Ultraviolet B (UVB) radiation is among the most harmful environmental eye stressors [1, 2]. Cornea is the most anterior and directly exposed tissue and acts as the primary barrier against ultraviolet light; nonetheless, it is also highly vulnerable to phototoxic damage [1]. Repeated or acute UVB exposure results in excessive reactive oxygen species production, mitochondrial dysfunction, and apoptotic pathway activation in corneal epithelial cells, potentially leading to the loss of epithelial cells, corneal opacity, and impaired vision [1]. Clinically, UV-induced keratitis and progressive ocular surface disorders are significant public health concerns, especially in populations with high sunlight exposure or occupational risks [2]. Nevertheless, effective therapeutic strategies for preventing or attenuating UVB-induced corneal apoptosis remain limited.

Previous approaches have largely focused on antioxidant supplementation or topical formulations using well-known compounds, such as vitamin C, vitamin E, or plant-derived flavonoids [3]. Although these agents exhibit some efficacy, their clinical utility is often restricted by their instability, limited bioavailability, and insufficient potency to counteract severe oxidative stress [3]. Therefore, identifying natural compounds with potent antioxidant activity, reliable safety, and strong cellular protective effects is warranted in ophthalmic research.

Peucedanum japonicum Thunb. (SBP) is a medicinal herb widely used in East Asian herbal pharmacology [4]. Historically, it has been used for its antipyretic, analgesic, and anti-inflammatory properties [5]. Phytochemical analyses revealed that SBP contains coumarins, chromones, and phenolic acids that exhibit strong antioxidant capacities and free radical scavenging properties [4, 6]. Recent pharmacological studies have suggested that SBP extract protects against inflammation-induced tissue injury, regulates immune responses, and modulates oxidative stress in different experimental models [5, 6]. Our previous reports demonstrated that it protected ocular tissues from urban particulate matter (UPM)-mediated oxidative stress and mitigated delayed wound healing following blue light irradiation [7, 8]. These studies establish SBP as a potential therapeutic agent for environmentally induced ocular surface damage.

However, its role in preventing UVB-induced corneal epithelial apoptosis, which is mechanistically distinct from UPM- or blue light-mediated oxidative injury, has not yet been systematically addressed. Because mitochondrial dysfunction is a central driver of UVB-induced apoptosis, we assessed whether SBP fractions, particularly the n-butanol (n-BuOH) fraction, could stabilize mitochondrial membrane potential, preserve mitochondrial morphology, and suppress apoptotic signaling in corneal epithelial cells. Additionally, chlorogenic acid, previously identified as a major bioactive component of SBP, was tested in vivo as a reference compound to validate its protective effects.

