Antioxidant activity of ethyl acetate and methanolic extracts of two marine algae, Nannochloropsis oculata and Gracilaria gracilis - an in vitro assay

ABSTRACT

The aim of this study was to evaluate the antioxidant activity of ethyl acetate and methanolic extracts of two marine algae, Nannochloropsis oculata and Gracilaria gracilis. The extracts were assayed for total phenol and flavonoid content, DPPH free radical scavenging capacity, nitric oxide activity, iron chelation activity, and reducing power activity. Total phenol and flavonoid content were found to be high in both algae. Ethyl acetate extracts of both algae were found to exhibit significant antioxidant activity. Ethyl acetate extract of N. oculata exhibited a good capacity for iron chelation, nitrate oxide, and scavenging DPPH free radicals (72.95±2.30, 73.73±1.76, and 39.03±0.97% inhibition at 400 µg mL^-1 respectively).

Keywords:
Marine algae/antioxidant activity; Nannochloropsis Oculata/total phenol/total flavonoid/ Gracilaria Gracilis/total phenol/total flavonoid

INTRODUCTION

Free radicals, which refer to any molecular species containing unpaired electron in an atomic orbital, are unstable elements amenable to take part in chemical reactions (Yangthong, Hutadilok-Towatana, Phromkunthong, 2009). Reactive oxygen, nitrogen and sulfur species are free that produce superoxide anion, hydroxyl radical, hydrogen peroxide and nitric oxide. These compounds may cause diseases as diverse as cancer, diabetes, Alzheimer, and Parkinson through inducing oxidative stresses, cell damage and death (Lü et al., 2010; Yangthong, Hutadilok-Towatana, Phromkunthong, 2009; Ebrahimzadeh, Safdari, Khalili, 2015). A huge number of natural compounds have been proven to exhibit antioxidant activity and be applicable for treatment of oxidative-damage related diseases. Large number of plants (e.g. Salvia virgate and Silybum marianum), fungi (e.g. Cantharellus cibarius) and alga (e.g. Ecklonia cava and Stoechospermum marginatum) are regarded as major sources of natural antioxidants (Ebrahimzadeh et al., 2016; Ebrahimzadeh, Safdari, Khalili, 2015; Khalili, Ebrahimzadeh, Kosaryan, 2015).

Recent studies have shown that marine algae are rich in bioactive compounds including polysaccharides, polyunsaturated fatty acids, polyphenolic compounds, antioxidants, peptides, essential vitamins and minerals (Kim et al., 2014; Fernando et al., 2017; Wang et al., 2017). These compounds exhibit wide range of pharmacological activities, including antioxidant, antibacterial, antifungal, anti-inflammatory, anti-aging, and anti-cancer activities (Mayer et al., 2009; Fernando et al., 2016; Agatonovic-Kustrin, Morton, 2017). In the current study, we evaluate the anti-oxidative activity of two marine alga, Nannochloropsis oculata and Gracilaria gracilis.

MATERIAL AND METHODS

Chemicals

Ferrozine, trichloroacetic acid, 1, 1-diphenyl-2-picryl hydrazyl (DPPH), and potassium ferricyanide were purchased from Sigma Chemical Co. (Germany). butylated hydroxyanisole, ascorbic acid, sulfanilamide, N-(1-naphthyl)ethylenediamine dihydrochloride, ethylenediaminetetraacetic acid, and ferric chloride were purchased from Merck (Germany).

Algae species

N. oculata was cultivated to Walne medium (Walne 1970) (Table I). G. gracilis samples were kindly provided by Dr. Dehpour (Department of Biology, Islamic Azad University, Branch of Qaemshahr, Qaemshahr, Iran). The samples have been gathered from the beach of Persian Gulf (Bostaneh, Central District of Bandar Lengeh County, Hormozgan Province, Iran. Coordinates: 26°30′37″N 54°39′20″E, 2015). G. gracilis samples were washed with water and then dried at 45 °C for 24 h using forced convection drying oven.

