A Multi-Target Phytotherapeutic Approach to Benign Prostatic Hyperplasia: Preclinical Characterization of a PhytoBPH-Mix

Abstract

Background: Benign prostatic hyperplasia (BPH) is a prevalent condition affecting over 50% of men aged 60 and above, often leading to lower urinary tract symptoms that significantly impact quality of life. Current pharmacological treatments, including α-adrenergic receptor antagonists and 5α-reductase inhibitors, are associated with adverse effects, prompting the exploration of alternative therapies. This study investigates the potential role of a novel multi-component phytocomplex (PhytoBPH-Mix) comprising Serenoa repens, Pygeum africanum, Urtica dioica, Epilobium angustifolium L., Protium heptaphyllum, lycopene, Vitamin E, zinc, and selenium. Methods: The anti-androgenic, anti-inflammatory, and antimicrobial properties of the mixture were evaluated in vitro. Results: The formulation significantly inhibited 5α-reductase activity, reduced the release of pro-inflammatory cytokines (IL-6 and TNF-α), and exhibited antibacterial effects against E. coli compared to individual extracts. Conclusions: These findings suggest that this specific mixture offers a promising natural alternative or an adjuvant for managing BPH by targeting multiple pathological mechanisms with minimal side effects and could also serve as an effective adjuvant in conventional therapy.

1. Introduction

The prostate is a male urogenital organ located beneath the bladder and encircling the urethra. Structurally, it consists of fibromuscular and glandular tissues. The fibromuscular component plays a crucial role in fluid compartmentalization during urination and ejaculation, while the glandular tissue serves as a reproductive function by producing prostatic fluid. This slightly acidic (pH 6.5) milky secretion nourishes spermatozoa and contains enzymes, ions (notably zinc and citrate), and prostate-specific antigen (PSA). Zinc and citrate enhance sperm motility, while PSA facilitates sperm movement by liquefying the sperm clot [1]. The prostate is susceptible to a range of disorders, including benign enlargement, inflammatory or bacterial conditions (prostatitis), and oncological diseases. Among these, benign prostatic hyperplasia (BPH) is a non-malignant overgrowth of prostate tissue affecting over 50% of men aged 60 and above. While BPH itself is not inherently harmful, it can lead to lower urinary tract symptoms (LUTS) such as weak urinary stream, intermittency, nocturia, urgency, and incomplete bladder emptying [2,3]. The precise mechanisms underlying BPH remain unclear, though several factors, including aging, growth factors, interleukins, and sex hormones, are implicated. These factors converge on epithelial–stromal crosstalk, chronic low-grade inflammation, and dysregulated androgen signaling, which together promote progressive tissue remodeling and hyperplasia [4]. The prevalence of BPH increases with age, affecting 50–60% of men in their 60s and 80–90% of men over 70. Prostate volume has been observed to increase annually by 2–2.5% [5]. Research by McDowell et al. revealed that inflammatory cells in the prostate release growth factors and interleukins, promoting the proliferation of prostate epithelial cells [6,7]. Interleukin-17 (IL-17), detected only in inflamed or BPH-affected prostates, stimulates the production of IL-6 and IL-8, thereby sustaining a pro-inflammatory microenvironment that promotes epithelial proliferation and stromal activation [8]. Hormones like estrogens and their modulators also contribute to prostate growth and differentiation, with elevated estradiol (E2) levels correlating with increased prostate volume [9]. Androgens, particularly testosterone and its metabolite dihydrotestosterone (DHT), are critical for normal prostate development and play a significant role in BPH. Testosterone is converted into DHT by the enzyme 5α-reductase, particularly isozyme type 2. DHT is essential for adult prostate growth, and its elevated levels in BPH tissues have led to the use of 5α-reductase inhibitors, such as finasteride and dutasteride, to alleviate lower urinary tract symptoms (LUTS) and reduce prostate size and PSA levels. However, these medications are often associated with adverse effects, including decreased libido, reduced ejaculate volume, impotence, and gynecomastia due to testosterone’s conversion to estrogen, which limits long-term compliance and highlights the need for safer, multi-target alternatives [3,10]. Additionally, α-adrenergic receptor antagonists, such as tamsulosin and silodosin, are used to improve urinary symptoms by relaxing smooth muscle in the prostate and bladder neck, although these drugs may cause side effects like retrograde ejaculation [11]. The combination of these therapies, as recommended by the Medical Therapy of Prostatic Symptoms (MTOPS) guidelines, can exacerbate side effects, necessitating the exploration of alternative treatments. In recent years, phytotherapeutics have emerged as potential alternatives due to their minimal side effects [12]. Serenoa repens (saw palmetto), native to coastal regions of the USA and tropical parts of the Americas, contains bioactive compounds such as phytosterols and fatty acids. Serenoa repens (commonly known as saw palmetto) is a small palm native to the southeastern United States. In phytotherapy, the main plant material used is its ripe berry, from which a lipido-sterolic hexanic extract is obtained. This extract, commercially known as Permixon^®, is widely used for the treatment of BPH. The therapeutic efficacy of this extract is attributed to the presence of bioactive compounds, including phytosterols and free or esterified fatty acids, which exhibit anti-inflammatory and anti-androgenic properties [13,14].

