Gastroprotective Effects of Tordylium trachycarpum Extract Against Ethanol-Induced Gastric Injury: Involvement of Antioxidant, Anti-Inflammatory, and Anti-Apoptotic Mechanisms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses an interesting topic and presents biologically plausible findings; however, it could be further strengthened in several aspects.

The abstract could better emphasize the innovativ nature of the study and include some key quantitative findings.

The introduction is well structured, but the literature gap should be defined more critically, also avoiding overly strong statements regarding pathways that were not directly investigated.

The results section is clear, although in some parts it remains too descriptive. It would be useful to provide a deeper interpretation of the findings, particularly in comparison with omeprazole, and to verify some possible inconsistencies in the parameters related to gastric mucus production. In addition, the figures could be made more readable and less overloaded with statistical annotations.

I did not understand why the Methods section is placed after the Discussion. In any case, it would be useful to better clarify randomization procedures, blinding, sample size justification, and statistical details.

The discussion requires a more critical and in-depth interpretation, particularly regarding the plausible biological mechanisms of the extract and the role of flavonoids and phenolic compounds.

Furthermore, a dedicated limitations section is missing, including aspects such as the absence of advanced molecular analyses, chronic experimental models, and isolation of the bioactive compounds.

The conclusions are coherentwith the findings, but they could be more balanced regarding the therapeutic potential of the plant, emphasizing more clearly the need for fuurther preclinical and translational studies.

Comments on the Quality of English Language

The manuscript would benefit from careful English language editing to improve fluency, clarity, and scientific readability.

Author Response

Response to Reviewer 1

We sincerely thank you for your careful evaluation of our manuscript and for your constructive comments and suggestions. We appreciate your positive assessment of the biological relevance and potential significance of our study. We have carefully revised the manuscript according to all comments raised and believe that the revised version has been substantially improved in terms of clarity, scientific rigor, and presentation.

Our detailed responses are provided below.

The manuscript addresses an interesting topic and presents biologically plausible findings; however, it could be further strengthened in several aspects.

Comments 1: The abstract could better emphasize the innovativ nature of the study and include some key quantitative findings.

Response 1:

Thank you for this valuable suggestion. The abstract has been revised to more clearly emphasize the novelty of this work as the first in vivo pharmacological evaluation of Tordylium trachycarpum in an ethanol-induced gastric ulcer model. [Abstract, lines 19-22 and 26-30 page 1]

Comments 2: The introduction is well structured, but the literature gap should be defined more critically, also avoiding overly strong statements regarding pathways that were not directly investigated.

Response 2:

We appreciate this important observation. The Introduction has been revised to better define the current literature gap concerning the limited pharmacological and toxicological evidence available for Tordylium trachycarpum. In addition, statements regarding NF-κB and MAPK signaling pathways were removed because these pathways were not directly investigated experimentally in the current study. [Introduction, lines 44-47 and 91-99, pages 2-3]

Comments 3: The results section is clear, although in some parts it remains too descriptive. It would be useful to provide a deeper interpretation of the findings, particularly in comparison with omeprazole, and to verify some possible inconsistencies in the parameters related to gastric mucus production. In addition, the figures could be made more readable and less overloaded with statistical annotations.

Response 3:

Thank you for these constructive suggestions. The Results section has been revised to provide clearer biological interpretation of the findings. The gastric mucus data were carefully rechecked. In addition, the figures and statistical annotations were reformatted to improve readability and presentation quality. [Results, Sections 2.7–2.13, Figures 4–8, pages 7–11]

Comments 4: I did not understand why the Methods section is placed after the Discussion. In any case, it would be useful to better clarify randomization procedures, blinding, sample size justification, and statistical details.

Response 4:

We thank the reviewer for this observation. The manuscript structure follows the journal formatting template; however, the Materials and Methods section has now been substantially revised to improve clarity. Additional details regarding randomization, blinding, sample size rationale, and statistical analysis were added according to ARRIVE recommendations and reviewer suggestions. [Materials and methods, Section 4.6, lines 393-408, page 15; Section 4.7, lines 422-435, page 16; Section 4.8, lines 466-473, pages 17]

Comments 5: The discussion requires a more critical and in-depth interpretation, particularly regarding the plausible biological mechanisms of the extract and the role of flavonoids and phenolic compounds.

