**1. Introduction**

Prostatitis and pelvic inflammatory disease (PID) are common chronic conditions in the population, caused by pathogenic infection [1,2]. Herbal extracts endowed with antioxidant/anti-inflammatory effects have been long considered as a reliable strategy to blunt the burden of oxidative stress and inflammation in prostate and ovary tissue [3–5]. Pollen extract represents an innovative approach for the management of the clinical symptoms related to prostatitis [6], being also able to relieve inflammation and hyperplasia of the prostate [7], with anticancer potential most likely associated with antioxidant and antimutagenic effects [8]. In this case, pollen appears to relieve pain in patients with benign prostatic hyperplasia, at least in the early stages. Its administration together with chemotherapeutic agents has been seen to increase the number of people who have experienced a significant therapeutic effect [9]. Due to its content in phytoestrogens, pollen has also been shown to improve the symptoms of polycystic ovary syndrome in rats [10], although there is still a lack of scientific literature about the effects of pollen in PID.

**Citation:** Chiavaroli, A.; Di Simone, S.C.; Acquaviva, A.; Libero, M.L.; Campana, C.; Recinella, L.; Leone, S.; Brunetti, L.; Orlando, G.;

Nilofar; et al. Protective Effects of PollenAid Plus Soft Gel Capsules' Hydroalcoholic Extract in Isolated Prostates and Ovaries Exposed to Lipopolysaccharide. *Molecules* **2022**, *27*, 6279. https://doi.org/10.3390/ molecules27196279

Academic Editor: Nour Eddine Es-Safi

Received: 31 August 2022 Accepted: 17 September 2022 Published: 23 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Pollen represents the set of microgametophytes produced by spermatophytes in the male cones, in the case of gymnosperms, and in the anthers, the fertile part of the stamens, in the case of angiosperms. Pollen has different shapes and colors and dimensions between 2.5 and 250 μm. Its composition varies mainly according to the geographical origins and the botanical species visited by the insect, together with less relevant but still important factors such as climatic conditions and the type of soil [8]. From the literature, it appears that pollen grains deriving from various plant species contain about 200 active substances, including proteins and amino acids, carbohydrates, lipids and fatty acids, enzymes and coenzymes, nucleic acids, phenolic compounds, vitamins, and minerals [9].

Pollen, as well as other bee products, has been used since ancient times as a food for its nutritional value and for a wide spectrum of therapeutic activities, of which the best known are antifungal, antibacterial, antiviral, antioxidant, and anti-inflammatory. Evidence has been reported on the activity of phenolic compounds in pollen extracts against Grampositive and Gram-negative bacteria, fungi, and yeasts [10–12]. The content in phenolic compounds has been also related to the anti-inflammatory and antioxidant properties of pollen [13].

In this context, the aims of the present work were to analyze the phenolic composition of an innovative formulation containing Graminex G60TM Flower Pollen Extract, a mixture of standardized and dry pollen of rye grass (*Secale cereale* L.), corn (*Zea mays* L.), and timothy (*Phleum pratense* L.), and NAXTM 7% paste, both suspended in extra virgin olive oil (EVO) as amber soft gel capsules (PollenAid Plus), and to evaluate cytotoxic activity of the hydroalcoholic extract from this formulation on immortalized human prostate cancer PC3 cells and human ovary cancer OVCAR-3 cells. The effect of the extract on cell viability was also investigated in a myoblast C2C12 cell line, which was chosen as a non-tumor comparison cell model. The protective effects of this extract were also investigated in isolated prostate and ovary tissues exposed to *Escherichia coli* lipopolysaccharide (LPS), a reliable experimental model of tissue inflammation [14]. In this context, we measured the gene expression of pro-inflammatory factors, including inteleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). The gene expression of superoxide dismutase (SOD) and catalase, which are deeply involved in antioxidant response, was measured in both tissues, as well. Finally, an in silico study was conducted for unraveling, albeit partially, the mechanisms of action underlying the observed effects and putative interactions against transient receptor potential vanilloid 1 (TRPV1), an ion channel present on sensory neurons and localized in particular on small neurons and type C amielin fibers responsible for nociceptive transmission. TRPV1 is activated by a number of harmful stimuli and its activity is regulated by numerous inflammatory mediators including prostaglandins, bradykinins, and serotonin. In addition to the sensitization of TRPV1 by inflammatory mediators, the activation of TRPV1 stimulates the release of inflammatory molecules associated with the transmission of pain such as substance P and bradykinin, which in turn contribute to the peripheral sensitization of TRPV1 as well as the activation of mast cells and the perpetuation of the state of neurogenic inflammation. The administration of substances capable of acting on some of the actors underlying the pathophysiology of pain, such as TRPV1, and at the same time counteracting neurogenic inflammation is a rational approach in the treatment of chronic pelvic pain pathologies.

