**1. Introduction**

Considering that traditional foods are increasingly believed healthy and wholesome, food manufacturers are developing new food products returning to natural products and traditional recipes that will be attractive to the widest potential consumers [1,2]

Blackthorn (*Prunus spinosa* L.), which belongs to the *Rosaceae* family, is a perennial plant originally growing in temperate continental climate of the northern hemisphere, particularly widespread in the Mediterranean countries and in the southeast regions of Italy. It is used for treatment of many diseases due to its diuretic, spasmolytic, antimicrobial, and antioxidant activities [3]. Moreover, *Prunus spinosa* L. fruit (PSF) is used for the production of various traditional jams and beverages such as juice, wine, tea, and distillates in food industry [4]. It contains substantial quantities of phenolic antioxidants, including, in particular, flavonols, phenolic acids, and coumarin derivatives [5].

Epidemiological investigations demonstrated that diets rich in plant polyphenols protect against diabetes, osteoporosis, cardiovascular, and neurodegenerative diseases [6]. Dietary compounds and specific polyphenol-rich foods could have a pivotal function in the prevention of diseases associated with oxidative stress by increasing the circulation of antioxidant compounds and neutralizing the reactive oxygen species, due to their number and position of hydroxyl groups [7]. Protein nitration, lipid peroxidation, chronic inflammation, and oxidative damage to DNA may be prevented by polyphenols, which results in vasodilatory, vasoprotective, anti-atherogenic, antithrombotic, and anti-apoptotic effects, as free radical scavengers, metal chelators, inhibitors of pro-inflammatory enzymes, and modifiers of cell signaling pathways [8].

Recently, new alternatives have become desirable, and plants metabolites have been screened for antimicrobial agents for treatment of infectious diseases, due to the development of antibiotic resistance by pathogenic bacteria [9]. For instance, in several studies, dietary polyphenols have been reported to exert an antibacterial activity [10].

In the present study, the potential biological activities of wild blackthorn fruit from southeast regions of Italy were investigated. Considering the PSF as a potential natural source of phenolic compounds, this work was designed to study its in vitro antimicrobial, antioxidant, and antihemolytic activities. Moreover, for the first time, the In Vivo protective e ffect and antioxidant capacity of PSF in high-fat diet (HFD) and streptozotocin (STZ)-induced oxidative stress has been studied.

#### **2. Materials and Methods**

Blackthorn fresh fruits were obtained from wild orchards of the Campobasso (Italy) area in October 2015. Taxonomic identification of plant material was confirmed by Prof. Elisabetta Brugiapaglia from Department of Agricultural, Environmental and Food Sciences, University of Molise, Campobasso, Italy.

#### *2.1. Chemicals and Reagents*

All solvents and chemicals were of analytical grade. Nutrient Broth (NB), Nutrient Agar (NA), Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), McFarland standard were purchased from Oxoid (Basingstone, UK). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), dinitrophenylhydrazine (DNPH), trichloroacetic acid (TCA), perchloric acid (PCA), thiobarbituric acid (TBA), 1,1,3,3-tetramethoxypropane (TEP), guanidine hydrochloride, ortho-phthalaldehyde (OPA), reduced glutathione (GSH), phosphoric acid, potassium dihydrogen phosphate (KH2PO4), hydrochloric acid (HCl), streptozotocin (STZ), ethanol, ethyl acetate, and methanol from Sigma-Aldrich (St. Louis, MO, USA). All HPLC analytical standards, including protocatechuic acid, syringic acid, rutin, ellagic acid, cynaroside, daidzein, neochlorogenic acid, chlorogenic acid, vitexin, trans *p*-coumaric acid, trans-sinapic acid, trans ferulic acid, rosmarinic acid, resveratrol, apigenin, myricetin, quercetin, and kaempferol, were bought from Sigma-Aldrich (St. Louis, MO, USA). Phosphate bu ffer saline (PBS) was bought from VWR (Radnor, PA, USA).

#### *2.2. Plant Material Preparation*

After the pits were removed, the frozen fruits were lyophilized and crushed in a mortar, and the powder was stored at −20 ◦C. Briefly, 1 g of sample was extracted with 10 mL of water for 2 h on a horizontal shaker Unimax 2010 (Heidolph Instruments, GmbH, Schwabach, Germany) for in vitro antioxidant activity, polyphenols quantification, and antimicrobial activity. PSF extracts were centrifuged (2300× g at 4 ◦C for 20 min) (Jouan CR3i centrifuge, Newport Pagnell, UK), and the supernatants were collected.

For the In Vivo experiment, the lyophilized and powdered fruit was dissolved in water.

#### *2.3. Total Phenolic Content (TPC) and Polyphenols Quantification by HPLC-DAD*

For the determination of total phenolic content in water extract, we followed the Singleton and colleagues protocol [11]. The concentration of polyphenols was expressed as mg of gallic acid equivalents (GAE)/g of dry weight (d.w.).

Prior to HPLC analysis, the extracts were filtered through syringe filters Q-Max (0.22 μm, 25 mm, PVDF) (Frisenette ApS, Knebel, Denmark) into the HPLC vials. The HPLC apparatus consisted of an Agilent 1260 Infinity HPLC (Agilent Technologies GmbH, Waldbronn, Germany) quaternary solvent manager coupled with degasser (G1311B), sampler manager (G1329B), Diode Array Detector (G1315C), column manager (G1316A). The analytical column was a Waters Cortecs endcapped RP-C18 column (150 mm × 4.6 mm × 2.7 μm particle size; Waters Corp., Milford, MA, USA). The analyses were carried out at 30 ◦C by a gradient system with a mobile phase of 0.1% ortho-phosphoric acid in deionised water (C) and acetonitrile gradient grade (D) at a flow rate of 0.60 mL/min, and the injection volume was 5 μL. The gradient elution was as follows: 0–1 min (90% C and 10% D), 1–5 min (85% C and 15% D), 5–10 min. (80% C and 20% D), 10–12 min. (80% C and 20% D), 12–20 min (30% C and 70% D), and 20–25 min (30% C and 70% D). The post-run was set at 3 min. The samples were kept at 4 ◦C in the sampler manager. The detection wavelengths were set at 265 nm (gallic acid, 4-hydroxibenzoic acid, rutin, and genistein), 320 nm (chlorogenic acid, ca ffeic acid, trans-*p*-coumaric acid, trans-sinapic acid, trans-ferulic acid, rosmarinic acid, resveratrol), and 372 nm (myricetin, quercetin and kaempferol). Data were analyzed by Agilent Open Lab Chem Station software for LC 3D systems.

#### *2.4. HPLC-DAD Method Validation*

Reference phenolic compounds were dissolved in HPLC purity methanol and diluted to appropriate concentration ranges (5–50 μg/mL). The linearity of each calibration curve was assessed by linear regression analysis. The limit of detection (LOD) and quantification (LOQ) were estimated by measuring signal-to-noise ratio of the individual peak of each standard compound. The LOD and LOQ were calculated according to the International Conference on Harmonisation guidelines [12].