Response 5:

We appreciate this insightful comment. The Discussion section has been extensively revised to provide a more balanced and critical interpretation of the findings. We also clarified that although flavonoids and phenolic compounds may contribute to the observed activity, the previously identified major constituents, including methoxsalen and triphenylphosphine oxide, may also play important biological roles. The revised discussion avoids unsupported mechanistic overinterpretation. [Discussion, Section 3.3, lines 279-295, pages 12-13]

Comments 6: Furthermore, a dedicated limitations section is missing, including aspects such as the absence of advanced molecular analyses, chronic experimental models, and isolation of the bioactive compounds.

Response 6:

Thank you for this valuable recommendation. A dedicated limitations paragraph has now been added to the Discussion section. [Discussion, Section 3.5, lines 334-348, pages 13-14]

Comments 7: The conclusions are coherentwith the findings, but they could be more balanced regarding the therapeutic potential of the plant, emphasizing more clearly the need for fuurther preclinical and translational studies.

Response 7:

We agree with the reviewer. The Conclusion section has been revised to provide a more balanced interpretation of the therapeutic implications of Tordylium trachycarpum. [Conclusions, lines 475-489, pages 17]

Comments 8: The manuscript would benefit from careful English language editing to improve fluency, clarity, and scientific readability.

Response 8:

Thank you for this recommendation. The manuscript has undergone extensive English language editing to improve grammar, clarity, fluency, and scientific readability throughout the text.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents the first in vivo pharmacological evaluation of Tordylium trachycarpum Boiss. (Apiaceae), a plant used in Kurdish ethnomedicine for gastrointestinal disorders. Using the ethanol-induced gastric ulcer model in Sprague-Dawley rats, the authors report that the methanolic extract (250 and 500 mg/kg) significantly reduces gastric lesion area, increases mucus production, stabilizes gastric pH, enhances antioxidant enzyme activity (SOD and CAT), reduces lipid peroxidation (MDA), modulates pro- and anti-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6, IL-10), and regulates apoptotic markers (HSP70, Bax). Acute oral toxicity testing at doses up to 5 g/kg shows no hepatotoxic or nephrotoxic effects in either sex. The higher dose (500 mg/kg) achieves gastroprotective efficacy comparable to omeprazole (20 mg/kg). Preliminary phytochemical screening identified flavonoids, coumarins, tannins, phenolics, terpenoids, and glycosides. However, the manuscript requires major revision before acceptance.

MAJOR COMMENTS

1. Sample Size Not Justified [Section 4.7 (p. 14, line 396); Section 4.8 (p. 15, lines 445-448)]: The study uses only 6 animals per group with no explanation for why this number was chosen. A power calculation or at least a scientific justification must be provided, as the current sample size may be too small to reliably detect differences between groups.
2. Randomization and Blinding Not Described [Section 4.6 (p. 14, lines 373-381); Section 4.7 (p. 14, lines 395-405)]: The manuscript does not explain how animals were randomly assigned to groups, and there is no mention of whether the researchers measuring outcomes were blinded to treatment. This is a basic requirement for animal studies and must be addressed.
3. Mechanistic Claims Exceed the Data [Introduction (p. 2, lines 45-48; p. 3, lines 97-101); Discussion (p. 12, lines 317-325)]: The Introduction and Discussion claim that the extract works through NF-κB and MAPK signaling pathways, but these pathways were never actually measured in the study. Claims must be limited to what was directly tested, or the relevant experiments must be added.
4. Mucus Data Description is Contradictory [Section 2.8 (p. 7, lines 174-178); Table 6 (p. 8)]: Section 2.8 states the extract "reduced mucus synthesis," but the data clearly show the extract increased mucus production compared to the ulcer group. This factual error must be corrected to avoid misleading readers.
5. Statistical Symbols Are Inconsistent [Figure 7 legend (p. 10, lines 218-223); Figure 8 legend (p. 10, lines 231-235)]: The significance symbols used in Figures 7 and 8 do not follow standard scientific convention and differ between the two figures. All statistical notation must be standardized and corrected throughout.
6. Numbering Errors and Conflicting Data [Sections 2.2-2.6 (pp. 4-6, lines 114-156); Table 3 (p. 6, lines 142-145)]: Section 2.2 appears twice, Section 2.3 is missing, and the female rat liver data shows a significant ALT increase in the table that the text incorrectly describes as non-significant. These errors must be corrected as they affect the safety conclusions.
7. Active Compounds Not Properly Identified [Section 3 Discussion (pp. 11-12, lines 282-287); Conclusions (p. 16, lines 457-461)]: The study attributes its effects to flavonoids and phenolics, but the group's own previous chemical analysis showed the main compounds are methoxsalen and triphenylphosphine oxide, which are neither. The discussion must be revised to honestly address which compounds are likely responsible for the observed activity.

