Dihydrokaempferol Supports Epidermal Barrier, Dermal Repair, and Enhances Post-Procedure Recovery

Abstract

The epidermal barrier is essential for skin function, resilience, and tolerance to environmental and procedural stress. Disruption of this barrier is common after cosmetic treatments such as chemical peeling, underscoring the need for ingredients with proven biological and clinical support for barrier recovery. This study evaluated dihydrokaempferol (DHK) using molecular, preclinical, and clinical approaches. Gene expression profiling in keratinocytes and dermal fibroblasts revealed that DHK enhanced barrier-related genes, reduced inflammation-associated markers, and modulated genes involved in extracellular matrix remodeling. Functional assays confirmed antioxidant activity, suppression of stress-induced interleukin 6 (IL-6) release, increased elastin production, and improved fibroblast migration. Clinically, a randomized, placebo-controlled, split-face study following standardized chemical peeling demonstrated that DHK-treated skin showed significant improvements in dermatologist-assessed skin attributes versus placebo. Together, these findings indicate that DHK supports epidermal barrier integrity, modulates dermal repair mechanisms, and enhances recovery from controlled skin stress. The effects observed at the molecular and cellular levels translated into measurable improvements in visible skin quality, highlighting DHK’s potential as a bioactive ingredient.

1. Introduction

The skin barrier plays a fundamental role in maintaining skin health and homeostasis by protecting the body from environmental, chemical, and biological insults. This barrier function primarily resides in the stratum corneum, whose integrity depends on a highly organized lipid matrix and the coordinated cellular activity of epidermal keratinocytes and dermal fibroblasts [1]. Stratum corneum integrity decreases with age, contributing to challenges such as atopic dermatitis [2]. This decline is multifactorial, driven by intrinsic factors such as reduced lipid production, altered skin surface pH, and diminished keratinocyte turnover, as well as extrinsic factors such as UV exposure, environmental pollutants, and mechanical stress [3]. Disruption of this barrier can lead to increased permeability, chronic inflammation, reduced lipid synthesis, and delayed tissue repair, all of which contribute to clinically relevant impairments in skin health and are targets for personal care intervention [4,5,6].

At the molecular level, ceramides and lipid metabolism enzymes are critical determinants of stratum corneum structure and function, as reduced ceramide levels correlate with a compromised barrier, dryness, and impaired recovery [7]. Epidermal keratinocytes contribute to this landscape through terminal differentiation and lipid synthesis, and dysregulated inflammatory signaling in these cells can exacerbate barrier dysfunction [8]. Dermal fibroblasts influence the dermal-epidermal interface and support skin repair processes through cell proliferation, migration, and the production of extracellular matrix (ECM) proteins, including elastin and collagen [9,10]. In addition to lipid homeostasis and ECM remodeling, cellular inflammation and oxidative stress are directly impacted by barrier disruption. Reactive oxygen species generated by various stressors can amplify inflammation and delay recovery, and antioxidants have been shown to modulate inflammatory markers and support the integrity of structural proteins in the skin [11]. Effective barrier restoration, therefore, requires a modulation of epidermal inflammation and lipid metabolism, alongside dermal repair mechanisms, and the reduction in oxidative stress.

Among cosmetic procedures, chemical exfoliation, such as acid peeling, is widely used to improve skin appearance but can also transiently compromise barrier integrity [12]. This controlled insult can trigger inflammatory responses, oxidative damage, and delayed recovery of epidermal lipids, underscoring the need for functional skincare ingredients that support barrier restoration. Topical approaches designed to restore the barrier have traditionally focused on delivering physiological lipids such as ceramides, and ceramide-containing formulations have demonstrated improvements in barrier integrity [13]. However, beyond lipid supplementation, multifunctional cosmetic active ingredients that not only improve visible skin attributes but also act on the biological mechanisms underlying barrier repair represent a promising area of research.

DHK (also known as aromadendrin) is a naturally occurring flavonoid found in a range of medicinal, edible, and woody plants, with bioactivity potential for a range of human disorders [14]. Flavonoids are a diverse set of plant secondary metabolites known primarily for their antioxidant and anti-inflammatory properties [15,16]. The mechanism of action of flavonoids includes scavenging free radicals and modulating inflammatory pathways, both of which are relevant to skin health and aging [17,18]. Consistent with these properties, previous studies found that DHK can modulate oxidative stress [19,20] and has anti-inflammatory activity [21,22]. These studies highlight DHK as a potential bioactive molecule for dermatological applications focused on the skin barrier.

