Atractylodes Macrocephala Koidz Extracts (AKE) Enhance Skin Barrier Repair via Synergistic Ceramide Upregulation, Hyaluronidase Inhibition, Anti-Inflammatory Action, and Proteins Modulation

Atractylodes Macrocephala Koidz Extracts (AKE) Enhance Skin Barrier Repair via Synergistic Ceramide Upregulation, Hyaluronidase Inhibition, Anti-Inflammatory Action, and Proteins Modulation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 22 November 2025 V1 Latest version Share on Atractylodes Macrocephala Koidz Extracts (AKE) Enhance Skin Barrier Repair via Synergistic Ceramide Upregulation, Hyaluronidase Inhibition, Anti-Inflammatory Action, and Proteins Modulation Authors : Huan Liu , Le Yu , Chang-Yin Yang , Yu-qiang Zhao , Lin Xu 0000-0002-6332-1376 , and Ying Zhou [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176380590.04686603/v1 167 views 83 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Although synthetic agents such as hydrocortisone and tretinoin are widely employed to treat skin disorders, their association with cutaneous irritation and adverse events limits utility in sensitive skin. In contrast, herbal ingredients offer gentle, effective, and less irritating alternative therapeutic options for sensitive skin in managing these conditions. Therefore, we investigated several medicinal plants extracts and found that AKE and PPE , derived from Atractylodes macrocephala Koidz. and Paeonia lactiflora Pall. respectively, demonstrated efficacy in promoting skin barrier repair. Further studies revealed that both extracts, particularly AKE , repaired the barrier through synergistic mechanisms, including enhancing ceramide synthesis, inhibiting hyaluronidase activity, reducing inflammatory responses and regulating skin-related proteins. Furthermore, in mouse damaged epidermal barrier models, AKE achieved an 81.2% recovery rate, significantly improving barrier restoration and relieving sensitive skin symptoms. Our findings offer novel insights into low-irritation skin barrier repair, potentially advancing Chinese herbal medicine development for cosmetic applications. Cite this paper: Chin. J. Chem. 2025 , 43 , XXX—XXX. DOI: 10.1002/cjoc.70XXX Atractylodes Macrocephala Koidz Extracts (AKE) Enhance Skin Barrier Repair via Synergistic Ceramide Upregulation, Hyaluronidase Inhibition, Anti-Inflammatory Action, and Proteins Modulation Huan Liu a, 1 , Le Yu a, 1 , Chang-Yin Yang b , Yu-qiang Zhao a , Lin Xu b ,* , Ying Zhou a,* * a Yunnan Characteristic Plant Extraction Laboratory, College of Chemical Science and Technology, Yunnan University, Kunming 650091, China. b Liwa Institute of Skin Health, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China Atractylodes macrocephala Koidz | Skin barrier | Ceramide | Hyaluronidase inhibition | Anti-inflammatory | Skin barrier-related proteins | Comprehensive Summary Although synthetic agents such as hydrocortisone and tretinoin are widely employed to treat skin disorders, their association with cutaneous irritation and adverse events limits utility in sensitive skin. In contrast, herbal ingredients offer gentle, effective, and less irritating alternative therapeutic options for sensitive skin in managing these conditions. Therefore, we investigated several medicinal plants extracts and found that AKE and PPE, derived from Atractylodes macrocephala Koidz. and Paeonia lactiflora Pall. respectively, demonstrated efficacy in promoting skin barrier repair. Further studies revealed that both extracts, particularly AKE, repaired the barrier through synergistic mechanisms, including enhancing ceramide synthesis, inhibiting hyaluronidase activity, reducing inflammatory responses and regulating skin-related proteins. Furthermore, in mouse damaged epidermal barrier models, AKE achieved an 81.2% recovery rate, significantly improving barrier restoration and relieving sensitive skin symptoms. Our findings offer novel insights into low-irritation skin barrier repair, potentially advancing Chinese herbal medicine development for cosmetic applications. [1]¿p#1 Background and Originality Content Skin, a large and complex organ, primary function as the first line of defense between the internal environment of the host and the external world. It protects the body from a wide range of harmful agents, including toxic chemicals, ultraviolet radiation, mechanical injury, allergens, and pathogenic microorganisms 1 . A key component of this protective role is the epidermal barrier, which maintains skin homeostasis. This barrier consists of several distinct anatomical layers: the stratum basale, stratum spinosum, stratum granulosum, and stratum corneum 2-3 . When the epidermal barrier is damaged, transepidermal water loss (TEWL) increases, leaving the skin more vulnerable to external insults. This leads to symptoms commonly associated with sensitive skin, such as dryness, flaking, roughness, wrinkles, erythema, burning, and swelling 4-5 . Over time, these disruptions may result in epidermal thickening and pruritus, potentially progressing to more serious dermatological conditions 6 . Since 1949, synthetic agents like hydrocortisone and tretinoin have been widely used to treat skin disorders such as atopic dermatitis, acne, and psoriasis 7-9 . While these drugs are highly effective and commonly used in clinical settings 10-11 , they are also associated with skin irritation and adverse effects, including burning, persistent erythema, edema, itching, and scaling, which limit their use in individuals with sensitive skin 6, 9 . In contrast, bioactive compounds derived from medicinal plants are known for being safer, gentler, and less irritating, making them appealing alternatives for alleviating skin barrier damage 6 . In this study, we selected several medicinal and edible plant-based herbs as the candidates of low-irritation skin barrier repair, including Atractylodes macrocephala Koidz. ( A. macrocephala ), Paeonia lactiflora Pall. ( P. lactiflora ), Dendrobium officinale Kimura et Migo ( D. officinale ), and Polygonatum odoratum (Mill.) Druce ( P. odoratum ). Nitric oxide (NO, key molecule in the occurrence of inflammation) inhibition assays identified only the active ingredients AKE derived from A. macrocephala and PPE from P. lactiflora as potent skin anti-inflammatory candidates 12 . Comprehensive evaluation revealed that both extracts, particularly AKE , significantly enhanced skin barrier repair through synergistic mechanisms: boosting ceramide biosynthesis; inhibiting hyaluronidase (HAase), collagenase, and elastase activities; suppressing pro-inflammatory cytokines IL-6 and IL-8; upregulating occludin expression; and normalizing keratin expression. Critically, in murine epidermal barrier damage models, AKE treatment achieved an 81.2 % recovery rate, surpassing PPE (79.3 %), highlighting its superior efficacy