Ultrasound‐assisted enzymatic extraction of citrus pomace yields dietary fiber‐rich extract with skin‐protective effects

Abstract

BACKGROUND

Citrus pomace is an abundant byproduct of citrus juice processing and represents a sustainable source of dietary fiber and bioactive polysaccharides. The present study aimed to valorize citrus pomace through ultrasound‐assisted enzymatic extraction (UAEE) and to evaluate the protective effects of the resulting extract obtained by Celluclast® treatment with ultrasound pretreatment (CCU) against particulate matter (PM)‐induced skin damage.

RESULTS

UAEE markedly improved the extraction yield of citrus pomace extract, with CCU showing a higher yield (55.17%) than distilled water extraction (15.67%) and Celluclast® extraction without ultrasound pretreatment (54.00%). CCU also showed the highest dietary fiber recovery based on extraction yield. In PM‐stimulated HaCaT keratinocytes, CCU increased cell viability from 75.26% to 94.12% and reduced intracellular reactive oxygen species production in a concentration‐dependent manner. CCU further suppressed PM‐induced pro‐inflammatory responses by reducing tumor necrosis factor‐α, interleukin‐1β and interleukin‐6 levels, as well as inducible nitric oxide synthase and cyclooxygenase‐2 protein expression. In addition, CCU restored the expression of skin barrier‐and hydration‐related proteins, including filaggrin, loricrin, involucrin and hyaluronic acid synthase 1. In vivo, oral administration of CCU also alleviated PM‐induced inflammation and water loss in mouse skin.

CONCLUSION

These findings demonstrate that UAEE is an effective approach for converting citrus pomace into a dietary fiber‐rich functional extract with anti‐inflammatory and skin barrier‐protective properties. CCU may therefore serve as a value‐added ingredient for nutraceutical or skin‐health‐related functional applications. © 2026 Society of Chemical Industry.

INTRODUCTION

The skin serves as the primary barrier against external environmental stressors, including ultraviolet radiation, pathogens and airborne particulate matter (PM). Impairment of epidermal homeostasis by environmental pollutants has been increasingly associated with inflammatory skin disorders and skin barrier dysfunction. ¹ , ² Recent dermatological investigations have emphasized the detrimental impacts of environmental pollutants, particularly airborne PM, on the progression of inflammatory skin diseases such as psoriasis, acne, and atopic dermatitis. A common pathological mechanism among these diseases involves the overproduction of reactive oxygen species (ROS) production, which triggers the release of pro‐inflammatory cytokines from human epidermal keratinocytes, subsequently leading to skin barrier impairment. ³ , ⁴ Given the increasing prevalence of air pollution‐related skin disorders, there is a growing demand for effective strategies to prevent or restore PM‐induced cutaneous damage. In this context, natural resources have gained attention as promising therapeutic candidates because of their low toxicity, biocompatibility and broad‐spectrum bioactivity. ⁵ , ⁶ Exploring bioactive compounds derived from natural sources may therefore offer valuable insights for the development of safe and effective skin‐protective agents.

Over the past decades, the increasing demand for eco‐friendly and health‐promoting products, alongside global population growth, has led to a significant rise in fruit processing byproducts. These agro‐industrial wastes‐such as seeds, shells, leaves, cores and pomace constitute approximately 20–25% of total fruit and vegetable production, posing environmental and economic challenges for sustainable waste management. ⁷ , ⁸ Among these, citrus pomace, a major byproduct of juice processing, has gained attention as a promising natural resource for valorization because of its abundance of phytochemicals and bioactive compounds. ⁷ , ⁹ It is also enriched with essential nutrients, including vitamins, minerals and dietary fibers, making it highly suitable for nutraceuticals, cosmeceuticals and pharmaceutical formulations. ¹⁰ Therefore, establishing sustainable strategies to valorize citrus processing waste not only contributes to circular economy goals, but also supports the development of novel functional ingredients with added value. In recent years, citrus‐derived polysaccharides and dietary fibers have also been investigated for their potential protective effects against oxidative stress and inflammation‐related disorders, suggesting their applicability in skin health‐related functional systems. ¹¹ , ¹²