Supercritical CO₂ (SC-CO₂) extracts of saw palmetto have shown greater efficacy in alleviating BPH symptoms compared to conventional extracts [15,16]. In Europe, Serenoa repens has been extensively used as a first-line phytotherapeutic option for managing LUTS associated with BPH, owing to its favorable safety profile and clinical effectiveness in symptom control [17]. Pygeum africanum, derived from the bark of Prunus africana, a tree native to African highlands, contains phytosterols, fatty acids, and triterpenoid compounds. Its pharmacological properties include anti-inflammatory, antiproliferative, and antiandrogenic activities, along with 5α-reductase inhibition [18]. Urtica dioica (nettles) contains lignans that inhibit androgen binding to sex hormone receptors and acids like salicylic acid with anti-inflammatory properties. Additionally, its polysaccharides and lectins block the epidermal growth factor receptor, limiting prostate growth. This extract also inhibits aromatase, an enzyme that converts testosterone to estrogen, further addressing BPH pathology [19,20]. Epilobium, a genus of herbaceous plants, contains a phytocomplex rich in flavonoids, phenolic acids, and tannins such as oenothein B. These compounds possess antioxidant and antibacterial properties, beneficial for managing urinary tract infections and prostatitis [21,22]. Protium heptaphyllum, a tree in the Burseraceae family, produces a resin rich in triterpenes, notably α and β-amyrin, which exhibit anti-inflammatory effects by inhibiting prostaglandins and tumor necrosis factor-alpha (TNF-α) [23]. Recent reviews have highlighted the mechanistic plausibility, particularly through modulation of NF-κB signaling, inhibition of 5α-reductase, and attenuation of oxidative stress pathways, for using plant-based compounds in BPH, underscoring their anti-inflammatory, anti-androgenic, and antioxidant effects [24,25]. Evidence from human and animal studies suggests that oxidative stress plays a key role in the onset of prostatic inflammation. Elevated levels of 8-OH deoxyguanosine, a marker of oxidative DNA damage, have been detected in BPH tissues, correlating with increased prostate weight and localized predominantly in epithelial cells. In transgenic mice overexpressing Nox4 in the prostate, enhanced oxidative damage was associated with epithelial hyperplasia, stromal thickening, and fibrosis, reinforcing the link between redox imbalance and the inflammatory mechanisms underlying LUTS [26]. In this context, oxidative stress has been identified as a key factor in the pathogenesis of chronic prostatitis and BPH, supporting the therapeutic use of antioxidants such as lycopene Vitamin E (α-tocopherol), zinc, and selenium to mitigate oxidative damage and enhance clinical therapeutic benefits [27,28].

This study investigates the potential role of an additive combination of natural herbal extracts of Serenoa repens, Pygeum africanum, Urtica dioica, Epilobium, Protium heptaphyllum, added with tomato-derived lycopene, Vitamin E, zinc, and selenium designed to simultaneously modulate androgen metabolism, inflammatory signaling, oxidative stress, and microbial burden within the prostate microenvironment, for the management of BPH. By harnessing the diverse properties of these bioactive substances, including anti-inflammatory, antioxidant, androgen-modulating, and 5α-reductase-inhibiting activities, this formulation offers a promising alternative therapeutic strategy. The mixture has not only the potential to address key pathological mechanisms of BPH but also to minimize the adverse effects commonly associated with conventional treatments, paving the way for safer and more comprehensive management options.