In this study, we investigated the biological and clinical effects of DHK, a functional skincare active ingredient designed to support skin barrier restoration. DHK activity was evaluated using a comprehensive approach that included gene expression profiling in human dermal fibroblasts and epidermal keratinocytes, alongside mechanistic in vitro assays assessing antioxidant capacity, inflammatory mediator release, and extracellular matrix production. Functional regeneration was further examined using a cellular migration assay, and clinical efficacy was assessed in a study conducted following a standardized acid peel insult. Collectively, this study evaluated whether DHK modulates key molecular and cellular pathways involved in skin barrier repair and whether these mechanistic effects translate into measurable improvements in skin quality under clinically relevant conditions.

2. Materials and Methods

2.1. Dihydrokaempferol

DHK (INCI: Aromadendrin, IUPAC: (2R, 3R)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one) was sourced from Debut Biotechnology, Inc. (DermCeutical DHK, San Diego, CA, USA) as a white powder with a purity of 99.6% (w/w) by qNMR analysis. This ingredient was produced through proprietary fermentation, extraction, and purification processes using engineered E. coli strains.

2.2. Cell Culture and Cell-Based Assays

Normal Human Epidermal Keratinocyte (NHEK) cells (ATCC) were grown in EpiLife Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with human keratinocyte growth supplement (HKGS) and gentamicin (Bio Basic, Amherst, NY, USA). Human Dermal Fibroblasts (HDFa) (Thermo Fisher Scientific) were grown in Fibroblast Basal Medium 2 (PromoCell, Heidelberg, Germany). Cryopreserved cells were grown until 80% confluency, and passaged up to 3 times.

2.3. Cell Viability Assay

Cell viability was determined using an assay which assesses cellular metabolic activity. Metabolically active cells reduce the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, producing a measurable colorimetric signal. Assays were performed using a commercially available kit (Sigma-Aldrich, St. Louis, MI, USA) according to the manufacturer’s instructions.

2.4. Transcriptomic Analysis

NHEK and HDFa cells were grown as described above and treated with DHK (Dermceutical DHK, Debut Biotechnology, San Diego, CA, USA) or dimethyl sulfoxide (DMSO) (Sigma-Aldrich) control for 24 h (n = 3). Following treatment, cells were detached from cell culture plates with TrypLE (Gibco, Thermo Fisher Scientific), rapidly cryopreserved, and stored at −80 °C until further processing. Frozen cell pellets were transported on dry ice to an external service provider (Genewiz, South Plainfield, NJ, USA) for total RNA extraction, library construction, and next-generation sequencing. Messenger RNA was enriched via poly-A selection during library preparation, and unique molecular identifiers (UMI) were incorporated to enable removal of duplicate reads. Sequencing was performed using paired-end reads (150 bp) with an average depth of 30 million reads per sample. Processed reads were aligned to the human reference genome (GRCh38) and annotated transcriptome (Gencode v44) using STAR (version 2.7.11a). Gene count was determined using featureCount (version 2.0.6). DESeq2 (version 1.44.0) was used to identify statistically significant differentially expressed genes (adjusted p ≤ 0.05 & absolute log2 fold change > 0.5). Pathway-level changes were evaluated by enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using gene set enrichment analysis (GSEA) algorithm implemented in ClusterProfiler (version 4.12.0). Enrichment of Gene Ontology Biological Processes was performed using the Over-Representation Analysis algorithm implemented in ClusterProfiler. Visualization of selected KEGG pathways with integrated gene expression data were generated with Pathview (version 1.44.0).

2.5. Elastase Inhibition & Elastin Production

Stock solutions were prepared as follows: 0.1 M Tris–HC (pH 7.5) as assay buffer, human neutrophil elastase (HNE) (1 U/mL in assay buffer), MeO-Suc-Ala-Ala-Pro-Val-pNA (MAAPVN) at 1.0 mM in DMSO as the enzyme substrate, oleanolic acid (200 µg/mL) as a positive control. DMSO served as the solvent for test compound and as the negative control. For the enzymatic assay, working solutions of HNE and the test compounds were combined and preinubated at 25 °C for 10 min. MAAPVN was then added and the mixture was incubated at 37 °C for 1 h. The enzymatic activity was determined by measuring the release of p-nitroaniline at 405 nm using a microplate reader (Spectramax M2, Molecular Devices, San Jose, CA, USA).