Maximizing the recovery of bioactive components is a key objective in the valorization of fruit and vegetable byproducts into high‐value functional ingredients. To achieve this, it is critical to adopt and optimize efficient, sustainable extraction methods, such as supercritical fluid, microwave‐assisted, enzyme‐assisted, ultrasound‐assisted and the use of green solvents. ¹³ , ¹⁴ Among these, enzyme‐assisted and ultrasound‐assisted, either individually or in combination, have demonstrated superior extraction efficacy, reduced solvent usage, and cost‐effectiveness, making those techniques widely studied for the recovery of bioactives from citrus processing residues. ¹⁵ , ¹⁶ , ¹⁷ In particular, ultrasound‐assisted enzymatic extraction (UAEE) has recently emerged as a promising approach for isolating polysaccharides and dietary fibers from agro‐industrial waste streams. This technique combines the cell wall‐degrading effects of ultrasound with the substrate‐specific hydrolytic action of enzymes, thereby improving yield, extraction kinetics and functionality of the recovered compounds. ¹⁸ , ¹⁹ , ²⁰ Citrus peel and pomace, being rich sources of polysaccharides such as pectin and other dietary fibers, are well‐suited for UAEE‐based recovery. ¹⁶ , ²¹ For this reason, applying UAEE to citrus byproducts can be a sustainable and efficient strategy for enhancing both the functional value and industrial utilization of these agro‐wastes. However, despite the growing interest in UAEE for recovering functional compounds from citrus byproducts, limited studies have explored the skin‐protective efficacy of dietary fiber‐rich citrus pomace extracts under PM‐induced skin stress conditions.

Dietary fiber, particularly pectin and other soluble fibers abundant in citrus pomace, consists of complex carbohydrates such as non‐starch polysaccharides and associated plant components. It is widely recognized for its diverse health‐promoting properties, including antioxidant, anti‐inflammatory, anti‐cancer, anti‐obesity and immunomodulatory effects, as well as the ability to modulate gut microbiota. ²² , ²³ For example, soluble dietary fiber extracted from citrus peel via ultrasound‐assisted alkaline extraction demonstrated strong antioxidant activity and positive regulatory effects on intestinal flora. ²⁴ These bioactivities position dietary fibers as promising candidates for use in nutraceutical and cosmeceutical applications. Recent studies have further revealed that dietary fibers from olive pomace and guar gum can reduce skin inflammation in murine models and alleviate skin dehydration in humans, respectively. ²⁵ , ²⁶ With increasing interest in the multifunctional roles of dietary fiber, modern extraction techniques such as UAEE have gained attention for their ability to efficiently recover high‐quality, bioactive fibers. ²⁷ , ²⁸ , ²⁹ , ³⁰ Thus, applying UAEE to extract functional dietary fibers from citrus pomace may provide a dual benefit‐waste valorization and the development of skin‐protective functional materials. Recent studies have suggested that plant‐derived polysaccharides can regulate oxidative stress and inflammatory signaling pathways involved in epidermal differentiation and skin barrier maintenance. ³¹ , ³² However, despite the growing interest in UAEE for recovering functional compounds from citrus byproducts, limited studies have investigated the protective effects of dietary fiber‐rich citrus pomace extracts against PM‐induced skin inflammation and barrier dysfunction.

Based on this background, the present study was designed to explore the potential of citrus pomace as a sustainable source of bioactive dietary fiber through UAEE. Specifically, this study aimed to evaluate the protective effects of the fiber‐rich extract against PM‐induced oxidative stress, inflammation, and skin barrier disruption in vitro and in vivo. To the best of our knowledge, this is the first study to evaluate the skin‐protective efficacy of a UAEE‐derived dietary fiber‐rich citrus pomace extract against PM‐induced oxidative stress, inflammation and barrier dysfunction using both in vitro and in vivo models.

MATERIALS AND METHODS

Materials and reagents

The particulate matter (PM; CRM No. 28) used in this study was purchased from the National Institute for Environmental Studies (NIES, Ibaraki, Japan) and was originally collected in Beijing, China. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and 2′,7′‐dichlorohydrofluorescein diacetate (DCFH‐DA) were obtained from Sigma‐Aldrich Co. (St Louis, MO, USA). Dulbecco's modified Eagle's medium, phosphate‐buffered saline (PBS, 1×), penicillin/streptomycin and fetal bovine serum were purchased from Gibco BRL (Grand Island, NY, USA). Primary and secondary antibodies used for western blot analysis were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and Cell Signaling Technology (Danvers, MA, USA), respectively.

