Anti-Inflammatory and Antimicrobial Activities of Portuguese Prunus avium L. (Sweet Cherry) By-Products Extracts

The bioactivity of natural by-products in food and pharmaceutical applications is the subject of numerous studies. Cherry production and processing generates large amounts of biowaste, most of which is not used. The recovery of these by-products is essential for promoting the circular economy and to improving sustainability in the food industry. In this work, we explored the anti-inflammatory and antimicrobial potential of two different extracts from stems, leaves, and flowers of Portuguese cherries. The anti-inflammatory potential was studied on lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW 264.7) by evaluating the effect of by-products on cellular viability and nitric oxide (NO) production. Disc diffusion and minimum inhibitory concentration (MIC) were used to determine antimicrobial activity. The cherry by-products had no cytotoxic effect on RAW 264.7 cells, and were able to inhibit nitrite production in a dose-dependent manner. Moreover, all aqueous infusions showed good antioxidant activity against NO radicals. Moreover, leaf extracts showed the best activity against most of the strains studied. The results revealed, for the first time, interesting anti-inflammatory and antimicrobial properties of cherry by-products. This could potentially be of interest for their therapeutic use in the treatment of inflammation-related diseases or in controlling the growth of microorganisms.


Introduction
Since ancient times, plants formed the basis for the development of many therapeutic products used in traditional medicine with significant pharmacological properties [1]. Natural products consist of various bioactive compounds that have been shown to have health-promoting properties, keep the body healthy, and protect against a variety of diseases, many of which are related to oxidative stress [2]. Nowadays, consumers are looking for healthier foods/products for their daily life. In this context, food and pharmaceutical industries are increasingly interested in finding compounds with good biological properties from natural sources [3,4]. Detailed knowledge of phytochemical composition, biological properties, safety profile, and environmental toxicity is essential for the characterization of herbal products.
Prunus avium L., commonly known as sweet cherry, is a high-value fruit of great nutritional and economic importance, widely distributed throughout the world [5][6][7]. The literature dealing with this specie has shown that the cherries possess interesting

Cell Culture
RAW 264.7 cell line, was cultured in a DMEM high glucose medium, supplemented with 10% (v/v) of non-inactivated FBS, 1% penicillin/streptomycin, and 1.5 g/L sodium bicarbonate, at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 . The cells were passaged using a cell scrapper, before reaching confluence and according to ATCC recommendations.

Effect of Cherry By-Products Extracts on Cellular Viability
To determine the effects of hydroethanolic extracts and aqueous infusions of P. avium by-products on cellular viability, the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed, as previously described [19]. Briefly, 2.5 × 10 4 cells per well were seeded in a 96-well plate and incubated with different concentrations (50, 100, 200, 400, and 800 µg/mL) of the extracts, for 24 h, according to the described method, with minor adjustments [19]. Then, the medium was then removed and the MTT solution was added to each well (1 mg mL −1 ) and the plates were incubated, in the dark at 37 • C for 4 h. The absorbance of formazan crystals was measured at 570 nm using an XmarckTM microplate absorbance spectrophotometer (Bio-Rad, Hercules, CA, USA). Six independent experiments were performed in triplicate.

Nitric Oxide (NO) Radicals Production
For nitrite accumulation in culture medium, cells were seeded at a density of 1 × 10 5 per well in a 96-well plate for 24 h, according to the method described by Oliveira and co-workers [20]. After that, the medium was removed, and the cells were incubated for 2 h with different dilutions, as described in the previous section. Subsequently, 1 µg mL −1 LPS was used to stimulate the cells, for 22 h. Production of NO radicals was determined by measuring nitrite accumulation in the supernatant, as previously described [20]. The Griess reagent (1% (w/v) sulfanilamide in 5% (w/v) phosphoric acid and 0.1% (w/v) N-(1-naphthyl)-ethylenediamine dihydrochloride) was mixed into the supernatants, and incubated at room temperature for 30 min. Absorbance was measured at 550 nm.

