Chemicals
Annexin V-FITC assay kit, Hoechst 33342, 5,5′6,6′-tetrachloro-1,1′3,3′tetraethylbenzimi-dazolycarbocyanine iodide (JC-1) staining solution, NBT, xanthine oxidase (EC 1.1.3.22, Grade IV), ABTS, xanthine, quercetin, β-carotene and ASC were obtained from Sigma-Aldrich, USA. Ferric chloride, potassium ferricyanide, Potassium persulfate and ferrozine were obtained from Sisco Research Laboratory Pvt. Ltd. India and EDTA from Hi-mediai, India. All solvents used in this study were of analytical grade.
Seaweed collection and preparation of solvent fractions
The brown seaweed of T. conoides (J. Agardh) Kuetz was collected from the intertidal zone of Mandapam, Gulf of Mannar, Southeast Coast of Tami Nadu, India. The algae samples (1 kg) were washed with fresh tap water followed by distilled water to remove salt and other debris along with necrotic parts. Samples were made into small pieces and shade dried after then powdered using a mixer grinder. The powdered sample was stored in a polyethylene bag at room temperature. The shade dried powder 250 g was immersed in a 2 L of 2 conical flasks with each 1.25 L of 95% ethanol and left for 24 h under constant stirring. After the 24 h, transfer from conical flask to Soxhlet apparatus and extracts was collected in bottom flask at 40 °C and this was repeated twice with 2.5 L of 95% ethanol. Final volume of 4.5 L ethanol extract was brought out into 25 g (10%) with rotary evaporator. The ethanol extract was transfer into a separating funnel and partitioned between distilled water and hexane with 1:6 ratio [18]. This mixture was thoroughly mixed for 15 min and then hexane fraction (HF) was collected 1.4 L after 1 h incubation. Similarly, aqueous layer 250 mL was further fractionated with dichloromethane (DMF) and ethyl acetate (EAF). All the fractions were concentrated in rotary evaporator. The yield of the fractions was 20, 16, and 52% respectively. The aqueous fraction (AF) was lyophilized and obtained 12% of yield.
Qualitative analysis of secondary metabolites
Based on the method described by Allen [19] and Harbone [20] the following major phytochemicals such as alkaloids, tannins, saponins, terpenoids, phenols, flavonoids, coumarin, glycosides, reducing sugar, steroids and resins were spectrophotometrically analyzed in ethanol extract of T. conoides.
Antibacterial assay using a disc diffusion method
Antibacterial activity was measured using a disc diffusion method described by Mackeen et al. [21]. Standard antibiotic, streptomycin was used as positive control. For antibacterial screening, 8 μg of solvent fractions was loaded onto each disc (6 mm diameter) and placed on previously inoculated agar plate with clinically important both gram positive and negative bacteria. The plates were incubated for 24 h at 37 °C. Diameter of zone inhibition around the disc were measured and expressed as in millimeter.
Quantification of total antioxidants in solvent fractions
The ABTS.+ radical preparation and antioxidant quantification were clearly given in the previous article 19. The assay was performed in one mL reaction volume. HF, DMF, EAF and AF were tested at concentration with range of 0–80 μg/mL. β-carotene and L-ascorbic were used as standard antioxidants. Appropriate blanks were run in each assay and all the experiments were carried out in five replications and the values were averaged. The TAA and RAA were calculated and expressed based on the formula in the previous study [18].
Superoxide radical scavenging activity
According to Tota and Kumura [22], the superoxide radical was generated by xanthine oxidase system. The reaction mixture consisted of 2.70 mL of 40 mM sodium carbonated buffer which included 0.1 mM EDTA (pH 10.0), 0.06 mL of 10 mM xanthine, 0.03 mL of 0.5% bovine serum albumin, 0.03 mL of 2.5 mM NBT, and 0.06 mL of EAF (0–80 μg/mL) or ascorbic acid (30 μg/mL). The mixture was kept at room temperature (25 °C) for 20 min and after 0.12 mL of xanthine oxidase (0.04 units) was added. The absorbance at 560 nm was recorded (formation of blue formazan) after 5 min with five readings. The results were expressed as percentage of radical scavenging activity.
Estimation of iron chelation activity
The reaction mixture contains 100 μL of 1 mM FeCl2, 3.7 mL of distilled water and 1 mL aliquot of the EAF (0–80 μg/mL)/standard, EDTA (3 mg/mL). The reaction was initiated by adding 200 μL of 5 mM ferrozine. The reaction mixture was incubated at room temperature for 20 min and the absorbance was recorded at 562 nm [23]. The iron chelating activity of EAF was calculated and expressed as percentage.
