Review Reports - Cytotoxic Activity and In Silico Study of Secondary Metabolites Derived from Dactylospongia elegans

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for the Authors

In this article, the authors have explored an intriguing subject that has been somewhat overlooked in recent Cytotoxic activity and in silico Study of Secondary Metabolites

derived from Dactylospongia elegans. Considering the limited availability of such articles, this manuscript holds considerable potential for publication in marinedrugs. However, further polishing is required to meet the marinedrugs journal's standards. With its concise content, this article promises to offer valuable insights to scientists. After undergoing minor revisions, I endorse the publication of this manuscript in marinedrugs where it can contribute significantly to the field.

Page 3, Line 132, authors should mention in the text, what different type of solvents were used in the liquid partition and column chromatography processes for extracting compounds from Dactylospongia elegans?
Page 4, Line 135, need to include the final yields (weights) of the ethyl acetate fraction and the water residue after partitioning?
Page 4, Line 142, what are secondary metabolite classes were identified in the ethyl acetate fraction of Dactylospongia elegans using TLC analysis?
Page 4, Line 152, What bioactive compounds were detected in the LC–MS/MS analysis of the ethyl acetate fraction, and which compound classes do they belong to?
Page 5, Line 152, At what retention times and m/z values were the identified compounds detected in the LC–MS/MS analysis of Dactylospongia elegans?
Page 5, Line 153, what are the identified compounds from the LC–MS/MS analysis have previously been reported from marine organisms, and how are their sources?
Page 7, Line 204, are there morphological changes were observed in MDA-MB-231 cells after treatment with the ethyl acetate fraction and F4 subfraction of Dactylospongia elegans?
Page 7, Line 213, what did the effects of the ethyl acetate fraction compare to cisplatin treatment in terms of cell number and cytotoxic impact and their modification?
Page 11, Line 246, what are the compounds exhibited the strongest binding affinity toward both 1T46 and 4OQ3 targets, and which type of were its binding energies and key interactions?
Page 11, Line 267, what do the binding affinities and interaction profiles of 5-epi-ilimaquinone and Andrastine D compare between the 1T46 and 4OQ3 protein targets and need more supporting data?
Page 12, Line 309, is there any evidence (morphological, spicular, and molecular) was used to confirm the identification of the sample as Dactylospongia elegans?
Page 12, Line 324, what type of secondary metabolite classes were detected in the ethyl acetate fraction using TLC analysis, and which class was not detected?
Page 12, Line 349, what does 5-epi-ilimaquinone contribute to cytotoxic activity against MDA-MB-231 cells based on its chemical likely properties?
Page 13, Line 347, what does the cytotoxic activity of the ethyl acetate fraction of Dactylospongia elegans compare to cisplatin based on IC₅₀ values?
Page 13, Line 374, which type of morphological changes were observed in MDA-MB-231 cells after treatment, and how do these changes indicate about the mechanism of cell death?
Page 13, Line 394, what might be natural product fractions, such as the F4 subfraction, show low cytotoxic potency compared to purified compounds?

Comments for author File: Comments.pdf

Author Response

The authors thank you and reviewer colleagues for your communication in connection with the above manuscript. We have read the evaluation report with great interest and are grateful for your time and courtesy. After consultation with all authors, we decided that our manuscript should be revised in line with the evaluation report and resubmitted.

Please allow us to advise the Reviewers that in the evaluation report, there were issues that we will fully agreed with as well as felt that revisions in line with such comments should significantly benefit the manuscript. All such issues have been addressed in the revised manuscript with additional data. Our response, therefore, includes items that we were happy to address and a minority of cases that we thought were better to be left of the reasons provided.

The manuscript has been thoroughly revised and the affected sections are marked in “blue”. The authors’ responses are briefly itemized below.

Thank you for Reviewer 1, please find our response for your comment below:

Comment 1:

Page 3, Line 132, authors should mention in the text, what different type of solvents were used in the liquid partition and column chromatography processes for extracting compounds from Dactylospongia elegans?

Response 1:

Thank you for the helpful comment. We have revised the text in Page 3 (Line 132) to explicitly specify the solvent systems used during both liquid-liquid partitioning (ethyl acetate–water) and normal-phase column chromatography (n-hexane–ethyl acetate gradient system followed by methanol)

Comment 2:

Need to include the final yields (weights) of the ethyl acetate fraction and the water residue after partitioning?

Response 2:

Thank you for this valuable suggestion. We have revised the manuscript to explicitly present both the final weights and the percentage yields (% w/w) of the ethyl acetate fraction and the aqueous residue after the liquid-liquid partitioning process.

