Perilla frutescens Britton var. acuta Kudo ameliorates OVA-induced allergic asthma via inhibiting NF-κB and p38 signaling pathways

Abstract

Allergic asthma, the most common phenotype of asthma, is a chronic inflammatory airway disease driven by Type 2 immune responses and characterized by eosinophilic infiltration, elevated IgE levels, and activation of inflammatory pathways. This study investigated the anti-inflammatory and immunomodulatory effects of Perilla frutescens var. acuta Kudo extract (PFE), a plant rich in rosmarinic acid (RA), using an ovalbumin (OVA)-induced mouse model of allergic asthma. Given RA’s well-documented anti-allergic properties, extraction conditions were optimized to maximize RA yield. PFE treatment significantly reduced immune cell infiltration in bronchoalveolar lavage fluid, suppressed Th2-associated cytokine production, and decreased serum IgE levels, thereby alleviating airway inflammation. Mechanistically, PFE inhibited activation of NF-κB and p38 MAPK pathways, key drivers of inflammatory responses in allergic asthma. These findings suggest that PFE attenuates allergic airway inflammation through both immune regulation and suppression of pro-inflammatory signaling, supporting its potential as a natural therapeutic agent for allergic asthma.

Introduction

Asthma is a chronic inflammatory respiratory disease that affects over 300 million people worldwide, with its prevalence continuing to rise, particularly in industrialized nations (Murdoch and Lloyd, 2010). Approximately 5% to 10% of the global population suffer from asthma, with variations influenced by regional and environmental factors (Wenzel, 2012). Among various types of asthma, allergic asthma is the predominant phenotype of asthma. It is estimated that up to 80% of childhood asthma cases and over 50% of adult cases have an allergic component (Johansson et al., 2001; Schatz and Rosenwasser, 2014). Clinically, asthma is characterized by bronchial hyperresponsiveness (BHR), excessive mucus production, and chronic inflammation, leading to recurrent episodes of wheezing, breathlessness, and coughing (Holgate, 2012; Lambrecht and Hammad, 2015). In allergic asthma, these respiratory symptoms are often accompanied by typical allergic manifestations such as sneezing, nasal congestion, rhinorrhea, and itchy or watery eyes, as the condition frequently coexists with comorbidities like allergic rhinitis or atopic dermatitis (Bousquet et al., 2008; Douwes and Pearce, 2002). The pathogenesis of allergic asthma is primarily driven by Type 2 inflammation, which involves a complex interplay among Th2 cells, eosinophils, and mast cells. This inflammatory response is mediated by key cytokines, including IL-4, IL-5, and IL-13, which contribute to airway inflammation and remodeling (Murphy and O’Byrne, 2010; Wenzel, 2012). Eosinophilic infiltration and elevated IgE levels are hallmarks features of allergic asthma, which is characterized by hypersensitivity to airborne allergens (Fahy, 2015).

Despite extensive research on allergic asthma, effective and long-term therapeutic options remain limited, particularly for patients with severe or steroid-resistant asthma (Adcock et al., 2008). Current treatments, including inhaled corticosteroids (ICS) and β2-adrenergic agonists, primarily focus on symptom management rather than addressing the underlying inflammatory mechanisms (Barnes, 2002). This has led to increasing interest in alternative and natural product-based treatments that offer anti-inflammatory and immunomodulatory benefits with fewer adverse effects (Subali et al., 2024). Perilla frutescens Britton var. acuta Kudo, commonly known as Perilla, has been widely used in traditional medicine and cuisine for its health-promoting effects (Adam et al., 2023; Lee et al., 2019; Seo and Baek, 2009). It is particularly noted for its anti-inflammatory, anti-allergic, and antibacterial properties (Huang et al., 2014; Kim et al., 2011; Liu et al., 2013). Among its numerous bioactive constituents, rosmarinic acid (RA) is especially abundant and has been extensively studied for its potent anti-inflammatory effects in conditions such as allergic rhinitis, atopic dermatitis, and asthma (Lee et al., 2008; Liang et al., 2016; Netala et al., 2024; Zhou et al., 2022). However, research optimizing its extraction and application for allergic asthma treatment remains insufficient.

