*3.2. Antimicrobial Activity*

The antimicrobial activity on selected Gram-negative (Figure 2A) and Gram-positive (Figure 2B) enteric bacteria was measured by evaluating the growth inhibition by increasing concentrations of PSF extract. The antimicrobial activities have been compared with the standard antibiotics, used as positive controls.

The lowest concentration of tested PSF extract (0.25 mg/mL) inhibited more than 50% of the Gram-negative bacteria *Escherichia coli* (70.19% ± 1.21%), *Salmonella typhimurium* (79.98% ± 0.54%), and *Enterobacter aerogenes* (83.02% ± 0.54%) growth (Figure 2A). The same concentration (0.25 mg/mL) was able to inhibit more than 50% of the Gram-positive bacteria *Enterococcus faecalis* (82.86% ± 1.94%) and *Staphylococcus aureus* (79.92% ± 1.23%) growth (Figure 2B). The antimicrobial activity of phenolic compounds occurring in plant foods has been widely studied against a wide range of microorganisms. The damage to the bacterial membrane and suppression of some virulence factors, including enzymes and toxins, are suggested to be the mechanisms of their antimicrobial action [26]. Some flavonoids (rutin, myricetin, and quercetin) and phenolic acids (gallic, caffeic, and ferulic acids) of PSF extract may be responsible for its antibacterial action [27,28].

**Figure 2.** Growth inhibition effect of PSF extract (0, 0.25, 0.50, 0.75, and 1.00 mg/mL) against Gram-negative bacteria (**A**) (*Escherichia coli* ATCC 25922, *Salmonella enterica* ser. *typhimurium* ATCC 14028, and *Enterobacter aerogenes* ATCC 13048) and Gram-positive bacteria (**B**) (*Enterococcus faecalis* ATCC 29212 and *Staphylococcus aureus* ATCC 25923). Significantly different from negative control (PSF 0 mg/mL): \*\*\* *p* ≤ 0.001. Results are reported as means (*n* = 3) values ± standard deviation.

#### *3.3. In Vitro Antioxidant Activity*

As shown in Figure 3A, pretreated erythrocytes with PSF aqueous extract (100 mg/mL) exhibited a significantly higher cellular antioxidant activity (CAA unit = 48.43 ± 1.68) compared with untreated cells (CAA = 0; *p* ≤ 0.001), comparable to 100 μM Trolox (CAA unit = 16.52 ± 3.60; *p* ≤ 0.001) and 500 μM Trolox (CAA unit = 36.67 ± 1.48; *p* ≤ 0.001). Taking these results into consideration, the EC50 of PSF extract for antioxidant activity in red blood cells was 100 mg/mL.

The antihemolytic activity of PSF extract was screened in erythrocytes exposed to high doses of AAPH, causing a strong oxidative hemolysis. Figure 3B shows that PSF extract exerted a significant inhibition of AAPH-induced hemolysis compared with the control erythrocytes (AAPH-treated). PSF extract (100 mg/mL) pretreated cells demonstrated a marked antihemolytic effect (84% hemolysis inhibition) compared with AAPH-treated cells (*p* ≤ 0.001), with a reduction of the hemolysis similar to that of the highest concentration of the reference standard (500 μM Trolox). The antihemolytic EC50 of PSF extract was 10 mg/mL (data not shown). We found that PSF exerted a potent ROS-scavenger activity. Indeed, when intact human erythrocytes were pre-incubated with a PSF aqueous extract, a strong protective effect against AAPH-generated ROS production and hemolysis was observed. These antioxidant and antihemolytic effects of PSF are probably due to the activity of gallic acid, rutin, and quercetin in red blood cell [29,30].

**Figure 3.** (**A**) Effects of PSF extract (100 mg/mL) on cellular antioxidant activity (CAA) in human erythrocytes. Significantly different from untreated cells (CAA unit = 0): \*\*\* *p* ≤ 0.001. (**B**) Effects of PSF extract (100 mg/mL) on dihydrochloride (AAPH)-induced oxidative hemolysis in human erythrocytes. Significantly different from CTR (AAPH-treated cells): \*\*\* *p* ≤ 0.001. Trolox was used as reference standard. Results are reported as means (*n* = 3) values ± standard deviation.

#### *3.4. In Vivo Experiment*

#### 3.4.1. The Effect of Blackthorn on Body Weight and Liver Weight

In comparison with CTR group, rats of the HFD group exhibited a significant lower final body weight (396.8 ± 40.6 vs. 307.5 ± 23.3 g/rat, respectively) (*p* ≤ 0.001). The administration of PSF did not induce significant changes in the final body weight, neither in PSF400 group (317.2 ± 27.6 g/rat), nor in PSF800 group (312.7 ± 42.5 g/rat), when compared with HFD group.