2. Materials and Methods

Extracts were kindly donated by Anvest Health s.r.l. (Castel San Giorgio, Italy). Specifically, Serenoa repens (Bartram) Small, ripe berries, with lipido-sterolic extract (85–95% fatty acids) obtained by supercritical CO₂ extraction; Prunus africana (Hook. f.) Kalkman (Pygeum), bark extract standardized to 2.5% β-sitosterol; Urtica dioica L. (Nettle), root extract standardized to 0.8% β-sitosterol; Epilobium angustifolium L., aerial parts; extract with drug/extract ratio of 4:1; Protium heptaphyllum (Aubl.) Marchand, resin extract standardized to 2.5% α-/β-amyrins; Lycopersicon esculentum Mill. (Tomato), fruit extract standardized to 18% lycopene; Vitamin E, Zinc; Selenium. All other solvents and chemicals were of analytical grade by Sigma-Aldrich (Milan, Italy).

For in vitro experiments, the formulation PhytoBPH-Mix was resuspended in dimethyl sulfoxide (DMSO) and diluted in the appropriate culture medium, with a final DMSO concentration not exceeding 0.1% v/v to avoid cytotoxic effects.

2.1. Preparation of PhytoBPH-Mix

The PhytoBPH-Mix was prepared using a geometric dilution method. Specifically, the components were weighed according to the amounts reported in

Table 1. Composition of powders.

Component	Quantity (mg)
Serenoa repens	400
Pygeum africanum	100
Urtica dioica	80
Epilobium angustifolium L.	50
Protium heptaphyllum	50
Tomato	18.5
Zinc	12.5
Vitamin E	10
Selenium	0.042

, then gradually and homogeneously mixed using a mortar and pestle to ensure uniform distribution of active ingredients. The Serenoa repens used in the formulation was a SC-CO₂ extract containing approximately 90% total free and esterified fatty acids (provided by Anvest Health s.r.l. Castel San Giorgio, Salerno, Italy). The relative proportions of the components were selected to reflect standardized extract potency and formulation ranges commonly used in experimental and nutraceutical preparations.

2.2. Measurement of 5-α-Reductase Inhibitory Activity

The inhibition activity of PhytoBPH-Mix on the 5α-reductase was evaluated through a biochemical method. Prostatic epithelial cells and prostatic fibroblasts (4 × 10⁴ cells per well in 24-well plates) were suspended in a medium containing 4 mM phosphate buffer (pH 6.5 for 5α-reductase type 1 or pH 5.0 for type 2) and 1.9 nCi [4-¹⁴C] testosterone. The incubation with extract Serenoa repens (10 µg/mL), Pygeum africanum (2.5 µg/mL), and the formulation containing the same amount of extracts was carried out for 30 min at 37 °C. Cells were resuspended in ethanol to extract 5α-reductase, and its activity was calculated from the percentage of the extent of the conversion of [4-¹⁴C] testosterone to [4-¹⁴C] dihydrotestosterone. The radioactively labeled substrate [4-^¹⁴C] testosterone used in the assay was purchased from PerkinElmer (Cat. No. NET370250UC; Waltham, MA, USA). All incubations were performed using prostatic epithelial and stromal cells as described below. Untreated cells were used as a control.

