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Article

Birch Sap Attenuates Inflammatory Cytokines and Improves Skin Parameters in Cellular and Animal Models of Skin Irritation

by
Chao-Hsien Sung
1,2,3,†,
Chien-Fen Huang
3,†,
Yu-Jou Hsu
3,
Chi-Ming Pu
4,
Chia-Chi Kung
1,
Thomas W. Chu
5,6,* and
Chi-Feng Hung
2,3,7,*
1
Department of Anesthesiology, Fu Jen Catholic University Hospital, Fu Jen Catholic University, New Taipei City 24205, Taiwan
2
School of Medicine, Fu Jen Catholic University, New Taipei City 24205, Taiwan
3
PhD Program in Pharmaceutical Biotechnology, Fu Jen Catholic University, New Taipei City 24205, Taiwan
4
Division of Plastic Surgery, Department of Surgery, Cathay General Hospital, Taipei 10630, Taiwan
5
Department of Dermatology, Far Eastern Memorial Hospital, New Taipei City 22060, Taiwan
6
Department of Dermatology, Eastern Virginia Medical School, Norfolk, VA 23507, USA
7
School of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Cosmetics 2025, 12(6), 282; https://doi.org/10.3390/cosmetics12060282
Submission received: 18 November 2025 / Revised: 8 December 2025 / Accepted: 9 December 2025 / Published: 11 December 2025
(This article belongs to the Section Cosmetic Dermatology)
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Abstract

Natural ingredients with soothing and skin-protecting effects are becoming increasingly popular in cosmetic science. Great interest has been directed to birch sap, a nutrient-rich fluid from Betula species. This study aimed to investigate whether birch sap can modulate inflammatory responses and maintain skin barrier functions in both cell and animal models. The polysaccharide composition of birch sap was characterized. TNF-α/IFN-γ-stimulated HaCaT keratinocytes were used to assess the effects of birch sap on inflammatory cytokine expression and activation of MAPK and NF-κB signaling pathways. An in vivo model of chemically induced skin irritation was further used to examine the effects of oral birch sap administration on skin hydration, transepidermal water loss, histological features, and cutaneous blood flow. Birch sap significantly reduced IL-1β, IL-6, and IL-8 expression and attenuated MAPK and NF-κB phosphorylation. In vivo, birch sap improved hydration, reduced transepidermal water loss, epidermal thickening and erythema, and decreased elevated skin blood flow associated with inflammation. These results demonstrate that birch sap provides modulatory effects on inflammation and barrier-supportive effects in both cellular and animal models, suggesting its potential as a naturally derived cosmetic ingredient for promoting skin comfort and maintaining epidermal barrier integrity.

Graphical Abstract

1. Introduction

Skin irritation is a common problem that affect skin comfort and appearance; it is associated with genetic factors, environmental exposures, and immune activation [1,2]. Heightened inflammatory responses, oxidative stress, and impaired skin barrier function are the main mechanisms in skin hypersensitivity and inflammatory skin diseases [3,4,5]. Although numerous topical agents are available to soothe irritated skin and support barrier recovery, many formulations offer only partial relief or may cause sensitivity with long-term use, creating a demand for naturally derived ingredients with favorable safety profiles [6]. Birch sap, a naturally occurring fluid collected from Betula species, has long been consumed in Northern Europe and Asia for general wellness and cosmetic applications [7,8]. The primary components of birch sap are polysaccharides, but it also contains numerous bioactive substances, despite their low concentration, which can regulate cell activity [7,9,10]. These substances include vitamins, organic acids, and essential minerals [9,11,12,13]. Birch sap from Betula Alba could reduce ultraviolet-induced cell damage in vitro [14]. However, little is known about the effect of birch sap on skin inflammation and hypersensitivity. Based on these findings, we evaluated the anti-inflammatory and antioxidative effects of birch sap in cell and animal models. Elucidating these mechanisms may facilitate the development of safe and naturally derived formulations based on birch sap.