However, when compared with CTR rats, HFD rats exhibited a statistically significant increase in liver weight (8.9 ± 1.4 vs. 13.4 ± 1.2 g, respectively) (*p* ≤ 0.001) and in relative liver weight (2.2 ± 0.2 vs. 4.1 ± 1.0 g liver/100 g b.w., respectively) (*p* ≤ 0.001). No significant difference in liver weight was found between HFD-fed rats and PSF-treated rats of PSF400 group (14.8 ± 1.7 g) and PSF800 group (14.6 ± 1.8 g). The same trend was found in relative liver weight between HFD-fed rats and PSF-treated rats of PSF400 group (4.5 ± 0.3 g liver/100 g b.w.) and PSF800 group (4.8 ± 0.7 g liver/100 g b.w.). HFD treatment caused hepatic lipid accumulation and increased liver weight and all the biochemical

parameters in serum [16,31]. However, the PSF extract did not improve the liver weight and serum and liver biochemical parameters linked to steatosis.

#### 3.4.2. The E ffect of PSF on Serum and Liver Biochemical Parameters

Serum AST, ALT, glucose, total cholesterol, triglycerides, and total hepatic lipid content were significantly higher in the HFD group compared with the normal diet group (CTR group), while serum insulin was significantly lower. After four weeks of treatment with 800 mg of PSF/kg b.w., rats of the PSF800 group showed a significant decrease of total hepatic lipids content compared with HFD group (162.15 ± 35.52 vs. 209.90 ± 11.91; *p* ≤ 0.05) (Table 3). Some studies have demonstrated that polyphenols decrease the hepatic lipid accumulation caused by high-fat diet [32]. Moreover, it was also reported that the single isolated polyphenol can improve the high liver lipids content due to a high-fat diet administration, as in the case of rutin [33], gallic acid [34], and quercetin [35]. The crude extracts can be more advantageous than the isolated components, since a single bioactive molecule can change its properties with the presence of other compounds in the extracts [36].

**Table 3.** Nutritional e ffect of PSF on biochemical parameters in serum and liver of rats (*n* = 7).


Analyses were performed through one-way ANOVA and Tukey's test for post-hoc. \*, *p* ≤ 0.05 vs. CTR; \*\*, *p* ≤ 0.01 vs. CTR; \*\*\*, *p* ≤ 0.001 vs. CTR; §, *p* ≤ 0.05 vs. HFD. AST, aspartate aminotransferase; ALT, alanine aminotransferase.

#### 3.4.3. The E ffect of PSF on Liver and Brain Oxidative Stress of Rats

The high-fat diet was probably responsible for the decrease of GSH (Figure 4A) content and the increase of protein carbonylation (Figure 4C) and MDA (Figure 4E) levels in the liver samples of HFD rats, compared with the CTR group. Moreover, while hepatic GSH content was not a ffected by PSF treatment (Figure 4A), administration of PSF improved the oxidative stress status of rats according to the protein carbonylation, at the higher concentration of treatment (800 mg/kg b.w.) (Figure 4C), and to the MDA content, in a dose-dependent manner (Figure 4E). It has been demonstrated that plant polyphenols are related to the improvement of hepatic oxidative stress caused by a high-fat diet through the e activation of Nrf2 transcription factor, which increases expression of antioxidant enzymes [37]. Moreover, it was reported that even the single polyphenol, if isolated, can improve the high-fat-diet-induced hepatic oxidative stress, as in the case of rutin [33] and gallic acid [38].

In comparison with the CTR group, HFD treatment promoted an increase of the brain oxidative stress parameters in rats, as shown by protein carbonylation (Figure 4D) and MDA assay (Figure 4F). The addition of 400 mg/kg b.w. and 800 mg/kg b.w. of PSF to the diet reversed the e ffect caused by the high-fat diet and, in particular, the MDA assay showed a dose-dependent response pattern. Nevertheless, both the HFD and the PSF treatments did not induce significant changes in rat brain GSH levels (Figure 4B). The intake of a high-fat diet is linked to an increased risk of neurodegenerative disease related to diabetes [39]. Considering that, the polyphenols-rich fruits could protect neurons against the oxidative stress induced by intake of saturated fatty acids [40]. Recently, Nabavi and colleagues demonstrated that gallic acid exerts a neuroprotective e ffect against sodium fluoride-induced oxidative stress in rat brain [41]. Moreover, it has been shown that other polyphenols contained in PSF, such as rutin, ferulic acid, and trans-sinapic acid, should contribute to the prevention of brain oxidative stress in rats [42–44].

**Figure 4.** Effect of PSF treatment at two different concentrations (PSF400 and PSF800) on GSH content of liver (**A**) and brain (**B**), protein carbonylation of liver (**C**) and brain (**D**), and malondyaldeide of liver (**E**) and brain (**F**). Results are reported as means (*n* = 7) values ± standard deviation. Values within each row of different letters are significantly different (*p* ≤ 0.05), *p* ≤ 0.05 vs. CTR; \*\*, *p* ≤ 0.001 vs. CTR; --, *p* ≤ 0.01 vs. HFD; ---, *p* ≤ 0.001 vs. HFD.

Our findings sugges<sup>t</sup> an improved liver and brain antioxidant defense in rats treated with PSF.
