## 3 Results
### Effect of Saffron Leaf Extracts on Proliferation of Caco-2 Cells  
The effect of safron leaf phenolic fraction (SLPF) on the proliferation of Caco-2 cells was evaluated in in vitro assays. Caco-2 cells, derived from human colon carcinoma, were cultured under standard conditions to evaluate the impact of these extracts on cell growth. The results, presented in Figure 3, express the inhibition of proliferation in percentages with respect to the control (EtOH), whereas Table 2 presents the EC\({}_{50}\) and EC\({}_{90}\) values, allowing for a direct comparison between the phenolic fraction of safron and the unprocessed extract.  
Figure 3: Antiproliferative effects of safron leaf extract (SLE; blue) and safron leaf polyphenol fraction (SLPF; brown) on Caco-2 cell proliferation over 8 days, expressed as a percentage relative to the control (EtOH). Cells were treated with three concentrations: 0.5 mg/mL, 0.125 mg/mL and 0.031 mg/mL. Data represent mean \(\pm\) SD. Different letters denote statistically significant differences between treatments and time points (Tukey’s test, \(p<0.01\)).  
The assayed extracts exhibited a clear dose- and time-dependent antiproliferative effect on Caco-2 cells (Figure 3), with SLPF consistently demonstrating stronger inhibitory activity than SLE, especially at intermediate concentrations. Statistical analysis revealed that SLE exerted limited cytotoxic activity on day 1, with robust effects from day 2 to day 4, especially at the highest concentration. In contrast, SLPF induced a progressive and significant inhibitory response from the first 24 h, supporting a more immediate and cumulative mechanism of action. At the concentration of 0.5 mg/mL, SLE reduced cell proliferation by nearly 90% on day 4, whilst for SLPF similar levels were achieved by day 8, suggesting a more sustained and potent inhibitory response for the SLPF. At 0.125 mg/mL, only SLPF induced a significant inhibitory effect, reinforcing the extract's greater antiproliferative capacity. In contrast, the lowest concentration (0.031 mg/mL) resulted in a modest but constant inhibition of about 20% for both extracts, which gradually decreased over time, and which can be attributed to the chemical degradation of the compounds. This suggests baseline biological activity even at minimal doses. Notably, the control Caco-2 cells continued to proliferate steadily throughout the eight-day incubation period, confirming their viability and normal growth kinetics in the absence of treatment. This observation enhanced the development of a multivariate model (Table 2) that incorporates both concentration (mg/L) and time (days) to statistically estimate the inhibitory potential of each concentration and extract. Otherwise, a logarithmic regression analysis was performed for each sample to mathematically calculate the percentage inhibition at each concentration. Based on these data, linear regression was applied to calculate and estimate the EC\({}_{50}\) and EC\({}_{50}\) values, thereby quantifying the superior inhibitory potency of SLPF relative to SLE. On day 4, when only a modest statistical increase in inhibitory activity was observed, the EC\({}_{50}\) value for SLE was calculated to be 0.31 mg/mL, while that of SLPF was 0.22 mg/mL, indicating an approximately 30% greater inhibitory effect for the phenolic fraction. Furthermore, the EC\({}_{90}\) values were calculated to be 0.71 mg/mL for SLE and 0.52 mg/mL for SLPF, further supporting the higher efficacy of SLPF in suppressing Caco-2 cell proliferation. Similarly, the multivariate model estimated EC\({}_{50}\) and EC\({}_{90}\) values very similar to those obtained from the empirical logarithmic regression data considering inhibition vs. time at the different concentrations, thus improving its reliability, particularly for the SLPF (R\({}^{2}\) = 0.82).