Comment 3:

What secondary metabolite classes were identified in the ethyl acetate fraction of Dactylospongia elegans using TLC analysis?

Response 3:

Thank you for this observation. We have revised the text to clarify the role of each reagent used in the TLC analysis. Sulfuric acid (H₂SO₄) served as a universal detection reagent for organic compounds, while specific reagents were mapped directly to their corresponding secondary metabolite classes

Comment 4:

Page 4, Line 152, What bioactive compounds were detected in the LC–MS/MS analysis of the ethyl acetate fraction, and which compound classes do they belong to?

Response 4:

Thank you for your confirmation. As we already write in Table 1, Bioactive compounds that we detected from LC-MS/MS analysis are Communesin A

which is Alkaloid class, Andrastine D is Meroterpenoid class, Cortisol is

Steroid class, Myricetin is Flavonoid class, 5-epi-ilimaquinone is Sesquiterpene quinone class, and Calciferol is Fat.

Comment 5:

Page 5, Line 152, At what retention times and m/z values were the identified compounds detected in the LC–MS/MS analysis of Dactylospongia elegans?

Response 5:

Thank you again for the confirmation. We already write in Table 1, the first column is the retention times and the next column is m/z value of the identified compounds detected in LC-MS/MS analysis. We also write down it in page 6 line 160-169. We highlight with yellow colour.

“At a retention time of 9.559 min, a compound with a measured mass of 456.2125 [M+H]+ was identified as Communesin A, previously reported from marine algae Enteromorpha intestinalis. Another peak was detected at 11.692 min, corresponding to Andrastine D (m/z 428.3112), a metabolite commonly derived from marine fungi such as Penicillium sp.. Additionally, the chromatogram indicated the presence of Cortisol (m/z 362.2028) at 11.741 min and Myricetin (m/z 319.2848) at 11.776 min, both of which have been previously reported from marine sources. A notable peak at 12.341 min showed a mass of 354.2745 [M+H]+, predicted as 5-epi-illimaquinone, a well-documented metabolite from Dactylospongia elegans (Figure 2a) [11]. Finally, a compound detected at 14.573 min with m/z 396.3203 was identified as Calciferol, a marine algae–derived metabolite (Figure 2b).”

Comment 6:

Page 5, Line 153, what are the identified compounds from the LC–MS/MS analysis have previously been reported from marine organisms, and how are their sources?

Response 6:

Thank you for your thoroughly observation. Although we write down the previously report identified compound source from what kind of marine organism in column 5, we get from only from databank from LC-MS/MS analysis system. We also write it down in the same page 5 line 160-169 as above question. We highlight with yellow colour.

“At a retention time of 9.559 min, a compound with a measured mass of 456.2125 [M+H]+ was identified as Communesin A, previously reported from marine algae Enteromorpha intestinalis. Another peak was detected at 11.692 min, corresponding to Andrastine D (m/z 428.3112), a metabolite commonly derived from marine fungi such as Penicillium sp.. Additionally, the chromatogram indicated the presence of Cortisol (m/z 362.2028) at 11.741 min and Myricetin (m/z 319.2848) at 11.776 min, both of which have been previously reported from marine sources. A notable peak at 12.341 min showed a mass of 354.2745 [M+H]+, predicted as 5-epi-illimaquinone, a well-documented metabolite from Dactylospongia elegans (Figure 2a) [11]. Finally, a compound detected at 14.573 min with m/z 396.3203 was identified as Calciferol, a marine algae–derived metabolite (Figure 2b).”

Comment 7:

Page 7, Line 204, are there morphological changes were observed in MDA-MB

231 cells after treatment with the ethyl acetate fraction and F4 subfraction of

Dactylospongia elegans?

Response 7:

Thank you for your observation in this figures, we change the figures color for make it easier to observed the cells morphology. Also due to late notification regarding the concentration of the F4 subfraction sample, we recalculated the IC₅₀ value and adjusted the photo we show in Figure 3c. Indeed, as shown in Figure 3 there is clear difference between before and after treatment in all groups. As we explain in page 7 line 204-215, untreated MDA-MB-231 cells (Figure 3a) appeared confluent, with a spindle-like shape and intact cell membranes. After treatment with 15.625 µg/ml of ethyl acetate fraction of Dactylospongia elegans (Figure 3b), F4 subfraction of Dactylospongia elegans (Figure 3c) and cisplatin (Figure 3d), the cells showed morphological changes such as blebbing (blue arrow), rounding, detachment, and membrane shrinkage (red arrow), suggesting cytotoxic effects consistent with cell death. The difference between MDA-MB-231 cells treated with the ethyl acetate fraction (Figure 3b) and F4 subfraction of Dactylospongia elegans (Figure 3c) and showed the numbers seems fewer in the ethyl acetate fraction, this phenomenon indicated besides apoptosis, cell cycle arrest also occurred after cells treated with the ethyl acetate fraction of Dactylospongia elegans, while not occurred in F4 subfraction treated MDA-MB-231 cells.

