Isolation and Identification of Dihydrophenanthrene Derivatives from Dendrobium virgineum with Protective Effects against Hydrogen-Peroxide-Induced Oxidative Stress of Human Retinal Pigment Epithelium ARPE-19 Cells

Oxidative stress is a significant factor in the development of age-related macular degeneration (AMD), which results from cell damage, dysfunction, and death in the retinal pigmented epithelium (RPE). The use of natural compounds with antioxidant properties to protect RPE cells from oxidative stress has been explored in Dendrobium, a genus of orchid plants belonging to the family Orchidaceae. Two new compounds and seven known compounds from the MeOH extract of the whole plant of Dendrobium virgineum were successfully isolated and structurally characterized. Out of all the compounds isolated, 2-methoxy-9,10-dihydrophenanthrene-4,5-diol (3) showed the highest protective effect against hydrogen peroxide (H2O2)-induced oxidative stress in human retinal pigment epithelial (ARPE-19) cells. Therefore, it was selected to evaluate its protective effect and mechanism on oxidative-stress-induced ARPE-19 cells. Cells were pre-treated with compound 3 at 25, 50, and 100 µg/mL for 24 h and then induced with 400 µM H2O2 for 1 h. The results demonstrated that compound 3 significantly (p < 0.05) increased cell viability by 10–35%, decreased ROS production by 10–30%, and reduced phosphorylation of p38, ERK1/2, and SAPK/JNK by 20–70% in a dose-dependent manner without toxicity. Furthermore, compound 3 significantly (p < 0.05) modulated the expression of apoptosis pathway proteins (cytochrome c, Bax and Bcl-2) by 20–80%, and enhanced SOD, CAT, and GPX activities, and GSH levels in a dose-dependent manner. These results suggest that compound 3 protects ARPE-19 cells against oxidative stress through MAPKs and apoptosis pathways, including the antioxidant system. Thus, compound 3 could be considered as an antioxidant agent for preventing AMD development by protecting RPE cells from oxidative stress and maintaining the retina. These findings open up new possibilities for the use of natural compounds in the treatment of AMD and other oxidative-stress-related conditions.


Introduction
The abnormality of the eyes in the macular area causes irreversible blindness and retinal disease, the so-called age-related macular degeneration (AMD) [1]. The prevalence such as polyphenols, carotenoids, flavonoids, and organosulfur compounds, which exhibited the preventive effects in AMD, have been reported in various studies [37][38][39][40][41][42][43][44][45]. Moreover, Dendrobium extract has been reported to improve vision [46]. Dendrobium virgineum Rchb.f. (Figure 1A), known in Thai as "Ueang Nang Chi", is usually founded in dry evergreen forests and mixed deciduous forests in Thailand's east and north-east regions, and in Myanmar, Laos, and Vietnam. Its flower consists of white petals and a red lip, and it was first discovered by Reichenbach in 1884 [47]. In this study, we aimed to isolate and determine the protective effect against H2O2induced oxidative stress in human retinal pigment epithelial (ARPE-19) cells of isolated compounds from the whole plant of Dendrobium virgineum. Among 9 isolated compounds, two new compounds (1 and 2) and seven known compounds (3)(4)(5)(6)(7)(8)(9) were identified. Compound 3, with the most potent antioxidant effect, was further investigated regarding its protective mechanism against H2O2-induced ARPE-19 cells.

Plant Material
The whole plants of D. virgineum were obtained from Chatuchak Market (Bangkok, Thailand) in September 2015. Authentication was performed by the author (Boonchoo Sritularak). A voucher specimen (BS-DVir-2558) was deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University.
Dendrovirginin (1)  cells were allowed to grow until they reached over 90% confluence before being utilized in subsequent experiments.

Cell Viability Assay
The viability of cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide tetrazolium (MTT) assay, which measures the conversion of MTT substrate to a purple-colored formazan product in viable cells. After cells reached their experimental time points, they were washed once with phosphate buffer saline (PBS) and incubated with MTT in PBS at 0.5 mg/mL for 4 h at 37 • C. After removal of the MTT solution, DMSO was added to dissolve the formazan crystals generated by viable cells. The absorbance was subsequently measured at 540 nm using a microplate reader (SPECTROstarNano, BMG LABTECH, Ortenberg, Germany).

