Total phenolic content of the ethanol extract of A. marina leaves was higher than the ethyl acetate extract
The ethanol extract of A. marina had a higher yield of extractions than the ethyl acetate extract of the A. marina. The total phenolic content of A. marina extracts was determined. The calibration curve generated from the analysis of the standard (gallic acid) was linear (Figure S1A). In detail, the ethanol extract of A. marina showed higher phenolic content (345 µg/ml gallic acid/0.01 g extract) than ethyl acetate extract (147.5 µg/ml gallic acid/0.01 g extract).
Total flavonoid content of the ethanol extract of A. marina leaves was higher than the ethyl acetate extract
The total flavonoid content of both ethanol and ethyl acetate extracts of A. marina was determined. The calibration curve generated from the analysis of the gallic acid was linear (Figure S1B). In addition, the equation and parameter were y = 0.0009x + 0.0155 and R2 = 0.9081, respectively. The results showed that the ethanol extract of A. marina had a higher flavonoid content (47.8 mM GAE) than ethyl acetate extract (38.6 mM GAE).
GC-MS profile of the ethanol and ethyl acetate extracts of A. marina leaves showed anti-cancer compounds
GC-MS analyses revealed several different molecules in the ethanol and ethyl acetate extracts of A. marina leaves (Figure S2). Sixty compounds were identified in the ethanol extract of A. marina leaves (Table S1). In detail, there were 26 compounds with anti-cancer and apoptotic effects based on the previous researches. In addition, 56 compounds were detected in the ethyl acetate extract of A. marina leaves (Table S2). Twenty of these compounds had anti-cancer and apoptotic effects based on the previous studies. The effects of both ethanol and ethyl acetate compounds on three cell lines (MCF-7, OVCAR3, and HeLa) were also evaluated in Tables S1 and S2. Moreover, other biological activities were observed in A. marina extracts including anti-oxidant, anti-inflammatory, antimicrobial, and anti-fungal activities are summarized in Tables S1 and S2.
MTT proliferation test of ethanol extracts of A. marina leaves had anti-proliferative effects on MCF-7
The anti-proliferative effect of 120 and 160 µg/mL concentrations of ethanol extract was observed on the MCF-7 cell line (p < 0.05, Fig. 1A). However, the extract had a proliferative effect on OVCAR3 cell lines at 40, 80, and 120 µg/mL concentrations (p < 0.05, Fig. 1B). In contrast, the ethanol extract of A. marina had no significant effects on the HeLa cell line at different concentrations (Fig. 1C). In addition, the ethanol extract of A. marina had also an anti-proliferative effect on the Vero cell line at the concentrations of 40 and 160 µg/mL (p < 0.05, Fig. 1D). Therefore, ethanol extract of A. marina leaves at 120 µg/mL had anti-proliferative effects on MCF-7 cancer cell lines comparing with normal Vero cells.
Moreover, the ethyl acetate extract had no significant effects on MCF-7 and OVCAR3 cell lines (Figs. 1E and 1F). The anti-proliferative effect of ethyl acetate extract was observed at the concentration of 160 µg/mL on the HeLa cell line (p = 0.01, Fig. 1G). The ethyl acetate extract had also anti-proliferative effects on the Vero cell line at the concentration of 120 and 160 µg/mL (p < 0.05, Fig. 1H). Therefore, ethyl acetate extract of A. marina leaves had no applied anti-proliferative effects on three cancer cell lines comparing with normal Vero cells.
A. marina leaves had cytotoxic activity on MCF-7 by ethanol extract and HeLa by ethyl acetate extract
The ethanol and ethyl acetate extracts of A. marina leaves potentially inhibited the viability of MCF-7 cells, OVCAR3 cells, HeLa cells, and Vero cells with CC50 values of 70 and 102 µg/mL, 1087 and 272 µg/mL, 189 and 67 µg/mL, and 382 and 242 µg/mL, respectively. As shown in Figs. 2A and 2B, these two extracts had significant dose-dependent inhibition on the proliferation and viability of the MCF-7 and HeLa cancer cells. The results showed that the cytotoxic activity of these extracts on MCF-7 and HeLa cancer cells was more active than OVCAR3 cancer cells and Vero normal cells.
Cancer cell count decreased by ethanol and ethyl acetate extract treatments
The ethanol extract decreased MCF-7 cell number at 7 days (p < 0.05, Figure S3A). Increasing the concentrations of ethanol extract 1.5 times increased the PDT of MCF-7 cells (Table 1). In addition, the ethanol extract decreased OVCAR3 cell number at five days (p < 0.05, Figure S3B). A dose-dependent increase of PDT to two to five times was seen on the effect of ethanol extract on OVCAR3 cell proliferation (Table 1). The ethanol extract decreased HeLa cell number from day 6 (p < 0.05, Figure S3C). Increasing the concentrations of ethanol extract 1.8 times increased PDT of HeLa cells (Table 1). The ethanol extract decreased Vero cell number at days 7 (p < 0.05, Figure S3D). A dose-dependent increase of PDT to 1.5 times was seen on the effect of ethanol extract on the Vero cell proliferation (Table 1).
