3.2.9. Prostate-Specific Anti-Inflammatory Activity

The prostate-specific anti-inflammatory activity of the bioaccessible fraction of tested formulations was evaluated in a prostatic epithelium in vitro model, based on tumoral prostatic cells (LNCaP). The prostate-specific anti-inflammatory activity was evaluated pre-treating the in vitro model for 2 h with the bioaccessible fractions corresponding to SRO concentrations reported in Table 8, and then exposing the model to inflamed conditions for 4 h. In particular, prostatic epithelium in vitro model was exposed either to normal monocytic/macrophage cell culture medium (uninflamed condition) or THP-1 cell culture conditioned medium (CM, inflamed condition).

**Table 8.** Absorption rate expressed as a percentage of SRO absorption ± standard error (SE) and concentration (μg/mL) at the basolateral compartment (serosal).


CM was obtained by stimulating overnight PMA-differentiated THP-1 cells with the pro-inflammatory compound LPS (1 ng/mL). LNCaP cells in normal monocytic/macrophage cell culture medium and CM were used as negative and positive controls of inflammation respectively. Diclofenac, a well-known anti-inflammatory drug, was used as a positive control of anti-inflammatory activity. A dose-response curve was performed with MTS assay to determine Diclofenac highest non-lethal concentration on prostatic epithelium in vitro model after 6 h exposure. Similarly, the impact of bioaccessible fractions on LNCaP viability was checked by using MTS assay and morphology. At the end of the treatment, in vitro prostate epithelia were washed with pre-warmed DPBS and detached in ice-cold PBS with a cell scraper. Following centrifugation (1000× *g* for 5 min), LNCaP cells were resuspendend in lysis buffer (protease inhibitor cocktail and 0.1% Triton X-100 in deionized water) and sonicated (5 s pulse-on at 10% amplitude and 25 sec pulse-off, total time 1.5 min) (Sonicator Q700, QSonica, Newtown, CT, USA). Finally, cell lysates were centrifuged for 15 min at 10,000× *g* and the surnatants recovered. Anti-inflammatory activity was determined by measuring the amount of pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) present in cell lysates. IL-1β and TNF-α were quantified by commercial ELISA (Enzyme-Linked Immunosorbent Assay) kits, following the manufacturer's instructions.

Anti-inflammatory effect of bioaccessible fractions was also evaluated in cell lysate by measuring cyclooxygenase (COX) enzyme 1 and 2 activity. Total COX and COX-2 activities were assessed with a commercial COX activity assay kit, following the manufacturer's instructions.

### 3.2.9.1. SRO Pro-Apoptotic Activity

To evaluate the pro-apoptotic activity of the bioaccessible fractions of SRO, a fluorimetric assay, based on caspases 3/7 activation, was performed (Apo-ONE® Homogeneous Caspase-3/7 Assay). This assay is based on the ability of activated caspases 3 and 7 to selectively cleave a specific substrate, making it fluorescent (excitation wavelenght 499 nm, emission wavelength 521 nm). Consequently, the produced fluorescence intensity is linked to the activation of the apoptotic process by cells. To correlate anti-inflammatory with pro-apoptotic activity, the same experimental setup described above for the anti-inflammatory activity was applied. Experiments were performed in triplicate and the assay conducted following manufacturer's instructions.

#### 3.2.9.2. Smooth Muscle Myorelaxing Activity

The myorelaxing activity of the bioaccessible fraction of tested formulations was evaluated in an in vitro model of smooth muscles, based on WPMY-1 human myofibroblast stromal cell. The myorelaxing effect was analyzed by means of a two-step gel-contraction assay, following the manufacturer's instructions. The gel contraction assay is characterized by two phases: mechanical stress-generating phase and floating phase. During the first phase, a mix of myofibroblast and collagen is seeded and left to develop mechanical stress for two days while, during the floating phase, gels are released and allowed to freely contract, dissipating the mechanical stress generated during the first phase. Fibroblast-containing gels were exposed to the bioaccessible fraction of the formulations for the duration of the experiment (24 h). The myorelaxation activity was calculated with the following formula:

$$\text{Myorelaxing Activity} \left( \% \right) = 100 - \left( \left( \text{C}\_{\text{expposed}} / \text{C}\_{\text{control}} \right) \times 100 \right) \tag{2}$$

where Cfibroblast is the contraction of the fibroblast-containing gels exposed to the bioaccessible fraction of the different formulations and Ccontrol the contraction of the untreated fibroblast-containing gel (positive control of contraction).

Gel contraction is calculated as follows:

$$\text{Contrraction} \left( \% \right) = 100 - \left( \text{( $D\_{\text{fibroblast}}$ ' $D\_{\text{colligen}}$ )} \times 100 \right) \tag{3}$$

where Dfibroblast represents the diameter of the fibroblast-containing gel and Dcollagen the diameter of the collagen gel in which fibroblast were not seeded (negative control of contraction). Experimental and control cells were plated in triplicate and the diameter of each collagen gel was photographed and measurement via images analysis, performed with ImageJ (University of Wisconsin-Madison, Madison, WI, USA).

#### 3.2.9.3. Measurement of Prostate Specific Antigen (PSA) Secretion by LNCaP Prostatic Cells

The effect of the different formulation bioaccessible fraction on the secretion of the androgen-induced prostate-specific antigen (PSA), commonly used as a marker for prostate tumors, was evaluated in LNCaP prostatic cells. After seeding and adhesion (48 h), normal cell culture medium was replaced with a medium containing low hormone level for 24 h (10% charcoal-stripped FBS and 1% Penicillin-Streptomycin mix in RPMI without phenol red). The pre-treatment medium was then removed and cells were incubated with the bioaccessible fraction of each formulation. A control was included in the assay by treating cells with low-hormones cell culture medium. Additionally, control and cells treated with LBF bioaccessible fraction were stimulated with dihydrotestosterone (DHT) (10 nM), known to stimulate the release of PSA [38]. After 24 h exposure, media were collected for measurement of secreted PSA using a commercial ELISA kit (Abcam), following the manufacturer's instructions. Results were expressed as percentage of secreted PSA in cells treated with different formulations compared to control.
