5. Discussion

Hospital-acquired infections represent a serious complication, often arising during the convalescence period in a medical facility [43]. It has been reported that HAIs amount to the first cause of death among hospitalized patients. To prevent these injurious events, the Centers for Disease Control and Prevention (CDC) constantly monitor the clinical practices correlated to an increased risk for microorganism contamination and invasion, including ventilator-associated pneumonia, catheter-associated urinary tract infections, bloodstream infections, and surgical site infections [44]. These illnesses can be caused by different pathogens, belonging to bacteria, fungi, or virus’s family. Accordingly, infectious diseases can significantly compromise the clinical outcome of the patients, including the oncologic ones. The increasing development of resistant mechanisms to the most commonly used drug promoted the research of new bioactive chemical compounds from phytoextracts. Concerning that literature data demonstrated the antimicrobial and the antiproliferative effects of walnut extracts [11,16,45,46,47]. However, despite the great attention paid to J. regia extracts, the knowledge on the biological effects of the edible pellicle is still limited.

In our previous work, we proved an antibacterial and antibiofilm activity of the present extract against CoNS. Furthermore, by UPLC-Ms/Ms, we chemically analyzed the phytoextract, revealing the presence of several biologically active molecules (Table 5) [18]. These results led us to hypothesize further antimicrobial properties. Indeed, in the present study, we found that WPE exerts an interesting activity against HSV-1 and HSV-2, with a concentration-dependent antiviral effect. It was demonstrated that natural extracts rich in polyphenols are capable to directly block virus attachment to host-cell [48,49] or interfering with the early phases of the replicative cycle [50]. Accordingly, the anti-herpes effect may be attributed to a synergistic effect of different compounds, belonging to phenols, flavonoids, and sterols. For example, among flavonoids, rutin and quercetin exerted potent anti-herpetic activity, both showing an EC₅₀ of 5 μM against HSV-1, although only quercetin inhibited HSV-2 infection, with an EC₅₀ of 35 μM [31,32]. A number of reports highlighted the antiviral role of quercetin against HSV. Hung et al. demonstrated the ability of quercetin to inhibit HSV-1 infection of Vero cells [42]. A further study showed that this molecule is able to modulate the expression of HSV proteins, such as the viral glycoprotein D (gD), an essential protein for the attachment of virus on host cell membrane during the infectious process, and ICP0, a viral protein expressed in the early phases of the HSV-1 replication cycle. Furthermore, the authors also demonstrated that quercetin acts at the genic level, downregulating the correlated gene-replication such as ICP0, UL13, and UL52 [51]. The WPE contains other compounds [18], including protocatechuic acid, myricetin [19], gallic acid, ellagic acid, kaempferol [22], and β-sitosterol [36], which were also associated with the anti-HSV activity. Interestingly, an in-silico analysis showed that tris-juglone, one of the previously determined constituents of WPE (Table 5) [18], showed antiviral activity against the SARS-CoV-2 virus by inhibiting cathepsin L, a lysosomal cysteine endopeptidase involved in the activation of heparinase [39]. The indirect block of this enzyme reduces the egress of the virus from the host cell. Since cathepsin L plays the same role in HSV-2, we can hypothesize that the HSV-inhibitory effect of WPE could be similarly mediated by tris-juglone [52].

Furthermore, WPE was more efficient in reducing bacterial growth of Gram-positive strains compared to those Gram-negative, as indicated by the lower MIC values. This dissimilar effect reflects differences in the cell wall composition between the two bacterial groups [16,53]. Indeed, Gram-negative bacteria are characterized by a highly selective external membrane to the passage of the molecules, which makes these bacteria more resistant to drugs [53,54,55]. Nevertheless, as reported by Saraiva et al., since several bacteria strains showed MIC values under the concentration of 500 µg/mL, WPE can be considered an active antimicrobial agent [56].

Candida species are also responsible for healthcare-related infections both in immunocompetent and immunocompromised hosts [57,58]. Although new synthetic drugs are generally active against resistant Candida species, their administration can cause toxicity, interactions with other drugs, and inadequate bioavailability [59,60,61]. Experimental evidence supported the anticandidal activity of different extracts from J. regia, including leaf, bark, and root [62,63,64,65]. In agreement with these data, our findings showed an antifungal activity of WPE against different Candida species, with MICs ranging from 17.18 to 275.00 µg/mL. This effect could be attributed to the presence of the flavonoids quercetin, myricetin, and rutin [34]. Besides, in our previous chromatographic analysis, we identified the flavonol avicularin. This compound was isolated from Juglans sinensis leaves [66]. Da Silva Sa et al. demonstrated that this compound is able to inhibit C. albicans growth in concentrations of 2 to 16 µg/mL [37]. Concerning non-albicans Candida species, the extract was less efficient in reducing the growth of these pathogens. This effect could be due to both a reduced permeability of these yeasts to natural extracts and to their higher capacity to produce biofilm [67,68].

Taken together, these results clearly showed good antimicrobial effects of WPE, revealing its efficacy against different pathogens. In the oncologic field, the development of opportunistic infections is considered a common side effect of chemotherapy [69]. According to this concept, we evaluated the potential antiproliferative action of the extract, by treating two different cancer cell lines (MCF-7 and Caco-2 cells) and a primary fibroblast cell line (HFF-1) with increasing concentrations of WPE, for 24 h, 48 h, and 72 h. Interestingly, after a prolonged period (48 h and 72 h) the treatment with WPE significantly reduced Caco-2 cell viability in a dose-dependent manner. Considering that the pellicle of walnut is edible, being concomitantly ingested with the fruit and that the gastrointestinal tract is the most exposed district to the action of dietary ingredients, it is possible to hypothesize a benefic effect of this portion of the walnut, rich in polyphenols, at the intestinal level [70]. In this respect, literature data showed the ability of a large variety of classes of dietary polyphenols, including rutin, quercetin, myricetin, ellagic acid [30], and avicularin [38], all present in our extract, to affect glucose transport in Caco-2 cells via both facilitated transport proteins (GLUT 1 and 2) and sodium-dependent glucose transporter 1 (SGLT1). Several natural extracts, usually rich in these molecules, were also found to inhibit glucose uptake in Caco-2 cells through these mechanisms [38,71]. Being the main cellular energetic source, glucose plays a key role also in cancer cell growth [72]. It has been demonstrated that the inhibition of glucose uptake is able to arrest cancer progression [73]. Therefore, in our case, the cytotoxic effect of WPE on Caco-2 cells could be attributed to the reduced glucose internalization, mediated by the synergistic action of polyphenolic compounds. Conversely, the inefficacy of WPE on MCF-7 cells confirmed the results obtained in our previous studies, which showed a greater drug resistance of these cells, induced by the breast cancer resistance protein (BCRP) expression [74]. Notably, WPE did not determine any cytotoxic effect on the primary HFF-1 cells.

The protective role of WPE was further explored by evaluating its potential scavenger activity against reactive oxygen species (ROS). As expected, the extract inhibited superoxide anion formation similarly to the SOD enzyme, also showing the ability to bleach the stable DPPH radical. The free radical-scavenging activity is strictly related to the high content of phenols and flavonoids, as determined by the Folin–Ciocalteu method (Table 4). These results are consistent with other studies focusing on J. regia pellicle extracts, which revealed a high amount of phenols and flavonoids, mostly in yellow pellicle than in red ones [75,76].