Determination of Radicals NO Scavenging Activity
NO radicals scavenging activity was evaluated according to the method explained by Jesus and co-workers [21]. Briefly, the same concentrations used in the cells were dissolved in potassium phosphate buffer (100 mM, pH 7.4) and mixed with SNP (20 mM), as described by Gonçalves and co-workers [11]. Plates were then incubated for 1 h at room temperature, under light. Griess reagent, composed by 1% sulfanilamide and 0.1% naphthylethylenediamine in 2% phosphoric acid (H 3 PO 4 ), was added to the wells, and the multiwell plates were incubated for 10 min, in the dark. Then, the absorbance was measured at 560 nm. The radical scavenging activity NO was determined by comparing the absorbance values between the extracts and the control and corresponded to the mean ± standard deviations of three independent experiments, performed in triplicate.

Disk Diffusion Method
The disk diffusion test was performed according to CLSI M02-A12 (2015) for bacteria [22]. Sterile Petri dishes (90 mm Ø) were used using 25 mL of medium. A 0.5 McFarland suspensions were made from bacterial cultures in sterile saline solution. Antimicrobial disks (Filtar Lab, 6 mm Ø) were impregnated with 10 µL each of a hydroethanolic extract or an aqueous infusion of P. avium leaves, stems, and flowers, following to the method previously described [23], with small modifications. Gentamycin was used as a positive control. MHA and with tryptic soy agar (TSA) medium were used for this experiment. The plates were incubated at 37 • C for 24 h. Posteriorly, the inhibition zone was visualized and measured in millimeters. The obtained results are reported as means ± standard deviations of three independent experiments.

Evaluation of the Minimum Inhibitory Concentration (MIC)
The susceptibility of bacteria to the hydroethanolic extracts and aqueous infusions of cherry leaves, stems, and flowers was determined using the broth microdilution method, as previously described [24]. Briefly, inoculums were prepared by suspension in NaCl 0.85% (w/v), and turbidity was adjusted to 0.5 McFarland to get a final concentration of approximately 5 × 106 CFU mL −1 . Experiments were performed in 96-well plate with a range of concentrations between 2 and 0.015 mg mL −1 , posteriorly incubated at 37 • C for 24 h. Then, 30 µL of a resazurin solution (0.01%) was added to each well and incubated for 2 h at 37 • C. The assay was performed in triplicate, and results were reported as modal values.

Evaluation of P. avium by-Products Biocompatibility
The cytotoxicity of hydroethanolic extracts and aqueous infusions of sweet cherry leaves, stems, and flowers was investigated using the normal human dermal fibroblasts (NHDF) cell line. This cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in high-glucose DMEM medium supplemented with 10% FBS and 1% antibiotic/antimycotic and 25 µg mL −1 amphotericin B (Sigma-Aldrich, Inc., St. Louis, MO, USA). 1 × 10 4 cells per well were seeded in 96-well plate and incubated at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 for 24 h. After, the cells were incubated with different concentrations of extracts of P. avium by-products (50, 100, 200, 400, and 800 µg mL −1 ), according to the method described previously [13]. After 24 h, the MTT assay described in Section 2.5.1 was performed. The absorbance was measured at 570 nm using an XmarckTM Microplate Absorbance Spectrophotometer (Bio-Rad, Hercules, CA, USA). Six independent experiments were performed in triplicate.

Statistical Analysis
The results were analyzed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Data were expressed as mean ± standard deviations or mean ± standard error of the mean. One-way analysis variance (ANOVA) with Tukey's multiple comparison test or Student's t-test were performed to compare the effect of each concentration of P. avium by-products extract with controls. A p value < 0.05 was considered statistically significant.

