α-Iso-Cubebene Alleviates AMD-like Retinal Injury Through Modulation of Oxidative Stress and Inflammatory Response

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a robust and multifaceted investigation into the protective effects of a-iso-cubebene against AMD, utilizing both in vitro (ARPE-19 cells) and in vivo (BALB/c mice) models. The study's core strengths lie in its comprehensive analysis of multiple biological pathways which demonstrates clear, dose-dependent responses that align well across the phenotypic and molecular data. Points below should be improved.
1. Spelling Errors spread thought out the manuscript should be corrected
2. The methodology states that the mice received an intravitreal injection of 100 µM a-iso-cubebene, but it completely omits the injection volume
3. While the in vitro experiments appropriately use Vitamin C as a positive control, the animal model lacks a comparable positive control group. Why ?
4. The authors must state their blinding protocols to rule out observer bias
5. The correlation matrix in Figure 8a occupies significant space but offers limited biological insight, as it essentially just restates mathematically that higher doses led to better outcomes across the assays. This figure should be moved to the supplementary materials
6. The discussion notes that a-iso-cubebene activates the Nrf2 pathway but does not hypothesize how it does so at the molecular level. Adding a brief mechanistic hypothesis would elevate the scientific depth of the discussion
7. Ithenticate similarity percent should be reduced by repharasing some paragraphs since its so high in its current form

Author Response

# Reviewer 1

1. Spelling Errors spread thought out the manuscript should be corrected.

☞ We thank the reviewer for this valuable comment. According to your comments, the manuscript has been carefully re-examined in its entirety, and all identified spelling, typographical, and grammatical errors have been corrected throughout the revised version. All corrected words and paragraphs have been indicated as red color.

 

2. The methodology states that the mice received an intravitreal injection of 100 µM a-iso-cubebene, but it completely omits the injection volume.

☞ According to your comments, the correct injection volume has been added as followings (line 270-272);

“After adapting to the dark cages for seven days, all mice in the BL-exposed groups were injected intravitreally with 100 μM of α-iso-cubebene (1 mL) or the same volume of 1× PBS once (1 mL) under the dark condition.”

 

3. While thein vitro experiments appropriately use Vitamin C as a positive control, the animal model lacks a comparable positive control group. Why?

☞ Thank you for your valuable comment. Vitamin C was included as a positive control in the in vitro experiments to validate the antioxidant responsiveness of the ARPE cells with AMD, and to provide a reference for the antioxidant activity of α-iso-cubebene. However, the primary objective of the in vivo study was to evaluate the therapeutic efficacy of α-iso-cubebene itself rather than to compare its effects with those of a known antioxidant. Therefore, a positive control group was not included in the animal experiment. Nevertheless, in order to overcome the issue you presented, we presented the inclusion of a positive control in animal model as a limitation of the study and future as follows (line 625-628);

“Also, another limitation of the present study is the absence of a positive control group in the animal model. Future studies comparing α-iso-cubebene with established antioxidants or approved AMD therapeutics will further clarify its relative therapeutic potential.”

 

4. The authors must state their blinding protocols to rule out observer bias.

☞ According to your comments, we have further described a sentence to solve above issue as followings (line 298-300);

“Also, the thickness analyses were performed by researchers blinded to the injection groups to minimize observer bias.”

 

5. The correlation matrix in Figure 8a occupies significant space but offers limited biological insight, as it essentially just restates mathematically that higher doses led to better outcomes across the assays. This figure should be moved to the supplementary materials

☞ According to your comments, Figure 8a has been reorganized as Supplementary Figure S3.

6. The discussion notes that a-iso-cubebene activates the Nrf2 pathway but does not hypothesize how it does so at the molecular level. Adding a brief mechanistic hypothesis would elevate the scientific depth of the discussion.

☞ According to your comments, we have further described a sentence to solve above issue as followings (line 541-545);

“Although the precise molecular mechanism remains unclear, one possible mechanism is that α-iso-cubebene may promote Nrf2 stabilization and nuclear translocation through modulation of the Keap1–Nrf2 regulatory axis, as reported for several antioxidant phyto-chemicals, thereby enhancing endogenous antioxidant enzymes such as SOD and pro-tecting retinal cells from oxidative injury [50,51].”

 

7. Authenticate similarity percent should be reduced by rephrasing some paragraphs since its so high in its current form.

