# Rosemary Leaf Extract Inhibits Glycation, Breast Cancer Proliferation, and Diabetes Risks
## 3 Results and Discussion
### Effect of Rosemary Extract on Proliferation of Breast Cancer Cell Line MCF-7  
In this study, the effect of rosemary extract on the proliferation of cancer cells was investigated by treating MCF-7 cell lines with doses at 0.025, 0.050, 1.000, and 2.000 mg/mL for 96 h. As shown in Figure 5, the survival rate of MCF-7 cells decreased with the increase of concentrations of rosemary extract. Approximately 90.12% and 68.53% of MCF-7 cells survived at RE concentrations of 0.125 and 0.25 mg/mL, respectively (Figure 5). However, it significantly dropped to 13.57% at the RE concentration of 0.5 mg/mL (Figure 5) and reached 6.02% for RE of 1.0 mg/mL and 2.16% for RE of 2.0 mg/mL (Figure 5). The IC50 value of RE was 0.317 mg/mL. The inhibition of proliferation in MCF-7 cells could result from the induction of apoptosis by RE. Thus, TUNEL and DAPI co-staining assay was used to determine DNA fragmentation and nuclear condensation as apoptotic markers. Apoptosis is defined by a series of characteristic changes in cell morphology during cell death [33]. Morphological changes in MCF-7 cells treated with RE were observed. Control cells maintained a typical intact appearance, nevertheless, RE treated cells showed morphological alterations such as cellular shrinkage, cell monolayer area reduction, less adherence, and floating shapes (Figure 6).  
The efficient inhibitory performance of RE might be due to the abundance of phenolics. For example, a positive correlation between the number of unsubstituted hydroxyl groups (free OH), and the anti-proliferation capability was found in the study of [PERSON] [34]. It has been reported that hydroxyl groups, especially in the form of ortho-diphenyl moieties in rosemarinic acid and carbonyl functions (COOH) in carnosic acid could enhance the cytotoxic effects on the MCF-7 cell [34]. The related mechanism might lead one to speculate that the increased toxicity was contributed by ortho-diphenyl through the chelation and reduction of transition metals (Fe, Cu, and Zn) which accelerate the cytotoxic effects on the MCF-7 cell [35]. A similar result has been reported in the study by [PERSON], in which the anti-proliferative activity of rosemarinic acid was also observed in breast cancer cells by the induction of mitochondrial-mediated cancer cell apoptosis [36]. Another hypothetical mechanism is the interactions between phenolics and cellular proteins. The phenol and carbonyl groups of phenolics and amine or amide residues of a protein easily generate soluble or insoluble complexes via the formation of hydrogen bonds [35]. However, those interactions and the related products might affect and destroy important cellular structures. Additionally, carnosic acid could also contribute to the anti-proliferation capability of RE, since it accounts for approximately 24% of the total phenolics. The mechanism of carnosic acid was based on the induction of the G2/M phase of the cell cycle which led to cancer cell apoptosis [37]. These observations suggest that RE was effective in inducing apoptosis in MCF-7 cells.  
Figure 5: Survival rates of MCF-7 cancer cells treated with different levels of RE.  
Figure 6: Images of DAPI staining (green color) and TUNEL (blue color) to indicate the MCF-7 cell apoptosis.