Anti-Inflammatory Potential
The anti-inflammatory process is the body's natural mechanism in response to infection or injury. However, if the inflammatory state is overestimated, it can contribute to the onset and development of numerous diseases, such as cancer, cardiovascular and neurological diseases [25]. Plant extracts consist of various phytochemicals, mainly phenolic compounds [6,7]. According to the literature, these compounds possess a plethora of health benefits, including anti-inflammatory properties [11,20].
Macrophages play a crucial function in the innate immune response, so in this work, we used RAW 264.7 cells as an in vitro inflammatory model to investigate the possible anti-inflammatory activity of P. avium stems, leaves, and flowers.

Effect of P. avium By-Products on the Viability of the Raw 264.7 Cell Line
To determine whether the hydroethanolic extracts and aqueous infusions of sweet cherry leaves, stems, and flowers affect macrophages viability of, the MTT assay was performed after 24 h of exposure. The concentration of the extracts was adjusted to 50, 100, 200, 400 and 800 µg mL −1 . As shown in Figure 1, measurement of cell viability after treatment with all tested concentrations of the two extracts from P. avium stems and flowers aqueous infusion resulted in cell viability ≥90% ( Figure 1C,D,F). The hydroethanolic extract and aqueous infusion both from leaves showed a cell viability ≥90% in a concentrations range of 50 to 400 µg mL −1 and 50 to 200 µg mL −1 , respectively ( Figure 1A,B). The hydroethanolic extract of flowers showed cell viability between 80% and 90% ( Figure 1E). Similar results were obtained by Orabona and co-workers [26] for Crocus sativus L. byproducts. Based on these results, we can assume that the concentrations used are safe and non-toxic for further studies.
The biological properties of secondary plant metabolites have been extensively studied [7,27,28]. According to previous studies about phytochemical composition of P. avium by-products, it is known that cherry leaves, stems, and flowers are composed by several phenolic compounds, namely hydroxycinnamic acid, flavonols, flavan-3-ols, and flavanones [13,21]. Among several phenolics present in these by-products, the 3-caffeoylquinic acid cis, 5-caffeoylquinic acid trans, quercetin 3-O-rutinoside, quercetin 3-O-glucoside, 3-coumaroyl-5-caffeoylquinic acid, and 3-coumaroyl-4-caffeoylquinic acid are the most abundant [13]. The maintenance of RAW 264.7 cells viability when exposed to several concentrations and different extracts may be related to the presence of these hydroxycinnamic acids and flavonoids. Furthermore, these phenolic compounds are described as being able to reduce inflammation [29]. In addition, they are molecules with low cytotoxicity [29,30]. leaves, stems, and flowers on RAW 264.7 macrophages. Cells were plated and submitted to increasing concentrations of extracts for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess cell viability. The presented data correspond to the means ± standard deviation of three independent assays and are represented as percentage (%) of control cells. Statistical analysis: One-way ANOVA was performed for each concentration compared to control; * p value < 0.05, ** p < 0.01, and # p < 0.001 were considered significant.

Effect of P. avium By-Products on NO Production
Numerous inflammatory mediators are released as part of the inflammatory process. In addition, macrophages produce NO in large quantities, and it is known that natural products have shown a good response to this increase [31]. Lipopolysaccharide are extracellular elements of Gram-negative bacteria and possess potent stimuli for monocytes and macrophages. When these cells are activated by a stimulus induced by LPS, they produce inflammatory mediators, including NO, and release them via the regulation of pro-inflammatory factors [32]. Therefore, in this work, we investigated the anti-inflammatory potential of hydroethanolic extracts and aqueous infusions of cherry leaves, stems, and flowers through their ability to inhibit the production of NO in stimulated RAW 264.7 macrophages. An inflammatory stimulus was elicited by LPS (1 μg mL −1 ), to simulate an inflammatory state. The production of nitrites (NO stable metabolites), was measured after cell exposure to P. avium by-products extracts by the colorimetric Griess reaction. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess cell viability. The presented data correspond to the means ± standard deviation of three independent assays and are represented as percentage (%) of control cells. Statistical analysis: One-way ANOVA was performed for each concentration compared to control; * p value < 0.05, ** p < 0.01, and # p < 0.001 were considered significant.