2. Materials and Methods

2.1. Materials

Birch sap samples, obtained from Betula platyphylla, were provided by Chihiro Kawase, Forest Works (Hokkaido, Japan). The samples were immediately frozen and air-shipped to Taiwan. The compositional analysis of polysaccharides followed the methodology detailed by Huang et al. [15]. Human keratinocyte cells (HaCaT) cells were provided by Professor Ming-Jen, Hsu from Department of pharmacology, Taipei Medical University, Taiwan. The following antibodies were used in the experiments: Phospho-p38 MAPK Antibody (Affinity Biosciences, Cincinnati, OH, USA, #AF4001), p38 MAPK Antibody (Cell Signaling Technology, Danvers, MA, USA, #9212), phospho-p44/42 MAPK (ERK1/2) Antibody (Cell Signaling, Danvers, USA, #4370), p44/42 MAPK (ERK1/2) Antibody (Cell Signaling, Danvers, USA, #9102), phospho-SAPK/JNK Antibody (Cell Signaling, Danvers, USA, #4668), SAPK/JNK Antibody (Cell Signaling, Danvers, USA, #9252), phosphor-IκBα Antibody (Cell Signaling, Danvers, USA, #9246), β-actin Antibody (Cell Signaling, Danvers, USA, #4970), phospho-NF-κB p65/RelA-S276 Antibody (ABclonal Technology, Woburn, MA, USA, #AP0123), and NF-κB p65 Antibody (Cell Signaling, Danvers, USA, #8242). Anti-rabbit IgG antibodies (Cell Signaling, Danvers, USA, #7074) and anti-mouse IgG antibodies (Cell Signaling, Danvers, USA, #7076) were used as secondary antibodies.

2.2. In Vitro Experiments

2.2.1. Cell Lines

HaCaT cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cultures were kept in a humidified incubator regulated at 37 °C with 5% CO2. Cells were allowed to grow until they reached approximately 90% confluence before being harvested and subcultured for experimental use. Sub-confluent cultures were consistently used for all subsequent assays.

2.2.2. Cell Viability Test

Cell viability was quantified using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium] colorimetric assay. HaCaT cells were initially seeded at 5 × 104 cells per well in 24-well microplates and subsequently treated with various concentrations of birch sap for 24 h. Following the treatment phase, 300 μL of MTT solution (prepared in a 1:9 ratio with culture medium) was added to each well and incubated at 37 °C for 2 h, during which metabolically active cells reduced the MTT to insoluble formazan crystals. The medium was then removed, and the crystals were dissolved by adding 200 μL of dimethyl sulfoxide (DMSO) to each well. Finally, the optical density was measured at 550 nm using an ELISA reader, where the resulting absorbance is directly proportional to the number of viable cells present.

2.2.3. Western Blot Assay

HaCaT cells were cultured in 3.5 cm dishes until 90% confluence was reached, followed by 24 h of nutrient deprivation. Cells were incubated with various concentrations of birch sap for 1 h and subsequently stimulated with TNF-α/IFN-γ for 30 min or 1 h. After cells were collected and lysed, the lysates were centrifuged at 13,200 revolutions per minute for 10 min at 4 °C, and protein quantification was performed using the Pierce BCA protein assay kit (Pierce, Rockford, IL, USA).
Equal amounts of protein (20–40 μg) were resolved on 10% SDS-polyacrylamide gels and subsequently transferred onto PVDF membranes. Non-specific binding was inhibited by incubating the membranes in a solution of 5% skimmed milk powder prepared in TBS–T (Tris-buffered saline with 0.05% Tween 20) under shaking conditions for 1 h. The membrane was then washed 3 times each for 10 min with TBS-T. Primary antibodies diluted at a ratio of 1:1000 were added and incubated overnight at 4 °C. The membrane was washed three more times with TBS-T. Secondary antibodies, diluted at a ratio of 1:1000, were added and incubated for 1 h. Membrane was washed again 3 times with TBS-T. Protein bands were visualized with addition of chemiluminescence substrate, and images were captured with a BIO-STEP Celvin® chemiluminescent detection system (Biostep GmbH, Burkhardtsdorf, Germany).