Cytotoxicity Assay of Compounds 1-9
Following a 24 h incubation of ARPE-19 cells, the media was removed and cells were washed with serum-free media. Cells were then treated with varying concentrations (10, 50, and 100 µg/mL) of compounds 1-9 for 24 h, with a 0.5% DMSO used as the control group. Cell viability was assessed using the MTT assay after the respective experimental time.

Determining the Optimal H 2 O 2 Concentration for Cytotoxicity Induction
ARPE-19 cells in a 96-well plate were treated by removing the media and washing with serum-free media. Subsequently, cells were treated with serum-free medium containing various concentrations of H 2 O 2 (200-1000 µM) for 30, 60, and 120 min at 37 • C. The control group consisted of cells cultured in serum-free medium without H 2 O 2 . Following the experimental treatment, cells were washed twice with PBS and evaluated for cell viability using the MTT assay.
2.8. Assessing the Effect of Compound 3 on ARPE-19 Cells Exposed to H 2 O 2 ARPE-19 cells in 6-well and 96-well plates were washed one time with serum-free media and pre-incubated with compound 3 (25, 50, and 100 µg/mL) in serum-free media for 24 h. A 0.5% DMSO served as the control group. After pre-treatment with compound 3, cells were washed with serum-free media and then treated with appropriate H 2 O 2 concentrations in serum-free medium at 37 • C for 1 h. Cell viability was measured using the MTT assay in the 96-well plate setup, while the 6-well plate setup was used for non-enzymatic and enzymatic antioxidant assays, caspase-9 and caspase-3 activities, and western immunoblot analysis.

Evaluation of Reactive Oxygen Species (ROS) Production
To determine intracellular ROS, ARPE-19 cells were seeded at 3.0 × 10 4 cells/well in black 96-well, clear bottom plates and cultured for 24 h. After washing with serum-free media, cells were pre-incubated for 24 h with compound 3 (25, 50 and 100 µg/mL) in serum-free media, with 0.5% DMSO as the control. Subsequently, cells were incubated with 10 µM DCFH-DA in serum-free media at 37 • C for 30 min, then treated with varying concentrations of H 2 O 2 in serum-free media at 37 • C for 1 h. Following PBS washes, ROS production was measured using a fluorescence microplate reader with excitation/emission wavelengths of 485/530 nm.

Western Blot Analysis
ARPE-19 cell lysates were prepared from a 6-well plate by centrifuging at 14,000 rpm at 4 • C for 10 min, and the protein concentrations were determined using the BCA protein assay kits. Equal amounts (40 µg) of protein samples were separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane, blocked with 5% dry milk, and then incubated with anti-p-p38, anti-p-ERK1/2, anti-p-SAPK/JNK, anti-Bax, anti-Bcl-2, or anti-cytochrome c at a ratio of 1:1000 (v/v) in TBST overnight. The membrane was washed with TBST and incubated with a 1:2000 species-specific horseradish peroxide conjugated secondary antibody for 2 h. The protein levels were detected using an enhanced chemiluminescent detection kit, followed by imaging. The densitometry values of the phosphorylated forms of p38 (p-p38), ERK1/2 (p-ERK1/2), and SAPK/JNK (p-SAPK/JNK) were normalized to the band intensity of the respective total forms of p38, ERK1/2, and SAPK/JNK. Similarly, the densitometry values of cytochrome c, Bax, and Bcl-2 were normalized to the band intensity of β-actin.

Caspase-9 and -3 Activities
Following pre-treatment with compound 3 and H 2 O 2 induction, as described in Section 2.8, the cells were homogenized in a hypotonic buffer to extract the supernatant. The supernatant was combined with a specific substrate (N-acetyl-Leu-Glu-His-Asp pnitroanilide or N-acetyl-Asp-Glu-Val-Asp p-nitroanilide for caspase-9 or caspase-3, respectively) at a concentration of 100 µmol/L. The mixture was incubated at 37 • C for 1 h., and the absorbance was measured at 450 nm using a microplate reader to detect the activity of the caspases.

SOD, GPx, CAT, and GSH Determination
After treating ARPE-19 cells with compound 3 and H 2 O 2 , as described in Section 2.8, the cells were harvested by scraping and incubated with 0.5% (v/v) Triton X-100 in cold PBS. The resulting cell solution was transferred to a 1.5-mL tube and subjected to sonication in an ultrasonic sonicator bath at 4 • C for 10 min. The cell lysate was then centrifuged at 14,000× g at 4 • C for 10 min, and the supernatant was collected to measure the levels of GSH and the activities of SOD, GPx, and CAT using assay kits.