Elastin production was quantified using an Elastin enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions. Briefly, HDFa were cultured as described and treated with the test articles. Following 24 h treatment, culture supernatants were collected and diluted for use in the assay. Elastin standards and samples were added in triplicate to commercial ELISA plates (Abcam ab239433, Cambridge, UK). Data are presented as the mean ± standard deviation of independent experiments.

2.6. Radical Scavenging Assay

Antioxidant activity was assessed using a radical scavenging assay. This method quantifies the reduction of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) by measuring a decrease in absorbance. Stock solutions were prepared on the day of analysis, and included 200 µM DPPH in ethanol (Cayman Chemical 14805, Ann Arbor, MI, USA), 400 µM Trolox (Cayman Chemical 10011659), for standard curve generation, and 0.01% (w/w) L-Ascorbic Acid as a positive control; ethanol served as the negative control. Reactions combined equal volumes of DPPH solution and the test sample, then incubated at 25 °C in the dark for 30 min. Absorbance was recorded at 517 nm using a plate reader (Spectramax M2, Molecular Devices).

2.7. Anti-Inflammatory Activity

NHEK cells were cultured as described above and seeded at a density of 7500–10,000/cm². Cells were maintained at 37 °C for 48–72 h to allow attachment and stabilization. Culture medium was then replaced with fresh, pre-warmed medium and cells were treated with an inflammatory cocktail consisting of lipopolysaccharide (LPS, 2.0 µg/mL from Escherichia coli O26:B6), and polyinosinic–polycytidylic acid (poly(I:C), 12.5 µg/mL). After one hour of inflammatory stimulation, test compounds were added, and cells were incubated for an additional 24 h. Conditioned media was collected for quantification of IL-6 levels using a commercially available ELISA kit (ELH-IL6-1, RayBiotech, Peachtree Corners, GA, USA) according to the manufacturer’s instructions. Absorbance was measured using a plate reader (Spectramax M2, Molecular Devices).

2.8. Barrier-Based Migration Assay

Primary HDFa were cultured in a 24-well plate with an insert that creates a 0.9 mm cell-free (wound) zone visible under the microscope (Abcam Wound Healing Assay, ab242285). 24 h after cells adhered, the insert was removed, and T0 photographs were taken. The spent media was replaced with test article media for an additional 48 h, imaging every 24 h. Test articles were DHK (Dermceutical DHK, Debut), ascorbic acid (Research Products International, Mount Prospect, IL, USA), copper tripeptide (GHK-Cu, Active Peptide, Cambridge, MA, USA), ceramide NP (DS-ceramide-Y30-HP, Croda, Snaith, UK), DMSO, and media (Fibroblast Basal Medium 2 supplemented with SupplementMix, gentamicin, and amphotericin B, PromoCell). Cellular migration across the empty field was captured using an ECHO Revolve microscope with an Olympus UPlanFL N 4×/0.13 PhL objective (ECHO, San Diego, CA, USA). The images were analyzed using CellProfiler (version 4.2.8) [22] to measure the area occupied by cells within the wound region. Migration was expressed as the percentage reduction in wound area relative to the T0 time point (% closure). All experiments were performed in triplicate.

2.9. Clinical Trial

2.9.1. Ethical Conduct

This study was conducted in accordance with the intent and purpose of Good Clinical Practice regulations described in Title 21 of the U.S. Code of Federal Regulations (CFR), and the Declaration of Helsinki, and approved by the Allendale Institutional Review Board (AIRB, Old Lyme, CT, USA), Protocol DCS-61-24.

2.9.2. Subject Information and Consent

A signed informed consent form was obtained from each subject prior to performing any study procedures. No study-related procedures or activities were performed until each subject was fully informed and had signed and dated the consent form.