2.2.4. Quantitative Polymerase Chain Reaction (qPCR)

Cells were pretreated with birch sap for 1 h and then stimulated with TNF-α/IFN-γ for an additional 1 h. Cells were collected and centrifuged at 16,000 revolutions per minute for 10 min at 4 °C, and the resulting pellet was used for RNA extraction. Total RNA was isolated and purified using the GeneDireX® Total RNA Isolation Kit (GeneDireX, Inc., Vegas, NV, USA), following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from total RNA using the iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the supplied protocol. PowerUp™ SYBR™ Green Master Mix (Applied Biosystems™, Foster City, CA, USA) was used in qPCR tests. Each 20-μL reaction contained 2 μL sample, 10 μL of SYBR Green Master Mix, 7.5 μL of nuclease-free water, and 0.25 μL each of forward and reverse primers. Amplification reactions were carried out on an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems™, Foster City, CA, USA). The primer sequences used for human genes are listed in Table 1.

2.3. In Vivo Experiments

2.3.1. Experiment Animals

Eight-week-old male BALB/c mice were obtained from the National Laboratory Animal Center. The experiments were approved, and the protocol number was A11128 (Approval on 20 July 2022). Mice were divided into groups of two to four per cage under standardized conditions (21 ± 2 °C, 55 ± 10% relative humidity, 12-h light/dark cycle, and 10–15 air exchanges per hour). Ambient noise was kept below 65 dB. Animals were acclimated for one week before experiments began. Alfalfa-free chow and water were provided ad libitum throughout the study. Right-ear thickness was measured using a Mitutoyo 500-196-30 ABSOLUTE Digimatic Caliper (Mitutoyo, Kawasaki, Japan), which was calibrated prior to each measurement.

2.3.2. Experiment Groups

2,4-Dinitrochlorobenzene (DNCB) is a well-established agent for inducing allergic dermatitis in murine models [16]. Mice were randomly assigned to five groups: (1) Control, (2) DNCB only, (3) birch sap 6 mL/kg with DNCB, (4) birch sap 10 mL/kg with DNCB, and (5) dexamethasone 0.2 mg/kg with DNCB (positive control). Dexamethasone served as the positive control and was administered by intraperitoneal injection, whereas DNCB was applied topically (100 μL to the dorsal skin and 20 μL to the right ear). Each experimental group consisted of three mice. Three days before the start of the experiment (Day−3), mice were anesthetized, and dorsal hair was removed using a razor followed by application of depilatory cream. After 3-day period of recovery and acclimatization (until Day 1), the general health status of the mice and the condition of the depilated dorsal skin were examined to confirm the absence of abnormalities before initiating the experiment. Skin physiological parameters were measured daily using a multifunctional skin analysis system (MPA-580; Courage & Khazaka, Cologne, Germany). Non-contact laser speckle contrast imaging (RWD Life Science, Shenzhen, China, RFLSI-ZW) quantified dorsal skin blood flow. Photographs of dorsal and ear skin and body weight data were also collected. Temperature and humidity were controlled during the measurements were performed. The experimental protocol consisted of two phases: a sensitization phase (Days 1–4) and a re-induction phase (Days 5–14). Baseline skin physiology measurements were performed and 1% DNCB was applied to skin in all DNCB-treated groups on Day 1. During the re-induction phase, mice received daily oral gavage of birch sap (6 mL/kg or 10 mL/kg) or dexamethasone (0.2 mg/kg) from Day 5 onward. Additionally, dorsal skin and right ear were treated with 0.5% DNCB on Days 8, 11, and 14. Physiological parameters and photographic records were collected on Day 1, Day 5 and on the day immediately following each DNCB challenge (Day 2, 9, 12, 15). On Day 15, the mice were sacrificed, and tissues were collected for further analysis. The complete timeline of experiments is illustrated in Figure 1.

2.3.3. Histopathological Analysis

Mouse skin tissues were fixed overnight at 4 °C in 4% paraformaldehyde (PFA), followed by dehydration, clearing, and paraffin embedding. The paraffin blocks were sectioned at 5 µm using a Leica microtome and stained with hematoxylin and eosin (H&E). Tissue sections were photographed with an EVOSTM FL Auto Imaging System (Version 1.6, Thermo Fisher Scientific, Waltham, MA, USA).

2.4. Data Analysis

All experimental data are expressed as the mean ± SEM (Standard Error of the Mean). Analysis were performed with Sigma-Plot (Version 14.0). An unpaired, two-tailed Student’s t-test was used to compare differences between groups. Results were considered statistically significant when the p-value was below 0.05.