Comment 8:

Page 7, Line 213, what did the effects of the ethyl acetate fraction compare to

cisplatin treatment in terms of cell number and cytotoxic impact and their

modification?

Response 8:

The ethyl acetate fraction appears to have a lower viable cell count (Figure 3b), indicating not only a cytotoxic effect but also a favorable cell cycle. This significantly impacts the IC₅₀value because the cell number is reduced compared to cells treated with cisplatin, which only acts through apoptosis (Figure 3d).

Comment 9:

Page 11, Line 246, what are the compounds exhibited the strongest binding affinity toward both 1T46 and 4OQ3 targets, and which type of were its binding energies and key interactions?

Response 9:

Thank you for this valuable comment. To address this query clearly, we have added a dedicated paragraph summarizing Calciferol as the compound with the strongest binding affinity toward both 1T46 and 4OQ3 target proteins. The added text explicitly outlines its binding energies, inhibition constants (Ki), and the specific interaction types (hydrogen bonds, hydrophobic, and alkyl/$\pi$-alkyl contacts) stabilizing its interaction with key active site residues.

Comment 10:

Page 11, Line 246, what do the binding affinities and interaction profiles of 5-epi-ilimaquinone and Andrastine D compare between the 1T46 and 4OQ3 protein targets and need more supporting data?

Response 10:

Thank you for this comment. We have added a concise comparative summary to the text highlighting that both 5-epi-ilimaquinone and Andrastine D show higher selectivity toward 4OQ3 over 1T46. This enhanced binding potency is driven by richer interaction networks in the 4OQ3 active site, including key H-bonds, $\pi$–sigma contacts, and multiple alkyl interactions.

Comment 11:

Page 12, Line 309, is there any evidence (morphological, spicular, and molecular) was used to confirm the identification of the sample as Dactylospongia elegans?

Response 11:

Thank you for the confirmation. As we write in page 3 line 95-97 and Figure 1b and 1c, we identified the sponge in its underwater condition and the results of its spicule specimens based on the sponge taxonomy guidebook, namely "SYSTEMA PORIFERA: A Guide to the Classification of Sponges" that written by John N. A. Hooper et al. Then we were more confident with the results of DNA barcoding analysis which obtained similarities with previously reported NCBI data that identified as Dactylospongia elegans sponges.

Comment 12:

Page 12, Line 324, what type of secondary metabolite classes were detected in the ethyl acetate fraction using TLC analysis, and which class was not detected?

Response 12:

As stated in the original manuscript, the TLC analysis revealed the presence of terpenoids, steroids, and alkaloids in the ethyl acetate fraction of Dactylospongia elegans, whereas phenolics were not detected.

To make this point crystal clear and avoid any ambiguity, we have revised the text in Page 12 (Line 324) to explicitly state the detected vs. non-detected classes at the beginning of the discussion.

Comment 13:

Page 12, Line 349, what does 5-epi-ilimaquinone contribute to cytotoxic activity against MDA-MB-231 cells based on its chemical likely properties?

Response 13:

Thank you for this insightful question. We have expanded the text in Page 12 (Line 352) to explicitly detail how the chemical properties of 5-epi-illimaquinone drive its cytotoxic effect against MDA-MB-231 cells.

Specifically, we highlighted its lipophilic sesquiterpene backbone, which promotes membrane permeability and cellular uptake, alongside its electrophilic quinonoid core, which acts as a redox-active agent (generating ROS via redox cycling) and an electrophilic Michael acceptor. Together, these chemical features trigger oxidative stress, mitochondrial impairment, and apoptosis in MDA-MB-231 cells.

Comment 14:

Page 13, Line 347, what does the cytotoxic activity of the ethyl acetate fraction of Dactylospongia elegans compare to cisplatin based on IC₅₀ values?

Response 14:

Thank you for your clarification. Based on the IC₅₀value, the cytotoxic activity of the ethyl acetate fraction of Dactylospongia elegans is 15.72 ± 3 µg/mL is more toxic than cisplatin which is 30.99 ± 3 µg/mL. This is because the ethyl acetate fraction of Dactylospongia elegans contains several compounds that work synergistically, resulting in a superior anticancer effect.

Comment 15:

Page 13, Line 374, which type of morphological changes were observed in MDA

MB-231 cells after treatment, and how do these changes indicate about the

mechanism of cell death?

