Nutraceutical Effects of Gastrodiae elata and Coenzyme Q10 on Oxidative Stress and Inflammatory Pathways in an In Vitro Gut–Prostate Axis Model

Abstract

Background/Objectives: Benign prostatic hyperplasia (BPH) is a multifactorial condition associated with androgen imbalance, oxidative stress, and chronic inflammation, leading to growing interest in food-derived bioactive compounds with multitarget activity. This study aimed to investigate the biological effects of a nutraceutical combination of Gastrodiae elata Blume extract and coenzyme Q10 (Q10), focusing on mechanisms relevant to prostate physiological balance using food-relevant in vitro models. Methods: An intestinal epithelial barrier model (Caco-2) was employed to assess intestinal tolerance and permeability of the tested compounds. Subsequently, a prostate epithelial–stromal co-culture exposed to dihydrotestosterone (DHT) was used to reproduce BPH-like cellular conditions. Oxidative stress, inflammatory mediators, androgen-related pathways, and markers of proliferation and apoptosis were evaluated following simulated intestinal passage. Results: The combined formulation showed no cytotoxic effects and demonstrated efficient intestinal permeability. After intestinal passage, the combination significantly reduced oxidative stress and inflammatory responses in the prostate co-culture, decreasing reactive oxygen species and pro-inflammatory mediators, including NF-κB, TNF-α, and IL-1β. In parallel, the formulation modulated androgen-related pathways by reducing 5-α-reductase activity and DHT levels while supporting testosterone homeostasis. Across some of the evaluated endpoints, the combined formulation tended to show more pronounced protective effects compared with the individual components. Conclusions: These results suggest that a combination of Gastrodiae elata and coenzyme Q10 may have a positive effect on prostate health. In the nutraceutical field, this food-based formulation could help support prostate health, probably through antioxidant, anti-inflammatory, and hormonal control mechanisms. Further studies using advanced experimental models are warranted.

1. Introduction

Benign prostatic hyperplasia (BPH) is characterised by the non-cancerous enlargement of prostate tissue in older men and is often associated with lower urinary tract symptoms (LUTS). The risk of developing BPH increases significantly with age, with prevalence rates estimated at 50–60% among men aged 60–70 years and rising to 80–90% in those over 70 [1]. The regulation of prostate growth and function is mainly governed by androgens, particularly testosterone and its active metabolite dihydrotestosterone (DHT). Within prostatic tissue, the enzyme 5-α-reductase converts testosterone to DHT, which, due to its higher affinity for the androgen receptor (AR), plays a key role in prostate physiology and the development of hyperplasia. Consequently, the actions of androgen and oestrogen receptors, along with 5-α-reductase activity, are vital factors in the pathogenesis of BPH [2,3].

Alongside androgen signalling, there is increasing evidence that ongoing inflammation plays a crucial biological role in the development of BPH. Histological analyses often reveal the coexistence of hyperplastic nodules and inflammatory infiltrates in prostate tissue [2,3,4]. Increased pro-inflammatory cytokine levels observed in BPH point to the significant contribution of innate immune mechanisms, such as those mediated by interleukin (IL)-1 and Toll-like receptor (TLR) signalling pathways [5,6,7]. Prostate cells express multiple TLRs, whose activation promotes the production of cytokines such as tumour necrosis factor alpha (TNFα), IL-1β, IL-6, IL-8 and IL-10 [4,7]. Nuclear factor kappa B (NF-κB) acts as a central regulator linking inflammatory signalling, apoptosis and cell proliferation. Additionally, oxidative stress has been extensively documented in BPH, contributing to excessive reactive oxygen species (ROS) production and interacting with androgen-driven proliferative pathways [8,9,10]. Current pharmacological strategies for BPH primarily aim to alleviate symptoms and reduce prostate volume. Alpha-1 adrenergic antagonists are frequently prescribed for the management of LUTS [11]. In contrast, 5-α-reductase inhibitors (5-ARIs), including finasteride and dutasteride, act by lowering DHT levels and promoting atrophy of the prostate epithelium, which progressively reduces prostate size and alleviates symptoms [12,13,14]. Despite their clinical effectiveness, these treatments may be associated with adverse effects, including sexual dysfunction, which can limit long-term adherence [15]. Consequently, alongside conventional pharmacological approaches, growing interest has been directed toward phytochemicals and natural compounds capable of modulating androgen-related pathways, inflammation and oxidative stress [16].

Within this framework, oxidative stress results from a disruption between increased production of reactive oxygen species (ROS) and the body’s natural antioxidant systems, such as the enzymes superoxide dismutase (SOD) and glutathione peroxidase 3 (GPX3). Ongoing oxidative stress activates redox-sensitive signalling pathways, particularly NF-κB, which then raises the production of pro-inflammatory mediators, including TNFα and IL-1β, maintaining the hyperplastic state of prostate tissue. The use of natural bioactive substances with antioxidant and anti-inflammatory properties could therefore impact these closely interconnected molecular pathways.

Gastrodiae elata Blume (named Gastrodiae), known as Tian Ma in traditional Asian dietary and medicinal practices, has attracted attention for its antioxidant and anti-inflammatory activities, largely due to its phenolic constituents, such as gastrodin and 4-hydroxybenzylic acid [17]. Although studies directly addressing prostate models are limited, experimental evidence indicates that Gastrodiae extracts can suppress inflammatory responses and modulate cell proliferation across diverse biological contexts. For instance, extracts have been shown to inhibit inflammatory signalling by modulating mitogen-activated protein kinase (MAPK) pathways in macrophages and animal models of arthritis [18], while anti-proliferative and pro-apoptotic effects have been observed in several in vitro systems [19]. These properties suggest potential relevance for biological processes underlying BPH and warrant further investigation.

Coenzyme Q10 (Q10), a lipophilic quinone present in eukaryotic cells, plays a vital role in mitochondrial energy production and cellular redox balance [20]. Q10 acts as a potent antioxidant by scavenging reactive oxygen species and preventing lipid peroxidation and oxidative damage to biomolecules [21]. Growing scientific interest in Q10 has emerged in conditions characterised by chronic inflammation, oxidative stress, and mitochondrial dysfunctionall of which are involved in BPH pathophysiology [22,23,24,25]. Within this framework, the combination of Gastrodiae and Q10 represents a promising nutraceutical strategy, as both compounds exhibit antioxidant and anti-inflammatory activities, acting through partially distinct and potentially complementary molecular pathways. Preclinical studies indicate that Gastrodiae influences MAPK- and NF-κB-related signalling and regulates apoptosis pathways [18,26,27], whereas Q10 sustains mitochondrial redox balance and reduces NF-κB-linked inflammatory responses [28,29]. Their combined use may thus enable coordinated regulation of multiple mechanisms essential for maintaining prostate physiological stability. From a food science and nutraceutical perspective, increasing attention has been devoted to food-derived or food-related bioactive compounds capable of supporting organ-specific functions through multitarget mechanisms rather than strictly pharmacological approaches. In this context, Gastrodiae and coenzyme Q10 are recognised as bioactives with well-established safety profiles and documented biological activities. The present study, therefore, aimed to investigate the biological effects of a formulation combining Gastrodiae extract and Q10 using food-relevant in vitro models, with the objective of elucidating its ability to modulate key processes involved in maintaining prostate physiological balance under conditions associated with prostatic hyperplasia.