3. Results

3.1. Monosaccharides Compositional Analysis in Birch Sap

Samples of the birch sap were analyzed according to the procedure described by Huang et al. [15]. The monosaccharides composition analysis of birch sap identified fucose, glucose, and fructose as main constituents with an approximate ratio of 3:5:8, respectively.

3.2. Analysis of the Effect of Birch Sap on Keratinocyte Viability

HaCaT cells were treated with birch sap at concentrations ranging from 0% to 10% for 24 h. Cell viability was determined by the MTT assay to assess cytotoxicity. Birch sap did not exhibit cytotoxicity against HaCaT cells at any concentration tested (Figure 2). The results demonstrate that birch sap is well-tolerated by keratinocytes. Therefore, concentrations of 1%, 3%, and 10% were selected for subsequent studies.

3.3. Effects of Birch Sap on Pro-Inflammatory Cytokines mRNA Expression After HaCaT Cells Stimulation with Tumor Necrosis Factor-α (TNF-α)/Interferon-γ(IFN-γ)

TNF-α and IFN-γ stimulation is routinely used to stimulate HaCaT cells, promoting the upregulation of cytokines and chemokines [17]. Cells were preincubated with birch sap for 1 h, followed by stimulation with TNF-α/IFN-γ for 1 h. RT-qPCR analysis demonstrated that TNF-α/IFN-γ stimulation markedly increased the expression of pro-inflammatory cytokines, whereas birch sap treatment significantly attenuated these effects. Specifically, birch sap reduced the expressions of interleukin-1β (IL-1β) and IL-8 at concentrations of 3% and 10% (Figure 3A,C), and suppressed IL-6 expression at 1%, 3%, and 10% (Figure 3B).

3.4. Effects of Birch Sap on MAPK Phosphorylation After HaCaT Cells Stimulation with TNF-α/IFN-γ

TNF-α and IFN-γ can activate MAPK signaling pathways in HaCaT keratinocytes and promote inflammatory responses [18,19]. To examine whether birch sap modulates these signaling pathways, cells were pretreated with birch sap at concentrations of 1%, 3%, and 10% (diluted in complete medium) for 1 h, followed by stimulation with TNF-α/IFN-γ for 30 min. Phosphorylation levels of p38, ERK, and JNK were assessed using Western blot analysis. Stimulation markedly increased the p38, ERK, and JNK phosphorylation. We observed a dose-dependent reduction in protein phosphorylation in cells treated with birch sap (Figure 4).

3.5. Effects of Birch Sap on NF-κB Pathway Phosphorylation After HaCaT Cells Stimulation with TNF-α/IFN-γ

Cells were pretreated with birch sap at concentrations of 1%, 3%, and 10% for 1 h, followed by stimulation with TNF-α/IFN-γ for an additional 1 h. IκBα and NF-κB phosphorylation were evaluated using Western blot analysis. Stimulation with TNF-α/IFN-γ significantly increased IκBα and NF-κB phosphorylation. Pretreatment with birch sap decreased the level of IκBα and NF-κB phosphorylation, suggesting inhibition of the NF-κB signaling cascade (Figure 5).

3.6. Effects of Birch Sap on Skin Lesions Induced by DNCB

Oral administration of birch sap (10 mL/kg) significantly alleviated erythema and reduced epidermal hyperplasia (Figure 6A). Histological analysis with hematoxylin and eosin (H&E) staining of ear tissues revealed marked morphological differences between the treatment groups (Figure 6B). As expected, DNCB application markedly increased epidermal thickness, indicative of severe inflammatory changes. In contrast, birch sap treatment significantly attenuated this epidermal thickening, demonstrating the ability to mitigate DNCB-induced epidermal hyperplasia and inflammation.

3.7. Effects of Birch Sap on Skin Blood Flow

The effect of birch sap on cutaneous blood flow was assessed using laser speckle contrast imaging (LSCI). A major hallmark of inflammation is the increased blood flow resulting from localized vasodilation. A reduction in the severity of inflammation is typically associated with normalization of skin blood flow [20]. Mice treated with DNCB showed markedly increased skin blood flow, while mice treated with birch sap at doses of 6 mL/kg and 10 mL/kg exhibited significantly lower skin blood flow (Figure 7). These results indicate that birch sap normalizes skin blood flow, likely by alleviating inflammation-induced vasodilation.

