# Propolis Hydroalcoholic Extracts: Biochemical Characterization and Antifungal Efficacy
## 3 Results  
### Bio-Assay: Effectiveness of Hydroalcoholic Propolis Against B. cinerea  
The assay showed the inhibitory activity against _B. cinerea_ mycelial growth of the different propolisik extracts, obtained by two methods of extraction, as reported in Section 2.3. Figure 1 shows a pronounced antifungal activity of propolisis extracted using ethanol solution at 90% and in particular that obtained by sonication and 1 d of incubation (Supplemental Figure S1). The greatest area of mycelial inhibition was recorded for the extract PU3 (331.2 mm\({}^{2}\)), followed by PFvg1 (246.2 mm\({}^{2}\)) and PFvg2 (227.2 mm\({}^{2}\)), all, respectively, obtained via the sonication method. The lowest inhibition halos (mm\({}^{2}\)), within the same extraction mode, were displayed by PV4 (182.9 mm\({}^{2}\)) and PT5 (168.1 mm\({}^{2}\)). Even in the case of the 7 d extraction, 90% ethanol solution was found to be more effective. No fungal inhibition activity was recorded for the solvent solutions (Supplemental Figure S1). Figure 1: Area (mm\({}^{2}\)) of halo inhibition of _Botrytis cinerea_ growth by different propolisis (PFvg1, PFvg2, PU3, PV4, PT5) extracts obtained, respectively, with 30%, 60% and 90% ethanol solution and with 7 and 1 days of extraction. The last method was preceded by 20 min of sonication. Data are the mean of six replicates \(\pm\) standard error. Different letters indicate significant differences within the propolisis according to [PERSON]’s test (\(\alpha\) = 0.05). Volatile and Non-Volatile Fraction of Propolis Extracts: Efficacy on B. cinerea Conidial Germination  
The experiments were conducted using propolis extracts obtained using ethanol 90% for 1 d of incubation following 20 min of sonication, which was the most effective method as reported in the preliminary bio-assay results. Nine concentrations, ranging between 0 (control) and 200 ppm, of each extract were subsequently tested for their efficacy through the production of volatile and non-volatile metabolites against the conidial germination of _B. cinerea_. In Figure 2, _B. cinerea_ colony-forming units (CFUs), affected by the volatile fraction of the propolis extracts at different concentrations, are reported. The highest inhibition on CFU growth was recorded at a concentration of 200 ppm for all the propolis extracts: 85.61% (PFvg1), 92.46% (PFvg2), 78.1% (PU3), 57.53% (PV4) and 69.86% (PT5). The volatile compounds of propolis extracts PV4 and PT5 were less effective than those of the other extracts. However, impressive results were displayed by PFvg1 and PFvg2 starting from the lowest tested concentration. In this regard, both propolis extracts inhibited CFUs, respectively, by 32.87% and 61.64% at a concentration of 3.12 ppm, compared to the control. A more scalar inhibition trend was observed for the other propolis. Regarding the non-volatile metabolites assay, four out of five propolis extracts were able at different concentrations to completely inhibit fungal CFUs. Propolis extract PFvg1 completely inhibited _B. cinerea_ CFU growth starting from 50 ppm. A total inhibition was recorded starting from a concentration of 100 ppm for PU3 and PV4. With PT5 propolis, 95% of inhibition was reached at 200 ppm. Both propolis PFvg1 and PU3, at 25 ppm, were able to inhibit _B. cinerea_ CFUs by almost 90% (Figure 3). Figure 2: Effect of volatile metabolites produced by PFvg1, PFvg2, PU3, PV4 and PT5 propolis extracts obtained using 90% ethanol solution and 1 day of extraction, preceded by 20 min of sonication. Nine concentrations, ranging between 0 ppm (control) and 200 ppm, were tested on _Botrytis cinerea_ colony-forming unit (CFU) growth. The data are the mean of six replicates \(\pm\) standard error. Different letters indicate significant differences within the propolis according to [PERSON]’s test (\(\alpha\) = 0.05). By the obtained results, the effective dose (EC50) of each propolis extract concerning the mode of action (volatile and non-volatile) was calculated using the probit values (Table 2).