Research Article

TNF-α Antagonism by Fermented Red Ginseng and Restoration of Skin-Barrier Gene Expression

Jung Yoon Lee^1,*,Jea-Hyuk Kim^2,*, Solomon Lee³, Suji Baek²

Abstract

Background/Objectives: Chronic skin inflammation disrupts skin barrier function and delays wound healing. Tumor necrosis factor-alpha (TNF-α) is a key inflammatory mediator that induces pro-inflammatory cytokines and suppresses critical barrier-related genes such as hy-aluronan synthase 2 (HAS-2) and filaggrin, impairing skin homeostasis.

Methods: Red ginseng fermented extract (GE) was prepared and analyzed via high-performance liquid chromatography (HPLC) to con-firm the presence of ginsenosides Rg1 and Rb1. In silico molecular docking was used to assess interactions between these compounds and TNF-α. In vitro experiments were conducted using a TNF-α-induced inflammatory skin model to evaluate the effects of GE on HAS-2 and filaggrin gene expression.

Results: HPLC analysis verified the presence of Rg1 and Rb1 in GE. Molecular docking revealed strong binding affinities between both ginsenosides and TNF-α, suggesting interference with TNF-α receptor binding. In vitro, GE treatment preserved or restored HAS-2 and filaggrin expression under inflammatory conditions, indicating protection of skin barrier integrity via inhibition of TNF-α signaling.

Conclusions: GE improves skin barrier function by blocking TNF-α signaling through its active components Rg1 and Rb1. These findings highlight the potential of GE as a natural therapeutic agent for inflammatory skin disorders and skin barrier repair.

Keywords

Red Ginseng extract, Ginsenoside Rb1, TNF-α, Skin Barrier, Inflammation

Introduction

Chronic inflammatory skin diseases—such as atopic dermatitis, psoriasis, and chronic eczema—affect a substantial portion of the global population and signifi-cantly impair quality of life due to symptoms like pruritus, dryness, exudation, and pain [1,2]. Recurrent disruption of the skin barrier facilitates the penetration of external antigens, which in turn induces inflammation and perpetuates a vicious cycle [3]. These conditions are not merely cosmetic concerns; they pose consider-able social and economic burdens through increased healthcare costs and reduced capacity to perform daily activities [4]. Consequently, the development of practical and integrative inflammation-control strategies using biocompatible and low-risk natural compounds is an urgent public health priority.

Tumor necrosis factor-alpha (TNF-α), a major pro-inflammatory cytokine secreted by immune cells, plays a central role in chronic inflammatory responses [5,6]. Upon binding to its receptor (TNF-α receptor), TNF-α activates key inflammatory signaling pathways such as NF-κB and MAPK, which stimulate the expression of various inflammatory mediators, including IL-1β, IL-6, and COX-2 [7,8]. Simultaneously, TNF-α suppresses the expression of critical genes responsible for maintaining skin homeostasis [9]. In particular, the downregulation of hyaluronan synthase 2 (HAS-2) and filaggrin—a structural protein essential for moisture retention and barrier recovery—is considered a major contributor to impaired skin hydration, weakened wound healing, and compromised barrier function [10]. TNF-α thus not only amplifies inflammatory cascades within the skin but also directly undermines regenerative capacity and skin barrier integrity, acting as a key pathological factor [11]. Therefore, therapeutic strategies that suppress TNF-α activity and restore the expression of skin barrier genes such as HAS-2 and filaggrin may offer effective interventions for chronic skin inflammation.

While current TNF-α inhibitors (e.g., infliximab, adalimumab) are highly effective, their long-term use is limited by high cost and adverse effects such as increased infection risk due to systemic immunosuppression [12, 13]. In this context, natural bioactive compounds have emerged as promising alternatives [14]. The field of cosmeceuticals, which bridges functional skincare and therapeutic intervention, provides a novel approach for managing skin inflammation through products that are both effective and suitable for regular use.

Panax ginseng, a traditional medicinal herb with scientifically validated effects, is well recognized for its immunomodulatory, anti-inflammatory, and antioxidant properties [15]. Among its active constituents, ginsenosides Rg1 and Rb1 have been reported to act on various inflammatory pathways, exerting potent anti-inflammatory effects [16]. This study aims to investigate whether these compounds can directly inhibit TNF-α activity and restore the expression of key skin barrier genes. The findings will provide a scientific basis for cosmeceutical strategies targeting inflammatory skin barrier dysfunction.

