## 3 Discussion  
Although conventional anticancer drugs exhibit high efficacy against different types of cancer, obvious side effects have been observed. Additionally, the resistance of cancer cells to these drugs as a consequence of cell mutations is considered an obstacle and an enormous crisis in terms of increasing morbidity and mortality rates. Consequently, multi-targeted approaches using medicinal plants and phytoconstituents in combination with synthetic drugs are becoming an effective method to overcome these limitations. Pine species are an interesting example of widespread medicinal plants, with over one hundred species worldwide and a long track record of medicinal uses. Pine species' needles, inner bark, and resin are rich with many natural compounds possessing potent biological activities, such as abietic acid [45]. Additionally, pine remedies have been proven effective as anti-infection therapies for urinary tract infections, sinus infections, and lung-associated illnesses or allergies such as coughs and colds [46]. Topically, pine has been reported as anti-inflammatory agent with arthritis and skin infections [47]. Abietic acid is the major constituent of _P. palustris_ from the resin acid [48]. In vitro, abietic acid has been shown to function as a testosterone 5\(\alpha\)-reductase inhibitor and can be used for treatment of benign tumors such as prostatic hyperplasia [49]. It was interesting to study its in vitro effects in comparison to pine extract in order to understand its potential anticancer activity and cytotoxicity against different cancer cell lines and normal cells. The antiproliferative and cytotoxicity results showed that the isolated abietic acid is more potent, compared to pine extract, against breast cancer cell lines (MCF-7), with minimal cytotoxicity to normal cells. This observation matched recent studies that have shed light on the ability of pine extracts and pure abietic acid prepared from various pine species to exert anticancer effects [50; 51; 52; 53]. However, data presented in this study are the first, to our knowledge, to demonstrate the ability IC\({}_{50}\) of pine extract and abietic acid to suppress the viability of human breast cancer MCF-7 cells, and the first study to elucidate the mechanism of their action. MCF-7 was put to use as the sensitive cell line and tested with both pine extract and abietic acid for further analysis, to demonstrate  
Figure 3: Total antioxidant activity (TOA) of IC\({}_{50}\) dose of both pine extract and abietic acid treatment of MCF-7 cells for 24 h. Both extract and abiotic acid induce significant increases in total antioxidant capacity as compared to negative control without (W/O) H\({}_{2}\)O\({}_{2}\). TOA calculated compared to negative control only (in case of without H\({}_{2}\)O\({}_{2}\)), or treated with 50 uM hydrogen peroxide for 1 h. Data represented as TOA \(\pm\) SEM, \(n=3\). Results are presented as \(\pm\) SEM, \(p\) value, for comparison with Doxorubicin control, is **\(p<0.01\), **\(p<0.001\)**. its possible anticancer activity and potency. These may be developed as candidates for chemoprevention or as a chemotherapeutic adjuvant for breast tumors. Cell cycle analysis showed abietic acid increased cell population arrest exclusively in the G\({}_{2}\)/M phase as well as the subGO-G\({}_{1}\) subpopulation of breast cancer cell cycle, suggesting that abietic acid-induced signaling leads to breast cancer cell antiproliferation [50; 54]. Indeed, our live/dead assay (apoptosis assay) confirmed that abietic acid was able to induce early apoptosis in a way similar to pine extract, as indicated by higher Annexin-V staining than propidium iodide in MCF-7 treated cells relative to the control. The combination of different bioactive ingredients in the extract may explain the difference seen between pine extract and abietic acid affecting cells in cell cycle phases [55].