☞ According to your comments, we have reviewed the manuscript in detail and corrected it carefully to reduce authenticate similarity percent. All corrected words and paragraphs have been indicated as red color.

 

 

# Reviewer 2

1. The authors should moderate several claims regarding AMD treatment or prevention. The current models represent AMD-like/photooxidative retinal injury rather than full AMD pathology. Statements implying direct therapeutic efficacy for AMD should be revised to more cautious wording.

☞ According to your comments, we have reviewed the manuscript in detail and corrected it carefully to solve above issue. The Abstract is presented as an example;

“(1) Background: Although oxidative stress plays a critical role in age-related macular de-generation (AMD) progression, natural product–derived single compounds against AMD remain largely unexplored. We investigated the protective effects and underlying mechanism of α-iso-cubebene against AMD-like retinal injury; (2) Methods: Alterations on key phenotypes for AMD were analyzed in AMD-mimicking models using ARPE-19 cells co-treated with blue light (BL) and N-retinylidene-N-retinylethanolamine (A2E), as well as BL-exposed BALB/c mice; (3) Results: In BL+A2E-treated ARPE-19 cells, α-iso-cubebene reduced intracellular reactive oxygen species (ROS) and nitric oxide (NO) production as well as restored superoxide dismutase (SOD) activity and nuclear factor erythroid 2–related factor 2 (Nrf2), indicating activation of the antioxidant defense system. Further-more, α-iso-cubebene improved cell viability, reduced apoptotic cell populations, and regulated apoptosis-related signaling pathways under oxidative stress conditions. It also attenuated cyclooxygenase-2 (COX-2)-mediated inducible nitric oxide synthase (iNOS) signaling, and inhibited inflammasome activation. Importantly, these protective effects were consistently observed on the protection of histopathological structure and normalization of inflammatory cytokines in the retina of BL-exposed BALB/c mice; (4) Conclusions: Collectively, our results demonstrate that α-iso-cubebene as a promising therapeutic candidate alleviates AMD-like retinal injury through activation of the Nrf2-mediated antioxidant pathway and suppression of inflammatory and apoptotic signaling.”

 

2. The in vivo dosing information needs clarification. Please specify the intravitreal injection volume, the actual amount delivered per eye, the vehicle/solubility conditions, and the rationale for using 100 µM α-iso-cubebene. The limitation of using only one dose and a short 24 h treatment should also be acknowledged.

☞ According to your comments, we have corrected the manuscript to solve above issue as followings;

(line 270-277)

“After adapting to the dark cages for seven days, all mice in the BL-exposed groups were injected intravitreally with 100 μM of α-iso-cubebene (1 mL) or the same volume of 1× PBS once (1 mL) under the dark condition. The optimal dosage of α-iso-cubebene including injection volume, the actual amount delivered per eye, the vehicle/solubility conditions were firstly determined based on previous studies because in vivo exposure was influenced by systemic distribution, tissue-specific uptake and rapid clearance [37,38]. However, this injection method had the limitation of using a single dose and a short treatment duration (24 h).”

 

3. The conclusion that α-iso-cubebene “activates Nrf2-mediated antioxidant signaling” should be better supported or phrased more cautiously. If available, data on nuclear Nrf2 translocation or downstream targets such as HO-1/NQO1 would strengthen this point. Otherwise, the authors should state that α-iso-cubebene was associated with increased Nrf2 expression.

☞ According to your comments, we have further described a sentence to solve above issue as followings (line 541-545);

“Although the precise molecular mechanism remains unclear, one possible mechanism is that α-iso-cubebene may promote Nrf2 stabilization and nuclear translocation through modulation of the Keap1–Nrf2 regulatory axis, as reported for several antioxidant phytochemicals, thereby enhancing endogenous antioxidant enzymes such as SOD and protecting retinal cells from oxidative injury [50,51].”

 

4. Please clarify the number of biological replicates versus technical replicates in each experiment, especially for the animal qPCR and histological analyses. The manuscript should also state whether retinal thickness measurements were performed in a randomized and blinded manner.

☞ According to your comments, we have further described a sentence to solve above issue as followings:

(line 298-300)

“Also, the thickness analyses were performed by researchers blinded to the injection groups to minimize observer bias.”

