Chemicals and solvents

All the chemicals and reagents used for this study were of standard analytical grade (NaOH, H2SO4, HCl, ferric chloride, ammonium molybdate, sodium phosphate, potassium ferricyanide, trichloroacetic acid, TCA, thiobarbituric acid, sodium pyruvate, ascorbic acid, Wagner reagent, Mayer's reagent were procured from Fisher Scientific (Chennai, India) and Hi Media Laboratories (Mumbai, India). Dulbecco’s modified Eagle’s medium (DMEM), 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid) (HEPES), DAPI (4',6-diamidino-2-phenylindole), beta-actin, MTT 3-(4, 5-dimethylthiozol-2-yl)-3, 5-diphenyl tetrazolium bromide) from Sigma Aldrich, protein assay kit from BioRad, India, nitrocellulose membrane (Millipore, Bangalore) solvents (methanol, benzene, chloroform were procured from Fisher Scientific (Chennai, India).

Collection, cultivation, and extraction of Nostoc calcicola

The Nostoc calcicola was obtained from NFMC (National Facility for Marine Cyanobacteria) in Tiruchirapalli and was cultivated in ASN III media to promote mass production. Freshly prepared media was sterilized, subjected to UV light exposure, and the media was replenished every 7 days to ensure the provision of essential minerals for optimal growth. Growth conditions included a controlled light/dark cycle of 16/8 h, a temperature of 22 ± 2 °C, and a light intensity of 3000 lx. Subculturing was conducted to sustain growth and attain substantial biomass production.

After 15 days, a significant quantity of Nostoc calcicola biomass was obtained, which was collected through centrifugation followed by drying, weighing, and extraction utilizing methanol. Filtration was employed to remove the extracted residue while the remaining solution was concentrated under reduced pressure using a rotary evaporator at a precisely controlled temperature of 60 ± 10 °C. The resulting concentrated extract enriched with bioactive substances served as the basis for further investigations [21].

Assessment of photochemical in Nostoc calcicola

Preliminary phytochemical screening of methanolic extract of Nostoc calcicola to identify the presence of bioactive secondary metabolites using standard methods as described by Uddin et al., in 2018 [22] with slight modification.

Flavonoids using alkaline reagent test where a mixture of 2 mL of 2.0% sodium hydroxide (NaOH) and the aqueous extract was prepared. This mixture displayed a concentrated yellow colour, which turned colourless upon the addition of dilute acid, confirming the presence of flavonoids.

Alkaloids using Wagner reagent—where two drops of Wagner reagent were added to 2 mL of the extract and thoroughly mixed. The development of a reddish colour indicated the presence of alkaloids. Further confirming using Mayer’s Test by adding few drops of Mayer's reagent to 1 mL of the extract, resulting in the formation of a yellowish or white precipitate, confirming the presence of alkaloids.

Saponins by Froth’s Test in which 2.0 mL of extract was shaken vigorously for 2 min. The appearance of frothing indicated the presence of saponins.

Terpenoids was identified using Salkowski’s Test by adding 5.0 mL of extract with 2.0 mL of chloroform followed by addition of concentrated sulfuric acid to create a layered solution. A reddish-brown coloration at the interface confirmed the presence of terpenoids.

Steroids by Libbermann Burchard’s Test in which 1.0 mL of extract along with 0.5 mL of acetic anhydride was taken and cooled using ice bath. Chloroform was added to the mixture followed by addition of 1.0 mL of concentrated sulfuric acid (H2SO4) at the sides of the tube. The presence of steroids was indicated by the formation of a reddish-brown ring at the junction of the two liquids.

Tannins were identified using ferric chloride test, where three drops of 5% ferric chloride was added to 1.0 mL of the extract, resulting in the formation of a greenish-black precipitate, indicating the presence of tannins. Further confirmed using lead acetate Test in which 5.0 mL of the extract and few drops of 1% lead acetate were added. The formation of a yellow precipitate indicated the presence of tannins.

