## 3 Biological Activities
### Antifungal Activity
Time response experiments showed that selected monoterenes rapidly reduce the viability of yeast cells in a time- and dose-dependent manner. Furthermore, reduced viability was associated with loss of cell membrane integrity [57]. The same authors determined the mode of action of citral against _S. cerevisiae_ using a flow cytometry study, finding evidence that the acute fungicidal activity of citral against _S. cerevisiae_ cells involves loss of membrane and cell wall integrity, resulting in dose-dependent apoptotic/necrotic cell death (Figure 3). However, yeast cells that escape this first alteration of the cell membrane, particularly evident in the sublethal concentration, die by metacaspase-mediated apoptosis induced by the accumulation of intracellular reactive oxygen species (ROS) [57]. Citral obtained from the _Litsea_ (L.) _cubeba_ plant was used in the formulation of food packaging films, showing that citral coatings achieved antifungal effects against _S. cerevisiae_ and _A. niger_, showing a reduction of 4.74 and 4.29 log, respectively, at the concentration of 1.8 \(\mu\)g/mL [45]. Citral significantly inhibited fungal growth and mycotoxin production in _A. ochraceus_. Specifically, 75, 125, 150, and 200 \(\mu\)L/L of citral suppressed mycelial growth by 33%, 46%, 50%, and 100%, respectively. Furthermore, 75 \(\mu\)L/L of citral inhibited ochratoxin-A accumulation by 25%. Through proteomic analysis, an attempt was made  
Figure 3: Mechanisms of action of citral as an antifungal agent [54; 56; 57]. to elucidate the inhibitory mechanism of citral on mycelial growth and the production of achratoxin A in subinhibitory concentrations (75 \(\mu\)L/L), and it was revealed that achratoxin production is a complex process that involves many associated factors related to various processes, including nutrient intake, sterol biosynthesis, ribosome biogenesis, energy metabolism, oxidative stress, and amino acid metabolism (Figure 3). Furthermore, citral at 75 \(\mu\)L/L downregulated achratoxin biosynthetic genes, including _p_ks and _n_r_ps, but slightly upregulated global regulatory factors _veA_, _velB_, and _laeA_ (Figure 3) [54]. The synergistic antifungal effect of citral and eugenol on _P. roqueforti_ was evaluated. The determination of the reactive oxygen species and the malondialdehydedehyde content indicated that the combination could induce the peroxidation of lipids of the membrane, with the contribution of citral being more significant than that of eugenol. Furthermore, the downregulation of the gene encoding a subunit of NADPH oxidase indicated that the explosion of reactive oxygen species induced by the compounding was mediated by NADPH oxidase (Figure 3). The combination of citral and eugenol disrupted the integrity of the cell membrane and internal structures, resulting in the degradation of cellular contents. The combination induced membrane lipid peroxidation and enhanced the ability to destroy the cell membrane. The combined agents eventually caused cell content leakage and cell death [16]. Citral at 2.0 or 4.0 \(\mu\)L/mL strongly damaged _P. digitatum_ mitochondria by causing matrix loss and an increase in irregular mitochondria. The extent of deformation mitochondria increased with increasing citral concentrations, with a decrease in the intracellular ATP content and an increase in the extracellular ATP content. Oxygen consumption showed that citral resulted in an inhibition in the tricarboxylic acid cycle pathway of _P. digitatum_ cells, inducing a decrease in the activities of citrate synthetase, isocitrate dehydrogenase, \(\alpha\)-ketoglutate dehydrogenase, succinodehydrogenase, and citric acid content while enhancing the activity of malic dehydrogenase in _P. digitatum_ cells [56]. [PERSON], [PERSON] [58] investigated the antifungal activity and mechanism of citral and other terpen against _Zygosaccharomyces rouxii_, with citral showing the most potent antifungal effect.