in barrier restoration and alleviating sensitive skin symptoms. In addition, LC-MS and molecular docking identified potential active compounds within AKE, providing insights for further mechanistic exploration. These findings demonstrate that specific medicinal plant extracts, notably AKE, effectively enhance skin barrier repair. Results and Discussion Medicinal plants selection and extraction Significant inflammation can result from elevated NO levels, which is a crucial biological mediator. Reducing NO production can lessen the inflammatory reaction 12 . Based on this, several medicinal plants, 1, 13-15 , including A. macrocephala , P. lactiflora , D. officinale , and P. odoratum were evaluated for their inhibitory activity against NO production in LPS-induced RAW264.7 cells. The results suggested that these herbs exhibited varying degrees of inhibitory activity. Among them, A. macrocephala and P. lactiflora showed significant NO production inhibition with inhibition rate 97.39 % and 77.06 %, respectively (Fig. S1). Therefore, extracts of A. macrocephala and P. lactiflora , prepared using optimized extraction methods, were selected for further investigation of their ability to repair the skin barrier. As shown in Fig. 1A, the powdered roots and rhizomes of P. lactiflora (300 g) were refluxed with 80% ethanol (3 × 3.0 L, 3 h each) at 60 °C. The combined extracts were concentrated under reduced pressure to obtain a crude extract (75.8 g), which was suspended in water and sequentially partitioned with petroleum ether and n-butanol. The n-butanol fraction was further concentrated to yield the optimized extract PPE (50.2 g). Meanwhile, A. macrocephala powder (300 g) was subjected to ultrasonic-assisted extraction using ethyl acetate (3 × 3 L, 24 h each) at room temperature. After solvent removal under reduced pressure, the optimized extract AKE was obtained (60.1 g) ( Fig. 1B ). Both extracts, AKE and PPE, obtained through optimized extraction procedures, were subsequently used for further investigations. [1]¿p#1 Figure 1 (A-B) Preparation process of AKE and PPE. (C-D) Effects of different concentrations of AKE (C) and PPE (D) on the viability of HaCaT cells after 24, 48, and 72 hours of treatment. Excellent biocompatibility of AKE and PPE As keratinocytes are the predominant cells in the epidermis and critical to barrier function 16 , we assessed the cytotoxicity of AKE and PPE on HaCaT cells to evaluate their safety for cosmetic applications. Cells were treated with AKE or PPE at concentrations of 0.5, 2.5, 12.5, and 62.5 μg/mL. As shown in Fig. 1C-1D, both extracts exhibited minimal cytotoxicity across the tested range, and promoted HaCaT cell proliferation were observed at concentrations up to 12.5 μg/mL (P < 0.05). These results suggested that both AKE and PPE were relatively safe for keratinocytes, meeting a key criterion for further cosmetic development. Up-regulation of ceramide level in HaCaT cells According to the ”brick and mortar” model, the skin’s barrier consists of corneocytes (”bricks”) embedded in a lipid matrix (”mortar”), predominantly composed of ceramides (~50 %), cholesterol (~25 %), and free fatty acids (~15 %) (Fig. 2A). Ceramides, as sphingolipids, function dually as structural elements and signaling mediators regulating skin homeostasis, immune responses, and cellular renewal 3-4 . Substantial evidence correlates ceramide depletion to increased TEWL, impaired capacitance, and barrier pathologies such as dryness, pruritus, acne, aging, atopic dermatitis, and psoriasis 3-4, 17 . To investigate the effects of AKE and PPE on ceramide synthesis, HaCaT cells were treated with varying concentrations of each extract. AKE markedly enhanced ceramide production across the range of 6.25-40 μg/mL, outperforming the commercial reference compound. The most pronounced effect was observed at 12.5 μg/mL, where AKE elevated ceramide levels by 1.61-fold compared to the control. In contrast, PPE showed a weaker effect, with its maximum increase of 1.15-fold occurring at 50 μg/mL (Fig. 2C). To further validate these findings, ceramide levels were assessed at 24, 48, and 72 hours using the optimal concentrations of each extract. Both extracts promoted time-dependent increases in ceramide synthesis, with AKE consistently demonstrating superior efficacy (Fig. 2D-2E). These results underscore AKE ’s potent regulatory effect on ceramide biosynthesis, highlighting its strong potential in supporting epidermal barrier homeostasis. Figure 2 (A) Schematic illustration of the ”Brick and Mortar” model of the skin epidermal barrier. (B) Mechanisms of hyaluronidase, elastase, and collagenase in skin barrier degradation. (C) Effect of AKE and PPE at different concentrations on ceramide content in HaCaT cells. (D~E) Time-dependent effects of AKE (12.5 μg/mL) and PPE (50.0 μg/mL) on ceramide content in HaCaT cells at 24, 48, and 72 hours. (F-H): Inhibitory activity of AKE and PPE on hyaluronidase (F), collagenase (G), and elastase (H) enzymes. *P < 0.05, **P < 0.01, ***P < 0.001. Inhibition of enzyme activity in vitro Hyaluronidase (HAase), a key extracellular matrix (ECM) enzyme widely present in body tissues, catalyzes the degradation of hyaluronic acid (HA), thereby reducing fluid viscosity and increasing tissue permeability. In the skin, HAase-induced HA breakdown can lead to inflammation, allergic reactions, accelerated aging, increased vascular permeability, and cancer cell migration. 18 . In addition to HA, collagen and elastin are essential structural components of the skin, contributing to its strength, elasticity, and hydration. These proteins are degraded by collagenase and elastase, respectively. Excessive enzyme activity can damage dermal structure and promote wrinkle formation 19 . Notably, elastase has broad substrate specificity, degrading multiple connective tissue proteins, while also inducing inflammation, increasing permeability, and impairing wound healing 20 (Fig. 2B). Therefore, inhibiting these enzymes is crucial for maintaining skin homeostasis. Due to concerns about the side effects of synthetic inhibitors, plant-derived enzyme inhibitors are increasingly explored for pharmaceutical and cosmetic use. In vitro enzyme inhibition assays are widely employed to evaluate the therapeutic potential of bioactive plant compounds. To assess the inhibitory effects of AKE and PPE on HAase, collagenase, and elastase, dose-dependent assays were conducted using concentrations ranging from 0.125 to 5.0 mg/mL. Enzyme activity was measured relative to control reactions, using appropriate synthetic substrates and reference compounds. The results revealed that both extracts inhibited all three enzymes in a concentration-dependent manner (Fig. 2F-2H). Notably, AKE exhibited superior inhibitory activity, consistently outperforming both PPE