Chemical reagents. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), aluminum chloride, ammonium molybdate, aqueous hydrochloric acid, chloroform, concentrated sulfuric acid, di-sodium hydrogen phosphate, ethanol, ethyl acetate, ferric chloride, ferrous sulfate, Folin-Ciocalteu’s reagent, gallic acid, glacial acetic acid, hydrogen peroxide, methanol, olive oil, potassium acetate, potassium dihydrogen phosphate, potassium persulfate, sodium acetate, sodium carbonate, sodium nitrite and sodium phosphate were purchased from Sigma. All the chemical reagents used in the study were of analytical grade.
Seaweed sample collection and preparation. Mature G. tenuistipitata and P. tetrastromatica sample were collected from the wild source at Saint Martin’s Island (92º28ʹ40.12ʺE and 20º65ʹ51.43ʺN) of Bay of Bengal of Bangladesh in March 2020. Saint Martin's Island is still considered a biologically diverse ecosystem free of external pollutants, with a dense growth of various seaweeds. Permission of sample collection was gained from the local government before harvesting seaweed. In this experiment, samples of two different species of seaweeds (one red and one brown) were commonly found in the rocky surfaces during low tide. Dr. Md. Enamul Hoq, Former Director of BFRI, authenticated the botanical identification of seaweed species as the voucher specimen has been previously deposited at BFRI herbarium. The entire plant was collected from the exposed rock to ensure that the holdfast would not be left out. The collected thallus was washed thoroughly with clean seawater to remove dirt, sand, and other impurities. The specimen was preserved in an icebox at 4℃ and transported to the laboratory to maintain the fresh quality. Fresh samples were then washed thoroughly with distilled water for further removal of any other remaining impurities. Cleaned seaweed was then kept in a freeze dryer (VaCo 2, Zirbus, UK) for 48 hours at -83℃ to remove the moisture. Dried samples were sealed in plastic bags and stored in a refrigerator at 4℃ for further analysis in the laboratory.
Preparation of seaweed extract. Dried seaweed sample was grounded to make fine powder as the finer the powder, the more efficient the extraction would be. Four gram of seaweed fine powder was soaked in 100 mL of solvent (water, methanol and ethanol) by maceration for the preparation of an extract by solvent extraction. The sample was kept in the dark for 24 h with intermittent shaking for better extraction. After incubation, the solution was filtered with Whatman filter paper No 4 (20-25 µm) retaining hygienic conditions. After filtration, the remaining wet powder was again extracted in their respective solvents for 12 h through sporadic shaking and filtered to get the maximum out of the sample powder. The methanol and ethanol extracts were then concentrated using a Rotary Vacuum Evaporator (LRE-702A, Labocon, UK) and Nitrogen Evaporator (AT-EV-50, Athena Technology, India) at 36°C and the water solvent was dried by the Freeze Dryer (VaCo 2, Zirbus, UK) at -83℃16. Finally working solutions were prepared as 1mg/mL, 3mg/mL, 5mg/mL and 7mg/mL for each extract.
Qualitative analysis of phytochemical substances. Newly prepared all crude extracts of seaweed were subjected to qualitative assessments for the identification of various classes of active phytochemical constituents such as saponin64, terpenoid65, cardiac glycosides66 and phlobatannin16 following standard methods. General reactions in these analyses exposed the presence or absence of these compounds in the crude extracts tested.
Test for saponin (frothing test). About 5mL of each extracts was taken in separate test tubes and 5mL distilled water added in each test tube. The mixture was shaken vigorously for a minute and observed for a stable persistent froth. After the froth was persistent for at least 10 minutes, 3 drops of olive oil was added in the mixture and shaken vigorously again for the formation of an emulsion, which indicates the presence of saponins in the sample64.
Test for terpenoids (Salkowski test). About 5mL of each extract was taken in a test tube. 2mL chloroform was added and mixed cautiously. 3mL of concentrated sulfuric acid was added carefully and slowly in the solution to form a layer. Formation of reddish brown color at the interface indicates the presence of terpenoids in the sample65.
Test for cardiac glycosides (Keller-Killani test). 5mL of each extracts was taken in separate test tubes. 2mL of prepared reagent (glacial acetic acid containing one drop of ferric chloride) was added in each sample. 1mL concentrated sulfuric acid was added to the solutions carefully. A brown ring at the interface indicates the presence of a deoxysugar, characteristic of cardenolides. A violet ring may appear below the brown ring (at the H2SO4 layer). In the acetic acid layer, a greenish ring may form just above the brown ring which will gradually spread throughout this layer66.
Test for phlobatannins. Five mL of each extracts was taken in separate test tubes. Few drops of 1% aqueous hydrochloric acid was added in each test tube and mixed. After mixing, it was observed for precipitation. Red colored precipitation indicates the presence of phobatannins in the extracts16.
FTIR spectroscopy. Fourier transform infrared (FTIR) spectroscopy is a technique to obtain the spectrum of absorption or transmission of a sample under infrared light67. Different crude extracts of P. tetrastromatica and G. tenuistipitata were used to determine the presence of characteristic peaks and their functional groups using FTIR spectroscopy (Perkin Elmer Spectrum 2)68-70. FTIR spectra were recorded within the wavelengths of 450 and 4,000 cm–1. Analysis was done in triplicate and confirmed the spectrum in case of all extracts.