3.8. Effects of Birch Sap on Skin Physiological Parameters

Skin irritation and barrier disruption weaken epidermal integrity and lead to increased transepidermal water loss (TEWL) [21]. These parameters (TEWL, skin hydration, erythema, and ear thickness) were assessed with a multifunctional skin analysis system, with baseline measurements taken prior to the commencement of experiment.
Following DNCB application, there was a marked increase in TEWL and erythema by Day 9. A significant right ear thickening was also observed in the DNCB-treated group, consistent with epidermal hyperplasia. In contrast, mice treated with birch sap (6 mL/kg or 10 mL/kg) or dexamethasone (0.2 mg/kg) demonstrated substantial improvement across all parameters (Figure 8). These findings indicate that birch sap effectively ameliorates barrier impairment and cutaneous inflammation induced by DNCB, supporting its potential role in restoring epidermal homeostasis.

4. Discussion

Skin irritation and barrier dysfunction result from interactions between various factors, such as impaired epidermal barrier integrity, altered immune regulation, and heightened inflammatory responses [4,22,23,24]. Barrier disruption prompts keratinocytes to release cytokines and chemokines, thereby exacerbating local inflammation and inducing additional damage of the stratum corneum [25,26,27]. These mediators are crucial in driving Th2- and Th1-associated inflammatory patterns and facilitate immune-cell infiltration [28]. MAPK family members, including ERK, JNK, and p38, play a pivotal role in transducing extracellular stress signals into intracellular responses [29]. Moreover, exposure to stimulants such as IFN-γ and TNF-α activates the NF-κB pathway, enabling nuclear translocation and transcription of downstream inflammatory genes [30,31]. Enhanced activity of MAPK and NF-κB has been reported in models of skin irritation, and suppression of these pathways is considered an effective strategy for reducing epithelial inflammatory responses [3,30,31,32,33,34,35]. In our study, birch sap demonstrated inhibitory effects on these canonical inflammatory pathways in keratinocytes activated by TNF-α and IFN-γ, as evidenced by reduced phosphorylation of p38, ERK, JNK, IκBα, and NF-κB. Since these pathways regulate secretions of IL-1β, IL-6, and IL-8 [36], the observed decrease in cytokine expression suggests that birch sap effectively modulates early inflammatory signaling events. The simultaneous influence of birch sap on multiple inflammatory signaling pathways contributes to the robust physiological improvements observed in vivo.
Another significant result is the normalization of cutaneous blood flow, assessed by LSCI. Vasodilation and increased blood flow are characteristics of inflammation in irritated skin. The ability of birch sap to reduce hyperemia provides functional evidence of its anti-inflammatory effect. Combined with improvements in skin physiological parameters, these outcomes demonstrate that birch sap not only attenuates inflammation but also promotes restoration of epidermal homeostasis and function. These benefits may be attributed to polysaccharides and trace minerals in birch sap. Plant-derived polysaccharides may contribute to anti-aging, wound healing and skin barrier repair through mechanisms such as scavenging free radicals, regulating telomerases activity, modulating the gut–skin axis, and improving skin tight junction function [37,38,39,40,41,42]. Oral supplementation of aloe polysaccharides may decrease skin thickness and improve expressions of genes related to tight junction in animal models of atopic dermatitis [43]. Orally administered polysaccharides from blackcurrants could improve skin hydration in ultraviolet-irradiated hairless mice [44]. These beneficial outcomes are associated with the suppression of key signaling cascades, specifically the MAPK pathway and NF-κB pathway, and the activity of matrix metalloproteinases (MMPs) [45,46,47]. Minerals contained in birch sap, including strontium, selenium, magnesium, and calcium, could help reduce inflammation in skin, reduce oxidative stress and promote skin barrier function [48,49]. Although we did not isolate individual constituents, the multi-component nature of birch sap may produce synergistic actions on barrier repair and inflammatory modulation. Preliminary clinical evidence strongly supports the relevance of birch-based formulations in cosmetic applications. In a randomized controlled study, a 28-day topical birch juice intervention significantly increased stratum corneum hydration, reduced TEWL, and diminished erythema and skin perfusion in individuals with sensitive skin [49].
There are several limitations in the current study. First, birch sap used in this study was an unrefined preparation. Therefore, future investigations should focus on detailed compositional analysis to determine specific bioactive compounds and their possible pharmacological actions. Standardized extraction procedures must also be established. Furthermore, stability testing of the birch sap must be conducted to determine its shelf life and influence on practical usability in cosmetic formulations. Second, although oral administration produced measurable physiological benefits, the pharmacokinetic characteristics of compounds in birch sap remain insufficiently defined. Comprehensive studies addressing absorption, systemic distribution, metabolic transformation, and bioavailability will be necessary to clarify the mechanism by which orally ingested birch sap influences skin physiology.

