Anti-Inflammation and Anti-Hypersensitivity Effects of Corchorus olitorius Extract in Cellular and NC/Nga Mouse Models of Atopic Dermatitis

Abstract

Atopic dermatitis (AD) is a chronic inflammatory skin disorder that is characterized by pruritus, erythema, and eczematous lesions, and pruritus is often regarded as the most burdensome symptom that affects quality of life. Current therapies, including corticosteroids and immunosuppressants, are limited by their adverse effects, thus highlighting the need for safer alternatives. Corchorus olitorius extract (CE), a polyphenol- and flavonoid-rich leafy vegetable, has been reported to possess anti-inflammatory properties. However, its potential as an anti-hypersensitivity agent has not been systematically evaluated. In this study, we investigated the anti-inflammation and anti-hypersensitivity properties of CE using RAW 264.7 macrophages, HaCaT keratinocytes, and human mast cell-1, as well as NC/Nga mice with 2,4-dinitrochlorobenzene-induced AD-like lesions. CE exhibited no cytotoxicity and significantly suppressed nitric oxide, tumor necrosis factor-α, interleukin (IL)-6, IL-1β, and thymus and activation-regulated chemokine production. Furthermore, CE inhibited the degranulation of mast cells by reducing histamine and β-hexosaminidase release, thereby demonstrating anti-hypersensitivity activity. The oral administration of CE attenuated the scratching behavior and improved skin severity scores in NC/Nga mice, with effects at the highest dose comparable to that of dexamethasone. Collectively, these findings provide the first comprehensive evidence that CE alleviates AD-like symptoms by regulating inflammatory mediators and hypersensitivity responses, thus supporting its potential as a safe oral therapeutic or functional food candidate for AD.

INTRODUCTION

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by persistent itching, erythema, xerosis, and eczematous lesions (Steinhoff et al., 2022; Jeskey et al., 2024). Among these symptoms, pruritus is particularly burdensome, as it promotes the scratching behavior that exacerbates skin barrier disruption and inflammation, thereby creating a vicious cycle of disease progression (Lavery et al., 2016; Tominaga and Takamori, 2022). Furthermore, patients often rate pruritus as the most distressing symptom of AD, as it is closely associated with sleep disturbances, impaired daily functioning, and reduced quality of life (Rams et al., 2024; Blauvelt et al., 2025). Previous studies have suggested that the pathogenesis and treatment of AD should not be confined solely to the regulation of inflammation but should also critically address the management of pruritus. Current medical approaches, including topical corticosteroids, calcineurin inhibitors, and systemic immunosuppressants, can provide short-term relief (Siwe et al., 2024). However, their use is limited by side effects such as immune suppression and skin thinning, or rebound flares following withdrawal (Ference and Last, 2009; Abraham and Roga, 2014; Coondoo et al., 2014). These limitations have promoted a growing interest in the identification of safer and more sustainable therapeutic strategies.

Among alternative approaches, natural products have garnered substantial attention because of their broad bioactivities and relatively favorable safety profiles (Yang et al., 2015; Deng et al., 2022). In addition to pharmacological agents, various food-derived materials and phytochemicals, such as polyphenols, probiotics, and herbal extracts, have been investigated for their potential to alleviate AD (Wu et al., 2021; Weber et al., 2023). For instance, resveratrol, curcumin, and catechins have been shown to reduce inflammatory cytokine expression and improve the skin barrier function, while certain probiotic strains have attenuated the T helper cell type 2 (Th2)-driven immune responses and pruritus in AD models (Beagles and Lerner, 2024; Mo et al., 2024; Cui and Wang, 2025).