(line 474-479)

“(a) Experimental schedule for BALB/c mice. (b) Representative H&E-stained retinal sections. The preparation of stained eye section was performed using three to five mice per group, and measurements of thickness were conducted twice per section. (c) Transcriptional levels of inflammatory cytokine genes. The preparation of total RNA sample was performed using two to three mice per group, and measurements of mRNA level was conducted twice per sample.”

 

5. The anti-angiogenic/neovascularization claims should be toned down. VEGF, MMP2, and MMP9 expression changes in ARPE-19 cells are indirect evidence and do not by themselves demonstrate suppression of angiogenesis or neovascularization.

☞ According to your comments, we have corrected few related sentences to solve above issue as followings (line 560-563):

“Although additional functional studies are required, the present findings provide preliminary evidence supporting the potential utility of α-iso-cubebene in suppressing angio-genesis-associated processes relevant to neovascular AMD.”

 

6. Examples include “BLAB/c” instead of BALB/c, “leutein” instead of lutein, several grammatical errors, and the ABTS abbreviation in the Figure 1 legend?? The manuscript requires careful language and formatting revision.

☞ We thank the reviewer for this valuable comment. According to your comments, the manuscript has been carefully re-examined in its entirety, and all identified spelling, typographical, and grammatical errors have been corrected throughout the revised version. All corrected words and paragraphs have been indicated as red color.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript reports the protective effects of α-iso-cubebene against blue light/A2E-induced retinal pigment epithelial injury and blue light-induced retinal damage in mice. Overall, the study is interesting and the data are generally consistent, showing reduced oxidative stress, inflammation, apoptosis-related signaling, and retinal structural damage. The topic is relevant to AMD-related retinal injury and the manuscript may be suitable for publication, however, this reviewer has several concern and suggestions as follows.

1. The authors should moderate several claims regarding AMD treatment or prevention. The current models represent AMD-like/photooxidative retinal injury rather than full AMD pathology. Statements implying direct therapeutic efficacy for AMD should be revised to more cautious wording.

2. The in vivo dosing information needs clarification. Please specify the intravitreal injection volume, the actual amount delivered per eye, the vehicle/solubility conditions, and the rationale for using 100 µM α-iso-cubebene. The limitation of using only one dose and a short 24 h treatment should also be acknowledged.

3. The conclusion that α-iso-cubebene “activates Nrf2-mediated antioxidant signaling” should be better supported or phrased more cautiously. If available, data on nuclear Nrf2 translocation or downstream targets such as HO-1/NQO1 would strengthen this point. Otherwise, the authors should state that α-iso-cubebene was associated with increased Nrf2 expression.

4. Please clarify the number of biological replicates versus technical replicates in each experiment, especially for the animal qPCR and histological analyses. The manuscript should also state whether retinal thickness measurements were performed in a randomized and blinded manner.

5. The anti-angiogenic/neovascularization claims should be toned down. VEGF, MMP2, and MMP9 expression changes in ARPE-19 cells are indirect evidence and do not by themselves demonstrate suppression of angiogenesis or neovascularization.

6. Examples include “BLAB/c” instead of BALB/c, “leutein” instead of lutein, several grammatical errors, and the ABTS abbreviation in the Figure 1 legend?? The manuscript requires careful language and formatting revision. 

Author Response

# Reviewer 1

1. Spelling Errors spread thought out the manuscript should be corrected.

☞ We thank the reviewer for this valuable comment. According to your comments, the manuscript has been carefully re-examined in its entirety, and all identified spelling, typographical, and grammatical errors have been corrected throughout the revised version. All corrected words and paragraphs have been indicated as red color.

 

2. The methodology states that the mice received an intravitreal injection of 100 µM a-iso-cubebene, but it completely omits the injection volume.

☞ According to your comments, the correct injection volume has been added as followings (line 270-272);

“After adapting to the dark cages for seven days, all mice in the BL-exposed groups were injected intravitreally with 100 μM of α-iso-cubebene (1 mL) or the same volume of 1× PBS once (1 mL) under the dark condition.”

 

3. While thein vitro experiments appropriately use Vitamin C as a positive control, the animal model lacks a comparable positive control group. Why?