2.9.3. Design and Controls

Thirty subjects (100% female, Fitzpatrick types I-II) were enrolled in this single-site split-face study. 30 subjects were chosen to satisfy assumptions of the Central Limit Theorm. Subjects who signed consent and met all inclusion criteria and none of the exclusion criteria were enrolled at the baseline visit. Subjects were screened for suitability and prepared for a 25% trichloroacetic acid (TCA) face peel by the dermatologist investigator. This procedure creates an environment to rapidly measure improvements in skin healing. Following the procedure subjects had a post-procedural dressing applied which was removed the next morning. Subjects received the study cream (base formulation + 0.3% DHK) for application to one randomized side of the face and a neutral cream (base formulation only) for application to the other size of the face. Subjects were instructed to apply the study cream and bland moisturizer twice daily until study completion. An independent blinded dispenser who was not associated with other study activities randomized the study product application site and maintained a locked randomization log until data lock was completed. The objective of this study was to assess the efficacy of a cream on both immediate and sustained recovery on post-procedural facial appearance after 48 h and 2 weeks of twice-daily use. DHK inclusion level was determined based on optimal concentrations identified in vitro.

2.9.4. Inclusion Criteria

Eligible participants were healthy male and female subjects aged 35–65 years of all skin types who were in generally good physical and mental health, dependable, and willing to comply with study requirements and visit schedules. Subjects were required to sign an informed consent form in accordance with 21 CFR Part 50, agree to use only the study cream, and refrain from introducing new skincare products or colored cosmetics (including lipsticks, eye shadows, facial foundations, blushes, and powders) for the duration of the study.

2.9.5. Exclusion Criteria

Subjects were excluded if they had a history of facial skin cancer on test areas, were undergoing cancer treatment, were pregnant, nursing, or planning pregnancy, or were participating in another facial clinical study. Additional exclusions included the presence of sunburn, moderate to severe suntan, asymmetrical aging, uneven skin tone, tattoos, scars, dilated vessels, or other facial conditions that could interfere with assessments; uncontrolled systemic disease; current moderate to severe inflammatory acne; known allergies to topical products; or use of facial prescription topical medications. Subjects were also excluded for recent use of medications that could confound results (including high-dose anti-inflammatory or immunosuppressive drugs), recent use of exfoliating or retinoid products (AHAs/BHAs within 2 weeks; retinoids within 8 weeks; isotretinoin within 1 year), and recent esthetic procedures (ablative laser, microneedling, chemical peels, or dermabrasion within 6 months; non-ablative laser or IPL within 3 months).

2.9.6. Study Measures

Skin attributes were graded by an expert investigator for efficacy: post-procedure erythema, tactile smoothness, visual smoothness, pigmentation, clarity, radiance, firmness, and overall healthy skin appearance. All assessments were made on a 5-point ordinal scale (0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe) at baseline, 48 h, and week 2. The standard 5-point ordinal scale represents a method for measuring improvements from baseline, with 4 indicating the strongest visibility of the parameter and 0 representing the absence of noticeable parameter. This scale was utilized for each individual parameter on either side of the face to score the placebo and active-treated sides separately. For parameters of erythema, tactile smoothness, visual smoothness, pigmentation, clarity, firmness, radiance, and overall skin appearance, a decrease in value represents an improvement from baseline. For tolerability endpoints, a standard 5-point ordinal scale is also used to assess redness, swelling, and dryness, with 0 indicating an absence of symptoms and 4 representing severe symptoms. Assessments were made separately for each side of the face.

2.9.7. Statistical Methods

Along with descriptive statistics (means, standard deviations, and percentages), investigator-assessed clinical grading scale outcomes were analyzed using nonparametric methods, including the Wilcoxon signed-rank test and sign test for paired comparisons across time points. Statistical significance was defined at an alpha level of 0.05.

3. Results

3.1. DHK Modulates Epidermal Barrier-Related Gene Expression

To determine whether DHK affected keratinocyte viability, cells were treated with DHK for 24 h and assessed by MTT assay. No significant reduction in metabolic activity was observed across the dose range tested, indicating that DHK is not cytotoxic from 0.001–0.01% (Figure S1a).

RNA-sequencing analysis revealed a pronounced anti-inflammatory transcriptional response to DHK. Gene set enrichment analysis demonstrated significant downregulation of inflammation-associated pathways, including tumor necrosis factor-α (TNF-α) and other cytokines (Figure 1a). Consistent with these results, several downstream targets of the nuclear factor kappa-B (NF-κB) signaling pathway were also downregulated (Figure 1a and Figure S2a).

DHK also significantly modulated genes associated with epidermal differentiation and barrier formation. Differentiation markers Desmocollin 1 (DSC1) and Claudin 1 (CLDN1) were downregulated, while transcriptional regulators from Notch receptor 1 (NOTCH1) and Grainyhead-like (GRHL) families were upregulated [23,24]. Notably, multiple tight junction-associated genes, claudin family members, and Occludin (OCLN) were upregulated in response to DHK, as highlighted in the volcano plot in Figure 1b and Figure S2b [25].