Corchorus olitorius L., commonly referred to as Molokhia, is a leafy vegetable that is widely consumed in tropical and subtropical regions and has long been used in traditional medicine for treating wounds, fevers, and inflammatory disorders (Handoussa et al., 2013; Biswas et al., 2022). Moreover, pharmacological research has revealed that C. olitorius extract (CE) is abundant in phytochemicals such as chlorogenic acid, which possess antioxidant, antibacterial, and anti-inflammatory properties (İlhan et al., 2007; Öztürk and Savaroğlu, 2010; Yan et al., 2013; Lee et al., 2023b). Evidence from in vitro studies has demonstrated that ethanolic extracts of this plant can inhibit nitric oxide (NO) and pro-inflammatory cytokine production while promoting the migration of fibroblasts and keratinocytes, which suggests potential wound-healing and skin-protective effects (Lee et al., 2023b). Furthermore, in vivo experiments using BALB/c mice have indicated that the oral administration of CE attenuated AD-like symptoms and decreased serum immunoglobulin (Ig) E and Th2 cytokine levels (Lee et al., 2024). However, these previous studies were limited to only evaluating the anti-inflammatory outcomes in vitro and employed BALB/c mice, which do not fully recapitulate the characteristics of human AD. To date, no study has comprehensively examined the anti-hypersensitivity potential of C. olitorius, particularly in the NC/Nga mouse model, which mirrors human AD pathology more closely than a BALB/c mouse model (Suto et al., 1999; Shiohara et al., 2004; Maskey et al., 2024).

In this study, we comprehensively evaluated the efficacy of CE across cellular and animal systems. Specifically, we investigated its ability to suppress NO, pro-inflammatory cytokines, and chemokine production in macrophages and keratinocytes, while also assessing mast cell stabilization via the inhibition of histamine and β-hexosaminidase release. In parallel, we employed an NC/Nga mouse model, which is a clinically relevant human AD model, to determine whether the oral administration of CE alleviated the hypersensitivity-driven scratching behavior and improved the skin condition. By combining molecular, cellular, and behavioral endpoints, this study provides a more comprehensive understanding of CE as a natural oral therapeutic option for AD treatment.

MATERIALS AND METHODS

Preparation of CE powder

Fresh leaves of C. olitorius were washed with distilled water and dried in a hot-air dryer at 60°C. The dried leaves were extracted twice with 70% ethanol at a ratio of 1:20 (w/v; Jinro Distillers) at 70°C for 6 h, and the combined extracts were filtered through a housing filter (1 µm pore size). The filtrate was concentrated under reduced pressure to 15°Bx (65°C, −600 mmHg), mixed with dextrin at a ratio of 1:1 (w/w; 50% solid content, Daesang), and sterilized at 90°C for 30 min. The mixture was then spray-dried (170°C inlet temperature and 80°C outlet temperature) to obtain the final CE powder (Azhar et al., 2020).

Measurement of cell viability

The cell viability was measured using the water-soluble tetrazolium (WST)-1 assay (Kamiloglu et al., 2020). The cells used in this study were murine-derived macrophages RAW 264.7 (KCLB), human keratinocytes HaCaT (KCLB), and human mast cell (HMC)-1 (Merck). The cells were seeded into 96-well plates and stabilized in an incubator at 37°C under 5% CO₂ conditions. After 24 h, the CE was diluted in Dulbecco’s Modified Eagle Medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Hyclone), which was then applied to the cells. Following a further 24 h of incubation, 20 µL of WST salt (DoGenBio) solution was added to each well and incubated for 1 h at 37°C in the CO₂ incubator. The absorbance was then measured at 450 nm using a microplate reader (VersaMax, Molecular Devices). Cell viability of the CE-treated groups was expressed as a percentage relative to that of the CE-untreated control group (set as 100%). For each condition, three wells were used per group, and the mean±standard deviation (SD) was calculated for analysis.

Measurement of inflammatory and allergic mediators in various cell lines

The NO production was measured using the Griess reaction assay (Sun et al., 2003). The RAW 264.7 cells were seeded in 24-well plates and stabilized as described above, followed by treatment with various concentrations of CE (0－200 µg/mL) and 1 µg/mL of lipopolysaccharide (LPS, Sigma-Aldrich) for 24 h. Then 100 µL of culture supernatant was transferred to a 96-well plate and combined with an equal volume of Griess reagent (Sigma-Aldrich). After incubation for 5 min in the dark, the absorbance was measured at 450 nm. Other pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β levels, were quantified in the culture supernatants using commercial ELISA kits (R&D Systems) according to the manufacturer’s instructions.