2. Materials and Methods

All experiments were designed to evaluate the biological effects of food-derived bioactive compounds using food-relevant in vitro models.

2.1. Gastrodiae Elata Blume Extract Phytochemical Characterisation

2.1.1. Determination of Total Polyphenols

As reported in the literature [30], the Folin–Ciocalteu method was used to evaluate the total polyphenol content in the raw material. Briefly, 1 mL of sample, previously diluted with water to at least 60 mL, was combined with 5 mL of 2 M Folin–Ciocalteu reagent and allowed to react for approximately 1–8 min. Subsequently, 15 mL of a 20% (w/v) Na₂CO₃ solution was added, and the mixture was brought to a final volume of 100 mL with water. The reaction mixture was kept at room temperature, and absorbance was measured at 765 nm using a cuvette with a 1 cm path length. Gallic acid (GA) served as the calibration standard. Results are reported as mean ± SD (%) from five independent experiments, each conducted in triplicate.

2.1.2. Determination of Total Triterpenes

Total triterpenes were quantified using the vanillin–perchloric acid colourimetric method [31]. The sample extract was reacted with perchloric acid and heated to promote the formation of the chromophore. After cooling, the vanillin solution in glacial acetic acid was added. The absorbance was recorded at 548 nm. Oleanolic acid was used as the calibration standard, and triterpene content was expressed as mean ± SD (%) of five independent experiments performed in triplicate.

2.1.3. Determination of Total Lignans by HPLC

Lignan content was analysed using high-performance liquid chromatography (HPLC) [32]. Extraction was performed using a suitable organic solvent system, followed by sample filtration. Separation was performed on a reversed-phase C18 column with gradient elution using acidified water and acetonitrile (or methanol) as the mobile phases. Detection was performed with a UV detector (Infinite 200 Pro MPlex plate reader, Tecan, Männedorf, Switzerland) set to 280–290 nm, corresponding to the specific lignan profile. Quantification was achieved through external calibration with secoisolariciresinol diglucoside (SDG) as the standard. Data are presented as mean ± SD (%) from five independent experiments carried out in triplicate

2.1.4. Determination of Anthraquinone Derivatives by HPLC

Total anthracene derivatives were quantified by HPLC after sample extraction and, when required, acid hydrolysis to convert glycosidic forms into their corresponding aglycones [33]. Chromatographic separation employed a reversed-phase C18 column with a gradient elution system using water and acetonitrile (or methanol) as the mobile phase. Detection was performed using a UV–Vis detector (Infinite 200 Pro MPlex plate reader, Tecan, Männedorf, Switzerland), typically set at 254 nm or 430 nm, depending on the specific anthraquinone compounds analysed. Emodin served as the external calibration standard, and total anthracene levels are reported as mean ± SD (%) from five independent experiments, each conducted in triplicate.

2.1.5. Determination of Total Carbohydrates by the Phenol–Sulphuric Acid Method

Polysaccharide content was measured using the phenol–sulphuric acid method [34]. After mixing the diluted sample with 5% phenol and concentrated sulphuric acid, absorbance at 490 nm was recorded (UV-Vis; Infinite 200 Pro MPlex plate reader, Tecan, Männedorf, Switzerland). Glucose served as the standard, and results are presented as mean ± SD (%) from five independent experiments performed in triplicate.

2.2. Sample Preparation

To evaluate the potency of a combination of Gastrodiae and Q10 in counteracting BPH, we tested both compounds individually and in combination. Specifically, the Gastrodia extract (extract/drug ratio: 10:1, referring to the ratio between the dried herbal drug and the obtained extract; dry, powdered Rhizoma extract, 100 mesh; containing polysaccharides, polyphenols, triterpenoids, and lignans; water-solvent extract) and Coenzyme Q10 (Q10, 99.2% purity) were investigated for their complementary and enhancing actions. Both substances under study were supplied by Nutra Futura s.r.l. (Legnano, Italy).

To ascertain the most efficacious concentrations for the experimental procedures, a dose–response screening was conducted for both Gastrodiae and Q10, utilising concentrations documented in the extant literature. Indeed, for Gastrodiae, the concentration range is 100 µg/mL to 300 µg/mL [35], whereas that of Q10 is 10 µg/mL to 100 µg/mL [36].

They were prepared in Dulbecco’s Modified Eagle’s Medium (DMEM, Merck Life Science, Rome, Italy), supplemented without foetal bovine serum (FBS), supplemented with 2 mM L-glutamine and 1% penicillin–streptomycin (all components of the preparation medium were supplied by Merck Life Science, Rome, Italy). A concentration of 10 nM DHT (Merck Life Science, Roma, Italy) was added directly to the prostate co-culture medium to induce the BPH condition [37,38]. The 10 mM stock solution (3.7 mg in 1 mL) of finasteride (Merck Life Science, Rome, Italy) was diluted in DMEM without phenol red and serum to obtain a concentration of 1 µM in the well [39]. Also, the 10 mM stock solution (5.3 mg in 1 mL) of dutasteride (Merck Life Science, Rome, Italy) was diluted in DMEM without phenol red and serum to obtain a concentration of 1 µM in the well [40]. Both chemical agents are 5α-reductase inhibitors that act on isoform 2 (finasteride) and on isoforms 1 and 2 (dutasteride), respectively, and both were used as positive controls in the following analyses: testosterone, DHT, AR, and 5α-reductase (isoforms 1 and 2).

For all tests, the negative control is represented by the control cells placed in the respective culture medium.

2.3. Cell Cultures

Caco-2 cells (ATCC, Manassas, VA, USA), a human colorectal adenocarcinoma line, were employed to model the intestinal epithelial barrier in vitro [41]. Cells at passages 26–32 were used to ensure representative paracellular permeability and nutrient absorption features [42]. Cultures were maintained in DMEM Advanced (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin–streptomycin (all from Merck Life Science, Rome, Italy) at 37 °C with 5% CO₂. For viability assays, 1 × 10⁴ cells were seeded per 96-well, while 2 × 10⁴ cells were plated on 6.5 mm Transwell^® inserts (0.4 μm pore, Corning^® Costar^®) in 24-well plates for barrier and absorption studies. The intestine–prostate axis was modelled by culturing Caco-2 cells on Transwell^® inserts above a co-culture of RWPE-1 and WPMY-1 prostate cell lines in the lower compartment. Cells were synchronised 8 h prior to stimulation in DMEM lacking phenol red and containing 0.5% FBS, 2 mM L-glutamine, and 1% penicillin–streptomycin.

RWPE-1 prostate epithelial cells (ATCC) were cultured in serum-free keratinocyte medium (K-SFM, Thermo Fisher) with 0.05 mg/mL bovine pituitary extract and 5 ng/mL epidermal growth factor (Merck Life Science), maintained at 37 °C and 5% CO₂.

WPMY-1 stromal cells (ATCC) were grown in DMEM (Merck Life Science) with 5% FBS, 1 mM sodium pyruvate, 4 mM L-glutamine, and 1% penicillin–streptomycin at 37 °C and 5% CO₂.

The prostate co-culture was established with RWPE-1 and WPMY-1 cells using a 1:1 mix of K-SFM and DMEM, supplemented with 2.5% FBS, 0.025 mg/mL BPE, 2.5 ng/mL EGF, 0.5 mM sodium pyruvate, 2 mM L-glutamine, and 0.5% penicillin–streptomycin, following literature protocols [37,38].