In this study, we aimed to maximize the yield of rosmarinic acid from Perilla frutescens by optimizing the extraction conditions, ensuring that the highest concentration of the active compound is utilized for therapeutic evaluation. The optimized extract was then applied to an OVA-induced allergic asthma mice model to investigate its potential anti-inflammatory and immunomodulatory effects.

Materials and methods

Chemicals and reagents

Rosmarinic acid was obtained from Sigma Aldrich (St. Louis, MO, USA), HPLC-grade methanol, water, and acetonitrile of were procured from Fisher Scientific (Hampton, NH, USA), and ACS grade Formic acid of was obtained from Sigma-Aldrich. This study focused on analyzing rosmarinic acid with a purity ≥ 98%. Perilla frutescens Britton var. acuta Kudo extract (PFE) was the plant material under investigation. HPLC-grade methanol, water, and acetonitrile of were utilized as solvents for both mobile phase preparation and sample solutions, while ACS grade formic acid was incorporated to enhance chromatographic performance.

Sample solution preparation and extraction

To extract bioactive compounds from freeze-dried PFE powder, 0.5 g of the sample was weighed and placed into a 50 mL conical tube. Subsequently, 10 mL of 100% methanol was added to the tube, which was then sealed with a lid. The sealed tube was subjected to ultrasonic extraction at room temperature for 30 min. Following sonication, the tube was allowed to cool to room temperature while being vortexed. The mixture was then centrifuged at 4000 rpm for 5 min. The supernatant was carefully transferred to a 25 mL volumetric flask, and the extraction process was repeated using the same procedure. The supernatant from the second extraction was combined with the first extract in the 25 mL volumetric flask and diluted to volume with 100% methanol. The resulting solution was subsequently filtered through a 0.45 μm membrane filter and further diluted 4- to 20-fold with 70% methanol.

Standard solution preparation

To prepare the standard solution, 5 mg of the standard substance was precisely weighed and transferred to a 50 mL volumetric flask. The substance was dissolved in 70% methanol, and ultrasonic treatment was applied to ensure complete dissolution. The solution was then diluted to volume with 70% methanol, yielding a standard stock solution with a concentration of 100 mg/L. The stock solution was further diluted with 70% methanol to prepare standard solutions at concentrations of 10, 25, 50, and 100 mg/L.

Chromatographic conditions

The analysis was performed on an Agilent HPLC 1200 Series system equipped with a DAD set at 330 nm for recording chromatograms. Chromatographic separation was conducted on a CAPCELL PAK C18 UG120 column (4.6 × 250 mm × 5 μm) at 25 °C. The mobile phase comprised a mixture of 0.1% formic acid solution (solvent A) and 100% acetonitrile (solvent B) whose composition was applied as follows: 0–30 min, 12% (B); 30.1–45 min, 20% (B); 45.1–60 min, 30% (B) at a flow rate of 1 mL/min. The injection volume for each sample was set at 10 μL.

Instruments

In this study, chromatographic analysis was conducted using an Agilent HPLC 1200 system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Diode Array Detector (DAD) for detection purposes. Chromatographic separation was achieved using a CAPCELL PAK C18 UG120 column with dimensions of 4.6 × 250 mm and a particle size of 5 μm (Osaka Soda Co., Ltd, Japan). Chromatograms were recorded at a wavelength of 330 nm using the DAD. Additionally, an ultrasonicator (JAC-5020, Kodo Technical Research Co., Ltd.) was used in the experimental setup.

Animal experiments

All animal experiments were approved by the Institutional Animal Care and Use Committee of Yonsei University (IACUC-202305–1683-01). Female 7-week-old BALB/c mice were obtained from DBL (Chung Buk, Korea). To establish an allergic airway inflammation model, mice were sensitized via intraperitoneal (IP) injection of 100 μg ovalbumin (OVA, Sigma-Aldrich) and 2 mg aluminum hydroxide (Alum, Sigma-Aldrich) on Day 0 and 7, followed by intranasal OVA challenges (50 μg OVA per dose) on Day 14, 15, 20, and 21. PFE was administered daily via oral gavage at low (200 mg/kg) and high (400 mg/kg) doses from Day 10 to Day 23. Mice were sacrificed on Day 23, and biological samples were collected for further analysis. Mice were divided into four experimental groups: Control (No OVA), OVA-induced group (OVA only), OVA + PFE 200 mg/kg, and OVA + PFE 400 mg/kg.