2.3. Anti-Inflammatory Activity

To evaluate the anti-inflammatory activity of the PhytoBPH-Mix, pro-inflammatory cytokines IL-6 and tumor necrosis factor-alpha TNF-α were revealed in cell-free supernatants using commercial enzyme-linked immunosorbent assay kits (ELISA). Both VCaP cells and immune-responsive cell lines (THP-1 and RAW 264.7) were used in parallel to assess anti-inflammatory activity. Cytokine levels were quantified in supernatants from both models. The use of prostate epithelial cells complements the immunological data and reflects the inflammatory context of BPH more closely. For the assay, immune-responsive cell lines such as RAW 264.7 murine macrophages or THP-1 human monocytes were seeded in 96-well plates at a density of approximately 1 × 10⁵ cells per well. These cells were then stimulated with lipopolysaccharide (LPS) to induce a robust inflammatory response prior to treatment with the PhytoBPH-Mix. Lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich, Cat. No. L4391, Merck KGaA, Darmstadt, Germany) was used at a final concentration of 1 µg/mL, a sufficient dose to induce a measurable pro-inflammatory response without compromising cell viability. After incubation, the supernatants were harvested and subjected to ELISA for cytokine quantification. ELISA kits for human IL-6 (R&D Systems, Cat. No. D6050) and TNF-α (R&D Systems, Cat. No.DTA00C) (Minneapolis, MN, USA) were used according to the manufacturer’s protocols. Plates were read using a Bio-Rad iMark^™ Microplate Reader (Bio-Rad, Hercules, CA, USA) at 450 nm with correction at 550 nm. After the addition of the TMB (3,3′,5,5′-tetramethylbenzidine) substrate, the enzymatic reaction yielded a color change, which was terminated using sulfuric or hydrochloric acid. The ELISA test used two antibodies and a detection system to identify the above cytokines in immunosorbent plates after a spectrophotometric reading at a wavelength of 450 nm (corrected to 550 nm). VCaP cells (prostate cells immortalized) were plated in multiwells and serially treated for 24 h with Serenoa repens, Pygeum africanum, Protium heptaphyllum, and the formulation in a concentration range of 0.5–3.0 µg/mL. The concentration range of 0.5–3.0 µg/mL was selected based on literature indicating that similar extracts maintain cell viability at these doses. The anti-inflammatory potential of PhytoBPH-Mix was then assessed based on its ability to significantly reduce these levels compared to the LPS-only control group.

2.4. Antibacterial Activity: Time-Killing Assay

To evaluate the antimicrobial activity of the PhytoBPH-Mix, time-killing studies were performed on E. coli (ATCC 15221) in co-culture with VCaP cells (prostate cells immortalized). The bacterial suspension (0.5 McFarland scale) in MHB, corresponding to 1.5 × 10⁶ CFU/mL (colony forming unit per ml) was prepared and incubated at 37 °C under constant shaking. Then, bacteria suspension in co-culture with VCaP cells (1 × 10⁵ cells) was inoculated in sterile 96-well plates (200 µL/well) and maintained at 37 °C overnight. After that, bacteria suspensions with cells were treated with a solution of Epilobium at 0.7 mg/mL and the formulation containing the same amount of Epilobium. A saline solution was used as a positive control. At specified time points (1, 3, and 7 days), viable counts were determined to count CFU/mL by plating serial dilutions on MHA incubated at 37 °C. Mueller–Hinton broth (MHB) and Mueller–Hinton agar (MHA) were obtained from Oxoid, Thermo Fisher Scientific (Basingstoke, UK). E. coli strain used in the assay was ATCC 15221. Graphics were obtained, plotting the time against the number of CFU recovered. Each antimicrobial assay was performed in triplicate on separate days.

2.5. Cell Culture Conditions

All cell lines used in this study were commercially sourced and maintained according to standard protocols.

To be more specific, for Prostatic Epithelial Cells (PECs) and Prostatic Fibroblasts (Stromal Cells), human primary prostate epithelial cells and stromal fibroblasts were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA; Cat. Nos. 4410 and 4420, respectively). Cells were cultured in Prostate Epithelial Cell Medium (PrEGM^™, Lonza, Walkersville, MD, USA) or Fibroblast Medium (FM, ScienCell Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL), at 37 °C in a humidified atmosphere with 5% CO₂.

In the case of VCaP cells, the VCaP human prostate cancer cell line was obtained from ATCC^® (CRL-2876^™). Cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium, high glucose, Thermo Fisher Scientific) supplemented with 10% FBS, 1% L-glutamine, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin).

When RAW 264.7 cells were used, murine macrophage RAW 264.7 cells were purchased from ATCC^® (TIB-71^™) and maintained in RPMI-1640 medium (Gibco, Grand Island, N.Y.) supplemented with 10% FBS and antibiotics.

In the case of THP-1 cells, human monocytic THP-1 cells were obtained from ATCC^® (TIB-202^™, Manassas, VA, USA). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol, and antibiotics.

All cell cultures were routinely maintained at 37 °C in a 5% CO₂ incubator and passed before reaching 80% confluency. Mycoplasma contamination was routinely checked using a PCR-based detection kit.