and the positive control across all tested concentrations. These findings highlight AKE ’s strong potential as a natural multi-enzyme inhibitor for maintaining skin integrity and preventing degradation of extracellular matrix components. Figure 3 (A) Schematic of the experimental workflow for the analysis of inflammatory factors, occludin, and keratin. (B~E) Inhibitory effects of different concentrations of AKE and PPE on IL-6 and IL-8 levels in HaCaT cells. (F) Effect of AKE and PPE at various concentrations on occludin content in HaCaT cells (24 h). (G~H) Time-dependent effects of AKE (50.0 μg/mL) and PPE (25.0 μg/mL) on occludin content in HaCaT cells at 24/48/72 h. (I) Effect of AKE and PPE at various concentrations on keratin content in HaCaT cells (24 h). (J–K) Time-dependent effects of AKE (50.0 μg/mL) and PPE (50.0 μg/mL) on keratin content in HaCaT cells at 24/48/72 h. *P < 0.05, **P < 0.01, ***P < 0.001; # P < 0.05, ## P < 0.01. Anti- hyaluronidase activity We specifically evaluated the ability of AKE and PPE to inhibit HAase activity, which is involved in the depolymerization of HA and contributes to skin inflammation, allergies, and aging 21 . As depicted in Fig. 2F, both extracts inhibited HAase activity in a dose-dependent manner, with inhibition approaching 80% at the highest tested concentration. AKE exhibited particularly strong inhibitory activity, with an IC₅₀ of 0.5544 mg/mL, outperforming the positive control ursolic acid (IC₅₀ = 0.6975 mg/mL). In contrast, PPE showed weaker potency, with an IC₅₀ of 1.293 mg/mL 22 ( Fig. S2 ). These results confirm the anti-hyaluronidase properties of both extracts, with AKE standing out as a potent natural inhibitor. Given its superior efficacy, AKE shows strong potential for use in cosmetic formulations targeting skin inflammation, allergic reactions, dehydration, and aging-related conditions. Anti- collagenase activity We investigated the ability of AKE and PPE to inhibit collagenase, a key enzyme involved in collagen degradation and dermal structural damage. As depicted in Fig. 2G, both extracts showed concentration-dependent inhibitory effects. Notably, AKE exhibited the strongest activity, with an IC₅₀ of 1.220 mg/mL, slightly outperforming both PPE (IC₅₀ = 1.012 mg/mL) and the positive control, ursolic acid (IC₅₀ = 1.228 mg/mL) (Fig. S2). At higher concentrations (2.00-5.00 mg/mL), AKE consistently demonstrated greater efficacy, whereas PPE and ursolic acid showed comparable but weaker effects (Fig. 2G). These results highlight AKE ’s potent anti-collagenase activity, supporting its potential as an effective skincare ingredient for preserving collagen integrity and maintaining skin structure and hydration. Anti- elastase activity To evaluate their ability to prevent elastin degradation, the elastase inhibitory activities of AKE , PPE , and ursolic acid were assessed. As depicted in Fig. 2H, all three showed concentration-dependent inhibition. Among them, AKE exhibited the most potent activity, with an IC₅₀ of 0.4304 mg/mL, significantly outperforming ursolic acid (IC₅₀ = 1.434 mg/mL) and PPE (IC₅₀ = 2.839 mg/mL). These findings highlight AKE ’s exceptional anti-elastase capacity, supporting its strong potential as a cosmetic ingredient for preventing skin dehydration, sagging, and wrinkle formation through effective protection of elastin integrity. Figure 4 (A-C): Fluorescence emission spectra of hyaluronidase in the presence of increasing concentrations of PPE at 310 K ( A); Stern–Volmer plots (B) and double-logarithmic Stern–Volmer plots (C) for fluorescence quenching of hyaluronidase by PPE at 300/310/320 K. (D-F): Fluorescence emission spectra of hyaluronidase in the presence of increasing concentrations of AKE at 310 K (D); Stern–Volmer plots (E) and double-logarithmic Stern–Volmer plots (F) for fluorescence quenching of hyaluronidase by AKE at at 300/310/320 K. (G-I): Fluorescence emission spectra of elastase in the presence of increasing concentrations of PPE at 310 K (G); Stern–Volmer plots (H) and double-logarithmic Stern–Volmer plots (I) for fluorescence quenching of elastase by PPE at at 300/310/320 K. (J-L): Fluorescence emission spectra of elastase in the presence of increasing concentrations of AKE at 310 K (J); Stern–Volmer plots (K) and double-logarithmic Stern–Volmer plots (L) for fluorescence quenching of elastase by AKE at at 300/310/320 K. (M-O): Fluorescence emission spectra of collagenase in the presence of increasing concentrations of PPE at 310 K (M); Stern–Volmer plots (N) and double-logarithmic Stern–Volmer plots (O) for fluorescence quenching of collagenase by PPE at at 300/310/320 K. (P-R): Fluorescence emission spectra of collagenase in the presence of increasing concentrations of AKE at 310 K (P); Stern–Volmer plots (Q) and double-logarithmic Stern–Volmer plots (R) for fluorescence quenching of collagenase by AKE at at 300/310/320 K. All fluorescence measurements: λ ex = 280 nm. Enzyme concentrations: hyaluronidase (160 U/mL), elastase (300 U/mL), collagenase (125 U/mL). AKE/PPE extract concentrations: 0, 3.3, 6.7, 10.0, 13.3, 16.7, 20.0, 23.3, 26.7, 30.0, and 33.3 μg/mL. Anti-inflammatory activity In addition to being a critical structural component of the epidermal lipid matrix, ceramides also act as second messengers involved in keratinocyte proliferation, differentiation, and regulation of inflammatory responses. Dysregulation of ceramide metabolism is associated with inflammatory skin disorders 3, 23 . IL-6 and IL-8 are key pro-inflammatory cytokines implicated in various skin inflammatory processe [1]¿p#1 23 . To evaluate the anti-inflammatory effects of AKE and PPE , we measured the levels of IL-6 and IL-8 in HaCaT cells treated with each extract (6.25~50 µg/mL) for 24 hours (Fig. 3B-3E). The results showed that PPE did not significantly reduce IL-8 levels at 6.25–25 µg/mL, but exhibited significant inhibition at 50 µg/mL (P < 0.01). Surprisingly, AKE effectively suppressed IL-8 production across all tested concentrations (6.25~50 µg/mL, P < 0.001). Moreover, both ingredients dose-dependently inhibited IL-6 production (P < 0.001), with AKE demonstrating greater potency than PPE . These findings indicated that AKE possessed significant anti-inflammatory activity in vitro . Importantly, their ceramide-enhancing effects did not trigger inflammatory responses, supporting their safety and efficacy as candidates for skin barrier repair. Regulation of occludin and keratin level in HaCaT cells Tight junction (TJ) proteins, critical for epidermal barrier integrity, function as a rescue system during the stratum corneum impairment 2 . Occludin, one of the critical TJ proteins, is essential for establishing the defensive skin barrier. Its levels are significantly decreased or absent in the stratum corneum when skin barrier impairment 24-26 . Keratin constitute the intermediate filament cytoskeleton of epithelial cells and are essential fibrous structural components of the epidermal barrier, including in hair, nails, and the outer layer of skin 27-28 . Notably, their overexpression may trigger keratinization disorders. 