TABLE I
Walne media for cultivated N. oculata

Stocks	Per 100 mL
(1) Trace metal solution (TMS)
ZnCl2	2.1 g
CoCl2.6HO2	2.0 g
(NH4)6Mo7O24.4HO2	0.9 g
CuSO4.5H2O	2.0 g
Make up to 100 mL with distilled water. This solution is normally cloudy. Acidify with a few drops of conc. HCl to give a clear solution.
(2) Vitamin solution
Vitamin B12. (Cyanocobalamin)	10.0 mg
Vitamin B1(Thiamine.HCl)	10.0 mg
Vitamin H (Biotin)	200.0 µg
Make up to 100 ml with distilled water.
(3) Nutrient solution	per litre
FeCl3.6H02	1.3 g
MnCl2.4H02	0.36 g
H3BO3	33.6 g
EDTA(Disodium salt)	45.0 g
NaH2PO4.2HO2	20.0 g
NaNO3	100.0 g
TMS (1 above)	1.0 ml
Make up to 1 litre with distilled water.
Medium	per litre
Nutrient solution (3)	1.0 mL
Vitamin solution (2)	0.1 mL
Sterilised seawater	1.0 L

• Dispense nutrient and vitamin solutions separately into 10 mL and 1 mL respectively and autoclave at 15 psi for 15 minutes. Add an aliquot of each aseptically 10 L of sterilised seawater.

Preparation of methanolic and ethyl acetate extracts from N. oculata and G. gracilis

Ethyl acetate extracts were obtained by percolating 10 g of powdered algae for 24 h at room temperature. The extracts were refined using Whatman filter paper (No. 1).The remaining samples were used to extract methanolic compounds. Both the extracts were filtered and then concentrated under reduced pressure at 40 °C using a rotary evaporator. All extracts were dried by vacuum freeze dryer.

Determination of total flavonoid and phenolic contents

To quantity total flavonoids in the algae species, 0.5 mL of each algal extract (Concentration: 800 µg mL^-1) was mixed with 1.5 mL methanol, 0.1 mL 1 M potassium acetate, 0.1 mL aluminum chloride10%, and 2.8 mL distilled water, and then incubated at room temperature for 30 min. The absorbance was measured at 415 nm by spectrophotometer. Total flavonoid content was calculated as quercetin equivalents (QE) from a calibration curve as described previously (Ebrahimzadeh, Safdari, Khalili, 2015). Total phenolic content was measured using Folin-Ciocalteu method. Summarily, the extracts (0.5 mL) were mixed with 2.5 mL of Folin-Ciocalteau reagent (0.2 N) and 2.0 mL sodium carbonate (concentration of 75g/L) and subjected to absorbance measuring at 760 nm (spectrophotometer (UV-Visible EZ201, Perkin Elmer, Norwalk, Connecticut). More details on the procedure can be found in our previously published article (Ebrahimzadeh, Safdari, Khalili, 2015). The results were expressed as gallic acid equivalents (GAE).

Metal chelating activity assay

Metal chelating activity assay has been described in our previous work (Ebrahimzadeh, Safdari, Khalili, 2015). Summarily, 1 mL of each extracts (800 μg mL^-1) was mixed with 0.5 mL of 2 mM FeCl (II) solution, to which 0.2 mL of ferrozine (5 mM) was added to initiate the reaction by adding. The absorbance of the solutions was measured at 562 nm. EDTA was used as a standard.

Nitric oxide scavenging activity assay

3 mL of each extract (800 µg mL^-1) was added to 1 mL of sodium nitroprusside (10 mM) and incubated at room temperature for 150 min. Afterward, 0.5 mL Griess reagent [1% sulfanilamide, 2% H₃PO₄, and 0.1% N-(1-naphthyl) ethylenediamine dihydro chloride] was added to the reaction and subjected to absorbance measuring at 546 nm. Quercetin (a flavonoid compound, PubChem CID: 5280343) was used as a positive control (Ebrahimzadeh, Safdari, Khalili, 2015).

DPPH radical scavenging activity

DPPH radical scavenging activity was assayed as described previously (Ebrahimzadeh, Safdari, Khalili, 2015). Summarily, 2 mL of DPPH solution (100 μM) was added to 2 mL of each fraction (concentration of 800 μg mL^-1) and then incubated at room temperature for 15 min, the absorbance was recorded at 517 nm. The reaction was then subjected to absorbance measuring at 517 nm. BHA was used as standard control.

Reducing power assay

Each extract (at different concentrations, ranging from 100 to 1600 μg mL^-1) was mixed with 1 mL potassium hexacyanoferrate and 1 mL phosphate buffer (0.2 M, pH 6.6) (1%), incubated at 50°C (in a water bath) for 20 min, and then received To terminate the reaction, 1 mL of TCA (10%) was added to each reaction. The upper portion of the solution (1 mL) was mixed with 1 mL distilled water and 0.2 mL iron (III) chloride solution (0.1%) and subjected to absorbance measuring at 700 nm (Ebrahimzadeh, Safdari, Khalili, 2015).