Effect of P. avium By-Products on NO Production
Numerous inflammatory mediators are released as part of the inflammatory process. In addition, macrophages produce NO in large quantities, and it is known that natural products have shown a good response to this increase [31]. Lipopolysaccharide are extracellular elements of Gram-negative bacteria and possess potent stimuli for monocytes and macrophages. When these cells are activated by a stimulus induced by LPS, they produce inflammatory mediators, including NO, and release them via the regulation of pro-inflammatory factors [32]. Therefore, in this work, we investigated the antiinflammatory potential of hydroethanolic extracts and aqueous infusions of cherry leaves, stems, and flowers through their ability to inhibit the production of NO in stimulated RAW 264.7 macrophages. An inflammatory stimulus was elicited by LPS (1 µg mL −1 ), to simulate an inflammatory state. The production of nitrites (NO stable metabolites), was measured after cell exposure to P. avium by-products extracts by the colorimetric Griess reaction.
As shown in Figure 2, all extracts of cherry by-products were able to decrease the production of NO, in LPS-stimulated RAW 264.7 cells, in a dose-dependent manner. In particular, the hydroethanolic extracts of leaves and stems and the aqueous infusions of flowers were able to decrease NO to below 50% at extract concentrations of 400 µg mL −1 or more (Figure 2A,C-F). Interestingly, both extracts of flowers were promising, and showed a very significant reduction in NO, at extract concentrations of 200 µg mL −1 or greater ( Figure 2E,F). To our knowledge, no similar studies have been performed with sweet cherry by-products. However, similar profiles were obtained for sweet cherry phenolic-rich fractions of the same variety of by-products [11]. As shown in Figure 2, all extracts of cherry by-products were able to decrease the production of NO, in LPS-stimulated RAW 264.7 cells, in a dose-dependent manner. In particular, the hydroethanolic extracts of leaves and stems and the aqueous infusions of flowers were able to decrease NO to below 50% at extract concentrations of 400 μg mL −1 or more (Figure 2A,C-F). Interestingly, both extracts of flowers were promising, and showed a very significant reduction in NO, at extract concentrations of 200 μg mL −1 or greater ( Figure 2E,F). To our knowledge, no similar studies have been performed with sweet cherry by-products. However, similar profiles were obtained for sweet cherry phenolic-rich fractions of the same variety of by-products [11]. leaves, stems, and flowers on macrophage nitric oxide (NO) production upon an inflammatory stimulus. Cells were plated and exposed to Ctrl medium exposed to increasing concentrations of extracts for 24 h, in the presence of lipopolysaccharide (LPS) (1 μg mL −1 ). Griess assay was performed to assess nitrites levels in the supernatant. The presented data correspond to the means ± standard error of the mean of three independent assays and are represented as percentage (%) of control cells (C) cells exposed to LPS (Ctrl LPS). Statistical analysis: One-way ANOVA was performed for each concentration compared to control with LPS; * p value < 0.05, ** p < 0.01, and # p < 0.001 were considered significant. leaves, stems, and flowers on macrophage nitric oxide (NO) production upon an inflammatory stimulus. Cells were plated and exposed to Ctrl medium exposed to increasing concentrations of extracts for 24 h, in the presence of lipopolysaccharide (LPS) (1 µg mL −1 ). Griess assay was performed to assess nitrites levels in the supernatant. The presented data correspond to the means ± standard error of the mean of three independent assays and are represented as percentage (%) of control cells (C) cells exposed to LPS (Ctrl LPS). Statistical analysis: One-way ANOVA was performed for each concentration compared to control with LPS; * p value < 0.05, ** p < 0.01, and # p < 0.001 were considered significant.