Glycosides by Borntrager’s Test—Ferric chloride was added to the extract, and the resulting solution was filtered. Dilute hydrochloric acid (HCl) and an organic solvent (benzene) were added to the filtrate, followed by dilute ammonia. The pink colour transitioned to red, indicating the presence of glycosides. Further confirmed using Killarkillani Test in which, this involved mixing 2 mL of the extract with glacial acetic acid, one drop of 5% ferric chloride (FeCl3), and concentrated sulphuric acid (H2SO4). A reddish-brown colour appeared at the interface of the two liquid layers, and the upper layer exhibited a bluish-green hue.

Phlobatannins was identified by adding 1% hydrochloric acid (HCl) to 10 mL of the extract, followed by heating in a boiling water bath. The formation of a red precipitate indicated the presence of phlobatannins.

Anthraquinones identification was done by taking equal volumes of extract and benzene were combined in a test tube and thoroughly mixed. Half the volume of 10% ammonia solution was added to this mixture. The presence of anthraquinones was confirmed by the development of red, pink, or violet coloration in the ammonia phase.

Phenol by Phthalein dye test—A mixture of 0.1 g of extract and 0.1 g of phthalic anhydride was heated with 1–2 drops of concentrated sulphuric acid. The resulting solution was then carefully poured into a beaker containing 15 mL of dilute sodium hydroxide solution. The presence of phenol groups was indicated by the appearance of a pink fluorescent colour.

Investigation of competence to attenuate oxidative damage by Nostoc calcicola

The radical scavenging capacity of the extracts was studied using.

DPPH radicals scavenging assay

The assessment of the ability of Nostoc calcicola extract to scavenge free radicals was conducted using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. A minor modification was employed following the method of Shimada et al. in 1992 [23]. The reaction mixture was prepared consisting of 1.0 mL of the Nostoc calcicola extract at various concentrations (ranging from 20 to 100 µg/mL) and 1.0 mL of a 0.8 mM/L DPPH solution. This mixture was then subjected to incubation under shaking conditions for 30 min, and the absorbance was recorded at 517 nm, using ascorbic acid as a control. The inhibition percentage, indicative of the DPPH radical scavenging capacity, was subsequently calculated.

$$\% } = \left[ }\;}}}}\;}}}} \right)} \right] \times 100$$

Phosphomolybdenum assay

A solution was prepared by dissolving 10 mg of Nostoc calcicola extract in 1 mL of DMSO. From this solution, 100μL was extracted and combined with 1 mL of a reagent solution. The reagent solution was prepared by mixing 0.588 mL of sulphuric acid, 0.049 g of ammonium molybdate, and 0.036 g of sodium phosphate, and then adjusting the final volume to 10 mL with distilled water. This mixture was then incubated in a boiling water bath at 95 °C for 90 min. After this incubation period, the absorbance of the solution was measured at 695 nm. As a standard, ascorbic acid (prepared at a concentration of 10 mg/mL in DMSO) was used. The Phosphomolybdenum reduction potential (PRP) of the tested extracts was reported as a percentage. Prieto et al., (1999) [24].

Ferric ions (Fe3 +) reducing antioxidant power assay (FRAP)

The ferric ion reducing power of Nostoc calcicola was evaluated following the methodology of Oyaizu et al. from 1986 [25], with slight modifications. Specifically, 20–100 µg/mL of Nostoc calcicola extract was blended with 2.5 mL of a 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of a 1% potassium ferricyanide [K3Fe(CN)6] solution, after which the mixture was incubated at 50 °C for a duration of 20 min. Following incubation, 2.5 mL of 10% trichloroacetic acid was added to the mixture, followed by centrifugation at 3000 rpm for 10 min. Subsequently, 2.5 mL of deionized water and 0.5 mL of a 0.1% FeCl3 solution were added to 2.5 mL of the resulting supernatant. The absorbance at 700 nm was measured in comparison to ascorbic acid, which served as a positive control.