For thymus and activation-regulated chemokine (TARC) measurement, HaCaT cells were cultured in 6-well plates and washed, and then the supernatant was removed. Next, the cells were irradiated with ultraviolet B (UVB, 10 mJ/cm²). Subsequently, the CE was applied, and the cells were incubated for 24 h. The TARC levels were quantified using a commercial ELISA kit (R&D Systems) following the manufacturer’s instructions.

The release of β-hexosaminidase from HMC-1 mast cells was quantified using a colorimetric assay as previously described by Kim et al. (2017a). The HMC-1 cells were seeded in 6-well plates and incubated, followed by centrifugation at 377 g for 5 min (Labogene, GYLZ-1248R) to remove the supernatant, and the cells were washed twice with Siraganian buffer (Biosolution). CE diluted in Siraganian buffer was applied at different concentrations and incubated for 30 min, after which compound 48/80 (Sigma-Aldrich) was added, and the cells were incubated for a further 30 min. The supernatant was collected, and 100 µL aliquots were transferred to a 96-well plate. Subsequently, 50 µL of 5 mM PN-(GlcNAc)₂ (Sigma-Aldrich) was added, and the mixture was incubated at 37°C for 1 h. The reaction was stopped by adding 50 µL of 0.1 M NaHCO₃ (Sigma-Aldrich), and absorbance was then measured at 405 nm. The histamine levels were quantified in the culture supernatants using commercial ELISA kits according to the manufacturer’s instructions.

For each condition, two wells were used per group, and the mean±SD was calculated for analysis from three independent experiments.

Induction of the AD NC/Nga mouse model

Three-week-old male NC/Nga mice (Central Lab Animal Inc.) were purchased and acclimated for 7 days in clean cages under controlled environmental conditions (temperature, 23±2°C; humidity, 55±10%; with a 12/12 h light/dark cycle). Food and water were provided ad libitum, and the mice were randomly assigned to groups (n=5 per group). The AD mouse model was established according to a previous study (Kim et al., 2014a). After acclimatization, the dorsal skin of each mouse was shaved from the lower part of the ears to the upper part of the tail. To induce skin sensitization, 200 µL of 1% 2,4-dinitrochlorobenzene (DNCB, Sigma-Aldrich) solution (acetone:olive oil=3:1) was applied topically to the shaved area. Four days later, 150 µL of 0.4% DNCB solution was reapplied three times per week for 5 weeks to induce AD-like lesions. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Sahmyook University (IACUC approval No. SYUIACUC 2023-018) and were performed in accordance with the guidelines for the care and use of laboratory animals.

Administration of CE in the AD animal model

Mice in the positive control group received dexamethasone (DEX, 1 mg/kg) once daily for 7 consecutive days, whereas those in the experimental groups were orally administered CE at doses of 50, 100, or 200 mg/kg following the same regimen.

Measurement of scratching counts and skin severity scoring in the AD animal model

Scratching behavior was evaluated on days 1, 3, and 7 after CE and DEX administration. Scratching counts were recorded for 1 h, beginning 30 min after administration. Skin condition severity was assessed on days 1, 3, and 7 after administration. The evaluation was performed using a standard clinical AD scoring system (Pang et al., 2023). Five parameters (erythema, pruritus and dry skin, edema and excoriation, erosion, and lichenification) were individually scored from 0 to 3 (0, none; 1, mild; 2, moderate; and 3, severe). The total score ranged from 0 to 15. Five NC/Nga mice per group were evaluated, and to minimize the influence of potential outliers, the highest and lowest scratching behavior counts and skin severity scores within each group were excluded from the statistical analysis.

Statistical analysis

All data were presented as the mean±SD. Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s post hoc test to compare the treatment groups with the control. Differences were considered statistically significant at P<0.05. Analyses were performed using GraphPad Prism software (version 10.6.0, GraphPad Software).

RESULTS

Effect of CE on cell viability in RAW 264.7, HaCaT, and HMC-1 cells

In RAW 264.7 macrophages, CE treatment did not induce cytotoxicity at any of the tested concentrations (Fig. 1A). Instead, a slight but significant increase in cell viability was observed, which reached approximately 112.6%－123.9% of that of the untreated control at concentrations of 50－200 µg/mL. In the HaCaT keratinocytes, cell viability was consistently maintained at 98.6%－101.8% across all treatment groups, exhibiting no statistical difference compared with that of the untreated control (Fig. 1B). Similarly, in HMC-1 mast cells, cell viability remained stable within the range of 97.5%－102.9%, with no cytotoxic effects detected up to 200 µg/mL (Fig. 1C). These results demonstrate that CE did not induce cytotoxicity in the tested cell lines.