Preparation of ultrasound‐ and enzyme‐assisted citrus pomace extracts

Citrus pomace was kindly provided by ILHAE Co., (Jeju, Republic of Korea). Frozen samples (18 g) were mixed with 900 mL of distilled water (DW) and pretreated using an ultrasonic processor (VCX750; Sonics & materials Inc, Newtown, CT, USA) under the conditions: 20 kHz frequency, 80% amplitude, 10 s on/10 s off pulses, 4 °C for 2 h. The 120 min of ultrasound pretreatment condition was selected based on previous studies reporting enhanced extraction efficiency and structural disruption of citrus byproducts under prolonged sonication condition. ²¹ , ³³ , ³⁴ Both ultrasound‐pretreated and non‐pretreated samples were subsequently incubated with 1% carbohydrate‐hydrolyzing enzyme (Celluclast® 1.5L FG; Novozymes, Bagsvaerd, Denmark; cellulase activity: approximately 700 EGU g⁻¹) at 50 °C for 24 h in a shaking incubator. After enzymatic hydrolysis, the reaction was terminated by heating at 100 °C for 10 min, followed by cooling to room temperature (RT). The unhydrolysed residues were removed by centrifugation at 3000 × g for 10 min at 4 °C. The collected supernatants were adjusted to pH 7.0 and lyophilized for subsequent analysis. The resulting extracts were designated as distilled water extract (CD), Celluclast‐treated extract without ultrasound pretreatment (CC) and ultrasound‐pretreated Celluclast extract (CCU), respectively.

Determination of dietary fiber contents

Total dietary fiber was quantified using Megazyme Total Dietary Fiber Assay Kit (K‐TDFR; Megazyme, Bray, Ireland) in accordance with the manufacturer's instruction. Briefly, 1 g of freeze‐dried sample was subjected to enzymatic digestion with heat‐stable α‐amylase (100 °C, pH 6.0), protease (60 °C, pH 7.5) and amyloglucosidase (60 °C, pH 4.5) to simulate gastrointestinal digestion. After enzymatic treatment, the digest was filtered under vacuum, and the residue was washed with ethanol (95%) and acetone to remove low‐molecular‐weight carbohydrates and lipids. The residue was dried and weighed to determine insoluble dietary fiber (IDF), while ethanol‐precipitated filtrate was used for soluble dietary fiber (SDF) analysis. The sum of IDF and SDF was calculated as total dietary fiber and expressed as g per 100 g.

Protective effects of extracts against PM‐induced cytotoxicity

The protective effects of the extracts against PM‐induced cytotoxicity were evaluated in HaCaT keratinocytes using the MTT assay, following a previously reported method with minor adjustments. ³⁵ HaCaT cells were seeded into 24‐well plates at a density of 1.5 × 10⁵ cells mL⁻¹ and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. After preincubation, the cells were treated with various concentrations of the extracts. One hour later, PM (CRM No. 28) was added, and the cells were further incubated for 24 h. Subsequently, MTT solution (2 mg mL⁻¹) was added to each well and incubated for 3 h. The supernatant was then removed, and 300 μL of dimethyl sulfoxide was added to dissolve the formazan crystals at RT. The absorbance was measured at 540 nm using a microplate reader (Synergy™ HTX Multi‐Mode Reader; BioTek Instruments, Winooski, VT, USA). Cell viability was expressed as a percentage relative to the untreated control group.

Evaluation of intracellular ROS scavenging activity in HaCaT cells

The inhibitory effects of the extracts on PM‐induced intracellular ROS generation were evaluated in HaCaT keratinocytes using a slightly modified DCFH‐DA assay adapted from a previously reported method. ³⁶ HaCaT cells were seeded into 96‐well black plates at a density of 1.5 × 10⁵ cells mL⁻¹ and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. After preincubation, the cells were treated with various concentrations of the extracts, followed by exposure to PM (CRM No. 28) 1 h later, and further incubated for 24 h. Subsequently, 10 μL of 1 mm DCFH‐DA solution was added to each well and incubated for 30 min. The cells were then washed twice with PBS and the fluorescence intensity of DCF was measured using a spectrofluorometer at excitation and emission wavelengths of 485 nm and 528 nm, respectively.

Determination of pro‐inflammatory cytokine inhibition in HaCaT cells

The inhibitory effects of the extracts on pro‐inflammatory cytokine production in PM‐stimulated HaCaT keratinocytes were evaluated. HaCaT cells were seeded into 24‐well plates at a density of 1.5 × 10⁵ cells mL⁻¹ and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. After preincubation, the cells were treated with various concentrations of the extracts, followed by exposure to PM (CRM No. 28) 1 h later, and further incubated for 24 h. The culture supernatants were collected by centrifugation at 3000 × g for 10 min at 4 °C, and the levels of tumor necrosis factor‐alpha (TNF‐α), interleukin‐1 beta (IL‐1β) and interleukin‐6 (IL‐6) were quantified using a commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer's instructions.