Hydroxyl radical scavenging assay

The hydroxyl radical scavenging activity was assessed using the method developed by Halliwell, Gutteridge, and Arouma in 1987, with slight adjustments [26]. To prepare stock solutions, EDTA (1 mM), FeCl3 (10 mM), ascorbic acid (1 mM), H2O2 (10 mM), and deoxyribose (10 mM) were dissolved in distilled deionized water. For the reaction mixture, the following components were combined sequentially: 0.1 mL of EDTA, 0.01 ml of FeCl3, 0.1 mL of H2O2, 0.36 mL of deoxyribose, 1.0 mL of Nostoc calcicola extract (at concentrations of 20–100 µg/mL), 0.33 ml of phosphate buffer (50 mM, pH 7.4), and 0.1 mL of ascorbic acid. The resulting mixture was then incubated at 37 °C for 1 h. Subsequently, 1.0 mL of this mixture was extracted separately and mixed with 1.0 mL of 10% TCA (trichloroacetic acid) and 1.0 mL of 0.5% TBA (thiobarbituric acid). The absorbance of this mixture was measured at 532 nm.

$$}\;}\;}(\% ) = \left( \right) \times \left. \right)$$

(A1 is the absorbance of standards or reaction mixture, A0 is the absorbance of the negative control).

Determination of cytotoxicityCell lines and culturing

The Human colorectal adenocarcinoma (HT-29) cells were sourced from the National Center for Cell Sciences (NCCS) in Pune, India. The cells were maintained and cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 2 mM l-glutamine and a balanced salt solution (BSS). The BSS composition was adjusted to incorporate 1.5 g/L Na2CO3, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L glucose, 10 mM (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid) (HEPES), 2 mM l-glutamine, 10% fetal bovine serum (GIBCO, USA) followed by the addition of 1 mL/L penicillin and streptomycin solution (100 IU/100 µg). The optimal growth and viability were ensured regularly providing 37 °C temperature with a 5% CO2 concentration in a humidified CO2 atmosphere throughout the experimental period.

Antiproliferative action against colorectal cancer

Cell viability of Nostoc calcicola was assessed using the MTT assay with slight modifications, as previously described by Khader et al. [27]. HT-29 cells were cultivated in a 96-well plate until reaching a confluence of 75–80% after 48 h. The medium was then replaced with a fresh complete medium along with Nostoc calcicola extract (10–50 µg/mL), and incubated for an additional 48 h. After the incubation period, the culture medium was removed, and each well was treated with 100µL of MTT (3-(4, 5-dimethylthiozol-2-yl)-3, 5-diphenyl tetrazolium bromide) solution (Hi-Media) and incubated again for 4 h at 37 °C to obtain formazan crystals. Further, the supernatant was discarded followed by the addition of 50µL of dimethyl sulfoxide (DMSO) to dissolve the formazan crystals in each well and incubated for 10 min. The optical density (OD) of each well was measured at 620 nm using an ELISA multiwell plate reader (Thermo Multiskan EX, USA). The results were compared with the standard control doxorubicin.

The OD value was used to calculate the percentage of viability using the following formula

$$\% \;}\;} = \frac}\;}\;}\;}}}}\;}\;}\;}}} \times 100$$

Morphological screening

The selected cells that were grown on coverslips (1 × 105 cells/coverslip) were incubated with Nostoc calcicola at different concentrations and then fixed in ethanol: acetic acid solution (3:1, v/v). The coverslips were gently mounted on glass slides for the morphometric analysis. Three monolayers per experimental group were micro-graphed and the morphological changes of the cells were analyzed using Nikon (Japan) bright field inverted light microscopy at 10× magnification [28]

Fluorescence microscopy analysis Acridine orange (AO) and ethidium bromide (EtBr) staining

To perform staining and visualization, a dye mixture consisting of acridine orange (AO) and ethidium bromide (EtBr) at concentrations of 100 mg/mL each in distilled water was prepared. In a clean microscope coverslip add 1 μL of this dye mixture with 90 μL of a cell suspension containing a concentration of 1 × 105 cells/mL. The cells were initially washed with phosphate-buffered saline (PBS) at pH 7.2, stained with 10 μL of the AO/EtBr dye mixture along with different concentrations (10, 25, 50 μg/mL) of Nostoc calcicola extract and incubated for 2 min. The cells were washed twice with PBS buffer to remove excess stain and visualized using a fluorescence microscope (Nikon Eclipse, Inc, Japan) at a magnification of × 400, with an excitation filter set at 480 nm [29].