5. Conclusions

Birch sap exhibited potent anti-inflammatory and skin barrier recovery effects in vitro and in vivo in a chemical-induced skin damage model. These effects are likely associated with the inhibition of MAPK and NF-κB pathways and the downregulation of inflammatory cytokines. Birch sap also helped normalize skin blood flow and improve skin physiological parameters. These preliminary findings on birch sap demonstrated its translational value as a natural cosmetic ingredient. Standardized preparations, chemical compositional analysis, thorough toxicological examinations, and rigorous clinical investigations are further needed to verify the potential of birch sap as a valuable, well-defined and safe agent in cosmetic products.

Author Contributions

Conceptualization, C.-H.S., C.-C.K., T.W.C. and C.-F.H. (Chi-Feng Hung); Methodology, Y.-J.H., C.-F.H. (Chien-Feng Huang); Validation, C.-H.S., C.-M.P., C.-C.K., T.W.C. and C.-F.H. (Chi-Feng Hung); Formal analysis, Y.-J.H., C.-F.H. (Chien-Feng Huang) and C.-F.H. (Chi-Feng Hung); Investigation, C.-H.S., Y.-J.H.; Resources, C.-H.S., T.W.C. and C.-F.H. (Chi-Feng Hung); Data curation, Y.-J.H., C.-F.H. (Chien-Feng Huang); Writing—original draft preparation, C.-H.S. and C.-F.H. (Chi-Feng Hung); Writing—review and editing, Y.-J.H., C.-H.S., T.W.C. and C.-F.H. (Chi-Feng Hung); Funding acquisition, C.-H.S., C.-M.P., C.-C.K., T.W.C. and C.-F.H. (Chi-Feng Hung). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Far Eastern Memorial Hospital (111-FEMH-FJU-02), Fu Jen Catholic University Hospital (FJUH-114-040), Cathay General Hospital (106-CGH-FJU-06), and Ministry of Science and Technology (113-2320-B-030-007-MY3) in Taiwan.

Institutional Review Board Statement

All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Fu Jen Catholic University (approval number A11128, approval date: 20 July 2022). The principles of the 3Rs (Replacement, Reduction, and Refinement) and ARRIVE guidelines were followed to optimize the experimental design.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is available upon reasonable request. The data supporting the findings of this study can be obtained from the corresponding author.

Acknowledgments

We thank Yun-Di Wu and Andrea Ssu-Chi Yu for their participation in this study. HaCaT cells were provided by Ming-Jen, Hsu from the Department of Pharmacology, Taipei Medical University, Taiwan.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
Nuclear factor kappa B NF-κB
Mitogen-activated protein kinases MAPK
Tumor necrosis factor-αTNF-α
Interferon-γINF-γ
Dulbecco’s modified eagle mediumDMEM
Fetal bovine serumFBS
Dimethyl sulfoxideDMSO
2,4-DinitrochlorobenzeneDNCB
Hematoxylin and eosin H&E
Standard error of the meanS.E.M.
Quantitative polymerase chain reactionqPCR
InterleukinIL
Laser speckle contrast imaging LSCI
Transepidermal water lossTEWL
ParaformaldehydePFA
Matrix metalloproteinaseMMP
Nuclear factor erythroid 2-related factors 2Nrf2