Uric acid formation inhibitory activity
The reaction mixture consisted of 2.76 mL of 40 mM sodium carbonate buffer which included 0.1 mM EDTA (pH 10.0), 0.06 mL of 10 mM xanthine, and 0.06 mL of EAF (0–80 μg/mL) or ascorbic acid (30 μg/mL). The reaction was started by the addition of 0.12 mL of xanthine oxidase (0.04 units) and the absorbance at 293 nm was recorded after 5 min with five readings at room temperature [22]. The results were expressed as percentage of uric acid formation by EAF.
Cytotoxicity assay
The cell line study was carried out at AU-KBC Research Centre, Anna University, Chennai, India. The cytotoxicity assay performed using MTT assay as described by Mosmann [24]. Tetrazolium salt MTT solution prepared freshly in concentration of 0.5 mg/mL of PBS. Cells seeded in 96 -well plates (1 × 105 cells/well) and allowed to adhere for 6 h. The cells were treated with different concentrations (0 - 320 μg/mL) of EAF /quercetin after filtered through 0.2 μ Millipore filter and kept for 24 and 48 h. Consequently, 100 μL of MTT dye (5 mg in 10 ml of serum free medium) added in each well. Control also maintained in the same manner. The plates were incubated at 5% CO2 incubator for 4 h. The formazan crystals dissolved in 100 μL of 20% SDS (in 50% dimethyl formamide) and the measured optical density (OD) using a 96 well microplate reader at 570 nm. The inhibition ratio (I %) calculated using the following equation: I% = (Acontrol-Atreated)/Acontrol × 100%.
Flow cytometric analysis of cell cycle
Cells seeded into 6-well culture plates and exposed to EAF/quercetin at the concentrations (0–320 μg/mL) for 48 h. After treatment, cells (1 × 106 cells/mL) were collected and fixed with 70% ice-cold ethanol (v/v) and stored at −20 °C overnight. The fixed cells washed twice with PBS and incubated for 30 min in the dark at room temperature with 1 mL of PBS containing PI (20 μg/mL), 0.1% (v/v) Triton X-100 and 0.1% RNase A [25]. A total of at least 30, 000 events were collected and analyzed by flowcytometer. The percentage of cells in G0/G1, S and G2/M phases was determined using the Cell Quest acquisition software.
Flow cytometric analysis of apoptosis
The annexin V-FITC assay was performed according to the manufacture’s protocol. Briefly, cells were seeded into 6-well plates and incubated for 48 h with different concentration (0–320 μg/mL) of EAF and quercetin. Cells (1 × 106 cells/mL) washed twice with PBS and resuspended in 500 μL of binding buffer containing 5 μL of FITC conjugated annexin-V and 5 μL of PI. The plates were incubated in the dark for 15 min at ambient temperature and after analyzed by flowcytometry within a 1 h period. The percentage of total apoptotic cells was calculated by the addition of both early and late apoptotic evens.
Detection of nuclear condensation by Hoechst assay
Cells treated with EAF of T. conoides (320 μg/mL) for 48 h and then cells were washed with PBS and fixed with methanol: acetic acid (3:1) for 15 min at room temperature. Fixed cells then washed with PBS and stained with 5 μg/mL of Hoechst 33342 stain for 10 min. Changes in the morphology of nuclei in the cells were observed using a fluorescence microscope [26].
Detection of mitochondrial membrane potential by JC-1 assay
The mitochondrial membrane potential (ΔΨm) was analyzed using the 5,5′6,6′-tetrachloro-1,1′3,3′tetraethylbenzimi-dazolycarbocyanine iodide (JC-1) by fluorescence microscopy. Cells treated with EAF of T. conoides (320 μg/mL) for 48 h and after 5 μl of the JC-1 staining solution with 1 ml culture of medium was added to each well. Samples incubated in 5% CO2 incubator at 37 °C for 20 min. Then, cells washed twice with PBS buffer solution and visualized under a fluorescence microscope [26].
Statistical analysis
The results were expressed as a mean ± SD/SE with three or five values. All the data were analyzed statistically by One-way ANOVA followed by Tukey’s multiple comparisons using SPSS software student’s version-16. A p value <0.05 was considered statistically significant.