Statistical Analysis
The results are expressed as mean ± standard deviation (SD) of at least three independent experiments. Statistical analysis was performed with SPSS software version 16.0 (SPSS inc., Chicago, IL, USA). The differences among groups were assessed by one-way analysis of variance (ANOVA). Statistical significance was set at p < 0.05.

Evaluation of the Effects of Compounds (1-9) on Viability of ARPE-19 Cells
To measure their non-toxic concentrations, compounds 1-9 were tested on ARPE-19 cells before assessing their protection against oxidative stress. The treatment was conducted for 24 h at 25, 50, and 100 µ g/mL. Compounds 1, 2, and 4 at 50 and 100 µ g/mL, as well as compounds 8 and 9 at 100 µ g/mL, exhibited cytotoxicity against ARPE-19 cells (Figure 3). To ensure efficient and continuous activity, the maximum concentration used in subsequent experiments was 25 µ g/mL.

Evaluation of the Effect of H 2 O 2 on Viability and ROS Production of ARPE-19 Cells
Various concentrations of H 2 O 2 (200-1000 µM) were applied to ARPE-19 cells for 30, 60, and 120 min to determine the concentration required for a roughly 50% reduction in viability. Results indicated that H 2 O 2 treatment caused a concentration and time-dependent decrease in cell viability and an increase in ROS production ( Figure 4A,B). Treatment with 400 µM of H 2 O 2 for 60 min caused a 50% reduction in cell viability ( Figure 4A). As a result, 400 µM of H 2 O 2 for 60 min was employed to generate oxidative stress in ARPE-19 cells.

Evaluation of the Effect of H2O2 on Viability and ROS Production of ARPE-19 Cells
Various concentrations of H2O2 (200-1000 µ M) were applied to ARPE-19 cells for 30, 60, and 120 min to determine the concentration required for a roughly 50% reduction in viability. Results indicated that H2O2 treatment caused a concentration and time-dependent decrease in cell viability and an increase in ROS production ( Figure 4A,B). Treatment with 400 µ M of H2O2 for 60 min caused a 50% reduction in cell viability ( Figure 4A). As a result, 400 µ M of H2O2 for 60 min was employed to generate oxidative stress in ARPE-19 cells.

Evaluation of the Effect of Compounds (1-9) on Cell Viability of Oxidative-Stress-Induced ARPE-19 Cells
To assess their protective effects against H2O2-induced oxidative stress in ARPE-19 cells, compounds 1-9 were evaluated by pre-incubating cells with each compound at 25 µ g/mL for 24 h. After washing with serum-free media, cells were treated with serum-free media containing 400 µ M of H2O2 for 60 min. Among the compounds, compound 3 showed the highest protective effect against oxidative stress in ARPE-19 cells ( Figure 5A)

Evaluation of the Effect of Compounds (1-9) on Cell Viability of Oxidative-Stress-Induced ARPE-19 Cells
To assess their protective effects against H 2 O 2 -induced oxidative stress in ARPE-19 cells, compounds 1-9 were evaluated by pre-incubating cells with each compound at 25 µg/mL for 24 h. After washing with serum-free media, cells were treated with serumfree media containing 400 µM of H 2 O 2 for 60 min. Among the compounds, compound 3 showed the highest protective effect against oxidative stress in ARPE-19 cells ( Figure 5A) without inducing toxicity in normal ARPE-19 cells ( Figure 5B). Therefore, compound 3 was selected to evaluate its protective mechanism in oxidative-stress-induced ARPE-19 cells. As shown in Figure 3, the cytotoxicity results revealed that the maximum concentration of compound 3 at 100 µg/mL had no significant impact on viability of ARPE-19 cells compared with the control. Consequently, concentrations of 25, 50, and 100 µg/mL of compound 3 were chosen for the protective mechanism studies.

