# Propolis Hydroalcoholic Extracts: Biochemical Characterization and Antifungal Efficacy
## 4 Discussion  
The present research confirms that propolis is chemically rich, and this fact, as well as its antimicrobial effectiveness, varies according to plant, environment, season and bee type [37]. The composition of propolis is crucial for understanding its biological role [8]. Until now, according to the available data, propolis' antimicrobial and antifungal efficacy has been attributed principally to polyphenols and flavonoids [38]. The propolisis extracts used in the present study displayed a wide range of polyphenols. Our results confirmed the data reported by [PERSON] et al. [39] that showed how the efficiency of extraction by maceration was less effective than that achieved by ultrasounds. Prolonged extraction by sonication leads to degradation of phenolics along with flavonoids, as reported by [PERSON] and [PERSON] [40]. Ethanol is an organic solvent with intermediate polarity, so exposure to higher temperature can contribute to increased substance solubilization [41]. However, ethanol has a higher efficiency than water in the extraction of phenols and polar compounds. Also, it is the substance most used for the extraction of bioactive compounds from propolisis [42]. In particular, Pfvg1, PFvg2 and PU3 reported a high amount of genistein, regardless of their geographical origin. [PERSON] et al. [43] reported that genistein and chrysin are commonly found in propolisis and are among the predominant bioactive constituents. In the present study, propolisis extracts PV4 and PT5 did not exhibit the presence of galangin and genestein and on average displayed a fair amount of content of apigenin, caffeic acid, chrysyn, ferulic acid, kaempfer, luteolin, \(p\)-coumaric acid and quercetin. [PERSON] et al. [44] attributed the possible mechanism of inhibition of protein synthesis and cell membrane functions to quercetin. Figure 6: OPLS (Orthogonal Projections to Latent Structures) model analysing propolis extracts to predict their bioactivity, represented by the EC50 values. (**Left Plot**): relationship between observed and predicted EC50 values for each propolisis sample. (**Right Plot**): coefficients of the model. Propolis hydroalcoholic extracts PFvg1, PFvg2 and PU3, during in vitro assays, showed a notable inhibitory effect on _B. cinerea_, compared to the other extracts. PFvg1 propolis extract displayed a high level of ferulic acid. This is known to exert an antimicrobial action that cause cell membrane disfunction and changes in cellular morphology [44]. However, together with the other polyphenols, it provides synergistic activity against microbial organisms. In the evaluation of the efficacy of propolis extracts in inhibiting conidial germination of _B. cinerea_, also the propolis volatile metabolites were considered. Conversely to non-VOCs, volatile metabolites showed a gradual inhibition against the pathogen, with the best results observed at higher concentrations for all the extracts. The antifungal effect of propolis increased with increasing doses [45]. Concerning this, [PERSON] et al. [45] have reported that higher propolis doses were effective against _Sclerotinia scleroorum_ and _Penicillium digitatum_ of citrus fruit. Usually, volatile compound concentration was low in the propolis. However, the volatilome is closely connected to propolis biological activity as well as it being a key element in its characterization [4]. From the VOC propolis analysis, the main detected compounds in the five propolis belong to the sesquiterpene alcohols class (\(\alpha\)-cadinol, \(\alpha\)-eudesmol, calamenene, cadinol) and to aromatic compounds (benzyl acetate, benzyl benzoate). Our results are confirmed by a study by [PERSON] et al. [8] that reported the abundance of sesquiterpenes in most of the studied European propolis. In addition, [PERSON] and [PERSON] [46] reported that \(\beta\)-eudesmol and benzyl benzoate were found to be the main constituents of the essential oil derived from _Populus nigra_, including nonanoic acid and tiglic acid, mainly found in _Geraniaceae_ and _Euphorbiaceae_ plants.