Assessment of airway inflammation

The mouse trachea was surgically opened, and a catheter was inserted into the upper airway. Bronchoalveolar lavage fluid (BALF) was obtained by instilling 700 μL of sterile phosphate-buffered saline (PBS) into the lung, followed by gentle aspiration. BALF samples were centrifuged to pellet immune cells, which were subsequently stained for flow cytometry analysis.

Antibodies

The following primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA): IκBα (#4812), phospho-IκBα (Ser32) (#2859), phospho-NF-κB p65 (Ser536) (#3033), NF-κB p65 (#8242), IKKα (#11930), phospho-IKKα/β (Ser176/180) (#2697), phospho-p38 (#9211), and total p38 (#9212). Vinculin (#sc-25336) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

For flow cytometry analysis, the following antibodies were used: anti-CD45.2 (APC, #109813), anti-Ly-6G (PE-Cy7, #127618), anti-CD45R (BV421, #103227), and anti-CD170 (PE, #155506) from BioLegend (San Diego, CA, USA), as well as anti-CD3ε (PerCP-Cy5.5, #65–0031-U025), anti-CD11b (VF500, #85–0112-U100), and Ghost dye (Red780, #13-0865-T100) from TONBO Biosciences (San Diego, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)

Cytokine and immunoglobulin levels were quantified using commercially available ELISA kits following the manufacturers’ protocols. IL-4, IL-13, and TNF-α concentrations were measured in bronchoalveolar lavage fluid (BALF) using ELISA kits from R&D Systems (Minneapolis, MN, USA). Serum IgE levels were determined using an ELISA kit from Invitrogen (Carlsbad, CA, USA). The absorbance was measured at 450 nm using a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific), and concentrations were calculated based on standard curves generated from the provided standards.

Immunoblot assay

Mouse lung superior and middle lobes were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors. The lysates were centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatant was collected. Protein concentrations were measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were separated via SDS-PAGE and transferred to a nitrocellulose membrane (GVS, Rome, Italy). After blocking with 5% skim milk in TBST, membranes were incubated with primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies. Protein bands were detected using WestGlow™ PICO PLUS ECL (Bio-Helix) and visualized with a suitable imaging system.

Histological analysis

The inferior lobe of the lung was collected for histological analysis. The tissues were fixed in 4% paraformaldehyde solution at room temperature, followed by paraffin embedding to generate tissue blocks. Thin sections were cut from the paraffin blocks and mounted on glass slides. Hematoxylin and eosin (H&E) staining was performed to examine general tissue morphology and inflammatory cell infiltration. Stained sections were analyzed under a light microscope and histological changes were assessed using CaseViewer software (v2.4.0).

Flow cytometry

BALF sample pellets were stained for surface markers using specific antibodies for mouse anti-CD45.2 (APC, #109813), anti-Ly-6G (PE-Cy7, #127618), anti-CD45R (BV421, #103227), anti-CD170 (PE, #155506), anti-CD3ε (PerCP-Cy5.5, #65–0031-U025), anti-CD11b (VF500, #85–0112-U100), Ghost dye (red780 #13–0865-T100). After gentle mixing, the mixture was incubated in the dark at RT for 30 min. Flow cytometry was performed using MACSQuant® Analyzer 16 Flow Cytometer instrument, and the data were analyzed using FlowJo V10.8 software.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software, version 8.0.1 (GraphPad Software, La Jolla, CA, USA). Data from animal experiments were expressed as mean ± standard error of the mean (SEM). Group differences were analyzed using an unpaired t-test to compare the control and treatment groups. Statistical significance was defined as (*p < 0.05, **p < 0.01, ***p < 0.001).