Fig. 1.

Effects of Corchorus olitorius extract (CE) on cell viability in various cell lines. (A) RAW 264.7 macrophages, (B) HaCaT keratinocytes, and (C) human mast cell-1 cells were treated with 0－200 µg/mL of CE for 24 h, and cell viability was measured using the water-soluble tetrazolium-1 assay. Data are presented as the mean±standard deviation (n=3 wells/group). Statistical analysis was performed using a one-way analysis of variance followed by Dunnett’s post hoc test. *P<0.05, **P<0.01, and ***P<0.001.

Inhibitory effect of CE on NO and pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 macrophages

LPS stimulation markedly increased NO production compared with the untreated control (Fig. 2A). CE treatment significantly reduced NO levels in a concentration-dependent manner, reaching 98.5% at 50 µg/mL, 89.9% at 100 µg/mL, and 66.4% at 200 µg/mL relative to the LPS-only treated group. A similar pattern was observed for TNF-α levels (Fig. 2B), where CE significantly and concentration-dependently suppressed the production of TNF-α to 65.2% at 50 µg/mL, 59.3% at 100 µg/mL, and 48.9% at 200 µg/mL. Furthermore, IL-6 production was reduced in a concentration-dependent manner, with levels decreased to 53.0%, 39.9%, and 17.4% at 50, 100 and 200 µg/mL of CE, respectively (Fig. 2C). Similarly, IL-1β was significantly attenuated by CE treatment, declining to 33.5%, 27.2%, and 16.0% at 50, 100, and 200 µg/mL of CE, respectively (Fig. 2D). These results reveal that CE effectively suppressed NO and pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 macrophages in a concentration-dependent manner.

Fig. 2.

Effects of Corchorus olitorius extract (CE) on nitric oxide (NO) and pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. RAW 264.7 cells were pretreated with 0－200 µg/mL of CE and stimulated with 1 µg/mL of LPS for 24 h. The production of (A) NO, (B) tumor necrosis factor (TNF)-α, (C) interleukin (IL)-6, and (D) IL-1β was measured and expressed as a percentage relative to the LPS-only stimulated control (as 100%). Data are presented as the mean±standard deviation (n=2 wells/group) from three independent experiments. Statistical significance was determined using a one-way analysis of variance with Dunnett’s post hoc test. **P<0.01 and ****P<0.0001.

Inhibitory effect of CE on TARC production in UVB-stimulated HaCaT keratinocyte

UVB irradiation (10 mJ/cm²) markedly increased the TARC secretion compared with that of the untreated control group (Fig. 3). In contrast, CE treatment significantly reduced the TARC levels in a concentration-dependent manner, decreasing to 63.3% at 50 µg/mL, 53.9% at 100 µg/mL, and 42.0% at 200 µg/mL relative to the UVB-only treated group. These results demonstrate that CE effectively inhibits UVB-induced TARC production in HaCaT keratinocytes in a concentration-dependent manner.

Fig. 3.

Inhibitory effect of Corchorus olitorius extract (CE) on thymus and activation-regulated chemokine (TARC) production in ultraviolet B (UVB) radiation-stimulated HaCaT keratinocytes. HaCaT cells were pretreated with 50, 100, and 200 µg/mL of CE and irradiated with UVB at 10 mJ/cm². After 24 h, the TARC levels in the culture supernatants were quantified and expressed as a percentage relative to the UVB-only stimulated control. Data are presented as the mean±standard deviation (n=2 wells/group) from three independent experiments. Statistical significance was determined using a one-way analysis of variance with Dunnett’s post hoc test. ****P<0.0001

Inhibitory effect of CE on histamine and β-hexosaminidase production in compound 48/80-stimulated HMC-1 mast cells