In all experiments, the mixture and the reference extracts were resuspended in dimethyl sulfoxide (DMSO) and diluted in the corresponding culture medium. The final concentration of DMSO in all treated samples did not exceed 0.1% v/v, a threshold known to be non-cytotoxic and commonly used in cell-based assays. This same concentration of DMSO (0.1% v/v) was used in the vehicle control group, which served as the solvent control across all assays.

2.6. Statistical Analysis

Statistical analysis was conducted using GraphPad Prism 10.1.0 (Boston, MA, USA) was used for statistical analysis. Specifically, for the experiment that evaluates anti-androgenic activity, comparisons were performed using multiple unpaired t-tests, while for the experiments on the activity of 5α-reductase type II on prostatic fibroblasts with different types of Serenoa repens extracts compared to PhytoBPH-Mix, the one-way ANOVA followed by Dunnett’s multiple comparisons test. The two-way ANOVA analysis of variance was used, and multiple comparisons were made by Tukey’s multiple tests have been applied for the antimicrobial activity against E. coli experiments.

3. Results

3.1. Anti-Androgenic Activity

Since both type I and II 5α-reductase are upregulated in benign prostatic hyperplasia (BPH) tissue, a novel phytoterapeutic formulation (PhytoBPH-Mix), containing both Serenoa repens and Pygeum africanum extracts and others, was compared to individual plant extracts. Experiments were conducted on prostatic epithelial and prostatic fibroblast cells to verify the ability to inhibit enzymes. This capability was evaluated as a reduction in the amount of converted dihytestosterone (DHT). As reported in Figure 1, both Serenoa repens and Pygeum africanum extracts significantly suppressed 5α-reductase activity. This effect was particularly notable in prostatic epithelial cells, which express both type I and type II isoforms (predominant isoenzyme in adult prostate tissue) [29]. Notably, the mixture of extracts exhibited superior inhibition of both isoforms compared to the single extracts, suggesting a functionally additive or combined mechanism likely due to multi-target interactions on distinct enzymatic and receptor-mediated pathways. Dual inhibition of 5α-reductase could lead to an advantage in the treatment of BPH since it has been demonstrated that dual inhibitors, such as dutasteride, are more efficacious than selective inhibition [20,30]. Thus, due to its broad enzymatic targeting the formulation could be a promising phytotherapeutic candidate for comprehensive BPH management. Serenoa repens, known as saw palmetto, is widely used as a phytotherapeutic agent in the treatment of BPH, inhibiting 5α-reductase type I and II, as well as fibroblast proliferation. Since extraction methods can alter the lipid and sterol profiles of botanical preparations, thereby affecting biological efficacy [31], in this work various Serenoa repens extracts were evaluated. Sc-CO₂ extraction yielded a product with approximately 90% free and esterified fatty acids, demonstrating superior 5α-reductase type II inhibition compared to hydroalcoholic extracts (55% or 90% lipid content). Statistical analysis confirmed that the SC-CO₂ Serenoa repens extract and the corresponding mixture PhytoBPH-Mix demonstrated significantly higher inhibition of 5α-reductase type II compared to hydroalcoholic extracts. No significant differences were observed in the inhibition of 5α-reductase type II among hydroalcoholic extracts with different fatty acid content (Figure 2). These findings align with previous studies reporting stronger type II inhibition for lipid-rich SC-CO₂ extracts. This enhanced effect persisted when the Sc-CO₂ extract was incorporated into PhytoBPH-Mix, which demonstrated about 15% greater efficacy in inhibiting 5α-reductase type II than the hydroalcoholic extract-based formulation. These findings corroborate previous studies [32] emphasizing the superior bioactivity of CO₂-extracted Serenoa repens. Moreover, PhytoBPH-Mix, which also includes Urtica dioica, exhibited even stronger inhibitory activity than any single extract probably due to a combined effect between Serenoa repens, Pygeum africanum and Urtica dioica. In fact, although the specific mechanism of action is not yet clear, it may involve combined inhibition of 5α-reductase catalytic activity, reduced androgen receptor activation, and modulation of intracellular steroid availability [33]. The additive action may involve bioactive phytosterols such as β-sitosterol, which are known to modulate androgen metabolism and inflammation signaling via interference with androgen receptor binding and suppression of NF-κB–dependent transcription [34,35]. The observed inhibition of 5α-reductase is greater with the PhytoBPH-Mix than with the individual extracts, likely reflecting an additive or complementary effect of PhytoBPH-Mix components.