Quantitative analysis of phytochemicals
Total phenolic content (TPC). This parameter was carried out in the crude extracts using Folin-Ciocalteu Phenol reagents and external calibration with Gallic acid following by71 with slight modification. Briefly, 0.5 mL extract solution was added with 0.1 mL of FC reagent solution. After 15 min, 2.5 mL of saturated Na2CO3 (75 g/L) was added in the solution and allowed to stand for 30 min at RT and absorbance was measured at 760 nm using the spectrophotometer (T80+ UV/VIS Spectrophotometer, UK). The concentration of total phenolics was calculated as mg of Gallic acid equivalent per gram. The calibration equation for Gallic acid was
Y= 0.0116X + 0.0162; R2 = 0.9987 (1)
Total flavonoid content (TFC). This parameter was computed in the crude extracts using the aluminum chloride colorimetric method with minor modifications72. Briefly, 1 mL extract solution was mixed with 3 mL methanol, 0.2 mL 10% aluminum chloride and 0.2 mL 1 M potassium acetate. The solution was then incubated at RT for 30 minutes and absorbance was measured at 420 nm. The concentration of total flavonoids was calculated as mg of quercetin equivalent per gram. The calibration equation for Quercetin was
Y=0.0102X − 0.0637; R² = 0.9693 (2)
Evaluation of total antioxidant capacity
DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay. The DPPH free-radical scavenging assay was carried out in triplicate with negligible modification73. Different concentrated (1, 3, 5, 7 mg/ml) aliquot extracts solution was mixed with 2.5 mL 0.15mM DPPH solution (prepared in ethanol) and vortexes well. After 30 min incubation in dark, the absorbance of the mixture was read at 517 nm using spectrophotometer (T80+ UV/VIS Spectrophotometer, UK). Different concentrations were tested for each sample to get IC50 value which is defined as the amount of antioxidant necessary to decrease the initial DPPH ion by 50%. Ascorbic acid was used as a positive control. The percent radical scavenging activity of the crude extracts was calculated using the following formula:
DPPH radical scavenging activity (%) = [(A0 – A1)/ (A0)] ×100 (3)
Where: A0 is the absorbance of DPPH radicals + methanol and A1 is the absorbance of DPPH radicals + sample extract.
ABTS radical scavenging assay. The antioxidant activities of different extracts were evaluated through the ABTS radical scavenging by the extracts ability to scavenge ABTS with slight modification74. Aliquot concentrations (1, 3, 5 and 7 mg/ml) of extracts (50 μL) was added with 950 μL of ABTS solution (7mM ABTS solution and 2.45mM Potassium persulfate) followed by incubation at RT for 16 h in dark. Spectrophotometer (T80+ UV/VIS Spectrophotometer, UK) was applied to evaluate the absorbance at 734 nm. IC50 values were tested for each sample at each concentration. Ascorbic acid was used as a positive control. The percentage of inhibition was calculated using the following formula,
ABTS scavenged (%) = [(Acontrol – Asample)/ (Acontrol)] × 100 (4)
Where: Acontrol is the absorbance of ABTS radicals + solvent and Asample is the absorbance of ABTS radicals + sample extract.
Reducing power assay. Antioxidant activity of different crude extracts reducing power at various concentrations with insignificant modification16. Briefly, 1.5μL of extracts was mixed with 1.5μL of phosphate buffer (0.2 M, pH 6.6) and 1.5μL of potassium hexacyanoferrate (1%, w/v). After incubation at 50ᴼC in a water bath for 20 min, 1.5μL of trichloroacetic acid solution (10%) was added and centrifuge at 800 × g for 10 min. The supernatant was collected and mixed with 3 mL of DW and 200 μL of ferric chloride solution (0.1%, w/v) and incubated at RT for 10 min for stable absorbance at 700 nm; as the more absorbance of the reaction mixture more the reducing power of the extracts will be. Here ascorbic acid was used as a positive control. Antioxidant activity was also expressed as equivalents of ascorbic acid.
Phosphomolybdenum assay. The antioxidant activity of different extract solution (water, ethanol and methanol) was evaluated by the green phosphomolybdenum complex formation with slight modification75. A reagent solution was prepared with 0.6M H2SO4, 28mM Sodium phosphate and 4mM Ammonium molybdate. Further, 1.8 mL reagent solution was mixed with 0.2 mL of dilute extract solution and placed in a boiling water bath for 90 minutes at 95°C. After cooling down, the absorbance of each sample was measured at 695 nm using spectrophotometer (T80+ UV/VIS Spectrophotometer, UK). Blank was run same procedure just replacing the extract with the equivalent solvent. Antioxidant activity was also expressed as equivalents of ascorbic acid.
Hydrogen peroxide scavenging activity. Extracts antioxidant activities were evaluated by the hydrogen peroxide scavenging activity with slight modification76. Briefly, aliquot extracts at various concentrations was added 0.3 mL hydrogen peroxide solution (40mM) and 1.2 mL phosphate buffer (40mM; pH 7.4) and vortexes well. After 10 min the absorbance was measured at 230 nm against a blank solution (phosphate buffer). Different concentrations were tested for each sample to get IC50 value. Ascorbic acid was used as a positive control. The percentage of inhibition of the crude extracts was calculated using the following formula:
Hydrogen peroxide scavenged (%) = [(A0 – A1)/ (A0)] × 100 (5)
Where: A0 is the Absorbance of control and A1 is the Absorbance of sample solution.
Statistical analysis. The obtained experimental data was analyzed through the standard statistical procedure. Data were analyzed using SPSS software (IBM Co., Chicago, IL). Analysis of variance (ANOVA) and Duncan’s multiple range method were used to compare solvents and samples. Values were expressed as means ± standard deviations. Differences were considered significant at p < 0.05. All analyses were performed in triplicate.