Stimulation with compound 48/80 markedly increased histamine release compared with that of the untreated control (Fig. 4A). The CE treatment significantly reduced the histamine levels in a concentration-dependent manner, decreasing to 76.5% at 50 µg/mL, 62.9% at 100 µg/mL, and 51.3% at 200 µg/mL relative to that of the compound 48/80-only treated group. Similarly, β-hexosaminidase release was significantly elevated after compound 48/80 stimulation (Fig. 4B). However, the levels of β-hexosaminidase were concentration-dependently suppressed by CE treatment, showing reductions to 81.8%, 75.6%, and 68.9% at 50, 100, and 200 µg/mL of CE, respectively. These results indicate that CE effectively inhibited mast cell degranulation by suppressing the release of histamine and β-hexosaminidase in a concentration-dependent manner.

Fig. 4.

Inhibitory effect of Corchorus olitorius extract (CE) on histamine and β-hexosaminidase release in compound 48/80-stimulated human mast cell (HMC)-1 mast cells. HMC-1 cells were pretreated with 50, 100, and 200 µg/mL of CE and stimulated with 10 µg/mL of compound 48/80 for 30 min. (A) Histamine and (B) β-hexosaminidase release was measured, and the results were expressed as a percentage relative to the compound 48/80-only stimulated control. Data are presented as the mean±standard deviation (n=2 wells/group) from three independent experiments. Statistical significance was determined using a one-way analysis of variance with Dunnett’s post hoc test. ***P<0.001 and ****P<0.0001.

Effect of CE on the scratching behavior in AD-induced mice

The AD non-induced control group (G1) consistently exhibited very low scratching counts throughout the experimental period (Fig. 5). In contrast, the untreated AD-induced group (G2) maintained the highest scratching frequency, with counts exceeding 200/h on day 1 and remaining above 170 counts/h on day 7. The oral administration of CE markedly reduced the scratching behavior in a dose-dependent manner. Mice treated with 50 mg/kg (G3) and 100 mg/kg (G4) exhibited a gradual decrease to 132.7 counts/h and 123.3 counts/h respectively, whereas the 200 mg/kg CE group (G5) revealed the most pronounced reduction to 110.7 counts/h by day 7. The positive control group that was treated with 1 mg/kg of DEX (G6) demonstrated a comparable decrease, reaching 93.0 counts/h by day 7. These results indicated that CE administration effectively attenuated the scratching behavior in AD-induced mice in a concentration-dependent manner.

Fig. 5.

Effect of Corchorus olitorius extract (CE) on scratching behavior in atopic dermatitis (AD)-induced mice. Scratching counts were measured for 1 h on days 1, 3, and 7 after treatment. G1, non-AD induced normal control; G2, AD-only induced control; G3, AD+CE (50 mg/kg); G4, AD+CE (100 mg/kg); G5, AD+CE (200 mg/kg); and G6, AD+dexamethasone (1 mg/kg). Data are presented as the mean±standard deviation (n=3/group). Statistical significance was determined using a one-way analysis of variance with Dunnett’s post hoc test.

Effect of CE on skin severity score in AD-induced mice

The non-AD-induced control group (G1) maintained a severity score of 0 throughout the experimental period (Fig. 6). In contrast, the untreated AD-induced group (G2) consistently exhibited the highest severity score, starting at 14.3 on day 1 and remaining above 8 on day 7. The oral administration of CE reduced skin severity in a dose-dependent manner. Mice treated with 50 mg/kg (G3) and 100 mg/kg (G4) CE groups exhibited gradual decreases to 4.7 and 3.7, respectively, while the 200 mg/kg CE group (G5) demonstrated the most pronounced improvement, with scores reduced to 2.3 by day 7. In addition, the DEX-treated group (G6) exhibited a comparable effect, reaching almost complete resolution with a score close to 1 by day 7. These results indicate that CE administration significantly attenuated skin severity in an AD-induced mouse model in a concentration-dependent manner.

Fig. 6.

Effect of Corchorus olitorius extract (CE) on the skin severity scores in atopic dermatitis (AD)-induced mice. The skin severity was scored on days 1, 3, and 7 using five parameters (erythema, pruritus/dry skin, edema/excoriation, erosion, and lichenification), which were each graded 0－3. G1, non-AD induced normal control; G2, AD-only induced control; G3, AD+CE (50 mg/kg); G4, AD+CE (100 mg/kg); G5, AD+CE (200 mg/kg); and G6, AD+dexamethasone (1 mg/kg). Data are presented as the mean±standard deviation (SD) (n=3/group). Statistical significance was determined using a one-way analysis of variance with Dunnett’s post hoc test.