Experimental animals

Fifteen male C57BL/6 mice (6 weeks old) were purchased and acclimated for 1 week in an animal facility maintained under controlled environmental conditions before the experiment. The mice were randomly divided into three groups (n = 5 per group), including a control group. All animals were housed under standard conditions with a temperature of 22 ± 2 °C, relative humidity of 50 ± 10% and a 12:12 h light/dark photocycle. PM (100 μg mL⁻¹) was topically applied to a 1 cm² area on the dorsal skin of each mouse once daily for seven consecutive days. The control and PM‐treated groups received physiological saline, whereas the treatment group was orally administered CCU at a dose of 400 mg kg⁻¹ body weight day⁻¹. All animal procedures were approved by the Laboratory Animal Administration Committee of Soonchunhyang University (Approval No: SCH25‐0002) and were conducted in accordance with the institutional guidelines for animal experimentation.

Determination of pro‐inflammatory cytokine inhibition in C57BL/6 mice

Dorsal skin tissues collected from the mice were homogenized and lysed in lysis buffer (20 mm Tris–HCl, pH 7.5, 150 mm NaCl, 1 mm Na₂EDTA, 1 mm EGTA, 1% NP‐40, 1% sodium deoxycholate, 2.5 mm sodium pyrophosphate, 1 mm β‐glycerophosphate, 1 mm Na₃VO₄ and 1 μg mL⁻¹ leupeptin). The lysates were centrifuged at 3000 × g for 10 min at 4 °C, and the supernatants were collected for analysis. Protein concentrations in the supernatants were quantified using a standard curve generated with bovine serum albumin (Sigma‐Aldrich). The pro‐inflammatory cytokine levels were then determined using a commercially available ELISA kit (R&D Systems) in accordance with the manufacturer's instructions.

Western blot assay

Western blotting was performed to determine the protein expression levels in fine dust‐treated HaCaT keratinocytes and C57BL/6 mouse dorsal skin. HaCaT cells and mouse skin tissues were homogenized and lysed in buffer (20 mm Tris–HCl, pH 7.5, 150 mm NaCl, 1 mm Na₂EDTA, 1 mm EGTA, 1% NP‐40, 1% sodium deoxycholate, 2.5 mm sodium pyrophosphate, 1 mm β‐glycerophosphate, 1 mm Na₃VO₄ and 1 μg mL⁻¹ leupeptin). The lysates were centrifuged at 13 000 × g for 10 min at 4 °C, and the supernatants were collected. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were separated by electrophoresis on 10% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris‐buffered saline containing 0.1% Tween‐20 (TBS‐T) for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies diluted 1:500. After washing three times with TBS‐T, the membranes were incubated with secondary antibodies (1:3000 dilution) for 1.5 h at RT. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific) and quantified using Fusion Capt Advance FX7 software (Vilber Lourmat, Paris, France).

Statistical analysis

All experiments were performed in triplicate, and the results are presented as the mean ± SD. Statistical analyses were conducted using SPSS, version 20.0 (IBM Corp., Armonk, NY, USA). Differences among groups were analysed using one‐way analysis of variance (ANOVA), followed by Duncan's multiple range test for post‐hoc comparisons. P < 0.05 was considered statistically significanct.

