Collection of seaweeds
Seaweeds are collected from Kovalam (covelong), Chennai – Coordinates: 12.7925°N 80.2530°E and Mandapam, Ramanathapuram district – Coordinates: 9.28°N 79.12°E. Seaweeds were first observed, then collected according to differentiation in morphology and colour. Collected seaweed samples are then transferred gently into Polythene zip-lock sample cover. Collected seaweed samples were quickly taken to a laboratory and then seaweed was gently washed in sterile water. Seaweed samples are then gently pressed with a sterile tissue paper to remove wetness from seaweed samples. Seaweed samples were further sterilized to remove external microorganisms for the isolation of endophytic fungi.

Isolation of endophytic fungi from seaweeds.
Seaweeds samples were cut into little fragments of exactly 1 x 1 cm and washed three times with sterile seawater to eliminate surface debris. Little fragment of seaweed sample was immersed in EtOH 70% for 60 – 80 seconds for surface sterilization. Small fragments of seaweed tissue were soaked dried with a sterile cotton cloth (or rinse with sterile artificial sea water) to cease the sterilization with EtoH 70%. Fragment of seaweed sample was kept carefully on the surface of a Petri plate containing potato dextrose agar medium with sterilized forceps so, that the freshly cut edges are in direct contact with the agar surface. Seal the Petri plates with parafilm, label and store it at 20 – 25°C. Fungal growth from the cut seaweed fragments usually initiate’s after 2-3 days until 14 days after the onset of the experiment [10]. Different fungal growth were observed from different seaweed sample. The individual fungal growth was isolated by removing hyphal tips growing out of the cut seaweed fragments with a sterile loop onto a fresh Petri plate containing fresh potato dextrose agar medium. The isolated marine endophytic fungi from the host marine algae were identified through microscopic observation and fungal genus were compared with standard monographs. They were distinguished from each other by their cultural characteristics such as colony morphology, growth rates, hyphal mat characteristics, and pigmentation of the fungal colony and medium^11-16. All the marine endophytic fungal isolates were documented and maintained in potato dextrose agar slants.

Large-scale cultivation of endophytic fungi from seaweeds.
Small fragments of fungal mycelium were transferred aseptically with a sterile bent inoculation loop in a 1000 ml Erlenmeyer flask containing the sterilized Potato Dextrose Broth medium. Fungal growth is carried out at room temperature under stationary condition. Based upon the fungal growth, Potato Dextrose Broth medium was incubated for 3–4 weeks.

Collection of crude extracts from endophytic fungi
Fungal cultured Potato Dextrose Broth was first filtered with a Büchner funnel and mycelium remains were discarded. Fungal cultured Potato dextrose broth was once again filtered through a glass funnel using Whatman qualitative filter paper, fungal culture filtrate collected in 1000 ml beaker. The Fungal cultured filtrate was then transferred into a separation funnel, equal volumes of ethyl acetate was added into a separation funnel and mixed rigorously. An aqueous phase and ethyl acetate phase was formed in a separating funnel, the aqueous phase was affably separated from the separation funnel and discarded. Ethyl acetate phase was then separated and collected in 1000 ml Erlenmeyer flasks, collected ethyl acetate extract was then transferred to 1000 ml Round-bottom flask. Ethyl acetate fungal extract was then dried under vacuum using a rotary evaporator at 40°C, this process takes about 40 minutes. At the end of the process Ethyl acetate solvent was collected in receiving round-bottom flask and the fungal extract was retained in evaporating round-bottom flask. Fungal extracted was observed as a dry solid or oily residue in evaporating round-bottom flask¹⁷.

Fungal extracted was removed from the round-bottom flask by eluting the fungal extract with ethyl acetate solvent. Eluted fungal extracted was dispensed in sterile vials and then fungal extracts were air dried at room temperature. After air drying, dried fungal extracts were stored in the freezer for furthered bio-activity screening.

1, 1-Diphenyl-2-picryl-hydrazyl (DPPH) Assay
Buffered methanol with acetic acid were used for the preparation of the DPPH stock solution, 40 ml of 0.1M acetate buffer (pH 5.5) added to 60 ml of methanol to prepare buffered methanol. All the chemical and solvents used in the study were analytical grade. The reaction tubes were wrapped in aluminium foil and kept at 30°C for 30 minutes in darkness, due to the photosensitivity of the chemical used in the study. The optical density of the sample was measured by spectrophotometric at 517 nm and the data obtained were presented as percentage scavenging activity¹⁸.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Gas chromatography –Mass spectrometry were performed by using a JEOL GCMATE II GC-MS spectrometer. The initial temperature of this instrument was at 50°C for 2 minutes. After this period the temperature of the oven was increased to 250°C. At the rate of 10°C/min maintained for 15 minutes. The temperature of the injection port was assured at 220°C and flow rate of helium at 1ml/min. Analyte ionization voltage was 70 electron volt (eV). The samples were injected in split less method. The Mass spectral scans range was at the rate of 50-650 (m/z). Application of computer in analysing GC-MS electron –ionization mass spectra, utilizing the National Institute of Standards and Technology (NIST) software Ver.2.1 MS data library, acquired spectrum were compared and to identify the compounds¹⁹.

Bioactivity Assay
Cell cytotoxicity assay in Vero cell line.
The weighed fungal crude extracts were first dissolved in 25µL of DMSO and then mixed on the vortex mixer. 5 ml of MEM was added to the DMSO dissolved fungal crude extracts. The extracts were filtered through Whatman syringe filter (pore size-0.2µm). The filtered crude extract was made up into 1 ml stock. 100µL of diluted fungal extracts was added in the 96 well plates. The 96 well plates were kept in 5% CO₂ incubator at 36°C. Vero cells observed subsequently 24 hours, 48 hours and 72 hours.

Anticancer activity in HEp2 and A549 cell lines.
The weighed fungal crude extracts were first dissolved in 25µL of DMSO and then mixed on a vortex mixer. 5 ml of MEM was added to the DMSO dissolved fungal crude extracts. The dissolved fungal crude extracts were filtered through Whatman syringe filter (pore size-0.2µm). The filtered crude extract was made up into 1ml stock. 100µL of diluted fungal extracts was added in the 96 well plates. The 96 well plates were kept in 5% CO₂ incubator at 36°C and HEp2 and A549 cells were observed after 24 hours, 48 hours and 72 hours.

MTT assay
MTT assay is one the prominent in-vitro assay utilised for the assessment of cell viability, proliferation of cells and cell cytotoxicity. The main principle of the MTT is that yellow coloured compound 3-(4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium Bromide) is converted into blue Formazan derivative (2E, AZ) – (4, 5-Dimethylthiazol-2-yl)-3, 5-diphenylformazan by mitochondrial reductase. Formazan crystals are then solubilised with Dimethyl Sulfoxide (CH₃)₂ SO and released solubilised formazan reagent is measured spectrophotometrically. Since the reduction of MTT can occur only in metabolically active cells, the level of activity is a measure of the viability of the cells^20,21.