Response 15:

Thank you for your confirmation. The morphological changes that marked mechanism of cell death apoptosis is a reduced number of adherent cells, increased rounding, shrinkage that showed in red arrows and the presence of condensed bodies or blebbing that showed in blue arrows (Figure 3 b-3d).

Comment 16:

Page 13, Line 394, what might be natural product fractions, such as the F4

subfraction, show low cytotoxic potency compared to purified compounds?

Response 16:

Thank you for affirmation.

Based on LC-MS/MS result, the predicted active compounds are Communesin A, Andrastine D, Cortisol, and 5-epi-ilimaquinone, which have anticancer activity.

Furthermore, Myricetin and Calciferol have antioxidant effects and are supplements for cancer prevention.

Subfraction F4 has an IC₅₀ of 41.76 ± 0.5 µg/mL, although has not yet been identified but NMR to confirm which compound it is. Based on previous research, we predicted that the most cytotoxic compound would be 5-epi-ilimaquinone, which might also contribute to cancer cell killing in the ethyl acetate fraction. Although the pure compound has not yet been obtained in our study because the source raw material is limited, but the ethyl acetate fraction of Dactylospongia elegans has been shown to have a stronger cytotoxic effect than the positive control, cisplatin.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors This manuscript investigates the cytotoxic activity and performs anin silico analysis of secondary metabolites derived from Dactylospongia elegans. After careful evaluation, the research topic is within the scope of this journal. However, several essential issues must be thoroughly revised before the manuscript can be accepted for publication. a）According to Table 2 (Line 152) and Figure 2 (Line 157), the authors identified secondary metabolites of Dactylospongia elegans merely based on LC‑MS/MS analysis without reference standard confirmation. The current evidence is insufficient to support the accuracy of compound identification. The authors should provide additional supporting data, such as structural deduction based on MS/MS fragmentation patterns of the detected compounds. b）The chemical structures presented in Figure 2 and Figure 4 are of low resolution, which compromises figure readability. It is recommended that all predicted compound structures be redrawn and compiled into a single high‑definition figure for clear presentation. c）The Discussion section starting at Line 273 is overly redundant and unstructured. To improve logical organization, the authors are suggested to add 2–3 subheadings in the Discussion section and concentrate similar analytical contents under corresponding subsections to streamline the overall narrative.

Author Response

The manuscript has been thoroughly revised and the affected sections are marked in “blue”. The authors’ responses are briefly itemized below.

Thank you for Reviewer 2, please find our response for your comment below:

Reviewer 2

Comment a:

According to Table 2 (Line 152) and Figure 2 (Line 157), the authors identified secondary metabolites of Dactylospongia elegans merely based on LC‑MS/MS analysis without reference standard confirmation. The current evidence is insufficient to support the accuracy of compound identification. The authors should provide additional supporting data, such as structural deduction based on MS/MS fragmentation patterns of the detected compounds.

Response a:

We thank the reviewer for this valuable comment. To strengthen compound identification in the absence of authentic reference standards, we have updated Table 2 by adding experimental MS/MS fragment ions (m/z) and comparing them directly with reference fragment ions (m/z). Specifically for 5-epi-ilimaquinone, as its spectral reference in public databases like MassBank is limited due to the general scarcity of marine natural product spectral libraries, its identification was supported by comparing precursor and available mass spectral data with published literature (Carpi et al, 2022). Accordingly, these metabolites are putatively annotated as MSI Level 2 compounds, as now clarified in the revised manuscript.

Comment b:

The chemical structures presented in Figure 2 and Figure 4 are of low resolution, which compromises figure readability. It is recommended that all predicted compound structures be redrawn and compiled into a single high‑definition figure for clear presentation.

Response b:

We sincerely thank the reviewer for this helpful recommendation. We agree that clear structural representation is essential for readability.

To address this, we have:

Redrawn all predicted chemical structures using dedicated chemical drawing software to ensure standard bond lengths, angles, and crystal-clear structural annotations.
Compiled all structures from the original Figure 2 and Figure 4 into a single, unified high-definition figure (now presented as Figure 2a-d, and Figure 4 in the revised manuscript) with a high resolution of 600 DPI (TIFF format).

Comment c:

The Discussion section starting at Line 273 is overly redundant and unstructured. To improve logical organization, the authors are suggested to add 2–3 subheadings in the Discussion section and concentrate similar analytical contents under corresponding subsections to streamline the overall narrative.

Response c:

Thank you for your valuable suggestions, we've added two sentences to the Discussion section page 12 line 297-300. Hopefully, this makes the narrative clearer.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The author has made revisions to my previous question. I suggest that the journal can accept it.