RESULTS AND DISCUSSION

Yield and dietary fiber content of extracts

Among the three extraction methods evaluated‐distilled water extraction (DW), enzyme‐assisted extraction using Celluclast (CC) and ultrasound‐assisted enzymatic extraction (CCU)‐distinct differences were observed in both extraction yield and dietary fiber recovery. The CCU method, which involved 120 min of ultrasonic pretreatment prior to enzymatic hydrolysis, achieved the highest extraction yield (55.17 ± 0.58%) and greatest dietary fiber efficiency relative to yield (3.70%) (Table 1). These values exceeded both the DW (15.67 ± 1.04%; 1.43%) and CC (54.00 ± 0.50%; 3.30%) groups, suggesting a synergistic enhancement effect from the combination process. In particular, the CCU process increased extraction yield approximately 3.5‐fold compared to DW extraction, highlighting the effectiveness of ultrasound pretreatment in enhancing enzymatic recovery of functional components from citrus pomace. Although the total dietary fiber content appeared slightly higher in the DW extract (9.10%), this likely reflects concentration because of its low extraction yield, rather than superior fiber recovery. By contrast, CCU facilitated the release of water‐soluble components at the same time as maintaining substantial dietary fiber content, thereby improving the overall fiber recovery efficiency. These findings suggest that ultrasound pretreatment may enhance substrate accessibility during enzymatic hydrolysis, contributing to more efficient recovery of dietary fiber‐rich extracts. This interpretation is supported by several recent studies involving citrus byproducts. For example, ultrasound‐assisted enzymatic extraction from orange peel pomace significantly improved dietary fiber yield and functional quality, including viscosity and antioxidant activity. ²¹ Similarly, extended sonication enhanced the solubility and antioxidant potential of pomelo peel‐derived fibers. ³³ Furthermore, prolonged ultrasonication for 120 min of sonication facilitated efficient nanocellulose extraction from orange peel by disrupting cell wall structures and promoting component release. ³⁴

Table 1.

Yield and dietary fiber content of CP extracts

Citrus pomace (CP) extracts
Extraction method	Yield (%)	Dietary fiber contents (DFCs) (g kg−1)	DFCs per yield
DW (CD)	15.67 ± 1.04	91.0	14.30
Celluclast (CC)	54.00 ± 0.50	61.2	33.05
Ultrasonic + Celluclast (CCU)	55.17 ± 0.58	67.1	37.02

These findings provide strong mechanistic and empirical justification for the CCU approach, highlighting its capacity to maximize both extraction yield and functional dietary fiber recovery. Thus, applying 120 min of ultrasound pretreatment in citrus pomace valorization not only improves extraction efficiency, but also aligns with sustainable bioprocessing strategies for high‐value functional material development.

Protective effects of extracts against PM‐induced cytotoxicity

PM is known to penetrate the epidermal barrier and induce cytotoxicity in keratinocytes, contributing to oxidative stress and inflammatory skin damage. ⁴ To evaluate the protective effects of CP extracts against PM‐induced cytotoxicity, an MTT assay was conducted using human HaCaT keratinocytes. As shown in Fig. 1, exposure to 200 μg mL⁻¹ of PM significantly decreased the cell viability to 75.26%. The PM concentration (200 μg mL⁻¹) was selected based on previous studies demonstrating significant oxidative stress and inflammatory responses in HaCaT keratinocytes without inducing excessive cytotoxicity. ³⁷ Although the PM concentration used in this study may not directly reflect environmental exposure levels, it has been widely employed in in vitro skin models to reproduce PM‐induced oxidative and inflammatory stress under controlled experimental conditions. Treatment with all extract types‐DW (CD), Celluclast (CC) and ultrasound‐assisted Celluclast (CCU)‐ameliorated PM‐induced cytotoxicity, with cell viability increasing to 81.48%, 83.38%, and 94.12%, respectively. Among these, the CCU extract showed the most pronounced protective effect, with a statistically significant difference compared to the PM group.

Figure 1.

Protective effects of citrus pomace extracts (CD, CC and CCU) against particulate matter (PM)‐induced cell damage in HaCaT keratinocytes. Data are expressed as the mean ± SD (n = 3). ###P < 0.001 indicates significant differences compared to the CON group. ***P < 0.001 indicates significant differences compared to the PM‐induced group.

This enhanced cytoprotective effect of the CCU extract may be attributed to the improved quality and accessibility of bioactive compounds, particularly dietary fibers, resulting from ultrasonic pretreatment. Consistent with the findings from extraction yield and fiber recovery, UAEE appears to not only increase the quantity of extractable components, but also improve their functional efficacy in protecting skin cells against environmental pollutants.

Inhibitory effects of CCU extract on PM‐induced ROS production

Accumulating evidence suggests that oxidative stress is a major contributor to PM‐induced skin damage, primarily through the overproduction of intracellular ROS. ³⁸ To assess the antioxidant potential of the CCU extract, ROS levels were quantified in PM‐stimulated HaCaT keratinocytes using a DCFH‐DA fluorescent probe assay. As shown in Fig. 2, treatment with PM (200 μg mL⁻¹) markedly elevated intracellular ROS levels to 166.83% relative to untreated control cells. Notably, co‐treatment with the CCU extract significantly attenuated ROS accumulation in a dose‐dependent manner. At concentrations of 50, 100 and 200 μg mL⁻¹, ROS levels were reduced to 144.93%, 130.86% and 111.38%, respectively. In particular, treatment with 200 μg mL⁻¹ CCU reduced ROS generation by approximately 33% compared to the PM‐treated group.

