰. They are widespread in coastal regions and rich in chemicals and secondary metabolites such as agar, carrageenan, alginate, alkaloids, phenolics and phlorotannins, all of which have several practical applications²¹. Hence in the present study, three different seaweeds (Gracileria corticata of red algae, Ulva lactuca of green algae and Sargassum ilicifolum of brown algae) from coastal regions of Visakhapatnam, Andhra Pradesh were selected. The objective of the study is to evaluate the antioxidant potential of three different seaweeds in chloroform, acetone and methanol extract.

MATERIALS AND METHODS:

Sample collection:

Three different seaweed samples (G. corticata, U. lactuca and S. ilicifolum) were collected from at the Bay of Bengal sea coast region of Visakhapatnam where it is located in North Eastern part of Andhra Pradesh between 17^o-15' and 18^o-32' Northern latitude and 18^o- 54' and 83^o-30' in Eastern longitudes.

Chemicals and reagents:

All chemicals and solvents used were of analytical grade, obtained from SD Fine Chemical Pvt. Ltd., Mumbai, India. Rotavapor was used for drying the samples under vacuum (Buchi, Switzerland), Model: R-210.

Preparation of the extracts:

The samples were collected and preserved in zip lock plastic bags after removing the sand particles and other fauna attached to the algae. Seaweed samples were identified with descriptions and the taxonomic keys were provided by Prasanna Lakshmi and Narasimha Rao²². The algal materials were thoroughly washed with running tap water, shade dried at room temperature and made into coarse powder. The powder was further subjected to maceration with chloroform, acetone, and methanol successively and collected filtrate was concentrated using vacuum evaporator, weighed and stored at ˗20°C.

Protein extraction and SDS-PAGE:

The algal proteins of all the three samples were extracted using standard protocol. 50 mg of each algal extract was homogenized separately with pre-cooled extraction buffer (62.5mM Tris-HCl; pH 6.8; 2.5% of SDS; 10% of glycerol; 5% 2-mercaptoetanol). The homogenate was incubated for 1 hour at room temperature in shaker and it was centrifuged at 15,000 rpm for 15 minutes at 4°C. The supernatant was collected and kept at -20°C, until further use.

SDS-PAGE was done using the method described by Fatchiyah et al.²³. 3.125ml of 30% acrylamide, 2.75ml of 1M Tris buffer (pH 8.8), 1.5ml of water was taken and to this 0.075ml of 10% SDS, 0.075 of 10% APS and 6.25μl of TEMED were added and mixed well. The protein samples from three algal species were dissolved in 1X 1M Tris-HCl (pH 6.8), 50% glycerol, 10% SDS, 5% β- merchaptoethanol and to this 1% bromophenol blue was added. The above sample mix was incubated for 5minutes at 100°C. The samples were then loaded into wells of stacking gel containing 0.45ml of 30% acrylamide, 0.38ml of 1M Tris buffer (pH 6.8), 2.1ml of water, 30μl of 10% SDS, 30μl of 10% APS, and 5μl of TEMED. After completing SDS PAGE it was stained with Coomassie brilliant blue and then destained to visualize the bands.

The protein profiles of three algal species were compared with each other and bands of protein fragments scored manually as (1) or (0) depending on the presence or absence of a particular band. The data was analysed using NTSYS software package. The SIMQUAL programme was used to calculate Jaccard’s coefficient. Clustering was done using Sequential Agglomerative Heirarchial Nested Clustering (SAHN) routine and a dendrogram was constructed using UPGMA with NTSYS package.

Antioxidant activity:

For the assessment of antioxidant activity, the chloroform, acetone and methanol extracts of selected algae were dissolved in deionized water or dimethyl sulphoxide (DMSO) respectively as per method of analysis.