29-31 . To investigate epidermal barrier-related protein alterations, occludin and keratin expression in HaCaT cells was quantified via enzyme-linked immunosorbent assay (ELISA) (Fig. 3F~3K). Experimental results demonstrated that both AKE and PPE significantly upregulated occludin expression and inhibited abnormal keratin expression within the concentration range of 6.25-50 μg/mL. AKE showed superior regulatory activity, with the strongest occludin upregulation observed at 50 μg/mL, compared to PPE ’s peak effect at 25 μg/mL (Fig. 3F). Both extracts also exhibited maximal keratin suppression at 50 μg/mL (Fig. 3I). At these optimal concentrations, AKE consistently outperformed PPE, and both extracts showed significant differences from the control group across multiple time points (24 h, 48 h, 72 h) (Fig. 3J-3K). These findings indicate that AKE , in particular, effectively promotes skin barrier repair and prevents epidermal hyperplasia by enhancing occludin expression and suppressing keratin—two key proteins critical to epidermal integrity. Table 1 Quenching constants ( K SV ), binding constants ( K a ) and relative thermodynamic parameters of the interaction between AKE / PPE and HAase at different temperatures. [1]¿p#1 Sample Temp. (K) K sv (mL/mg) K a (mL/mg) n \[\mathbf{\mathrm{\Delta}}\mathbf{S}^{\mathbf{0}}\mathbf{\ }\] (J/mol·K） \[\mathbf{\mathrm{\Delta}}\mathbf{H}^{\mathbf{0}}\mathbf{\ }\] (KJ/mol) \[\mathbf{\mathrm{\Delta}}\mathbf{G}^{\mathbf{0}}\mathbf{\ }\] (KJ/mol) PPE 300 27.3 867.0 0.642 124.338 32.070 -5.232 310 33.4 1084.7 0.622 -6.475 320 34.8 1944.5 0.814 -7.719 AKE 300 23.4 2321.9 1.007 -68.232 -23.793 -3.323 310 23.0 902.7 0.712 -2.641 320 22.8 591.4 0.560 -1.959 [1]¿p#1 Fluorescence quenching reveals direct enzyme-ligand interactions Fluorescence quenching spectroscopy was used to assess the interactions between AKE/PPE and the enzymes HAase, elastase, and collagenase. These enzymes contain intrinsic fluorophores, primarily tryptophan (Trp) and tyrosine (Tyr), which emit fluorescence at ~340 nm upon excitation at 280 nm. Trp, in particular, serves as a sensitive probe for monitoring changes in the protein microenvironment during ligand binding or conformational shifts 32-33 . As depicted in Fig. 4, increasing concentrations of AKE and PPE led to a gradual decrease in enzyme fluorescence intensity, accompanied by red shifts in emission maxima at 300 K, 310 K, and 320 K. These red shifts suggest that Trp residues became increasingly exposed to a polar environment, indicating direct interaction with the extracts 34 . AKE induced more pronounced fluorescence quenching and spectral shifts than PPE, implying a stronger binding affinity to enzyme active sites. These results demonstrate that AKE , in particular, can effectively bind to and modulate key skin-degrading enzymes, supporting its role as a potent natural inhibitor for maintaining tissue homeostasis. Additional fluorescence spectra at 300 K and 320 K are provided in the Supplementary Material. (Fig. S3-S5). Fluorescence quenching arises from factors such as molecular rearrangements, excited-state reactions, or interactions with small molecules. It is generally classified as either dynamic quenching, caused by collisions between fluorophores and quenchers, or static quenching, resulting from the formation of non-fluorescent ground-state complexes 35 . These mechanisms show opposite temperature dependencies: static quenching weakens with increasing temperature due to complex destabilization, whereas dynamic quenching is enhanced as higher temperatures promote molecular collisions 36 . To further explore the quenching mechanism of AKE / PPE on each enzyme, we applied the Stern-Volmer (S-V) equation to calculated the quenching constants ( K sv ). As shown in Fig. 4, the S-V plot of F 0 /F versus [Q] demonstrated a linear relationship for all three enzymes, and the K sv values derived from the slopes. The calculated K q values for each group demonstrated strong linearity (R² > 0.98), thus validating the applicability of the S-V model. The K sv for AKE / PPE -HAase interactions at 300 K, 310 K, and 320 K are listed in Table 1, while those for elastase and collagenase are provided in the Supplementary Material (Table. S1, S2). The results demonstrated that increased K sv values with rising temperature for the PPE –HAase and AKE –collagenase complexes, consistent with dynamic quenching mechanism. In contrast, other complexes showed decreased K sv values at elevated temperatures, indicating static quenching . Figure 5 (A) Experimental design. Mice were divided into five groups (n = 3 per group); the normal group received no treatment; the water-treated group served as the model control; AKE- and PPE-treated groups received topical application of extract-based creams; the vehicle group received the cream base alone. (B–E): Comparison of TEWL (B), skin pH (C), elasticity (D), and hydration (E) measured at days 0, 2, 4, and 8 following treatments. ‘###’ indicates significantly different from the normal group at the p < 0.001 level. ‘*’, ‘**’, and ‘***’ indicate significantly different from the model group at the p < 0.05, p < 0.01, and p < 0.001 levels, respectively. Figure 6 (A) Histological analysis of skin lesions using hematoxylin and eosin (H&E) staining. (B) Representative images of dorsal skin from mice on days 0, 2, 4, and 8 following treatments. (C) Comparison of epidermal thickness, as well as ceramide, occludin, and keratin content in the dorsal skin of mice across different treatment groups. ‘***’ indicate significantly different from model group at the p < 0.001 level. Calculation of the binding parameters and identification of the binding sites When small molecules bind independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules scan be evaluated using the equation: log[(F 0 −F)/F] = log K a + n log [Q ], where K a is the binding constant and n represents the number of binding sites. A plot of log[( F 0 − F)/F] versus log[Q] yields a straight line whose slope corresponds to n and whose Y- intercept equals log K a 37 (Fig. 4). The corresponding values of K a and n at different temperatures were obtained and are listed in Table 1 and Tables S1, S2 . Analysis of the results revealed that the K a values for the PPE-HAase and AKE-collagenase complexes increased with rising temperature, suggesting that higher temperatures reduced diffusion but enhanced binding stability. Conversely, other complexes exhibited the opposite trend. Notably, the pattern of K a values closely mirrored that of