Figure 2.

Effects of CCU on scavenging intracellular reactive oxygen species (ROS) in PM‐induced HaCaT keratinocytes. Data are expressed as the mean ± SD (n = 3). ###P < 0.001 indicates significant differences compared to the CON group. ***P < 0.001 indicates significant differences compared to the PM‐induced group.

Excessive ROS production is known to activate redox‐sensitive inflammatory pathways, including nuclear factor‐kappa B and mitogen‐activated protein kinase signaling, ultimately contributing to cytokine release and epidermal barrier disruption. ³⁹ Therefore, the ROS‐scavenging activity of CCU may play a critical role in suppressing downstream inflammatory responses induced by PM exposure. These findings suggest that the antioxidant activity of the CCU extract may contribute to its protective role against PM‐induced oxidative stress, potentially mediated by functional fibers retained or enhanced through the UAEE process.

Anti‐inflammatory effects of CCU extract on PM‐induced cytokine production

PM exposure has been shown to activate inflammatory signaling pathways in epidermal keratinocytes, leading to the overexpression of key pro‐inflammatory cytokines such as TNF‐α, IL‐1β and IL‐6, which are central mediators in the pathogenesis of inflammatory skin conditions. ³⁸ , ⁴⁰ To evaluate the anti‐inflammatory potential of the CCU extract, the levels of TNF‐α, IL‐1β and IL‐6 were quantified in PM‐stimulated HaCaT cells. As illustrated in Fig. 3, PM treatment significantly increased the secretion of all three cytokines. However, CCU extract treatment effectively reduced their expression in a dose‐dependent manner, suggesting that the extract modulates PM‐induced inflammatory responses. This result aligns with the observed reductions in ROS levels and indicates that the CCU extract not only attenuates oxidative stress, but also inhibits downstream inflammatory cascades.

Figure 3.

Effects of CCU on inhibiting pro‐inflammatory cytokine (TNF‐α, IL‐1β and IL‐6) levels in PM‐induced HaCaT keratinocytes. Data are expressed as the mean ± SD (n = 3). ###P < 0.001 indicates significant differences compared to the CON group. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significant differences compared to the PM‐induced group.

TNF‐α, IL‐1β and IL‐6 are key mediators involved in PM‐induced epidermal inflammation and barrier dysfunction. Therefore, the suppression of these cytokines by CCU extract suggests that citrus‐derived dietary fibers and polysaccharides may contribute to the regulation of keratinocyte‐mediated inflammatory responses under environmental stress conditions.

Inhibitory effects of CCU extract on PM‐induced inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) expression

Following the observed downregulation of pro‐inflammatory cytokines, the effects of CCU extract on inflammatory enzyme expression were evaluated to further validate its anti‐inflammatory efficacy. Western blot analysis revealed that PM exposure markedly upregulated the expression of iNOS and COX‐2, which are key downstream mediators in the oxidative and inflammatory response. ⁴¹ However, CCU extract treatment significantly reduced the expression of both proteins in a dose‐dependent manner (Fig. 4), suggesting effective inhibition of the PM‐induced inflammatory cascade at the protein level. Because iNOS and COX‐2 contribute to excessive nitric oxide and prostaglandin production during inflammatory conditions, ⁴² , ⁴³ their suppression may alleviate oxidative damage and epidermal dysfunction associated with PM exposure.

Figure 4.

Effects of CCU on inflammation‐related protein expression levels in PM‐induced HaCaT keratinocytes. The expression levels of iNOS and COX‐2 were analysed by western blotting, and the quantitative results are displayed. ###P < 0.001 indicates significant differences compared to the CON group. ***P < 0.001 indicates significant differences compared to the PM‐induced group.

This is consistent with previous studies reporting the anti‐inflammatory potential of dietary fibers, particularly citrus‐derived polysaccharides. For example, citrus pectin with low degree of esterification significantly suppressed iNOS and COX‐2 expression in lipopolysaccharide‐stimulated macrophages. ⁴⁴ In another study, a polysaccharide isolated from Citrus medica significantly downregulated iNOS and ROS in RAW 264.7 cells, further supporting the anti‐inflammatory properties of citrus polysaccharides. ⁴⁵ These findings suggest that citrus‐derived polysaccharides and dietary fibers may modulate inflammatory signaling pathways associated with PM‐induced skin inflammation.