☞ Thank you for your valuable comment. Vitamin C was included as a positive control in the in vitro experiments to validate the antioxidant responsiveness of the ARPE cells with AMD, and to provide a reference for the antioxidant activity of α-iso-cubebene. However, the primary objective of the in vivo study was to evaluate the therapeutic efficacy of α-iso-cubebene itself rather than to compare its effects with those of a known antioxidant. Therefore, a positive control group was not included in the animal experiment. Nevertheless, in order to overcome the issue you presented, we presented the inclusion of a positive control in animal model as a limitation of the study and future as follows (line 625-628);

“Also, another limitation of the present study is the absence of a positive control group in the animal model. Future studies comparing α-iso-cubebene with established antioxidants or approved AMD therapeutics will further clarify its relative therapeutic potential.”

 

4. The authors must state their blinding protocols to rule out observer bias.

☞ According to your comments, we have further described a sentence to solve above issue as followings (line 298-300);

“Also, the thickness analyses were performed by researchers blinded to the injection groups to minimize observer bias.”

 

5. The correlation matrix in Figure 8a occupies significant space but offers limited biological insight, as it essentially just restates mathematically that higher doses led to better outcomes across the assays. This figure should be moved to the supplementary materials

☞ According to your comments, Figure 8a has been reorganized as Supplementary Figure S3.

6. The discussion notes that a-iso-cubebene activates the Nrf2 pathway but does not hypothesize how it does so at the molecular level. Adding a brief mechanistic hypothesis would elevate the scientific depth of the discussion.

☞ According to your comments, we have further described a sentence to solve above issue as followings (line 541-545);

“Although the precise molecular mechanism remains unclear, one possible mechanism is that α-iso-cubebene may promote Nrf2 stabilization and nuclear translocation through modulation of the Keap1–Nrf2 regulatory axis, as reported for several antioxidant phyto-chemicals, thereby enhancing endogenous antioxidant enzymes such as SOD and pro-tecting retinal cells from oxidative injury [50,51].”

 

7. Authenticate similarity percent should be reduced by rephrasing some paragraphs since its so high in its current form.

☞ According to your comments, we have reviewed the manuscript in detail and corrected it carefully to reduce authenticate similarity percent. All corrected words and paragraphs have been indicated as red color.

 

 

# Reviewer 2

1. The authors should moderate several claims regarding AMD treatment or prevention. The current models represent AMD-like/photooxidative retinal injury rather than full AMD pathology. Statements implying direct therapeutic efficacy for AMD should be revised to more cautious wording.

☞ According to your comments, we have reviewed the manuscript in detail and corrected it carefully to solve above issue. The Abstract is presented as an example;

“(1) Background: Although oxidative stress plays a critical role in age-related macular de-generation (AMD) progression, natural product–derived single compounds against AMD remain largely unexplored. We investigated the protective effects and underlying mechanism of α-iso-cubebene against AMD-like retinal injury; (2) Methods: Alterations on key phenotypes for AMD were analyzed in AMD-mimicking models using ARPE-19 cells co-treated with blue light (BL) and N-retinylidene-N-retinylethanolamine (A2E), as well as BL-exposed BALB/c mice; (3) Results: In BL+A2E-treated ARPE-19 cells, α-iso-cubebene reduced intracellular reactive oxygen species (ROS) and nitric oxide (NO) production as well as restored superoxide dismutase (SOD) activity and nuclear factor erythroid 2–related factor 2 (Nrf2), indicating activation of the antioxidant defense system. Further-more, α-iso-cubebene improved cell viability, reduced apoptotic cell populations, and regulated apoptosis-related signaling pathways under oxidative stress conditions. It also attenuated cyclooxygenase-2 (COX-2)-mediated inducible nitric oxide synthase (iNOS) signaling, and inhibited inflammasome activation. Importantly, these protective effects were consistently observed on the protection of histopathological structure and normalization of inflammatory cytokines in the retina of BL-exposed BALB/c mice; (4) Conclusions: Collectively, our results demonstrate that α-iso-cubebene as a promising therapeutic candidate alleviates AMD-like retinal injury through activation of the Nrf2-mediated antioxidant pathway and suppression of inflammatory and apoptotic signaling.”

 

2. The in vivo dosing information needs clarification. Please specify the intravitreal injection volume, the actual amount delivered per eye, the vehicle/solubility conditions, and the rationale for using 100 µM α-iso-cubebene. The limitation of using only one dose and a short 24 h treatment should also be acknowledged.

