Unraveling Potential Compounds of Uncaria gambir (W.Hunter) Roxb. as Antikeloid Agent: In Silico, In Vitro and Ex Vivo Experimental Validation

Response to Reviewer 1 Comments

1. Summary

Thank you very much for your feedback on this manuscript. We appreciate the time you have dedicated to reviewing our work. Please find the detailed responses below and the corresponding revisions in track changes in the resubmitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Is the research design appropriate?

Must be improved

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Must be improved

Are the conclusions supported by the results?

Must be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: How did hexane show the same phenolic content as in the ethanol fraction, although hexane is non polar solvent?

Response 1: Thank you for your insightful comment. We suggest that the presence of phenol in the hexane fraction might be due to lipophilic phenolic derivatives or emulsion formation during partitioning process. This study employed liquid-liquid fractionation which practically has limited selectivity in separating compounds strictly based on polarity. As a result, overlapping distribution of active compounds across different solvents may have occurred. We respectfully acknowledge this as the limitation of this study and suggest that future studies consider employing advanced separation techniques which may offer greater resolution and improved effectiveness in isolating specific bioactive compounds. This statement and its relevant citation have been added in the discussion section in the revised manuscript (line 347-349 and line 397-400)

Comments 2: The LC/MS chromatogram showed the presence of many compounds which the authors did not reveal?

Response 2: Thank you for your valuable comment. The full chromatogram and the presence of compounds have been provided in the supplementary materials.

Comments 3: In Table 3. The binding energy values have been depicted with a comma?

Response 3: Thank you for pointing this out. The binding energy values in Table 3 are currently presented using commas as decimal separators, which may cause confusion. We will revise the table to use periods as decimal points in accordance with standard scientific notation.

Comments 4: Figure 2. must accompany molecular docking visualization of all the compounds for more clarity and authenticity.

Response 4: Thank you for your valuable comment. We agree to include molecular docking visualizations for all compounds. Accordingly, we have updated Figure 2 in the revised manuscript.

Comments 5: The authors performed docking simulations with TGFβ1 and -Akt as the presumed target. However, no evidence has been provided to confirm that these selected proteins are indeed the target of the tested compounds.

Response 5: Thank you for your insightful comment. TGFβ1 and Akt were selected as the presumed target regarding to those proteins were belonging to the key targets with the highest interaction scores (based on our previous study) and relying to the same signaling pathway which is the non-canonical TGFβ pathway. This statement has been clarified in the result section (line 274-277).

Comments 6: In Table 3. What is a native ligand? And which standard reference has been used for the comparison?

Response 6: Thank you for pointing this out, the native ligand in Table 3 refers to the co-crystalized ligand originally bound to the target proteins structure as obtained from the Protein Data Bank (PDB). It serves a benchmark to validate the docking protocol and to compare the binding affinities of the test compounds. We have clarified this point by including the relevant PDB IDs and native ligand names in the result section (line 280-282).

Comments 7: The authors have mostly discussed their results with general statements and are not clear.

Response 7: Thank you for your valuable feedback. We have carefully revised the discussion to enhance clarity. We hope these improvements address your concern.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for your feedback on this manuscript. We appreciate the time you have dedicated to reviewing our work. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Can be improved

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Inconsistent Terminology and Grammar. [64] Write “and is widely found in West Sumatera”.

Response 1: Thank you for your comment, we have revised the sentence as your suggestion.

Comments 2: Although Gambir’s chemical groups (flavonoids, alkaloids, phenolics) are mentioned, no quantitative or specific compositional analysis is summarized.

Response 2: Thank you for your valuable comment. We have conducted LCMS analysis to identify the chemical constituents present in the Gambir extract and fractions. We have added the summary of the result to the supplementary materials of this manuscript.

Comments 3: Provide a table or paragraph summarizing Gambir’s major phytochemicals and known effects.  

Response 3: Thank you for your insightful comment. We have added the explanation in paragraph 2 of introduction section (line 65-76)

Comments 4: Add a schematic illustrating proposed mechanisms of Gambir action against keloid formation.