2.4. Experimental Design

This study was divided into three phases to assess the beneficial effects of Gastrodiae and Q10 in reducing BPH after crossing the intestinal barrier (the scheme is shown in Figure 1). In phase 1, a dose–response screening was performed to identify the optimal and effective concentration for the next phase. To achieve this, a series of integrity tests was conducted, including measurements of trans-epithelial electrical resistance (TEER). Additionally, absorption tests were performed using a fluorescent probe on a 3D model of the intestinal barrier. These tests were conducted over a period of 1 to 6 h [43]. In the second phase, selected concentrations were tested after intestinal transit in a prostate co-culture under physiological conditions, creating the gut–prostate axis. The intestinal barrier model was treated with compounds for 6 h. The basolateral medium, containing crossed compounds or metabolites, was then collected and added to the prostatic co-culture, where prostate cells were exposed for 24 h. Cell viability was assessed via MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), together with ROS production (Cytochrome c reduction) and lipid peroxidation (TBARS assay), also analysed.

Figure 1.

A schematic representation of the experimental protocol is provided below. This study was conducted in three sequential phases: dose–response screening and intestinal barrier assessment (Phase 1), evaluation of selected concentrations after intestinal transit using a gut–prostate co-culture model (Phase 2), and investigation of the effects on an in vitro model of benign prostatic hyperplasia (BPH) induced by DHT (Phase 3).

During phase 3, the prostate co-culture system was employed to assess the impact of tested compounds on a BPH-like in vitro model following simulated intestinal passage, with BPH conditions induced by treating cells with 10 nM DHT for four days [37,38]. In this model, a series of experiments were performed to examine cell proliferation, levels of proliferating cell nuclear antigen (PCNA) by ELISA kit, oxidative stress parameters such as ROS production by Cytochrome C reduction, Superoxide dismutase (SOD) and Glutathione Peroxidase 3 (GPX3) levels by specific ELISA kit, and the production of key pro-inflammatory cytokines, including NF-κB, TNFα, and IL-1β (all analysed by ELISA kit), after treatment with intestinal conditioned medium for 24 h. In addition, the apoptotic pathway (BAX and Bcl-2) was also analysed through densitometric analysis after Western blot. Principal intracellular pathways associated with BPH, including testosterone and dihydrotestosterone (DHT) levels, androgen receptor (AR), 5-α-reductase isoforms 1 and 2, luteinising hormone (LH), follicle-stimulating hormone (FSH), and the serotonin receptor hydroxytryptamine receptor 1A (HTR1A), were evaluated using ELISA assays.

2.5. In Vitro Intestinal Barrier Model

Following a literature-based protocol recognised by both the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) [41,44,45], an in vitro intestinal barrier model was established using the Transwell^® system (Corning^® Costar^®, Merck Life Science, Rome, Italy) to evaluate the permeability of various substances. Caco-2 cells were seeded and cultured in complete medium for 21 days according to standard methods, with regular medium replacement in both the apical and basolateral chambers to promote optimal growth and differentiation. This procedure ensured the formation of an intact monolayer, replicating physiological conditions relevant for further analyses [41]. On day 21, when transepithelial electrical resistance (TEER) reached ≥400 Ω·cm², absorption studies commenced [46]. Prior to stimulation, the apical medium was adjusted to pH 6.5 (reflecting small intestinal conditions), while the basolateral side was set to pH 7.4, similar to blood [41]. Cells were equilibrated at 37 °C with 5% CO₂ for 15 min before the addition of test substances. Fluorescence was measured with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 490/514 nm. Cells were exposed to test compounds for 2–6 h before analysing absorption rates. Results were calculated as the percentage of the initial amount permeating the monolayer, and the permeation rate (J, [nmol min (mg protein)]) was determined as follows [47]:

J = Jmax [C]/(Kt + [C]) (1)

where

C: the initial concentration of fluorescein.

Jmax: the maximum permeation rate.

Kt: the Michaelis–Menten constant.

Results are expressed as the means ± SD (%).

2.6. Prostate Epithelial–Stromal Co-Culture Model

The effects of the tested substances on BPH in vitro, after simulated intestinal absorption, were evaluated using a prostate co-culture model as reported in the literature [37,38]. RWPE-1 and WPMY-1 cells were seeded in a 1:1 ratio (600,000 cells/well) in 12-well plates and cultured in a 1:1 mix of K-SFM and DMEM, supplemented with 2.5% FBS, 0.025 mg/mL BPE, 2.5 ng/mL EGF, 0.5 mM sodium pyruvate, 2 mM L-glutamine, and 0.5% penicillin–streptomycin (all from Merck Life Science, Rome, Italy). Hydrogels with 2% agarose were prepared using the Small Spheroid 12–256 system (Microtissues Inc., Providence, RI, USA). After four days, the co-culture was treated with 10 nM DHT for another four days to induce a BPH-like condition [37,38].

2.7. MTT-Based Cell Viability Assay

The cytotoxicity of all tested substances was evaluated in vitro using the MTT assay (Merck Life Science, Rome, Italy), following established protocols [48]. After the treatment period, cell viability was measured spectrophotometrically (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) by recording absorbance at 570 nm and correcting at 690 nm. Results were compared to untreated controls (set as 0%) and are shown as mean ± SD from five independent experiments, each conducted in triplicate.

2.8. Oxidative Stress Panel Evaluation

2.8.1. Assessment of ROS Production

Superoxide anion generation was assessed using the Cytochrome C reduction assay [48] by measuring absorbance at 550 nm in culture supernatants with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Results for O₂⁻ were expressed as mean ± SD (%) nanomoles of reduced cytochrome C per microgram of protein, based on five independent experiments conducted in triplicate versus control.

2.8.2. Lipid Peroxidation Assessment by TBARS Assay

Lipid peroxidation in prostate co-culture lysates was assessed using the TBARS assay kit (Cayman Chemical, Tallinn, Estonia) [49]. Absorbance was measured at 530–540 nm with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) and compared to a 0–50 µM standard curve. Results are reported as mean ± SD (%) relative to control (0 line), based on five independent experiments in triplicate.

2.8.3. Superoxide Dismutase (SOD) Activity Assessment

Superoxide dismutase (SOD) levels in prostate co-culture lysates were measured using Cayman’s Superoxide Dismutase Assay Kit, which detects Cu/Zn, Mn, and Fe isoforms [50]. Samples were compared to a standard curve ranging from 0.05 to 0.005 U/mL, and absorbance was read at 480 nm with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Results are expressed as mean ± SD (%) relative to the control (0 line), from five independent experiments conducted in triplicate.

2.8.4. Assessment of Glutathione Peroxidase 3 (GPX3) Levels

GPX3 levels were measured using the Human GPX3 (Glutathione peroxidase 3) ELISA Kit (FineTest, Wuhan, China) according to the instructions. GPX3 concentrations in prostate co-culture lysates were measured using a standard curve (1.563–100 ng/mL), with absorbance readings at 480 nm on a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Data are presented as mean ± SD (%) relative to control (0 line), based on five independent experiments conducted in triplicate.

2.9. Inflammatory Panel Evaluation

2.9.1. Assessment of NF-κB (p65) Activation

NF-κB (p65) levels in prostate co-culture lysates were measured using the NF-κB Transcription Factor Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) following the manufacturer’s protocol [47]. Nuclear extracts were prepared, and NF-κB was identified with a specific primary antibody. Absorbance was read at 450 nm with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland), and concentrations were calculated from a standard curve. Results are expressed as mean ± SD (%) relative to control (0 line), based on five independent experiments performed in triplicate.