Taken together, these results highlight the potential of CCU extract, rich in dietary fiber and citrus polysaccharides, to regulate inflammatory mediator expression and protect against PM‐induced skin inflammation.

Restoration of skin barrier and moisturizing proteins by CCU extract

Because PM is known to disrupt epidermal homeostasis by downregulating structural proteins and natural moisturizing factors, we next examined the effects of CCU extract on skin barrier‐associated proteins including filaggrin, involucrin, loricrin and hyaluronic acid synthase 1 (HAS1). PM treatment (200 μg mL⁻¹) significantly reduced the expression of these key proteins, consistent with previous findings showing that PM impairs skin barrier function by inhibiting filaggrin, involucrin, and loricrin via TNF‐α mediated pathways. ⁴ , ⁴⁶ However, treatment with CCU extract restored the expression of these proteins in a dose‐dependent manner (Fig. 5). Filaggrin, involucrin and loricrin are essential epidermal differentiation markers involved in barrier formation and maintenance, where as HAS1 contributes to skin hydration through hyaluronic acid synthesis. ⁴⁷ Therefore, restoration of these proteins by CCU extract suggests its potential to preserve epidermal integrity and alleviate PM‐induced barrier dysfunction and dehydration. At a 200 μg mL⁻¹ concentration of CCU, the expression levels of filaggrin, involucrin and HAS1 were nearly restored to those of the control group.

Figure 5.

Effects of CCU on skin structural and moisturizing protein expression levels in PM‐induced HaCaT keratinocytes. The expression levels of filaggrin (FLG), involucrin (IVL), loricrin (LOR) and hyaluronic acid synthase 1 (HAS1) were analysed by western blotting, and the quantitative results are displayed. #P < 0.05 and ###P < 0.001 indicate significant differences compared to the CON group. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significant differences compared to the PM‐induced group.

Supporting our findings, one study demonstrated that plant‐derived polysaccharide‐rich extracts could restore PM‐induced reduction of skin barrier proteins including filaggrin and loricrin. ⁴⁸ Moreover, poly‐γ‐glutamic acid, a microbial‐derived polysaccharide similar in function to dietary fibers, has been reported to enhance mRNA levels of skin barrier markers and hyaluronic acid synthases, ⁴⁹ reinforcing the potential of polysaccharide‐based fiber materials from citrus by‐products in skin barrier recovery.

Together, these findings indicate that CCU extract, potentially enriched with citrus‐derived polysaccharides and dietary fibers, may mitigate PM‐induced impairment of skin barrier integrity by modulating structural and moisturizing proteins critical for epidermal homeostasis. These results further highlight the potential applicability of dietary fiber‐rich citrus byproducts as functional ingredients for protecting the skin against environmentally induced oxidative and inflammatory stress.

Anti‐inflammatory and moisturizing effects of CCU extract on PM‐treated skin

Following the in vitro confirmation of anti‐inflammatory and hydration‐protective effects of CCU extract in PM‐stimulated human keratinocytes, its protective efficacy was further evaluated in vivo using a murine skin model. As illustrated in Fig. 6(A), 100 μg mL⁻¹ PM was topically applied, while CCU extract was orally administered (400 mg kg⁻¹) daily for 7 consecutive days. One day after the final treatment, dorsal skin tissues were collected and analysed for skin hydration and inflammatory responses. PM was topically applied to induce localized skin inflammation and barrier disruption, whereas CCU extract was orally administered to evaluate its potential as a dietary fiber‐rich functional ingredient capable of exerting systemic protective effects against environmentally induced skin damage.

Figure 6.

Effects of CCU administration on skin inflammation and hydration in PM‐treated C57BL/6 mice. (A) The schematic diagram of animal experiments. (B) The levels of pro‐inflammatory cytokines, hyaluronic acid and skin water contents were analysed and quantified in the skin tissues. ##P < 0.05 and ###P < 0.001 indicate significant differences compared to the CON group. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significant differences compared to the PM‐induced group.