Response 4: Thank you for your comment. While we agree that schematic illustration of proposed mechanisms would enhance clarity, we believe that such a diagram would be more appropriately presented following a more comprehensive analysis of the key target proteins involved in targeted signaling pathway. Therefore, we plan to construct the proposed mechanism diagram in our subsequent manuscript after finishing the protein expression analysis. We have added a brief statement in the discussion section (line 395-397) to acknowledge this limitation and to outline our intention for future work.

Comments 5: “Dry rubber powder" should be replaced with "dried plant powder".

Response 5: Agree, we have replaced the words as your comment.

Comments 6: [108] Write "extracted using 96% ethanol."

Response 6: Thank you for your comment, we have revised the sentence as your comment (line 114).

Comments 7: Provide ratio of sample to solvent.

Response 7: Agree, we have added the ratio of sample to solvent as your comment (line 114).

Comments 8: Fractionation procedure is superficially described.

Response 8: Thank you for your comment. Agree, we have revised the fractionation procedure (line 116-123)

Comments 9: For Folin–Ciocalteu, 760 or 765 nm is standard, not 725 nm as stated in the text.

Response 9: Thank you for pointing this out, we have revised the wavelength from 725 to 765 (line 132) in the method section to align with the standard protocol for Folin-Ciocalteu assay.

Comments 10: For flavonoid assays (AlCl₃ method), 415 nm is the standard wavelength used, 376 nm seems unusually low unless a different method was used, if used kindly mention that.

Response 10: Thank you for your comment. While 415 nm is commonly used for flavonoid assay, the absorbance in our study was measured at 376nm based on the maximum absorbance of the quercetin-AlCl₃ complex under our experimental conditions. We have added a citation to support the chosen wavelength (line 132).

Comments 11: Total run time (min) for one sample analysis is missing.

Response 11: Thank you for pointing this out, we have added the total run time in the revised manuscript (line 141)

Comments 12: A cone flow rate of 0 L/h suggests either an error in writing or an atypical setup that needs justification.

Response 12: Thank you for your comment. The use of 0 L/h cone gas flow rate was based on the method reported by Ismed et al, 2021. We have cited the reference (line 145-147)

Comments 13: [136] "SMILES" (Simplified Molecular Input Line Entry System).

Response 13: Thank you for your comment, the term has been revised as your comment (line 149).

Comments 14: Incubation with CCK-8 solution for "2–4 hours" is too broad. Needs to be fixed at a specific time for consistency.

Response 14: Thank you for your comment, we have been revised the time of incubation (l. 201).

Comments 15: No positive cytotoxic control (DMSO, H₂O₂) is mentioned.

Response 15: Thank you for your valuable comment. In this study, DMSO was employed as the solvent for Gambir extract and fractions. We optimized that the DMSO concentration remained below the toxicity level, confirming that any observed effects were attributed to the treatment, not the solvent. We have clarified this in the method section (line 192-194).

Comments 16: What constitutes the "complete media"?

Response 16: Thank you for your valuable comment. Complete media was made from DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic, as stated in our previous study (line 194-195).

Source: Ningsih SS. Efficient protocol for isolating human fibroblast from primary skin cell cultures: application to keloid, hypertrophic scar, and normal skin biopsies. 2024;9(September).

Comments 17: No cytotoxic positive control was included to validate assay performance.

Response 17: Thank you for your insightful feedback. In this study, positive control was not included due to the currently limited understanding of Gambir’s mechanism of action in keloid. Therefore, it is not feasible to select positive control with a comparable mode of action. Accordingly, the effects of treatments were assessed relative to untreated keloid tissue explant as negative control to establish a baseline response. The inclusion of such a control will be considered in future studies once the exact mechanism action of Gambir to keloid is identified and validated. This clarification has been added to Materials and Methods section for transparency (line 223-227).

Comments 18: Key findings are repeated multiple times.

Response 18: Thank you for your valuable feedback. We have revised the manuscript to eliminate unnecessary redundancy of the key findings.

Response to Reviewer 3 Comments

1. Summary

Thank you very much for your feedback on this manuscript. We appreciate the time you have dedicated to reviewing our work. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Is the research design appropriate?

Must be improved

Are the methods adequately described?

Yes

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: 1) The entire manuscripts be check for grammar errors and mistakes

Response 1: Thank you for your comment. We thoroughly read and revised the manuscript. We hope the updated manuscript now meets the journal’s standard.