2.9.2. Quantification of Tumour Necrosis Factor Alpha (TNFα)

The Human TNFα ELISA Kit (Merck Life Science, Milan, Italy) was used on the supernatant of the prostatic co-culture, following the standard protocol [48]. Sample absorbance was measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) and quantified in pg/mL with a 0–6000 pg/mL standard curve. Results are reported as mean ± SD (%) versus control (0 line), based on five independent triplicate experiments.

2.9.3. Quantification of Interleukin-1 Beta (IL-1β)

The Human IL-1β ELISA Kit (R&D Systems, Minneapolis, MN, USA) was used to determine the presence of IL-1β in prostatic co-culture cell lysates, following the instructions [48]. Absorbance was measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) and compared to a standard curve (3.906–250 pg/mL). Data are presented as mean ± SD relative to control (0 line) from five independent experiments in triplicate.

2.10. Assessment of Cell Proliferation by Crystal Violet Staining

Crystal violet staining was employed to evaluate cell proliferation in the prostate co-culture model following treatment, in line with established protocols [41]. Absorbance at 595 nm was recorded with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland), and cell counts were normalised to T0 and compared with controls. Results are shown as data from five independent triplicate experiments.

2.11. Assessment of Proliferating Cell Nuclear Antigen (PCNA) Levels

The Human Proliferating Cell Nuclear Antigen (PCNA) ELISA Kit (Thermo Fisher Scientific, Milan, Italy) was used on the supernatant of the prostatic co-culture, following the standard protocol. Absorbance at 450 nm was measured using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) and expressed in ng/mL against a standard curve ranging from 2.5 to 600 ng/mL. Results are presented as mean ± SD (%) compared to control (0 line) from five independent experiments conducted in triplicate.

2.12. Western Blot Analysis

For analysis, prostate co-cultures were lysed on ice using RIPA buffer (50 mM HEPES, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 10% glycerol, and 1%), supplemented with 5 mM MgCl₂, 1 mM EGTA, 1 mM NaF, 2 mM sodium orthovanadate, and a 1:100 protease inhibitor cocktail (all reagents from Merck Life Science, Rome, Italy). Protein samples (40 μg) were separated on 8% and 15% SDS-PAGE gels, transferred to PVDF membranes (GE Healthcare Europe GmbH, Milan, Italy), and incubated overnight at 4 °C with primary antibodies against BAX and Bcl-2 (both 1:500, Santa Cruz, CA, USA). Protein levels were normalised to β-actin (1:5000, Merck Life Science) and reported as mean ± SD (%).

2.13. Testosterone Conversion Panel

2.13.1. Measurement of Testosterone Levels

Following incubation with primary and secondary antibodies, absorbance was measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Concentrations were determined using a 3.9–500 ng/mL standard curve, and results are presented as mean ± SD relative to control (0 line) from five independent triplicate experiments.

2.13.2. Measurement of Dihydrotestosterone (DHT) Levels

Dihydrotestosterone (DHT) in prostatic co-culture supernatants was measured using a DHT ELISA Kit (Cusabio, Houston, TX, USA) according to the standard protocol [51]. Absorbance at 450 nm was recorded with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland), and concentrations (pg/mL) were calculated from a 10–2000 pg/mL standard curve. Data are expressed as mean ± SD (%) compared to control (0 line) based on five independent triplicate experiments.

2.13.3. Assessment of 5-α-Reductase Type 1 (SRD5A1) Levels

5-alpha-reductase 1 levels in prostate co-culture lysates were measured with an ELISA Kit (Cusabio, Houston, TX, USA) according to the manufacturer’s protocol. Absorbance was read at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland), and concentrations (pg/mL) were determined using a 12–2400 pg/mL standard curve. Results are presented as mean ± SD relative to control (0 line) from five independent experiments, each performed in triplicate.

2.13.4. Assessment of 5-α-Reductase Type 2 (SRD5A2) Levels

5-alpha-reductase 2 concentrations in prostate co-culture lysates were measured with an ELISA Kit (Cusabio, Houston, TX, USA) following the manufacturer’s instructions. Absorbance at 450 nm was read using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Switzerland), and pg/mL values were derived from a 12–2400 pg/mL standard curve. Data are presented as mean ± SD vs. control (0 line) from five triplicate experiments.

2.13.5. Assessment of Androgen Receptor (AR) Levels

The Androgen Receptor (AR) ELISA Kit (MyBioSource, San Diego, CA, USA) was used to quantify androgen receptor concentrations in prostate co-culture lysates. OD was measured at 450 nm, and concentration was determined by interpolation from an AR standard curve (0.312–20 ng/mL). Results are reported as mean ± SD (%) relative to control (0 line) from five experiments, each in triplicate.

2.14. Measurement of Luteinizing Hormone (LH) Levels

The Human Luteinizing Hormone (LH) QuickTest ELISA Kit (FineTest, Wuhan, China) was used to measure LH levels in prostate co-culture lysates. Optical density at 450 nm was measured, and concentrations were determined using an LH standard curve (0.312–20 ng/mL). Data are presented as mean ± SD (%) relative to control (0 line) from five independent triplicate experiments.

2.15. Measurement of Follicle-Stimulating Hormone (FSH) Levels

The Human Follicle-Stimulating Hormone (FSH) ELISA Kit (FineTest, Wuhan, China) was used to measure FSH levels in prostate co-culture lysates. FSH levels were calculated from a 1.563–100 mIU/mL standard curve after measuring absorbance at 450 nm. Data are shown as mean ± SD (%) versus control from triplicate experiments.

2.16. Measurement of Serotonin (5-Hydroxytryptamine, 5-HT) Levels

Serotonin levels in prostate lysates were determined using the ST/5-HT ELISA kit (FineTest, Wuhan, China) following the manufacturer’s protocol. Absorbance at 450 nm was measured, and concentrations (1.563–100 ng/mL) were derived from a standard curve. Results are expressed as mean ± SD (%) compared to the control from five triplicate experiments.

2.17. Assessment of Serotonin Receptor 1A (5-HTR1A) Levels

5HTR1A levels in co-culture prostate model supernatants were determined using the Human HTR1A ELISA Kit (FineTest, Wuhan, China) according to the manufacturer’s instructions. Absorbance at 450 nm was measured, and concentrations (1.56–10 ng/mL) were calculated from a standard curve. Results are reported as mean ± SD (%) versus control from five independent triplicate experiments.

2.18. Statistical Analysis

For all experimental methods except Western blot, data are presented as mean ± SD from at least five biological replicates, each with three technical replicates. Western blot results are shown as mean ± SD of optical density from at least 3 independent biological replicates, each performed in triplicate; representative blots are provided. Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test in GraphPad Prism 10.2.3 (GraphPad Software, La Jolla, CA, USA), with significance set at p < 0.05. Results from other experiments were normalised to a control value of 0%.