Evaluation of the Effect of Compound 3 on Cell Viability and ROS Production in Oxidative-Stress-Induced ARPE-19 Cells
The protective effects of compound 3 against oxidative-stress-induced cell death in ARPE-19 cells were investigated by pre-incubation with compound 3 at 25, 50, and 100 µg/mL for 24 h followed by induction of oxidative stress with 400 µ M H2O2 for 60 min. The protective effect of compound 3 against H2O2-induced oxidative stress was supported by inverted microscopic analysis ( Figure 6A). H2O2 treatment caused a 50% decrease in cell viability compared with the control group ( Figure 6B). However, compound 3 significantly (p < 0.05) protected the cell viability of ARPE-19 cells in a dose-dependent manner when compared with the H2O2 group ( Figure 6B). In terms of ROS production, H2O2 significantly (p < 0.05) increased ROS production compared with the control group (Figure

Evaluation of the Effect of Compound 3 on Cell Viability and ROS Production in Oxidative-Stress-Induced ARPE-19 Cells
The protective effects of compound 3 against oxidative-stress-induced cell death in ARPE-19 cells were investigated by pre-incubation with compound 3 at 25, 50, and 100 µg/mL for 24 h followed by induction of oxidative stress with 400 µM H 2 O 2 for 60 min. The protective effect of compound 3 against H 2 O 2 -induced oxidative stress was supported by inverted microscopic analysis ( Figure 6A). H 2 O 2 treatment caused a 50% decrease in cell viability compared with the control group ( Figure 6B). However, compound 3 significantly (p < 0.05) protected the cell viability of ARPE-19 cells in a dose-dependent manner when compared with the H 2 O 2 group ( Figure 6B). In terms of ROS production, H 2 O 2 significantly (p < 0.05) increased ROS production compared with the control group ( Figure 6C). On the other hand, compound 3 significantly (p < 0.05) decreased ROS production in a dose-dependent manner when compared with the H 2 O 2 group ( Figure 6C). These findings suggest that compound 3 protects ARPE-19 cells against oxidative-stress-induced cytotoxicity by reducing ROS production dose-dependently.

Evaluation of the Effect of Compound 3 on MAPKs Protein Expression in Oxidative-Stress-Induced ARPE-19 Cells
The protective effects of compound 3 on ARPE-19 cells under oxidative stress were investigated to determine the underlying molecular mechanisms. Previous research indicated that phosphorylation of MAPK signaling pathways (p38, ERK1/2, and SAPK/JNK) promoted H2O2-induced apoptosis [57]. The current study explored whether the same (B) viability of cells was determined using an MTT assay; (C) production of ROS was measured by a DCFH-DA assay. Results present average cell viability and ROS production as mean ± SD (n = 4). Different superscript letters (a-e) indicate significant differences between values in the column (p < 0.05).

Evaluation of the Effect of Compound 3 on MAPKs Protein Expression in Oxidative-Stress-Induced ARPE-19 Cells
The protective effects of compound 3 on ARPE-19 cells under oxidative stress were investigated to determine the underlying molecular mechanisms. Previous research indicated that phosphorylation of MAPK signaling pathways (p38, ERK1/2, and SAPK/JNK) promoted H 2 O 2 -induced apoptosis [57]. The current study explored whether the same pathway contributed to H 2 O 2 -induced cell damage and death. Immunoblotting was used to analyze protein expression and revealed that incubation of ARPE-19 cells with H 2 O 2 at 400 µM for 1 h significantly increased the phosphorylation of p38 (p-p38), ERK1/2 (p-ERK1/2), and SAPK/JNK (p-SAPK/JNK) in comparison with the control group ( Figure 7A-C). This finding suggests that H 2 O 2 -induced cell death occurs via the p-p38, p-ERK1/2, and p-SAPK/JNK pathways. Pre-incubation with compound 3 (25, 50, and 100 µg/mL) for 24 h reduced the expression of the phosphorylation form of p38, ERK1/2, and SAPK/JNK in comparison with the H 2 O 2 -induced ARPE-19 cell group (p < 0.05), indicating that compound 3 protects against oxidative stress via a dose-dependent modulation of the MAPKs signaling pathway. pathway contributed to H2O2-induced cell damage and death. Immunoblotting was used to analyze protein expression and revealed that incubation of ARPE-19 cells with H2O2 at 400 µ M for 1 h significantly increased the phosphorylation of p38 (p-p38), ERK1/2 (p-ERK1/2), and SAPK/JNK (p-SAPK/JNK) in comparison with the control group ( Figure 7A-C). This finding suggests that H2O2-induced cell death occurs via the p-p38, p-ERK1/2, and p-SAPK/JNK pathways. Pre-incubation with compound 3 (25, 50, and 100 µ g/mL) for 24 h reduced the expression of the phosphorylation form of p38, ERK1/2, and SAPK/JNK in comparison with the H2O2-induced ARPE-19 cell group (p < 0.05), indicating that compound 3 protects against oxidative stress via a dose-dependent modulation of the MAPKs signaling pathway.

