*2.8. Statistical Analysis*

Statistical analysis has been carried out using GraphPad Prism, version 6.00 for Windows (GraphPad software, San Diego, CA, USA). Results were expressed as mean values ± standard deviation (SD) of analysis of data deriving from extractions and assays made in triplicate. Di fferences between samples were analyzed by one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test; *p*-value lower than 0.05 was considered statistically significant.

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

#### *3.1. Polyphenolic Composition of Opuntia ficus-indica Cladode Extracts*

The polyphenolic extracts from immature and mature cladodes revealed a predominant presence of piscidic and eucomic acids. These phenolic acids were identified upon their retention time, UV-Visible spectra and fragmentation pattern; also their mass spectral characteristics (by TOF LC/MS) were compared with the available literature [18,19] (Figure 1).

**Figure 1.** High performance liquid chromatography time-of-flight mass spectrometry (HPLC/MS-TOF) chromatogram of *Opuntia ficus-indica* extract from immature cladodes (*imm*). The chromatographic profile of mature cladode ( *m*) extract was similar, except for isorhamnetin glucoside (peak 9), only present in *imm*. The numbers indicating peaks refer to the identified compounds reported in Table 1.

The presence of piscidic acid and eucomic acid in *Opuntia* cladode has been reported [18,20]; others reported the occurrence of hydroxybenzoic acids derivatives (2- or 4- or 3,4-dihydroxy benzoic acids) [21].

Δ In the mature cladodes, we found a higher content of both piscidic and eucomic acids in respect to the value reported (on a similar development stage) by [18], threefold and tenfold, respectively (Table 2).


**Table 1.** Identification of phenolic acids and flavonols in *Opuntia ficus-indica* extracts (from *imm* and *m*) by means of High performance liquid chromatography time-of-flight mass spectrometry HPLC/TOF (M − H)<sup>−</sup>.

**Table 2.** Phenolic acids and flavonols content of *Opuntia ficus-indica* extracts, from immature and mature cladodes. Phenolic acids were quantified using the calibration curve of *p*-hydroxybenzoic acid. Flavonols were determined using calibration curves with the closest appropriate standard (rutin, isorhamnetin glucoside or isorhamnetin rutinoside). Mean values (*n* = 3) ± standard deviation (SD) are expressed as dry weight (DW) or fresh weight (FW). The same letters (*a–h*) in the column indicate that mean values are not significantly different (*p* < 0.05).


In the immature cladodes, we found a much higher value, as sum of piscidic and eucomic acids, even if the ratio is inverted: in immature cladode the predominant form is eucomic acid, instead in mature cladode piscidic acid is predominant (Table 2). Anyway, the amount of piscidic and eucomic acids found in *O. ficus-indica* cladode, particularly at immature stage, is exceptionally high.

Flavonol compounds (mainly isorhamnetin derivatives and rutin) were also identified (Figure 1 and Table 1). Isorhamnetin derivatives and rutin have been already described to be present in cladode extracts of di fferent *Opuntia* species [18–23]. In those papers, isorhamnetin was reported as predominant core aglycone for flavonoids present in *Opuntia* sp., with low occurrence of quercetin and kaempferol. We confirmed these findings (Figure 1 and Table 2).

The content of isorhamnetin glucoside in our immature sample was similar to that one reported by Guevara-Figueroa and co-workers [21], in a wild variety (Amarillo) of *Opuntia ficus-indica*, instead our reported content of narcissin was much higher, as well as the rutin content (Table 2). In a mature cladode sample, the content of isorhamnetin derivatives (totaling 1.76 mg/g DW) was similar to that one reported in the above study (1.69 mg/g DW) [21]. The total flavonols content in our immature samples (6.78 mg/g DW) was in agreemen<sup>t</sup> with the content (8.82 mg/g DW) reported in young cladodes of *Opuntia ficus-indica* [23]. In mature sample, the total flavonols content (totalling 2.51 mg/g DW) was again in agreemen<sup>t</sup> with [21] who found ~3.5 mg /g DW.

Isorhamnetin derivatives, piscidic and eucomic acids present in such a high amount in cladode of *Opuntia ficus-indica* indicate that nopal can be considered a promising plant for the development of polyphenol-based commercial products. The e ffect of these phenolic compounds has been evaluated in vitro for contrasting hypercholesterolemia [24], and against UVA-induced oxidative stress on human keratinocytes [25], suggesting a possible pharmaceutical use of cladode extracts.

#### *3.2. In Vitro Antioxidant Activity*

Cladode extracts analysis revealed a much higher total phenol content, as well as antioxidant capacity, at immature stage of development, than mature cladode (Table 3). Therefore, the habit of Mexican population to eat 'nopalitos' (that is young prickly pads of 3–4 weeks of age) relays on a scientific basis of higher health benefit.

**Table 3.** Total phenols and antioxidant capacity, expressed as Oxygen Radical Absorbance Capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC), of *Opuntia ficus-indica* extracts, from immature and mature cladodes. Total phenols are expressed as gallic acid equivalents (GAE), TEAC and ORAC assays are expressed as Trolox Equivalent (TE). Mean values (*n* = 11) ± standard deviation (SD) are expressed on a dry weight (DW) or fresh weight (FW) basis. The same letters (a,b) in the same column indicate that mean values are not significantly di fferent (*p* < 0.05).


Total phenol content (Table 3) was higher than that one reported in some studies [21,26] but in agreemen<sup>t</sup> with [23] (for immature cladode), and similar to that one reported in mature cactus pads from Tenerife [27] (for mature cladode); in fact, the phenol level results higher in immature cladodes if compared with mature, e.g., about 4 g/kg in our immature cladode samples from Apulia, and 2.6 g/kg in mature cladodes as reported by Rocchetti et al. for Sicilian *Opuntia ficus-indica* [26].