Statistical analysis

All the experiments included in this study were done in triplicate and the results expressed as means ± SD. The data were analyzed through one-way analysis of variance (ANOVA) using GrphPad Prism. The means were compared using Tukey multiple comparisons test (p<0.05).

RESULTS AND DISCUTION

Ethyl acetate and methanolic extracts of N. oculata and G. gracilis

The maximum and the minimum extraction efficiencies belonged to ethyl acetate extract of G. gracilis and methanolic extract of G. gracilis, 0.016 and 0.059 g/g DW, respectively. Extraction efficiencies for ethyl acetate and methanolic extracts of N. oculata were calculated to be 4.54 and 5.15 %, respectively.

Total flavonoid and phenolic contents

Total flavonoid content of all the extracts is shown in Table II. Ethyl acetate extracts of both N. oculata and G. gracilis species were found to be rich in flavonoid compounds (71.79±2.32 and 66.48±1.87 mg quercetin equivalent g^-1 of extract, respectively) while their methanolic extracts were found to contain less amounts of flavonoid compounds (42.08±1.09 and 26.47±1.203 mg quercetin equivalent g^-1 of extract, respectively).

TABLE II
Total phenol and flavonoid contents and antioxidant activities of ethyl acetate and methanolic extract of N. oculata and G. gracilis

Sample name		N. oculata		G. gracilis
Extract		Ethyl acetate extract	Methanolic extract	Ethyl acetate extract	Methanolic extract
EFFICIENCY (g/g DW)		0.045	0.051	0.016	0.059
Total Flavonoid content (mg quercetin equivalent g-1 of extract)		71.79±2.32	42.08±1.09	66.48±1.87	26.47±1.203
Total Phenol content (mg gallic acid equivalent g-1 of extract)		41.45±1.42	30.94±1.61	35.53±1.47	29.39±2.01
Iron chelation activity*	percentage of inhibition at 400 µg mL-1	72.95±2.30	29.71±1.98	49.71±1.93	19.49±1.46
Nitric Oxide activity **		73.73±1.76	65.6±1.54	65.33±2.54	34.8±1,59
DPPH***		39.03±0.97	21.68±1.41	20.48±0.38	17.4±1.34

• Each value is the mean of three replicate determinations ± SD; * EDTA was used as control (IC50 =18.27 ± 0.09 μg mL^-1); ** Quercetin was used as control (IC50 =5.28 ± 0.2 μg mL^-1); *** BHA was used as control (IC50 = 53.96 ± 3.1 μg mL^-1).

Total phenol content was calculated using Folin-Ciocalteau method and linear gallic acid standard curve (y = 0.0054x +0.0623; r = 1). The content of total phenols in the algal species is shown in Table II. The maximum and the minimum amounts of phenolic compounds belonged to ethyl acetate extract of N. oculata and methanolic extract of the G. gracilis 41.45±1.42 and 29.39±2.01mg gallic acid equivalent g^-1 of extract, respectively (Table II). Total phenol content of methanolic extract of N. oculata and ethyl acetate extract of G. gracilis were calculated to be 30.94±1.61 and 35.53±1.47 mg gallic acid equivalent g^-1 of extract, respectively.

We found the maximum phenolic compounds in ethyl acetate extract of algal species. Our results are consistent with the results of Duan et al. (2006). However, our findings are inconsistent with the reports of Farasat et al. (2014). They evaluated the total phenol and flavonoid content of some algae species driven from northern coasts of the Persian Gulf, and showed that methanolic extracts contain higher levels of flavonoid content.

Both phenolic and flavonoid compounds have been demonstrated to exhibit wide range of advantageous biological activities, including antioxidant activity, free radical scavenging activity, anti-cancer and anti- atherosclerotic activities, and so forth (Balasundram, Sundram, Samman, 2006; Cox, Abu-Ghannam, Gupta, 2010; Duan et al., 2006; Li et al., 2007; Fernando et al., 2016).

Iron chelation activity

Ethyl acetate extracts of both algae species were found to have higher iron chelation activity than their methanolic extracts. . The maximum and the minimum iron chelation activities belonged to ethyl acetate extract of N. oculata (and the methanolic extract of G. gracilis - 72.95±2.30 and 19.49±1.46% inhibition at 400 µg mL^-1, respectively. The correlation coefficient (r²) between iron chelation activity and either “total phenol” or “flavonoid content “was calculated to b 0.99 and 0.89, respectively.

Studies show that algae are rich in compounds with significant iron chelation activity (Bermejo, Piñero, Villar, 2008). Metal chelation ability depends on the type (flavonoid and phenolic acid), structure, and the number of available hydroxyl groups (Wang, Jónsdóttir, Ólafsdóttir, 2009).

