**3. Material and Methods**

#### *3.1. Samples*

PollenAid Plus soft gel capsules were kindly provided by IdiPharma (Catania, Italy). The formulation contains Graminex G60™ Flower Pollen Extract (45.86%), NAX™ 7% paste (2.29%), soy lecithin (3.06%), yellow beeswax (2.93%), and extra virgin olive oil (45.86) as the vehicle. All the ingredients are contained in oval dark amber capsules. Graminex G60TM Flower Pollen Extract is a water-soluble extract of rye pollen grown in the USA. It is standardized to 6% amino acids and exhibits both antioxidant and anti-inflammatory activities. It is also non-allergenic, solvent-free, vegan, and non-GMO. Graminex G60™ Flower Pollen Extract was studied in our previous paper [17]. NAX™ 7% paste is a lipidsoluble paste of rye pollen grown in the USA. It is standardized to 7% phytosterols including β-sitosterol plus essential fatty acids such as ω-3 and ω-6. Applications include women's health, skin health, and heart health, and it can be directly formulated into soft gels. It is non-allergenic, solvent-free, vegan, and non-GMO. The extraction of this formulation was carried out by diluting 100 μL in 1 mL of a hydroalcoholic solution, constituted by 500 μL of ultrapure water and 500 μL of methanol. Subsequently, ultrasound-assisted extraction (UAE) was carried out. The operative conditions were 60 ◦C for 20 min at full power.

### *3.2. HPLC-DAD-MS*

The identification and quantification of phenolic compounds were conducted through HPLC-DAD-MS analysis. The HPLC apparatus consisted of two PU-2080 PLUS chromatographic pumps, a DG-2080-54 line degasser, a mix-2080-32 mixer, UV, diode array (DAD) detector, a mass spectrometer (MS) detector (expression compact mass spectrometer (CMS), Advion, Ithaca, NY, USA), an AS-2057 PLUS autosampler, and a CO-2060 PLUS column thermostat (all from Jasco, Tokyo, Japan). Integration was performed by ChromNAV2 chromatography software. The separation was conducted within 60 min of the chromatographic run, starting from the following separation conditions: 97% water with 0.1% formic acid, 3% methanol with 0.1% formic acid. Details of the gradient are reported in Table 2. The separation was performed on an Infinity lab Poroshell 120-SB reverse phase column (C18, 150 × 4.6 mm i.d., 2.7 μm) (Agilent, Santa Clara, CA, USA). Column temperature was set at

30 ◦C. The injection volume was 5 μL. Quantitative determination of phenolic compounds was performed via DAD detector at 254 nm, through 7-point calibration curves, with linearity coefficients (R2) > 0.999, in the concentration range 2–140 μg/mL. The area under the curve from the HPLC chromatogram was used to quantify the analyte concentrations in the extract. Details of the phytochemical identification are included in Table 3. The extract was also qualitatively analyzed with an MS detector in positive and negative ion mode. MS signal identification was realized through comparison with a standard solution and MS spectra present in the MassBank Europe database. The statistical analysis was performed using GraphPad Prism version 5.01 software (San Diego, CA, USA).


**Table 2.** Gradient elution conditions.

**Table 3.** Mass to charge (*m*/*z*) ratios, retention times, and quantities of the investigated phenolic compounds. DAD detector was set at 254 nm.



### *3.3. Cell Cultures*

The effects of the extract (100–500 μg/mL) on myocyte C2C12, human prostate PC3 cancer, and human ovary OVCAR-3 cancer cell viability were determined through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The experimental conditions are fully described in our previous papers [35,36].

#### *3.4. Ex Vivo Studies*

Adult C57/BL6 and female mice (3-month-old, weight 20–25 g) were housed in Plexiglass cages (2–4 animals per cage; 55 cm × 33 cm × 19 cm) and maintained under standard laboratory conditions (21 ± 2 ◦C; 55 ± 5% humidity) on a 14/10 h light/dark cycle, with ad libitum access to water and food. Housing conditions and experimentation procedures were strictly in agreement with the European Community ethical regulations (EU Directive no. 26/2014) on the care of animals for scientific research. In agreement with the recognized principles of "replacement, refinement and reduction in animals in research", colon specimens were obtained as residual material from vehicle-treated mice randomized in our previous experiments, approved by the local ethical committee ('G. d'Annunzio' University, Chieti, Italy) and Italian Health Ministry (Project no. 885/2018-PR).

Isolated prostate and ovary specimens were maintained in a humidified incubator with 5% CO2 at 37 ◦C for 4 h (incubation period), in RPMI buffer with added bacterial LPS (10 μg/mL), as previously described [17]. During the incubation period, the tissues were challenged with scalar concentrations of the extract (10–2000 μg/mL).

#### *3.5. Gene Expression Analysis*

Total RNA was extracted from both prostate and ovary specimens using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's protocol, and reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Waltman, MA, USA). Gene expression of TNF-α, IL-6, CAT, and SOD was determined by quantitative real-time PCR using TaqMan probe-based chemistry, as previously described [14]. PCR primers and TaqMan probes were purchased from Thermo Fisher Scientific Inc. The elaboration of data was conducted with the Sequence Detection System (SDS) software version 2.3 (Thermo Fischer Scientific). Relative quantification of gene expression was performed by the comparative 2−ΔΔCt method [37].