Results and discussion

Optimization of rosmarinic acid content in Perilla frutescens extract using HPLC analysis

To determine the optimal extraction conditions, Perilla frutescens Britton var. acuta Kudo was extracted under 39 different extraction conditions, and the resulting Perilla frutescens extract (PFE) was evaluated using high-performance liquid chromatography (HPLC). Rosmarinic acid (RA) was selected as the key marker compound due to its high concentration in PFE and its well-established anti-allergic effects (Hong and Kim, 2010; Ko et al., 2010). HPLC analysis confirmed the presence of RA in PFE by comparing its retention time with that of the standard RA solution. Both the standard and sample solutions exhibited an identical retention time at approximately 45 min, verifying the accuracy of detection (Fig. 1A and B). The method specificity was validated by ensuring clear peak separation, and the standard calibration curve demonstrated excellent linearity (R² = 1.00), confirming the reliability of the analysis (Table 1). Among the 39 extraction conditions, the highest RA content was obtained using 50% ethanol at 50 °C for 4 h, yielding 6.28 mg/g of PFE (Table 2). Based on these results, this condition was selected as the optimal extraction method for further studies.

Fig. 1.

Chromatographic analysis of RA content in PFE using HPLC. A Chromatogram of the standard rosmarinic acid (RA) solution. B Chromatogram of the Perilla frutescens extract (PFE) sample. HPLC analysis was performed to confirm the presence of RA in PFE by comparing retention times between the standard and sample solutions

Table. 1.

Calibration curve preparation using rosmarinic acid standard solution

Table 2.

Rosmarinic acid content in PFE results

Dilution conditions	Extract condition			Area	Concentration analysis (μg/mL)	Rosmarinic acid content (mg/g)
	Ethanol concentration (%)	Time (h)	Temperature (°C)
0.5 g freeze-dried PFE powder + 25 mL solution > 4times dilution	0	2	20	17.07087	1.02928	0.205856
			40	39.37844	2.04019	0.408038
			60	51.24931	2.57814	0.515628
		4	20	22.97535	1.29685	0.25937
			40	39.87135	2.06253	0.412506
			50	74.83063	3.72251	0.744502
			60	30.5294	1.63918	0.327836
		6	20	74.31049	3.6232	0.72464
			40	3.20396	0.400878	0.080176
			60	16.7985	1.01694	0.203388
	10	2	20	0	0	0
			40	0	0	0
			60	0	0	0
		4	20	0	0	0
			40	0	0	0
			60	0	0	0
		6	20	0	0	0
			40	6.87199	0.567101	0.11342
			60	0	0	0
	30	2	20	0	0	0
			40	0	0	0
			60	7.19652	0.581808	0.116362
		4	20	0	0	0
			40	4.24023	0.447838	0.089568
			50	380.2712	17.37492	3.474984
			60	0	0	0
		6	20	0	0	0
			40	0	0	0
			60	0	0	0
	50	2	20	0	0	0
			40	0	0	0
			60	0	0	0
		4	20	0	0	0
			40	609.2266	27.86388	5.572776
			50	631.0265	28.58304	5.716608
			60	687.3131	31.40251	6.280502
		6	20	584.3874	26.73825	5.34765
			40	590.3464	27.00829	5.401658
			60	642.531	29.37313	5.874626

PFE reduces allergy-associated immune cell airway infiltration in OVA-induced asthma

To establish an allergic airway inflammation model, mice were sensitized via intraperitoneal (IP) injection of ovalbumin (OVA) and aluminum hydroxide (Alum) on Day 0 and 7, followed by intranasal OVA challenges on Day 14, 15, 20, and 21 (Fig. 2A). OVA (ovalbumin)-induced asthma model, have been widely utilized to mimic the immunological and pathological characteristics of allergic asthma. Repeated OVA sensitization and challenge induce eosinophilic infiltration, airway inflammation, excessive mucus secretion, and increased airway hyperresponsiveness (AHR), resembling clinical asthma symptoms (Casaro et al., 2019). Perilla frutescens leaf extract (PFE) was administered daily via oral gavage at low (200 mg/kg) and high (400 mg/kg) doses from Day 10 to Day 23. Mice were sacrificed on Day 23, and biological samples were collected for further analysis. Throughout the experiment, no significant differences in body weight were observed among the experimental groups (Fig. 2B). Flow cytometry analysis revealed that total cell numbers were significantly reduced in the low-dose PFE group compared to the untreated allergic control group (Fig. 2C). Further analysis showed a significant reduction in B cells, while T cell and neutrophil counts remained unchanged (Fig. 2D, E and F). Notably, eosinophils were also significantly reduced in the low-dose PFE group (Fig. 2G). These findings suggest that PFE treatment effectively mitigates allergic inflammation by modulating immune cell populations, particularly reducing B cell and eosinophil infiltration.