Genes involved in cell cycle progression and proliferation were broadly downregulated following DHK treatment. KEGG pathway mapping revealed suppression of multiple nodes within the cell cycle pathway (Figure 1c). Importantly, this reduction in proliferative gene expression occurred without cytotoxicity, suggesting a shift toward later stages of differentiation [26]. The transcriptomic profile induced by DHK is consistent with a keratinocyte state of reduced inflammation, regulated proliferation, and epidermal barrier formation [27,28].

3.2. DHK Primes Fibroblasts for Extracellular Matrix Remodeling

Similar to cell viability results in keratinocytes, DHK was not cytotoxic to fibroblasts at the tested concentration range (Figure S1b). RNA-seq analysis of fibroblasts revealed regulation of genes associated with ECM remodeling and tissue repair (

Table 1. Gene Ontology Biological Processes enriched in DHK-treated fibroblasts.

Gene Ontology ID	Description	Adj. p-Value
GO:0030198	Extracellular matrix organization	2.07 × 10−3
GO:0043062	Extracellular structure organization	2.07 × 10−3
GO:0001659	Temperature homeostasis	2.07 × 10−3
GO:0045229	External encapsulating structure organization	2.07 × 10−3
GO:0002385	Mucosal immune response	1.17 × 10−2
GO:0045444	Fat cell differentiation	1.17 × 10−2
GO:0030199	Collagen fibril organization	1.67 × 10−2
GO:0010035	Response to inorganic substance	1.67 × 10−2
GO:0002251	Organ or tissue specific immune response	1.67 × 10−2
GO:0042730	Fibrinolysis	1.67 × 10−2

).

As seen in the volcano plot in Figure 2a, genes involved in matrix synthesis and remodeling readiness, such as Matrix Metallopeptidase 3 (MMP3) and Hyaluronan synthase 1 (HAS1), were upregulated following DHK treatment [29,30]. In contrast, genes associated with matrix crosslinking and structural stabilization (e.g., Lysyl Oxidase Like 1 (LOXL1), Lysyl Oxidase Like 4 (LOXL4), ADAM Metallopeptidase with Thrombospondin Type 1 Motif 5 (ADAMTS5)) were downregulated, suggesting a shift toward a more dynamic and permissive ECM state [31]. Several genes implicated in early wound response and tissue remodeling were also differentially regulated. DHK reduced the expression of genes associated with late-stage remodeling and inflammatory processes (e.g., Thrombospondin 1 (THBS1), Plasminogen Activator, Urokinase (PLAU), and Serpin Family B Member 2 (SERPINB2)) suggesting a transcriptional state favoring wound preparedness rather than fibrosis [32,33].

Figure 2a is focused on the observed regulation of genes associated with fibroblast proliferation and migration. DHK upregulated genes associated with cell motility, matrix interaction, and growth responsiveness (e.g., Aquaporin 1 (AQP1), Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), Integrin Subunit Beta 1 (ITGB1)), and cell cycle regulators (e.g., Cell Division Cycle 6 (CDC6), CDC28 Protein Kinase Regulatory Subunit 1B (CKS1B), Cyclin A (CCNA)) [34]. Conversely, expression of genes such as AKT Serine/Threonine Kinase 1 (AKT1), THBS1, and CD248 Molecule (CD248) was downregulated, indicating selective proliferative signaling rather than global activation [35]. Together, the DHK-induced gene expression profile is characterized by ECM remodeling, regulated protease activity, and modulation of migration and proliferation-associated pathways.

3.3. The Activities of DHK Are Confirmed in Preclinical Skin-Related Assays

3.3.1. Protection Against Matrix Degradation and Structural Stress

Healthy, flexible, intact skin depends on elastin production and barrier repair, along with suppression of internal degradative processes, such as elastase activation. Additionally, a compromised barrier provides less resistance to external stressors, further accelerating the breakdown of elastin fibers [36]. Figure 3 highlights two roles DHK plays in maintaining a resilient structural matrix. DHK inhibited elastase in a dose-dependent manner, with concentrations of 0.5% and 0.1% significantly inhibiting elastase activity compared with solvent controls (Figure 3a).