3. Results

3.1. Phytochemical Characterisation of Gastrodiae Elata Blume

The phytochemical characterisation (Table 1) of a dry extract of Gastrodiae (drug–extract ratio 10:1) revealed a compositional profile mainly characterised by polysaccharides, with a quantified content of 10.5% (w/w). The extract also contained measurable amounts of total triterpenes (approximately 2% w/w) and total polyphenols (approximately 0.5% w/w). HPLC analysis further indicated that lignans and anthraquinone derivatives were present at very low levels, below 0.05% (w/w). Overall, these results show that the phytochemical profile of the extract is primarily composed of primary metabolites and selected phenolic compounds, consistent with the traditional use of Gastrodiae and with previous reports on its biological activities. The low levels of specific secondary metabolite classes, together with the defined drug-to-extract ratio and measured polysaccharide content, support the potential for further research within nutraceutical and food bioactive frameworks.

Table 1.

The table summarises the phytochemical characterisation of a dry extract of Gastrodiae.

Component	Method	Content (%)
Polysaccharides	UV-Vis Method	10.5 ± 0.85
Triterpenoids	UV-Vis Method	2 ± 0.37
Polyphenols	UV-Vis Method	0.5 ± 0.03
Lignans	HPLC	0.05 ± 0.002
Anthracene	HPLC	0.05 ± 0.002

The analysed component, the analytical method, and the content expressed as a percentage weight-by-weight of the extract (% w/w) are reported. All data are presented as mean ± SD from five independent experiments performed in triplicate. HPLC analysis was employed to identify lignans and anthracene.

3.2. Dose–Response and Time-Dependent Screening in an In Vitro Intestinal Barrier Model

In the initial phase, intestinal barrier integrity and permeability were assessed using Caco-2 cell monolayers to determine appropriate concentrations for subsequent experiments. Cells grown on Transwell^® supports were exposed to various concentrations of Gastrodiae extract (100, 200, and 300 µg/mL) and coenzyme Q10 (10, 50, and 100 µg/mL) for incubation periods ranging from 1 to 6 h, with transepithelial electrical resistance (TEER) measured at each time point. As shown in Figure 2A,B, treatment with Gastrodiae extract resulted in higher TEER values compared to untreated control cells throughout the experiment, indicating an effect on intestinal barrier integrity. Of the tested concentrations, 100 µg/mL produced the most significant effect compared to 200 and 300 µg/mL (p < 0.05), particularly between 3 and 6 h. Both TEER and permeability assessments showed a peak response around 4 h of treatment with Gastrodiae at 100 µg/mL. Similarly, as shown in Figure 2A,B, exposure to Q10 increased TEER values compared to control cells throughout the incubation period (p < 0.05). Among the tested concentrations, Q10 at 100 µg/mL demonstrated a greater ability to preserve intestinal barrier integrity than 10 and 50 µg/mL. Based on these preliminary results, 100 µg/mL was chosen as the optimal concentration for both Gastrodiae extract and Q10 in subsequent experiments.

Figure 2.

Screening of integrity and absorption profile in an in vitro intestinal barrier model (dose–response study and time-dependent study). In (A), the TEER values of Gastrodiae; in (B), the absorption rate of Gastrodiae; in (C), the TEER values of Q10; in (D), the absorption rate of Q10. Gastrodiae = Gastrodiae elata Blume; Q10 = Coenzyme Q10. Data are expressed as mean ± SD (%) of 5 independent experiments performed in triplicate, normalised to the control line (0%). In (A–C), p < 0.05 vs. Control (untreated cells); * p < 0.05 vs. other concentrations. In (D), * p < 0.05 vs. Control; α p < 0.05 vs. other concentrations.

Following concentration selection, additional barrier integrity and permeability analyses were conducted to compare the effects of individual compounds with those of their combination. As shown in Figure 3A, treatment with each individual compound resulted in higher TEER values compared to control cells (p < 0.05). Notably, the combined formulation of Gastrodiae and Q10 further increased TEER values relative to both the control group and the individual treatments (p < 0.05). Consistent with these findings, permeability analysis confirmed the trans-epithelial passage of all tested samples (Figure 3B), with the combined formulation displaying enhanced permeability profiles compared to the single components (p < 0.05). Meanwhile, the stability of the main phytochemical constituents of Gastrodiae following simulated intestinal passage was evaluated using UV–Vis analysis (Figure 3C). The polysaccharide content, initially quantified at 10.5% (w/w) in the native extract, decreased to 7.6% after intestinal passage of Gastrodiae alone, corresponding to an approximate recovery of 72%. In contrast, co-administration with Q10 resulted in a higher retained polysaccharide content (8.9%), equivalent to approximately 85% recovery. Similarly, total polyphenols, initially quantified at 0.5% (w/w), decreased to 0.33% following intestinal passage of Gastrodiae alone (around 66% recovery). When Gastrodiae was combined with Q10, the post-passage polyphenol content was higher (0.39%), representing an approximate recovery of 78%, indicating improved stability of phenolic constituents during simulated intestinal transit.

Figure 3.

Effects of the combination on integrity and absorption on 3D intestinal in vitro model. In (A), TEER values of all agents tested; in (B), the absorption of all agents tested; and in (C), the main component content of Gastrodiae after intestinal transit. Gastrodiae = Gastrodiae elata Blume; Q10 = Coenzyme Q10. Data are expressed as mean ± SD (%) of 5 independent experiments performed in triplicate, normalised to the control line (0%). In (A), p < 0.05 vs. Control (untreated cells); * p < 0.05 vs. other concentrations. In (B), p < 0.05 vs. Control; * p < 0.05 vs. single agents.

In conclusion, the data indicated a possible additive effect of Q10 on the activity of Gastrodiae at the intestinal level, with regard to both integrity and absorption.

3.3. Effects of Gastrodiae Elata, Q10, and Their Combination on the Intestine–Prostate Axis

Based on the preliminary screening results, the effects of individual compounds and their combination on the intestine–prostate axis were evaluated by assessing cell viability, ROS production, and lipid peroxidation. As shown in Figure 4A, treatment with Gastrodiae extract or Q10 resulted in increased cell viability compared with the control group (p < 0.05). Notably, the combined formulation of Gastrodiae and Q10 was associated with a further increase in cell viability, corresponding to an increase of approximately 24% compared with Gastrodiae alone (p < 0.05) and approximately 34% compared with Q10 alone (p < 0.05). The effects on cell viability were accompanied by changes in oxidative status, as evaluated by ROS analysis. As shown in Figure 4B, treatment with Gastrodiae and Q10 maintained ROS production at levels comparable to those of control cells (approximately 3% and 4.75%, respectively; p < 0.05). Compared with the individual compounds, the combined formulation resulted in a further reduction in ROS levels relative to control values (p < 0.05), corresponding to approximately 5.71-fold and 7.33-fold reductions compared with Gastrodiae and Q10 alone, respectively (p < 0.05). These findings were further supported by lipid peroxidation analysis (Figure 4C). Treatment with Gastrodiae alone was associated with a greater reduction in lipid peroxidation compared with Q10 (approximately 70%, p < 0.05). The combined formulation of Gastrodiae and Q10 led to a statistically significant decrease in lipid peroxidation levels compared with control cells (p < 0.05). Additionally, lipid peroxidation levels were lower in cells exposed to the combined formulation than in those treated with the individual compounds, with reductions of approximately 1.77-fold and 2.31-fold compared with Gastrodiae and Q10 alone, respectively (p < 0.05). Overall, the data suggest that the combination of Gastrodiae and Q10 is associated with a different modulation of cell viability and oxidative stress-related parameters along the gut-prostate axis compared to the individual compounds. The combined formulation exerted coordinated effects on cell viability, ROS production, and lipid peroxidation, supporting its relevance for further investigation in the context of nutraceutical and food bioactive research.

