In-vitro Antioxidant activity of Marine Red Algae Gracilaria foliifera

 

Jenifer. P ¹, C.P. Balakrishnan¹*, S. Chidambaram Pillai²

¹Department of Botany, Aditanar College of Arts and Science, Virapandianpatnam, Tiruchendur - 628216, Tamil Nadu, India

² PG and Research Department of Botany, V.O. Chidambaram College, Tuticorin- 628 008, Tamil Nadu, India

 

ABSTRACT:

Seaweeds are potential as natural sources of antioxidants and have gained recent attention.  The present study was made on antioxidant activity was performed by organic solvent extracts like methanol and aqueous of marine red algae Gracilaria foliifera (Forssk). In-vitro antioxidant analysis like DPPH, H₂O₂, OH, NO radical scavenging activity and total antioxidant activity also carried out by using standard procedures. In this study, radical scavenging activity was observed in different concentrations range from 100, 250, 500,750, 1000 μg/ml.  The performances of scavenging activity of above extracts were comparing with standard ascorbic acid. An IC₅₀ value of methanol and aqueous extract of DPPH radical were recorded at 679.27µg/ml and 801.12µg/ml, H₂O₂ radical were recorded at 790 µg/ml and 935.95µg/ml, hydroxyl radical at 687.92μg/ml and 159.56μg/ml respectively. Whereas nitric oxide the IC₅₀ of methanol and aqueous were recorded at 812.75μg/ml and 1107.17μg/ml respectively. They were compared with IC₅₀ value of standard ascorbic acid. In-vitro antioxidant results clearly indicated that the beneficial effect of G. foliifera exhibited significant value was compared with standard.

 

 

 

INTRODUCTION:

In human system at end of metabolism the consumable O₂ capable the potential to damage the cell molecules. They are called “Free Radicals”. They are capable of attacking the healthy cells of the body, consuming them to lose their structure and function. Antioxidants to protect the cells and organ system of the body against reactive oxygen species (ROS), human have evolved a highly sophisticated and complex antioxidant protection system that functions interactively to neutralize free radicals.

 

Free radicals are produced as a part of normal metabolic processes. Reactive oxygen species (ROS) include free radical for example, hydroxyl radical (OH), superoxide anion (O₂) and non free radical species such as hydrogen peroxide (H₂O₂), nitric oxide (NO) are various forms of activated oxygen and are destructive to various physiologically important molecules including protein, lipids, cell membrane, DNA and other cellular constituents (Wijeratne et al., 2005). Marine algae are produced a wide variety of chemically active metabolites in their surroundings, these active metabolites also known as biogenic compounds such as halogenated compounds, alcohols, aldehydes, terpenoids are produced by several species of marine algae and have antioxidant, antibacterial, antialgal and antifouling properties.  Seaweeds are considered to be a rich source of antioxidants (Devi et al., 2011 and Kelman et al., 2012). Natural antioxidants found in algae play an important role against various diseases and ageing processes by protecting the cell from oxidative damage (Karawita et al., 2007). In-vitro antioxidant activity of marine red algae Chondrococcus hornemanni and Spyridia fusiformis at different solvent namely chloroform, methanol and aqueous extract were reported by Bhuvaneswari et al., 2013. Therefore, the present study was to evaluate the antioxidant activities of marine red algae Gracilaria foliifera using different concentrations of organic solvents like methanol and aqueous extract.

 

MATERIALS AND METHODS:

Collection of Seaweed:

Marine red algae Gracilaria foliifera (Forssk) was collected from Manapad coast of Tamil Nadu, India (8.3775°N; 78.0522°E) at low tide. Specimen was washed thoroughly in seawater to remove extraneous matter such as epiphytes and sand. After collection, fresh samples were taken into plastic jar and brought back to the laboratory immediately.  Samples were washed by tape water for several times, then gently brushed and rinsed with distilled water and then dried at room temperature.  Dried sample was pulverized using domestic blender and stored in air tight container for further use.

 

Antioxidant Activities:

Preparation and Processing of the extracts and Standard:

10g of powdered sample was subjected to extract with methanol and aqueous using Soxhlet extractor for six hours and the extraction was repeated twice. The extracts were then concentrated to dryness under reduced pressure and controlled temperature (40-50^oC). The stock solutions were serially diluted with respective solvents to get lower concentrations (1000, 750, 500, 250, 100 μg/ml). Each concentration was prepared in triplicate. These were subjected to in-vitro assay of total antioxidant activity by the method of phosphomolybdate using α- tocopherol as the standard (Prieto et al., 1999). The total antioxidant capacity was expressed as µg g^-1 equivalents of Vitamin E α-tocopherol by using the standard α-tocopherol graph. Nitric Oxide scavenging method can be determined by the use of the Griess Illosvoy reaction (Garrat 1964), Hydrogen peroxide by method of Dehpour et al., 2009, 1, 1-diphenyl-2-Picrylhydrazyl (DPPH) by Mensor et al., 2001 and Hydroxyl radical scavenging methods by Chung et al., 1997. Vitamin C (Ascorbic acid) was used as a standard and was dissolved in respective solvent and diluted quantitatively to obtain a concentration of 100µg ml^-1.

 

Scavenging activity (%) = [(A – B) / A] x 100

Where A is the absorbance of control, B is the absorbance of the sample (extract/ ascorbic acid).

A percent inhibition versus concentration curve was plotted and the concentration of sample required for 50% inhibition was calculated by linear regression analysis.

 

Calculation of IC₅₀ :

IC₅₀ value was calculated by using the formula to determine the regression equation Y= mx + c in MS Office excel version 2007

 

Statistical Analysis:

All experiments were carried out in triplicate (n=3). The results were presented as mean± SE standard error using SPSS statistics 17.0 software. The datas were statistically analyzed by one-way ANOVA and Tukey post-hoc test. The level of statistical significance was set at p < 0.05.