Nitric oxide activity

Nitric oxide activity of the algal extracts at concentration of 400 µg mL^-1 for ethyl acetate extract of N. oculata, the methanolic extract of N. oculata, the ethyl acetate extract of G. gracilis, and ethyl acetate extract of G. gracilis were calculated to be 73.73±1.7 %, 65.6±1.54, 65.33±2.54 and 34.8±1.59% inhibition respectively. There was a low correlation between nitric oxide activity and either total phenol or flavonoid contents (r²= 0.56 and r²= 0.69 respectively). Reactive oxygen and nitrogen species are two main sources of oxidative substances, including hydrogen peroxide, superoxide, nitric oxide and peroxynitrite (ONOO⁻). These substances can damage proteins, lipids and DNA. (Thanigaivel et al., 2016).

DPPH radical scavenging activity

DPPH color change from purple -to- yellow is an index for radical-scavenging activity, the more intense color transformation the higher scavenging activity is expected (Gontijo et al., 2012). As shown Table II, the highest DPPH radical scavenging activity belonged to the ethyl acetate extract of N. oculata (39.03±0.97% inhibition at 400 µg mL^-1) and the lowest activity belonged to methanolic extract of G. gracilis (17.4±1.34% inhibition at 400 µg mL^-1). Methanolic extract of N. oculata and ethyl acetate extract of G. gracilis (at concentration of 400 µg mL^-1) were found to inhibit DPPH free radicals up to 21.68±1.41 and 20.48±0.38%, respectively. DPPH was found to have a high correlation (r²= 0.81) with total phenolic compounds but not with total flavonoid compounds (r² = 0.49). These findings are consistent with the results of our previous study (carried out on Cantharellus cibarius) and also with the results of Orhan and Üstün who investigated the correlation of phenol content and antioxidant activity of some Turkey mushrooms (Ebrahimzadeh, Safdari, Khalili, 2015; Orhan, Üstün, 2011; Duan et al., 2006).

Reducing power activity

Reducing power, which is an index for antioxidant activity, can be measured using a reaction in which Fe ³⁺ in potassium ferricyanide is converted into Fe ²⁺. The reaction product is measured using spectrophotometer at λ = 700 nm (Sharma, Gujral, Singh, 2012; Gontijo et al., 2012). As shown in Figure 1, reducing power of all the extracts increased in a dose dependent manner. In both N. oculata and G. gracilis species, ethyl acetate extracts werefound to have higher reduction capacity than methanolic extract (Figure 1). The least reduction power belonged Methanolic extract of G. gracilis. Vitamin C (the control) reached a plateau at concentration of 800µg/ml^-1 (OD = 0.401), whereas the ethyl acetate extract of N. oculata and methanolic extract of G. gracilis exceeded this value and produced OD values of 0.73 and 0.531, respectively. At concentration of 1600µg/ml^-1 reducing power was found to have a potent coloration with total phenolic (r²=0.98) and flavonoid (r²=0.92) compounds. Phenolic compounds and flavonoids are electron donor substances that play an important role in exhibiting reduction capacity (Sharma, Gujral, Singh, 2012). Therefore, high coloration between reducing power and total phenolic and flavonoid content may be a reason for high reducing power activity.

FIGURE 1
Reducing power of ethyl acetate and methanolic extracts of algal species. Each point on the graphs represents the mean of three repetitions.

Irudayaraj and colleagues (2012) reported that ethyl acetate extract driven from leaves of Toddalia asiatica has potential anti-diabetic and antioxidant capacities. Al-Saeedi, Al- Ghafri, Hossain (2016) reported that the ethyl acetate extract of Ziziphus jujuba L. has high levels of phenolic and flavonoid compounds. Therefore, the ethyl acetate extracts of N. oculata and G. gracilis could be used as a natural antioxidant for the treatment of different diseases related with free radicals and oxidative stress.

CONCLUSION

Ethyl acetate extracts of Nannochloropsis oculata and Gracilaria gracilis algae have considerable amounts of phenolic and flavonoid compounds. Ethyl acetate extracts driven from the both algae species exhibit significant antioxidant activity. There is a good correlation between total phenols and flavonoids content and iron chelation activity

ACKNOWLEDGMENTS

This research was supported by a grant from the research council of Medical Sciences University of Mazandaran, Iran.

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Publication Dates

• Publication in this collection
  2018

History

• Received
  16 May 2017
• Accepted
  07 Aug 2017