MINOR COMMENTS

1. The word "immunohistochemical" in the abstract is incorrect — the study actually used a blood-based ELISA test, not tissue staining. Correct this throughout.
2. Some references cited to support key claims in the Introduction are unrelated to those claims and should be replaced with appropriate sources.
3. The actual p-values for liver and kidney parameters should be reported in the tables instead of only stating "no significant differences."
4. A 34% increase in urea levels in male rats, even if statistically non-significant, deserves reporting with exact values rather than being dismissed in one sentence.
5. The Figure 4 caption incorrectly implies both extract doses performed worse than omeprazole, when the high dose showed comparable results. Revise for accuracy.
6. The pH value quoted in the text for the treated group appears to actually belong to the ulcer control group. Check and correct.
7. The speculative sentence suggesting the extract may have enhanced liver function should be removed as it is unsupported by the data.
8. The extraction method is described as maceration but actually involved ultrasonication — the correct term should be used. Also clarify whether the crude or purified extract was tested biologically.
9. The OECD toxicity guideline cited (No. 425) does not match the experimental design used. Verify and cite the correct guideline.
10. There is no vehicle-matched control group for the extract, meaning the effect of the solvent alone cannot be ruled out. Acknowledge this as a limitation.
11. The pH meter was calibrated between pH 4 and 7, but gastric pH values near 2.7 fall outside this range. Confirm measurement accuracy or note it as a limitation.
12. Reference numbering contains errors starting from Reference 17, including a misplaced URL. Correct the entire reference list from that point.
13. IL-1β, IL-6, and TNF-α are described as "anti-inflammatory cytokines" when they are in fact pro-inflammatory. Correct this throughout the manuscript.
14. Several sentences contain redundant phrasing, grammatical errors, and unclear language. A professional English language edit is strongly recommended before resubmission.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2

We sincerely thank you for your careful evaluation of our manuscript and for the constructive comments and suggestions. We have revised the manuscript extensively to improve its scientific quality, clarity, and methodological transparency. All revisions have been highlighted in the revised manuscript.

Below, we provide a detailed point-by-point response to all comments.

Major Comments

Comments 1: Sample Size Not Justified [Section 4.7 (p. 14, line 396); Section 4.8 (p. 15, lines 445-448)]: The study uses only 6 animals per group with no explanation for why this number was chosen. A power calculation or at least a scientific justification must be provided, as the current sample size may be too small to reliably detect differences between groups.

Response 1:

Thank you for this important comment. We have now clarified the rationale for the sample size in the revised manuscript. The sample size was selected based on previous published studies using ethanol-induced gastric ulcer models and in accordance with standard experimental practice for preliminary in vivo pharmacological investigations (References 2 and 10). We also aimed to minimize animal use in compliance with ethical principles and ARRIVE recommendations. In the acute oral toxicity study, animals were allocated at n = 10 per group, whereas in the ethanol-induced gastric ulcer model, n = 6 animals per group were used, consistent with commonly accepted practice in similar gastroprotective studies. A statement explaining the sample size rationale has been added to the Materials and Methods section. [Materials and Methods, Section 4.6, lines 393-408, page 15; Section 4.7, lines 422-435, page 16; Section 4.8, lines 466-473, page 17].

Comments 2: Randomization and Blinding Not Described [Section 4.6 (p. 14, lines 373-381); Section 4.7 (p. 14, lines 395-405)]: The manuscript does not explain how animals were randomly assigned to groups, and there is no mention of whether the researchers measuring outcomes were blinded to treatment. This is a basic requirement for animal studies and must be addressed.

Response 2:

We appreciate this observation. The manuscript has been revised to clarify that animals were randomly allocated into experimental groups. We also clarified that blinding was not implemented during treatment administration or outcome assessment, which has now been acknowledged as a study limitation. Additional details regarding randomization and blinding have been included in the Materials and Methods section. [Materials and methods, Section 4.6, lines 393-408, page 15; Section 4.7, lines 422-435, page 16; Section 4.8, lines 466-473, page 17; Discussion, Section 3.5, lines 335-348, pages 13-14].