3.2. Anti-Inflammatory Activity

To evaluate anti-inflammatory effects, VCaP cells (immortalized prostate cells) were treated with increasing concentrations (0.5–3.0 µg/mL) of individual plant extracts (Serenoa repens, Pygeum africanum, Protium heptaphyllum), and the combined PhytoBPH-Mix. These concentrations were selected to maintain cellular viability and avoid confounding effects related to cytotoxicity or non-specific stress responses, as well as physical artifacts in the culture medium [36,37,38]. As reported in Figure 3, all extracts significantly decreased IL-6 and TNF-α secretion. At the lowest tested concentration (0.5 µg/mL), reductions in TNF-α were not statistically significant for some individual extracts, whereas the mixture demonstrated the most potent and dose-dependent suppression of inflammatory activity at all tested concentrations. As reported in the literature Serenoa repens reduces the expression of several inflammatory mediators and inhibits upstream signaling pathways such as NF-κB and MAPK [39]. In addition, selenium and lycopene components known to synergize with Serenoa repens may further suppress inflammatory cascades by scavenging reactive oxygen species and reducing redox-dependent activation of inflammatory transcription factors [40]. Moreover, co-administration of Serenoa repens and Urtica dioica has been shown to reduce IL-6 production and inhibit nuclear translocation of NF-κB [41]. Based on these data, PhytoBPH-Mix may exert a similar mechanism of action of single components, although this hypothesis should be confirmed by future experimental studies.

3.3. Antimicrobial Activity

Given the frequent occurrence of chronic bacterial inflammation in BPH histopathology, a time-killing assay on E. coli to assess the antimicrobial activity of PhytoBPH-Mix was performed. The E. coli time-killing assay was carried out in the presence of VCaP prostate epithelial cells to reproduce a more physiologically relevant environment accounting for host–pathogen interactions that may influence bacterial survival and antimicrobial efficacy and reflecting the interaction between bacteria and prostatic tissue often observed in BPH with chronic bacterial inflammation. The activity of PhytoBPH-Mix was compared with an equivalent dose (0.7 mg/mL) of isolated Epilobium extract [42,43]. Epilobium was used as a reference due to its well-documented antimicrobial activity, particularly against Escherichia coli, a key pathogen in chronic prostatic inflammation and urinary tract infections to evaluate the ability of the complete mixture to exert a broader or additive antibacterial effect. As shown in Figure 4, the novel mixture reduced E. coli viability more rapidly than Epilobium alone, indicating a stronger antimicrobial effect under the experimental conditions. Specifically, PhytoBPH-Mix was able to outperform the same dose of Epilobium extract alone by 40% on days 1 and 3, and by 25% on day 7. These findings suggest a cumulative, broad-spectrum antibacterial effect likely due to the phenolic constituents present in Epilobium and other plant extracts enhanced by the full phytocomplex present in the mixture. Polyphenols such as ellagitannins disrupt bacterial membranes, leading to intracellular oxidative stress and cytotoxicity [44,45]. The observed efficacy of PhytoBPH-Mix therefore points to a novel integrative strategy that combines anti-androgenic, anti-inflammatory, and antimicrobial activities addressing multiple BPH pathophysiological pathways simultaneously.

