*2.7. Animal Study*

#### 2.7.1. In Vivo experiment

Male Wistar rats (200–230 g b.w.) were maintained with ad libitum access to food and drinking water for a 12 h light/dark cycle in cages at room temperature with the 55% relative humidity. Rats were divided into two groups: the control (CTR) group (*n* = 5), fed a standard diet (64% carbohydrates, 19% proteins, 7% minerals and vitamins, 6% fibers, and 4% fats; the fats percentage corresponds to the 11% of the diet-derived energy) and the high-fat diet (HFD) group, fed a high fat/cholesterol diet (48.7% carbohydrates, 28% fats, including 2% cholesterol, 13.8% proteins, 4.4% fibers, 5.1% minerals and vitamins, the fats percentage corresponds to the 55% of diet-derived energy). After 5 weeks, the animals of HFD group were treated with a single i.p. injection of streptozotocin (40 mg/kg) [16]. Twenty four rats, resulted to be diabetic with a plasma glucose concentration >250 mg/dL, continued to be fed a HFD diet for a further 4 weeks and were randomly divided into three groups: HFD (n = 8) group, PSF400 (*n* = 8) group, and PSF800 (*n* = 8) group (Figure 1). Rats from CTR and HFD groups were intragastrically administered the same volume of water; rats from PSF400 and PSF800 groups were intragastrically administered lyophilized PSF at different doses (400 mg/kg b.w. and 800 mg/kg b.w., respectively). The weight gain was calculated by initial and final weights. The rats were sacrificed and blood samples were collected by cardiac puncture under general anesthesia. Liver and brain tissues were stored at −80 ◦C. Hepatic lipids were quantified and oxidative stress markers were analyzed in liver and brain. Blood was centrifuged (2300× g for 15 min) to obtain serum samples for laboratory analysis. Local Ethical Committee approved all animal procedures in accordance with the European Communities Council Directive of 24 November, 1986 (86/609/EEC).

**Figure 1.** Study design of the In Vivo experiment. CTR, control group; HFD, high-fat diet group; PSF400, PSF800.

## 2.7.2. Biochemical Analysis

Serum analyses were performed by a semi-automatic analyzer for clinical chemistry (model ARCO, Biotecnica Instruments SPA, Rome, Italy) for aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, and triglycerides. Glucose levels were measured with a glucose meter (Accu-Chek® Roche, Mannheim, Germany), and insulin using a Rat Insulin ELISA kit (Mercodia, Uppsala, Sweden).

#### 2.7.3. Hepatic Lipids Quantification

Fat content of the liver samples was determined by Folch and colleagues protocol [17], slightly modified. Liver samples from rats were homogenized with equal volumes of water and methanol. The resulting homogenate was subjected to three subsequent extractions in chloroform, followed by two washes with KCl 1 M and water. After complete evaporation and prolonged drying of the chloroform, fat content was weighed and expressed as mg/g tissue.

## 2.7.4. Oxidative Stress

Malondialdehyde (MDA) concentration of liver and brain samples was analyzed according to Seljeskog and colleagues [18], with some adaptations. An aliquot of 100 μL of homogenate sample was mixed with 0.1125 N PCA (300 μL) and 40 mM TBA (300 μL) for 10 sec and placed in a boiling water bath for 60 min. Methanol (600 μL) and 20% TCA (*w*/*v*) (200 μL) were added to the suspension and mixed for 10 sec, after cooling in a freezer at −20 ◦C for 20 min. The MDA content was quantified in the supernatant (7000× g for 6 min) by fluorimeter (Perkin Elmer LS-45, Perkin Elmer, Walham, MA, USA) (λex = 525, λem = 560). A standard curve was prepared by dissolving hydrolyzed TEP in water at different concentrations (33.5, 16.8, 8.4, 4.20, 2.10, 1.05, and 0.52 μM). The results have been expressed as nmol MDA/g tissue.