Fig. 2.

Effect of PFE on airway immune cell infiltration in OVA-induced asthma. A Schematic of the experimental design. Mice were divided into four groups: Control (no OVA sensitization or challenge, n = 2), OVA-induced group (OVA only, n = 4), OVA + PFE 200 mg/kg (n = 4), and OVA + PFE 400 mg/kg (n = 4). B Body weight measurements across experimental groups. C Quantification of total CD45.2⁺ immune cells in BALF. (D, E) Flow cytometry dot plots of lymphocyte populations (CD45.2⁺ CD11b⁻ cells) and corresponding quantifications. D B cells (B220⁺ lymphocytes) and E T cells (CD3ε⁺ lymphocytes). F, G Flow cytometry dot plots of granulocyte populations (CD45.2⁺ CD11b⁺ cells) and corresponding quantifications. (F) Neutrophils (Ly-6G⁺ granulocytes) and G Eosinophils (CD170⁺ granulocytes). Data are expressed as mean ± SEM. Statistical significance was analyzed using an unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001 versus the OVA-induced group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus No OVA group

PFE reduces OVA-induced airway inflammation and IgE levels

The cytokines IL-4, IL-13, TNF-α (BALF), and IgE (serum) play central roles in Th2-driven allergic inflammation, contributing to eosinophil recruitment, IgE production, airway inflammation, and mucus hypersecretion (Boonpiyathad et al., 2019; Gans and Gavrilova, 2020). Given their involvement in allergic asthma pathogenesis, their levels were measured to assess the effect of PFE treatment (Fig. 3A). OVA exposure significantly increased IL-4, IL-13, TNF-α, and IgE levels compared to the control group, whereas PFE treatment resulted in a dose-dependent reduction, with the 400 mg/kg group exhibiting the most pronounced suppression. The observed decrease in IgE levels suggests that PFE inhibits mast cell activation, a key driver of allergen-induced airway hyperresponsiveness (Hong et al., 2015) (Fig. 3A). To evaluate structural changes associated with airway inflammation, histological analysis was conducted on lung tissue sections stained with hematoxylin and eosin (H&E). The OVA-induced group exhibited severe peribronchiolar and perivascular inflammatory cell infiltration, indicative of airway remodeling and immune cell accumulation. However, PFE treatment significantly reduced inflammatory infiltrates in a dose-dependent manner, with the 400 mg/kg group showing the most notable improvement (Fig. 3B).These findings suggest that PFE effectively mitigates airway inflammation and IgE production in OVA-induced allergic asthma, likely by suppressing Th2 cytokine signaling and mast cell activation.

Fig. 3.

Effect of PFE on allergic phenotypes in OVA-induced asthma. A Protein levels of IL-4, IL-13, TNF-α (BALF) and IgE (serum) were evaluated using ELISA. Mice were divided into four groups: Control (no OVA, n = 3), OVA-induced group (OVA only, n = 6), OVA + PFE 200 mg/kg (n = 6), and OVA + PFE 400 mg/kg (n = 6). B H&E-stained lung sections showing peribronchiolar and perivascular inflammatory cell infiltration. Data are expressed as mean ± SEM. Statistical significance was analyzed using an unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001 versus the OVA-induced group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the control group

PFE mitigates OVA-induced allergic asthma by attenuating NF-κB and p38 MAPK activation

To examine the molecular mechanisms through which PFE exerts its anti-inflammatory effects, we analyzed the activation of NF-κB and p38 MAPK signaling pathways in lung tissue. Both pathways play a crucial role in allergic asthma pathogenesis, as NF-κB regulates the transcription of pro-inflammatory cytokines, while p38 MAPK contributes to immune cell recruitment and airway remodeling – processes that drive disease progression (Lee et al., 2022; Li et al., 2020). In the OVA-induced allergic asthma group, the p-IKK/IKK, p-IκB/IκB, and p-NF-κB/NF-κB ratios were significantly increased, indicating NF-κB pathway activation. PFE treatment at 400 mg/kg reduced these phosphorylation ratios, suggesting suppression of NF-κB-associated allergic signaling (Fig. 4A and B). Similarly, the OVA-induced allergic asthma group exhibited a marked increase in p38 MAPK activation, as evidenced by the elevated p-p38/p38 ratio in lung tissue. However, PFE treatment significantly inhibited p38 phosphorylation in a dose-dependent manner, with the 400 mg/kg group displaying the strongest effect (Fig. 4C and D). Since p38 MAPK activation contributes to airway inflammation and Th2 cytokine production, its inhibition by PFE suggests a potential mechanism through which PFE alleviates allergic airway responses (Zhou et al., 2023).