Effect of P. avium By-Products on NO Scavenging Activity
To determine whether the decrease in production of NO was due to the scavenging activity of extracts from P. avium leaves, stems, and flowers, the NO scavenging activity was considered. As shown in Figure 3, all hydroethanolic extracts and aqueous infusions of cherry by-products showed significant scavenging activity in a concentration-dependent manner. The aqueous infusions of cherry stems, flowers, and leaves were the most active ( Figure 3B,D,F) showing a scavenging activity greater than 50% (IC 50 values of 111.4 ± 0.31, 149.3 ± 0.94, and 183.5 ± 0.72 µg mL −1 , respectively). Hydroethanolic extracts and aqueous infusions of leaves, stems, and flowers demonstrated an excellent scavenging activity against NO radicals compared to ascorbic acid (IC 50 = 767.67 ± 4.04 µg mL −1 ) (data not shown). The results obtained are in agreement with previous studies [21]. leaves, stems, and flowers on NO scavenging activity. Griess assay was performed to assess NO scavenging in supernatant. The presented data correspond to the means ± standard deviation of three independent assays and are represented as percentage (%) of control. Statistical analysis: Oneway ANOVA was performed for each concentration compared to control; # p < 0.001 was considered significant.

Antimicrobial Potential
Screening of antimicrobial activity of hydroethanolic extracts and aqueous infusions of P. avium leaves, stems, and flowers was evaluated using several Gram-positive and Gram-negative bacteria. For this purpose, the disk-diffusion method was first performed, L. leaves, stems, and flowers on NO scavenging activity. Griess assay was performed to assess NO scavenging in supernatant. The presented data correspond to the means ± standard deviation of three independent assays and are represented as percentage (%) of control. Statistical analysis: One-way ANOVA was performed for each concentration compared to control; # p < 0.001 was considered significant. The phenolic compounds are natural molecules with a relevant antioxidant potential. Oxidative stress can contribute to the inflammatory process through the activation of several anti-inflammatory mediators. In this context, phenolics appear as quite useful compounds in the inhibition of oxidative stress. The antioxidant properties of P. avium byproducts previously studied [13,21] are associated with the capacity to capture free radicals. The chemical structure of phenolic compounds in sweet cherry leaves, stems, and flowers is capable of transferring hydrogen atoms to radicals, thus reducing their concentration [11]. In addition, the NO scavenging activity of the extracts may help to break the chain of reactions triggered by excessive formation of NO and, thus, prevent the development of diseases, which also favors their use in the treatment of inflammatory disorders.

Antimicrobial Potential
Screening of antimicrobial activity of hydroethanolic extracts and aqueous infusions of P. avium leaves, stems, and flowers was evaluated using several Gram-positive and Gram-negative bacteria. For this purpose, the disk-diffusion method was first performed, which measures the influence of the P. avium extracts on the normal growth of bacteria on agar plates. The larger the diameter without growing bacteria around the paper disc impregnated with the extract, the higher the growth inhibitory properties of the extract. The diameters of the inhibition zones are shown in Table 1. Thus, higher antimicrobial activity against B. cereus (ATCC 11778) was observed with the hydroethanolic extract of stems, followed by stems aqueous infusion (inhibition halo of 7.24 ± 0.14 mm and 4.06 ± 0.2 mm, respectively) ( Table 1). In addition, the hydroethanolic extract of stems was also able to inhibit the growth of S. aureus (ATCC 25923), with an inhibition halo of 3.04 ± 0.16 mm. According to the study conducted by Afonso and co-workers [10], extracts from cherry stems were also found to be more effective, for Gram-positive bacteria. The strain P. mirabilis (CECT 17) was the most resistant to the hydroethanolic extract from leaves, with an inhibition halo of 0.32 ± 0.15 mm. In general, the hydroethanolic extracts of P. avium by-products were those that showed the highest microbial activity against Gram-positive bacteria. These by-products showed almost no antimicrobial activity against Gram-negative bacteria ( Table 1).
The antimicrobial activity of P. avium by-products has been associated with the content of phenolic compounds. Indeed, several authors reported that phenolics, such as p-coumaric acid, p-coumaroylquinic acid derivative, sakuranetin, neochlorogenic and chlorogenic acids, and catechin are some compounds involved in the antimicrobial properties of plant extracts [11,[38][39][40]. Aromatic hydroxyl groups present in many phenolics have high affinity for bacterial membranes, interfering with membrane and cytoplasmic organelles, which may lead to bacterial death [10,41,42].