the K sv , further supporting the inferred quenching mechanisms 34, 38 . Furthermore, the number of binding sites ( n ) at all three temperatures was approximately equal to 1, indicating that AKE and PPE each occupied a specific inhibitory site on their respective targets 37, 39 . Thermodynamic parameters and binding forces Non-covalent interactions between bioactive molecules and macromolecules such as proteins include hydrophobic interactions, hydrogen bonding, van der Waals forces, and electrostatic forces 34, 36 . The signs and magnitudes of thermodynamic parameters (∆G 0 , ∆H 0 , ∆S 0 ) can be used to clarify the binding forces between the biomacromolecule and small chemical molecule. According to the literature 34, 36, 38, 40-42 , If both ΔH⁰ > 0 and ΔS⁰ > 0, the binding is mainly driven by hydrophobic interactions. If ΔH⁰ > 0 and ΔS⁰ < 0, the binding is mainly through electrostatic interactions. If ΔH⁰ 0, the binding involves both hydrophobic and electrostatic interactions. Lastly, if ΔH⁰ < 0 and ΔS⁰ < 0, the dominant forces are van der Waals interactions and hydrogen bonding. ΔH⁰ and ΔS⁰ values were obtained from the slope and intercept of the plot of ln K a versus 1/T ( Fig. S6-S7 ). The thermodynamic parameters for AKE/PPE-enzymes interactions are summarized in Table 1 and Tables S1–S2. The negative ΔG⁰ values at all three temperatures, indicating that the binding of AKE/PPE to the enzymes was spontaneous. For the PPE–HAase and AKE–collagenase complexes, positive ΔH⁰ and ΔS⁰ suggested hydrophobic interactions were the dominant binding forces. A positive ΔS⁰ is commonly interpreted as a sign of hydrophobic interaction, since ligand binding often leads to the burial of hydrophobic protein surfaces and the release of bound water molecules, producing favorable entropic changes 38 . In contrast, other complexes exhibited negative ΔH⁰ and ΔS⁰ values, implying that their interactions were primarily driven by van der Waals forces and hydrogen bonds. Despite non-covalent interactions are generally weak and non-specific, their collective action can induce conformational and functional changes in target proteins 34, 37 . [1]¿p#1 In vivo assessment of AKE on skin barrier repair in a mouse model To evaluate the effects of AKE on epidermal barrier repair after physical damage, the dorsal skin of mice was tape-stripped repeatedly. Cream (used as vehicle) containing 10% AKE or PPE (used as positive control) was applied topically, and skin parameters including TEWL, pH, elasticity, and hydration were measured on days 0, 2, 4, and 8. TEWL, an indicator of moisture loss and barrier damage, significantly decreased following treatment with either AKE or PPE cream, indicating faster skin recovery ( Fig. 5A ). After 8 days, AKE-treated skin showed a healing rate of 81.2%, slightly higher than PPE (79.3%), and significantly better than the vehicle group (51.8%, Fig. 5B ). Other skin parameters, including pH, elasticity, and hydration, also showed significant improvement, approaching normal levels ( Fig. 5C-5E ). These results demonstrate that both extracts enhanced skin barrier repair, with AKE showing superior efficacy. Furthermore, to explore the mechanism, expression levels of ceramide, occludin, and keratin in mouse dorsal skin were analyzed. AKE significantly upregulated ceramide and occludin barrier-related proteins to near-normal levels (p < 0.001), while suppressing aberrant keratin expression more effectively than PPE ( Fig. 6C ). These findings align with in vitro and histological results, confirming AKE’s robust role in skin barrier restoration. In summary, AKE effectively accelerates barrier repair by boosting ceramide and occludin levels while reducing keratin overexpression, thereby alleviating epidermal hyperkeratosis. These results support its potential as an active ingredient in cosmetics targeting skin barrier dysfunction. Figure 7 The LC-MS/MS analysis results about the chemical structures of principal components identified in AKE. [1]¿p#1 Qualitative analysis of AKE by LC-MS/MS The LC-MS/MS technique is widely applied for analyzing the chemical constituents of herbal medicines due to its reliability and sensitivity in measuring retention time, accurate mass, and characteristic fragment ions. 16 . To identify the potential active compounds in AKE, UHPLC-ESI-HRMS/MS analysis was performed in positive ion mode. This analysis successfully revealed the presence of atractylenolides I and II, which are constituents potentially involved in skin barrier repair. Compound identification was based on accurate mass measurements, with mass errors maintained within 5 ppm, and confirmed by comparison with literature and public mass spectral databases such as MassBank, http://www.massbank.jp/QuickSearch.html 43 . As shown in Fig. 7, in AKE, fragment ions at m/z 231.1384 [M+H]⁺ and m/z 253.1203 [M+Na]⁺ indicated the presence of atractylenolide I, while ions at m/z 233.1537 [M+H]⁺ and m/z 255.1354 [M+Na]⁺ confirmed atractylenolide II. A similar analysis conducted on PPE identified paeoniflorin and albiflorin as its potential active components (Fig. S8). Atractylenolides I and II are well-known bioactive volatile constituents of A. macrocephala, noted for their anti-inflammatory and anti-ulcer activities 44-45 . Based on these properties and their presence in AKE, atractylenolides I and II are hypothesized to be the primary candidate compounds potentially contributing to the observed skin barrier repair effects. Molecular docking analysis Molecular docking enables theoretical prediction of the binding modalities between inhibitors (ligands) and receptors (proteins), quantifying interaction affinity and elucidating binding mechanisms 46-48 . To further investigate whether atractylenolides I and II are the active components in AKE, molecular docking analyses were conducted with three skin-related enzymes: HAase, elastase, and collagenase (Fig. 8) . As shown in Fig. 8A and 8B, atractylenolides I and II formed stable docking poses with HAase through hydrogen bonding and hydrophobic interactions involving residues such as Tyr202(A), Trp130(A), Ala116(A), Val59(A), Trp42(A), Val322(A), Thr72(A), and Asn61(A). Notably, more negative binding energy values indicate stronger ligand–protein interactions . 