#### **2. Results**

In the present study, 17 compounds were identified in the hydroalcoholic extract and quantified through HPLC-DAD-MS. The quantification was carried out by comparison with pure standards (Figure 1). Among assayed compounds, 3-hydroxytyrosol, catechin, gentisic acid, and chlorogenic acid were the main phytochemicals (Table S1). The quantification of such compounds in the extract is consistent with their previous identification in the plants of origin of the pollen [15,16], despite the presence of the vehicle (extra-virgin olive oil: EVO); thus, indicating the EVO as a reliable vehicle which displays multiple advantages: biocompatibility, health-promoting effects, and sustainability. The determination

of phenolic compounds is consistent with our previous study of Graminex pollen using different analytical conditions [17]. The presence of phenolic compounds in the extract makes rational the evaluation of protective effects in prostate and ovary cells and tissues, as described below.

**Figure 1.** (**A**) Chromatogram related to the analysis of the hydroalcoholic extract from Graminex pollen. (**B**) Phenolic compounds identified and quantified in the extract. 3-Hydroxytyrosol (peak #2), catechin (peak #4), gentisic acid (peak #5), and chlorogenic acid (peak #8) were the most abundant phenolic compounds present in the extract.

Regarding the pharmacological study, the extract (10–2000 μg/mL) was tested on different cell lines, namely human prostate cancer PC3 cells and human ovary cancer OVCAR-3 cells, to investigate cytotoxic properties against tumor cells. Additionally, the extract was also added to the medium of myoblast C2C12 cells, to determine the susceptibility of a non-tumor cell line to scalar concentrations. Intriguingly, all three cell lines displayed a similar response after exposure to the extract. Indeed, the cell viability was slightly reduced at the highest tested concentration (2000 μg/mL). However, the cell viability was >70% compared to the control (ctrl) group, in all three cell models (Figure 2); thus, ruling out any significant cytotoxic effect towards both tumor and non-tumor cells.

**Figure 2.** Effects of the extract on human prostate cancer PC3 cells, human ovary cancer OVCAR-3 cells, and the non-tumoral C2C12 myoblast cell line. At the highest tested concentration, the extract induced a mild reduction of cell viability in all tested cell lines (ANOVA, *p* < 0.05; \* *p* < 0.05 vs. ctrl group). However, the cell viability was always over 70% compared to the respective ctrl group; thus, suggesting biocompatibility in the concentration range 10–2000 μg/mL. This range was considered as biocompatible for the subsequent ex vivo determination in the prostate and ovary tissues.

The extract (10–2000 μg/mL) was also tested in isolated prostate and ovary specimens challenged with *E. coli* LPS, chosen as pro-inflammatory stimulus [14,17]. In this context, it is notable that *E. coli* infection has been related to both prostatitis and PID [18,19]. The LPS stimulus induced the upregulation of TNF-α and IL-6 both in prostate and ovary tissues (Figures 3 and 4). The extract treatment was effective in reverting the increased gene expression of both cytokines; thus, demonstrating anti-inflammatory effects in both tissues. In the case of prostate tissues, this study is also consistent with previous clinical observations about the capability of Graminex pollen to contrast the inflammatory component of prostatitis [20,21]. The anti-inflammatory effects also agree with previous studies highlighting the inhibition of IL-8 production [22] and cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) activities, measured as prostaglandin E2 and nitrites levels, respectively [23–25].