This study demonstrated the anti-apoptotic effect of SBP extract on corneal epithelial cells, thereby identifying a novel natural substance capable of protecting the eye from UV-induced damage [1]. These findings provide an important basis for the development of new therapeutic agents to maintain ocular health. By elucidating apoptosis through mitochondrial damage as the major mechanism, this research reinforces the scientific validity of using SBP as a protective agent. When corneal epithelial cells were exposed to UVB irradiation, cell viability markedly decreased, accompanied by the loss of mitochondrial membrane potential and morphological abnormalities characteristic of apoptosis [1, 2]. Moreover, JC-1 staining revealed a pronounced shift from red to green fluorescence in the UVB group, indicating mitochondrial depolarization [9]. To exclude cytotoxicity and identify the bioactive fractions, we screened SBP fractions using an NADH dehydrogenase activity assay. The n-BuOH fraction demonstrated no mitotoxicity, displaying clear protective effects against UVB-induced damage (Fig. 1A–L). Given that the MTT assay primarily reflects mitochondrial activity, SBP may act through mitochondrial regulation. Subsequent analyses with JC-1 and MitoTracker confirmed this hypothesis [10]. SBP treatment restored the red/green fluorescence ratio in a dose-dependent manner, with the n-BuOH fraction exhibiting the most potent protective effect (Fig. 1M–Q). Furthermore, morphological imaging with MitoTracker revealed that the mitochondria in UVB-exposed cells were fragmented and punctate, reflecting oxidative stress and apoptotic signaling initiation. Conversely, SBP-treated cells maintained elongated tubular mitochondria, indicating mitochondrial integrity preservation (Fig. 1M–Q). Overall, SBP may prevent mitochondrial dysfunction, which is a central trigger of apoptosis. To further strengthen our mechanistic insights, an apoptotic protein array was performed. UVB irradiation increased the expression of pro-apoptotic proteins, including Bcl-2–associated X protein (Bax), Bcl-2–interacting mediator of cell death (Bim), Cysteine-aspartic protease-3 (Caspase-3), CD40 ligand (CD40L), Heat shock protein 60 (HSP60), Insulin-like growth factor-1 (IGF-1), and Cyclin-dependent kinase inhibitor 1A (p21), while the anti-apoptotic protein Survivin was dysregulated (Fig. 2). In particular, the upregulation of Bax and Bim promotes mitochondrial outer membrane permeabilization, leading to cytochrome c release and subsequent activation of Caspase-9 and Caspase-3, a hallmark of the intrinsic (mitochondria-mediated) apoptotic pathway [11]. Additional factors such as CD40L, HSP60, IGF-1, and p21 further reflect stress-induced apoptotic signaling and cross-talk with mitochondrial processes [12]. Dysregulation of Survivin, an inhibitor of apoptosis, further amplifies this pro-apoptotic cascade [12]. Importantly, SBP treatment reversed or attenuated these molecular alterations, restoring the balance between pro- and anti-apoptotic signaling and thereby counteracting UVB-induced mitochondrial apoptosis (Fig. 2). Thus, SBP may protect against UVB-induced corneal apoptosis by maintaining mitochondrial potential and influencing the expression of key regulators of the intrinsic apoptotic cascade. These protective effects were validated in vivo. Fluorescein staining of the corneal surfaces revealed that UVB exposure induced severe epithelial damage, characterized by diffuse punctate staining, whereas SBP-treated mice demonstrated markedly reduced fluorescein uptake, indicating preservation of corneal barrier integrity (Fig. 3A). This reduction in staining suggests that SBP protected epithelial tight junctions and maintained barrier function against UVB-induced disruption (Fig. 3A). Given that fluorescein staining is a clinical indicator of ocular surface health, these findings further highlight the translational potential of SBP in preventing UVB-related ocular surface disorders [13]. Histological examination of the corneal tissues from UVB-irradiated mice revealed epithelium thinning, disorganization of the cell layers, and stromal swelling. Contrastingly, the SBP-treated corneas retained their epithelial structures and displayed reduced tissue damage, particularly in the n-BuOH group. Although the present study did not include a drug-only (without UVB) control group, previous in vitro and in vivo data have confirmed that SBP and its n-BuOH fraction are non-toxic to normal cells and tissues [7]. Future studies must include such drug-only groups to further evaluate the ocular safety and baseline effects of SBP under physiological conditions. The TUNEL assay provided additional evidence that numerous TUNEL-positive nuclei were observed in the UVB-exposed corneal epithelium; nonetheless, these apoptotic signals were significantly reduced in the SBP-treated groups (Fig. 3B). Quantitative HPLC analysis from our previous study [7] revealed that it contained high levels of chlorogenic acid (2.30 $\pm$ 0.03 mg/g), along with its related isomers, neochlorogenic acid (2.27 $\pm$ 0.03 mg/g) and cryptochlorogenic acid (2.57 $\pm$ 0.04 mg/g). These data confirm that the n-BuOH fraction is enriched in chlorogenic acid derivatives, which are the principal phenolic components responsible for its antioxidant and anti-apoptotic activities. In line with our previous phytochemical studies, three chlorogenic acid isomers (chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid) were tested for cytotoxicity in vitro (Supplementary Fig. 1). Among these, chlorogenic acid was the only compound that showed no detectable mito-toxicity at the tested concentrations, whereas cryptochlorogenic acid and neochlorogenic acid caused dose-dependent reductions in NADH dehydrogenase activity, consistent with their negative effects on cell viability shown in Supplementary Fig. 2. Accordingly, chlorogenic acid was selected as the reference compound for in vivo validation. Notably, previous studies have also identified chlorogenic acid as a major bioactive constituent of SBP, demonstrating its efficacy in protecting against oxidative stress and cellular injury [7, 8]. Consistent with these findings, SBP treatment in the present study exerted protective effects comparable to those of chlorogenic acid, further supporting its potential as a natural therapeutic candidate. Notably, this comparison was based on nominal dose (mg/kg) rather than molar equivalence, as the n-BuOH fraction represents a complex phytochemical mixture enriched in chlorogenic acid and related phenolic compounds. Therefore, the comparable efficacy observed in vivo likely reflects a bioactivity-based equivalence rather than an exact molar match.