DPPH (2,2-diphenyl-1-picrylhydrazyl)radical scavenging activity:

Free radical scavenging activities of different algal extracts were evaluated by DPPH free radical activity²⁴. 1.0ml of 0.1mM DPPH in methanol was added to 3ml of different extracts in dimethyl sulphoxide (DMSO) at different concentrations (200, 400, 600, 800 and 1000 µg/ml). The mixture was shaken vigorously and allowed to stand at room temp for 30 min, then, the absorbance was measured at 517nm by using spectrophotometer (UV-VIS Shimadzu). Ascorbic acid was used as reference standard and the experiment was done in triplicates. Lower absorbance of the reaction mixture indicated higher free radical activity. The percent DPPH scavenging effect was calculated by using following equation.

DPPH scavenging effect (%) or Percent inhibition

= A₀– A₁ / A₀ × 100.

Where A₀ was the Absorbance of control reaction and A₁ was the Absorbance in presence of test or standard sample.

FRAP (Ferric Antioxidant Power) assay:

The assay was carried out for different extracts as demonstrated by Limet al.²⁵with slight modifications. 300mM acetate buffer (pH 3.6), 20mM 2,3,5-Triphenyltetrazolium chloride (TPTZ) solution in 40 mM HCl, and 20mM FeCl₃ were mixed together in the ratio of 10:1:1 to make FRAP solution and tested against extracts by allowing it to react with the FRAP solution in the ratio of 1:30 for 30min in dark at 37°C. The blue coloured product (Ferrous tripyridyltriazine complex) was formed and the absorbance was taken at 593nm spectrophotometrically. Ascorbic acid was used as reference standard.

Hydrogen peroxide (H₂O₂) Scavenging Activity:

For assessing the scavenging activity of hydrogen peroxide of three seaweeds the method described by Ruch et al.²⁶ with light modification was used.H₂O₂with a concentration of 4mMol/L was prepared in phosphate buffer solution (PBS) (pH 7.4). Different concentrations of Algal extract were prepared in distilled water. Approximately, 4ml of algal extract and 0.6ml of 4 mMol/L H₂O₂solutions were mixed and incubated for 10 minutes at room temperature. Then the absorbance was measured at 230nm against a blank solution containing the algal extract in PBS without H₂O₂.

H₂O₂ radical scavenging activity

=(A_control-A_test)/A_control×100

Where A_control is the absorbance of H₂O₂ radical+methanol; A_test is the absorbance of H₂O₂ radical+sample extract or standard.

Metal Chelating Assay:

The chelation of ferrous ions by sample was estimated by method of Dinisetal.²⁷. About 50µl of 2mM FeCl₂ was added to 1ml of different concentrations (200 to 1000µg/ml) of the test sample. The reaction was initiated by the addition of 0.2ml of 5mM ferrozine solution. The mixture was shaken vigorously and allowed to stand at room temperature for 10min. The absorbance of the solution was measured at 562nm. Na₂ EDTA was used as positive control. Ascorbic acid was used as standard drug. The percentage inhibition of ferrozine-Fe²⁺ complex formation was calculated using formula.

% Inhibition= (A_control-A_test)/A_control×100

Reducing Power Assay:

Different concentrations (200 to 1000μg/ml) test sample extracts were taken and mixed well with 2.5ml of phosphate buffer (pH 6.6) and 2.5ml of (1%) potassium ferricyanide²⁸and were incubated for 20min at 50°C. To this, 2.5ml of (10%) trichloroacetic acid was added and then centrifuged at 3000rpm for 10min. From this, 2.5 ml of supernatant was taken and was mixed with 2.5ml distilled water and 0.5ml of (0.1%) ferric chloride solution. The absorbance was measured at 700nm. A blank without extract was prepared simultaneously. Different concentrations of Ascorbic acid (20 to 100 μg/ml) were used as standard.

Data analysis:

A complete randomized design (CRD) was used in all experiments where ever necessary and the data was subjected to one way analysis of variance (ANOVA) using Minitab 15 software. Duncan’s Multiple Range Test (DMRT) was used to separate the means for significant effect. A significance level of 0.05 used for all statistical tests.

RESULTS:

SDS-PAGE analysis:

SDS-PAGE banding pattern of the three algal extracts was shown in figure 1. A Total of 23 different protein bands were observed. In U.lactuca16 protein bands were observed, in S. ilicifolium 14 protein bands were observed and in G. Corticata (red algae) 17 protein bands were observed (figure.1a).