Figure 4.

Effects on the intestine–prostate axis. In (A), analysis of cell viability assessed by MTT test; in (B), analysis of ROS production by reduction in Cytochrome C; and in (C), lipid peroxidation analysis determined by a specific kit. Gastrodiae = Gastrodiae elata Blume 100 µg/mL; Q10 = Coenzyme Q10 100 µg/mL. Data are expressed as mean ± SD (%) of five independent experiments performed in triplicate, normalised to the control line (0%). * p < 0.05 vs. Control; α p < 0.05 vs. Q10; β p < 0.05 vs. Gastrodiae.

3.4. Effects on a Prostatic Co-Culture Model Under BPH Conditions

Compounds that crossed the intestinal barrier model were subsequently applied to a prostate epithelial–stromal co-culture system under BPH-like conditions to evaluate their effects on oxidative stress, inflammatory markers, and cellular pathways associated with prostatic hyperplasia. A series of experiments was therefore performed to assess ROS production, antioxidant enzyme levels (SOD and GPX3), inflammatory mediators, and intracellular signalling events that are typically altered by DHT stimulation.

Under BPH-like conditions, increased oxidative stress and impaired antioxidant defences were observed. As shown in Figure 5A,B, exposure to 10 nM DHT resulted in increased ROS production and reduced SOD levels compared with untreated control cells (p < 0.05). Treatment with Gastrodiae extract or Q10 alone attenuated DHT-induced ROS accumulation and partially restored SOD levels (p < 0.05 vs. DHT). Notably, the combined formulation of Gastrodiae and Q10 was associated with a more pronounced modulation of these parameters, showing increases of approximately 18% and 2.79-fold compared with Gastrodiae alone, and 51% and 4.80-fold compared with Q10 alone, respectively (p < 0.05).

Figure 5.

Antioxidant and anti-inflammatory effects on a prostatic co-culture model under BPH. In (A), analysis of ROS production by reduction in Cytochrome C; in (B), SOD quantification determined by a specific kit; in (C), GPX3 levels quantified by ELISA Kit; in (D), NF-κB production was detected with a specific ELISA Kit; in (E), TNFα production was detected with a specific ELISA Kit; and in (F), IL-1β production was detected with a specific ELISA Kit. DHT = 10 nM; Gastrodiae = Gastrodiae elata Blume 100 µg/mL; Q10 = Coenzyme Q10 100 µg/mL. Data are expressed as mean ± SD (%) of 5 independent experiments performed in triplicate, normalised to the control line (0%). * p < 0.05 vs. Control; φ p < 0.05 vs. DHT; α p < 0.05 vs. Q10; β p < 0.05 vs. Gastrodiae.

GPX3 levels were further evaluated as an indicator of oxidative stress regulation and redox balance (Figure 5C). DHT treatment significantly reduced GPX3 levels compared with the untreated control (p < 0.05). In contrast, exposure to Gastrodiae, Q10, or their combination resulted in increased GPX3 levels under DHT-stimulated conditions (p < 0.05 vs. DHT). Gastrodiae alone induced a greater increase in GPX3 levels than Q10, corresponding to an increase of approximately 87% (p < 0.05). The combined formulation further enhanced GPX3 levels, showing approximately 1.2- and 2.51-fold increases compared with Gastrodiae and Q10 alone, respectively (p < 0.05).

The inflammatory profile of the prostatic co-culture under BPH-like conditions was subsequently assessed by analysing NF-κB activation and the production of the pro-inflammatory cytokines TNFα and IL-1β. As shown in Figure 5D–F, DHT stimulation significantly increased NF-κB, TNFα and IL-1β levels compared with the untreated control (p < 0.05), with increases of approximately 15%, 16% and 20.5%, respectively. Treatment with Gastrodiae or Q10 alone reduced the levels of all investigated inflammatory markers under DHT-stimulated conditions (p < 0.05 vs. DHT). Gastrodiae was associated with a greater reduction in NF-κB levels than Q10, corresponding to approximately a 67% decrease (p < 0.05).

The combined formulation of Gastrodiae and Q10 resulted in a further decrease in NF-κB levels compared with Q10 alone (approximately 71%, p < 0.05), while no statistically significant difference was observed relative to Gastrodiae alone. Consistent trends were observed for TNFα and IL-1β production (Figure 5E,F). DHT significantly increased TNFα and IL-1β levels (p < 0.05), whereas treatment with the individual compounds reduced cytokine production. Gastrodiae alone showed a greater reduction in TNFα and IL-1β levels compared with Q10 (approximately 68% and 74%, respectively; p < 0.05). The combined formulation exhibited reductions comparable to those observed with Gastrodiae alone and greater than those observed with Q10, resulting in approximately 72% decreases in TNFα and 79% decreases in IL-1β (p < 0.05).

In BPH-like conditions, increased cell proliferation is often accompanied by decreased apoptotic activity. Based on this rationale, proliferation- and apoptosis-related parameters were analysed to assess the effects of the tested compounds in the prostatic co-culture model. Cell proliferation and proliferating cell nuclear antigen (PCNA) levels were therefore evaluated following DHT stimulation.

As shown in Figure 6A, pretreatment with 10 nM DHT significantly increased cell proliferation compared to the physiological control (untreated cells, 0% line; p < 0.05). Exposure to Gastrodiae extract or Q10 alone markedly attenuated the DHT-induced proliferative response. Specifically, Gastrodiae and Q10 reduced cell proliferation by about 80% and 74%, respectively, compared with DHT-treated cells (p < 0.05). The combination of Gastrodiae and Q10 led to a further reduction in DHT-induced cell proliferation by approximately 82%, although this decrease was not statistically different from the effects observed with each compound individually.

Figure 6.

Anti-proliferative and apoptotic effects on a prostatic co-culture model under BPH. In (A), cell proliferation is evaluated by Crystal violet staining; in (B), PCNA levels are measured using an ELISA kit; in (C,D), BAX and Bcl-2 densitometric analyses are shown; and in (E), an example of a Western blot lane derived from three experimental tests performed in triplicate is reported. All abbreviations are shown in Figure 5. Data are expressed as mean ± SD (%) of 5 independent experiments performed in triplicate, normalised to the control line (0%). * p < 0.05 vs. Control; φ p < 0.05 vs. DHT; α p < 0.05 vs. Q10.

These observations were supported by PCNA analysis (Figure 6B). DHT stimulation caused a significant increase in PCNA levels compared to control cells (p < 0.05). Treatment with all tested agents significantly lowered PCNA levels under BPH-like conditions (p < 0.05 vs. DHT). Gastrodiae alone was linked to a greater reduction in PCNA levels compared to Q10, with an approximate decrease of 19.5% (p < 0.05). The combined formulation further lowered PCNA levels, with reductions of about 19% compared to Gastrodiae alone (p < 0.05) and approximately 32% compared with Q10 alone (p < 0.05).

