*3.5. Anti-Inflammatory Activity*

We investigated the impact of PEP on the inflammatory process in RAW264.7 cells stimulated with LPS. During an inflammatory event, the inducible enzyme nitric oxide synthase (iNOS) is responsible for an exacerbated production of NO, which can lead to tissue lesions, organ dysfunction, and inflammation-related diseases [43]. As shown in Figure 3, LPS significantly (*p* < 0.05) stimulated the production of NO in macrophages (Control+) with respect to non-stimulated cells (Control−). The percentage of NO production was significantly (*p* < 0.05) reduced by 24–39% in RAW264.7 cells pre-treated with PEP. Pre-treatment of LPS-stimulated cells with VD 80 ◦C and VD 60 ◦C powders led to greater attenuation of NO production with an inhibition range between 33% and 39%. The rest of the extracts had a similar behavior (*p* < 0.05), regardless of the drying treatment conducted, and caused an attenuation in the production of NO in a range between 24% and 30%. Apparently, a greater anti-inflammatory capacity would be influenced both, by the type of treatment used (VD > SPD > FD) and by the temperature used in each treatment. Overall, this behavior is similar to that observed in the results obtained from the analysis of antioxidant activity (ROS inhibition), where VD 80 ◦C was the most active sample in both determinations. Some investigators have demonstrated that the cellular inflammatory responses are caused because of ROS production [17,44]. Consequently, a reduction in intracellular ROS levels could lead to an inhibition of the inflammation process through the reduction of NO production. This postulate agrees with our results, since the samples that showed high antioxidant activity by reducing intracellular ROS (Figure 2) also showed relevant anti-inflammatory properties by reducing NO production (Figure 3).

**Figure 3.** Effect of PEP (1 mg/mL) on nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophage cells. Values are expressed as a percentage relative to the LPS-stimulated control group and are represented by mean ±SD (*n* = 3). Bars with different letters indicate significant differences on NO production by ANOVA LSD Tukey test (*p* ≤ 0.05). Freeze drying (FD); Vacuum drying 40 ◦C (VD 40 ◦C), 60 ◦C (VD 60 ◦C), 80 ◦C (VD 80 ◦C); Spray drying (SPD).

However, it is difficult to precise the main polyphenolic compounds that would be most involved in the anti-inflammatory response observed, although apparently, several of them appear to be involved. Previous studies have demonstrated that the anti-inflammatory effects of plum phenolic compounds [1,18], mainly due to the presence of phenolic acids, can decrease the expression of inflammatory mediators, such as nuclear factor κB (NF-κB), vascular cell adhesion molecule 1 (VCAM-1), cyclooxygenase-2 (COX-2), and iNOS mRNA [45]. In addition, plum phenolic compounds can contribute to the modulation of the inflammatory responses in human cells by inhibiting various inflammatory factors, such as cytokines IL-6 and IL-8 [17]. In addition, the potential anti-inflammatory activity of the PEP could be particularly important, considering that activation inflammatory pathways can stimulate proliferation of cancer cells [38].

#### **4. Conclusions**

This study has shown that plum extract powders gained after freeze-, vacuum-, and spray-drying have promising antibacterial, antioxidant, and anti-inflammatory properties that have been tested in different biological models. The drying processes significantly influences both, the physical properties and the composition of polyphenols, and thus, the bioactive properties plum juice extract powders. The drying techniques moderated the content of polyphenolic compounds in the powders, which influence the different levels of growth inhibition against the foodborne pathogens, evidencing a strain-dependent effect being the most relevant for FD extract. This extract inhibited significantly (*p* < 0.05) the growth of all the bacteria studied, except *E. coli* strain. It was observed that *L. monocytogenes* was inhibited by all the dried plum juice extracts in the range of 17–46%, regardless of the drying process used. These powders could also induce cellular protection against oxidative stress by preventing intracellular ROS accumulation, but the level of antioxidant capacity may be determined by the conditions applied during the drying process. It was shown that VD 80 ◦C and SPD caused the highest protection against oxidative damage in stressed cells, inducing an inhibition percentage of ROS production close to 37% respect to the oxidative control cells. Moreover, plum extracts powders exhibited a greater anti-inflammatory capacity, which would be influenced both, by the type of treatment used and by the temperature used in each treatment, being the VD 80 ◦C - the sample with the highest protection level. The results demonstrate that the drying method selected can be an effective tool for modulating the composition, physical, and bioactive properties of plum extracts powders.

**Author Contributions:** Conceptualization, J.M.S., A.M.-C., and A.J.M.-R.; data curation, A.M.-C., and A.J.M.-R.; Formal analysis, J.M.S. and A.M.-C.; funding acquisition, A.J.M.-R.; investigation, J.M.S., A.M.-C., and A.J.M.-R.; Methodology, J.M.S. and A.M.-C.; project administration, A.J.M.-R.; resources, A.J.M.-R.; software, J.M.S., and A.M.-C.; supervision, A.J.M.-R.; writing-original draft, J.M.S., A.M.-C., and A.J.M.-R.; writing-review and editing, J.M.S., A.M.-C., and A.J.M.-R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by AGL2017-89566-R project from the Consejo Superior de Investigaciones Científicas (Spain).

**Acknowledgments:** This work was founded through Project AGL2017-89566-R from the Consejo Superior de Investigaciones Científicas (Spain). Authors are grateful to Krzysztof Lech and Adam Figiel from Institute of Agricultural Engineering (Wroclaw University of Environmental and Life Sciences) for possibility to perform water activity and color measurements.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