According to the Sequential Agglomerative Hierarchical Nested Clustering (SAHN) routine and a dendrogram was constructed using UPGMA. According to this G. corticata and U. lactuca were closely related species and they were situated in single cluster and S. ilicifolium was placed in other cluster (figure.1b).

Figure 1: SDS-PAGE analysis.

(a) Lane-1: Green algae (U. lactuca); Lane-2: Brown algae (S. ilicifolium); Lane-3: Red algae (G. corticata) and Protein marker (100 kDa), (b) UPGMA dendrogram of three algae.

Anti-oxidant activity:

DPPH free radical scavenging activity:

The chloroform, acetone and methanolic extracts of three algae showed the concentration dependent DPPH free radical scavenging activity. The G. corticata extracts showed DPPH free radicalscavenging activity from 5.51±0.42 to 50.58±0.30. Among three extracts, methanolic extract have more (50.587±0.30), acetone extract has moderate (46.89±0.32) and chloroform extract have poor (19.84±0.27) DPPH free radical scavenging activity (Figure.2a). The IC₅₀ values for the three extracts were 2860.5µg/ml, 902.09µg/ml and 794.61µg/ml respectively (figure.2f).

The U. lactuca extracts showed moderate DPPH free radical scavenging activity ranging from 9.63±0.13 to 43.62±0.56 (Figure 2a). The IC₅₀ values for the three extracts were 1267.00µg/ml, 1196.36µg/ml and 1082.11 µg/ml respectively (figure.2f).

S. ilicifolium extract showed more DPPH free radical scavenging activity ranging from 12.02±0.19 to 48.93±0.22 (Figure. 2a). The IC₅₀ values for all the three extracts were 1139µg/ml, 1499.15µg/ml and 989.17µg/ml respectively (figure.2f).

Among three algal species G.corticata extracts showed high activity compared to other two algae and U. lactuca showed less DPPH free radical scavenging activity. The order of DPPH free radical scavenging activity for different algae was as follows: G. corticata >S. ilicifolum>U. lactuca. The order of activity for different solvent extracts was as follows: methanolic extract > acetone extract >chloroform extract.

Ferric Antioxidant Power (FRAP) assay:

FRAP assay was used to determine the antioxidant capacity of the selected algal extracts and the tested extracts showed concentration dependent reducing potential on ferric ion to ferrous ion. The results of FRAP assay indicated that, tested algal extracts at different concentration possess moderate activity as donation of hydrogen ion to ferric tripyridyltriazine (Fe³⁺-TPTZ) complex and producing a coloured ferrous tripyridyltriazine (Fe²⁺-TPTZ).

The extracts of G. corticata reducing potential ranges from 2.58±0.17 to 31.03±0.64 (figure. 2b). The IC₅₀ values for chloroform, acetone and methanol extracts was 1844.28µg/ml, 2489.05µg/ml and 1540.32µg/ml (figure. 2f).The extracts of U. lactuca showed moderate reducing potential ranges from 2.73±0.06 to 23.37±0.64 (figure.2b). The IC₅₀values for chloroform, acetone and methanol extracts were 2474.42µg/ml, 2740.35µg/ml and 2274.71µg/ml respectively(figure. 2f).

The S. ilicifolum showed less activity compared to U. lactuca and G. Corticata extracts. The reducing potential of chloroform, acetone and methanol extract ranges from 13.33±0.33-18.04±0.31 respectively (figure.2b). The IC₅₀values for chloroform, acetone and methanol extracts were 3658.5µg/ml, 3250.75µg/ml and 3239.86µg/ml respectively (figure.2f).

Among three algae Gracileria corticata showed more activity compare to other two algal species. The order of activity was as follows: G. corticata > U. lactuca > S. ilicifolum.The order of activity for different solvent extract was as follows: methanolic extract >acetone extract > chloroform extract in U. lactuca and S. ilicifolum but in G. corticata it was methanolic extract > chloroform extract >acetone extract.