48 . Atractylenolide I (-7.76 kcal/mol) and II (-8.00 kcal/mol) showed stronger binding affinities to HAase compared with paeoniflorin (-5.81 kcal/mol) and albiflorin (-5.40 kcal/mol) (Fig. S9A-9B), consistent with the higher HAase inhibition rate observed for AKE over PPE (Fig. 2F). Similar docking trends were observed with collagenase and elastase (Fig. 8C-8F and Fig. S9C-9F). These results, in agreement with in vitro enzyme inhibition assays, further suggesting that atractylenolides I and II are likely the bioactive constituents of AKE . More importantly, identifying such potential active compounds lays the foundation for deeper exploration of the mechanisms by which AKE promotes skin barrier repair. Figure 8 Molecular docking results illustrating the binding poses and interactions between HAase (A-B), collagenase (C-D) and elastase (E-F) and key compounds: atractylenolide I and atractylenolide II. Green dashed lines represent hydrogen bonds; semicircles indicate hydrophobic interactions. Conclusions In this study, we evaluated the potential of two plant-derived extracts, AKE and PPE , for repairing the skin barrier and alleviating symptoms associated with sensitive skin. Among them, AKE demonstrated superior efficacy. AKE significantly promoted ceramide biosynthesis in keratinocytes, a key process for maintaining skin hydration and barrier integrity. It also inhibited skin-degrading enzymes (HAase, collagenase, and elastase), thereby contributing to the prevention of inflammation, dehydration, and premature aging. Moreover, AKE suppressed pro-inflammatory cytokines (IL-6 and IL-8), enhanced the expression of the tight junction protein occludin, and normalized keratinocyte differentiation markers, suggesting comprehensive skin barrier restoration effects. Spectrofluorometric analyses confirmed target-specific interactions between AKE and the cutaneous enzymes, supporting its inhibitory mechanism. In the mouse model of skin barrier disruption, topical application of AKE significantly improved barrier function (achieved 81.2% recovery rate), further validating its effectiveness in vivo . Finally, LC-MS and molecular docking analyses identified atractylenolides I and II as the major bioactive constituents in AKE . Collectively, these findings support AKE as a promising candidate for future cosmetic and dermatological applications aimed at restoring skin barrier function and managing sensitive skin conditions. Experimental [1]¿p#1 Materials and Instruments All solvents and reagents used were of analytical grade. The enhanced Cell Counting Kit-8 (CCK-8), Bicinchoninic Acid assay Kit-8, NO assay kit, Bovine Serum Albumin (BSA, > 98 %) were acquired from Beyotime Biotechnology (Shanghai, China). Lipopolysaccharide (LPS) were purchased from TargetMol (Shanghai, China). Human ceramide ELISA Kit, Human occludin ELISA Kit, and Human keratin ELISA Kit were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). Mouse keratin ELISA Kit was provided from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). Mouse ceramide ELISA Kit and Mouse occludin ELISA Kit were provided from Shanghai Keqiao Biotechnology Co., Ltd. (Shanghai, China). Elastase was bought from Beijing Aobosen Biotechnology Co., Ltd. (Beijing, China). Human IL-6 ELISA Kit and Human IL-8 ELISA Kit were provided from Elabscience Biotechnology Co., Ltd. (Wuhan, China). N-Succinyl-Ala-Ala-Ala-p-Nitroanilide (98 %) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China). N-[3-(2-Furyl) acryloyl]-Leu-Gly-Pro-Ala, Tris-HCl buffer solution (0.05 mol/L, pH 7.5 and 1mol/L, pH 8.0), Hyaluronidase were purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Ursolic Acid (≥ 98 %), Sodium Hyaluronate (≥ 93 %, MW:1800000) and Collagenase (from Clostridium histolyticum, Type I, 125 U/mg) were bought from Shanghai Titan Scientific Co., Ltd (Shanghai, China). [1]¿p#1 Plant source information Air-dried roots and rhizomes of D. officinale , P. odoratum , P. lactiflora and A. macrocephala were purchased from a traditional Chinese medicine market in Luosiwan International Trade City, Kunming, China, in April 2023. According to the supplier, the plant materials were harvested in September 2022 from Yulong County (Lijiang), Malipo County (Wenshan) and Tengchong County (Baoshan), respectively. The species were identified by Dr. Ying Zhou from the School of Chemical Science and Technology, Yunnan University. Voucher specimens of D. officinale, P. odoratum, P. lactiflora and A. macrocephala (YNU20230402, YNU20230403, YNU20230404 and YNU20230405) were deposited at the Laboratory of Intelligent Molecular Synthesis and Functionalization, Yunnan University. Cell culture The human HaCaT keratinocyte cell line was obtained from Meisen CTCC (Zhejiang Meisen Cell Technology Co., Ltd.). The RAW264.7 cells were purchased from FUHENG BIOLOGY (Shanghai Fuheng Biotechnology Co., Ltd.). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin (Zhejiang Meisen Cell Technology Co., Ltd.). Cells were maintained in a humidified incubator at 37 °C with 5 % CO₂ until reaching the logarithmic growth phase. Cytotoxicity assay The cytotoxicity of PPE and AKE extracts was assessed using the Cell Counting Kit-8 (CCK-8). HaCaT cells (1 × 10⁵ cells/well) were seeded into 96-well plates and incubated for 24 h. The extracts, dissolved in dimethyl sulfoxide (DMSO), were applied at varying concentrations (final DMSO concentration: 2 %). Cells were treated for 24, 48, and 72 h in five replicate wells per condition. After incubation, 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 2 h. Absorbance was measured at 450 nm using a microplate reader 49 . Nitric Oxide inhibition activity measure To investigate the nitric oxide (NO) inhibitory activity of four herbs extracts, 8.0 × 104 RAW 264.7 cells/well were seeded in a 24-well plate, incubated in a 37 °C, 5% CO 2 incubator for 24 h, and then simultaneously treated with 2 µg/mL LPS and 25 μg/mL. After 24 h, the culture supernatants were collected and centrifuged to measure NO levels using NO assay kits, per manufacturer instructions. ELISA assay When HaCaT cells reached ~80 % confluence, they were treated with varying concentrations of PPE, AKE (6.25–50 μg/mL), or a positive control. After 24 h, the culture supernatants were collected and centrifuged to measure IL-6 and IL-8 levels using ELISA kits, per manufacturer instructions. For protein detection, HaCaT cells were treated with PPE and AKE (6.25–50 μg/mL) for 24 h, harvested, and lysed using RIPA buffer. Protein concentrations were determined using the BCA method, and ELISA kits were used to quantify ceramide, occludin, and keratin. The same procedure was applied to assess time-dependent effects at 24, 48, and 72 h. Inhibition of hyaluronidase activity in vitro HAase inhibition was evaluated by measuring N-acetyl-glucosamine produced from sodium hyaluronate 50-51 . The assay system included 30 μL of sample (0.125–5.00 mg/mL), 30 μL HAase (10 U/mL), and 10 μL phosphate buffer (300 mM, pH 5.35), incubated sequentially at 37 °C. The reaction was initiated by adding 30 μL hyaluronic acid substrate (0.03 %), followed by 45 min incubation. The reaction was stopped with 100 μL acid albumin solution (0.1 % BSA, pH 3.75) and incubated for another 10 min at 37 °C. Absorbance was measured at 600 nm. Ursolic acid served as the positive control. Inhibition was calculated as 52 : Enzyme inhibition