Comments 2: Introduction; Limited information on the previous research done on this plant material, secondary metabolites and bioactivities.

Response 2: Thank you for your valuable comment. In response, we have revised the introduction section and relevant references have been added to strengthen the scientific basis of our study. (line 65-76)

Comments 3: Materials and Methods; check line 141-142 for errors/reference, for chemical compounds, use IUPAC naming conventions, use superscripts and/or subscripts appropriately.

Response 3: Thank you for your feedback. We have checked line 141-142 and confirmed that it is a reference citation, which has been revised as the journal format (line 155).

Comments 4: Materials and Methods; check line 187-189, the % cell viability formular or SI formular was not defined in the text.

Response 4: Thank you for your comment. We have defined and clarified the % viability and SI formula in the revised manuscript (line 202-205 and line 211-215)

Comments 5: Materials and Methods; for the ex-vivo analysis, which compound, or drug was used as positive or negative control? N.B. not talking about the NC for fluorescence background purpose.

Response 5: Thank you for your insightful feedback. In this study, positive control was not included due to the currently limited understanding of Gambir’s mechanism of action in keloid. Therefore, it is not feasible to select positive control with a comparable mode of action. Accordingly, the effects of treatments were assessed relative to untreated keloid tissue explant as negative control to establish a baseline response. The inclusion of such a control will be considered in future studies once the exact mechanism action of Gambir to keloid is identified and validated. This clarification has been added to Materials and Methods section for transparency (line 223-227).

Comments 6: Result; What was the percentage yield of each fraction or extracts as mentioned in line 112-112 of the material and method section.

Response 6: Thank you for your comment. The percentage yield of each extract/fraction has now been calculated and included in the Materials and Methods section (line 122-125) and result section (Table 1, line 253)

Comments 7: Result; line 222 to 224, talks about this study and some compounds identified and then refenced (8). in line 225 for a previous study, please clarify your report for better understanding.

Response 7: Thank you for your valuable feedback. We appreciate your attention to this point. The original sentence may have lacked clarity in distinguishing between the findings of the present study and the supporting information cited from reference (8). We have revised the sentence to ensure that it more clearly indicates which compounds were identified in this study and which are supported by previous literature. This clarification has been incorporated into the Results section to enhance the readability (Line 248-252).

Comments 8: Result; IUPAC, subscripts be respected.

Response 8: Thank you for your careful observation. We have carefully reviewed the chemical names and formulas presented in the Results section to ensure they follow IUPAC nomenclature (Table 2).

Comments 10: Result; Figure 1 is not clear and maybe not so important, because table 2 alone summarizes figure 1 with additional details.

Response 10: Thank you for your thoughtful comment. We appreciate your observation regarding Figure 1. Upon review, we agree to Figure 1 to improve clarity and avoid redundancy. While Table 2 provides a detailed summary, we believe that Figure 1 remains important for visually representing the overall profile of the compounds. To improve clarity and streamline the main manuscript, Figure 1 and the detailed compound information have been moved to the supplementary materials. The manuscript text has been revised accordingly to maintain coherence and focus (line 247 and supplementary).

Comments 11: Result: Figure 5. E, how or what tool and method was use for the cell number determination.

Response 11: Cells were stained with DAPI (Abcam, ab104139) on day 6 post-treatment. The fluorescence image was acquired using inverted fluorescence microscope (ZOE Fluorescent Cell Imager-Bio-Rad) under standardized settings. Cell quantification was performed by analyzing the images with ImageJ apps, which allowed for accurate counting of DAPI-stained nuclei to estimate cell numbers. This procedure has been detailed in the Materials and Methods section for clarity (line 231-234).

Comments 12: Discussion and conclusion; The discussion looks great, and the conclusion well summarized.

Response 12: We sincerely appreciate your positive feedback. It is encouraging to know that the discussion and conclusion sections meet your expectations.

Comments 13: Comments on the Quality of English Language; Entire manuscripts be check for grammar errors and mistakes

Response 13: Thank you for your helpful suggestion. We have carefully reviewed and professionally proofread the manuscript to improve grammar and language clarity.