Hydrogen peroxide (H₂O₂) scavenging activity:

H₂O₂ scavenging activity is one of the often-used methanol to measure the antioxidant activity of natural extracts, because H₂O₂can be formed in the body from different metabolic reactions. The H₂O₂ is independently not much harmful but it is the main precursor for highly reactive oxygen species (ROS) that is OH^̇̇.The H₂O₂ scavenging activity is important method to measure antioxidant activity of natural extracts by their percentage inhibition (%inhibition) of produced OH^̇ free radicals through Fenton reaction.

The extracts of Gracileria corticata showed moderated H₂O₂ scavenging activityand ranging from 6.60±0.51 to 35.87±0.64 (figure. 2c). The IC₅₀values of chloroform, acetone and methanolic extracts were 2860.5µg/ml, 902.09µg/ml and 794.61µg/ml respectively (figure.2f). The Ulva lactuca extracts showed less H₂O₂ scavenging activity and ranges from 7.46±0.19 to 23.82±0.33 (figure.2c). The IC₅₀ values of chloroform, acetone and methanolic extracts were 1267.00µg/ml, 1196.36µg/ml and 1082.11µg/ml respectively(figure.2f).

The S. ilicifolum extracts showed more activity than other two algae extracts and ranging from 13.57±0.30 to 64.40±0.60 (Figure. 2c). The IC₅₀ values of chloroform, acetone and methanolic extracts were 1139.00µg/ml, 1499.15µg/ml and 989.17µg/ml (Figure.2f). Among three algal species, S. ilicifolum extracts showed more activity compared to other two algal species extracts. The order of H₂O₂ scavenging activity was S. ilicifolum> G. corticata > U. lactuca and the order for different solvent extracts wasmethanolic extract >acetone extract > chloroform extract.

Metal chelating assay:

Metal chelating activity is one the commonly used method for evaluation of antioxidant activity of different extracts with ferrozine solution. The extracts of G. corticata showed metal chelating activity ranging from 2.53±0.33 to 34.10±0.22 (Figure 2d). The IC₅₀ values of chloroform, acetone and methanolic extracts were 2339.13µg/ml, 2570.05µg/ml and 1325.64µg/ml respectively (figure.2f).

The U. Lactuca showed less activity compared to other two algal extracts and ranges from 3.47±0.20 to 25.61±0.57 (figure.2d). The IC₅₀ values of chloroform, acetone and methanolic extracts were 2952.94µg/ml, 1863.07µg/ml and 2026.08µg/ml respectively (figure.2f).

The extracts of S. ilicofolum showed good activity. The activity of S. ilicifolium ranges from 5.08±0.14 to 25.35±0.28 (figure.2d). The IC₅₀ values of chloroform, acetone and methanolic extracts were 1983.8µg/ml, 2368.95µg/ml and 2110.83µg/ml respectively (figure.2f).

In the present study the selected algal extracts showed concentration dependent inhibition of Fe³⁺ions. Among three algae the methanolic extract of Gracileria corticata showed more activity when compared to methanol extract of S. ilicifolum.

The order of metal chelating activity was G. corticata > U. lactuca = S. ilicifolum and the order for different solvent extracts was methanolic extract >acetone extract > chloroform extract.

Reducing Power Assay:

The reducing power assay is mainly based on the evaluation of absorbance of testing extracts on reduction of the potassium ferricyanide (Fe³⁺) to potassium ferrocyanide.

The reducing power of G. corticata extracts ranges from 1.70±0.21 to 2.61±0.11µg/ml (figure 2e). The absorbance of U. lactuca extracts ranges from 2.24±0.12 to 2.87±0.13µg/ml (figure 2e). The absorbance of S. ilicifolum extracts ranges from 1.82±0.10 to 3.48±0.17µg/ml (figure.2e).

Among three algal species S. ilicifolum extracts showed more reducing potential. Among all extracts of three algal species methanolic extracts of the three showed more potential except in U. lactuca. The order of reducing power assay was S. ilicifolum > Ulva lactuca > G. corticata and the order of solvent extracts was methanolic extract = Acetone extract > chloroform extract.