Materials & Methods 

Quantitative Analysis of Ginsenosides Rg1 and Rb1 Using HPLC

High-performance liquid chromatography (HPLC) was used to quantify the major ginsenosides Rg1 and Rb1 in the red ginseng extract. The analysis was performed using a Waters 2695 Separation Module equipped with a photodiode array (PDA) detector (Model 2996), and a reverse-phase C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of distilled water (A) and acetonitrile (B), with the following gradient program: starting at 17% B, increasing to 35% B over 30 minutes, then maintained at 50% B until 60 minutes, followed by re-equilibration. The flow rate was set at 1.0 mL/min, with detection at 203 nm. The injection volume was 10 μL, and the column temperature was maintained at 30°C. The sample solution was prepared by dissolving the freeze-dried red ginseng extract in 70% ethanol, followed by filtration through a 0.45 μm syringe filter prior to injection. Standard curves were constructed using commercially available Rg1 and Rb1 standards (≥98% purity, Sigma-Aldrich). Quantification was based on retention time and peak area com-parison with the standards. Each sample was analyzed in triplicate, and the mean values were reported.

Molecular Docking Analysis Between Ginsenosides and TNF-α

The 3D structures of ginsenosides Rg1 and Rb1 were obtained from the PubChem database and subjected to energy minimization using Chem3D software. The crystal structure of the TNF-α protein was retrieved from the RCSB Protein Data Bank (PDB ID: 2AZ5). Preprocessing of the protein structure was performed using AutoDock Tools 1.5.7, during which water molecules and co-crystallized ligands were removed, and polar hydrogens and Gasteiger charges were added. Molecular docking was carried out using AutoDock Vina. A grid box was defined around the receptor binding site of TNF-α to focus the docking analysis. Each docking simulation was performed in triplicate, and the conformation with the lowest binding energy was selected as the representative pose. Binding affinity was reported as binding energy (kcal/mol), which was used to evaluate the potential interactions between TNF-α and the ginsenosides Rg1 and Rb1. Docked complexes were visualized using PyMOL and LigPlot+ to identify interaction sites. Hydrogen bonds and hydrophobic inter-actions were analyzed to elucidate the molecular mechanisms underlying the binding affinity.

Preparation of Fermented red Ginseng Extract (GE)

To produce a high-functional fermented red ginseng extract (GE), a four-step pro-cessing procedure was employed. First, 100 g of 6-year-old red ginseng powder was subjected to hot water extraction at 90°C for 2 hours to obtain water-soluble com-ponents. Subsequently, the extract was fermented at 37°C for 48 hours using Lac-tobacillus rhamnosus and Bacillus subtilis strains. After fermentation, the mixture was pasteurized at 80°C to inactivate enzymatic activity. The fermented solution was then autoclaved under high-temperature and high-pressure conditions (121°C, 1.5 atm, 30 minutes). Following this, solvent extraction was performed using 70% ethanol, and the extract was concentrated under reduced pressure and freeze-dried. Through this process, an GE enriched in ginsenosides Rg1 and Rb1 was obtained.

Evaluation of the Effects of Fermented Red Ginseng Extract Using an In Vitro Wound Healing Model

HaCaT human keratinocyte cells were seeded into 6-well plates at a density of 5 × 10⁵ cells per well and cultured until they reached over 90% confluence. Once the cells became confluent, a single straight-line scratch was created across the cell monolayer using a sterile 200 μL pipette tip to simulate a wound. Detached cells and debris were removed by washing with PBS, and the culture medium was replaced with serum-free DMEM. GE was then applied to each group at various concentra-tions (e.g., 50, 100, and 200 μg/mL). Images of the wound area were captured at 0, 12, 24, and 48 h post-treatment using a phase-contrast microscope. The wound closure area was quantitatively analyzed using ImageJ software. The wound closure rate was calculated using the following formula: Wound closure (%) = [(wound width at 0 h – wound width at time t) / wound width at 0 h] × 100.

Semi-Quantitative RT-PCR and Gel Electrophoresis for HAS-2 and Filaggrin

Total RNA was extracted from treated HaCaT cells using TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. One microgram (1 μg) of total RNA was used to synthesize complementary DNA (cDNA). RT-PCR was then performed using gene-specific primers for HAS-2, filaggrin, and GAPDH (as an internal con-trol). PCR amplification was carried out under the following conditions: initial de-naturation at 95 °C for 3 minutes, followed by 30–35 cycles of denaturation at 95 °C for 30 seconds, annealing at 58–60 °C for 30 seconds, and extension at 72 °C for 30 seconds. The amplified PCR products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. Bands were visualized under a UV transillu-minator, and band intensities were quantified using ImageJ software. The relative expression levels of HAS-2 and filaggrin were calculated as ratios to GAPDH band intensity.