Fig. 4.

Effect of PFE on NF-κB and p38 MAPK signaling in lung tissue from OVA-induced allergic asthma mice. A Representative immunoblots showing expression levels of phosphorylated and total IKK, IκB, and NF-κB in lung tissues. Vinculin was used as a loading control. Protein samples were obtained from four experimental groups: Control (no OVA, n = 2), OVA-induced group (OVA only, n = 3), OVA + PFE 200 mg/kg (n = 3), and OVA + PFE 400 mg/kg (n = 3). B Densitometric quantification of p-IKK/IKK, p-IκB/IκB, and p-NF-κB/NF-κB ratios, analyzed using ImageJ software. C Representative immunoblot showing phosphorylated and total p38 MAPK in lung tissues from the same biological replicates used in (A) (Control: n = 2; OVA: n = 3; OVA + PFE 200 mg/kg: n = 3; OVA + PFE 400 mg/kg: n = 3). D Densitometric quantification of p-p38/p38 ratio, analyzed using ImageJ software. Data are expressed as mean ± SEM. Statistical significance was analyzed using an unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001 versus the OVA-induced group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the control group

In this study, we demonstrated that Perilla frutescens leaf extract (PFE) treatment alleviates allergic airway inflammation in an OVA-induced mouse model. Flow cytometry analysis revealed that low-dose PFE treatment significantly reduced total cell numbers, with a notable decrease in B cells and eosinophils, suggesting an attenuation of the allergic response. T cell analysis showed no statistically significant reduction in T cell count following PFE treatment; however, a decreasing trend was observed. T cell subsets were not analyzed in this study, making it necessary to investigate whether PFE affects specific T cell populations involved in allergic inflammation. Furthermore, the antigen specificity of these T cells remains unclear, and future experiments should include OVA-specific T cell analysis to better understand the role of PFE in modulating adaptive immune responses. Although IgE levels were assessed, it remains to be determined whether the antibodies detected were OVA-specific, highlighting the need for further investigation into antigen-specific immune responses. These additional analyses will help clarify the immunomodulatory mechanisms of PFE in allergic conditions.

While this study focused on the therapeutic efficacy of Perilla frutescens extract (PFE) as a whole, it remains unclear whether the observed anti-inflammatory effects are primarily attributable to rosmarinic acid (RA) or to the synergistic action of multiple phytochemicals within the extract. Although RA content was optimized and quantified, we did not conduct comparative studies between pure RA, PFE, and RA-depleted fractions. Such experiments are necessary to clarify the mechanistic contribution of RA to the observed biological outcomes. Future investigations using purified RA and chemically fractionated extracts will be essential to verify the role of RA and potentially identify additional active components responsible for the anti-asthmatic effects of PFE.

Although the higher dose of PFE (400 mg/kg) exhibited overall trends toward reduced airway inflammation, certain immune parameters, such as eosinophil counts, showed more pronounced statistical significance at the lower dose (200 mg/kg). This observation may reflect a biphasic dose–response, which is commonly observed with plant-derived compounds, where moderate doses elicit maximal biological effects while higher concentrations may lead to plateaued or diminished responses. Mechanistically, this may involve receptor desensitization, target saturation, or the activation of negative feedback pathways. Although we did not observe signs of overt toxicity, this study did not assess toxicological endpoints. Therefore, future studies evaluating a broader dose range and mechanistic analysis are warranted to clarify the optimal therapeutic window for PFE.

Funding

National Research Foundation of Korea, RS-2023-NR076822, Sanguine Byun, RS-2024-00442483, Sung-Wook Hong, The Food Industry Promotional Agency of Korea, Korea Institute for Advancement of Technology, RS-2024-00418203, Sung-Wook Hong.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.