## 3 Results and Discussion
### In Vitro Experiments
#### 3.2.2 Reactive Oxygen Species (ROS) Generation  
To determine ROS formation, 50 \(\upmu\)M and 250 \(\upmu\)M curcumin prepared in ethanol and propylene glycol solution, respectively, were selected. The former was the optimum working concentration in vitro, whereas the latter, however, was selected since, although  
Figure 2: Effect of curcumin-based photosensitization at different curcumin concentrations, prepared in ethanol and propylene glycol, and light doses on reduction of _Aspergillus flavus_ spores in vitro. Data are means \(\pm\) SE (\(t=9\)); means with different lowercase letters indicate significant (\(p<0.05\)) differences at different light doses and means with uppercase letters indicate significant (\(p<0.05\)) differences at the same light dose. the 1000 \(\upmu\)M curcumin in propylene glycol resulted in the highest photoinactivation of _A. flavus_, the absorbance was above the maximum detection limit of the spectrophotometer used (absorbance over 2.5). Therefore, a lower concentration of curcumin in propylene glycol was used in the experiment. The ROS generation was indirectly monitored using DPBF, a reactive oxygen species quencher [36]. The absorption intensity of DPBF at 410 nm provides a qualitative estimation of ROS generated during photosensitization, and its decrease indicates the generation of ROS. Light intensity of 36.2 mW\(\cdot\)cm\({}^{-2}\) led to a higher and more rapid reduction in DPBF absorption intensity, which in turn represents a swift generation of ROS (Figure 3). Additionally, illumination at 36.2 mW\(\cdot\)cm\({}^{-2}\) (k\({}_{36.2}\) = 2.2277) gave rise to 1.4 times faster ROS generation in ethanolic curcumin solution than, illumination at 28.9 mW\(\cdot\)cm\({}^{-2}\) (k\({}_{28.9}\) = 1.622). For curcumin dissolved in propylene glycol, the ROS were generated 1.6 times faster at 36.2 mW\(\cdot\)cm\({}^{-2}\), than at 28.9 mW\(\cdot\)cm\({}^{-2}\) (k\({}_{36.2}\) = \(-\)0.7561 and k\({}_{28.9}\) = \(-\)0.4841, respectively). The DPBF decay in ethanolic curcumin solution followed second-order kinetics (Figure 3c). However, first-order kinetics was observed when using propylene glycol as the solvent (Figure 3d). The use of ethanolic curcumin solution ensures a generation of a higher amount of ROS than curcumin in propylene glycol, though higher concentration was used in the latter solvent. These results correlate with the observed photoinactivation efficacy of curcumin in ethanol and propylene glycol against _A. flavus_ spores. To achieve an effective microbial inactivation, higher curcumin concentrations were required using propylene glycol (>500 \(\upmu\)M). Whereas curcumin concentrations as low as 25 \(\upmu\)M were effective against the spores using ethanol as solvent. No significant change (\(p\) > 0.05) was observed in the absorption intensity of DPBF through dark incubation with the photosensitizer. Curcumin is highly soluble in ethanol but showed partial solubility in propylene glycol. Therefore, it is reasonable to assume that all curcumin dissolved in the ethanolic solution is readily available to absorb light and produce ROS, resulting in a high efficiency at a low concentration. Nevertheless, using propylene glycol, a large amount of curcumin is needed to produce enough quantum yield to photoinactivate the spores, as it was previously reported. Using a propylene glycol solution of curcumin to inactivate several fungi species, including _Aspergillus_ spp., [PERSON] et al. [24] observed that dye concentrations over 600 \(\upmu\)M were required to inactivate roughly 90% of fungal spores. However, concentrations between 100 and 400 \(\upmu\)M inactivated only up to 60% of the spores. The same authors evaluated the efficacy of curcumin in propylene glycol to inactivate the naturally occurring microflora in date fruits and reported 1400 \(\upmu\)M of curcumin as the most effective concentration against various microorganisms [8].