Fig. 1.

Protective effects of SBP extract and its solvent fractions against UVB-induced mito-toxicity in corneal epithelial cells. Cell viability after treatment with (A,G) SBP extract at the indicated concentrations, with or without UVB irradiation; (B,H) hexane fraction; (C,I) ethyl acetate fraction; (D,J) n-BuOH fraction; (E,K) chloroform fraction; (F,L) water fraction. (M) Quantitative analysis of mitochondrial morphology and fluorescence intensity. (N–P) Ratio of red to green fluorescence indicating mitochondrial membrane potential ($\mathrm{\Delta }$$\mathrm{\Psi }$m). (Q) Representative fluorescence images of corneal epithelial cells stained with JC-1 (red: aggregated, intact mitochondria; green: monomeric, depolarized mitochondria). SBP extract and its n-BuOH fraction significantly restored mitochondrial membrane potential and preserved elongated mitochondrial morphology compared with the UVB-only group. Scale bars: top two rows = 500 µm; bottom two rows = 100 µm. Data are expressed as mean $\pm$ SD from at least three independent experiments. *p 0.05, **p 0.01, ***p 0.001 versus control; ##p 0.01, ###p 0.001 versus UVB group. UVB, ultraviolet B; SBP, Peucedanum japonicum Thunb; n-BuOH, n-butanol.

Fig. 2.

Effects of SBP extract and its n-BuOH fraction on UVB-induced apoptosis-related protein expression in corneal epithelial cells. (A) Layout of the apoptosis antibody array showing the positions of 43 apoptosis-related proteins, with red boxes marking protein spots that exhibited notable expression changes among the groups. (B) Representative protein array blots from each group: control, UVB, UVB + SBP, and UVB + n-BuOH fraction. Red boxes indicate protein spots that showed detectable and significant expression changes between groups. Quantification of (C) Bax expression levels. (D) Bim expression levels. (E) Caspase-3 expression levels. (F) CD40L expression levels. (G) HSP60 expression levels. (H) IGF-1 expression levels. (I) p21 expression levels. (J) Survivin expression levels. UVB exposure markedly increased pro-apoptotic protein expression, which was attenuated by SBP extract and its n-BuOH fraction. SBP and n-BuOH treatments reduced UVB-induced upregulation of Bax, Bim, Caspase-3, CD40L, HSP60, IGF-1, and p21, while partially restoring Survivin levels. Data are presented as mean $\pm$ SD from three independent experiments. UVB, ultraviolet B; SBP, Peucedanum japonicum Thunb; Bax, Bcl-2–associated X protein; Bim, Bcl-2–interacting mediator of cell death; Caspase-3, Cysteine-aspartic protease-3; CD40L, CD40 ligand; IGF-1, Insulin-like growth factor-1; p21, Cyclin-dependent kinase inhibitor 1A (CDKN1A).

Fig. 3.

Protective effects of SBP extract and its n-BuOH fraction against UVB-induced corneal damage in mice. (A) Representative fluorescein staining images of corneal surfaces. UVB exposure induced severe epithelial damage and punctate keratopathy, which were markedly reduced by SBP extract and n-BuOH fraction treatment. Due to the curved nature of the corneal surface and the imaging method used (fluorescence photography), exact spatial calibration was not feasible. Therefore, scale bars were not included. (B) Hematoxylin and eosin staining of corneal tissues. UVB irradiation caused epithelial thinning, disorganization, and stromal edema, whereas SBP-treated groups exhibited preserved epithelial structure and reduced tissue injury. Scale bar = 100 µm. TUNEL assay of corneal sections. Numerous TUNEL-positive nuclei were detected in the UVB group, indicating extensive apoptotic cell death. SBP extract and the n-BuOH fraction significantly decreased the number of TUNEL-positive cells, demonstrating their anti-apoptotic effects in vivo. UVB, ultraviolet B; SBP, Peucedanum japonicum Thunb; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