☞ According to your comments, we have corrected the manuscript to solve above issue as followings;

(line 270-277)

“After adapting to the dark cages for seven days, all mice in the BL-exposed groups were injected intravitreally with 100 μM of α-iso-cubebene (1 mL) or the same volume of 1× PBS once (1 mL) under the dark condition. The optimal dosage of α-iso-cubebene including injection volume, the actual amount delivered per eye, the vehicle/solubility conditions were firstly determined based on previous studies because in vivo exposure was influenced by systemic distribution, tissue-specific uptake and rapid clearance [37,38]. However, this injection method had the limitation of using a single dose and a short treatment duration (24 h).”

 

3. The conclusion that α-iso-cubebene “activates Nrf2-mediated antioxidant signaling” should be better supported or phrased more cautiously. If available, data on nuclear Nrf2 translocation or downstream targets such as HO-1/NQO1 would strengthen this point. Otherwise, the authors should state that α-iso-cubebene was associated with increased Nrf2 expression.

☞ According to your comments, we have further described a sentence to solve above issue as followings (line 541-545);

“Although the precise molecular mechanism remains unclear, one possible mechanism is that α-iso-cubebene may promote Nrf2 stabilization and nuclear translocation through modulation of the Keap1–Nrf2 regulatory axis, as reported for several antioxidant phytochemicals, thereby enhancing endogenous antioxidant enzymes such as SOD and protecting retinal cells from oxidative injury [50,51].”

 

4. Please clarify the number of biological replicates versus technical replicates in each experiment, especially for the animal qPCR and histological analyses. The manuscript should also state whether retinal thickness measurements were performed in a randomized and blinded manner.

☞ According to your comments, we have further described a sentence to solve above issue as followings:

(line 298-300)

“Also, the thickness analyses were performed by researchers blinded to the injection groups to minimize observer bias.”

(line 474-479)

“(a) Experimental schedule for BALB/c mice. (b) Representative H&E-stained retinal sections. The preparation of stained eye section was performed using three to five mice per group, and measurements of thickness were conducted twice per section. (c) Transcriptional levels of inflammatory cytokine genes. The preparation of total RNA sample was performed using two to three mice per group, and measurements of mRNA level was conducted twice per sample.”

 

5. The anti-angiogenic/neovascularization claims should be toned down. VEGF, MMP2, and MMP9 expression changes in ARPE-19 cells are indirect evidence and do not by themselves demonstrate suppression of angiogenesis or neovascularization.

☞ According to your comments, we have corrected few related sentences to solve above issue as followings (line 560-563):

“Although additional functional studies are required, the present findings provide preliminary evidence supporting the potential utility of α-iso-cubebene in suppressing angio-genesis-associated processes relevant to neovascular AMD.”

 

6. Examples include “BLAB/c” instead of BALB/c, “leutein” instead of lutein, several grammatical errors, and the ABTS abbreviation in the Figure 1 legend?? The manuscript requires careful language and formatting revision.

☞ We thank the reviewer for this valuable comment. According to your comments, the manuscript has been carefully re-examined in its entirety, and all identified spelling, typographical, and grammatical errors have been corrected throughout the revised version. All corrected words and paragraphs have been indicated as red color.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, to enhance the scientific impact of your manuscript and facilitate the peer-review process without requiring additional laboratory experiments, i recommend implementing several strategic textual revisions. 
First, since the findings regarding Nrf2 activation and inflammasome suppression are not corroborated by mechanistic tests such as gene knockdown, i suggest toning down the definitive causal statements in the Discussion and Conclusions. It would be more appropriate to use cautious language, indicating that the observed effects "may be associated with" or "partially mediated by" these pathways. 
Second, to address the limitations in the animal model, the rationale behind selecting the single 100 µM dose and the short 24-hour evaluation period should be clearly justified with references to previous studies, framing this design explicitly as a proof-of-concept approach. 
Third, to compensate for the lack of an in vivo positive control, i recommend theoretically comparing the protective effects of alpha-iso-cubebene on retinal layers with the outcomes of other agents tested in similar models in the current literature. 
Fourth, grouping the lack of pharmacokinetic/pharmacodynamic data and the absence of a positive control under Study Limitations subheading will transparently position this research as a preliminary study, thereby strengthening the manuscript's academic credibility.