Apoptotic pathways were further assessed by analysing through densitometric analysis BAX and Bcl-2 protein expression, which are commonly used indicators of apoptosis regulation in prostatic cells. An altered BAX/Bcl-2 balance in favour of Bcl-2 is generally associated with decreased apoptotic signalling under BPH-like conditions. Figure 6C,D indicate that stimulation with DHT lowered BAX expression and increased intracellular Bcl-2 levels compared to untreated controls (p < 0.05). Treatment with Gastrodiae, Q10, or both together partially reversed these alterations, resulting in higher BAX and lower Bcl-2 expression compared with cells exposed to DHT alone (p < 0.05). Among the individual compounds, Gastrodiae showed greater modulation of apoptosis-related markers than Q10, increasing BAX expression by approximately 46% and reducing Bcl-2 expression by approximately 26% (p < 0.05). Notably, the combined formulation of Gastrodiae and Q10 showed a more pronounced modulation of the BAX/Bcl-2 balance compared with either compound alone, with additional increases in BAX densitometric intracellular presence of approximately 50% and 96.5% and decreases in Bcl-2 expression of approximately 34% and 69% relative to Gastrodiae and Q10, respectively (p < 0.05).

Under BPH-like conditions, increased 5-α-reductase activity causes higher dihydrotestosterone (DHT) production, leading to more androgen receptor (AR) activation within prostate cells and supporting greater cell proliferation [3]. Based on this biological framework, the effects of the tested compounds on androgen-related parameters were assessed in the prostatic co-culture model.

As shown in Figure 7A,B, exposure to 10 nM DHT significantly increased the activity of 5-α-reductase isoforms 1 and 2 compared to the untreated control (p < 0.05). Treatment with Gastrodiae extract or Q10 alone significantly decreased the activity of both 5-α-reductase isoforms relative to DHT-stimulated conditions (p < 0.05). Notably, the combined formulation of Gastrodiae and Q10 caused a more substantial reduction in 5-α-reductase isoform activity compared with Q10 alone, amounting to approximately 81% (p < 0.05).

Figure 7.

Mechanism analyses for prostate hyperplasia on the prostatic co-culture model. In (A), 5-α-reductase 1 levels were evaluated by ELISA Kit; in (B), 5-α-reductase 2 levels were evaluated by ELISA Kit; in (C), DHT quantification was measured by ELISA Kit; in (D), testosterone levels were detected with ELISA Kit; and in (E), AR levels were measured by ELISA Kit. All abbreviations are shown in Figure 5. Data are expressed as mean ± SD (%) of 5 independent experiments performed in triplicate, normalised to the control line (0%). * p < 0.05 vs. Control; φ p < 0.05 vs. DHT; α p < 0.05 vs. Q10; β p < 0.05 vs. Gastrodiae.

For a mechanistic reference, finasteride and dutasteride were included in the experimental design to contextualise the modulation of androgen-related pathways within the in vitro model. Their inclusion was not intended to suggest therapeutic equivalence but rather to offer a comparative framework for interpreting changes in 5-α-reductase activity induced by the tested food-derived bioactive compounds. Under these conditions, the inhibitory effects of Gastrodiae alone and in combination with Q10 on 5-α-reductase activity fell within the range observed for the reference compounds.

Consistent with changes in 5-α-reductase activity, DHT levels rose while testosterone levels fell under DHT-stimulated conditions (Figure 7C,D). Treatment with Gastrodiae or Q10 alone partially reversed these effects, resulting in lower DHT levels and higher testosterone levels compared to DHT-treated cells (p < 0.05). Combining Gastrodiae and Q10 produced a greater impact than Q10 alone, reducing DHT levels by about 68% and increasing testosterone levels by roughly 73% (p < 0.05).

To further assess androgen pathways, levels of androgen receptor (AR), luteinising hormone (LH), and follicle-stimulating hormone (FSH) were measured after treatment. As shown in Figure 7E, DHT exposure significantly increased AR levels compared to controls (p < 0.05). All tested compounds reduced AR expression relative to DHT (p < 0.05). Notably, the combination of Gastrodiae and Q10 caused a greater AR reduction than Q10 alone (about 74%, p < 0.05), with no significant difference from Gastrodiae alone.

To further characterise the effects of the tested compounds on endocrine- and signalling-related parameters, luteinising hormone (LH), follicle-stimulating hormone (FSH), serotonin, and the serotonin receptor Htr1A were analysed following treatment in the prostatic co-culture model (Figure 8A–D).

Figure 8.

LH, FSH, Serotonin, and 5-Htr1a levels in the prostatic co-culture model. In (A), LH determination by ELISA Kit; in (B), FSH quantified by ELISA Kit; in (C), serotonin production was measured by an ELISA kit; and in (D), 5-HT1a levels were measured by an ELISA kit. All abbreviations are shown in Figure 5. Data are expressed as mean ± SD (%) of 5 independent experiments performed in triplicate, normalised to the control line (0%). * p < 0.05 vs. Control; φ p < 0.05 vs. DHT; α p < 0.05 vs. Q10; β p < 0.05 vs. Gastrodiae.

As shown in Figure 8A,B, treatment with the combined formulation of Gastrodiae and Q10 resulted in increased LH and FSH levels compared with Q10 alone, corresponding to increases of approximately 1.46-fold and 1.26-fold, respectively (p < 0.05). No statistically significant differences were observed between Gastrodiae alone and the DHT-stimulated condition for these parameters.

DHT stimulation significantly reduced serotonin levels and Htr1A receptor expression compared with untreated control cells (p < 0.05; Figure 8C,D). Treatment with Gastrodiae, Q10, or their combination was associated with increased serotonin levels and receptor expression relative to DHT-treated cells (p < 0.05). Importantly, treatment with the combination of Gastrodiae and Q10 resulted in a more significant increase in both serotonin levels and Htr1A expression compared to Gastrodiae alone (by approximately 72% and 70.5%, respectively; p < 0.05) and Q10 alone (by about 78% and 80%, respectively; p < 0.05).

4. Discussion

BPH poses a significant health concern for a large segment of the ageing male population. Lower urinary tract symptoms (LUTS) associated with BPH are known to negatively impact quality of life, emphasising the need for effective, well-tolerated management strategies [52,53]. Current pharmacological treatments, including 5-α-reductase inhibitors such as finasteride and dutasteride, are commonly used to reduce prostate size and relieve symptoms [54]. However, their clinical use may be restricted by side effects, including sexual dysfunction, gynaecomastia, and mood changes, which can adversely affect long-term adherence [55]. These limitations have driven greater interest in complementary and supportive approaches, including food-derived bioactive compounds and nutraceuticals, which may provide favourable safety profiles and multitarget biological activities [56,57,58]. This study examined the effects and mechanisms of specific natural compounds, both alone and with coenzyme Q10, using in vitro models relevant to BPH-like conditions. A salient aspect of this research pertained to the evaluation of the combined formulation of Gastrodiae and Q10, which exhibited an overall divergent biological modulation effect in comparison to the individual components. One potential consequence of the combination could be the integrated modulation of multiple biological processes involved in prostatic physiology, including oxidative stress, inflammatory signalling, and cell turnover. The analysis of androgen-related parameters, inflammatory mediators (NF-κB, TNFα, and IL-1β), and serotonin-related signalling contributed to a more integrated understanding of the pathways influenced by these food-derived bioactives.