The protein carbonylation was determined using the method adapted from Terevinto and colleagues [19]. Liver and brain samples were homogenized and incubated with 0.02 M DNPH in 2 M HCl. Proteins were then precipitated by adding 20% TCA and recovered by centrifugation (625× g for 10 min). Pellets were washed three times with ethanol:ethyl acetate (1:1, *v*/*v*), melted in 6 M guanidine HCl in 0.02 M KH2PO4 (pH 6.5), and centrifuged. The absorbance of the supernatant was measured at 390 nm. The results have been expressed as nmol/g tissue.

The GSH content in liver and brain samples was evaluated according to Browne and Armstrong [20], with slight modifications. Proteins were then precipitated by adding 10% TCA (*w*/*v*) at 4 ◦C for 30 min. An aliquot of 150 μL of the sample was incubated with an equal volume of *o*–phthaldehyde (1 mg/mL) in 10% methanol (*v*/*v*) by 15 min at 37 ◦C. After centrifugation (625× g for 3 min), fluorescence was measured (Perkin Elmer LS-45, Perkin Elmer, Walham, MA, USA) (λex = 350, λem = 420). A calibration curve has been performed by dissolving GSH in water at different concentrations (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 μM), and GSH concentrations have been calculated as μmol GSH/g tissue.

## *2.8. Statistical Analysis*

The statistical analyses have been performed by Statistical Package for Social Science (SPSS) 17 for Windows (SPSS, Inc., Chicago, IL, USA). The results are shown as the mean value ± standard deviation (s.d.) and analyzed through a one-way ANOVA and Tukey's test for post-hoc with significance at *p* ≤ 0.05.

#### **3. Results and Discussion**

#### *3.1. Quantification of Total Polyphenols*

The TPC of wild Italian blackthorn fruit extract was quantified by a spectrophotometric method, and the content was 5.50 ± 0.19 mg GAE/g d.w. To our knowledge, any other results have been found about total phenolic content of blackthorn fruit on dry weight, but some authors reported that TPC in blackthorn fruit on fresh weight ranged from 0.42–4.13 mg GAE/g [21–23].

The HPLC-DAD method validation was estimated by quantifying the limit of detection (LOD), limit of quantification (LOQ), and the recovery. All parameters indicate that the method exhibits a good sensitivity for identification as well as quantification of the polyphenols. All the parameters are listed in Table 1.

**Table 1.** Retention time (Rt), LOD (limit of detection), LOQ (limit of quantification), and recovery of phenolic compound quantification method by HPLC-DAD in the *Prunus spinosa* L. fruit (PSF) aqueous extract (*n* = 3).


The quality of phenolic profile of wild Italian blackthorn and the concentrations of single compounds are shown in Table 2. Rutin (183.94 mg/kg d.w.) was the principal phenolic component, followed by 4-hydroxybenzoic acid, gallic acid, trans-sinapic acid, quercetin, trans-ferulic acid, caffeic acid, rosmarinic acid, trans cumaric acid, genistin, and myricetin. Our findings are partially in accordance with those of some other authors that showed considerable quantities of phenolic acids (quercetin and caffeic acid) in blackthorn fruits from Southeast Serbia [3,24]; by contrast we did not find either neochlorogenic or kaempferol. HPLC-UV analysis of the methanolic extract of fresh blackthorn plums from Turkey recently allowed Baltas and colleagues to detect five phenolic acids, namely protocatechuic acid, p-OH benzoic acid, vanillic acid, syringic acid, and p-coumaric acid, as well as flavonoids, such as epicatechin and luteolin [25]. Another recent study about quantification of phenolic compounds by HPLC-UV in methanolic extract of frozen blackthorn fruits from Romania showed high chlorogenic and neochlorogenic acid levels, followed by glycosides of quercetin [23]. Considering that the solubility of polyphenols in solvent of different polarity is determined by their structure, different types of extraction solvent and procedures may influence the efficiency of phenolic compounds extraction and their resultant content [3].


**Table 2.** Concentrations of phenolic compounds in the PSF aqueous extract.