Evaluation of the Effect of Compound 3 on Apoptosis Protein Expression in Oxidative-Stress-Induced ARPE-19 Cells
To understand how compound 3 protects against oxidative stress, we assessed its molecular mechanisms by analyzing the apoptosis pathway. Specifically, we evaluated the levels of downstream targets of the MAPK pathways, such as cytochrome c, Bax (pro-apoptotic), and Bcl-2 (anti-apoptotic) proteins [58,59]. We determined the expression of cytochrome c, Bax, and Bcl-2 through immunoblotting in ARPE-19 cells induced with oxidative stress (Figure 8A-C). Our results demonstrated that H 2 O 2 incubation significantly increased cytochrome c and Bax levels and decreased Bcl-2 levels compared with the control group. were normalized to the total form bands of p38, ERK1/2, and SAPK/JNK, respectively. Results are presented as mean ± SD values (n = 4). Different superscript letters (a-d) indicate significant differences between values in the column (p < 0.05).

Evaluation of the Effect of Compound 3 on Apoptosis Protein Expression in Oxidative-Stress-Induced ARPE-19 Cells
To understand how compound 3 protects against oxidative stress, we assessed its molecular mechanisms by analyzing the apoptosis pathway. Specifically, we evaluated the levels of downstream targets of the MAPK pathways, such as cytochrome c, Bax (proapoptotic), and Bcl-2 (anti-apoptotic) proteins [58,59]. We determined the expression of cytochrome c, Bax, and Bcl-2 through immunoblotting in ARPE-19 cells induced with oxidative stress ( Figure 8A-C). Our results demonstrated that H2O2 incubation significantly increased cytochrome c and Bax levels and decreased Bcl-2 levels compared with the control group. However, pre-incubating cells with compound 3 at concentrations of 25, 50, and 100 µg/mL for 24 h resulted in a significant dose-dependent decrease in the levels of cytochrome c and Bax proteins, and a significant dose-dependent increase in the level of Bcl-2 protein compared with the H2O2 group ( Figure 8A-C). Thus, in a dose-dependent manner, the protective effect of compound 3 against oxidative-stress-induced cell death is mediated by regulating the cytochrome c, Bax, and Bcl-2 proteins in the apoptosis pathway. However, pre-incubating cells with compound 3 at concentrations of 25, 50, and 100 µg/mL for 24 h resulted in a significant dose-dependent decrease in the levels of cytochrome c and Bax proteins, and a significant dose-dependent increase in the level of Bcl-2 protein compared with the H 2 O 2 group (Figure 8A-C). Thus, in a dose-dependent manner, the protective effect of compound 3 against oxidative-stress-induced cell death is mediated by regulating the cytochrome c, Bax, and Bcl-2 proteins in the apoptosis pathway.

Effect of Compound 3 on Caspase-9 and Caspase-3 Activities in ARPE-19 Cells under Oxidative Stress
To investigate the anti-apoptotic effect of compound 3 on oxidative-stress-induced ARPE-19 cells, we examined its impact on caspase-9 and caspase-3 activities. H 2 O 2 exposure significantly (p < 0.05) increased the activity of both caspases compared with the control group ( Figure 9A,B). However, pre-incubation with compound 3 at 25, 50, and 100 µg/mL for 24 h significantly decreased caspase-9 and caspase-3 activities in a dosedependent manner compared with the H 2 O 2 group ( Figure 9A,B). These findings indicate that compound 3 can mitigate H 2 O 2 -induced apoptosis from oxidative stress by modulating the apoptosis pathway via caspase-9 and caspase-3 activities.