Comments 3: Mechanistic Claims Exceed the Data [Introduction (p. 2, lines 45-48; p. 3, lines 97-101); Discussion (p. 12, lines 317-325)]: The Introduction and Discussion claim that the extract works through NF-κB and MAPK signaling pathways, but these pathways were never actually measured in the study. Claims must be limited to what was directly tested, or the relevant experiments must be added.

Response 3:

We agree with the reviewer. The manuscript has been revised to avoid overinterpretation of the data. Statements referring to direct modulation of NF-κB and MAPK pathways have been removed throughout the Introduction, and Discussion sections. The revised text now limits mechanistic interpretations to antioxidant, anti-inflammatory, and anti-apoptotic effects supported directly by the experimental findings. [Introduction, lines 44-47, page 2; lines 91-99, page 3; Discussion, Section 3.4, lines 326-333, page 13].

Comments 4: Mucus Data Description is Contradictory [Section 2.8 (p. 7, lines 174-178); Table 6 (p. 8)]: Section 2.8 states the extract "reduced mucus synthesis," but the data clearly show the extract increased mucus production compared to the ulcer group. This factual error must be corrected to avoid misleading readers.

Response 4:

The statement has been corrected. The revised text now correctly states that the extract increased gastric mucus production compared with the ulcer control group. The incorrect description in Section 2.8 has been corrected. [Results, Section 2.8, lines 177-179, page 8].

Comments 5: Statistical Symbols Are Inconsistent [Figure 7 legend (p. 10, lines 218-223); Figure 8 legend (p. 10, lines 231-235)]: The significance symbols used in Figures 7 and 8 do not follow standard scientific convention and differ between the two figures. All statistical notation must be standardized and corrected throughout.

Response 5:

We appreciate the reviewer’s careful observation. Performed, statistical symbols and significance annotations in all figures and figure legends have now been standardized according to conventional scientific notation. [Figure 7 legend, lines 222-226, page 10; Figure 8 legend, lines 234-239, page 11]

Comments 6: Numbering Errors and Conflicting Data [Sections 2.2-2.6 (pp. 4-6, lines 114-156); Table 3 (p. 6, lines 142-145)]: Section 2.2 appears twice, Section 2.3 is missing, and the female rat liver data shows a significant ALT increase in the table that the text incorrectly describes as non-significant. These errors must be corrected as they affect the safety conclusions.

Response 6:

Thank you for highlighting these issues. Section numbering throughout the Results section has been corrected. We also carefully rechecked all biochemical data and corrected the description regarding ALT values in female rats to ensure consistency between the text and tables. [Results, Section 2.3, line 122; Section 2.4, lines 135-137, page 5].

Comments 7: Active Compounds Not Properly Identified [Section 3 Discussion (pp. 11-12, lines 282-287); Conclusions (p. 16, lines 457-461)]: The study attributes its effects to flavonoids and phenolics, but the group's own previous chemical analysis showed the main compounds are methoxsalen and triphenylphosphine oxide, which are neither. The discussion must be revised to honestly address which compounds are likely responsible for the observed activity.

Response 7:

Thank you for this valuable comment. We agree that our previous GC–MS analysis identified methoxsalen and triphenylphosphine oxide among the major detected constituents. However, GC–MS mainly detects volatile and low-molecular-weight compounds, whereas our preliminary phytochemical screening also confirmed the presence of flavonoids, phenolics, coumarins, terpenoids, and glycosides. Accordingly, we have revised the Discussion and Conclusions sections to avoid attributing the observed activity solely to flavonoids and phenolics. We now state that the gastroprotective effects may result from the combined action of multiple phytochemical constituents present in the extract. We also acknowledge that further studies are needed to identify the specific compounds responsible for the observed biological activity. [Discussion, Section 3.3, lines 280-295, pages 12–13; Section 3.5, lines 335-348, pages 14-15; Conclusions, lines 475-489, page 17].

Minor Comments

1. The word "immunohistochemical" in the abstract is incorrect — the study actually used a blood-based ELISA test, not tissue staining. Correct this throughout.

Corrected. The term “immunohistochemical” was replaced with “ELISA-based analysis” where appropriate.

2. Some references cited to support key claims in the Introduction are unrelated to those claims and should be replaced with appropriate sources.

The Introduction section was carefully reviewed, and unrelated or weak references were replaced with more appropriate and relevant citations.

3. The actual p-values for liver and kidney parameters should be reported in the tables instead of only stating "no significant differences."