activity (%) = [(OD sample - OD control) / OD sample] ×100 % [1]¿p#1 Inhibition of elastase activity in vitro Following established protocols 37, 53 , the assay mixture included 50 μL of sample (0.125–5.00 mg/mL), 50 μL elastase (0.5 U/mL), and 50 μL Tris-HCl buffer (0.1 mM, pH 8.0), incubated for 15 min at 37 °C. Then, 50 μL of N-Succinyl-Ala-Ala-Ala-p-nitroanilide (1 mM) was added and incubated for 20 min. Absorbance was recorded at 410 nm. Ursolic acid was used as a reference; 5 % DMSO served as the negative control. [1]¿p#1 Inhibition of collagenase activity in vitro This spectrophotometric assay 54-55 . involved 50 μL collagenase (1 U/mL), 50 μL Tris-HCl buffer (0.05 mM, pH 7.5), and 50 μL of sample (0.125–5.00 mg/mL), incubated at 37 °C for 15 min. Then, 50 μL of N-[3-(2-Furyl) acryloyl]-Leu-Gly-Pro-Ala (0.48 mg/mL) was added for another 20 min incubation. Absorbance was measured at 335 nm. Ursolic acid and 5 % DMSO were used as reference and control, respectively 51 . Fluorescence quenching of three enzymes by AKE / PPE Quantitative analysis of the interactions between the plant extracts (AKE and PPE) and three enzymes, hyaluronidase, elastase, and collagenase, was performed via fluorimetric titration. Enzyme solutions (3.0 mL) were prepared at concentrations of 160 U/mL (hyaluronidase), 300 U/mL (elastase), and 125 U/mL (collagenase). Each enzyme solution was titrated with successive additions of a 2.0 mg/mL plant extract solution, added in 5 μL increments, yielding a final concentration range of 0–33.3 μg/mL. After each addition, the mixtures were allowed to equilibrate for 5 minutes at room temperature. Fluorescence emission spectra were then recorded at three different temperatures, 300 K, 310 K, and 320 K, using a spectrofluorometer. The excitation wavelength was set to 280 nm, and emission was scanned over the range of 300–500 nm. Both excitation and emission bandwidths were set to 10 nm, and the scan speed was 3000 nm/min. Temperature control was maintained using a circulating water bath 34, 38, 56 . The fluorescence quenching mechanism was calculated by Stern-Volmer equation 35 : \(\frac{F_{0}}{F}\)\(=\ 1+Kq\ \tau 0\ [Q]\ =\ 1\ +\ KSV\ [Q]\) (1) In the Eq. (1), where F0 and F are the fluorescence intensities of enzymes before and after the addition of the quencher, respectively; Kq is the quenching rate constant of biomolecule; [Q] is the concentration of quencher; τ0 is the average lifetime of biomolecule without quencher and the value of τ0 of biopolymer is 10-8 s; KSV is the Stern-Volmer dynamic quenching constant. Therefore, the KSV at different temperatures can be determined by linear regression plot of F0/F versus [Q]. [1]¿p#1 Binding constants & number of binding sites Binding constants (Ka) and number of binding sites (n) were obtained using the double-logarithmic equation: log\(\frac{F_{0}-F}{F}\) \(=\ log\ Ka\)\(+\ nlog\ [Q]\) (2) [1]¿p#1 Thermodynamic characteristics of interactions The thermodynamic analysis can further clarify the binding force of the interactions between quencher and enzymes. In consideration of the dependence of association constant (Ka) on temperature, a thermodynamic process was considered to be responsible for this interaction. Therefore, the thermodynamic parameters were calculated in order to further characterize the acting forces between enzymes and quencher. The thermodynamic parameters including enthalpy (\(\mathrm{\Delta}H^{0}\)), entropy change (\(\mathrm{\Delta}S^{0}\)) and free energy change (\(\mathrm{\Delta}G^{0}\)) were calculated by the van’t Hoff equation as followed: log Ka = - \(\frac{\mathrm{\Delta}H^{0}}{2.303\text{RT}}\)\(+\) \(\frac{\mathrm{\Delta}S^{0}}{2.303R}\) (3) where R is the gas constant, and T is the different experimental temperature (300, 310 and 320 K). The values of\(\mathrm{\Delta}H^{0}\ \)and \(\mathrm{\Delta}S^{0}\)were obtained from the slope and intercept of linear plot of logKa versus 1/T. The value of free energy change \(\mathrm{\Delta}G^{0}\) can be calculated from the following equation 56 : \(\mathrm{\Delta}G^{0}\) = \(\mathrm{\Delta}H^{0}\)- T\(\mathrm{\Delta}S^{0}\) (4) Animal model and treatments Healthy male SPF CD-1/ICR mice (six weeks old, 18–22 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). All animal experiments were conducted in accordance with international ethical guidelines for the care and use of laboratory animals. Mice were housed under controlled conditions at 24 ± 2 °C with 55 ± 10 % relative humidity and a 12-hour light/dark cycle. They were provided with a standard laboratory diet and had ad libitum access to food and water. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Yunnan Luoyu Biotechnology Co., Ltd. (Approval No. SL20240403). After a one-week acclimation period, mice were randomly divided into five groups (n = 3 per group). Prior to the experiment, a 2 × 2 cm² area of dorsal hair was removed using a hair shaver. Except for the normal control group, epidermal barrier disruption was induced in all other groups by repeatedly stripping the dorsal skin with adhesive tape for five consecutive days, until transepidermal water loss (TEWL) exceeded 35 mg/cm²/h. TEWL was measured using a VapoMeter (Tewameter TM300, Courage Khazaka, Germany). TEWL refers to the amount of water that passively evaporates from inside the body through the epidermal layer. It is a widely accepted parameter for evaluating skin barrier function in humans and animals, with higher TEWL values indicating greater barrier damage 17 . The normal group received no treatment, while the other four groups were treated topically with 200 μL of either: distilled water, 10 % PPE extract-based cream, 10 % AKE extract-based cream, or cream base alone. Treatments were applied to the dorsal skin twice daily for eight consecutive days. Epidermal barrier recovery was assessed by measuring TEWL, pH, skin elasticity, and hydration on days 0, 2, 4, and 8 after treatment initiation. Recovery rate was calculated using the following formula 17 : Epidermal barrier recovery (100 %) = (TEWL immediately after barrier disruption - TEWL at the indicated timepoint) / (TEWL immediately after barrier disruption -baseline TEWL) ×100 % Histological examination Histological analysis of the dorsal skin of mice was conducted following previously published methods 17 . At the end of the treatment period, the animals were euthanized, and the dorsal skin was immediately excised. Each skin sample was divided into two portions: one for morphological evaluation and the other for biochemical analysis of ceramide, occludin, and keratin content. For morphological analysis, the tissue samples were fixed in 10 % neutral buffered formalin, embedded in paraffin, and sectioned at a thickness of 6 μm. The sections were stained with hematoxylin and eosin (H&E) and examined for epidermal