DISCUSSION

Previous studies have detailed the anti-inflammatory properties of C. olitorius in either in vitro or in vivo settings, thus demonstrating its ability to suppress inflammatory mediators and ameliorate AD-like symptoms (Lee et al., 2023b; Dania et al., 2024; Lee et al., 2024). However, these studies were largely confined to BALB/c mouse models or focused exclusively on the inflammatory endpoints such as cytokine production and serum IgE levels. None of these studies have systematically evaluated the hypersensitivity-related outcomes, despite the fact that pruritus and scratching are hallmark features of AD and are closely associated with impaired sleep and reduced quality of life of patients (Legat, 2021; Armario-Hita et al., 2024). Therefore, the most important novelty of the present study lies in demonstrating the anti-hypersensitivity potential of CE, which complements and extends on its previously demonstrated anti-inflammatory activities.

Our findings revealed that CE inhibited mast cell degranulation, as evidenced by the concentration-dependent reduction of histamine and β-hexosaminidase release in HMC-1 cells (Fig. 4). These two markers are indicators of mast cell and basophil degranulation, which are rapidly released within minutes of IgE-dependent or independent stimulation to mediate acute hypersensitivity reactions (Galli and Tsai, 2012; Lv et al., 2018). Accordingly, their reduction in this study provides functional evidence of the inhibition of mast cell degranulation, blockade of acute mediator release, and subsequent anti-hypersensitivity activity (Kuehn et al., 2010; Schulman et al., 2023). From a mechanistic perspective, phytochemical and pharmacological reviews have reported that C. olitorius contains quercetin, quercetin derivatives, and caffeoylquinic acids, which suggests that the anti-hypersensitivity effect of CE is consistent with the flavonoid-mediated suppression of mast cell signaling (Biswas et al., 2022; Cao and Gao, 2024). Moreover, quercetin and kaempferol have been shown to inhibit the FceεI-SYK-ERK axis in human and murine mast cells, thereby reducing the degranulation and release of mediators such as histamine and β-hexosaminidase (Kim et al., 2014b; Kaag and Lorentz, 2023). The concomitant reduction of histamine and β-hexosaminidase observed in this study aligns with these flavonoid-mediated mast cell-stabilizing mechanisms. These in vitro observations directly reflected our in vivo assessment, where the oral administration of CE led to a marked decrease in scratching frequency in NC/Nga mice (Fig. 5). As scratching behavior is a sensitive and clinically relevant endpoint of pruritus (Akiyama et al., 2010; Yassky and Kim, 2024), this provides compelling evidence that CE exerts systemic anti-hypersensitivity effects via the stabilization of mast cells. This is the first report to link the suppression of mast cell-facilitated mediator release with behavioral improvements in an AD mouse model treated with CE.