Thank you for this valuable comment. To improve transparency, the exact p-values for liver and kidney biochemical parameters have now been provided in the Supplementary Materials (Figures S1–S4).

4. A 34% increase in urea levels in male rats, even if statistically non-significant, deserves reporting with exact values rather than being dismissed in one sentence.

Thank you for this comment. Urea levels increased dose-dependently from 27.66 ± 2.11 mg/dL in the control group to 37.02 ± 3.82 mg/dL in the high-dose group; however, the change was not statistically significant (p > 0.05). Creatinine and uric acid levels remained unchanged among groups. The text revised and changed. [Section 2.9, line 181-188, page 8]

5. The Figure 4 caption incorrectly implies both extract doses performed worse than omeprazole, when the high dose showed comparable results. Revise for accuracy.

Thank you for this important observation. We agree that the original Figure 4 caption may have inaccurately implied that both extract-treated groups were less effective than omeprazole. The caption has now been revised to clarify that the higher dose (500 mg/kg) of T. trachycarpum demonstrated gastroprotective effects comparable to the omeprazole-treated group. [Section 2.7, Figure 4, lines 170-175, page 7] 

6. The pH value quoted in the text for the treated group appears to actually belong to the ulcer control group. Check and correct.

Thank you for identifying this error. The gastric pH values were carefully rechecked against the original dataset, and a typographical error was corrected in the Results section. The corrected value is now reported consistently throughout the manuscript and tables. This correction does not affect the statistical analysis or study conclusions. [Section 2.9, lines 181-188, page 8; Table 6, page 8]

7. The speculative sentence suggesting the extract may have enhanced liver function should be removed as it is unsupported by the data.

Thank you for this observation. We agree that this statement was not directly supported by the presented data. Therefore, the speculative sentence suggesting enhanced liver function has been removed from the Discussion section.

8. The extraction method is described as maceration but actually involved ultrasonication — the correct term should be used. Also clarify whether the crude or purified extract was tested biologically.

The extraction procedure has been clarified. The method is now described as ultrasound-assisted extraction. We also clarified that the biologically tested material was the purified methanolic extract. [Section 4.2, lines 362-375, pages 14-15]

9. The OECD toxicity guideline cited (No. 425) does not match the experimental design used. Verify and cite the correct guideline.

Thank you for this important observation. We carefully reviewed the OECD guideline citation and agree that OECD Guideline No. 425 did not accurately correspond to the experimental design used in this study. The manuscript has been corrected to cite OECD Guideline No. 423 (Acute Oral Toxicity – Acute Toxic Class Method), which more appropriately reflects the grouped dosing protocol and acute toxicity assessment performed. [Section 4.6, line 393, page15]. 

Reference: Organization for Economic Co-operation and Development (OECD) (2002) Test No. 423: Acute Oral Toxicity – Acute Toxic Class Method. OECD Guidelines for the Testing of Chemicals, Section 4. Paris: OECD Publishing. Available at: OECD Guideline 423 .

10. There is no vehicle-matched control group for the extract, meaning the effect of the solvent alone cannot be ruled out. Acknowledge this as a limitation.

We acknowledge this limitation and have now included it in the Discussion section.

11. The pH meter was calibrated between pH 4 and 7, but gastric pH values near 2.7 fall outside this range. Confirm measurement accuracy or note it as a limitation.

Thank you for pointing this out. We have carefully checked the manuscript and found that the reported gastric pH value of 2.7 was a typographical error. The correct value is 4.7. The manuscript has been corrected accordingly. This correction does not affect the results, statistical analysis, or conclusions of the study. [Table 6, page 8] 

12. Reference numbering contains errors starting from Reference 17, including a misplaced URL. Correct the entire reference list from that point.

The entire reference list was carefully revised and corrected.

13. IL-1β, IL-6, and TNF-α are described as "anti-inflammatory cytokines" when they are in fact pro-inflammatory. Correct this throughout the manuscript.

Corrected throughout the manuscript. These cytokines are now properly described as pro-inflammatory cytokines.

14. Several sentences contain redundant phrasing, grammatical errors, and unclear language. A professional English language edit is strongly recommended before resubmission.

The manuscript has undergone extensive English language editing to improve grammar, clarity, readability, and scientific presentation.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No additional comments

Comments on the Quality of English Language

The manuscript would benefit from careful English language editing to improve fluency, clarity, and scientific readability.

Reviewer 2 Report

Comments and Suggestions for Authors

The author has modified the manuscript as per suggestion.