**Figure 3.** Inhibitory effects of the hydroalcoholic extract (10–2000 μg/mL) on TNF-α gene expression in isolated prostate (**A**) and ovary (**B**) specimens. ANOVA, *p* < 0.0001; \*\*\* *p* < 0.001 vs. ctrl group.

**Figure 4.** Inhibitory effects of the hydroalcoholic extract (10–2000 μg/mL) on IL-6 gene expression in isolated prostate (**A**) and ovary (**B**) specimens. ANOVA, *p* < 0.0001; \*\*\* *p* < 0.001 vs. ctrl group.

In prostate and ovary specimens, LPS stimulus (50 μg/mL) was also effective in increasing the gene expression of both CAT and SOD (Figures 5 and 6), which are deeply involved in the antioxidant response. Indeed, the extract was able to prevent the LPS-induced upregulation of CAT and SOD gene expression. Additionally, after extract administration, the gene expression of both enzymes was even lower than the one displayed by the control (ctrl) group. Previously, LPS stimulus has been found to alter CAT and SOD levels, with both inhibitory and stimulatory effects. We cannot exclude that these discrepancies could depend, albeit partially, on the employed experimental models [26,27]. Therefore, also considering the intrinsic scavenging/reducing properties and ability of Graminex to blunt LPS-induced lipoperoxidation in isolated prostate [17], we hypothesize that the extract effects on SOD and CAT gene expression could be related to antioxidant effects, which can be mediated, albeit partially, by polyphenolic compounds.

**Figure 6.** Inhibitory effects of the hydroalcoholic extract (10–2000 μg/mL) on CAT gene expression in isolated prostate (**A**) and ovary (**B**) specimens. ANOVA, *p* < 0.0001; \*\*\* *p* < 0.001 vs. ctrl group.

In order to explore the mechanisms of action underlying the observed effects, an in silico study was conducted on the platform STITCH, considering the main phytochemicals present in the extract; namely catechin, 3-hydroxytyrosol, chlorogenic acid, and gentisic acid (2,5-dihydroxybenzoic acid). Catechin was predicted to interact with IL-6, cyclooxygenase-2 (COX-2, PGTS2), and with iNOS (Figure 7). This is partly consistent with our findings of anti-inflammatory effects by the extract, in both prostate and ovary tissue, and with the literature data [5]. Intriguingly, 3-hydroxytyrosol and chlorogenic acid were predicted to interact with BCL-2 and caspase-3, respectively. Previous studies showed the capability of 3-hydroxytyrosol and chlorogenic acid to reduce BCL-2 and caspase-3 gene and protein expression, respectively [28,29], while gentisic acid could interact with fibroblast growth factor 1 (FGF1), whose levels are increased in prostate cancer [30]. This could explain, albeit partially, the mild reduction (<30%) of cell viability in all considered cell lines at the highest tested concentration.

**Figure 7.** *Cont*.

**Figure 7.** Target component analysis conducted on the STITCH bioinformatics platform for unraveling putative interactions between prominent extracts' phytochemicals and putative proteins involved in inflammatory and cytotoxicity effects.

Finally, a docking approach was conducted to explore the putative interactions between the extract's phytochemicals and the TRPV1 receptor, whose expression is increased in prostate inflammation [31]. Among phytochemicals detected in the extract, chlorogenic acid and catechin showed micromolar affinity (10–12 μM) towards the TRPV1 receptor (Figure 8); thus, suggesting direct interactions that could be crucial in mediating the observed anti-inflammatory properties. According to these predictions, further in vitro studies are needed to unravel the effects of catechin and chlorogenic acid on TRPV1 expression and activity.

**Figure 8.** (**A**) Putative interactions between catechin and TRPV1 receptor (PDB ID: 7LR0). Free energy binding (ΔG) and putative affinity (*K*i) are −6.8 kcal/mol and 10.5 μM, respectively. (**B**) Putative interactions between chlorogenic acid and TRPV1 receptor (PDB ID: 7LR0). Free energy binding (ΔG) and putative affinity (*K*i) are −6.7 kcal/mol and 12.5 μM, respectively.
