Sample preparation

Ipomoea tuba sample was collected from Nizampatnam mangroves, Guntur, Andhra Pradesh, India. The sample was prepared from leaves of I. tuba by soaking 50 g of powdered sample in 50 mL of absolute methanol for 72 h. The sample was filtered through Whatman No. 42 filter paper and then methanol was evaporated from the test sample by rotary vacuum evaporator (EV11, Equitron Medica Pvt Ltd, Mumbai, India). The final crude extract was dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich Chemicals Pvt Ltd, Merck, Bangalore, India), made to final concentration of 100 mg/mL and used for antiproliferative studies. The concentration of DMSO maintained in the wells was less than 1%, which is not toxic to the cell lines (8, 9).

Identification of compounds by GC-MS analysis

Bioactive compounds in leaf extract of I. tuba were identified by GC-MS (6890 series; Agilent, Santa Clara, CA, USA). The following chromatographic conditions were maintained: initial column temperature 30 °C, heated up to 300 °C at 10 °C/5 min, flow rate 1.0 mL/min and helium was used as carrier gas in split mode. The bioactive compounds were identified based on retention times and quantified by integration of peak area. Similarity of compounds was compared with known compounds using NIST based AMDIS software (10).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability

Ipomoea tuba leaf extract is screened for in vitro cytotoxicity activity on MCF-7 and HeLa cells (5x10³ cell/well) using MTT (Sigma-Aldrich, Merck, St. Louis, MO, USA) assay. The sample (100 µL diluted plant extract) was added to 100 µL of Dulbecco`s Modified Eagle’s medium (DMEM), then the cell lines were added to the 96-well microtiter plate, and incubated for 48 h at 37 °C. The MTT was added and allowed to incubate for 2 h until the purple precipitate was formed. Then, absorbance values were measured at 520 nm using UV-Vis spectrophotometer (Cary 60; Agilent Technologies, Selangor Darul Ehsan, Malaysia). The dose-response curve was plotted for evaluation of IC₅₀ values (11).

Statistical analysis

The experimental data of both cell lines were statistically analyzed using ANOVA method. The value p<0.05 is considered statistically significant for the analysis of the percentage of inhibition of cell viability.

GC-MS analysis for compound identification

The chromatogram confirmed the presence of compounds such as fatty acids: docosanoic, octadecatrienoic and cis-9-octadecanoic acids, triterpenoid γ-sitosterol, and terpene alcohol in the leaf extract of mangrove plant Ipomoea tuba. The compounds were identified based on retention times. Table 1 gives the molecular mass and retention times of each compound. Angaye et al. (12) reported various bioactive compounds in the extracts of mangroves Rhizophora mangle, Rhizophora racemosa, Avicennia germinans and Laguncularia racemosa. Phytochemical compounds such as β-sitosterol, eicosanol and taraxerol are found in Bruguiera cylindrica extract and showed their cytotoxicity against neuro2A cancer cell lines (13).

The bioactive compounds from Avicennia marina, Salvadora persica and Avicennia officinalis contain several types of secondary metabolites like flavonoids, tannins, alkaloids and saponins (14, 15). In previous study, GC-MS analysis of the extracts of Acrostichum aureum confirmed the presence of bioactive compounds such as stigmasterol, γ-sitosterol, campesterol and 24-methylene cycloartenol with potential activity against adenocarcinoma, carcinoma and other human cancer cell lines (16). Ganesh and Vennila (17) reported the presence of different bioactive compounds, namely terpenoids, steroids, saponins, catechol and phenols in the methanol extracts of Acanthus ilicifolius and Avicennia officinalis. Report from Basyuni et al. (18) showed that the mangrove species Acanthus ilicifolius, Rhizophora apiculata, Sonneratia caseolaris contain wide range of phytoconstituents like triterpenoids, taraxerol, germanicol and tannins, with anti-inflammatory, anticarcinogenic, antimicrobial and antiprotozoan activities. The mangrove plant Rhizophora mucronata leaf extract contains vindoline, catharanthine and serpentine, the major alkaloids with free radical scavenging and cytotoxicity activity (19). The findings of Grozav et al. (20) proved that the derivative of thiazole synthesized from mangrove plant leaves has potential anticancer effect against ovarian cancer cell lines A2780 and HeLa cell lines.

In vitro antiproliferative activity of I. tuba leaf extract on MCF-7 cells

Extract of I. tuba leaves showed significant in vitro antiproliferative effect on MCF-7, and the viability of MCF-7 was reduced with the increase in the concentration of the sample. High reduction of MCF-7 cells was observed at the concentration of I. tuba leaf extract of 100 µg/mL (Table 2) and the IC₅₀ value against MCF-7 cells was found to be (40.4±0.1) μg/mL. During experiments, it was observed that the increase in sample concentration altered the morphology of MCF-7 cells, leading to cell death (Fig. 1)^{. Similar findings are reported for in vitro cytotoxicity effect of Avicennia marina extracts on different cancerous cells (21-23)}. Patra and Thatoi (24) reported the antiproliferative activity of the methanol extract of Heritiera fomes leaves against melanoma cell lines and achieved 40% inhibition. The methanol extract of Avicennia marina leaf showed anticancer activity against MDA-MB 231 and MCF-7 cell (25).

Fig. 1

Morphology of MCF-7 cells after the treatment with the extract of Ipomoea tuba leaf: a) untreated MCF-7 cell lines, and b-g) treated with different concentrations (5, 10, 25, 50, 75 and 100 µg/mL respectively) of I. tuba leaf extract

In vitro antiproliferative activity of I. tuba leaf extracts on HeLa cells

HeLa cell viability was decreased with the increased concentration of leaf extract and the maximum reduction in HeLa cells was observed at 100 µg/mL (Table 2), with the IC₅₀ value of (39.4±0.1) μg/mL. It was observed that after the treatment with I. tuba extract, the HeLa cells slowly detached from one another. Fig. 2 shows the change in morphology of HeLa cells. Khajure and Rathod (26) reported that the extract of A. ilicifolius had cytotoxic activity against HeLa and KB cells. Rajeswari et al. (27) also reported that the flavone molecule from Excoecaria agallocha has the cytotoxic activity against HeLa cells.

Fig. 2

Morphology of HeLa cells after the treatment with the extract of Ipomoea tuba leaf: a) untreated HeLa cell lines, and b-g) treated with different concentrations (5, 10, 25, 50, 75, and 100 µg/mL) of I. tuba leaf extract