4. Discussion

This study investigated the anti-androgenic, anti-inflammatory, and antimicrobial activities of the multi-component mixture, which contains well-characterized extracts of Serenoa repens (supercritical CO₂ extract), Pygeum africanum, Epilobium, selenium, lycopene, and other botanicals. The formulation was designed to integrate ingredients with complementary properties relevant to BPH and associated with LUTS acting on the modulation of multiple BPH-related pathways within a single phytocomplex. In the anti-androgenic assays, both PhytoBPH-Mix and the SC-CO₂ extract of Serenoa repens showed significantly greater inhibition of 5α-reductase type II compared to hydroalcoholic extracts, confirming previous reports that lipid-rich extracts are particularly effective in modulating androgen metabolism. No significant differences were detected for type I inhibition, suggesting that fatty acid enrichment mainly impacts type II activity. The results reflect additive effects, with the possibility of limited synergy at specific molecular targets. This reinforces the value of combining high-quality extracts within a single mixture. In the anti-inflammatory models, performed in both prostate epithelial (VCaP) and immune-responsive (THP-1 and RAW 264.7) cells, PhytoBPH-Mix reduced LPS-induced IL-6 and TNF-α release in a concentration-dependent manner. Experiments conducted in THP-1 and RAW 264.7 were exploratory and were therefore not included in the quantitative dataset but were used to confirm the responsiveness of the mixture in immune-competent models. Reductions were generally greater than with the individual extracts tested, underscoring the potential of combining phytoconstituents such as fatty acids, phytosterols, and polyphenols present across all botanical components to target inflammation through complementary mechanisms to achieve broader pathway coverage than single-agent approaches. Selenium and lycopene, included for their documented antioxidant and anti-inflammatory roles, may further enhance these effects. The antimicrobial time-kill assay against E. coli revealed a faster and more pronounced effect for the mixture than for Epilobium alone at equivalent concentrations. Epilobium was chosen as a comparator due to its known antibacterial properties, allowing assessment of the additional benefit provided by the full formulation. Conducting the assay in the presence of VCaP cells created a model closer to the prostate microenvironment, capturing host–pathogen interactions that may influence antimicrobial efficacy in vivo. While the individual properties of the botanical extracts and supplements used here are well-documented, the novelty of this work lies in their specific combination within PhytoBPH-Mix. In fact, it integrates components with complementary pharmacological profiles, potentially producing additive effects. Given that plant extracts naturally contain a rich variety of bioactive molecules, their biological actions are likely multifactorial, involving endocrine, immune, oxidative, and microbial pathways. Bioavailability of the key constituents of PhytoBPH-Mix is expected to vary, as reported for similar phytocomplexes, and this may shape their overall clinical activity. Direct extrapolation between in vitro concentrations and human plasma levels is inherently limited, particularly for complex phytotherapeutic formulations. However, plasma exposure does not necessarily reflect prostate tissue availability. Several constituents of PhytoBPH-Mix, including fatty acids, phytosterols, triterpenes, and lycopene, are highly lipophilic and have been shown to accumulate within prostatic tissue at levels exceeding plasma concentrations [36,40]. The µg/mL range used here is consistent with mechanistic in vitro studies on individual BPH-related phytocompounds and was selected to assess target modulation rather than systemic exposure. The present results suggest that the formulation has the potential to address multiple mechanistic targets in BPH, including androgen conversion, inflammatory cytokine production, oxidative stress, and bacterial persistence. While these models are appropriate for mechanistic investigations, in vivo validation will be required to confirm these results. Future research, expanding into phytochemical profiling, comparative pharmacology, and clinical evaluation, will further define its role alongside established therapeutic strategies.

5. Conclusions

This study demonstrates the preclinical efficacy of a novel multi-component phytocomplex formulation (PhytoBPH-Mix), combining Serenoa repens, Pygeum africanum, Urtica dioica, Epilobium, and Protium heptaphyllum, along with key antioxidant cofactors (lycopene, selenium, zinc, and vitamin E). The rationale of PhytoBPH-Mix lies in pathway coverage, supporting a multitarget strategy aligned with the multifactorial pathophysiology of BPH. The mixture has shown to simultaneously inhibit both isoforms of 5α-reductase, achieving reductions of up to 35–40% in DHT conversion. In addition, PhytoBPH-Mix significantly attenuated the release of inflammatory cytokines (IL-6 by 45% and TNF-α by 50%) in prostate cell lines and exhibited a time-dependent antimicrobial effect against E. coli, surpassing the activity of single-extract controls. These effects are due to the contribution of different phytocomponents, likely mediated through the modulation of androgen signaling, inflammatory responses, and microbial growth via complementary pathways involved in BPH. Importantly, this is the first integrated report to show the simultaneous inhibition of 5α-reductase, modulation of inflammatory cytokines, and microbial suppression by a specific phytocomplex mixture in vitro. In summary, PhytoBPH-Mix demonstrated anti-androgenic, anti-inflammatory, and antimicrobial activities in vitro, consistent with the complementary contributions of its botanical and supplemental components. These findings provide a preliminary basis for future investigations through controlled clinical trials to validate its efficacy, optimal dosage, and safety profile. If confirmed in vivo, this mixture could represent a safe and effective alternative or adjuvant to conventional pharmacotherapy in the management of BPH, potentially minimizing the adverse effects associated with α-blockers and synthetic 5α-reductase inhibitors.