Author Response

# Reviewer 1

1.First, since the findings regarding Nrf2 activation and inflammasome suppression are not corroborated by mechanistic tests such as gene knockdown, i suggest toning down the definitive causal statements in the Discussion and Conclusions. It would be more appropriate to use cautious language, indicating that the observed effects "may be associated with" or "partially mediated by" these pathways.

☞ According to your comments, we have corrected some related sentences in Discussion and Conclusion as followings;

(line 526-535)

“These effects may be partially mediated, at least in part, by direct free radical scavenging and by enhancement of endogenous antioxidant defenses through activation of the Nrf2 signaling pathway. Taken as a whole, the current data support the possibility that α-iso-cubebene may be associated with the reduction of oxidative retinal injury by simultaneously modulating several antioxidant and cytoprotective pathways. Although the precise molecular mechanism remains unclear, it is conceivable that α-iso-cubebene may influence the Keap1–Nrf2 regulatory axis, as reported for several antioxidant phytochemicals. However, further mechanistic studies are required to determine whether Nrf2 stabilization and nuclear translocation contribute to the observed increase in antioxidant defenses and protection against oxidative retinal injury [50,51].”

(line 617-624)

“α-Iso-cubebene effectively protected ARPE-19 cells against A2E and BL co-treatment, and was associated with reduced reactive oxygen species accumulation, enhanced cellular antioxidant defenses, and alterations in angiogenic, inflammatory, and apoptotic signaling markers. Furthermore, α-iso-cubebene alleviated retinal structural disorganization and reduced inflammatory cytokine expression in BL-exposed BALB/c mice (Figure 8). Therefore, the present findings suggest that α-iso-cubebene may represent a promising natural product-derived single compound for the prevention and management of AMD.”

2. Second, to address the limitations in the animal model, the rationale behind selecting the single 100 µM dose and the short 24-hour evaluation period should be clearly justified with references to previous studies, framing this design explicitly as a proof-of-concept approach.

☞ According to your comments, we have corrected some-related sentences in Materials and methods as followings (line 270-278);

“The dose of α-iso-cubebene (100 μM) and the evaluation period for 24 h were selected based on previous studies employing acute BL-induced retinal injury models and intravitreal administration of antioxidant compounds [37,38]. As the primary objective of this study was to provide proof-of-concept evidence for the protective effects of α-iso-cubebene against AMD-like retinal injury, a single-dose design with short-term evaluation was adopted. Nevertheless, we acknowledge that additional studies investigating dose–response relationships and long-term therapeutic effects are required to further verify its efficacy and molecular mechanism.”

 

3. Third, to compensate for the lack of an in vivo positive control, i recommend theoretically comparing the protective effects of alpha-iso-cubebene on retinal layers with the outcomes of other agents tested in similar models in the current literature.

☞ According to your comments, we have corrected some-related sentences in Discussion as followings (line 590-597);

“Although direct comparisons should be interpreted with caution because of differences in experimental models and treatment protocols, the degree of retinal structural preservation observed following α-iso-cubebene treatment appears to be broadly consistent with the protective effects reported for other natural compounds, including resveratrol, quercetin, and curcumin, in similar retinal degeneration models [22,24]. In particular, α-iso-cubebene effectively preserved the thickness of multiple retinal layers and attenuated retinal disorganization, supporting its potential efficacy within the range of responses previously reported for established antioxidant compounds.”

 

4. Fourth, grouping the lack of pharmacokinetic/pharmacodynamic data and the absence of a positive control under Study Limitations subheading will transparently position this research as a preliminary study, thereby strengthening the manuscript's academic credibility.

☞  According to your comments, we have corrected some-related sentences in Discussion as followings (line 624-635);

“Despite these encouraging findings, the present study should be considered a proof-of-concept investigation and has several limitations. First, only a single dose of α-iso-cubebene and a relatively short evaluation period (24 h) were examined in the ani-mal model. Second, in vivo study, a positive control was not included, limiting direct comparisons with established retinal protective agents. Third, pharmacokinetic and pharmacodynamic analyses were not performed, restricting a comprehensive assessment of the biological relevance, tissue exposure, and therapeutic efficacy of α-iso-cubebene under physiological conditions. Therefore, future studies are required to investigate dose–response relationships, long-term efficacy, pharmacokinetic properties, bioavailability, and safety profiles. Such investigations will be essential for validating the therapeutic potential of α-iso-cubebene and facilitating its translation toward clinical applications for AMD and related retinal degenerative disorders.”