The antioxidant capacity measured by ABTS assay resulted higher for immature cladode than mature (12.5 vs 8.2 μmol TE/g DW, respectively) (Table 3). Only one study dealt with TEAC of cladode extracts reporting a several-fold higher value [28]. The ORAC values for our immature and mature cladode (Table 3) agreed with the literature: ~770 μmol TE/g DW [28]; a range from 260–380 μmol TE/g DW, in young pads [19]; 657 μmol TE/g DW [29]. The ORAC value for immature cladode extracts was comparable to the value reported for blueberry; instead, that one for mature cladode was comparable to strawberry/raspberry values [30].

Therefore, from the experiments assessing the antioxidant capacity of cladodes extracts, we can conclude that both immature and mature cladodes are a good source of antioxidant functional compounds.

#### *3.3. Ex Vivo Antioxidant Activity*

3.3.1. Cellular Antioxidant Activity Assay in Red Blood Cells (CAA-RBC)

As shown in Figure 2A, cladodes pre-treated erythrocytes exhibited a significantly higher cellular antioxidant activity (CAA unit = 30 ± 1) compared to untreated cells (control, CAA = 0; *p* < 0.01), but lower than Trolox (CAA unit = 45 ± 0.79) used as standard. The cellular antioxidant activity of mature and immature cladodes was similar for both samples (30 ± 1 and 29 ± 0.9, respectively).

**Figure 2.** (**A**) Effects of *Opuntia ficus-indica* cladode extracts on the cellular antioxidant activity (CAA) in human erythrocytes. Trolox was used as reference standard. (**B**) Effects of *Opuntia ficus-indica* cladodes extracts on 2,2-Azobis (2-methylpropionamidine) dihydrochloride (AAPH)-induced hemolysis in human erythrocytes. Trolox was used as reference standard. Assays were carried out in triplicate and the results were expressed as mean values ± SD. Different letters indicate significant differences (*p* ≤ 0.05). One-way ANOVA with Bonferroni's multiple comparison test.

## 3.3.2. Haemolysis Assay

Since erythrocyte is a unique cell model with simple metabolism and sensitivity to oxidation, oxidative haemolysis is widely used to evaluate antioxidant activity. Therefore, cladode extracts were tested on human erythrocytes to counteract the oxidative hemolysis induced by peroxyl radicals produced by AAPH thermal decomposition. The anti-hemolytic activity of cladode extracts was compared with that from erythrocytes exposed to AAPH alone (control). As shown in Figure 2B both *Opuntia* cladodes extracts exhibited a strong anti-hemolytic e ffect (about 80%). This e ffect was statistically significant (*p* ≤ 0.001) compared to the control (that induced 100% of hemolysis) but lower than the Trolox (about 90% hemolysis inhibition). No statistically significant di fferences were found between mature and immature cladodes anti-hemolytic e ffects.

## *3.4. Antimicrobial Activity*

The antimicrobial activity against selected enteric bacterial strains was measured evaluating the O.D. at 600 nm in the presence of increasing doses of cladode extract. Di fferent dilutions of methanol did not a ffect the bacterial growth (data not shown).

Gram-negative microorganisms (*Escherichia coli* ATCC 25922, *Salmonella typhimurium* ATCC 14028 and *Enterobacter aerogenes* ATCC 13048) were inhibited at a concentration of 2000 μg/mL of mature cladode extract, whereas immature cladode extract was more e ffective inhibiting at a concentration of 1500 μg/mL (Table 4). The two Gram-positive bacteria (*Staphylococcus. aureus* ATCC 25923 and *Enterococcus. faecalis* ATCC 29212) showed MIC values of 1500 μg/mL for mature and 1000 for immature cladode extract (Table 4). The MIC against planktonic cells of the biofilm producer *Staphylococcus aureus* ATCC 35556 was 1000 and 700 μg/mL for mature and immature extracts, respectively (Table 4).


**Table 4.** Minimal inhibitory concentrations (MIC) of *Opuntia ficus-indica* mature ( *m*) and immature cladode extracts (*imm*) against selected bacterial strains.

Our results agree with several other studies showing that the inhibitory e ffect of phenolic compounds from natural extracts are more e ffective to Gram-positive than Gram-negative bacteria [7]. The susceptibility of bacteria to drugs depends on the characteristics of the drug (hydrophobicity or hydrosolubility) and on the microbial membrane composition [31].

The antimicrobial activity of plant phenolics has been extensively investigated against many di fferent microorganisms [6]. We previously reported the antimicrobial activity of bergamot whole-fruit powder against potentially pathogenic bacteria [32]. In general, phenolic compounds are involved in membrane damage, protein and cell wall binding and enzyme inactivation; they can act as pro-oxidants, leading to damage of DNA, lipids and other biological molecules [33].

Few authors have reported the antimicrobial activity of *Opuntia ficus-indica*. Ginestra and co-workers [18] reported that di fferent phytochemical fractions of *Opuntia ficus-indica* did not show antimicrobial activity against the tested bacterial strains. On the other hand, it has been reported the antimicrobial activity of alcoholic and aqueous extracts of *Opuntia* cladodes against *Vibrio cholerae* and *Proteus mirabilis* [34]. Moreover, other authors [35] described the antimicrobial activity of *Opuntia*

cladodes against *Escherichia coli* and *Staphylococcus aureus*, with a minimum bactericidal concentration (MBC) of 4 mg/mL and 1 mg/mL, respectively.

The antimicrobial activity of *Opuntia* cladodes extracts may be related to its high content of polyphenols, especially isorhamnetin that has been already reported exerting antimicrobial activity [36].