In summary, this study demonstrated that the SBP extract exerts significant protective effects against UVB-induced corneal epithelial apoptosis, with the n-BuOH fraction showing the strongest efficacy both in vitro and in vivo. This observed protection appeared to be closely linked to mitochondrial function preservation, suggesting that the active constituents within the n-BuOH fraction may enhance mitochondrial activity and prevent depolarization-induced apoptotic signaling. Based on previous phytochemical analyses and supporting evidence from earlier studies, chlorogenic acid has emerged as a plausible candidate that contributes to this effect. Our previous HPLC profiling confirmed that the n-BuOH fraction contained the highest concentrations of chlorogenic acid and its related derivatives, including neochlorogenic and cryptochlorogenic acids [7]. However, this fraction also contains minor coumarin- and flavonoid-type compounds, which possess antioxidant and anti-apoptotic activities. Therefore, synergistic interactions among multiple constituents may collectively contribute to the overall protective effects of SBP against UVB-induced corneal injury. Nevertheless, further investigations are warranted to confirm whether chlorogenic acid is the major bioactive compound or whether synergistic interactions among multiple n-BuOH fraction components are responsible. Future studies should focus on isolating the active constituents, clarifying mitochondria-related molecular pathways in greater detail, and assessing the long-term safety and clinical applicability in preclinical models. Furthermore, evaluating the ocular bioavailability and tissue distribution of SBP-derived compounds particularly chlorogenic acid and its derivatives will be essential for future translational development. Pharmacokinetic and corneal penetration studies are planned to determine the absorption, distribution, and retention of these compounds within ocular tissues. One limitation of this study is that only male mice were used in the in vivo experiments to minimize hormonal variability. However, sex-related physiological differences may affect oxidative stress responses and drug efficacy. Therefore, future studies should include both male and female mice of matching age and weight to comprehensively evaluate potential sex-dependent variations and enhance the translational relevance of our findings.

SBP extract, particularly its n-BuOH fraction, effectively protected corneal epithelial cells against UVB-induced apoptosis by preserving mitochondrial function and modulating apoptosis-related proteins. Both in vitro and in vivo evidence support its potential as a safe natural candidate for preventing or treating UV-related ocular surface disorders. Therefore, SBP is a promising therapeutic target for ophthalmic development. Further studies are warranted to isolate the active compounds and evaluate their long-term efficacy.

NADH, nicotinamide adenine dinucleotide; n-BuOH, n-butanol; PBS, phosphate-buffered saline; SBP, Peucedanum japonicum Thunb.; SD, standard deviation; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; UVB, ultraviolet B; $\mathrm{\Delta }$$\mathrm{\Psi }$m, mitochondrial membrane potential.

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Conceptualization, HL, YY, GP, SK; methodology, GP, HL; formal analysis, GP, HL; investigation, GP, SK; data curation, GP, HL; writing—original draft preparation, HL, GP; writing—review and editing, HL, YY, GP, SK; visualization, HL, GP, SK; supervision, GP, SK. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The Institutional Animal Care Committee of the Korea Institute of Oriental Medicine (KIOM) approved the experimental protocols KIOM: #25-010, which were performed according to the guidelines of the Animal Care and Use Committee at KIOM.

This work was supported by the Jeollanam-do R&D program operated by Jeonnam Technopark (KIOM management No. ERN2411030) for the project titled, “Functional eye health food made from Peucedanum japonicum Thunb. extract to improve visual acuity deterioration caused by phototoxicity.” and the Korea Institute of Oriental Medicine (KSN2511030) through the Development of Innovative Technologies for the Future Value of Herbal Medicine Resources project.

The authors declare no conflict of interest. Young-Sik Yoo is an employee of Mediverse Co., Ltd. Sunoh Kim is an employee of the Central R&D Center of B&Tech Co., Ltd. The authors declare that these affiliations did not influence the study design, data interpretation, or manuscript preparation.

During preparation of this manuscript, the authors used ChatGPT [5.0 version, OpenAI] for language editing and grammar checking. After using this tool, the authors carefully reviewed and revised the text and take full responsibility for the content of the publication.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/FBL46498.