In parallel, CE treatment consistently suppressed NO, TNF-α, IL-6, IL-1β, and TARC production in macrophages and keratinocytes, thereby confirming CE’s broad anti-inflammatory activity (Fig. 2 and 3). These effects are attributable, at least in part, to bioactive phytochemicals such as chlorogenic acid, quercetin, and caffeoylquinic acids, which have been demonstrated to modulate the nuclear factor-κB, janus kinase/signal transducer and activator of transcription, and mitogen-activated protein kinase signaling pathways while enhancing antioxidant defenses via nuclear factor erythroid 2-related factor 2 (Nrf2) activation (Yin et al., 2021; Ge et al., 2023; Nguyen et al., 2024). Notably, chlorogenic acid has been reported as a major constituent of CE, and numerous studies have demonstrated its contribution to the suppression of NO/inducible nitric oxide synthase activity as well as the downregulation of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β (Kim et al., 2017b; Huang et al., 2023). However, no studies have directly compared the therapeutic efficacy of chlorogenic acid with that of the whole CE extract. The present findings, which reveal the broader effects on cytokine suppression, chemokine regulation, and hypersensitivity outcomes, suggest that CE may exert enhanced bioactivity through synergistic interactions among its various phytochemicals rather than through chlorogenic acid alone. Thus, further comparative studies are required to delineate the relative contributions of chlorogenic acid versus those of the complex phytochemical composition of CE. In addition, quercetin and its derivatives are known to exert anti-histaminic and anti-hypersensitivity effects by inhibiting the degranulation of mast cells and blocking the FceεI-mediated signaling pathways (Alam et al., 2022). In keratinocytes, TARC is a key chemokine that mediates Th2 cell recruitment and contributes to the pathogenesis of AD, as it is strongly induced by TNF-α/interferon-γ stimulation but is downregulated by several natural products and probiotics (Ju et al., 2009; Lee et al., 2023a). Therefore, this observed reduction in TARC following CE treatment suggests that CE may attenuate keratinocyte-driven Th2 chemotactic signaling, thereby suppressing the amplification of cutaneous inflammatory responses. Importantly, this dual regulation, i.e., anti-inflammatory on one axis and anti-hypersensitivity on the other, suggests that CE addresses immune-driven inflammation as well as hypersensitivity-driven pruritus, the two major pathological pillars of AD.

The use of an NC/Nga mouse model further enhances the translational value of our findings. Unlike BALB/c mice, NC/Nga mice spontaneously develop chronic dermatitis with severe pruritus, and thus more closely recapitulate human AD pathology (Suto et al., 1999; Ye et al., 2025). Our demonstration that the oral administration of CE in this model improved the skin severity scores (Fig. 6) and scratching behavior (Fig. 5) emphasizes its potential applicability as an oral therapeutic or functional food ingredient for the treatment of AD. The ability to reduce hypersensitivity is particularly meaningful given that patients often indicate that pruritus is the most burdensome symptom, surpassing even visible lesions because of its effect on their quality of life.

The present findings that CE treatment did not reduce cell viability in the various cell lines (Fig. 1) indicate that its anti-inflammatory effects, including the suppression of NO, TNF-α, IL-6/IL-1β, and TARC production, were unlikely to result from nonspecific cytotoxicity but rather from genuine pharmacological activity. This interpretation was supported by a previous study, which showed that ethanolic extracts of C. olitorius leaves exerted no cytotoxic effects on RAW 264.7 cells, normal human dermal fibroblasts, and HaCaT cells at concentrations of up to 200 µg/mL and, in some cases, even enhanced cell viability, while concomitantly improving the anti-inflammatory and wound-healing marker levels (Lee et al., 2023b). In line with these results, C. olitorius is known to contain abundant bioactive compounds, including polyphenols and flavonoids, which enhance the antioxidant defenses via free radical scavenging and Nrf2 activation, thereby modulating inflammation without affecting cell survival (Abdel-Razek et al., 2022). Notably, other studies have demonstrated that CE can induce anti-proliferative or cytotoxic effects against tumor cell lines, such as human melanoma, gastric cancer, and pancreatic cancer (Tosoc et al., 2021; Alshabi et al., 2022; Sameh et al., 2025). These observations collectively suggest that CE exerts a dual profile, providing protective, non-cytotoxic effects in normal or nontumor cells, while selectively inducing cytotoxicity in malignant cells.

Taken together, this study provides the first comprehensive demonstration that CE exerts a dual regulatory effect against AD by simultaneously suppressing inflammatory mediator levels and alleviating the hypersensitivity responses. By integrating molecular, cellular, and behavioral evidence, we establish CE as a promising candidate for the development of an orally available therapeutic or functional food for the treatment of AD.

In conclusion, this study provides the first comprehensive evidence that CE induces dual mechanisms against AD by suppressing the levels of inflammatory mediators and alleviating the hypersensitivity responses. CE reduced cytokine and chemokine production as well as stabilized mast cells, which resulted in marked improvements in scratching behavior and skin severity scores in NC/Nga mice. These findings highlight CE as a promising oral therapeutic or functional food candidate for AD. Further studies are warranted to explore its molecular mechanisms, to compare its efficacy with key phytochemicals, such as chlorogenic acid, and to evaluate the long-term safety and pharmacokinetic profiles to facilitate clinical translation.