Effect of Compound 3 on Caspase-9 and Caspase-3 Activities in ARPE-19 Cells under Oxidative Stress
To investigate the anti-apoptotic effect of compound 3 on oxidative-stress-induced ARPE-19 cells, we examined its impact on caspase-9 and caspase-3 activities. H2O2 exposure significantly (p < 0.05) increased the activity of both caspases compared with the control group (Figure 9A,B). However, pre-incubation with compound 3 at 25, 50, and 100 µg/mL for 24 h significantly decreased caspase-9 and caspase-3 activities in a dose-dependent manner compared with the H2O2 group ( Figure 9A,B). These findings indicate that compound 3 can mitigate H2O2-induced apoptosis from oxidative stress by modulating the apoptosis pathway via caspase-9 and caspase-3 activities. Figure 9. Effect of compound 3 on caspase-9 and caspase-3 activities in oxidative-stress-induced ARPE-19 cells. Cells were pre-treated with compound 3 at 25, 50 and 100 µ g/mL for 24 h, followed by incubation with H2O2 at 400 µ M for 60 min. The treated cells were homogenized in a hypotonic buffer to obtain the part of the supernatant. The supernatant was determined on (A) caspase-9 and (B) caspase-3 activities. Results are presented as mean ± SD values (n = 4). Different superscript letters (a-e) indicate significant differences between values in the column (p < 0.05).

Evaluation of the Effect of Compound 3 on SOD, CAT, and GPx Activities as well as GSH Levels in ARPE-19 Cells under Oxidative Stress
To determine whether compound 3 modulates enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as the non-enzymatic antioxidant glutathione (GSH), we examined their activities and levels in ARPE-19 cells under oxidative stress. Results showed that H2O2 incubation significantly decreased SOD, CAT, and GPx activities, and GSH levels compared with the control group (p < 0.05) ( Figure 10A-D). However, pre-incubation of cells with compound 3 at concentrations of 25, 50, and 100 µ g/mL for 24 h significantly improved SOD, CAT, and Figure 9. Effect of compound 3 on caspase-9 and caspase-3 activities in oxidative-stress-induced ARPE-19 cells. Cells were pre-treated with compound 3 at 25, 50 and 100 µg/mL for 24 h, followed by incubation with H 2 O 2 at 400 µM for 60 min. The treated cells were homogenized in a hypotonic buffer to obtain the part of the supernatant. The supernatant was determined on (A) caspase-9 and (B) caspase-3 activities. Results are presented as mean ± SD values (n = 4). Different superscript letters (a-e) indicate significant differences between values in the column (p < 0.05).

Evaluation of the Effect of Compound 3 on SOD, CAT, and GPx Activities as well as GSH Levels in ARPE-19 Cells under Oxidative Stress
To determine whether compound 3 modulates enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as the non-enzymatic antioxidant glutathione (GSH), we examined their activities and levels in ARPE-19 cells under oxidative stress. Results showed that H 2 O 2 incubation significantly decreased SOD, CAT, and GPx activities, and GSH levels compared with the control group (p < 0.05) ( Figure 10A-D). However, pre-incubation of cells with compound 3 at concentrations of 25, 50, and 100 µg/mL for 24 h significantly improved SOD, CAT, and GPx activities, and GSH levels in a dose-dependent manner in ARPE-19 cells induced with oxidative stress (Figure 10A-D). Interestingly, incubation with only compound 3 at a concentration of 100 µg/mL significantly increased SOD, CAT, and GPx activities, and GSH levels compared with the control group ( Figure 10A-D). These findings suggest that pre-treatment with compound 3 could enhance the antioxidant system in RPE cells and protect against potential oxidative stress inducers. The findings suggest that prior administration of compound 3 could enhance the antioxidant system of ARPE-19, thus providing protection against potential triggers of oxidative stress.
GPx activities, and GSH levels in a dose-dependent manner in ARPE-19 cells induced with oxidative stress (Figure 10A-D). Interestingly, incubation with only compound 3 at a concentration of 100 µ g/mL significantly increased SOD, CAT, and GPx activities, and GSH levels compared with the control group ( Figure 10A-D). These findings suggest that pretreatment with compound 3 could enhance the antioxidant system in RPE cells and protect against potential oxidative stress inducers. The findings suggest that prior administration of compound 3 could enhance the antioxidant system of ARPE-19, thus providing protection against potential triggers of oxidative stress.

Discussion
Plants in the genus Dendrobium have been traditionally used for medicinal purposes, and one of the benefits of Dendrobium extract is vision improvement [46]. In this study, we initially investigated the protective effects of the MeOH extract from the whole plants of D. virgineum on H2O2-induced oxidative stress in ARPE-19 cells. Chromatographic isolation of this plant led to the isolation of two new compounds, dendrovirginin (compound 1) and dendrovirginone (compound 2), along with seven known compounds (compounds 3-9). The isolated compounds from the plant were subsequently evaluated for their cytotoxicity and protective effects on ARPE-19 cells under oxidative stress. Among the isolated compounds, only compound 3 showed the highest protective effect without inducing toxicity. Therefore, compound 3 could be a potential candidate for treating oxidative-stressrelated eye diseases and was chosen for further protective effect and mechanism evaluation.