To complement the preservation of elastin fibers, DHK treatment in fibroblasts also stimulated elastin production to a level comparable to that of vitamin C and significantly higher than in untreated cells (Figure 3b). Elastin production and elastase inhibition act as both mechanical and inflammatory buffers in skin [37]. By preserving elastin fiber integrity and limiting matrix degradation, DHK’s activities help establish a dermal state conducive to remodeling.

3.3.2. Reduction in Molecular Damage and Inflammatory Signaling

The antioxidant capacity of DHK was evaluated using a DPPH radical scavenging assay. DHK significantly reduced DPPH radical levels in a dose-dependent manner, with a significant reduction vs. solvent control observed across the concentration range of 0.016–0.063% (Figure 4a). These findings suggest that DHK limits the availability of free radicals, a key contributor to molecular damage in skin [38].

To assess the impact of DHK on inflammatory signaling, IL-6 secretion was measured by ELISA following pro-inflammatory stimulation. As seen in Figure 4b, DHK significantly reduced IL-6 production compared to stimulated controls in a dose-dependent manner. This inhibitory effect is consistent with the RNA-seq data and was observed to progressively reduce IL-6 at concentrations ranging from 0.001% to 0.003%, outperforming the industry benchmark niacinamide at a much higher concentration.

3.3.3. Enhanced Tissue Repair Dynamics

The effects of DHK on fibroblast migration and proliferation were evaluated using an in vitro wound healing assay, with cellular influx into a defined cell-free zone quantified as percent closure. At 24 h, DHK treatment at 0.003% resulted in a significant increase in wound closure compared with untreated control cells. However, other industry-standard ingredients (vitamin C, tripeptide, ceramide NP) and the vehicle control (DMSO) did not significantly improve closure at this time point (Figure 5).

At 48 h, DHK continued to demonstrate superior wound closure performance with the most complete and monodisperse distribution of cells at the site of the wound. Both DHK and ceramide NP achieved significantly greater percent closure compared to DMSO-treated controls, whereas vitamin C and tripeptide did not. These findings indicate that DHK supports the early and later phases of wound repair and are consistent with the data above, suggesting a concept of a dermal state primed for regeneration.

3.4. DHK Improves Clinical Skin Response Following Chemical Peeling

3.4.1. Tolerability and Safety

30 participants completed the study with 0 subject withdrawals. DHK was well tolerated throughout the 2-week study period. Investigator assessments indicated no evidence of treatment-related skin irritation at any time point. No adverse events were reported during the study, and both predefined tolerability and safety endpoints were met.

3.4.2. Efficacy Against a Controlled Dermatologic Stressor

The clinical performance of DHK was evaluated in a randomized, split-face design following a standardized dermatologist-administered acid peel. Investigator-graded assessments were conducted at 48 h and 2 weeks post-procedure to characterize acute recovery and sustained improvements in visible skin quality. Blinded clinical grading included erythema, tactile smoothness, visual smoothness, pigmentation, clarity, radiance, firmness, and overall healthy skin appearance. Changes from baseline for DHK-treated and control sides are summarized in

Table 2. Investigator-graded efficacy of DHK and placebo formulations.

	48 h Post-Peel			2 Weeks Post-Peel
Measurement	DHK	Placebo	p-Value	DHK	Placebo	p-Value
Erythema	1.07	0.90	0.319	1.87	1.77	0.79
Tactile Smoothness	0.83	0.27	<0.001 *	1.53	0.87	<0.001 *
Visual Smoothness	0.80	0.37	0.002 *	1.57	0.93	<0.001 *
Pigmentation	0.07	0.00	0.354	0.23	0.23	1.00
Clarity	0.50	0.20	0.017 *	1.03	0.50	<0.001 *
Radiance	0.70	0.30	0.002 *	1.23	0.57	<0.001 *
Firmness	0.07	0.00	0.212	0.10	0.07	0.66
Overall	0.40	0.07	0.003 *	0.70	0.13	<0.001 *

* = p value < 0.05, threshold for statistical significance of DHK vs. placebo.

. Positive values reflect greater improvement relative to baseline.

At 48 h post-procedure, the DHK-treated side demonstrated statistically significant improvements versus placebo across multiple parameters, including tactile smoothness, visual smoothness, clarity, radiance, and overall appearance (Table 2; Figure 6). These early differences are consistent with recovery during the acute post-procedural phase, a period characterized by barrier disruption, inflammation, and transepidermal water loss. The observed improvements in smoothness and clarity suggest accelerated restoration of epidermal structural integrity and surface cohesion.