DISCUSSION:

The preliminary phytochemical analysis revealed the presence of all the three major classes of plant secondary metabolites namely terpenoids, alkaloids and phenolic compounds. The high antioxidant activity of the seaweeds tested in the present study indicates they are potentially prolific source of highly bioactive secondary metabolites.

The present study reveals that G. corticata showed high DPPH radical scavenging activity, FRAP activity, metal chelating activity among all three selected seaweeds whereas S. ilicifolum showed high H₂O₂ Radical scavenging activity and reducing power assay among three seaweeds used in the present study. Generally, the reducing properties are associated with the presence of compounds, which exert their action by breaking the free radical chain by donating a hydrogen atom or a single electron²⁹. Compounds with structures containing two or more of the following functional groups: –OH, –SH, –COOH, –PO₃H₂, >C=O, –NR₂, –S– and –O– in a favourable structure-function configuration will have chelation activity and polyphenols derived from seaweeds are potent Fe²⁺ chelators³⁰. Metal chelating potency of phenolic compounds is also dependent upon their unique phenolic structure, and the number and location of –OH groups. From the present study it was understood that high antioxidant activities of G. corticata followed by S. ilicifolum might be due to high phenolic content. Similar results where the extracts of red seaweeds, G. verrucosa, G. textorii, Grateloupia filicina and Polysiphonia japonica were also reported to have high DPPH radical scavenging activity, FRAP activity, metal chelating activity was reported by Heo et al.³¹; Chakraborty et al.³². The significant correlation of total phenolic content with different antioxidant activities indicates that phenolic principles present in the red seaweed extracts are endowed with potential antiradical properties. The presence of polyphenolic compounds such as phlorotannins in brown seaweeds was reported by Gómez-Guzmánet al.³³.

Extraction is the important step to recover and isolate bioactive compounds from the materials. Efficiency of the extraction is strongly affected by the extraction method and the solvent used³⁴. Since antioxidant compounds have different polarities different solvents were used to test their antioxidant potential. Among the different solvent extracts used in the present study methanol extracts showed high antioxidant activities followed by acetone. This might be due to significantly higher quantities of phenolic compounds in methanol extract and it shows most of the compounds in the selected seaweeds are more polar. Similar finding was also reported by Kuppusamy et al.³⁵. Similar studies where methanolic extract having more antioxidant activity was reported by Gonget al.³⁶, Wanget al.³⁷. Methanol was identified as the most effective solvent for extraction resulting in the highest antioxidant and in vitroanti-inflammatory activities³⁸.

CONCLUSION:

Seaweeds of marine ecosystem are rich in bioactive molecules and widely used in food medicine and pharmaceutical industries. Marine seaweeds have been recognized as unique for its structural and functional components when compared to terrestrial organisms. In the present study seaweeds collected from Visakhapatnam coast were screened for antioxidant potential in different seaweed extracts.

This study shows that the methanolic extracts of G. corticata have high levels of antioxidants with high DPPH, FRAP, reducing power activities and S. ilicifolum have high H₂O₂ radical scavenging, and metal chelating activities. The in vitro assay conducted in this study showed high cytotoxic activity. This is attributed to their higher nutrient source and alkaloid content. Methanolic extract contains significantly high amount phenolic content when compare to other extracts and showed higher antioxidant activity. From our study the selected seaweeds G. corticata, U. lactuca and S. ilicifolum was also be used as a source of potent bioactive compounds for various therapeutic applications. The study confirms that the nutraceutical potential seaweeds and also recommends the seaweeds to be a part of daily diet because of their beneficial health properties.

CONFLICTS OF INTEREST:

The authors declare no conflict of interests including the financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence, the present work.

ACKNOWLEDGMENTS:

The authors acknowledge the Head, Department of Biochemistry, Andhra University for providing the facilities to carry out the research work.

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Received on 31.01.2023 Modified on 27.04.2023

Research J. Pharm. and Tech 2024; 17(5):2018-2024.

DOI: 10.52711/0974-360X.2024.00319