 

RESULTS AND DISCUSSION:

The absorbance values, % inhibition, IC₅₀ values of radical scavenging ability such as DPPH, H₂O₂, OH and NO at different concentrations (100, 250, 500, 750, 1000 μg/ml) in methanol and aqueous extract of red algae G. foliifera is shown in table 1. Total antioxidant activity of methanol and aqueous extracts of G. foliifera equivalent with Vitamin E standard (α- tocopherol) and results were presented in Table-2. The standard regression curve present in fig.2 and the linear regression equation [y=0.0004x + 0.0807 (R²= 0.9814)] obtained by using Microsoft office excel. DPPH radical scavenging activity of different concentrations (100, 250, 500, 750, 1000 µg/ml) of methanol and aqueous extracts result a colour change from purple to yellow, the absorbance decreased when the DPPH was scavenged by an antioxidant through donation of hydrogen to form a stable DPPH molecule. The DPPH antioxidant assay is based on the ability of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) a stable free radical to decolorize in the presence of antioxidants (Kumarasamy et al., 2007).

 

In DPPH radical, the IC₅₀ value of the methanol, aqueous extract and standard were showed at 679.27µg/ml, 801.12µg/ml and 486.99µg/ml respectively. In H₂O₂ radical the IC₅₀ value of the methanol, aqueous extract and standard were showed at 790 µg/ml, 935.95µg/ml and 855.38µg/ml respectively. H₂O₂ itself is not very reactive; it can sometimes cause cytotoxicity by giving rise to hydroxyl radicals in the cell. Thus, removing H₂O₂ is very important throughout food systems (Nabavi et al., 2009). The identification of compounds that have excellent hydroxyl scavenging activity would be significant for some diseases caused by oxidative stress. It has been demonstrated that plants contain many natural antioxidants compounds which have been identified as hydroxyl radical scavengers (Zheng and Wang 2001). In hydroxyl radical scavenging activity, the IC₅₀ values of the methanol and water extracts were recorded at 687.92μg/ml and 159.56μg/ml. Whereas, IC₅₀ value of the reference ascorbic acid was 68.24μg/ml. Nitric oxide is a free radical that is generated when sodium nitroprusside reacts with oxygen to form nitrite, induces the inflammatory response and its toxicity multiplies if it reacts with O₂ radicals to form peroxynitrite (Stadler 2011). In nitric oxide scavenging of the IC₅₀ values of the methanol and water extracts were recorded at 812.75μg/ml and 1107.17μg/ml respectively. Whereas, IC₅₀ value of the reference ascorbic acid was 163.05μg/ml. Significant amount of total antioxidant activity was obtained from methanol extract 150 to 305 µg/g equivalent of vitamin E followed by water 167.5 to 352.5 µg/g. Amoung these, the IC₅₀ value of aqueous extract of NO radical activity showed poor significant value compared with standard ascorbic acid. Naturally seaweeds contain large amounts of antioxidant compounds which control the free radical formation from metabolic reaction.  In earlier, many workers reported seaweeds are considered to be a rich source of antioxidants (Cahyana et al., 1992, Devi et al., 2011, Kelman et al., 2012 and Kim et al., 2012).

 

Table 1: Different methods of radical scavenging activity of G. foliifera in different extract compare with standard Ascorbic acid

^*Values are expressed as mean ± SEM (Standard Error Mean) n=3 which, with different letters (within column), indicate significant difference (p<0.05). Post Hoc Test (Tukey HSD)

 

 

Table 2: Total antioxidant activity of G. foliifera in different extract equivalent of vitamin E standard (a-Tocopherol)

	Concentration (µg/ml)	Standard	Methanolic Extract		Water extract
		a-Tocopherol (Vitamin E) Absorbance (λ 695 nm)*	Absorbance*	µg/ml equivalent of vitamin E standard (a-Tocopherol)	Absorbance*	µg/ml equivalent of vitamin E standard (a-Tocopherol)
Total antioxidant activity	100	0.105±0.001a	0.060±0.002a	150	0.067±0.003a	167.5
	250	0.177±0.006b	0.073±0.003ab	182.5	0.073±0.002a	182.5
	500	0.259±0.010c	0.089±0.004bc	222.5	0.106±0.003b	265
	750	0.378±0.015d	0.099±0.005c	247.5	0.121±0.004c	302.5
	1000	0.421±0.011e	0.122±0.007d	305	0.141±0.002d	352.5

^*Values are expressed as mean ± SE (n=3) which, with different letters (within column), indicate significant difference (p<0.05). Post Hoc Test (Tukey HSD)

 

 

Fig 1: Standard curve for total antioxidant activity assay using α-tocopherol (100-1000 µg/ml)

 

CONCLUSION:

The present study has shown the antioxidant activity of marine red algae. The antioxidant content of the seaweeds varies significantly with organic solvents. Thus the G. foliifera is a potential source for natural antioxidant which will be beneficial as nutraceuticals and pharmaceuticals. The findings of the current report are useful for further research aiming at isolating specific phenolic compounds responsible for the antioxidant activity of this alga. The results have showed that the methanol and aqueous extracts of G. foliifera of marine red algae exhibit antioxidant potential and they are sources of natural antioxidant compounds.

 

ACKNOWLEDGEMENT:

The authors are gratefully acknowledges the University Grants Commission (UGC), New Delhi for the financial assistance of this project (Ref. No. 42-935/2013) under MRP scheme.

 

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Received on 31.03.2017       Accepted on 19.06.2017     

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