Discussion
Plants in the genus Dendrobium have been traditionally used for medicinal purposes, and one of the benefits of Dendrobium extract is vision improvement [46]. In this study, we initially investigated the protective effects of the MeOH extract from the whole plants of D. virgineum on H 2 O 2 -induced oxidative stress in ARPE-19 cells. Chromatographic isolation of this plant led to the isolation of two new compounds, dendrovirginin (compound 1) and dendrovirginone (compound 2), along with seven known compounds (compounds 3-9). The isolated compounds from the plant were subsequently evaluated for their cytotoxicity and protective effects on ARPE-19 cells under oxidative stress. Among the isolated compounds, only compound 3 showed the highest protective effect without inducing toxicity. Therefore, compound 3 could be a potential candidate for treating oxidative-stress-related eye diseases and was chosen for further protective effect and mechanism evaluation.
This study represents the first demonstration of the protective effects of the natural bioactive compound, compound 3, belonging to the dihydrophenanthrene group, isolated from D. virgineum, against oxidative stress in ARPE-19 cells. Compound 3 exhibited its protective effect by modulating the key apoptotic mitogen-activated protein kinases (MAPKs), namely p38, extracellular-signal-regulated kinases 1/2 (ERK1/2 or p44/42), and stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK), as well as the apoptotic signaling pathway, encompassing Bax, Bcl-2, and cytochrome c. Furthermore, compound 3 could protect ARPE-19 under oxidative stress by enhancing the activities of enzymatic antioxidant systems, including SOD, CAT, and GPx, and the non-enzymatic antioxidant GSH. Previous studies have demonstrated that natural bioactive compounds belonging to the dihydrophenanthrene group possess direct antioxidant activities in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and cupric ion reducing antioxidant capacity (CUPRAC) assays [60]. Hence, it is plausible that compound 3 may confer its protective effect on oxidative stress in ARPE-19 cells via direct antioxidant activity. Taken together, these findings emphasize the potential utility of compound 3 as a prophylactic agent for AMD.
Moreover, we found that compound 3 exerts a protective effect via the modulation of phosphorylation of MAPKs signaling, namely p38, ERK1/2, and SAPK/JNK. These MAPKs play essential roles in cellular functions such as apoptosis and proliferation [58,59]. Transient or acute stimulation of this pathway is crucial for normal cell survival, whereas sustained or chronic stimulation can lead to cell death. Such studies show that stimulating these MAPKs can cause downstream expression of apoptotic regulators, including Bax (pro-apoptotic), Bcl-2 (anti-apoptotic), and cytochrome c. In addition, the phosphorylation of MAPKs is strongly related to the promotion of H 2 O 2 -induced cell apoptosis and death in RPE cells [59,61,62]. Hence, assessing the activation of MAPK and the expression of downstream molecules such as Bax, Bcl-2, and cytochrome c provides insights into the mechanism of H 2 O 2 -induced apoptosis in ARPE-19. The treatment of H 2 O 2 led to an increase in the phosphorylation of p38, ERK1/2, and SAPK/JNK in ARPE-19. As a result, there was an increase in Bax and cytochrome c expression, along with a decrease in Bcl-2 expression, indicating that cell death following H 2 O 2 treatment occurred via the apoptotic signaling pathway. Pre-incubation with compound 3 prevented these changes, leading to increased cell viability under oxidative stress by H 2 O 2 . These findings highlight the potential of compound 3 as a preventive agent for oxidative-induced cell dysfunction and death in RPE cells, particularly in the case of AMD.
Additionally, pre-incubation with compound 3 increased the activities of key enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as the levels of the non-enzymatic antioxidant glutathione (GSH). These results suggest that compound 3 can function indirectly as an antioxidant by enhancing the activities and levels of these critical antioxidants. Under oxidative stress conditions induced by incubation with H 2 O 2 , we observed a decrease in the activities of SOD, CAT, and GPx, as well as the levels of GSH. However, pre-incubation with compound 3 improved the activities of these enzymes and the level of GSH in comparison with the cells under oxidative stress. Furthermore, under normal conditions, where the cells were pre-incubated with compound 3 without H 2 O 2 , we observed a significant increase in SOD, CAT, and GPx activities, as well as GSH levels compared with the control group. These results indicate that the pre-treatment of compound 3 could support oxidative stress protection by enhancing the SOD, CAT, and GPx activities, and the levels of GSH. A previous study showed that bioactive compounds from the Dendrobium extract protected HaCaT keratinocytes cells from oxidative stress by activating non-enzymatic and enzymatic antioxidant systems, leading to reduced ROS production [63]. Another study showed that the bioactive compounds in the dihydrophenanthrene group increased enzymatic antioxidant activity in polymorphonuclear leukocytes [64].
The exact mechanisms by which compound 3 influences SOD, CAT, and GPx activities, as well as the GSH level, were not evaluated in this study. However, previous studies have reported that antioxidant compounds can protect RPE cells against oxidative stress by activating the signaling pathway of Akt/Nrf2 [65,66]. This pathway relates the translocation of Nrf2 into the nucleus, which leads to the expression of various non-enzymatic and enzymatic antioxidants. Numerous studies have reported that dihydrophenanthrene compounds, such as compound 3, exert protective effects against oxidative stress by activating the Nrf2 signaling pathway [67,68]. Therefore, it is likely that compound 3 can influence the levels of the non-enzymatic and enzymatic antioxidants through this pathway. Our findings suggest that compound 3 can protect RPE cells from oxidative stress by enhancing the activities of key enzymatic antioxidants, such as SOD, CAT, and GPx, and the level of GSH. Further studies are needed to elucidate the precise mechanisms by which compound 3 influences these antioxidants' activities and levels.