Statistical Analysis

All experiments were performed in triplicate (n = 3), and data are presented as mean ± standard deviation. Statistical significance was determined using one-way analysis of variance, followed by Tukey’s post hoc test for multiple comparisons. A p-value of less than 0.05 was considered statistically significant. Statistical analyses were conducted using GraphPad Prism.

Results

Qualitative Analysis of Ginsenosides Rg1 and Rb1 in Red Ginseng Extract

To qualitatively confirm the presence of the major active components ginsenoside Rg1 and Rb1 in the red ginseng extract, HPLC was used. As shown in Fig.1 A, multiple peaks were observed, indicating the complex composition of saponins in the extract. Among them, distinct peaks appeared at retention times of 22.457 min and 38.716 min, corresponding to standard ginsenosides Rg1 and Rb1, respectively. These retention times matched those of authentic standards, suggesting the presence of these ginsenosides in the sample.

As shown in Fig.1 B, a single strong peak was detected at 38.751 min. Although close to the retention time of Rb1, the slight shift suggests that this peak may correspond to Rb1, especially considering the sample was likely a high-purity Rb1 standard or a post-fermentation enriched fraction.

As shown in Fig.1 C, the graph showed a clear single peak at 22.6 min, which closely aligned with the retention time of the Rg1 standard, indicating the presence of Rg1 in high purity within the sample. These chromatographic results provide qualitative evidence that the fermented and re-extracted red ginseng extract prepared in this study contains meaningful levels of ginsenosides Rg1 and Rb1, supporting the effectiveness of the applied extraction and fermentation process.

Figure 1. HPLC analysis for the identification of ginsenosides Rg1 and Rb1. (A) Chromatogram of red ginseng extract showing peaks corresponding to ginsenosides Rg1 and Rb1. (B) Chromatogram of the ginsenoside Rb1 standard. (C) Chromatogram of the ginsenoside Rg1 standard.

 Molecular docking simulation revealed that ginsenosides Rg1 and Rb1 directly interacted with the TNF-α protein.

Rg1 was stably docked within the receptor-binding site, maintained through hy-drogen bonding and hydrophobic interactions. In contrast, Rb1 was docked into a structurally rotated pocket and exhibited a binding energy of −7.483 kcal/mol, in-dicating a stronger affinity compared with Rg1 (−6.911 kcal/mol). Molecular surface rendering showed that the binding region of Rb1 corresponded to a high-affinity site (red), whereas that of Rg1 was localized to a moderate-affinity site (orange/yellow). These findings suggest that Rb1 binds more effectively to the active site of TNF-α (Fig.2). Collectively, both ginsenosides may contribute to anti-inflammatory effects through TNF-α inhibition, providing fundamental evidence for the therapeutic po-tential of red ginseng-derived constituents in inflammatory disorders.

Figure 2. This schematic illustrates the predicted molecular interactions between TNF-α protein and the red ginseng-derived saponins Rg1 and Rb1.

(A) Ribbon and surface representation of TNF-α showing the location of the ligand-binding pocket. (B) Three-dimensional chemical structure of ginsenoside Rg1. (C) Predicted docking pose of Rg1 within the TNF-α binding pocket, demonstrating stable interactions. (D) Rotated structural view of TNF-α to visualize the binding orientation of Rb1. (E) Three-dimensional chemical structure of gin-senoside Rb1. (F) Predicted docking pose of Rb1 within the TNF-α moderate-affinity site, demonstrating stable interactions. The surface rendering is color-coded to represent binding affinity: red indicates high-affinity in-teractions, orange and yellow represent moderate affinity, and green to blue correspond to low binding probability or structural surfaces.

Skin Regenerative and Barrier-Enhancing Effects of GE

To evaluate whether GE has skin regeneration and barrier strengthening effects, a scartch wound healing test and PCR were performed. As shown in Fig.3A, treatment with 0.1% and 1% GE resulted in more than a three-fold increase in cell migration compared with the untreated control (UN), showing a wound closure capacity comparable to that of the EGF-treated group. These findings suggest that GE pro-motes cell migration, thereby contributing to skin regeneration. Furthermore, mRNA analysis revealed a significant upregulation of HAS-2 and FLG, genes associated with skin hydration and barrier formation, in response to GE treatment. Notably, in the 1% GE-treated group, HAS-2 expression increased by approximately 450%, while FLG expression rose by about 300%.