Effect of P. avium By-Products on NHDF Cell Line
To understand the biocompatibility of P. avium by-product extracts with human cells, the effects of hydroethanolic extracts and aqueous infusions of leaves, stems, and flowers were studied in NHDF cells (Figure 4). The results show that, both extracts from stems increased significantly the cellular viability of these cells ( Figure 4C,D). Although aqueous infusions of leaves and flowers slightly reduced the viability of NDHD, and considering ISO 1093:5-2009, it is possible that the concentrations used did not present cytotoxicity when compared to untreated cells (Control) (Figure 4B,F).
The biological properties of the extracts are related to their composition of phenolic compounds. In the literature, several studies have demonstrated that these phytochemicals, isolated or in synergy, contribute to the elimination of ROS and, consequently to the reduction of oxidative stress, promote the maintenance of healthy skin through collagen production [44][45][46][47]. Detailed knowledge of the chemical composition and biological properties of P. avium by-products in conjugation with its biosafety profile, biocompatibility, and use is essential for biomedical applications [48]. leaves, stems, and flowers on the normal human dermal fibroblast (NHDF) cell line. Cells were plated and exposed to increasing concentrations of extracts for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess cell viability. The presented data correspond to the means ± standard deviation of three independent assays and are represented as percentage (%) of control cells. Statistical analysis: One-way ANOVA was performed for each concentration compared to control; * p value < 0.05, ** p < 0.01, and # p < 0.001 were considered significant. Cells were plated and exposed to increasing concentrations of extracts for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess cell viability. The presented data correspond to the means ± standard deviation of three independent assays and are represented as percentage (%) of control cells. Statistical analysis: One-way ANOVA was performed for each concentration compared to control; * p value < 0.05, ** p < 0.01, and # p < 0.001 were considered significant.
The biological properties of the extracts are related to their composition of phenolic compounds. In the literature, several studies have demonstrated that these phytochemicals, isolated or in synergy, contribute to the elimination of ROS and, consequently to the reduction of oxidative stress, promote the maintenance of healthy skin through collagen production [44][45][46][47]. Detailed knowledge of the chemical composition and biological properties of P. avium by-products in conjugation with its biosafety profile, biocompatibility, and use is essential for biomedical applications [48].

Conclusions
This work is the first to report the anti-inflammatory and antimicrobial activity of leaves, stems, and flowers of P. avium from the Fundão region (Portugal). Both sweet cherry by-products extracts were found to be safe and non-cytotoxic to RAW 264.7 and NDHF cell lines. The hydroethanolic extracts of leaves and stems and the aqueous infusion of flowers showed promising results suppressing inflammation by reducing the production of NO by LPS-activated RAW 264.7 macrophages. In addition, all aqueous infusions of the by-products, especially cherry flowers, showed excellent scavenging activity against NO radicals. In addition, cherry leaves, stems, and flowers were found to be more effective against Gram-positive bacteria. Moreover, the hydroethanolic extract of the leaves and the aqueous infusion of the leaves showed great antimicrobial capacity for most of the tested bacteria. Our results demonstrate, for the first time, that P. avium by-products are a good source of phenolics with relevant anti-inflammatory and antimicrobial potential, highlighting the importance of using these biowastes as bioactive compounds for therapeutic use in inflammation and control the microbial growth.