At 2 weeks, DHK maintained statistically significant improvement over placebo across the same graded endpoints (Table 2; Figure 6). The persistence of improvements in radiance, clarity, and smoothness supports both barrier normalization and downstream dermal remodeling, both of which contribute to overall skin quality.

Representative subject images (Figure 7) visually corroborate the measured outcomes. The DHK-treated profiles show reduced surface irregularity and improved radiance relative to the placebo control, consistent with the dermatologist-graded improvements. These images confirm that DHK-mediated biological activity translates into meaningful esthetic improvement following chemical exfoliation.

4. Discussion

This study demonstrates that DHK supports skin resilience and recovery through coordinated modulation of oxidative stress, inflammatory signaling, epidermal differentiation, and dermal repair dynamics. Across transcriptomic, molecular, and clinical assessments, DHK was associated with an enhanced adaptive response to skin stress and improvements in visible skin quality.

Oxidative stress and inflammation are central drivers of skin aging and post-procedural dysregulation [39]. DHK demonstrated radical scavenging activity and significantly reduced stimulus-induced IL-6 secretion, supporting its capacity to limit molecular damage and downstream inflammatory signaling, consistent with previous studies [19]. These functional data align with our RNA-seq analysis in keratinocytes, which show downregulation of TNF, IL6, and NF-kB-associated pathways (Figure 1 and Figure S2). Together, these results suggest that DHK attenuates early stress signaling, which may help to prevent excessive inflammatory responses following environmental or procedural insults. Keratinocyte transcriptomics also revealed that DHK selectively modulates differentiation programs and barrier-associated genes. Downregulation of desmosomal components, coupled with upregulation of Notch signaling, spinous layer differentiation, and tight junction genes, suggests a shift toward epidermal renewal and barrier organization [23,24,25].

In fibroblasts, DHK induced a gene expression signature indicative of ECM remodeling and wound-preparatory signaling. The regulation of HAS1, TGFBR1, integrins, and ECM-interacting genes suggests a fibroblast state supportive of tissue repair and dermal–epidermal communication [30]. Maintenance of extracellular matrix integrity is important for preserving dermal mechanical function. The functional elastin homeostasis data for DHK combined with gene expression regulation of matrix remodeling genes suggest DHK supports matrix adaptability while limiting degradation. Such a remodeling profile may reduce the release of inflammatory mediators and help maintain dermal-epidermal signaling during the recovery process [40]. Functional validation using a wound healing assay confirmed that DHK significantly enhanced fibroblast migration and closure dynamics compared with untreated cells and benchmark ingredients, supporting a role for DHK in promoting tissue repair.

Post-procedural care is an important aspect of recovery after facial rejuvenation procedures, particularly chemical exfoliation treatments such as acid peels. These treatments are associated with epidermal injury and require appropriate skin care to support barrier repair and mitigate inflammation and erythema. Active ingredients with enhanced biological activity may help reduce discomfort, accelerate recovery, and improve clinical outcomes following standard procedures. The objective of this study was to investigate the efficacy of DHK on post-procedural facial appearance after 2 weeks of twice-daily use. DHK significantly improved investigator-graded measures of smoothness, clarity, radiance, and overall appearance at both 48 h and 2 weeks compared to placebo. These sustained benefits reflect activity beyond the immediate recovery period and into the remodeling phase of skin renewal, both of which are consistent with our mechanistic data for DHK. Reduction in oxidative stress, upregulation of barrier-associated genes, attenuation of cytokine-driven inflammation, and enhanced fibroblast migration and matrix remodeling provide a mechanistic basis for the accelerated recovery and improved visible skin quality observed following the peel.

The trajectory of future research could include expanding clinical evaluation to more thoroughly characterize DHK’s effects by analyzing lipid composition. In addition, extending the study duration could provide deeper insight into the long-term effects of DHK on both healthy and post-procedure skin.

5. Conclusions

In conclusion, data from this study suggest that DHK elicits molecular and cellular responses aligned with effective skin recovery, including attenuation of inflammatory pathways and support of accelerated epidermal and dermal repair. These responses were observed in parallel with improvements in post-procedure recovery and visible skin quality following a standardized dermatologic challenge. Taken together, these findings support further investigation of DHK as an active ingredient with potential relevance for skin resilience with implications for wound healing, harsh dermal rejuvenation procedures, and aging.