DAPI staining

DAPI (4′,6-diamidino-2-phenylindole) assay was performed in a clean glass microscope coverslip seeded with cells, placed in a 24-well plate, and treated with different concentrations of the Nostoc calcicola extract (10, 25, 50 μg/mL) for a period of 24 h. Further, the fixed cells were permeabilized using 0.2% triton X-100 (50 μL) for 10 min at room temperature and incubated with 10 μL of DAPI stain for 3 min and another coverslip is placed over the cells to ensure the uniform spreading of the stain. The stained cells were observed under a fluorescent inverted microscope (× 400), as described by Phull et al. [30].

Cell cycle arrest analysis

Cell cycle analysis was conducted to assess the effects of the Nostoc calcicola extract on HT-29 cells. The cells were initially seeded in a 96-well plate and incubated at 37 °C with 5% CO2 for 24 h. Subsequently, the medium in each well was replaced with fresh medium supplemented with varying concentrations of the Nostoc calcicola extract (10–50 μg/mL), while a control group received an unsupplemented medium and incubated for 24 h. The cells were harvested using trypsin, washed with PBS, treated with 70% ethanol to fix the cell, and stored at − 20 °C for 1 h. For staining the cellular nuclear DNA, propidium iodide (PI) was employed. The ethanol-fixed cells were washed following centrifugation and resuspended in 50 μg/mL PI, 100 μg/mL RNase in PBS, and incubated at 37 °C for 30 min. Flow cytometry analysis was performed using a BD FACS flow cytometer, with duplicate samples analyzed. A total of 10,000 events were collected from each sample, and the fluorescence signal intensity was recorded during the analysis. These experimental procedures closely follow the methodology outlined by Khader et al. [31].

Western blot assay of anticancer and apoptosis-related proteins

To assess the regulation of apoptotic and anti-apoptotic proteins in the treated cells, Western blotting was performed. HT-29 cells were seeded onto 100 mm culture dishes at a density of 1.5 × 106 cells and treated with Nostoc calcicola extract for 24 h. Following treatment, the medium was removed, and the cells were washed multiple times with PBS (0.01 M, pH 7.2). Subsequently, the cells were lysed with lysis buffer (0.1 mL per plate) for 20 min and the lysate was obtained by centrifugation at 10,000× g for 5 min at 4 °C, and the protein concentration in the harvested lysate was quantified using a protein assay kit. Equal amounts of protein (100 μg) from each lysate were loaded onto a 12% SDS polyacrylamide gel for electrophoresis. The proteins were then transferred onto a nitrocellulose membrane (Millipore, Bangalore) and blocked with 10% skimmed milk in water for 1 h. After washing the membrane with PBS containing 0.1% Tween 20, primary antibodies against caspase-9 and beta-actin were added at a volume-to-volume ratio of 1:1000. The membrane was incubated overnight at 4 °C with the primary antibodies. Following overnight incubation, the primary antibodies were washed off, and secondary antibodies were added to the membrane for 1 h of incubation at room temperature. This step allowed for the detection of the primary antibodies bound to their respective target proteins [32]. The Intensity of blots were quantified with densitometric analysis and Amersham Image Quant™ 800, Image J software, instrument was used for densitometric analysis.

GC–MS analysis

The gas chromatography-mass spectrometry (GC–MS) profiling was performed using Clarus 680 GC fused with silica column packed with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250 μm df). Helium was employed as carrier gas at a constant flow of 1 mL/min and the injector temperature was set at 250 °C during the chromatographic run. The analysis was carried out by injecting 1μL of extract sample into the instrument and the oven temperature was maintained at 50 °C (1 min) followed by 280 °C at the rate of 10. The mass detector (transfer line temperature 280 °C; ion source temperature 200 °C and ionization mode electron impact at 70 eV) was conducted with a scan time 0.2 s and scan interval of 0.1 s [33, 34]. The spectrums of the components were compared with the database of spectrum of known components in the GC–MS NIST (2008) library.

Statistics

All the in vitro experiments were carried out in triplicate, and the experiments were repeated at least thrice and expressed as the Mean ± S.D. and all the grouped data were statistically evaluated with SPSS\17.0 software.