Table 1
Mean of population doubling time of MCF-7, OVCAR3, HeLa and Vero cell lines after exposure to different concentrations (µg/mL) of ethanol and ethyl acetate extracts of Avicennia marina leaves
Extracts | Concentrations (µg/mL) | Population doubling time (PDT) (days) |
MCF-7 | OVCAR3 | HeLa | Vero |
Ethanol | 0 | 1.1 | 2.5 | 1.5 | 1.1 |
| 40 | 1.3 | 6 | 1.7 | 1.6 |
| 80 | 1.3 | 4.2 | 2 | 1.6 |
| 120 | 1.4 | 10.6 | 2 | 1.6 |
| 160 | 1.6 | 5 | 2.8 | 1.6 |
Ethyl acetate | 0 | 1.1 | 2.5 | 1.4 | 1.1 |
| 40 | 1.5 | 3.4 | 1.5 | 1.3 |
| 80 | 1.6 | 2.8 | 1.6 | 1.5 |
| 120 | 1.4 | 2.7 | 2.9 | 1.4 |
| 160 | 2 | 6.8 | 2.8 | 1.7 |
Moreover, the ethyl acetate extract decreased MCF-7 cell number at 5 days (p < 0.05, Figure S3E). Increasing the concentrations of ethyl acetate extract 1.8 times increased PDT of MCF-7 cells (Table 1). In addition, the ethyl acetate extract decreased OVCAR3 cell number at five days (p < 0.05, Figure S3F). A dose-dependent increase of PDT to 2.7 times was seen on the effect of ethyl acetate extract on the OVCAR3 cell proliferation (Table 1). The ethyl acetate extract decreased HeLa cell number from days 7 (p < 0.05, Figure S3G). Increasing the concentrations of ethyl acetate extract 2 times increased PDT of HeLa cells (Table 1). The ethyl acetate extract decreased Vero cell number at 3 days (p < 0.05, Figure S3H). A dose-dependent increase of PDT to 1.5 times was seen on the effect of ethyl acetate extract on the Vero cell proliferation (Table 1).
A. marina ethanol extracts reduced the viability of OVCAR3 and HeLa cells
160 µg/mL ethanol extract of A. marina leaves decreased OVCAR3 and HeLa cells viability from day 2 (p < 0.05, Figs. 3B and 3C). The 160 µg/mL ethyl acetate extract of A. marina leaves decreased the viability of OVCAR3, HeLa and Vero cells from days 6, 5, and 5, respectively (p < 0.05, Figs. 3F and 3G).
Cell cycle analysis of HeLa cell lines showed an increase of dead cells
Based on the findings of the cell MTT assay, PDT assay, and cell viability test, the most effective concentrations of the extracts were determined and used for cell cycle assay (Fig. 4). The MCF-7 cell line was treated with 120 µg/mL concentration of ethanol extract of A. marina leaves that 2-times increased the number of cells in the S phase. The OVCAR3 cell line was treated with 160 µg/mL concentration of ethyl acetate extract. The HeLa cell line was treated with 120 µg/mL concentration of ethyl acetate extract. The proportion of dead cells increased in HeLa cells after treatment with ethyl acetate extract of A. marina leaves (p < 0.05, Fig. 4I).
Cell apoptosis analysis of MCF-7, OVCAR3, and HeLa cell lines showed increase of apoptotic cells by A. marina leaves extracts
Based on the findings of the cell MTT assay, PDT assay, and cell viability test, the most effective concentrations of the extracts were determined and used for cell apoptosis assay (Fig. 5). The MCF-7 cell line was treated with 120 µg/mL concentration of ethanol extract of A. marina leaves. The OVCAR3 and HeLa cell lines were treated with 160 µg/mL and 120 µg/mL of ethyl acetate extract, respectively. After treating the HeLa, MCF-7, and OVCAR3 cell lines, the proportion of apoptotic cells increased (p < 0.05, Figs. 5G-5I).
Western blot analysis showed an increase of BAX, caspase-1, -3, and − 7 expressions in MCF-7, OVCAR3, and HeLa cell lines by A. marina leaves extracts
The MCF-7 cell line was treated with 120 µg/mL concentration of ethanol extract of A. marina leaves. The OVCAR3 and HeLa cell lines were treated with 160 µg/mL and 120 µg/mL of ethyl acetate extract, respectively. Expressions of BAX, cleaved-caspase-1, -3, and − 7 increased in MCF-7, OVCAR3, and HeLa, and cell lines after treatment (Fig. 6). However, expressions of BCL-2, pro-caspase-1, -3, and − 7 decreased in MCF-7, OVCAR3, and HeLa cell lines after treatment (Fig. 6).