As shown in Fig. 6(B), PM exposure significantly reduced skin hydration, whereas CCU extract effectively restored hyaluronic acid production and water content to near‐normal levels. These improvements may be associated with the high‐quality dietary fiber content in the extract, which is known to possess water‐holding capacity and swelling properties, as previously reported for UAEE‐derived citrus fibers. ⁵⁰ Furthermore, biochemical analyses demonstrated a marked reduction in PM‐induced overexpression of pro‐inflammatory cytokines (IL‐1β and IL‐6), indicating that the oral administration of CCU not only attenuated inflammatory responses, but also contributed to skin barrier homeostasis. These findings are consistent with previous reports demonstrating that dietary supplementation with natural polysaccharides can alleviate environmentally induced skin damage by improving hydration and maintaining tissue integrity. ⁵¹

Restorative effects of CCU extract on skin inflammation and barrier proteins in PM‐exposed mice

To investigate the skin health‐improving potential of CCU extract in vivo, we evaluated its regulatory effects on both inflammation‐related and skin barrier‐related proteins in the dorsal skin of PM‐exposed mice. As expected, PM exposure led to a substantial increase in the expression of inflammatory markers, iNOS and COX‐2, at the same time as reducing the levels of key structural proteins including filaggrin, loricrin and involucrin, indicating skin barrier disruption and inflammatory stress. Notably, oral administration of CCU extract for 7 consecutive days effectively suppressed the expression of iNOS and COX‐2, at the same time as restoring the expression of filaggrin, loricrin and involucrin (Fig. 7(A), (B)), demonstrating dual functionality of CCU extract in mitigating cutaneous inflammation and enhancing barrier resilience.

Figure 7.

Effects of CCU administration on inflammatory and skin barrier protein expression levels in PM‐treated C57BL/6 mice. (A) The expression levels of iNOS, COX‐2, (B) FLG, IVL and LOR were analysed by western blotting, and the quantitative results are displayed. ##P < 0.05 and ###P < 0.001 indicate significant differences compared to the CON group. *P < 0.05 and **P < 0.01 indicate significant differences compared to the PM‐induced group.

These outcomes are in line with previous findings indicating the skin barrier‐enhancing potential of plant‐derived oligosaccharides and polysaccharides. For example, raffinose, a plant‐derived trisaccharide, was shown to upregulate filaggrin, loricrin and involucrin expression in both keratinocyte cultures and in vivo mouse skin models. ⁵² Although that study primarily focused on epidermal differentiation, other plant‐derived polysaccharides and/or dietary fibers have also shown immunomodulatory activity by suppressing iNOS and COX‐2 in inflammatory conditions. ⁵³ , ⁵⁴ Our findings extend this understanding by demonstrating that a UAEE‐derived citrus pomace extract rich in dietary fiber may modulate both inflammatory responses and skin barrier‐associated proteins under PM‐induced stress conditions.

Although the precise molecular mechanisms remain to be fully elucidated, plant‐derived polysaccharides and dietary fibers have been reported to regulate oxidative stress‐ and inflammation‐related signaling pathways involved in keratinocyte activation and epidermal barrier maintenance. ⁵⁵ , ⁵⁶ In addition, the potential involvement of dietary fiber‐mediated systemic regulation, including gut–skin interactions, ²⁶ warrants further investigation.

Together, these results suggest that CCU extract has strong potential as a dietary fiber‐rich functional ingredient for alleviating environmentally induced skin inflammation and dehydration through both anti‐inflammatory and skin barrier supportive mechanisms.

CONCLUSIONS

In conclusion, the ultrasound‐assisted enzymatic extract (CCU) derived from citrus pomace demonstrated a significant protective effect against PM‐induced skin inflammation and barrier disruption. Among the tested extracts, CCU showed the highest extraction yield and improved dietary fiber recovery, supporting its potential as a value‐added functional ingredient obtained through sustainable extraction processes. In PM‐stimulated HaCaT keratinocytes, CCU extract effectively enhanced cell viability, suppressed intracellular ROS production, and reduced the expression of pro‐inflammatory cytokines and inflammatory mediators. In addition, CCU treatment restored the expression of key skin barrier‐associated proteins and improved skin hydration‐related responses in both in vitro and in vivo models. Taken together, these findings suggest that CCU, potentially enriched with citrus‐derived dietary fibers and polysaccharides, may alleviate PM‐induced oxidative and inflammatory stress while preserving epidermal homeostasis and barrier function. Overall, the present study highlights the potential of citrus processing byproducts as sustainable sources of functional dietary fiber and provides a promising strategy for their biocatalytic valorization toward nutraceutical and skin health‐related applications.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.