Results from the intestinal barrier model indicated that the tested compounds did not adversely affect intestinal integrity and exhibited adequate in vitro permeability at the concentrations investigated. Following simulated intestinal passage, the compounds modulated cell viability, oxidative stress, and lipid peroxidation in the prostatic co-culture model, supporting the relevance of a gut–organ axis approach. Recent evidence suggests the existence of a biologically plausible gut–prostate axis, whereby intestinal barrier integrity, gut microbiota composition, and systemic inflammatory and metabolic mediators may influence prostate physiology and BPH development [59,60]. Although causal relationships in humans remain to be fully established, alterations in microbial diversity, circulating cytokines, and barrier dysfunction have been associated with prostate volume and inflammatory status [61]. Within this conceptual context, our in vitro model represents a simplified reconstruction of one mechanistic component of this interaction, focusing on epithelial translocation and downstream prostate responses, while not reproducing the full complexity of in vivo microbiota–immune–endocrine crosstalk.

Within this experimental framework, reference compounds such as finasteride and dutasteride were included to contextualise androgen-related mechanisms, in line with the existing literature describing their ability to reduce DHT levels and modulate prostate growth. Regarding Gastrodiae elata Blume, the available literature describes its antioxidant and anti-inflammatory properties, with reported effects on cell proliferation and inflammatory mediators in non-prostatic models [62,63,64]. The present findings expand on these observations by demonstrating, for the first time, the ability of Gastrodiae to modulate key molecular processes associated with BPH-like conditions, including oxidative stress, inflammatory signalling, and androgen-related pathways. Specifically, decreases in 5-α-reductase activity, DHT levels, and AR expression, along with increased testosterone and serotonin levels, were observed. These results suggest that Gastrodiae, alone or combined with Q10, may influence prostate-related cellular processes within a nutraceutical research context. Coenzyme Q10 also displayed consistent antioxidant and anti-inflammatory effects in the prostatic co-culture model, evidenced by reduced ROS production and lower NF-κB and TNFα levels, aligning with previous reports [65,66,67,68]. Additionally, Q10 affected hormonal parameters, including LH, FSH, and testosterone levels, indicating a broader role in maintaining cellular homeostasis. Importantly, the contribution of Q10 was selective and complementary, rather than homogeneous across all endpoints analysed. In particular, the most evident effect was observed in relation to the levels of LH, FSH and serotonin, as well as the expression of the serotonin receptor, while on the other parameters the integration did not show a systematic enhancement compared to the treatment with Gastrodiae alone. While Gastrodiae directly modulated inflammatory and androgen-related pathways, Q10 primarily supported mitochondrial-linked redox and endocrine mechanisms. As a mitochondrial electron carrier and antioxidant, Q10 stabilises intracellular redox balance and energy metabolism, particularly relevant in BPH-like conditions where oxidative stress drives hyperplastic growth [69]. Its effects on LH, FSH, and testosterone suggest modulation of steroidogenic homeostasis via mitochondrial support. Therefore, Q10 adds value primarily in outcomes linked to mitochondrial–redox–endocrine regulation, acting as a complementary component of the combined formulation rather than a dominant agent. Notably, the combined formulation of Gastrodiae and Q10 enhanced several of these effects, indicating complementary interactions between mitochondrial redox regulation and phytochemical-mediated signalling. These observations are aligned with prior in vivo and clinical studies [70].

The combined formulation also showed effects on cell proliferation and apoptosis-related markers, evidenced by reduced PCNA levels and modulation of the BAX/Bcl-2 balance. These results agree with previous evidence showing that oxidative stress, inflammation, and disrupted apoptotic signalling contribute to prostate hyperplasia [71,72,73,74,75,76,77]. The coordinated modulation of these pathways by the combination of Gastrodiae and Q10 supports the idea of a multitarget approach based on food-derived bioactives rather than single-mechanism intervention. Importantly, the differential effects observed under physiological versus DHT-stimulated conditions reflect context-dependent modulation rather than contradictory outcomes. Indeed, several natural compounds exerted cytoprotective antioxidant effects in healthy cells while suppressing inflammation- and androgen-driven hyperproliferation in pathological models [78,79,80,81]. These findings support our interpretation of a homeostatic, rather than purely anti-proliferative, mechanism of action.

In conclusion, in several endpoints, Gastrodiae alone produced effects comparable to the combination, suggesting a complementary rather than uniformly enhanced profile.

Although in vitro models offer a controlled, reproducible platform for studying specific molecular and cellular processes, they do not fully replicate the complexity of the human physiological environment. Therefore, the current results should be seen as preliminary and hypothesis-generating. Further studies using more advanced experimental models will be needed to confirm the relevance of these findings and to explore their potential translational implications.

From a food science and nutraceutical perspective, the present findings support investigating combined formulations of Gastrodiae and coenzyme Q10 as food-related bioactive compounds capable of modulating multiple pathways relevant to prostate physiological balance under conditions associated with BPH.

5. Conclusions

This study demonstrates that the combination of Gastrodiae elata Blume extract and coenzyme Q10 produces synergistic biological effects relevant to prostate health in food-relevant in vitro models. The formulation showed intestinal compatibility and, after simulated passage, impacted oxidative stress, inflammatory signalling, and androgen-related pathways in a prostate epithelial–stromal co-culture system.

Notably, the combined formulation generally exhibited more pronounced effects than the individual components, indicating functional complementarity and multitarget activity. These findings suggest that combining these bioactive compounds could be a promising approach within food-derived nutraceutical research aimed at supporting prostate physiological balance. However, further in vivo and clinical studies are needed to establish its translational relevance.

Abbreviations

The following abbreviations are used in this manuscript:

AR	Androgen receptor
BPE	Bovine pituitary extract
BPH	Benign prostatic hyperplasia
DHT	Dihydrotestosterone
DMEM	Dulbecco’s Modified Eagle’s Medium
DMEM-Adv	Advanced Dulbecco’s Modified Eagle’s Medium
EGF	Epidermal Growth Factor
ELISA	Enzyme-Linked Immunosorbent Assay
FBS	Foetal bovine serum
FSH	Follicle-stimulating hormone
GPX3	Glutathione Peroxidase 3
HTR1A	Hydroxy-tryptamine
IL-1	Interleukin 1
IL-10	Interleukin 10
IL-1β	Interleukin 1β
IL-6	Interleukin 6
IL-8	Interleukin 8
K-SFM	Serum-free keratinocyte medium
LH	Luteinising hormone
LUTS	Lower urinary tract symptoms
MAPK	Mitogen-activated protein kinase
MTT	3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NF-κB	Nuclear factor kappa-light-chain-enhancer of activated B cells
PCNA	Cell nuclear antigen
ROS	Reactive Oxygen Species
SOD	Superoxide dismutase
SRD5A1	3-oxo-5-alpha-steroid 4-dehydrogenase 1
SRD5A2	3-oxo-5-alpha-steroid 4-dehydrogenase 2
TEER	Trans-epithelial electrical resistance
TLR	Toll-Like Receptor
TNFα	Tumour necrosis factor α

Author Contributions

Conceptualization, R.G., S.M., and F.U.; methodology, R.G., S.M., and F.P.; software, R.G., S.M., and F.P.; validation, R.G., S.M., and F.U.; formal analysis, R.G., S.M., and F.U.; investigation, R.G., S.M., and F.U.; resources, F.U.; data curation, R.G., S.M., and F.U.; writing—original draft preparation, R.G., S.M., F.P., and F.U.; visualisation, F.U.; supervision, F.U.; project administration, F.U.; funding acquisition, F.U. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The Laboratory of Physiology stores raw data in a secure system to ensure permanent retention. This study’s data are available from the corresponding author upon reasonable request.

Conflicts of Interest

Authors Rebecca Galla and Francesca Parini were employed by the company Noivita Srls. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding Statement

This research received no external funding.