Five bioactive molecules in A. marina leaves extracts had the highest affinity to apoptotic peptides
Thirty-three compounds derived from ethanol and ethyl acetate extracts of the A. marina leaves (Table 2) have been selected based on the previous studies as anti-cancer compounds (Tables S1 and S2) to study their binding affinity to apoptotic proteins BAX, BCL-2, caspase-1, -3, and − 7 through the docking process. Though, the ligand-protein complexation is controlled by conformations and intermolecular interactions such as electrostatic and Van der Waals forces [30]. The most stable complex has the lower negative energy or binding affinity, ΔG [U total in kcal/mol], as the lower negative energy indicates the more favorable ligand-protein interaction. The results of the docking process are shown in Table 2. According to Table 2, the binding affinity is in the range of -3.6 to -10.8 kcal/mol. Among selected compounds, ergosta-5,22-dien-3-ol, acetate shows the best affinity for caspase-7. Stigmasterol shows the best affinity for BAX. Beta amyrin shows the best binding affinity for BCL-2, caspase-1 and caspase-7. Moreover, it was found that three compounds of alpha amyrin, beta amyrin and cholesta-22, 24-dien-5-ol,4,4-dimethyl have the same affinity to caspase-3. The intermolecular interactions of these compounds are shown in Fig. 7.
Table 2
The results of the docking process of anti-cancer compounds in ethanol and ethyl acetate extracts of Avicennia marina leaves and their interactions with apoptotic peptides in cancer cells
Ligands | Binding affinity (Kcal/mol) |
BAX | BCL-2 | Caspase 1 | Caspase 3 | Caspase 7 |
(R)-3-hydroxydecanoic acid | -4.8 | -4.8 | -4.9 | -4.3 | -5.6 |
2,4-Di-Tert-Butylphenol | -6.4 | -6 | -5.7 | -4.8 | -7.3 |
2-Tridecanol | -4.8 | -5 | -4.5 | -4.1 | -5.6 |
4-Butoxy-2-methyl-2-pentanyl acetate | -4.6 | -5.2 | -4.7 | -4.5 | -6.1 |
9,12-Octadecadienoyl chloride | -5.6 | -5.8 | -4.2 | -3.9 | -6.4 |
Alpha amyrin | -8.4 | -8.4 | -7.3 | -6.7 | -8.3 |
Beta amyrin | -7.7 | -9.5 | -7.6 | -6.7 | -6.6 |
Betulin | -7.8 | -7.4 | -6.5 | -6.2 | -8.1 |
Cholesta-22, 24-dien-5-ol,4,4-dimethyl | -8.4 | -8.2 | -6.9 | -6.7 | -10.5 |
Cyclohexanol 1-methyl-4-(1-methylethyl)- | -5.3 | -6 | -5.4 | -4.7 | -5.8 |
Decanoic acid | -4.7 | -4.8 | -4.5 | -3.9 | -5.3 |
Dihydrocarveol | -5.3 | -5.8 | -5.2 | -4.3 | -6.1 |
Dodecane | -4.5 | -4.6 | -4.3 | -4.2 | -5.2 |
Ergosta-5,22-dien-3-ol, acetate | -8.7 | -8.1 | -6.8 | -6.2 | -10.8 |
Ethyl oleate | -5 | -5.7 | -4.4 | -4.5 | -6.7 |
Gamma-Sitosterol | -5 | -8.5 | -6.2 | -6 | -10.1 |
Hexadecane | -4.8 | -4.7 | -3.6 | -3.7 | -6.1 |
Hexadecanoic Acid 2-Hydroxy-1-(Hydroxymethyl) Ethyl Ester | -5.6 | -5.4 | -4.7 | -4.1 | -6.2 |
Levoglucosan | -5.3 | -4.8 | -4.2 | -4.2 | -4.9 |
Linoleic acid | -5.6 | -5.6 | -4.8 | -4.2 | -6.7 |
Lupeol | -8.3 | -7.9 | -6.8 | -6.5 | -8.4 |
Myristic acid | -5.1 | -5 | -4.8 | -4 | -6.1 |
Myristoleic acid | -5.2 | -5.4 | -4.8 | -3.9 | -6.4 |
Norspermidine | -3.3 | -3.6 | -4 | -3.4 | -4.1 |
Octadecanoic acid | -5.1 | -5 | -4.6 | -4 | -6.3 |
Octadecanoic acid, ethyl ester | -5.2 | -5.1 | -4.4 | -4.1 | -6.1 |
Palmitic acid | -5.1 | -5 | -4.9 | -4 | -6.1 |
Pentadecanoic acid | -5.1 | -5.3 | -4.1 | -3.6 | -6.2 |
Phenylmethyl ester | -6 | -5.4 | -4.5 | -4 | -7.7 |
Phytol | -5.9 | -5.8 | -4.5 | -4.5 | -6.9 |
Squalene | -7.3 | -7.8 | -4.7 | -4.4 | -9.5 |
Stigmasterol | -8.8 | -8.4 | -6.6 | -6 | -10 |
Vitamin E | -7.5 | -5.8 | -5.8 | -4.5 | -9.3 |