structure and pathology. To assess skin barrier repair, epidermal thickness was measured using a Pannoramic MID II scanner. Measurements were performed individually for each animal and used for statistical analysis. Qualitative analysis of PPE and AKE by LC-MS Chromatographic separation was conducted using a Hypersil GOLD Phenyl C4 column (100 mm × 2.1 mm, 1.9 μm particle size). The gradient elution program employed a mobile phase consisting of 0.1 % formic acid in water (2 %) and methanol (98 %) over a 5-minute run time. The injection volume was 5 μL, and the flow rate was maintained at 0.3 mL/min. Mass spectrometry data were acquired using an electrospray ionization (ESI) source in positive ion mode. The operating conditions were as follows: sheath gas flow rate of 13.5 L/min, auxiliary gas flow rate of 3 L/min, spray voltage of 3 kV, and capillary temperature of 350 °C. The scan range for mass detection was set from m/z 80 to 1200. Molecular docking study Molecular docking was employed to investigate the interactions between four candidate compounds, paeoniflorin, albiflorin, atractylenolide I, and atractylenolide II, and three target enzymes: hyaluronidase, elastase, and collagenase. The three-dimensional (3D) structures of these enzymes were obtained from the Protein Data Bank (PDB; https://www.rcsb.org/): hyaluronidase (PDB ID: 2PE4), collagenase (PDB ID: 4AR1), and elastase (PDB ID: 3EST). Prior to docking, the protein structures were preprocessed using Visual Molecular Dynamics (VMD) software to remove existing ligands and solvent molecules, ensuring structural integrity and consistency 57 . The 3D structures of the ligands (paeoniflorin, albiflorin, atractylenolide I, and II) were optimized by energy minimization using Gaussian 09 software 58 , applying the B3LYP functional and 6-31G basis set. Docking simulations were conducted using AutoDock 4.2 59 , with the docking box set to encompass the entire enzyme structure to allow for a comprehensive assessment of potential binding sites. A flexible docking approach was used to account for conformational changes in both ligands and proteins during binding. The docking grid parameters for each enzyme were defined as follows: for hyaluronidase, the center was set at x = 38.54, y = -25.778, z = -8.639, with a size of 126 Å in all dimensions; for elastase, the center was at x = -4.525, y = 28.35, z = 43.101, with a size of 108 Å in the x-axis, 90 Å in the y-axis, and 100 Å in the z-axis; and for collagenase, the center was at x = -7.175, y = -29.506, z = 13.012, with a size of 108 Å in all dimensions. Docking results were ranked according to binding energy, with the lowest-energy conformation representing the most favorable ligand–protein interaction. LigPlot+ was used to generate 2D interaction diagrams, identifying key hydrogen bonds and hydrophobic contacts 60 . PyMOL was used to visualize the 3D structures of the most stable ligand–enzyme complexes, providing detailed insights into molecular interactions. Statistical analysis GraphPad Prism 6.0 software was used for all statistical analyses. The statistical differences between groups were performed using one-way analysis of variance (ANOVA). All quantitative data were expressed as mean ± SEM from at least three independent experiments. Statistical significance was considered at the P < 0.05. Supporting Information The supporting information for this article is available on the WWW under https://doi.org/10.1002/cjoc.70XXX. Acknowledgement We thank Advanced Analysis and Measurement Center of Yunnan University for the sample testing service. Funding: This work was financially supported by the National Natural Science Foundation of China (No. 22367023), the Project of Yunnan Characteristic Plant Screening and R&D Service CXO Platform ( 2022YKZY001), Postgraduate Scientific Research Innovation Project of Yunnan University (KC-24249631). Authors thank Advanced Analysis and Measurement Center of Yunnan University for the sample testing service. References 60. Li, P.-P.; Jiang, X.-M.; Shi, J.-Y.; Zhang, W.; Ding, Y.-F.; Xie, S.-Z.; Wu, D.-L., Structural characterization and anti-inflammatory activity of a novel polysaccharide from Paeonia lactiflora Pall. J Funct Foods 2024, 120 , 106392. Ying, W.; Jia-Ying, W.; Jing-Xuan, B.; Xiao-Qi, W.; Amy Sze-Man, L.; Xiao-Yun, F.; Rui-Xuan, H.; Li, W.; Xiaobing, D.; Xiu-Qiong, F.; Zhi-Ling, Y., Macrophage and keratinocyte cell assays suggest that the supercritical CO2 extract of black soybean possesses anti-inflammatory and skin barrier-protective effects. 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[1]¿p#1 Manuscript received: XXXX, 2025 Manuscript revised: XXXX, 2025 Manuscript accepted: XXXX, 2025 Version of record online: XXXX, 2025 Left to Right: Authors Names You will be invited to submit the most recent photos of all the authors upon acceptance of the manuscript Entry for the Table of Contents Atractylodes Macrocephala Koidz Extracts (AKE) Enhance Skin Barrier Repair via Synergistic Ceramide Upregulation, Hyaluronidase Inhibition, Anti-Inflammatory Action, and Proteins Modulation Huan Liu a, 1 , Le Yu a, 1 , Chang-Yin Yang b , Yu-qiang Zhao a , Lin Xu b,* , Ying Zhou a,* * Chin. J. Chem. 2025 , 43 , XXX—XXX. DOI: 10.1002/cjoc.70XXX Herbal extracts AKE ( Atractylodes macrocephala Koidz.) demonstrated efficacy in promoting skin barrier repair, with AKE achieving 81.2% recovery in mouse models via synergistic mechanisms, including enhancing ceramide synthesis, inhibiting hyaluronidase activity, reducing inflammatory responses and regulating skin-related proteins. This study provides novel insights into low-irritation barrier repair, advancing herbal for cosmetic development. Information & Authors Information Version history V1 Version 1 22 November 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords anti-inflammatory atractylodes macrocephala koidz ceramide hyaluronidase inhibition skin barrier skin barrier-related proteins Authors Affiliations Huan Liu Yunnan University View all articles by this author Le Yu Yunnan University View all articles by this author Chang-Yin Yang East China Normal University View all articles by this author Yu-qiang Zhao Yunnan University View all articles by this author Lin Xu 0000-0002-6332-1376 East China Normal University View all articles by this author Ying Zhou [email protected] Yunnan University View all articles by this author Metrics & Citations Metrics Article Usage 167 views 83 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Huan Liu, Le Yu, Chang-Yin Yang, et al. Atractylodes Macrocephala Koidz Extracts (AKE) Enhance Skin Barrier Repair via Synergistic Ceramide Upregulation, Hyaluronidase Inhibition, Anti-Inflammatory Action, and Proteins Modulation. Authorea . 22 November 2025. 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