Conclusions
In conclusion, the study shows that compound 3, a dihydrophenanthrene group compound isolated from Dendrobium virgineum, has a protective effect against oxidativestress-induced damage in ARPE-19 cells by modulating key apoptotic signaling pathways and enhancing the activities of enzymatic and non-enzymatic antioxidants ( Figure 11). These findings suggest that compound 3 has potential as a prophylactic agent for AMD and other oxidative-stress-related eye diseases. The study also highlights the importance of evaluating the activation of MAPKs and the apoptosis pathway to understand how oxidative stress induces apoptosis in RPE cells. The discovery of new natural bioactive compounds such as compound 3 provides opportunities for the development of novel therapeutic agents to prevent and treat oxidative-stress-induced diseases.
have reported that antioxidant compounds can protect RPE cells against oxidative stress by activating the signaling pathway of Akt/Nrf2 [65,66]. This pathway relates the translocation of Nrf2 into the nucleus, which leads to the expression of various non-enzymatic and enzymatic antioxidants. Numerous studies have reported that dihydrophenanthrene compounds, such as compound 3, exert protective effects against oxidative stress by activating the Nrf2 signaling pathway [67,68]. Therefore, it is likely that compound 3 can influence the levels of the non-enzymatic and enzymatic antioxidants through this pathway. Our findings suggest that compound 3 can protect RPE cells from oxidative stress by enhancing the activities of key enzymatic antioxidants, such as SOD, CAT, and GPx, and the level of GSH. Further studies are needed to elucidate the precise mechanisms by which compound 3 influences these antioxidants' activities and levels.

Conclusions
In conclusion, the study shows that compound 3, a dihydrophenanthrene group compound isolated from Dendrobium virgineum, has a protective effect against oxidativestress-induced damage in ARPE-19 cells by modulating key apoptotic signaling pathways and enhancing the activities of enzymatic and non-enzymatic antioxidants ( Figure 11). These findings suggest that compound 3 has potential as a prophylactic agent for AMD and other oxidative-stress-related eye diseases. The study also highlights the importance of evaluating the activation of MAPKs and the apoptosis pathway to understand how oxidative stress induces apoptosis in RPE cells. The discovery of new natural bioactive compounds such as compound 3 provides opportunities for the development of novel therapeutic agents to prevent and treat oxidative-stress-induced diseases. Author Contributions: conceptualization, C.M., P.R. and B.S.; data curation, P.P., R.P. and C.M.; formal analysis, P.P., C.M., P.R. and B.S.; investigation, P.P., R.P., P.M., V.K. and W.M.; methodology, P.P., R.P., V.K., C.M., K.L. and B.S.; resources, P.R. and B.S.; supervision, P.R., K.L. and B.S.; writing-original draft, V.K., C.M. and B.S.; writing-review & editing, C.M., P.R. and B.S. All authors have read and agreed to the published version of the manuscript.