Figure 3. Effects of Red Ginseng Extract (GE) on Wound Healing and Barrier-Related Gene Expression in HaCaT Cells.

(A) HaCaT cells were seeded into 6-well plates, and a scratch wound was generated using a sterile pipette tip. Cells were then cultured for 24 h under different conditions: untreated (UN), GE 0.1%, GE 1%, and EGF. The cells were observed using contrast microscope. Quantification of wound healing area was performed using ImageJ software. (B) Semi-quantitative RT-PCR analysis of HAS-2 and FLG expression following GE treatment. GAPDH served as the internal control. (D) Band intensity was quantified using ImageJ. The UN group was set to 100%. *P < 0.05 vs. UN.

Discussion

Chronic skin inflammation disrupts skin barrier function and delays wound healing [17]. Among inflammatory mediators, TNF-α plays a central role by inducing the secretion of cytokines such as IL-1β and IL-6 while suppressing the expression of barrier-related genes including HAS-2 and filaggrin, thereby impairing skin home-ostasis [18-20]. In this study, molecular docking analysis and cell-based assays demonstrated that ginsenosides Rg1 and Rb1, the major saponins derived from red ginseng, directly interact with TNF-α and may inhibit its activity. Both compounds docked stably into the binding site of TNF-α, with Rb1 showing stronger binding affinity than Rg1. These molecular findings suggest that ginsenosides can attenuate TNF-α signaling, thereby suppressing inflammatory gene expression and restoring barrier-related gene activity.

Consistent with the docking results, functional assays further supported the potential of GE. The WST-8 assay indicated that GE was not cytotoxic and enhanced cell viability, while the wound healing assay demonstrated that GE treatment promoted keratinocyte migration to a level comparable with EGF. Moreover, semi-quantitative RT-PCR revealed significant upregulation of HAS-2 and filaggrin in GE-treated HaCaT cell, uggesting that GE can restore skin barrier function through modulation of inflammatory pathways.

The implications of these findings extend beyond cosmetic science. Skin diseases, including atopic dermatitis, psoriasis, and acne, are increasingly recognized as chronic conditions that impair quality of life (QoL) on a global scale [21].  According to WHO (2016), more than 30% of the global population is affected by chronic skin disorders, with significant social and psychological burdens [22]. The Global Burden of Disease (GBD) study by Hay et al. (2014) identified skin diseases as the fourth leading cause of nonfatal disease burden worldwide [23]. Furthermore, epidemiological patterns vary across socioeconomic contexts: while infectious skin conditions prevail in low-income countries due to limited hygiene and healthcare access, high-income countries experience higher prevalence of chronic inflammatory skin diseases driven by urbanization, environmental pollution, and stress [25]. These perspectives indicate that inflammatory skin diseases should be addressed not only as biomedical disorders but also as global public health challenges.

Within this context, the results of the present study provide scientific evidence for the use of natural cosmeceutical ingredients, particularly red ginseng saponins, in modulating inflammation and promoting skin barrier recovery. Specifically, HAS-2 contributes to epidermal hydration and tissue regeneration via hyaluronic acid synthesis, while filaggrin plays a key role in keratinocyte cohesion and barrier integrity. TNF-α inhibition by ginsenosides may therefore contribute to maintaining homeostasis in inflammatory skin conditions such as atopic dermatitis.

Conclusion

In summary, this study demonstrated that red ginseng extract and its major saponins, Rg1 and Rb1, directly interact with TNF-α, attenuating its pro-inflammatory sig-naling and supporting the recovery of skin barrier-related genes. Molecular docking simulations revealed stable binding of both compounds to TNF-α, with Rb1 exhib-iting higher affinity. In vitro assays confirmed that GE enhances keratinocyte via-bility, accelerates wound closure, and upregulates HAS-2 and filaggrin expression.

These results suggest that GE holds promise as a preventive and complementary approach for inflammatory skin disorders. Beyond cosmetic applications, GE may serve as a functional ingredient in the development of advanced cosmeceuticals aimed at alleviating the burden of chronic skin diseases. The findings provide a scientific foundation for future translational research and product development, positioning GE as a potential alternative strategy in global public health to reduce the impact of skin-related diseases.

Acknowledgments

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

JYL and SJB conceived and designed the experiments. JHK and SML performed the molecular docking analyses using AutoDock. JYL and SJB conducted the experiments and collected the data. JYL and SJB wrote the manuscript. All authors reviewed and approved the final version of the manuscript.

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