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Figure 1. Summary of experimental timeline. Red star signs indicated skin physiology tests, including TEWL, ear thickness, erythema, and hydration. TEWL: Transepidermal water loss.
Figure 1. Summary of experimental timeline. Red star signs indicated skin physiology tests, including TEWL, ear thickness, erythema, and hydration. TEWL: Transepidermal water loss.
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Figure 2. Cell viability in human keratinocytes (HaCaT) after treatment with different concentrations of birch sap using the MTT Assay. -: Not exposed to birch sap.
Figure 2. Cell viability in human keratinocytes (HaCaT) after treatment with different concentrations of birch sap using the MTT Assay. -: Not exposed to birch sap.
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Figure 3. Effects of Birch sap on mRNA expression levels of (A) IL-1β, (B) IL-6 and (C) IL-8 in TNF-α/IFN-γ-stimulated HaCaT cells. Cells were treated with different concentrations of birch sap for 1 h and stimulated with TNF-α/IFN-γ (10 ng/mL) for 1 h. Total RNA was isolated, and the mRNA expression levels were determined using qPCR. Values are presented as mean ± SEM from three independent experiments. -: Not exposure to birch sap or TNF-α/IFN-γ; ## p < 0.01 compared to the control condition; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment condition.
Figure 3. Effects of Birch sap on mRNA expression levels of (A) IL-1β, (B) IL-6 and (C) IL-8 in TNF-α/IFN-γ-stimulated HaCaT cells. Cells were treated with different concentrations of birch sap for 1 h and stimulated with TNF-α/IFN-γ (10 ng/mL) for 1 h. Total RNA was isolated, and the mRNA expression levels were determined using qPCR. Values are presented as mean ± SEM from three independent experiments. -: Not exposure to birch sap or TNF-α/IFN-γ; ## p < 0.01 compared to the control condition; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment condition.
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Figure 4. Effects of Birch sap on the phosphorylation of (A) p38, (B) ERK, (C) JNK in TNF-α/IFN-γ-stimulated HaCaT cells. After 1 h of birch sap treatment, cells were stimulated with TNF-α/IFN-γ (10 ng/mL) for 0.5 h. Western blot was performed and quantitatively analyzed. Values are presented as mean ± SEM from the three independent experiments. -: Not exposure to birch sap or TNF-α/IFN-γ; ## p < 0.01 compared to the control group; ** p < 0.01 compared with the TNF-α/IFN-γ treatment group.
Figure 4. Effects of Birch sap on the phosphorylation of (A) p38, (B) ERK, (C) JNK in TNF-α/IFN-γ-stimulated HaCaT cells. After 1 h of birch sap treatment, cells were stimulated with TNF-α/IFN-γ (10 ng/mL) for 0.5 h. Western blot was performed and quantitatively analyzed. Values are presented as mean ± SEM from the three independent experiments. -: Not exposure to birch sap or TNF-α/IFN-γ; ## p < 0.01 compared to the control group; ** p < 0.01 compared with the TNF-α/IFN-γ treatment group.
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Figure 5. Effect of Birch sap on (A) IκB, (B) NF-κB phosphorylation in TNF-α/IFN-γ-stimulated HaCaT cells. After 1 h of birch sap treatment, cells were stimulated with TNF-α/IFN-γ (10 ng/mL) for 1 h. Western blot was performed and quantitatively analyzed. Values are presented as mean ± SEM from the three independent experiments. -: Not exposure to birch sap or TNF-α/IFN-γ; ## p < 0.01 compared to the control group; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment group.
Figure 5. Effect of Birch sap on (A) IκB, (B) NF-κB phosphorylation in TNF-α/IFN-γ-stimulated HaCaT cells. After 1 h of birch sap treatment, cells were stimulated with TNF-α/IFN-γ (10 ng/mL) for 1 h. Western blot was performed and quantitatively analyzed. Values are presented as mean ± SEM from the three independent experiments. -: Not exposure to birch sap or TNF-α/IFN-γ; ## p < 0.01 compared to the control group; * p < 0.05 and ** p < 0.01 compared with the TNF-α/IFN-γ treatment group.
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Figure 6. Phenotypic and histological analysis to assess the effect of birch sap on DNCB-induced skin lesion. (A) Phenotypic appearance of mouse skin after 15 days of treatment; (B) Histological analysis of ear tissue sections after H&E staining from each treatment group. The red arrow indicates the epidermal layer of skin. DNCB application increased the epidermal thickness, which was significantly reduced by oral birch sap treatment.
Figure 6. Phenotypic and histological analysis to assess the effect of birch sap on DNCB-induced skin lesion. (A) Phenotypic appearance of mouse skin after 15 days of treatment; (B) Histological analysis of ear tissue sections after H&E staining from each treatment group. The red arrow indicates the epidermal layer of skin. DNCB application increased the epidermal thickness, which was significantly reduced by oral birch sap treatment.
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Figure 7. Effect of birch sap on skin blood flow assessed by Laser Speckle Contrast Imaging. Increased cutaneous blood flow was observed after treatment with DNCB. Marked reduction in blood flow was observed in oral birch sap group compared to DNCB group. Warm colors (red/yellow) depict higher blood flow, whereas cool colors (blue) represent lower blood flow.
Figure 7. Effect of birch sap on skin blood flow assessed by Laser Speckle Contrast Imaging. Increased cutaneous blood flow was observed after treatment with DNCB. Marked reduction in blood flow was observed in oral birch sap group compared to DNCB group. Warm colors (red/yellow) depict higher blood flow, whereas cool colors (blue) represent lower blood flow.
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Figure 8. Effects of birch sap on physiological parameters. Values are presented as mean ± SEM from at least three independent experiments. ## p < 0.01 compared with the control group; * p < 0.05 and ** p < 0.01 compared with the DNCB-induced group.
Figure 8. Effects of birch sap on physiological parameters. Values are presented as mean ± SEM from at least three independent experiments. ## p < 0.01 compared with the control group; * p < 0.05 and ** p < 0.01 compared with the DNCB-induced group.
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Table 1. Human primer sequences for RT-PCR.
Table 1. Human primer sequences for RT-PCR.
GenesPrimersSequence (5′-3′)
IL-1βForwardCTC TCA CCT CTC CTA CTC ACT
ReverseATC AGA ATG TGG GAG CGA AT
IL-6ForwardCGA GCC CAC CGG GAA CGA AA
ReverseGGA CCG AAG GCG CTT GTG GAG
IL-8ForwardACT GAG AGT GAT TGA GAG TGG AC
ReverseAAC CCT CTG CAC CCA GTT TTC
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MDPI and ACS Style

Sung, C.-H.; Huang, C.-F.; Hsu, Y.-J.; Pu, C.-M.; Kung, C.-C.; Chu, T.W.; Hung, C.-F. Birch Sap Attenuates Inflammatory Cytokines and Improves Skin Parameters in Cellular and Animal Models of Skin Irritation. Cosmetics 2025, 12, 282. https://doi.org/10.3390/cosmetics12060282

AMA Style

Sung C-H, Huang C-F, Hsu Y-J, Pu C-M, Kung C-C, Chu TW, Hung C-F. Birch Sap Attenuates Inflammatory Cytokines and Improves Skin Parameters in Cellular and Animal Models of Skin Irritation. Cosmetics. 2025; 12(6):282. https://doi.org/10.3390/cosmetics12060282

Chicago/Turabian Style

Sung, Chao-Hsien, Chien-Fen Huang, Yu-Jou Hsu, Chi-Ming Pu, Chia-Chi Kung, Thomas W. Chu, and Chi-Feng Hung. 2025. "Birch Sap Attenuates Inflammatory Cytokines and Improves Skin Parameters in Cellular and Animal Models of Skin Irritation" Cosmetics 12, no. 6: 282. https://doi.org/10.3390/cosmetics12060282

APA Style

Sung, C.-H., Huang, C.-F., Hsu, Y.-J., Pu, C.-M., Kung, C.-C., Chu, T. W., & Hung, C.-F. (2025). Birch Sap Attenuates Inflammatory Cytokines and Improves Skin Parameters in Cellular and Animal Models of Skin Irritation. Cosmetics, 12(6), 282. https://doi.org/10.3390/cosmetics12060282

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